Combined effects of elevated CO2, N fertilizer and water deficit stress on diazotrophic community in sub-humid tropical paddy soil

Elevated carbon dioxide (eCO₂), higher dose of recommended nitrogen (HDN), and water deficit stress (WDS) are three important factors which influence individually on diazotrophic community in paddy soil, thereby affecting crop yield and its nitrogen use efficiency. However, the combined effects of eCO₂, HDN, and WDS on soil diazotrophic community remains inadequate particularly in rice. Hence, the present study was conducted in CO₂-maintained open-top chamber to assess the diazotrophic community under combined influence of eCO₂ (550 ± 20 μmol mol⁻¹ as compared to ambient CO₂i.e. 400 ± 10 μmol mol⁻¹) and HDN (120 kg N ha⁻¹, 50% higher than recommended dose of N for rice i.e. 80 kg N ha⁻¹, in sub-humid tropical region of eastern India) with and without WDS (−60 kPa) at two growth stages of rice (tillering, TI and panicle initiation, PI) and correlated with soil carbon (C) pools and enzyme activities. The results indicated that soil organic C, readily mineralizable C, microbial biomass C, acid and alkaline phosphatase activities were significantly (p < 0.05) increased under combined influence of eCO₂ and HDN at both TI and PI stages of rice without WDS. The results also showed that abundance of nifH varied from ambient CO₂ (aCO₂) to eCO₂ and found higher in aCO₂ under recommended dose of nitrogen (RDN) without WDS, whereas under eCO₂, nifH copy number was found higher under HDN at both TI and PI stages of rice without WDS. Biplot analysis revealed that abundance of nifH and nitrifiers were highly correlated under aCO₂ coupled with RDN without WDS. Illumina MiSeq®-based targeted nifH metagenome showed that eCO₂ encouraged the relative abundance of Proteobacteria and Cyanobacteria, whereas suppressed the other diazotrophic phyla like Actinobacteria, Firmicutes, and Chlorobi, but the reverse trend was observed in HDN treatments. Heatmap analysis revealed a unique differentiation of Rhizobiales and Pseudomonadales under eCO₂ coupled with RDN without WDS. Moreover, three-way interactions of eCO₂, HDN, and WDS also showed significant (p < 0.05) difference of soil C pools and enzymatic activities at both growth stages of rice. Overall, the present study showed that diazotrophic community in paddy soil was significantly (p < 0.05) altered by the additive effects of eCO₂, HDN, and WDS.