Skip to main content
SpringerLink
Log in

Physiological and molecular characterization of locally adapted Rhizobium strains of lentil (Lens culinaris Medik.) having restricted phage sensitivity

  • Original Article
  • Published:
Annals of Microbiology Aims and scope Submit manuscript

Abstract

Indigenous rhizobia were isolated from root nodules of lentil plants collected from various agro-climatic regions of India. These isolates together with four standard lentil Rhizobium strains were screened for sensitivity against eight phages. Four strains, USDA 2431, BHULR 104, BHULR 113 and BHULR 115 having restricted sensitivity to lytic phages LRP-1, LRP-4, LRP-13 and LRP-15 respectively, were characterized for both physiological and molecular characters. All strains had a generation time of between 3.8 and 5.6 h in yeast extract–mannitol (YM) broth, and colonies on YM agar plates showed an acidic reaction. Compared to other strains, strain USDA 2431 grew poorly when sucrose was the sole carbon source and showed maximum growth in arabinose-containing medium. The intrinsic antibiotic resistance level in all strains was tested against seven antibiotics and found to be high with ampicillin and kanamycin (50–60 μg ml-1) but very low with neomycin (0.03 μg ml-1). With the exception of strain BHULR 113, all strains expressed ex planta nitrogenase activity, with strain USDA 2431 showing the maximum activity (26.8 nmol C2H4 h-1 mg-1 protein) after 6 h of incubation. Genomic and phylogenetic relationships among the strains were examined by randomly amplified polymorphic DNA and 16S rRNA sequence analysis. Genetic distance varied from 0.09 to 0.23 among the strains, and the primer OPL-11 was found to be suitable for the discrimination of these strains. The 16S rRNA sequence analysis revealed 99–100% similarity with Rhizobium leguminosarum bv. viciae. These results clearly indicate that phage sensitivity is a useful marker for discriminating locally soil-adapted rhizobial strains forming effective nodules in lentil.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • Andersson AF, Banfield JF (2008) Virus population dynamics and acquired virus resistance in natural microbial communities. Science 320:1047–1050

    Article  PubMed  CAS  Google Scholar 

  • Appunu C, Dhar B (2006) Phage typing of indigenous soybean-rhizobia and relationship of a phage group strains for their asymbiotic and symbiotic nitrogen fixation. Indian J Exp Biol 44:1006–1011

    PubMed  CAS  Google Scholar 

  • Arrese-Igor C, Estavillo JM, Peńa JI, Gonazalez-Murua G, Aparicio-Tejo PM (1989) Effect of low nitrate supply on nitrogen fixation in alfalfa root nodules induced by Rhizobium meliloti strains with varied nitrate reductase activity. J Plant Physiol 135:207–211

    Article  CAS  Google Scholar 

  • Athar M (1998) Drought tolerance by lentil rhizobia (Rhizobium leguminosarum) from arid and semiarid areas of Pakistan. Lett Appl Microbiol 26:38–42

    Article  Google Scholar 

  • Ausubel FM, Roger B, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl (eds) (1994) Current protocols in molecular biology. John Wiley and Sons, New York

  • Bell T, Newman JA, Silverman BW, Turner SL, Lilley AK (2005) The contribution of species richness and composition to bacterial services. Nature 436:1157

    Article  PubMed  CAS  Google Scholar 

  • Beynon JL, Josey DP (1980) Demonstration of heterogeneity in a natural population of Rhizobium phaseoli using variation in intrinsic antibiotic resistance. J Gen Microbiol 118:437–442

    Google Scholar 

  • Bremer E, Kessel C, Nelson L, Rennie RJ, Rennie DA (1990) Selection of Rhizobium leguminosarum strains for lentil (Lens culinaris) under growth room and field conditions. Plant Soil 121:47–56

    Article  Google Scholar 

  • Dhar B, Ramkrishna K (1987) Morphology and general characteristics of phages of chickpea rhizobia. Arch Microbiol 147(2):121–125

