First report of Ceratocystis ficicola causing wilt disease on common fig (Ficus carica) in Italy.

Common fig (Ficus carica L.) in is one of the most important crops in the Mediterranean area. In Italy, it is grown on a total area of 2118 ha. In Apulia (South-eastern Italy), the annual production of fig exceeds 3200 tons annually and together with olive and grapevine, they characterize the Apulian agricultural panorama. In September 2021, symptoms of a vascular wilt disease and, in severe cases, decline tree mortality were observed in Salento area (Apulia). Symptomatic Affected plants showed symptoms of leaf wilt and different stages of disease expression, which begins with leaf chlorosis on shoots, followed by wilting, extensive defoliation and twig dieback. On the main branches andlower part of the trunk and in some cases on lateral branches, bark cracks and cankers were observed and extended wood discoloration was detected in cross sections. In two orchards located in Salice Salentino (Site 1) and Squinzano (Site 2), where disease incidence exceeded 80%, 3-5 wood discs per tree were gathered from affected tissues from two (Site 1) and four (Site 2) trees. Isolations were performed on malt extract agar 2% amended with 0.5 g L-1 streptomycin sulfate. A Ceratocystis species was recovered from all samples trees with high frequencies (83.3%). Two-week-old colonies on potato dextrose agar showed black ascomata with 300-600 µm wide bases and 1100-2250 µm long necks. On the tips of the necks, ascospores (5-6x4-5 µm) exuded in creamy white sticky masses. Endoconidia (5-9x4.5-7 µm) were abundant, cylindrical, aseptate, and produced in chains. Two monoconidial representative strains CRSFA.Cer.033 (Site 1) and CRSFA.Cer.035 (Site 2) were deposited in the DISSPA Di.S.S.P.A. collection of the University of Bari. Species identification was done through sequence analyses of rDNA internal transcribed spacer region (ITS) using ITS5/ITS4 primers (White et al. 1990), elongation factor 1 alpha gene (TEF) using EF1/EF2 (O'Donnell et al. 1998) and RNA polymerase II gene (RPB2) gene using RPB2-5F/FRPB2fRPB2-7cR (Liu et al. 1999). Sequences were deposited in NCBI GenBank (accession numbers: OQ329983-OQ335969 (ITS), OQ352265-OQ352266 (TEF), OQ352268-OQ352267 (RPB2)). The sequences of both Apulian isolates were identical. BLAST searches revealed high similarity to the sequences of two isolates of Ceratocystis ficicola Kajitani and Masuya from Japan: ex-type CMW38543 and CMW38544, specifically 98.41% identity matching with KY685076 (ITS), 100% with KY685079 (TEF), and 99.87% with KY685083 and KY685082 (RPB2). Pathogenicity tests were conducted, on six common fig 'Dottato' seedlings by inoculating one year-old twigs with mycelium plugs (Bolboli et al. 2022). Control plants were inoculated with PDA plugs without mycelium. After one month, all inoculated twigs showed symptoms of wilt. Forty days post-inoculation, the bark of inoculated twigs was removed, and longitudinal and transverse sections revealed wood discolorations extending above and below the inoculation point. Ceratocystis ficicola was consistently reisolated from symptomatic tissues and identified as described above, thus fulfilling Koch's postulates. Neither symptoms nor positive isolations were observed in control seedlings. The pathogen was first described in 2011 in Japan (Kajitani and Masuya, 2011) where it currently affects all fig-producing areas. In 2018, it was reported for the first time in the EPPO region in Greece (Tsopelas et al., 2021), and to our knowledge, this is the first report on its detection in Italy. Since February 2022, C. ficicola has been included in the EPPO alert list because of its potential to cause tree mortality and the difficulty of its eradication. Regional national surveys are therefore urgently needed to determine its distribution in the fig growing areas of Italy and limit its spread. References Bolboli, Z., et al. 2022. Mycol. Prog. 21:89. doi: 10.1007/s11557-022-01834-9 Kajitani, Y., and Masuya, H., 2011. Mycoscience 52:349. doi: 10.1007/s10267-011-0116-5 Liu, Y. J., et al. 1999. Mol. Biol. and Evol. 16:1799. doi: 10.1093/oxfordjournals.molbev.a026092 O'Donnell, K., et al. 1998. Proc Natl Acad Sci USA 95:2044. doi: 10.1073/pnas.95.5.2044 Tsopelas, P., et al. 2021. Phytopathol. Mediterr. 60:337. doi: 10.36253/phyto-12794 White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego. doi: 10.1016/0307-4412(91)90165-5.

