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Identification and Characterization of Resistance to Phytophthora capsici within Squash (Cucurbita Spp.)

Permanent Link: http://ufdc.ufl.edu/UFE0022890/00001

Material Information

Title: Identification and Characterization of Resistance to Phytophthora capsici within Squash (Cucurbita Spp.)
Physical Description: 1 online resource (74 p.)
Language: english
Creator: Padley, Leslie
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: capsici, cucurbita, cucurbitaceae, cucurbits, moschata, pepo, phytophthora, resistance, squash, summer, winter
Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Phytophthora capsici Leonian causes several disease syndromes on squash (Cucurbita spp.) including crown rot, foliar blight, and fruit rot, which can lead up to 100% crop loss. Currently, there are no summer or winter squash cultivars resistant to this pathogen which can aid in disease management strategies. I evaluated 115 summer squash (C. pepo) accessions for their response to crown inoculation with a suspension of P. capsici isolates. Replicates of each accession were rated on a scale ranging from 0 (no symptoms) to 5 (plant death). Mean disease rating scores (DRS) and standard deviations ranged from 1.3 to 5.0 and 0 to 2.0, respectively. Accessions with the lowest mean DRS were rescreened and paralleled those of the initial study with PI 181761 exhibiting the lowest mean DRS at 0.5. Further screening and selection from the C. pepo germplasm collection will aid in the development of summer squash cultivars with P. capsici crown rot resistance. A series of interspecific hybridizations of two wild Cucurbita species with winter squash (C. moschata) led to the development of Cucurbita breeding line #394 which segregated for resistance to P. capsici crown rot. Additional selections of #394 for resistance to P. capsici crown rot were performed. Breeding line #394-1-27-12 was created and is homozygous resistant to P. capsici crown rot. The inheritance of resistance to P. capsici found within #394-1-27-12 was determined through pollination with ?Butterbush? a susceptible butternut-type winter squash (C. moschata). Segregation ratios of the F2 and BC progeny of this cross support a model in which resistance to P. capsici crown rot, within #394-1-27-12, is conferred by three dominant genes. Introgression of P. capsici crown rot resistance from #394-1-27-12 into the morphologically diverse domesticates within Cucurbita is currently underway. My research identified sources of resistance to P. capsici within summer squash (C. pepo), developed a Cucurbita breeding line with P. capsici crown rot resistance, and determined the inheritance of P. capsici crown rot resistance introgressed from two Cucurbita wild species. Summer and winter squash breeding material developed from this project will aid in the disease management of P. capsici.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Leslie Padley.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Kabelka, Eileen.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022890:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022890/00001

Material Information

Title: Identification and Characterization of Resistance to Phytophthora capsici within Squash (Cucurbita Spp.)
Physical Description: 1 online resource (74 p.)
Language: english
Creator: Padley, Leslie
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: capsici, cucurbita, cucurbitaceae, cucurbits, moschata, pepo, phytophthora, resistance, squash, summer, winter
Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Phytophthora capsici Leonian causes several disease syndromes on squash (Cucurbita spp.) including crown rot, foliar blight, and fruit rot, which can lead up to 100% crop loss. Currently, there are no summer or winter squash cultivars resistant to this pathogen which can aid in disease management strategies. I evaluated 115 summer squash (C. pepo) accessions for their response to crown inoculation with a suspension of P. capsici isolates. Replicates of each accession were rated on a scale ranging from 0 (no symptoms) to 5 (plant death). Mean disease rating scores (DRS) and standard deviations ranged from 1.3 to 5.0 and 0 to 2.0, respectively. Accessions with the lowest mean DRS were rescreened and paralleled those of the initial study with PI 181761 exhibiting the lowest mean DRS at 0.5. Further screening and selection from the C. pepo germplasm collection will aid in the development of summer squash cultivars with P. capsici crown rot resistance. A series of interspecific hybridizations of two wild Cucurbita species with winter squash (C. moschata) led to the development of Cucurbita breeding line #394 which segregated for resistance to P. capsici crown rot. Additional selections of #394 for resistance to P. capsici crown rot were performed. Breeding line #394-1-27-12 was created and is homozygous resistant to P. capsici crown rot. The inheritance of resistance to P. capsici found within #394-1-27-12 was determined through pollination with ?Butterbush? a susceptible butternut-type winter squash (C. moschata). Segregation ratios of the F2 and BC progeny of this cross support a model in which resistance to P. capsici crown rot, within #394-1-27-12, is conferred by three dominant genes. Introgression of P. capsici crown rot resistance from #394-1-27-12 into the morphologically diverse domesticates within Cucurbita is currently underway. My research identified sources of resistance to P. capsici within summer squash (C. pepo), developed a Cucurbita breeding line with P. capsici crown rot resistance, and determined the inheritance of P. capsici crown rot resistance introgressed from two Cucurbita wild species. Summer and winter squash breeding material developed from this project will aid in the disease management of P. capsici.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Leslie Padley.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Kabelka, Eileen.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022890:00001


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IDENTIFIC ATION AND CHARACTERIZATION OF RESISTANCE TO PHYTOPHTHORA CAPSICI WITHIN SQUASH (CUCURBITA SPP.) By LES DEAN PADLEY, JR. A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1

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2008 Les Dean Padley, Jr. 2

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To m y wife, Michelle Cook Padley; my son, Jona than Edwin Padley; and both of my families I would not have accomplished so much or be where I am today without your love and guidance. Thank You All. 3

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ACKNOWL EDGMENTS I would like to thank Dr. Eil een Kabelka for everything she has done for me. Her guidance will help me in the years to come. I would also like to thank Dr. Jose Chaparro, Dr. Pamela Roberts, and Dr. Steven Sargent for giving me the opportunity to obtain this degree. I would finally like to thank the late Dr. Leandro Ra mos, who initiated the search for sources of resistance to Phytophthora capsici in squash, more than a decade ago. I am grateful to everyone. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 ABSTRACT .....................................................................................................................................9 CHAPTER 1 LITERATURE REVIEW.......................................................................................................11 Introduction .............................................................................................................................11 Squash ( Cucurbita spp.) .........................................................................................................12 Taxonomy ........................................................................................................................12 Production ........................................................................................................................13 Cultivation .......................................................................................................................14 Postharvest Practices .......................................................................................................15 Phytophthora capsici ..............................................................................................................16 Host Range ......................................................................................................................17 Disease Symptoms ...........................................................................................................17 Reproduction ...................................................................................................................18 Disease Management .......................................................................................................19 Host Resistance ...............................................................................................................20 2 EVALUATION OF CUCURBITA PEPO ACCESSIONS FOR CROWN ROT RESISTANCE TO SQUASH ISOLATES OF PHYTOPHTHORA CAPSICI .....................24 Introduction .............................................................................................................................24 Materials and Methods ...........................................................................................................25 Plant Material ..................................................................................................................25 Phytophthora capsici Isolates and Inoculum Preparation ...............................................25 Greenhouse Studies, Inoculation, and Sc oring for Response to Inoculation ..................26 Results and Discussion ...........................................................................................................27 3 A CUCURBITA BREEDING LINE WI TH CROWN ROT RESISTANCE TO PHYTOPHTHORA CAPSICI DERI VED FR OM WILD CUCURBITA SPECIES.............37 Introduction .............................................................................................................................37 Materials and Methods ...........................................................................................................38 Plant Material ..................................................................................................................38 Greenhouse Studies and Single Plant Selections for Hom ozygosity to P. capsici Crown Rot Resistance ..................................................................................................38 Phytophthora capsici Isolates and Inoculum Preparation ...............................................40 5

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Phytophthora capsici Crown Inoculation, Scoring for Response and Data Analysis .....41 Results and Discussion ...........................................................................................................41 4 INHERITANCE OF RESISTANCE TO CROWN ROT CAUSED BY PHYTOPHTHORA CAPSICI IN CUCURBITA. .................................................................50 Introduction .............................................................................................................................50 Materials and Methods ...........................................................................................................51 Plant Material ..................................................................................................................51 Phytophthora capsici Isolates and Inoculum Preparation ...............................................51 Experimental Design and Data Analysis .........................................................................52 Results and Discussion ...........................................................................................................53 5 OVERALL CONCLUSIONS.................................................................................................57 APPENDIX A RESPONSE OF CUCURBITA PEPO ACCESSIONS TO CROWN INOCULATION W ITH SUSPENSIONS OF PHYTOPHT HORA CAPSICI ISOLATED FROM TOMATO AND PEPPER......................................................................................................61 B RESPONSE TO CROWN INOCULAT ION USING THREE DIFFERENT PHYTOPHTHORA CAPSICI ZOO SPORE CONCENTRATIONS.....................................66 LIST OF REFERENCES ...............................................................................................................68 BIOGRAPHICAL SKETCH .........................................................................................................74 6

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LIST OF TABLES Table page 1-1 Plant species susceptible to Phytophthora capsici. ................................................................22 2-1 Response of Cucurbita pepo accessions to a suspension of Phytophthora capsici squash isolates fro m Florida. Acce ssions are ranked according to their mean disease rating score (DRS). .......................................................................................................................30 2-2 Response of eight selected accessions of Cucurbita p epo to a suspension of Phytophthora capsici isolates from Florida. ......................................................................34 3-1 Description and response of Cucurbita lundelliana PI 438542, C. okeechobeeness sbsp. okeechobeenesis 19 Cu curbita breeding lines, and suscep tible controls Yellow Summer Squash ( C. pepo ) and Early Prolific Straightneck ( C. pepo) to a suspension of Phytophthora capsici isolates.z...................................................................44 3-2 Response of Cucurbita breeding line #394, #394-1, a nd susceptible control EarlyProlific Straightneck ( C. pepo ) to a suspension of Phytophthora capsici isolates. ...............................................................................................................................45 3-3 Response of Cucurbita breeding line #394-1-27, rooted cuttings of #394-1-27, and susceptible control Butterbush ( C. moschata ) to a suspension of Phytophthora capsici iso lates. ..................................................................................................................46 3-4 Response of Cucurbita breeding lines #394-1-27, #394-1-27-12 and susceptible control Butterbush ( C. moschata) to a suspension of Phytophthora capsici isolates. .................47 4-1 Segregation for resistance to Phytophthora capsici crown inoculation in Cucurbita breeding line #394-1-27-12, Butterbush, F F and BC progeny.1 2.................................55 A-1 Response of Cucurbita pepo accessions to a suspension of Phytophthora capsici isola ted from tomato in Florida. Acce ssions are ranked accord ing to their mean disease rating score (DRS). ................................................................................................62 A-2 Response of Cucurbita pepo accessions to a suspension of Phytophthora capsici isolated from pepper in Florida. Acce ssions are ranked acco rding to their mean disease rating score (DRS). ................................................................................................64 B-1 Response of Cucurbita pepo PI 179267, PI 181761 and their selfed progeny to a suspension of Phytophthora capsici squash isolates at 100,000, 50,000 and 25,000 zoospore co ncentrations. ....................................................................................................67 7

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LIST OF FI GURES Figure page 2-1 Geographic location of 115 Cucurbita pepo accessions representing 24 countries. ...............35 2-2 Disease rating scale (0-5) for Phytophthora capsici crown inoculation on Cucurbita p epo ...................................................................................................................36 3-1 Disease rating scale (0-5) for Phytophthora capsici crown inoculation on Cucurbita moschata ..........................................................................................................48 3-2 Pedigree of Phytophthora capsici crown rot resistant Cuc urbita breeding line #394-127-12. .................................................................................................................................49 4-1 Response of Cucurbita breeding line #394-1-27-12 a nd Butterbush to crown inoculation with a suspension of Phytophthora capsici isolates. ......................................56 8

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy IDENTIFICATION AND CHARACTERIZATION OF RESISTANCE TO PHYTOPHTHORA CAPSICI WITHIN SQUASH (CUCURBITA SPP.) By Les Dean Padley, Jr. December 2008 Chair: Eileen Kabelka Major: Horticultural Science Phytophthora capsici Leonian causes several disease syndromes on squash ( Cucurbita spp.) including crown rot, foliar blight, and fru it rot, which can lead up to 100% crop loss. Currently, there are no summer or winter squash cultivars resistant to this pathogen which can aid in disease management strategies. I evaluated 115 summer squash ( C. pepo ) accessions for their response to crown inoculation with a suspension of P. capsici isolates. Replicates of each accession were rated on a scale ranging from 0 (no symptoms) to 5 (plant death). Mean disease rating scores (DRS) and standard deviations ranged from 1.3 to 5.0 and 0 to 2.0, respectively. Accessions with the lowest mean DRS were rescreened a nd paralleled those of the initial study with PI 181761 exhibiting the lowest mean DRS at 0.5. Further screening and selection from the C. pepo germplasm collection will aid in the development of summer squash cultivars with P. capsici crown rot resistance. A series of interspecific hybridizations of two wild Cucurbita species with winter squash ( C. moschata) led to the development of Cucurbita breeding line #394 which segregated for resistance to P. capsici crown rot. Additional selec tions of #394 for resistance to P. capsici crown rot were performed. Breeding line #394-127-12 was created and is homozygous resistant 9

