Populations of Phytophthora rubi Show Little Differentiation and High Rates of Migration Among States in the Western United States.

Population genetics is a powerful tool to understand patterns and evolutionary processes that are involved in plant-pathogen emergence and adaptation to agricultural ecosystems. We are interested in studying the population dynamics of Phytophthora rubi, the causal agent of Phytophthora root rot in raspberry. P. rubi is found in the western United States, where most of the fresh and processed raspberries are produced. We used genotyping-by-sequencing to characterize genetic diversity in populations of P. rubi sampled in the United States and other countries. Our results confirm that P. rubi is a monophyletic species with complete lineage sorting from its sister taxon P. fragariae. Overall, populations of P. rubi show low genetic diversity across the western United States. Demographic analyses suggest that populations of P. rubi from the western United States are the source of pathogen migration to Europe. We found no evidence for population differentiation at a global or regional (western United States) level. Finally, our results provide evidence of migration from California and Oregon into Washington. This report provides new insights into the evolution and structure of global and western United States populations of the raspberry pathogen P. rubi, indicating that human activity might be involved in moving the pathogen among regions and fields.

Development and Application of qPCR and RPA Genus- and Species-Specific Detection of Phytophthora sojae and P. sansomeana Root Rot Pathogens of Soybean.

Phytophthora root rot of soybean, caused by Phytophthora sojae, is one of the most important diseases in the Midwestern United States, and is estimated to cause losses of up to 1.2 million metric tons per year. Disease may also be caused by P. sansomeana; however, the prevalence and damage caused by this species is not well known, partly due to limitations of current diagnostic tools. Efficient, accurate, and sensitive detection of pathogens is crucial for management. Thus, multiplex qPCR and isothermal RPA (recombinase polymerase amplification) assays were developed using a hierarchical approach to detect these Phytophthora spp. The assays consist of a genus-specific probe and two species-specific probes that target the atp9-nad9 region of the mitochondrial genome that is highly specific for the genus Phytophthora. The qPCR approach multiplexes the three probes and a plant internal control. The RPA assays run each probe independently with a plant internal control multiplexed in one amplification, obtaining a result in as little as 20 mins. The multicopy mitochondrial genome provides sensitivity with sufficient variability to discern among different Phytophthora spp. The assays were highly specific when tested against a panel of 100 Phytophthora taxa and range of Pythium spp. The consistent detection level of the assay was 100 fg for the qPCR assay and 10 pg for the RPA assay. The assays were validated on symptomatic plants collected from Michigan (U.S.) and Ontario (Canada) during the 2013 field season, showing correlation with isolation. In 2014, the assays were validated with samples from nine soybean producing states in the U.S. The assays are valuable diagnostic tools for detection of Phytophthora spp. affecting soybean.

Systematic Development of Phytophthora Species-Specific Mitochondrial Diagnostic Markers for Economically Important Members of the Genus.

The genus Phytophthora contains many invasive species to the U.S.A. that have the potential to cause significant damage to agriculture and native ecosystems. A genus and species-specific diagnostic assay was previously reported based on mitochondrial gene order differences that allowed for the systematic development of 14 species-specific TaqMan probes for pathogen detection ( Bilodeau et al. 2014 ). In this study, an additional 32 species-specific TaqMan probes for detection of primarily invasive species have been validated against 145 Phytophthora taxa as well as a range of Pythium and plant DNA samples. All validated probes were found to be species-specific and could be multiplexed with a genus-specific probe. The lower limit of linear detection using purified genomic DNA ranged from 1 to 100 fg in all assays. In addition, 124 unique TaqMan probes for Phytophthora spp. developed in silico are presented, which, if testing confirms they are species-specific, will provide diagnostic capabilities for approximately 89% of the genus. To enhance sensitivity of detection for several species that contained a single nucleotide polymorphism (SNP) in the reverse primer, a second primer was developed that is added in a small amount to the master mix. Furthermore, a PCR-RFLP system was developed that could be used to identify individual species when multiple species are present in a sample, without requiring cloning or sequencing. Several experiments were also conducted to compare various qPCR thermal cyclers and independent validation experiments with another research laboratory to identify possible limitations when the assays are used on a range of equipment in different labs. This system represents a comprehensive, hierarchal approach to increase the detection capability and provide tools to help prevent the introduction of invasive Phytophthora species.

