Present study was conducted at the Department of Biochemistry, Quaid-i-Azam University Islamabad, Pakistan to characterize 19 chickpea (Cicer arietinum L.) and 11 green pea (Pisum sativum L.) rhizobia from root nodules using soils collected from 27 different localities of Pakistan for phenotypic, molecular and symbiotic characterization.
Commercial strain of rhizobia for both chickpea and green pea was isolated from Bio-power (manufactured by National Institute of Molecular Biology and Genetics (NIBGE), Faisalabad) through root nodules. Fresh cultures were used for Gram's staining to confirm as gram-negative rods. Soil isolates were confirmed as rhizobia by inoculating 5 day old seedling grown in sterile mixture of sand and soil (1:1).
All the isolated rhizobia were gram negative. The coloration of rhizobial colonies were either milky-white translucent or shiny with circular shape. 25% of the chick pea isolates were fast growers (50 min < GT < 3 h), 25% were slow growers (3 < G = 9) and 50% were very-slow growing while incase of green pea rhizobia 33.3% were fast growers, 50% slow growers and 16.66% were very slow growers.
Chick pea rhizobia survived between pH 4.0 to 9.5. 25% of them were tolerant to slight acid pH and 95% to neutral pH and 25% to pH 9.5. Over all eight strains (8cp, 9cp, 12cp, 13cp, 16cp, 24cp, 26cp, 27cp) showed wide tolerance against extreme pH. Green pea rhizobia survived between pH 5.0 to 9.5. Approximately 1% isolate sustained pH 4 and 4.5.100 % grew in slight acidic to alkaline pHs (5.5 to pH 8. Five strains (7gp, 8gp, 9gp, 23gp, 24gp) showed wide tolerance against extreme pH. In chickpea survival rang varied from 0% to 3.5%.. 100% isolates were able to grow at 0.3% to 1.5% NaCl, while 85% showed positive response from 0% to 2% NaCl. Eleven strains (2cp, 5cp, 6cp, 7cp, 8cp, 9cp, 11cp, 12cp, 25cp, 26cp, 27cp) showed wide tolerance for different NaCl concentrations used in this experiment. Survival rang of green pea rhizobia against NaCl stress varies from 0% to 3.5%.100% isolate were able to grow at 0.5% to 0.8% NaCl, 75% isolates continued to grow at 0.1% while 50% up to 1.5% NaCl concentration. 3gp, 7gp, 8gp, 12gp, 27gp compete with check strain for high salt concentrations. Survival rang of chickpea rhizobia against temperature varies from 5°C to 50°C. All the isolates were able to grow at 25°C while 85% continued to grow at 40°C . 25% of the isolates tolerated temperature of 45°C. Two strains (11cp and 25cp) ranked top for temperature tolerance from 5°C to 50°C.Rang of survival of green pea rhizobia for temperature varies from 5°C to 45°C. All the isolates were able to grow at 25°C while 75% continue to grow at 45°C (3gp, 8gp, 9gp, 12gp, 26gp, 27gp). No strain survived at 50°C. Present work revealed four major clusters among chickpea rhizobia for physiological traits at the distance of 8.9. In case of green pea two major clusters were found at distance of 6.5. PCA analysis revealed basic pH (6.5 to 9) and NaCl treatment with Eigenvalue 12.04 were the major source of variation among chickpea rhizobia. All the coefficients were significant in first factor except 0% salt concentration and pH 9.5. for green pea rhizobia. Most of them showed inhibition at higher concentrations. Maximum isolates (40%) were resistant against erythrosine, 30% for penicillin G, 25% for chloramphenicol and amphicillin, 20% for streptomycin and only 15% isolates were resistant for kanamycin sulfate. Most of them showed inhibition at moderate concentrations. Maximum isolates (35%) were resistant against erythrosine and chloramphenicol, 30% for penicillin G, 26% for streptomycin and amphicillin, only 17% isolates were resistant for penicillin and kanamycin sulfate. Maximum green pea isolates (33%) were resistant against chloramphenicol and erythromycin, 25% for streptomycin and amphicillin. However, 17% isolates were found to be resistant to penicillin and kanamycin. Two isolates viz; 13 cp and 24cp were found resistant for all heavy metals while 7cp was found sensitive for all heavy metals tested in this experiment. According to present study 95% isolates were found resistant against Ag, Pb, Mn, Cu and Zn where 85% were resistant to Co, 30% for Ni and 10% for Hg. Again differential response was observed within green pea isolates. 24gp was resistant for all the heavy metals tested. According to present study 95% isolates were found resistant against Ag, Pb, Mn, Cu and Zn where 85% were resistant to Co, 30% for Ni and 10% for Hg . Chickpea rhizobia explores that four main clusters at the distance of 0.5. The cluster diagram of green pea rhizobia reveals two major clusters at the distance of 1.4. According to principle components for antibiotic and heavy metals of chickpea rhizobia. First factor shared 42.95% variation with Eigenvalue 6.01. Kanamyin, Ag, Pb, Mn, Cu, Co, Zn were most important coefficients. In case of antibiotics and heavy metal resistance the first factor contributed 26% of the total variation with Eigenvalue 2.34. The most important coefficients in this factor were Chloramphenicol and Amphicillin.