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to P. capsici crown rot. The inher itance of resis tance to P. capsici found within #394-1-27-12 was determined through pollination with Butterbus h a susceptible butternut-type winter squash ( C. moschata). Segregation ratios of the F2 and BC progeny of this cross support a model in which resistance to P. capsici crown rot within #394-1-27-12, is conf erred by three dominant genes. Introgression of P. capsici crown rot resistance from #394-1-27-12 into the morphologically diverse domesticates within Cucurbita is currently underway. My research identified sources of resistance to P. capsici within summer squash ( C. pepo), developed a Cucurbita breeding line with P. capsici crown rot resistance, and determined the inheritance of P. capsici crown rot resistance introgressed from two Cucurbita wild species. Summer and winter squash breedin g material developed from this project will aid in the disease management of P. capsici 10

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11 CHAPTER 1 LITERATURE REVIEW Introduction Squash, pumpkins and gourds ( Cucurbita spp.) rank among the t op producing vegetable crops in the world (FAOSTAT, 2007a). From this worldwide production, the United States ranked 5th, in 2006, producing 861,000 metric tons of which squash represented 474,100 metric tons (FAOSTAT, 2007b). This 474,100 metric t ons of yield grossed over $229 million in sales for the United States squash industry making th is vegetable crop a multimillion dollar business (USDA, 2007a). Squash is affected by many pathogens and pest s (Zitter et al., 1996). One of the most devastating is the oomycetous pathogen, Phytophthora capsici The incidence of disease caused by P. capsici on cucurbits has increased with reported yield loss as high as 100% (Hausbeck and Lamour, 2004; Tian and Babadoost, 2004). Phytophthora capsici can infect cucurbits at any growth stage and is capable of causing crown rot, foliar blight, and fruit rot (Zitter et al., 1996; Roberts et al., 2001). Given optimal conditions an entire field of cucurbits can be devastated by P. capsici in a matter of days (Bab adoost, 2004; Hausbeck and Lamour, 2004; Lee et al., 2001; Roberts et al., 2001). With the increased occurrence and severity of P. capsici research for management alternatives, including breeding cucu rbits for resistance, is key in effectively managing this pathogen (Hausbeck and Lam our, 2004; French-Monar et al., 2005; Keinath, 2007). Currently, there are no summer or winter squash cultivars re sistant to the various disease syndromes caused by P. capsici Germplasm collections, represen ting wild and exotic materi al, are valuable sources of beneficial genes and have been used to iden tify sources of resistan ce to numerous plant pathogens (Herrington et al., 2001; Paris and Cohen, 2000; Stephens, 2003; USDA, 2006c,d).

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For summer squash, a C. pepo germ plasm collection, mainta ined at the USDA-ARS North Central Regional Plant Introduction Station (NCRPIS), Ames, Iowa, has more than 900 C. pepo accessions available for evaluation. For winter squash, resistance to P. capsici derived from two Cucurbita wild species, C. lundelliana and C. okeechobeenesis sbsp. okeechobeenesis has recently been introgressed into a C. moschata genetic background (Kabelka et al., 2007). One particular breeding line, designate d #394, exhibits heterozygosity fo r resistance to the crown rot syndrome of P. capsici and is of particular interest for continued selection to produce a homozygous crown rot resistant winter squash breeding line. This research was initiated to identify sources of resistance to P. capsici for introgression into summer squash ( C. pepo ) and to better understand the nature of P. capsici resistance recently introgressed into winter squash ( C. moschata). While there are several disease syndromes of P. capsici this research focuses on crown rot with the long-term goal of developing advanced breeding material with resistance. The specific objectives of this research projec t were: (1) identify sources of resistance to P. capsici crown rot within the C. pepo germplasm collection; (2) develop a homozygous P. capsici crown rot resistant Cucurbita breeding line; and (3) char acterize the resistance to P. capsici crown rot found within Cucurbita breeding line #394. The successful completion of this research will not only aid in the disease management of P. capsici within squash but will result in continued productivity and profitabi lity of both summer and winter squash. Squash ( Cucurbita spp.) Taxonomy The genera Cucurbita within the family Cucurbitaceae, consists of 27 species native to the Western hemisphere (Wilson, 1990). Of these 27 species, five are cultivated and include C. pepo, C. moschata C. maxima C. argyrosperma (formerly C. mixta) and C. ficifolia 12

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(Robinson and Decker-Walters, 1999). Cucurbita cultivars are categorized as summ er squash or winter squash. Summer squash are eaten immature when tender and seeds are small and soft. Winter squash are generally eaten when rind and seeds are fully mature. Summer squash cultivars are C. pepo while winter squash cultivars can be C. pepo C. maxima C. moschata, or C. argyrosperma Fruit shape within C. pepo is the basis for ten horticultural classifications of this species (Paris, 1986; 1996). These include eight edible-fruited cultivar groups designated pumpkin, vegetable marrow, cocozelle, zucchini, acorn, sc allop, crookneck, and straightneck and two nonedible-fruited ornamental cultivar groups design ated orange gourd and ovifera gourd. There is considerable morphol ogical diversity of C. moschata and horticultural groupings, based on market-type cultivars, include neck, cheese, tr opical, and japonica (Bates et al., 1990; Robinson and Decker-Walters, 1999). Cucurbita maxima exhibits greater divers ity of fruit types than C. pepo (Bates et al., 1990; Robinson and Decker-W alters, 1999). Severa l horticultural groups based on market-type describe this species and include Australian blue, banana, buttercup, hubbard, mammoth, and turban. Cultivars of C. argyrosperma and C. ficifolia are produced primarily for their seed which provides a c onsiderable amount of oil and protein. Production In 2006, squash, pumpkins and gourds were ranked 11th in terms of production among worldwide vegetable crops with a total yi eld of 21,003,464 tons (FAOSSTAT, 2007b). The top producing countries for squash, pumpkins and gourds were China (6,060,250 tons), India (3,678,413 tons), Russian Federation (1,184,670 tons ), Ukraine (1,064,000 t ons) and the United States (861,870 tons). From the 861,870 tons of squash, pumpkins and gourds produced in the United States, squash composed 55% (474,100 tons) of this total with a va lue of approximately $229 million. Of this $229 million industry, Flor ida ranked second in the nation earning 38 13

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m illion dollars in revenue. Other top states, in terms of revenue from squash production, include Georgia ($49,920,000), California ($37,929,000), New York ($28,274,000) and Michigan ($14,994,000) (USDA, 2007b). Cultivation Squash are annual herbaceous bus hes or vines that ar e typically grown in the fall or spring when the threat of freeze or frost is over (Pee t, 2001a). Seeds are planted in bare ground, or raised beds, with a soil high in organic ma tter and a ph between 6 and 6.5. Optimum growing temperature for squash is between 24 to 29 C during the day and 16 to 21C at night. Temperatures below 4.4C for several days can ca use severe damage to the plants, and exposure to temperatures above 29C can cause fruit dr op along with small fruit size (Peet, 2001b). Like other cucurbits, squash can be transplanted fr om greenhouse to field to increase earliness and decrease the chance of frost damage. Flowers on squash plants are monoecious, produced just above the axil of the leaf, and are conspicuously bright yellow to orange in color (Swiader & Ware, 2002). Female flowers are easily distingu ished from male flowers due to the developing ovary at the base of the flower. Typically male flowers are produced 3-4 days before the female flowers with a ratio of 3:1 male to female flowers, developing as the plant grows. Changes in photoperiod and temperature can a ffect the flower ratio and bl ooming time (Stephens, 2003). Winter squash are grown for a period of 80 to 140 days until fruit reach full maturity (Swiader & Ware, 2002). Summer squash are grown for 40-60 days before producing marketable fruit after the first pollinations. Sunny dry weather is needed throughout the growing season for optimum fruit production. For pollination, honey bees are required for squash due to the large pollen size and the short pollination window, 8-10 a.m., in which the female flowers are open. The size and shape of the fruit, along with the quant ity and thickness of the seed is directly proportional to the 14

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am ount of pollen placed onto the stigma. If ad equate pollen is not deposited on the female flower the fruit will become misshapen or po ssibly abort. One honey bee hive per acre is recommended to insure fruit set (Peet, 2001b; Swiader and Ware, 2002). High levels of fertilization are required to grow squash. The amount and frequency of fertilizer used is based on soil type, climate, plant spacing and cultural management of the crop. Postharvest Practices Maintaining the postharvest quality of summer a nd winter squash from harvest to the retail level has few similarities and many differences (Kader, 2002). Both summer and winter squash are non-climacteric fruit, meani ng they do not undergo a final ripening caused by the release of ethylene. This lack of color change and conve rsion of starch to suga rs during final ripening allows squash fruit to be at horticultural mature when harvested. Once harvested the fruit of summer and winter squash are ready to eat. Beyo nd these similarities summer and winter squash differ from harvest through re tail level in the maintenan ce of postharvest quality. Summer squash are harvested several times throughout the season at an immature stage 4060 days after planting (Swiader and Ware, 2002). Since the fruit are harvested at a young stage the rind is still very soft making the fruit more sus ceptible to damage and deterioration. Due to their soft rinds and immature state, summer squa sh are harvested by hand and the fruit are cooled immediately after harvest. Fru it are stored in a cooler at 7 C 10C with a relative humidity (RH) of 95% (Kader, 2002). The high humidity in st orage is used to compensate for the soft rind of the fruit that allows for greater water loss. Under these storage conditions summer squash can be kept at commercial standards for a maximu m of 2 weeks (Kader, 2002). Chilling injury can occur if the fruit are stored below 5C or they undergo one light freeze. Summer squash are displayed for retail sale as fresh or cut fruit in a refrigerated area due to their high perishability (McCollum, 2004). 15

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W inter squash are harvested once, by hand, at physiological matur ity, usually between 80 to 140 days after planting (Brecht, 2004). At physiological matu rity, the fruit have a strong flesh color with a hard, non-glossy, rind that is resistant to damage and deterioration (Olson et al., 2007). After harvest the fruit may be cured for 10 to 20 days at a temperature of 24C to 27C with a RH of 80%. This process allows for woun ds to heal, the rind to harden, and any immature fruit to ripen for a longer stor age life (Swiader and Ware, 2002). Fruit are room cooled and stored at 12C 15C with a RH of 50% 70% (Kader, 2002). Winter squash are less chilling sensitive then summer squash and are able to with stand one to two light fr eezes without injury. At the retail level winter s quash are displayed at room temperature (Brecht, 2004). Phytophthora capsici Phytophthora capsici Leonian is a devastating plant pathogen in the Phylum oomycete (Babadoost, 2004). This fungal-like pathogen produces mycelium that branch at 90 degree angles and reproduces asexuall y by means of sporangium and zoospores and sexually by means of oospores. Phytophthora capsici was first discovered in the fall of 1918 by Leon H. Leonian at the New Mexico Agricultural Research Station in Les Cruces. In a report published in 1922, he described a new species of phytophthor a as the cause of considerable damage to a field of chili peppers in 1918 which then reappeared in the same field and surrounding farms the next year (Leonian, 1922). Since its discovery, P. capsici has caused severe epidemics of many vegetable crops in Central and South America, Europe, As ia, Korea and the United States (Roberts et al., 2001). In the United States, P. capsici has been identified in many of the vegetable producing states including California, North and South Caro lina, Florida, Georgia, Illinois, New Jersey, Michigan and Texas (Babadoost and Islam, 2003; Caf-Filho et al., 1995; Isaleit, 2007; Lamour and Hausbeck, 2003; Ristaino, 1990). In the state of Florida, P. capsici has been known to cause 16

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severe outbreaks during unusually w et and warm w eather. During one of the last outbreaks, a survey of growers in Manatee County showed losses from P. capsici ranging from 35% in tomato, 65% in cantaloupe, 42% in bell and jalapeno peppers, 100% in squash, and 36% in watermelon (Roberts et al., 2001). Host Range Phytophthora capsici has a wide host range that incl udes a minimum of 53 susceptible species in 24 different families (Table 1-1). The families Cucurbitaceae and Solanaceae contain many horticulturally significant crops worldwide that are susceptible to P. capsici (Babadoost, 2004; Hausbeck and Lamour, 2004). The level of virulence an isolate has on a host plant can vary greatly depending on the pathogenicity of the isolate and the host/isolate interaction (Hausbeck and Lamour, 2004). Determining the virulence among P. capsici isolates is the key to developing an effective way of c ontrolling this pathogen in the fiel d. Host-specific isolates have been found in tomato and pepper (Babadoost et al., 2008; Hausbeck and Lamour, 2004; Lee et al., 2001; Ristaino, 1990). Isolates also exist th at can infect multiple hosts allowing them to survive from one growing season to another. Disease Symptoms Phytophthora capsici can cause disease on susceptible plants at any grow th stage; although immature plants of some species are more sus ceptible thin mature plants (Roberts et al., 1999; Tiam and Babadoost, 2004; Lee et al., 2001). Symptoms caused by P. capsici include seed rot (pre-emergence damping-off), seedling blight (post-emergence damping-off), root rot, and crown rot which can cause plant stunting, wilting and/or death of the entire plant in a very short period of time. Stem lesions can occur along any part of the stem in a host plant and appear as dark brown, water-soaked lesions that can girdle the plant. Leaf spot s develop when infected water lands on a leaf causing dark brown s pots to appear that will range fr om one-half to several inches 17

