Tuber melanosporum: mating type distribution in a natural plantation and dynamics of strains of different mating types on the roots of nursery-inoculated host plants


Author for correspondence:
Francesco Paolocci
Tel: +39 075 5014861


  • In light of the recent finding that Tuber melanosporum, the ectomycorrhizal ascomycete that produces the most highly prized black truffles, is a heterothallic species, we monitored the spatial distribution of strains with opposite mating types (MAT) in a natural truffle ground and followed strain dynamics in artificially inoculated host plants grown under controlled conditions.
  • In a natural truffle ground, ectomycorrhizas (ECMs), soil samples and fruit bodies were sampled and genotyped to determine mating types. Simple sequence repeat (SSR) markers were also used to fingerprint ECMs and fruit bodies. The ECMs from nursery-inoculated host plants were analysed for mating type at 6  months and 19 months post-inoculation.
  • In open-field conditions, all ECMs from the same sampling site showed an identical mating type and an identical haploid genotype, based on SSR analysis. Interestingly, the gleba of fruit bodies always demonstrated the same genotype as the surrounding ECMs. Although root tips from nursery-grown plants initially developed ECMs of both mating types, a dominance of ECMs of the same MAT were found after several months.
  • The present study deepens our understanding of the vegetative and sexual propagation modes of T. melanosporum. These results are highly relevant for truffle cultivation.


Truffles are edible fruiting bodies produced by symbiotic ascomycetes belonging to the genus Tuber. Fruiting is a crucial step in the fungal life cycle. Fruiting in Tuber spp. occurs only upon interaction of fungi with host plants, but the determinants that modulate the transition from the vegetative to the reproductive phase are still largely unknown. Thus far, only a few studies have focused on fungal species richness and distribution in truffle plantations (Murat et al., 2005; Baciarelli-Falini et al., 2006; Bertini et al., 2006; Iotti et al., 2010; Napoli et al., 2010; Rubini et al., 2010a).

The commonly held notion of Tuber spp. sexual reproduction considered these fungi to be homothallic or even exclusively selfing species (Bertault et al., 1998, 2001). Because homothallic strains can successfully complete the sexual cycle without a mating partner, fungal fructification in truffle plantations should depend only on the favorability of environmental and nutritional conditions under this model.

More recently, however, genetic evidence has emerged in support of the hypothesis that both Tuber magnatum and T. melanosporum, the two most profitable truffle species, can indeed outcross (Rubini et al., 2005; Paolocci et al., 2006; Riccioni et al., 2008). Furthermore, the work of Martin et al. (2010) and the companion paper to this study (Rubini et al., 2010b), demonstrated that T. melanosporum is a heterothallic species. Sexual development in filamentous ascomycetes is controlled by the mating type (MAT) locus (Coppin et al., 1997; Butler, 2007). In heterothallic ascomycetes, there are two alleles at this locus with highly dissimilar DNA sequences, known as idiomorphs (Metzenberg & Glass, 1990). As with many other filamentous ascomycetes, the T. melanosporum MAT1-1 idiomorph (herein also referred to as MAT(−)) contains a gene, MAT1-1-1, encoding a protein with an α-domain, whereas the MAT1-2 idiomorph (MAT(+)) carries a gene, MAT1-2-1, encoding a protein with an HMG-box domain (Martin et al., 2010; Rubini et al., 2010b). Heterothallic ascomycetes possess two different diffusible pheromone peptides and corresponding G protein-coupled receptors that function in the recognition and chemo-attraction of strains of opposite mating types (Bistis, 1981; Bölker & Kahmann, 1993; Dohlman & Thorner, 2001). The particular pheromone and receptor expressed depends on the MAT locus allele, although neither the pheromone nor the receptor is encoded at this locus. Analysis of genes involved in the mating process, including pheromone response, meiosis and fruiting body development, showed that the majority of sex-related components identified in other ascomycetes are also present in T. melanosporum (Martin et al., 2010).

Thus, strains of T. melanosporum are not self-compatible. Rather, in this fungal species, sexual reproduction occurs only between strains of opposite mating type.

A further layer of complexity is therefore added to investigating the determinants that control fructification processes in T. melanosporum, as the distribution of truffle strains carrying opposite mating types in natural sites must also be assessed. It is of paramount interest in artificial truffle plantations to encourage a balance of strains of both mating types. Thus, host inoculation procedures should be targeted to ensure the presence of both mating types on nursery inoculated plants used to establish artificial truffle plantations.

