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Two new species of Ophiostomatales (Sordariomycetes) associated with the bark beetle Dryocoetes alni from Poland
expand article infoBeata Strzałka, Robert Jankowiak, Piotr Bilański, Nikita Patel§, Georg Hausner§, Riikka Linnakoski|, Halvor Solheim
‡ University of Agriculture, Kraków, Poland
§ University of Manitoba, Winnipeg, Canada
| Natural Resources Institute Finland, Helsinki, Finland
¶ Norwegian Institute of Bioeconomy Research, Ås, Norway
Open Access

Abstract

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.

Keywords

Bark beetle, Ceratocystiopsis, hardwoods, Leptographium, ophiostomatoid fungi, taxonomy, two new species

Introduction

Bark beetles in the genus Dryocoetes (Coleoptera: Curculionidae: Scolytinae) are mainly secondary pests infesting dead, injured, and felled or windthrown conifer- and hardwood hosts. For this reason, most members of Dryocoetes have no or only minor economic importance, although Dryocoetes confusus, the most destructive species in the genus, may cause extensive mortality of subalpine fir (Abies lasiocarpa) in North America (Bright 1963; Negrón and Popp 2009). The biology of hardwood-infesting Dryocoetes species is poorly understood in Poland. One of them is Dryocoetes alni (Georg), which has a wide geographical distribution, extending from France in the west to Siberia in the east, and from Fennoscandia in the north to Italy and Asia Minor in the south (Dodelin 2010). In Poland it occurs rarely, but probably it is widespread. This beetle species attacks weakened or dead trees of Alnus spp., Populus spp. and Corylus avellana (Gutowski and Jaroszewicz 2001; Borowski et al. 2012).

Dryocoetes beetles live in close association with fungi; most notably with members of the Ophiostomatales (Ascomycota, Sordariomycetes) that are well-recognized associates of bark- and wood-dwelling beetles (Kirisits 2004; Wingfield et al. 2017). According to Six (2012), associations among bark beetles and fungi range from mutualistic to commensal, and from facultative to obligate. Some fungi are highly specific and associated only with a single beetle species, while others can be associated with many beetle species. The majority of fungi vectored by Dryocoetes cause sapstain but some are responsible for serious tree diseases, such as the pathogenic species of Leptographium, Grosmannia dryocoetidis which is involved in A. lasiocarpa mortality (Molnar 1965). Ophiostomatales is comprised of two families, Kathistaceae and Ophiostomataceae, the latter comprising several several phylogenetic lineages that include, among others, Ceratocystiopsis, Graphilbum, Leptographium, Ophiostoma, Raffaelea and Sporothrix (Hyde et al. 2020). Members of these lineages have similar morphological and ecological characteristics. These fungi are also referred to as so-called ophiostomatoid fungi, a polyphyletic grouping characterized by the production of sticky spore masses at the apices of the flask-shaped sexual fruiting structures and their association with different arthropods (De Beer and Wingfield 2013; De Beer et al. 2013a, b, 2016; Hyde et al. 2020).

Leptographium sensu lato is a broadly defined polyphyletic group of morphologically similar species (De Beer and Wingfield 2013). To date, Leptographium sensu lato includes ten species complexes and some smaller lineages with uncertain taxonomic positions (De Beer and Wingfield 2013; Jankowiak et al. 2017). The genus Leptographium contains more than 150 described taxa, most of which are associated with phloem- and wood breeding beetles that affect a wide range of plants worldwide (Jacobs and Wingfield 2001). Leptographium species colonizing the roots of conifers may cause tree health problems, such as members of the Leptographium wageneri species complex that are responsible for black stain root disease (BSRD) on conifers in western North America (Goheen and Cobb 1978). Morphologically, species of Leptographium sensu lato are characterized by mononematous, darkly pigmented conidiophores terminating in penicillate branches. In addition, species belonging to the Grosmannia olivacea species complex also form synnematous conidiophores. Some members of Leptographium sensu lato produce sporothrix-like or hyalorhinocladiella-like synanamorphs. Many Leptographium sensu lato also form sexual morphs characterized by globose ascomata with elongated necks (Jacobs and Wingfield 2001) and these were often included in the genus Grosmannia Goid. (Goidànich 1936).

Leptographium species have historically been classified into various genera including Grosmannia, Ceratocystis Ellis and Halst. (Upadhyay 1981), and Ophiostoma Syd. and P. Syd. (Seifert et al. 1993). Phylogenetic analyses based on the ribosomal large subunit (LSU) and beta-tubulin sequence data carried out by Zipfel et al. (2006) documented distinct differences between Ophiostoma and Grosmannia, and redefined the latter genus to include all Leptographium with sexual morphs. De Beer and Wingfield (2013) re-evaluated the taxonomy of Leptographium and Grosmannia, considering all available DNA sequence data for all species. The authors concluded that sequence data for additional gene regions would be necessary to fully resolve the delineation of Leptographium and Grosmannia. De Beer and Wingfield (2013) suggested that all known Leptographium and Grosmannia placed in Leptographium sensu lato based on phylogenetic inference, should be treated in their current genera (Leptographium or Grosmannia). However, new species, excluding those residing in the G. penicillata species complex, should provisionally be treated in Leptographium, irrespective of their sexual or asexual morphs.

In contrast to species of Leptographium sensu lato, members of Ceratocystiopsis are less widespread globally. The genus Ceratocystiopsis currently includes nearly 20 taxa, most of which are collected from plants infested by phloem and wood-breeding beetles. Ceratocystiopsis species have short-necked perithecia, elongated ascospores, and hyalorhinocladiella-like asexual morphs (Upadhyay 1981; De Beer and Wingfield 2013).

Surveys of hardwood-infesting bark beetles in Poland have recently led to the recovery of an unknown Leptographium species from Dryocoetes alni (Jankowiak et al. 2019a). In addition, several isolates resembling Ceratocystiopsis have also been isolated from D. alni in association with Populus tremula L. In this study, all known Leptographium and Ceratocystiopsis species as well as the newly collected isolates were compared based on morphology and DNA sequence data for six nuclear loci, with the overall aim of providing accurate identifications for these fungi.

