Comparison of structural and antioxidant activity of polysaccharide extracted from truffles.

As the main component of truffles, polysaccharides have a variety of biological activities such as anti-oxidation, anti-tumor, and hypoglycemic activity, and these activities are closely related to its structure. In this study, Tuber Aestivum crude polysaccharide (TACP) and Tuber Melanosporum crude polysaccharide (TMCP) were obtained from Tuber Aestivum and Tuber Melanosporum by using microwave-assisted hot water, and then the Sephadex G-200 column was utilized to further separate and purify Tuber Aestivum polysaccharide (TAP) and Tuber Melanosporum polysaccharide (TMP) from TACP and TMCP. The structural characterization results showed that the molecular weight of TAP was 2.18 × 104  kDa, while TMP was 8.79 × 103  kDa. Although the two polysaccharide components were mainly composed of mannose (Man) and glucose (Glc), the molar ratio of Man and Glc in TAP was 14.76: 12.31, with a molar ratio of 5.43:10.94 in TMP. Furthermore, the antioxidant activity of two polysaccharide components was evaluated. TAP and TMP could protect porcine jejunal epithelial (IPEC-J2) cells from oxidative damage by H2 O2 , but TAP exhibited stronger antioxidant effects. It was mainly reflected that TAP could increase the secretion level of intracellular antioxidant enzymes (superoxide dismutase and catalase) in IPEC-J2 cells, and had a significant effect on the total antioxidant capacity of cells. The reactive oxygen species and malondialdehyde had better scavenging ability at the concentration of 20 µg/ml. The difference between TAP and TMP may be due to the dissimilar structure. Its structure-activity relationship needs further study. PRACTICAL APPLICATION: The structure of TAP and TMP were different, and TAP had higher molecular weight. Besides, TAP and TMP can protect IPEC-J2 cells from oxidative stress, providing a theoretical basis for developing potential antioxidant drugs of practical significance.

Headspace Solid-Phase Microextraction Followed by Gas Chromatography-Mass Spectrometry as a Powerful Analytical Tool for the Discrimination of Truffle Species According to Their Volatiles.

This study provides the first assessment of the volatile metabolome map of Tuber Aestivum and Tuber Borchii originating from Greece using headspace solid-phase micro-extraction (HS-SPME) coupled to gas chromatography-mass spectrometry (GC-MS). For the extraction of the volatile fraction, the SPME protocol was optimized after examining the effects of sample mass, extraction temperature, and extraction time using the one-variable at-a-time approach (OVAT). The optimum parameters involved the extraction of 100 mg of homogenized truffle for 45 min at 50°C. Overall, 19 truffle samples were analyzed, and the acquired data were normalized and further processed with chemometrics. Agglomerative hierarchical clustering (HCA) was used to identify the groups of the two species. Partial least squares-discriminant analysis (PLS-DA) was employed to develop a chemometric model that could discriminate the truffles according to the species and reveal characteristic volatile markers for Tuber Aestivum and Tuber Borchii grown in Greece.

Larval Morphological Adaptations of Leiodes cinnamomea (Panzer, 1793) (Coleoptera: Leiodidae: Leiodinae)-Obligatory Feeder of Tuber Species.

Detailed redescription of morphology for three larval instars of truffle-feeder Leiodes cinnamomea, documented on scanning micrographs and illustrations, is provided. Systematic context, observations on biology and unique characteristics of larvae of the only well-described representative of Leiodini are discussed. Exoskeletal invaginations (pseudomycangia), granulation on the head and the median longitudinal row of sclerotized plates on tergites VII-IX-the unique structures among leiodid larvae-were confirmed, described and documented. A mosaic strategy for beetles living in ephemeral and in solid fungi is discussed.

Application of Pressurized Liquid Extractions to Obtain Bioactive Compounds from Tuber aestivum and Terfezia claveryi.

