Sepideh Massoumi Alamouti

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.

Population structure and migration pattern of a conifer pathogen, Grosmannia clavigera, as influenced by its symbiont, the mountain pine beetle

We investigated the population structure of Grosmannia clavigera (Gc), a fungal symbiont of the mountain pine beetle (MPB) that plays a crucial role in the establishment and reproductive success of this pathogen. This insect–fungal complex has destroyed over 16 million ha of lodgepole pine forests in Canada, the largest MPB epidemic in recorded history. During this current epidemic, MPB has expanded its range beyond historically recorded boundaries, both northward and eastward, and has now reached the jack pine of Alberta, potentially threatening the Canadian boreal forest. To better understand the dynamics between the beetle and its fungal symbiont, we sampled 19 populations in western North America and genotyped individuals from these populations with eight microsatellite markers. The fungus displayed high haplotype diversity, with over 250 unique haplotypes observed in 335 single spore isolates. Linkage equilibria in 13 of the 19 populations suggested that the fungus reproduces sexually. Bayesian clustering and distance analyses identified four genetic clusters that corresponded to four major geographical regions, which suggested that the epidemic arose from multiple geographical sources. A genetic cluster north of the Rocky Mountains, where the MPB has recently become established, experienced a population bottleneck, probably as a result of the recent range expansion. The two genetic clusters located north and west of the Rocky Mountains contained many fungal isolates admixed from all populations, possibly due to the massive movement of MPB during the epidemic. The general agreement in north–south differentiation of MPB and G. clavigera populations points to the fungal pathogen’s dependence on the movement of its insect vector. In addition, the patterns of diversity and the individual assignment tests of the fungal associate suggest that migration across the Rocky Mountains occurred via a northeastern corridor, in accordance with meteorological patterns and observation of MPB movement data. Our results highlight the potential of this pathogen for both expansion and sexual reproduction, and also identify some possible barriers to gene flow. Understanding the ecological and evolutionary dynamics of this fungus–beetle association is important for the modelling and prediction of MPB epidemics.

The genome and transcriptome of the pine saprophyte Ophiostoma piceae , and a comparison with the bark beetle-associated pine pathogen Grosmannia clavigera

BackgroundOphiostoma piceae is a wood-staining fungus that grows in the sapwood of conifer logs and lumber. We sequenced its genome and analyzed its transcriptomes under a range of growth conditions. A comparison with the genome and transcriptomes of the mountain pine beetle-associated pathogen Grosmannia clavigera highlights differences between a pathogen that colonizes and kills living pine trees and a saprophyte that colonizes wood and the inner bark of dead trees.ResultsWe assembled a 33 Mbp genome in 45 scaffolds, and predicted approximately 8,884 genes. The genome size and gene content were similar to those of other ascomycetes. Despite having similar ecological niches, O. piceae and G. clavigera showed no large-scale synteny. We identified O. piceae genes involved in the biosynthesis of melanin, which causes wood discoloration and reduces the commercial value of wood products. We also identified genes and pathways involved in growth on simple carbon sources and in sapwood, O. piceae’s natural substrate. Like the pathogen, the saprophyte is able to tolerate terpenes, which are a major class of pine tree defense compounds; unlike the pathogen, it cannot utilize monoterpenes as a carbon source.ConclusionsThis work makes available the second annotated genome of a softwood ophiostomatoid fungus, and suggests that O. piceae’s tolerance to terpenes may be due in part to these chemicals being removed from the cells by an ABC transporter that is highly induced by terpenes. The data generated will provide the research community with resources for work on host-vector-fungus interactions for wood-inhabiting, beetle-associated saprophytes and pathogens.

Rapid identification and detection of pine pathogenic fungi associated with mountain pine beetles by padlock probes.

Fifteen million hectares of pine forests in western Canada have been attacked by the mountain pine beetle (Dendroctonus ponderosae; MPB), leading to devastating economic losses. Grosmannia clavigera and Leptographium longiclavatum, are two fungi intimately associated with the beetles, and are crucial components of the epidemic. To detect and discriminate these two closely related pathogens, we utilized a method based on ligase-mediated nucleotide discrimination with padlock probe technology, and signal amplification by hyperbranched rolling circle amplification (HRCA). Two padlock probes were designed to target species-specific single nucleotide polymorphisms (SNPs) located at the inter-generic spacer 2 region and large subunit of the rRNA respectively, which allows discrimination between the two species. Thirty-four strains of G. clavigera and twenty-five strains of L. longiclavatum representing a broad geographic origin were tested with this assay. The HRCA results were largely in agreement with the conventional identification based on morphology or DNA-based methods. Both probes can also efficiently distinguish the two MPB-associated fungi from other fungi in the MPB, as well as other related fungi in the order Ophiostomatales. We also tested this diagnostic method for the direct detection of these fungi from the DNA of MPB. A nested PCR approach was used to enrich amplicons for signal detection. The results confirmed the presence of these two fungi in MPB. Thus, the padlock probe assay coupled with HRCA is a rapid, sensitive and reproducible method for the identification and detection of these ophiostomatoid fungi.

