Microbial Ecology

, Volume 52, Issue 3, pp 436–443 | Cite as

Endophytic Occupation of Root Nodules and Roots of Melilotus dentatus by Agrobacterium tumefaciens

  • Ling Ling Wang
  • En Tao Wang
  • Jie Liu
  • Ying Li
  • Wen Xin Chen


Agrobacterium strains have been frequently isolated from the root nodules of different legumes. Various possible mechanisms have been proposed to explain the existence of these bacteria in nodules, but there is no sufficient experimental evidence to support the estimations. In this work, we proved that the Agrobacterium strain CCBAU 81181, which was originally isolated from the root nodules of Onobrychis viciaefolia, and a symbiotic strain of Sinorhizobium meliloti CCBAU 10062 could coinhabit the root nodules of Melilotus dentatus. Analyses were performed by using a fluorescence marker, reisolation of bacteria from nodules, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of whole cellular proteins, and polymerase chain reaction amplification of symbiotic genes. The inoculation of A. tumefaciens CCBAU 81181 did not affect the growth and nodulation of plants. CCBAU 81181 and 24 other Agrobacterium strains isolated from nodules were incapable of nodulating on their original or alternative host and 22 strains of these strains were endophytes in the roots and stems of their hosts. Also, the tumor-inducing A. tumefaciens strains IAM 13129T and C58 were found capable of entering the roots of Glycyrrhiza pallidiflora, but did not cause pathogenic symptoms. With these results, we conclude that A. tumefaciens strains could be endophytic bacteria in the roots, stems, and root nodules. This finding partially explains why Agrobacterium strains were frequently isolated from the surface-sterilized nodules.


Rhizobium Root Nodule Agrobacterium Endophytic Bacterium Agrobacterium Strain 
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 the State Key Basic Research and Development Plan of China (2006CB100206, 2004DKA30560-1 and 2001CB108905). E.T.W. thanks CONACyT, Mexico, for their financial support (grant number 34123-N).


