Molecular Markers for Identification of P. ramorum and other Phytophthora spp. from Diseased Tissue

Frank N. Martin, USDA-ARS, 1636 East Alisal St, Salinas, CA, 93905; (831) 755-2873; fmartin@pw.ars.usda.gov and P.W. Tooley, USDA-ARS, 1301 Ditto Ave, Ft. Detrick, MD, 21702

The use of molecular methods for detection of plant pathogens can reduce the time required to process samples as well as improve the accuracy of pathogen identification. Rather than focus on identifying a single species, methods that focus first on identification at a genus level followed by a nested species-specific detection could facilitate identification of a broader number of pathogens. For example, while P. ramorum is the causal agent of SOD, it is not uncommon to recover P. nemorosa or P. pseudosyringae from forest samples exhibiting symptoms similar to those caused by P. ramorum. The diversity of Phytophthora spp. recovered from nursery samples causing similar symptoms would be even greater. Having a marker system that would first accurately determine if a Phytophthora spp. was present and then allow identification to a species level would simplify sample processing as well as provide additional data on the diversity of Phytophthora spp. present on the hosts under investigation.

A Species-specific PCR detection system was developed based on amplification of the spacer region between the mitochondrially-encoded cox I and II genes of Phytophthora using genus-specific primers developed from conserved regions of the flanking genes (Martin, F.N., Tooley, P.W. and Blomquist, C., 2004, Phytopathology 94: 621-631). A two-step multiplex amplification procedure is used for determining if a Phytophthora sp. is present in symptomatic plant tissue and clarifying if it is P. ramorum, P. nemorosa or P. pseudosyringae. The first round multiplex amplification contains two primer pairs, one for amplification of plant sequences to serve as an internal control to ensure that extracted DNA is of sufficient quality to allow for PCR amplification and a second primer pair specific for amplification of sequences from Phytophthora spp. The products of the first round amplification are diluted and amplified with primer pairs nested within the genus-specific amplicon that are specific for either P. ramorum, P. nemorosa or P. pseudosyringae. Using purified pathogen DNA the limit of detection for P. ramorum using this marker system was approximately 2.0 fg of total DNA. More recent work with this marker system has modified it for use with real-time PCR and identified species-specific primers for 5 other Phytophthora spp. To facilitate the identification of species other than P. ramorum, P. nemorosa or P. pseudosyringae, several restriction enzymes were identified that digest the genus-specific amplicon and RFLP analysis can be used to differentiate most species.

An RFLP technique was developed for identification of unknown isolates. PCR primers spanning the mitochondrially-encoded cox I and II genes have been developed that are capable of amplifying most of the gene cluster in the genus Phytophthora (Phytopathology 94: 983-991). Digestion of the amplicons with restriction enzymes generated species-specific RFLP banding profiles that were effective for isolate classification to a species level.

Additional details on these marker systems can be found at http://pwa.ars.usda.gov/salinas/cipru/frank/phyto.htm

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