    Article  Google Scholar 

  • Dhar B, Singh BD, Singh RB, Srivastava JS, Singh VP, Singh RM (1979) Occurrence and distribution of rhizobiophages in Indian soils. Acta Microbiol Pol 28(4):319–324

    PubMed  CAS  Google Scholar 

  • Dhar B, Upadhyay KK, Singh RM (1993) Isolation and characterization of bacteriophages specific for Rhizobium leguminosarum bivor phaseoli. Can J Microbiol 39:775–779

    Article  Google Scholar 

  • Dooley JJ, Harrison SP, Mytton LR, Dye M, Cresswell A, Skot L, Beeching JR (1993) Phylogenetic grouping and identification of Rhizobium isolates on the basis of random amplified polymorphic DNA profiles. Can J Microbiol 39:665–673

    Article  PubMed  CAS  Google Scholar 

  • Eaglesham ARJ (1987) The use of intrinsic antibiotic resistance for Rhizobium study. In: Elkan GH (ed) Symbiotic nitrogen fixation technology. Dekker, New York, pp 185–204

  • Giannakis C, Nicholas DJD, Wallace W (1988) Utilization of nitrate by bacteroids of Bradyrhizobium japonicum in the soybean root nodule. Planta 174:51–58

    Article  CAS  Google Scholar 

  • Glenn AR, Dilworth MJ (1981) The uptake and hydrolysis of disaccharides by fast and slow growing species of Rhizobium. Arch Microbiol 129:233–239

    Article  CAS  Google Scholar 

  • Jaiswal SK, Dhar B (2010) Morphology and general characteristics of phage specific to Lens culinaris rhizobia. Biol Fertil Soils 46:681–687

    Article  CAS  Google Scholar 

  • Jaiswal SK, Dhar B (2011) Identification and assessment of symbiotic effectiveness of phage-typed Rhizobium leguminosarum strains on lentil (Lens culinaris Medik) cultivars. Curr Microbiol 62:1503–1509

    Article  PubMed  CAS  Google Scholar 

  • Kaneshiro T, Cromwell CD, Hanrahan RF Jr (1978) Acetylene reduction activity in free-living cultures of Rhizobia. Int J Syst Bacteriol 28:27–31

    Article  CAS  Google Scholar 

  • Kurz WGW, La Rue TA (1975) Nitrogenase activity in Rhizobia in absence of plant host. Nature 256:407–408

    Article  CAS  Google Scholar 

  • Lesley SM (1982) A bacteriophage typing system for Rhizobium meliloti. Can J Microbiol 28:180–189

    Article  Google Scholar 

  • Lindstrom EK, Jarvis BDW, Lindstrom PE, Patel JJ (1983) DNA homology, phage-typing and cross-nodulation studies of rhizobia infecting Galegae species. Can J Microbiol 29:781–789

    Article  Google Scholar 

  • Lindstrom K, Lipsanen P, Kaijalainen S (1990) Stability of markers used for identification of two Rhizobium galgae inoculant strains after five years in the field. Appl Environ Microbiol 56:444–450

    PubMed  CAS  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with Folin-phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  • Maheshwari DK, Saraf M (1994) Effect of carbaryl and 2,4-D to nitrogenase and uptake hydrogenase in agar cultures and root nodules formed by Rhizobium leguminosarum. J Gen Appl Microbiol 40:569–574

    Article  CAS  Google Scholar 

  • Manhart JR, Wong PP (1979) Nitrate reductase activity of rhizobia and the correlation between nitrate reduction and nitrogen fixation. Can J Microbiol 25:1169–1174

    Article  PubMed  CAS  Google Scholar 

  • McComb JA, Elliott J, Dilworth MJ (1975) Acetylene reduction by Rhizobia in pure culture. Nature 256:409–410

    Article  CAS  Google Scholar 

  • McNeil DL, Hill GD, Materne M, McKenzie BA (2007) Global production and world trade. In: Yadav SS, McNeil D, Stevenson PC (eds): lentil: an ancient crop for modern times. Kluwer, Dordrecht, pp. 95–106

