Crop diversification drives microbial-mediated nitrogen accumulation via fungal necromass stabilization

While microbial-derived nitrogen (N) constitutes a significant reservoir of soil organic N, the mechanisms driving its production and persistence under diversified cropping systems remain elusive. Leveraging a 43-year field experiment comparing conventional wheat-rice rotation (ConCrop) and a diversified barley/cotton–wheat/rice–fava bean/maize rotation (DivCrop), we quantified microbial necromass N via amino sugar biomarkers and coupled with 18 O-H 2 O isotopic tracing to assess microbial growth and N use efficiency (NUE). Our 43-year diversified cropping trial revealed that accelerated biomass turnover (+116–123%) and enhanced microbial NUE (+28–42%) drive a 47–89% increase in microbial necromass N accumulation compared to ConCrop. This was caused by increased labile N, as well as the enrichment of copiotrophic taxa favoring rapid biomass turnover. These shifts amplified fungal and bacterial necromass production, with fungal necromass N dominating (26–51% of total necromass N vs. 5.2–10% bacterial). The elevated fungi/bacteria ratio both in living biomass and necromass pools, indicating preferentially stabilized fungal N. Necromass stabilization was further mediated by exchangeable Mg²⁺ and Ca²⁺, which enhanced organo-mineral coordination, contributing to 42–61% of total N retention under crop diversification. Vertical stratification analyses uncovered 27–42% greater necromass N contributions at 15–30 cm depths. Our findings establish that crop diversification reinforces soil N persistence by synchronizing microbial life-death dynamics with geochemical protection pathways, offering a mechanistic roadmap for climate-smart agroecosystem management.