Mirco Iotti

Périgord black truffle genome uncovers evolutionary origins and mechanisms of symbiosis

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 ∼125 megabases is the largest and most complex fungal genome sequenced so far. This expansion results from a proliferation of transposable elements accounting for ∼58% of the genome. In contrast, this genome only contains ∼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.

The ectomycorrhizal community in natural Tuber borchii grounds

Although Tuber borchii is a commercially valuable truffle, its habitat has been virtually ignored. Here, we examine the ectomycorrhizal fungal communities in natural T. borchii grounds. Ectomycorrhizas under T. borchii ascomata and up to 1 m away were collected and morphologically assigned to pine or oak host plants. They were then morphotyped and molecular typed using internal transcribed spacer regions. Seventy ectomycorrhizal taxa were identified, many of which were rare. Tuber borchii dominated, forming 20% of ectomycorrhizas, with Thelephoraceae, Inocybaceae and Sebacinaceae being the other main species. Species composition was markedly affected by the host plant, although community structure and composition was also influenced by the location from which the soil cores were collected. Tuber dryophilum, an edible truffle, but without commercial value, shared the habitat with T. borchii. Its mycorrhizas were never found together with those of T. borchii. Tuber borchii was present on both oaks and pines, but was more abundant in soil cores where the roots of both hosts were present. It is suggested that the presence of young oaks contributed to the maintenance of T. borchii colonization on pines.

Interactions between Tuber borchii and other ectomycorrhizal fungi in a field plantation

The cultivation and competitiveness of Tuber borchii was investigated. Seedlings of Pinus pinea were infected either with T. borchii, Laccaria bicolor (strain S238), Hebeloma sinapizans, T. borchii and L. bicolor , or T. borchii and H. sinapizans. Uninoculated seedlings weakly infected with Sphaerosporella brunnea were used as controls. In 1990 the seedlings were transplanted to a coastal site at Marina di Ravenna, Italy. Examination of seedling roots in 1994 and 1995 showed that L. bicolor, H. sinapizans and S. brunnea ectomycorrhizas were replaced by T. borchii and other ectomycorrhizal fungi. The DNA sequence of the ITS region was analysed using the specific pair of primers, ITS1—ITS4, to type the ectomycorrhizas of Tuber borchii. The ectomycorrhizas were also identified by digestion of the PCR products using different restriction enzymes. T. borchii truffle production began in 1994 and for the next four years an average of 7 kg of truffles ha −1 were harvested. This study describes a simple method for the cultivation of T. borchii.

Assessment of ectomycorrhizal fungal communities in the natural habitats of Tuber magnatum (Ascomycota, Pezizales)

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.

Self/nonself recognition in Tuber melanosporum is not mediated by a heterokaryon incompatibility system.

Vegetative incompatibility is a widespread phenomenon in filamentous ascomycetes, which limits formation of viable heterokaryons. Whether this phenomenon plays a role in maintaining the homokaryotic state of the hyphae during the vegetative growth of Tuber spp. Gene expression, polymorphism analysis as well as targeted in vitro experiments allowed us to test whether a heterokaryon incompatibility (HI) system operates in Tuber melanosporum. HI is controlled by different genetic systems, often involving HET domain genes and their partners whose interaction can trigger a cell death reaction. Putative homologues to HI-related genes previously characterized in Neurospora crassa and Podospora anserina were identified in the T. melanosporum genome. However, only two HET domain genes were found. In many other ascomycetes HET domains have been found within different genes including some members of the NWD (NACHT and WD-repeat associated domains) gene family of P. anserina. More than 50 NWD homologues were found in T. melanosporum but none of these contain a HET domain. All these T. melanosporum paralogs showed a conserved gene organization similar to the microexon genes only recently characterized in Schistosoma mansoni. Expression data of the annotated HI-like genes along with low allelic polymorphism suggest that they have cellular functions unrelated to HI. Moreover, morphological analyses did not provide evidence for HI reactions between pairs of genetically different T. melanosporum strains. Thus, the maintenance of the genetic integrity during the vegetative growth of this species likely depends on mechanisms that act before hyphal fusion.

Multilocus phylogenetic and coalescent analyses identify two cryptic species in the Italian bianchetto truffle, Tuber borchii Vittad.

Tuber borchii (Ascomycota, Pezizales) is a highly valued truffle sold in local markets in Italy. Despite its economic importance, knowledge on its distribution and genetic structure is scarce. The objective of this work was to investigate the factors shaping the genetic structure of T. borchii using 61 representative specimens with a broad distribution throughout Italy. In spite of the lack of morphological differences, phylogenetic and coalescent-based analyses using four loci identified two genetically isolated groups sympatrically distributed. The low levels of divergence between the two clades may be the result of recent range expansions from geographically distinct refugia, potentially mediated by reforestation using coniferous species that are common ectomycorrhizal symbionts for both groups.

