Skip to main content
SpringerLink
Log in

Comparison of specificity and sensitivity of immunochemical and molecular techniques for determination of Clavibacter michiganensis subsp. michiganensis

  • Published:
Folia Microbiologica Aims and scope Submit manuscript

Abstract

Detection of Clavibacter michiganensis subsp. michiganensis (Cmm), causing bacterial canker of tomato, was verified using PTA-ELISA and IFAS with PAbs of Neogen Europe Ltd. (UK), and with published and also laboratory-generated PCR primers from the Cmm tomatinase gene. The specificity of this technique was determined with 15 plant-pathogenic and 4 common, saprophytic bacteria. With IFAS, crossreactions were found for Pantoea dispersa, P. agglomerans and Rahnella aquatilis, and with PTA-ELISA for Curtobacterium flaccumfaciens, Pectobacterium atrosepticum and Dickeya sp. Cross-reactions with subspecies other than michiganensis were also found using both methods. Molecular methods were optimized by verification of annealing temperatures and times for both primers. Conditions were finally adjusted to 30 s at 65 °C for Dreier’s and 10 s at 69 °C for our primer set. After this optimization, both primer pairs produced positive reaction only with Cmm. By means of PTA-ELISA and IFAS, Cmm strains were detected at a concentration up to 105 CFU/mL and 103 CFU/mL, respectively. The PCR test with bacterial cell suspensions reached a sensitivity of 103 CFU/mL with our designed primers and 104 CFU/mL with Dreier’s primer pair.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

Cmm :

Clavibacter michiganensis subsp. michiganensis

ELISA:

enzyme-linked immunosorbent assay

IFAS:

indirect fluorescent antibody stain

MAb:

monoclonal antibody

PAb:

polyclonal antibody

PBS:

phosphate-buffered saline

PCR:

polymerase chain reactions

PFGE:

pulse-field gel electrophoresis

PTA-ELISA:

plate-trapped enzyme-linked immunosorbent assay

rep-PCR:

repetitive sequence-based polymerase chain reactions

SA:

slide agglutination

References

  • Alvarez A.M.: Integrated approaches for detection of plant pathogenic bacteria and diagnosis of bacterial diseases. Ann.Rev.Phytopathol.42, 339–366 (2004).

    Article  CAS  Google Scholar 

  • Chang R.J., Ries S.M., Pataky J.K.: Dissemination of Clavibacter michiganensis subsp. michiganensis by practices used to produce tomato transplants. Phytopathology81, 1276–1281 (1991).

    Article  Google Scholar 

  • Dreier J., Bermpohl A., Eichenlaub R.: Southern hybridization and PCR for specific detection of phytopathogenic Clavibacter michiganensis subsp. michiganensis. Phytopathology85, 462–468 (1995).

    Article  CAS  Google Scholar 

  • EPPO/CABI: Quarantine Pests for Europe, 2nd. ed. (J.M. Smith, D.G. McNamara, P.R. Scott, M. Holderness, Eds), pp. 981–984. CAB International, Wallingford (UK) 1997.

    Google Scholar 

  • Firrao G., Locci R.: Identification of Clavibacter michiganensis subsp. sepedonicus using polymerase chain reaction. Can.J.Microbiol.40, 148–151 (1994).

    Article  CAS  PubMed  Google Scholar 

  • Gitaitis R.D., Walcott R.: The epidemiology and management of seedborne bacterial diseases. Ann.Rev.Phytopathol.45, 371–397 (2007).

    Article  CAS  Google Scholar 

  • Gleason M.L., Braun E.J., Carlton W.M., Peterson R.H.: Survival and dissemination of Clavibacter michiganensis subsp. michiganensis in tomatoes. Phytopathology81, 1519–1523 (1991).

    Article  Google Scholar 

  • Gleason M.L., Gitaitis R.D., Ricker M.D.: Recent progress in understanding and controlling bacterial canker of tomato in Eastern North America. Plant Dis.77, 1069–1075 (1993).

    Google Scholar 

  • Henson J.M., French R.: The polymerase chain reaction and plant disease diagnosis. Ann.Rev.Phytopathol.31, 81–109 (1993).

    Article  CAS  Google Scholar 

  • Jahr H., Bahro R., Burger A., Ahlemeyer J., Eichenlaub R.: Interactions between Clavibacter michiganensis subsp. michiganensis and its host plants. Environ.Microbiol.1, 113–118 (1999).

    Article  CAS  PubMed  Google Scholar 

  • Janse J.D., Kokošková B.: Indirect immunofluorescence microscopy for the detection and identification of plant pathogenic bacteria (in particular for Ralstonia solanacearum), pp. 89–100 in R. Burns (Ed.): Plant Pathology. Techniques and Protocols. Scottish Agricultural Science Agency, Diagnostics and Molecular Biology Section, Edinburg 2009.

    Google Scholar 

  • Kaneshiro W.S., Alvarez A.M.: Specificity of PCR and ELISA assays for hypovirulent and avirulent Clavibacter michiganensis subsp. michiganensis. Phytopathology91, 46 (2001).

    Google Scholar 

  • Kaneshiro W.S., Mizumoto C.Y., Alvarez A.M.: Differentiation of Clavibacter michiganensis subsp. michiganensis from seedborne saprophytes using ELISA, Biolog and 16S rDNA sequencing. Eur.J.Plant Pathol.116, 45–56 (2006).

