Objectives This study investigates the effects of drought stress memory on the microbial community assembly in the rhizosphere, endosphere, and phyllosphere compartments of soybean, to elucidate the dynamics of microbial communities across distinct ecological niches and their potential impacts on soybean growth and yield.

Methods A pot experiment was conducted using the soybean cultivar ‘Fendou 93’. Fifteen days of drought treatment were initiated 14 days after seedling emergence, with well-watered plants serving as controls. Following 15 days of rewatering, the plants entered the flowering stage. At both the flowering and maturity stages, rhizosphere soil, root-associated, and middle leaf samples were collected for high- throughput sequencing using the Illumina PE250 platform. Bioinformatic analyses were performed to characterize bacterial and fungal α-diversity, β-diversity, taxonomic composition, and co-occurrence networks. Differential abundance analysis combined with random forest modeling identified key bacterial and fungal taxa beneficial to plant drought resistance and yield improvement.

Results Drought stress markedly reduced soybean biomass by 75.71% and hundred-grain weight by 19.19%, while increasing the height of the lowest pod by 22.90%. From the rhizosphere to the endosphere and phyllosphere, both bacterial and fungal α-diversity progressively declined. Compared with the control, microbial diversity in the root and leaf compartments decreased significantly under drought, whereas rhizosphere diversity remained largely unchanged. β-diversity analysis revealed significant shifts in bacterial and fungal community composition across all three compartments. Dominant bacterial phyla included Proteobacteria and Actinobacteriota, while Ascomycota predominated among fungi. Following drought stress, the relative abundance of Actinobacteriota in the rhizosphere increased by 43.75%, and that of Proteobacteria in the root endosphere increased by 14.44%. For fungi, Mortierellomycota showed significant increases in the rhizosphere (49.04%) and root endosphere (31.34%). Co-occurrence network analysis indicated that drought reduced the number of nodes and edges, as well as overall network complexity, suggesting weakened microbial interactions. While fungal community modularity, closeness centrality, and betweenness centrality remained largely unchanged under drought, the complexity of the root-associated fungal network declined markedly, indicative of community restructuring. Differential abundance analysis combined with random forest modeling identified drought-responsive keystone taxa, including the bacterial genus Streptomyces and the fungal genus Paraphoma. These rhizosphere-associated taxa represent potential beneficial groups that may contribute to the regulation of soybean drought adaptation and yield formation.

Conclusions Drought stress at the seedling stage persistently alters soybean microbiome structures across rhizosphere, endosphere, and phyllosphere compartments, significantly reducing both microbial α-diversity and network complexity. The structural changes likely weaken microbial interactions, consequently affecting soybean physiological processes and drought resistance. Notably, keystone taxa including Streptomyces and Paraphoma show significant correlations with hundred-grain weight, suggesting their potential as targets for microbial-mediated drought resilience enhancement.