Synchronized navigation of current and prior studies using image registration improves radiologist's efficiency.

PURPOSE: The purpose of this study was to investigate how the use of multi-modal rigid image registration integrated within a standard picture archiving and communication system affects the efficiency of a radiologist while performing routine interpretations of cases including prior examinations. METHODS: Six radiologists were recruited to read a set of cases (either 16 neuroradiology or 14 musculoskeletal cases) during two crossover reading sessions. Each radiologist read each case twice, one time with synchronized navigation, which enables spatial synchronization across examinations from different study dates, and one time without. Efficiency was evaluated based upon time to read a case and amount of scrolling while browsing a case using Wilcoxon signed rank test. RESULTS: Significant improvements in efficiency were found considering either all radiologists simultaneously, the two sections separately and the majority of individual radiologists for time to read and for amount of scrolling. The relative improvement for each individual radiologist ranged from 4 to 32% for time to read and from 14 to 38% for amount of scrolling. CONCLUSION: Image registration providing synchronized navigation across examinations from different study dates provides a tool that enables radiologists to work more efficiently while reading cases with one or more prior examinations.

Development of Rapid Isothermal Amplification Assays for Detection of Phytophthora spp. in Plant Tissue.

Several isothermal amplification techniques recently have been developed that are tolerant of inhibitors present in many plant extracts, which can reduce the need for obtaining purified DNA for running diagnostic assays. One such commercially available technique that has similarities with real-time polymerase chain reaction (PCR) for designing primers and a labeled probe is recombinase polymerase amplification (RPA). This technology was used to develop two simple and rapid approaches for detection of Phytophthora spp.: one genus-specific assay multiplexed with a plant internal control and the other species-specific assays for Phytophthora ramorum and P. kernoviae. All assays were tested for sensitivity (ranging from 3 ng to 1 fg of DNA) and specificity using DNA extracted from more than 136 Phytophthora taxa, 21 Pythium spp., 1 Phytopythium sp., and a wide range of plant species. The lower limit of linear detection using purified DNA was 200 to 300 fg of DNA in all pathogen RPA assays. Six different extraction buffers were tested for use during plant tissue maceration and the assays were validated in the field by collecting 222 symptomatic plant samples from over 50 different hosts. Only 56 samples were culture positive for Phytophthora spp. whereas 91 were positive using the Phytophthora genus-specific RPA test and a TaqMan real-time PCR assay. A technique for the generation of sequencing templates from positive RPA amplifications to confirm species identification was also developed. These RPA assays have added benefits over traditional technologies because they are rapid (results can be obtained in as little as 15 min), do not require DNA extraction or extensive training to complete, use less expensive portable equipment than PCR-based assays, and are significantly more specific than current immunologically based methods. This should provide a rapid, field-deployable capability for pathogen detection that will facilitate point-of-sample collection processing, thereby reducing the time necessary for accurate diagnostics and making management decisions.

Development of a multiplex assay for genus- and species-specific detection of Phytophthora based on differences in mitochondrial gene order.

A molecular diagnostic assay for Phytophthora spp. that is specific, sensitive, has both genus- and species-specific detection capabilities multiplexed, and can be used to systematically develop markers for detection of a wide range of species would facilitate research and regulatory efforts. To address this need, a marker system was developed based on the high copy sequences of the mitochondrial DNA utilizing gene orders that were highly conserved in the genus Phytophthora but different in the related genus Pythium and plants to reduce the importance of highly controlled annealing temperatures for specificity. An amplification primer pair designed from conserved regions of the atp9 and nad9 genes produced an amplicon of ≈340 bp specific for the Phytophthora spp. tested. The TaqMan probe for the genus-specific Phytophthora test was designed from a conserved portion of the atp9 gene whereas variable intergenic spacer sequences were used for designing the species-specific TaqMan probes. Specific probes were developed for 13 species and the P. citricola species complex. In silico analysis suggests that species-specific probes could be developed for at least 70 additional described and provisional species; the use of locked nucleic acids in TaqMan probes should expand this list. A second locus spanning three tRNAs (trnM-trnP-trnM) was also evaluated for genus-specific detection capabilities. At 206 bp, it was not as useful for systematic development of a broad range of species-specific probes as the larger 340-bp amplicon. All markers were validated against a test panel that included 87 Phytophthora spp., 14 provisional Phytophthora spp., 29 Pythium spp., 1 Phytopythium sp., and 39 plant species. Species-specific probes were validated further against a range of geographically diverse isolates to ensure uniformity of detection at an intraspecific level, as well as with other species having high levels of sequence similarity to ensure specificity. Both diagnostic assays were also validated against 130 environmental samples from a range of hosts. The only limitation observed was that primers for the 340 bp atp9-nad9 locus did not amplify Phytophthora bisheria or P. frigida. The identification of species present in a sample can be determined without the need for culturing by sequencing the genus-specific amplicon and comparing that with a reference sequence database of known Phytophthora spp.

