Effect of mixed inhibitor application on N2O production pathways in paddy soil

In flooded paddy soils, quantifying and discerning nitrous oxide (N2O) production by four biological pathways (nitrifier nitrification (NN), nitrifier denitrification (ND), nitrification-coupled denitrification (NCD), and heterotrophic denitrification (HD)) are essential for developing innovative strategies to mitigate the greenhouse effect. Soils were collected from Shenyang Experimental Station of the Institute of Applied Ecology, Liaoning Province, China, and were sealed and incubated in the dark at 25 °C and submerged for 48 h. The amount of N2O produced by each of the four pathways and the abundance of corresponding functional genes were determined by dual-isotope (15N-18O) labeling technique combined with quantitative PCR (qPCR). In our incubation experiment, 15N isotope tracing showed that not urea but paddy soil was the largest contributor of N2O within 48 h after urea application. Combined application of urea and mixed inhibitors (N-(n-butyl) thiophosphoric triamide (NBPT) + phenylphosphorodiamidate (PPD) + 3,4-dimethylpyrazole phosphate (DMPP)) could reduce total N2O production by 26.69%. Through dual-isotope (18O-15N) labeling technology, it was found that N2O production mainly came from HD pathway, accounting for 77% of total N2O production, and N2O produced by ammonia oxidation (NN, ND, and NCD) after urea application accounted for 23% of total N2O production. The largest proportion of N2O production among the ammonia oxidation pathways was the ND pathway (0–23.00%), followed by the NN pathway (0–19.21%) and NCD pathway (0–3.79%). Application of mixed inhibitors significantly reduced the N2O produced by the HD pathway by more than 15%; reduced the N2O produced by the ammonia oxidation pathway from 23.00 to 10.39%; and reduced the N2O produced by the NN, ND, and NCD pathways by more than 50%. This is probably caused by the decreased ammonium level and reduced gene copies of AOB amoA, narG, and nirK, which are key N2O producing genes. Incubation experiment showed that ammonia oxidation pathway is also an important pathway for N2O production in flooded paddy soil. Mixed inhibitors have inhibitory effects on N2O produced by NN, ND, NCD, and HD pathways. The future development of mixed inhibitor application strategies suitable for paddy fields is of great significance for mitigating the global greenhouse effect.