Unveiling the dominant role of soil pH in shaping nitrogen cycling microbial gene abundances: Insights from 65-years of chemical fertilizer selection in acidic grassland meadow

Abstract Understanding the microbial processes driving the nitrogen (N) cycle is crucial for enhancing plant productivity and mitigating environmental pollution. The long-term application of synthetic fertilizers induces significant alterations in the microbial community functions. Microbes inhabiting acidic soils may exhibit distinct responses to chemical fertilizer application compared to soils with neutral pH. However, the chronic or occasional changes resulting from repeated nutrient flushes due to fertilizer application remain insufficiently elucidated, especially in acidic grasslands. Therefore, our study was on an acidic semi-natural grassland, where the soil was subjected to chemical fertilizer P (superphosphate), K (potassium sulfate), PK, N (ammonium nitrate), NPK, PK+N (PK applied in spring and N applied once in summer) over a span of 65 years. Gene abundances associated with the N-cycle ( amoA , nifH , nirK , nirS , and nosZ ) were quantified at seven different time points throughout the year considering the temporal effect caused by fertilizer application. Our findings reveal that in the long term, soil pH emerged as the predominant factor influencing the gene abundance related to N-fixation and denitrification outweighing the impact of nutrient availability. Notably, the application of N fertilizer had a positive effect on the abundance of nitrifiers, while the abundance of denitrifiers decreased due to soil acidification induced by fertilizer application. In summary, our study highlights that the microbial community involved in N cycling is more sensitive to the difference in soil pH shaped by long-term fertilizer application rather than to the direct impact of fertilizer application. Highlight Seasonal sampling confirmed the long-term effect of fertilizer on microbial N-cycling genes Fertilizer types shaped soil pH, consequently impacted on diazotrophs and denitrifies abundances Abundance of denitrifying microbe did not explain the increase of N 2 O emission by N fertilizer N 2 O emission was positively correlated with AOA abundance