Bacterial spot caused by Xanthomonas phaseoli pv. manihotis (Xpm) is considered the main bacterial disease that affects cassava, causing significant losses when not properly managed. In the present study, a fast, sensitive, and easy-to-apply method to detect Xpm via colorimetric loop-mediated isothermal amplification (LAMP) was developed. In order to ensure the use of a unique to the target pathovar core region for primer design, 74 complete genomic sequences of Xpm together with different bacterial species and pathovars were used for comparative genomics. A total of 42 unique genes were used to design 27 LAMP primer sets, from which nine primers were synthesized and only one (Xpm_Lp1 primer set) showed sufficient efficiency in preliminary tests. The sensitivity, assessed by a serial dilution of the type strain (IBSBF 278) DNA, yielded high sensitivity, detecting up to 100 fg. The LAMP primers showed high specificity, not cross-reacting with other bacterial species or other pathovars tested, and amplifying only the Xpm isolates. Tests confirmed the high efficiency of the protocol using infected or inoculated macerated cassava leaves, without the need for additional sample treatment. The LAMP test developed in this study was able to detect Xpm in a fast, simple, and sensitive way, and it can be used to monitor the disease under laboratory and field conditions.
Keywords: Causal Agent; Pathogen detection; Prokaryotes; Subject Areas; Techniques.