A comparative study of three detection techniques for Leifsonia xyli subsp. xyli, the causal pathogen of sugarcane ratoon stunting disease

Authors: WU, QIBIN - Fujian Agricultural & Forestry University; Pan, Yong-Bao; ZHOU, DINGGANG - Fujian Agricultural & Forestry University; Grisham, Michael; GAO, SHIWU - Fujian Agricultural & Forestry University; SU, YACHUN - Fujian Agricultural & Forestry University; GUO, JINLONG - Fujian Agricultural & Forestry University; QUE, YOUXIONG - Fujian Agricultural & Forestry University; XU, LIPIING - Fujian Agricultural & Forestry University

Submitted to: BioMed Research International
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/23/2018
Publication Date: 5/23/2018
Publication URL: https://handle.nal.usda.gov/10113/6740177
Citation: Wu, Q., Pan, Y.-B., Zhou, D., Grisham, M.P., Gao, S., Su, Y., Guo, J., Que, Y., Xu, L. 2018. A comparative study of three detection techniques for Leifsonia xyli subsp. xyli, the causal pathogen of sugarcane ratoon stunting disease. BioMed Research International. 2018(2786458):1-11. https://doi.org/10.1155/2018/2786458.
DOI: https://doi.org/10.1155/2018/2786458

Interpretive Summary: Sugarcane ratoon stunting disease (RSD) is one of the most serious diseases causing significant yield losses and variety degradation. The disease is caused by a tiny bacterium Leifsonia xyli subsp. xyli (Lxx) that is very difficult to isolate, culture, and detect. Because Lxx-infected sugarcane plants do not exhibit external symptoms, disease diagnosis is challenging and is done through immunological and molecular assays. In this study, the specificity and sensitivity of three molecular detection techniques, namely, conventional polymerase chain reaction (PCR), Lxx-loop mediated isothermal amplification (Lxx-LAMP) and real-time quantitative PCR (RT-qPCR), were compared for the detection of Lxx DNA from two types of experimental samples, i.e., infected sugarcane juice and a recombinant pMD18-T plasmid containing a DNA fragment of Lxx. The results showed that all these techniques could detect Lxx with great reproducibility. When the DNA extracted from Lxx-infected sugarcane juice was used as a template, Lxx-LAMP had the highest detection sensitivity, which was 10-fold higher than RT-qPCR and 100-fold higher than conventional PCR. When the Lxx-pMD18-T recombinant plasmid was used as the template, Lxx-LAMP was as sensitive as RT-qPCR, but was 10 times more sensitive than conventional PCR. Furthermore, adding 0.4 µM loop primers (LF/LP) can accelerate the reaction and reduce the total reaction time and the optimal amount of PCR enzyme was determined to be 6.0 Units per Lxx-LAMP reaction. The results presented herein will provide experimental support for the detection of the RSD pathogen Lxx and a theoretical foundation for further application.

Technical Abstract: Ratoon stunting disease (RSD), which is caused by the bacterium Leifsonia xyli subsp. xyli (Lxx), is now recognized worldwide as the most economically devastating disease impacting sugarcane. RSD causes significant yield losses and variety degradation. Diagnosis of RSD is challenging because it does not exhibit any discernible internal and external symptoms. Moreover, the Lxx bacteria are very small and difficult to isolate, cultivate and detect. In this study, conventional polymerase chain reaction (PCR), Lxx-loop mediated isothermal amplification (Lxx-LAMP) and real-time quantitative PCR (RT-qPCR) were utilized to specifically detect the presence of pathogens in Lxx+ sugarcane juice of Yue-gan 18 and Lxx-pMD18-T recombinant plasmid. The results showed that Lxx is a highly specific causal pathogen for RSD. All three techniques provided great reproducibility, while Lxx-LAMP had the highest sensitivity. When DNA extracted from Lxx+ sugarcane juice was used as a template, the sensitivity of Lxx-LAMP was 10- and 100-fold higher than that of RT-qPCR and conventional PCR, respectively. When Lxx-pMD18-T recombinant plasmid was used as a template, Lxx-LAMP was as sensitive as RT-qPCR, but was 10 times more sensitive than conventional PCR. Based on the Lxx-LAMP detection system established in our lab, adding 0.4 µmol/L loop primers (LF/LP) can accelerate the reaction and reduce the total time required. In addition, the amount of Bst DNA polymerase large fragment was further optimized and the optimal amount for Lxx-LAMP reaction was determined to be 6.0 U. The results presented herein provide experimental support for the detection of RSD Lxx pathogen and a theoretical foundation for further application.