Two new species of Leptographium associated with Tomicus spp. infesting Pinus spp. in Southwestern China.

Leptographium panxianense and L. puerense are proposed as new taxa based on sequence data and morphological characters. The phylogenetic analyses based on ITS2-partial LSU rDNA region, ß-tubulin and elongation factor 1-α genes showed that L. panxianense and L. puerense formed well-supported clades and were closely related to L. yunnanense, L. wushanense and L. conjunctum, and then nested within the L. lundbergii complex. The two species differ in their conidial size and shape. The conidia of L. panxianense are larger than those of L. puerense while the conidial shape of L. puerense is more ovovoid. The optimal growth temperature of both L. panxianense and L. puerense is at 20 °C, which is different from those of L. yunnanense, L. wushanense and L. conjunctum. Comparison of sequence data and morphological characters confirmed the placement of the two undescribed taxa in the genus of Leptographium.

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

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

Molecular assays to detect the presence and viability of Phytophthora ramorum and Grosmannia clavigera.

Wood and wood products can harbor microorganisms that can raise phytosanitary concerns in countries importing or exporting these products. To evaluate the efficacy of wood treatment on the survival of microorganisms of phytosanitary concern the method of choice is to grow microbes in petri dishes for subsequent identification. However, some plant pathogens are difficult or impossible to grow in axenic cultures. A molecular methodology capable of detecting living fungi and fungus-like organisms in situ can provide a solution. RNA represents the transcription of genes and can become rapidly unstable after cell death, providing a proxy measure of viability. We designed and used RNA-based molecular diagnostic assays targeting genes essential to vital processes and assessed their presence in wood colonized by fungi and oomycetes through reverse transcription and real-time polymerase chain reaction (PCR). A stability analysis was conducted by comparing the ratio of mRNA to gDNA over time following heat treatment of mycelial cultures of the Oomycete Phytophthora ramorum and the fungus Grosmannia clavigera. The real-time PCR results indicated that the DNA remained stable over a period of 10 days post treatment in heat-treated samples, whereas mRNA could not be detected after 24 hours for P. ramorum or 96 hours for G. clavigera. Therefore, this method provides a reliable way to evaluate the viability of these pathogens and offers a potential way to assess the effectiveness of existing and emerging wood treatments. This can have important phytosanitary impacts on assessing both timber and non-timber forest products of commercial value in international wood trade.

Phylogenetic re-evaluation of the Grosmannia penicillata complex (Ascomycota, Ophiostomatales), with the description of five new species from China and USA.

The Grosmannia penicillata complex (Ophiostomatales, Ascomycota) is one of the major species complexes in Leptographium sensu lato. Most of these are wood staining fungi associated with conifer-infesting bark beetles, and the complex encompasses the type species of the genus Grosmannia. Yet the phylogenetic relationships of species within the complex is unresolved. The aim of this study was to re-evaluate the circumscriptions of all known species in the G. penicillata complex, as well as isolates resembling G. penicillata obtained from a recent survey in China. Phylogenetic analyses of four gene regions: Internal transcribed spacer 2 and large subunit (ITS2-LSU), beta-tubulin (TUB), calmodulin (CAL), and translation elongation factor 1 alpha (TEF-1α) resolved the relationships of 15 species, including four new species (Grosmannia xianmiense sp nov., Grosmannia purpurea sp. nov., Grosmannia crassifolia sp. nov. and Grosmannia maixiuense sp. nov.), from China. Some isolates from pine in the USA that had previously been identified as Grosmannia abietina, represented a distinct taxon that is described here as Grosmannia xeno-abietina sp. nov.

A pine wood sample preparation method for high target and quality DNA extraction for detection of Esteya vermicola by PCR from living pine.

