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

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 and functions. However, there is still limited research on how long-term application of N, P and K fertilizers over 60 years, either individually or in combination, especially in acidic grasslands, influences the abundance of microbial N-cycling genes and N 2 O emissions. Therefore, our study was conducted 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 65 years. Gene abundances associated with the N-cycle ( nifH , amoA , nirK , nirS , nosZ , and nrfA ) were quantified at seven different time points throughout the year considering the temporal effect caused by fertilizer application. Our findings reveal that soil pH emerged as the predominant factor influencing the gene abundance related to N-fixation and denitrification outweighing the effect of the temporal nutrient increases induced by fertilizer application. N 2 O emissions were significantly positively correlated with ammonia-oxidizing archaea (AOA) abundance, while no correlation was found with denitrifiers and nitrate ammonifiers. This suggests that further investigation into the mechanisms of N 2 O production by AOA in acidic grasslands is warranted. Our study highlights that the microbial community involved in N-cycling is shaped by the difference in soil pH resulting from long-term chemical fertilizer application rather than by the direct and temporal impact of fertilizer application. • Fertilizer type shaped soil pH, which consequently affected the abundances of diazotrophs and denitrifiers. • Long-term application of N fertilizer increased AOA abundance, whereas K fertilizer reduced AOA. • The ratio of ( nirK + nirS )/ nosZ gene did not correlate with N 2 O emissions. • N 2 O emissions were positively correlated with AOA abundance.