Fine Structure of Ascosporogenesis in Nematospora coryli Peglion, a Pathogenic Yeast

Observations with the electron microscope of the early stages of ascospore formation in Nematospora coryli Peglion reveal the following sequence of events. Partial dissociation of the nuclear membrane is followed by the appearance of unit membranes. Each unit membrane gives rise to two pairs of double membranes delimiting the ascospores from the epiplasm of the ascus. Enlarged mitochondria which have a granular matrix and limited cristae development are also regularly seen.     

Ascospore aggregation and oxylipin distribution in the yeast Dipodascopsis tothii

Upon cultivation of the yeast Dipodascopsis tothii in its sexual stage, small ascospores are released individually from the ascus tip, which then assemble in sheathed cluster balls. In contrast to Dipodascopsis uninucleata, this yeast produced smooth bean shaped ascospores with sheath-like appendages that assemble in a disordered sheathed ball of ascospores outside the ascus. Strikingly, upon release, the ascus tip contained 3-hydroxy oxylipins, while the released ascospore clusters contained little or no 3-hydroxy oxylipins as indicated by immunofluorescence microscopy. In D. uninucleata, these oxylipins are concentrated on the spore surface and interspore matrix, but not on the ascus tip.     

The fine structure of ascospore shape and development inCeratocystis fimbriata

Ascospore development inCeratocystis fimbriata Ell. & Halst. commenced in an eight-nucleate ascus. A single vesicle formed along the periphery of the ascus from fragments of ascospore delimiting membranes, surrounded all eight nuclei and eventually invaginated, first forming pouches with open ends, then finally enclosing each of the eight nuclei in a separate sac, thus delimiting ascospores. Pairing of the ascospores followed and brim formation occurred at the contact area between two ascospores. Osmiophilic bodies contributed to the formation of brim-like appendages by fusing to the ascospore walls. Additional brims were observed at opposite ends of the ascospores giving them a double-brimmed appearance.Abbreviations AV ascus vesicle - DM delimiting membrane - EV electron translucent bodies - G granules - M mitochondria - N nucleus - OB osmiophilic bodies - PMV plasmamembrane vesicles - PW primary wall - SW secondary wall     

The release of elongated, sheathed ascospores from bottle-shaped asci in Dipodascus geniculatus

Yeasts use different mechanisms to release ascospores of different lengths from bottle-shaped asci. Round to oval-shaped ascospores are enveloped in oxylipin-coated compressible sheaths, enabling ascospores to slide past each other when they reach the narrowing ascus neck. However, more elongated ascospores do not contain sheaths, but are linked by means of oxylipin-coated interlocked hooked ridges on the surfaces of neighboring ascospores, thereby keeping them aligned while they are pushed towards the ascus tip by turgor pressure. In this study, we found elongated, oxylipin-coated sheathed ascospores in Dipodascus geniculatus that are released effectively from bottle-shaped asci without alignment. This is possible because the ascus neck and opening have a diameter that is the same as the length of the ascospore, thus allowing the ascospores to turn sideways without blocking the ascus when they are released. We found that increased concentrations of acetylsalicylic acid inhibit both ascospore release and 3-hydroxy oxylipin production in this yeast, thereby implicating this oxylipin in sexual reproduction.     

Meiosis and ascospore development in Cochliobolus heterostrophus

Cochliobolus heterostrophus produces eight filiform ascospores per ascus, following meiosis and a postmeiotic mitosis. Early ascus development and nuclear divisions in C. heterostrophus resemble those of the prototypic Pyrenomycete Neurospora crassa. However, the two fungi differ in several important details owing to differences in ascus and ascospore shape, spindle pole body (SPB) behavior during spore delimitation, and ascospore development. In C. heterostrophus, the two spindles at meiosis II, and the four spindles at the postmeiotic mitosis are aligned irregularly, unlike the tandem or ladder rung-like orientation of spindles of N. crassa. Prior to ascospore delimitation, all eight nuclei reorient themselves and their SPB plaques migrate toward the base of the ascus. The SPB plaques facilitate demarcation of the lower end of each incipient ascospore. The filiform ascospores are uninucleate and unsegmented at inception but they become highly multinucleate, multisegmented, and helically coiled when mature. An account of ascus development, nuclear divisions, and ascospore delimitation and maturation is presented here and supported by a series of photomicrographs.     

