Kocuria flava induced growth and chromium accumulation in Cicer arietinum L.

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Kocuria Flava Induced Growth and Chromium Accumulation in Cicer Arietinum L a

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N. K. Singh , U. N. Rai , D. K. Verma & G. Rathore

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Plant Ecology and Environmental Science Division , CSIR-National Botanical Research Institute , Lucknow , India b

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Exotic and Qurantine Management Division , National Bureau of Fish Genetic Resources , Lucknow , India Accepted author version posted online: 19 Apr 2013.Published online: 10 Sep 2013.

To cite this article: N. K. Singh , U. N. Rai , D. K. Verma & G. Rathore (2014) Kocuria Flava Induced Growth and Chromium Accumulation in Cicer Arietinum L, International Journal of Phytoremediation, 16:1, 14-28, DOI: 10.1080/15226514.2012.723065 To link to this article: http://dx.doi.org/10.1080/15226514.2012.723065

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International Journal of Phytoremediation, 16:14–28, 2014 C Taylor & Francis Group, LLC Copyright ISSN: 1522-6514 print / 1549-7879 online DOI: 10.1080/15226514.2012.723065

KOCURIA FLAVA INDUCED GROWTH AND CHROMIUM ACCUMULATION IN CICER ARIETINUM L N. K. Singh,1 U. N. Rai,1 D. K. Verma,2 and G. Rathore2 Downloaded by [Archives & Bibliothèques de l'ULB] at 03:43 05 February 2015

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Plant Ecology and Environmental Science Division, CSIR-National Botanical Research Institute, Lucknow, India 2 Exotic and Qurantine Management Division, National Bureau of Fish Genetic Resources, Lucknow, India In the present investigation a chromate tolerant rhizobacterium Kocuria flava was isolated and inoculated to the Cicer arietinum L to evaluate its effects on growth and chromium accumulation upon exposure of different concentration of chromium (1–10 μg ml−1) as Cr (VI) for 24 d. K. flava inoculated plant of C. arietinum demonstrated luxuriant growth as compared to non inoculated plant at respective concentration of Cr (VI). K. flava found to ameliorate chromium induced phytotoxicity in terms of chlorophylls, carotenoid and protein contents and thus helps the plant in acquiring higher biomass with high chromium concentration. After 24 d, maximum concentration of chromium recorded in root of C. arietinum (4892.39 μg g−1 dw) inoculated with K. flava as compared to non inoculated plant (1762.22 μg g−1 dw) upon exposure of 5 μg ml−1 Cr (VI). Therefore, application of C. arietinum in association with K. flava could be more efficient in decontamination of chromium polluted site. Moreover, K. flava may be used as a bioresource for developing microbes assisted phytoremediation system due to its compatibility. KEY WORDS: chromium, rhizobacterium, bioaccumulation, phytoremediation, growth promotion

INTRODUCTION Chromium is generated from various industrial processes like electroplating, leather tanning, wood preservations, manufacturing of dye, paint and paper (Khezami and Capart 2005). The maximum concentration allowed for chromium (VI) ions in drinking water is 0.05 mg l−1. The US EPA allows solutions containing heavy metals to be discharged if the concentration is usually less than 5.0 mg l−1. The chromate [Cr(VI)] is highly soluble and therefore can overcome the cellular permeability barrier, entering via sulphate transport pathways since it bears structural similarity with SO4 . Unless it is rapidly reduced it can oxidatively damage the DNA via the production of free radicals. Hexavalent chromium causes lung cancer (De Flora 2000), chromate ulcer, perforation of nasal septum and kidney damage in humans and toxic to other organisms as well. Due to leakage, poor storage and improper disposal, hexavalent chromium has become one of the most frequently detected contaminant at the waste disposal sites. Conventionally, hexavalent chromium containing

Address correspondence to U.N. Rai, Plant Ecology and Environmental Science Division, CSIR-National Botanical Research Institute, Lucknow – 226 001, India. E-mail: rai [email protected] 14


