A new multiplex TaqMan qPCR targetingClavibacter michiganensisvirulence-related genes

ABSTRACT Bacterial canker of tomato caused by Clavibacter michiganensis ( Cm ) is one of the most devastating bacterial diseases affecting the tomato industry worldwide. The ability of Cm to infect seeds and plants without causing symptoms makes it a particularly dangerous pathogen. Unfortunately, there are no resistant tomato cultivars or effective chemical or biological control methods available to growers against Cm . Its control relies heavily on prevention. The implementation of a rapid and accurate detection tool is imperative to monitor the presence of Cm and prevent its spread. In this study, we developed a specific and sensitive multiplex TaqMan qPCR assay to detect Cm and distinguish it from related bacterial species that affect tomatoes. Two Cm chromosomal virulence-related genes, rhuM and tomA , were used as specific targets. The plant internal control tubulin alpha 3 was included in each of the multiplexes to improve the reliability of the assay. Specificity was evaluated with 12 Cm strains and 14 bacterial strains, including other Clavibacter spp. and bacterial pathogens of tomato from different geographic locations and sources. Results showed that the assay was able to detect all Cm strains but not other related bacteria. The assay was validated using plant tissue and seed samples following artificial infection and accurately detected the presence of Cm in all tested samples. The tool described here is highly specific, sensitive, and reliable for the detection of Cm and allows the quantification of Cm in seeds, roots, stems, and leaves, finding a lower abundance of Cm in the roots compared to the other parts of the plant. This new diagnostic assay can also be adapted for multiple purposes such as seed certification programs, surveillance, biosafety, the effectiveness of control methods, border protection, and epidemiological studies.