, Volume 251, Issue 4, pp 943–953 | Cite as

Phenotypic and molecular characterization of native Azospirillum strains from rice fields to improve crop productivity

  • Ranjan K Sahoo
  • Mohammad W Ansari
  • Madhusmita Pradhan
  • Tushar K Dangar
  • Santanu Mohanty
  • Narendra TutejaEmail author
Original Article


Beneficial microorganisms have been considered as an important tool for crop improvement. Native isolates of Azospirillum spp. were obtained from the rhizospheres of different rice fields. Phenotypic, biochemical and molecular characterizations of these isolates led to the identification of six efficient strain of Azospirillum. PCR amplification of the nif genes (nifH, nifD and nifK) and protein profile of Azospirillum strains revealed inter-generic and inter-specific diversity among the strains. In vitro nitrogen fixation performance and the plant growth promotion activities, viz. siderophore, HCN, salicylic acid, IAA, GA, zeatin, ABA, NH3, phosphorus metabolism, ACC deaminase and iron tolerance were found to vary among the Azospirillum strains. The effect of Azospirillum formulations on growth of rice var. Khandagiri under field condition was evaluated, which revealed that the native formulation of Azospirillum of CRRI field (As6) was most effective to elevate endogenous nutrient content, and improved growth and better yield are the result. The 16S rRNA sequence revealed novelty of native Azospirillum lipoferum (As6) (JQ796078) in the NCBI database.


Azospirillum Biofertilizer Crop improvement Plant growth Rice rhizosphere Plant yield 



Work on plant stress tolerance in NT's laboratory is partially supported by the Department of Science and Technology (DST) and the Department of Biotechnology (DBT), Government of India.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

709_2013_607_MOESM1_ESM.doc (110 kb)
ESM 1 (DOC 109 kb)
709_2013_607_MOESM2_ESM.ppt (202 kb)
ESM 2 (PPT 202 kb)


