Nitrification inhibitors reduce N2O emissions from Mollisols by potentially targeting Nitrosospira cluster 3a and denitrifiers

Nitrification inhibitors (NIs) are widely used to mitigate nitrous oxide (N₂O) emissions from agricultural soils. However, its efficiency is highly uncertain owing to varying environmental conditions and still-debated inhibition mechanisms, especially the responses of the nitrifiers and denitrifiers responsible for N₂O production. Here, we conducted microcosm incubations to investigate the comparative effectiveness of three NIs (DCD, DMPP, and nitrapyrin) on N₂O emissions from cultivated Mollisols under contrasting moisture levels (60 % and 90 % water-filled pore space, WFPS). The soil nitrification rate, N₂O-related gene abundance, and community composition of ammonia-oxidizing bacteria (AOB) were determined. The results showed that all NIs effectively inhibited nitrification, with DCD and DMPP having higher efficacy than nitrapyrin, regardless of soil moisture conditions. Interestingly, a greater decrease in N₂O emissions was observed under 90 % WFPS than 60 % WFPS (39.0–47.6 % vs. 26.8–31.9 %). NIs selectively decreased the amoA gene abundance in AOB rather than in ammonia-oxidizing archaea. The most abundant AOB (> 90 %) belonged to Nitrosospira. RDA analysis revealed that the AOB Nitrosospira cluster 3a had the greatest relationship with N₂O emissions, and its abundance was significantly decreased with NI amendments. Moreover, non-target denitrifying genes (nirS and nirK) were suppressed, particularly at high moisture levels, which further contributed to reduced N₂O emissions. Overall, our findings highlight the significant role of environmental conditions, keystone species of AOB, and non-targeted effects on denitrifiers on the efficacy of NIs. Additionally, these results imply that NIs are a potent option for fertilizer management to mitigate N₂O emissions and that DCD and DMPP have promising prospects for the cultivated Mollisols agro-system under climate change with intensifying extreme rainfall events.