Introduction

Legumes are well known for their important roles in maintaining productivity in agricultural systems (1). They are rich in proteins, minerals, vitamins and sugars in seeds (2). Persian clover (Trifoliumresupinatum) is an annual plant of the legume family and it has a high nutritive value as pasture or hay (3). Persian clover is among the most important forage crops native from the temperate regions cultivated in this regions to produce seeds (4). Persian clover can establish a symbiotic relation with the soil Rhizobium (5).

The Rhizobium is a genus of gram-negative, aerobic, rod-shaped bacteria that activate plant root nodulation in leguminous plants. Members of this genus are nitrogen-fixing and common soil inhabitants (6). Rhizobium is one of the most prominent Plant growth promoting rhizobacteria (PGPR) members. PGRP are a group of bacteria that can actively colonize plant roots and increase plant growth. These PGPR can prevent the deleterious effects of phytopathogenic organisms and environmental stressors (7). The use of PGPR to promote plant growth has increased in various parts of the world. PGPR can affect plant growth by producing and releasing secondary metabolites and facilitate the availability and uptake of certain nutrients from the root environment (8).

Sulfur dioxide (SO₂) is a major air pollutant that its concentrations increase in many metropolitan and industrial areas. It is a primary product of fossil fuels power plants combustion or from refining of sulfur-containing ores; it influences human health and theglobal ecology (9).SO₂occures normally 0.05–0.5 ppm in the urban areas and up to 2.0 ppm or more around point sources of air pollutionand it is the major source of atmospheric sulfur (10). The result of second world war that followed by the post war economic expansion is an unprecedented absolute rate of SO₂ emissions. Global emissions peaked in the 1970s, and have declined overall since 1990, with an increase between 2002 and 2005, largely due to strong growth of emissions in China. The 40% of SO₂ global emissions originated from Asia and it is growing (11).

In very low concentrations, SO₂ especially in sulfur-deficient soils can cause positive effects on physiological and growth characteristics of plants (13). But, high concentrations of SO₂ causes toxicity and growth reduction due to sulphite and sulphate accumulation within the plant. The main factors that determinethe phytotoxicity of SO₂ are: environmental conditions, duration of exposure, atmospheric SO₂ concentration, sulphur status of the soil and the genetic constitution of the plant (13, 14). SO₂ can easily penetrate into chloroplasts and affect plant growth and development. Even when stomata are closed, SO₂ can react with water to produce bisulfite and enter the leaf through the cuticle. In the chloroplasts, SO₂ is mainly converted into sulfite, which causes a reduction of net CO₂ assimilation, inhibits photosynthetic enzymes and decreases the photosynthetic electron transport rate (9). SO₂-toxicity is mainly attributed to produce high reactive intermediates such as the sulphur trioxide radical (HSO₃^-), the superoxide radical and the hydroxyl radical, which are generated during the radical-initiated oxidation of SO₂. Chloroplasts can initiate oxidation of SO₂ and hence, may be a primary site of radical production during SO₂ treatment. To counteract the toxicity of reactive oxygen species (ROS), a high efficient antioxidative defence system, composed of both non-enzymatic (e.g. α-tocopherol, β-carotene, glutathione and ascorbate) and enzymatic constituents (e.g. superoxide dismutase SOD, catalase, peroxidase and enzymes of the ascorbate-glutathione cycle) is present in all plant cells (15). In the present study, effects of Rhizobium inoculation on antioxidant activity and capacity of Trifolium resupinatum under different concentrations of SO₂ (0, 0.5, 1, 1.5 and 2 ppm) were evaluated.

Material and methods

Bacterial strains and inoculant preparation

In this research two Rhizobium strains were used. A local strain isolated from Persian clover nodules of Arak region in central of Iran, a city of Iran that high levels of SO₂ has been reported (12), as native strain. Physiological and biochemical characters of the local isolated bacteria were examined according to methods described in Bergey's manual of systematic bacteriology (16). For this purpose, clover roots were sterilized with 70% ethanol and were washed with sterile distilled water (17). Then the pink nodules (containing active bacteria) isolated from roots, crushed in distilled water and cultured in solid medium of YMA (18). These cultures were transferred to incubator at 25^oC. After incubation, gram reaction and morphology of bacteria were studied under the microscope. Formation of convex prominent semi-transparent slimy and mucilage colonies and gram-negative reaction were considered a sign of successful isolation of Rhizobium (17).

Standard strain of Rhizobium (Rhizobium meliloti PTCC 1684) were obtained from the Persian type culture collection (PTCC, Iran). To activate these bacteria, 1 ml of liquid medium of YMA under sterile conditions added to bacteria. For proliferation of bacteria, one inoculation loop of these bacteria dissolved in 100 ml of liquid YMA and incubated on an orbital shaker at 200 rpm for 24 h.

