Skip to main content
Log in

Endophytic nitrogen-fixing Klebsiella variicola strain DX120E promotes sugarcane growth

Biology and Fertility of Soils Aims and scope Submit manuscript

Abstract

Klebsiella variicola bacteria are found in association with plants. Little is known about their colonization patterns, roles, and mechanisms during association with the plant hosts. Here, we identified a nitrogen-fixing bacterium, DX120E, which was isolated from surface-sterilized roots of the ROC22 sugarcane cultivar, as K. variicola by phylogenetic analyses of its 16S rRNA gene, RNA polymerase β-subunit gene, and DNA gyrase subunit A gene sequences. gfp-tagged DX120E was found to colonize at the roots and aerial parts of micropropagated sugarcane plantlets by fluorescence microscopy and confocal microscopy. DX120E was able to survive in soils and colonize in root epidermal cells, intercellular spaces in root cortices, and leaf mesophyll and vascular tissues. DX120E preferentially colonized at root maturation and elongation zones and entered roots via cracks at the emergence site of lateral roots and at disrupted epidermis. DX120E may penetrate root epidermal cells with the aid of their cellulose-degrading enzymes. 15N isotope dilution assay demonstrated that DX120E was able to fix N2 in association with ROC22 sugarcane plants under gnotobiotic condition. DX120E was also able to promote GT21 cultivar growth and plant uptake of N, P, and K under greenhouse condition. Together, this study for the first time shows that a K. variicola strain is able to colonize in its sugarcane plant hosts, to fix N2 in association with plants, and to promote plant growth.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  • An Q, Kuang B, Mu X, Li JD (1999) Colonization and nitrogenase production by Klebsiella oxytoca SA2 in rice roots. Prog Nat Sci 9:1262–1268

    Google Scholar 

  • An Q, Dong Y, Wang W, Li Y, Li J (2007) Constitutive expression of the nifA gene activates associative nitrogen fixation of Enterobacter gergoviae 57–7, an opportunistic endophytic diazotroph. J Appl Microbiol 103:613–620

    Article  CAS  PubMed  Google Scholar 

  • An Q, Yang X, Feng L, Mao B, Kuang B, Li J (2010) Using orthogonal sectioning with a laser scanning confocal microscopic system to determine endophytic colonization of plant roots by GFP-tagged bacteria. J Chin Electr Microsc Soc 29:354–360

    CAS  Google Scholar 

  • Baldani JI, Baldani VLD (2005) History on the biological nitrogen fixation research in graminaceous plants: special emphasis on the Brazilian experience. Ann Brazil Acad Sci 77:549–579

    Article  CAS  Google Scholar 

  • Bao S-D (2000) Soil and agricultural chemistry analysis, 3rd edn. China Agriculture Press, Beijing, pp 263–271

    Google Scholar 

  • Barret M, Morrissey JP, O'Gara F (2011) Functional genomics analysis of plant growth-promoting rhizobacterial traits involved in rhizosphere competence. Biol Fertil Soils 47:729–743

    Article  CAS  Google Scholar 

  • Bashan Y, Kamnev AA, de-Bashan LE (2013) Tricalcium phosphate is inappropriate as a universal selection factor for isolating and testing phosphate-solubilizing bacteria that enhance plant growth: a proposal for an alternative procedure. Biol Fetil Soils 49:465–479

    Article  CAS  Google Scholar 

  • Compant S, Clément C, Sessitsch A (2010) Plant growth-promoting bacteria in the rhizo- and endosphere of plants: their role, colonization, mechanisms involved and prospects for utilization. Soil Biol Biochem 42:669–678

    Article  CAS  Google Scholar 

  • de Santi Ferrara FI, Oliveira ZM, Gonzales HHS, Floh EIS, Barbosa HR (2012) Endophytic and rhizospheric enterobacteria isolated from sugar cane have different potentials for producing plant growth-promoting substances. Plant Soil 353:409–417

    Article  CAS  Google Scholar 

  • Döbereiner J (1992) History and new perspectives of diazotrophs in association with non-leguminous plants. Symbiosis 13:1–13

    Google Scholar 

  • Fouts DE, Tyler HL, DeBoy RT, Daugherty S, Ren Q, Badger JH, Durkin AS, Huot H, Shrivastava S, Kothari S, Dodson RJ, Mohamoud Y, Khouri H, Roesch LFW, Krogfelt KA, Struve C, Triplett EW, Methé BA (2008) Complete genome sequence of the N2-fixing broad host range endophyte Klebsiella pneumoniae 342 and virulence predictions verified in mice. PLoS Genet 4(7):e1000141

    Article  PubMed Central  PubMed  Google Scholar 

  • Govindarajan M, Kwon S-W, Weon H-Y (2007) Isolation, molecular characterization and growth-promoting activities of endophytic sugarcane diazotroph Klebsiella sp. GR9. World J Microbiol Biotechnol 23:997–1006

