The narrow footprint of ancient balancing selection revealed by heterokaryon incompatibility genes in Aspergillus fumigatus.

In fungi, fusion between individuals leads to localized cell death, a phenomenon termed heterokaryon incompatibility. Generally, the genes responsible for this incompatibility are observed to be under balancing selection resulting from negative frequency-dependent selection. Here, we assess this phenomenon in Aspergillus fumigatus, a human pathogenic fungus with a very low level of linkage disequilibrium as well as an extremely high crossover rate. Using complementation of auxotrophic mutations as an assay for hyphal compatibility, we screened sexual progeny for compatibility to identify genes involved in this process, called het genes. In total, 5/148 (3.4%) offspring were compatible with a parent and 166/2142 (7.7%) sibling pairs were compatible, consistent with several segregating incompatibility loci. Genetic mapping identified five loci, four of which could be fine mapped to individual genes, of which we tested three through heterologous expression, confirming their causal relationship. Consistent with long-term balancing selection, trans-species polymorphisms were apparent across several sister species, as well as equal allele frequencies within A. fumigatus. Surprisingly, a sliding window genome-wide population-level analysis of an independent dataset did not show increased Tajima's D near these loci, in contrast to what is often found surrounding loci under balancing selection. Using available de novo assemblies, we show that these balanced polymorphisms are restricted to several hundred base pairs flanking the coding sequence. In addition to identifying the first het genes in an Aspergillus species, this work highlights the interaction of long-term balancing selection with rapid linkage disequilibrium decay.

The human fungal pathogen Aspergillus fumigatus can produce the highest known number of meiotic crossovers.

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.

Allorecognition genes drive reproductive isolation in Podospora anserina.

Allorecognition, the capacity to discriminate self from conspecific non-self, is a ubiquitous organismal feature typically governed by genes evolving under balancing selection. Here, we show that in the fungus Podospora anserina, allorecognition loci controlling vegetative incompatibility (het genes), define two reproductively isolated groups through pleiotropic effects on sexual compatibility. These two groups emerge from the antagonistic interactions of the unlinked loci het-r (encoding a NOD-like receptor) and het-v (encoding a methyltransferase and an MLKL/HeLo domain protein). Using a combination of genetic and ecological data, supported by simulations, we provide a concrete and molecularly defined example whereby the origin and coexistence of reproductively isolated groups in sympatry is driven by pleiotropic genes under balancing selection.

Azole-Resistance Development; How the Aspergillus fumigatus Lifecycle Defines the Potential for Adaptation.

In order to successfully infect or colonize human hosts or survive changing environments, Aspergillus fumigatus needs to adapt through genetic changes or phenotypic plasticity. The genomic changes are based on the capacity of the fungus to produce genetic variation, followed by selection of the genotypes that are most fit to the new environment. Much scientific work has focused on the metabolic plasticity, biofilm formation or the particular genetic changes themselves leading to adaptation, such as antifungal resistance in the host. Recent scientific work has shown advances made in understanding the natural relevance of parasex and how both the asexual and sexual reproduction can lead to tandem repeat elongation in the target gene of the azoles: the cyp51A gene. In this review, we will explain how the fungus can generate genetic variation that can lead to adaptation. We will discuss recent advances that have been made in the understanding of the lifecycle of A. fumigatus to explain the differences observed in speed and type of mutations that are generated under different environments and how this can facilitate adaptation, such as azole-resistance selection.

Cytoplasmic Mixing, Not Nuclear Coexistence, Can Explain Somatic Incompatibility in Basidiomycetes.

Nonself recognition leading to somatic incompatibility (SI) is commonly used by mycologists to distinguish fungal individuals. Despite this, the process remains poorly understood in basidiomycetes as all current models of SI are based on genetic and molecular research in ascomycete fungi. Ascomycete fungi are mainly found in a monokaryotic stage, with a single type of haploid nuclei, and only briefly during mating do two genomes coexist in heterokaryotic cells. The sister phylum, Basidiomycota, differs in several relevant aspects. Basidiomycete fungi have an extended heterokaryotic stage, and SI is generally observed between heterokaryons instead of between homokaryons. Additionally, considerable nuclear migration occurs during a basidiomycete mating reaction, introducing a nucleus into a resident homokaryon with cytoplasmic mixing limited to the fused or neighboring cells. To accommodate these differences, we describe a basidiomycete model for nonself recognition using post-translational modification, based on a reader-writer system as found in other organisms. This post-translational modification combined with nuclear migration allows for the coexistence of two genomes in one individual while maintaining nonself recognition during all life stages. Somewhat surprisingly, this model predicts localized cell death during mating, which is consistent with previous observations but differs from the general assumptions of basidiomycete mating. This model will help guide future research into the mechanisms behind basidiomycete nonself recognition.

