A resourceful methodology to profile indolic auxins produced by rhizo-fungi using spectrophotometry and HPTLC

Abstract

Plant growth-promoting fungi play an important role in development of sustainable agriculture. In the current study, 13 fungal strains were isolated from the rhizosphere of healthy (wheat) plant and screened for their indolic auxin production potential. strain PGFW, strain BFW and strain DGFW were amongst the most efficient indolic auxin-producing strains. Indolic auxins such as indole 3 acetate (IAA), indole 3 butyrate (IBA) and indole 3 propionate (IPA) are produced by fungi. The conventional method to assess the IAA production is through a spectrophotometric assay using Salkowski's reagent, which quantifies all indolic auxins and not individual auxins. Moreover, it was also observed that the absorption maxima () of the samples, when compared to that of standard indole-3-acetic acid, showed deviation from the latter, indicative of production of a mixture of indolic derivatives by the fungi. Hence, for further profiling of these indolic compounds, high-performance thin layer chromatography (HPTLC) based protocol was standardized to precisely detect and quantify individual indolic auxins like IAA, IBA and IPA in the range of 100-1000 ng per spot. HPTLC analysis also showed that the fungal strains produce different indolic auxins in media with and without fortification of tryptophan, with the production of indolic auxins being enhanced in presence of tryptophan. Thus, this standardized HPTLC protocol is an efficient and sensitive methodology to separate and quantify the indolic derivatives.

Test

“…Among these three solvent systems, ethyl acetate/n-butanol/acetic acid (5 + 5 + 1), n-hexane/acetone/ethyl acetate (4 + 6 + 2), and methanol/water (8 + 2) provided satisfactory separations. These results indicate that HPTLC ingerprinting has great potential for rapid qualitative screening of various herbal medicines containing extracts for the presence of bioactive compounds [8].…”

Diaporthe species associated with the maritime grass Festuca rubra subsp. pruinosa

“…Siderophore activity was determined using the chrome azurol S agar plate assay as described by Schwyn and Neilands (1987) . The production of indole-3-acetic acid (IAA) was determined quantitatively using both spectrophotometric ( Gordon and Weber, 1951 ) and chromatographic ( Patel et al, 2018 ) assays. The ability of Diaporthe strains to produce amylase, cellulase and protease was analyzed in vitro in the supplemented media described by Hankin and Anagnostakis (1975) .…”

Plant growth promoting and antimicrobial functionalities of rhizosphere Pseudomonas species in flooded rice soils of coastal Odisha

“…Indole acetic acid (IAA) production was estimated by inoculating 3 × 10 7 cfu/ml bacterial isolates in 10 ml tryptone broth (g/l: peptone 20, NaCl 5, pH 7.4) containing 100 µg/ml L‐tryptophan, incubated for 72 h. IAA concentration in the culture supernatant was estimated as IAA equivalent (µg/ml culture) by recording OD (A530 nm) spectrophotometrically (Patel et al., 2018) after mixing 1 ml culture supernatant and 1 ml Salkowoski reagent (35% perchloric acid 50 ml, 0.5 M ferric chloride 1 ml).…”