A hyphal release-capture soil microcosm for recovering hyphosphere bacterial communities

Fungal hyphae form spatially confined interfaces in soil that mediate close associations with bacteria, collectively referred to as the hyphosphere. Despite its recognized ecological importance, experimental access to hyphosphere-associated microbial communities under realistic soil and plant-associated conditions has remained limited. Here, we present a soil-mimetic microcosm that enables controlled recovery of hyphosphere bacterial communities embedded within plant-associated soil. The system integrates field-derived soil, a native soil microbial inoculum, living cotton seedlings, and a spatially constrained fungal inoculum housed within sterile cell-strainer assemblies, permitting hyphal extension into soil while preserving a recoverable fungal-soil boundary. Using the soil-borne plant pathogen Fusarium oxysporum f. sp. vasinfectum as a model filamentous fungus, we show that the microcosm enables reproducible recovery of hypha-associated soil microaggregates containing physically attached bacterial cells. Full-length 16S rRNA profiling revealed pronounced reductions in bacterial richness and evenness in hyphosphere samples relative to bulk and rhizosphere soils (Shannon diversity, Kruskal-Wallis H = 15.25, P = 0.0016, with genus richness declining by 87% in bulk soil and 24% in rhizosphere soil contexts), consistent with recruitment of a restricted subset of the surrounding microbiota. Ordination analyses demonstrated clear compositional separation between soil and hyphosphere compartments (PERMANOVA F = 7.14, R 2 = 0.572, P = 0.001), with hyphosphere communities of bulk and rhizosphere origin converging to similar composition despite differing starting soils ( R 2 = 0.128, P = 0.307). Phylogenetic turnover analyses (βNTI) indicated phylogenetic structuring (exceeding the +2 threshold), whereas taxonomic analyses identified a conserved set of bacterial genera consistently associated with hyphae, alongside compartment-specific taxa influenced by soil and plant context. Together, these findings establish the novel hyphal release-and-capture microcosm as a reproducible, ecologically grounded platform for studying hyphosphere-associated bacterial communities in plant-associated soils. IMPORTANCE Soil fungi recruit distinct bacterial communities along their hyphae, forming a specialized microhabitat known as the hyphosphere. Despite its ecological importance, studying hyphosphere-associated bacteria under realistic plant-soil conditions has remained experimentally difficult. Here, we present a soil-mimetic hyphal release-capture microcosm that enables controlled reconstruction and recovery of bacterial communities assembled along fungal hyphae in intact plant-associated soils. Using the cotton wilt pathogen Fusarium oxysporum f. sp. vasinfectum , we show that fungal hyphae reproducibly recruit reduced and compositionally distinct bacterial assemblages from surrounding soils through strong deterministic selection. Because this pathogen is a regulated quarantine organism for which field inoculation is restricted, this system provides a tractable experimental platform for studying hyphosphere assembly under controlled yet ecologically relevant conditions. This approach provides new opportunities to investigate fungal-bacterial interactions, microbial community assembly, and plant-associated soil microbiomes at spatially resolved hyphal interfaces.