Reduced nitrogen application alleviates nodulation inhibition in intercropped soybean by reshaping root exudates and enhancing cross-kingdom bacteria-fungi interactions

Maize-soybean intercropping enhances resource-use efficiency but exposes soybean to canopy shading, which suppresses photosynthetic carbon assimilation and inhibits nodulation. Although nitrogen (N) management is known to regulate nodulation, whether reduced N application can alleviate shade-induced nodulation inhibition and whether root exudate-mediated rhizosphere microbial responses are involved remain unclear. We examined the effects of three N levels (NN: no N application; RN: reduced N application; CN: conventional N application) and two planting patterns (MS: monocropping; IS: intercropped with maize) on soybean nodulation, root exudate profiles, and rhizosphere cross-kingdom microbial networks. Compared with MS, IS significantly reduced nodule number; however, under RN, nodule weight and average nodule weight were comparable between IS and MS. Nodule starch content under ISRN was 144.00% and 23.04% higher than under ISCN and ISNN, respectively. Metabolomic analysis revealed that differential metabolites under intercropping were predominantly enriched in lipid-related compounds, particularly N-Tetradecanoyl-DL-homoserine lactone (N-C14-HSL) and Panaquinquecol 1, both of which were significantly and positively correlated with nodulation traits and nodule carbon metabolism indicators. Neither bacterial nor fungal α-diversity differed significantly among treatments, whereas community structure was significantly shaped by both planting pattern and N level. Cross-kingdom co-occurrence network analysis revealed that bacterial-fungal linkages increased by 48.6% under IS relative to MS. Module 19 was significantly enriched under RN and positively correlated with nodulation indicators and the relative abundances of N-C14-HSL and Panaquinquecol 1. Five core OTUs, belonging to the orders Phycisphaerales, Spizellomycetales, Sordariales, Rokubacteriales, and Aggregatilineales, showed consistent positive correlations with nodulation traits and both lipid compounds. Collectively, these findings demonstrate that reduced N application alleviates shade-induced nodulation inhibition in intercropped soybean by reshaping root exudate composition and enhancing cross-kingdom bacterial-fungal interactions in the rhizosphere.