The evidence that T. melanosporum is an obligately outcrossing fungus (Martin et al., 2010; Rubini et al., 2010b) calls for new investigations on the factors that affect mating and fruiting in this truffle species. Thus, we tracked the distribution patterns of mycelia harboring complementary MAT genes beneath a T. melanosporum truffle ground. Here, we show that strains with different mating type are not evenly distributed beneath productive soil patches, that host roots are colonized by a single fungal genet and that ectomycorrhizal strains likely behave as the ‘maternal’ partner in the mating process, with ‘paternal’ partners not necessarily present as ECM within the same soil patches. We also show that strain competition and replacement occur on roots of artificially inoculated plants grown in pots.
Mapping the presence of T. melanosporum mating types in a natural truffle field shows uneven distribution and sheds light on T. melanosporum biology
Many studies carried out on symbiotic fungal species have revealed that the species composition of ectomycorrhizal communities can differ greatly from that of sporocarp communities (Gardes & Bruns, 1996; Horton & Bruns, 2001; Hirose et al., 2004; Zampieri et al., 2010). In this study, we monitored the distribution of T. melanosporum strains with different mating types in a natural black truffle ground by performing parallel genotyping of ascocarps, ECMs and soil samples.
The first finding from the fingerprinting of T. melanosporum ECMs is that each mycorrhizal tip, regardless of whether it was collected from naturally or artificially inoculated plants, always produced only a single allele when PCR amplified with either MAT- specific or SSR-specific primer pairs. This is the first genetic evidence in support of the hypothesis that T. melanosporum ECMs result from the colonization of host roots by haploid mycelia (Rubini et al., 2007). This finding is also consistent with previous results obtained on ECMs collected from Q. pubescens plants nursery-inoculated with T. magnatum (Paolocci et al., 2006).
The distribution analysis of strains with different mating types provides an intriguing scenario, whereby both MAT are not equally represented in the soil of productive areas. Strains of opposite mating type indeed tend to be far apart. The minimal distance between sites where ECMs with different mating types were detected was 50 m (Fig. 1 and Table S1).
The finding that not only single host plants but also delimited ground areas showed the presence of ECMs with the same mating type is consistent with the idea of a vegetative spread of a single strain that may compete and displace all other strains, ultimately leading to the formation of a genet. To test this hypothesis, all T. melanosporum ECMs collected in the Borgo Cerreto field were genotyped using seven SSR loci. This analysis allowed us to sort the black truffle ECMs into seven genetic classes. The findings that all ECMs beneath a host plant, or beneath close plants, displayed the same mating type and the same multilocus genotype at SSR loci supports our hypothesis that all of these ECMs resulted from plant colonization by a single genet. Embracing this thesis, it can be pointed out that seven genets were present within the Borgo Cerreto ground. Molecular markers have previously been used to identify genets of many ectomycorrhizal fungi using the allelic configuration of sporocarps, ECMs or both (Bastide et al., 1994; Anderson et al., 1998; Gherbi et al., 1999; Sawyer et al., 1999; Selosse et al., 1999; Guidot et al., 2001; Redecker et al., 2001; Kretzer et al., 2003). By examining the distribution pattern of T. melanosporum genets in Borgo Cerreto, it appears that single genets are confined to a small number of host plants. For example, within the area delimited by the sampling sites 236, 238 and 241, three genets (III, IV and V) were characterized, each specific to one sampling site.
T. melanosporum mycelia spread and colonize the soil in late spring, when sexual reproduction is hypothesized to occur (Sourzat, 1997). Consequently, fruit body formation appears to be a long-lasting process that may begin in late spring but is not completed until winter. Soil samples were collected in late spring to monitor the distribution of T. melanosporum strains with opposite mating types. As a general rule, soil samples showed the presence of strains that shared the same mating type with surrounding ECMs. However, soil strains with a mating type different from that of the neighboring ectomycorrhizal strains were also detected (Table 2). Fungal structures (i.e. hyphae, spores) in the soil that yielded DNA with mating type opposite to that of the nearby ECMs remain to be elucidated. Given that specific procedures for breaking spore walls are generally needed to isolate DNA from spores within truffle fruit bodies (Paolocci et al., 2006), it is plausible that DNA was isolated from mycelia produced by ascospores or mitospores (Urban et al., 2004) or, alternatively, from mycelia developing from ECMs of trees not sampled in this study because they were unproductive. It is also plausible that soil DNA of opposite mating type with respect to the surrounding ECMs is contributed by mycelia developed from ECMs of plants meters apart. Under this scenario, present data suggest that T. melanosporum mycelia originating from ECMs could spread over relatively long distances in the soil (50–80 m or more). Other ectomycorrhizal fungi can indeed spread over tens of meters (Dahlberg, 1997; Bonello et al., 1998; Selosse et al., 1999; Hirose et al., 2004).
