Development and Validation of a Field-Based Colorimetric LAMP Assay for the Detection of Clavibacter michiganensis in Tomato Plants

Point-of-care diagnostics are revolutionizing the detection of plant pathogens by enabling rapid, on-site identification without the need for specialized laboratories. One of the tools used for this purpose is loop-mediated isothermal amplification (LAMP). LAMP is a powerful molecular technique increasingly used in pathogen control for its rapid, sensitive, and specific detection of plant pathogens. The aim of this study was the development of a novel, easy-to-use portable colorimetric LAMP (cLAMP) assay that could be used by inexperienced personnel for the detection of the pathogen <i>Clavibacter michiganensis</i>. The assay was combined with a newly constructed device in which LAMP can be performed in 30 min. Initially, a new set of LAMP primers targeting the <i>micA</i> gene was designed and evaluated the sensitivity (100 fg/reaction) and specificity of the assay. Next, the limit of detection (LoD) of two different commercial LAMP kits was compared with common laboratory detection techniques (DAS-ELISA, immunofluorescence, quantitative PCR, and PCR) using the same samples. Additionally, the LoD of the developed cLAMP assay was evaluated in bacterial suspensions and plant extracts spiked with <i>C. michiganensis</i> and validated the effect on the LoD of plant extracts from different tomato varieties. Lastly, its efficacy for <i>C. michiganensis</i> detection was assessed in experimentally inoculated tomato seedlings. The developed method for <i>C. michiganensis</i> detection can be used as a reliable tool for the early detection of the pathogen for field-based applications by untrained personnel.