Ophiostomatoid fungi associated with the northern spruce engraver, Ips perturbatus, in western Canada

A number of ophiostomatoid fungi were isolated from the spruce-infesting bark beetle, Ips perturbatus and its galleries collected from felled spruce trees and logs in northern BC and the Yukon Territory. Isolates were identified to species using morphological characteristics, nuclear ribosomal DNA and partial β-tubulin gene sequences. Thirteen morphological and phylogenetic species were identified among the isolates. Leptographium fruticetum, Leptographium abietinum, Ophiostoma bicolor, Ophiostoma manitobense, O. piceaperdum, and eight undescribed species of the genus Ophiostoma and the anamorph genera Leptographium, Hyalorhinocladiella, Ambrosiella and Graphium. A number of these species, i.e. L. fruticetum, Hyalorhinocladiella sp. 2, O. bicolor and O. manitobense, were isolated repeatedly from I. perturbatus, while others, i.e. Graphium sp. 1 and O. piceaperdum, seemed to be␣sporadic associates. Among all the isolates, L. fruticetum had the highest relative dominance in this survey. A high frequency of occurrence of this species with the beetle may indicate a specific relationship between the two partners.

Target-specific PCR primers can detect and differentiate ophiostomatoid fungi from microbial communities associated with the mountain pine beetle Dendroctonus ponderosae

The aim of this study was to develop DNA probes that could identify the major fungal species associated with mountain pine beetles (MPB). The beetles are closely associated with fungal species that include ophiostomatoid fungi that can be difficult to differentiate morphologically. The most frequently isolated associates are the pine pathogens Grosmannia clavigera and Leptographium longiclavatum, the less pathogenic Ophiostoma montium, and an undescribed Ceratocystiopsis species (Cop. sp.). Because growing, isolating and extracting DNA from fungi vectored by MPB can be time and labour intensive, we designed three rDNA primer sets that specifically amplify short rDNA amplicons from O. montium, Cop. sp. and the pine Leptographium clade. We also designed two primer sets on a gene of unknown function that can differentiate G. clavigera and L. longiclavatum. We tested the primers on 76 fungal isolates that included MPB associates. The primers reliably identified their targets from DNA obtained from pure fungal cultures, pulverized beetles, beetle galleries, and tree phloem inoculated with G. clavigera. The primers will facilitate large-scale work on the ecology of the MPB-fungal-lodgepole pine ecosystem, as well as phytosanitary/quarantine sample screening.

Genetic and genomic evidence of niche partitioning and adaptive radiation in mountain pine beetle fungal symbionts

Bark beetles form multipartite symbiotic associations with blue stain fungi (Ophiostomatales, Ascomycota). These fungal symbionts play an important role during the beetles 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.

An Evaluation of British Columbian Beetle-Killed Hybrid Spruce for Bioethanol Production

The development of bioconversion technologies for production of fuels, chemicals, and power from renewable resources is currently a high priority for developed nations such as the United States, Canada, and the European Union as a way to improve national energy security and reduce greenhouse gas emissions. The widespread implementation of such technologies will require a sustainable supply of biomass from forestry and agriculture. Forests are a major source of feedstocks for biofuels production in Canada. Woody biomass includes residues from logging and forest thinning, and from wood processing and pulp production.More recently, damaged wood caused by beetle infestations has become available on a large scale in Western Canada. This study evaluates beetle-killed British Columbian hybrid spruce (HS) (Picea glauca × P. engelmannii) as a feedstock for the production of bioethanol. In the past 30 yr, attack by the beetle Dendroctonus rufipennis and associated fungi has resulted in estimated losses of more than three billion board feet in British Columbia alone. Here we describe the chemical and some physical characteristics of both healthy (HHS) and beetle-killed (BKHS) British Columbian HS and evaluate the technical feasibility of using these feedstocks as a source of biomass for bioethanol production. Untreated HHS and BKHS did not differ significantly in chemical composition except for the moisture content, which was significantly lower in BKHS (approx 10%) compared with HHS (approx 18%). However, the yields of carbohydrates in hydrolyzable and fermentable forms were higher at mild pretreatment conditions (H-Factor <1000) for BKHS compared with HHS. At medium (H-Factor 1000–2000) and severe (H-Factor >2000) pretreatment conditions HHS and BKHS behaved similarly. Organosolv pretreated HHS and BKHS demonstrated good ethanol theoretical yields, approx 70 and 80%, respectively.

Comparative Genomics of the Pine Pathogens and Beetle Symbionts in the Genus Grosmannia

Studies on beetle/tree fungal symbionts typically characterize the ecological and geographic distributions of the fungal populations. There is limited understanding of the genome-wide evolutionary processes that act within and between species as such fungi adapt to different environments, leading to physiological differences and reproductive isolation. Here, we assess genomic evidence for such evolutionary processes by extending our recent work on Grosmannia clavigera, which is vectored by the mountain pine beetle and jeffrey pine beetle. We report the genome sequences of an additional 11 G. clavigera (Gc) sensu lato strains from the two known sibling species, Grosmannia sp. (Gs) and Gc. The 12 fungal genomes are structurally similar, showing large-scale synteny within and between species. We identified 103,430 single-nucleotide variations that separated the Grosmannia strains into divergent Gs and Gc clades, and further divided each of these clades into two subclades, one of which may represent an additional species. Comparing variable genes between these lineages, we identified truncated genes and potential pseudogenes, as well as seven genes that show evidence of positive selection. As these variable genes are involved in secondary metabolism and in detoxifying or utilizing host-tree defense chemicals (e.g., polyketide synthases, oxidoreductases, and mono-oxygenases), their variants may reflect adaptation to the specific chemistries of the host trees Pinus contorta, P. ponderosa, and P. jeffreyi. This work provides a comprehensive resource for developing informative markers for landscape population genomics of these ecologically and economically important fungi, and an approach that could be extended to other beetle-tree-associated fungi.