Contribution of active drug efflux to enhanced tolerance to new fungicides in the wheat pathogen Zymoseptoria tritici

Active drug efflux is a widespread resistance mechanism in the wheat pathogen Zymoseptoria tritici as in other plant pathogenic fungi, but also in human pathogens. Active drug efflux has been demonstrated using radioactively labeled fungicides or fluorescent compounds. However, both technologies do not easily allow testing efflux of molecules that are neither radioactive nor fluorescent. We therefore designed a miniaturized cellular test to quantify active drug efflux in Z. tritici that will permit rapid analyses in restricted reaction volumes of unlabeled molecules. We incubated isogenic strains of IPO323 with normal, enhanced or reduced efflux in 24-well-micro-titer plates along with a succinate dehydrogenase inhibitor (SDHI) fungicide known to be affected by active drug efflux, under previously determined conditions of temperature, light exposure and shaking. After a week of growth, we extracted the fungicide from the growth medium (extracellular), from the fungal cells (intracellular) and from both to determine the extracellular, intracellular and total fungicide concentrations respectively by LC/MS/MS analyses. We showed that the ratios between the intracellular quantity and the total quantity of the tested SDHI perfectly correlated to the determined EC50. This approach will allow to easily and rapidly assay molecules for active drug efflux.