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During a study of yeast diversity in Azorean vineyards, four strains were isolated which were found to represent a novel yeast species based on the sequences of the internal transcribed spacer (ITS) region (ITS1-5.8S–ITS2) and of the D1/D2 domain of the large subunit (LSU) rRNA gene, together with their physiological characteristics. An additional strain isolated from Drosophila suzukii in Italy had identical D1/D2 sequences and very similar ITS regions (five nucleotide substitutions) to the Azorean strains. Phylogenetic analysis using sequences of the ITS region and D1/D2 domain showed that the five strains are closely related to Clavispora lusitaniae, although with 56 nucleotide differences in the D2 domain. Intraspecies variation revealed between two and five nucleotide differences, considering the five strains of Clavispora santaluciae. Some phenotypic discrepancies support the separation of the new species from their closely related ones, such as the inability to grow at temperatures above 35 °C, to produce acetic acid and the capacity to assimilate starch. Neither conjugations nor ascospore formation were observed in any of the strains. The name Clavispora santaluciae f.a., sp. nov., is proposed to accommodate the above noted five strains (holotype, CBS 16465T; MycoBank no., MB 835794).

In this study, yeasts were isolated from an indigenous starter culture for cachaca production (Brazilian spirit) and brewer’s spent grain and characterized through a series of phenotypic assays: the killer profile, ascospore formation capacity, growth at high temperatures, resistance to natamycin and actidione, and exoenzyme production. One hundred thirty-four (n = 134) yeasts were isolated and identified as belonging to 6 genera and 10 different species. The ascospore formation in potassium acetate agar was observed in 86% of the isolates (73 Saccharomyces cerevisiae, 3 Torulaspora delbrueckii, and 1 Wickerhamomyces anomalus) highlighting their potential for microencapsulation. Only S. cerevisiae LMQA SNR 70 expressed an effective killer factor (ability to destroy sensitive strains), while only 3 S. cerevisiae, 1 Clavispora lusitaniae, and 1 Pichia kudriavzevii isolates were able to grow up at 45 °C. At 50 and 100 mg/L, actidione showed selectivity action for Candida parapsilosis, while natamycin inhibited the growth of all isolates. The non-Saccharomyces isolates stood out in the qualitative and quantitative tests for amylolytic, pectinolytic, cellulolytic, and xylanolytic activities. A predominance of the species W. anomalus and T. delbrueckii was observed. Besides, quantitative tests revealed that 23% of the isolates exhibited exoenzymatic activity in a nutrient-poor environment, and W. anomalus LMQA CSC 5 e LMQA CSC 43 stood out for the multi-enzymatic in agar and liquid medium response. The phenotypic profile showed that 16% of the isolates expressed two or more characteristics studied, with 2/3 represented by the non-Saccharomyces species. These results highlight the importance of studying yeast species for new biotechnological purposes.

A total of 21 yeast isolates were recovered as part of a research project on biodiversity of yeasts in traditional dairy products in Alborz province, Iran. Standard protocols were used to carry out phenotypic, biochemical, physiological characterization and the phylogenetic analysis of combined the D1/D2 domain of the large ribosomal subunit (26S or LSU) and ITS region sequences. Five strains represented a potential new ascomycetous yeast species. Ascospore formation was not observed in these strains, and they did not ferment the examined carbon sources. Phylogenetic analysis placed these isolates in a well-supported sub-clade in the genus Saccharomycopsis. Here, we describe this novel yeast as Saccharomycopsis oxydans sp. nov.

The endoplasmic reticulum (ER) is an elaborate organelle composed of distinct structural and functional domains. ER structure and dynamics involve membrane-shaping proteins of the reticulon and Yop1/DP1 families, which promote membrane curvature and regulate ER shaping and remodeling. Here, we analyzed the function of the reticulon (RTN1) and Yop1 proteins (YOP1 and YOP2) of the model fungus Podospora anserina and their contribution to sexual development. We found that RTN1 and YOP2 localize to the peripheral ER and are enriched in the dynamic apical ER domains of the polarized growing hyphal region. We discovered that the formation of these domains is diminished in the absence of RTN1 or YOP2 and abolished in the absence of YOP1 and that hyphal growth is moderately reduced when YOP1 is deleted in combination with RTN1 and/or YOP2. In addition, we found that RTN1 associates with the Spitzenkorper. Moreover, RTN1 localization is regulated during meiotic development, where it accumulates at the apex of growing asci (meiocytes) during their differentiation and at their middle region during the subsequent meiotic progression. Furthermore, we discovered that loss of RTN1 affects ascospore (meiotic spore) formation, in a process that does not involve YOP1 or YOP2. Finally, we show that the defects in ascospore formation of rtn1 mutants are associated with defective nuclear segregation and spindle dynamics throughout meiotic development. Our results show that sexual development in P. anserina involves a developmental remodeling of the ER that implicates the reticulon RTN1, which is required for meiotic nucleus segregation. IMPORTANCE Meiosis consists of a reductional cell division, which allows ploidy maintenance during sexual reproduction and which provides the potential for genetic recombination, producing genetic variation. Meiosis constitutes a process of foremost importance for eukaryotic evolution. Proper partitioning of nuclei during this process relies on accurate functioning and positioning of the spindle, the microtubule cytoskeletal apparatus that conducts chromosome segregation. In this research, we show that in the model fungus Podospora anserina this process requires a protein involved in structuring the endoplasmic reticulum (ER)-the reticulon RTN1. The ER is a complex organelle composed of distinct structural domains, including different peripheral domains and the nuclear envelope. Our findings suggest that spindle dynamics during meiosis relies on remodeling of the ER membrane, which involves the activity of RTN1. Our research discloses that the proteins implicated in shaping the ER are main contributors to the regulation of nuclear dynamics during the sexual cycle.

Four yeast strains (RIFY 10001T, RIFY 10002, RIFY 10003, and RIFY 10004) were isolated from flowers growing in fields of mustard and broad beans in Japan. Ascospore formation was not observed. Sequence analysis of the D1/D2 domain of the large subunit ribosomal RNA (LSU rRNA) gene of the four strains indicated that they belong to the genus Metschnikowia and are closely related to Metschnikowia hawaiiana strain CBS 9146T and Metschnikowia orientalis strain CBS 10331T. The D1/D2 domain of the LSU rRNA gene and internal transcribed spacer regions of strain RIFY 10001T were 85.7% identical to those of M. hawaiiana strain CBS 9146T. All four strains were distinguished from the M. hawaiiana strain CBS 9146T by their inability to ferment glucose. Hence, these four strains are novel species and were named as Metschnikowia miensis (holotype: RIFY 10001T; isotypes: NBRC 112445T = CBS 14749T).

