Fungal Mycelia Growth Inhibition Assay

Fungal growth inhibition assays were performed following a similar protocol to Pedras et al.⁷ Spores of Botrytis cinerea SF1 or Alternaria brassicicola FSU218 were grown on PDA at room temperature in the dark for 3 days (B. cinerea) or 7 days (A. brassicicola). Serial dilutions of phytoalexins were made in DMSO and added to warm potato dextrose agar to final concentrations of 20, 50, 100, 200, and 500 μM and poured into 6-well plates (2 mL/well). The same procedure was used for pyrimethanil containing media, but with concentrations of 0.1, 1, 10, 25, 50, and 100 μM. 5 cm circular plugs of growing fungal mycelia were transferred to cooled plates and grown at room temperature in the dark for 24 h (B. cinerea) or 75 h (A. brassicicola). At this time the longest diameter of fungal growth was measured and compared to plugs grown on PDA with 1% DMSO to calculate percent growth inhibition. IC₅₀ values and significance were calculated from percent growth inhibition of all replicates using a nonlinear regression model in GraphPad Prism.