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industrial effluent is treated by physico-chemical methods like reduction, precipitation, ion exchange, reverse osmosis and electro-dialysis. However, these processes are costly and not-ecofriendly. Further, maximum achievable chromate removal efficiency by conventional methods is not sufficient to attain the desired treated effluent quality standard for disposal by the industries. Some methods, such as soil washing, can pose an adverse effect on biological activity, soil structure and fertility, and incur significant engineering costs (Pulford and Watson 2003). Phytoremediation, using plants to remove metal pollutants from contaminated soil is being developed as new methods for the remediation of contaminated land (AbouShanab et al. 2006). This environment friendly, cost effective and plant based technology is expected to have significant economic, aesthetic and technical advantages over traditional engineering techniques (Susarla et al. 2002). However, phytoextraction is frequently slower than traditional remediation techniques, requiring several growing seasons for site cleanup, because of small biomass of hyperaccumulator plants or poor capacity to take up metals of non-hyperaccumulator plants etc. (McIntyre 2003). Metals at supra optimal concentrations affect growth, development and yield of the plants (Sinha and Gupta 2005). Several practices to enhance phytoextraction efficiency have been suggested, such as use of chelators to increase bioavailability of low-solubility metals, genetic engineering and production of transgenic plants with strong tolerance and metal accumulation ability, use of rhizosphere microbes to enhance biomass of hyperaccumulator plants, and other agronomic practices (Prasad and Freitas 2003). One way to relieve toxicity of heavy metals to plants might involve use of plant growth promoting bacteria or free-living soil bacteria that exert some beneficial effect on plant development when they are either applied to seeds or incorporated into the soil. While plants may protect bacteria from toxic metals in the surrounding medium by providing a surface for attachment as well as nutrients (Andrews and Harris 2000), bacteria may also protect plants from metal toxicity, by mechanisms such as depositing metals outside of bacterial cell walls (Lodewyckx et al. 2002). Therefore, the potential use of alternative methods that exploit rhizosphere microbes to enhance phytoextraction potential of hyperaccumulating plants has been investigated (Abou-Shanab et al. 2003; Pal et al. 2005). Despite importance of rhizobacteria in the metal remediation, introduced inoculation of indigenous microbes for increasing phytoremediation potential of plants through biostimulation has largely been ignored (Singh et al. 2010). In the context of above, improving plant- microbe interaction and introduction of beneficial rhizospheric microorganism are vital for increased biomass production and tolerance of plant to heavy metals (Glick 2003). Therefore, in this study, we investigated indigenous chromate tolerant rhizobacterium K. flava from the rhizosphere of Scirpus lacustris, its effects on growth and chromium accumulation in Cicer arietinum for implications these plant-associated bacteria for enhanced chromium removal.

MATERIALS AND METHODS Isolation of Chromate Tolerant Rhizobacteria For isolation of chromate tolerant rhizobacterial strain the plant Scirpus lacustris was collected from chromium contaminated area of Unnao U.P., India and brought to the laboratory in sterilized polythene bags. Root of S. lacustris washed thoroughly with tap water for 2 min followed by sterile 0.85% (w/v) saline milli Q water (MQW) (Millipore Corporation, USA). Washed root macerated in 0.85% saline MQW with a mortar and pistal

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for homogenization. Serial dilutions (0.1 ml) of the homogenate was poured on nutrient agar plate (Hi Media Laboratories Pvt. Ltd., India) containing different concentration of chromium (as K2 Cr2 O7 ) and incubated at 28 ± 2◦ C as described earlier (Nautiyal 1997). The most efficient chromate tolerant rhizobacterium tolerated high concentration of chromate was screened, purified, designated as RZB-03 and stored at 4◦ C for further tests (Singh et al. 2010).


