Arboriculture & Urban Forestry 47(3): May 2021 (Fraedrich 2008; Harrington et al. 2008). Although R. lauricola is a secondary pest of dying and unhealthy trees in its native Asian range (Shih et al. 2018), in North America the fungus is extremely pathogenic to many native lauraceous species (Hughes et al. 2015), including those currently outside of impacted areas, such as California laurel (Umbellularia californica [Hook. & Arn.] Nutt.)(e.g., Fraedrich 2008). Upon being introduced to Georgia and South Carolina by 2002 (Fraedrich et al. 2008), laurel wilt disease quickly spread south throughout Florida, west to Texas (Menard et al. 2016), along the Atlantic coast up to North Car- olina (North Carolina Forest Service 2012), and has recently crossed the Appalachian Mountains into Tennessee and Kentucky (Loyd et al. 2020). Since the disease is already widely established in the southeastern United States, eradication efforts are no longer viable, and current management suggestions include early detection and removal of infected trees in an effort to slow the spread of laurel wilt disease to new areas (Hughes et al. 2015). Early detection, in particular, is the most crucial and currently the most inefficient step for the successful management of the disease (Hughes et al. 2015). The guidelines in place to confirm the disease involve a lengthy process, includ- ing the shipment of a suspected sample to an equipped laboratory and the isolation of a pure fungal culture, followed by DNA extraction and species-specific PCR (Hughes et al. 2015). This is a time intensive process, often taking a week or more, and it signifi- cantly delays any disease management options. Fur- thermore, there are currently no PCR protocols that can reliably detect R. lauricola directly from host tis- sues, thus necessitating the isolation of the fungus before any molecular test can be performed (Dreaden et al. 2014) and emphasizing the need for a diagnostic test that can quickly and accurately detect R. lauricola directly from host tissues, ideally in the field. Loop-mediated isothermal amplification (LAMP) is a relatively recent molecular technique (Notomi et al. 2000) that has the capability of providing point-of- care diagnostic testing (Niessen 2015). LAMP is less sensitive to inhibitors than PCR (Francois et al. 2011), allowing for the use of crude DNA extracts, which can be rapidly obtained under field conditions (Kogovsek et al. 2015; Colombari et al. 2016). LAMP reactions are also isothermal, thereby removing the require- ment for a thermal cycler (Notomi et al. 2000). While basic LAMP reactions can be performed on a simple 99 hot plate, portable devices that feature a heating ele- ment along with fluorescence-based detection are also available for real-time monitoring (Ebert et al. 2010; Jenkins et al. 2011). The use of portable LAMP devices fulfills the criteria for Early Detection and Rapid Response as outlined by the US Department of the Interior for safeguarding natural resources (US Department of the Interior 2016) and would be a use- ful tool for the rapid detection of invasive pathogens such as R. lauricola. LAMP assays have been shown to successfully diagnose plant diseases, including those caused by bacterial (e.g., Keremane et al. 2015; Ocenar et al. 2019), viral (e.g., Congdon et al. 2019), and fun- gal pathogens (e.g., Tomlinson et al. 2010; Villari et al. 2013; Aglietti et al. 2019). We recently designed a species-specific LAMP assay for the molecular detection of R. lauricola directly from host tissues in less than 20 minutes of reaction time and as early as 12 days after inoculation when using crude DNA extracts (Hamilton et al. 2020). However, the assay has been only tested with artifi- cially inoculated and otherwise clean redbay samples, and only on a benchtop instrument in a laboratory set- ting. Further testing is needed to ensure the approach is suitable when used in the field, when samples might be colonized by multiple organisms, and when laboratory equipment is not available. Building upon our previous work, the aim of this study was to eval- uate the performance of the recently designed R. lau- ricola species-specific LAMP assay for use under field conditions comparable to those actually encoun- tered by laurel wilt surveillance personnel, arborists, or orchard managers. With respect to this, we divided the study into 2 successive phases with the specific objectives of (I) testing the suitability of the LAMP assay when using crude DNA extracts from naturally infected sassafras and redbay wood samples in a lab- oratory setting, and (II) verifying the feasibility of the LAMP assay when conducted directly in the field on portable equipment. MATERIALS AND METHODS Objective I: Testing of Naturally Infected Samples Sample Collection Redbay and sassafras samples were collected in the summer and autumn of 2018, respectively, from 6 dif- ferent locations in 6 different states of the southeastern ©2021 International Society of Arboriculture
May 2021
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