Distinct responses of fungal and bacterial denitrification genes to seasonal changes, nitrogen deposition and precipitation reduction in subtropical forest soils

China's subtropical forests are widely recognized as one of the world's largest natural sources of nitrous oxide (N₂O), primarily due to high nitrogen (N) deposition from anthropogenic activities. Climate change has made precipitation reduction increasingly common in subtropical regions, significantly influencing N₂O emissions. Denitrification is the main process contributing to N₂O emissions in subtropical forest soils; however, most previous studies have focused on bacterial denitrification, often overlooking fungal denitrification. In this study, a factorial experiment was conducted using a randomized complete block design with four replicates per treatment, in a subtropical forest soil. We examine the effects of simulated N deposition, precipitation reduction, and their combination on the abundance of genes encoding nitrite reductase enzymes, including bacterial (nirK, nirS) and fungal (nirK) variants, with a focus on their seasonal dynamics during summer and winter. N deposition and precipitation reduction treatments showed distinct effects. N deposition significantly reduced fungal and bacterial nirK abundance in winter and decreased bacterial nirS abundance in summer. Precipitation reduction further suppressed bacterial nirK and nirS abundance in winter but had no effect on fungal nirK. In addition to treatment effects, seasonal variation also shaped gene abundances, with higher fungal nirK levels in winter and higher bacterial nirK in summer, while bacterial nirS remained seasonally stable. Predictor analysis using random forest models identified available phosphorus (AP) as the strongest driver of fungal nirK abundance. In contrast, bacterial nirK was primarily influenced by soil pH and AP, while ammonium was the key regulator of bacterial nirS. These results highlight the distinct responses of fungal and bacterial denitrifiers to seasonal changes, nitrogen deposition, and precipitation reduction, emphasizing the need to consider both microbial groups when examining biogeochemical cycles and their environmental controls under future climate scenarios. These insights are crucial for refining predictive models of N₂O fluxes and for designing informed management practices in subtropical forest ecosystems.