European Journal of Soil Biology 75 (2016) 142e150

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European Journal of Soil Biology

journal homepage: http://www.elsevier.com/locate/ejsobi

Original article

Role of soil associated Exiguobacterium in reducing arsenic toxicity and

promoting plant growth in Vigna radiata
Neha Pandey*, Renu Bhatt
Department of Biotechnology, Guru Ghasidas Vishwavidyalaya (A Central University), Bilaspur, Chhattisgarh, 495009, India

a r t i c l e i n f o a b s t r a c t

Article history: The present work describes the effects of inoculating metal resistant and plant growth promoting bac-
Received 8 January 2016 teria (PGPB) on the growth of Vigna radiata under arsenic (As) stress. The Exiguobacterium sp. strain, As-9
Received in revised form was isolated from arsenic contaminated soil, which showed resistance to exceptionally high concen-
24 May 2016
trations of As(V) and As(III). Assessment of plant growth promoting parameters revealed the ability of the
Accepted 31 May 2016
strain for the solubilization of phosphate, production of indole-3-acetic acid (IAA) and exopolysaccharide
(EPS). Soil inoculated with this arsenic resistant PGPB in the presence of high concentrations of As(V) and
Handling Editor: C.C. Tebbe As(III) was used for the germination of seeds of V. radiata under controlled conditions. The in vitro ex-
periments proved that Exiguobacterium significantly (p < 0.05) increased the shoot and root biomass of
Keywords: V. radiata in the presence of As(V) and As(III) together with increase in the plant height, survival index
Exiguobacterium and chlorophyll content after 15 days of inoculation. It also protected the plants from the detrimental
Arsenate effects of arsenic by reducing its uptake and translocation by colonizing the root surface. In addition,
Arsenite assay of antioxidant enzymes and lipid peroxidation test revealed significant (p < 0.01) reduction in
Plant growth promotion
arsenic-induced oxidative stress in V. radiata in the presence of bacteria. Owing to its wide action
Oxidative stress
spectrum, the arsenic resistant PGPB could provide a new insight into the remediation of arsenic
contaminated soil and serve as an effective growth promoting bioinoculant for plants in metal stressed
soil.
© 2016 Elsevier Masson SAS. All rights reserved.

1. Introduction Arsenic is a nonessential element for plant; however depression

Arsenic (As) is a toxic metalloid which is globally distributed in
the environment [1] and poses a major health problem [2]. Toxicity
and chemical behaviour of arsenic compounds are largely influ-
enced by the form and speciation, the inorganic arsenite [As(III)]
and arsenate [As(V)] being the dominant and toxic species [3].
Moreover, unlike organic pollutants, arsenic cannot be degraded to
harmless product instead persist indefinitely in the environment
and so is the focus of public attention. The existence of arsenic
residues in different oxidation states in rock, soil and ground water
[4] contributes to major health hazards not only to humans but to
plants and animals as well. The distribution of arsenic in soils may
vary with soil type, although concentrations ranging from 0.2 to
40 mg kg
1 have been reported [5] but the concentration tolerated
by plants ranges from 1 to 50 mg kg
1 depending upon the crop
variety [6].
* Corresponding author.
E-mail address: pandey.neha02@gmail.com (N. Pandey).
of plant growth usually occurs at higher levels of arsenic applica-
tion which leads to various phytotoxic symptoms such as inhibition
of seed germination, necrosis and wilting, decrease in plant height,
stunted root and shoot growth, lower fruit and grain yield, reduced
enzymatic activity and replacement of phosphorous in reactions
[7]. Bioavailability, uptake and phytotoxicity of arsenic in plants are
influenced by factors like arsenic concentration in soil, arsenic
species, plant species and soil properties, like the redox potential,
pH and soil phosphorus content [8]. Uptake of arsenic by plants
occurs primarily through the root system, and the highest arsenic
concentrations are reported in roots and tubers. It is assumed that
plants take up As(V) through phosphate transporters due to their
similarity with phosphorous [9], while As(III) is incorporated by
aquaporin channels [10]. As(III) reacts with sulfhydryl groups (-SH)
of enzymes thereby inhibiting cellular function and causing death
[11]. Even though arsenic is not a redox metal, there is significant
evidence that exposure of plants to inorganic arsenic results in the
generation of reactive oxygen species (ROS) [12] which may result
in damage to DNA, proteins and lipids [13]. To combat such loss,