Selective Flamingo Medium for the Isolation of Aspergillus fumigatus.

For various studies in the clinic as well as the environment, it is essential to be able to selectively isolate Aspergillus fumigatus from samples containing bacteria as well as various other fungi (mainly Mucorales). Six agar media were compared for effectiveness in selectively isolating Aspergillus fumigatus from agricultural plant waste, woodchip waste, green waste, soil, grass and air samples collected in The Netherlands at a 48 °C incubation. The Flamingo Medium incubated at 48 °C, provided the most effective condition for the isolation of A. fumigatus from environmental samples, since it effectively inhibited the growth of competing fungi (mainly Mucorales) present in the environmental samples. Flamingo Medium reduced the number of colonies of Mucorales species by 95% and recovered an average of 20-30% more A. fumigatus colonies compared to the other media. We further confirmed that Flamingo Medium can inhibit the growth of clinical Mucorales, which occasionally present in patient's tissue and can also be used for clinical applications. We suggest the use of Flamingo Medium as an efficient method for the study of A. fumigatus from important environmental niches for which there is increasing interest. Additionally, it can also be used in the clinic to isolate A. fumigatus especially from tissue contaminated with Mucorales.

Somatic deficiency causes reproductive parasitism in a fungus.

Some multicellular organisms can fuse because mergers potentially provide mutual benefits. However, experimental evolution in the fungus Neurospora crassa has demonstrated that free fusion of mycelia favours cheater lineages, but the mechanism and evolutionary dynamics of this exploitation are unknown. Here we show, paradoxically, that all convergently evolved cheater lineages have similar fusion deficiencies. These mutants are unable to initiate fusion but retain access to wild-type mycelia that fuse with them. This asymmetry reduces cheater-mutant contributions to somatic substrate-bound hyphal networks, but increases representation of their nuclei in the aerial reproductive hyphae. Cheaters only benefit when relatively rare and likely impose genetic load reminiscent of germline senescence. We show that the consequences of somatic fusion can be unequally distributed among fusion partners, with the passive non-fusing partner profiting more. We discuss how our findings may relate to the extensive variation in fusion frequency of fungi found in nature.

Flower Bulb Waste Material is a Natural Niche for the Sexual Cycle in Aspergillus fumigatus.

With population genetic evidence of recombination ongoing in the natural Aspergillus fumigatus population and a sexual cycle demonstrated in the laboratory the question remained what the natural niche for A. fumigatus sex is. Composting plant-waste material is a known substrate of A. fumigatus to thrive and withstand temperatures even up to 70°C. Previous studies have shown indirect evidence for sexual reproduction in these heaps but never directly demonstrated the sexual structures due to technical limitations. Here, we show that flower bulb waste material from stockpiles undergoing composting can provide the conditions for sexual reproduction. Direct detection of ascospore structures was shown in agricultural flower bulb waste material by using a grid-based detection assay. Furthermore, we demonstrate that ascospores can germinate after exposure to 70°C for up to several days in contrast to asexual conidia that are unable to survive a two-hour heat shock. This indicates a sufficient time frame for ascospores to survive and escape composting stockpiles. Finally, sexual crosses with cleistothecium and viable ascospore formation could successfully be performed on flower bulb waste material. Recombination of A. fumigatus can now be explained by active sexual reproduction in nature as we show in this study that flower bulb waste material provides an environmental niche for sex.

Dynamics of Aspergillus fumigatus in Azole Fungicide-Containing Plant Waste in the Netherlands (2016-2017).

