Molecular diagnostics of Phytophthora Spp. from natural ecosystems
Crop Improvement and Protection Research
2013 Annual Report
1a.Objectives (from AD-416):
To validate molecular diagnostic tools for identification of Phytophthora ramorum, the pathogen responsible for sudden oak death.
1b.Approach (from AD-416):
Two different molecular diagnostic techniques will be evaluated for their effectiveness in identifying Phytophthora species in infected plat tissue using samples collected from different regions of the USA. One procedure is a TaqMan real time PCR diagnostic marker that exhibits specificity at a genus level (it can identify if a Phytophthora spp. is present) in addition to a species level (different probes can differentiate P. ramorum or P. kernoviae from other species). The second procedure is for determination of mitochondrial haplotype of P. ramorum, which so far has been correlated with the nuclear genotype. The technique uses melt curve analysis to make the determination, which takes much less time than conventional techniques for genotype determinations.
This agreement was established in support of objective 2 of the parent project, the goal being to develop molecular diagnostic tools for the identificatioin of emerging diseases of vegetables and strawberry, and use these tools in the development of management strategies as alternatives to methyl bromide. The objective of the project is to optimize and fully validate a new molecular diagnostic technique for Phytophthora spp. that was developed as part of CRIS 5305-22000-12-06R, "Development of a systematic approach for marker selection in Phytophthora using mitochondrial genomic sequences." DNA from field samples collected as part of the state's ongoing survey for P. ramorum have been obtained from a collaborator at the California Department of Agriculture, Sacramento, from a diagnostician at Oregon State University who is managing the national survey of P. ramorum for the west coast, and a diagnostician at Penn State University who is managing the samples from the national survey of P. ramorum for the eastern part of the country. These samples were used to validate the diagnostic technique have undergone sequence analysis to confirm the identification of the species that are present and we are in currently writing the manuscript. A single tube PCR-melt curve analysis technique for haplotyping P. ramorum isolates was developed but not validated. An isothermal amplification technique was developed for rapid detection of the genus Phytophthora and is in the final stages of validation. A second isothermal amplification marker system for detection of P. ramorum was developed and is currently being validated.