In Press at Mycologia, published on February 1, 2010 as doi:10.3852/09-018

Short title: Pythium aphanidermatum Genetic structure and distribution of Pythium aphanidermatum populations in Pennsylvania greenhouses based on analysis of AFLP and SSR markers Seonghee Lee Department of Plant Pathology, Pennsylvania State University, University Park, Pennsylvaia 16802 Carla D. Garzón Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, Oklahoma 74078 Gary W. Moorman Department of Plant Pathology, Pennsylvania State University, University Park, Pennsylvania 16802 Abstract: Pythium aphanidermatum is one of the most aggressive species in the genus and has a wide host range, but little is known about its population genetic structure. We tested 123 P. aphanidermatum isolates with six AFLP primer combinations and four SSR markers. The genetic diversity of P. aphanidermatum was 0.34 with AFLP and 0.55 with SSR markers. SSR genotypes totaled 3–8 for each locus, and a total of 14 SSR genotypes were found among all isolates. Three major genetic groups were identified with the combination of AFLP and SSR marker data. The genetic structure observed among P. aphanidermatum isolates was related to location and mefenoxam fungicide resistance instead of host. Four genotypes (PA1, PA2, PA5 and PA7) were found in the population from a commercial greenhouse, and the genetic diversity of a greenhouse population was similar to that found in the whole sample. The molecular tools for P. aphanidermatum

Copyright 2010 by The Mycological Society of America.

isolates identified the possible gene flow within and among populations in Pennsylvania greenhouses. Key words: fungicide resistance, Pythium aphanidermatum INTRODUCTION Pythium aphanidermatum (Edson) Fitzp. causes many economically important diseases and is one of the most pathogenic species in the genus. This pathogen reduces vigor, quality and yield of crops, often killing a large percentage of plants affected. P. aphanidermatum is particularly devastating for greenhouse crops grown at warm temperatures and is the species most frequently associated with poinsettia (Euphorbia pulcherrima Willd.ex Klotzsch) root rot in Pennsylvania (Moorman et al. 2002). Several studies have been conducted on the management of P. aphanidermatum (Amer and Utkhede 2000, Ben-Yephet and Nelson 1999, Boehm and Hoitink 1992, Chen et al. 1998, Goldberg et al. 1992, Heungens and Parke 2000, Mansoori and Jaliani 1996), but little is known about its spatial distribution or movement in production systems (Roberts et al. 2005). Most of the genetic studies that included P. aphanidermatum isolates have concentrated on diagnostic issues (Herrero and Klemsdal 1998, Lévesque et al. 1998, Moorman et al. 2002). The three studies that examined the genetic diversity of P. aphanidermatum (Chen et al. 1991, Garzón et al. 2005, Herrero and Klemsdal 1998) had a relatively small sample size, and the limited use of molecular marker techniques hampered characterization of the genetic structure of the population. A study of soluble proteins and isozymes (Chen et al. 1991) found high levels of variation among isolates of P. aphanidermatum. However Herrero and Klemsdal (1998) found little genetic diversity in a sample of 20 isolates from nine countries with three random amplified polymorphic DNA

(RAPD) primers. Also Garzón et al. (2005) found little genetic variation in a sample of 23 isolates from eight countries on four continents with a single amplified fragment length polymorphism (AFLP) primer combination. AFLP (Vos et al. 1995) and simple sequence repeats (SSR), also called microsatellites (Queller et al. 1993), are highly reproducible molecular markers that have been widely applied to population genetic studies in many organisms. The major disadvantage of AFLP is that these dominant markers are less informative than codominant markers such as SSR. This limitation may be overcome by increasing the number of AFLP bands or increasing the sample size (Jorde et al. 1999). We developed SSR markers for P. aphanidermatum, P. cryptoirregulare and P. irregulare (Lee and Moorman 2008), and in an analysis of P. aphanidermatum isolates (Lee and Moorman 2008) we identified highly reproducible and polymorphic SSR loci with potential use in genetic studies. Geographic barriers, hosts and fungicide resistance can directly affect genetic diversity in species. Although it generally is recognized that species of Pythium are not host specific (Sutton et al. 2006), population genetic studies of P. aphanidermatum from different hosts and locations might provide insights on genetic variability and the movement of genes within and among populations. The greenhouse industry is highly interconnected, and plants grown in Pennsylvania in fact might have been of local origin, grown in another state or grown at an off-shore production facility. Knowledge of the exact genetic identity of species isolated from infected plants and characterization of populations within species are required to assess the significance of finding particular isolates in a crop production system and for determining

