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

## TL;DR

This study explores the bacterial communities inside Chinese truffles, revealing how they vary by species and location, and their role in truffle health and sustainability.

## Contribution

The study provides new insights into the biogeographical patterns and functional roles of endophytic bacteria in Chinese truffles.

## Key findings

- Bradyrhizobium is the dominant genus, with significant abundance variation across truffle species and regions.
- Shannon diversity indices show strong species- and altitude-related bacterial diversity differences.
- Environmental factors and host species are primary drivers of bacterial community composition.

## 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.

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.