A Field-Portable Diagnostic Approach Confirms Laurel Wilt Disease Diagnosis in Minutes Instead of Days
Jeffrey L. Hamilton, Stephen W. Fraedrich, Campbell J. Nairn, Albert E. Mayfield, and Caterina Villari
Abstract: Background: Laurel wilt disease has caused the extensive mortality of lauraceous species in the southeastern United States. The causal agent is an invasive fungus, Raffaelea lauricola, which is a symbiont of the beetle Xyleborus glabratus and causes a rapid, fatal vascular wilt. Early diagnosis of laurel wilt is imperative for efficient disease management. The current diagnostic process, however, is slow due to the lengthy laboratory procedures required to confirm pathogen presence. Methods: We tested the robustness and field-portability of a recently developed, species-specific, loop-mediated isothermal amplification (LAMP) assay for R. lauricola, with the overall goal of eliminating the need for a laboratory confirmation of the diagnosis. We tested the robustness of the assay using benchtop equipment with naturally infected samples. We then tested the assay directly in the field using a portable device. Results: The assay successfully detected R. lauricola directly from symptomatic wood tissue using crude DNA extracts. Furthermore, the assay readily allowed users to distinguish between symptoms caused by R. lauricola infection and similar symptoms caused by other agents. In-field, we assayed wood samples from symptomatic redbay (Persea borbonia [L.] Spreng) and sassafras (Sassafras albidum [Nutt.] Nees) across the Southeast and successfully detected R. lauricola-infected trees in less than an hour. Conclusion: Results of this study confirmed that the field-deployable LAMP assay is robust and can rapidly and accurately detect R. lauricola in infected trees directly on-site. LAMP technology is well suited for in-field implementation, and these results serve as an incentive for further development and use of this technology in the field of forest pathology.
Keywords: Crude DNA; Early Detection and Rapid Response; In-Field; Loop-Mediated Isothermal Amplification (LAMP); Raffaelea