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Ecological and Phylogenetic Characterization of Phytophthora capsici in Florida

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Ecological and Phylogenetic Characterization of Phytophthora capsici in Florida
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2008

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African Americans ( jstor )
Inoculum ( jstor )
Oospores ( jstor )
Pathogens ( jstor )
Peppers ( jstor )
Phytophthora ( jstor )
Soil science ( jstor )
Soils ( jstor )
Species ( jstor )
Weeds ( jstor )

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University of Florida
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ECOLOGICAL AND PHYLOGENETIC CHARACTERIZATION OF Phytophthora capsici IN FLORIDA By RONALD DAVID FRENCH 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 2004

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Copyright 2004 by Ronald David French

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To GOD, for keeping me spiritually str ong and in good physical and mental health. To my parents, Edward Ronald French and Delia Monar de French, for their love and support throughout th ese many years. To all my loved ones who were th ere for me during this journey. To all those who encouraged me to stay in the discipline and stay the course.

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iv ACKNOWLEDGMENTS I would like to express my sincere gratitu de and appreciation to my co-advisors, Drs. Jeffrey B. Jones and Pamela D. Robert s, for having embraced me as their student, and giving me the independence to express the ideas and objectives that have allowed me to conduct my doctoral studies. I have valu ed their guidance, co mments, patience, and motivation that have allowed me to grow as a plant pathologist. I am grateful to Drs. James W. Kimbrough, Robert J. McGovern, Jef frey A. Rollins, and Edward L. Braun for being a part of my supervisory committee. Sincere gratitude goes to Ken Shuler, Extension Agent IV Palm Beach County, who allo wed me to travel with him in several opportunities to meet growers and sample fi eld locations which otherwise would have been difficult to accomplish on my own. I am also grateful to Dr. David J. Mitchell, for providing me a strong foundation in soilborne plant pathology, es pecially in what relates to the oomycetes in the genera Pythium and Phytophthora . I wish to thank Gerald Minsavage in Gainesville, and Dr. Rama Urs in Immokalee for their technical expertise. I wish to thank the plant pa thology office staff for making my departmental stay as smooth as possible. For their patience and continual support, I would like to thank my parents and loved ones for being there for me.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES...........................................................................................................ix ABSTRACT....................................................................................................................... ..x CHAPTER 1 REVIEW OF LITERATURE.......................................................................................1 2 SURVIVAL OF INOCULUM OF Phytophthora capsici IN SOIL THROUGH TIME UNDER DIFFERENT SOIL TREATMENTS..................................................6 Introduction................................................................................................................... 6 Materials and Methods.................................................................................................8 Results........................................................................................................................ .13 Discussion...................................................................................................................17 3 NATURAL INFESTATIONS OF Phytophthora capsici ARE ASSOCIATED WITH LOCAL WEED POPULATIONS IN CONVENTIONAL VEGETABLE FARMS.......................................................................................................................21 Introduction.................................................................................................................21 Materials and Methods...............................................................................................23 Results........................................................................................................................ .27 Discussion...................................................................................................................34 4 INCREASED RESISTANCE TO MEFENOXAM AND THE OCCURRENCE OF BOTH MATING TYPES OF phytophthora capsici IN FLORIDA...........................36 Introduction.................................................................................................................36 Materials and Methods...............................................................................................38 Results........................................................................................................................ .41 Discussion...................................................................................................................52

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vi 5 SINGLE POPULATION OF phytophthora capsici IN FLORIDA INFERRED FROM THE MOLECULAR PHYLOGENY OF ISOLATES FROM DIFFERENT CROPS, GEOGRAPHIES, AND YEAR OF ISOLATION.......................................57 Introduction.................................................................................................................57 Materials and Methods...............................................................................................59 Results........................................................................................................................ .62 Discussion...................................................................................................................66 LIST OF REFERENCES...................................................................................................70 BIOGRAPHICAL SKETCH.............................................................................................76

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vii LIST OF TABLES Table page 2-1. Average soil moisture content of soil in an envelope at time of sampling for each treatment in two separate years of experimentation.................................................14 2-2. Percentage of soil samples with viable inoculum of Phytophthora capsici as determined by soil dilution plating (SDP), modified soil dilution plating-overlay (mSDPO), and lemon leaf baiting (LLB).................................................................15 2-3. Average number of co lony forming units of Phytophthora capsici for each treatment per sampling period..................................................................................................17 3-1. Recovery of Phytophthora capsici from common weed species present in vegetable grower farms in Palm Beach County, Florida..........................................................28 3-2. The effect of soil infestations with Phytophthora capsici isolates obtained from weed species in Palm Beach County, Florida, on percent mortality of bell pepper seedlings...................................................................................................................30 3-3. Pathogenicity tests on black nightshade ( Solanum nigrum ) using isolates of Phytophthora capsici ................................................................................................30 3-4. Pathogenicity tests on common purslane ( Portulaca oleracea ) using isolates of Phytophthora capsici ...............................................................................................31 3-5. Pathogenicity tests on Carolina wild geranium ( Geranium carolinianum ) using isolates of Phytophthora capsici ..............................................................................32 3-6. Pathogenicity tests on eastern black nightshade ( Solanum ptycanthum ) using isolates of Phytophthora capsici ...........................................................................................32 3-7. Pathogenicity tests on Carolina horsenettle ( Solanum carolinense ) using isolates of Phytophthora capsici ................................................................................................33 3-8. Pathogenicity tests on Am erican black nightshade ( Solanum americanum ) using isolates of Phytophthora capsici ..............................................................................33 4-1. Characterization by mating type and in vitro sensitivity to mefenoxam of isolates of Phytophthora capsici from Palm Beach County isolated between 1982 and 2003 from various crops and soils.....................................................................................43

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viii 4-2. Characterization by mating type and in vitro sensitivity to mefenoxam of isolates of Phytophthora capsici from Martin County..............................................................46 4-3. Characterization by mating type and in vitro sensitivity to mefenoxam of isolates of Phytophthora capsici from Dade County.................................................................46 4-4. Characterization by mating type and in vitro sensitivity to mefenoxam of isolates of Phytophthora capsici from Collier County..............................................................47 4-5. Characterization by mating type and in vitro sensitivity to mefenoxam of isolates of Phytophthora capsici from Manatee County...........................................................48 4-6. Characterization by mating type and in vitr o sensitivity to mefenoxam of isolates of Phytophthora capsici from unspecified locations in Florida isolated between 1993 and 1997 from several host plants............................................................................49 4-7. Isolates of Phytophthora capsici classified by mating type and sensitivity to mefenoxam from ten vegetable farms between 2001 and 2003...............................50 4-8. Characterization by mating type and in-vitr o sensitivity to mefenoxam of isolates of Phytophthora capsici isolated in 2002 from three weed species in Palm Beach County......................................................................................................................51 5-1. Summary information for isolates of Phytophthora capsici used for PCR amplification of ITS (I) or mitochondrial (M) sequences........................................60

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ix LIST OF FIGURES Figure page 2-1. Modified soil dilution pl ating-overlay assay plates....................................................11 2-2. Lemon leaf baiting of soil for Phytophthora capsici ..................................................12 2-3. Open face agar plate with colonies of Phytophthora capsici obtained only via the modified soil dilution plating-overlay assay............................................................16 2-4. Center of origin of a colony of Phytophthora capsici that originated from a germinating oospore.................................................................................................18 3-1. Papillate sporangia of Phytopthora capsici CWG1-3 isolated from Carolina wild geranium ( Geranium carolinianum) in Palm Beach County...................................27 3-2. DNA amplified from isolates of Phytophthora spp. recovered from weeds in an initial screening on PARPH plates using primers PCAP and ITS 1........................29 3-3. The effect of Phytophthora capsici on black nightshade ( Solanum nigrum ) inoculated with isolate NS h7-2 (left) in comparison to the non-inoculated black nightshade (right).....................................................................................................31 4-1. DNA amplified from isolates of Phytophthora capsici recovered Carolina wild geranium in an initial screening on PA RPH plates using primers PCAP and ITS 1.........................................................................................................................4 2 4-2. Effects of mefenoxam concentr ations on radial growth of Phytophthora capsici resistant isolate PBC1-5...........................................................................................45 4-3. Frequency of mefenoxam sensitivity in Phytophthora capsici for isolates collected during the 1980s, 1990s, and 2000-2003 based on in vitro studies at 100 g/ml....52 5-1. Rooted parsimony tree based on the inte rnal transcribed spacer sequences of 23 isolates of Phytophthora capsici ..............................................................................64 5-2. Rooted parsimony tree based on intergenic m itochondrial sequences of 30 isolates of Phytophthora capsici ................................................................................................65

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x 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 ECOLOGICAL AND PHYLOGENETIC CHARACTERIZATION OF Phytophthora capsici IN FLORIDA By RONALD DAVID FRENCH May 2004 Chair: Jeffrey B. Jones Cochair: Pamela D. Roberts Major Department: Plant Pathology Root, crown, and fruit rot, as well as foliar blight, caused by the oomycete Phytophthora capsici L., are limiting factors for ve getable production in Florida. Although P. capsici has been reported since 1931, re latively minor damage had been observed until 1982 when this pathogen cause d devastating losses to bell pepper, eggplant, and squash. Although it is assumed that rainfall was res ponsible for the worst disease outbreaks caused by this pathogen in Florida, the incidence of this disease has increased dramatically where other factors may be importa nt. Such factors may include survival of inoculum in soil and weeds, increase in f ungicide resistance, sexual recombination, and the introduction of new pathogen populations. Previous studies on survival of oospores in soil determined that the viability of this spore was of about 84 days. This study showed that after 343 days viable oospores were

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xi still detected by a modified soil dilution plating assay and by baiting soil with lemon leaves. Weeds were sampled in commercial farms where P. capsici had been recently reported. Common purslane, Carolina wild geranium, and American black nightshade harbored P. capsici in their root system. Greenhouse st udies confirmed that these three weeds were alternative hosts for this pathogen. This is the first report on Carolina wild geranium and American black nightshade. Increased resistance to the fungicide mefenoxam by P. capsici was reported in Michigan and North Carolina. In this study, resistance to mefenoxam in Florida was observed in isolates of P. capsici from Palm Beach, Martin, Dade, Collier, and Manatee counties. A strong population shift from se nsitivity to resistance was confirmed for P. capsici between 1982 and 2003. Both mating types of P. capsici occurred in all counties surveyed and in the same field. Therefor e, oospore production and sexual recombination might be taking place. A molecular phylogenetic study was conducte d on isolates of P. capsici to determine if there is more than one popul ation present in Florida. Using ITS and mitochondrial sequence analyses, little genetic diversity in isolates of P. capsici from Florida was revealed suggesti ng that a single population of P. capsici exists in Florida. These findings have assessed othe r factors that may explain why P. capsici has become a major problem for vegetable pr oduction during the past twenty years. Selection seems to be the driving force behind P. capsici in Florida rather than the introduction of new and more aggressive pathogen populations.

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1 CHAPTER 1 REVIEW OF LITERATURE Pepper ( Capsicum annuum L.) is an important cash crop for farmers in the United States and around the world. In the United States alone, pepper production in 1998 was approximately 650,000 metric tons (Eddy, 1999). Florida ranks second, behind California, in fresh market vegetable production, not only in area under cultivation (10%) but also in production (10%) and value (15 %) of the crops. On a value basis for vegetables, pepper production in Florida in 1997-98 accounted for 16% of the stateÂ’s total, second only to tomato (28%) (Mayna rd and Hochmuth, 1999). In 1997, Florida led the country in fresh and processed bell pepp ers, growing approximately 26,326 hectares with a value of close to $503 million. In Florida, field production of vegetable crops is base d on high input, intensively managed production systems which utilize broa d spectrum fumigants, such as methyl bromide, to manage soilborne pests (Can tliffe et al., 1995; Chellemi et al., 1997). Another alternative is soil solarization, whic h has been shown to be cost effective and may be an alternative to preplant fumi gation with methyl bromide (Stapleton, 1982; Chellemi et al., 1997; McGovern et al., 2000). Root, crown, and fruit rots, as well as foliar blight, caused by Phytophthora capsici Leonian, are limiting factors for production of several vegetable and fruit crops including bell pepper, cucumber, eggplant, pumpkin, squash, and tomato. P. capsici , an oomycete, is more closely related to photosynthetic algae than true fungi and is currently placed in the kingdom Chromista (Erwin and Ribeiro, 1996). Phytophthora spp. are water molds,

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2 and water is critical to the disease cycle of this pathoge n (Pernezny et al., 2003). This pathogen has been a major problem in Florid a and other states such as California, Michigan, New Jersey, New Mexico, New York, and North Carolina (Ristaino and Johnston, 1999). Diseases caused by this pa thogen are most common in the subtropics, tropics, and in the summer months of temp erate regions (Ploetz et al., 2002). The incidence of P. capsici has increased in recent years in FloridaÂ’s pepper and squash production areas (McGovern et al., 1998; Roberts et al., 1999 ; Ploetz and Haynes, 2000). Many Phytophthora spp. do not survive extended periods away from their host unlike many other fungal plant pathogens such as Fusarium oxysporum and the oomycete Pythium aphanidermatum (Erwin and Ribeiro, 1996). Mycelium and zoospores produced by Phytophthora spp. survive for only a few weeks, chlamydospores may survive for up to 6 years in some species, and oospores may la st up to 4 years in nonsterile soil (Erwin and Ribeiro, 1996). In P. capsici , there is no production of chlamydospores except for those isolates of tropical crops, wh ich have been reclassified as P. tropicalis novo species by Aragaki and Uchida (2001). Bowers et al. (1990) determined that 9% of oospores of P. capsici tested as viable after 27 weeks using a vital staining procedure. Viability may not translate to germination and therefore, in the same study, this form of inoculum was only able to cause disease on pepper seedlings with inoculum as old as 16 weeks. In Brazil, Ansani and Matsuoka (1983) determined that mycelium of P. capsici survived fewer than 120 days in infected hypocotyl a nd root tissue of pepper buried in the soil, while sporangia and zoospores survived in soil for fewer than 75 days. Since 1931, when Weber (1932) first observed P. capsici in Florida, this pathogen has been prevalent in Florida vegetable pr oduction. However, relatively minor damage

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3 had been observed until 1982 when this pathog en caused devastating losses to pepper in Palm Beach County as well as in eggplant ( Solanum melongena L.) and squash ( Cucurbita pepo L. var. esculentum Nees) (Ploetz and Haynes,2000; McGovern et al., 1998). Other major outbreaks occurred in 1993 and 1998 in pepper, cucurbit and other vegetable production. Although it is assumed that rainfall was responsible for the worst disease outbreaks caused by this pathogen in Florida, the in cidence of this disease has increased dramatically where additional fact ors may be of importa nce (Ploetz et al., 2002). Species that have recently occupied new regions often l ack genetic diversity as a result of the population bottleneck associated with colonization (Nei et al., 1975). If new populations of P. capsici have been introduced in Florida, they might be distinguishable from one another. Currently, phylogenetic and population genetic methods that compare nucleic acid variation are bei ng used to identify species and populations of fungi; understanding nucleic acid variation and its an alysis by cladistics has greatly changed the way taxonomic inferences have been made (Taylor et al., 1999). Oospores are considered the primary survival propagules of P. capsici due to their thick wall, which enables them to overwinte r in the soil (Hwang and Kim, 1995; Ristaino and Johnston, 1999). This sexual phase occurs when isolates of opposite compatibility or mating type (designated A1 and A2) are in cl ose proximity, leading to the formation of antheridia and oogonia and the subsequent form ation of oospores. During this process, genetic recombination may occur and oospores may represent new gene combinations (Erwin and Ribeiro, 1996).

