Assessment of Alternative Candidate Subcortical Insect Vectors From Walnut Crowns in Habitats Quarantined for Thousand Cankers Disease

Thousand cankers disease (TCD) results from the combined activity of the fungal pathogen, Geosmithia morbida Kolarik, Freeland, Utley, and Tisserat and its principle vector, Pityophthorus juglandis (Blackman) (Coleoptera: Curculionidae: Scolytinae) in Juglans L. spp. and Pterocarya Kunth spp. host plants. TCD has been reported from the eastern and western United States. To evaluate potential for other beetle species to vector the fungus in east Tennessee, specimens were collected using ethanol-baited traps that were suspended beneath crowns of TCD-symptomatic trees. Associations of G. morbida with insect species collected in traps were assessed in an unsuccessful, preliminary culture-based fungal assay, and then with a molecular-based detection method. For culture-based assays, rinsate from washed, individual insects was plated on nutrient media and growing colonies were subcultured to obtain axenic G. morbida cultures for identification. For the molecular-based method, G. morbida presence was detected by amplifying the previously developed, species-specific microsatellite locus GS004. Capillary electrophoresis was used to detect the amplified amplicons and representative reactions were validated using Sanger sequencing. Eleven beetle species were found to carry G. morbida, including Cnestus mutilatus (Blandford), Dryoxylon onoharaensum (Murayama), Hylocurus rudis (LeConte), Monarthrum fasciatum (Say), Monarthrum mali(Fitch), Xyleborinus saxesenii (Ratzeburg), Xylosandrus crassiusculus (Motschulsky), Xylosandrus germanus (Blandford) (all Coleoptera: Curculionidae: Scolytinae), Stenomimus pallidus (Boheman) (Coleoptera: Curculionidae: Cossoninae), Oxoplatypus quadridentatus (Olivier) (Coleoptera: Curculionidae: Platypodinae), and Xylops basilaris (Say) (Coleoptera: Bostrichidae). These findings raise concerns that alternative subcortical insect species that already occur within quarantined habitats can sustain incidence of introduced G. morbida and contribute to spread within the native range of black walnut, Juglans nigra L, in the eastern United States.