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.

Summary Truffles (Tuber spp.) are symbiotic fungi that develop underground in association with plant roots. Food connoisseurs describe their scent as sensual, seductive and unique. These mysterious fungi, however, do not produce their aroma for the mere pleasure of humans. Truffle volatiles act as odorant cues for mammals and insects which are thus able to locate the precious fungi underground and spread their spores. They also freely diffuse in the soil and mediate interactions with microorganisms and plant roots, potentially regulating a complex molecular dialogue among soil fauna and flora. The aim of this review is to synthesize 30 yr of research on truffle volatiles, spanning fields of study from chemical ecology to aroma biosynthesis. Specific aspects of truffle volatile ecology and biology will be discussed, including which species have been studied so far and for what purpose, what ecological role has been demonstrated or speculated to exist for specific truffle volatiles, which volatiles are common or unique to certain species and what their biosynthetic route might be. Future challenges in truffle aroma research will also be addressed, focusing on how high‐throughput post‐genomic technologies may advance our understanding of truffle aroma biosynthesis and chemical ecology.

Truffles are hypogeous fungi which live in symbiosis with plant host roots in order to accomplish their life cycle. Some species, such as Tuber magnatum Pico, the 'white truffle', and Tuber melanosporum Vittad., the 'black truffle', are highly appreciated in many countries because of their special taste and smell. The great demand for the black and white truffles, the increasing attention towards other species of local interest for the rural economy (such as T. aestivum) together with a drop in productivity, have stimulated researchers to develop projects for a better understanding of the ecology of truffles by exploiting the new approaches of environmental microbiology and molecular ecology. Specific primers have been developed to identify many morphologically similar species, the distribution of T. magnatum has been followed in a selected truffle-ground, the phylogeography of T. melanosporum and T. magnatum has been traced, and the microorganisms associated with the truffles and their habitats have been identified.

In a recent study pyrosequencing of the ribosomal internal transcribed spacer-1 (ITS-1) has validated the effectiveness of such technology in the survey of soil fungal diversity. Here we compare the two ITS regions, ITS-1 and ITS-2, of the fungal populations occurring in Tuber melanosporum/Quercus pubescens truffle grounds and sampled in two areas, one devoid of vegetation (''burned'', brulé in French) where T. melanosporum fruiting bodies are usually collected, and outside the brulé. TS1F/ITS2 and ITS3/ITS4 were used respectively for the amplification of the ITS-1 and ITS-2 regions. Two amplicon libraries were built, one for inside and the other for outside. A set of 15.788 reads was obtained. After the removal of low quality sequences, 3568 and 3156 sequences were obtained from inside the brulé with the ITS-1 and ITS-2 primers respectively. The sequences obtained from outside the brulé were 4490 with the ITS-1 primers and 2432 with the ITS-2 primers. Most of the sequences obtained for both ITS fragments could be attributed to fungal organisms.The pair of primers, ITS1-F/ITS2, was more selective, producing fewer non-fungal sequences (1% inside, 3% outside), in addition to a higher number of sequences, than the pair ITS3/ITS4 (6% inside, 11% outside). Although differences are present in the taxa percentages between ITS-1 and ITS-2, both reveal that Ascomycota were the dominant fungal phylum and that their number decreased moving from inside the brulé to outside, while the number of Basidiomycota increased. Taken together, both the short ITS-1 and ITS-2 reads obtained by the high throughput 454 sequencing provide adequate information for taxon assignment and are suitable to correlate the dynamics of the fungal populations to specific environments.

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

The aims of the work were to elucidate the distribution of the ectomycorrhizal fungus Tuber magnatum Pico during its symbiotic stage, and to identify the root-associated fungi in a natural truffle-ground located in North Italy. Ectomycorrhizal root tips were harvested in the truffle ground, sorted in morphotypes and analyzed by ITS. Morphological and molecular analyses revealed that (i) T. magnatum mycorrhizae were rare and independent on the fruitbody productions and (ii) the dominant fungal species belonged to Thelephoraceae, followed by Tuberaceae and Sebacinaceae.

Genetic variability is one of the major survival strategies developed by symbiotic fungi. We focused on the ectomycorrhizal fungus Tuber uncinatum Chatin that produces edible ascomata. In order to understand the degree of its variability and its relatedness to another morphologically-similar truffle, T. aestivum Vittad., ascomata of T. uncinatum were collected from a single natural truffle-ground located in the north of Italy and compared with samples from other Italian sites, as well as with T. aestivum ascomata from other European regions. We used multi-locus approaches, such as microsatellite-primed PCR (polymerase chain reaction), and single locus markers, such as mitochondrial and nuclear ribosomal DNA on 30 samples. The results demonstrate that the level of genetic polymorphism among isolates of T. uncinatum was higher than in other Tuber species, like T. melanosporum. Neighbour-joining analyses were carried out on a binary data matrix on 12 ascomata of T. uncinatum and T. aestivum, and on 15 internal transcribed spacer (ITS) sequences of these species and 5 from other Tuber species. Taken together, they clustered T. uncinatum and T. aestivum in two separate groups. The mitochondrial rDNA primers, NMS1 and NMS2, were not able to differentiate morphologically related and unrelated truffles. Moreover, a pair of primers, intentionally designed to differentiate isolates of T. aestivum and T. uncinatum from other Tuber species, successfully amplified DNA from all the samples of T. aestivum and T. uncinatum considered in our analysis. In conclusion, different molecular approaches separate T. aestivum and T. uncinatum according to their spore reticulum and their taste and smell.

Summary• The fruiting bodies of the ectomycorrhizal (ECM) fungus Tuber melanosporum are usually collected in an area devoid of vegetation which is defined as a 'burnt area' (brulé in French).• Here, the soil fungal populations of inside and outside brulé were compared in order to understand whether the scanty plant cover was related to a change in fungal biodiversity. Both denaturing gradient gel electrophoresis (DGGE) and molecular cloning of the internal transcribed spacer (ITS) marker were employed on soil DNA to obtain profiles from nine truffle grounds and fungal sequences from one selected truffle ground sampled in two years.• Denaturant gradient gel electrophoresis profiles from the two areas formed two distinct clusters while molecular cloning allowed 417 fungal sequences to be identified. T. melanosporum was the dominant fungus within the brulé . There were nine new haplotypes, which had never been detected in fruiting bodies. The Basidiomycota ECM fungi decreased within the brulé , indicating a competitive effect of T. melanosporum on the other ECM fungi.• Among other factors, the dynamics of fungal populations seems to be correlated to brulé formation.