Members of the fungal kingdom serve as models for numerous cellular processes, among them sexuality.1 In heterothallic ascomycetes, mating-type systems ensure that only compatible isolates fuse to enter the sexual phase.2,3,4,5,6 This includes reciprocal secretion and recognition of pheromones, commonly termed α-factor and a-factor, which are processed from peptide precursors.7,8,9,10 Identification of fungal mating pheromones and their cognate receptors has been achieved by homology searches11,12,13,14,15,16,17; however, this approach had failed to detect a-factor-like pheromones from Eurotiomycetes,5,18,19,20,21 a fungal group including medically and economically important species.22 Sexuality of the opportunistic pathogen Aspergillus fumigatus23,24,25 is genetically determined by a bipolar mating-type system encoding MAT1-1-1 and MAT1-2-1 regulators.16,26,27,28,29,30 By analyzing transcriptome data from strains overexpressing the corresponding MAT genes,31 we identified a candidate pheromone precursor gene B (ppgB) to encode the elusive Eurotiomycete a-factor pheromone. Its deduced peptide is 24 aa in length and features a canonical CaaX farnesylation motif. Further analyses provided supporting evidence that PpgB is a prototype for the a-factor-like pheromone of the aspergilli, including expression of ppgB in a MAT1-2-1-dependent manner, and that an A. fumigatus ppgBΔ deletion strain was unable to mate and form fruiting bodies with a compatible partner. Inspection of Aspergillus genomes from members of the section Fumigati revealed high conservation of PpgB sequence as well as of the α-factor-like PpgA, indicating that incompatibility factors other than solely pheromone discrimination are responsible for speciation. The identification of the A. fumigatusa-factor-like pheromone closes a substantial knowledge gap with respect to cellular recognition and sexual propagation of Eurotiomycete fungi.

Fungi are essential for a wide variety of food products and processes. They have a major role in the production of many fermented foodstuffs, may be eaten directly as fruit bodies and mycelium, and are used to produce food additives. They contribute to food production worldwide, even in cultures which do not typically consume mushrooms, because yeasts and edible moulds are utilised in a great variety of fermentation processes. Most fungal cultures used in food production at industrial scale show evidence of selection and domestication. However, other strains may still be obtained from the wild, either through incidental colonisation of a fermentation substrate as a result of a traditional preparation method or due to the inability to reliably cultivate a given organism, necessitating collection from its native habitat. This review provides an overview of the uses of fungi, both yeasts and filamentous fungi, in food production with a focus on research findings over the past decade. This includes a review of the production of foodstuffs through the fermentation of a wide variety of substrates, particularly dairy, but also including meat and plant matter. In addition, the use of fungi in the production of secreted enzymes and food additives is considered. Finally, the cultivation and harvesting of fungal fruiting bodies and mycoprotein are reviewed. The review aims to capture the breadth of the field by covering examples from every inhabited continent, including reference to fungal food systems which have historically been under-studied.

Aspergillus nidulans is an important model organism for eukaryotic biology and the reference for the section Nidulantes in comparative studies. In this study, we de novo sequenced the genomes of 25 species of this section. Whole-genome phylogeny of 34 Aspergillus species and Penicillium chrysogenum clarifies the position of clades inside section Nidulantes. Comparative genomics reveals a high genetic diversity between species with 684 up to 2433 unique protein families. Furthermore, we categorized 2118 secondary metabolite gene clusters (SMGC) into 603 families across Aspergilli, with at least 40 % of the families shared between Nidulantes species. Genetic dereplication of SMGC and subsequent synteny analysis provides evidence for horizontal gene transfer of a SMGC. Proteins that have been investigated in A. nidulans as well as its SMGC families are generally present in the section Nidulantes, supporting its role as model organism. The set of genes encoding plant biomass-related CAZymes is highly conserved in section Nidulantes, while there is remarkable diversity of organization of MAT-loci both within and between the different clades. This study provides a deeper understanding of the genomic conservation and diversity of this section and supports the position of A. nidulans as a reference species for cell biology.