their origins, thus preventing their introduction into and facilitating their elimination from crop production areas. The objectives of the present study were to study the population genetic structure of P. aphanidermatum isolates from Pennsylvania greenhouses to determine the genetic variation and movement of those isolates and the genetic distribution within and among populations. MATERIALS AND METHODS Fungal isolates and DNA extractions.—Isolates (n = 123) identified by morphology and ITS sequence analysis as P. aphanidermatum were included in this study. The majority of isolates (n = 87) were collected from greenhouse crops in Pennsylvania to determine the genetic distribution of P. aphanidermatum isolates within and among greenhouses. Isolates from seven other states in USA and seven other countries were included to compare genetic diversity (TABLE I). All isolates were stored at room temperature in pieces of water agar in vials containing sterile tap water. For DNA extraction isolates were grown on potato dextrose agar 1 d, transferred to 10 cm diam Petri dishes with 15 mL 20% clarified V8 juice broth (Campbell, New Jersey) and grown 3–7 d at 23 C in the dark. Mycelial masses were harvested with a Buchner funnel and rinsed with tap water. Each mycelial mass was transferred to a 2 mL centrifuge tube, frozen, freeze-dried and stored at −20 C until used. DNA was extracted from approximately 100 µg dehydrated mycelium with QIAGEN DNeasy Plant Mini Kits (QIAGEN, Valencia, California) according to manufacturer instructions. Freeze-dried mycelium in 1.5 mL microcentrifuge tubes was pulverized at room temperature with micro pestles and sterile quartz sand. Purified DNA was quantified with a spectrophotometer at OD 260 nm/280 nm (Beckman Coulter, California). All DNA samples were diluted to 25 ng per µL and stored −20 C until used. Genetic analysis with AFLP markers.—A total 500 ng DNA was digested 3 h at 37 C with 5 U MseI (New England Biolabs Inc., Ipswich, Massachusetts) and 5 U EcoRI (Promega Corp., Madison, Wisconsin), followed by inactivation at 70 C for 15 min. Ligation mixture (5 µL) containing 50 pmol MseI adapters, 5 pmol EcoRI adapters and 1 U T4 DNA ligase (Promega, Madison, Wisconsin) was added to 25 µL digested DNA, and the reaction was incubated overnight at room temperature. Pre-amplification was performed with

EcoRI-A/MseI-C primer pairs (Integrated DNA Technology, Iowa): 30 cycles at 94 C for 30 s, 56 C for 1 min and 72 C for 1 min, followed by 10 min at 72 C. Each 25 µL of the reaction contained 5 µL restriction/ligation DNA, 1× PCR buffer (New England Biolabs, Ipswich, Massachusetts), 2.5 mM dNTP (Promega Corp., Madison, Wisconsin), 1 U Taq DNA polymerase and 25 mM each primer. PCR product was diluted (1:10) and 2 µL dilution was used for the selective amplification with six primer combinations (EcoRI-AG/MseI-AC, EcoRI-AT/MseI-CC, EcoRI-AA/MseI-GT, EcoRI-AC/MseI-GT, EcoRI-AC/MseI-TC and EcoRI-AC/MseI-TT). PCR reaction was one cycle of 2 min at 94 C, 12 cycles of 30 s at 94 C, 30 s at 65 C (decrease annealing temperature by 0.7 each cycle) and 1 min at 72 C. The remaining 25 cycles were carried out 30 s at 94 C, 30 s at 56 C, 1 min at 72 C and the final extension step was 72 C for 2 min. The PCR products (6 µL) were separated on a 5% polyacrylamide denaturing sequencing gel in 1× TBE buffer for 3 h at 75 v. The gel was stained with the Silver Sequence DNA Sequence System (Promega Corp., Madison, Wisconsin), dried at room temperature overnight, and an image was produced with a flatbed scanner. Only amplified bands 200–800 bp were scored for AFLP analysis. AFLP fragments were coded as binary strings, presence (1) and absence (0) of a band. Unclear bands due to weak PCR amplification were scored as missing data. Genetic analysis with SSR markers.—Four polymorphic SSR markers (P18GAA1-71, P18GAA1-72, P18CAT1-74 and P18TCC3-25) developed for P. aphanidermatum were used in this study (Lee and Moorman 2008). PCR was conducted with a 20 µL reaction containing 1× PCR buffer (New England Biolabs, Ipswich, Massachusetts), 2.5 mM dNTP (Promega, Madison, Wisconsin), 1 U Taq DNA polymerase (New England Biolabs, Ipswich, Massachusetts), 3 µM each primer with this PCR program: 5 min initial denaturation at 94 C followed by 30–35 cycles of 94 C for 30 s, annealing temperatures for 30 s (TABLE III), 72 C for 30 s, and a final extension 10 min at 72 C. PCR products were separated on a 6% non-denaturing polyacrylamide gel and viewed with ethidium bromide staining. The isolates showing different genotypes for each SSR locus were chosen to determine allele sizes, and PCR was performed with fluorescent dye (5′6FAM®; Integrated DNA Technologies, Coralville, Iowa) labeled primer for further detection on an automatic ABI Prism 3100 capillary sequencing system (Pennsylvania State University, Nucleic Acid Facility). In addition the DNA of different alleles for each SSR locus were purified from the gel with a GenElute PCR Clean-Up Kit (Sigma-Aldrich, St Louis, Missouri) and sequenced with an ABI Hitachi 3730XL DNA

