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The pine wood nematode (PWN), Bursaphelenchus xylophilus, is a destructive invasive species that exerts devastating effects on most native pines in invaded regions, while many of the non-native pines have resistance to PWN. Recently, increasingly more research is focused on how microbial communities can improve host resistance against pathogens. However, the relationship between the microbial community structures and varying levels of pathogen resistance observed in different pine tree species remains unclear. Here, the bacterial and fungal communities of introduced resistant pines Pinus elliottii, P. caribaea, and P. taeda and native susceptible pines healthy and wilted P. massoniana infected by PWN were analyzed. The results showed that 6057 bacterial and 3931 fungal OTUs were annotated. The pine samples shared 944 bacterial OTUs primarily in the phyla Proteobacteria, Acidobacteria, Firmicutes, Bacteroidetes, and Chloroflexi and 111 fungal OTUs primarily in phyla Ascomycota and Basidiomycota, though different pines had unique OTUs. There were significant differences in microbial community diversity between different pines, especially between the bacterial communities of resistant and susceptible pines, and fungal communities between healthy pines (resistant pines included) and the wilted P. massoniana. Resistant pines had a greater abundance of bacteria in the genera Acidothermus (class unidentified_Actinobacteria) and Prevotellaceae (class Alphaproteobacteria), but a lower abundance of Erwinia (class Gammaproteobacteria). Healthy pines had a higher fungal abundance of Cladosporium (class Dothideomycetes) and class Eurotiomycetes, but a lower abundance of Graphilbum, Sporothrix, Geosmithia (class Sordariomycetes), and Cryptoporus (classes Agaricomycetes and Saccharomycetes). These differences in microbial abundance between resistant and healthy pines might be associated with pathogen resistance of the pines, and the results of this study contribute to the studies exploring microbial-based control of PWN.

The kingdom Fungi encompasses an estimated 2.2 to 6.2 million species that occupy diverse environments, including aquatic, extremely dry, and hot or frosty habitats all over the world. To cope with adverse environmental conditions, fungi have developed numerous adaptations and life strategies, including symbiosis with other organisms, ranging from close, reciprocally beneficial (mutualistic) associations to severe pathogenic infestations. These interactions have an enormous impact on ecosystem functioning, with implications for agriculture and human health. For this reason, understanding the mechanisms enabling the successful development of fungal interactions is necessary for their efficient management. Recent advances in different 'omics' approaches have enabled us to compare species responses to the environment in a more complex way than before and to gain deeper insights into the adaptive mechanisms underlying specific life strategies. My thesis is divided into four main sections. In the first section, I sum up findings about adaptations of fungal symbionts of plants and animals. Then, I introduce two fungal genera, Geosmithia and Pseudogymnoascus, to which I applied comparative methods for tracking adaptive traits. The ecological diversity of the genus Geosmithia allows to trace adaptive...

Fungi of the genus Geosmithia are frequently associated with bark beetles that feed on phloem on various woody hosts. Most studies on Geosmithia were carried out in North and South America and Europe, with only two species were reported from Taiwan, China. The aim of this study was to investigate the diversity of Geosmithia species in southern China. Field surveys in Guangdong, Guangxi, Hunan, Jiangxi and Shanghai yielded a total of 76 fungal isolates from six beetle species. Isolates were grouped based on morphology. The ITS, β-tubulin and elongation factor 1-α gene regions of representatives of each group were sequenced. Phylogenetic trees were constructed based on those sequences. In total five species were identified, with one previously described species G. putterillii and four new species which were described as G. jiulianshanensis, G. jiangxiensis, G. formosana, and G. pulverea (Geosmithia sp. 3 and Geosmithia sp. 23) sp. nov., in this paper.

A phenotype is the expression of interactions between species genotype and environment. We quantified the contributions of ecological and phylogenetic associations to phenotypic variation in Geosmithia fungi. Geosmithia are symbiotic beetle-associated saprotrophs with a range of life histories and host specificities, including obligate nutritional beetle mutualists (ambrosia fungi) and phytopathogens. We hypothesized that: (1) species phenotypes are better explained by their ecology than by their phylogenetic relationships; (2) niche specialization was accompanied by enzymatic capability losses; and (3) ambrosia Geosmithia species have higher nutritional quality and antibiotic capabilities than species with facultative symbioses. Our results confirmed that long-term co-evolved specialists have reduced metabolic breadth in comparison to generalists. Phytopathogenic G. morbida produces unique enzyme suites with affinity to ligno-cellulose. Mycelia of ambrosia fungi contain large amounts of oleic fatty acid with nutritive and possibly allelopathic function. Overall, our results indicate that Geosmithia ecology have greater effect on species phenotype than their phylogenetic relationships.

The fungal genus Geosmithia is best known due to one species, G. morbida, which is vectored by the walnut twig beetle (WTB) and contributes to Thousand Cankers Disease (TCD) on walnut and wingnut trees. However, the genus is globally very diverse and abundant, and dominates a ubiquitous but understudied niche – the twig-infesting, phloem-feeding bark beetle mycobiome. The Geosmithia community in North America is only now beginning to be described. Very limited information is available for the South East, despite the region's potential to be a Geosmithia diversity hotspot. To survey the Geosmithia community in the subtropical USA, to assess their beetle and tree associations, and to test for the presence of G. morbida, we systematically deployed branch sections of nine tree species, including three Juglandaceae, in North Florida. We recovered 55 Geosmithia isolates from 195 beetle specimens from 45 exposed branch units. Neither G. morbida nor its beetle vector were detected. We identified 14 Geosmithia species; those in the G. pallida species complex were the most prevalent. Four undescribed phylogenetic species were recovered, indicating that the Geosmithia diversity in North America remains under-documented. Analysis of the association of Geosmithia with beetles and trees suggested that most species are generalists, five display preference for certain tree species, and none is specific to any beetle species.

