Long's (Braya longii Fernald) and Fernald's (B. fernaldii Abbe) Braya are endemic to the Great Northern Peninsula of Newfound-land, Canada. These herbaceous perennials are endangered due to habitat loss and degradation, as well as ongoing threats from fungal infections and insect herbivory. While recovery plans are being implemented, the identity of pathogenic fungi and their origins remain largely unknown. This study was initiated to describe the associated fungal community and infection symptoms. Plant tissues from three populations were sampled over 5 years yielding 326 fungal isolates identified primarily by nucleotide sequence analysis of the internal transcribed spacer rDNA region. Isolates included 36 taxa, 16 of which were detected once and 5 detected three times or less. In contrast, 12 taxa were detected across multiple sites, sampling intervals and years. These included two species of Alternaria, Boeremia exigua, closely related species of the Didymellaceae, Botrytis cinerea, Fusarium sp., Colletotrichum sp., Stagonosporopsis sp., Pleospora sp., and Truncatella angustata. Some common taxa showed seasonal trends. The isolated fungi included known pathogens of Brassicaceae. The plants in this study frequently showed symptoms indicative of fungal infection and insect herbivory. These findings improve our knowledge of Braya endophyte communities and will inform recovery efforts for these endangered species.

We examined the effects of temperature and salt concentration on growth of the freshwater oomycete Saprolegnia parasitica that has recently (since 2013) been found to infect an important subsistence fish (in Iñupiaq, Aanaakłiq; broad whitefish, Coregonus nasus) on the Colville River in Nuiqsut, Alaska. Using two confirmed isolates (one from the Colville River and another from a southern British Columbia aquaculture facility), we tested the following hypotheses: (1) the isolate from Alaska will grow at a greater rate than the isolate from British Columbia at lower temperatures, (2) the isolate from British Columbia will grow at a greater rate at higher temperatures than the Alaska isolate, and (3) increasing salinity will reduce the growth rate of both isolates similarly at all temperatures. In addition, we used local observations—subsistence fishers and observations associated with scientific monitoring—to assist in interpreting the potential implications of our experimental results in the context of these environmental observations. In the habitat relevant to this study, water temperature ranges between <0°C and 18°C, and salinity ranges between 0 and 30 parts per thousand due to a seasonal (and occasional west wind-driven) saltwater intrusions. No statistically significant differences were detected in growth rate or salt tolerance between the two isolates at the temperatures and salinities tested; high temperature (24°C) and low salt concentration are associated with the highest growth rate for both isolates. From our lab study, one might conclude that the peak host colonization would occur during the seasonal period of warmest water temperature; however, the observations by local fishers and biologists show this not to be the case. We conclude that, at this time, we do not have evidence that peak warm water is the primary cause of an increased incidence of infection by this freshwater mold. Although indirect and lag analysis of temperature and timing of infection were not part of this study, we note that there is a greater role of complex interactions among biotic and abiotic factors (including temperature) that may predispose some individuals in the population to become infected during spawning season.

Lodgepole pine (Pinus contorta var. latifolia Engelm. ex S. Watson) is an important lumber species in Canada, and seed orchards are expected to meet the increased demand for seed. However, seed production has been consistently low in the Okanagan region orchards of British Columbia, Canada. To determine whether the fungal microbiome contributes to seed loss, histological and molecular approaches were used. Seed production was studied at seven Okanagan orchards, all outside the natural range of lodgepole pine, and at one near Prince George, within its natural range. Seed losses were highest in the Okanagan, compared with Prince George. The role of fungal colonizers in consuming seed during the last stages of maturation is described. Fungal hyphae were frequently observed at all locations in developing seed, particularly once storage substances accumulated. Fungi identified from host tissues using molecular and morphological techniques included Alternaria, Cladosporium, Fusarium, Penicillium, and Sydowia. The opportunistic foliar pathogen Sydowia polyspora, which is known to have a variable biotrophic status, was detected at most orchards within different host tissues (seeds, needles, and conelets), in association with pollen, and in the air column. Reduced seed viability observed in Okanagan orchards is most likely due to a combination of factors, including composition of the fungal microbiome.

