Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Specific and Sensitive Detection of Mycocentrospora acerina (Hart.) Causing Round Leaf Spot Disease in Sanqi (Panax notoginseng)

Abstract

Round leaf spot, caused by Mycocentrospora acerina, is one of the most destructive diseases in Sanqi (Panax notoginseng) plantations in China. Accurate and timely detection of M. acerina is critical for developing effective integrated disease management strategies. Therefore, we developed a loop-mediated isothermal amplification (LAMP) assay for detection of M. acerina with primers targeting the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA). The LAMP reaction products were visually assessed using SYBR Green I and agarose gel electrophoresis. The ideal reaction temperature and time of LAMP assay were optimized to 64.5 °C and 45 min, respectively. The specificity of the developed LAMP assay was validated using 78 isolates belonging to 26 species, including M. acerina, Mycocentrospora species, and other plant pathogens. The LAMP assay was highly specific for M. acerina. Positive reactions were obtained only with the genomic DNA of M. acerina, and no cross-reaction was obtained with DNA extracted from other species. The detection limit of the LAMP assay for M. acerina was 10 fg genomic DNA per 25-μL reaction mixture. The LAMP assay successfully detected M. acerina in both symptomatic and latently infected leaf samples. The results indicate that the LAMP assay has the potential to be an efficient, highly specific, and sensitive method for diagnosing P. notoginseng round leaf spot disease caused by M. acerina in both the symptomatic and latent stages in the field and might be useful for disease management.