Genetische Untersuchungen zur Fruchtkörper- und Artbildung bei Basidiomyceten: Genetische Kontrolle der Fruchtkörperbildung beiPolyporus ciliatus

Using mainly the basidiomycetePolyporus ciliatus, we were able to produce fully developed fruitbodies not only in the normal meiotic cycle (teleomorphic = perfect) but in some stocks also in the mitotic cycle (anamorphic = imperfect) of exclusively haploid mycelia. These haploid and mitotic fruitbodies can be distinguished from dikaryotic ones only by microscopic examination, i.e. by their lack of clamp connections, karyogamy and meiosis, the production of two-spored basidia with few, but larger basidiospores. Using three other stocks of isogenic mycelial strains that did not form any fruiting structures from either haploid or dikaryotic mycelia with clamp connections, we could not find any evidence for a “fruiting initiation gene” as was postulated byEsser & al. (1977). No fruitbodies occurred in compatible combinations within one isogenic stock, but normal fruitbodies are produced abundantly in crossings of different isogenic stocks. We were able to confirm these results with the help of the non-fruiting strains originally used byStahl & Esser (1976).—Our experimental results are compatible with the hypothesis suggested byKniep (1930): The genetic control of the sexual cycle by the mating type factors A and B on the one hand, and of the fruitbody formation on the other, are essentially independent. For the latter, a multitude of different genes has been proposed. Fruitbody formation thus is a typical polygenic character. The results of experiments on two other morphological characters i.e. primordia and coralloid structures, also correspond with this genetic concept.     

长根小奥德蘑双孢菌株与四孢菌株核相变化的比较

用双苯并咪唑（Hoechst 33258）染色法分别对长根小奥德蘑Oudemansiella radicata双孢菌株和四孢菌株的菌丝、子实体、担孢子进行染色观察,结果表明：双孢长根小奥德蘑菌丝细胞多为单核,无锁状联合;原担子中单核进行一次有丝分裂形成两个横向或纵向排列的子核,这2个子核分别进入2个担孢子中,留下无核的空担子;成熟担孢子具有一个核。四孢长根小奥德蘑菌丝细胞大多数为双核,具有锁状联合;进入原担子中的两个单倍性细胞核先发生核配,形成一个二倍性的核,再经过减数分裂形成四个染色体减半的单倍性子核,     

Nuclear fusion,meiosis and the origin of dikaryotic hyphae in Armillariella mellea

The behaviour of nuclei during the growth and differentiation of basidiocarp primordia of Armillariella mellea (Vahl) Karst. is described. The primordial initials which arose from monokaryotic rhizomorphs were also monokaryotic. In older primordia, at the site of initiation of gill folds, multinucleate cells formed at the tips of monokaryotic hyphae and gave rise to the dikaryotic hyphae bearing clamp connections. These formed the gills of the older primordia. Cytological studies suggested that the nuclei in monokaryotic cells were diploid. In young basidial primordia haploidization occurred in the cells which were to become multinucleate prior to giving rise to dikaryotic hyphae of the gills. In mature basidia after nuclear fusion and meiosis had occurred, each of the four haploid daughter nucleic migrated into a basidiospore and then divided mitotically. One of the mitotic daughter nucleic migrated from each spore back into the basidium so that mature spores were uninucleate.Abbreviations M.T.O.C. microtubule organizing centre     

THE BASIDIA OF EXIDIA NUCLEATA. II. DEVELOPMENT

The basidia of Exidia nucleate, were studied by light and electron microscopy. The basidium arises from a basal clamp-connection as a subcylindrical, dikaryotic structure but soon becomes sphaeropedunculate. Mitochondria, endoplasmic reticulum, and compact lamellar systems are more abundant and more evenly dispersed in diploid probasidia than in dikaryotic probasidia, whereas vacuoles in the apical regions are larger in dikaryotic probasidia. Oil globules are few or absent in the earlier stages but increase in size and number throughout basidial development. Karyogamy results in a diploid nucleus approximately twice the volume of haploid nuclei. Although indications were noted of reorganization of the nuclear envelope during the terminal stages of reduction division, available evidence does not suggest a prolonged loss of the nuclear envelope during division. Internal wall formation is centripetal, at least in those stages observed. In areas of active wall formation, a conspicuous zone devoid of mitochondria, oil globules, and endoplasmic reticulum was noted consistently. The circumscribing wall of a hypobasidial segment is composed of 2 lamellae of contrasting electron-density and is separated from the walls of adjacent hypobasidial segments and the wall delimiting the enucleate stalk by a median electron-transparent lamella. Although extensive vacuolation occurs in the hypobasidial segments as the nuclei and most of the cytoplasm pass through the epibasidia into the basidiospores, evidence of active nuclear migration was observed. Each basidiospore contains a nucleus, mitochondria, endoplasmic reticulum, and numerous oil globules.     