MORPHOLOGICAL AND BIOCHEMICAL CHARACTERIZATION Morphological characterization was performed by using a standardized method (Murray et al. 1994). Biochemical test of chromate tolerant rhizobacterium was done by using KBOO3 Hi 25TM Identification kit (Hi Media Laboratories Pvt. Ltd., India). Susceptibility for different antibiotics of the RZB-03 strain was determined by the disc diffusion method (Bauer et al. 1996). Antibiotic impregnated discs were placed on freshly prepared lawn of each isolate on Muller Hinton agar plates and examined for the diameter of inhibition zone. Identification of Chromate Tolerant Rhizobacterium For the 16S rDNA based identification genomic DNA was extracted and amplified in PCR using the genomic DNA as template and bacterial universal primers, 20F 5 AGAGTTTGATC(AC)TGGCTCAG-3 (position at 8-27 E. coli numbering) and 1500R 5 -CGATCCTACTTGCGTAG-3 (position at 1510-1492 E. coli numbering) were used (Weisburg et al. 1991). PCR amplification was performed with final volumes of 50 μl, using 0.4 μl of Taq DNA polymerase (5 U μl−1 fermentas), 10 mM dNTPs (fermentas), 1.5 mM MgCl2 (fermentas), 10 pM of each primer and 400 ng of purified DNA. Subsequently sample was subjected to a pre-heating cycle at 95◦ C for 5 min. Each amplification cycle consisted of DNA denaturation at 95◦ C for 1 min, primer annealing to the template at 52◦ C for 1 min, followed by a primer extension at 72◦ C for 1.5 min. The 35th cycle involved a primer extension at 72◦ C for 10 min followed by an infinite period at 4◦ C. PCR products were separated on a 0.8% agarose gel with ethidium bromide (0.5 μg mL−1) at a constant voltage of 7V cm−1. The amplification product was purified using a DNA purification kit (Qiagen). Sequencing of the purified PCR product was performed by Ocimum Biosolutions Pvt. Ltd. (India). Sequence data were compiled and sequence similarities were calculated with the DNASTAR Lasergene software. The nucleotide substitution rate was calculated and a distance matrix tree was constructed by the neighbor-joining method (Saitou and Nei 1987), using the program CLUSTAL W (Thompson et al. 1994). Alignment positions with gaps and unidentified bases were not considered in the calculations. Phylogenetic tree was constructed by using MEGA ver 4.1 software (http://www.megasoftware.net). The topology of the tree was evaluated by bootstrap analysis with 1,000 replicates. The nucleotide sequence of 16S rDNA gene deposited to GenBank database under accession number JF750332. Experimental Setup For this study, seeds of the Cicer arietinum L. (var. CSG-8962) were procured from Indian Institute of Pulse Research, Kanpur (India). To evaluate the effects of chromate tolerant rhizobacteria K. flava on growth and chromium accumulation in C. arietinum the



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seeds were inoculated with K. flava. The chromate tolerant strain K. flava previously isolated form S. lacustris was used to inoculate the seed of C. arietinum. For seed inoculation pure culture of K. flava was taken from agar plates into nutrient broth and allowed to grow up to 108–1010 cfu ml−1 on shaker for 48 h at 28◦ C (40% moisture). The resultant bacterial culture (108–1010 cfu ml−1) mixed with autoclaved talc and seeds of C. arietinum previously sterilized by 70% ethanol (5 min.), 20% Bleach chlorox (10 min.), followed by three rinses in sterile MQW wetted in gum arabic (10%) and incubated for 24 h at 28–30◦ C (Singh et al. 2010). After incubation seeds were soaked with distilled water separately and covered by black carbon paper and kept in dark for germination. After germination young seedlings were transferred and acclimatized in a culture room for further treatments. Acid washed beakers (250 ml) covered with muslin cloths was used to provide base for growing seedlings of C. arietinum in 10% Hoagland nutrient solution (Hoagland and Arnon 1950). The stock solution of Cr was made-up by using K2 Cr2 O7 in 10% Hoagland solution and different concentration (0.0, 1.0, 5.0, and 10.0 μg ml−1) of Cr were made by diluting the stock solution with 10% nutrient’s solution. Seeds mixed with talc and broth without K. flava served as control. The seedlings (5 nos.) of C. arietinum were placed on muslin cloth which covered 250 ml beaker containing 0.0, 1.0, 5.0, and 10.0 μg ml−1 concentrations of chromate in 10% Hoagland solution in triplicate. Plants were provided with a light intensity of 115 μ mol m−2 s−1 for 14 h L / 10 h D at 25 ± 2◦ C.

