Isolation of Saccharomycopsis species from plant material.

Saccharomycopsis species are natural organic sulphur auxotrophs. Their genomes do not encode genes for the uptake and assimilation of sulphate and thus these species cannot grow on media lacking e.g. methionine. Due to the similarity between sulphate and selenate, uptake and assimilation of selenate occurs through the same pathway starting from sulphate transporters encoded by the homologs of the SUL1 and SUL2 genes in S. cerevisiae. Lack of these transporters renders Saccharomycopsis species resistant to selenate levels that are toxic to other microorganisms. We used this feature to enrich environmental samples for Saccharomycopsis species. This led to the isolation of S. schoenii, S. lassenensis and a hitherto undescribed Saccharomycopsis species with limited by-catch of other yeasts, mainly belonging to Metschnikowia and Hanseniaspora. We performed growth and predation assays to characterize the potential of these new isolates as predacious yeasts. Most Saccharomycopsis species are temperature sensitive and cannot grow at 37°C; with the exception of S. lassenensis strains. Predation assays with S. schoenii and S. cerevisiae as prey indicated that predation was enhanced at 20°C compared to 30°C. We crossed an American isolate of S. schoenii with our German isolate using marker directed breeding. Viable progeny indicated that both strains are interfertile and belong to the same biological species. S. lassenensis is heterothallic, while S. schoenii and the new Saccharomycopsis isolate, for which we suggest the name S. geisenheimensis sp. nov., are homothallic.

Genome Analysis of a Newly Discovered Yeast Species, Hanseniaspora menglaensis.

Annual surveys of Irish soil samples identified three isolates, CBS 16921 (UCD88), CBS 18246 (UCD443), and CBS 18247 (UCD483), of an apiculate yeast species within the Hanseniaspora genus. The internal transcribed spacer (ITS) and D1/D2 region of the large subunit (LSU) rRNA sequences showed that these are isolates of the recently described species Hanseniaspora menglaensis, first isolated from Southwest China. No genome sequence for H. menglaensis is currently available. The genome sequences of the three Irish isolates were determined using short-read (Illumina) sequencing, and the sequence of one isolate (CBS 16921) was assembled to chromosome level using long-read sequencing (Oxford Nanopore Technologies). Phylogenomic analysis shows that H. menglaensis belongs to the fast-evolving lineage (FEL) of Hanseniaspora. Only one MAT idiomorph (encoding MATα1) was identified in all three sequenced H. menglaensis isolates, consistent with one mating type of a heterothallic species. Genome comparisons showed that there has been a rearrangement near MATα of FEL species compared to isolates from the slowly evolving lineage (SEL).

Diversity and Clonality in Populations of Phytophthora citrophthora and P. syringae Causing Brown Rot of Citrus in California.

Phytophthora citrophthora and P. syringae are currently the primary causal organisms of brown rot of citrus fruits in California. To possibly find an explanation for the prevalence of the previously minor species P. syringae, we determined the population structures of both pathogens in California using next-generation sequencing and population genomics analyses. Whole-genome sequencing and aligning with newly assembled reference genomes identified 972,266 variants in 132 isolates of P. citrophthora and 422,208 variants in 154 isolates (including 24 from noncitrus tree crops) of P. syringae originating from three major growing regions. The resulting data sets were visualized using principal component analysis, discriminant analysis of principal components, unweighted pair-group method with arithmetic mean dendrograms, fastStructure, and minimum spanning networks, and we obtained the index of association, diversity summary statistics, and genetic distance statistics values GST, G''ST, and Jost's D. Subpopulations of both species were mostly defined by their geographic origin indicating restricted dispersal of inoculum. Except for five isolates, the population structure of P. citrophthora (that is heterothallic and unlikely to reproduce sexually) was clonal to semi-clonal, with very little genetic diversity within and among subgroups. In contrast, the population structure of P. syringae was also clonal to semi-clonal, but isolates were placed into four main clusters of much higher diversity. Clonality in both species can be explained by a high level of asexual reproduction. The higher diversity in the homothallic P. syringae is likely due to commonly occurring sexual reproduction. One distinct cluster of P. syringae consisted solely of isolates from noncitrus hosts; therefore, the origin of P. syringae in citrus could not be resolved.

Engineering heterothallic strains in fission yeast.

