European Journal of Plant Pathology

, Volume 140, Issue 4, pp 757–768 | Cite as

Characterization and phylogenetic diversity of Agrobacterium vitis from Serbia based on sequence analysis of 16S-23S rRNA internal transcribed spacer (ITS) region

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


Serious outbreaks of grapevine crown gall disease were observed in major Serbian viticultural regions during the last five years. Tumorigenic Agrobacterium vitis was identified as a causal agent by using conventional bacteriological and molecular tests. The 36 studied strains of A. vitis showed homogeneous biochemical and physiological characteristics, but were heterogeneous in their pathogenic properties, especially on tomato and sunflower. Furthermore, genetic differences related to chromosomal and plasmid DNA were observed. The Ti plasmid of 35 strains was classified as the octopine/cucumopine (O/C) type, whereas one was classified as the vitopine (V) type. The O/C strains were further divided into O/C-1 and O/C-2 groups based on PCR analysis. Moreover, the sequence analysis of the 16S-23S rRNA ITS region provided robust and precise delineation of studied strains. Although a high level of genetic diversity in A. vitis strains from Serbia was revealed by using this approach, their genotypic relatedness with the strains from other countries suggested their common origin. Also, association between the chromosomal and plasmid DNA was determined for some phylogenetic groups and clusters.


Crown gall Grapevine 16S-23S rRNA ITS region Ti plasmid Opine type 



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 Thomas J. Burr, Desen Zheng, Sandor Süle and Enrico Biondi for kindly providing Agrobacterium strains. We also thank Enrico Biondi for helpful discussions and for critical reading of the manuscript.


