Mature Asci Research Articles

Morphological characterization of Gibberella coronicola sp. nov., obtained through mating experiments of Fusarium pseudograminearum

Mating experiments were performed among 18 strains ofFusarium pseudograminearum, formerly recognized as the Group 1 population ofF. graminearum. Heterothallic production of perithecia was observed in eight out of all 153 possible combinations. Mature asci and viable ascospores were recovered in seven of the eight combinations. Perithecia in the fertile pairings were subglobose to ovoid, dark, 120–370 μm diam and formed directly on the surface of rice stems placed on the culture media. Asci were unitunicate and 8-spored when mature. Mature ascospores were primarily hyaline, fusoid, straight or curved, with rounded ends and (1-)3-septate. Dimensions of the teleomorph obtained forF. pseudograminearum were different from those of theG. zeae teleomorph ofF. Graminearum. A new species ofGibberella, G. coronicola, is described and illustrated for the teleomorph ofF. pseudograminearum. The Group 1 and Group 2 populations recognized previously withinF. graminearum differ in their anamorphic and teleomorphic morphology, ecological habitats, pathogenicity, mode of sexual reproduction and phylogenetic relationships.

First Report of Powdery Mildew on Cornus florida in Connecticut Caused by Microsphaera pulchra.

Powdery mildew of dogwood (Cornus florida L.) has been observed on landscape trees since 1992, and has been increasing in severity recently. In 1998, mildewed leaves (n = 100) were collected from each of four locations in Connecticut. White mildew colonies first appeared in the early summer, 4 to 6 weeks after leaf emergence, and the initial colonies rapidly coalesced to cover the entire adaxial leaf surface. Incidence on observed trees reached nearly 100%. Premature autumn coloration and defoliation occurred on those trees where incidence was highest; tree mortality due solely to powdery mildew was not observed. Cleistothecia were apparent on both adaxial and abaxial leaf surfaces within 4 to 6 weeks of initial infection. Mycelium was white in color, and conidiophores were three-celled with a slightly bent foot cell. Fibrosin bodies were not present in conidia (n = 400), which measured 14.1 m ± 0.80 × 28.1 m ± 1.60 (mean + standard error of the mean). Cleistothecia measured 114.7 m ± 1.77 in diameter, and had 4 to 10 dichotomously branched appendages, which measured 140.7 m ± 3.95 in length (1.23× cleistothecial diameter). The fungus was identified as Microsphaera pulchra based on mycelial, conidial, and cleistothecial characteristics (1). In addition, cleistothecia were found on twigs and on fallen leaves of dogwood; these overwintering cleistothecia contained mature asci and mature ascospores in March 1999. Microsphaera penicillata recently was redescribed to include only that species causing powdery mildew on Alnus spp., and having appendages 1.5× cleistothecial diameter (2). The fungus causing powdery mildew in Connecticut previously had been reported to be M. penicillata, which was not found in the present study.

SWM1, a developmentally regulated gene, is required for spore wall assembly in Saccharomyces cerevisiae.

Meiosis in Saccharomyces cerevisiae is followed by encapsulation of haploid nuclei within multilayered spore walls. Formation of this spore-specific wall requires the coordinated activity of enzymes involved in the biosynthesis of its components. Completion of late events in the sporulation program, leading to spore wall formation, requires the SWM1 gene. SWM1 is expressed at low levels during vegetative growth but its transcription is strongly induced under sporulating conditions, with kinetics similar to those of middle sporulation-specific genes. Homozygous swm1Delta diploids proceed normally through both meiotic divisions but fail to produce mature asci. Consistent with this finding, swm1Delta mutant asci display enhanced sensitivity to enzymatic digestion and heat shock. Deletion of SWM1 specifically affects the expression of mid-late and late sporulation-specific genes. All of the phenotypes observed are similar to those found for the deletion of SPS1 or SMK1, two putative components of a sporulation-specific MAP kinase cascade. However, epistasis analyses indicate that Swm1p does not form part of the Sps1p-Smk1p-MAP kinase pathway. We propose that Swm1p, a nuclear protein, would participate in a different signal transduction pathway that is also required for the coordination of the biochemical and morphological events occurring during the last phase of the sporulation program.

F-actin distribution and function during sexual differentiation in Schizosaccharomyces pombe.

