Stage‐specific biological nitrogen fixation depends on distinct carbon and nitrogen availability in long‐term fertilized paddy fields

Abstract Industrial Haber–Bosch provides 32 Tg nitrogen (N) per year to global croplands. Such large amounts of N fertilization will reshape soil N cycles. One uncertainty is whether and how these long‐term N inputs impact soil biological N 2 fixation (BNF) associated with plant growth, a crucial microbial‐driven process to decipher the balance between soil N and plant N in paddy fields. To fill this gap, we conducted a 10‐year field experiment to systematically evaluate the impacts of three N fertilization rates (0, 75 and 150 kg N ha −1 ) on soil BNF activity, diazotrophic communities and environmental dynamics at two typical rice growth stages (tillering and maturation) in paddy fields. Moderate N fertilization at a 75 kg N ha −1 rate significantly stimulated BNF activity and nif H gene abundance at the tillering stage, with increases of 20.9% and 49.7%, respectively. This enhancement was primarily due to the supply of available carbon (C) sources from organic acids, which promoted the BNF process to meet the N requirement from rice at the early stage. In contrast, BNF activity and nif H gene abundance decreased with increasing N levels at the maturation stages, reflecting the negative impact of N enrichment on diazotrophs. The accumulation of inorganic N and amino acids reduced the dependence on the BNF process. Stage‐specific key diazotrophs, such as Rhizobium at tillering and Azoarcus at maturation, played pivotal roles in the soil BNF. Soil‐dissolved organic C and inorganic N were positively correlated with BNF at the tillering stage, but negatively at the maturation stage. Random Forest analysis further revealed that the BNF was primarily regulated by available nutrients at the tillering stage, while closely associated with the key diazotrophs at the maturation stage. This study highlights that the stage‐specific BNF process depends on C availability and key diazotrophs with long‐term N fertilization, providing a basis for optimized N management in paddy fields. Read the free Plain Language Summary for this article on the Journal blog.