analyzer (Pennsylvania State University, Nucleic Acid Facility) to determine whether the allele variations are from the differences of simple sequence repeats. Data analysis.—In AFLP data analysis only the markers in 200–800 bp that could be scored unambiguously and showed clear presence or absence patterns in all P. aphanidermatum isolates were included. The isolates with the same AFLP fingerprint (genotype) were considered clones. To prevent a statistical bias the clonecorrected dataset was used for all population genetic analyses. Nei's genetic diversity (H) (Nei 1973) was calculated for analyzing the genetic structure within and among geographical locations and hosts. Allele frequencies and heterozygosity of each SSR locus were calculated with PopGene and PowerMarker 3.25 (Liu and Muse 2005). The genetic relationship among isolates was investigated by the unweighted pair-group method with arithmetic average (UPGMA) cluster analysis with the bootstrap 1000 replications with PAUP 4.0. Reaction to mefenoxam.—The sensitivity of 71 isolates to the fungicide mefenoxam, an isomer of metalaxyl, was tested in vitro and scored the resistance to mefenoxam as described by Moorman et al. (2002). Cornmeal agar (BBL cornmeal agar, Becton, Dickinson & Co., Sparks, Maryland) was amended with 100 μg mefenoxam/mL by suspending Subdue MAXX (21.3% mefenoxam; Syngenta) in molten agar before pouring it into Petri plates. Plates were inoculated by inverting a 5 mm diam block of colonized agar onto the center of each plate. Each isolate was plated on two fungicide-free plates and two plates containing fungicide, then incubated in the dark at 25 C approximately 40 h. Growth was measured from the edge of the inoculum block to the edge of the colony along two random radii per plate. Each isolate was tested at least twice. If 100 μg/mL mefenoxam did not slow isolate growth by 50% as compared to growth in the absence of fungicide, it was considered mefenoxam resistant (Sanders 1984).

RESULTS Genetic variation of AFLP.—A total of 232 unambiguously amplified bands were produced by the combination of six AFLP primers in 123 P. aphanidermatum isolates. AFLP markers produced an average of 39 reproducible bands 200–800 bp. Among all AFLP fragments 103 AFLP loci were reproducible and polymorphic. The average percentage of polymorphisms among six AFLP primer combinations was 44%, and two AFLP

combinations, MseI-CC / EcoRI-AT (64%) and MseI-GT/EcoRI-AC (62%), were highly polymorphic. The average genetic diversity (H) of six AFLP markers was 0.344 (TABLE II). AFLP genotypes were associated with sampling locations instead of with host or to fungicide resistance, and only unique genotypes were included in genetic analyses. Three genetic groups (A, B and C) were observed in the dendrogram derived from multiple AFLP loci (FIG. 1 and SUPPLEMENTAL FIG. 1). The major genetic group A contained 87 isolates sampled from the different locations and hosts. Fourteen of the 21 isolates included in group B were collected from Greenhouse X, a commercial facility in Pennsylvania. Most isolates from that facility were in genetic groups A and B. Genetic group C, which contained 15 P. aphanidermatum isolates and outgroup P. deliense isolate 42142-99, was distinct from the other two genetic groups. Genetic variations of SSR.—Four SSR markers (P18GAA1-71, P18GAA1-72, P18CAT174 and P18TCC3-25; Lee and Moorman 2008) were examined in 123 P. aphanidermatum isolates and a total of 12 alleles were recorded (TABLE III). The observed and expected heterozygosities were 0.925 and 0.498 to 0.966 and 0.597. Polymorphic information contents (PIC) among 123 isolates were 0.374–0.514. Genotypes totaled 3–8 for each SSR locus. In all SSR loci heterozygosity was high (TABLE III), and the major genotype was heterozygous containing the most frequent alleles (TABLE IV). Sequence analysis of SSR regions showed that the allele variations in SSR loci were due mostly to differing simple sequence repeat numbers. In SSR locus P18CAT1-74 the sequence alignment of alleles 147 and 150 revealed that the insertion or deletion of the trinucleotide repeat, CAT, modified the allele size (data not shown). Similar to AFLP three genetic groups (A, B and C) were observed in the dendrogram derived from four SSR loci (SUPPLEMENTAL FIG. 1). The

