Geosmithia associated with hardwood-infesting bark and ambrosia beetles, with the description of three new species from Poland.

Geosmithia species (Hypocreales, Ascomycota) are associates of bark beetles and other arthropods. One species, Geosmithia morbida, is a virulent tree pathogen of Juglans nigra. To date, 10 Geosmithia spp. from conifer-infesting, and at least 23 species from hardwood associated bark beetles have been reported from Europe. The aim of this study was to survey Geosmithia spp. associated with 18 bark and ambrosia beetle species in hardwood ecosystems in Poland. In addition, we evaluated the pathogenicity of the six Geosmithia species by inoculating Acer, Fagus, Quercus, Tilia and Ulmus seedlings. Our surveys yielded a total of 1060 isolates from 2915 beetles and 1887 galleries. We identified isolates using morphology and ITS, ß-tubulin and TEF1-α sequences. Altogether we identified 11 species including nine previously known and two new species described here as Geosmithia fagi sp. nov. and G. pazoutovae sp. nov. In addition, a sister species G. longistipitata sp. nov., associated with Picea trees, is described here. Bark beetles from hardwoods, with exeption of Dryocoetes alni, D. villosus, Scolytus ratzeburgi and ambrosia beetles, appear to be regular vectors of Geosmithia spp. Like in other parts of the world, most Geosmithia taxa exhibited a distinct level of vector/host specificity. None of Geosmithia isolates induced any disease symptoms under the conditions of our experiment. This study highlights the need for more intensive surveys across additional areas of Central and Northern Europe, insect vectors and host tree species in order to elucidate the Geosmithia species diversity in this region.

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.

Three new Leptographium spp. (Ophiostomatales) infecting hardwood trees in Norway and Poland.

Species of Leptographium are characterized by mononematous or synnematous conidiophores and are commonly associated with different arthropods. Some of them also produce a sexual state characterised by globose ascomata with elongated necks. Compared to investigations on coniferous trees, the occurrence of Leptographium species on hardwood trees has been poorly studied in Europe. During a survey of ophiostomatoid fungi on various hardwood tree species in Norway and Poland, three unusual species, which fit in the broader morphological description of Leptographium spp., were found in association with Trypodendron domesticum, Trypodendron signatum and Dryocoetes alni, and from wounds on a variety of hardwoods. Phylogenetic analyses of sequence data for six different loci (ITS1-5.8 S-ITS2, ITS2-LSU, ACT, ß-tubulin, CAL, and TEF-1α) showed that these Leptographium species are phylogenetically closely related to the species of the Grosmannia olivacea complex. The first species forms a well-supported lineage that includes Ophiostoma brevicolle, while the two other new taxa resided in a separate lineage; possibly affiliated with Grosmannia francke-grosmanniae. All the new species produce perithecia with necks terminating in ostiolar hyphae and orange-section shaped ascospores with cucullate, gelatinous sheaths. These species also produce dark olivaceous mononematous asexual states in culture. In addition, two of the newly described species have a second type of conidiophore with a short and non-pigmented stipe. The new Leptographium species can be easily distinguished from each other by their appearance and growth in culture. Based on novel morphological characters and distinct DNA sequences, these fungi were recognised as new taxa for which the names Leptographium tardum sp. nov., Leptographium vulnerum sp. nov., and Leptographium flavum sp. nov. are provided.

Two new Leptographium spp. reveal an emerging complex of hardwood-infecting species in the Ophiostomatales.

Species of Leptographium are generally characterized by mononematous conidiophores and are commonly associated with bark beetles and weevils. These species are responsible for sapstain and in some cases serious diseases on a range of primarily coniferous trees. In comparison with coniferous trees, the occurrence of Leptographium species on hardwood trees has been poorly studied in Europe. During a survey of ophiostomatoid fungi on various tree species in Norway and Poland, three unusual species, which fit the broader morphological description of Leptographium spp., were found in association with Scolytus ratzeburgi, Dryocoetes alni and Trypodendron domesticum on a variety of hardwoods, and from wounds on Tilia cordata. Phylogenetic analyses of sequence data for three gene regions (ITS2-LSU, ß-tubulin, and TEF1-α) showed that these Leptographium species are phylogenetically closely related to each other and form a well-supported lineage that included Grosmannia grandifoliae and Leptographium pruni. The first species could be distinguished from the other Leptographium species based on conidiophores arising from spiral hyphae, chlamydospore-like structures and a hyalorhinocladiella-like synanamorph in culture. The second species differs from the previous one by having distinctly shorter conidiophores and smaller conidia. This species also produces a well-developed sporothrix-like synanamorph with denticulate conidiogenous cells. Based on these unusual morphological characteristics and distinct DNA sequences, these fungi were recognised as new taxa for which the names Leptographium trypodendri sp. nov. and L. betulae sp. nov. are provided. The third group of isolates belonged to Grosmannia grandifoliae, representing the first report of this species outside of the USA. The newly defined G. grandifoliae complex is the first species complex in Leptographium s.l. consisting of only hardwood-infecting species.