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4 Weeds have been shown to be alterna tive hosts for several plant pathogens (Hepperly et al., 1980; Black et al., 1996; Dissanayake et al., 1996; Barak et al., 2001; Adkins and Rosskopf, 2002; Hollowell et al., 2003). Recent field and shadehouse experiments in Dade County, Florid a, found that common purslane ( Portulaca oleracea ) was colonized by P. capsici . This was the first report of a naturally occurring weed serving as an alternative host for P. capsici (Ploetz and Haynes, 2000; Ploetz et al., 2002). For disease management, the principal prac tices include cultural control to avoid build-up of inoculum and the use of fungici des (Ristaino et al., 1992). The phenylamide class of fungicides, which includes metalaxyl (Ridomil), has been used on pepper and cucurbit production in Florida for over tw o decades. However, the intensive use of metalaxyl leads or may lead to rapid se lection of metalaxyl-resistant strains of Phytophthora spp., as it did with P. infestans in Europe, where selection occurred within a year of fungicide appli cation (Dowley and OÂ’Sullivan, 1981; Goodwin et al., 1998). Mefenoxam (Ridomil Gold EC), the active enantiomer contained in the racemic fungicide metalaxyl, has been used as the ne west formulation of metalaxyl for over six years (Parra and Ristaino, 2001). Selec tion of mefenoxam-resistant strains of P. capsici may be rapidly occurring in Florida as well, since production of peppers and cucurbits is dependent on fungicide applications to ensure a quality harvest. In 1997, such selection occurred in North Carolina when mefenoxam was sprayed for the first time in pepper fields (Parra and Ristaino, 2001) ; almost 60% of isolates of P. capsici from diseased plants were resistant to mefenoxam. The gr eatest proportion of resi stant isolates came

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5 from fields where mefenoxam was used alone rather than in combination with other fungicides. Although the etiology of P. capsici is well understood, there is still a lack of ecological understand ing of how the pathogen persis ts when not provided with a susceptible host plant. It has been difficult, if not impossible, to detect the pathogen in soil prior to the growing season, especially when only one crop is planted per year, as is the case with temperate regions. Characterizing P. capsici in the ecological sense might help answer epidemiological questions and provide a more solid foundation for disease control.

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6 CHAPTER 2 SURVIVAL OF INOCULUM OF Phytophthora capsici IN SOIL THROUGH TIME UNDER DIFFERENT SOIL TREATMENTS Introduction Many Phytophthora spp . do not survive extended periods away from their hosts unlike many other fungal plant pathogens such as Fusarium oxysporum and the oomycete Pythium aphanidermatum (Erwin and Ribeiro, 1996). Mycelium and zoospores produced by most Phytophthora spp. survive only for a fe w weeks, chlamydospores may survive for up to 6 years, and oospores may la st up to 4 years in nonsterile soil (Krober, 1980; Erwin and Ribeiro, 1996). In Phytophthora. capsici , there is no production of chlamydospores except for those isolates of tr opical crops, which have been reclassified as P. tropicalis novo species by Aragaki and Uchida (2001). Bowers et al. (1990) determined that 9% of oospores of P. capsici tested viable after 27 weeks using a vital staining procedure. Viability may not translate to germination and therefore, in the same study, this form of inoculum was only able to cause disease on pe pper seedlings with inoculum as old as 16 weeks. In Brazil, Ansani and Matsuoka ( 1983) determined that mycelium of P. capsici survived fewer than 120 days in infected hypocotyl and root tissue of pepper buried in the soil, while sporangia and zoospores survived in soil for fewer than 75 days. The failure to detect P. capsici in soil does not mean that the pathogen is no longer there. According to Mitchell and Kannwi scher-Mitchell (1992), in areas where Phytophthora species are endemic, initial inoc ulum, most likely in the form of

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7 chlamydospores or oospores, is often not de tected. Low or undetectable levels of inoculum have been observed to increase to high concentrations when environmental conditions favor population growth. In Florida, field production of vegetable crops is base d on high input, intensively managed production systems which utilize broa d spectrum fumigants, such as methyl bromide, to manage soilborne pests (Can tliffe et al., 1995; Chellemi et al., 1997). Another alternative is soil solarization, whic h has been shown to be cost effective and may be an alternative to preplant fumi gation with methyl bromide (Stapleton, 1982; Chellemi et al., 1997; McGovern et al., 2000). The ability of a fungal plant pathogen to pe rsist in soil depends on its efficiency for survival in diseased host tissue buried in soil, sensitivity to temperature and temperature fluctuations, soil moisture, and microbial ac tivity. The depth at which inoculum was buried in soil was not a significant determin ant for survival (Ansani and Matsuoka, 1983; Bowers, 1990). Sampling techniques for detection of propagules of P. capsici may include soil dilution plating, baiting soil with pepper leaf discs, and soil saturation followed by dilution plating (Larkin et al., 1995). No single technique is suitable for all types of inoculum (Ristaino and Johnston, 1999). McGovern et al. (2000) reported a reduction of Phytophthora blight of Madagascar periwinkle in three separate years with so il solarization in autumn. Soil solarization suppressed the development and final inciden ce of Phytophthora blight in periwinkle but did not eliminate the pathogen. Barbercheck and Von Broembsen (1986) determined that solarization for 3 weeks eliminated P. cinnamomi from 91% of buried infested wheat grains and completely eradicat ed the pathogen after 6 weeks.

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8 The objective of this study was to evaluate the effect s of soil solarization and fumigation with methyl bromide on the survival of P. capsici in soil. In the process, assessing how long inoculum may survive in soil may indicate potential of initial inoculum survival from year to year. Materials and Methods Isolates and Inoculum Production Two isolates of P. capsici , Cp30 (A1 compatibility type) and Cp32 (A2 compatibility type), were isolated from bell pepper ( Capsicum annuum L.) in 1997 and 1998, respectively, and used for oospore production in this study. Isolates were provided from the Phytophthora Collection, Plant Pathology Depart ment, University of Florida, Gainesville. Both isolates were maintained on BBL corn meal agar (CMA). Approximately 25 g of red berry wheat seeds ( Triticum aestivum L.) were added to a 250-ml flask containing 25 ml of deionized water (Mitche ll and Kannwischer-Mitchell, 1992). Seeds were soaked for 24 h and autocl aved twice for 20 min on two consecutive days. Each flask was inoculated with five 5-mm disks from a 4-day-old culture of each isolate, Cp30 and Cp32, which were grown se parately on 10% V-8 juice agar. Cultures were incubated for 21 days at 25° C in the dark and shaken every 3 days to prevent clumping. Field Site Field plots were located at the Southwes t Florida Research and Education Center (SWFREC) in Immokalee, Florida. Raised beds were spaced 150 cm apart on centers and were 75 cm in width. Irrigation was carried out thr ough drip tubes. Soil used for this study was Immokalee fine sand from the SWFREC. Its taxonomic class is sandy, siliceous, hypertherm ic Arenic Alaquods. Immokalee fine sand

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9 is typically 97% sand, 2.5% si lt, 0.3% clay, with an organi c matter content of 1.96% and pH 4.2 at a depth of 0-13 cm (Roberts et al., 1999). The top 15 cm of soil was typically dark gray, a combination of organic matter a nd lightly gray sand. The next 15 cm of soil consisted of loose, gray, fine sand. Usi ng soil dilution plating and pepper seedling bioassays, the soil was tested and found not to contain propagules of P. capsici or any other Phytophthora spp. (data not shown). Soil Treatments Three treatments were arranged in a random ized complete block design and were replicated six times. Treatments we re 1) soil solarization with a 30m-thick, clear, gasimpermeable film, 2) plasti c mulch (control) using a 30m-thick, white-on-black, lowdensity polyethylene film (Edison Plastics, L ee Hall, VA) and 3) soil fumigation using a tarped treatment (white-on-black film) fumi gated with a 67:33 formulation of methyl bromide:chloropicrin at 39.2 g/m2. Solarization treatment was stopped after 28 days by applying the white-on-black plastic mulch over the solarization film. Infested wheat seed was shredded and a dded to microwaved Immokalee fine sand soil by mixing 20g of shredded seed per kilogram of soil or the equi valent of 20 mg of infested seed per gram of soil (Coelho, 1997) . Approximately 12 g of infested soil was added to 25 cm2 envelopes made from Versapor 3000 (Pall Corporation, Ann Arbor, Michigan) acrylic membranes with 3-µm pore size. Envelopes were sealed with 3M® Scotch ™600 transparent adhesive tape a nd kept under moist conditions. Within 18 h, envelopes containing infested so il were buried in soil at a depth of 15 cm prior to each treatment. The experiment was conducted from September 2001 until August 2002 and repeated a second year from September 2002 through August 2003. Six envelopes were

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10 randomly sampled after 28, 63, 119, 175, 245, and 343 days and assayed for P. capsici as described below. At the first sampling date, thirty 5-week-old Capsicum annuum L. “Enterprise” bell pepper seedlings were transplanted, double-cropped and spaced 30 cm apart. Pepper seedlings were planted in th e middle of each 10-meter experimental block, leaving approximately 2 meters on each side wi th no plantings. This non-planted area is where envelopes were buried. Standard Soil Dilution Plating As say for Detection of Inoculum Four grams of soil from each envelope we re added to 16 ml of 0.25% sterile water agar and stirred for 2 min; 1-ml samples we re then plated on PA RPH medium, for a 1:5 soil dilution plating (SDP) (L arkin et al., 1995). This me dium consists of 17 g of cornmeal agar (Difco) in 1 L of deionized water amended with 5 mg of pimaricin, 250 mg of ampicillin, 10 mg of rifampicin, 100 mg of pentachloronitr obenzene (PCNB), and 50 mg of hymexazol per liter of medium (Mitchell and Kannwische r-Mitchell, 1992). A total of ten PARPH plates were used for each soil sample. Plates were incubated in the dark at 24 C for 72-96 h, and then rinsed under running water to remove the soil from the surface of the medium. Suspect colonies of P. capsici were counted by circling individual colonies on the bottom of the petri plate with a permanent marker to delineate colony growth. Modified Soil Dilution Plating As say for Detection of Inoculum For the modified soil dilution platingagar overlay method (mSDPO), a 15-ml second layer of agar consisting of 10% clarif ied V8 juice agar, amended with the same chemicals as in PARPH medium, was poured ove r the SDP plates (Fig. 2-1) immediately after the first colony reading or observati on (French-Monar, 2002). Delaying the pouring

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11 of the overlay for more than 24 h promot es the formation of bacterial lawns and germination of non-targeted fungi no longer inhibited by the antibio tics and fungicides, respectively. The mSDPO plates were incuba ted in the dark for an additional 72 h at 24 C. Plates were visualized for new colony growth and mycelia was observed under the microscope at 40X and 100X magnification fo r the presence of sporangia and mycelia characteristic of P. capsici . For year 2, colony counts were facilitated by gently poking through the middle of the overla y, cracking the agar overlay, a nd sliding it off the plate while leaving the original layer intact. Figure 2-1. Modified soil diluti on plating-overlay assay plat es. V-8juice agar amended with chemical concentrations as in PARPH medium was gently poured over the soil dilution plates once they were read. The two layers of agar can be differentiated by color as observed in the top plate. Leaf Baiting of Soil for Detection of Inoculum A baiting technique using lemon leaves wa s utilized for soil samples collected 119, 175, 245, and 343 days after burial in soil. This lemon leaf baiting (LLB) technique was used since detection of viable propagules by SDP previously was show n to decline or was

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12 undetectable in soil 12 week s after soil infestation (Bow ers et al., 1990; Erwin and Ribeiro, 1996). For baiting, the perimeter of rough lemon leaves was cut with scissors and at least seven cuts per side were made at a 45 angle towards the mid-vein (Grimm and Alexander, 1973; Von Broembsen, 1998). For each sample, a total of 4 g of sampled soil was added to an Arrow® 355-ml 2-Ply styrofoam bowl. Approximately 100 ml of sterile deionized water amended with pimaricin, ampicillin, rifampicin, PCNB, and hymexazol, at the same concentrations as in PARPH medium, were added to each bowl. One pre-cut rough lemon leaf was gently floate d on the surface of the water in each bowl (Fig. 2-2). Figure 2-2. Lemon leaf baiting of soil for Phytophthora capsici . Soil was added to water amended with the same chemical concen trations as in PARPH medium. Precut lemon leaf was gently floated in th e water, bowl was covered and kept in the dark for five days. Bowls were covered with a black pl astic and kept in the dark at 24 C for 120 h; leaves were then rinsed with sterile deionized water, blotted dry, and five leaf sections of approximately 1 cm2 each were plated onto two plates of PARPH medium for a total of ten leaf sections per sample. PARPH pl ates were incubated in the dark at 24 C and observed after 72 h for colony growth. Colony observations and readi ngs were continued

PAGE 24

13 every 24 h for up to 120 h. Mycelial growth emanating from the leaf sections was transferred to a fresh PARPH plate fo r observation and species confirmation. Statistical Analysis Values obtained for each assay were an alyzed by ANOVA. Mean comparisons were conducted using FisherÂ’s protecte d least significant difference (FLSD, P 0.05) test. Data were subjected to analysis usi ng the Statistical Analysis System (Release 8.02 Level 02M0 for Windows; SAS Institute, Cary, NC, USA). Results Soil Parameters During soil solarization, the average high te mperature of soil per day at a 15 cm depth was 36 C and 35 C during 2001 and 2002, respectively as monitored at 4 p.m. For the non-treated plastic mulch and fumigation pl ots, the average high temperature of soil at a 15 cm depth was 24 C for both years. The average soil moisture content in the envelope soil varied during sampling peri ods but there was no significant variation between treatments during each sampling period (Table 2-1). Effect of Soil Treatment on Recovery of Phytophthora capsici through Time No viable inoculum was detected from a ny soil samples from the fumigated plots at any sampling date in both years by the SDP, mSDPO, or LLB assay which was introduced during the last four sampling peri ods. Following solarization, at 28 days, soil samples were assayed and no P. capsici was detected in the solarized samples in year 1. In year 2, P. capsici was not detected using SDP but was detected in all samples via the mSDPO assay. The non-treated soil samples ha d viable inoculum as determined by both assays (Table 2-2).

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14 Table 2-1. Average soil moisture content of soil in an envelo pe at time of sampling for each treatment in two separate years of experimentation. Means are based on the average of six envelopes per treatment. Average Soil Moisture Content of Envelope Soil at Time of Sampling (%)a Sampling Time Year 1 (in days) Solarization Soil Fumigation Plastic Mulch 0 12.7a 12.7a 12.7a 28 14.8a 13.4a 12.2a 63 16.4a 13.0a 16.8a 119 6.6a 5.7a 8.6a 175 16.0a 13.0a 11.3a 245 9.0a 12.8a 8.8a 343 14.7a n.t.b 14.2a Sampling Time Year 2 (in days) Solarization Soil Fumigation Plastic Mulch 0 13.1a 13.1a 13.1a 28 13.4a 15.4a 12.2a 63 10.7a 13.8a 11.7a 119 8.9a 11.1a 10.7a 175 9.7a 9.8a 9.3a 245 9.5a 13.1a 9.8a 343 3.8a n.t. 6.0a aMeans followed by the same letter within each row for each sampling period are not significantly different accord ing to FisherÂ’s LSD test. bn.t.= not tested Sixty-three days after burying th e inoculum, SDP failed to detect P. capsici in the soil solarized samples but mSDPO had detected the organism in 50 and 83% of the samples in year 1 and year 2, respectively. The non-treated plots (white plastic mulch) had viable inoculum in as much as 83% of samples for both years, although SDP failed to detect any P. capsici in year 2. After 119 days, SDP faile d to detect viable inoculum in both years in the solarized samples, although mSDPO detected inoculum of P. capsici in 100 and 83% of the samples for year 1 and year 2, respectively (Fig. 2-3). Lemon leaf baiting was more variable but still detected inoculum in 100% and 17% of samples for

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15 year 1 and year 2, respectively. The non-trea ted soil samples contained viable inoculum in both years using all three techniques. Table 2-2. Percentage of soil samp les with viable inoculum of Phytophthora capsici as determined by soil dilution plating (SDP ), modified soil dilution platingoverlay (mSDPO), and lemon leaf baiting (LLB). aMeans followed by the same letter within each row for each treatment are not significantly different accord ing to FisherÂ’s LSD Test. bn.t.= not tested After 175 days, SDP was successful in detecting P. capsici only in year 1 from the solarized plots but only in 17% of samp les (Table 2-2). In year 2, mSDPO was significantly bette r in detecting P. capsici in 100% of samples while LLB only had 33% detection success. The LLB assay was more sensitive than mSDPO in year 1 but equally sensitive in year 2 for the non-treated soil samples. P. capsici was still recovered after 245 days from both the solarized and non-tr eated white plastic mulch samples using mSDPO and LLB; however, P. capsici was not detected in solarized soil using SDP in both years. The mSDPO assay detected P. capsici in all solarized and non-treated Recovery of Phytophthora capsici (%)a Soil Solarization White Plastic Mulch Year Days post transplant SDP mSDPO LLB SDP mSDPO LLB 1 28 0a 0a n.t.b 33a 100b n.t. 1 63 0a 50b n.t. 83.3a 83.3a n.t. 1 119 0a 100b 100b 50a 100b 83.3b 1 175 16.7a 33.3a 100b 33.3a 50a 100b 1 245 0a 100b 50ab 0a 100b 83.3b 1 343 0a 100b 16.7a 0a 16.7a 16.7a 2 28 0a 100b n.t. 100a 100a n.t. 2 63 0a 83.3b n.t. 0a 83.3b n.t. 2 119 0a 83.3b 16.7a 33.3a 100b 50ab 2 175 0a 100b 33.3a 0a 100b 66.7c 2 245 0a 100b 83.3b 16.7a 100b 83.3b 2 343 0a 100b 33.3a 0a 100b 16.7a

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16 samples for both years, and LLB detected P. capsici in 83% of white plastic mulch soil samples for both years but only 50% in y ear 1 and 83% in ye ar 2 (Table 2-2). Figure 2-3. Open face agar plate with colonies of Phytophthora capsici obtained only via the modified soil dilution plating-overl ay assay. Sporulation can be observed with the naked eye and sporangia can be observed microscopically at 40X or 100X magnification. At the last sampling date, at 343 days, viab le inoculum was still detected in both solarized and non-treated soils but only by using mSDPO and LLB (Table 2-2). The mSDPO assay was more sensitive than LLB. Population levels were signi ficantly higher when using the mSDPO assay (Table 23). Inoculum was not detected at all in both years with SDP in the solarized plots except for sampling in year 1 at 175 days when onl y 0.5 colony forming units (cfu) per gram of soil were assessed. The plastic mulch control soil assayed with SDP had populations that ranged from 0 to 38 cfu/g of soil in y ear 1 and 0 to 33 cfu/g in year 2.

