Sergio Arcioni

Two repeated DNA sequences of European strains of the symbiotic fungus Tuber melanosporum were isolated and characterized. One of these, SS14, representing about 0.05% of the fungal genome, was shown to be a T. melanosporum -specific sequence by Southern and dot-blot hybridization. The second one, named SS15, is about 0.0025% of the entire genome, and it is specific not only to T. melanosporum but also to the Asian black truffle Tuber indicum . Neither of these two fragments hybridizes with any of the other European truffle species tested. By sequence analysis of these two fragments, PCR primers were designed and used to selectively amplify DNA from T. melanosporum ascocarps and ectomycorrhizae by simple and multiplex PCR. No amplification products were obtained with DNA from either mycorrhizal roots or fruit bodies of other ectosymbiotic fungi. The two identified genomic traits also provided useful information for a better understanding of the phylogenetic relationships among black truffle species and for testing T. melanosporum intraspecific variability.

Protocols for isolating and amplifying fungal DNA from ectomycorrhizae, overcoming the presence of PCR inhibitors, were established to ensure a fast and reliable truffle mycorrhizal identification. By using species-specific ITS primers in multiplex PCR, morphologically very similar Tuber melanosporum, Tuber indicum and Tuber brumale ectomycorrhizae were identified in a single step of amplification, irrespective of their physiological stage. The molecular method assures a more accurate and reliable monitoring of nursery-inoculated plants both before and after their in-field planting, than any morphological or ITS/RFLP analyses so far performed. It should also prevent frauds, already a problem, and the potential for environmental damage due to the incorrect typing of the allochthonous species T. indicum as European truffle species, when the identity of ectomycorrhizae is based solely on morphology.