Optimum amount of Rhizobium to stimulate clover growth was reported 10⁵cells/mL (19). For this purpose two strains of Rhizobium (native and standard) were cultured separately in liquid medium of YMA (18) and incubated on an orbital shaker at 200 rpm for 24 h at 25^oC (20). Then these cultures were centrifuged at 1000g for 10 min and were resuspended with phosphate buffer. If, the optical density (OD₆₂₀) of this solution was 0.1 it means 10⁸cells/mL (21). To prepare optimum amount of inoculants (10⁵cells/mL), this solution was diluted by phosphate buffer.

Seed preparation and its inoculation

The seeds of Persian clover (Trifolium resupinatum cv. Alashtar Lorestan) were prepared from Arak Agriculture Research Center. They were surface-sterilized by 70% ethanol for 2 min and 1% sodium hypochlorite for 5 min, after that washed with distilled water 5 times (22). There after, seeds were divided into three groups. First group of seeds inoculated with native inoculants (10⁵cells/mL), second group of seeds inoculated with standard inoculants (10⁵cells/mL) and third group soaked in sterile phosphate buffer. All of the groups were placed under vacuum and ambient temperature for 2 h (23).

Hydroponic cultivation of seed

Inoculated and non-inoculated seeds were placed in plates system containing nutrient solution (without nitrogen) in the darkness for 24 h. The germinated seeds transferred to sterile microtubes in plastic container containing 2 L of Half-Hoagland solution (without nitrogen) (24). Containers were oxygenated by the air compressor. Each container was considered as a treatment. These containers were maintained under 12 h photoperiod, at 25^oC during day and 20^oC during night. The nutrient solution changed every five days (23).

SO₂ injection to plant

Sulfur dioxide 0.1 % gas prepared from Shazand Petrochemical Co. injected in different concentrations 0 (as control), 0.5, 1, 1.5 and 2 ppm into 31 days plants. Gas injection was performed by syringe for 5 days and 2 h daily to closed plastic containers (25).

Measurement of root fresh and dry weight

Root fresh weight of 41-days plants was measured, then these roots were placed in oven at 75 2 ⁰C for 24h and dry weight was measured.

Enzyme assays

A) Extraction

Leaf fresh materials (0.1g) was powdered by liquid nitrogen and homogenized in 1 ml of 50 mM phosphate buffer (pH=7) containing 1 mM ethylene diamine tetra acetic acid (EDTA) by a homogenizer into microtubes. Insoluble materials removed by Beckman refrigerated centrifuge at 13000 g for 20 min at 4°C, and the supernatant used as the source of enzyme extraction.

B) Assays

All activities of the enzymes determined with a spectrophotometer (PG T80 UV/VIS, Oasis Scientific Inc.).

Superoxide dismutase (SOD) assay

SOD activity was measured by monitoring the inhibition of photochemical reduction of nitro blue tetrazolium according to the method described by Giannopolitis and Ries (1977), using a reaction mixture (3 mL) containing 50mM phosphate buffer (pH=7.8), 13mM methionine, 75 µM nitro blue tetrazolium, 20 µM riboflavin, 0.1 mM EDTA and 100 µl of the enzyme extract in absence of light. The reaction mixtures were illuminated for 15 min under fluorescent light. One unit of superoxide dismutase activity is defined as the amount of enzyme required to cause 50% inhibition of nitro blue tetrazolium reduction, which was monitored at 560 nm (26).

Catalase (CAT) assay

Catalase activity was assayed by measuring the initial amount of hydrogen peroxide disappearance using the method of Cakmak and Marschner (1992) in a reaction mixture containing 2 ml of 25 mM phosphate buffer (pH=7.0, containing 10 mM H₂0₂) and 100 µl of the enzyme extract. Decomposition of 1 µmol H₂O₂/min is equal to one unit of catalase activity (27).