A combined mitochondrial and nuclear multilocus phylogeny of the genus Phytophthora.

The most recent phylogenetic analysis of the genus Phytophthora was completed in 2008 (Blair et al., 2008) and utilized 8.1 kb of sequence data from seven nuclear loci. Given the large number of species that have recently been described, this study was undertaken to broaden the available information on the phylogeny of the genus. A total of 166 isolates representing 92 recognized species and 17 provisional species were analyzed, including many of the same isolates used in the nuclear multilocus study of Blair et al. (2008). Four mitochondrial genes (cox2, nad9, rps10 and secY) were sequenced with a total of 2373 bp used in the analysis; the species relationships recovered with mitochondrial data were largely consistent with those observed previously in the nuclear analysis. Combining the new mitochondrial data with the nuclear data from Blair et al. (2008) generated a dataset of 10,828 bp representing 11 loci, however resolution of basal clade relationships was still low. We therefore implemented a modified multispecies coalescent approach with a subset of the data, and recovered increased resolution and moderate to high support for clade relationships. A more detailed analysis of species from clades 2 and 8 identified an additional seven phylogenetic lineages that warrant further investigation to determine if they represent distinct species. As has been reported in other phylogenetic studies of the genus, there was no consistent correlation between phylogenetic relatedness and morphological features or ecology.

Phytophthora database 2.0: update and future direction.

The online community resource Phytophthora database (PD) was developed to support accurate and rapid identification of Phytophthora and to help characterize and catalog the diversity and evolutionary relationships within the genus. Since its release in 2008, the sequence database has grown to cover 1 to 12 loci for ≈2,600 isolates (representing 138 described and provisional species). Sequences of multiple mitochondrial loci were added to complement nuclear loci-based phylogenetic analyses and diagnostic tool development. Key characteristics of most newly described and provisional species have been summarized. Other additions to improve the PD functionality include: (i) geographic information system tools that enable users to visualize the geographic origins of chosen isolates on a global-scale map, (ii) a tool for comparing genetic similarity between isolates via microsatellite markers to support population genetic studies, (iii) a comprehensive review of molecular diagnostics tools and relevant references, (iv) sequence alignments used to develop polymerase chain reaction-based diagnostics tools to support their utilization and new diagnostic tool development, and (v) an online community forum for sharing and preserving experience and knowledge accumulated in the global Phytophthora community. Here we present how these improvements can support users and discuss the PD's future direction.

Analyses of the population structure in a global collection of Phytophthora nicotianae isolates inferred from mitochondrial and nuclear DNA sequences.

Genetic variation within the heterothallic cosmopolitan plant pathogen Phytophthora nicotianae was determined in 96 isolates from a wide range of hosts and geographic locations by characterizing four mitochondrial (10% of the genome) and three nuclear loci. In all, 52 single-nucleotide polymorphisms (SNPs) (an average of 1 every 58 bp) and 313 sites with gaps representing 5,450 bases enabled the identification of 50 different multilocus mitochondrial haplotypes. Similarly, 24 SNPs (an average of 1 every 69 bp), with heterozygosity observed at each locus, were observed in three nuclear regions (hyp, scp, and ß-tub) differentiating 40 multilocus nuclear genotypes. Both mitochondrial and nuclear markers revealed a high level of dispersal of isolates and an inconsistent geographic structuring of populations. However, a specific association was observed for host of origin and genetic grouping with both nuclear and mitochondrial sequences. In particular, the majority of citrus isolates from Italy, California, Florida, Syria, Albania, and the Philippines clustered in the same mitochondrial group and shared at least one nuclear allele. A similar association was also observed for isolates recovered from Nicotiana and Solanum spp. The present study suggests an important role of nursery populations in increasing genetic recombination within the species and the existence of extensive phenomena of migration of isolates that have been likely spread worldwide with infected plant material.