Among the Saprolegnia species found in aquaculture facilities, S. parasitica is recognized as the primary fish pathogen and remains an ongoing concern in fish health management. Until recently, these pathogens were kept in check by use of malachite green; due to its toxicity, this chemical has now been banned from use in many countries. It is difficult to predict and control S. parasitica outbreaks in freshwater systems and there is a need to understand the population genetic structure of this pathogen. Genetic characterization of this species in aquaculture systems would provide information to track introductions and determine possible sources of inoculum. Degenerate PCR primers containing short sequence repeats were used to create microsatellite-associated genetic markers (random amplified microsatellites) for the comparison of S. parasitica isolates collected primarily from commercial Atlantic salmon aquaculture systems in British Columbia, Canada, over a 15 mo period to describe their spatial and temporal variability. The frequencies of amplified products were compared and the population genetic diversity was measured using Nei's genetic distance and Shannon's information index, while the species population structure was evaluated by phylogenetic analysis. S. parasitica was detected in all facilities sampled. Genetic diversity was low but not clonal, most likely due to repeated introduction events and a low level of sexual recombination over time. A better understanding of pathogen population structure will assist the development of effective preventative measures and targeted treatments for disease outbreaks.

The conspecificity of Finnish and western Canadian isolates of the decay fungus Chondrostereum purpureum was investigated by several approaches, including the assessment of genetic variability, mating and progeny analysis, and the analysis of selected phenotypic traits. Eight second-generation single spore strains per fungal isolate pairing were investigated with specific genetic markers developed for both Finnish and Canadian parental isolates. Tests of linkage disequilibrium were used to analyze whether these markers assorted independently among single spore strains. This procedure was similarly applied to the third-generation spore progeny. Finally, global non-metric multidimensional scaling was used to analyze independent random amplified microsatellite marker data to assess the genetic variability of the parental Finnish and Canadian isolates, and their second- and third-generation progeny. Our results revealed that the parental isolates from Finland and western Canada were genetically divergent, but no interfertility barriers were identified between these geographically distant fungi. Furthermore, parental genetic markers used in mating studies demonstrated that second- and third-generation spore progenies underwent normal meiosis and genetic recombination without linkage disequilibrium. Based on this work, the studied C. purpureum isolates from Finland and Canada can be considered as belonging to a single biological species, although genetic and limited phenotypic differentiation was observed.

The ITS region of the rDNA gene was compared for Saprolegnia spp. in order to improve our understanding of nucleotide sequence variability within and between species of this genus, determine species composition in Canadian fin fish aquaculture facilities, and to assess the utility of ITS sequence variability in genetic marker development. From a collection of more than 400 field isolates, ITS region nucleotide sequences were studied and it was determined that there was sufficient consistent inter-specific variation to support the designation of species identity based on ITS sequence data. This non-subjective approach to species identification does not rely upon transient morphological features. Phylogenetic analyses comparing our ITS sequences and species designations with data from previous studies generally supported the clade scheme of Diéguez-Uribeondo et al. (2007) and found agreement with the molecular taxonomic cluster system of Sandoval-Sierra et al. (2014). Our Canadian ITS sequence collection will thus contribute to the public database and assist the clarification of Saprolegnia spp. taxonomy. The analysis of ITS region sequence variability facilitated genus- and species-level identification of unknown samples from aquaculture facilities and provided useful information on species composition. A unique ITS-RFLP for the identification of S. parasitica was also described.

Ophiostoma novo-ulmi is the causative agent of Dutch elm disease (DED). It is an ascomycetous filamentous fungus that ranks as the third most devastating fungal pathogen in Canada. The disease front has spread eastward and westward from the epicentre in Ontario and Quebec and is threatening elm populations across the country. Numerous mitigation strategies have been tried to eradicate this pathogen, but success has thus far been limited. An alternative approach might utilize double-stranded RNA (dsRNA) mycoviruses which have been reported to induce hypovirulence in other fungi.