A PLE (pressurized liquid extraction) method was adjusted following a full-factorial experimental design to obtain bioactive-enriched fractions from Tuber aestivum and Terfezia claveryi. Temperature, time and solvent (water, ethanol and ethanol-water 1:1) parameters were investigated. The response variables investigated were: obtained yield and the levels of total carbohydrate (compounds, ß-glucans, chitin, proteins, phenolic compounds and sterols). Principal component analysis indicated water solvent and high temperatures as more adequate parameters to extract polysaccharide-rich fractions (up to 68% of content), whereas ethanol was more suitable to extract fungal sterols (up to 12.5% of content). The fractions obtained at optimal conditions (16.7 MPa, 180 °C, 30 min) were able to protect Caco2 cells from free radical exposure, acting as antioxidants, and were able to reduce secretion of pro-inflammatory cytokines in vitro: IL-6 (50%), and TNFα (80% only T. claveryi ethanol extract), as well as reduce high inhibitory activity (T. aestivum IC50: 9.44 mG/mL).

Eco-archaeological excavation techniques reveal snapshots of subterranean truffle growth.

Despite its status as a highly-prized and coveted fungi in gastronomy, many aspects of the subterranean life cycle of the Burgundy truffle (Tuber aestivum) are still unknown, because in situ observations of the formation and maturation of truffle fruitbodies remain difficult. Here, we adopted a suite of archaeological fine-scale excavating techniques to provide unique spatiotemporal snapshots of Burgundy truffle growth at three sites in southern Germany. We also recorded the relative position, fresh weight, maturity level and genotype composition of all excavated fruitbodies. Varying by a factor of thousand, the fresh weight of 73 truffle ranged from 0.1 to 103.2 g, with individual maturity levels likely representing different life cycle stages from completely unripe to fully ripe and even decaying. While only a slightly positive relationship between fruitbody weight and maturity level was found, our results suggest that genetically distinct specimens can exhibit different life cycle stages at the same period of time and under the same environmental conditions. We therefore argue that truffles are likely able to grow, mature and ripe simultaneously between early summer and late winter of the following year. Our case study should encourage further eco-archaeological truffle excavations under different biogeographic settings and at different seasons of the year to gain deeper insights into the fungi's subterranean ecology. The expected cross-disciplinary findings will help truffle hunters and farmers to improve their harvest practices and management strategies.

Ectomycorrhiza resilience and recovery to extreme flood events in Tuber aestivum and Quercus robur.

Very little is known about the impact of flooding and ground saturation on ectomycorrhizal fungi (EcM) and increasing flood events are expected with predicted climate change. To explore this, seedlings inoculated with the EcM species Tuber aestivum were exposed to a range of flood durations. Oak seedlings inoculated with T. aestivum were submerged for between 7 and 65 days. After a minimum of 114-day recovery, seedling growth measurements were recorded, and root systems were destructively sampled to measure the number of existing mycorrhizae in different zones. Number of mycorrhizae did not display correlation with seedling growth measurements. Seven days of submersion resulted in a significant reduction in mycorrhizae numbers and numbers reduced most drastically in the upper zones. Increases in duration of submersion further impacted mycorrhizae numbers in the lowest soil zone only. T. aestivum mycorrhizae can survive flood durations of at least 65 days. After flooding, mycorrhizae occur in higher numbers in the lowest soil zone, suggesting a mix of resilience and recovery. The results will aid in furthering our understanding of EcM but also may aid in conservation initiatives as well as providing insight for those whose livelihoods revolve around the collection of EcM fruiting bodies or cropping of the plant partners.

White mulch and irrigation increase black truffle soil mycelium when competing with summer truffle in young truffle orchards.