Ascospores of Talaromyces.macrosporus belong to the most stress resistant eukaryotic cells and show a constitutive dormancy, i.e., no germination occurs in the presence of rich growth medium. Only an extreme trigger as very high temperature or pressure is able to evoke synchronized germination. In this study, several changes within the thick cell wall of these cells are observed after a heat treatment: (i.) a change in its structure as shown with EPR and X-ray diffraction, (ii.) a release of an abundant protein into the supernatant, which is proportional to the extent of heat activation, (iii.) a change in the permeability of the cell wall as judged by fluorescence studies in which staining of the interior of the cell wall correlates with germination of individual ascospores. The gene encoding the protein, dubbed ICARUS, was studied in detail and was expressed under growth conditions that showed intense ascomata (fruit body) and ascospore formation. It encodes a small 7–14 kD protein. Blast search exhibits that different Talaromyces species show a similar sequence, indicating that the protein also occurs in other species of the genus. Deletion strains show delayed ascomata formation, release of pigments into the growth medium, higher permeability of the cell wall and a markedly shorter heat activation needed for activation. Further, wild type ascospores are more heat-resistant. All these observations suggest that the protein plays a role in dormancy and is related to the structure and permeability of the ascospore cell wall. However, more research on this topic is needed to study constitutive dormancy in other fungal species that form stress-resistant ascospores.

Enhanced commercial application of silver nanoparticles (AgNPs) is increasing the chance of their release into aquatic environments, potentially putting non-target microorganisms at risk. Impacts of AgNPs and Ag+ on two fungal ecotypes of Articulospora tetracladia, collected from a metal-polluted (At61) and a non-polluted (At72) stream, were assessed based on antioxidant enzymatic and proteomic responses. At61 showed more tolerance to AgNPs than At72 (EC20, 158.9 vs 7.5 µg L-1, respectively). Antioxidant enzyme activities were induced by AgNPs or Ag+ in both fungal ecotypes. Proteomic responses to AgNPs or Ag+ revealed that 41.3% of the total altered proteins were common in At72, while 27.3% were common in At61. In At72, gene ontology enrichment analyses indicated that Ag+ increased mainly the content of proteins involved in proteostasis and decreased the content of those related to vesicle-mediated transport; whereas the key group of proteins induced by AgNPs had functions in DNA repair and energy production. In At61, AgNPs induced proteins involved in energy production and protein biosynthesis, while both Ag forms induced proteins related to cell-redox and protein homeostasis, ascospore formation, fatty acid biosynthesis and nucleic acids metabolism. Both Ag forms induced stress-responsive proteins, and this was consistent with the responses of antioxidant enzymes. The negligible quantity of Ag+ released from AgNPs (, programme UID/ BIA/04050/2019 and the Emergemix project (PTDC/BIABMA/30922/2017) funded by national funds through the Portuguese Foundation for Science and Technology (FCT) I.P. and by the ERDF through the COMPETE2020 – Programa Operacional Competitividade e Internacionalização

Inducer of meiosis 2 (Ime2), a protein kinase that has been identified in diverse fungal species, functions in the regulation of various cellular processes, such as ascospore formation, pseudohyphal growth, and sexual reproduction. In this study, AoIme2, an ortholog of Saccharomyces cerevisiae Ime2, was characterized in the nematode-trapping fungus Arthrobotrys oligospora. Disruption of the gene Aoime2 caused defective growth, with slower mycelial growth in ΔAoime2 mutants than the wild type (WT) strain, and in the mutants, the number of hyphal septa in mycelia was higher and the number of cell nuclei in mycelia and conidia was considerably lower than in the WT strain. The conidial yields of the ΔAoime2 mutants were decreased by ∼33% relative to the WT strain, and the transcription of several sporulation-related genes, including abaA, fluG, rodA, aspB, velB, and vosA, was markedly downregulated during the conidiation stage. The ΔAoime2 mutants were highly sensitive to the osmotic stressors NaCl and sorbitol, and the cell wall of partial hyphae in the mutants was deformed. Further examination revealed that the cell wall of the traps produced by ΔAoime2 mutants became loose, and that the electron-dense bodies in trap cells were also few than in the WT strain. Moreover, Aoime2 disruption caused a reduction in trap formation and serine-protease production, and most hyphal traps produced by ΔAoime2 mutants did not form an intact hyphal loop; consequently, substantially fewer nematodes were captured by the mutants than by the WT strain. In summary, an Ime2-MAPK is identified here for the first time from a nematode-trapping fungus, and the kinase is shown to be involved in the regulation of mycelial growth and development, conidiation, osmolarity, and pathogenicity in A. oligospora.

The wheat head blight fungus Fusarium graminearum has two highly similar beta-tubulin genes with overlapping functions during vegetative growth but only TUB1 is important for sexual reproduction. To better understand their functional divergence during ascosporogenesis, in this study we characterized the sequence elements important for stage-specific functions of TUB1. Deletion of TUB1 blocked the late but not initial stages of perithecium formation. Perithecia formed by tub1 mutant had limited ascogenous hyphae and failed to develop asci. Silencing of TUB1 by MSUD also resulted in defects in ascospore formation. Interestingly, the 3'-UTR of TUB1 was dispensable for growth but essential for its function during sexual reproduction. RIP mutations that specifically affected Tub1 functions during sexual reproduction also were identified in two ascospore progeny. Furthermore, site-directed mutagenesis showed that whereas the non-editable mutations at three A-to-I RNA editing sites had no effects, the N347D (not T362D or I368V) edited mutation affected ascospore development. In addition, the F167Y, but not E198K or F200Y, mutation in TUB1 conferred tolerance to carbendazim and caused a minor defect in sexual reproduction. Taken together, our data indicate TUB1 plays an essential role in ascosporogenesis and sexual-specific functions of TUB1 require stage-specific RNA processing and Tub1 expression.

