Ophiostomatoid fungi associated with hardwood-infesting bark and ambrosia beetles in Poland: Taxonomic diversity and vector specificity

Fungi in the orders Ophiostomatales and Microascales (Ascomycota), often designated as ophiostomatoid fungi, are frequent associates of scolytine bark and ambrosia beetles that colonize hardwood and coniferous trees. Several species, e.g., Ophiostoma novo-ulmi, are economically damaging pathogens of trees. Because little is known regarding the ophiostomatoid fungi in Europe, we have explored the diversity of these fungi associated with hardwood-infesting beetles in Poland. This study aims to clarify the associations between fungi in the genera Ambrosiella, Graphium (Microascales), Graphilbum, Leptographium, Ophiostoma and Sporothrix (Ophiostomatales) and their beetle vectors in hardwood ecosystems. Samples associated with 18 bark and ambrosia beetle species were collected from 11 stands in Poland. Fungi were isolated from adult beetles and galleries. Isolates were identified based on morphology, DNA sequence comparisons for five gene regions (ITS, LSU, ßT, TEF 1-α, and CAL) and phylogenetic analyses. In total, 36 distinct taxa were identified, including 24 known and 12 currently unknown species. Several associations between fungi and bark and ambrosia beetles were recorded for the first time. In addition, associations between Dryocoetes alni, D. villosus, Hylesinus crenatus, Ernoporus tiliae, Pteleobius vittatus and ophiostomatoid fungi were reported for the first time, and Sporothrix eucastanea was reported for the first time outside of the USA. Among the species of Ophiostomatales, 14 species were in Ophiostoma s. l., two species were in Graphilbum, nine species were in Sporothrix, and seven species were in Leptographium s. l. Among the species of Microascales, three species were in Graphium, and one was in Ambrosiella. Twenty taxa were present on the beetles and in the galleries, twelve only on beetles, and four only in galleries. Bark and ambrosia beetles from hardwoods appear to be regular vectors, with ophiostomatoid fungi present in all the beetle species. Most ophiostomatoid species had a distinct level of vector/host specificity, although Ophiostoma quercus, the most frequently encountered species, also had the greatest range of beetle vectors and tree hosts. Plant pathogenic O. novo-ulmi was found mainly in association with elm-infesting bark beetles (Scolytus multistriatus, S. scolytus, and P. vittatus) and occasionally with H. crenatus on Fraxinus excelsior and with Scolytus intricatus on Quercus robur.     

Ophiostomatoid fungi associated with the northern spruce engraver, <Emphasis Type="Italic">Ips perturbatus</Emphasis>, in western Canada

A number of ophiostomatoid fungi were isolated from the spruce-infesting bark beetle, Ips perturbatus and its galleries collected from felled spruce trees and logs in northern BC and the Yukon Territory. Isolates were identified to species using morphological characteristics, nuclear ribosomal DNA and partial β-tubulin gene sequences. Thirteen morphological and phylogenetic species were identified among the isolates. Leptographium fruticetum, Leptographium abietinum, Ophiostoma bicolor, Ophiostoma manitobense, O. piceaperdum, and eight undescribed species of the genus Ophiostoma and the anamorph genera Leptographium, Hyalorhinocladiella, Ambrosiella and Graphium. A number of these species, i.e. L. fruticetum, Hyalorhinocladiella sp. 2, O. bicolor and O. manitobense, were isolated repeatedly from I. perturbatus, while others, i.e. Graphium sp. 1 and O. piceaperdum, seemed to be␣sporadic associates. Among all the isolates, L. fruticetum had the highest relative dominance in this survey. A high frequency of occurrence of this species with the beetle may indicate a specific relationship between the two partners.     

Ophiostomatoid fungi associated withIps cembrae in Japan and their pathogenicity of Japanese larch

Six ophiostomatoid fungi, i.e.,Ceratocystiopsis minuta, Ceratocystis laricicola, Ophiostoma brunneo-ciliatum, O. laricis, O. piceae andOphiostoma sp., were isolated from the galleries ofIps cembrae on Japanese Iarch (Larix kaempferi) logs in central Honshu, Japan. Japanese larch trees approximately 30 yr old were inoculated with all six fungi.Ceratocystis laricicola produced the largest lesions on the inner bark around the inoculation point and the largest dry zone in the sapwood. Furthermore,C. laricicola was the only fungus associated withI. cembrae that had the ability to kill Japanese larch, death occurring in 30-yr-old trees within 3.5 mo after inoculation.     

