Development and application of a PCR-based 'molecular tool box' for the identification of Phytophthora species damaging forests and natural ecosystems

A PCR-based 'molecular tool box', based on a region of the ras-related protein gene Ypt1, was developed for the identification of 15 Phytophthora species that damage forests and trees: P. cactorum, P. cambivora, P. cinnamomi, P. citricola, P. europaea, P. inundata, P. lateralis, P. megasperma, P. nemorosa, P. kernoviae, P. pseudosyringae, P. psychrophila, P. quercina, P. ramorum and P. ilicis. Most primers proved highly specific in BLAST analyses and in tests with DNA from 72 isolates of 35 species of Phytophthora and nine species representative of Pythium. Exceptions were primers designed for P. cactorum and P. ilicis, which cross-reacted with P. idaei and P. nemorosa, respectively. Amplification with Phytophthora-genus-specific primers before amplification with the various species-specific primers (nested PCR) increased the sensitivity of detection over amplification with species-specific primers only: detection limits ranged between 100 and 10 pg target DNA mu L-1 in the latter, compared with 100 fg mu L-1 in nested PCR. Using existing methods for rapid extraction and purification of DNA, single-round amplification was appropriate for detection of target Phytophthora species in leaves, but nested PCR was required for soil and water samples. The quarantine pathogens P. ramorum and P. kernoviae were detected in a number of naturally infected leaves collected in England and Wales, whereas P. citricola was commonest in water and soil samples from natural Scottish ecosystems.