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.     

A diverse assemblage of <Emphasis Type="Italic">Ophiostoma</Emphasis> species,including two new taxa on eucalypt trees in South Africa

Fungi in the Ophiostomatales include important pathogens of trees as well as agents of wood stain, reducing the economic value of timber. They rely on insects, such as bark beetles, for dispersal and are commonly associated with wounds on trees. Although Ophiostoma spp. have been reported from eucalypt wood chips in South Africa, very little is known about the diversity of the Ophiostomatales, or their insect associates, on plantation-grown Eucalyptus spp. The aim of this study was to consider the diversity and distribution of the Ophiostomatales infecting fresh wounds on Eucalyptus trees in the country. Additionally, knowledge regarding their association with nitidulid beetles, which have previously been shown to carry Ophiostoma spp., was sought. Surveys were conducted in five provinces where Eucalyptus spp. are commonly grown, and the fungi collected were identified using morphological comparisons and multigene sequence phylogenies. Of the 139 isolates collected, five Ophiostoma spp. were identified including O. quercus, O. tsotsi and O. tasmaniense. These were from cut stumps as well as from the nitidulid beetles Brachypeplus depressus and Carpophilus spp. In addition, two new taxa in the O. stenoceras–Sporothrix schenckii complex were identified from Eucalyptus trees infested by Phoracantha semipunctata. The two new taxa are described as O. candidum sp. nov., and O. fumeum sp. nov., respectively. The results of this study clearly show that the diversity and ecology of Ophiostoma spp. on Eucalyptus trees in South Africa is poorly understood and that further studies are required to determine the possible economic relevance of these fungi.     

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.     

Ophiostoma arduennense sp. nov. (Ophiostomatales, Ascomycota) from Fagus sylvatica in southern Belgium

Ophiostoma arduennense sp. nov. is described from several cultures isolated from Fagus sylvatica in southern Belgium. The species is mainly characterized by globose perithecia with small button-like bases ornamented with brown hyphal hairs of variable length and, long cylindrical necks ending in ostiolate hyphae. It is homothallic with small reniform ascospores and no apparent anamorph. It is closely associated with the ambrosia beetles Xyloterus domesticus and X. signatus. Its phylogenetic relationships within Ophiostoma are discussed and the species is compared with other Ophiostoma species associated with European beech or other broad-leaved trees in Europe, especially species of the O. quercus–O. piceae complex. The species is responsible for a dark brown staining of the sapwood and its role in the decline of beech in Southern Belgium is also discussed.     

Ophiostoma gemellus and Sporothrix variecibatus from mites infesting Protea infructescences in South Africa

Ophiostoma (Ophiostomatales) represents a large genus of fungi mainly known from associations with bark beetles (Curculionidae: Scolytinae) infesting conifers in the northern hemisphere. Few southern hemisphere native species are known, and the five species that consistently occur in the infructescences of Protea spp. in South Africa are ecologically unusual. Little is known about the vectors of Ophiostoma spp. from Protea infructescences, however recent studies have considered the possible role of insects and mites in the distribution of these exceptional fungi. In this study we describe a new species of Ophiostoma and a new Sporothrix spp. with affinities to Ophiostoma, both initially isolated from mites associated with Protea spp. They are described as Ophiostoma gemellus sp. nov. and Sporothrix variecibatus sp. nov. based on their morphology and comparisons of DNA sequence data of the 28S ribosomal, beta-tubulin and internal transcribed spacer (ITS1, 5.8S, ITS2) regions. DNA sequences of S. variecibatus were identical to those of a Sporothrix isolate obtained from Eucalyptus leaf litter in the same area in which S. variecibatus occurs in Protea infructescences. Results of this study add evidence to the view that mites are the vectors of Ophiostoma spp. that colonize Protea infructescences. They also show that DNA sequence comparisons are likely to reveal additional cryptic species of Ophiostoma in this unusual niche.     

