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Comparative Study of Plant Growth Promoting Bacteria in Minimizing Toxic Effects of Chromium on Growth and Metabolic Activities in Wheat (Triticum aestivum).

Byline: Sadia Naseem, Muhammad Yasin, Muhammad Faisal and Ambreen Ahmed

Summary: In this study, effect of inoculation of five bacterial strains i.e., Kushneria avicenniae AHT, Halomonas sp. AST, Bacillus sp. AMP2, Halomonas venusta APA and Arthrobacter mysorens AHA on the growth of Triticum aestivum var. Inqilab 97 was observed under various concentrations (0, 10 and 20 ugml-1) of different chromium salts (CrCl3, K2CrO4, K2Cr2O7). Bacterial inoculation caused reduction in the chromium uptake (22-32, 5-22 and 2-18%) of seedlings both at 10 and 20 ug ml-1 CrCl3, K2CrO4 and K2Cr2O7 when compared with respective non- inoculated treatment. Also increase in acid phosphatase and peroxidase contents was recorded due to bacterial inoculations compared to control.

Keywords: Chromium, Rhizobacteria, Triticum aestivum, Acid phosphatase, Peroxidase.

Introduction

Heavy metals like Zn, Cd, Pb, Ba, Cr, etc. show toxic effects by altering biological processes through metal-ions displacement or blockage and are hazardous in concentrations higher than the permissive limits. The effect of chromium (Cr) on plants is well pronounced. Chromium is a group VI-B transition metal with atomic number 24 and average atomic mass 51.998g. In natural terrestrial environment, Chromium commonly exists in the hexavalent or trivalent oxidation state. Chromium is a metal that is commonly found in the industrial effluents and is quite toxic to human beings via oral exposure causing kidney injury, lung cancer, etc. The effluents from electroplating factories, leather tanneries and textile manufacturing factories are responsible for addition of Cr in water.

Chromium occurs in various oxidation states (Cr+2 to Cr+6 ) but Cr+3 and Cr+6 are most abundant forms and are of biological significance. Cr (III) is less harmful and less mobile while Cr (VI) is more toxic, mutagenic and carcinogenic [1]. Hexavalent chromium is a known carcinogen. It causes inflammation of the skin and exposure to chromium (VI) results in increased incidence of lung cancer. On the other hand, trivalent chromium is not known as toxic for humans.

Chromium-resistant bacterial strains can survive in the presence of heavy metal stress through development of various strategies. The toxic Cr (VI) should be necessarily converted by several bacterial strains which are able to reduce toxic hexavalent chromium into trivalent chromium [2]. Chromate tolerance in chromium-resistant bacteria occurred by different mechanisms such as decreasing chromate accumulation by resistant cells or reducing toxic hexavalent (Cr+6) to trivalent (Cr+3) chromium [3]. Bacterial interactions with metals have been very helpful for treatment of heavy metals in contaminated waste water. Inoculation of microbes in the soil proved to be very successful for phytostimulation through improvement of soil structure, suppression of pathogens and degradation of toxic substances.

Rhizobacteria present at the chromium contaminated sites have high chromium reduction potential and use of these plant growth promoting bacteria (PGPB) with chromium-resistant potential generally improves plant growth efficiently with simultaneous reduction in chromium toxicity. Presently the use of rhizobacteria for plant growth promotion is attracting considerable attention as an alternative approach for bioremediation of heavy metal contamination.

To evaluate the impact of bacterial inoculations (Kushneria avicenniae AHT, Halomonas sp. AST, Bacillus sp. AMP2, Halomonas venusta APA and Arthrobacter mysorens AHA) and effect of chromium salt (CrCl3, K2CrO4, K2Cr2O7) stresses (10ugml-1, 20ugml-1) on seed germination and early growth of Triticum aestivum, present work was carried out. Effect of inoculation on germination and growth parameters such as root length, shoot length and fresh weight were studied along with biochemical analysis (peroxidase content and acid phosphatase content) and chromium content estimation.

Experimental

Preparation of Bacterial Inoculum:

Five bacterial strains i.e., Kushneria avicenniae AHT, Halomonas sp. AST, Bacillus sp. AMP2, Halomonas venusta APA and Arthrobacter mysorens AHA isolated by Afrasiab [4] were used in this study. Bacterial strains from 24 hours incubated fresh cultures were grown on L-Agar plates. Plates were then kept for 24 hours at 37C. Cells from fresh cultures were harvested. The pellets were redissolved in sterilized distilled water. The optical density (OD) was monitored at 600 nm and was adjusted to the same value i.e., 105 CFU ml-1.