http://dx.doi.org/10.1016/j.ejsobi.2016.05.007
1164-5563/© 2016 Elsevier Masson SAS. All rights reserved.
 N. Pandey, R. Bhatt / European Journal of Soil Biology 75 (2016) 142e150
plants use their scavenging system to control ROS (peroxide, su-
peroxide, hydroxyl ion etc) which comprises of enzymatic anti-
oxidative systems including superoxide dismutase (SOD), guaiacol
peroxidase (GPX), catalase (CAT) and ascorbate peroxidase (APX).
These antioxidant enzymes are in charge of keeping the balance
between ROS production and cellular destruction [14].
Despite the long history of interest in arsenic resistant plants
and microbes, the attention of microbiologists towards plant-
associated bacteria from heavy metal enriched habitats is more
recent [15]. The diversity of soil microbial communities is exceed-
ingly rich and is known to develop different arsenic resistance and
detoxification mechanisms thereby redistributing arsenic in soil
[16]. Many of the metal resistant bacteria and fungi are able to
transform arsenic compounds by oxidation, reduction, methylation
and demethylation [17] and known to promote plant growth by
direct or indirect mechanisms [18]. They produce iron chelators and
siderophores, solubilize metal phosphates and produce growth
hormones [19]. Soil microorganisms affect arsenic mobility and
availability to the plant; they act as a filter to maintain low arsenic
concentrations in plant tissues, while improving P nutrition of the
host plant [8]. Application of metal resistant plant growth pro-
moting microbes therefore may be vital in improving nutrient
availability for plants and enhancing the bioremediation of heavy
metal contaminated soils [20]. These microorganisms may also
prove to be beneficial for the revegetation/phytostabilisation of
arsenic polluted sites.
Vigna radiata L. Wilczek (mung bean) is a major legume culti-
vated for its edible seeds and sprouts across India. The mature seeds
provide an invaluable source of digestible protein for humans.
Owing to its popularity and rapid growth characteristics, mung
bean seedlings were used as a model plant for the study. The ob-
jectives were (i) to evaluate the plant growth promoting activity of
arsenic resistant bacterium (Exiguobacterium sp.), (ii) to investigate
the effect of arsenic on growth and on antioxidant enzymes of
mung bean plants, and (iii) to examine the protective role of Exi-
guobacterium against arsenic toxicity in mung bean plants under
in vitro conditions.
2. Materials and methods
2.1. Bacterial strain, arsenic resistance and removal assays
An arsenic resistant bacterium (As-9), isolated from the arsenic
rich soil of Rajnandgaon, Chhattisgarh and identified as Exiguo-
bacterium sp. based on16S rDNA gene sequence analysis (Gene
Bank accession number: KC894600.1) [21] was used in the present
study. The isolate was Gram positive, rod-shaped and resistant to
high concentrations of As(V) (700 mM) and As(III) (180 mM). In an
attempt to determine the arsenic removal efficiency of the bacte-
rium, it was cultured in Basal Salt Medium (BSM), which was
modified from that described by Aksornchu et al. [22] and consisted
of the following components per litre: yeast extract (1.0 g),
(NH4)2SO4 (0.3 g), MgSO4$7H2O (0.14 g), CaCl2$2H2O (0.2 g), NaCl
(0.1 g), H3BO3 (0.6 mg), glucose (10.0 g) with the pH adjusted to 8.0.
Just prior to use, the medium was supplemented with a concen-
tration of either 100 mg L
1 of Sodium arsenate (Na2HAsO4$7H2O,
PubChem CID: 61460) or Sodium arsenite (NaAsO2, PubChem CID:
443495) and incubated at 30  C. After an interval of 24 h, 5 mL
aliquots of bacterial culture was removed and centrifuged at 8000g
for 10 min to obtain the cell free supernatant. The concentration of
arsenic was then investigated in the supernatant against control
(non-inoculated broth amended with arsenic) using Hydride Gen-
eration Atomic Absorption Spectrophotometer (HG-AAS) (AA-
7000, Shimadzu) by the method described by Wang et al. [23].
143
2.2. Plant growth promoting (PGP) activities of bacteria under
arsenic stress
The PGP activities of the bacterial strain was assayed under
in vitro conditions in the presence and absence of arsenic. Phos-
phate solubilizing activity was quantitatively estimated in NBRIP
media containing tricalcium phosphate amended with
0e1000 mg L
1 of As(V). The media was inoculated with the bac-
terial strain and incubated at 28  C for 144 h at 170 rpm. The sol-
ubilized phosphate in the culture supernatant was quantified as
detailed by Fiske and Subbarow [24].
For the IAA analysis, the isolate was grown in LB broth amended
with L-tryptophan (0.5 mg mL
1) and 0e1000 mg L
1 concentra-
tion of As(V). After 120 h of incubation, cells were removed by
centrifugation at 10,000g and the supernatant (1 mL) was mixed
vigorously with 2 mL of Salkowski’s reagent (1.0 mL 0.5 M FeCl3 and
50 mL 35% HClO4). Absorbance of the pink colour was read after
25 min at 560 nm and concentration of IAA was calculated from the
standard curve [25].
The production of exopolysaccharide (EPS) was determined
as suggested by Kazy et al. [26] with minor modifications. The
strain As-9 was grown in BSM supplemented with 5% sucrose
and 0e1000 mg L
1 of As(V) with continuous shaking
(100 rpm) at 28  C for 72 h. For the extraction and purification
of bacterial EPS, cells were completely removed from the me-
dium by centrifugation at 15,000g for 30 min and the super-
natant was added to a double volume of ice-cold ethanol (95%),
thoroughly mixed and allowed to stand for 24 h at 4  C. The
precipitated EPS was collected by centrifugation (18,000g,
30 min, 4  C) and repeatedly washed with 95% ethanol, trans-
ferred to a filter paper and weighed after overnight drying at
room temperature.
2.3. Plant material and experimental design
For in vitro experiments, soil samples were collected from Guru
Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh, India. Soil pH
was 6.77 and it contained 0.32% organic carbon, 143.2 kg ha
1 ni-
trate (NO3), 15.6 kg ha
1 phosphorous (P2O5), 187.4 kg ha
1 po-
tassium (K2O) and 46% moisture. Concentrations of As, Fe, Hg, Cu,
Pb, Mn and Zn were 0.82, 2.32, 0.91, 0.69, 8.27, 3.82 and
0.98 mg kg
1 respectively.
About 50 g of sieved soil were transferred to 250 mL conical
flasks and sterilized by autoclaving to kill all the microbial popu-
lation as to observe the effect of a selective bacterium upon the
growth of plants. Exiguobacterium (E) was used as a bioinoculant
[cultured in BSM broth, enumerated and serially diluted with 0.9%
(w/v) saline] and mixed with soil at a level of 106 CFU g
1 soil. Seeds
of mung bean were procured from the Krishi Vigyan Kendra,
Bilaspur, Chhattisgarh and used in the study. Fresh and healthy
seeds were soaked in water for 24 h; thereafter the germinated
seeds with 3e5 mm radicles were surface sterilized using 0.1%
HgCl2 and sown in soil. Six treatments were used: (a) untreated and
non-inoculated blank [soil/-As(V)/-As(III)/
E], (b) untreated and
inoculated with As-9, control [soil/-As(V)/-As(III)/þE], (c) As(V)
treated and non-inoculated test [soil/þAs(V)/
E], (d) As(V) treated
and inoculated test [soil/þAs(V)/þE], (e) As(III) treated and non-
inoculated test [soil/þAs(III)/
E], and (f) As(III) treated and inocu-
lated test [soil/þAs(III)/þE]. In all the test flasks, sodium arsenate
[As(V)] and sodium arsenite [As(III)] were added aseptically to
achieve the final concentration of 100 mg kg
1 soil. The flasks were
incubated at 25  C ± 2  C and 16/8 h photoperiod for a period of 15
days.
144 N. Pandey, R. Bhatt / European Journal of Soil Biology 75 (2016) 142e150
2.4. Measurement of plant growth and chlorophyll content
After 15 days of cultivation, plants were carefully removed from
soil and the roots were washed several times with distilled water to
remove any traces of soil. Length of the roots and shoots
(stems þ leaves) were measured and biomass was determined by
measuring their fresh weight. The survival index (SI) was calculated
by dividing the number of plants survived in each treatment by the
sample size.
Number of plants survived
SI ¼  100
Sample size
The tolerance index (TI) was measured by dividing the length of
shoot/root of the plants exposed to arsenic by that of control. The
equation used was:
Mean length of root or shoot of arsenic treated plant
TI ¼
Mean length of root or shoot of control plant
 100
Total chlorophyll content in the leaf tissues of mung bean plants
grown in arsenic stressed and metal free soil was determined. The
samples were homogenized with 10 mL of acetone (80% v/v) fol-
lowed by centrifugation at 15,000g for 10 min. The absorbance was
measured with a spectrophotometer at 663 and 645 nm and
chlorophyll content was calculated using the equations given by
Arnon [27].
2.5. Arsenic content in plant tissues
The arsenic concentration in the roots and shoots of all the
plants were estimated separately after 15 days of treatment. Sam-
ples were oven dried at 65  C for 72 h and grounded to a fine
powder. Subsamples (50 mg) were then digested with aquaregia
(conc. HCl and HNO3 in the ratio of 3:1) for 1 h and filtered through
2.5 mm filter paper. Total arsenic concentration was determined
quantitatively using HG-AAS as described by Abdel-Lateef et al. [28]
and the arsenic Accumulation factor (AF) was calculated using the
following equation:
Concentration of arsenic in plant tissues
AF ¼
Initial concentration of arsenic in soil
2.6. Arsenic induced anatomical changes
Stem and root samples were harvested from 1 cm above and
1 cm below the stem-root intersection and cross sections were cut
with razor blades. Samples were immediately immersed in distilled
water and stained using 0.05% safranin (w/v in 50% ethanol). Sec-
tions were mounted in 20% glycerine as temporary preparation and
observed under light microscope equipped with a digital camera
(Leica ED4 HD) for the structural changes.
2.7. Root colonization by Exiguobacterium
Seedlings of 15 days old mung bean plants which were non-
