Antonietta Mello

Périgord black truffle genome uncovers evolutionary origins and mechanisms of symbiosis

The Périgord black truffle (Tuber melanosporum Vittad.) and the Piedmont white truffle dominate today’s truffle market. The hypogeous fruiting body of T. melanosporum is a gastronomic delicacy produced by an ectomycorrhizal symbiont endemic to calcareous soils in southern Europe. The worldwide demand for this truffle has fuelled intense efforts at cultivation. Identification of processes that condition and trigger fruit body and symbiosis formation, ultimately leading to efficient crop production, will be facilitated by a thorough analysis of truffle genomic traits. In the ectomycorrhizal Laccaria bicolor, the expansion of gene families may have acted as a ‘symbiosis toolbox’. This feature may however reflect evolution of this particular taxon and not a general trait shared by all ectomycorrhizal species. To get a better understanding of the biology and evolution of the ectomycorrhizal symbiosis, we report here the sequence of the haploid genome of T. melanosporum, which at ∼125 megabases is the largest and most complex fungal genome sequenced so far. This expansion results from a proliferation of transposable elements accounting for ∼58% of the genome. In contrast, this genome only contains ∼7,500 protein-coding genes with very rare multigene families. It lacks large sets of carbohydrate cleaving enzymes, but a few of them involved in degradation of plant cell walls are induced in symbiotic tissues. The latter feature and the upregulation of genes encoding for lipases and multicopper oxidases suggest that T. melanosporum degrades its host cell walls during colonization. Symbiosis induces an increased expression of carbohydrate and amino acid transporters in both L. bicolor and T. melanosporum, but the comparison of genomic traits in the two ectomycorrhizal fungi showed that genetic predispositions for symbiosis—‘the symbiosis toolbox’—evolved along different ways in ascomycetes and basidiomycetes.

ITS-1 versus ITS-2 pyrosequencing: a comparison of fungal populations in truffle grounds

In a recent study pyrosequencing of the ribosomal internal transcribed spacer-1 (ITS-1) has validated the effectiveness of such technology in the survey of soil fungal diversity. Here we compare the two ITS regions, ITS-1 and ITS-2, of the fungal populations occurring in Tuber melanosporum/Quercus pubescens truffle grounds and sampled in two areas, one devoid of vegetation (“burned”, brulé in French) where T. melanosporum fruiting bodies are usually collected, and outside the brulé. TS1F/ITS2 and ITS3/ITS4 were used respectively for the amplification of the ITS-1 and ITS-2 regions. Two amplicon libraries were built, one for inside and the other for outside. A set of 15.788 reads was obtained. After the removal of low quality sequences, 3568 and 3156 sequences were obtained from inside the brulé with the ITS-1 and ITS-2 primers respectively. The sequences obtained from outside the brulé were 4490 with the ITS-1 primers and 2432 with the ITS-2 primers. Most of the sequences obtained for both ITS fragments could be attributed to fungal organisms. The pair of primers, ITS1-F/ITS2, was more selective, producing fewer non-fungal sequences (1% inside, 3% outside), in addition to a higher number of sequences, than the pair ITS3/ITS4 (6% inside, 11% outside). Although differences are present in the taxa percentages between ITS-1 and ITS-2, both reveal that Ascomycota were the dominant fungal phylum and that their number decreased moving from inside the brulé to outside, while the number of Basidiomycota increased. Taken together, both the short ITS-1 and ITS-2 reads obtained by the high throughput 454 sequencing provide adequate information for taxon assignment and are suitable to correlate the dynamics of the fungal populations to specific environments.

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 beta-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.

Interactions of fungi with other organisms

Living organisms establish complex networks of mutualistic and antagonistic interactions in nature, which impact strongly on their own survival and on the stability of the whole population. Fungi, in particular, can shape natural as well as man-managed ecosystems due to their ubiquitous occurrence and the range of interactions they establish with plants, animals and other microbes. This review describes some examples of mutualistic and antagonistic fungal interactions that are of particular interest for their ecological role, or because they can be exploited by man to improve plant health and/or productivity in sustainable agriculture and forestry.

