Fertilization practices affect biological nitrogen fixation by modulating diazotrophic community in acidic soil in southern China

Biological nitrogen (N) fixation (BNF) driven by diazotrophs is an important pathway for N input in agricultural ecosystems. However, free-living BNF and its associated diazotrophic communities under different fertilization practices in acidic soils are poorly studied. Here, we conducted a long-term fertilization experiment (29 years), including an unfertilized control (CK), chemical N, P and K fertilizer (NPK), NPK plus lime (NPKL), NPK plus straw (NPKS), NPK plus straw and lime (NPKSL) and NPK plus manure (NPKM), and explored how fertilization practices affect free-living BNF by changing biotic and abiotic variables. Compared with CK (1.51 nmol C₂H₂ d⁻¹ g⁻¹), BNF rates were significantly increased in NPKM (1.99 nmol C₂H₂ d⁻¹ g⁻¹) but reduced in NPK (0.55 nmol C₂H₂ d⁻¹ g⁻¹), NPKL (0.61 nmol C₂H₂ d⁻¹ g⁻¹) and NPKS (0.69 nmol C₂H₂ d⁻¹ g⁻¹). Similarly, chemical fertilization, regardless of the addition of lime or straw, reduced the abundance (0.71-1.18 × 10⁸ gene copies g⁻¹) and α-diversity (1.11-2.43) of the diazotrophic community, while manure application had a positive effect on diazotrophic abundance (3.23 × 10⁸ gene copies g⁻¹) and α-diversity (3.36). Non-parametric multivariate analysis of variance (PERMANOVA) suggested that manure application (R² = 0.212, p = 0.001) showed the stronger influence on diazotrophic community composition than additions of lime (R² = 0.115, p = 0.019) and straw (R² = 0.064, p = 0.161). Random forest model analysis revealed that BNF rates can be significantly (p < 0.05) explained by soil pH (9.9%), diazotrophic community attributes (composition, 8.5%; Chao1, 8.1%; abundance, 6.0%; Shannon, 5.7%), and soil total carbon (5.1%). Partial least squares path modeling (PLS-PM) suggested that diazotrophic community and soil properties mainly provided direct and indirect contributions to the variations of BNF rates, respectively. The dominant genera Pelomonas, Azospirillum and Dechloromonas were positively associated with BNF rates, with their members observed as keystone species in the community network. Together, chemical fertilizer combined with manure is an effective practice for improving BNF in acidic soil by affecting soil diazotrophic community.