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Phenotypic and molecular characterisation of efficient nitrogen-fixing Azotobacter strains from rice fields for crop improvement

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Abstract

Biological nitrogen fixation (BNF) is highly effective in the field and potentially useful to reduce adverse effects chemical fertilisers. Here, Azotobacter species were selected via phenotypic, biochemical and molecular characterisations from different rice fields. Acetylene reduction assay of Azotobacter spp. showed that Azotobacter vinelandii (Az3) fixed higher amount of nitrogen (121.09 nmol C2H4 mg-1 bacteria h-1). Likewise, its plant growth functions, viz. siderophore, hydrogen cyanide, salicylic acid, IAA, GA3, zeatin, NH3, phosphorus solubilisation, ACC deaminase and iron tolerance, were also higher. The profile of gDNA, plasmid DNA and cellular protein profile depicted inter-generic and inter-specific diversity among the isolates of A. vinelandii. The PCR-amplified genes nifH, nifD and nifK of 0.87, 1.4 and 1.5 kb , respectively, were ascertained by Southern blot hybridisation in isolates of A. vinelandii. The 16S rRNA sequence from A. vinelandii (Az3) was novel, and its accession number (JQ796077) was received from NCBI data base. Biofertiliser formulation of novel A. vinelandii isolates along with commercial one was evaluated in rice (Oriza sativa L. var. Khandagiri) fields. The present finding revealed that treatment T4 (Az3) (A. vinelandii) are highly efficient to improved growth and yield of rice crop.

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References

  • Akhter MS, Hossain SJ, Amir-Hossain SK, Datta RK (2012) Isolation and characterization of salinity tolerant Azotobacter sp. Greener J Biol Sci 2:42–051

    Google Scholar 

  • Banerjee A, Padhi S, Adhya TK (1999) Persistence and biodegradation of vinclozolin in tropical rice soils. Pest Sci 55:1177–1181

    Article  CAS  Google Scholar 

  • Barua S, Tripathi S, Chakraborty A, Ghosh S, Chakrabarti K (2012) Characterization and crop production efficiency of diazotrophic bacterial isolates from coastal saline soils. Microbiol Res 167:95–102

    Article  PubMed  Google Scholar 

  • Bashan Y, de-Bashan LE (2010) How the plant growth-promoting bacterium Azospirillum promotes plant growth—a critical assessment. Adv Agron 108:77–136

    Article  CAS  Google Scholar 

  • Bhaduri S, Demchick PH (1983) Simple and rapid method for disruption of bacteria for protein studies. Appl Environ Microbial 46:941–943

    CAS  Google Scholar 

  • Bhattacharjee RB, Singh A, Mukhopadhyay SN (2008) Use of nitrogen-fixing bacteria as biofertilizer for non-legumes: prospects and challenges. Appl Microbiol Biotechnol 80:199–209

    Article  PubMed  CAS  Google Scholar 

  • Bhattacharyya P, Roy KS, Neogi S, Adhya TK, Rao KS, Manna MC (2012) Effects of rice straw and nitrogen fertilization on greenhouse gas emissions and carbon storage in tropical flooded soil planted with rice. Soil Till Res 124:119–130

    Article  Google Scholar 

  • Bohme L, Langer U, Bohme F (2005) Microbial biomass, enzyme activities and microbial community structure in two European long-term field experiments. Agric Ecosyst Environ 109:141–152

    Article  Google Scholar 

  • Chapman HD, Pratt PF (1982) Method and of analysis of soil, plant and water, 2nd edn. California University Agricultural Division, California, p 170

    Google Scholar 

  • Chaudry IA, Cornfield AH (1966) The determination of total sulfur in soil and plant material. Analyst 91:528–530

    Article  Google Scholar 

  • Choudhury ATMA, Kennedy IR (2004) Prospects and potentials for systems of biological nitrogen fixation in sustainable rice production. Biol Fertil Soils 39:219–227

    Article  Google Scholar 

  • 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 141–160

    Google Scholar 

  • Govindan M, Bagyaraj DJ (1995) Field response of wetland rice to Azospirillum inoculation. J Soil Biol Ecol 15:17–22

