Microbial Ecology

, Volume 63, Issue 4, pp 813–821 | Cite as

Genetic Diversity of Endophytic Diazotrophs of the Wild Rice, Oryza alta and Identification of the New Diazotroph, Acinetobacter oryzae sp. nov.

  • Hassan Javed Chaudhary
  • Guixiang Peng
  • Mei Hu
  • Yumei He
  • Lijuan Yang
  • Yan Luo
  • Zhiyuan Tan
Environmental Microbiology


Thirty-three endophytic diazotrophs were isolated from surface-sterilized leaves, stem, and roots of wild rice Oryza alta. The SDS–PAGE profile of total protein and insertion sequence-based polymerase chain reaction (IS-PCR) fingerprinting grouped the isolates into four clusters (I–IV). The 16S rRNA gene sequence homology of the representative strains B21, B31, B1, and B23 of clusters I, II, III, and IV were assigned to Pseudomonas oleovorans (99.2% similarity), Burkholderia fungorum (99.4% similarity), Enterobacter cloacae (98.9% similarity), and Acinetobacter johnsonii (98.4% similarity), respectively. The results showed wide genetic diversity of the putative diazotrophic strains of the wild rice, O. alta, and the strains of cluster IV are the first report of nitrogen-fixing Acinetobacter species. The cell size, phenotypic characters, total protein profile, genomic DNA fingerprinting, DNA–DNA hybridization, and antibiotic resistance differentiated strain B23T from its closest relatives A. johnsonii LMG999T and Acinetobacter haemolyticus LMG996T. The DNA–DNA hybridization also distinguished the strain B23T from the closely related Acinetobacter species. Based on these data, a novel species, Acinetobacter oryzae sp. nov., and strain B23T (=LMG25575T = CGMCC1.10689T) as the type strain were proposed.


Acinetobacter Wild Rice Enterobacter Cloaca nifH Gene Strain B23T 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This work was supported by National Basic Research Program of China (973 Program, 2010CB126502), National Natural Science Foundation of China (NSFC, 30770001), Doctoral Fund of Ministry of Education of China (20094404110006), Science and Technology Program of Guangdong Province (2010B060200017, 2010B090400450), and Program for New Century Excellent Talents in University (NCET-07-0315).

Supplementary material

248_2011_9978_MOESM1_ESM.doc (130 kb)
Supplementary Table 1 (DOC 130 kb)


