Potato dextrose broth (pdb)

Cultures of strains Fo4287, Fo5276, Fo47, Foii5, and Fo32931 were grown in potato dextrose broth (Becton, Dickinson and Company, Sparks, MD), strain MN25 was grown on potato dextrose agar (Becton, Dickinson and Company, Sparks, MD) for 5 days, The strains were spotted into the center of plates containing either minimal medium alone (74 ) or minimal medium supplemented with rapamycin (final concentration of 50 ng/ml). The plates were stored at 28° for 48 h and then transferred to room temperature. After the transfer to room temperature, the diameter of each colony was measured at 24-h intervals. Growth rate was determined as the average increase in size (in millimeters) per 24 h. Reduction in growth rate was calculated as follows: 100 − [(growth rate on rapamycin-containing plate/growth rate on control plate) × 100]. In order to generate a range of error, all nine comparisons of growth rate between the three control plates and three rapamycin-containing plates were used to calculate reduction in growth. All species/condition plates were done in triplicate.

Potato dextrose broth (PDB) and potato dextrose agar (PDA) came from Becton, Dickinson, and Company (Franklin Lakes, NJ, USA). Tween^® 20 (CAS No. 9005-64-5) was bought from Sigma Aldrich Quimica S.A. (Madrid, Spain).
To conduct the in vitro experiments, we used certain fungicides as positive controls. These included Ortiva^® (azoxystrobin 25%; Syngenta, Basel, Switzerland), Vondozeb^® (mancozeb 75%; UPL Iberia, Barcelona, Spain), and Fesil^® (fosetyl-Al 80%; Bayer, Leverkusen, Germany), kindly provided by the Plant Health and Certification Center (CSCV) of the Gobierno de Aragón.
The fungal isolates of A. alternata (CRD 41/37/2019), C. coccodes (CRD 246/190), and R. solani (CRD 207/99) were obtained from the Regional Diagnostic Center of Aldearrubia (Junta de Castilla y León). S. sclerotiorum (MYC-799) and V. dahliae (MYC-1134) were acquired from the Centre for Agrifood Research and Technology of Aragon (CITA). Additionally, F. oxysporum f. sp. lycopersici (CECT 2866) was obtained from the Spanish Type Culture Collection (Valencia, Spain).

We used 17 strains isolated from bread wheat (T. aestivum L.), durum wheat (T. turgidum ssp. durum (Desf.) Husn.), or Agropyron sp. registered as P. tritici-repentis at the Research Center of Genetic Resources, National Agriculture and Food Research Organization (NARO) (Table 1). Each strain was grown in potato dextrose broth (Becton, Dickinson and Co., Franklin Lake, NJ, USA), and genomic DNA was extracted in DNAs-ici-F extraction buffer (Rizo Inc., Tsukuba, Japan).

For the antifungal susceptibility assay, the yeast Candida spp. was sown with disposable handles in Petri dishes (90 × 15 mm) containing 65 g/L of Sabouraud dextrose agar (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), and the plates were incubated at 37 °C for 48 h. Candida spp. isolates were cultured in 5 mL of 25 g/L of PDB medium (Potato Dextrose Broth, Becton, Dickinson and Company), at 37 °C for 18 h in a shaker model New Brunswick™ Innova^® 43 (VWR International LLC, Radnor, PA, USA). The in vitro antifungal activity of crotamine and other native peptides was evaluated essentially as described in Yamane et al. [15 (link)]. Crotamine was tested in the concentrations of 10, 20, 40, 80, and 160 µM, while other natural peptides were tested in a single concentration (1 mM each), as also described in the Figure 1 legend. These native peptides were added in a 96-well plate containing the isolates of Candida spp. (about 2.5–5.0 × 10³ cells/100 µL) in PDB medium, before incubation at 37 °C for 24 h. The Candida growth rate was determined by measurements at 595 nm using a plate reader SpectraMax (Molecular Devices LLC, San Jose, CA, USA). Briefly, the inhibition of growth was calculated by subtracting the density of the strain in the presence of the peptides studied from the maximum density value of this strain growth in the absence of peptides.

The non-inducing PDB-BD medium (Potato Dextrose Broth) came from Becton, Dickinson and Company (NJ, USA). Leucinostatin-inducing PDB medium was prepared in the lab. Briefly, 200 g of potatoes were boiled for 30 min, and then 20 g of glucose were dissolved into the filtrate and diluted to 1 L. The rye agar medium contained 50 g of crushed rye, 20 g of sucrose and 15 g of agar per liter. PDB cultures with 1×10⁵ conidia per mL of PLBJ-1 were grown at 28°C on a shaker at 150 rpm for 8 days before DNA/RNA isolation.

Antifungal assays were performed as described in (Bleackley et al., 2014 (link)). S. cerevisiae cells were prepared by diluting to an OD₆₀₀ = 0.01 and C. albicans cells were diluted to OD₆₀₀ = 0.0002 in ½ strength potato dextrose broth (1/2 PDB) (Becton Dickinson). Assays investigating the combined effect of chemical cell wall synthesis inhibitors with defensins were performed as described in (Bleackley et al., 2017 ). Synergy was assessed using the fractional inhibitory concentration (FIC) equation. $\mathit{FIC}=\left(\frac{{\mathit{MIC}}_{A}combination}{{\mathit{MIC}}_{A}alone}\right)+\left(\frac{{\mathit{MIC}}_{B}combination}{{\mathit{MIC}}_{B}alone}\right)$
Synergy is defined as an FIC value of less than 0.5. Assays investigating the activity of defensins in salt were set up using the same checkerboard method as the synergy assays but with NaCl in place of one of the antifungal molecules.