Ambrosia beetle Premnobius cavipennis (Scolytinae: Ipini) carries highly divergent ascomycotan ambrosia fungus,Afroraffaelea ambrosiae gen. nov. et sp. nov. (Ophiostomatales)

Ambrosia beetles and fungi represent an interesting and economically important symbiosis, but the vast majority of ambrosia fungi remain unexplored, hindering research, management of pathogens, and mitigation of invasive species. Beetles in the subtribe Premnobiini are one example of an entire beetle lineage whose fungal symbionts have never been studied. Here, we identify one dominant fungal symbiont of Premnobius cavipennis by using fungus culturing, community sequencing, microtome sectioning and micro-CT scanning of mycangia. Phylogenetic analyses of combined 18S and 28S rDNA and β-tubulin sequences revealed a highly divergent fungal lineage within Ophiostomatales, Afroraffaelea ambrosiae gen. nov. et sp. nov. The newly described fungal lineage represents another origin of the symbiosis within the Kingdom Fungi, adding to our understanding of the geographic ancestry of ambrosia fungi. P. cavipennis possesses pharyngeal mycangia which appear restrictive in fungus selection. This ambrosia beetle-fungus association has remained stable even after invasions into non-native regions.     

Leptographium tereforme sp. nov. and other Ophiostomatales isolated from the root-feeding bark beetle Hylurgus ligniperda in California

The redhaired pine bark beetle Hylurgus ligniperda (F.) is native to Europe but was discovered in Los Angeles, California, in 2003. This root-and stump-feeding beetle is a common vector of Ophiostomatales, which are potential tree pathogens or causes of blue stain of conifer sapwood. In this study Ophiostomatales were isolated on a cycloheximide-amended medium from 118 adult H. ligniperda collected from infested logs of Pinus halepensis and P. pinea at two sites in California. In total eight species of Ophiostomatales were identified and seven species that occasionally were isolated were unidentified. The most frequently isolated species were Ophiostoma ips and Grosmannia galeiforme, which were isolated respectively from 31% and 23% of the 118 beetles. The other species isolated included O. piceae (isolated from 9% of the beetles), O. querci (8%) and Leptographium tereforme sp. nov. (6%). Grosmannia huntii, L. serpens, three Sporothrix species, O. floccosum, O. stenoceras, two unidentified Hyalorhinocladiella sp. and a sterile fungus each were isolated from fewer then 5% of beetles. Most of the identified species already were known in USA and have been found in association with H. ligniperda in other countries. However the new species, L. tereforme, and G. galeiforme were recorded from USA for the first time, and this is the first report of L. serpens from western North America.     

Evidence for a new lineage of primary ambrosia fungi in Geosmithia Pitt (Ascomycota: Hypocreales)

Geosmithia is a genus of mitosporic filamentous fungi typically associated with phloeophagous bark beetles world-wide. During this study, the fungal associates of ambrosia beetles Cnesinus lecontei, Eupagiocerus dentipes, and Microcorthylus sp. from Costa Rica, were studied using morphology and DNA sequences. Fungal associates belonged to four undescribed Geosmithia species. Geosmithia eupagioceri sp. nov. and G. microcorthyli sp. nov. are evidently primary ambrosia fungi of their respective vectors E. dentipes and Microcorthylus species. They both have convergently evolved distinct morphological adaptations including the production of large, solitary and globose conidia, and yeast-like cells. Tunnels of C. lecontei contained an undescribed Geosmithia species, but its nutritional importance for its vector is unclear. An auxiliary ambrosia fungus, Geosmithia rufescens sp. nov., was found associated with both G. eupagioceri and the Geosmithia species associated with C. lecontei. G. microcorthyli is genetically quite similar to the phloem-associated Geosmithia sp. 8 from Europe. Large differences in morphology between these two species suggest the rapid co-evolution resulting from the close symbiosis of the former with its beetle host. The ITS rDNA sequences of G. microcorthyli and Geosmithia sp. 8 were not diagnostic, suggesting that alternative markers such as EF-1α, IGS rDNA or β-tubulin should be used, together with morphological and ecological data, for species delimitation in this genus. The primary ambrosia fungi described here are derived from phloem-associated ancestors, and represent two independent lineages of ambrosia fungi in the Hypocreales and a new ecological strategy within Geosmithia.     