Small RNA profiling reveals phosphorus deficiency as a contributing factor in symptom expression for citrus huanglongbing disease.

Huanglongbing (HLB) is a devastating citrus disease that is associated with bacteria of the genus 'Candidatus Liberibacter' (Ca. L.). Powerful diagnostic tools and management strategies are desired to control HLB. Host small RNAs (sRNA) play a vital role in regulating host responses to pathogen infection and are used as early diagnostic markers for many human diseases, including cancers. To determine whether citrus sRNAs regulate host responses to HLB, sRNAs were profiled from Citrus sinensis 10 and 14 weeks post grafting with Ca. L. asiaticus (Las)-positive or healthy tissue. Ten new microRNAs (miRNAs), 76 conserved miRNAs, and many small interfering RNAs (siRNAs) were discovered. Several miRNAs and siRNAs were highly induced by Las infection, and can be potentially developed into early diagnosis markers of HLB. miR399, which is induced by phosphorus starvation in other plant species, was induced specifically by infection of Las but not Spiroplasma citri that causes citrus stubborn-a disease with symptoms similar to HLB. We found a 35% reduction of phosphorus in Las-positive citrus trees compared to healthy trees. Applying phosphorus oxyanion solutions to HLB-positive sweet orange trees reduced HLB symptom severity and significantly improved fruit production during a 3-year field trial in south-west Florida. Our molecular, physiological, and field data suggest that phosphorus deficiency is linked to HLB disease symptomology.

Membrane-based oligonucleotide array developed from multiple markers for the detection of many Phytophthora species.

Most Phytophthora spp. are destructive plant pathogens; therefore, effective monitoring and accurate early detection are important means of preventing potential epidemics and outbreaks of diseases. In the current study, a membrane-based oligonucleotide array was developed that can detect Phytophthora spp. reliably using three DNA regions; namely, the internal transcribed spacer (ITS), the 5' end of cytochrome c oxidase 1 gene (cox1), and the intergenic region between cytochrome c oxidase 2 gene (cox2) and cox1 (cox2-1 spacer). Each sequence data set contained ≈250 sequences representing 98 described and 15 undescribed species of Phytophthora. The array was validated with 143 pure cultures and 35 field samples. Together, nonrejected oligonucleotides from all three markers have the ability to reliably detect 82 described and 8 undescribed Phytophthora spp., including several quarantine or regulated pathogens such as Phytophthora ramorum. Our results showed that a DNA array containing signature oligonucleotides designed from multiple genomic regions provided robustness and redundancy for the detection and differentiation of closely related taxon groups. This array has the potential to be used as a routine diagnostic tool for Phytophthora spp. from complex environmental samples without the need for extensive growth of cultures.

Species tree estimation for the late blight pathogen, Phytophthora infestans, and close relatives.

To better understand the evolutionary history of a group of organisms, an accurate estimate of the species phylogeny must be known. Traditionally, gene trees have served as a proxy for the species tree, although it was acknowledged early on that these trees represented different evolutionary processes. Discordances among gene trees and between the gene trees and the species tree are also expected in closely related species that have rapidly diverged, due to processes such as the incomplete sorting of ancestral polymorphisms. Recently, methods have been developed for the explicit estimation of species trees, using information from multilocus gene trees while accommodating heterogeneity among them. Here we have used three distinct approaches to estimate the species tree for five Phytophthora pathogens, including P. infestans, the causal agent of late blight disease in potato and tomato. Our concatenation-based "supergene" approach was unable to resolve relationships even with data from both the nuclear and mitochondrial genomes, and from multiple isolates per species. Our multispecies coalescent approach using both Bayesian and maximum likelihood methods was able to estimate a moderately supported species tree showing a close relationship among P. infestans, P. andina, and P. ipomoeae. The topology of the species tree was also identical to the dominant phylogenetic history estimated in our third approach, Bayesian concordance analysis. Our results support previous suggestions that P. andina is a hybrid species, with P. infestans representing one parental lineage. The other parental lineage is not known, but represents an independent evolutionary lineage more closely related to P. ipomoeae. While all five species likely originated in the New World, further study is needed to determine when and under what conditions this hybridization event may have occurred.

DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer.

Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes.

The receptor-like kinase SERK3/BAK1 is required for basal resistance against the late blight pathogen phytophthora infestans in Nicotiana benthamiana.

BACKGROUND: The filamentous oomycete plant pathogen Phytophthora infestans causes late blight, an economically important disease, on members of the nightshade family (Solanaceae), such as the crop plants potato and tomato. The related plant Nicotiana benthamiana is a model system to study plant-pathogen interactions, and the susceptibility of N. benthamiana to Phytophthora species varies from susceptible to resistant. Little is known about the extent to which plant basal immunity, mediated by membrane receptors that recognise conserved pathogen-associated molecular patterns (PAMPs), contributes to P. infestans resistance. PRINCIPAL FINDINGS: We found that different species of Phytophthora have varying degrees of virulence on N. benthamiana ranging from avirulence (incompatible interaction) to moderate virulence through to full aggressiveness. The leucine-rich repeat receptor-like kinase (LRR-RLK) BAK1/SERK3 is a major modulator of PAMP-triggered immunity (PTI) in Arabidopsis thaliana and N. benthamiana. We cloned two NbSerk3 homologs, NbSerk3A and NbSerk3B, from N. benthamiana based on sequence similarity to the A. thaliana gene. N. benthamiana plants silenced for NbSerk3 showed markedly enhanced susceptibility to P. infestans infection but were not altered in resistance to Phytophthora mirabilis, a sister species of P. infestans that specializes on a different host plant. Furthermore, silencing of NbSerk3 reduced the cell death response triggered by the INF1, a secreted P. infestans protein with features of PAMPs. CONCLUSIONS/SIGNIFICANCE: We demonstrated that N. benthamiana NbSERK3 significantly contributes to resistance to P. infestans and regulates the immune responses triggered by the P. infestans PAMP protein INF1. In the future, the identification of novel surface receptors that associate with NbSERK3A and/or NbSERK3B should lead to the identification of new receptors that mediate recognition of oomycete PAMPs, such as INF1.

Phytophthora-ID.org: A Sequence-Based Phytophthora Identification Tool.

Contemporary species identification relies strongly on sequence-based identification, yet resources for identification of many fungal and oomycete pathogens are rare. We developed two web-based, searchable databases for rapid identification of Phytophthora spp. based on sequencing of the internal transcribed spacer (ITS) or the cytochrome oxidase (cox) 1 and 2 spacer region, followed by BLAST searching the databases. Both databases are highly selective. For ITS, only sequences associated with published Phytophthora spp. descriptions or classic Phytophthora phylogenetics references are included. For the cox spacer region, only data obtained by resequencing select isolates reported in published work were included. Novel taxa tentatively named are selectively included in the database and labeled as Phytophthora taxon "X"; as in, for example, P. taxon "asparagi". The database was validated with 700 Phytophthora isolates collected from nursery environments during 2006 to 2009. This resource, found at www.Phytophthora-ID.org , is a robust and validated tool for molecular identification of Phytophthora spp. and is regularly being updated.

The promise and pitfalls of sequence-based identification of plant-pathogenic fungi and oomycetes.

Sequences of selected marker loci have been widely used for the identification of specific pathogens and the development of sequence-based diagnostic methods. Although such approaches offer several advantages over traditional culture-based methods for pathogen diagnosis and identification, they have their own pitfalls. These include erroneous and incomplete data in reference databases, poor or oversimplified interpretation of search results, and problems associated with defining species boundaries. In this letter, we outline the potential benefits and drawbacks of using sequence data for identification and taxonomic deduction of plant-pathogenic fungi and oomycetes, using phytophthora as a primary example. We also discuss potential remedies for these pitfalls and address why coordinated community efforts are essential to make such remedies more efficient and robust.

Viability of Oomycete Propagules Following Ingestion and Excretion by Fungus Gnats, Shore Flies, and Snails.

