Structure and function of the mating-type locus in the homothallic ascomycete, Didymella zeae-maydis

Homothallic Didymella zeae-maydis undergoes sexual reproduction by selfing. Sequence analysis of the mating type (MAT) locus from this fungus revealed that MAT carries both MAT1-1-1 and MAT1-2-1 genes found in heterothallic Dothideomycetes, separated by ～1.0 kb of noncoding DNA. To understand the mechanistic basis of homothallism in D. zeae-maydis, each of the MAT genes was deleted and the effects on selfing and on ability to cross in a heterothallic manner were determined. The strain carrying an intact MAT1-1-1 but defective MAT1-2-1 gene (MAT1-1-1;ΔMAT1-2-1) was self-sterile, however strains carrying an intact MAT1-2-1 but defective MAT1-1-1 gene (ΔMAT1-1-1;MAT1-2-1), when selfed, showed delayed production of a few ascospores. Attempts to cross the two MAT deletion strains yielded fewer ΔMAT1-1-1;MAT1-2-1 than MAT1-1-1;ΔMAT1-2-1 progeny and very few ascospores overall compared to WT selfs. This study demonstrates that, as in the other homothallic Dothideomycetes, both MAT genes are required for full fertility, but that, in contrast to other cases, the presence of a single MAT1-2-1 gene can induce homothallism, albeit inefficiently, in D. zeae-maydis.     

Mating type gene analyses in the genus Diplodia: From cryptic sex to cryptic species

Cryptic species are common in Diplodia, a genus that includes some well-known and economically important plant pathogens. Thus, species delimitation has been based on the phylogenetic species recognition approach using multigene genealogies. We assessed the potential of mating type (MAT) genes sequences as phylogenetic markers for species delimitation in the genus Diplodia. A PCR-based mating type diagnostic assay was developed that allowed amplification and sequencing of the MAT1-1-1 and MAT1-2-1 genes, and determination of the mating strategies used by different species. All species tested were shown to be heterothallic. Phylogenetic analyses were performed on both MAT genes and also, for comparative purposes, on concatenated sequences of the ribosomal internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1-α) and beta-tubulin (tub2). Individual phylogenies based on MAT genes clearly differentiated all species analysed and agree with the results obtained with the commonly used multilocus phylogenetic analysis approach. However, MAT genes genealogies were superior to multigene genealogies in resolving closely related cryptic species. The phylogenetic informativeness of each locus was evaluated revealing that MAT genes were the most informative loci followed by tef1-α. Hence, MAT genes can be successfully used to establish species boundaries in the genus Diplodia.     

Presence and expression of the mating type locus in Paracoccidioides brasiliensis isolates

Paracoccidioides brasiliensis has been classified in the phylum Ascomycota, order Onygenales, family Ajellomycetaceae, even in the absence of a known sexual cycle or mating system. The objective of this work was to determine the presence of the mating type locus in 71 P. brasiliensis isolates from various sources. A PCR assay using specific primers for the MAT 1 gene was developed and applied for the detection of such genes. Two heterothallic groups (MAT1-1 or MAT1-2) were recognized and, in some isolates, gene expression was confirmed indicating the existence of a basal gene expression. The distribution of two mating type loci in the studied population suggested that sexual reproduction might occur in P. brasiliensis. This finding points towards the possibility of applying a more precise definition of the concept of biological species to P. brasiliensis. Further studies should be conducted to confirm the sexual capacity of this fungus and its implications among phylogenetic species and geographical distribution.     

Sexual reproduction in aflatoxin-producing Aspergillus nomius

Sexual reproduction was examined in the aflatoxin-producing fungus Aspergillus nomius. Crosses between sexually compatible strains resulted in the formation of multiple nonostiolate ascocarps within stromata, which places the teleomorph in genus Petromyces. Ascocarp and ascospore morphology in Petromyces nomius were similar to that in P. flavus and P. parasiticus, and differences between teleomorphs were insufficient for species separation. Formation of mature ascocarps was infrequent, with only 24% of the 83 crosses producing viable ascospores. The majority of P. nomius strains contained a single mating-type gene (MAT1-1 or MAT1-2), but several strains contained both genes. MAT1-1/MAT1-2 strains were self-sterile and capable of mating with both MAT1-1 and MAT1-2 strains; hence P. nomius appears to be functionally heterothallic.     

