Deciphering the Relationships Between Soil Enzymatic Activities and N- and P-Cycling Functional Genes Under Long-Term Fertilization

Long-term fertilization profoundly influences soil biochemical processes and microbial functionality, yet the coupling mechanisms between soil enzyme activities and functional genes in nutrient cycling remain unclear. This study investigated the effects of different fertilization regimes-nitrogen alone (N), nitrogen-phosphorus-potassium fertilizer (NPK), organic fertilizer (M), and combined organic-inorganic fertilizer (MNPK)-on soil properties, enzyme activities, N- and P-cycling-related functional gene abundances, and faba bean (<i>Vicia faba</i> L.) yield in a 45-year ongoing field experiment in subtropical eastern China. Results showed that long-term fertilization significantly affected soil pH, electrical conductivity, nutrient contents, and crop yield. Organic fertilizer addition (M and MNPK) markedly improved soil organic matter, total and available nutrients, and enhanced faba bean grain yield by 75.07-92.79% compared with NPK, whereas NPK had limited benefits on total and available soil nutrients compared with N-only application. Soil enzyme activity analysis revealed that the MNPK treatment achieved the highest urease and neutral protease activities, while acid and alkaline protease activities responded inconsistently. Phosphorus-related enzymes (acid, neutral, and alkaline phosphatases) were strongly stimulated by organic inputs, reflecting enhanced P mineralization potential. Functional gene analysis showed that N-fixation and assimilatory nitrate reduction genes increased under M and MNPK, while N assimilation, N mineralization, anammox, nitrification, denitrification, and dissimilatory nitrate reduction genes were enriched under N treatment. Phosphate uptake and transport genes were upregulated under NPK, M, and MNPK, whereas inorganic P solubilization genes were highest under N. Significant positive correlations were observed among soil enzyme activities, nutrient contents, and faba bean yield, whereas acid and alkaline protease activities showed opposite trends. The relative abundances of N- and P-cycling functional genes exhibited distinct yet coordinated relationships with soil fertility indicators and enzyme activities. These findings provide mechanistic insights into the long-term regulation of soil-microbe interactions and nutrient cycling, offering a scientific basis for sustainable fertilization strategies in agroecosystems.