Clonal integration drives divergent assembly of rhizosphere bacteria and fungi in Hydrocotyle vulgaris under cadmium heterogeneity.

Clonal plants enhance their fitness in heterogeneous environments through clonal integration, which enables resource sharing among interconnected ramets. However, how clonal integration shapes rhizosphere microbiome assembly and function remains poorly understood. In this study, we constructed patchy heterogeneous soils with cadmium-polluted and unpolluted substrates to simulate fragmented polluted habitats. Apical ramets were grown in cadmium-polluted soil and basal positions were cultivated in unpolluted soil, with stolons left either intact or severed. Rhizosphere and root-zone soils were sampled, and bacterial and fungal communities were characterized via 16 S rRNA and ITS amplicon sequencing. Our results demonstrated that stolon integration significantly altered rhizosphere microbial composition, with elevated relative abundances of Acidobacteriota, Actinobacteriota and Ascomycota and a reduction in Firmicutes. Beneficial genera Streptomyces and Savoryella were enriched under intact stolon treatments. Bacterial α-richness (Chao1) was significantly higher in the rhizosphere of stolon-intact plants, especially at the basal positions, whereas fungal α- richness showed no significant difference among treatments. Beta-diversity analysis revealed that bacterial communities were more sensitive to stolon integration than fungal communities, with fungal community structural shifts observed only in the basal rhizosphere. Amplicon sequence variant (ASV) analysis showed that intact stolons increased unique bacterial ASVs but reduced unique fungal ASVs in the basal rhizosphere. Collectively, these findings demonstrate that clonal integration differentially regulates the assembly of bacterial and fungal communities in the rhizosphere. This study highlights the distinct ecological roles of bacteria and fungi in plant-soil feedback and emphasizes multi-kingdom assessments of clonal integration under heterogeneous Cd condition.