Esteya vermicola is an endophytic fungus of pine wood nematode with demonstrated biocontrol efficacy. At present, the methods for detection of this fungus from pine wood is still inconvenient and inefficient such as thin-section microscopic observations. In the present study, a simple protocol was developed for wood sample preparation for effective extraction of fungal DNA from wood samples for PCR detection. The protocol of preparation of the sample involves washing in sterile water overnight on a shaking table followed by filtration and centrifugation to obtain the extracted DNA. The result indicates that with this sample preparation protocol, any proper DNA extraction method can be effectively used for the rapid and reliable detection of E. vermicola from pine wood. This method can provide valuable support for follow-up studies with practical applications, such as investigation of the growth rate of E. vermicola and how long it remains viable inside a pine tree.

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

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

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

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

A novel application of RNase H2-dependent quantitative PCR for detection and quantification of Grosmannia clavigera, a mountain pine beetle fungal symbiont, in environmental samples.

Mountain pine beetle (Dendroctonus ponderosae Hopkins; MPB) is an economically and ecologically important pest of pine species in western North America. Mountain pine beetles form complex multipartite relationships with microbial partners, including the ophiostomoid fungi Grosmannia clavigera (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingfield, Ophiostoma montium (Rumbold) von Arx, Grosmannia aurea (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingfield, Leptographium longiclavatum (Lee, Kim, and Breuil) and Leptographium terebrantis (Barras and Perry). These fungi are vectored by MPB to new pine hosts, where the fungi overcome host defenses to grow into the sapwood. A tree's relative susceptibility to these fungi is conventionally assessed by measuring lesions that develop in response to fungal inoculation. However, these lesions represent a symptom of infection, representing both fungal growth and tree defense capacity. In order to more objectively assess fungal virulence and host tree susceptibility in studies of host-pathogen interactions, a reliable, consistent, sensitive method is required to accurately identify and quantify MPB-associated fungal symbionts in planta. We have adapted RNase H2-dependent PCR, a technique originally designed for rare allele discrimination, to develop a novel RNase H2-dependent quantitative PCR (rh-qPCR) assay that shows greater specificity and sensitivity than previously published PCR-based methods to quantify MPB fungal symbionts in pine xylem and MPB whole beetles. Two sets of assay probes were designed: one that amplifies a broad range of ophiostomoid species, and a second that amplifies G. clavigera but not other MPB-associated ophiostomoid species. Using these primers to quantify G. clavigera in pine stems, we provide evidence that lesion length does not accurately reflect the extent of fungal colonization along the stem nor the quantity of fungal growth within this colonized portion of stem. The sensitivity, specificity, reproducibility, cost effectiveness and high-throughput potential of the rh-qPCR assay makes the technology suitable for identification and quantification of a wide array of pathogenic and beneficial microbes that form associations with plants and other organisms, even when the microbial partner is present in low abundance.

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

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

Putative origins of the fungus Leptographium procerum.

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

Hawksworthiomyces gen. nov. (Ophiostomatales), illustrates the urgency for a decision on how to name novel taxa known only from environmental nucleic acid sequences (ENAS).

