Mycosphere microbiome assembly and metabolic reprogramming in Phallus rugulosus revealed by multi-omics integration

The mycosphere, defined as the soil zone influenced by fungal hyphae, hosts a distinct microbiome that plays a critical role in host development. Nevertheless, the assembly rules and metabolic functions in macrofungi remain poorly understood. This study aims to disentangle these processes in the edible fungus Phallus rugulosus . We integrated metagenomics, metatranscriptomics, and untargeted metabolomics to compare mycosphere soil (MS) and bulk soil (BS) of P. rugulosus . No significant differences in alpha-diversity were detected between MS and BS, whereas beta diversity analysis showed significant separation. The MS harbored significantly enriched saprotrophic fungi and bacteria whose functional gene profiles pointed to involvement in anaerobic carbon, nitrogen, and sulfur cycling (e.g., Patescibacteria, Fibrobacterota, Thermodesulfobacteriota). Functional profiling indicated a metabolic shift from aerobic respiration to anaerobic fermentation and coupled nitrification–denitrification within the mycosphere. Metabolomics identified 254 up-regulated metabolites in the MS, including a signaling-molecule (indole-3-carboxaldehyde) and defense-related compounds (dihydrodaidzein). Network analysis identified Module 1, dominated by Patescibacteria and Fibrobacterota, as a core discriminative signature (AUC = 0.92). Partial least squares path-modeling (PLS-PM, GOF = 0.836) revealed strong positive associations among the mycosphere effect, soil fertility, microbial restructuring, functional gene expression, and metabolite accumulation, revealing a hierarchical cascade propagating from soil properties through microbial communities and functional genes to metabolite profiles. These findings are consistent with the three hypotheses that P. rugulosus enriches specific microbial taxa, that functional gene expression shows coordinated changes with these taxa, and that these components form an interconnected microbe–gene–metabolite network. This multi-omics framework advances our understanding of fungus-microbe interactions and provides reference information for future optimization of edible-mushroom cultivation systems.