inoculated and those treated with As-9, both were selected to
study root colonization. Plants were gently removed from the soil
with forceps and washed with distilled water to remove the soil not
strongly adhering to the roots. The roots were then separated from
the plants using a sterilized blade and bacterial counts were
measured as CFU/g root. For Scanning electron microscopic (SEM)
examination, approximately 2 cm root segments were cut and
processed immediately as described by Kim and Kremer [29] with
minor modifications. Tissue samples (roots) from inoculated and
non-inoculated seedlings were fixed in 2.5% (v/v) glutaraldehyde
and 4% (v/v) paraformaldehyde in 0.1 M phosphate buffer saline
(PBS) (pH 7.2) at 4  C for 5e6 h. Samples were washed thrice with
PBS and post fixed in 1% (w/v) osmium tetroxide in PBS for 2 h at
4  C. Tissues were dehydrated in a 50e100% (v/v) gradient ethanol
series, and dried. Samples were subsequently mounted and sput-
tered with gold particle and observed under Scanning Electron
Microscope (JSM-6360).
[1]
2.8. Effect of arsenic on antioxidant enzymes
2.8.1. Enzyme extraction
Fresh roots and shoots (200 mg) were homogenized separately
in 2 mL of cold potassium phosphate buffer (50 mM, pH 7.0) con-
taining 1 mM EDTA using a chilled mortar and pestle. The ho-
mogenate was centrifuged at 20,000g at 4  C for 15 min and the
[2] clear supernatant was used for enzyme assays. For measuring APX
(ascorbate peroxidase) activity, the tissues were separately ground
in homogenizing medium containing 2.0 mM ascorbate in addition
to the other ingredients.
2.8.2. Enzyme assays
Superoxide dismutase (SOD) activity was determined by nitro
blue tetrazolium (NBT) photochemical assay following Beyer and
Fridovich [30]. SOD activity was expressed as U (unit) mg
1 protein.
One unit was equivalent to the amount of enzyme that gave 50%
inhibition of NBT reduction in one minute. APX activity was assayed
by the methods of Nakano and Asada [31]. The reaction mixture
(3 mL) contained 50 mM phosphate buffer (pH 7.0), 0.1 mM EDTA,
0.5 mM ascorbate, 0.1 mM H2O2 and 0.1 mL enzyme extract. The
hydrogen peroxide dependent oxidation of ascorbate was followed
by a decrease in the absorbance at 290 nm. Specific activity was
calculated with extinction coefficient value of 2.8 mM
1 cm
1.
Glutathione reductase (GR) activity was determined as described
by Smith et al. [32] by monitoring the increase in absorbance at
412 nm when 5, 50 -dithiobis (2-nitrobenzoic acid) (DTNB) is
reduced by GSH. Catalase (CAT) activity was assayed by measuring
[3] the reduction of H2O2 at 240 nm (ε ¼ 039.4 mM
1 cm
1) as
described by Chance and Maehly [33]. Guaiacol peroxidase (GPX)
was measured by monitoring the oxidation of guaiacol (8.26 mM,
ε ¼ 26.6 mM
1 cm
1) in the presence of 8.8 mM H2O2 in 25 mM
sodium acetate buffer (pH 5.0) at 470 nm [34].
2.9. Lipid peroxidation
Malondialdehyde (MDA) is an end product of lipid peroxidation
and was quantified by the thiobarbituric acid (TBA) test as
described by Du and Bramlage [35]. Fresh roots and shoots
(200 mg) of mung plants were separately homogenized in 4 mL of
1% (w/v) trichloroacetic acid (TCA) and centrifuged at 18,000g for
10 min. To the supernatant was added 1 mL of 0.5% (w/v) TBA in
20% TCA. The mixture was incubated in boiling water bath for
30 min and then transferred to an ice bath to stop the reaction. The
mixture was centrifuged at 18,000g for 5 min and supernatant was
collected. Absorbance was recorded at 532 nm and corrected for
specific turbidity by subtracting the absorbance at 600 nm. The
concentration of MDA which forms a red adduct with two mole-
cules of TBA (MDA-TBA2) was calculated from the extinction co-
efficient of 155 mM
1 cm
1.
 N. Pandey, R. Bhatt / European Journal of Soil Biology 75 (2016) 142e150
2.10. Statistical analysis
All the experiments were conducted in triplicates. The mean
values are presented with the SD. The significance of differences
between control and treatment was determined by Student’s t-test
using Minitab 17 software package. Differences at p  0.05 were
considered statistically significant.
3. Results
3.1. Arsenic removal by Exiguobacterium
The isolate As-9 was quite efficient in removing both As(V) and
As(III) from the growth medium. Maximum removal of arsenic took
place during the initial phase of growth (48 h) with the concen-
tration ranging between 50.2 and 76.9 mg L
1 for As(V) and
13.3e26.5 mg L
1 for As(III) (Fig. 1). However, the removal effi-
ciency was considerably decreased with the increasing time period.
Further, results of the study revealed that the strain was more
efficient in removing As(V) than As(III). At the end of the experi-
ment (168 h), Exiguobacterium successfully removed about 99% of
As(V) from the medium while only 90% removal was achieved in
case of As(III). It is clear from the results that arsenic removal de-
pends upon the type of oxyanion enrichment which leads to some
physiological adaptations in the bacterium to adjust with the
changing environment. The study also suggested that arsenic
concentration had specific equilibrium, after which there was no
significant effect on removal even by increasing the time of
incubation.
3.2. Plant growth promoting features of Exiguobacterium
The arsenic resistant Exiguobacterium used in the present study
revealed considerable production of PGP substances both in the
absence and presence of arsenic (Table 1). The strain solubilized
substantial amount of P (53.4e67.7 mg mL
1) which indicates its
ability to utilize tricalcium phosphate as the source of P even in the
presence of As(V); however, there was a noticeable decrease with
the increasing concentration. Results also showed that Exiguo-
bacterium produced good amount of IAA in the presence and
absence of As(V) with a concentration ranging between 10 and
15 mg mL
1. This indicated that the bacterium was able to utilize L-
tryptophan as a precursor for growth and IAA production. Further
investigation of the PGP activities of the strain revealed enhanced
production of EPS (20e28 mg mL
1) with 400e600 mg L
1 As(V)
Fig. 1. Removal of As(V) and As(III) by Exiguobacterium. Data are presented as
mean ± SD; error bars represent the standard error of three replicate experiments.
145
concentration. The results clearly indicated that the presence of
As(V) did not affect the ability of the strain to show PGP activities.
However, at a concentration of 1000 mg L
1 As(V), P solubilization
and production of IAA and EPS decreased by 21.13%, 35.09% and
59.50% respectively.
3.3. Effect of inoculation of Exiguobacterium on plant growth and
chlorophyll content under arsenic stress
Results showed that the seeds grown in soil amended with
100 mg kg
1 concentration of As(V) or As(III) together with plant
growth promoting Exiguobacterium showed better growth
compared to non-inoculated plants. In contrast, a considerable
decrease in the growth of mung plants was observed with the
application of As(V) and As(III) alone and the effects been more
prominent in case of As(III). A decrease in the length of roots and
shoots was observed in the presence of As(V) (36.9% and 53.9%) and
As(III) (65.7% and 90.9%) respectively. Similarly, reduction in the
biomass of roots and shoots was also observed in the presence of
As(V) (40% and 52.17%) and As(III) (57.1% and 91.3%) (Table 2).
However, the inoculation of Exiguobacterium increased the biomass
of shoots and roots respectively by 22.23% and 39% in the presence
of As(V) and 29% and 78% in the presence of As(III) (Table 2). In
addition, appreciable increase in the length of roots and shoots was
also observed upon application of bioinoculant which correspond
to 18.15% and 29.6% in the presence of As(V) while 12.33% and
30.81% in the presence of As(III). The survival index and tolerance
level of the plants towards As(V) and As(III) also increased to a
significant level when grown in the presence of bioinoculant.
Further, the application of Exiguobacterium increased the chloro-
phyll content of the plants even in the presence of arsenic when
compared to the non-inoculated tests where, a significant decrease
(p < 0.01) in the total chlorophyll content was observed with As(V)
and As(III) treatments (Table 3).
3.4. Arsenic content in plant tissues
Total arsenic in tissues of non-inoculated and inoculated plants
grown in soil supplemented with 100 mg kg
1 of As(V) or As(III)
was quantified. Results of HG-AAS analysis revealed an increase in
uptake and accumulation of arsenic in the roots and shoots of non-
inoculated plants. It was found that the arsenic concentration was
significantly higher (p < 0.001) in roots than in shoots with a
considerable differences in the amount of arsenic in As(V) and
As(III) treatments (Table 3). The study also showed that the uptake
and accumulation of As(III) was appreciably high in all the plants
when compared to As(V). Interestingly, arsenic resistant and plant
growth promoting Exiguobacterium used as a bioinoculant caused
a substantial decrease (5 fold and 3 fold respectively) in the con-
centrations of both As(V) and As(III) in roots as well as shoots
when compared to non-inoculated plants. The decreased accu-
mulation of arsenic in the presence of bioinoculant might be due to
the bacterial uptake of arsenic from its surrounding. However,
comparatively high amount of arsenic was found in roots than
shoots of all the mung plants both in the absence and presence of
bioinoculant.
3.5. Arsenic induced anatomical changes
Ultra-structural changes in the roots and shoots of mung bean
plants were observed under arsenic stress condition. The trans-
verse section of the roots of the arsenic treated plants was marked
by the blackening of the cells all along the walls of the cortex
indicating severe arsenic toxicity (Fig. 2iii, v). Further, the changes
were more prominent in the plants exposed to As(III) than As(V).
146 N. Pandey, R. Bhatt / European Journal of Soil Biology 75 (2016) 142e150

