A New Multiplex TaqMan qPCR for Precise Detection and Quantification ofClavibacter michiganensisin Seeds and Plant Tissue

Abstract

ABSTRACTBacterial canker of tomato caused byClavibacter michiganensis(Cm) is one of the most devastating bacterial diseases affecting the tomato industry worldwide. As the result ofCmcolonization of the xylem, the susceptible host shows typical symptoms of wilt, marginal leaf necrosis, stem cankers, and ultimately plant death. However, is the ability ofCmto 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 againstCm. Its control relies heavily on prevention. The implementation of a rapid and accurate detection tool is imperative to monitor the presence ofCmand prevent its spread. In this study, we developed a specific and sensitive multiplex TaqMan qPCR assay to detectCmand distinguish it from related bacterial species that affect tomato plants. TwoCmchromosomal virulence-related genes,rhuM andtomA, were used as specific targets. The plant internal controltubulin alpha-3was included in each of the multiplexes to improve the reliability of the assay. Specificity was evaluated with 37 bacterial strains and more than 120 samples, including otherClavibacterspp. and related and unrelated bacterial pathogens from different geographic locations affecting a wide variety of hosts. Results showed that the assay was able to screen allCmstrains against other related bacteria. The assay was validated on tissue and seed samples following artificial infection and all tested samples accurately detected the presence ofCm. The tool described here is highly specific, sensitive, and reliable for the detection ofCmand allows the quantification ofCmin seeds, roots, stems, and leaves, finding a lower abundance ofCmin the roots compared to the other parts of the plant. 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.