Introduction

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 [4]. 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 [10]. 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′) [27]. 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 [20]. 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 [14]. 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

Indian mustard (B. nigra) was used to check the effect of isolate and its purified IAA for their plant growth-promoting activity. Mustard seeds were sterilized using 0.01 % HgCl2 and washed with sterile distilled water four times. The seeds were then allowed to germinate in sterile petriplates containing filter paper soaked in sterile distilled water for 2 days. These germinated seeds were then transferred to sterile 30 ml of MS [22] medium (Hi-Media) without IAA in the following two different sets.

  1. 1.

    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).

  2. 2.

    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

All the 9 isolates of P. putida UB1, tested for their IAA production showed a significant amount of IAA production in tryptophan-supplemented medium (Table 1), whereas no IAA production was observed in the medium devoid of tryptophan. The production of IAA by the isolates only in the presence of l-tryptophan indicates that the tested strains utilize l-tryptophan as a precursor for IAA production during their growth in the medium. Maximum IAA production was obtained with isolate UB1.

Table 1 Indole acetic acid production by cultures isolated from the rhizospheric soil of alfalfa
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Identification of the Isolate

The isolate was identified by 16S rRNA gene analysis. The 16S rRNA NCBI BLAST analysis showed 99 % similarity with the strain P. putida strain NB2011. Further phylogenetic analysis using software MEGA 4 revealed that the strain finds closest relationship with P. putida and hence was confirmed to be a putida strain (Fig. 1) (NCBI GenBank accession JF 304108).

Fig. 1
figure 1

Phylogenetic analysis based on 16S rRNA gene sequences available from National Center for Biotechnology information data library constructed after multiple alignments of data by ClustalX. The evolutionary history was inferred using the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. Phylogenetic analyses were conducted in MEGA software version 4.0

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IAA Production Profile

To investigate the effect of incubation period on IAA production, the samples were withdrawn from the production media at 12-h interval up to 144 h. The data obtained suggest there was growth-associated IAA production, and maximum production was observed at 96 h (Fig. 2). The results indicated that l-tryptophan from the medium is taken up at more or less constant rate and transform it to IAA. After 96 h, the growth and IAA production gradually decreased. Previous study by Swain et al. [35] showed that IAA production increased linearly from 2 to 8 days and decreased later with a decrease in the growth of organisms in l-tryptophan-supplemented medium. Patten and Glick [25, 26] have also reported increase in IAA production up to 96 h and attributed to the greater availability of the precursor. Datta and Basu [9] studied the IAA-degrading enzymes responsible for decrease in IAA production after 96-h incubation period. The decrease in IAA production might be due to the production of IAA-degrading enzymes.

Fig. 2
figure 2

Effect of incubation period on IAA production

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Effect of l-tryptophan Concentration on IAA Production

l-tryptophan is considered as a precursor for IAA production because its addition to medium increases IAA production [1, 29, 32]. Different concentrations of l-tryptophan between 0.05 and 0.25 mg/ml were selected to see the effect on IAA production. The spectrophometric analysis showed gradual increase in the IAA production with the increase in l-tryptophan concentration. 0.2 mg/ml of l-tryptophan concentration in the medium showed maximum IAA production (Fig. 3). Our results are in agreement with the work of Khalid et al. [16] who studied the effect of l-tryptophan concentration for the production of IAA and observed that l-tryptophan-derived auxin biosynthesis was enhanced several folds. Khalid et al. [16] showed variable amount of auxins produced by the rhizobacteria in vitro, and amendment of the culture media with l-tryptophan (l-TRP) further stimulated auxin biosynthesis. As per our studies, l-tryptophan concentration used (ranging from 0.05 to 0.25 mg/ml) which resulted in an increase in IAA production where 0.2 mg/ml tryptophan concentration gave maximum IAA production.