    Article  CAS  Google Scholar 

  • Haahtela K, Laakso T, Korhonen TK (1986) Associative nitrogen fixation by Klebsiella spp.: adhesion sites and inoculation effects on grass roots. Appl Environ Microbiol 52:1074–1079

    CAS  PubMed Central  PubMed  Google Scholar 

  • Iniguez AL, Dong Y, Triplett EW (2004) Nitrogen fixation in wheat provided by Klebsiella pneumonia 342. Mol Plant Microbe Interact 17:1078–1085

    Article  CAS  PubMed  Google Scholar 

  • James EK (2000) Nitrogen fixation in endophytic and associative symbiosis. Field Crops Res 65:197–209

    Article  Google Scholar 

  • James EK, Reis VM, Olivares FL, Boiardi JI, Döbereiner J (1994) Infection of sugarcane by the nitrogen-fixing bacterium Acetobacter diazotrophicus. J Exp Bot 45:757–766

    Article  CAS  Google Scholar 

  • James EK, Gyaneshwar P, Mathan N, Barraquio WL, Reddy PM, Iannetta PPM, Olivares FL, Ladha JK (2002) Infection and colonization of rice seedlings by the plant growth-promoting bacterium Herbaspirillum seropedicae Z67. Mol Microbe Plant Interact 15:894–906

    Article  CAS  Google Scholar 

  • Kovtunovych G, Lar O, Kamalova S, Kordyum V, Kleiner D, Kozyrovska N (1999) Correlation between pectate lyase activity and ability of diazotrophic Klebsiella oxytoca VN13 to penetrate into plant tissues. Plant Soil 215:1–6

    Article  CAS  Google Scholar 

  • Li Y-R (2010) Current knowledge of sugarcane. Chinese Agriculture Publishing, Beijing

    Google Scholar 

  • Lima E, Boddey RM, Döbereiner J (1987) Quantification of biological nitrogen fixation associated with sugar cane using a 15N-aided nitrogen balance. Soil Biol Biochem 19:165–170

    Article  CAS  Google Scholar 

  • Lin L, Guo W, Xing Y, Zhang X, Li Z, Hu C, Li S, Li Y, An Q (2012a) The actinobacterium Microbacterium sp.16SH accepts pBBR1-based pPROBE vectors, forms biofilms, invades roots, and fixes N2 associated with micropropagated sugarcane plants. Appl Microbiol Biotechnol 93:1185–1195

    Article  CAS  PubMed  Google Scholar 

  • Lin L, Li Z, Hu C, Zhang X, Chang S, Yang L, Li Y, An Q (2012b) Plant growth-promoting nitrogen-fixing enterobacteria are in association with sugarcane plants growing in Guangxi, China. Microbes Environ 27:391–398

    Article  PubMed  Google Scholar 

  • Lindow SE, Brandl MT (2003) Microbiology of the phyllosphere. Appl Environ Microbiol 69:1875–1883

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Luo T, Ouyang X, Yang L, Li Y (2010) Effect of nitrogen-fixing bacteria inoculation on biological nitrogen fixation in sugarcane by 15N isotope dilution technique. J Nucl Agric Sci 24:1026–1031

    Google Scholar 

  • Magnani GS, Didonet CM, Cruz LM, Picheth CF, Pedrosa FO, Souza EM (2010) Diversity of endophytic bacteria in Brazilian sugarcane. Genet Mol Res 9:250–258

    Article  CAS  PubMed  Google Scholar 

  • Mehnaz S, Baig DN, Lazarovits G (2010) Genetic and phenotypic diversity of plant growth promoting rhizobacteria isolated from sugarcane plants growing in Pakistan. J Microbiol Biotechnol 20:1614–1623

    Article  CAS  PubMed  Google Scholar 

  • Mirza MS, Ahmad W, Latif F, Haurat J, Bally R, Normand P, Malik KA (2001) Isolation, partial characterization, and the effect of plant growth-promoting bacteria (PGPB) on micropropagated sugarcane in vitro. Plant Soil 237:47–54

    Article  CAS  Google Scholar 

  • Oliveira ALM, Stoffels M, Schmid M, Reis VM, Baldani JI, Hartmann A (2009) Colonization of sugarcane plantlets by mixed inoculations with diazotrophic bacteria. Eur J Soil Biol 45:106–113

    Article  CAS  Google Scholar 

  • Pinto-Tomás AA, Anderson MA, Suen G, Stevenson DM, Chu FS, Cleland WW, Weimer PJ, Currie CR (2009) Symbiotic nitrogen fixation in the fungus gardens of leaf-cutter ants. Science 326:1120–1123

    Article  PubMed  Google Scholar 

  • Podschun R, Ullmann U (1998) Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 11:589–603