<Emphasis Type="Italic">Geosmithia</Emphasis> Fungi are Highly Diverse and Consistent Bark Beetle Associates: Evidence from their Community Structure in Temperate Europe

Geosmithia spp. (Ascomycota: Hypocreales) are little-studied, dry-spored fungi that occur in galleries built by many phloeophagous bark beetles. This study mapped the distribution and environmental preferences of Geosmithia species occurring in galleries of temperate European bark beetles. One hundred seven host tree samples of 16 tree species infested with 23 subcortical insect species were collected from across Europe during the years 1997–2005. Over 600 Geosmithia isolates from the beetles were sorted into 17 operational taxonomic units (OTUs) based on their phenotype similarity and phylogeny of internal transcribed spacer (ITS) region of rDNA (ITS1-5.8S-ITS2). The OTUs represent six known species and eight undescribed taxa. Ninety-two samples infested with subcortical insects were characterized by the presence/absence of OTUs and the similarity among the samples was evaluated. Geographically distant populations of the same beetle species host relatively uniform Geosmithia communities across large geographic areas (ranging from southern Bulgaria to the Czech Republic). This suggests effective dispersal of Geosmithia spp. by bark beetles. Clustering of similar samples in ordination analysis is correlated predominantly with the isolation source (bark beetles and their respective feeding plant), but not with their geographical origin. The composition of the Geosmithia OTU community of each bark beetle species depends on the degree of isolation of the species’ niches. Thus, Geosmithia communities associated with regularly co-occurring bark beetle species are highly similar. The similarity decreases with decreasing frequency of beetle species’ co-occurrence, a pattern resembling that of entomochoric ophiostomatoid fungi. These findings suggest that: 1) communities of Geosmithia spp. are vector-specific; 2) at least in some cases, the association between Geosmithia OTUs and bark beetles may have been very stable and symbioses are likely to be a fundamental factor in the speciation of Geosmithia fungi; and 3) that even nonsticky spores of Geosmithia are suitable for maintaining an insect–fungus association, contrary to previous hypotheses. An erratum to this article can be found at     

Metabarcoding of fungal communities associated with bark beetles

Many species of fungi are closely allied with bark beetles, including many tree pathogens, but their species richness and patterns of distribution remain largely unknown. We established a protocol for metabarcoding of fungal communities directly from total genomic DNA extracted from individual beetles, showing that the ITS3/4 primer pair selectively amplifies the fungal ITS. Using three specimens of bark beetle from different species, we assess the fungal diversity associated with these specimens and the repeatability of these estimates in PCRs conducted with different primer tags. The combined replicates produced 727 fungal Operational Taxonomic Units (OTUs) for the specimen of Hylastes ater, 435 OTUs for Tomicus piniperda, and 294 OTUs for Trypodendron lineatum, while individual PCR reactions produced on average only 229, 54, and 31 OTUs for the three specimens, respectively. Yet, communities from PCR replicates were very similar in pairwise comparisons, in particular when considering species abundance, but differed greatly among the three beetle specimens. Different primer tags or the inclusion of amplicons in separate libraries did not impact the species composition. The ITS2 sequences were identified with the Lowest Common Ancestor approach and correspond to diverse lineages of fungi, including Ophiostomaceae and Leotiomycetes widely found to be tree pathogens. We conclude that Illumina MiSeq metabarcoding reliably captures fungal diversity associated with bark beetles, although numerous PCR replicates are recommended for an exhaustive sample. Direct PCR from beetle DNA extractions provides a rapid method for future surveys of fungal species diversity and their associations with bark beetles and environmental variables.     

