Fertilization practices affect biological nitrogen fixation by modulating diazotrophic communities in an 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 (29 years) fertilization experiment to explore how fertilization affected free-living BNF via changing biotic and abiotic variables. The fertilization treatments included an unfertilized control (CK), chemical N, phosphorus (P), and potassium (K) fertilizers (NPK), NPK plus lime (NPKL), NPK plus straw (NPKS), NPK plus straw and lime (NPKSL), and NPK plus manure (NPKM). Compared with CK (1.51 nmol C2H2 g-1 d-1), BNF rate was significantly (P < 0.05) higher in NPKM (1.99 nmol C2H2 g-1 d-1) but lower in NPK (0.55 nmol C2H2 g-1 d-1), NPKL (0.61 nmol C2H2 g-1 d-1), and NPKS (0.69 nmol C2H2 g-1 d-1). Similarly, chemical fertilization treatments without manure reduced the gene abundance (0.71 x 108-1.18 x 108 copies g-1) and alpha-diversity (Shannon index, 1.11-2.43) of diazotrophic communities, whereas the treatment with manure had a positive effect on diazotrophic abundance (3.23 x 108 copies g-1) and Shannon index (3.36). Non-parametric multivariate analysis of variance (PERMANOVA) suggested that manure application (R2 = 0.212, P = 0.001) had a stronger influence on diazotrophic community composition than the addition of lime (R2 = 0.115, P = 0.019) or straw (R2 = 0.064, P = 0.161). Random forest modeling revealed that BNF rates can be significantly (P < 0.05) explained by soil pH (9.9%), diazotrophic community attributes (composition, 8.5%; Chao 1 index, 8.1%; abundance, 6.0%; Shannon index, 5.7%), and soil total carbon (5.1%). Partial least squares path modeling (PLS-PM) suggested that the diazotrophic community attributes and soil properties mainly provided direct and indirect contributions to the variations in BNF rates, respectively. The dominant genera, Pelomonas, Azospirillum, and Dechloromonas, were positively associated with BNF rates, with their members being observed as keystone species in the community network. Application of chemical fertilizers combined with manure is an effective practice for improving BNF in acidic soils by affecting soil diazotrophic communities.