Scott DiGuistini

De novo genome sequence assembly of a filamentous fungus using Sanger, 454 and Illumina sequence data

Sequencing-by-synthesis technologies can reduce the cost of generating de novo genome assemblies. We report a method for assembling draft genome sequences of eukaryotic organisms that integrates sequence information from different sources, and demonstrate its effectiveness by assembling an approximately 32.5 Mb draft genome sequence for the forest pathogen Grosmannia clavigera, an ascomycete fungus. We also developed a method for assessing draft assemblies using Illumina paired end read data and demonstrate how we are using it to guide future sequence finishing. Our results demonstrate that eukaryotic genome sequences can be accurately assembled by combining Illumina, 454 and Sanger sequence data.

Genome and transcriptome analyses of the mountain pine beetle-fungal symbiont Grosmannia clavigera, a lodgepole pine pathogen

In western North America, the current outbreak of the mountain pine beetle (MPB) and its microbial associates has destroyed wide areas of lodgepole pine forest, including more than 16 million hectares in British Columbia. Grosmannia clavigera (Gc), a critical component of the outbreak, is a symbiont of the MPB and a pathogen of pine trees. To better understand the interactions between Gc, MPB, and lodgepole pine hosts, we sequenced the ∼30-Mb Gc genome and assembled it into 18 supercontigs. We predict 8,314 protein-coding genes, and support the gene models with proteome, expressed sequence tag, and RNA-seq data. We establish that Gc is heterothallic, and report evidence for repeat-induced point mutation. We report insights, from genome and transcriptome analyses, into how Gc tolerates conifer-defense chemicals, including oleoresin terpenoids, as they colonize a host tree. RNA-seq data indicate that terpenoids induce a substantial antimicrobial stress in Gc, and suggest that the fungus may detoxify these chemicals by using them as a carbon source. Terpenoid treatment strongly activated a ∼100-kb region of the Gc genome that contains a set of genes that may be important for detoxification of these host-defense chemicals. This work is a major step toward understanding the biological interactions between the tripartite MPB/fungus/forest system.

Gene genealogies reveal cryptic species and host preferences for the pine fungal pathogen Grosmannia clavigera

Grosmannia clavigera is a fungal pathogen of pine forests in western North America and a symbiotic associate of two sister bark beetles: Dendroctonus ponderosae and D. jeffreyi. This fungus and its beetle associate D. ponderosae are expanding in large epidemics in western North America. Using the fungal genome sequence and gene annotations, we assessed whether fungal isolates from the two beetles inhabiting different species of pine in epidemic regions of western Canada and the USA, as well as in localized populations outside of the current epidemic, represent different genetic lineages. We characterized nucleotide variations in 67 genomic regions and selected 15 for the phylogenetic analysis. Using concordance of gene genealogies and distinct ecological characteristics, we identified two sibling phylogenetic species: Gc and Gs. Where the closely related Pinus ponderosa and P. jeffreyi are infested by localized populations of their respective beetles, Gc is present. In contrast, Gs is an exclusive associate of D. ponderosae mainly present on its primary host‐tree P. contorta; however, in the current epidemic areas, it is also found in other pine species. These results suggest that the host‐tree species and the beetle population dynamics may be important factors associated with the genetic divergence and diversity of fungal partners in the beetle‐tree ecosystems. Gc represents the original G. clavigera holotype, and Gs should be described as a new species.

Unequal Recombination and Evolution of the Mating-Type (MAT) Loci in the Pathogenic Fungus Grosmannia clavigera and Relatives

Sexual reproduction in fungi is regulated by the mating-type (MAT) locus where recombination is suppressed. We investigated the evolution of MAT loci in eight fungal species belonging to Grosmannia and Ophiostoma (Sordariomycetes, Ascomycota) that include conifer pathogens and beetle symbionts. The MAT1-2 idiomorph/allele was identified from the assembled and annotated Grosmannia clavigera genome, and the MAT locus is flanked by genes coding for cytoskeleton protein (SLA) and DNA lyase. The synteny of these genes is conserved and consistent with other members in Ascomycota. Using sequences from SLA and flanking regions, we characterized the MAT1-1 idiomorph from other isolates of G. clavigera and performed dotplot analysis between the two idiomorphs. Unexpectedly, the MAT1-2 idiomorph contains a truncated MAT1-1-1 gene upstream of the MAT1-2-1 gene that bears the high-mobility-group domain. The nucleotide and amino acid sequence of the truncated MAT1-1-1 gene is similar to its homologous copy in the MAT1-1 idiomorph in the opposite mating-type isolate, except that positive selection is acting on the truncated gene and the alpha(α)-box that encodes the transcription factor has been deleted. The MAT idiomorphs sharing identical gene organization were present in seven additional species in the Ophiostomatales, suggesting that the presence of truncated MAT1-1-1 gene is a general pattern in this order. We propose that an ancient unequal recombination event resulted in the ancestral MAT1-1-1 gene integrated into the MAT1-2 idiomorph and surviving as the truncated MAT1-1-1 genes. The α-box domain of MAT1-1-1 gene, located at the same MAT locus adjacent to the MAT1-2-1 gene, could have been removed by deletion after recombination due to mating signal interference. Our data confirmed a 1:1 MAT/sex ratio in two pathogen populations, and showed that all members of the Ophiostomatales studied here including those that were previously deemed asexual have the potential to reproduce sexually. This ability can potentially increase genetic variability and can enhance fitness in new, ecological niches.

