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Soil fungi belonging to different functional guilds, such as saprotrophs, pathogens, and mycorrhizal symbionts, play key roles in forest ecosystems. To date, no study has compared the actual gene expression of these guilds in different forest soils. We used metatranscriptomics to study the competition for organic resources by these fungal groups in boreal, temperate, and Mediterranean forest soils. Using a dedicated mRNA annotation pipeline combined with the JGI MycoCosm database, we compared the transcripts of these three fungal guilds, targeting enzymes involved in C- and N mobilization from plant and microbial cell walls. Genes encoding enzymes involved in the degradation of plant cell walls were expressed at a higher level in saprotrophic fungi than in ectomycorrhizal and pathogenic fungi. However, ectomycorrhizal and saprotrophic fungi showed similarly high expression levels of genes encoding enzymes involved in fungal cell wall degradation. Transcripts for N-related transporters were more highly expressed in ectomycorrhizal fungi than in other groups. We showed that ectomycorrhizal and saprotrophic fungi compete for N in soil organic matter, suggesting that their interactions could decelerate C cycling. Metatranscriptomics provides a unique tool to test controversial ecological hypotheses and to better understand the underlying ecological processes involved in soil functioning and carbon stabilization.

The cherry spruce rust caused by Thekopsora areolata (Fr.) Magnus results in significant losses in spruce seed production in the forest industry. The pathogen is present in Asia and Europe but absent from North America where it has been considered as a potential threat and listed as a quarantine organism by the United States Department of Agriculture. A comprehensive list and in-depth information regarding the alternate hosts of this pathogen are important for conducting epidemiological studies and for optimal disease control. Prunus padus L. is the main alternate host reported for T. areolata. In this study, we investigated the susceptibility of domestic and exotic Prunus spp. and other potential alternate host-plant species native to Scandinavia to T. areolata infection through a field survey and aeciospore inoculation experiments in the greenhouse and laboratory. No new susceptible species were found. In Sweden, a new record of Prunus grayana Maxim. with low susceptibility to T. areolata was found. In addition, we updated the list of currently confirmed alternate hosts of T. areolata according to field observations and inoculation results. Prunus padus and Prunus serotina Ehrh., as well as their hybrids and subspecies of Prunus padus, are highly susceptible, while Prunus depressa Pursh, Prunus grayana, Prunus spinosa L., and Prunus tenella Batsch are considered slightly susceptible. Key words: rust fungi, Picea abies (L.) H.Karst, cherry spruce rust, susceptibility, Prunus. La rouille de l'epinette causee par Thekopsora areolata (Fr.) Magnus entraine des pertes importantes dans la production de semences d'epinette dans l'industrie forestiere. L'agent pathogene est present en Asie et en Europe, mais il est absent en Amerique du Nord ou il a ete considere comme une menace et inscrit comme organisme de quarantaine par le departement de l'agriculture des Etats-Unis. Une liste complete et des informations approfondies concernant les hotes relais de cet agent pathogene sont importantes pour realiser des etudes epidemiologiques et pour un controle optimal de la maladie. Prunus padus L. est le principal h[delta]te relais signale pour T. areolata. Dans cette etude, les auteurs ont etudie la sensibilite de Prunus spp. domestiques et exotiques et d'autres especes vegetales hotes relais potentiels originaires de Scandinavie, a l'infection par T. areolata, au moyen d'observations sur le terrain et d'experiences d'inoculation d'ecidiospores en serre et en laboratoire. Aucune nouvelle espece sensible n'a ete trouvee. En Suede, un nouveau signalement de Prunus grayana Maxim. avec une faible sensibilite a T. areolata a ete trouve. De plus, les auteurs ont mis a jour la liste des hotes relais actuellement confirmes de T. areolata selon les observations de terrain et les resultats d'inoculation. Prunus padus, Prunus serotina Ehrh., ainsi que leurs hybrides et sous-especes de Prunus padus, sonttressensibles, tandisque Prunus depressa Pursh, Prunus grayana, Prunus spinosa L. et Prunus tenella Batsch sont consideres comme legerement sensibles. [Traduit par la Redaction] Mots-cles : champignons de rouille, Picea abies (L.) H.Karst, rouille de l'epinette, sensibilite, Prunus., Introduction Norway spruce, Picea abies (L.) H.Karst, is a significant coniferous species in natural forests throughout Europe, particularly important in Scandinavia. It has been introduced to and planted in North [...]

