Rapid In Situ Assay for Indoleacetic Acid Production by Bacteria Immobilized on a Nitrocellulose Membrane

Abstract

We have developed a new assay that differentiates between indoleacetic acid (IAA)-producing and-nonproducing bacteria on a colony plate lift. Medium supplemented with 5 mM L-tryptophan is inoculated with isolates of interest, overlaid with a nitrocellulose membrane, and then incubated until bacterial colonies reach 1 to 2 mm in diameter. The membrane is removed to a filter paper saturated with Salkowski reagent and incubated until distinct red haloes form around the colonies. The colorimetric reaction to IAA is limited to a region immediately surrounding each colony, is specific to isolates producing IAA, occurs within 1 h after the membrane is placed in the reagent, and is sensitive to as little as 50 pmol of IAA in a 2-mm2 spot. We have used this assay for quantifying epiphytic and endophytic populations of IAA-producing isolates of Pseudomonas syringae subsp. savastanoi and for detecting IAA-producing colonies of other pseudomonads and Erwinia herbicola. The assay provides a rapid and convenient method to screen large numbers of bacteria.

Growth and nutrition of cowpea (Vigna unguiculata) under water deficit as influenced by microbial inoculation via seed coating

“…Regarding the seed coating procedure, the R. irregularis inoculum was sieved through a 500μm mesh and mixed with starch/silicon dioxide mixture (coating material) in the proportion of 1:1 (w/w) (the inoculum-coating material mixture was provided by Symbiom Ltd., Czech Republic). Pseudomonas putida strain GP was isolated from an agricultural soil in central Portugal used to grow Lupinus albus L. and tested positively for indoleacetic acid (IAA) (Brick, Bostock, & Silverstone, 1991), ammonia (Cappuccino & Sherman, 1992) and siderophores production (Schwyn & Neilands, 1987), phosphate solubilisation (Gaur, 1990), N fixation (Dobereiner, Marriel, & Nery, 1976), biofilm formation in the presence of different salt concentrations, 0.5 to 2.5 M (Christensen et al, 1985) and water stress tolerance (Ma, Rajkumar, Zhang, & Freitas, 2016). For the seed coating with bacteria, P. putida was grown in LB media for 17 hr at 28-30°C and 150 rpm, centrifuged at 3,500 rpm for 15 min and re-suspended in ringer solution with 1% carboxymethylcellulose (as an adhesive agent).…”

Glyphosate affects micro-organisms in rhizospheres of glyphosate-resistant soybeans

“…Rhizosphere soils were screened for índoleacetic acid-producing (IAA) bacteria by plating on S1 agar medium supplemented with 5 mmol l )1 l-tryptophan. A nitrocellulose membrane (90 mm diameter) was placed directly on the agar surface after plating prior to incubation (Bric et al 1991). After 48 h, the nitrocellulose membrane was removed from each plate and placed on filter paper (Whatman 42) saturated with Salkowski reagent (2% 0AE5 mol l )1 FeCl 3 in 35% HClO 4 ) in a clean petri plate for up to 4 h (Gordon and Weber 1951).…”

The plant growth‐promoting potential of the mesophilic wood‐rot mushroomPleurotus pulmonarius

“…The production of IAA by P. pulmonarius was detected by the Salkowski's method. The supernatants were analysed using the Salkowski reagent in a colorimetric test (Bric et al 1991). Tryptophan (TRP), a precursor or a building block of IAA, is recommended as a precursor for IAA biosynthesis in micro-organisms especially in fungi (Sarwar et al 1992;Normanly et al 1993;Spaepen et al 2007;Jogaiah et al 2013;Fu et al 2016).…”