Terbinafine resistance in Trichophyton mentagrophytes and Trichophyton rubrum in the Czech Republic: A prospective multicentric study.

BACKGROUND: Terbinafine, an allylamine antifungal, is crucial for treating dermatophytosis by inhibiting squalene epoxidase (SQLE) in the ergosterol biosynthetic pathway. However, resistance is emerging, particularly in India and Southeast Asia, but reports of resistance spread worldwide. Despite this, comprehensive studies on terbinafine resistance in Trichophyton are still limited. OBJECTIVES: This research aimed to determine the prevalence of terbinafine resistance in the Czech Republic, with a focus on Trichophyton rubrum and Trichophyton mentagrophytes, and investigate the underlying molecular mechanisms. PATIENTS/METHODS: A total of 514 clinical strains of T. rubrum and 240 T. mentagrophytes collected from four Czech clinical institutions were screened for terbinafine resistance. Molecular investigations included DNA sequencing, specifically the ITS rDNA region and SQLE gene, as well as antifungal susceptibility testing following EUCAST guidelines. RESULTS: While no resistance was observed in T. rubrum, 2.5% of T. mentagrophytes strains exhibited resistance, marked by the F397L mutation in SQLE. Notably, resistance surged from 1.2% in 2019 to 9.3% in 2020 but reverted to 0% in 2021. All resistant strains were identified as T. mentagrophytes var. indotineae. Resistant strains exhibited high MICs for terbinafine (≥4 mg L-1 ) but low MICs to the other seven antifungals tested except for fluconazole. CONCLUSIONS: This study highlights the emergence of terbinafine-resistant T. mentagrophytes strains in the Czech Republic, with the F397L mutation being pivotal. Due to the relatively low resistance level, the current guidelines for dermatomycosis treatment in the Czech Republic remain effective, but ongoing surveillance is essential for timely adaptations if resistance patterns change.

Mycobiomes of two distinct clades of ambrosia gall midges (Diptera: Cecidomyiidae) are species-specific in larvae but similar in nutritive mycelia.

IMPORTANCE: Ambrosia gall midges are endophagous insect herbivores whose larvae live enclosed within a single gall for their entire development period. They may exhibit phytomycetophagy, a remarkable feeding mode that involves the consumption of plant biomass and mycelia of their cultivated gall symbionts. Thus, AGMs are ideal model organisms for studying the role of microorganisms in the evolution of host specificity in insects. However, compared to other fungus-farming insects, insect-fungus mutualism in AGMs has been neglected. Our study is the first to use DNA metabarcoding to characterize the complete mycobiome of the entire system of the gall-forming insects as we profiled gall surfaces, nutritive mycelia, and larvae. Interestingly, larval mycobiomes were significantly different from their nutritive mycelia, although Botryosphaeria dothidea dominated the nutritive mycelia, regardless of the evolutionary separation of the tribes studied. Therefore, we confirmed a long-time hypothesized paradigm for the important evolutionary association of this fungus with AGMs.

Concentration-dependent effect of plant secondary metabolites on bacterial and fungal microbiomes in caterpillar guts.

IMPORTANCE: The caterpillar gut is an excellent model system for studying host-microbiome interactions, as it represents an extreme environment for microbial life that usually has low diversity and considerable variability in community composition. Our study design combines feeding caterpillars on a natural and artificial diet with controlled levels of plant secondary metabolites and uses metabarcoding and quantitative PCR to simultaneously profile bacterial and fungal assemblages, which has never been performed. Moreover, we focus on multiple caterpillar species and consider diet breadth. Contrary to many previous studies, our study suggested the functional importance of certain microbial taxa, especially bacteria, and confirmed the previously proposed lower importance of fungi for caterpillar holobiont. Our study revealed the lack of differences between monophagous and polyphagous species in the responses of microbial assemblages to plant secondary metabolites, suggesting the limited role of the microbiome in the plasticity of the herbivore diet.

GlobalAMFungi: a global database of arbuscular mycorrhizal fungal occurrences from high-throughput sequencing metabarcoding studies.

