106 Hamilton et al: A Portable Diagnostic Approach Confirms Laurel Wilt Disease in Minutes quality. In our experiment, for instance, we had 3 false negatives. When samples were retested in the laboratory, however, samples tested positive, which was the correct diagnosis as confirmed by isolation of the fungus in culture. The only difference between the protocol in the field and the protocol in the lab was that in the lab we used less starting material for the extraction. This suggests that the reactions in the field failed due to the presence of inhibitors in the template. The possibility that the false negatives observed in-field were due to degradation of the reagents, on the other hand, was excluded because the positive controls amplified as expected both in terms of time and intensity. The presence of inhibitors in naturally infected, darkly pigmented samples was a common issue we encountered as we were trouble- shooting the crude DNA extraction protocol. Ulti- mately, this is the reason we decreased the amount of wood used during extractions and reduced the volume of template per reaction from 5 µL to 1 µL for in-field LAMP tests, compared to the protocol described in Hamilton et al. (2020). Despite the presence of inhib- itors, however, extracting from discolored wood tis- sues is still preferred, as it maximizes the probability of pathogen detection when the fungus is present. Three of the sassafras samples that tested positive for the presence of R. lauricola, both in the field and with further assessment in the laboratory, failed to exhibit growth when plated on a media. This might be because the fungus was no longer viable, even while its DNA was still present. The in-field testing phase of this study was conducted in the autumn and winter seasons, when sassafras has already shed its leaves, which made it difficult to determine how long sampled trees had been dead. Over time, the sapwood of trees with laurel wilt dries out, and it becomes increasingly difficult to isolate R. lauricola from samples (Fraedrich 2020, unpublished data). The 3 samples in question may have come from trees that had been dead for many months, and sample moisture content and colonization by secondary organisms may have affected the ability to isolate R. lauricola. Yet, the LAMP assay was still able to detect the pres- ence of R. lauricola DNA in samples. Future work should focus on further optimization of in-field LAMP-based diagnostic procedures. This includes the development of commercially available lyophilized, ready-to-use reagents and a more efficient crude DNA extraction protocol, preferably without ©2021 International Society of Arboriculture the need of a dedicated power source (for instance, as in Aglietti et al. 2019). Current LAMP technology requires minimal training for personnel performing the assay (Tomlinson et al. 2013; Thiessen et al. 2018). Additional improvements could further reduce the need for technical expertise and increase the ease and portability of the assays. In conclusion, this study demonstrates the success- ful use of LAMP-based technology to rapidly diagnose laurel wilt disease and possibly other tree pathogens directly in-field. These results should foster the in-field use of this technology for other forest pathogens as well, ultimately enabling better disease management. The capability of LAMP to provide rapid and accu- rate in-field confirmation of forest and other tree dis- eases contributes to an effective early detection and rapid response system to mitigate the impact of these damaging agents (Luchi et al. 2020). LITERATURE CITED Aglietti C, Luchi N, Pepori AL, Bartolini P, Pecori F, Raio A, Capretti P, Santini A. 2019. Real-time loop-mediated isothermal amplification: An early-warning tool for quarantine plant pathogen detection. AMB Express. 9(1):50. https://doi.org/10 .1186/s13568-019-0774-9 Aukema JE, Leung B, Kovacs K, Chivers C, Britton KO, Englin J, Frankel SJ, Haight RG, Holmes TP, Liebhold AM, McCullough DG, Von Holle B. 2011. Economic impacts of non-native forest insects in the continental United States. PLoS One. 6(9):e24587. https://doi.org/10.1371/journal.pone.0024587 Chong JH, Reid L, Williamson M. 2009. Distribution, host plants, and damage of the black twig borer, Xylosandrus compactus (Eichhoff), in South Carolina. Journal of Agricul- tural and Urban Entomology. 26(4):199-208. https://doi.org/ 10.3954/1523-5475-26.4.199 Colombari F, Villari C, Simonato M, Cascone P, Ferracini C, Alma A, Guerrieri E, Battisti A. 2016. Rapid on-site identifi- cation of the biocontrol agent of the Asian chestnut gall wasp. Biocontrol Science and Technology. 26(9):1285-1297. https://doi.org/10.1080/09583157.2016.1195335 Congdon B, Matson P, Begum F, Kehoe M, Coutts B. 2019. Application of loop-mediated isothermal amplification in an early warning system for epidemics of an externally sourced plant virus. Plants (Basel). 8(5):139. https://doi.org/10.3390/ plants8050139 Dreaden TJ, Davis JM, Harmon CL, Ploetz RC, Palmateer AJ, Soltis PS, Smith JA. 2014. Development of multilocus PCR assays for Raffaelea lauricola, causal agent of laurel wilt dis- ease. Plant Disease. 98(3):379-383. https://doi.org/10.1094/ PDIS-07-13-0772-RE Ebert K, Andreou M, Millington S, King DP. 2010. Evaluation of a portable amplification platform for loop-mediated iso- thermal amplification (LAMP) of foot-and-mouth disease virus (FMDV) and African swine fever (ASFV). Horsham,
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