Ophiostoma novo-ulmi is the third most devastating fungal pathogen in Canada, affecting native Ulmus spp. Control efforts are pursuing a gene regulation approach, using RNAi cassettes driven by constitutive heterologous promoters to evaluate virulence-related genes. A homologous carbon-catabolite regulated promoter (alcA) was developed for a cassette that regulates endopolygalacturonase (EPG) expression. Using the YFP reporter to assess promoter functionality, expression was repressed under glucose, released with its depletion and not repressed under glycerol. The EPG-alcA-RNAi cassette was similarly inactive under glucose, but highly expressed following glucose depletion, reducing EPG expression by 80–100%. Transfer to glucose medium restored native EPG expression as RNAi was down-regulated. This demonstration of RNAi regulation by carbon source offers a promising tool to evaluate gene functionality.

As part of the DNA Sequencing Research Group of the Association of Biomolecular Resource Facilities, we have tested the reproducibility of the Roche/454 GS-FLX Titanium System at five core facilities. Experience with the Roche/454 system ranged from <10 to >340 sequencing runs performed. All participating sites were supplied with an aliquot of a common DNA preparation and were requested to conduct sequencing at a common loading condition. The evaluation of sequencing yield and accuracy metrics was assessed at a single site. The study was conducted using a laboratory strain of the Dutch elm disease fungus Ophiostoma novo-ulmi strain H327, an ascomycete, vegetatively haploid fungus with an estimated genome size of 30-50 Mb. We show that the Titanium System is reproducible, with some variation detected in loading conditions, sequencing yield, and homopolymer length accuracy. We demonstrate that reads shorter than the theoretical minimum length are of lower overall quality and not simply truncated reads. The O. novo-ulmi H327 genome assembly is 31.8 Mb and is comprised of eight chromosome-length linear scaffolds, a circular mitochondrial conti of 66.4 kb, and a putative 4.2-kb linear plasmid. We estimate that the nuclear genome encodes 8613 protein coding genes, and the mitochondrion encodes 15 genes and 26 tRNAs.

Engineering of glycan specific expression systems is being undertaken in numerous different expression systems, including fungal expression systems. N-glycosylation, the post-translational attachment of specific oligosaccharides to asparagine residues in newly formed proteins, has been shown to be important in modulating the biochemical and/or pharmacological properties of proteins and may affect half-life, immunogenicity, activity, and/or efficacy of glycoproteins. The fidelity of expressed proteins may thus be maintained by engineering expression systems to produce native N-glycans. The alpha-1,2-mannosidases are key in the development of N-glycans in both higher and lower eukaryotes. Understanding their biochemistry and substrate specificity has important applications in the development of humanized protein expression systems. Here we report an analysis and biochemical characterization of alpha-1,2-mannosidase enzymes from the filamentous fungus Aspergillus nidulans. Two Class 1 alpha-1,2-mannosidases from A. nidulans are shown to be capable of reducing the oligosaccharide Man(9)GlcNAc(2) to Man(5)GlcNAc(2), the precursor for further complex N-glycan structures-this indicates that these enzymes may have overlapping and possibly redundant activities. The alpha-1,2-mannosidase IC enzyme appeared to digest these oligosaccharides more readily than the alpha-1,2-mannosidases IB enzyme even though the alpha-1,2-mannosidase IB had a higher specific activity towards the synthetic substrate Man-alpha-1,2-Man-OCH3. This difference may be related to certain structural differences which limit the access of these intermediates within the binding pocket to the catalytic region of the enzyme. Manipulation of the expression of these components of the N-glycan processing pathway will be an important step in glyco-engineering this important fungal expression strain.

The fungal pathogen, Ophiostomo novo-ulmi, has been responsible for the rapid decline of American elm (Ulmus americana) across North America and remains a serious threat to surviving elm populations. The production of pectinolytic polygalacturonase enzymes has been implicated as a virulence factor for many fungal pathogens, including O. novo-ulmi. Previous work has shown that the targeted disruption of the endopolygalacturonase gene locus epg1 of O. novo-ulmi reduced, but did not eliminate pectinase activity. In the present study, we evaluated the use of RNA interference (RNAi) as a method of suppressing expression of the epg1 locus in O. novo-ulmi and compared its efficiency to the gene disruption method. While there was a reduction in epg1-specific mRNA transcripts and in the amount of polygalacturonase enzyme secreted for both methods of gene regulation, neither method completely suppressed the expression of pectinase activity. There was, however, a significantly greater reduction in both transcript levels and secreted enzyme observed for some of the RNAi transformants. As the first demonstration of RNAi in O. novo-ulmi, this method of gene regulation shows promise in future studies of gene expression and pathogenicity.

