Detection of Tuber melanosporum DNA in soil

Our objectives were (i) to develop a molecular method to detect mycelia of Tuber melanosporum (black truffle) in soil and (ii) to test for mycelial distribution around two truffle-bearing Quercus ilex trees in a truffle orchard. Isolation of total DNA from soil was performed, followed by PCR amplification with T. melanosporum-specific primers and restriction analysis. To address the detection sensitivity level, soil samples were inoculated with known amounts of gleba of T. melanosporum. The detection limit was >/=11.4 mug of hyphae g(-1) of soil. Mycelium was detected primarily within the area defined by the truffle burn and within the top 35 cm of the soil in all directions from the trees.     

Quantification of extraradical mycelium of Tuber melanosporum in soils from truffle orchards in northern Spain

Quantification of extraradical mycelium of black truffle (Tuber melanosporum) has been carried out in a natural truffle ground and in seven truffle orchards (around 20 years old) established in Tierra Estella and Valdorba sites, within the natural distribution area of the black truffles in Navarre (northern Spain). Specific primers and a Taqman® probe were designed to perform real-time PCR with DNA extracted from soil samples. Amplification of T. melanosporum DNA was obtained from 131 out of the 160 soil samples. The detection limit of the technique was 1.48 μg mycelium/g of soil. The extraradical mycelium biomass detected in the soil from the natural truffle ground was significantly greater (up to ten times higher) than the mycelium biomass detected in any of the orchards. Soil from productive, nonirrigated orchards in the Tierra Estella site contained significantly more extraradical mycelium than the rest of orchards irrigated, productive of T. brumale, or nonproductive. The comparison of soil mycelium biomass in nonirrigated evergreen oak orchards in both sites showed significantly more mycelium biomass in the Tierra Estella site. This study is the first attempt to quantify extraradical mycelium of T. melanosporum in the soil using Taqman® probes. The obtained quantitative results are of special interest to evaluate the fungal response to cultural treatments and to monitor the dynamics of the extraradical mycelium of T. melanosporum in the soil.     

Occurrence of Tuber melanosporum in relation to soil surface layer properties and soil differentiation

An intensive survey was carried out on a 12-year-old experimental truffle bed of Tuber melanosporum Vitt. located in the central Apennines. The aim of the investigation was to relate the presence and carpophore production of T. melanosporum to changes in soil structure, aeration and fertility — expressed in terms of 0.25–2.00 mm aggregate fraction, total organic carbon, DTPA-extractable Mn and host plant height — and to determine if these modifications, whenever present, could be ascribed to soil differentiation within the truffle bed. The occurrence of pianelli — i.e. areas with little herbaceous ground cover created by T. melanosporum — showed a close relationship with host plant height and aeration of soil surface layers. Where pianelli occurred, the height of symbiont trees increased and the content of reduced Mn, indicating the presence of a well-aerated soil environment, decreased. The variation of host plant height was attributable not only to the increased absorption of nutrients related to the ectomycorrhizal partnership, but also to soil differentiation. The soils of the investigated area were characterized by a relatively low slope gradient, a rigid framework of gravel and a homogeneous physico-chemical behaviour, due to the predominance of Ca among exchangeable bases. In these environmental conditions, T. melanosporum was present in the rather thick soil belonging to Typic Rendolls, whereas it was absent in the area characterized by thin Lithic Rendolls. In the latter case, the plant cover was probably too scarce to protect T. melanosporum from summer dryness, and consequently the more resistant T. aestivum species prevailed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparison of ectomycorrhizal communities in natural and cultivated Tuber melanosporum truffle grounds