In the course of two independent studies three yeasts have been isolated from temperate deciduous trees in Hungary and Germany. Analyses of nucleotide sequences of D1/D2 domains of the 26S rRNA gene (LSU) suggested that these strains belong to the Meyerozyma clade in Debaryomycetaceae (Saccharomycetales). The phylogenetic analysis of a concatenated alignment of the ITS region and LSU gene sequences confirmed the placement of the three strains in the Meyerozyma clade close to Candida elateridarum. If mixed in proper combinations, the strains formed one to two hat shaped ascospores in deliquescent asci. In addition to the ascospore formation, the three studied strains differed from Candida elateridarum and other members of the Meyerozyma clade in terms of ribosomal gene sequence and some physiological properties. To accommodate the above-noted strains, we describe the new species as Meyerozyma amylolytica sp. nov. (holotype: DSM 27310T; ex-type cultures: NCAIM Y.02140T=MUCL 56454T, allotype: NCAIM Y.01955A; ex-allotype culture: DSM 27468), MB 821663. Additionally, we propose the transfer of five non-ascosporic members of the Meyerozyma clade to the genus Meyerozyma as the following new taxonomic combinations Meyerozyma athensensis f.a., comb. nov. (MB 821664), Meyerozyma carpophila f.a., comb. nov. (MB 821665), Meyerozyma elateridarum f.a., comb. nov. (MB 821666), Meyerozyma neustonensis f.a., comb. nov. (MB 821667), and Meyerozyma smithsonii f.a., comb. nov. (MB 821668).

Age-based inheritance of centrosomes in eukaryotic cells is associated with faithful chromosome distribution in asymmetric cell divisions. During Saccharomyces cerevisiae ascospore formation, such an inheritance mechanism targets the yeast centrosome equivalents, the spindle pole bodies (SPBs) at meiosis II onset. Decreased nutrient availability causes initiation of spore formation at only the younger SPBs and their associated genomes. This mechanism ensures encapsulation of nonsister genomes, which preserves genetic diversity and provides a fitness advantage at the population level. Here, by usage of an enhanced system for sporulation-induced protein depletion, we demonstrate that the core mitotic exit network (MEN) is involved in age-based SPB selection. Moreover, efficient genome inheritance requires Dbf2/20-Mob1 during a late step in spore maturation. We provide evidence that the meiotic functions of the MEN are more complex than previously thought. In contrast to mitosis, completion of the meiotic divisions does not strictly rely on the MEN whereas its activity is required at different time points during spore development. This is reminiscent of vegetative MEN functions in spindle polarity establishment, mitotic exit, and cytokinesis. In summary, our investigation contributes to the understanding of age-based SPB inheritance during sporulation of S. cerevisiae and provides general insights on network plasticity in the context of a specialized developmental program. Moreover, the improved system for a developmental-specific tool to induce protein depletion will be useful in other biological contexts.

Ascospores are the primary inoculum in Fusarium graminearum. Interestingly, 70 of its genes have premature stop codons (PSC) and require A-to-I editing during sexual reproduction to encode full-length proteins, including the ortholog of yeast Ama1, a meiosis-specific activator of APC/C. In this study, we characterized the function of FgAMA1 and its PSC editing. FgAMA1 was specifically expressed during sexual reproduction. The Fgama1 mutant was normal in growth and perithecium formation but defective in ascospogenesis. Instead of forming four-celled, uninucleate ascospores, Fgama1 mutant produced oval, single-celled, binucleated ascospores by selfing. Some mutant ascospores began to bud and underwent additional mitosis inside asci. Expression of the wild-type or edited FgAMA1 but not the uneditable allele complemented Fgama1. In the Fgama1 x mat-1-1 outcross, over 60% of the asci had eight Fgama1 or intermediate (elongated but single-celled) ascospores, suggesting efficient meiotic silencing of unpaired FgAMA1. Deletion of FgPAL1, one of the genes upregulated in Fgama1 also resulted in defects in ascospore morphology and budding. Overall, our results showed that FgAMA1 is dispensable for meiosis but important for ascospore formation and discharge. In F. graminearum, whereas some of its targets are functional during meiosis, FgAma1 may target other proteins that function after spore delimitation.

International audience; Peroxisomes are versatile and dynamic organelles that are required for the development of diverse eukaryotic organisms. We demonstrated previously that in the fungus Podospora anserina different peroxisomal functions are required at distinct stages of sexual development, including the initiation and progression of meiocyte (ascus) development and the differentiation and germination of sexual spores (ascospores). Peroxisome assembly during these processes relies on the differential activity of the protein machinery that drives the import of proteins into the organelle, indicating a complex developmental regulation of peroxisome formation and activity. Here we demonstrate that peroxisome dynamics is also highly regulated during development. We show that peroxisomes in P. anserina are highly dynamic and respond to metabolic and environmental cues by undergoing changes in size, morphology and number. In addition, peroxisomes of vegetative and sexual cell types are structurally different. During sexual development peroxisome number increases at two stages: at early ascus differentiation and during ascospore formation. These processes are accompanied by changes in peroxisome structure and distribution, which include a cell-polarized concentration of peroxisomes at the beginning of ascus development, as well as a morphological transition from predominantly spherical to elongated shapes at the end of the first meiotic division. Further, the mostly tubular peroxisomes present from second meiotic division to early ascospore formation again become rounded during ascospore differentiation. Ultimately the number of peroxisomes dramatically decreases upon ascospore maturation. Our results reveal a precise regulation of peroxisome dynamics during sexual development and suggest that peroxisome constitution and function during development is defined by the coordinated regulation of the proteins that control peroxisome assembly and dynamics.

In eukaryotes, the retromer is an endosome-localized complex involved in protein retrograde transport. However, the role of such intracellular trafficking events in pathogenic fungal development and pathogenicity remains unclear. The role of the retromer complex in Fusarium graminearum was investigated using cell biological and genetic methods. We observed the retromer core component FgVps35 (Vacuolar Protein Sorting 35) in the cytoplasm as fast-moving puncta. FgVps35-GFP co-localized with both early and late endosomes, and associated with the trans-Golgi network (TGN), suggesting that FgVps35 functions at the donor endosome membrane to mediate TGN trafficking. Disruption of microtubules with nocodazole significantly restricted the transportation of FgVps35-GFP and resulted in severe germination and growth defects. Mutation of FgVPS35 not only mimicked growth defects induced by pharmacological treatment, but also affected conidiation, ascospore formation and pathogenicity. Using yeast two-hybrid assays, we determined the interactions among FgVps35, FgVps26, FgVps29, FgVps17 and FgVps5 which are analogous to the yeast retromer complex components. Deletion of any one of these genes resulted in similar phenotypic defects to those of the ΔFgvps35 mutant and disrupted the stability of the complex. Overall, our results provide the first clear evidence of linkage between the retrograde transport mediated by the retromer complex and virulence in F. graminearum.

