Enzymatic-Driven Responses of Soil Fertility and Crop Yields to Different Long-Term Organic Substitution Regimes Under Wheat–Maize Rotation

Long-term excessive chemical fertilization threatens the sustainability of wheat–maize rotation systems in the North China Plain. Organic substitution is a promising alternative to sustain crop productivity and soil health, yet its underlying mechanisms require clarification. This study investigated the effects of six fertilization treatments (unfertilized [CK], chemical nitrogen [N] alone at 180 kg N ha−1 season−1 [NPK], chemical N 25% substituted by chicken manure per season [NPKM], full manure substitution per season [CM], chemical N 25% substituted by straw return under no tillage per season [NT] and chemical N 25% substituted by straw return under rotary tillage per season [ST]) on soil fertility and crop productivity in a long-term wheat–maize rotation field experiment initiated in 2007. All treatments followed a randomized complete block design with three replicates per treatment. Wheat and maize plants were randomly collected from each plot at the harvest stage of each season, and weighed and measured for yield and N uptake, while soil samples were randomly collected from each plot at maize harvest stage for chemical and enzyme activity analyses. Compared to NPK, organic substitution maintained grain yields while significantly enhancing key soil fertility indicators: soil organic carbon (C) (up to 53.8%), and labile C and N pools including readily oxidizable C (by 120.0%), ammonium N (by 23%) and microbial biomass C (up to 164.5%). It also strongly stimulated the activities of C-acquiring (e.g., β-glucosidase and cellobiohydrolase) and N-cycling (e.g., β-N-acetylglucosaminidase and urease) enzymes by up to 278.7% and 256.3%, respectively. Multivariate analyses identified these enzymes as primary drivers of soil C and N dynamics, with direct positive links to crop yield. In conclusion, long-term organic substitution, particularly full manure substitution, improved yield stability and soil fertility predominantly through an enzymatic-driven stimulation of nutrient cycling and organic matter accumulation, offering a viable strategy to reduce chemical fertilizer inputs and enhance crop production sustainability.