  1. Argun, N., Momol, M. T., Maden, S., Momol, E. A., Reid, C. L., Çelek, H., et al. (2002). Characterization of Agrobacterium vitis strains isolated from Turkish grape cultivars in the Central Anatolia region. Plant Disease, 86(2), 162–166. doi: 10.1094/PDIS.2002.86.2.162.CrossRefGoogle Scholar
  2. Arsenijević, M., Stojić, J., & Panić, M. (1974). Contribution to the study of bacterial canker of the grapevine (Agrobacterium tumefaciens). Plant Protection, 25, 257–264.Google Scholar
  3. Bautista-Zapanta, J. N., Arafat, H. H., Tanaka, K., Sawada, H., & Suzuki, K. (2009). Variation of 16S-23S internally transcribed spacer sequence and intervening sequence in rDNA among the three major Agrobacterium species. Microbiological Research, 164(6), 604–612. doi: 10.1016/j.micres.2007.08.003.PubMedCrossRefGoogle 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. Burr, T. J., & Otten, L. (1999). Crown gall of grape: biology and disease management. Annual Review of Phytopathology, 37, 53–80. doi: 10.1146/annurev.phyto.37.1.53.PubMedCrossRefGoogle Scholar
  6. Burr, T. J., Bazzi, C., Süle, S., & Otten, L. (1998). Crown gall of grape: biology of Agrobacterium vitis and the development of disease control strategies. Plant Disease, 82(12), 1288–1297. doi: 10.1094/PDIS.1998.82.12.1288.CrossRefGoogle Scholar
  7. Burr, T. J., Reid, C. L., Adams, C. E., & Momol, E. A. (1999). Characterization of Agrobacterium vitis strains isolated from feral Vitis riparia. Plant Disease, 83(2), 102–107. doi: 10.1094/PDIS.1999.83.2.102.CrossRefGoogle Scholar
  8. Genov, I., Atanassov, I., Tsvetkov, I., & Atanassov, A. (2006a). Isolation and characterization of Agrobacterium strains from grapevines in Bulgarian vineyards and wild grapes, V. vinifera ssp. silvestris. Vitis, 45, 97–101.Google Scholar
  9. Genov, I., Atanassov, I., Yordanov, Y., Tsvetkov, I., & Atanassov, A. (2006b). Genetic diversity of Agrobacterium vitis strains, isolated from grapevines and wild grapes in Bulgaria, assessed by Cleaved Amplified Polymorphic Sequences analysis of 16S-23S rDNA. Vitis, 45, 125–130.Google Scholar
  10. 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.PubMedPubMedCentralGoogle Scholar
  11. Knauf, V. C., Panagopoulos, C. G., & Nester, E. W. (1982). Genetic factors controlling the host range of Agrobacterium tumefaciens. Phytopathology, 72, 1545–1549.Google Scholar
  12. Kumagai, L., & Fabritius, A.-L. (2008) Detection and differentiation of pathogenic Agrobacterium vitis and Agrobacterium tumefaciens in grapevine using multiplex Bio-PCR. In 2nd Annual National Viticulture Research Conference, Davis, California (pp. 42–47)Google Scholar
  13. Momol, E. A., Burr, T. J., Reid, C. L., Momol, M. T., Hseu, S. H., & Otten, L. (1998). Genetic diversity of Agrobacterium vitis as determined by DNA fingerprints of the 5′-end of the 23S rRNA gene and Random Amplified Polymorphic DNA. Journal of Applied Microbiology, 85(4), 685–692. doi: 10.1111/j.1365-2672.1998.00581.x.CrossRefGoogle Scholar
  14. 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
  15. Normand, P., Ponsonnet, C., Nesme, X., Neyra, M., & Simonet, P. (1996). ITS analysis of prokaryotes. In A. D. L. Akkermans, J. D. van Elsas, & F. J. de Bruijn (Eds.), Molecular microbial ecology manual (pp. 1–12). Dordrecht: Kluwer Academic Publishers.Google Scholar
  16. Otten, L., de Ruffray, P., Momol, E. A., Momol, M. T., & Burr, T. J. (1996). Phylogenetic relationship between Agrobacterium vitis isolates and their Ti plasmids. Molecular Plant-Microbe Interactions, 9, 782–786.CrossRefGoogle Scholar
  17. Palacio-Bielsa, A., González-Abolafio, R., Álvarez, B., Lastra, B., Cambra, M. A., Salcedo, C. I., et al. (2009). Chromosomal and Ti plasmid characterization of tumorigenic strains of three Agrobacterium species isolated from grapevine tumours. Plant Pathology, 58(3), 584–593. doi: 10.1111/j.1365-3059.2008.01984.x.CrossRefGoogle Scholar
  18. Panić, M. (1973). Bakteriozni rak vinove loze. Jugoslovensko Vinogradarstvo i Voćarstvo, 8, 19–22.Google Scholar
  19. Paulus, F., Huss, B., Bonnard, G., Ridé, M., Szegedi, E., Tempé, J., et al. (1989). Molecular systematics of biotype III Ti plasmids of Agrobacterium tumefaciens. Molecular Plant-Microbe Interactions, 2, 64–74.CrossRefGoogle Scholar
  20. 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
  21. 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
  22. 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
  23. Sawada, H., Ieki, H., Kobayashi, S., & Oiyama, I. (1992). Grouping of tumorigenic Agrobacterium spp. Based on Ti plasmid-related phenotypes. Japanese Journal of Phytopathology, 58(2), 244–252. doi: 10.3186/jjphytopath.58.244.CrossRefGoogle Scholar
  24. Schroth, M. N., McCain, A. H., Foott, J. H., & Huisman, O. C. (1988). Reduction in yield and vigor of grapevine caused by crown gall disease. Plant Disease, 72, 241–246.CrossRefGoogle Scholar
  25. 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
  26. Szegedi, E., & Bottka, S. (2002). Detection of Agrobacterium vitis by polymerase chain reaction in grapevine bleeding sap after isolation on a semiselective medium. Vitis, 41, 37–42.Google Scholar
  27. Szegedi, E., Czakó, M., Otten, L., & Koncz, C. S. (1988). Opines in crown gall tumours induced by biotype 3 isolates of Agrobacterium tumefaciens. Physiological and Molecular Plant Pathology, 32(2), 237–247. doi: 10.1016/S0885-5765(88)80020-1.CrossRefGoogle Scholar
  28. Szegedi, E., Bottka, S., Mikulas, J., Otten, L., & Sule, S. (2005). Characterization of Agrobacterium tumefaciens strains isolated from grapevine. Vitis, 44, 49–54.Google Scholar
  29. 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. Molecular Biology and Evolution, 28(10), 2731–2739. doi: 10.1093/molbev/msr121.PubMedCrossRefPubMedCentralGoogle Scholar
  30. 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

Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2014

Authors and Affiliations

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

Personalised recommendations