Abstract
Fungicides are extensively used in agriculture to control fungal pathogens which are responsible for significant economic impact on plant yield and quality. The conventional antifungal screening techniques, such as water agar and 96-well plates, are based on laborious protocols and bulk analysis, restricting the analysis at the single spore level and are time consuming. In this study, we present a droplet-based microfluidic platform that enables antifungal analysis of single spores of filamentous fungus Alternaria alternata. A droplet-based viability assay was developed, allowing the germination and hyphal growth of single A. alternata spores within droplets. The viability was demonstrated over a period of 24 h and the antifungal screening was achieved using Kunshi/Tezuma as antifungal agent. The efficacy results of the droplet-based antifungal analysis were compared and validated with the results obtained from conventional protocols. The percentage inhibitions assessed by the droplet-based platform were equivalent with those obtained by the other two methods, and the Pearson correlation analysis showed high correlation between the three assays. Taken together, this droplet-based microfluidic platform 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.
Highlights
Fungicides are extensively used in agriculture to control fungal pathogens which are responsible for significant economic impact on plant yield and quality
We have successfully developed a droplet-based microfluidic platform to perform antifungal analysis on single encapsulated spores of phytopathogenic fungus, Alternaria alternata
This work relied on a series of key novel advances in the field of plant pathology and fungicide analysis methods, for instance: (1) the development of an on-chip method for single spore encapsulation, (2) the demonstration of a biocompatible droplet-based assay using A. alternata spore as a model system of filamentous fungi that survives up to 24 h inside a droplet, and (3) the ability to perform antifungal analysis using a microfluidics droplet-based system
Summary
Fungicides are extensively used in agriculture to control fungal pathogens which are responsible for significant economic impact on plant yield and quality. Droplet-based microfluidics has emerged as an increasingly interesting alternative to conventional microtiter plate approaches for enzymatic HT screening of fungi[22,23,24] These methods can provide an alternative approach for single spore e ncapsulation[25] and have the potential to become a powerful tool for large-scale antifungal analysis. A droplet-based approach tackling these challenges and using biocompatible reagents might hold a potential to progressively transition to single spore analysis for antifungal screening for routine assays, which could revolutionize how fungicides are developed and how fungicide resistance is controlled in fungi. We demonstrate the design and characterization of a droplet-based antifungal analysis platform with precise fluidic control, as a new benchmark for rigorous, reproducible, single spore encapsulation, and antifungal analysis
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