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1.  Phenotypic heterogeneity is a selected trait in natural yeast populations subject to environmental stress 
Environmental Microbiology  2013;16(6):1729-1740.
Populations of genetically uniform microorganisms exhibit phenotypic heterogeneity, where individual cells have varying phenotypes. Such phenotypes include fitness-determining traits. Phenotypic heterogeneity has been linked to increased population-level fitness in laboratory studies, but its adaptive significance for wild microorganisms in the natural environment is unknown. Here, we addressed this by testing heterogeneity in yeast isolates from diverse environmental sites, each polluted with a different principal contaminant, as well as from corresponding control locations. We found that cell-to-cell heterogeneity (in resistance to the appropriate principal pollutant) was prevalent in the wild yeast isolates. Moreover, isolates with the highest heterogeneity were consistently observed in the polluted environments, indicating that heterogeneity is positively related to survival in adverse conditions in the wild. This relationship with survival was stronger than for the property of mean resistance (IC50) of an isolate. Therefore, heterogeneity could be the major determinant of microbial survival in adverse conditions. Indeed, growth assays indicated that isolates with high heterogeneities had a significant competitive advantage during stress. Analysis of yeasts after cultivation for ≥ 500 generations additionally showed that high heterogeneity evolved as a heritable trait during stress. The results showed that environmental stress selects for wild microorganisms with high levels of phenotypic heterogeneity.
doi:10.1111/1462-2920.12243
PMCID: PMC4231229  PMID: 24000788
2.  Evolutionary Relationships between Rhynchosporium lolii sp. nov. and Other Rhynchosporium Species on Grasses 
PLoS ONE  2013;8(10):e72536.
The fungal genus Rhynchosporium (causative agent of leaf blotch) contains several host-specialised species, including R. commune (colonising barley and brome-grass), R. agropyri (couch-grass), R. secalis (rye and triticale) and the more distantly related R. orthosporum (cocksfoot). This study used molecular fingerprinting, multilocus DNA sequence data, conidial morphology, host range tests and scanning electron microscopy to investigate the relationship between Rhynchosporium species on ryegrasses, both economically important forage grasses and common wild grasses in many cereal growing areas, and other plant species. Two different types of Rhynchosporium were found on ryegrasses in the UK. Firstly, there were isolates of R. commune that were pathogenic to both barley and Italian ryegrass. Secondly, there were isolates of a new species, here named R. lolii, that were pathogenic only to ryegrass species. R. lolii was most closely related to R. orthosporum, but exhibited clear molecular, morphological and host range differences. The species was estimated to have diverged from R. orthosporum ca. 5735 years before the present. The colonisation strategy of all of the different Rhynchosporium species involved extensive hyphal growth in the sub-cuticular regions of the leaves. Finally, new species-specific PCR diagnostic tests were developed that could distinguish between these five closely related Rhynchosporium species.
doi:10.1371/journal.pone.0072536
PMCID: PMC3797698  PMID: 24146740
3.  Discovery of a Sexual Cycle in Aspergillus lentulus, a Close Relative of A. fumigatus 
Eukaryotic Cell  2013;12(7):962-969.
Aspergillus lentulus was described in 2005 as a new species within the A. fumigatus sensu lato complex. It is an opportunistic human pathogen causing invasive aspergillosis with high mortality rates, and it has been isolated from clinical and environmental sources. The species is morphologically nearly identical to A. fumigatus sensu stricto, and this similarity has resulted in their frequent misidentification. Comparative studies show that A. lentulus has some distinguishing growth features and decreased in vitro susceptibility to several antifungal agents, including amphotericin B and caspofungin. Similar to the once-presumed-asexual A. fumigatus, it has only been known to reproduce mitotically. However, we now show that A. lentulus has a heterothallic sexual breeding system. A PCR-based mating-type diagnostic detected isolates of either the MAT1-1 or MAT1-2 genotype, and examination of 26 worldwide clinical and environmental isolates revealed similar ratios of the two mating types (38% versus 62%, respectively). MAT1-1 and MAT1-2 idiomorph regions were analyzed, revealing the presence of characteristic alpha and high-mobility-group (HMG) domain genes, together with other more unusual features such as a MAT1-2-4 gene. We then demonstrated that A. lentulus possesses a functional sexual cycle with mature cleistothecia, containing heat-resistant ascospores, being produced after 3 weeks of incubation. Recombination was confirmed using molecular markers. However, isolates of A. lentulus failed to cross with highly fertile strains of A. fumigatus, demonstrating reproductive isolation between these sibling species. The discovery of the A. lentulus sexual stage has significant implications for the management of drug resistance and control of invasive aspergillosis associated with this emerging fungal pathogen.
doi:10.1128/EC.00040-13
PMCID: PMC3697472  PMID: 23650087
4.  Molecular Epidemiology of Aspergillus fumigatus Isolates Harboring the TR34/L98H Azole Resistance Mechanism 
Journal of Clinical Microbiology  2012;50(8):2674-2680.
