DETECTION AND QUANTIFICATION OF SPONGOSPORA SUBTERRANEA F. SP. SUBTERANNEA BY SPECIFIC DNA AMPLIFICATION. K.S. BELL, J.R. CLAXTON, J. ROBERTS, D. CULLEN, N.A. WILLIAMS, J.G. HARRISON, 1.K. TOTH, D.E.L. COOKE AND J.M. DUNCAN. Scottish Crop Research Institute, lnvergowrie, Dundee, DD2 5DA, UK. Background and objectives Spongospora subterranea f. sp. subterannea ( S. subterranea ) causes the potato blemish disease powdery scab. This disease is increasingly common and reduces the market value of potato crops [1]. The aim of this project was to develop methods based on the polymerase chain reaction (PCR) for the identification, detection and quantification of S. subterranea in tuber washings, plant material and soil.
Materials and methods A PCR-amplified 0.55 kb fragment of ribosomal DNA from cystosori of S. subterranea was sequenced and specific PCR primers (Spsl and Sps2) from within the internal transcribed spacer regions (ITS1 and ITS2) were designed. The specific primers were used in PCR sensitivity tests with DNA from S. subterranea
  • and in specificity tests with DNA from a range of soil-borne microorganisms, including the taxonomically related Plasmodiophora brassicae, Polymyxa betae, Polymyxa graminis and Ligniera sp., and with potato DNA. Quantitative PCR was developed by comparison of the ratio of products obtained after coamplification of known amounts of a control competitor DNA fragment and a dilution series of S. subterranea DNA with Spsl and Sps2. DNA was extracted and purified from soil samples by a rapid method employing a bead beater for disintegration of cystosori and cetyltrimethylammonium bromide for removal of humic substances. Samples were further purified by passage through Sephadex and polyvinylpolypyrolidone spin columns.
    Results and conclusions PCR using primers Spsl and Sps2 amplified a fragment of correct size when tested against S. subterranea DNA but no amplification occurred with DNA from other organisms. When DNA extracted from approximately 105 cystosori or 2 x 105 zoospores was diluted, amplification occurred at a concentration approximately equivalent to 25 x 10-5 cystosori or 1 zoospore per 25 ml PCR reaction. Amplification was detected from peel and washings of infected and apparently healthy tubers, but not from peel of Scottish classified seed potatoes or axenic micropropagated tubers or leaves. The bead beating method yielded high molecular weight DNA extracts from soil samples and, following spun column purification, these were sufficiently pure to serve as templates in PCR thus allowing detection of S. subterranea in soil. Detection of S. subterranea by PCR is more sensitive than previously reported serological techniques [1] and is quicker and more reliable than conventional bait plant bioasssays. A PCR-based assay could be useful for developing disease risk assessments of field sails and seed stock potato stocks.
    References 1. Walsh JA, Merz, U, Harrison, JG 1996. Plant Pathology 45, 884-895.