Members of Cdc14 phosphatases are common in animals and fungi, but absent in plants. Although its orthologs are conserved in plant pathogenic fungi, their functions during infection are not clear. In this study, we showed that the CDC14 ortholog is important for pathogenesis and morphogenesis in Fusarium graminearum. FgCDC14 is required for normal cell division and septum formation and FgCdc14 possesses phosphatase activity with specificity for a subset of Cdk-type phosphorylation sites. The Fgcdc14 mutant was reduced in growth, conidiation, and ascospore formation. It was defective in ascosporogenesis and pathogenesis. Septation in Fgcdc14 was reduced and hyphal compartments contained multiple nuclei, indicating defects in the coordination between nuclear division and cytokinesis. Interestingly, foot cells of mutant conidia often differentiated into conidiogenous cells, resulting in the production of inter-connected conidia. In the interphase, FgCdc14-GFP localized to the nucleus and spindle-pole-body. Taken together, our results indicate that Cdc14 phosphatase functions in cell division and septum formation in F. graminearum, likely by counteracting Cdk phosphorylation, and is required for plant infection.

Aspergillus nidulans StuA and Neurospora crassa ASM-1 are orthologous APSES (ASM-1, PHD1, SOK2, Efg1, StuA) transcription factors conserved across a diverse group of fungi. StuA and ASM-1 have roles in asexual (conidiation) and sexual (ascospore formation) development in both organisms. To address the hypothesis that the last common ancestor of these diverse fungi regulated conidiation with similar genes, asm-1 was introduced into the stuA1 mutant of A. nidulans. Expression of asm-1 complemented defective conidiophore morphology and restored conidia production to wild type levels in stuA1. Expression of asm-1 in the stuA1 strain did not rescue the defect in sexual development. When the conidiation regulator AbaA was tagged at its C-terminus with GFP in A. nidulans, it localized to nuclei in phialides. When expressed in the stuA1 mutant, AbaA::GFP localized to nuclei in conidiophores but no longer was confined to phialides, suggesting that expression of AbaA in specific cell types of the conidiophore was conditioned by StuA. Our data suggest that the function in conidiation of StuA and ASM-1 is conserved and support the view that, despite the great morphological and ontogenic diversity of their condiphores, the last common ancestor of A. nidulans and N. crassa produced an ortholog of StuA that was involved in conidiophore development.

Five closely related yeast strains were isolated from soil in Kharg Island, Persian Gulf, Iran, and from fallen fruits in Galle, Sri Lanka, during separate projects. Morphologically, the strains produced white-coloured yeast colonies, with cells that were ovoid to ellipsoidal, making branched, true hyphae and pseudohyphae. Ascospore formation was not observed. Biochemically, the strains were able to ferment d-glucose and weakly ferment d-galactose. The strains could use a wide variety of carbon sources except methanol and hexadecane. Phylogenetic analyses using combined sequences of the small ribosomal subunit and the D1/D2 domains of the LSU, as well as the internal transcribed spacer regions, suggested that these strains belong to the Wickerhamomyces clade and that together they form one strongly supported phylogenetic clade. Differences in their sequences, biochemistry and morphology suggest they are representatives of distinct species of the genus Wickerhamomyces. Therefore, the name Wickerhamomyces orientalis f.a., sp. nov. is proposed to accommodate these novel strains; the type strain is IBRC-M 30103T (=CBS 13306T). The MycoBank number is MB 807323.

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.

The polyol mannitol is one of the main compatible solutes in Neosartorya fischeri and accumulates in conidia and ascospores. Here, it is shown that biosynthesis of mannitol in N. fischeri mainly depends on mannitol 1-phosphate dehydrogenase (MpdA). Reporter studies and qPCR analysis demonstrated that mpdA is moderately expressed in vegetative hyphae and conidiophores, while it is highly expressed during development of ascospores. Deletion of mpdA reduced mannitol in whole cultures as much as 85% of the wild type, while trehalose levels had increased more than 4-fold. Decreased mannitol accumulation had no effect on mycelial growth irrespective of heat- or oxidative stress. Notably, conidia of the ΔmpdA strain had higher mannitol and lower trehalose levels. They were more sensitive to heat stress. The most distinct phenotype of mpdA deletion was the absence of full development of ascospores. Formation of cleistothecia, and asci was not affected. The ascus cell wall, however, did not dissolve and asci contained incompletely formed or aborted ascospores. Addition of the Mpd inhibitor nitrophenide to the wild type strain also resulted in disturbed ascospore formation. Taken together, these results show that mannitol has a role in sexual development of N. fischeri and in stress resistance of conidia.

Eleven yeast strains representing two hitherto undescribed species were isolated from different kinds of meat samples in Hungary and one from the sediment of a tropical freshwater river in Southeastern Brazil. The analysis of the sequences of their large subunit rRNA gene D1/D2 domain and the internal transcribed spacer (ITS) regions placed the two new species in the Yarrowia clade. Some of the seven strains representing the first new species can mate and give rise to asci and form ascospores embedded in capsular material, which qualifies it as the third teleomorph species of the Yarrowia clade. The name Yarrowia porcina sp. nov. (type strain: NCAIM Y.02100(T) = CBS 12935(T) = NRRL Y-63669(T), allotype strain UFMG-RD131(A) = CBS 12932(A)) is proposed for this new yeast species, which, based on physiological characters, is indistinguishable from Yarrowia lipolytica and some other species of the genus. Considerable intraspecific variability was detected among the sequences of the large subunit rRNA gene D1/D2 domains of the seven strains. The variability among the D1/D2 sequences exceeded the divergence observed among the ITS sequences and in some cases more than 1 % substitution among the D1/D2 sequences was detected. The conspecificity of these strains was supported by the low (0-3 substitutions) sequence divergence among their ITS sequences, the result of a parsimony network analysis utilizing the concatenated ITS and D1/D2 sequences and also by the fingerprint patterns generated by microsatellite primed PCR. No ascospore formation was observed in the group of the other five strains representing the second new species. These strains shared identical D1/D2 and ITS sequences. Yarrowia bubula f.a., sp. nov. (type strain: NCAIM Y.01998(T) = CBS 12934(T) = NRRL Y-63668(T)) is proposed to accommodate these strains.