Soil samples collected in winter (i.e. samples S-18 and S-20) yielded the same MAT amplicon as the surrounding ECMs, consistent with the view that the DNA was contributed by mycelia spread from ECMs in the vicinity. The alternative hypothesis that this DNA originated from free-living mycelia could also be embraced. However, the evidence that T. melanosporum presents a restricted repertoire of Carbohydrate Active enZymes (CAZymes) able to degrade plant cell wall polysaccharides suggests that the saprobic ability of this fungus is low and the survival of free-living mycelium likely very limited (Martin et al., 2010).
The detection of T. melanosporum in soil from areas where truffles were produced during the last season but where T. melanosporum ECMs were not found in this study (e.g. sites 245, 246 and 242) is similar to the pattern of sporocarp and vegetative spread of several other ectomycorrhizal fungi, including Tuber spp. (Zhou et al., 2001; Lian et al., 2006; Zampieri et al., 2010). This confirms that even when sporocarps are collected, collecting the corresponding ECMs is not a trivial matter (Horton & Bruns, 2001).
A number of interesting findings related to the genetic diversity shown by the ascocarps collected from the same sites emerged when we superimposed the soil and ECM analyses.
First, the genotype exhibited by the gleba of truffles was always identical to that of the corresponding ECMs. This result cannot be explained by the lack of informative markers, as pool of spores from the same ascocarps can show additional SSR alleles to those of the respective gleba. Rather, it can be interpreted that ectomycorrhizal strains make a ‘maternal’ contribution in the mating process. We have previously shown that the gleba of Tuber ascocarp is a uniparental tissue formed by haploid mycelium (Paolocci et al., 2006; Riccioni et al., 2008). We can now extend our conclusion to infer that this ‘maternal’ tissue is preferentially or, presumably, solely derived from the strains that have colonized the host roots. With this model in mind, the ectomycorrhizal strain should allocate carbon resources from the host plant to the primordia of the fruit body to sustain its development. It is also possible that mating occurs between non-ectomycorrhizal strains, or that non-ectomycorrhizal strains behave as ‘maternal’ partners. However, in all cases, the development of the nascent fruit body would be seriously compromised by the lack of nutritional resources provided by the plant (Zeller et al., 2008). Furthermore, in the present study and in the companion paper (Rubini et al., 2010b), we show that the gleba can be formed either by MAT(+) or MAT(−) mycelia, indicating that truffle mycelia are equally competent to form male and female reproductive structures, regardless of their mating type.
Second, spores of some fruit bodies display alleles never detected among the ECMs in the truffle ground. By comparing the SSR alleles displayed by the gleba with those present in the corresponding pool of spores, the putative genotype of the ‘paternal’ partner can be inferred. Following this approach, we were able to identify up to 16 genotypes in the site under investigation, but only seven at the ectomycorrhizal level. The spores of some ascocarps showed the same SSR allelic configuration as the corresponding gleba (i.e. fruit bodies FB-591, FB-579 and FB-590). Because these ascocarps are derived from outcrossing, as demonstrated by the presence of both mating types in DNA isolated from their spores, the only difference between their genotypes is at the MAT locus. Thus, genotypes I, II and VI, found in the gleba of the above-mentioned truffles, differ from genotypes X, IV and XIII, as inferred from the analysis of the corresponding spores, only for the allelic configuration at the MAT locus, respectively (Table 3). These results suggest that these truffles may have resulted from biparental inbreeding. Mating between closely-related parents has been shown to give rise to progeny that may not display marker segregation aside from the mating type (Marra & Milgroom, 2001).
Our extensive genotyping of ECMs and fruit bodies suggests that the alleles unique to spores could be provided by ‘erratic’ strains in the sampled field. Monitoring soil biodiversity throughout seasons should enhance our understanding of the dynamics of truffle strains in the soil and, in turn, provide valuable insights into Tuber life cycle.
