Genetic Variation and Phylogeny of Spongospora subterranea f.sp. subterranea Based on Ribosomal DNA Sequence Analysis

American Journal of Potato Research, Nov/Dec 2004 by Qu, Xinshun, Christ, Barbara J

Spongospora subterranea is considered a member of the order Plasmodiophorida (Karling 1968), which are characterized as having cruciform nuclear division, multinucleate plasmodia, biflagellate zoospores and resting spores. Other economically important members of plasmodiophorids include Plasmodiophora brassicae Woronin, the cause of club root disease of brassicas; Polymyxa species, the vectors of several plant viruses (Adams 1991); and S. subterranea (Wallr.) Lagerh f.sp. nasturtii Tomlinson, the cause of crook root disease of watercress (Tomlinson 1958). The taxonomic affinities of the Plasmodiophorida have been unclear for a long time, and traditionally mycologists have placed these organisms within fungi (Alexopoulos and Mims 1979; Sparrow 1958), whereas, Barr (1992) and Alexopoulos et al. (1996) considered that the plasmodiophorids may have had their origin in the protozoans. Recently, DNA sequences obtained from small-subunit rRNA gene (SSU rRNA) have been used to predict phylogeny for species in the Plasmodiophorida, but the results were conflicting. Based on the phylogenetic analysis of SSU rDNA sequences of P. brassicae and Polymyxa spp., Castlebury and Domier (1998), Ward and Adams (1998), and K�hn et al. (2000) reported that the plasmodiophorids are a distinct group and are not closely related to any other eukaryotes. Down et al. (2002) examined the SSU rDNA sequences of P. brassicae and S. subten-anea f.sp. nasturtii, and concluded that they are not closely related to a range of protists and fungi. However, Cavalier-Smith and Chao (1997) and Bulman et al. (2001) found that, the plasmodiophorids are related to the Rhizopoda based on phylogenetic analyses of SSU rDNA sequences. Since the number of available SSU rDNA sequences from a variety of other eukaryotes has increased quickly during the last few years, further studies on the phylogeny of S. subterranea and other species in the Plasmodiophorida using SSU rDNA sequences may therefore contribute new information on its taxonomic position and relationships with other eukaryotes.

Our objective was to investigate genetic variation in isolates of & subterranea from the British Isles and North America using ITS1/2 including 5.8S rDNA sequences and to confirm phylogenetic relationships of S. sublerranea using SSU rDNA sequences.

MATERIALS AND METHODS

Sources of S. subterranea

Fifty-two field isolates of S. subterranea were obtained from the Republic of Ireland, Northern Ireland, Scotland, Canada, and USA (Table 1) either from different fields or from the same field with samples taken over several years. These locations were chosen because of access to potato production areas with powdery scab and because this work had not been carried out previously in these areas. Each isolate was prepared by the removal of single spore balls from powdery scab lesions of naturally infected potato tubers by the method of Qu et al. (2001).

DNA Extraction

Genomic DNA of S. subterranea was extracted from spore balls using the modified method of M�ller and Harling (1996). A single spore ball pellet was crushed between a sterile microscope slide and a cover slip that had been coated with Sigmacote (Sigma). Spore rupture was monitored with the aid of a compound microscope. After removal of the cover slip, squashed spore balls or cystosori (resting spores) were washed directly into 1.5-mL microcentrifuge tubes with 10 �L TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). This procedure was repeated five times for each collection and extracts were transferred to a 1.5-mL microcentrifuge tube, centrifuged at 10,000 g for 2 min and the supernatant subjected to PCR amplification immediately, or stored at -20 C.