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in diam eter. If these leaf spots coalesce, foliar blight can occur. Symptoms of fruit infection on a susceptible host are easily recognized. It begins as a dark sunken lesion that can develop into a fine white powder-like layer of spores which can cover the entire surface of the fruit. Fruit infection can occur several days before the symp toms are visible allowing fruit rot to develop postharvest. Reproduction Phytophthora capsici can reproduce through means of sexual or asexual spores (Babadoost, 2004). There are tw o types of asexual spores; sporangium and zoospores. Sporangia are lemon-shaped spores that are prod uced on the surface of the plant or fruit. These spores can be spread through rain water, irri gation water or wind blown rain. In a moist environment, the sporangium can either directly germinated to infect a host or release smaller biflagellate swimming spores calle d zoospores. Only one zoospore is needed to infect a plant. Zoospores of this pathogen are attracted to the root exudates of a host a nd can travel for several hours through water in search of new host to infe ct (Babadoost, 2004). The zoospores ability to move through water, along with multiple infection cycles in one season, allows P. capsici to begin as a small infection that can expand to a larg e epidemic in a relatively short period of time (Hausbeck and Lamour, 2004). The sexual spores of P. capsici are called oospores (Babadoost, 2004). Oospores are produced when two compatible mating types, A1 and A2, come together and undergo sexual recombination. Oospores are formed when a ma le gametangium, called an antheridium, and a female gametangium, called an oogomium, undergo meiosis and fuse through plasmogamy and karyogamy to produce an oospore with half the genetic material from each parent. The oospores of P. capsici are thick-walled, resistant to desiccation a nd cold temperatures, and can survive in the soil for many years (Keinath, 2004). Once triggered by the proper environmental condition, 18

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the oospore can germ inate to produce a germ tube, sporangium, and/or zoospores to infect new plants (Babadoost, 2004). Disease Management Phytophthora capsici has been controlled mainly by use of fungicides. However, this practice has changed as A1 and A2 mating types have been introduced into fields allowing for mutation and recombination of the parental types which creates resistance to the various fungicides. Isolates of P. capsici from many states are now resi stant to mefenoxam, the active ingredient in a common fungicide used to control oomycetes (Hausbeck and Lamour, 2004; Roberts et al., 1999). A combination of fact ors, including water management, crop rotation, cultural practices, fumagation and resistant/tolerant crop varie ties, along with fungicides are needed to properly control the level and spread of this pathogen (Tian and Babadoost, 2004; Keinath, 2004). Proper drainage in the field is a key factor for controlling P. capsici in the field (Ristaino and Johnston, 1999). A level, well drained, field with no low lying areas will help prevent focal points for the development of epidemics. A proper irrigation plan will also help reduce the incidence of disease in the field. A study condu cted by Caf-Filho et al. (1995) showed that avoiding excessive irrigation reduc ed the loss in yield due to P. capsici in a furrow field. Furrow irrigation should also be limite d due to the easy spread of P. capsici in water from the point of origin down the field to non-infected plants (C af-Filho et al., 1995) Crop rotation is used to decrease the level of P. capsici in the field between planting of susceptible crops (Tian and Ba badoost, 2004). A minimum three years crop rotation of plants not susceptible to P. capsici is recommended to decrease th e level of oospores in the field (Hausbeck and Lamour, 2004). Rotating a crop be tween two susceptible hosts, such as pepper and cucurbits, can result in severe disease problems in the field (Ristaino, 1990) 19

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There are s everal cultural prac tices that will help manage P. capsici in the field (Babadoost, 2004). These include: growing susceptible crops on raised beds (6 inch minimum) in fields with no history of th e disease; selecting fields that are isolated from known P. capsici infected fields; scouting fields for symptoms of P. capsici ; plowing under the parts of a field with diseased plants including the healthy plan ts that border the diseased area; removing diseased fruit from the field; and cleaning farm equipment of soil when traveling between fields. The newest tool in disease scouting is the use of PCR based methods to identify P. capsici at the early stage of infection (Babadoost, 2004; Ti an and Babadoost, 2004; Hausbeck and Lamour, 2004). A proper fungicide rotation can be effective in controlling P. capsici under normal field conditions (Caf-Filho, et al., 1995 ). However, in a conducive environment, these fungicides have been proven inadequate in controlling th is pathogen. Mefenoxam, a systemic phenylamide chemical used in many fungicides by grower s, has become ineffective in controlling P. capsici due to a possible single gene mutation in th e pathogen (Lamour and Hausbeck, 2003). The fumigant, methyl bromide, has been used extensively to control P. capsici throughout the United States but due to its deleterious effects on th e ozone it is being phased out (USDA, 2006a). Alternative fungicides are being tested to manage P. capsici ; however, efficancy is limited under conductive conditions, nor does there appear to be a simple broad-spectrum fumigant to replace mefenoxam and methyl bromide. Host Resistance Resistance to P. capsici exists within certain plant spec ies. In pepper, resistance to P. capsici comes from two different sources: PI 201232 which has intermediate levels of resistance and the Mexican land race called Cri ollo de Morelos 334 which has resistance to foliar blight, stem blight, and root rot (Alcantara and Bosland, 1994; Ortega et al., 1995; Sy et 20

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al., 2005). In waterm elon, Lee et al. (2001) sc reened nine Korean and Japanese pumpkin cultivars which showed a quantit ative level of resistance to P. capsici with variety Danmatmaetdol being the most resistant. In squash, there are no known public sources of resistance to P. capsici 21

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Table 1-1. Plant species susceptible to Phytophthora capsici Fa mily Common Name Scientific Name Aloaceae Aloe Aloe sp. Apiaceae Carrot Daucus carota Araceae Flamingo lily; oilcloth flower Antherium andreanum Asteraceae Cosmos Cosmos Cav. s p. Safflower Carthamus tinctorius L Brassicaceae Cauliflower Brassica oleracea L Radish Raphanus sativus Turnip Brassica rapa Cactaceae Indian Fig Opuntia ficus-indica Mill. Caryophyllaceae Carnation Dianthus barbathus L Chenopodiaceae Beet Beta vulgaris Spinach Spinacia oleracea Swiss-chard Beta vulgaris var. cicla Cucurbitaceae Acorn squash Cucurbita moschata Blue Hubbard squash Cucurbita pepo Cantaloupe Cucumis melo Cucumber Cucumis sativus Gourd Cucurbita pepo Honeydew Melon Cucumis melo Melon Pisum melo Muskmelon Cucumis melo Pumpkin Cucurbita maxima Red Bryony; wild hop Bryonia dioica Jacq. Watermelon Citrullus lanatus Yellow squash Cucurbita pepo Zucchini squash Cucurbita pepo Ebenaceae Persimmon Diospyros kaki L. Fabaceae Alfalfa; lucerne Medicago sativa L. Broadbean Vicia faba L. Butter or civet bean Phaseolus lunatus L. Green bean Phaseolus vulgaris Lima bean Phaseolus lunatus Snow Pea Pisum sativus Lauraceae Avocado Persea americana Mill. Liliaceae Carolina geranium Geranium carolinianum Onion Allium cepa L. Linaceae Flax Linum sp. Malvaceae Cotton Gossypium hirsutum L. 22

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Table 1-1. C ontinued. Family Common Name Scientific Name Malvaceae Okra Abelmoschus esculentus Velvet-leaf Abutilon theophrasti Moraceae Fig Ficus carica Orchidaceae Vanilla Vanilla planifolia Andr. Piperaceae Betle Piper betle L. Black pepper Piper nigrum Portulacaceae Common Purslane Portulaca oleracea Proteaceae Macadamia nut Macadamia integrifolia Pincushion flower Leucospermum R. Br. Rosaceae Apple Malus pumila Mill. Hawthorn Crataegus oxyacantha L. Peach Prunus persica (L.) Batsch Rutaceae Citrus Citrus sp. Solanaceae American Black Nightshade Solanum americanum Bell pepper Capsicum annuum L. Eggplant Solanum melongena Hot pepper Capsicum annuum & Capsicum frutescens Jimson weed Datura stramonium L. Tobacco Nicotiana tabacum Tomato Lycopersicon esculentum Sterculiaceae Cocoa Theobroma cacao (Bittenbender et al., 1992; Hausbeck and Lamour, 2004; Holmes et al., 2001; Kellam and Zentmyer, 1986; Lamour and Hausbeck, 2003; Lee et al., 2001; Roberts et al., 2001; FrenchMoroa et al., 2006; Tian and Babadoost, 2004; Zentmyer, 1983). 23

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24 CHAPTER 2 EVALUATION OF CUCURBITA PEPO AC CESSIONS FOR CROWN ROT RESISTANCE TO SQUASH ISOLATES OF PHYTOPHTHORA CAPSICI Introduction The oomycetous pathogen, Phytophthora capsici Leonian, infects a wide range of plant taxa involving more than 49 sp ecies (Erwin and Ribeiro, 1996). Oospores, the sexual stage of P. capsici can survive in the soil, in crop debris, and in certain weeds for long periods of time (Zitter et al., 1996; Hausbeck and Lamour, 2004; French-Monar et al., 2006). The asexual zoospores of P. capsici contained in sporangia can be dispersed across a field by rain drops and irrigation water in a relatively short period of ti me. Given optimal conditions an entire field of crops can be devastated by P. capsici in a matter of days (Zitter et al., 1996; Roberts et al., 2001). The incidence of disease caused by P. capsici on cucurbits has increased in vegetable production regions of the U.S. with reported yiel d loss as high as 100% (Hausbeck and Lamour, 2004; Tian and Babadoost, 2004). The in creased occurrence and severity of P. capsici has prompted research for fungicide management alte rnatives and interest in breeding cucurbits for resistance or tolerance (Babadoost, 2000; Stevenson et al., 2000; 2001; Seebold and Horten, 2003; Hausbeck and Lamour, 2004; McGrath, 2004; Tian and Babadoost, 2004; Waldenmaier, 2004; French-Monar et al., 2005; Keinath, 2007). Cucurbita pepo L. (pumpkin, squash, and gourd) is an economically im portant group of the Cucurbitaceae (Paris et al., 2003). Eight cultivar-groups of edible-fruited domesticates of C. pepo have been described (Paris, 1986) which includes pumpkin, cocozelle, vegetable marrow, zucchini, acorn, scallop, crookneck, and straightneck. Phytophthora capsici can infect C. pepo at any growth stage and is ca pable of causing crown rot, folia r blight and fruit rot (Zitter et al., 1996; Roberts et al., 2001). Crown rot appears at the soil li ne causing stems to turn dark

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brown, becom e water-soaked, and quickly collapse causing plant de ath. Foliar symptoms appear as rapidly expanding, water-soaked lesions. Dieback of shoot tips, wilting, shoot rot, and plant death quickly follows initial infection. Fruit, wh ich can be infected at any stage of maturity, may exhibit sunken, brown, water-soaked areas which ar e rapidly covered by white sporangial growth under moist environmental conditions. Currently, there are no C. pepo cultivars resistant or tolerant to P. capsici (Hausbeck and Lamour, 2004). Germplasm collections are valuable sources of beneficial genes in cluding resistance or tolerance to numerous plant pathogens. The C. pepo germplasm collection maintained at the USDA-ARS North Central Regi onal Plant Introduction Statio n (NCRPIS), Ames, Iowa, has more than 900 C. pepo accessions available for evaluation (USDA, 2006b). While P. capsici causes several disease syndromes on C. pepo, the objective of this study was to evaluate a select group of C. pepo accessions for resistance to the crown rot syndrome of P. capsici Materials and Methods Plant Material Because no core collection representing C. pepo has been established to date, accessions selected for this study were based on two criter ia: fruit type and geographic location. Based on NCRPIS descriptors, accessions with oblong yello w fruit were chosen. In addition, randomly chosen representatives from each geographic lo cation of the collecti on with at least two accessions were selected. Overall, the 115 accessions selected represented 24 countries (Fig. 2-1). Susceptible controls used in th is study were two open pollinated commercial C. pepo cultivars Early Prolific Straight neck and Yellow Summer Squash. Phytophthora capsici Isolates and Inoculum Preparation Three highly virulent P. capsici mating type A1 isolates (01-1938A, RJM98-730 and RJM98-805) collected from squash were obtained from Dr. P. Roberts (University of Florid a, 25

