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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.

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.

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.

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.

Cell wall polysaccharides have been used as chemotaxonomic markers in a number of fungal genera. In this study, alkali-extractable and water-soluble cell wall polysaccharides F1SS were purified from cell walls of species of the genus Geosmithia. Chemical and 1H-NMR analyses of these polysaccharides revealed three different structures: (i) the polysaccharide from Geosmithia namylowskii was composed of β-(1[Formula: see text]5)-galactofuranose chains attached to an α-(1[Formula: see text]2)-(1[Formula: see text]6)-mannan core identical to that isolated in several species of Eupenicillium, (ii) the polysaccharide from Geosmithia viridis was a glucomannogalactan similar to that obtained in Talaromyces flavus, and (iii) the polysaccharide from Geosmithia putterillii contained β-(1[Formula: see text]5)-(1[Formula: see text]6)-galactofuranose with some units of the β-(1[Formula: see text]5) residues substituted at position O-6 by single residues of galactofuranose; this galactan was attached to an α-(1[Formula: see text]2)-(1[Formula: see text]6)-mannan core. The taxonomy of the genus is discussed according to the polysaccharides F1SS, and our results are compared with the results of rDNA analyses in this genus.Key words: Eurotiales, Geosmithia, Eupenicillium, Talaromyces, cell wall polysaccharides, chemotaxonomy.

Since 2001, widespread mortality of black walnut (Juglans nigra) has been reported in Colorado, USA. Affected trees initially show a yellowing and thinning of leaves in the upper crown, followed by twig and branch dieback and ultimately tree death. We report that this mortality is the result of a combination of an expanded geographic range of the walnut twig beetle (Pityophthorus juglandis), its aggressive feeding behavior on black walnut, and extensive cankering caused by an unnamed Geosmithia fungus associated with the beetle. Geosmithia was consistently recovered from the bodies of P. juglandis and this insect introduces the fungus into healthy trees during gallery formation. This is the first report of Geosmithia as a pathogen of black walnut. We propose the name Thousand Cankers to describe this disease because mortality is the result of bark necrosis caused by an enormous number of coalescing branch and trunk cankers. A second pathogen, Fusarium solani, was isolated from the margins of elongate trunk cankers during the final stages of decline, but not from cankers surrounding beetle galleries. Thousand Cankers Disease is eliminating black walnut along the Front Range of Colorado and poses a grave risk to this species in its native range in eastern North America should the insect/Geosmithia complex be introduced. Accepted for publication 26 May 2009. Published 11 August 2009.

Using high-performance liquid chromatography, the ubiquinone (Coenzyme Q) systems were determined for 335 isolates assigned to 118 species of Penicillium, and related teleomorph and anamorph genera. In the genus Eupenicillium, Q-9 occurred in all 21 species tested. In the genus Penicillium, isolates belonging to subgenera Aspergilloides, Furcatum, and Penicillium were of Q-9 except for the two species P. megasporum (Q-10) and P. asperosporum (Q-10(H2)) placed in series Megaspora. In subgenus Biverticillium, species in section Coremigena had Q-9, but Q-10(H2) was found in section Simplicia, and also in the genus Geosmithia, except for G. argillacea (Q-10). Almost all isolates in Talaromyces had Q-10(H2), but sometimes Q-10(H4), except for T. thermophilus, T. leycettanus, T. avellaneus and T. striatus, all of which had Q-10. Eladia, Penicilliopsis and Dendrosphaera had Q-9, but Q-10(H2) combined with Q-10(H4) was observed in all isolates of Trichocoma. The ubiquinone system is considered to be useful as a chemotaxonomic criterion in solving taxonomic problems in Penicillium and related fungi, and should be taken into consideration in future taxonomic treatments.

