A Rapid and Portable EmDEA-Based Diagnostic System for On-Site Detection of Erwinia amylovora, the Causal Agent of Fire Blight.

Fire blight, caused by Erwinia amylovora, is a devastating bacterial disease that primarily affects rosaceous fruit trees, including apple (Malus spp.) and pear (Pyrus spp.), and poses a significant threat to global fruit production and orchard management. Effective control depends on precise, sensitive, and practical diagnostic technologies that can be deployed directly in the field. In this study, we developed a portable diagnostic system that enables the rapid and accurate detection of E. amylovora under on-site conditions. The system targets a highly conserved hypothetical protein gene specific to the pathogen, for which six primer pairs and three probes were designed and evaluated. The optimized probe-primer combination demonstrated high specificity, yielding no amplification in fifteen non-target microbial strains or in host plant tissues, and exhibited a sensitivity equivalent to that of quantitative PCR, reliably detecting as little as 100 femtograms of genomic DNA or 20 colony-forming units per milliliter of bacteria. To streamline sample processing, we employed cellulose filter paper strips to extract DNA within one minute, achieving detection from leaf surfaces at a sensitivity of 32 colony-forming units per square centimeter. The diagnostic kit is stable at room temperature and, when combined with a palm-sized heating device, allows the entire process from sample preparation to result interpretation to be completed in 5-21 min. Field trials further confirmed the suitability of this system for rapid pathogen detection. This study introduces a sensitive, portable, and user-friendly diagnostic platform that enhances fire blight management and provides a framework for developing similar tools against other plant diseases.