Plant Harvest The harvesting of plants was carried out after the each treatment duration. Whole plant was uprooted and washed with deionized water and rinsed repeatedly, blotted dried and then roots and shoots were separated manually for estimation of various parameters.

Biomass Estimation Plants were blotted dry and weighed after each harvesting period and biomass was calculated on dry weight basis (g dw). For dry weight determinations fresh plant tissues were kept separately in oven at 105◦ C till a constant weight is obtained.

Estimation of Chlorophyll Content Treated plant material (100 mg) was crushed in 5 ml of (80%, v/v) chilled acetone following the method of Arnon (1949) and estimated according to Machlachan and Zalik (1963). Extract was centrifuged at 10,000 g for 10 min and absorbance of supernatant was read at 663 and 645 nm using UVICON spectrophotometer.

Carotenoid Estimation Known amounts of plant samples (100 mg) were extracted in 80% chilled acetone and absorbance was read on UVICON spectrophotometer at wavelengths 480 and 510 nm. Amount of carotenoid was calculated in mg g−1 (FW) by the formula given earlier (Duxbury and Yentsch 1956).

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Estimation of Protein Content Plant material (100 mg) crushed in 5 ml of 10% of chilled trichloroacetic acid (TCA) and centrifuged at 10000 g for 10 min. After decanting the supernatant pellets were washed and resuspend in 5 ml of 1 N NaOH and heated for 15 min, cooled and again centrifuged at 10000 g for 10 min. Protein content was estimated following the method of Lowry et al. (1951) using bovine serum albumin (Sigma) as standard.


Chromium Estimation 100 mg of dried plant materials were taken separately and digested in HNO3 :HClO4 (3:1) mixture and diluted to 10 ml with double distilled water and the solution was filtered through whatman filter paper No. 40 (ashless) and final volume was made 100 ml. Analysis of Cr was done by using GBC Avanta Atomic Absorption Spectrophotometer. Statistical Analysis The all experiments were setup in the randomized block design. To confirm the variability of data and validity of results, all data were subjected to two way analysis of variance (ANOVA) and to determine significance between treatments. For group wise comparison of means Duncan’s Multiple Range Test (DMRT) was applied to see the significant level (Gomez and Gomez 1984). RESULTS AND DISCUSSION Isolation and Characterization of Chromate Tolerant Rhizobacteria Amongst thirty seven isolates isolated form plant, S. lacustris growing in chromium contaminated area, one strain (RZB-03) tolerated high concentration of chromate (600 μg ml−1). The strain (RZB-03) was characterized as Gram-positive, aerobic, non-motile and coccoid. The strain (RZB-03) responded positive test for catalase, nitrate reduction, amylase, phosphatase and urease, however, negative for oxidase, gelatinase, β-glucuronidase, production of indole and acetoin (Voges-Proskauer reaction). The strain grew in the absence of NaCl and in the presence of up to 10% NaCl, at 28–45◦ C and pH 7–9 and found sensitive to kanamycin, ampicillin, carbenicillin, chlortetracycline and erythromycin. The isolated chromate tolerant strain (RZB-03) was identified as Kocuria flava (JF750332) by 16S rDNA sequencing (Fig. 1) and selected for study on the basis of its high degree of chromate tolerance. The Kocuria sp. was also recovered from varied environments (Zhou et al. 2008) and higher Cr (VI) resistance and Cr (VI) reduction capability of Kocuria sp. were reported from higher Cr contaminated site (Desai et al. 2009). Effect of Inoculation of K. flava on Growth of C. arietinum Rhizobacterium K. flava inoculated and non inoculated plants of C. arietinum were grown under different Cr concentration for 24 d along with control. The growth response of C. arietinum was evaluated by determining various growth parameters. Root length, shoot length and biomass of the C. arietinum was reduced against increase Cr (VI) concentration in the medium, however, an increase was found in K. flava inoculated plant at respective Cr concentrations (Fig. 2A-B & 3A). There was 50% reduction in root length at 5 μg ml−1 of