Arbuscular mycorrhizal (AM) fungi are crucial mutualistic symbionts of the majority of plant species, with essential roles in plant nutrient uptake and stress mitigation. The importance of AM fungi in ecosystems contrasts with our limited understanding of the patterns of AM fungal biogeography and the environmental factors that drive those patterns. This article presents a release of a newly developed global AM fungal dataset (GlobalAMFungi database, https://globalamfungi.com) that aims to reduce this knowledge gap. It contains almost 50 million observations of Glomeromycotinian AM fungal amplicon DNA sequences across almost 8500 samples with geographical locations and additional metadata obtained from 100 original studies. The GlobalAMFungi database is built on sequencing data originating from AM fungal taxon barcoding regions in: i) the small subunit rRNA (SSU) gene; ii) the internal transcribed spacer 2 (ITS2) region; and iii) the large subunit rRNA (LSU) gene. The GlobalAMFungi database is an open source and open access initiative that compiles the most comprehensive atlas of AM fungal distribution. It is designed as a permanent effort that will be continuously updated by its creators and through the collaboration of the scientific community. This study also documented applicability of the dataset to better understand ecology of AM fungal taxa.

Microbiome specificity and fluxes between two distant plant taxa in Iberian forests.

BACKGROUND: Plant-associated microbial communities play important roles in host nutrition, development and defence. In particular, the microbes living within internal plant tissues can affect plant metabolism in a more intimate way. Understanding the factors that shape plant microbial composition and discovering enriched microbes within endophytic compartments would thus be valuable to gain knowledge on potential plant-microbial coevolutions. However, these interactions are usually studied through reductionist approaches (in vitro models or crop controlled systems). Here, we investigate these ecological factors in wild forest niches using proximally located plants from two distant taxa (blueberry and blackberry) as a model. RESULTS: Although the microbial communities were quite similar in both plants, we found that sampling site had a high influence on them; specifically, its impact on the rhizosphere communities was higher than that on the roots. Plant species and sample type (root vs. rhizosphere) affected the bacterial communities more than the fungal communities. For instance, Xanthobacteraceae and Helotiales taxa were more enriched in roots, while the abundance of Gemmatimonadetes was higher in rhizospheres. Acidobacteria abundance within the endosphere of blueberry was similar to that in soil. Several taxa were significantly associated with either blackberry or blueberry samples regardless of the sampling site. For instance, we found a significant endospheric enrichment of Nevskia in blueberry and of Sphingobium, Novosphingobium and Steroidobacter in blackberry. CONCLUSIONS: There are selective enrichment and exclusion processes in the roots of plants that shapes a differential composition between plant species and sample types (root endosphere-rhizosphere). The special enrichment of some microbial taxa in each plant species might suggest the presence of ancient selection and/or speciation processes and might imply specific symbiosis. The selection of fungi by the host is more pronounced when considering the fungal trait rather than the taxonomy. This work helps to understand plant-microbial interactions in natural ecosystems and the microbiome features of plants.

New insight into the bark beetle ips typographus bacteriome reveals unexplored diversity potentially beneficial to the host.

