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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. [ABSTRACT FROM AUTHOR]

Grosmannia clavigera is a fungal pathogen associated with the mountain pine beetle ( Dendroctonus ponderosae) which is devastating large areas of western Canada’s conifer forests. This fungus also produces a dark melanin pigment that discolors pine sapwood. We have generated the draft genome of G. clavigera. However, functional characterization of genes identified in the genome sequence requires an efficient gene disruption method. In this work, we report a gene replacement strategy for G. clavigera using the Agrobacterium-mediated transformation in conjunction with linear or split-marker deletion cassettes. In addition, we used long flanking regions up to 3 kb from both sides of the targeted genes in our deletion cassettes. We assessed this gene disruption method with two genes from the melanin biosynthesis pathway that produce easily detectable white and red/brown mutant phenotypes: polyketide synthase and scytalone dehydratase. The approach yielded G. clavigera gene replacements with homologous recombination rates between 65 and 82%. For filamentous fungi, this is the first report showing that split-markers can be used with Agrobacterium-mediated transformation to generate appropriate mutants. This method can now be applied to efficiently identify genes involved in G. clavigera fungal pathogenicity and will facilitate understanding how the fungus overcomes the host defence system. [ABSTRACT FROM AUTHOR]

The article reports on a study of the differences in pathogenicity of Grosmannia clavigera, a blue-staining fungus associated with the mountain pine beetle, on young lodgepole pine in British Columbia. It made inoculations of Grosmannia clavigera fungi into young lodgepole pine trees. It founds out that Grosmannia clavigera isolates differed in terms of lesions, occluded larger areas, and sapwood moisture content.

Ophiostoma clavigerum is a destructive pathogen of lodgepole pine ( Pinus contorta) forests in western North America. It is therefore a relevant system for a genomics analysis of fungi vectored by bark beetles. To begin characterizing molecular interactions between the pathogen and its conifer host, we created an expressed sequence tag (EST) collection for O. clavigerum. Lodgepole pine sawdust and oleoresin media were selected to stimulate gene expression that would be specific to this host interaction. Over 6500 cDNA clones, derived from four normalized cDNA libraries, were single-pass sequenced from the 3′ end. After quality screening, we identified 5975 high-quality reads with an averagephred 20 of greater than 750 bp. Clustering and assembly of this high-quality EST set resulted in the identification of 2620 unique putative transcripts.blastx analysis revealed that only 67% of these unique transcripts could be matched to known or predicted protein sequences in public databases. Functional classification of these sequences provided initial insights into the transcriptome of O. clavigerum. Of particular interest, our ESTs represent an extensive collection of cytochrome P450 s, ATP-binding-cassette-type transporters and genes involved in 1,8-dihydroxynaphthalene-melanin biosynthesis. These results are discussed in the context of detoxification of conifer oleoresins and fungal pathogenesis. [ABSTRACT FROM AUTHOR]

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A new species of the Ophiostoma piceae-complex was isolated from bark beetles and the insect galleries of Larix kaempferi in Japan. This species was described as Ophiostoma breviusculum. The fungus was morphologically similar to O. piceae and O. quercus. However the average length of the perithecial necks and synnemata were shorter than for O. piceae and O. quercus. Synnemata morphological characteristics also differentiated O. breviusculum from the other species of the Ophiostoma piceae-complex isolated from conifers. Mating tests demonstrated that this fungus did not produce perithecia with O. floccosum, O. piceae and O. quercus. In phylogenetic trees using rDNA ITS O. breviusculum was placed in a clade with O. canum, O. piceae and O. subalpinum, but when using β-tubulin it was placed into a separate clade. [ABSTRACT FROM AUTHOR]

Copyright of Canadian Journal of Botany is the property of Canadian Science Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Canadian Journal of Forest Research is the property of Canadian Science Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Genomic DNA was extracted from 129 isolates of Ceratocystis resinifera, a species belonging to the C. coerulescens complex, and 19 polymorphic random amplified polymorphic DNA markers were used to study the population genetic structure of this fungus. The analysis suggested a moderate value for genetic diversity (HS= 0.209). However, when monomorphic markers and rare alleles, representing 89 markers, also were included in the calculation, the genetic diversity of Canadian populations of C. resinifera appeared to be much lower (HS = 0.045). This could be explained by two hypotheses: (i) recent introduction of this species into North America and (ii) clonal reproduction (by selfing). No specialization by C. resinifera for coniferous tree species was observed based on genetic differentiation index between isolates sampled from Pinus and Picea spp. and on phylogenetic analysis using Dice coefficient of association. In spite of a low genetic diversity, a very high genetic differentiation was observed among the nine geographical populations studied (FST = 20.8%). The genetic differences were especially striking when populations from Eastern Canada were compared with populations from Western Canada (Φst = 0.27%; P < 0.001), suggesting that a geographic reproductive barrier occurs in Central Canada. This barrier may be the consequence of a weak migration of insect vectors of C resinifera due to reduced presence of hosts in the Canadian Great Plains, where extensive agriculture occurs. However, results from pairwise FST matrix and phylogeny of haplotypes suggest that the barrier is not totally impenetrable because some gene flow occurred from the west and from the east in the Big River (Saskatchewan) population located in the middle of the Great Plains. [ABSTRACT FROM AUTHOR]

