Abstract
Truffles are hypogeous ascomycetes, belonging to the Tuber genus (true truffles) that form ectomycorrhizal symbiosis with the roots of different plant species. The Italian white truffle (Tuber magnatum Pico) is the world's most expensive mushroom. Despite its economic value, it has yet to be successfully cultivated, due to knowledge gaps in its biology and ecology. The aim of the project is to highlight T. magnatum relationships with soil microbiome and plants. To reach these objectives, four work packages (WP) will be considered: WP1 will investigate the interaction between T.magnatum and soil microorganisms in vitro; WP2, will study the interaction between T. magnatum and soil microorganisms and viruses in the field; WP3 will investigate the interactions between T. magnatum and host/non-host plants and WP4 is dedicated to project management and dissemination. Researches will be carried out in lab, greenhouse and field. Taking advantage of the T. magnatum genome sequencing and of studies on other Tuber species carried out by the research groups involved in this project, transcriptomic studies will be carried out on T. magnatum pure cultures, in association with mycelium growth-promoting bacteria and plant roots. The role of microbial community towards T. magnatum development will be studied through Illumina MiSeq technology in productive and not productive patches of a truffle ground, as well as its answer towards the inoculum of bacteria promoting mycelium growth of T. magnatum. Viruses will be also considered as component of the complex soil microbiota for the first time. Lastly, the possible endophytism of T. magnatum with ectomycorrhizal host plants and herbaceous plants will be investigated by fluorescent in situ hybridization. The results of this project aim to expand knowledge on T. magnatum soil ecology, laying the foundation for its cultivation. They will also indicate the most suitable techniques for the management and conservation of natural truffle grounds. Moreover, this study will give new insights on how to improve the mycorrhization techniques with T. magnatum and how to evaluate the quality of mycorrhized plants. Besides the commercial value of T. magnatum ascomata, the presence of areas intended for truffle production is now more important than ever for an eco-friendly management of agroforestry environments and it can represent a promising socio-economic opportunity for marginal lands. The PI of the project has a long experience in coordinating truffle projects and the other three research groups involved in this project have been collaborating in truffle research for more than 20 years.
Results achieved
The project successfully achieved its overall objective of advancing knowledge on the biology and ecology of Tuber magnatum Pico, the Italian white truffle, by elucidating key interactions between the fungus, soil microorganisms, viruses, and plants. The generated results significantly reduced major knowledge gaps that have historically limited the cultivation of this economically valuable species. A major achievement was obtained within WP1, which investigated the interactions between T. magnatum and soil bacteria under controlled laboratory conditions. For the first time, a stable and reproducible system for the in vitro cultivation of T. magnatum mycelium was developed through the identification of a highly specific association with bacteria belonging to the genus Bradyrhizobium (Graziosi et al., 2024). Experimental evidence demonstrated a strict mutualistic relationship between the fungus and these bacteria. Mycelial growth was significantly enhanced in the presence of the bacterial isolates, whereas fungal development was strongly impaired when bacterial partners were removed. Phylogenetic analyses showed that all isolates belonged to a previously undescribed lineage within the Bradyrhizobium jicamae supergroup and possessed the nitrogen-fixation gene nifH, suggesting a potential role in fungal nutrition. These findings provide a scientific basis for the future development of microbial inocula and improved cultivation strategies. Within WP2, the project investigated the role of soil microbial communities and viruses associated with natural white truffle grounds. Field experiments evaluated the effects of inoculating productive truffle grounds with Bradyrhizobium japonicum. Quantitative PCR analyses revealed an increase in T. magnatum mycelial biomass in two of the four treated plots one year after inoculation. Metabarcoding analyses also showed shifts in bacterial community