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We tested the effect of leaching on the concentration of western red cedar (WRC; Thuja plicata Donn ex D. Don) heartwood extractives that are known to exhibit antimicrobial activity and correlated this with fungal growth and decay. We assessed the extractive tolerance of the following fungal species: Acanthophysium lividocaeruleum, Coniophora puteana, Heterobasidion annosum, Pachnocybe ferruginea, Phellinus sulphurascens, and Phellinus weirii by measuring their growth rate (mm/day) on media with or without WRC leachate. These data were correlated with the ability of the fungal species to grow on and decay leached versus nonleached WRC. We used an ergosterol assay to estimate growth and a standard soil-block test to assess decay. We estimated that leaching reduced the concentration of 5 major extractives: (-)-plicatic acid, beta-thujaplicin, gamma-thujaplicin, beta-thujaplicinol, and thujic acid by approximately 80%. Phellinus sulphurascens exhibited the lowest extractive-tolerance in vitro, grew poorly on and caused minimal decay in nonleached WRC, but it grew well on and decayed pine and leached WRC. Coniophora puteana, H. annosum, and P. weirii displayed moderate to high tolerance to leachate, grew on and caused decay in nonleached as well as leached WRC, but their growth and decay were always greatest on leached WRC and pine, suggesting that leaching enhances decay by these fungi. Acanthophysium lividocaeruleum and Pachnocybe ferruginea exhibited high extractive-tolerance. Whereas A. lividocaeruleum clearly caused decay on all types of wood, no decay was observed with Pachnocybe ferruginea, which grew very slowly in the different wood species, and it may or may not be able to decay wood.

The work reported here investigated the fungal community inhabiting western redcedar fence material with a focus on species colonizing wood below the surface, of which little is known. From seven pieces of fence material, twenty-three different fungal species were isolated and characterized using both traditional morphology and molecular identification methods. The species identified included thirteen ascomycetous and ten basidiomycetous fungi. Isolates were tested for their resistance to beta-thujaplicin - one of the principle fungicidal agents of western redcedar heartwood extractives. Generally, ascomycetous fungi exhibited greater resistance to beta-thujaplicin than basidiomycetous fungi. Interestingly, three ascomycetous and two basidiomycetous species frequently isolated had high tolerance to this compound. These species could be candidate 'pioneer' species that invade and detoxify western redcedar extractives, paving the way for colonization by decay fungi.

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. [ABSTRACT FROM PUBLISHER]

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.

Characterization of the virulence of bark beetle-vectored fungi is important for assessing potential impacts of beetle outbreaks. Massive inoculation of trees with a cork borer appears to give the most accurate estimate of fungal virulence, but cork borer inoculation is time and labor intensive. In October 2003, 18 Pinus contorta var. latifolia were inoculated with a beetle-associated fungus, Grosmannia clavigera (Robinson-Jeffrey and Davidson) Zipfel et al., at densities of 200 and 800 cork borer holes m-2. In July 2004 nine trees were inoculated using bark flap inoculations. The fungal-induced moisture content reduction, sapwood occlusion area and needle discoloration were similar for the 800 cork borer holes m-2 density and for bark flap inoculations, while pathogenicity symptoms induced by the 200 cork borer holes m-2 were less intense. Bark flap inoculations were three times faster to perform than high-density cork borer inoculations, but differences in incubation time and yearly weather variation highlight the need for further studies. The bark flap method may be an efficient alternative to using massive inoculation densities when testing the ability of specific fungi to kill hosts, while the cork borer method may be a better method to assess pathogenic symptoms or the aggressiveness of specific fungi. [ABSTRACT FROM AUTHOR]

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]

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.)

Wood sapstaining fungi produce multiple proteases that break down wood protein. Three groups of subtilases have been identified in sapstaining fungi; however, it is not known if these groups have distinct physiological roles (B. Hoffman and C. Breuil, Curr. Genet. 41:168-175, 2002). In this work we examined the role of the subtilase Albinl from Ophiostoma piliferum. Reamplification of cDNA ends PCR was used to obtain the albinl gene sequence. The encoded subtilase is probably extracellular and involved in nutrient acquisition. This gene was disrupted with an Agrobacterium tumefacients-mediated transformation system. Two of the disruptants obtained had significantly lower levels of proteolytic activity, slower growth in bovine serum albumin, and significantly reduced growth on wood. Thus, albin1 plays an important role in O. piliferum's ability to acquire nitrogen from wood proteins. [ABSTRACT FROM AUTHOR]

