Duplex real-time PCR assay for the simultaneous detection of Ophiostoma novo-ulmi and Geosmithia spp. in elm wood and insect vectors

Dutch elm disease (DED) is a destructive tracheomycosis caused by Ophiostoma novo-ulmi, an ascomyceteprobably originating in East-Asia that is devastating natural elm populations throughout Europe, NorthAmerica and Asia. The fungus is mainly spread by elm bark beetles that complete their life cycle betweenhealthy and diseased elms. Recently, it has been highlighted that some fungi of the genus Geosmithia,which are similarly well associated with bark beetles, seem to also play a role in the DED pathosystem acting as mycoparasites of O. novo-ulmi. Although some relationship between the fungi is clear, the biologicalcycle of Geosmithia spp. within the DED cycle is still partly unclear, as is the role of Geosmithia spp. in association with the bark beetles. In this work, we tried to clarify these aspects by developing a qPCR duplexTaqMan assay to detect and quantify DNA of both fungi. The assay is extremely sensitive showing a limitof detection as low as 2 fg ?l-1 for both fungi. We collected woody samples from healthy and infected elmtrees throughout the beetle life cycle. All healthy elm samples were negative for both Geosmithia spp. andO. novo-ulmi DNA. Geosmithia spp. are never present in infected, but living trees, while they are presentin frass of elm bark beetles (EBB - Scolytus spp.) and at each stage of the EBB life cycle in much higherquantities than O. novo-ulmi. This work provides a better understanding of the role and interactions occurring amongst the main players of the DED pathosystem.