Molecular basis of cycloheximide resistance in the Ophiostomatales revealed.

Resistance to the antibiotic Cycloheximide has been reported for a number of fungal taxa. In particular, some yeasts are known to be highly resistant to this antibiotic. Early research showed that this resulted from a transition mutation in one of the 60S ribosomal protein genes. In addition to the yeasts, most genera and species in the Ophiostomatales are highly resistant to this antibiotic, which is widely used to selectively isolate these fungi. Whole-genome sequences are now available for numerous members of the Ophiostomatales providing an opportunity to determine whether the mechanism of resistance in these fungi is the same as that reported for yeast genera such as Kluyveromyces. We examined all the available genomes for the Ophiostomatales and discovered that a transition mutation in the gene coding for ribosomal protein eL42, which results in the substitution of the amino acid Proline to Glutamine, likely confers resistance to this antibiotic. This change across all genera in the Ophiostomatales suggests that the mutation arose early in the evolution of these fungi.

Ascarosides Promote the Prevalence of Ophiostomatoid Fungi and an Invasive Pathogenic Nematode, Bursaphelenchus xylophilus.

Understanding the coevolution of pathogens and their associated mycoflora depend upon a proper elucidation of the basis of their chemical communication. In the case of pine wilt disease, the mutual interactions between cerambycid beetles, invasive pathogenic nematodes, (Bursaphelenchus xylophilus) and their symbiotic ophiostomatoid fungi provide a unique opportunity to understand the role of small molecules in mediating their chemical communication. Nematodes produce ascarosides, a highly conserved family of small molecules that serve essential functions in nematode biology and ecology. Here we demonstrated that the associated fungi, one of the key natural food resources of pine wood nematodes, can detect and respond to these ascarosides. We found that ascarosides significantly increase the growth of L. pini-densiflorae and Sporothrix sp. 1, which are native fungal species in China that form a symbiotic relationship with pinewood nematodes. Hyphal mass of L. pini-densiflorae increased when treated with asc-C5 compared to other ophiostomatoid species. Field results demonstrated that in forests where higher numbers of PWN were isolated from beetle galleries, L. pini-densiflorae had been prevalent; the same results were confirmed in laboratory studies. Furthermore, when treated with asc-C5, L. pini-densiflorae responded by increasing its production of spores, which leads to a higher likelihood of dispersal by insect vectors, hence explaining the dominance of L. pini-densiflorae over S. sp. 1 in the Tianwang and Nanlu Mountains within the Northern Forestry Centre of China. These findings provide an emphatic representation of coevolution of pine wood nematode and its associated fungi. Our results lay a broader foundation for a better understanding of inter-kingdom mutualisms and the chemical signals that mediate their establishment.

Two new Sporothrix species from Protea flower heads in South African Grassland and Savanna.

The inflorescences and infructescences of African Protea trees provide habitat for a large diversity of Sporothrix species. Here we describe two additional members, Sporothrix nsini sp. nov. and Sporothrix smangaliso sp. nov., that are associated with the infructescences of various Protea species from grasslands and savannas in the KwaZulu-Natal, North-West, Gauteng and Mpumalanga provinces of South Africa. Their description raises the number of described Protea-associated Sporothrix species to twelve. S. smangaliso sp. nov. is distantly related to other Protea-associated species and, in phylogenies using multiple markers (ITS, beta-tubulin and calmodulin), groups with taxa such as Sporothrix bragantina from Brazil and Sporothrix curviconia from the Ivory Coast. S. nsini sp. nov. resolved as sister to a clade containing four other Protea-associated species within the Sporothrix stenoceras complex. S. nsini sp. nov. was collected from within the same infructescences of Protea caffra that also contained the closely related S. africana and S. protearum. This highlights the need to study and understand the factors that influence host selection and speciation of Sporothrix in this atypical niche.

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.

Defence syndromes in lodgepole - whitebark pine ecosystems relate to degree of historical exposure to mountain pine beetles.