To address these issues, we monitored the spatio-temporal distribution of T. melanosporum strains in a natural plantation and on nursery-inoculated host plants. To achieve the first aim, truffle, mycorrhizal and soil samples were collected from a natural truffle ground located in central Italy and genetically typed using mating type-specific primers and primers to seven T. melanosporum-specific simple sequence repeat (SSR) loci. To achieve the second aim, host plants that were nursery-inoculated with spores from selected truffles were monitored over time to analyse the dynamics of plant colonization by different strains.

These studies showed a prevalence of ectomycorrhizas (ECMs) with the same mating type on single host plants. In addition, sexual distribution in a natural field site was patchy and unbalanced, with plants colonized by single ectomycorrhizal strains. All of these results help to elucidate truffle biology and are also of considerable practical impact for optimizing host plant inoculation and increasing production in truffle fields.

Materials and Methods

Sampling of ECMs, fruit bodies and soil samples from a natural truffle ground

The natural T. melanosporum ground investigated is located in Borgo Cerreto, near Spoleto, central Italy (Fig. 1). Soil and mycorrhizal samples were collected from productive areas (hereinafter referred to as sites) in late spring 2009 and in winter 2009–2010, following the indications of the truffle ground owners (Fig. 1, Tables 1 and 2). The host species present in the sampled sites were Quercus pubescens and Ostrya carpinifolia. Host species were of different age and size. Other non-host trees and shrubs such as Fraxinus ornus, Acer sp., Spartium junceum and Juniperus sp. were also present. Details about sample positions and putative host plants in each sampled site are given in the Supporting Information, Fig. S1.

Figure 1.

 Map of Borgo Cerreto truffle ground showing the sampling sites. The collection sites of roots (r), soil (s) and ascocarps (fb) are indicated with closed circles; those of roots and soil are indicated with open circles; sampling sites of roots only are indicated with open triangles. Tinted areas indicate cultivated sites.

Table 1.   Mating type- and simple sequence repeat (SSR)-based fingerprinting of Tuber melanosporum ectomycorrhizas (ECMs) from a natural truffle ground
SiteSample nameECMs1MAT2SSR (bp)Genotypes
  1. 1Number of ECMs analysed.

  2. 2(+), MAT1-2-1; (−), MAT1-1-1.

  3. 3Samples collected in winter 2009–2010, all the other samples were collected in Spring 2009.

  4. nd, Not detected.

Table 2.   Mating type characterization of Tuber melanosporum from the soil in a natural truffle ground
SiteSample nameMAT
  1. 1Samples collected in winter 2009–2010, all the other samples were collected in spring 2009.

  2. nd, Not detected.


Soil and root samples were collected using a soil corer at c. 15–20 cm depth and were stored in sterile plastic bags for no > 3 d at 4°C. The root samples were soaked in water and sieved to separate root fragments and ECMs from the soil. Fine roots were analysed under a stereomicroscope to evaluate the presence of T. melanosporum ECMs according to morphological markers (Zambonelli et al., 1993; Rauscher et al., 1995). From each root sample, 5–15 single T. melanosporum ECMs were randomly collected under the stereomicroscope and stored in microcentrifuge tubes at −80°C for molecular analyses (Table 1). The identity of T. melanosporum ECMs was confirmed by molecular analysis using species-specific internal transcribed spacer (ITS) primers (Paolocci et al., 1999).

Truffle ascocarps were harvested with the help of a trained dog during the winter of 2009–2010. Additional soil and root samples were gathered near ascocarps collected from sites other than those sampled in spring 2009 (see Tables 1 and 2).

The ECM sample names, collection sites, soil samples and ascocarps are given in Tables 1, 2 and 3, respectively, and in Fig. 1. Geographic coordinates and pairwise distances among sites are given in Table S1.

Table 3.   Mating type- and simple sequence repeat (SSR)-based fingerprinting of Tuber melanosporum ascocarps
  1. 1Samples collected in December 2009 (d) and January 2010 (j).

  2. 2Spore samples always produced both MAT(+) and MAT(−) PCR amplicons.

  3. sp, Spore samples; g, gleba samples; M, maternal; P, paternal.

