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Members of the family Trichomeriaceae, belonging to the Chaetothyriales order and the Ascomycota phylum, are known for their capability to inhabit hostile environments characterized by extreme temperatures, oligotrophic conditions, drought, or presence of toxic compounds. The genus Knufia encompasses many polyextremophilic species. In this report, the genomic and morphological features of the strain FJI-L2-BK-P2 presented, which was isolated from the Mars 2020 mission spacecraft assembly facility located at the Jet Propulsion Laboratory in Pasadena, California. The identification is based on sequence alignment for marker genes, multi-locus sequence analysis, and whole genome sequence phylogeny. The morphological features were studied using a diverse range of microscopic techniques (bright field, phase contrast, differential interference contrast and scanning electron microscopy). The phylogenetic marker genes of the strain FJI-L2-BK-P2 exhibited highest similarities with type strain of Knufia obscura (CBS 148926T) that was isolated from the gas tank of a car in Italy. To validate the species identity, whole genomes of both strains (FJI-L2-BK-P2 and CBS 148926T) were sequenced, annotated, and strain FJI-L2-BK-P2 was confirmed as K. obscura. The morphological analysis and description of the genomic characteristics of K. obscura FJI-L2-BK-P2 may contribute to refining the taxonomy of Knufia species. Key morphological features are reported in this K. obscura strain, resembling microsclerotia and chlamydospore-like propagules. These features known to be characteristic features in black fungi which could potentially facilitate their adaptation to harsh environments.

Most fungal bone and joint infections (arthritis) are caused by Mucormycosis (Mucor indicus). These infections may be difficult to treat and may lead to chronic bone disorders and disabilities, thus the use of new antifungal materials in bone disorders is vital, particularly in immunocompromised individuals, such as those who have contracted coronavirus disease 2019 (COVID-19). Herein, we reported for the first time the preparation of nitrogen-doped carbon quantum dots (N/CQDs) and a nitrogen-doped mesoporous carbon (N/MC) using a quick micro-wave preparation and hydrothermal approach. The structure and morphology were analysed using X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM) and surface area analyser. Minimum inhibitory concentration (MIC), disc diffusion tests, minimum fungicidal concentration (MFC) and antifungal inhibitory percentages were measured to investigate the antifungal activity of N/CQDs and N/MC nanostructures. In addition to the in vivo antifungal activity in rats as determined by wound induction and infection, pathogen count and histological studies were also performed. According to in vitro and in vivo testing, both N/CQDs with small size and N/MC with porous structure had a significant antifungal impact on a variety of bone-infecting bacteria, including Mucor infection. In conclusion, the present investigation demonstrates that functional N/CQDs and N/MC are effective antifungal agents against a range of microbial pathogenic bone disorders in immunocompromised individuals, with stronger and superior fungicidal activity for N/CQDs than N/MC in vitro and in vivo studies.

Cryomyces antarcticus, a melanized cryptoendolithic fungus endemic to Antarctica, can tolerate environmental conditions as severe as those in space. Particularly, its ability to withstand ionizing radiation has been attributed to the presence of thick and highly melanized cell walls, which—according to a previous investigation—may contain both 1,8‐dihydroxynaphthalene (DHN) and L‐3,4 dihydroxyphenylalanine (L‐DOPA) melanin. The genes putatively involved in the synthesis of DHN melanin were identified in the genome of C. antarcticus. Most important is capks1 encoding a non‐reducing polyketide synthase (PKS) and being the ortholog of the functionally characterized kppks1 from the rock‐inhabiting fungus Knufia petricola. The co‐expression of CaPKS1 or KpPKS1 with a 4′‐phosphopantetheinyl transferase in Saccharomyces cerevisiae resulted in the formation of a yellowish pigment, suggesting that CaPKS1 is the enzyme providing the precursor for DHN melanin. To dissect the composition and function of the melanin layer in the outer cell wall of C. antarcticus, non‐melanized mutants were generated by CRISPR/Cas9‐mediated genome editing. Notwithstanding its slow growth (up to months), three independent non‐melanized Δcapks1 mutants were obtained. The mutants exhibited growth similar to the wild type and a light pinkish pigmentation, which is presumably due to carotenoids. Interestingly, visible light had an adverse effect on growth of both melanized wild‐type and non‐melanized Δcapks1 strains. Further evidence that light can pass the melanized cell walls derives from a mutant expressing a H2B‐GFP fusion protein, which can be detected by fluorescence microscopy. In conclusion, the study reports on the first genetic manipulation of C. antarcticus, resulting in non‐melanized mutants and demonstrating that the melanin is rather of the DHN type. These mutants will allow to elucidate the relevance of melanization for surviving extreme conditions found in the natural habitat as well as in space. [ABSTRACT FROM AUTHOR]

Meristematic fungi are mainly defined as having aggregates of thick-walled, melanised cells enlarging and reproducing by isodiametric division. Dothideomycetes black meristematic and meristematic-like fungi have been allied to Myriangiales, which currently has two accepted families, Myriangiaceae and Elsinoaceae, with fungi mainly regarded as pathogens, parasites, saprobes and epiphytes of different plant species. This study aimed to verify the phylogenetic position using four nuclear markers (SSU, LSU, ITS and RPB2) of the incertae sedis genera associated with Myriangiales, namely Endosporium, Gobabebomyces, Lembosiniella and Phaeosclera, and the new genus, Endophytium gen. nov. (including E. albocacti sp. nov. and E. cacti sp. nov.), established for endophytic fungi occurring in cacti in Brazil. Based on morphology, lifestyle and phylogenetic inferences, these black meristematic and meristematic-like fungi cannot be accommodated in Myriangiales. Combining these results, three new orders and two new families are introduced: Endophytiales ord. nov. (including Endophytiaceae fam. nov. for Endophytium gen. nov.), Endosporiales ord. nov. (including Endosporiaceae for Endosporium) and Phaeosclerales ord. nov. (including Phaeoscleraceae fam. nov. for Phaeosclera). Gobabebomyces and Lembosiniella remained incertae sedis due to their disposition in the phylogenetic tree, that moved among clades accordingly with the gene analysed. Our results show that the inclusion of endophytic fungi obtained from plants in dry forests can contribute to the discovery of new taxa, clarify the phylogenetic position of allied taxa and confer information to the estimation of national and global fungal diversity. [ABSTRACT FROM AUTHOR]