Parasitism and saprotrophic wood decay are two fungal strategies fundamental for succession and nutrient cycling in forest ecosystems. An opportunity to assess the trade-off between these strategies is provided by the forest pathogen and wood decayer Heterobasidion annosum sensu lato.We report the annotated genome sequence and transcript profiling, as well as the quantitative trait loci mapping, of one member of the species complex: H. irregulare. Quantitative trait loci critical for pathogenicity, and rich in transposable elements, orphan and secreted genes, were identified.A wide range of cellulose-degrading enzymes are expressed during wood decay. By contrast, pathogenic interaction between H. irregulare and pine engages fewer carbohydrate-active enzymes, but involves an increase in pectinolytic enzymes, transcription modules for oxidative stress and secondary metabolite production.Our results show a trade-off in terms of constrained carbohydrate decomposition and membrane transport capacity during interaction with living hosts. Our findings establish that saprotrophic wood decay and necrotrophic parasitism involve two distinct, yet overlapping, processes.

Author(s): Ãke Olson (corresponding author) [1]; Jan Stenlid [1] Hybrid species of fungal pathogens that infect wild and cultivated plants are emerging with new virulence and host ranges, posing a [...]

Studies the mitochondrial control of fungal hybrid virulence. Threat of hybrid species of fungal pathogens to agriculture and forestry; Discovery of hybrid species of fungal plant pathogens; Mitochondria of the hybrid isolates.

Sizes of mitochondrial genomes vary extensively between fungal species although they typically contain a conserved set of core genes. We have characterised the mitochondrial genome of the conifer root rot pathogen Heterobasidion irregulare and compared the size, gene content and structure of 20 Basidiomycete mitochondrial genomes. The mitochondrial genome of H. irregulare was 114, 193 bp and contained a core set of 15 protein coding genes, two rRNA genes and 26 tRNA genes. In addition, we found six non-conserved open reading frames (ORFs) and four putative plasmid genes clustered in three separate regions together with 24 introns and 14 intronic homing endonuclease genes, unequally spread across seven of the core genes. The size differences among the 20 Basidiomycetes can largely be explained by length variation of intergenic regions and introns. The Agaricomycetes contained the nine largest mitochondrial genomes in the Basidiomycete group and Agaricomycete genomes are significantly (p < 0.001) larger than the other Basidiomycetes. A feature of the Agaricomycete mitochondrial genomes in this study was the simultaneous occurrence of putative plasmid genes and non-conserved ORFs, with Cantharellus cibarius as only exception, where no non-conserved ORF was identified. This indicates a mitochondrial plasmid origin of the non-conserved ORFs or increased mitochondrial genome dynamics of species harbouring mitochondrial plasmids. We hypothesise that two independent factors are the driving forces for large mitochondrial genomes: the homing endonuclease genes in introns and integration of plasmid DNA.

Incompatibility systems in which individuals bearing identical alleles reject each other favor the maintenance of a diversity of alleles. Mushroom mating type loci (MAT) encode for dozens or hundreds of incompatibility alleles whose loss from the population is greatly restricted through negative frequency selection, leading to a system of alleles with highly divergent sequences. Here, we use DNA sequences of homeodomain (HD) encoding genes at the MAT locus of five closely related species of the root rot basidiomycete Heterobasidion annosum sensu lato to show that the extended coalescence time of MAT alleles greatly predates speciation in the group, contrasting loci outside of MAT that show allele divergences largely consistent with the species phylogeny with those of MAT, which show rampant trans-species polymorphism. We observe a roughly 6-fold greater genealogical depth and polymorphism of MAT compared with non-MAT that argues for the maintenance of balanced polymorphism for a minimum duration of 24 My based on a molecular-clock calibrated species phylogeny. As with other basidiomycete HD genes, balancing selection appears to be concentrated at the specificity-determining region in the N-terminus of the protein based on identification of codons under selection and the absence of recombination within the region. However, the elevated polymorphism extends into the nonspecificity determining regions as well as a neighboring non-MAT gene, the mitochondrial intermediate peptidase (MIP). In doing so, increased divergence should decrease recombination among alleles and as a by-product create incompatibilities in the functional domains not involved in allele recognition but in regulating sexual development.

