Four new Ophiostoma species associated with conifer- and hardwood-infesting bark and ambrosia beetles from the Czech Republic and Poland.

Fungi under the order Ophiostomatales (Ascomycota) are known to associate with various species of bark beetles (Coleoptera: Curculionidae: Scolytinae). In addition this group of fungi contains many taxa that can impart blue-stain on sapwood and some are important tree pathogens. A recent survey that focussed on the diversity of the Ophiostomatales in the forest ecosystems of the Czech Republic and Poland uncovered four putative new species. Phylogenetic analyses of four gene regions (ITS1-5.8S-ITS2 region, ß-tubulin, calmodulin, and translation elongation factor 1-α) indicated that these four species are members of the genus Ophiostoma. All four newly described species can be distinguished from each other and from closely related species based on DNA sequence comparisons, morphological characters, growth rates, and their insect associations. Based on this study four new taxa can be circumscribed and the following names are provided: Ophiostoma pityokteinis sp. nov., Ophiostoma rufum sp. nov., Ophiostoma solheimii sp. nov., and Ophiostoma taphrorychi sp. nov. O. rufum sp. nov. is a member of the Ophiostoma piceae species complex, while O. pityokteinis sp. nov. resides in a discrete lineage within Ophiostoma s. stricto. O. taphrorychi sp. nov. together with O. distortum formed a well-supported clade in Ophiostoma s. stricto close to O. pityokteinis sp. nov. O. solheimii sp. nov. groups within a currently undefined lineage A, which also includes Ophiostoma grandicarpum and Ophiostoma microsporum. This study highlights the need for more intensive surveys that should include additional countries of Central Europe, insect vectors and host tree species in order to elucidate Ophiostoma species diversity in this region.

Ophiostoma quercus: An unusually diverse and globally widespread tree-infecting fungus.

Ophiostoma quercus (Ascomycota, Ophiostomatales) is a globally widespread, insect-vectored fungus that colonizes a wide diversity of hardwood and conifer hosts. Although the fungus is considered to be non-pathogenic, it is closely related to the fungi that cause Dutch elm disease. We examined the global diversity of O. quercus based on a ribosomal RNA marker and three unlinked gene regions. The fungus exhibited substantial morphological diversity. In addition, O. quercus had high genetic diversity in every continent from which it was collected, although the fungus was most diverse in Eurasia. There was no evidence of geographical clustering of haplotypes based on phylogenetic and network analyses. In addition, the phylogenetic trees generated based on the different markers were non-congruent. These results suggest that O. quercus has been repeatedly moved around the globe, because of trade in wood products, and that the fungal species most likely outcrosses regularly. The high genetic diversity of the fungus, as well as its ability to utilize a wide variety of arthropod vectors and colonize a tremendous diversity of tree host species makes O. quercus truly unique among ophiostomatoid fungi.

Ophiostomatoid fungi associated with mangroves in South Africa, including Ophiostoma palustre sp. nov.

Mangrove trees are continuously under stress due to environmental and/or anthropogenic pressures, which expose them to attack by pathogens, compromising their survival. Ophiostomatoid fungi cause sap stain and diseases of a wide spectrum of tree species globally. These fungi infect trees through natural, insect, animal and/or human made wounds. During routine surveys of mangrove trees in South Africa, wounds on branches and stems of Avicennia marina were regularly monitored for the presence of ophiostomatoid fungi at ten study sites in the country. The stems of four mangrove species, A. marina, Bruguiera gymnorrhiza, Rhizophora mucronata and Barringtonia racemosa were also wounded and evaluated for the appearance of these fungi. Ophiostomatoid fungi were obtained from the mangrove associate B. racemosa, but not from any of the true mangroves. Analyses of DNA sequence data for the internal transcribed spacer, ß-tubulin, calmodulin and translation elongation factor gene regions revealed that the fungi isolated from the wounds on B. racemosa belong to three species in the Ophiostomataceae, including a new taxon described here as Ophiostoma palustre sp. nov. These results suggest that the mangrove associate B. racemosa is more prone to colonization by ophiostomatoid fungi than the true mangroves.

The Ophiostoma clavatum species complex: a newly defined group in the Ophiostomatales including three novel taxa.