PAGE 26

Southwest Florida Research and Education Center Immokalee, FL). Inoculum was prepared using a modified procedure based on Mitchell, 1978, Mitchell et al., 1978, and Mitchell and Kannwischer-Mitchell, 1992. For each P. capsici isolate, one 5-mm mycelial plug fro m cornm eal agar was transferred to a 20% clarified V8 agar plate. After 7 days of growth at room temperature, ten 5-mmV8 agar mycelial plugs from each plate were placed into a 20% clarified V8 broth plate to grow for an a dditional 7 days in a 28C incubat or. The V8 broth was then drained and each plate w as washed two times with st erilized distilled water. Sterilized distilled water was added to cover mycelial growth in all plates which were then placed under incandescent lights at 28 -30C to induce spora ngial development. After 24 h, sporangia were chilled at 4 C for 45 min to induce zoospore release. The mycelia from each plate were strained through cheesecloth and a 1-ml encysted zoospore sample was counted using a hemacytometer. A suspension of the three isolates containing equal portions of each, was prepared at a concentration of 2x104 zoospores/ml. Greenhouse Studies, Inoculation, and Sc oring for Response to Inoculation The selected C. pepo accessions were evaluated in two separate studies. The first study evaluated accessions based on fruit type (71 acce ssions). The second study evaluated accessions based on geographic location (44 accessions). For each study, a randomized complete block design was used. Eight blocks containing a single seed of each accession and the two susceptible controls were sown in standard 18 cell flats containing Fafard #3S potting mix (Fafard Inc., Agawam, MA). Not all of the 115 accessions germ inated in all eight replications. Greenhouse temperatures were maintained between 19C to 34C. Seedlings were watered daily and at the cotyledon stage each received 1 g of slow-release fertilizer (Osmocote 14-14-14 NPK, Grace Sierra Horticulture Products, Milpitas, CA). At the second to third true-leaf-stage, each seedling was inoculated at its crown with 5 ml of the 2x104 zoospores/ml suspension of P. capsici Prior 26

PAGE 27

to inoculation, the potting m ix was watered and remained saturated for 24-36 hours to optimize the zoospore infection process. Fourteen days af ter inoculation, the plants were visually rated based on a scale ranging from 0 to 5; where 0 = no symptoms, 1 = small brown lesion at base of stem, 2 = lesion has progressed up to the cotyledons causing constr iction at the base, 3 = plant has partially collapsed with apparent wilting of leaves, 4 = plant has completely collapsed with severe wilting present, and 5 = plant death (F ig. 2-2). A mean disease rating score (DRS), calculated as a weighted average, and standa rd deviations (SD) were calculated for each accession and the susceptible controls. A third test was performed to rescreen accessions from the first two studies exhibiting a mean DRS of less than 2. Eight replications consisting of one plant of each of these accessions and the susceptible control Yellow Summer Squash were planted in a randomized complete block design, inoculated, and visually rated for their response to P. capsici as above. Mean DRSs and SDs were calculated for each of the rescreened accessions and the susceptible control. Results and Discussion Mean DRSs for crown inoculation with P. capsici among the 115 accessions ranged from 1.3 to 5 (Table 2-1). Average standard devia tion of DRSs within accessions was 1.1 and ranged from 0 to 2. Thirteen accessions (11.3%) had at l east 50% of their replicat es with a DRS of 0 or 1. Eight of these had a mean DRS of less than 2. These eight accessions were chosen for rescreening (Table 2-2). Results of the rescreen study paralleled those of the initial study in that the mean DRSs among the accessions remained less than 2 and the average SD was 1.4. PI 181761 exhibited the lowest mean DRS at 0.5 with all plants in this acce ssion rated as either 0 or 1. These findings suggest that accessions within the C. pepo collection are potential sources of resistance to P. capsici 27

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In this study, all accessions collected in the United States were susceptible to the suspension of P. capsici isolates from Florida. Based on the origin of th e eight accessions chosen for rescreening, four were obtained from Germany and Turkey. Evaluating additional accessions from Asia, Europe, and Mexico in the future might be worthwhile. The findings from these tests suggest th at many of the C. pepo accessions exhibited ssegergation for resistance to P. capsici crown rot. Many cucurbit accessions are maintained by open or sib-pollination, therefore, segergation for a particular trait may occur. Screening and continuous selection of individuals originati ng from cucurbit accessions can lead to breeding lines homogeneous for particular trait(s). This approach was used to develop the melon race one powdery mildew [ Podosphaera xanthii (syn. Sphaerotheca fuliginea auct. p.p.)] resistant watermelon (Citrullus lanatus var. lanatus ) line PI 525088-PMR (Davis et al., 2006). Phytophthora capsici can cause disease on all plant tissu e of susceptible hosts. Disease on each of these tissues can be considered a separa te disease syndrome, i.e., crown rot, root rot, foliar blight, and fruit rot. Different genetic me chanisms may be responsible for host resistance to the various syndromes. This is the case with root rot and foliar blight resistance in pepper ( Capsicum annuum var. annuum ) (Walker and Bosland, 1999). A si milar situation exists in the host-pathogen in teraction of P. infestans and potato ( Solanum tuberosum L.). Different genes are responsible for the resistances in tubers, vines, and foliage of the potato plant (Budin et al., 1978; Howard, 1978). Physiological races within the P. capsici C. annuum interaction have been identified (Oelke et al., 2003; Glosier et al., 2008). Pat hogen races are important in pepper breeding as cultivars resistant to P. capsici isolates found in specific grow ing regions continue to be developed. While we have tentatively identified resistance to isolates of P. capsici from Florida 28

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in C. pepo, addition al studies will be performed to ev aluate the Phytophthora crown rot resistant lines against isolates from around the wo rld. If physiological races within the P. capsici C. pepo interaction are identified, it will play an important role in breeding for Phytopht hora resistance of the edible-fruited domesticates of C. pepo. Results from this study indicate that there is potential resistance to P. capsici crown rot within C. pepo accessions. Through screening and selection, the development of C. pepo lines homozygous for P. capsici resistance will allow us to st udy the inheritance of resistance, evaluate the P. capsici C. pepo interaction, and create Phytophthora crown rot resistant cultivars to aid in disease management of this pathogen. Further studies are also ne cessary to evaluate the Phytophthora crown rot resistant C. pepo breeding lines developed from this study for their response to Phytophthora foliar blight and fruit rot. 29

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Table 2-1. Response of Cucurbita p epo accessions to a suspension of Phytophthora capsici squash isolates from Florida. Accessions are ranked according to their mean disease rating score (DRS). Accession or Cultivarz Mean DRS (0-5 scale) SD % plants with disease rating 1 Speciesy Seed origin PI 181761 1.3 0.9 75 Cp Lebanon PI 615132 1.3 0.5 75 Cp Mexico PI 174185 1.4 0.5 63 Cp Turkey PI 615142 1.4 0.5 63 Cp Kazakhstan PI 169417 1.6 1.6 71 Cp Turkey PI 266925 1.8 1.5 75 Cp Germany PI 209783 1.9 1.4 50 Cp Germany PI 512709 1.9 2.0 75 Cp Spain PI 169450 2.1 1.4 38 Cp Turkey PI 181944 2.1 1.8 63 Cp Syria PI 167053 2.3 1.8 38 Cp Turkey PI 169476 2.3 1.9 50 Cp Turkey PI 179267 2.3 1.9 50 Cp Turkey PI 181878 2.3 1.3 25 Cp Syria PI 288240 2.3 1.8 50 Cp Egypt PI 299574 2.5 1.9 50 Cp South Africa PI 234252 2.6 1.7 38 Cp Argentina PI 173684 2.7 1.6 14 Cp Turkey PI 136448 2.8 1.6 25 Cp China PI 285611 2.8 1.4 0 Cp Poland PI 169469 2.9 2.0 43 Cp Turkey PI 177377 2.9 1.9 38 Cp Syria PI 193501 2.9 1.4 0 Cp Ethiopia PI 368592 2.9 1.5 13 Cp Macedonia PI 458731 2.9 1.6 25 Cp Argentina PI 507885 2.9 1.6 25 Cp Hungary PI 163232 3.0 1.9 38 Cp India PI 165018 3.0 1.5 25 Cp Turkey PI 169448 3.0 1.9 29 Cp Turkey PI 222721 3.0 1.7 13 Cp Iran PI 311102 3.0 1.2 0 Cp Guatemala PI 311741 3.0 1.6 25 Cp Poland PI 532355 3.0 1.9 20 Cpf Mexico PI 167199 3.1 1.5 13 Cp Turkey PI 193502 3.1 1.6 25 Cp Ethiopia 30

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Table 2-1. C ontinued. Accession or Cultivarz Mean DRS (0-5 scale) SD % plants with disease rating 1 Speciesy Seed origin PI 304061 3.1 1.7 25 Cp Pakistan PI 355054 3.1 1.6 13 Cp Iran PI 169418 3.2 2.0 33 Cp Greece PI 169442 3.2 1.6 0 Cp Turkey PI 169443 3.3 1.9 25 Cp Turkey PI 257287 3.3 1.3 0 Cp Spain PI 234615 3.3 1.7 14 Cp South Africa PI 169425 3.4 1.8 25 Cp Turkey PI 318826 3.4 1.3 13 Cp Mexico PI 169426 3.5 1.7 13 Cp Turkey PI 181760 3.5 1.9 25 Cp Lebanon PI 274787 3.5 1.2 0 Cp India PI 357940 3.5 1.7 13 Cp Yugoslavia PI 175705 3.6 1.9 29 Cp Turkey PI 169472 3.6 1.9 25 Cp Turkey PI 212060 3.6 1.7 13 Cp Greece PI 269483 3.6 1.5 13 Cp Pakistan PI 364241 3.6 1.9 25 Cp Hungary PI 379307 3.6 1.6 13 Cp Yugoslavia PI 176964 3.7 1.7 14 Cp Turkey PI 183678 3.8 1.8 13 Cp Turkey PI 169475 3.8 1.8 20 Cp Turkey PI 175704 3.8 1.8 17 Cp Turkey PI 532354 3.9 1.5 0 Cpf Mexico Ames 26619 3.9 1.2 0 Cpo United States PI 169461 3.9 1.6 13 Cp Turkey PI 172860 3.9 1.6 13 Cp Turkey PI 183232 3.9 1.8 25 Cp Egypt PI 385970 3.9 1.6 0 Cp Kenya PI 532356 3.9 1.8 13 Cpf Mexico PI 93458 3.9 1.5 14 Cp China PI 135394 4.0 1.3 0 Cp Afghanistan PI 169429 4.0 1.9 14 Cp Turkey PI 169453 4.0 1.5 0 Cp Turkey PI 357929 4.0 1.4 13 Cp Macedonia Ames 26871 4.1 1.6 13 Cpo United States PI 169477 4.1 1.2 0 Cp Turkey 31

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Table 2-1. C ontinued. Accession or Cultivarz Mean DRS (0-5 scale) SD % plants with disease rating 1 Speciesy Seed origin PI 614683 4.1 1.6 13 Cpf Mexico PI 167042 4.3 1.4 0 Cp Turkey PI 171631 4.3 1.5 13 Cp Turkey PI 181763 4.3 1.2 0 Cp Lebanon PI 234616 4.3 1.3 0 Cp South Africa PI 169435 4.4 1.0 0 Cp Turkey PI 169445 4.5 1.0 0 Cp Turkey PI 169458 4.5 0.9 0 Cp Turkey PI 169478 4.5 0.9 0 Cp Turkey PI 181758 4.5 1.1 0 Cp Lebanon Ames 26873 4.8 0.7 0 Cpo United States Ames 26882 4.8 0.7 0 Cpo United States PI 167084 4.8 0.7 0 Cp Turkey PI 172868 4.8 0.7 0 Cp Turkey PI 274336 4.8 0.7 0 Cp Guatemala Ames 26608 4.9 0.4 0 Cpo United States Ames 26622 4.9 0.4 0 Cpo United States PI 135398 5.0 0.0 0 Cp Afghanistan PI 379314 5.0 0.0 0 Cp Macedonia PI 615141 5.0 0.0 0 Cp Kazakhstan Ames 26607 5.0 0.0 0 Cpo United States Ames 26609 5.0 0.0 0 Cpo United States Ames 26610 5.0 0.0 0 Cpo United States Ames 26612 5.0 0.0 0 Cpo United States Ames 26616 5.0 0.0 0 Cpo United States Ames 26617 5.0 0.0 0 Cpo United States Ames 26620 5.0 0.0 0 Cpo United States Ames 26624 5.0 0.0 0 Cpo United States Ames 26833 5.0 0.0 0 Cpo United States Ames 26872 5.0 0.0 0 Cpo United States Ames 26875 5.0 0.0 0 Cpo United States Ames 26876 5.0 0.0 0 Cpo United States Ames 26877 5.0 0.0 0 Cpo United States Ames 26879 5.0 0.0 0 Cpo United States Ames 26883 5.0 0.0 0 Cpo United States Ames 26884 5.0 0.0 0 Cpo United States Ames 26885 5.0 0.0 0 Cpo United States 32

