Integrated metagenomic–metabolomic insights into plant–microbe interactions mediated by <i>Bacillus</i> volatile compounds

Modulation of plant-microbe interactions with signaling molecules offers a promising strategy to promote plant growth and stress adaptation. However, identifying effective signaling molecules and elucidating the mechanisms for regulating the rhizosphere microbiome remain major challenges. In this study, the roles and mechanisms of <i>Bacillus</i> volatile compounds as potential signaling molecules in plant-microbe interactions were investigated. First, the genome and metabolism of a novel <i>Bacillus subtilis</i> strain capable of producing acetoin and 2,3-butanediol were studied, and the titers of the two compounds were increased to 86.76 g/L by sequential metabolic engineering. Subsequently, the effects of volatile compounds on the growth of vegetables (<i>Brassica rapa</i> and <i>Solanum lycopersicum</i> var.) were studied. Plant growth, nutrient (nitrogen, phosphorus, and potassium) utilization efficiency, and salt stress resistance were improved significantly. Compared with water as a control, significant changes in the abundance of 109 microbial genera of <i>B. rapa</i>'s rhizosphere microbiome were identified with volatile compound application. Notably increased microbes included nitrogen-fixing, phosphate- and potassium-solubilizing, stress-resistant, plant growth-promoting, and auxin-secreting microbes. Additionally, genes involved in nitrogen, phosphorus, and potassium utilization in the rhizosphere microbiome were significantly increased, and corresponding metabolism was found. Finally, metabolomic analyses of <i>S. lycopersicum</i> var.'s roots and leaves revealed 67 significantly upregulated compounds with the application of volatile compounds. These compounds were primarily involved in stress resistance, oxidative stress alleviation, free radical scavenging, and auxin-related plant growth promotion. This work demonstrates that <i>Bacillus</i> volatile compounds regulate rhizosphere microbiome and plant-microbe interactions and enhance plant nutrient utilization efficiency, stress tolerance, and growth.IMPORTANCEPlant productivity and stress resilience are strongly influenced by interactions between plants and the rhizosphere microbiome, yet practical strategies to rationally modulate native soil microbial communities remain limited. This study demonstrates that <i>Bacillus</i> volatile compounds, specifically acetoin and 2,3-butanediol, function as effective signaling molecules that coordinate plant-microbe interactions in the rhizosphere. By integrating plant physiology, metagenomics, and metabolomics, we show that these volatile compounds not only enhance plant growth and nutrient use efficiency but also reprogram rhizosphere microbial communities toward functions that benefit nitrogen, phosphorus, and potassium acquisition and stress adaptation. Notably, volatile application improved plant salt tolerance, highlighting their strong ecological and physiological impact. This work provides mechanistic evidence that <i>Bacillus</i>-derived volatiles act as signaling molecules to activate the rhizosphere microbiome and plant metabolic responses. The findings offer a scalable and environmentally friendly strategy for improving crop performance and soil health, with broad implications for sustainable agriculture.