The black truffle (Tuber melanosporum Vittad.) and the summer truffle (Tuber aestivum Vittad.) are two of the most appreciated edible fungi worldwide. The natural distributions of both species partially overlap. However, the interspecific interactions between these truffles and how irrigation and mulching techniques impact the dynamics between them are still unknown. Here, an experimental truffle plantation with Quercus ilex was established in Maials (Catalonia, Spain), combining three soil mulch treatments (white mulch, black mulch and bare soil as a control) and two irrigation regimes (irrigated and non-irrigated as a control) to investigate truffle mycelial dynamics in soil when both truffle species co-occur. The development of truffle mycelium in two different seasons (spring and autumn) in two consecutive years (2017 and 2018) was quantified using qPCR. Truffle mycelia of both species showed greatest development under white mulch. When mycelia of both truffle species co-occurred in soil, irrigation combined with white mulch resulted in greater quantities of T. melanosporum mycelial biomass, whereas the control irrigation treatment favoured the development of T. aestivum. Mulch treatments were also advantageous for seedling growth, which was expressed as root collar diameter and its increment during the study period. Significant relationships between root collar diameter and root growth and the amount of mycelial biomass in the soil were observed for both truffle species. Our results indicate the potential advantages of using white mulch to support irrigation in truffle plantations located in areas with dry Mediterranean climatic conditions to promote the development of Tuber mycelium.

Anti-angiogenic and anti-inflammatory activity of the summer truffle (Tuber aestivum Vittad.) extracts and a correlation with the chemical constituents identified therein.

Fungi are a huge source of unexplored bioactive compounds. Owing to their biological activities, several fungi have shown commercial application in the health industry. Tuber aestivum Vittad. is one such edible fungi with an immense scope for practical biological applications. In the present study, the anti-angiogenic activity of petroleum ether and ethanol extracts of T. aestivum was investigated using the chick chorioallantoic membrane assay and compared to the positive controls silibinin and lenalidomide. Both the extracts showed a dose-dependent anti-angiogenic response. The extracts were also assessed for their anti-inflammatory potential by lipoxygenase-inhibition assay. The IC50 values for LOX inhibition assay, computed by the Boltzmann plot, were 368.5, 147.3 and 40.2 µg/mL, for the petroleum ether extract, ethanol extract, and the positive control ascorbic acid, respectively. The ethanol extract of T. aestivum showed superior anti-angiogenic and anti-inflammatory activity than the petroleum ether extract. Compositional investigation of the extracts by GC-MS revealed the presence of various bioactive compounds. The compounds were correlated to their anti-angiogenic and anti-inflammatory activity based on a meticulous literature search.

A study on the fatty acid composition of lipids in truffles selected from Europe and Africa.

The gas chromatography of hexane extracts from ascocarps of Tuber maculatum (mostly the first report), T. aestivum/unicantum, T. borchii, T. melanosporum and Tirmania nivea dominantly showed palmitic, stearic, oleic and linoleic acids followed by traces of polyunsaturated fatty acids. The fatty acid content varied from ca. 8-61 mg g- 1, dry-weight-basis with species with highest for T. maculatum. Polyunsaturated fatty acids contributions varied from ca. 42-59%. The dominant fatty acid varied with the species. A comparison with existing reports on same species cultivated in different regions showed differences in contributions by saturated, monounsaturated and polyunsaturated fatty acids as well as dominant fatty acids detected. Lesser explored species such as T. borchii, T. maculatum call for further research. This is a preliminary study that indicates fatty acid composition as a potential tool for distinction like aroma between truffle species and geographies of cultivation. This forms the basis for further studies in different species and regions.

Identification of bacteria and fungi inhabiting fruiting bodies of Burgundy truffle (Tuber aestivum Vittad.).

