Rapid diagnosis of Ralstonia solanacearum infection sweet potato in China by loop-mediated isothermal amplification.

Abstract

Bacterial wilt of sweet potato is caused by Ralstonia solanacearum, which is distributed in southern China and causes significant economic losses each year. The pathogen is soil- and rhizome-borne, and thus its rapid detection may prevent the occurrence and spread of the disease. R. solanacearum has been listed as a quarantine disease in China. With the advent of molecular biology, many novel tools have been explored for the rapid identification of plant pathogens. In this study, a strain-specific detection method was developed for this specific pathogen that infects sweet potato using loop-mediated isothermal amplification (LAMP). A set of new LAMP-specific primers was designed from the orf428 gene, which can specifically detect the R. solanacearum bacterium that infect sweet potato. The LAMP reaction consisted of 8.0mmol·L-1Mg2+, 1.4mmol·L-1 dNTPs, and 0.32UμL-1 Bst 2.0 DNA polymerase and was performed at 65°C for 1h. The amplification products were detected by visualizing a mixture of color changes using SYBR Green I dye and assessing ladder-like bands by electrophoresis. Our method has specificity, i.e., it only detected R. solanacearum in sweet potato, and it has high sensitivity, with a detection limit of 100fg·μL-1 genomic DNA and 103CFU·mL-1 of bacterial fluid. In addition, R. solanacearum could be directly detected in infected sweet potato tissues without the need for DNA extraction. The LAMP method established in this study is a highly specific, sensitive, and rapid tool for the detection of bacterial wilt in sweet potato caused by R. solanacearum.