Truffles are hypogeous ectomycorrhizal (EM) fungi belonging to the genus Tuber. Although outplanting of truffle-inoculated host plants has enabled the realization of productive orchards, truffle cultivation is not yet standardized. Therefore, monitoring the distribution of fungal species in different truffle fields may help us to elucidate the factors that shape microbial communities and influence the propagation and fruiting of Tuber spp. In this study, we compared the fungal biodiversity in cultivated and natural Tuber melanosporum truffle fields located in Central Italy. To this end, ectomycorrhizas (ECM) and soil samples were molecularly analyzed, and an inventory of the fungi associated with Quercus pubescens plants colonized by T. melanosporum, Tuber aestivum or Tuber brumale was compiled. T. melanosporum and T. aestivum were dominant on the cultivated plants, and the number of EM species was markedly lower in the cultivated sites than in the natural sites. However, in the same site, EM biodiversity was higher in T. brumale-colonized plants than in T. melanosporum-colonized plants. These results suggest that different Tuber spp. may have different competitive effects on the other mycobionts. Additionally, in keeping with our previous findings, we found that the number of T. melanosporum genotypes recovered from the soil samples was higher than that of the underlying ECM.     

Genetic Diversity and Mating Type Distribution of Tuber melanosporum and Their Significance to Truffle Cultivation in Artificially Planted Truffieres in Australia

Tuber melanosporum is a truffle native to Europe and is cultivated in countries such as Australia for the gastronomic market, where production yields are often lower than expected. We assessed the genetic diversity of T. melanosporum with six microsatellite loci to assess the effect of genetic drift on truffle yield in Australia. Genetic diversity as assessed on 210 ascocarps revealed a higher allelic diversity compared to previous studies from Europe, suggesting a possible genetic expansion and/or multiple and diverse source populations for inoculum. The results also suggest that the single sequence repeat diversity of locus ME2 is adaptive and that, for example, the probability of replication errors is increased for this locus. Loss of genetic diversity in Australian populations is therefore not a likely factor in limiting ascocarp production. A survey of nursery seedlings and trees inoculated with T. melanosporum revealed that <70% of seedlings and host trees were colonized with T. melanosporum and that some trees had been contaminated by Tuber brumale, presumably during the inoculation process. Mating type (MAT1-1-1 and MAT1-2-1) analyses on seedling and four- to ten-year-old host trees found that 100% of seedlings but only approximately half of host trees had both mating types present. Furthermore, MAT1-1-1 was detected significantly more commonly than MAT1-2-1 in established trees, suggesting a competitive advantage for MAT1-1-1 strains. This study clearly shows that there are more factors involved in ascocarp production than just the presence of both mating types on host trees.     

Spatio-Temporal Dynamic of Tuber magnatum Mycelium in Natural Truffle Grounds

Tuber magnatum produces the world''s 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.     

Phylogenetic relationships between Tuber pseudoexcavatum, a Chinese truffle, and other Tuber species based on parsimony and distance analysis of four different gene sequences

Phylogenetic relationships between Tuber pseudoexcavatum and other Tuber species were investigated by studying the sequences of four genes: 5.8S-ITS2, beta-tubulin, protein kinase C and elongation factor 1alpha. The four phylogenetic trees allowed to differentiate the black truffle clade, composed of two subclades, one comprising the Asian black truffles (T. indicum, T. sinense, T. himalayense) and the Perigord black truffle (T. melanosporum), the second comprising T. pseudoexcavatum and T. brumale. These two subclades diverged relatively early. We propose a common ancestor, located between Europe and China, to all the black truffles. The T. brumale/pseudoexcavatum subclade would have started to diverge and migrate first, T. brumale towards Europe through a northern route and T. pseudoexcavatum towards China. Later the T. melanosporum subclade would have started to migrate through the same route, T. melanosporum towards Europe and T. indicum towards China, leading to vicariant species.     

Bacterial and fungal communities associated with Tuber magnatum‐productive niches

Abstract

Truffles are hypogeous ectomycorrhizal fungi of ecological interest for forestry in soils of the northern hemisphere, and of economical relevance for food markets worldwide. The molecular mechanisms that control truffle body formation are largely unknown, as well as the environmental factors that are likely involved. Among the latter, it has been hypothesized that soil‐borne communities may have an impact on truffle production. To address this question, we investigated bacterial and fungal communities resident in productive versus adjacent non‐productive grounds of the white truffle Tuber magnatum by using PCR‐DGGE. Although bacterial communities were generally highly similar across all samples within the grounds, profiles did cluster according to the productivity of circumscribed niches, and a Moraxella osloensis population appeared to be associated with productive sites. Fungal communities revealed several populations, yet showed no obvious patterns in relation to productivity, although Mortierella and Fusarium oxysporum appeared to be more abundant in the productive area. Our results offer a first glimpse into microbial communities thriving in truffle productive niches, and open the question as to whether microbe‐mediated mechanisms may facilitate/inhibit truffle fruiting‐body production or, vice versa, i.e. whether truffle sporocarps have an impact on the microbes living in the rhizosphere.     

