Alena Kubátová

Increasing Incidence of Geomyces destructans Fungus in Bats from the Czech Republic and Slovakia

Background White-nose syndrome is a disease of hibernating insectivorous bats associated with the fungus Geomyces destructans. It first appeared in North America in 2006, where over a million bats died since then. In Europe, G. destructans was first identified in France in 2009. Its distribution, infection dynamics, and effects on hibernating bats in Europe are largely unknown. Methodology/Principal Findings We screened hibernacula in the Czech Republic and Slovakia for the presence of the fungus during the winter seasons of 2008/2009 and 2009/2010. In winter 2009/2010, we found infected bats in 76 out of 98 surveyed sites, in which the majority had been previously negative. A photographic record of over 6000 hibernating bats, taken since 1994, revealed bats with fungal growths since 1995; however, the incidence of such bats increased in Myotis myotis from 2% in 2007 to 14% by 2010. Microscopic, cultivation and molecular genetic evaluations confirmed the identity of the recently sampled fungus as G. destructans, and demonstrated its continuous distribution in the studied area. At the end of the hibernation season we recorded pathologic changes in the skin of the affected bats, from which the fungus was isolated. We registered no mass mortality caused by the fungus, and the recorded population decline in the last two years of the most affected species, M. myotis, is within the population trend prediction interval. Conclusions/Significance G. destructans was found to be widespread in the Czech Republic and Slovakia, with an epizootic incidence in bats during the most recent years. Further development of the situation urgently requires a detailed pan-European monitoring scheme.

Taxonomic revision of Eurotium and transfer of species to Aspergillus.

Aspergillus section Aspergillus contains economically important, xerophilic fungi that are widely distributed in nature and the human environment and are known for their ability to grow on substrates with low water activity. The taxa were revised based on sequence data from four loci, PCR fingerprinting, micro- and macromorphology, and physiology. The number of taxa was reduced to 17 species, all of which can be distinguished with sequence data from either the caM or RPB2 locus. The original description of A. proliferans was supplemented by a description of its teleomorph. This species seems to be relatively common and often has been confused with A. glaucus. In addition, green sporulating isolates of A. niveoglaucus isolated from food and several other substrates are indistinguishable in phenotype from A. glaucus. A dichotomous key based on ascospore size and ornamentation and the ability to grow at specific combinations of temperature and water activity is provided for identification of species. In response to recent changes in the botanical code, we transferred the Eurotium species to Aspergillus and selected one name for each species.

Geosmithia Fungi are Highly Diverse and Consistent Bark Beetle Associates : Evidence from their Community Structure in Temperate Europe

Geosmithia spp. (Ascomycota: Hypocreales) are little-studied, dry-spored fungi that occur in galleries built by many phloeophagous bark beetles. This study mapped the distribution and environmental preferences of Geosmithia species occurring in galleries of temperate European bark beetles. One hundred seven host tree samples of 16 tree species infested with 23 subcortical insect species were collected from across Europe during the years 1997–2005. Over 600 Geosmithia isolates from the beetles were sorted into 17 operational taxonomic units (OTUs) based on their phenotype similarity and phylogeny of internal transcribed spacer (ITS) region of rDNA (ITS1-5.8S-ITS2). The OTUs represent six known species and eight undescribed taxa. Ninety-two samples infested with subcortical insects were characterized by the presence/absence of OTUs and the similarity among the samples was evaluated. Geographically distant populations of the same beetle species host relatively uniform Geosmithia communities across large geographic areas (ranging from southern Bulgaria to the Czech Republic). This suggests effective dispersal of Geosmithia spp. by bark beetles. Clustering of similar samples in ordination analysis is correlated predominantly with the isolation source (bark beetles and their respective feeding plant), but not with their geographical origin. The composition of the Geosmithia OTU community of each bark beetle species depends on the degree of isolation of the species’ niches. Thus, Geosmithia communities associated with regularly co-occurring bark beetle species are highly similar. The similarity decreases with decreasing frequency of beetle species’ co-occurrence, a pattern resembling that of entomochoric ophiostomatoid fungi. These findings suggest that: 1) communities of Geosmithia spp. are vector-specific; 2) at least in some cases, the association between Geosmithia OTUs and bark beetles may have been very stable and symbioses are likely to be a fundamental factor in the speciation of Geosmithia fungi; and 3) that even nonsticky spores of Geosmithia are suitable for maintaining an insect–fungus association, contrary to previous hypotheses.

Rare and new etiological agents revealed among 178 clinical Aspergillus strains obtained from Czech patients and characterized by molecular sequencing

A collection of 178 Aspergillus isolates, recovered from Czech patients, mostly from 2007-2011, was subjected to multilocus DNA sequence typing using the ITS region, β-tubulin, and calmodulin genes. An unusually wide spectrum of etiologic agents that included 36 species of Aspergillus is discussed in the context of recent taxonomic and clinical reports. Invasive aspergillosis (IA), onychomycosis, and otitis externa were the predominant clinical entities. Five cases due to species newly proven as etiologic agents of human mycoses, as well as cases with unique clinical manifestations caused by unusual agents are discussed in more detail. Three species (i.e., A. insulicola, A. westerdijkiae and A. tritici) were identified as the confirmed etiologic agents of non-dermatophytic onychomycosis. Emericella rugulosa was recovered from a premature newborn with a fatal necrotising disseminated infection and is reported for only the second time as the cause of IA. Furthermore, we document the first infection due to A. calidoustus in a patient with chronic granulomatous disease. The infection manifested as a latent brain aspergilloma with an unusual clinical-laboratory finding. In addition to the well-known agents of human mycosis, several rarely isolated or poorly documented species were identified. An undescribed cryptic species related to A. versicolor was found to be common among isolates linked to proven and probable onychomycosis. An isolate representing A. fresenii, or an unnamed sister species, were causal agents of otomycosis. Three well defined, and tentative new species belonging to section Cervini, Candidi and Aspergillus (Eurotium spp.), were associated with cases of probable onychomycosis.

