Sporulation et mycosporines chez un champignon imparfait: Ascochyta fabae

Sporalation and mycosporins in the Deuteromycete Ascochyta fabae .
To investigate the sporogenic activity of mycosporins, a strain of Ascochyta fabae Speg., in which conidiogenesis and mycosporin synthesis are easily induced, has been selected. In darkness, few mature pycnidia and little mycosporin are produced. Dry weight and mycosporin production were followed during mycelial growth and the conidiogenic phase, in both illuminated and non-illuminated cultures: Mycosporin production appears closely linked with the morphogenic differentiation; its synthesis starts when the exponential growth is over. There is a good correlation between conidiogenesis and mycosporin production.
Irrespective of illumination, there is no increase in the rate of sporulation when maycosporins are added at different concentration levels. This cannot be explained by impermeability of the hyphae to mycosporins since uptake starts after a lag phase of 7 days and then proceeds until 15 to 20 days; at the same time the mycelium of A. fabae keeps its sporulation competence until 16 days after transfer to complete darkness. Mycosporin accumulated into conidiospores – 3 to 5% of dry weight – is not metabolized during the germination; on the contrary, a slight synthesis occurs after 20 h of germination.
Mycosporins thus seem to be secondary metabolites appearing as products from a type of metabolism that occurs at a very low level during mycelial growth and increases its activity during reproductive morphogenesis in numerous species of fungi.     

Perithecium morphogenesis in Sordaria macrospora

The perithecium of the self-fertile ascomycete Sordaria macrospora provides an excellent model in which to analyse fungal multicellular development. This study provides a detailed analysis of perithecium morphogenesis in the wild type and eight developmental mutants of S. macrospora, using a range of correlative microscopical techniques. Fundamentally, perithecia and other complex multicellular structures produced by fungi arise by hyphal aggregation and adhesion, and these processes are followed by specialization and septation of hyphal compartments within the aggregates. Perithecial morphogenesis can be divided into the ascogonial, protoperithecial, and perithecial stages of development. At least 13 specialized, morphologically distinct cell-types are involved in perithecium morphogenesis, and these fall into three basic classes: hyphae, conglutinate cells and spores. Conglutinate cells arise from hyphal adhesion and certain perithecial hyphae develop from conglutinate cells. Various hypha-conglutinate cell transitions play important roles during the development of the perithecial wall and neck.     

Composition sterolique et morphogeneses reproductrices chez Gnomonia leptostyla

Sterol components of Gnomonia leptostyla mycelia have been investigated from in vitro cultures in which sexual and asexual morphogenesis are induced by temperature and light conditions. The nature and content of free sterols and sterol esters were determined by MIKE spectrometry. Relations between sterol composition (total sterols; Δ^5,7 and Δ⁵ sterols) and reproductive morphogenesis are discussed, particularly with respect to the degree of sexuality induced.     

Scanning Electron Microscopy on the Perithecial Development of Phyllactinia corylea on Mulberry-II. Sexual Stage

Development of perithecia of Phyllactinia corylea (Pers.) Karst. on mulberry (Morus spp.) leaves was examined by scanning electron microscopy. Two short specialized structures, antheridium and ascogonium, emerged from two separate hyphae, were fused with each other and developed into an egg‐shaped perithecial primordia. These primordia later developed into globose immature perithecia, which covered with protruded wall cells with clear margins. A large number of hyphae emerged from near the base of globose perithecia, which radiated on the leaf surface and thus helped the perithecia to fix to the surface. Specific characteristic penicillate cells and acicular appendages originated from the immature perithecia. The penicillate cells developed with apical sterigma‐like projections from the wall cells of the upper part of immature perithecia, whereas the acicular appendages originated from the shrunken wall cells at the perithecial equatorial planes. On maturation of perithecia, the acicular appendages bent down and pushed the perithecia above the substrate and thus helped them to liberate out. The sterigma‐like projections were covered with paste‐like granular substance, which help the dispersed perithecia to attach to mulberry leaves and branches.     