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Kocuria himachalensis (AY987383)

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Kocuria rosea (X87756)

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Kocuria polaris (AJ278868)

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Kocuria aegyptia (DQ059617)

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Kocuria turfanensis (DQ531634) Kocuria flava (EF602041) 100

Kocuria flava strain RZB-03 (JF750332)


Kocuria palustris (Y16263) Kocuria rhizophila (Y16264)

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Kocuria marina (AY211385)

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Kocuria carniphila (AJ622907) Micrococcus luteus (AJ536198)

0.005 Figure 1 Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequences, showing the position of strain RZB-03 among species of the genus Kocuria. Numbers at nodes represent bootstrap support (%) analysis of 1000 resampled datasets (only values>50% are indicated) Bar, 0.005 substitutions per nucleotide position. Micrococcus luteus was used as an out group.

Cr, in contrast to 10% increase observed in K. flava inoculated plants at 24 d of exposure. Similarly, minimum shoot length observed at 10 μg ml−1 of Cr(VI), however, an increase of 10% in shoot length was recorded in K. flava inoculated plant. Cr affects growth of the test plant as evidenced by decrease in root and shoot length by increasing Cr concentration in the medium. Similarly, root elongation of B. napus has also been reported by PGPR (Sheng and Xia 2006) as well as non- identified rhizobacteria on the B. juncea root (Belimov et al. 2005). Bacteria associated with roots of the plants can strongly act on the plant nutrition and provide resistance (Dimkpa et al. 2009; Belimov et al. 2009). Shoot length was found maximum, i.e., 23.43 cm in K. flava inoculated plant and minimum (7.29 cm) in noninoculated plant at 24 d of Cr(VI) exposure. Similarly, Egamberdieva D. (2008) reported increased the root and shoot growth of wheat and peas when inoculated with Kocuria sp. along with other bacteria and demonstrated production of indole-3 acetic acid (IAA) by inoculated strains, which most probably accounted for the overall synergistic effect on growth. Biomass recorded maximum i.e. 1.97 g dw and 1.83 g dw in K. flava inoculated plants after 24 d and 12 d of treatment, respectively, as compared to noninoculated plants, i.e., 1.66 g dw and 1.15 g dw at 0 μg ml−1 of Cr concentration. A significant increase in biomass of C. arietinum by K. flava inoculation has been observed at 5 and 10 μg ml−1 of Cr(VI) concentration as compared to noninoculated plant (DMRT, P < 0.05) (Fig. 3A). Numerous mechanisms involved in mutualistic relationships that PGPR able to modify the plant growth and development by increasing nutrient and water uptake and therefore, the plant biomass (Belimov et al. 2004). In general the metal resistant rhizosphere bacteria have exceptional ability to protect the host plants from metal toxicity by several possible mechanisms. The best known mechanism is the utilization ACC by rhizosphere bacteria. The other mechanisms or regulators driven by PGPR, such as siderophores, specific enzymes, organic acids involved in phosphorus


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Figure 2 Effect of K. flava (RZB-03) inoculation on [A] root length (cm) and [B] shoot length (cm) of C. arietinum grown under different Cr concentration (μg ml−1) at different treatment duration. All values are mean of three replicate ± S.D. ANOVA significant at P < 0.01. Different letters indicate significantly different value (DMRT, P < 0.05).