Meiosis and ascospore development in Cochliobolus heterostrophus.

Cochliobolus heterostrophus produces eight filiform ascospores per ascus, following meiosis and a postmeiotic mitosis. Early ascus development and nuclear divisions in C. heterostrophus resemble those of the prototypic Pyrenomycete Neurospora crassa. However, the two fungi differ in several important details owing to differences in ascus and ascospore shape, spindle pole body (SPB) behavior during spore delimitation, and ascospore development. In C. heterostrophus, the two spindles at meiosis II, and the four spindles at the postmeiotic mitosis are aligned irregularly, unlike the tandem or ladder rung-like orientation of spindles of N. crassa. Prior to ascospore delimitation, all eight nuclei reorient themselves and their SPB plaques migrate toward the base of the ascus. The SPB plaques facilitate demarcation of the lower end of each incipient ascospore. The filiform ascospores are uninucleate and unsegmented at inception but they become highly multinucleate, multisegmented, and helically coiled when mature. An account of ascus development, nuclear divisions, and ascospore delimitation and maturation is presented here and supported by a series of photomicrographs.     

Electron Micrograph Study of the Asci and Ascospores of Metschnikowia Kamienski

Internal and surface structures of asci and ascospores were studied by transmission electron microscopy (TEM) and by scanning electron microscopy (SEM) to establish the character and number of ascospores within the ascus of Metschnikowia krissii. Enzyme digestion with snail gut enzymes and SEM examination suggested the presence of a single ascospore enclosed in a thick sheath of epiplasmic materials. Two closely associated ascospores without an epiplasmic sheath were clearly distinguishable from asci of M. bicuspidata var. chathamia when similarly treated. Ultramicrotomy and TEM established conclusively that M. krissii produced a single ascospore per ascus. Neither SEM nor TEM revealed any morphological detail of the ascospores of taxonomic significance.     

冬虫夏草子囊孢子的发育

经光学显微镜观察,冬虫夏草[Cordyceps sinensis(Berk.)Sacc.]子囊孢子的发育可分为三个阶段:(1)原子囊孢子期:孢子近球形,椭圆形,卵圆形或长圆形,4.8—10×3.6—6μm。(2)孢子伸长期:从孢子一端突出,伸长生长,出现分隔,隔细胞较长,30—37.5μm。(3)子囊孢子形成期:孢子继续伸长并不断出现分隔,隔间长6—12μm。一般仅有两个孢子成熟,其余多数原子囊孢子败育,不萌发或形成极纤细的丝状物。基于子囊孢子发育的过程,讨论了冬虫夏草及其近缘种的分类地位。     

Incubation of excised apothecia enhances ascus maturation of Sclerotinia sclerotiorum

Synchronized maturation of ascospores of Sclerotinia sclerotiorum is desirable for establishing a transformation system, conducting genetic analyses of the pathogen, defining the precise epidemiological roles of ascospores and screening plant germplasm for resistance. In general, fresh apothecia collected from germinated sclerotia contained primarily immature or discharged asci. This study was undertaken to investigate whether maturation of asci and ascospores could be enhanced by incubation of excised apothecia and to determine the effects of factors such as temperature, excision time, light and ventilation on maturation of asci and ascospores in excised apothecia. Maturation of asci was compared between intact and excised apothecia that were incubated under similar conditions. Results demonstrated that temperature was an important factor affecting ascus maturation of S. sclerotiorum during incubation of excised apothecia, and the optimum temperature was around 21 C. After incubation at 21 C for 30 h, the percentage of undischarged mature asci in excised apothecia increased up to 70-80%. This increase was accompanied by a significant increase in ascospore production of up to 5 x 10(5) ascospores per apothecium. Detailed time course studies indicated that mature asci peaked at 30-36 h of postexcision incubation. Mature asci and the number of ascospores were higher in open incubation than in closed incubation, suggesting that accumulation of volatile substances was not required for ascus/ascospore maturation during postexcision incubation and ventilation could enhance the maturation process. Light also did not affect the maturation of asci during the incubation of excised apothecia. Germination rates for ascospores from excised apothecia under various treatments were similar to those from untreated apothecia but declined slightly with time postexcision. The incubation of excised apothecia promoted ascus maturation compared with intact apothecia.     

