Symbiosis Among Naematelia aurantialba, Stereum hirsutum, and Their Associated Microbiome in the Composition of a Cultivated Mushroom Complex JinEr

The JinEr mushroom ("Golden Ear"), a globally rare edible and medicinal macrofungus, comprises a symbiotic complex formed by the symbiotic association of <i>Naematelia aurantialba</i> (<i>Tremellomycetes</i>) and <i>Stereum hirsutum</i> (<i>Agaricomycetes</i>). However, the interactions between these fungi and their associated microbiome remain poorly understood. This study employed high-throughput amplicon sequencing, in situ microbial isolation and culture, and microbial confrontation assays to analyze microbial diversity, community structure, and potential functional roles of the endomycotic bacterial community within JinEr basidiomata and its cultivation substrate. Molecular analysis confirmed the heterogenous composition of the basidiomata, revealing <i>N. aurantialba</i> constitutes less than 20% of the fungal biomass, while <i>S. hirsutum</i> predominates, accounting for approximately 80%. Endomycotic fungi accounted for 0.33% (relative abundance) of the fungal community. Prokaryotic analysis identified <i>Delftia</i> and <i>Sphingomonas</i> as the dominant endomycotic bacterial genera within basidiomata, comprising 85.42% of prokaryotic sequences. Endomycotic bacterial diversity differed significantly (<i>p</i> < 0.05) between basidiomata and substrate, indicating host-specific selection. Cultivation-based approaches yielded 140 culturable bacterial isolates (spanning four families and seven genera) from basidiomata core tissues. In vitro co-culture experiments demonstrated that eight representative bacterial strains exhibited compatible growth with both hosts, while one <i>Enterobacteriaceae</i> strain displayed antagonism towards them. These findings confirm that the heterogeneous JinEr basidiomata harbor a specific prokaryotic assemblage potentially engaged in putative symbiotic or commensal associations with the host fungi. This research advances the understanding of microbial ecology in this unique fungal complex and establishes a culture repository of associated bacteria. This collection facilitates subsequent screening for beneficial bacterial strains to enhance the JinEr cultivation system through the provision of symbiotic microorganisms.