Development of a Loop-mediated Isothermal Amplification Detection Assay for Verticillium dahliae Infection in Chrysanthemum

Chang-Gi Back^1,* Mi-Jeong Park ¹ Kyung-Sook Han² Jong-Han Park¹

¹Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Wanju 55365, Korea
²Planning and Coordination Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Wanju 55365, Korea

Abstract

Verticillium wilt disease is caused by a fungal plant pathogen Verticillium dahliae, which attacks commercial crops such as chrysanthemum. The conventional methods so far used to identify this fungal pathogen require high expertise and are time-consuming. Therefore, in this study, we developed an assay for the rapid and specific detection of V. dahliae infection using loop-mediated isothermal amplification (LAMP) method. For this assay, four primers for LAMP were designed for targeting cellulose-growth-specific protein partial mRNA gene in Verticillium dahliae . Under standard condition, the optimum reaction temperature for amplification is around 60 ℃ within 60 minutes. This LAMP assay was designed to amplify only present in V. dahliae. When this LAMP assay applied to the DNAs for four other soilborne fungi and host plants, no amplification was detected. Therefore, this LAMP assay we developed for V. dahliae is expected to do detection at the early stage of its infection. The fast and reliable detection method will allow us to develop effective management system to monitor and control infection of this pathogen in chrysanthemum plant.

Figures & Tables

Fig. 1. Specificity test of loop-mediated isothermal amplification assay for detection of species.Lane M: 100 bp DNA Ladder, 1-3: V. (NIHHS 11-027, 13-118, 13-290) isolated from chrysanthemum (Chrysanthemum morifolium), 4-5: V. (NIHHS 13-252, 14-324) isolated by Chinese cabbage (Brassica rapa subsp. pekinensis), 6: V. chlamydosporium (KACC No. 40859), 7: sp. (KACC No. 42493), 8: Negative control.