Establishment of a rapid detection technology system for Neofusicoccum laricinum based on CEEA isothermal amplification

Neofusicoccum laricinum (Sawada) Y. Hattori & C. Nakash is a quarantine pest of imported plants and a forestry quarantine pest in China. It is also one of only two fungal plant pathogens listed in the Catalogue of Key Managed Invasive Alien Species. Developing rapid, accurate, and visual detection method is crucial for effective prevention and control of this disease. In this study, a method combining composite enzyme index amplification （CEEA） technique with lateral flow dipstick （LFD） technology was developed. Using specifically designed CEEA primer sets and probes for N. laricinum, followed by optimization of reaction conditions （39 ℃ for 15 minutes）, the method could specifically detect N. laricinum strains from diverse regions with a limit of detection (LOD) as low as 100.0 fg·µL⁻¹, the entire process to be completed in 30 minutes. This technology requires no expensive instruments and does not identify other fungi on Larch, demonstrating good applicability. With its high efficiency, speed, and sensitivity, this technology provides strong support for in-situ rapid detection and identification of pathogens in the forest, as well as for the early warning of disease.