The treatment of patients suffering from Aspergillus diseases is hampered due to infections with Aspergillus fumigatus that are already resistant to medical azoles. Previous work has suggested that A. fumigatus likely gains resistance through environmental azole exposure in so-called hot spots. Here, we investigated A. fumigatus resistance dynamics over time at three sites at which farmers used azole fungicides for crop protection. Over 16 months, 114 samples were taken from stockpiles of decaying plant waste. A. fumigatus and azole fungicide residues were ubiquitously present in the plant waste. On average, 105A. fumigatus CFU/g was recovered, of which roughly half were itraconazole and tebuconazole resistant. Similar tandem repeat-mediated resistance mechanisms were found in colonies cultured from plant waste as reported in clinical azole-resistant isolates. Our results show a consistent high burden of azole-resistant A. fumigatus in azole-containing plant waste and underscores the need to further investigate resistance-reducing interventions and transmission routes.IMPORTANCEAspergillus fumigatus is consistently present independently on season at a high abundance in plant waste material throughout the sampling period. Our study confirmed that long-term storage sites of azole-containing decaying plant material can indeed be considered hot spots, which can sustain resistance development and maintenance in A. fumigatus Roughly half of individual isolates were azole resistant and carried genetic mutations that are highly similar to those found in patients with azole-resistant invasive aspergillosis. Our work suggests that environmental sources of azole resistance in A. fumigatus may be important, underscoring the need for further studies on environment-to-patient transmission routes.

Parasexual recombination enables Aspergillus fumigatus to persist in cystic fibrosis.

Aspergillus fumigatus is a saprobic fungus that causes a range of pulmonary diseases, some of which are characterised by fungal persistence such as is observed in cystic fibrosis (CF) patients. Creation of genetic variation is critical for A. fumigatus to adapt to the lung environment, but biofilm formation, especially in CF patients, may preclude mutational supply in A. fumigatus due to its confinement to the hyphal morphotype. We tested our hypothesis that genetic variation is created through parasexual recombination in chronic biofilms by phenotypic and genetic analysis of A. fumigatus isolates cultured from different origins. As diploids are the hallmark of parasex, we screened 799 A. fumigatus isolates obtained from patients with CF, chronic pulmonary lung disease and acute invasive aspergillosis, and from the environment for spore size. Benomyl sensitivity, nuclear content measurements through fluorescence-activated cell sorting and scanning electron microscopy were used to confirm the diploid state of large size spores. Whole genome sequencing was used to characterise diploid-associated genetic variation. We identified 11 diploids in isolates recovered from six of 11 (55%) CF patients and from one of 24 (4%) chronic aspergillosis patients, but not in 368 isolates from patients with acute Aspergillus infection and the environment. Diploid formation was associated with accumulation of mutations and variable haploid offspring including a voriconazole-resistant isolate. Parasexual recombination allows A. fumigatus to adapt and persist in CF patients, and plays a role in azole resistance development. Our findings are highly significant for understanding the genetics and biology of A. fumigatus in the human lung.

The taxonomy of the model filamentous fungus Podospora anserina.

The filamentous fungus Podospora anserina has been used as a model organism for more than 100 years and has proved to be an invaluable resource in numerous areas of research. Throughout this period, P. anserina has been embroiled in a number of taxonomic controversies regarding the proper name under which it should be called. The most recent taxonomic treatment proposed to change the name of this important species to Triangularia anserina. The results of past name changes of this species indicate that the broader research community is unlikely to accept this change, which will lead to nomenclatural instability and confusion in literature. Here, we review the phylogeny of the species closely related to P. anserina and provide evidence that currently available marker information is insufficient to resolve the relationships amongst many of the lineages. We argue that it is not only premature to propose a new name for P. anserina based on current data, but also that every effort should be made to retain P. anserina as the current name to ensure stability and to minimise confusion in scientific literature. Therefore, we synonymise Triangularia with Podospora and suggest that either the type species of Podospora be moved to P. anserina from P. fimiseda or that all species within the Podosporaceae be placed in the genus Podospora.

The Medical Triazole Voriconazole Can Select for Tandem Repeat Variations in Azole-Resistant Aspergillus Fumigatus Harboring TR34/L98H Via Asexual Reproduction.

