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Abstract Background Along bacteria, yeasts are common in forages and forage fermentations as spoilage microbes or as additives, yet few studies exist with species-level data on these fungi’s occurrence in feedstuff. Active dry yeast and other yeast-based products are also common feed additives in animal husbandry. Here, we aimed to characterize both fermented and non-fermented milking cow feedstuff samples from Hungary to assess their microbial diversity in the first such study from Central Europe. Results We applied long-read bacterial metabarcoding to 10 fermented and 25 non-fermented types of samples to assess bacterial communities and their characteristics, surveyed culturable mold and yeast abundance, and identified culturable yeast species. Fermented forages showed the abundance of Aerococcaceae, Bacillaceae, Brucellaceae, Lactobacillaceae, Staphylococcaceae, and Thermoactinomycetaceae, non-fermented ones had Cyanothecaceae, Enterobacteriaceae, Erwiniaceae, Gomontiellaceae, Oxalobacteraceae, Rhodobiaceae, Rickettsiaceae, and Staphylococcaceae. Abundances of bacterial families showed mostly weak correlation with yeast CFU numbers, only Microcoleaceae (positive) and Enterococcaceae and Alcaligenaceae (negative correlation) showed moderate correlation. We identified 14 yeast species, most commonly Diutina rugosa, Pichia fermentans, P. kudriavzevii, and Wickerhahomyces anomalus. We recorded S. cerevisiae isolates only from animal feed mixes with added active dry yeast, while the species was completely absent from fermented forages. The S. cerevisiae isolates showed high genetic uniformity. Conclusion Our results show that both fermented and non-fermented forages harbor diverse bacterial microbiota, with higher alpha diversity in the latter. The bacterial microbiome had an overall weak correlation with yeast abundance, but yeasts were present in the majority of the samples, including four new records for forages as a habitat for yeasts. Yeasts in forages mostly represented common species including opportunistic pathogens, along with a single strain of Saccharomyces used as a feed mix additive.

Measurement(s) species associations Technology Type(s) digital curation Factor Type(s) associations between Carabidae and Fungi Sample Characteristic - Organism Carabidae • Fungi • Laboulbeniales Sample Characteristic - Location global Machine-accessible metadata file describing the reported data: https://doi.org/10.6084/m9.figshare.14602770

Hesperomyces virescens (Ascomycota, Laboulbeniales), a fungal ectoparasite, is thus far reported on Harmonia axyridis from five continents: North and South America, Europe, Africa, and Asia. While it is known that He. virescens can cause mortality of Ha. axyridis under laboratory conditions, the role of biotic and abiotic factors in influencing the distribution of He. virescens in the field is unknown. We collected and screened 3,568 adult Ha. axyridis from 23 locations in seven countries in Central Europe between October and November 2018 to test the effect of selected host characters and climate and landscape variables on the infection probability with He. virescens. Mean parasite prevalence of He. virescens on Ha. axyridis was 17.9%, ranging among samples from 0 to 46.4%. Host sex, climate, and landscape composition did not have any significant effect on the infection probability of He. virescens on Ha. axyridis. Two color forms, f. conspicua and f. spectabilis, had a significantly lower parasite prevalence compared to the common Ha. axyridis f. novemdecimsignata.

The use of yeast-containing probiotics is on the rise; however, these products occasionally cause fungal infections and possibly even fungemia among susceptible probiotic-treated patients. The incidence of such cases is probably underestimated, which is why it is important to delve deeper into the pathomechanism and the adaptive features of S. ‘boulardii’. Here in this study, the potential role of the gene heme oxygenase-1 (HMX1) in probiotic yeast bloodstream-derived infections was studied by generating marker-free HMX1 deletion mutants with CRISPR/Cas9 technology from both commercial and clinical S. ‘boulardii’ isolates. The six commercial and clinical yeasts used here represented closely related but different genetic backgrounds as revealed by comparative genomic analysis. We compared the wild-type isolates against deletion mutants for their tolerance of iron starvation, hemolytic activity, as well as kidney burden in immunosuppressed BALB/c mice after lateral tail vein injection. Our results reveal that the lack of HMX1 in S. ‘boulardii’ significantly (p < 0.0001) increases the kidney burden of the mice in most genetic backgrounds, while at the same time causes decreased growth in iron-deprived media in vitro. These findings indicate that even a single-gene loss-of-function mutation can, surprisingly, cause elevated fitness in the host during an opportunistic systemic infection. Our findings indicate that the safety assessment of S. ‘boulardii’ strains should not only take strain-to-strain variation into account, but also avoid extrapolating in vitro results to in vivo virulence factor determination.

