Development of a Next-Generation Sequencing screening method for exotic forest pathogens from fungal spores in air and occurring on insect vectors

Anthropogenic practices, namely agriculture and horticulture, are responsible for the dispersal of phytopathogenic fungal spores in the environment. While the volume of international trading increases yearly, so does the introduction and establishment of invasive and exotic fungal phytopathogens. Similarly, wood-boring insects threaten forests by facilitating spore movement to host trees. To protect forest resources, early detection of exotic fungi is the key in managing invasive species. Our new approach uses next-generation sequencing (NGS) and metagenomics for rapid and high-throughput species screening near high-risk areas in Canada. Based on a list of several regulated invasive species, we screened 398 air and insect trap samples from 2013 to 2015. We designed barcoded PCR primers and produced tagged amplicons by amplifying the ITS1 from fungi and oomycetes. We amplified the ATP9-NAD9 region from oomycetes detected to increase Phytophthora spp. species resolution. In parallel to the 15 NGS runs (~45 million reads) performed on the Ion Torrent platform, we validated the results using species-specific qPCR assays and observed similar results comparing both techniques. We detected economically important pine pathogens Heterobasidion annosum s.s. and H. abietinum/parviporum and several Phytophthora species, which highlights the benefits of our powerful approach to increase surveying capacity to screen for phytopathogens.