Application of an Automated Quantitative Method to Determine Fungicide Resistance inBotrytis cinerea

An automated quantitative (AQ) assay used for measuring fungal growth with a microplate reader was compared with the linear growth method to determine fungicide resistance in Botrytis cinerea. The AQ assay uses absorbance in the range of 0.0-0.6 units as a measure of fungal biomass. This technique was successfully used to establish EC 50 values (the concentration of fungicide that reduces absorbance by half) to iprodione and a mixture (1:5) of carbendazim and diethofencarb in an economical and rapid way. The AQ assay used 100 times less medium than did the conventional method of measuring radial growth (RG) of mycelium on fungicide-amended medium, and up to 96 samples (one microplate) could be processed at once. The assay was also performed with conidia stored at -20 C in a 20% glycerol solution for 4 mo, and EC 50 values did not differ significantly from EC 50 values using fresh conidia. Growth inhibition was measured most accurately when the spore concentration in a well of the microplate was 10 or 20 spores per microliter. EC 50 values determined by the AQ assay were compared with EC 50 values obtained by the RG method, and both were positively correlated. Regression lines to predict EC 50 values by the RG method from AQ values were: logY = - 0.392 + 1.04 logX for iprodione, and logY = 0.742 + 1.38 logX for the mixture of carbendazim and diethofencarb where Y = EC 50 value determined by the RG method and X = EC 50 value determined by the AQ assay