4.6.4. Indole-3- Acetic Acid (IAA) Production

The bacterial isolates were screened for the production of the auxin IAA by using the Salkowski colorimetric assay as originally described by Bric et al. [82] and modified by Ribeiro et al. [83]. Isolates were cultured in 15-mL Falcon tubes containing 3 mL of Luria Bertani (LB) medium supplemented with tryptophan (1 mg mL₋₁) as an auxin precursor. Bacterial isolates were grown in a shaker (120 rpm) for 1 day at 28 °C. A 1-mL aliquot of bacterial cultures was then centrifuged at 9500× g for 2 min and 100 μL of supernatant were added to 96 micro-titre plate wells followed by the addition of 100 μL of Salkowski’s reagent (150 mL of 98% H₂SO₄, 7.5 mL of 0.5 M FeCl₃·6H₂O, and 250 mL distilled water) and the 96 micro-plate was incubated in the dark for 30 min at room temperature. Bacterial isolates producing IAA were characterized by the formation of a distinct red color [82]. An autoclaved bacterial culture was used as a negative control. The experiments were repeated twice, each with three replicates for each isolate.