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Screening of plant growth-promoting rhizobacteria from maize (Zea mays) and wheat (Triticum aestivum).


Soil contains a wide diversity of microbes. Microbes are concentrated in nutrient-rich soil regions, including the topsoil layer and the region around the plant root. Roots secrete rhizodeposits that stimulate microbial life and allow fast spreading of microbes through soils. Plants can benefit from soil microbes in many ways. Certain microbes stimulate plant growth, enrich soils, degrade pollutants, or protect plants against pathogens. Analysis of the genotypic and phenotypic characteristics of indigenous rhizobacteria can help in understanding the mechanism of interactions between them and plant roots [1]. Plant growth-promoting rhizobacteria (PGPR) are free-living, soil-borne bacteria, which, when applied to seeds or crops, enhance the growth of the plant or reduce the damage from soil-borne plant pathogens [2]. The rhizosphere is the soil portion found around the root and under the influence of the root. It is the site with complex interaction between the root and associated microorganisms [3]. It has been noted by many workers that Pseudomonas, Bacillus, Arthrobacter, Azospirillum, Klebsiella, and Enterobacter, isolated from the rhizosphere of various crops, showed synergistic effects on plant growth [2, 4, 5]. Beneficial effects of micro-organisms have often been evaluated based on faster seed germination, better seedling emergence, and increased plant growth. Plant growth-promoting rhizobacteria enhance plant growth either by direct or indirect mechanisms [5]. The indirect mechanisms of plant growth promotion by PGPR include i) antibiotic production ii) depletion of iron from the rhizosphere iii) synthesis of antifungal metabolites iv) production of fungal cell wall lysing enzymes v) competition for sites on roots and vi) induced systemic resistance [2, 6, 7]. The production of phytohormones has been suggested to be one of the mechanisms by which PGPR bacteria stimulate plant growth [4]. Aslantas et al. [8] reported that the PGPR of apple showed the growth-promoting effect with possible involvement of the plant growth regulators indole-3-acetic acid and cytokinin. The objectives of the study were to isolate plant growth promoting rhizosphere bacteria from maize and wheat, and evaluate their beneficial effects on plant growth properties.


Pot experiments

Soil was collected from Forest Research Institute, Dehradun. This soil had pH 6.7 and contained 7.3 g/kg organic C, 590 mg/kg N, 60 mg/kg P, and 71 mg /kg K. Kjeldahl method was used to determine the total nitrogen content in the soil. For determination of the total organic carbon content, elementary analysis was used [9], while K and P content were also determined by flame photometric method and molybdenum blue method, respectively [10, 11]. For pot experiments, maize (Zea mays) and wheat (Triticum aestivum) were used as experimental plants.

Isolation and characterization of soil microorganisms

For the isolation of bacterial population, both maize and wheat plants were grown in the field. Plants were harvested 30 days after seed emergence were harvested. After harvesting, the roots and shoots were separated. To isolate the rhizospheric bacteria, loosely held soil to fresh roots were gently shaken with 9 ml of sterile distilled water containing 1 ml of 40% tetramethylthiuramdisulphide solution for 30 min. From the suspension, a serial dilution (1:100) was prepared, and the resulting suspensions were spread over the surface of a succinate agar medium (sodium glutamate 1.00 g, sodium succinate 5.00 g, ammonium sulphate 0.40 g, magnesium sulphate 0.20 g, dipotassium hydrogen sulphate 0.50 g, agar agar 15.0 g). Six inoculated plates per dilution were incubated at 28[degrees]C and the bacterial colony forming units (CFUs) were counted after 72 hours of incubation. After getting the pure culture of isolated bacterial population, identification and characterization was done by using morphological, physiological, Biochemical and bacterial detection kit methods (BIOCON INC., INDIA).

IAA production

After the confirmation of IAA production activity by isolated bacterial isolates, bacterial strains were grown in succinate broth with and without tryptophan (500 mg/ml) and incubated at 28[degrees]C for 72 hours. A 2ml culture was taken from each tube and centrifuged at 10,000 rpm for 15 min (REMI RM-12C DX). One milliliter of the supernatant was taken in a tube to which 100 ml of 10 mM orthophosphoric acid and 2 ml of reagent (1 ml of 0.5 M Fe[Cl.sub.3] in 50 ml of 35% HCl[O.sub.4]) were added. After 25 minutes (after color density reached its maximum), the mixture was read in UV-spectrophotometer at 530nm absorbance. The quantity of IAA was determined by comparison with a standard curve using pure IAA [12].

