Intercropping and relay intercropping systems, which significantly improve land use efficiency, are used worldwide to increase crops yield. The wheat-maize-soybean relay intercropping system has been widely employed by famers in Southwestern China for years, but the detailed mechanisms through which the nitrogen fertilizer use efficiency reach the high level in this system remain unclear. In the present study, two separate pot experiments were performed by 15N isotope dilution (ID) labeling and direct ¹⁵N foliar feeding (FF) assays, and a solid barrier was employed to prevent the roots intergrowth and N movement among crops in the first experiment, using no barrier as the control. The results showed that, under the no-barrier condition, the grain yields, ¹⁵N uptake and ¹⁵N recovery efficiency of wheat and maize were significantly increased, but those measures in soybean were decreased compared to the solid barrier condition. Furthermore, bi-directional N transfer was detected during the co-growing stage of crops, the amount (N_transfer) and percentage (%NT) of ¹⁵N transferred varied significantly with the fertilizer-N rate, and the maximum reached at 150 – 300 kg N ha–1 level. The N_transfer from maize to wheat was 16.1% – 163.0% higher than that from wheat to maize; the N_transfer from soybean to maize was 1.7 – 6.0 times higher than those from maize to soybean, while the %NT from soybean to maize were 6.7 – 22.2 times higher than those from maize to soybean. Conclusively, this study revealed that the interaction of the roots among crops significantly increased the uptake efficiency and recovery efficiency, and further, the positive N competition and bi-directional N transfer of each crops were the main contributors to improve the N use efficiency in the wheat-maize-soybean relay intercropping system.

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