Detection and identification of Phytophthora alni

In 2004 Brasier et al. described new species--Phytophthora alni, which was especially aeggressive to alder. Now, this Phytophthora disease of alder is widely distributed in Europe as well as in Poland. In this research note we report on identification and detection of P. alni from water and soil samples using PCR method with species-specific primers. Dilution series of P. alni zoospore were used to test the potential sensitivity of the PCR detection methods. Zoospores of P. alni were produced by flooding of 1-week-old Frozen Pea Medium (FPM) cultures in Petri dishes with 30 ml distilled water. The dishes were incubated at 20 degrees C. After 5 days, sporangial production was checked using a binocular microscope and plates were placed at 4 degrees C for 1 h to enhance zoospore release. Zoospores were counted under the microscope using Burker's cabin. A dilution series of zoospores ranging from 5 to 5000 per 200 microl was prepared in autoclaved distilled water and in 1 g samples of autoclaved soil. DNA was extracted from artificially infected water and soil, and purified using the CleanUp Kit (A&A Biotechnology). Zoospores of P. alni in the water were detected by PCR in 5 x 10(3), 5 x 10(2), 5 x 10(1) concentrations. In case of detecting spores in the artificially infected soil it succeeded only for two highest concentrations, i.e. 5 x 10(3), 5 x 10(2) and only when the DNA was additionally purified.