  1. Ahmad F, Ahmad I, Khan MS (2005) Indole acetic acid production by the indigenous isolates of Azotobacter and fluorescent Pseudomonas in the presence and absence of tryptophan. Turkish J Biol 29:29–34Google Scholar
  2. Alexander M (1991) Introduction to soil microbiology. Krieger, NY, USA, 467pGoogle Scholar
  3. Baldani JI, Krieg NR, Baldani VLD, Hartmannand A, Dobereiner J (2005) Genus II. Azospirillum. In: Brenner DJ, Krieg NR, Staley JT (eds) Bergey's manual of systematic bacteriology, vol 2C. Springer, New York, USA, pp 7–26Google Scholar
  4. Bashan Y, de-Bashan LE (2010) How the plant growth-promoting bacterium Azospirillum promotes plant growth—a critical assessment. Adv Agron 108:77–136CrossRefGoogle Scholar
  5. Bhaduri S, Demchick PH (1983) Simple and rapid method for disruption of bacteria for protein studies. Applied Environmental Microbiology 46:941–943PubMedPubMedCentralGoogle Scholar
  6. Bhattacharjee RB, Singh A, Mukhopadhyay SN (2008) Use of nitrogen-fixing bacteria as biofertilizer for non-legumes: prospects and challenges. Appl Microbiol Biotechnol 80:199–209CrossRefPubMedGoogle Scholar
  7. Briones AM, Okabe S, Umemiya Y, Ramsingh NB, Reichardt W, Okuyama H (2002) Effect of different cultivars on population of ammonia-oxidizing bacteria in root environment of rice. Appl Environ Microbial 68:3067–3075CrossRefGoogle Scholar
  8. Caballero-Mellado J, Lopez-Reyes L, Bustillos-Cristales R (1999) Presence of 16S rRNA genes in multiple replicons in Azospirillum brasilense. FEMS Microbiol Lett 178:283–288CrossRefGoogle Scholar
  9. Choudhury ATMA, Kennedy IR (2004) Prospects and potentials for systems of biological nitrogen fixation in sustainable rice production. Biol Fertil Soils 39:219–227CrossRefGoogle Scholar
  10. Collee JG, Miles PS (1989) Tests for identification of bacteria. In: Collee JG, Duguid JP, Fraser AG, Marmion BP (eds) Practical medical microbiology. Churchil Livingstone, NY, USA, pp 141–160Google Scholar
  11. Dangar TK, Basu PS (1991) Abscisic acid production in culture by some Rhizobium spp. of leguminous trees and pulses. Folia Microbiol 36:527–532Google Scholar
  12. Freitas ADS, Vieira CL, Santos CERS, Stamford NP, Lyra MCCP (2007) Caracterizac¸a˜o de rizo´bios isolados de Jacatupe´ cultivado em solo salino no Estado de Pernanbuco, Brasil. Bragantia 66:497–504Google Scholar
  13. Hardy RWF, Holsten RD, Jackson EK, Burns RC (1968) The acetylene-ethylene assay for N2 fixation: laboratory and field evaluation. Plant Physiol 43:1185–1207CrossRefPubMedPubMedCentralGoogle Scholar
  14. Hartmann A (1988) Ecophysiological aspects of growth and nitrogen fixation in Azospirillum spp. Plant and Soil l l0:225–238Google Scholar
  15. Hayat R, Ali S, Amara U, Khalid R, Ahmed I (2010) Soil beneficial bacteria and their role in plant growth promotion: a review. Ann Microbiol 60:579–598CrossRefGoogle Scholar
  16. Hill S, Sawers G (2000) Azotobacter. In: Lederberg J (ed) Encyclopedia of microbiology, vol. 1A-Cth edn. Academic, NY, USA, pp 359–371Google Scholar
  17. Jensen RB, Dam M, Gerdes K (1994) Partitioning of plasmid R1. The parA operon is autoreguated by ParR and its transcription is highly stimulated by a downstream activating element. J Mol Biol 236:1299–1309Google Scholar
  18. Jimenez DJ, Montana JS, Martinez MM (2011) Characterization of free nitrogen fixing bacteria of the genus Azotobacter in organic vegetable-grown colombian soils. Brazilian J Microbiol 42:846–858.Google Scholar
  19. Kaneko T, Minamisawa K, Isawa T, Nakatsukasa H, Mitsui H, Kawaharada Y, Nakamura Y, Watanabe A, Kawashima K, Ono A, Shimizu Y, Takahashi C, Minami C, Fujishiro T, Kohara M, Katoh M, Nakazaki N, Nakayama S, Yamada M, Tabata S, Sato S (2010) Complete genomic structure of the cultivated rice endophyte Azospirillum sp. B510. DNA Res 17:37–50CrossRefPubMedPubMedCentralGoogle Scholar
