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

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 8.0 mmol·L-1Mg2+, 1.4 mmol·L-1 dNTPs, and 0.32U μL-1 Bst 2.0 DNA polymerase and was performed at 65 °C for 1 h. 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 100 fg·μL-1 genomic DNA and 103 CFU·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.