Waters 486 detector

Quantification of IAA was performed by spectrophotometry [23] (link) and confirmed by HPLC according to Torres et al. [15] (link). Briefly, aliquots of 500 µl of bacterial culture were centrifuged at 11.300 x g for 10 min. Subsequently, 250 µl of supernatant was filtered (0.2 µm), mixed with 250 µl of Salkowski´s reagent (7.9 M H 2 SO 4 and 12.5 gl -1 FeCl 3 ) and gently shaken in inverted position at least 10 times. Samples were incubated in the dark for 30 min and the absorbance at 530 nm was measured. An aliquot of filtered supernatants was also injected with a final volume of 20 µl in an HPLC Waters 600-MS device (Waters Inc., USA) equipped with an U6K injector and C18 reverse phase column (Purospher STAR RP C-18, 3 mm, Lichrocart 55-4) heated at 30 °C, coupled to a system with UV-VIS Waters 486 detector (Waters Inc., USA) set at 265 nm. The elution was performed with a mixture of ethanol: acetic acid: water (Et-OH/H-Ac/H20) (12: 1: 87) as mobile phase at a flow rate of 1 ml.min -1 at 30 °C. The retention time for IAA was 10.1-10.3 minutes and it was previously identified using an appropriate standard solution of IAA (Sigma Aldrich, Germany). Quantification was performed by integration of the peak area corresponding to the retention time (RT) using an integration software (Waters Inc. USA). The IAA concentration was expressed in μg.ml -1