Table 1
Plant growth promoting (PGP) activities of the bacterial strain at different concentrations of As(V).

As(V)a doze (mg L
1) Phosphate solubilization (mg mL
1) IAAb production (mg mL
1) EPSc production (mg mL
1)

0 67.7 ± 2.5 15.1 ± 0.7 4.02 ± 0.6

200 65.9 ± 1.6 14.2 ± 1.0 20.7 ± 1.5
400 62.3 ± 1.6 13.8 ± 0.9 23.8 ± 0.9
600 58.5 ± 2.2 12.1 ± 1.0 28.4 ± 1.1
800 56.1 ± 1.7 10.9 ± 0.3 18.6 ± 0.9
1000 53.4 ± 2.0 9.8 ± 0.9 11.5 ± 0.9

Values indicate ± S.D. of three replicates.

a
Sodium arsenate.
b
Indole acetic acid.
c
Exopolysaccharide.

Table 2 with bioinoculant showed no visible modifications even in the

Effect of arsenic on the growth of mung bean plants after 15 days treatment. presence of arsenic (Fig. 3 iv, vi) which was comparable to control
Treatments Length (cm) Biomass (g) (Fig. 3i, ii).
Shoot Root Shoot Root

Blank 19.5 ± 2.21* 6.2 ± 0.6ns 0.27 ± 0.08ns 0.19 ± 0.03ns 3.6. Root colonization by Exiguobacterium
Control 24.3 ± 2.63 7.3 ± 1.0 0.35 ± 0.04 0.23 ± 0.01
As(V)/
E 11.2 ± 2.32** 4.6 ± 1.01* 0.21 ± 0.04ns 0.11 ± 0.04* The ability of the Exiguobacterium to colonize roots of mung
As(V)/þE 18.4 ± 1.98* 6.0 ± 1.21* 0.27 ± 0.09ns 0.18 ± 0.08*
bean plants was assessed after 15 days of inoculation. The total
As(III)/
E 2.2 ± 0.2*** 2.5 ± 0.3** 0.15 ± 0.01* 0.02 ± 0.01**
As(III)/þE 9.7 ± 1.14** 3.4 ± 0.6* 0.21 ± 0.03* 0.09 ± 0.02* culturable bacteria recovered from the roots of the inoculated
plants was approximately, 3.6  104 CFU/g of fresh weight of roots.
Each value is a mean ± SD of three replicates (n ¼ 3) where each replicate consti-
tuted five plants/flask. Value within a column are significantly different according to
Root sections examined by SEM revealed strong proliferation and
one way ANOVA with respect to control, where, ns ¼ non-significant, ***p < 0.001, distribution of the isolate preferentially on the surface of the roots
**p < 0.01, *p < 0.05. (E ¼ Exiguobacterium). (Fig. 4a). Visual examination of the roots inoculated with the isolate

Table 3
Effect of arsenic and inoculation of Exiguobacterium on leaf chlorophyll content and accumulation in Vigna radiata after 15 days of treatment with 100 mg kg
1 As(V) and As(III).

