Inventory and Comparative Evolution of the ABC Superfamily in the Genomes of <Emphasis Type="Italic">Phytophthora ramorum</Emphasis> and <Emphasis Type="Italic">Phytophthora sojae</Emphasis>

Automated and manual annotation of the ATP binding cassette (ABC) superfamily in the Phytophthora ramorum and P. sojae genomes has identified 135 and 136 members, respectively, indicating that this family is comparable in size to the Arabidopsis thaliana and rice genomes, and significantly larger than that of two fungal pathogens, Fusarium graminearum and Magnaporthe grisea. The high level of synteny between these oomycete genomes extends to the ABC superfamily, where 108 orthologues were identified by phylogenetic analysis. The largest subfamilies include those most often associated with multidrug resistance. The P. ramorum genome contains 22 multidrug resistance-associated protein (MRP) genes and 49 pleiotropic drug resistance (PDR) genes, while P. sojae contains 20 MRP and 49 PDR genes. Tandem duplication events in the last common ancestor appear to account for much of the expansion of these subfamilies. Recent duplication events in the PDR and ABCG families in both the P. ramorum and the P. sojae genomes indicate that selective expansion of ABC transporters may still be occurring. In other kingdoms, subfamilies define both domain arrangements and proteins having a common phylogenetic origin, but this is not the case for several subfamilies in oomycetes. At least one ABCG type transporter is derived from a PDR transporter, while transporters in the ABCB-half family cluster with transporters from bacterial, plant, and metazoan genomes. Additional examples of transporters that appear to be derived from horizontal transfer events from bacterial genomes include components of transporters associated with iron uptake and DNA repair. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A five-minute DNA extraction method for expedited detection of Phytophthora ramorum following prescreening using Phytophthora spp. lateral flow devices

In a direct comparison with established methods for Phytophthora ramorum detection (isolation followed by morphological identification, or conventional DNA extraction followed by TaqMan real-time PCR) a rapid, simplified detection method in which membranes of lateral flow devices (LFDs) are added directly to TaqMan real-time PCR reactions was used to test 202 plant samples collected by plant health inspectors in the field. P. ramorum prevalence within the 202 samples was approximately 40% according to routine testing by isolation or TaqMan real-time PCR. The diagnostic sensitivity and specificity of the rapid detection method were 96.3% and 91.2%, respectively. This method can be used in conjunction with Phytophthora spp. lateral flow devices to reduce the number of samples requiring testing using more laborious conventional methods. The effect of combining prescreening for Phytophthora spp. with P. ramorum-specific tests is discussed in terms of the positive and negative predictive values of species-specific detection when testing samples collected in different inspection scenarios.     

The Pectin Methylesterase Gene Complement of Phytophthora sojae: Structural and Functional Analyses,and the Evolutionary Relationships with Its Oomycete Homologs

Phytophthora sojae is an oomycete pathogen that causes the disease known as root and stem rot in soybean plants, frequently leading to massive economic damage. Additionally, P. sojae is increasingly being utilized as a model for phytopathogenic oomycete research. Despite the economic and scientific importance of P. sojae, the mechanism by which it penetrates the host roots is not yet fully understood. It has been found that oomycetes are not capable of penetrating the cell wall solely through mechanical force, suggesting that alternative factors facilitate breakdown of the host cell wall. Pectin methylesterases have been suggested to be important for Phytophthora pathogenicity, but no data exist on their role in the P. sojae infection process. We have scanned the newly revised version of the annotated P. sojae genome for the presence of putative pectin methylesterases genes and conducted a sequence analysis of all gene models found. We also searched for potential regulatory motifs in the promoter region of the proposed P. sojae models, and investigated the gene expression levels throughout the early course of infection on soybean plants. We found that P. sojae contains a large repertoire of pectin methylesterase-coding genes and that most of these genes display similar motifs in the promoter region, indicating the possibility of a shared regulatory mechanism. Phylogenetic analyses confirmed the evolutionary relatedness of the pectin methylesterase-coding genes within and across Phytophthora spp. In addition, the gene duplication events that led to the emergence of this gene family appear to have occurred prior to many speciation events in the genus Phytophthora. Our results also indicate that the highest levels of expression occurred in the first 24 hours post inoculation, with expression falling after this time. Our study provides evidence that pectin methylesterases may be important for the early action of the P. sojae infection process.     

