Objectives The community variation of bacteria and fungi are crucial factors deciding soil ecological functions. We studied the response of bacterial and fungal communities and their functional roles to long-term nitrogen application levels in Loess Plateau, China.

Methods The soil samples were collected in 2022 from the five N application treatment plots of a long-term experiment, which was in the State Key Agro-Ecological Experimental Station in Changwu County, established in 1984. The N application rates in the five selected treatments were 0, 45, 90, 135, and 180 kg/hm² (denoted as N0, N45, N90, N135, and N180). The bacterial and fungal community structures, soil enzyme activities related to carbon cycling, and soil properties were determined, and the changes relative to the initial of experiment were calculated.

Results N application led to significant changes in soil chemical properties and microbial population and community structures. Compared to the initials, long term N application increased soil microbial biomass carbon from 113.0 to 177.5−234.0 mg/kg, microbial activity from 4.92 to 5.83−7.26 g/(m²·s), and plant root biomass from 1.73 to 2.32−2.98 t/hm², decreased bacterial community richness and diversity index (Chao1 and Shannon) by 4.0%−12.3% and 1.6%−1.8%, respectively, did not changed fungal communities obviously. N application significantly decreased the relative abundance of Proteobacteria, Gemmatimonadetes, Bacteroidetes, Dothideomycetes, and Agaricomycetes by 2.4%−16.4%, 2.1%−26.3%, 24.1%−50.0%, 29.4%−58.8%, and 44.4%−88.9%, respectively, but increased the relative abundance of Actinobacteria, Acidobacteria, Chloroflexi, Ascomycota, and Tremellomycetes by 10.4%−34.7%, 37.8%−54.1%, 14.3%−28.6%, 8.3%−73.8%, and 18.2%−45.5%. N application elevated soil organic C (SOC) from initial 6.50 g/kg to 7.00−7.35 g/kg, dissolved organic C (DOC) from 22.43 mg/kg to 27.60−34.87 mg/kg, NO₃⁻-N from 1.38 mg/kg to 1.78−2.25 mg/kg, and decreased C/N from 8.90 to 8.64−7.74. With the increase of N application rate, the soil nitrification increased by 14.3% to 39.6%, aerobic ammonia oxidation functions increased by 25.1%−48.2%, and the activities of xylanase increased by 13.5% to 39.3%, and that of cellobiose hydrolase increased by 50.3% to 126.8%. The changes of soil bacterial richness and diversity were correlated with that of SOC, DOC, NO₃⁻-N, and C/N ratios. The Proteobacteria abundance was negatively linked to DOC levels, Actinobacteria abundance was positively associated with SOC, DOC, and NO₃⁻-N contents, Ascomycota and Basidiomycota abundances were not significantly correlated with soil physiochemical properties.

Conclusions In Loess Plateau, bacteria structures are more sensitive than fungal structure to N application rate. The changes of bacterial community composition are positively correlated with soil organic matter, dissolved organic carbon and NO₃⁻-N, and negatively correlated with total nitrogen and C/N. Nitrogen application increased the abundance of functional bacteria related to carbon cycle in soil and promoted the secretion of xylosidase and cellobiohydrolase.