3.4. Antifungal Bioassay

All synthesized compounds 1–20 were screened for their in vitro antifungal activities against four fungi, including R. solani, F. oxysporum, A. tenuissima, and A. solani. by the mycelium growth rate method. All screened compounds were dissolved in acetone (1 mL), and the solutions were diluted with aqueous 1% Tween 60, and then were added to sterile potato dextrose agar (PDA, 49 mL). For primary screening, the compounds were tested at a concentration of 50 mg/L. All fungi species were cultivated in PDA at 28 ± 1 °C, for 2 days, to produce new mycelium before the antifungal assays were conducted. Next, mycelia dishes of about 8 mm diameter were cut from the culture medium and were picked up with a sterilized inoculating needle and inoculated in the center of the PDA plates. The inoculated plates were incubated at 28 ± 1 °C, for 24 h. Acetone in sterile aqueous 1% Tween 60 served as the negative control, whereas thifluzamide served as the positive control. Each sample was screened in three replicates and each colony diameter of the three replicates was measured four times by the cross-bracketing method. The data was statistically analyzed and the corrected inhibitory rates (I) were calculated using the following formula: I (%) = [(C-T)/(C-8 mm)] × 100, where C represents the average diameter of mycelia in the blank test, T represents the average diameter of mycelia on treated PDA media. The results of the antifungal tests are summarized in Table 1 and Table 3, and the EC₅₀ values of some compounds were calculated and are listed in Table 2.