VIEW ARTICLE

Molecular Plant Pathology

Restriction Fragment Length Polymorphisms in Enzymatically Amplified Ribosomal DNAs of Three Heterothallic Pythium Species. Weidong Chen, Center for Biodiversity, Illinois Natural History Survey, and Department of Plant Pathology, University of Illinois at Urbana-Champain, 607 East Peabody Drive, Champaign 61820; Phytopathology 82:1467-1472. Accepted for publication 1 September 1992. Copyright 1992 The American Phytopathological Society. DOI: 10.1094/Phyto-82-1467.

Three heterothallic Pythium species, P. heterothallicum, P. splendens, and P. sylvaticum, were studied by examining restriction fragment length polymorphisms in polymerase chain reaction-amplified nuclear and mitochondrial ribosomal DNAs. Two different sizes of the internal transcribed spacer (ITS) from the nuclear rDNA were observed. The ITS for P. heterothallicum and P. splendens was about 850 base pair (bp) long, and the ITS for P. sylvaticum was about 1,020 bp long. Each length variant showed distinct banding patterns after restriction enzyme digestions. In addition, digestion with three enzymes differentiated P. heterothallicum and P. splendens. Restriction enzyme digestions of the nuclear small subunit rDNA and an 800-bp DNA from the 5’ end of the nuclear large subunit rDNA also showed species-specific banding patterns. In contrast, a 1,000-bp DNA from the mitochondrial large subunit rDNA did not show as much interspecific variation as did the nuclear rDNA. Little intraspecific variation was observed with nuclear rDNA. The species-specific banding patterns could be used to identify heterothallic Pythium species in the absence of mating reactions. The restriction sites in the ITS region were determined, and the maps were aligned to previously published data for five homothallic species. In phylogenetic analysis based on the variable restriction sites in the ITS region, the heterothallic species did not form a monophyletic group, suggesting that heterothallism does not represent a distinct species lineage in Pythium.