Chronic Nitrogen Deposition Alters Diazotrophic Community Composition, Reduces Biological Nitrogen Fixation, and Restructures Fungal-Diazotroph CO-Occurrence Networks in Deadwood

Biological nitrogen fixation (BNF) by diazotrophs contributes to increasing nitrogen (N) availability in nutrient-poor deadwood during the decomposition process. However, chronically elevated atmospheric N deposition may increase N availability, thereby reshaping diazotrophic community and suppressing BNF. We simulated high N deposition by repeatedly applying ammonium-nitrate solution to deadwood of 13 tree species over 9 years (N addition) and compared diazotrophic community composition and BNF rates with untreated controls. Deadwood N concentrations increased over time in both control and N addition, with N addition resulting in higher N concentrations at the final sampling, although significant treatment effects were detected only in Tilia and Pinus. Chronic high-N addition was associated with reduced BNF activity, with significant suppression primarily observed in coniferous deadwood, while responses among broadleaved species were weak, variable, or absent. The N addition altered diazotroph richness and community composition by increasing the abundance of Bradyrhizobium and by reducing Methylocapsa across all tree species. Under N addition, BNF correlated positively with nifH gene copy numbers in broadleaved deadwood but negatively in coniferous deadwood. Co-occurrence networks were more interconnected and modular under N addition, with diazotrophs (e.g., Azospirillum) central in broadleaved deadwood and fungi (e.g., Meliniomyces, Athelia) central in coniferous deadwood. Tree clade (coniferous vs. broadleaved) strongly shaped richness and community response, with broadleaved and coniferous species showing distinct patterns.Overall, the largely robust diversity and community composition of diazotrophs and BNF activity under high N addition suggest that moderately increasing N deposition has little influence on fungal deadwood decomposition and the function of deadwood as a carbon pool in forest ecosystems.