Background <p>The identification of complex spatial patterns of microbial communities in relation to their ecological niches is fundamental to understanding the mechanisms of ecological interactions among diverse organisms. This study introduces a novel three-dimensional (3D) sampling approach to examine the spatial dynamics of microbial populations and niche differentiation influenced by plant-mediated effects in natural ecosystem.</p> Results <p>Microbial communities across horizontal and vertical dimensions were systematically mapped, and we found that the total microbial diversity, particularly among eukaryotes, increased more than ten-fold compared to that obtained via single-grid sampling, emphasizing the importance of spatial heterogeneity in shaping microbial dynamics. Moreover, the 3D framework enabled us to identify taxa specifically associated with particular plants, offering insights into plant–microbe interactions, pathogen prevalence, and ecological consequences of plant-driven effects on local communities.</p> Conclusions <p>Collectively, these findings demonstrate that 3D sampling approach provides a reproducible and scalable methodology for investigating microbial spatial heterogeneity, pathogen ecology, and niche differentiation in natural environments.</p>