Endophytic nitrogen-fixing Klebsiella variicola strain DX120E promotes sugarcane growth
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.
KeywordsColonization Endophytic bacteria Klebsiella variicola Nitrogen fixation Plant growth-promoting bacteria Sugarcane
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).
- An Q, Kuang B, Mu X, Li JD (1999) Colonization and nitrogenase production by Klebsiella oxytoca SA2 in rice roots. Prog Nat Sci 9:1262–1268Google 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 29:354–360Google Scholar
- Bao S-D (2000) Soil and agricultural chemistry analysis, 3rd edn. China Agriculture Press, Beijing, pp 263–271Google Scholar
- Döbereiner J (1992) History and new perspectives of diazotrophs in association with non-leguminous plants. Symbiosis 13:1–13Google 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):e1000141PubMedCentralPubMedCrossRefGoogle Scholar
- Li Y-R (2010) Current knowledge of sugarcane. Chinese Agriculture Publishing, BeijingGoogle 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 93:1185–1195PubMedCrossRefGoogle 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 24:1026–1031Google 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 21:122–128CrossRefGoogle 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 356:35–49CrossRefGoogle 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 43:65–68Google Scholar