An official website of the United States government.
The ability to accurately and rapidly identify the causal agent of a disease is essential for implementing control and regulatory measures to prevent major crop disease epidemics. The destructive potential of Phytophthora diseases is well illustrated by late blight (caused by P. infestans) and sudden oak death (P. ramorum). Phytophthora also exhibits the high potential of emergence of new species and strains through hybridization. Considering that late blight and sudden oak death are unlikely to be the last major disease outbreaks of economic and/or environmental significance, as well as increasing concerns about agricultural bioterrorism, systematically cataloging the genetic and phenotypic diversity of Phytophthora in global agroecosystems warrants immediate attention.
<P>
To enhance the ability to detect, diagnose, monitor, and manage Phytophthora diseases, we propose to catalog genetic and phenotypic diversity of Phytophthora species in a database format that can be easily accessed, utilized, and compared by the global community of plant health professionals, and to provide an array of molecular diagnostic tools. This project will establish a baseline for monitoring the emergence of new/foreign pathogens and help us track the movement of Phytophthora via agricultural trades. Specifically, this collaborative project will accomplish the following three objectives:
<ul><LI>
The first is to establish a comprehensive phylogenetic framework for Phytophthora species. Given the vast diversity of Phytophthora in nature and the limited resolution of morphological traits for identification, the use of genetic markers to complement pathogen identification is critical for prompt implementation of suitable control and regulatory measures. Genus-wide phylogenetic analyses will be carried out using global Phytophthora strain collections. The resulting data will serve as an invaluable resource for monitoring the dynamics of Phytophthora species and their populations in the context of global agroecosystems.
<LI>Based on data from the phylogenetic analyses, we will also develop and test a number of molecular diagnostic tools/protocols for detecting and differentiating major Phytophthora pathogens at both the species and population levels. The sensitivity and specificity of these methods will be evaluated in multiple states.
<LI> To build a comprehensive genotype, phenotype, and specimen archive for Phytophthora, a relational internet database will be constructed. Phylogenetic data will be linked to the available phenotypes (morphology, virulence, mating type, host, fungicide resistance, geographic origin, etc.) of individual isolates in a format that can be easily accessed, analyzed, and visualized.
NON-TECHNICAL SUMMARY: Crop loss from plant disease poses a serious threat to global food/fiber/feed security. In addition to natural outbreak of various diseases, the threat to US agriculture from the deliberate release of pathogens should not be underestimated. High virulence of Phytophthora species and their ability to spread rapidly throughout the world establishes Phytophthora as one of the most important groups of plant pathogens. Recent discoveries of inter-specific hybridization among Phytophthora spp. in nature, which could yield novel pathogens and play an important role in the exploitation of new host plant species, further underscore the threat posed by Phytophthora species. Toward the goal of enhancing our ability to detect, diagnose, monitor, and manage Phytophthora diseases, we propose to systematically catalog genotypic and phenotypic data of Phytophthora spp. in a database format that can be easily accessed and utilized by the global community of plant health professionals, and to develop and optimize molecular diagnostic tools for Phytophthora at both the species and population levels. The use of genotyping to identify strains and species of interest will greatly assist the study of newly isolated pathogens by researchers who have limited experience in morpho-taxonomy, or by regulatory agency scientists who must quickly assess the threat of a new isolate for rapid deployment of containment and/or eradication measures.
<P>
APPROACH: This project will be a team effort of the World Phytophthora Collection (WPC) at UC-Riverside, Penn State, Pennsylvania Department of Agriculture (PDA), NCSU, and the USDA-ARS NCAUR and CIPRU. Genomic DNA will be extracted from Phytophthora isolates stored at the WPC and the PDA, and sent to Penn State. At Penn State, PCR and DNA sequence reactions will be performed. Standard PCR protocols will be used to amplify at least five different gene regions, the ITS region and 5 end of the 28S nuclear rRNA (D1 and D2 domains), at least three phylogenetically informative protein-coding genes (e.g., translation elongation factor 1-alpha and beta-tubulin) and/or intergenic regions, and the mitochondrially encoded cox II gene. Primers will be designed utilizing the three Phytophthora genomes being sequenced, including P. infestans, P. sojae and P. ramorum, which represent three divergent clades in the genus Phytophthora. Markers will be tested on a phylogenetically diverse set of Phytophthora species to ensure their utility. Clean DNA sequence reactions will then be sent to the USDA NCAUR, where they will be processed on a capillary DNA sequencer at that facility. The resulting DNA sequence files will be uploaded via FTP and accessed back at Penn State for databasing. In the database, genotypic data will serve as the main anchor for linking available phenotypic information for Phytophthora at both the species and isolate levels. Basic search functions will allow users to search the database using species name, genetic marker, or host name, and search capabilities based on specific morphological keys will be added. At the species level, users will be able to access varied information useful for dealing with selected species: (i) taxonomic information, (ii) general biology (disease cycle, isolation and culture media, current control strategies, images of disease symptoms and the various morphological keys used to identify the pathogen and the disease, etc.), (iii) available molecular diagnostic tools, including protocols for amplifying markers for phylogenetic analysis and identification by PCR, including primer sequences, (iv) a map indicating which states/countries the species has been reported to exist, and (v) a literature reference guide. Genotype data archived in the database will be used to develop and evaluate molecular diagnostic tools/protocols for detecting and differentiating pathogens at both the species and population levels. Potential methods to be evaluated include conventional and real-time PCR, pathogen-specific macro/micro arrays for detecting and differentiating a mixture of strains, PCR coupled with ELISA, PCR-SSCP analysis, and PCR-RFLP approaches based on single nucleotide variation in different populations. The sensitivity and specificity of these methods will be evaluated at Penn State/PDA, UC-Riverside, NCSU, and the USDA-ARS CIPRU using reference cultures as well as Phytophthora samples taken from field surveys. Results and recommendations from these tests will be provided via the database.