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Table 2-1. C ontinued. Accession or Cultivarz Mean DRS (0-5 scale) SD % plants with disease rating 1 Speciesy Seed origin Ames 26887 5.0 0.0 0 Cpo United States Ames 26888 5.0 0.0 0 Cpo United States Ames 26890 5.0 0.0 0 Cpo United States Ames 26891 5.0 0.0 0 Cpt United States Ames 26892 5.0 0.0 0 Cpt United States Ames 26893 5.0 0.0 0 Cpt United States EPS 5.0 0.0 0 Cp United States YSS 5.0 0.0 0 Cp United States zSusceptible C. pepo controls EPS, 'Early Prolific St raightneck' and YSS, 'Yellow Summer Squash'. yCp, Cucurbita pepo ; Cpf, Cucurbita pepo subsp. fraterna ; Cpo, Cucurbita pepo var. ozarkana; Cpt, Cucurbita pepo var. texana. 33

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Table 2-2. Response of eight selected accession s of Cucurbita pepo to a suspension of three Phytophthora capsici isolates from Florida. Disease Rating Scale (DRS)y Number of plants within each category Accession or Cultivarz 0 1 2 3 4 5 Mean DRS SD PI 181761 4 4 0 0 0 0 0.5 0.5 PI 615132 4 1 0 2 1 0 1.4 1.7 PI 174185 2 3 3 0 0 0 1.1 0.8 PI 615142 1 2 4 1 0 0 1.3 0.9 PI 169417 5 0 1 1 0 1 1.3 1.9 PI 266925 2 4 0 1 0 1 1.5 1.7 PI 209783 3 1 2 0 0 2 1.9 2.1 PI 512709 2 0 5 0 0 1 1.9 1.6 YSS 0 0 0 0 0 8 5.0 0.0 zSusceptible C. pepo control YSS, 'Yellow Summer Squash'. yDisease rating based on a scale ranging from 0 (no symptoms) to 5 (plant death). 34

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Figure 2-1. Geographic location of 115 Cucurbita pepo accessions representing 24 countries (USDA, 2006b). 35

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Figure 2-2. Disease rating scale (0-5) for Phytophthora capsici crown inoculation on Cucurbita pepo 36

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37 CHAPTER 3 A CUCURBITA BREEDING LINE WI TH CROWN ROT RESISTANCE TO PHYTOPHTHORA CAPSICI DERIVED FR OM WILD CUCURBITA SPECIES. Introduction Phytophthora capsici Leonin is a devastating oomycet ous pathogen that affects many vegetable crops. Since its discovery in 1918, the incidence of disease caused by P. capsici has increased throughout the United States with repo rts of yield loss from many vegetable producing states including California, North and South Caro lina, Florida, Georgia, Illinois, New Jersey, Michigan and Texas (Babadoost and Islam, 2003; Caf-Filho et al., 1995; Ristaino, 1990; Hausbeck and Lamour, 2004; Isaleit, 2007; Lam our and Hausbeck, 2003; Leonian, 1922; Tian and Babadoost, 2004). With the incr eased occurrence and severity of P. capsici within the U.S., researchers have begun looking into alternativ e forms of managing th is pathogen, including breeding for resistance (Hausbeck and Lam our, 2004; French-Monar et al., 2005; Keinath, 2007). In cucurbits, P. capsici can infect at any growth stag e and given optimum conditions an entire field can be destroyed in a matter of days (Roberts et al., 2001). The disease syndromes of P. capsici on cucurbits includes crown rot, foliar blight and fruit rot (Zitter et al., 1996; Roberts et al., 2001). Crown rot appears at the soil line where stems tu rn dark brown, become watersoaked, and quickly collapse causi ng plant death. Foliar sympto ms appear as rapidly expanding, water-soaked lesions. Dieback of shoot tips, wilt ing, shoot rot, and plant death quickly follows initial infection. Fruit, which can be infected at any stage of maturity, may exhibit sunken, brown, water-soaked areas, rapidly covered by white sporangial growth under moist environmental conditions. In squash ( Cucurbita spp.), resistance to P. capsici had recently been found within two wild gourd species, C. lundelliana PI 438542 and C. okeechobeenesis sbsp. okeechobeenesis

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(Kabelka et al., 2007). Resistance to the crown rot syndrom e caused by P. capsici derived from the two wild species, was intr ogressed through a series of hybridizations providing breeding material 62.5% C. moschata, 25% C. lundelliana PI 438542 and 12.5% C. okeechobeenesis sbsp. okeechobeenesis From this series, 19 lines were tested for response to P. capsici and all were found to be segregating for resist ance (Kabelka et al., 2007). Th e objective of this study was to develop, from this material, a Cucurbita breeding line homozygous resi stant to the crown rot syndrome of P. capsici Materials and Methods Plant Material The development of Cucurbita breeding material, with P. capsici resistance, was accomplished through a series of in terspecific hybridizations of C. lundelliana C. okeechobeensis sbsp. okeechobeenesis and C. moschata At each hybridization event, selections for horticultural characteristics of fruit shape and ri nd and flesh color were made. Nineteen lines, exhibiting desirable horticultural traits, were selected and evaluated for response to P. capsici inoculation and all segregat ed for resistance based on replicated greenhouse trials (Table 3-1). Of the lines ev aluated, #394, which has pear-shaped fruit and dark orange flesh color, was chosen for further evaluation and selection for resistance to crown rot caused by P. capsici Greenhouse Studies and Single Plan t Selections for Homozygosity to P. capsici Crown Rot Resistance A series of greenhouse studies, with selections for resistance to P. capsici crown rot inoculation, were performed to develop a homozygous P. capsici crown rot resistant Cucurbita breeding line. Throughout all stud ies, greenhouse temperatures were maintained between 19C to 34C, plants were watered daily, and at the cotyledon stage the plants received 1 g of slow38

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release fertilizer (Osm ocote, 14-14-14 NPK, Grace Sierra Horticulture Products, Milpitas, CA). Fafard #3S potting mix (Fafard Inc., Agawam, MA) was used throughout. Phytophthora capsici isolates and inoculum preparation, the crown in oculation protocol, scoring for response to inoculation, and analysis of data are described below. U pon completion of each study, and for the purpose of developing next generation progeny, selected asymptomatic plants were transplanted to 9.5 L plastic pots for further growth and develo pment, self-pollination, fruit harvest and seed extraction. Each transplant received an additional 5 g of slow release fertilizer (Osmocote, 14-14-14 NPK). The first of the series of greenhous e studies evaluated breeding line #394 (F4) and progeny from the self-pollination of an asymptomatic single plant selection from #394, designated #394-1 (F5). Using a completely randomized design, 10 seed of #394, 20 seed of #394-1, and 5 seed of the susceptible cont rol Early Prolific Straightneck (C. pepo) were sown into standard 18 cell flats. At the second to third true-leaf-stage, each seedling was inoculated at its crown with 5 ml of the 2x104 zoospores/ml suspension of P. capsici Twenty one days after inoculation, plants were visual ly rated for response and asym ptomatic individuals of #394-1 were transplanted for the development of F6 generation seed. The second greenhouse study evaluated proge ny from an asymptomatic individual, designated #394-1-27 (F6), using a randomized complete bl ock design. Eight replications consisting of one plant each of #391-1-27 and of the susceptible control, an open pollinated commercial cultivar Butterbush ( C. moschata ), were sown into 152.4 mm diameter plastic azalea pots. At the second to third true-leaf-stag e, each seedling was inocul ated at its crown with 5 ml of the 2x104 zoospores/ml suspension of P. capsici Twenty one days after inoculation, 39

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plants were visually rated for response a nd asymptom atic individuals of #394-1-27 were transplanted for th e development of F7 generation seed. A third greenhouse study was performed to test the accuracy of my P. capsici crown inoculation protocol was in identifying asympt omatic individuals and to rule out possible escapes. For this test, I utili zed rooted cuttings made from the seven asymptomatic #394-1-27 (F6) transplants, from the second greenhouse st udy, including a non-inoc ulated susceptible control Butterbush plant. The stem end of three to four cuttings from each of the seven asymptomatic plants, for a total of 26, and four cuttings from Butterbush were dipped into indole-3-butyric acid (0.1%) to enhance root development and planted into 152.4 mm diameter plastic azalea pots arranged in a randomized co mplete block design. All cuttings were watered daily and at root development each cutting received 1 g of slow-release fertilizer (Osmocote, 1414-14 NPK). Two weeks later, each rooted cutting was inoculated at its crown with 5 ml of the 2x104 zoospores/ml suspension of P. capsici Scoring for response and data analyses were performed as described below. A final greenhouse study eval uated seed of #394-1-27 (F6) and #394-1-27-12 (F7), progeny from an asymptomatic selection from the second greenhouse study. Eight replications consisting of one plant of each and the suscepti ble control Butterbush were planted in a randomized complete block design. At the second to third true-leaf-stage, each seedling was inoculated at its crown with 5 ml of the 2x104 zoospores/ml suspension of P. capsici Twenty one days after inoculation, plants were visually rated for their response to P. capsici Phytophthora capsici Isolates and Inoculum Preparation Three highly virulent P. capsici mating type A1 isolates (01-1938A, RJM98-730 and RJM98-805), collected from squash, were obtained from Dr. Pamela Roberts (Southwest Florida Research and Education Center, Immokalee, FL). A suspension of the three isolates, containing 40

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equal portions of each, was prepared as descri bed in Chapter 2 of this dissertation, at a concentration of 2x104 zoospores/ml. Phytophthora capsici Crow n Inoculation, Scoring for Response and Data Analysis At the second to third true-leaf-stage, each seedling was inoculated at its crown with 5 ml of the 2x104 zoospores/ml suspension of P. capsici Prior to inocula tion, the potting mix was watered and remained saturated for 24-36 hours to optimize the zoospore infection process. Twenty one days after inoculation, the plants were visually rated based on a scale ranging from 0 to 5; where 0 = no symptoms, 1 = small brown le sion at base of stem, 2 = lesion has progressed up to the cotyledons causing constriction at the base, 3 = plant has partially collapsed with apparent wilting of leaves, 4 = plant has complete ly collapsed with severe wilting present, and 5 = plant death (Fig. 3-1). A mean disease rating score (DRS), calculated as a weighted average, and standard deviations (SD) were calculated for each line and the susceptible controls. Results and Discussion Evaluation of breeding line #349 (F4) and progeny from the self-pollination of an asymptomatic single plant sel ection from #394, designated #394-1 (F5), revealed each to be segregating for resistance to crown rot caused by P. capsici (Table 3-2). By day 21, both lines had greater than 50% of their progeny symptoma tic with disease ratings of greater than 1. Breeding line #394 had a mean DRS of 1.0 and a SD of 1.5 while #394-1 had a mean DRS and SD of 0.7. All replicati ons of the susceptible open pollinated commercial control, Early Prolific Straightneck, rapidly developed tan-brown water-soaked lesions at their crowns which rapidly collapsed and caused plant death. Evaluation of breeding line #394-1-27 (F6), an asymptomatic indivi dual selected from the previous study, revealed seven out of eight of its progeny to be asymptomatic 21 days after P. capsici crown inoculation, with a m ean DRS of 0.1 and SD of 0.4 (T able 3-3). In this study, 41

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all replica tions of the susceptible control, Butterbush, developed tan-brown water-soaked lesions at their crowns which rapidly caused plant death. The next study tested the accuracy of my P. capsici crown inoculation protocol in identifying asymptomatic individuals. For this te st, I utilized rooted cuttings made from the seven asymptomatic #394-1-27 (F6) transplants including a non-i noculated susceptible control Butterbush plant. Evaluation of 26 rooted cuttings revealed all to be asymptomatic post P. capsici crown inoculation (Table 3-3) All rooted cuttings from a non-inoculated susceptible control Butterbush plant quickly died following P. capsici crown inoculation. This study reveals that my crown inoculat ion protocol is accurate in de termining response to crown rot caused by P. capsici in Cucurbita breeding material. A final study evaluated breeding lines #394-1-27 (F6) and #394-1-27-12 (F7) and revealed all plants within each to be asym ptomatic to crown inoculation with P. capsici (Table 3-4). As above, all plants of the susceptible control Butterbush died following P. capsici crown inoculation. This final study suggest s I have successfully developed a Cucurbita breeding line homozygous resistant to P. capsici crown rot. Resistance to P. capsici has been found in other vegetable crops. In pepper ( Capsicum annuum L.), resistance to P. capsici comes from two different s ources; PI 201232 and a Mexican landrace called Criollo de More los 334 (Alcanta ra and Bosland, 1994; Ortega et al., 1995; Sy et al., 2005). In watermelon (Citrullus lanatus L.), a screen of nine Ko rean and Japanese cultivars by Lee et al. (2001), showed a variety named D anmatmaetdol as having the highest level of resistance to P. capsici With potential resistance to P. capsici within C. pepo identified in Chapter 1 of this dissertation and with the germplasm developed in this Chapter, breeding for resistance to P. capsici within Cucurbita will aid in the disease management of this pathogen. 42

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A Cucurbita breeding line, designated #394-1-27-12, was developed w ith resistance to the crown rot syndrom e caused by P. capsici (see Figure 3-2 for its pedi gree). At maturity, #394-127-12 produces smooth, medium-green, striped, obovate -shaped fruit, with medium orange flesh color. Growth habit is vine; leav es are shallow-lobed and mottled. Cucurbita breeding line #394-1-27-12 will be a useful source of P. capsici crown rot resistance for introgression into the morphologically diverse edible-f ruited domesticates within Cucurbita. Further studies are needed to evaluate #394-1-27-12 for its response to foliar blight and fruit rot and to determine the inheritance of resist ance to crown rot caused by P. capsici 43