Two species of blue-stain fungi with similar morphologies, Ophiostoma brunneo-ciliatum and Ophiostoma clavatum, are associates of bark beetles infesting Pinus spp. in Europe. This has raised questions whether they represent distinct taxa. Absence of herbarium specimens and contaminated or mistakenly identified cultures of O. brunneo-ciliatum and O. clavatum have accentuated the uncertainty regarding their correct identification. The aim of this study was to reconsider the identity of European isolates reported as O. brunneo-ciliatum and O. clavatum by applying DNA-based identification methods, and to provide appropriate type specimens for them. Phylogenetic analyses of the ITS, ßT, TEF-1α and CAL gene sequences revealed that the investigated isolates represent a complex of seven cryptic species. The study confirmed that ITS data is insufficient to delineate species in some Ophiostoma species clusters. Lectotypes and epitypes were designated for O. clavatum and O. brunneo-ciliatum, and three new species, Ophiostoma brunneolum, Ophiostoma macroclavatum and Ophiostoma pseudocatenulatum, are described in the newly defined O. clavatum-complex. The other two species included in the complex are Ophiostoma ainoae and Ophiostoma tapionis. The results suggest co-evolution of these fungi in association with specific bark beetles. The results also confirm the identity of the fungus associated with the pine bark beetle Ips acuminatus as O. clavatum, while O. brunneo-ciliatum appears to be mainly associated with another pine bark beetle, Ips sexdentatus.

Multigene phylogenies and morphological characterization of five new Ophiostoma spp. associated with spruce-infesting bark beetles in China.

Ophiostoma spp. (Ophiostomatales, Ascomycota) are well-known fungi associated with bark beetles (Coleoptera: Scolytinae). Some of these are serious tree pathogens, while the majority is blue-stain agents of timber. In recent years, various bark beetle species have been attacking spruce forests in Qinghai province, China, causing significant damage. A preliminary survey was done to explore the diversity of the ophiostomatoid fungal associates of these beetles. The aims of the present study were to identify and characterize new Ophiostoma spp. associated with spruce-infesting bark beetles in Qinghai Province, and to resolve phylogenetic relationships of Ophiostoma spp. related to the Chinese isolates, using multigene phylogenetic analyses. Results obtained from four gene regions (ribosomal internal transcribed spacer regions, ß-tubulin, calmodulin, translation elongation factor-1α) revealed five new Ophiostoma spp. from Qinghai. These included O. nitidus sp. nov., O. micans sp. nov., and O. qinghaiense sp. nov. in a newly defined O. piceae complex. The other two new species, O. poligraphi sp. nov. and O. shangrilae sp. nov., grouped in the O. brunneo-ciliatum complex. Based on DNA sequence and morphological comparisons, we also show that O. arduennense and O. torulosum are synonyms of O. distortum, while O. setosum is a synonym of O. cupulatum.

Novel ophiostomatalean fungi from galleries of Cyrtogenius africus (Scolytinae) infesting dying Euphorbia ingens.

Euphorbia ingens trees have been dying in large numbers in the Limpopo Province of South Africa for approximately 15 years. The ambrosia beetle Cyrtogenius africus is often found infesting diseased and dying trees. The aim of this study was to identify the ophiostomatoid fungi occurring in the galleries of C. africus. Logs infested with this beetle were collected from the KwaZulu-Natal, Limpopo, Mpumalanga, and North West Provinces of South Africa. Fungi belonging to the Ophiostomatales were identified based on morphology and comparison of sequence data for the ß-tubulin, ITS1-5.8S-ITS2 and LSU gene regions. A novel species of Ophiostoma and a novel genus in the Ophiostomatales were identified. Inoculation studies with these fungi produced lesions in the branches of healthy E. ingens trees.

High-resolution DNA melt-curve analysis for cost-effective mass screening of pairwise species interactions.

Ecological studies of pairwise interactions are constrained by the methods available for rapid species identification of the interacting organisms. The resolution of data required to characterize species interaction networks at multiple spatio-temporal scales can be intensive, and therefore laborious and costly to collect. We explore the utility of high-resolution DNA melt-curve analysis (HRM) as a rapid species identification method. An approach was developed to identify organisms at the pairwise interaction level, with particular application to cryptic species interactions that are traditionally difficult to study. Here, we selected a challenging application; to identify the presence/absence of pathogenic fungi (Sporothrix inflata, Ophiostoma nigrocarpum and Ophiostoma galeiforme) transported by bark beetle vectors (Hylastes ater and Hylurgus ligniperda). The technique was able to distinguish between different species of DNA within a single, pooled sample. In test applications, HRM was effective in the mass screening and identification of pathogenic fungal species carried by many individual bark beetle vectors (n = 455 beetles screened) across large geographic scales. For two of the fungal species, there was no difference in the frequency of association with either of their vectors, but for the third fungal species there was a shift in vector-pathogen associations across locations. This technique allows rapid, mass screening and characterization of species interactions at a fraction of the time and cost of traditional methods. It is anticipated that this method can be readily applied to explore other cryptic species interactions, or other studies requiring rapid generation of large data sets and/or high-throughput efficiency.

