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Genetic diversity of Azotobacter strains isolated from soils by amplified ribosomal DNA restriction analysis

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Abstract

Strains of Azotobacter mediate in the nitrogen fixation process by reducing of N2 to ammonia. In this study, 50 strains were isolated from different rhizospheric soil in central Iran, by using soil paste-plate method. These strains were biochemically identified and characterized on differential LG medium based on morphological and physiological properties. Results obtained showed that identified strains were belonging to three species, namely A. chroococcum, A. vinelandii and A. beijernckii. In order to molecular analysis, the 16S rRNA gene was amplified using 27f and 1495r primers and PCR products were subsequently restricted with RsaI, HpaII and HhaI. Cluster analysis based on amplified ribosomal DNA restriction analysis were revealed intraspecific polymorphism and differentiated strains into two mains clusters, clusters A and B. Cluster A strains were related to the A. vinelandii, whereas cluster B strains were related to the A. chroococcum and A. beijerinckii. The results show that amplified ribosomal DNA restriction analysis is a powerful and discriminatory tool for the identification of members of the genus Azotobacter.

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References

  1. Becking, J.H., The family Azotobacteraceae, in The Procaryotes: A Handbook on Habitats, Isolation, and Identification of Bacteria, Heidelberg: Springer, 1981, p. 2440.

    Google Scholar 

  2. Tchan, Y.T., Azotobacteraceae, in Bergey’s Manual of Systematic Bacteriology, Kreig, N.R. and Holt, J.G., Eds., Baltimore: Williams and Wilkins Press, 1984, pp. 220–229.

    Google Scholar 

  3. Tejera, N., Lluch, C., Martinez-Toledo, M.V., and Gonzalez-Lopez, M.V., Isolation and characterization of Azotobacter and Azospirillum strains from the sugarcane rhizosphere, Plant Soil, 2005, vol. 270, no. 1, pp. 223–232.

    Article  CAS  Google Scholar 

  4. Holt, J.G., Krieg, N.R., Sneath, P.H.A., et al., Bergey’s Manual of Determinative Bacteriology, Philadelphia: Williams and Wilkins Press, 1994.

    Google Scholar 

  5. De Smedth, J., Bauwens, M., Tytgat, R., and De Ley, J., Intraand intergeneric similarities of ribosomal ribonucleic acid cistrons of free-living, nitrogenfixing bacteria, Int. J. Syst. Bacteriol., 1980, vol. 30, no. 1, pp. 106–122.

    Article  Google Scholar 

  6. Jensen, V. and Petersen, E.J., Taxonomic studies on Azotobacter chroococcum Bejjerinck and Azotobacter beijerinckii Lipman, J. R. Vet. Agric. Coll., 1995, vol. 84, no. 4, pp. 107–126.

    Google Scholar 

  7. Clementi, F., Alginate production by Azotobacter vinelandii, Biotechnology, 1997, vol. 17, no. 4, pp. 327–361.

    CAS  Google Scholar 

  8. Spinosa-Victoria, D., Lopez-Reyes, L., and Cruz-Benitez, A.D.L., Use of 16S rRNA gene for characterization of phosphate-solubilizing bacteria associated with corn, Articulo Cientiffico, Rev. Fitotec. Mex., 2009, vol. 32, no. 1, pp. 31–37.

    Google Scholar 

  9. Bahri, S., Wahyudi, A.T., and Mubarik, N.R., Genetic diversity of plant growth promoting rhizobacteria of Bacillus sp. based on 16S rRNA sequence and amplified rDNA restriction analysis, Microbiology, 2009, vol. 3, no. 1, pp. 12–16.

    Google Scholar 

  10. Turner, G.L. and Gibson, A.H., Measurement of nitrogen fixation by indirect means, in Methods for Evaluating Biological Nitrogen Fixation, Bergersen, F.J., Ed., Chichester: Wiley, 1980, pp. 111–158.

    Google Scholar 

  11. Rademaker, J.L. and W. de Bruijn, F.J., Characterization and classification of microbes by rep-PCR genomic finger printing and computer-assisted pattern analyses, Int. J. Syst. Evol. Microbiol., 1997, vol. 50, no. 1, pp. 665–667.

    Google Scholar 

  12. Weisburg, W.G., Barns, S.M., Pelletier, D.A., and Lane, D.J., 16S Ribosomal DNA amplification for phylogenetic study, J. Bacteriol., 1991, vol. 173, no. 2, pp. 697–703.

    PubMed  CAS  PubMed Central  Google Scholar 

  13. Aqquilanti, L., Cavalca, L., Clementi, F., et al., Amplified ribosomal DNA restriction analysis for the characterization of Azotobacteraceae: a contribution to the study of these free-living nitrogen-fixing bacteria, Microbiol. Meth., 2004, vol. 57, no. 1, pp. 197–206.

    Article  Google Scholar 

  14. Sokal, R.R. and Sneath, P.H.A., Principle of Numerical taxonomy, San Francisco: W.H. Freeman, 1963.

    Google Scholar 

  15. Rohlf, F.J., NTSYS-pc. Numerical Taxonomy and Multivariate Analysis System, version 1.8 Applied Biostatistics, New York: Setauket Press, 1998.

