Depth-Dependent Responses of Soil Microbial Communities to Nitrogen Application Gradients Under Rice Straw Return

Crop straw return is widely used to maintain soil fertility in intensive rice systems. How soil microbial communities respond to nitrogen rate under full straw return, and whether these responses differ between soil layers, remains poorly resolved. In this study, a field experiment with five nitrogen application rates (0 [RF0], 53 [RF1], 93 [RF2], 133 [RF3], and 173 [RF4] kg N ha−1) under uniform full rice straw return (7500 kg straw ha−1) was conducted in a cold-region paddy ecosystem. Soil physicochemical properties, bacterial community structure and functional potential (metagenomics) were systematically analyzed in surface (0–10 cm) and subsurface (10–20 cm) layers. Results showed that moderate nitrogen application (133 kg N ha−1) optimally enhanced total nitrogen, total organic carbon, labile carbon fractions, and available phosphorus and potassium in both soil layers. However, excessive nitrogen application (173 kg N ha−1) provided no further benefits and significantly decreased available phosphorus and potassium relative to their peak values observed under optimal nitrogen inputs. A combined PCoA of both soil layers showed no separation by depth or by nitrogen rate. PERMANOVA detected a weak but significant overall model (R2 = 0.241, p = 0.0002) without a significant depth × nitrogen interaction (R2= 0.065, p = 0.328). Bacterial taxonomic composition exhibited strong resistance to nitrogen disturbance, with a large core microbiome (dominated by Proteobacteria, Chloroflexi, and Acidobacteria) remaining stable across treatments and depths. In contrast, functional genes and metabolic pathways involved in carbon, nitrogen, and sulfur cycling displayed high plasticity. Moderate nitrogen activated complex carbohydrate degradation and dark carbon fixation in deeper soil, while high nitrogen shifted surface communities from autotrophic carbon fixation (Calvin-Benson-Bassham cycle) toward heterotrophic fermentation and strongly stimulated denitrification pathways, increasing potential gaseous nitrogen loss. Overall, this study reveals a clear taxonomic-functional decoupling and demonstrates that 133 kg N ha−1 represents the optimal threshold for balancing soil nutrient accumulation, microbial functional activity, and environmental risk under full rice straw return. These findings provide a theoretical basis for precision nitrogen management in straw-return systems.