There have been many recent studies using environmental nucleic acid sequences (ENAS) to assess fungal diversity. As a result, more than a third of all fungal sequences in GenBank are of environmental origin. But inconsistent annotation of the thousands of undescribed taxa represented by these sequences limits access to these data. Consequently, these ENAS and the taxa they represent are rarely considered in other studies, and especially not in taxonomic treatments. This problem is confounded by the fact that the current version of the International Code of Nomenclature for Algae, Fungi, and Plants (Melbourne Code) prohibits the description of novel taxa known only from ENAS. There have been suggestions to emend the Code to allow a systematic nomenclatural treatment of these currently 'orphan' taxa but this has yet to occur. In this study, we considered the feasibility of using sequences from environmental studies to resolve the generic status of Sporothrix lignivora. This species forms a single lineage distinct from Sporothrix and other genera in the Ophiostomatales. BLAST searches in GenBank using LSU and ITS sequences of S. lignivora corresponded with several sequences from environmental studies. This also led to the discovery of isolates collected in diversity studies based on culturable fungi, with high similarity to S. lignivora. Phylogenetic analyses including taxa representing all major genera and lineages in the Ophiostomatales revealed a distinct, well-supported lineage that included S. lignivora and the ENAS. This confirmed the presence of a new genus in the Ophiostomatales described here as Hawksworthiomyces gen. nov., with S. lignivora as type species. Whereas only one described species was known in the so-called S. lignivora complex, our analyses revealed nine additional lineages in what is now Hawksworthiomyces. For three of these lineages, we were able to obtain isolates and these are described as Hawksworthiomyces taylorii sp. nov., Hawksworthiomyces crousii sp. nov., and Hawksworthiomyces hibbettii sp. nov. Five of the lineages each included one or more sequences from single studies, and thus remain unnamed. The remaining lineage included two sequences from separate studies of fungi inhabiting conifer wood. One of these sequences was an uncultured fungus clone from a spruce log in Sweden. The other sequence was for an isolate from a western red cedar fencepole in British Columbia, Canada, that was subsequently lost. These two ITS sequences differ in only two nucleotide positions. We are confident that they represent the same taxon and meet the criteria for an ENAS species, for which we provide the name, Hawksworthiomyces sequentia sp. nov. ENAS, and designate a DNA sequence as type in the absence of a type specimen. This case study makes it clear that environmental sequences and those from lost isolates can be extremely valuable in phylogeny-based taxonomic studies. It emphasises the fact that the Code should be emended to enable the naming of such taxa in a manner that will facilitate their incorporation in other studies.

Ophiostomatoid fungi associated with mangroves in South Africa, including Ophiostoma palustre sp. nov.

Mangrove trees are continuously under stress due to environmental and/or anthropogenic pressures, which expose them to attack by pathogens, compromising their survival. Ophiostomatoid fungi cause sap stain and diseases of a wide spectrum of tree species globally. These fungi infect trees through natural, insect, animal and/or human made wounds. During routine surveys of mangrove trees in South Africa, wounds on branches and stems of Avicennia marina were regularly monitored for the presence of ophiostomatoid fungi at ten study sites in the country. The stems of four mangrove species, A. marina, Bruguiera gymnorrhiza, Rhizophora mucronata and Barringtonia racemosa were also wounded and evaluated for the appearance of these fungi. Ophiostomatoid fungi were obtained from the mangrove associate B. racemosa, but not from any of the true mangroves. Analyses of DNA sequence data for the internal transcribed spacer, ß-tubulin, calmodulin and translation elongation factor gene regions revealed that the fungi isolated from the wounds on B. racemosa belong to three species in the Ophiostomataceae, including a new taxon described here as Ophiostoma palustre sp. nov. These results suggest that the mangrove associate B. racemosa is more prone to colonization by ophiostomatoid fungi than the true mangroves.

Wounds on Rapanea melanophloeos provide habitat for a large diversity of Ophiostomatales including four new species.

Rapanea melanophloeos, an important canopy tree in Afromontane forests, is commonly utilised for medicinal bark harvesting. Wounds created from these activities provide entrance for many fungi, including arthropod-associated members of the Ophiostomatales and Microascales (ophiostomatoid fungi). In this study we assessed the diversity of wound-associated Ophiostomatales on storm-damaged R. melanophloeos trees in the Afromontane forests of South Africa. Five species were identified based on micro-morphological and molecular phylogenetic analyses. These included Ophiostoma stenoceras and four newly described taxa Sporothrix itsvo sp. nov., S. rapaneae sp. nov., S. uta sp. nov. and O. noisomeae sp. nov. Four of these are members of the S. schenckii-O. stenoceras complex (O. stenoceras, S. itsvo sp. nov., S. rapaneae sp. nov., S. uta sp. nov.) while O. noisomeae groups basal in the Ophiostomatales alongside the S. lignivora complex and Graphilbum. In addition to other taxa known from this host, the present study shows that there is a rich, yet still poorly explored, diversity of Ophiostomatales associated with R. melanophloeos in Afromontane forests. More taxa are likely to be discovered with increased research effort. These must be assessed in terms of pathogenicity towards this ecologically and economically important tree.