Unequal recombination and evolution of the mating-type (MAT) loci in the pathogenic fungus Grosmannia clavigera and relatives.

Sexual reproduction in fungi is regulated by the mating-type (MAT) locus where recombination is suppressed. We investigated the evolution of MAT loci in eight fungal species belonging to Grosmannia and Ophiostoma (Sordariomycetes, Ascomycota) that include conifer pathogens and beetle symbionts. The MAT1-2 idiomorph/allele was identified from the assembled and annotated Grosmannia clavigera genome, and the MAT locus is flanked by genes coding for cytoskeleton protein (SLA) and DNA lyase. The synteny of these genes is conserved and consistent with other members in Ascomycota. Using sequences from SLA and flanking regions, we characterized the MAT1-1 idiomorph from other isolates of G. clavigera and performed dotplot analysis between the two idiomorphs. Unexpectedly, the MAT1-2 idiomorph contains a truncated MAT1-1-1 gene upstream of the MAT1-2-1 gene that bears the high-mobility-group domain. The nucleotide and amino acid sequence of the truncated MAT1-1-1 gene is similar to its homologous copy in the MAT1-1 idiomorph in the opposite mating-type isolate, except that positive selection is acting on the truncated gene and the alpha(α)-box that encodes the transcription factor has been deleted. The MAT idiomorphs sharing identical gene organization were present in seven additional species in the Ophiostomatales, suggesting that the presence of truncated MAT1-1-1 gene is a general pattern in this order. We propose that an ancient unequal recombination event resulted in the ancestral MAT1-1-1 gene integrated into the MAT1-2 idiomorph and surviving as the truncated MAT1-1-1 genes. The α-box domain of MAT1-1-1 gene, located at the same MAT locus adjacent to the MAT1-2-1 gene, could have been removed by deletion after recombination due to mating signal interference. Our data confirmed a 1:1 MAT/sex ratio in two pathogen populations, and showed that all members of the Ophiostomatales studied here including those that were previously deemed asexual have the potential to reproduce sexually. This ability can potentially increase genetic variability and can enhance fitness in new, ecological niches.

Sequencing and annotation of the Ophiostoma ulmi genome.

BACKGROUND: The ascomycete fungus Ophiostoma ulmi was responsible for the initial pandemic of the massively destructive Dutch elm disease in Europe and North America in early 1910. Dutch elm disease has ravaged the elm tree population globally and is a major threat to the remaining elm population. O. ulmi is also associated with valuable biomaterials applications. It was recently discovered that proteins from O. ulmi can be used for efficient transformation of amylose in the production of bioplastics. RESULTS: We have sequenced the 31.5 Mb genome of O.ulmi using Illumina next generation sequencing. Applying both de novo and comparative genome annotation methods, we predict a total of 8639 gene models. The quality of the predicted genes was validated using a variety of data sources consisting of EST data, mRNA-seq data and orthologs from related fungal species. Sequence-based computational methods were used to identify candidate virulence-related genes. Metabolic pathways were reconstructed and highlight specific enzymes that may play a role in virulence. CONCLUSIONS: This genome sequence will be a useful resource for further research aimed at understanding the molecular mechanisms of pathogenicity by O. ulmi. It will also facilitate the identification of enzymes necessary for industrial biotransformation applications.

Use of loop-mediated isothermal amplification for detection of Ophiostoma clavatum, the primary blue stain fungus associated with Ips acuminatus.

