Optimization of Indole Acetic Acid Production by Pseudomonas putida UB1 and its Effect as Plant Growth-Promoting Rhizobacteria on Mustard (Brassica nigra)
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Indole-3-acetic acid (IAA) production was monitored in nine plant growth-promoting rhizobacteria isolated from the rhizospheric soil of alfalfa (Medicago sativa). The isolate producing maximum amount of IAA was identified as Pseudomonas putida UB1 by 16S rRNA partial gene sequencing. Further investigations were carried out for optimal l-tryptophan concentration and other growth parameters for IAA production by P. putida. IAA production increased when the l-tryptophan medium for IAA production was supplemented with sucrose 0.5 %, (NH4)2SO4 10 mg/ml, and tryptophan 0.2 mg/ml at pH 7.5. Maximum IAA production was achieved at 96 h. Production of IAA was confirmed by extraction of crude IAA and subsequent thin-layer chromatography analysis. The specific spot from the extracted IAA was found to correspond with a spot of standard IAA with the same R f value (0.36). The crude IAA was further purified by passing it through a silica gel column. P. putida UB1 and its purified IAA were demonstrated to show stimulatory effect on mustard (Brassica nigra) plants in vitro with their incorporation in MS medium as a novel approach.
KeywordsIndole-3-acetic acid Pseudomonas putida Plant growth-promoting rhizobacteria (PGPR) Mustard (Brassica nigra)
Plant growth-promoting rhizobacteria (PGPR) are a heterogeneous group of bacteria found in rhizosphere in association with roots and root surfaces or free living in soil and influences plant growth directly or indirectly. A large number of bacteria including species of Pseudomonas, Azospirillum, Azotobacter, Klebsiella, Enterobacter, Alcaligenes, Arthobacter, Burkholderia, Bacillus, and Serratia have been reported to enhance plant growth [12, 17, 24]. The direct promotion of plant growth involves mechanisms like nitrogen fixation, solubilization of phosphorous and iron from the soil, and production of phytohormones like indole-3-acetic acid (IAA), gibberellins, cytokinins. The indirect mechanisms involve inhibition of phytopathogens . The bacteria that stimulate plant growth are referred to as plant growth-promoting bacteria (PGPB) [3, 8, 13]. Some mycorrhizal fungi and other fungi like Trichoderma are also known to stimulate plant growth [21, 37, 39].
Microorganisms inhabiting rhizosphere of plants utilize the rich source of substrates from the roots and are expected to synthesize and release auxins as secondary metabolites [6, 11, 15, 34]. Indole-3-acetic acid is a naturally occurring and main auxin in plants as it controls many important physiological processes like cell enlargement and division, tissue differentiation, and responses to light and gravity [18, 36]. Bacterial auxins have the potential to change any of these processes by altering the plant auxin pool. It depends on the amount of IAA produced and the sensitivity of plant tissue to changing levels of IAA. The roots are the most sensitive organs and respond to the changing levels of IAA by elongation of primary roots, formation of adventitious and lateral roots, or cessation of growth . Indole-3-acetic acid does not function as a hormone in bacterial cells but their ability to produce the same may have evolved as it is important in plant–bacteria relationship [25, 26, 31].
The present study reports optimization of growth parameters for IAA production by Pseudomonas putida UB1 isolated from the rhizospheric soils of alfalfa (Medicago sativa) plants and its plant growth-promoting effect on Mustard (Brassica nigra).
Materials and Methods
Isolation and Identification of Pseudomonas putida
Rhizospheric soil of alfalfa (M. sativa) field located at the experimental farm of Anand Agriculture University, Anand, Gujarat, India, was used for isolation of PGPR organisms. After removal of plant from soil, root portion was cut and packed in sterile plastic bags. The rhizosphere soil was collected in a separate bag. The bags were transported to laboratory under cold conditions for immediate processing. Adhering soil was carefully brushed off, and the plant roots were vigorously shaken and washed off with sterile saline solution so as to remove microorganisms closely associated with roots. To prepare soil suspension, approximately 1 gm of soil was suspended in sterile distilled water and vortexed. This was then serially diluted. 100 μl aliquot of dilution was plated in triplicate on nutrient agar and yeast extract mannitol agar medium. Plates were incubated at 28 °C for 3 days. Well-isolated colonies were selected, purified, and maintained on nutrient agar and yeast extract mannitol agar. IAA-producing isolates were selected by growing them in IAA production medium as described below. The isolate producing maximum amount of IAA was further selected for the optimization of IAA production.
