Analysis of Indole-3-Acetic Acid and Related Indoles in Culture Medium from Azospirillum lipoferum and Azospirillum brasilense

Abstract

Analysis of neutral and acidic ethyl acetate extracts from culture medium of Azospirillum brasilense 703Ebc by high-performance liquid chromatography (HPLC) and combined gas chromatography-mass spectrometry demonstrated the presence of indole-3-acetic acid (IAA), indole-3-ethanol, indole-3-methanol, and indole-3-lactic acid. IAA in media of 20 strains of A. brasilense and Azospirillum lipoferum was analyzed quantitatively by both the colorimetric Salkowski assay and HPLC-based isotopic dilution procedures. There was little correlation between the estimates obtained with the two procedures. For instance, the Salkowski assay suggested that the culture medium from A. brasilense 703Ebc contained 26.1 μg of IAA ml −1 , whereas HPLC revealed the presence of only 0.5 μg of IAA ml −1 . Equivalent estimates with A. brasilense 204Ed were 10.5 and 0.01 μg of IAA ml −1 , respectively. The data demonstrate that the Salkowski assay is not a reliable method for measuring the IAA content of Azospirillum culture medium and that estimates in excess of 10 μg of IAA ml −1 should be viewed with particular caution. Metabolism of [2′- 14 C]IAA by A. brasilense 703Ebc yielded radiolabeled indole-3-methanol, whereas roots of maize ( Zea mays L.) seedlings gave rise to [ 14 C]oxindole-3-acetic acid and an array of polar metabolites. Metabolism of [2′- 14 C]IAA by maize roots inoculated with A. brasilense 703Ebc produced a metabolic profile characteristic of maize rather than Azospirillum species.