Orientador: Marcos José Salvador Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: As plantas e os micro-organismos estão entre os recursos naturais que apresentam considerável importância para a busca de novos metabólitos bioativos. Plantas da família Gesneriaceae são uma interessante fonte de metabólitos secundários com diversidade em termos químicos e relatos de algumas moléculas com atividades antioxidante, anti-inflamatória, analgésica e anticâncer. No entanto, poucas espécies de Sinningia Nees (Gesneriaceae) foram quimicamente estudadas, não sendo conhecida a diversidade de endofíticos a elas associada e o impacto desses micro-organismos na biossíntese de substâncias bioativas. Portanto, é um universo químico e biológico inexplorado que precisa ser estudado, dado o potencial dessas plantas e seus simbiontes como fonte de moléculas com aplicações terapêuticas e tecnológicas. Neste contexto, este estudo objetivou avaliar as condições de cultivo, a bioatividade e o potencial metabólico de Sinningia speciosa (Lodd.) Hiern (Gesneriaceae) "grupo Fyfiana" (planta in natura, plântula in vitro e calos) e de Geosmithia sp. SSP7, endófito oriundo de suas folhas. Para tanto, procedeu-se o estabelecimento das condições de cultivo do material vegetal (planta in natura, plântulas in vitro e calos); o cultivo, a identificação morfológica e molecular de Geosmithia sp. SSP7 e a obtenção de extratos metanólicos da biomassa da planta e das culturas desse fungo, os quais foram submetidos à análises por UHPLC/ESI-MS (de alta e baixa resolução) e à avaliação das atividades antioxidante, antimicrobiana, anti-Leishmania e do efeito citotóxico. Plântulas in vitro foram obtidas tanto de sementes, quanto por via organogênese indireta em meio MS suplementado com fitorreguladores 1,0 mg/L BAP x 0,2 mg/L ANA, resultando em uma alta eficiência de regeneração após 4 semanas. Os calos foram obtidos a partir de um balanço relativamente baixo em concentração (0,05 mg/L) de BAP e de 2,4-D. Os resultados da caracterização morfológica e molecular sugerem que a cepa SSP7 pertence ao gênero Geosmithia. Em termos de produção de biomassa, o meio de cultura caldo MID foi o que melhor favoreceu o crescimento fúngico, enquanto a maior quantidade de extrato metanólico em massa (mg) foi obtida a partir do cultivo no meio CZP. Quando comparado o perfil cromatográfico do extrato metanólico da planta in natura, plântula in vitro, cultura de calos e de Geosmithia sp. SSP7, verificou-se a ocorrência de regiões com similaridade de picos cromatográficos e outras em que os picos diferiram bastante entre a amostra da planta e do fungo. Por UHPLC/ESI-MS/MS foram identificados e quantificados um glicosídeo fenólico, Calceolariosídeo B, e uma antraquinona, ¿ Pustulina 7-hidroxi-6-metoxi-tectoquinona, ambas substâncias detectadas na maioria dos extratos provenientes da planta e em co-cultura in vitro da plântula ou cultura de calos com Geosmithia sp. SSP7. Os resultados mostraram que a associação endófito-planta levou a aumento da concentração destas duas substâncias, sugerindo modulação positiva da associação simbiótica na biossíntese destas moléculas. Ainda, verificou-se que fatores como a luminosidade e balanço de fitorreguladores nas etapas de cultivo, bem como o estímulo biológico por parte de Geosmithia sp. SSP7, impactaram positivamente no desenvolvimento das culturas da planta in vitro, no teor de Calceolariosídeo B e de Pustulina e na capacidade antioxidante dos extratos estudados. Os extratos apresentaram uma capacidade antioxidante promissora, com valores ? 800 µmol TE/g. Por outro lado, não foi detectada atividade antimicrobiana e frente a promastigotas de Leishmania amazonensis (PH8) para os extratos analisados nas condições experimentais utilizadas e, também a maioria dos extratos não apresentou efeito citotóxico na viabilidade celular da linhagem proveniente de fibroblasto de embrião de camundongo 3T3 com valores de IC50 ? 250,0 µg/mL Abstract: Plants and microorganisms are among the natural resources presenting substantial importance in the search for new bioactive metabolites. Plants belonging to Gesneriaceae¿s family are an interesting source of secondary metabolites which possess a great chemical diversity and scientific reports of some bioactive molecules (antioxidant, anti-inflammatory, analgesic and anticancer activity). However, few species of Sinningia Ness (Gesneriaceae) were chemically studied, and the diversity of endophytes associated with them as well as the impact of these microorganisms on the biosynthesis of bioactive substances are unknown. Therefore, it is an unexplored chemical and biological universe that needs to be studied, given the potential of these plants and their symbionts as a source of molecules with therapeutic and technological applications. In this sense, this study aimed to evaluate the cultivation conditions, bioactivity and metabolic potential of Sinningia speciosa (Lodd.) Hiern (Gesneriaceae) "Fyfiana group", (in natura plant, in vitro seedling and callus culture) and of Geosmithia sp. SSP7, endophyte from its leaves. For this, we established the conditions for the cultivation of the plant material mentioned above, and morphological and molecular identification of the endophytic strain SSP7. Methanolic extracts of the plant biomass and the fungal cultures were obtained, which were submitted to UHPLC/ESI-MS analysis and the evaluation of antioxidant, antimicrobial, anti-Leishmania and cytotoxic activities. In vitro seedlings were obtained from both seeds and indirect organogenesis in MS medium supplemented with 1.0 mg/L BAP x 0.2 mg/L NAA phyto-regulators, resulting in a high regeneration efficiency after 4 weeks. Callus was obtained from a relatively low concentration balance (0.05 mg/L) of BAP and 2,4-D. The results of fungal morphological and molecular characterization suggest that the SSP7 strain belongs to the genus Geosmithia. In terms of biomass production, the MID broth culture medium was the one that most favored fungal growth, while the largest amount of mass methanolic extract (mg) was obtained from cultivation in CZP medium. When comparing the chromatographic profiles of the methanolic extract from in natura, in vitro seedling and callus culture with Geosmithia sp. SPP7, regions with similar chromatographic peaks and others in which peaks significantly differed between plant and fungus samples were found. Through UHPLC/ESI-MS/MS, a phenolic glycoside-Calceolarioside B and an anthraquinone-Pustuline (7-hydroxi-6-methoxy-tectoquinone) were identified and quantified. Both substances were detected in most extracts from plant and co-culture in vitro of seedling or callus culture with Geosmithia sp. SSP7. The results showed that the endophyte-plant association increased the concentration of these two substances, suggesting a positive modulation of the symbiotic association in the biosynthesis of these molecules. In addition, factors such as luminosity and phyto-regulator balance in the cultivation stages as well as biological stimulation by the Geosmithia sp. SSP7 had a positive impact on the development of the in vitro plant cultures, on Calceolarioside B and Pustuline contents, and in the antioxidant capacity of the extracts. The extracts showed a promising antioxidant capacity with values ? 800 µmol TE/g. On the other hand, antimicrobial activity and effect against Leishmania amazonensis (PH8) promastigotes for the extracts analyzed under the experimental conditions used were not detected, and most of the extracts also did not show a cytotoxic effect on cell viability of the lineage from 3T3 mouse embryo fibroblast with IC50 ? 250.0 µg/mL Mestrado Fármacos, Medicamentos e Insumos para Saúde Mestre em Ciências CAPES 88882.435462/2019-01