Influence de la lumière et de la mycosporine sur la composition stérolique du Nectria galligena au cours de la morphogenese sexuée

The nature and content of free sterols, analyzed by MIKE spectrometry, were investigated in in vitro cultures submitted to different light regimes with or without the addition of exogenous mycosporin. The sterol composition was related to varying degrees of induced sexual morphogenesis, particularly when mycosporin was added to the nutrient medium.     

A Glomerella species phylogenetically related to Colletotrichum acutatum on Norway maple in Massachusetts

A fungus isolated from Norway maple (Acer platanoides) in the Boston, Massachusetts, area was determined to be a species of Glomerella, the teleomorph of Colletotrin chum acutatum. Pure cultures of the fungus were obtained from discharged ascospores from perithecia in leaf tissue. This fungus was determined to be homothallic based on the observation of perithecial development in cultures of single-spore isolates grown on minimal salts media and with sterile toothpicks. A morphological and molecular analysis was conducted to determine the taxonomic position of this fungus. Parsimony analyses of a combined nucleotide dataset of the ITS and LSU rDNA region, and of the D1-D2 LSU rDNA region, indicated that this species has phylogenetic affinities with Colletotrichum acutatum, C. acutatum f. sp. pineum, C. lupini, C. phormii and G. miyabeana. These results are significant because C. acutatum has not been reported on Acer platanoides. In addition the consistent presence of perithecia on leaf tissue and in culture is unusual for Colletotrichum, suggesting that the teleomorphic state is important in the life cycle of this fungus.     

Effect of Trichoderma harzianum on perithecial production of Gibberella zeae on wheat straw

Conidial suspensions and cell-free filtrates of Trichoderma harzianum isolates were evaluated for their effectiveness in reducing perithecial and ascospore production of Gibberella zeae on wheat straw. Isolate T-22, which is registered in the US as a biological control agent (Plant Shield™), was included in the study as a positive control. When co-inoculated with G. zeae all 11 isolates of T. harzianum significantly reduced perithecial numbers on wheat straw. Five T. harzianum isolates, including T-22, reduced perithecial formation by 70% or greater. All isolates of G. zeae, varied in their ability to produce perithecia. Isolate 192132 produced the greatest number of perithecia and was used to further evaluate the effect of application time of the T. harzianum isolates. Perithecial reduction was highest (96-99%) when T. harzianum conidial suspension or cell-free filtrate was applied to straw 24 h prior to inoculation with G. zeae. Control was less effective when T. harzianum was applied at the same time (co-inoculated) or 24 h after G. zeae. Treatments which reduced perithecial numbers also reduced ascospore numbers; however, the average numbers of ascospores per perithecia were not significantly lowered. Field trials showed significant reduction of perithecia on residues treated with T. harzianum prior to placement on the soil surface. Both T. harzianum and G. zeae were re-isolated from residues sampled in July and August after 30 and 60 days of exposure to the environment.     

Nutrient-dependent inhibition of perithecial development due to sequential crosses on the same mycelium of Neurospora tetrasperma

Summary An explanation of perithecial inhibition in the second of two sequential crosses at different locations on the same mycelium of Neurospora tetrasperma was sought by (1) assaying media that had supported inhibited and uninhibited portions of the mycelium which contained no developing perithecia, (2) determining the effect of these media on perithecial development, (3) adding nutrients to inhibited portions of the mycelium, and (4) assaying carbon sources in media that had supported portions of the mycelium which contained developing perithecia, and portions, both inhibited and uninhibited, which contained no developing perithecia. Different kinds and volumes of media and various intervals of time between sequential crosses were used to aid in determining limits of perithecial inhibition. Perithecial inhibition was observed to be independent of volatile metabolites and pH, independent of non-volatile metabolites, reversible by addition of nutrients, dependent upon nutrient volume, and correlated with the concentration of the carbon source in the medium. It is proposed that second crosses are inhibited because of a previous lowering of the concentration of nutrients in the medium in second-cross locations, owing to prior demand upon those nutrients by the developing perithecia in first-cross locations. The possibility of an activation signal between first- and second-cross locations is discussed. No inhibitory substance in inhibited locations was detected.Supported in part by a National Science Foundation Traineeship.     