Developing a taxonomic identification system of Phytophthora species based on microsatellites

BackgroundPhytophthora is the most important genus of the Oomycete plant pathogens. Nowadays, there are 117 described species in this genus, most of them being primary invaders of plant tissues. The different species are causal agents of diseases in a wide range of crops and plants in natural environments. In order to develop control strategies against Phytophthoraspecies, it is important to know the biology, ecology and evolutionary processes of these important pathogens.AimsThe aim of this study was to propose and validate a low cost identification system for Phytophthora species based on a set of polymorphic microsatellite (SSRs) markers.MethodsThirty-three isolates representing Phytophthora infestans, Phytophthora andina, Phytophthora sojae, Phytophthora cryptogea, Phytophthora nicotianae, Phytophthora capsici and Phytophthora cinnamomi species were obtained, and 13 SSRs were selected as potentially transferable markers between these species. Amplification conditions, including annealing temperatures, were standardized for several markers.ResultsA subset of these markers amplified in all species, showing species-specific alleles.ConclusionsThe adaptability and impact of the identification system in Colombia, an Andean agricultural country where different Phytophthora species co-exist in the same or in several hosts grown together, are discussed.     

Gene Duplication and Fragment Recombination Drive Functional Diversification of a Superfamily of Cytoplasmic Effectors in Phytophthora sojae

Phytophthora and other oomycetes secrete a large number of putative host cytoplasmic effectors with conserved FLAK motifs following signal peptides, termed crinkling and necrosis inducing proteins (CRN), or Crinkler. Here, we first investigated the evolutionary patterns and mechanisms of CRN effectors in Phytophthora sojae and compared them to two other Phytophthora species. The genes encoding CRN effectors could be divided into 45 orthologous gene groups (OGG), and most OGGs unequally distributed in the three species, in which each underwent large number of gene gains or losses, indicating that the CRN genes expanded after species evolution in Phytophthora and evolved through pathoadaptation. The 134 expanded genes in P. sojae encoded family proteins including 82 functional genes and expressed at higher levels while the other 68 genes encoding orphan proteins were less expressed and contained 50 pseudogenes. Furthermore, we demonstrated that most expanded genes underwent gene duplication or/and fragment recombination. Three different mechanisms that drove gene duplication or recombination were identified. Finally, the expanded CRN effectors exhibited varying pathogenic functions, including induction of programmed cell death (PCD) and suppression of PCD through PAMP-triggered immunity or/and effector-triggered immunity. Overall, these results suggest that gene duplication and fragment recombination may be two mechanisms that drive the expansion and neofunctionalization of the CRN family in P. sojae, which aids in understanding the roles of CRN effectors within each oomycete pathogen.     

Are the polygenic architectures of resistance to <Emphasis Type="Italic">Phytophthora capsici</Emphasis> and <Emphasis Type="Italic">P. parasitica</Emphasis> independent in pepper?

The pepper accession Criollo de Morelos 334 is the most efficient source of resistance currently known to Phytophthora capsici and P. parasitica. To investigate whether genetic controls of resistance to two Phytophthora species are independent, we compared the genetic architecture of resistance of CM334 to both Phytophthora species. The RIL population F5YC used to construct the high-resolution genetic linkage map of pepper was assessed for resistance to one isolate of each Phytophthora species. Inheritance of the P. capsici and P. parasitica resistance was polygenic. Twelve additive QTLs involved in the P. capsici resistance and 14 additive QTLs involved in the P. parasitica resistance were detected. The QTLs identified in this progeny were specific to these Phytophthora species. Comparative mapping analysis with literature data identified three colocations between resistance QTLs to P. parasitica and P. capsici in pepper. Whereas this result suggests presence of common resistance factors to the two Phytophthora species in pepper, which possibly derive from common ancestral genes, calculation of the colocation probability indicates that these colocations could occur by chance.     