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.

The role of chemical properties of the material deposited in nests of white stork in shaping enzymatic activity and fungal diversity.

Organic debris accumulated in bird nests creates a unique environment for organisms, including microbes. Built from various plant materials that are typically enriched by animal residues, bird nest favours the development of various fungal groups. The aim of this study was to investigate the chemical properties of the material deposited in the white stork Ciconia ciconia nests and the link between extracellular enzyme activity and the diversity and composition of culturable fungi. Our findings revealed low C/P and N/P ratio values in the nest materials, which indicate a high P availability. Nest material C/N/P ratio ranged from 67/8/1 to 438/33/1. Enzymatic activity strongly correlated with the content of carbon, nitrogen, and pH of the material deposited in the nests. A total of 2726 fungal isolates were obtained from the nests, from which 82 taxa were identified based on morphology and DNA sequence data. The study indicates that white stork nests are microhabitat characterised by diverse chemical and biochemical properties. We found relationship between the fungal richness and diversity and the C/P and N/P ratios of materials from the nests. Our study showed that culturable fungi occurred frequently in materials with high levels of C, N, and P, as well as high concentrations of base alkaline elements (Ca, Mg, and K).

Vector affinity and diversity of Geosmithia fungi living on subcortical insects inhabiting Pinaceae species in central and northeastern Europe.

Fungi from the genus Geosmithia (Ascomycota: Hypocreales) are associated with bark beetles (Coleoptera: Scolytinae), though little is known about ecology, diversity, and distribution of these fungi across beetle and its host tree species. This study surveyed the diversity, distribution and vector affinity of Geosmithia isolated from subcortical insects that colonized trees from the family Pinaceae in Central and Northeastern Europe. Twelve Geosmithia species were isolated from 85 plant samples associated with 23 subcortical insect species (including 14 bark beetle species). Geosmithia community composition was similar across different localities and vector species; although the fungal communities associated with insects that colonized Pinus differed from that colonizing other tree species (Abies, Larix, and Picea). Ten Geosmithia species from four independent phylogenetic lineages were not reported previously from vectors feeding on other plant families and seem to be restricted to the vectors from Pinaceae only. We conclude that presence of such substrate specificity suggests a long and stable association between Geosmithia and bark beetles.

Five new Graphium species from hardwood trees in Poland.

Graphium species form a well-supported monophyletic lineage within the Microascales (Ascomycota). Members of this genus can be found in association with bark beetles, as well as on tree wounds and in soils. During surveys of bark and ambrosia beetle-associated fungi and cavities made by woodpeckers on hardwood trees in Poland, many isolates with an affinity to Graphium were recovered. They were identified based on their morphological characters and sequence data for the internal transcribed spacer (ITS), 28S rDNA, ß-tubulin (TUB2), and translation elongation factor 1-α (TEF1) gene regions. The results revealed five new species, described here as G. brachiatum, G. longistipitatum, G. polonicum, G. radicatum, and G. trypophloei.

Soil-borne Ophiostomatales species (Sordariomycetes, Ascomycota) in beech, oak, pine, and spruce stands in Poland with descriptions of Sporothrixroztoczensis sp. nov., S.silvicola sp. nov., and S.tumida sp. nov.