    Google Scholar 

  • Haefele SM, Wopereis MCS, Schloebohm AM, Wiechmann H (2004) Long-term fertility experiments for irrigated rice in the West African Sahel: effect on soil characteristics. Field Crops Res 85:61–77

    Article  Google Scholar 

  • Hanlon EA, Devore JM (1989) IFAS extension soil testing laboratory chemical procedures and training manual. Circ. No. 812. Fla. Coop. Ext. Ser., IFAS, Univ. Of Florida, Gainesville, FL

    Google Scholar 

  • Hardy RWF, Holsten RD, Jackson EK, Burns RC (1968) The acetylene–ethylene assay for N2 fixation: laboratory and field evaluation. Plant Physiol 43:1185–1207

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • 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 60:579–598

    Article  Google Scholar 

  • Hill S, Sawers G (2000) Azotobacter. In: Lederberg J (ed) Enclyopedia of microbiology, vol 1A–C. Acad Press, NY, pp 359–371

    Google Scholar 

  • Hong JS, Geun TP, Mi SC, Moon SH (2006) Solubilization of insoluble inorganic phosphates by a novel salt- and pH-tolerant Pantoea agglomerans R-42 isolated from soybean rhizosphere. Biores Technol 97:204–210

    Google Scholar 

  • Jackson TL (1973) Soil chemical analysis. Prentice-Hall, New Delhi, India

    Google Scholar 

  • Jen-Hshuan C (2006) The combined use of chemical and organic fertilizers and/or biofertilizer for crop growth and soil fertility. Int Work Sustained Manag Soil-rhizosphere Syst Efficient Crop Prod Fertil Use 16:1–10

    Google Scholar 

  • Jensen HL (1954) The Azotobacteriaceae. Bacteriol Rev 18:195–214

    PubMed Central  PubMed  CAS  Google Scholar 

  • Jimenez DJ, Montana JS, Martinez MM (2011) Characterization of free nitrogen fixing bacteria of the genus Azotobacter in organic vegetable-grown colombian soils. Braz J Microbiol 42:846–858

    PubMed Central  PubMed  CAS  Google Scholar 

  • Kader MA, Mamun SMA, Hossain SMA, Hasna MK (2000) Effect of Azotobacter application on the growth and yield of transplanted aman rice and nutrient status of post-harvest soil. Pakistan J Biol Sci 3:1144–1147

    Article  Google Scholar 

  • Kanimozhi K, Panneerselvam (2011) A Investigation of soil characters and Azospirillum isolated from paddy soils of Thanjavur district, East Coast of Tamilnadu, India. Arch Appl Sci Res 3:525–536

    CAS  Google Scholar 

  • Kannan T, Ponmurugan P (2010) Response of paddy (Oryza sativa L.) varieties to Azospirillum brasilense inoculation. J Phytol 2:8–13

    Google Scholar 

  • Kanungo PK, Panda D, Adhya TK, Ramakrishnan B, Rao VR (1997) Nitrogenase activity and nitrogen-fixing bacteria associated with rhizosphere of rice cultivars with varying N absorption efficiency. J Sci Food Agric 73:485–488

    Article  CAS  Google Scholar 

  • Karadeniz A, Topcuoglu SF, Inan S (2006) Auxin, gibberellin, cytokinin and abscisic acid production in some bacteria. World J Microbiol Biotechnol 22:1061–1064

    Article  CAS  Google Scholar 

  • Kennedy IR, Tcha YT (1992) Biological nitrogen fixation in non-leguminous field crops: recent advances. Plant Soil 141:93–118

    Article  CAS  Google Scholar 

  • 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 384–402

    Google Scholar 

  • Keyeo F, Noor O, Amir HG (2011) The effects of nitrogen fixation activity and phytochrome production of diazotroph in promoting growth of rice seedlings. Biotechnology 10:267–273

    Article  CAS  Google Scholar 

  • 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. Ind J Biotechnol 2:246–250

    Google Scholar 

  • Kluepfel DA (1993) The behaviour and tracking of bacteria in the rhizosphere. Annu Rev Phytopathol 31:441–472

    Article  Google Scholar 

  • Kumar K, Kannaiyan S (2006) In: John William S (ed) Molecular characterization of nif genes of heterotrophic and endophytic diazotrophs in rice. Biodiversity: life to our mother earth, Chennai, India, p 467