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17 Table 2-3. Average number of colony forming units of Phytophthora capsici for each treatment per sampling period. Soil was assayed using standard soil dilution plating (SDP) and a modified soil di lution plating-agar overlay (mSDPO) assay. zMeans followed by the same letter within each row for each treatment are not significantly different accord ing to FisherÂ’s LSD Test. Discussion In this study, the first sa mpling from the solarization experiment did not contain any viable propagules in year 1 using bot h SDP and mSDPO. But in year 2, only mSDPO detected viable inoculum in the sample s. This would indicate that inoculum such as mycelium was controlled and oospores of P. capsici were reduced or injured by fall solarization. The plastic mulch controls had viable inoculum detected with both assays in both years. Lack of initial de tection of inoculum in the solarized plots may be due to the initial raising of soil temperatures that ma y cause thermal death, membrane disruption, or inactivation of pathogenic microorganism s (Katan, 1981; Katan and De Vay, 1991). Although during fall solarization temperatures may have reached an average high of Average Number of Colony Forming Units (cfu)/g soilz Soil Solarization White Plastic Mulch Year Sampling Time (in days) SDP mSDPO SDP mSDPO 1 28 0.0a 0.0a 1.6a 6.6b 1 63 0.0a 1.4a 38.1a 60.0a 1 119 0.0a 17.1b 2.4a 34.7b 1 175 0.5a 3.6a 0.4a 1.7a 1 245 0.0a 29.1b 0.0a 61.0b 1 343 0.0a 14.0b 0.0a 1.7b 2 28 0.0a 86.6b 32.5a 172.3b 2 63 0.0a 41.8b 0.0a 70.6b 2 119 0.0a 15.0b 0.6a 158.7b 2 175 0.0a 35.5b 0.0a 108.6b 2 245 0.0a 25.3b 0.2a 82.5b 2 343 0.0a 14.7b 0.0a 32.9b

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18 36 C, such temperature is enough to halt the growth of many isol ates, which normally have optimum temperatures between 24 C and 33 C (Tsao, 1991; Erwin and Ribeiro, 1996). The failure to detect Phytophthora spp. following soil solarization may be attributed both to the SDP technique a nd delayed germination of propagules. No detection with SDP might explain why inoculum of Phytophthora spp. was not detected in previous studies (McGove rn et al., 2000; Chellemi et al., 1994). More time was required for growth or germination of a pr opagule and that is what the mSDPO assay allowed for. Figure 2-4. Center of origin of a colony of Phytophthora capsici that originated from a germinating oospore. This picture, ta ken at 400X magnification, was obtained only by the modified soil d ilution plating-overlay assay. Since other propagules such as zoospores, mycelia, and sporangia are expected to survive fewer than 120 days, the last three sa mpling periods should have consisted of soil containing only oospores. Visual observations under the microscope in fact revealed oospores as the source of origin of some colonies (Fig. 2-4). These oospores were aplerotic and had germinated to form a co lony but, in some instances, the initial mycelium had produced a terminal sporangium . Most colonies may have lost their

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19 oospore when soil dilution plates were washed to remove the soil overlay or were not easily observed under the microscope at 40X or 100X magnification. Although indirectly, since it occurs post-solariza tion, another mechanism responsible for decreasing inoculum levels in the solarized soil in this study may be biological control. Soil te mperatures that were 12 C higher in the solarized plots at a 15cm depth may have promoted steady growth of microorganisms such as thermotolerant fungi, actinomycetes, and bacteria (Ho itink and Boehm, 1999). These edaphic microorganisms may play an important co mponent in reducing inoculum levels by competition, antibiosis, and parasitism or pr edation. Based on the results obtained only by SDP, fall soil solarization was important at eradicating inoculum. Only once was inoculum detected by SDP and at a very lo w population of 0.5 cfu/g soil. However, the mSDPO may have allowed for delayed germ ination and colony formation during the extra days 3-4 days of incubation. That is w hy population levels in solarized soil samples were, in two sampling periods, equal or sligh tly greater than that of the plastic mulch control. In the plastic mulch control, viable inoc ulum was detected in all 12 sampling dates with mSDPO but only seven times with SDP. After the 175 day sampling date and beyond, SDP determined population levels of P. capsici to be 0 to 0.6 cfu/g soil while mSDPO determined populations to be 1.7 to 108.6 cfu/g of soil. The fact that inoculum was not detected at all at 343 days with SD P but was detected with mSDPO could explain why inoculum is not detected in soil prior to the growing seas on. In year 2, high levels of inoculum were still detected after 343 days as the mSDPO assay estimated populations of P. capsici to be on, average, 32.9 cfu/g of soil.

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20 P. capsici was detected for at least 343 days, lo nger than in previous studies with the pathogen (Ansani and Matsuoka; 1983; Bowers et al., 1990). Inoculum was grown in wheat seed, which may have protected P. capsici and increased its longevity. Inoculum of P. capsici may survive in soil by infecting seed and other plant tissue that may have come from a field crop or a weed. Oospore su rvival in soil is assumed to be the main source of initial inoculum of P. capsici (Ristaino and Johns ton, 1999; Lamour and Hausbeck, 2000) In Florida, this study was able to determine the survival of oospores of P. capsici from one year to another. This propa gule represents a potential source of initial inoculum that could result in peppe r and other vegetable disease outbreaks in commercial fields.

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21 CHAPTER 3 NATURAL INFESTATIONS OF Phytophthora capsici ARE ASSOCIATED WITH LOCAL WEED POPULATIONS IN CONVENTIONAL VEGETABLE FARMS Introduction Phytophthora blight is an important plant dis ease causing significant losses in bell pepper and other fruit and vegetable crops such as cucumber, pumpkin, squash and tomato (Parra and Ristaino, 2001). Phytophthora capsici , an oomycete, is more closely related to photosynthetic algae than true fungi and is currently placed in the kingdom Chromista (Erwin and Ribeiro, 1996). Phytophthora spp. are water molds, and water is critical to the diseas e cycle of this pathogen (Ristaino, 2003). This pathogen has been a major problem in Florida and other states su ch as California, Mich igan, North Carolina, New Jersey, and New Mexico (Ristaino a nd Johnston, 1999). Diseases caused by this pathogen are most common in the subtropics , tropics, and in the summer months of temperate regions (Ploetz et al., 2002). The incidence of P. capsici has increased in recent years in FloridaÂ’s pepper and squash producti on areas (McGovern et al., 1998; Roberts et al., 1999; Ploetz and Haynes, 2000). Phytophthora spp. require wet conditions and ar e usually very poor saprophytes (Erwin and Ribeiro, 1996). Without availability of host plants to allow for survival of the organism, P. capsici must overseason as mycelium, sporangia, zoospores, or as oospores if both compatibility or mating types are present. In an overwintering field experiment on survival of P. capsici in New Jersey soil, Bowers et al. (1990) showed that 9% of oospores were still viable after 189 days by us ing a vital stain assay for viability. In

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22 Brazil, Ansani and Matsuoka (1983) reported that sporangia and zoospores survived fewer than 75 days in soil while oospores survived for up to 120 days. Weeds have been shown to be alterna tive hosts for several plant pathogens (Hepperly et al., 1980; Black et al., 1996; Dissanayake et al., 1996; Barak et al., 2001; Adkins and Rosskopf, 2002; Hollowell et al., 2003). Recent field and shadehouse experiments tested 15 common weed species that commonly occur in squash fields in the Homestead area of south Florida by inocula tion and examination for root colonization by P. capsici (Ploetz and Haynes, 2000; Ploetz et al., 2002). The authors found that common purslane (Portulaca oleracea) was colonized by P. capsici in 2 out of 25 plants sampled from infested fields. The other fourteen weed species were not colonized by the pathogen in either field or shadehouse (seedling) studies. Seedlings tested in the shadehouse experiments by Ploetz and Haynes (2000) we re: common beggarÂ’s tick ( Bidens alba ), Lepidium sp., Pangolagrass ( Digitaria decumbens ), jungle rice ( Echinocloa colonum ), panic grass ( Panicum adspersum ), foxtail ( Setaria verticillata ), partridge pea ( Cassia fasiculata ), and phaesy bean ( Macroptilium sp.). Plants tested in field experiments were: spiny amaranth ( Amaranthus spinosum ), Santa Maria ( Parthenium hysterophorus ), sweetpotato ( Ipomea batatas ), Virginia pepperweed ( Lepidium virginicum ), knotgrass ( Paspalum distichum ), Black medic ( Medicago lupulina ), and common purslane ( Portulaca oleracea ). The objectives of this study were to identify natural populations of P. capsici occurring on weed populations in commerc ial vegetable farms and to assess their importance as alternative hosts for this pathogen.

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23 Materials and Methods Field Sites Commercial vegetable fields were sa mpled in Palm Beach County, located in southeast Florida. A total of nine locati ons were sampled once each during August and December 2001, and March 2002. Fields were located in Dubois farms, Whitworth farms, Green Cay Farm, Midway Farm , Hobe Sound Farm and Jill Farm. Sampling of Weeds Weed sampling focused on prevalent weed species that were present in close proximity to vegetable field beds, and which ha d not been previously tested by Ploetz and Haynes (2000). Since these authors reported co mmon purslane as an alternative host for P. capsici , this weed species might pose the sa me threat in Palm Beach County and therefore was sampled in Palm Beach County. Other weeds sampled for this study were: American black nightshade ( Solanum americanum ), Carolina wild geranium ( Geranium carolinianum ), common ragweed ( Ambrosia artemisiifolia ), purple nutsedge ( Cyperus rotundus ), and yellow nutsedge ( C. esculentus ). Weeds were sampled from fi elds with incidence of P. capsici during the 2001-2002 growing season. Whole weed plants between 15 and 40 cm long were dug out from the soil to keep the root system intact, bagged, labeled and taken back to the laboratory for immediate processing. Weed plant sample s were non-symptomatic and roots looked healthy and with little or no r oot discolorations or lesions. Roots were thoroughly rinsed under high pre ssure water, surfa ce-sterilized with 70% ethanol for about 45 s, and blotted dr y. Crown and root ti ssue were plated on PARPH, a selective medi um for isolation of Phytophthora spp. This medium consists of 17 g of cornmeal agar in 1 L of distilled water and amended with 5 mg of pimaricin, 250

PAGE 35

24 mg of ampicillin, 10 mg of rifampicin, 100 mg of pentachloronitrob enzene, and 50 mg of hymexazol (Mitchell and Kannwischer-Mitchell, 1992). Plates were incubated for 3 to 8 days in the dark at 24 C (Lamour and Hausbeck, 2000). Suspect colonies were cleaned of bacter ial contamination using a Van Tieghem cell (Erwin and Ribeiro, 1996). Isolate confirmation as P. capsici was based on colony morphology, sporangial charac teristics, oogonial and oospor e morphometrics, and the production of oospores when paired with an opposite mating type (Erwin and Ribeiro, 1996; Aragaki and Uchida, 2001). PCR Identification of Isolates Isolates were tested using the Polyme rase Chain Reaction (PCR) to validate morphological identification as P. capsici . DNA was extracted from mycelium of the isolates using a modified phenol: chloroform extraction method (Goodwin et al., 1992). DNA was amplified using the primer PCAP in combination with the universal primer ITS 1 (Ristaino et al., 1998 ). Confirmation of P. capsici was obtained when amplification of an approximately 172-bp fragment visualized on 1% agarose gels containing a molecular size standard for size approximation. This primer combination will also amplify P. citricola and P. citrophthora . However, the amplification product for P. citrophthora is closer to 200-bp and this speci es can be differentiated from P. capsici by morphological characteristics such as sporangia bei ng noncaducous (Erwin and Ribeiro, 1996). Although the amplification product of P. citricola is of similar size to P. capsici, this species can be differentiated from P. capsici by being homothallic and having different morphological characteristics such as non caducous, semipapillate sporangia.

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25 Pathogenicity Tests on Pepper To further characterize any isolates of P. capsici recovered from weeds as being plant pathogenic, pathogenicit y tests were carried out in the greenhouse on bell pepper ( Capsicum annuum L.) cultivar “Sakata Hybrid”. Inoculum of P. capsici was produced on autoclaved seeds of wheat ( Triticum aestivum L.) according to Mitchell and Kannwischer-Mitchell (1992). Approximately 20 grams of seed plus 25 ml of deionized water were added to a 250-ml flask and a llowed to soak overnight. Flasks were autoclaved two times on two c onsecutive days. Isolates of P. capsici were individually grown on V-8 agar for 5 days. At this point, 5-mm diameter disk plugs were added to the watersoaked seeds and incubated for 3 weeks at 25 C. Infested seed was ground in a Black & Decker® HC3000 Mincer/Chopper and mixe d at 1g per kg of autoclaved soil. One 5-week-old pepper seedling was added to infested soil in each of four 10-cm diameter pots per isolate per experiment. E xperiments were repeated twice. Disease assessments were conducted daily and seedlings were monitored for mortality for up to 4 weeks. Samples of diseased seedlings were plated immediately after mortality onset, while any surviving plants were plated after four weeks. Pathogenicity Tests on Weeds Any isolate or sub-gr oup of isolates of P. capsici obtained from a specific weed were tested on such weed in greenhouse e xperiments. In addition, one or two known isolates of P. capsici were tested on the weeds to asse ss for differential pathogenicity or root colonization. Approximately 8-week-old weed seedlings were inoculated using the same inoculation method describe d above. Known isolates of P. capsici that were used were: Cp30, an A1 compatibility type isolated from bell pepper in Palm Beach County in

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26 1997, and Cp32, an A2 compatibility type is olated from bell pepper in Palm Beach County in 1998. Four pots per isolate were utilized per experiment. The experiment was repeated once. Diseased root and cr own tissue was plated on PARPH medium immediately upon plant mortality or af ter 7 weeks for non-diseased plants. In case the taxonomic identification to species level of the American black nightshades sampled might have been erroneous, isolates of P. capsici were tested in the greenhouse on 7-week-old seedlings from four other related Solanum spp. of importance to Florida or the Unites States: cockroachberry ( S. capsicoides ), Carolina horsenettle ( S. carolinense ), black nightshade ( S. nigrum ), and eastern black nightshade ( S. ptycanthum ). Seeds of common purslane and eastern blac k nightshade were obtained commercially from Valley Seed Service (Fresno, Californi a), seeds for American black nightshade, cockroachberry, Carolina horsenettle, and bl ack nightshade were obtained from Mark Elliot (Dr. Raghavan Charudattan laboratory, University of Florida), and seeds for Carolina wild geranium were obtained from Daniela Bell (Dr. Laura Galloway laboratory, University of Virginia). Statistical Analysis Pathogenicity experiments on pepper and w eeds were arranged in a completely randomized block design with four and eight replications, respectively. Experiments were repeated once and data from each expe riment were pooled because homogeneity of variance was confirmed by Bar tlettÂ’s test (Gomez and Go mez, 1994). Data from each variable was arcsine transformed before analysis of variance (ANOVA) was conducted using SAS (Release 8.02 Level 02M0 for Windows ; SAS Institute Inc., Cary, NC, USA).

