Identification of the Ectomycorrhizal Basidiomycete Tylospora fibrillosa Donk by RFLP Analysis of the PCR-Amplified ITS and IGS Regions of Ribosomal DNA

Sitka spruce mycorrhizas, macroscopically identified as being formed by Tylospora fibrillose Donk, were sampled from a young and an 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 rDNA 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 ITS 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. fibrillose from other ectomycorrhizal species but there were only minor differences between the T. fibrillose isolates. PCR amplification of the ITS region, digestion with the endonuclease Hinf I and examination of the RFLPs produced proved to be a rapid method by which to distinguish T. fibrillose from a large number of other basidiomycetes. The method was also applied to DNA extracted from single mycorrhizal root tips. The intergenic spacer region (IGS) 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. fibrillose isolates within this region were limited and RFLPs were not useful in discriminating between isolates, suggesting a low genetic variability in this species.