Species of the highly diverse fungal genus Aspergillus are well-known agricultural pests, and, most importantly, producers of various mycotoxins threatening food safety worldwide. Mycotoxins are studied predominantly from the perspectives of human and livestock health. Meanwhile, their roles are far less known in nature. However, to understand the factors behind mycotoxin production, the roles of the toxins of Aspergilli must be understood from a complex ecological perspective, taking mold-plant, mold-microbe, and mold-animal interactions into account. The Aspergilli may switch between saprophytic and pathogenic lifestyles, and the production of secondary metabolites, such as mycotoxins, may vary according to these fungal ways of life. Recent studies highlighted the complex ecological network of soil microbiotas determining the niches that Aspergilli can fill in. Interactions with the soil microbiota and soil macro-organisms determine the role of secondary metabolite production to a great extent. While, upon infection of plants, metabolic communication including fungal secondary metabolites like aflatoxins, gliotoxin, patulin, cyclopiazonic acid, and ochratoxin, influences the fate of both the invader and the host. In this review, the role of mycotoxin producing Aspergillus species and their interactions in the ecosystem are discussed. We intend to highlight the complexity of the roles of the main toxic secondary metabolites as well as their fate in natural environments and agriculture, a field that still has important knowledge gaps.

Aflatoxins are wide-spread harmful carcinogenic secondary metabolites produced by Aspergillus species, which cause serious feed and food contaminations and affect farm animals deleteriously with acute or chronic manifestations of mycotoxicoses. On farm, both pre-harvest and post-harvest strategies are applied to minimize the risk of aflatoxin contaminations in feeds. The great economic losses attributable to mycotoxin contaminations have initiated a plethora of research projects to develop new, effective technologies to prevent the highly toxic effects of these secondary metabolites on domestic animals and also to block the carry-over of these mycotoxins to humans through the food chain. Among other areas, this review summarizes the latest findings on the effects of silage production technologies and silage microbiota on aflatoxins, and it also discusses the current applications of probiotic organisms and microbial products in feeding technologies. After ingesting contaminated foodstuffs, aflatoxins are metabolized and biotransformed differently in various animals depending on their inherent and acquired physiological properties. These mycotoxins may cause primary aflatoxicoses with versatile, species-specific adverse effects, which are also dependent on the susceptibility of individual animals within a species, and will be a function of the dose and duration of aflatoxin exposures. The transfer of these undesired compounds from contaminated feed into food of animal origin and the aflatoxin residues present in foods become an additional risk to human health, leading to secondary aflatoxicoses. Considering the biological transformation of aflatoxins in livestock, this review summarizes (i) the metabolism of aflatoxins in different animal species, (ii) the deleterious effects of the mycotoxins and their derivatives on the animals, and (iii) the major risks to animal health in terms of the symptoms and consequences of acute or chronic aflatoxicoses, animal welfare and productivity. Furthermore, we traced the transformation and channeling of Aspergillus-derived mycotoxins into food raw materials, particularly in the case of aflatoxin contaminated milk, which represents the major route of human exposure among animal-derived foods. The early and reliable detection of aflatoxins in feed, forage and primary commodities is an increasingly important issue and, therefore, the newly developed, easy-to-use qualitative and quantitative aflatoxin analytical methods are also summarized in the review.

Abstract Background Bat flies (Diptera: Nycteribiidae and Streblidae) are obligate, blood-sucking ectoparasites of bats with specialized morphology, life-cycle and ecology. Bat flies are occasionally infected by different species of Laboulbeniales (Fungi: Ascomycota), microscopic fungal ectoparasites belonging to three genera: Arthrorynchus spp. are restricted to the Eastern Hemisphere, while species of Gloeandromyces and Nycteromyces occur on Neotropical bat flies. Little is known about the distribution and host specificity of Arthrorynchus spp. on bat flies. In this study, we focused on sampling bat flies from the cave-dwelling bat species Miniopterus schreibersii. Bat and ectoparasite collection took place in Albania, Croatia, Hungary, Italy, Portugal, Slovakia, Spain and Switzerland. Flies were inspected for Laboulbeniales infections. Results Six hundred sixty seven bat flies of five species were collected: Nycteribia latreillii, N. pedicularia, N. schmidlii, Penicillidia conspicua, and P. dufourii. Laboulbeniales infection was observed on 60 specimens (prevalence = 9%). Two Laboulbeniales species, Arthrorhynchus eucampsipodae and A. nycteribiae, were present on three bat fly species. All observations of A. eucampsipodae were on N. schmidlii, and A. nycteribiae was present on P. conspicua and P dufourii. Arthrorhynchus eucampsipodae is, for the first time, reported from Slovakia and Spain. Arthrorhynchus nycteribiae represents a new country record for Portugal and Slovakia. There were no significant differences among infection rates in different countries. Females of N. schmidlii showed a higher infection rate than males with an observable trend (P = 0.0502). No sex differences in infection rate for P. conspicua and P. dufourii were detected. Finally, thallus density was significantly lower in N. schmidlii compared to P. conspicua and P. dufourii. Conclusions With this study, we contribute to the knowledge of the geographical distribution and host specificity of Laboulbeniales fungi associated with ectoparasitic bat flies within Europe. We discuss parasite prevalence and host specificity in the light of our findings and the available literature. Penicillidia conspicua is unambiguously the main host species for A. nycteribiae based on our and previous findings. Differences in parasite intensity and sex-biased infections of the fungi are possible depending on the species.

