European Journal of Plant Pathology

, Volume 142, Issue 4, pp 701–713 | Cite as

Genetic diversity of tumorigenic bacteria associated with crown gall disease of raspberry in Serbia

  • Nemanja KuzmanovićEmail author
  • Anđelka Prokić
  • Milan Ivanović
  • Nevena Zlatković
  • Katarina Gašić
  • Aleksa Obradović


During the last 3 years, crown gall disease was observed in some young raspberry plantations throughout Serbia, causing considerable economic losses. Based on biochemical and physiological tests, PCR targeting the 23S rRNA gene, and 16S rRNA and recA gene sequence analysis, at least two different species were identified as causal agents of disease. Out of 14 strains isolated from raspberry tumors, 12 were identified as tumorigenic Rhizobium rhizogenes, one belonged to Agrobacterium tumefaciens genomic species G8, while the remaining strain formed a separate phylogenetic lineage within A. tumefaciens species complex, different from all known genomic species. All strains investigated harbored nopaline-type of Ti plasmid and showed identical pathogenic properties by inoculating several test plants. However, they were divided into two genetic groups based on PCR-RFLP analysis of Ti plasmid virA-virB2 region. Furthermore, total of nine unique ERIC-PCR profiles were identified among the strains studied. Although strains of R. rhizogenes exhibited similar ERIC-PCR profiles, they were differentiated into six distinct genetic groups. Based on the fact that some genetic groups were composed of strains originating from different geographic areas, it can be assumed that they have a common origin and were probably disseminated by movement of infected plant material.


Crown gall Raspberry Agrobacterium Rhizobium Ti plasmid 



This research was supported by the project III46008 financed by Ministry of Education, Science and Technological Development, Republic of Serbia, and EU Commission project AREA, No 316004. The authors gratefully acknowledge Enrico Biondi, Gerald V. Minsavage, Jeffrey B. Jones, Joanna Puławska, Sandor Süle and Subrata K. Das for kindly providing bacterial strains. We also thank Joanna Puławska for critical reading of the manuscript and helpful comments.