Three new <Emphasis Type="Italic">Ophiostoma</Emphasis> species isolated from Japanese red pine

Three new species of Ophiostoma found on Japanese red pine are described as Ophiostoma pusillum sp. nov., O. botuliforme sp. nov., and O. nigrogranum sp. nov. Ophiostoma pusillum is characterized by oblong ascospores and a Hyalorhinocladiella anamorph. Ophiostoma botuliforme has ostioles covered with a hyaline gelatinous cap, allantoid ascospores, and a Pesotum anamorph with hyaline to pale brown stipes. Ophiostoma nigrogranum has hyaline ostiolar hyphae with rounded tips, allantoid ascospores, and sclerotium-like structures.Contribution no. 172, Laboratory of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba     

Identification of the ambrosia fungus of <Emphasis Type="Italic">Xyleborus monographus</Emphasis> and <Emphasis Type="Italic">X. dryographus</Emphasis> (Coleoptera: Curculionidae,Scolytinae)

The scolytid ambrosia beetles Xyleborus monographus and X. dryographus were investigated to identify their nutritional ambrosia fungi. The examination of the oral mycetangia of the beetles, the specialized organs for fungal transport, revealed the dominant occurrence of Raffaelea montetyi, a fungus that was also predominant in the beetle tunnels in the immediate vicinity of the feeding larvae. R. montetyi was previously known only as the ambrosia fungus of the platypodid ambrosia beetle, Platypus cylindrus. These beetle species inhabit the same habitat, mainly trunks of oaks in the Western Palaeartic. The possibility of an exchange of the symbiotic fungus between the ambrosia beetles within their common breeding place is discussed. Consequently, the previous hypothesis of a species-specific association of a single ambrosia fungus with a single beetle species is questioned. A phylogenetic analysis based on DNA sequences classified R. montetyi within the Ophiostomatales of the ascomycetes. The investigation of conidiogenesis of R. montetyi by SEM supported this taxonomic placement and showed the development of the conidia by annellidic percurrent proliferation, identical to the conidiogenesis reported for many anamorph states of the Ophiostomatales.     

Discovery of fungus-mite mutualism in a unique niche

The floral heads (infructescences) of South African Protea L. represent a most unusual niche for fungi of the economically important genus Ophiostoma Syd. and P. Syd. emend. Z.W. de Beer et al. Current consensus holds that most members of Ophiostoma are vectored by tree-infesting bark beetles. However, it has recently been suggested that mites, phoretic on these bark beetles, may play a central role in the dispersal of Ophiostoma. No bark beetles are known from Protea. Therefore, identifying the vectors of Ophiostoma in Protea infructescences would independently evaluate the role of various arthropods in the dispersal of Ophiostoma. Infructescence-colonizing arthropods were tested for the presence of Ophiostoma DNA using polymerase chain reaction (PCR) and for reproductive propagules by isolation on agar plates. PCR tests revealed that few insects carried Ophiostoma DNA. In contrast, various mites (Proctolaelaps vandenbergi Ryke, two species of Tarsonemus Canestrini and Fonzago, and one Trichouropoda Berlese species) frequently carried Ophiostoma propagules. DNA sequence comparisons for 28S ribosomal DNA confirmed the presence of O. splendens G. J. Marais and M. J. Wingf., O. palmiculminatum Roets et al., and O. phasma Roets et al. on these mites. Two apparently undescribed species of Ophiostoma were also identified. Light and scanning electron microscopy revealed specialized structures in Trichouropoda and one Tarsonemus sp. that frequently contained Ophiostoma spores. The Trichouropoda sp. was able to complete its life cycle on a diet consisting solely of its identified phoretic Ophiostoma spp. This study provides compelling evidence that mites are the primary vectors of infructescence-associated Ophiostoma spp. in South Africa.     

Characterization of fungal communities associated with the bark beetle Ips typographus varies depending on detection method, location, and beetle population levels