<P>
PROGRESS: 2005/03 TO 2008/02<BR>
OUTPUTS: Due to their high virulence and ability to spread rapidly, Phytophthora is one of the most important groups of plant pathogens. To enhance our capability of rapid detection, diagnosis, and monitoring of Phytophthora species, we archived genotypic and phenotypic diversity of Phytophthora in a highly integrative cyberinfrastructure, named as the Phytophthora Database (PD; www.phytophthoradb.org). The PD can easily be accessed, searched, and updated and offers mechanisms for data sharing to facilitate collaboration. To characterize the evolutionary relationships among Phytophthora species, which are critical to support molecular diagnosis, informative molecular markers at multiple nuclear and mitochondrial loci were identified using the available genome sequences for P. infestans, P. ramorum, P. capsici, and P. sojae. We have sequenced up to eight nuclear loci of >1,500 isolates that represent most of the known species within Phytophthora (>90 species, including several novel species). In addition to those regions, four mitochondrially-encoded genes are currently being sequenced from selected isolates representing the known species to construct an evolutionary framework based on mitochondrial gene sequences. Sequence data from additional 1,000 isolates are currently being curated. In addition to systematically cataloging genotypic and phenotypic data on Phytophthora in a web-accessible format, the PD provides a number of data search, analysis and visualization tools to support identification, detection, and risk assessment of Phytophthora. The home page of the PD provides menus that lead users to: (i) an overview of the PD, including a guide on how to use it, (ii) current statistics on the stored data, (iii) databases, and (iv) data analysis tools. To support the identification of an unknown isolate by querying the sequence database using one or more of the marker sequences, a BLAST search tool was installed. Additional data search and analysis tools include 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 position of each species within a genus-wide phylogenetic tree is shown to illustrate its evolutionary context (i.e., relationships with other Phytophthora species). For many species, the following information is also available, including nomenclature, morphological and growth characteristics, hosts and disease symptoms, life cycle, control/management strategies, diagnostic methods, and selected references. <BR>PARTICIPANTS: Seogchan Kang, David Geiser and Izabela Makalowska at Penn State participated in the design and development of the Phytophthora Database, sequence data generation and analysis, and writing papers. Sook-Young Park and Ekaterina Nikolaeva, postdoctoral associates in Kang's lab, sequenced ITS regions of Phytophthora isolates from samples collected in nurseries and streams. Jaime Blair, a postdoctoral associate co-supervised by Kang and Geiser, developed a set of nuclear markers for genus-wide phylogenetic analyses and characterized selected strains representing know species diversity in the genus. Seong H. Kim at PA Department of Agriculture isolated Phytophthora from plant and stream samples and extracted genomic DNA from these cultures. Mike Coffey at UC-Riverside curated ~6,000 Phytophthora accessions, extracted genomic DNA from more than 3,000 accessions, sequenced their ITS region, and participated in analyzing phylogenetic data. Yong-Hwan Lee at Seoul National University and his students participated in the development of the Phytophthora Database. Frank Martin at USDA-ARS identified new mitochondrial genetic markers and characterized representative isolates using these markers. Kelly Ivors at NCSU generated genotypic data from selected species and prepared species information pages for several species. <BR>TARGET AUDIENCES: The use of genotyping for pathogen identification will greatly assist the study of newly isolated pathogens by researchers who have limited experience in taxonomy, or by regulatory agency scientists who must quickly assess the threat of a new isolate for rapid deployment of containment and/or eradication measures. Data and information from the project, which are archived in the PD, support research, extension, and education on Phytophthora diseases. Because the PD provides applied and molecular information on Phytophthora species, we feel the PD has more utility than GenBank. Besides, the data stored at the PD is provided by specialists actively working with Phytophthora, hence proper identification of reference cultures should be less prone to error. <BR><BR>
IMPACT: 2005/03 TO 2008/02<BR>
The Phytophthora Database (PD) serves as a model that can be easily adopted to develop databases for important pathogen groups, which are critical to adequately support efforts to study and manage pathogen threats. The rapid expansion of global commerce and human travel has greatly accelerated the introduction of non-indigenous pathogens. This expansion, coupled with global climate change, is reshaping North American pathogen communities. If history is a guide, these forces will lead to the appearance of new diseases and reemergence of quiescent ones. In addition to direct production losses and environmental damages from disease, plant pathogens can incur huge indirect costs due to trade disruption and restriction. Phytophthora species pose a grave threat to agricultural and ecological systems. The destructive potential of Phytophthora diseases is well illustrated by late blight (P. infestans), which was responsible for the Irish potato famine and has again become globally problematic due to the introduction of new, fungicide-resistant lineages of P. infestans. Sudden oak death (SOD) in the U.S. and diseases on ornamental plants in the U.S. and Europe, caused by P. ramorum, are examples of the threat to horticultural crops and forest ecosystems. Two other potentially destructive taxa not yet reported in the U.S. are P. kernoviae and P. alni. The likelihood that these invasive species will not be the last threat to U.S. forest and the nursery industry signifies the importance of continuously monitoring the diversity, distribution and dynamics of Phytophthora. Discoveries of inter-specific hybridization among Phytophthora species in nature, which could yield novel pathogens, also underscore the importance of this monitoring. Genotypic and phenotypic data of Phytophthora spp., which have been systematically cataloged in the PD, allow members of the global community of plant health professionals to easily access and utilize archived data to detect, diagnose, monitor, and manage Phytophthora diseases. A main contribution of this project is the establishment of a baseline for monitoring the emergence of new/foreign Phytophthora. In addition, the PD offers a new paradigm for dealing with other pathogen threats to agricultural and environmental systems.