The Effect of Intercropping Leguminous Crops in the Winter Fallow Season of Mulberry Plantations on Soil Nitrogen and Phosphorus Status

To explore the effects of legume intercropping on soil nitrogen (N) and phosphorus (P) cycling processes in mulberry plantations during the winter fallow period, three treatments were set up: control (no intercropping, CK), single-legume intercropping (T1: Medicago polymorpha), and mixed-legume intercropping (T2: Medicago polymorpha + Astragalus sinicus). The variations in soil available nutrients, enzyme activities, microbial community structure, and functional gene abundances were investigated, with a focus on the influence of spatial heterogeneity (middle of inter-row as position A, and mulberry-adjacent as position B). Compared with CK, both T1 and T2 significantly altered soil microbial community composition and enhanced β-diversity (R2 = 0.747, p < 0.001), and these effects were more pronounced at position A. For soil N cycling, T1 and T2 reduced available N content especially at position B; however, T2 significantly increased urease activity, while T1 decreased urease activity at position A. Both treatments elevated the abundances of N-cycling functional genes (e.g., glnA, ureC, nifD) and symbiotic nitrogen-fixing rhizobia, with T1 increasing Bradyrhizobium and T2 increasing Sinorhizobium. For soil P cycling, T1 and T2 enhanced phosphatase activity to promote organic P conversion; T1 strengthened the entire P metabolic chain by upregulating multiple P-related functional genes, while T2 specifically increased the abundance of the organic P transport gene ugpb. Structural equation modeling showed that soil microbial Shannon diversity promoted the relative abundances of N-cycling and P-cycling functional genes, and cascading pathways among nutrient-cycling genes and enzyme activities governed soil available N and P. The distinct effects of T1 and T2 highlight the importance of optimizing intercropping patterns, and spatial heterogeneity should be considered in agronomic effect evaluation. Leguminous green manure intercropping should be encouraged in mulberry plantations to boost soil nutrient-cycling processes driven by microorganisms.