Ophiostomatales (Ascomycota) contains many species, most of which are associated with bark beetles. Some members of this order are plant or animal pathogens, while others colonize soil, different plant tissues, or even carpophores of some Basidiomycota. However, little is known about soil-inhabiting Ophiostomatales fungi. A survey of these fungi associated with soil under beech, oak, pine, and spruce stands in Poland yielded 623 isolates, representing 10 species: Heinzbutiniagrandicarpa, Leptographiumprocerum, L.radiaticola, Ophiostomapiliferum, O.quercus, Sporothrixbrunneoviolacea, S.dentifunda, S.eucastaneae, and two newly described taxa, namely Sporothrixroztoczensissp. nov. and S.silvicolasp. nov. In addition, isolates collected from fallen shoots of Pinussylvestris that were pruned by Tomicus sp. are described as Sporothrixtumidasp. nov. The new taxa were morphologically characterized and phylogenetically analyzed based on multi-loci sequence data (ITS, ß-tubulin, calmodulin, and translation elongation factor 1-α genes). The Ophiostomatales species were especially abundant in soil under pine and oak stands. Leptographiumprocerum, S.silvicola, and S.roztoczensis were the most frequently isolated species from soil under pine stands, while S.brunneoviolacea was the most abundant in soil under oak stands. The results highlight that forest soil in Poland has a wide diversity of Ophiostomatales taxa, but further studies are required to uncover the molecular diversity and phylogenetic relationships of these fungi, as well as their roles in soil fungal communities.

Six new species of Sporothrix from hardwood trees in Poland.

Sporothrix (Sordariales, Ascomycota) is a well-supported monophyletic lineage within the Ophiostomatales, species of which occur in a diverse range of habitats including on forest trees, in the soil, associated with bark beetles and mites as well as on the fruiting bodies of some Basidiomycota. Several species have also been reported as important human and animal pathogens. During surveys of insect- and wound-associated Ophiostomatales from hardwood trees in Poland, many isolates with affinity to Sporothrix were recovered. In the present study, six undescribed Sporothrix spp. collected during these surveys are characterized based on their morphological characteristics and multi-locus phylogenenetic inference. They are described as Sporothrixcavum, Sporothrixcracoviensis, S.cryptarchum, S.fraxini, S.resoviensis, and S.undulata. Two of the Sporothrix spp. reside in the S.gossypina-complex, while one forms part of the S.stenoceras-complex. One Sporothrix sp. is a member of lineage F, and two other species grouped outside any of the currently defined species complexes. All the newly described species were recovered from hardwood habitats in association with sub-cortical insects, wounds or woodpecker cavities. These species were morphologically similar, with predominantly asexual states having hyaline or lightly pigmented conidia, which produce holoblastically on denticulate conidiogenous cells. Five of the new taxa produce ascomata with necks terminating in long ostiolar hyphae and allantoid ascospores without sheaths. The results suggest that Sporothrix species are common members of the Ophiostomatales in hardwood ecosystems of Poland.

Biological and physicochemical properties of the nests of White Stork Ciconia ciconia reveal soil entirely formed, modified and maintained by birds.

The physiological and behavioural activities of animals have far-reaching impacts on the characteristics and functioning of soil. This includes vertebrates, which are capable of modifying the physicochemical and biochemical properties of soil. To date, however, no species is known to be responsible for the entire process of soil formation, modification and maintenance. Large-bodied birds build nests which they then use for several years or even decades. During nest construction or renovation, birds gather and transport to the nesting site organic and mineral matter that includes tree branches of various sizes, twigs, turf, straw and hay. Over time, during subsequent breeding events, adult birds supply further loads of organic matter to the nest, such as food remains, excrement, pellets, feathers, egg shells and other materials. Taking the White Stork Ciconia ciconia as an example, we have shown that the materials deposited in the nests of large-bodied birds gradually produce ornithogenic soils over the years, with distinguishable layers having different physicochemical characteristics and biochemical activities. The tested nesting substrate met the criteria for ornithogenic material; the layers had appropriate thickness and phosphorus pentoxide (P2O5) content. Results of the study indicates that the material contained in White Stork nests have the characteristics of Histosols. Moreover, such nests harbour assemblages of fungi and arthropods that contain species typical of soil mycobiota and fauna, respectively. This study is the first to describe a soil that is formed, modified and maintained entirely by vertebrates and is physically isolated from the ground. Our results highlight the fact that the nests of large birds are unique structures in ecosystems and provide a habitat for a rich and diverse assemblage of organisms.

Two new species of Ophiostomatales (Sordariomycetes) associated with the bark beetle Dryocoetes alni from Poland.