Dynamics of fungal mating types distribution on nursery-inoculated host plants
Host plants artificially inoculated to grow truffle species are now produced worldwide to sustain natural production and/or initiate ex novo truffle production, even in geographic areas where these fungi are not endemic (Hall et al., 2003). The method that uses a spore suspension as the inoculum has been the most widely used procedure since its development three decades ago (Fontana, 1967; Chevalier & Grente, 1978).
In light of T. melanosporum’s heterothallism, this inoculation procedure at first appears to be more appropriate than inoculation by in vitro-cultivated mycelium or by root contact between uninoculated and inoculated host plants, as it ensures the presence of both mating types in mycorrhizal plants. The successful inoculation of plants from spore inoculum depends on many factors, one of which is the ripening stage of the fruit body used as the spore donor, which should provide as many spores that are competent to germinate as possible. In a previous experiment in which we inoculated host plants with spores from T. magnatum ascocarps, we showed that some plants developed genetically different ECMs in equal ratios, whereas in others, ECMs produced by a single strain were prevalent (Paolocci et al., 2006). As these ECMs showed a genetic profile identical to that of the gleba of the truffle used as the spore donor, this result has been interpreted to mean that the mycelium of the gleba also is accountable for host root colonization, with spores unable to germinate or to compete with the ‘maternal’ mycelium. These findings fueled our interest in tracking the development of ECMs obtained under controlled conditions on host plants treated with spore suspensions from donor ascocarps to experimentally verify whether ECMs of both mating types would actually be produced. Of the 12 plants analyzed, only one (P10) had ECMs of the same mating type at 6 months PI. This suggests that the simultaneous development of ECMs of different mating type on the same root apparatus is indeed possible and confirms that most ECMs were derived from spore-germinated primary mycelia. However, just over 1 yr later, seven of the 12 plants screened showed all sampled ECMs with the same mating type, and a marked prevalence of one mating type in two of the remaining five plants (P2 and P3). Notably, the dominant mating type was not necessarily the same as the gleba of the fruit bodies used as the spore donor. These results suggest that the plant-colonizing capacity of a given strain over time does not result from gleba-derived mycelia performing better than those originating from spores.
Taken together, these experimental data overlap nicely with that of the field study. They strongly support the idea that competition among genetically different ECM strains occurs on a given host plant, ultimately leading to the prominence of a single strain and the displacement of all others. This phenomenon may be related to or result from mechanisms that control vegetative incompatibility. Such a phenomenon has been documented between in vitro-cultured strains of Tuber borchii (Sbrana et al., 2007). To the best of our knowledge, no studies have been carried out to test this phenomenon between T. melanosporum mycelia.
Strain displacement at the root level might also negatively interfere with mycelia spreading and/or viability in the soil. In most of the soil samples collected from pot-grown plants with ECMs of a single mating type or with a biased representation of the two genetic classes, a marked prevalence of a mating type-specific band relative to the dominant or prevalent strains was observed.
The present data provide substantial insight into truffle strain dynamics on host roots. They also suggest that to improve T. melanosporum production or to establish ex novo black truffle plantations, nursery-inoculated host plants harboring ECMs of both mating types should be used. In light of these findings, it cannot be taken for granted that spore-inoculated host plants can sustain ectomycorrhizal strains of both mating types on their roots. As the screening of each inoculated seedling to ascertain the mating type of their ECMs does not seem economically feasible, the use of plant plots inoculated with either MAT(+) or MAT(−) in vitro-cultivated mycelia strains appears to be a promising alternative to produce host plants with strains of certified mating type on their roots. Productive orchards can then be established by outplanting close to other seedlings harboring ECMs of different mating type. Studies aimed at determining the optimal spacing and ratios between plants with strains of different mating types should be performed in the next future.
In conclusion, although these results need to be corroborated by further molecular analyses on a larger number of host nursery-inoculated plants and on different natural and cultivated truffle stands, this study provides a breakthrough in the understanding of the distribution and dynamics of T. melanosporum strains of opposite mating types in open-field conditions and on host plants grown under controlled conditions. Our results are of both basic and applicative relevance. Future investigations will allow us to determine whether the biased distribution of mating types is a factor that truly limits truffle fructification. Finally, because it is possible that other economically important Tuber spp. are also heterothallic, this study paves the way for similar investigations in other truffle species.