Sterilization and Inoculation of Seeds

Certified seeds of Triticum aestivum var. Inqilab 97 were obtained from Punjab Seed Corporation, Lahore, Pakistan. Healthy seeds of Triticum aestivum var-97 were treated aseptically with 0.1% HgCl2 solution. Seeds were then repeatedly washed with autoclaved distilled water to completely remove HgCl2. Sterilized seeds were then treated with bacterial cultures adjusted to same optical density for twenty mins. For control treatment, seeds were treated with autoclaved distilled water for the same interval of time.

Experimental Set Up

The effect of bacterial strains i.e., Kushneria avicenniae AHT, Halomonas sp. AST, Bacillus sp. AMP2, Halomonas venusta APA and Arthrobacter mysorens AHA was studied on the germination and growth of Triticum aestivum seedlings in the presence and absence of various concentrations (10 ugml-1, 20 ugml-1) of three different chromium salts [CrCl3, K2CrO4, K2Cr2O7] . Non-inoculated treatments in the presence and absence of chromium were also used for the current study.

The experimental set up was as follows:

Petriplates of 120 mm diameter were washed and oven dried. Two layers of Whatman filter paper No. 1 were placed in each plate. Then the plates were autoclaved at 121 C for 15 mins (15lb/in2) and oven dried. Plates were properly labeled for each strain and control (without inoculation) with different concentrations of working solutions i.e., CrCl3 (0 ugml-1, 10 ugml-1, 20 ugml-1), K2CrO4 (0 ugml-1, 10 ugml-1, 20 ugml-1), K2Cr2O7 (0 ugml-1, 10 ugml-1, 20 ugml-1). 10ml of sterilized respective salt concentration (CrCl3, K2CrO4 and K2Cr2O7) was supplied to each respective plate so that the filter papers were well moistened. In the non- inoculated treatment without any chromium stress, 10ml of sterilized distilled water was used. Previously treated and inoculated seeds of plants (25 for each petriplate) were uniformly spread on the moistened filter paper with the help of sterilized forceps.

Three replicates were used for each treatment and control. All petriplates were kept in dark at 25 + 2 C for 3 days. After three days, germination of seeds was noted. After germination, germinated seedlings were transferred to the labeled pots each containing 140 gm sieved soil and stress solution (0, 10 and 20 ugml-1) of chromium salts (CrCl3, K2CrO4 and K2Cr2O7) were given to the respective pots. The pots were shifted to light (10 K lux, 16 hours duration) at 25+ 2 C. Seedlings were grown for 10-15 days. Daily observations were made. The experiment was repeated thrice.

Estimation of Chromium Uptake

Humphries's (1985) method was used for digestion of plant material and chromium content estimation was done following Rand et al. method [5]. For this purpose, oven dried plant material i.e., plant leaves were weighed and taken in respective labeled flasks. In each flask, two ml of HClO4 and ten ml of HNO3 were added and the flasks were then heated on sand bath. Brown fumes of HNO3 were initially released from flask which became dense white later. After removing the flask from the sand bath when solution became clear, the final volume was made upto 15 ml using distilled water after cooling. Then samples were used for chromium estimation following Rand et al., 1979. For chromium estimation, 2-3 drops of methyl orange were added (as an indicator) in one ml of digested sample taken in labeled flasks. Appearance of pink colour indicated acidity of the solution. Ammonium hydroxide was then added till pink colour turned to yellow indicating alkaline nature of the solution.

Now 50% H2SO4 was added dropwise which resulted in the appearance of pink colour. When pink colour reappeared, 1ml of 50% H2SO4 was added in excess. Final volume was then raised to 40ml using distilled water and solution was then heated till boiling of solution. At this point, 2-3 drops of KMnO4 were added which resulted in purple coloration. In case this colour persists then 1ml of sodium azide was mixed in each flask and boiled for two mins. The solution became colourless at the end in which 0.25 ml of H3PO4 was added after cooling and the final volume was raised to 100ml by using distilled water. In each flask, 2ml of diphenyl carbazide was then added and the flasks were kept for half an hour in dark. Reaction of chromium with diphenyl carbazide resulted in purple colouration. Then the O.D. was monitored at 510 nm using spectrophotometer (200- D) and chromium content was calculated by using the following formula:

Chromium (mg/l) = A x 100/B x C [A = ug Cr, B = ml of original sample, C = ml obtained from 100 ml digested sample]

Estimation of Peroxidases

David and Murray method [6] was followed for estimation of peroxidases. Already weighed and frozen leaves of treated plants were crushed. Phosphate buffer (0.1M; pH 7) was used in a ratio of 1:4. The samples were centrifuged for 10 min at 4C. Crude enzyme extract (0.2 ml) was mixed with 2.5 ml of 0.1 M phosphate buffer and 0.2 ml of 1% guaicol solution. Guaicol solution was not added in control set of test tubes. The tubes were kept for several mins at room temperature. After few mins, 0.3% H2O2 solution was added and solution was stirred well. Optical density was recorded at 470nm on spectrophotometer (200-D). The optical density was taken using blank which is prepared by adding and mixing 2.5 ml phosphate buffer, 0.2 ml water and 0.1 ml of 0.3 % H2O2.

