Mating strategy and mating type distribution in six global populations of the Eucalyptus foliar pathogen Teratosphaeria destructans.

Teratosphaeria destructans is an aggressive fungal pathogen causing leaf and shoot blight on young Eucalyptus trees in plantations. The disease occurs across tropical and subtropical regions of South East Asia and has recently been found in South Africa. Asexual structures of the pathogen are produced on infected tissues, but sexual structures have never been observed. The aim of this study was to investigate the reproductive biology of T. destructans by characterising its mating type (MAT1) locus and investigating its potential for sexual recombination. We found that T. destructans has a heterothallic mating system, with either the MAT1-1-1 and MAT1-1-10 genes (MAT1-1 idiomorph) or the MAT1-2-1 and MAT1-2-12 genes (MAT1-2 idiomorph) present in a single individual. With a multiplex PCR assay, it was possible to distinguish the two MAT idiomorphs in several Teratosphaeria species and this approach was applied to six global populations of T. destructans. Although both mating types occurred in the South East Asian populations, a single mating type dominated each population. Isolates from the recent disease outbreak in South Africa comprised only a single mating type. Attempts to induce a sexual cycle in vitro using strains of opposite mating type were not successful. The uneven distribution of mating types in populations of T. destructans and the presence of only an asexual state on infected tissues suggests the absence of or at least a minor role for sexual reproduction where the pathogen occurs on non-native Eucalyptus in plantations.

Frequency of the Mating-Type (MAT1) in Histoplasma capsulatum Isolates from Buenos Aires, Argentina.

Sex is genetically determined in Histoplasma capsulatum, governed by a sex-specific region in the genome called the mating-type locus (MAT1). We investigate the distribution of isolates of two H. capsulatum mating types in the clades circulating in Buenos Aires, Argentina. Forty-nine H. capsulatum isolates were obtained from the culture collection of the Mycology Center. The MAT1 locus was identified by PCR from the yeast suspension. The analysis of forty-eight isolates from clinical samples exhibited a ratio of 1.7 (MAT1-1:MAT1-2) and the only isolate from soil was MAT1-1. Forty-five H. capsulatum isolates belonged to the LAm B clade (H. capsulatum from Latin American group B clade) and showed a ratio of 1.8 (MAT1-1:MAT1-2). These results suggest an association between the mating types in isolates belonging to the LAm B clade. It remains to be defined whether a greater virulence should be attributed to the differences between the strains of the opposite mating type of the LAm B clade.

Evolution of the mating types and mating strategies in prominent genera in the Botryosphaeriaceae.

Little is known regarding mating strategies in the Botryosphaeriaceae. To understand sexual reproduction in this fungal family, the mating type genes of Botryosphaeria dothidea and Macrophomina phaseolina, as well as several species of Diplodia, Lasiodiplodia and Neofusicoccum were characterized from whole genome assemblies. Comparisons between the mating type loci of these fungi showed that the mating type genes are highly variable, but in most cases the organization of these genes is conserved. Of the species considered, nine were homothallic and seven were heterothallic. Mating type gene fragments were discovered flanking the mating type regions, which indicates both ongoing and ancestral recombination occurring within the mating type region. Ancestral reconstruction analysis further indicated that heterothallism is the ancestral state in the Botryosphaeriaceae and this is supported by the presence of mating type gene fragments in homothallic species. The results also show that at least five transitions from heterothallism to homothallism have taken place in the Botryosphaeriaceae. The study provides a foundation for comparison of mating type evolution between Botryosphaeriaceae and other fungi and also provides valuable markers for population biology studies in this family.

The novel Aspergillus fumigatus MAT1-2-4 mating-type gene is required for mating and cleistothecia formation.