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Table 3-1. Description and response of Cucurbita lundellian a PI 438542, C. okeechobeeness sbsp. okeechobeenesis 19 Cucurbita breeding lines, and suscep tible controls Yellow Summer Squash ( C. pepo ) and Early Prolific Straightneck ( C. pepo) to a suspension of Phytophthora capsici isolates.z No. of Plantsy Line Fruit Shape Fruit Color Flesh Color R S C. lundelliana PI 438542 Oval Dark Green w/ stripes Light Yellow 8 0 C. okeechobeenes s sbsp. okeechobeenesisw Oval Dark Green w/ stripes Light Yellow 6 0 Breeding line #322 Elongate Green w/ stripes Orange 11 7 Breeding line #381 Oblate Green Orange 20 9 Breeding line #382 Round Dark Green Orange 9 9 Breeding line #383 Oblate Green w/ stripes Orange 15 6 Breeding line #384 Round Green Orange 7 2 Breeding line #385 Round Dark Green Dark Orange 10 18 Breeding line #387 Pear Green Orange 12 6 Breeding line #388 Oblate Green Orange 13 3 Breeding line #389 Oblate Green Light Yellow 10 1 Breeding line #390 Oblate Green Dark Orange 20 6 Breeding line #391 Oblate Green Orange 7 4 Breeding line #393 Round Green Orange 22 3 Breeding line #394 Pear Green w/ stripes Dark Orange 19 9 Breeding line #395 Round Green Orange 11 5 Breeding line #396 Round Green Orange 14 10 Breeding line #397 Round Dark Green Dark Orange 9 18 Breeding line #398 Round Dark Green Orange 16 4 Breeding line #399 Oblate Green w/stripes Orange 11 8 Breeding line #400 Oblate Dark Green Orange 14 10 Yellow Summer Squash 0 10 Early Prolific Straightneck 0 10 zData from Kabelka et al., 2007. yDisease rating based on a scale ranging from 0 (no symptoms) to 5 (plant de ath). Plants scored as 0 were classified as resistan t (R) while those scored 1-5 were classified as susceptible (S). wSeed source collected from Torrey Island, Okee chobee, FL and provided by T.W. Walters and D.S. Decker-Walters, Fairchild Tr opical Garden, Miami, FL. 44

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Table 3-2. Response of Cucurbita breeding line #394, #394-1, and susceptible control EarlyProlific Straightneck ( C. pepo ) to a suspension of Phytophthora capsici isolates. Disease Rating Scale (D RS)z No. of plants within each category Line Generation 0 1 2 3 4 5 Mean DRS SD #394 F4 4 5 0 0 0 1 1.0 1.5 #394-1 F5 8 10 2 0 0 0 0.7 0.7 Early Prolific Straightneck 0 0 0 0 0 5 5.0 0.0 zDisease rating based on a scale ranging from 0 (no symptoms) to 5 (plant death). 45

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Table 3-3. Response of Cucurbita breeding line #394-1-27, rooted cuttings of #394-1-27, and susceptible control Butterbush ( C. moschata ) to a suspension of Phytophthora capsici isolates. Disease Rating Scale (D RS)z No. of plants within each category Line Generation 0 1 2 3 4 5 Mean DRS SD #394-1-27 F6 7 1 0 0 0 0 0.1 0.4 Butterbush 0 0 0 0 0 8 5.0 0.0 #394-1-27 rooted cuttingsy F6 26 0 0 0 0 0 0.0 0.0 Butterbush rooted cuttingsx 0 0 0 0 0 4 5.0 0.0 zDisease rating based on a scale ranging from 0 (no symptoms) to 5 (plant death). yThree to four cuttings taken from each of seven asymptomatic mature plants of #394-1-27 (F ). 6 xCuttings taken from an asymptomatic mature plant of Butterbush. 46

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Table 3-4. Response of Cucurbita breeding lines #394-1-27, #394-1-27-12 and susceptible control Butterbush (C. moschata ) to a suspension of Phytophthora capsici isolates. Disease Rating Scale (DRS)z No. of plants within each category Line Generation 0 1 2 3 4 5 Mean DRS SD #394-1-27 F6 8 0 0 0 0 0 0.0 0.0 #394-1-27-12 F7 8 0 0 0 0 0 0.0 0.0 Butterbush 0 0 1 0 0 7 4.6 1.1 zDisease rating based on a scale ranging from 0 (no symptoms) to 5 (plant death). 47

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Figure 3-1. Disease rating scale (0-5) for Phytophthora capsici crown inoculation on Cucurbita moschata 48

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49 Figure 3-2. Pedigree of Phytophthora capsici crown rot resistant Cucurbita breeding line #394-1-27-12.

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50 CHAPTER 4 INHERITANCE OF RESISTANCE TO CR OWN ROT CAUSED BY PHYTOPHTHORA CAPSICI IN CUCURBITA. Introduction The oomycetous pathogen, Phytophthora capsici Leonian, is capable of causing several disease syndromes in cucurbits in cluding crown rot, folia r blight and fruit ro t (Zitter et al., 1996; Roberts et al., 2001). Crown rot appears at the soil line on the plant as a dark brown, watersoaked lesion that quickly collapses the stem causing plant death. Foli ar blight appears as rapidly expanding, water-soaked lesions on the leav es that eventually cau ses dieback of shoot tips, wilting, shoot rot, and plant death. Fruit rot appears as sunken, brown, water-soaked areas which are rapidly covered by white sporangial growth under moist enviro nmental conditions. The incidence of disease caused by P. capsici in cucurbit production areas of the United States has increased with reported yield loss as high as 100% (Hausbeck and Lamour, 2004; Tian and Babadoost, 2004). Given optimal environmental conditions, an entire field of cucurbits can be destroyed by P. capsici in a matter of days (Zitter et al., 1996; Roberts et al., 2001). The increased occurrence and severity of P. capsici has prompted research for fungicide management alternatives and interest in br eeding cucurbits for resistance (Babadoost, 2000; Stevenson et al., 2000, 2001; Seebold and Horten, 2003; Hausbeck and Lamour, 2004; McGrath, 2004; Tian and Babadoost, 2004; Waldenmaier, 2004; French -Monar et al., 2005; Keinath, 2007). Cucurbita are considered to be one of the most morphologically variable genera in the plant kingdom (Whitaker and Robinson, 1986; Robi nson and Decker-Walters, 1999). There are 22 wild and five cultivated species of Cucurbita The cultivated species, grown around the world, include C. pepo, C. moschata, C. maxima, C. argyrosperma (formerly C mixta ) and C. ficifolia Cucurbita cultivars are categorized as summer or winter squash. Summer squash are eaten immature when tender and seeds are sma ll and soft. Winter squash are generally eaten

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when rind and seeds are fully m ature. Summer squash cultivars are C. pepo while winter squash cultivars can be C. pepo, C. maxima C. moschata, or C. argyrosperma A search for sources of resistance within Cucurbita, to the various syndromes of P. capsici had been performed and included representatives from C. maxima C. moschata C. pepo, and three wild species, C. ecuadorensis C. lundelliana and C. okeechobeensis (Kabelka et al., 2007). From this scree n, resistance to the cr own rot syndrome of P. capsici was identified in the wild species, C. lundelliana PI 438542 and C. okeechobeenesis subsp. okeechobeenesis This resistance was in trogressed, through a series of hybridizations, selfpollinations, and single plant selections, into a winter squash ( C. moschata) background. One line, designated #394-1-27-12, was advanced to the F7 generation and is homozygous for P. capsici crown rot resistance. The obj ective of this study was to char acterize the inheritance of resistance to crown rot caused by P. capsici within the Cucurbita breeding line #394-1-27-12. Materials and Methods Plant Material The Cucurbita breeding line #394-1-27-12, resistant to crown rot caused by P. capsici was crossed with Butterbush (BB), a butternut-type winter squash ( C. moschata) highly susceptible to P. capsici Controlled pollinations were carried out in the greenhouse to generate F1 (BB x 394-1-27-12), F2 and reciprocal backcross (BC) progenies. The susceptible control used in all studies was an open pollinated comm ercial cultivar Butterbush. Phytophthora capsici Isolates and Inoculum Preparation Three highly virulent P. capsici mating type A1 isolates (01-1938A, RJM98-730 and RJM98-805), collected from squash, were obtained from Dr. Pamela Roberts (Southwest Florida Research and Education Center, Immokalee, FL). A suspension of the three isolates, containing 51

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equal portions of each, was prepared as descri bed in Chapter 2 of this dissertation, at a concentration of 2x104 zoospores/ml. Experimental Design and Data Analysis Evaluation of #394-1-27-12, Butterbush, F1, F2 and BC progenies for response to P. capsici crown inoculation was performed in gree nhouse studies using completely randomized designs. Seed were sown in 15.2 cm azalea plastic pots containing Fafard #3S potting mix (Fafard Inc., Agawam, MA). Seedlings were wa tered daily and greenhouse temperatures were maintained between 19C to 34C. At the coty ledon stage, each seedling received 1 g of slowrelease fertilizer (14-14-14 NP K, Grace Sierra Horticulture Products, Milpitas, CA). The F2 progeny test consisted of 200 individuals plus 10 replicates of both parents. The four BC progeny tests, performed separately, cons isted of 100 individuals each of BB x F1 and F1 x BB plus 10 replicates of both parents and 50 individuals each of 394-1-27-12 x F1 and F1 x 394-127-12 plus 8 replicates of both parents and the F1. At the second to third true-leaf-stage, each seedling was inoculated at its crown with 5 ml of the 2x104 zoospores/ml suspension of P. capsici Prior to inocula tion, the potting mix was watered and remained saturated for 24-36 hours to optimize the zoospore infection process. Twenty-one days after inoculation, the plants were visually rated based on a scale ranging from 0 to 5; where 0 = no symptoms, 1 = small brown lesi on at base of stem, 2 = lesion progressed up to the cotyledons causing constriction at the base, 3 = plant has partially collapsed with apparent wilting of leaves, 4 = plant has completely collap sed with severe wilting present, and 5 = plant death. In all studies, plants scored as 0 were cl assified as resistant while those scored 1-5 were classified as susceptible. Segregation ratios were analyzed by Chi-square analysis. 52

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Results and Discussion The Cucurbita breeding line #394-1-27-12 exhibited no symptoms of crown rot caused by P. capsici under the conditions of this study (Fig. 4-1) However, five days post-inoculation, the susceptible cultivar Butterbush developed a tanbrown water-soaked lesion at its crown that rapidly expanded, caused stem collapse, and plant death. The F1 of the cross between Butterbush and #394-1-27-12 reacted similarly to that of the resistant parent, #394-1-27-12, remaining asymptomatic (Table 4-1). The F2 progeny segregated in 27:37 [resistant (R):susceptible (S)] ratio while the backcrosses to the susceptible parent, Butterbush segregated in a 1:7 (R:S) ratio. Progeny of the backcrosses to the parent #394-1-27-12 were all resistant. Collectively, the segregation ratios support a model in which resistance to the crown rot syndrome caused by P. capsici is conferred by three dominant genes. In pepper ( Capsicum annuum L.), it has been shown that resistance to root rot, stem blight, and foliar blight, caused by P. capsici are under different genetic mechanisms (Sy et al., 2005). This is also suggested for potato ( Solanum tuberosum L.) where tuber, vine, and foliage resistance to another Phytophthora species, P. infestans (Mont.) de Bar y, are controlled by separate genes (Bonde et al., 1940; Rudorf et al., 1950). Recently, #394-1-27-12 has been found to possess resistance to the foliar blight syndrome of P. capsici Studies are currently underway to determine if the genetic mechanisms for crow n rot and foliar blight resistance within #394-127-12 are the same or are different (K abelka, personal communication, 2008). Physiological races of P. capsici have been identified within the P. capsici C. annuum interaction (Oelke et al., 2003; Gl osier et al., 2008). This plays an important role in developing pepper cultivars w ith resistance to P. capsici isolates found in specific growing regions. The resistance in #394-1-27-12 is cu rrently being tested against P. capsici isolates from different regions of the United States and Europe to test for specificity. If physiological races within the 53