Peroxidases were calculated by using the following formula:

Peroxidase content (unit mg-1) = O.D of test - O.D of control/O.D of control x mg. of plant material

Estimation of Acid Phosphatases

Acid phosphatases were estimated following Iqbal and Rafique [7]. Already frozen and weighed leaves of various treated and control plants were crushed in cold pestle and mortar with 0.1 M Tris- HCl buffer and were then placed at 4C. Estimation of acid phosphatases was carried out by using the supernatant obtained. For this purpose, two sets of tubes were prepared, one set "for test" and second "for control". In the set "for test", 1 ml of substrate disodium pheny1 phosphate was mixed with 1 ml of citrate buffer [pH 4.9] in each test tube. It was placed in a water bath for three mins at 37C. After three mins, 0.2 ml of enzyme extract was added, mixed gently and kept at 37C for one hour. After one hour of incubation at 37C, 0.5N NaOH (one ml) was added to stop further reaction. In the other set that is "for control", 1 ml of substrate disodium phenyl phosphate was mixed with 1 ml of citrate buffer in each test tube and was kept in a water bath at 37C for one hour.

After heating at 37C for one hour, 1ml of 0.5N NaOH was added and mixed. Then 0.2ml of enzyme extract was added. In addition, one test tube was taken as standard and one as blank. In blank tube, 1.2 ml of citrate buffer, one ml of distilled water and one ml of 0.5N NaOH were mixed in a test tube. In standard tube, 1.2 ml of citrate buffer, 1 ml of phenol, and 1 ml of 0.5N NaOH were mixed together in a test tube. Now in all above test tubes, 1 ml of 0.5N NaHCO3 was added and then 1 ml of 4-amino antipyrin solution was also added. Then 1 ml of potassium ferricyanide was added. Contents in all test tubes were mixed thoroughly. Intensity of reddish brown colour was recorded against water using spectrophotometer (200D) at 510nm.

Following formula was used for estimating acid phosphatases:

Acid phosphatases (K.A. Units/100 ml)=

T-C/(S - B) x W

where K.A. unit is the liberation of 1 mg of phenol in one hour. [T = OD of test, C = OD of control, S = OD of standard, B = OD of blank, W =Weight of plant material (mg)]

Statistical Analysis

Data obtained was analyzed statistically following Steel and Torrie [8]. Mean, standard error of the mean and least significant difference were calculated.

Results

Effects of inoculation of five bacterial strains (Kushneria avicenniae AHT, Halomonas sp. AST, Bacillus sp. AMP2, Halomonas venusta APA and Arthrobacter mysorens AHA) on the growth of Triticum aestivum var. Inqilab 97 were observed under various stress concentrations (0, 10, 20 ug ml-1) of different chromium salts (CrCl3, K2CrO4, K2Cr2O7).

Percentage Germination

In non-inoculated seeds, as concentration increased from 0-20 ug ml-1 (CrCl3, K2CrO4, K2Cr2O7), there was decrease (except CrCl3, K2CrO4 ; 10 ug ml-1) in percentage germination of Triticum aestivum seeds over control treatment (Table-1).

Bacterial inoculations stimulated germination over control treatment. Under CrCl3 stress, with increase in salt concentration, bacterial inoculation either had no effect or caused reduction (except Kushneria avicenniae AHT -10 ug ml-1; Halomonas venusta APA-20 ug ml-1 CrCl3) in percentage germination. At 10 ug ml-1 CrCl3, bacterial inoculations enhanced percentage germination (except Arthrobacter mysorens AHA) over non-inoculated respective treatments (Table-1). In case of K2CrO4 stress, as concentration increased from 0-20 ugml-1, generally, reduction in germination percentage of Triticum aestivum seeds (except Arthrobacter mysorens AHA 10 ug ml-1) was observed. In general, inoculation of bacterial strains at 10 ugml-1 and 20 ugml-1 K2CrO4 enhanced percentage germination (except Kushneria avicenniae AHT at 10 ugml-1; Halomonas sp. AST at 20 ugml-1) over non-inoculated respective treatments.

