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. 2011 Jan 13;17(1):25–32. doi: 10.1007/s12298-010-0041-7

Production of indole acetic acid by Pseudomonas sp.: effect of coinoculation with Mesorhizobium sp. Cicer on nodulation and plant growth of chickpea (Cicer arietinum)

Deepak K Malik 1, Satyavir S Sindhu 1,✉
PMCID: PMC3550561  PMID: 23572992

Abstract

Pseudomonas isolates obtained from the rhizosphere of chickpea (Cicer arietinum L.) and green gram (Vigna radiata) were found to produce significant amount of indole acetic acid (IAA) when grown in a LB medium broth supplemented with L-tryptophan. Seed bacterization of chickpea cultivar C235 with different Pseudomonas isolates showed stunting effect on the development of root and shoot at 5 and 10 days of seedling growth except the strains MPS79 and MPS90 that showed stimulation of root growth, and strains MPS104 and MRS13 that showed shoot growth stimulation at 10 days. Exogenous treatment of seeds with IAA at 0.5 and 1.0 μM concentration caused similar stunting effects on root and shoot growth compared to untreated control both at 5 and 10 days of observation, whereas higher concentration of IAA (10.0 μM) inhibited the growth of seedlings. Coinoculation of chickpea with IAA-producing Pseudomonas strains increased nodule number and nodule biomass by Mesorhizobium sp. Cicer strain Ca181. The plant dry weights of coinoculated treatments showed 1.10 to 1.28 times increase in comparison to Mesorhizobium-inoculated plants alone and 3.62 to 4.50 times over uninoculated controls at 100 days of plant growth. The results indicated the potential usefulness of allelopathic rhizosphere bacteria and growth-mediating IAA in enhancement of nodulation and stimulation of plant growth in chickpea.

Keywords: Indole-acetic acid (IAA), Pseudomonas sp., Mesorhizobium sp. Cicer, Seedling growth, Nodulation, Chickpea

Introduction

A rich diversity of microorganisms, varying from pathogens to beneficial, continuously interact with higher plants in soil ecosystem and influences the development of plant root in the soil (Ahmad et al.2008; Taghavi et al.2009). Potential to exploit beneficial plant-microbe interactions to enhance plant growth and nutrient uptake has been well documented by inoculation of plant growth promoting rhizobacteria (PGPR). These PGPR strains improve plant growth and soil quality by different mechanisms, including increased mobilization of insoluble nutrients (Lifshitz et al.1987; Ahmad et al.2008), biocontrol of phytopathogenic organisms (Weller 2007) and/or by production of phytohormones (Dubeikovsky et al. 1993; Spaepen et al.2007). However, yield reductions have also been reported following continuous cultivation of a single crop species on the same area for long periods (Alstrom 1992; Kirkegaard et al.1993).

In the rhizosphere of some crops, the accumulation of undesirable groups of microorganisms and production of phytotoxic allelochemicals has been found to cause soil sickness (Schippers et al.1987; Karen et al.2001). Their metabolites negatively influence the enzymatic activity, plant physiological processes and reduce the availability of some plant nutrients and their uptake by crop plants. Wide ranges of allelochemicals have been isolated from plant growth-mediating rhizosphere bacteria and even a single species of bacteria could produce several allelochemicals, e.g., geldanamycin, nigericin and hydanthocidin are produced by Streptomyces hygroscopicus. These allelochemicals inhibited radicle growth of Lepidium sativum by 50% in Petri dishes at 1 to 2 ppm (Heisey and Putnam 1986). Similarly, Pseudomonas syringae strain 3366 produced the metabolite phenazine-1-carboxylic acid that suppressed germination of seeds and reduced root and shoot growth of winter wheat and other weeds in agar diffusion assays (Gealy et al.1996). Other allelochemicals such as succinic acid and lactic acid were produced from Pseudomonas putida (Yoshikawa et al.1993), cyanide by Pseudomonas spp. (Bakker and Schippers 1987) and indole acetic acid by Pseudomonas fluorescens (Dubeikovsky et al. 1993; Barazani and Friedman 1999).

IAA biosynthesis has been correlated with stimulation of root proliferation by rhizosphere bacteria (Persello-Cartieaux et al.2003; Spaepen et al.2007), which enhanced uptake of nutrients by the associated plants (Lifshitz et al. 1987). The effect of IAA has been found to depend on the concentration, that is, low concentrations of exogenous IAA can promote, whereas high concentrations can inhibit root growth (Arshad and Frankenberger 1992). Moreover, inoculation with an Azospirillum brasilense Sp245 mutant strain, strongly reduced in auxin biosynthesis or addition of increasing concentrations of exogenous auxin to the plant growth medium, indicated that the differential response to A. brasilense Sp245 among the common bean (Phaseolus vulgaris L.) genotypes is related to the bacterial produced auxin (Remans et al.2008). For various PGPR, the promotion of plant growth after inoculation with rhizobacteria has been attributed to biosynthesis and secretion of IAA in case of Azospirillum brasilense (Okon and Vanderleyden 1997), Rhizobium species (Hirsch and Fang 1994) as well as in Xanthomonas and Pseudomonas (Patten and Glick 1996; Zhang et al.1997).

