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Role of soil associated Exiguobacterium in reducing arsenic toxicity and promoting plant growth in Vigna radiata
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: Received 8 January 2016 Received in revised form 24 May 2016 Accepted 31 May 2016
The present work describes the effects of inoculating metal resistant and plant growth promoting bacteria (PGPB) on the growth of Vigna radiata under arsenic (As) stress. The Exiguobacterium sp. strain, As-9 was isolated from arsenic contaminated soil, which showed resistance to exceptionally high concentrations of As(V) and As(III). Assessment of plant growth promoting parameters revealed the ability of the 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 As(III) was used for the germination of seeds of V. radiata under controlled conditions. The in vitro experiments proved that Exiguobacterium significantly (p < 0.05) increased the shoot and root biomass of V. radiata in the presence of As(V) and As(III) together with increase in the plant height, survival index and chlorophyll content after 15 days of inoculation. It also protected the plants from the detrimental effects of arsenic by reducing its uptake and translocation by colonizing the root surface. In addition, assay of antioxidant enzymes and lipid peroxidation test revealed significant (p < 0.01) reduction in arsenic-induced oxidative stress in V. radiata in the presence of bacteria. Owing to its wide action 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.
Handling Editor: C.C. Tebbe Keywords: Exiguobacterium Arsenate Arsenite Plant growth promotion Oxidative stress
1. Introduction 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 influenced 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 kg1 have been reported [5] but the concentration tolerated by plants ranges from 1 to 50 mg kg1 depending upon the crop variety [6].
* Corresponding author. E-mail address: [email protected] (N. Pandey). http://dx.doi.org/10.1016/j.ejsobi.2016.05.007 1164-5563/© 2016 Elsevier Masson SAS. All rights reserved.
Arsenic is a nonessential element for plant; however depression of plant growth usually occurs at higher levels of arsenic application 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,
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plants use their scavenging system to control ROS (peroxide, superoxide, hydroxyl ion etc) which comprises of enzymatic antioxidative 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 plantassociated bacteria from heavy metal enriched habitats is more recent [15]. The diversity of soil microbial communities is exceedingly 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 promoting 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 cultivated 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 objectives 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 Exiguobacterium 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 Exiguobacterium 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 bacterium, 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 concentration of either 100 mg L1 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 Generation Atomic Absorption Spectrophotometer (HG-AAS) (AA7000, Shimadzu) by the method described by Wang et al. [23].
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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. Phosphate solubilizing activity was quantitatively estimated in NBRIP media containing tricalcium phosphate amended with 0e1000 mg L1 of As(V). The media was inoculated with the bacterial strain and incubated at 28 C for 144 h at 170 rpm. The solubilized 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 mL1) and 0e1000 mg L1 concentration 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 L1 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 medium by centrifugation at 15,000g for 30 min and the supernatant 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, transferred 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 ha1 nitrate (NO3), 15.6 kg ha1 phosphorous (P2O5), 187.4 kg ha1 potassium (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 kg1 respectively. About 50 g of sieved soil were transferred to 250 mL conical flasks and sterilized by autoclaving to kill all the microbial population 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 g1 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 noninoculated test [soil/þAs(III)/E], and (f) As(III) treated and inoculated 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 kg1 soil. The flasks were incubated at 25 C ± 2 C and 16/8 h photoperiod for a period of 15 days.
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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.
SI ¼
Number of plants survived 100 Sample size
[1]
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 [2] 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) followed 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. Samples 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:
AF ¼
Concentration of arsenic in plant tissues Initial concentration of arsenic in soil
[3]
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). Sections 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 noninoculated 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 sputtered with gold particle and observed under Scanning Electron Microscope (JSM-6360).
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) containing 1 mM EDTA using a chilled mortar and pestle. The homogenate was centrifuged at 20,000g at 4 C for 15 min and the 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) mg1 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 mM1 cm1. 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 the reduction of H2O2 at 240 nm (ε ¼ 039.4 mM1 cm1) 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 mM1 cm1) 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 molecules of TBA (MDA-TBA2) was calculated from the extinction coefficient of 155 mM1 cm1.
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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 concentration ranging between 50.2 and 76.9 mg L1 for As(V) and 13.3e26.5 mg L1 for As(III) (Fig. 1). However, the removal efficiency 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 experiment (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 depends 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 mL1) 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 Exiguobacterium produced good amount of IAA in the presence and absence of As(V) with a concentration ranging between 10 and 15 mg mL1. This indicated that the bacterium was able to utilize Ltryptophan as a precursor for growth and IAA production. Further investigation of the PGP activities of the strain revealed enhanced production of EPS (20e28 mg mL1) with 400e600 mg L1 As(V)
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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 L1 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 kg1 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 chlorophyll 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 kg1 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 noninoculated 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 concentrations of both As(V) and As(III) in roots as well as shoots when compared to non-inoculated plants. The decreased accumulation 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
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.
