Truffle Microbiome Is Driven by Fruit Body Compartmentalization Rather than Soils Conditioned by Different Host Trees

Truffles are among the most expensive edible mushrooms; their value is worth billions of U.S. dollars annually in international markets. They establish ectomycorrhizal symbiotic relationships with diverse host tree roots and produce hypogeous ascomata. Their whole life cycle is closely related to their associated microbiome. However, whether truffle-associated compartments or host tree rhizospheres are the vital driver for truffle ascomata microbiome is unclear. To identify and compare fungal and bacterial communities in four truffle-associated compartments (Tuber indicum: bulk soil, adhering soil to peridium, peridium, and gleba) from three host trees, we sequenced their ITS (fungal) and 16S (bacterial) ribosomal DNA using the Illumina MiSeq high-throughput platform. We further applied the amplicon data to analyze the core microbiome and microbial ecological networks. Tuber indicum microbiome composition was strongly driven by its associated compartments rather than by their symbiotic host trees. Truffle microbiome was bacteria dominated, and its bacterial community formed a substantially more complex interacting network compared to that of the fungal community. The core fungal community changed from Basidiomycota dominated (bulk soil) to Rozellomycota dominated (interphase soil); the core bacterial community shifted from Bacteroidetes to Proteobacteria dominance from truffle peridium to gleba tissue. Especially, at the truffle and soil interphase, the niche-based selection of truffle microbiome was verified by (i) a clear exclusion of four bacterial phyla (Rokubacteria, Nitrospirae, Chloroflexi, and Planctomycetes) in gleba; (ii) a significant decrease in alpha-diversity (as revealed by Chao 1, Shannon, and Simpson indices); and (iii) the complexity of the network substantially decreased from bulk soil to soil-truffle interphase and further to the peridium and gleba. The network analysis of microbiome showed that the microbial positive interactions were higher in truffle tissues than in both bulk soil and peridium-adhering soil and that Cupriavidus, Bradyrhizobium, Aminobacter, and Mesorhizobium spp. were the keystone network hubs in the truffle gleba. This study provides insights into the factors that drive the truffle microbiome dynamics and the recruitment and function of the microbiome components. IMPORTANCE Currently, the factors that drive the microbiome associated with truffles, the most highly prized fungi in the world, are largely unknown. We demonstrate for the first time here that truffle microbiome composition is strongly driven by associated compartments rather than by symbiotic host trees. The truffle microbiome was bacteria dominated, and its bacterial community formed a substantially more complex (with the higher numbers of nodes, links, and modules) interacting network compared to that of the fungal community. Network analysis showed a higher number of positive microbial interactions with each other in truffle tissues than in both bulk soil and peridium-adhering soil. For the first time, the fungal community structure associated with truffles using high-throughput sequencing, microbial networks, and keystone species analyses is presented. This study provides novel insights into the factors that drive the truffle microbiome dynamics and the recruitment and function of the microbiome components, showing that they are more complex than previously thought.