UC Davis

Geosmithia morbida, the fungus that causes thousand cankers disease, forms localized cankers on the trunk and branches of various walnut species following inoculation of spores carried by the phloem-feeding walnut twig beetle (Pityophthorus juglandis). Diagnosis currently requires expertise in fungal isolation and morphological identification or laboratory PCR. A molecular diagnostic technique using crude tissue extracts and interpreted in-field will simplify diagnoses. We developed a qualitative recombinase polymerase amplification (RPA) assay for G. morbida in which dual-labeled translation elongation factor 1-alpha (TEF1-α) amplicons are detected with a lateral flow strip. A consensus sequence for the TEF1-α gene was generated for G. morbida and RPA parameters established. The selected primer/probe combination successfully identifies all tested isolates of G. morbida at DNA concentrations of 100 pg/µL. One isolate was chosen for serial dilution and was identified at concentrations as low as 100 fg/µL. The assay has also identified the closely related, nonpathogenic species Geosmithia fassatiae when DNA is purified and in a high concentration, albeit with reduced sensitivity. Furthermore, the assay can be used successfully to test crude extracts from lesioned walnut bark.