Aspergillus fumigatus is a saprotrophic fungus whose spores are ubiquitous in the atmosphere. It is also an opportunistic human pathogen in immunocompromised individuals, causing potentially lethal invasive infections, and is associated with severe asthma and sinusitis. The species is only known to reproduce by asexual means, but there has been accumulating evidence for recombination and gene flow from population genetic studies, genome analysis, the presence of mating-type genes and expression of sex-related genes in the fungus. Here we show that A. fumigatus possesses a fully functional sexual reproductive cycle that leads to the production of cleistothecia and ascospores, and the teleomorph Neosartorya fumigata is described. The species has a heterothallic breeding system; isolates of complementary mating types are required for sex to occur. We demonstrate increased genotypic variation resulting from recombination between mating type and DNA fingerprint markers in ascospore progeny from an Irish environmental subpopulation. The ability of A. fumigatus to engage in sexual reproduction is highly significant in understanding the biology and evolution of the species. The presence of a sexual cycle provides an invaluable tool for classical genetic analyses and will facilitate research into the genetic basis of pathogenicity and fungicide resistance in A. fumigatus, with the aim of improving methods for the control of aspergillosis. These results also yield insights into the potential for sexual reproduction in other supposedly 'asexual' fungi.

Major insights into sexual development and cryptic sexuality within filamentous fungi have been gained from investigations using Aspergillus species. Here, an overview is first given into sexual morphogenesis in the aspergilli, describing the different types of sexual structures formed and how their production is influenced by a variety of environmental and nutritional factors. It is argued that the formation of cleistothecia and accessory tissues, such as Hülle cells and sclerotia, should be viewed as two independent but co-ordinated developmental pathways. Next, a comprehensive survey of over 75 genes associated with sexual reproduction in the aspergilli is presented, including genes relating to mating and the development of cleistothecia, sclerotia and ascospores. Most of these genes have been identified from studies involving the homothallic Aspergillus nidulans, but an increasing number of studies have now in addition characterized 'sex-related' genes from the heterothallic species Aspergillus fumigatus and Aspergillus flavus. A schematic developmental genetic network is proposed showing the inter-relatedness between these genes. Finally, the discovery of sexual reproduction in certain Aspergillus species that were formerly considered to be strictly asexual is reviewed, and the importance of these findings for cryptic sexuality in the aspergilli as a whole is discussed.

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 TR 34 /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 TR 34 / 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 TR 34 /L98H isolates could outcross with azole-susceptible isolates of different genetic backgrounds, suggesting that TR 34 / L98H isolates can undergo the sexual cycle in nature. Overall, our findings suggest a common ancestor of the TR 34 /L98H mechanism and subsequent migration of isolates harboring TR 34 /L98H across Europe.A spergillus fumigatus is a saprophytic fungus that is capable of causing a wide range of diseases in various hosts. Invasive aspergillosis is the most severe manifestation of Aspergillus infection in humans, and this disease is associated with substantial mortality and morbidity. Medical triazoles, such as itraconazole, voriconazole, and posaconazole, play an important role in the management of Aspergillus diseases. However, azole resistance is an emerging problem in A. fumigatus and has been shown to be associated with increased probability of treatment failure (10,11,19,20,30,35,37,39,41).Azole resistance is commonly due to mutations in the cyp51A gene, which encodes 14-␣-demethylase in the ergosterol biosynthesis pathway. In azole-resistant clinical A. fumigatus isolates, a wide variety of cyp51A mutations, such as substitutions at codons G54, G138, P216, F219, M220, and G448, have been found (5,11,29). This is in contrast with a different pattern of resistance observed in isolates from The Netherlands. Here, a resistance mechanism consisting of the L98H substitution together with a 34-bp tandem repeat (TR 34 ) in the promoter region of this gene (TR 34 / L98H) was found to be present in over 90% of itraconazole-resistant isolates, which also showed reduced susceptibility to voriconazole and posaconazole (30,36). TR 34 /L98H isolates were recovered primarily from azole-naïve patients and were also recovered from the environment (28, 36). These observations suggest that azole-resistant Aspergillus is acquired by patients from an environmental source rather than arising through azole therapy. Recently, we provided evidence that exposure of A. fumigatus to 14-␣-demethylase inhibitor (DMI) fungicides might provide a selective pressure leading to the emergence of TR 34 /L98H resistant isolates in the environment (27). On the basis of in vitro crossresistance, molecule alignment studies, and docking simulations, five triazole fungicides that ...

Penicillium chrysogenum is a filamentous fungus of major medical and historical importance, being the original and present-day industrial source of the antibiotic penicillin. The species has been considered asexual for more than 100 y, and despite concerted efforts, it has not been possible to induce sexual reproduction, which has prevented sexual crosses being used for strain improvement. However, using knowledge of mating-type (MAT) gene organization, we now describe conditions under which a sexual cycle can be induced leading to production of meiotic ascospores. Evidence of recombination was obtained using both molecular and phenotypic markers. The identified heterothallic sexual cycle was used for strain development purposes, generating offspring with novel combinations of traits relevant to penicillin production. Furthermore, the MAT1-1-1 mating-type gene, known primarily for a role in governing sexual identity, was also found to control transcription of a wide range of genes with biotechnological relevance including those regulating penicillin production, hyphal morphology, and conidial formation. These discoveries of a sexual cycle and MAT gene function are likely to be of broad relevance for manipulation of other asexual fungi of economic importance.sexual recombination | secondary metabolism | ascomycete

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.

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