Long-term organic fertilization enhances paddy soil nitrogen availability through direct nutrient stimulation and complementary diazotroph-mediated pathway

Abstract

Long-term organic fertilization is known to improve soil fertility, but the mechanisms by which it enhances soil nitrogen (N) availability, particularly the relative importance of direct nutrient stimulation versus diazotroph-mediated pathways, remain unclear. We examined the effects of four fertilization regimes (without fertilizer, CK; 100% chemical fertilizer, NPK; rice straw plus chemical fertilizer, RS; and 30% organic manure plus 70% chemical fertilizer, OM) on soil properties, diazotrophic community composition ( nif H gene amplicon sequencing), N 2 fixation activity (potential BNF and nitrogenase concentration), and soil N pools (NH 4 + –N, NO 3 − –N, and MBN) in a 37 year double-cropping paddy rice field experiment. Long‑term organic fertilization, particularly OM, significantly increased soil organic carbon, total nitrogen and microelements (Mo, Fe, V and AMo). Relative to CK, OM treatment increased soil potential BNF rates by 342% and nitrogenase concentration by 178%, and maintained the highest soil inorganic nitrogen and MBN pools. Soil diazotrophic community composition (PCoA Axis1), rather than alpha diversity or nif H gene abundance, was the strongest correlate of potential BNF rates ( r  = 0.70, p  < 0.001) and nitrogenase concentration ( r  = 0.85, p  < 0.001). Partial least squares path modeling revealed that soil nutrients influenced soil N availability through dual pathways: a dominant direct effect on soil N fixation potential (0.734, p = 0.002), and a significant indirect effect mediated by soil diazotrophic community composition and N fixation activity (0.330). The model explained 98.8% of the variance in soil N availability with a goodness of fit of 0.842. In contrast, soil microelements showed negligible direct or indirect effects. These findings demonstrate that long-term organic fertilization enhances paddy soil N availability through coupled mechanisms: a dominant direct stimulation of N fixation by increased carbon and nutrient availability, complemented by a secondary pathway involving shifts in diazotrophic community composition and their concomitant N fixation activity. This study provides mechanistic evidence that sustaining soil N fertility in intensive rice agriculture depends on both adequate organic nutrient inputs and the maintenance of functional diazotrophic communities.