genetic group A (n = 79) and B (n = 31) contained most of the commercial greenhouse samples and were from diverse hosts. Isolates in C, including outgroup P. deliense isolate 42142-99, was distant from the other two groups. The same SSR genotypes were observed frequently in the same sampling location as found in AFLP. Phylogenetic relationships among isolates with AFLP and SSR genotypes.—The phylogenetic results obtained with AFLP and SSR were highly correlated, but not all the same isolates were in each group for both analysis. The difference between AFLP and SSR genotypes among isolates probably resulted from the limited number of SSR markers. Three genetic groups, namely A, B and C, were observed in the phylogenetic analyses (UPGMA) performed with both genetic marker systems (FIG. 1). A is the major group and contained most of isolates sampled from Pennsylvania, including those from Greenhouse X. While AFLP analysis divided A and B into two subgroups each, SSR lacked discrimination power to resolve this structure (SUPPLEMENTARY FIG. 1). The P. deliense isolate (42142-99) used as outgroup species clustered inside group C with other distant isolates. Genetic structure and distribution of Pennsylvania isolates.—Isolates (n = 48) collected from Greenhouse X were analyzed to monitor the genetic movement and distributions in the greenhouse. Four SSR genotypes, PA1 (n = 43), PA2 (n = 3), PA5 (n = 1) and PA7 (n = 1), were found in the population (TABLE I). Isolates in the major genotype, PA1, were found in several hosts grown in that greenhouse including, poinsettia, impatiens and chrysanthemum as well as in soil and debris in the facility. The genetic diversity of all isolates from Greenhouse X was 0.52, 43 isolates having the PA1 SSR genotype. Three isolates (P125, 82445-01 and 82543-01) were in PA2 and one isolate (216E) was in PA7.

Each genotype was associated with a different genetic group. PA1 was closely related to PA10 (isolate P143 was collected from a location 140 km from the commercial greenhouse). Isolates in PA2 were closely related to more genotype groups (data not shown). Evaluating isolates from around the world, one from India was included in PA1 and isolates from Tanzania, Zambia and Venezuela were included in PA2 (TABLE I). Isolates containing the same AFLP and SSR genotypes were selected for determining genetic distributions of P. aphanidermatum from a small scale (Greenhouse X) to a large scale (Pennsylvania and other states). Isolates sampled from the same locations, either in Pennsylvania or Greenhouse X, shared the same genotypes (TABLE V). Samples collected from different locations in Pennsylvania shared the same AFLP and SSR genotypes, indicating genetic movement among isolates from different geographical locations. The same genotypes were found in the Greenhouse X population of P. aphanidermatum collected from infected plants, loose soil and plant debris, suggesting a wide distribution of inoculum in the entire production system. Of interest, the same genotypes were found in the irrigation system, which suggests this is the major distribution path for inoculum inside the greenhouse. Isolates MR42-U from Kentucky, Zen98-5-U from California, G-6 and H-29 from New York and 141749R from India were the same genotypes as isolates from Pennsylvania or Greenhouse X, indicating genetic movement nationally and internationally. Genetic differentiation among P. aphanidermatum isolates.—P. aphanidermatum isolates were divided into 14 different genotype groups (PA1–PA14) by SSR markers (TABLE IV). Group PA1 included the most isolates (n = 84), mostly from Greenhouse X. PA2 included isolates of wide geographical origin.

Based on comparison of AFLP and SSR markers poinsettias (n = 58) contained 11 genotypes and chrysanthemums (n = 7) and turf grasses (n = 17) had two and five different genotypes respectively. All three populations share the same genotypes, and no significant correlations with host were found (TABLE I). The genetic and genotypic similarities were closely related with sampling locations. Mefenoxam resistance.—Thirty-one isolates of P. aphanidermatum were found to be resistant to mefenoxam. Twenty-six resistant isolates were in genetic group A, while the remaining five isolates were in groups B or C. All worldwide isolates were not resistant to mefenoxam, but resistant isolates were found in Pennsylvania and other states. Moreover mefenoxam-resistant isolates were from different hosts including, poinsettia, chrysanthemum and turf grass. Eighty-four percent of mefenoxam resistant isolates were found in genetic group A and SSR genotype group PA1. DISCUSSION The degree of polymorphism in the P. aphanidermatum population was similar in both AFLP and SSR. Although SSR revealed higher degrees of polymorphism than AFLP in Garcia et al. (2004) and Jones et al. (1997), in this study a large number of highly polymorphic AFLP primer combinations were used that improved the discrimination among genetic clusters (SUPPLEMENTARY FIG. S). The results from both AFLP and SSR datasets were similar and differentiated genetically distant isolates, suggesting that P. aphanidermatum is a genetically diverse species. Moreover both marker data determined three major genetic groups in P. aphanidermatum isolates even though not all groups in each analysis contained the same isolates. The use of more SSR markers might reveal more genetic diversity that might minimize the discrepancies between the results with the two