Bark beetles belonging to the genus Dryocoetes (Coleoptera, Curculionidae, Scolytinae) are known vectors of fungi, such as the pathogenic species Grosmannia dryocoetidis involved in alpine fir (Abies lasiocarpa) mortality. Associations between hardwood-infesting Dryocoetes species and fungi in Europe have received very little research attention. Ectosymbiotic fungi residing in Ceratocystiopsis and Leptographium (Ophiostomatales, Sordariomycetes, Ascomycota) were commonly detected in previous surveys of the Dryocoetes alni-associated mycobiome in Poland. The aim of this study was to accurately identify these isolates and to provide descriptions of the new species. The identification was conducted based on morphology and DNA sequence data for six loci (ITS1-5.8S, ITS2-28S, ACT, CAL, TUB2, and TEF1-α). This revealed two new species, described here as Ceratocystiopsis synnemata sp. nov. and Leptographium alneum sp. nov. The host trees for the new species included Alnus incana and Populus tremula. Ceratocystiopsis synnemata can be distinguished from its closely related species, C. pallidobrunnea, based on conidia morphology and conidiophores that aggregate in loosely arranged synnemata. Leptographium alneum is closely related to Grosmannia crassivaginata and differs from this species in having a larger ascomatal neck, and the presence of larger club-shaped cells.

Seven new species of Graphilbum from conifers in Norway, Poland, and Russia.

During surveys of insect-associated mycobiomes in Norway, Poland, and Russia, isolates with affinity to Graphilbum (Ophiostomatales, Ascomycota) were recovered. In this study, eight known Graphilbum species as well as the newly collected isolates were compared based on morphology and DNA sequence data for four gene regions. The results revealed seven new species, described here as G. acuminatum, G. carpaticum, G. curvidentis, G. furuicola, G. gorcense, G. interstitiale, and G. sexdentatum. In addition to these species, G. crescericum and G. sparsum were commonly found in Norway. All new species were recovered from conifers in association with bark beetles, cerambycid beetles, and weevils and were morphologically similar, predominantly with pesotum-like asexual morphs. Where sexual morphs were present, these were small ascomata with short necks and rod-shaped ascospores having hyaline sheaths. The results suggest that Graphilbum species are common members of the Ophiostomatales in conifer ecosystems.

Four new Ophiostoma species associated with hardwood-infesting bark beetles in Norway and Poland.

Ophiostoma spp. (Ophiostomatales, Ascomycota) are well-known fungi associated with bark and ambrosia beetles (Curculionidae: Scolytinae, Platypodinae). Fungi in the Ophiostomatales include serious tree pathogens as well as agents of timber blue-stain. Although these fungi have been extensively studied in the northern hemisphere, very little is known regarding their occurrence on hardwoods in Europe. The aims of the present study were to identify and characterize new Ophiostoma spp. associated with bark and ambrosia beetles infesting hardwoods in Norway and Poland, and to resolve phylogenetic relationships of Ophiostoma spp. related to the Norwegian and Polish isolates, using multigene phylogenetic analyses. Results obtained from five gene regions (ITS, LSU, ß-tubulin, calmodulin, translation elongation factor 1-α) revealed four new Ophiostoma spp. These include Ophiostoma hylesinum sp. nov., O. signatum sp. nov., and O. villosum sp. nov. that phylogenetically are positioned within the Ophiostoma ulmi complex. The other new species, Ophiostoma pseudokarelicum sp. nov. reside along with Ophiostoma karelicum in a discrete, well-supported phylogenetic group in Ophiostoma s. stricto. The results of this study clearly show that the diversity and ecology of Ophiostoma spp. on hardwoods in Europe is poorly understood and that further studies are required to enrich our knowledge about these fungi.

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.

Putative origins of the fungus Leptographium procerum.

Appropriate management of invasive fungi requires adequate understanding of their global diversities and movement histories. The fungus Leptographium procerum is associated with root-colonizing forest insects in pine forests throughout the world, and may have contributed to the aggressive behaviour of the red turpentine beetle (Dendroctonus valens) in the beetle's invasive range in China. We used microsatellites and mating type loci to investigate the global diversity of L. procerum and the source population of L. procerum associated with D. valens in China. Clustering analyses supported the separation of the fungal data set into three genetically and geographically-distinct clusters: Europe, North America, and China. The fungus had the highest genetic diversity in Europe, followed by North America and China. Analyses using Approximate Bayesian Computation supported Europe as the most likely source of the North American and Chinese populations. Overall, the results suggested that Europe is the global centre of diversity of L. procerum. Furthermore, they suggested that L. procerum most likely arrived in China independently of D. valens and adopted this beetle as a vector after its introduction.