Loop-mediated isothermal amplification (LAMP) is an alternative amplification technology which is highly sensitive and less time-consuming than conventional PCR-based methods. Three LAMP assays were developed, two for detection of species of symbiotic blue stain fungi associated with Ips acuminatus, a bark beetle infesting Scots pine (Pinus sylvestris), and an additional assay specific to I. acuminatus itself for use as a control. In common with most bark beetles, I. acuminatus is associated with phytopathogenic blue stain fungi involved in the process of exhausting tree defenses, which is a necessary step for the colonization of the plant by the insect. However, the identity of the main blue stain fungus vectored by I. acuminatus was still uncertain, as well as its frequency of association with I. acuminatus under outbreak and non-outbreak conditions. In this study, we employed LAMP technology to survey six populations of I. acuminatus sampled from the Southern Alps. Ophiostoma clavatum was detected at all sampling sites, while Ophiostoma brunneo-ciliatum, reported in part of the literature as the main blue stain fungus associated with I. acuminatus, was not detected on any of the samples. These results are consistent with the hypothesis that O. clavatum is the main blue stain fungus associated with I. acuminatus in the Southern Alps. The method developed in the course of this work provides a molecular tool by which it will be easy to screen populations and derive important data regarding the ecology of the species involved.

The relative abundance of mountain pine beetle fungal associates through the beetle life cycle in pine trees.

The mountain pine beetle (MPB) is a native bark beetle of western North America that attacks pine tree species, particularly lodgepole pine. It is closely associated with the ophiostomatoid ascomycetes Grosmannia clavigera, Leptographium longiclavatum, Ophiostoma montium, and Ceratocystiopsis sp.1, with which it is symbiotically associated. To develop a better understanding of interactions between beetles, fungi, and host trees, we used target-specific DNA primers with qPCR to assess the changes in fungal associate abundance over the stages of the MPB life cycle that occur in galleries under the bark of pine trees. Multivariate analysis of covariance identified statistically significant changes in the relative abundance of the fungi over the life cycle of the MPB. Univariate analysis of covariance identified a statistically significant increase in the abundance of Ceratocystiopsis sp.1 through the beetle life cycle, and pair-wise analysis showed that this increase occurs after the larval stage. In contrast, the abundance of O. montium and Leptographium species (G. clavigera, L. longiclavatum) did not change significantly through the MPB life cycle. From these results, the only fungus showing a significant increase in relative abundance has not been formally described and has been largely ignored by other MPB studies. Although our results were from only one site, in previous studies we have shown that the fungi described were all present in at least ten sites in British Columbia. We suggest that the role of Ceratocystiopsis sp.1 in the MPB system should be explored, particularly its potential as a source of nutrients for teneral adults.

The highly variable mitochondrial small-subunit ribosomal RNA gene of Ophiostoma minus.

Mitochondrial genomes in the true fungi are highly variable both in size and organization. Most of this size variation is due to the presence of introns and intron-encoded open reading frames (ORFs). The objectives for this work were to examine the mitochondrial small-subunit ribosomal RNA (rns) gene of strains of Ophiostoma minus for the presence of introns and to characterize such introns and their encoded ORFs. DNA sequence analysis showed that among different strains of O. minus various rns gene exon/intron configurations can be observed. Based on comparative sequence analysis and RNA secondary structure modeling group I introns with LAGLIDADG ORFs were uncovered at positions mS569 and mS1224 and group II introns were present at positions mS379 and mS952. The mS379 group II intron encoded a fragmented reverse transcriptase (RT)-like ORF and the mS952 group II intron encoded a LAGLIDADG-type ORF. Examples of intron ORF degeneration due to frameshift mutations were observed. The mS379 group II intron is the first mitochondrial group II intron to have an ORF inserted within domain II, typically RT-like ORFs are inserted in domain IV. The evolutionary dynamics of the intron-encoded ORFs have also been examined.

Ophiostoma tsotsi sp. nov., a wound-infesting fungus of hardwood trees in Africa.

Polymorphic sequence-characterised marker assays from a recent diversity study on the Ascomycete fungus Ophiostoma quercus reported that some isolates from Africa were genetically distinct from O. quercus. In the present study, these African isolates were compared with authentic O. quercus isolates by evaluating morphological characters, growth in culture, mating compatibility and DNA sequence data. The isolates from Africa were morphologically similar to O. quercus, presenting Pesotum and Sporothrix synanamorphs in culture. Phylogenetic analyses of the ribosomal internal transcribed spacer regions 1 and 2, beta-tubulin and translation elongation factor 1-alpha gene regions confirmed that the African group represents a distinct species within the hardwood lineage of the O. piceae complex, closely related to O. ulmi and O. himal-ulmi. Mating studies between O. quercus and the African isolates showed that isolates mated predominantly with those of their own group, although there were rare cases of fertile crosses between the groups. Isolates residing in the African lineage are described here as a new species, O. tsotsi sp. nov.