In poor nitrogen conditions, fission yeast cells mate, undergo meiosis and form spores that are resistant to deleterious environments. Natural isolates of Schizosaccharomyces pombe are homothallic. This allows them to naturally switch between the two h- and h+ mating types with a high frequency, thereby ensuring the presence of both mating partners in a population of cells. However, alteration of the mating type locus can abolish mating type switching or reduce it to a very low frequency. Such heterothallic strains have been isolated and are common in research laboratories due to the simplicity of their use for Mendelian genetics. In addition to the standard laboratory strains, a large collection of natural S. pombe isolates is now available, representing a powerful resource for investigating the genetic diversity and biology of fission yeast. However, most of these strains are homothallic, and only tedious or mutagenic strategies have been described to obtain heterothallic cells from a homothallic parent. Here, we describe a simple approach to generate heterothallic strains. It takes advantage of an alteration of the mating type locus that was previously identified in a mating type switching-deficient strain and the CRISPR-Cas9 editing tool, allowing for a one-step engineering of heterothallic cells with high efficiency.

Effects of MAT1-2 Spore Ratios on Fruiting Body Formation and Degeneration in the Heterothallic Fungus Cordyceps militaris.

The medicinal mushroom Cordyceps militaris is widely exploited in traditional medicine and nutraceuticals in Asian countries. However, fruiting body production in C. militaris is facing degeneration through cultivation batches, and the molecular mechanism of this phenomenon remains unclear. This study showed that fruiting body formation in three different C. militaris strains, namely G12, B12, and HQ1, severely declined after three successive culturing generations using the spore isolation method. PCR analyses revealed that these strains exist as heterokaryons and possess both the mating-type loci, MAT1-1 and MAT1-2. Further, monokaryotic isolates carrying MAT1-1 or MAT1-2 were successfully separated from the fruiting bodies of all three heterokaryotic strains. A spore combination of the MAT1-1 monokaryotic isolate and the MAT1-2 monokaryotic isolate promoted fruiting body formation, while the single monokaryotic isolates could not do that themselves. Notably, we found that changes in ratios of the MAT1-2 spores strongly influenced fruiting body formation in these strains. When the ratios of the MAT1-2 spores increased to more than 15 times compared to the MAT1-1 spores, the fruiting body formation decreased sharply. In contrast, when MAT1-1 spores were increased proportionally, fruiting body formation was only slightly reduced. Our study also proposes a new solution to mitigate the degeneration in the heterokaryotic C. militaris strains caused by successive culturing generations.

Large-Scale Field Cultivation of Morchella and Relevance of Basic Knowledge for Its Steady Production.

Morels are one of the most highly prized edible and medicinal mushrooms worldwide. Therefore, historically, there has been a large international interest in their cultivation. Numerous ecological, physiological, genetic, taxonomic, and mycochemical studies have been previously developed. At the beginning of this century, China finally achieved artificial cultivation and started a high-scale commercial development in 2012. Due to its international interest, its cultivation scale and area expanded rapidly in this country. However, along with the massive industrial scale, a number of challenges, including the maintenance of steady economic profits, arise. In order to contribute to the solution of these challenges, formal research studying selection, species recognition, strain aging, mating type structure, life cycle, nutrient metabolism, growth and development, and multi-omics has recently been boosted. This paper focuses on discussing current morel cultivation technologies, the industrial status of cultivation in China, and the relevance of basic biological research, including, e.g., the study of strain characteristics, species breeding, mating type structure, and microbial interactions. The main challenges related to the morel cultivation industry on a large scale are also analyzed. It is expected that this review will promote a steady global development of the morel industry based on permanent and robust basic scientific knowledge.

Predominant Clonal Reproduction with Infrequent Genetic Recombination of Phaeoacremonium minimum in Western Cape Vineyards.

Phaeoacremonium minimum is an important esca and Petri disease pathogen that causes dieback of grapevines in South Africa. Little is known regarding the reproductive strategy of the pathogen. Sexual reproduction could lead to a better adaptation of the pathogen to disease management strategies by combining alleles through recombination. The study aimed to investigate the genetic diversity and recombination potential of eight populations in the Western Cape, from six commercial vineyards and two nursery rootstock mother blocks. This was achieved by developing and applying nine polymorphic microsatellites and mating-type-specific markers. Thirty-seven genotypes were identified from 295 isolates. Populations were characterised by the same dominant genotype (MLG20 occurring 65.43%), low genotypic diversity (H) and high numbers of clones (81.36% of dataset). However, genotypes from the same sampling sites were not closely related based on a minimum spanning network and had high molecular variation within populations (94%), suggesting that multiple introductions of different genotypes occurred over time. Significant linkage disequilibrium among loci (r̅d) further indicated a dominant asexual cycle, even though perithecia have been observed in these four populations. The two rootstock mother blocks had unique genotypes and genotypes shared with the vineyard populations. Propagation material obtained from infected rootstock mother blocks could lead to the spread of more genotypes to newly established vineyards. Based on our results, it is important to determine the health status of rootstock mother blocks. Management strategies must focus on reducing aerial inoculum to prevent repeated infections and further spread of P. minimum genotypes.