composition following inoculation, particularly within Pseudomonadota, Planctomycetota, Actinomycetota and Chloroflexota, suggesting that bacterial inoculation can influence the soil microbial environment and favour fungal development (Ranocchi et al., under review). An additional innovative outcome of WP2 was the characterization of the viral component of T. magnatum ecosystems. Through metagenomic sequencing and dedicated bioinformatic analyses, viral genomes were reconstructed from truffle fruiting bodies and truffle-ground soils. Preliminary results revealed an unexpectedly high diversity of viruses, including mycoviruses belonging to at least ten different taxonomic groups. This represents the first exploration of the T. magnatum virome and opens new perspectives for understanding ecological processes regulating truffle productivity. Significant achievements were also obtained within WP3, which focused on interactions between T. magnatum and plants. The project provided the first direct evidence that the Italian white truffle can colonize non-ectomycorrhizal herbaceous plants as an endophyte. Novel species-specific Fluorescence In Situ Hybridization (FISH) probes were designed and validated to visualize T. magnatum within plant tissues (Graziosi et al., 2025). Field surveys conducted in natural truffle grounds detected T. magnatum DNA in the roots of several herbaceous species, including Carex pendula, Hedera helix, Primula vulgaris, and Arum italicum. Most importantly, confocal microscopy combined with FISH unequivocally demonstrated the presence of viable T. magnatum hyphae inside the root cortical tissues of Carex pendula, providing the first direct microscopic evidence of endophytic colonization by the Italian white truffle. These findings expand current knowledge of the ecological strategy of T. magnatum, suggesting that non-host plants may contribute to its persistence and dispersal in natural ecosystems. The project generated important scientific outputs. Results were published in international peer-reviewed journals, including the description of the mutualistic interaction between T. magnatum and Bradyrhizobium spp. (Graziosi et al., 2024) and the discovery of endophytic colonization of herbaceous plants by T. magnatum (Graziosi et al., 2025). An additional manuscript describing the effects of Bradyrhizobium inoculation on soil microbiota and T. magnatum growth is currently under review (Ranocchi et al., under review). Furthermore, sequencing data generated during the project were deposited in the NCBI Sequence Read Archive (PRJNA1269443), ensuring compliance with Open Access and FAIR principles. Overall, the integration of results from all work packages generated a comprehensive framework describing the ecological network associated with T. magnatum. The identification of specific Bradyrhizobium–T. magnatum interactions, the demonstration of fungal endophytism in herbaceous plants, the characterization of soil microbial responses to bacterial inoculation, and the first exploration of the white truffle virome collectively represent substantial scientific advances. These results significantly expand current knowledge of T. magnatum ecology and establish a solid scientific foundation for the development of improved mycorrhization techniques, sustainable management of natural truffle grounds, conservation of white truffle habitats, and future efforts aimed at the cultivation of the Italian white truffle. References: Graziosi S., Puliga F., Iotti M., Amicucci A., Zambonelli A. (2024). In vitro interactions between Bradyrhizobium spp. and Tuber magnatum mycelium. Environmental Microbiology Reports, 16(3), e13271. Graziosi S., Deloche L., Januario M., Selosse M.A., Deveau A., Bach C., Chen Z., Murat C., Iotti M., Rech P., Zambonelli A. (2025). Newly Designed Fluorescence In Situ Hybridization Probes Reveal Previously Unknown Endophytic Abilities of Tuber magnatum in Herbaceous Plants. Microbial Ecology, 88, 42. Ranocchi B., Ghignone S., Iotti M., Stocchi V., Zambonelli A., Mello A., Amicucci A. Bradyrhizobium Inoculation as a Sustainable Strategy to Enhance Tuber magnatum Mycelium and Modulate Soil Microbiota in Natural Truffle Grounds. Mycorrhiza (under review).Dettagli del progetto
Responsabile scientifico: Alessandra Zambonelli
Strutture Unibo coinvolte:
Dipartimento di Scienze e Tecnologie Agro-Alimentari
Coordinatore:
Università Telematica San Raffaele Roma(Italy)
Contributo totale Unibo: Euro (EUR) 56.062,00
Durata del progetto in mesi: 24
Data di inizio
12/10/2023
Data di fine:
28/02/2026