On the other hand, the inhibitory effect of some deleterious rhizobacteria through IAA secretion has been related to various bacterial species including Enterobacter taylorae, Klebsiella planticola, Alcaligenes faecalis, Xanthomonas maltophila, Pseudomonas sp. and Flavobacterium sp. (Sarwar and Kremmer 1995; Suzuki et al.2003). Mutants of Pseudomonas putida that produced high levels of IAA inhibited root growth of seedlings of canola (Brassica campestris) by ca. 33% (Xie et al.1996). Thus, ambiguity about effect of IAA on growth of root, shoot and rate of seedling emergence has been reported (de Freitas and Germida 1990; Sarwar and Kremmer 1995; Barazani and Friedman 1999). Despite the potential of allelopathic bacteria and growth-mediating allelochemicals in agriculture, it is one of the poorly understood areas of plant-microbe interactions. Further work is needed to characterize bacteria and allelochemicals from the rhizosphere soil and to study their effect on the crop plants.

Chickpea (Cicer arietinum L.), the world’s third most important food legume, is grown during the winter season in arid and semi-arid zones in Asia. South and South-East Asia region contributes about 80% to the global chickpea production and India is the principal chickpea producing country with production of 5,970,000 tonnes (83% share in the region) in 2008. The present studies showed that IAA-producing Pseudomonas strains showed stunting effect on the development of root and shoot at 5 and 10 days of seedling growth in chickpea (Cicer arietinum L.). However, Coinoculation of IAA-producing Pseudomonas strains with Mesorhizobium sp. Cicer strain in chickpea increased nodule number and plant dry weights in comparison to Mesorhizobium-inoculated plants and uninoculated plants.

Materials and methods

Bacterial cultures and seeds

Standard strains of Pseudomonas sp. MRS13 and effective Mesorhizobium sp. Ca181 used in the present studies were taken from the Department of Microbiology, CCS Haryana Agricultural University, Hisar and maintained on Luria-Bertani (LB) medium (Sambrook et al. 1989) and yeast-extract mannitol agar (YEMA) medium (Vincent 1970), respectively by periodic transfers. Seeds of chickpea (Cicer arietinum L.) cv. C235 were obtained from Pulses Section, Department of Plant Breeding, CCS Haryana Agricultural University, Hisar.

Isolation of Pseudomonas strains from rhizosphere

Soil samples from the rhizosphere of chickpea and green gram were collected from different locations at pre-flowering stage of plant growth. From each location, 10-15 healthy plants were uprooted along with adhered soil and brought to the laboratory in polythene bags. From pooled samples of each location, 10 g adhered rhizosphere soil along with roots (cut to 2-3 cm pieces) was used for serial dilutions and 10-3 to 10-5 dilutions were plated on solidified King’s B (KB) agar medium plates. The plates were incubated at 28 ± 1°C for 3-4 days. Pseudomonas colonies giving fluorescence against reflected light were picked up and purified further by streaking on the KB medium plates. Selected isolates were identified upto the genus level by morphological and biochemical characteristics (Palleroni 1984) and were transferred on KB medium slopes.

Determination of IAA production

IAA production by different Pseudomonas isolates was determined using Salkowski’s reagent (Gordon and Weber 1951; Mayer 1958). The purified and freshly grown cultures on Luria-Bertani (LB) medium slopes were transferred into tubes containing 5 ml LB broth supplemented with 100 μg ml-1 L-tryptophan and were incubated at 28 ± 1°C for 2 and 4 days. The broth was then centrifuged for 5 min at 10,000 rpm and in the supernatant equal volume of Salkowski’s reagent was added. The contents were mixed and allowed to stand at room temperature for 30 min to develop colour. The optical density was then recorded at 500 nm. Uninoculated broth served as control. Standard curve was prepared with 5-100 μg ml-1 of IAA (Sigma Chemicals) for quantification.

Effect of Pseudomonas strains on seedling growth

Healthy seeds of chickpea cv. C235 were surface sterilized with acidic alcohol (H2SO4: ethanol, 7:3, v/v) for 3 min followed by thorough washing with repeated changes of sterilized distilled water (Sindhu et al. 1999). The surface sterilized seeds were inoculated with broth culture of different Pseudomonas isolates and allowed to be adsorbed for 30 min. Inoculated seeds were germinated on water agar germination plates (10 g agar L-1 distilled water) at 28 ± 1°C in a BOD incubator. Uninoculated seeds treated with LB broth alone were sown as control. The root and shoot lengths were measured at 5 and 10 days after sowing.

Effect of exogenous IAA on root and shoot growth

Stock solution of IAA was prepared in ethanol at a concentration of 0.1 M and subsequently different concentrations of IAA (0.5, 1.0 and 10.0 μM) were made in distilled water. Surface sterilized seeds of chickpea were treated with different concentrations for 30 min. The IAA-treated seeds were germinated on water agar plates at 28 ± 1°C. Root and shoot lengths of the germinated seedlings were measured at 5 and 10 days for comparison with bacterial treatments.

Coinoculation of Pseudomonas strains with Mesorhizobium sp. Cicer strain Ca181 under chillum jar conditions

For preparing chillum jar assemblies, thoroughly washed and dried coarse river sand was used to fill the upper assembly while the lower assembly was filled with quarter-strength of Sloger's nitrogen-free mineral salt solution (Sloger 1969). The whole assembly was then autoclaved at 15 lbs pressure for 3 h. Surface-sterilized seeds of chickpea cv. C235 were inoculated with broth culture of Mesorhizobium sp. Cicer strain Ca181 alone or as coinoculants with Pseudomonas isolates by mixing the broth of the two in a ratio of 1:1 (v/v). Two ml of the mixed inoculum was inoculated on 15 seeds and left for 30 min for adsorption. In case of strain Ca181 alone, 1 ml of broth and 1 ml water was added to have relatively the same level of inoculum. Seeds were then sown in sterilized chillum jar assemblies. Uninoculated seeds were sown as control, keeping 9 replications for each treatment. In each chillum jar, 5 seeds were sown and after germination, 3 seedlings were kept. The jars were kept in a net house under day light conditions. Quarter strength Sloger's nitrogen-free mineral salt solution was used for watering as and when required. The plants were uprooted after 60, 80 and 100 days of sowing and observations were taken for nodule number, nodule fresh weight, nitrogenase activity and plant dry weights.