Sexual propagation accompanied by recombination and the formation of spore-containing fruiting bodies is a cornerstone of fungal genetics and biology. In the human pathogen Aspergillus fumigatus sexual identity has previously been shown to be determined by MAT1-1-1 or MAT1-2-1 genes which act as transcriptional regulators and are present within idiomorphs found at the MAT locus. We here report the identification and first characterization of a further novel gene, termed MAT1-2-4, that is present in the MAT1-2 idiomorph of A. fumigatus. A mating-type swapping strategy was used to achieve an unbiased deletion of the MAT1-2-4 gene with no impact on MAT1-2-1 gene expression. Phenotypical characterization of the resulting strain revealed an inability to mate with the compatible MAT1-1 progenitor, demonstrating that the MAT1-2-4 gene product is a genuine mating-type factor required for correct sexual development. A GPI-anchored protein of unknown function was identified as interaction partner. However, no functional role in the mating process or ascosporogenesis could be demonstrated by deletion analysis for this latter protein, although a role in heterokaryon formation is suggested. Bioinformatic analysis also demonstrated the presence of MAT1-2-4 homologues in some, but not all, other Aspergillus species and the evolutionary origins and implications of the MAT1-2-4 gene are discussed.

Functional convergence and divergence of mating-type genes fulfilling in Cordyceps militaris.

Fungal sexual lives are considerably diversified in terms of the types of mating systems and mating-control gene functions. Sexual fruiting bodies of the ascomycete fungus Cordyceps militaris have been widely consumed as edible and medicinal mushrooms, whereas the regulation of fruiting-body development and sex in this fungus remain elusive. Herein, we performed the comprehensive functional analyses of mating-type (MAT) genes in C. militaris. Interspecies functional convergence was evident that MAT1-1 and MAT1-2-1 null mutants were sterile and lost the ability to produce stromata in outcrosses with the opposite mating-type partner. In contrast to other fungal species, functional divergence of MAT1-1-1 and MAT1-1-2 was also observed that ΔMAT1-1-1 produced barren stromata in outcrosses, whereas ΔMAT1-1-2 generated fruiting bodies morphologically similar to that of the parental strain but with sterile perithecia. The homothallic-like transformants MAT1-2::MAT1-1-1 (haploidic MAT1-2 isolate transformed with the MAT1-1-1 gene) produced sterile stromata, whereas the MAT1-1::MAT1-2-1 (haploidic MAT1-1 isolate transformed with the MAT1-2-1 gene) mutant was determined to be completely fruitless. The findings relating to the fully fertile gene-complementation mutants suggest that the genomic location is not essential for the MAT genes to fulfill their functions in C. militaris. Comparison of the production of bioactive constituents cordycepin and adenosine provides experimental support that the fungal sexual cycle is an energy consuming process. The results of the present study enrich our knowledge of both convergent and divergent controls of fungal sex.

MAT gene idiomorphs suggest a heterothallic sexual cycle in a predominantly asexual and important pine pathogen.

Diplodia pinea (=Sphaeropsis sapinea) is a well-known and economically important latent pathogen of Pinus spp. in many parts of the world. Despite intensive scrutiny, its sexual state has never been observed and the fungus has thus been considered exclusively asexual. It was, therefore, surprising that a recent population genetics study showed high genotypic diversity and random association of alleles in a number of populations, suggesting that the pathogen has a cryptic sexual stage. Using the genome sequence of two individual D. pinea isolates, we interrogated the structure of the MAT locus in this fungus. The results suggested that D. pinea is heterothallic (self-sterile) with complete and apparently functional copies of the MAT genes containing the α-1 and HMG domains present in different isolates. In addition to the MAT1-2-1 and MAT1-1-1 genes, we found a MAT1-1-4 gene in the MAT1-1 idiomorph and a novel MAT1-2-5 gene in the MAT1-2 idiomorph. Importantly, the frequencies of occurrence of both idiomorphs in populations examined were not significantly different from a 1:1 ratio, which would be expected in sexually reproducing populations. Although the sexual state has never been observed, the results strongly suggest that D. pinea has a cryptic, heterothallic sexual cycle.

Genetic architecture and evolution of the mating type locus in fusaria that cause soybean sudden death syndrome and bean root rot.

