Rapid Identification and Detection of Xanthomonas phaseoli pv. manihotis, Causing Bacterial Blight Disease in Cassava by Real-Time PCR using LNA Probe

Effective detection and early monitoring of Xanthomonas phaseoli pv. manihotis (Xpm) causing Cassava Bacterial Blight (CBB) disease using rapid DNA-based method is paramount in promoting curative management of the disease. The traditional methods of identification such as pathogen culturing-based upon morphological, biochemical and physiological approaches, traditional PCR involving post-PCR reaction processing gel electrophoresis are more often labour intensive, time-consuming and require exceptional classical taxonomical dexterity. Owing to these limitations of the traditional detection methods, we developed primer and Locked Nucleic Acid probe (LNA probe), and with the aid of Real-time PCR, Xpm was reliably detected from the extracted DNA and directly from the infected cassava plant tissues without DNA extraction within a relatively shorter time (20-25 min). The designed probe has a fluorescent amide (FAM) as reporter dye and calboxytetramethylrodamine (TAMRA) as repressive dye. The selectivity of the developed probe and primer sets were also tested against different Xanthomonas species, and other plant pathogenic bacteria from different genera as well as host cassava genomic DNA. In the end, only Xpm was sensitively and selectively detected by the developed probe, confirming reliable detection and identification of Xpm as the true causal agent of CBB of the infected cassava samples taken from various agroecological zones of Ghana This is the first study, which uses LNA probe, which is mostly used for human pathogens in the identification of cassava plant pathogen. (C) 2020 Friends Science Publishers