Journal of General Plant Pathology

, Volume 78, Issue 2, pp 121–126 | Cite as

Novel PCR primers for detection of genetically diverse virulent Agrobacterium tumefaciens biovar 1 strains

  • Lani E. Yakabe
  • M. Malendia Maccree
  • Padma Sudarshana
  • Ali E. McClean
  • Shane R. Parker
  • William P. Wechter
  • Gernot Presting
  • Mizuri Marutani-Hert
  • Daniel A. KluepfelEmail author
Bacterial and Phytoplasma Diseases


Novel PCR primers were developed to amplify a 243-bp fragment of an intergenic region between gene 5 and tms2 on the T-DNA of Agrobacterium tumefaciens biovar 1. These primers exhibit 100% positive correlation with strain virulence, 100% negative correlation with avirulence, and did not generate extraneous bands, thus facilitating robust real-time PCR detection.


Agrobacterium tumefaciens Crown gall Species-specific Virulence-specific PCR detection Ti-plasmid 



We thank Dr. R.M. Davis for critical reading of this manuscript and Lynne Pearson for administrative support. This work was supported in part by the USDA-ARS CRIS project 5306-22000-14-00D, the California Walnut Board and the CDFA, Fruit Tree, Nut Tree and Grapevine Improvement Advisory Board.


