High-throughput sequencing reveals endophytic bacterial differentiation of common truffles ( Tuber spp.) in China: diversity, biogeographical patterns, and fungal health implications

ABSTRACT As valuable hypogeous fungi, truffles depend on fruiting-body-associated microorganisms for lifecycle functions like growth and nutrient cycling. This study sampled fruiting bodies of 10 Tuber species from 16 sites across six major truffle-producing provinces in China, characterizing endophytic bacterial communities via high-throughput sequencing and multivariate analysis. Proteobacteria dominated the endophytic bacteria, with Bradyrhizobium as the prevalent genus. Significant genus-level compositional differences occurred across provenances and species: Bradyrhizobium reached 99.80% relative abundance in Tuber sinense from Mengzi, Yunnan, versus 7.90% in Tuber shii from Dali (12.6-fold difference). Shannon diversity indices ( n = 48) revealed striking species- and altitude-related variations ( P < 0.001): Tuber lijiangense (5.111) and T. shii (5.091) had the highest diversity, while T. sinense (1.336) had the lowest (3.8-fold gap). Subtropical Dali samples exhibited a sevenfold higher diversity compared to those from the Mengzi region, which is geographically closer to the tropics. Non-metric scaling and principal coordinates analysis identified environmental factors (soil, climate) and host species as primary drivers, with species effects potentially overriding environment. Five core taxa (all Rhizobiales ) suggested nitrogen-fixing roles, while Variovorax (via linear discriminant analysis effect size) emerged as an external-disturbance-sensitive opportunist. This study clarifies endophytic bacterial variation patterns and drivers, identifies key taxa, and informs truffle ecological interactions, providing a scientific basis for sustainable resource management and conservation. IMPORTANCE This study underscores the critical importance of truffle endophytic bacteria in mediating fungal health and ecological resilience, addressing a major knowledge gap in hypogeous fungal microbiome research. By integrating high-throughput sequencing across 10 Tuber species in China, we reveal how bacterial communities (dominated by Bradyrhizobium ) shape biogeographical patterns and functional roles like nitrogen fixation. These findings advance understanding of microbe-fungal symbioses, with direct applications for sustainable truffle cultivation (e.g., microbial inoculants) and climate-resilient management—aligning perfectly with AEM's focus on applied microbial ecology and biotechnological relevance.