Nuclear and Non-nuclear Factors influencing Clamp Connection Formation in Coprinus disseminatus

Matings between sister single spore lines of Coprinus disseminatusshowed a cryptic tetrapolar pattern. The two groups of matingsthat resulted in formation of mycelia with clamp connections(apparent dikaryons) differed in rate of nuclear penetrationduring mating. In one group penetration occurred at rates comparablewith nuclear migration in other species and in the other groupit was often extensive but at a rate similar to, or less than,the dikaryon growth-rate. No differences were detected betweenthese two groups in stability, colony extension rate, frequencyof clamp connections, proportions of true clamp connectionsand pseudoclamps, or number of nuclei per hyphal tip cell. Cytological studies and the isolation of hyphal tips showedthat both groups of apparent dikaryons were heterokaryotic di-or trikaryons. The di- and trikaryotic conditions co-existedin the same mycelium, but adjacent cells of individual branchingsystems usually contained equal numbers of nuclei. Within apparentdikaryons the number and kinds of nuclei per cell were similarin hyphae with clamp connections and those with simple septa.Treatments that prevented clamp connection formation did notalter the nuclear status of most of the hyphae. Irregularities in nuclear distribution were infrequent and mostwere associated with pseudoclamps. Forty per cent of nodes withprobable pseudoclamps yielded homokaryotic branches, which wereof either constituent mating type. There was some indicationthat irregularities in nuclear distribution could also occurduring divisions associated with simple septa.     

Griseofulvin-induced alterations in site of dividing nuclei and structure of septa in a dikaryon ofSchizophyllum commune

Summary Ultrastructural study of a dikaryon of the basidiomyceteSchizophyllum commune showed that treatment with griseofulvin affected the site of the dividing nuclei and the location and structure of the septa. The microtubules were considered to be the primary target of griseofulvin, since they participate in nuclear division and movement in the hyphae, and their assembly is known to be in other organisms than fungi inhibited by griseofulvin. It is pointed out that dikaryotic hyphae with two nuclei and a clamp connection per cell are more sensitive indicators of the effect of griseofulvin than homokaryotic hyphae, whose structure is less complex.     

THE SEPTAL PORES OF COPFINUS LAGOPUS IN RELATION TO NUCLEAR MIGRATION

The septal pores of homokaryotic and dikaryotic mycelia of Coprinus lagopus have the complex structure characteristic of certain Basidiomycetes. The edge of the pore is thickened and covered on either side by a dome-shaped, perforated, membranous cap. However, in heterokaryotic mycelia in which nuclear migration was believed to be taking place, in addition to the complex pores, simple pores were also observed as well as various intermediate stages interpreted as steps in the breakdown of complex to simple pores. The simple pores may facilitate nuclear migration.     

Light and electron microscope study of unilateral mating between a secondary mutant and a wild-type strain ofSchizophyllum commune

Summary After hyphal fusions between the secondary mutant and wild-type strains ofSchizophyllum commune some of the fused hyphae show several nuclei per cell and dissolved septa. These hyphae are designated migration hyphae because they are evidently the main routes of nuclear exchange between the two strains. On the wild-type side of the mating the nuclei spread gradually from the main part of the migration hyphae into the side branches, which develop into an extensive network with many anastomoses. The first cells with pseudoclamps or clamp connections are observed in this network.On the mutant side of the mating the branching is less developed and the number of anastomoses is smaller. The cross walls of the septa are poorly dissolved, and nuclear aggregations occur in the hyphae. No development of clamp connections or pseudoclamps is observed. It is suggested that the unilateral mating response of the secondary mutant strain might possibly be caused by the failure of the septa in the mutant hyphae to dissolve, which inhibits the distribution of the nuclei into the side branches of migration hyphae.     

