Abstract Root exudation is one of the primary processes that mediate interactions between plant roots, microorganisms, and the soil matrix. Previous research has shown that plant root exudate profiles vary between species and genotypes which can likely support different microbial associations. Here, utilizing distinct sorghum genotypes as a model system, we characterized the chemical heterogeneity between root exudates and the effects of that variability on soil microbial membership and metabolisms. Distinct exudate chemical profiles were quantified and used to formulate synthetic root exudate treatments, a High Organic acid Treatment (HOT) and a High Sugar Treatment (HST). Root exudate treatments were added to laboratory soil reactors and 16S rRNA gene profiling illustrated distinct microbial membership in response to HST or HOT amendments. Alpha and beta diversity metrics were significantly different between treatments, (Shannon’s, p &lt; 0.0001, mrpp = 0.01, respectively). Exometabolite production was highest in the HST, with increased production of key organic acids, non-proteinogenic amino acids, and three plant growth-promoting phytohormones (benzoic acid, salicylic acid, indole-3-acetic acid), suggesting plant-derived sugars fuel microbial carbon metabolism and contribute to phytohormone production. Linking the metabolic capacity of metagenome-assembled genomes in the HST to the exometabolite patterns, we identified potential plant growth-promoting microorganisms that could produce these phytohormones. Our findings emphasize the tractability of high-resolution multi-omics tools to investigate soil microbiomes, opening the possibility of manipulating native microbial communities to improve specific soil microbial functions and enhance crop production. Importance Understanding interactions between plant root exudates and the soil microbiome provides an avenue for a more comprehensive appreciation for how plant roots modulate their microbial counterparts to promote an environment favorable to plant fitness. Although these dynamics are appreciated as indispensable, mechanisms controlling specific rhizobiome membership and complexity are not fully understood. In this study, we investigate how variability in root exudation, modeled after differences observed between distinct sorghum genotypes, contributes to altered microbial membership and metabolisms. The results demonstrate how microbial diversity is influenced by root exudates of differing chemical composition and how changes in microbial membership correspond to modifications in carbon utilization and enhance production of plant-relevant metabolites. Our findings suggest carbon substrate preferences among bacteria in semi-arid climate soils and mechanisms for root exudate utilization. These findings provide new information on plant-soil environments useful for the development of efficient and precise microbiota management strategies in agricultural systems.

Variation in root exudate composition influences soil microbiome membership and function