Importance of appropriate genome information for the design of mating type primers in black and yellow morel populations.

Morels are highly prized edible fungi where sexual reproduction is essential for fruiting-body production. As a result, a comprehensive understanding of their sexual reproduction is of great interest. Central to this is the identification of the reproductive strategies used by morels. Sexual reproduction in fungi is controlled by mating-type (MAT) genes and morels are thought to be mainly heterothallic with two idiomorphs, MAT1-1 and MAT1-2. Genomic sequencing of black (Elata clade) and yellow (Esculenta clade) morel species has led to the development of PCR primers designed to amplify genes from the two idiomorphs for rapid genotyping of isolates from these two clades. To evaluate the design and theoretical performance of these primers we performed a thorough bioinformatic investigation, including the detection of the MAT region in publicly available Morchella genomes and in-silico PCR analyses. All examined genomes, including those used for primer design, appeared to be heterothallic. This indicates an inherent fault in the original primer design which utilized a single Morchella genome, as the use of two genomes with complementary mating types would be required to design accurate primers for both idiomorphs. Furthermore, potential off-targets were identified for some of the previously published primer sets, but verification was challenging due to lack of adequate genomic information and detailed methodologies for primer design. Examinations of the black morel specific primer pairs (MAT11L/R and MAT22L/R) indicated the MAT22 primers would correctly target and amplify the MAT1-2 idiomorph, but the MAT11 primers appear to be capable of amplifying incorrect off-targets within the genome. The yellow morel primer pairs (EMAT1-1 L/R and EMAT1-2 L/R) appear to have reporting errors, as the published primer sequences are dissimilar with reported amplicon sequences and the EMAT1-2 primers appear to amplify the RNA polymerase II subunit (RPB2) gene. The lack of the reference genome used in primer design and descriptive methodology made it challenging to fully assess the apparent issues with the primers for this clade. In conclusion, additional work is still required for the generation of reliable primers to investigate mating types in morels and to assess their performance on different clades and across multiple geographical regions.

Indirect Evidence Based on Mating-Type Ratios for the Role of Sexual Reproduction in European and Chinese Populations of Plenodomus biglobosus (Blackleg of Oilseed Rape).

Blackleg (Phoma) disease, caused by the ascomycete fungi Plenodomus biglobosus and P. lingam, threatens oilseed rape (OSR; Brassica napus) crops internationally. In many parts of the world, both species co-occur, but in China only P. biglobosus has so far been reported. Plenodomus biglobosus reproduces asexually (pycnidiospores), but also sexually (pseudothecia-yielding ascospores), via a heterothallic mating system requiring MAT1-1 and MAT1-2 genotypes. However, the roles of airborne ascospore inoculum in driving blackleg disease outbreaks in China are less well understood compared to elsewhere in the world. This is despite the very different agronomic cropping practices in parts of China, in which paddy rice and OSR are often grown in rotation; OSR stubble is often submerged under water for long periods potentially affecting pseudothecial development. Here, we indirectly investigate the potential role of sexual reproduction by developing new polymerase chain reaction (PCR) -based mating-type diagnostics for P. biglobosus and subsequently screening an international collection of 59 European and 157 Chinese isolates. Overall, in both Europe and China, P. biglobosus mating types did not deviate from a 1:1 ratio, such as is generally thought to occur under frequency-dependent selection in sexually reproducing pathogen populations. Both mating types were balanced in all the individual European countries tested (Austria, France, Poland, UK). Conversely, in China, mating types were only balanced in the eastern region; in the northern and southwestern regions there were skewed ratios, more typical of predominantly asexual reproduction, towards MAT1-1 and MAT1-2, respectively. The implications of these findings and future research directions for improved understanding of P. biglobosus epidemiology on OSR, particularly in China, are considered.

Strains and approaches for genetic crosses in the oleaginous yeast Lipomyces starkeyi.