BACKGROUND: Ips typographus (European spruce bark beetle) is the most destructive pest of spruce forests in Europe. As for other animals, it has been proposed that the microbiome plays important roles in the biology of bark beetles. About the bacteriome, there still are many uncertainties regarding the taxonomical composition, insect-bacteriome interactions, and their potential roles in the beetle ecology. Here, we aim to deep into the ecological functions and taxonomical composition of I. typographus associated bacteria. RESULTS: We assessed the metabolic potential of a collection of isolates obtained from different life stages of I. typographus beetles. All strains showed the capacity to hydrolyse one or more complex polysaccharides into simpler molecules, which may provide an additional carbon source to its host. Also, 83.9% of the strains isolated showed antagonistic effect against one or more entomopathogenic fungi, which could assist the beetle in its fight against this pathogenic threat. Using culture-dependent and -independent techniques, we present a taxonomical analysis of the bacteriome associated with the I. typographus beetle during its different life stages. We have observed an evolution of its bacteriome, which is diverse at the larval phase, substantially diminished in pupae, greater in the teneral adult phase, and similar to that of the larval stage in mature adults. Our results suggest that taxa belonging to the Erwiniaceae family, and the Pseudoxanthomonas and Pseudomonas genera, as well as an undescribed genus within the Enterobactereaceae family, are part of the core microbiome and may perform vital roles in maintaining beetle fitness. CONCLUSION: Our results indicate that isolates within the bacteriome of I. typographus beetle have the metabolic potential to increase beetle fitness by proving additional and assimilable carbon sources for the beetle, and by antagonizing fungi entomopathogens. Furthermore, we observed that isolates from adult beetles are more likely to have these capacities but those obtained from larvae showed strongest antifungal activity. Our taxonomical analysis showed that Erwinia typographi, Pseudomonas bohemica, and Pseudomonas typographi species along with Pseudoxanthomonas genus, and putative new taxa belonging to the Erwiniaceae and Enterobacterales group are repeatedly present within the bacteriome of I. typographus beetles, indicating that these species might be part of the core microbiome. In addition to Pseudomonas and Erwinia group, Staphylococcus, Acinetobacter, Curtobacterium, Streptomyces, and Bacillus genera seem to also have interesting metabolic capacities but are present in a lower frequency. Future studies involving bacterial-insect interactions or analysing other potential roles would provide more insights into the bacteriome capacity to be beneficial to the beetle.

Taxonomic analysis reveals host preference of rare fungi in endophytes of Vitis vinifera from the Czech Republic.

This paper represents the results of screening a diversity of fungal endophytes associated with Vitis vinifera leaves and canes in the Czech Republic. The characterization of strains is based on morphological and phylogenetic analyses of ITS, EF1α and TUB2 sequence data. Our strain selection covers 16 species and seven orders belonging to Ascomycota and Basidiomycota. Together with ubiquitous fungi, we report on several poorly known plant-associated fungi, Angustimassarina quercicola (= A. coryli, a synonym proposed in this study) and Pleurophoma pleurospora. Other species, such as Didymella negriana, D. variabilis, Neosetophoma sp. (species identical or sister to N. rosae), Phragmocamarosporium qujingensis and Sporocadus rosigena, have so far been little known and rarely found, but are frequent on V. vinifera in different parts of the world and obviously belong to a microbiota with a strong preference for this plant. Detailed taxonomical identification allowed us to identify species with apparent stable associations with V. vinifera, for which further interactions with V. vinifera can be expected. Our study is the first to focus on V. vinifera endophytes in Central Europe and expands the knowledge about their taxonomy, ecology and geography.

Proportions of taxa belonging to the gut core microbiome change throughout the life cycle and season of the bark beetle Ips typographus.

The European spruce bark beetle, Ips typographus, is a serious pest of spruce forests in Europe, and its invasion and development inside spruce tissues are facilitated by microorganisms. We investigated the core gut bacterial and fungal microbiomes of I. typographus throughout its life cycle in spring and summer generations. We used cultivation techniques and molecular identification in combination with DNA and RNA metabarcoding. Our results revealed that communities differ throughout their life cycle and across generations in proportion of dominantly associated microbes, rather than changes in species composition. The bacteriome consisted mostly of the phylum Gammaproteobacteria, with the most common orders and genera being Enterobacteriales (Erwinia and Serratia), Pseudomonadales (Pseudomonas), and Xanthomonadales. The fungal microbiome was dominated by yeasts (Saccharomycetes-Wickerhamomyces, Kuraishia, and Nakazawaea), followed by Sordariomycetes (Ophiostoma bicolor and Endoconidiophora polonica). We did not observe any structure ensuring long-term persistence of microbiota on any part of the gut epithelium, suggesting that microbial cells are more likely to pass through the beetle's gut with chyme. The most abundant taxa in the beetle's gut were also identified as dominant in intact spruce phloem. Therefore, we propose that these taxa are acquired from the environment rather than specifically vectored between generations.

