P-163 - Developing a LAMP assay for the detection of Harringtonia lauricola in avocado trees and beetle vectors

Harringtonia lauricola (HL), the causal agent of laurel wilt, has killed millions of Lauraceae trees in the southeastern U.S. The disease impacts avocado production in South Florida and threatens other producing areas due to its wide range of hosts and beetle vectors. We aimed to develop a field-deployable LAMP assay to detect HL from avocado trees and beetle vectors. Genome sequencing of 19 HL isolates from various sources identified 158 HL unique sequences absent in closely related species. Five sequences with no significant homology in GenBank were selected for LAMP primer design. Preliminary evaluation using kit purified DNA showed that three primer sets detected as little as 4 pg/ml of HL DNA, but only the HL_0354 set detected HL at that low concentration when HL DNA was mixed with avocado DNA. The three sets detected HL in the same DNA samples from naturally infected and inoculated trees that were positive with the traditional detection method (PCR with IFW primers), and none cross-reacted with DNA from healthy avocado trees, closely related species, or co-habitants in avocado trees (n=15).  Due to its sensitivity, the HL_0354 set was further evaluated using crude DNA extracts and a portable LAMP device. The HL_0354 set detected HL in 66% of tissue samples that yielded HL in culture media, and 81% of beetle samples were accurately diagnosed, as was confirmed with the IFW primers. On-field validation is underway, but our findings indicate that the HL_0354 set shows promise for developing a sensitive, specific, and robust method for HL field detection and surveillance.