  1. Agrios GN (2005) Plant pathology, 5th edn. Elsevier Academic Press, BurlingtonGoogle Scholar
  2. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410PubMedGoogle Scholar
  3. Bini F, Geider K, Bazzi C (2008a) Detection of Agrobacterium vitis by PCR using novel virD2 gene-specific primers that discriminate two subgroups. Eur J Plant Pathol 122:403–411CrossRefGoogle Scholar
  4. Bini F, Kuczmog A, Putnoky P, Otten L, Bazzi C, Burr TJ, Szegedi E (2008b) Novel pathogen-specific primers for the detection of Agrobacterium vitis and Agrobacterium tumefaciens. Vitis 47:181–189Google Scholar
  5. Bliss FA, Almedhi AA, Dandekar AM, Schuerman PL, Bellaloui N (1999) Crown gall resistance in accessions of 20 Prunus species. HortSci 34:326–330Google Scholar
  6. Burr TJ, Otten L (1999) Crown gall of grape: biology and disease management. Annu Rev Phytopathol 37:53–80PubMedCrossRefGoogle Scholar
  7. Cubero J, Martínez MC, Llop P, López MM (1999) A simple and efficient PCR method for the detection of Agrobacterium tumefaciens in plant tumors. J Appl Microbiol 86:591–602PubMedCrossRefGoogle Scholar
  8. Dong L-C, Sun C-W, Thies KL, Luthe DS, Graves CH Jr (1992) Use of polymerase chain reaction to detect pathogenic strains of Agrobacterium. Phytopathology 82:434–439CrossRefGoogle Scholar
  9. Epstein L, Kaur S, McKenna JR, Grant JA, Olson WH, Reil WO (2008) Crown gall can spread between walnut trees in nurseries and reduce future yields. Calif Agric 62:111–115CrossRefGoogle Scholar
  10. Farrand SK, van Berkum PB, Oger P (2003) Agrobacterium is a definable genus of the family Rhizobiaceae. Int J Syst Evol Microbiol 53:1681–1687PubMedCrossRefGoogle Scholar
  11. Gans J, Wolinsky M, Dunbar J (2005) Computational improvements reveal great bacterial diversity and high metal toxicity in soil. Science 309:1387–1390PubMedCrossRefGoogle Scholar
  12. Haas JH, Moore LW, Ream W, Manulis S (1995) Universal PCR primers for the detection of phytopathogenic Agrobacterium strains. Appl Environ Microbiol 61:2879–2884PubMedGoogle Scholar
  13. Holmes B, Roberts P (1981) The classification, identification and nomenclature of agrobacteria. J Appl Bacteriol 50:443–467CrossRefGoogle Scholar
  14. Kawaguchi A, Sawada H, Inoue K, Nasu H (2005) Multiplex PCR for the identification of Agrobacterium biovar 3 strains. J Gen Plant Pathol 71:54–59CrossRefGoogle Scholar
  15. Keane PJ, Kerr A, New PB (1970) Crown gall of stone fruit. II. Identification and nomenclature of Agrobacterium isolates. Aust J Biol Sci 23:585–595Google Scholar
  16. Kerr A, Panagopoulos CG (1977) Biotypes of Agrobacterium radiobacter var. tumefaciens and their biological control. Phytopathol Z 90:172–179CrossRefGoogle Scholar
  17. Nesme X, Leclerc M-C, Bardin R (1990) PCR detection of an original endosymbiont: the Ti plasmid of Agrobacterium tumefaciens. In: Nardon P, Gianinazzi-Pearson V, Grenier AM, Margulis L, Smith DC (eds) Endocytobiology IV. Institut National de la Recherche Agronomique, Paris, pp 47–50Google Scholar
  18. Ophel K, Kerr A (1990) Agrobacterium vitis sp. nov. for strains of Agrobacterium biovar 3 from grapevines. Int J Syst Bacteriol 40:236–241CrossRefGoogle Scholar
  19. Palacio-Bielsa A, Cambra MA, López MM (2009) PCR detection and identification of plant-pathogenic bacteria: updated review of protocols (1989–2007). J Plant Pathol 91:249–297Google Scholar
  20. Pulawska J, Sobiczewski P (2005) Development of a semi-nested PCR based method for sensitive detection of tumorigenic Agrobacterium in soil. J Appl Microbiol 98:710–721PubMedCrossRefGoogle Scholar
  21. Pulawska J, Willems A, Sobiczewski P (2006) Rapid and specific identification of four Agrobacterium species and biovars using multiplex PCR. Syst Appl Microbiol 29:470–479PubMedCrossRefGoogle Scholar
  22. Puopolo G, Raio A, Zoina A (2007) Early detection of Agrobacterium tumefaciens in symptomless artificially inoculated Chrysanthemum and peach plants using PCR. J Plant Pathol 89:185–190Google Scholar
  23. Riker AJ, Banfield WM, Wright WH, Keitt GW, Sagen HE (1930) Studies on infectious hairy root of nursery trees of apples. J Agric Res 41:507–540Google Scholar
  24. Sachadyn P, Kur J (1997) A new PCR system for Agrobacterium tumefaciens detection based on amplification of T-DNA fragment. Acta Microbiol Pol 46:145–156PubMedGoogle Scholar
  25. Sawada H, Ieki H, Matsuda I (1995) PCR detection of Ti and Ri plasmids from phytopathogenic Agrobacterium strains. Appl Environ Microbiol 61:828–831PubMedGoogle Scholar
  26. Smith EF, Townsend CO (1907) A plant-tumor of bacterial origin. Science 25:671–673PubMedCrossRefGoogle Scholar
  27. Suzaki K, Yoshida K, Sawada H (2004) Detection of tumorigenic Agrobacterium strains from infected apple saplings by colony PCR with improved PCR primers. J Gen Plant Pathol 70:342–347CrossRefGoogle Scholar
  28. Tan BS, Yabuki J, Matsumoto S, Kageyama K, Fukui H (2003) PCR primers for identification of opine types of Agrobacterium tumefaciens in Japan. J Gen Plant Pathol 69:258–266CrossRefGoogle Scholar
  29. Tighe SW, de Lajudie P, Dipietro K, Lindström MK, Nick G, Jarvis BDW (2000) Analysis of cellular fatty acids and phenotypic relationships of Agrobacterium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium species using the Sherlock Microbial Identification System. Int J Syst Evol Microbiol 50:787–801PubMedCrossRefGoogle Scholar
  30. Tzfira T, Citovsky V (eds) (2008) Agrobacterium: from biology to biotechnology. Springer, New YorkGoogle Scholar
  31. Vogel J, Normand P, Thioulouse J, Nesme X, Grundmann GL (2003) Relationship between spatial and genetic distance in Agrobacterium spp. in 1 cubic centimeter of soil. Appl Environ Microbiol 69:1482–1487PubMedCrossRefGoogle Scholar
  32. Weller SA, Stead DE (2002) Detection of root mat associated Agrobacterium strains from plant material and other sample types by post-enrichment TaqMan PCR. J Appl Microb 92:118–126CrossRefGoogle Scholar
  33. Wood DW, Setubal JC, Kaul R, Monks DE, Kitajima JP, Okura VK, Zhou Y et al (2001) The genome of the natural genetic engineer Agrobacterium tumefaciens C58. Science 294:2317–2323PubMedCrossRefGoogle Scholar
  34. Yakabe LE, Parker SR, Kluepfel DA (2010) Effect of pre-plant soil fumigants on Agrobacterium tumefaciens, pythiaceous species, and subsequent soil recolonization by A. tumefaciens. Crop Protect 29:583–590CrossRefGoogle Scholar
  35. Young JM, Kerr A, Sawada H (2005) Genus II. Agrobacterium Conn 1942, 359AL. In: Garrity GM (ed) Bergey’s manual of systemic bacteriology, part C, 2nd edn, vol II. Springer Science, New York, pp 340–345Google Scholar

Copyright information

© The Phytopathological Society of Japan and Springer (Outside the USA) 2012

Authors and Affiliations

  • Lani E. Yakabe
    • 1
  • M. Malendia Maccree
    • 2
  • Padma Sudarshana
    • 3
  • Ali E. McClean
    • 1
  • Shane R. Parker
    • 1
  • William P. Wechter
    • 4
  • Gernot Presting
    • 5
  • Mizuri Marutani-Hert
    • 1
  • Daniel A. Kluepfel
    • 1
    Email author
  1. 1.USDA/ARS Crops Pathology and Genetics Research UnitDavisUSA
  2. 2.Environmental Health and SafetyUniversity of California, DavisDavisUSA
  3. 3.Monsanto CompanyWoodlandUSA
  4. 4.U.S. Vegetable LaboratoryUSDA/ARSCharlestonUSA
  5. 5.Department of Molecular Biosciences and BioengineeringUniversity of HIHonoluluUSA

Personalised recommendations