Methods for evaluating bacterial dispersal on hyphal networks

Abstract In heterogeneous environments, the hyphae of filamentous fungi and oomycetes can facilitate the dispersal of other microorganisms. The use of these “fungal highways” (FH) is regulated by both physical and biological factors with their interplay resulting in variable capabilities of different microbes to establish FH. Several devices have been developed to test the movement of bacteria across mycelium. However, these methods are usually time-consuming and cannot be applied at a large scale. In this study, we developed 3D‑printed experimental devices that physically separate two environments while allowing hyphal networks to act as bridges for bacterial movement. The final design allows for the simultaneous testing of up to 10 pairs and the inclusion of any culturing media. With these devices, we investigated how fungal–bacterial pairing, nutrient conditions, and inoculation strategies influence FH formation. Bacterial transport was limited in nutrient‑rich media but increased under poorer nutrient conditions, consistent with enhanced exploratory growth of the mycelium. Both cis‑ and trans‑inoculation supported FH formation, although bacterial arrival was delayed in the absence of co‑inoculation. The devices were used to demonstrate that transport of bacteria by FH was relevant for the colonization of a natural substrate. Finally, we established a novel in planta assay to evaluate FH formation during host colonization. This assay demonstrated that Fusarium graminearum can transport bacteria during wheat spike colonization. Together, these results provide accessible, scalable tools to study hyphal‑mediated bacterial dispersal and highlight the combined role of biological specificity and nutrient context in the establishment of FH.