    CAS  PubMed Central  PubMed  Google Scholar 

  • Quecine MC, Araújo WL, Rossetto PB, Ferreira A, Tsui S, Lacava PT, Mondin M, Azevedo JL, Pizzirani-Kleiner AA (2012) Sugarcane growth promotion by the endophytic bacterium Pantoea agglomerans 33.1. Appl Environ Microbiol 78:7511–7518

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Reis VM, Olivares FL, Oliveira ALM, dos Reis FB, Baldani JI Jr, Döbereiner J (1999) Technical approaches to inoculate micropropagated sugarcane plants with Acetobacter diazotrophicus. Plant Soil 206:205–211

    Article  Google Scholar 

  • Rosenblueth M, Martínez-Romero E (2006) Bacterial endophytes and their interactions with hosts. Mol Plant Microbe Interact 19:827–837

    Article  CAS  PubMed  Google Scholar 

  • Rosenblueth M, Martínez L, Silva J, Martínez-Romero E (2004) Klebsiella variicola, a novel species with clinical and plant-associated isolates. System Appl Microbiol 27:27–35

    Article  CAS  Google Scholar 

  • Santi C, Bogusz D, Franche C (2013) Biological nitrogen fixation in non-legume plants. Ann Bot 111:743–767

    Article  CAS  PubMed  Google Scholar 

  • 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 194:25–36

    Article  CAS  Google Scholar 

  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tanaka K, Shimizu T, Zakria M, Njoloma J, Saeki Y, Sakai M, Yamakawa T, Minamisawa K, Akao S (2006) Incorporation of a DNA sequence encoding green fluorescent protein (GFP) into endophytic diazotroph from sugarcane and sweet potato and the colonizing ability of these bacteria in Brassica oleracea. Microbes Environ 21:122–128

    Article  Google Scholar 

  • Taulé C, Mareque C, Barlocco C, Hackembruch F, Reis VM, Sicardi M, Battistoni F (2012) The contribution of nitrogen fixation to sugarcane (Saccharum officinarum L.), and the identification and characterization of part of the associated diazotrophic bacterial community. Plant Soil 356:35–49

    Article  Google Scholar 

  • Urquiaga S, Cruz KHS, Boddey RM (1992) Contribution of nitrogen fixation to sugar cane: nitrogen-15 and nitrogen-balance estimates. Soil Sci Soc Am J 56:105–114

    Article  Google Scholar 

  • Urquiaga S, Xavier RP, de Morais RF et al (2012) Evidence from field nitrogen balance and 15N natural abundance data for the contribution of biological N2 fixation to Brazilian sugarcane varieties. Plant Soil 356:5–21

    Article  CAS  Google Scholar 

  • Wang LW, Li YR, He WZ, Xian W, Liang J, Tan YM (2007) The detection of activity for endophytic nitrogen fixing bacteria in sugarcane (Saccharum officinarum L.) by stem apical culture seedlings. Plant Physiol Commun 43:65–68

    CAS  Google Scholar 

  • Webster G, Gough C, Vasse J, Batchelor CA, O'Callaghan KJ, Kothari SL, Davey MR, Dénarié J, Cocking EC (1997) Interactions of rhizobia with rice and wheat. Plant Soil 194:115–122

    Article  CAS  Google Scholar 

  • Zakria M, Udonishi K, Ogawa T, Yamamoto A, Saeki Y, Akao S (2008a) Influence of inoculation technique on the endophytic colonization of rice by Pantoea sp. isolated from sweet potato and by Enterobacter sp. isolated from sugarcane. Soil Sci Plant Nutr 54:224–236

    Article  Google Scholar 

  • Zakria M, Udonishi K, Saeki Y, Yamamoto A, Akao S (2008b) Infection, multiplication and evaluation of the nitrogen-fixing ability of Herbaspirillum sp. strain B501gfp1 in sugarcane stems inoculated by the vacuum infiltration method. Microbes Environ 23:128–133

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Dr. Duli Zhao for reviewing the manuscript. This study was supported by the National Natural Science Foundation of China (31171504 and 31101122), Natural Science Foundation of Guangxi Provence (2011GXNSFF018002 and 2011GXNSFA018076), Guangxi Key Laboratory of Sugarcane Genetic Improvement (13-A-03-07), Guangxi Key Laboratory Construction Program (13-051-29), and Science and Technology Development Foundation of Guangxi Academy of Agricultural Sciences (2011YT01 and 2012YZ09).

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Li-Tao Yang, Yang-Rui Li or Qianli An.

Additional information

C.-Y. Wei and L. Lin contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOC 10319 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wei, CY., Lin, L., Luo, LJ. et al. Endophytic nitrogen-fixing Klebsiella variicola strain DX120E promotes sugarcane growth. Biol Fertil Soils 50, 657–666 (2014). https://doi.org/10.1007/s00374-013-0878-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00374-013-0878-3

Keywords

Navigation