solubilization, fixation of atmospheric N2 (Pattern and Glick 1996) or in plant protection against heavy metal toxicity (Burd et al. 1998). Similarly, chlorophyll a content was found maximum i.e. 1.41 and 1.77 mg g−1 fw after 12 and 24 d of growth in non inoculated plants at 0 μg ml−1 of Cr concentration, respectively. However, minimum chlorophyll a content was recorded, i.e., 0.80 mg g−1 fw in noninoculated plants followed by 1.61 mg g−1 fw in inoculated plants after 24 d of exposure at 10 μg ml−1 of Cr indicating a concentration and duration dependent inhibition of chlorophyll a by Cr and promotion by K. flava (Fig. 3B). Chlorophyll b content was



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Figure 3 Effect of K. flava (RZB-03) inoculation on [A] biomass (g) and [B] chlorophyll a (mg g−1 fw) content of C. arietinum grown under different concentration of Cr (μg ml−1) and at different treatment durations. All values are mean of three replicate ± S.D. ANOVA significant at P < 0.01. Different letters indicate significantly different value (DMRT, P < 0.05).

also found maximum i.e. 0.88 mg g−1 fw at 0 μg ml−1 of Cr concentration and minimum i.e. 0.19 mg g−1 fw in noninoculated plant followed by 0.37 mg g−1 fw in inoculated plant after 24 d of treatment at 10 μg ml−1 of Cr concentration. (Fig. 4A). In this case, it is interesting to note that although K. flava inoculation enhanced chlorophyll b content under both control and Cr supplemented medium but at higher concentration of Cr the effect was insignificant (DMRT, P < 0.05). As shown in Fig. 4B maximum total chlorophyll content (2.96 mg g−1 fw) was recorded at 0 μg ml−1 of Cr concentration in plants inoculated with

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Figure 4 Effect of K. flava (RZB-03) inoculation on [A] chlorophyll b (mg g−1 fw) and [B] total chlorophyll (mg g−1 fw) content of C. arietinum grown under different concentration of Cr (μg ml−1) at different treatment durations. All values are mean of three replicate ± S.D. ANOVA significant at P < 0.01. Different letters indicate significantly different value (DMRT, P < 0.05).

K. flava. However, total chlorophyll content decreased to 0.83 mg g−1 fw in non-inoculated plant after 24 d of exposure at 10 μg ml−1 of Cr concentration. Chlorophyll a, b and total content increased in K. flava inoculated C. arietinum, as compared to non inoculated plants under Cr stress, however, a decrease in chlorophyll content was observed at higher