Most fungal bone and joint infections (arthritis) are caused by Mucormycosis (Mucor indicus). These infections may be difficult to treat and may lead to chronic bone disorders and disabilities, thus the use of new antifungal materials in bone disorders is vital, particularly in immunocompromised individuals, such as those who have contracted coronavirus disease 2019 (COVID-19). Herein, we reported for the first time the preparation of nitrogen-doped carbon quantum dots (N/CQDs) and a nitrogen-doped mesoporous carbon (N/MC) using a quick micro-wave preparation and hydrothermal approach. The structure and morphology were analysed using X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM) and surface area analyser. Minimum inhibitory concentration (MIC), disc diffusion tests, minimum fungicidal concentration (MFC) and antifungal inhibitory percentages were measured to investigate the antifungal activity of N/CQDs and N/MC nanostructures. In addition to the in vivo antifungal activity in rats as determined by wound induction and infection, pathogen count and histological studies were also performed. According to in vitro and in vivo testing, both N/CQDs with small size and N/MC with porous structure had a significant antifungal impact on a variety of bone-infecting bacteria, including Mucor infection. In conclusion, the present investigation demonstrates that functional N/CQDs and N/MC are effective antifungal agents against a range of microbial pathogenic bone disorders in immunocompromised individuals, with stronger and superior fungicidal activity for N/CQDs than N/MC in vitro and in vivo studies. [ABSTRACT FROM AUTHOR]

Abstract The black fungus Exophiala causes a wide range of infections from superficial to subcutaneous, but also invasive fungal infections in immunocompromised patients as well as healthy individuals. In addition, Exophiala, is a common colonizer of the air ways of patients with cystic fibrosis. However, the source of infection and mode of transmission is still unclear. The aim of this study was to investigate the presence of Exophiala in samples collected from Swedish indoor environments. We found that the Exophiala species were commonly found in dishwashers and that Exophiala dermatitidis was the most common Exophiala species, being identified in 70% (26 out of the 37) of samples. Almost all E. dermatitidis isolates had the ability to grow at 42 °C (P = 0.0002) and were catalase positive. Voriconazole and posaconazole exhibited the lowest MICs, while caspofungin and anidulafungin lack the antifungal activities in vitro. Future studies are needed to illuminate the transmission mode of the fungi.

The black fungus Exophiala causes a wide range of infections from superficial to subcutaneous, but also invasive fungal infections in immunocompromised patients as well as healthy individuals. In addition, Exophiala, is a common colonizer of the air ways of patients with cystic fibrosis. However, the source of infection and mode of transmission is still unclear. The aim of this study was to investigate the presence of Exophiala in samples collected from Swedish indoor environments. We found that the Exophiala species were commonly found in dishwashers and that Exophiala dermatitidis was the most common Exophiala species, being identified in 70% (26 out of the 37) of samples. Almost all E. dermatitidis isolates had the ability to grow at 42 °C (P = 0.0002) and were catalase positive. Voriconazole and posaconazole exhibited the lowest MICs, while caspofungin and anidulafungin lack the antifungal activities in vitro. Future studies are needed to illuminate the transmission mode of the fungi. [ABSTRACT FROM AUTHOR]

Abstract Members of the family Trichomeriaceae, belonging to the Chaetothyriales order and the Ascomycota phylum, are known for their capability to inhabit hostile environments characterized by extreme temperatures, oligotrophic conditions, drought, or presence of toxic compounds. The genus Knufia encompasses many polyextremophilic species. In this report, the genomic and morphological features of the strain FJI-L2-BK-P2 presented, which was isolated from the Mars 2020 mission spacecraft assembly facility located at the Jet Propulsion Laboratory in Pasadena, California. The identification is based on sequence alignment for marker genes, multi-locus sequence analysis, and whole genome sequence phylogeny. The morphological features were studied using a diverse range of microscopic techniques (bright field, phase contrast, differential interference contrast and scanning electron microscopy). The phylogenetic marker genes of the strain FJI-L2-BK-P2 exhibited highest similarities with type strain of Knufia obscura (CBS 148926T) that was isolated from the gas tank of a car in Italy. To validate the species identity, whole genomes of both strains (FJI-L2-BK-P2 and CBS 148926T) were sequenced, annotated, and strain FJI-L2-BK-P2 was confirmed as K. obscura. The morphological analysis and description of the genomic characteristics of K. obscura FJI-L2-BK-P2 may contribute to refining the taxonomy of Knufia species. Key morphological features are reported in this K. obscura strain, resembling microsclerotia and chlamydospore-like propagules. These features known to be characteristic features in black fungi which could potentially facilitate their adaptation to harsh environments.