Warming climate is allowing tree-killing bark beetles to expand their ranges and access naïve and semi-naïve conifers. Conifers respond to attack using complex mixtures of chemical defences that can impede beetle success, but beetles exploit some compounds for host location and communication. Outcomes of changing relationships will depend on concentrations and compositions of multiple host compounds, which are largely unknown. We analysed constitutive and induced chemistries of Dendroctonus ponderosae's primary historical host, Pinus contorta, and Pinus albicaulis, a high-elevation species whose encounters with this beetle are transitioning from intermittent to continuous. We quantified multiple classes of terpenes, phenolics, carbohydrates and minerals. Pinus contorta had higher constitutive allocation to, and generally stronger inducibility of, compounds that resist these beetle-fungal complexes. Pinus albicaulis contained higher proportions of specific monoterpenes that enhance pheromone communication, and lower induction of pheromone inhibitors. Induced P. contorta increased insecticidal and fungicidal compounds simultaneously, whereas P. albicaulis responses against these agents were inverse. Induced terpene accumulation was accompanied by decreased non-structural carbohydrates, primarily sugars, in P. contorta, but not P. albicaulis, which contained primarily starches. These results show some host species with continuous exposure to bark beetles have more thoroughly integrated defence syndromes than less-continuously exposed host species.

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.

The Ophiostoma clavatum species complex: a newly defined group in the Ophiostomatales including three novel taxa.

Two species of blue-stain fungi with similar morphologies, Ophiostoma brunneo-ciliatum and Ophiostoma clavatum, are associates of bark beetles infesting Pinus spp. in Europe. This has raised questions whether they represent distinct taxa. Absence of herbarium specimens and contaminated or mistakenly identified cultures of O. brunneo-ciliatum and O. clavatum have accentuated the uncertainty regarding their correct identification. The aim of this study was to reconsider the identity of European isolates reported as O. brunneo-ciliatum and O. clavatum by applying DNA-based identification methods, and to provide appropriate type specimens for them. Phylogenetic analyses of the ITS, ßT, TEF-1α and CAL gene sequences revealed that the investigated isolates represent a complex of seven cryptic species. The study confirmed that ITS data is insufficient to delineate species in some Ophiostoma species clusters. Lectotypes and epitypes were designated for O. clavatum and O. brunneo-ciliatum, and three new species, Ophiostoma brunneolum, Ophiostoma macroclavatum and Ophiostoma pseudocatenulatum, are described in the newly defined O. clavatum-complex. The other two species included in the complex are Ophiostoma ainoae and Ophiostoma tapionis. The results suggest co-evolution of these fungi in association with specific bark beetles. The results also confirm the identity of the fungus associated with the pine bark beetle Ips acuminatus as O. clavatum, while O. brunneo-ciliatum appears to be mainly associated with another pine bark beetle, Ips sexdentatus.

High-resolution melting curve analysis: A detection assay for Ceratocystis eucalypticola and C. manginecans in infected Eucalyptus.

Eucalyptus spp. in plantations are negatively affected by canker and wilt diseases caused by several species of Ceratocystis, particularly those in the Latin American Clade (LAC). Ceratocystis eucalypticola and Ceratocystis manginecans are of particular concern where disease epidemics are reported globally, with recent outbreaks emerging in South African and Indonesian Eucalyptus plantations. Consequently, a rapid screening protocol is required for these pathogens. In this study, a high-resolution melting curve analysis (HRMA) was developed to detect C. eucalypticola and C. manginecans that bypasses time-consuming isolation and post-PCR procedures. Primers targeting a 172 bp region of the cerato-platanin (CP) gene were designed. Using these primers, the accuracy of HRMA to detect and distinguish between these two LAC species was assessed using pure fungal DNA, and DNA extracted directly from Eucalyptus samples naturally infected with C. eucalypticola. The assay accurately detected the presence of C. eucalypticola and C. manginecans and quantifies their DNA, both from cultures, and directly from wood samples. HRMA further differentiated these two species from all other tested LAC individuals. This assay was also able to detect the presence of all the tested LAC species and distinguish seven of these, including C. fimbriata, to species level. Ceratocystis polyconidia was the only non-LAC off-target species detected. Based on these results, the developed assay can be used to rapidly identify C. eucalypticola and C. manginecans directly from infected plant material or fungal cultures, with the potential to also screen for several other LAC species.

Pathogenicity and induced resistance in Larix kaempferi and Larix olgensis inoculated with Endoconidiophora fujiensis.