Materials and methods

Isolates and herbarium specimens

Isolations were made from the bark beetle D. alni and its galleries established in P. tremula logs. Strains were collected in beech-alder stand in southern Poland (Paprocice: 50°48'56.10"N, 21°2'51.23"E) during March–September 2018. The isolation procedures were the same as described by Jankowiak et al. (2019a). Isolates were also collected from Alnus incana (L.) Moench and P. tremula infested by D. alni and from Malus sylvestris (L.) Mill. infested by Scolytus mali (Bechstein) during studies conducted by Jankowiak et al. (2019a).

All fungal isolates used in this study are listed in Table 1. The isolates are maintained in the culture collection of the Department of Forest Pathology, Mycology and Tree Physiology; University of Agriculture in Krakow, Poland, and in the culture collection of the Natural Resources Institute Finland (Luke), Finland. The ex-type isolates of the new species described in this study were deposited in the Westerdijk Fungal Biodiversity Institute (CBS), Utrecht, the Netherlands, and in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa. Herbarium specimens have been deposited in the Herbarium of the University of Turku (TUR), Finland. Three reference strains were obtained from collections. These included a living culture of Ceratocystiopsis pallidobrunnea (WIN(M)51) from the culture collection of University of Manitoba (Canada), and cultures of Grosmannia crassivaginata (CBS 119444) and Leptographium piriforme (CMW 52066) (Table 1). Taxonomic descriptions and nomenclatural data have been registered in MycoBank (www.MycoBank.org) (Robert et al. 2013).

Table 1.

Fungal isolates used in the present study.

Species1 Isolate no2 Herbarium no3 Host Insect vector Origin GenBank accession no4
CMW CBS KFL and NRIF TUR ITS1-5.8S-ITS2-28S ITS2-28S TUB2 TEF1-α ACT CAL
Taxon 1
Ceratocystiopsis synnemata sp. nov. 16216DA http://mus.utu.fi/TFU.207991 Alnus incana Dryocoetes alni Resko MN900984 MN901005 MN901014 not obtained not obtained
13418DA http://mus.utu.fi/TFU.207992 Populus tremula Dryocoetes alni Paprocice MN900985 MN901006 MN901015 not obtained not obtained
149a18DA http://mus.utu.fi/TFU.207993 Populus tremula Dryocoetes alni Paprocice MN900986 MN901007 MN901016 not obtained not obtained
149b18DA http://mus.utu.fi/TFU.207994 Populus tremula Dryocoetes alni Paprocice MN900987 MN901008 MN901017 not obtained not obtained
16918DAH http://mus.utu.fi/TFU.207995 Populus tremula Dryocoetes alni Paprocice MN900988 MN901009 MN901018 not obtained not obtained
17718DA http://mus.utu.fi/TFU.207996 Populus tremula Dryocoetes alni Paprocice MN900989 MN901010 MN901019 not obtained not obtained
Taxon 2
Leptographium alneum sp. nov. 52067 144905 13116RJDA http://mus.utu.fi/TFU.207559 Alnus incana Dryocoetes alni Resko MN900990 MH283185 MH283218 MH283406 MN901029 MN901041
52072 144904 16016bRJDA http://mus.utu.fi/TFU.207997 Alnus incana Dryocoetes alni Resko MN900991 MH283219 MH283407 MN901030 MN901042
144903 16216bRJDA http://mus.utu.fi/TFU.207998 Alnus incana Dryocoetes alni Resko MN900992 MH283186 MH283220 MH283408 MN901031 MN901043
52070 7617RJDA Populus tremula Dryocoetes alni Paprocice MN900993 MH283221 MN901020 MN901032 MN901044
52075 144902 7717RJDA http://mus.utu.fi/TFU.207558 Populus tremula Dryocoetes alni Paprocice MN900994 MH283222 MN901021 MN901033 MN901045
52069 8417RJDA Populus tremula Dryocoetes alni/ Paprocice MN900995 MH283223 MN901022 MN901034 MN901046
8617RJDA Populus tremula Dryocoetes alni Paprocice MN900996 MH283224 MN901023 MN901035 MN901047
52076H 144901H 8917RJDAH http://mus.utu.fi/TFU.207557 Populus tremula Dryocoetes alni Paprocice MN900997 MH283225 MN901024 MN901036 MN901048
9117RJDA Populus tremula Dryocoetes alni Paprocice MN900998 MH283226 MN901025 MN901037 MN901049
88616RJSM Malus sylvestris. Scolytus mali Rozpucie MN900999 MH283187 MH283227 MH283409 MN901038 MN901050
52071 144900 88716aRJSM http://mus.utu.fi/TFU.207556 Malus sylvestris Scolytus mali Rozpucie MN901000 MH283188 MH283228 MH283410 MN901039 MN901051
Leptographium piriforme 52066 297NBRZ16AO Betula pendula Wound Żohatyn MN901001 MH740931 MH740984 MH741134 not obtained not obtained
10618DA Alnus incana Dryocoetes alni Paprocice MN901002 MN901011 MN901026 not obtained not obtained
Grosmannia crassivaginata 134 119144 unknown unknown unknown MN901003 MN901012 MN901027 MN901040 not obtained
Ceratocystiopsis pallidobrunnea WIN(M) 51 Populus tremuloides unknown Duck Mountain5 MN901004 MN901013 MN901028 not obtained not obtained

DNA extraction, PCR and sequencing

DNA extractions were done as described by Jankowiak et al. (2018). For sequencing and phylogenetic analyses, six loci were amplified: internal transcribed spacer 1 and 2 (ITS1-5.8S-ITS2), internal transcribed spacer 2 and large subunit (ITS2-28S), actin (ACT), beta-tubulin (TUB2), calmodulin (CAL) and the translation elongation factor 1-alpha (TEF1-α) using the primers listed in Table 2.

Table 2.

Information on PCR primers used in this study.