CYTOLOGICAL STUDIES IN LACCARIA (AGARICALES). II. ASSESSING PHYLOGENETIC RELATIONSHIPS AMONG LACCARIA,HYDNANGIUM, AND OTHER AGARICALES

Laccaria and Hydnangium typically undergo a postmeiotic mitotic nuclear division in basidiospores, basidia, or less commonly, in sterigmata resulting in eight nuclei. Basidiospores formed on tetrasterigmate basidia are binucleate, and those on bisterigmate basidia are tetranucleate in both genera. While Hydnangium and Laccaria likely comprise a monophyletic group—a hypothesis based on a number of shared characters—the presence of multinucleate basidiospores in several putative outgroups precludes using this shared character state as a justification for recognizing these two genera in a separate family, Hydnangiaceae, as had been proposed. Based on a review of the literature, the presence and location of postmeiotic mitotic divisions in basidia and basidiospores of Agaricales do not appear to be appropriate for assessing phylogenetic relationships among genera or families because of a high level of homoplasy.     

NUCLEAR BEHAVIOR IN THE BASIDIOMYCETE,VOLVARIELLA VOLVACEA

The basidiospores of the straw mushroom are typically uninucleate and its vegetative hyphae are generally multinucleate. There is a marked reduction of nuclear number in the trama and subhymenium. Interphase nuclei exist in two forms, each of which undertakes a particular mode of division. The “diffused” nuclei divide by conventional mitosis while the “constricted” ones divide amitotically. In metaphase of mitosis nine chromosomes were seen both in polar and lateral view. This haploid number confirms the nine bivalents found in basidia during meiosis. A unique characteristic of this fungus is that the diploid nucleus, the two postkaryotic nuclei and the four postkaryotic nuclei may be enclosed by a well-defined nuclear envelope during division.     

Stereum sanguinolentum: Is it an amphithallic basidiomycete?

Monobasidiospore isolates were prepared from basidiocarps of Stereum sanguinolentum. Five isolates per basidiome were paired with each other and with isolates from the trama. Interbasidiome pairings of the trama isolates and of a selection of single-spore isolates also were performed. Thin sections of the hymenium were stained with DAPI and examined by fluorescence microscopy to study the nuclei in the basidia. Spore prints were stained with DAPI to count the number of nuclei per spore. SEM was used to determine the number of basidiospores per basidium. All intrabasidiome pairings were compatible. In contrast, interbasidiome pairings, except one, were incompatible, independent of whether single-spore or trama isolates were paired. Fertile basidiomes were formed in single-basidiospore cultures. Basidia were regularly four-spored. On average, 5% of the basidiospores possessed one nucleus, 82% two, 2% three and 1% four nuclei. Ten percent of the spores appeared to be empty. Karyogamy, meiosis and postmeiotic mitosis were observed in the basidia. Nuclei resulting directly from meiosis, i.e., without having undergone postmeiotic mitosis, sometimes were observed in the sterigmata or spore primordia. The high number of vegetative compatibility groups (VCG) of S. sanguinolentum observed in this study and earlier studies is difficult to explain without sexual or parasexual recombination. We suppose that the majority of spores with ≥2 nuclei are amphithallic, possessing at least one nucleus of each mating type. Recombination could occur by exchange of nuclei among VCGs via anastomoses between homothallic compartments. Transfer of nuclei from heterothallic to homothallic mycelia or matings between homothallic mycelia, which originate from monokaryotic spores, might be other paths for gene exchange.     

Alternation of nuclear phase in the filamentous basidiomycete,Helicobasidium mompa

Variation in the number of nuclei and cellular ploidy were observed in eight strains ofHelicobasidium mompa. The basidiospores, single-spore isolates and field-isolated strains were all dikaryons. The cellular ploidy, which was assessed by analyzing the fluorescence emitted by DAPI-stained nuclei, was unstable: monokaryotic strains derived from the original dikaryotic strains by successive subcultures were mainly tetraploid, although the original dikaryon was in most cases diploid. On the other hand, a dikaryotic strain derived by treatment with benomyl was haploid. These results suggest that diploid dikaryon is a normal nuclear phase ofH. mompa in nature, and the alternation of ploidy may be due to a feature of the mating system of this fungus.     

