Techniques for isolation, identification and molecular characterization of Moko disease-related Ralstonia solanacearum strains

Abstract

Ralstonia solanacearum is one of the most destructive phytopathogens in the agriculture. In banana cultivation, the race 2 causes Moko disease, which is present in some areas in Mexico, and when epidemic outbreaks burst there are losses. Race 2 comprises subgroups (sequevars) which are usually identified by Multiplex PCR reactions. Isolation of R. solanacearum is usually hard, even on semiselective media, because it grows slowly and it is frequently contaminated with Pseudomonas, Klebsiella and Erwinia bacteria, which show similar phenotypes. This work shows a strategy that make easy the isolation, identification and classification of R. solanacearum race 2. The protocol comprises immunodiagnosis of plant suspicious samples, confirmation of diagnosis by PCR, isolation on semiselective media SMSA and B-King, and genotyping of sequevars by single, specific PCR reactions. In the present work 25 strains were isolated from Tabasco, and they genotyped in sequevar 6. Their previous genotyping by Multiplex yielded confuse results, which make evident that Multiplex can result in incorrect conclusions. We discourage to propose genetic divergence and emergency of new strains based on Multiplex results, as done in some recent reports.