Novel ophiostomatalean fungi from galleries of Cyrtogenius africus (Scolytinae) infesting dying Euphorbia ingens.

Euphorbia ingens trees have been dying in large numbers in the Limpopo Province of South Africa for approximately 15 years. The ambrosia beetle Cyrtogenius africus is often found infesting diseased and dying trees. The aim of this study was to identify the ophiostomatoid fungi occurring in the galleries of C. africus. Logs infested with this beetle were collected from the KwaZulu-Natal, Limpopo, Mpumalanga, and North West Provinces of South Africa. Fungi belonging to the Ophiostomatales were identified based on morphology and comparison of sequence data for the ß-tubulin, ITS1-5.8S-ITS2 and LSU gene regions. A novel species of Ophiostoma and a novel genus in the Ophiostomatales were identified. Inoculation studies with these fungi produced lesions in the branches of healthy E. ingens trees.

Rapid Induction of Multiple Terpenoid Groups by Ponderosa Pine in Response to Bark Beetle-Associated Fungi.

Ponderosa pine (Pinus ponderosa) is a major and widely distributed component of conifer biomes in western North America and provides substantial ecological and economic benefits. This tree is exposed to several tree-killing bark beetle-microbial complexes, including the mountain pine beetle (Dendroctonus ponderosae) and the phytopathogenic fungus Grosmannia clavigera that it vectors, which are among the most important. Induced responses play a crucial role in conifer defenses, yet these have not been reported in ponderosa pine. We compared concentrations of terpenes and a phenylpropanoid, two phytochemical classes with strong effects against bark beetles and their symbionts, in constitutive phloem tissue and in tissue following mechanical wounding or simulated D. ponderosae attack (mechanical wounding plus inoculation with G. clavigera). We also tested whether potential induced responses were localized or systemic. Ponderosa pines showed pronounced induced defenses to inoculation, increasing their total phloem concentrations of monoterpenes 22.3-fold, sesquiterpenes 56.7-fold, and diterpenes 34.8-fold within 17 days. In contrast, responses to mechanical wounding alone were only 5.2, 11.3, and 7.7-fold, respectively. Likewise, the phenylpropanoid estragole (4-allyanisole) rose to 19.1-fold constitutive levels after simulated attack but only 4.4-fold after mechanical wounding. Overall, we found no evidence of systemic induction after 17 days, which spans most of this herbivore's narrow peak attack period, as significant quantitative and compositional changes within and between terpenoid groups were localized to the wound site. Implications to the less frequent exploitation of ponderosa than lodgepole pine by D. ponderosae, and potential advantages of rapid localized over long-term systemic responses in this system, are discussed.

New species of Ophiostomatales from Scolytinae and Platypodinae beetles in the Cape Floristic Region, including the discovery of the sexual state of Raffaelea.

Olea capensis and Rapanea melanophloeos are important canopy trees in South African Afromontane forests. Dying or recently dead individuals of these trees are often infested by Scolytinae and Platypodinae (Curculionidae) beetles. Fungi were isolated from the surfaces of beetles emerging from wood samples and their galleries. Based on micro-morphological and phylogenetic analyses, four fungal species in the Ophiostomatales were isolated. These were Sporothrix pallida and three taxa here newly described as Sporothrix aemulophila sp. nov., Raffaelea vaginata sp. nov. and Raffaelea rapaneae sp. nov. This study represents the first collection of S. pallida, a species known from many environmental samples from across the world, from Scolytinae beetles. S. aemulophila sp. nov. is an associate of the ambrosia beetle Xyleborinus aemulus. R. rapaneae sp. nov. and R. vaginata sp. nov. were associated with a Lanurgus sp. and Platypodinae beetle, respectively, and represent the first Raffaelea spp. reported from the Cape Floristic Region. Of significance is that R. vaginata produced a sexual state analogous with those of Ophiostoma seticolle and O. deltoideosporum that also grouped in our analyses in Raffaelea s. str., to date considered an asexual genus. The morphology of the ossiform ascospores and anamorphs of the three species corresponded and the generic circumscription of Raffaelea is thus emended to accommodate sexual states. The two known species are provided with new combinations, namely Raffaelea seticollis (R.W. Davidson) Z.W. de Beer and T.A. Duong comb. nov. and Raffaelea deltoideospora (Olchow. and J. Reid) Z.W. de Beer and T.A. Duong comb. nov.