  1. Alippi, A., López, A., & Balatti, P. (2012). Diversity among agrobacteria isolated from diseased plants of blueberry (Vaccinium corymbosum) in Argentina. European Journal of Plant Pathology, 134(2), 415–430. doi: 10.1007/s10658-012-0001-x.CrossRefGoogle Scholar
  2. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., et al. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25(17), 3389–3402.PubMedCentralPubMedCrossRefGoogle Scholar
  3. Arsenijević, M. (1989). Agrobacterium tumefaciens parazit pitome kupine (Rubus sp.). Paper presented at the Kongres mikrobiologov Jugoslavije (VI), Maribor, 11–15.09.Google Scholar
  4. Bini, F., Kuczmog, A., Putnoky, P., Otten, L., Bazzi, C., Burr, T. J., et al. (2008). Novel pathogen-specific primers for the detection of Agrobacterium vitis and Agrobacterium tumefaciens. Vitis, 47, 181–189.Google Scholar
  5. Bouzar, H., & Moore, L. W. (1987). Isolation of different Agrobacterium biovars from a natural oak savanna and tallgrass prairie. Applied and Environmental Microbiology, 53(4), 717–721.PubMedCentralPubMedGoogle Scholar
  6. Burr, T. J., Reid, C. L., Katz, B. H., Tagliati, M. E., Bazzi, C., & Breth, D. I. (1993). Failure of Agrobacterium radiobacter strain K-84 to control crown gall on raspberry. HortScience, 28(10), 1017–1019.Google Scholar
  7. Conn, H. J. (1942). Validity of the Genus Alcaligenes. Journal of Bacteriology, 44(3), 353–360.PubMedCentralPubMedGoogle Scholar
  8. Costechareyre, D., Rhouma, A., Lavire, C., Portier, P., Chapulliot, D., Bertolla, F., et al. (2010). Rapid and efficient identification of Agrobacterium species by recA allele analysis: Agrobacterium recA diversity. Microbial Ecology, 60(4), 862–872. doi: 10.1007/s00248-010-9685-7.PubMedCrossRefGoogle Scholar
  9. Cubero, J., Lastra, B., Salcedo, C. I., Piquer, J., & Lopez, M. M. (2006). Systemic movement of Agrobacterium tumefaciens in several plant species. Journal of Applied Microbiology, 101(2), 412–421. doi: 10.1111/j.1365-2672.2006.02938.x.PubMedCrossRefGoogle Scholar
  10. De Ley, J. (1974). Phylogeny of procaryotes. Taxon, 23, 291–300.CrossRefGoogle Scholar
  11. De Ley, J., Tijtgat, R., De Smedt, J., & Michiels, M. (1973). Thermal stability of DNA: DNA hybrids within the genus Agrobacterium. Journal of General Microbiology, 78(2), 241–252. doi: 10.1099/00221287-78-2-241.CrossRefGoogle Scholar
  12. du Plessis, H. J., van Vuuren, H. J. J., & Hattingh, M. J. (1984). Biotypes and phenotypic groups of strains of Agrobacterium in South Africa. Phytopathology, 74, 524–529.CrossRefGoogle Scholar
  13. Farrand, S. K., van Berkum, P. B., & Oger, P. (2003). Agrobacterium is a definable genus of the family Rhizobiaceae. International Journal of Systematic and Evolutionary Microbiology, 53(5), 1681–1687. doi: 10.1099/ijs. 0.02445-0.PubMedCrossRefGoogle Scholar
  14. Haas, J. H., Moore, L. W., Ream, W., & Manulis, S. (1995). Universal PCR primers for detection of phytopathogenic Agrobacterium strains. Applied and Environmental Microbiology, 61(8), 2879–2884.PubMedCentralPubMedGoogle Scholar
  15. Hildebrand, E. M. (1940). Cane gall of brambles caused by Phytomonas rubi n. sp. Journal of Agricultural Research, 61, 685–696.Google Scholar
  16. Hobolth, L. A. (1973). Agrobacterium radiobacter var. tumefaciens biotype 2 found on Rubus insularis in Denmark. Botanisk-Tiddskrif, 68, 160–164.Google Scholar
  17. Holmes, B., & Roberts, P. (1981). The classification, identification and nomenclature of agrobacteria. Journal of Applied Bacteriology, 50(3), 443–467. doi: 10.1111/j.1365-2672.1981.tb04248.x.CrossRefGoogle Scholar
  18. Jones, J. B., & Raju, B. C. (1988). Systemic Movement of Agrobacterium tumefaciens in symptomless stem tissue of Chrysanthemum morifolium. Plant Disease, 72, 51–54.CrossRefGoogle Scholar