Locus Primers Fungi
ITS1-5.8S ITS1-F (Gardes and Bruns 1993), ITS4 (White et al. 1990) Ceratocystiopsis, Leptographium
28S LR0R, LR5 (Vilgalys and Hester 1990) Ceratocystiopsis
ITS2-28S ITS3 (White et al. 1990), LR3 (Vilgalys and Hester 1990) Leptographium
TUB2 Bt2a, Bt2b (Glass and Donaldson 1995) Ceratocystiopsis
T10 (O’Donnell and Cigelnik 1997), Bt2b (Glass and Donaldson 1995) Leptographium
ACT Lepact-F, Lepact-R (Lim et al. 2004) Leptographium
CAL CL3F, CL3R (De Beer et al. 2016) Leptographium
TEF1-α F-728F (Carbone and Kohn 1999), EF2 (O’Donnell et al. 1998) Ceratocystiopsis
EF1F, EF2R (Jacobs et al. 2004) Leptographium

DNA fragments were amplified in a 25 µL reaction mixture containing 0.25 µL of Phusion High-Fidelity DNA polymerase (Finnzymes, Espoo, Finland), 5 µL Phusion HF buffer (5x), 0.5 µL of dNTPs (10 mM), 0.75 µL DMSO (100%) and 0.5 µL of each primer (25 µM). Amplification reactions were performed in the LabCycler Gradient thermocycler (Sensoquest Biomedical Electronics GmbH, Germany). Amplification of the various loci was performed under the following conditions: a denaturation step at 98 °C for 30 s was followed by 35 cycles of 5 s at 98 °C, 10 s at 52–64 °C (depending on the primer melting temperature and fungal species) and 30 s at 72 °C, and a final elongation step at 72 °C for 8 min. The PCR products were visualized under UV light on a 2% agarose gel stained with Midori Green DNA Stain (Nippon Genetic Europe).

Amplified products were sequenced with the BigDye Terminator v 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and the products were resolved with an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems), at the DNA Research Centre (Poznań, Poland) using the same primers that were used for the PCR. The sequences (Table 1) were compared with sequences retrieved from GenBank using the BLASTn algorithm (Altschul et al. 1990). Newly obtained sequences were deposited in NCBI GenBank and added to previous alignments for the ITS1-5.8S-ITS2-28S and ITS2-28S regions (Jankowiak et al. 2019a). Alignments were adjusted to accommodate the new sequences and the data sets were used to obtain consensus sequences and the two data sets were concatenated.

Phylogenetic analyses

BLAST searches (Altschul et al. 1990) using the BLASTn algorithm were performed to retrieve similar sequences from GenBank (http://www.ncbi.nlm.nih.gov) and accession numbers for these sequences are presented in the corresponding phylogenetic trees (Figs 14). Datasets were curated with the Molecular Evolutionary Genetic Analysis (MEGA) v6.06 program (Tamura et al. 2013).

The phylogenetic position of Taxon 1 was determined from their concatenated ITS1-5.8S-ITS2-28S sequences within a dataset that covered all ITS1-5.8S and ITS2-28S sequences of Ceratocystiopsis available in GenBank, as well as sequences of C. pallidobrunnea obtained in this study (Fig. 1). The outgroup taxa for the ITS1-5.8S-ITS2-28S dataset analysis were Ophiostoma karelicum and O. quercus. The TUB2 dataset included all available sequences for reference species in Ceratocystiopsis that could be retrieved from GenBank and six of our isolates (Fig. 2) in order to identify isolates to the species level.

In the case of Taxon 2, the ITS2–28S dataset included most of the available sequences for reference species in Leptographium sensu lato that could be retrieved from GenBank (Fig. 3) to show the placement of our isolates within this group. The outgroup taxa for the ITS2–28S dataset analysis was O. karelicum and O. novo-ulmi. The concatenated constructs of sequences for multiple loci (ITS1-5.8S-ITS2–28S + TUB2 + TEF1-α + ACT) were also used for 11 of our isolates, Grosmannia crassivaginata, and L. piriforme (Fig. 4). Before individual data sets for the ITS2-28S, ACT, TUB2, and the TEF1-α gene regions were used for 11 of our isolates, Grosmannia crassivaginata, and L. piriforme. The outgroup taxa for the ITS1-5.8S-ITS2–28S + TUB2 + TEF1-α + ACT datasets analysis were Leptographium flavum and L. vulnerum. Datasets concerning the protein coding sequences were concatenated. Sequence alignments were performed using the online version of MAFFT v7 (Katoh and Standley 2013). The ITS1-5.8S, ITS2-28S, ACT, TUB2, and TEF1-α datasets were aligned using the E-INS-i strategy with a 200PAM/k=2 scoring matrix, a gap opening penalty of 1.53 and an offset value of 0.00. The alignments were checked manually with BioEdit v.2.7.5 (Hall 1999). The resulting alignments and trees were deposited into TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S25615). Aligned data sets of the protein-coding genes were compared to gene maps constructed by Yin et al. (2015) to determine the presence or absence of introns and to confirm that introns and exons were appropriately aligned (Suppl. material 1: Tables S1–S3). Single nucleotide polymorphisms (SNPs) for different gene regions between the new taxa and the phylogenetically closest related species were also identified by comparative sequence analysis.

Phylogenetic trees were inferred for each of the datasets using three different methods: Maximum likelihood (ML), Maximum Parsimony (MP) and Bayesian inference (BI). For ML and BI analyses, the best-fit substitution models for each aligned dataset were established using the corrected Akaike Information Criterion (AICc) in jModelTest 2.1.10 (Guindon and Gascuel 2003; Darriba et al. 2012). ML analyses were carried out with PhyML 3.0 (Guindon et al. 2010), utilizing the Montpelier online server (http://www.atgc-montpellier.fr/phyml/). The ML analysis included bootstrap analysis (1000 bootstrap pseudoreplicates) in order to assess node support values and the overall reliability of the tree topology.

The best evolutionary substitution model for ITS2-28S (Leptographium) and the ITS1-5.8S-ITS2–28S (Ceratocystiopsis) was GTR+I+G. The best evolutionary substitution model for TUB2 (Ceratocystiopsis) and for the combined ITS1-5.8S-ITS2–28S, ACT, TUB2, and TEF1-α, datasets for Leptographium was GTR+G.

MP analyses were performed with PAUP* 4.0b10 (Swofford 2003). Gaps were treated as fifth state. Bootstrap analysis (1000 bootstrap replicates) was conducted to determine the levels of confidence for the nodes within the inferred tree topologies. Tree bisection and reconnection (TBR) was selected as the branch swapping option. The tree length (TL), Consistency Index (CI), Retention Index (RI), Homoplasy Index (HI) and Rescaled Consistency Index (RC) were recorded for each analysed dataset after the trees were generated.

BI analyses using Markov Chain Monte Carlo (MCMC) methods were carried out with MrBayes v3.1.2 (Ronquist and Huelsenbeck 2003). The four MCMC chains were run for 10 million generations applying the best-fit model for each data set. Trees were sampled every 100 generations, resulting in 100,000 trees. The Tracer v1.4.1 program (Rambaut and Drummond 2007) was utilized to determine the burn-in value for each dataset. The remaining trees were utilized to generate a 50% majority rule consensus tree, which allowed for calculating posterior probability values for the nodes.