In order to grow in wood, sapstaining fungi produce multiple proteases. Previously we have shown that three groups of subtilases appear to be present in sapstaining fungi; however, it is unknown whether these groups have distinct physiological roles. A representative gene from each of the three groups was chosen and the copy number and presence of homologous genes in other sapstaining fungi were determined. As well, the expressional regulation of these genes was determined in response to available nutrients, exogenous pH, and culture age. Gene homologues in the Ofloc1 group were common in Ophiostoma species. However, homologues from the Opic group were found in only certain Ophiostoma species. Cr group homologues were found in all of the species tested, except for Ophiostoma piceae. The expression of opil1, an Ofloc1 group gene, was induced by BSA, regulated by pH, and expressed within 12 h of induction by BSA. The expression of the opic gene, an Opic group gene, was induced by BSA but required the removal of either nitrogen or carbon repression, was also regulated by pH, and was expressed within 24 h of BSA induction. The Cr group gene opil2 was expressed under all conditions tested. [Copyright &y& Elsevier]

Presents a study which described a transformation protocol mediated by Agrobacterium tumefaciens for the sapstaining fungus Ophiostoma piceae. Review of related literature; Materials and methods used; Results and discussion.

Wood sapstain, a cosmetic defect that results in significant economical loss to forest-products industries, is caused by mycelial melanization of the wood-colonizing ophiostornatoid fungi. To improve our understanding of how melanin biosynthesis is regulated in the cosmopolitan sapstaining fungus, Ophiostoina piceae, we used insertional mutagenesis. Insertional mutants were generated by restriction enzyme-mediated integration (REMI) and Agrobacterium-mediated integration (AMI). We screened 1,053 REMI and 1,083 AMI transformants and found 30 mutants with impaired growth or pigmentation. We characterized four AM! transformants in more detail, in which the 1-DNA integrated at a single locus. The albino mutant TOPA45 had incorporated the 1-DNA in a polyketide synthase gene (PKSI). The mutants TOPA! and TOPAIO76 displayed reduced pigmentation. In TOPA I, the 1-DNA was inserted into a gene that encodes a putative protein kinase activator whereas, for TOPA 1076, it was inserted into a gene that encodes a protein with unknown function. Finally, the vegetative hyphac of mutant TOPA8I4 were not melanized, whereas the synnemata displayed the same level of pigmentation as the wild type. In the TOPA8I4 mutant, segregation analysis revealed that the mutant phenotype was not linked to the T-DNA insertion locus but to a translocation from the PIGI locus to the left border of the T-DNA. The protein predicted for the PIGI locus had a middle homology region that was specific to fungal transcription factors. [ABSTRACT FROM AUTHOR]

We recovered a spontaneous albino strain from ascospores of Ceratocystis resinifera, a sapstain fungus that grows deeply and rapidly in freshly felled conifer trees. This albino strain, named Kasper, was tested for its ability to prevent discoloration of spruce sapwood caused by wild-type sapstain fungi and compared with Cartapip 97, a commercially available biological control agent of sapstain in lodgepole pine and red pine logs. In a laboratory trial, Kasper reduced sapstain of white spruce logs as much as 94.4% and was more efficient than Cartapip 97. In field trials conducted in an area north of Qu6bec City, Kasper reduced sapstain of black spruce as much as 80%. In three of four field trials, Kasper was significantly more efficient than Cartapip 97 in reducing sapstain development. The exception was encountered in a 2003 trial conducted in a sawmill yard where Kasper did not reduce sapstain. In a field trial conducted in western Canada, at Aleza Lake forest near Prince George, Kasper almost totally prevented the development of sapstain, even after 24 weeks. These results suggest albino strains derived from C. resinifera might be an additional source of potential biocontrol agents against sapstain. [ABSTRACT FROM AUTHOR]

Abstract: Grosmannia clavigera is a fungal associate of the mountain pine beetle (Dendroctonus ponderosae) and a pathogen of lodgepole pine (Pinus contorta) that must overcome terpenoid oleoresin and phenolic defenses of host trees. G. clavigera responds to monoterpene influx with complementary mechanisms that include export and the use of these compounds as a carbon source. Cytochromes P450 (CYPs) may also be involved in the metabolism of host defense compounds. We have identified and phylogenetically classified G. clavigera CYPs (CYPome). We show that although the G. clavigera CYPome has contracted in evolution, certain CYP families have expanded by duplication. We analyzed RNA-seq data for CYP expression following treatment with terpenes and pine phloem extracts to identify CYPs potentially involved in detoxification of these pine defense compounds. We also used transcriptome analysis of G. clavigera grown on monoterpenes, triglycerides or oleic acid as a carbon source to identify up-regulated CYPs that may be involved in the utilization of these compounds to support fungal growth. Finally, we identify secondary metabolite biosynthetic gene clusters that contain CYPs, and CYPs in clusters that may be involved in conversion of host chemicals. [Copyright &y& Elsevier]

Abstract: 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. [ABSTRACT FROM AUTHOR]