Typing truffle species by PCR amplification of the ribosomal DNA spacers

Variation within the internal transcribed spacer (ITS) and the intergenic spacer (IGS) of the ribosomal RNA genes of European species of Tuber was examined by polymerase chain reaction (PCR) and coupled restriction fragment length polymorphism (RFLP) analysis. Ribosomal DNA spacers were successfully amplified from mycelium, ectomycorrhiza, and fruit bodies of a wide range of truffle species ( Tuber aestivum, T. albidum, T. brumale, T. excavatum, T. ferrugineum, T. magnatum, T. melanosporum, T. rufum and T. uncinatum). Interspecific variation in the length and number of restriction sites of the amplified ITS and IGS was observed and most truffles could thus be reliably distinguished by PCR-RFLP. In contrast, the degree of intraspecific rDNA variation observed was low within T. melanosporum, but sufficient to discriminate several isolates. These results demonstrate that the PCR-RFLP analysis of rDNA spacers provides an efficient alternative for typing pure fungal cultures and fruit bodies for the food industry and a versatile tool for strain fingerprinting of ectomycorrhizas in ecosystems.