243FB-576d(+)292292/285147147141141134134/116141141312312463463II (+)VIII (−)
FB-591j(+)292292147147141141134134141141312312463463II (+)IV (−)
245FB-577d(−)292292147147159159/141134134141141312312463463IX (−)II (+)
244FB-578d(+)289289/292147147141141134134141141310310463463I (+)VI (−)
FB-579d(+)289289147147141141134134141141310310463463I (+)X (−)
FB-580d(+)289289147147141141134134141141310310/312463463I (+)XI (−)
FB-581d(+)289289147147141141134134141141310310463463I (+)X (−)
FB-592j(+)289289147147141141134134141141310310463463I (+)X (−)
FB-593j(+)289289147147141141134134141141310310463463I (+)X (−)
241FB-582d(−)290290147147159159/141134134141141312312463463III (−)XII (+)
235FB-590j(−)292292147147141141134134141141310310463463VI (−)XIII (+)
236FB-583d(−)290290147147/144141141134134141141310310/312463463V (−)XIV (+)
FB-584d(−)290290147147141141134134141141310310/312463463V (−)XII (+)
FB-585d(−)290290147147141141134134/130141141310310463463/467V (−)XV (+)
FB-594j(−)290290/292147147141141134134/138141141310310/312463463/465V (−)XVI (+)

Glasshouse host plant inoculation and sampling of soil and ECMs

Tuber melanosporum-inoculated plants were prepared as follows: seeds of Q. pubescens were treated with a 5% NaOCl solution for 15 min, washed in sterile water and placed in sterile agriperlite (VIC, Milan, Italy) in a glasshouse at 25°C. After c. 1 month, the young plantlets were transferred to plastic bags containing an autoclaved (121°C for 180 min) peat–sand–loamy soil mixture (2 : 1 : 5, v : v : v). Each plant was inoculated by covering the roots with c. 200 ml of potting media previously mixed with 3 g of spore slurry obtained from fresh T. melanosporum ascocarps. In total, four plots consisting of 10 plants each were prepared, and each plot was inoculated with spores from a single T. melanosporum ascocarp. Before being used for plant inoculation, the ascocarps were checked with T. melanosporum species-specific ITS primers according to Paolocci et al. (1997) and MAT-specific primers (Table S2) to ascertain the species identity and the mating type of their gleba, respectively. Two truffles with gleba formed by hyphae harboring the MAT(−) idiomorph and two with gleba containing the MAT(+) idiomorph (Table 4) were selected for plant inoculation.

Table 4.   Mating type characterization of Tuber melanosporum ectomycorrhizas (ECMs) from inoculated plants
PlotAscocarpPlantOctober 20081November 20091
MAT (+)MAT (−)MAT (+)MAT (−)
  1. 1Number of ECM tips for each mating type is reported.

Imel1 (−)P173140
IImel6 (−)P473014
IIImel2 (+)P764150
IVmel4 (+)P1090150

After inoculation, the plots were spaced in the glasshouse to avoid cross-contamination, and pots were placed on a metallic grid to avoid any contact with the ground. Plants were grown at 25°C under natural light conditions and irrigated with tap water purified with active charcoal.

Roots from three plants in each plot were collected 6 months (October 2008) and 19 months (November 2009) post inoculation (PI). The first sampling was performed by cutting portions of root branches using a sterile scalpel and randomly collecting the ECMs. After the first sampling, the plants were grown in a glasshouse until the second sampling. Soil samples were collected in November 2009 by taking three replicates per plant at c. 120° and at c. 5 cm from the stem. Absence of root parts in the soil samples was ascertained using a stereomicroscope. Tables 4 and 5 provide names and collection dates of the ECMs and soil samples, respectively.

Table 5.   Mating type characterization of Tuber melanosporum DNA from the soil of inoculated plants
PlotPlantSoil samples
  1. Values indicate the proportion of MAT(+)- and MAT(−)-specific PCR products.


DNA isolation and molecular analyses

Genomic DNA was isolated from single mycorrhizal root tips as described by Paolocci et al. (1999). Soil DNA was isolated from 0.25 g of soil using a PowerSoil DNA Isolation Kit (Mo Bio Laboratories, Solana Beech, CA, USA) according to the manufacturer’s instructions. DNA from ascocarps and pools of spores were isolated according to Paolocci et al. (2006).

Mating type analyses were carried out using the T. melanosporum mating type-specific primers according to Rubini et al. (2010b). The SSR analyses were carried out with seven markers (Table S2) derived from the analysis of the T. melanosporum genome (Martin et al., 2010) and selected on the basis of showing polymorphic alleles in a preliminary screen carried out on truffles from different populations (C. Murat et al., unpublished). In Table S2 the genomic position of each locus investigated is also given.