  1. 1.
    Khush G (2003) Productivity improvements in rice. Nutr Rev 61:114–116CrossRefGoogle Scholar
  2. 2.
    Ladha JK, Reddy PM (2003) Nitrogen fixation in rice systems: state of knowledge and future prospect. Plant Soil 252:151–167CrossRefGoogle Scholar
  3. 3.
    Döbereiner J (1992) History and new perspectives of diazotrophs in association with non-leguminous plants. Symbiosis 13:1–13Google Scholar
  4. 4.
    Boddey RM (1995) Biological nitrogen fixation in sugar cane: a key to energetically viable biofuel production. Crit Rev Plant Sci 14:263Google Scholar
  5. 5.
    You CB, Zhou FY (1989) Non-nodular endorhizospheric nitrogen fixation in wetland rice. Can J Microbiol 35:403–408CrossRefGoogle Scholar
  6. 6.
    Elbeltagy A, Nishioka K, Sato T, Suzuki H, Ye B, Hamada T, Isawa T, Mitsui H, Minamisawa K (2001) Endophytic colonization and in planta nitrogen fixation by a Herbaspirillum sp. isolated from wild rice species. Appl Environ Microbiol 67:5285–5293PubMedCrossRefGoogle Scholar
  7. 7.
    Baldani VLD, Baldani JI, Döbereiner J (2000) Inoculation of rice plants with the endophytic diazotrophs Herbaspirillum seropedicae and Burkholderia spp. Biol Fertil Soils 30:485–491CrossRefGoogle Scholar
  8. 8.
    Bergogne-Berezin E, Towner KJ (1996) Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features. Clin Microbiol Rev 9:148–165PubMedGoogle Scholar
  9. 9.
    Nishimura Y, Ino T, Iizuka H (1988) Acinetobacter radioresistens sp. nov. Isolated from cotton and soil. Int J Syst Bacteriol 38:209–211CrossRefGoogle Scholar
  10. 10.
    Bouvet PJM, Grimont PAD (1986) Taxonomy of the genus Acinetobacter with the recognition of Acinetobacter baumannii sp. nov., Acinetobacter haemolyticus sp. nov., Acinetobacter johnsonii sp. nov., and Acinetobacter junii sp. nov. and emended descriptions of Acinetobacter calcoaceticus and Acinetobacter lwoffii. Int J Syst Bacteriol 36:228–240CrossRefGoogle Scholar
  11. 11.
    Anandham R, Weon HY, Kim SJ, Kim YS, Kim BY, Kwon SW (2010) Acinetobacter brisouii sp. nov., isolated from a wetland in Korea. J Microbiol 48:36–39PubMedCrossRefGoogle Scholar
  12. 12.
    Kim D, Baik KS, Kim MS, Park SC, Kim SS, Rhee MS, Kwak YS, Seong CN (2008) Acinetobacter soli sp. nov., isolated from forest soil. J Microbiol 46:396–401PubMedCrossRefGoogle Scholar
  13. 13.
    Carr EL, Kampfer P, Patel BK, Gurtler V, Seviour RJ (2003) Seven novel species of Acinetobacter isolated from activated sludge. Int J Syst Evol Microbiol 53:953–963PubMedCrossRefGoogle Scholar
  14. 14.
    Vaneechoutte M, Nemec A, Musilek M, van der Reijden TJ, van den Barselaar M, Tjernberg I, Calame W, Fani R, De Baere T, Dijkshoorn L (2009) Description of Acinetobacter venetianus ex Di Cello et al. 1997 sp. nov. Int J Syst Evol Microbiol 59:1376–1381PubMedCrossRefGoogle Scholar
  15. 15.
    Stoltzfus JR, So R, Malarvithi PP, Ladha JK, de Bruijn FJ (1997) Isolation of endophytic bacteria from rice and assessment of their potential for supplying rice with biologically fixed nitrogen. Plant Soil 194:25–36CrossRefGoogle Scholar
  16. 16.
    Wang HR, Peng GX, Zhang GX (2006) Characterization of endophytic diazotrophs isolated from molasses grass. Acta Ecologica Sinica 26:2566–2571Google Scholar
  17. 17.
    Eckert B, Weber OB, Kirchhof G, Halbritter A, Stoffels M, Hartmann A (2001) Azospirillum doebereinerae sp. nov., a nitrogen-fixing bacterium associated with the C4-grass Miscanthus. Int J Syst Evol Microbiol 51:17–26PubMedGoogle Scholar
  18. 18.
    Baldani VLD, Alvarez MADB, Baldani JI, Döbereiner J (1986) Establishment of inoculated Azospirillum spp. in the rhizosphere and in roots of field grown wheat and sorghum. Plant Soil 90:35–46CrossRefGoogle Scholar
  19. 19.
    Peng GX, Chen WX, Tan ZY (2004) Identification and phylogenetic analysis of closely related Rhizobium species by rRNA gene intergenic spacer sequence. J South China Agric Univ 23:58–62Google Scholar
  20. 20.
    Zehr JP, McReynolds LA (1989) Use of degenerate oligonucleotides for amplification of the nifH gene from the marine cyanobacterium Trichodesmium thiebautii. Appl Environ Microbiol 55:2522–2526PubMedGoogle Scholar
  21. 21.
    Tan Z, Hurek T, Gyaneshwar P, Ladha JK, Reinhold-Hurek B (2001) Novel endophytes of rice form a taxonomically distinct subgroup of Serratia marcescens. Syst Appl Microbiol 24:245–251PubMedCrossRefGoogle Scholar
  22. 22.
    Sneath PHA, Sokal RR (1973) Numerical taxonomy. The principles and practices of numerical classification. Freeman, San FranciscoGoogle Scholar
  23. 23.
    Peng G, Wang H, Zhang G, Hou W, Liu Y, Wang ET, Tan Z (2006) Azospirillum melinis sp. nov., a group of diazotrophs isolated from tropical molasses grass. Int J Syst Evol Microbiol 56:1263–1271PubMedCrossRefGoogle Scholar
  24. 24.
    Pearson WR (2000) Flexible sequence similarity searching with the FASTA3 program package. Methods Mol Biol 132:185–219PubMedGoogle Scholar
  25. 25.
    Van de Peer Y, De Wachter R (1994) TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Appl Biosci 10:569–570PubMedGoogle Scholar