Oxylipin-coated hat-shaped ascospores of Ascoidea corymbosa

We previously implicated 3-hydroxy oxylipins and ascospore structure in ascospore release from enclosed asci. Using confocal laser scanning microscopy on cells stained with fluorescein-coupled, 3-hydroxy oxylipin-specific antibodies, we found that oxylipins are specifically associated with ascospores and not the vegetative cells or ascus wall of Ascoidea corymbosa. Using gas chromatography--mass spectrometry the oxylipin 3-hydroxy 17:0 could be identified. Here, we visualize for the first time the forced release of oxylipin-coated, hat-shaped ascospores from terminally torn asci, probably through turgor pressure. We suggest that oxylipin-coated, razor-sharp, hat-shaped ascospore brims may play a role in rupturing the ascus to affect release.     

Programmed ascospore death in the homothallic ascomycete Coniochaeta tetraspora

Immature asci of Coniochaeta tetraspora originally contain eight uninucleate ascospores. Two ascospore pairs in each ascus survive and mature, and two die and degenerate. Arrangement of the two ascospore types in individual linear asci is what would be expected if death is controlled by a chromosomal gene segregating at the second meiotic division in about 50% of asci. Cultures originating from single homokaryotic ascospores or from single uninucleate conidia are self-fertile, again producing eight-spored asci in which four spores disintegrate, generation after generation. These observations indicate that differentiation of two nuclear types occurs de novo in each sexual generation, that it involves alteration of a specific chromosome locus, and that the change occurs early in the sexual phase. One, and only one, of the two haploid nuclei entering each functional zygote must carry the altered element, which is segregated into two of the four meiotic products and is eliminated when ascospores that contain it disintegrate. Fusion of nuclei cannot be random-a recognition mechanism must exist. More study will be needed to determine whether the change that is responsible for ascospore death is genetic or epigenetic, whether it occurs just before the formation of each ascus or originates only once in the ascogonium prior to proliferation of ascogenous hyphae, and whether it reflects developmentally triggered alteration at a locus other than mating type or the activation of a silent mating-type gene that has pleiotropic effects. Similar considerations apply to species such as Sclerotinia trifoliorum and Chromocrea spinulosa, in which all ascospores survive but half the spores in each ascus are small and self-sterile. Unlike C. tetraspora, another four-spored species, Coniochaetidium savoryi, is pseudohomothallic, with ascus development resembling that of Podospora anserina.     

Cataractispora receptaculorum, a new freshwater ascomycete from Hong Kong

A new species of Cataractispora, C. receptaculorum, is described from freshwater habitats. This species is characterized by triseptate verruculose ascospores and polar appendages that unfurl in water. The ascospores lack polar chambers that enclose the appendages as in C. bipolaris and C. viscosa. An ultrastructural study of this species revealed that the ascus wall and apical ring of this species is typical of the Annulatascaceae, while the ascospore wall with verruculose ornamentations and the ontogeny of the ascospore polar appendages are similar to the other species of Cataractispora. Cataractispora receptaculorum is illustrated with interference light, scanning and transmission electron micrographs.     

