Rapid diagnosis of pathogenic Phytophthora species in soil by real-time PCR

Real-time PCR assays based on the TaqMan system and using ITS sequences were developed for the identification of Phytophthora species, including P.cactorum, P.megasperma, P.plurivora, P.pseudosyringae and P.quercina, all of which are currently causing significant damage to roots of forest trees in both managed stands and natural ecosystems. Total genomic DNA was extracted from mycelia of aforementioned Phytophthora isolates. Species-specific primers for P.cactorum, P.megasperma, P.plurivora, P.pseudosyringae and P.quercina were designed based on ITS sequences of rDNA. The amplification efficiency of target DNA varied from 93.1% (P.pseudosyringae) to 106.8% (P.quercina). The limit of the detection was calculated as 100 - 1,000fg DNA, depending on the Phytophthora species. In mixed soil samples, all Phytophthora species were detected for Ct values shifted by 0.7 - 2.1 cycles. Based on these real-time PCR assays we were able to identify the five Phytophthora species. These techniques will be of value in the identification of these pathogens, which may cause up to 80 - 90% fine root loss in oak stands.