types of markers. The genotypes shared by different genetic groups and geographic origins indicate a possible gene flow among the different populations. However the distribution and genetic flow among P. aphanidermatum strains from geographically distinct locations and hosts still need to be examined with more samples from a broader geographic area. Sexual reproduction is a factor that could contribute to the geographic structure within the species. Although P. aphanidermatum is considered to be primarily a homothallic species, it is possible that out-crossing is occurring in nature. No studies of outcrossing have been performed on P. aphanidermatum; however out-crossing was found in P. ultimum and in P. irregulare in vitro (Francis and St Clair 1997) and in fields (Harvey et al. 2001). Morphological traits in P. aphanidermatum that might favor out-crossing, for example oogonia can be terminal or intercalary and antheridia can be diclinous (van der Plaats-Niterink 1981), originating from distinct mycelia. Out-crossing could occur more frequently in P. aphanidermatum than in species with hypogynous antheridia, such as P. ultimum. Francis and St Clair (1993) reported 20% hybrid oospores from in vitro crosses of P. ultimum. Thus it is possible that the rate of out-crossing in P. aphanidermatum could be higher in similar conditions. The results obtained in this investigation support this hypothesis because most of the 123 isolates analyzed were heterozygous for one or more SSR loci (i.e. 96% of isolates were heterozygous for P18CAT1-74, TABLE III). The SSR analysis was repeated twice, and the presence of alleles was consistently reproducible. Our results show the high value of observed heterozygosity in all the four SSR loci (TABLE IV), suggesting the potential existence of a high rate of random mating in nature. Also our study (Lee and Moorman 2008) reported that the observed level of heterozygosity in P. irregulare and P. cryptoirregulare was much lower than that in P. aphanidermatum.

Pythium aphanidermatum has a wide host range, including many annuals and bedding plants. Our findings indicate that sample location instead of host might be the main factor contributing to the genetic diversity of P. aphanidermatum isolates. The same marker genotypes and genetic cluster were closely related to the sampling locations (FIG. 1), while different hosts were observed in the same genetic group. A partial genetic association with host was found in turf grass isolates; however this does not indicate that host is the factor contributing to the genetic diversity of the population. Because the same genotypes were found in other genetic groups it is possible that the genetic exchange among turf populations is more frequent than with greenhouse populations, by means still unknown. This result was found consistently in both AFLP and SSR genotype data. Mefenoxam is extensively used in floricultural greenhouses and on turf and is likely having a strong impact in the selection for resistant genotypes (Moorman et al. 2002). In that study we found that resistance to mefenoxam could be associated with a population structure. Among the 31 mefenoxam-resistance isolates, 26 belonged to the major SSR genotype PA1. However in isolates sensitive to mefenoxam no genetic relationships associated with a population structure was observed; 10 SSR genotypes were found in 38 mefenoxam-sensitive isolates. A similar result was found in the AFLP data. The 26 mefenoxam-resistant isolates collected from eight locations were placed in the major genetic group A, based on both AFLP and SSR analyses. This finding suggests that the use of mefenoxam might be the selection force increasing the prevalence of fungicide resistance in the population. As a result more numbers of fungicide-resistance isolates might be found in the major genotype or population. However this finding may be a reflection of the population structure of our samples alone. An intensive study with a large

number of samples collected from a single location will be necessary to understand the relationship between fungicide resistance and a specific genotype. While geographic location might be influencing the genetic structure in P. aphanidermatum, it is more likely that the sharing of isolates among greenhouses through the movement of infected plants accounts for the fact that most Pennsylvania isolates fell into the genotype group PA1. Other Pennsylvania isolates, identified as PA2, share the same genotype with isolates from California, Venezuela, Tanzania and Zambia. Also an isolate from India shared the same genotype with the isolates from the commercial greenhouse in Pennsylvania. This finding suggests that new isolates are being introduced to Pennsylvania and other greenhouse crop production facilities on imported plants that harbor P. aphanidermatum. In summary our study identified genetic structure in P. aphanidermatum populations and revealed the distribution of major genotypes in Pennsylvania greenhouses with the combination of AFLP and SSR markers. An important finding was that molecular tools could be used to monitor the movement and distribution of particular isolates within the production system. The early characterization of possible inoculum sources with molecular marker techniques might help trace the pathogen to its origin, thus allowing its elimination before crop losses occur. Moreover we also identified that potential inoculum may be widespread once the pathogen is introduced into the production system and is likely to move among and within greenhouses in infected plant material or through the irrigation system. This might have important implications regarding the management of the pathogen in greenhouses. The use of a larger number of samples and SSR markers might increase our

understanding of the spatial distribution of P. aphanidermatum isolates within and among greenhouse production systems. ACKNOWLEDGMENTS This work was supported by the USDA-ARS Floriculture and Nursery Crops Research Initiative and Pennsylvania State University Agricultural Experiment Station. We thank Rachel Leonard for technical assistance throughout the study.