Defining the relationship between phylogeny, clinical manifestation, and phenotype for Trichophyton mentagrophytes/interdigitale complex; a literature review and taxonomic recommendations.

This study looked for correlations between molecular identification, clinical manifestation, and morphology for Trichophyton interdigitale and Trichophyton mentagrophytes. For this purpose, a total of 110 isolates were obtained from Czech patients with various clinical manifestations of dermatophytosis. Phenotypic characters were analyzed, and the strains were characterized using multilocus sequence typing. Among the 12 measured/scored phenotypic features, statistically significant differences were found only in growth rates at 37 °C and in the production of spiral hyphae, but none of these features is diagnostic. Correlations were found between T. interdigitale and higher age of patients and between clinical manifestations such as tinea pedis or onychomychosis. The MLST approach showed that internal transcribed spacer (ITS) genotyping of T. mentagrophytes isolates has limited practical benefits because of extensive gene flow between sublineages. Based on our results and previous studies, there are few taxonomic arguments for preserving both species names. The species show a lack of monophyly and unique morphology. On the other hand, some genotypes are associated with predominant clinical manifestations and sources of infections, which keep those names alive. This practice is questionable because the use of both names confuses identification, leading to difficulty in comparing epidemiological studies. The current identification method using ITS genotyping is ambiguous for some isolates and is not user-friendly. Additionally, identification tools such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fail to distinguish these species. To avoid further confusion and to simplify identification in practice, we recommend using the name T. mentagrophytes for the entire complex. When clear differentiation of populations corresponding to T. interdigitale and Trichophyton indotineae is possible based on molecular data, we recommend optionally using a variety rank: T. mentagrophytes var. interdigitale and T. mentagrophytes var. indotineae.

Species in the T. mentagrophytes complex lack support from usual taxonomic methods and simple identification tools are missing or inaccurate. To avoid recurring confusions, we propose naming the entire complex as T. mentagrophytes and optionally use rank variety to classify the observed variability.

Amesia hispanica sp. nov., Producer of the Antifungal Class of Antibiotics Dactylfungins.

During a study of the diversity of soilborne fungi from Spain, a strain belonging to the family Chaetomiaceae (Sordariales) was isolated. The multigene phylogenetic inference using five DNA loci showed that this strain represents an undescribed species of the genus Amesia, herein introduced as A. hispanica sp. nov. Investigation of its secondary metabolome led to the isolation of two new derivatives (2 and 3) of the known antifungal antibiotic dactylfungin A (1), together with the known compound cochliodinol (4). The planar structures of 1-4 were determined by ultrahigh performance liquid chromatography coupled with diode array detection and ion mobility tandem mass spectrometry (UHPLC-DAD-IM-MS/MS) and extensive 1D and 2D nuclear magnetic resonance (NMR) spectroscopy after isolation by HPLC. All isolated secondary metabolites were tested for their antimicrobial and cytotoxic activities. Dactylfungin A (1) showed selective and strong antifungal activity against some of the tested human pathogens (Aspergillus fumigatus and Cryptococcus neoformans). The additional hydroxyl group in 2 resulted in the loss of activity against C. neoformans but still retained the inhibition of As. fumigatus in a lower concentration than that of the respective control, without showing any cytotoxic effects. In contrast, 25″-dehydroxy-dactylfungin A (3) exhibited improved activity against yeasts (Schizosaccharomyces pombe and Rhodotorula glutinis) than 1 and 2, but resulted in the appearance of slight cytotoxicity. The present study exemplifies how even in a well-studied taxonomic group such as the Chaetomiaceae, the investigation of novel taxa still brings chemistry novelty, as demonstrated in this first report of this antibiotic class for chaetomiaceous and sordarialean taxa.

Insight into the genomes of dominant yeast symbionts of European spruce bark beetle, Ips typographus.