Resistance to azole antifungals in Aspergillus fumigatus, a WHO Critical Priority fungal pathogen, is rising worldwide and threatens the efficacy of frontline therapies for invasive aspergillosis. This resistance has emerged largely through environmental selection driven by the extensive use of azole fungicides in agriculture and horticulture, yet its evolutionary origins remain obscure. Here, we analysed whole-genome sequences from 1,220 isolates collected over a century from 34 countries, revealing five distinct but interfertile genetic clusters spanning the global population. Azole resistance alleles were highly concentrated within particular clusters and were associated with mutator genotypes carrying defects in DNA repair, known to accelerate the accumulation of adaptive mutations. Two clusters exhibited resistance to multiple fungicide classes, including azoles, benzimidazoles, strobilurins and succinate dehydrogenase inhibitors, indicating the emergence of stacked multidrug resistance with no measurable fitness cost in vitro or in murine models. Molecular dating situates the expansion of resistant lineages in the latter half of the twentieth century, coinciding with the introduction and intensification of modern fungicide use. Phylogeographic reconstruction points to Western Europe—particularly intensive horticultural systems and the international bulb trade—as a major source of globally disseminated azole-resistant A. fumigatus, underscoring the urgent need for coordinated One Health surveillance and intervention.

Members of the lichen-forming fungal genus Oxneriaria are known to occur in cold polar and high altitudinal environments. Two new species, Oxneriaria crittendenii and O. deosaiensis, are now described from the high altitude Deosai Plains, Pakistan, based on phenotypic, multigene phylogenetic and chemical evidence. Phenotypically, O. crittendenii is characterised by orbicular light-brown thalli 1.5–5 cm across, spot tests (K, C, KC) negative, apothecia pruinose, hymenium initially blue then dark orange in response to Lugol’s solution. Oxneriaria deosaiensis is characterised by irregular areolate grey thalli 1.5–2 cm across, K test (light brown), KC test (dark brown), apothecia epruinose, hymenium initially blue then dark blue in response to Lugol’s solution. Both species share the same characters of thalli with black margins and polarilocular ascospores. The closest previously reported species, O. pruinosa, differs from O. crittendenii and O. deosaiensis in having non-lobate margins, thin thalline exciple (45–80 μm thick), short asci (55–80 × 25–42 μm) and K positive (yellow) and KC negative tests and divergent DNA sequence in the ITS, LSU and mt SSU regions. The newly-described Oxneriaria species add to growing evidence of the Deosai Plains as a region of important arctic-alpine biodiversity.

Penicillium roqueforti is used worldwide in the production of blue-veined cheese. The blue-green colour derives from pigmented spores formed by fungal growth. Using a combination of bioinformatics, targeted gene deletions, and heterologous gene expression we discovered that pigment formation was due to a DHN-melanin biosynthesis pathway. Systematic deletion of pathway genes altered the arising spore colour, yielding white to yellow-green to red-pink-brown phenotypes, demonstrating the potential to generate new coloured strains. There was no consistent impact on mycophenolic acid production as a result of pathway interruption although levels of roquefortine C were altered in some deletants. Importantly, levels of methyl-ketones associated with blue-cheese flavour were not impacted. UV-induced colour mutants, allowed in food production, were then generated. A range of colours were obtained and certain phenotypes were successfully mapped to pathway gene mutations. Selected colour mutants were subsequently used in cheese production and generated expected new colourations with no elevated mycotoxins, offering the exciting prospect of use in future cheese manufacture.

Sexual reproduction involving meiosis is essential in most eukaryotes. This produces offspring with novel genotypes, both by segregation of parental chromosomes as well as crossovers between homologous chromosomes. A sexual cycle for the opportunistic human pathogenic fungus Aspergillus fumigatus is known, but the genetic consequences of meiosis have remained unknown. Among other Aspergilli, it is known that A. flavus has a moderately high recombination rate with an average of 4.2 crossovers per chromosome pair, whereas A. nidulans has in contrast a higher rate with 9.3 crossovers per chromosome pair. Here, we show in a cross between A. fumigatus strains that they produce an average of 29.9 crossovers per chromosome pair and large variation in total map length across additional strain crosses. This rate of crossovers per chromosome is more than twice that seen for any known organism, which we discuss in relation to other genetic model systems. We validate this high rate of crossovers through mapping of resistance to the laboratory antifungal acriflavine by using standing variation in an undescribed ABC efflux transporter. We then demonstrate that this rate of crossovers is sufficient to produce one of the common multidrug resistant haplotypes found in the cyp51A gene (TR34/L98H) in crosses among parents harboring either of 2 nearby genetic variants, possibly explaining the early spread of such haplotypes. Our results suggest that genomic studies in this species should reassess common assumptions about linkage between genetic regions. The finding of an unparalleled crossover rate in A. fumigatus provides opportunities to understand why these rates are not generally higher in other eukaryotes.