  26. 26.
    Marmur J (1961) A procedure for the isolation of DNA from microorganisms. J Mol Biol 3:171–173Google Scholar
  27. 27.
    Marmur J, Doty P (1962) Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5:109–118PubMedCrossRefGoogle Scholar
  28. 28.
    De Ley J, Cattoir H, Reynaerts A (1970) The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12:133–142PubMedCrossRefGoogle Scholar
  29. 29.
    Chen WX, Yan GH, Li JL (1988) Numerical taxonomic study of fast-growing soybean rhizobia and proposal that Rhizobium fredii be assigned to Sinorhizobium gen. nov. Int J Syst Evol Microbiol 38:392–397Google Scholar
  30. 30.
    Gao JL, Sun JG, Li Y, Wang ET, Chen WX (1994) Numerical taxonomy and DNA relatedness of tropical rhizobia isolated from Hainan province of China. Int J Syst Evol Microbiol 44:151–158Google Scholar
  31. 31.
    Tan ZY, Wang ET, Peng GX, Zhu ME, Martinez-Romero E, Chen WX (1999) Characterization of bacteria isolated from wild legumes in the north-western regions of China. Int J Syst Bacteriol 49:1457–1469PubMedCrossRefGoogle Scholar
  32. 32.
    Pei Q, Shahir S, Santhana Raj A, Zakaria Z, Ahmad W (2009) Chromium (VI) resistance and removal by Acinetobacter haemolyticus. World J Microbiol Biotechnol 25:1085–1093CrossRefGoogle Scholar
  33. 33.
    Wayne LG, Brenner DJ, Colwell RR, Grimont PAD, Kandler O, Krichevsky MI, Moore LH, Moore WEC, Murray RGE, Stackebrandt E, Starr MP, Truper HG (1987) Report of the Ad Hoc Committee on Reconciliation of Approaches to Bacterial Systematics. Int J Syst Bacteriol 37:463–464CrossRefGoogle Scholar
  34. 34.
    Hallmann J, Quadt-Hallmann A, Mahaffee WF, Kloepper JW (1997) Bacterial endophytes in agricultural crops. Can J Microbiol 43:895–914CrossRefGoogle Scholar
  35. 35.
    Reinhardt ÉL, Ramos PL, Manfio GP, Barbosa HR, Crodowaldo P, Filho CAM (2008) Molecular characterization of nitrogen-fixing bacteria isolated from brazilian agricultural plants at São Paulo State. Braz J Microbiol 39:414–422CrossRefGoogle Scholar
  36. 36.
    Singh R, Mishra R, Jaiswal H, Kumar V, Pandey S, Rao S, Annapurna K (2006) Isolation and identification of natural endophytic Rhizobia from rice (Oryza sativa L.) through rDNA PCR–RFLP and sequence analysis. Curr Microbiol 52:345–349PubMedCrossRefGoogle Scholar
  37. 37.
    Lovell CR, Piceno YM, Quattro JM, Bagwell CE (2000) Molecular analysis of diazotroph diversity in the rhizosphere of the smooth cordgrass, Spartina alterniflora. Appl Environ Microbiol 66:3814–3822PubMedCrossRefGoogle Scholar
  38. 38.
    Zehr JP, Mellon MT, Zani S (1998) New nitrogen-fixing microorganisms detected in oligotrophic oceans by amplification of nitrogenase (nifH) genes. Appl Environ Microbiol 64:3444–3450PubMedGoogle Scholar
  39. 39.
    Singh M, Kushwaha C, Singh R (2009) Studies on endophytic colonization ability of two upland rice endophytes, Rhizobium sp. and Burkholderia sp., using green fluorescent protein reporter. Curr Microbiol 59:240–243PubMedCrossRefGoogle Scholar
  40. 40.
    Dortet L, Legrand P, Soussy C, Cattoir V (2006) Bacterial identification, clinical significance, and antimicrobial susceptibilities of Acinetobacter ursingii and Acinetobacter schindleri, two frequently misidentified opportunistic pathogens. J Clin Microbiol 44:4471–4478PubMedCrossRefGoogle Scholar
  41. 41.
    Nemec A, Musilek M, Maixnerova M, De Baere T, van der Reijden TJ, Vaneechoutte M, Dijkshoorn L (2009) Acinetobacter beijerinckii sp. nov. and Acinetobacter gyllenbergii sp. nov., haemolytic organisms isolated from humans. Int J Syst Evol Microbiol 59:118–124Google Scholar
  42. 42.
    Nemec A, Musilek M, Sedo O, De Baere T, Maixnerova M, van der Reijden TJ, Zdrahal Z, Vaneechoutte M, Dijkshoorn L (2010) Acinetobacter bereziniae sp. nov. and Acinetobacter guillouiae sp. nov., to accommodate Acinetobacter genomic species 10 and 11, respectively. Int J Syst Evol Microbiol 60:896–903PubMedCrossRefGoogle Scholar
  43. 43.
    Towner KJ, Bergogne-Berezin E, Fewson CA (1991) The biology of Acinetobacter. Taxonomy. Plenum, New YorkGoogle Scholar
  44. 44.
    Mak NK, Mok YK, Chui VW, Wong MH (1990) Removal of lead from aqueous solution by Acinetobacter calcoaceticus. Biomed Environ Sci 3:202–210PubMedGoogle Scholar
  45. 45.
    Xie S, Liu J, Li L, Qiao C (2009) Biodegradation of malathion by Acinetobacter johnsonii MA19 and optimization of cometabolism substrates. J Environ Sci 21:76–82CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Hassan Javed Chaudhary
    • 1
  • Guixiang Peng
    • 2
  • Mei Hu
    • 3
  • Yumei He
    • 1
  • Lijuan Yang
    • 1
  • Yan Luo
    • 1
  • Zhiyuan Tan
    • 1
  1. 1.Provincial Key Laboratory of Plant Molecular Breeding, College of AgricultureSouth China Agricultural UniversityGuangzhouChina
  2. 2.College of Resources and EnvironmentSouth China Agricultural UniversityGuangzhouChina
  3. 3.Agro-Environment Protection InstituteMinistry of AgricultureTianjinChina

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