    Google Scholar 

  • Kyriazakis I, Oldham JD (1993) Diet selection in sheep: the ability of growing lambs to select a diet that meets their crude protein (nitrogen × 6.25) requirements. Br J Nutr 69:617–629

    Article  PubMed  CAS  Google Scholar 

  • Li JC, Shi J, Zhao XL, Wang GYU, Ren YJ (1994) Separation and determination of three kinds of plant hormone by high performance liquid chromatography. Chin J Anal Chem 22:801–804

    Google Scholar 

  • Macmillan J, Suter PJ (1963) Thin-layer chromatography for the gibberellins. Nature London 197:790

    Article  CAS  Google Scholar 

  • Malik KA, Rakhshanda B, Samina M, Rasul G, Mirza MS, Ali S (1997) Association of nitrogen-fixing, plant-growth promoting rhizobacteria (PGPR) with kallar grass and rice. Plant Soil 194:37–44

    Article  CAS  Google Scholar 

  • Marianna M, Szilvia V, Eva G, Nora B, Brigitta T, Laszlo L (2005) The possible role of biofertilizers in agriculture. Ratarstvo 12:585–588

    Google Scholar 

  • Meng L, Ding WX, Cai ZC, Qin SW (2005) Storage of soil organic C and soil respiration as affected by long-term quantitative fertilization (in Chinese). Adv Earth Sci 20:687–692

    Google Scholar 

  • Mot RD, Vanderhyden J (1989) Application of two-dimensional protein analysis for strain finger printing and mutant analysis of Azospirillum sp. Can J Microbiol 35:960–967

    Article  Google Scholar 

  • Nayak DR, Jagadeeshbabu Y, Adhya TK (2007) Long-term application of compost influences microbial biomass and enzyme activities in a tropical aeric endoaquept planted to rice under flooded condition. Soil Biol Biochem 39:1897–1906

    Article  CAS  Google Scholar 

  • Needleman BA, Gburek WJ, Sharpley AN, Peterson GW (2001) Environmental management of soil phosphorus: Modeling spatial variability in small fields. Soil Sci Soc Am J 65:1516–1522

    Article  Google Scholar 

  • Patel PH, Patel JP, Bhatt SA (2013) Characterization and phylogenetic relatedness of Azotobacter Salinestris. J Microbiol Biotechnol Res 3:65–70

    Google Scholar 

  • Pathak H, Byjesh K, Chakrabarti B, Aggarwal PK (2011) Potential and cost of carbon sequestration in Indian agriculture:estimates from long-term field experiments. Field Crops Res 120:102–111

    Article  Google Scholar 

  • Patil V (2011) Production of indole acetic acid by Azotobacter sp. Recent Res Sci Technol 3:14–16

    Google Scholar 

  • Pedraza RO, Bellone CH, de-Bellone SC, Sorte PMFB, Teixeira KRDS (2009) Azospirillum inoculation and nitrogen fertilization effect on grain yield and on the diversity of endophytic bacteria in the phyllosphere of rice rainfed crop. Eur J Soil Biol 45:36–43

    Google Scholar 

  • 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 75:1143–1150

    Article  PubMed  CAS  Google Scholar 

  • Prajapati K, Yami KD, Singh A (2008) Plant growth promotional effect of Azotobacter chroococcum, Piriformospora indica and vermicompost on rice plant. Nepal J Sci Technol 9:85–90

    Google Scholar 

  • Rai M (2004) International year of rice—an overview. Indian Farming 54:3–6

    Google Scholar 

  • Rajaee S, Alikhani HA, Raiesi F (2007) Effect of plant growth promoting potentials of Azotobacter chroococcum native strains on growth, yield and uptake of nutrients in wheat. J Sci Technol Agric Nat Resour 11:285–297

    CAS  Google Scholar 

  • Reddy BP, Reddy KRN, Rao MS, Rao KS (2008) Efficacy of antimicrobial metabolites of Pseudomonas fluorescens against rice fungal pathogens. Curr Trends Biotechnol Pharm 2:178–182

    CAS  Google Scholar 

  • Saharan BS, Nehra V (2011) Plant growth promoting rhizobacteria: a critical review. Life Sci Med Res 21:1–30

