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Development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of Hemileia vastatrix in coffee plantations

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Abstract

Coffee leaf rust (CLR) caused by Hemileia vastatrix is a devastating worldwide disease. Early monitoring is crucial for controlling CLR quickly and efficiently. However, accurately identifying CLR in its early stages via the naked eye is challenging. Moreover, detecting H. vastatrix using PCR-based methods is time-consuming, labour-intensive, and occasionally exhibits low sensitivity. Loop-Mediated Isothermal Amplification (LAMP) technology is known for its speed, specificity, and sensitivity to identifying many pathogens accurately. Therefore, in this study, we conducted a comparative analysis of ITS sequences from H. vastatrix and other H. vastatrix and Uredinales strains available in the National Center for Biotechnology Information (NCBI) database using the BLASTn tool. Based on this analysis, we designed specific primers that target the unique region and its flanking regions within the ITS sequences of H. vastatrix. Using SYBR Green I dye, we established a LAMP technique for rapid and sensitive detection of H. vastatrix. Moreover, we optimised the LAMP protocol to enhance sensitivity and specificity for H. vastatrix detection. Under the optimised conditions, the established LAMP protocol detected as little as 1pg/μL of H. vastatrix DNA within 60min at 63°C. This sensitivity is approximately 100 times higher than that achieved using conventional PCR. Our method proved effective in detecting H. vastatrix at the early stages of CLR symptom development on the coffee leaves in field conditions.

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Data availability

DNA sequences are available in the GenBank database, with the accession numbers listed in the Results. All other relevant data are within the paper.

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Conflicts of interest

The authors declare no conflicts of interest.

Funding

This research was funded by the National Key R & D Program of China, grant number 2018YFD0201100; Special Funds for Efficient Tropical Agriculture Development of Hainan Province (UF37721); FAO/IAEA Collaborative Research Project, grant number No. 20380; Hainan Provincial Natural Science Foundation of China, grant number 320QN187; the Central Public-Interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences, grant number No. 1630042017021.

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  1. Weihuai Wu, Guihua Wang and Han Wang contributed equally to this work.

Authors and Affiliations

  1. Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, China

    Weihuai Wu, Guihua Wang, Han Wang, Thomas Gbokie Jr, Chunping He, Xing Huang, Yanqiong Liang, Rui Li & Kexian Yi

  2. Yunnan International Joint Laboratory of Coffee Research, Mangshi, Yunnan, China

    Weihuai Wu

  3. School of Tropical Agriculture and Forestry, Hainan University, Haikou, China

    Guihua Wang

  4. College of Plant Protection, Nanjing Agricultural University, Nanjing, China

    Thomas Gbokie Jr

  5. Sanya Research Institute, Chinese Academy of Tropical Agricultural Sciences, Sanya, People’s Republic of China

    Kexian Yi

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  1. Weihuai Wu
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Contributions

Conceptualization, Kexian Yi; Methodology, Weihuai Wu; Validation, Han Wang; Formal analysis, Weihuai Wu, Han Wang, and Guihua Wang; Investigation, Thomas Gbokie, Jr., Yanqiong Liang, and Rui Li; Data curation, Weihuai Wu, Han Wang; Writing-original draft preparation, Weihuai Wu, Guihua Wang; Writing-review and editing, Guihua Wang, Weihuai Wu; Review, Kexian Yi; Supervision, Kexian Yi; and Project administration, Chunping He and Xing Huang. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Kexian Yi.

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Wu, W., Wang, G., Wang, H. et al. Development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of Hemileia vastatrix in coffee plantations. Trop. plant pathol. (2024). https://doi.org/10.1007/s40858-023-00627-z

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