CYP51 encodes the cytochrome P450 sterol 14α-demethylase, an enzyme essential for sterol biosynthesis and the target of azole fungicides. In Fusarium species, including pathogens of humans and plants, three CYP51 paralogues have been identified with one unique to the genus. Currently, the functions of these three genes and the rationale for their conservation within the genus Fusarium are unknown. Three Fusarium graminearum CYP51s (FgCYP51s) were heterologously expressed in Saccharomyces cerevisiae. Single and double FgCYP51 deletion mutants were generated and the functions of the FgCYP51s were characterized in vitro and in planta. FgCYP51A and FgCYP51B can complement yeast CYP51 function, whereas FgCYP51C cannot. FgCYP51A deletion increases the sensitivity of F. graminearum to the tested azoles. In ΔFgCYP51B and ΔFgCYP51BC mutants, ascospore formation is blocked, and eburicol and two additional 14-methylated sterols accumulate. FgCYP51C deletion reduces virulence on host wheat ears. FgCYP51B encodes the enzyme primarily responsible for sterol 14α-demethylation, and plays an essential role in ascospore formation. FgCYP51A encodes an additional sterol 14α-demethylase, induced on ergosterol depletion and responsible for the intrinsic variation in azole sensitivity. FgCYP51C does not encode a sterol 14α-demethylase, but is required for full virulence on host wheat ears. This is the first example of the functional diversification of a fungal CYP51.

Four yeast strains were isolated from rotting wood samples collected from two sites in the Baotianman Nature Reserve and the Laojieling Nature Reserve in China. DNA sequence comparison and other taxonomic characteristics identified the strains as a single novel species of the genus Metschnikowia. The name Metschnikowia henanensis sp. nov. is proposed to accommodate these highly divergent organisms with the type strain BY-97(T) (= CICC 1982(T) = CBS 12677(T)). The novel species produced chlamydospores, but it did not exhibit ascospore formation in sporulation media for 4 weeks. Molecular phylogeny from the D1/D2 domains of the large subunit (LSU) rRNA gene sequences placed this new species in a basal position to the Metschnikowia viticola/Candida kofuensis/Metschnikowia noctiluminum subclade, and an undescribed Candida species namely strains IMB-EMP4 and IMB-EMP5 was a close sister to M. henanensis.

Three novel D-xylose-fermenting yeast species of Spathaspora clade were recovered from rotting wood in regions of the Atlantic Rainforest ecosystem in Brazil. Differentiation of new species was based on analyses of the gene encoding the D1/D2 sequences of large subunit of rRNA and on 642 conserved, single-copy, orthologous genes from genome sequence assemblies from the newly described species and 15 closely-related Debaryomycetaceae/Metschnikowiaceae species. Spathaspora girioi sp. nov. produced unconjugated asci with a single elongated ascospore with curved ends; ascospore formation was not observed for the other two species. The three novel species ferment D-xylose with different efficiencies. Spathaspora hagerdaliae sp. nov. and Sp. girioi sp. nov. showed xylose reductase (XR) activity strictly dependent on NADPH, whereas Sp. gorwiae sp. nov. had XR activity that used both NADH and NADPH as co-factors. The genes that encode enzymes involved in D-xylose metabolism (XR, xylitol dehydrogenase and xylulokinase) were also identified for these novel species. The type strains are Sp. girioi sp. nov. UFMG-CM-Y302(T) (=CBS 13476), Sp. hagerdaliae f.a., sp. nov. UFMG-CM-Y303(T) (=CBS 13475) and Sp. gorwiae f.a., sp. nov. UFMG-CM-Y312(T) (=CBS 13472).

Diversity of yeasts in association with bees and their food sources has been explored during the last decade. In Thailand, there has been no study of yeast identification in honey and bees. Hence, a total of 186 yeast strains were isolated from 37 honey samples of 12 different bee species. On the basis of morphological and physiological characteristics, 55 representative strains were chosen and identified by sequence analysis of the 26S rDNA D1/D2 domain and the ITS region. The data were compared with the published sequences and the results showed the occurrence of 19 ascomycetous and 1 basidiomycetous yeast species. Six strains of the new species were isolated. Phylogenetic analysis of the 26S rDNA D1/D2 sequence revealed that they were conspecific and most closely related to Zygosaccharomyces mellis. Based on the ITS sequence, the new species was clustered with the type β and clearly distinguished from the type α. Sequence analysis of combined ITS-26S rDNA D1/D2 showed similar results. The occurrence of these two types, with a divergence of more than 1% in their sequences, and low DNA relatedness among them suggested that members of the type β can be regarded as separate species. An analysis of the morphological and physiological characteristics was performed. Ascospore formation was observed on acetate agar and Gorodkowa agar. The new Zygosaccharomyces species differed physiologically from Z. mellis in 4 assimilation tests. This data supports the hypothesis that the new species, Zygosaccharomyces siamensis, is a novel ascosporogenous yeast. The type strain is JCM 16825T (=CBS 12273T) and a description is given here.

Gibberella zeae (anamorph: Fusarium graminearum), a homothallic (self-ferile) ascomycete with ubiquitous geographic distribution, causes serious diseases in several cereal crops. Ascospores (sexual spores) produced by this fungal pathogen have been suggested as the main source of primary inoculum in disease development. Here, we report the function of a gene designated GzRUM1, which is essential for ascospore formation in G. zeae. The deduced product of GzRUM1 showed significant similarities to the human retinoblastoma (tumor suppressor) binding protein 2 and a transcriptional repressor, Rum1 in the corn smut fungus (Ustilago maydis). The transcript of GzRUM1 was detected during the both vegetative and sexual stages, but was more highly accumulated during the latter stage. In addition, no GzRUM1 transcript was detected in a G. zeae strain lacking a mating-type gene (MAT1-2), a master regulator for sexual development in G. zeae. Targeted deletion of GzRUM1 caused no dramatic changes in several traits except ascospore formation. The GzRUM1 strain produced perithecia (sexual fruit bodies) but not asci nor ascospores within them. This specific defect leading to an arrest in ascospore development suggests that GzRUM1, as Rum1 in U. maydis, functions as a transcriptional regulator during sexual reproduction in G. zeae.

Four yeast strains, RS42, SSK10, ST-520 and ST-521, isolated from water in a mangrove forest, bark of a tree and fruit of Ficus sp., respectively, were found to represent a hitherto undescribed anamorphic species. The four strains are related to Candida sinolaborantium in the D1/D2 domain of the large subunit rRNA gene, but differed by eight nucleotide substitutions and two indels, and for this reason are regarded as members of a separate species. Because ascospore formation was not detected, it is described as a new species of Candida, Candida nonsorbophila sp. nov. The type strain is RS42(T) (BCC 25963(T)=NBRC 103860(T)=CBS 10862(T)). This species is distinguished from C. sinolaborantium by the inability to assimilate L-sorbose, L-rhamnose and galactitol, and a higher maximum growth temperature.

