Development of a multiplex nested PCR method for detection of Xanthomonas axonopodis pv. manihotis in Cassava

Cassava (Manihot esculenta), an important multipurpose crop, represents a food source for millions of people and a source of industrial raw material in tropical regions. A limitation in its production is cassava bacterial blight (CBB), a disease caused by Xanthomonas axonopodis pv. manihotis (Xam). CBB is spread mainly by propagation of infected cuttings, therefore early pathogen detection and disease diagnosis are crucial to prevent its dispersal and the consequent negative impact on the crop. A previously reported detection method was inefficient at detecting isolates of Xam more recently collected in cassava fields in Colombia. Consequently, a new method for pathogen detection was developed using multiplex nested PCR. A set of primers was selected amongst a group designed for five housekeeping gene sequences (rpoB, gltA, ftsZ, gyrB and groEL), and for the region of the gene encoding for the C-terminal portion of TALE1 (Xam) , a protein involved in virulence of Xam. In this work, a multiplex nested PCR based on the set of primers RpoB and Cterm was optimized to achieve an effective detection of Xam. The developed method successfully detects the presence of Xam in cassava plants collected in infected fields.