Gnotobiotic assay of Growth Pouch Study (GPS)

The gnotobiotic assay was performed as described by Lifshitz et al. [13]. Seeds were surface-sterilized by treatment with 95% ethanol (v/v) for 10 to 20 s, followed by soaking in 20% bleach (v/v) for 10 min. Seeds were washed with sterile distilled water 5-7 times in order to remove excess bleach. The seeds were then air dried by placing them in the laminar flow hood for 24 h. Surface-sterilized seeds were picked at random and placed onto half- strength tryptic soy agar (TSA) plates and incubated at 27[degrees]C for 24 h to further check for any contamination. Seeds were considered to be free from contamination if there were neither bacterial nor fungal growth occurring on plates incubated with surface-sterilized seeds.

A 100-fold dilution of the bacterial cells grown in half strength tryptic soy broth (TSB) was performed using 0.1 M MgS[O.sub.4]. Surface-sterilized seeds were soaked in 10 ml of bacterial suspension for 10-15 min with gentle agitation.

Growth pouch experiments were conducted in order to determine the ability of PGPR strains to produce the phytohormone, indole-3-acetic acid (IAA) in the presence of maize and wheat roots. The effect of PGPRs (plant growth promoting rhizobacteria) inoculation on maize and wheat (individually) and the concentration of IAA in the rhizosphere were measured at different time intervals (24h, 48h, 72h and 96h). The growth pouches were filled with 25 ml of Hoagland's nutrient solution and seeds treated with bacterial strains (maize and wheat, individually) were transferred aseptically to growth pouches (3 seeds per pouch and 3 pouches per treatment) [14]. Seeds treated with 0.1 M MgS[O.sub.4] were considered as controls. The growth pouches were incubated in a growth cabinet with gentle shaking at 100 rpm to create aerobic conditions for growth of PGPR strains. Supernatants from growth pouches (10 ml) were obtained by centrifugation at 4000 rpm for 20 min at 4[degrees]C (REMI RM-12C DX) and filtration using 0.22 [micro]m membrane filters. The phytohormone IAA was assayed using ELISA kits (Phytodetek, Agdia Inc, Elkhart, IN, USA). Stock solutions of the IAA (10 [micro]mole/ml) were prepared in absolute methanol. Standard concentrations of 78-2500 pmoles/ml (IAA) were used. One hundred microlitres of standard or the sample were used for each assay.

The intensity of the color was read at 405nm using an ELISA plate reader and related to phytohormone concentrations by means of a standard curve.

Plant growth promotion

The effect of isolated strains on plant growth was studied in pot experiments. The inoculation treatments were setup in a randomized design with six replicates.

The day before sowing, pots were filled with 350 g soil. The soil was moistened with water and maintained at 60% of its moisture holding capacity. Four seeds of maize and 4 of wheat were sown per pot. After germination, plants were thinned to two per pot. The bacteria were cultured in glycerin-peptone-medium. Tubes were secured on a rotary shaker on 120rpm for 72 hours at 23[degrees]C. Seedlings of these plants were inoculated with 1 ml of the bacterial suspension, which resulted in an inoculum's density of [10.sup.6]cfu/ml. The control was considered as un-inoculated plants. Plants were placed in a temperature-regulated growth chamber at a light intensity 20 kLux for 16 hours at 16[degrees]C during the day and 12[degrees]C at night.

Data analysis

The data were analysed for significant differences (P < 0.05) of main effects using 2-way ANOVA and Student-Newman-Keuls tests.


All sixty isolates were screened for their PGPR activities and fifteen of them were selected for further studies due to their higher IAA production ability than the rest of isolates. Among the isolates, the representative bacterial strains in the rhizosphere were identified as Bacillus lentus AK1, Bacillus sp. AK21, Azospirillum brasilense AK11, Bacillus subtilis AK31, Bacillus lentus AK8, Bacillus sp. AK2, Pseudomonas fluorescens AK30, Azotobacter diazotrophicus AK14, Microbacterium sp. AK19, Bacillus lentus AK17, Pseudomonas fluorescens AK32, Bacillus sp. AK3 and Bacillus halodurans AK15.