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LEGENDS FIG. 1. Relationships among 123 P. aphanidermatum isolates with six AFLP primers and four SSR primers. The P. deliense isolate (42142-99) was used as outgroup. The tree was obtained by combining AFLP and SSR datasets and was inferred with the UPGMA method. The genetic distances were computed with the maximum composite likelihood method (Tamura et al. 2004) with the bootstrap test (1000 replicates). SUPPLEMENTARY FIG. 1. A comparison of phylogenetic trees obtained from AFLP and SSR datasets. The P. deliense isolate (42142-99) was used as outgroup.

FOOTNOTES Submitted 2 Sep 2009; accepted for publication 11 Dec 2009. 1

Corresponding author. E-mail: [email protected]

TABLE I. Description of 123 P. aphanidermatum isolates used in this study Isolates 141749R a P47 340458 a 100439R a P12 P102 P112 P126 P127 P128 G-6 346952 a 82569-01 82570-01 82585-01 82586-01 82587-01 82588-01 82589-01 82590-01 82592-01 82593-01 82594-01 P02 P14 72063-96 81192-96 P16 P18 P20 P22 P23 P24 54138-98 71009-98 81728-98 81729-98 P32 P33 P35 45041-99 P163 45044-99 66011-99 66012-99 81931-99 81954-99 81955-99 81969-99 P45 P52 10-1E

Source Atropa belladonna (Belladonna) Cactus Carica papaya (Papaya) Carica papaya (Papaya) Chrysanthemum (Mums) Chrysanthemum (Mums) Chrysanthemum (Mums) Chrysanthemum (Mums) Chrysanthemum (Mums) Chrysanthemum (Mums) Chrysanthemum (Mums) Cucumis melo (Muskmelon) Debris in greenhouse Debris in greenhouse Debris in greenhouse Debris in greenhouse Debris in greenhouse Debris in greenhouse Debris in greenhouse Debris in greenhouse Debris in greenhouse Debris in greenhouse Debris in greenhouse Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia)

Date -b 1999 1990 1995 2002 2002 2003 2003 2003 1991 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 1986 1995 1996 1996 1996 1996 1996 1997 1997 1997 1998 1998 1998 1998 1998 1998 1998 1999 2005 1999 1999 1999 1999 1999 1999 1999 1999 1999 2000

Location India Greenhouse, Michigan Malaysia Tanzania Paradise, PA Greenhouse, McClure, PA Retail store, Lebanon, PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse, unidentified, NY Israel Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse, Bloomsburg, PA Greenhouse, Rehrersburg, PA Greenhouse, unidentified, PA Greenhouse, unidentified, PA Greenhouse, Bloomsburg, PA Greenhouse X , Lititz PA Greenhouse, unidentified, PA Greenhouse X , Lititz PA Greenhouse, Danville, PA Greenhouse, Danville, PA Greenhouse, unidentified, PA Greenhouse, unidentified, PA Greenhouse, unidentified, PA Greenhouse, unidentified, PA Greenhouse X , Lititz PA Greenhouse, Zionsville, PA Greenhouse X , Lititz PA Greenhouse, unidentified, PA Greenhouse X , Lititz PA Greenhouse, unidentified, PA Greenhouse, unidentified, PA Greenhouse, unidentified, PA Greenhouse, unidentified, PA Greenhouse, unidentified, PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse, McClure, PA Greenhouse, Sunbury, PA Greenhouse X , Lititz PA

Reactionc S S S R R S S I R S I R R S S R S S R S R S R S R S S S S R R S R

SSRd PA1 PA4 PA3 PA2 PA1 PA1 PA2 PA1 PA1 PA2 PA1 PA12 PA1 PA1 PA1 PA1 PA1 PA1 PA1 PA1 PA1 PA1 PA1 PA1 PA1 PA1 PA1 PA11 PA1 PA1 PA1 PA1 PA1 PA1 PA1 PA1 PA1 PA1 PA9 PA1 PA2 PA1 PA1 PA14 PA2 PA1 PA1 PA5 PA1 PA1 PA1 PA1

Groupe A A A A A B A A B A A A B B B B B B A B A A A C C A A A A A C A A B B A A A A A A A A A A A A B A A A A

Isolates 10-2E 10-3E 12-1E 12-2E 12-3E 13-2E 22162-98 82505-01 82506-01 82559-01 82560-01 82568-01 H-29 H-48 P92 P103 P104 P105 P106 P107 P114 P132 83269-04 P143 184E 199E 200E 202E P157 P155 P162 82543-01 P115 216E 82482-01 82537-01 82561-01 82426-01 82445-01 370995R a 58847R a H-56 81949-99 363669R a 62163-98 61163-98 80E aphan TN P42 P1-U P20-U

