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The microbial community associated with ascocarps of the ectomycorrhizal fungus Tuber borchii Vittad. was studied by both cultivation and direct extraction of bacterial 16S rRNA gene (rDNA) sequence approaches. The inner part of six T. borchii ascoma collected in North-Central Italy was used to establish a bacterial culture collection and to extract the total genomic DNA to obtain a library of 16S rDNAs representative of the truffle bacterial community. Most of the isolates were affiliated to the gamma-Proteobacteria, mainly Fluorescent pseudomonads; some isolates were members of the Bacteroidetes group and Gram-positive bacteria, mostly Bacillaceae. The majority of the clones from the library were alpha-Proteobacteria showing significant similarity values, of greater than 97%, with members of the Sinorhizobium/Ensifer Group, Rhizobium and Bradyrhizobium spp. not previously identified as Tuber-associated bacteria. Only a few bacterial strains belonging to this bacterial subclass were found in the culture collection and isolated on a medium specific for Rhizobium-like organisms. A few clones were members of the beta- and gamma-Proteobacteria; as well as low and high G+C Gram-positive bacteria. Our findings clearly indicate that a dual approach increases the information obtained on the structural composition of a truffle bacterial community as compared to that derived via cultivation or direct recovery of 16S rDNA sequences alone.

Truffles are known worldwide for their peculiar taste, aroma, and nutritious properties, which increase their economic value. However, due to the challenges associated with the natural cultivation of truffles, including cost and time, submerged fermentation has turned out to be a potential alternative. Therefore, in the current study, the cultivation of in submerged fermentation was executed to enhance the production of mycelial biomass, exopolysaccharides (EPSs), and intracellular polysaccharides (IPSs). The mycelial growth and EPS and IPS production was greatly impacted by the choice and concentration of the screened carbon and nitrogen sources. The results showed that sucrose (80 g/L) and yeast extract (20 g/L) yielded maximum mycelial biomass (5.38 ± 0.01 g/L), EPS (0.70 ± 0.02 g/L), and IPS (1.76 ± 0.01 g/L). The time course analysis of truffle growth revealed that the highest growth and EPS and IPS production was observed on the 28th day of the submerged fermentation. Molecular weight analysis performed by the gel permeation chromatography method revealed a high proportion of high-molecular-weight EPS when 20 g/L yeast extract was used as media and the NaOH extraction step was carried out. Moreover, structural analysis of the EPS using Fourier-transform infrared spectroscopy (FTIR) confirmed that the EPS was β-(1-3)-glucan, which is known for its biomedical properties, including anti-cancer and anti-microbial activities. To the best of our knowledge, this study represents the first FTIR analysis for the structural characterization of β-(1-3)-glucan (EPS) produced from grown in submerged fermentation.

$\\bullet$ TbSP1 is a secreted and surface-associated phospholipase A2 previously found to be up-regulated in C-or N-deprived free-living mycelia from the ectomycorrhizal ascomycete Tuber borchii. As nutrient limitation is considered an important environmental factor favouring the transition to symbiotic status, TbSP1 was suggested to be involved in the formation of mycorrhizas. $\\bullet$ An in vitro symbiosis system between Cistus incanus and T. borchii was set up: TbSP1 mRNA levels in free-living mycelia and in mycorrhizas sampled in different districts of the plant-fungus interaction were examined. In the same samples, TbSP1 protein expression was analysed by immunoelectron microscopy. $\\bullet$ A substantially enhanced TbSP1 mRNA expression, compared with nutrient-limited but free-living mycelia, was detected in the presence of the plant and reached maximal levels in fully developed mycorrhizas. A similar expression trend was revealed by immunolocalization experiments. $\\bullet$ We have shown that TbSP1 appears to respond to two partially overlapping yet distinct stimuli: nutrient starvation and mycorrhiza formation.