Nitrogenase assay

Nitrogenase activity in nodules was determined by measuring acetylene reduction activity (ARA) at different stages of plant growth (Hardy et al. 1968). The plants from chillum jars were uprooted and the adhered soil was removed by shaking the plants gently. The root and shoot portions were separated. Roots along with nodules were transferred to 250-ml conical flasks fitted with B24 joint and serum stoppers. In each flask, 10% of the air was replaced with freshly prepared acetylene and the flasks were incubated at 28 ± 1°C for 1 h. Ethylene formed was determined by Gas Liquid Chromatograph (Nucon Aimil 5700, New Delhi, India) using dual flame ionization detector (FID) and Porapak N columns (2 M length x 2 mm diameter). Nitrogen at a flow rate of 40 ml min-1 was used as a carrier gas and hydrogen at a flow rate of 20 ml min-1 was used as fuel gas. Oven, detector and injector temperatures were kept at 105, 110 and 110°C, respectively. Standard ethylene was used for calibration and nitrogenase activity was expressed as μM of acetylene reduced h-1 plant-1.

Nodule fresh weight and plant dry weight

The nodules were detached from the roots after determination of nitrogenase activity, washed with water and blotted in folds of filter paper. The nodules were counted and fresh weight was taken. Shoot portion was dried in oven at 90°C for 24 h and weighed.

Results

Screening of Pseudomonas isolates for IAA production in broth

Out of 40 Pseudomonas isolates obtained from the rhizosphere of chickpea and green gram, only 11 isolates produced indole-acetic acid in LB broth (Table 1). The amount of IAA produced varied from 10.2 to 31.2 μg ml-1 of supernatant in different isolates. Isolates CPS59, CPS63, CPS67, CPS72, MPS77, MPS78 and MPS94 produced significant amount of IAA (18.1–31.2 μg ml-1) at 2 days of growth. At 4 days of culture growth, the amount of IAA released in the supernatant increased. Isolates CPS59, CPS63, CPS72, MPS77, MPS78 and MPS94 excreted 22.2 to 40.6 μg ml-1 of IAA. Maximum amount of IAA production was observed in Pseudomonas isolates CPS72 and MPS77.

Table 1.

Production of indole acetic acid by different Pseudomonas isolates. Control treatment contains LB broth and the Salkowski’s reagent. IAA production was calculated on the basis of equal 1.0 optical density of bacterial growth suspension

Pseudomonas isolate IAA production (μg ml-1 supernatant)
2 days 4 days
Control 0.0 0.0
CPS10 10.2 10.8
CPS59 18.2 22.8
CPS63 18.1 22.2
CPS67 18.4 19.7
CPS72 27.7 30.3
MPS77 31.2 40.6
MPS78 19.2 24.0
MPS79 11.4 16.8
MPS90 14.0 20.0
MPS94 19.7 25.1
MPS104 16.7 19.8
MRS13 13.2 19.5
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Effect of seed inoculation and exogenous application of IAA on root and shoot growth

All the IAA producing Pseudomonas isolates showed stunting effect on root and shoot growth at 5 days (Table 2). Maximum stunting effect on root and shoot growth was observed with isolate MPS77 followed by MRS13, CPS59 and MPS94. Isolates CPS67, MPS79 and MPS90 showed maximum stunting effect on shoot at both 5 and 10 days. However, all the isolates comparatively had elongation effect on root growth at 10 days in comparison to 5 days observation. A little shoot elongation effect was observed with isolates MRS13 and MPS104 at 10 days.

Table 2.

Effect of Pseudomonas isolates on seedling growth of chickpea. Means of five replications ± SE

Treatment Root length (cm) Shoot length (cm)
5 days 10 days 5 days 10 days
Control 14.35 ± 0.44 17.12 ± 0.82 8.02 ± 0.44 10.40 ± 1.1
CPS10 12.46 ± 0.50 16.96 ± 0.50 7.88 ± 0.41 9.38 ± 0.85
CPS59 9.45 ± 0.51 15.90 ± 0.75 6.30 ± 0.33 10.30 ± 1.1
CPS63 9.86 ± 0.51 15.70 ± 0.51 6.96 ± 0.56 10.40 ± 1.9
CPS67 9.50 ± 0.55 14.24 ± 0.05 7.64 ± 0.30 7.94 ± 1.0
CPS72 11.52 ± 0.33 16.60 ± 0.29 8.10 ± 0.29 9.66 ± 1.7
MPS77 8.95 ± 0.36 16.20 ± 0.62 6.12 ± 0.62 10.40 ± 1.6
MPS78 11.24 ± 0.43 16.20 ± 0.19 7.46 ± 0.49 9.14 ± 1.8
MPS79 11.35 ± 0.72 17.70 ± 0.71 7.50 ± 0.20 8.30 ± 1.1
MPS90 9.85 ± 0.18 17.40 ± 0.28 7.20 ± 0.40 8.80 ± 0.3
MPS94 9.50 ± 0.50 11.30 ± 0.05 6.40 ± 0.19 9.25 ± 2.1
MPS104 10.26 ± 0.88 12.10 ± 0.89 7.26 ± 0.80 11.00 ± 1.7
MRS13 9.04 ± 0.45 16.74 ± 0.20 6.20 ± 0.20 11.14 ± 2.2
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The exogenous application of IAA at 0.5 μM on seeds showed stunting effect on both root and shoot as compared to untreated control seedlings at 5 and 10 days of observation (Table 3). The taproot growth was inhibited but it caused lateral root development. At higher levels of IAA (10 μM), there was complete inhibition of root growth at 5 days but little root growth was observed at 10 days. At 10 μM concentration, shoot growth was completely inhibited even at 10 days of observation.

