ISOLATION AND CHARACTERIZATION OF STRESS TOLERANT STRAINS OF RHIZOBIUM

Abstract

The present work was undertaken to isolate and characterize salt, low pH and cold tolerant strains of Rhizobium. Five hundred and thirty strains of Rhizobium trifolii were isolated from root nodules of host plant berseem (Trifolium alexandrinum), collected from different agroclimatic regions of Uttar Pradesh and Haryana. After purification, these strains were subjected to stresses of salinity, acidity, alkalinity and low temperature. Preliminary screening was done in petridishes on the basis of production of single colonies on mannitol salt yeast extract (MSY) agar medium under different stresses. Out of 530 only 30 strains were found to form single colonies on MSY agar supplemented with 3.0% sodium chloride (NaCl). For further screenig these 30 strains were grown in MSY broths containing 4.0% NaCl and after 72 hrs culture was spread on MSY agar with same concentration of NaCl. Though all 30 strains showed growth in liquid medium in presence of 4.0% NaCl, only 11 strains produced single colonies on solid medium containing the same percentage of NaCl. Tolerance to chlorides and sulphates of Mg++ and K for these 11 strains was also determined. Lesser toxicity of Mg+4 as compared to K and a trend of more tolerance towards sulphates than chlorides was observed. Most of the strains (88%) out of 530 strains tested were found to form single colonies on MSY agar (pH 10) but on 4.5 pi I medium only 15 strains produced single colonies. Further, after 72 hrs of growth in broth of pH 4.0 only five strains out of these 15 strains formed single colonies on pH 4.0 medium No strain showed growth in pH 3.5 medium CO Screening for tolerance to low temperature was done in petri dishes by streaking 530 strains on MSY agar medium and incubating at I8°C. After 6 days 310 strains were found to form single colonies. Growth of these strains was again observed at 8°C and five strains were found to show growth at this temperature. Antibiotic resistance patterns for chloramphenicol, rifampicin, streptomycin, kanamycin and tetracycline were obtained for the 35 strains which were found tolerant to different stresses in preliminary screening. Multiple resistances were observed in most of the strains which may be important for the competitive ability of the strains in the rhizosphere All the strains which were found tolerant to different stresses were tested for their authentication and symbiotic properties by inoculating them on seedlings of berseem {Trifolium alexandrinum cv.S-29), grown aseptically on nitrogen free agar medium slants. Appearance of first nodule was found between 13 to 26 days. Marked variation in shoot length (9.3 to 22.1 cm) and shoot dry weight (9.3 to 21.6 mg) indicated genetic variation in symbiotic properties of these stress tolerant strains. Twenty genotypes of host (T. alexandrinum) were also screened for tolerance to different stresses on the basis of germination percentage, growth and dry weight of the seedlings. Under different stresses maximum germination was observed in genotype JHB-146 (38.58%), JHB-93-3 (48.33%) and HFB-480 (85.13%) in 0.5% NaCl, pH 4.0 and at 8°C, respectively. The genotypes of host cultivars were found more sensitive than Rhizobium to stress conditions Less deleterious effect of acidic pi I and low temperature was observed than salt stress. pH changes during the growth were observed after inoculation in normal and stress media. All the 15 strains tolerant to different stresses decreased the pH of the medium from 7.0 and the range of decrease was (ii) from 6.75 to 5.48. Slight decrease in pH (6.68 to 6.09) was observed when salt tolerant strains were grown in presence of 4.0% NaCl. In contrast, acidic pH tolerant strains raised the pH of the acidic medium from 4.0 and the increased pH varied from 5.12 to 6.41. Decrease in pH in normal medium may be due to yield of acidic end products and increase in pH of the acidic medium may be due to alkalizing end products production by the tolerant rhizobia. Differences in colony forming units (CFU) of salt and acid tolerant strains under normal and stress conditions were monitored. Significant differences between CFU under normal and stress were found in nine salt tolerant and four acid tolerant strains. Least difference of CFU between stress and normal condition was recorded in strains UK 113 i.e. 7.7xl08 and 9.2x10 cells/ml respectively . In strain UK181, the cell counts in pH 4.0 and pH 7.0 media were 7.46xl08 and 16. lxlO8 cells/ml respectively Though both O.D. and CFU values were affected considerably under different stress conditions correlation between O.D. and CFU values could not be drawn due to strain specific production of exopolysaccharides. The tolerant Rhizobium trifolii strains were tested for their ability to produce cell surface molecules. Production of cell surface molecules was determined by growing the strains on normal and stress medium containing aniline blue for (3(1->3) glucans, congo red for cellulose fibrils and sodium deoxycholate for lipopolysaccharides. Motility of the strains was observed in normal and stress media by swarm plates. All the different stress tolerant strains were found to produce LPS and cellulose fibrils on normal medium Production of cellulose fibrils was not found in presence of 4.0% NaCl, in pH 4.0 and at 8°C whereas production of LPS was not found in presence of 4.0% NaCl while all the acidic pH and low temperature tolerant strains were producing LPS except UK200 at 8°C. Except two strains at 8°C all (Si) the stress tolerant strains were producing (3(1->3) glucans under their respective stresses while in normal condition production of this molecule was unclear or absent in several strains. Out of 15 tolerant strains, only two strains were immotile on normal swarm plates while six strains in presence of 4.0% NaCl, three strains in pH 4.0 medium and 4 strains at 8°C were found motile. Total proteins from six salt tolerant and three acid tolerant strains after growing them in normal and respective stress conditions were analyzed by SDS-PAGE. Some differences have been observed in protein patterns under stress and normal conditions indicating the synthesis of some stress induced proteins.