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.

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.

Fungicolous Fungi on Pseudosclerotial Plates and Apothecia of Hymenoscyphus fraxineus and Their Biocontrol Potential.

In the present work, research tasks were carried out in the search for fungi with potential biocontrol possibilities in relation to the ash dieback pathogen, Hymenoscyphus fraxineus. In the years 2012-2021, dead petioles of F. excelsior and F. mandshurica were collected, on which morphological structures of H. fraxineus showed unusual symptoms of dying (apothecia) and signs of colonization by other fungi (pseudosclerotial plates). Based on morphological and molecular phylogenetic data, 18 fungal taxa were identified. Thirteen of them belong to Ascomycota: Clonostachys rosea, Cl. solani, Cordyceps sp., Minimidochium sp., Nemania diffusa, Fusarium sp., Pestalotiopsis sp., Trichoderma atroviride, T. harzianum, T. polysporum, T. rodmanii, T. tomentosum, Trichoderma sp., and five other taxa are represented by Basidiomycota: Corticiales sp., Cyathus olla, Efibula sp., Gymnopus sp. and Polyporales sp. In 108 dual cultures in vitro, three different types of interactions were distinguished: (i) physical colony contact (5.6%), (ii) presence of an inhibition zone between the colonies (0.9%), and (iii) copartner overgrowth of H. fraxineus colonies and partial or complete replacement of the pathogen (93.5%). In the dual cultures, various morphological deformations of H. fraxineus hyphae were observed: the development of apical or intercalary cytoplasmic extrusions, development of internal hyphae of the test fungi in pathogens' hyphae, the deformation and disruption of significant sections of H. fraxineus hyphae via lysis and mycoparasitism, complete desolation of H. fraxineus cells and breakdown of hyphae into short fragments, and disappearing of pigment in the affected hyphae of H. fraxineus. The inoculation tests performed in vivo or in glass Petrie dishes showed that all the identified taxa were able to lead to pathological changes in H. fraxineus apothecia, and the mycelium of some of them completely covered pseudosclerotial plates of H. fraxineus. It was emphasized in the discussion that such activity of these fungi in forest stands may contribute to the reduction in the H. fraxineus inoculum reservoir.

Fungal endophytes in Fraxinus excelsior petioles and their in vitro antagonistic potential against the ash dieback pathogen Hymenoscyphus fraxineus.

Fungal endophytes were isolated from 250 asymptomatic leaf petioles of Fraxinus excelsior collected from trees showing symptoms of ash dieback in five forest sites in southern Poland. Fungal isolations yielded 1646 colonies representing 97 taxa, including 92 Ascomycota and 5 Basidiomycota species. The most common Ascomycota comprised Nemania serpens (38.0 % of colonized petioles), Diaporthe eres (33.6 %), Venturia fraxini (26.4 %), Diaporthe sp. 1 (20.4 %), Alternaria sp. 1 (14.8 %), Colletotrichum acutatum (14.8 %), Nemania diffusa (14.0 %), Colletotrichum gloeosporioides (12.4 %) and Colletotrichum sp. (12.4 %). The occurrence of all these taxa except Alternaria sp. 1 was significantly different between the studied forest sites. Two yeast species, Vishniacozyma foliicola (4.8 %) and Cystobasidium pinicola (2.8 %), dominated among the Basidiomycota endophytes detected. All the fungal endophytes were tested in dual culture antagonistic assays against two strains of Hymenoscyphus fraxineus, resulting in the development of four interaction types. The interactions included the physical contact of co-partners' mycelia (41.8 %), development of an inhibition zone (47.4 %), growth of endophyte mycelia over H. fraxineus colonies (9.3 %) and growth of H. fraxineus mycelia over endophyte colonies (1.5 %). The strongest antibiotic activity against H. fraxineus, measured by the width of the inhibition zone, was observed for Cytospora pruinosa, Fusarium lateritium, Phoma sp. 2, Pleosporales sp. 2 and Thielavia basicola. A variety of morphophysiological deformations of H. fraxineus hyphae were observed under endophyte pressure: spiral twist of the hyphae, formation of cytoplasmic extrusions, development of torulose hyphae and excessive lateral branching of the hyphae. The strongest antagonistic effects, coupled with the potential to overgrow H. fraxineus colonies, was shown by Clonostachys rosea, Nemania diffusa, N. serpens, Peniophora cinerea, Rosellinia corticium and Xylaria polymorpha. Some of these species were able to attack H. fraxineus hyphae in a mycoparasitic manner. The antagonistic activities included the physical penetration of H. fraxineus hyphae, dissolution of hyphal cell walls, disappearance of pigmentation, disintegration of hyphae and degradation of other fungal structures. In contrast, one of the most commonly detected endophytes in ash leaves, Venturia fraxini, did not show in vitro antagonistic potential against H. fraxineus. Finally, we discuss the potential of the detected fungal endophytes to combat H. fraxineus invasion, the cause of ash decline in Europe.

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.

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 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.

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.

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.

Virulence of Hymenoscyphus albidus and H. fraxineus on Fraxinus excelsior and F. pennsylvanica.

European ash (Fraxinus excelsior) is currently battling an onslaught of ash dieback, a disease emerging in the greater part of its native area, brought about by the introduction of the ascomycete Hymenoscyphus fraxineus (= Hymenoscyphus pseudoalbidus). The closely-related fungus Hymenoscyphus albidus, which is indigenous to Europe, is non-pathogenic when in contact with F. excelsior, but could pose a potential risk to exotic Fraxinus species. The North American green ash (Fraxinus pennsylvanica) is planted widely throughout Europe and regenerates naturally within this environment but little is known about the susceptibility of this species to ash dieback. We performed wound inoculations with both fungi (nine strains of H. fraxineus and three strains of H. albidus) on rachises and stems of F. excelsior and F. pennsylvanica under field conditions in Southern Poland. Necrosis formation was evaluated after two months on the rachises and after 12 months on the stems. After inoculation of H. albidus, only small lesions (of up to 1.3 cm in length) developed on the F. excelsior and F. pennsylvanica rachises, but with no significant distinction from the controls. Hymenoscyphus albidus did not cause necrotic lesions on the stems of either Fraxinus species. In contrast, H. fraxineus induced necroses on all inoculated rachises of both ash species with mean lengths of 8.4 cm (F. excelsior) and 1.9 cm (F. pennsylvanica). Necroses also developed on all of the inoculated F. excelsior stems (mean length 18.0 cm), whereas on F. pennsylvanica such lesions only occurred on about 5% of the stems (mean length 1.9 cm). The differences between strains were negligible. No necroses were observed on the control plants. Reisolations of H. albidus were only successful in around 8-11% of the cases, while H. fraxineus was reisolated from 50-70% of the inoculated organs showing necrotic lesions. None of the Hymenoscyphus species were isolated from the control plants. Our data confirm H. fraxineus' high virulence with regards to F. excelsior and demonstrate a low virulence in relation to F. pennsylvanica under field conditions in Poland. Hymenoscyphus albidus did not express any perceivable pathogenicity on both host species.