Divulging diazotrophic bacterial community structure in Kuwait desert ecosystems and their N2-fixation potential

Autoři: M. K. Suleiman aff001;  A. M. Quoreshi aff001;  N. R. Bhat aff001;  A. J. Manuvel aff001;  M. T. Sivadasan aff001
Působiště autorů: Desert Agriculture and Ecosystems Program, Environment and Life Sciences Research Center, Kuwait Institute for Scientific Research, Safat, Kuwait aff001
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0220679


Kuwait is a semi-arid region with soils that are relatively nitrogen-poor. Thus, biological nitrogen fixation is an important natural process in which N2-fixing bacteria (diazotrophs) convert atmospheric nitrogen into plant-usable forms such as ammonium and nitrate. Currently, there is limited information on free-living and root-associated nitrogen-fixing bacteria and their potential to fix nitrogen and aid natural plant communities in the Kuwait desert. In this study, free living N2-fixing diazotrophs were enriched and isolated from the rhizosphere soil associated with three native keystone plant species; Rhanterium epapposum, Farsetia aegyptia, and Haloxylon salicornicum. Root-associated bacteria were isolated from the root nodules of Vachellia pachyceras. The result showed that the strains were clustered in five groups represented by class: γ-proteobacteria, and α-proteobacteria; phyla: Actinobacteria being the most dominant, followed by phyla: Firmicutes, and class: β-proteobacteria. This study initially identified 50 nitrogen-fixers by16S rRNA gene sequencing, of which 78% were confirmed to be nitrogen-fixers using the acetylene reduction assay. Among the nitrogen fixers identified, the genus Rhizobium was predominant in the rhizosphere soil of R. epapposum and H. salicornicum, whereas Pseudomonas was predominant in the rhizosphere soil of F. aegyptia, The species Agrobacterium tumefaciens was mainly found to be dominant among the root nodules of V. pachyceras and followed by Cellulomonas, Bacillus, and Pseudomonas genera as root-associated bacteria. The variety of diazotrophs revealed in this study, signifying the enormous importance of free-living and root-associated bacteria in extreme conditions and suggesting potential ecological importance of diazotrophs in arid ecosystem. To our knowledge, this study is the first to use culture-based isolation, molecular identification, and evaluation of N2-fixing ability to detail diazotroph diversity in Kuwaiti desert soils.

Klíčová slova:

Agrobacterium tumefaciens – Bacteria – Deserts – Diazo compounds – Nitrogen fixation – Rhizosphere – Rhizobium – Root nodules


1. Köberl J, Prettenthaler F, Bird DN. Modelling climate change impacts on tourism demand: A comparative study from Sardinia (Italy) and Cap Bon (Tunisia). Sci Total environ. 2016; 543: 1039–1053. doi: 10.1016/j.scitotenv.2015.03.099 25891683

2. Hsu SF, Buckley DH. Evidence for the functional significance of diazotroph community structure in soil. The ISME Journal. 2009; 3: 124–136. doi: 10.1038/ismej.2008.82 18769458

3. Martínez-Hidalgo P, Hirsch AM. The nodule microbiome: N2-fixing rhizobia do not live alone. Phytobiomes. 2017; 1: 70–82. https://doi.org/10.1094/PBIOMES-12-16-0019-RVW.

4. Gulati A, Sood S, Rahi P, Thakur P, Chauhan S, Chawla I. Diversity Analysis of Diazotrophic Bacteria Associated with the Roots of Tea (Camellia sinensis (L.) O. Kuntze). J Microbiol Biotechn. 2011; 21(6): 545–55. doi: 10.4014/jmb.1012.12022

5. Simon HM, Smith MW, Herfort L. Metagenomic insights into particles and their associated microbiota in a coastal margin ecosystem. Front Microbiol. 2014; 5: 466. doi: 10.3389/fmicb.2014.00466 25250019

6. Pajares S, Bohannan BJM. Ecology of nitrogen fixing, nitrifying, and denitrifying microorganisms in tropical forest soils. Front Microbiol. 2016; 7: 1045. doi: 10.3389/fmicb.2016.01045 27468277

7. Dixon R, Kahn D. Genetic regulation of biological nitrogen fixation. Nat Rev Microbiol. 2004; 2(8): 621–31. doi: 10.1038/nrmicro954 15263897

8. Ladha JK, Reddy PM. 2000. Steps towards nitrogen fixation in rice: Quest for nitrogen fixation in rice. Proceedings of the 3rd Working Group Meeting on Assessing Opportunities of Nitrogen Fixation in Rice, 2000; August 9–12, 1999, Los Banos, Phillipines, pp: 33–46.