Tuber species may be regarded as complex microhabitats hosting diverse microorganisms inside their fruiting bodies. Here, we investigated the structure of microbial communities inhabiting the gleba of wild growing (in stands) T. aestivum, using Illumina sequencing and culture-based methods. The two methods used in combination allowed to extract more information on complex microbiota of Tuber aestivum gleba. Analysis of the V3-V4 region of 16S rDNA identified nine phyla of bacteria present in the gleba of T. aestivum ascomata, mostly Proteobacteria from the family Bradyrhizobiaceae. Our results ideally match the earlier data for other Tuber species where the family Bradyrhizobiaceae was the most represented. The ITS1 region of fungal rDNA represented six alien fungal species belonging to three phyla. To complement the metagenomic analysis, cultivable fungi and bacteria were obtained from the gleba of the same T. aestivum fruiting bodies. The identified fungi mostly belong to the phylum Basidiomycota and same to Ascomycota. Analysis of cultivable bacteria revealed that all the specimens were colonized by different strains of Bacillus. Fungal community inhabiting T. aestivum fruiting bodies was never shown before.

Orchard Conditions and Fruiting Body Characteristics Drive the Microbiome of the Black Truffle Tuber aestivum.

Truffle fungi are well known for their enticing aromas partially emitted by microbes colonizing truffle fruiting bodies. The identity and diversity of these microbes remain poorly investigated, because few studies have determined truffle-associated bacterial communities while considering only a small number of fruiting bodies. Hence, the factors driving the assembly of truffle microbiomes are yet to be elucidated. Here we investigated the bacterial community structure of more than 50 fruiting bodies of the black truffle Tuber aestivum in one French and one Swiss orchard using 16S rRNA gene amplicon high-throughput sequencing. Bacterial communities from truffles collected in both orchards shared their main dominant taxa: while 60% of fruiting bodies were dominated by α-Proteobacteria, in some cases the ß-Proteobacteria or the Sphingobacteriia classes were the most abundant, suggesting that specific factors (i.e., truffle maturation and soil properties) shape differently truffle-associated microbiomes. We further attempted to assess the influence in truffle microbiome variation of factors related to collection season, truffle mating type, degree of maturation, and location within the truffle orchards. These factors had differential effects between the two truffle orchards, with season being the strongest predictor of community variation in the French orchard, and spatial location in the Swiss one. Surprisingly, genotype and fruiting body maturation did not have a significant effect on microbial community composition. In summary, our results show, regardless of the geographical location considered, the existence of heterogeneous bacterial communities within T. aestivum fruiting bodies that are dominated by three bacterial classes. They also indicate that factors shaping microbial communities within truffle fruiting bodies differ across local conditions.

New insights into black truffle biology: discovery of the potential connecting structure between a Tuber aestivum ascocarp and its host root.

According to isotopic labeling experiments, most of the carbon used by truffle (Tuber sp.) fruiting bodies to develop underground is provided by host trees, suggesting that trees and truffles are physically connected. However, such physical link between trees and truffle fruiting bodies has never been observed. We discovered fruiting bodies of Tuber aestivum adhering to the walls of a belowground quarry and we took advantage of this unique situation to analyze the physical structure that supported these fruiting bodies in the open air. Observation of transversal sections of the attachment structure indicated that it was organized in ducts made of gleba-like tissue and connected to a network of hyphae traveling across soil particles. Only one mating type was detected by PCR in the gleba and in the attachment structure, suggesting that these two organs are from maternal origin, leaving open the question of the location of the opposite paternal mating type.

Antimicrobial activity of gallic acid against food-related Pseudomonas strains and its use as biocontrol tool to improve the shelf life of fresh black truffles.