  20. Kannan T, Ponmurugan P (2010) Response of paddy (Oryza sativa L.) varieties to Azospirillum brasilense inoculation. J Physiol 2:08–13Google Scholar
  21. Kenmore P, (2003) Sustainable rice production, food security and enhanced livelihoods in “Rice Science: In: Mew TW, Brar DS, Peng S, Dawe D, Hardy B (eds.). Innovations and Impact for Livelihood. Beijing, China, p 27–34Google Scholar
  22. Kennedy C (2005a) Genus I. Beijernckia. In: Staley JT, Boone DR, Brenner DJ, de Vos P, Garriety GM, Goodfellow M, Krieg NR, Rainey FA, Schlifer KH (eds) Bergey's manual of systematic bacteriology, vol 2C. Springer, New York, USA, pp 423–432CrossRefGoogle Scholar
  23. Kennedy C (2005b) Genus IV. Derxia. In: Staley JT, Boone DR, Brenner DJ, de Vos P, Garriety GM, Goodfellow M, Krieg NR, Rainey FA, Schlifer KH (eds) Bergey's manual of systematic bacteriology, vol 2C, eds. Springer, New York, USA, pp 671–674CrossRefGoogle Scholar
  24. Kennedy C, Rudnick P, MacDonald ML, Melton T (2005) Genus III. Azotobacter. In: Brenner DJ, Krieg NR, Staley JT (eds) Bergey's manual of systematic bacteriology, vol 2B. Springer, New York, USA, pp 384–402Google Scholar
  25. Khan MR, Talukdar NC, Thakur D (2003) Detection of Azospirillum and PSB in rice rhizosphere soil by protein and antibiotic resistance profile and their effect on grain yield of rice. Indian J Biotechnol 2:246–250Google Scholar
  26. Kluepfel DA (1993) The behaviour and tracking of bacteria in the rhizosphere. Annu Rev Phytopathol 31:441–472CrossRefGoogle Scholar
  27. Lakshmi-Kumari M, Lakshmi V, Nalini PA, Subba-Rao NS (1980) Reactions of Azospirillum to certain dyes and their usefulness in enumeration of the organisms. Curr Sci 49:438–439Google Scholar
  28. Martin-Didonet CCG, Chubatsu LS, Souza EM, Kleina M, Rego FGM, Rigo LU, Yates MG, Pedrosa FO (2000) Genome structure of the genus Azospirillum. J Bact 182:4113–4116CrossRefPubMedPubMedCentralGoogle Scholar
  29. Mot RD, Vanderhyden J (1989) Application of two-dimensional protein analysis for strain finger printing and mutant analysis of Azospirillum sp. Canadian J Microbiol 35:960–967CrossRefGoogle Scholar
  30. Okon Y, Albrecht SL, Burris RH (1977) Methods for growing Spirillum lipoferum and for counting it in pure culture and in association with plants. Appl Environ Microbiol 3:85–88Google Scholar
  31. Palleroni NJ (2005) Genus I. Pseudomonas. In: Brenner DJ, Krieg NR, Staley JT, Garrity GM (eds) Bergey’s manual of systematic bacteriology. Part B. The gammaproteobacteria. Springer, New York, p 323–379Google Scholar
  32. Patel PH, Patel JP, Bhatt SA (2013) Characterization and phylogenetic relatedness of Azotobacter Salinestris. J Microbiol Biotechnol Res 3:65–70Google Scholar
  33. Perrig D, Boiero ML, Masciarelli OA, Penna C, Ruiz OA, Cassan FD, Luna MV (2007) Plant-growth-promoting compounds produced by two agronomically important strains of Azospirillum brasilense, and implications for inoculants formulation. Appl Microbiol Biotechnol 75:1143–1150CrossRefPubMedGoogle Scholar
  34. Pot B, Gills M (2005) Genus III. Aquaspirillum. In: Staley JT, Boone DR, Brenner DJ, de Vos P, Garriet GM, Goodfellow M, Krieg NR, Rainey FA, Schlifer KH (eds) Bergey's manual of systematic bacteriology, 2cth edn. Springer, New York, USA, pp 801–823CrossRefGoogle Scholar
  35. Postgate JR (1982) The fundamentals of Nitrogen fixation. Cambridge University Press. p 60–102Google Scholar
  36. Reddy BP, Reddy KRN, Rao MS, Rao KS (2008) Efficacy of antimicrobial metabolites of pseudomonas fluorescens against rice fungal pathogens. Curr Trends Biotechnol Pharm 2:178–182Google Scholar
