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ANALYSIS OF RELATIONSHIPS AMONG THE PLASMODIOPHORIDS USING RIBOSOMAL DNA SEQUENCES AND DEVELOPMENT OF PCR PRIMERS FOR THE SPECIFIC DETECTION OF POLYMYXA SPECIES
ANALYSIS OF RELATIONSHIPS AMONG THE PLASMODIOPHORIDS USING RIBOSOMAL DNA SEQUENCES AND DEVELOPMENT OF PCR PRIMERS FOR THE SPECIFIC DETECTION OF POLYMYXA SPECIES E WARD and M J ADAMS IACR-Rothamsted, Harpenden, Herts, AL5 2JQ, UK. Background and objectives Polymyxa species are obligate parasites of plant roots that are important as vectors of plant viruses. Polymyxa graminis infects cereals and grasses and can transmit a variety of viruses including barley yellow mosaic virus and soil-borne wheat mosaic virus. Polymyxa betae infects sugar beet and related plants and can transmit several viruses including beet necrotic yellow vein virus, the causal agent of rhizomania in sugar Piasmodiophora,Spongospora and Ligniera are collectively known as plasmodiophodds. Although traditionally placed among the fungi and studied by plant pathologists, the wider taxonomic affinities of this group have recently been questioned and they are now often classified among the protozoa. We have therefore determined RDNA sequences for a number of Polymyxa isolates and for some related fungi, both to investigate the relationships within the plasmodiophodds and to determine their affinities with other eukaryote groups. Routine microscopic examination of plant roots for the presence of Polymyxa (e.g. for epidemiological studies or screening breeding lines) is a skilled and tedious task. Over the past few years we therefore have developed several molecular techniques to simplify and improve the detection of these fungi. These include RFLP analysis of PCR amplified RDNA and a specific probe and PCR primers for Polymyxa betae [1], [2]. Further details on molecular studies of Polymyxa and other plasmodiophodds are also available on our web site (http:/.res.bbsrc.ac.uklcdmlpiantpathladams/plasmod). However, no method was available for simple and specific detection of either Polymyxa graminis or both Polymyxa species. The rDNA sequence data was also therefore used to develop PCR primers for these purposes. Results and conclusions A region of about 800bp of ribosomal DNA was sequenced in 10 isolates of Polymyxa and one isolate each of Plasmodiophora brassicae and the chytrid Oipidium brassicae. This region consists of about 330bp at the 3' end of the 18S gene, the 5.8S DNA and the two internal transcribed spacers. A shorter region was also sequenced from one isolate of Spongospora subterranea and a Ligniera species. In phylogenetic analyses, the Ligniera and Spongospora isolates grouped with the other plasmodiophodds and the Oipidium isolate with the fungi. The plasmodiophodds appeared as a distinct group and were not closely related to any other eukaryotes (including a range of fungi and protozoa). The DNA sequences were used to design primers for specific amplification of either Polymyxa or P. graminis DNA. References 1. Ward E, Adams MJ, Mutasa ES, Collier CR and Asher MJC, 1994. Plant Pathology 43, 872-877. 2. Mutasa ES, Chvvarszynska DM, Adams MJ, Ward E and Asher MJC, 1995. Physiological and Molecular Plant Pathology 47, 303-313.