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.

False positive Ralstonia solanacearum real‐time PCR results in routine potato tuber samples caused by Advenella species: adaptations to avoid these cross‐reactions

“…14-33, Table 2) in this study. As expected, the MultiRaso real-time PCR assessment by Massart et al (2014) and the B2-system by Weller et al (2000) generated negative results, because of non-sequential homologies within the corresponding target DNA sequence in the cross-reacting reference strains, in the isolates from selected routine potato tuber samples (2013 and 2015) and in the routine potato tuber samples of 2015. The use of the RSP-55T MGB Probe, from Vreeburg et al (2016), in combination with the RS-primer set from Weller et al (2000), reduced false positive cases of the original RS primer-probe system from Weller et al (2000) by 100%.…”

“…Different real-time PCR tests for the detection of Ralstonia solanacearum were developed over the last two decades, including Weller et al (2000), Massart et al (2014) and Vreeburg et al (2016). Each of these tests for R. solanacearum has its advantages and disadvantages.…”

Performance of real‐time PCR and immunofluorescence for the detection of Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum in potato tubers in routine testing

“…Neither IF nor PCR were able to distinguish between R. solanacearum and R. syzigii in this study ( Table 2); for PCR this corresponds to the description by Weller et al (2000). Ralstonia solanacearum is stated to be distinguished from R. syzigii by the Massart PCR technique (Massart et al, 2014), but this was not tested in this study.…”

“…(). Ralstonia solanacearum is stated to be distinguished from R. syzigii by the Massart PCR technique (Massart et al ., ), but this was not tested in this study.…”

Validation of four real‐time TaqMan PCRs for the detection of Ralstonia solanacearum and/or Ralstonia pseudosolanacearum and/or Clavibacter michiganensis subsp. sepedonicus in potato tubers using a statistical regression approach

“…This new PCR has been validated together with three other TaqMan PCRs, including the multiplex TaqMan of Massart et al (2014) (detecting R. solanacearum and C. michiganensis subsp. sepedonicus.…”