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Development of the simultaneous detection of Ralstonia solanacearum race 3 and Clavibacter michiganensis subsp. sepedonicus in potato tubers by a multiplex real-time PCR assay

Abstract

Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum (Smith) Yabuuchi et al. race 3 are the causal agents of ring-rot and brown-rot of potato respectively. These diseases represent a serious threat to potato production in temperate climates. Both bacteria are listed as A2 pests in the EPPO region and as zero-tolerance quarantine organisms in the European Union. All the detection tests developed so far were only focused on the detection of a single pathogen while the absence of both bacteria has to be certified in the seed tubers. We have therefore developed a new multiplex real-time PCR assay to simultaneously detect both bacteria in a single assay. Additionally, the reliability of this molecular diagnostic test has been improved by the simultaneous amplification of an internal control, corresponding to a potato gene co-extracted from the sample. The polyvalence and the specificity of each set of bacterial primers and probes were evaluated on more than 90 bacterial strains. The limit of detection of this triplex real-time protocol was similar to those observed with other molecular protocols previously developed for the individual detection of one of these bacteria. A concordance of 100 % was obtained in a blind test mimicking the routine application of the technology. In conclusion, this new protocol represents a straightforward and convenient method potentially adapted to primary screening of potato tubers.

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Acknowledgments

We thank Dr. Johan Van Vaerenbergh and Dr. Rachid Tahzima (ILVO, Gent, Belgium) for fruitful collaboration and for sending the blinded samples. We are grateful to Sylvie Van Mellaert for excellent technical assistance. We also thank MM. Walter Van Ormelingen et David Michelante from the Federal Agency for the safety of the Food Chain (AFSCA - Belgium) for the external coordination of the blind-test and for fruitful discussions throughout the project. This work was supported by AFSCA and the General Management of Agriculture (DGA) of the Walloon Region Ministry (Belgium).

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Correspondence to Sébastien Massart.

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Massart, S., Nagy, C. & Jijakli, M.H. Development of the simultaneous detection of Ralstonia solanacearum race 3 and Clavibacter michiganensis subsp. sepedonicus in potato tubers by a multiplex real-time PCR assay. Eur J Plant Pathol 138, 29–37 (2014). https://doi.org/10.1007/s10658-013-0294-4

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  • DOI: https://doi.org/10.1007/s10658-013-0294-4

Keywords

  • Ralstonia solanacearum
  • Clavibacter michiganensis
  • Real-time PCR
  • Diagnostic
  • Quarantine pathogen
  • Multiplex detection