Morphology, growth studies and mating tests

Morphological characters were examined for selected isolates and for the herbarium specimens chosen to represent the type specimens for the newly proposed species. Cultures were grown on 2% MEA agar [MEA: 20 g Bacto malt extract (Becton Dickinson and Company, Franklin Lakes, USA), 20 g agar (Bacto agar powder from Becton Dickinson and Company, Franklin Lakes, USA), 1 l deionized water] with or without host tree twigs to induce potential ascocarp formation. Autoclaved twigs with bark were positioned in the centre of the MEA agar plates. Fungal cultures were derived from single spores, and crossings were made following the technique described by Grobbelaar et al. (2010). To encourage the production of ascomata for species descriptions, single conidial isolates were crossed in all possible combinations. Cultures were incubated at 25 °C and monitored regularly for the appearance of fruiting structures.

Morphological features were examined by mounting materials in 80% lactic acid on glass slides, and observing various fruiting structures using a Nikon Eclipse 50i microscope (Nikon Corporation, Tokyo, Japan) with an Invenio 5S digital camera (DeltaPix, Maalov, Denmark) to capture photographic images. Microscopy was done as previously described by Kamgan Nkuekam et al. (2011). Color designations were based on the charts of Kornerup and Wanscher (1978).

For each taxonomically relevant structure fifty measurements were made, whenever possible, with the Coolview 1.6.0 software (Precoptic, Warsaw, Poland). Averages, ranges and standard deviations were calculated for the measurements, and these are presented in the format ‘(min–)(mean-SD)–(mean+SD)(–max)’.

Growth characteristics for the two newly proposed species and Grosmannia crassivaginata (isolate CBS 119144) were determined by analyzing the radial growth for five isolates in pure culture that represent each of the studied species (Table 1). Agar disks (5 mm diam.) were cut from actively growing margins of fungal colonies for each of the tested isolates and these disks were placed in the center of plates containing 2% MEA. Four replicate plates for each of the proposed new species and G. crassivaginata were incubated at 5, 10, 15, 20, 25, 30 and 35 °C. The radial growth (two measurements per plate) were determined 7 d (Taxon 1) and 4 d (Taxon 2, and G. crassivaginata) after inoculation, and growth rates were calculated as mm/d.

Results

Morphological characteristics

The two new taxa showed differences with regards to growth rates in culture and color differences ranging from white (Taxon 1) to brownish gray (Taxon 2). Taxon 1 produced a hyalorhinocladiella-like asexual morph with simple and highly branched conidiophores, which often aggregate in loosely synnemata that were arranged either singly or in groups topped with white mucilaginous spore drops. Taxon 2 produced short mononematous conidiophores with allantoid conidia, and stalked club-shaped cells. A sexual morph could be induced in all isolates of Taxon 2; the most distinct features observed in both the herbarium specimens and the studied isolates were the short ascomatal necks and falcate ascospores with gelatinous sheaths. Sexual morph was not observed for Taxon 1 in any of the crosses done between different isolates. Morphological differences among these new taxa and the most closely related species are listed in Tables 3, 4, and discussed in the Notes under the new species descriptions in the Taxonomy section.

The optimal growth temperatures were 25 °C for Taxon 1 and 30 °C for Taxon 2. No growth was observed at 5 °C for Taxon 2.

Phylogenetic analyses

Alignments for the Ceratocystiopsis data set of ITS1-5.8S-ITS2-28S contained 1278 characters and for the TUB2 512 characters (including gaps). Alignments for the ITS2-28S and the concatenated combined Leptographium data set of ITS1-5.8S-ITS2-28S+ACT+TUB2, TEF1-α, contained 637, and 13276 characters (including gaps), respectively. The exon/intron arrangement of the TUB2 Ceratocystiopsis species complex data included exons 3, 4, 5 and 6, interrupted with introns 3, 4, and 5. The exon/intron arrangement of the TUB2 Leptographium data included exons 3, 4, and 5/6, interrupted with introns 3 and 4, but lacking intron 5. The aligned TEF1-α gene region consisted of introns 3, 5 and exons 4/5, 6, while lacking intron 4. The alignment of the ACT dataset contained exons 5 and 6, interrupted with intron 5.

The ITS1-5.8S-ITS2-28S (Fig. 1) and ITS2-28S (Fig. 3) trees show the placement of the Polish isolates (referred to as Taxon 1 and Taxon 2) within the Ophiostomatales. Taxon 1 resided among sequences representing species that are members of Ceratocystiopsis (Fig. 1), while Taxon 2 grouped with other species in the Leptographium sensu lato (Fig. 3). Taxon 1 was closely related with C. pallidobrunnea (Fig. 1), while Taxon 2 formed a separate lineage within Leptographium sensu lato that included Leptographium piriforme and Grosmannia crassivaginata (Fig. 3). Taxon 1 had unique ITS1-5.8S-ITS2-28S sequences compared with other Ceratocystiopsis species (Fig. 1), while isolates of Taxon 2 had ITS2-28S sequences that were almost identical with sequences noted in G. crassivaginata (Fig. 3).

Figure 1. 

Phylogram obtained from Maximum Likelihood (ML) analyses of the ITS1-5.8S-ITS2-28S data for the Ceratocystiopsis spp. Sequences obtained during this study are presented in bold type. The Bootstrap values ≥ 75% for ML and Maximum Parsimony (MP) analyses are presented at nodes as follows: ML/MP. Bold branches indicate posterior probabilities values ≥ 0.95 obtained from Bayesian Inference (BI) analyses. * Bootstrap values <75%. The tree is drawn to scale (see bar) with branch length measured in the number of substitutions per site. Ophiostoma karelicum and Ophiostoma quercus represent the outgroup.

Figure 2. 

Phylogram obtained from Maximum Likelihood (ML) analyses of TUB2 data for the Ceratocystiopsis spp. Sequences obtained during this study are presented in bold type. The Bootstrap values ≥ 75% for ML and Maximum Parsimony (MP) analyses are presented at nodes as follows: ML/MP. Bold branches indicate posterior probabilities values ≥ 0.95 obtained from Bayesian Inference (BI) analyses. * Bootstrap values <75%. The tree is drawn to scale (see bar) with branch length measured in the number of substitutions per site.

