Loop-mediated isothermal amplification combined with alkaline lysis to detect Colletotrichum fructicola infection on oil-tea plants.

Abstract

BACKGROUND Camellia oleifera anthracnose (COA), caused by Colletotrichum fructicola, is a serious threat to both the yield and quality of Camellia oleifera oil (COO). To detect and distinguish incipient C. fructicola infection on asymptomatic plants is essential for COA control. Therefore, an efficient and rapid diagnostic method for detection of C. fructicola is desperately needed for making management strategies promptly. RESULTS We created a loop-mediated isothermal amplification (LAMP) method in conjunction with alkaline lysis (AL) to differentiate C. fructicola from other COA-related Colletotrichum species. Compared to the conventional polymerase chain reaction (PCR) method, the LAMP method not only exhibited higher sensitivity and specificity in the detection of C. fructicola but also required less complicated equipment (just a dry bath), less operating time (30 min for plant crude DNA lysis and 15 min for LAMP assay), and could be used for on-site diagnosis. The minimum detectable concentration of the C. fructicola DNA using LAMP was 10 pg, which was 100 times lower than the conventional PCR. Furthermore, the AL method of extracting crude genomic DNAs (gDNAs) from oil tea leaves could be applied for the LAMP detection under field conditions with a sensitivity of up to 5 × 103 spores mL-1 of C. fructicola infection, which was ten times more sensitive than conventional PCR. CONCLUSION We developed a LAMP assay combined with an AL DNA extraction method (LAMP-AL) to accurately detect and differentiate the C. fructicola infection on oil-tea leaves. The assays specifically detailed here are portable and simple to apply in the field conditions with limited resources. © 2025 Society of Chemical Industry.