Ophiostomatoid fungi including two new fungal species associated with pine root-feeding beetles in northern Spain

Many bark beetles live in a symbiosis with ophiostomatoid fungi but very little is known regarding these fungi in Spain. In this study, we considered the fungi associated with nine bark beetle species and one weevil infesting two native tree species (Pinus sylvestris and Pinus nigra) and one non-native (Pinus radiata) in Cantabria (Northern Spain). This included examination of 239 bark beetles or their galleries. Isolations yielded a total of 110 cultures that included 11 fungal species (five species of Leptographium sensu lato including Leptographium absconditum sp. nov., five species of Ophiostoma sensu lato including Ophiostoma cantabriense sp. nov, and one species of Graphilbum). The most commonly encountered fungal associates of the bark beetles were Grosmannia olivacea, Leptographium procerum, and Ophiostoma canum. The aggressiveness of the collected fungal species was evaluated using inoculations on two-year-old P. radiata seedlings. Leptographium wingfieldii, Leptographium guttulatum, and Ophiostoma ips were the only species capable of causing significant lesions.     

Fungi Vectored by the Bark Beetle Ips typographus Following Hibernation Under the Bark of Standing Trees and in the Forest Litter

The bark beetle Ips typographus has different hibernation environments, under the bark of standing trees or in the forest litter, which is likely to affect the beetle-associated fungal flora. We isolated fungi from beetles, standing I. typographus-attacked trees, and forest litter below the attacked trees. Fungal identification was done using cultural and molecular methods. The results of the two methods in detecting fungal species were compared. Fungal communities associated with I. typographus differed considerably depending on the hibernation environment. In addition to seven taxa of known ophiostomoid I. typographus-associated fungi, we detected 18 ascomycetes and anamorphic fungi, five wood-decaying basidomycetes, 11 yeasts, and four zygomycetes. Of those, 14 fungal taxa were detected exclusively from beetles that hibernated under bark, and six taxa were detected exclusively from beetles hibernating in forest litter. The spruce pathogen, Ceratocystis polonica, was detected occasionally in bark, while another spruce pathogen, Grosmannia europhioides, was detected more often from beetles hibernating under the bark as compared to litter. The identification method had a significant impact on which taxa were detected. Rapidly growing fungal taxa, e.g. Penicillium, Trichoderma, and Ophiostoma, dominated pure culture isolations; while yeasts dominated the communities detected using molecular methods. The study also demonstrated low frequencies of tree pathogenic fungi carried by I. typographus during its outbreaks and that the beetle does not require them to successfully attack and kill trees.     

Broadscale Specificity in a Bark Beetle–Fungal Symbiosis: a Spatio-temporal Analysis of the Mycangial Fungi of the Western Pine Beetle

Whether and how mutualisms are maintained through ecological and evolutionary time is a seldom studied aspect of bark beetle–fungal symbioses. All bark beetles are associated with fungi and some species have evolved structures for transporting their symbiotic partners. However, the fungal assemblages and specificity in these symbioses are not well known. To determine the distribution of fungi associated with the mycangia of the western pine beetle (Dendroctonus brevicomis), we collected beetles from across the insect’s geographic range including multiple genetically distinct populations. Two fungi, Entomocorticium sp. B and Ceratocystiopsis brevicomi, were isolated from the mycangia of beetles from all locations. Repeated sampling at two sites in Montana found that Entomocorticium sp. B was the most prevalent fungus throughout the beetle’s flight season, and that females carrying that fungus were on average larger than females carrying C. brevicomi. We present evidence that throughout the flight season, over broad geographic distances, and among genetically distinct populations of beetle, the western pine beetle is associated with the same two species of fungi. In addition, we provide evidence that one fungal species is associated with larger adult beetles and therefore might provide greater benefit during beetle development. The importance and maintenance of this bark beetle–fungus interaction is discussed.     