Public awareness has been raised on the importance of natural history and academic collections for science and society in a time when reduced financial support and staff cuts are prevalent. In the field of biology, new species and new interspecies associations are constantly discovered by making use of museum collections, digitalised materials or citizen science programs. In our study, the Myrmica Latreille, 1804 image collection of AntWeb.org was screened for fungal ectoparasites. A total of 397 imaged specimens from 133 species were visually investigated. A single specimen of M. hellenica Finzi, 1926, collected in Greece by U. Sahlberg, showed a conspicuous fungal infection. The parasite was identified using microscopic methods as Rickia wasmannii Cavara, an ectoparasitic fungal species specialised to Myrmica ants. This finding represents a new country record and a new Myrmica species for the host spectrum of R. wasmannii. According to our results, online entomological databases can be screened relatively easily for ectoparasitic fungal infections from new hosts and new regions. However, depending on quality of the insect voucher photos, additional investigation of the material could be needed to confirm the identity of the parasite.

Saccharomyces yeast probiotics (S. ‘boulardii’) have long been applied in the treatment of several gastrointestinal conditions. Despite their widespread use, they are rare opportunistic pathogens responsible for a high proportion of Saccharomyces mycosis cases. The potential virulence attributes of S. ‘boulardii’ as well as its interactions with the human immune system have been studied, however, no information is available on how these yeasts may change due to in-host evolution. To fill this gap, we compared the general phenotypic characteristics, cell morphology, virulence factors, epithelial and immunological interactions, and pathogenicity of four probiotic product samples, two mycosis, and eight non-mycosis samples of S. ‘boulardii’. We assessed the characteristics related to major steps of yeast infections. Mycosis and non-mycosis isolates both displayed novel characters when compared to the product isolates, but in the case of most virulence factors and in pathogenicity, differences were negligible or, surprisingly, the yeasts from products showed elevated levels. No isolates inflicted considerable damage to the epithelial model or bore the hallmarks of immune evasion. Our results show that strains in probiotic products possess characteristics that enable them to act as pathogens upon permissive conditions, and their entry into the bloodstream is not due to active mechanisms but depends on the host. Survival in the host is dependent on yeast phenotypic characteristics which may change in many ways once they start evolving in the host. These facts call attention to the shortcomings of virulence phenotyping in yeast research, and the need for a more thorough assessment of probiotic use.

The polyphyletic group of black fungi within the Ascomycota (Arthoniomycetes, Dothideomycetes, and Eurotiomycetes) is ubiquitous in natural and anthropogenic habitats. Partly because of their dark, melanin-based pigmentation, black fungi are resistant to stresses including UV- and ionizing-radiation, heat and desiccation, toxic metals, and organic pollutants. Consequently, they are amongst the most stunning extremophiles and poly-extreme-tolerant organisms on Earth. Even though ca. 60 black fungal genomes have been sequenced to date, [mostly in the family Herpotrichiellaceae (Eurotiomycetes)], the class Dothideomycetes that hosts the largest majority of extremophiles has only been sparsely sampled. By sequencing up to 92 species that will become reference genomes, the “Shed light in The daRk lineagES of the fungal tree of life” (STRES) project will cover a broad collection of black fungal diversity spread throughout the Fungal Tree of Life. Interestingly, the STRES project will focus on mostly unsampled genera that display different ecologies and life-styles (e.g., ant- and lichen-associated fungi, rock-inhabiting fungi, etc.). With a resequencing strategy of 10- to 15-fold depth coverage of up to ~550 strains, numerous new reference genomes will be established. To identify metabolites and functional processes, these new genomic resources will be enriched with metabolomics analyses coupled with transcriptomics experiments on selected species under various stress conditions (salinity, dryness, UV radiation, oligotrophy). The data acquired will serve as a reference and foundation for establishing an encyclopedic database for fungal metagenomics as well as the biology, evolution, and ecology of the fungi in extreme environments.

