Production of siderophores was determined by plate assay using chrome azurol S (CAS) (44 (link), 84 (link)) as previously described by Zajc et al. (19 , 35 (link)). CAS agar was prepared as follows: first, the mixture of 10 mL of 1 mM FeCl3 × 6H2O (Sigma-Aldrich, USA) in 10 mM HCl (Merck, Germany), 50 mL of CAS solution (Acros Organics, USA), and 40 mL hexadecyltrimethylammonium bromide (CTAB) (Sigma-Aldrich, USA) was prepared. Then, the medium of 30.24 g piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) (Acros Organics, USA), 12 g 50% (wt/wt) NaOH (Sigma-Aldrich, USA), 20 g malt extract (Biolife, Italy), 1 g peptone (Merck, Germany), 20 g glucose, and 20 g agar in 900 mL deionized water was prepared. Both solutions were autoclaved separately and carefully combined after cooling. Plates were inoculated with 5 µL of the cell suspension or with one mycelial plug and incubated at 25°C for 14 days. After the incubation, yellow, orange, or pink discoloration around the colonies was observed and regarded as production of siderophores.
The relative amount of produced siderophore was calculated according to Zajc et al. (35 (link)):
The relative amount of produced siderophore was calculated according to Zajc et al. (35 (link)):
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