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DEVELOPMENT OF A PCR ASSAY FOR THE DETECTION OF SPONGOSPORA SUBTERRANEA F. SP. SUBTERRANEA IN HOST TISSUES AND SOIL
DEVELOPMENT OF A PCR ASSAY FOR THE DETECTION OF SPONGOSPORA SUBTERRANEA F. SP. SUBTERRANEA IN HOST TISSUES AND SOIL JA KAVANAGH l, QU XINSHUN l and D EGAN 1 l Department of Environmental Resource Mana-ement, University College Dublin, Dublin 4, Ireland Background and objectives Spongospora subterranea f. sp. subterranea is a soil-borne biotrophic fungus that causes powdery scab of potato and also transmits Potato Mop Top Virus. Although a bioassay [1] and an ELISA system [2] have been developed for the detection of this pathogen, problems relating to their use for the rapid and sensitive identification of all vegetative and reproductive structures of this pathogen in host tissues and soil, still exist. The aims of this study are to desi.n primers for the specific identification of S. subterranea by the polymerase chain reaction (PCR) and to test their sensitivity for the detection of this patho-en in host tissues and soil. Results and conclusions The primer pair SBITS4/SBITS5 was designed following extraction of DNA from clean spore balls (cystosori) isolated from cvs. Saturna and Cara, PCR amplification of the ITSI and ITS2 RDNA regions usin. the universal primer pair ITS4/ITS5 and sequencing the resultant 600 bp product in both directions. The specificity of this primer pair was confirmed when a single 303 bp product was generated following PCR amplifications with DNA from clean spore balls and zoospores originating from four potato cultivars. No product was obtained from DNA extracted from potato tissues, a wide range of filamentous fungi or taxonomically related fungi: Plasmodiophora brassicae, Polymyxa graminis and Polymyxa betae. A single 303 bp product was also amplified by PCR using the SBITS4/SBITS5 primer pair with DNA extracted from a single spore ball by Gene ReleaserTM. The primers were then used for the detection of S. subterranea in host tissues. After PCR amplifications with DNA from S. subterranea infected tomato seedling roots inoculated with spore balls taken from four different potato cultivars, a single 303 bp product was generated, but not from non-infected control seedlings. Followino, PCR with DNA from S. subterranea infected tubers orioinatinu from eight sources: Ireland (5), Scotland (2) and N. Ireland (1), a 303 bp product was also amplified but not from non-infected control micro-tubers. S. subterranea was also detected in infested field soil, but not in non-infested soil usino, this PCR assay. These results demonstrate that the desioned primer pair SBITS4/SBITS5 BITS5 is specific for the identification of vegetative and reproductive structures in the life cycle of S. subterranea f. sp. subterranea and for its detection in host tissues and soil. The sensitive and specific PCR amplification of S. subterranea DNA, without interference by DNA from host plants and contaminating microorganisms, provides a relatively simple and effective routine method for the detection of this patho-en in tubers and soil. References 1. Flett SP, 1983. Transactions of the British Mycological Society 81, 424-5. 2. Harrison JG, Rees EA, Barker H, Lowe R, 1993. Plant Pathology 42, 181-6.