Droplet-based microfluidics platform for antifungal analysis against filamentous fungi

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.

Highly flexible and accurate serial picoinjection in droplets by combined pressure and flow rate control

“…Our picoinjection methodology is complementary to these systems since combinations of reagents are added only after droplet formation, which enables to test concentration gradients of reagents on cells or reactions that require a preceding droplet incubation step. Applications of interest might include the study of components that affect hyphae in filamentous fungi, which first need to be incubated to allow hyphal growth, 47 to optimize assay component concentrations for the detection of secreted cell products such as cytokines or antibodies, 48 to find the optimal concentration of a component in multistep chemical synthesis, 29 or other, multistep protocols. Although combinations of reagents can also be added to incubated droplets by fusing them with preformed droplet libraries, 15 our method provides a higher degree of flexibility since for example the length of the concentration sweep can be adjusted ( e.g.…”

Droplet microfluidics for single‐molecule and single‐cell analysis in research, diagnosis, and therapy

“…Antifungal analysis has been performed after the single spore encapsulation of Alternaria alternata, a filamentous fungus capable of surviving for up to 24 h inside droplets. 44 Analysis of microalgae capable of producing oils and biofuels can be improved by controlling the growth and oil content of the cultivated strains. The recent adaptation of droplet microfluidics enables investigations with a single-cell resolution of both factors, serving as a high-throughput tool for analyzing oil production by microalgae.…”

“…The detection of fungi and bacteria in environmental samples has improved compared with that using traditional assays, thereby improving resistance studies. Antifungal analysis has been performed after the single spore encapsulation of Alternaria alternata , a filamentous fungus capable of surviving for up to 24 h inside droplets 44 . Analysis of microalgae capable of producing oils and biofuels can be improved by controlling the growth and oil content of the cultivated strains.…”

Development and Validation of a HTS Platform for the Discovery of New Antifungal Agents against Four Relevant Fungal Phytopathogens

“…Plant pathogens are one of the major problems affecting agriculture worldwide. They are responsible for the loss of one third of the crops produced annually [ 1 , 2 ]. Fungal phytopathogens are widely distributed and can impose plant diseases on a large variety of plants, including cereals, vegetables, and fruit.…”