Anthropogenic activities have a major impact on the global environment. Canada’s natural resources are threatened by the spread of fungal pathogens, which is facilitated by agricultural practices and international trade. Fungi are introduced to new environments and sometimes become established, in which case they can cause disease outbreaks resulting in extensive forest decline. Here, we describe how a nationwide sample collection strategy coupled to next-generation sequencing (NGS) (i.e., metagenomics) can achieve fast and comprehensive screening for exotic invasive species. This methodology can help provide guidance to phytopathology stakeholders such as regulatory agencies. Several regulated invasive species were monitored by processing field samples collected over 3 years (2013 to 2015) near high-risk areas across Canada. Fifteen sequencing runs were required on the Ion Torrent platform to process 398 samples that yielded 45 million reads. High-throughput screening of fungal and oomycete operational taxonomic units using customized fungi-specific ribosomal internal transcribed spacer 1 barcoded primers was performed. Likewise, Phytophthora-specific barcoded primers were used to amplify the adenosine triphosphate synthase subunit 9–nicotinamide adenine dinucleotide dehydrogenase subunit 9 spacer. Several Phytophthora spp. were detected by NGS and confirmed by species-specific quantitative polymerase chain reaction (qPCR) assays. The target species Heterobasidion annosum sensu stricto could be detected only through metagenomics. We demonstrated that screening target species using a variety of sampling techniques and NGS—the results of which were validated by qPCR—has the potential to increase survey capacity and detection sensitivity, reduce hands-on time and costs, and assist regulatory agencies to identify ports of entry. Considering that early detection and prevention are the keys in mitigating invasive species damage, our method represents a substantial asset in plant pathology management.

A total of 252 aerial spore samples from the provinces of Quebec and Ontario were obtained during an extensive research programme focusing on aerobiology of fungal spores. Samples were collected on 95 sticky rods from rotary arm spore collectors and 157 filters from passive rain collectors. DNA from samples was PCR-amplified for fungal ribosomal ITS and sequenced using High Throughput Sequencing (HTS) methods. After bioinformatics analysis of DNA sequences, the presence of Diplodia corticola, an emerging tree pathogen in North America, was observed. In total, 313 DNA sequence reads from aerial spores of D. corticola were found in the Illumina data set, and 199 DNA sequence reads were obtained from the Ion Torrent data set. DNA of D. corticola was found in 16 of the 32 sampled sites, always less than 10 reads per site, with the exception of three sites – Quebec City, Aylmer and Ottawa – where 287, 125 and 73 DNA reads, respectively, were detected. This is not a first report of the presence of D. corticola causing a tree disease in Canada as symptomatic trees have not been identified. Typically, like many other species of the Botryosphaeriaceae, this fungal pathogen is believed to be an opportunistic plant endophyte capable of living asymptomatically for several years before showing up as a pathogen when conducive conditions arise. Its presence as singletons in nearly half the sampled sites in Quebec and Ontario may be the result of long-distance spore dispersal originating from known infected sites in Massachusetts, Maine and other unknown sites in the north-eastern USA. However, its much higher read counts in three sites may indicate the possibility of a few trees being asymptomatically infected and spreading conidia locally.

Soil fungi are important components of boreal forest ecosystems; for example, saprotrophic fungi regulate nutrient cycling, and mycorrhizal species facilitate nutrient uptake by plants. This study aimed to assess soil fungal communities in a reclaimed area and an adjacent natural mixedwood forest and to identify the distribution of taxa available for seedling colonization. Soil fungal microbiomes were assessed along three transects (from 10 m inside the interior of the undisturbed forest to 40 m inside the reclaimed area) and in the roots of small aspen within the natural forest. Using high-throughput deoxyribonucleic acid (DNA) sequencing of internal transcribed spacer amplicons, a total of 2796 unique fungal taxa were detected across fine roots, forest floor, and mineral soils collected along the transects, whereas 166 taxa were detected in the aspen roots from the natural forest. Within the interior of the forest, ectomycorrhizal fungi were more common, whereas in the reclaimed areas, arbuscular mycorrhizae and saprophytes were more common. This survey showed that natural areas of adjacent undisturbed forest can act as a source of ectomycorrhizal fungi for dispersal into reclaimed areas. Notably, soil fungal taxa colonizing the root systems of small aspen included species that are specifically associated with soils from the undisturbed forest (primarily ectomycorrhizae) or the reclaimed clearing (saprotrophs and plant pathogens).