Geosmithia members are mitosporic filamentous fungi commonly recorded and isolated from bark beetles of the Scolytinae subfamily and their respective host&rsquo, s species. This genus includes 18 species formally described and 38 phylogenetic species recorded in several localities from Africa, Asia, Australia, Europe, and North and South America, where they exhibit frequent associations with phloeophagous and wood-boring bark beetles. Among phloephagous bark beetle species, specifically, in members of the genus Phloeosinus Chapuis, almost 10% of Geosmithia strains have been isolated. By its physiographic elements and high bark beetle and conifer species richness, Mexico is a potential region to host a high diversity of Geosmithia species and potential new species. In the present study, we systematically sampled and isolated, cultured, and molecularly identified members of the Geosmithia species associated with Phloeosinus spp. and their Juniperus spp. host trees at the north of Sierra Madre Oriental, at Nuevo Leon State, Mexico. Phylogenetic analyses based on 378 internal transcribed spacer region (ITS) sequences supported the presence of strains from Geosmithia langdonii-Geosmithia sp. 32 clade associated with Phloeosinus serratus vector and with Juniperus coahuilensis (JC) host, and the presence of strains from Geosmithia sp. 21-Geosmithia xerotolerans clade with Phloeosinusdeleoni and Juniperus flaccida (JF)in this geographical region. The genetic and morphological differences found in our strains with respect to those previously described in the species from both clades (Geosmithia langdonii-Geosmithia sp. 32 and Geosmithia sp. 21-G. xerotolerans) suggest that both Geosmithia lineages from Nuevo Leon correspond to two potential new species in the genus.

Geosmithia species (Hypocreales, Ascomycota) are associates of bark beetles and other arthropods. One species, Geosmithia morbida, is a virulent tree pathogen of Juglans nigra. To date, 10 Geosmithia spp. from conifer-infesting, and at least 23 species from hardwood associated bark beetles have been reported from Europe. The aim of this study was to survey Geosmithia spp. associated with 18 bark and ambrosia beetle species in hardwood ecosystems in Poland. In addition, we evaluated the pathogenicity of the six Geosmithia species by inoculating Acer, Fagus, Quercus, Tilia and Ulmus seedlings. Our surveys yielded a total of 1060 isolates from 2915 beetles and 1887 galleries. We identified isolates using morphology and ITS, β-tubulin and TEF1-α sequences. Altogether we identified 11 species including nine previously known and two new species described here as Geosmithia fagi sp. nov. and G. pazoutovae sp. nov. In addition, a sister species G. longistipitata sp. nov., associated with Picea trees, is described here. Bark beetles from hardwoods, with exeption of Dryocoetes alni, D. villosus, Scolytus ratzeburgi and ambrosia beetles, appear to be regular vectors of Geosmithia spp. Like in other parts of the world, most Geosmithia taxa exhibited a distinct level of vector/host specificity. None of Geosmithia isolates induced any disease symptoms under the conditions of our experiment. This study highlights the need for more intensive surveys across additional areas of Central and Northern Europe, insect vectors and host tree species in order to elucidate the Geosmithia species diversity in this region.

