Antifungal Activity Assays

Growth inhibition assays of Fusarium proliferatum were in 96-well flat-bottom microtiter plates as previously described, with minor changes (López-García et al., 2015). Basically, 50 μl of fungal conidia (5 × 10⁴ conidia/ml), in 1/10 diluted potato dextrose broth (PDB) containing 0.02% (w/v) chloramphenicol, were mixed in each well with 50 μl of LD fractions. The Ficoll buffer II of LD fractions was exchanged with sterile deionized H₂O containing 0.02% Tween-20 using Zeba^TM Spin Desalting columns (Thermo Fisher Scientific, Spain). Samples were prepared in triplicate. Plates were incubated with agitation for 72 h at 28°C. Fungal growth was monitored every 24 h by measuring the OD₆₀₀ using a Spectramax M3 reader (Molecular Devices, United States), and mean values and standard deviation (SD) were calculated. Experiments were repeated twice with similar results.