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Development of a recombinase polymerase amplification assay with qualitative end-point detection for diagnosis of thousand cankers disease in walnut

Jason Simmons: University of California

<div><em>Geosmithia morbida, </em>the causal fungus of thousand cankers disease, forms cankers on the trunk and branches of trees of various walnut species following attack by the walnut twig beetle, which vectors the pathogen. Diagnosis typically requires isolating the fungus from bark tissue on media, which can be confounded by the presence of morphologically similar, nonpathogenic <em>Geosmithia</em> species and other fungi. A molecular technique for direct analysis of infected walnut tissue will simplify and expedite diagnoses. A recombinase polymerase amplification assay for <em>G. morbida </em>was developed in which amplicons were designed for lateral flow strip detection. Based on a consensus sequence for the <em>G. morbida</em> <em>Ef 1-alpha</em> gene, a probe was designed with the 5’ end labeled with FAM, the 3’ end with a C3 spacer, and an internal abasic nucleotide replacement. Forward and reverse primers were designed to flank the probe, and the primer in the opposite orientation to the probe was labeled at the 5’ end with biotin. All combinations of forward and reverse primers and probes were screened against 1 µM DNA of <em>G. morbida, Geosmithia fassatiae, </em>and <em>Geosmithia lavendula</em>, and further tested against DNA of <em>Juglans regia</em> and <em>Fusarium solani</em>. The selected primer/probe combination identifies all tested isolates of <em>G. morbida</em> with species-level specificity. Experiments are underway to optimize sensitivity and adapt for field application.</div>