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 Geo-smithia morbida and vectored by the walnut twig beetle Pityophthorus juglandis. In infected walnut and butternut (Juglans spp.) hosts and wingnut species (Pterocarya spp.) hosts, tree decline and death results in ecological disruption and economic losses. A rapid molecular detec-tion protocol for TCD using microsatellite markers can confirm the presence of insect vector or fungal pathogen DNA, but it requires spe-cialized expensive equipment and technical expertise. Using four differ-ent experimental approaches, capillary and conventional gel electrophoresis, and traditional polymerase chain reaction (PCR) and quantitative PCR (qPCR), we describe simplified and inexpensive pro-cesses for diagnostic confirmation of TCD. The improved and rapid detection protocols reported in this study 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 allow visualization of positive test results for infected host plant tissue samples. Conventional gel elec-trophoresis and TaqMan molecular probe protocols detected presence of DNA from TCD-Associated fungal and insect samples. These proce-dural 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.