Background: The rhizosheath, a cohesive soil layer firmly adhering to plant roots, plays a vital role in facilitating water and mineral uptake. In pearl millet, rhizosheath formation is genetically controlled and influenced by root exudates. Here, we investigated the impact of root exudates on the microbiota composition, interactions, and assembly processes, and rhizosheath structure in pearl millet using four distinct lines with contrasting soil aggregation abilities. Results: Utilizing 16S rRNA gene and ITS metabarcoding for microbiota profiling, coupled with FTICR-MS metabonomic analysis of metabolite composition in distinct plant compartments and root exudates, we revealed substantial disparities in microbial diversity and interaction networks. The ß-NTI analysis highlighted bacterial rhizosphere turnover driven primarily by deterministic processes, showcasing prevalent homogeneous selection in root tissue (RT) and root-adhering soil (RAS). Conversely, fungal communities were more influenced by stochastic processes. In bulk soil assembly, a combination of deterministic and stochastic mechanisms shapes composition, with deterministic factors exerting a more pronounced role. Metabolic profiles across shoots, RT, and RAS in different pearl millet lines mirrored their soil aggregation levels, emphasizing the impact of inherent plant traits on microbiota composition and unique metabolic profiles in RT and exudates. Notably, exclusive presence of antimicrobial compounds, including DIMBOA and H-DIMBOA, emerged in root exudates and RT of low aggregation lines. Conclusions: This research underscores the pivotal influence of root exudates in shaping the root-associated microbiota composition across pearl millet lines, entwined with their soil aggregation capacities. These findings underscore the interconnectedness of root exudates and microbiota, which jointly shape rhizosheath structure, deepening insights into soil–plant-microbe interactions and ecological processes shaping rhizosphere microbial communities. Deciphering plant–microbe interactions and their contribution to soil aggregation and microbiota dynamics holds promise for the advancement of sustainable agricultural strategies.

Current agricultural practices rely heavily on synthetic fertilizers, which not only consume a lot of energy but also disrupt the ecological balance. The overuse of synthetic fertilizers has led to soil degradation. In a more sustainable approach, alternative methods based on biological interactions, such as plant growth-promoting bacteria (PGPRs), are being explored. PGPRs, which include both symbiotic and free-living bacteria, form mutualistic relationships with plants by enhancing nutrient availability, producing growth regulators, and regulating stress responses. This study investigated the potential of Sphingomonas sediminicola Dae20, an α-Proteobacteria species commonly found in the rhizosphere, as a beneficial PGPR. We observed that S. sediminicola Dae20 stimulated the root system and growth of three different plant species in the Brassicaceae family, including Arabidopsis thaliana, mustard, and rapeseed. The bacterium produced auxin, nitric oxide, siderophores and showed ACC deaminase activity. In addition to activating an auxin response in the plant, S. sediminicola Dae20 exhibited the ability to modulate other plant hormones, such as abscisic acid, jasmonic acid and salicylic acid, which are critical for plant development and defense responses. This study highlights the multifunctional properties of S. sediminicola Dae20 as a promising PGPR and underscores the importance of identifying effective and versatile beneficial bacteria to improve plant nutrition and promote sustainable agricultural practices.