    Google Scholar 

  • Sahrawat KL (2000) Macro and micronutrients removed by upland and lowland rice cultivars in West Africa. Commun Soil Sci Plant Anal 31:717–723

    Article  CAS  Google Scholar 

  • Saikia SP, Jain V (2007) Biological nitrogen fixation with non-legumes: an achievable target or a dogma? Curr Sci 92:317–322

    CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Samuel S, Muthukkaruppan SM (2011) Characterization of plant growth promoting rhizobacteria and fungi associated with rice, mangrove and effluent contaminated soil. Curr Bot 2:22–25

    CAS  Google Scholar 

  • Seck PA, Diagne A, Mohanty S, Wopereis MCS (2012) Crops that feed the world 7: rice. Food Sec 4:7–24

    Article  Google Scholar 

  • Setubal JC et al (2009) Genome sequence of Azotobacter vinelandii, an obligate aerobe specialized to support diverse anaerobic metabolic processes. J Bacteriol 191:4534–4545

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Shrestha RK, Maskey SI (2005) Associative nitrogen fixation in lowland rice. Nepal Agric Res J 6:112–121

    Google Scholar 

  • Singh MS (2006) Cereal crops response to Azotobacter—a review. Agric Rev 27:229–231

    Google Scholar 

  • Smibert R, Krieg NR (1995) Phenotypic characterization. In: Gerhardt P, Murray RGE, Wood W, Krieg E (eds) Methods for general and molecular bacteriology. American Society for Microbiology, Washington DC, pp 607–654

    Google Scholar 

  • Strzelczyk E, Kampert M, Li CY (1994) Cytokinin like substances and ethylene production by Azospirillum in media with different carbon sources. Microbiol Res 149:55–60

    Article  CAS  Google Scholar 

  • Subba Rao NS (2007) Soil microorganisms and plant growth. Oxford IBH publication, New Delhi

    Google Scholar 

  • Syono K, Newcomb W, Torrey JG (1976) Cytokinin production in relation to the development of pea root nodules. Can J Bot 54:2155–2162

    Google Scholar 

  • Tejera N, Luch C, Martinez-Toledo MV, Gonzalez-Lopez J (2005) Isolation and characterization of Azotobacter and Azospirillum strains from the sugarcane rhizosphere. Plant Soil 270:223–232

    Article  CAS  Google Scholar 

  • 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 37:1016–1024

    CAS  Google Scholar 

  • Upper CD, Helgeson JP, Kemp JD, Schmidt CJ (1970) Gas–liquid chromatographic isolation of cytokinins from natural sources: 6-(3-methyl-2-butenylamino) purine from Agrobacterium tumefaciens. Plant Physiol 45:543–547

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Vessey JK (2003) Plant growth promoting rhizobacteria as biofertilizers. Plant Soil 255:571–586

    Article  CAS  Google Scholar 

  • You CB, Song N, Wang HX, Li JP, Lin M, Hai WL (1991) Association of Alcaligenes faecalis with wetland rice. Plant Soil 137:81–85

    Article  Google Scholar 

  • Yushmanov SV, Chumakov KM (1988) Algorithms of the maximum topological similarity phylogenetic trees construction. Mol Genet Microbiol Virusol 3:9–15

    Google Scholar 

  • 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 5:185–190

    CAS  Google Scholar 

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Acknowledgments

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

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Authors and Affiliations

  1. Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, 110067, New Delhi, India

    Ranjan K. Sahoo, Mohammad W. Ansari & Narendra Tuteja

  2. Division of Crop Production, Central Rice Research Institute, Cuttack, Odisha, India

    Tushar K. Dangar

  3. Department of Soil Science and Agricultural Chemistry, College of Agriculture, Orissa University of Agriculture and Technology, Bhubaneswar, Odisha, India

    Santanu Mohanty

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  1. Ranjan K. Sahoo
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Correspondence to Narendra Tuteja.

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Sahoo, R.K., Ansari, M.W., Dangar, T.K. et al. Phenotypic and molecular characterisation of efficient nitrogen-fixing Azotobacter strains from rice fields for crop improvement. Protoplasma 251, 511–523 (2014). https://doi.org/10.1007/s00709-013-0547-2

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