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27 Mean comparisons were conducted using Fisher Â’s protected least si gnificant difference (FLSD, P 0.05) test. Results Isolation and Identification of Phytophthora spp. from Weed Roots Out of a total of 187 samples representi ng six weed species, a total of 16 root systems contained Phytophthora spp. growing out on PARPH medium (Table 3-1). Morphological features were used to identify isolates of Phytophthora spp. recovered from weed roots plated on PARPH medium. Thirteen isol ates were classified as P. capsici based on morphological characteristics de scribed by Erwin and Ribeiro (1996) and Aragaki and Uchida (2001). These isolates had semi-papill ate to papillate sporangia, caducous sporangia with pedicels that exceeded 35 m in length, exhibited heterothallism, and did not produce chlamydospores (Fig. 3-1) . When paired with an opposite mating type, oospores produced were mostly aplero tic; oogonia averaged between 36-38 microns in diameter and were amphyginous in their anth eridial attachment. Except for one isolate from common ragweed, the other fift een isolates were heterothallic. Figure 3-1. Papillate sporangia of Phytopthora capsici CWG1-3 isolated from Carolina wild geranium ( Geranium carolinianum) in Palm Beach County.

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28 P. capsici was isolated from the roots of two common purslane plants, seven Carolina wild geranium plants, and four Amer ican black nightshade plants. Twelve out of thirteen isolates of P. capsici isolates were of compatibil ity type A1. One isolate from Carolina wild geranium, CWG1-4, had the A2 compatibility type. Table 3-1. Recovery of Phytophthora capsici from common weed species present in vegetable grower farms in Palm Beach County, Florida. No. of isolates recovered/ no. plants assayeda Host (Common name) Host (Binomial) Aug 2001 Dec 2001 Mar 2002 American black nightshade Solanum americanum 4/14 Carolina wild geranium Geranium carolinianum 1/3 6/18 b Common purslane Portulaca oleracea 0/35 0/4 2/26 Common ragweed Ambrosia artemisifolia 0/7 0/8 c 0/12 d Purple nutsedge Cyperus rotundus 0/18 0/12 Yellow nutsedge Cyperus esculentus 0/18 0/12 aFor each sampling date, the number of plants of a given species from which Phytophthora capsici was recovered is listed over the number that was assayed. bOne isolate identified as P. capsici was not able to be sub-cultured and was lost. cTwo isolates of Phytophthora spp. were recovered. dOne isolate of Phytophthora sp. was recovered. PCR Identification of Isolates Two isolates from common purslane, si x from Carolina wild geranium, four isolates from American black nightshade, and one from ragweed produced a PCR product with primers PCAP and ITS 1. All amplifi cation products were of the expected 172-bp except for the ragweed isolate which amplified a slightly la rger region of about 200 bp. Based on fragment size (Ristai no et al., 1998) and morphological characteristics such as non-caducous sporangia (Erwin and Ribeiro, 1996) , this isolate was te ntatively identified as P. citrophthora . Isolates not amplified by PCR in two separate reactions were not used for further experimentation.

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29 Figure 3-2. DNA amplified from isolates of Phytophthora spp. recovered from weeds in an initial screening on PARPH plates us ing primers PCAP and ITS 1. Isolates from American black nightshade (lanes 1-4), common purslane (lanes 5-6), common ragweed (lane 7), P. capsici Cp10 (lane 8), and P. capsici Cp32 (lane 9) were screened. Lane 10 contains a no-template c ontrol; lane 11 contains a 100-bp ladder. Pathogenicity Tests on Pepper Only heterothallic isolates which produced a PCR product were used for pathogenicity tests on bell pepper seedlings. All isolates from common purslane and American nightshade, and 5 of 6 isolates from Carolina wild geranium, caused 100% mortality on pepper (Table 3-2). One isolate from Carolina wild geranium caused mortality in only 38% of the pepper seedlings after 4 weeks. Based on th ese data, a sub-group of isolates recovered from each weed species was used for furthe r experimentation on their host of origin. Isolates chosen were: CoPur3-1 from common purslane; NSh71, NSh7-2, and NSh7-3 from American black nightshade; and CW G1-2, CWG1-3, and CWG1-4 from Carolina wild geranium. Pathogenicity Tests on Weeds Black nightshade ( S. nigrum ) was the only weed species where plant mortality occurred when soil was infested with P. capsici . Mortality and pe rcent recovery of P. 1234567891011500 200 1234567891011500 200

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30 capsici from the roots of black nightshade inoculated with NSh7-2 and Cp32 was 100% (Table 3-3). Table 3-2. The effect of soil infestations with Phytophthora capsici isolates obtained from weed species in Palm Beach County, Florida, on percent mortality of bell pepper seedlings. Isolate Host (Common Name) Host (Binomial ) Mortality (%) CoPur3-1 Common purslane Portulaca oleracea 100.00a CoPur3-3 Common purslane Portulaca oleracea 100.00a CWG1-1 Carolina wild geranium Geranium carolinianum 37.50b CWG1-2 Carolina wild geranium Geranium carolinianum 100.00a CWG1-3 Carolina wild geranium Geranium carolinianum 100.00a CWG1-4 Carolina wild geranium Geranium carolinianum 100.00a CWG1-5a Carolina wild geranium Geranium carolinianum 100.00a CWG1-5b Carolina wild geranium Geranium carolinianum 100.00a NSh7-1 American black nightshade Solanum americanum 100.00a NSh7-2 American black nightshade Solanum americanum 100.00a NSh7-3 American black nightshade Solanum americanum 100.00a NSh7-4 American black nightshade Solanum americanum 100.00a a Values for each variable were arcsine transformed before analysis. Data for each variable within each species were pooled for statistical analysis because homogeneity of variance was confirmed by BartlettÂ’s test. Table 3-3. Pathogenicity tests on black nightshade ( Solanum nigrum ) using isolates of Phytophthora capsicia. Isolate Tested Original Host Mortality (%) Incidence (%) Seed control 0.00 a 0.00 a Cp30 Bell pepper 62.50 b 75.00 b NSh7-2 American black nightshade 100.00 b 100.00 c Cp32 Bell pepper 100.00 b 100.00 c a Values for each variable within weed species were arcsine transformed before analysis. Data for each variable within weed species were pooled for statistic al analysis because homogeneity of variance was confirmed by BartlettÂ’s test. All isolates were aggressive in causing disease and plant mort ality on bell pepper within fourteen days of soil infestation with P. capsici. Symptoms of this pathogen on black nightshade were dark lesions around the crown, wilting, leaf drop, and the spread of crown lesions upwards along the main stem (Fig. 3-1). Roots of moribund or dead black nightshade plants were co mpletely thinned out and rotted.

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31 Figure 3-3. The effect of Phytophthora capsici on black nightshade ( Solanum nigrum ) inoculated with isolate NSh7-2 (left) in comparison to the non-inoculated black nightshade (ri ght). Extensive leaf drop and st em rot can be visualized in the pot containing the diseased plant material. None of the P. capsici isolates tested on common purslane ( P. oleracea ) caused plant mortality (Table 3-4). The incidence of P. capsici in roots using three isolates was not significant compared to th e non-infested control. Isolate Cp30 was not detected at all from the root systems of this plant. Table 3-4. Pathogenicity tests on common purslane ( Portulaca oleracea ) using isolates of Phytophthora capsici a. Isolate Tested Original Host Mortality (%) Incidence (%) Seed control 0.00 a 0.00 a Cp30 Bell pepper 0.00 a 0.00 a CoPur3-1 Common purslane 0.00 a 12.50 ab Cp32 Bell pepper 0.00 a 25.00 ab a Values for each variable within weed species were arcsine transformed before analysis. Data for each variable within weed species were pooled for statistic al analysis because homogeneity of variance was confirmed by BartlettÂ’s test. Carolina wild geranium ( G. carolinensis ) had no mortality among plants tested with six different isolates of P. capsici (Table 3-5). Great vari ability was found in root colonization by each isolate. Isolates grouped into three different categories of differential colonization, ranging from 31% to 81% of plant roots tested. The two highest

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32 colonization rates were from isolates CWG1-2 and CWG14, both originally isolated from Carolina wild geranium. Table 3-5. Pathogenicity tests on Carolina wild geranium ( Geranium carolinianum ) using isolates of Phytophthora capsicia. Isolate TestedOriginal Host Mortality (%) Incidence (%) Seed Control 0.00 a 0.00 a CWG1-3 Carolina wild geranium 0.00 a 31.25 b Cp30 Bell pepper 0.00 a 31.25 b Cp32 Bell pepper 0.00 a 56.25 c CWG1-2 Carolina wild geranium 0.00 a 81.25 d CWG1-4 Carolina wild geranium 0.00 a 81.25 d a Values for each variable within weed species were arcsine transformed before analysis. Data for each variable within weed species were pooled for statistic al analysis because homogeneity of variance was confirmed by BartlettÂ’s test. None of the six isolates of P. capsici tested on eastern black nightshade ( Solanum ptycanthum ) were capable of causing plant mortality or disease symptoms (Table 3-6). Table 3-6. Pathogenicity test s on eastern black nightshade ( Solanum ptycanthum ) using isolates of Phytophthora capsicia. Isolate Tested Original Host Mortality (%) Incidence (%) Seed control 0.00 a 0.00 a Cp30 Bell pepper 0.00 a 37.50 b NSh7-1 American black nightshade 0.00 a 75.00 c NSh7-3 American black nightshade 0.00 a 75.00 c Cp32 Bell pepper 0.00 a 87.50 c NSh7-2 American black nightshade 0.00 a 100.00 c a Values for each variable within weed species were arcsine transformed before analysis. Data for each variable within weed species were pooled for statistic al analysis because homogeneity of variance was confirmed by BartlettÂ’s test. There were differential levels of root col onization by the isolates tested. Except for isolate Cp30 which was only recovered from 38% of root samples, all other isolates tested were detected in at least 75% of the root samples plated for P. capsici . Isolate NSh7-2 was recovered from the root systems of all root samples.

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33 Cockroachberry ( S. capsicoides ) had no plant mortality from the three isolates (Cp30, Cp 32, and NSh7-2) used in pathoge nicity studies (Data not shown). P. capsici was not isolated from the root systems of cockroachberry (Data not shown). Carolina horsenettle ( S. carolinense ) had no plant mortality but P. capsici did colonize some of the root samples inoculated with isolates Cp32 and NSh7-2 (Table 3-7). Samples with root colonization did not exceed 25%. Table 3-7. Pathogenicity tests on Carolina horsenettle ( Solanum carolinense ) using isolates of Phytophthora capsicia. Isolate Tested Original Host Mortality (%) Incidence (%) Control 0.00 a 0.00 a NSh7-2 American black nightshade 0.00 a 12.50 a Cp32 Bell pepper 0.00 a 25.00 a a Values for each variable within weed species were arcsine transformed before analysis. Data for each variable within weed species were pooled for statistic al analysis because homogeneity of variance was confirmed by BartlettÂ’s test. Plants of American black nightshade ( S. americanum ) also had 0% mortality rate among the plants tested w ith three isolates of P. capsici (Table 3-8). However, there was a significant difference in root colonization levels by P. capsici depending on the isolate used. Isolates recovered from roots ranged fr om 0% for isolate Cp30 to 63% for isolate Cp32. Table 3-8. Pathogenicity tests on American black nightshade ( Solanum americanum ) using isolates of Phytophthora capsici a. Isolate Tested Original Host Mortality (%) Incidence (%) Seed control 0.00 a 0.00 a Cp30 Bell pepper 0.00 a 0.00 a NSh7-2 American black nightshade 0.00 a 12.50 a Cp32 Bell pepper 0.00 a 62.50 b aValues for each variable within weed species were arcsine transformed before analysis. Data for each variable within weed species were pooled for statistic al analysis because homogeneity of variance was confirmed by BartlettÂ’s test.

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34 Discussion Weeds as alternative hosts for P. capsici in Florida and in the Unites States were first examined by Ploetz and Haynes (2000). These authors were successful in identifying common purslane as an alternative host for this pathogen, and low levels of root colonization were confirmed by this study. Even low levels of inoculum may survive season to season. As with diseases caused by many Phytophthora spp., low levels of initial inoculum are still signifi cant in future disease outbreaks (Erwin and Ribeiro, 1996). However, if common pursl ane is well managed, as was the case in several Palm Beach County farms surveyed, this weed may not pose a major problem. Under natural field conditions, Carolina wild geranium and American black nightshade were also found to be alternative hosts for P. capsici . Five isolates of P. capsici from Carolina wild geranium did not cau se any mortality in this plant but the pathogen was able to colonize the root system of this pl ant. Although roots appeared healthy when visualized with the naked eye, root colonization was detected in up to 81% of root samples. Therefore, P. capsici can use this weed as a mechanism of survival when a host crop is unavailable. In greenhouse studies, besides American black nightshade, other Solanum spp. were found to be reservoirs of P. capsici . Isolate Cp32 efficiently colonized up to 63% of root tissue samples of American blac k nightshade. Black nightshade ( S. nigrum ) was the only weed species in which P. capsici caused high levels of mortality (63-100%) and root colonization (75-100%). Black nightshade may represent a significant source of inoculum for P. capsici in Florida if introduced. Carolin a horsenettle was still colonized, althoughat very low levels, by P. capsici . Although cockroachberry roots were not

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35 colonized by P. capsici in greenhouse experiments, other isolates of P. capsici may be able to colonize this plant. The current study and that conducted in Ho mestead (Ploetz et al., 2002) have only tested 20 weed species. In many field sites sampled in this study, it was difficult to find weeds close to pepper beds due to intensive weed management prac tices carried out by these growers. However, other farms where weeds are less intensively managed, including organic operations, coul d potentially harbor weeds that have not been tested for susceptibility to P. capsici . The use of herbicides, tillage, fumigation, and other practices, made it difficult to examine weeds until December 2001. During March 2002, P. capsici was detected in all three weed species that were determ ined to be alternative hosts of P. capsici . Therefore, weed management should begin as soon as the growing season is over, since any inoculum found in weed plants at this time can potentially survive until August or September, when the next growing season be gins. Even if soil is tilled and weeds incorporated into the soil, undecomposed plant material may contain inoculum that will persist in soil. This is the first study wher e natural populations of P. capsici have been detected in two naturally occurr ing weed species: G. carolinianum , and S. americanum . The survival of inoculum in weeds may be an important mechanism by which P. capsici is able to persist from year to year.