Spruce bark beetle Ips typographus can trigger outbreaks on spruce that results in significant losses in the forest industry. It has been suggested that symbiotic microorganisms inhabiting the gut of bark beetles facilitate the colonization of plant tissues as they play a role in the detoxification of plant secondary metabolites, degrade plant cell wall and ameliorate beetle's nutrition. In this study, we sequenced and functionally annotated the genomes of five yeasts Kuraishia molischiana, Cryptococcus sp., Nakazawaea ambrosiae, Ogataea ramenticola, and Wickerhamomyces bisporus isolated from the gut of Ips typographus. Genome analysis identified 5314, 7050, 5722, 5502, and 5784 protein coding genes from K. molischiana, Cryptococcus sp., N. ambrosiae, O. ramenticola, and W. bisporus, respectively. Protein-coding sequences were classified into biological processes, cellular and molecular function based on gene ontology terms enrichment. Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation was used to predict gene functions. All analyzed yeast genomes contain full pathways for the synthesis of essential amino acids and vitamin B6, which have nutritional importance to beetle. Furthermore, their genomes contain diverse gene families related to the detoxification processes. The prevalent superfamilies are aldo-keto reductase, ATP-binding cassette and the major facilitator transporters. The phylogenetic relationships of detoxification-related enzymes aldo-keto reductase, and cytochrome P450 monooxygenase, and ATP-binding cassette are presented. Genome annotations also revealed presence of genes active in lignocellulose degradation. In vitro analyses did not confirm enzymatic endolytic degradation of lignocellulose; however, all species can utilize and pectin and produce a large spectrum of exolytic enzymes attacking cellulose, chitin, and lipids.

Higher white-nose syndrome fungal isolate yields from UV-guided wing biopsies compared with skin swabs and optimal culture media.

BACKGROUND: North American bat populations have suffered severe declines over the last decade due to the Pseudogymnoascus destructans fungus infection. The skin disease associated with this causative agent, known as white-nose syndrome (WNS), is specific to bats hibernating in temperate regions. As cultured fungal isolates are required for epidemiological and phylogeographical studies, the purpose of the present work was to compare the efficacy and reliability of different culture approaches based on either skin swabs or wing membrane tissue biopsies for obtaining viable fungal isolates of P. destructans. RESULTS: In total, we collected and analysed 69 fungal and 65 bacterial skin swabs and 51 wing membrane tissue biopsies from three bat species in the Czech Republic, Poland and the Republic of Armenia. From these, we obtained 12 viable P. destructans culture isolates. CONCLUSIONS: Our results indicated that the efficacy of cultures based on wing membrane biopsies were significantly higher. Cultivable samples tended to be based on collections from bats with lower body surface temperature and higher counts of UV-visualised lesions. While cultures based on both skin swabs and wing membrane tissue biopsies can be utilised for monitoring and surveillance of P. destructans in bat populations, wing membrane biopsies guided by UV light for skin lesions proved higher efficacy. Interactions between bacteria on the host's skin also appear to play an important role.

Seasonal Shifts in Bacterial and Fungal Microbiomes of Leaves and Associated Leaf-Mining Larvae Reveal Persistence of Core Taxa Regardless of Diet.