Chitin, one of the most important carbohydrates of the fungal cell wall, is synthesized by chitin synthases (CHS). Seven sequences encoding CHSs have been identified in the genome of Neurospora crassa. Previously, CHS-1, -3 and -6 were found at the Spitzenkörper (Spk) core and developing septa. We investigated the functional importance of each CHS in growth and development of N. crassa. The cellular distribution of each CHS tagged with fluorescent proteins and the impact of corresponding gene deletions on vegetative growth and sexual development were compared. CHS-2, -4, -5 and -7 were also found at the core of the Spk and in forming septa in vegetative hyphae. As the septum ring developed, CHS-2-GFP remained at the growing edge of the septum until it localized around the septal pore. In addition, all CHSs were located in cross-walls of conidiophores. A partial co-localization of CHS-1-m and CHS-5-GFP or CHS-2- GFP occurred in the Spk and septa. Analyses of deletion mutants suggested that CHS-6 has a role primarily in hyphal extension and ascospore formation, CHS-5 in aerial hyphae, conidia and ascospore formation, CHS-3 in perithecia development and CHS-7 in all of the aforementioned. We show that chs-7/ csmB fulfills a sexual function and chs-6/chsG fulfills a vegetative growth function in N. crassa but not in Aspergillus nidulans, whereas vice versa chs-2/chsA fulfills a sexual function in A. nidulans but not in N. crassa. This suggests that different classes of CHSs can fulfill distinct developmental functions in various fungi. Immunoprecipitation followed by mass spectrometry of CHS-1-GFP, CHS-4-GFP and CHS-5- GFP identified distinct putative interacting proteins for each CHS. Collectively, our results suggest that there are distinct populations of chitosomes, each carrying specific CHSs, with particular roles during different developmental stages.

Strains of Neurospora crassa exist as two alternative mating type forms, A and a; differences in mating type are required for the initiation of the sexual cycle (Shiu and Glass, 2000). The mating-type (mat) locus also acts as a heterokaryon incompatibility (het) locus, such that hyphal fusion between A and a strains results in a heterokaryon that shows extremely inhibited growth, absence of conidiation, and hyphal compartmentation and death (Glass et al., 2000). The A and a mating type sequences occupy the same locus in A and a strains, but are highly dissimilar in sequence. The mat a-1 gene, which encodes a putative HMG (high mobility group) type of transcriptional regulator, provides all the functions for the a mating type, including mating, ascospore formation, and heterokaryon incompatibility (Chang and Staben, 1994). The mat A locus encodes three proteins. MAT A-2 and MAT A-3 are responsible for ascospore formation (Ferreira et al., 1998); MAT A-3 is a putative HMG type of transcriptional regulator. MAT A-1 is predicted to be a a-domain type of transcriptional regulator and is both necessary and sufficient to confer A mating specificity and trigger heterokaryon incompatibility with a strains (Glass et al., 1990). Mutations in an unlinked locus, tol, suppress mating-type incompatibility such that tol A and tol a strains are capable of forming a vigorous heterokaryon (Newmeyer, 1970; Shiu and Glass, 1999).

The homothallic, filamentous ascomycete Sordaria macrospora is self-fertile and produces sexual fruiting bodies (perithecia) without a mating partner. Even so, S. macrospora transcriptionally expresses two pheromone-precursor genes (ppg1 and ppg2) and two pheromone-receptor genes (pre1 and pre2). The proteins encoded by these genes are similar to alpha-factor-like and a-factor-like pheromones and to G-protein-coupled pheromone receptors of the yeast Saccharomyces cerevisiae. It has been suggested that in S. macrospora, PPG1/PRE2 and PPG2/PRE1 form two cognate pheromone-receptor pairs. To investigate their function, we deleted (delta) pheromone-precursor genes (delta ppg1, delta ppg2) and receptor genes (delta pre1, delta pre2) and generated single- as well as double-knockout strains. No effect on vegetative growth, fruiting-body, and ascospore development was seen in the single pheromone-mutant and receptor-mutant strains, respectively. However, double-knockout strains lacking any compatible pheromone-receptor pair (delta pre2/delta ppg2, delta pre1/delta ppg1) and the double-pheromone mutant (delta ppg1/delta ppg2) displayed a drastically reduced number of perithecia and sexual spores, whereas deletion of both receptor genes (delta pre1/delta pre2) completely eliminated fruiting-body and ascospore formation. The results suggest that pheromones and pheromone receptors are required for optimal sexual reproduction of the homothallic S. macrospora.

Thirty-two strains, many of them isolated from wood-associated habitats, and designated as Kuraishia (Pichia) capsulata and Candida molischiana according to their phenotype, exhibited two types of HaeIII restriction fragment patterns of their small subunit rDNA with the neighboring ITS. One fragment pattern corresponded to that of the type strain of K. capsulata, whereas the other pattern was unique to the typestrain of C. molischiana. Sequencing of the D1/D2 domain of the large subunit rDNA confirmed that the different HaeIII restriction fragment patterns of small subunit rDNA with the neighboring ITS reliably distinguished K. capsulata from C. molischiana. Ascospore formation was observed in several C. molischiana strains and K. molischiana (type strain: NCAIM Y.01725, CBS 9993) is proposed as the teleomorphic state of Candida molischiana.

Three heterothallic, haplontic yeast species,Metschnikowia hamakuensis,Metschnikowia kamakouanaandMetschnikowia mauinuiana, are described from isolates associated with endemic nitidulid beetles living on various endemic plants on three Hawaiian islands. As morphospecies, they are similar toMetschnikowia hawaiiensis, but based on mating compatibility and ascospore formation, they can be assigned clearly to distinct biological species. Analysis of ITS/5·8S and D1/D2 large subunit rDNA sequences shows that, withM. hawaiiensisand two other isolates, these species form a distinct subclade within the large-sporedMetschnikowiaspecies, indicating that they are Hawaiian endemics. Type cultures are:M. hamakuensis, UWOPS 04-207.1T=CBS 10056T=NRRL Y-27834T(type, h+) and UWOPS 04-204.1=CBS 10055=NRRL Y-27833 (allotype, h−);M. kamakouana, UWOPS 04-112.5T=CBS 10058T=NRRL Y-27836T(type, h+) and UWOPS 04-109.1=CBS 10057=NRRL Y-27835 (allotype, h−); andM. mauinuiana, UWOPS 04-190.1T=CBS 10060T=NRRL Y-27838T(type, h+) and UWOPS 04-110.4=CBS 10059=NRRL Y-27837 (allotype, h−).