9. Rondon MA, Lehmann J, Ramírez J, Hurtado M. Biological nitrogen fixation by common beans (Phaseolus vulgaris L.) increases with bio-char additions. Biol Fertil Soils. 2007; 43: 699–708. doi: 10.1007/s00374-006-0152-z

10. de Souza R, Beneduzi A, Ambrosini A, Costa PB, Meyer J, Vargas LK. et al. The effect of plant growth-promoting rhizobacteria on the growth of rice (Oryza sativa L.) cropped in southern Brazilian fields. Plant Soil. 2013; 366: 585–603. doi: 10.1007/s11104-012-1430-1

11. Estrada GA, Baldani VID, de Oliveria DM, Urquiaga S, Baldani JI. Selection of phosphate solubilizing diazotrophic Herbaspirillum and Burkholderia strains and their effect on rice crop yield and nutrient uptake. Plant Soil. 2013; 369: 115. https://doi.org/10.1007/s11104-012-1550-7.

12. Köberl M, Müller H, Ramadan EM, Berg G. Desert farming benefits from microbial potential in arid soils and promotes diversity and plant health. PLoS ONE. 2011; 6(9): e24452. doi: 10.1371/journal.pone.0024452 21912695

13. Abdal MS, Suleiman MK. Soil conservation as a concept to improve Kuwait environment. Journal Archives of Nature Conservation and Landscape Research. 2002; 41(3–4): 125–130. https://doi.org/10.1080/0003930022000043419.

14. Khosravia M, Boulangera RW, Wilson DW, Olgun CG, Tamura S. et al. Dynamic centrifuge tests of structures with shallow foundations on soft clay reinforced by soil-cement grids. Solis and Foundations. 2017; 57(4): 501–513. https://doi.org/10.1016/j.sandf.2017.06.002.

15. El-Sheikh MA, Saleh H, El-Quosy DE,.Mahmoud AA. Improving water quality in polluated drains with free water surface constructed wetlands. Ecol Eng. 2010; 36(10): 1478–1484. https://doi.org/10.1016/j.ecoleng.2010.06.030.

16. Eida AA, Ziegler M, Lafi FF, Michell CT, Voolstra CR, Hirt H, et al. Desert plant bacteria reveal host influence and beneficial plant growth properties. PLoS ONE. 2018; 13(12): e0208223. doi: 10.1371/journal.pone.0208223 30540793

17. Ortiz N, Armada E, Duque E, Roldán A, Azcón R. Contribution of arbuscular mycorrhizal fungi and/or bacteria to enhancing plant drought tolerance under natural soil conditions: effectiveness of autochthonous or allochthonous strains. J plant physiol. 2015; 174: 87–96. doi: 10.1016/j.jplph.2014.08.019 25462971

18. Friesen ML, Porter SS, Stark SC, von Wettberg EJ, Sachs JL, Martinez-Romero E. Microbially mediated plant functional traits. Annu Rev Ecol Evol S. 2011; 42: 23–46. https://doi.org/10.1146/annurev-ecolsys-102710-145039.

19. de Zelicourt A, Al-Yousif M, Hirt H. Rhizosphere microbes as essential partners for plant stress tolerance. Mol plant. 2013; 6(2): 242–5. doi: 10.1093/mp/sst028 23475999

20. Mengual C, Schoebitz M, Azcón R, Roldán A. Microbial inoculants and organic amendment improves plant establishment and soil rehabilitation under semiarid conditions. J environ manage. 2014; 134: 1–7. doi: 10.1016/j.jenvman.2014.01.008 24463051

21. Kieft T, Skujinš J. Soil microbiology in reclamation of arid and semiarid lands. In: Skujins J, editor. Semiarid lands and deserts: soil resource and reclamation. New York: Marcel Dekker; 1991. p. 209–56.

22. Eckford R, Cook FD, Saul D, Aislabie J, Foght J. Free-living heterotrophic nitrogen-fixing bacteria isolated from fuel-contaminated Antarctic soils. Appl Environ Microbiol. 2002; 68(10): 5181–5185. doi: 10.1128/AEM.68.10.5181-5185.2002 12324373

23. Küçük Ç, M. Kivanç M, Kinaci E. Characterization of Rhizobium sp. isolated from bean. Turk J Biol. 2006; 30: 127–132.

24. Gothwal RK, Nigam VK, Mohan MK, Sasmal D, Ghosh P. Screening of nitrogen fixers from rhizospheric bacterial isolates associated with important desert plants. Appl Ecolo Env Res. 2007; 6(2): 101–109.