The fungal pathogens Ophiostoma ulmi (Buisman) Nannf. and O. novo-ulmi Brasier, the causal agents of Dutch elm disease, are responsible for decimation of elms in the Northern Hemisphere during the twentieth and twentyfi rst centuries. Pectinolytic enzymes, including polygalacturonase, have been implicated as virulence factors in the life history of many fungi. To determine the role of polygalacturonase in Dutch elm disease, we cloned and characterised the polygalacturonase gene from the highly aggressive O. novo-ulmi. This gene was similar to polygalacturonase genes of other fungi, and was present as a single copy in the genome. Genetic disruption of the gene was not lethal to the pathogen but led to a reduction of pectinolytic activity in vitro. It appears, therefore, that polygalacturonase has a limited role in fungal virulence and parasitic fi tness in the life history of O. novo-ulmi.

ABSTRACT This study assessed the potential effects of transgenic aspen overexpressing a polyphenol oxidase gene on diversity in rhizosphere communities. Cultivation-independent methods were used to better delineate bacterial and fungal populations associated with transgenic and nontransgenic trees. Gene libraries for the bacterial component of the rhizosphere were established using 16S rRNA and chaperonin-60 (CPN-60) gene sequences, while the fungal community was characterized using 18S rRNA gene sequences. The 16S rRNA gene libraries were dominated by alphaproteobacterial sequences, while the CPN-60 gene libraries were dominated by members of the Bacteroidetes / Chlorobi group. In both the CPN-60 and 16S rRNA libraries, there were differences in only minor components of the bacterial community between transgenic and unmodified trees, and no significant differences in species diversity were observed. Compared to the bacterial gene libraries, greater coverage of the underlying population was achieved with the fungal 18S rRNA libraries. Members of the Zygomycota, Chytridiomycota, Ascomycota , and Basidiomycota were recovered from both libraries. The dominant groups of fungi associated with each tree type were very similar, although there were some qualitative differences in the recovery of less-abundant fungi, likely as a result of the underlying heterogeneity of the fungal population. The methods employed revealed only minor differences between the bacterial and fungal communities associated with transgenic and unmodified trees.

We have identified the Kluyveromyces lactis maltase (KlMAL22) and maltose permease (KlMAL21) intergenic region as a candidate bi-directional promoter for heterologous gene expression. The expressions of cyan and yellow fluorescent proteins from, respectively, the KlMAL22 and KlMAL21 orientations of the promoter, were compared between two promoter variants during growth in media containing glucose, galactose or glycerol. Expression from both orientations of the native promoter was repressed during growth in glucose and galactose and was induced during growth in glycerol. Disruption of a putative Mig1p binding site caused some de-repression of the maltase orientation of the promoter by 48 h of growth in glucose. The KlMAL21-KlMAL22 bi-directional promoter can be used to carry out regulated expression of heterologous gene products.

The causal agent of Dutch elm disease, Ophiostoma ulmi s. lat., has been spreading across North America since the 1920s. The population of the pathogen in Winnipeg, Manitoba, Canada was surveyed in 1993 and 2002 using a combination of RAPD marker analysis, vegetative compatibility tests, and surveys for viral double-stranded RNA (dsRNA). The data presented here show that the population, based on the vegetative compatibility and RAPD analysis, was highly genetically uniform, and has remained so for the nine-year duration of the study. The pathogen population was also monitored for the presence and spread of dsRNA molecules that, depending on identity, can be associated with a diseased phenotype and reduction of virulence in the pathogen. The current study found a very low incidence of dsRNA, and did not find any evidence for spread of these molecules through the population, even though there appeared to be no barriers to the transfer of the dsRNA between Winnipeg isolates. Despite the observation that isolates infected with the Winnipeg dsRNA showed no obvious phenotypic differences, the low incidence of dsRNA in general suggests that infected isolates do not compete as successfully as the uninfected isolates. The highly clonal nature of the pathogen population may be exploited in a control strategy.