Tuber borchii forms ectomycorrhiza with oaks, hazel, and pines, including Pinus sylvestris. However, its ectomycorrhiza morphotype with P. sylvestris was not comprehensively described so far, and molecular analyses are missing despite a high danger of misidentification of T. borchii ectomycorrhiza with other closely related and less valuable truffle species. We described for the first time the morphology and anatomy of T. borchii-P. sylvestris ectomycorrhiza using differential interference contrast technique and semi-thin sections in combination with molecular confirmation of identity. Color of ectomycorrhiza is reddish to dark brown, and morphotypes are unevenly but densely covered by warts-bearing pin-like cystidia. All layers of the hyphal mantle are pseudoparenchymatous with outer mantle layer formed of epidermoid cells. T. borchii ectomycorrhiza was identified by a molecular comparison with fruitbodies used for inoculation and its respective ectomycorrhizae. T. borchii has a wide ecological amplitude. To get a better insight in mycorrhization requirements, we investigated growth of P. sylvestris and its ectomycorrhiza infection rate with T. borchii in substrate with different lime content. The mycorrhization of P. sylvestris with T. borchii in the mycorrhization substrate and cultivation in greenhouse conditions was successful, with colonization of P. sylvestris varying between 36.5 and 48.1%. There was no significant correlation of mycorrhization to applied lime contents, and consequently to pH in substrate, while the increased levels of lime improved growth of the P. sylvestris seedlings.

In filamentous ascomycete fungi, the utilization of alternate carbon sources is influenced by the zinc finger transcription factor CreA/CRE-1, which encodes a carbon catabolite repressor protein homologous to Mig1 from Saccharomyces cerevisiae. In Neurospora crassa, deletion of cre-1 results in increased secretion of amylase and β-galactosidase. Here we show that a strain carrying a deletion of cre-1 has increased cellulolytic activity and increased expression of cellulolytic genes during growth on crystalline cellulose (Avicel). Constitutive expression of cre-1 complements the phenotype of a N. crassa Δcre-1 strain grown on Avicel, and also results in stronger repression of cellulolytic protein secretion and enzyme activity. We determined the CRE-1 regulon by investigating the secretome and transcriptome of a Δcre-1 strain as compared to wild type when grown on Avicel versus minimal medium. Chromatin immunoprecipitation-PCR of putative target genes showed that CRE-1 binds to only some adjacent 5'-SYGGRG-3' motifs, consistent with previous findings in other fungi, and suggests that unidentified additional regulatory factors affect CRE-1 binding to promoter regions. Characterization of 30 mutants containing deletions in genes whose expression level increased in a Δcre-1 strain under cellulolytic conditions identified novel genes that affect cellulase activity and protein secretion. Our data provide comprehensive information on the CRE-1 regulon in N. crassa and contribute to deciphering the global role of carbon catabolite repression in filamentous ascomycete fungi during plant cell wall deconstruction.

Stone pine (Pinus pinea) produces a highly demanded dried fruit, the pine nuts; Tuber borchii produces a well-known commercialized truffle. Thus, the co-culture of P. pinea and T. borchii may represents an interesting productive option. This work evaluated the effect of T. borchii inoculation on stone pine tree growth, survival, health, entry into cone production, and mycorrhization level during the first 3 years after establishment along a 2000 km gradient in Chile. We tested two treatments (inoculation and non-inoculation) in a multi-environment trial (MET) involving seven sites along Chile. Plantations were repeatedly measured during 3 years after establishment. In all environments, inoculation enhanced tree height, root collar diameter (RCD) and crown diameter (6.9, 10 and 8.3% higher for inoculated than for non-inoculated plants). Vigor was also favored (14.1% more vigorous trees). Stone pine performance was enhanced in all sites, but mainly in those with extreme environmental conditions. T. borchii mycorrhizae were abundant (over 60% of mycorrhized root apexes) 3 years after establishment. The truffle-host plant combination was not previously evaluated in Chile. The results showed high root colonization levels and the persistence of mycorrhization 3 years after establishment, and an enhanced effect of mycorrhization on tree growth and vigor under different soil and climate conditions, confirming the high plasticity of both species.

Submerged fermentation of Tuber borchii and T. maculatum was performed to produce mycelia towards evaluating their bioactive potential and probable toxicity. Truffle mycelia had been mostly explored for biopolymers previously. Methanolic extraction yield, polyphenol content, and flavonoid content varied with species and period of fermentation. In vitro DPPH, FRAP, ABTS and ORAC radical scavenging activity of mycelial extracts of T. maculatum and T. borchii at 7 and 10 days of bioreactor fermentation corroborated with phenolics content. Absence of toxicity and antimutagenicity in lag and log phases in Saccharomyces cerevisiae D7 cells supported possibly safe nutraceutical use of mycelia of both truffles. Mineral content showed variation with species and fermentation time for macro- (calcium, potassium, phosphorus, sodium, iron, magnesium, and manganese) and trace elements (zinc, selenium, copper, chromium, and nickel). Mineral bioaccumulation can be explored for food-fortification applications. The demonstrated activities and detected constituents show potential applications in food, pharmaceutical, cosmetics and nutraceuticals.

