2.6. Indole-3-Acetic Acid (IAA) and ACC Deaminase Production

The quantification method for IAA of the endophytes was adopted as previously described [22]. To measure the production of IAA, bacterial isolates were inoculated into 0.5 mg L-tryptophan/mL containing medium and at 37 °C with continuous shaking at 125 rpm for 48 h, as described [23]. Then, the 2 mL culture was centrifuged at 15,000 rpm for 1 min, and a 1 mL aliquot of the supernatant was mixed with 2 mL of Salkowski’s reagent, incubated for 20 min in darkness at room temperature. The absorbance was measured on a spectrophotometer at 530 nm, and the concentration was determined using a standard curve of pure IAA, as previously described [22].

The ACC deaminase activity of the chlorpyrifos-degrading bacteria was determined according to the modified methods [24,25,26], which measure the amount of α-ketobutyrate produced upon the hydrolysis of ACC. The endophytic bacterial strains were separately grown in tryptic soy broth medium (TSB) for 18 h at 28 °C to determine the ACC deaminase activity as described in our study [21]. The cell suspension without ACC was used as a negative control, and the one with (NH₄)₂SO₄ (0.2% w/v) was used as a positive control. The number of μmol of α-ketobutyrate produced by this reaction was determined by comparing the absorbance at 540 nm of a sample to a standard curve of α-ketobutyrate ranging between 10 and 200 μmol [24,26].