All 15 bacterial strains produced IAA (Table 3). The highest concentration of IAA was produced by bacterial strain Bacillus subtilis AK31, Pseudomonas fluorescens AK32 and Bacillus sp. AK21.

Gnotobiotic assay or growth pouch study (GPS) with maize revealed that with control experiment less amount of IAA production were recorded (0.15, 0.99, 1.51 and 2.43 pmol/ml at 24h, 48h, 72h and 96h, respectively), while PGPR inoculated seeds showed a good level of IAA production at different time intervals (Table 1). Similar results were also recorded with wheat seeds (Table 2). Furthermore, control experiments also revealed a deficient level of seed germination and radical length in maize (0.1, 0.4, 1.1 and 2.0 cm) and wheat (0.0, 0.2, 0.9 and 1.8 cm) at different time intervals (24h, 48h, 72h and 96h) and good germination ability as well as increased root length were recorded in maize and wheat (Tables 1 and 2) with bacterial inoculated seeds. These results may provide partial evidence that PGPRs and IAA play an important role in plant growth promotion.

Results of pot experiments showed that the bacterial strains increased the root dry weight and shoot dry weight of maize (30% and 26%) and wheat (40% and 10%) as compared to the control (Table 4).


Plant growth-promoting rhizobacteria colonize plant roots and exert beneficial effects on plant growth and development by a wide variety of mechanisms. To be an effective PGPR, bacteria must be able to colonize root environment because they need to establish themselves in the rhizosphere at population densities sufficient to produce the beneficial effects. Our results have demonstrated that selected plant growth-stimulating rhizosphere bacterial isolates were able to increase the growth of maize and wheat. Plant growth-promoting rhizobacteria promote plant growth by producing plant growth regulators or phytohormones [5]. These promote seed germination, root elongation, and stimulation of leaf expansion. Eighty percent of microorganisms, including pseudomonads, isolated from the rhizosphere of various crops have the ability to produce auxins as secondary metabolites [15, 16]. Researchers showed that root surface area of maize seedlings was significantly increased after inoculation with Azospirillum [17, 18, 19, 20, 21, 22]. Similar results revealed that Pseudomonas inoculation (P. fluorescens PsIA12 and Agrobacterium rhizogenes A1A4) stimulated wheat growth [23, 24].

Many scientists reported that plant growth promoting rhizobacteria might enhance plant height and productivity by synthesizing phytohormones [25, 26, 27]. In our experiments, all selected bacterial strains were able to produced IAA at different concentrations in comparison to controls. In summary, the bacteria-associated plant growth properties shown in our experiments suggested that PGPR bacteria were able to produce phytohormone auxin (IAA) that stimulated the growth of maize and wheat crops.

It was hypothesized that IAA and other plant hormones were responsible for increased growth of canola, tomato and wheat in non-sterile soil inoculated with Azotobacter paspali [28]. The experiment carried out in present work has demonstrated that Plant growth-promoting isolates have capacity to modify the growth of maize and wheat. Auxins produced by rhizobacteria can influence plant growth, including root development which improve uptake of essential nutrients thus increasing plant growth [29]. In the pot experiment, it was observed that inoculation with PGPR strains significantly promoted growth and increased the root-shoot dry weight of maize and wheat. These results are consistent with the findings of other researchers who assessed the inoculation effect of PGPR Azospirillum brasilense on growth and dry weight of spring wheat [18, 30]. Similarly, promotion in growth parameters and yields of various crop plants in response to inoculation with PGPR were reported by other workers [19, 20, 21, 27].


The greater root and shoot dry weight response to all inoculants compared to control clearly showed the beneficial role of these rhizobacteria. Such an improvement might be attributed to produce growth promoting substances. Based on these results, it can be concluded that, inoculation of Bacillus lentus, Bacillus sp., Azospirillum brasilense, Bacillus subtilis, Pseudomonas fluorescens, Azotobacter diazotrophicus, Bacillus halodurans and Pseudomonas strains as biofertilizer will be useful in increasing cereal productivity and contribute to tackling food deficits in the world.