The survival of pathogenic fungi in the host after invasion depends on their ability to obtain nutrients, which include the transition metal zinc. This essential micronutrient is required to maintain the structure and function of various proteins and, therefore, plays a critical role in various biological processes. The host's nutritional immunity limits the availability of zinc to pathogenic fungi mainly by the action of calprotectin, a component of neutrophil extracellular traps. Here we investigated the adaptive responses of Fonsecaea pedrosoi to zinc-limiting conditions. This black fungus is the main etiological agent of chromoblastomycosis, a chronic neglected tropical disease that affects subcutaneous tissues. Following exposure to a zinc-limited environment, F. pedrosoi induces a high-affinity zinc uptake machinery, composed of zinc transporters and the zincophore Pra1. A proteomic approach was used to define proteins regulated by zinc deprivation. Cell wall remodeling, changes in neutral lipids homeostasis, and activation of the antioxidant system were the main strategies for survival in the hostile environment. Furthermore, the downregulation of enzymes required for sulfate assimilation was evident. Together, the adaptive responses allow fungal growth and development and reveals molecules that may be related to fungal persistence in the host. [ABSTRACT FROM AUTHOR]

In the harshest environmental conditions of the Antarctic desert, normally incompatible with active life, microbes are adapted to exploit the cryptoendolithic habitat (i.e., pore spaces of rocks) and represent the predominant life-forms. In the rocky niche, microbes take advantage of the thermal buffering, physical stability, protection against UV radiation, excessive solar radiation, and water retention-of paramount importance in one of the driest environments on Earth. In this work, high-throughput sequencing and culture-dependent approaches have been combined, for the first time, to untangle the diversity and distribution of black fungi in the Antarctic cryptoendolithic microbial communities, hosting some of the most extreme-tolerant microorganisms. Rock samples were collected in a vast area, along an altitudinal gradient and opposite sun exposure-known to influence microbial diversity-with the aim to compare and integrate results gained with the two approaches. Among black fungi, Friedmanniomyces endolithicus was confirmed as the most abundant taxon. Despite the much stronger power of the high-throughput sequencing, several species were not retrieved with DNA sequencing and were detectable by cultivation only. We conclude that both culture-dependent and -independent analyses are needed for a complete overview of black fungi diversity. The reason why some species remain undetectable with molecular methods are speculated upon. The effect of environmental parameters such as sun exposure on relative abundance was clearer if based on the wider biodiversity detected with the molecular approach.

Black fungi are a group of melanotic microfungi characterized by remarkable polyextremotolerance. Due to a broad ecological plasticity and adaptations at the cellular level, it is predicted that they may survive in a variety of extreme environments, including harsh niches on Earth and Mars, and in outer space. However, the molecular mechanisms aiding survival, especially in space, are yet to be fully elucidated. Based on these premises, the rock-inhabiting black fungus Knufia chersonesos (Wt) and its non-melanized mutant (Mut) were exposed to simulated microgravity-one of the prevalent features characterizing space conditions-by growing the cultures in high-aspect-ratio vessels (HARVs). Qualitative and quantitative proteomic analyses were performed on the mycelia and supernatant of culture medium (secretome) to assess alterations in cell physiology in response to low-shear simulated microgravity (LSSMG) and to ultimately evaluate the role of cell-wall melanization in stress survival. Differential expression was observed for proteins involved in carbohydrate and lipid metabolic processes, transport, and ribosome biogenesis and translation via ribosomal translational machinery. However, no evidence of significant activation of stress components or starvation response was detected, except for the scytalone dehydratase, enzyme involved in the synthesis of dihydroxynaphthalene (DNH) melanin, which was found to be upregulated in the secretome of the wild type and downregulated in the mutant. Differences in protein modulation were observed between K. chersonesos Wt and Mut, with several proteins being downregulated under LSSMG in the Mut when compared to the Wt. Lastly, no major morphological alterations were observed following exposure to LSSMG. Similarly, the strains' survivability was not negatively affected. This study is the first to characterize the response to simulated microgravity in black fungi, which might have implications on future astrobiological missions.

The survival of living organisms depends on iron, one of the most abundant metals in the Earth's crust. Nevertheless, this micronutrient is poorly available in our aerobic atmosphere as well as inside the mammalian host. This problem is circumvented by the expression of high affinity iron uptake machineries, including the production of siderophores, in pathogenic fungi. Here we demonstrated that F. pedrosoi , the causative agent of the neglected tropical disease chromoblastomycosis, presents gene clusters for siderophore production. In addition, ten putative siderophore transporters were identified. Those genes are upregulated under iron starvation, a condition that induces the secretion of hydroxamates, as revealed by chrome azurol S assays. RP-HPLC and mass spectrometry analysis allowed the identification of ferricrocin as an intra- and extracellular siderophore. F. pedrosoi can grow in different iron sources, including the bacterial ferrioxamine B and the host proteins ferritin, hemoglobin and holotransferrin. Of note, addition of hemoglobin, lactoferrin and holotransferrin to the growth medium of macrophages infected with F. pedrosoi enhanced significantly fungal survival. The ability to produce siderophores in iron limited conditions added to the versatility to utilize different sources of iron are strategies that certainly may contribute to fungal survival inside the host. [ABSTRACT FROM AUTHOR]

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.

Iron is a micronutrient required by almost all living organisms. Despite being essential, the availability of this metal is low in aerobic environments. Additionally, mammalian hosts evolved strategies to restrict iron from invading microorganisms. In this scenario, the survival of pathogenic fungi depends on high-affinity iron uptake mechanisms. Here, we show that the production of siderophores and the reductive iron acquisition system (RIA) are employed by Cladophialophora carrionii under iron restriction. This black fungus is one of the causative agents of chromoblastomycosis, a neglected subcutaneous tropical disease. Siderophore biosynthesis genes are arranged in clusters and, interestingly, two RIA systems are present in the genome. Orthologs of putative siderophore transporters were identified as well. Iron starvation regulates the expression of genes related to both siderophore production and RIA systems, as well as of two transcription factors that regulate iron homeostasis in fungi. A chrome azurol S assay demonstrated the secretion of hydroxamate-type siderophores, which were further identified via RP-HPLC and mass spectrometry as ferricrocin. An analysis of cell extracts also revealed ferricrocin as an intracellular siderophore. The presence of active high-affinity iron acquisition systems may surely contribute to fungal survival during infection. [ABSTRACT FROM AUTHOR]

