Rapid and simple colorimetric detection of quiescent Colletotrichum in harvested fruit using reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) technology

Anthracnose, caused by the fungus Colletotrichum gloeosporioides, is one of the major causes of postharvest decay of fruits and vegetables. Detection of the pathogen at an early stage of infection is crucial to developing a disease management strategy. In this work, a loop-mediated isothermal amplification (LAMP) assay was developed for the rapid detection of C. gloeosporioides targeting the transcript enoyl-CoA hydratase (ECH) that significantly upregulates only during C. gloeosporioides quiescent stage. The assay enabled a naked-eye detection of C. gloeosporioides RNA within 23 min based on a color change of LAMP products from pink to yellow. The detection limit of the LAMP assay was 1 pg of total RNA extracted from fruit peel in a 25 μL reaction. Positive results were obtained only in samples carrying the ECH gene, whereas no cross-reaction was observed for a different quiescent marker (histone deacetylase (HDAC)) or an appressorium marker (scytalone dehydratase, (SD)), indicating the high specificity of the method. Hence, the results indicate that the developed LAMP assay is a rapid, highly sensitive, and specific tool for the early detection of quiescent C. gloeosporioides and could be employed to manage postharvest diseases.