25. Muyzer G, de Waal EC, Uitterlinden AG. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain-amplified gene coding for 16S rRNA. Appl Environ Microbiol. 1993; 59: 695–700. 7683183

26. Lane DJ, Pace B, Olsen GJ, Stahl DA, Sogin ML, Pace NR. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci USA. 1985; 82:6955–6959. doi: 10.1073/pnas.82.20.6955 2413450

27. Hall T. Biological sequence alignment editor for Win95/98//NT/2K/XP. Available from: http://www.mbio.ncsu.edu/BioEdit/bioedit.html

28. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. 1999; 41: 95–98.

29. BLAST: The Basic Local Alignment Search Tool. Available from: http://www.ncbi.nlm.nih.gov/BLAST/.

30. Thompson K, Bakker JP, Bekker RM. Soil seed banks of north west Europe: methodology, density and longevity. 1997. Cambridge: Cambridge University Press.

31. MEGA: Molecular Evolutionary Genetics Analysis. Available from: http://www.megasoftware.net.

32. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016; 33(7): 1870–1874. doi: 10.1093/molbev/msw054 27004904

33. Saitou N, Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Bio Evol. 1987; 4: 406–425. https://doi.org/10.1093/oxfordjournals.molbev.a040454.

34. Kimura M. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 1980; 16: 111–120. doi: 10.1007/bf01731581 7463489

35. Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution. 1985; 39: 783–791. doi: 10.1111/j.1558-5646.1985.tb00420.x 28561359

36. Ronquist F, Huelsenbeck JP. Mr.Bayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 2003; 19(12): 1572–1574. doi: 10.1093/bioinformatics/btg180 12912839

37. Roper MM, Gupta VVSR. Enhancing non-symbiotic N2 fixation in agriculture. Open Agr J. 2016; 10: 7–27. doi: 10.2174/1874331501610010007

38. Ren M, Zhang Z, Wnag X, Zhou Z, Chen D. et al. Diversity and contributions to nitrogen cycling and carbon fixation of soil salinity shaped microbial communities in Tarim Basin. Front Microbiol. 2018; 9: 431 doi: 10.3389/fmicb.2018.00431 29593680

39. Cary SC, McDonald IR, Barrett JE, Cowan DA. On the rocks: the microbiology of Antarctic dry valley soils. Nat Rev Microbiol. 2010; 8: 129–138. doi: 10.1038/nrmicro2281 20075927

40. Bais HP, Weir TL, Perry LG, Gilroy S, Vivanco JM. The role of root exudates in rhizosphere interactions with plants and other organisms. Annu Rev Plant Biol. 2006; 57: 233–66. doi: 10.1146/annurev.arplant.57.032905.105159 16669762

41. Berg G, Smalla K. Plant species and soil type cooperatively shape the structure and function of microbial communities in the rhizosphere. FEMS Microbiol Ecol. 2009; 68: 1–13. doi: 10.1111/j.1574-6941.2009.00654.x 19243436

42. Bulgarelli D, Rott M, Schlaeppi K, van Themaat EVL, Ahmadinejad N, Assenza F, et al. Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota. Nature. 2012; 488(7409): 91–5. doi: 10.1038/nature11336 22859207

43. Zhan J, Sun Q. Diversity of free-living nitrogen-fixing microorganisms in the rhizosphere and non-rhizosphere of pioneer plants growing on wastelands of copper mine tailings. Microbiol Res. 2012 20;167(3): 157–65. doi: 10.1016/j.micres.2011.05.006 21665448

44. Bulgarelli D, Schlaeppi K, Spaepen S, van Themaat EVL, Schulze-Lefert P. Structure and functions of the bacterial microbiota of plants. Annu Rev Plant Biol. 2013; 64: 807–38. doi: 10.1146/annurev-arplant-050312-120106 23373698

45. Bulgarelli D, Garrido-Oter R, Münch Philipp C, Weiman A, Dröge J, Pan Y, et al. Structure and function of the bacterial root microbiota in wild and domesticated barley. Cell Host & Microbe. 2015; 17(3): 392–403. https://doi.org/10.1016/j.chom.2015.01.011.

46. Lundberg DS, Lebeis SL, Paredes SH, Yourstone S, Gehring J, Malfatti S, et al. Defining the core Arabidopsis thaliana root microbiome. Nature. 2012; 488: 86. doi: 10.1038/nature11237 22859206

47. Yeoh YK, Dennis PG, Paungfoo-Lonhienne C, Weber L, Brackin R, Ragan MA, et al. Evolutionary conservation of a core root microbiome across plant phyla along a tropical soil chronosequence. Nature Communications. 2017; 8. https://doi.org/10.1038/s41467-017-00262-8.

48. Lester ED, Satomi M, Ponce A. Microflora of extreme arid Atacama desert soils. Soil Biol. Biochem. 2007; 39(2): 704–708. https://doi.org/10.1016/j.soilbio.2006.09.020.

