Predicting the influence of fertilization regimes on potential N fixation through their effect on free-living diazotrophic community structure in double rice cropping systems.

Free-living nitrogen fixation (FLNF) by diazotrophs is a ubiquitous renewable resource and may constitute an attractive viable solution to the problem of environmental degradation caused by nitrogen over-fertilization. However, the biotic and abiotic control mechanisms of free-living nitrogen fixation are poorly understood. Moreover, there is rare information to link the composition and functions of diazotrophic communities; additionally, there are insufficient or inadequate predictors of the potential N fixation rate (PNFR) to help improve fertilization strategies. Here, through high-throughput sequencing, network analysis, and 15N 2 labelling methods, we investigated the long-term effects of partial substitution of mineral N by organic fertilizers on diazotroph abundance and composition in double rice cropping systems. The field trial was conducted for 34 years and included five treatments: control without fertilizer (CK); only mineral NPK fertilizer (NPK); 70% inorganic N + 30% organic N + PK (NPKM 1); 50% inorganic N + 50% organic N + PK (NPKM 2); 30% inorganic N + 70% organic N + PK (NPKM 3). Our results revealed that at the same nutrient input level, increasing the percentage of organic N substitution reduced diazotrophic abundance. Compared with the CK and NPK treatments, the organic substitution regimes reduced the fluctuation range of the PNFR between the early and late rice growing seasons. NH 4 +-N was the primary factor that negatively correlated (p < 0.01) with diazotrophic abundance, while the potential N fixation rate was positively correlated (p < 0.01) with soil C/N. Among abiotic factors, the models incorporating soil C/N and Fe2+/Mo could accurately predict the potential N fixation rate. High-throughput sequencing and network analyses revealed that Bradyrhizobium and Geobacter were the dominant genera in diazotrophic co-occurrence and keystone taxa patterns under all fertilization regimes. Soil pH and NH 4 +-N were the most important factors in shaping the diazotrophic co-occurrence and keystone patterns in paddy soils, respectively. Diazotrophic keystone composition was more suitable for predicting and characterising the potential N fixation rate than the co-occurrence pattern of diazotrophs. Moreover, compared with the CK and NPK regimes, partial substitution of mineral N with organic N fertilizer improved the prediction accuracy of potential N fixation rate more effectively. Overall, we highlight the benefits of organic substitution fertilizer regime in stabilizing and improving the potential N fixation rate. Our findings provide relevant insights into the potential of key microbial taxa for predictive N fixation for optimising chemical N fertilizer application strategies in paddy soils. • Bradyrhizobium and Geobacter were dominant genera in diazotrophic co-occurrence. • Collaborative diazotrophic community relationships outweighed competing ones. • Diazotrophic keystone taxa were suitable for predicting potential N fixation. • Organic substitution regime facilitated to stabilize and improve N fixation rate. [ABSTRACT FROM AUTHOR]