Geosmithia morbida is an emerging fungal pathogen which serves as a model for examining the evolutionary processes behind pathogenicity because it is one of two known pathogens within a genus of mostly saprophytic, beetle-associated, fungi. This pathogen causes thousand cankers disease in black walnut trees and is vectored into the host via the walnut twig beetle. Geosmithia morbida was first detected in western United States and currently threatens the timber industry concentrated in eastern United States. We sequenced the genomes of G. morbida in a previous study and two nonpathogenic Geosmithia species in this work and compared these species to other fungal pathogens and nonpathogens to identify genes under positive selection in G. morbida that may be associated with pathogenicity. Geosmithia morbida possesses one of the smallest genomes among the fungal species observed in this study, and one of the smallest fungal pathogen genomes to date. The enzymatic profile in this pathogen is very similar to its nonpathogenic relatives. Our findings indicate that genome reduction or retention of a smaller genome may be an important adaptative force during the evolution of a specialized lifestyle in fungal species that occupy a specificniche, such as beetle vectored tree pathogens. We also present potential genes under selection in G. morbida that could be important for adaptation to a pathogenic lifestyle.

Geosmithia are fungi associated with wood-boring bark beetles. Most Geosmithia species do no harm to host trees, but the canker-causing Geosmithia morbida and its beetle vector, the walnut twig beetle, cause the disease complex known as thousand cankers disease on walnut trees. Continuous surveys in Florida have found neither Geosmithia morbida nor its beetle vector in the state, but many native Geosmithia species have been recovered. These native species look similar to the pathogenic fungus but are harmless to their plant hosts. This 4-page fact sheet written by Yin-Tse Huang and Jiri Hulcr and published by the UF/IFAS School of Forest Resources and Conservation provides basic guidelines to sample Geosmithia species in the field and information for distinguishing the plant pathogenic Geosmithia morbida from other Geosmithia species. http://edis.ifas.ufl.edu/fr412

Geosmithia species (Ascomycota: Hypocreales) are common ectosymbionts of scolytine bark and ambrosia beetles that feed on coniferous and deciduous trees in different forest ecosystems. Geosmithia morbida is the canker pathogen that causes extensive mortality of Juglans nigra. Because little is known regarding the Geosmithia species on European silver fir (Abies alba), we have investigated the diversity and abundance of these fungi associated with insects infesting European silver fir in Poland. Samples associated with eight beetle species were collected from three fir forests. Fungi were isolated from beetles and galleries. Isolates were identified based on morphological characteristics, DNA sequence comparison for three gene regions (ITS, ßT, TEF1-α), and phylogenetic analyses. Geosmithia was detected in 33% of the total 531 beetle samples obtained from A. alba. Two undescribed species of Geosmithia were distinguished, Geosmithia sp. 9 and Geosmithia sp. 16. Associations of Pityokteines spp. with Geosmithia fungi were recorded for the first time. Pityokteines vorontzowi and Pityophthorus pityographus appear to be regular vectors for Geosmithia sp. 9 and Geosmithia sp. 16, respectively. Pityokteines curvidens and Cryphalus piceae were associated with Geosmitha sp. 9 at lower frequencies.

The mortality of a young elm Ulmus minor in 2014 in Geneva prompted a search for the microorganisms potentially involved. Symptoms included foliar chlorosis and wilting followed by defoliation of branches. Wood symptoms included a brown streaking of sap wood and brown stains in trunk and branches. The comparison of the resulting ITS rDNA sequences to the NCBI Nucleotide database allowed to identify 10 different organisms. The genus Geosmithia represented 48% of the isolates belonging to three species: Geosmithia langdonii (7 isolates) and 2 unknown morphologically and genetically different Geosmithia sp. 1 and sp. 2 (4 isolates). Geosmithia species are very little known ascomycetes, which have been recently shown to be opportunistic pathogens on broadleaved trees and conifers, living as saprobes in galleries of many bark beetle species. In the case described here, Geosmithia langdonii, and the unknown Geosmithia species were found in symptomatic wood while bark beetle galleries were found in close regions of the symptomatic wood. Geosmithia langdonii was the major fungus retrieved from the symptomatic wood and could have contributed, along with other identified fungal species, to a pathogenic complex producing symptoms similar to the ones of the Dutch Elm Disease and led to the dieback of this elm tree. Geosmithia langdonii and 2 yet unknown Geosmithia species (sp. 1 and sp. 2), different from any other reported Geosmithia species are reported from an elm tree in Switzerland for the first time.

Species of Geosmithia are cosmopolitan but understudied fungi, and most are associated with phloem-feeding bark beetles on various woody hosts. We surveyed 207 bark and ambrosia beetles from 37 species in the eastern USA for associated fungi. The community is dominated by species in the G. pallida species complex (GPSC) and included several Geosmithia isolates that appear to be new to science. The new Geosmithia isolates exhibited the characteristic brownish-colored colonies typical for the G. pallida species complex and were phylogenetically resolved as two genealogically exclusive lineages based on a concatenated multilocus data set based on the internal transcribed spacers (ITS) of the nuc rDNA (ITS1-5.8S-ITS2 = ITS), and the translation elongation factor 1-α (TEF1-α), β-tubulin (TUB2), and RNA polymerase II second largest subunit (RPB2) genes. Two new Geosmithia species, G. brunnea and G. proliferans, are proposed, and their morphological traits and phylogenetic placements are presented.