Ophiostoma piceae is a sap-staining fungus that colonizes and discolours wood. It has been established that the mycelium form but not the yeast form becomes pigmented in vitro. Suppressive subtractive hybridization PCR was used to isolate transcripts specifically upregulated in either the yeast or mycelial forms of O. piceae. Subtracted cDNAs were cloned and transformed into Escherichia coli. The yeast and mycelium specific clones were then screened by differential screening to reduce the possibility of isolating false positive clones and the differential expression of the two sets of cDNAs was confirmed by reverse Northern hybridization. Numerous genes appear to be specifically expressed in either the yeast or mycelial forms. Sequence analysis identified several cDNAs similar to known genes. However, a few cDNAs showed no similarity to sequences in the public databases. [Copyright &y& Elsevier]

Leptographium pyrinum, Leptographium terebrantis, Ophiostoma aureum, Ophiostoma clavigerum, and Ophiostoma robustum are very similar in morphology, host trees choice, and the way they are disseminated by bark beetles. Their phylogenetic relationships were clarified using rDNA and protein coding genes including actin, β-tubulin, and translation elongation factor-1α. Protein coding gene trees showed better resolution than the rDNA tree, which generated three clades: O. clavigerum, L. terebrantis/L. pyrinum, and O. robustum/O. aureum. A combined gene phylogenetic tree, which was supported by high bootstrap values, showed that O. aureum, L. pyrinum, O. robustum, and O. clavigerum each formed distinct clades while L. terebrantis was paraphyletic to O. clavigerum. The higher variability of the protein coding genes and the congruity in their phylogenetic results suggested that these genes may be better markers for identifying closely related species. These gene trees have also facilitated the description of the evolutionary relationships among these species. [Copyright &y& Elsevier]

Copyright of Canadian Journal of Botany is the property of Canadian Science Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Two synonymous sapstain species, Ophiostoma montium and Ophiostoma ips, which are vectored by Dendroctonus ponderosae and various bark beetles, respectively, were differentiated into separate species using growth and molecular characteristics. Analysis of 32 isolates of the two species from different countries showed that O. ips was able to grow at 35°C while O. montium was not. This growth-based differentiation was strongly supported by sequence data for the internal transcribed spacer (ITS), 5.8S and partial 28S rDNA, and the β-tubulin genes. The β-tubulin gene sequence data indicate that the two species can easily be differentiated with a single polymerase chain reaction (PCR) assay. [Copyright &y& Elsevier]

The genus Ceratocystis includes several morphologically similar species commonly found as agents of sapstain in coniferous trees. In this paper we describe a simple and reliable polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique that aids in the identification and differentiation of these fungi. PCR was used to amplify a 1.3-kb fragment of the β-tubulin gene from C. coerulescens, C. pinicola, C. douglassi, C. resinifera, C. rufipenni, C. polonica and C. adiposa. The PCR amplicon from representative isolates was sequenced. This information was utilized to select restriction enzymes that generated species-specific RFLP patterns. This approach was tested on our collection of over 200 Ceratocystis isolates and identified the fungi with a high level of confidence, reducing the time needed to identify these species by classical methods. [Copyright &y& Elsevier]

Thielavia terrestris 255B, a thermophilic ascomycete, produced two major forms of xylanase with pIs of 4.6 (xylanase I) and 6.1 (xylanase II). The latter enzyme could be purified to > 99% homogeneity using anion-exchange chromatography and gel filtration. Xylanase II had a mol wt of 25.7 kDa (SDS-PAGE) and a pH and a temperature optimum of 3.6-4.0 and 60-65°C, respectively. The ratio of the enzyme's activity against xylan and carboxymethylcellulose was 500-1000 to 1, indicating a possible application of this enzyme in biobleaching processes. The amino acid sequence of this protein is being determined, and initial data suggest that the enzyme belongs to a group of low-mol wt xylanases that have been isolated from both bacteria and fungi. [ABSTRACT FROM AUTHOR]