  19. Kerr, A., Manigault, P., & Tempe, J. (1977). Transfer of virulence in vivo and in vitro in Agrobacterium. Nature, 265(5594), 560–561.PubMedCrossRefGoogle Scholar
  20. King, E. O., Ward, M. K., & Raney, D. E. (1954). Two simple media for the demonstration of pyocyanin and fluorescin. Journal of Laboratory and Clinical Medicine, 44(2), 301–307.PubMedGoogle Scholar
  21. Kuzmanović, N., Ivanović, M., Prokić, A., Gašić, K., Blagojević, N., Puławska, J., et al. (2013). Identification and characterization of Agrobacterium spp. isolated from apricot in Serbia. European Journal of Plant Pathology, 137(1), 11–16. doi: 10.1007/s10658-013-0229-0.CrossRefGoogle Scholar
  22. Kuzmanović, N., Ivanović, M., Prokić, A., Gašić, K., Zlatković, N., & Obradović, A. (2014). Characterization and phylogenetic diversity of Agrobacterium vitis from Serbia based on sequence analysis of 16S-23S rRNA internal transcribed spacer (ITS) region. European Journal of Plant Pathology, 140, 757–768. doi: 10.1007/s10658-014-0507-5.CrossRefGoogle Scholar
  23. Lamovšek, J., Geric Stare, B., & Urek, G. (2014). Isolation of non-pathogenic Agrobacterium spp. biovar 1 from agricultural soils in Slovenia. 2014, 53, 130–139.Google Scholar
  24. Lassalle, F., Campillo, T., Vial, L., Baude, J., Costechareyre, D., Chapulliot, D., et al. (2011). Genomic species are ecological species as revealed by comparative genomics in Agrobacterium tumefaciens. Genome Biology and Evolution, 3, 762–781. doi: 10.1093/gbe/evr070.PubMedCentralPubMedCrossRefGoogle Scholar
  25. Lindström, K., & Young, J. P. W. (2011). International committee on systematics of prokaryotes subcommittee on the taxonomy of Agrobacterium and Rhizobium: minutes of the meeting, 7 September 2010, Geneva, Switzerland. International Journal of Systematic and Evolutionary Microbiology, 61(12), 3089–3093. doi: 10.1099/ijs. 0.036913-0.PubMedCrossRefGoogle Scholar
  26. Michel, M. F., Brasileiro, A. C., Depierreux, C., Otten, L., Delmotte, F., & Jouanin, L. (1990). Identification of different Agrobacterium strains isolated from the same forest nursery. Applied and Environmental Microbiology, 56(11), 3537–3545.PubMedCentralPubMedGoogle Scholar
  27. Milijašević, S., Gavrilović, V., Živković, S., Trkulja, N., & Pulawska, J. (2007). First report of tumorigenic Agrobacterium radiobacter on raspberry in Serbia. Pesticides and Phytomedicine, 22, 113–119.Google Scholar
  28. Moore, L. W., Bouzar, H., & Burr, T. J. (2001). Agrobacterium. In N. W. Schaad, J. B. Jones, & W. Chun (Eds.), Laboratory guide for identification of plant pathogenic bacteria (3rd ed., pp. 17–35). St Paul: APS Press.Google Scholar
  29. Mougel, C., Cournoyer, B., & Nesme, X. (2001). Novel tellurite-amended media and specific chromosomal and Ti plasmid probes for direct analysis of soil populations of Agrobacterium biovars 1 and 2. Applied and Environmental Microbiology, 67(1), 65–74. doi: 10.1128/aem. 67.1.65-74.2001.PubMedCentralPubMedCrossRefGoogle Scholar
  30. Mougel, C., Thioulouse, J., Perriere, G., & Nesme, X. (2002). A mathematical method for determining genome divergence and species delineation using AFLP. International Journal of Systematic and Evolutionary Microbiology, 52(Pt 2), 573–586.PubMedGoogle Scholar
  31. Nesme, X., Michel, M. F., & Digat, B. (1987). Population heterogeneity of Agrobacterium tumefaciens in galls of Populus L. from a single nursery. Applied and Environmental Microbiology, 53(4), 655–659.PubMedCentralPubMedGoogle Scholar
  32. Nesme, X., Ponsonnet, C., Picard, C., & Normand, P. (1992). Chromosomal and pTi genotypes of agrobacterium strains isolated from Populus tumors in two nurseries. FEMS Microbiology Letters, 101(3), 189–196. doi: 10.1016/0378-1097(92)90815-6.CrossRefGoogle Scholar
