Development and Evaluation of a Loop-Mediated Isothermal Amplifcation (LAMP) Assay for Specific and Sensitive Detection of <i>Puccinia melanocephala</i> Causing Brown Rust in Sugarcane
Weihuai Wu,
Guihua Wang,
Han Wang,
Liqian Zhu,
Yanqiong Liang,
Thomas Gbokie,
Ying Lu,
Xing Huang,
Chunping He,
Jianfeng Qin,
Kexian Yi
Affiliations
Weihuai Wu
Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
Guihua Wang
Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
Han Wang
Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
Liqian Zhu
Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
Yanqiong Liang
Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
Thomas Gbokie
Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
Ying Lu
Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
Xing Huang
Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
Chunping He
Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
Jianfeng Qin
Guangxi Subtropical Crops Research Institute, Nanning 530001, China
Kexian Yi
Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
Sugarcane brown rust (SCBR), caused by Puccinia melanocephala, is a destructive fungal disease that has extensively spread in the sugarcane-cultivating regions across the world. Early monitoring plays an important role in predicting the P. melanocephala epidemic and managing SCBR. However, accurately identifying SCBR based on symptoms and urediniospore morphology at the initial stage is a challenge. Further, it is tedious, time-consuming, labor-intensive, and requires expensive equipment to detect P. melanocephala using PCR-based methods. Loop-mediated isothermal amplification (LAMP) technology is renowned for its speed, simplicity, and low equipment requirements for specifically and sensitively identifying many pathogens. Therefore, in this study, a novel and highly sensitive LAMP assay was developed for the specific detection of P. melanocephala in sugarcane. Here, the internal transcribed spacer (ITS) sequence of P. melanocephala was selected as the target gene for LAMP primer design. Based on the color change of SYBR Green I and gel electrophoresis, specific LAMP primers were screened. Further, the optimal reaction conditions for the LAMP assay were determined at 63 °C for 60 min. The LAMP assay showed a high degree of specificity for the detection of P. melanocephala in sugarcane, with no cross-reactivity with other fungal pathogens. The established LAMP protocol was highly sensitive and can be used to detect as low as 1 pg/μL of P. melanocephala plasmid DNA, which is comparable to that of nested PCR and ~100 times more sensitive than conventional PCR. Finally, the detection rate of the LAMP method was higher than that of conventional and nested PCR in field samples.
