Rapid diagnosis of Ralstonia solanacearum infection sweet potato in China by loop-mediated isothermal amplification
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ORIGINAL PAPER
Rapid diagnosis of Ralstonia solanacearum infection sweet potato in China by loop‑mediated isothermal amplification Huawei Li1,2 · Hong Zhang1,2 · Zhonghua Liu1,2 · Zhijian Lin1,2 · Yongxiang Qiu1,2 · Hao Tang1,2 · Sixin Qiu1,2 Received: 17 November 2019 / Revised: 7 September 2020 / Accepted: 24 September 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020
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 strainspecific 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 1 03 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. Keywords Sweet potato · Bacterial wilt · R. solanacearum · LAMP Sweet potato [Ipomoea batatas (L.) Lam.] is an important crop and is a high-calorie food due to its readily digestible carbohydrates (Clark et al. 2012; Clark and Moyer 1988). More than 100 countries produce sweet potato, and countries in Asia produce over 80% of the world’s total volume (Chen et al. 2012). The bacterial wilt of sweet potato is a quarantine disease that is caused by soil-borne R. solanacearum and largely affects crop production (Chen et al. 2014; Clark et al. 1992; He et al. 1983). To date, this disease has only been reported in southern China in the provinces of Fujian, Guangdong, and Taiwan (Chen et al. 2011; Huang et al. Communicated by Erko Stackebrandt. * Sixin Qiu [email protected] 1
Institute of Crop Sciences, Fujian Academy of Agricultural Sciences, Fuzhou 350013, Fujian, China
Scientific Observing and Experimental Station of Tuber and Root Crops in South China, Ministry of Agriculture, Fuzhou 350013, Fujian, China
2
1956). In recent years, bacterial wilt of swee
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