The combined influence of microbiome and soil environment contributes to the chemotype differentiation in <i>Atractylodes lancea</i>

<i>Atractylodes lancea</i>, a traditional Chinese medicinal herb, is divided into two chemotypes based on the production of volatile bioactive compounds: the geo-authentic Maoshan chemotype (MSA, rich in atractylon and atractylodin) and the non-authentic Hubei chemotype (HBA, dominated by hinesol and β-eudesmol). However, the mechanisms underlying the differentiation of these chemotypes remain poorly understood. By sequencing analysis of wild and cultivated <i>A. lancea</i> samples from multiple provinces in China, we revealed that the genetic divergence into Maoshan-Dabie Mountains group (MA) and Qinling-Taihang Mountains group (SA) was occurred along altitudinal and climatic gradients, but the chemotype was not solely genetically determined. Interestingly, both MA and SA genotypes in the MSA-favorable or HBA-conducive soil could develop into the MSA or the HBA chemotype, respectively, indicating a role of the soil microbiome. Critically, specific rhizosphere microbiomes were indicated as key mediators-core in this process, including <i>Streptomyces</i> in MSA formation and <i>Paenibacillus</i> in HBA formation. Shared endophytic core genera, such as <i>Rhodococcus</i>, <i>Ralstonia</i>, <i>Sphingomonas</i>, and <i>Bradyrhizobium</i>, further contributed to this divergence through species-level functional variation. Using piecewise structural equation modeling, we further confirmed that altitude, climate, and soil properties directly or indirectly influenced the chemotype formation via genotype-microbiome interactions. Taken together, this study highlights the central role of soil and microbes in the chemotype differentiation of <i>A. lancea</i> and provides new insights into its underlying mechanisms. The regulatory role of microbes in the production of volatile bioactive compounds offers a theoretical foundation for the microbial breeding strategies to improve medicinal quality and clinical efficacy.