The analogy that guided the research team, led by Miao Liu and Yang Wang of Northwest A&F University with senior author Minglei Li, comes from plant science. Just as plant roots sculpt the rhizosphere, the soil zone immediately influenced by roots, fungi sculpt the mycosphere. For macrofungi, this zone is far more than real estate. Previous work on cultivated species such as the button mushroom Agaricus bisporus and morels has shown that casing soil microbes are essential triggers for primordium formation and fruiting body development. Under sterile, microbe-free conditions, many macrofungi simply fail to produce mushrooms at all. Yet the underlying mechanisms, specifically which microbial groups matter and which metabolic pathways they operate, have remained largely opaque, in part because traditional single-gene surveys suffer from primer bias and limited community coverage.

To break through that bottleneck, the team collected five intact P. rugulosus fruiting bodies from a wild bamboo forest in November 2025 and sampled two soil types for each: mycosphere soil, the material tightly adhering to the stipe and surrounding mycelial zone, and bulk soil taken in a five-point pattern at a ten-centimeter radius. They then layered three omics approaches on top of one another. Amplicon sequencing with synthetic spike-in standards provided absolute quantification of bacterial 16S rRNA and fungal ITS copy numbers. Metatranscriptomics, sequenced on an Illumina platform, captured which functional genes were actually being expressed. Untargeted liquid chromatography mass spectrometry mapped the small-molecule chemistry of both soil zones. Five biological replicates per group anchored the statistics.

The first surprise was what did not change. Alpha diversity metrics, including the Shannon and Simpson indices, showed no significant differences between mycosphere and bulk soil for either bacteria or fungi. The mycosphere effect, it turns out, is not about adding or losing species richness. It is about wholesale compositional restructuring. Non-metric multidimensional scaling based on Bray-Curtis distances revealed completely non-overlapping fungal communities and partially overlapping but clearly diverging bacterial communities, a separation confirmed by permutational analysis of variance. Fungi responded even more strongly than bacteria, with an F-statistic of 4.284 and an R-squared of 0.349, compared with 3.006 and 0.273 for bacteria. The fungus, in other words, rewrites who lives in its neighborhood without changing how many kinds of residents there are.

Linear discriminant analysis effect size screening identified the specific taxa being favored. Among bacteria, the mycosphere was significantly enriched in Patescibacteria, Fibrobacterota, and Thermodesulfobacteriota, three phyla whose functional profiles point squarely toward anaerobic carbon, nitrogen, and sulfur cycling. Families enriched near the fungus included anaerobic chemoorganotrophs such as Williamwhitmaniaceae, denitrifiers in Rhodocyclaceae, and organic matter degraders in Thalassobaculales. Meanwhile, strictly aerobic methanotrophs like Methylomonadaceae declined, consistent with the emergence of microaerophilic or anaerobic conditions as fungal metabolism consumes local oxygen. On the fungal side, saprotrophic Mortierellomycota and Basidiobolomycota rose while obligately symbiotic Glomeromycota fell, and lignocellulose-degrading families such as Lentitheciaceae and Niessliaceae expanded as potential pathogens in Diatrypaceae and Hypoxylaceae receded. The mycosphere, the data suggest, is a deliberately curated guild of decomposers and element cyclers, with competitors and pathogens pushed out.

Redundancy analysis and Mantel tests tied these shifts to soil chemistry. Mycosphere soil had markedly lower pH but elevated soil organic matter, organic carbon, total nitrogen, total phosphorus, total potassium, available phosphorus, and alkaline-hydrolyzable nitrogen, while available potassium dropped. The first two RDA axes explained 86.9 percent of bacterial and 80.3 percent of fungal community variation, with organic matter, nitrogen, phosphorus, potassium, and available nutrients acting as significant drivers. The pattern suggests a clear ecological partition: macronutrient pools, likely modified by fungal secretion and mycelial activity, favor saprotrophic and element-cycling functional groups near the fungus, whereas pH and available potassium act as filters maintaining oligotrophic, aerobic communities farther away.

