Rapid detection of the E198A mutation of carbendazim-resistant isolates in Colletotrichum gloeosporioides by loop-mediated isothermal amplification

Apple bitter rot is a serious agricultural disease caused by Colletotrichum gloeosporioides. In recent years, carbendazim-resistant C. gloeospori-oides strains bearing an E198A point mutation in the beta-tubulin gene (GAG to GCG) have emerged, threatening global apple production. As such, rapidly detecting the presence of this E198A mutation in C. gloeosporioides isolates is essential in order to monitor the spread of this pathogen and to prevent outbreaks of disease. Herein, we developed a simple loop-mediated isothermal amplification (LAMP) approach to detecting the E198A mutation in C. gloeosporioides isolates from 'Gala' apple samples. This optimized LAMP protocol was sufficient to establish the E198A genotype of a given isolate following a 60 min incubation at 63 degrees C by using four specific primers. The results of this reaction could be interpreted visually based on a fluorescent yellow-green color change upon the addition of the SYBR Green I dye, and were additionally confirmed via gel elec-trophoresis. Importantly, this LAMP assay was capable of rapidly and reliably detecting apples that were infected with carbendazim-resistant isolates harboring this E198A mutation. In conclusion, this LAMP assay in this study can rapidly, specifically, and sensitively detect cases of apple bitter rot caused by C. gloeosporioides isolates harboring the E198A mutation.

Automated summary

In this study, a simple LAMP approach was developed to rapidly detect the E198A mutation in apple bitter rot-causing C. gloeosporioides isolates. The optimized LAMP protocol allowed for the genotype determination of a given isolate within 60 minutes of incubation at 63 degrees C using four specific primers. The results of the reaction could be visually interpreted based on a fluorescent yellow-green color change and were further confirmed via gel electrophoresis. Importantly, this LAMP assay was capable of rapidly and reliably detecting apples infected with carbendazim-resistant isolates harboring the E198A mutation.