Background: The skin is the first line of defence against communities of resident viruses, bacteria and fungi. The composition of the microbiome might change with factors related to the environment and host. The microbiome is dominated by bacteria. Dermatophytes and yeasts are the predominant fungi that are also involved in opportunistic infections of skin, hair and nails. Among environmental fungi, Chaetothyriales (black yeasts and relatives) are enriched by hydrocarbon pollution in domesticated habitats and comprise numerous species that cause mild‐to‐severe disease. Methods: We investigated the presence of black fungi in the skin microbiome by conducting an analysis in the publicly available metagenomic SRA database (NCBI). We focused on the causative agents of chromoblastomycosis and phaeohyphomycosis and used barcodes and padlock probe sequences as diagnostic tools. Results: A total of 132,159,577 MB was analysed and yielded 18,360 reads that matched with 24 species of black fungi. Exophiala was the most prevalent genus, and Cyphellophora europaea was the most abundant species. Conclusion: This study reveals the abundant presence of Chaetothyriales on the skin without necessarily being associated with infection. Most of the detected causal agents are known from mild skin diseases, while also species were revealed that had been reported from CARD9‐deficient patients. [ABSTRACT FROM AUTHOR]

Dematiaceous fungi are pigmented molds with a high content of melanin in their cell walls that can cause fatal infections in immunocompromised hosts. Direct microscopy is the main method for the rapid diagnosis of dematiaceous fungi in clinical specimens. However, it is often difficult to distinguish their hyphae from non-dematiaceous hyphae and yeast pseudohyphae. Our aim was to develop a fluorescence staining method that targets melanin for the detection of dematiaceous molds in clinical specimens. Glass slide smears of clinical samples and sterile bronchoalveolar lavage spiked with dematiaceous and non-dematiaceous fungi were treated with hydrogen peroxide, and digital images were recorded using direct microscopy with different fluorescent filters. The images of fungi were compared for their fluorescence intensity using the NIS-Elements software. The fluorescent signal between dematiaceous and non-dematiaceous fungi demonstrated a markedly increased mean intensity for dematiaceous molds following hydrogen peroxide treatment (7510.3 ± 10,427.6 vs. 0.3 ± 3.1, respectively, p < 0.0001). No fluorescent signal was detected in the absence of hydrogen peroxide. "Staining" fungal clinical specimens with hydrogen peroxide, followed by fluorescence microscopy examination, can differentiate between dematiaceous and non-dematiaceous fungi. This finding can be used for the detection of dematiaceous molds in clinical specimens and enables the early and appropriate treatment of infections. [ABSTRACT FROM AUTHOR]

Abstract Some members of Chaetothyriales, an order containing potential agents of opportunistic infections in humans, have a natural habitat in nests of tropical arboreal ants. In these black fungi, two types of ant symbiosis are known, i.e. occurrence in domatia inside living plants, or as components of carton constructions made of ant-chewed plant tissue. In order to explain differences between strains from these types of association, we sequenced and annotated genomes of two newly described carton species, Incumbomyces lentus and Incumbomyces delicatus, and compared these with genomes of four domatia species and related Chaetothyriales. General genomic characteristics, CYP genes, carbohydrate-active enzymes (CAZymes), secondary metabolism, and sex-related genes were included in the study.

Black fungi are an ecological group of melanized fungi specialized in extremotolerance and assumed to be among the most stress-resistant eukaryotes on Earth. Multi-omics studies have provided significant evidence that they have a peculiar response to stress that differs considerably from that of common mesophilic hyphomycetes. Survival strategies displayed by these organisms have situated them as attractive models for astrobiology and, in general, for studies directed towards the definition of the actual limits for life. Moreover, the ascertained aptitude of black fungi for degradation of hazardous volatile pollutants and for plastic breakdown suggests prospective application of several species.

The Cross of the Inquisition, sculpted in 1903 and raised on a column with a fluted shaft and ornamented with vegetable garlands, is located in a corner of the Plateresque façade of the Seville City Hall. The Cross was vandalized in September 2019 and the restoration concluded in September 2021. A geological and microbiological study was carried out in a few small fragments. The data are consistent with the exposure of the Cross of the Inquisition to an urban environment for more than 100 years. During that time, a lichen community colonized the Cross and the nearby City Hall façades. The lichens, bryophytes and fungi colonizing the limestone surface composed an urban community, regenerated from the remains of the original communities, after superficial cleaning of the limestone between 2008 and 2010. This biological activity was detrimental to the integrity of the limestone, as showed by the pitting and channels, which evidence the lytic activity of organisms on the stone surface. Stone consolidation was achieved with Estel 1000. Preventol RI80, a biocide able to penetrate the porous limestone and active against bacteria, fungi, lichens, and bryophytes, was applied in the restoration. [ABSTRACT FROM AUTHOR]

Featured Application: This is an updated review on black fungi as main biodeteriogens of cultural heritage stone artifacts. Colonization pattern, taxonomy, and methods to eradicate their settlement are discussed here. Black fungi are considered as one of the main group of microorganisms responsible for the biodeterioration of stone cultural heritage artifacts. In this paper, we provide a critical analysis and review of more than 30 years of studies on black fungi isolated from stone-built heritage from 1990 to date. More than 109 papers concerning the fungal biodeterioration activity of stone were analysed. The main findings were a check list of the black fungal taxa involved in the biodeterioration of stone-built heritage, with a particular reference to meristematic black fungi, the main biodeterioration pattern attributed to them, and the methods of study including the new molecular advances. A particular focus was to discuss the current approaches to control black fungi from stone-built heritage and future perspectives. Black fungi are notoriously hard to remove or mitigate, so new methods of study and of control are needed, but it is also important to combine classical methods with new approaches to improve current knowledge to implement future conservation strategies. [ABSTRACT FROM AUTHOR]