  33. Panday, D., Schumann, P., & Das, S. K. (2011). Rhizobium pusense sp. nov., isolated from the rhizosphere of chickpea (Cicer arietinum L.). International Journal of Systematic and Evolutionary Microbiology, 61(Pt 11), 2632–2639. doi: 10.1099/ijs. 0.028407-0.PubMedCrossRefGoogle Scholar
  34. Peluso, R., Raio, A., Morra, F., & Zoina, A. (2003). Physiological, biochemical and molecular analyses of an Italian collection of Agrobacterium tumefaciens strains. European Journal of Plant Pathology, 109(4), 291–300. doi: 10.1023/A:1023556108085.CrossRefGoogle Scholar
  35. Picard, C., Ponsonnet, C., Paget, E., Nesme, X., & Simonet, P. (1992). Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Applied and Environmental Microbiology, 58(9), 2717–2722.PubMedCentralPubMedGoogle Scholar
  36. Ponsonnet, C., & Nesme, X. (1994). Identification of Agrobacterium strains by PCR-RFLP analysis of pTi and chromosomal regions. Archives of Microbiology, 161(4), 300–309. doi: 10.1007/BF00303584.PubMedGoogle Scholar
  37. Popoff, M. Y., Kersters, K., Kiredjian, M., Miras, I., & Coynault, C. (1984). Taxonomic position of Agrobacterium strains of hospital origin. Ann Microbiol (Paris), 135a(3), 427–442.Google Scholar
  38. Portier, P., Fischer-Le Saux, M., Mougel, C., Lerondelle, C., Chapulliot, D., Thioulouse, J., et al. (2006). Identification of Genomic Species in Agrobacterium Biovar 1 by AFLP Genomic Markers. Applied and Environmental Microbiology, 72(11), 7123–7131. doi: 10.1128/aem. 00018-06.PubMedCentralPubMedCrossRefGoogle Scholar
  39. Puławska, J. (2010). Crown gall of stone fruits and nuts, economic significance and diversity of its causal agents: tumorigenic Agrobacterium spp. Journal of Plant Pathology, 92, S1.87–S81.98.Google Scholar
  40. Puławska, J., & Sobiczewski, P. (2005). Development of a semi-nested PCR based method for sensitive detection of tumorigenic Agrobacterium in soil. Journal of Applied Microbiology, 98(3), 710–721. doi: 10.1111/j.1365-2672.2004.02503.x.PubMedCrossRefGoogle Scholar
  41. Puławska, J., Willems, A., & Sobiczewski, P. (2006). Rapid and specific identification of four Agrobacterium species and biovars using multiplex PCR. Systematic and Applied Microbiology, 29(6), 470–479. doi: 10.1016/j.syapm.2005.11.002.PubMedCrossRefGoogle Scholar
  42. Puławska, J., Willems, A., De Meyer, S. E., & Sule, S. (2012a). Rhizobium nepotum sp. nov. isolated from tumors on different plant species. Systematic and Applied Microbiology, 35(4), 215–220. doi: 10.1016/j.syapm.2012.03.001.PubMedCrossRefGoogle Scholar
  43. Puławska, J., Willems, A., & Sobiczewski, P. (2012b). Rhizobium skierniewicense sp. nov., isolated from tumours on chrysanthemum and cherry plum. International Journal of Systematic and Evolutionary Microbiology, 62(Pt 4), 895–899. doi: 10.1099/ijs. 0.032532-0.PubMedCrossRefGoogle Scholar
  44. Rademaker, J., & De Bruijn, F. (1997). Characterization and classification of microbes by rep-PCR genomic fingerprinting and computer assisted pattern analysis. DNA markers: protocols, applications and overviews, 151–171.Google Scholar
  45. Rhouma, A., Boubaker, A., Nesme, X., & Dessaux, Y. (2006). Plasmid and chromosomal diversity of a Tunisian collection of Agrobacterium tumefaciens strains. Tunisian Journal of Plant Protection, 1, 73–84.Google Scholar
  46. Shams, M., Campillo, T., Lavire, C., Muller, D., Nesme, X., & Vial, L. (2012). Rapid and efficient methods to isolate, type strains and determine species of Agrobacterium spp. in pure culture and complex environments. In J. C. Jimenez-Lopez (Ed.), Biochemical Testing. InTech.
  47. Shams, M., Vial, L., Chapulliot, D., Nesme, X., & Lavire, C. (2013). Rapid and accurate species and genomic species identification and exhaustive population diversity assessment of Agrobacterium spp. using recA-based PCR. Systematic and Applied Microbiology, 36(5), 351–358. doi: 10.1016/j.syapm.2013.03.002.PubMedCrossRefGoogle Scholar