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36 CHAPTER 4 INCREASED RESISTANCE TO MEFENOXAM AND THE OCCURRENCE OF BOTH MATING TYPES OF Phytophthora capsici IN FLORIDA Introduction Phytophthora capsici Leonian, an oomycete, is a so ilborne pathogen that causes root rot, crown rot, fruit rot and foliar blig ht on several solanaceous and cucurbit hosts. This pathogen has adversely affected pe pper, tomato, pumpkin, squash, and watermelon production in the United States and throughout the world (Caf é-Filho et al., 1995; Hwang and Kim, 1995; Ristaino and J ohnston, 1999). The incidence of P. capsici has increased in recent years in Florida’s pepper and squa sh production areas (McGovern et al., 1998; Roberts et al., 1999; Ploetz and Haynes, 2000). Since 1931, when Weber (1932) first observed P. capsici in Florida, this pathogen has been prevalent in Florida vegetable pr oduction. However, relatively minor damage had been observed until 1982 when this pat hogen caused devastating losses in Palm Beach County to pepper ( Capsicum annuum L.), eggplant ( Solanum melongena L.) and squash ( Cucurbita pepo L. var esculentum Nees) (McGovern et al., 1998; Ploetz and Haynes, 2000). Other major outbreaks also occurred in 1993, 1998, and 2003 in pepper, cucurbits and other vegetable crops (p ersonal communication, Pamela Roberts). Oospores are considered the primary survival propagules of P. capsici due to their thick wall, which enables them to overwinte r in the soil (Hwang and Kim, 1995; Ristaino and Johnston, 1999). This sexual phase occurs when isolates of opposite compatibility or mating types (designated A1 and A2) are in cl ose proximity, leading to the formation of

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37 antheridia and oogonia and the subsequent form ation of oospores. During this process, genetic recombination may occur and oospores may represent new gene combinations (Erwin and Ribeiro, 1996). For disease management, the principal prac tices include cultural control to avoid build-up of inoculum (Ristaino et al., 1992) and the use of fungicides. The phenylamide class of fungicides, which includes metalaxyl (Ridomil), has been used on pepper and cucurbit production in Florida for over tw o decades. However, the intensive use of metalaxyl may lead to rapid selectio n of metalaxyl-resistant strains of Phytophthora spp., as it did with P. infestans in Europe, where selection occurr ed within a year of fungicide application (Dowley and OÂ’Sulliv an, 1981; Goodwin et al., 1998). Mefenoxam (Ridomil Gold EC), the active en antiomer of metalaxyl, has been used as the newest formulation of metalaxyl fo r over six years. Selection of mefenoxamresistant strains of P. capsici may be rapidly occurring in Florida as well, since production of peppers and cucurbits is depende nt on fungicide applic ations to ensure a good quality harvest (Ploetz and Haynes, 2000) . In 1997, such selection occurred in North Carolina when mefenoxam was sprayed fo r the first time in pepper fields (Parra and Ristaino, 2001); almost 60% of isolates of P. capsici from diseased plants were resistant to mefenoxam. The greatest proportion of resistant isolates came from fields where mefenoxam was used alone rather than in combination with other fungicides. Since both compatibility types were present among resistant isolates, oospore production may be occurring and any inoculum surviving as oospores will be able to carry such resistance into the next growing season.

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38 The objectives of this study were to 1) collect isolates of P. capsici from several geographic regions of Florida from current or recent outbreaks and fr om collections that date back several years; 2) test the in vitro sensitivity to mefenoxam to assess if selection of mefenoxam–resistant strains of P. capsici has occurred throughout Florida; and 3) characterize isolates from fiel ds by mating type to assess if both types are present, a sign that oospore production, geneti c recombination, long-term survival, and build-up of fungicide resistant proge ny may be taking place. Materials and Methods Pathogen Sampling and Isolation Diseased plant material and soil were collected from fields where P. capsici had just been observed within a two-week period. Soil was collected from the rhizosphere or in close proximity to diseased plant material . Weeds were sampled in close proximity to beds with infected pepper plan tings. None of the weeds were diseased but were chosen for their common occurrence in fields sample d. Samples were bagged and transported in a cooler back to the laboratory. Stems and roots were thoroughly rinsed under high pressure water and surfacesterilized with 70% EtOH for about 45 s and small pieces (150 x 40 mm) of actively expanding lesions or roots were plated on PA RPH medium. This medium consist of 17 g of cornmeal agar (CMA) amended with 5 mg of pimaricin, 250 mg of ampicillin, 10 mg of rifampicin, 100 mg of pent achloronitrobenzene, and 50 mg of hymexazol per liter of distilled water (Mitchell and Kannwischer-Mitch ell, 1992). Plates were wrapped with parafilm and incubated for 3 to 8 days in the dark at 24 C (Lamour and Hausbeck, 2000).

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39 Soil was processed by adding approximatel y 40 g of infested soil to 160 ml of 0.25% sterile water agar and stirred for 2 min; 1-ml samples were then plated on PARPH medium, for a 1:5 soil dilution plating (Larki n et al., 1995). A total of ten PARPH plates were used for each soil sample. Plates were incubated in the dark at 24 C for 72-96 h, and then rinsed under running water to remove the soil from the surface of the medium. Suspect colonies were sub-cultured and initial confirmation was based on colony morphology and sporangial characteristics (Erwin and Ribeiro, 1996; Aragaki and Uchida, 2001). PCR Identification of Isolates A subset of all isolates was tested us ing PCR (Polymerase Chain Reaction) to confirm identification as P. capsici . DNA was extracted from mycelium of the isolates by a modified phenol: chloroform extracti on method (Goodwin et al., 1992). DNA was amplified using the P. capsici primer, PCAP (5Â’-TAATCAGTTTTGTGAAATGG), in combination with the universal primer ITS1 (5Â’-TCCGTAGGTGAACCTGCGG) (Lee et al., 1993; Ristaino et al., 1998). Any isolates that did not amplify in two separate reactions, or the PCR product was greater than 172-bp, were not used for compatibility type experiments and mefenoxam sensitivity test s. Amplification of an approximate 172bp fragment was visualized on 1% agarose ge ls containing a molecular size standard for size approximation. Culture Collection and Maintenance Isolates of P. capsici from Florida spanning from 1982 to 2001 were obtained from Drs. David J. Mitchell (Unive rsity of Florida-Gainesville), Randy C. Ploetz (University of Florida TREC-Homestead), Pamela D. Roberts (University of Florida SWFREC-

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40 Immokalee), and Robert J. McGovern (University of Fl orida GCREC-Bradenton). Isolates from 2001 to 2003 were collected from field sites throughout Florida. All isolates were maintained in CMA plates and stored in the dark at 24 C. Cultures were transferred every two months Compatibility Type Determination Isolates of P. capsici were grown on 10% clarified V8 juice (CV8) agar and crossed with known testers for A1 (isolate Cp-30) and A2 (isolate Cp-32). A 5-mmdiameter plug of non-colonized 10% CV8 agar was sandwiched between a plug of tester mycelium and the isolate of unknown compatibil ity type. After 4 days incubation at 25 C, the middle plug was examined under the microscope for oospore formation (Mitchell and Kannwis cher-Mitchell, 1992). In Vitro Fungicide Sensitivity Cultures of the isolates were grown on CV 8 agar for 4-6 days. Agar disks (approx. 6 mm diameter) from actively growing edges of the colony were placed at the center of clarified 10% CV8 juice agar media previously amended with 0, 5, and 100 g/ml of mefenoxam (Ridomil Gold EC; 48% a.i., susp ended in sterile deionized water), and added to 10% CV8 juice agar cooled to 50 C (Lamour and Hausbeck, 2000). Inoculated plates were incubated at 24 C for 72 h and colony diameters were measured. Subtracting the inoculation plug diameter from the diam eter growth of each colony and dividing the average diameter of the amended plates by the average diameter of the unamended control plates was used to calculate the pe rcent growth of each isolate on the amended media (Deahl et al., 1995). Isolates were ch aracterized as sensitive (S) if colony growth on media amended with 5 g/ml of mefenoxam was less than 40% of the isolates growth

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41 on non-amended media. Intermediate (I) isolates grew greater than 40% of the control at 5 g/ml but less than 40% of th at on non-amended media at 100 g/ml. Resistant (R) isolates grew greater than 40% of that on nonamended media at 100 g/ml (Parra and Ristaino, 2001). Results Morphological Identificati on of Isolates Recovered All isolates recovered in PARPH me dium were observed for morphological characteristics typical of P. capsici . Isolates of P. capsici produced ovoid to ellipsoid, papillate to semi-papillate, caducous spor angia that had pedicels exceeding 35 m in length (Erwin and Ribeiro, 1996). No chlam ydospores were observed, isolates were heterothallic and when paired with opposite mating types, oogonia were on average 36 m in diameter, antheridia were amphyginous in their oogonial attachment, and oospores were aplerotic. Sporangia did not exhibit a tapered base and mean sporangial diameter was less than 26 m (Aragaki and Uchida, 2001). Isolates identified as P. capsici are listed in Tables 4-1 to 4-6 and 4-8. PCR Identification of Weed Isolates All isolates from weeds originally identified as P. capsici through the use of selective media, colony morphology, and sporan gial and mycelial characteristics, were further validated as P. capsici through the use of the PCAP primer. Isolates from common purslane ( Portulaca oleracea ), nightshade ( Solanum sp.), and Carolina wild geranium ( Geranium carolinianum ) had PCR products of a pproximately 172-bp (Fig. 41). One isolate from common ragweed ( Ambrosia artemisifolia ) had a PCR product of approximately 200-bp and was not co nsidered for further studies

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42 123456789500 200 123456789500 200 Figure 4-1. DNA amplified from isolates of Phytophthora capsici recovered Carolina wild geranium in an initial screeni ng on PARPH plates us ing primers PCAP and ITS 1. Isolates tested were CWG1-1 (lanes 4), CWG1-2 (lane 5), CWG 13 (lane 6), CWG1-4 (lane 7), CWG1-5a (lane 8), and CWG1-5b (lane 9). Lane 1 contains a 100-bp ladder, lane 2 contai ns a no-template control, and lane 3 represents P. capsici isolate Cp32 from pepper. Mefenoxam Sensitivity and Compatibility Type Among Isolates in Florida In Palm Beach County, approximately 30% of isolates from crop and soil isolations were resistant to mefenoxam, but none of them dated prior to the ye ar 2000 (Table 4-1). The oldest resistant isolate wa s Cp-38, dating back to 2000. Fi ve isolates grew at 90% or more on mefenoxam-amended media at 100 g/ml compared to the non-amended control plates. No isolates tested at 100 g/ml grew between 30 and 70% of the non-amended control plates.

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43Table 4-1. Characteriza tion by mating type and in vitro sensitivity to mefenoxam of isolates of Phytophthora capsici from Palm Beach County isolated between 1982 and 2003 from various crops and soils. Isolate Host Year Mating type Source Sensitivity to Mefenoxam at 100 g/ml (%)a Cp-7 Bell Pepper 1984 A2 D.J. Mitchell Sensitive (0) Cp-11 Bell Pepper 1984 A2 D.J. Mitchell n.t.b Cp-12 Bell Pepper 1983 A1 D.J. Mitchell n.t. Cp-13 Bell Pepper 1983 A2 D.J. Mitchell Sensitive (0) Cp-14 Bell Pepper 1983 A1 D.J. Mitchell Sensitive (23) Cp-15 Bell Pepper 1983 A2 D.J. Mitchell Sensitive (0) Cp-16 Bell Pepper 1982 A1 D.J. Mitchell Sensitive (18) Cp-17 Bell Pepper 1982 A1 D.J. Mitchell Sensitive (23) Cp-32 Bell Pepper 1998 A2 D.J. Mitchell Sensitive (11) Cp-36 Tomato 1999 A2 D.J. Mitchell Sensitive (11) Cp-38 Bell Pepper 2000 A2 D.J. Mitchell Resistant (76) Cp-48 Bell Pepper 2000 A1 D.J. Mitchell Sensitive (13) PBC1-2 Bell Pepper 2001 A1 This study Resistant (96) PBC1-3 Bell Pepper 2001 A1 This study Resistant (103) PBC1-5 Bell Pepper 2001 A1 This study Resistant (114) PBC6-5 Bell Pepper 2001 A1 This study Resistant (85) PBC11-1 Bell Pepper 2001 A1 This study Sensitive (11) PBC11-2 Bell Pepper 2001 A2 This study Sensitive (9) PBC12-1 Bell Pepper 2001 A1 This study Sensitive (5) PBC12-3 Bell Pepper 2001 A1 This study Sensitive (0) PBC13-1 Bell Pepper 2001 A2 This study Sensitive (8) PBC14-1 Bell Pepper 2001 A2 This study Sensitive (5) PBC14-2 Bell Pepper 2001 A1 This study Resistant (68) PBC14-3 Bell Pepper 2001 A1 This study Resistant (72) PBC14-4 Bell Pepper 2001 A1 This study Sensitive (9)

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44Table 4-1. Continued Isolate Host Year Mating type Source Sensitivity to Mefenoxam at 100 g/ml (%)a PBCP10-1 Bell Pepper 2001 A1 This study Sensitive (7) PBCP10-2 Bell Pepper 2001 A1 This study Sensitive (9) PBCP10-3 Bell Pepper 2001 A1 This study Sensitive (6) PBCP10-4 Bell Pepper 2001 A1 This study Sensitive (13) PBCS1-4 Soil 2001 A2 This study Sensitive (10) PBCS8-4 Soil 2001 A2 This study Resistant (83) PBCS11-3 Soil 2001 A2 This study Sensitive (4) PBCS12-3 Soil 2001 A1 This study Sensitive (6) PBCS13-1 Soil 2001 A1 This study Sensitive (20) WPBM1 Bell Pepper 2001 A1 P.D. Roberts Resistant (100) WPBM2 Bell Pepper 2001 A1 P.D. Roberts Resistant (66) WPBM3 Bell Pepper 2001 A2 P.D. Roberts Sensitive (25) WPBM4 Bell Pepper 2001 A2 P.D. Roberts Resistant (86) WPBM5 Bell Pepper 2001 A2 P.D. Roberts Sensitive (15) WPBM6 Bell Pepper 2001 A1 P.D. Roberts Resistant (108) WPBM7 Bell Pepper 2001 A1 P.D. Roberts Sensitive (18) WPBM8 Squash 2001 A2 P.D. Roberts Sensitive (13) aIsolates were characterize d as sensitive to mefenoxam if colony growth at 5 g ml-1 was less than 40% of the isolateÂ’s growth on the nonamended control media. Intermediate isolates (I) exhibited growth greater than 40% of the nonamended media control at 5 g ml-1, but less than 40% of the nonmended me dia control with mefenoxam at 100 g ml-1 . Isolates that were re sistant exhibited growth greater than 40% of the nonamended me dia control with mefenoxam at 100 g ml-1. bn.t.= not tested. Isolates that were not tested were a result of isolate loss during storage in the cu lture collection prior to mefenoxam tests.

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45 In Palm Beach County, four isolates were insensitive to mefenoxam, as they were able to grow faster (between 100 and 114%) in mefenoxam-amended plates at 100 g/ml in comparison to the non-amended plates. Is olate PBC1-5 was fully resistant (Fig. 4-2). Figure 4-2. Effects of mefenoxam con centrations on radial growth of Phytophthora capsici resistant isolate PBC1-5. Th is isolate grew equally in 10% clarified V8 agar medium amended with 100 g/ml (left), 5 g/ml (center), and 0 g/ml (right) of mefenoxam. In Martin County, 33% of isolates were resistant to mefenoxam and were all isolated from soil in both 2001 and 2002 (Table 4-2). One resistant isolate, MCS 14-3, grew equally in the 100 g/ml mefenoxam-amended plates as in the non-amended control plates. Most of the sensitiv e isolates grew 10% or less in comparison to the control. In Dade County, the three levels of sens itivity to mefenoxam tested were found. Although most isolates were sensitive to mefen oxam, intermediate and resistant levels of sensitivity were also found (Table 4-3). Howe ver, no isolate grew at greater than 51% of the control in mefenoxam-amended plates at 100 g/ml. Three isolates did not grow at all at this concentration.