The effect of K2Cr2O7 showed that with increase in concentration from 0 ug ml-1 to 20 ug ml-1, there was decrease in percentage germination (except Arthrobacter mysorens AHA at 10 ug ml-1). At 10-20 ug ml-1 concentration, all bacterial strains (except Bacillus sp. AMP2 at 10 ug ml-1) significantly enhanced percentage germination (Table-1).

Shoot Length

Shoot lengths of seedlings was maximum at 0 ugml-1 chromium. With increasing concentration of salt stress from 0-20 ug ml-1, reduction in shoot length was observed when compared with 0 ugml-1 treatment. Under CrCl3, K2CrO4 and K2Cr2O7 stress, increasing salt concentration (0-20 ug ml-1) reduced shoot length. In general, bacterial inoculations enhanced shoot length both at 10 ugml-1 and 20 ugml- 1 salt concentration when compared with non- inoculated respective treatments (Table-2).

Root length

In non-inoculated seedlings, reduction in root lengths (except CrCl3 at 10 ug ml-1) was observed with increasing salt concentrations (CrCl3, K2CrO4 and K2Cr2O7) from 0 to 20 ug ml-1. Bacterial inoculations stimulated root length in comparison with the non-inoculated control (Table-3). Under CrCl3 stress, increase in the root length was observed at 10 ug ml-1 salt concentration while at 20 ug ml-1, root length of seedlings was decreased when compared with 0 ugml-1 treatment. In case of K2CrO4 and K2Cr2O7 stress, increasing salt concentration (0- 20 ug ml-1) reduced root length. In general, bacterial inoculations enhanced root length both at 10 ug ml-1 and 20 ug ml-1 salt concentrations in comparison to the respective non-inoculated treatments (Table-3).

Seedling Length

With increasing salt concentration from 0 to 20 ug ml-1, seedling length was found to be reduced (except CrCl3, 20 ug ml-1) when compared with 0 ug ml-1 treatment. All bacterial strains stimulated the seedling growth over non-inoculated treatments.

Under K CrO and K Cr O stress, increase in salt concentration from 0 to 20 ug ml-1 had adverse effects on seedling length. At 10 and 20 ug ml-1 CrCl3, K2CrO4 and K2Cr2O7 salt concentration, bacterial strains stimulated the growth of seedlings (except Bacillus sp. AMP2 at 10 ug ml-1 K2Cr2O7; Halomonas venusta APA at 10, 20 ug ml-1 CrCl3 and 10 ug ml-1 K2CrO4 and Arthrobacter mysorens AHA at 20 ug ml-1 CrCl3) when compared with respective non-inoculated treatments (Table-4).

Table-1: Effects of bacterial inoculations on percentage germination of Triticum aestivum grown under different concentrations of Chromium salts (CrCl3, K2CrO4, K2Cr2O7).

###Cr Salt ug ml-1

###Sr no.###0###10###20

###Strains

###CrCl3###K2CrO4###K2Cr2O7###CrCl3###K2CrO4###K2Cr2O7

###1###Control###68###76.00###70.00###64.00###68.00###60.00###56.00

###2###AHT###86###88.00###68.00###74.00###80.00###62.00###70.00

###3###AST###100###96.00###92.00###88.00###72.00###60.00###68.00

###4###AMP2###86###80.00###72.00###60.00###80.00###68.00###58.00

###5###APA###80###80.00###70.00###76.00###88.00###66.00###64.00

###6###AHA###76###72.00###88.00###76.00###70.00###68.00###70.00

L S D (P=0.05)

###For Strains###9.2###9.8

###For Treatments###8.6###12.0

Table-2: Effects of bacterial inoculations on shoot length (cm) of Triticum aestivum grown under different concentrations of Chromium salts (CrCl3, K2CrO4, K2Cr2O7), (Means SE of three replicates).