Table 3.

Effect of exogenously applied IAA on root and shoot growth of chickpea seedlings on water agar germination plates. Means of five replications ± SE

Treatment Root length (cm) Shoot length (cm)
5 days 10 days 5 days 10 days
Control 10.85 ± 0.44 18.7 ± 0.69 7.5 ± 0.24 15.0 ± 0.37
0.5 μM 8.4 ± 0.58 7.2 ± 0.27 4.3 ± 0.14 5.1 ± 0.12
1.0 μM 2.0 ± 0.40 4.9 ± 0.48 2.5 ± 0.05 2.9 ± 0.08
10.0 μM 0.0 1.7 ± 0.06 0.0 0.0
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Effect of coinoculation of Pseudomonas with Mesorhizobium sp. Cicer strain Ca181

Seed inoculation of chickpea with Mesorhizobium sp. Cicer Ca181 alone or on coinoculation with Pseudomonas isolates increased the plant dry weights in comparison to uninoculated controls under chillum jar conditions at all the stages of plant growth (Table 4). The plant dry weight gains varied from 1.14 to 1.80 times to those of Mesorhizobium-inoculated treatments and 4.50 to 7.10 times to those of uninoculated controls at 60 days of plant growth. Five Pseudomonas isolates i.e., CPS10, CPS67, MPS77, MPS78 and MPS104 caused maximum gain in plant dry weight ratios i.e., 1.48, 1.77, 1.80, 1.49 and 1.61 times to those of Mesorhizobium-inoculated plants, respectively. The nodule promoting effect was evident with only five Pseudomonas isolates CPS67, MPS77, MPS94, MPS104 and MRS13, and coinoculation resulted in increased nodule weight. Pseudomonas strain-dependent variations in acetylene reduction activity were observed in nodules of various treatments.

Table 4.

Effect of coinoculation of chickpea with Pseudomonas strains and Mesorhizobium strain Ca181 on symbiotic parameters at 60 and 80 days of plant growth under sterile conditions. Data are average values of three plants

Treatments Plant growth (days) Nodule number (plant-1) Nodule fresh weight (mg plant-1) Nitrogenase activity (μM C2H2 reduced plant-1 h-1) Plant dry weight (mg plant-1)
Control 60 – – – 102
80 – – – 205
CPS63 60 – – – 86
80 – – – 188
MPS94 60 – – – 78
80 – – – 214
Mesorhizobium strain Ca181 60 25 462 3.17 407
80 35 1,208 3.52 536
Ca181 + CPS10 60 22 565 3.79 604
80 59 1,662 3.48 1,035
Ca181 + CPS59 60 20 286 4.35 534
80 43 1,432 3.97 732
Ca181 + CPS63 60 18 364 3.12 562
80 37 1,164 4.14 768
C181 + CPS67 60 30 765 2.59 724
80 34 1,035 3.70 964
Ca181 + CPS72 60 21 508 2.62 532
80 45 1,456 4.61 972
Ca181-MPS77 60 36 802 3.48 735
80 40 1,318 2.85 946
Ca181 + MPS78 60 21 468 3.78 609
80 39 1,285 4.18 756
Ca181 + MPS79 60 24 402 2.71 568
80 36 1,202 1.36 954
Ca181 + MPS90 60 24 406 2.01 504
80 41 1,342 3.86 628
Ca181 + MPS94 60 27 537 4.36 465
80 48 1,472 3.96 936
Ca181 + MPS104 60 28 564 3.24 656
80 34 956 2.43 962
Ca181 + MRS13 60 33 712 3.92 564
80 60 1,664 3.87 907
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At 80 days of plant growth, plant dry weight ratio of coinoculated plants varied from 3.06 to 5.0 times over uninoculated control and 1.19 to 1.93 times in comparison to Mesorhizobium-inoculated plants (Table 4). More plant growth enhancement was observed on coinoculation with Pseudomonas cultures CPS10, CPS67, CPS72, MPS77, MPS79, MPS94 and MPS104. Coinoculation with Pseudomonas cultures CPS10, CPS59, CPS72, MPS77, MPS90, MPS94 and MRS13 significantly increased nodule biomass and nodule number, indicating stimulation of nodulation by Mesorhizobium on coinoculation with Pseudomonas strains.

At later stages of plant growth (100 days), coinoculation with six Pseudomonas strains CPS10, CPS59, CPS72, MPS77, MPS79 and MRS13 increased the plant dry weights of chickpea 3.62 to 4.50 times to control (Table 5). Three Pseudomonas strains MPS78, MPS90 and MPS94 showed very little (1.10 to 1.28 times) increase in the symbiotic effectiveness. Most of the Pseudomonas strains promoted nodulation by Mesorhizobium sp. Cicer strain Ca181 except the strain MPS104. The acetylene reduction activity (ARA) in nodules at 100th day declined in most of the coinoculation treatments as compared to ARA observed at 80 days of plant growth.

Table 5.