Truffles are symbiotic fungi in high demand for the aroma of their fruiting bodies which are colonized by a diverse microbial flora. Specific sulfur containing volatiles (thiophene derivatives) characteristic of the white truffle Tuber borchii were recently shown to be derived from the bacterial community inhabiting truffle fruiting bodies. Our aim here was to investigate whether thiophene derivatives contributed to the human-sensed aroma of T. borchii. Furthermore, we questioned whether the concentration of thiophene volatiles was affected by freezing or whether it differed in truffles from distinct geographical origins. Gas chromatography–olfactometry (GC-O) analysis revealed that thiophene derivatives were major contributors to the aroma of T. borchii. Of four thiophene derivatives detected in this study, 3-methyl-4,5-dihydrothiophene was the most important one in terms of its contribution to the overall aroma. The relative concentration of thiophene derivatives was unaffected by freezing; however, it differed in samples collected in distinct geographical locations (Italy versus New Zealand). The causes of this variability might be differences in storage conditions and/or in bacterial community composition of the fruiting bodies; however, further work is needed to confirm these hypotheses. Overall, our results demonstrate that thiophene derivatives are major contributors to the human-sensed aroma of T. borchii.

Pinus pinea is an interesting species due to its pine nuts. Tuber borchii, a fungus that produces truffles, is a delicacy. Both species association offers a new productive opportunity. This work evaluated the effect of fertilization and inoculation with T. borchii on stone pine seedling growth in nursery and one year after field establishment in Chile, as well as mycorrhizal persistence. A factorial trial with two factors, inoculation and fertilization, was conducted in nursery. Mycorrhization, seedling growth, biomass and needle nutrient concentration after one growing season were measured. Mycorrhization and plant growth were measured one year after establishment a multi-environment trial involving seven sites. In nursery, inoculated seedlings presented over 50% of mycorrhized root apexes, with no differences between fertilized and non-fertilized plants. Under fertilization, inoculated plants had 16.5% higher height than non-inoculated ones. Fertilization increased seedling growth independently of inoculation. Inoculation did not modify the aerial to root biomass ratio. Inoculated and non-fertilized plants showed statistically higher P and lower K in needles than non-inoculated seedlings. One year after establishment, inoculation favored plant growth, and plants presented on average 88% of root apexes mycorrhized with T. borchii. Fertilization did not affect plant growth or mycorrhization levels. Stone pine inoculation with T. borchii was successful to produce abundantly mycorrhized roots in nursery. One year after field establishment, plant height and crown diameter were enhanced by inoculation, and high mycorrhization level suggests future truffle harvests. Both P. pinea and T. borchii showed high adaptability to different environments.Key messageStone pine (Pinus pinea) seedlings non-inoculated and inoculated with Tuber borchii were produced in nursery with and without fertilization, and established in seven field trials in central-southern Chile. Before planting, inoculated seedlings presented abundantly mycorrhized root systems, and fertilized plants showed higher height and root collar diameter, independently of inoculation, than non-fertilized ones. One year after plant field establishment, mycorrhization had a positive effect on plant growth, and T. borchii colonization was higher than in nursery. Fertilization did not affect growth or mycorrhization level. The results have practical implications for trufficulture and pine nut production in non-native habitats.

• Aroma variability in truffles has been attributed to maturation (Tuber borchii), linked to environmental factors (Tuber magnatum), but the involvement of genetic factors has been ignored. We investigated aroma variability in Tuber uncinatum, a species with wide distribution. Our aim was to assess aroma variability at different spatial scales (i.e. trees, countries) and to quantify how aroma was affected by genotype, fruiting body maturity, and geographical origin. • A volatile fingerprinting method was used to analyze the aroma of 223 T. uncinatum fruiting bodies from seven European countries. Maturity was estimated from spore melanization. Genotypic fingerprinting was performed by amplified fragment length polymorphism (AFLP). • Discriminant analysis revealed that, regardless of the geographical origin of the truffles, most of the aroma variability was caused by eight‐carbon‐containing volatiles (C8‐VOCs). In an orchard of T. uncinatum, truffles producing different concentrations of C8‐VOCs clustered around distinct host trees. This clustering was not associated with maturity, but was associated with fungal genotype. • These results indicate that the variation in C8‐VOCs in truffles is most likely under genetic control. They exemplify that understanding the factors behind aroma variability requires a holistic approach. Furthermore, they also raise new questions regarding the ecological role of 1‐octen‐3‐ol in truffles.