Microsatellite markers can provide valuable information about gene flow and population history. We developed and tested new microsatellites for the nitrophilic lichenized fungus Xanthoria parietina and studied its genetic diversity and structure within the urban area of Munich, Bavaria. We compared its local genetic pattern with that of its photobiont partner Trebouxia decolorans, for which existing microsatellites were applied. For comparison, a reference site with clean air was included in the sampling. We found support for three genetic clusters in the fungus X. parietina, which occurred intermingled in collecting sites. There was a high degree of admixture within fungal populations and individuals, and analysis of molecular variance revealed a lack of population structure in the mycobiont. The Trebouxia photobiont, in contrast, exhibited structured populations which grouped into two to five genetic clusters, and individuals showed less admixture than in the mycobiont. This indicates that the two lichen partners differ in their ability to move around in the landscape. The microsatellite markers we report are polymorphic and are suitable for population genetic studies.

Using a citizen science approach, we identify a country-wide exposure to aerosolized spores of a human fungal pathogen, Aspergillus fumigatus , that has acquired resistance to the agricultural fungicide tebuconazole and first-line azole clinical antifungal drugs. Genomic analysis shows no distinction between resistant genotypes found in the environment and in patients, indicating that at least 40% of azole-resistant A. fumigatus infections are acquired from environmental exposures. Hotspots and coldspots of aerosolized azole-resistant spores were not stable between seasonal sampling periods. This suggests a high degree of atmospheric mixing resulting in an estimated per capita cumulative annual exposure of 21 days (±2.6). Because of the ubiquity of this measured exposure, it is imperative that we determine sources of azole-resistant A. fumigatus to reduce treatment failure in patients with aspergillosis.

AbstractA novel lichen species occurring on rocks was collected from three different localities within Deosai National Park, Gilgit-Baltistan, Pakistan. Phylogenetic analyses of the nrDNA ITS and nuLSU regions revealed that it clustered within the genus Anamylopsora. Further chemical and morpho-anatomical analyses confirmed its uniqueness, and it is described here as a new species under the name A. pakistanica. The distinguishing characters are: an irregularly squamulose appressed thallus on rocks without rhizines; an epinecral layer up to 25 μm thick; ascospores that are hyaline, simple, thick-walled with a smooth surface; septate paraphyses with a pigmented apical cell in a gel-like matrix; globose to subglobose pycnidia with hyaline and bacilliform pycnidiospores. In particular, the species is distinguished from other members of the genus by morpho-anatomical features including the coloration of the thalli, the presence of a thick lower cortex (up to 100 μm), and the presence of simple, thick-walled ascospores. Specimens were found at altitudes up to 4587 m, the highest elevation yet reported for Anamylopsora. A key and comparison to all existing species of the genus Anamylopsora is also given.

Infections caused by the fungal pathogen Aspergillus fumigatus are increasingly resistant to first-line azole antifungal drugs. However, despite its clinical importance, little is known about how susceptible patients acquire infection from drug-resistant genotypes in the environment. Here, we present a population genomic analysis of 218 A. fumigatus isolates from across the UK and Ireland (comprising 153 clinical isolates from 143 patients and 65 environmental isolates). First, phylogenomic analysis shows strong genetic structuring into two clades (A and B) with little interclade recombination and the majority of environmental azole resistance found within clade A. Second, we show occurrences where azole-resistant isolates of near-identical genotypes were obtained from both environmental and clinical sources, indicating with high confidence the infection of patients with resistant isolates transmitted from the environment. Third, genome-wide scans identified selective sweeps across multiple regions indicating a polygenic basis to the trait in some genetic backgrounds. These signatures of positive selection are seen for loci containing the canonical genes encoding fungicide resistance in the ergosterol biosynthetic pathway, while other regions under selection have no defined function. Lastly, pan-genome analysis identified genes linked to azole resistance and previously unknown resistance mechanisms. Understanding the environmental drivers and genetic basis of evolving fungal drug resistance needs urgent attention, especially in light of increasing numbers of patients with severe viral respiratory tract infections who are susceptible to opportunistic fungal superinfections.

