European Journal of Plant Pathology

, Volume 137, Issue 1, pp 11–16 | Cite as

Identification and characterization of Agrobacterium spp. isolated from apricot in Serbia

  • Nemanja Kuzmanović
  • Milan Ivanović
  • Anđelka Prokić
  • Katarina Gašić
  • Nevena Blagojević
  • Joanna Puławska
  • Aleksa Obradović
Short Communications

Abstract

Crown gall, caused by tumorigenic strains of Agrobacterium spp., is considered one of the most important diseases in stone fruit nurseries throughout the world. Since the crown gall disease has not been studied extensively in Serbia for more than 30 years, the objective of this study was to isolate, identify and characterize the bacterium associated with crown gall symptoms on one-year-old apricot trees. Samples were collected from a nursery in central Serbia and subjected to laboratory analysis. Bacteria were isolated from tumour tissue on yeast mannitol agar (YMA) and six gram-negative isolates were selected for further study. PCR assay using primers specific for virD2, virC, ipt and tms2 pathogenicity-associated plasmid genes revealed that isolates harbour Ti plasmids. All studied strains carrying Ti plasmids were classified as nopaline-type based on further molecular analysis. Using a multiplex PCR assay, targeting 23S rRNA gene sequences, and physiological and biochemical tests, five strains were assigned as A. rhizogenes/biovar 2 and the remaining one as A. tumefaciens/biovar 1. Identity of the strains was confirmed by sequence analysis of the 16S rRNA gene. In pathogenicity assay, all six strains caused tumour formation on inoculated carrot root discs, young tomato and sunflower plants.

Keywords

Crown gall Apricot Tumorigenic Pathogenic plasmid Opines 

References

  1. Costechareyre, D., Rhouma, A., Lavire, C., Portier, P., Chapulliot, D., Bertolla, F., Boubaker, A., Dessaux, Y., & Nesme, X. (2010). Rapid and efficient identification of Agrobacterium species by recA allele analysis. Microbial Ecology, 60, 862–872.PubMedCrossRefGoogle Scholar
  2. Garrett, C. M. E. (1987). The effect of crown gall on growth of cherry trees. Plant Pathology, 36, 339–345.CrossRefGoogle Scholar
  3. Haas, J. H., Moore, L. W., & Ream, W. (1995). Universal PCR primers for detection of phytopathogenic Agrobacterium strains. Applied and Environmental Microbiology, 61, 2879–2884.PubMedGoogle Scholar
  4. Hooykaas, P. J. J., Den Dulk-Ras, H., Ooms, G., & Schilperoort, R. A. (1980). Interactions between octopine and nopaline plasmids in Agrobacterium tumefaciens. Journal of Bacteriology, 143, 1295–1306.PubMedGoogle Scholar
  5. Kuzmanović, N., Ćalić, A., Ivanović, M., Gašić, K., Puławska, J., & Obradović, A. (2012). First report of Agrobacterium vitis as the causal agent of grapevine crown gall in Serbia. Plant Disease, 96, 286.CrossRefGoogle Scholar
  6. Lassalle, F., Campillo, T., Vial, L., Baude, J., Costechareyre, D., Chapulliot, D., Shams, M., Abrouk, D., Lavire, C., Oger-Desfeux, C., Hommais, F., Gueguen, L., Daubin, V., Muller, D., & Nesme, X. (2011). Genomic species are ecological species as revealed by comparative genomics in Agrobacterium tumefaciens. Genome Biology and Evolution, 3, 762–781.PubMedCrossRefGoogle Scholar
  7. Milijašević, S., Gavrilović, V., Živković, S., Trkulja, N., & Puławska, J. (2007). First report of tumorigenic Agrobacterium radiobacter on raspberry in Serbia. Pesticides and Phytomedicine, 22, 113–119.Google Scholar
  8. Moore, L., Warren, G., & Strobel, G. (1979). Involvement of a plasmid in the hairy root disease of plants caused by Agrobacterium rhizogenes. Plasmid, 2, 617–626.PubMedCrossRefGoogle Scholar
  9. Moore, L. W., Bouzar, H., & Burr, T. (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
  10. Mougel, C., Thioulouse, J., Perrière, G., & Nesme, X. (2002). A mathematical method for determining genome divergence and species delineation using AFLP. International Journal of Systematic and Evolutionary Microbiology, 52, 573–586.PubMedGoogle Scholar
  11. Otten, L., Burr, T., & Szegedi, E. (2008). Agrobacterium: a disease-causing bacterium. In T. Tzfira & V. Citovsky (Eds.), Agrobacterium: from biology to biotechnology (pp. 1–46). New York: Springer.Google Scholar
  12. Petit, A., & Tempé, J. (1985). The function of T-DNA in nature. In L. Van Vloten- Doting, G. S. P. Groot, & T. C. Hall (Eds.), Molecular form and function of the plant genome (pp. 625–636). New York: Plenum Press.Google Scholar
  13. 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–S1.98.Google Scholar
  14. 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, 710–721.PubMedCrossRefGoogle Scholar
  15. 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, 470–479.PubMedCrossRefGoogle Scholar
  16. Puławska, J., Willems, A., De Mayer, S., & Sule, S. (2012a). Rhizobium nepotum sp. nov. isolated from tumours on different plant species. Systematic and Applied Microbiology, 35, 215–220.PubMedCrossRefGoogle Scholar
  17. 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, 895–899.CrossRefGoogle Scholar
  18. 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, 342–347.CrossRefGoogle Scholar
  19. Szegedi, E., Czakό, 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, 237–247.CrossRefGoogle Scholar
  20. 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
  21. 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, 2731–2739.PubMedCrossRefGoogle Scholar
  22. 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, 258–266.CrossRefGoogle Scholar
  23. Weisburg, W. G., Barns, S. M., Pelletier, D. A., & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, 173, 697–703.PubMedGoogle Scholar

Copyright information

© KNPV 2013

Authors and Affiliations

  • Nemanja Kuzmanović
    • 1
  • Milan Ivanović
    • 1
  • Anđelka Prokić
    • 1
  • Katarina Gašić
    • 2
  • Nevena Blagojević
    • 1
  • Joanna Puławska
    • 3
  • Aleksa Obradović
    • 1
  1. 1.Faculty of AgricultureUniversity of BelgradeBelgradeSerbia
  2. 2.Department of Plant PathologyInstitute for Plant Protection and EnvironmentBelgradeSerbia
  3. 3.Pomology DivisionResearch Institute of HorticultureSkierniewicePoland

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