Development of a PCR-based assay for the detection of Plasmodiophora brassicae in soil

A single-tube nested polymerase chain reaction (STN PCR) method was develop ed for detecting the causal agent of clubroot disease, Plasmodiophora brass icae. Outer primer PBTZS-2 (5'-CCGAATTCGCGTCAGCGTGA-3') to amplify a 1457 b p-fragment from P. brassicae DNA and nested primers, PBTZS-3 (5'-CCACGTCGAT CACGTTGCAAT-3') and PBTZS-4 (5'-GCTGGCGTTGATCTACTGGAA-TT-3'), to amplify a 398 bp-fragment internal of the 1457 bp-fragment were used for the STN PCR. The 398 bp-fragment was amplified from as little as 1 fg of P. brassicae D NA with the STN PCR. A protocol for extracting P. brassicae DNA directly fr om soil was developed. By using the protocol, DNA was extracted from artifi cially infested soil containing various numbers of P. brassicae resting spo res and the resulting DNA was used as template for the STN PCR. As little a s one resting spore of P. brassicae per g of soil was detectable with the S TN PCR. The STN PCR was applied to naturally infested soil from 3 fields an d one canal bed. The 398 bp-fragment was amplified from soil of 2 fields an d the canal bed. To improve the detection of P. brassicae, the STN PCR prod ucts were subjected to second PCR amplification (double PCR) using the nest ed primers PBTZS-3 and PBTZS-4. The double PCR amplification generated a si ngle 398 bp-DNA band which was visualized clearly on the agarose gel for al l the 4 soil samples tested. A combination of the STN PCR and the double PC R appears a useful assay method for detecting P, brassicae resting spores i n field soil.