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Abstract
Laurel wilt is an invasive, fatal vascular wilt disease that is devastating lauraceous species in the southeastern United States. It is caused by Raffaelea lauricola, the mycangial symbiont of Xyleborus glabratus, and trees infected with the pathogen experience wilted foliage, sapwood discoloration, and rapid mortality. Since its introduction in 2002 near Savannah, Georgia, laurel wilt disease has quickly spread as far as Florida, Kentucky, North Carolina, and Texas. Current operations to prevent the spread of laurel wilt disease are delayed by a week or more by laboratory procedures to confirm the diagnosis. To mitigate damage and improve disease management, we developed a loop-mediated isothermal amplification (LAMP) assay for the rapid in-field detection of the pathogen. Our specie-specific assay is capable of amplifying as little as 0.5 pg of fungal DNA, as few as 50 spores, and can detect the pathogen directly from wood tissue in as little as 12 days post inoculation, even when testing crude DNA extracts. It can also detect the pathogen directly on the beetle host. To verify the robustness of the assay, we tested naturally infected wood samples from across the Southeast and successfully confirmed the presence or absence of the disease, even in those cases where visual symptoms were ambiguous. Finally, we validated the assay for use on a portable device directly at point-of-care, showing how the use of LAMP allows rapid disease confirmation within an hour of locating a potentially diseased plant. This study provides a successful example that should foster the use of LAMP based technology to rapidly diagnose laurel wilt disease directly in-field, ultimately enabling better disease management.