The MP, ML and BI analyses of the individual dataset (ITS2-28S, ACT, TUB2, TEF1-α) provided trees with similar topologies (data not shown). In the TUB2 tree (Fig. 2), Taxon 1 formed a well-supported lineage that clearly separated this newly proposed species from all the other known species in Ceratocystiopsis and the most closely related species C. pallidobrunnea (Fig. 2). The combined analyses of the ITS1-5.8S-ITS2-28S+TUB2+ACT+TEF1-α data grouped isolates of Taxon 2 in a lineage together with L. piriforme and G. crassivaginata, in agreement with the ITS2-28S tree. However, this taxon formed a well-supported lineage next to a clade containing G. crassivaginata (Fig. 4).

Figure 3. 

Phylogram obtained from Maximum Likelihood (ML) analyses of the ITS2-28S for selected species of Leptographium sensu lato. Sequences obtained during this study are presented in bold type. The Bootstrap values ≥ 75% for ML and Maximum Parsimony (MP) analyses are presented at nodes as follows: ML/MP. Bold branches indicate posterior probabilities values ≥ 0.95 obtained from Bayesian Inference (BI) analyses. * Bootstrap values <75%. The tree is drawn to scale (see bar) with branch length measured in the number of substitutions per site. Ophiostoma karelicum and O. quercus represents the outgroup in analyses of ITS2-28S.

Figure 4. 

Phylogram obtained from Maximum Likelihood (ML) analyses of the combined datasets of ITS1-5.8S-ITS2-28S+ACT+TUB2+TEF1-α for selected species of Leptographium sensu lato. Sequences obtained during this study are presented in bold type. The Bootstrap values ≥ 75% for ML and Maximum Parsimony (MP) analyses are presented at nodes as follows: ML/MP. Bold branches indicate posterior probabilities values ≥ 0.95 obtained from Bayesian Inference (BI) analyses. * Bootstrap values <75%. The tree is drawn to scale (see bar) with branch length measured in the number of substitutions per site. Leptographium flavum and L. vulnerum represents the outgroup in analyses of the combined datasets of ITS1-5.8S-ITS2-28S+ACT+TUB2+TEF1-α.

The six isolates of Taxon 1 obtained in this study were distinguished from C. pallidobrunnea using SNP analyses for each of the ITS1-5.8S-ITS2-28S, TUB2, TEF1-α gene region sequences. The total number of SNP differences between the six isolates and C. pallidobrunnea for all three genes was 166 (26 for ITS1-5.8S-ITS2-28S, 60 for TUB2, and 80 for TEF1-α). Little intraspecific sequence variation was found within 6 isolates of Taxon 1. Intraspecific variability of the ITS1-5.8S-ITS2-28S, TUB2 and TEF1-α genes was detected for Taxon 1 in one position, i.e. 387, two positions, i.e. 212, 217, and one position i.e. 482, respectively (Suppl. material 1: Tables S1, S2).

The 11 isolates of Taxon 2 obtained in this study were distinguished from G. crassivaginata using SNP analyses for each of the ITS1-5.8S-ITS2-28S, TUB2, TEF1-α, ACT gene region sequences. The total number of SNP differences between the 11 isolates and G. crassivaginata for all four genes was 59 (8 for ITS1-5.8S-ITS2-28S, 16 for TUB2, 25 for TEF1-α, and 10 for ACT). The intraspecific sequence variation was greater for 11 isolates of Taxon 2 than for Taxon 1. Intraspecific variability of the TUB2, TEF1-α, and ACT genes was detected for Taxon 2 in eight positions, i.e. 36, 82, 83, 87, 215, 230–232; nine positions, i.e. 14, 21, 31, 46, 101, 196, 272, 352, 549; and five positions, i.e. 402, 749, 754, 755, 766, respectively (Suppl. material 1: Table S3).

These results indicate that the six isolates of Taxon 1 within Ceratocystiopsis and the 11 isolates of Taxon 2 within Leptographium represent novel species.

Taxonomy

The morphological characterization and phylogenetic comparisons based on six genetic loci, showed that two taxa associated with D. alni from Poland are distinct from each other and from other known taxa in the Ophiostomatales. Therefore, they are described here as new species:

Taxon 1

Ceratocystiopsis synnemata B. Strzałka, R. Jankowiak & G. Hausner, sp. nov. MycoBank No: 835151

Fig. 5

Etymology

The epithet (synnemata) refers to the synnematous conidiomata formed by this fungus.

Type

Poland, Paprocice, from Dryocoetes alni beetle infesting Populus tremula, 5 Oct 2018, K. Miśkiewicz (TUR 207995 http://mus.utu.fi/TFU.207995 holotype, ex-holotype cultures: NRIF 16918DA = KFL 16918DA).

Description

Sexual morph : not observed. Asexual morph: hyalorhinocladiella-like. Conidiophores micronematous or macronematous. The micronematous conidiophores, hyaline, consist of conidiogenous cells arising singly from the vegetative hyphae (6–)8.6–16.4(–23.2) × (0.6–)0.9–1.3(–1.6) µm. The macronematous conidiophores are much larger, (14.5–)17.3–39.8(–76.9) µm long than the preceding forms and from a basal cell, (3.1–)5.3–11.2(–17) × (0.9–)1.1–1.9(–2.6) µm. The basal cells branch lateral or penicillate and form 1–5 branches (mostly 1–2) producing conidiogenous cells at their apices. Conidiophores often aggregate in loosely synnemata, (43.2–)52.3–86.4(–114.7) µm long, (2.4–)3.6–8.2(–12.9) µm wide at the tip. Conidia hyaline, smooth, unicellular, oblong-elliptical, (2.4–)2.8–3.5(–4) × (1–)1.1–1.3(–1.4) µm. Cultural characteristics: Colonies with optimal growth at 25 °C on 2% MEA with radial growth rate 1.4 (± 0.1) mm/d, growth very well at 30 °C (1.3 mm/d) and 35 °C (1.0 mm/d). Colonies yellowish gray, margin smooth. Hyphae pale gray in color, smooth, submerged in the medium and aerial mycelium rare, not constricted at the septa, 0.4–2.6 (mean 1.1±0.6) µm diam., asexual morph moderately abundant, very abundant after adding twigs.

