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 b y Tylospora fibrillosa Donk, were sampled from a young and an old plan tation and the mycobionts were isolated into pure culture. DNA was ext racted and fragments of the ribosomal DNA (rDNA) were amplified using the polymerase chain reaction (PCR). The primers were directed to cons erved regions of fungal rDNA and hybridize to a wide range of fungi. O ne amplified region includes the internal spacer (ITS) region which ha s a low degree of conservation. The ITS amplification products, which were approximately 600 base pairs (bp), were digested with a variety o f restriction endonucleases in order to detect restriction fragment le ngth polymorphisms (RFLPs). The RFLPs clearly separated T. fibrillosa from other ectomycorrhizal species but there were only minor differenc es between the T. fibrillosa isolates. PCR amplification of the ITS re gion, digestion with the endonuclease Hinf I and examination of the RF LPs produced proved to be a rapid method by which to distinguish T. fi brillosa from a large number of other basidiomycetes. The method was a lso applied to DNA extracted from single mycorrhizal root tips. The in tergenic 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 inter genic region between the 25S and the 5S genes were amplified and analy zed as above. Polymorphisms between T. fibrillosa isolates within this region were limited and RFLPs were not useful in discriminating betwe en isolates, suggesting a low genetic variability in this species.