Conifers have dominated forests for more than 200 million years and are of huge ecological and economic importance. Here we present the draft assembly of the 20-gigabase genome of Norway spruce (Picea abies), the first available for any gymnosperm. The number of well-supported genes (28,354) is similar to the >100 times smaller genome of Arabidopsis thaliana, and there is no evidence of a recent whole-genome duplication in the gymnosperm lineage. Instead, the large genome size seems to result from the slow and steady accumulation of a diverse set of long-terminal repeat transposable elements, possibly owing to the lack of an efficient elimination mechanism. Comparative sequencing of Pinus sylvestris, Abies sibirica, Juniperus communis, Taxus baccata and Gnetum gnemon reveals that the transposable element diversity is shared among extant conifers. Expression of 24-nucleotide small RNAs, previously implicated in transposable element silencing, is tissue-specific and much lower than in other plants. We further identify numerous long (>10,000 base pairs) introns, gene-like fragments, uncharacterized long non-coding RNAs and short RNAs. This opens up new genomic avenues for conifer forestry and breeding.

Fungal peroxidases (ClassII) have a key role in degrading recalcitrant polyphenolic compounds in boreal forest wood, litter and humus. To date, their occurrence and activity have mainly been studied in a small number of white-rot wood decomposers. However, peroxidase activity is commonly measured in boreal forest humus and mineral soils, in which ectomycorrhizal fungi predominate. Here, we used degenerate PCR primers to investigate whether peroxidase-encoding genes are present in the genomes of a wide phylogenetic range of ectomycorrhizal taxa. Cloning and sequencing of PCR products showed that ectomycorrhizal fungi from several different genera possess peroxidase genes. The new sequences represent four major homobasidiomycete lineages, but the majority is derived from Cortinarius, Russula and Lactarius. These genera are ecologically important, but consist mainly of non-culturable species from which little ecophysiological information is available. The amplified sequences contain conserved active sites, both for folding and substrate oxidation. In some Cortinarius spp., there is evidence for gene duplications during the evolution of the genus. ClassII peroxidases seem to be an ancient and a common feature of most homobasidiomycetes, including ectomycorrhizal fungi. Production of extracellular peroxidases may provide ectomycorrhizal fungi with access to nitrogen sequestered in complex polyphenolic sources.

To investigate functional differences in the recognition and response mechanisms of conifer roots to fungi with different trophic strategies, Pinus sylvestris L. was challenged with a saprotrophic fungus Trichoderma aureoviride Rifai. The results were compared with separate studies investigating pine interactions with a pathogen, Heterobasidion annosum (Fr.) Bref. sensu stricto and an ectomycorrhizal symbiont, Laccaria bicolor Maire (Orton). Global changes in the expression of 2109 conifer genes were assayed 1, 5 and 15 days after inoculation. Gene expression data from a cDNA microarray were analyzed by the 2-interconnected mixed linear model statistical approach. The total number of genes differentially expressed compared with the uninfected control was similar after challenge with the pathogen and the ectomycorrhizal symbiont, but the number of differentially expressed genes increased over time for H. annosum, and decreased for L. bicolor. Inoculation of pine roots with T. aureoviride resulted overall in a much lower number of genes with changed transcript levels compared with inoculation with H. annosum or L. bicolor. Functional classification of the differentially expressed genes revealed that the ectomycorrhizal fungus triggered transient induction of defence-related genes. The response and induction of defence against the pathogen was delayed and the magnitude increased over time. Thus, there were specific transcriptional responses depending on whether the conifer roots were challenged with mutualistic, saprotrophic or pathogenic fungi. This suggests that pine trees are able to recognize diverse fungal species and specifically distinguish whether they are pathogenic, neutral or beneficial microbial agents.

