Long-Term Organic Fertilization Enhances Soil Fertility and Reshapes Microbial Community Structure with Decreasing Effects Across Soil Depth

Sustaining agricultural productivity and soil health under intensive cultivation requires a comprehensive understanding of fertilization effects, particularly on deeper soil layers, which has received limited attention compared to surface soils. This study investigated how different fertilization regimes (inorganic, organic, and combined organic-inorganic fertilizers) influence soil physicochemical properties, microbial diversity, community structure, and functional gene abundances at three soil depths (0-20 cm, 20-40 cm, and 40-60 cm) in a 40-year fertilization experiment. Organic fertilization significantly improved topsoil fertility indicators such as soil organic matter (56.6-109.2%), total nitrogen (66.7-122.0%), total phosphorus (198.6-413.2%), and available phosphorus (984.8-1622.1%) and potassium (35.3-438.1%). Compared with the unfertilized control and nitrogen-only treatment, rice yield increased by 97.1-130.5% under NPK and sole organic fertilization, and further increased by 184.1-255.9% under combined organic-inorganic fertilization. However, fertilization effects diminished with soil depth due to limited nutrient mobility. Microbial diversity significantly decreased with depth and was minimally influenced by fertilization treatments. Microbial community structure varied notably among fertilization treatments at the surface layer, mainly driven by soil nutrients, whereas soil depth had a dominant effect on microbial community structure and compositions. Co-occurrence networks showed the highest complexity in surface soil microbial communities, which declined with soil depth, reflecting potential synergistic and mutualistic relationships in topsoil and the adaptation of microbial communities to nutrient-limited conditions in subsoil. Microbial functional gene analyses highlighted clear depth-dependent distributions, with surface layers enriched in decomposition-related genes, while deeper layers favored anaerobic processes. Overall, long-term fertilization exerted strong depth-dependent effects on soil fertility, microbial community structure, and functional potential in paddy soils.