Sexual differentiation in Schizosaccharomyces pombe is induced from the G1 phase of the cell cycle by nitrogen starvation and the presence of mating pheromones. We describe the distribution of F-actin during sexual differentiation. Cortical F-actin dots have previously been shown to be restricted to one end of the rod shaped cell during the G1 phase of the cell cycle. Within half an hour of nitrogen starvation the distribution of cortical F-actin dots switched from being monopolar to bipolar. This was then reversed as the F-actin cytoskeleton repolarized so that cortical F-actin dots accumulated towards the projection tip at one end of the cell. Following cell fusion, F-actin dots were randomly scattered during the horsetail movement that precedes meiosis I and remained scattered until prometaphase or metaphase of meiosis II, when they concentrated around the nucleus. F-actin was seen on the lagging face of the nuclei which faced the partner nucleus during anaphase B of meiosis II. Early on in this anaphase F-actin was also seen on the opposite side of the nucleus, near the spindle pole body. F-actin accumulated within the spores in the mature ascus. Treatment with the actin depolymerising drug Latrunculin A showed that F-actin is required for cell fusion and spore formation. Latrunculin A treatment extended all stages from karyogamy to meiosis I. The S. pombe homologue of the actin binding protein profilin, Cdc3, was shown to be required for conjugation. Cdc3 co-localized with the formin related molecule Fus1 at the projection tip. The polarization of F-actin cortical dots to the projection tip was unaffected in the cdc3.124 mutant, but cdc3.124 mutant cells were unable to break down the cell walls between the two cells following agglutination.

G protein alpha subunit genes control growth, development, and pathogenicity of Magnaporthe grisea.

Three G protein alpha subunit genes have been cloned and characterized from Magnaporthe grisea: magA is very similar to CPG-2 of Cryphonectria parasitica; magB is virtually identical to CPG-1 of Cryphonectria parasitica, to gna1 of Neurospora crassa, and to fadA of Emericella nidulans; and magC is most similar to gna2 of Neurospora crassa. Homologous recombination resulting in targeted deletion of magA had no effect on vegetative growth, conidiation, or appressorium formation. Deletion of magC reduced conidiation, but did not affect vegetative growth or appressorium formation. However, disruption of magB significantly reduced vegetative growth, conidiation, and appressorium formation. magB- transformants, unlike magA- and magC- transformants, exhibited a reduced ability to infect and colonize susceptible rice leaves. G protein alpha subunit genes are required for M. grisea mating. magB- transformants failed to form perithecia, whereas magA- and magC- transformants did not produce mature asci. These results suggest that G protein alpha subunit genes are involved in signal transduction pathways in M. grisea that control vegetative growth, conidiation, conidium attachment, appressorium formation, mating, and pathogenicity.

A Putative Rhamnogalacturonase Required for Sexual Development of Neurospora crassa

In previous work, the asd-I (ascus development) gene of the filamentous fingus Neurospora crassa was identified as a gene expressed preferentially during the sexual cycle and shown to be essential for normal sexual development. The asd-I gene has been sequenced and further characterized. It contains two introns, the first of which is in-frame and inefficiently or differentially spliced. The predicted ASD-I protein has extensive homology with rhamnogalacturonase B of Aspergillus aculeatus, which cleaves the backbone within the ramified hairy regions of pectin. In homozygous asd-I crosses, sexual development is initiated and large numbers of normal-sized asci are formed. Ascospore delineation does not occur, however, and no sexual progeny are produced. As most asd-I asci contain eight nuclei, the two meiotic divisions and subsequent mitotic division typical of normal crosses seem to occur, but the haploid nuclei are not partitioned into ascospores. In wild-type crosses, the ASD-I protein is present in large amounts in croziers and young asci, but it is only faintly detectable in more mature asci containing developing ascospores. Models to explain the possible role of a rhamnogalacturonase in sexual development are presented.

Ascus and ascospore morphogenesis

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.