Variation in pathogenicity of a mountain pine beetle–associated blue-stain fungus, Grosmannia clavigera, on young lodgepole pine in British Columbia

Grosmannia clavigera is the most pathogenic blue-staining fungal associate of the mountain pine beetle (Dendroctonus ponderosae). In contrast to its importance as a primary invader of lodgepole pine (Pinus contorta) sapwood, intraspecific variability in pathogenicity of G. clavigera on lodgepole pine, the predominant host of mountain pine beetles in British Columbia, has not been investigated in detail. The present work reports on pathogenicity indicators induced by five G. clavigera isolates inoculated into lodgepole pines and growth characteristics of the isolates on artificial media. Fungi were inoculated at 200 inoculations/m2 into young lodgepole pine trees. Phloem lesion length, sapwood occlusion area, and sapwood moisture content were measured after 7 or 48 weeks. Three isolates produced long lesions, occluded larger areas, and reduced more the moisture content after 48 weeks compared with the remaining two isolates. Isolate ATCC 18086 induced the strongest pathogenic symptoms after 7 weeks and grew the fastest up to 22.5 °C but grew the slowest at 27.5 °C. In a low-oxygen environment, most isolates grew faster than under ambient conditions. Significant intraspecific variation was observed among G. clavigera isolates for all parameters tested. Key words: Grosmannia clavigera, Ophiostoma clavigerum, mountain pine beetle, blue-stain fungus, intraspecific variation, pathogenicity. Grosmannia clavigera est le plus pathogénique des champignons du bleuissement associés au dendroctone du pin ponderosa (Dendroctonus ponderosae). Contrairement à son importance en tant qu’envahisseur primaire de l’aubier du pin tordu latifolié (Pinus contorta), la variabilité intraspécifique de la pathogénicité de G. clavigera envers ce dernier, hôte principal du dendroctone du pin ponderosa en Colombie-Britannique, n’a pas été étudiée en détail. Les présents travaux font état d’indicateurs de pathogénicité produits par cinq isolats de G. clavigera appliqués sur des pins tordus latifoliés ainsi que des caractéristiques de croissance de ces isolats cultivés en milieu artificiel. De jeunes pins tordus latifoliés ont été inoculés avec le champignon à raison de 200 inoculations/m2. Au bout de 7 ou 48 semaines, la longueur des lésions subies par le phloème, la surface d’occlusion de l’aubier ainsi que son degré d’humidité ont été mesurés. Au bout de 48 semaines, comparativement aux deux autres, trois des cinq isolats avaient produit de longues lésions, occlus de plus larges surfaces et avaient davantage réduit le degré d’humidité. Au bout de 7 semaines, l’isolat ATCC 18086 avait provoqué les symptômes pathogènes les plus aigus. Son taux de croissance avait également été le plus rapide, et ce, jusqu’à 22,5 °C, pour devenir le plus lent à 27,5 °C. La plupart des isolats ont crû plus rapidement dans un environnement faible en oxygène que dans les conditions de milieu ambiant. Une variation intraspécifique significative a été observée chez les isolats de G. clavigera, et ce, relativement à tous les paramètres analysés. Mots-clés : Grosmannia clavigera, Ophiostoma clavigerum, dendroctone du pin ponderosa, champignon du bleuissement, variation intraspécifique, pathogénicité.

Characterization of microsatellite loci in the fungus, Grosmannia clavigera, a pine pathogen associated with the mountain pine beetle

The largest forest pest epidemic in Canadian history caused by the mountain pine beetle (MPB) and its fungal associates has killed over 15 million hectares of forest. Sixty simple sequence repeat regions were identified from Grosmannia clavigera, an MPB associated fungus. Eight loci genotyped in 53 isolates from two populations in British Columbia, Canada revealed three to 10 alleles per locus and gene diversities of 0 to 0.79. All but two of these loci showed length polymorphism in Leptographium longiclavatum, a related MPB fungal associate. These microsatellites will be useful in population genetic studies of these fungi.