Filamentous fungi have a crucial impact on the food safety and technological quality of malting barley. Commonly used techniques for the detection of seed-borne fungi are based on cultivation and identification by morphological criteria. In contrast, this study established a quantitative real-time polymerase chain reaction (PCR) assay based on SYBR green technology for the detection and quantification of black fungal species (Alternaria spp., Epicoccum nigrum, Cladosporium cladosporioides, Penicillium verrucosum and Aspergillus niger) on brewing barley and compares it with the traditional cultivation technique and visual assessment. To screen the fungal spectrum over different barley varieties and harvest years, naturally infected samples of malting barley and corresponding malts (Hordeum vulgare L.) were analyzed over four consecutive years (2018–2021), grown under different climatic conditions in Germany. Alternaria and Cladosporium spp. DNA were present in all examined barley samples, even without visible contamination. In contrast, detection via culture-based methods does not reliably cover all species. Molecular analysis showed that there was less fungal biomass after malting, by 58.57% in the case of A. alternata, by 28.27% for Cladosporium spp. and by 12.79% for Epicoccum nigrum. Correlation analysis showed no causal relationship between fungal DNA and the number of black kernels. The qPCR provides a highly sensitive and time-saving screening method for detecting latent fungal infections in brewing grains to identify batches that are potentially highly contaminated with toxigenic fungi. [ABSTRACT FROM AUTHOR]

Some members of Chaetothyriales, an order containing potential agents of opportunistic infections in humans, have a natural habitat in nests of tropical arboreal ants. In these black fungi, two types of ant symbiosis are known, i.e. occurrence in domatia inside living plants, or as components of carton constructions made of ant-chewed plant tissue. In order to explain differences between strains from these types of association, we sequenced and annotated genomes of two newly described carton species, Incumbomyces lentus and Incumbomyces delicatus, and compared these with genomes of four domatia species and related Chaetothyriales. General genomic characteristics, CYP genes, carbohydrate-active enzymes (CAZymes), secondary metabolism, and sex-related genes were included in the study. [ABSTRACT FROM AUTHOR]

Definition: Black fungi are an ecological group of melanized fungi specialized in extremotolerance and assumed to be among the most stress-resistant eukaryotes on Earth. Multi-omics studies have provided significant evidence that they have a peculiar response to stress that differs considerably from that of common mesophilic hyphomycetes. Survival strategies displayed by these organisms have situated them as attractive models for astrobiology and, in general, for studies directed towards the definition of the actual limits for life. Moreover, the ascertained aptitude of black fungi for degradation of hazardous volatile pollutants and for plastic breakdown suggests prospective application of several species. [ABSTRACT FROM AUTHOR]

Subterranean Cultural Heritage sites are frequently subject to biological colonization due to the high levels of humidity, even in conditions of low irradiance and oligotrophy. Here microorganisms form complex communities that may be dangerous through mineral precipitation, through the softening of materials or causing frequent surface discolorations. A reduction of contamination's sources along with the control of microclimatic conditions and biocide treatments (overall performed with benzalkonium chloride) are necessary to reduce microbial growths. Dark discolorations have been recorded in the painted Etruscan tombs of Tarquinia, two of which have been analyzed to collect taxonomical, physiological, and ecological information. Eighteen dark-pigmented fungi were isolated among a wider culturable fraction: nine from blackening areas and nine from door sealings, a possible route of contamination. Isolates belonged to three major groups: Chaetothyriales , Capnodiales (Family Cladosporiaceae), and Acremonium -like fungi. Exophiala angulospora and Cyphellophora olivacea , a novelty for hypogea, were identified, while others need further investigations as possible new taxa. The metabolic skills of the detected species showed their potential dangerousness for the materials. Their tolerance to benzalkonium chloride-based products suggested a certain favouring effect through the decreasing competitiveness of less resistant species. The type of covering of the dromos may influence the risk of outer contamination. Fungal occurrence can be favoured by root penetration. • Tolerance to biocides affects fungal spreading. • Metabolic screenings show fungal dangerousness for materials. • Subterranean environments show unknown fungal taxa. [ABSTRACT FROM AUTHOR]

Black fungi (Auricularia auricula) was known as one of the major mushrooms grown and cultivated all over the world, especially northeast Asia. It has been proved that black fungi had tremendous biological functions, and thus was being remarked to be a potential ingredient for the functional food. This review comprises a comprehensive research for the isolation and characterization of bioactive components in black fungi and their potent physiological activities. Meanwhile, chemical and metabolic methodologies are introduced to clarify the specified mechanism and future challenges. Integrated research results showed that black fungi contain some bioactive components such as polysaccharide, melanin and phenolic. According to origin and isolation method, the structural features of polysaccharide and melanin were significantly different. Health benefits such as antioxidant, hypoglycemic, antitumor and immunostimulation could be exerted with black fungi components and their derivatives. Besides, biological activity and physicochemical property of black fungi enabled the application in the drug delivery and food production. Detailed mechanisms confirmed that oxidative damage removal, immune signal transduction and gut microbiota enhancement supports the promising functionality. However, present research of black fungi still demands the more efficient extraction and purification including subcritical water, ohmic, high hydrostatic pressure (HHP) and two-phase partitioning. Challenges to some health benefits such as gut microbiota-immune, dosage suitable for clinical practice and especially structure-function relationship should also be worthwhile to increase the total functional value of black fungi. • Polysaccharide, melanin and polyphenol are identified as black fungi biomolecules. • ROS removal by black fungi is a key governor of whole health benefits. • Black fungi provoke the immune-gut microbiota axis for physiological functions. • Physicochemical property of black fungi is applied for food and drug delivery. [ABSTRACT FROM AUTHOR]