With climate warming and economic globalization, insect-microbe assemblages are becoming increasingly responsible for various devastating forest diseases worldwide. Japanese larch (Larix kaempferi) is extensively cultivated in China because of its high survival rate, rapid maturation and robust mechanical properties. Endoconidiophora fujiensis, an ophiostomatoid fungus associated with Ips subelongatus, has been identified as a lethal pathogen of L. kaempferi in Japan. However, there is a dearth of research on the pathogenicity of E. fujiensis in larches in China. Therefore, we investigated the pathogenicity of E. fujiensis in introduced L. kaempferi and indigenous larch (Larix olgensis) trees and compared the induced resistance responses to the pathogen in both tree species in terms of physiology and gene expression. Five-year-old saplings and 25-year-old adult trees of L. olgensis and L. kaempferi were inoculated in parallel during the same growing season. Endoconidiophora fujiensis exhibited high pathogenicity in both larch species, but particularly in L. kaempferi compared with L. olgensis adult trees; adult L. olgensis was more resistant to E. fujiensis than adult L. kaempferi, which was reflected in higher accumulation of defensive monoterpenes, such as myrcene, 3-carene and limonene and the earlier induction of defense genes catalase (CAT) and pathogenesis-related protein 1 (PR1). This study contributes to our understanding of the interactions between bark beetle-associated ophiostomatoid fungi and host larches, from phenotypic responses to alterations in secondary metabolites via defense- and metabolism-related gene activation, providing a valuable foundation for the management of larch diseases and pests in the future.

Effect of Huntiella decorticans and drought on Nothofagus dombeyi seedlings.

In the temperate forests of Patagonia, Argentina, Nothofagus dombeyi, commonly known as Coihue, has shown sensitivity to intense drought events, leading to mortality. Studies have been conducted on Coihue decline and mortality using a variety of approaches, including the role of extreme heat waves and drought, pests and pathogens, particularly the fungus Huntiella decorticans. This work aimed to evaluate survival, vitality, necrosis extension and growth response of inoculated and non-inoculated Coihue seedlings from different provenances exposed to different soil moisture levels. To achieve this goal, 96 Coihue seedlings from 2 different provenances were assigned to 8 different experimental treatments. Treatments were composed of the presence or absence of H. decorticans and different soil moisture content conditions, dry, wet and the exposure to dry condition at different times of the experiment. Both dry conditions and H. decorticans had negative effects on the survival and growth rate of Coihue. The combination of both factors resulted in 100 % mortality, regardless of the plants' geographical provenances. Mortality began to be observed 3 months after pathogen inoculation, during the warmest month. Necrosis extension produced by the pathogen was similar for most of the inoculated treatments. The treatment under wet condition during the experiment but subjected to dry condition in the previous growing season presented lower necrosis extension (8.4 ±â€…3.2 %), than the other treatments. The radial increase was also affected by the treatments and geographical provenance, being those plants exposed to dry conditions which grew less (0.19 ±â€…0.21 mm). This study enhances our understanding of the plant-pathogen interaction. According to our results, Coihue may become more susceptible to mortality, when H. decorticans and water deficit conditions act synergistically.

Early colonization of Protea flowers enable dominance of competitively weak saprobic fungi in seed cones, benefitting their hosts.

Sporothrix and Knoxdaviesia fungi use pollinators to colonize Protea flowers at anthesis. These saprobes remain dominant in the nutrient-rich, fire-retardant Protea seed-cones (infructescences) for at least a year after flowering. We tested the hypothesis that they competitively exclude potentially detrimental fungi from infructescences during this time. We compared seed set and longevity of infructescences containing Sporothrix and Knoxdaviesia vs. those that contain 'contaminant' saprobes. Hereafter we evaluated their competitive abilities against the 'contaminant' saprobes. Infructescences devoid of Sporothrix and Knoxdaviesia were dominated by Penicillium cf. toxicarium, Cladosporium cf. cladosporoides and Fusarium cf. anthophilum. Sporothrix and Knoxdaviesia presence did not affect seed viability, but infructescences persisted longer than those colonised by 'contaminant' fungi. The 'contaminant' species were stronger competitors than Sporothrix and Knoxdaviesia. However, Sporothrix and Knoxdaviesia could defend captured space well against 'contaminant' species. This effect was enhanced when fungal taxa grew on media prepared from their usual Protea host species, clarifying their dominance and host consistency observed in the field. Sporothrix and Knoxdaviesia from Protea are therefore weak competitors against common saprobes, especially when growing on alternative hosts, and need to colonise flowers very early (before colonization by other fungi) to dominate in this environment. They may delay seed release from infructescences longer than if these are colonised by other saprobes, increasing chances of seed release to occur after fire, when conditions are more favourable for Protea recruitment.