Spatio-temporal dynamic of Tuber magnatum mycelium in natural truffle grounds.

Tuber magnatum produces the worlds most expensive truffle. This fungus produces very rare ectomycorrhizas which are difficult or even impossible to detect in the field. A “real-time” PCR assay was recently developed to quantify and to track T. magnatum mycelium in soil. Here, this technique was used to investigate the spatial distribution of T. magnatum extra-radical mycelium in soil productive patches and its dynamic across seasons. This study was carried out in four different natural T. magnatum truffle grounds located in different Italian regions. During the fruiting seasons, the amount of T. magnatum mycelium was significantly higher around the fruiting points and decreased going farther away from them. Moreover, T. magnatum mycelium inside the productive patches underwent seasonal fluctuations. In early spring, the amount of T. magnatum mycelium was significantly higher than in summer. In summer, probably due to the hot and dry season, T. magnatum mycelium significantly decreased, whereas in autumn it increased again and was concentrated at the putative fruiting points. These results give new insights on T. magnatum ecology and are useful to plan the most appropriate sampling strategy for evaluating the management of a truffle ground.

Development and validation of a real-time PCR assay for detection and quantification of Tuber magnatum in soil

BackgroundTuber magnatum, the Italian white truffle, is the most sought-after edible ectomycorrhizal mushroom. Previous studies report the difficulties of detecting its mycorrhizas and the widespread presence of its mycelium in natural production areas, suggesting that the soil mycelium could be a good indicator to evaluate its presence in the soil. In this study a specific real-time PCR assay using TaqMan chemistry was developed to detect and quantify T. magnatum in soil. This technique was then applied to four natural T. magnatum truffières located in different regions of Italy to validate the method under different environmental conditions.ResultsThe primer/probe sets for the detection and quantification of T. magnatum were selected from the ITS rDNA regions. Their specificity was tested in silico and using qualitative PCR on DNA extracted from 25 different fungal species. The T. magnatum DNA concentration was different in the four experimental truffières and higher in the productive plots. T. magnatum mycelium was however also detected in most of the non-productive plots. Ascoma production during the three years of the study was correlated with the concentration of T. magnatum DNA.ConclusionsTaken together, these results suggest that the specific real-time PCR assay perfected in this study could be an useful tool to evaluate the presence and dynamics of this precious truffle in natural and cultivated truffières.

Characterization of Tuber borchii and Arbutus unedo mycorrhizas.

For the first time, arbutoid mycorrhizas established between Tuber borchii and Arbutus unedo were described. Analyzed mycorrhizas were from one T. borchii natural truffle ground, dominated by Pinus pinea, as well as synthesized in greenhouse conditions. A. unedo mycorrhizas presented some typical characteristics of ectomycorrhizas of T. borchii. However, as in arbutoid mycorrhizas, ramification was cruciform and intracellular colonization in epidermal cells was present. The ability of T. borchii to form ectomycorrhizas with A. unedo opens up the possibility to also use this fruit plant for truffle cultivation. This represents an important economic opportunity in Mediterranean areas by combining both the cultivation of precious truffles and the production of edible fruits which are used fresh or in food delicacies.

Techniques for Host Plant Inoculation with Truffles and Other Edible Ectomycorrhizal Mushrooms

Large-scale production of high-quality mycorrhizal plants in the greenhouse is mainstay for the modern cultivation of edible ectomycorrhizal mushrooms (EEMMs). Success at this step not only depends on the reliability of the fungal inoculum used for plantlet mycorrhization but also on the environmental conditions attending symbiosis establishment. Methods developed 40–50 years ago for inoculating host plants with EEMMs are still largely used today, with slight modifications. Mycelial inoculations are used commercially only for some edible ectomycorrhizal (EEM) basidiomycetes, while inoculation with spores is the most common method for producing seedlings colonized with truffle mycorrhizas. However, pure cultures and ectomycorrhizas of Tuber have also been used to obtain mycorrhizal plants mainly for scientific purposes. Mycelium-based inoculum offers many advantages, such as lower contamination risks and more reliable root colonization and provides opportunities for genetic selection of EEMM strains. The long-time preservation of EEM cultures and the creation of germ plasm banks would be an important step to support mycelium-based technologies. To this aim, we demonstrate successful cryopreservation of strains of Tuber borchii and Tuber aestivum. Recent advances in genetics and biotechnology of EEMs and their hosts have the potential to transform the current EEMM nursery trade.