Weathering processes seriously affect the durability of outdoor marble monuments. In urban environments, a very common deterioration phenomenon is the dark discoloration or blackening of marble. This paper describes a multidisciplinary study on the state of conservation of white marbles of the Florence Cathedral and the microbial community involved in their deterioration. The study is focused on the widespread dark discoloration of marble analyzed in two differently exposed sites of the Cathedral. It aims to provide information useful for future interventions to control the microbial growth. By chemical and petrographic analysis, in situ and ex situ microscopy, and cultivation and identification of microorganisms, it was found that (i) the darkening is mainly due to the growth of black fungi and dark cyanobacteria and (ii) the state of conservation of marble and the growth pattern of microorganisms seems to be linked to the microclimatic conditions, in particular to solar radiation exposure. This is the first report on the lithobiontic community inhabiting the Florence Cathedral marbles, with a more detailed investigation of the culturable mycobiota. [ABSTRACT FROM AUTHOR]

Dischidia major is an epiphyte with pitcher leaves that serve as nests for ants. We investigated this ant-plant symbiosis in two sites in southeastern Thailand, Rayong and Trat, using a morphological and molecular approach. In our study sites, D. major was colonized by one monomorphic ant species of genus Philidris. The inner surface of the pitcher leaves had a black and green lining composed of intermingled coccoid cells and filaments of algae and fungi, reminiscent of a biofilm structure. Microscopic investigation of the algae suggested they belonged to Trebouxia (coccoid cells) and Trentepohliaceae (filaments). Molecular investigation of environmental samples and pure cultures of the fungi revealed five species of Chaetothyriales and four species of Capnodiales, among which two have already been isolated from ant-plant symbioses in Africa and South America and five were described species known from various environments around world. One appears to be an undescribed species. Thus, most fungal associates were likely ubiquitous species. Our study highlights the need to include the identity and functional ecology of microbes in studies of the evolutionary and functional ecology of ant-plant symbioses. [ABSTRACT FROM AUTHOR]

Black fungi are a group of melanotic microfungi characterized by remarkable polyextremotolerance. Due to a broad ecological plasticity and adaptations at the cellular level, it is predicted that they may survive in a variety of extreme environments, including harsh niches on Earth and Mars, and in outer space. However, the molecular mechanisms aiding survival, especially in space, are yet to be fully elucidated. Based on these premises, the rock-inhabiting black fungus Knufia chersonesos (Wt) and its non-melanized mutant (Mut) were exposed to simulated microgravity—one of the prevalent features characterizing space conditions—by growing the cultures in high-aspect-ratio vessels (HARVs). Qualitative and quantitative proteomic analyses were performed on the mycelia and supernatant of culture medium (secretome) to assess alterations in cell physiology in response to low-shear simulated microgravity (LSSMG) and to ultimately evaluate the role of cell-wall melanization in stress survival. Differential expression was observed for proteins involved in carbohydrate and lipid metabolic processes, transport, and ribosome biogenesis and translation via ribosomal translational machinery. However, no evidence of significant activation of stress components or starvation response was detected, except for the scytalone dehydratase, enzyme involved in the synthesis of dihydroxynaphthalene (DNH) melanin, which was found to be upregulated in the secretome of the wild type and downregulated in the mutant. Differences in protein modulation were observed between K. chersonesos Wt and Mut, with several proteins being downregulated under LSSMG in the Mut when compared to the Wt. Lastly, no major morphological alterations were observed following exposure to LSSMG. Similarly, the strains’ survivability was not negatively affected. This study is the first to characterize the response to simulated microgravity in black fungi, which might have implications on future astrobiological missions.

Among agents of chromoblastomycosis, Fonsecaea pugnacius presents a unique type of infection because of its secondary neurotropic dissemination from a chronic cutaneous case in an immunocompetent patient. Neurotropism occurs with remarkable frequency in the fungal family Herpotrichiellaceae, possibly associated with the ability of some species to metabolize aromatic hydrocarbons. In an attempt to understand this new disease pattern, were conducted genomic analysis of Fonsecaea pugnacius (CBS 139214) performed with de novo assembly, gene prediction, annotation and mitochondrial genome assembly, supplemented with animal infection models performed with Tenebrio molitor in Mus musculus lineages BALB/c and C57BL/6. The genome draft of 34.8 Mb was assembled with a total of 12,217 protein-coding genes. Several proteins, enzymes and metabolic pathways related to extremotolerance and virulence were recognized. The enzyme profiles of black fungi involved in chromoblastomycosis and brain infection were analyzed with the Carbohydrate-Active Enzymes (CAZY) and peptidases database (MEROPS). The capacity of the fungus to survive inside Tenebrio molitor animal model was confirmed by histopathological analysis and by presence of melanin and hyphae in host tissue. Although F. pugnacius was isolated from brain in a murine model following intraperitoneal infection, cytokine levels were not statistically significant, indicating a profile of an opportunistic agent. A dual ecological ability can be concluded from presence of metabolic pathways for nutrient scavenging and extremotolerance, combined with a capacity to infect human hosts.

The Cross of the Inquisition, sculpted in 1903 and raised on a column with a fluted shaft and ornamented with vegetable garlands, is located in a corner of the Plateresque façade of the Seville City Hall. The Cross was vandalized in September 2019 and the restoration concluded in September 2021. A geological and microbiological study was carried out in a few small fragments. The data are consistent with the exposure of the Cross of the Inquisition to an urban environment for more than 100 years. During that time, a lichen community colonized the Cross and the nearby City Hall façades. The lichens, bryophytes and fungi colonizing the limestone surface composed an urban community, regenerated from the remains of the original communities, after superficial cleaning of the limestone between 2008 and 2010. This biological activity was detrimental to the integrity of the limestone, as showed by the pitting and channels, which evidence the lytic activity of organisms on the stone surface. Stone consolidation was achieved with Estel 1000. Preventol RI80, a biocide able to penetrate the porous limestone and active against bacteria, fungi, lichens, and bryophytes, was applied in the restoration.