Microorganisms are key mediators of interactions between insect herbivores and their host plants. Despite a substantial interest in studying various aspects of these interactions, temporal variations in microbiomes of woody plants and their consumers remain understudied. In this study, we investigated shifts in the microbiomes of leaf-mining larvae (Insecta: Lepidoptera) and their host trees over one growing season in a deciduous temperate forest. We used 16S and ITS2 rRNA gene metabarcoding to profile the bacterial and fungal microbiomes of leaves and larvae. We found pronounced shifts in the leaf and larval microbiota composition and richness as the season progressed, and bacteria and fungi showed consistent patterns. The quantitative similarity between leaf and larval microbiota was very low for bacteria (~9%) and decreased throughout the season, whereas fungal similarity increased and was relatively high (~27%). In both leaves and larvae, seasonality, along with host taxonomy, was the most important factor shaping microbial communities. We identified frequently occurring microbial taxa with significant seasonal trends, including those more prevalent in larvae (Streptococcus, Candida sake, Debaryomyces prosopidis, and Neoascochyta europaea), more prevalent in leaves (Erwinia, Seimatosporium quercinum, Curvibasidium cygneicollum, Curtobacterium, Ceramothyrium carniolicum, and Mycosphaerelloides madeirae), and frequent in both leaves and larvae (bacterial strain P3OB-42, Methylobacterium/Methylorubrum, Bacillus, Acinetobacter, Cutibacterium, and Botrytis cinerea). Our results highlight the importance of considering seasonality when studying the interactions between plants, herbivorous insects, and their respective microbiomes, and illustrate a range of microbial taxa persistent in larvae, regardless of their occurrence in the diet. IMPORTANCE Leaf miners are endophagous insect herbivores that feed on plant tissues and develop and live enclosed between the epidermis layers of a single leaf for their entire life cycle. Such close association is a precondition for the evolution of more intimate host-microbe relationships than those found in free-feeding herbivores. Simultaneous comparison of bacterial and fungal microbiomes of leaves and their tightly linked consumers over time represents an interesting study system that could fundamentally contribute to the ongoing debate on the microbial residence of insect gut. Furthermore, leaf miners are ideal model organisms for interpreting the ecological and evolutionary roles of microbiota in host plant specialization. In this study, the larvae harbored specific microbial communities consisting of core microbiome members. Observed patterns suggest that microbes, especially bacteria, may play more important roles in the caterpillar holobiont than generally presumed.

Pre-invasion assessment on African invasive grasses revealed five new species of ergot fungi, Claviceps section Pusillae.

Ergot, the genus Claviceps comprises several deeply diverged lineages, recently classified as sections. Among them, the section Pusillae, is the most speciose, with a centre of distribution in Africa but occurring worldwide, often as a consequence of its invasive potential. This section includes the most severe plant pathogens such as Claviceps africana and C. gigantea, responsible for toxicoses and a significant reduction in the seed yields of Sorghum and Zea. In this study we surveyed ergot diversity in South Africa, focusing on grasses native to this region, but known for their high potential of invasiveness. The revision based on molecular and phenotypic markers revealed 16 species, with a high proportion of undescribed diversity, confirming Africa as a hot spot for this section. Five new species, Claviceps tulasnei, Claviceps eulaliae, Claviceps hypertheliae, Claviceps fredericksoniae and Claviceps arundinellae were described from Setaria, Eulalia, Hyperthelia, Miscanthus and Arundinella respectively. Claviceps texensis infecting Cenchrus, previously only identified from the same host in Texas, USA, was confirmed to be present in Africa, which is assumed to be its primary area of distribution. In addition, the host grass genus Anthephora is newly reported as a host of Claviceps digitariae. The most of the taxa were negligible concerning alkaloid production, with the exception of C. fredericksoniae, which is a sister of potent alkaloid producer C. africana, and produces mainly DH-ergosine, together with traces of DH-ergocornine. The host/parasite associations within Pusillae section is very narrow, suggesting that co-speciation is the major speciation driver in this group. Host grasses of the described species are already recognised invasive species and their ovarial parasites need to be monitored. This is highlighted by the fact that all Pusillae produced air-borne secondary conidia, which is autapomorphy of this section and considered to be important for their invasive abilities.

Fungi are more transient than bacteria in caterpillar gut microbiomes.

Despite an increasing number of studies on caterpillar (Insecta: Lepidoptera) gut microbiota, bacteria have been emphasized more than fungi. Therefore, we lack data on whether fungal microbiota is resident or transient and shaped by factors similar to those of bacteria. We sampled nine polyphagous caterpillar species from several tree species at multiple sites to determine the factors shaping leaf and gut bacterial and fungal microbiota as well as the extent to which caterpillars acquire microbiota from their diet. We performed 16S and ITS2 DNA metabarcoding of the leaves and guts to determine the composition and richness of the respective microbiota. While spatial variables shaped the bacterial and fungal microbiota of the leaves, they only affected fungi in the guts, whereas the bacteria were shaped primarily by caterpillar species, with some species harboring more specific bacterial consortia. Leaf and gut microbiota significantly differed; in bacteria, this difference was more pronounced. The quantitative similarity between leaves and guts significantly differed among caterpillar species in bacteria but not fungi, suggesting that some species have more transient bacterial microbiota. Our results suggest the complexity of the factors shaping the gut microbiota, while highlighting interspecific differences in microbiota residency within the same insect functional group.