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Cr concentration. Metals have a potential to alter the rate of photosynthesis by disturbing the structure of chloroplast leading to the changes in the fatty acid composition, inhibiting photosynthetic pigment and enzymes of Calvin cycle (Vazquez et al. 1987). Similarly, concentration-duration dependent reduction in carotenoid content was evident; however, this reduction in carotenoid content was restored in the plants inoculated with K. flava (Fig. 5A). Maximum carotenoid content was found in inoculated control plants, i.e., 0.45 mg g−1 fw after 24 d of growth, however, the minimum concentration of carotenoid was recorded i.e. 0.01 mg g−1 fw after 12 d in noninoculated followed by 0.075 mg g−1 fw in inoculated plants. An increase in the amount of carotenoids was found at higher concentration of Cr in the present study, which is concomitant to previous studies Vallisneria spiralis exposed to Cr (Vajpayee et al. 2001) but not in case of Nymphaea, which could be ascribed due to variations in metal toxicity and tolerance in the plants. There was an increase in protein content of the plant up to 5 μg ml−1 concentration of the Cr at both the treatment durations (Fig. 5B), however, it reduced significantly at 10 μg ml−1 of Cr concentration. It is interesting to mention that there was insignificant (DMRT, P < 0.05) reduction in protein content of the plant inoculated with K. flava, under different Cr concentrations. Protein content in K. flava inoculated plants increased by 5–10% at different Cr concentration. Maximum protein content was found, i.e., 41.65 mg g−1 fw at 5 μg ml−1 Cr concentration after 12 d of treatment in inoculated plant, while minimum protein content i.e. 24.82 mg g−1 fw was recorded at 10 μg ml−1 of Cr treatment after 24 d of exposure. Higher concentration of Cr had toxic effect on the protein content. A decrease in protein content in presence of Cr or other metals may be due to the breakdown of soluble protein or due to the increased activity of protease or other catabolic enzymes, which were activated and destroyed the proteins. Inoculation with rhizobacterial strain K. flava most likely mitigated the toxicity of the metals and thus increased biomass and protein content in presence of Cr. Similar observation have been reported in Brassica juncea under metal stress (Wu et al. 2006). Effect of Inoculation K. flava on Cr Accumulation in C. arietinum The Cr accumulation in shoot and root part of C. arietinum growing at different concentration of Cr with and without K. flava inoculation is depicted in Fig. 6A & 6B. Maximum Cr accumulation in shoot of C. arietinum was recorded i.e. 61.15 μg g−1 dw in inoculated plant followed by 59.73 μg g−1 dw in noninoculated plant at 10 μg ml−1 Cr concentration after 24 d of treatment. In contrast, root accumulated significantly higher amount of Cr showing less transportation in the above ground part of plant at 10 μg ml−1 Cr concentration. There was less accumulation of Cr in the root at higher concentration of Cr which might be ascribed due to phytotoxicity of Cr at higher concentration. The maximum accumulation of Cr was found in the root of inoculated plant i.e. 4892.39 μg g−1 dw as compared to noninoculated plant, i.e., 1762.22 μg g−1 dw at 5 μg ml−1 Cr concentration after 24 d of treatment. K. flava inoculation greatly affected Cr accumulation in plants C. arietinum grown in Cr supplemented nutrient solution. Cr accumulation was more in roots as compared to shoots, as roots of the plants act as a barrier against heavy metal translocation and this may be a potential tolerance mechanism operating in the roots (Ernst et al. 1992; Singh et al. 2010). The degree of Cr accumulation in roots was more as compared to shoot suggest that C. arietinum is capable of well balanced uptake and translocation under Cr stress. Presence of rhizobacteria has been reported to increase accumulation of Zn (Whiting et al. 2001) and Ni (Abou-Shanab et al. 2003) in T. caerulensces and A. murale

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Figure 5 Effect of K. flava (RZB-03) inoculation on [A] carotenoid (mg g−1 fw) and [B] protein content (mg g−1 fw) of C. arietinum grown under different concentration of Cr (μg ml−1) at different treatment durations. All values are mean of three replicate ± S.D. ANOVA significant at P < 0.01. Different letters indicate significantly different value (DMRT, P < 0.05).

respectively. Similarly, Rai et al. (2004) reported inoculation of plant with a fly ash tolerant Rhizobium strain (PJ-1), which conferred tolerance for the plant to grow under fly ash stress conditions with more translocation of metals to the above ground parts. Plant growth promoting rhizobacterial strains K3 and S 32 reported to promote growth of Brassica

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Figure 6 Effect of K. flava (RZB-03) inoculation on chromium accumulation (μg g−1 dw) in [A] shoot and [A] root of C. arietinum grown under different chromium concentrations (μg ml−1) at different treatment duration. All values are mean of three replicate ± S.D. ANOVA significant at P < 0.01. Different letters indicate significantly different value (DMRT, P < 0.05).

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juncea under Cr stress (Rajkumar et al. 2005). Similarly, improvement of rape plant growth and Cd uptake by Cd resistant bacteria has been reported (Sheng et al. 2008). It is also reported that K. flava removed 97% of copper at 1000 mg L−1 initial Cu concentration and produced urease, an enzyme that leads to microbially induced calcite precipitation (Achal et al. 2011). Therefore, in our study, plant growth promotion and increased Cr accumulation in K. flava inoculated plants may be due synergistic consequence of production of plant growth promotory products (PGP) and reduction in phytotoxicity. However, the specific mechanisms can be determined by assaying the production of PGP by K. flava in the presence and absence of Cr(VI).