Heterothallism revealed in the root rot fungi Berkeleyomyces basicola and B. rouxiae

Berkeleyomyces basicola and Berkeleyomyces rouxiae, two sister species previously treated collectively as Thielaviopsis basicola, reside in the Ceratocystidaceae (Microascales, Ascomycota). Both species are important root pathogens of many important agricultural crops and ornamental plants. Although T. basicola has been known for more than 150y, a sexual state has never been found and it has been assumed to be an asexual pathogen. The aim of this study was to determine the mating strategy of the two Berkeleyomyces species. Investigation of the genome sequences of two B. basicola isolates allowed for the complete characterization of the MATlocus, revealing that it has a typical heterothallic mating system with the MAT1-1andMAT1-2 idiomorphs occurring in different isolates. PCR amplification using mating type primers developed in this study, showed that the MAT1-1-1andMAT1-2-1 genes were also present in different isolates of B. rouxiae. Pairing of isolates representing the two mating types of both species,using a variety of techniques failed to produce sexual structures. Although we have found no direct evidence that they reproduce sexually, these fungi are clearly heterothallic with both mating types occurring in some countries suggesting that a cryptic sexual cycle could exist for them.     

Shifting fungal reproductive mode by manipulation of mating type genes: obligatory heterothallism of Gibberella zeae

Fungi capable of sexual reproduction use heterothallic (self-sterile) or homothallic (self-fertile) mating strategies. In most ascomycetes, a single mating type locus, MAT, with two alternative forms (MAT1-1 and MAT1-2) called idiomorphs, controls mating ability. In heterothallic ascomycetes, these alternative idiomorphs reside in different nuclei. In contrast, most homothallic ascomycetes carry both MAT1-1 and MAT1-2 in a single nucleus, usually closely linked. An example of the latter is Gibberella zeae, a species that is capable of both selfing and outcrossing. G. zeae is a devastating cereal pathogen of ubiquitous geographical distribution, and also a producer of mycotoxins that threaten human and animal health. We asked whether G. zeae could be made strictly heterothallic by manipulation of MAT. Targeted gene replacement was used to differentially delete MAT1-1 or MAT1-2 from a wild-type haploid MAT1-1; MAT1-2 strain, resulting in MAT1-1; mat1-2, mat1-1; MAT1-2 strains that were self-sterile, yet able to cross to wild-type testers and, more importantly, to each other. These results indicated that differential deletion of MAT idiomorphs eliminates selfing ability of G. zeae, but the ability to outcross is retained. They also indicated that both MAT idiomorphs are required for self-fertility. To our knowledge, this is the first report of complete conversion of fungal reproductive strategy from homothallic to heterothallic by targeted manipulation of MAT. Practically, this approach opens the door to simple and efficient procedures for obtaining sexual recombinants of G. zeae that will be useful for genetic analyses of pathogenicity and other traits, such as the ability to produce mycotoxins.     

Sexuality and Genetic Identity in the Agaricus Section Arvenses

Twelve wild collections and one commercial strain were used to characterize breeding systems and to develop molecular identities in the Arvenses section of the genus Agaricus, which includes the “horse mushroom” A. arvensis. Two morphotypes were identified based on macro- and micromorphological features. However, not all collections could be delimited by conventional taxonomic characters. Sequencing of the small subunit intergenic spacer (ITS) region (368 to 370 bp) of the rRNA genes clearly resolved the 13 collections into two clusters consistent with the identified morphotypes. Single-spore progenies and mating type testers were established and used to test intra- and interstock compatibility. The two compatibility groups identified were consistent with ITS clusters. Compatibility group I stocks readily interbred within the constraints of a unifactorial heterothallic system with a multiallelic mating type factor. Compatibility group II had a more restricted breeding pattern, and interactions were difficult to predict on the basis of mating type. Morphological data, ITS sequences, and the ability to interbreed suggest that these collections are part of a complex of interrelated species. Single-spore, homokaryotic isolates from both compatibility groups were able to fruit in compost culture, and two of the collections may represent natural homokaryotic fruiting. We conclude that species from the section Arvenses have versatile unifactorial heterothallic life cycles that permit both interbreeding and homokaryotic fruiting.     

