The Périgord black truffle (Tuber melanosporum Vittad.) and the Piedmont white truffle dominate today's truffle market. The hypogeous fruiting body of T. melanosporum is a gastronomic delicacy produced by an ectomycorrhizal symbiont endemic to calcareous soils in southern Europe. The worldwide demand for this truffle has fuelled intense efforts at cultivation. Identification of processes that condition and trigger fruit body and symbiosis formation, ultimately leading to efficient crop production, will be facilitated by a thorough analysis of truffle genomic traits. In the ectomycorrhizal Laccaria bicolor, the expansion of gene families may have acted as a 'symbiosis toolbox'. This feature may however reflect evolution of this particular taxon and not a general trait shared by all ectomycorrhizal species. To get a better understanding of the biology and evolution of the ectomycorrhizal symbiosis, we report here the sequence of the haploid genome of T. melanosporum, which at approximately 125 megabases is the largest and most complex fungal genome sequenced so far. This expansion results from a proliferation of transposable elements accounting for approximately 58% of the genome. In contrast, this genome only contains approximately 7,500 protein-coding genes with very rare multigene families. It lacks large sets of carbohydrate cleaving enzymes, but a few of them involved in degradation of plant cell walls are induced in symbiotic tissues. The latter feature and the upregulation of genes encoding for lipases and multicopper oxidases suggest that T. melanosporum degrades its host cell walls during colonization. Symbiosis induces an increased expression of carbohydrate and amino acid transporters in both L. bicolor and T. melanosporum, but the comparison of genomic traits in the two ectomycorrhizal fungi showed that genetic predispositions for symbiosis-'the symbiosis toolbox'-evolved along different ways in ascomycetes and basidiomycetes.

ruffle fungi differentiate into subterranean fruiting bodies bearing spores sequestered in an inconspicuous globouslike mass of hyphae. Truffle-forming species have evolved in nearly every major group of fleshy fungi over 100 times independently within the Ascomycota and Basidiomycota and the majority of evolutionary transitions to a truffle morphology have occurred in lineages establishing ectomycorrhizal mutualistic symbiosis with plants 1. This pattern suggests that symbiosis has been a major driver in the evolution of truffle diversity. The evolution of the hypogeous lifestyle across a diversity of truffle lineages also suggests that the transition from epigeous to hypogeous fruiting is driven by strong selection for traits, for example pungent odours, that promote

Summary• Pure cultures of Tuber maculatum , Tuber melanosporum , Tuber aestivum , Tuber macrosporum , Tuber rufum and Tuber brumale were isolated and characterized by morphological and molecular methods.• The Tuber mycelia were isolated from fruit bodies and molecular identification was performed using specific primers, restriction fragment length polymorphism and/or sequence analyses of the ITS region.• The species grew between 1.1 mm wk − 1 and 14 mm wk − 1 on the selected medium. The mycelium of different Tuber species showed several common morphological features such as hyphal anastomoses, vesicle formation and hyphal aggregation. Differences were found in the frequency of these morphological features and in the hyphal pattern. The isolated mycelia also showed differences in the hyphal branch angle, septal distance, hyphal diameter and rate of growth of the hyphae.• This result opens the possibility of using pure mycelial cultures of Tuber spp. for experimental purposes and for the commercial production of infected truffle plants.

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The ectomycorrhizal (ECM) fungal communities of four natural Tuber magnatum truffle grounds, located in different Italian regions (Abruzzo, Emilia-Romagna, Molise, and Tuscany), were studied. The main objective of this study was to characterize and compare the ECM fungal communities in the different regions and in productive (where T. magnatum ascomata were found) and nonproductive points. More than 8,000 (8,100) colonized root tips were counted in 73 soil cores, and 129 operational taxonomic units were identified using morphological and molecular methods. Although the composition of the ECM fungal communities studied varied, we were able to highlight some common characteristics. The most plentiful ECM fungal taxa belong to the Thelephoraceae and Sebacinaceae families followed by Inocybaceae and Russulaceae. Although several ectomycorrhizas belonging to Tuber genus were identified, no T. magnatum ectomycorrhizas were found. The putative ecological significance of some species is discussed.