Organic manure rather than phosphorus fertilization primarily determined asymbiotic nitrogen fixation rate and the stability of diazotrophic community in an upland red soil

Biological nitrogen fixation (BNF) plays a vital role in nitrogen supply in agricultural ecosystem but is generally impaired by agricultural fertilization. Understanding the trade-off between fertilization and BNF, and the underlying mechanisms are essential to optimize fertilization management for sustainable agriculture production. In this study, we examined the potential rate of asymbiotic N-2 fixation, nifH gene abundance, the composition and co-occurrence network of diazotrophic community in an acidic red soil received different organic and inorganic fertilization regimes for more than 40 years. Our results showed that long-term chemical fertilizer application drastically decreased soil microbial biomass, nitrogenase activity and nifH gene abundance in comparison to the unfertilized control. Organic manure application showed significantly positive effect on soil nitrogenase activity and crop yield via regulating soil pH and the key ecological cluster of diazotrophic community (module #2 which was mainly represented by Bradyrhizobium, Pseudacidovorax and Azospirillum), while phosphorus fertilization showed no obvious promotion effect. Organic manure amendment significantly increased the diversity of diazotrophs and enriched some diazotrophic taxa, particularly Pseudacidovorax and Rhodopseudomonas which were likely responsible for the high N-2-fixation potential. The control and organic manure amendment treatments possessed a more complex and stable diazotrophic network than the chemical fertilizer treatments did, which greatly facilitated the resistance of diazotrophic community to environmental stress and thus sustained a high N-2 fixation potential. Together, our study demonstrated that organic manure application can effectively alleviate the inhibitory effect of nitrogen fertilization on N-2 fixation via regulating soil property and shaping a more stable diazotrophic network.