Piperazine-Modified Ketoconazole Derivatives Show Increased Activity against Fungal and Trypanosomatid Pathogens.

Ketoconazole (KTZ) is an imidazole drug applied topically to treat numerous skin infections. However, as a systemic antifungal, KTZ' efficacy and safety no longer justify its use as a first-line treatment. Azole conjugates often display higher solubility and better antifungal activities than their parent azoles. Accordingly, we aimed at developing suitable linkers for clickable azole conjugation with a second antifungal molecule, and targeted drug delivery towards improving antifungal activity. For its low price and high availability, we selected KTZ as a molecular scaffold to introduce such chemical modifications. We prepared a series of piperazine-modified KTZ derivatives and we evaluated their in vitro antifungal and antitrypanosomal activity against fourteen strains of pathogenic fungi and two strains of Trypanosoma parasites. Several compounds were more effective against the pathogens than KTZ. Compound 5 was 24 times more potent against Aspergillus flavus and 8 times more potent against A. fumigatus than KTZ, with similarly low cytotoxicity to HEK cells up to 100 µM. Derivative 6 had 9- and 7-fold higher activity against T. brucei gambiense and T. brucei brucei than KTZ, respectively, and inhibited trypanosoma growth at single micromolar EC50 values. Combined, our findings will foster further research of piperazine-modified KTZs as promising antifungal and antiparasitic drugs towards enhancing the properties of both KTZ and other azole derivatives.

A re-assessment of Taxomyces andreanae, the alleged taxol-producing fungus, using comparative genomics.

The monotypic "bulbilliferous hyphomycete" genus Taxomyces was erected in 1993 for a fungal endophyte isolated from the Yew tree Taxus brevifolia and named Taxomyces andreanae. This fungus was reported to produce the plant-derived anti-cancer drug taxol. The original description of the fungus was not conclusive as to its taxonomic position because no sporulation or other salient morphological features were reported. Consequently, the taxonomic affinities of this fungus have remained obscure. However, a full genome sequence of this strain was generated by a German research group in 2013, in an unsuccessful attempt to detect the biosynthesis genes encoding for taxol. This prompted us to search for phylogenetic marker genes and compare those with the data that recently have become available from state-of-the-art polyphasic taxonomic studies. Surprisingly, the strain turned out to belong to the phlebioid clade of wood-destroying Basidiomycota as inferred from a comparison of its partial ITS, the 28S rDNA (LSU), the RNA polymerase II largest subunit (rpb1), the RNA polymerase II second largest subunit (rpb2), and the translation elongation factor 1-α (tef1) sequences. A multi gene genealogy based on these loci revealed that the closest relative is Ceriporiopsis (syn. Mycoacia) gilvescens. Even though such wood-destroying Basidiomycota are regularly encountered among the endophytic isolates after surface-disinfection of plant organs, the vast majority of the reported endophytic fungi belong to the Ascomycota. Nevertheless, the data available now allow for synonymizing Taxomyces with Ceriporiopsis, and the necessary new combination is made.

Characterization and microsatellite marker development for a common bark and ambrosia beetle associate, Geosmithia obscura.

