Spatial heterogeneity of the microbiota in Cypripedium franchetii and Its correlation with organ-specific metabolomes

Introduction Cypripedium franchetii , a plant of ornamental and medicinal value, is designated as a Grade II Protected Species in China. The C. franchetii population inhabiting the fragile ecosystem at Galongla Pass exhibits unclear patterns in microbial composition across rhizosphere soil, root, stem, and leaf tissues, as well as metabolite distribution and plant tissue-specific microbe-metabolite relationships. Methods This study focused on C. franchetii , a plant growing in the fragile ecosystem of Galongla Pass in Tibet. We employed an integrated approach combining high-throughput amplicon sequencing (targeting bacterial 16S rRNA genes and fungal ITS regions) and untargeted metabolomics (LC-MS/MS) to systematically analyze the microbial community structure and metabolite distribution in its rhizosphere soil and root, stem, and leaf tissues. We further explored the associations between endophytic microorganisms and metabolites within plant tissues. Results The results revealed significant differences in microbial composition across plant compartments: distinct variations were observed between rhizosphere soil and plant tissues, while stem and leaf microbial communities exhibited greater similarity. At the phylum level, Pseudomonadota dominated among bacteria, while Basidiomycota and Ascomycota were the predominant fungal phyla. At the genus level, dominant taxa showed tissue specificity: Cronobacter and Lactobacillus were dominant bacterial genera in roots, whereas Acinetobacter dominated in stems, and Acinetobacter and Agrobacterium were prominent in leaves. For fungi, Tulasnella was the dominant genus in rhizosphere soil and roots, while Dioszegia prevailed in stems and leaves. Metabolite analysis indicated significant differences in metabolic profiles among tissues, with stem and leaf metabolite compositions being relatively similar. Correlation analysis further revealed statistically significant correlations between differential microorganisms and differential metabolites in roots and stems, identifying 31 microbial genera significantly correlated with 48 high-abundance metabolites. Discussion This study systematically unveils the tissue-specific microecological and metabolic characteristics of C. franchetii during its post-flowering nutrient accumulation phase. Key findings include: microbial community assembly involves cooperative mechanisms between core conserved taxa (e.g., Tulasnella ) and habitat-specific taxa; microbial diversity exhibits a gradient decline from the rhizosphere into plant tissues accompanied by functional group succession; and extensive yet specific potential interaction networks (based on statistical covariation) exist between microorganisms and host metabolites, indicating potential microbial involvement in regulating plant secondary metabolism. These findings not only provide guidance for the conservation of C. franchetii (requiring consideration of both core symbiotic and habitat-specific taxa) and constructing synthetic microbial communities during artificial propagation, but also offer a new theoretical basis for the targeted regulation of medicinal active ingredient synthesis through the microbiome.