Effect of coinoculation of Pseudomonas strains and Mesorhizobium sp. Cicer strain Ca181 on symbiotic parameters of chickpea at 100 days of plant growth. Nitrogenase activity (μM C2H2 reduced plant-1 h-1). Data are average values of three plants

Treatments Nodule number (plant-1) Nodule fresh weight (mg plant-1) Nitrogenase activity (μM) Plant dry weight (mg plant-1)
Control 5 94 – 432
Mesorhizobium strain Ca181 43 1,362 3.68 938
CPS63 – – – 465
MPS94 – – – 462
Ca181 + CPS10 80 2,875 3.60 1,948
Ca181 + CPS59 48 1,512 2.58 1,564
Ca181 + CPS63 54 1,768 3.35 1,262
Ca181 + CPS67 51 1,632 4.36 1,305
Ca181 + CPS72 74 2,704 3.17 1,764
Ca181 + MPS77 55 1,864 2.29 1,608
Ca181 + MPS78 48 1,566 3.43 1,035
Ca181 + MPS79 65 2,536 2.91 1,936
Ca181 + MPS90 50 1,608 2.78 1,124
Ca181 + MPS94 52 1,674 3.64 1,205
Ca181 + MPS104 41 1,335 2.84 1,266
Ca181 + MRS13 64 2,508 2.04 1,705
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Discussion

The plant rhizosphere is a dynamic ecological environment in soil for plant-microbe interactions (Benizri et al.2001; Somers et al.2004). Beneficial microbial allelopathies in the root zone are a key agent of change in soil ecosystems and affect crop health, yield and soil quality (Sturz and Christie 2003; Taghavi et al.2009). The release of allelochemicals such as phenolic acids, phytotoxins, cyanide, phenazine-1-carboxylic acid and excess amount of IAA by rhizosphere bacteria were found to suppress germination of seeds and reduced root as well as shoot growth in different crops (Bakker and Schippers 1987; Gealy et al.1996; Karen et al.2001). Production of allelochemicals, particularly IAA has been considered as an important attribute of PGPR strains that can affect plant growth in diverse ways, varying from pathogenesis and growth inhibition to plant growth stimulation (Prikryl et al. 1985; Somers et al.2004; Spaepen et al.2007).

In the present study, out of 40 Pseudomonas isolates obtained from chickpea and green gram rhizosphere, only 11 isolates were found to produce IAA. The amount of IAA produced varied from 10.2 to 31.2 μg ml-1 of supernatant in different Pseudomonas isolates at 2 days and from 10.8 to 40.6 μg ml-1 at 4 days of bacterial growth (Table 1). The production of phytohormones in chemically defined media has also been reported in other PGPR strains including Azotobacter chroococcum (Muller et al.1989), Azospirillum (Bar and Okon 1992; Remans et al.2008), Rhizobium species (Hirsch and Fang 1994), Bacillus polymyxa (Holl et al.1988), Pseudomonas fluorescens (Dubeikovsky et al. 1993) and Pseudomonas putida (Taghavi et al.2009).

Inoculation of IAA-producing Pseudomonas isolates on seeds of chickpea showed initial stunting effect on root and shoot growth except in a few cases where little stimulation of root and shoot growth was observed. Maximum stunting effect on root as well as shoot growth was observed at 5 days with the strain MPS77 followed by strains MRS13, CPS59 and MPS94 (Table 2). Pseudomonas isolates CPS67, MPS79 and MPS90 showed maximum stunting effect on shoot at both the stages of observations. All the Pseudomonas strains comparatively increased the root growth at 10 days than to 5 days observation. The initial stunting effect on seedlings could be due to contact of bacterial cell with legume seeds, due to synthesis or secretion of excessive amount of IAA and/or some inhibitory agent produced by the bacterium grown in synthetic medium (Loper and Schroth 1986; Bolton and Elliott 1989). Similar results were obtained when cuttings of sour cherry (Prunus cerasus) and black-currant (Ribes nigrum) were inoculated with a recombinant strain of Pseudomonas fluorescens that produced increased amount of IAA. A high density of bacterium inoculum on the roots of cherry cuttings inhibited root growth, whereas lower densities on black-currant promoted growth (Dubeikovsky et al.1993).

Exogenous application of IAA was made on chickpea seeds to correlate the inhibitory or growth promoting effects on seedling growth with IAA production in defined medium. The exogenous application of IAA at 0.5 μM concentration showed stunting effect on both root and shoot growth of chickpea seedlings in comparison to untreated seeds (Table 3). The tap root growth was inhibited but it caused lateral root formation. At higher concentrations of IAA (10.0 μM), there was complete inhibition of root growth. Similarly, high concentrations of IAA caused stunting of shoot and inhibited shoot emergence even at 10 days. Arshad and Frankenberger (1992) also reported that the effect of IAA is concentration dependent, that is, low concentrations of exogenous IAA can promote, whereas high concentration can inhibit root growth. Loper and Schroth (1986) observed a significant linear relationship between IAA accumulation of the rhizobacterial strains and decreased root elongation of sugar beet seedlings. Similarly, inoculation of canola (Brassica campestris) seeds with Pseudomonas putida GR12-2, which produced low level of IAA, resulted in 2 to 4-fold increase in the length of seedling roots, whereas an IAA over producing mutant inhibited root growth of seedlings by 33% (Xie et al.1996). In contrast, Astrom et al. (1993) reported that treatment with a cell-free culture filtrate of P. fluorescens caused a strong inhibitory effect on root elongation of wheat seedlings. In other studies also, inhibitory effect of some deleterious rhizobacteria (DRB) was related to the high amount of IAA excretion (Sarwar and Kremmer 1995; Barazani and Friedman 1999; Suzuki et al.2003).