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Karnwal A * (1)

* Corresponding Author:

(1) Reader, Bhojia Institute of Life Sciences, Teh. Nalagarh, Budh, Baddi, Solan, H.P. 173205.
Table 1: Effect of isolated PGPRs (plant growth-promoting
rhizobacterias) on maize root growth and IAA production in
Gnotobiotic study (Growth Pouch Study)

Bacterial          Experiment             Incubation Time (h)
Strains              Detail
                                      24      48      72      96

Control       IAA Conc. (pmol/ml)    0.15    0.99    1.51    2.43
              Root length (cm)       0.10    0.40    1.10    2.00

AK1           IAA Conc. (pmol/ml)    1.90    2.78    4.24    6.86
              Root length (cm)       0.40    1.30    2.50    4.10

AK21          IAA Conc. (pmol/ml)    2.00    2.88    4.40    7.11
              Root length (cm)       0.42    1.35    2.59    5.00

AK11          IAA Conc. (pmol/ml)    0.80    1.65    2.52    4.08
              Root length (cm)       0.17    0.77    1.49    4.50

AK31          IAA Conc. (pmol/ml)    2.00    2.88    4.40    7.11
              Root length (cm)       0.42    1.35    2.59    5.00

AK8           IAA Conc. (pmol/ml)    2.10    2.99    4.56    7.36
              Root length (cm)       0.44    1.40    2.68    3.80

AK2           IAA Conc. (pmol/ml)    1.10    1.96    2.99    4.83
              Root length (cm)       0.23    0.92    1.76    4.00

AK30          IAA Conc. (pmol/ml)    1.40    2.27    3.46    5.59
              Root length (cm)       0.29    1.06    2.04    3.00

AK14          IAA Conc. (pmol/ml)    1.00    1.86    2.84    4.58
              Root length (cm)       0.21    0.87    1.67    5.20

AK19          IAA Conc. (pmol/ml)    0.90    1.76    2.68    4.33
              Root length (cm)       0.19    0.82    1.58    5.00

AK17          IAA Conc. (pmol/ml)    1.30    2.17    3.31    5.34
              Root length (cm)       0.27    1.01    1.95    4.50

AK32          IAA Conc. (pmol/ml)    1.50    2.37    3.62    5.85
              Root length (cm)       0.32    1.11    2.13    5.30

AK3           IAA Conc. (pmol/ml)    1.00    1.86    2.84    4.58
              Root length (cm)       0.21    0.87    1.67    4.80

AK15          IAA Conc. (pmol/ml)    0.90    1.76    2.68    4.33
              Root length (cm)       0.19    0.82    1.58    3.30

AK9           IAA Conc. (pmol/ml)    1.50    2.37    3.62    5.85
              Root length (cm)       0.32    1.11    2.13    3.60

AK25          IAA Conc. (pmol/ml)    1.90    2.78    4.24    6.86
              Root length (cm)       0.40    1.30    2.50    4.80

Table 2: Effect of isolated PGPRs (plant growth-promoting
rhizobacterias) on wheat root growth and IAA production in
Gnotobiotic study (Growth Pouch Study)

Bacterial   Experiment Detail          Incubation Time (h)
                                   24      48      72      96

Control     IAA Conc. (pmol/ml)   0.10    0.97    1.47    2.25
            Root length (cm)      0.00    0.20    0.90    1.80

AK1         IAA Conc. (pmol/ml)   0.60    1.63    2.49    3.80
            Root length (cm)      0.20    0.70    1.70    3.50

AK21        IAA Conc. (pmol/ml)   0.90    2.03    3.10    4.73
            Root length (cm)      0.30    0.87    2.12    4.36

AK11        IAA Conc. (pmol/ml)   0.30    1.23    1.88    2.87
            Root length (cm)      0.10    0.53    1.28    2.64

AK31        IAA Conc. (pmol/ml)   1.50    2.83    4.32    6.59
            Root length (cm)      0.50    1.21    2.95    6.07

AK8         IAA Conc. (pmol/ml)   1.00    2.17    3.31    5.04
            Root length (cm)      0.33    0.93    2.26    3.60

AK2         IAA Conc. (pmol/ml)   1.20    2.43    3.71    5.66
            Root length (cm)      0.40    1.04    2.53    4.00

AK30        IAA Conc. (pmol/ml)   0.90    2.03    3.10    4.73
            Root length (cm)      0.30    0.87    2.12    3.00

AK14        IAA Conc. (pmol/ml)   1.10    2.30    3.51    5.35
            Root length (cm)      0.37    0.99    2.39    5.20

