Responses of a soil fungal community to severe windstorm damages in an old silver fir stand.

Forests are increasingly threatened by climate change and the Anthropocene seems to have favored the emergence and adaptation of pathogens. Robust monitoring methods are required to prevent biodiversity and ecosystems losses, and this imposes the choice of bioindicators of habitat health. Fungal communities are increasingly recognized as fundamental components in nearly all natural and artificial environments, and their ecosystem services have a huge impact in maintaining and restoring the functionality of ecosystems. We coupled metabarcoding and soil analyses to infer the dynamics of a fungal community inhabiting the old silver fir stand in Vallombrosa (Italy), which is known to be afflicted by both Armillaria and Annosum root rot. The forest was affected in 2015, by a windstorm which caused a partial falling and uprooting of trees. The remaining stand, not affected by the windstorm, was used as a comparison to infer the consequences of the ecosystem disturbance. We demonstrated that the abundance of pathogens alone is not able to explain the soil fungal differences shown by the two areas. The fungal community as a whole was equally rich in the two areas, even if a reduction of the core ectomycorrhizal mycobiome was observed in the wind-damaged area, accompanied by the increase of wood saprotrophs and arbuscular mycorrhizas. We hypothesize a reshaping of the fungal community and a potentially ongoing re-generation of its functionalities. Our hypothesis is driven by the evidence that key symbiotic, endophytic, and saprotrophic guilds are still present and diversified in the wind-damaged area, and that dominance of single taxa or biodiversity loss was not observed from a mycological point of view. With the present study, we aim at providing evidence that fungal communities are fundamental for the monitoring and the conservation of threatened forest ecosystems.

Metabolites from a Pleurotus tuber-regium collected in Madagascar.

Pleurotus tuber-regium was isolated from a dead trunk of Raphia farinifera (Arecaceae) in a lowland moist forest in Antsohihy, Madagascar, and the species was confirmed by molecular analysis and morphological observations. The main bioactive metabolites of the mycelium extracts were identified by mass spectrometry techniques. Five structural diverse metabolites, tryptophol, pyroglutamic acid, prolyldiketopiperazine B, sporol and RKS-1778, were characterised by LC-MS qTOF analysis of the hydro-alcoholic extract. GC-MS analysis of both chloroform and ethyl acetate extracts revealed the presence of several saturated and -unsaturated fatty acids and their esters derivatives.

Fungal Patterns from Soils in Madagascar: an Insight from Maromizaha Forest (Evergreen Humid Forest) to Outside (Deciduous Forest).

Soil fungal diversity was studied by next-generation sequencing and compared in two different Malagasy ecosystems, the first a New Protected Area (Maromizaha NAP) that is a rich humid evergreen forest and the second a degraded and declined deciduous forest (Andaravina) whose area has been also eroded. Both areas, however, have comparable annual rainfalls and soil pH values. So it was of interest to examine the soil fungal diversity in each system and compare them. We detected 1,817,658 reads representing Ascomycota, which were dominant in both habitats (55.9%), followed by unidentified fungi (21.5%), Basidiomycota (12.7%) and Mortierellomycota (6.7%), with Mucoromycota, Chytridiomycota, Glomeromycota and other phyla accounting for less than 5% in total. In detail, 1,142 OTUs out of 1,368 constitute the common core shared by both sampling areas, which are characterized by tropical climate, whereas 185 are Maromizaha specific and 41 Andaravina specific. The most represented guilds involve fungi related to saprotrophic behaviour, with a greater tendency towards pathotrophic mode. A significant variability in terms of richness and abundance is present within Maromizaha, which is a heterogeneous environment for fungi but also for plant composition, as it emerged from the vegetational survey of the investigated sites. A few fungal sequences match taxa from Madagascar, highlighting the scarce representativeness of fungi from this island in the fungal databases and their still low knowledge. Enlarging studies in Madagascar will help not only to unravel its largely unknown fungal biodiversity but also to give a contribution for studies on the reconstruction of the diversity of soil fungi worldwide.

Role and potentialities of bacteria associated with Tuber magnatum: A mini-review.