Morphological and molecular characterisation of Geosmithia putterillii, G. pallida comb. nov. and G. flava sp. nov., associated with subcorticolous insects.

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.

A complex of three new white-spored, sympatric, and host range limited Geosmithia species

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.

Diversity of fungal communities in saline and acidic soils in the Soos National Natural Reserve, Czech Republic

During 2003–2005, the diversity of culturable filamentous soil microfungi in saline and acidic soils of the Soos National Natural Reserve (Czech Republic) was studied. Altogether, 28 soil samples were collected from four sampling sites and were processed by various approaches. In total, 92 fungal taxa were identified using classical and molecular markers. Several detected species were known from similar substrata worldwide; however, the overall fungal spectrum was distinct, as shown by comparison to similar studies. All methodological approaches increased the observed fungal diversity. The different fungal communities observed on the four sampling sites were influenced by the complex effects of environmental factors. The growth response of selected strains to different salinities and pH values was determined. The results of the growth tests showed high adaptability of all tested species to the extreme conditions of the studied substrate. Two acidophilic species (Acidomyces acidophilus, Sporothrix sp.) were isolated.

New species in Aspergillus section Fumigati from reclamation sites in Wyoming (U.S.A.) and revision of A. viridinutans complex

The Aspergillus viridinutans complex includes morphologically similar, soil-inhabiting species. Although its species boundaries have not been fully defined, many isolates from the complex have been isolated as opportunistic human and animal pathogens. In the present study, these species were dominant in spoil sites subjected to various types of reclamation management after coal mining. These species were characterised using two different PCR-fingerprinting methods, sequence data from the β-tubulin (benA) and calmodulin (caM) genes, macro- and micromorphology (optical and scanning electron microscopy), maximum growth temperatures and mating experiments. In addition, RNA polymerase II gene (RPB2), actin (act1) and ITS sequences were deposited for the ex-type isolates of newly described species. The mating experiment results, phylogenetic analyses and ascospore morphology suggested the presence of five species in the A. viridinutans complex. Aspergillus aureolus (syn. Neosartorya aureola) was the only homothallic species. Three species, A. felis, A. udagawae (syn. N. udagawae) and A. wyomingensis sp. nov., were heterothallic and their morphologically distinguishable teleomorph was induced by systematic mating experiments. Aspergillus viridinutans s. str. seems to be a very rare species and was represented only by the ex-type isolate in which the MAT1-1 locus was amplified. Aspegillus viridinutans and A. aureolus were typified in accordance with the rules of the new botanical code. Other species outside the A. viridinutans complex isolated from the reclamation sites were A. fumigatiaffinis and A. lentulus as well as two new sister species, A. brevistipitatus sp. nov. and A. conversis sp. nov. which were closely related each to other and to N. papuensis. Both new species are phylogenetically distant from all anamorphic species and resemble A. brevipes, A. duricaulis and A. unilateralis in micromorphology and are distinguishable from each other by the slower growth of A. conversis on all tested media. Interestingly, no isolate from the reclamation sites represented A. fumigatus s. str. which is usually reported as the dominant species from the section Fumigati in soil.

Molecular Characterization of a Heterothallic Mating System in Pseudogymnoascus destructans, the Fungus Causing White-Nose Syndrome of Bats

White-nose syndrome (WNS) of bats has devastated bat populations in eastern North America since its discovery in 2006. WNS, caused by the fungus Pseudogymnoascus destructans, has spread quickly in North America and has become one of the most severe wildlife epidemics of our time. While P. destructans is spreading rapidly in North America, nothing is known about the sexual capacity of this fungus. To gain insight into the genes involved in sexual reproduction, we characterized the mating-type locus (MAT) of two Pseudogymnoascus spp. that are closely related to P. destructans and homothallic (self-fertile). As with other homothallic Ascomycota, the MAT locus of these two species encodes a conserved α-box protein (MAT1-1-1) as well as two high-mobility group (HMG) box proteins (MAT1-1-3 and MAT1-2-1). Comparisons with the MAT locus of the North American isolate of P. destructans (the ex-type isolate) revealed that this isolate of P. destructans was missing a clear homolog of the conserved HMG box protein (MAT1-2-1). These data prompted the discovery and molecular characterization of a heterothallic mating system in isolates of P. destructans from the Czech Republic. Both mating types of P. destructans were found to coexist within hibernacula, suggesting the presence of mating populations in Europe. Although populations of P. destructans in North America are thought to be clonal and of one mating type, the potential for sexual recombination indicates that continued vigilance is needed regarding introductions of additional isolates of this pathogen.

Cryopreservation of filamentous micromycetes and yeasts using perlite

The viability, growth and morphology of 48 strains ofAscomycota (including 17 yeasts) and 20 strains ofZygomycota were determined after a 2-d and then after 1-year storage in liquid nitrogen using a new cryopreservation method with perlite as a particulate solid carrier. In case ofAscomycota, 45 strains (94 %) out of 48 survived both 2-d and 1-year storage in liquid nitrogen, respectively. In case ofZygomycota, all 20 strains survived both storage. In addition, 3 strains ofBasidiomycota counted among yeasts were tested and all survived the 1 year storage. In all surviving cultures no negative effects of cryopreservation by this method have been observed after 1-year of storage in liquid nitrogen. The results indicate that the perlite protocol can be successfully used for cryopreservation of taxonomically different groups of fungi and also for fungi which failed to survive other routinely used preservation procedures.