Source Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Euphorbia pulcherrima (Poinsettia) Soil in greenhouse pot Soil outside greenhouse Greenhouse floor soil Greenhouse water Greenhouse water Greenhouse water Impatiens (Balsams) Impatiens (Balsams) Nicotiana tabacum (Tabacco) Nicotiana tabacum (Tabacco) Pelargonium hortorum (Geranium) Phaseolus lunatus (Legume) Pisum sativum (Snow pea) Schlumbergera sp. (Christmas cactus) Schlumbergera sp. (Christmas cactus) Sediment from water Turf grass Turf grass Turf grass Turf grass

Date 2000 2000 2000 2000 2000 2000 2000 2001 2001 2001 2001 2001 2001 2001 2001 2002 2002 2002 2002 2002 2002 2003 2004 2004 2005 2005 2005 2001 2002 2006 2001 2001 2001 2001 2001 2001 1999 1998 2000 2001 1999 1974 1975

Location Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse, unidentified, PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse, Utica, NY Greenhouse, Utica, NY Greenhouse, Zionsville, PA Greenhouse X , Lititz PA Greenhouse, Danville, PA Greenhouse, Zionsville, PA Greenhouse, Zionsville, PA Greenhouse, Zionsville, PA Greenhouse, Zionsville, PA Greenhouse X , Lititz PA Greenhouse, unidentified, PA Greenhouse, Danville, PA Greenhouse X in PA Greenhouse X in PA Greenhouse X in PA Greenhouse X in PA Zionsville, PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse, Zionsville, PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Greenhouse X , Lititz PA Venezuela Zambia Greenhouse, Utica, NY Outdoors, unidentified, PA Taiwan Greenhouse, unidentified, PA Greenhouse, unidentified, PA Greenhouse X , Lititz PA TN State College, PA University Park, PA Jonestown, PA

Reactionc R R R R R S S R S R R S R R R R R S S S S S S R S

SSRd PA1 PA1 PA1 PA1 PA1 PA1 PA1 PA1 PA1 PA1 PA1 PA1 PA1 PA3 PA1 PA1 PA1 PA2 PA1 PA2 PA4 PA1 PA1 PA10 PA1 PA1 PA1 PA1 PA1 PA1 PA1 PA2 PA2 PA7 PA1 PA1 PA1 PA1 PA2 PA2 PA2 PA8 PA1 PA13 PA2 PA2 PA1 PA3 PA1 PA1

Groupe A A A A A A A A B B B B A A C B A B B A B A A A A A A A B B A A B C A A A B B A A B A B A A A A A C A

Isolates P21-U P3-U P7-U P27-U P31-U P32-U P38-U MR40-U MR42-U P41-U P39A-U P39B-U P40-U Zen97-35-U Zen97-70-U Zen97-71-U Zen97-375-U Zen97-378-U Zen98-5-U P41

Source Turf grass Turf grass Turf grass Turf grass Turf grass Turf grass Turf grass Turf grass Turf grass Turf grass Turf grass Turf grass Turf grass Turf grass Turf grass Turf grass Turf grass Turf grass Turf grass Turf grass

Outgroup species (Pythium deliense) Pelargonium hortorum (Geranium) 42142-99 a

Date 1975 1975 1975 1976 1986 1986 1988 1991 1991 1991 1994 1994 1994 1997 1997 1997 1998 1998 1998 1999

Location Freeport, PA University Park, PA University Park, PA Radnor, PA Washington, PA Altoona, PA Chambersburg, PA Cincinnati, OH Louisville, KY University Park, PA University Park, PA University Park, PA University Park., PA Maryland Illinois California California Lewistown, PA

1999

Greenhouse, Connellsville, PA

Reactionc S S S S S R R S R R S S -

SSRd PA1 PA1 PA6 PA3 PA1 PA1 PA1 PA1 PA1 PA1 PA4 PA2 PA1 PA1 PA2 PA1

S

CABI, Centre for Agriculture and Biosciences International, UK, accession number. No information is available. c Mefenoxam sensitive (S), resistant (R) and intermediate (I) resistance. d Genotype groups from four SSR loci. e Genetic groups determined by the combination of six AFLP primers. b

Groupe A A C A B A A B A A A A A B B B A A A A

TABLE II. Descriptions of AFLP polymorphisms using 6 primer combinations across 123 P. aphanidermatum isolates. Genetic diversity (H) of each AFLP combination was calculated with Nei's (1973) gene diversity. Primer combination MseI-AC / EcoRI-AG MseI-CC / EcoRI-AT MseI-GT / EcoRI-AA MseI-GT / EcoRI-AC MseI-TC / EcoRI-AC MseI-TT / EcoRI-AC Average a

Total band numbera 40 25 61 29 47 30 38.7

Polymorphic band 16 16 21 18 17 15 17.2

Polymorphism (%) 40 64 34 62 36 50 44

H 0.329 0.349 0.356 0.342 0.366 0.318 0.344

Only unambiguously amplified band across all isolates were included for AFLP analysis.