Among the hypogeous ectomycorrhizal fungi, the white truffle Tuber magnatum Picco is the species of greatest interest, both from an ecological and economic point of view. The increasing market demand of the precious white truffle along with the fall in its natural production led to a growing interest in cultivation techniques and encouraged truffle growers and researchers to deeper investigate factors that could affect and improve T. magnatum productivity. In this context, microbial communities play a central role. Indeed, in the last few years, the hypothesis of a potential link between microbial community composition and truffle orchard productivity is arousing a greater attention. Moreover, since the value of the prized T. magnatum can vary in relation to its provenience, the need to define a reliable tracking system is also emerging and bacteria appear to be a promising tool. Accordingly, the present mini-review summarises the knowledge currently available on T. magnatum microbial communities, focusing on the role of truffle-associated bacteria and highlighting similarities and differences between samples of different origin, to address the following issues: (i) Is there a correlation between microbial taxa and truffle ground productivity? (ii) Can bacteria actually be used as markers of T. magnatum geographic origin? The identification of microorganisms able to promote T. magnatum formation may represent an important advance in the field of truffle farming. Similarly, the detection of bacterial taxa that can be used as markers of T. magnatum origin could have a considerable impact on truffle industry and trade, even at local scale.

Systemic effects of Tuber melanosporum inoculation in two Corylus avellana genotypes.

Roots of the European hazelnut (Corylus avellana L.), i.e., one of the most economically important nut species, form symbiosis with ectomycorrhizal (ECM) fungi, including truffles. Although physical interactions only occur in roots, the presence of mycorrhizal fungi can lead to metabolic changes at a systemic level, i.e., in leaves. However, how root colonization by ECM fungi modifies these processes in the host plant has so far not been widely studied. This work aimed to investigate the response in two C. avellana genotypes, focusing on leaves from plants inoculated with the black truffle Tuber melanosporum Vittad. Transcriptomic profiles of leaves of colonized plants were compared with those of non-colonized plants, as well as sugar and polyphenolic content. Results suggested that T. melanosporum has the potential to support plants in stressed conditions, leading to the systemic regulation of several genes involved in signaling and defense responses. Although further confirmation is needed, our results open new perspectives for future research aimed to highlight novel aspects in ECM symbiosis.

Localized reshaping of the fungal community in response to a forest fungal pathogen reveals resilience of Mediterranean mycobiota.

Mediterranean forests are facing the impact of pests such as the soilborne Phytophthora cambivora, the causal agent of Ink disease, and this impact is made more severe by global changes. The status and resilience of the soil microbial ecosystem in areas with such a disturbance are little known; however, the assessment of the microbial community is fundamental to preserve the ecosystem functioning under emerging challenges. We profile soil fungal communities in a chestnut stand affected by ink disease in Italy using metabarcoding, and couple high-throughput sequencing with physico-chemical parameters and dendrometric measurements. Since the site also includes an area where the disease symptoms seem to be suppressed, we performed several analyses to search for determinants that may contribute to such difference. We demonstrate that neither pathogen presence nor trees decline associate with the reduction of the residing community diversity and functions, but rather with microbial network reshaping through substitutions and new interactions, despite a conservation of core taxa. We predict interactions between taxa and parameters such as soil pH and C/N ratio, and suggest that disease incidence may also relate with disappearance of pathogen antagonists, including ericoid- and ectomycorrhizal (ECM) fungi. By combining metabarcoding and field studies, we infer the resilient status of the fungal community towards a biotic stressor, and provide a benchmark for the study of other threatened ecosystems.

Different patterns in root and soil fungal diversity drive plant productivity of the desert truffle Terfezia claveryi in plantation.

The desert truffle Terfezia claveryi is one of the few mycorrhizal fungi currently in cultivation in semiarid and arid areas. Agroclimatic parameters seem to affect its annual yield, but there is no information on the influence of biotic factors. In this study, fungal diversity was analysed by high-throughput sequencing of the ITS2 rDNA region from soil and root samples to compare productive and non-productive mycorrhizal plants in a 4-years old plantation (Murcia, Spain). The fungal metaprofile was dominated by Ascomycota phylum. Desert truffle productivity was driven by different patterns of fungal species composition in soil (species replacement) and root (species richness differences). Moreover, positive associations for ectomycorrhizal and negative for arbuscular mycorrhizal guilds were found in productive roots, and positive associations for fungal parasite-plant pathogen guild in non-productive ones. Soil samples were dominated by pathotroph and saprotroph trophic modes, showing positive associations for Aureobasidium pullulans and Alternaria sp. in productive areas, and positive associations for Fusarium sp. and Mortierella sp. were found in non-productive soils. Finally, some significant OTUs were identified and associated to ascocarp producing patches, which could serve as predictive and location markers of desert truffle production.

First Report of Neofusicoccum parvum Associated with Chestnut Nut Rot in Italy.

