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.

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.

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.

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.

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.

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.

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.