Meiotic silencing in the homothallic fungus Gibberella zeae

The homothallic ascomycete fungus Gibberella zeae is an important pathogen on major cereal crops. The objective of this study was to determine whether meiotic silencing occurs in G. zeae. Cytological studies demonstrated that GFP and RFP-fusion proteins were not detected during meiosis, both in heterozygous outcrosses and homozygous selfings. The deletion of rsp-1, a homologue used for studies on meiotic silencing of Neurospora crassa, triggered abnormal ascospores from selfing, but outcrosses between the mutant and wild-type strain resulted in some ascospores with mutant phenotype (low occurrence of ascus dominance). When the ectopic mutants that carried an additional copy of rsp-1 were selfed, they primarily produced ascospores with normal shape but a few ascospores (0.23 %) were abnormal, in which both endogenous and ectopically integrated genes contained numerous point mutations. The ectopic mutants showed low occurrence of ascus dominance in outcrosses with strains that carried the wild-type allele. Approximately 10 % of ascospores were abnormal but all of the single-ascospore isolates produced normal-shaped ascospores from selfing. However, no ascus dominance was observed when the mutants were outcrossed with a sad-1 deletion mutant, which lacks the putative RNA-dependent RNA polymerase essential for meiotic silencing in N. crassa. All results were consistent with those generated from an additional gene, roa, required for ascospore morphogenesis. This study demonstrated that G. zeae possesses a functional meiotic silencing mechanism which is triggered by unpaired DNA, as in N. crassa.     

Ultrastructure of ascospore delimitation in freeze substituted samples ofAscodesmis nigricans (Pezizales)

Summary Freeze substitution proved to be a valuable technique for studying the early stages of ascosporogenesis inAscodesmis nigricans. Our observations indicate that the ascus vesicle originated from the ascus plasma membrane. Invaginations of the plasma membrane produced ascus vesicle initials consisting of two closely spaced unit membranes. The appearance of the outer leaflet of each of these membranes was identical to that of the inner leaflet of the ascus plasma membrane. Apparent points of continuity between ascus vesicle initials and the plasma membrane were observed. Ascus vesicle initials accumulated in the ascus cytoplasm near the plasma membrane and then coalesced to form the ascus vesicle, a peripheral, cylinder-like structure consisting of two closely spaced unit membranes that extended from the ascus apex to the ascus base. The ascus vesicle then became invaginated in a number of regions and subsequently gave rise to eight sheet-like segments, or ascosporedelimiting membranes, that encircled uninucleate segments of cytoplasm forming ascospore initials. Like the ascus vesicle, each ascospore-delimiting membrane consisted of two closely spaced unit membranes, the inner of which became the ascospore plasma membrane. The ascospore wall then developed between the spore plasma membrane and the outer membrane. Many details of ascospore maturation were clearly visible in freeze substituted samples.     

Mapping 3-hydroxy oxylipins on ascospores of <Emphasis Type="Italic">Eremothecium sinecaudum</Emphasis>

3-Hydroxy oxylipins were uncovered on ascospores of Eremothecium sinecaudum using immunofluorescence microscopy. This was confirmed by gas chromatography mass spectrometry. These oxylipins were observed only on ascospore parts characterised by nano-scale surface ornamentations simulating a corkscrew as demonstrated by scanning electron microscopy. Conventional ascospore staining further confirms its hydrophobic nature. Using confocal laser scanning microscopy we found that the corkscrew part with spiky tip of needle-shaped ascospores may play a role in rupturing the ascus in order to affect its release. Through oxylipin inhibition studies we hypothesise a possible role for 3-hydroxy oxylipins in facilitating the rupturing process.     

An acetylsalicylic acid-sensitive aggregation phenomenon in Dipodascopsis uninucleata

Aggregation of ascospores has been discovered in the yeast Dipodascopsis uninucleata. When this yeast is cultivated to reach the sexual reproductive stage, small ascospores are individually released from the tip of a sac-like ascus which then aggregate in orderly clusters. Acetylsalicylic acid (ASA) inhibited ascospore release and subsequent ordered aggregation process. We suggest that novel ASA-sensitive oxidised fatty acids (3 R-hydroxy-oxylipins) and small hooks located on the surface of these ascospores, are involved.     