Inoculation of legumes and cereal plants with PGPR strains has been found to show a wide range of effects on plant growth that varied among different strains of PGPR. Chickpea plants inoculated with Pseudomonas strains i.e., CPS10, CPS67, MPS77, MPS78 and MPS104 caused increase in plant dry weight ratios i.e., 1.14 to 1.80 times to those of Mesorhizobium-inoculated plants, respectively at 60 days of plant growth. Plant dry weight ratio of coinoculated plants varied from 3.06 to 5.0 over control and 1.19 to 1.93 times in comparison to Mesorhizobium-inoculated plants at 80 days of plant growth (Table 4). At later stages of plant growth (100 days), coinoculation with Pseudomonas strains CPS10, CPS59, CPS72, MPS77, MPS79 and MRS13 increased the plant dry weights of chickpea 3.62 to 4.50 times over the uninoculated control (Table 5). Similar effect of coinoculation of rhizobacteria with Rhizobium on symbiotic parameters have been reported in other legumes like alfalfa (Knight and Langston-Unkeffer 1988), chickpea (Parmar and Dadarwal 1999), green gram (Sindhu et al. 1999), pea (Bolton et al. 1990; Berggren et al.2001) and soybean (Dashti et al. 1997).

Coinoculation with most of the IAA-producing Pseudomonas strains with Mesorhizobium sp. Cicer strain Ca181 also resulted in increased nodule number and nodule fresh weight (Table 4, 5), indicating stimulation of nodulation by Mesorhizobium sp. on coinoculation. Rhizobacteria as well as mycorrhizal fungi have been found to enhance the production of flavonoid-like compounds or phytoalexins in roots of several crop plants (Parmar and Dadarwal 1999; Goel et al.2001) that induce the transcription of rhizobial nodulation (nod) genes. The localized plant hormone auxins have also been shown to participate in the fundamental responses of nodule morphogenesis. Similar nodule-promoting effects of Pseudomonas sp. on coinoculation with Rhizobium strains have been reported in soybean (Nishijima et al.1988; Zhang et al. 1996) and green gram (Sindhu et al.1999).

The initial stunting effects of Pseudomonas strains on root and shoot growth under controlled conditions, however, did not show adverse effect on nodulation and plant growth in these studies when these bacteria were used as coinoculants with Mesorhizobium. For example, coinoculation with Pseudomonas isolates MPS79 and CPS10, which showed maximum stunting effect on shoot under cultural conditions at 10 days (Table 2), resulted in significant gain in shoot dry weight at 100 days of plant growth (Table 5) in comparison to the high IAA producer Pseudomonas isolate MPS77. It is, therefore, apparent that IAA production by the rhizobacteria beyond a critical limit may not be desired for plant growth promotion. Because the relative concentration of microbial allelochemicals may result in different response of higher plants, therefore, inoculation tests under field conditions are essential for evaluating the allelopathic impact of soil-borne microorganisms.