In Europe as in North America, elms are devastated by Dutch elm disease (DED), caused by the alien ascomycete Ophiostoma novo-ulmi. Pathogen dispersal and transmission are ensured by local species of bark beetles, which established a novel association with the fungus. Elm bark beetles also transport the Geosmithia fungi genus that is found in scolytids’ galleries colonized by O. novo-ulmi. Widespread horizontal gene transfer between O. novo-ulmi and Geosmithia was recently observed. In order to define the relation between these two fungi in the DED pathosystem, O. novo-ulmi and Geosmithia species from elm, including a GFP-tagged strain, were grown in dual culture and mycelial interactions were observed by light and fluorescence microscopy. Growth and sporulation of O. novo-ulmi in the absence or presence of Geosmithia were compared. The impact of Geosmithia on DED severity was tested in vivo by co-inoculating Geosmithia and O. novo-ulmi in elms. A close and stable relation was observed between the two fungi, which may be classified as mycoparasitism by Geosmithia on O. novo-ulmi. These results prove the existence of a new component in the complex of organisms involved in DED, which might be capable of reducing the disease impact. Electronic supplementary material The online version of this article (10.1007/s00248-017-1062-3) contains supplementary material, which is available to authorized users.

Fungi associated with the bark beetles Orthotomicus erosus, Tomicus destruens and Pityogenes calcaratus were sampled in various pine forests throughout Israel. Three ophiostomatoid fungi, Ophiostoma ips, Graphilbum rectangulosporium and Leptographium wingfieldii, and a fourth non-ophiostomatoid fungus, Geosmithia sp. 24, were identified by using morphological characteristics and molecular genetic analyses. O. ips, the most common fungus, was mainly isolated from O. erosus. The least common fungus, G. rectangulosporium, was frequently isolated from all three studied scolytids, while L. wingfieldii was almost exclusively associated with T. destruens. The fourth fungus, Geosmithia sp. 24, was isolated from both O. erosus and P. calcaratus. This is the first time that an association between O. erosus and a Geosmithia sp. has been reported. Our findings also suggest that Geosmithia sp. 24 can be separated into two distinct sub-groups by molecular analyses. Pathogenicity was demonstrated only for L. wingfieldii, both on Aleppo and brutia pine, exclusively under controlled conditions (25 ± 5 °C) but not at elevated temperatures.

Geosmithia belongs among fungi living in symbiosis with phloem-feeding bark beetles. Several species have altered their ecology to that of obligatory symbiosis with ambrosia beetles, which has led to a shift in their phenotype and caused formation of large spherical conidia. In this study, we pose the following questions; (1) Is the conidial DNA content of Geosmithia correlated with conidial volume?; (2) Is the DNA content of Geosmithia related to the degree of mutual dependence between Geosmithia and their vector? There was a positive and strong correlation between conidial DNA content and conidial volume in Geosmithia. Also species more narrowly associated with the vector tend to have a larger conidial DNA content and volume than less narrowly associated species. Ambrosia fungi achieved the biggest conidial DNA content and volume compared to other species. We suppose that polyploidisation occurred during the evolution of ambrosia species in the genus Geosmithia. a 2014 Elsevier Ltd and The British Mycological Society.

Numerous dead and dying individuals of the Western Cape endemic tree Virgilia oroboides (Fabaceae) were recently observed within a South African national botanical garden. Root-rot fungi and fungi symbiotic with bark beetles (Curculionidae; Scolytinae) from diseased trees were assessed for their respective roles in V. oroboides mortality. Disease progression was also monitored over 1 year. Fungi were isolated from surface sterilized bark and root samples from diseased trees and provisionally identified using data from the internal transcribed spacer regions (ITS1, ITS2), including the 5·8S rRNA gene (ITS). Pathogenicity of selected fungi towards V. oroboides was tested under field conditions. The pathogenicity of various bark beetle-associated Geosmithia (Hypocreales: Hypocreomycetidae) spp. from V. oroboides were similarly assessed. The only fungus consistently isolated from lesions on the roots and bark of declining V. oroboides, and never from healthy individuals, represented an undescribed Diaporthe (Diaporthales, Diaporthaceae) species that was characterized using molecular (using data from the ITS marker and part of the β-tubulin gene, TUB), cultural and morphological characters. It is an aggressive pathogen of V. oroboides, newly described here as Diaporthe virgiliae sp. nov. Trees of all ages are susceptible to this pathogen with subsequent bark beetle attack of mature trees only. All Geosmithia spp. from beetles and/or infected trees were nonpathogenic towards V. oroboides. Diaporthe virgiliae caused a severe decline in the health of the monitored V. oroboides population over a period of only 1 year and should be considered as a significant threat to these trees.

