Arboriculture & Urban Forestry 47(3): May 2021 Verification of Field Results Results of the fungal isolations were mostly consistent with the results of the LAMP assay (Table 3), but dis- crepancies in some of the results were observed. Spe- cifically, 2 of the sassafras samples that tested positive at the Georgia site and 1 of the sassafras samples that tested positive in the South Carolina site did not result in growth of R. lauricola once plated. High quality DNA of these wood samples was extracted in the labora- tory and tested again with a LAMP assay on benchtop equipment, and a positive result was observed in all cases (Figure 2), confirming that the results previ- ously observed in the field were a true positive. On the other hand, the 2 redbay samples that tested negative in the North Carolina site and 1 of the redbay samples that tested negative in the South Carolina site resulted in growth of R. lauricola once plated, indicating that the results previously observed in the field were a false negative. To further assess if the false negative was potentially due to inhibition of the reaction, a new crude extraction with less starting plant material was per- formed in the laboratory, followed by a LAMP reaction on the portable device. Results of this second reaction were all positives for the presence of R. lauricola (Table 3), confirming that the false negative results observed in the field were probably due to inhibition of the reaction. Overall true positive rate of the LAMP assay performed in field conditions was 90%. Figure 2. Example of a laboratory assessment of discording results between in-field LAMP assay using a portable device and fungal plating. Positive results are identifiable by a rapidly increasing rate of fluorescence over time, indicating the suc- cessful amplification of target DNA. Samples that tested posi- tive in the field, but that did not result in growth of Raffaelea lauricola when plated in growing media, were further assessed by high quality DNA extraction followed by a LAMP assay on benchtop equipment. Each reaction was run in 3 technical replicates. GA, Georgia; SC, South Carolina; NTC, no template control; PC, positive control. DISCUSSION This study demonstrates that a LAMP-based molecular assay can rapidly and accurately detect the laurel wilt disease pathogen R. lauricola directly in-field without the reliance on an external laboratory. Building upon previous work that developed the species-specific LAMP primers (Hamilton et al. 2020), we were able to show that the assay is capable of correctly detect- ing the pathogen directly in naturally infected sam- ples, even when multiple organisms are likely to be colonizing the tissues. Moreover, we were able to determine the reliability of the LAMP assay when conducted directly in the field on portable equipment. The actual positive rate of the in-field testing was 90%, and the test provides great advantages when one considers that the diagnostic response (which included sampling of identified trees, crude DNA extraction, and LAMP reaction) was completed within an hour of arriving on site. This is an improvement when com- pared to previous confirmation strategies that could take a week or more (Dreaden et al. 2014; Hughes et al. 2015) and exemplifies how the implementation of LAMP could provide a robust detection tool that could improve integrated pest management of laurel wilt disease. By providing immediate and accurate diagnoses at infection locations, management prac- tices such as the sanitation removals of infected trees could be made without delay. Due to the generic features of laurel wilt symptoms (Fraedrich et al. 2008), the disease can be misdiag- nosed. We received several putatively infected redbay and sassafras samples collected by different personnel involved in the disease surveillance operations. All of these putatively infected samples displayed some degree of wilting and sapwood discoloration, which resembled symptoms of laurel wilt, yet many tested negative, and R. lauricola could not be isolated, illus- trating how diagnosis based on visual symptoms still requires pathogen detection for disease confirmation, which to date relies on external laboratories. This is particularly important when the symptoms observed in lauraceous species are associated with colonization by different Asian ambrosia beetles, such as Xylosan- drus compactus and Xylosandrus crassiusculus, which do not carry R. lauricola or similar pathogens, but can also result in sapwood discoloration and dieback of small branches (Chong et al. 2009; Ranger et al. 2016). One of the challenges of performing point-of-care diagnostic testing is the limited control over sample ©2021 International Society of Arboriculture 105
May 2021
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