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.

De novo sequence assembly

A method for de novo assembly of a eukaryotic genome using Illumina, 454 and Sanger generated sequence data

No abstract

Summary Grosmannia clavigera is a bark beetle‐vectored pine pathogen in the mountain pine beetle epidemic in western North America. Grosmannia clavigera colonizes pines despite the trees' massive oleoresin terpenoid defences. We are using a functional genomics approach to identify G. clavigera's mechanisms of adaptation to pine defences. We annotated the ABC transporters in the G. clavigera genome and generated RNA‐seq transcriptomes from G. clavigera grown with a range of terpenes. We functionally characterized GcABC‐G1, a pleiotropic drug resistance (PDR) transporter that was highly induced by terpenes, using qRT‐PCR, gene knock‐out and heterologous expression in yeast. Deleting GcABC‐G1 increased G. clavigera's sensitivity to monoterpenes and delayed development of symptoms in inoculated young lodgepole pine trees. Heterologous expression of GcABC‐G1 in yeast increased tolerance to monoterpenes. G. clavigera but not the deletion mutant, can use (+)‐limonene as a carbon source. Phylogenetic analysis placed GcABC‐G1 outside the ascomycete PDR transporter clades. G. clavigera appears to have evolved two mechanisms to survive and grow when exposed to monoterpenes: GcABC‐G1 controls monoterpene levels within the fungal cells and G. clavigera uses monoterpenes as a carbon source. This work has implications for understanding adaptation to host defences in an important forest insect–fungal system, and potentially for metabolic engineering of terpenoid production in yeast.

Several media designed for use in a most probable number (MPN) determination of petroleum-degrading microorganisms were compared. The best results, i.e., largest numbers, were obtained using a buffered (32 mM PO4≡) liquid medium containing 1% hydrocarbon substrate. Of 104 presumptive oil degraders tested, 20 grew on oil agar medium but did not utilize oil or a mixture of pure paraffinic hydrocarbons (C10 to C16n-alkanes) in liquid (MPN) medium. Visible turbidity in the liquid medium was correlated with hydrocarbon utilization. Counts of petroleum degraders obtained using liquid medium (MPN) were in most cases higher than those obtained on an oil-amended silica gel medium. Both procedures yield an estimation of oil degraders, and the oil-amended agar permits growth of organisms which do not degrade crude oil. All strains of oil-degrading microorganisms examined in this study were lipolytic, but the converse was not always true.