Microassay validation for bacterial IAA estimation as a new fine-tuned PGPR screening assay

Abstract

The detection and quantification of Indole -3 Acetic Acid (IAA) produced by Plant growth promoting rhizobacteria (PGPR) rely on a standard well-documented assay, which remains time-consuming, laborious, and costly. These drawbacks led to sway interest to economic and reliable assays. The aim of this work is to validate and standardize a fast, reliable, and cost-effective microassay to quantify IAA produced by bacteria with an easy microplate method. In order to validate the accuracy of the IAA microplate assay, bacterial samples from different genera were assayed using two methods: the conventional IAA estimation assay and the IAA micro- assay. The microassay shows a prominent reduction in used bacterial supernatant volume as well as Salkowski reagent volume of about 92.5%. It is considerably cheaper than the conventional one of around 56%. The newly performed microplate assay is 23 times faster. The result of IAA quantitative analysis for 13 bacterial strains showed that Bacillus muralis and Bacillus toyonensis produced the highest IAA concentration (23.64±0.003μg/ml and 23.35±0.006μg/ml, respectively). The obtained data from both methods were highly correlated with an R-value of 0.979. The microassay offers the ability to read the optical density of all samples simultaneously since used volumes of bacterial supernatants and Salkowski reagent were minimized to place the mixture in 96-well microplates, which reduces greatly required labor. Furthermore, the application of the IAA micro-plate assay reduces drastically the reagent waste and toxicity hazard of Salkowski reagent in the environment, thus, we can classify it as eco-friendly respecting the Green Chemistry concept according to Environmental Protection Agency (EPA). The IAA microassay is a, reliable, rapid and cost-effective and eco-friendly method to screen plant growth promoting potential of more than 23 bacterial strains by microplate. It could be an alternative for the conventional IAA assay as a routine research tool.