Long-Term Fertilization Differentially Reshapes Bacterial CNPS Cycling Potential in Bulk and Rhizosphere Soils

Long-term fertilization can reshape soil microbial communities and alter soil ecosystem functioning. However, few studies have comprehensively investigated changes in microbial functional potential for carbon, nitrogen, phosphorus, and sulfur (CNPS) cycling in bulk and rhizosphere soils in response to long-term fertilization, or the relationships between microbial taxonomic shifts and functional responses. We combined 16S rRNA gene amplicon sequencing with high-throughput quantitative PCR targeting 71 CNPS cycling-related genes in a 32-year field experiment comprising an unfertilized control (CK), mineral fertilizer (NPK), manure (M), and NPK combined with manure (NPKM). The results showed that both mineral NPK fertilizer and manure increased soil nutrient availability but had contrasting effects on soil pH. Relative to CK, NPK, M, and NPKM increased rapeseed grain yield by 303.9%, 108.7%, and 397.8%, respectively, and biomass by 303.0%, 99.6%, and 410.5%, respectively. Fertilization also reshaped bacterial community composition, and the responses were similar between bulk and rhizosphere soils. In contrast, functional-gene abundances showed significant soil compartment-dependent responses to fertilization. NPK fertilization reduced most rhizosphere gene groups associated with C degradation, N transformation, P mobilization, and S cycling while increasing nitrification-related genes, whereas manure increased genes involved in carbohydrate degradation, methane metabolism, multiple N transformations, organic P mineralization, phosphate solubilization, and sulfate reduction. Network analysis showed that genes mediating similar processes clustered within common modules and were associated with shared bacterial taxa. Crop productivity was associated with variation in genes involved in N fixation and organic P mineralization in rhizosphere soil. Our results revealed associations between functional-gene abundances, soil nutrients, crop productivity, and bacterial community changes under long-term fertilization, highlighting the importance of quantitative functional-gene analysis as an indicator for evaluating soil multifunctionality.