Assessment of intra-specific variability in Saprolegnia parasitica populations of aquaculture facilities in British Columbia, Canada.

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.

Two novel mitoviruses from a Canadian isolate of the Dutch elm pathogen Ophiostoma novo-ulmi (93-1224).

BACKGROUND: 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. METHODS: Using a modified single primer amplification technique (SPAT) in combination with chromosomal walking, we have determined the genome sequence of two RdRp encoding dsRNA viruses from an O. novo-ulmi isolate (93-1224) collected from the disease front in Winnipeg. RESULTS: We propose that these viruses, which we have named OnuMV1c and OnuMV7 based on sequence similarity to other Ophiostoma mitoviruses, are two new members of the genus Mitovirus in the family Narnaviridae. CONCLUSIONS: The discovery of such dsRNA elements raises the potential for engineering these viruses to include other genetic elements, such as anti-sense or interfering RNAs, to create novel and highly specific biological controls. Naïve fungal hosts could be infected with both the engineered molecule and a helper mitovirus encoding an RdRp which would provide replication capacity for both molecules.

Suppression of polygalacturonase gene expression in the phytopathogenic fungus Ophiostoma novo-ulmi by RNA interference.

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.

Effects of transgenic hybrid aspen overexpressing polyphenol oxidase on rhizosphere diversity.

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.

Recent Asian origin of chytrid fungi causing global amphibian declines.

Globalized infectious diseases are causing species declines worldwide, but their source often remains elusive. We used whole-genome sequencing to solve the spatiotemporal origins of the most devastating panzootic to date, caused by the fungus Batrachochytrium dendrobatidis, a proximate driver of global amphibian declines. We traced the source of B. dendrobatidis to the Korean peninsula, where one lineage, BdASIA-1, exhibits the genetic hallmarks of an ancestral population that seeded the panzootic. We date the emergence of this pathogen to the early 20th century, coinciding with the global expansion of commercial trade in amphibians, and we show that intercontinental transmission is ongoing. Our findings point to East Asia as a geographic hotspot for B. dendrobatidis biodiversity and the original source of these lineages that now parasitize amphibians worldwide.

Species composition of the genus Saprolegnia in fin fish aquaculture environments, as determined by nucleotide sequence analysis of the nuclear rDNA ITS regions.

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.

Heterologous expression of cyan and yellow fluorescent proteins from the Kluyveromyces lactis KlMAL21-KlMAL22 bi-directional promoter.

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.

A nine-year genetic survey of the causal agent of Dutch elm disease, Ophiostoma novo-ulmi in Winnipeg, Canada.

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.

Endopolygalacturonase is encoded by a multigene family in the basidiomycete Chondrostereum purpureum.

The basidiomycete Chondrostereum purpureum produces several plant cell wall-degrading enzymes, including endopolygalacturonase (endoPG). Degenerate oligonucleotide primers were designed according to conserved regions of endoPG genes from various fungi, plants, and bacteria and used to amplify members of this gene family from C. purpureum. Four different amplification products showed significant similarity to known endoPGs and were used as hybridization probes to screen a library of genomic DNA sequences and to retrieve five full-length endoPG genes (epgA, epgB1, epgB2, epgC, and epgD). The identities between the deduced polypeptides for epgA, epgB1, epgC, and epgD ranged from 61.8 to 80.0%, while the deduced polypeptides for epgB1 and epgB2 shared 97.1% identity. Phylogenetic analysis suggested that the duplication of existing endoPG genes occurred after the divergence of the ascomycetes and basidiomycetes. C. purpureum is the first basidiomycete fungus for which the endoPG gene family has been described.

A retrotransposon-like element and its occurrence in British Columbia populations of Chondrostereum purpureum.

Repetitive DNA elements discovered in the basidiomycete Chondrostereum purpureum were characterized and validated for use as genetic markers. Regions of these marker sequences were similar to retrotransposon and retrotransposon-like sequences, as indicated by BLAST searches of NCBI databases. These sequences occur in multiple DNA fragments of variable length in a given C. purpureum isolate, and thus can serve as strain-specific genetic markers. The segregation of the markers within a progeny set demonstrated their stability through meiosis. The population structure of C. purpureum was assessed using the markers. There was no evidence of a barrier to gene flow between C. purpureum populations separated by 1400 km and no indication of population sub-structuring based on host or geographical source of isolate. Repetitive fragments were amplified from four other species, suggesting the occurrence of these retrotransposon-like elements in other basidiomycetes and the potential utility of these markers for other fungi.