Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    The medical triazole voriconazole can select for tandem repeat variations in azole-resistant aspergillus fumigatus harboring tr34 /l98h via asexual reproduction
    Zhang, Jianhua ; Zoll, Jan ; Engel, Tobias ; Heuvel, Joost van den; Verweij, Paul E. ; Debets, Alfons J.M. - \ 2020
    Journal of Fungi 6 (2020)4. - ISSN 2309-608X
    Antifungal agents - Asexual reproduction - Aspergillus fumigatus - Azole resistance - Tandem repeats variants

    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.

    Data from: Combinations of Spok genes create multiple meiotic drivers in Podospora
    Vogan, Aaron A. ; Ament-Velásquez, S.L. ; Granger-Farbos, Alexandra ; Svedberg, Jesper ; Bastiaans, Eric ; Debets, Fons ; Coustou, Virginie ; Yvanne, Hélène ; Clavé, Corinne ; Saupe, Sven J. ; Johannesson, Hanna - \ 2019
    Dryad
    podospora anserina - meiotic drive - genomics - gene drive - Podospora pauciseta - Spore-killing - Spok - genomic conflict
    Meiotic drive is the preferential transmission of a particular allele during sexual reproduction. The phenomenon is observed as spore killing in multiple fungi. In natural populations of Podospora anserina, seven spore killer types (Psks) have been identified through classical genetic analyses. Here we show that the Spok gene-family underlies the Psks. The combination of Spok genes at different chromosomal locations defines the spore killer types and creates a killing hierarchy within the same population. We identify two novel Spok homologs located within a large (74-167 kbp) region (the Spok block) that resides in different chromosomal locations in given strains. We confirm that the SPOK protein performs both killing and resistance functions and show that these activities are dependent on distinct domains, a predicted nuclease and kinase domain. Genomic and phylogenetic analyses across ascomycetes suggest that the Spok genes disperse via cross-species transfer, and evolve by duplication and diversification within lineages.
    Combinations of Spok genes create multiple meiotic drivers in Podospora
    Vogan, Aaron A. ; Ament-Velásquez, S.L. ; Granger-Farbos, Alexandra ; Svedberg, Jesper ; Bastiaans, Eric ; Debets, Alfons J.M. ; Coustou, Virginie ; Yvanne, Hélène ; Clavé, Corinne ; Saupe, Sven J. ; Johannesson, Hanna - \ 2019
    eLife 8 (2019). - ISSN 2050-084X
    evolutionary biology - fungi - gene drive - genetics - genomic conflict - genomics - Podospora - selfish genetic element - spore killer

    Meiotic drive is the preferential transmission of a particular allele during sexual reproduction. The phenomenon is observed as spore killing in multiple fungi. In natural populations of Podospora anserina, seven spore killer types (Psks) have been identified through classical genetic analyses. Here we show that the Spok gene family underlies the Psks. The combination of Spok genes at different chromosomal locations defines the spore killer types and creates a killing hierarchy within a population. We identify two novel Spok homologs located within a large (74-167 kbp) region (the Spok block) that resides in different chromosomal locations in different strains. We confirm that the SPOK protein performs both killing and resistance functions and show that these activities are dependent on distinct domains, a predicted nuclease and kinase domain. Genomic and phylogenetic analyses across ascomycetes suggest that the Spok genes disperse through cross-species transfer, and evolve by duplication and diversification within lineages.

    Environmental hotspots for azole resistance selection of aspergillus fumigatus, the netherlands
    Schoustra, Sijmen E. ; Debets, Alfons J.M. ; Rijs, Antonius J.M.M. ; Zhang, Jianhua ; Snelders, Eveline ; Leendertse, Peter C. ; Melchers, Willem J.G. ; Rietveld, Anton G. ; Zwaan, Bas J. ; Verweij, Paul E. - \ 2019
    Emerging Infectious Diseases 25 (2019)7. - ISSN 1080-6040 - p. 1347 - 1353.