In autumn 2018, during a study on the pathogens involved in the etiology of chestnut nut rot symptoms observed in three of the main sweet chestnut (Castanea sativa) growing areas in Sardinia (Site 1: 39°56'55"N/09°11'45"E; site 2: 39°58'20"N/09°09'41"E; site 3: 40°52'50"N/09°08'45"E), Gnomoniopsis smithogilvyi was found to be the main causal agent. In addition to G. smithogilvyi, 15 out of 450 nuts processed, yielded on potato dextrose agar (PDA, 39 g/L) at 22°C white colonies with dense aerial mycelium becoming dark grey after 4 to 7 days. Pycnidia were produced within 4 weeks in half-strength PDA incubated at room temperature under natural daylight. The hyaline, ellipsoid to fusiform and aseptate conidia measured 13.4-19.2 × 4.8-7.7 µm (n = 50). All morphological characters matched those reported for Neofusicoccum parvum by Phillips et al. (2013). Identity of isolates was confirmed by DNA sequence analysis of the internal transcribed spacer region (ITS) and part of the translation elongation factor 1-alpha gene (tef1-α). DNA extraction, PCR amplification reactions and DNA sequencing were carried out according to Linaldeddu et al. (2016). In the phylogenetic analysis based on combined ITS and tef1-α gene sequences the N. parvum isolates clustered within two well-supported subclades. In the first subclade (ML bootstrap = 88%) three isolates clustered together with the ex-type culture of N. parvum (CMW9081) while in the second subclade (ML bootstrap = 95%) three isolates clustered together with the ex-type culture of Neofusicoccum algeriense (CBS 137504), a species recently synonymised with N. parvum by Lopes et al. (2016). Sequences of six representative isolates were deposited in GenBank (MK968559-MK968564 and MT010339-MT010344 for ITS and tef1-α, respectively). The pathogenicity of six isolates, belonging to the two haplotypes, was undertaken by inoculating five asymptomatic nuts per isolate. After disinfecting the nut surface with 70% ethanol and removing a piece of shell (5 mm diameter) with a sterile cork borer, the nuts were inoculated with a same-sized agar-mycelium plug cut from the margin of a 5-day-old PDA colony. Ten control nuts were inoculated with a sterile PDA plug applied as described above. Inoculated nuts were kept in thermostat at 22 °C in the dark for 18 days. All nuts inoculated with N. parvum showed light-brown to dark necrosis of kernel associated with loss of tissue consistency. The symptoms were congruent with those observed in nature. All N. parvum isolates were successfully reisolated from all the inoculated nuts, fulfilling Koch's postulates. No lesions were observed on controls. N. parvum is recognized as an emerging plant pathogen worldwide. In particular, several studies report N. parvum as a growing threat to agricultural and forest ecosystems in the Mediterranean area (Larignon et al., 2015; Manca et al., 2020). This is the first report of N. parvum causing chestnut nut rot in Italy.

Heterogeneity of the white truffle Tuber magnatum in a limited geographic area of Central-Southern Italy.

Molise region (Central-Southern Italy) is one of the Italian richest areas of truffles and contributes significantly to the national production of the precious Tuber magnatum. Nevertheless, Molise truffle has received little scientific attention. Accordingly, in the present study, two T. magnatum populations collected in two different sites of Molise region were characterised from a morphological, genetic and microbiological point of view. A considerable variability between and within the two analysed groups emerged, suggesting an interesting heterogeneity of Molise white truffle populations. Ascocarps of the two groups significantly differed in size and maturation degree, although no linear correlation between weight and maturity was found. Genetic investigations focused on the Sequence-Characterised Amplified Region SCAR A21-inf. Three haplotypes, randomly distributed within the two truffle groups regardless of their collection sites, were detected. The 16S rRNA gene amplicon high-throughput sequencing provided an overview of the composition of the ascocarp-associated bacterial communities. A predominance of α-Proteobacteria was observed, with Bradyrhizobium among the main genera. However, some truffles showed unusual microbial profiles, with Pedobacter, Polaromonas and other bacterial genera as dominant taxa.

Soil Fungal Communities Investigated by Metabarcoding Within Simulated Forensic Burial Contexts.

