Development of a loop-mediated isothermal amplification assay for sensitive and specific detection of Fusarium oxysporum f. sp. cucumerinum Owen

Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum Owen (FOC), is a destructive disease affecting cucumber production worldwide. Developing an accurate and reliable method for detection of FOC is important for disease prediction and control. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated for specific and sensitive detection of FOC. Four LAMP primers were designed based on the sequence of the FOC-specific random amplified polymorphic DNA (RAPD) marker OPZ-12₈₆₅. LAMP reactions were performed at different temperatures and for different durations, and the optimal temperature and duration were 63 °C for 60 min, respectively. Hence, a LAMP assay for detection of FOC was established. The specificity of the LAMP method was evaluated against 119 isolates of FOC and other pathogens, and only FOC isolates yielded positive results. In sensitivity tests, the lowest concentration of genomic DNA required for the LAMP assay was 10 fg in a 25 μL reaction. The LAMP assay was successfully applied to detect FOC in cucumber tissues and soil from infested fields, and the positive ratios of LAMP, PCR, and traditional tissue isolation for detecting FOC from diseased cucumber root samples were100%, 86.6 and 83.3%, respectively. Therefore, the LAMP assay developed herein should serve as a simple, cost-effective, rapid, highly specific, and sensitive tool for the visual detection of FOC and contribute to improved disease management.