Roles of small RNAs in soybean defense against Phytophthora sojae infection

The genus Phytophthora consists of many notorious pathogens of crops and forestry trees. At present, battling Phytophthora diseases is challenging due to a lack of understanding of their pathogenesis. We investigated the role of small RNAs in regulating soybean defense in response to infection by Phytophthora sojae, the second most destructive pathogen of soybean. Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are universal regulators that repress target gene expression in eukaryotes. We identified known and novel small RNAs that differentially accumulated during P. sojae infection in soybean roots. Among them, miR393 and miR166 were induced by heat‐inactivated P. sojae hyphae, indicating that they may be involved in soybean basal defense. Indeed, knocking down the level of mature miR393 led to enhanced susceptibility of soybean to P. sojae; furthermore, the expression of isoflavonoid biosynthetic genes was drastically reduced in miR393 knockdown roots. These data suggest that miR393 promotes soybean defense against P. sojae. In addition to miRNAs, P. sojae infection also resulted in increased accumulation of phased siRNAs (phasiRNAs) that are predominantly generated from canonical resistance genes encoding nucleotide binding‐leucine rich repeat proteins and genes encoding pentatricopeptide repeat‐containing proteins. This work identifies specific miRNAs and phasiRNAs that regulate defense‐associated genes in soybean during Phytophthora infection.     

Combined use of bulked segregant analysis and microarrays reveals SNP markers pinpointing a major QTL for resistance to Phytophthora capsici in pepper

Key message

Bulked segregant analysis (BSA) using Affymetrix GeneChips revealed candidate genes underlying the major QTL for Phytophthora capsici resistance in Capsicum . Using the candidate genes, reliable markers for Phytophthora resistance were developed and validated.

Abstract

Phytophthora capsici L. is one of the most destructive pathogens of pepper (Capsicum spp.). Resistance of pepper against P. capsici is controlled by quantitative trait loci (QTL), including a major QTL on chromosome 5 that is the predominant contributor to resistance. Here, to maximize the effect of this QTL and study its underlying genes, an F₂ population and recombinant inbred lines were inoculated with P. capsici strain JHAI1-7 zoospores at a low concentration (3 × 10³/mL). Resistance phenotype segregation ratios for the populations fit a 3:1 and 1:1 (resistant:susceptible) segregation model, respectively, consistent with a single dominant gene model. Bulked segregant analysis (BSA) using Affymetrix GeneChips revealed a single position polymorphism (SPP) marker mapping to the major QTL. When this SPP marker (Phyto5SAR) together with other SNP markers located on chromosome 5 was used to confirm the position of the major QTL, Phyto5SAR showed the highest LOD value at the QTL. A scaffold sequence (scaffold194) containing Phyto5SAR was identified from the C. annuum genome database. The scaffold contained two putative NBS-LRR genes and one SAR 8.2A gene as candidates for contributing to P. capsici resistance. Markers linked to these genes were developed and validated by testing 100 F₁ commercial cultivars. Among the markers, Phyto5NBS1 showed about 90 % accuracy in predicting resistance phenotypes to a low-virulence P. capsici isolate. These results suggest that Phyto5NBS1 is a reliable marker for P. capsici resistance and can be used for identification of a gene(s) underlying the major QTL on chromosome 5.     

Genetic characterization of Phytophthora nicotianae by the analysis of polymorphic regions of the mitochondrial DNA

A new method based on the analysis of mitochondrial intergenic regions characterized by intraspecific variation in DNA sequences was developed and applied to the study of the plant pathogen Phytophthora nicotianae. Two regions flanked by genes trnY and rns and trnW and cox2 were identified by comparing the whole mitochondrial genomes of Phytophthora infestans, Phytophthora ramorum, and Phytophthora sojae and amplified using primers designed from the flanking conserved genes. These regions were sequenced from 51 isolates of P. nicotianae of both A1 and A2 mating type recovered from different hosts and geographic regions. Amplicon length varied from 429 bp to 443 bp (trnY/rns) and 322 bp to 373 bp (trnW/cox2) with intraspecific variation due to single nucleotide polymorphisms and indels. Seventeen, seven and 20 different haplotypes were detected by individually analyzing regions trnY-rns, trnW-cox2 and the combined data set of sequences from both regions, respectively. Phylogenetic analysis inferred with three different methods enabled the grouping of isolates in five clades, each containing different mitochondrial haplotypes and revealed diversity in the mitochondrial genome of P. nicotianae. The majority of isolates from citrus grouped in a single clade indicating either movement of isolates on planting stock or an association of particular isolates with this host. Phylogenetic groups were not correlated with the radial growth rate of the isolates or the rapidity of apple flesh colonization. The method developed in the present study represents an innovative molecular tool for the characterization of natural populations of P. nicotianae and should be easily expanded to other species of Phytophthora as well as other plant pathogens. It can be used to track specific haplotypes and, thanks to its high genetic resolution, it could be standardized and applied in a DNA barcoding like strategy for the precise identification of sub-specific taxa. Compared to alternative molecular methods, a major advantage is that results are unbiased (a list of nucleotides) and highly reproducible, thus enabling the comparison of data from different laboratories and time periods. Furthermore, the method could be further enhanced by the identification of additional variable mitochondrial and/or nuclear genomic regions.     