Ultra-structural changes in the roots and shoots of mung bean plants were observed under arsenic stress condition. The transverse 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).
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Table 1 Plant growth promoting (PGP) activities of the bacterial strain at different concentrations of As(V). As(V)a doze (mg L1)
Phosphate solubilization (mg mL1)
0 200 400 600 800 1000
67.7 65.9 62.3 58.5 56.1 53.4
± ± ± ± ± ±
2.5 1.6 1.6 2.2 1.7 2.0
IAAb production (mg mL1)
EPSc production (mg mL1)
15.1 ± 0.7 14.2 ± 1.0 13.8 ± 0.9 12.1 ± 1.0 10.9 ± 0.3 9.8 ± 0.9
4.02 20.7 23.8 28.4 18.6 11.5
± ± ± ± ± ±
0.6 1.5 0.9 1.1 0.9 0.9
Values indicate ± S.D. of three replicates. a Sodium arsenate. b Indole acetic acid. c Exopolysaccharide.
Table 2 Effect of arsenic on the growth of mung bean plants after 15 days treatment. Treatments
Length (cm)
Biomass (g)
Shoot
Root
Blank Control As(V)/E As(V)/þE As(III)/E As(III)/þE
19.5 ± 2.21* 24.3 ± 2.63 11.2 ± 2.32** 18.4 ± 1.98* 2.2 ± 0.2*** 9.7 ± 1.14**
6.2 7.3 4.6 6.0 2.5 3.4
± ± ± ± ± ±
Shoot 0.6ns 1.0 1.01* 1.21* 0.3** 0.6*
0.27 0.35 0.21 0.27 0.15 0.21
± ± ± ± ± ±
with bioinoculant showed no visible modifications even in the presence of arsenic (Fig. 3 iv, vi) which was comparable to control (Fig. 3i, ii).
Root 0.08ns 0.04 0.04ns 0.09ns 0.01* 0.03*
0.19 0.23 0.11 0.18 0.02 0.09
± ± ± ± ± ±
0.03ns 0.01 0.04* 0.08* 0.01** 0.02*
Each value is a mean ± 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).
3.6. Root colonization by Exiguobacterium The ability of the Exiguobacterium to colonize roots of mung bean plants was assessed after 15 days of inoculation. The total culturable bacteria recovered from the roots of the inoculated plants was approximately, 3.6 104 CFU/g of fresh weight of roots. Root sections examined by SEM revealed strong proliferation and distribution of the isolate preferentially on the surface of the roots (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 kg1 As(V) and As(III). Treatments
Blank Control As(V)/E As(V)/þE As(III)/E As(III)/þE
Chlorophyll (mg mg1)
1.1 1.3 0.7 1.1 0.3 0.6
± ± ± ± ± ±
0.1ns 0.2 0.15* 0.2ns 0.1** 0.15*
As accumulation (mg Kg1) Shoot
Root
0.001ns 0 0.088 ± 0.016 ± 0.180 ± 0.057 ±
0.003* 0.001 0.145 ± 0.030 ± 0.338 ± 0.124 ±
0.005** 0.004* 0.045*** 0.003**
Accumulation factor (AF) (mg Kg1)
0.006*** 0.003*** 0.008*** 0.004***
0.04 0.01 2.33 0.46 5.18 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 endodermis 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) treatments 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
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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.
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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 kg1 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 metabolites [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 Exiguobacterium 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 solubilization of phosphate in the rhizosphere and IAA production makes a major contribution to plant growth by stimulating elongation of the
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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 adaptation 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 kg1) 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 chlorophyll 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 morphological 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, inoculation of bioinoculant caused considerable decrease in arsenicinduced 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 hypothesis was confirmed by SEM analysis which showed high level colonization of Exiguobacterium on the root surface of the inoculated mung bean plants. This proved that the successful colonization of the bacterium resulted in the decreased uptake of arsenic in tissues and helped the plants to grow and propagate at high concentrations 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) alterations 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 pronounced 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 indicator of oxidative damage produced during peroxidation of membrane 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 bioinoculant, there were many reductions in the antioxidative enzyme activity and MDA concentration in V. radiata to the extent comparable to control, indicating the lower production of ROS thereby reducing oxidative damage. In conclusion, the results of the study indicated that Exiguobacterium 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 ultimate 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, Chemosphere 72 (2008) 1548e1552. [4] P.L. Smedley, D.G. Kinniburgh, A review of the source, behaviour and distribution 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.