Roles for receptors, pheromones, G proteins, and mating type genes during sexual reproduction in Neurospora crassa

Here we characterize the relationship between the PRE-2 pheromone receptor and its ligand, CCG-4, and the general requirements for receptors, pheromones, G proteins, and mating type genes during fusion of opposite mating-type cells and sexual sporulation in the multicellular fungus Neurospora crassa. PRE-2 is highly expressed in mat a cells and is localized in male and female reproductive structures. Δpre-2 mat a females do not respond chemotropically to mat A males (conidia) or form mature fruiting bodies (perithecia) or meiotic progeny (ascospores). Strains with swapped identity due to heterologous expression of pre-2 or ccg-4 behave normally in crosses with opposite mating-type strains. Coexpression of pre-2 and ccg-4 in the mat A background leads to self-attraction and development of barren perithecia without ascospores. Further perithecial development is achieved by inactivation of Sad-1, a gene required for meiotic gene silencing. Findings from studies involving forced heterokaryons of opposite mating-type strains show that presence of one receptor and its compatible pheromone is necessary and sufficient for perithecial development and ascospore production. Taken together, the results demonstrate that although receptors and pheromones control sexual identity, the mating-type genes (mat A and mat a) must be in two different nuclei to allow meiosis and sexual sporulation to occur.     

Scanning electron microscopy of surface and internal features of developing perithecia of Neurospora crassa.

Stages in the development of perithecia of Neurospora crassa, designated by the time elapsed after crossing, were investigated with the scanning electron microscope, from protoperithecia through perithecia. The usual examination of external features of whole specimens with this instrument was augmented by a freeze-fracture technique which allowed the viewing of development internally as well. Rapid increases in perithecial size soon after crossing were followed by the appearance, in section, of a centrum, at first undifferentiated but subsequently developing ascogenous hyphae. The perithecial beak appeared as a compact mass easily distinguishable in whole specimens from the surrounding hyphae by means of texture as well as shape. Two ascospores were photographed during emergence from an ostiole, but ostioles were found more frequently closed than open.     

Experiments using sterilized apple-leaf discs to study the mode of action of urea in suppressing perithecia of Venturia inaequalis (Cke.) Wint.

Laboratory experiments using sterilized apple-leaf discs showed that treatment of leaves with urea during the early stages of perithecial initiation induced a high nitrogen content of the leaves, which prevented further development of perithecia although mycelial growth was unaffected. Treatments applied at a later stage of fungal development inhibited both perithecial and mycelial growth. Some of the bacteria isolated from urea-treated leaves in the field restricted perithecial development, particularly when applied in the first month after inoculation with suspensions of conidia from sexually compatible strains of the fungus. One isolate, a Pseudomonas sp., was shown to be important in the decomposition of apple leaves.     

Barbatosphaeria gen. et comb. nov., a new genus for Calosphaeria barbirostris

The new genus Barbatosphaeria is described for a perithecial ascomycete known as Calosphaeria barbirostris occurring on decayed wood of deciduous trees under the periderm. The fungus produces nonstromatic perithecia with hyaline, 1-septate ascospores formed in unitunicate, nonamyloid asci. Anamorphs produced in vitro belong to Sporothrix and Ramichloridium with holoblastic-denticulate conidiogenesis; conidiophores of the two types were formed in succession during the development of the colony. Phylogenetic analyses of nuLSU rDNA sequences indicate that this fungus is distinct from morphologically similar Lentomitella, tentatively placed in the Trichosphaeriales. It groups with freshwater Aquaticola and Cataractispora and terrestrial Cryptadelphia in maximum parsimony analysis; the same grouping but without Cryptadelphia was inferred from Bayesian analysis. Cultivation, morphology and phylogenetic studies of the nuLSU rDNA support the erection of a new genus for C. barbirostris.     

Media for identification of Gibberella zeae and production of F-2-(Zearalenone).

Media are described for the isolaton of Fusarium graminearum in the perithecial state, Gibberella zeae, and for the production of F-2 (zearalenone) by Fusarium species. On soil extract-corn meal agar isolated medium, G. Zeae produced perithecia in 9 to 14 days under a 12-h photoperiod. Species of Fusarium were screened for F-2 production on a liquid medium. From strains that produced F-2, the yields, from stationary cultures of G. zeae and F. culmorum after 12 days of incubation, ranged from 22 to 86 mg/liter. Three strains produced no F-2. Glumatic acid, starch, yeast extract,and the proper ratio of medium volume-to-flask volume were necessary for F-2 synthesis.     

