Isolation and molecular characterisation of diazotrophic growth-promoting bacteria from wheat rhizospheric soils of Punjab
S. K. Gosal A E , G. S. Saroa B , Y. Vikal C , S. S. Cameotra D , Neemisha Pathania A and A. Bhanot A
+ Author Affiliations
- Author Affiliations
A Department of Microbiology, Agricultural University, Ludhiana – 141 004, Punjab, India.
B Department of Soils, Agricultural University, Ludhiana – 141 004, Punjab, India.
C School of Agricultural Biotechnology, Agricultural University, Ludhiana – 141 004, Punjab, India.
D Institute of Microbial Technology, Chandigarh, India.
E Corresponding author. Email: skgosal@rediffmail.com
Soil Research 49(8) 725-732 https://doi.org/10.1071/SR11136
Submitted: 9 June 2011 Accepted: 21 September 2011 Published: 20 December 2011
Abstract
Diazotrophs are nitrogen-fixing bacteria which possess the nifH gene that codes for the nitrogenase enzyme involved in reduction of atmospheric dinitrogen to ammonia. Seventy-two diazotrophic bacteria were isolated using eight nitrogen-free media from wheat rhizospheric soil. The diazotrophic population was found to be negatively related to soil nitrogen, whereas a positive correlation was observed with organic carbon and electrical conductivity of soil. The isolates were initially identified on the basis of cultural, morphological, and biochemical characterisation. Various diazotrophic isolates were screened for functional activities. Thirty-seven isolates were acetylene reduction assay positive, among which 28 isolates exhibited nitrogenase activity ranging from 22.3 to 72.0 nmol C2H4/h. The majority of isolates were able to produce indole acetic acid ranging from 11.2 to 23.0 µg/mL and only a few diazotrophs could solubilise phosphate. These isolates showed amplification with two nifH primers (nifH1 and nifH2), thereby confirming their diazotrophic potential. The positive nifH isolates were further characterised using restriction fragment length polymorphism of 16S rDNA to reveal diversity among them. Based on UPGMA clustering and partial sequencing of 16S rDNA, the isolates were identified as Azotobacter sp., Azospirillum sp., Stenotrophomonas maltophilia, Stenotrophomonas sp., Sphingomonas paucimobilis, Rhizobium larrymoorei, Pseudomonas aeruginosa, and Xanthomonas oryzae.
Additional keywords: diazotrophs, diversity analysis, nifH, physicochemical, RFLP, 16S rDNA sequencing.
References
Adhikari TB, Joseph CM, Yang GP, Phillips DA, Nelson LM (2001) Evaluation of bacteria isolated from rice for plant growth promotion and biological control of seedling disease of rice. Canadian Journal of Microbiology 47, 916–924.| Evaluation of bacteria isolated from rice for plant growth promotion and biological control of seedling disease of rice.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXotl2nsLg%3D&md5=8d696ff12aba6536ff3aaa868bdde86cCAS |
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. Journal of Molecular Biology 215, 403–410.
Andrade G, Esteban E, Velascol L, Maria JL, Bedmar EJ (1997) Isolation and identification of N2-fixing microorganisms from the rhizosphere of Capparis spinosa (L.). Plant and Soil 197, 19–23.
| Isolation and identification of N2-fixing microorganisms from the rhizosphere of Capparis spinosa (L.).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXjs1yksA%3D%3D&md5=d045b5c9dd43f5d23523672de13976cbCAS |
Borneman J, Skroch PW, O’Sullivan KM, Palus JA, Rumjanek NG, Jansen JL, Nienhuis J, Triplett EW (1996) Molecular microbial diversity of an agricultural soil in Wisconsin. Applied and Environmental Microbiology 62, 1935–1943.
Clerc A, Manceau C, Nesme X (1998) Comparison of randomly amplified polymorphic DNA with amplified fragment length polymorphism to assess genetic diversity and genetic relatedness within genospecies III. Applied and Environmental Microbiology 64, 1180–1187.