Azole-resistant Aspergillus fumigatus isolates recovered at high frequency from patients, harbor mutations that are associated with variation of promoter length in the cyp51A gene. Following the discovery of the TR34/L98H genotype, new variations in tandem repeat (TR) length and number of repeats were identified, as well as additional single nucleotide polymorphisms (SNPs) in the cyp51A gene, indicating that the diversity of resistance mutations in A. fumigatus is likely to continue to increase. Investigating the development routes of TR variants is critical to be able to design preventive interventions. In this study, we tested the potential effects of azole exposure on the selection of TR variations, while allowing haploid A. fumigatus to undergo asexual reproduction. Through experimental evolution involving voriconazole (VOR) exposure, an isolate harboring TR343/L98H evolved from a clinical TR34/L98H ancestor isolate, confirmed by whole genome sequencing. TR343/L98H was associated with increased cyp51A expression and high VOR and posaconazole MICs, although additional acquired SNPs could also have contributed to the highly azole-resistant phenotype. Exposure to medical azoles was found to select for TR343, thus supporting the possibility of in-host selection of TR34 variants.

Environmental Hotspots for Azole Resistance Selection of Aspergillus fumigatus, the Netherlands.

Azole resistance is a major concern for treatment of infections with Aspergillus fumigatus. Environmental resistance selection is a main route for Aspergillus spp. to acquire azole resistance. We investigated the presence of environmental hotspots for resistance selection in the Netherlands on the basis of the ability of A. fumigatus to grow and reproduce in the presence of azole fungicide residues. We identified 3 hotspots: flower bulb waste, green waste material, and wood chippings. We recovered azole-resistant A. fumigatus from these sites; all fungi contained cyp51A tandem repeat-mediated resistance mechanisms identical to those found in clinical isolates. Tebuconazole, epoxiconazole, and prothioconazole were the most frequently found fungicide residues. Stockpiles of plant waste contained the highest levels of azole-resistant A. fumigatus, and active aerobic composting reduced Aspergillus colony counts. Preventing plant waste stockpiling or creating unfavorable conditions for A. fumigatus to grow in stockpiles might reduce environmental resistance burden.

Mutation-rate plasticity and the germline of unicellular organisms.

The mutation rate is a fundamental factor in evolutionary genetics. Recently, mutation rates were found to be strongly reduced at high density in a wide range of unicellular organisms, prokaryotic and eukaryotic. Independently, cell division was found to become more asymmetrical at increasing density in diverse organisms; some 'mother' cells continue dividing, while their 'offspring' cells do not divide further. Here, we investigate how this increased asymmetry in cell division at high density can be reconciled with reduced mutation-rate estimates. We calculated the expected number of mutant cells due to replication errors under various modes of segregation of template-DNA strands and copy-DNA strands, both under symmetrical (exponential) and asymmetrical (linear) growth. We show that the observed reduction in the mutation rate at high density can be explained if mother cells preferentially retain the template-DNA strands, since new mutations are then confined to non-dividing daughter cells, thus reducing the spread of mutant cells. Any other inheritance mode results in an increase in the number of mutant cells at higher density. The proposed hypothesis that patterns of DNA-strand segregation are density-dependent fundamentally challenges our current understanding of mutation-rate estimates and extends the distinction between germline and soma to unicellular organisms.

Relevance of heterokaryosis for adaptation and azole-resistance development in Aspergillus fumigatus.

Aspergillus fumigatus causes a range of diseases in humans, some of which are characterized by fungal persistence. Aspergillus fumigatus, being a generalist saprotroph, may initially establish lung colonization due to its physiological versatility and subsequently adapt through genetic changes to the human lung environment and antifungal treatments. Human lung-adapted genotypes can arise by spontaneous mutation and/or recombination and subsequent selection of the fittest genotypes. Sexual and asexual spores are considered crucial contributors to the genetic diversity and adaptive potential of aspergilli by recombination and mutation supply, respectively. However, in certain Aspergillus diseases, such as cystic fibrosis and chronic pulmonary aspergillosis, A. fumigatus may not sporulate but persist as a network of fungal mycelium. During azole therapy, such mycelia may develop patient-acquired resistance and become heterokaryotic by mutations in one of the nuclei. We investigated the relevance of heterokaryosis for azole-resistance development in A. fumigatus. We found evidence for heterokaryosis of A. fumigatus in patients with chronic Aspergillus diseases. Mycelium from patient-tissue biopsies segregated different homokaryons, from which heterokaryons could be reconstructed. Whereas all variant homokaryons recovered from the same patient were capable of forming a heterokaryon, those from different patients were heterokaryon-incompatible. We furthermore compared heterokaryons and heterozygous diploids constructed from environmental isolates with different levels of azole resistance. When exposed to azole, the heterokaryons revealed remarkable shifts in their nuclear ratio, and the resistance level of heterokaryons exceeded that of the corresponding heterozygous diploids.