Nous avons évalué plusieurs fongicides inhibiteurs de stérols lors de programmes de vaporisation en pleine saison ou en post-récolte en vue de l'inhibition de la formation d'ascospores par le Venturia inaequalis, agent pathogène de la tavelure de la pomme. Les traitements post-récolte avec le flusilazole et le diniconazole étaient comparables ou supérieurs à ceux utilisant le benomyl, avec une suppression de la production d'ascospores de 55 à 99 % ; cependant, le bitertanol a stimulé la production d'ascospores jusqu'à 52%. Lorsqu'appliqués neuf fois lors des programmes en pleine saison, le bitertanol, le flusilazole et le triflumizole ont réduit la formation d'ascospores à un niveau similaire ou supérieur à celui obtenu avec le dodine. Les programmes en pleine saison ont été efficaces à réduire la production d'ascospores., Several sterol-inhibiting (SI) fungicides were tested in post-harvest and full-season spray programs for the inhibition of ascospore formation by Venturia inaequalis, the causal fungus of apple scab. Post-harvest treatments with flusilazole and diniconazole were comparable to or better than those with benomyl and suppressed ascospore production by 55 to 90 %, although bitertanol stimulated ascospore production by up to 52 %. When applied nine times in full-season programs, bitertanol, flusilazole, and triflumizole reduced ascospore formation to a degree similar to or greater than that achieved with dodine. Full-season programs with SI fungicides in combination with mancozeb were highly effective for reducing ascospore production.

Kinesins are motor proteins which are classified into 11 different families. We identified 11 kinesin-like proteins in the genome of the filamentous fungus Aspergillus nidulans . Relatedness analyses based on the motor domains grouped them into nine families. In this paper, we characterize KipB as a member of the Kip3 family of microtubule depolymerases. The closest homologues of KipB are Saccharomyces cerevisiae Kip3 and Schizosaccharomyces pombe Klp5 and Klp6, but sequence similarities outside the motor domain are very low. A disruption of kipB demonstrated that it is not essential for vegetative growth. kipB mutant strains were resistant to high concentrations of the microtubule-destabilizing drug benomyl, suggesting that KipB destabilizes microtubules. kipB mutations caused a failure of spindle positioning in the cell, a delay in mitotic progression, an increased number of bent mitotic spindles, and a decrease in the depolymerization of cytoplasmic microtubules during interphase and mitosis. Meiosis and ascospore formation were not affected. Disruption of the kipB gene was synthetically lethal in combination with the temperature-sensitive mitotic kinesin motor mutation bimC4 , suggesting an important but redundant role of KipB in mitosis. KipB localized to cytoplasmic, astral, and mitotic microtubules in a discontinuous pattern, and spots of green fluorescent protein moved along microtubules toward the plus ends.

Selfing in the chestnut blight fungus, Cryphonectria parasitica , occurs by two different genetic mechanisms. Most self-fertile isolates of C. parasitica are heterokaryotic for mating type, and the progeny from selfing segregate for mating type. Further, we resolved mating-type ( MAT ) heterokaryons into homokaryons of both mating types by isolating uninucleate asexual spores (conidia). However, because ascospore progeny, with rare exceptions, are not MAT heterokaryons, C. parasitica must lack a regular mechanism to maintain heterokaryosis by selfing. We hypothesize that heterokaryon formation may occur either because of recurrent biparental inbreeding, or by mating-type switching, possibly one involving some kind of parasexual process. The second mechanism found for selfing in C. parasitica occurred less frequently. Three single-conidial isolates ( MAT-1 and MAT-2 ) selfed and produced progeny that did not segregate for mating type. It is currently not known if meiosis occurs during ascospore formation by this mechanism. Index descriptors: Cryphonectria parasitica , Heterokaryosis, MAT , Mating type, Self-fertility, Self-incompatibility, Self-sterility

Schizosaccharomyces pombe (fission yeast) is essentially a haploid organism. The cylindrical cells grow at the poles and divide symmetrically at a central septum. This chapter reviews the current understanding of the fission yeast mating-type system. Facilitated by the repetitive arrangement of homology boxes at three closely spaced subloci, the mat region of S. pombe is prone to genetic rearrangements, which can yield various types of heterothallic derivatives. The activation of mating-type genes requires profound changes in the transcriptional program in prestationary cells. P-factor is a 23-amino-acid unmodified peptide, made by proteolytic cleavage while a larger precursor protein is transported along the conventional secretory pathway. Pheromone signaling induces transcription of a large number of genes required for mating and meiosis. The nutritional state is being monitored by additional 7TM-protein-coupled receptor components. More recently, the biased guidance of strand invasion has been attributed to a mat-specific protein complex, Swi5-Swi2, assumed to be equivalent to the Swi5-Sfr1 complex required for the homology search in general recombination. The donor cassettes are embedded in a 20-kb stretch of heterochromatin, which is devoid of coding genes other than the internal parts of mat2-M and mat3-P themselves. The critical inducer of meiosis in S. pombe is the RNA-binding Mei2 protein, but the mechanism of Mei2 action is still unclear. The tight coupling to ascospore formation puts meiosis in a critical position as to long-term survival in a dormant state.

During ascospore formation in Saccharomyces cerevisiae , the secretory pathway is reorganized to create new intracellular compartments, termed prospore membranes. Prospore membranes engulf the nuclei produced by the meiotic divisions, giving rise to individual spores. The shape and growth of prospore membranes are constrained by cytoskeletal structures, such as septin proteins, that associate with the membranes. Green fluorescent protein (GFP) fusions to various proteins that associate with septins at the bud neck during vegetative growth as well as to proteins encoded by genes that are transcriptionally induced during sporulation were examined for their cellular localization during prospore membrane growth. We report localizations for over 100 different GFP fusions, including over 30 proteins localized to the prospore membrane compartment. In particular, the screen identified IRC10 as a new component of the leading-edge protein complex (LEP), a ring structure localized to the lip of the prospore membrane. Localization of Irc10 to the leading edge is dependent on SSP1 , but not ADY3 . Loss of IRC10 caused no obvious phenotype, but an ady3 irc10 mutant was completely defective in sporulation and displayed prospore membrane morphologies similar to those of an ssp1 strain. These results reveal the architecture of the LEP and provide insight into the evolution of this membrane-organizing complex.