Fungi in the genus Geosmithia (Ascomycota: Hypocreales) are frequent associates of bark beetles and woodborers that colonize hardwood and coniferous trees. One species, Geosmithia morbida, is an economically damaging invasive species. The authors surveyed the Geosmithia species of California and Colorado, USA, to (i) provide baseline data on taxonomy of Geosmithia and beetle vector specificity across the western USA; (ii) investigate the subcortical beetle fauna for alternative vectors of the invasive G. morbida; and (iii) interpret the community composition of this region within the emerging global biogeography of Geosmithia. Geosmithia was detected in 87% of 126 beetle samples obtained from 39 plant species. Twenty-nine species of Geosmithia were distinguished, of which 13 may be new species. Bark beetles from hardwoods, Cupressus, and Sequoia appear to be regular vectors, with Geosmithia present in all beetle gallery systems examined. Other subcortical insects appear to vector Geosmithia at lower frequencies. Overall, most Geosmithia have a distinct level of vector specificity (mostly high, sometimes low) enabling their separation to generalists and specialists. Plant pathogenic Geosmithia morbida was not found in association with any other beetle besides Pityophthorus juglandis. However, four additional Geosmithia species were found in P. juglandis galleries. When integrated with recent data from other continents, a global pattern of Geosmithia distribution across continents, latitudes, and vectors is emerging: of the 29 Geosmithia species found in the western USA, 12 have not been reported outside of the USA. The most frequently encountered species with the widest global distribution also had the broadest range of beetle vectors. Several Geosmithia spp. with very narrow vector ranges in Europe exhibited the similar degree of specialization in the USA. Such strong canalization in association could reflect an ancient origin of each individual association, or a recent origin and a subsequent diversification in North America.

Bark beetles (Coleoptera, Scolytinae) are known to be associated with fungi, particularly species of the orders Ophiostomatales and Microascales. However, very little is known about other ectosymbionts of phloeophagous bark beetles on Pinaceae. In this study, we examined the Geosmithia species associated with eight bark beetle species infesting Picea abies and Pinus sylvestris branches in Poland. Fungi were isolated from 1 731 samples collected from 14 study sites. We identified a total of 653 isolates that were sorted into nine taxa based on their phenotypic similarity and phylogeny of their ITS-LSU regions of rDNA, β-tubulin, elongation factor 1α and the second-largest subunit of the RNA polymerase II gene. They represented nine species without formal names. There were large quantitative and qualitative differences in the composition of Geosmithia communities between P. sylvestris and P. abies trees. The proportion of samples infested with Geosmithia species suggests that this association is more widespread among bark beetles infesting branches of P. sylvestris . In addition, these beetles were vectors of different Geosmithia species compared with than the beetles that colonize P. abies . In mixed-conifer forests, the Geosmithia communities were more diverse and richer than in pure spruce or pine stands, where the insects Pityogenes chalcographus and Pityophthorus pityographus with low host-specificity play a distributing role for various Geosmithia species. Among eight bark beetle species examined, only P. bidentatus , P. pityographus , P. chalcographus and Polygraphus poligraphus acted as effective vectors for Geosmithia species. The following hypothesis emerges from these studies: changes in the composition of ectosymbionts of pine- and spruce-infesting bark beetles in Central Europe run along a gradient of thickness of the wood substrata preferred by insects.

Previous work had shown that a sequence homologous to the gene encoding class II hydrophobin cerato-ulmin from the fungus Ophiostoma novo-ulmi, the causal agent of Dutch Elm Disease (DED), was present in a strain of the unrelated species Geosmithia species 5 (Ascomycota: Hypocreales) isolated from Ulmus minor affected by DED. As both fungi occupy the same habitat, even if different ecological niches, the occurrence of horizontal gene transfer was proposed. In the present work we have analysed for the presence of the cerato-ulmin gene 70 Geosmithia strains representing 29 species, isolated from different host plants and geographic locations. The gene was found in 52.1% of the strains derived from elm trees, while none of those isolated from nonelms possessed it. The expression of the gene in Geosmithia was also assessed by real time PCR in different growth conditions (liquid culture, solid culture, elm sawdust, dual culture with O. novo-ulmi), and was found to be extremely low in all conditions tested. On the basis of these results we propose that the cerato-ulmin gene is not functional in Geosmithia, but can be considered instead a marker of more extensive transfers of genetic material as shown in other fungi. © 2014 The British Mycological Society.

Genome size has played an important role in the evolution of plants and animals because changes in genome size seem to accompany if not facilitate evolutionary adaptation to environmental conditions. Flow cytometry (FCM) is a widespread method for determining genome size thanks to its high accuracy and speed of measurements. Nevertheless, only a few comparative studies of FCM methods exist in the field of mycology, and reviews are absent. In this study, we compared the suitability of several concentrations and RNAse A incubation times, fixatives and buffers for estimating genome size in fungi. We chose the genus Geosmithia as a model filamentous fungus. We also introduced a new standard, Aspergillus fumigatus CEA10, to determine absolute genome size. We found FCM to be an appropriate method for measuring genome size in fungi, but optimization steps showed that incorrect propidium iodide staining of nuclei can overestimate genome size due to cytoplasmic staining. We identified fixation with methanol:glacial acetic acid (3:1 v/v), 10% DMSO, 0.1% Triton-X 100, and 5 mM EDTA in combination with Tris-MgCl2 buffer as the best treatment. © 2014 International Society for Advancement of Cytometry

SummaryGeosmithia morbida is an emerging fungal pathogen which serves as a paradigm for examining the evolutionary processes behind pathogenicity because it is one of two known pathogens within a genus of mostly saprophytic, beetle-associated, fungi. This pathogen causes thousand cankers disease in black walnut trees and is vectored into the host via the walnut twig beetle. G. morbida was first detected in western US and currently threatens the timber industry concentrated in eastern US.We sequenced the genomes of G. morbida and two non-pathogenic Geosmithia species and compared these species to other fungal pathogens and nonpathogens to identify genes under positive selection in G. morbida that may be associated with pathogenicity.G. morbida possesses one of the smallest genomes among the fungal species observed in this study, and one of the smallest fungal pathogen genomes to date. The enzymatic profile is this pathogen is very similar to its relatives.Our findings indicate that genome reduction is an important adaptation during the evolution of a specialized lifestyle in fungal species that occupy a specific niche, such as beetle vectored tree pathogens. We also present potential genes under selection in G. morbida that could be important for adaptation to a pathogenic lifestyle.