The oleaginous yeast Lipomyces starkeyi is a powerful lipid producer with great industrial potential. Recent studies have reported the isolation of mutant L. starkeyi cells with higher lipid producing capacity. Although genetic engineering strategies have been applied to L. starkeyi, classical genetic approaches are lacking. The development of tools that facilitate genetic crosses in L. starkeyi would not only make it possible to build improved lipid-producing strains but also facilitate molecular biological analysis of this species. In this study, I report a set of strains and approaches useful for performing genetic crosses with L. starkeyi. The homothallic L. starkeyi reportedly forms an ascus containing two to 20 spores. These spores were resistant to glusulase and could be dissected using a micromanipulator, suggesting that random spore and tetrad (spore dissection) analysis can be adapted for L. starkeyi. Additionally, to isolate a pair of heterothallic strains useful for genetic crosses, the homothallic strain was exposed to UV irradiation, and 10 self-sterile strains were crossed with one another. One of these combinations, Ls75 and Ls100, sporulated stably. Moreover, to detect genetic recombination, I introduced a different drug resistance marker into each strain and crossed them. The resulting progeny exhibited Mendelian segregation of the resistance markers. Altogether, the work reported here provides a powerful resource for genetic analysis in L. starkeyi.

Mating-Type Locus Organization and Mating-Type Chromosome Differentiation in the Bipolar Edible Button Mushroom Agaricus bisporus.

In heterothallic basidiomycete fungi, sexual compatibility is restricted by mating types, typically controlled by two loci: PR, encoding pheromone precursors and pheromone receptors, and HD, encoding two types of homeodomain transcription factors. We analysed the single mating-type locus of the commercial button mushroom variety, Agaricus bisporus var. bisporus, and of the related variety burnettii. We identified the location of the mating-type locus using genetic map and genome information, corresponding to the HD locus, the PR locus having lost its mating-type role. We found the mip1 and ß-fg genes flanking the HD genes as in several Agaricomycetes, two copies of the ß-fg gene, an additional HD2 copy in the reference genome of A. bisporus var. bisporus and an additional HD1 copy in the reference genome of A. bisporus var. burnettii. We detected a 140 kb-long inversion between mating types in an A. bisporus var. burnettii heterokaryon, trapping the HD genes, the mip1 gene and fragments of additional genes. The two varieties had islands of transposable elements at the mating-type locus, spanning 35 kb in the A. bisporus var. burnettii reference genome. Linkage analyses showed a region with low recombination in the mating-type locus region in the A. bisporus var. burnettii variety. We found high differentiation between ß-fg alleles in both varieties, indicating an ancient event of recombination suppression, followed more recently by a suppression of recombination at the mip1 gene through the inversion in A. bisporus var. burnettii and a suppression of recombination across whole chromosomes in A. bisporus var. bisporus, constituting stepwise recombination suppression as in many other mating-type chromosomes and sex chromosomes.

Characterization of mating type genes in heterothallic Neonectria species, with emphasis on N. coccinea, N. ditissima, and N. faginata.

Neonectria ditissima and N. faginata are canker pathogens involved in an insect-fungus disease complex of American beech (Fagus grandifolia) in North America commonly known as beech bark disease (BBD). In Europe, both N. ditissima and N. coccinea are involved in BBD on European beech (Fagus sylvatica). Field observations across the range of BBD indicate ascospores to be the dominant spore type in the environment. Several studies report a heterothallic (self-sterile) mating strategy for Neonectria fungi, but one study reported homothallism (self-fertility) for N. ditissima. As such, investigations into mating strategy are important for understanding both the disease cycle and population genetics of Neonectria. This is particularly important in the United States given that over time N. faginata dominates the BBD pathosystem despite high densities of nonbeech hosts for N. ditissima. This study utilized whole-genome sequences of BBD-associated Neonectria spp. along with other publicly available Neonectria and Corinectria genomes and in vitro mating assays to characterize mating type (MAT) locus and confirm thallism for select members of Neonectria and Corinectria. MAT gene-specific primer pairs were developed to efficiently characterize the mating types of additional single-ascospore strains of N. ditissima, N. faginata, and N. coccinea and several other related species lacking genomic data. These assays also confirmed the sexual compatibility among N. ditissima strains from different plant hosts. Maximum likelihood phylogenetic analyses of both MAT1-1-1 and MAT1-2-1 sequences recovered trees with similar topology to previously published phylogenies of Neonectria and Corinectria. The results of this study indicate that all Neonectria and Corinectria tested are heterothallic based on our limited sampling and, as such, thallism cannot help explain the inevitable dominance of N. faginata in the BBD pathosystem.

Sporangiospore Viability and Oospore Production in the Spinach Downy Mildew Pathogen, Peronospora effusa.

