The rhizosphere and hyphosphere differentially regulate microbiomes and nutrient cycling within soil aggregates in an ectomycorrhiza-dominated forest

• Microbiomes were aggregate-size specific and varied significantly in rhizosphere and hyphosphere soils. • Bacterial and fungal communities were regulated by aggregate size and rhizosphere effects, respectively. • Hyphosphere promoted C degradation gene abundance, while rhizosphere enhanced N and P cycling genes. • Microbial network complexities increased with decreasing aggregate sizes. • The core EcM genus Sebacina was consistently enriched in both the rhizosphere and hyphosphere. Plant roots and associated extraradical hyphae of ectomycorrhizal (EcM) fungi are known to harbor niche-specific microbiome and functionality in mobilizing soil carbon mobilization and nutrient cycling. However, the differences in soil microbiomes at the aggregate level between two important niches (rhizosphere versus hyphosphere) remain unclear. We sequenced microbial communities and quantified functional genes from soil adjacent to root tips (rhizosphere), soil adjacent to hyphal tips (hyphosphere), and bulk soil across three aggregate sizes in an EcM-dominant coniferous forest. The bacterial communities were influenced primarily by aggregate size, whereas the fungal communities were highly responsive to rhizosphere and hyphosphere effects. The abundance of microbial functional genes associated with carbon degradation and denitrification increased only in the hyphosphere but not in the rhizosphere. Both the rhizosphere and hyphosphere increased the abundance of genes involved in nitrogen and phosphorus cycling, such as those involved in nitrogen fixation, organic phosphorus mineralization, and inorganic phosphorus solubilization, but with varing intensities. In comparison, genes associated with ammoxidation decreased in both the rhizosphere and hyphosphere relative to those in the bulk soil. The complexity, connectivity, and compactness of bacterial networks were greater in the hyphosphere, whereas the rhizosphere shaped centralized fungal networks. With decreasing aggregate size, the microbial network complexity increased. The core EcM taxa Sebacina and Laccaria were prevalent in the hyphosphere, whereas Sebacina and Mortierella were persistently enriched in the rhizosphere. These findings suggest that the contrasting effects of rhizosphere and hyphosphere microbiomes and functional genes in soil aggregates and the importance of keystone EcM taxa should be considered when ecosystem C, N and P budgets are predicted under changing environments.