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.

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.

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.

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.

Multilocus Phylogeography of the Tuber mesentericum Complex Unearths Three Highly Divergent Cryptic Species.

Tuber mesentericum is an edible European black truffle, apparently easy to recognize, but showing a high degree of genetic variability. In this study, we performed an integrative taxonomic assessment of the T. mesentericum complex, combining a multilocus phylogeographic approach with morphological analyses, and including authentic specimens of Vittadini, and Berkeley and Broome. We performed maximum likelihood phylogenetic analyses, based on single and concatenated gene datasets (ITS rDNA, ß-tubulin, elongation factor 1-α), and including all available sequences from previous studies. Phylogenetic analyses consistently recovered three reciprocally monophyletic and well-supported clades: clade I, with a wide range across Europe; clade II, specimens collected mainly in the Iberian, Italian, and Balkan peninsulas; and clade III, specimens collected almost exclusively in central Italy. Genetic distance between clades ranged from 10.4% to 13.1% at the ITS region. We also designed new primer pairs specific for each phylogenetic lineage. Morphology of spores, asci, and peridium were investigated on specimens representing the three lineages. Macro- and micromorphological analyses of ascomata revealed only a few, but not diagnostic, differences between the three phylogenetic lineages, thus, confirming that they are morphologically cryptic. By studying authentic specimens of Vittadini, and Berkeley and Broome, it was possible to identify the three clades as T. mesentericum, Tuber bituminatum, and Tuber suave sp. nov., and to designate an epitype for T. mesentericum s.s. and a lectotype for T. bituminatum. Future investigations on volatile organic compound (VOC) composition are needed to define the aroma repertoires in this species complex.

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.

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.

Synthesis and ultrastructural observation of arbutoid mycorrhizae of black truffles (Tuber melanosporum and T. aestivum).

Arbutus unedo (the strawberry tree) is a Mediterranean shrub which forms arbutoid mycorrhizae with a variety of Asco- and Basidiomycetes. After the discovery of the mycorrhizal symbiosis between A. unedo and Tuber borchii, in this study, arbutoid mycorrhizae were synthetized in greenhouse with Tuber aestivum and Tuber melanosporum. Six months after inoculation, both species colonized the roots of all inoculated A. unedo seedlings, but mature mycorrhizae were only observed after 12 months. Ultrastructure analysis of Tuber arbutoid mycorrhizae was described for the first time, showing, as observed in typical endosymbiosis, a rearrangement of host cells and the creation of an interface compartment with both truffle species. Immunolabelling experiments suggested that pectins are not present in the interface matrix surrounding the intracellular hyphae. Thus, the ability to establish symbiosis with A. unedo seems to be a common feature in the genus Tuber, opening up the possibility to use this plant for mycorrhization with valuable truffles. This could represent an important economic opportunity in Mediterranean areas by combining the production of truffles, edible fruits and valued honey.

Tuber iranicum, sp. nov., a truffle species belonging to the Excavatum clade.

Truffles in the genus Tuber are hypogeus fungi that have a worldwide distribution. Despite this, knowledge about their diversity in the Middle East is very limited. In recent years, large quantities of truffles have been imported from Iran for being sold in Italy. While analyzing certain commercial batches of T. aestivum from Iran, we found some ascomata that resembled T. excavatum but had macro- and micromorphological features that were distinct from this species. They were subglobose, or depressed to slightly irregular, with a conspicuous basal cavity, grayish brown, brown, or pinkish gray, with a minutely papillose peridium. The gleba was pinkish gray in youth, brown at maturity, marbled with cream branched veins. Ascospores were broadly ellipsoid, with an irregular reticulum and distinctive long crests along the longitudinal axis, up to 9 µm high. Analysis of internal transcribed spacer (ITS) and large subunit (LSU) rDNA sequences showed that these specimens form a monphyletic and well-supported taxon within the Excavatum clade. Morphological and molecular analyses supported the proposal of the new species T. iranicum.

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.

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.

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.

Ultra-Low Freezing to Preserve the Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Agaricomycetes).

Ganoderma lucidum (Curtis) P. Karst., commonly used in traditional Chinese medicine, is characterized by strong genetic and phenotypic variability that reflects its active components. To preserve such a source of pharmacologically active metabolites, specimens must be collected from different geographic regions and their genetic integrity ensured during storage. To this aim, we tested the effect of ultra-low freezing (ULF) at -120°C on the vitality, mycelial growth rate, and fruiting ability of 3 Italian strains of G. lucidum. Results showed that all strains reacted positively to ULF, demonstrating an ability to recover after 3 months of storage without morphological or physiological changes occurring, regardless of treatment. The successful storage of G. lucidum at -120°C opens up the possibility to create a germplasm bank to collect strains of this medicinal fungus from throughout Europe, thereby contributing to the maintenance of its diversity.