TABLE III. Allele size, frequency, and genetic information of four SSR loci analyzed for 123 P. aphanidermatum isolates.

a b

SSR locus

Allele length (bp)

Allele frequency

P18GAA1-71

172 181 193 196

0.016 0.484 0.402 0.098

P18GAA1-72

184 194 206 209

0.015 0.485 0.405 0.095

P18CAT1-74

147 150

0.500 0.500

P18TCC3-25

138 151

0.469 0.531

Genotype 172 172/193 181 181/193 181/196 196 184 184/206 184/209 194 194/206 194/209 206 209 147 147/150 150 138 138/151 151

HE: expected heterozygosity. HO: observed heterozygosity. PIC: polymorphic information content.

Genotype frequency 0.008 0.017 0.008 0.782 0.168 0.017 0.008 0.008 0.008 0.016 0.787 0.148 0.008 0.016 0.016 0.960 0.024 0.008 0.925 0.067

HE/HOa

PICb

0.597/0.966

0.514

0.592/0.951

0.508

0.500/0.960

0.375

0.498/0.925

0.374

TABLE IV. Description of SSR genotypes of 116 P. aphanidermatum isolates. Five isolates (P1-U, P7-U, P21-U, P38-U, and P39A-U) and P. deliense (42142-99) were excluded from this study because of the presence of missing alleles. Genotype PA1 PA2 PA3 PA4 PA5 PA6 PA7 PA8 PA9 PA10 PA11 PA12 PA13 PA14

Isolate 85 isolates 16 isolates 4 isolates P47, P114, Zen9770-U 81955-99 P31-U 216E H-56 P33 P143 P16 346952 363669R 66011-99

P18GAA1-71 181/193 181/196 181/193 181/196

SSR loci P18GAA1-72 P18CAT1-74 194/206 147/150 194/209 147/150 194/206 147/150 194/206 147/150

P18TCC3-25 138/151 138/151 151 138/151

172 172/193 172/193 181 181/193 181/193 181/196 181/196 196 196

184 184/206 194/206 194 194/209 206 184/209 194/209 209 209

151 138/151 138/151 151 138/151 138/151 138/151 138/151 138 138/151

150 147/150 147/150 147/150 147/150 147 147/150 147 147/150 147/150

TABLE V. Descriptions of isolates showing same AFLP and SSR genotypes. The genotypes from all isolates were obtained with six highly polymorphic AFLP primer combinations and four SSR loci. Sampling location

Source

P12, P33, G-6, 81931-99, 81949-99

Isolates

Pennsylvania, New York

Phaseolus lunatus, Euphorbia pulcherrima, Chrysanthemum

P23, P24, 81728-98, H-29

Pennsylvania, New York

Euphorbia pulcherrima

P132, P162, P163, 81969-99, 82482-01, 82505-01, 82561-01, 82589-01

CGH

81192-96, 82592-01, 82594-01

Pennsylvania, CGH

Euphorbia pulcherrima, Debris

Zen97-375, Zen97-378, P41-U

—b

Turf grass

a

Euphorbia pulcherrima, Water, Debris

P20-U, 45044-99

Pennsylvania

Euphorbia pulcherrima, Turf grass

22162-98, 81729-98, 81954-99,

Pennsylvania

Euphorbia pulcherrima

P35, 184E, 200E

CGH

Euphorbia pulcherrima

61163-98, 45041-99, 82543-01

Pennsylvania, CGH

Euphorbia pulcherrima, Schlumbergera sp., Pot

10-1E, 12-3E

CGH

Euphorbia pulcherrima

P45, 12-1E, 80E, 199E, 202E, MR42-U, 72063-96

Pennsylvania, Kentucky, CGH Euphorbia pulcherrima, Sediment, Turf grass

P52, P3-U, 10-2E, 10-3E, 141749R, 8253701, 83269-04

Pennsylvania, India, CGH

Euphorbia pulcherrima, Atropa belladonna, Turf grass, Water

66012-99, Zen98-5-U

Pennsylvania, California

Euphorbia pulcherrima, Turf grass

82560-01, 82568-01, 82585-01, 82586-01, 82587-01, 82590-01

CGH

Euphorbia pulcherrima, Debris

82426-01, 82559-01

CGH

Euphorbia pulcherrima, Impatiens

82570-01, 82588-01 a

CGH

Debris

Commercial Greenhouse X in Pennsylvania. b No information is available.