A rapid emergence of azole resistance has been observed in Aspergillus fumigatus in The Netherlands over the past decade. The dominant resistance mechanism appears to be of environmental origin and involves the TR34/L98H mutations in cyp51A. This resistance mechanism is now also increasingly being found in other countries. Therefore, genetic markers were used to gain more insights into the origin and spread of this genotype. Studies of 142 European isolates revealed that those with the TR34/L98H resistance mechanism showed less genetic variation than azole-susceptible isolates or those with a different genetic basis of resistance and were assigned to only four CSP (putative cell surface protein) types. Sexual crossing experiments demonstrated that TR34/L98H isolates could outcross with azole-susceptible isolates of different genetic backgrounds, suggesting that TR34/L98H isolates can undergo the sexual cycle in nature. Overall, our findings suggest a common ancestor of the TR34/L98H mechanism and subsequent migration of isolates harboring TR34/L98H across Europe.
doi:10.1128/JCM.00335-12
PMCID: PMC3421523  PMID: 22675126
5.  Presence and Functionality of Mating Type Genes in the Supposedly Asexual Filamentous Fungus Aspergillus oryzae 
The potential for sexual reproduction in Aspergillus oryzae was assessed by investigating the presence and functionality of MAT genes. Previous genome studies had identified a MAT1-1 gene in the reference strain RIB40. We now report the existence of a complementary MAT1-2 gene and the sequencing of an idiomorphic region from A. oryzae strain AO6. This allowed the development of a PCR diagnostic assay, which detected isolates of the MAT1-1 and MAT1-2 genotypes among 180 strains assayed, including industrial tane-koji isolates. Strains used for sake and miso production showed a near-1:1 ratio of the MAT1-1 and MAT1-2 mating types, whereas strains used for soy sauce production showed a significant bias toward the MAT1-2 mating type. MAT1-1 and MAT1-2 isogenic strains were then created by genetic manipulation of the resident idiomorph, and gene expression was compared by DNA microarray and quantitative real-time PCR (qRT-PCR) methodologies under conditions in which MAT genes were expressed. Thirty-three genes were found to be upregulated more than 10-fold in either the MAT1-1 host strain or the MAT1-2 gene replacement strain relative to each other, showing that both the MAT1-1 and MAT1-2 genes functionally regulate gene expression in A. oryzae in a mating type-dependent manner, the first such report for a supposedly asexual fungus. MAT1-1 expression specifically upregulated an α-pheromone precursor gene, but the functions of most of the genes affected were unknown. The results are consistent with a heterothallic breeding system in A. oryzae, and prospects for the discovery of a sexual cycle are discussed.
doi:10.1128/AEM.07034-11
PMCID: PMC3318824  PMID: 22327593
6.  Identification and Characterization of an Aspergillus fumigatus “Supermater” Pair 
mBio  2011;2(6):e00234-11.
Abstract
The mating efficiency of 50 Aspergillus fumigatus isolates from both clinical and environmental sources was analyzed. Forty isolates completed the sexual cycle in 4 weeks with variable levels of fertility designated high, medium, or low. Two opposite-mating-type strains exhibiting the highest fertility, AFB62 (MAT1-1), isolated from a case of invasive aspergillosis, and AFIR928 (MAT1-2), isolated from the environment, were chosen as the supermater pair. Single cleistothecia obtained from a cross of the two strains harbored a minimum of 1 × 104 ascospores. The viability of ascospores increased with the age of the fruiting body, 17% at 4 weeks and reaching 95% at 20 weeks. AFB62 and AFIR928 were equally virulent in two different murine models, despite differences in their sources. High recombination frequencies were observed when the closely linked genes alb1 (AFUA_2G17600) and abr2 (AFUA_2G17530) were used as genetic markers. Comparative genome hybridization analyses revealed that only 86 genes (ca. 0.86% of the genome) are significantly diverged between AFB62 and AFIR928. The high fertility in a relatively short period, combined with a high degree of virulence and a high recombination frequency, demonstrates that the mating pair AFB62 and AFIR928 provides an excellent tool for genetic studies of A. fumigatus.