References

  1. Ahmad F, Ahmad I, Khan MS. Screening of free-living rhizosphere bacteria for their multiple plant growth promoting activities. Microbiol Res. 2008;163:173–181. doi: 10.1016/j.micres.2006.04.001. [DOI] [PubMed] [Google Scholar]
  2. Alstrom S. Saprophytic soil microflora in relation to yield reductions in soil repeatedly cropped with barley (Hordeum vulgare L.) Biol Fertil Soils. 1992;14:145–150. doi: 10.1007/BF00336265. [DOI] [Google Scholar]
  3. Arshad M, Frankenberger WT., Jr . Microbial production of plant growth regulators. In: Metting FB Jr, editor. Soil microbial ecology, applications in agricultural and environmental management. New York: Dekker; 1992. pp. 27–32. [Google Scholar]
  4. Astrom B, Gustafsson A, Gerhardson B. Characteristics of a plant deleterious rhizosphere pseudomonad and its inhibitory metabolite(s) J Appl Bacteriol. 1993;74:20–28. [Google Scholar]
  5. Bakker AW, Schippers B. Microbial cyanide production in the rhizosphere in relation to potato yield reduction and Pseudomonas spp.-mediated plant growth stimulation. Soil Biol Biochem. 1987;19:451–457. doi: 10.1016/0038-0717(87)90037-X. [DOI] [Google Scholar]
  6. Bar T, Okon Y. Induction of indole-3-acetic acid synthesis and possible toxicity of tryptophan in Azospirillum brasilense Sp7. Symbiosis. 1992;13:191–198. [Google Scholar]
  7. Barazani O, Friedman J. Is IAA the major growth factor secreted from plant growth mediating bacteria? J Chem Ecol. 1999;25:2397–2406. doi: 10.1023/A:1020890311499. [DOI] [Google Scholar]
  8. Benizri E, Boudoin E, Guckert A. Root colonization by inoculated plant growth-promoting rhizobacteria. Biocontrol Sci Technol. 2001;11:557–574. doi: 10.1080/09583150120076120. [DOI] [Google Scholar]
  9. Berggren I, van Vuurde JWL, Martensson AM. Factors influencing the effect of deleterious Pseudomonas putida rhizobacteria on initial infection of pea roots by Rhizobium leguminosarum bv. viciae. Appl Soil Ecol. 2001;17:97–106. doi: 10.1016/S0929-1393(01)00130-5. [DOI] [Google Scholar]
  10. Bolton H, Jr, Elliott LF. Toxin production by a rhizobacterial Pseudomonas sp. that inhibits wheat root growth. Plant Soil. 1989;114:269–278. doi: 10.1007/BF02220807. [DOI] [Google Scholar]
  11. Bolton H, Jr, Elliott LF, Turco RF, Kennedy AC. Rhizoplane colonization of pea seedlings by Rhizobium leguminosarum and a deleterious root colonizing Pseudomonas sp. and effects on plant growth. Plant Soil. 1990;123:121–124. [Google Scholar]
  12. Dashti N, Zhang F, Hynes RK, Smith DL. Application of plant growth promoting rhizobacteria to soybean [Glycine max (L.) Merr.] increases protein and dry matter yield under short season conditions. Plant Soil. 1997;188:33–41. doi: 10.1023/A:1004295827311. [DOI] [Google Scholar]
  13. de Freitas JR, Germida JJ. Plant growth promoting rhizobacteria for winter wheat. Can J Microbiol. 1990;36:265–272. doi: 10.1139/m90-046. [DOI] [Google Scholar]
  14. Dubeikovsky AN, Mordukhova EA, Kochetkov VV, Polikarpova FY, Boronin AM. Growth promotion of blackcurrant softwood cuttings by recombinant strain Pseudomonas fluorescens BSP53a synthesizing an increased amount of indole-3-acetic acid. Soil Biol Biochem. 1993;25:1277–1281. doi: 10.1016/0038-0717(93)90225-Z. [DOI] [Google Scholar]
  15. Gealy DR, Gurusiddaiah S, Ogg AG., Jr Isolation and characterization of metabolites from Pseudomonas syringae strain and their phytotoxicity against certain weed and crop species. Weed Sci. 1996;44:383–392. [Google Scholar]
  16. Goel AK, Sindhu SS, Dadarwal KR. Seed bacterization with fluorescent Pseudomonas enhances the synthesis of flavonoid-like compounds in chickpea (Cicer arietinum L.) Physiol Mol Biol Plants. 2001;6:195–198. [Google Scholar]
  17. Gordon SA, Weber RP. Colorimetric estimation of indole acetic acid. Plant Physiol. 1951;26:192–195. doi: 10.1104/pp.26.1.192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hardy RWF, Holsten RD, Jackson EK, Burns RC. The acetylene-ethylene assay for N2 fixation: laboratory and field evaluation. Plant Physiol. 1968;43:1185–1205. doi: 10.1104/pp.43.8.1185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Heisey RM, Putnam AR. Herbicidal effects of geldamycin and nigericin, antibiotics from Streptomyces hygroscopicus. J Nat Prod. 1986;49:859–865. doi: 10.1021/np50047a016. [DOI] [PubMed] [Google Scholar]
  20. Hirsch AM, Fang Y. Plant hormones and nodulation: what is the connection? Plant Mol Biol. 1994;26:5–9. doi: 10.1007/BF00039514. [DOI] [PubMed] [Google Scholar]
  21. Holl FB, Chanway CP, Turkington R, Radley RA. Response of crested wheatgrass (Agropyron cristatum L.), perennial ryegrass (Lolium perenne) and white clover (Trifolium repens L.) to inoculation with Bacillus polymyxa. Soil Biol Biochem. 1988;20:19–24. doi: 10.1016/0038-0717(88)90121-6. [DOI] [Google Scholar]
  22. Karen S, Udo B, Frank L, Dominique R. Can simultaneous inhibition of seedling growth and stimulation of rhizosphere bacterial populations provide evidence for phytotoxin transfer from plant residues in the bulk soil to the rhizosphere of sensitive species? J Chem Ecol. 2001;27:807–829. doi: 10.1023/A:1010362221390. [DOI] [PubMed] [Google Scholar]
  23. Kirkegaard JW, Munns R, James RA, Gardener PA, Angus JF. Reduced growth and yield of wheat with conservation cropping. II. Soil biological factors limit under direct drilling. Aust J Agric Res. 1993;46:75–88. doi: 10.1071/AR9950075. [DOI] [Google Scholar]
  24. Knight TJ, Langston-Unkeffer PJ. Enhancement of symbiotic dinitrogen fixation by a toxin-releasing plant pathogen. Science. 1988;241:951–954. doi: 10.1126/science.241.4868.951. [DOI] [PubMed] [Google Scholar]
  25. Lifshitz R, Kloepper JW, Kozlowski M. Growth promotion of canola (rapeseed) seedlings by a strain of Pseudomonas putida under gnotobiotic conditions. Can J Microbiol. 1987;33:390–395. doi: 10.1139/m87-068. [DOI] [Google Scholar]
  26. Loper JE, Schroth MN. Influence of bacterial sources of indole-3-acetic acid on root elongation of sugar beet. Plant Pathol. 1986;76:386–389. [Google Scholar]