Vegetative nuclear structures and their mode of division inCyathus species

Vegetative nuclear division in the homokaryotic and dikaryotic hyphae ofCyathus olla Brodie,C. setosus Brodie andC. bulleri Brodie was investigated. In the homokaryotic hyphae a nucleolus develops within a globular condensed nucleus consisting of a folded up filament. As the nucleolus increases in size, the nucleus unfolds and can assume a ring, horseshoe or filament configuration. The filament duplicates and (usually when unwound from the nucleolus) divides longitudinally. Occasionally, strand separation occurs while the filament is wrapped in the form of a ring around the nucleolus. The daughter nuclei may condense before the next division. In the dikaryotic hyphae the same nuclear cycle occurs as in the homokaryons except that an extra nuclear condensation to the globular form can occur in both the clamp and tube nuclei. The division of these two nuclei is not always synchronous and, moreover, the stage of karyokinesis of the clamp nucleus is not closely synchronized with the formation of the clamp connection. A deeply stained granule is associated with the nucleus. Some granules can be observed to be connected to the nucleus by a faintly Feulgen positive thread-like structure but other granules are sessile. The granule or centriole-like body is thought to direct the nuclear unfolding process. It may divide prior to, or after nuclear division.     

Intranucleäre Mitosen in homokaryotischen und dikaryotischen Mycelien der Basidiomyceten

Zusammenfassung In homokaryotischen und in dikaryotischen Mycelien der Basidiomyceten wird das Volumen des prämitotischen Kernes dadurch reduziert, daß der eigentliche Teilungskern von einem membranumgebenen Restkern abgetrennt wird. Da die Kernmembran während der gesamten Mitose persistiert, arbeitet der Spindelapparat intranucleär.

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Intranuclear mitosis in homokaryotic and dikaryotic mycelia of basidiomycetes

Summary In homokaryotic as well as in dikaryotic mycelia of Basidiomycetes the volume of the premitotic nucleus is reduced by separating the division nucleus from a residual nucleus which is surrounded by a membrane. For the nuclear membrane persists throughout the mitosis the spindle apparatus acts intranuclear.

     

FINE STRUCTURE OF MYCOTA. 7. OBSERVATIONS ON SEPTA OF ASCOMYCETES AND BASIDIOMYCETES

Moore , R. T., and J. H. Mc Alear . (Cornell U., Ithaca, N. Y.) Fine structure of mycota. 7. Observations on septa of Ascomycetes and Basidiomycetes. Amer. Jour. Bot. 49(1): 86–94. Illus. 1962.—Electron microscopy of 13 carpomycete species (5 Ascomycetes, 5 Basidiomycetes, and 3 Deuteromycetes) presents evidence that suggests there is an Ascomycete- and a Basidiomycete-type septum. The observed Ascomycete septa taper slightly or not at all toward a simple, clear-channel, central pore, while the observed Basidiomycete septa flare sharply and broadly at the center to produce what is termed a dolipore septum. On each side of this septum is a double membrane structure that is crescent-shaped in section. This is given the name parenthesome. These dolipore-parenthesome septa were found to be characteristic of basidiocarpic, dikaryotic, hyphae. Interpretative drawings of both types of septa are presented. From these and the micrographs, it is not difficult to see how materials, including nuclei, could pass through the Ascomycete-type septum, but it would appear that the modifications of the Basidiomycete-type septum while maintaining cytoplasmic continuity would prohibit nuclear migration in dikaryotic hyphae. It is postulated that as the plant and animal phylogenetic lines have developed actual diploidism, the terminal group of the fungi may have achieved functional diploidism. Of the 3 species of Deuteromycetes examined, 2, Aspergillus variecolor and Stilbum zacalloxanthum, have typical Ascomycete-type septa; the third, QM 7739, has Basidiomycete-type septa. This fungus is a species of Sclerotium, but the present confusion and lack of definitive information regarding the taxonomy, genetics, and perfect stage of this genus prohibit the enlarging at present on the possible significance of this observation.     

