Transcriptome Metabolic Characterization of Tuber borchii SP1-A New Spanish Strain for In Vitro Studies of the Bianchetto Truffle.

Truffles are ascomycete hypogeous fungi belonging to the Tuberaceae family of the Pezizales order that grow in ectomycorrhizal symbiosis with tree roots, and they are known for their peculiar aromas and flavors. The axenic culture of truffle mycelium is problematic because it is not possible in many cases, and the growth rate is meager when it is possible. This limitation has prompted searching and characterizing new strains that can be handled in laboratory conditions for basic and applied studies. In this work, a new strain of Tuber borchii (strain SP1) was isolated and cultured, and its transcriptome was analyzed under different in vitro culture conditions. The results showed that the highest growth of T. borchii SP1 was obtained using maltose-enriched cultures made with soft-agar and in static submerged cultures made at 22 °C. We analyzed the transcriptome of this strain cultured in different media to establish a framework for future comparative studies, paying particular attention to the central metabolic pathways, principal secondary metabolite gene clusters, and the genes involved in producing volatile aromatic compounds (VOCs). The results showed a transcription signal for around 80% of the annotated genes. In contrast, most of the transcription effort was concentrated on a limited number of genes (20% of genes account for 80% of the transcription), and the transcription profile of the central metabolism genes was similar in the different conditions analyzed. The gene expression profile suggests that T. borchii uses fermentative rather than respiratory metabolism in these cultures, even in aerobic conditions. Finally, there was a reduced expression of genes belonging to secondary metabolite clusters, whereas there was a significative transcription of those involved in producing volatile aromatic compounds.

The bianchetto truffle (Tuber borchii) a lead-resistant ectomycorrhizal fungus increases Quercus cerris phytoremediation potential.

Tuber borchii is a European edible truffle which forms ectomycorrhizas with several soft- and hardwood plants. In this article, the effects of high level of Pb on the in vitro growth of five T. borchii strains and the molecular mechanisms involved in Pb tolerance were studied. Moreover, the effects of the Pb treatment on T. borchii ectomycorrhizas and on the growth, element uptake and distribution in different organs of Quercus cerris seedlings were investigated. The results showed an extraordinary tolerance of T. borchii mycelium to Pb: all the tested strains were able to grow at Pb concentration over 4000 mg L-1 . The mechanisms of tolerance seem related to Pb sequestration in the vacuole and its immobilization as crystal of Pb oxalate outside the hyphae rather than detoxification processes, considering the low expression of glutaredoxin and thioredoxin genes. T. borchii-Q. cerris mycorrhizas tolerate a soil concentration of Pb from 1869 to 4030 mg kg-1 although, at these Pb concentrations, T. borchii showed a reduced ability to colonize roots. T. borchii mycorrhization increased the uptake of Pb by Q. cerris. Mycorrhization and Pb treatment also significantly influenced the uptake and translocation in the plant of other elements.

Co-occurrence of true truffle mycelia in Tuber magnatum fruiting sites.

Tuber magnatum (the white truffle) is the most precious species of the genus Tuber which comprises the hypogeous ectomycorrhizal species called "true truffle." Despite its high economic value, the knowledge on its ecology is scant, principally due to the difficulty to find its mycorrhizas in the soil. The possibility to detect its mycelium by DNA extracted from soil has given a new chance for studying this truffle species. In this work, the co-occurrence of other Tuber species with T. magnatum mycelium was investigated by using species-specific primers in several productive areas located in central and northern Italy. Most (82%) of the examined soil samples showed at least one other Tuber species in addition to T. magnatum. The most common was T. maculatum (72% of soil samples) followed by T. borchii, T. rufum, T. brumale, T. dryophilum, T. macrosporum, and T. melanosporum (40%, 37%, 22%, 19%, 12%, and 1% of soil samples, respectively). Tuber aestivum was never detected in T. magnatum productive patches. Analysis of species co-occurrence showed that the pairwise associations between T. dryophilum-T. brumale, T. brumale-T. borchii, and T. borchii-T. dryophilum was significant. The results suggest that Tuber mycelial network in white truffle grounds is much more extensive than the distribution of their ectomycorrhizas and competitive exclusion between different Tuber species seems to take place only for root colonization.