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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: Received 8 January 2016 Received in revised form 24 May 2016 Accepted 31 May 2016
The present work describes the effects of inoculating metal resistant and plant growth promoting bacteria (PGPB) on the growth of Vigna radiata under arsenic (As) stress. The Exiguobacterium sp. strain, As-9 was isolated from arsenic contaminated soil, which showed resistance to exceptionally high concentrations of As(V) and As(III). Assessment of plant growth promoting parameters revealed the ability of the 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 As(III) was used for the germination of seeds of V. radiata under controlled conditions. The in vitro experiments proved that Exiguobacterium significantly (p < 0.05) increased the shoot and root biomass of V. radiata in the presence of As(V) and As(III) together with increase in the plant height, survival index and chlorophyll content after 15 days of inoculation. It also protected the plants from the detrimental effects of arsenic by reducing its uptake and translocation by colonizing the root surface. In addition, assay of antioxidant enzymes and lipid peroxidation test revealed significant (p < 0.01) reduction in arsenic-induced oxidative stress in V. radiata in the presence of bacteria. Owing to its wide action 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.
Handling Editor: C.C. Tebbe Keywords: Exiguobacterium Arsenate Arsenite Plant growth promotion Oxidative stress
1. Introduction 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 influenced 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 kg1 have been reported [5] but the concentration tolerated by plants ranges from 1 to 50 mg kg1 depending upon the crop variety [6].
* Corresponding author. E-mail address: [email protected] (N. Pandey). http://dx.doi.org/10.1016/j.ejsobi.2016.05.007 1164-5563/© 2016 Elsevier Masson SAS. All rights reserved.
Arsenic is a nonessential element for plant; however depression of plant growth usually occurs at higher levels of arsenic application 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,
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plants use their scavenging system to control ROS (peroxide, superoxide, hydroxyl ion etc) which comprises of enzymatic antioxidative 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 plantassociated bacteria from heavy metal enriched habitats is more recent [15]. The diversity of soil microbial communities is exceedingly 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 promoting 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 cultivated 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 objectives 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 Exiguobacterium 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 Exiguobacterium 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 bacterium, 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 concentration of either 100 mg L1 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 Generation Atomic Absorption Spectrophotometer (HG-AAS) (AA7000, Shimadzu) by the method described by Wang et al. [23].
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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. Phosphate solubilizing activity was quantitatively estimated in NBRIP media containing tricalcium phosphate amended with 0e1000 mg L1 of As(V). The media was inoculated with the bacterial strain and incubated at 28 C for 144 h at 170 rpm. The solubilized 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 mL1) and 0e1000 mg L1 concentration 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 L1 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 medium by centrifugation at 15,000g for 30 min and the supernatant 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, transferred 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 ha1 nitrate (NO3), 15.6 kg ha1 phosphorous (P2O5), 187.4 kg ha1 potassium (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 kg1 respectively. About 50 g of sieved soil were transferred to 250 mL conical flasks and sterilized by autoclaving to kill all the microbial population 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 g1 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 noninoculated test [soil/þAs(III)/E], and (f) As(III) treated and inoculated 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 kg1 soil. The flasks were incubated at 25 C ± 2 C and 16/8 h photoperiod for a period of 15 days.
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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.
SI ¼
Number of plants survived 100 Sample size
[1]
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 [2] 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) followed 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. Samples 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:
AF ¼
Concentration of arsenic in plant tissues Initial concentration of arsenic in soil
[3]
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). Sections 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 noninoculated 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 sputtered with gold particle and observed under Scanning Electron Microscope (JSM-6360).
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) containing 1 mM EDTA using a chilled mortar and pestle. The homogenate was centrifuged at 20,000g at 4 C for 15 min and the 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) mg1 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 mM1 cm1. 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 the reduction of H2O2 at 240 nm (ε ¼ 039.4 mM1 cm1) 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 mM1 cm1) 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 molecules of TBA (MDA-TBA2) was calculated from the extinction coefficient of 155 mM1 cm1.
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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 concentration ranging between 50.2 and 76.9 mg L1 for As(V) and 13.3e26.5 mg L1 for As(III) (Fig. 1). However, the removal efficiency 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 experiment (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 depends 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 mL1) 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 Exiguobacterium produced good amount of IAA in the presence and absence of As(V) with a concentration ranging between 10 and 15 mg mL1. This indicated that the bacterium was able to utilize Ltryptophan as a precursor for growth and IAA production. Further investigation of the PGP activities of the strain revealed enhanced production of EPS (20e28 mg mL1) with 400e600 mg L1 As(V)
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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 L1 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 kg1 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 chlorophyll 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 kg1 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 noninoculated 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 concentrations of both As(V) and As(III) in roots as well as shoots when compared to non-inoculated plants. The decreased accumulation 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
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.
