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Microfluidic Approaches for High-Throughput Fungicide Screening and Sensitivity Testing

Periodic Reporting for period 1 - MAHT-FunSST (Microfluidic Approaches for High-Throughput Fungicide Screening and Sensitivity Testing)

Reporting period: 2019-04-24 to 2021-04-23

Fungicides are extensively used in agriculture to control fungal pathogens which are responsible for significant economic impact on plant yield and quality. Discovery and development of new fungicides face great challenges including a) screening of large libraries of antifungal candidates to evaluate their efficacy, phytotoxicity, and other possible effects, b) high cost of product development driven by the extensive studies, and c) complex in vitro and in vivo experiments. The conventional antifungal screening techniques, such as water ager and 96-well plates, are based on laborious protocols and bulk analysis, are time consuming and have closed system designs (no flow through reagents or culture medium). The classical approaches are also limiting in terms of single spore analysis and particular difficulties, such as handling and reproducibility can lead to considerable variability between observations within experimental runs. Therefore, a droplet-based microfluidic platform tackling these challenges was developed to progressively transition to single spore analysis for fungicide screening for routine assays.

The developed technology can be used not only in the context of any large-scale fungicide screening studies, but is also well-suited to analyze the mode of action of antifungal agents and resistance-development in fungi. The platform can be further extended for analysis of the molecular mechanisms of fungicides which play a crucial role in cellular mechanisms. A deeper understanding of the mode of action of fungicide will help to optimize their application, which in turn contributes to their successful use in food and feed production. The optimized fungicide application can reduce the cost of growers’ budget, lowers the risk of resistance development and pesticide residue in harvest and can positively impact the environmental and beneficial organisms.

The main objectives of this MSCA project were to establish a simple microfluidic device capable of (a) encapsulating single spores of fungi, (b) performing antifungal screening, and (c) quantifying gradient-based antifungal-dose response.
The droplet-based microfluidics fungicide screening platform developed during the project allows evaluation of the direct effect of antifungal agents on spore germination in a confined microenvironment, with high reproducibility and enabling the quantification of spore germination using widely accessible microscope image analysis. The design incorporates micron-sized traps that confine the encapsulated spore, allowing their individual visualization and assessment over time by bright-field microscopy. To validate the screening results using the microfluidic platform, the screening experiments with two conventional methods i.e. water agar (WA) and 96-microtiter plate, were also performed. In the present work, the design and characterization of a droplet-based fungicide screening platform with precise fluidic control was demonstrated, as a new benchmark for rigorous, reproducible, single spore analysis and fungicide screening.

Scientific results and fundamental microfluidic concepts were exploited and disseminated to a diversity of audiences in the form of detailed protocols and application notes (single spore encapsulation and fungicide screening using droplet microfluidics), a webinar, market survey, conference presentations, and manuscript (submitted for publication).
The droplet-based microfluidic platform for single spore encapsulation and fungicide screening provides a wide range of potential applications for the analysis of fungicide resistance development as well as combinatorial screening of other antimicrobial agents and even antagonistic fungi. The platform has a potential to progressively transition to single spore analysis for fungicide screening for routine assays, which could ultimately revolutionize how fungicides are developed and how fungicide resistance is controlled in fungi. This can provide new opportunities for the development of urgently needed novel fungicides and plant disease control strategies. The cost of fungicide development will be reduced while increasing the efficiency of fungicide screening, the resulting technology is foreseen to address the current economic burden related to fungicide development within the EU area.
Microfluidic setup for single spore encapsuilation using pressure-driven flow control from Elveflow.