The isolate producing maximum IAA was characterized based on the 16S rRNA partial sequencing using universal primer set 16F (5′AGA GTT TGA TCC TGG CTG 3′) and 16R (5′GGT TAC CTT GTT ACG ACT 3′) . Amplification was performed in thermocycler with following PCR conditions: 35 cycles of 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min with initial denaturation at 95 °C for 5 min and final extension at 72 °C for 10 min. The isolate was identified as P. putida.
IAA Production and Optimization of Production Parameters
For IAA production, the culture medium was inoculated with overnight grown P. putida UB1 (1 O.D cells/100 ml); the IAA production medium consisted of the following components (g/l): peptone 10, l-tryptophan 1, NaCl 5, yeast extract 6, and pH adjusted to 7.6 . The cultures, in 500 ml flasks containing 200 ml medium each, were incubated at 30 °C, 150 rpm for 72 h. Five ml of the medium was withdrawn regularly at 12-h interval and assayed for IAA production by Salkowski’s method . Two ml of supernatant was mixed with 2 drops of orthophosphoric acid and 4 ml of Salkowski’s reagent (50 ml of 35 % perchloric acid and 1 ml 0.5 M FeCl3 solution) and incubated in dark for 1 h. Absorbance was measured at 535 nm using spectrophotometer (Helios).
IAA Production Profile
The fermentation medium with precursor l-tryptophan was prepared and inoculated with culture (1 O.D cells/100 ml) and incubated at 30 °C in an environmental shaker at 150 rpm. Samples of 5 ml each were withdrawn at 12-h intervals for up to 144 h and analyzed for IAA production. Similar incubation conditions were used in other experiments. Concentration of l-tryptophan as a precursor greatly influences the IAA production. The precursor concentration was used in the range of 0.05–0. 25 mg/ml in the medium. To determine the effect of static and shaking condition on IAA production, 2 sets of flasks with similar production media were inoculated. One set was kept in the environmental shaker at 150 rpm, and the other set was maintained in static condition. pH is one of the most important physicochemical parameters, which affects the overall production of IAA. pH range 6–8 was examined for its effect on IAA production. The initial pH of the medium was adjusted using 1 N HCl or 1 N NaOH. Four different sugars viz. sucrose, glucose, lactose, and fructose were used in the fermentation medium at concentration of 0.5 % to analyze their effect on IAA production. The media containing different sugars with the same amount of precursor were inoculated with the organism. After incubation, the supernatants were subjected to IAA estimation. IAA production was studied in the presence of additional five different nitrogen sources like NaNO3, KNO3, NH4NO3, (NH4)2SO4, and urea at the concentration of 10 mg/ml in l-tryptophan-supplemented production media.
Extraction, Purification, and Characterization of IAA
Two flasks containing 300 ml of IAA production media with 15 mg/ml of l-tryptophan were inoculated with the organism. After incubation, the media was centrifuged at 5000g for 15 min, and the supernatant was acidified to pH 2.5 with 1 N HCl and extracted twice with equal volume of ethyl acetate. The ethyl acetate fraction was evaporated to dryness at 40 °C using rotavapor. The dried extract was dissolved in 3 ml of methanol and kept at 4 °C. The production of IAA was confirmed by spotting 10 μl of the IAA extract on TLC plate (10 × 10 cm silica gel 60 F254, Merck, Germany) along with 50 μg/ml of IAA (MW 175.19) standard (Hi-Media) in methanol. The solvent system used was n-butanol: ammonia: water (10:1:10), and the plates were developed using Salkowski’s reagent.
Partial purification of IAA from crude extract was carried out by silica gel column chromatography (22 × 5 cm), and fractions were collected with solvent system, n-butanol: ammonia: water (10:1:10). 10 μl of each fraction was tested for IAA using Salkowski’s reagent and thin-layer chromatography as discussed earlier. The fractions showing presence of purified IAA were pooled together and dried. The IAA thus obtained was used for further experiments.
