Development and Validation of Molecular Assays for Detection of Root- and Butt-Rot Diseases in Western Redcedar

Western redcedar (WRC; Thuja plicata) root- and butt-rot diseases are caused by a set of wood-decay fungal pathogens, which have posed a significant threat to forest health and resulted in substantial economic losses of WRC production. Traditional approaches for disease detection are labor-intensive and more suitable on mature trees at late infection stages. This study developed and validated internal transcribed spacer region next-generation sequencing (ITS-NGS) and quantitative polymerase chain reaction (qPCR) assays for detecting decay-disease infections in WRC seedlings with high sensitivity and specificity. The efficiencies of ITS-PCR amplification were in silico predicted and validated through ITS-NGS using a pure fungal DNA mixture. For diagnosis of decay pathogens in WRC seedlings, fine root and root collar samples were collected from greenhouse inoculation trials. ITS-NGS identified positive infection rates of 100% for Armillaria ostoyae, Heterobasidion occidentale, and Poriella subacida in diseased seedlings, but the diagnostic efficiency for Coniferiporia weirii was affected by the types of sampled tissues. Species-specific qPCR assays were developed for C. weirii and revealed positive infection rates up to 100% in inoculated seedlings. Relative fungal abundances measured by ITS-NGS and qPCR were highly comparable, with significant correlation, demonstrating the reliability of both molecular diagnostic approaches. Moreover, qPCR provided higher quantification accuracy for a trace amount of the pathogen in total DNA extracted from host tissues. These results provided evidence for the application of ITS-NGS and qPCR assays as robust and reliable molecular tools for detecting early and latent infections of fungal pathogens in complex tissue samples for enhancing WRC disease management. [Figure: see text] Copyright © 2026 His Majesty the King in Right of Canada, as represented by the Minister of Natural Resources Canada. This is an open access article distributed under the CC BY 4.0 International license.