Symbioses between Geosmithia fungi and wood-boring and bark beetles seldom result in disease induction within the plant host. Yet, exceptions exist such as Geosmithia morbida, the causal agent of Thousand Cankers Disease (TCD) of walnuts and wingnuts, and Geosmithia sp. 41, the causal agent of Foamy Bark Canker disease of oaks. Isolates of G. obscura were recovered from black walnut trees in eastern Tennessee and at least one isolate induced cankers following artificial inoculation. Due to the putative pathogenicity and lack of recovery of G. obscura from natural lesions, a molecular diagnostic screening tool was developed using microsatellite markers mined from the G. obscura genome. A total of 3256 candidate microsatellite markers were identified (2236, 789, 137 di-, tri-, and tetranucleotide motifs, respectively), with 2011, 703, 101 di-, tri-, and tetranucleotide motifs, respectively, containing markers with primers. From these, 75 microsatellite markers were randomly selected, screened, and optimized, resulting in 28 polymorphic markers that yielded single, consistently recovered bands, which were used in downstream analyses. Five of these microsatellite markers were found to be specific to G. obscura and did not cross-amplify into other, closely related species. Although the remaining tested markers could be useful, they cross-amplified within different Geosmithia species, making them not reliable for G. obscura detection. Five novel microsatellite markers (GOBS9, GOBS10, GOBS41, GOBS43, and GOBS50) were developed based on the G. obscura genome. These species-specific microsatellite markers are available as a tool for use in molecular diagnostics and can assist future surveillance studies.

Geosmithia Species Associated With Bark Beetles From China, With the Description of Nine New Species.

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 being reported from Taiwan, China. This study aimed to investigate the diversity of Geosmithia species in China. Field surveys in Fujian, Guangdong, Guangxi, Hunan, Jiangsu, Jiangxi, Shandong, Shanghai, and Yunnan yielded a total of 178 Geosmithia isolates from 12 beetle species. The isolates were grouped based on morphology. The internal transcribed spacer, ß-tubulin, and elongation factor 1-α gene regions of the representatives of each group were sequenced. Phylogenetic trees were constructed based on those sequences. In total, 12 species were identified, with three previously described species (Geosmithia xerotolerans, G. putterillii, and G. pallida) and nine new species which are described in this paper as G. luteobrunnea, G. radiata, G. brevistipitata, G. bombycina, G. granulata (Geosmithia sp. 20), G. subfulva, G. pulverea (G. sp. 3 and Geosmithia sp. 23), G. fusca, and G. pumila sp. nov. The dominant species obtained in this study were G. luteobrunnea and G. pulverea. This study systematically studied the Geosmithia species in China and made an important contribution to filling in the gaps in our understanding of global Geosmithia species diversity.

Fungal Endophytes of Vitis vinifera-Plant Growth Promoters or Potentially Toxinogenic Agents?

Fungal endophytes occurring in grapevine (Vitis vinifera L.) are usually important sources of various compounds with biological activities with great potential for use in agriculture. Nevertheless, many species isolated from this plant belong to the genera Fusarium, Alternaria, or Aspergillus, all of which are well-known to produce mycotoxins. Our study is focused on the assessment of the toxinogenic potential of fungal endophytes isolated from vineyards in the Czech Republic. In total, 20 endophytic fungal species were cultivated in wine must, and 57 mycotoxins of different classes were analysed by liquid chromatography coupled with mass spectrometry. As a result, alternariol, tentoxin, meleagrin, roquefortine C, gliotoxin, and verruculogen were detected in the culture medium, of which verruculogen followed by gliotoxin were the most frequent (present in 90 and 40% of samples, respectively) and most concentrated (up to thousands ng/mL). The alternaria mycotoxins alternariol and tentoxin were detected not only in Alternaria sp. cultures, but traces of these mycotoxins were also quantified in the Diatripe and Epicoccum cultures. Meleagrin and roquefortine C were detected in Didymella sancta and Penicillium crustosum, gliotoxin was detected in Alternaria sp., Didymella sp., Aureobasidium pullulans, Cladosporium herbarum, Penicillium crustosum and Pleurophoma ossicola, and verruculogen was quantified in 99% of endophytic isolates investigated. The potential of endophytes to produce mycotoxins should be carefully checked, specifically in cases where they are intended for the purpose of V. vinifera growing.