Flavonoids produced by legume roots act as signaling molecules that induce the expression of nod genes in symbiotic rhizobia. However, the role of flavonoids in root exudates under intercropping systems in promoting soybean nodulation remains unclear. Two consecutive years of field experiments were conducted using maize–soybean strip intercropping with interspecific row spacings of 30cm (MS30), 45cm (MS45), and 60cm (MS60), along with sole cropping of soybean(SS) and maize (MM). Root interactions were manipulated using either no root barrier (NB) or a polyethylene plastic barrier (PB) to assess the relationship between flavonoids in root exudates and soybean nodulation. We found that root–root interaction between soybean and maize increased nodule number and fresh weight in intercropped soybean, with enhancement gradually increasing as interspecific distancewidened. The proportion of nodules with diameters exceeding 0.4cm was higher in intercropped soybean under NB compared to PB. Additionally, the expression of nodule-related genes-GmENOD40, GmNIN2b, and GmEXPB2-was up-regulated. Furthermore, compared to monocropping, isoflavone secretion by soybean roots decreased, whereas flavonoid and flavonol secretion by both maize and soybean roots increased under intercropping. The abundance of differentially secreted flavonoid metabolites in the rhizosphere of both species declined when rootcontact was prevented by the barrier. In soybean roots, the expression of GmCHS8and GmIFS1was up-regulated, while GmICHGwas down-regulated under root interaction. Most flavonoid and flavonol compounds showed positive correlations with nodule diameter. Nodule number, fresh weight, and the proportion of nodules larger than 0.2cm increased in diverse soybean genotypes treated with maize root exudates, which contributed to enhanced nitrogen fixation capacity. Therefore, maize–soybean strip intercropping, combined with optimal row spacing, enhances the positive effects of underground root interactions and improves nodulation and nitrogen fixation in intercropped soybean.

maize hybrids  /  planting density  /  photosynthetic characteristics  /  photosynthetic N use efficiency  /  grain yield