Monitoring the impact of confinement on hyphal penetration and fungal behavior

Abstract

Through their expansive mycelium network, soil fungi alter the physical arrangement and chemical composition of their local environment. This can significantly impact bacterial distribution and nutrient transport and can play a dramatic role in shaping the rhizosphere around a developing plant. However, direct observation and quantitation of such behaviors is extremely difficult due to the opacity and complex porosity of the soil microenvironment. In this study, we demonstrate the development and use of an engineered microhabitat to visualize fungal growth in response to varied levels of confinement. Microfluidics were fabricated using photolithography and conventional soft lithography, assembled onto glass slides, and prepared to accommodate fungal cultures. Selected fungal strains across three phyla (Ascomycota: Morchella sextalata, Fusarium falciforme; Mucoromycota: Linnemannia elongata, Podila minutissima, Benniella; Basidiomycota: Laccaria bicolor, and Serendipita sp.) were cultured within microhabitats and imaged using time-lapse microscopy to visualize development at the mycelial level. Fungal hyphae of each strain were imaged as they penetrated through microchannels with well-defined pore dimensions. The hyphal penetration rates through the microchannels were quantified via image analysis. Other behaviors, including differences in the degree of branching, peer movement, and tip strength were also recorded for each strain. Our results provide a repeatable and easy-to-use approach for culturing fungi within a microfluidics platform and for visualizing the impact of confinement on hyphal growth and other fungal behaviors pertinent to their remodeling of the underground environment.

Fig 1. Microfluidics design and its geometry.

(Left) Microhabitats consist of a central buffer region and two inoculation ports connected by a series of 15 microchannels. (Center) Optical micrographs show the entry region of the microchannels that connect the inoculation ports to the buffer channel. 5 and 10 μm channels were prepared with 50μm markers along their length. (Right) Channels were etched to a depth of 6 μm as shown in the scanning electron micrograph.

Fig 2

Fungal microfluidics setup (A) Fluidic devices bonded to a glass slide are inoculated with a plug of fungal agar sealed with a small PDMS plug. (B) The devices are then fixed to the bottom of a petri dish with scotch tape. Water or damp tissues are added around the devices, a cover is added, and the device is sealed with parafilm. (C) Devices are placed into a 3D-printed dish holder and imaged at set intervals in an inverted microscope for the duration of the experiment.

Fig 3

Characterization of fungi in engineered microhabitats (A) Fungal isolates inoculated on PDA plates. Mycelial network of fungal isolates entering microhabitat channels from open geometries (B) Representative images of mycelial characteristics from selected species.

Fig 4. Histogram plots of instantaneous growth rates.

Changes in instantaneous growth rates are evident and illustrate the impact of increasing confinement on fungal hyphal penetration rates.