5S ribosomal RNA gene spacers as species-specific probes for eight species of Pythium

Some species of Pythium have tandem arrays of 5S genes unlinked to the ribosomal DNA (rDNA) repeat unit. The gene spacers from 8 such species (P. intermedium, P. macrosporum, P. sylvaticum, P. ultimum, P. okanogenense, P. anandrum, P. acanthicum, and P. mastophorum) were amplified and used as probes against the genomic DNA of 92 species of Pythium. Separate probes were prepared from P. ultimum var. ultimum and P. ultimum var. sporangiiferum. All nine of the probes were species or variety specific. Six of the probes also were tested against three or more isolates of the same species and recognized all of them, except in the case of two P. ultimum var. sporangiiferum isolates, which were recognized by the P. ultimum var. ultimum probe. Four of the probes also were species specific when amplified DNA was used as the target for hybridization. These results show that the 5S rRNA gene spacer sequence diverged rapidly after speciation and may be useful in defining species boundaries in the genus Pythium