Comparative study between Salkowski reagent and chromatographic method for auxins quantification from bacterial...

Area of Science:

• Microbiology
• Plant Science
• Biochemistry

Background:

• Auxins, particularly indole-3-acetic acid (IAA), are crucial plant hormones produced by bacteria.
• The Salkowski reagent is a common colorimetric method for estimating IAA, but its accuracy at higher concentrations is questionable.
• Overestimation by the Salkowski method can arise from detecting other indole compounds besides IAA.

Purpose of the Study:

• To compare the accuracy of the Salkowski colorimetric method against a precise chromatographic method (LC-MS/MS) for quantifying auxin production in plant growth-promoting bacteria (PGPB).
• To evaluate potential overestimation of indole-acetic acid (IAA) by the Salkowski assay.
• To analyze auxin profiles of ten PGPB strains from three different sources, with and without tryptophan supplementation.

Main Methods:

• Ten PGPB strains were cultured in modified TSA medium for 96 hours, with and without tryptophan.
• Bacterial supernatants were analyzed using both the Salkowski colorimetric reagent and ultra-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).
• Quantification of indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), and indole-3-butyric acid (IBA) was performed.

Main Results:

• The Salkowski method yielded IAA concentrations ranging from 5.4 to 27.4 mg L⁻¹ without tryptophan and 4.4 to 160 mg L⁻¹ with tryptophan.
• LC-MS/MS confirmed IAA as the principal auxin, with significantly higher concentrations observed upon tryptophan addition.
• The Salkowski reagent overestimated IAA by 41-1042 folds without tryptophan and 7-16330 folds with tryptophan, with indole-3-propionic acid (IPA) also detected.

Conclusions:

• The Salkowski reagent significantly overestimates auxin (IAA) concentrations in PGPB.
• Chromatographic techniques like LC-MS/MS are essential for accurate auxin quantification.
• Further research should employ sensitive methodologies to precisely determine IAA and other related auxin compounds in bacterial cultures.