Filamentous fungi have a crucial impact on the food safety and technological quality of malting barley. Commonly used techniques for the detection of seed-borne fungi are based on cultivation and identification by morphological criteria. In contrast, this study established a quantitative real-time polymerase chain reaction (PCR) assay based on SYBR green technology for the detection and quantification of black fungal species (Alternaria spp., Epicoccum nigrum, Cladosporium cladosporioides, Penicillium verrucosum and Aspergillus niger) on brewing barley and compares it with the traditional cultivation technique and visual assessment. To screen the fungal spectrum over different barley varieties and harvest years, naturally infected samples of malting barley and corresponding malts (Hordeum vulgare L.) were analyzed over four consecutive years (2018–2021), grown under different climatic conditions in Germany. Alternaria and Cladosporium spp. DNA were present in all examined barley samples, even without visible contamination. In contrast, detection via culture-based methods does not reliably cover all species. Molecular analysis showed that there was less fungal biomass after malting, by 58.57% in the case of A. alternata, by 28.27% for Cladosporium spp. and by 12.79% for Epicoccum nigrum. Correlation analysis showed no causal relationship between fungal DNA and the number of black kernels. The qPCR provides a highly sensitive and time-saving screening method for detecting latent fungal infections in brewing grains to identify batches that are potentially highly contaminated with toxigenic fungi.

Black fungi are considered as one of the main group of microorganisms responsible for the biodeterioration of stone cultural heritage artifacts. In this paper, we provide a critical analysis and review of more than 30 years of studies on black fungi isolated from stone-built heritage from 1990 to date. More than 109 papers concerning the fungal biodeterioration activity of stone were analysed. The main findings were a check list of the black fungal taxa involved in the biodeterioration of stone-built heritage, with a particular reference to meristematic black fungi, the main biodeterioration pattern attributed to them, and the methods of study including the new molecular advances. A particular focus was to discuss the current approaches to control black fungi from stone-built heritage and future perspectives. Black fungi are notoriously hard to remove or mitigate, so new methods of study and of control are needed, but it is also important to combine classical methods with new approaches to improve current knowledge to implement future conservation strategies.

Among agents of chromoblastomycosis, Fonsecaea pugnacius presents a unique type of infection because of its secondary neurotropic dissemination from a chronic cutaneous case in an immunocompetent patient. Neurotropism occurs with remarkable frequency in the fungal family Herpotrichiellaceae, possibly associated with the ability of some species to metabolize aromatic hydrocarbons. In an attempt to understand this new disease pattern, were conducted genomic analysis of Fonsecaea pugnacius (CBS 139214) performed with de novo assembly, gene prediction, annotation and mitochondrial genome assembly, supplemented with animal infection models performed with Tenebrio molitor in Mus musculus lineages BALB/c and C57BL/6. The genome draft of 34.8 Mb was assembled with a total of 12,217 protein-coding genes. Several proteins, enzymes and metabolic pathways related to extremotolerance and virulence were recognized. The enzyme profiles of black fungi involved in chromoblastomycosis and brain infection were analyzed with the Carbohydrate-Active Enzymes (CAZY) and peptidases database (MEROPS). The capacity of the fungus to survive inside Tenebrio molitor animal model was confirmed by histopathological analysis and by presence of melanin and hyphae in host tissue. Although F. pugnacius was isolated from brain in a murine model following intraperitoneal infection, cytokine levels were not statistically significant, indicating a profile of an opportunistic agent. A dual ecological ability can be concluded from presence of metabolic pathways for nutrient scavenging and extremotolerance, combined with a capacity to infect human hosts. [ABSTRACT FROM AUTHOR]

Free-living and mycorrhizal fungi are able to enhance the weathering of rock and other solid substrates. Deciphering the exact mechanisms of these natural processes requires their experimental simulation. Moreover, by performing these simulations with genetically amenable rock-weathering fungi, one can knock-out certain fungal traits and consequently identify their weathering-relevant function. Here, the effect of the rock-inhabiting fungus, Knufia petricola A95, on the dissolution kinetics of an Fe-bearing olivine (Mg 1.86 Fe 0.19 SiO 4) is investigated at 25 °C and pH 6 using reproducible batch and mixed flow experiments. The availability of a melanin-deficient mutant (Δ Kppks) of K. petricola A95, which produces more extracellular polymeric substances (EPS) than the wild type (WT), enables the comparative study of the role of melanin and EPS in olivine dissolution. In abiotic dissolution experiments, the olivine dissolution rate decreased considerably over time at pH 6 but not at pH 3.5. This inhibition of abiotic olivine dissolution at pH 6 was most likely caused by the in-situ oxidation of ferrous Fe and/or the precipitation of ferric hydroxides at the olivine surface. In corresponding biotic experiments at pH 6, both the wild type K. petricola and its melanin-deficient mutant Δ Kppks solubilised and bound significant amounts of Fe released by olivine dissolution. Fe oxidation and precipitation were thus prevented and olivine dissolution proceeded faster than in the abiotic experiments. By sequestering Fe directly at the olivine surface, the attached wild type K. petricola cells were particularly efficient at preventing the oxidation of Fe at the mineral surface: the slowdown of olivine dissolution almost completely disappeared. The attachment capacity of these wild type cells is most likely mediated by wild type-specific EPS. Our presented experimental systems allow the oxidation of mineral-released Fe and include a rock-inhabiting fungus, thus simulating chemical, physical and biological conditions that set dissolution rates in a way that is relevant to natural ecosystems. [ABSTRACT FROM AUTHOR]