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P. capsici Cucurbita interac tion are identified, it will pl ay an important role in breeding for P. capsici resistance within Cucurbita. Different screening methods have been developed to examine plants for their response to the various disease syndromes caused by P. capsici The greenhouse assay used in this study allowed for precise observations of plant response to crown inocul ation and for the determination of inheritance of resistance to crown rot caused by P. capsici This assay provides a standardized test environment. It also allows for th e screening of test material using defined P. capsici inoculum sources. This assay will aide in the introgression of P. capsici crown rot resistance from #394-1-27-12 into the morphologically di verse edible-fruited domesticates within Cucurbita Molecular linkage maps are usef ul tools to facilitate breed ing efforts providing molecular markers for marker-assisted-selection and to increase our knowledge of Cucurbita genetics. Using molecular markers, instead of phenotypic a ssays, can increase the precision and efficiency of subsequent selection steps applied in plant breeding. Co -dominant PCR-based molecular markers tightly linked (<5 cM) to P. capsici crown rot resistance would provide the most benefit allowing distinction between homozygous resist ant and heterozygous resistant individuals. Studies are currently underway to create a mo lecular linkage map of the segregating progeny developed in this study to identify markers linked to P. capsici crown rot resistance. As the genes for resistance to P. capsici crown rot within #394-1-27-12 may be from either C. lundelliana PI 438542 or C. okeechobeenesis or both, molecular analysis may also shed light as to the contributor of this resistance. 54

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Table 4-1. Segregation for resistance to Phytophthora capsici crown inoculation in C ucurbita breeding line #394-1-27-12, Butterbush, F1, F2 and BC progeny. No. of plantsz Genetic Models Generation R S One-gene Expected ratio (R:S)y X2 Two-gene Expected ratio (R:S)y X2 Three-gene Expected ratio (R:S)y X2 #394-1-27-12 28 0 Butterbush (BB) 0 38 F1 (BB x 394-1-27-12) 8 0 F2 (BB x 394-1-27-12) 92 108 3:1 89.7*** 9:7 8.5*** 27:37 1.2ns BC1 (BB x F1) 17 83 1:1 43.6*** 1:3 3.4ns 1:7 1.9ns BC1 (F1 x BB) 10 85 1:1 59.2*** 1:3 10.6*** 1:7 0.3ns BC1 (394-1-27-12 x F1) 50 0 1:0 1:0 1:0 BC1 (F1 x 394-1-27-12) 50 0 1:0 1:0 1:0 zR=resistant, S=susceptible. yRatio based on data classified as either resistant (0) or susceptible (1,2,3,4,5). nsX2 value not significant P 0.05. ***Significant at 0.0001 probability level. 55

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Figure 4-1. Response of Cucurbita breeding line #394-1-27-12 a nd Butterbush to crown inoculation with a suspension of Phytophthora capsici isolates. A & B) Breeding line #394-1-27-12 remained asymptomatic post crown inoculation. C) Butterbush develops a tan-brown water-soaked lesion at its crown that ra pidly expands causing stem collapse. D) plant death. 56

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CHAP TER 5 OVERALL CONCLUSIONS Squash ( Cucurbita spp.) is a multimillion dollar industr y in the United states producing over $229 million in sales for the year 2006 alone (USDA, 2007b). Phytophthora capsici Leonian is a fungal-like pathogen th at has causes several disease syndromes in squash including crown rot, foliar blight and fru it rot. In the United States, P. capsici has been identified in many of the vegetable producing states including Cali fornia, North and South Carolina, Florida, Georgia, Illinois, New Jersey, Michigan, and Te xas (Babadoost and Islam, 2003; Caf-Filho et al., 1995; Isaleit, 2007; Lamour and Hausbeck, 2003; Ristaino, 1990). The objectives of this research project were: (1) iden tify sources of resistance to P. capsici crown rot within the C. pepo germplasm collection; (2) develop a homozygous P. capsici crown rot resistant Cucurbita breeding line; and (3) characterize the resistance to P. capsici crown rot found within Cucurbita breeding line #394. For the first objective, 150 accessions from 24 different countries were chosen from the United States C. pepo germplasm collection and screened for their response to a three isolate suspension of Phytophthora capsici in three independent studies. From the 115 accessions thirteen (11.3%) had at least 50% of their replicates with a DR S of 0 or 1. Eight of these accessions had a mean DRS of less than 2 and were rescreened. Results of the rescreen study paralleled those of the initial study in that the mean DRSs among the accessions remained less than 2 and the average SD was 1.4. PI 181761 exhibited the lowest mean DRS at 0.5 with all plants in this accession rated as either 0 or 1. The findings fr om these screens suggest that accessions within the C. pepo collection are potential sources of resistance to P. capsici Through screening and selection, the development of C. pepo lines homozygous for P. capsici resistance will allow us to study the inheritance of resistance, evaluate the P. capsici-C. pepo 57

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interaction, and create P hytophthora crown rot resistant cultivars to aid in disease management of this pathogen. Based on the origin of the eight accessions chosen, evaluating additional accessions from Asia, Europe, and Mexico in the future might be worth considering. Further studies are also necessary to evaluate the Phytophthora crown rot resistant C. pepo breeding lines developed from this study for their response to Phytophthora foliar blight and fruit rot. In the second objective, resistance to P. capsici was found within two wild gourd species, C. lundelliana PI 438542 and C. okeechobeenesis sbsp. okeechobeenesis (Kabelka et al., 2007). Resistance to the crown rot syndrome caused by P. capsici derived from the two wild species, was introgressed through a se ries of hybridizations providing breeding material 62.5% C. moschata, 25% C. lundelliana PI 438542 and 12.5% C. okeechobeenesis sbsp. okeechobeenesis From this series, breeding line #394 was tested for response to P. capsici and was found to be segregating for resistance (Kabel ka et al., 2007). The objective of this study was to develop, from this material, a Cucurbita breeding line hom ozygous resistant to the crown rot syndrome of P. capsici Breeding line #349 (F4) and three generations of progeny from the self-pollination of an asymptomatic single plant selection were screened for resistance to the P. capsici Results from these screened revealed the mean DRS and SD of each generation decr ease with selection. Breeding line #394 had a mean DRS of 1.0 and a SD of 1.5 while #394-1 had a mean DRS and SD of 0.7. Evaluation of breeding line #394-1-27 (F6) revealed seven out of eight of its progeny to be asymptomatic with a mean DRS of 0.1 and SD of 0.4. A final study evaluating breeding lines #394-1-27-12 (F7) revealed all plants to be asymptomatic to crown inoculation with P. capsici. Cucurbita breeding line #394-1-27-12 was determined to be homozygous resistant to the crown rot syndrome caused by P. capsici At maturity, #3941-27-12 produces smooth, 58

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m edium-green, striped, obovate-shaped fruit, with me dium orange flesh color. Growth habit is vine; leaves are shallow-lobed and mottled. Cucurbita breeding line #394-1-27-12 will be a useful source of P. capsici crown rot resistance for intr ogression into the morphologically diverse edible-fruited domesticates within Cucurbita Further studies are needed to evaluate #394-1-27-12 for its response to foliar blight and fruit rot and to determ ine the inheritance of resistance to crown rot caused by P. capsici The third objective of my research was to ch aracterize the inherita nce of resistance to crown rot caused by P. capsici within the Cucurbita breeding line #394-1-27-12. The Cucurbita breeding line #394-1-27-12 was crosse d with Butterbush (BB), a butternut-type winter squash ( C. moschata) highly susceptible to P. capsici Controlled pollinations were carried out in the greenhouse to generate F1 (BB x 394-1-27-12), F2 and reciprocal backcross (BC) progenies. Twenty-eight plants of Cucurbita breeding Line #394-1-27-12, 38 pl ants of Butterbush, eight F1s, 200 F2s, 100 plants each of BB x F1 and F1 x BB and 50 plants each of 394-1-27-12 x F1 and F1 x 394-1-27-12 were screened for their response to a three isolate suspension of Phytophthora capsici. The Cucurbita breeding line #394-1-27-12 and the F1 exhibited no symptoms of crown rot caused by P. capsici under these conditions. The F2 progeny segregated in 27:37 [resistant (R):suscepti ble (S)] ratio while the backcrosses to the susceptible parent, Butterbush segregated in a 1:7 (R:S) ratio. Progeny of the backcrosses to the parent #394-1-2712 were all resistant. Collectively, the segregatio n ratios support a model in which resistance to the crown rot syndrome caused by P. capsici is conferred by three dominant genes. The resistance in #394-1-27-12 is now currently being tested against P. capsici isolates from different regions of the United States and Europe to test for specificity. Studies are also being conducted 59

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to create a molecular linkage m ap of the segreg ating progeny developed in this study to identify markers linked to P. capsici crown rot resistance. 60

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61 APPENDIX A RESPONSE OF CUCURBITA PEPO ACCESSIONS TO CROWN INOCULATION WITH SUSPENSIONS OF PHYTOPHTHORA CAPSICI ISOLATED FROM TOMATO AND PEPPER. Previous studies have shown that P. capsici from one host, such as tomato, can be pathogenic on other susceptible hosts, such as pepper (Ristaino, 1990). Two common susceptible hosts that are grown in ratation with squash in Florida are tomatoes and peppers. I evaluated 46 accessions from the C. pepo germplasm collection for thei r response, in separate studies, to crown inocula tion using suspensions of P. capsici isolated from tomato and pepper. Phytophthora capsici isolates from tomato (M29, Cp-27 and Cp-36) and pepper (Cp-32, Cp-30 and Imm018) were obtained from Dr. Pamela Roberts (Southwest Florida Research and Education Center, Immokalee, FL). Inoculum preparation, experimental design, the crown inoculation protocol, scoring for response to inoculation, and analys is of data were followed as described in Chapter 2 of this dissertation. Mean disease rating scores (DRSs) to crown inoculation with P. capsici isolated from tomato among the 46 accessions ranged from 1.5 to 5 (Table A-1). Average standard deviation of DRSs within accessions was 1.3 and ranged fro m 0 to 2. Eight accessions (17.4%) had at least 50% of their replicates with disease ratings of 0 or 1. Six of these had a mean DRS of less than 2. Mean disease rating scores (DRSs) to crown inoculation with P. capsici isolated from pepper among the 46 accessions ranged from 1.5 to 5 (Table A-2). Average standard deviation of DRSs within accessions was 1.3 and ranged from 0 to 2.2. Four accessions (8.7%) had at least 50% of their replicates with di sease ratings of 0 or 1. Three of these had a mean DRS of less than 2. These findings suggest that accessions within the C. pepo collection are worth considering as potential host differentials to the isolates of P. capsici collected from tomato and pepper.

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Table A-1. Response of Cucurbita p epo accessions to a suspension of Phytophthora capsici isolated from tomato in Florida. Acce ssions are ranked accord ing to their mean disease rating score (DRS). Accession or Cultivarz Mean DRS (0-5 scale)y SD % plants with disease rating 1 Seed Origin PI 209783 1.5 0.8 63 Germany PI 299574 1.5 0.8 63 South Africa PI 512709 1.5 1.5 63 Spain PI 174185 1.8 0.5 25 Turkey PI 266925 1.9 1.1 50 Germany PI 355054 1.9 0.6 25 Iran PI 163232 2.0 1.4 50 India PI 181761 2.0 1.3 38 Lebanon PI 318826 2.0 1.3 38 Mexico PI 179267 2.1 1.5 38 Turkey PI 458731 2.1 1.6 50 Argentina PI 285611 2.3 1.3 38 Poland PI 136448 2.4 1.7 38 China PI 173684 2.4 1.7 38 Turkey PI 181944 2.4 1.8 50 Syria PI 311741 2.4 1.7 38 Poland PI 304061 2.6 1.6 25 Pakistan PI 181763 2.8 1.8 38 Lebanon PI 183232 2.8 1.4 25 Egypt PI 288240 2.8 1.7 33 Egypt PI 615142 2.8 1.3 13 Kazakhstan PI 257287 2.9 1.6 14 Spain PI 222721 2.9 1.8 25 Iran PI 311102 2.9 1.6 25 Guatemala PI 379307 3.0 1.7 50 Yugoslavia PI 507885 3.0 2.0 25 Hungary PI 615132 3.0 1.7 13 Mexico PI 234615 3.2 1.5 0 South Africa PI 274787 3.3 1.5 13 India PI 269483 3.4 1.8 25 Pakistan PI 234252 3.5 1.6 13 Argentina PI 193502 3.6 1.7 13 Ethiopia PI 357940 3.6 1.9 25 Yugoslavia PI 357929 3.8 1.8 13 Macedonia PI 177377 3.9 1.6 13 Syria PI 364241 3.9 1.2 0 Hungary PI 212060 4.0 1.7 17 Greece 62

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Table A-1 Continued. Accession or Cultiva rz Mean DRS (0-5 scale)y SD % plants with disease rating 1 Seed Origin PI 368592 4.0 1.5 13 Macedonia PI 193501 4.1 1.4 0 Ethiopia PI 169418 4.3 1.5 14 Greece PI 093458 4.6 0.7 0 China PI 135394 4.8 0.7 0 Afghanistan PI 135398 5.0 0.0 0 Afghanistan PI 181758 5.0 0.0 0 Lebanon PI 274336 5.0 0.0 0 Guatemala PI 615141 5.0 0.0 0 Kazakhstan EPS 5.0 0.0 0 United States YSS 5.0 0.0 0 United States zSusceptible C. pepo controls EPS, 'Early Prolific St raightneck' and YSS, 'Yellow Summer Squash'. YDisease rating based on a scale ranging fr om 0 (no symptoms) to 5 (dead plant). 63

