Field-Deployable Detection of Chestnut Blight Pathogen Cryphonectria parasitica Using Enzyme-Mediated Duplex Exponential Amplification.

Chestnut blight caused by Cryphonectria parasitica poses a major threat to chestnut plantations worldwide. Rapid, field-deployable diagnostic tools are essential for effective disease surveillance and management. Here, we developed and validated an enzyme-mediated duplex exponential amplification (EmDEA) assay for the specific and sensitive detection of C. parasitica. The assay successfully distinguished C. parasitica from non-target fungal species with a detection limit of 10 pg of genomic DNA per reaction. Application to naturally infected bark samples yielded results consistent with those of conventional qPCR. The complete workflow, including crude DNA extraction and fluorescence-based detection under isothermal conditions, was completed within 35 min. Our findings demonstrate that the EmDEA assay is a sensitive, robust, and field-adaptable tool that can be used for the early detection of chestnut blight, with significant potential for deployment in resource-limited environments.