Fungal Growth Inhibition Assay

The peptides were tested for antifungal activity toward the filamentous fungi F. graminearum by performing hyphal growth inhibition assays according to the method of Bleackley et al. (2017) with slight modifications. Fungal isolates were grown under constant agitation on a carboxymethyl cellulose (CMC) sporulation medium (Cappellini and Peterson, 1965) at 170rpm and 25°C for 5–7days until spores were abundantly produced. Macroconidia were collected after centrifugation at 5000min⁻¹ and 4°C for 15min, resuspended in half-strength potato dextrose broth (PD Broth) culture medium and adjusted to ≈5 × 10⁴ spores/ml using a hemocytometer. Aliquots (90μl) of the spore suspension were incubated for 48h at 25°C in a 96-well microplate with filter–sterilized peptide solutions (10μl) at different concentrations in water. Germination of spores was evaluated by measuring the optical density at 595nm using a microplate reader Infinite M200 Pro (Tecan, Männedorf, Switzerland) after 0, 19, 24, 43, and 48h of incubation. Each test was performed in triplicate. The minimum inhibitory concentration (MIC) was determined as the peptide concentration that completely inhibits fungal growth. Inhibition data were analysed by one-way ANOVA and the mean differences were evaluated at p < 0.05 using the Tukey test. Statistical analyses were performed using the InfoStat software (Di Rienzo et al., 2018).

For the most active peptides, a time-to-kill experiment was performed amending half strength PD Broth media with the peptides at their MIC. Half strength PD Broth (5ml) was inoculated with macroconidia from F. graminearum SP1 to a final concentration of 1 × 10⁴ conidia/ml. Peptides were added at their MIC and the amended PD Broth was cultured for different periods: 0.5, 1, 3, and 6h. A growth control was performed by incubating the conidia with water instead of peptide at 25°C in the dark for 48h. After each time period, 100μl of the 5ml culture were added to 900μl of sterilized water. This dilution was vortexed for 10s and 100μl were plated onto three different half strength potato dextrose agar (PD Agar) plates and incubated for 3days at 25°C in the dark before the colonies were counted. Each incubation time was replicated twice.