Host trees

Alnus incana, Populus tremula

Insect vector

Dryocoetes alni

Distribution

Poland

Note

Ceratocystiopsis synnemata can be distinguished from C. pallidobrunnea by the shape and size of the conidia. Ceratocystiopsis synnemata has shorter and oblong-elliptical conidia in contrast to the allantoid conidia of C. pallidobrunnea (C. synnemata: 2.4–4 ×1–1.4 µm; C. pallidobrunnea: 2.5–5 × 0.7–1.2 µm (Olchowecki and Reid 1974), 2–7 × 0.7–2.5 µm (Upadhyay 1981) (Table 3). In addition, C. synnemata produces conidiophores that aggregate into loosely arranged synnemata.

Figure 5. 

Ceratocystiopsis synnemata sp. nov. (NRIF 16918DA=KFL 16918DA) a, b micronematous conidiophores c–e macronematous conidiophores f, g conidiophores aggregate in synnemata h conidia i fourteen-day-old culture on MEA. Scale bars: 25 μm (a), 10 μm (b), 25 μm (c–g), 10 μm (h).

Table 3.

Morphological comparisons of closely related species to Ceratocystiopsis synnemata sp. nov.

Species Ceratocystiopsis pallidobrunnea (Olchowecki and Reid 1974) Ceratocystiopsis pallidobrunnea (Upadhyay 1981) Ceratocystiopsis synnemata sp. nov.
Sexual state Present Present unknown
Ascomata base 40–60 40–75
Ascomatal neck length (μm) 15–60 21.2–66
Ascospore shape allantoid or falcate with truncate ends in side view, cylindrical or fusiform with truncate ends in face view falcate with truncate or obtuse ends in side view, fusiform or ellipsoid-fusiform in face view
Ascospore size (in face view, μm) (-3.5)4.5–7.5 × 0.7–1 excluding sheath 14–17.5(-22.5) × 1–1.5(-1.8) including sheath
Conidial shape allantoid or oblong with obtuse ends cylindrical, allantoid oblong-elliptical
Conidial size (μm) 2.5–5 × 0.7–1.2 2–7 × 0.7–2.5 2.4–4 × 1–1.4
Branched conidiophores present, to 50 μm long present present, 76.9 μm long
Conidiophores aggregate into synnemata absent absent present
Optimal growth temp on MEA
Growth rate at optimum
Host Populus tremuloides Populus tremuloides Alnus incana, Populus tremula
Arthropods unknown unknown Dryocoetes alni,
Distribution Manitoba, Canada Manitoba, Canada Poland

Taxon 2

Leptographium alneum B. Strzałka, R. Jankowiak & P. Bilański, sp. nov.

MycoBank No: MycoBank No: 835146
Fig. 6

Etymology

The epithet (alneum) refers to the species name of the bark beetle vector of this fungus, Dryocoetes alni.

Type

Poland, Paprocice, from Dryocoetes alni beetle infesting Populus tremula, 2 Nov 2017, K. Miśkiewicz, (TUR 207557 http://mus.utu.fi/TFU.207557 holotype, ex-holotype cultures: CBS 144091 = CMW 51789).

Figure 6. 

Leptographium alneum sp. nov. (CBS 144901) a Dryocoetes alni-infested Populus tremula tree b galleries of D. alni with ascomata c ascoma d ascomatal base e ascospores f ostiolar hyphae g–i conidiophores, black arrow indicates barrel-shaped cells j conidiogenous k conidia l club-shape cells m fourteen-day-old culture on MEA. Scale bars: 50 μm (c), 25 μm (d), 10 μm (e), 10 μm (f), 25 μm (g), 25 μm (h), 50 μm (i), 10 μm (j), 10 μm (k), 50 μm (l).

Description

Sexual morph : Ascomata developing after 30 d on sterilized Populus twigs when two mating types were paired: superficially or partly embedded in the agar or wood, single. Bases light brown to dark brown, globose, unornamented, (59–)66–90(–108) μm in diameter, necks dark brown, cylindrical, straight or curved, (58–)68–88(–114) μm long (excluding ostiolar hyphae), (18.7–)20.7–27.9(–31) μm wide at base, (10.2–)11.8–15.3(–17.8) μm wide at the tip. Ostiolar hyphae present, pale brown, straight, non-septate or sporadically one-septate, numerous, divergent, pointed at the tip, (14.6–)15.9–19(–22.7) μm long, 5 to 12 in number. Asci not seen. Ascospores one-celled, hyaline, falcate in side view, (7.4–)8.1–11.1(–14.3) × (1.2–)1.5–2.1(–2.4) μm; fusiform in face view, (6.9–)7.4–8.8(–10.3) × (1.8–)2–2.8(–3.3) μm; end view not seen, excluding hyaline gelatinous sheath, (8.9–)10–11.5(–12.2) × (4.5–)5.5–6.7(–7) μm in face view including sheath, accumulated in orange yellow-colored mass at the tip of the neck. Gelatinous sheath 0.5–3 μm thick, oval in face view.

Asexual morph: conidiophores macronematous, arising directly from hyphae, single solitary, without rhizoidal hyphae at the bases, often with barrel-shaped or globose cells, (48.1–)59.3–84.2(–102.9) μm in length. Stipes erect, light olivaceous, 1–4 septate (mostly 2), (7.6–)14.3–39.2(–48.5) μm long (from first basal septum to below primary branches), (2–)2.4–5.4(–15.6) μm wide below primary branches, apical cell often strongly swollen, (3.2–)3.8–5.2(–6.1) μm wide at base, basal cell rarely swollen. Conidiophores often composed of barrel or globose cells. Conidiogenous apparatus (20–)26.5–38.6(–48.7) μm long (excluding conidial mass) consisting of 2–3 series of branches-type B (more than two branches) (Jacobs and Wingfield 2001). Primary branches light olivaceous, cylindrical or swollen, smooth, (5.1–)5.8–12.7(–23.1) × (1.2–)1.6–4.2(–6.8) μm. Conidiogenous cells hyaline, tapering from base to apex, (11.6–)13.2–19(–23.7) × (0.8–)0.9–2(–3.5) μm. Conidia hyaline, mostly allantoid, sometimes oblong to obovoid (3.2–)3.7–5.9(–9.7) × (0.8–)1–1.8(–2.8) μm, accumulating around the conidiogenous apparatus as a creamy mucilaginous mass.

