Short-term intercropping reduces total symbiotic N2 fixation and alters the fate of nitrogen derived from free-living fixation

Symbiotic (SNF) and free-living (FLN) N2 fixation are key bioavailable nitrogen (N) sources in agroecosystems, yet their integrated response to intercropping remains inadequately understood. We conducted a field experiment with intercropped silage maize (Zea mays L.) and Chinese milk vetch (Astragalus sinicus L.), assessing the intercropping effects on N dynamics by examining SNF and FLN in conjunction with soil properties and the composition of rhizosphere microbial communities, thereby linking plant performance with belowground processes. Intercropping increased silage maize total dry matter yield by 14.36% but reduced the total amount of symbiotically fixed N (Ndfa). This reduction was due to the suppressed biomass and lower tissue N concentration of the intercropped Chinese milk vetch, while its fixation efficiency (%Ndfa) remained stable. Crucially, while the gross rate of soil FLN was unaltered, intercropping reprogrammed the partitioning of FLN-derived 15N in a species-specific manner: allocation to the microbial biomass N pool increased in the Chinese milk vetch rhizosphere (from 13.85% to 25.37%), whereas allocation to the plant–available nitrate (NO3–-N) pool increased in the silage maize rhizosphere (from 2.67% to 15.67%). Several dominant genera of diazotrophs (Sinorhizobium, Bradyrhizobium), other bacteria (Microvirga, Bacillus), and fungi (e.g. Fungi gen Incertae sedis) were positively correlated with the proportion of inorganic N derived from FLN. We conclude that short-term intercropping reduces total symbiotic N input but enhances system performance by species-specifically redirecting FLN-derived N, a process mediated by crop-specific rhizosphere microbiomes to optimize N bioavailability for the cereal crop.