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

Western redcedar (Thuja plicata, WRC) 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 labour-intensive and more suitable on matured 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. Efficiencies of ITS-PCR amplification were in silico predicted and validated through ITS-NGS using 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 they 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 pathogen in total DNA extracted from host tissues. These results provided evidence for application of ITS-NGS and qPCR assay as robust and reliable molecular tools for detecting early and latent infections of fungal pathogens in complex tissue samples for enhancing WRC disease management.