DOI: https://doi.org/10.29363/nanoge.eimc.2021.005
Publication date: 5th July 2021
Clonostachys rosea is a filamentous fungi used as a bio-control agent (BCA) with natural fungicidal properties. While already widely applied in agriculture, many of the underlying metabolic mechanisms are still unknown. Studying filamentous fungi in high-throughput fashion using laboratory automation systems remains a challenging task due to their complex life cycle with multicellular states, and hyphal growth. Microfluidics, especially droplet-based systems, is a robust approach to study single cell organisms or conidia (fungal spores) since they allow the study of their germination behavior in an in-vivo like environment and the identification of enzymes secreted by the filamentous fungi. However, current microfluidic methods are not well adapted to host filamentous fungi due to their hyphal growth, which can cause the droplets to easily burst after long droplet incubation times and under high-field dielectrophoretic (DEP)sorting conditions (e.g., 1.3kVpp). Moreover, their polydispersity in droplet size after incubation also requires careful tuning of the sorting conditions.[1]
In this work, we have developed a low voltage electrostatics based co-planar binary sorter that reliably sorts a C. rosea filamentous fungi droplet library (> 87.6% +- 2.7) using a field as low as 12.5 VRMS. The combination of a low voltage sorter, the use of an optical fiber based detection system, a portable mini-spectrometer, and open-source software suite drastically reduce the footprint of this system compared to gold-standard techniques.[2] Using our system, a mutant library of C. rosea was incubated for at least 36 h under several test conditions and subsequently screened for production of chitinases and b-1,3-glucanases. After sorting, we will test recovered temperature resistant BCA candidates for their activity against a plant pathogen (Botrytis cinerea). Since both of these enzymes are responsible for plant pathogen cell wall breakdown, we could measure reduction in growth of B. cinerea, while the wild type C. rosea showed less growth and activity under the test conditions.[3] We anticipate this accessible system adds to the available toolkit for researchers to study and screen other relevant industrial or agricultural filamentous fungi.
We thank Lallemand Inc. for donating the C.rosea strain. We thank the Canadian Govt. Dept. of Agriculture and Agri-Food Canada Canadian Collection of Fungal Cultures (DAOMC) for donating plant pathogen strains. We thank the Natural Sciences and Engineering Research Council (NSERC), the Fonds de Recherche Nature et technologies (FRQNT), and the Canadian Foundation of Innovation (CFI) for funding. KS thanks FRQNT B2Xs & NSERC CREATE SynBioApps program for graduate funding. KS., CLA. thank Concordia University Dept. of Electrical and Computer Engineering for FRS Funding and the Biology Department for academic resources. SCCS thanks Concordia for a University Research Chair.
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