RAPID AND VISUAL DETECTION OF COLLETOTRICHUM GLOEOSPORIOIDES ON ANOECTOCHILUS USING LOOP-MEDIATED ISOTHERMAL AMPLIFICATION ASSAY

Abstract

Anthracnose, caused by Colletotrichum gloeosporioides, is among the most destructive diseases to the plant genus Anoectochilus in China. C. gloeosporioides can enter and spread through a commercial field via asymptomatic infected plants. Early and accurate detection of C. gloeosporioides is essential to controlling this disease in the field. To enable rapid detection of the pathogen on symptomless Anoectochilus plants, we developed a convenient, cost-effective, and highly sensitive loop-mediated isothermal amplification (LAMP) method. Four LAMP primers were designed based on the internal transcribed spacer (ITS) sequence of C. gloeosporioides. All 25 C. gloeosporioides isolates collected from geographically distinct counties in China yielded positive results in the LAMP assay. No cross-reaction was observed with other fungal pathogens. A sensitivity assay showed that the PCR and LAMP assays had detection limits of 10 pg and 10 fg of genomic DNA per 25-μL reaction, respectively. Furthermore, we used the PCR and LAMP assays for the detection of C. gloeosporioides DNA from naturally infected Anoectochilus plants. The LAMP assay developed in this study is simple, fast, sensitive, and specific, and can be used in the field to detect C. gloeosporioides in infected plant tissue.