Giovanni Pacioni

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.

Effects of Tuber metabolites on the rhizospheric environment

Several species of Tuber (Ascomycotina, Tuberales) produce a ‘burned’ area around their symbiotic plants as a result of a phytotoxic action. The volatile substances from Tuber sporophores, truffles, have been previously identified by gas chromatography coupled to gas mass spectrometry and then tested for their toxic effects on higher plants as well as soil micro-organisms. All tested organisms appeared to suffer a great inhibition when exposed to some of these substances evaporated into desiccators. Three aldehydes (2-methyl propanal, 2-methyl butanal and 3-methyl butanal) produced these effects on the two soil fungi tested ( Penicillium vinaceum and Aspergillus alliaceus ). In addition to some of these, two alcohols (2-methyl butanol and 3-methyl butanol) are also active on plants ( Triticum vulgare and Lens culinaris ). 2-Methyl butanol generally affects plants to a high degree, but the strongest effects to microfungi are caused by 3-methyl butanal. However, a strain of Pseudomonas isolated from truffle surface tolerated much higher concentrations of the three aldehydes. The substances affecting the soil micro-aerobic environment, where seeds germinate and roots live, are normal compounds of fungal metabolism produced via pyruvate pathway. Their toxic action could be a consequence of alterations caused both to the cellular membrane and to nucleic acids. The new term ‘hydnosphere’ has been proposed to define this peculiar case of soil relationships between truffle and neighbouring coenosis.

Insect attraction by Tuber: a chemical explanation

Volatile substances, identified as components of the truffle odour, were tested as to their ability to attract truffle-eating insects. Arthropods were trapped by the thousand in all traps, but the truly mycetophilous insects, as well as those species strictly bound to truffles in their biology, appear clearly attracted only by dimethyl sulphide.

Lactarius ectomycorrhizae on Abies alba: morphological description, molecular characterization, and taxonomic remarks.

To date, the ectomycorrhizae formed by silver fir (Abies alba), an ecologically valuable and in- digenous tree species in many European mountain forests, have been poorly investigated. We character- ized the mycorrhizae formed by three Lactarius spe- cies (Lac. subsericatus, Lac. intermedius, Lac. salmon- icolor) on silver fir, on the basis of material originat- ing from central Italy. The identification of the fun- gal symbiont was achieved by means of morphoanatomical observations of mycorrhizae, and by comparison of ITS sequences obtained from my- corrhizae and sporocarps of putative fungal partners. Sequences also were obtained from specimens of the same species but from different geographic origin or from closely related Lactarius species. A maximum likelihood analysis of the data was performed. On the whole, the resultant tree is in good agreement with sporocarp and mycorrhiza morphology. RFLP pat- terns were calculated from sequence data. A discus- sion on the main morphoanatomical characters dis- tinguishing the Lactarius ectomycorrhizae reported in this study from those already described belonging to related species, is also included. The accuracy of different methods to identify mycorrhizae formed by closely related Lactarius species on silver fir, are dis-

Odour composition of the Tuber melanosporum complex

Odour composition of some taxa related to Tuber melanosporum has been studied by gas chromatography coupled to a mass spectrometer (GC-MS). Although qualitative differences have been observed, the relative amounts of substances present may be more significant for their chemical taxonomy. A concept of the Tuber odour according to its ecological function is discussed

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.

Truffle tyrosinase: Properties and activity

Abstract The present paper investigates the l -3,4-dihydroxyphenylalanine oxidase (EC 1.14.18.1) and l -tyrosine 3-monooxygenase (EC 1.14.18.1) activities of truffles of the genus Tuber , a highly pigmented group of Ascomycetes. The laccase (EC 1.10.2.1) activity has also been explored in the homogenate supernatants from these mushrooms. The effects of various inhibitors of tyrosinase, of buffer concentration, temperature and pH on the tyrosinase activity of truffle cytosols have been investigated. The K m values of l -3,4-dihydroxyphenylalanine and l -tyrosine have been calculated and are in the range of those found in other mushrooms (i.e. 0.37 mM and 2.70 mM respectively).The polyacryamide gel electrophoretic pattern of the l -3,4-dihydroxyphenylalanine oxidase activities of different species of truffles have been obtained. Moreover, the truffle pigment formation and localization have been histochemically investigated and correlated with the reproductive differentiation.

Fungi in ectomycorrhizal associations of silver fir (Abies alba Miller) in Central Italy

Abstract Naturally occurring ectomycorrhizas of silver fir (Abies alba Miller) were studied in two stands, one natural and one artificial, situated in Central Italy. A total of 25 mycorrhizal types was classified, for eight of which the mycobiont was identified at the species level. Analysis of macroscopic and microscopic features and matching of field-collected carpophores with associated mycorrhizas led to the tentative identification of several other types encountered during this study, at least at the genus level. No significant differences were noticed between natural and artificial stands in the relative richness of mycorrhizal types found on A. alba, indicating the maturity of the artificial stand with regard to succession of ectomycorrhizal fungi. Confocal laser scanning microscopy was used for visualization of mycorrhizal structures formed by Lactarius spp., without the need for specific staining with a fluorochrome, thanks to latex autofluorescence. This technique allowed observation of several structures in greater detail than with conventional light microscopy.

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.

Characterization of Lactarius tesquorum ectomycorrhizae on Cistus sp. and molecular phylogeny of related European Lactarius taxa

Lactarius is one of the larger genera of ectomycorrhizal Basidiomycota, with about 400 species recognized worldwide. The ectomycorrhizae formed by Lactarius tesquorum on Cistus sp., one of the most common and ecologically relevant shrubs in the semi-arid regions in the Mediterranean basin, are described here in terms of morphological, anatomical and molecular features. An ITS rDNA sequence-based phylogenetic analysis was performed on the related European Lactarius taxa (L. mairei, L. pubescens, L. scoticus, L. spinosulus, L. torminosulus and L. torminosus) currently classified together with L. tesquorum in the subgenus Piperites section Piperites. Piperites s.s. could be divided into two main clusters; L. mairei and especially L. spinosulus were related less closely to the other taxa. This study is part of a broader effort to extend our knowledge of the distribution, phylogeny and ectomycorrhizal biology of Lactarius species in selected ecosystems.