DEVELOPMENT OF SPECIFIC PRIMERS FOR 16S rRNA GENE ANALYSIS IN THE DETECTION OF Ralstonia solanacearum SPECIES COMPLEX

Abstract

Members of Ralstonia solanacearum species complex (RSSC) are causal agents of vascular wilt disease in more than 450 crop species, including solanaceous plants such as potatoes, tomatoes, bell pepper, eggplant, etc. These phytopathogens cause serious yield loss mostly in solanaceous crops which are grown in tropical, subtropical, and temperate regions of the world. Yield losses comprise 80%–100% in potato, up to 91% for tomato, 10%–30% in tobacco, 33%–90% in banana, and reduce crop productivity and yield. PCR-methods are specific, sensitive and cost-effective approaches for the detection and identification of RSSC members. The objective of this study was to compare specificity of routinely used primer mix for PCR RSSC detection with the newly developed pairs of species-specific primers for ease of use diagnostics in a laboratory. Materials and Methods. The conserved genomic regions of the 16S rRNA sequences of R. solanacearum, R. pseudosolanacearum, and R. syzygii were selected for the design of primers for this study. Newly created primer species specificity was tested in PCR using the DNA of the two targets and 13 non-target strains of bacteria. Results. Three pairs of newly created primers Rs-28(F)/Rs-193(R), Rs-28(F)/OLI-160(R), Rs28(F)/OLI248(R) produced single specific fragments for bacterial strains of Ralstonia solanacearum: 166 bp, 132 bp, and 220 bp. products respectively. No PCR products were obtained during amplification with the negative control or non-target DNA templates from other bacterial species. Conclusion. Designed primers can be used for the development of PCR system for the qualitative and quantitative detection of RSSC members.