100 Hamilton et al: A Portable Diagnostic Approach Confirms Laurel Wilt Disease in Minutes Table 1. Results of laboratory LAMP assays and corresponding validation via fungal isolation to detect Raffaelea lauricola from wood tissue samples collected from sites with laurel wilt disease in the southeastern US. Results are reported as number of positive assays or fungal isolations out of the total samples tested. State Collecting agency Site name Host species Arkansas Georgia Louisiana North Carolina South Carolina Texas Arkansas Department of Agriculture, Forestry Division Georgia Forestry Commission United States Forest Service North Carolina Forest Service South Carolina Forestry Commission United States Forest Service John H. Kirby State Park Redbay 0/5 0/5 0/5 0/5 Conway Cemetery Historic State Park Toomsboro Kisatchie National Forest Cabin Lake Road Sesquicentennial State Park Sassafras Redbay Sassafras Redbay Redbay Symptomatic samples 3/5 3/5 0/3 4/5 4/5 3/5 3/5 0/3 4/5 4/5 Non-symptomatic samples LAMP Fungal LAMP Fungal isolation 0/5 0/5 0/3 0/5 0/5 0/5 0/3 0/5 0/5 isolation 0/5 United States, including Arkansas, Georgia, Louisi- ana, North Carolina, South Carolina, and Texas. At each location, 2 sites were selected: one where laurel wilt disease was known to occur, and one where the disease did not occur. From each site with diseased trees, 5 symptomatic and 5 non-symptomatic trees were sampled (Table 1), while at sites with no dis- ease, only 5 non-symptomatic trees were sampled (Table 2). The only exceptions to this sampling design were that (1) in South Carolina, no non-diseased site could be found, and (2) at the Louisiana location, only 3 trees were sampled per symptom type per site. Samples consisted of 10-cm-long branch or stem sec- tions, ranging between 4 mm and 15 mm in diameter. Sampling was accomplished with the assistance of local state and federal forestry agencies, who identi- fied and collected the samples during their routine surveillance operations and then shipped them over- night on ice to the University of Georgia. Once received, samples were stored at ˗20 °C until further processing and analyses. DNA Extraction in the Laboratory Crude DNA extracts were obtained following the protocol described in Hamilton et al. (2020). Briefly, 15 mg to 20 mg of wood shavings were removed from samples with a scalpel after debarking and added to a 1.5 mL micro-centrifuge tube containing 300 mL of 5% Chelex 100 (BioRad, California, USA). The wood shavings were then boiled for 5 minutes, vor- texed for 15 seconds, boiled again for 5 minutes, and vortexed again for 15 seconds, before being spun ©2021 International Society of Arboriculture down in a mini-centrifuge (VWR, Radnor, PA, USA) at 3,884 × g for 30 seconds. The supernatant was used as template in the LAMP reactions. LAMP Assay on a Benchtop Instrument LAMP reactions were performed as per Hamilton et al. (2020) with one modification, whereby less DNA template (i.e., the total DNA that goes in every reac- tion) was used in order to reduce background noise and potential inhibition of the LAMP assays. Each 25 mL reaction contained 15 mL 1× no-dye Isothermal Master Mix (Optigene, Horsham, UK), 0.7 mL of each internal primer BT-FIP and BT-BIP (100 mM), 0.7 mL of each external primer BT-F3 and BT-B3 (10 mM), 0.2 mL of F-Loop primer (100 mM), 0.24 mL of assimilating probe fluorescent (FAM) strand (10 mM), 0.46 mL of quencher strand (10 mM), 5.3 mL of molec- ular grade water (Thermo Fisher Scientific, Waltham, MA, USA), and 1 mL of crude DNA template. All primers were diluted in molecular grade water. Reac- tions were carried out in 0.2-mL optically clear PCR eight-tube strips (Thermo Fisher Scientific) and run on a benchtop StepOnePlus Real-Time PCR system (Thermo Fisher Scientific) programmed with the fol- lowing conditions: preheat to 65 °C for 1 minute, 65 °C for 30 minutes with fluorescence reading every minute, followed by a denaturing step at 85 °C for 5 minutes to halt the reaction and deactivate the poly- merase. Each sample was run in triplicate, and each reaction included a tube with 1 ng of high quality DNA of R. lauricola isolate FS-0009 (Hamilton et al. 2020) as a positive control, in addition to a
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