Guaiacol peroxidase (GPX) assay

Guaiacol peroxidase activity was measured by Polle et al. (1994). The reaction mixture contained 100 mM phosphate buffer (pH=7.0), 20 mM guaiacol, 10 mM H₂0₂ and 50 µl of the enzyme extract. Activity was determined by increasing absorbance at 470 nm due to guaiacol oxidation (28).

c) Measurement of DPPH-radical scavenging activity

Determination based on DPPH (1, 1-diphenyl-2-picryl hydrazyl) radical scavenging activity Abe et al. (1998) method (29). Leaf fresh materials (100 mg) was powdered by liquid nitrogen, homogenized in 1 ml of 90% ethanol and then maintained at 4°C for 24 h. Insoluble materials removed by centrifuge at 3500 g for 5 min. 20 µl of extracting solution was mixed with 800 µl of DPPH (0.5 mM in ethanol). The absorbance of the resulting solution was measured at 517 nm after 30 min in darkness. The antiradical capacity (three replicates per treatment) was expressed as IC₅₀ (mg ml^-1), the antiradical dose required to cause a 50% inhibition. A lower IC₅₀ value corresponds to a higher antioxidant capacity of plant extract (30). The ability to scavenge the DPPH^.radical was calculated by:

I% =  100

Where A0 is the absorbance of the control at 30 min, and A1 is the absorbance of the sample at 30 min.

Statistical analysis

All data were analyzed by variance analysis using SPSS 16. Experiments were tested using completely randomized design in factorial for three replicates. Mean comparisons were conducted using Duncan’s test.

Results

Effect of bacterial inoculation and SO₂ pollution on root

Study of root morphology of inoculated clover plants indicated pink nodules on the roots of these plants. Root fresh and dry weight of inoculated plants with native Rhizobium increased significantly as compared with inoculated plants with standard Rhizobium and non-inoculated palnts. In inoculated plants with native Rhizobium, root fresh and dry weight increased until 85.2% and 87.57% respectively in compared with non-inoculated palnts. Also, in inoculated plants with standard Rhizobium, root fresh and dry weight increased until 44.44% and 35.71% respectively in compared with non-inoculated palnts (Table 1).

Root fresh and dry weight of Persian clover showed a significant increase in 0.5 ppm concentration of SO₂ but decreased significantly in high concentrations of SO₂ (1, 1.5 and 2 ppm) as compared with control plants (Table 2).

Interaction between bacterial inoculation and SO₂ on root fresh and dry weight was significant statistically. The highest levels of root fresh and dry weight were obtained at inoculated plants with native Rhizobium under 0.5 ppm of SO₂. The lowest levels of root fresh and dry weight were obtained at non-inoculated plants under 2 ppm SO₂. Reduction of 71.88% and 85% in fresh and dry weight of non-inoculated roots under 2 ppm SO₂ was changed to 25% and 45% respectively in inoculation with native Rhizobium and to 56% and 56% respectively in inoculation with standard Rhizobium. Namely, inoculation improves stress effects of high consentrations of SO₂ (Fig.1).

Table 1- Effects of bacterial inoculation (no-inoculation (-R), inoculation with native and standard Rhziobium) on root fresh and dry weight in 41-days plants. Similar words indicate not significantly difference according to Duncan's test. The data are the means of three replicates±SE and comparisons were performed separately for each index.

Table 2- Effects of SO₂ pollution (0, 0.5, 1, 1.5 and 2 ppm) on root fresh and dry weight in 41-days plants. Similar words indicate not significantly difference according to Duncan's test. The data are the means of three replicates and comparisons were performed separately for each index.

Fig.1- Interaction between of bacterial inoculation [no-inoculation (-R), inoculation with native (R_t) and standard Rhizobium (R_s)] and SO₂ on root weight, fresh (a) and dry (b), in 41 days Trifolium resupinatum. The data are the means of three replicates. Means followed by different letters are significantly difference (P < 0.01) as determined by Duncan’s test.

Effects of bacterial inoculation and SO₂ pollution on total antioxidants:

In this study, the total antioxidant activities (I%) and capacities (IC₅₀) of the samples were determined by DPPH-radical scavenging activity test. The results showed that bacterial inoculation had no significant effect on total antioxidant activity and capacity.

The DPPH radical scavenging activity and capacity didn’t change in 0 and 0.5 ppm concentrations of SO₂ gas. Increasing SO₂ stress changed I% and IC₅₀ significantly. I% increased and IC₅₀ decreased in high concentration of SO₂ (1, 1.5 and 2 ppm) as compared with control plant. I% was at its highest value in 2 ppm of SO₂ with 80.58% increase in comparison with the controls (Fig. 2a) whereas IC₅₀ was at its lowest value in 2 ppm with 44.13% decrease in comparison with the controls (Fig 2b).

Fig.2- Effect of different concentrations of SO₂ on I% (a) and IC₅₀ (b) of 41 days Trifolium resupinatum. The data are the means of three replicates. Means followed by different letters are significantly different (P < 0.01) as determined by Duncan’s test.