All PCR amplifications were carried out in a Gene Amp 9700 PCR system (Applied Biosystems, Foster City, CA, USA) using the cycling parameters reported by Rubini et al. (2010b) for the MAT loci and by C. Murat et al. (unpublished) for the SSR loci. The use of MAT sequences is covered by EU patent application n°. 10 175517.1. Polymerase chain reaction reactions were performed in a 50-μl reaction mixture containing 200 μM of each dNTP, 10 pmol of each primer, 4 mM MgCl2, 10 mM Tris-HCl pH 9.0, 50 mM KCl, 2.5 units of Taq polymerase (GE Healthcare, Roosendal, Netherlands) and 5–10 ng of target DNA. For DNA from ECM samples, 0.35 mg of bovine serum albumin (BSA) was added to the PCR reactions according to Paolocci et al. (1999). All PCR experiments included a negative control (no DNA template).

When both mating type-specific PCR products were obtained from soil samples, the two PCR bands were separated by agarose gel electrophoresis in the presence of ethidium bromide and then photographed under a UV transilluminator. The intensity of PCR bands was then measured from the acquired digital images using ImageJ densitometry software (Version 1.42, National Institutes of Health, Bethesda, MD, USA). The relative band intensity within each sample was calculated by dividing the intensity value of each band by the sum of the intensity values of both bands.


Mating type- and SSR-based genetic fingerprinting of T. melanosporum ECMs from a natural truffle ground

A total of 32 root samples were collected from 15 productive sites in a natural truffle ground of Borgo Cerreto (Table 1). Multiple root samples were collected from short distance (1–2 m) within the ‘burned areas’ typically characterized by the absence of understory vegetation (Fasolo-Bonfante & Fontana, 1971). Tuber melanosporum ECMs were found in at least one root sample per site, with the exception of sites 242, 245 and 247, despite performing two samplings in site 247 (ECM-07 and ECM-08). Overall, of the 32 root samples analysed, 19 showed the presence of T. melanosporum ECMs (Table 1). A total of 155 single T. melanosporum ECMs, distributed among these 19 root samples, were genotyped using MAT- and seven SSR-specific primer pairs. Remarkably, each single mycorrhizal sample yielded only a single amplicon per primer pair, regardless of the primer pairs used. Each mycorrhiza displayed either the presence of the MAT(+) or MAT(−) idiomorph. Furthermore, different ECMs from the same root sample, as well as ECMs from the same collection site, always displayed the same mating type. Fig. 2 shows the mating type of ECMs from across the truffle ground. It is notable that ECMs from sites close each other (in the range of 3–30 m) shared the same mating type, suggesting the prevalence of a single strain or, alternatively, of multiple strains with the same mating type beneath each soil patch. In particular, ECMs from the three southernmost collection sites (236, 238 and 241) all showed the MAT(−) idiomorph, as did ECMs from the proximal sites 237, 248 and 249 in the northernmost area of the truffle field. Ectomycorrhizas harboring the MAT(+) idiomorph were predominantly located in the central zone of the field. The shortest distance between sites displaying ECMs with different mating types was c. 50 m (from site 235 to sites 244 and 239).

Figure 2.

 Map of Borgo Cerreto truffle ground showing the distribution of mating types and genets among ectomycorrhizas (ECMs). Tinted areas indicate cultivated sites.

All 155 ECMs were genotyped using the SSR markers. Polymorphisms were detected at locus ssrme09 (three alleles) and at loci ssrme03, ssrme15 and ssrme29 (two alleles each) (Table 1). Overall, by combining results from MAT and SSR analyses, seven different genotypes were identified (Table 1, Fig. 2). This analysis also showed that all of the ECMs from a given root sample not only shared the same mating type but also an identical SSR multilocus genotype. For example, the 60 ECMs from six independent samplings (ECM-01 to ECM-06) within collection site 244 all shared the same genotype (I), which suggests that this area is colonized by a single strain that likely represents a genet. Notably, all ECMs from site 239, the collection site closest to 244, also showed genotype I. Conversely, SSR genotyping revealed that ECMs from nearby sites (3–7 m further apart) were colonized by different genets, all with the same mating type. For example, ECMs from sites 249 and 248 showed mycelial strains harboring the MAT(−) idiomorph but different allelic configurations at the SSR loci, with ECMs from site 249 exhibiting genotype VII and those from site 248 genotype IV. The distribution of mating types and genets of all ECM samples are given in Figs 2 and S1.

