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.

Bark beetles form multipartite symbiotic associations with blue stain fungi (Ophiostomatales, Ascomycota). These fungal symbionts play an important role during the beetle's life cycle by providing nutritional supplementation, overcoming tree defences and modifying host tissues to favour brood development. The maintenance of stable multipartite symbioses with seemingly less competitive symbionts in similar habitats is of fundamental interest to ecology and evolution. We tested the hypothesis that the coexistence of three fungal species associated with the mountain pine beetle is the result of niche partitioning and adaptive radiation using SNP genotyping coupled with genotype-environment association analysis and phenotypic characterization of growth rate under different temperatures. We found that genetic variation and population structure within each species is best explained by distinct spatial and environmental variables. We observed both common (temperature seasonality and the host species) and distinct (drought, cold stress, precipitation) environmental and spatial factors that shaped the genomes of these fungi resulting in contrasting outcomes. Phenotypic intraspecific variations in Grosmannia clavigera and Leptographium longiclavatum, together with high heritability, suggest potential for adaptive selection in these species. By contrast, Ophiostoma montium displayed narrower intraspecific variation but greater tolerance to extreme high temperatures. Our study highlights unique phenotypic and genotypic characteristics in these symbionts that are consistent with our hypothesis. By maintaining this multipartite relationship, the bark beetles have a greater likelihood of obtaining the benefits afforded by the fungi and reduce the risk of being left aposymbiotic. Complementarity among species could facilitate colonization of new habitats and survival under adverse conditions.

The karyotype is shaped by different chromosome rearrangements during species evolution. However, determining which rearrangements are responsible for karyotype changes is a challenging task and the combination of a robust phylogeny with refined karyotype characterization, GS measurements and bioinformatic modelling is necessary. Here, this approach was applied in Heterotaxis to determine what chromosome rearrangements were responsible for the dysploidy variation. We used two datasets (nrDNA and cpDNA, both under MP and BI) to infer the phylogenetic relationships among Heterotaxis species and the closely related genera Nitidobulbon and Ornithidium. Such phylogenies were used as framework to infer how karyotype evolution occurred using statistical methods. The nrDNA recovered Ornithidium, Nitidobulbon and Heterotaxis as monophyletic under both MP and BI; while cpDNA could not completely separate the three genera under both methods. Based on the GS, we recovered two groups within Heterotaxis: (1) "small GS", corresponding to the Sessilis grade, composed of plants with smaller genomes and smaller morphological structure, and (2) "large GS", corresponding to the Discolor clade, composed of plants with large genomes and robust morphological structures. The robust karyotype modeling, using both nrDNA phylogenies, allowed us to infer that the ancestral Heterotaxis karyotype presented 2n = 40, probably with a proximal 45S rDNA on a metacentric chromosome pair. The chromosome number variation was caused by ascending dysploidy (chromosome fission involving the proximal 45S rDNA site resulting in two acrocentric chromosome pairs holding a terminal 45S rDNA), with subsequent descending dysploidy (fusion) in two species, H. maleolens and H. sessilis. However, besides dysploidy, our analysis detected another important chromosome rearrangement in the Orchidaceae: chromosome inversion, that promoted 5S rDNA site duplication and relocation.

Pythium species are ubiquitous organisms known to be pathogens to terrestrial plants and marine algae. While several Pythium species (hereafter, Pythium) are described as pathogens to marine red algae, little is known about the pathogenicity of Pythium on marine green algae. A strain of a Pythium was isolated from a taxonomically unresolved filamentous Ulva collected in an intertidal area of Oslo fjord. Its pathogenicity to a euryhaline Ulva intestinalis collected in the same area was subsequently tested under salinities of 0, 15, and 30 parts per thousand (ppt). The Pythium isolate readily infected U. intestinalis and decimated the filaments at 0 ppt. Mycelium survived on U. intestinalis filaments for at least 2 weeks at 15 and 30 ppt, but the infection did not progress. Sporulation was not observed in the infected algal filaments at any salinity. Conversely, Pythium sporulated on infected grass pieces at 0, 15, and 30 ppt. High salinity retarded sporulation, but did not prevent it. Our Pythium isolate produced filamentous non-inflated sporangia. The sexual stage was never observed and phylogenetic analysis using internal transcribed spacer suggest this isolate belongs to the clade B2. We conclude that the Pythium found in the Oslo fjord was a pathogen of U. intestinalis under low salinity.

The mountain pine beetle (MPB; Dendroctonus ponderosae Hopkins, 1902) forms beneficial symbiotic associations with fungi. Here we explored the fine-scale spatial genetic structure of three of those fungi using single nucleotide polymorphism. We found that single mated pairs of beetles carry not only multiple fungal species, but also multiple genotypes of each species into their galleries. We observed genetic diversity at a fine spatial scale. Most of the diversity was found within and among galleries with nonsignificant diversity among trees. We observed clonal propagation almost exclusively within galleries. Ophiostoma montium (Rumbold) Arx possessed a larger expected number of multilocus genotypes and lower linkage disequilibrium than Grosmannia clavigera (Rob.-Jeffr. & R.W. Davidson) Zipfel, Z.W. de Beer & M.J. Wingf. and Leptographium longiclavatum S.W. Lee, J.J. Kim & C. Breuil. More than 80% of fungal samples were genetically unrelated, a result that parallels what has been observed in the beetles. The proportion of genetically related samples within galleries was higher in O. montium (40%) than in G. clavigera (20%) or L. longiclavatum (6%), likely the consequence of within-gallery sexual recombination in O. montium. The underlying genetic diversity reported here and the differences among fungal species could enable the symbiont community to quickly respond to new environmental conditions or changes in the host, enhancing the maintenance of this multipartite relationship and allowing the MPB to colonize new habitats.