Research on basal stem rot (BSR) of ornamental palms caused by basidiospores from <Emphasis Type="Italic">Ganoderma boninense</Emphasis>

Basidiospores were isolated from the fruiting bodies of Ganoderma infecting oil palms from an estate in Johor and from ornamental palms (including oil palms) from Singapore. The spores were then germinated to obtain homokaryotic mycelia. Based on clamp connection formation in paired hyphal fusions, tester strains were identified from the homokaryons isolated. Compatibility tests were then carried out using these testers to determine the relatedness of the homokaryotic Ganoderma isolates, both from Johor and from Singapore. Results from the compatibility tests showed that Ganoderma from both locations belong to the same species, while the Ganoderma isolates from Singapore share some common alleles. The pathogenicity tests carried out on Chrysalidocarpus lutescens seedlings using inoculum growing on rubber wood blocks showed that dikaryotic mycelia can cause basal stem rot infection.     

Origin and ultrastructure of complex septa in Schizophyllum commune development

Summary The mode of origin of cross-walls in living cells of S. commune was studied in basidiospore germinants, vegetative homokaryotic mycelium and the dikaryon of this mushroom. The de novo origin of septa in basidiospore germinants always involved annular ingrowth of cross-walls. The restriction of intercalary growth was observed in the transition from a two-celled hypha to a three-celled unit. Primary branch formation occurred in nonseptate germinants, subterminal cells of the young hypha and in the hyphal apex. Septum formation in purely vegetative homokaryotic mycelium as well as the main cross-wall of the dikaryon was also by annular ingrowth. Ultrastructure studies of the septal pore apparatus revealed no differences in those of germinants, wild-type or morphologically aberrant unilateral diploidizing strains of vegetative mycelium or in the clamp connection of the dikaryon. Simple septa of two general types prevailed in the common-A heterokaryon of S. commune.     

Expression of Proteins and Glycoproteins Encoded by the Haploid Nuclei in the Dikaryotic State in the Basidiomycete Agrocybe aegerita

The total proteins and concanavalin A-binding glycoproteins of the cultivated mushroom Agrocybe aegerita were studied in homokaryotic siblings and in dikaryotic strains. The glycoproteins exhibited considerable variability compared with the proteins; the genetic diversity detected in homokaryons in the glycoprotein analysis was 30-fold higher than the genetic diversity revealed by protein analysis, and the glycoprotein patterns could be used to characterize individual genotypes. We found that the expression of glycoproteins in haploid nuclei was significantly asymmetric when the nuclei were paired in dikaryons. The expression levels of the two component nuclei depended on their genotypes, and each haploid nucleus was characterized by its level of expression. Furthermore, some specific glycoproteins that were not detected in all of the homokaryons were newly synthesized in the dikaryotic strains. Among these was a glycoprotein designated gpAa-65, which was identified in all of the dikaryotic strains and appeared to be a good molecular marker of the dikaryotic state.     

Direct studies of nuclear movements in Schizophyllum commune

Summary Measurements of nuclear positions in apical cells of homokaryotic mycelia and dikaryotic mycelium of Schizophyllum commune showed that nuclei occupied a near central position in most cases. Forward nuclear movements observed in living hyphal apices occurred at rates within the range of hyphal growth and could account for the maintenance of centrally located nuclei. Opposed nuclear movements followed mitosis and greatly exceeded the rate of hyphal growth. Septum disruption and rapid nuclear movements characterized an A _xB_mut homokaryon. Neither cytoplasmic streaming nor actively participating granules or filaments could account for any of these nuclear movements.     

Anatomy and phylogenetic significance of Eoconularia loculata, a conulariid from the Silurian of Gotland

The type material of Eoconularia loculata (Wiman, 1895), a conulariid with high, bifurcated septa, was originally found in an erratic boulder, hence the source-rock is as yet unidentified. Recently, a rich material of the species has been discovered at eleven localities in the Silurian Hemse Beds of Gotland Another two localities in the Slite Beds (Wenlockan, Sheinwoodian) revealed what is assumed to be the ancestor of E loculata. This ancestral form probably constitutes a separate taxon distinguished from E. loculata in having simple, unbranched septa. E. loculata is re-described, and four ontogenetic stages in septal development are recognized. During stage 1, the most juvenile stage, the septa are simple. The septa in stages 3 and 4, the adult stages, are coarse and bifurcated. The affinities of conulariids are discussed, with the conclusion that the group shares a number of similarities with modem scyphomans. The microstructures of the exoskeleton show several similarities with coronatids, and the septa are interpreted to be homologous with the internal structures of stauromedusids. The stratigraphical distribution of all currently known septate conulariids suggests that septa were a primitive morphologic feature ranging from early? Ordovician to late Silurian. The simplest type, however, persisted at least into the early Permian. Five of the eleven described septa types have been found only among the conulariids from Gotland. □Conulariida, EOCONULARIA LOCULATA, bifurcated septa, taxonomy, ecology, morphology, Scyphozoa, Sweden, Gotland, Silurian.     

