Faculty of Agriculture, Gifu University, Gifu 501-1193, Japan

Background and objectives
In Pythium species, the use of a selective medium is well established for isolation from soil. However, if diagnosis of a specific species is necessary, the colonies that appear on the medium must be individually transferred to culture media for identification, because more than one species of Pythium usually persist in a soil. PCR for amplification of diagnostic molecular markers is useful for identification and detection of plant pathogens. Detection signals usually are recognized as bands of PCR products after electrophoresis, but the sensitivity is not so high. Thus, Southern hybridization must be done to obtain a higher sensitivity, and can increase specificity using a specific probe. Although nitrocellulose or nylon membranes are used for Southern hybridization, a well of a 96-well microplate instead of the membranes can be used to bind DNA on a solid phase, and the DNA is hybridized on the well with the probe [1]. This microplate hybridization method enables us to determine DNA concentration as an absorbance, as with ELISA. The authors developed a species-specific primer of Pythium ultimum and detected the fungus from single, naturally infected seedlings of sugar beet, cucumber and Chinese cabbage using PCR [2]. In this study, we report the detection of P. ultimum from soil using PCR microplate hybridization.

Materials and methods
For DNA extraction from soil, 0.1 g of soil was put into a 1.5-ml eppendorf tube with 0.2 g of 1-mm glass beads. The tube was added with 10 l skim milk (0.2 g/ml), 250 l extraction buffer (100 mM Tris-HCl pH 9.0, 40 mM EDTA), 50 l 10% SDS and 5 l RNase A (10 mg/ml) followed by vigorous vortexing for 1 min. An equal volume of benzyl chloride was added to the tube. The tube was vortexed again for 1 min, incubated at 50C for 60 min, and added with 150 l of 3 M NaOAc. After 15 min incubation in ice, the tube was centrifuged at 15,000 g for 5 min. The supernatant was transferred to a new tube and DNA was precipitated with cold ethanol. After dissolving DNA in TE buffer, DNA was purified with DNA purification kit (Toyobo, Japan). In PCR, a total volume of 25 l of reaction mixture contained 1 M of each primers, 1.25 units of rTaq DNA polymerase, 0.2 mM dNTP mixture, 400 ng/l of BSA, 1xPCR buffer and 1 l of DNA from soil. Primer set consisted of K1 (5'-ACGAAGGTTGGTCTGTTG-3') and K3 (5'-TCTCTACGCAACTAAATGC-3') [2]. The amplification conditions were: an initial denaturation at 94C for 3 min, followed by 40 cycles of denaturation at 94C for 1 min, annealing at 60C for 1 min, and extension at 72C for 2 min, with final extension at 72C for 10 min. PCR products were fixed on the well of a 96-well microplate in DNA-fixing solution (10xSSC, 10 mM EDTA). PCR products were hybridized with Dig-labelled probe prepared from PCR product of P. ultimum, following treatment of the anti-Dig antibody with alkaline phosphatase. The visualization was performed using p-nitrophenyl phosphate in 10% diethanolamine buffer, and the absorbance was determined by the microplate reader.

Results and conclusions
Ten oospores of P. ultimum were detectable from artificially infested soil using PCR microplate hybridization. Thirteen soil samples were collected from different crop fields and soil types throughout Japan. In PCR microplate hybridization, P. ultimum was detected from all ten soils from which the fungi were isolated on the Pythium-selective medium, but not from the other three soils without P. ultimum. Further investigations will be necessary to quantitatively determine the population in soil.

1. Inouye S, Hondo R, 1990. Journal of Clinical Microbiology 28, 1469-1472.
2. Kageyama K, Ohyama A, Hyakumachi M, 1997. Plant Disease 81, 1155-1160.