Comparison of qPCR and Metabarcoding Methods as Tools for the Detection of Airborne Inoculum of Forest Fungal Pathogens

Abstract

Forest diseases caused by invasive fungal pathogens are becoming more common, sometimes with dramatic consequences to forest ecosystems. The development of early detection systems is necessary for efficient surveillance and to mitigate the impact of invasive pathogens. Windborne spores are an important pathway for introduction of fungal pathogens into new areas; the design of spore trapping devices adapted to forests, capable of collecting different types of spores, and aligned with development of efficient molecular methods for detection of the pathogen, should help forest managers anticipate new disease outbreaks. Two types of Rotorod samplers were evaluated for the collection of airborne inoculum of forest fungal pathogens with a range of spore sizes in five forest types. Detection was by specific quantitative PCR (qPCR) and by high-throughput sequencing (HTS) of amplified internal transcribed spacer sequences using a new bioinformatic pipeline, FungiSearch, developed for diagnostic purposes. Validation of the pipeline was conducted on mock communities of 10 fungal species belonging to different taxa. Although the sensitivity of the new HTS pipeline was lower than the specific qPCR, it was able to detect a wide variety of fungal pathogens. FungiSearch is easy to use, and the reference database is updatable, making the tool suitable for rapid identification of new pathogens. This new approach combining spore trapping and HTS detection is promising as a diagnostic tool for invasive fungal pathogens.