Cultural characteristics : Colonies with optimal growth at 30 °C on 2% MEA with radial growth rate 8.8 (± 0.9) mm/d, good growth observed at 35 °C (8.3 mm/d) and better than at 25 °C (7.9 mm/d). Colonies brownish gray with distinct silvery gloss, margin smooth. Hyphae olive yellow in color, smooth, submerged in the medium and aerial mycelium abundant, not constricted at the septa, 1.1–5.5 (mean 2.5±1) µm diam., asexual morph very abundant, which gives a shade of gray. Club-shaped cells terminal on septate hyphal branches present, (11.5–)14.8–25.6(–33.3) × (7.7–)11.3–15.1(–18.2) μm, born on a multicelled stalk, (7.2–)14.7–82.4(–124.2) μm long, (4.4–)5.1–7.7(–9.7) μm wide below primary septa, (2.9–)4–6(–7.4) μm wide at base. Perithecia and asexual morph co-occur in culture.

Host trees

Alnus incana, Malus sylvestris, Populus tremula

Insect vector

Dryocoetes alni, Scolytus mali

Distribution

Poland

Note

Morphologically, Leptographium alneum differs from Grosmannia crassivaginata in having longer ascomatal necks (L. alneum: 58–114 µm: G. crassivaginata: 40–60 µm), and the presence of larger club-shaped cells (L. alneum: 11.5–33.3 × 7.7–18.2 µm; G. crassivaginata: 12–20 × 8–12 µm (Griffin 1968), 6.5–18.5 × 5–13.5 µm (CBS 119144), (Table 4). In addition, L. alneum has aseptate or sporadically 1-septate ostiolar hypahe, which are septate in G. crassivaginata. Leptographium alneum frequently produces conidiophores with barrel-shaped or globose cells, while in G. crassivaginata the cells of the conidiophore are slightly swollen at most. In contrast to G. crassivaginata (CBS 119144), L. alneum has larger conidia, especially in regard to width (L. alneum: (3.2–)3.7–5.9(–9.7) × (0.8–)1–1.8(–2.8) µm: G. crassivaginata: (2.4–)3.2–5(–8.1) × (0.7–)0.9–1.3(–1.7) µm) (Table 4). Leptographium alneum has brownish gray colony with silvery gloss cultures in contrast to the olive brown colored colonies of G. crassivaginata (isolate CBS 119144). The optimal growth on MEA for L. alneum and G. crassivaginata (isolate CBS 119144) is 30 °C. However, L. alneum grows much faster than G. crassivaginata (L. alneum 8.8 mm/d, G. crassivaginata 6.9 mm/d) and grows faster at 35 °C (8.3 mm/d) than at 25 °C (7.9 mm/d). In contrast, G. crassivaginata grows much faster at 25 °C (5.6 mm/d) than 35 °C (4.4 mm/d).

Table 4.

Morphological comparisons of closely related species to Leptographium alneum sp. nov.

Species* G. crassivaginata (Griffin 1968), holotype DAOM 110144) G. crassivaginata (Jacobs and Wingfield 2001), holotype DAOM 110144) G. crassivaginata (Upadhyay 1981), RWD 858, WIN(M) 69-12 G. crassivaginata (this study, CBS119144) L. piriforme (Greif et al. 2006) L. alneum sp. nov.
Sexual state Present Present Present Absent Unknown Present
Ascomata base 40–90 40–90 35–110 59–108
Ascomatal neck length (μm) 40–60 40–60 37–70 including ostiolar hyphae 58–114 excluding ostiolar hyphae
Ostiolar hyphae length (μm) 10–25, septate septate 14.6–22.7, non-septate
Ascospore shape Falcate in side view, fusiform in face view Fusiform, Falcate in side view, fusiform in face view Falcate in side view, fusiform in face view
Ascospore size (in face view, μm) 5–7 × 1.excluding sheath, 10–11.5×5–6.5 including sheath 10–11 × 5–6 including sheath 9–12 × 5–7 including sheath 6.9–10.3 × 1.8–3.3 excluding sheath, 8.9–12.2 × 4.5–7 including sheath
Conidiophores length (μm) to 50 25–105 (-120) to 85 (28.6–)33.2–63.2(–109.1) (48.1–)59.3–84.2(–102.9)
Stipe length (μm) 8–60(-85) (5.6–)3.7–26(–58.6) 7–45.6 (7.6–)14.3–39.2(–48.5)
Conidiogenous apparatus length (μm) 15–55 (-60) (22.3–)27–41.3(–54.7) (20–)26.5–38.6(–48.7)
Conidial shape Cylindrical to allantoid Oblong to obovoid Clavate, curved Oblong to allantoids, often clavate Curved Cylindrical to allantoid
Conidial size (μm) 3–6 × 1–1.5 4–10×1–2 2.5–12 × 1–2 (2.4–)3.2–5(–8.1) × (0.7–)0.9–1.3(–1.7) 2.4–4.6 × 1.0–1.4 (3.2–)3.7–5.9(–9.7) × (0.8–)1–1.8(–2.8)
Club-shaped cells size (μm) 12–20 × 8–12 on short hyphal branches 9–23 × 7–14 on immersed hyphal branches (6.5–)8.5–14.1(–18.5) × (5–)6.5–10.8(–13.5), born terminally or laterally on a non-septate or 2–3 septate stalk, 4.8–41.5 long, 2.5–7.3 wide below primary septa 14.4–31.2 × 7.2–16., borne on a one- to four celled stalk, 7.2–45.6 × 4.8–7.2 (11.5–)14.8–25.6(–33.3) × (7.7–)11.3–15.1(–18.2), born terminally on a multicelled stalk, 7.2–124.2 long , 4.4–9.7 wide below primary septa
Colony color and optimal growth temp on MEA Brown, - Olivaceous, 30, Pale to dark brown or chaetura brown, - Olive brown, 30 Light brown, 35 Brownish grey, 30
Radial growth rate (mm/d) at optimum 6.9 mm 8.8 mm
Host Picea mariana, Populus grandidentata, P. tremuloides Picea mariana, P. glauca, Pinus resinosa, P. sylvestris, P. strobus, Fraxinus nigra, Populus grandidentata, P. tremuloides Populus tremuloides unknown Unknown (Greif et al. 2006); Pinus sylvestris (Jankowiak and Kolařík 2010); Betula verrucosa (Jankowiak et al. 2019c); Populus tremula (this study) Populus tremula, Malus sylvestris (Jankowiak et al. 2019a, this study)
Arthropods Unknown Unknown Coleoptera, Diptera, Araneae, Acari, Hemiptera, Lepidoptera, Collembola, Psocoptera, Trichoptera, and Hymenoptera: Formicidae, D. alni (this study) Dryocoetes alni, Scolytus mali
Distribution Ontario, Canada Ontario, Canada,USA Fort Collins, Colarado (USA), Monitoba (Canada) Unknown Alberta, Canada,
Poland
Poland