    Article  CAS  Google Scholar 

  • Kokošková B., Mráz I., Hýblová J.: Comparison of specificity and sensitivity of immunochemical and molecular techniques for reliable detection of Erwinia amylovora. Folia Microbiol.52, 175–182 (2007).

    Article  Google Scholar 

  • Krämar I., Griesbach E.: Use of ELISA for detection of Clavibacter michiganensis subsp. michiganensis in tomato. OEPP/EPPO Bull.25, 185–193 (1995).

    Google Scholar 

  • Lopez M.M., Cambra M., Aramburu J.M., Bolinches J.: Problems of detecting phytopathogenic bacteria by ELISA. OEPP/EPPO Bull.17, 113–117 (1987).

    Google Scholar 

  • Louws F.J., Schneider M., De Brujin F.J.: Assessing genomic diversity of microbes using repetitive-sequence based PCR (rep-PCR), pp. 63–94 in G. Tranzos (Ed.): Nucleic Acid Amplification Methods of Environmental Samples. Technomic Publishing Co., Lancaster (PA, USA) 1996.

    Google Scholar 

  • Louws F.J., Bell J., Medina-mora C.M., Smart C.D., Opgenorth D., Ishimaru C.A., Hausbeck M.K., De Bruijn F.J., Fulbright D.W.: Rep-PCR-mediated genomic fingerprinting: a rapid and effective method to identify Clavibacter michiganensis. Phytopathology88, 862–868 (1998).

    Article  CAS  PubMed  Google Scholar 

  • Luo L.X., Walters C., Bolkan H., Liu X.L., Li J.Q.: Quantification of viable cells of Clavibacter michiganensis subsp. michiganensis using a Dann binding dye and a real-time PCR assay. Plant Pathol.57, 332–337 (2008).

    Article  CAS  Google Scholar 

  • Nario K., Fumihiro Y., Takahiro K., Yuji N.: Detection of Clavibacter michiganensis subsp. michiganensis by enzyme-linked immunosorbent assay (ELISA) using the biotin-avidin system. Proc.Assoc.Plant Prot.Kyushu47, 9–12 (2001).

    Google Scholar 

  • OEPP/EPPO: Clavibacter michiganensis subsp. michiganensis. OEPP/EPPO Bull.35, 275–283 (2005).

  • Pánková I., Kokošková B.: Sensitivity and specificity of monoclonal antibody Mn-Cs1 for detection and determination of Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot of potato. Plant Protect.Sci.38, 117–124(2002).

    Google Scholar 

  • Salm H., Geider K.: Real-time PCR for detection and quantification of Erwinia amylovora, the causal agent of fireblight. Plant Pathol.53, 602–610 (2004).

    Article  CAS  Google Scholar 

  • Santos M.S., Cruz L., Norskov P., Rasmussen O.F.: Rapid and sensitive detection of Clavibacter michiganensis subsp. michiganensis in tomato seeds by polymerase chain reaction. Seed Sci.Technol.25, 581–584 (1997).

    Google Scholar 

  • Schaad N.W., Frederik R.D.: Real-time PCR and its application for rapid plant disease diagnostics. Can.J.Plant Pathol.24, 250–258 (2002).

    CAS  Google Scholar 

  • Schaad N.W., Sule S., Van Vuurde J.W.L., Vruggink H., Alvarez A.M., Benedict A.A., De Wael L., van Laere O.: Serology, pp. 153–190 in F. Klement, K. Rudolf, D.C. Sands (Eds): Methods in Phytobacteriology. Akademiai Kiadó, Budapest 1990.

    Google Scholar 

  • Schaad N.W., Jones J.B., Chun W.: Laboratory Guide for Identification of Plant Pathogenic Bacteria, 3rd ed. American Phytopathological Society, St.Paul (MN, USA) 2001.

    Google Scholar 

  • Schneider B.J., Zhao J., Orser C.S.: Detection of Clavibacter michiganensis subsp. sepedonicus by DNA amplification. FEMS Microbiol.Lett.190, 207–212 (1993).

    Google Scholar 

  • Versalovic J., Schneider M., Lupskij R.: Genomic fingerprinting of bacteria using repetitive sequence based PCR (rep-PCR). Meth. Mol.Cell.Biol.5, 25–40 (1994).

    CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratory of Diagnostics and Epidemiology of Microorganisms, Plant Medicine Division, Crop Research Institute, 161 06, Prague 6, Czech Republic

    B. Kokošková

  2. Department of Plant Virology, Institute of Plant Molecular Biology, Biological Centre of the Academy of Sciences of the Czech Republic, 370 05, České Budějovice, Czech Republic

    I. Mráz & J. Fousek

Authors
  1. B. Kokošková
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. I. Mráz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. Fousek
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to B. Kokošková.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kokošková, B., Mráz, I. & Fousek, J. Comparison of specificity and sensitivity of immunochemical and molecular techniques for determination of Clavibacter michiganensis subsp. michiganensis . Folia Microbiol 55, 239–244 (2010). https://doi.org/10.1007/s12223-010-0035-4

Download citation

  • Received:

  • Revised:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12223-010-0035-4

Keywords

Search

Navigation