Experimental evidence of bark beetle adaptation to a fungal symbiont

The importance of symbiotic microbes to insects cannot be overstated; however, we have a poor understanding of the evolutionary processes that shape most insect–microbe interactions. Many bark beetle (Coleoptera: Curculionidae, Scolytinae) species are involved in what have been described as obligate mutualisms with symbiotic fungi. Beetles benefit through supplementing their nutrient‐poor diet with fungi and the fungi benefit through gaining transportation to resources. However, only a few beetle–fungal symbioses have been experimentally manipulated to test whether the relationship is obligate. Furthermore, none have tested for adaptation of beetles to their specific symbionts, one of the requirements for coevolution. We experimentally manipulated the western pine beetle–fungus symbiosis to determine whether the beetle is obligately dependent upon fungi and to test for fine‐scale adaptation of the beetle to one of its symbiotic fungi, Entomocorticium sp. B. We reared beetles from a single population with either a natal isolate of E. sp. B (isolated from the same population from which the beetles originated), a non‐natal isolate (a genetically divergent isolate from a geographically distant beetle population), or with no fungi. We found that fungi were crucial for the successful development of western pine beetles. We also found no significant difference in the effects of the natal and non‐natal isolate on beetle fitness parameters. However, brood adult beetles failed to incorporate the non‐natal fungus into their fungal transport structure (mycangium) indicating adaption by the beetle to particular genotypes of symbiotic fungi. Our results suggest that beetle–fungus mutualisms and symbiont fidelity may be maintained via an undescribed recognition mechanism of the beetles for particular symbionts that may promote particular associations through time.     

Ophiostomatoid fungi (Ascomycota) associated with <Emphasis Type="Italic">Pinus tabuliformis</Emphasis> infested by <Emphasis Type="Italic">Dendroctonus valens</Emphasis> (Coleoptera) in northern China and an assessment of their pathogenicity on mature trees

Dendroctonus valens is an invasive pest in coniferous forests of northern China. It was suspected of being responsible for the death of more than three million Pinus tabuliformis trees. The present study sought to identify the ophiostomatoid fungi associated with D. valens in northern China and understand the possible role of these fungi in the pine decline. On the basis of morphology, physiology, mating compatibility and phylogenetic analyses of multiple DNA sequences, seven species of ophiostomatoid fungi were isolated from and around D. valens galleries: Leptographium alethinum, Grosmannia koreana (teleomorph of L. koreanum), L. procerum, L. sinoprocerum, L. truncatum, Pesotum aureum and P. pini. All have been recorded for the first time in China. Among them, the occurrence of the dominant species L. procerum is positively linked to attack intensities of D. valens. The pathogenicity of four species (L. koreanum, L. procerum, L. sinoprocerum and L. truncatum) was tested on mature P. tabuliformis trees by stem inoculation. All inoculated strains caused significant necrotic lesions on the inner bark. However, L. koreanum and L. truncatum induced more extensive lesions than L. procerum and L. sinoprocerum. Their association with D. valens and the P. tabuliformis decline is discussed.     

Two species of <Emphasis Type="Italic">Leptographium</Emphasis> isolated from blue-stained sapwood of <Emphasis Type="Italic">Pinus khasya</Emphasis> and bark beetles in Thailand

Two species of Leptographium were isolated from blue-stained sapwood of Pinus khasya and bark beetle galleries in pine trees near Chiang Mai, Thailand. Based on morphological observations, these two species were identified as L. pini-densiflorae and L. yunnanense. This is the first record of these fungi in Thailand. Leptographium yunnanense appeared to be associated with Polygraphus major. This is contribution No. 206, Laboratory of Plant Parasitic Mycology, Graduate School of Life & Environmental Sciences, University of Tsukuba     

Constant association of ophiostomatoid fungi with the bark beetle <Emphasis Type="Italic">Ips subelongatus</Emphasis> invading Japanese larch logs

Ceratocystiopsis, Ceratocystis, Grosmannia, and Ophiostoma species were isolated from Ips subelongatus and beetle-infested Japanese larch logs collected at several areas in central and northern Honshu Island, Japan, to determine constant associates of I. subelongatus. Ceratocystiopsis minuta, two species of Ceratocystis, three species of Grosmannia, and four species of Ophiostoma were isolated. Of the fungi isolated in the present study, G. laricis, O. brunneociliatum, and O. piceae were constant associates of the beetles. Ceratocystis fujiensis, Ceratocystiopsis minuta, and Ophiostoma sp. F were occasionally isolated with high frequencies of occurrence but were not consistent associates. Ceratocystis fujiensis was most often isolated as the leading fungal invasion in the sapwood of Japanese larch logs invaded by I. subelongatus, confirming that the fungus acts as a primary invader of sapwood in beetle-attacked logs. Contribution No. 221, Laboratory of Plant Parasitic Mycology, Graduate School of Life and Environmental Sciences, University of Tsukuba     

