Detection and quantification of Spongospora subterranea f. sp subterranea in soils and on tubers using specific PCR primers

PCR-based methods were developed for the detection and quantification of th e potato pathogen Spongospora subterranea f. sp. subterranea (S. subterrane a) in peel, tuber washings and soil. A partial sequence was obtained for S. subterranea ribosomal DNA and specific PCR primers (Sps1 and Sps2) were ch osen from the internal transcribed spacer regions. These primers amplified a 391 bp product from S. subterranea DNA but did not amplify DNA from potat o or a range of soil-borne microbes, including related species. Diluted S. subterranea DNA was detected at a concentration equivalent to 25 x 10(-5) c ystosori or 1 zoospore per PCR. Amplification was detected from peel and wa shings of infected and apparently healthy tubers, but not from peel of Scot tish classified seed potatoes or axenically micropropagated potatoes. A rap id method for extracting S. subterranea DNA from soils was developed. This yielded DNA pure enough for PCR within 3 h and facilitated the detection of 1-5 cystosori per gram of soil. A PCR quantification technique was develop ed involving comparison of product ratios obtained after co-amplification o f S. subterranea DNA along with an internal standard (competitor DNA fragme nt). This quantitative technique was also adapted for use in soil. PCR dete ction of S. subterranea in soil was considerably more sensitive than previo usly reported immunoassays and was quicker and easier than conventional bai t plant bioassays. Such an assay could be useful for developing disease ris k assessments for field soils and seed potato stocks and for future studies on the ecology and control of S. subterranea.