Mating type characterization of T. melanosporum in the soil in the Borgo Cerreto truffle ground

A total of 29 soil samples were collected from 14 sites (Table 2, Fig. S1) and examined by PCR using MAT-specific primers. Twenty-six samples yielded one or two MAT-specific amplicons, but the remaining samples did not yield any (Table 2). The identity of the amplicons was confirmed by direct sequence analysis of products from sites that yielded only the MAT(−)-specific (i.e. S-09) or only the MAT(+)-specific (i.e. S-02) band (data not shown). At least one soil sample per site produced a T. melanosporum-specific MAT amplicon, suggesting that black truffle was always present in productive soil patches, despite the fact that some soil samples were collected independently and were relatively distant from the position of the ECMs. In the majority of the cases (20 of the 26), soil samples showed a single amplicon, generally corresponding to the mating type displayed by ECMs from the same collection site (Table 1). Specifically, for collection site 244 (from which 60 ECMs and seven soils samples were collected and analysed), most of the soil samples (five of seven) exhibited the MAT(+) amplicon, as did all ECMs from this site. The remaining two soil samples (S-01 and S-06) showed both MAT(+)-specific and MAT(−)-specific amplicons. Similarly, the two soil samples from site 243 and one (S-29) of the two samples from site 250 showed the presence of both MAT(+) and MAT(−) in the soil, but all of the ECMs from these sites were exclusively MAT(+). Of the two soil samples (S-25 and S-26) that produced T. melanosporum mating type-specific amplicons from site 249, one (S-26) showed both MAT(+) and MAT(−) bands, but all mycorrhizas from this site displayed the MAT(−) idiomorph. It is interesting to note that T. melanosporum was detected beneath some productive sites (245, 247 and 242) where no T. melanosporum ECMs were found (Table 2, Fig. S1).

Genetic fingerprinting of ascocarps from the natural truffle ground

A total of 15 T. melanosporum ascocarps were harvested from six sites during the winter collection season of 2009–2010 (Table 3). For each of these ascocarps, pools of spores were isolated from the surrounding gleba, and DNA from both the spores and the corresponding gleba was analysed by PCR using primers specific for the MAT locus and the seven SSR loci used to genotype ECMs. The genotype of each gleba was defined on the basis of SSR and MAT screenings. In contrast to the gleba, pools of spores always displayed both MAT-specific amplicons, and some also displayed additional SSR alleles with respect to the corresponding gleba (Table 3). Thus, by integrating the information from the analysis of the gleba, which is considered ‘maternal’ (M) tissue (Paolocci et al., 2006), with that derived from the spores, the putative genotype of the ‘paternal’ (P) partner for each truffle was defined (Table 3). Intriguingly, the gleba of these truffles always had the same genotype as the ECMs sampled from the same areas. For example, the gleba of all truffles collected from site 244 (FB-578 to FB-581, FB592 and FB-593) were genotype I, as were the 60 ECMs sampled in this area. However, truffles harvested from the same areas and collected simultaneously showed different ‘paternal’ partners. This was the case for truffles FB-578, FB-579 and FB-580 (site 244) and truffles FB-583, FB-584 and FB-585 (site 236). Despite being productive, no T. melanosporum ECMs were recorded in collection site 245, and soil samples exhibited only the MAT(−) idiomorph, the same mating type displayed by the gleba of the single truffle collected there.

By combining the data from the gleba and spores within each truffle with that of the ECMs at the MAT locus and at six polymorphic SSR loci, 16 genotypes were identified (I–XVI, Table 6). The I/X, II/IV and VI/XIII genotypes shared identical SSR alleles but had different configurations at the mating type locus. Genotypes I–VII were derived from analysis of ECMs. With the sole exception of genotype VII, all of these genotypes were also recorded in the gleba of the truffles. Genotype VII was only exhibited by ECMs from collection site 249, which unfortunately did not produce any truffles during the last productive season. Different SSR alleles from those displayed by the gleba and ECM samples were recorded in the spore pools. Spores of FB-576 harbored alleles 285 and 116 at the ssrme09 and ssrme17 loci, respectively, which were never detected in ECMs or soil DNA. Similarly, none of the ECMs showed alleles 130 or 138 at locus ssrme17, or alleles 467 and 465 at locus ssrme30, which were only found in the spores of samples FB-585 and FB-594, respectively.

