Rapid Detection of <i>Colletotrichum siamense</i> from Infected Tea Plants Using Filter-Disc DNA Extraction and Loop-Mediated Isothermal Amplification
Huayan Zou, Taiwen Li, Jing Zhang, Huijuan Shao, Koji Kageyama, Wenzhuo Feng
Abstract
The pathogen Colletotrichum siamense causes tea anthracnose, resulting in economic losses to the Chinese tea industry. To effectively diagnose this pathogen in the field, we developed a loop-mediated isothermal amplification (LAMP) method using highly specific primers with a sensitivity of 1 pg/μl designed for amplifying the CAL gene, which was 10 times higher than that of conventional PCR. Additionally, to improve the method for obtaining DNA samples required for on-site diagnosis, we used the filter-disc DNA extraction method, which does not require special instruments and can be completed in a few minutes, and found that it effectively meets the requirements for the LAMP reaction. Finally, we combined LAMP with a filter-disc DNA extraction method (FDE-LAMP) to diagnose different degrees of disease in inoculated samples and 20 samples from the field. The results showed that the procedure had sufficient sensitivity for pathogen detection. Therefore, the FDE-LAMP procedure could greatly contribute to managing and preventing tea anthracnose in the field.