Molecular biology of yeast exoglucanases

Three exoglucanase (Exg) genes have been reported in Saccharomyces cerevisiae. Gene EXG1 encodes the major isoenzyme (Exgl). Differential glycosylation of the primary translation product throughout the secretory pathway results in the secretion of several glycoforms. The major glycoform (Exglb) contains two short carboxypeptidase Y-like oligosaccharides attached to both potential glycosylation sites present in the molecule. A minor glycoform (Exgla) arises from the former by elongation of the second oligosaccharide. The protein portion is processed in the secretory pathway by the Kex2 protease. Gene EXG2 encodes a 63 kDa polypeptide with 12 potential glycosylation sites. The predicted protein, Exgll, carries a signal peptide at the amino terminus and a glycosyl-phosphatidyl inositol anchoring motif at the carboxyl end. The latter appears responsible for the particulate nature of this isoenzyme, since its elimination results in the secretion of this activity into the culture medium. Gene SSG1 encodes a 52 kDa polypeptide which is specifically synthesized during sporulation of diploids. SSC1 expression is under control of both sexual (a1-α2 element) and nutritional control. Although homozygous ssg1 / ssg1 diploid strains are still able to complete sporulation, they exhibited a delay in the appearance of mature asci. Single or double disruption of EXG1 and EXG2 did not result in any relevant phenotype and the triple mutant behaved as ssg1 /ssg1. A Exgl-related enzyme is secreted by Candida albicans. All these four enzymes share 8 highly conserved regions in the same relative positions, indicating that they derive from a common ancestor. However, no clear function has so far been demonstrated for them.

Factors Affecting Maturation and Release of Ascospores ofVenturia pirinain Oregon

The first mature ascospores of Venturia pirina, the causal agent of pear scab, were observed at bud swell in late February to late March. The percentage of asci that contained mature ascospores within a pseudothecium reached a maximum during the bloom period. Ascospore maturity during three seasons was described with linear regression. The accumulated degree-days with a base temperature of 0 C was the independent variable, and ln(1/1-proportion of mature asci) was the dependent variable. The maturity model was validated at two diverse locations in 1993. Ascospore release usually was associated with rain (nine of 19 events) or dew (eight of 19 events). Light stimulated the discharge of ascospores, but night release of ascospores occurred in all 3 yr of the study [...]

SSG1, a gene encoding a sporulation-specific 1,3-beta-glucanase in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the meiotic process is accompanied by a large increase in 1,3-beta-glucan-degradative activity. The molecular cloning of the gene (SSG1) encoding a sporulation-specific exo-1,3-beta-glucanase was achieved by screening a genomic library with a DNA probe obtained by polymerase chain reaction amplification using synthetic oligonucleotides designed according to the nucleotide sequence predicted from the amino-terminal region of the purified protein. DNA sequencing indicates that the SSG1 gene specifies a 445-amino-acid polypeptide (calculated molecular mass, 51.8 kDa) showing extensive similarity to the extracellular exo-1,3-beta-glucanases encoded by the EXG1 gene (C. R. Vazquez de Aldana, J. Correa, P. San Segundo, A. Bueno, A. R. Nebreda, E. Mendez, and F. del Rey, Gene 97:173-182, 1991). The N-terminal domain of the putative precursor is a very hydrophobic segment with structural features resembling those of signal peptides of secreted proteins. Northern (RNA) analysis reveals a unique SSG1-specific transcript, 1.7 kb long, which can be detected only in sporulating diploids (MATa/MAT alpha) but does not appear in vegetatively growing cells or in nonsporulating diploids (MAT alpha/MAT alpha) when incubated under nitrogen starvation conditions. The meiotic time course of SSG1 induction indicates that the gene is transcribed only in the late stages of the process, beginning at the time of meiosis I and reaching a maximum during spore formation. Homozygous ssg1/ssg1 mutant diploids are able to complete sporulation, although with a significant delay in the appearance of mature asci.

Ultrastructural observations onNimbospora bipolaris(Halosphaeriaceae, Ascomycetes)

The ultrastructure of the ascus, ascospores and appendages ofNimbospora bipolariswere examined at the transmission electron microscope level. The mature ascus wall comprises a single narrow electron-opaque layer which deliquesces and lacks an apical elaboration. Ascospore walls comprise a bipartite mesosporium, a thinner electron-opaque episporium and an outer mucilaginous sheath. Before release from the ascus the mucilaginous sheath is compact and folded. The fibrillar, trailing ascospore appendages are arranged in eight equidistant tufts around the circumference of the septum, originate from the mesosporium and emerge through pores in the episporium. In early stages of development the appendages are retained within the mucilaginous sheath but upon release from the ascus the sheath expands and the appendages pierce the sheath and radiate out to form their characteristic appearance. Ascospore ontogeny inN. bipolarisis compared with other appendaged members of the Halosphaeriaceae.

