1260 uplc

A crude methanol-extract of A. zeylanica leaf was provided in lyophilized form from the University of Mississippi National Center for Natural Products Research (NCNPR, University, MS) and was stored in the dark at 4 °C in a vacuum chamber. Dried extract was dissolved in 5% acetonitrile in water to a final concentration of 0.1 mg/ml. Sample (1 μl for MS, 3 μl for MS/MS) was injected into an Agilent 1260 UPLC and chromatographed on an Agilent Poroshell 120 EC-18 column (3.0 × 50 mm, 2.7-micron particle size). Solvent A was 0.1% formic acid in water, and solvent B was 0.1% formic acid in acetonitrile. The initial solvent was 5% B with a 3 min hold. The solvent mix was then programmed to 30% B at 7 min, then to 95%B at 10 min. Column effluent was analyzed on an Agilent 6520 QTof, high resolution mass spectrometer. Sample was analyzed in positive ESI mode with a capillary voltage of 4000 V. Compounds were identified from exact mass and MS/MS spectra by comparison with published data [9] , [24] (link).

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.