Diagnosis of P. ramorum was initially attempted via direct isolation from symptomatic plant tissue. However, Phytophthora species are often difficult to culture from plants, which can lead to false-negative isolations and misdiagnosis of infected plant material. The detection and extent of P. ramorum colonization in various host plant tissues was estimated with real-time quantitative polymerase chain reaction (PCR) using TaqMan chemistry. Pathogen DNA was amplified by species-specific primers and a fluorogenic TaqMan probe based on a 74 base pair sequence of the internal transcribed spacer (ITS2) of nuclear ribosomal DNA (rDNA). To check for specificity, the primers (Pram5 and Pram6) and probe (Pram7) were applied to DNA dilutions of 10 P. ramorum isolates and 14 other Phytophthora species. This assay was positive for all dilutions of P. ramorum and P. lateralis, and negative for all other species tested with quantification of P. ramorum cells linear over a range of 4 orders of magnitude.
Recent observations indicate that another new and undescribed Phytophthora species (temporarily referred to as P. ilicis-like) is often co-isolated with P. ramorum from infected plant material. To help discern the distribution of this new species and its relationship with P. ramorum within host plants, another set of species-specific TaqMan primers and probe were developed for P. ilicis-like. This protocol amplifies a 64 base pair sequence of the ITS2 region of the rDNA. To check for specificity, the primers (Pi-like1 and Pi-like2) and probe (Pi-like3) were applied to DNA dilutions of 10 P. ilicis-like isolates and 14 other Phytophthora species.
Using this technique with a multiplex approach, both P. ramorum and P. ilicis-like DNA can be simultaneously amplified in the same reaction tube, but individually detected by species-specific primers and TaqMan probes labeled with different fluorescent dyes. The total assay can be completed in less than 3 hours without the need to run additional rounds of PCR, agarose gels, or sequencing reactions. The TaqMan probes and primer sets designed in this study can be used as a rapid screening tool for the detection and quantification of P. ramorum and P. ilicis-like DNA in pure-culture and environmental plant extracts without prior isolation and characterization of the organisms by traditional microbiological methods.