To understand the protective action of antioxidants against SO₂ stress, Persian clover plants were treated with native and standard Rhizobium followed by measurement of the level of I% and IC₅₀. The effect of interaction between bacterial inoculation and SO₂ pollution on I% was significant as compared with control plants (no bacteri, no SO₂). Increasing SO₂ stress increased I% and decreased IC₅₀ of Persian clover leaves significantly in comparison with control plants (Fig. 3). Inoculation of Persian clover plant with native and standard Rhizobium reduced the stress effects of high concentrations of SO₂ on I% and IC₅₀ significantly. Among bacterial inoculation and SO₂ pollution treatments, non-inoculated plants under 2 ppm SO₂ and inoculated plants with native Rhizobium under 0 ppm SO₂ showed higher and lower levels of I% respectively. I% was lower in inoculated treatments compared with control treatments (Fig. 3a). Interaction between bacterial inoculation and SO₂ pollution indicated significant effects (p≤0.01) on IC₅₀. Increasing doses of SO₂ decreased IC₅₀ significantly. The IC₅₀ in the leaves of non-inoculated Persian clover plant under 2 ppm of SO₂ was 50.59% lower than control treatments. The level of decreasing in IC₅₀ was slightly higher in inoculated plants. The effect of interaction between bacterial inoculation and SO₂ pollution indicated a decline in IC₅₀ by 41.58% and 42.10% on inoculated plants with native and standard Rhizobium under 2 ppm SO₂ respectively. IC₅₀ levels were lower in inoculated plants than non-inoculated plants (Fig. 3b).

Fig.3- Interaction between bacterial inoculation [no-inoculation (-R), inoculation with native (R_t) and standard Rhizobium (R_s)] and SO₂ on values of I% (a) and IC₅₀ (b) in 41 days Trifolium resupinatum. The data are the means of three replicates. Means followed by different letters are significantly different (P < 0.01) as determined by Duncan’s test.

Effects of bacterial inoculation and SO₂ pollution on antioxidant activity (SOD, CAT, GPX)

The results showed that inoculation didn’t create any significant effect on antioxidant activity (SOD, CAT, GPX) and there was no significant difference among inoculated and non-inoculated plants.

There was significant difference among treatments under different concentrations of SO₂ pollution in SOD, CAT and GPX activity. The change in SO₂ concentration caused an increase in antioxidant activity (Fig 4). SOD activity increased significantly with 0.5, 1, 1.5 and 2 ppm concentrations of SO₂. For instance, 2 ppm treatment showed a maximum effect (99.44% increase) on SOD activity, whereas 1.5, 1 and 0.5 ppm concentrations increased by 65.17%, 30% and 10.82% as compared with control plants (under 0 ppm) (Fig. 4a). The effects of SO₂ pollution on Catalase activity was significant. CAT activity was affected by SO₂ treatments in comparison with the controls and was highest in the 2 ppm dose whereas it didn’t change in other doses. The CAT activity in the leaves of Persian clover plant under 2 ppm 78.26% was higher than control treatments (0 ppm) (Fig. 4b). The antioxidant activity of Guaiacol peroxidase was not different under the influence of 0 and 0.5 ppm of SO₂ pollution, but increased in higher doses of SO₂ pollution. Among SO₂ pollution treatments, 2 ppm concentration showed highest level of GPX activity.The GPX activity in the leaves of Persian clover at 1, 1.5 and 2 ppm of SO₂ pollution was 20.58%, 50.33% and 68.23 % higher than 0 ppm of SO₂ pollution treatments, respectively (Fig 4c).

Fig. 4- Effect of different concentrations of SO₂ on activity of SOD (a), CAT (b) and GPX (c) of 41 days Trifolium resupinatum. The data are the means of three replicates. Means followed by different letters are significantly different (P < 0.01) as determined by Duncan’s test.

Interaction of bacterial inoculation and SO₂ gas on antioxidant activity of SOD, CAT and GPX was significant statistically. The highest levels of SOD, CAT and GPX activity were obtained at non-inoculated plants under 2 ppm SO₂. In inoculated plants under high concentrations of SO₂ (1, 1.5 and 2 ppm) antioxidant activity was lower than non-inoculated plants. For instance, non-inoculated plants under 2 ppm of SO₂ gas treatment showed 84.98% increase in the GPX activity but inoculated plants with native and standard Rhizobium under 2 ppm of SO₂ gas treatment showed 64.27% and 63.13% increase, respectively. The level of increase in antioxidant activity under SO₂ pollution stress in inoculated plants was lower than in the non-inoculated plants (Fig. 5).

Fig. 5- Interaction between bacterial inoculation [no-inoculation (-R), inoculation with native (R_t) and standard Rhizobium (R_s)] and SO₂ on SOD (a), GPX (b) and CAT (c) activity of 41 days Trifolium resupinatum. The data are the means of three replicates. Means followed by different letters are significantly different (P < 0.01) as determined by Duncan’s test.