AK19        IAA Conc. (pmol/ml)   1.00    2.17    3.31    5.04
            Root length (cm)      0.33    0.93    2.26    4.60

AK17        IAA Conc. (pmol/ml)   0.40    1.37    2.09    3.18
            Root length (cm)      0.13    0.59    1.42    2.90

AK32        IAA Conc. (pmol/ml)   1.80    3.23    4.93    7.53
            Root length (cm)      0.60    1.39    3.37    6.90

AK3         IAA Conc. (pmol/ml)   0.80    1.90    2.90    4.42
            Root length (cm)      0.27    0.81    1.98    4.10

AK15        IAA Conc. (pmol/ml)   1.20    2.43    3.71    5.66
            Root length (cm)      0.40    1.04    2.53    5.20

AK9         IAA Conc. (pmol/ml)   1.60    2.97    3.50    5.34
            Root length (cm)      0.53    1.27    2.39    3.20

AK25        IAA Conc. (pmol/ml)   1.20    2.43    3.71    5.66
            Root length (cm)      0.40    1.04    2.53    4.80

Table 3: Indole-3-acetic acid (IAA) production by isolated bacterial
strains ([micro]g [ml.sup.-1] filtrate)

Origin               Bacterial strains                  IAA

Wheat rhizosphere    Bacillus lentus AK1                1.45
Wheat rhizosphere    Bacillus sp. AK21                  1.90
Wheat rhizosphere    Azospirillum brasilense AK11       0.60
Wheat rhizosphere    Bacillus subtilis AK31             2.62
Wheat rhizosphere    Bacillus lentus AK8                1.36
Wheat rhizosphere    Bacillus sp. AK2                   1.50
Wheat rhizosphere    Pseudomonas fluorescens AK30       1.30
Maize rhizosphere    Azotobacter diazotrophicus AK14    1.49
Maize rhizosphere    Microbacterium sp. AK19            1.30
Maize rhizosphere    Bacillus lentus AK17               1.11
Maize rhizosphere    Pseudomonas fluorescens AK32       2.70
Maize rhizosphere    Bacillus sp. AK3                   1.40
Maize rhizosphere    Bacillus halodurans AK15           1.55
Maize rhizosphere    Pseudomonas AK9                    1.25
Maize rhizosphere    Pseudomonas AK25                   1.50

Table 4: Influence of isolated bacterial strains on shoot and root
dry weight of maize and wheat

Bacterial strains                  Dry matter
                                   of Maize

                                   Root        Shoot

Control                            100         100
                                   (0.2021)    (0.1623)
Bacillus lentus AK1                110         101
Bacillus sp. AK21                  123 *       116
Azospirillum brasilense AK11       115         110
Bacillus subtilis AK31             130 *       126 *
Bacillus lentus AK8                110         101
Bacillus sp. AK2                   116         108
Pseudomonas fluorescens AK30       104         100
Azotobacter diazotrophicus AK14    121         116 *
Microbacterium sp. AK19            120         114
Bacillus lentus AK17               119         114
Pseudomonas fluorescens AK32       124 *       120 *
Bacillus sp. AK3                   116         110
Bacillus halodurans AK15           109         101
Pseudomonas AK9                    109         101
Pseudomonas AK25                   116         100

Bacterial strains                  Dry matter
                                   of Wheat

                                   Root        Shoot

Control                            100         100
                                   (0.1956)    (0.2205)
Bacillus lentus AK1                120         105
Bacillus sp. AK21                  130 *       105
Azospirillum brasilense AK11       106         100
Bacillus subtilis AK31             135 *       106
Bacillus lentus AK8                116         108
Bacillus sp. AK2                   120         101
Pseudomonas fluorescens AK30       109         100
Azotobacter diazotrophicus AK14    125 *       110 *
Microbacterium sp. AK19            118         102
Bacillus lentus AK17               110         101
Pseudomonas fluorescens AK32       140 *       110 *
Bacillus sp. AK3                   119         102
Bacillus halodurans AK15           123 *       106
Pseudomonas AK9                    115         101
Pseudomonas AK25                   120         103

* Significantly different from the control for P < 0.05
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Author:Karnwal, A.
Publication:African Journal of Food, Agriculture, Nutrition and Development
Article Type:Report
Geographic Code:9INDI
Date:May 1, 2012
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