Geosmithia spp. ( Ascomycota : Hypocreales ) are dry-spored fungi that occur in galleries built by many phloeophagous bark beetles. This study mapped the diversity, host spectrum and area of distribution of Geosmithia spp. occurring in galleries of bark beetle species with a Mediterranean distribution. Eighty-six wood samples of 19 tree species infested by 18 subcortical insect species were collected from across the Mediterranean Basin during the years 2003–2006. Geosmithia spp. were found in 82 samples of angiosperms and two host trees from the family Juniperaceae infested by 14 bark beetles and the bostrichid Scobicia pustulata , suggesting that the association of Geosmithia and phloeophagous bark beetles is very widespread in the Mediterranean. Geosmithia isolates were sorted into 13 operational taxonomic units (OTUs) based on their phenotype similarity and phylogeny of their ITS regions of rDNA (ITS1–5.8S–ITS2). The OTUs represent five known species ( G. flava , G. langdonii , G. lavendula , G. pallida , G. putterillii ) and seven undescribed taxa. Most of the bark beetles were associated with on average 1–2.5 OTUs per sample. G. lavendula , considered very uncommon in nature, was found as a common associate of bark beetles. Six out of 13 OTUs were found to be distributed in the Mediterranean but not in neighbouring areas of temperate Europe suggesting that Geosmithia spp. have a geographically limited distribution, probably due to their dependency on the geographically limited area of their vectors. The proportion of generalists and specialists among Geosmithia spp. was smaller compared with data from temperate Europe. A possible explanation is the effective dispersal of Geosmithia by polyphagous bostrichids across the niches defined by mutually exclusive bark beetles.

All hypocrealean species of the genus Geosmithia are anamorphic fungi with connections to bark beetles. G. fassatiae, G. langdonii and G. obscura are described as new sympatric species associated with Scolytus carpini, S. intricatus and S. rugulosus in Central Europe. The species represent a complex of three sister taxa with affinities to G. flava that may be distinguished by differences in morphology, unique RAPD patterns and by sequences of ITS region rDNA. Intraspecific variability and habitat specificity of new species is described and discussed. The high morphological, genetic and ecological uniformity suggest that these Geosmithia spp. are recently derived. A key to all accepted hypocrealean species of the genus is provided.

Geosmithia putterillii is an anamorphic fungus with connections to bark beetles. Genetic variability of 89 isolates traditionally grouped in G. putterillii and G. lavendula isolated from different geographical regions from subcorticolous insects and from other unspecific substrata was assessed using RAPD, sequencing of the ITS region (ITS1-5.8SrDNA-ITS2) and morphological characters. RAPD analysis revealed eight distinct groups. One group was represented by G. lavendula type strain and showed no relations to any other isolate. Five RAPD-types with similar ITS sequences and phenotype were related to the ex-type strain of Penicillium pallidum (generally given as a synonym of G. putterillii). Because of unique phylogenetic position and a phenotype markedly different from G. putterillii, the new combination G. pallida is made here. For another group of isolates formerly identified as G. putterillii the new species G. fiava is described based on a characteristic RAPD-type, a unique ITS sequences and a different phenotype. These newly recognized species are stable in culture and with worldwide distribution.

Merimbla ingelheimense (van Beyma) Pitt is described for the anamorphic state of Talaromyces avellaneus C.R. Benjamin, a holomorphic fungus. Diagnostic features are discussed, together with a key for distinguishing Merimbla from Aspergillus, Geosmithia, Paecilomyces, Penicillium, and Raperia.

Geosmithia Pitt is erected to accommodate species, previously placed in Penicillium, with the following combination of characters: colonies with conidia in colors other than grey–blue or grey–green, penicilli with all elements roughened, and with both phialides and conidia cylindroidal. Geosmithia lavendula (Raper & Fennell) Pitt, G. putterillii (Thom) Pitt, G. namyslowskii (Zaleski) Pitt, G. cylindrospora (G. Smith) Pitt, G. argillacea (Stolk, Evans & Nilsson) Pitt, and G. emersonii (Stolk) Pitt are described as new combinations; G. swiftii is a new species for the anamorphic state of Talaromyces bacillisporus (Swift) C. R. Benjamin. A key is provided.