Compost is an ecological niche for Aspergillus fumigatus due to its role as a decomposer of organic matter and its ability to survive the high temperatures associated with the composting process. Subsequently, composting facilities are associated with high levels of A. fumigatus spores that are aerosolized from compost and cause respiratory illness in workers. In the UK, gardening is an activity enjoyed by individuals of all ages, and it is likely that they are being exposed to A. fumigatus spores when handling commercial compost or compost they have produced themselves. In the present study, 246 citizen scientists collected 509 soil samples from locations in their gardens in the UK, from which were cultured 5,174 A. fumigatus isolates. Of these isolates, 736 (14%) were resistant to tebuconazole: the third most-sprayed triazole fungicide in the UK, which confers cross-resistance to the medical triazoles used to treat A. fumigatus lung infections in humans. These isolates were found to contain the common resistance mechanisms in the A. fumigatus cyp51A gene TR34/L98H or TR46/Y121F/T289A, as well as the less common resistance mechanisms TR34, TR53, TR46/Y121F/T289A/S363P/I364V/G448S, and (TR46)2/Y121F/M172I/T289A/G448S. Regression analyses found that soil samples containing compost were significantly more likely to grow tebuconazole-susceptible and tebuconazole-resistant A. fumigatus strains than those that did not and that compost samples grew significantly higher numbers of A. fumigatus than other samples. IMPORTANCE The findings presented here highlight compost as a potential health hazard to individuals with predisposing factors to A. fumigatus lung infections and as a potential health hazard to immunocompetent individuals who could be exposed to sufficiently high numbers of spores to develop infection. Furthermore, we found that 14% of A. fumigatus isolates in garden soils were resistant to an agricultural triazole, which confers cross-resistance to medical triazoles used to treat A. fumigatus lung infections. This raises the question of whether compost bags should carry additional health warnings regarding inhalation of A. fumigatus spores, whether individuals should be advised to wear facemasks while handling compost, or whether commercial producers should be responsible for sterilizing compost before shipping. The findings support increasing public awareness of the hazard posed by compost and investigating measures that can be taken to reduce the exposure risk.

EDITORIAL article Front. Cell. Infect. Microbiol., 28 June 2022Sec. Fungal Pathogenesis https://doi.org/10.3389/fcimb.2022.934267

AbstractWe demonstrate country-wide exposures to aerosolized spores of a human fungal pathogen,Aspergillus fumigatus, that has acquired resistance to first line azole clinical antifungal drugs. Assisted by a network of citizen scientists across the United Kingdom, we show that 1 in 20 viable aerosolized spores of this mold are resistant to the agricultural fungicide tebuconazole and 1 in 140 spores are resistant to the four most used azoles for treating clinical aspergillosis infections. Season and proximity to industrial composters were associated with growth ofA. fumigatusfrom air samples, but not with the presence of azole resistance, and hotspots were not stable between sampling periods suggesting a high degree of atmospheric mixing. Genomic analysis shows no distinction between those resistant genotypes found in the environment and in patients, indicating that ~40% (58/150 sequenced genomes) of azole-resistantA. fumigatusinfections are acquired from environmental exposures. Due to the ubiquity of this measured exposure, it is crucial that we determine source(s) of azole-resistantA. fumigatus, who is at greatest risk of exposure and how to mitigate these exposures, in order to minimize treatment failure in patients with aspergillosis.One sentence summaryUK-wide citizen science surveillance finds a ubiquitous exposure to aerosolized spores of a human fungal pathogen that have evolved in the environment cross-resistance to essential clinical antifungal drugs