Importance Aspergillus fumigatus is a heterothallic fungal pathogen that causes life-threatening infections in immunocompromised hosts. Although heterothallism facilitates genetic study via recombinational analysis, previous work showed that a 6-month incubation period is required for the completion of sexual reproduction in this species. Such a long incubation period impedes progress in genetic research. To discover a highly fertile (supermater) pair that can complete the sexual cycle in a considerably shorter period, we screened 50 strains collected from various geographic regions for mating efficiency. We identified a highly virulent pair of supermaters that can be an invaluable tool for genetic study.
Importance
Aspergillus fumigatus is a heterothallic fungal pathogen that causes life-threatening infections in immunocompromised hosts. Although heterothallism facilitates genetic study via recombinational analysis, previous work showed that a 6-month incubation period is required for the completion of sexual reproduction in this species. Such a long incubation period impedes progress in genetic research. To discover a highly fertile (supermater) pair that can complete the sexual cycle in a considerably shorter period, we screened 50 strains collected from various geographic regions for mating efficiency. We identified a highly virulent pair of supermaters that can be an invaluable tool for genetic study.
doi:10.1128/mBio.00234-11
PMCID: PMC3225970  PMID: 22108383
7.  Genomic Analysis of the Necrotrophic Fungal Pathogens Sclerotinia sclerotiorum and Botrytis cinerea 
Amselem, Joelle | Cuomo, Christina A. | van Kan, Jan A. L. | Viaud, Muriel | Benito, Ernesto P. | Couloux, Arnaud | Coutinho, Pedro M. | de Vries, Ronald P. | Dyer, Paul S. | Fillinger, Sabine | Fournier, Elisabeth | Gout, Lilian | Hahn, Matthias | Kohn, Linda | Lapalu, Nicolas | Plummer, Kim M. | Pradier, Jean-Marc | Quévillon, Emmanuel | Sharon, Amir | Simon, Adeline | ten Have, Arjen | Tudzynski, Bettina | Tudzynski, Paul | Wincker, Patrick | Andrew, Marion | Anthouard, Véronique | Beever, Ross E. | Beffa, Rolland | Benoit, Isabelle | Bouzid, Ourdia | Brault, Baptiste | Chen, Zehua | Choquer, Mathias | Collémare, Jérome | Cotton, Pascale | Danchin, Etienne G. | Da Silva, Corinne | Gautier, Angélique | Giraud, Corinne | Giraud, Tatiana | Gonzalez, Celedonio | Grossetete, Sandrine | Güldener, Ulrich | Henrissat, Bernard | Howlett, Barbara J. | Kodira, Chinnappa | Kretschmer, Matthias | Lappartient, Anne | Leroch, Michaela | Levis, Caroline | Mauceli, Evan | Neuvéglise, Cécile | Oeser, Birgitt | Pearson, Matthew | Poulain, Julie | Poussereau, Nathalie | Quesneville, Hadi | Rascle, Christine | Schumacher, Julia | Ségurens, Béatrice | Sexton, Adrienne | Silva, Evelyn | Sirven, Catherine | Soanes, Darren M. | Talbot, Nicholas J. | Templeton, Matt | Yandava, Chandri | Yarden, Oded | Zeng, Qiandong | Rollins, Jeffrey A. | Lebrun, Marc-Henri | Dickman, Marty
PLoS Genetics  2011;7(8):e1002230.
Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38–39 Mb genomes include 11,860–14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea–specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.
Author Summary
Sclerotinia sclerotiorum and Botrytis cinerea are notorious plant pathogenic fungi with very wide host ranges. They cause vast economic damage during crop cultivation as well as in harvested produce. These fungi are typical examples of necrotrophs: they first kill host plant cells and then colonize the dead tissue. The genome sequences of the two fungi were determined in order to examine commonalities in structure and content and in order to find unique features that may distinguish them from other pathogenic fungi and from saprotrophic fungi. The genomes show high sequence identity and a similar arrangement of genes. S. sclerotiorum and B. cinerea differ in their regulation of sexual reproduction, and the genetic basis and its evolution could be explained from the genome sequence. The genome sequence revealed a striking difference in the number and diversity of secondary metabolism gene clusters, which may be involved in the adaptation to different ecological niches. Altogether, there were no unique features in the genomes of S. sclerotiorum and B. cinerea that could be identified as “silver bullets,” which distinguish these aggressive pathogens from other pathogenic and non-pathogenic fungi. These findings reinforce the quantitative, multigenic nature of necrotrophic pathogenesis.