###Cr Salt ug ml-1

Sr. no.###0###10###20

###Strains###CrCl3###K2CrO4###K2Cr2O7###CrCl3###K2CrO4###K2Cr2O7

###1###control###13.15 0.15###12.90 0.25###12.60 0.35###12.15 1.02###11.95 0.24###10.05 0.36###10.03 0.12

###2###AHT###17.85 0.14###16.50 0.87###14.55 0.34###15.95 0.23###15.10 0.25###9.65 0.35###12.55 0.14

###3###AST###21.85 0.26###18.90 0.35###17.25 0.36###18.00 0.24###18.40 0.26###16.50 0.24###15.80 0.15

###4###AMP2###18.10 0.28###14.20 0.34###13.70 0.34###11.45 0.49###14.00 0.15###10.10 0.35###11.00 0.34

###5###APA###15.55 0.26###13.88 0.34###9.70 0.24###11.90 0.34###12.90 0.34###11.20 0.34###11.00 0.15

###6###AHA###18.05 0.24###16.350.34###15.55 0.29###14.50 0.34###12.30 0.15###12.10 0.24###11.30 0.35

L S D (P=0.05)

###For Strains###13.1###0.7

###For Treatments###16.0###2.1

Table-3: Effects of bacterial inoculations on root length (cm) of Triticum aestivum grown under different concentrations of Chromium salts (CrCl3, K2CrO4, K2Cr2O7), (Means SE of three replicates).

###Cr Salt ug ml-1

Sr.No.###0###10###20

###Strains###CrCl3###K2CrO4###K2Cr2O7###CrCl3###K2CrO4###K2Cr2O7

###1###control###4.750.12###4.850.24###4.200.26###3.400.24###4.500.26###2.500.24###2.250.12

###2###AHT###6.850.24###5.900.34###4.000.89###4.800.24###6.200.34###3.650.24###3.070.32

###3###AST###6.370.34###4.990.34###4.420.38###5.500.26###4.390.34###3.050.34###3.620.12

###4###AMP2###5.250.32###3.950.35###3.910.23###3.400.35###2.650.34###2.650.26###2.800.35

###5###APA###5.450.36###4.550.34###2.620.25###2.950.32###4.250.35###2.450.21###3.180.25

###6###AHA###5.720.35###5.150.36###4.820.25###4.370.26###4.820.24###3.670.23###2.511.02

L S D (P=0.05)

###For Strains###0.8###0.8

###For Treatments###0.9###1.0

Table-4: Effects of bacterial inoculations on seedling length (cm) of Triticum aestivum grown under different concentrations of Chromium salts (CrCl3, K2CrO4, K2Cr2O7), (Means SE of three replicates).

###Cr Salt ug ml-1

Sr.no###0###10###20

###Strains###CrCl3###K2CrO4###K2Cr2O7###CrCl3###K2CrO4###K2Cr2O7

###1###control###19.90 0.12###18.750.21###16.80 0.35###15.550.35###20.00 0.25###13.070.24###12.55 2.05

###2###AHT###25.70 0.35###25.400.25###17.770.15###20.750.25###21.352.03###13.270.12###15.62 1.02

###3###AST###28.22 0.36###26.890.35###23.670.24###23.500.23###24.800.37###21.000.23###22.12 0.21

###4###AMP2###23.35 0.24###22.400.36###21.610.24###14.850.24###21.000.34###18.750.24###13.70 0.25

###5###APA###26.99 0.35###18.430.15###16.320.28###17.000.25###18.150.24###15.000.24###15.58 0.25

###6###AHA###25.77 0.36###21.500.57###20.370.21###19.400.25###17.120.34###17.920.25###14.86 0.21

L S D (P=0.05)

###For Strains###2.7###4.2

###For Treatments###3.3###3.6

Fresh Weight per seedling

In non-inoculated seedlings, with increasing concentration of salts from 0 to 20 ug ml-1, fresh weight per seedling decreased as compared to the 0 ug ml-1 treatment. Bacterial inoculation either reduced or had no effect on fresh weight per seedling when compared with the non-inoculated treatment (Table-5). Under CrCl3 stresses, increase in salt concentration (0 to 20 ug ml-1) reduced fresh weight per seedling as compared to the control treatment. Bacterial inoculations reduced (except Bacillus sp. AMP2 at 10 ug ml-1) fresh weight per seedling both at 10 and 20 ug ml-1 CrCl3 concentration when compared with respective non- inoculated treatments (Table-5).

Under K2CrO4 stress, reduction in fresh weight per seedling was observed with increasing salt concentration (0- 20 ug ml-1). All bacterial strains reduced (except Bacillus sp. AMP2) fresh weight per seedling at both concentrations of salt stress (10 and 20 ug ml-1) when compared with respective non- inoculated treatment (Table-5). Under K2Cr2O7 salt stress, increase in salt concentration (0-20 ug ml-1) reduced fresh weight as compared to 0 ug ml-1 treatment. At 10 and 20 ug ml-1 (K2Cr2O7), bacterial inoculations either reduced (except Bacillus sp. AMP2 both at 10 and 20 ug ml-1; Kushneria avicenniae AHT at 20 ug ml-1) or had no effect on fresh weight per seedling when compared with respective non-inoculated treatments.