Treatments Chlorophyll (mg mg
1) As accumulation (mg Kg
1) Accumulation

factor (AF) (mg Kg
1)
Shoot Root

Blank 1.1 ± 0.1ns 0.001ns 0.003* 0.04

Control 1.3 ± 0.2 0 0.001 0.01
As(V)/
E 0.7 ± 0.15* 0.088 ± 0.005** 0.145 ± 0.006*** 2.33
As(V)/þE 1.1 ± 0.2ns 0.016 ± 0.004* 0.030 ± 0.003*** 0.46
As(III)/
E 0.3 ± 0.1** 0.180 ± 0.045*** 0.338 ± 0.008*** 5.18
As(III)/þE 0.6 ± 0.15* 0.057 ± 0.003** 0.124 ± 0.004*** 1.81

Values indicate ± SD of three replicates (n ¼ 3) where each replicate constituted five plants/flask. Value within a column are significantly different according to one way
ANOVA with respect to control, where, ns ¼ non-significant, ***p < 0.001, **p < 0.01, *p < 0.05. (E ¼ Exiguobacterium).

The light micrographs of arsenic treated roots showed breakdown
of cortex and endodermal cells (Fig. 2iii, v) compared to the control
(Fig. 2i, ii). Changes in the sizes, shapes and arrangements of
cortical parenchyma cell were also observed in the roots of arsenic
treated plants. Anatomical study revealed decreased root hairs,
damaged vascular bundles and endodermis together with distorted
pericycle in roots which was even prominent in seedlings treated
alone with As(III). However, the symptoms of arsenic toxicity were
successively decreased in the roots of plants grown in the presence
of bioinoculant (Fig. 2iv, vi) which showed well developed cortex
with minimum structural changes in both epidermis and endo-
dermis which is consistent with the control.
The light micrographs of stem cross sections of arsenic treated
plants also showed blackening of the walls of cortex cells but were
less intense when compared to the roots (Fig. 3iii, v). The results
also revealed the blackening of the endodermal cells of the plants in
the presence of As(III) which was not observed in As(V) treatments.
The transverse sections of the As(V) and As(III) treated stems
showed large pith, distorted epidermis along with decreased and
damaged vascular bundles. The arsenic treated plants also showed
decreased intercellular spaces and break down of the cells
compared to the control. In contrast, the shoots of seedlings treated
appeared to be distorted compared to the non-inoculated plants.
The results also revealed that the colonization of bacteria occurred
in rhizosphere soil. In contrast, roots of the non-inoculated plants
typically revealed a smooth, undamaged surface where the cell wall
was distinct and continuous with the complete absence of bacterial
colonies (Fig. 4b).
3.7. Effect of arsenic on antioxidant enzymes
Arsenic showed inhibitory effects on the growth of plants by
activating oxidative damage in the tissues. As(V) and As(III) treat-
ments significantly (p < 0.05) increased the antioxidative enzymatic
activity (SOD, APX, GR, CAT and GPX) in V. radiata by a considerable
extent which was even higher in the presence of As(III). Further, the
oxidative damage was more prominent in roots than in shoots. As(V)
and As(III) exposure increased the SOD activity in roots by 82.43%
and 77.18% while in shoots by 76.08% and 70.82% respectively
compared to control (Fig. 5a). Similarly, the activity of APX increased
to 89.98% and 87.24% in roots and 86.32% and 79.8% in shoots when
exposed to As(III) and As(V) (Fig. 5b). As shown in Fig. 5c, the activity
of GR in roots and shoots rose significantly (p < 0.05) in presence of
As(V) (74.57% and 66.86%) and As(III) (80.06% and 74.57%) over
 N. Pandey, R. Bhatt / European Journal of Soil Biology 75 (2016) 142e150 147

Fig. 2. Microscopic examination of ultra structural changes in root under arsenic stress. (i) Blank (ii) Control (iii) As(V)/
E (iv) As(V)/þE (v) As(III)/
E (vi) As(III)/þE.