doi:10.1371/journal.pgen.1002230
PMCID: PMC3158057  PMID: 21876677
8.  The Amsterdam Declaration on Fungal Nomenclature 
Hawksworth, David L. | Crous, Pedro W. | Redhead, Scott A. | Reynolds, Don R. | Samson, Robert A. | Seifert, Keith A. | Taylor, John W. | Wingfield, Michael J. | Abaci, Özlem | Aime, Catherine | Asan, Ahmet | Bai, Feng-Yan | de Beer, Z. Wilhelm | Begerow, Dominik | Berikten, Derya | Boekhout, Teun | Buchanan, Peter K. | Burgess, Treena | Buzina, Walter | Cai, Lei | Cannon, Paul F. | Crane, J. Leland | Damm, Ulrike | Daniel, Heide-Marie | van Diepeningen, Anne D. | Druzhinina, Irina | Dyer, Paul S. | Eberhardt, Ursula | Fell, Jack W. | Frisvad, Jens C. | Geiser, David M. | Geml, József | Glienke, Chirlei | Gräfenhan, Tom | Groenewald, Johannes Z. | Groenewald, Marizeth | de Gruyter, Johannes | Guého-Kellermann, Eveline | Guo, Liang-Dong | Hibbett, David S. | Hong, Seung-Beom | de Hoog, G. Sybren | Houbraken, Jos | Huhndorf, Sabine M. | Hyde, Kevin D. | Ismail, Ahmed | Johnston, Peter R. | Kadaifciler, Duygu G. | Kirk, Paul M. | Kõljalg, Urmas | Kurtzman, Cletus P. | Lagneau, Paul-Emile | Lévesque, C. André | Liu, Xingzhong | Lombard, Lorenzo | Meyer, Wieland | Miller, Andrew | Minter, David W. | Najafzadeh, Mohammad Javad | Norvell, Lorelei | Ozerskaya, Svetlana M. | Öziç, Rasime | Pennycook, Shaun R. | Peterson, Stephen W. | Pettersson, Olga V. | Quaedvlieg, William | Robert, Vincent A. | Ruibal, Constantino | Schnürer, Johan | Schroers, Hans-Josef | Shivas, Roger | Slippers, Bernard | Spierenburg, Henk | Takashima, Masako | Taşkın, Evrim | Thines, Marco | Thrane, Ulf | Uztan, Alev Haliki | van Raak, Marcel | Varga, János | Vasco, Aida | Verkley, Gerard | Videira, Sandra I.R. | de Vries, Ronald P. | Weir, Bevan S. | Yilmaz, Neriman | Yurkov, Andrey | Zhang, Ning
The Amsterdam Declaration on Fungal Nomenclature was agreed at an international symposium convened in Amsterdam on 19–20 April 2011 under the auspices of the International Commission on the Taxonomy of Fungi (ICTF). The purpose of the symposium was to address the issue of whether or how the current system of naming pleomorphic fungi should be maintained or changed now that molecular data are routinely available. The issue is urgent as mycologists currently follow different practices, and no consensus was achieved by a Special Committee appointed in 2005 by the International Botanical Congress to advise on the problem. The Declaration recognizes the need for an orderly transitition to a single-name nomenclatural system for all fungi, and to provide mechanisms to protect names that otherwise then become endangered. That is, meaning that priority should be given to the first described name, except where that is a younger name in general use when the first author to select a name of a pleomorphic monophyletic genus is to be followed, and suggests controversial cases are referred to a body, such as the ICTF, which will report to the Committee for Fungi. If appropriate, the ICTF could be mandated to promote the implementation of the Declaration. In addition, but not forming part of the Declaration, are reports of discussions held during the symposium on the governance of the nomenclature of fungi, and the naming of fungi known only from an environmental nucleic acid sequence in particular. Possible amendments to the Draft BioCode (2011) to allow for the needs of mycologists are suggested for further consideration, and a possible example of how a fungus only known from the environment might be described is presented.
doi:10.5598/imafungus.2011.02.01.14
PMCID: PMC3317370  PMID: 22679594
Anamorph; Article 59; BioCode; Candidate species; Environmental sequences; International Code of Botanical Nomenclature; MycoCode; Pleomorphic fungi; Teleomorph
9.  Genomic Islands in the Pathogenic Filamentous Fungus Aspergillus fumigatus 
PLoS Genetics  2008;4(4):e1000046.
