Rapid Molecular Detection and Population Genetics of <i>Pityophthorus juglandis</i>, a Vector of Thousand Cankers Disease in <i>Juglans</i> spp.

Thousand Cankers Disease (TCD) is a disease complex involving the fungal pathogen Geosmithia morbida, an insect vector Pityophthorus juglandis, and the hosts, Juglans spp. and Pterocarya spp. Signs and symptoms of TCD include crown thinning due to branch dieback, yellowing and wilting of the leaves, appearance of epicormic shoots, numerous entrance/exit holes, gallery formation by P. juglandis, and the development of small, dark brown cankers underneath the bark. TCD originally described from western U.S., has now expanded to eastern U.S. and northwestern Italy. The disease complex is often difficult to diagnose due to the absence of symptoms or signs on the bark surface. Furthermore, disease symptoms can be confused with the impact of other abiotic or biotic agents. As a result, rapid molecular detection of TCD is necessary to improve our detection methods and prevent massive die-offs of these important trees. We also have limited knowledge regarding the genetic diversity of the TCD complex members. Therefore, understanding population dynamics, gene flow, and the spread of this disease is important in combating future outbreaks and potential large scale epidemics. In this study, we focused on two objectives: developing rapid molecular detection protocol for TCD, and evaluation of genetic diversity, spatial structure, and distribution of P. juglandis from subpopulations in the U.S. and Europe using microsatellite loci. Using previously developed species specific microsatellite loci for G. morbida and P. juglandis, our results provided a successful protocol with a high degree of sensitivity and outlined evidence that rapid molecular detection of TCD is feasible, effective, and time efficient. For population studies, P. juglandis specimens (n=839) from 40 subpopulations across northwestern, southwestern, and eastern U.S., and Italy were genotyped using twelve highly polymorphic P. juglandis microsatellite loci. Our results indicated high genetic diversity, presence of population structure, and limited gene flow among these groups. Also, high levels of genetic diversity across all groups were explained by human mediated movement of infested plant material from multiple sources on multiple occasions. This supports an earlier hypothesis that the disease has been established in these areas for a longer period of time than previously expected.