Fruiting Body Formation from Regenerated Mycelium of Pleurotus ostreatus Protoplasts

Fruiting bodies were induced from mycelium regenerated from Pleurotus ostreatus protoplasts. Mycelia originated from protoplasts conformed to parental strain mycelia in morphology. Six strains selected at random from the dikaryotic regenerants were able to form normal fruiting bodies, yielding 18% more than the parent.     

Dikaryotic fruiting body development in a single dikaryon of <Emphasis Type="Italic">Agrocybe aegerita</Emphasis> and the spectrum of monokaryotic fruiting types in its monokaryotic progeny

Using monokaryotic offspring from several dikaryotic parental strains, the phenomenon of monokaryotic fruiting has been previously analysed in the commercially cultivated high-quality edible mushroom Agrocybe aegerita, revealing a variety of monokaryotic fruiting types. Here, we report a single dikaryotic A. aegerita strain, A. aegerita AAE-3, and 40 monokaryons derived from it, which exhibit a wide spectrum of monokaryotic fruiting types, including a rare, previously unknown type. Advantageously, the selected parental strain A. aegerita AAE-3 completes its life cycle within three weeks by the formation of dikaryotic fruiting bodies of typical agaric morphology on malt extract agar plates. In order to morphologically compare normal dikaryotic fruiting to monokaryotic fruiting, histology was performed from all dikaryotic fruiting body development stages and all fruiting types of monokaryotic origin. No clamp connections or dikaryotic hyphae were observed within the plectenchyma of monokaryotic fruiting stages. Among the monokaryotic fruiting types of the A. aegerita AAE-3-derived monokaryons, we also characterised the rare ‘stipe type’ here described as ‘elongated initials type’ as no differentiation into a future cap and stipe was seen. The two mating-compatible monokaryotic strains representing the extremes of the fruiting type spectrum observed, A. aegerita AAE-3-13 (‘mycelium type’) and A. aegerita AAE-3-32 (‘abortive?+?true homokaryotic fruiting fruiter type, AHF?+?THF fruiter type’), were also found to readily produce oidia (arthrospores). In order to obtain a set of mating-compatible monokaryons covering the whole observed spectrum of monokaryotic fruiting, the two monokaryons A. aegerita AAE-3-40 (‘initials type’) and A. aegerita AAE-3-37 (‘elongated initials type’) have been selected for their mating compatibility with A. aegerita AAE-3-32 and A. aegerita AAE-3-13, respectively. Together with the parental dikaryotic strain A. aegerita AAE-3, this set of standard monokaryons could prove useful for studies exploring the factors regulating monokaryotic fruiting in comparison to dikaryotic mushroom formation.     

Control of fungal development. II. Kinetics of growth during dikaryosis in Schizophyllum commune

This paper describes a method for mating colonies of Schizophyllum commune so that the mating process in sets of individual colonies occurs synchronously. This technique has permitted us to define the kinetics of growth during dikaryosis, the transition from the homokaryotic to the dikaryotic stage of the life cycle. Homokaryons and established dikaryons have essentially the same exponential growth rates. We find that, during the transition from the homokaryon to the dikaryon, there is a period of distinctly slower growth, and it is during this period that the morphology typical of the dikaryon first becomes apparent. Growth kinetics for the transition from the homokaryon to the common-A heterokaryon are also established. This method will permit us to study sequential biochemical and physiological events occurring during these two developmental transitions.     

