3.3.67
DEVELOPMENT OF SPECIES-SPECIFIC PRIMERS FOR IDENTIFICATION AND DETECTION OF PYTHIUM ACANTHICUM, P. DIMORPHUM, P. MYROTYLUM AND P. ROSTRATUM
PH WANG,YT WANG, CY CHUNG Department of Microbiology, Soochow University, Taipei, Taiwan, 11102, Peoples' Republic of China Background and objecties Pythium spp. include many important plant pathogens causing damping-off, root rot and replant diseases. We developed a highly specific method for the identification and detection of Pythium acanthicum, P. dimorphum, P. myriotylum and P. rostratum based on amplification of a fragment from the ITSI region by PCR. This was achieved by sequencing the internal transcribed spacer (ITS) I region of the nuclear ribosomal repeat unit of thirty five Pythium species (unpublished). The nucleotide sequence variation observed has provided informatin on interspecies variability within Pythium. The species-specific primer was designed from these sequences. Materials and methods DNA was extracted from mycelium using a method based on CTAB extraction [1]. Dichloromethane was substituted for chloroform in the extraction procedure. PCR mixtures (100 ~) contained genomic DNA (50 ng), PCR buffer (Boehringer Mannhein), 50 ~M of each dNTP, 0.1 ~M of each primer and 4 units Taq DNA polymerase. Reactions were subjected to denaturation at 94 0C, 2 mm for the first cycle and 20 sec for subsequent 39 cycles, 20 sec at selected temperature for primer annealing, 3 sec at 72 0C for extension, and a final cycle of 5 sec at 72 0C. The reaction took about 150 mm. Ten ~l of the PCR products were checked on agarose gels (1.2 %, wlv) by ethidium bromide staining (0.5 ~gIml). Results and conclusions Having established the intra-specific divergence and inter-specific homology levels in ITSI among 35 species of Pythium. PCR primers of Pad, Pdil, Pmyl, and Prol were designed specifically for Pythium acanthicum, P. dimorphum, P. myriotylum and P. rostratum. Experiments with annealing temperatures ranging from 54 ~ to 68 0C revealed that as the temperature of annealing rose, the non-specific back ground diminished without adversely affecting the amplification of the expected fragment, so PCR amplification experiments were conducted at higher, more stringent, temperatures. Thus, the annealing temperature for primer pairs Pacl/1T52, Pdil/1T54, PmylIlTS2, and ProIIITS2 were selected as 68, 56, 57 and 68 0C, respectively. A portion of the ITS I region could be amplified from their genomic DNA when specific primer was used with 1T52 or 1T54, but did not produce products with DNA from any other 34 Pythium species. Amplification of the P. myriotylum product by PCR from 12.5 fg of fungal DNA exemplified the sensitivity of the test, which is of particular importance with low level infections. References I Doyle, J J; Doyle, J L (1990) Focus. 12,13. 2 White, T J; Bruns, T; Lee, S; Taylor, J (1990) In: PCR Protocols, A Guide to Methods and ApplicaUons, Innis, M A; Gelfand, K H; Sninsky, J J; White, T J (eds), San Diego: Academic Press, 315-322.