Sex and truffles: first evidence of Perigord black truffle outcrosses

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(*Author for correspondence:
tel +33 (0)3 83 39 40 40; fax +33 (0)3 83 39 40 69; email claude.murat@nancy.inra.fr)

The Perigord truffle (Tuber melanosporum Vittad.) is a ‘cult-food’, one of the worldwide recognized icons of European gastronomy and culture, for which genomic and genetic information could act as a knowledge platform to improve its production and environmental persistence. The fruiting body of T. melanosporum is an edible truffle (= hypogeous ascocarp), which is a delicacy highly appreciated for its delicate organoleptic properties (i.e. taste and perfumes). This fungus belongs to the Ascomycota (Pezizales; Tuberaceae). It is endemic to calcareous soils in southern Europe and found in symbiotic association with roots of deciduous trees, mostly oaks and hazelnut trees, but also poplars. In this symbiotic relationship – the ectomycorrhizal association – long, branching fungal filaments known as hyphae ramify between cells of the root's outer layers, form a sheath around the root and radiate outwards into the surrounding soil and litter. In late Summer, extramatrical hyphae aggregate to form fruit body initials, from which the fruiting bodies then develop during Fall and early Winter. In truffles, the fruit body (or ascocarp) is formed by sterile hyphae (gleba) and fertile hyphae in which are found the ascospores. The spores released from mature truffles germinate in the following Spring, producing a homokaryotic vegetative mycelium (Paolocci et al., 2006), which results in colonization of tree root tips and further development of the symbiosis completing the truffle life cycle.

‘Even if direct evidence is still lacking, outcrossing is probably a common strategy used in all (or almost all) Tuber sp.’

Within ascomycetous fungi, sexually reproducing species usually follow one of the three basic sexual reproductive strategies: homothallism, pseudohomothallism (secondary homothallism) and heterothallism (Pöggeler, 2001). Sexual reproduction in filamentous ascomycetes is controlled by idiomorphic mating-type alleles, MAT1-1 and MAT1-2 (Pöggeler, 2001). Homothallic species contain both mating types, while for heterothallic species the two mating types carry one of the two idiomorphs.

It is of primary importance to characterize the reproductive mode of fungal species because the mating-type genes play a role in virulence (Mandel et al., 2007), survival (Houbraken et al., 2008) and fruiting body formation (Nolting & Pöggeler, 2006). Characterizing the reproduction mode of truffle species will therefore help to further our understanding of their biology and ecology.

As yet, experimental procedures to follow the whole life cycle of Tuber sp. in the laboratory are not available and hence this precludes any comprehensive study of Tuber genetics. Based on the available molecular markers, T. melanosporum was thought to be homothallic or even exclusively selfing (Bertault et al., 1998; Murat et al., 2004). Recently, genetic analyses using simple sequence repeats (SSR) carried out on the white truffle, Tuber magnatum, showed that outcrossing occurs for this species (Paolocci et al., 2006). In this issue of New Phytologist, Riccioni et al. (pp. 466–478) elegantly demonstrated that the Perigord black truffle also outcrosses. They identified additional alleles in the asci beside those present in the surrounding uniparental gleba. In the same study, Riccioni and colleagues, using multiloci and single loci molecular markers, highlighted a genetic structure among T. melanosporum populations and found that the southern-most populations have the highest allele richness. Similar results have been found for oaks, the main hosts of truffles, suggesting that the evolutionary history of ectomycorrhizal fungi and their host trees is linked (Petit et al., 2003; Murat et al., 2004).

In 2007, in their New Phytologist Letter, Rubini and colleagues raised some key questions about the biology of truffles:

  • 1Can all Tuber sp. outcross?
  • 2Are truffles prevalently outcrossing or heterothallic species?
  • 3What is the morphology of the mating structures in these fungi?

So far, evidence for outcrossing in the genus Tuber has been obtained for T. magnatum and T. melanosporum (Paolocci et al., 2006; Ricionni et al., 2008). Both species are phylogenetically divergent (Fig. 1) and their split probably occurred more than 180 million years ago (Jeandroz et al., 2008). Even if direct evidence is still lacking, outcrossing is probably a common strategy used in all (or almost all) Tuber sp. We recently pointed out that the Chinese black truffle (Tuber indicum Cook and Massee) is a potentially invasive species threatening T. melanosporum in Europe (Murat et al., 2008). Indeed, T. indicum is more competitive than T. melanosporum and both species are phylogenetically closely related (Fig. 1). It remains to be demonstrated whether the two are able to breed. The paper by Riccioni et al. confirms that we cannot exclude breeding between both species, especially if outcrossing occurs for both truffles.