The 26S proteasome is the multiprotein complex that degrades proteins that have been marked for destruction by the ubiquitin pathway. It is made up of two multisubunit complexes, the 20S catalytic core and the 19S regulatory complex. We describe the isolation and characterization of conditional mutants in the regulatory complex and their use to investigate interactions between different subunits. In addition we have investigated the localization of the 26S proteasome in fission yeast, by immunofluorescence in fixed cells and live cells with the use of a GFP-tagged subunit. Surprisingly, we find that in mitotic cells the 26S proteasome occupies a discrete intracellular compartment, the nuclear periphery. Electron microscopic analysis demonstrates that the complex resides inside the nuclear envelope. During meiosis the localization showed a more dynamic distribution. In meiosis I the proteasome remained around the nuclear periphery. However, during meiosis II there was a dramatic relocalization: initially, the signal occupied the area between the dividing nuclei, but at the end of mitosis the signal dispersed, returning to the nuclear periphery on ascospore formation. This observation implies that the nuclear periphery is a major site of proteolysis in yeast during mitotic growth and raises important questions about the function of the 26S proteasome in protein degradation.

Ascospore formation was observed in the type strain of Candida thermophila Shin K-S, Shin YK, Yoon and Park on some yeast sporulation media. In addition, a further sporulating strain was found that proved to be conspecific with C. thermophila on the basis of sequences of the D1/D2 domain of the large-subunit (26S) rRNA gene and the internal transcribed spacer (ITS)1-5.8S rRNA gene - ITS2 region. Therefore, Ogataea thermophila Péter, Tornai-Lehoczki, Shin K-SDlauchy sp. nov. is proposed as the teleomorph of C. thermophila. The type strain is Y94(T)=JCM 10994(T)=KCCM 50661(T)=KCTC 17233(T).

Ascospore formation of Arthroderma simii was observed using electron microscopy. Using scanning electron microscopy, many round asci were observed inside the gymnothecia obtained by 4 weeks of incubation after mating. Ascospores were produced inside the asci, then discoid-shaped mature ascospores were observed through the degenerate and disrupted ascus cell wall. Disseminated ascospores were observed outside the gymnothecium 7 weeks after inoculation. Using transmission electron microscopy, the enveloping membrane system of two unit membranes invaginated and delimited the cytoplasm. The inner membranes were found to have changed into ascospore plasma membranes, whereas the outer membranes and intercisternal space were changed into ascus cell wall. The subcellular events of ascospore formation were found to be essentially similar to those of other dermatophytes.

Approximately 500 species of ascomycetous yeasts, including members of Candida and other anamorphic genera, were analyzed for extent of divergence in the variable D1/D2 domain of large subunit (26S) ribosomal DNA. Divergence in this domain is generally sufficient to resolve individual species, resulting in the prediction that 55 currently recognized taxa are synonyms of earlier described species. Phylogenetic relationships among the ascomycetous yeasts were analyzed from D1/D2 sequence divergence. For comparison, the phylogeny of selected members of the Saccharomyces clade was determined from 18S rDNA sequences. Species relationships were highly concordant between the D1/D2 and 18S trees when branches were statistically well supported.

The diagnostic feature for members of the subdivision Ascomycotina is that sexual reproduction results in the production of endogenous ascospores within asci. A range of ascus and ascospore types is found within the group. Traditionally, asci have been classified as unitunicate, bitunicate or prototunicate, although a spectrum of intermediate types of asci also occurs. Variations occur in the structure of ascus and ascospore walls, ascus apical structures, septal pore structures, ascospore size and shape, ascospore pigmentation, the number of cell compartments within each ascospore, the number of ascospores per ascus and the arrangement of ascospores within an ascus. In filamentous ascomycetes, asci arise from ascogenous hyphae and this usually takes place asynchronously within an ascoma. Crozier formation is involved in ascus initiation in many, but not all, filamentous species. Asci exhibit determinate growth and usually represent highly specialized hyphal elements. Following ascus extension a simple to complex apical structure is commonly formed in those ascomycetes which discharge their ascospores actively. Ascospore formation involves cytoplasmic compartmentalization by double delimiting membranes between which the ascospore wall develops. Confusion in the literature about the origin of delimiting membranes is resolved by using the endomembrane concept which comprises one membrane-generating system with different levels of membrane transformation between the nuclear envelope and plasma membrane. Spindle pole bodies and microtubules play important roles in the organization of nuclei and the assembly of the delimiting membranes. Major problems with ascospore wall terminology are overcome by using a simple scheme in which wall material is either primary or secondary. Ascospore liberation from mature asci may be either an active or passive process. In actively discharging species, the motive force for discharge commonly arises from an increase in internal hydrostatic pressure arising from water uptake. To date most studies on ascus and ascospore morphogenesis have been descriptive involving light microscopy and/or electron microscopy. However, much useful information has also been obtained from mutant analysis and significant progress has recently been made in understanding the role of the mat locus in controlling dikaryon formation. Of significance in future studies will be the increased integration of traditional approaches with newer cell biological techniques to study ascus and ascospore morphogenesis.

The teleomorph of Candida kunwiensis Hong, Bae, Herzberg, Titze, Lachance, Metschnikowia kunwiensis, is described. Repeated attempts to obtain ascospore formation succeeded using modified V8 sporulation media and extended incubation times. The asci are ovoid, with only a small protrusion caused by the spore(s). The species is diplontic, possibly homothallic, with one or two ascospores per ascus. Aside from having atypical ovoid asci, the acicular shape of the spores is characteristic of the genus Metschnikowia. The type strain is CBS 9676T.

Neurospora crassa and related ascomycetes such as Podospora anserina exist in two mating types, encoded in a unique region of one chromosome. Classical genetic analysis outlined the nature of the questions and provided important materials for further work. In the mating type region, there is little DNA sequence resemblance between the two mating types. They are, therefore, called idiomorphs rather than alleles. There are no silent copies of these sequences in the genome, so mating type switching is impossible. Cloning, sequence analysis, and complementation studies involving these idiomorphs has begun to shed light on their function. One of the idiomorphs contains three reading frames; one is essential for fertilization and fruiting body formation and the other two are involved in post-fertilization functions including ascus and ascospore formation. In various species of the genus Neurospora, the centromere-proximal flank of the idiomorphs is highly variable in DNA sequence among species, and in some cases, between mating types. The similarities and differences in these flanking sequences allow some conclusions to be drawn about the possible phylogenetic relationship of these species. Key words: Neurospora, ascomycetes, mating, evolution, compatibility, HMG proteins.