4.5.4. Antifungal Activity in vitro

The antifungal activity was made by a microdilution technique according to a previous report [48], with some modifications. Briefly, extracts were diluted with 100 µL of sterile PDB to obtain different doses and were added into a sterile 96-well microplate. Then, 100 µL of the spore’s suspension of R. stolonifer or F. oxysporum at a concentration of 10⁴ spores/mL, was added. Fungal sporulation was monitored by changes in the optical density (OD) in fully automatic microplate lector (BIOBASE-EL 10A, Jinan, SHG, China) at 530 nm during 36 h (12 h intervals, at an incubation temperature of 25 ± 2 °C). A positive control was prepared by mixing 100 µL of sterile PDB with 100 µL of spore suspension. The percentage of growth inhibition (%) was calculated by the following equation:

where ODsample represents the optical density of each treatment and ODcontrol represents the optical density of the control. All treatments were replicated four times. The inhibition results were used to estimate the minimum inhibitory concentration (MIC) of extract that causes a 50% and 90% of reduction in fungal growth (MIC₅₀ and MIC₉₀, respectively).