DEVELOPMENT OF A SPECIES-SPECIFIC PROBE FOR PYTHIUM-ULTIMUM USING AMPLIFIED RIBOSOMAL DNA

The internal transcribed spacer (ITS) of nuclear ribosomal DNA (rDNA) was amplified by the polymerase chain reaction (PCR) with universal primers and used to differentiate species of Pythium that are difficult to identify by morphological criteria. The restriction sites on the ITS region of an isolate of Pythium ultimum were mapped. Digoxigenin-labeled probes (100-200 bp) representing different sequences along the entire ITS region were prepared from gel-purified restriction fragments of rDNA amplified by PCR. The restriction fragment probes from the ITS I spacer between the small ribosomal DNA (SrDNA) subunit and the 5.8S gene had a high degree of species specificity to P. ultimum when tested by dot blot hybridization against 24 other Pythium species. Similar results were obtained when the entire ITS I of P. ultimum was used as a probe. The quantities of total DNA on dot blots were standardized at 5 or 50 ng per dot. No difference in hybridization could be detected among the 13 isolates of P. ultimum (var, ultimum and var. sporangiferum from eight countries) and two isolates of Pythium group G recently classified as P. ultimum.