Influence of Temperature on Apothecial Development and Ascospore Discharge byBlumeriella jaapii

Cherry leaves bearing apothecia of Blumeriella jaapii with either immature or mature asci were collected from a sweet cherry (Prunus avium) orchard near East Lansing, Michigan, for experiments on apothecial development and ascospore discharge, respectively. Disks cut from the leaves were incubated at six temperatures in experiments on apothecial development (4-24 C), and on ascospore discharge (8-30 C). Microscopic examination of crushed apothecia was used to evaluate changes in maturity. The number of asci per apothecium and the percentage of mature plus discharged asci after 7 days of incubation were related to temperature with quadratic regression analyses

Meiotic Drive in Neurospora and Other Fungi

When a gene complex called Spore killer is heterozygous, ascospores representing two of the four products of each meiosis are killed. Only those that receive the killer complex survive. This article reviews what is known of the mode of action of the Neurospora Spore killers, their chromosomal basis, and their occurrence in nature. Similar genes or gene com- plexes have been found in other fungi. An example from Podospora anserina is analyzed. The best-studied example of meiotic drive in fungi is Spore killer in Neuro- spora, which has much in common with the Segregation Distorter (SD) complex in Drosophila and the t complex in mice. The effects are manifested postmeioti- cally in all three systems, but the results of drive are seen most directly in Neuro- spora. With the SD complex and with the t haplotypes, it is necessary to produce a heterozygous male and then to obtain and analyze diploid progeny in order to assess the effects of that heterozygosity in the father. Electron microscopy of heterozygous SD crosses can be used to confirm that about half of the sperm deteriorate, but this procedure is far too demanding for routine use. In Neuro- spora, by contrast, Spore killer is manifested in ascospores that can be seen under low magnification with a dissecting microscope. These ascospores are the immediate products of meiosis; yet they differ from sperm in being themselves the progeny. In Neurospora crassa, each fertilization event in a cross results in a fruiting body within which haploid nuclei proliferate in dikaryotic fashion, producing many pairs of nuclei of opposite mating type that fuse and go into meiosis, each in a separate ascus. As with male meiosis in plants and animals, all four meiotic products normally survive. Each mature ascus normally contains eight viable black ascospores, which originated from the same meiosis (the four immediate meiotic products undergo an additional mitosis before ascospore walls enclose the eight daughter nuclei). The result is essentially four sets of identical twins. A feature of ascospore formation that is important for Spore killer is the timing of cell-wall formation. Meiosis and the postmeiotic mitosis occur in a common cytoplasm. No cell walls are present in the developing ascus until the ascospore walls are formed. (For a diagram of the Neurospora life cycle, see Perkins and Barry 1977; for an illustrated account of events in the ascus, see Raju 1980.) Spore killer was discovered because of its dramatic effect on the appearance of ascospores. Viable ascospores are black, but inviable ascospores remain small and unpigmented. When a Spore-killer strain is crossed with the standard wild

Scanning electron microscopy of Tuber sporocarps and associated bacteria

The relationship between bacteria and the sporocarpic structures of truffles of the genus Tuber has been studied by means of scanning electron microscopy. Pieces of the truffles, critical-point dried then broken by traction, showed bacteria on hyphae of the ‘venae externae’ as well as inside the mature asci. Possibly the bacteria initially aid development of sporocarpic hyphae through the interchange of metabolites and may subsequently be associated with activation of ascospores.

Sporogenesis and septum schizolysis in Dipodascus aggregates

Asci, ascospores, and arthroconidia of Dipodascus aggregatus were examined by electron microscopy. Freeze-drying of mature asci caused rupture of the ascal walls and made it possible to observe capsulated ascospores in situ. Two types of septa occurred: (i) one having a remarkably thickened cross wall which protruded to form a circumferential ridge on the hyphae, and (ii) the other without such a bulging ridge. Schizolysis of these septa resulted in the formation of morphologically distinct arthroconidia. The scizolytic process of the ridged septa was demonstrated in detail. There was no evidence to support that the plasmadesmal canals in the septa function as disjunctive pegs in the process of arthroconidial separation.

Presence of abnormal synaptonemal complexes in heterothallic species of Neurospora.

Synaptonemal complex abnormalities are frequent in reconstructed meiotic prophase nuclei of Neurospora crassa and Neurospora intermedia. Three kinds of synaptonemal complex anomalies were seen: lateral component splits, lateral component junctions, and multiple complexes. The anomalies apparently are formed during or after the pairing process, as they were not seen in the largely unpaired early zygotene chromosomes. Their presence at all the other substages from mid-zygotene to late pachytene indicates that they are not eliminated before the synaptonemal complex decomposes at diplotene. Abnormal synaptonemal complexes were seen in all 19 crosses of N. crassa and N. intermedia that were examined, including matings between standard laboratory strains, inversions, Spore killers, and strains collected from nature. The frequency of affected nuclei and degree of abnormality within a nucleus varied in different matings. No abnormalities were present in the homothallic species Neurospora africana and Neurospora terricola. Structural chromosome aberrations, introgression, and heterozygosity have been eliminated as causes for pairing disorder. The abnormal synaptonemal complexes seemingly do not interfere with normal ascus development and ascospore formation. The affected nuclei are not aborted during meiotic prophase, nor are they eliminated by abortion of mature asci. The abnormal meiocytes do not lead to aneuploidy, as judged by the low frequency of white ascospores in crosses between wild type strains that have many abnormalities. Thus, the abnormal synatonemal complexes do not appear to prevent chiasma formation between homologues.