Rapid typing of truffle mycorrhizal roots by PCR amplification of the ribosomal DNA spacers

DNA analyses were developed to type mycorrhizas of two Tuber species of commercial value (T. melanosporum, T. borchii) and a competitive fungus (Sphaerosporella brunnea) which forms ectomycorrhizas with plants usually considered hosts for truffles. Polymerase chain reaction (PCR) amplification of DNA isolated from fruitbodies, mycelia, mycorrhizas and leaves of host plants, was performed with a primer pair for an internal transcribed spacer ITS1-4. ITS amplification followed by restriction fragment length polymorphism (RFLP) analysis of the amplified products clearly distinguished the two Tuber species at the fruitbody, mycorrhiza and mycelium levels.

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.

Tuber borchii versus Tuber maculatum: Neotype studies and DNA analyses

Two similar truffle species described by Vittadini, Tuber borchii and T. maculatum, were compared. We designate neotypes for both species, which were described microscopically and compared with fre...

Truffle Brûlés Have an Impact on the Diversity of Soil Bacterial Communities

Background The development of Tuber melanosporum mycorrhizal symbiosis is associated with the production of an area devoid of vegetation (commonly referred to by the French word ‘brûlé’) around the symbiotic plants and where the fruiting bodies of T. melanosporum are usually collected. The extent of the ecological impact of such an area is still being discovered. While the relationship between T. melanosporum and the other fungi present in the brûlé has been assessed, no data are available on the relationship between this fungus and the bacteria inhabiting the brûlé. Methodology/Principal Findings We used DGGE and DNA microarrays of 16S rRNA gene fragments to compare the bacterial and archaeal communities inside and outside of truffle brûlés. Soil samples were collected in 2008 from four productive T. melanosporum/Quercus pubescens truffle-grounds located in Cahors, France, showing characteristic truffle brûlé. All the samples were analyzed by DGGE and one truffle-ground was analyzed also using phylogenetic microarrays. DGGE profiles showed differences in the bacterial community composition, and the microarrays revealed a few differences in relative richness between the brûlé interior and exterior zones, as well as differences in the relative abundance of several taxa. Conclusions/Significance The different signal intensities we have measured for members of bacteria and archaea inside versus outside the brûlé are the first demonstration, to our knowledge, that not only fungal communities, but also other microorganisms are affected by T. melanosporum. Firmicutes (e.g., Bacillus), several genera of Actinobacteria, and a few Cyanobacteria had greater representation inside the brûlé compared with outside, whereas Pseudomonas and several genera within the class Flavobacteriaceae had higher relative abundances outside the brûlé. The findings from this study may contribute to future searches for microbial bio-indicators of brûlés.

Molecular and morphological characterization of Tuber magnatum mycorrhizas in a long-term survey.

Tuber magnatum Pico is an ectomycorrhizal fungus whose mycorrhizas can be barely distinguished morphologically from those of other related white truffles. Here we describe the use of specific primers based on the T. magnatum ITS sequence for screening mycorrhizas from a large number of growth chambers, greenhouse and nursery samples taken in a long-term survey. This molecular identification technique enabled a new morphological characterization to be set up for T. magnatum mycorrhizas.

Soil metaproteomics reveals an inter-kingdom stress response to the presence of black truffles

For some truffle species of the Tuber genus, the symbiotic phase is often associated with the presence of an area of scant vegetation, commonly known as the brûlé, around the host tree. Previous metagenomics studies have identified the microorganisms present inside and outside the brûlé of a Tuber melanosporum truffle-ground, but the molecular mechanisms that operate in this ecological niche remain to be clarified. To elucidate the metabolic pathways present in the brûlé, we conducted a metaproteomics analysis on the soil of a characterized truffle-ground and cross-referenced the resulting proteins with a database we constructed, incorporating the metagenomics data for the organisms previously identified in this soil. The soil inside the brûlé contained a larger number of proteins and, surprisingly, more proteins from plants, compared with the soil outside the brûlé. In addition, Fisher’s Exact Tests detected more biological processes inside the brûlé; these processes were related to responses to multiple types of stress. Thus, although the brûlé has a reduced diversity of plant and microbial species, the organisms in the brûlé show strong metabolic activity. Also, the combination of metagenomics and metaproteomics provides a powerful tool to reveal soil functioning.