98 Hamilton et al: A Portable Diagnostic Approach Confirms Laurel Wilt Disease in Minutes Arboriculture & Urban Forestry 2021. 47(3):98–109 https://doi.org/10.48044/jauf.2021.010 URBAN FORESTRY ARBORICULTURE Scientific Journal of the International Society of Arboriculture & A Field-Portable Diagnostic Approach Confirms Laurel Wilt Disease Diagnosis in Minutes Instead of Days By Jeffrey L. Hamilton, MS, Stephen W. Fraedrich, PhD, Campbell J. Nairn, PhD, Albert E. Mayfield, PhD, and Caterina Villari, PhD 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 symp- tomatic 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 incen- tive 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 lauricola. INTRODUCTION Invasive diseases are one of the greatest threats to for- ests and individual trees, and pathogenic fungi and fungus-like organisms in particular cause the greatest amount of damage (Santini et al. 2013), such as in the case of the pathogens that cause chestnut blight and Dutch elm disease, each of which has resulted in the deaths of millions of trees (Freinkel 2007; Martín et al. 2018). In addition to their devastating effects on ecosystems, the cost of managing invasive species can reach into the billions of dollars annually (Pimentel et al. 2005; Aukema et al. 2011; National Invasive Species Council 2016). Efforts to protect trees from invasive fungal diseases are paramount to ensure the preservation of the benefits they provide. Early detec- tion, followed by a rapid response, are among the ©2021 International Society of Arboriculture crucial steps of frontline defense to contain invasive species and mitigate their damage (Lamarche et al. 2015; National Invasive Species Council 2016; Eschen et al. 2018). Laurel wilt is an invasive, fatal vascular disease that has resulted in extensive mortality among North American members of the Lauraceae family (Fraed- rich et al. 2008). The species most commonly affected are redbay (Persea borbonia [L.] Spreng), sassafras (Sassafras albidum [Nutt.] Nees), and avocado (Per- sea americana Mill.), which collectively include eco- logically and economically important forest, urban, and agriculture trees (Fraedrich et al. 2008; Ploetz et al. 2012). The disease is caused by an ambrosia fun- gus, Raffaelea lauricola, which is carried by a beetle vector, Xyleborus glabratus (redbay ambrosia beetle)
May 2021
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