    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
    Aanen, Duur K. ; Debets, Alfons J.M. - \ 2019
    Proceedings of the Royal Society. B: Biological Sciences 286 (2019)1902. - ISSN 0962-8452 - 7 p.
    asymmetrical cell division - density-associated mutation-rate plasticity - germline–soma distinction - immortal strand hypothesis - mutation rate - 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.

    Data from: Relevance of heterokaryosis for adaptation and azole-resistance development in Aspergillus fumigatus
    Zhang, J. ; Snelders, E. ; Zwaan, B.J. ; Schoustra, S.E. ; Kuijper, Ed J. ; Arendrup, Maiken C. ; Melchers, Willem J.G. ; Verweij, Paul E. ; Debets, A.J.M. - \ 2019
    Wageningen University & Research
    heterokaryon incompatibility - azole resistance - flexible nuclear ratio - Aspergillus fumigatus
    Aspergillus fumigatus causes a range of diseases in humans, some of which are characterized by fungal persistence. A. fumigatus, being a generalist saprotroph, may initially establish lung colonisation 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.
    Relevance of heterokaryosis for adaptation and azole-resistance development in Aspergillus fumigatus
    Zhang, Jianhua ; Snelders, Eveline E. ; Zwaan, Bas J. ; Schoustra, Sijmen E. ; Kuijper, Ed J. ; Arendrup, Maiken C. ; Melchers, Willem J.G. ; Verweij, Paul E. ; Debets, Alfons J.M. - \ 2019
    Proceedings of the Royal Society. B: Biological Sciences 286 (2019)1896. - ISSN 0962-8452
    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.
    Asymmetrical template-DNA strand segregation can explain density-associated mutation-rate plasticity
    Aanen, D.K. ; Debets, A.J.M. - \ 2018
    BioRxiv - 9 p.
    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; in yeast, 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 exponential and under 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.
    The obligate alkalophilic soda-lake fungus Sodiomyces alkalinus has shifted to a protein diet
    Grum-Grzhimaylo, A. ; Falkoski, D.L. ; Heuvel, Joost van den; Valero Jimenez, C.A. ; Min, B. ; Choi, I.G. ; Lipzen, A. ; Daum, C.G. ; Aanen, D.K. ; Tsang, A. ; Henrissat, B. ; Bilanenko, E.N. ; Vries, R.P. de; Kan, J.A.L. van; Grigoriev, I.V. ; Debets, A.J.M. - \ 2018
    Molecular Ecology 27 (2018)23. - ISSN 0962-1083 - p. 4808 - 4819.
    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
    Heidarian, Reza ; Fotouhifar, Khalil Berdi ; Debets, Alfons J.M. ; Aanen, Duur K. - \ 2018
    PLoS ONE 13 (2018)7. - ISSN 1932-6203

    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, β-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.

    Mosaic structure of the fungal community in the Kislo-Sladkoe Lake that is detaching from the White Sea
    Grum-Grzhimaylo, Olga A. ; Debets, Alfons J.M. ; Bilanenko, Elena N. - \ 2018
    Polar Biology 41 (2018)10. - ISSN 0722-4060 - p. 2075 - 2089.
    Brackish lake - Coastal rising - Fungal diversity - Glacioisostatic movement

