Arboriculture & Urban Forestry 47(3): May 2021 ice packs until testing. Prior to testing, samples were centrifuged 2,000 × g for 10 seconds on a Corning LSE #6770 mini-centrifuge (Corning, NY, USA) connected to the same power source previously used for the kettle, and only the supernatant was used as template in the LAMP reactions. LAMP Assay on a Portable Device Preliminary testing was performed using a BioRanger™ Platform (Diagenetix, Honolulu, HI, USA), but because the device functioning was not consistent (data not shown), we decided to proceed with a Genie® III (Optigene) portable LAMP device. LAMP reactions in the field were performed using the same crude DNA reaction mix as described above, which had been prepared beforehand (24 hours to 36 hours prior to testing) and already aliquoted into strips. Ready-to-use reaction strips, with each including a positive control and a negative control as described above, were double wrapped in aluminum foil to avoid photodegradation of the probes and stored at 4 °C until use. During travel, reagents were constantly kept on ice. After adding 1 mL of template, LAMP reactions were performed on the Genie® III (Optigene) device programmed with the following conditions: 65 °C for 30 minutes with the default fluorescence reading (15 seconds) followed by an inactivation step at 85 °C for 5 minutes to halt the reaction and deactivate the polymerase. Verification of Field Results Field collected samples were transported to the labo- ratory to further verify the presence of R. lauricola. A portion of tissue of each sample was plated onto amended malt extract agar as previously described, and the presence of mycelial growth of R. lauricola or its absence was recorded. Discrepancies among field LAMP assay results and plating were further assessed as follows: (1) if a sample tested positive according to the in-field LAMP assay, but R. lauricola was not recovered when plated, a high quality DNA extraction followed by a LAMP assay on the benchtop equipment was performed following the protocol previously described; (2) if a sample tested negative according to the in-field LAMP assay, but R. lauricola was recov- ered when plated, a crude DNA extraction with less starting material, to reduce potential inhibition, was performed in the laboratory, followed by a LAMP assay on the portable device, as previously described. For high quality DNA extractions, samples were ground in liquid nitrogen and extracted with a Qiagen 103 DNeasy plant mini kit (QIAGEN, Germantown, MD, USA) as per the manufacturer’s protocol. Statistical Analysis To determine the accuracy of the in-field LAMP assay compared to the actual presence of R. lauricola in the samples, the true positive rate (i.e., sensitivity) was calculated as the number of correct positive pre- dictions (i.e., positive in-field LAMP assays con- firmed by either laboratory isolations or high quality DNA testing) divided by the total number of positives (i.e., positive laboratory isolations and high quality DNA testing)(Yerushalmy 1947) using Microsoft Excel® 2013 (Version 15.0.5215.1000). RESULTS Objective I: Testing of Naturally Infected Samples LAMP Testing in the Laboratory The results of the laboratory validation of naturally infected samples collected in diseased locations are reported in Table 1, whereas the samples collected in the non-diseased locations are reported in Table 2. Of the symptomatic host samples received from cooper- ators, 4 out of 5 samples from North Carolina and South Carolina and 3 out of 5 samples from Arkansas and Georgia tested positive for R. lauricola, whereas none of the symptomatic samples collected in Louisi- ana or Texas tested positive for the pathogen (Table 1). None of the samples from non-symptomatic trees gave a positive result with the LAMP assay, regard- less of whether or not laurel wilt was present at the location (Tables 1 and 2). Fungal Isolation Results of the fungal isolations were all consistent with the results of the LAMP assays (Tables 1 and 2). Growth of R. lauricola was only observed for those samples that tested positive with the LAMP assay, while no growth was observed when the results of the LAMP assay were negative. Objective II: In-Field Testing of the LAMP Assay LAMP Assay on a Portable Device The results of the in-field testing of symptomatic samples are reported in Table 3, and an example of individual runs results from each state is shown in ©2021 International Society of Arboriculture
May 2021
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