  1. 1.
    Alazard, D, Duhoux, E (1990) Development of stem nodules in a tropical forage legume Aeschynomene afraspera. J Exp Bot 41: 1199–1206Google Scholar
  2. 2.
    Anyango, B, Wilson, KJ, Beynon, JL, Giller KE (1995) Diversity of rhizobia nodulating Phaseolus vulgaris L. in two Kenyan soils of contrasting pHs. Appl Environ Microbiol 61: 4016–4021PubMedGoogle Scholar
  3. 3.
    Bala, A, Giller, KE (2001) Symbiotic specificity of tropical tree rhizobia for host legumes. New Phytol 149: 495–507CrossRefGoogle Scholar
  4. 4.
    Boogerd, FC, van Rossum, D (1997) Nodulation of groundnut by Bradyrhizobium: a simple infection process by crack entry. FEMS Microbiol Rev 21: 5–27CrossRefGoogle Scholar
  5. 5.
    Chen, LS, Figueredo, A, Pedrosa, FO, Hungria, M (2000) Genetic characterization of soybean rhizobia in Paraguay. Appl Environ Microbiol 66: 5099–5103PubMedCrossRefGoogle Scholar
  6. 6.
    Chen, WM, James, EK, Prescott, AR, Kierans, M, Sprent, JI (2003) Nodulation of Mimosa spp. by the β-Proteobacterium Ralstonia taiwanensis. Mol Plant–Microb Interact 16: 1051–1061Google Scholar
  7. 7.
    De Lajudie, P, Willems, A, Nick, G, Mohamed, TS, Torck, U, Filai-Maltouf, A, Kersters, K, Dreyfus, B, Lindström, K, Gillis, M (1999) Agrobacterium bv. 1 strains isolated from nodules of tropical legumes. Syst Appl Microbiol 22: 119–132Google Scholar
  8. 8.
    De Ley, J, Cattoir, H, Reynaerts, A (1970) The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12: 133–142PubMedCrossRefGoogle Scholar
  9. 9.
    Gao, JL, Turner, SL, Kan, FL, Wang, ET, Tan, ZY, Qiu, YH, Gu, J, Terefework, Z, Young, JP, Lindström, K, Chen, WX (2004) Mesorhizobium septentrionale sp. nov. and Mesorhizobium temperatum sp. nov., isolated from Astragalus adsurgens growing in the northern regions of China. Int J Syst Evol Microbiol 54: 2003–2012PubMedCrossRefGoogle Scholar
  10. 10.
    Hallmann, J, Quadt-Hallmann, A, Mahaffee, WF, Kloepper, JW (1997) Bacterial endophytes in agricultural crops. Can J Microbiol 43: 895–914Google Scholar
  11. 11.
    Han, SZ, Wang, ET, Chen, WX (2005) Diverse bacteria isolated from root nodules of Phaseolus vulgaris and species within the genera Campylotropis and Cassia grown in China. Syst Appl Microbiol 28: 265–276PubMedCrossRefGoogle Scholar
  12. 12.
    Haukka, K, Lindström, K, Young, JPW (1998) Three phylogenetic groups of nodA and nifH genes in Sinorhizobium and Mesorhizobium isolates from leguminous trees growing in Africa and Latin America. Appl Environ Microbiol 64: 419–426PubMedGoogle Scholar
  13. 13.
    Hiroyuki, S, Hiroyuki, I, Izumi, M (1995) PCR detection of Ti and Ri plasmids from phytopathogenic Agrobacterium strains. Appl Environ Microbiol 61: 828–831Google Scholar
  14. 14.
    Hurek, T, Wagner, B, Reihold-Hurek, B (1997) Identification of N2-fixing plant- and fungus-associated Azoarcus species by PCR-based genomic fingerprints. Appl Environ Microbiol 63: 4331–4339PubMedGoogle Scholar
  15. 15.
    Hynes, MF, McGregor, NF (1990) Two plasmids other than the nodulation plasmid are necessary for formation of nitrogen-fixing nodules by Rhizobium leguminosarum. Mol Microbiol 4: 567–574PubMedCrossRefGoogle Scholar
  16. 16.
    Imshenetskii, AA, Pariiskaia, AN, Gorelova, OP (1976) The presence of Agrobacterium tumefaciens in lucerne root nodules. Mikrobiol 45: 561–563 (in Russian)Google Scholar
  17. 17.
    Khbaya, B, Neyra, M, Normand, P, Zerhari, K, Filali-Maltouf, A (1998) Genetic diversity and phylogeny of rhizobia that nodulate Acacia spp. in Morocco assessed by analysis of rRNA genes. Appl Environ Microbiol 64: 4912–4917PubMedGoogle Scholar
  18. 18.
    Liu, J, Wang, ET, Chen, WX (2005) Diverse rhizobia associated with woody legumes Wisteria sinensis, Cercis racemosa and Amorpha fruticosa grown in the temperate zone of China. Syst Appl Microbiol 28: 465–477PubMedCrossRefGoogle Scholar
  19. 19.
    Marmur, J (1961) A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3: 208–218CrossRefGoogle Scholar
  20. 20.
    Mhamdi, R, Lagrerre, G, Aouani, ME, Mars, M, Amarger, N (2002) Different species and symbiotic genotypes of field rhizobia can nodulate Phaseolus vulgaris in Tunisian soils. FEMS Microbiol Ecol 41: 77–84CrossRefPubMedGoogle Scholar
  21. 21.
    Mhamdi, R, Mrabet, M, Laguerre, G, Tiwari, R, Aouani, ME (2005) Colonization of Phaseolus vulgaris nodules by Agrobacterium-like strains. Can J Microbiol 51: 105–111PubMedCrossRefGoogle Scholar
  22. 22.
    Odee, DW, Haukka, K, McInroy, SG, Sprent, JI, Sutherland, JM, Young, JPW (2002) Genetic and symbiotic characterization of rhizobia isolated from tree and herbaceous legumes grown in soils from ecologically diverse sites in Kenya. Soil Biol Biochem 34: 801–811CrossRefGoogle Scholar
  23. 23.
    Sohail, H, Sumera, Y, Kauser, AM, Yusuf, Z, Fauzia, YH (2004) Rhizobium, Bradyrhizobium and Agrobacterium strains isolated from cultivated legumes. Biol Fertil Soils 39: 179–185CrossRefGoogle Scholar
  24. 24.
    Stuurman, N, Bras, CP, Schlaman, H.R.M., Wijfjes, A.H.M., Bloemberg, G, Spaink, HP (2000) Use of green fluorescent protein color variants expressed on stable broad-host-range vectors to visualize rhizobia interacting with plants. Mol Plant–Microb Interact 13: 1163–1169Google Scholar
  25. 25.
    Tan, ZY, Wang, ET, Peng, GX, Zhu, ME, Martinez-Romero, E, Chen, WX (1999) Characterization of bacteria isolated from wild legumes in the northwestern region of China. Int J Syst Bacteriol 49: 1457–1469PubMedGoogle Scholar
  26. 26.
    Tan, ZY, Xu, XD, Wang, ET, Gao, JL, Martínez-Romero, E, Chen, WX (1997) Phylogenetic and genetic relationships of Mesorhizobium tianshanense and related rhizobia. Int J Syst Bacteriol 47: 874–879PubMedCrossRefGoogle Scholar
  27. 27.
    Vega-Hernández, MC, Pérez-Galdona, R, Dazzo, FB, Jarabo-Lorenzo, A., Alfayate, MC, León-Barrios, M (2001) Novel infection process in the indeterminate root nodule symbiosis between Chamaecytisus proliferus (tagasaste) and Bradyrhizobium sp. New Phytol 150: 707–721CrossRefGoogle Scholar
  28. 28.
    Vincent, JM (1970) A Manual for the Practical Study of Root Nodule Bacteria. Blackwell Scientific, OxfordGoogle Scholar
  29. 29.
    Wolde-Meskel, E, Terefework, Z, Frostegård, A, Lindström, K (2005) Genetic diversity and phylogeny of rhizobia isolated from agroforestry legume species in southern Ethiopia. Int J Syst Evol Microbiol 55: 1439–1452PubMedCrossRefGoogle Scholar
  30. 30.
    Yanagi, M, Yamasato, K (1993) Phylogenetic analysis of the family Rhizobiaceae and related bacteria by sequencing of 16S rRNA gene using PCR and DNA sequencer. FEMS Microbiol Lett 107: 115–120PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • Ling Ling Wang
    • 1
    • 2
  • En Tao Wang
    • 1
    • 3
  • Jie Liu
    • 1
    • 4
  • Ying Li
    • 1
  • Wen Xin Chen
    • 1
  1. 1.Key Laboratory of Agro-Microbial Resource and Application, Ministry of Agriculture/College of Biological SciencesChina Agricultural UniversityBeijingChina
  2. 2.College of Life SciencesSouth China Agricultural UniversityGuangzhouChina
  3. 3.Departamento de Microbiología, Escuela Nacional de Ciencias BiológicasInstituto Politécnico NacionalMexicoMexico
  4. 4.Department of Biological and Pharmaceutical Engineering, College of Chemical EngineeringQingdao University of Science and TechnologyQingdaoChina

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