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

• Published in: Archives of microbiology Vol. 203; no. 2; pp. 777 - 785
• Main Authors: Li, Huawei, Zhang, Hong, Liu, Zhonghua, Lin, Zhijian, Qiu, Yongxiang, Tang, Hao, Qiu, Sixin
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.03.2021; Springer Nature B.V
• Subjects: Agriculture - methods; Aquatic plants; Bacteria; Biochemistry; Biomedical and Life Sciences; Biotechnology; Cell Biology; China; Deoxyribonucleic acid; DNA; DNA polymerase; DNA Primers; DNA-directed DNA polymerase; Ecology; Economic impact; Electrophoresis; Gene amplification; Ipomoea batatas; Ipomoea batatas - microbiology; Life Sciences; Microbial Ecology; Microbiology; Molecular biology; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques; Original Paper; Pathogens; Plant bacterial diseases; Plant Diseases - microbiology; Potatoes; Ralstonia solanacearum; Ralstonia solanacearum - genetics; Rhizomes; Sweet potatoes; Vegetables; Wilt; China; Sweet potato; Bacterial wilt; LAMP; R. solanacearum
• ISSN: 0302-8933; 1432-072X
• DOI: 10.1007/s00203-020-02059-8

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 −1 Mg 2+ , 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 10 3  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 .