Phytophthora sojae apoplastic effector AEP1 mediates sugar uptake by mutarotation of extracellular aldose and is recognized as a MAMP

Diseases caused by Phytophthora pathogens devastate many crops worldwide. During infection, Phytophthora pathogens secrete effectors, which are central molecules for understanding the complex plant–Phytophthora interactions. In this study, we profiled the effector repertoire secreted by Phytophthora sojae into the soybean (Glycine max) apoplast during infection using liquid chromatography–mass spectrometry. A secreted aldose 1-epimerase (AEP1) was shown to induce cell death in Nicotiana benthamiana, as did the other two AEP1s from different Phytophthora species. AEP1 could also trigger immune responses in N. benthamiana, other Solanaceae plants, and Arabidopsis (Arabidopsis thaliana). A glucose dehydrogenase assay revealed AEP1 encodes an active AEP1. The enzyme activity of AEP1 is dispensable for AEP1-triggered cell death and immune responses, while AEP-triggered immune signaling in N. benthamiana requires the central immune regulator BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1. In addition, AEP1 acts as a virulence factor that mediates P. sojae extracellular sugar uptake by mutarotation of extracellular aldose from the α-anomer to the β-anomer. Taken together, these results revealed the function of a microbial apoplastic effector, highlighting the importance of extracellular sugar uptake for Phytophthora infection. To counteract, the key effector for sugar conversion can be recognized by the plant membrane receptor complex to activate plant immunity.

Phytophthora sojae apoplastic effector AEP1 triggers pattern-triggered immunity in nonhost plants and contributes to P. sojae virulence by promoting the uptake of extracellular sugar.     

Survival and recovery of Phytophthora capsici and oomycetes in tailwater and soil from vegetable fields in Florida

A current trend in Florida agriculture to conserve water is to irrigate with surface runoff water (tailwater) recovered in retention ponds and canals. Water filtration and lemon leaf baiting recovered Phytophthora capsici and other plant pathogenic Oomycetes in runoff water from ponds and canals. A total of 196 isolates of Phytophthora spp. and 471 isolates of Pythium spp. were recovered. Phytophthora spp. included P. capsici, P. cinnamomi, P. lateralis, P. nicotianae, P. citricola, P. cryptogea and P. erythroseptica. Species of Pythium were P. aphanidermatum, P. catenulatum, P. helicoides, P. irregulare, P. myriotylum, and Pythium‘group F’. Isolates of P. aphanidermatum, P. irregulare, P. myriotylum, and Pythium‘group F’ were pathogenic on pepper and tomato. Recovery of P. capsici propagules was related to soil moisture‐holding capacity and time interval but not temperature. Recovery of P. capsici propagules at 100% soil moisture‐holding capacity and 30° C was 57 days. In tailwater, recovery of propagules of P. capsici was 63 days at 24°C to 25°C. The potential exists to reintroduce and disseminate species of Phytophthora and Pythium when using tailwater for irrigation or other practices.     

Proteins related to green algal striated fiber assemblin are present in stramenopiles and alveolates

In green algae, striated fiber assemblin (SFA) is the major protein of the striated microtubule-associated fibers that are structural elements in the flagellar basal apparatus. Using Basic Local Alignment Search Tool (BLAST) searches of recently established databases, SFA-like sequences were detected in the genomes not only of green algal species but also of a range of other protists. These included species in two alveolate subgroups, the ciliates (Tetrahymena thermophila, Paramecium tetraurelia) and the dinoflagellates (Perkinsus marinus), and two stramenopile subgroups, the oomycetes (Phytophthora sojae, Phytophthora ramorum, Phytophthora infestans) and the diatoms (Thalassiosira pseudonana, Phaeodactylum tricornutum). Together with earlier identification of SFA-like sequences in the apicomplexans, these results indicate that homologs of SFA are present across the alveolates and stramenopiles. Antibodies raised against SFA from the green alga, Spermatozopsis similis, react in immunofluorescence assays with the two basal bodies and an anteriorly directed striated fiber in the flagellar apparatus of biflagellate Phytophthora zoospores.     

