Deciphering bacterial community composition and function at critical interfaces of plant-arbuscular mycorrhizal fungi-bacterial holobiont

Arbuscular mycorrhizal (AM) fungi connect plant roots and soil bacteria, forming a cross-kingdom holobiont driven by plant-derived carbon flux and soil-derived nutrient flux. This holobiont encompasses not only roots and rhizosphere but also interfaces expanded by slender AM fungal hyphae. Our understanding of the microbiomes across these interconnected interfaces remains limited and fragmented. We used a split-root microcosm to inoculate the same maize root system with three AM fungal species, establishing a simplified holobiont. Amplicon sequencing, Biolog-ECO plates, and 13CO₂ labelling revealed the bacterial diversity and community-level physiological profiles (CLPPs). Bacterial communities colonizing non-mycorrhizal roots, mycorrhizal roots, and hyphae exhibited distinct structures and CLPPs. AM fungal species significantly influenced these bacterial communities, particularly in the mycorrhizosphere and hyphosphere, where notable changes occurred in key nutrient-cycling groups, such as phosphate solubilizers and nitrogen fixers. The diversity of the hyphosphere microbiome was closely aligned with fungal phylogeny. Additionally, a stable core microbiome persisted across all interfaces within the plant-AM fungi-bacterial holobiont, with key taxa such as Pseudomonas and Bacillus harboring the P-mobilizing genes encoding quinoprotein glucose dehydrogenase (gcd) and β-propeller phytase (bpp), highlighting their functional importance in nutrient cycling. Our study provides a comprehensive and precise hyphal-scale characterization of microbial communities across key interfaces, offering detailed insights into plant-microbial dynamics driving nutrient cycling and ecosystem functioning.