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46 Table 4-2. Characteriza tion by mating type and in vitro sensitivity to mefenoxam of isolates of Phytophthora capsici from Martin County. Isolate Host Year Mating type Source Sensitivity to Mefenoxam at 100 g/ml (%)a FtPi1-1 Bell Pepper 2001 A2 This study Sensitive (6) FtPi1-2 Bell Pepper 2001 A2 This study Sensitive (10) FtPi1-3 Bell Pepper 2001 A2 This study Sensitive (7) MC1A-1 Bell Pepper 2001 A2 This study Sensitive (8) MC1A-1p Bell Pepper 2001 A2 This study Sensitive (8) MC1A-5s Soil 2001 A2 This study Sensitive (9) MCS14-3 Soil 2001 A2 This study Resistant (100) MCS14-4 Soil 2001 A2 This study Sensitive (9) MCSo13-2 Soil 2002 A1 This study Sensitive (23) MCSo15-1 Soil 2002 A2 This study Resistant (74) MCSo15-2 Soil 2002 A2 This study Resistant (68) MCSo15-3 Soil 2002 A2 This study Resistant (65) a Isolates were characterized as sensitiv e to mefenoxam if colony growth at 5 g ml-1 was less than 40% of the isolateÂ’s growth on the nonamended control media. Intermediate isolates (I)exhibited growth greater than 40% of the nonamended media control at 5 g ml-1 , but less than 40% of the nonmende d media control with mefenoxam at 100 g ml-1 . Isolates that were resistant exhibited growth greater th an 40% of the nonamended media control with mefenoxam at 100 g ml-1 . Table 4-3. Characteriza tion by mating type and in vitro sensitivity to mefenoxam of isolates of Phytophthora capsici from Dade County. Isolate Host YearMating type Source Sensitivity to Mefenoxam at 100 g/ml (%)a I-2LedfordSQ2cc Squash 1999 A1 R.C. PloetzSensitive (0) I-2LedfordSQ3Br Squash 1999 A2 R.C. PloetzSensitive (14) III LedfordSQ3F Squash 1999 A1 R.C. PloetzSensitive (0) Iori SQ1A Squash 1999 A1 R.C. PloetzResistant (43) Iori SQ2D-2 Squash 1999 A2 R.C. PloetzResistant (51) Iori SQ2D-3A Squash 1999 A2 R.C. PloetzSensitive (12) Iori SQ2D-3B Squash 1999 A2 R.C. PloetzSensitive (0) Strano SQ1A-1 Squash 1999 A2 R.C. PloetzSensitive (15) Strano SQ1A-2 Squash 1999 A1 R. C. PloetzIntermediate (23) Strano SQ2B-1 Squash 1999 A1 R. C. PloetzIntermediate (25) Strano SQ3A-2 Squash 1999 A1 R.C. PloetzSensitive (7) a Isolates were characterized as sensitiv e to mefenoxam if colony growth at 5 g ml-1 was less than 40% of the isolateÂ’s growth on the nonamended control media. Intermediate isolates (I) exhibited growth greater than 40% of the nonamended media control at 5 g ml-1 , but less than 40% of the nonmende d media control with mefenoxam at 100 g ml-1 . Isolates that were resistan t exhibited growth greater th an 40% of the nonamended media control with mefenoxam at 100 g ml-1 .

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47 In Collier County, isolates collected between 1993 and 2001were sensitive to mefenoxam, whereas all 11 isolates of P. capsici collected from production fields in 2003 were resistant to mefenoxam (Table 4-4) . Growth of these isolates on mefenoxamamended plates at 100 g/ml ranged between 63 and 76% of the growth on the nonamended control plates. Growth of sensit ive isolates on mefenoxam-amended plates at 100 g/ml was less than 19% in comparison to the non-amended plates. Table 4-4. Characteriza tion by mating type and in vitro sensitivity to mefenoxam of isolates of Phytophthora capsici from Collier County. Isolate Host Year Mating type Source Sensitivity to Mefenoxam at 100 g/ml (%)a RJM93-378 Squash 1993 A2 R.J. McGovernSensitive (19) RJM93-420 Watermelon 1993 A2 R.J. McGovernSensitive (8) ImmM29 Tomato 2001 A1 P.D. Roberts Sensitive (10) ImmM32 Tomato 2001 A2 P.D. Roberts Sensitive (0) ImmM33 Bell Pepper 2001 A1 P.D. Roberts Sensitive (8) Imm10 Bell Pepper 2002 A2 This study Sensitive (0) Imm10b Bell Pepper 2002 A2 This study Sensitive (0) Imm018 Bell Pepper 2003 A1 This study Resistant (63) Imm0424-1 Bell Pepper 2003 A1 This study Resistant (66) Imm0424-2 Bell Pepper 2003 A1 This study Resistant (63) Imm0424-3 Bell Pepper 2003 A1 This study Resistant (68) Imm0424-4 Bell Pepper 2003 A1 This study Resistant (76) Imm0424-5 Bell Pepper 2003 A1 This study Resistant (67) Imm0424-6 Bell Pepper 2003 A1 This study Resistant (66) Imm0424-7 Bell Pepper 2003 A1 This study Resistant (63) Imm0424-8 Bell Pepper 2003 A1 This study Resistant (67) Imm0424-9 Bell Pepper 2003 A1 This study Resistant (65) Imm0424-10 Bell Pepper 2003 A1 This study Resistant (68) a Isolates were characterized as sensitiv e to mefenoxam if colony growth at 5 g ml-1 was less than 40% of the isolateÂ’s growth on the nonamended control media. Intermediate isolates (I) exhibited growth greater than 40% of the nonamended media control at 5 g ml-1 , but less than 40% of the nonmende d media control with mefenoxam at 100 g ml-1 . Isolates that were resistan t exhibited growth greater th an 40% of the nonamended media control with mefenoxam at 100 g ml-1.

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48 All eleven isolates collecte d in Collier in 2003 were hi ghly resistant to mefenoxam at 100 g/ml. None grew at less than 63% of the growth of the isolate on the nonamended plates. In previous years, some is olates were fully sensitive to mefenoxam and did not grow at all at 100 g/ml. Table 4-5. Characteriza tion by mating type and in vitro sensitivity to mefenoxam of isolates of Phytophthora capsici from Manatee County. Isolate Host Yea r Mating type Source Sensitivity to Mefenoxam at 100 g/ml (%)a RJM97-274 Squash 1997 A1 R.J.McGovern Sensitive (18) RJM98-285 Bell Pepper 1998 A2 R.J.McGovern Sensitive (0) RJM98-662 Squash 1998 A1 R.J.McGovern Sensitive (0) RJM98-698 Watermelon 1998 A2 R.J.McGovern Sensitive (7) RJM98-727 Watermelon 1998 A2 R.J.McGovern Intermediate (15) RJM98-728 Watermelon 1998 A2 R.J.McGovern Sensitive (4) RJM98-730 Squash 1998 A2 R.J.McGovern Sensitive (12) RJM98-765 Watermelon 1998 A2 R.J.McGovern Sensitive (11) RJM98-767 Bell Pepper 1998 A2 R.J.McGovern n.t.b RJM98-788 Squash 1998 A2 R.J.McGovern Sensitive (4) RJM98-794 Tomato 1998 A2 R.J.McGovern Resistant (43) RJM98-800 Squash 1998 A2 R.J.McGovern Sensitive (6) RJM98-805 Squash 1998 A1 R.J.McGovern Sensitive (10) RJM98-819B Watermelon 1998 A2 R.J.McGovern Sensitive (0) RJM98-825 Squash 1998 A2 R.J.McGovern Sensitive (15) RJM99-254 Squash 1999 A2 R.J.McGovern Sensitive (15) RJM99-1265 Squash 1999 A2 R.J.McGovern Sensitive (16) RJM99-323 Tomato 1999 A2 R.J.McGovern Sensitive (16) RJM01-1938 Calabaza 2001 A1 R.J.McGovern Resistant (47) CalSq-1 Calabaza 2001 A1 This study Sensitive (35) a Isolates were characterized as sensitiv e to mefenoxam if colony growth at 5 g ml-1 was less than 40% of the isolat eÂ’s growth on the nonamended control media. Intermediate isolates (I) exhibited growth greater than 40% of the nonamended media control at 5 g ml-1, but less than 40% of the nonmende d media control with mefenoxam at 100 g ml-1. Isolates that were resistan t exhibited growth greater th an 40% of the nonamended media control with mefenoxam at 100 g ml-1. bn.t.= not tested. Isolates that were not tested were a result of isolate loss during storage in the culture collection prior to mefenoxam tests. Isolates from Manatee County exhibited a ll three sensitivity levels to mefenoxam (Table 4-5). Approximately 84% of all isol ates from Manatee tested were sensitive to

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49 mefenoxam but the degree of sensitivity varied. Most sensitive isolates grew at less than 18% compared to the control in mefenoxam-am ended plates (Table 4-5). One sensitive isolate, CalSq-1, grew as high as 35% of the cont rol. Both resistant is olates had values at the lower threshold for resistance as th ey both grew at less than 50% at 100 g/ml of mefenoxam compared to the non-amended plates. Seven isolates from Florida with unknown geographical orig in were also tested for sensitivity and all were sensitive to mefenoxam. None were able to grow at greater than 23% of the control in the me fenoxam-amended plates at 100 g/ml (Table 4-6). All isolates were originally isolated between 1993 and 1997. Table 4-6. Characterization by mating type and in vitro sensitivity to mefenoxam of isolates of Phytophthora capsici from unspecified locations in Florida isolated between 1993 and 1997 from several host plants. Isolate Host Year Mating type Source Sensitivity to Mefenoxam at 100 g/ml (%)a Cp-22 Watermelon 1993 A1 D.J. MitchellSensitive (20) Cp-25 Watermelon 1993 A2 D.J. MitchellSensitive (11) Cp-26 Watermelon 1993 A1 D.J. Mitchelln.t.b Cp-27 Tomato 1995 A1 D.J. MitchellSensitive (6) Cp-29 Watermelon 1997 A2 D.J. MitchellSensitive (0) Cp-30 Bell Pepper 1997 A1 D.J. MitchellSensitive (0) Cp-30a Bell Pepper 1997 A2 D.J. MitchellSensitive (23) a Isolates were characterized as sensitiv e to mefenoxam if colony growth at 5 g ml-1 was less than 40% of the isolateÂ’s growth on the nonamended control media. Intermediate isolates (I) exhibited growth greater than 40% of the nonamended media control at 5 g ml-1, but less than 40% of the nonme nded media control with mefenoxam at 100 g ml1. Isolates that were resi stant exhibited growth greater than 40% of the nonamended media control with mefenoxam at 100 g ml-1. bn.t.= not tested. Isolates that were not tested were a resu lt of isolate loss during storage in the culture collection prior to mefenoxam tests. All five counties included in this study had both mating types present. In Palm Beach County, 65% of isolates were of t ype A1. Approximately 74% of isolates collected since 2001 were of A1 type. In Ma rtin County, 92% of isolates were of A2

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50 type, all isolated within the past three y ears. The sample size, however, was small (N=12). For this study, A2 mating types we re collected only in 2002 while A1 mating types were collected in 2003. Approximately 85% of isolates collected since 2002 have been A1 mating types. In Manatee County, 25% of all isolates were of A1 mating type. Forty-five isolates collected between 2001 and 2003 from diverse growers and geographical locations had almost a 1:1 ratio of isolates that were sensitive and resistant to mefenoxam (Table 4-7). Approximately 58% of sensitive isolates were of A2 mating type. Approximately 92% of resistant isolates collected from vegetable farms were of A1 mating type. Table 4-7. Isolates of Phytophthora capsici classified by mating type and sensitivity to mefenoxam from ten vegetable farms between 2001 and 2003. Number of isolates Sensitive Intermediate Resistant Grower (Year) Field A1 A2 A1 A2 A1 A2 1 (2001) 1 1 2 0 0 3 1 1 0 3 0 0 0 0 2 (2001) 2 0 3 0 0 0 0 3 (2001) 1 4 0 0 0 0 0 4 (2001) 1 1 0 0 0 1 0 5 (2001) 1 1 0 0 0 0 0 6 (2001) 1 1 1 0 0 0 0 7 (2002) 1 0 2 0 0 0 0 8 (2003) 1 0 0 0 0 1 0 9 (2003) 1 0 0 0 0 10 0 1 0 0 0 0 4 0 10 (2002) 2 0 0 0 0 5 1 Total 8 11 0 0 24 2 Mefenoxam Sensitivity and Mating Type among Phytophthora capsici Isolates Recovered from Weeds Isolates of P. capsici were recovered from Carolina wild geranium ( Geranium carolinianum ), American black nightshade ( Solanum americanum ), and common purslane ( Portulaca oleracea ). All but one of the twelve isolates tested were of A1

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51 compatibility type (Table 4-8). The only A2 compatibility type was found on Carolina wild geranium. Table 4-8. Characterization by mating type and in-vitro se nsitivity to mefenoxam of isolates of Phytophthora capsici isolated in 2002 from three weed species in Palm Beach County. Isolate Host Mating type Source Sensitivity to Mefenoxam at 100 g/ml (%)a CWG1-1 Wild Geranium A1 This study Resistant (78) CWG1-2 Wild Geranium A1 This study Resistant (83) CWG1-3 Wild Geranium A1 This study Resistant (85) CWG1-4 Wild Geranium A2 This study Resistant (91) CWG1-5a Wild Geranium A1 This study Resistant (68) CWG1-5b Wild Geranium A1 This study Resistant (86) CoPur3-1 Common purslane A1 This study Resistant (74) CoPur3-2 Common purslane A1 This study Resistant (94) NSh7-1 Nightshade A1 This study Resistant (57) NSh7-2 Nightshade A1 This study Resistant (100) NSh7-3 Nightshade A1 This study Resistant (107) NSh7-4 Nightshade A1 This study Resistant (76) a Isolates were characterized as sensitiv e to mefenoxam if colony growth at 5 g ml-1 was less than 40% of the isolat eÂ’s growth on the nonamended control media. Intermediate isolates (I) exhibited growth greater than 40% of the nonamended media control at 5 g ml-1, but less than 40% of the nonmende d media control with mefenoxam at 100 g ml-1. Isolates that were resistan t exhibited growth greater th an 40% of the nonamended media control with mefenoxam at 100 g ml-1. All isolates of P. capsici from weeds were resistant to mefenoxam. Ten isolates actually grew faster than the control on mefenoxam-amended media at 5ppm and ranged in growth diameter ratio from 101% of the control to 118%. At 100 g/ml, two isolates from nightshade had growths of 100% and 107% compared to the control. Mefenoxam sensitivity during the past twenty years Isolates that date back to the 1980s we re all fully sensitive to mefenoxam; none growing at greater than 23% of the control at 100 g/ml (Table 4-1). Sample size was

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52 small (N=6) in comparison to those from the 1990s and 2000-2003. In the 1990s, isolates that were resistant to mefenoxam did not gr ow at more than 70% of the non-amended control plates when tested at 100 g/ml. Isolates from the years 2000-2003 were either strongly resistant to mefenoxam or strongly sensitive to this fungicide. However all ranges of growth at 100 g/ml were found (Fig. 4-3). Both resistant and sensitive isolates were commonly found among the isolates tested. 0 5 10 15 20 25 0-10>10>20>30>40>50>60>70>80>90>100 Growth on 100 ppm of mefenoxam amended media (% of control)Frequency of isolates 1980s 1990s 2000-2003 Figure 4-3. Frequency of mefenoxam sensitivity in Phytophthora capsici for isolates collected during the 1980s , 1990s, and 2000-2003 based on in vitro studies at 100 g/ml. Discussion Both mating types were present in Pa lm Beach County and all other counties surveyed. Isolates collected from 1982 until 1997 were all sensitive to mefenoxam. In 1997, it was the first or second year this form ulation was used as the replacement for

