Mycelial Effects on Phage Retention during Transport in a Microfluidic Platform

Abstract

Phages (i.e. viruses that infect bacteria) have been considered as good tracers for the hydrological transport of colloids and (pathogenic) viruses. Little, however, is known about interactions of phages with (fungal) mycelia as the prevalent soil microbial biomass. Forming extensive and dense networks, mycelia provide significant surfaces for phage-hyphal interactions. Here, we for the first time quantified the mycelial retention of phages in a microfluidic platform that allowed for defined fluid exchange around hyphae. Two common lytic tracer phages (Escherichia coli phage T4 and marine phage PSA-HS2) and two mycelia of differing surface properties (Coprionopsis cinerea, Pythium ultimum) were employed. Phage-hyphal interaction energies were approximated by the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) approach of colloidal interaction. Our data show initial hyphal retention of phages of up to ≈ 4 × 10 7 PFU mm-2 (≈ 2550 PFU mm-2 s-1) with a retention efficiency depending on the hyphal and, to a lesser extent, the phage surface properties. Experimental data were supported by XDLVO calculations, which revealed the highest attractive forces for the interaction between hydrophobic T4 phages and hydrophobic C. cinerea surfaces. Our data suggest that mycelia may be relevant for the retention of phages in the subsurface and need to be considered in subsurface phage tracer studies. Mycelia-phage interactions may further be exploited for the development of novel strategies to reduce or hinder the transport of undesirable (bio-)colloidal entities in environmental filter-systems.

Platforms for High-Throughput Screening and Force Measurements on Fungi and Oomycetes

“…Compared to a conventional HTS platform, LOC technology facilitates the screening at the single hypha or spore level, especially for the high-resolution monitoring of morphogenesis. While cultivation microchamber platforms [33,34,[36][37][38][39][40] are easy to fabricate and operate, they suffer from control issues of spore distribution inside the chambers and cell immobilization during media or reagent exchange. Microfluidic droplet techniques [42,43] address some of these problems and allow for high-throughput detection and screening.…”

“…Motile zoospores swam randomly in the microchannels and then started to settle close to the root tip up to 2 h after inoculation. In other more integrated examples, microfluidic platforms were used to study the interactions of phages with fungal mycelia [39] and the defense response to spatially confined predation by a fungivorous nematode [40].…”

Mycelia-Assisted Isolation of Non-Host Bacteria Able to Co-Transport Phages

“…However, much less is known about their ability to co-transport phages. Although often enabling bacterial dispersal through water-unsaturated zones, mycelia can retain the waterborne transport of phages [40,41]. Phage adsorption to (bio-)surfaces is mainly driven by the properties (e.g., hydrophobicity [40,42,43], surface charge [44]) of the phages and their interacting surfaces [20,40,45,46], phage morphology, and size [47] as well as environmental factors [48,49].…”

“…It is based on the observation that phages can interact with non-host bacteria and that phage-carrying bacteria can move along mycelia out of soil and develop separable colonies in plaques formed by their co-transported phages in host biofilms. We chose two model phages of differing surface hydrophobicity (i.e., hydrophobic Escherichia virus T4 (T4) and hydrophilic Pseudoalteromonas phage HS2 (HS2)), as hydrophobic phages are often found to be more adsorptive to (bio-)surfaces than hydrophilic phages [40,43]. To validate our system, we first mixed T4 or HS2 with gfp-labeled Pseudomonas putida KT2440 as inoculum and screened for plaques containing green fluorescent colonies that formed during the mycelia-assisted colonization of phage-carrying P. putida KT2440 in biofilms of the respective phages' host strains.…”

Phage co-transport with hyphal-riding bacteria fuels bacterial invasion in water-unsaturated microbial ecosystems

“…They thereby serve as pathways for bacteria to efficiently disperse (fungal highways [5,6]), forage [7] and colonize new habitats [8,9]. Hyphae however may reduce dispersal of intrinsically non-motile yet abundant ( >10 8 g -1 soil [10]) soil virus-like particles as they have been shown to retain waterborne phages [11,12]. Considering the slow diffusion (~ 0.034 mm/d [13]) and enhanced inactivation at dry conditions [14,15], transport of phages seems particularly restricted in water-unsaturated habitats.…”