A New Multiplex TaqMan qPCR for Precise Detection and Quantification of Clavibacter michiganensis in Seeds and Plant Tissue

Abstract

Bacterial canker of tomato caused by Clavibacter michiganensis (Cm) is one of the most devastating bacterial diseases affecting the tomato industry worldwide. As the result of Cm colonization of the xylem, the susceptible host shows typical symptoms of wilt, marginal leaf necrosis, stem cankers, and ultimately plant death. However, is the ability of Cm to infect seeds and plants without causing symptoms what makes it an even more dangerous pathogen. Unfortunately, there are no resistant 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 tomato plants. 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 37 bacterial strains including other Clavibacter spp. and related and unrelated bacterial pathogens from different geographic locations affecting a wide variety of hosts. Results showed that the assay is able to discriminate Cm strains from other related bacteria. The assay was validated on tissue and seed samples following artificial infection and all tested samples accurately detected the presence of Cm. 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, and roots. The 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.