STUDIES ON ARSENIC RESISTANCE PROPERTIES OF SYMBIOTICALLY ACTIVE RHIZOBIAL STRAINS

Abstract

Rising concentration ofthe toxic metalloid arsenic (As) in underground water due to anthropogenic activities has been a problem since a long time. Irrigation with arsenic contaminated groundwater not only reduces the crop yield but also contaminates the edible parts ofthe crops. Arsenic enters the food chain through drinking ofcontaminated water and consumption of contaminated foods. Decrease in legume yield has been reported due to arsenic contamination in the agricultural fields. Rhizobia, a collective term for the genera Rhizobium, Sinorhizobium, Mesorhizobium, Azorhizobium and Bradyrhizobium, play an important role in maintaining the nitrogen status ofthe soil by undergoing symbiosis with legumes. Various types ofstress conditions including arsenic stress adversely affect the Rhizobium-legime symbiosis. Thus evaluation of rhizobial strains of an agricultural field and their symbiotic efficiencies is pre-requisite. In the present study three rhizobial strains were assessed for arsenic resistance properties and symbiotic efficiencies under arsenic stress. A symbiotic study has also been done to propose a model for alleviation of arsenic toxicity in alfalfa by inoculating the plants with an aquaglyceroporin (AqpS) disrupted strain, namely, Sinorhizobium meliloti smk956. Two out of three rhizobial strains were isolated from the root nodules of alfalfa and cowpea plants grown in local agricultural fields and designated as Sinorhizobium sp. DP10 and Rhizobium sp. DP99 after 16S rDNA study. Phylogenetic analysis showed that the alfalfa isolate formed a monophyletic clade with Sinorhizobium meliloti Lma-x and shared maximum sequence similarity with it (97.3 %) and the cowpea isolate formed a monophyletic clade with Rhizobium leguminosarum bv. viciae strain BKVLV17 and showed maximum similarity with it (95.7%). Third rhizobial strain used in this study was Rhizobium leguminosarum bv. trifoliiMTCC905. All the rhizobial strains were grown individually in MSY liquid medium containing various concentrations of either arsenite or arsenate. The rhizobial strains showed more resistance to As (V) than As (III). The LD50 values of the strains S. meliloti Rml021, S. meliloti smk956, Sinorhizobium sp. DP10, Rhizobium sp. DP99 and R. leguminosarum bv. trifolii MTCC905 for arsenite are 0.524, 0.685, 1.22, 1.505 and 1.174 mM, and for arsenate are 6.872, 4.392, 4.323, 6.659 and 6.467 mM, respectively. From the LD50values it is evident that the S. meliloti Rml021 and 5. meliloti smk956 were less resistant to both arsenite and arsenate than the native strains Sinorhizobium sp. DP10, Rhizobium sp. DP99 and R. leguminosarum bv. trifolii MTCC905. The AaqpS strain S. meliloti smk956 showed more resistance to As (III) and less resistance to As (V) than the S. meliloti strain Rml021. Thearsenic uptake study by the rhizobial strains included bioadsorption of arsenic on the cell surface, bioaccumulation of arsenic in the cell and total arsenic uptake (bioaccumulation + bioadsorption). All the aspects of arsenic uptake were significantly higher by the AaqpS strain S. meliloti smk956. The bioaccumulation capacity of S. meliloti smk956 was 26.3, 39.6, 88.08 and 82.27% more than the strains S. meliloti Rml021, Sinorhizobium sp. DP10, Rhizobium sp. DP99 and R. leguminosarum bv. trifolii MTCC905, respectively. The total arsenic uptake of the strain S. meliloti smk956 was 41.85, 68.16, 76.28 and 73.5 %than the strains S. meliloti Rml021, Sinorhizobium sp. DP10, Rhizobium sp. DP99 and R. leguminosarum bv. trifolii MTCC905, respectively. 11 Rhizobium sp. DP99 showed least bio-adsorption and bio-accumulation capacity followed byR. leguminosarum bv. trifolii MTCC905. The arsC genes of all the strains were amplified using degenerate primers (Sa Pereira et al., 2007) to detect the presence of ars operon. A strong band at 400 bp was detected after amplification. The nucleotide sequences of the amplified DNA products (sequenced through Ocimum Biosolution India Pvt. Ltd., Hyderabad) were subjected to analysis through blastx programme. The analysis revealed similarities of all the PCR amplified sequences with ArsC proteins ofvarious bacteria belonging to class a and p proteobacteria. The phylogenetic analysis showed clustering ofthe ArsC sequences with that of other rhizobial strains. Plant studies were carried out to study the effect of arsenic (supplied as sodium arsenate) on symbioses ofnative rhizobial strains with their respective host plants. The arsenic concentrations in roots were exceedingly more than the shoots in all sets ofplants. The arsenic concentrations in roots and shoots increased with increase in the concentration of arsenic in the nutrient medium. At 5 mg/L arsenic level, the shoot arsenic concentrations were 72.12, 8.98 and 9.88 ug/gm tissue and root arsenic concentrations were 1130.71, 425.84 and 1401.6 ug/gm tissue in cowpea, alfalfa and clover plants, respectively. The average shoot length and dry weight of the cowpea plants decreased significantly at 1mg/L level ofarsenic than those of the plants growing with 0 mg/L arsenic but in case of alfalfa and clover the shoot length and dry weight of the plants decreased significantly only at 5 mg/L arsenic level. Significant reductions in average in nodule number by a value of 46.93%, 44.4% and 31.3 %, were also observed in cowpea, alfalfa and clover plants, respectively. The mean total chlorophyll content of the cowpea, alfalfa and clover plants decreased significantly by 22.9%, 33.4% and 36.19% at 5 mg/L level. In comparison to the plants growing with 0 mg/L arsenic the mean shoot nitrogen contents of the cowpea and clover plants did not differ significantly at 1 mg/L arsenic concentration but significantly decreased by 20.81% (F= 18.05, P=0.013) and 63.7% at 5 mg/L arsenic concentration. In case of alfalfa, the mean shoot nitrogen contents of the plants growing with 1 and 5 mg/L arsenic were severely affected and reduced significantly by 16.52% (F= 50.03, P=0.002) and 46.42% (F=921.34, P<0.001) than those of the plants growing in control condition, respectively. The mean nitrogenase activities of cowpea, clover and alfalfa plants growing with 5 mg/L arsenic significantly decreased by 47.7% (F=l 11.74, PO.001), 52.17% (F=739.36, PO.001) and 10.52% (F=9.70, P= 0.035) in comparison to those of plants growing with 0 mg/L arsenic, respectively. The root hair curling was found to be defective even at 1 mg/L of arsenic stress condition. Zones of bulged root hairs were more in case of seedlings growing under arsenic stress in comparison to proper root hair bandings in the seedlings of control condition. In cowpea, clover and alfalfa plants, the antioxidant enzymes like superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and glutathione reductase (GR) activity increased significantly at 1 mg/L level. At 5 mg/L arsenic level the POD, CAT and GR activities decreased significantly. The SOD activity was less affected in all the plant types.

