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

We measured traits (anatomical, chemical, and morphological) of lower order (absorption-dominated) and higher order (transportation-dominated) roots of 37 herbaceous species (13 monocots and 24 dicots) in a temperate grassland. Furthermore, we employed high-throughput quantitative polymerase chain reaction and 16S rRNA gene sequencing to investigate bacterial nitrogen-transformation gene abundance, diversity, and community assembly along the soil-root continuum (rhizosphere, rhizoplane, and endosphere) and analyzed their relationships with root traits.

Monocot roots exhibited greater bacterial diversity and nitrogen-transformation gene abundances than dicots. Within dicots, lower order roots showed higher bacterial diversity and nitrogen-transformation gene abundances than higher order roots, a pattern not observed in monocots. Lower order roots, characterized by higher cortex proportion, facilitated the enrichment of diverse bacteria and recruitment of nitrogen-transformation microorganisms. These patterns were associated with a decrease in homogeneous selection from lower order to higher order roots.

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