    The major part of the north polar region is intensely rising by postglacial crustal movement. This process gives rise to the separation of different basins from seas and oceans, which affects a combination of freshwater and marine organisms. Gradually losing contact with the seas, many near-shore lakes of the Arctic are mostly desalted and form bogs. Fungi as decomposers play an important role in all ecosystems. However, the diversity and role of fungi in Arctic aquatic ecosystems is largely unknown. It is also not clear how the taxonomic structure of the fungal community is affected by the process of gradual desalinization and waterlogging. We investigated the diversity of filamentous culturable fungi in different parts of the brackish Kislo-Sladkoe Lake (White Sea, Russia). Annually, 42 samples of the bottom and coastal soils have been collected at the lake from which fungi were recovered on standard and selective media. Based on morphological and molecular markers, a total of 127 taxa have been identified. The fungal community appeared to be influenced by its sea origin and comprised both marine (Paradendryphiella salina, Acremonium spp.) and terrestrial soil species of Penicillium, Talaromyces, Mucor, Umbelopsis, Cladosporium, Cadophora, Sistotrema, Helotiales, Pleosporales, sphagnum moss destructors (Oidiodendron spp.) and insect-associated species of Tolypocladium. The results indicate that the composition of the fungal community in the rising polar White Sea region reflects the dynamics of global changes in physical–chemical parameters and animal and plant associations because of separation from the sea.

    Why some fungi senesce and others do not: An evolutionary perspective on fungal senescence
    Maas, Marc F.P.M. ; Debets, Alfons J.M. ; Zwaan, Bas J. ; Diepeningen, Anne D. van - \ 2017
    In: The Evolution of Senescence in the Tree of Life Cambridge University Press - ISBN 9781107078505 - p. 341 - 361.

    Fungi are generally considered to be modular organisms with no clear distinction of a germ line: With the expansion of the mycelium, chances for reproduction are expected to increase, and each unit under favourable circumstances may produce offspring. Fungi with such modular body plans are expected to be long-lived, as most fungi indeed seem to be. However, fungi exist that do senesce, and their growth often seems to be limited by space or time. For these fungi, we can consider the term 'pseudo-unitary', as life history details and ecological conditions constrain the size of the soma and the opportunities for reproduction. We may predict the life history traits and ecological conditions that favour such evolution of fungal senescence. Known proximate mechanisms of fungal senescence can be viewed in the light of this evolutionary context.

    Data from: Evolution of cross-resistance to medical triazoles in Aspergillus fumigatus through selection pressure of environmental fungicides
    Zhang, J. ; Heuvel, Joost van den; Debets, A.J.M. ; Verweij, Paul E. ; Melchers, Willem J.G. ; Zwaan, B.J. ; Schoustra, S.E. - \ 2017
    Wageningen University & Research
    triazole resistance - cross-resistance - experimental evolution
    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.
    The alkalophilic fungus Sodiomyces alkalinus hosts beta- and gammapartitiviruses together with a new fusarivirus
    Hrabáková, Lenka ; Grum-Grzhimaylo, Alexey A. ; Koloniuk, Igor ; Debets, Alfons J.M. ; Sarkisova, Tatiana ; Petrzik, Karel - \ 2017
    PLoS ONE 12 (2017)11. - ISSN 1932-6203
    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
    Zhang, Jianhua ; Heuvel, Joost van den; Debets, Fons ; Verweij, Paul E. ; Melchers, Willem J.G. ; Zwaan, Bas J. ; Schoustra, Sijmen E. - \ 2017
    Proceedings of the Royal Society. B: Biological Sciences 284 (2017)1863. - ISSN 0962-8452
    Aspergillus fumigatus - Evolutionary trajectory - Experimental evolution - Sterol-biosynthesis-inhibiting (SI) fungicides and medical triazoles - Triazole resistance

    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
    Zhang, Jianhua ; Snelders, Eveline ; Zwaan, Bas J. ; Schoustra, Sijmen E. ; Meis, Jacques F. ; Dijk, Karin van; Hagen, Ferry ; Beek, Martha T. van der; Kampinga, Greetje A. ; Zoll, Jan ; Melchers, Willem J.G. ; Verweij, Paul E. ; Debets, Fons - \ 2017
    mBio 8 (2017)3. - ISSN 2161-2129
    Ascospores - Aspergillus fumigatus - Azole resistance - Compost heap - Conidiospores - Hot spot for resistance development - Novel mutation - Sexual reproduction

    This study investigated the dynamics of Aspergillus fumigatus azoleresistant 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-triazoleresistant mutation (TR46 3/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 TR46 3 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 TR46 3 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 azoleresistant 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.