Dalmastri C, Chiarini L, Cantale C, Bevivino A, Tabacchioni S (1999) Soil type and maize cultivars affect the genetic diversity of maize root-associated Burkholderia cepacia populations. Microbial Ecology 38, 273–284.
| Soil type and maize cultivars affect the genetic diversity of maize root-associated Burkholderia cepacia populations.Crossref | GoogleScholarGoogle Scholar |
Deslippe JR, Egger KN (2006) Molecular diversity of nifH genes from bacteria associated with high arctic dwarf shrubs. Microbial Ecology 51, 516–525.
| Molecular diversity of nifH genes from bacteria associated with high arctic dwarf shrubs.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XmtFGqtb8%3D&md5=4b225f45cac172cb3e832c5070e49b73CAS |
Edi-Promono M, Moawad AM, Vlek PLG (1996) Effect of phosphate solubilizing Pseudomonas putida on the growth of maize and its survival in the rhizosphere. Indonesian Journal of Crop Science 11, 13–23.
Flores-Mireles AL, Winans SC, Holguin G (2007) Molecular characterization of diazotrophic and denitrifying bacteria associated with mangrove roots. Applied and Environmental Microbiology 73, 7308–7321.
| Molecular characterization of diazotrophic and denitrifying bacteria associated with mangrove roots.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXnsFyhsg%3D%3D&md5=f6813586799df1491c1c04992dee0dd9CAS |
Galdagi R, Krishnaraj PU, Kulkarni JH, Tangmin SA (2002) Biodiversity of Azorhizobium in ornamental rhizosphere soils of Karnataka. In ‘Proceedings 17th World Congress of Soil Science’. Thailand, p. 537. (International Union of Soil Sciences)
Glick BR (2005) Modulation of plant ethylene levels by the bacterial enzyme ACC deaminase. FEMS Microbiology Letters 251, 1–7.
| Modulation of plant ethylene levels by the bacterial enzyme ACC deaminase.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhtVWhu7jI&md5=a3a458f9d39a3e632452dabb566eb93dCAS |
Gordon AS, Weber RP (1951) Colorimetric estimation of indole acetic acid Plant Physiology 26, 192–195.
| Colorimetric estimation of indole acetic acidCrossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaG3MXis1Glsw%3D%3D&md5=9039290d69f9092b6eda86c61b8b3b5aCAS |
Grayston SJ, Wang S, Campbell CD, Edwards AC (1998) Selective influence of plant species on microbial diversity in the rhizosphere. Soil Biology & Biochemistry 30, 369–378.
| Selective influence of plant species on microbial diversity in the rhizosphere.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXhvFWrsro%3D&md5=74981e920439e92203f181726a8f44aeCAS |
Gulati A, Rahi P, Vyas P (2008) Characterization of phosphate solubilizing fluorescent pseudomonad from rhizosphere of seabuckthorn growing in the cold deserts of Himalayas. Current Microbiology 56, 73–79.
| Characterization of phosphate solubilizing fluorescent pseudomonad from rhizosphere of seabuckthorn growing in the cold deserts of Himalayas.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhsVKrsrrF&md5=1d07b19c0ad219b53f3bfcddd9dd1ce6CAS |
Hardy RWF, Holsten RD, Jackson EK (1968) The acetylene-ethylene assay for N2-fixation—laboratory and field evaluation. Plant Physiology 43, 1185–1207.
| The acetylene-ethylene assay for N2-fixation—laboratory and field evaluation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaF1cXkslCnsbw%3D&md5=850413deacb1afed2debd351cedbc6c4CAS |
Holt JK, Kreig NR, Sneath PHA, Stanley JT, William ST (1994) ‘Bergey’s Manual of Systematic Bacteriology.’ (Williams and Wilkins: Baltimore, MD)
Jackson ML (1973) ‘Soil chemical analysis.’ Vol. 60. pp. 7–33. (Prentice Hall of India: New Delhi)
Lata, Saxena AK (2003) Characterization of plant growth promoting rhizobacteria. In ‘Training manual on biofertilizer technology’. (Ed. AK Saxena) pp. 24–25. (IARI: New Delhi)
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 and Soil 194, 37–44.
| Association of nitrogen-fixing, plant growth promoting rhizobacteria (PGPR) with kallar grass and rice.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2sXnt1Wjurc%3D&md5=a63c8648f89bcd5cd927d7109e880f6aCAS |
Nesme X, Vanecchoutte M, Orso S, Hoste B, Swings J (1995) Diversity and genetic relatedness with genera Xanthomonas and Stenotrophomonas using restriction endonuclease site divergences of PCR amplified 16S rRNA gene. Systematic and Applied Microbiology 18, 127–135.
