A novel application of RNase H2-dependent quantitative PCR for detection and quantification of Grosmannia clavigera, a mountain pine beetle fungal symbiont, in environmental samples

• DOI: 10.1093/treephys/tpx147
• Publication Date: 2017-10-30T20:12:23Z
• Authors: Chandra H McAllister; Colleen E Fortier; Kate R St Onge; Bianca M Sacchi; Meaghan J Nawrot; Troy Locke; Janice E K Cooke
• Keywords: Mountain pine beetle

Mountain pine beetle (Dendroctonus ponderosae Hopkins; MPB) is an economically and ecologically important pest of species in western North America. beetles form complex multipartite relationships with microbial partners, including the ophiostomoid fungi Grosmannia clavigera (Robinson-Jeffrey Davidson) Zipfel, de Beer Wingfield, Ophiostoma montium (Rumbold) von Arx, aurea Leptographium longiclavatum (Lee, Kim, Breuil) terebrantis (Barras Perry). These are vectored by MPB to new hosts, where overcome host defenses grow into sapwood. A tree's relative susceptibility these conventionally assessed measuring lesions that develop response fungal inoculation. However, represent a symptom infection, representing both growth tree defense capacity. In order more objectively assess virulence studies host–pathogen interactions, reliable, consistent, sensitive method required accurately identify quantify MPB-associated symbionts planta. We have adapted RNase H2-dependent PCR, technique originally designed for rare allele discrimination, novel quantitative PCR (rh-qPCR) assay shows greater specificity sensitivity than previously published PCR-based methods xylem whole beetles. Two sets probes were designed: one amplifies broad range species, second G. but not other species. Using primers stems, we provide evidence lesion length does reflect extent colonization along stem nor quantity within this colonized portion stem. The sensitivity, specificity, reproducibility, cost effectiveness high-throughput potential rh-qPCR makes technology suitable identification quantification wide array pathogenic beneficial microbes associations plants organisms, even when partner present low abundance.