Decomposition of animal bodies in the burial environment plays a key role in the biochemistry of the soil, altering the balance of the local microbial populations present before the introduction of the carcass. Despite the growing number of studies on decomposition and soil bacterial populations, less is known on its effects on fungal communities. Shifts in the fungal populations at different post-mortem intervals (PMIs) could provide insights for PMI estimation and clarify the role that specific fungal taxa have at specific decomposition stages. In this study, we buried pig carcasses over a period of 1- to 6-months, and we sampled the soil in contact with each carcass at different PMIs. We performed metabarcoding analysis of the mycobiome targeting both the internal transcribed spacer (ITS) 1 and 2, to elucidate which one was more suitable for this purpose. Our results showed a decrease in the fungal taxonomic richness associated with increasing PMIs, and the alteration of the soil fungal signature even after 6 months post-burial, showing the inability of soil communities to restore their original composition within this timeframe. The results highlighted taxonomic trends associated with specific PMIs, such as the increase of the Mortierellomycota after 4- and 6-months and of Ascomycota particularly after 2 months, and the decrease of Basidiomycota from the first to the last time point. We have found a limited number of taxa specifically associated with the carrion and not present in the control soil, showing that the major contributors to the recorded changes are originated from the soil and were not introduced by the carrion. As this is the first study conducted on burial graves, it sets the baseline for additional studies to investigate the role of fungal communities on prolonged decomposition periods and to identify fungal biomarkers to improve the accuracy of PMI prediction for forensic applications.

Metabarcoding to investigate changes in soil microbial communities within forensic burial contexts.

The estimation of the time elapsed since death (post-mortem interval, or PMI) is one of the key themes that forensic scientists have to address frequently. However, the estimation of PMI still suffers from poor accuracy and biases especially when decomposition stages are prolonged, so further improvements in methods for PMI estimation are desirable. Soil microbial communities associated with decomposing bodies have been shown to be good candidates for the estimation of the PMI of exposed bodies. Nevertheless, further research is required to better understand the bacterial succession associated with decomposition of buried carcasses in order to test its reliability and applicability for the estimation of PMI and to better understand the dynamics involved with decomposition within this particular scenario. Therefore we explored the succession of soil microbial communities associated with four decomposing pig carcasses (from one to six months PMI) using a metabarcoding approach. The sequencing of the bacterial 16S rRNA variable region 4 (V4) revealed trends linking particular microbial taxa with specific PMIs, and notably an increase in Proteobacteria, Firmicutes and Bacteroidetes at specific PMIs as well as a decrease in Acidobacteria. Our results, in accordance with previous studies conducted on exposed bodies of different mammalian species (including humans), also showed a general reduction of the taxonomic richness from two months PMI onwards, as well as an incomplete re-establishment of the starting soil microbial conditions after six months PMI. We also found specific mammal-derived taxa, such as Bacteroides spp., being still present in the soil after six months PMI. As such, this study serves as a baseline for additional research to allow the characterisation of biomarkers associated with specific PMIs. Due to the similarity between the results presented here and those reported in other types of decomposition studies we believe that the metabarcoding approach has considerable potential in the estimation of the PMI, particularly to clarify cases involving heavily skeletonised bodies or for the investigation of clandestine graves in which the carcass has been moved from its original place of deposition.

Pezizomycetes genomes reveal the molecular basis of ectomycorrhizal truffle lifestyle.

Tuberaceae is one of the most diverse lineages of symbiotic truffle-forming fungi. To understand the molecular underpinning of the ectomycorrhizal truffle lifestyle, we compared the genomes of Piedmont white truffle (Tuber magnatum), Périgord black truffle (Tuber melanosporum), Burgundy truffle (Tuber aestivum), pig truffle (Choiromyces venosus) and desert truffle (Terfezia boudieri) to saprotrophic Pezizomycetes. Reconstructed gene duplication/loss histories along a time-calibrated phylogeny of Ascomycetes revealed that Tuberaceae-specific traits may be related to a higher gene diversification rate. Genomic features in Tuber species appear to be very similar, with high transposon content, few genes coding lignocellulose-degrading enzymes, a substantial set of lineage-specific fruiting-body-upregulated genes and high expression of genes involved in volatile organic compound metabolism. Developmental and metabolic pathways expressed in ectomycorrhizae and fruiting bodies of T. magnatum and T. melanosporum are unexpectedly very similar, owing to the fact that they diverged ~100 Ma. Volatile organic compounds from pungent truffle odours are not the products of Tuber-specific gene innovations, but rely on the differential expression of an existing gene repertoire. These genomic resources will help to address fundamental questions in the evolution of the truffle lifestyle and the ecology of fungi that have been praised as food delicacies for centuries.

Recent Insights on Biological and Ecological Aspects of Ectomycorrhizal Fungi and Their Interactions.

