Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Anthracnose Disease Caused by Colletotrichum asianum and Colletotrichum musae

Anthracnose is a plant disease that causes significant post-harvest damage to agricultural products. It is primarily caused by fungi of the Colletotrichum genus, which can infect plants at any growth stage. Notably, Colletotrichum can exist in a latent form, infecting immature fruits without showing symptoms until ripening. This leads to undesirable fruit appearance, reduced shelf life, and hindered export potential. This research aimed to develop a Loop-Mediated Isothermal Amplification (LAMP) assay for the detection of anthracnose disease caused by Colletotrichum asianum. Fruits of mango, guava, dragon fruit, and banana showing disease symptoms were collected from markets around Naresuan University and cultured using a tissue transplant technique. The study isolated 11 fungal strains, which were grouped into three categories based on morphological characteristics such as mycelial color and conidial shape. Internal Transcribed Spacer (ITS) sequencing identified five fungal species: three Colletotrichum species (C. musae, C. fructicola, and C. asianum), along with Fusarium incarnatum and Pestalotiopsis sydowiana from other genera. The developed LAMP assay successfully detected C. asianum and C. musae at a constant temperature of 65 °C within 45 minutes. The detection sensitivity for C. asianum was as low as 1 ng/µL of DNA, while C. musae required a minimum of 25 ng/µL. When compared to conventional PCR,the LAMP assay demonstrated up to 10-fold higher sensitivity. In conclusion, the LAMP technique proves to be an efficient and rapid method for detecting anthracnose disease, allowing for more effective disease management and a reduction in post-harvest losses, including those affecting fruit exportation.