Truffle fruiting occurs in chemically and microbially distinct soil niches

Ectomycorrhizal fungi (EMF) play critical roles in nutrient exchange, soil processes, and plant community structure, yet the mechanisms linking their environmental interactions to reproduction remain poorly understood. The black truffle (Tuber melanosporum) provides a model for studying these dynamics through its formation of brûlés-vegetation-free zones around a host plant and associated with fructification. Although prior studies have associated truffle production with factors such as pH, carbonates, and specific microbial groups, most work has been limited by narrow parameter ranges, uncontrolled confounders, and an absence of direct fruiting measurements, leaving unresolved whether these patterns facilitate reproduction or merely accompany it. Here we address these limitations using a broad paired design sampling 93 trees with and without brûlés across productive and unproductive North American orchards. We show that fruiting consistently aligns with distinct soil chemical profiles -including reduced organic matter and nitrogen, and elevated iron and magnesium-and with highly structured microbial communities. Fruiting soils exhibited the highest bacterial and fungal diversity, strong beta-divergence from non-fruiting soils, and enrichment of chemoheterotrophic guilds capable of mobilising nutrients and accelerating organic matter turnover. Network analyses revealed that productive soils support more cohesive and functionally integrated microbial consortia, despite only modest shifts in taxonomic composition. These patterns suggest that T. melanosporum reproduction depends not solely on favourable edaphic conditions, but on active niche construction at the soil chemical-microbial interface. Our findings highlight a previously underappreciated reproductive dimension of EMF ecosystem engineering, with implications for truffle cultivation, sustainable land management, and broader mycorrhizal ecology.