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Title: Validation of a TaqMan diagnostic assay for the systematic development of Phytophthora genus and species specific markers

item Miles, Timothy
item Martin, Frank
item BILODEAU, GUILLAUME - Canadian Food Inspection Agency
item COFFEY, MICHAEL - University Of California

Submitted to: American Phytopathological Society Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 5/12/2014
Publication Date: N/A
Citation: N/A

Interpretive Summary: A comprehensive hierarchal approach to detect several non-native Phytophthora spp. has been developed. These plant pathogens have the potential to cause damage to agriculture and native ecosystems. These assays, which were developed, were tested for sensitivity and specificity, and were validated against the purified DNA of over 96 valid species and 14 provisional species.

Technical Abstract: The genus Phytophthora contains many species that are not native to the USA and have the potential to cause significant damage to agriculture and native ecosystems. A genus and species-specific diagnostic assay was developed based on mitochondrial gene order differences that allowed for the systematic development of species-specific TaqMan probes for their identification. Previous research validated the marker system for 14 species. Here we will discuss over 130 unique probes developed in silico for additional Phytophthora spp., with the focus on non-native species. Additional species-specific TaqMan probes (including P. austrocedrae, P. cajani, P. parvispora, P. melonis, P. multivesiculata, P. pinifolia, P. pistaciae, P. primulae, P. quercina, and P. vignae) were validated with 96 valid Phytophthora spp. and 14 provisional species. All probes were found to be species-specific and could be multiplexed with a genus-specific probe and a plant internal control (IC). The lower limit of linear detection using purified DNA was 100 fg in all assays. An IC was developed to check for PCR inhibitors common in soil DNA extractions. Based on the interspecific sequence variation observed it should be possible to develop probes capable of distinguishing >85% of the Phytophthora spp. investigated; we are currently in the process of validating an additional 18 probes. This system represents a comprehensive, hierarchal approach to increase detection capability and reduce the introduction of these non-native Phytophthora species.