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 aesfivum, T. albidum, T. brumale, T. excavarnm, 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 rONA variation observed was low within T. melanosporum, but sufficient to discriminate several isolates. These results demonstrate that the PCR-RFLP analysis of rONA 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. The commercial inoculation of oak and hazel tree seedlings with ecologically adapted truffles is presently being developed in France and Italy (Chevalier & Grente, 1979; Bencivenga, 1982). Although many experiments have shown that truffle inoculation is beneficial in terms of fruit-body production, results have been somewhat erratic on outplanting sites where indigenous truffles and ectomycorrhizal fungi communities are large and competitive. How this natural population could interact with introduced truffle isolates needs to be determined to select potential candidates for large scale inoculation programs. This requires tracking the environmental fate of the introduced truffles to determine their survival, growth, and dissemination within a microbial community. The identification of mycorrhizas and fruit bodies of truffles, based on morphological features, is reliable for most species. However, the ectomycorrhizas of several morphological species, such as Tuber aestivum Vittad. and T. uncinatum Ch., T. aibidum Pico and T. magnatum Pico, are morphologically similar, but only T. uncinatum, T. aestivum and T. magnatum are gastronomically and thus economically valuable (Chevalier et al., 1986). Biophysical (Papa et ai. 1987; Papa & Polimeni, 1990) and biochemical (protein patterns, isoenzyme electrophoresis, scent composition) (Dupre & Chevalier, 1991; Dupre et ai., 1992; Mouches et ai., 1981; Pacioni et al., 1989; Pacioni & Pompini, 1989; Palenzona et al., 1990) techniques have been