Genes within genes: multiple LAGLIDADG homing endonucleases target the ribosomal protein S3 gene encoded within an rnl group I intron of Ophiostoma and related taxa.

In some ascomycete fungi, ribosomal protein S3 (Rps3) is encoded within a group I intron (mL2449) that is inserted in the U11 region of the mitochondrial large subunit rDNA (rnl) gene. Previous characterization of the mL2449 intron in strains of Ophiostoma novo-ulmi subspecies americana (Dutch Elm Disease) revealed a complex genes-within-genes arrangement whereby a LAGLIDADG homing endonuclease gene (HEG) is inserted into the RPS3 gene near the 3' terminus, creating a hybrid Rps3-LAGLIDADG fusion protein. Here, we examined 119 additional strains of Ophiostoma and related taxa representing 85 different species by a polymerase chain reaction- based survey and detected both short (approximately 1.6 kb) and long (>2.2 kb) versions of the mL2449 intron in 88 and 31 strains, respectively. Among the long versions encountered, 21 were sequenced, revealing the presence of either intact or degenerated HEG-coding regions inserted within the RPS3 gene. Surprisingly, we identified two new HEG insertion sites in RPS3; one near the original C-terminal insertion site and one near the N-terminus of RPS3. In all instances, the HEGs are fused in-frame with the RPS3-coding sequences to create fusion proteins. However, comparative sequence analysis showed that upon insertion, the HEGs displaced a portion of the RPS3-coding region. Remarkably, the displaced RPS3-coding segments are duplicated and fused in-frame to the 3' end of RPS3, restoring a full-length RPS3 gene. We cloned and expressed the LAGLIDADG portion of two Rps3-HEG fusions, and showed that I-OnuI and I-LtrI generate 4 nucleotide (nt), 3' overhangs, and cleave at or 1 nt upstream of the HEG insertion site, respectively. Collectively, our data indicate that RPS3 genes are a refuge for distinct types of LAGLIDADG HEGs that are defined by the presence of duplicated segments of the host gene that restore the RPS3 gene, thus minimizing the impact of the HEG insertion on Rps3 function.

Fungal radiation in the Cape Floristic Region: an analysis based on Gondwanamyces and Ophiostoma.

The Cape Floristic Region (CFR) displays high levels of plant diversity and endemism, and has received focused botanical systematic attention. In contrast, fungal diversity patterns and co-evolutionary processes in this region have barely been investigated. Here we reconstruct molecular phylogenies using the ITS and beta-tubulin gene regions of the ophiostomatoid fungi Gondwanamyces and Ophiostoma associated with southern African Protea species. Results indicate that they evolved in close association with Protea. In contrast to Protea, Ophiostoma species migrated to the CFR from tropical and subtropical Africa, where they underwent subsequent radiation. In both Gondwanamyces and Ophiostoma vector arthropods probably facilitated long-distance migration and shorter-distance dispersal. Although ecological parameters shaped most associations between ophiostomatoid fungi and Protea, there is congruence between fungal-host-associations and the systematic classification of Protea. These results confirm that the entire biotic environment must be considered in order to understand diversity and evolution in the CFR as a whole.

Fungi, including Ophiostoma karelicum sp. nov., associated with Scolytus ratzeburgi infesting birch in Finland and Russia.

Several elm-infesting bark beetles belonging to the genus Scolytus (Coleoptera: Scolytinae) are vectors of Ophiostoma spp., most notably the Dutch elm disease fungi. A related bark beetle species, Scolytus ratzeburgi, is known to infest birch in various parts of Europe, but it is unknown whether fungi are associated with this beetle. The aim of this study was to identify several fungal species isolated from S. ratzeburgi. Beetles and their galleries were collected from Betula pendula at three different sites in the boreal forests of the Karelia region, on both the Finnish and Russian sides of the border. Three ophiostomatoid fungi were isolated from the beetles and their galleries. One Penicillium and one Bionectria species were isolated only from the Finnish material and, based on DNA sequences, were identified as P. brevicompactum and a species close to the anamorph of B. zelandianovae. Two Ophiostoma species present in low numbers included O. quercus and a species closely related to O. catonianum. Only one Ophiostoma species was isolated consistently from all the galleries and beetles considered in the study. Comparison of DNA sequences and morphological characterization showed that this fungus represents an undescribed taxon, described here as O. karelicum sp. nov.