Chromium Content

No chromium content was detected at 0 ug ml-1 treatment either in control or treatments inoculated with bacteria (Table-6). Under CrCl3, K2CrO4 and K2Cr2O7 stress, increase in salt concentration from 0 to 20 ug ml-1 enhanced chromium uptake over control treatment. Bacterial inoculation caused reduction in the chromium uptake of seedlings both at 10 and 20 ug ml-1 salt concentration when compared with respective non inoculated treatment (Table-6).

Acid Phosphatases

Generally with increasing concentration of salt stress from 0-20 ug ml-1, acid phosphatases also increased. At 0 ug ml-1, all bacterial inoculations (except Bacillus sp. AMP2) enhanced acid phosphatases relative to the non-inoculated control treatment. Under CrCl3, K2CrO4 and K2Cr2O7 stresses, acid phosphatases increased with increasing salt concentration. In general, bacterial inoculations caused increase in acid phosphatases except Kushneria avicenniae AHT at 20 ug ml-1 CrCl3; Bacillus sp. AMP2 at 20 ug ml-1 K2CrO4 and Arthrobacter mysorens AHA at 20 ug ml-1 CrCl3 , K2CrO4 and K2Cr2O7 (Table-7).

Table-5: Effects of bacterial inoculations on fresh weight per seedling (g) of Triticum aestivum grown under different concentrations of Chromium salts (CrCl3, K2CrO4, K2Cr2O7), (Means SE of three replicates).

###Cr Salt ug ml-1

Sr. No.###0###10###20

###Strains###CrCl3###K2CrO4###K2Cr2O7###CrCl3###K2CrO4###K2Cr2O7

1###control###0.200.02###0.140.02###0.090.01###0.080.01###0.130.001###0.080.002###0.060.001

2###AHT###0.180.001###0.120.00###0.060.00###0.080.001###0.100.00###0.050.00###0.070.001

3###AST###0.150.001###0.080.00###0.060.00###0.070.00###0.080.001###0.050.00###0.020.00

4###AMP2###0.200.00###0.160.00###0.100.00###0.100.001###0.120.002###0.090.00###0.080.001

5###APA###0.130.00###0.100.00###0.060.00###0.080.00###0.090.001###0.070.002###0.060.00

6###AHA###0.110.001###0.110.001###0.060.00###0.070.002###0.100.00###0.040.00###0.050.00

L S D (P=0.05)

###For Strains###0.04###0.04

For Treatments###0.05###0.05

Table-6: Effects of bacterial inoculations on chromium uptake (ug g-1) of Triticum aestivum grown under different concentrations of Chromium salts (CrCl3, K2CrO4, K2Cr2O7).

###Cr Salt ug ml-1

Sr. no.###0###10###20

###Strains###CrCl3###K2CrO4###K2Cr2O7###CrCl3###K2CrO4###K2Cr2O7

###1###Control###0.00###2.24###11.8###10.43###1.48###13.50###14.44

###2###AHT###0.00###1.02###10.59###10.20###1.06###11.00###12.74

###3###AST###0.00###1.12###10.44###9.48###1.04###12.89###12.44

###4###AMP2###0.00###1.04###11.20###9.90###1.00###10.40###13.99

###5###APA###0.00###0.04###11.00###10.30###1.15###12.39###11.94

###6###AHA###0.00###0.11###9.90###10.10###1.13###12.49###14.10

L S D (P=0.05)

###For Strains###0.6###1.0

###For Treatments###0.8###1.2

Table-7: Effects of bacterial inoculations on activity of acid phosphatase enzyme (units g-1) of Triticum aestivum grown under different concentrations of Chromium salts (CrCl3, K2CrO4, K2Cr2O7)=

###Cr Salt ug ml-1

Sr. No.###0###10###20

###Strains###CrCl3###K2CrO4###K2Cr2O7###CrCl3###K2CrO4###K2Cr2O7

###1###Control###1.73###1.67###2.47###2.47###2.81###4.00###4.05

###2###AHT###2.50###2.47###3.15###4.43###2.63###6.88###5.31

###3###AST###2.69###2.80###3.81###4.31###3.50###4.19###5.06

###4###AMP2###1.50###2.39###2.53###3.21###3.55###3.43###4.77

###5###APA###2.39###2.34###3.82###3.86###2.80###4.00###4.00

###6###AHA###2.00###2.10###3.09###2.64###2.33###3.55###3.09

L S D (P=0.05)