Both mating types in the heterothallic fungus Ophiostoma quercus contain MAT1-1 and MAT1-2 genes

In heterothallic Ascomycota, two opposite but distinct mating types control all sexual processes. Using mating crosses, mating types were assigned to ten isolates of the heterothallic fungal species Ophiostoma quercus. Primers were subsequently designed to target the MAT1-1-1, MAT1-1-3 (of the mating type 1 idiomorph), and MAT1-2-1 (of the mating type 2 idiomorph) genes in these isolates. Results showed that all isolates contained the full gene sequence for the MAT1-2-1 gene. In addition, fragments of the MAT1-1-1 and MAT1-1-3 genes were sequenced from all isolates. These results were unexpected, as each isolate from a heterothallic species would typically contain only one of the two possible MAT idiomorphs.     

Characterization of the mating-type genes in Leptographium procerum and Leptographium profanum

Leptographium procerum and the closely related species Leptographium profanum, are ascomycetes associated with root-infesting beetles on pines and hardwood trees, respectively. Both species occur in North America where they are apparently native. L. procerum has also been found in Europe, China New Zealand, and South Africa where it has most probably been introduced. As is true for many other Leptographium species, sexual states have never been observed in L. procerum or L. profanum. The objectives of this study were to clone and characterize the mating type loci of these fungi, and to develop markers to determine the mating types of individual isolates. To achieve this, a partial sequence of MAT1-2-1 was amplified using degenerate primers targeting the high mobility group (HMG) sequence. A complete MAT1-2 idiomorph of L. profanum was subsequently obtained by screening a genomic library using the HMG sequence as a probe. Long range PCR was used to amplify the complete MAT1-1 idiomorph of L. profanum and both the MAT1-1 and MAT1-2 idiomorphs of L. procerum. Characterization of the MAT idiomorphs suggests that the MAT genes are fully functional and that individuals of both these species are self-sterile in nature with a heterothallic mating system. Mating type markers were developed and tested on a population of L. procerum isolates from the USA, the assumed center of origin for this species. The results suggest that cryptic sexual reproduction is occurring or has recently taken place within this population.     

Discovery of a Sexual Cycle in Aspergillus lentulus,a Close Relative of A. fumigatus

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.     

Frequency and Genetic Diversity of the MAT1 Locus of Histoplasma capsulatum Isolates in Mexico and Brazil

The MAT1-1 and MAT1-2 idiomorphs associated with the MAT1 locus of Histoplasma capsulatum were identified by PCR. A total of 28 fungal isolates, 6 isolates from human clinical samples and 22 isolates from environmental (infected bat and contaminated soil) samples, were studied. Among the 14 isolates from Mexico, 71.4% (95% confidence interval [95% CI], 48.3% to 94.5%) were of the MAT1-2 genotype, whereas 100% of the isolates from Brazil were of the MAT1-1 genotype. Each MAT1 idiomorphic region was sequenced and aligned, using the sequences of the G-217B (+ mating type) and G-186AR (− mating type) strains as references. BLASTn analyses of the MAT1-1 and MAT1-2 sequences studied correlated with their respective + and − mating type genotypes. Trees were generated by the maximum likelihood (ML) method to search for similarity among isolates of each MAT1 idiomorph. All MAT1-1 isolates originated from Brazilian bats formed a well-defined group; three isolates from Mexico, the G-217B strain, and a subgroup encompassing all soil-derived isolates and two clinical isolates from Brazil formed a second group; last, one isolate (EH-696P) from a migratory bat captured in Mexico formed a third group of the MAT1-1 genotype. The MAT1-2 idiomorph formed two groups, one of which included two H. capsulatum isolates from infected bats that were closely related to the G-186AR strain. The other group was formed by two human isolates and six isolates from infected bats. Concatenated ML trees, with internal transcribed spacer 1 (ITS1) -5.8S-ITS2 and MAT1-1 or MAT1-2 sequences, support the relatedness of MAT1-1 or MAT1-2 isolates. H. capsulatum mating types were associated with the geographical origin of the isolates, and all isolates from Brazil correlated with their environmental sources.     