The obligate alkalophilic soda-lake fungus Sodiomyces alkalinus has shifted to a protein diet.

Sodiomyces alkalinus is one of the very few alkalophilic fungi, adapted to grow optimally at high pH. It is widely distributed at the plant-deprived edges of extremely alkaline lakes and locally abundant. We sequenced the genome of S. alkalinus and reconstructed evolution of catabolic enzymes, using a phylogenomic comparison. We found that the genome of S. alkalinus is larger, but its predicted proteome is smaller and heavily depleted of both plant-degrading enzymes and proteinases, when compared to its closest plant-pathogenic relatives. Interestingly, despite overall losses, S. alkalinus has retained many proteinases families and acquired bacterial cell wall-degrading enzymes, some of them via horizontal gene transfer from bacteria. This fungus has very potent proteolytic activity at high pH values, but slowly induced low activity of cellulases and hemicellulases. Our experimental and in silico data suggest that plant biomass, a common food source for most fungi, is not a preferred substrate for S. alkalinus in its natural environment. We conclude that the fungus has abandoned the ancestral plant-based diet and has become specialized in a more protein-rich food, abundantly available in soda lakes in the form of prokaryotes and small crustaceans.

Phylogeny of Paecilomyces, the causal agent of pistachio and some other trees dieback disease in Iran.

One of the most important fungal agents of pistachio dieback disease belongs to the ascomycete genus Paecilomyces that has been identified as P. variotii. In 2012-2014, 700 plant samples from pistachio trees and 27 other plant species with dieback symptoms were collected from 10 provinces of Iran. Of the 567 pistachio samples, 277 Paecilomyces strains were obtained and from the 133 samples of other plants (except pistachio and including Pistacia mutica, Punica granatum, Prunus amygdalus, Caesalpinia gilliesii, Nerium oleander, Tamarix aphylla, Tamarix hispida and Haloxylon sp.), 23 fungal isolates were recovered and five isolates were obtained from the air of infected pistachio orchards. Based on morphology, all 305 isolates were identified as P. variotii. Physiological studies revealed that 299 isolates belong to P. formosus. Three isolates were assigned to P. variotii, while three isolates could not be assigned to any of the known species. Of the 305 isolates, 62 were selected for phylogenetic analysis based on DNA variation (ITS, ß-tubulin and calmodulin). This analysis showed that all of our isolates form a clade with P. formosus. P. formosus consists of the three former species P. formosa, P. lecythidis and P. maximus. This study shows that our isolates form a strongly supported clade with strains of P. lecythidis. So, the causal agent of dieback disease of pistachio and other examined trees is P. formosus which is closely related to the former species P. lecythidis and has some differences with the former species P. formosa and P. maximus. Based on phylogenetic studies P. formosus thus seems to be a species complex that could be divided into three separate species.

The alkalophilic fungus Sodiomyces alkalinus hosts beta- and gammapartitiviruses together with a new fusarivirus.

Mixed infection by three dsRNA viruses, a novel betapartitivirus, a gammapartitivirus, and a novel fusarivirus, has been identified in four isolates of the obligate alkalophilic fungus Sodiomyces alkalinus. The first, Sodiomyces alkalinus partitivirus 1 (SaPV1), is placed within the genus Betapartitivirus and is related to Ustilaginoidea virens partitivirus 2. The taxonomic position of the second virus is less clear as it shares high (85%) amino acid sequence identity but significantly low (77%) nucleotide sequence identity of the capsid protein with Colletotrichum truncatum partitivirus 1. The third, the novel Sodiomyces alkalinus fusarivirus 1 (SaFV1), is related to Fusarium poae fusarivirus 1. All the viruses show efficient vertical transmission through asexual and sexual spores. These novel coexisting viruses do not evoke apparent phenotypic alteration to their fungal host. This is the first description of a viral infection in an alkalophilic fungus.