###For Strains###1.9###2.1

###For Treatments###0.5###2.6

Table-8: Effects of bacterial inoculations on activity of peroxidase enzyme (units g-1) of Triticum aestivum grown under different concentrations of Chromium salts (CrCl3, K2CrO4, K2Cr2O7)

###Cr Salt ug ml-1

Sr No.###0###10###20

###Strains###CrCl3###K2CrO4###K2Cr2O7###CrCl3###K2CrO4###K2Cr2O7

###1###Control###6.10###7.07###8.48###11.50###9.00###9.02###15.66

###2###AHT###8.80###9.64###10.20###12.47###11.33###12.15###14.00

###3###AST###8.03###9.07###13.00###15.84###10.20###15.31###16.62

###4###AMP2###9.01###12.38###14.14###16.25###12.00###16.11###17.50

###5###APA###8.65###10.00###15.60###17.17###13.00###14.11###19.24

###6###AHA###9.90###10.34###14.04###18.40###11.00###11.28###18.00

L S D (P=0.05)

###For Strains###0.4###1.0

###For Treatments###0.5###1.2

Peroxidases

With increasing salt concentration from 0-20 ug ml-1, increase in peroxidases was observed relative to the control treatment. Bacterial inoculations enhanced peroxidase content when compared with non-inoculated control treatment (Table-8). Under CrCl3 stresses, it was evident that increasing CrCl3 concentration from 0-20 ug ml-1 increased peroxidases over control treatment. All bacterial strains caused increase in peroxiodases both at 10 and 20 ug ml-1 salt stresses when compared with respective non-inoculated treatments. Under K2CrO4 stresses, generally, peroxidases increased with increase in salt concentration from 0-20 ug ml-1. Bacterial inoculations caused increase in peroxidases both at 10 and 20 ug ml-1 salt stress as compared to the respective non-inoculated treatments (Table-8).

In case of K2Cr2O7, increase in salt concentration (0-20 ug ml-1) increased peroxidases. At both 10 and 20 ug ml-1 stress, all bacterial strains caused increase (except Kushneria avicenniae AHT at 20 ug ml-1 K2Cr2O7) in peroxidases when compared with respective non-inoculated treatments.

Discussion

Rapid industrialization, dramatic increase in population and urbanization in the last few decades have added massive loads of pollutants in the water resources and agricultural land. Eco-friendly and cost-effective remediation technologies are required for such unprecedented pollution in aquatic ecosystems. A large number of industries including paper, pulp and textile, electroplating, printing, iron- steel, pesticide, petroleum, solvent, paint, and pharmaceutical etc., utilize large quantity of organic chemicals and water which vary in their toxicity and composition. The discharge of industrial effluents to various water bodies (lakes, canals and rivers etc.) leading to water pollution and agricultural land pollution is a matter of great concern. Biological methods for remediation of toxic metals provide an eco-friendly approach [9]. Phytoremediation of heavy metal pollution in the environment has been accomplished through the use of bacteria, yeast, fungi etc.

In this study, the role of bacterial strains (Kushneria avicenniae AHT, Halomonas sp. AST, Bacillus sp. AMP2, Halomonas venusta APA, Arthrobacter mysorens AHA) on the growth of Triticum aestivum var. Inqilab 97, was observed under various stress concentrations (0, 10, 20 ug ml-1) of different chromium salts (CrCl3, K2CrO4, K2Cr2O7). We found that in non-inoculated seeds, as concentration increased from 0-20 ug ml-1 (CrCl3, K2CrO4, K2Cr2O7), there was decrease (except (CrCl3, K2CrO4 ;10 ug ml-1) in percentage germination of Triticum aestivum seeds compared to control treatment. Similar effects of increased Cr concentration over seedling germination were found by other researchers [10, 11].

With increasing concentration of salt stress from 0-20 ug ml-1, reduction in seedling length, shoot length and root length was observed when compared with 0 ug Cr ml-1 treatment. Moreover, bacterial inoculations stimulated root growth of seedlings as compared to non-inoculated treatments. Diwan et al. [12] have also reported similar results i.e., growth of Brassica juncea (Indian mustard) got reduced and root length also reduced [13] with increased concentration of Cr. All bacterial strains stimulated shoot growth when compared with non-inoculated treatments. Faisal et al. [14] have also found a similar positive effect of bacterial inoculation over plant growth under Cr stress. Generally, with increase in concentration of salt from 0 to 20 ug ml-1, decrease in fresh biomass per seedling was observed when compared with 0 ug ml-1 treatment. Bacterial inoculation either enhanced or had no effect on fresh biomass when compared with non-inoculated treatment.