49. Fierer N, Strickland MS, Liptzin D, Bradford MA, Cleveland CC. Global patterns in belowground communities. Ecol. Lett. 2009; 12: 1238–1249. https://doi.org/10.1111/j.1461-0248.2009.01360.x

50. Dahal B, NandaKafle G, Perkins L, Brözela VS. Diversity of free-Living nitrogen fixing Streptomyces in soils of the badlands of South Dakota. Microbiological Research. 2017; 195: 31–39. doi: 10.1016/j.micres.2016.11.004 28024524

51. Cleveland CC, Townsend AR, Taylor P, Alvarez-Clare S, Bustamante MMC. et al. Relationships among net promary productivity, nutrients and climate in tropical rain forest: a pan-tropical analysis. Ecol Lett. 2011; 14: 939–947 doi: 10.1111/j.1461-0248.2011.01658.x 21749602

52. Brookshire ENJ, Hedin E, Newbold JD, Sigman DM, Jackson JK. Sustained losses of bioavailable nitrogen from montane tropical forests. Nat Geosci. 2012; 5: 123–126. doi: 10.1038/ngeol372

53. Reed SC, Cleveland CC, Townsend AR. Controls over leaf litter and soil nitrogen fixation in two lowland tropical rain forests. Biotropica. 2007; 39: 585–592. https://doi.org/10.1111/j.1744-7429.2007.00310.x.

54. Cusack DF, Silver W, McDowell WH. Biological nitrogen fixation in two tropical forests: ecosystem level patterns and effects of nitrogen fertilization. Ecosystems. 2009; 12: 1299–315. https://doi.org/10.1007/s10021-009-9290-0

55. Reed SC, Townsend AR, Cleveland CC, Nemergut DR. Microbial community shifts influence patterns in tropical forest nitrogen fixation. Oecologia. 2010; 164(2): 521–31. doi: 10.1007/s00442-010-1649-6 20454976

56. Mirza BS, Potisap C, Nüsslein K, Bohannan BJM, Rodriguesa JLM. Response of Free-Living Nitrogen-Fixing Microorganisms to Land Use Change in the Amazon Rainforest. Appl Environ Microbiol. 2014; 80(1): 281–288. doi: 10.1128/AEM.02362-13 24162570

57. Kifle MH, Laing MD. Effects of Selected Diazotrophs on Maize Growth. Front Plant Sci. 2016; 7: 1429. doi: 10.3389/fpls.2016.01429 27713756

58. Xu J, Kloepper JW, Huang P, McInroy JA, Hu CH. Isolation and characterization of N2‐fixing bacteria from giant reed and switchgrass for plant growth promotion and nutrient uptake J Basic Microbiol. 2018; 58(5): 459–471. doi: 10.1002/jobm.201700535 29473969

59. Koivunen ME, Morisseau C, Horwath WR, Hammock BD. Isolation of a strain of Agrobacterium tumefaciens (Rhizobium radiobacter) utilizing methylene urea (ureaformaldehyde) as nitrogen source. Can. J. Microbiol. 2004; 50: 167–174. doi: 10.1139/w04-001 15105883

60. Velázquez E, Peix A, Zurdo-Pineiro JL, Palomo JL, Mateos PF, et al. The coexistence of symbiosis and pathogenicity-determining genes in Rhizobium rhizogenes strains enables them to reduce nodules and tumors or hairy roots in plats. Mol Plant Microbe Interact. 2005; 18(12): 1325–1332. doi: 10.1094/MPMI-18-1325 16478052

61. Young JM, Kuykendall LD, Martinz-Romero E, Kerr A, Sawada H. A revision of Rhizobium Frank 1889, with an emended description of the genus, and the inclusion of all species of Agrobacterium Conn 1942 and Allorhizobium undicola de Lajudie et al. 1998 as new combinations: Rhizobium radiobacter, R. rhizogenes, R. rubi, R. undicola and R. vitis. Int J Syst Evol Microbiol. 2001; 51: 89–103. doi: 10.1099/00207713-51-1-89 11211278

62. Kanvinde L, Sastry GRK. Agrobacterium tumefaciens is a diazotrophic bacterium. Appl Environ Microbiol. 1990; 56(7): 2087–2092. 16348237

63. My PT, Manucharova NA, Stepanov AL, Pozdyakov LA, Selitskaya OV, Emtsev VT. 2015. Agrobacterium tumefaciens as associative nitrogen-fixing bacteria. Moscow University Soil Science Bulletin. 2015; 70(3): 133–138. https://doi.org/10.3103/S0147687415030047.

Článek vyšel v časopise


2019 Číslo 12
Nejčtenější tento týden