The internal transcribed spacer (ITS) region (ITS1, 5.8S rDNA, and ITS2) separates the genes coding for the SSU 18S and the LSU 26S genes in the rDNA units which are organized into long tandem arrays in the overwhelming majority of fungi. As members of a multigenic family, these units are subject of concerted evolution, which homogenizes their sequences. Exceptions have been observed in certain groups of plants and in a few fungal species. In our previous study we described exceptionally high degree of sequence diversity in the D1/D2 domains of two pulcherrimin-producing Metschnikowia (Saccharomycotina) species which appeared to evolve by reticulation. The major goals of this study were the examination of the diversity of the ITS segments and their evolution. We show that the ITS sequences of these species are not homogenized either, differ from each other by up to 38 substitutions and indels which have dramatic effects on the predicted secondary structures of the transcripts. The high intragenomic diversity makes the D1/D2 domains and the ITS spacers unsuitable for barcoding of these species and therefore the taxonomic position of strains previously assigned to them needs revision. By analyzing the genome sequence of the M. fructicola type strain, we also show that the rDNA of this species is fragmented, contains pseudogenes and thus evolves by the birth-and-death mechanism rather than by homogenisation, which is unusual in yeasts. The results of the network analysis of the sequences further indicate that the ITS regions are also involved in reticulation. M. andauensis and M. fructicola can form interspecies hybrids and their hybrids segregate, providing thus possibilities for reticulation of the rDNA repeats.

Evolution and genome stabilization have mostly been studied on the Saccharomyces hybrids isolated from natural and alcoholic fermentation environments. Genetic and phenotypic properties have usually been compared to the laboratory and reference strains, as the true ancestors of the natural hybrid yeasts are unknown. In this way the exact impact of different parental fractions on the genome organization or metabolic activity of the hybrid yeasts is difficult to resolve completely. In the present work the evolution of geno- and phenotypic properties is studied in the interspecies hybrids created by the cross-breeding of S. cerevisiae with S. uvarum or S. kudriavzevii auxotrophic mutants. We hypothesized that the extent of genomic alterations in S. cerevisiae × S. uvarum and S. cerevisiae × S. kudriavzevii should affect the physiology of their F1 offspring in different ways. Our results, obtained by amplified fragment length polymorphism (AFLP) genotyping and karyotyping analyses, showed that both subgenomes of the S. cerevisiae x S. uvarum and of S. cerevisiae × S. kudriavzevii hybrids experienced various modifications. However, the S. cerevisiae × S. kudriavzevii F1 hybrids underwent more severe genomic alterations than the S. cerevisiae × S. uvarum ones. Generation of the new genotypes also influenced the physiological performances of the hybrids and the occurrence of novel phenotypes. Significant differences in carbohydrate utilization and distinct growth dynamics at increasing concentrations of sodium chloride, urea and miconazole were observed within and between the S. cerevisiae × S. uvarum and S. cerevisiae × S. kudriavzevii hybrids. Parental strains also demonstrated different contributions to the final metabolic outcomes of the hybrid yeasts. A comparison of the genotypic properties of the artificial hybrids with several hybrid isolates from the wine-related environments and wastewater demonstrated a greater genetic variability of the S. cerevisiae × S. kudriavzevii hybrids. Saccharomyces cerevisiae × S. uvarum artificial and natural hybrids showed considerable differences in osmolyte tolerance and sensitivity to miconazole, whereas the S. cerevisiae × S. kudriavzevii hybrids exhibited differences also in maltotriose utilization. The results of this study suggest that chromosomal rearrangements and genomic reorganizations as post-hybridization processes may affect the phenotypic properties of the hybrid progeny substantially. Relative to their ancestors, the F1 segregants may generate different phenotypes, indicating novel routes of evolution in response to environmental growth conditions.

