Conventional Gel Electrophoresis and TaqMan Probes Enable Rapid Confirmation of Thousand Cankers Disease from Diagnostic Samples.

Abstract

Thousand cankers disease (TCD) is caused by the fungal pathogen, Geosmithia morbida,a, and vectored by the walnut twig beetle, Pityophthorus juglandis. In infected walnut and butternut (Juglans spp.) hosts, tree decline and death results in ecological disruption and economic losses. A rapid molecular detection protocol for TCD using microsatellite markers can confirm the presence of insect vector or fungal pathogen DNA, but it requires specialized expensive equipment, and technical expertise. Using four different experimental approaches, capillary and conventional gel electrophoresis, and traditional PCR and qPCR, we describe simplified and inexpensive processes for diagnostic confirmation of TCD. The improved and rapid detection protocols reported here reduce time and equipment costs associated with detection of molecular pest and pathogen DNA by: 1) using conventional gel electrophoresis or TaqMan molecular probes to elucidate the detection limits for G. morbida and P. juglandis DNA; and 2) identifying resources that allows visualization of positive test results for infected host plant tissue samples. Both conventional gel electrophoresis and TaqMan molecular probe protocols detected presence of DNA from TCD-associated fungal and insect samples. These procedural improvements can be readily adopted by diagnostic end-users and adapted for use with other complex disease systems to enable rapid pest and pathogen detection.