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53 metalaxyl in Florida. That year, isolate R JM98-794 from Manatee County (west Florida), isolated from tomato, was re sistant to mefenoxam (Table 45). Another isolate, RJM98727, from watermelon, was intermediate in its sensitivity to mefenoxam. Isolates from Dade County, located in sout h Florida, had been previously tested by Ploetz and Haynes (2000) a nd Ploetz et al. (2002) and resistance was found to be common among isolates collected and tested. This study, using 11 isolates from Dade County provided by R.C. Ploetz, reconfirms such findings from isolates that date back to 1999. In Palm Beach County, located in southeast Florida, this study found the first isolate of P. capsici resistant to mefenoxam. This isolat e, Cp38, was originally isolated from tomato in the year 2000. Since compatibility type A1 and A2 are present throughout Florida, sexual recombination may have played a role in fitness levels of th e pathogen and besides weather factors, might help explain the occu rrence of epidemics and losses in vegetable production on a yearly basis. Resistance to metalaxyl and mefenoxam might have promoted a population shift where even intermedia te levels of resistan ce may be effective in allowing epidemics to take place (Parra and Ristaino, 2001). The dependence on these fungicides since the late 1970s and changes in production systems and cultural practices may have also contributed to outbreak s never before seen prior to 1982 (Ploetz et al., 2002). Since m oderate to full resistance to mefenoxam has been found throughout Florida in P. capsici populations, management strategies should take this into account. Although high rainfall ma y be attributed to the major epidemics in Florida (Ploetz et al., 2002) caused by this pa thogen, reduction in ini tial inoculum levels

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54 of P. capsici in grower fields could have reduced su ch outbreaks if cultural practices that reduce inoculum levels were carried out. Continuous use of mefenoxam will select and increase the population numbers of resistant isolates to this fungi cide. In Florida, a populati on shift has occu rred where the proportion of resistant isolates to mefenoxam is increasing similar to what was observed by Ristaino and Parra (2001) in North Carolina. This may be due to continuous use of mefenoxam, which has selected for resistant isolates and created a shift in populations from mostly sensitive to increasing levels of resistant isolates. Some growers use this fungicide on transplants in th e plant houses, under the plastic mulch in the fields, and as a drench and foliar spray (per sonal communication, Ken Shule r). Each application can increase the possibility of select ion for fungicide resistance in P. capsici populations. The equal or faster growth of isolates on mefenoxam-amended media in comparison to the non-amended media is a sign that insensitiv ity to this fungicide has clearly developed. This has been previously observed in stat es such as Michigan and North Carolina (Lamour and Hausbeck, 2000; Parra and Rist aino, 2001). Resistant and non-resistant isolates are likely to survive since many gr owers do not rotate with a non-susceptible host crop. Some field sites tested had been pl anted with cucumbers and tomatoes six and twelve months, respectively, before the peppe r growing season. These three crops are susceptible to P. capsici (personal communication, Ken S huler). The potential for inoculum survival, build-up, and selection is enhanced by continuous cropping with susceptible crops. Since resistance to mefenoxam appears to be quickly building up year after year, soon this fungicide will be ineff ective as a preventive control for P. capsici outbreaks in

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55 Florida. Currently, seed companies are developing new varieties of pepper with resistance to the blight phase of the dis ease and some already ha ve been released. However, the soilborne phase of Phytophthora blight still depends on chemical control (Ristaino, 2003). Since complete resistance to P. capsici is not available in commercial cultivars of pepper, tomato, cucumber, and eggplant, growers will have to adopt other strategies to reduce population leve ls of this pathogen so that initial inoculum is reduced to a minimum from season to season. The survival of isolates in weed sp ecies, poses anothe r problem. Although common purslane had been found to harbor inoculum of P. capsici at low levels in studies conducted by Ploetz et al. (2002) in Dade County, the occurrence of P. capsici in Carolina wild geranium, American black nightshade, and common purslane in Palm Beach County adds to the number of potential alternative hosts for this pathogen. All isolates recovered from weed s were resistant to mefenoxam and all but one isolate were of A1 mating type. Not only do weeds pose a problem as sources of initial inoculum from one growing season to another, but they also harbor genotypes resistant to mefenoxam. Since both compatibility type s are present in both weed and non-weed isolates, sexual recombination may take pl ace and oospores produced. Such spores may persist in soil and render fungicide applications in effective the following season. Although it is very common to find both A1 and A2 compatibility types present in a field, it usually takes a populat ion shift to reach a 1:1 ratio among isolates within a field site (Ploetz and Haynes, 2000; Parra and Ristaino, 2001). Produc tion of oospores is likely when both compatibility types are pr esent and such sexual recombination may influence population structure. Studies by Lamour and Hausbeck (2000) report up to six

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56 mefenoxam sensitivity-compatibility type co mbinations present in oospore progeny and within single fields. Resistance to mefenoxam has increased during the past twenty years in Florida. Both mating types are not only present in Dade County but in other major vegetableproducing counties as well. Since both mating types have been found to be present in a field, this may lead to su rvival of inoculum of P. capsici from season to season in the form of oospores. Therefore, initial inoc ulum may be a result of survival of P. capsici as oospores. Such inoculum not only may be resi stant to mefenoxam but may have acquired other beneficial characteristics as a result of genetic recombination.

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57 CHAPTER 5 SINGLE POPULATION OF Phytophthora capsici IN FLORIDA INFERRED FROM THE MOLECULAR PHYLOGENY OF IS OLATES FROM DIFFERENT CROPS, GEOGRAPHIES, AND YEAR OF ISOLATION Introduction Phytophthora capsici Leonian, an oomycete, is a soilborne pathogen that causes root rot, crown rot, fruit rot and foliar blig ht on several solanaceous and cucurbit hosts. This pathogen causes significant losses in bell pepper, tomato, pumpkin, squash, and watermelon production in the United States a nd throughout the world (Café-Filho et al., 1995; Hwang and Kim, 1995; Rist aino and Johnston, 1999). Weber (1932) first observed P. capsici in Florida, and this pathogen has been prevalent in Florida vegetable production ever since. However, relatively minor damage had been attributed to infection by P. capsici until 1982. That year, this pathogen caused devastating losses to bell pepper ( Capsicum annuum L.), eggplant ( Solanum melongena L.), and squash ( Cucurbita pepo L. var. esculentum Nees) in Palm Beach County (Ploetz and Haynes 2000, McGovern et al., 1998). Ot her major outbreaks also occurred in 1993 and 1998 in pepper, cucurbits and other vegetable crops. The incidence of P. capsici has increased in recent years in bell pepper and squash production areas of Florida (McGovern et al., 1998; Roberts et al., 1999; Ploetz and Haynes, 2000). Although it is assumed that abnormally high rainfall was responsible for the most disease outbreaks caused by this pathogen in Florida, the in cidence of this disease has increased dramatically pointing to additiona l causative factors (Ploetz et al., 2002). One factor that might account for a difference in pathogen behavior dur ing the last twenty

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58 years could be the introduc tion of new populations of th is pathogen that are more aggressive and ecologically better fit. Similar analyses have been applied to epidemics of human diseases. A study of an epidemic of coccidiomycosis, caused by Coccidiodes immitis in California between 1991 and 1994, determin ed that environmental rather than genetic factors were responsible for this disease outbreak (Fishe r et al., 2000). The epidemic was related to the length of drought preceding rainfall and not just rainfall alone. Population genetic analyses ruled out a new pathogenic strain as being responsible for the epidemic in California. Species that have recently immigrated to new regions often lack genetic diversity as a result of the population bottleneck associated with colonization (Nei et al., 1975). If new populations of P. capsici have been introduced in Florida, they might be distinguishable from one another. Cu rrently, phylogenetic and population genetic methods that compare variation in nucleic acid sequences are bei ng used to identify species and populations of fung i; understanding nucleic acid va riation and its analysis by cladistics has greatly changed the way taxonomic inferences have been made (Taylor et al., 1999). One approach to understanding the demogra phic history of a population from genetree data involves examining two different m easures of haplotype variation. According to Avise (2000), haplotype diversity anal yzes information based on the numbers and frequencies of different alle les at a locus, regardless of sequence relationships. Nucleotide diversity is a weighted seque nce divergence between individuals in a population, regardless of different haplotypes.

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59 The use of multilocus sequence typing (MLST), which uses nucleotide sequences of each of several genes to characterize dive rsity, has uncovered new, cryptic species in in fungi such as Candida albicans and Coccidiodes imitis (Taylor and Fisher, 2003). In fungal plant pathogens, one example of a taxa based on phenotype which was proven wrong with this approach is Fusarium oxysporum f. sp. cubense . This plant pathogen is a mix of four clades which independently jumped to banana (OÂ’Donnell et al., 1998). Using both nuclear and mitochondrial sequen ces have proven useful in resolving relationships among and within species. Ba sed on the nuclear phylogenetic analysis of internal transcribed spacer (ITS) sequence data (ITS1, 5.8S rRNA gene, ITS2), Goodwin et al. (2001) concl uded that the genus Mycosphaerella is monophyletic and contains numerous polyphyletic anamorph genera. OÂ’ Donnell et al. (1998) used mitochondrial small subunit ribosomal RNA genes for the Fusarium oxysporum complex and also found that they were monophyletic although many forma speciales were polyphyletic. The objectives of this study were to: 1) conduct molecula r phylogenetic analyses of isolates of P. capsici in Florida obtained from diffe rent hosts, different geographical locations, and of diverse ages; and 2) dete rmine if there is a single population of P. capsici present in Florida. Materials and Methods Sources of Isolates and Culture Methods The ITS region (ITS1, 5.8S rRNA gene, ITS2) was sequenced from 23 isolates of P. capsici (Table 5-1). Isolates we re either received from collaborators or were specifically isolated for this study. Cultures were grown on clarifie d 10% V-8 juice broth for 1 week.

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60 Table 5-1. Summary information for isolates of Phytophthora capsici used for PCR amplification of ITS (I) or mitochondrial (M) sequences. Isolate County, State Host Year Source Brim6Tift (M) Tift, GA Squash 2001 K. Seebold CoPur3-2 (I, M) Palm Beach, FL Common purslane2002 This study Cp-1 (I, M) Brazil Cacao 1986 D.J. Mitchell Cp-2 (I, M) Puerto Rico Black Pepper 1986 D.J. Mitchell Cp-3 (I, M) Brazil Black Pepper 1986 D.J. Mitchell Cp-9 (M) New Jersey Squash 1978 D.J. Mitchell Cp-10 (I, M) California Bell Pepper 1985 D.J. Mitchell Cp-11 (I, M) Palm Beach, FL Bell pepper 1984 D.J. Mitchell Cp-17 (M) Palm Beach, FL Bell Pepper 1982 D.J. Mitchell Cp-18 (I, M) New Jersey Eggplant 1986 D.J. Mitchell Cp-19 (I, M) New Jersey Summer Squash 1987 D.J. Mitchell Cp-21 (I, M) Brazil Rubber 1989 D.J. Mitchell Cp-25 (M) Florida Watermelon 1994 D.J. Mitchell Cp-27 (M) Florida Tomato 1995 D.J. Mitchell Cp-38 (I, M) Palm Beach, FL Bell Pepper 2000 D.J. Mitchell CWG1-2 (I) Palm Beach, FL Wild Geranium 2002 This study CWG1-3 (I, M) Palm Beach, FL Wild Geranium 2002 This study CWG1-4 (I, M) Palm Beach, FL Wild Geranium 2002 This study CWG1-5a (M) Palm Beach, FL Wild Geranium 2002 This study IoriSQ1A (I, M) Dade, FL Squash 1999 R.C. Ploetz IoriSQ2D-3B (I, M) Dade, FL Squash 1999 R.C. Ploetz Imm0424-4 (M) Collier, FL Bell Pepper 2003 This study Imm0424-6 (M) Collier, FL Bell Pepper 2003 This study LedfordSQ3Br (I, M) Dade, FL Squash 1999 R.C. Ploetz LedfordSQ3F (I, M) Dade, FL Squash 1999 R.C. Ploetz MC1A-1 (M) Martin, FL Bell Pepper 2001 This study MS11 (M) Massachusetts Unknown R. L. Wick NSh7-1 (I, M) Palm Beach, FL American Black Nightshade 2002 This study NSh7-2 (I, M) Palm Beach, FL American Black Nightshade 2002 This study NSh7-3 (I, M) Palm Beach, FL American Black Nightshade 2002 This study NSh7-4 (I) Palm Beach, FL American Black Nightshade 2002 This study Ptrop2 a (M) Orange, FL Pothos 2001 R. Leahy PtropAY207010b (I) GenBankAY207010 StranoSQ1A-2 (I, M) Dade, FL Squash 1999 R.C. Ploetz aThis isolate of the closely related P. tropicalis was sequenced and served as an outgroup for mtDNA phylogenetic analyses. bThe ITS sequence of the closely related P. tropicalis was obtained from GenBank and served as an outgroup for ITS phylogenetic analysis.

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61 Cultures were grown at 24 C on a shaken platform at 120 rpm, harvested by vacuum filtration, lyophilized overni ght, and stored at -80 C (Goodwin et al., 2001). The mitochondrial DNA (mtDNA) intergenic regi on was sequenced from 30 isolates of P. capsici , twenty-one of which were also used for ITS sequencing (Table 5-1). DNA Extraction, PCR amplific ation, and Sequencing DNA was extracted from lyophilized myce lium by a modified phenol: chloroform method (Goodwin et al., 1992). The comple te ITS region for each isolate tested was amplified with primers ITS 4 (5Â’-TCCTCC GCTTATTGATATGC) paired with primer ITS 5 (5Â’-GGAAGTAAAAGTCGTAACAAGG). For mtDNA amplification, an intergenic region was chosen ba sed on the mtDNA sequence of P. infestans (Paquin et al., 1997). Primers were designed to amplify this intergenic region located between the trnY gene and the small subunit ribosomal RNA gene. The size of this region was 786-bp in length. Primer MT4 (5Â’-CCAAGGTTA ATGTTGAGGGTTCG) was paired with MT5 (5Â’-CGATAGCATTTATTCTGAGCCAGG). The amplification of the ITS region was conducted in a thermocycler (PTC-100; MJ Research Inc., Waltham, MA) as descri bed by Nakasone (1996) with the following cycle parameters: 94 C for 2 min, 30 cycles of 93 C for 30 s, 53 C for 2 min, 72 C for 2 min, and a final extension of 10 min at 72 C. For mitochondrial am plification, the cycle parameters were: 95 C for 5 min, 30 cycles of 95 C for 30 s, 54 C for 30 s, 72 C for 1 min 30 s, and a final extension of 5 min at 72 C. Amplification of products of the correct size was verified on 1% agarose gels. The remaining product was purified with a polymerase chain reaction (PCR) prep kit (QI AGEN Inc., Valencia, CA). Purified PCR

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62 products were submitted for sequencing to the Interdisciplinary Center for Biotechnology Research DNA Sequencing Laboratory, Univers ity of Florida, Gainesville, Florida. DNA Sequence Alignment and Analyses All ITS sequences were edited to include th e complete ITS1, 5.8S ribosomal RNA gene, and ITS2 sequences. The mtDNA sequences were edited to include the complete amplified sequences minus primers. The DNA sequences were aligned by a three-step process with the profil e mode of ClustalX (Thompson et al ., 1997). Each profile was also checked and edited manually. Phylogenetic analyses were done with PAUP Portable Version 4.0.0d55 for Unix, which was part of GCG ver. 10.0. Phylogenetic relationships based on DNA sequencing data was inferred by maximum parsimony analyses with a heuristic tree search using KimuraÂ’s two-parameter method (Kimura, 1980) for estimating evolutionary distances and impl ements the neighbor joining algorithm of Saitou and Nei (1987). To determine support for the various clades of the trees, the analysis was bootstrapped with 1000 replications (Martin, 2000). Results ITS and Mitochondrial Sequencing and Alignment The ITS sequences from P. capsici were of the expected 752-bp length, although two isolates, CoPur3-2 and Cp3, were 751bp in length. The mtDNA sequences amplified from P. capsici were smaller than the exp ected 786-bp predicted from P. infestans : approximately 500-bp, as visualized in a 1% agarose gel. Once primer sequences were subtracted, amplification products were between 451 and 461-bp, depending on the isolate. This length included parts of the flanking sequences of the regions used for primer design. Although 21 isolates had mtDN A intergenic sequences of 460-bp, isolates

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63 Cp9, Cp10, Cp17, Cp18, Nsh7-1, CWG1-4, and CWG1-5a had sequences of 459-bp, Cp1 was 451-bp and Cp2 was 461-bp. The sequence for P. tropicalis isolate PtropAY207010 used as an outgroup for ITS analysis was 752-bp while the sequence fo r the outgroup isolat e for mitochondrial analysis, P. tropicalis isolate Ptrop2, was 460-bp. Phylogenetic Analyses with ITS Sequences All isolates of P. capsici from Florida and the United States tested formed a major group with a very high bootstrap support of 100% (Fig. 5-1). Excluded, although not grouped together, were P. capsici isolates Cp1, Cp2, and Cp3 that were isolated from cacao or black pepper. Within the major group, all isolates were strongly supported (95%) and only one was excluded: P. capsici isolate Cp19, which was originally isolated in New Jersey from summer squash in 1987. All but one Florida isolate clustered together with a bootstrap support of 64%. Excl uded were isolates Cp10 (California) and Cp18 (New Jersey), a nd CWG1-5a (Florida). The closely related P. tropicalis isolate PtropAY207010 fell outside the Florida cl ade. Another tropical isolate of P. capsici which was isolated from rubber in Brazil in 1989 was the exception among the foreign tropical isolates and clustered wi th the clade containing all but one of the Florida isolates. All sixteen isolates clustered together ha d the same sequence except for isolate NSh7-1. The ITS sequence differed by only one base. The strict consensus of th is tree is not well resolved, with few internal branches and most isolates falling into a polycotomy of 16 isolates.