Morphogenesis of Ascospores in Saccharomyces cerevisiae

Ultrastructural changes associated with ascospore formation in Saccharomyces cerevisiae were investigated by using freeze-etching and thin-sectioning techniques. The first nuclear division (meiosis I) is indicated by the appearance of spindle fibers within the nucleus. The nucleus subsequently elongates and eventually assumes a barbell shape; the second nuclear division (meiosis II) occurs before nuclear separation. The spindle fibers involved in meiosis II appear to be oriented perpendicular to those observed in meiosis I. A discrete bilaminar structure (forespore wall) progressively delineates each ascospore nucleus and encloses cytoplasmic material including mitochondria and endoplasmic reticulum. The forespores then elongate, close off, and become separated from the ascus cytoplasm by membranes. The ascospores assume a spherical shape as spore coat material is laid down; the latter stages of ascospore formation are characterized by thickening of the ascospore wall and disintegration of the ascus cytoplasm. No structures which could be identified as chromosomes were observed.     

Physiological and environmental aspects of ascospore discharge in Gibberella zeae (anamorph Fusarium graminearum)

We investigated ascospore discharge in the perithecial fungus, Gibberella zeae. In a wind tunnel study that simulated constant rain and varying day and night lengths, the rate of ascospore release was approximately 8-30% greater under light than in complete darkness. Under constant light, ascospore discharge occurred at maximal rates at relative humidity levels greater than 92%. When perithecia were placed under conditions of high external osmolarity, ascospore discharge was significantly reduced. Ascospores were discharged from asci along with droplets of fluid, the epiplasm, from within the ascus. Analysis of discharged epiplasmic fluid by GC-MASS Spectrometry revealed that mannitol was the major simple sugar component of the fluid. Activity of mannitol dehydrogenase, which catalyzes the conversion of fructose to mannitol, was higher in protein extracts from mature perithecia than in extracts from vegetative tissue. Several inhibitors of K(+) and Ca(++) ion channels inhibited ascospore discharge, which suggested that ascospore discharge resulted from the buildup of turgor pressure generated by ion fluxes and mannitol accumulation.     

Abnormal ascospore morphology in the bud mutant of Neurospora tetrasperma

A recessive ascospore mutant of Neurospora tetrasperma, named bud, was isolated from a wild-collected heterokaryotic strain with four different nuclear components. bud segregates as a single mendelian gene. When bud is homozygous, meiosis is apparently normal but postmeiotic events are not. Abnormal orientation of spindles at the postmeiotic mitosis often results in failed pair-wise association of nuclei and their irregular distribution along the length of the ascus prior to spore delimitation. Consequently, many asci cut out more than four ascospores; some contain no nuclei while others contain more than two. The most dramatic effect of bud is on ascospore delimitation itself. Many ascospores are irregularly shaped and are often interconnected, because of incomplete spore delimitation. Ascospores also show one or two lobes or bud-like extensions of varying sizes. Over 75% of ascospores from bud x bud remain white or tan and are inviable. The interaction of bud with a dominant Eight-spore mutant (E) was examined in both heterozygous and homozygous crosses. When both bud and E are heterozygous, bud has no effect on ascospore delimitation or on the phenotype of E because bud is recessive; many asci produce 5-8 ascospores just as in E x E(+). And when bud is homozygous and E is heterozygous, ascospore delimitation is less affected than when E is absent. Moreover, when both bud and E are homozygous, the effect on ascospore development is less extreme than when E is homozygous singly.     

印度块菌子囊果内部解剖结构的扫描电镜观察

利用扫描电镜对成熟印度块菌子囊果的内部结构进行了观察,以系统揭示其子囊果内部组织特征,为块菌属的分类以及块菌属真菌子囊果的生理研究奠定基础。观察结果进一步证明,成熟印度块菌子囊果横切面上的白色迷宫状脉络是由不育的侧丝构成,而暗脉则是被侧丝缠绕并包裹着的可育的菌丝组织,即产孢组织,这些白色脉络和暗脉就构成了印度块菌子囊果横切面上迷宫状的纹脉;产孢组织中,可观察到正在发育的大大小小的子囊被缠绕在一起的大量产囊丝与侧丝包裹着,形成密密麻麻微小的类似蜂巢状结构;子囊孢子游离于子囊中,成熟子囊孢子表面有刺状纹饰,刺的顶端有小弯钩。单个子囊内含的子囊孢子大小与其内含的子囊孢子数目有关,子囊内所含的子囊孢子越多,子囊孢子就越小。