2.8. Chrome Azurol S (CAS) Assay

To measure the total siderophore activity of each strain, the chrome azurol S (CAS) colorimetric method was used as described previously [18]. Briefly, 1 × 10⁶ conidia of each fungal strain were inoculated into ANM broth and culture at 25 °C for 2 days. The cells and culture supernatant were harvested after centrifugation at 10,000 rpm for 20 min. The cells were mechanically broken with 0.1 mm glass beads in a bead beater (Biospec, Bartlesville, OK, USA). Total protein concentration was determined in cell lysate and culture supernatant and then adjusted to 2 mg/mL. The siderophore activity in 2 mg of total protein was measured by adding 0.15 mM chrome azurol S, 0.015 mM FeCl₃, 1.5 mM hexadecyl trimethyl ammonium bromide (HDTMA) and 1 M piperazine (pH 5.6), determining the absorbance at 630 nm. An EDTA (Sigma-Aldrich, St. Louis, MO, USA) was used to generate a standard curve. Using this standard curve, the siderophore production of each fungal strain was calculated in terms of micromoles per gram of total protein.