  27. Mayer AM. Determination of indole acetic acid by the Salkowsky reaction. Nature. 1958;182:1670–1671. doi: 10.1038/1821670a0. [DOI] [PubMed] [Google Scholar]
  28. Muller F, Deigele C, Ziegler H. Hormonal interactions in the rhizosphere of maize (Zea mays L.) and their effects on plant development. Zournal Pflanzen Bordennk. 1989;152:247–254. doi: 10.1002/jpln.19891520217. [DOI] [Google Scholar]
  29. Nishijima F, Evans WR, Vesper SJ. Enhanced nodulation of soybean by Bradyrhizobium in the presence of Pseudomonas fluorescens. Plant Soil. 1988;111:149–150. doi: 10.1007/BF02182049. [DOI] [Google Scholar]
  30. Okon Y, Vanderleyden J. Root associated Azospirillum species can stimulate plants. Am Soc Microbiol News. 1997;63:366–370. [Google Scholar]
  31. Palleroni NJ. Family 1. Pseudomonadaceae. In: Krieg NR, Holt JG, editors. Bergey’s manual of systemic bacteriology. Baltimore: Williams and Wilkins; 1984. pp. 143–213. [Google Scholar]
  32. Parmar N, Dadarwal KR. Stimulation of nitrogen fixation and induction of flavonoid-like compounds by rhizobacteria. J Appl Microbiol. 1999;86:36–44. doi: 10.1046/j.1365-2672.1999.00634.x. [DOI] [Google Scholar]
  33. Patten CL, Glick BR. Bacterial biosynthesis of indole-3-acetic acid. Can J Microbiol. 1996;42:207–220. doi: 10.1139/m96-032. [DOI] [PubMed] [Google Scholar]
  34. Persello-Cartieaux F, Nussaume L, Robaglia C. Tales from the underground: molecular plant-rhizobacterial interactions. Plant Cell Environ. 2003;26:189–199. doi: 10.1046/j.1365-3040.2003.00956.x. [DOI] [Google Scholar]
  35. Prikryl Z, Vancura V, Wurst M. Auxin formation by rhizosphere bacteria as a factor of root growth. Biol Plant. 1985;27:159–163. doi: 10.1007/BF02902155. [DOI] [Google Scholar]
  36. Remans R, Bebee S, Manrique MB, Tovar E, Rao I, Croonenborghs A, Torres-Gutierrez R, El-Howeity M, Michiels J, Vanderleyden J. Physiological and genetic analysis of root responsiveness to auxin-producing plant growth-promoting bacteria in common bean (Phaseolus vulgaris L.) Plant Soil. 2008;302:149–161. doi: 10.1007/s11104-007-9462-7. [DOI] [Google Scholar]
  37. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning. Cold Spring Harbor: A Laboratory Manual; 1989. [Google Scholar]
  38. Sarwar M, Kremmer RJ. Enhanced suppression of plant growth through production of L-tryptophan compounds by deleterious rhizobacteria. Plant Soil. 1995;172:261–269. doi: 10.1007/BF00011328. [DOI] [Google Scholar]
  39. Schippers AB, Bakker AW, Bakker PAHM. Interaction of deleterious and beneficial microorganism and effect on cropping practices. Annu Rev Phytopathol. 1987;25:339–358. doi: 10.1146/annurev.py.25.090187.002011. [DOI] [Google Scholar]
  40. Sindhu SS, Gupta SK, Dadarwal KR. Antagonistic effect of Pseudomonas spp. on pathogenic fungi and enhancement of plant growth in green gram (Vigna radiata) Biol Fertil Soils. 1999;29:62–68. doi: 10.1007/s003740050525. [DOI] [Google Scholar]
  41. Sloger C. Symbiotic effectiveness and nitrogen fixation in nodulated soybean. Plant Physiol. 1969;44:1666–1668. doi: 10.1104/pp.44.12.1666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Somers E, Vanderleyden J, Srinivasan M. Rhizosphere bacterial signaling: a love parade beneath our feet. Crit Rev Microbiol. 2004;30:205–240. doi: 10.1080/10408410490468786. [DOI] [PubMed] [Google Scholar]
  43. Spaepen S, Vanderleyden J, Remans R. Indole-3-acetic acid in microbial and microorganism-plant signaling. FEMS Microbiol Rev. 2007;31:425–448. doi: 10.1111/j.1574-6976.2007.00072.x. [DOI] [PubMed] [Google Scholar]
  44. Sturz AV, Christie BR. Beneficial microbial allelopathies in the root zone: the management of soil quality and plant disease with rhizobacteria. Soil Tillage Res. 2003;72:107–123. doi: 10.1016/S0167-1987(03)00082-5. [DOI] [Google Scholar]
  45. Suzuki S, Yuxi H, Oyaizu H, He Y. Indole-3-acetic acid production in Pseudomonas fluorescens HP72 and its association with suppression of creeping bentgrass brown patch. Curr Microbiol. 2003;47:138–143. doi: 10.1007/s00284-002-3968-2. [DOI] [PubMed] [Google Scholar]
  46. Taghavi S, Garafola C, Monchy S, Newman L, Hoffman A, Weyens N, Barac T, Vangronsveld J, van der Lelie D. Genome survey and characterization of endophytic bacteria exhibiting a beneficial effect on growth and development of poplar trees. Appl Environ Microbiol. 2009;75:748–757. doi: 10.1128/AEM.02239-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Vincent JM. A manual for the practical study of root nodule bacteria. International biological programme handbook 15. Oxford: Blackwell Scientific Publisher; 1970. [Google Scholar]
  48. Weller DM. Pseudomonas biological control agents of soilborne pathogens: Looking back over 30 years. Phytopathology. 2007;97:250–256. doi: 10.1094/PHYTO-97-2-0250. [DOI] [PubMed] [Google Scholar]
  49. Xie H, Pasternack JJ, Glick BR. Isolation and characterization of mutants of plant growth promoting rhizobacterium Pseudomonas putida GR12-2 that overproduce indole acetic acid. Curr Microbiol. 1996;32:67–71. doi: 10.1007/s002849900012. [DOI] [Google Scholar]
  50. Yoshikawa M, Hirai N, Wakabayashi K, Sugizaki H, Iwamura H. Succinic and lactic acids as plant growth promoting compounds produced by rhizospheric Pseudomonas putida. Can J Microbiol. 1993;39:1150–1154. doi: 10.1139/m93-173. [DOI] [Google Scholar]
  51. Zhang F, Dashti N, Hynes RK, Smith DL. Plant growth-promoting rhizobacteria and soybean [Glycine max (L.) Merr.] nodulation and nitrogen fixation at suboptimal root zone temperatures. Ann Bot. 1996;77:453–459. doi: 10.1006/anbo.1996.0055. [DOI] [Google Scholar]
  52. Zhang F, Dashti N, Hynes RK, Smith DL. Plant growth-promoting rhizobacteria and soybean [Glycine max (L.) Merr.] growth and physiology at suboptimal root zone temperatures. Ann Bot. 1997;79:243–249. doi: 10.1006/anbo.1996.0332. [DOI] [Google Scholar]

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