L- arginine and urea were the most utilized carbon sources (100%) followed by L- arabinose (95%) and potassium gluconate, D- maltose (90%) by chickpea isolates. L- arginine and D-glucose and malic acid were the most utilized carbon sources (100%) followed by L- arabinose, D-mannose, D-mannitol, D-maltose and potassium gluconate (92%). In case of green pea again D- glucose was the least utilized carbon source (17%)by green pea isolates.Cluster analysis of carbon source utilization divide 20 isolates in to for major groups at the distance of 1.3. At the distance of 3.8 green pea isolates were divided in to three major clusters.PCA analysis of chickpea isolates revealed 44.84% of total variations for first factor with Eigenvalue 8.96. In this factor capric acid, tri-sodium citrate was the most important coefficient. In case of green pea first factor shared 42.64% of total variation with Eigenvalue of 5.11.
Rang of plasmid numbers varied from 1 to 3 for chickpea rhizobia, while size of plasmid varied from 2.3kb to 33kb for chickpea isolates. At the distance of 0.3 four clusters were evident in chickpea isolates. Rang of plasmid numbers for green pea varied from 1 to 2 and size of plasmid varied from 9.4kb to 33kb. five main clusters are evident at the distance of 1.5.
The RAPD banding profile of chick pea rhizobia generated by primers B8 and B10. At the distance of 1.5, three major clusters were evident. The RAPD banding patterns of green pea rhizobia generated by primers A5, E4, and F8. At the distance of 1, six clusters were obvious.
Significant positive correlation was recorded in chickpea for nodule number and nodule DW (.965). Nodule size exhibited positive correlation for nodule DW (.358) and negative association was observed for total shoot nitrogen (%) (-.308). Root DW was positively correlated with shoot dry weight (.341). Root length showed positive association with stem length (308). Shoot DW and stem length also exhibited positive correlation (.378).
In green pea nodule number showed positive association with nodule DW (0.455), root DW (0.346), shoot DW (0.784) and negative correlation was found with nodule size (-0.401). Nodule size was positively correlated with total shoot nitrogen (0.369). Nodule DW also positively associated with shoot DW (0.416) and total shoot total nitrogen (0.374). Root DW exhibited strong positive association with shoot DW (0.620) and total shoot nitrogen (%).
21cp was the most efficient isolate with 98% dry matter yield and 14.25% total shoot protein and 16cp was the least efficient isolate with only 22% relative effectiveness. Present work revealed that 18gp and 26gp were the most efficient isolates with 99% dry matter yield and 14.25%, 7.85% total shoot protein, respectively while 8gp was the least efficient isolate with only 57% dry matter and 8.8% total shoot protein.In unstressed conditions nodulation and growth parameters of both the crops varied according to the inoculated rhizobia strains. It was observed in remaining isolates that although they successfully nodulated chick pea and green pea seedlings but salt treatment limited nodulation and growth significantly.Nodule dry weight/plant was the most adversely affected parameter (74.1%) in chickpea and shoot dry weight (88.14%) in case of green pea after. Green pea genotype (Meteor) was more sensitive to salt stress for total shoot nitrogen than chickpea genotype (NCS 0305) used in present study.Over all 29.8% reduction was recorded for total shoot nitrogen in salt treated chickpea seedlings and 19.4% in green pea.Mean nitrogenase activity decreased (71.6%) in salt treated chickpea seedlings and 55.9% in green pea. Reduction in chlorophyll a was 29.7% and 10.7%. for chlorophyll b in chickpea. While chlorophyll a decline in green pea was 30.5% and 26.4% for chlorophyll b.
Chickpea seedlings over all exhibited 47.47% reduction for total nodule soluble proteins, 71.06% in catalase, 57.22% in Ascorbate peroxidase,while Peroxidase activity increased by 26.97% , Superoxide dismutase by 21.96%, ).Green pea seedlings over all exhibited 45.11% reduction for total nodule soluble proteins 71.63% in catalase, 9.29% in Ascorbate peroxidase, Glutathione activity by 198.42%, LPO by 61.65%, while Peroxidase activity increased by 26.97% , Superoxide dismutase by 21.96%, Glutathione activity by 185.75%, LPO by 96.81%.It is clear from the correlation study that under no salt stress catalase was involved in primary defense against (ROS). A strong positive association between SOD, POD and GSH was observed under salt stress. The results suggest that these enzymes act to protect nodular tissue against salt mediated oxidative damage and probably participate with other component of the intrinsic detoxifying system of the cells.
SDS-PAGE studies revealed expression of 6 proteins in chick pea rhizobia (approximately 10, 15, 27, 35, 56 and 65 kDa) and 11 in green pea rhizobia (approximately 12, 35, 40, 55, 57, 65, 70, 95, 115, 130 and 150 kDa) under salt stress.
Four chickpea (21cp, 22cp, 26cp and 27cp) and five green pea (3gp, 12gp, 24gp, 26gp and 27gp) isolates were better in relative effectiveness (in comparison to T(N) control and commercial strain (BP)) and total shoot nitrogen, tolerant to basic pHs, high salt concentrations, elevated temperatures, resistance for majority of antibiotic and heavy metals tested and were able to utilizes wide rang of carbon sources and efficient symbionts under salt stress with strong antioxidant defense system and salt stress gene expression; are most suitable candidate inoculants. Therefore all the isolates that perform better in broth media as well as pot experiment under NaCl stress expressed stress tolerant genes and could be used as successful inoculum for corresponding crop.