Straw incorporation increases stage-specific biological nitrogen fixation by stimulating microbial carbon turnover and nitrogenase gene expression in paddy soil

• Straw incorporation stimulated soil BNF, but the effect decreased with rice growth stages. • Straw incorporation increased soil diazotrophic diversity and network density in paddy fields. • Microbial carbon turnover was the key factor affecting soil BNF under straw incorporation. Straw amendments can improve soil fertility by loading organic carbon (C) into soils, but whether and how biological nitrogen fixation (BNF) occurs in long-term rice straw (RS)-incorporated paddy fields remain poorly understood. To fill this gap, we explored the effects of three rates of straw incorporation (0, 3 and 6 t ha -1 ; RS 0 , RS 3 and RS 6 ) on soil BNF activities inferred from acetylene reduction assay (ARA) and diazotrophic communities at three rice growth stages (tillering, elongation, and maturation) based on a 10-year field experiment. The ARA activities increased significantly in response to increasing straw incorporation rates across all three rice growth stages, while the effect decreased as rice growth progressed. Soil BNF was associated with key diazotrophs, such as Dechloromonas, Bradyrhizobium , and Azospirillum . Straw incorporation increased diazotrophic abundance, diversity and interactions, which consequently improved soil BNF activities and rice yields. Straw incorporation increased rice yield by 12.6% in RS 3 and by 15.5% in RS 6 compared with the control. Structural equation models (SEMs) suggested that microbial C turnover and nitrogenase gene expression were the key factors affecting soil stage-specific BNF associated with the decomposition of straw. These results revealed that C-rich straw incorporation reconfigured soil N dynamics, enabling simultaneous improvement of soil fertility and rice yields through demand-driven BNF patterns in paddy fields.