Refrigeration alone or in combination with other technologies represents the main tool used in the last decades to preserve the freshness of black truffles. This is principally due to the delicateness and vulnerability of this edible hypogeous fungus, so that other invasive preservation practices cannot be adopted. However, the proliferation of some microbial species during the cold storage still represents an unsolved problem. Pseudomonads are among the main spoiler bacteria responsible for the deterioration of refrigerated black truffles. Their growth ability at low temperatures requires the use of additional hurdles to prolong the shelf-life of truffles without altering their major features. The use of natural compounds may represent an alternative system for the biocontrol of this kind of product. Specifically, gallic acid (GA) is a phenolic acid naturally present in different foods, whose effectiveness was in vitro demonstrated against Pseudomonas spp. In our study, we reported the antimicrobial activity expressed by GA not only in vitro, using as target bacteria Pseudomonas putida DSMZ 291T, P. fluorescens DSMZ 50090T, P. fragi DSMZ 3456T and Pseudomonas spp. P30-4, previously isolated from black truffles, but also in situ on fresh black truffles stored at 4°C for 28days. Our results showed Minimum Inhibitory Concentrations (MIC) of 2.5mg/mL GA for all tested strains, except for P. fluorescens DSMZ 50090T, having a MIC corresponding to 5mg/mL GA. The Minimum Bactericidal Concentration (MBC) was 10mg/mL for all strains. The analysis of kinetic parameters showed that the survival declined passing from 2.5 to 10mg/mL GA concentrations, with P. fluorescens confirmed to be the most resistant strain. Moreover, images obtained from Scanning Electron Microscopy revealed that Pseudomonas cells were strongly injured by the treatment with GA at 2.5mg/mL concentration, displaying visible pores on the cellular surfaces, absence of flagella and lysis with loss of cytoplasmic material. The storage test performed on fresh black truffles confirmed in situ the GA antimicrobial activity observed in vitro, with a drastic reduction not only of Pseudomonas spp., but also of the other assessed microbial groups, including Enterobacteriaceae and Eumycetes. Finally, sensory analysis established the absence of off-flavours and the preservation of positive features in black truffles treated with 2.5mg/mL GA and stored for 28 d at 4°C. The results obtained in this study suggest that GA is a potential biocontrol tool to decontaminate and preserve fresh black truffles during refrigerated storage.

Multi-cropping edible truffles and sweet chestnuts: production of high-quality Castanea sativa seedlings inoculated with Tuber aestivum, its ecotype T. uncinatum, T. brumale, and T. macrosporum.

The plantation and management of sweet chestnut (Castanea sativa Mill.) orchards is a common and traditional land use system in many areas of Europe that offers the advantage of simultaneous production of nuts and timber. During the last decades, sweet chestnut has declined dramatically in many regions because of the profound social changes in rural areas coupled with pathogen attacks. Truffles, the hypogeous ascocarps of the ectomycorrhizal genus Tuber, are currently cultivated using host trees inoculated with these fungi for improving production in truffle orchards. The production of good forestry quality chestnut seedlings inoculated with European truffles in nurseries is essential for multi-cropping plantation establishment, but so far, it has not been implemented in agroforestry practices. Moreover, it is necessary to assess the physiological condition of the seedlings due to the high calcium amendment needed for the growth of Tuber spp. mycelium that can become toxic for the host plants. In this study, seedlings of C. sativa were inoculated with Tuber aestivum and its ecotypes T. uncinatum, T. brumale, and T. macrosporum and were grown in a greenhouse using culture conditions favorable for the production of high-quality plants for forestry purposes. At the end of the assay, levels of root colonization and morphological and physiological parameters of the seedlings were measured. The colonization of C. sativa with T. aestivum, its ecotype T. uncinatum, and T. brumale was successful, and the seedlings showed normal growth. Inoculation protocols with T. macrosporum need to be improved. Tuber species formed well-developed ectomycorrhizae on C. sativa in nursery conditions.

Comparison of Chemical Composition in Tuber aestivum Vittad. of Different Geographical Origin.

Truffles are prized and nutrition-rich edible hypogeous fungi. The aim of this study was a comprehensive investigation of chemical composition of Burgundy truffle (Tuber aestivum Vittad.). We tried to answer the question: what is the impact of the environment on the truffle quality. To know the nutritional value of Burgundy truffle we compared lipids, proteins, saccharides, polyphenolics, flavonoids, total sterols, ergosterol, volatile flavour and aroma compounds content in fruit bodies of the fungus collected in three different geographical regions, i.e., Poland, Slovakia, and Italy. A comparison of the above mentioned compounds is especially interesting due to environmental and climatic differences among the studied geographical regions. Results revealed that fruit bodies of T. aestivum from Poland and Slovakia possessed nearly similar content of proteins, total sterols, and saccharides. The fruiting bodies from Italy contained significantly larger amounts of most of the investigated compounds. In turn, Polish specimens had higher content of lipids and polyphenolics than Slovak and Italian ones. We have found higher similarity of volatile compounds composition between Polish and Italian specimens than those of Polish and Slovak origin.