    Genome of the obligately alkaliphilic fungus Sodiomyces alkalinus reveals its adaptations to high pH.
    Grum-Grzhimaylo, A. ; Falkoski, D.L. ; Heuvel, Joost van den; Min, B. ; Choi, I.G. ; Henrissat, Bernard ; Franssen, H.G.J.M. ; Bilanenko, E.N. ; Vries, Ronald P. De; Kan, J.A.L. van; Grigoriev, I.V. ; Debets, A.J.M. - \ 2017
    In: Abstract Book 29th Fungal Genetics Conference Asilomar 17, Pacific Grove, CA, USA 14-19 March 2017. - Genetics Society of America - p. 152 - 152.
    Alkaliphilic fungi, i.e. fungi that grow optimally at high pH, are exceptional with only a handful of species described to date. Here, we sequenced the obligate alkaliphilic ascomycete S. alkalinus isolated from alkaline soda soils, and explored the features responsible for its unusual life-style. We found that PacC, the major regulator of alkaline-related genes, is up-regulated at higher pH values than PacC orthologues of neutrophilic species. To assess how this fungus can degrade carbohydrate sources under extremely alkalic conditions, we searched for the CAZymes encoded in the genome of S. alkalinus and performed enzyme assays. We revealed many cellulases, but their overall activity was low presumably because of slower induction. Although cellulolytic and hemicellulolytic activity was optimal at pH 6, there still was some activity at pH 10, at which pH it was completely absent in the neutrophilic A. oryzae. Excellent in vitro growth on xylan indicates that grasses are the preferred nutritional source for S. alkalinus in nature. We detected potent proteolytic activity at alkaline pH, compared to the neutrophilic A. oryzae, which may reflect the need for obtaining extra nitrogen, as this essential element becomes limiting at alkaline conditions. Proteins of bacterial cells, which are present in bulk at soda lakes, seem the likely source of nitrogen. In support of this hypothesis, we found several instances of horizontal transfer of prokaryotic genes into the genome of S. alkalinus, encoding enzymes that degrade bacterial cell walls. The genome of S. alkalinus will provide a valuable source to further study the biology and evolution of alkaliphilic trait in fungi with respect to neutrophilic species. In addition, it may provide alkaline-active metabolites of commercial interest.
    Fungus-associated bacteriome in charge of their host behavior
    Schulz-Bohm, Kristin ; Tyc, Olaf ; Boer, Wietse de; Peereboom, Nils ; Debets, Fons ; Zaagman, Niels ; Janssens, Thierry K.S. ; Garbeva, Paolina - \ 2017
    Fungal Genetics and Biology 102 (2017). - ISSN 1087-1845 - p. 38 - 48.
    Fungal bacteriome - Fungal behavior - Interactions - Mucor hiemalis - Volatiles
    Bacterial-fungal interactions are widespread in nature and there is a growing number of studies reporting distinct fungus-associated bacteria. However, little is known so far about how shifts in the fungus-associated bacteriome will affect the fungal host's lifestyle. In the present study, we describe for the first time the bacterial community associated with the saprotrophic fungus . Mucor hiemalis, commonly found in soil and rhizosphere. Two broad-spectrum antibiotics that strongly altered the bacterial community associated with the fungus were applied. Our results revealed that the antibiotic treatment did not significantly reduce the amount of bacteria associated to the fungus but rather changed the community composition by shifting from initially dominating . Alpha-Proteobacteria to dominance of . Gamma-Proteobacteria. A novel approach was applied for the isolation of fungal-associated bacteria which also revealed differences between bacterial isolates obtained from the original and the antibiotic-treated . M. . hiemalis. The shift in the composition of the fungal-associated bacterial community led to significantly reduced fungal growth, changes in fungal morphology, behavior and secondary-metabolites production. Furthermore, our results showed that the antibiotic-treated isolate was more attractive and susceptible to mycophagous bacteria as compared to the original isolate. Overall, our study highlights the importance of the fungus-associated bacteriome for the host's lifestyle and interactions and indicate that isolation with antibacterials is not sufficient to eradicate the associated bacteria.
