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Poster: Diseases of Plants: Disease Detection & Diagnosis


Utilizing mitochondrial loci to develop TaqMan and recombinase polymerase amplification assays for the genus Pythium
T. MILES (1), A. Burkhardt (2), F. Martin (2) (1) California State University Monterey Bay, U.S.A.; (2) USDA-ARS, U.S.A.

Mitochondrial marker systems have been utilized in other oomycete systems to develop novel molecular diagnostic tools in both laboratory and field settings. For Pythium a mitochondrial gene order difference was identified that allowed for genus specific amplification of more than 75 species across a wide range of clades within the genus. PCR results for this locus tested negative on Phytophthora and several Phytopythium spp. A TaqMan diagnostic assay was developed using a FAM labeled probe and validated on environmental samples from over 40 host plants (nursery and field samples). A sequencing protocol was also developed to confirm a positive, however, if multiple species are present amplicon cloning will be needed to get clean data. In addition to the TaqMan assay an assay using the isothermal technology known as recombinase polymerase amplification (RPA) was developed. Using the same purified and field samples the RPA technique was effective at detecting Pythium species in less than 20 minutes without a laborious DNA extraction. The use of this marker system will not only help confirm Pythium infection in plants, it may also be useful in environmental samples and it will complement the other marker system developed for Phytophthora. Sequencing of additional species is in progress to evaluate species specific detection/identification capabilities. Due to the amplicon size, this locus may also be useful in future metagenomic studies of Pythium diversity.