We present the genome sequences of a new clinical isolate of the important human pathogen, Aspergillus fumigatus, A1163, and two closely related but rarely pathogenic species, Neosartorya fischeri NRRL181 and Aspergillus clavatus NRRL1. Comparative genomic analysis of A1163 with the recently sequenced A. fumigatus isolate Af293 has identified core, variable and up to 2% unique genes in each genome. While the core genes are 99.8% identical at the nucleotide level, identity for variable genes can be as low 40%. The most divergent loci appear to contain heterokaryon incompatibility (het) genes associated with fungal programmed cell death such as developmental regulator rosA. Cross-species comparison has revealed that 8.5%, 13.5% and 12.6%, respectively, of A. fumigatus, N. fischeri and A. clavatus genes are species-specific. These genes are significantly smaller in size than core genes, contain fewer exons and exhibit a subtelomeric bias. Most of them cluster together in 13 chromosomal islands, which are enriched for pseudogenes, transposons and other repetitive elements. At least 20% of A. fumigatus-specific genes appear to be functional and involved in carbohydrate and chitin catabolism, transport, detoxification, secondary metabolism and other functions that may facilitate the adaptation to heterogeneous environments such as soil or a mammalian host. Contrary to what was suggested previously, their origin cannot be attributed to horizontal gene transfer (HGT), but instead is likely to involve duplication, diversification and differential gene loss (DDL). The role of duplication in the origin of lineage-specific genes is further underlined by the discovery of genomic islands that seem to function as designated “gene dumps” and, perhaps, simultaneously, as “gene factories”.
Author Summary
Aspergillus is an extremely diverse genus of filamentous ascomycetous fungi (molds) found ubiquitously in soil and decomposing vegetation. Being supreme opportunists, aspergilli have adapted to overcome various chemical, physical, and biological stresses found in heterogeneous environments. While most species in the genus are saprophytes, a surprising number are able to infect wounded plants and animals. Remarkably, the allergic human host also responds abnormally to the aspergilli with lung and sinus disease. The advent of immunosuppressive agents and other medical advances have created a large worldwide pool of human hosts susceptible to some Aspergillus species, including the world's most harmful mold and the causative agent of invasive aspergillosis, Aspergillus fumigatus. In this study, we have used the power of comparative genomics to gain insight into genetic mechanisms that may contribute to the metabolic versatility and pathogenicity of this important human pathogen. Comparison of the genomes of two A. fumigatus clinical isolates and two closely related, but rarely pathogenic species showed that their genomes contain several large isolate- and species-specific chromosomal islands. The metabolic capabilities encoded by these highly labile regions are likely to contribute to their rapid adaptation to heterogeneous environments such as soil or a living host.
doi:10.1371/journal.pgen.1000046
PMCID: PMC2289846  PMID: 18404212
11.  Genetic Control of Resistance to the Sterol 14α-Demethylase Inhibitor Fungicide Prochloraz in the Cereal Eyespot Pathogen Tapesia yallundae 
Applied and Environmental Microbiology  2000;66(11):4599-4604.
Sexual crosses were used to determine the genetic basis of resistance to the sterol 14 α-demethylase inhibitor fungicide prochloraz in the cereal eyespot pathogen Tapesia yallundae. Three different crosses between sensitive parental strains (22-432 and 22-433 [the concentration required to inhibit growth by 50% {IG50} for each was ≤0.03 mg/liter]) and field isolates from France and New Zealand with differing levels of resistance (PR11 [IG50 = 0.5 mg/liter], PR1 [IG50 = 1.0 mg/liter], and 11-3-18 [IG50 = 2.4 mg/liter]) yielded progeny showing a bimodal distribution, with an even number of sensitive and resistant progeny. This indicated the segregation of a single major gene for resistance in each cross, which was confirmed by the use of backcrosses, crosses between F1 progeny, and control crosses between sensitive parents. However, there was also evidence of additional quantitative genetic components responsible for the increased IG50s of the more resistant isolates. A further cross was made between isolate PR11 and an F1 progeny arising from isolate 11-3-18, and this also yielded progeny which were entirely prochloraz resistant. This suggested that resistance genes were allelic in these two isolates, with resistance conferred by a gene at the same locus (or closely linked loci), despite the fact that the isolates (PR11 and 11-3-18) originated from different continents.
PMCID: PMC92355  PMID: 11055899

Results 1-11 (11)