Sporangia of Phytophthora capsici and P. nicotianae, as well as hyphal swellings of Pythium splendens, P. sylvaticum, and P. ultimum, were ingested by adult shore flies but none were viable after passing through the digestive tract. Oospores of Pythium aphanidermatum retained their viability following ingestion by adult shore flies. Larval stages of fungus gnats and shore flies ingested sporangia of Phytophthora capsici, P. nicotianae, and P. ramorum, but they were not viable upon excretion. In contrast, hyphal swellings of Pythium splendens, P. sylvaticum, and P. ultimum, chlamydospores of Phytophthora ramorum, and oospores of Pythium aphanidermatum, retained their viability after passage through the digestive tract of these larvae. Snails were capable of ingesting and excreting viable sporangia and chlamydospores of P. ramorum, which upon excretion infected detached leaves. Although the impact of larvae and snails in the rapid dissemination of pathogen propagules is unknown, this work does highlight the possibility that some often-ignored animal-fungus interactions should be considered in long-range dispersal of pathogen propagules via food webs.

Phytophthora bisheria sp. nov., a new species identified in isolates from the Rosaceous raspberry, rose and strawberry in three continents.

A homothallic semipapillate slow growing Phytophthora species associated with root rot of strawberries from greenhouse-grown plants in North Carolina, USA, root rot of roses in the Netherlands, and root rot of raspberry in Knoxfield, Australia, was identified. The main character of this organism is the production of paragynous antheridia with broad attachment to the oogonial wall. The morphology of the pathogen does not match that of any of the more than 85 described Phytophthora species. Phylogenetic analysis based on sequences of the internal transcribed spacer rDNA region (ITS1-5.8S-ITS2) of this taxon and those from other Phytophthora species from GenBank supports the conclusion that this organism is an unreported new species. In the phylogenetic tree with other reported Phytophthora species at the GenBank, the new species is more closely related to others in ITS clade 2 comprising semipapillate taxa including P. botryosa, P. citrophthora, P. colocasiae, P. meadii, P. citricola, P. inflata, P.tropicalis, P. capsici, Phytophthora sp. 'glovera' and P. multivesiculata. The most closely related species is P. multivesiculata isolated from Cymbidium orchid in the Netherlands. In this paper we describe the morphological characteristics and the phylogenetic relationships that support the description of this taxon as a new species Phytophthora bisheria sp. nov.

A multi-locus phylogeny for Phytophthora utilizing markers derived from complete genome sequences.

Phytophthora species are devastating plant pathogens in both agricultural and natural environments. Due to their significant economic and environmental impact, there has been increasing interest in Phytophthora genetics and genomics, culminating in the recent release of three complete genome sequences (P. ramorum, P. sojae, and P. infestans). In this study, genome and other large sequence databases were used to identify over 225 potential genetic markers for phylogenetic analyses. Here, we present a genus-wide phylogeny for 82 Phytophthora species using seven of the most informative loci (approximately 8700 nucleotide sites). Our results support the division of the genus into 10 well-supported clades. The relationships among these clades were rigorously evaluated using a number of phylogenetic methods. This is the most comprehensive study of Phytophthora relationships to date, and many newly discovered species have been included. A more resolved phylogeny of Phytophthora species will allow for better interpretations of the overall evolutionary history of the genus.

Phytophthora Database: A Forensic Database Supporting the Identification and Monitoring of Phytophthora.

Phytophthora spp. represent a serious threat to agricultural and ecological systems. Many novel Phytophthora spp. have been reported in recent years, which is indicative of our limited understanding of the ecology and diversity of Phytophthora spp. in nature. Systematic cataloging of genotypic and phenotypic information on isolates of previously described species serves as a baseline for identification, classification, and risk assessment of new Phytophthora isolates. The Phytophthora Database (PD) was established to catalog such data in a web-accessible and searchable format. To support the identification of new Phytophthora isolates via comparison of their sequences at one or more loci with the corresponding sequences derived from the isolates archived in the PD, we generated and deposited sequence data from more than 1,500 isolates representing the known diversity in the genus. Data search and analysis tools in the PD include BLAST, Phyloviewer (a program for building phylogenetic trees using sequences of selected isolates), and Virtual Gel (a program for generating expected restriction patterns for given sequences). The PD also provides a customized means of storing and sharing data via the web. The PD serves as a model that easily can be adopted to develop databases for other important pathogen groups.