Small molecule metabolites drive plant rhizosphere microbial community assembly patterns.

The assembly of rhizosphere microbial communities is essential for maintaining plant health, yet it is influenced by a wide range of biotic and abiotic factors. The key drivers shaping the composition of these communities, however, remain poorly understood. In this study, we analyzed 108 plant samples and evaluated root traits, plant growth characteristics, soil enzyme activities, rhizosphere metabolites, and soil chemical properties to identify the primary determinants of rhizosphere community assembly. Across 36 soil samples, we obtained 969,634 high-quality sequences, clustering into 6,284 ASVs predominantly classified into Proteobacteria (57.99%), Actinobacteria (30%), and Bacteroidetes (5.13%). Our findings revealed that rhizosphere metabolites accounted for more variance in microbial community composition compared to chemical properties (ANOVA,  = 1.53,  = 0.04), enzyme activities, or root traits (ANOVA,  = 1.04,  = 0.001). Seven small molecule metabolites, including glycerol, sorbitol, phytol, and alpha-ketoglutaric acid, were significantly correlated with βNTI, underscoring their role as critical drivers of microbial community assembly. The genus , significantly associated with βNTI (R = 0.25,  = 0.009), emerged as a keystone taxon shaping community structure. Soil culture experiments further validated that small molecule metabolites can modulate microbial community assembly. The ST treatment, enriched with these metabolites, produced 1,032,205 high-quality sequences and exhibited significant shifts in community composition (Adonis,  = 0.001, R = 0.463), with showing higher abundance compared to the control (CK). Variable selection (βNTI >2) drove phylogenetic turnover in ST, while stochastic processes (|βNTI| < 2) dominated in CK. This study provides quantitative insights into the role of rhizosphere metabolites in shaping microbial community assembly and highlights their potential for targeted modulation of rhizosphere microbiomes.