Inhibitory effects of xenocoumacin 1 on the different stages of <Emphasis Type="Italic">Phytophthora capsici</Emphasis> and its control effect on Phytophthora blight of pepper

Phytophthora species cause enormous economic loss every year worldwide. Xenocoumacin 1 (Xcn1), isolated from the bacterium Xenorhabdus nematophilus, is a broad-spectrum antibiotic against agricultural pathogens, especially Phytophthora. To understand the inhibitory mode of Xcn1 toward Phytophthora pathogens, we determined the inhibitory effects of Xcn1 on Phytophthora capsici both in vitro and in vivo. In vitro, Xcn1 inhibited different stages in the life cycle of P. capsici, including sporangium formation, zoospore germination, and mycelial growth, with 50% effective concentration (EC₅₀) values of 0.037, 0.81, and 2.44 μg ml^?1, respectively. Xcn1 also reduced zoospore motility. In vivo, Xcn1 efficiently controlled the Phytophthora blight of pepper with a disease reduction of 99% at a concentration of 5 μg ml^?1 assessed on the third day after incubation of wound stem plants. In addition, Xcn1-treated P. capsici mycelia exhibited increased mycelial branch spacing, evident plasmolysis, and leakage of intracellular components. In conclusion, in the presence of Xcn1, several stages in the life cycle of P. capsici were inhibited, and the hyphae exhibited obvious morphological changes.     

Laccase activity and putative laccase genes in marine-derived basidiomycetes

Studies of laccases from marine-derived fungi are limited. In the present work, putative laccase genes from three marine-derived basidiomycetes and their laccase activities were evaluated. High amounts of laccase were produced by the fungal strains Marasmiellus sp. CBMAI 1062 (971.2 U L⁻¹) and Peniophora sp. CBMAI 1063 (709.03 U L⁻¹) when grown for 21 d at 28 °C in MA2ASW medium prepared with artificial seawater. Marine-derived basidiomycetes produced multiple distinct laccase sequences of about 200 bp with 73–90 % similarity to terrestrial basidiomycete laccases. Marasmiellus sp. CBMAI 1062 and Tinctoporellus sp. CBMAI 1061 showed the greatest laccase gene diversity with three and four distinct putative laccase sequences, respectively. This is the first report of laccase genes from marine-derived fungi, and our results revealed new putative laccases produced by three basidiomycetes.     

Molecular Distinctions Between Phytophthora capsici and Ph. tropicalis Based on ITS Sequences of Ribosomal DNA

Among four species of Phytophthora tested, only Ph. capsici and Ph. tropicalis showed the same length for DNA sequence for both internal transcribed spacer (ITS)1 and ITS2 of ribosomal DNA. Phytophthora palmivora and P. nicotianae have lengths different from each other, and from the other two species. Although A1 and A2 types of Ph. capsici differ from each other by only one nucleotide, there are 10 different nucleotides between A1 and A2 types of Ph. tropicalis. Phylogenetic analysis of combined ITS sequences identified four clades each consisting A1 and A2 mating types of same species. The neighbor‐joining and maximum parsimony trees show that Ph. tropicalis (A2) is clustered with the clade of two isolates of Ph. capsici before joining the clade of A1 and two other isolates of Ph. tropicalis from GenBank. Our results support the separation of Ph. tropicalis and demonstrate the need to sequence more than a single isolate of a species in the study of molecular phylogeny of Phytophthora. The phylogenetic trees also suggest that Ph. tropicalis (A2) may represent a transitional isolate in the process of species evolution.     

