Quantifying Indole-3-Acetic Acid Production

Indole-3-acetic acid production was examined for the identified bacteria using the method of Yan et al. (2018) (link). Tryptophan was formulated into a 2.5 mg ml⁻¹ solution, filtered through a 0.22-μm Millipore filter for sterilization and added to nitrogen-containing liquid medium to a final tryptophan concentration of 0.5 mg ml⁻¹. The bacteria were inoculated into the above solution according to 1% of the inoculum and were shaken at 28 °C for 48–72 h. Centrifugation was performed for 15 min, and the supernatant was added to a C18 extraction column to extract IAA. Afterwards, IAA was eluted with 100% methanol, and the final 0.5 ml extract was obtained as the sample. Chromatographic separation was performed on a reverse-phase column (Phenomenex Kinetex® XB-C18 2.6 μm 4.6 × 100 mm) in a liquid chromatography system (Agilent 1,260 UPLC) equipped with an ultraviolet detector monitoring the wavelength of 261 nm. The injection volume was 5 μl at a flow rate of 0.4 ml min⁻¹ with a mobile phase composed of solvent A (5 mM ammonium formate in 0.1% formic acid) and solvent B (100% methanol) for separation in gradientmode.