Validation and Application of a Real-time PCR Protocol for the Specific Detection and Quantification of Clavibacter michiganensis subsp. sepedonicus in Potato

Abstract

Clavibacter michiganensis subsp. sepedonicus (Cms) multiplies very rapidly, passing through the vascular strands and into the stems and petioles of a diseased potato. Therefore, the rapid and specific detection of this pathogen is highly important for the effective control of the pathogen. Although several PCR assays have been developed for detection, they cannot afford specific detection of Cms. Therefore, in this study, a computational genome analysis was performed to compare the sequenced genomes of the C. michiganensis subspecies and to identify an appropriate gene for the development of a subspecies-specific PCR primer set (Cms89F/R). The specificity of the primer set based on the putative phage-related protein was evaluated using genomic DNA from seven isolates of Cms and 27 other reference strains. The Cms89F/R primer set was more specific and sensitive than the existing assays in detecting Cms in in vitro using Cms cells and its genomic DNA. This assay was also able to detect at least 1.47×102 copies/μl of cloned-amplified target DNA, 5 fg of DNA using genomic DNA or 10−6 dilution point of 0.12 at OD600 units of cells per reaction using a calibrated cell suspension.

Development of SCAR markers for rapid and specific detection of Pseudomonas syringae pv. morsprunorum races 1 and 2, using conventional and real-time PCR

“…actinidiae (Gallelli et al 2014 ) and Clavibacter michiganensis subsp. sepedonicus (Cho et al 2015 ). The high sensitivity of the developed assay (obtained in our hands) will be invaluable for detecting the target bacteria in the early latent period of the disease, allowing growers to undertake appropriate prevention or protection programmes.…”

Detection of Potato ring rot Pathogen Clavibacter michiganensis subsp. sepedonicus by Loop-mediated isothermal amplification (LAMP) assay

“…In addition, in the study by Cho et al . 13 , the sensitivity of CMS detection with a real-time PCR method was found to be 5 fg. But according to the CMS detection guide issued by EPPO, the reference method can detect 10 3 –10 4 cell ml −1 CMS, which means at least 10 4 copy DNA, and it is also the typical bacterial load for CMS on potato plants 51 .…”

“…Polymerase chain reaction (PCR) is undoubtedly the most commonly used method of DNA-based diagnostic tests 9,10 . PCR methods are used for the detection of CMS in many studies 11–13 . Mills 14 reported that the limit of detection was 100 CFU ml −1 using PCR methods.…”

Pest categorisation of Clavibacter sepedonicus

“…Pest distribution outside the EU C. sepedonicus is distributed in Europe, North America and Asia (Figure 1; CABI, 2019; EPPO; 2019). One or more strains from Argentina were included in some studies (Mirza et al, 1993;Fousek et al, 2002;Koko skov a et al, 2005;Cho et al, 2015), but these are likely to be from an interception and the pathogen is not officially reported from that country (CABI, 2019; EPPO, 2019). One or more strains from Argentina were included in some studies (Mirza et al, 1993;Fousek et al, 2002;Koko skov a et al, 2005;Cho et al, 2015), but these are likely to be from an interception and the pathogen is not officially reported from that country (CABI, 2019; EPPO, 2019).…”

“…A finding has been reported in Egypt (Seleim et al, 2014) but not confirmed (EPPO, 2015). One or more strains from Argentina were included in some studies (Mirza et al, 1993;Fousek et al, 2002;Koko skov a et al, 2005;Cho et al, 2015), but these are likely to be from an interception and the pathogen is not officially reported from that country (CABI, 2019;EPPO, 2019). The pathogen was first described in northern Europe and has been traditionally seen as a disease restricted to cool northern temperate countries of the world, but is becoming increasingly widespread (EPPO, 2006;CABI, 2019).…”