Development of an Improved Isolation Approach and Simple Sequence Repeat Markers To Characterize Phytophthora capsici Populations in Irrigation Ponds in Southern Georgia

Phytophthora capsici, the causal agent of Phytophthora blight, is a major concern in vegetable production in Georgia and many other states in the United States. Contamination of irrigation water sources by P. capsici may be an important source of inoculum for the pathogen. A simple method was developed in this study to improve the efficiency of recovering P. capsici from fruits used as baits in irrigation ponds. In contrast to direct isolation on agar plates, infected fruit tissues were used to inoculate stems of pepper seedlings, and the infected pepper stems were used for isolation on agar plates. With isolation through inoculation of pepper stems, the frequency of recovering P. capsici from infected eggplant and pear fruits increased from 13.9% to 77.7% and 8.1% to 53.5%, respectively, compared with direct isolation on agar plates. P. capsici was isolated from seven out of nine irrigation ponds evaluated, with most of the ponds containing both A1 and A2 mating types and a 4:5 ratio of A1 to A2 when isolates from all ponds were calculated. All P. capsici isolates were pathogenic on squash plants, and only a small proportion (8.2%) of the isolates were resistant or intermediately sensitive to mefenoxam. Simple sequence repeats (SSRs) were identified through bioinformatics mining of 55,848 publicly available expressed sequence tags of P. capsici in dbEST GenBank. Thirty-one pairs of SSR primers were designed, and SSR analysis indicated that the 61 P. capsici isolates from irrigation ponds were genetically distinct. Cluster analysis separated the isolates into five genetic clusters with no more than two genetic groups in one pond, indicating relatively low P. capsici genetic diversity in each pond. The isolation method and SSR markers developed for P. capsici in this study could contribute to a more comprehensive understanding of the genetic diversity of this important pathogen.Phytophthora capsici, the causal agent of Phytophthora blight, is a widespread and destructive plant pathogen that causes root rot, crown rot, fruit rot, and foliar blight on many economically important crops in the United States and throughout the world (1). A number of important vegetable crops are susceptible to this pathogen, including peppers, squash, cucumber, watermelon, cantaloupe, zucchini, eggplant, pumpkin, tomatoes, and snap beans. The pathogen causes significant yield reductions and quality losses to vegetable industries and has become a major concern in vegetable production in the United States in recent years. The efficacies of current strategies for management of the disease are limited. No single fungicide has consistently and effectively suppressed losses caused by P. capsici epidemics. While fungicides containing the active ingredient mefenoxam provide some level of control of P. capsici, mefenoxam-resistant isolates that challenge the usefulness of the compound have developed (3, 8).It is critical to understand the ecology and epidemiology of P. capsici in order to design more effective disease management strategies. Studies conducted in recent years indicate that P. capsici survives in irrigation water in the United States, and irrigation water may serve as an important inoculum source. Roberts et al. (14) reported that P. capsici was isolated from tailwater (surface runoff water) in Florida using water filtration and lemon leaf baiting techniques. Gevens et al. (3) used pear and cucumber fruits as baits and isolated P. capsici from irrigation water sources in Michigan. It was unknown, however, if irrigation water sources in Georgia could be significant sources of primary inoculum. Earlier studies using water filtration or direct isolation from water and bottom sediment did not identify P. capsici in surface irrigation ponds in Georgia (16).Since surface water can be a significant source of P. capsici, it is critical to use appropriate methods to isolate the pathogen from irrigation water and to facilitate characterization of the isolates. Fruit, especially pears, is often used as bait to recover Phytophthora spp. from water (3, 21). In comparison to water filtration, the baiting technique is easier and less labor intensive. However, direct isolation from infected fruit bait is often hampered by other microorganisms. Isolation of Phytophthora spp. is often affected by Pythium spp. that overgrow fruit and agar plates. Hence, development of a more efficient isolation method is needed to increase the frequency of P. capsici recovery to facilitate the detection and characterization of isolates associated with water sources.The objectives of this study were to develop an efficient method to isolate P. capsici from irrigation ponds in southern Georgia and to develop simple sequence repeat (SSR) markers to analyze the genetic diversity of P. capsici populations in irrigation ponds. SSRs are tandemly repeated motifs of 1 to 6 bases found in the nuclear genomes of all eukaryotic organisms and are often abundant and evenly dispersed (7). They are highly polymorphic, multiallelic, and codominant and are believed to be a more efficient marker system than restriction fragment length polymorphisms and randomly amplified polymorphic DNAs (18, 23). SSR markers have been derived from publicly available expressed sequence tags (ESTs) of a few plant pathogens, including Phytophthora infestans, Phytophthora sojae, and Magnaporthe grisea (5, 10, 23); however, no SSRs for P. capsici have been developed. Development of EST-SSR markers may provide an effective molecular marker system for analysis of genetic variation within P. capsici populations.