Ascoma Morphology of Pseudopeziza Trifolii Forma Specialis Medicaginis-Sativae (Dermateaceae) on Alfalfa

Ascomatal initials of Pseudopeziza trifolii f. sp. medicaginis-sativae consist of ascogonial coils forming on intracellular hyphae in parenchyma cells of living alfalfa leaflets. In some young ascomata, the ascogonial coils are surrounded by an aggregation of hyphae, while in other initials, such hyphae are sparse. Ascogenous hyphae and paraphyses develop and grow through the surrounding mycelium toward the host epidermis. Croziers proliferate from the binucleate ascogenous cells. Asci are eventually produced, with each mature ascus containing eight uninucleate ascospores. Ascosporogenesis is similar to that described in a number of Ascomycetes. Until the time of ascospore discharge, the hymenium of an ascoma is covered by hyphae in and under the infected host epidermis, and by periphysoids arising from these hyphae. The host epidermis ruptures when the ascoma is mature. A pseudoparenchymatous excipulum is present. Vegetative mycelium generally fills most of the host cells under the ascoma, and somatic hyphae appear to radiate outward from the ascoma to surrounding host cells. Septa in the hyphae have pores, with Woronin bodies in close proximity to the pores. Some pores have electron-dense plugs. The aggregation of hyphae observed throughout ascoma development is often referred to as a stroma; however, this aggregation is not always well developed, and it is not known whether or not it arises as a result of a sexual stimulus. Ascomata of this fungus can therefore be interpreted as either stromatic or nonstromatic.

Subcellular differentiation in sporulating yeast cells

We have previously described the induction of two sets of sporulation-specific mRNAs in Saccharomyces cerevisiae. Herein we correlate the appearance of these RNAs with the major morphogenic events of sporulation, and we analyze the spatial distribution of the RNAs within the ascus. Several observations suggest that the first set of messages is involved in spore wall synthesis. In fractionation experiments, these mRNAs are detected in the ascal cytoplasm but not in developing spores, indicating that the proteinaceous component of the spore wall is synthesized from the external compartment. The second set of messages is induced later in the course of spore maturation. These mRNAs accumulate within the spores and, unlike the first set of mRNAs, are retained in mature asci until the early stages of germination. We conclude that the development of ascospores proceeds through the differentiation of functionally distinct subcellular compartments.

Ascospore discharge in Pleospora herbarum (Pers.) Rabenh. (Dothideales) stimulated by near-ultraviolet radiation

An isolate of P. herbarum from beet seed failed to discharge ascospores in darkness but did so when exposed to light either continuously or cyclically (12 h light/12 h dark). When colonies with mature asci were subjected to a regime of alternating light and darkness for 54 days at a constant temperature of 20°C, ascospores were discharged over the entire period. Maximum discharge occurred on the 23rd day; few spores were liberated towards the end of the period. Light-induced spore discharge occurred over a wide temperature range (10–30°C) with the optimum being approximately 14–23°C. When light of different wavelengths (300 nm-infrared) was tested, only near-ultraviolet (310–330 nm) radiation stimulated ascospore discharge. Vertical height of ascospore discharge was also determined. When ascospores were trapped above colonies over a range of heights (2–80 mm), most spores were caught at 2 mm; none was caught at heights above 30 mm. The number of spores trapped at 30 mm was only 1.3% of the capture at 2 mm.

A meiotic mutant of the fission yeast Schizosaccharomyces pombe that produces mature asci containing two diploid spores.

A mutant of the fission yeast Schizosaccharomyces pombe grew normally in the mitotic cycle but produced two-spored asci in the meiosis cycle. These spores were diploid, and the segregation of centromere-linked markers in the dyads was mostly reductional. Only the first meiotic division appears to occur in this tws1 mutant, resulting in enclosure of diploid nuclei into spores.