Mulberry-soybean intercropping reshapes rhizosphere metabolites and microbiota alongside enhanced soybean nodulation

Woody perennial-legume intercropping may improve nitrogen-use efficiency, but the belowground links among root-zone configuration, soybean nodulation, rhizosphere chemistry, microbial assembly and nitrogen movement remain unclear. We conducted a greenhouse pot experiment with soybean monoculture (Mono-S), mulberry monoculture (Mono-M), full-contact mulberry-soybean intercropping (Int), and root-separated intercropping with a 30 μm nylon-mesh barrier (Int + B). Physiological and N-related traits were assessed with six pot-level biological replicates, whereas untargeted rhizosphere metabolomics, bacterial full-length 16S rRNA profiling and fungal full-length ITS profiling used three pot-level replicates per treatment-plant component. Relative to Mono-S, Int increased soybean total nodule number by 12.3%, nitrogenase activity by 21.6%, whole-plant N accumulation by 9.1% and shoot dry weight by 26.5%. GmNIN and GmENOD40 transcript abundance was 2.60- and 2.22-fold higher, respectively, in Int-S than in Mono-S. Metabolomics revealed broad, host-specific treatment-associated shifts across multiple chemical classes, while selected putatively annotated soybean isoflavonoids and mulberry flavonoids showed contrasting response patterns. Host-specific PERMANOVA revealed significant differences in bacterial and fungal community composition. ¹⁵N label applied to soybean-side soil was recovered in mulberry roots, stems and leaves under both intercropping configurations, indicating recovery of soil-applied ¹⁵N label in mulberry. Mean land equivalent ratios exceeded 1 in both treatments and differed significantly. Cross-omics associations were largely treatment-structured, highlighting the need for targeted validation of specific metabolite-microbe relationships. Overall, full root-zone continuity was associated with the strongest coordinated soybean symbiotic and growth responses, while both intercropping configurations produced distinct rhizosphere restructuring. These findings provide a framework for understanding rhizosphere-mediated interactions in woody perennial-legume intercropping systems.