The Eurasian spruce bark beetle Ips typographus and their fungal associates can cause severe damage to Norway spruce forests. In this paper, by using both molecular and cultural methods, we compared fungal assemblages on bark beetles from different locations, characterized by different beetle population levels. Ips typographus was trapped in the western Alps in two outbreak and in two control areas. Sequencing of clone libraries of Internal Transcribed Spacer (ITS) identified 31 fungal Operational Taxonomic Units (OTUs), while fungal isolations yielded 55 OTUs. Only three OTUs were detected by both molecular and cultural methods indicating that both methods are necessary to adequately describe fungal richness. Fungal assemblages on insects from these four and from an additional 12 study sites differed among stands in response to varying ecological conditions and to the limited spreading ability of I. typographus. Ophiostomatoid fungi showed higher diversity in outbreak areas; the pathogenic Ophiostoma polonicum was relatively uncommon, while O. bicolor was the most abundant species. This result was not unexpected, as insects were trapped not at the peak but at the end of the outbreaks and supports the hypothesis of a temporal succession among Ophiostoma species. Ubiquitous endophytes of trees or common airborne fungi were present both in outbreak and in control areas. Wood decaying basidiomycetes were almost never detected on beetles. Yeasts were detected only by molecular analysis, and the OTUs detected matched those reported elsewhere in Europe and in the world, suggesting a very long association between some yeasts and bark beetles.     

Species of Ophiostomataceae isolated from Picea abies infested by the bark beetle Ips typographic

Isolations of microorganisms were made from the sapwood of Norway spruce ( Picea abies ) (L.) Karst. infested by the spruce bark beetle Ips typographus L. The most important group of fungi isolated, the species of Ophiostomataceae, is treated. Ten species were isolated, of which Ophiostoma polonicum Siem. appears to be the most active invader of the sapwood. Three new species are described, Ophiostoma ainoae sp. nov., O. flexuosum sp. nov. and O. cucullatum sp. nov. and three new combinations are proposed, O. davidsonii (Olchow. & Reid.) comb. nov., O. europhioides (Wright & Cain) comb. nov. and O. sagmatospora (Wright & Cain) comb. nov.     

Mycobiota associated with the ambrosia beetle Scolytodes unipunctatus (Coleoptera: Curculionidae, Scolytinae)

Ambrosia beetles (Coleoptera: Scolytinae) are associated with strictly entomochoric and mutualistic fungi. We studied the mycobiota associated with Scolytodes unipunctatus, ambrosia beetles that infest Cecropia trees in Central America. Isolates were characterized using morphology and rDNA sequences (ITS region, LSU, and SSU rDNA). Four species are described here: Raffaelea scolytodis sp. nov. (Ophiostomatales), Gondwanamyces scolytodis sp. nov., Custingophora cecropiae sp. nov., and Graphium sp. (Microascales). The genus Custingophora is emended to include Knoxdaviesia anamorphs of Gondwanamyces based on uniformity of DNA sequences and phenotype.     

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.     

Feeding by Scolytus bark beetles to test for differently susceptible elm varieties

Dutch elm disease (DED), caused by the fungi Ophiostoma ulmi and O. novo‐ulmi, has reduced elm populations severely in Europe and North America. Breeding programmes are in action to find less susceptible elm varieties suitable for re‐establishing elm stands. Bark beetles, mainly Scolytus spp., are the only known natural vectors of DED. During twig feeding, beetles transfer Ophiostoma spores to healthy elms. Thus, less palatable elms should run a lower risk of DED infections. In feeding preference bioassays, we offered twigs from elms exhibiting different degree of susceptibility to O. novo‐ulmi, together with non‐host trees to Scolytus beetles. Scolytus multistriatus preferred wych elm, Ulmus glabra, to 100% in two‐choice tests, whereas S. laevis did not discriminate between a tolerant and a susceptible variety of field elm, U. minor. We suggest that the feeding assay is useful as a low‐tech method in breeding programmes for evaluating the suitability of promising elm genotypes to vector insects.     

Three new <Emphasis Type="Italic">Ophiostoma</Emphasis> species with <Emphasis Type="Italic">Pesotum</Emphasis> anamorphs associated with bark beetles infesting <Emphasis Type="Italic">Abies</Emphasis> species in Nikko,Japan

Three species of Ophiostoma possessing Pesotum anamorphs isolated from bark beetles and their galleries infesting Abies species in Nikko, Japan, are described as new species. Ophiostoma nikkoense is characterized by brush-shaped synnemata producing long septate clavate conidia, perithecia with neck, and allantoid ascospores. Ophiostoma microcarpum has smaller perithecia with hyphoid ostiolar hyphae on the neck, and the ascospores are cylindrical or ossiform in side and face views. Ophiostoma abieticola has perithecia without ostiolar hyphae on the neck and produces orange-section-shaped or reniform ascospores.Contribution no. 187, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba