Species-specific polymorphisms in transcribed ribosomal DNA of five Pythium species

Abstract

Twenty-five isolates representing five Pythium species collected from diverse hosts and geographic origins were evaluated using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis. DNA regions coding for the small-subunit ribosomal RNA (SrDNA) and the internal transcribed spacer (ITS) were amplified and analyzed by restriction enzyme digestion. The amplified SrDNA was about 1800 bp long and uniform in length among the five species. However, restriction digestion revealed three polymorphic groups. They are P. arrhenomanes and P. graminicola, P. irregulare and P. spinosum, and P. ultimum. The amplified-ITS region showed three different lengths which corresponded to the three polymorphic groups of SrDNA. Each length variant of the ITS showed distinct banding patterns after restriction enzyme digestion. In addition, P. irregulare and P. spinosum each showed distinct banding patterns after digestion with enzymes HinfI and MboI. Physical maps of the restriction sites in the SrDNA and the ITS were determined. Length variation occurred primarily in the spacer between the SrDNA and 5.8 S rDNA; although, it also was detected in the ITS-2 region. Little intraspecific variation was observed in the SrDNA and ITS, and species could be reliably distinguished by RFLP analysis of the amplified rDNA regions. Data presented do not support the maintenance of P. arrhenomanes and P. graminicola as distinct species. Results indicate that PCR-RFLP can be used as a simple and speedy taxonomical tool for ecological studies of Pythium species.