In Vitro Assay for Plant Growth Promotion
Tubes containing sterile MS medium inoculated with the isolate (100 μl of cell suspension inoculated from a suspension containing 1 O.D cells/100 ml).
Tubes containing sterile MS medium containing IAA (16 μg/l) produced by the isolate.
The above tubes were incubated at 30 °C and were exposed to 12-h light/dark period (relative humidity maintained at 40 % and shelves illuminated by cool, white fluorescent lamps (40 W) to obtain the light intensity of 30–50 m.e.m 2 s−1). After 15 days, the seedlings were collected and measured for shoot length, root length, number of leaves, number of adventitious roots, wet weight of plants, shoots: root ratio, and chlorophyll content.
Results and Discussion
Indole acetic acid production by cultures isolated from the rhizospheric soil of alfalfa
Name of the isolate
Production of IAA (μg/ml)
Identification of the Isolate
IAA Production Profile
Effect of l-tryptophan Concentration on IAA Production
Effect of Static and Shaking Conditions on IAA Production
Effect of pH on IAA Production
Effect of Carbon Source on IAA Production
Effect of Nitrogen Source on IAA Production
Chromatographic Detection and Partial Purification of IAA
Indole-3-acetic acid produced was subject to detection by thin-layer chromatography using the solvent system n-butanol: ammonia: water (10:1:10). The chromatograms were observed under visible and UV light (254 nm). Upon spraying with the developing agent (Salkowski’s reagent), pink-colored spots were observed with the R f values 0.36 similar to the authentic IAA. Thin-layer chromatography and comparison with R f value of the standard revealed purified compound to be IAA. Ahmad et al.  have used the extract of the cultures and standard IAA and tested them in the solvent system hexane: ethyl acetate (80:20).
In Vitro Assay of Pseudomonas putida UB1 for its Plant Growth-Promoting Activity
Changes in various growth parameters during growth of B. nigra inoculated with a) P. putida UB1 b) purified IAA from P. putida UB1
Inoculation with P. putida UB1
Purified IAA from P. putida UB1
Average chlorophyll content
0.44 ± 0.015
2.916 ± 0.03
0.55 ± 0.02
1.5 ± 0.02
Average shoot length
3.26 ± 0.030
4.23 ± 0.035
3.56 ± 0.026
3.8 ± 0.005
Average root length
2.4 ± 0.015
2.4 ± 0.0152
2.4 ± 0.025
2.5 ± 0.005
Average no. of Adventitious roots
2.4 ± 1.154
13.66 ± 0.036
4.33 ± 0.577
8 ± 0.577
Average no. of leaves
5.33 ± 0.577
5.6 ± 0.152
4 ± 0.577
6 ± 0.577
In this study, nine isolates were tested for their ability to produce IAA. The isolate producing maximum amount of IAA was identified as P. putida UB1. Studies on optimization of IAA production suggest that maximum amount of IAA is produced at 96 h of incubation in IAA production media. The isolate produces maximum IAA at 0.2 mg/ml of tryptophan concentration, using sucrose and ammonium sulfate as carbon and nitrogen source, respectively. The presence of IAA in the rhizosphere influences the growth of the plant. Results of the plants harvested from the tubes indicated that the treatments helped in increasing the chlorophyll content, shoot length, and in the number of leaves as compared to control. The root length was not much affected but an increase in number of adventitious roots was observed which provides more surface area to the roots for better utilization of nutrients. Microorganisms in the rhizosphere utilize the root exudates and synthesize various metabolites utilizable by plants. Presence of P. putida UB1 in the medium showed an increase in the shoot length, root length and number of adventitious roots, hence it can be said that P. putida UB1 is able to utilize the root exudates and synthesize plant growth regulator like IAA. P. putida has been used for bioremediation as soil inoculant, as biocontrol agent, and for plant growth promotion [2, 7, 23]. We have demonstrated the incorporation of the organism and its product during the growth of the plant in tissue culture media as a novel approach resulting in better growth of the plant. Similar approach may help in obtaining healthy and better plants with tissue culture technique resulting in better survival of the plants when transferred to fields from laboratory. Thus, besides many other applications of Pseudomonas in biotechnology, it may find its importance in agricultural biotechnology as well.
Umang Bharucha is thankful to University Grants Commission (Delhi, India) for Teacher Fellowship.
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