Evolution of cross-resistance to medical triazoles in Aspergillus fumigatus through selection pressure of environmental fungicides.

Resistance to medical triazoles in Aspergillus fumigatus is an emerging problem for patients at risk of aspergillus diseases. There are currently two presumed routes for medical triazole-resistance selection: (i) through selection pressure of medical triazoles when treating patients and (ii) through selection pressure from non-medical sterol-biosynthesis-inhibiting (SI) triazole fungicides which are used in the environment. Previous studies have suggested that SI fungicides can induce cross-resistance to medical triazoles. Therefore, to assess the potential of selection of resistance to medical triazoles in the environment, we assessed cross-resistance to three medical triazoles in lineages of A. fumigatus from previous work where we applied an experimental evolution approach with one of five different SI fungicides to select for resistance. In our evolved lines we found widespread cross-resistance indicating that resistance to medical triazoles rapidly arises through selection pressure of SI fungicides. All evolved lineages showed similar evolutionary dynamics to SI fungicides and medical triazoles, which suggests that the mutations inducing resistance to both SI fungicides and medical triazoles are likely to be the same. Whole-genome sequencing revealed that a variety of mutations were putatively involved in the resistance mechanism, some of which are in known target genes.

A Novel Environmental Azole Resistance Mutation in Aspergillus fumigatus and a Possible Role of Sexual Reproduction in Its Emergence.

This study investigated the dynamics of Aspergillus fumigatus azole-resistant phenotypes in two compost heaps with contrasting azole exposures: azole free and azole exposed. After heat shock, to which sexual but not asexual spores are highly resistant, the azole-free compost yielded 98% (49/50) wild-type and 2% (1/50) azole-resistant isolates, whereas the azole-containing compost yielded 9% (4/45) wild-type and 91% (41/45) resistant isolates. From the latter compost, 80% (36/45) of the isolates contained the TR46/Y121F/T289A genotype, 2% (1/45) harbored the TR46/Y121F/M172I/T289A/G448S genotype, and 9% (4/45) had a novel pan-triazole-resistant mutation (TR463/Y121F/M172I/T289A/G448S) with a triple 46-bp promoter repeat. Subsequent screening of a representative set of clinical A. fumigatus isolates showed that the novel TR463 mutant was already present in samples from three Dutch medical centers collected since 2012. Furthermore, a second new resistance mutation was found in this set that harbored four TR46 repeats. Importantly, in the laboratory, we recovered the TR463 mutation from a sexual cross between two TR46 isolates from the same azole-containing compost, possibly through unequal crossing over between the double tandem repeats (TRs) during meiosis. This possible role of sexual reproduction in the emergence of the mutation was further implicated by the high level of genetic diversity of STR genotypes in the azole-containing compost. Our study confirms that azole resistance mutations continue to emerge in the environment and indicates compost containing azole residues as a possible hot spot. Better insight into the biology of environmental resistance selection is needed to retain the azole class for use in food production and treatment of Aspergillus diseases.IMPORTANCE Composting of organic matter containing azole residues might be important for resistance development and subsequent spread of resistance mutations in Aspergillus fumigatus In this article, we show the dominance of azole-resistant A. fumigatus in azole-exposed compost and the discovery of a new resistance mutation with clinical relevance. Furthermore, our study indicates that current fungicide application is not sustainable as new resistance mutations continue to emerge, thereby threatening the use of triazoles in medicine. We provide evidence that the sexual part of the fungal life cycle may play a role in the emergence of resistance mutations because under laboratory conditions, we reconstructed the resistance mutation through sexual crossing of two azole-resistant A. fumigatus isolates derived from the same compost heap. Understanding the mechanisms of resistance selection in the environment is needed to design strategies against the accumulation of resistance mutations in order to retain the azole class for crop protection and treatment of Aspergillus diseases.