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Title: Systematic development of Phytophthora species-specific mitochondrial diagnostic markers for economically important members of the genus

item MILES, TIM - California State University
item Martin, Frank
item ROBIDEAU, GREGG - Canadian Food Inspection Agency
item BILODEAU, GUILLAUME - Canadian Food Inspection Agency
item COFFEY, MIKE - University Of California

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/27/2017
Publication Date: 3/1/2017
Citation: Miles, T.D., Martin, F.N., Robideau, G., Bilodeau, G., Coffey, M.D. 2017. Systematic development of Phytophthora species-specific mitochondrial diagnostic markers for economically important members of the genus. Plant Disease. doi: 10.1094/PDIS-09-16-1224-RE.

Interpretive Summary: The genus Phytophthora contains many species that are important pathogens of economic crop plants and destructive of natural ecosystems. A number of species are invasive to North America, so having the ability to rapidly detect and identify them would benefit regulatory and research labs, thereby preventing their introduction. A highly specific detection technique was developed that is capable of pathogen detection at the genus as well as species level; this manuscript describes the validation of markers for 32 species, many of them for detection of species that are not native to the United States.

Technical Abstract: The genus Phytophthora contains many invasive species to the USA that have the potential to cause significant damage to agriculture and native ecosystems. A genus and species-specific diagnostic assay was previously reported based on mitochondrial gene order differences that allowed for the systematic development of 14 species-specific TaqMan probes for pathogen detection (Bilodeau et al., 2014). In this study an additional 32 species specific TaqMan probes for detection of primarily invasive species have been validated against 145 Phytophthora taxa as well as a range of Pythium and plant DNA samples. All validated probes were found to be species-specific and could be multiplexed with a genus-specific probe. The lower limit of linear detection using purified genomic DNA ranged from 1-100 fg in all assays. In addition, 124 unique TaqMan probes for Phytophthora spp. developed in silico are presented which, if testing confirms they are species specific, will provide diagnostic capabilities for approximately 89% of the genus. To enhance sensitivity of detection for several species that contained a SNP in the reverse primer, a second primer was developed that is added in a small amount to the master mix. Furthermore, a PCR-RFLP system was developed which could be used to identify species when multiple species are present in a sample without requiring cloning or sequencing. Several experiments were also conducted to compare various qPCR thermal cyclers and technology transfer experiments with another research laboratory to identify possible limitations when the assays are used on a range of equipment in different labs. This system represents a comprehensive, hierarchal approach to increase the detection capability and provide tools to help prevent the introduction of invasive Phytophthora species.