Table 6.   Genotypes identified within the natural truffle ground
  1. 1Genotypes inferred only from ascocarp analysis.

  2. 2Alleles detected in ascocarps only.


Assortment and spatiotemporal dynamics of T. melanosporum mycelia of opposite mating types on nursery-inoculated host plants

The presence and spatiotemporal distribution of mycelia of opposite mating types was evaluated in ECMs and soils from pot-grown plants inoculated with spores obtained from four mature T. melanosporum ascocarps (see the Materials and Methods section). As a control, pools of spores from the selected truffles were genotyped to verify that mating and meiosis in these samples had occurred. As expected the pools of spores always showed the presence of both MAT(+)-specific and MAT(−)-specific bands of approximately equal intensity, regardless of the mating type exhibited by the corresponding gleba (data not shown).

Six months PI, all inoculated plants showed the presence of T. melanosporum mycorrhizas with almost all root tips colonized by the inoculating fungus. Other mycorrhizal species were not detected. Three plants from each plot were then selected for the collection of c. 10 T. melanosporum ECMs per plant (Table 4). The DNA from each ECM tip produced a single amplicon from the MAT locus, either MAT(+) or MAT(−). With the sole exception of plant P10, whose ECMs all displayed the MAT(+) genotype, ECMs of both mating types were found in all the other plants.

For plots I and II, which were inoculated with MAT(−) ascocarps, ECMs of both mating types were almost equally distributed in plants P2 and P3 (plot I). Plant P1 and plants from plot II showed a slight prevalence of one of the two mating types.

For plots III and IV, which were inoculated with MAT(+) ascocarps, plants P7–P9 showed a balance of both MAT(+) and MAT(−) ECMs. Conversely, plants from plot IV showed a prevalence of ECMs of a given mating type. Plant P10 displayed only MAT(+) ECMs, whereas in P11 and P12 a prevalence of MAT(−) (7 : 3) or MAT(+) (7 : 2) ECMs was recorded, respectively.

In the second sampling (19 months PI), root density increased and, similar to the first sampling, almost all of the tips resulted to be colonized by T. melanosporum. No ECMs of other species were present. At this point, 15 ECMs per plant were randomly collected and analysed. Remarkably, in 7 of the 12 plants, ECMs with only one of the two mating types were detected. In addition to P10, plants P1, P4, P7, P9, P11 and P12 exhibited ECMs with only one mating type. Interestingly, of the two plants from the same plot (P7 and P9) that displayed a balance of ECMs of both mating types at 6 months PI, P7 showed only MAT(+) and P9 only MAT(−) ECMs by 19 months PI. Furthermore, at this point, a strong prevalence of one mating type on two (P2 and P3) of the five plants with ECMs of both mating types was observed (Table 4).

Next, soil samples were collected from plants displaying ECMs with exclusively one or one predominant mating type to test whether the prevalence of a given mating type on host roots could affect the presence or viability of T. melanosporum mycelia of the opposite sexual polarity in the soil. For this purpose, DNA was isolated from three soil samples per plant and PCR amplified with T. melanosporum-specific MAT primers. When PCR analysis showed both mating types in the same soil sample, the relative intensities of MAT(+)- and MAT(−)-specific bands were measured (Table 5). The PCR analysis showed a good agreement between the mating type detected in the soil and that of the ECMs.

Specifically, in the soil samples collected from plants P7, P9 and P10, whose ECMs harbored a single mating type, only the amplicon corresponding to that mating type was amplified (Table 5). From plants P1, P3, P5 and P11, at least one of the three soil samples produced a single MAT amplicon corresponding to that detected in the majority of the ECMs from the same plants. Conversely, the remaining soil samples yielded both MAT amplicons, although with different intensities. The amplicon corresponding to the mating type not detected, or less prevalent, on the ECMs was in fact far less intense than the other. An exception to this trend was seen in sample P4 where all ECMs displayed the MAT(−) genotype at 19 months PI, but a balanced presence of both MAT(+) and MAT(−) strains was detected in the soil.


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.


BB and CR were supported by Regione Umbria. The authors are grateful to Mr Dominici Amedeo and Mr Francesco Pontani, the owners of the truffle ground investigated.