Forty-three years of partial organic substitution shapes microbial assembly and multifaceted network stability in a paddy soil.

Partial substitution of chemical fertilizers with organic amendments is a promising strategy to sustain soil productivity while reducing chemical inputs. However, the ecological mechanisms by which organic substitution and inorganic reduction reshape soil microbial community assembly, life‑history strategies, and nutrient cycling potential remain poorly understood. A 43-year field experiment with different proportions of organic fertilizer substituting for inorganic nitrogen was conducted. Microbial community structure, assembly processes, keystone taxa, and functional genes involved in nitrogen (N) and sulfur (S) cycles were investigated with high-throughput amplicon and metagenomic sequencing. Our results showed that organic substitution significantly reshapes microbial community composition, increasing community evenness while maintaining species richness. It significantly reduced the proportion of transient and persistent microorganisms while increasing intermittent taxa. Organic substitution significantly reduced (p < 0.05) the contribution of stochastic processes in soil microbes in comparison to those treated only with chemical fertilizers. This shift was accompanied by the enrichment of specific functional phyla such as Actinomycetota (class Thermoleophilia), Myxococcota, and Gemmatimonadota, which served as keystone species in co‑occurrence networks. Functionally, organic substitution significantly upregulated genes involved in organic nitrogen mineralization (glnAB&ureABC&gdhA&GLUL) and anaerobic ammonium oxidation (anammox), while downregulating nitrification, dissimilatory nitrate reduction to ammonium (DNRA), and assimilatory nitrate reduction. Likewise, organic substitution reduced organic sulfur mineralization and hydrogen sulfide production (sreAB) but enhanced assimilatory sulfate reduction. The findings of this study provide new insights into the ecological mechanisms through which organic substitution regulates soil microbiomes and nutrient cycling.