糙皮侧耳染色体计数和担孢子形成过程中细胞核行为的研究

糙皮侧耳(Pleurotus ostreatus)菌褶经有丝分裂阻断剂预处理、原生质体铺片和Gie-msa 染色并结合苏木精染色后,经过多次反复实验观察,证明糙皮侧耳的染色体条数为 9(n=9);糙皮侧耳从菌褶分化完成到子实体完全成熟的过程中,不断有少量新的双核担子产生,发生核配直到释放担孢子。其减数分裂同步性不高。减数分裂后,4个子核分别进入4个担孢子中,留下中空的担子。     

Spore formation and karyological characterization of basidiosporogenesis,meiosis, post-meiotic and nuclear migration mitosis in Coprinus comatus

In Coprinus comatus, we sampled and observed different maturity stages of basidium to find the possible causes of binucleate basidiospores by microscopy using the HCI-Giemsa and DAPI staining methods. In basidium, following karyogamy, a single nucleolus was observed. We found evidence suggesting post-meiotic mitosis following nuclear migration in the spores. Post-meiotic mitosis occurred in the basidiospores, resulting in four binucleate basidiospores. This indicates that the basidiospore is a monokaryon, and that C. comatus shows the same pattern of basidiospore formation as Coprinopsis cinerea. This type of nuclear behavior was defined as pattern D, one of six distinct patterns (pattern A–F) of nuclear behavior during basidiosporogenesis have been described for basidiomycetes by Campos and Costa in 2010.     

草菇减数分裂和担孢子形成细胞核行为

本文用苏木精染色和双苯并咪唑(Hoechst 33258)染色法,从草菇子实体“纽期”菌褶分化完开始,每3小时对同一个子实体连续切取菌褶进行染色观察。结果表明草菇子实体“纽期”菌褶形成时,约10％的担子发生了核配;在子实体发育过程中,尤其是子实体成熟期后,不断有少量新的双核担子产生,并发生核配,使草菇减数分裂的同步性不高;草菇从菌褶分化完成(此时已有10％担子发生核配)到子实体完全成熟,菌褶变成深粉红至褐色(此时约70％担子完成减数分裂)需要28—30小时;担子减数分裂的持续时间为18小时,其中细线期和偶线期5.9小时、粗线期6.2小时、双线期和终变期3.4小时、中期10.5小时、后期Ⅰ到四分体2小时;经过对粗线期、双线和终变期以及中期Ⅰ染色体条数的多次反复观察,认为草菇的染色体条数为11(n=11);减数分裂后,4个子核分别进入4个担孢子中,留下无核的担子;绝大部分担孢子是单核的,有约5％的担孢子是双核的。     

Mal3, the Schizosaccharomyces pombe homolog of EB1, is required for karyogamy and for promoting oscillatory nuclear movement during meiosis

Two successive rounds of chromosome segregation following a single round of DNA replication enable the production of haploid gametes during meiosis. In the fission yeast Schizosaccharomyces pombe, karyogamy is the process where the nuclei from 2 haploid cells fuse to create a diploid nucleus, which then undergoes meiosis to produce 4 haploid spores. By screening a collection of S. pombe deletion strains, we found that the deletion of 2 genes, mal3 and mto1, leads to the production of asci containing up to 8 spores. Here, we show that Mal3, the fission yeast member of the EB1 family of conserved microtubule plus-end tracking proteins, is required for karyogamy, oscillatory nuclear movement, and proper segregation of chromosomes during meiosis. In the absence of Mal3, meiosis frequently initiates before the completion of karyogamy, thus producing up to 8 nuclei in a single ascus. Our results provide new evidence that fission yeast can initiate meiosis prior to completing karyogamy.     

Bulleromyces genus novum (Tremellales), a teleomorph for Bullera alba,and the occurrence of mating in Bullera variabilis

Mating is observed in Bullera alba and B. variabilis, resulting in the formation of dikaryotic mycelium with clamps, haustorial branches, and lateral and terminal dikaryotic, clavate, lageniform or subglobose cells. These cells develop in B. alba into tremellaceous phragmobasidia. Karyogamy has been observed in young non-divided basidia. Germination of the phragmobasidia occurred by acropetal chains of yeast cells, ballistospores or hyphae. Septal pores are dolipores with parenthesomes made up of U-shaped vesicles (Tremellales type). For the teleomorph of B. alba a new genus, Bulleromyces, is proposed, with only one species, viz. Bulleromyces albus.     