| Diversity and genetic relatedness with genera Xanthomonas and Stenotrophomonas using restriction endonuclease site divergences of PCR amplified 16S rRNA gene.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2MXnt1Cnsrc%3D&md5=d8c2adadffc8f29198edb9db580e847bCAS |
Normand P, Simonet P, Bardin R (1988) Conservation of nif sequences in Frankia. Molecular & General Genetics 213, 238–246.
| Conservation of nif sequences in Frankia.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL1MXhvVKrtb4%3D&md5=e591dcf86655f8f289980b20d82bbc66CAS |
Ozawa T, Ohwaki A, Okumura K (2003) Isolation and characterization of diazotrophic bacteria from the surface sterilized roots of some legumes. Scientific Report of the Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, No. 55, pp. 29–36.
Page AL, Miller RH, Koeney DR (1982) ‘Methods of soil analysis. Part 2: Chemical and microbiological properties.’ Agronomy Monograph No. 9. (American Society of Agronomy Inc., Soil Science Society of America Inc.: Madison, WI)
Park M, Kim C, Yang J, Lee H, Shin W, Kim S, Sa T (2005) Isolation and characterization of diazotrophic growth promoting bacteria from rhizosphere of agricultural crops of Korea. Microbiological Research 160, 127–133.
| Isolation and characterization of diazotrophic growth promoting bacteria from rhizosphere of agricultural crops of Korea.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXls1Gju7c%3D&md5=818358c5e68dec15444256623c943d4eCAS |
Richard LA (1954) Diagnosis and improvement of saline and alkali soils. Agriculture Hand Book No. 60. (USDA: Washington, DC)
Rohlf FJ (1998) ‘NTSYS-pc numerical taxonomy and multivariate analysis system. Version 2.02.’ (Exeter Publications: Setauket, NY)
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4, 406–425.
Suckstorff I, Berg G (2003) Evidence for dose dependent effects on plant growth by Stenotrophomonas strains from different origins. Journal of Applied Microbiology 95, 656–636.
| Evidence for dose dependent effects on plant growth by Stenotrophomonas strains from different origins.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXoslWru7s%3D&md5=2b4ef5e06ae84d0c2a3eb20cac1e3ba7CAS |
Tilak KVBR, Ranganayaki N, Pal KK, De R, Saxena AK, Nautiyal CS, Mittal S, Tripathi AK, Johri BN (2005) Diversity of plant growth and soil health supporting bacteria. Current Science 89, 136–150.
Ueda T, Yuga Y, Yahiro N, Matsuguchi T (1995) Remarkable N2 fixing bacterial diversity detected in rice roots by molecular evolutionary analysis of nifH gene sequences. Journal of Bacteriology 177, 1414–1417.
Velazco A, Castro R, Napoles MC (1999) Azospirillum sp. diazotroph predominant in the cultivation of rice (Oryza sativa L.) in the province of Pinar de Rio. Cultivos Tropicales 20, 5–9.
Vessey JK (2003) Plant growth promoting rhizobacteria as biofertilizer. Plant and Soil 255, 571–586.
| Plant growth promoting rhizobacteria as biofertilizer.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXns1Ojsr0%3D&md5=9e9fb188188efa88b4270eef32e7a899CAS |
Walkley W, Black CA (1934) An examination of the digtiareff method for determination soil organic matter and a proposed modification of the chromic acid titration method. Soil Science 37, 29–38.
| An examination of the digtiareff method for determination soil organic matter and a proposed modification of the chromic acid titration method.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaA2cXitlGmug%3D%3D&md5=8f8216c8a32203f6de7c69e24a34e97bCAS |
Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology 173, 697–703.
Yohalem DS, Lorbeer JW (1994) Intraspecific metabolic diversity among strains of Burkholderia cepacia isolated from decayed onions, soils and the clinical environment. Antonie van Leeuwenhoek 65, 111–131.
| Intraspecific metabolic diversity among strains of Burkholderia cepacia isolated from decayed onions, soils and the clinical environment.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2MXhtlChsL0%3D&md5=ae0a79bb1ed53ae22c6c50c711578d85CAS |
Zehr JP, Capone DG (1996) Problems and promises of assaying the genetic potential for nitrogen fixation in the marine environment. Microbial Ecology 32, 263–281.
| Problems and promises of assaying the genetic potential for nitrogen fixation in the marine environment.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK28XmtlymtLw%3D&md5=4253210f04f888faf9464c63f9cc0025CAS |