A major symbiont shift supports a major niche shift in a clade of tree-killing bark beetles

1. One small clade of bark beetles, out of thousands of species worldwide, has shifted from using phloem to using a combination of phloem and outer bark, or to completely using outer bark. 2. The shift to outer bark has been accompanied by a shift to non-typical bark beetle mutualist fungi in Entomocorticium (Basidiomycota) and Ceratocystiopsis (Ascomycota). 3. This study compared the growth and metabolic capabilities of fungi associated with a nearly phloem-independent species, Dendroctonus brevicomis, with those of mutualist fungi of Dendroctonus ponderosae, a completely phloem-colonising beetle in a sister clade associated with more typical Ascomycota in Grosmannia and Ophiostoma. 4. Only the basidiomycete associated with D. brevicomis could degrade cellulose and lignin, whereas both the ascomycete and basidiomycete could grow in outer bark. Ascomycetes associated with D. ponderosae could not degrade cellulose or lignin or grow in outer bark. 5. Beetles and fungi in these mutualisms may best be considered as co-niche constructors. For niche construction, one organism must modify a resource in a way that enhances its fitness while also influencing fitness of other organisms using the resource. Here, the beetles kill the tree, transport the fungi into the tree, and modify the woody substrate for use by the fungi. They have also evolved mycangia to ensure vertical dissemination of the fungi. In turn, the fungi modify tree tissues and provision nutrients to the host and have evolved traits that support their acquisition and transport by the beetle host in mycangia.     

Contribution of symbiotic mycangial fungi to larval nutrition of a leaf-rolling weevil

Some phytophagous insects have been known to inoculate certain fungi on plant substrates. In many cases of such insect–fungi relationships it has been considered that fungi contribute to insects by decomposing lignin or polysaccharides, and that the insects feed on the decomposition products or fungi themselves. Females of the leaf-rolling weevil in the genus Euops (Attelabidae) store spores of symbiotic fungi in the mycangia and inoculate them on leaf rolls. To determine the effect of mycangial fungi on larval nutrition in E. lespedezae, the nutritional value was compared between leaves with and without mycangial fungi. Two Penicillium species were isolated from the mycangia. These mycangial fungi showed little effect on the decomposition of lignin and polysaccharides, and showed little effect on enhancement of soluble sugars within leaves. Thus, the mutualism between Euops and its mycangial fungi contrasts with the mainly nutritional mutualisms between wood-infesting insects (termites, bark/ambrosia beetles, and wood wasps) and lignin/polysaccharide-decomposing fungi.     

Fungal associates of the lodgepole pine beetle, <Emphasis Type="Italic">Dendroctonus murrayanae</Emphasis>

Bark beetles are well known vectors of ophiostomatoid fungi including species of Ophiostoma, Grosmannia and Ceratocystis. In this study, the most common ophiostomatoid fungi associated with the lodgepole pine beetle, Dendroctonus murrayanae, were characterized. Pre-emergent and post-attack adult beetles were collected from lodgepole pines at four sites in British Columbia, Canada. Fungi were isolated from these beetles and identified using a combination of morphology and DNA sequence comparisons of five gene regions. In all four populations, Grosmannia aurea was the most common associate (74–100% of all beetles) followed closely by Ophiostoma abietinum (29–75%). Other fungi isolated, in order of their relative prevalence with individual beetles were an undescribed Leptographium sp. (0–13%), Ophiostoma ips (0–15%), Ophiostoma piliferum (0–11%), a Pesotum sp. (0–11%) and Ophiostoma floccosum (0–1%). Comparisons of the DNA sequences of Leptographium strains isolated in this study, with ex-type isolates of G. aurea, Grosmannia robusta, Leptographium longiclavatum, and Leptographium terebrantis, as well as with sequences from GenBank, revealed a novel lineage within the Grosmannia clavigera complex. This lineage included some of the D. murrayane isolates as well as several isolates from previous studies referred to as L. terebrantis. However, the monophyly of this lineage is not well supported and a more comprehensive study will be needed to resolve its taxonomic status as one or more novel taxa.     

