The Effects of Long-Term Manure and Grass Mulching on Microbial Communities, Enzyme Activities, and Soil Organic Nitrogen Fractions in Orchard Soils of the Loess Plateau, China

Organic manure and grass mulching are widely recognized as modifiers of soil microbial communities and nutrient dynamics; however, the combined effects of these practices on nitrogen fractionation and microbial functionality in orchard ecosystems remain poorly understood. This study conducted a comprehensive evaluation of soil nitrogen fractions, enzymatic activity, microbial diversity and functional traits in walnut orchards under three management practices: organic manure (OM), grass mulching combined with manure (GM), and chemical fertilization (CF) in China’s Loess Plateau. The results revealed that OM and GM significantly enhanced soil nutrient pools, with GM elevating total nitrogen by 1.96-fold, soil organic carbon by 97.79%, ammonium nitrogen by 128%, and nitrate nitrogen by 54.56% relative to CF. Furthermore, the OM significantly increased the contents of total hydrolysable nitrogen, amino sugar nitrogen, amino acid nitrogen, ammonia nitrogen, hydrolysable unidentified nitrogen, non-acid-hydrolyzable nitrogen compared to the CF and GM treatments. Meanwhile, ASN and AN had significant effects on mineral and total nitrogen. The OM and GM had higher activities of leucine aminopeptidase enzymes (LAP), α-glucosidase enzyme, β-glucosidase enzyme (βG), and N-acetyl-β-D-glucosidase enzyme (NAG). Microbial community analysis revealed distinct responses to different treatments: OM and GM enhanced bacterial Shannon index, while suppressing fungal diversity, promoting the relative abundance of copiotrophic bacterial phyla such as Proteobacteria and Chloroflexi . Moreover, GM favored the enrichment of lignocellulose-degrading Ascomycota fungi. Functional annotation indicated that Chemoheterotrophy (43.54%) and Aerobic chemoheterotrophy (42.09%) were the dominant bacterial metabolic pathways. The OM significantly enhanced the abundance of fermentation-related genes. Additionally, fungal communities under the OM and GM showed an increased relative abundance of saprotrophic taxa, and a decrease in the relative abundances of potential animal and plant pathogenic taxa. The Random forest model further confirmed that βG, LAP, and NAG, as well as Basidiomycota , Mortierellomycota , and Ascomycota served as pivotal mediators of soil organic nitrogen fraction. Our findings demonstrated that combined organic amendments and grass mulching can enhance soil N retention capacity, microbial functional redundancy, and ecosystem stability in semi-arid orchards. These insights support the implementation of integrated organic management as a sustainable approach to enhance nutrient cycling and minimize environmental trade-offs in perennial fruit production systems.