Fusarium tucumaniae is the only known sexually reproducing species among the seven closely related fusaria that cause soybean sudden death syndrome (SDS) or bean root rot (BRR). In a previous study, laboratory mating of F. tucumaniae yielded recombinant ascospore progeny but required two mating-compatible strains, indicating that it is heterothallic. To assess the reproductive mode of the other SDS and BRR fusaria, and their potential for mating, whole-genome sequences of two SDS and one BRR pathogen were analyzed to characterize their mating type (MAT) loci. This bioinformatic approach identified a MAT1-1 idiomorph in F. virguliforme NRRL 22292 and MAT1-2 idiomorphs in F. tucumaniae NRRL 34546 and F. azukicola NRRL 54364. Alignments of the MAT loci were used to design PCR primers within the conserved regions of the flanking genes APN1 and SLA2, which enabled primer walking to obtain nearly complete sequences of the MAT region for six MAT1-1 and five MAT1-2 SDS/BRR fusaria. As expected, sequences of the highly divergent 4.7 kb MAT1-1 and 3.7 kb MAT1-2 idiomorphs were unalignable. However, sequences of the respective idiomorphs and those that flank MAT1-1 and MAT1-2 were highly conserved. In addition to three genes at MAT1-1 (MAT1-1-1, MAT1-1-2, MAT1-1-3) and two at MAT1-2 (MAT1-2-1, MAT1-2-3), the MAT loci of the SDS/BRR fusaria also include a putative gene predicted to encode for a 252 amino acid protein of unknown function. Alignments of the MAT1-1-3 and MAT1-2-1 sequences were used to design a multiplex PCR assay for the MAT loci. This assay was used to screen DNA from 439 SDS/BRR isolates, which revealed that each isolate possessed MAT1-1 or MAT1-2, consistent with heterothallism. Both idiomorphs were represented among isolates of F. azukicola, F. brasiliense, F. phaseoli and F. tucumaniae, whereas isolates of F. virguliforme and F. cuneirostrum were only MAT1-1 and F. crassistipitatum were only MAT1-2. Finally, nucleotide sequence data from the RPB1 and RPB2 genes were used to date the origin of the SDS/BRR group, which was estimated to have occurred about 0.75 Mya (95% HPD interval: 0.27, 1.68) in the mid-Pleistocene, long before the domestication of the common bean or soybean.

Human adaptation and diversification in the Microsporum canis complex.

The Microsporum canis complex consists of one zoophilic species, M. canis, and two anthropophilic species, M. audouinii and M. ferrugineum. These species are the most widespread zoonotic pathogens causing dermatophytosis in cats and humans worldwide. To clarify the evolutionary relationship between the three species and explore the potential host shift process, this study used phylogenetic analysis, population structure analysis, multispecies coalescent analyses, determination of MAT idiomorph distribution, sexual crosses, and macromorphology and physicochemical features to address the above questions. The complex of Microsporum canis, M. audouinii and M. ferrugineum comprises 12 genotypes. MAT1-1 was present only in M. canis, while the anthropophilic entities contained MAT1-2. The pseudocleistothecia were yielded by the mating behaviour of M. canis and M. audouinii. Growth rates and lipase, keratinolysis and urea hydrolytic capacities of zoophilic M. canis isolates were all higher than those of anthropophilic strains; DNase activity of M. ferrugineum exceeded that of M. canis. The optimum growth temperature was 28 °C, but 22 °C favoured the development of macroconidia. Molecular data, physicochemical properties and phenotypes suggest the adaptation of zoophilic M. canis to anthropophilic M. ferrugineum, with M. audouinii in an intermediate position.

Organization and Unconventional Integration of the Mating-Type Loci in Morchella Species.

True morels (Morchella spp.) are a group of delicious fungi in high demand worldwide, and some species of morels have been successfully cultivated in recent years. To better understand the sexual reproductive mechanisms of these fungi, we characterized the structure of the mating-type loci from ten morel species, and seven of them were obtained using long-range PCR amplification. Among the studied species, eight were heterothallic, two were homothallic, and four types of composition were observed in the MAT loci. In three of the five black morel species, the MAT1-1-1, MAT1-1-10, and MAT1-1-11 genes were in the MAT1-1 idiomorph, and only the MAT1-2-1 gene was in the MAT1-2 idiomorph, while an integration event occurred in the other two species and resulted in the importation of the MAT1-1-11 gene into the MAT1-2 idiomorph and survival as a truncated fragment in the MAT1-1 idiomorph. However, the MAT1-1-11 gene was not available in the four yellow morels and one blushing morel species. M. rufobrunnea, a representative species of the earliest diverging branch of true morels, along with another yellow morel Mes-15, were confirmed to be homothallic, and the MAT1-1-1, MAT1-1-10, and MAT1-2-1 genes were arranged in a tandem array. Therefore, we hypothesized that homothallism should be the ancestral reproductive state in Morchella. RT-PCR analyses revealed that four mating genes could be constitutively expressed, while the MAT1-1-10 gene underwent alternative splicing to produce different splice variants.

