Cryptic microheterogeneity of the casing-layer microbiome and metabolome is associated with primordium fate in Phallus rubrovolvatus.

Commercial cultivation of Phallus rubrovolvatus relies on casing layers, yet yields remain unstable, with primordium development varying among individual microsites. This cryptic microheterogeneity is masked by conventional bulk sampling, limiting insight into casing-layer performance. Here, we combined phenotype-guided back-tracing with multi-omics to characterize microecological signatures associated with primordium fate. We identified two contrasting ecological regimes associated with reproductive outcomes. Productive microsites were characterized by a "Cooperative state" enriched in oligotrophic bacteria (e.g., Hyphomicrobium) and basidiomycetes (e.g., Clathrus columnatus). Productive microsites also showed lower available potassium (K+) and elevated catalase activity. Conversely, non-productive microsites were characterized by a Rhizopus-dominated "Disruptive state". This state was associated with elevated urease activity, lower catalase activity, and stress-associated metagenomic functional signatures. Overall, primordium fate was associated with distinct local microbial, functional, and metabolic configurations that were not fully explained by bulk physicochemical properties. We propose the "Cooperative vs. Disruptive" model as a conceptual framework and identify candidate microbial and metabolic features for future validation in microbiome-informed cultivation management.