Preventing the introduction and establishment of forest invasive alien species (FIAS) such as the Asian gypsy moth (AGM) is a high-priority goal for countries with extensive forest resources such as Canada. The name AGM designates a group of closely related Lymantria species (Lepidoptera: Erebidae: Lymantriinae) comprising two L. dispar subspecies (L. dispar asiatica, L. dispar japonica) and three closely related Lymantria species (L. umbrosa, L. albescens, L. postalba), all considered potential FIAS in North America. Ships entering Canadian ports are inspected for the presence of suspicious gypsy moth eggs, but those of AGM are impossible to distinguish from eggs of innocuous Lymantria species. To assist regulatory agencies in their identification of these insects, we designed a suite of TaqMan® assays that provide significant improvements over existing molecular assays targeting AGM. The assays presented here can identify all three L. dispar subspecies (including the European gypsy moth, L. dispar dispar), the three other Lymantria species comprising the AGM complex, plus five additional Lymantria species that pose a threat to forests in North America. The suite of assays is built as a “molecular key” (analogous to a taxonomic key) and involves several parallel singleplex and multiplex qPCR reactions. Each reaction uses a combination of primers and probes designed to separate taxa through discriminatory annealing. The success of these assays is based on the presence of single nucleotide polymorphisms (SNPs) in the 5’ region of mitochondrial cytochrome c oxidase I (COI) or in its longer, 3’ region, as well as on the presence of an indel in the “FS1” nuclear marker, generating North American and Asian alleles, used here to assess Asian introgression into L. dispar dispar. These assays have the advantage of providing rapid and accurate identification of ten Lymantria species and subspecies considered potential FIAS.

Hybridization and introgression are pervasive evolutionary phenomena that provide insight into the selective forces that maintain species boundaries, permit gene flow, and control the direction of evolutionary change. Poplar trees (Populus L.) are well known for their ability to form viable hybrids and maintain their distinct species boundaries despite this interspecific gene flow. We sought to quantify the hybridization dynamics and postzygotic fitness within a hybrid stand of balsam poplar (Populus balsamifera L.), eastern cottonwood (P. deltoides Marsh.), and their natural hybrids to gain insight into the barriers maintaining this stable hybrid zone. We observed asymmetrical hybrid formation with P. deltoides acting as the seed parent, but with subsequent introgression biased toward P. balsamifera. Native hybrids expressed fitness traits intermediate to the parental species and were not universally unfit. That said, native hybrid seedlings were absent from the seedling population, which may indicate additional selective pressures controlling their recruitment. It is imperative that we understand the selective forces maintaining this native hybrid zone in order to quantify the impact of exotic poplar hybrids on this native system.

Invasive alien tree pathogens can cause significant economic losses as well as large-scale damage to natural ecosystems. Early detection to prevent their establishment and spread is an important approach used by several national plant protection organizations (NPPOs). Molecular detection tools targeting 10 of the most unwanted alien forest pathogens in Canada were developed as part of the TAIGA project (http://taigaforesthealth.com/). Forest pathogens were selected following an independent prioritization. Specific TaqMan real-time PCR detection assays were designed to function under homogeneous conditions so that they may be used in 96- or 384-well plate format arrays for high-throughput testing of large numbers of samples against multiple targets. Assays were validated for 1) specificity, 2) sensitivity, 3) precision, and 4) robustness on environmental samples. All assays were highly specific when evaluated against a panel of pure cultures of target and phylogenetically closely-related species. Sensitivity, evaluated by assessing the limit of detection (with a threshold of 95% of positive samples), was found to be between one and ten target gene region copies. Precision or repeatability of each assay revealed a mean coefficient of variation of 3.4%. All assays successfully allowed detection of target pathogen on positive environmental samples, without any non-specific amplification. These molecular detection tools will allow for rapid and reliable detection of 10 of the most unwanted alien forest pathogens in Canada.