Discussion

This study identified two new species of ophiostomatoid fungi associated with Dryocoetes alni on Alnus incana and Populus tremula in hardwood ecosystems in Poland. DNA sequence comparisons and morphological features supported these as novel. The species were named Ceratocystiopsis synnemata and Leptographium alneum. The results confirm earlier findings that many species of the Ophiostomatales are associated with hardwood-infesting bark beetles in Poland (Jankowiak et al. 2017, 2018, 2019a; Aas et al. 2018).

The results of this study revealed one new species of Ceratocystiopsis bringing the total number of species in the genus to 17. The newly described species is morphologically similar to other species of Ceratocystiopsis, with hyalorhinocladiella-like asexual morph (De Beer et al. 2013a). In contrast to other Ceratocystiopsis species, C. synnemata produces simple as well as highly branched conidiophores reminiscent of C. pallidobrunnea (Olchowecki and Reid 1974; Upadhyay 1981) or C. rollhanseniana (Hausner et al. 2003). In addition, C. synnemata forms synnemata with loosely packed conidiophores that appear to be a unique feature of Ceratocystiopsis.

Ascomata in Ceratocystiopsis tended to be globose with short necks, and falcate ascospores surrounded by a gelatinous sheath (De Beer and Wingfield 2013). Generally, Ceratocystiopsis species produce perithecia in varying degrees of abundance and maturity (Upadhyay 1981; Plattner et al. 2009). Ceratocystiopsis synnemata did not form a sexual state in crosses between different isolates. That would suggest that this species is heterothallic or produces perithecia very sparsely.

Most of the formally described species of Ceratocystiopsis are known only from Pinaceae including those in the genera Picea, Pinus and Pseudotsuga. For example, in Poland, two species of Ceratocystiopsis have previously been reported: C. minuta and species of uncertain status, C. alba, which both have been isolated from spruce-infesting bark beetles (Jankowiak et al. 2009). Only C. pallidobrunnea was collected from hardwood tree species, Populus tremuloides in Canada (Olchowecki and Reid 1974; Plattner et al. 2009). The discovery of C. synnemata on Populus tremula in this study clearly show that Ceratocystiopsis species are also distributed in hardwood forest ecosystems in Europe.

A new species of Leptographium was discovered from Dryocoetes alni in this study. This new taxon is closely related to Leptographium piriforme and Grosmannia crassivaginata forming a well-supported lineage distinct from other species of Leptographium sensu lato. All these three species have the curved conidia formed on short conidiophores, club-shaped cells, short-necked perithecia, and falcate, sheathed ascospores. These features clearly distinguish them from the other species recognized in the various species complexes currently recognized within Leptographium sensu lato.

Based on DNA sequence comparisons, L. alneum described in this study is closely related to G. crassivaginata, a species described from Picea mariana, Populus grandidentata and P. tremuloides in Canada (Griffin 1968). Morphologically, L. alneum most closely resembles G. crassivaginata. The asexual morph of L. alneum produced short conidiophores, and stalked club-shaped cells, similar to those of G. crassivaginata. Other similarities are the presence of short-necked perithecia, and fusiform-falcate, sheathed ascospores. Leptographium alneum, however, differs from G. crassivaginata, in having longer ascomatal neck and larger club-shaped cells. Moreover, L. alneum has larger conidia than G. crassivaginata (CBS 119144), especially with regard to the width of the conidia. Other differences are the presence of barrel-shaped or globose cells that make up the conidiophores, which in G. crassivaginata are occasionally only slightly swollen. There are also differences in characteristics of cultures. Leptographium alneum has brownish gray cultures in contrast to olive brown culture of G. crassivaginata (isolate CBS 119144). Both species belong to fast-growing species on MEA, however L. alneum grows much faster than G. crassivaginata, especially at 35 °C.

Leptographium species are generally considered as saprotrophs or pathogens of conifer trees (Jacobs and Wingfield 2001). However, the results of the present study confirm previous Polish investigations that some of the Leptographium species have a close affinity to hardwoods in Europe. Recently, L. betulae, L. tardum, and L. trypodendri were collected from hardwood-infesting bark beetles in Poland (Jankowiak et al. 2017, 2018), while three other Leptographium species have been isolated and formally described from hardwood wounds (Jankowiak et al. 2018, 2019c).

There was no information on D. alni-associated fungi before 2019. Recent Polish research reported that only L. alneum (named as Leptographium sp. 7) is an associate of D. alni (Jankowiak et al. 2019a). However, the additional isolations conducted in 2018 demonstrated that this beetle species apart from L. alneum, was also associated with other ophiostomatoid species including C. synnemata and L. piriforme. Among them, only C. synnemata and L. alneum were commonly found in association with D. alni. The common occurrence of these species suggests their important role as fungal associates of D. alni in Poland. The results of the present study and other Polish findings (Jankowiak et al. 2019a) indicated that C. synnemata and L. alneum have been found only occasionally from other beetle species and therefore can be considered as regular and possible specific associates of D. alni. Leptographium piriforme is less specific and can be found with other beetle species (Jankowiak and Kolařík 2010) or hardwood wounds (Jankowiak et al. 2019c) in low numbers.

This work represents the most detailed survey of Ophiostomatales associated with D. alni in Europe. Two new species were described. Ophiostomatoid fungi on hardwoods have been relatively well investigated in Poland (Jankowiak et al. 2017, 2018, 2019a, b, c; Aas et al. 2018), but in other parts of Europe they are still poorly studied. In addition, many ophiostomatoid species have not been formally described and our study has contributed to filling this knowledge gap. The findings of this study clearly showed that the diversity and taxonomic placement of many members of the Ophiostomatales associated with hardwoods-infesting bark beetles in Europe are still poorly understood.

Acknowledgements

This work was supported by the National Science Centre, Poland (contract No. UMO-2014/15/NZ9/00560) and the Ministry of Science and Higher Education of the Republic of Poland.

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