Protein phosphatase Z is a "novel type" fungus specific serine/threonine protein phosphatase. Previously our research group identified the CaPPZ1 gene in the opportunistic pathogen Candida albicans and reported that the gene deletion had several important physiological consequences. In order to reveal the protein targets and the associated mechanisms behind the functions of the phosphatase a proteomic method was adopted for the comparison of the cappz1 deletion mutant and the genetically matching QMY23 control strain. Proteins extracted from the control and deletion mutant strains were separated by two-dimensional gel electrophoresis and the protein spots were stained with RuBPS and Pro-Q Diamond in order to visualize the total proteome and the phosphoproteome, respectively. The alterations in spot intensities were determined by densitometry and were analysed with the Delta2D (Decodon) software. Spots showing significantly different intensities between the mutant and control strains were excised from the gels and were digested with trypsin. The resulting peptides were identified by LC-MS/MS mass spectrometry. As many as 15 protein spots were found that exhibited significant changes in their intensity upon the deletion of the phosphatase and 20 phosphoproteins were identified in which the level of phosphorylation was modified significantly in the mutant. In agreement with previous findings we found that the affected proteins function in protein synthesis, oxidative stress response, regulation of morphology and metabolism. Among these proteins we identified two potential CaPpz1 substrates (Eft2 and Rpp0) that may regulate the elongation step of translation. RT-qPCR experiments revealed that the expression of the genes coding for the affected proteins was not altered significantly. Thus, the absence of CaPpz1 exerted its effects via protein synthesis/degradation and phosphorylation/dephosphorylation. In addition, our proteomics data strongly suggested a role for CaPpz1 in biofilm formation, was confirmed experimentally. Thus our unbiased proteomic approach lead to the discovery of a novel function for this phosphatase in C. albicans.

Rickia wasmannii Cavara (Ascomycota: Laboulbeniales) is an ectoparasitic fungus infecting Myrmica ants. Ant-parasitic Laboulbeniales and their interactions with the hosts have been in the focus of several studies. To assess the effects of these fungi, comparison of infected and uninfected or completely treated ants are needed. So far, treating Laboulbeniales infection was only achieved with cockroaches, but not with ants. We present a simple, yet relatively long, AgNP topical treatment that reduces or eliminates Rickia infection from Myrmica scabrinodis ants without affecting their lifespan. We discuss the possibilities of the proposed treatment in the light of the biology of Rickia.

Rickia lenoirii has been reported in seven localities in the Carpathian Basin, six in Hungary and one in Romania, on Messor structor (Hymenoptera: Formicidae) host specimens. This is the first occurrence of this fungus in two new (Pannonian and Continental) biogeographic regions. According to our findings, the northernmost (47°31'33.01"N) known occurrence of R. lenoirii is Ferenc-hegy (Ferenc Hill) in Budapest. These results demonstrate that ant-parasitic Laboulbeniales fungi may have wider distribution areas than previously expected by mycologists and myrmecologists.

Laboulbenia camponoti Batra, 1963 (Ascomycetes: Laboulbeniales), has been found on Camponotus aethiops (Latreille, 1798) (Hymenoptera: Formicidae) workers in the Carpathian Basin: in Baziaş, Caraş-Severin (Romania), and Vienna (Austria). Vienna is the northernmost known locality of this fungus (48°12' N). These new observations expand the area of L. camponoti from regions with Mediterranean and subtropical climatic influences to the common borders of the Continental and Pannonian regions. These results show that Camponotus samples from other climatic regions should be examined more closely for this fungal parasite.

Modern taxonomy of yeasts is mainly based on phylogenetic analysis of conserved DNA and protein sequences. By far the most frequently used sequences are those of the repeats of the chromosomal rDNA array. It is generally accepted that the rDNA repeats of a genome have identical sequences due to the phenomenon of sequence homogenisation and can thus be used for identification and barcoding of species. Here we show that the rDNA arrays of the type strains of Metschnikowia andauensis and M. fructicola are not homogenised. Both have arrays consisting of diverse repeats that differ from each other in the D1/D2 domains by up to 18 and 25 substitutions. The variable sites are concentrated in two regions that correspond to back-folding stretches of hairpin loops in the predicted secondary structure of the RNA molecules. The substitutions do not alter significantly the overall hairpin-loop structure due to wobble base pairing at sites of C-T transitions and compensatory mutations in the complementary strand of the hairpin stem. The phylogenetic and network analyses of the cloned sequences revealed that the repeats had not evolved in a vertical tree-like way but reticulation might have shaped the rDNA arrays of both strains. The neighbour-net analysis of all cloned sequences of the type strains and the database sequences of different strains further showed that these species share a continuous pool of diverse repeats that appear to evolve by reticulate evolution.