Pathophysiology and transcriptomic analysis of Picea koraiensis inoculated by bark beetle-vectored fungus Ophiostoma bicolor.

Ophiostomatoid fungi exhibit a complex relationship with bark beetles; exhausting of host tree defenses is traditionally regarded as one of the key benefits provided to beetle vectors. Ophiostoma bicolor is one of the dominant species of the mycobiota associated with Ips genus bark beetles which infect the spruce trees across the Eurasian continent. Host spruce trees resist fungal invasion through structural and inducible defenses, but the underlying mechanisms at the molecular level, particularly with respect to the interaction between bark beetle-associated fungi and host trees, remain unclear. The aim of this study was to observe the pathological physiology and molecular changes in Picea koraiensis seedlings after artificial inoculation with O. bicolor strains (TS, BH, QH, MX, and LWQ). This study showed that O. bicolor was a weakly virulent pathogen of spruce, and that the virulent of the five O. bicolor strains showed differentiation. All O. bicolor strains could induce monoterpenoid release. A positive correlation between fungal virulence and release of monoterpenoids was observed. Furthermore, the release rate of monoterpenoids peaked at 4 days post-inoculation (dpi) and then decreased from 4 to 90 dpi. Transcriptomic analysis at 4 dpi showed that many plant-pathogen interaction processes and mitogen-activated protein kinase (MAPK) metabolic processes were activated. The expression of monoterpenoid precursor synthesis genes and diterpenoid synthesis genes was upregulated, indicating that gene expression regulated the release rate of monoterpenoids at 4 dpi. The enriched pathways may reveal the immune response mechanism of spruce to ophiostomatoid fungi. The dominant O. bicolor possibly induces the host defense rather than defense depletion, which is likely the pattern conducted by the pioneers of beetle-associated mycobiota, such as Endoconidiophora spp.. Overall, these results facilitate a better understanding of the interaction mechanism between the dominant association of beetles and the host at the molecular level.

Interactions among Norway spruce, the bark beetle Ips typographus and its fungal symbionts in times of drought.

Resilience and functionality of European Norway spruce forests are increasingly threatened by mass outbreaks of the bark beetle Ips typographus promoted by heat, wind throw and drought. Here, we review current knowledge on Norway spruce and I. typographus interactions from the perspective of drought-stressed trees, host selection, colonisation behaviour of beetles, with multi-level effects of symbiotic ophiostomatoid fungi. By including chemo-ecological, molecular and behavioural perspectives, we provide a comprehensive picture on this complex, multitrophic system in the light of climate change. Trees invest carbon into specialised metabolism to produce defence compounds against biotic invaders; processes that are strongly affected by physiological stress such as drought. Spruce bark contains numerous terpenoid and phenolic substances, which are important for bark beetle aggregation and attack success. Abiotic stressors such as increased temperatures and drought affect composition, amounts and emission rates of volatile compounds. Thus, drought events may influence olfactory responses of I. typographus, and further the pheromone communication enabling mass attack. In addition, I. typographus is associated with numerous ophiostomatoid fungal symbionts with multiple effects on beetle life history. Symbiotic fungi degrade spruce toxins, help to exhaust tree defences, produce beetle semiochemicals, and possibly provide nutrition. As the various fungal associates have different temperature optima, they can influence the performance of I. typographus differently under changing environmental conditions. Finally, we discuss why effects of drought on tree-killing by bark beetles are still poorly understood and provide an outlook on future research on this eruptive species using both, field and laboratory experiments.

Fungal Communities Vectored by Ips sexdentatus in Declining Pinus sylvestris in Ukraine: Focus on Occurrence and Pathogenicity of Ophiostomatoid Species.