In the course of a global survey of the indoor mycobiota, we sampled and analysed settled dust from 87 buildings from 14 countries, using both a modified dilution-to-extinction method and 454-pyrosequencing. Rasamsonia is a recently established genus including thermotolerant or thermophilic species, five of which have been isolated from humans, including the emerging pathogen R. argillacea. A new species, R. pulvericola, was recovered from one residence in Songkhla, Thailand, and is morphologically characterised and compared phylogenetically with other members of the genus. Rasamsonia pulvericola forms a clade with R. brevistipitata and shares morphological characters such as usually biverticillate and never terverticillate conidiophores, and subglobose to ellipsoidal conidia. It has a lower maximum growth temperature and is the first mesophilic species added to the genus. The ITS sequence of R. pulvericola was not detected in the 454-pyrosequencing data for Thailand or other countries, but a similar ITS sequence was detected in Micronesia, probably representing another undescribed Rasamsonia species.

The early diagnosis of invasive pulmonary aspergillosis (IPA) is challenging. Fibered confocal fluorescence microscopy (FCFM) is a new technique that allows in vivo imaging of the lung microstructure during bronchoscopy. In this study, we investigated the ability of FCFM to detect a fluorescent peptide-tracer bound to Aspergillus fumigatus in experimental IPA in 13 immunosuppressed, non-neutropenic rats. Subpleural IPA microabscesses were imaged through a transthoracic window using FCFM in vivo after i.v. injection of the c(CGGRLGPFC)-NH2([FITC]) peptide (n = 7) or saline. Results were compared to 10 immunosuppressed, non-infected rats and to six immunosuppressed Geosmithia argillacea-infected rats with and without i.v. injection of the peptide. The peptide in vitro specifically labeled A. fumigatus grown under biofilm growth conditions but not G. argillacea. In vivo, FCFM showed a local infiltration of fluorescent host cells in both Aspergillus and Geosmithia infections. Lung/inner thoracic wall fluorescence intensity ratio (FI) did not differ before and after peptide administration on healthy lung areas, on non-specific inflammatory areas, or on Geosmithia micro-abscesses. In contrast, FI increased from 1.05 without peptide to 1.83 after peptide injection on Aspergillus micro-abscesses (p < 0.0001). In peptide-injected rats, FI from IPA foci was higher than from non-specific inflammation or from Geosmithia abscesses (p ≤ 0.002). Using c(CGGRLFPC)-NH2([FITC]) peptide, FCFM allows the in vivo specific imaging of pulmonary aspergillosis. These data provide the basis for the in vivo diagnosis of human pulmonary aspergillosis using alveolar confocal endomicroscopy.

Fungi from the genus Geosmithia (Ascomycota: Hypocreales) are associated with bark beetles (Coleoptera: Scolytinae), though little is known about ecology, diversity, and distribution of these fungi across beetle and its host tree species. This study surveyed the diversity, distribution and vector affinity of Geosmithia isolated from subcortical insects that colonized trees from the family Pinaceae in Central and Northeastern Europe. Twelve Geosmithia species were isolated from 85 plant samples associated with 23 subcortical insect species (including 14 bark beetle species). Geosmithia community composition was similar across different localities and vector species; although the fungal communities associated with insects that colonized Pinus differed from that colonizing other tree species (Abies, Larix, and Picea). Ten Geosmithia species from four independent phylogenetic lineages were not reported previously from vectors feeding on other plant families and seem to be restricted to the vectors from Pinaceae only. We conclude that presence of such substrate specificity suggests a long and stable association between Geosmithia and bark beetles.

