Establishment of the Recombinase Polymerase Amplification-Lateral Flow Dipstick Detection Technique for Fusarium oxysporum.

Abstract

Fusarium oxysporum causes crown rot, wilt, root rot, and many other major plant diseases worldwide. During the progression of strawberry crown rot disease, the pathogen is transmitted from the mother plant to the seedling through the stolon, with obvious characteristics of latent infection. Therefore, rapid and timely detection of F. oxysporum is important for efficient disease management. In this study, a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) detection technique was developed for the rapid detection of F. oxysporum on strawberry plants by targeting the CYP51C gene, which is unique to Fusarium spp. Because this RPA-LFD detection technique was highly specific to F. oxysporum, other Fusarium and non-Fusarium fungi were not detected. The optimal reaction temperature and time for this technique were 39°C and 8 min, respectively. The detection limit was 1 pg of F. oxysporum genomic DNA in a 50-μl reaction system. A total of 46 strawberry plants with or without crown rot symptoms collected from Jiande, Changxing, and Haining in Zhejiang Province were further assessed for F. oxysporum infection using both RPA-LFD and traditional tissue isolation techniques. The RPA-LFD test showed that 32 of the 46 strawberry plants tested were positive for F. oxysporum, while in the traditional isolation technique, F. oxysporum was isolated from 30 of the 46 strawberry plants. These results suggest that our established RPA-LFD method is rapid, sensitive, and highly specific in detecting F. oxysporum infection in strawberry plants.