The roots of most terrestrial plants are colonized by mycorrhizal fungi. They play a key role in terrestrial environments influencing soil structure and ecosystem functionality. Around them a peculiar region, the mycorrhizosphere, develops. This is a very dynamic environment where plants, soil and microorganisms interact. Interest in this fascinating environment has increased over the years. For a long period the knowledge of the microbial populations in the rhizosphere has been limited, because they have always been studied by traditional culture-based techniques. These methods, which only allow the study of cultured microorganisms, do not allow the characterization of most organisms existing in nature. The introduction in the last few years of methodologies that are independent of culture techniques has bypassed this limitation. This together with the development of high-throughput molecular tools has given new insights into the biology, evolution, and biodiversity of mycorrhizal associations, as well as, the molecular dialog between plants and fungi. The genomes of many mycorrhizal fungal species have been sequenced so far allowing to better understanding the lifestyle of these fungi, their sexual reproduction modalities and metabolic functions. The possibility to detect the mycelium and the mycorrhizae of heterothallic fungi has also allowed to follow the spatial and temporal distributional patterns of strains of different mating types. On the other hand, the availability of the genome sequencing from several mycorrhizal fungi with a different lifestyle, or belonging to different groups, allowed to verify the common feature of the mycorrhizal symbiosis as well as the differences on how different mycorrhizal species interact and dialog with the plant. Here, we will consider the aspects described before, mainly focusing on ectomycorrhizal fungi and their interactions with plants and other soil microorganisms.

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.

Arbuscular mycorrhizal fungal diversity in the Tuber melanosporum brûlé.

The development of the fruiting body (truffle) of the ectomycorrhizal fungus Tuber melanosporum is associated with the production of an area (commonly referred to with the French word brûlé) around its symbiotic plant that has scanty vegetation. As truffles produce metabolites that can mediate fungal-plant interactions, the authors wondered whether the brûlé could affect the arbuscular mycorrhizal fungi (AMF) that colonize the patchy herbaceous plants inside the brûlé. A morphological evaluation of the roots of plants collected in 2009 from a T. melanosporum/Quercus pubescens brûlé in France has shown that the herbaceous plants are colonized by AMF to a great extent. An analysis of the 18S rRNA sequences obtained from roots and soil inside the brûlé has shown that the AMF community structure seemed to be affected in the soil inside the brûlé, where less richness was observed compared to outside the brûlé.

Gene expression and metabolite changes during Tuber magnatum fruiting body storage.

The aim of this study was to investigate the impact of different 4 °C post-harvest storage periods on the quality of the white truffle Tuber magnatum. The expression of selected genes and the profiles of non-volatile metabolites have been analyzed. The up-regulation of genes related to cell wall metabolism and to a putative laccase points to cell wall modifications and browning events during cold storage. Time course RT-qPCR experiments have demonstrated that such transcription events probably depend on the ripening status, since this is delayed in partially ripe fruiting bodies. Changes in the concentrations of linoleate-derived metabolites occur during the first 3 days of considered cold storage, while the other metabolites, such as the amino acids, do not change. Taken together, the results demonstrate that complex molecular events occur in white truffles in the post-harvest period and before they are used as fresh products.

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.

Proteins from Tuber magnatum Pico fruiting bodies naturally grown in different areas of Italy.

BACKGROUND: A number of Tuber species are ecologically important. The fruiting bodies of some of these also have value as a cooking ingredient due to the fact that they possess exceptional flavor and aromatic properties. In particular, T. magnatum fruiting bodies (commonly known as truffles), are greatly appreciated by consumers. These grow naturally in some parts of Italy. However, the quality of these fruiting bodies varies significantly depending on the area of origin due to differences in environmental growth conditions. It is therefore useful to be able to characterize them. A suitable method to reach this goal is to identify proteins which occur in the fruiting bodies that are specific to each area of origin. In this work protein profiles are described for samples coming from different areas and collected in two successive years. To our knowledge this is the first time that proteins of T. magnatum have been thoroughly examined. RESULTS: Using two dimensional electrophoresis, reproducible quantitative differences in the protein patterns (total 600 spots) of samples from different parts of Italy (accession areas) were revealed by bioinformatic analysis. 60 spots were chosen for further analysis, out of which 17 could probably be used to distinguish a sample grown in one area from a sample grown in another area. Mass spectrometry (MS) protein analysis of these seventeen spots allowed the identification of 17 proteins of T. magnatum. CONCLUSIONS: The results indicate that proteomic analysis is a suitable method for characterizing those differences occurring in samples and induced by the different environmental conditions present in the various Italian areas where T. magnatum can grow. The positive protein identification by MS analysis has proved that this method can be applied with success even in a species whose genome, at the moment, has not been sequenced.

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.