Phytophthora ramorum, the causal agent of sudden oak death (SOD), has killed large numbers of coast live oaks (Quercus agrifolia), California black oaks (Q. kelloggii), canyon live oaks (Q. chrysolepis), and Shreve oaks (Q. parvula var. Shrevei) in coastal California, and tanoaks (Notholithocarpus densiflorus) in California and Oregon. Native and introduced bark and ambrosia beetles selectively tunnel into stem cankers caused by the pathogen. Beetle attacks have been shown to considerably shorten survival of infected coast live oaks. We hypothesized that this behavior facilitates the introduction into, and/or stimulates the activity of, decay and pathogenic fungi, which, in turn, accelerate tree death. The associations of infected coast live oaks, beetles, and sapwood-inhabiting fungi in two California forests were investigated to: (1) determine whether beetles introduce fungi into P. ramorum-infected coast live oaks, (2) catalog the community of fungal species isolated in pure culture from the sapwood of infected and uninfected trees, with and without beetle colonization, and (3) identify the fungi found on beetles reared from infected trees. We inoculated coast live oaks, and monitored symptom development (i.e., bleeding cankers and colonization by beetles) for 2 years. Trees were categorized as (1) live inoculated, bleeding, and still alive, (2) inoculated, bleeding with beetle attacks, and still alive, (3) inoculated with beetle attacks, and dead, (4) felled, asymptomatic before beetle attacks, and (5) felled, asymptomatic after beetle attacks. Trees were felled at 6-month intervals, sapwood samples were plated on four types of growth media, and the ITS sequences of the rDNA regions of morphologically unique fungal cultures were determined. Thirteen species were identified in the sapwood of inoculated trees prior to beetle attacks and 26 species were identified in living inoculated oaks after beetle colonization. The known oak pathogens Botryosphaeria corticola and B. sarmentorum were isolated from all categories of inoculated trees as well as from beetles. Fungi reported elsewhere as pathogens that were isolated from beetle-colonized trees include an Aureobasidium sp., Bartalinia robillardoides, Geosmithia fassatiae, a Monochaetia sp., Pleurostomophora richardsiae, Truncatella angustata, and Stereum hirsutum. Nineteen fungal taxa were isolated from beetles emerging from infected logs, including B. corticola, B. sarmentorum, a Pestalotiopsis sp., G. fassatiae, a Geosmithia sp., an Ophiostoma sp., and an uncultured Pleosporaceae sp. Preventing beetle attacks might limit introduction of fungi into infected coast live oaks, increasing the effectiveness of resistance mechanisms against P. ramorum. Where the disease is not fatal, beetles attacking trees infected by this introduced pathogen may facilitate new ecological associations between fungi, beetles, and a native host tree species.

Geosmithia argillacea, an anamorph of Talaromyces eburneus, is a thermophilic filamentous fungus that has a phenotype similar to that of the Penicillium species, except for the creamy-white colonies and cylindrical conidia. Recently, a new genus called Rasamsonia has been proposed, which is to accommodate the Talaromyces and Geosmithia species. Here, we report the first Korean case of G. argillacea isolated from a patient with a fungal ball. The patient was a 44-yr-old Korean man with a history of pulmonary tuberculosis and aspergilloma. The newly developed fungal ball in his lung was removed and cultured to identify the fungus. The fungal colonies were white and slow-growing, and the filaments resembled those of Penicillium. Molecular identification was carried out by sequencing the internal transcribed spacer (ITS) region of the 28S rDNA and the β-tubulin genes. A comparative sequence analysis using the GenBank (http://blast.ncbi.nlm.nih.gov/) database was performed with the basic local alignment search tool (BLAST) algorithm. The results revealed a 97-100% similarity with the G. argillacea ITS sequence. This case should increase awareness among physicians about the pathogenic potential of G. argillacea in humans and help them accurately identify this fungus, because it can be easily confused with Penicillium and Paecilomyces species owing to their similar phenotypic and microscopic characteristics. A molecular approach should be employed to enable accurate identification of G. argillacea.

In recent years, Geosmithia argillacea has been increasingly reported in humans and animals and can be considered an emerging pathogen. The taxonomy of Geosmithia was recently studied, and Geosmithia argillacea and related species were transferred to the new genus Rasamsonia . The diversity among a set of Rasamsonia argillacea strains, including 28 clinical strains, was studied, and antifungal susceptibility profiles were generated. Data obtained from morphological studies and from phylogenetic analyses of internal transcribed spacer (ITS) and partial β-tubulin and calmodulin sequences revealed the presence of four species in the Rasamsonia argillacea complex, two of which are newly described here: R. piperina sp. nov. and R. aegroticola sp. nov. In contrast to other related genera, all Rasamsonia species can be identified with ITS sequences. A retrospective identification was performed on recently reported clinical isolates from animal or human patients. Susceptibility tests showed that the antifungal susceptibility profiles of the four members of the R. argillacea complex are similar, and caspofungin showed significant activity in vitro , followed by amphotericin B and posaconazole. Voriconazole was the least active of the antifungals tested. The phenotypically similar species R. brevistipitata and R. cylindrospora had different antifungal susceptibility profiles, and this indicates that correct species identification is important to help guide appropriate antifungal therapy.

Geosmithia morbida is a filamentous ascomycete that causes Thousand Cankers Disease in the eastern black walnut tree. This pathogen is commonly found in the western U.S.; however, recently the disease was also detected in several eastern states where the black walnut lumber industry is concentrated. G. morbida is one of two known phytopathogens within the genus Geosmithia, and it is vectored into the host tree via the walnut twig beetle. We present the first de novo draft genome of G. morbida. It is 26.5 Mbp in length and contains less than 1% repetitive elements. The genome possesses an estimated 6,273 genes, 277 of which are predicted to encode proteins with unknown functions. Approximately 31.5% of the proteins in G. morbida are homologous to proteins involved in pathogenicity, and 5.6% of the proteins contain signal peptides that indicate these proteins are secreted. Several studies have investigated the evolution of pathogenicity in pathogens of agricultural crops; forest fungal pathogens are often neglected because research efforts are focused on food crops. G. morbida is one of the few tree phytopathogens to be sequenced, assembled and annotated. The first draft genome of G. morbida serves as a valuable tool for comprehending the underlying molecular and evolutionary mechanisms behind pathogenesis within the Geosmithia genus. Keywords: de novo genome assembly, pathogenesis, forest pathogen, black walnut, walnut twig beetle.