  48. Silvestro, D., & Michalak, I. (2012). raxmlGUI: a graphical front-end for RAxML. Organisms Diversity & Evolution, 12(4), 335–337. doi: 10.1007/s13127-011-0056-0.CrossRefGoogle Scholar
  49. Süle, S. (1978). Biotypes of Agrobacterium tumefaciens in Hungary. Journal of Applied Bacteriology, 44(2), 207–213. doi: 10.1111/j.1365-2672.1978.tb00792.x.CrossRefGoogle Scholar
  50. Suzaki, K., Yoshida, K., & Sawada, H. (2004). Detection of tumorigenic Agrobacterium strains from infected apple saplings by colony PCR with improved PCR primers. Journal of General Plant Pathology, 70(6), 342–347. doi: 10.1007/s10327-004-0133-8.CrossRefGoogle Scholar
  51. Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 30, 2725–2729. doi: 10.1093/molbev/mst197.PubMedCentralPubMedCrossRefGoogle Scholar
  52. Tan, B. S., Yabuki, J., Matsumoto, S., Kageyama, K., & Fukui, H. (2003). PCR primers for identification of opine types of Agrobacterium tumefaciens in Japan. Journal of General Plant Pathology, 69(4), 258–266. doi: 10.1007/s10327-003-0044-0.CrossRefGoogle Scholar
  53. Tarbah, F. A., & Goodman, R. N. (1987). Systemic spread of Agrobacterium tumefaciens biovar 3 in the vascular system of grapes. Phytopathology, 77, 915–920.CrossRefGoogle Scholar
  54. Tindall, B. J., Rossello-Mora, R., Busse, H. J., Ludwig, W., & Kampfer, P. (2010). Notes on the characterization of prokaryote strains for taxonomic purposes. International Journal of Systematic and Evolutionary Microbiology, 60(Pt 1), 249–266. doi: 10.1099/ijs. 0.016949-0.PubMedCrossRefGoogle Scholar
  55. Van Larebeke, N., Engler, G., Holsters, M., Van den Elsacker, S., Zaenen, I., Schilperoort, R. A., et al. (1974). Large plasmid in Agrobacterium tumefaciens essential for crown gall-inducing ability. Nature, 252(5479), 169–170.PubMedCrossRefGoogle Scholar
  56. Versalovic, J., Koeuth, T., & Lupski, J. R. (1991). Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Research, 19(24), 6823–6831.PubMedCentralPubMedCrossRefGoogle Scholar
  57. Weisburg, W. G., Barns, S. M., Pelletier, D. A., & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, 173(2), 697–703.PubMedCentralPubMedGoogle Scholar
  58. Weller, S. A., Stead, D. E., & Mazzucchi, U. (2004). Crown and cane gall of a blackberry-raspberry hybrid caused by Agrobacterium rhizogenes in Northern Italy. Journal of Plant Pathology, 86, 161–165.Google Scholar
  59. Yakabe, L. E., Parker, S. R., & Kluepfel, D. A. (2012). Role of systemic Agrobacterium tumefaciens populations in crown gall incidence on the walnut hybrid rootstock ‘Paradox’. Plant Disease, 96(10), 1415–1421. doi: 10.1094/PDIS-05-11-0364-RE.CrossRefGoogle Scholar
  60. Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., & Madden, T. (2012). Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics, 13(1), 134.PubMedCentralPubMedCrossRefGoogle Scholar
  61. Young, J. M., Kuykendall, L. D., Martinez-Romero, E., Kerr, A., & Sawada, H. (2001). 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. International Journal of Systematic and Evolutionary Microbiology, 51(Pt 1), 89–103.PubMedGoogle Scholar
  62. Zoina, A., Raio, A., Peluso, R., & Spasiano, A. (2001). Characterization of agrobacteria from weeping fig (Ficus benjamina). Plant Pathology, 50(5), 620–627. doi: 10.1046/j.1365-3059.2001.00603.x.CrossRefGoogle Scholar

Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2015

Authors and Affiliations

  • Nemanja Kuzmanović
    • 1
    Email author
  • Anđelka Prokić
    • 1
  • Milan Ivanović
    • 1
  • Nevena Zlatković
    • 1
  • Katarina Gašić
    • 2
  • Aleksa Obradović
    • 1
  1. 1.University of Belgrade-Faculty of AgricultureBelgradeSerbia
  2. 2.Department of Plant PathologyInstitute for Plant Protection and EnvironmentBelgradeSerbia

Personalised recommendations