Studying the means of dispersal of plant pathogens is crucial to better understand the dynamic interactions involved in plant infections. On one hand, entomologists rely mostly on both traditional molecular methods and morphological characteristics, to identify pests. On the other hand, high-throughput sequencing (HTS) is becoming the go-to avenue for scientists studying phytopathogens. These organisms sometimes infect plants, together with insects. Considering the growing number of exotic insect introductions in Canada, forest pest-management efforts would benefit from the development of a high-throughput strategy to investigate the phytopathogenic fungal and oomycete species interacting with wood-boring insects. We recycled formerly discarded preservative fluids from the Canadian Food Inspection Agency annual survey using insect traps and analysed more than one hundred samples originating from across Canada. Using the Ion Torrent Personal Genome Machine (PGM) HTS technology and fusion primers, we performed metabarcoding to screen unwanted fungi and oomycetes species, including Phytophthora spp. Community profiling was conducted on the four different wood-boring, insect-attracting semiochemicals; although the preservative (contained ethanol) also attracted other insects. Phytopathogenic fungi (e.g., Leptographium spp. and Meria laricis in the pine sawyer semiochemical) and oomycetes (mainly Peronospora spp. and Pythium aff. hypogynum in the General Longhorn semiochemical), solely associated with one of the four types of semiochemicals, were detected. This project demonstrated that the insect traps’ semiochemical microbiome represents a new and powerful matrix for screening phytopathogens. Compared to traditional diagnostic techniques, the fluids allowed for a faster and higher throughput assessment of the biodiversity contained within. Additionally, minimal modifications to this approach would allow it to be used in other phytopathology fields.

Heterobasidion irregulare is a basidiomyceteous forest pathogenic fungus which causes a root disease capable of killing large trees. Infection probability is proportional to the density of aerial basidiospores infecting freshly cut stumps. The purpose of this study was to quantify for the first time H. irregulare aerial basidiospore density in southern Québec and to determine the importance of a local basidiospore load from a nearby infested plantation. Spore counts from automated rotary arm spore collectors were assayed using a ribosomal ITS TaqMan real-time PCR detection assay. Cumulative basidiospore deposits on a 30-cm stump were estimated to be highest in the infected plantation with 2.37 basidiospores per stump, with decreasing count of 0.52 basidiospores at a 500-m distance and 0.46 basidiospores at a 1.5-km distance. At 5 km from the infected plantation, the number of basidiospores deposited on a stump was 0.22, a value similar to those at distances of 25 and 140 km. Since a single Heterobasidion basidiospore may travel as far as 500 km, the level detected at 5 km may represent the background basidiospore load resulting from long-distance dispersal, and basidiospores from a nearby infected plantation are unlikely to be an added risk of infection to plantations located more than 5 km away. Long distance aerial basidiospore dispersal will remain for now the most important constant source of infectious H. irregulare in Québec. Despite the very low risk level per stump in this study, the large number of trees thinned annually warrants that stump treatment with Rotstop^®C is still the best strategy, especially in valuable plantations.

Heterobasidion irregulare (Underw.) Garbel. & Otrosina is an important fungal parasitic basidiomycete of coniferous trees throughout temperate regions of the world. In Canada, the disease was first reported on red pine in Ontario in 1955, then in Quebec in 1989. As infection probability is proportional to the density of its aerial basidiospores infecting freshly cut pine stumps, the purpose of this study was to quantify the H. irregulare aerial basidiospore deposition along a southern Quebec transect. Spore counts from automated rotary arm collectors were determined by using a ribosomal ITS TaqMan real-time PCR detection assay. Total count was performed on rods at each location, with the highest score of 706.3 spores in Harrington, then 459.6 spores in Cowansville, rapidly decreasing to 52.3 spores in Compton and reaching lows in St-Aubert and Rimouski, with an eastward direction on the transect. Clearly, plantations in southwestern Quebec are at a higher risk of infection than anywhere else in the province. With few exceptions, weekly spore depositions at all sites other than Harrington were greatly below 0.2 per m⁻² h⁻¹. Despite this very low spore deposition rate observed in one season, the large number of trees thinned annually warrants that stump treatment with Rotstop® C is still the best strategy, especially in valuable plantations, to prevent against infection by Heterobasidion.