Phenotypic and molecular characterization of rhizobia nodulating Cicer arietinum L. and Pisum sativum L. isolated from different areas of Pakistan

Abstract

Legumes are important crop of Asia that fix nitrogen in association with genus Rhizobium. Chickpea (Cicer arietinum L.) and green pea (Pisum sativum L.) are the two most utilized food legumes in Pakistan. Nitrogen fixing bacteria can be used as alternative to chemical fertilizer without having deleterious effects on soil and microbes in addition to low cost. For this purpose isolation and characterization of various rhizobia is utmost important for symbiotic effectiveness. 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. Bulk samples of soil were collected from 8–22 cm depth and were analyzed for different physico-chemical characteristics and micronutrients. 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, Pakistan) 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 chickpea isolates showed fast growth, 25% slow growth and 50% very-slow growth. while in case of green pea rhizobia 33.3% grew fast, 50% slow and 16.66% grew very slow.. Various isolates obtained showed marked phenotypic variations. 25% of chickpea were tolerant to slight acid pH and 25% to basic pH. Overall eight strains (8cp, 9cp, 12cp, 13cp, 16cp, 24cp, 26cp, 27cp) were tolerant against extreme pH. Approximately 1% isolates sustained acidic pH. 100 % grew in slight acidic to alkaline pHs (5.5 to 8). Five isolates (7gp, 8gp, 9gp, 23gp, 24gp) tolerated extreme pHs. 85% chickpea isolates showed positive response from 0% to 2% NaCl. Eleven strains (2cp, 5cp, 6cp, 7cp, 8cp, 9cp, 11cp, 12cp, 25cp, 26cp, 27cp) performed best for NaCl stress. 100% green pea isolate were able to grow profusely at 0.5% to 0.8% NaCl and 50% up to 1.5% NaCl concentration. 3gp, 7gp, 8gp, 12gp, 27gp compete with check strain for high salt concentrations. All the chickpea isolates were able to grow at 25°C . 25% of the isolates tolerated temperature of 45°C. Two strains (11cp and 25cp) tolerated from 5°C to 50°C. All the green pea 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 isolates for physiological traits at the distance of 8.9 and two major clusters for green pea isolates at distance of 6.5. PCA analysis of chickpea isolates revealed basic pH (6.5 to 9) and NaCl treatment were the major source of variation while all other coefficients were significant except 0% NaCl and pH 9.5. for green pea rhizobia. Isolated rhizobia showed different levels of sensitivity for tested antibiotics. Most of chickpea isolate were inhibited (MIC) at higher antibiotic concentrations. Maximum isolates (40%) were resistant against erythrosine, while were resistant for penicillin G. Majority of green pea isolates were inhibited at moderate antibiotic concentrations. Maximum isolates (35%) were resistant against erythrosine and chloramphenicol followed by penicillin G (30%). Studied rhizobial isolates were diverse for heavy metal tolerance. Among chickpea nodulating rhizobia 95% were found resistant against Ag, Pb, Mn, Cu and Zn where 85% were resistant to Co, 30% for Ni and 10% for Hg. 13 cp and 24cp were found resistant for all heavy metals while 7cp was sensitive for all heavy metals tested. In case green pea isolates, 100% isolates were resistant against Ag, Pb, Mn, Cu and Zn, where 75% were resistant to Co, 50% for Ni and 18% for Hg. The cluster analysis for antibiotics and heavy metals explored four main clusters among chickpea rhizobia at the distance of 0.5 while two major clusters are evident at the distance of 1.4 among green pea rhizobia. According to principle components analysis for antibiotic and heavy metals, kanamycin, Ag, Pb, Mn, Cu, Co, Zn were most important coefficients found for chickpea rhizobia while chloramphenicol and amphicillin were the major source of variation for green pea rhizobia. Variable response was observed for carbon source utilization in rhizobia of chickpea as well as green pea. 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 while L-arginine and malic acid were the most utilized carbon sources (100%) were as D-glucose was the least utilized carbon source (17%) by green pea isolates. Cluster analysis of carbon source utilization divide 20 chickpea isolates in to for major groups at the distance of 1.3 while three major clusters were observed in green pea isolates at the distance of 3.8. PCA analysis of chickpea isolates for carbon source revealed that capric acid, tri- sodium citrate were the most important coefficients. In case of green pea most of the variations were contributed by L-tryptophane, D-glucose, D-mannose, D-mannitol, N-acetyl- glucosamine, D-maltose, potassium gluconate and adipic acid.