Fig. 3. Microscopic examination of ultra structural changes in shoot under arsenic stress. (i) Blank (ii) Control (iii) As(V)/
E (iv) As(V)/þE (v) As(III)/
E (vi) As(III)/þE.
148 N. Pandey, R. Bhatt / European Journal of Soil Biology 75 (2016) 142e150
Fig. 4. SEM micrograph of Vigna radiata root colonization by Exiguobacterium (a) root surface covered with bacteria (b) roots of non-inoculated control. Note that no bacteria are
present on the root.
Fig. 5. Activities of antioxidative enzymes (a) SOD (b) APX (c) GR (d) CAT (e) GPX and MDA content (f) in shoots and roots of V. radiata treated with 100 mg kg
1 of As(V) and As(III)
along with the bioinoculant Exiguobacterium. Values are mean ± S.D.
control. The seedlings supplemented with As(V) increased the CAT
activity in roots and shoots by 96.91% and 93.98% while 97.1% and
95.91% increase was observed in the presence of As(III) (Fig. 5d). The
activity of GPX was also enhanced with arsenic treatments. It was
found to be increased in roots by 89.52% and 92.71% and in shoots by
82.91% and 87.66% upon exposure to As(V) and As(III) respectively
(Fig. 5e). By contrast, significant (p < 0.01) reduction in the activities
of antioxidant enzymes was noticed when seedlings were grown in
the presence of bioinoculant and the values were found comparable
to control.
3.8. Lipid peroxidation
In our study, lipid peroxidation, measured as MDA levels
showed a significant (p < 0.001) increase in the roots and shoots of
mung bean plants in the presence of arsenic when compared to
control. The increase in MDA concentration was recorded to be
50.2% and 82.8% in shoots while 42.85% and 84.52% in roots
respectively, when the seedlings were exposed to As(V) or As(III)
alone (Fig. 5f). Further, the MDA concentration in the seedlings also
sharply differed upon exposure to different species of arsenic with
the effect being higher in case of As(III). Inoculation of arsenic
resistant Exiguobacterium reduced the levels of MDA in the shoots
to 28.02% and 45.24% and in roots to 35.11% and 59.52% respectively
even in the presence of As(V) and As(III).
4. Discussion
Presence of arsenic in soil is a global problem as it is considered
phytotoxic [7] which adversely affects plant growth thereby
reducing crop productivity. In recent years, the inoculation of
certain metal resistant PGPB is employed to enhance the survival of
plants by alleviating metal stress [19]. The use of PGPB might prove
to be effective because they are degradable, non-toxic and play an
important role in the growth and establishment of plants on the
contaminated soils by producing plant growth beneficial metabo-
lites [36].
The results of the present study showed that the strain As-9
could efficiently remove >90% of arsenic in a relatively short
period of time (168 h) (Fig. 1). A number of reports are available
which have shown the ability of the bacterial species including
PGPR in removing arsenic from their surrounding environment
[37,38]. This suggest the possible use of bacterial biomass for the
treatment of arsenic contaminated water, soil and sediments.
Interestingly, in our experiments, the arsenic resistant Exiguo-
bacterium was also found to solubilize phosphate and produced IAA
and EPS in the presence of arsenic (Table 1), which might have
played a key role in promoting plant growth in differentially
stressed environment [39]. It is well documented that solubiliza-
tion of phosphate in the rhizosphere and IAA production makes a
major contribution to plant growth by stimulating elongation of the
 N. Pandey, R. Bhatt / European Journal of Soil Biology 75 (2016) 142e150
plant cells or increasing cell division [40]. Other PGP trait viz.
production of EPS by bacteria also significantly increases plant
growth by facilitating biological nitrogen fixation and fulfilling the
carbohydrate requirement of the plant [41]. Reports also suggest
that microbial EPS binds metal with varying degrees of specificity
and affinity which could have far reaching implications on adap-
tation of bacteria to environmental stress and in metal removal
from contaminated sites [42].
The growth and metabolism of plants is adversely affected by
the presence of arsenic in the environment [43]. It was also
observed in the present study that the growth of the plants
decreased progressively in the presence of arsenic. However, the
application of bioinoculant caused a substantial increase in the
performance of the mung bean plants even in the presence of high
concentration (100 mg kg
1) of arsenic (Table 2). Similar results of
increase in plant growth and yield due to the application of metal
resistant PGPB has been reported by Rajkumar and Freitas [44];
Huang et al. [45] and Wani and Khan [46]. Further, the plants also
appeared to be selectively tolerant to different arsenic species.
Since As(III) is more toxic than As(V), more plants have evolved
tolerance capabilities to As(V). The study revealed that As(V) stress
had no effect on plant survival and all the plants were alive at the
end of the experiment; however plant biomass was affected when
compared to control. Furthermore, a progressive decrease in the
chlorophyll content was measured in V. radiata in the absence of
bioinoculant. In comparison, Exiguobacterium increased the chlo-
rophyll content in the plants by 46e84% even in the soil amended
with arsenic. This is in consistent with the study of Oves et al. [47].
Arsenic toxicity not only leads to physiological and morpho-
logical disorders in plants but also impart changes in its anatomical
features. In the present study, defects in the cortex cells together
with alteration in the epidermis, endodermis and vascular bundles
were observed in the plants exposed to arsenic. In contrast, inoc-
ulation of bioinoculant caused considerable decrease in arsenic-
induced toxicity in roots and shoots of V. radiata (Figs. 3 and 4).
The uptake and accumulation of arsenic in the roots and shoots
of V. radiata significantly (p < 0.001) increased in the absence of
bioinoculant (Table 3) which is likely to be associated with the
availability of arsenic in the soil. The results also showed that the
roots accumulated more arsenic compared to shoots. Roots are in
direct contact with the soil and therefore uptake more arsenic
which might be the possible reason that arsenic concentration
decreases in the order of roots > stems [48]. Moreover, the presence
of bioinoculant significantly decreased the arsenic accumulation in
roots and shoots when compared to non-inoculated but arsenic
amended plants. The decreased accumulation of arsenic in plant
tissues might be due to the bacterial uptake of metalloid. The hy-
pothesis was confirmed by SEM analysis which showed high level
colonization of Exiguobacterium on the root surface of the inocu-
lated mung bean plants. This proved that the successful coloniza-
tion of the bacterium resulted in the decreased uptake of arsenic in
tissues and helped the plants to grow and propagate at high con-
centrations of arsenic [49]. It is well established that inoculation of
the beneficial bacteria not only colonize the roots of the plants but
also prevent the rate and extent of pathogen colonization in roots
[50]. Another reason for the growth and survival of plants in the
arsenic rich soil could be the production of plant growth promoting
