Siderophore Production

To measure the siderophore production by bacterial isolates, both qualitative and quantitative methods were used. The capacity of bacterial isolates to produce siderophores was evaluated using the universal Chrome azurol S (CAS) agar medium (Schwyn and Neilands, 1987). The pure freshly produced all bacterial isolates were dotted on Petri plates with CAS medium and cultured for 4–5 days at 30 ± 2°C. The formation of an orange zone (hydroxamate-type siderophore) or a purple zone (catechol-type siderophore) surrounding the bacterial colonies on the plates was used to determine siderophore production by endophytic bacteria.

Quantitative estimation (hydroxamate-type siderophore) was completed by taking the supernatant of bacterial cultures grown in a broth of LB medium (Hu and Xu, 2011). Screwcap tubes (20 mL) containing 5 mL of LB broth were autoclaved. Afterward, 10 μL of freshly grown bacterial suspension (≈10⁸ CFU mL^–1) was inoculated, and the uninoculated broth was maintained as the control. After incubation at 30 ± 2°C for 3 days, bacterial cultures were pelleted by centrifuging at 12,000 rpm for 10 min. at 5°C, and the clear supernatant was used to determine the production of siderophore. 0.5 mL supernatant of each bacterial isolate was mixed with 0.5 mL CAS solution, kept in dark condition for 20 min, and measured its optical density at 630 nm by SPARK^® multimode microplate reader (Model- SW Sparkctl. Magellan V2.2 STD 2PC, Austria). Payne (1993) method was used to determine siderophore production in percent siderophore unit (PSU).