Host and nonhost bacteria support bacteriophage dissemination along mycelia and abiotic dispersal networks.

Bacteriophages play a crucial role in shaping bacterial communities, yet the mechanisms by which nonmotile bacteriophages interact with their hosts remain poorly understood. This knowledge gap is especially pronounced in structured environments like soil, where spatial constraints and air-filled zones hinder aqueous diffusion. In soil, hyphae of filamentous microorganisms form a network of 'fungal highways' (FHs) that facilitate the dispersal of other microorganisms. We propose that FHs also promote bacteriophage dissemination. Viral particles can diffuse in liquid films surrounding hyphae or be transported by infectable (host) or uninfectable (nonhost) bacterial carriers coexisting on FH networks. To test this, two bacteriophages that infect Pseudomonas putida DSM291 (host) but not KT2440 (nonhost) were used. In the absence of carriers, bacteriophages showed limited diffusion on 3D-printed abiotic networks, but diffusion was significantly improved in Pythium ultimum-formed FHs when the number of connecting hyphae exceeded 20. Transport by both host and nonhost carriers enhanced bacteriophage dissemination. Host carriers were five times more effective in transporting bacteriophages, particularly in FHs with over 30 connecting hyphae. This study enhances our understanding of bacteriophage dissemination in nonsaturated environments like soils, highlighting the importance of biotic networks and bacterial hosts in facilitating this process.