Establishment of a LAMP Assay for Rapid Detecting Diaporthe citri on Pomelo

  Objective  A loop-mediated isothermal amplification (LAMP) method for visual, rapid, and accurate detection of Diaporthe citri on pomelos in early stage of black spot disease was developed.

  Method  Based on the elongation factor-1α (EF-1α) sequence of D. citri, a set of LAMP primers was designed. Template DNA from the infected leaves was used to establish the optimal temperature and time for the LAMP operation. Assay specificity and sensitivity were verified by positive detection of the infected plants in the field.

  Result  The newly established LAMP method could effectively and specifically detect D. citri on pomelo at the optimal temperature of 65 ℃ with 60 m for the reaction time. The detection limit of the assay on D. citri was 10 fg·μL⁻¹. On 15 field samples with the typical black spot symptoms, a 100% positive detection rate was achieved by the LAMP assay.

  Conclusion  The established visual and rapid LAMP assay demonstrated a high specificity, sensitivity, and perfect detection of D. citri on diseased pomelo. It was considered appropriate for field application.