  • Mendum TA, Clark IM, Hirsch PR (2001) Characterization of two novel Rhizobium leguminosarum bacteriophages from a field release site of genetically-modified rhizobia. Antonie van Leeuwenhock 79:189–197

    Article  CAS  Google Scholar 

  • Monza J, Delgado MJ, Bedmar EJ (1992) Nitrate reductase and nitrite reductase activity in free-liing cells and bacteroids of Rhizobium loti. Plant Soil 139:203–207

    Article  CAS  Google Scholar 

  • O’Gara F, Shanmugam KT (1976) Control of symbiotic nitrogen fixation in rhizobia. Regulation of NH4 + assimilation. Biochem Biophys Acta 451:342–352

    Article  PubMed  Google Scholar 

  • Pagan JD, Child JJ, Scowcroft WR, Gibson AH (1975) Nitrogen fixation by Rhizobium cultured on a defined medium. Nature 256:406–407

    Article  CAS  Google Scholar 

  • Shah NH, Hafeez FY, Hussain A, Malik KA (1996) Influence of seasonal variation on the indigenous population of Rhizobium leguminosarum bv. viciae and competitive ability of introduced rhizobia in lentil. LENS Newsl 23:32–37

    Google Scholar 

  • Sontheimer EJ, Marrafini LA (2010) Slicer for DNA. Nature 468:45–46

    Article  PubMed  CAS  Google Scholar 

  • Stam H, van Verseveld HW, Stouthamer AH (1983) De-repression of Nitrogenase in chemostat cultures of fast growing Rhizobium leguminosarum. Arch Microbiol 135:199–204

    Article  CAS  Google Scholar 

  • Staniewski R (1970) Typing of Rhizobium by phages. Can J Microbiol 16:1003–1009

    Article  PubMed  CAS  Google Scholar 

  • Subba Rao NS (1976) Field response of legumes in India to inoculation and fertilizer application. In: Nutman PS (ed) Symbiotic nitrogen fixation in plants. International Biological Programme, vol. 7. Cambridge University Press, London, pp 255–268

  • Vincent JM (1970) A manual for the practical study of root nodule bacteria. IBP handbook No. 15. Blackwell Scientific, Oxford and Edinburgh

  • Vincent JM (1974) Root nodule symbiosis with Rhizobium. In: Quispel A (ed) The biology of nitrogen fixation. North Holland Research Monographs, Frontiers of Biology, vol. 33. North Holland, Amsterdam

  • Vos M, Birkett PJ, Birch E, Griffiths RI, Buckling A (2009) Local adaptation of bacteriophages to their bacterial hosts in soil. Science 325:833

    Article  PubMed  CAS  Google Scholar 

  • Yao ZY, Kan FL, Wang ET, Wei GH, Chen WX (2002) Characterization of rhizobia that nodulate legume species of the genus Lespedeza and description of Bradyrhizobium yuanmingense sp. nov. Int J Syst Evol Microbiol 52:2219–2230

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

The work was completed with the financial support of the Department of Biotechnology, Ministry of Science and Technology, Govt. of India. We thank Prof. A. Vaishampayan for help in discussion and suggestions in the manuscript

Author information

Authors and Affiliations

  1. Microbiology Laboratory, Department of Genetics and Plant Breeding, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, 221005, India

    Sanjay Kumar Jaiswal & Banshi Dhar

  2. Department of Biochemistry, Faculty of Sciences, Banaras Hindu University, Varanasi, 221005, India

    Ashutosh Kumar Rai

Authors
  1. Sanjay Kumar Jaiswal
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Banshi Dhar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ashutosh Kumar Rai
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sanjay Kumar Jaiswal.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jaiswal, S.K., Dhar, B. & Rai, A.K. Physiological and molecular characterization of locally adapted Rhizobium strains of lentil (Lens culinaris Medik.) having restricted phage sensitivity. Ann Microbiol 62, 1453–1459 (2012). https://doi.org/10.1007/s13213-011-0398-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13213-011-0398-z

Keywords

Search

Navigation