Development of a Duplex Droplet Digital Polymerase Chain Reaction Assay for Simultaneous Quantitative Detection of Two Erwinia Species

Fire blight and black shoot blight are two major bacterial diseases that affect apple and pear production in Korea and are caused by Erwinia amylovora and E. pyrifoliae, respectively. These diseases have recently reached epidemic levels, heightening the risk of coinfection within orchards and even individual trees. Traditional detection methods often fail to distinguish between these pathogens due to their similar characteristics and disease symptoms creating an urgent need for improved detection tools. In this study, we developed a novel droplet digital polymerase chain reaction (ddPCR) technique that specifically quantifies E. amylovora, with a detection range of 103 to 107 cfu/ml for cell culture templates and copies/ml for genomic DNA templates. The ddPCR platform is equipped with two fluorescence channels (FAM and HEX/VIC) and was further applied to develop a duplex ddPCR method for the simultaneous detection and absolute quantification of both pathogens. The method was tested on mixed DNA and cell cultures of E. amylovora and E. pyrifoliae and successfully quantified both pathogens within a single reaction. Moreover, duplex ddPCR effectively identified both pathogens in fruits artificially inoculated with various bacterial mixtures. This study provides valuable insights for addressing the cooccurrence of Erwinia diseases in orchards and offers a promising approach for precise diagnosis in plant disease management.