Spatial Variation of Bacterial Diversity in Shiro-Associated and Non-Mycorrhizal Microhabitats of Tuber sinense–Quercus aliena Symbiosis

The rhizospheric microbial community plays a crucial role in the growth and ecological adaptation of truffles. Although extensive research has been conducted on bacterial communities in truffle habitats, the spatial variation and functional implications across different regions and soil compartments remain poorly understood in the current literature. In this study, soil bacterial communities were analyzed using 16S rRNA high-throughput sequencing across truffle-producing (Tuber sinense) and non-producing sites in Quercus aliena forests in Panzhihua, China. To capture microhabitat-level variation, soils were classified into three compartments: rhizosphere soil tightly adhering to ectomycorrhizal roots (TRS), rhizoplane soil loosely attached to roots (TRE), and bulk soil from truffle zones without visible roots (TBS), with corresponding controls (RS, RE, BS) collected from truffle-free forests. An alpha diversity analysis revealed that truffle-producing soils harbored significantly higher bacterial richness than control soils, while beta diversity indicated more clustered community composition in truffle-associated soils. A taxonomic analysis showed that T. sinense occurrence was associated with enrichment of specific bacterial taxa, including Chloroflexi, Anaeromyxobacteraceae, and Bradyrhizobium, whereas widespread taxa such as Firmicutes and Bacteroidota were more abundant in control soils. To further identify microbial indicators closely associated with truffle presence, we employed random forest modeling, which highlighted Pseudomonas, Streptomyces, and Bradyrhizobium as key genera distinguishing truffle-associated rhizospheres. These findings suggest that T. sinense may influence the composition of rhizospheric microbial communities, thereby constructing a favorable rhizospheric microenvironment. This work provides new insights into the microbial ecology of T. sinense and lays a foundation for future truffle domestication and cultivation efforts.