Plants have evolved numerous belowground strategies to capture nutrients. While root functional differentiation between absorptiontransportation has been inferred from order-based traits, the role of microbiomes in mediating this differentiation remains unclear.

We measured traits (anatomical, chemical,morphological) of lower(absorption-dominated)higher(transportation-dominated) roots of 37 herbaceous species (13 monocots24 dicots) in a temperate grassland. Furthermore, we employed high-throughput quantitative polymerase chain reaction16S rRNA gene sequencing to investigate bacterial nitrogen-transformation gene abundance, diversity,community assembly along the soil-root continuum (rhizosphere, rhizoplane,endosphere)analyzed their relationships with root traits.

Monocot roots exhibited greater bacterial diversitynitrogen-transformation gene abundances than dicots. Within dicots, lowerroots showed higher bacterial diversitynitrogen-transformation gene abundances than higherroots, a pattern not observed in monocots. Lowerroots, characterized by higher cortex proportion, facilitated the enrichment of diverse bacteriarecruitment of nitrogen-transformation microorganisms. These patterns were associated with a decrease in homogeneous selection from lowerto higherroots.

This study reveals the mechanisms of functional differences among herbaceous root orders from a microbiome perspective, offering further insightshow root–microbe interactions underpin nitrogen-transformation potential in terrestrial ecosystems.