Identification of the ectomycorrhizal basidiomycete Tylospora fibrillosa Donk by RFLP analysis of the PCR-amplified ITS and IGS regions of ribosomal DNA.

Abstract

Sitka spruce mycorrhizas, macroscopically identified as being formed by Tylospora fibritiosa Donk, were sampled from a young and on old plantation and the mycobionts were isolated into pure culture. DNA was extracted and fragments of the ribosomal DNA (rDNA) were amplified using the polymerase chain reaction (PCR). The primers were directed to conserved regions of fungal rDXA and hybridize to a wide range of fungi. One amplified region includes the internal spacer (ITS) region which has a low degree of conservation. The JTS amplification products, which were approximately 600 base pairs (bp), were digested with a variety of restriction endonucleases in order to detect restriction fragment length polymorphisms (RFLPs). The RFLPs clearly separated T. fibrillosa from other ectomycorrhizal species but there were only minor differences between the T. fibrillosa isolates. PCR amplification of the ITS region, digestion with the endonudeasc HinfI and examination of the RFLPs produced proved to be a rapid method by which to distinguish T. fibriHosa from a large number of other basidiomyictes. The method was also applied to DNA extracted, from single mycotrhizal root tips. The imergenic spacer region (1GS) of the rDNA is more variable than the ITS region in several fungal species. The 5'end of the 25S and the intergenic region between the 25S and the 5S genes were amplified and analyzed as above. Polymorphisms between T. fibritiosa isolates within this region were limited and RFLPs were not useful m discriminating between isolates, suggesting a low genetic variability in this species.