Quantifying the number of spores of the thousand cankers disease pathogen, Geosmithia morbida, on individual walnut twig beetles, Pityophthorus juglandis, within California

Thousand cankers disease of walnuts, caused by the fungal pathogen, Geosmithia morbida, has led to the decline and death of native and introduced walnut trees, Juglans spp., throughout the USA. The pathogen is vectored as conidia by the walnut twig beetle (WTB), Pityophthorus juglandis, as it bores through the bark and enters the phloem. We trapped both sexes of adult WTB with aggregation pheromone-baited Lindgren funnel traps and developed a real-time quantitative polymerase chain reaction assay (TaqMan Procedure) to calculate the number of G. morbida spores on individual beetles.To do this we compared quantification cycle (Cq) values of our samples to Cq values of a plasmid reference curve (pCR2.1, Eurofins Genomics, Huntsville, AL). Identification and quantification of G. morbida was based on species-specific primers and hydrolysis probes from the β-tubulin gene. Beetles were collected during May/June and August (2012-2014) at six study sites in California (Butte, Los Angeles, Solano, Sutter, Tulare, and Yolo Cos.) with known infestations of WTB. Sites were chosen to represent several species of walnut trees and environments (orchard, urban, and native riparian stands).