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<OL> <LI> Develop tools for detection of intentionally or accidentally released high-threat plant pathogens utilizing real-time PCR-based detection systems specific to the biovar or race levels. <LI> Develop hyperspectral signatures for diseases caused by high-threat crop pathogens such that they might provide goals for instrument development to detect crop infestations from satellite platforms, and provide methods by which the large majority of agricultural lands can be frequently and accurately surveyed.<LI> Develop a database supporting accurate diagnosis and response strategies for confirmed and probable infestations by high threat crop pathogens. <LI> Integrate results of Objectives 1-3 into a comprehensive decision support system that will support the earliest possible detection, verification, and appropriate responses to the release of high threat pathogens into American agriculture.
NON-TECHNICAL SUMMARY: Recent incidents of terrorism have revealed that United States agriculture could become a target for bioterrorists. Further, we have poor capabilities to detect and respond to either intentional and accidental releases of high threat crop or forest pathogens. This project will develop a comprehensive system for detecting and responding to incidents of high-threat crop pathogen release.
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APPROACH: Because rapid detection can greatly minimize damage by high-risk pathogens and enhance the probabilities of achieving containment and eradication, the development of tools for rapidly and accurately detecting their presence and identity is an essential component of the proposed D.S.S. system. We propose to develop quick (1-2 hours), easy-to-operate, and highly accurate diagnostic tools for high-risk pathogens, which are based on PCR (presently deployed only for anthrax) and microarrays with pathogen-specific markers. Since plant pathologists can artificially test crops with pathogens, this would be a key place to start to secure spectral signatures of key crop pests. In a similar manner, later field trials would evaluate the ability to identify the presence of one key disease when the plant was infected with multiple pests. Similar experiments would be conducted with various cultivars (plant varieties) to see if horticultural differences might obscure the signatures detected. We are proposing to use the potato crop for initial studies, and the late blight disease as key crop pathogen, and early blight disease, insect damage, pesticides and potato cultivars as probable confusing issues. Other crop/pathogen systems will be evaluated in later years as funding is developed. One final element of remote detection is narrowing the area of the US that should be monitored. We anticipate the development of a key area list for each host crop. An expert system will weight multiple factors such as intensity of crop production, ports of entry, cardinal climate limitations, etc. to derive the key area list. During the year the number of these scanned will be further limited based on whether the local climate would support the development of infections. Typically, these would be derived from expert systems utilizing Doppler radar and/or weather station measurements of rain and temperature. The identity of a particular isolate at the species level is often not sufficient to predict its traits (virulence, host, chemical resistance, toxin production, etc.) because these traits are often variable within pathogen species. Since such information is critical to formulating control and regulatory measures for dealing with pathogens, we will generate and archive the information in a format that can be easily searched. Toward this goal, we propose to establish an internet-based database for high-risk pathogens. responders, and provide the latest information for effective eradication and containment of invasive or purposely released pathogens. BLASTN program will allow users to input a query sequence in FASTA format and receive the best matches in the database.
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PROGRESS: 2007/10 TO 2008/09 <BR>
OUTPUTS: During this period, we have focused on two areas, one of which is pathogenesis mechanisms of Rhodococcus fascians, a Gram-positive phytopathogenic bacterium that infects a wide range of monocotyledonous and dicotyledonous plants. The symptoms caused by R. fascians infection include leaf deformation, thickened hypocotyls, stunted growth, arrested root development, multi-shoot formation, and initiation of leafy galls. The isopentyl transferase (fas-1) gene of R. fascians is involved in the synthesis of a cytokinin-like product and is required for virulence, supporting the hypothesis that abnormal growth in infected hosts is caused by disruption of hormonal balance. A pea bioassay has been widely used in identifying virulent strains of R. fascians. However, different pea cultivars or even the same cultivar grown under different conditions varied in their susceptibility to infection, resulting in inconsistent results within and between different studies. To understand the basis of variability in symptom development, we investigated genetic and phenotypic differences among R. fascians strains collected by PDA from several species of symptomatic plants between 1984 and 2005. Amplification of the plasmid-borne fas-1 virulence gene by polymerase chain reaction (PCR) among randomly chosen single colonies of these R. fascians strains showed that many strains consisted of two populations of cells with or without the fas-1 gene. The number of cells carrying the gene was highly correlated to virulence. Inoculation of pea seedlings with several mixtures of fas-1 positive and negative cells that originated from same strains also demonstrated a strong correlation between the proportion of cells carrying the gene and the severity of disease symptoms. A protocol for detecting the fas-1 gene by Real time-PCR was developed to identify virulent strains of R. fascians. The other area of research is phenotypic and genotypic characterization of novel Phytophthora species identified among ~700 Phytophthora isolates collected during the 2006 P. ramorum survey. Because the sequences of internal transcribed spacer (ITS) regions of ribosomal RNA encoding genes are not sufficient for classification of new species, we sequenced an additional seven genetic markers in order to rigorously compare the evolutionary relationships between the putative new species and the known species. Additional research is being conducted on the use of endophytic Bacillus spp. bacteria that can stimulate host defenses against invasive pathogens. This research, conducted on cocoa trees has resulted in suppression of the invasive basidiomycete Moniliophthora spp. and Phytophthora under field conditions in Ecuador. <BR> PARTICIPANTS: Pennsylvania Dept. of Agriculture Plant Pathologists US. Department of Agriculture, Agricultural Research Service, Sustainable Perennial Crops Lab. Ecuador Dept. of Agriculture (INIAP, Pichilingue)<BR> TARGET AUDIENCES: State and Federal regulatory agencies Andean lowland farmers. <BR> PROJECT MODIFICATIONS: Work on invasive diseases of cacao has been undertaken following seed funding from USAID. This adds diseases that are having impact on the U.S. chocolate industry, and that follow similar entry and dispersal avenues as for U.S. crops. This modification adds INIAP in Ecuador, as a participant, and Andean region farmers as a target audience. <BR> <BR>
IMPACT: 2007/10 TO 2008/09<BR>
The research of Rhodococcus and Phytophthora species will allow state and national laboratories to accurately detect novel species of these genera that are important for crop biosecurity. The research on endophytes will allow suppression of important invasive diseases of cocoa trees without compromising the Agroforestry ecosystem that cocoa is produced in. A symposium is planned for January in Ecuador that will be an important step in technology transfer.