Metamorphosis of the Basidiomycota Ustilago maydis: Transformation of yeast-like cells into basidiocarps

Ustilago maydis (DC) Cda., a phytopathogenic Basidiomycota, is the causal agent of corn smut. During its life cycle U. maydis alternates between a yeast-like, haploid nonpathogenic stage, and a filamentous, dikaryotic pathogenic form that invades the plant and induces tumor formation. As all the members of the Subphylum Ustilaginomycotina, U. maydis is unable to form basidiocarps, instead it produces teliospores within the tumors that germinate forming a septate basidium (phragmobasidium). We have now established conditions allowing a completely different developmental program of U. maydis when grown on solid medium containing auxins in dual cultures with maize embryogenic calli. Under these conditions U. maydis forms large hemi-spheroidal structures with all the morphological and structural characteristics of gastroid-type basidiocarps. These basidiocarps are made of three distinct hyphal layers, the most internal of which (hymenium) contains non-septate basidia (holobasidia) from which four basidiospores develop. In basidiocarps meiosis and genetic recombination occur, and meiotic products (basidiospores) segregate in a Mendelian fashion. These results are evidence of sexual cycle completion of an Ustilaginomycotina in vitro, and the demonstration that, besides its quasi-obligate biotrophic pathogenic mode of life, U. maydis possesses the genetic program to form basidiocarps as occurs in saprophytic Basidiomycota species.     

A DNA Damage Checkpoint Pathway Coordinates the Division of Dikaryotic Cells in the Ink Cap Mushroom Coprinopsis cinerea

The fungal fruiting body or mushroom is a multicellular structure essential for sexual reproduction. It is composed of dikaryotic cells that contain one haploid nucleus from each mating partner sharing the same cytoplasm without undergoing nuclear fusion. In the mushroom, the pileus bears the hymenium, a layer of cells that includes the specialized basidia in which nuclear fusion, meiosis, and sporulation occur. Coprinopsis cinerea is a well-known model fungus used to study developmental processes associated with the formation of the fruiting body. Here we describe that knocking down the expression of Atr1 and Chk1, two kinases shown to be involved in the response to DNA damage in a number of eukaryotic organisms, dramatically impairs the ability to develop fruiting bodies in C. cinerea, as well as other developmental decisions such as sclerotia formation. These developmental defects correlated with the impairment in silenced strains to sustain an appropriated dikaryotic cell cycle. Dikaryotic cells in which chk1 or atr1 genes were silenced displayed a higher level of asynchronous mitosis and as a consequence aberrant cells carrying an unbalanced dose of nuclei. Since fruiting body initiation is dependent on the balanced mating-type regulator doses present in the dikaryon, we believe that the observed developmental defects were a consequence of the impaired cell cycle in the dikaryon. Our results suggest a connection between the DNA damage response cascade, cell cycle regulation, and developmental processes in this fungus.     

The Life Cycle of Cryptochlora perforans (Chlorarachniophyta)

Observations on the behaviour of different life cycle stages, gamete fusions, and measurements of nuclear DNA contents in Cryptochlora perforans resulted in a first concept concerning life histories in Chlorarachniophyta: the life cycle of Cr. perforans is diplohaplontic (gamete fusion with karyogamy - mitosis - meiosis - mitosis). In the haploid as well as in the diploid life cycle phases amoeboid and coccoid stages occur. The isomorphic gametes are modified amoebae frequently without filopodia. Only haploid flagellate stages are known representing mito- or meiozoospores. Diploid coccoid stages have a granular cytoplasmic structure and may be somewhat larger than haploid ones. Nevertheless, positive identification of haploid (gametophytic) and diploid (sporophytic) stages is only possible on the basis of nuclear DNA contents.     

Light microscopy of basidia, basidiospores, and nuclei in spores and hyphae of Filobasidiella neoformans (Cryptococcus neoformans).

Three hypha-forming strains of Cryptococcus neoformans were induced to form basidia and basidiospores. Light microscopy showed that basidia formed at the ends of terminal hyphal cells and were able to produce from a few to many basidiospores. The morphology of the sexual structures indicated that these strains belonged to the recently described perfect state of C. neoformans, Filobasidiella neoformans. The average dimensions of the basidiospores were 1.9 mum in width by 2.7 mum in length. Giemsa staining revealed that dikaryotic cells were formed in all three strains. Only one strain had both terminal and subterminal dikaryons, indicating functional clamp connections, whereas the two remaining strains had dikaryons restricted to the terminal cells. Basidiospores of two strains were mononucleate, and yeast cell clones derived from single basidiospores of these two strains were able to complete the sexual life cycle, thus indicating their primary homothallic nature.     

Two underscribed heterobasidiomycetes from Ontario

Specimens of two heterobasidiomycetous species, collected in Ontario and studied respectively by H. S. Jackson and R. F. Cain, are described as new; they arePlatygloea jacksonii andSigmogloea tremelloidea. P. jacksonii is characterized by small basidia and basidiospores and by sympodially developing conidia.Sigmogloea tremelloidea is an anomalous taxon with cylindric, mostly transversely septate (“auricularioid”) basidia, but some features suggest a possible relationship to the Tremellales.