The functional layer of the analysis revealed something more dramatic: a systematic metabolic reprogramming. In the carbon cycle, genes for ethanol fermentation, including ald and adh/mdh, were markedly upregulated in the mycosphere, alongside enrichment of methanogenesis and carbon fixation pathways, indicating a shift from aerobic respiration toward facultative anaerobic fermentation. Compensatory upregulation of gluconeogenesis genes fbp and pck suggested microbes were maintaining tricarboxylic acid cycle intermediates to keep biosynthesis running. In the nitrogen cycle, both nitrification genes amoABC and denitrification genes nirK/S were upregulated, pointing to coupled nitrification-denitrification rather than the storage-oriented nitrogen fixation and assimilatory nitrate reduction seen in bulk soil. Sulfur metabolism told a parallel story: the mycosphere dominated oxidative pathways including sulfide, thiosulfate, and sulfite oxidation, and in the dimethylsulfoniopropionate cycle it controlled downstream dimethyl sulfide oxidation, DMSO reduction, and methanethiol oxidation. High hydrogenase expression, with DMSO serving as an alternative electron acceptor, implies that mycosphere microbes exploit reduced sulfur compounds as electron donors to squeeze out extra energy in oxygen-poor conditions.

Metabolomics provided the chemical confirmation. Orthogonal partial least squares discriminant analysis cleanly separated the two soil zones, and differential screening identified 254 up-regulated metabolites in the mycosphere against only 58 down-regulated ones. The upregulated compounds clustered into nucleotide metabolism, with elevated 2-prime-deoxyguanosine, 2-prime-deoxyinosine, and xanthine signaling enhanced nucleic acid turnover; amino acid biosynthesis, with accumulation of L-phenylalanine, L-proline, L-tryptophan, and L-tyrosine supporting microbial proliferation; and cofactor metabolism, with niacinamide and pyridoxine rising. More intriguingly, the mycosphere accumulated signaling and defense chemistry, including indole-3-carboxaldehyde, a tryptophan-derived molecule that modulates bacterial quorum sensing at low concentrations and interferes with fungal growth at high ones, and dihydrodaidzein, an isoflavone with documented antifungal and antibacterial activity. Together with 2-naphthol, trimethoprim, and sulfalene, these compounds sketch a coordinated chemical-ecology strategy: recruit beneficial partners while suppressing competitors.

The integrative analysis then showed how these layers connect. Spearman correlation network analysis identified three microbe-gene-metabolite modules, and lasso logistic regression singled out Module 1, dominated by Patescibacteria and Fibrobacterota and linked to genes such as tcyM and tgl and metabolites including 2-methylglutaric acid and dihydrodaidzein, as the strongest discriminator between the two soil types, achieving an area under the ROC curve of 0.92. The authors propose a cross-feeding relationship: Fibrobacterota degrade cellulose into soluble sugars that Patescibacteria, ultra-small bacteria with reduced genomes lacking complete glycolytic and TCA pathways, ferment anaerobically. Causal mediation analysis added a crucial insight, showing that roughly 59.66 percent of the statistical association between microbiota and metabolites operated indirectly through functional genes, with the direct effect becoming non-significant once the mediator was accounted for. Partial least squares path modeling, with a goodness of fit of 0.836, formalized the full cascade: the mycosphere effect strongly shaped soil properties and both bacterial and fungal communities, bacterial communities drove functional gene expression, and gene expression was strongly associated with metabolite accumulation, while the direct fungal-to-metabolite path was negligible.

The authors are careful to note the limits of the work. The study was cross-sectional at a single site, all associations are correlational rather than causal, and a single time point cannot capture the dynamic succession of the mycosphere across the fungal life cycle. Isotope tracing, stable isotope probing, and co-culture experiments will be needed to confirm the inferred roles of the enriched taxa. Still, the framework itself is the headline. It demonstrates that the functional identity of a fungal microhabitat is not a simple sum of individual species or genes but the coordinated output of tightly integrated microbe-gene-metabolite modules, extending the well-studied rhizosphere paradigm of plant root systems to macrofungi. For growers of edible mushrooms, where casing soil microbiomes determine yield and continuous cropping obstacles remain stubborn, the practical implications are immediate: cultivating the right microbial consortia, and the anaerobic, sulfur-oxidizing, fermentation-driven conditions they require, may prove as important as the fungus itself.

Subject of Research: Mycosphere microbiome assembly and metabolic reprogramming in the edible fungus Phallus rugulosus

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

Article References: Liu, M., Wang, Y., Huang, K., Meng, Q., You, X., Tian, R., Zhang, X., Zhao, B., Li, L., Jiang, W., & Li, M. (2026). Mycosphere microbiome assembly and metabolic reprogramming in Phallus rugulosus revealed by multi-omics integration. Plant and Soil. https://doi.org/10.1007/s11104-026-09177-0

Image Credits: AI Generated

DOI: 10.1007/s11104-026-09177-0

Keywords: Phallus rugulosus, mycosphere, microbiome, metagenomics, metatranscriptomics, metabolomics, Patescibacteria, Fibrobacterota, anaerobic fermentation, nitrogen cycling, sulfur oxidation, edible mushroom cultivation