A Rapid Colorimetric Microtiter Bioassay to Evaluate Fungicide Sensitivity Among Verticillium dahliae Isolates

Abstract

Management of Verticillium wilt relies on the adoption of integrated control strategies. The effects of long-term chemical use may be negated by the development of fungicide resistance. A rapid colorimetric bioassay was developed to evaluate sensitivity of V. dahliae isolates to differently acting fungicides. This assay capitalizes on the advantages of a 96-well microtiter plate format and the nontoxic cell viability dye, Alamar Blue (AB). Analysis of variance revealed that incubation time, spore density, and media type were important parameters that must be optimized for the AB assay. The effective linear range of the assay was dependent on incubation time and spore density. Survival of 107 spores/ml of each of 10 isolates in the presence of a range of serial dilutions of nine commercial fungicides was assessed. Effective concentrations at 50 and 90% inhibition of growth were calculated for each fungicide. A comparison of the percent growth inhibition for each fungicide at 0.6 mg/ml, as determined by AB and amended-agar assays, revealed a strong positive correlation for six of the fungicides. The optimized AB assay proved to be a rapid and reproducible method of testing the efficacy of fungicides with the option of deriving quantitative or qualitative data.