Structural variation and phylogenetic analysis of the mating-type locus in the genus Morchella.

True morels (Morchella spp.) are a group of edible fungi that are in high demand worldwide. However, this group of fungi remains poorly understood in terms of its genetic structure, life cycle, and mating system. In the present study, we cloned the MAT locus in Morchella sp. Mes-20 using long-range polymerase chain reaction (PCR) amplification. Our results showed that the MAT1-2 idiomorph in the single-ascospore isolate YAASMCB-3 was 7.5 kb in length, harboring a single MAT1-2-1 gene, whereas the MAT1-1 idiomorph in the isolate YAASMCB-15 was 7.8 kb, carrying both MAT1-1-1 and MAT1-1-10 genes. Mating-type diagnostic assays of single-ascospore populations confirmed that Mes-20 is heterothallic. In addition, 42 collections belonging to 17 morel species (7 black morel species [Elata clade], 9 yellow morel species [Esculenta clade], and 1 species from the Rufobrunnea clade) were used to clone and characterize the MAT1-1-1 and MAT1-2-1 genes for development of the mating gene genealogies. In concordance with the multilocus phylogenetic trees, collections for the respective species were readily distinguished by well-supported lineages in mating gene genealogies. The topologies were consistent within the Elata clade, whereas the branching order and sister-group relationships slightly varied within the Esculenta clade. Our results show that species in the genus Morchella could be delimited by mating gene genealogies.

Cloning and analysis of Ophiocordyceps xuefengensis mating type (MAT) loci.

The entomopathogenic fungus Ophiocordyceps xuefengensis, a recently described species and identified as the sister taxon of Ophiocordyceps sinensis, is a desirable alternative to O. sinensis. The mating systems of fungi play a vitally important role in the regulation of sexual reproduction and evolution, but the mating type loci of O. xuefengensis were completely unknown. In this study, the mating systems of O. xuefengensis were analyzed. The conserved α-box region of the MAT1-1-1 and HMG-box of MAT1-2-1 were successfully obtained by PCR amplification. The distribution of both mating types in different tissues of wild and cultivated O. xuefengensis growth was detected and analyzed. The results showed that the asci always harbored both mating types, whereas the sclerotium, the stipe and each isolated strain of wild O. xuefengensis always had only one idiomorph, either MAT1-1 or MAT1-2, which confirmed that O. xuefengensis is heterothallic. The MAT1-1 locus of O. xuefengensis harbors MAT1-1-1, MAT1-1-2 and MAT1-1-3, and MAT1-2 contains the MAT1-2-1 gene. Southern blot analysis showed the MAT-1-1-1 and MAT-1-2-1 genes were single-copy in O. xuefengensis. These results will help to understand its life cycle and support artificial cultivation of O. xuefengensis.

Heterothallic sexual reproduction in three canker-inducing tree pathogens within the Fusarium torreyae species complex.

Fusarium zanthoxyli and F. continuum are sister taxa that are the etiological agents of canker disease of prickly ash (Zanthoxylum bungeanum) in northern China. These two pathogens, together with F. torreyae, the causal agent of canker disease of the critically endangered conifer Florida torreya (Torreya taxifolia) from northern Florida and southwestern Georgia, constitute a novel clade, the F. torreyae species complex. To assess their reproductive mode, a polymerase chain reaction (PCR) assay targeting the MAT1-1 and MAT1-2 idiomorphs was designed and validated, using MAT sequences mined from the whole-genome sequence of the three F. torreyae clade pathogens and several closely related fusaria. Results of the MAT idiomorph PCR assay indicated that isolates of the three pathogens were MAT1-1 or MAT1-2. When MAT1-1 and MAT1-2 isolates of each species were crossed on carrot agar, all of the F. zanthoxyli (N = 30) and F. continuum (N = 3) isolates tested were female fertile, yielding mature perithecia with viable ascospores. By comparison, only one pairing of the five different isolates of F. torreyae produced perithecia; however, the majority of the asci in this cross aborted or produced fewer than eight ascospores. Of the three temperatures tested (i.e., 22, 25, and 27 C), the optimal temperature for perithecium production was 22-25 C in F. zanthoxyli and 25 C in F. continuum and F. torreyae. Ascospore progeny from three separate crosses of F. zanthoxyli and F. continuum and one cross of F. torreyae were genotyped to assess whether they were the products of genetic recombination and sexual reproduction. Genotyping of 34-40 progeny from the F. zanthoxyli and F. continuum crosses confirmed that they were the products of sexual reproduction. However, only 36% of the progeny in the F. torreyae cross were recombinant, which was roughly half of the nonparental progeny expected with three markers segregating.

