Influence of nitrogen and phosphorus additions on N2-fixation activity, abundance, and composition of diazotrophic communities in a Chinese fir plantation

Highlights: • P addition increased N₂-fixation activity while N addition suppressed it in the subtropical forest. • P application could alleviate the inhibitory effect of low N input on N₂-fixation activity. • N addition inhibited diazotrophic growth while P input could promote it. • Soil mineral N availability rather than pH was the main factor in determining diazotrophic community composition. Although biological nitrogen (N) fixation (BNF) is an important N input process in subtropical forest ecosystems, how the diazotrophic communities related to this process respond to N and phosphorus (P) inputs is largely unknown. We investigated the effects of exogenous N and/or P inputs on N₂-fixation activity, diazotrophic abundance and community composition using a continuous application of fertilizers over 5 years experiment in a Chinese fir plantation. The fertilization regimes included control (CK), P treatment (P), low N addition treatment (N1), high N addition treatment (N2), low N and P addition treatment (N1P) and high N with P addition treatment (N2P). N₂-fixation activity was determined using the acetylene reduction assay (ARA). Quantitative PCR and Illumina Miseq sequencing of nifH gene were performed to analyze diazotrophic abundance and community composition, respectively. Our results showed that P addition increased N₂-fixation activity and nifH gene abundance by 189.07 n mol C₂H₄ and 1.02 × 10⁷ copies g^{− 1} dry soil, respectively, while were reduced by 1.19 n mol C₂H₄ and 2.04 × 10⁶ copies g^{− 1} dry soil when N was added. The application of P with low N (N1P) effectively alleviated the inhibitory effect of N input on N₂-fixation activity. N-related treatments resulted in significant decreases in operational taxonomic unit (OTU) richness and shifts in diazotrophic community structure. N₂-fixation activity and nifH gene abundance were strongly and positively correlated with soil pH and negatively correlated with mineral N (NH₄⁺-N and NO₃⁻-N) contents, while mineral N concentrations rather than soil pH appeared to be the main factor altering diazotrophic community structure. These results revealed that P addition played a positive role in regulating biological nitrogen fixation in subtropical forest ecosystems.