Downy mildew of spinach, caused by the obligate pathogen Peronospora effusa, remains the most important constraint in the major spinach production areas in the United States. This disease can potentially be initiated by asexual sporangiospores via "green bridges", sexually derived oospores from seed or soil, or dormant mycelium. However, the relative importance of the various types of primary inoculum is not well known. The ability of P. effusa sporangiospores to withstand abiotic stress, such as desiccation, and remain viable during short- and long-distance dispersal and the ability of oospores to germinate and infect seedlings remain unclear. Thus, the primary objectives of this research were to evaluate the impact of desiccation on sporangiospore survival and infection efficiency and examine occurrence, production, and germination of oospores. Results indicate that desiccation significantly reduces sporangiospore viability as well as infection potential. Leaf wetness duration of 4 h was needed for disease establishment by spinach downy mildew sporangiospores. Oospores were observed in leaves of numerous commercial spinach cultivars grown in California in 2018 and Arizona in 2019. Frequency of occurrence varied between the two states-years. The presence of opposite mating types in spinach production areas in the United States was demonstrated by pairing isolates in controlled crosses and producing oospores on detached leaves as well as intact plants. Information from the study of variables that affect sporangiospore viability and oospore production will help in improving our understanding of the epidemiology of this important pathogen, which has implications for management of spinach downy mildew.

An endophyte of Macrochloa tenacissima (esparto or needle grass) from Tunisia is a novel species in the Fusarium redolens species complex.

Here, we report on the morphological, molecular, and chemical characterization of a novel Fusarium species recovered from the roots and rhizosphere of Macrochloa tenacissima (halfa, esparto, or needle grass) in central Tunisia. Formally described here as F. spartum, this species is a member of the Fusarium redolens species complex but differs from the other two species within the complex, F. redolens and F. hostae, by its endophytic association with M. tenacissima and its genealogical exclusivity based on multilocus phylogenetic analyses. To assess their sexual reproductive mode, a polymerase chain reaction (PCR) assay was designed and used to screen the three strains of F. spartum, 51 of F. redolens, and 14 of F. hostae for mating type (MAT) idiomorph. Genetic architecture of the MAT locus in the former two species suggests that if they reproduce sexually, it is via obligate outcrossing. By comparison, results of the PCR assay indicated that 13/14 of the F. hostae strains possessed MAT1-1 and MAT1-2 idiomorphs and thus might be self-fertile or homothallic. However, when the F. hostae strains were selfed, 11 failed to produce perithecia and one only produced several small abortive perithecia. Cirrhi with ascospores, however, were only produced by 8/28 and 4/84 of the variable size perithecia, respectively, of F. hostae NRRL 29888 and 29890. The potential for the three F. redolens clade species to produce mycotoxins, pigments, and phytohormones was assessed by screening whole genome sequence data and by analyzing extracts on cracked maize kernel cultures via liquid chromatography-mass spectrometry.

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.

Design and validation of a robust multiplex polymerase chain reaction assay for MAT idiomorph within the Fusarium fujikuroi species complex.

We discovered that published polymerase chain reaction (PCR) assays for determining mating type (MAT) idiomorph failed to genotype some of the Fusarium fujikuroi species complex (FFSC) isolates recovered from Mangifera indica (mango), Swietenia macrophylla (big-leaf mahogany), Annona muricata (soursop), Bursera sp., and Tabebuia sp. in Mexico. Thus, the primary objective of this study was to design and validate a robust multiplex PCR-based diagnostic for typing MAT within the FFSC. To accomplish this objective, we mined the MAT1-1 or MAT1-2 locus from the genomes of 60 FFSC isolates, representing 56 phylospecies, and from four species in its sister group, the F. nisikadoi species complex (FNSC). Bioinformatic searches were facilitated by targeting DNA lyase (SLA2) and apurinic endonuclease (APN1), the genes that flank the MAT locus in Fusarium. As expected, three genes were identified within MAT1-1 (MAT1-1-1, MAT1-1-2, and MAT1-1-3) and two in MAT1-2 (MAT1-2-1 and MAT1-2-9), using the ab initio prediction tool AUGUSTUS. Of the three multiplex PCR assays we designed and tested, the one targeting MAT1-1-2 and MAT1-2-1 successfully genotyped the entire 71-isolate validation panel, which included 56 FFSC and 4 FNSC phylospecies. By contrast, the published PCR assays we tested produced positive genotypes for only 46.5-59% of the 71-isolate validation panel, but only when they were run as a uniplex assay. Although only one-fifth of the FFSC/FNSC are known to reproduce sexually, our results suggest that if they possess a sexual cycle, it is heterothallic (self-sterile).