  37. Rubio MB, Hermosa MR, Keck E, Monte E (2005) Specific PCR assays for the detection and quantification of DNA from the biocontrol strain Trichoderma harzianum 2413 in soil. Microb Ecol 49:25–33Google Scholar
  38. Saharan BS, Nehra V (2011) Plant growth promoting rhizobacteria: a critical review. Life Sci Medic Res 21:1–30Google Scholar
  39. Sahoo RK, Ansari MW, Dangar TK, Mohanty S, Tuteja N (2013) Phenotypic and molecular characterization of efficient nitrogen fixing Azotobacter strains from rice fields for crop improvement. Protoplasma. doi: 10.1007/s00709-013-0547-2 PubMedGoogle Scholar
  40. Sahrawat KL (2000) Macro and micronutrients removed by upland and lowland rice cultivars in West Africa. Commun Soil Sci Plant Anal 31:717–723CrossRefGoogle Scholar
  41. Saikia SP, Jain V (2007) Biological nitrogen fixation with non-legumes: an achievable target or a dogma? Curr Sci 92:317–322Google Scholar
  42. Samuel S, Muthukkaruppan SM (2011) Characterization of plant growth promoting rhizobacteria and fungi associated with rice, mangrove and effluent contaminated soil. Curr Bot 2:22–25Google Scholar
  43. SantAnna FH, Almeida LGP, Cecagno R, Reolon LA, Siqueira FM, Machado MRS, Vasconcelos ATR, Schrank IS (2011) Genomic insights into the versatility of the plant growth-promoting bacterium Azospirillum amazonense. BMC Genomics 12:409–423CrossRefGoogle Scholar
  44. Shrestha RK, Maskey SI (2005) Associative nitrogen fixation in lowland rice. Nepal Agric Res J 6:112–121Google Scholar
  45. Singh MS (2006) Cereal crops response to Azotobacter—a review. Agric Rev 27:229–231Google Scholar
  46. Smibert R, Krieg NR (1995) Phenotypic characterization. In: Gerhardt P, Murray RGE, Wood W, Krieg E (eds) Methods for general and molecular bacteriology. American Soc Microbiol, Washington DC, pp 607–654Google Scholar
  47. Strzelczyk E, Kampert M, Li CY (1994) Cytokinin like substances and ethylene production by Azospirillum in media with different carbon sources. Microbiol Res 149:55–60Google Scholar
  48. Subbarao NS (2007) Soil microorganisms and plant growth. Oxford IBH, New DelhiGoogle Scholar
  49. Tien TM, Gaskins MH, Hubbell DH (1979) Plant growth substances produced by Azospirillum brasilense and their effect on the growth of pearl millet (Pennisetum americanum L.). Appl Microbiol 37:1016–1024Google Scholar
  50. Watanabe A, Kawashima K, Ono A, Shimizu Y, Takahashi C, Minami C, Fujishiro T, Kohara M, Katoh M, Nakazaki N, Nakayama S, Yamada M, Tabata S, Sato S (2010) Complete genomic structure of the cultivated rice endophyte Azospirillum sp. B510. DNA Res 17:37–50CrossRefPubMedPubMedCentralGoogle Scholar
  51. Yushmanov SV, Chumakov KM (1988) Algorithms of the maximum topological similarity phylogenetic trees construction. Mol Genet Microbiol Virol 3:9–15Google Scholar
  52. Zaki N, Gomaa AM, Galal A, Farrag AA (2009) The associative impact of certain diazotrophs and farmyard manure on two rice varieties grown in a newly cultivated land. Res J Agric Biol Sci 5:185–190Google Scholar
  53. Zeigler RS, Adams A (2008) The relevance of rice. Rice 1:3–10CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2014

Authors and Affiliations

  • Ranjan K Sahoo
    • 1
  • Mohammad W Ansari
    • 1
  • Madhusmita Pradhan
    • 2
  • Tushar K Dangar
    • 3
  • Santanu Mohanty
    • 2
  • Narendra Tuteja
    • 1
    Email author
  1. 1.Plant Molecular Biology GroupInternational Centre for Genetic Engineering and BiotechnologyNew DelhiIndia
  2. 2.Department of Soil Science and Agricultural Chemistry, College of AgricultureOrissa University of Agriculture and TechnologyBhubaneswarIndia
  3. 3.Division of Crop ProductionCentral Rice Research InstituteCuttackIndia

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