A versatile microfluidic platform measures hyphal interactions between Fusarium graminearum and Clonostachys rosea in real-time

Alejandro Gimeno,Claire E Stanley,Pilar Junier,Saskia Bindschedler,Stefanie S Schmieder,Zacharie Ngamenie,Florian Walder,Susanne Vogelgsang,Beat Keller,Ming-Hui Hsung

doi:10.1038/s42003-021-01767-1

Abstract

Routinely, fungal–fungal interactions (FFI) are studied on agar surfaces. However, this format restricts high-resolution dynamic imaging. To gain experimental access to FFI at the hyphal level in real-time, we developed a microfluidic platform, a FFI device. This device utilises microchannel geometry to enhance the visibility of hyphal growth and provides control channels to allow comparisons between localised and systemic effects. We demonstrate its function by investigating the FFI between the biological control agent (BCA) Clonostachys rosea and the plant pathogen Fusarium graminearum. Microscope image analyses confirm the inhibitory effect of the necrotrophic BCA and we show that a loss of fluorescence in parasitised hyphae of GFP-tagged F. graminearum coincides with the detection of GFP in mycelium of C. rosea. The versatility of our device to operate under both water-saturated and nutrient-rich as well as dry and nutrient-deficient conditions, coupled with its spatio-temporal output, opens new opportunities to study relationships between fungi.

Highlights

Fungal–fungal interactions (FFI) are studied on agar surfaces
We developed the fungal–fungal interactions (FFI) device to direct and constrain the growth of two fungal cultures (F1 and fungal inoculum 2 (F2)) towards one another, making it possible to study their interaction on the cellular level (Fig. 1)
The FFI device was constructed by bonding a layer of poly(dimethylsiloxane) (PDMS), having microchannels embossed into the surface at a depth of 10 μm, to a glass-bottomed Petri dish

Summary

Introduction

Fungal–fungal interactions (FFI) are studied on agar surfaces. this format restricts high-resolution dynamic imaging. We demonstrate its function by investigating the FFI between the biological control agent (BCA) Clonostachys rosea and the plant pathogen Fusarium graminearum. Filamentous fungi, characterised by their formation of hyphal networks, have been investigated widely on agar plates or in liquid cultures to observe their growth behaviour and genetic or metabolic responses in FFI7–10 These studies yield valuable information on the colony level, the emergence of microfluidic technology has brought new opportunities to study microbes at the cellular and intra-cellular levels. Difficulties associated with sampling using these traditional approaches can lead to considerable variability between observations within experimental runs[25] Considering these limitations, we developed a microfluidic platform to explore interactions between filamentous fungi at the cellular level. We provide new evidence on this FFI with a first insight into the dynamics of a potential uptake of green fluorescence protein (GFP) by C. rosea from hyphae of fluorescently tagged F. graminearum in a time-lapse experiment

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Conclusion