Fig. 3
figure 3

Effect of l-tryptophan concentration on IAA Production

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Effect of Static and Shaking Conditions on IAA Production

Analysis after incubation showed higher production in shaking condition compared to the production in the static condition (Fig. 4). Higher production under shaking condition might be due to good oxygen transfer rate as oxygen availability influences the activity of enzymatic transformations of tryptophan to auxins. Reinecke and Bandurski [28] showed the importance of oxygen concentration for IAA production where shaking resulted in better levels of IAA excretion. Sarwar et al. [30] also demonstrates that IAA production in shaking conditions increases up to twofold as compared with static incubation under the optimal assay conditions.

Fig. 4
figure 4

Effect of agitatic and static condition on IAA production

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Effect of pH on IAA Production

Physicochemical conditions of the media used are always specific for the organisms to biosynthesize the products. One of the most important parameter for the growth of IAA-producing organisms and their metabolic activity is the pH of the IAA production media [40]. In our investigation, maximum IAA production was observed at pH 7.5 (Fig. 5). Previous studies by Santi et al. [29] also suggest that Rhizobium sp isolated from the root nodules of Vigna mungo (L.) also varied with the IAA production in different pH ranging from 6.4 to 7.8 where maximum IAA production was found at pH 7.2. It has also been demonstrated by Sarwar et al. [30] that soil pH has a significant effect on l-tryptophan-derived IAA production. Yuan et al. [40] studied the effect of different fertilization treatment on soil leads to change in the pH of soils which in turn affects the IAA production by soil bacteria.

Fig. 5
figure 5

Effect of pH on IAA production on IAA production

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Effect of Carbon Source on IAA Production

The carbon sources that are used in the biosynthesis of plant hormone during their growth in liquid culture media contribute to the overall efficiency of biosynthesis. Four different sugars (sucrose, fructose, lactose, and glucose) were studied for their effect on IAA production. Sucrose in the medium gave maximum production as compared to other carbon sources (Fig. 6). Studies on IAA production by bacteria suggest that individual carbon sources effects the IAA production by bacteria [29, 33, 38]. Studies by Sridevi et al. [33] revealed that mannitol and l-glutamic acid were the best promoters for IAA production by Rhizobium isolates. Basu and Ghosh [5] have reported biomass to carbon source ratio playing very important role in cell yield and related IAA production where the preferred carbon source by Rhizobium species was glucose at the rate of one percent.

Fig. 6
figure 6

Effect of carbon source on IAA production

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Effect of Nitrogen Source on IAA Production

Impact of nitrogen sources on IAA production was studied by addition of various nitrogenous compounds like urea, NaNO3, KNO3, NH4NO3, and (NH4)2SO4 to the tryptophan-supplemented medium. Among all the nitrogen sources used, (NH4)2SO4 was found to be the best nitrogen source for IAA production (Fig. 7). The nitrogen source of the production medium affects IAA production [19, 29, 33]. Studies on IAA biosynthesis by root nodule bacteria of various leguminous plants revealed that L-asparagine and glutamic acid when used as nitrogen source stimulate the IAA production.

Fig. 7
figure 7

Effect of nitrogen source on IAA production

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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. [1] 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

Mustard (B. nigra) seeds were selected to check the plant growth-promoting activity of P. putida UB1. The germinated seeds of mustard were placed in tubes containing MS medium supplemented with (a) P. putida UB1 and (b) IAA purified from the broth inoculated with P. putida UB1 as a novel approach of incorporating the organism and its product in tissue culture media (Table 2). Uninoculated tubes were kept as control for each set of experiment. Analysis of the seedlings after 15 day’s growth showed that both the treatments helped in increasing the chlorophyll content, shoot length, and in the number of leaves as compared to control; the increase was more pronounced in seedlings grown in the medium containing P. putida compared to the medium supplemented with IAA. The root length was not much affected but an increase in the 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, which are utilized 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 concluded that P. putida UB1 is able to utilize the root exudates and synthesize plant growth regulator like IAA [6, 11].

Table 2 Changes in various growth parameters during growth of B. nigra inoculated with a) P. putida UB1 b) purified IAA from P. putida UB1
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Conclusions

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.