CONCLUSIONS Application of microbial isolates to remediate metal contaminated site is emerging as one of the strategies in environmental remediation. Although, various investigations have been done on decontamination of metals using metal tolerant and accumulator plants, limited understanding of role and impacts of microbes in metal contaminated environment during remediation processes needs to be taken to make phytoremediation technology more feasible and efficient. The present study is a attempt to understand potential of chromate tolerant rhizobacteria in promoting growth, tolerance and Cr accumulation in plant growing under Cr stress. The data obtained during the present investigation clearly indicate that inoculation of K. flava facilitated growth and Cr accumulation in plant Cicer arietinum under high chromate concentration and thus increased phytoremediation potential. Therefore, significance of exploiting suitable combination of indigenous bacterial strains with hyper-accumulator plant for alleviation of metals from environment is envisaged for rehabilitation of metal polluted habitat to improve soil. Further, studies are warranted for field trials in order to directly assess suitability of the rhizobacterial inoculants for the efficient bioremediation and producing safer crop in Cr contaminated area.

ACKNOWLEDGMENTS The authors would like to thank Dr. C.S. Nautiyal, Director, National Botanical Research Institute, Lucknow for providing facilities. The author NKS is grateful to Council of Scientific and Industrial Research, New Delhi, India for award of Research Associateship.

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Colchicine induced polyploidy in chickpeas (Cicer arietinum L.).

Quantitative inheritance and barriers to crossability in Cicer arietinum L.

Determination of Photoperiod-Sensitive Phase in Chickpea (Cicer arietinum L.).

Genetic transformation in the grain legume Cicer arietinum L. (chickpea).

Dissecting the Root Nodule Transcriptome of Chickpea (Cicer arietinum L.).

Global transcriptome analysis of developing chickpea (Cicer arietinum L.) seeds.

Trichoderma inoculation augments grain amino acids and mineral nutrients by modulating arsenic speciation and accumulation in chickpea (Cicer arietinum L.).

Adaptive responses and genetic divergence in a world germplasm collection of chick pea (Cicer arietinum L.).

Molecular Mapping of Flowering Time Major Genes and QTLs in Chickpea (Cicer arietinum L.).

Immunohistochemical localization of β-glucosidases in lignin and isoflavone metabolism in Cicer arietinum L. seedlings.

Hormonal regulation of growth of Cicer arietinum L. epicotyls. changes in the chemical composition of the cell wall.

Synergistic effect of Pseudomonas putida and Bacillus amyloliquefaciens ameliorates drought stress in chickpea (Cicer arietinum L.).

Somatic embryogenesis and plant regeneration from callus cultures of chickpea (Cicer arietinum L.).

Cold stress alters transcription in meiotic anthers of cold tolerant chickpea (Cicer arietinum L.).

Nuclear phosphoproteome of developing chickpea seedlings (Cicer arietinum L.) and protein-kinase interaction network.

Study of the Hypoglycemic Activity of Derivatives of Isoflavones from Cicer arietinum L.

Early generation yield testing versus visual selection in chickpea (Cicer arietinum L.).

A trypsin and chymotrypsin inhibitor from chick peas (Cicer arietinum).

Preliminary characterization of a cholinesterase from roots of Bengal gram - Cicer arietinum L.

Efficient transgenic plant regeneration throughAgrobacterium-mediated transformation of Chickpea (Cicer arietinum L.).

Isolation of sodium chloride-tolerant callus line of Cicer arietinum L. cv. BG-203.

Siderophore biosynthesis genes of Rhizobium sp. isolated from Cicer arietinum L.

An advanced draft genome assembly of a desi type chickpea (Cicer arietinum L.).

Colloidal Nanomolybdenum Influence upon the Antioxidative Reaction of Chickpea Plants (Cicer arietinum L.).