Drought-induced stress and attacks by bark beetle Ips sexdentatus currently result in a massive dieback of Pinus sylvestris in eastern Ukraine. Limited and fragmented knowledge is available on fungi vectored by the beetle and their roles in tree dieback. The aim was to investigate the fungal community vectored by I. sexdentatus and to test the pathogenicity of potentially aggressive species to P. sylvestris. Analysis of the fungal community was accomplished by combining different methods using insect, plant, and fungal material. The material consisted of 576 beetles and 96 infested wood samples collected from six sample plots within a 300 km radius in eastern Ukraine and subjected to fungal isolations and (beetles only) direct sequencing of ITS rDNA. Pathogenicity tests were undertaken by artificially inoculating three-to-four-year-old pine saplings with fungi. For the vector test, pine logs were exposed to pre-inoculated beetles. In all, 56 fungal taxa were detected, 8 exclusively by isolation, and 13 exclusively by direct sequencing. Those included nine ophiostomatoids, five of which are newly reported as I. sexdentatus associates. Two ophiostomatoid fungi, which exhibited the highest pathogenicity, causing 100% dieback and mortality, represented genera Graphium and Leptographium. Exposure of logs to beetles resulted in ophiostomatoid infections. In conclusion, the study revealed numerous I. sexdentatus-vectored fungi, several of which include aggressive tree pathogens.

Ceratocystis quercicola sp. nov. from Quercus variabilis in Korea.

During a survey of putative fungal pathogens infecting oak trees in the Gangwon Province of the Republic of Korea, a fungus resembling a Ceratocystis sp. was repeatedly isolated from natural wounds on Quercus variabilis. Morphological comparisons and DNA sequence comparisons based on partial ß-tubulin and TEF-1α gene regions showed that the fungus resided in a distinct lineage. This novel Ceratocystis species is described here as C. quercicola sp. nov. This is the first novel species of Ceratocystis to be reported from Korea. A pathogenicity test showed that it can cause lesions on inoculated trees but that it had a very low level of aggressiveness. The discovery of this fungus suggests that additional taxa residing in Ceratocystis are likely to be discovered in Korea in the future.

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.

Nitrogen and Ergosterol Concentrations Varied in Live Jack Pine Phloem Following Inoculations With Fungal Associates of Mountain Pine Beetle.

Bark beetles form symbiotic associations with multiple species of fungi that supplement their metabolic needs. However, the relative contributions of each symbiont to the nutrition of bark beetles have been largely unexplored. Thus, we evaluated the ability of three fungal symbionts of mountain pine beetle to concentrate nitrogen and produce ergosterol while infecting phloem of a novel host jack pine. Ergosterol was used as proxy to determine the fungal biomass (hyphal density) in the current study. We inoculated 80 trees in two forest stands with one of the three fungal species or a non-fungal (control) agar. Six weeks later, we collected phloem from the necrotic lesions induced by the fungi, uninfected tissues adjacent to lesions, and non-inoculated control trees. We found that nutritional contributions varied with fungal species. Nitrogen in lesions was higher in trees inoculated with Ophiostoma montium or control trees, relative to Grosmannia clavigera or Leptographium longiclavatum. Furthermore, concentrations of ergosterol were higher in O. montium lesions compared to other tissues or treatments. These results suggest that O. montium differs from G. clavigera and L. longiclavatum in terms of acquiring nitrogen from host tissues and producing ergosterol.

Two new species of Ophiostomatales (Sordariomycetes) associated with the bark beetle Dryocoetes alni from Poland.

Bark beetles belonging to the genus Dryocoetes (Coleoptera, Curculionidae, Scolytinae) are known vectors of fungi, such as the pathogenic species Grosmannia dryocoetidis involved in alpine fir (Abies lasiocarpa) mortality. Associations between hardwood-infesting Dryocoetes species and fungi in Europe have received very little research attention. Ectosymbiotic fungi residing in Ceratocystiopsis and Leptographium (Ophiostomatales, Sordariomycetes, Ascomycota) were commonly detected in previous surveys of the Dryocoetes alni-associated mycobiome in Poland. The aim of this study was to accurately identify these isolates and to provide descriptions of the new species. The identification was conducted based on morphology and DNA sequence data for six loci (ITS1-5.8S, ITS2-28S, ACT, CAL, TUB2, and TEF1-α). This revealed two new species, described here as Ceratocystiopsis synnemata sp. nov. and Leptographium alneum sp. nov. The host trees for the new species included Alnus incana and Populus tremula. Ceratocystiopsis synnemata can be distinguished from its closely related species, C. pallidobrunnea, based on conidia morphology and conidiophores that aggregate in loosely arranged synnemata. Leptographium alneum is closely related to Grosmannia crassivaginata and differs from this species in having a larger ascomatal neck, and the presence of larger club-shaped cells.

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.