Application of droplet digital PCR in detection of seed-transmitted pathogen Acidovorax citrulli

Abstract

Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide. The pathogen is seed-transmitted, so seed detection to prevent distribution of contaminated seed is crucial in disease management. In this study, we adapted a quantitative real-time PCR (qPCR) assay to droplet digital PCR (ddPCR) format for A. citrulli detection by optimizing reaction conditions. The performance of ddPCR in detecting A. citrulli pure culture, DNA, infested watermelon/melon seed and commercial seed samples were compared with multiplex PCR, qPCR, and dilution plating method. The lowest concentrations detected (LCD) by ddPCR reached up to 2 fg DNA, and 102 CFU mL−1 bacterial cells, which were ten times more sensitive than those of the qPCR. When testing artificially infested watermelon and melon seed, 0.1% infestation level was detectable using ddPCR and dilution plating method. The 26 positive samples were identified in 201 commercial seed samples through ddPCR, which was the highest positive number among all the methods. High detection sensitivity achieved by ddPCR demonstrated a promising technique for improving seed-transmitted pathogen detection threshold in the future.

Keywords

• bacterial fruit blotch
• Acidovorax citrulli
• droplet digital PCR
• seed detection
• quantitative real-time PCR