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Table A-2. Response of Cucurbita p epo accessions to a suspension of Phytophthora capsici isolated from pepper in Florida. Acce ssions are ranked accord ing to their mean disease rating score (DRS). Accession or Cultivarz Mean DRS (0-5 scale)y SD % plants with disease rating 1 Seed origin PI 181763 1.5 0.5 50 Lebanon PI 355054 1.9 1.5 63 Iran PI 364241 1.9 1.4 50 Hungary PI 181944 2.0 1.4 50 Syria PI 181761 2.1 1.9 38 Lebanon PI 368592 2.2 1.5 33 Macedonia PI 311102 2.4 1.4 25 Guatemala PI 615142 2.4 1.1 0 Kazakhstan PI 512709 2.5 1.7 38 Spain PI 318826 2.6 1.6 25 Mexico PI 458731 2.6 0.7 0 Argentina PI 169418 2.9 2.1 14 Greece PI 135398 2.9 1.8 25 Afghanistan PI 173684 2.9 1.4 0 Turkey PI 274336 2.9 1.4 13 Guatemala PI 174185 3.0 1.8 25 Turkey PI 179267 3.0 1.8 25 Turkey PI 266925 3.0 1.7 25 Germany PI 209783 3.1 2.1 38 Germany PI 357929 3.1 1.6 13 Macedonia PI 234252 3.3 1.9 25 Argentina PI 288240 3.3 1.9 25 Egypt PI 615132 3.3 2.2 25 Mexico PI 234615 3.4 1.8 13 South Africa PI 136448 3.5 1.5 13 China PI 379307 3.5 1.7 13 Yugoslavia PI 212060 3.8 1.8 13 Greece PI 257287 4.0 1.5 14 Spain PI 163232 4.1 1.4 0 India PI 222721 4.3 1.5 13 Iran PI 285611 4.3 1.2 0 Poland PI 311741 4.3 1.4 13 Poland PI 181758 4.4 1.4 13 Lebanon PI 299574 4.4 1.4 13 South Africa PI 177377 4.5 1.4 13 Syria PI 615141 4.5 0.8 0 Kazakhstan PI 093458 4.6 1.1 0 China PI 357940 4.6 0.7 0 Yugoslavia PI 507885 4.6 1.1 0 Hungary PI 193502 4.9 0.4 0 Ethiopia 64

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Table A-2. Continued. Accession or Cultiva rz Mean DRS (0-5 scale)y SD % plants with disease rating 1 Seed origin PI 135394 5.0 0.0 0 Afghanistan PI 183232 5.0 0.0 0 Egypt PI 193501 5.0 0.0 0 Ethiopia PI 269483 5.0 0.0 0 Pakistan PI 274787 5.0 0.0 0 India PI 304061 5.0 0.0 0 Pakistan EPS 5.0 0.0 0 United States YSS 5.0 0.0 0 United States zSusceptible Cucurbita pepo controls EPS, 'Early Prolific Straightneck' and YSS, 'Yellow Summer Squash'. yDisease rating based on a scale ranging fr om 0 (no symptoms) to 5 (dead plant). 65

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66 APPENDIX B RESPONSE TO CROWN INOCULATION USI NG THREE DIFFERENT PHYTOPHTHORA CAPSICI ZOOSPORE CONCENTRATIONS. Many P. capsici crown inoculation studi es have been conducted at different zoospore concentrations with no standard inoculum le vel determined (Alcantara and Bosland, 1994; Ortega et al., 1995; Lee et al., 2001). A study was conducted to compare the response of C. pepo to crown inoculation using a suspension of P. capsici squash isolates at 100,000, 50,000 and 25,000 zoospore concentrations. Two C. pepo accessions, PI 179267 and PI 181761, progeny from previously selected asymptomatic plants from these two accessions, 179267-17 and 181761-18, and susceptible controls Early Prolific Straightneck and Yellow Summer Squash were used. Phytophthora capsici type A1 isolates (01-1938A, RJM98-730 and RJM98-805) collected from squash were obtained from Dr. P. Roberts (University of Florid a, Southwest Florida Research and Education Center, Imm okal ee, FL). Inoculum preparation, experimental design, the crown inoculation protoc ol, scoring for response to inoc ulation, and analysis of data were followed as described in Chapter 2 of this dissertation. Results of this study showed no significant difference in the response of each PI, th eir progeny, or susceptible controls to the three different concentrations of P. capsici zoospores.

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Table B-1. Response of Cucurbita p epo PI 179267, PI 181761 and their selfed progeny to a suspension of Phytophthora capsici squash isolates at 100,000, 50,000 and 25,000 zoospore concentrations. PI 179267 179267-17Z Disease Rating Scale (DRS)Y Disease Rating Scale (DRS) Zoospore Concentration 0 1 2 3 4 5 Mean DRSX Zoospore Concentration 0 1 2 3 4 5 Mean DRS 100,000 1 4 2 0 0 0 1.1a 100,000 1 5 0 0 1 1 1.8a 50,000 1 4 1 0 1 0 1.4a 50,000 1 6 0 1 0 0 1.1a 25,000 0 4 2 0 0 1 1.9a 25,000 0 6 1 0 0 1 1.6a Pooled 2 12 5 0 1 1 1.5 Pooled 2 17 1 1 1 2 1.5 PI 181761 181761-16Z Disease Rating Scale (DRS) Disease Rating Scale (DRS) Zoospore Concentration 0 1 2 3 4 5 Mean DRS Zoospore Concentration 0 1 2 3 4 5 Mean DRS 100,000 0 3 2 0 0 3 2.8a 100,000 4 2 1 0 0 1 1.1a 50,000 0 0 6 1 1 0 2.4a 50,000 3 3 1 0 0 1 1.3a 25,000 0 2 1 1 0 3 3.1a 25,000 5 2 0 0 1 0 0.8a Pooled 0 5 9 2 1 6 2.8 Pooled 12 7 2 0 1 2 1.1 Early Prolific StraightneckW Yellow Summer SquashW Disease Rating Scale (DRS) Disease Rating Scale (DRS) Zoospore Concentration 0 1 2 3 4 5 Mean DRS Zoospore Concentration 0 1 2 3 4 5 Mean DRS 100,000 0 0 0 0 0 8 5.0 100,000 0 0 0 0 0 8 5.0 50,000 0 0 0 0 0 8 5.0 50,000 0 0 0 0 0 8 5.0 25,000 0 0 0 0 0 8 5.0 25,000 0 0 0 0 0 8 5.0 Pooled 0 0 0 0 0 24 5.0 Pooled 0 0 0 0 0 24 5.0 zProgeny of a self-pollinated plant from PI with disease ra ting score of 0. yDisease rating based on a scale ranging from 0 (no symptoms) to 5 (dead plant). Number of plants scored within each category listed. xMeans followed by the same letter are not significantly different at p=0.05. wCucurbita pepo susceptible controls. 67

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Paris, H.S. 1986. A proposed subspecific classification for Cuc urbita pepo Phytologia, 61:133138. Paris H.S. 1996. Summer Squash: History, Divers ity, and Distrubtion. HortTechnology 6(1):613. Paris, H.S. and S. Cohen. 2000. Oligogenic inherita nce for resistance to Zu cchini yellow mosaic virus in Cucurbita pepo. Ann. Applies Biology 136:209-214. Paris, H.S., N. Yonash, V. Portnoy, N. Mozes-D aube, G. Tzuri and N. Katzir. 2003. Assessment of genetic relationships in Cucurbita pepo ( Cucurbita ceae) using DNA markers. Theor. Appl. Genet. 106:971-978. Peet, Mary. 2001a. Squash, Gourd a nd Pumpkin: Sustainable Pract ices for Vegetable Production in the South Production Practices. NCSU. Raleigh, North Carolina. http://www.cals.ncsu.edu/sustainable/peet/profiles/ppsquash.html Oct. 2008. Peet, Mary. 2001b. Squash, Gourd and Pum pkin: Sustainable Pract ices for Vegetable Production in the South Botany. NCSU. Raleigh, North Carolina. http://www.cals.ncsu.edu/sustain able/peet/profiles/botsquas.htm l Oct 2008. Ristaino, J. B. 1990. Intraspeci fic variation isolates of Phytophthora capsici from pepper and cucurbit fields in North Caro lina. Phytopathology 80(11)1253 1259. Ristaino, J.B. and S. A. Johns ton. 1999. Ecologically based approaches to management of Phytophthora blight on bell pepper s. Plant Disease 83(12):1080-1089. Roberts, P.D., R.J. McGovern, T.A. He rt, C.S. Vavina and R.R. Urs. 1999. Phytophthora capsici in tomato: survival, severity, age, variety and insensitivity to mefenoxam. Florida Tomato Institute Proceedings. 41-43. Roberts, P.D. R.J. McGovern, T.A. Kuchar ek and D.J. Mitchell. 2001. 10 December 2007. Vegetable diseases caused by Phytophthora capsici in Florida, PP-176. UF/IFAS EDIS publication. Gainesville, Florida. ht tp://edis.ifas.ufl.edu/VH045 Oct 2008. Robinson R.W. and D.S. Decker-Walters. 1999. Cucurbits. CAB Intl., New York.Sitterly, W. R. 1972. Breeding for disease resistance in cu curbits. Annual Review of Phytophthora. 10:471-490. Rudorf, W., P. Schaper, H. Ross, M. Baereck e and M. Torka. 1950. The breeding of resistant varieties of potatoes. The Amer. Potato J. 27:222-235. Seebold, K.W. and T.B. Horten. 2003. Evaluati on of fungicides for control of Phytophthora crown and fruit rot of summer squash, 2002. Fungicide Nematicide Tests. 58:V098. University of Georgia, Tifton, Georgia. http://www.plantmanagementnetwork.or g/pub/trial/fntests /reports/2003/V098.pdf Oct 2008. 71

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Sitterly, W .R. 1972. Breeding for disease resi stance in cucurbits. Annual Review of phytophthora. 10:471-490. Stephens, J.M. 2003. Squash, ZucchiniCucurbita pepo L. HS675 IFAS, University of Florida. Gainesville, Florida. http://edis.ifas.ufl.edu/mv142 Oct 2008. Stevenson, W.R. R.V., Jam es and R.E. Rand. 2000. Ev aluation of selected fungicides to control Phytophthora blight and fruit rot of cucu mber. Fungicide Nematacide Tests. 55:163. University of Wisconsin-Madison. Madison, Wisconsin. http://www.plantmanagementnetwork.or g/pub/trial/fntests /reports/2001/V16.pdf Oct 2008. Stevenson, W.R., R.V. Jam es and R.E. Rand. 2001. Ev aluation of selected fungicides to control Phytophthora blight and fruit rot of cucumb er. Fungicide Nematacide Tests. 56:V16. University of Wisconsin-Madison. Madison, Wisconsin. http://www.plantmanagementnetwork.or g/pub/trial/fntests /reports/2001/V16.pdf Oct 2008. Swiader, J.M. and G.W. W are 2002. Producing Vegetable Crops. 5th ed. Interstate Publishers, Inc. Danville, Illinois. Sy, O., P.W. Bosland and R. St einer. 2005. Inheritance of phytophthor a stem blight resistance as compared to phytophthora root rot and phytophthora foliar blight resistance in Capsicum annuum L. J. Amer. Soc. Hort. Sci.130(1):75-78. Tian, D. and M. Babadoost. 2004. Host range of Phytophthora capsici from pumpkin and pathogenicity of isolates. Plant Disease 88(5):485-489. USDA. 2006a. ARS National programs. Methyl bromide alternatives, Action Plan. National Germplasm Resources Laborat ory, Beltsville, Maryland. http://www.ars.usda.gov/research/program s/programs.htm?np_code=308&docid=264. Oct 2008. USDA. 2006b. ARS GRIN database. Summery statis tics of holding as of Composition for Cucurbita National Germplasm Resources La boratory, Beltsville, Maryland. http://www.ars-grin.gov/cgi-bin/npgs/htm l/stats/genus.pl? Cucurbita Oct 2008. USDA. 2006c. ARS, Na tional Genetic Resources Program. Germplasm Resources Information Network (GRIN). [Online Database] Na tional Germplasm Resources Laboratory, Beltsville, Maryland. http://www.ars-grin.gov/cgi-b in/npgs/acc/display.pl? 1264792 Oct 2008. USDA. 2006d. ARS, Na tional Genetic Resources Program. Germplasm Resources Information Network (GRIN). [Online Database] Na tional Germplasm Resources Laboratory, Beltsville, M aryland. http://www.ars-grin.gov/cgi-b in/npgs/acc/display.pl?1021977 Oct 2008. 72

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BIOGRAPHICAL SKETCH Les Padley Jr. was born in Largo, Florida, in January 9, 1980, to Les and Pam Padley. He graduated high school from the Center for Adva nced Technologies in 1998, and received his Bachelor of Science degree from Florida Southern College in environmental horticultural and business. In 2005 he obtained his Ma ster of Science degree in plan t breeding from the University of Florida. Currently, he is now working on his Doctor of Philosophy in plant breeding under Dr. Eileen Kabelka and has begun training as the NAFTA squash breed er for Syngenta.