Molecular characterization and mating type analysis of oyster mushroom (<Emphasis Type="Italic">Pleurotus</Emphasis> spp<Emphasis Type="Italic">.</Emphasis>) using single basidiospores for strain improvement

Existence of variability in morphological traits and growth rate of mycelium of homokaryotic single basidiospores can be exploited for the development of inter-strainal hybrids. We isolated 182 single basidiospores from mushroom bodies of P. sajor-caju, P. florida, P. eous and one wild relative of Pleurotus called Hypsizygus ulmaris. The single spores were isolated using a new technique that is less prone to contamination and more efficient than the common techniques used by earlier workers. All the isolates showed a varied range of cultural morphology. Mating types of all the isolates within the species were identified on the basis of hyphal fusion via anastomosis with the tester strains. Four compatible pairs of isolates with well prominent tuft in the contact zone were selected for dikaryon isolation. Dikaryons were used for spawn preparation and mushroom cultivation. The dikaryotic isolates with their replicates were evaluated for spawn run period, yield and biological efficiency. 42 isolates (10 di- and 32 mono-karyotic isolates) were analyzed with RAPD genetic markers. Phenotypic characters and mating types of all the 42 isolates analyzed genetically were correlated with their genetic polymorphism data. The isolates showed very large diversity both at the phenotypic and the genotypic level. Available phenotypic and genotypic data can further help in the selection of monosporous isolates for developing inter-strainal hybrids which can lead to better prospects for genetic improvement in different species of Pleurotus.     

A homologue of the yeast SHE4 gene is essential for the transition between the syncytial and cellular stages during sexual reproduction of the fungus Podospora anserina.

The Podospora anserina cro1 gene was identified as a gene required for sexual sporulation. Crosses homozygous for the cro1-1 mutation yield fruiting bodies which produce few asci due to the formation of giant plurinucleate cells instead of dikaryotic cells after fertilization. This defect does not impair karyogamy, but meioses of the resultant polyploid nuclei are most often abortive. Cytological studies suggest that the primary defect of the mutant is its inability to form septa between the daughter nuclei after each mitosis, a step specific for normal dikaryotic cell divisions. The cro1-1 mutant would thus be unable to leave the syncytial vegetative state while abiding by the meiotic programme. cro1-1 also shows defects in ascospore germination and growth rate. GFP-tagging of the CRO1 protein reveals that it is a cytosolic protein mainly expressed at the beginning of the dikaryotic stage and at the time of ascospore maturation. The CRO1 protein exhibits significant similarity to the SHE4 protein, which is required for asymmetric mating-type switching in budding yeast cells. Thus, a gene involved in asymmetric cell divisions in a unicellular organism plays a key role at the transition between the syncytial (vegetative) state and the cellular (sexual) state in a filamentous fungus.     

Ectomycorrhiza formation between Pseudotsuga menziesii seedling roots and monokaryotic and dikaryotic isolates of Laccaria bicolor

Seedling roots of Pseudotsuga menziesii were colonized with three monokaryotic isolates and one dikaryotic isolate of Laccaria bicolor to assess the effect of fungal genotype on ectomycorrhiza formation. Ectomycorrhizas resulting from colonization by the dikaryotic isolate had a multilayered mantle and a cortical Hartig net. One monokaryotic isolate (ss7) formed ectomycorrhizas comparable in anatomy to those induced by the dikaryotic isolate. Two other monokaryotic isolates (ss5, ss1) failed to form mantles or Hartig nets. Roots colonized by these isolates developed characteristics indicating an incompatible reaction.     

A NEW BASIDIOMYCETOUS SEPTAL TYPE: THE MULTIPERFORATE SEPTUM IN KRIEGERIA ERIOPHORI

Septa from metabasidia and clamp connections of the heterobasidiomycetous sedge parasite Kriegeria eriophori were studied with light and electron microscopy. Septa from clamp connections subtending probasidia were reconstructed from serial sections. Septa at clamp connections are perforated by multiple, simple pores, while metabasidial septa possess single, central swellings which probably represent pores occluded by wall material. The occurrence of multiperforate septa in the fungi is reviewed. The septal morphology of K. eriophori is compared to that of simple-septate heterobasidiomycetes, and the systematic and functional implications are discussed.