Hymenopterous parasitoids of bark beetles [Scolytidae] in Israel: Host relation,host plant,abundance and seasonal history

Twenty six species of parasitoids were recorded from 15 species of bark beetles in Israel. Parasitoids were collected from infested cut branches of broadleaves and trap-trees of pine and cypress. Most of the parasitoids are oligophagous and range fromEcphylus caudatus, a monophagous species exclusive toHypoborus ficus, toHeydenia pretiosa, which develops on 11 species.Scleroderma domesticus, Agrilocida ferrieri andEupelmus nearurozonus are occasional parasitoids of bark beetles.Cephalonomia hypobori, Israelius carthami, S. domesticus, Entedon ergias, Cerocephala eccoptogasteri andRoptocerus xylophagorum are cryptoparasitoids and search for their host in the galleries. Known parasitoids are listed from 15 tree genera belonging to 8 botanical families. A typical range for the common parasitoids is presented with within-tree distribution of pine parasitoids. Relatively monophagous species are usually dominant parasitoids in a specific beetle population, whereas relatively polyphagous parasitoids vary in abundance among different host populations.Calosota spp., 2Eurytoma spp. andDendrosoter hartigii are usually present in small numbers. Information concerning the seasonal history of the parasitoids suggests that the major species may raise about 5 to 7 annual generations. Contribution from the Agricultural Research Organization, Bet Dagan, Israel N^o 1331-E, 1984 series     

Ophiostomatoid fungi can emit the bark beetle pheromone verbenone and other semiochemicals in media amended with various pine chemicals and beetle-released compounds

Fungal volatile compounds can mediate fungal-insect interactions. Whether fungi can emit insect pheromones and how volatile chemicals change in response to chemicals the fungi naturally encounter is poorly understood. We analyzed volatiles emitted by Grosmannia clavigera (symbiont of the mountain pine beetle) and Ophiostoma ips (symbiont of the pine engraver beetle) growing in liquid media amended with compounds that the fungi naturally encounter: (−)-α-pinene, (+)-α-pinene, (−)-trans-verbenol, verbenone, or ipsdienol. Nine volatile compounds were identified among the fungal and amendment treatments. Volatiles qualitatively and quantitatively differed between fungal species and among amendment treatments. The bark beetle anti-aggregation pheromone (−)-verbenone was detected from both fungi growing in (−)-trans-verbenol-amended media. G. clavigera and O. ips can emit beetle pheromones and other beetle semiochemicals, suggesting that ophiostomatoid fungi could contribute to the chemical ecology of bark beetles. However, such contributions could be modulated by the presence of other environmental chemicals.     

Individual variation in two host plants of the ladybird beetle,Epilachna pustulosa (Coleoptera: Coccinellidae)

Individual variation in two species of host plants (thistle,Cirsium kamtschaticum, and blue cohosh,Caulophyllum robustum) of the herbivorous ladybird beetleEpilachna pustulosa was examined under laboratory conditions for their acceptability to adult beetles as a food resource, for adult preference and for larval performance. When clones of these plants were subjected to non-choice tests using posthibernating female beetles, there was found to be significant intraspecific variation among clones in terms of their acceptability, but interspecific variation was not detected. Significant intraspecific as well as interspecific variation were frequently detected in the two host plants when clones of these plants were subjected to choice tests using posthibernating female beetles; the magnitude of interspecific plant variation for beetle preference is not necessarily larger than that of intraspecific plant variation. Individual variation across plant species with respect to beetle larval performance was also significant. A positive correlation between adult preference and larval performance is suggested across the two taxonomically remote host plant species, thistle and blue cohosh, although this needs further investigation.