The symbiotic ability of the S. meliloti strain smk956 was studied under different arsenic stress conditions andcompared with that of wild type strain S. meliloti Rml021. In both sets of experiments, the arsenic concentration in shoots was much less than that of roots. At highest arsenic concentration the roots and shoots of the plants inoculated with S. meliloti smk956 accumulated 5.3 and 27.5% less arsenic per gram tissue than the plants inoculated with S. meliloti Rml021,respectively. The mean shoot length of the plants inoculated with S. meliloti strain smk956 increased by 25.1% than that of the plants inoculated with S. meliloti Rm1021 at 5 mg/L arsenic stress condition. In both sets of experiments, the mean dry weights of the plants decreased significantly with increase in arsenic concentration. In plants inoculated with S. meliloti Rml021, the mean dry weight of the plants decreased significantly by 43.23% (F=12.22, P=0.002) with addition of5mg/L arsenic but in the plants inoculated with S. meliloti smk956, it decreased by 26.74% (F=38.55, P<0.001) in comparison to their respective untreated plants. At 5mg/L arsenic concentration level the mean dry weight of the plants inoculated with S. meliloti smk956 was 19.1% (F=5.26, P=0.033) more than thatof plants inoculated with S. meliloti Rml021. With addition of 5 mg/L arsenic in the nutrient medium, the total chlorophyll content of the plants inoculated with S. meliloti Rml021 decreased steeply by 47.2 % (F= 17324.45, P<0.001) whereas in plants inoculated with S. meliloti smk956 it decreased only by 10.5% (F= 316.23, PO.0.001) in comparison to their respective untreated plants. The mean shoot nitrogen content of the plants growing with 5 mg/L arsenic was 41.29% (F=33.78, P=0.004) less in comparison to the untreated plants when inoculated with S. meliloti Rml021 but the decrease was only 5.4% when inoculated with S. meliloti smk956. The root hair curling was less affected under arsenic stress in case of alfalfa plants inoculated with S. meliloti smk956 than S. meliloti Rml021. The activities of the antioxidant enzymes increased at 1 mg/L and decreased at 5 mg/L arsenic stress conditions in both sets of experiments. At 5 mg/L arsenic the superoxide dismutase activity of the plants inoculated with S. meliloti smk956 decreased 53.5% (F=750.95, PO.001) but the catalase, peroxidase and glutathione reductase activities increased by 218.5% (F=750.95, PO.001) 218.4% (F=65.79, PO.001) and 24.3% (F=25.81, PO.007) than those of the plants inoculated with S. meliloti Rml021, respectively. From the above studies it can be concluded that the strains Rhizobium sp. DP99 and R. leguminosarum bv. trifolii MTCC905 are more tolerant to arsenic than Sinorhizobium sp. DP 10. The symbiosis between the rhizobial strains and their respective host plants was affected at 5 mg/L of arsenic stress condition which might be due to effect of arsenic on root hair curling. Moreover, the AaqpS strain S. meliloti smk956 with high bioaccumulation capacity has a better symbiotic ability than the wild type strain S. meliloti Rml021, thus strains devoid of arsenite efflux proteins could be a better option to alleviate arsenic toxicity in leguminous cropsgrown in arsenic contaminated fields.