  16. Shannon, C.E. and Weaver, W., The Mathematical Theory of Communication, Urbana: Univ. Illinois Press, 1949.

    Google Scholar 

  17. González-Lypez, J., Microorganismos Diazotrofos Asociados a Rances De Plantas No Leguminosas, Madris: Rueda Press, 1992.

    Google Scholar 

  18. Esquivel, G., Calva-Calva, G., Ferrera-Cerrato, R., et al., Encystment of Azotobacter nigricans grown diazotrophically on kerosene as sole carbon source, Arch. Microbiol., 2009, vol. 191, no. 3, pp. 275–281.

    Article  Google Scholar 

  19. Ahmad, F., Ahmad, I., and Khan, M.S., Screening of freeliving rhizospheric bacteria for their multiple plant growth promoting activities, Microbiol. Res., 2006, vol. 163, no. 1, pp. 173–181.

    PubMed  Google Scholar 

  20. Aquilanti, L., Favilli, F., and Clementi, F., Comparison of different strategies for isolation and preliminary identification of Azotobacter from soil samples, Soil Biol. Biochem., 2004, vol. 36, no. 1, pp. 1475–1483.

    Article  CAS  Google Scholar 

  21. Amos, W. and Harwoud, J., Factors affecting levels of genetic diversity in natural population, Biol. Sci., 1998, vol. 353, no. 1366, pp. 177–186.

    Article  CAS  Google Scholar 

  22. Baere, T.D., Mendoca, R., Claeys, G., et al., Evaluation of amplified rDNA restriction analysis (ARDRA) for the identification of cultured mycobacteria in diagnostic laboratory, BMC Microbiol., 2002, vol. 2, no. 4, pp. 1–22.

    Google Scholar 

  23. Jayarao, B.M., Dorer, J.R., and Oliver, S.P., Restriction fragment length polymorphism analysis of 16S ribosomal DNA of streptococcus and Enterococcus species of bovine origin, J. Clin. Microbiol., 1992, no. 9, pp. 2235–2240.

    Google Scholar 

  24. Sasaki, E., Osawa, R., Nishitani, Y., and Whiley, R.A., ARDRA and RAPD analysis of human and animal isolates of Streptococcus gallolyticus, J. Vet. Med. Sci., 2004, vol. 66, no. 11, pp. 1467–1470.

    Article  PubMed  CAS  Google Scholar 

  25. Tiedje, J.M., Asuming-Brempong, S., Nusslein, K., et al., Opening the black box of soil microbial diversity, Appl. Soil. Ecol., 1999, vol. 13, no. 2, pp. 109–122.

    Article  Google Scholar 

  26. Moyer, C.L., Tiedje, J.M., Dobbs, F.C., and Karl, D.M., A computer-simulated restriction fragment length polymorphism analysis of bacterial smallsubunit rRNA genes: efficacy of selected tetrameric restriction enzymes for studies of microbial diversity in nature, Appl. Environ. Microbiol., 1996, vol. 62, no. 7, pp. 2501–2507.

    PubMed  CAS  PubMed Central  Google Scholar 

  27. Vaneechoutte, M., Rossau, R., and De Vos, P., Rapid identification of the Comamonadaceae with amplified ribosomal restriction analysis (ARDRA), FEMS Microbiol. Letts., 1992, vol. 93, no. 3, pp. 227–234.

    Article  CAS  Google Scholar 

  28. Chéneby, D., Philippot, L., Hartmann, A., et al., 16s rRNA gene analysis for characterization of denitrifying bacteria isolated from three agricultural soils, FEMS Microbiol. Ecol., 2000, vol. 34, no. 2, pp. 121–128.

    Article  PubMed  Google Scholar 

  29. Jang, J., Bongjoon, K., Jongho, L., et al., Identification of Weissella species by the genus-specific amplified ribosomal DNA restriction analysis, FEMS Microbiol. Letts., 2002, vol. 212, no. 1, pp. 29–34.

    Article  CAS  Google Scholar 

  30. Ventura, M., Elli, M., Reniero, R., and Zink, R., Molecular microbial analysis of bifidobacterium isolates from different environments by the species-specific amplified ribosomal DNA restriction analysis (ARDRA), FEMS Microbiol. Ecol., 2001, vol. 36, no. 2, pp. 113–121.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Biotechnology, Science and Research Branch, Islamic Azad University, Tehran, Iran

    Z. Mazinani

  2. Department of Soil Biology, Soil and Water Researches Institute, Karaj, Iran

    A. Asgharzadeh

Authors
  1. Z. Mazinani
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  2. A. Asgharzadeh
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Correspondence to Z. Mazinani.

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Mazinani, Z., Asgharzadeh, A. Genetic diversity of Azotobacter strains isolated from soils by amplified ribosomal DNA restriction analysis. Cytol. Genet. 48, 293–301 (2014). https://doi.org/10.3103/S0095452714050041

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  • DOI: https://doi.org/10.3103/S0095452714050041

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