New data on ectomycorrhizae and soils of the Chinese truffles Tuber pseudoexcavatum and Tuber indicum, and their impact on truffle cultivation

Chinese truffles serve as a good complement to the market for Tuber melanosporum (Périgord black truffle). However, Chinese truffles could be introduced accidentally or fraudulently into the plantations of Mediterranean truffles, and they could have a negative effect on truffle production and natural ecosystems. The study of Tuber species from China which are commercialized in Europe began 14 years ago. Tuber pseudoexcavatum was proposed as a new species, and this has been validated by some authors based on molecular and phylogenetic studies. We synthesize their ectomycorrhizae using samples from the type collection, and we compare T. pseudoexcavatum and Tuber indicum ectomycorrhizae. The ectomycorrhizae of these species have a morphology which is related to the ectomycorrhizae of T. melanosporum. We provide useful information for the rapid screening of the above-mentioned Chinese truffles ectomycorrhizae, for the quality control of commercial plants mycorrhized with Tuber. Moreover, we analyze the soil tolerance and the host plant affinity of T. pseudoexcavatum and T. indicum, in order to assess the capacity of both Chinese truffles to penetrate T. melanosporum plantations and habitats.     

Discrimination of truffle fruiting body versus mycelial aromas by stir bar sorptive extraction

Stir bar sorptive extraction (SBSE) was applied in head space mode (HS), coupled with GC/MS, to compare the aroma profile of three truffle species. A total of 119 volatile organic compounds (VOCs) were identified from the fruiting bodies, of which 70 were not yet described in truffles and 60 in fungi. VOCs profile showed a high intra- and inter-specific variability, with alcohols and sulfur compounds dominating the HS of Tuber borchii and, alcohols, aldehydes and aromatic compounds the HS of T. melanosporum and T. indicum. Despite these variations, eight VOCs markers could be identified allowing the discrimination of the three species. Additionally, T. borchii and T. melanosporum both distinguished themselves from T. indicum due to higher aroma content and larger variety of sulfur containing compounds. Mycelial VOCs production was also investigated under two cultural conditions and led to the identification of eight VOCs. On one side, seven of them were also detected in the fruiting body, confirming their mycelial origin. On the other side, the total absence of some class of compounds (i.e. sulfur) in the mycelium raises questions about their origins in the fruiting bodies and confirms deep metabolic changes between the reproductive (fruiting body) and vegetative (mycelium) stages.     

Mycorrhizal inoculation of cloned hazels by Tuber melanosporum: effect of soil disinfestation and co-culture with Festuca ovina

A pot experiment was conducted to determine the effects of soil disinfestation and presence of Festuca ovina L. on the formation of Tuber melanosporum Vitt. mycorrhizae by Corylus avellana L. Festuca ovina reduced colonization of young hazels by T. melanosporum. Co-culture with F. ovina resulted in necrosis of hazel roots, leading to 33% plant mortality. The surviving hazels were poorly colonized by T. melanosporum and highly colonized by other symbionts. Roots of F. ovina did not seem to attract mycelium or to be infected by T. melanosporum. Relatively poor mycorrhizal colonization developed in non-disinfested soil containing native inoculum of Tuber melanosporum. Mycorrhization by T. melanosporum developed very efficiently in disinfested soil inoculated with a spore suspension with relatively little colonization by competing symbionts. Growth of host plant was also affected.     

