A quantitative and molecular examination of Tuber melanosporum mycorrhizae in Quercus ilex seedlings from different suppliers in Spain

Aim of study: The aim of the work was to determine the degree of mycorrhization of Quercus ilex L. subsp. ballota (Desf.) Samp. by the black truffle fungus T. melanosporum Vittad. by quantitative and molecular analyses.Area of study: seedlings inoculated by different Spanish suppliers.Material and methods: The internal transcribed spacers (ITS) of mycorrhizae from different plants were amplified by nested PCR involving fluorescently-labelled primers, and the amplicons either directly sized by ARISA or analysed by TRFLP following their digestion with restriction endonucleases. TRFLP analysis distinguished between mycorrhizae of T. melanosporum, T. indicum Cooke & Massee and T. borchii Vittad., as suggested possible by virtual (in silico) TRFLP analysis and real TRFLP analysis of the ascomata of these species.Main results: Significant differences between suppliers were detected in terms of the mean number of mycorrhizae established per plant and percentage mycorrhization. These results allowed the following quality standards for 2 year-old plants to be proposed: a) good quality: >3000 mycorrhizae/plant, >40% mycorrhization, b) medium (acceptable): >3000 mycorrhizae/plant, >30% mycorrhization, c) low quality: <3000 mycorrhizae/plant or <30% mycorrhization, always supposing the mycorrhizae counted represent the species of interest as confirmed by the presence of its DNA and the absence of DNA belonging to contaminating species. Finally, a new microsatellite allelic map obtained from the analysis of several T. melanosporum populations across Spain was used to provide a tool capable of determining the geographic origin of the fungi used to inoculate plants.Research highlights: The proposed quality standards can be useful for the evaluation and certification of commercialized Q. ilex plants mycorrhized with T. melanosporum.Keywords: mycorrhiza; certification; truffle; TRFLP; fungi