Isotope Labeling Reveals Complex Microbial Interactions during Agaricus bisporus Compost Colonization

Microbial interactions strongly influence carbon and nitrogen flows in mushroom compost, yet their functional roles during Agaricus bisporus colonization remain unresolved. We combined PLFA-SIP and nanoSIMS imaging with ITS amplicon sequencing to follow resource flows and microbial activity across spatial scales. Stable-isotope tracers (¹³C- glucose and ¹⁵N-ammonium) revealed that A. bisporus simultaneously facilitates and suppresses bacterial populations: fungal activity increased glucose assimilation by bacteria yet reduced overall bacterial biomass. NanoSIMS visualized nutrient-rich microenvironments along hyphae where bacterial ¹³C and ¹⁵N assimilation was elevated. Sequencing showed the fungal community to comprise essentially two organisms, A. bisporus and Mycothermus thermophilus , which differ approximately elevenfold in their content of the fungal biomarker C18:2ω6,9c. Total fungal PLFA therefore tracks which of the two dominates as much as it tracks fungal biomass. Together these findings reveal coupled fungal–bacterial nutrient processing and show that biomarker-based estimates of fungal biomass require community composition to be known. Multi-scale isotope probing provides a framework for resolving microbial interactions in complex detrital systems.