Plant diseases caused by fungi and Oomycetes represent worldwide threats to crops and forest ecosystems. Effective prevention and appropriate management of emerging diseases rely on rapid detection and identification of the causal pathogens. The increase in genomic resources makes it possible to generate novel genome-enhanced DNA detection assays that can exploit whole genomes to discover candidate genes for pathogen detection. A pipeline was developed to identify genome regions that discriminate taxa or groups of taxa and can be converted into PCR assays. The modular pipeline is comprised of four components: (1) selection and genome sequencing of phylogenetically related taxa, (2) identification of clusters of orthologous genes, (3) elimination of false positives by filtering, and (4) assay design. This pipeline was applied to some of the most important plant pathogens across three broad taxonomic groups: Phytophthoras (Stramenopiles, Oomycota), Dothideomycetes (Fungi, Ascomycota) and Pucciniales (Fungi, Basidiomycota). Comparison of 73 fungal and Oomycete genomes led the discovery of 5,939 gene clusters that were unique to the targeted taxa and an additional 535 that were common at higher taxonomic levels. Approximately 28% of the 299 tested were converted into qPCR assays that met our set of specificity criteria. This work demonstrates that a genome-wide approach can efficiently identify multiple taxon-specific genome regions that can be converted into highly specific PCR assays. The possibility to easily obtain multiple alternative regions to design highly specific qPCR assays should be of great help in tackling challenging cases for which higher taxon-resolution is needed.

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Bacterial communities mediate many of the processes in boreal forest floors that determine the functioning of these ecosystems, yet it remains uncertain whether the composition of these communities is distributed nonrandomly across the landscape. In a study performed in the southern boreal mixed wood forest of Québec, Canada, we tested the hypothesis that stand type (spruce/fir, aspen, paper birch), stand age (57, 78-85, and 131 years old), and geologic parent material (clay and till) were correlated with forest floor bacterial community composition. Forest floors in 54 independent forest stands were sampled to comprise a full factorial array of the three predictor variables. Bacterial community structure was examined by terminal restriction fragment (T-RF) length polymorphism analysis of genes encoding for 16S rRNA. Distance-based redundancy analysis of T-RF assemblages revealed that each predictor variable, as well as their interaction terms, had a significant effect on bacterial community composition, geologic parent material being the most discriminating factor. A survey of the 15 T-RFs with the highest percentage fit on the first two ordination axes describing the main effects indicated that each landscape feature correlated to a distinct group of bacteria. A survey of the most discriminant T-RFs describing the effect of stand type within each combination of stand age and geologic parent material indicated a strong dependency of several T-RFs on geologic parent material. Given the possible link between bacterial community composition and forest floor functioning, we also assessed the effects of the same three landscape features on community-level catabolic profiles (CLCP) of the extractable forest floor microbiota. Geologic parent material and stand type had significant effects on CLCPs. On clay plots, the effects of landscape features on T-RF patterns were highly consistent with their effects on CLCPs. In light of our results, we suggest that future research examine whether bacterial community composition or CLCPs can be used to detect latent environmental changes across landscape units.

Summary A set of quantitative hierarchical real‐time PCR assays was developed for the detection of Heterobasidion irregulare, H. occidentale, H. annosum sensu stricto and of the entire Heterobasidion annosum complex. These assays enable specific and accurate detection and quantification of the target species from DNA extracted on airborne collected spores. Heterobasidion‐specific TaqMan™ real‐time PCR detection assays were designed to function under homogeneous conditions so that they may be used in 96‐ or 384‐well plate format arrays for high‐throughput testing of large numbers of samples against multiple targets. Assays were validated for (i) specificity, (ii) sensitivity and (iii) repeatability. All assays were highly specific when evaluated against a panel of pure cultures of target and phylogenetically closely related species. Sensitivity, evaluated by assessing the limit of detection (with a threshold of 95% of positive samples), was found to be between one and a hundred ITS gene region copies or one conidia count equivalent. Precision or repeatability of each assay revealed a mean coefficient of variation of 5.9%. These molecular tools are now available for rapid and reliable monitoring of one of the most significant pathogen species complex of temperate northern coniferous forest around the world.

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