Itaconic acid is a versatile renewable industrial chemical, but its widespread use is limited by high production costs relative to petroleumbased alternatives such as acrylic acid. Dilute acid hydrolysates of red bran and white bran were produced from waste brans of red and white sorghum, respectively, and their suitability for itaconic acid production by Aspergillus terreus was investigated. Forty-six A. terreus isolates were screened on a defined medium, and the highest itaconic acid producers were employed in batch fermentation of the hydrolysates. Up to 7.8 g/L and 5.2 g/L itaconic acid (22.9% and 15.1% of stoichiometric yield) were obtained from red bran dilute acid hydrolysate and white bran dilute acid hydrolysate, respectively. The titres were lower than were obtained from a defined glucose medium where up to 64 g/L (78% of the theoretical yield) was produced. Several conditioning processes were then employed. Activated charcoal treatment was the best hydrolysate treatment method, with the removal of 42% of phenolic compounds and 38% of condensed tannins. It also resulted in a significant rise to 9.2 g/L itaconic acid (24.6% of the % theoretical yield possible). This is the first report of itaconic acid production from sorghum bran and provides a route to valorizing this waste.

Eyespot, caused by the related fungal pathogens Oculimacula acuformis and O. yallundae, is an important cereal stem-base disease in temperate parts of the world. Both species are dispersed mainly by splash-dispersed conidia but are also known to undergo sexual reproduction, yielding apothecia containing ascospores. Field diagnosis of eyespot can be challenging, with other pathogens causing similar symptoms, which complicates eyespot management strategies. Differences between O. acuformis and O. yallundae (e.g., host pathogenicity and fungicide sensitivity) require that both be targeted for effective disease management. Here, we develop and apply two molecular methods for species-specific and mating-type (MAT1-1 or MAT1-2) discrimination of O. acuformis and O. yallundae isolates. First, a multiplex PCR-based diagnostic assay targeting the MAT idiomorph region was developed, allowing simultaneous determination of both species and mating type. This multiplex PCR assay was successfully applied to type a global collection of isolates. Second, the development of loop-mediated isothermal amplification (LAMP) assays targeting β-tubulin sequences, which allow fast (<9 min) species-specific discrimination of global O. acuformis and O. yallundae isolates, is described. The LAMP assay can detect very small amounts of target DNA (1 pg) and was successfully applied in planta. In addition, mating-type-specific LAMP assays were also developed for rapid (<12 min) genotyping of O. acuformis and O. yallundae isolates. Finally, the multiplex PCR-based diagnostic was applied, in conjunction with spore trapping in field experiments, to provide evidence of the wind dispersal of ascospores from a diseased crop. The results indicate an important role of the sexual cycle in the dispersal of eyespot.

In 2019, during May to September a unique lichen occurring on soil was collected from four different localities in Deosai National Park, Gilgit-Baltistan, Pakistan. Phylogenetic analysis of the nrDNA ITS and LSU regions revealed that it clustered within the genus Placidium. Further morpho-anatomical and chemical analyses proved its novelty, and it is here described as a new species under the name P. deosaiense. The distinguishing characters of this novel taxon are brown to blackish 2-7 mm wide squamules, undulating in the center, epruinose at margins, epinecral layer up to 70 mu m, cylindrical asci with ellipsoid to narrowly ellipsoid ascospores and clavate to bacilliform pycnidiospores.

The fungal zinc finger transcription factor NsdC is named after, and is best known for, its essential role in sexual reproduction (never in sexual development). In previous studies with Aspergillus nidulans it was also shown to have roles in promotion of vegetative growth and suppression of asexual conidiation. In this study the function of the nsdC homologue in the opportunistic human pathogen A. fumigatus was investigated. NsdC was again found to be essential for sexual development, with deletion of the nsdC gene in both MAT1-1 and MAT1-2 mating partners of a cross leading to complete loss of fertility. However, a functional copy of nsdC in one mating partner was sufficient to allow sexual reproduction. Deletion of nsdC also led to decreased vegetative growth and allowed conidiation in liquid cultures, again consistent with previous findings. However, NsdC in A. fumigatus was shown to have additional biological functions including response to calcium stress, correct organization of cell wall structure and response to the cell wall stressors. Furthermore, virulence and host immune recognition was affected. Gene expression studies involving chromatin immunoprecipitation (ChIP) of RNA polymerase II (PolII) coupled to sequencing (Seq) revealed that deletion of nsdC resulted in changes in expression of over 620 genes under basal growth conditions. This demonstrated that this transcription factor mediates the activity of a wide variety of signalling and metabolic pathways and indicates that despite the naming of the gene, the promotion of sexual reproduction is just one among multiple roles of NsdC.