Draft Genome Sequence of Tuber borchii Vittad., a Whitish Edible Truffle.

The ascomycete Tuber borchii (Pezizomycetes) is a whitish edible truffle that establishes ectomycorrhizal symbiosis with trees and shrubs. This fungus is ubiquitous in Europe and is also cultivated outside Europe. Here, we present the draft genome sequence of T. borchii strain Tbo3840 (97.18 Mb in 969 scaffolds, with 12,346 predicted protein-coding genes).

Crested porcupines (Hystrix cristata): mycophagist spore dispersers of the ectomycorrhizal truffle Tuber aestivum.

Truffles, as hypogeous, ectomycorrhizal fungi, have no means to actively discharge spores into the environment and thus depend on mycophagists for spore dispersal. After consumption of fruiting bodies by animals and passage through the digestive tract, the spores are released in faecal pellets. Recently, in the Abruzzo region (Italy), Hystrix cristata has been spotted inside private truffières, but its role in spore dispersal has never been investigated. Here, we report our research on the occurrence of Tuber aestivum spores in porcupine's faecal contents in a truffière in L'Aquila, Italy, where a H. cristata specimen was photographed. The spores were isolated from faeces by using a suspension of 0.7 M ZnSO4. We also verified degradation and disfiguration of the digested spores' reticular ornamentation compared to that of fresh spores from ascomata collected inside the truffière, through measurements performed by scanning electron microscopy. A few truffle spores had germinated within the faeces.

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.

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.

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.

Genetic resources and mycelial characteristics of several medicinal polypore mushrooms (Polyporales, Basidiomycetes).

Mycelial characteristics of dikaryotic collections of 6 medicinal polypore mushrooms (Fomes fomentarius, Fomitopsis pinicola, Ganoderma adspersum, G. applanatum, G. lucidum, and G. resinaceum) with different geographical origins (Armenia, China, France, Iran, Italy, and Russia) were screened. A total of 42 polypore collections were molecularly identified by sequencing the internal transcribed spacer region of the ribosomal RNA genes' cluster, and a phylogenetic tree was constructed. Morphological characteristics of 37 cultures were observed on agar media (malt extract agar, potato dextrose agar) at different temperatures (25, 30, 35, and 38°C) at a pH of 6.0. Colony morphology, pigmentation of mycelium and agar, mycelial growth rate, in vitro teleomorph formation, and other macromorphological characteristics were thoroughly described and illustrated. Micromorphological features of mycelia, such as different hyphal structures, clamp cells, presence and type of asexual sporulation, chlamydospores, and others were observed. The taxonomic significance of the mycelial characteristics revealed was estimated. The obtained results will assist further biotechnological cultivation of medicinal polypore mushrooms to develop novel health care biotechnological products.

Biochemical characterization and antioxidant and antiproliferative activities of different Ganoderma collections.

The aim of this study was to conduct a molecular and biochemical characterization and to compare the antioxidant and antiproliferative activities of four Ganoderma isolates belonging to Ganoderma lucidum (Gl-4, Gl-5) and Ganoderma resinaceum (F-1, F-2) species. The molecular identification was performed by ITS and IGS sequence analyses and the biochemical characterization by enzymatic and proteomic approaches. The antioxidant activity of the ethanolic extracts was compared by three different methods and their flavonoid contents were also analyzed by high-performance liquid chromatography. The antiproliferative effect on U937 cells was determined by MTT assay. The studied mycelia differ both in the enzymatic activities and protein content. The highest content in total phenol and the highest antioxidant activity for DPPH free radical scavenging and chelating activity on Fe(2+) were observed with the Gl-4 isolate of G. lucidum. The presence of quercetin, rutin, myricetin, and morin as major flavonoids with effective antioxidant activity was detected. The ethanolic extracts from mycelia of G. lucidum isolates possess a substantial antiproliferative activity against U937 cells in contrast to G. resinaceum in which the antiproliferative effects were insignificant. This study provides a comparison between G. lucidum and G. resinaceum mycelial strains, and shows that G. resinaceum could be utilized to obtain several bioactive compounds.