Cornuvesica: A little known mycophilic genus with a unique biology and unexpected new species.

Little is known about the biology of the monotypic genus Cornuvesica (Microascales), apart from that isolates are notoriously difficult to culture on artificial media. A recent collection of material resembling this genus from freshly made wounds on Gmelina arborea in Indonesia, provided an opportunity to reconsider all available material of Cornuvesica falcata, type species of the genus. In addition to morphological comparisons, multigene phylogenetic analyses were made using sequences of the SSU, ITS, LSU and TEF-1α genes. Our results showed that the holotype of Cor. falcata from pine in Canada differed from all other material previously considered to represent this species and also from the new Indonesian collections. The collections considered represented three additional species that we describe here as new. Three New Zealand isolates and an isolate from UK were respectively described as Cor. acuminata and Cor. crypta, while the Indonesian isolates were described as Cor. magnispora. Phylogenies based on the SSU and LSU data sets showed that Cornuvesica spp. do not belong in the Ceratocystidaceae as previously suggested, but represent a distinct lineage in the Microascales that has yet to be named. Results showed that culture filtrates from other fungi or ferric chloride markedly stimulated the growth of Cor. magnispora.

Taxonomy and phylogeny of the Leptographium procerum complex, including Leptographium sinense sp. nov. and Leptographium longiconidiophorum sp. nov.

Leptographium procerum (Ophiostomatales, Ascomycota) is a well-known fungal associate of pine root-infesting bark beetles and weevils, occurring in several countries of the world. The fungus is not a primary pathogen but has been associated with white pine root decline in the USA and with serious damage caused by the introduced red turpentine beetle (RTB) Dendroctonus valens in China. Several species closely related to L. procerum have been described during the past decade. The aim of this study was to reevaluate species boundaries in the L. procerum complex using multigene phylogenetic analyses and morphological comparisons. Phylogenetic analyses of seven gene regions (ITS2-LSU, actin, ß-tubulin, calmodulin, translation elongation factor 1-α, and the mating type genes MAT1-1-3 and MAT1-2-1) distinguished between nine species in the complex. These included L. procerum, L. bhutanense, L. gracile, L. profanum, L. pini-densiflorae, L. sibiricum, L. sinoprocerum, as well as two new species described here as Leptographium sinense sp. nov. from Hylobitelus xiaoi on Pinus elliottii in China, and Leptographium longiconidiophorum sp. nov. from Pinus densiflora in Japan. Leptographium latens is reduced to synonymy with L. gracile, and an epitype is designated for L. procerum, because a living culture associated with the holotype of L. procerum did not exist. Amplification patterns of the mating type genes suggest that all known species in the L. procerum complex are heterothallic, although sexual states have not been observed for any of the species. The results also suggest that Eastern Asia is most probably the centre of species diversity for the L. procerum complex.

Phylogeny of ambrosia beetle symbionts in the genus Raffaelea.

The genus Raffaelea was established in 1965 when the type species, Raffaelea ambrosia, a symbiont of Platypus ambrosia beetles was described. Since then, many additional ambrosia beetle symbionts have been added to the genus, including the important tree pathogens Raffaelea quercivora, Raffaelea quercus-mongolicae, and Raffaelea lauricola, causal agents of Japanese and Korean oak wilt and laurel wilt, respectively. The discovery of new and the dispersal of described species of Raffaelea to new areas, where they can become invasive, presents challenges for diagnosticians as well as plant protection and quarantine efforts. In this paper, we present the first comprehensive multigene phylogenetic analysis of Raffaelea. As it is currently defined, the genus was found to not be monophyletic. On the basis of this work, Raffaelea sensu stricto is defined and the affinities of undescribed isolates are considered.

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.