Ascoma genotyping and mating type analyses of mycorrhizas and soil mycelia of Tuber borchii in a truffle orchard established by mycelial inoculated plants.

Tuber borchii (the Bianchetto truffle) is a heterothallic Ascomycete living in symbiotic association with trees and shrubs. Maternal and paternal genotype dynamics have already been studied for the black truffles Tuber melanosporum and Tuber aestivum but not yet for T. borchii. In this study, we analysed maternal and paternal genotypes in the first truffle orchard realized with plants inoculated with five different T. borchii mycelia. Our aims were to test the persistence of the inoculated mycelia, if maternal and/or paternal genotypes correspond to inoculated mycelia and to assess the hermaphroditism of T. borchii. The mating type of each isolate as well as those of mycorrhizas, ascomata and extraradical soil mycelia was determined. Moreover, simple sequence repeat (SSR) profiles of maternal and paternal genotypes were assessed in 18 fruiting bodies to investigate the sexual behaviour of this truffle. The maternal genotypes of the fruiting bodies corresponded to those of the inoculated mycelia with only two exceptions. This confirmed that the inoculated mycelia persisted 9 years after plantation. As regards paternal partner, only two had the same genotype as those of the inoculated mycelia, suggesting hermaphroditism. Most of the new paternal genotypes originated from a recombination of those of inoculated mycelia.

Effects of biogenerated ferric hydroxides nanoparticles on truffle mycorrhized plants.

Truffles are highly valuable ectomycorrhizal fungi that grow naturally in alkaline, calcareous soils. Iron deficiency chlorosis is a common problem in truffle (Tuber spp.) cultivation due to the high quantity of lime added to increase the pH of acidic soils. In this work, the effects of ferric hydroxide nanoparticles embedded in an exopolysaccharide (Fe-EPS NPs), extracted from cultures of Klebsiella oxytoca DSM 29614, were investigated on Quercus robur seedlings under greenhouse conditions. The plants were inoculated with Tuber borchii (the bianchetto truffle) and were cultivated with and without iron nanoparticle additions and compared with non-inoculated control plants. The seedlings were grown in limed soil in order to induce iron deficiency. Low doses of Fe-EPS NPs had a beneficial effect on the growth of the plants inoculated with T. borchii, increasing their height and reducing their leaf chlorosis 5 months after the first Fe-EPS NP treatment. Moreover, Fe-EPS NP treatments significantly increased the level of T. borchii mycorrhizal colonization and the ectomycorrhizal mantle thickness. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) applied to cross sections of mycorrhizas showed that Fe accumulated in the fungal mantle and apparently was slowly released serving as a resilient reservoir of iron for the plant. The results suggest that the application of Fe-EPS NPs is a promising technique in the production of Tuber mycorrhized plants in the nursery and could have future applications in the field.

Degradative Ability of Mushrooms Cultivated on Corn Silage Digestate.

The current management practice of digestate from biogas plants involves its use for land application as a fertilizer. Nevertheless, the inadequate handling of digestate may cause environmental risks due to losses of ammonia, methane and nitrous oxide. Therefore, the key goals of digestate management are to maximize its value by developing new digestate products, reducing its dependency on soil application and the consequent air pollution. The high nitrogen and lignin content in solid digestate make it a suitable substrate for edible and medicinal mushroom cultivation. To this aim, the mycelial growth rate and degradation capacity of the lignocellulosic component from corn silage digestate, undigested wheat straw and their mixture were investigated on Cyclocybe aegerita, Coprinus comatus, Morchella importuna, Pleurotus cornucopiae and Pleurotus ostreatus. The structural modification of the substrates was performed by using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. Preliminary in vitro results demonstrated the ability of P. ostreatus, P. cornucopiae and M. importuna to grow and decay hemicellulose and lignin of digestate. Cultivation trials were carried out on C. aegerita, P. cornucopiae and P. ostreatus. Pleurotus ostreatus showed the highest biological efficiency and fruiting body production in the presence of the digestate; moreover, P. ostreatus and P. cornucopiae were able to degrade the lignin. These results provide attractive perspectives both for more sustainable digestate management and for the improvement of mushroom cultivation efficiency.

Bacteria-produced ferric exopolysaccharide nanoparticles as iron delivery system for truffles (Tuber borchii).

Iron exopolysaccharide nanoparticles were biogenerated during ferric citrate fermentation by Klebsiella oxytoca DSM 29614. Before investigating their effects on Tuber borchii ("bianchetto" truffle) mycelium growth and morphology, they were tested on human K562 cell line and Lentinula edodes pure culture and shown to be non-toxic. Using these nanoparticles as iron supplement, the truffles showed extremely efficient iron uptake of over 300 times that of a commercial product. This avoided morphological changes in T. borchii due to lack of iron during growth and, with optimum nanoparticle dosage, increased growth without cell wall disruption or alteration of protoplasmatic hyphal content, the nuclei, mitochondria, and rough endoplasmic reticula being preserved. No significant modifications in gene expression were observed. These advantages derive from the completely different mechanism of iron delivery to mycelia compared to commercial iron supplements. The present data, in fact, show the nanoparticles attached to the cell wall, then penetrating it non-destructively without damage to cell membrane, mitochondria, chromatin, or ribosome. Low dosage significantly improved mycelium growth, without affecting hyphal morphology. Increases in hyphal diameter and septal distance indicated a healthier state of the mycelia compared to those grown in the absence of iron or with a commercial iron supplement. These positive effects were confirmed by measuring fungal biomass as mycelium dry weight, total protein, and ergosterol content. This "green" method for biogenerating iron exopolysaccharide nanoparticles offers many advantages, including significant economic savings, without toxic effects on the ectomycorrhizal fungus, opening the possibility of using them as iron supplements in truffle plantations.

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 approximately 125 megabases is the largest and most complex fungal genome sequenced so far. This expansion results from a proliferation of transposable elements accounting for approximately 58% of the genome. In contrast, this genome only contains approximately 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.

Tuber melosporum smooth spores: an anomalous feature in the genus Tuber.

This paper adds new insights on ecology and micromorphology of Tuber melosporum, a rare species with smooth spores. Eight T. melosporum ascomata collected in a 50 y old Pinus halepensis and Quercus ilex plantation in Sardinia, Italy, represent the first recovery of this species outside Spain. In comparison to the T. melosporum holotype, Italian specimens revealed differences in the number of spores in asci and spore shape. We propose an emended description of Tuber to include species without spore ornamentation.

First evidence for truffle production from plants inoculated with mycelial pure cultures.

Truffle (Tuber spp.) cultivation is based on raising mycorrhizal trees in greenhouses that have been inoculated with suspensions of ascospores. The problem with this is that pests, pathogens, and other mycorrhizal fungi can contaminate the trees. Furthermore, because ascospores are produced sexually, each plant potentially has a different genetic mycorrhizal makeup from each other so tailoring the mycorrhizal component of plants to suit a particular set of soil and climatic conditions is out of the question. Here, we report on the production of Tuber borchii-mycorrhized plants using pure cultures, establishing a truffière with these and subsequent production of its fruiting bodies. This study opens up the possibility of producing commercial numbers of Tuber-mycorrhized trees for truffle cultivation using mycelial inoculation techniques. It also poses questions about the mechanism of fertilization between the different strains which were located in different parts of the experimental truffière.

Characterization of Tuber borchii and Arbutus unedo mycorrhizas.

For the first time, arbutoid mycorrhizas established between Tuber borchii and Arbutus unedo were described. Analyzed mycorrhizas were from one T. borchii natural truffle ground, dominated by Pinus pinea, as well as synthesized in greenhouse conditions. A. unedo mycorrhizas presented some typical characteristics of ectomycorrhizas of T. borchii. However, as in arbutoid mycorrhizas, ramification was cruciform and intracellular colonization in epidermal cells was present. The ability of T. borchii to form ectomycorrhizas with A. unedo opens up the possibility to also use this fruit plant for truffle cultivation. This represents an important economic opportunity in Mediterranean areas by combining both the cultivation of precious truffles and the production of edible fruits which are used fresh or in food delicacies.

Antimicrobial Activity of Three Italian Strains of Morchella esculenta (Ascomycota).

Three genetically identified and morphologically characterized strains (MesAQ2-C, MesAQ6-2 and MesFI2-3) of the culinary-medicinal ascomycete mushroom Morchella esculenta (L.) Pers. collected in central-north Italy have been studied for their antifungal and antibacterial activities. The obtained data showed that mycelium of M. esculenta possess variable antimicrobial activity against four test fungi (Chrysosporium keratinophilum, Microsporum gypseum, Trichophyton terrestre, Penicillium griseofulvum), as well as one Gram positive (Staphylococcus aureus) and three Gram negative (Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa) test bacteria potentially pathogenic for humans and animals. Up to 20.4% of inhibition of the average mycelial growth rate (GRavr) of test fungi in dual culture experiment was detected. The samples of cultural liquid (CL) and mycelial extract (ME) obtained by static cultivation of M. esculenta strains showed up to 13.9 and 23.0% of GRavr inhibition of test fungi, respectively. Similarly, the inhibition of the bacterial colonies by CL and ME samples was 34.1 and 32.3%, respectively in comparison with the control with streptomycin indicating almost equal secretion of both intra- and extracellular antimicrobial compounds by M. esculenta mycelium. As a producer of antimicrobial compounds among tested M. esculenta strains, MesAQ2-C was the most effective. It may be considered for further myco-pharmacological research to develop mushroom-based antimicrobial biotech products with biomedical significance.

In vitro interactions between Bradyrhizobium spp. and Tuber magnatum mycelium.

Tuber magnatum is the most expensive truffle, but its large-scale cultivation is still a challenge compared to other valuable Tuber species. T. magnatum mycelium has never been grown profitably until now, which has led to difficulties to studying it in vitro. This study describes beneficial interactions between T. magnatum mycelium and never before described bradyrhizobia, which allows the in vitro growth of T. magnatum mycelium. Three T. magnatum strains were co-isolated on modified Woody Plant Medium (mWPM) with aerobic bacteria and characterised through microscopic observations. The difficulties of growing alone both partners, bacteria and T. magnatum mycelium, on mWPM demonstrated the reciprocal dependency. Three bacterial isolates for each T. magnatum strain were obtained and molecularly characterised by sequencing the 16S rRNA, glnII, recA and nifH genes. Phylogenetic analyses showed that all nine bacterial strains were distributed among five subclades included in a new monophyletic lineage belonging to the Bradyrhizobium genus within the Bradyrhizobium jicamae supergroup. The nifH genes were detected in all bacterial isolates, suggesting nitrogen-fixing capacities. This is the first report of consistent T. magnatum mycelium growth in vitro conditions. It has important implications for the development of new technologies in white truffle cultivation and for further studies on T. magnatum biology and genetics.

Effect of Bacteria Inoculation on Colonization of Roots by Tuber melanosporum and Growth of Quercus ilex Seedlings.

Tuber melanosporum is an ascomycete that forms ectomycorrhizal (ECM) symbioses with a wide range of host plants, producing edible fruiting bodies with high economic value. The quality of seedlings in the early symbiotic stage is important for successful truffle cultivation. Numerous bacterial species have been reported to take part in the truffle biological cycle and influence the establishment of roots symbiosis in plant hosts and the development of the carpophore. In this work, three different bacteria formulations were co-inoculated in Quercus ilex L. seedlings two months after T. melanosporum inoculation. At four months of bacterial application, the T. melanosporum ECM root tip rate of colonization and bacterial presence were assessed using both morphological and molecular techniques. A 2.5-fold increase in ECM colonization rate was found in the presence of Pseudomonas sp. compared to the seedlings inoculated only with T. melanosporum. The same treatment caused reduced plant growth either for the aerial and root part. Meanwhile, the ECM colonization combined with Bradyrhizobium sp. and Pseudomonas sp. + Bradyrhizobium sp. reduced the relative density of fibrous roots (nutrient absorption). Our work suggests that the role of bacteria in the early symbiotic stages of ECM colonization involves both the mycorrhizal symbiosis rate and plant root development processes, both essential for improve the quality of truffle-inoculated seedlings produced in commercial nurseries.

Exploring the canal environment in terms of water, bed sediments and vegetation in a reclaimed floodplain of Northern Italy.

The Po plain (Italy) is one of the largest floodplains in Europe that needs environmental restoration. To achieve this goal, the knowledge of the 'environment' (water, bed sediments and vegetation) of the canals crossing such floodplain is necessary. The water flow of the canals was kept low for hydraulic safety purposes from October to March (NIR), and high for irrigation purposes from April to September (IR). Within this framework, this study aimed to assess in 9 sites of the east part of Po plain 1) the canals' environment quality in terms of vegetation diversity, and water and bed sediment physicochemical properties; and 2) how these features are influenced by canal managements and landscape properties. Water was monthly sampled both in NIR and IR periods, the bed sediments were sampled in summer and winter periods, while the vegetation was recorded in spring and autumn. The low water flow during NIR worsened the water quality by increasing the concentrations of nutrients and salts. A higher salt and nutrient concentrations were observed both in water and bed sediments of canals crossing areas with fine texture alluvial deposits than in those flowing through medium texture alluvial deposits. Further, higher nutrient and salt concentrations were observed for the canals used as collectors of the water coming from other canals. Despite the differences observed for the bed sediments and water quality, the vegetation type and biodiversity did not show differences among the study sites probably because affected by the land use of the surrounding landscape. Indeed, the canals cross agricultural land which limit the developments of natural vegetation and do not promote plant biodiversity. Overall, the present study found out the key role of landscape properties and canal managements on 'canal environment' quality which need to be considered to perform an appropriate reclamation of such environments.

Ectomycorrhizal Fungal Community and Ascoma Production in a Declining Tuber borchii Plantation.

Tuber borchii is an edible ectomycorrhizal mushroom of considerable economic value. Its cultivation has become popular in recent years, but there are few studies on the factors affecting its productivity. In this work, the ascoma production and the ectomycorrhizal (ECM) community of a T. borchii plantation, established in an intensive farming area where this truffle is not naturally present, were studied. Tuber borchii production drastically declined from 2016 to 2021, and ascomata of other Tuber species (T. maculatum and T. rufum) were found from 2017. Molecular characterization of ectomycorrhizae carried out in 2016 identified 21 ECM fungal species, of which T. maculatum (22%) and Tomentella coerulea (19%) were the most abundant. Tuber borchii ectomycorrizae (16%) were almost entirely confined to the fruiting points. The diversity and structure of the ECM community on Pinus pinea were significantly different from those observed on hardwood trees. The obtained results suggest that T. maculatum (a native of the study site) tends to replace T. borchii through a mechanism of competitive exclusion. Although T. borchii cultivation is possible in suboptimal environments, particular care should be taken to limit competition with ECM fungi more suitable for local conditions.

Development and validation of a real-time PCR assay for detection and quantification of Tuber magnatum in soil.

BACKGROUND: Tuber magnatum, the Italian white truffle, is the most sought-after edible ectomycorrhizal mushroom. Previous studies report the difficulties of detecting its mycorrhizas and the widespread presence of its mycelium in natural production areas, suggesting that the soil mycelium could be a good indicator to evaluate its presence in the soil. In this study a specific real-time PCR assay using TaqMan chemistry was developed to detect and quantify T. magnatum in soil. This technique was then applied to four natural T. magnatum truffières located in different regions of Italy to validate the method under different environmental conditions. RESULTS: The primer/probe sets for the detection and quantification of T. magnatum were selected from the ITS rDNA regions. Their specificity was tested in silico and using qualitative PCR on DNA extracted from 25 different fungal species. The T. magnatum DNA concentration was different in the four experimental truffières and higher in the productive plots. T. magnatum mycelium was however also detected in most of the non-productive plots. Ascoma production during the three years of the study was correlated with the concentration of T. magnatum DNA. CONCLUSIONS: Taken together, these results suggest that the specific real-time PCR assay perfected in this study could be an useful tool to evaluate the presence and dynamics of this precious truffle in natural and cultivated truffières.

Effect of 300 mT static and 50 Hz 0.1 mT extremely low frequency magnetic fields on Tuber borchii mycelium.

The present work aimed to investigate whether exposure to static magnetic field (SMF) and extremely low frequency magnetic field (ELF-MF) can induce biomolecular changes on Tuber borchii hyphal growth. Tuber borchii mycelium was exposed for 1 h for 3 consecutive days to a SMF of 300 mT or an ELF-MF of 0.1 mT 50 Hz. Gene expression and biochemical analyses were performed. In mycelia exposed to ELF-MF, some genes involved in hyphal growth, investigated using quantitative real-time polymerase chain reaction, were upregulated, and the activity of many glycolytic enzymes was increased. On the contrary, no differences were observed in gene expression after exposure to SMF treatment, and only the activities of glucose 6-phosphate dehydrogenase and hexokinase increased. The data herein presented suggest that the electromagnetic field can act as an environmental factor in promoting hyphal growth and can be used for applicative purposes, such as the set up of new in vitro cultivation techniques.

First report of European truffle ectomycorrhiza in the semi-arid climate of Saudi Arabia.

Tuber melanosporum Vittad. (Black or Périgord truffle) is a truffle native to the Mediterranean Southern Europe, popular for its unique flavor, and has great economic importance. The present work focused on assessing the possibility of cultivating T. melanosporum associated with Quercus robur L. in the desert climate of Saudi Arabia. The plantation was initiated in November 2018 by planting 271 oak seedlings in the Al-Qassim desert area and checked for survival and ectomycorrhiza development after 1.5 years of plantation maintenance. Amongst the 271 seedlings planted, 243 plants survived two harsh seasons (2019 and 2020), and the randomly selected and tested seedlings were still mycorrhized with T. melanosporum. The mycorrhization level with T. melanosporum was between 5 and 35% of all fine roots, and the share of contaminant ectomycorrhiza was low. In comparison to other areas where T. melanosporum is successfully cultivated, the Al-Qassim desert area has 10-15 °C higher average summer temperatures and a low total annual precipitation, which necessitates regular irrigation of the plantation. This work opens the avenue for an adapted, yet sustainable cultivation of T. melanosporum-inoculated oak tree in a desert climatic condition and introduces new opportunities of the agro-forest business in Saudi Arabia and GCC region.

Effect of slug mycophagy on Tuber aestivum spores.

Truffles in the genus Tuber produce subterranean fruiting bodies that are not able to actively discharge their spores in the environment. For this reason, truffles depend on mycophagous animals for reproduction. Fungus consumption (mycophagy) is a behaviour typical of both vertebrates and invertebrates. Mammals, especially rodents, are the most studied group of mycophagists and have been found to consume a great variety of fungi. Among invertebrates, mycophagy is documented in arthropods, but rarely in molluscs. In our study we assessed the effect on the morphology and mycorrhizal colonization of Tuber aestivum spores after passage through the gut of slugs (Deroceras invadens) and, for comparison, of a house mouse (Mus musculus). Light, scanning electron and atomic force microscopy revealed that the digestion, especially by slugs, freed spores from the asci and modified their morphology. These are believed to be the reasons why we observed an improvement in oak mycorrhization with the slug and rodent ingested spores in comparison to a fresh spore inoculation. We also demonstrated by molecular barcoding that slugs' guts sampled on a Tuber melanosporum truffle ground contain spores from this species and Tuber brumale, further suggesting that some invertebrates are efficient Tuber spore dispersers.