    In-host adaptation and acquired triazole resistance in Aspergillus fumigatus: a dilemma for clinical management
    Verweij, P. ; Zhang, J. ; Debets, A.J.M. ; Meis, J.F. ; Schoustra, S.E. ; Veerdonk, F.L. van de; Zwaan, B.J. ; Melchers, W.J.G. - \ 2016
    The Lancet Infectious Diseases 16 (2016)11. - ISSN 1473-3099 - p. e251 - e260.
    Aspergillus fumigatus causes a range of diseases in human beings, some of which are characterised by fungal persistence. A fumigatus can persist by adapting to the human lung environment through physiological and genomic changes. The physiological changes are based on the large biochemical versatility of the fungus, and the genomic changes are based on the capacity of the fungus to generate genetic diversity by spontaneous mutations or recombination and subsequent selection of the genotypes that are most adapted to the new environment. In this Review, we explore the adaptation strategies of A fumigatus in relation to azole resistance selection and the clinical implications thereof for management of diseases caused by Aspergillus spp. We hypothesise that the current diagnostic tools and treatment strategies do not take into account the biology of the fungus and might result in an increased likelihood of fungal persistence in patients. Stress factors, such as triazole exposure, cause mutations that render resistance. The process of reproduction-ie, sexual, parasexual, or asexual-is probably crucial for the adaptive potential of Aspergillus spp. As any change in the environment can provoke adaptation, switching between triazoles in patients with chronic pulmonary aspergillosis might result in a high-level pan-triazole-resistant phenotype through the accumulation of resistance mutations. Alternatively, when triazole therapy is stopped, an azole-free environment is created that could prompt selection for compensatory mutations that overcome any fitness costs that are expected to accompany resistance development. As a consequence, starting, switching, and stopping azole therapy has the risk of selecting for highly resistant strains with wildtype fitness. A similar adaptation is expected to occur in response to other stress factors, such as endogenous antimicrobial peptides; over time the fungus will become increasingly adapted to the lung environment, thereby limiting the probability of eradication. Our hypothesis challenges current management strategies, and future research should investigate the genomic dynamics during infection to understand the key factors facilitating adaptation of Aspergillus spp.
    The diversity of microfungi in peatlands originated from the White Sea
    Grum-Grzhimaylo, Olga A. ; Debets, Fons ; Bilanenko, Elena N. - \ 2016
    Mycologia 108 (2016)2. - ISSN 0027-5514 - p. 233 - 254.
    fungal diversity - fungi decomposing Sphagnum - fungi in sediments - molecular identification - psychrotolerants

    The diversity of culturable filamentous microfungi in peat and sediments of four peatlands at the coastal zone of Kandalaksha Bay of the White Sea (Murmansk region, Russia) was studied by culture methods on standard and selective media. Annually 100 samples were collected from the bogs 2007-2010. Based on morphological, molecular markers and cultural features, 211 taxa were identified. Fungal communities observed at the peatlands were influenced mostly by their sea origin. We discovered a large difference between fungal communities from the peat and the sediments of the peatlands. In contrast to the sediments, the fungal community of the peat was found to be consistent throughout sampling sites. Fungi with specific ecophysiology, such as Sphagnum-decomposing species (Oidiodendron griseum, O. tenuissimum. Penicillium spinulosum, P. thomii, Talaromyces funiculosus), psychrotolerant and associated with insects species (Pseudogymnoascus pannorum, Tolypocladium spp.), typical marine species (Acremonium spp.) were found. In addition, different types of sterile mycelia were characteristic for the researched peatlands.

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