Biological nitrogen fixation is crucial for sustainable crop production. It occurs through free-living bacteria and rhizobial symbiosis with legumes. Chemical signaling between plants and soil microbiota, particularly rhizodeposition (root exudates), plays a key role in shaping the rhizosphere microbiome and its functions. While the mechanisms of rhizodeposition are well-understood for individual species, less is known about how interspecific plant interactions influence these processes, especially in diversified cropping systems. Diversified cropping, including intercropping and crop rotation, often leads to improved crop performance, particularly for legumes, due to enhanced nitrogen availability. This study aims to understand how intercropping affects the chemical signaling between peanuts (a legume) and their rhizosphere microbiota, and how this influences nitrogen fixation. The researchers hypothesized that intercropping alters peanut rhizodeposition, leading to changes in the rhizosphere microbiome and, consequently, improved nitrogen fixation and peanut growth. This mechanistic understanding is critical for designing effective crop combinations for sustainable agriculture.

The literature review highlights the importance of chemical signaling in plant-microbe interactions and the role of root exudates in shaping the rhizosphere microbiome. Existing research demonstrates that specific metabolites attract and filter microbial taxa, including those involved in nitrogen fixation. However, knowledge on how interspecific plant interactions influence these processes is limited. Studies on diversified cropping systems have shown improved crop performance, particularly legumes, due to enhanced nitrogen availability, but the underlying mechanisms remain unclear. The authors emphasize the need for a mechanistic understanding of how interspecific interactions influence legume nitrogen fixation by examining the chemical dialogue between plants and their rhizosphere microbiota.

This study utilized a long-term (8-year) field experiment comparing three cropping systems: peanut monoculture (PP), peanut-oilseed rape rotation (P-R), and peanut-maize intercropping rotated with oilseed rape (PM-R). Rhizosphere and bulk soil samples were collected to analyze soil chemical properties, including pH, organic carbon, total nitrogen, available phosphorus and potassium. Plant height, biomass, nodule density, and nodule-to-root mass ratio were measured. Nontargeted metabolomics (UHPLC-MS/MS) was used to profile peanut rhizosphere metabolites. Peanut root transcriptomics was performed to identify differentially expressed genes related to metabolite biosynthesis. Bacterial communities were characterized using 16S rRNA gene amplicon sequencing. Bacterial isolates were obtained from the PM-R rhizosphere, and their growth and nitrogen fixation activity were tested in microplate assays with specific metabolites. Finally, the effect of selected metabolites on *Bradyrhizobium* nodulation of peanut seedlings was investigated using gene expression analysis and nodulation counts.

Crop diversification significantly enhanced peanut production, with the PM-R system showing the highest peanut height, biomass, fruit weight, and nitrogen uptake compared to PP and P-R. Nodule density and nodule-to-root mass ratio were significantly higher in PM-R. Soil ¹⁵N fixation was also significantly greater in PM-R. Metabolite analysis revealed that the PM-R rhizosphere was enriched in flavonoids (quercetin, hyperoside), coumarins (scopoletin), and syringaldehyde. Transcriptomic analysis showed that phenylpropanoid biosynthesis pathways were upregulated in peanut roots under PM-R. Bacterial community analysis indicated that ASVs belonging to Gammaproteobacteria were enriched in PM-R. Microplate assays showed that the PM-R-enriched metabolites positively influenced the growth and nitrogen fixation activity of free-living bacterial isolates. *Bradyrhizobium* isolates showed increased nodulation gene expression (*nodDI*, *nodC*) in response to flavonoids and coumarins, leading to enhanced nodulation in peanut seedlings. These results demonstrated a mechanistic link between plant diversity and belowground functioning.

The findings demonstrate that intercropping with maize significantly enhances peanut performance and nitrogen fixation. This enhancement is attributed to specific changes in peanut rhizodeposition, including increased flavonoids and coumarins. These metabolites selectively promote the growth and nitrogen fixation activity of free-living bacteria and enhance the symbiotic interaction between peanut and *Bradyrhizobium*, ultimately improving nitrogen availability in the rhizosphere. The authors suggest that shade from maize canopy and competition for light might trigger peanut to produce these metabolites as an adaptive response, attracting beneficial microbes and improving its fitness. The study provides a mechanistic understanding of the positive effects of crop diversification on legume nitrogen fixation and offers insights for designing crop combinations that optimize soil fertility and sustainable agricultural practices.

This study reveals a mechanistic link between crop diversification, specifically intercropping with maize, and enhanced nitrogen fixation in peanut. The increased production of flavonoids and coumarins by peanuts in response to intercropping influences the rhizosphere microbiome, promoting both free-living and symbiotic nitrogen fixation. This improved nitrogen availability contributes to increased peanut biomass and yield. Future research could explore the role of other plant species and investigate the optimal combinations for maximizing the positive effects on legume nitrogen fixation in various agroecosystems.

The study is limited to one location and soil type. The effects observed might not be generalizable to other environments. The study focused on a limited set of metabolites and bacterial isolates. A more comprehensive analysis of the rhizosphere microbiome and its functional diversity would strengthen the conclusions. The loss of some samples in the metabolic analysis also presents a limitation to the study.