Phylogenetic relationships between European and Chinese truffles based on parsimony and distance analysis of ITS sequences

Phylogenetic relationships among truffle species from Europe and China were investigated through parsimony analysis of the ITS sequences. Three major clades were obtained among the species analysed. The so-called white truffles appeared polyphyletic since Tuber magnatum was grouped with brown truffles and not with the other white species (T. maculatum, T. borchii, T. dryophilum, T. puberulum). The black truffles investigated in this study, T. brumale, T. melanosporum, T. indicum and T. himalayense, were grouped in an independent clade. The Périgord black truffle T. melanosporum and the Chinese black truffles T. indicum and T. himalayense, were very closely related. The delimitation of these species was estimated by a distance analysis on several isolates collected from different geographic areas. In spite of intraspecific variations of the internal transcribed spacers (ITS) sequences, T. melanosporum and the Chinese black truffles can be unambiguously attributed to distinct taxa.     

Viscosinamide-producing Pseudomonas fluorescens DR54 exerts a biocontrol effect on Pythium ultimum in sugar beet rhizosphere

Two repeated DNA sequences of European strains of the symbiotic fungus Tuber melanosporum were isolated and characterized. One of these, SS14, representing about 0.05% of the fungal genome, was shown to be a T. melanosporum-specific sequence by Southern and dot-blot hybridization. The second one, named SS15, is about 0.0025% of the entire genome, and it is specific not only to T. melanosporum but also to the Asian black truffle Tuber indicum. Neither of these two fragments hybridizes with any of the other European truffle species tested. By sequence analysis of these two fragments, PCR primers were designed and used to selectively amplify DNA from T. melanosporum ascocarps and ectomycorrhizae by simple and multiplex PCR. No amplification products were obtained with DNA from either mycorrhizal roots or fruit bodies of other ectosymbiotic fungi. The two identified genomic traits also provided useful information for a better understanding of the phylogenetic relationships among black truffle species and for testing T. melanosporum intraspecific variability.     

Biochemical, electrophoretic and immunohistochemical aspects of malate dehydrogenase in truffles (Ascomycotina)

The malate dehydrogenase (MDH; EC 1.1.1.37; L-malate-NAD(+)-oxidoreductase) activities of truffles of the genus Tuber (Tuber melanosporum Vittad., Tuber brumale Vittad., Tuber aestivum Vittad., Tuber magnatum Pico, Tuber rufum Pico) have been characterized with regard to the K(m) and V(max) values in the direct and reverse reactions. The isoelectrofocusing has revealed bands showing pI values ranging from pH 5.85 to 7.8. The MDH of T. melanosporum has been partially purified by hydroxyapatite treatment, DEAE-cellulose and Sephadex G-75 columns. With the partially purified T. melanosporum MDH activity polyclonal anti-T. melanosporum MDH antibodies have been prepared and used to localize MDH in the mycorrhizae and ascocarps of T. melanosporum. These antibodies inhibit T. melanosporum MDH activity as well as that of T. magnatum but not that of rabbit liver; this supports the specificity of the MDH antibodies used to localize MDH in truffle tissues.     

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

? 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.     

Distribution and localization of microsatellites in the Perigord black truffle genome and identification of new molecular markers

The level of genetic diversity and genetic structure in the Perigord black truffle (Tuber melanosporum Vittad.) has been debated for several years, mainly due to the lack of appropriate genetic markers. Microsatellites or simple sequence repeats (SSRs) are important for the genome organisation, phenotypic diversity and are one of the most popular molecular markers. In this study, we surveyed the T. melanosporum genome (1) to characterise its SSR pattern; (2) to compare it with SSR patterns found in 48 other fungal and three oomycetes genomes and (3) to identify new polymorphic SSR markers for population genetics. The T. melanosporum genome is rich in SSRs with 22,425 SSRs with mono-nucleotides being the most frequent motifs. SSRs were found in all genomic regions although they are more frequent in non-coding regions (introns and intergenic regions). Sixty out of 135 PCR-amplified mono-, di-, tri-, tetra, penta, and hexa-nucleotides were polymorphic (44%) within black truffle populations and 27 were randomly selected and analysed on 139 T. melanosporum isolates from France, Italy and Spain. The number of alleles varied from 2 to 18 and the expected heterozygosity from 0.124 to 0.815. One hundred and thirty-two different multilocus genotypes out of the 139 T. melanosporum isolates were identified and the genotypic diversity was high (0.999). Polymorphic SSRs were found in UTR regulatory regions of fruiting bodies and ectomycorrhiza regulated genes, suggesting that they may play a role in phenotypic variation. In conclusion, SSRs developed in this study were highly polymorphic and our results showed that T. melanosporum is a species with an important genetic diversity, which is in agreement with its recently uncovered heterothallic mating system.     

