Thousand Cankers Disease Diagnosis with Conventional Electrophoresis and TaqMan Probes

Juglans nigra and related Juglans and Pterocarya species of trees are threatened by the fungus Geosmithia morbida, and the bark beetle Pityophthorus juglandis that serves as the primary vector for the plant pathogen. Together, this species complex causes thousand cankers disease (TCD) in susceptible host plants. When infection is severe, tree death can occur within three to four years, resulting in severe ecological and economic damage. Although the previously developed TCD rapid detection molecular protocol can quickly identify the presence of the vector or the pathogen, it requires specialized and costly equipment, and highly trained personnel. To make this protocol more user friendly this study aims to 1) to optimize the current molecular protocol by evaluating the lowest detection limits of G. morbida and P. juglandis on a conventional gel and 2) reduce the materials and equipment costs associated with the PCR result through a direct visualization method. As a result, reliance on the QIAxcel Advanced capillary electrophoresis system has been negated, and replaced by more affordable diagnostic methods, which included either conventional agarose gels or molecular probe protocols. The expensive QIAxcel Advanced capillary electrophoresis system (costing several tens of thousands of dollars US) was replaced with two less substantial costs; a $3,000 piece of equipment in the conventional gel protocol or a $200 piece of equipment in the molecular probe assay. Both the conventional gel and molecular probe protocols successfully detected the presence of the fungal and insect components of the disease. These new protocols will make the testing of J. nigra samples more accessible to regulators and diagnosticians.