Ultra-structural changes in the roots and shoots of mung bean plants were observed under arsenic stress condition. The transverse 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).
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Table 1 Plant growth promoting (PGP) activities of the bacterial strain at different concentrations of As(V). As(V)a doze (mg L1)
Phosphate solubilization (mg mL1)
0 200 400 600 800 1000
67.7 65.9 62.3 58.5 56.1 53.4
± ± ± ± ± ±
2.5 1.6 1.6 2.2 1.7 2.0
IAAb production (mg mL1)
EPSc production (mg mL1)
15.1 ± 0.7 14.2 ± 1.0 13.8 ± 0.9 12.1 ± 1.0 10.9 ± 0.3 9.8 ± 0.9
4.02 20.7 23.8 28.4 18.6 11.5
± ± ± ± ± ±
0.6 1.5 0.9 1.1 0.9 0.9
Values indicate ± S.D. of three replicates. a Sodium arsenate. b Indole acetic acid. c Exopolysaccharide.
Table 2 Effect of arsenic on the growth of mung bean plants after 15 days treatment. Treatments
Length (cm)
Biomass (g)
Shoot
Root
Blank Control As(V)/E As(V)/þE As(III)/E As(III)/þE
19.5 ± 2.21* 24.3 ± 2.63 11.2 ± 2.32** 18.4 ± 1.98* 2.2 ± 0.2*** 9.7 ± 1.14**
6.2 7.3 4.6 6.0 2.5 3.4
± ± ± ± ± ±
Shoot 0.6ns 1.0 1.01* 1.21* 0.3** 0.6*
0.27 0.35 0.21 0.27 0.15 0.21
± ± ± ± ± ±
with bioinoculant showed no visible modifications even in the presence of arsenic (Fig. 3 iv, vi) which was comparable to control (Fig. 3i, ii).
Root 0.08ns 0.04 0.04ns 0.09ns 0.01* 0.03*
0.19 0.23 0.11 0.18 0.02 0.09
± ± ± ± ± ±
0.03ns 0.01 0.04* 0.08* 0.01** 0.02*
Each value is a mean ± 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).
3.6. Root colonization by Exiguobacterium The ability of the Exiguobacterium to colonize roots of mung bean plants was assessed after 15 days of inoculation. The total culturable bacteria recovered from the roots of the inoculated plants was approximately, 3.6 104 CFU/g of fresh weight of roots. Root sections examined by SEM revealed strong proliferation and distribution of the isolate preferentially on the surface of the roots (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 kg1 As(V) and As(III). Treatments
Blank Control As(V)/E As(V)/þE As(III)/E As(III)/þE
Chlorophyll (mg mg1)
1.1 1.3 0.7 1.1 0.3 0.6
± ± ± ± ± ±
0.1ns 0.2 0.15* 0.2ns 0.1** 0.15*
As accumulation (mg Kg1) Shoot
Root
0.001ns 0 0.088 ± 0.016 ± 0.180 ± 0.057 ±
0.003* 0.001 0.145 ± 0.030 ± 0.338 ± 0.124 ±
0.005** 0.004* 0.045*** 0.003**
Accumulation factor (AF) (mg Kg1)
0.006*** 0.003*** 0.008*** 0.004***
0.04 0.01 2.33 0.46 5.18 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 endodermis 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) treatments 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
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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.
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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 kg1 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 metabolites [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 Exiguobacterium 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 solubilization of phosphate in the rhizosphere and IAA production makes a major contribution to plant growth by stimulating elongation of the
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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 adaptation 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 kg1) 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 chlorophyll 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 morphological 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, inoculation of bioinoculant caused considerable decrease in arsenicinduced 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 hypothesis was confirmed by SEM analysis which showed high level colonization of Exiguobacterium on the root surface of the inoculated mung bean plants. This proved that the successful colonization of the bacterium resulted in the decreased uptake of arsenic in tissues and helped the plants to grow and propagate at high concentrations 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
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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) alterations 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 pronounced 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 indicator of oxidative damage produced during peroxidation of membrane 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 bioinoculant, there were many reductions in the antioxidative enzyme activity and MDA concentration in V. radiata to the extent comparable to control, indicating the lower production of ROS thereby reducing oxidative damage. In conclusion, the results of the study indicated that Exiguobacterium 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 ultimate 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, Chemosphere 72 (2008) 1548e1552. [4] P.L. Smedley, D.G. Kinniburgh, A review of the source, behaviour and distribution 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.
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