Soil analysis reveals the presence of an extended mycelial network in a Tuber magnatum truffle-ground

Truffles are hypogeous ectomycorrhizal fungi. They belong to the genus Tuber and are currently considered a hot spot in fungal biology due to their ecological and economic relevance. Among all the species, Tuber magnatum is the most appreciated because of its special taste and aroma. The aim of this work was to set up a protocol to detect T. magnatum in soil and to assess its distribution in a natural truffle-ground. We used the β-tubulin gene as a marker to identify T. magnatum in the soil. This gene allowed us to trace the distribution of the fungus over the entire truffle-ground. Tuber magnatum was found, in one case, 100 m from the productive host plant. This study highlights that T. magnatum mycelium is more widespread than can be inferred from the distribution of truffles and ectomycorrhizas. Interestingly, a new haplotype – never described from fruiting body material – was identified. The specific detection of T. magnatum in the soil will allow to unravel the ecology of this fungus, following its mycelial network. Moreover, this new tool may have practical importance in projects aimed to increase large-scale truffle production, checking for T. magnatum persistence in plantations.     

Mycorrhization of Pecan trees (Carya illinoinensis) with commercial truffle species: Tuber aestivum Vittad. and Tuber borchii Vittad

Pecan (Carya illinoinensis) is an economically important nut tree native to the Mississippi basin and cultivated worldwide. In North America, species of truffles are regularly found fruiting in productive pecan orchards and the truffle genus Tuber appears to be abundant in pecan ectomycorrhizal (EM) communities. As an initial step to determine the feasibility of co-cropping European truffle species with pecan, we evaluated whether mycorrhizae of highly esteemed European truffle species (Tuber aestivum Vittad. T. borchii and T. macrosporum) could be formed on pecan seedlings. Seedlings were inoculated with truffle spores and were grown in a greenhouse for 10?months. Levels of EM colonization were estimated visually and quantified by counting EM tips. Ectomycorrhizae were identified both morphologically and molecularly with species-specific amplification and by sequencing of the ITS region of the nuclear ribosomal DNA (nrDNA). Both T. borchii and T. aestivum spores produced well-formed ectomycorrhizae on pecan seedlings with average root colonization levels of about 62% and 42%, respectively, whereas no ectomycorrhizae of T. macrosporum were formed. The anatomy and morphology of these truffle ectomycorrhizae on pecan was characterized. The co-cropping of T. aestivum and T. borchii may hold promise as an additional stream of revenue to pecan growers, although, further studies are needed to assess whether this symbiosis is maintained after planting in the field and whether truffle production can be supported by this host species.     

Five years investigation of female and male genotypes in périgord black truffle (Tuber melanosporum Vittad.) revealed contrasted reproduction strategies

The Périgord black truffle (Tuber melanosporum Vittad.) is a heterothallic ascomycete that establishes ectomycorrhizal symbiosis with trees and shrubs. Small‐scale genetic structures of female genotypes in truffle orchards are known, but it has not yet been studied in male genotypes. In this study, our aim was to characterize the small‐scale genetic structure of both male and female genotypes over five years in an orchard to better understand the T. melanosporum sexual reproduction strategy, male genotype dynamics, and origins. Two‐hundred forty‐one ascocarps, 475 ectomycorrhizas, and 20 soil cores were harvested and genotyped using microsatellites and mating type genes. Isolation by distance analysis revealed pronounced small‐scale genetic structures for both female and male genotypes. The genotypic diversity was higher for male than female genotypes with numerous small size genotypes suggesting an important turnover due to ascospore recruitment. Larger and perennial female and male genotypes were also detected. Only three genotypes (1.5%) were found as both female and male genotypes (hermaphrodites) while most were detected only as female or male genotype (dioecy). Our results suggest that germinating ascospores act as male genotypes, but we also proposed that soil mycelium could be a reservoir of male genotypes.