4.5. Fungal Spore Germination Inhibition Assay

Spore suspensions (2 × 10³ spores) of C. gloeosporioides and F. oxysporum were sowed in 100 μL of PDY medium in 96-well flat-bottom plates. To obtain half maximal inhibitory concentration (IC₅₀) for each fungus, several concentrations of recombinant snakin were used: 50, 75, 100, 150, 200, and 300 μg/mL for C. gloeosporioides and 30, 50, and 100 μg/mL for F. oxysporum. Commercial fungicide Tecto60 was used as the positive control (30 μg/mL) and water and MBP (300 μg/mL) were used as negative controls. Plates were incubated at 28 °C for three days, and each 24 h, we measured the absorbance at 595 nm in a BioTek™ Epoch Microplate Spectrophotometer System at 24, 48, and 72 h. We used the average absorbance data of three replicas of each treatment to generate a concentration–response curve. Finally, the inhibitory activity on fungal spore germination was visualized by light microscopy at 72 h incubation.