The mating system of the Eucalyptus canker pathogen Chrysoporthe austroafricana and closely related species.

Fungi in the genus Chrysoporthe are economically important canker pathogens of commercially grown Eucalyptus species and native Myrtales. Before the current study, homothallism was widely accepted as the mating system of these species, but this hypothesis could not be fully tested. Using whole genome sequences, we characterized the MAT locus of two C. austroafricana isolates and its sibling species, C. cubensis and C. deuterocubensis. A unique MAT1-2 idiomorph containing a truncated MAT1-1-1 gene, and a MAT1-1-2 gene, was identified in one isolate of C. austroafricana and a MAT1-1 idiomorph was found in the other. The presence of a single idiomorph in each isolate suggests that this fungus is heterothallic. Screening for MAT genes in 65 C. austroafricana isolates revealed a bias towards MAT1-2 idiomorphs suggesting a recent introduction in Eucalyptus species. Chrysoporthe cubensis and C. deuterocubensis are apparently homothallic since all the expected MAT genes were identified in their genome sequences. These findings were corroborated by the expression profiles of pheromone genes and their receptors, which conformed to the expected patterns observed in heterothallic and homothallic isolates. Long terminal repeat sequences (LTRs) and specifically retrotransposons were identified in the MAT locus of C. deuterocubensis and C. cubensis, indicating that the evolution of mating systems in Chrysoporthe species could be mediated by these elements.

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.

Unexpected placement of the MAT1-1-2 gene in the MAT1-2 idiomorph of Thielaviopsis.

Sexual reproduction in the Ascomycota is controlled by genes encoded at the mating-type or MAT1 locus. The two allelic versions of this locus in heterothallic species, referred to as idiomorphs, are defined by the MAT1-1-1 (for the MAT1-1 idiomorph) and MAT1-2-1 (for the MAT1-2 idiomorph) genes. Both idiomorphs can contain additional genes, although the contents of each is typically specific to and conserved within particular Pezizomycotina lineages. Using full genome sequences, complemented with conventional PCR and Sanger sequencing, we compared the mating-type idiomorphs in heterothallic species of Thielaviopsis (Ceratocystidaceae). The analyses showed that the MAT1-1 idiomorph of T. punctulata, T. paradoxa, T. euricoi, T. ethacetica and T. musarum harboured only the expected MAT1-1-1 gene. In contrast, the MAT1-2 idiomorph of T. punctulata, T. paradoxa and T. euricoi encoded the MAT1-2-1, MAT1-2-7 and MAT1-1-2 genes. Of these, MAT1-2-1 and MAT1-2-7 are genes previously reported in this idiomorph, while MAT1-1-2 is known only in the MAT1-1 idiomorph. Phylogenetic analysis showed that the Thielaviopsis MAT1-1-2 groups with the known homologues of this gene in other Microascales, thus confirming its annotation. Previous work suggests that MAT1-1-2 is involved in fruiting body development, a role that would be unaffected by its idiomorphic position. This notion is supported by our findings for the MAT1 locus structure in Thielaviopsis species. This also serves as the first example of a MAT1-1-specific gene restricted to only the MAT1-2 idiomorph.

Creation of Stable Heterothallic Strains of Komagataella phaffii Enables Dissection of Mating Gene Regulation.

The methylotrophic yeast Komagataella phaffii (Pichia pastoris) is homothallic and has been reported to switch mating type by an ancient inversion mechanism. Two mating-type (MAT) loci include homologs of the MATa and MATα transcription factor genes, with the expression from one locus downregulated by telomere position effects. However, not much is known about mating gene regulation, since the mixture of mating types complicates detailed investigations. In this study, we developed K. phaffii strains with stable mating types by deletion of the inverted-repeat region required for mating-type switching. These heterothallic strains retain their ability to mate with cells of the opposite mating type and were used to further elucidate mating gene regulation. Functional analysis of MAT mutant strains revealed the essential role of MATa2 and MATα1 in diploid cell formation. Disruption of MATa1 or MATα2 did not affect mating; however, in diploid cells, both genes are required for sporulation and the repression of shmoo formation. The heterothallic strains generated in this study allowed the first detailed characterization of mating gene regulation in K. phaffii They will be a valuable tool for further studies investigating cell-type-specific behavior and will enable in-depth genetic analyses and strain hybridization in this industrially relevant yeast species.