The homothallic mating-type locus of the conifer needle endophyte Phialocephala scopiformis DAOMC 229536 (order Helotiales).

We describe the complete mating-type (MAT) locus for Phialocephala scopiformis Canadian Collection of Fungal Cultures (DAOMC) 229536 - a basal lineage within Vibrisseaceae. This strain is of interest due to its ability to produce the important antiinsectan rugulosin. We also provide some of the first insights into the genome structure and gene inventory of nonclavicipitalean endophytes. Sequence was obtained through shotgun sequencing of the entire P. scopiformis genome, and the MAT locus was then determined by comparing this genomic sequence to known MAT loci within the Phialocephala fortinii s.l.-Acephala applanata species complex. We also tested the relative levels of sequence conservation for MAT genes within Vibrisseaceae (n = 10), as well as within the Helotiales (n = 27). Our results: (1) show a homothallic gene arrangement for P. scopiformis [MAT1-1-1, MAT1-2-1, and MAT1-1-3 genes are present], (2) increase the genomic survey of homothallism within Vibrisseaceae, (3) confirm that P. scopiformis contains a unique S-adenosyl-l-methionine-dependent methyltransferase (SAM-Mtase) gene proximal to its MAT locus, while also lacking a cytoskeleton assembly control protein (sla2) gene, and (4) indicate that MAT1-1-1 is the more useful molecular marker amongst the MAT genes for phylogenetic reconstructions aimed at tracking evolutionary shifts in reproductive strategy and/or MAT loci gene composition within the Helotiales.

Isolation of the MAT1-1 mating type idiomorph and evidence for selfing in the Chinese medicinal fungus Ophiocordyceps sinensis.

Ophiocordyceps sinensis is one of the most valued medicinal fungi in China. Research on the mating system and sexual development is vitally important to this endangered species. Previous efforts devoted to investigate the mating type (MAT) locus of O. sinensis, however, resulted in an incomplete understanding. In this study, the MAT1-1 locus of O. sinensis was investigated. The conserved α-box and HMG-box regions of the MAT1-1-1 and MAT1-1-3 genes, respectively, and a conserved region of the DNA lyase gene were successfully amplified using degenerate PCR. A combination of TAIL-PCR and long-range PCR were used to connect these genes and obtain the sequence of the MAT1-1 locus. Screening of 22 single spore isolates by PCR demonstrated that both the MAT1-1-1 and MAT1-2-1 genes cooccurred within the same isolate. Additionally, both MAT1-1-1 and MAT1-2-1 are expressed in vegetative mycelia, providing evidence that O. sinensis is likely capable of selfing. DAPI (4,6-diamidino-2-phenylindole) staining of ascospores and hyphae showed that a majority of hyphal compartments are binucleate, suggesting that O. sinensis may be pseudohomothallic. Analyses of sequence diversity showed lower levels of genetic diversity in MAT1-1-1 compared to MAT1-2-1, indicating the possibility that different selective pressures act on the two MAT idiomorphs. The MAT1-1-1 sequences of O. sinensis and Tolypocladium inflatum cluster as a monophyletic group consistent with phylogenetic classification of Ophiocordycipitaceae. Comparison of the structure of the MAT1-1 locus across hypocrealean taxa showed that O. sinensis contains all three mating type genes (MAT1-1-1, MAT1-1-2, and MAT1-1-3) and supported previous observations that of the four families in Hypocreales, MAT1-1-3 has undergone a lineage specific loss only in some members of the Cordycipitaceae.