PERITHECIAL FORMATION IN GELASINOSPORA RETICULISPORA. I. EFFECTS OF LIGHT AT TWO DIFFERENT GROWTH STATES

Hyphae of Gelasinospora reticulispora were cultured on corn meal agar in a growth tube at 25 ± 0.4°C under different light conditions. While the hyphal tip was growing, perithecia were not formed under continuous white light (ca. 2000 ergs cm^?2 sec^?1), but some perithecia were initiated in total darkness. However, when white light was given after a dark period, perithecial formation was greatly promoted. In these cases, perithecial formation occurred in older portion of the culture (the portion nearest the point of inoculation) at first, and then gradually spread to the younger portion. Immediately after the tip of hyphae reached the other end of the growth tube, perithecia were induced in the youngest portion of the hyphae irrespective of the photoconditions; then formation proceeded toward the older portion. This induction was not age-dependent, because in growth tubes with different lengths, perithecia always became visible ca. 24 hr after the tip of hyphae reached the other end of growth tube. The photoinhibitory effect was no longer observed thereafter, but photopromotive effect was still evident.     

Media for identification of Gibberella zeae and production of F-2-(Zearalenone).

Media are described for the isolaton of Fusarium graminearum in the perithecial state, Gibberella zeae, and for the production of F-2 (zearalenone) by Fusarium species. On soil extract-corn meal agar isolated medium, G. Zeae produced perithecia in 9 to 14 days under a 12-h photoperiod. Species of Fusarium were screened for F-2 production on a liquid medium. From strains that produced F-2, the yields, from stationary cultures of G. zeae and F. culmorum after 12 days of incubation, ranged from 22 to 86 mg/liter. Three strains produced no F-2. Glumatic acid, starch, yeast extract,and the proper ratio of medium volume-to-flask volume were necessary for F-2 synthesis.     

Scanning electron microscopy observations of the interaction between Trichoderma harzianum and perithecia of Gibberella zeae

Chronological events associated with the interaction between a strain of Trichoderma harzianum, T472, with known biological control activity against perithecial production of G. zeae, were studied with scanning electron microscopy to investigate the mechanisms of control. Large clusters of perithecia consisting of 5-15 perithecia formed on the autoclaved, mulched wheat straw inoculated with G. zeae alone (control) with an average of 157 perithecia per plate. Small clusters consisting of 3-6 and an average of 15 perithecia per plate perithecia formed on straw that was treated with T. harzianum. The mature perithecia from straw treated with T. harzianum produced less pigment and were lighter in color than those from the control plates. Furthermore the cells of the outer wall of these perithecia were abnormal in appearance and unevenly distributed across the surface. Immature perithecia were colonized by T. harzianum approximately 15 d after inoculation (dai) with the biocontrol agent and pathogen. Few perithecia were colonized at later stages. The affected perithecia collapsed 21 dai, compared to the perithecia in the control samples that began to collapse 28 dai. Abundant mycelium of T. harzianum was seen on the perithecia of treated samples. Perithecial structures may be resistant to penetration by the mycelium because direct penetration was not observed. Trichoderma harzianum colonized the substrate quickly and out-competed the pathogen, G. zeae.     

Field and laboratory studies of the effect of urea on ascospore production of Venturia inaequalis (Cke.) Wint

Applications of urea after harvest but before leaf-fall restricted perithecial production by Venturia inaequalis. Immersion of detached leaves in urea appeared to be the most effective method of preventing perithecial formation, although spraying attached leaves was equally effective when leaf abscission occurred within a week of treatment. A high nitrogen content within the leaf was one of the major factors contributing to suppression. Urea-treated leaves decomposed rapidly, thus destroying the overwintering substrate for the fungus. When apple plants (clone M. 111) were sprayed in autumn with 5 % urea, followed by a second (pre-bud-burst) application at 2 %, ascospore production in the spring was suppressed. The second treatment appeared to prevent the release of ascospores from mature perithecia.