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.     

Improvement of fruiting body production in Cordyceps militaris by molecular assessment

Cordyceps militaris is a heterothallic ascomycetous fungus that has been cultivated as a medicinal mushroom. This study was conducted to improve fruiting body production by PCR assessment. Based on single-ascospore isolates selected from wild and cultivated populations, the conserved sequences of α-BOX in MAT1-1 and HMG-BOX in MAT1-2 were used as markers for the detection of mating types by PCR. PCR results indicated that the ratio of mating types is consistent with a theoretical ratio of 1:1 (MAT1-1:MAT1-2) in wild (66:70) and cultivated (71:60) populations. Cross-mating between the opposite mating types produced over fivefold more well-developed fruiting bodies than self- or cross-mating between strains within the same mating type. This study may serve as a valuable reference for artificial culturing of C. militaris and other edible and medicinal mushrooms and may be useful to develop an efficient process for the selection, domestication, and management of strains for industrial-scale production.     

Non-Mendelian segregation influences the infection biology and genetic structure of the African tree pathogen Ceratocystis albifundus

The African fungal tree pathogen, Ceratocystis albifundus, undergoes uni-directional mating type switching, giving rise to either self-fertile or self-sterile progeny. Self-sterile isolates lack the MAT1-2-1 gene and have reduced fitness such as slower growth and reduced pathogenicity, relative to self-fertile isolates. While it has been hypothesized that there is a 1:1 ratio of self-fertile to self-sterile ascospore progeny in relatives of C. albifundus, some studies have reported a significant bias in this ratio. This could be due to the fact that either fewer self-sterile ascospores are produced or that self-sterile ascospores have low viability. We quantified the percentage of self-sterile and self-fertile ascospores from ascospore masses in C. albifundus using real-time PCR. Primers were designed to distinguish between spores that contained the MAT1-2-1 gene and those where this gene had been deleted. A significant bias towards the self-fertile mating type was observed in all single ascospore masses taken from sexual structures produced in haploid-selfed cultures. The same result was observed from a disease outbreak situation in an intensively managed field of cultivated native trees, and this was coupled with very low population diversity in the pathogen. This was in contrast to the results obtained from ascospore masses taken from the crosses performed under laboratory conditions or ascomata on native trees in a non-disease situation, where either self-fertile or self-sterile ascospores were dominant. The results suggest that reproductive strategies play a significant role in the infection biology and genetic structure of C. albifundus populations.     

Cylindrocladium buxicola, a new species affecting Buxus spp., and its phylogenetic status

A leaf and twig blight disease of Buxus spp. was found to be associated with a new species of Cylindrocladium. The novel species status was confirmed using morphological characters, sequencing of the ribosomal 5.8S RNA gene and the flanking internal transcribed spacers (ITS), the β-tubulin gene, and the high mobility group (HMG) of the MAT2 mating type gene. Cylindrocladium buxicola is proposed as a new name. Fifteen isolates from the UK and one isolate from New Zealand were paired in all combinations but no fertile perithecia were obtained suggesting that C. buxicola is heterothallic and all isolates belonged to one mating type. AFLP analysis showed that the isolates collected in the UK and New Zealand are genetically homogenous. Phylogenetic analyses indicated that this species falls within a new lineage.