Rapid detection of the plant pathogenic fungi Verticillium longisporum using species-specific fragment and enzyme mediated duplex exponential amplification (EmDEA) assay.

Verticillium longisporum is a highly destructive soil-borne pathogen responsible for verticillium wilt, a significant disease impacting numerous brassicaceous crops in various regions. Accurate species-level identification of V. longisporum is crucial for developing effective management strategies against verticillium wilt. However, the high sequence identity between V. longisporum and Verticillium dahliae, particularly at commonly used molecular markers like the Internal Transcribed Spacer (ITS) and Actin (ACT) genes, presents a significant challenge for accurate species-level differentiation. Here, we conducted a comprehensive comparative genomics analysis for Verticillium species. This approach, combined with targeted PCR validation, led to the identification of a novel V. longisporum-specific genomic fragment, designated VLspe2. Leveraging the sequence of VLspe2, we developed a precise Enzyme Mediated Duplex Exponential Amplification (EmDEA) assay for the rapid detection of V. longisporum, integrated with a fast DNA extraction protocol. The developed EmDEA assay demonstrated a minimum detection limit of 5 fg in a 20 µL reaction volume. This isothermal EmDEA assay offers accurate and rapid detection of V. longisporum at a low constant temperature (37–42 °C), providing invaluable guidance for on-site detection in agricultural fields and ports, ultimately contributing to plant disease management and food safety.