SSR-based identification of genetic groups within European populations of Tuber aestivum Vittad.

Tuber species are ectomycorrhizal ascomycetes establishing relationships with different host trees and forming hypogeous fruiting bodies known as truffles. Among Tuber species, Tuber aestivum Vittad. has a wide distributional range being found naturally all over Europe. Here, we performed large-scale population genetic analyses in T. aestivum to (i) investigate its genetic diversity at the European scale, (ii) characterize its genetic structure and test for the presence of ecotypes and (iii) shed light into its demographic history. To reach these goals, 230 ascocarps from different populations were genotyped using 15 polymorphic simple sequence repeat markers. We identified 181 multilocus genotypes and four genetic groups which did not show a clear geographical separation; although, one of them was present exclusively in Southeast France, Italy and Spain. Fixation index values between pairs of genetic groups were generally high and ranged from 0.29 to 0.45. A significant deficit of heterozygosity indicated a population expansion instead of a recent population bottleneck, suggesting that T. aestivum is not endangered in Europe, not even in Mediterranean regions. Our study based on a large-scale population genetic analysis suggests that genetically distinct populations and likely ecotypes within T. aestivum are present. In turn, this study paves the way to future investigations aimed at addressing the biological and/or ecological factors that have concurred in shaping the population genetic structure of this species. Present results should also have implications for the truffle market since defining genetic markers are now possible at least for some specific T. aestivum genetic groups.

Phenolic profile, antioxidant, anti-inflammatory and cytotoxic activities of black (Tuber aestivum Vittad.) and white (Tuber magnatum Pico) truffles.

The aim of this study was a comprehensive investigation on phenolic profile, antioxidant, anti-inflammatory and cytotoxic activity of the still insufficiently explored black summer truffles (Tuber aestivum Vittad.) and white (Tuber magnatum Pico) truffles. Methanol and water extracts, obtained by maceration and Soxhlet extraction, were examined. Forty-five phenolics was studied using LC-MS/MS: presence of 14 compounds was confirmed, with the most dominant being p-hydroxybenzoic acid, baicalein and kaempferol (T. aestivum), epicatechin and catechin (T. magnatum). Moderate antioxidant activity of both species was determined through several assays. Only T. magnatum showed anti-inflammatory potential by inhibiting COX-1 and 12-LOX pathway products synthesis. Methanol extracts exerted cytotoxicity against some tumour cell lines (HeLa, MCF7, HT-29), besides the prominent activity of water extracts towards breast adenocarcinoma (MCF7). To conclude, these results support further investigations of phytochemicals and biological activity towards verification of nutraceutical use of both species.

First identification of polymorphic microsatellite markers in the Burgundy truffle, Tuber aestivum (Tuberaceae).

PREMISE OF THE STUDY: Tuber aestivum, the most common truffle in Europe, plays an important role in the commercial truffle market. For the first time, microsatellite primers were developed to investigate polymorphism within this species. • METHODS AND RESULTS: Using direct shotgun pyrosequencing, 15 polymorphic microsatellites were identified out of the 7784 perfect microsatellites present in the 534620 reads obtained. Tested on 75 samples, these microsatellites were highly polymorphic. The number of alleles varied from four to 15, and the expected heterozygosity ranged from 0.266 to 0.620. A multilocus analysis allowed the identification of 63 genotypes over the 75 samples analyzed. • CONCLUSIONS: Direct shotgun pyrosequencing is a fast and relatively low-cost technique allowing identification of microsatellites in nonmodel species. The microsatellites developed in this study will be useful in population genetic studies to infer the evolutionary history of this species.