substances by the bacterial species [46]. Present study is the first
report on the application of arsenic resistant Exiguobacterium in
promoting growth of Vigna radiata under arsenic stress by
decreasing the uptake of metalloid in seedlings. Similar results of
increase in plant growth in the presence of other heavy metals have
been reported by Wani and Khan [51] and Rajkumar et al. [52].
Arsenic uptake induces toxicity that has been linked to the
production of reactive oxygen species (ROS), thereby inducing lipid
149
peroxidation and damage to DNA and proteins [53]. ROS production
in plants is regulated by antioxidative enzymes which remove free
radicals thereby protecting the cells from oxidative damage. In the
present study, arsenic treatment caused significant (p < 0.01) al-
terations in antioxidantive enzyme activities in mung bean plants
with a marked (p < 0.05) difference between the two arsenic
species. A considerable increase in the activities of SOD, CAT, GPX,
APX and GR was observed both in roots and shoots of the plants
under arsenic stress (Fig. 5aee). The changes were more pro-
nounced in the roots as compared to shoots and with As(III)
treatment. This corresponds to the more toxic behaviour of As(III)
than As(V). Similar observations with increased enzyme activities
in response to arsenic stress have also been reported by Duquesnoy
et al. [14], in Vicia faba and Zea mays; Mishra et al. [54], in Bacopa
monnieri L., Talukdar and Talukdar [55] in Wedelia chinensis Merrill
and Siddiqui et al. [56] in Withania somnifera. It is worth
mentioning that inability of the antioxidative enzymes generate
ROS which ultimately led to huge enhancement of MDA, an indi-
cator of oxidative damage produced during peroxidation of mem-
brane lipid by decomposition of polyunsaturated fatty acid [57].
Significant (p < 0.01) increase in the concentration of MDA was
observed in the mung bean plants with the application of arsenic
(Fig. 5f). Previous studies also reported severe lipid peroxidation in
different plant species under arsenic stress [56,58]. The increase in
activity of these antioxidative enzymes and MDA content is quite
obvious for the efficient recycling of ROS to ensure normal plant
growth. Our results also revealed that with the application of bio-
inoculant, there were many reductions in the antioxidative enzyme
activity and MDA concentration in V. radiata to the extent compa-
rable to control, indicating the lower production of ROS thereby
reducing oxidative damage.
In conclusion, the results of the study indicated that Exiguo-
bacterium sp. As-9 is an efficient arsenic accumulator and a highly
efficient PGPB which promoted the growth of V. radiata even in the
presence of relatively high concentration of arsenic. Further, it
protected the mung bean plants against the toxic effects of arsenic
by limiting the transfer of metalloid to the plant, while improving
plant nutrition. This could open new avenues in managing the toxic
metals in the environment and would offer a promising strategy to
promote plant growth in metal contaminated soil. Future research
is needed to develop this strain as a bioinoculant to verify its ulti-
mate beneficial effects in plant growth promotion and subsequent
enhancement of yield for agricultural crops under long term field
conditions.
Acknowledgements
We are thankful to Department of Science and Technology
(DST), New Delhi for funding the work through INSPIRE fellowship
and to Sophisticated Analytical Instrument Facility of North Eastern
Hill University, Shillong for SEM analysis.
References
[1] J. Matschullat, Arsenic in the geosphere-a review, Sci. Total Environ. 249
(2000) 297e312.
[2] S. Kapaj, H. Peterson, K. Liber, P. Bhattacharya, Human health effects from
chronic arsenic poisoning-a review, J. Environ. Sci. Health A Tox. Hazard.
Subst. Environ. Eng. 41 (2006) 2399e2428.
[3] L. Ascar, I. Ahumada, P. Richter, Influence of redox potential (Eh) on the
availability of arsenic species in soils and soils amended with biosolid, Che-
mosphere 72 (2008) 1548e1552.
[4] P.L. Smedley, D.G. Kinniburgh, A review of the source, behaviour and distri-
bution of arsenic in natural waters, Appl. Geochem. 17 (2002) 517e568.
[5] E. Smith, R. Naidu, A.M. Alston, Arsenic in the soil environment: a review, Adv.
Agron. 64 (1998) 149e195.
[6] L.M. Walsh, M.E. Sumner, D.R. Keeney, Occurrence and distribution of arsenic
in soils and plants, Environ. Health Perspect. 19 (1977) 67e71.
150 N. Pandey, R. Bhatt / European Journal of Soil Biology 75 (2016) 142e150
[7] F.J. Zhao, J.F. Ma, A.A. Meharg, S.P. McGrath, Arsenic uptake and metabolism in
plants, New Phytol. 181 (2009) 777e794.
[8] W.J. Fitz, W.W. Wenzel, Arsenic transformations in the soil-rhizosphere-plant
system: fundamentals and potential application to phytoremediation,
J. Biotechnol. 99 (2002) 259e278.
[9] A.A. Meharg, M.R. Macnair, An altered phosphate uptake system in arsenate
tolerant Holcus lanatus L, New Phytol. 116 (1990) 29e35.
[10] J.F. Ma, N. Yamaji, N. Mitani, X.Y. Xu, Y.H. Su, S.P. McGrath, F.J. Zhao, Trans-
porters of arsenite in rice and their role in arsenic accumulation in rice grain,
Proc. Natl. Acad. Sci. U.S.A. 105 (2008) 9931e9935.
[11] P.M. Finnegan, W. Chen, Arsenic toxicity: the effects on plant metabolism,
Front. Physiol. 3 (2012) 1e18.
[12] R. Requejo, M. Tena, Proteome analysis of maize roots reveals that oxidative
stress is a main contributing factor to plant arsenic toxicity, Phytochemistry
66 (2005) 1519e1528.
[13] Y. Kumagai, D. Sumi, Arsenic: signal transduction, transcription factor, and
biotransformation involved in cellular response and toxicity, Annu. Rev.
Pharmacol. Toxicol. 47 (2007) 243e262.
[14] I. Duquesnoy, G.M. Champeau, G. Evray, G. Ledoigt, A. Piquet-Pissaloux,
Enzymatic adaptations to arsenic-induced oxidative stress in Zea mays and
genotoxic effect of arsenic in root tips of Vicia faba and Zea mays, C. R. Biol. 333
(2010) 814e824.
[15] A. Sessitsch, M. Kuffner, P. Kidd, J. Vangronsveld, W.W. Wenzel, K. Fallmann,
M. Puschenreiter, The role of plant associated bacteria in the mobilization and
phytoextraction of trace elements in contaminated soils, Soil Biol. Biochem. 60
(2013) 182e194.
[16] C.S. Sheik, T.W. Mitchell, F.Z. Rizvi, Y. Rehman, M. Faisal, S. Hasnain,
M.J. McInerney, L.R. Krumholz, Exposure of soil microbial communities to
chromium and arsenic alters their diversity and structure, PLoS One 7 (2012)
e40059.
[17] J.F. Stolz, P. Basu, R.S. Oremland, Microbial transformation of elements: the
case of arsenic and selenium, Int. Microbiol. 5 (2002) 201e207.
[18] M. Rajkumar, S. Sandhya, M.N.V. Prasad, H. Freitas, Perspectives of plant
associated microbes in heavy metal phytoremediation, Biotech. Adv. 30
(2012) 1562e1574.
[19] L. Cavalca, R. Zanchi, A. Corsini, M. Colombo, C. Romagnoli, E. Canzi,
V. Andreoni, Arsenic-resistant bacteria associated with roots of the wild Cir-
sium arvense (L.) plant from an arsenic polluted soil, and screening of potential
plant growth promoting characteristics, Syst. Appl. Microbiol. 33 (2010)
154e164.
[20] H.I. Tak, F. Ahmad, O.O. Babalola, Advances in the application of plant growth
promoting rhizobacteria in phytoremediation of heavy metals, Rev. Environ.
Contam. Toxicol. 223 (2013) 33e52.