Weathering processes seriously affect the durability of outdoor marble monuments. In urban environments, a very common deterioration phenomenon is the dark discoloration or blackening of marble. This paper describes a multidisciplinary study on the state of conservation of white marbles of the Florence Cathedral and the microbial community involved in their deterioration. The study is focused on the widespread dark discoloration of marble analyzed in two differently exposed sites of the Cathedral. It aims to provide information useful for future interventions to control the microbial growth. By chemical and petrographic analysis, in situ and ex situ microscopy, and cultivation and identification of microorganisms, it was found that (i) the darkening is mainly due to the growth of black fungi and dark cyanobacteria and (ii) the state of conservation of marble and the growth pattern of microorganisms seems to be linked to the microclimatic conditions, in particular to solar radiation exposure. This is the first report on the lithobiontic community inhabiting the Florence Cathedral marbles, with a more detailed investigation of the culturable mycobiota.

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.

One of the primary current astrobiological goals is to understand the limits of microbial resistance to extraterrestrial conditions. Much attention is paid to ionizing radiation, since it can prevent the preservation and spread of life outside the Earth. The aim of this research was to study the impact of accelerated He ions (150 MeV/n, up to 1 kGy) as a component of the galactic cosmic rays on the black fungus C. antarcticus when mixed with Antarctic sandstones—the substratum of its natural habitat—and two Martian regolith simulants, which mimics two different evolutionary stages of Mars. The high dose of 1 kGy was used to assess the effect of dose accumulation in dormant cells within minerals, under long-term irradiation estimated on a geological time scale. The data obtained suggests that viable Earth-like microorganisms can be preserved in the dormant state in the near-surface scenario for approximately 322.000 and 110.000 Earth years within Martian regolith that mimic early and present Mars environmental conditions, respectively. In addition, the results of the study indicate the possibility of maintaining traces within regolith, as demonstrated by the identification of melanin pigments through UltraViolet-visible (UV-vis) spectrophotometric approach.

[Display omitted] • Fungal pigments melanin and carotenoids were investigated with Raman microscopy. • Wild-type, and three genetically modified strains of Knufia petricola A95 were analysed. • Wild fungal cells attached to ancient rock were also studied. • The Raman microscopes were equipped with lasers of 633 nm and 488 nm wavelengths. • This study contributes to a library of Raman spectra of natural fungi and their melanin. Fungal pigments such as melanin and carotenoids are distinctive markers of animal and plant pathogenic fungi as well as their environmental relatives. These complex pigments play important roles in pathogenicity and stress tolerance while also being useful as biomarkers. Accordingly, it is important to be able to identify in situ the pigments in black fungi, a group of clinical and environmental importance. In this study, wild-type and genetically modified strains of Knufia petricola A95 and wild fungal cells attached to ancient rock were investigated for their spectroscopic and microscopic Raman features and morphological appearance. Knockout mutants of melanin synthesis genes pks1 (polyketide synthase), sdh1 (scytalone dehydratase), and both pks1 and the carotenoid synthesis gene phd1 (phytoene desaturase) were studied We applied two different Raman microscopes using two lasers, with 633 nm and 488 nm wavelengths. We analyzed and compared Raman spectra between the measured reference substances and the mutant and wild-type strains. In the wild strain WT:A95, the peaks close to melanin peals were found at 1353 cm−1 and 1611 cm−1. There are no characteristic melanin peaks at 1580–1600 cm−1 and around 1350 cm−1 at the spectrum of the Δpks1/Δphd1 mutant and the Δsdh1 mutant. The Δpks1 mutant spectrum has the peaks at the beta-carotene v2 C-C in-plane stretch at 1155 cm−1 and v3 C-CH3 deformation at 1005 cm−1. The peaks of carotenoids and melanin were found in all mutants and the wild strain, except the Δpks1/Δphd1 mutant. Raman spectra allow for discrimination between the various pigments. Hence, interactions between natural fungal melanin, as well as other protective pigments, and complex environmental matrices can be characterized on a range of spatial and temporal scales. [ABSTRACT FROM AUTHOR]

In restoration and conservation practices, biocide treatments are considered one of the most practical approaches to remove biological colonization on artworks, including stone. Numerous studies have focused on the short- and long-term effects of these treatments and recently many alternative methods to reduce their potential hazards to human health and the environment have been proposed. In this study, a solvent gel containing dimethyl sulfoxide (DMSO), already used to clean paintings, was applied on colonized marble artifacts at the monumental cemetery of Bonaria (Cagliari – Italy) to remove biological patinas. The protocol efficiency was evaluated by scanning electronic microscopy, rugosimetric and colorimetric measurements and growth tests. A comparative study also was performed to validate the method using biocides currently used in conservation. The results demonstrate that DMSO solvent gel is efficient at removing patinas on stone, of low impact, easy to use, inexpensive and can be considered a more practical alternative to biocide treatments. [ABSTRACT FROM AUTHOR]

Melanins are ancient biological pigments found in all kingdoms of life. In fungi, their role in microbial pathogenesis is well established; however, these complex biomolecules also confer upon fungal microorganisms the faculty to tolerate extreme environments such as the Earth's poles, the International Space Station and places contaminated by toxic metals and ionizing radiation. A remarkable property of melanin is its capacity to interact with a wide range of electromagnetic radiation frequencies, functioning as a protecting and energy harvesting pigment. Other roles of fungal melanin include scavenging of free radical, thermo-tolerance, metal ion sequestration, cell development, and mechanical-chemical cellular strength. In this review, we explore the various functions ascribed to this biological pigment in fungi and its remarkable physicochemical properties. [ABSTRACT FROM AUTHOR]