With increase in Cr salt concentration from 0 to 20 ug ml-1 enhanced chromium uptake over control treatment. Bacterial inoculation caused reduction in the chromium uptake of seedlings both at 10 and 20 ug ml-1 salt concentrations when compared with respective non-inoculated treatment.

Our results also correlate with the findings of Diwan et al. [12]. In the present study, bacterial inoculations caused increase in acid phosphatases of the plants compared to the non-inoculated control in the absence of chromium stress. In the presence of chromium stress in non-inoculated treatments,increase in acid phosphatases was recorded with increasing salt stress from 0-20 ug ml-1 indicating their role to improve plant growth under stress conditions (Table-7). Bacterial inoculations, in the presence of chromium stress, was recorded to cause further enhancement in acid phosphatase activity in comparison with the respective control treatment which is a clear manifestation of bacterial involvement in promoting acid phosphatase activity especially in the presence of stress to help the plant to survive under stress conditions. With reference to acid phosphatases, various bacterial strains have shown variable potential to increase acid phosphatase activity.

For instance in the present study, two of the isolates Halomonas sp. APA and Arthrobacter mysorens AHA did not affect acid phosphatase activity in the presence of higher concentrations (20ugml-1) of chromium. Acid phosphatases are the enzymes found in plants which help in the growth of plants by making nutrients especially phosphates, available to the plants. These enzymes help in plant growth improvement under stress condition [15]. Similarly bacterial inoculations helped to increase peroxidase activity compared to non-inoculated control in the presence and absence of chromium stress (Table-8). All the isolates caused an increase in peroxidase activity compared to respective control treatment. With increasing salt concentration from 0-20 ug ml-1, increase in peroxidases was observed relative to the control treatment. Increase in peroxidase activity caused reduction in the adverse effects of toxic substances, thereby improving plant growth under chromium stress.

Peroxidases are the enzymes found in plants which help the plant to get rid of the toxic substances synthesized as a result of various biological processes. These help to oxidize toxic substances into harmless products or less toxic forms thus helping in plant growth improvement. These can be used for bioremediation purposes in the heavy metal contaminated areas [16, 17].

Conclusion

From literature, it is proven that microbes play an important role in promoting plant growth and survival in metal-polluted soils. The information generated in recent research has enhanced the understanding of bacteriology and of the metal tolerance of bacteria and plants; however, further studies in this area are necessary for improving and implementing the use of bacteria in phytoremediation programs. Moreover, this study shows that plant growth promoting bacteria can be used to detoxify toxic concentration of Cr in agricultural land. Future researchers on this topic should focus on the process optimization including the determination of the physiological mechanisms involved in metal translocation, absorption, and metabolism by the plants.

Acknowledgements

We acknowledge University of the Punjab for providing us funds to complete this research work.

Conflict of interest

All the authors have no conflict of interest.

References

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9. R. Saratale, G. Saratale, J. Chang and S. Govindwar, Bacterial Decolorization and Degradation of Azo Dyes: A review, J. Taiwan Inst. Chem. Eng., 142, 138 (2011).

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11. I. E. Akinci and S. Akinci, Effect of Chromium Toxicity on Germination and Early Seedling Growth in Melon (Cucumis melo L.), Afr. J. Biotechnol., 9, 4589 (2010).

12. H. Diwan, I. Khan, A. Ahmad and M. Iqbal, Induction of Phytochelatins and Antioxidant Defence System in Brassica juncea and Vigna radiata in Response to Chromium Treatments, Plant Growth Regul., 61, 97 (2010).

13. M. N. V. Prasad, M. Greger and T. Landberg, Acacia nilotica L. Bark Removes Toxic Elements from Solution: Corroboration from Toxicity Bioassay Using Salix viminalis L. in Hydroponic System, Int. J. Phytorem., 3, 289 (2001).

14. M. Faisal, A. Hameed and S. Hasnain, Chromium-Resistant Bacteria and Cyanobacteria: Impact on Cr(VI) Reduction Potential and Plant Growth, J. Ind. Microbiol. Biotechnol., 32, 615 (2005).

15. R. S Yadav and J.C. Tarafdar Influence of Organic and Inorganic Phosphorus Supply on the Maximum Secretion of Acid Phosphatase by Plants, Biol Fertil Soils, 34, 140 (2001).

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Author:Naseem, Sadia; Yasin, Muhammad; Faisal, Muhammad; Ahmed, Ambreen
Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Geographic Code:9PAKI
Date:Jun 30, 2016
Words:6364
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