[21] N. Pandey, R. Bhatt, Arsenic resistance and accumulation by two bacteria
isolated from a natural arsenic contaminated site, J. Basic Microbiol. 55 (2015)
1275e1286.
[22] P. Aksornchu, P. Prasertsan, V. Sobhon, Isolation of arsenic tolerant bacteria
from arsenic contaminated soil, Songklanakarin J. Sci. Technol. 30 (2008)
95e102.
[23] D.M. Wang, J. Feng, F.J. Qin, Y.S. Mo, Determination of six heavy metal ele-
ments in Zanthoxylum nitidum in twelve habitats of guangxi by ICP-AES,
Zhong Yao Cai 35 (2012) 366e368.
[24] C.H. Fiske, Y. Subbarow, A colorimetric determination of phosphorus, J. Biol.
Chem. 66 (1925) 375e400.
[25] S.A. Gordon, R.P. Weber, Colorimetric estimation of indoleacetic acid, Plant
Physiol. 26 (1951) 192e195.
[26] S.K. Kazy, P. Sar, S.P. Singh, A.K. Sen, S.F. D’Souza, Extracellular polysaccharides
of a copper-sensitive and a copper-resistant Pseudomonas aeruginosa strain:
synthesis, chemical nature and copper binding, World J. Microbiol. Biotechnol.
18 (2002) 583e588.
[27] D.T. Arnon, Copper enzymes in isolated chloroplast polyphenol oxidase in
Beta vulgaris, Appl. Environ. Microbiol. 55 (1949) 1665e1669.
[28] A.M. Abdel-Lateef, R.A. Mohamed, H.H. Mahmoud, Determination of arsenic
(III) and (V) species in some environmental samples by atomic absorption
spectrometry, Adv. Chem. Sci. 2 (2013) 110e113.
[29] S. Kim, R.J. Kremer, Scanning and transmission electron microscopy of root
colonization of morningglory (Ipomoea spp.) seedlings by rhizobacteria,
Symbiosis 39 (2005) 117e124.
[30] W.F. Beyer, I. Fridovich, Assaying for superoxide dismutase activity: some
large consequences of minor changes in conditions, Anal. Biochem. 161 (1987)
559e566.
[31] Y. Nakano, K. Asada, Hydrogen peroxide is scavenged by ascorbate specific
peroxidase in spinach chloroplast, Plant Cell Physiol. 22 (1981) 867e880.
[32] I.K. Smith, T.L. Vierheller, C.A. Thorne, Assay of glutathione reductase in crude
tissue homogenates using 5, 50 -dithiobis(2-nitrobenzoic acid), Anal. Biochem.
175 (1988) 408e413.
[33] B. Chance, A.C. Maehly, Assay of catalases and peroxidises, Methods Enzymol.
2 (1955) 764e817.
[34] A. Mika, S. Lüthje, Properties of guaiacol peroxidase activities isolated from
corn root plasma membranes, Plant Physiol. 132 (2003) 1489e1498.
[35] Z. Du, W.J. Bramlage, Modified thiobarbituric acid assay for measuring lipid
oxidation in sugar-rich plant tissue extracts, J. Agric. Food Chem. 40 (1992)
1566e1570.
[36] K.V. Kumar, S. Srivastava, N. Singh, H.M. Behl, Role of metal resistant plant
growth promoting bacteria in ameliorating fly ash to the growth of Brassica
juncea, J. Hazard. Mater. 170 (2009) 51e57.
[37] Q. Wang, D. Xiong, P. Zhao, X. Yu, B. Tu, G. Wang, Effect of applying an arsenic-
resistant and plant growth-promoting rhizobacterium to enhance soil arsenic
phytoremediation by Populus deltoides LH05-17, J. Appl. Microbiol. 11 (2011)
11065e11074.
[38] A. Rahman, N. Nahar, N.N. Nawani, J. Jass, P. Desale, B.P. Kapadnis, K. Hossain,
A.K. Saha, S. Ghosh, B. Olsson, A. Mandal, Isolation and characterization of a
Lysinibacillus strain B1-CDA showing potential for bioremediation of arsenics
from contaminated water, J. Environ. Sci. Health A Tox. Hazard Subst. Environ.
Eng. 49 (2014) 1349e1360.
[39] A. Kumar, P. Bhargava, L.C. Rai, Isolation and molecular characterization of
phosphate solubilizing Enterobacter and Exiguobacterium species from paddy
fields of eastern Uttar Pradesh, India, Afr. J. Microbiol. Res. 4 (2010) 820e829.
[40] K. Minamisawa, K. Fukai, Production of indole-3-acetic acid by Bradyrhizobium
japonicum: a correlation with genotype grouping and rhizobitoxine produc-
tion, Plant Cell Physiol. 32 (1991) 1e9.
[41] C.A. Bomfeti, L.A. Florentino, A.P. Guimar~ aes, P.G. Cardoso, M.C. Guerreiro,
F.M. de Souza, Exopolysaccharides produced by the symbiotic nitrogen-fixing
bacteria of leguminosae, Rev. Bras. Cie ^nc. Solo 35 (2011) 657e671.
[42] A. Pal, A.K. Paul, Microbial extracellular polymeric substances: central ele-
ments in heavy metal bioremediation, Indian J. Microbiol. 48 (2008) 49e64.
[43] M. Shri, S. Kumar, D. Chakrabarty, P.K. Trivedi, S. Mallick, P. Misra, D. Shukla,
S. Mishra, S. Srivastava, R.D. Tripathi, R. Tuli, Effect of arsenic on growth,
oxidative stress, and antioxidant system in rice seedlings, Ecotoxicol. Environ.
Saf. 72 (2009) 1102e1110.
[44] M. Rajkumar, H. Freitas, Influence of metal resistant-plant growth-promoting
bacteria on the growth of Ricinus communis in soil contaminated with heavy
metals, Chemosphere 71 (2008) 834e842.
[45] G.H. Huang, H.H. Tian, H.Y. Liu, X.W. Fan, Y. Liang, Y.Z. Li, Characterization of
plant-growth-promoting effects and concurrent promotion of heavy metal
accumulation in the tissues of the plants grown in the polluted soil by Bur-
kholderia strain LD-11, Int. J. Phytoremediat. 15 (2013) 991e1009.
[46] P.A. Wani, M.S. Khan, Nickel detoxification and plant growth promotion by
multi metal resistant plant growth promoting Rhizobium species RL9, Bull.
Environ. Contam. Toxicol. 91 (2013) 117e124.
[47] M. Oves, M.S. Khan, A. Zaidi, Chromium reducing and plant growth promoting
novel strain Pseudomonas aeruginosa OSG41 enhance chickpea growth in
chromium amended soils, Eur. J. Soil Biol. 56 (2013) 72e83.
[48] S.N. Selamat, S.R. Abdullah, M. Idris, Phytoremediation of lead (Pb) and arsenic
(As) by Melastoma malabathricum L. from contaminated soil in separate
exposure, Int. J. Phytoremediat. 16 (2014) 694e703.
[49] I. Mallick, S.T. Hossain, S. Sinha, S.K. Mukherjee, Brevibacillus sp. KUMAs2, a
bacterial isolate for possible bioremediation of arsenic in rhizosphere, Eco-
toxicol. Environ. Saf. 107 (2014) 236e244.
[50] R. Jog, M. Pandya, G. Nareshkumar, S. Rajkumar, Mechanism of phosphate
solubilization and antifungal activity of Streptomyces spp. isolated from wheat
roots and rhizosphere and their application in improving plant growth,
Microbiology 160 (2014) 778e788.
[51] P.A. Wani, M.S. Khan, Bacillus species enhance growth parameters of chickpea
(Cicer arietinum L.) in chromium stressed soils, Food Chem. Toxicol. 48 (2010)
3262e3267.
[52] M. Rajkumar, Y. Ma, H. Freitas, Improvement of Ni phytostabilization by
inoculation of Ni resistant Bacillus megaterium SR28C, J. Environ. Manag. 128
(2013) 973e980.
[53] O. Aksakal, N. Esim, Evaluation of arsenic trioxide genotoxicity in wheat
seedlings using oxidative system and RAPD assays, Environ. Sci. Pollut. Res.
Int. 22 (2015) 7120e7128.
[54] S. Mishra, S. Srivastava, S. Dwivedi, R.D. Tripathi, Investigation of biochemical
responses of Bacopa monnieri L. upon exposure to arsenate, Environ. Toxicol.
28 (2013) 419e430.
[55] T. Talukdar, D. Talukdar, Response of antioxidative enzymes to arsenic
induced phytotoxicity in leaves of a medicinal daisy, Wedelia chinensis Merrill,
J. Nat. Sci. Biol. Med. 4 (2013) 383e388.
[56] F. Siddiqui, P.K. Tandon, S. Srivastava, Analysis of arsenic induced physiolog-
ical and biochemical responses in a medicinal plant, Withania somnifera,
Physiol. Mol. Biol. Plants 21 (2015) 61e69.
[57] R. Mittler, Oxidative stress, antioxidants and stress tolerance, Trends Plant Sci.
7 (2002) 405e410.
[58] D. Talukdar, Arsenic-induced oxidative stress in the common bean legume,
Phaseolus vulgaris L. seedlings and its amelioration by exogenous nitric oxide,
Physiol. Mol. Biol. Plants 19 (2013) 69e79.