Figure 1.

Neighbour-Joining phylogenetic tree generated from the 5.8S-ITS2 rRNA region of 108 Tuber specimens. The tree was created with mega 4.0 using Kimura-2-parameter distances, pairwise deletion and bootstrap values corresponding to 500 replicates. Some clades have been concatenated according to Jeandroz et al. (2008). A star highlights the two species for which outcrossing has been demonstrated.

The demonstration that T. melanosporum outcrosses is an important finding that has several consequences for species management, such as its introduction through inoculated seedlings and its conservation. Indeed, since the 1970s, seedlings have been inoculated with T. melanosporum spore suspensions and implanted worldwide to generate artificial truffle grounds (Chevalier & Grente, 1979). The results presented by Riccioni et al. call for a better control of genotypes used as inoculum. Traditionally, spore suspensions are prepared from a handful of ascocarps, allowing different mating types to be present. Currently, new techniques are being developed to inoculate seedlings based on mycelia produced in pure culture (Zambonelli & Iotti, 2004). In this case, it is recommended that the mating type of the different mycelia is characterized in order to inoculate seedlings with compatible strains.

It is known that T. melanosporum prefers open forest ecosystems and that canopy closure leads to a rapid decline in the production of ascocarps. Using amplified length fragment polymorphism (AFLP), Riccioni et al. identified nine genets out of 11 samples and seven genets out of seven samples in two truffle grounds located at Capodacqua (Italy) and Cerreto di Spoleto (Italy), respectively. This unexpected genetic diversity revealed that T. melanosporum is able to form numerous genets and, consequently, T. melanosporum can be considered as an ‘early stage fungus’ favouring the sexual reproduction. This contention needs to be verified by population surveys of various natural truffle grounds over several years.

Truffles: homothallic or heterothallic species? The genome of the Perigord black truffles has recently been sequenced (Tuber Genome Consortium) and will soon be available. In this genome, the mating type genes have been identified, confirming that T. melanosporum is heterothallic (A. Rubini & F. Paolocci, pers. comm.). The analysis of these genes will allow a better understanding of the T. melanosporum lifecycle and the formation of ascocarps. On the other hand, the mating-type genes will be used as molecular markers to investigate the population genetics of this species, as already carried out for Coccidioides spp. (Mandel et al., 2007).

The second important finding of Riccioni and colleagues concerns the genetic structure of T. melanosporum populations. Analysing population genetics of a species provides information about (1) its history, (2) the factors that have generated the genetic differentiation among populations and (3) the occurrence of hot spots of genetic diversity. In 1998, Bertault and colleagues claimed that there was no genetic structure in T. melanosporum populations. In contrast, a strong geographic pattern for T. melanosporum has been identified more recently (Murat et al., 2004) using single nucleotide polymorphisms (SNPs) of the nuclear rRNA internal transcribed spacer (ITS). In their paper, Riccioni and colleagues confirmed that there is a significant genetic structure among T. melanosporum populations. Moreover, they analysed a higher number of samples from southern Europe than any previous studies and they found that the southern-most populations have a higher allelic richness. As already shown for oaks, the main host of truffles, the populations with the highest allele richness are indicative of the potential species refuge during the last glaciation (Petit et al., 2003). This is the first experimental evidence which indicates that T. melanosporum probably took refuge in southern Europe during the last glaciation. Interestingly, the two genotyped Spanish populations showed the highest values of allele richness. A more extensive sampling of the Spanish populations is required for confirming whether the Iberic peninsula represented another potential species refuge during the last glaciations.

Truffles provide an important source of income for truffle hunters and traders in different regions (the selling price of truffles can be as high as ı1000 per kg). Consequently, local administrations aim to promote and market their local truffle populations using specific appellations, for example ‘Truffe du Tricastin’, ‘Tartufo nero pregiato di Norcia’. However, it is currently impossible to differentiate ascocarps harvested in different regions or countries by genetic fingerprinting. The genetic differentiation, highlighted by Murat et al. (2004) and Riccioni et al., among T. melanosporum populations suggested that the characterization of molecular markers to identify the regional origin of ascocarps is within reach. In the fungal genome several thousand SSR motifs can be identified (Lim et al., 2004) given a large set of polymorphic markers (Kim et al., 2008). A T. melanosporum genome survey identified a large set of polymorphic SSR (C. Murat et al., unpublished) thus providing new molecular markers to analyse the natural populations of this truffle.

Sex will save truffles? The study by Riccioni et al., together with the first data from the T. melanosporum genome, are providing new and important data about the life cycle and population genetics of this species; however, we can be sure that T. melanosporum has not finished delivering all its secrets.

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