The infection-related expression of a Heterobasidion annosum (Fr.) Bref. sensu lato (s.l.) putative cytochrome P450 gene (CPM2) was analysed with realtime quantitative PCR. CPM2 was highly expressed after 20 days of growth in bark of living spruce trees, and up-regulated by nitrogen starvation on artificial media. Infection of pine seedlings in the presence of high-carbon medium results in low expression levels of CPM2, thus indicating that starvation is the primary regulatory factor for induction of this gene. The predicted cpm2 protein contains 507 amino acids with an estimated molecular mass of 56.1 kDa, and display all conserved amino acids of the cytochrome P450 protein family. The protein has a high similarity to the ord1/ordA O-methylsterigmatocystin oxidoreductases from Aspergillus flavus/A. parasiticus, responsible for catalysing the final step in aflatoxin biosynthesis. Results indicate that cpm2 is potentially important for pathogenicity in H. annosum s.l.

The mechanisms underlying defence reactions to a pathogen attack, though well studied in crop plants, are poorly understood in conifers. To analyze changes in gene transcript abundance in Pinus sylvestris L. root tissues infected by Heterobasidion annosum (Fr.) Bref. s.l., a cDNA microarray containing 2109 ESTs from P. taeda L. was used. Mixed model statistical analysis identified 179 expressed sequence tags differentially expressed at 1, 5 or 15 days post inoculation. In general, the total number of genes differentially expressed during the infection increased over time. The most abundant group of genes up-regulated upon infection coded for enzymes involved in metabolism (phenylpropanoid pathway) and defence-related proteins with antimicrobial properties. A class III peroxidase responsible for lignin biosynthesis and cell wall thickening had increased transcript abundance at all measurement times. Real-time RT-PCR verified the microarray results with high reproducibility. The similarity of the expression profiling pattern observed in this pathosystem to those documented in crop pathology suggests that angiosperms and gymnosperms use similar genetic programs in responding to invasive growth by microbial pathogens.

Identification of virulence factors of phytopathogens is important for the fundamental understanding of infection and disease progress in plants and for the development of control strategies. We have identified quantitative trait loci (QTL) for virulence on 1-year-old Pinus sylvestris and 2-year-old Picea abies seedlings and positioned them on a genetic linkage map of the necrotrophic phytopathogen Heterobasidion annosum sensu lato (s.l.), a major root rot pathogen on conifers. The virulence of 102 progeny isolates was analysed using two measurements: lesion lengths and fungal growth in sapwood from a cambial infection site. We found negative virulence effects of hybridization although this was contradicted on a winter-hardened spruce. On P. abies, both measurements identified several partially overlapping QTLs on linkage group (LG) 15 of significant logarithm of odds (LOD) values ranging from 2.31 to 3.85. On P. sylvestris, the lesion length measurement also identified a QTL (LOD 3.09) on LG 15. Moreover, QTLs on two separate smaller LGs, with peak LOD values of 2.78 and 4.58 were identified for fungal sapwood growth and lesion lengths, respectively. The QTL probably represent loci important for specific as well as general aspects of virulence on P. sylvestris and P. abies.

Heterobasidion annosum causes root and butt-rot in trees and is the most serious forest pathogen in the northern hemisphere. We developed a rapid and simple Agrobacterium-mediated method of gene delivery into H. annosum to be used in functional studies of candidate genes and for visualization of mycelial interactions. Heterobasidion annosum TC 32-1 was cocultivated at pH 5.6 and 20 degrees C in Hagems medium with Agrobacterium tumefaciens C58 carrying plasmids with hygromycin B resistance as the selectable marker and green fluorescent protein as a visual marker. We obtained 18 mitotically stable transformed isolates showing green fluorescence protein activity.

Using mRNA differential display we have identified differentially expressed genes in non-self-interacting vs. single mycelia of the conifer pathogen Heterobasidion annosum and the wood decomposing basidiomycete Physisporinus sanguinolentus. Altogether 39 differentially displayed bands were cloned and sequenced, corresponding to 21 unique genes, which were confirmed by semi-quantitative RT-PCR to be differentially expressed. Further confirmation of differential gene expression was made by real time RT-PCR. All 10 genes identified from P. sanguinolentus had lower expression, while in H. annosum three genes had higher and eight lower expression in non-self-interacting mycelia vs. single mycelia. One of the induced genes showed high similarity to the Coprinus cinereus recA/RAD51 homolog (rah1) which is essential for homologous recombination, DNA repair and stress responses.