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64 Figure 5-1. Rooted parsimony tree based on the internal transc ribed spacer sequences of 23 isolates of Phytophthora capsici . All bootstrap values above 50 (percentage of 1,000 replic ations) are indicated and rounded to the nearest integer. P. tropicalis isolate PtropAY207010 was used as an outgroup. Phylogenetic Analyses with mtDNA Sequences A single group with a very high bootstrap support value of 93% was validated for all isolates. Excluded from th is group were three isolat es: the tropical, non-Florida isolates Cp1, Cp2, and Cp3 (Fig.5-2). The other tropical isolate from rubber, Cp21, was positioned within the major group of isolates of P. capsici . Within this highly supported (93%), all isolates could not be resolved with the exception of five isolates, which formed two se parate clusters. In one of these clusters, Cp9 (New Jersey, 1978) and Cp10 (California, 1985) clustered with a bootstrap support

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65 of 63%. In the other cluster, weed isol ates NSh7-1, CWG1-4, and CWG1-5a clustered with a bootstrap support of 64% Figure 5-2. Rooted parsimony tree based on interg enic mitochondrial sequences of 30 isolates of Phytophthora capsici . All bootstrap values above 50 (percentage of 1,000 replications) are indicated a nd rounded to the nearest integer. Phytophthora tropicalis isolate Ptrop2 was used as an outgroup. The remaining 22 isolates with identical or almost identical sequences for all 22 isolates, was comprised of isolates from American black nightshade, bell pepper, Carolina wild geranium, common purslane , squash, tomato, and watermelon. Out-ofstate isolates from New Jersey, Georgia, and Massachusetts grouped together with Floridian isolates from Dade, Martin, Collie r, and Palm Beach counties. The strict

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66 consensus of this tree is not well resolved, w ith few internal branches and most isolates falling into a polycotomy of 22 isolates. Discussion All ITS sequences in this study were 752-bp in length ex cept for two that were 751bp in length. Sequences were highl y conserved and only a single group of P. capsici could be elucidated based on phylogenetic an alysis and a solid bootstrap support of 100% (Fig. 5-1). Excluded were isolates Cp1, C p2, and Cp3 that were isolated from cacao, black pepper, and black pepper, respec tively. Based on morphological studies by Aragaki and Uchida (2001), isolates of P. capsici from tropical crops have been assigned to a new species, P. tropicalis . Molecular phylogenetic st udies using both nuclear and mtDNA have confirmed this distinct groupi ng (Levesque et al., 1999; Abad and Abad, 2003; Bowers et al., 2003). Although tropical isolate Cp21 from rubber did cluster with P. capsici isolates from Florida, mo rphological studies would have to be utilized to assist in classifying this isolate and conf irm whether this isolate is indeed P. capsici . Although isolates Cp10, Cp18, and Cp19 were excluded from the rest of the isolates and separated on their own in the main clade, they repres ent an isolate from Ca lifornia and two from New Jersey. These three isolates from outsi de of Florida might be somewhat different due to their geographic location or time of isolation. Excluded, as well, was isolate CWG1-5a, a Florida isolate from Carolina wild geranium. This isolate could well represent a different sub-population of P. capsici present in low fre quency in Florida or the same population adapting to this weed. The internal transcribed spacer (ITS) re gions of rDNA have been useful in taxonomic differentiation between ge nera and species of fungi. Th is is due to the rate of accumulation of mutations in these regions th at often mirrors the rate of speciation

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67 (Bruns et al., 1991; Lee and Taylor, 1992). However, thes e sequences may not always distinguish between species and therefore their use in intraspecies differentiation is even less likely. According to Cooke et al. (2000), the problem with ITS data is that it is unclear how many differences th ere are between closely rela ted species compared with variation within species. Based on this study, ITS sequences faile d to show genetic variability among isolates of P. capsici in Florida although host, geographical location, and year of isolation of isolates were taken into account to incr ease the chances for greater differences in sequences in the isolates tested. New popul ations were not revealed by ITS phylogeny. This is to be expected since most ITS and mtDNA phylogenetic work with the genus Phytophthora is geared towards interspecies diffe rentiation rather than intraspecies differentiation (Cooke and Duncan, 1997; Cooke et al., 2000; Hudspeth et al., 2000; Abad and Abad, 2003; Bowers et al., 2003). Using mtDNA, most isolates had mitochond rial sequences that were mostly 460-bp in length. As with ITS sequenc ing, the tropical isolates of P. capsici were excluded from the rest of the isolates, except for isolate Cp21 from rubber. The separate clustering of isolates Cp9 and Cp10 represented isolates geographically remove d from Florida and may account for the observed variation. A sec ond cluster was comprised of one isolate of American black nightshade, NSh7-1, and two is olates of Carolina wild geranium, CWG14 and CWG 1-5a. Recently, both weeds found to harbor P. capsici (French-Monar, 2003). Unresolved, but grouped together wi th a 93% bootstrap support value, were isolates from weeds, several crops and countie s, as well as non-Flor ida isolates from New Jersey, Georgia, and Massachusetts.

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68 Mitochondrial DNA (mtDNA) is the most used genetic marker in population studies, but the reduction in diversity asso ciated with colonization bottlenecks is worsened by mitochondrial genes (Davies et al., 1999). According to Nei (1996), since the rate of sequence evolution varies exte nsively with gene or DNA segment, one can study evolutionary relationships at all levels of classifica tion, even at the species or intraspecies level with mitochondrial se quences. In this study, by focusing on an intergenic region of the mtDNA, greater variability in sequen ce data was expected. The three isolates of P. capsici recovered from weeds which clustered separately from the rest of isolates, might represent a haplotype that may be new in origin, only present in small frequencies, or prevalent where susceptible weed populations are present in vegetable farms. Although both ITS and mtDNA phylogenetic anal yses were not able to validate the existence of more than one population of P. capsici in Florida, the mtDNA data did find some minor sequence differences in a few isolates from Florida that resulted in separate clustering, especially in the mtDNA tree. In populations that are recent, as may be the case of P. capsici in Florida, populations may have a single high frequency and a single low frequency genotype or haplotype. This was the case with medfly ( Ceratitis capitata ) populations in the United States (Gasparich et al., 1997). P hylogenetic analyses of more isolates of P. capsici could help in determining if within the same population, new geneotypes or haplotypes might be occurring. The occurrence of major outbreaks in Fl orida might not be explained by the introduction of new pathogen populations in Flor ida. It could be linked to other factors that might have been acquired or selected fo r in this potentially uniform population. Such

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69 factors may include: prolonged survival of inoc ulum in soil, climat e changes, fungicide resistance, lack of crop rotations, host range expansion to weeds, and possible ecological adaptations from the pathogen (Bowers et al., 1990; Parra and Ristaino, 1998; Ristaino and Johnston, 1999; Ploetz et al., 2002). The ability to colonize weed species may allow for P. capsici to survive in nature in the absence of a susceptible hos t crop, and possibly evolve into new genot ypes or haplotypes.

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70 LIST OF REFERENCES Abad, Z. G., and Abad, J. A. 2003. Advances in the integration of morphological and molecular characterization in the genus Phytophthora : The case of P. niederhauseria sp. nov. Phytopathology 93:S1. Adkins, S., and Rosskopf, E.N. 2002. Key West nightshade, a new experimental host for plant viruses. Plant Dis. 86:1310-1314. Ansani, C.V., and Matsuoka, K. 1983. Sobrevivencia de Phytophthora capsici Leonian no solo (Survival of Phytophthora capsici Leonian in soil). Fitopatol. Bras. 8:269276. Aragaki, M., and Uchida, J. Y. 2001. Morphological dis tinctions between Phytophthora capsici and P. tropicalis sp. nov. Mycologia 93:137-145. Avise, J.C. 2000. Phylogeography: The hist ory and formation of species. Harvard University Press. Cambridge, Massachusetts, U.S.A. 447 pp. Barak, J.D., Koike, S. T., and Gilbertson, R. L. 2001. The role of crop debris and weeds in the epidemiology of bacterial leaf spot of lettuce in California. Plant Dis. 85:169-178. Barbercheck, M. E., and Von Broembsen, S. L. 1986. Effects of Soil Solarization on Plant-Parasitic Nematodes and Phytophthora cinnamomi in South Africa. Plant Dis. 70:945-950. Black, B. D., Padgett, G. B., Russin, J. S., Gri ffin, J. L., Snow, J. P., and Berggren, G. T., Jr. 1996. Potential weed hosts for Diaporthe phaseolorum var . caulivora , causal agent for soybean stem ca nker. Plant Dis. 80:763-765. Bowers, J.H., Papavizas, G.C., and Johnston, S.A. 1990. Effect of soil temperature and soil-water matric potential on the survival of Phytophthora capsici in natural soil. Plant Dis. 74:771-777. Bowers, J. H., Bailey, B. A., and Martin, F. N. 2003. Genetic diversity of temperate and tropical isolates of Phytophthora capsici . Phytopathology 93:S10. Bruns, T. D., White, T.J., and Taylor, J.W. 1991. Fungal molecular systematics. Annu. Rev. Ecol. Syst. 22:525-564.

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73 Hudspeth, D. S. S., Nadler, S. A., and Hudspeth, M. E. S. 2000. A COX2 molecular phylogeny of the Peronosporomycetes. Mycologia 92:674-684. Hwang, B. K. and Kim, C. H. 1995. Phytopht hora blight of peppe r and its control in Korea. Plant Dis. 79:221-227. Katan, J. 1981. Solar heating (solarization) of soil for control of soilborne pests. Ann. Rev. Phytopath. 19:211-236. Katan, J., and De Vay, J. E. (Eds.). 1991. Soil solarization. CRC Press, Boca Raton, Fl. 256 pp. Kimura, M. 1980. Sample method for estimating evolutionary rates of base substitutions through comparative studies of nucleotid e sequences. J. Mol. Evol. 16:111-120. Krober, H. 1980. Uberdauerung einiger Phytophthora -Arten im Boden (S urvival of some Phytophthora species in soil). Z. Pflan zenkr. Pflanzenschutz 87:227-235. Lamour, K. H., and Hausbeck, M. K. 2000. Mefenoxam insensitivity and the sexual stage of Phytophthora capsici in Michigan cucurbit fi elds. Phytopathology 90:396-400. Larkin, R. P., Ristaino, J. B., and Campbell, C. L. 1995. Detection and Quantification of Phytophthora capsici in Soil. Phytopathology 85:1057-1063. Lee, S. B., and Taylor, J. W. 1992. P hylogeny of five fungus-like protoctistan Phytophthora species, inferred from the internal transcribed spacers or ribosomal DNA. J. mol. Evol. 9:636-653. Lee, S.B., White, T.J., and Taylor. 1993. Detection of Phytophthora species by oligonucleotide hybridization to am plified ribosomal DNA spacers. Phytopathology 83:177-181. Levesque, C.A., Cock, A.W.A.M., Quail, A., and OÂ’Gorman, D. 1999. Molecular phylogeny, evolution and groupings of Pythium and Phytophthora species based on nuclear ribosomal DNA. Phytopathology 89:S45. Martin, F. N. 2000. Phylogene tic relationships among some Pythium species inferred from sequence analysis of the mitochondrially encoded cytochrome oxidase II gene. Mycologia, 92:711-727. Maynard, D. N., and Hochmuth, G. J. (Eds .). 1999. Vegetable Production Guide for Florida SP170. University of Florida. 247 pp. McGovern, R. J., Roberts, P. D., Kuchar ek, T. A., and Gilreath, P. R. 1998. Phytopthora capsici : New problems from an old enemy. Pages 9-16 in: 1998 Florida Tomato Institute Proceedings. Univ. Florida, Gainesville.

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75 Ristaino, J. B., Hord, M. J ., and Gumpertz, M. L. 1992. Population densities of Phytophthora capsici in field soils in relation to drip irrigation, rainfall, and disease incidence. Plant Dis. 76:1017-1024. Ristaino, J. B., Madritch, M., Trout, C. L., and Parra, G. 1998. PCR amplification of ribosomal DNA for species identifica tion in the plant pathogen genus Phytophthora . Appl. Environ. Microbiol. 64:948-954. Ristaino, J. B., and Johnston, S. A. 1999. Ecologically Based Approaches to Management of Phytophthora Blight on Bell Pepper. Plant Dis. 83: 1080-1088. Roberts, P.D., McGovern, R.J., Hert, A., Vavrina, C.S., and Urs, R.R. 1999. Phytophthora capsici on tomato: Survival, severity, age, variety, and insensitivity to mefenoxam. Pages 41-43 in: C.S. Vavrina (Ed.). Florida Tomato Institute Proceedings. University of Florida a nd Citrus & Vegetable Magazine PRO 516. Saitou, N., and Nei, M. 1987. The ne ighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Boil. Evol. 4:406-425. Stapleton, J. J. 1982. Effect of soil solari zation on populations of selected soilborne microorganisms and growth of deciduous fruit tree seedlings. Phytopathology 72:323-326. Taylor, J. W., Jacobson, D. J., and Fisher, M. C. 1999. The evolution of asexual fungi: reproduction, speciation and classifica tion. Annu. Rev. Phytopathol. 37:197-246. Thompson, J. D., Gibson, T. J., Plewniak, F. Jeanmougin, F., and Higgins, D. G. 1997. The CLUSTAL-X windows interface: flexib le strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25:4876-4882. Tsao, P.H. 1991. The identities, nomenclature and taxonomy of Phytophthora isolates from black pepper. Pages 185-211 in: Sarma, Y.R., and Premkumar, T.(eds). Diseases of black pepper. Proc. Int. Pepper Comm. Workshop on Black Pepper Diseases. Goa, India. Von Broembsen, S. L. 1998. Capturing and re cycling irrigation water to protect water supplies. Pages 27-29 in: Water Quality Handbook for Nurseries, Publication E951, Oklahoma Cooperative Extension Serv ice, Oklahoma State University. Weber, G.F. 1932. Blight of peppers in Florida caused by Phytophthora capsici . Phytopathology 22: 775-780.

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76 BIOGRAPHICAL SKETCH Ronald David French was born on July 10, 1970, to Edward Ronald French and Delia Monar de French, in San Isidro, Lima, Peru. He earned his Bachelor of Science in plant science from Cornell University, It haca, New York, in 1992. Upon completion of his undergraduate studies, Ronald worked as a research assistant in the James W. Moyer plant virology laboratory at North Carolina State University. In 1993, he began his Master of Agriculture studies, with an empha sis in soils and soilborne plant pathology. Upon graduation in 1995, he returned to Peru and was offered the opportunity to work for the International Potato Center and set up the first Latin American agricultural display at Epcot, Walt Disney World, Florida during the Spring 1996 Interna tional Flower and Garden Festival. Upon completion, he was o ffered an opportunity to stay at Epcot and work in a compost project that tested co mpost for disease suppression of soilborne pathogens at Epcot. In Fall 1998, he was offered a research assistantship at the University of Florida to pursue his studies toward a Doctor of Philosophy degree in plant pathology.