Mapping the distribution of 3-hydroxy oxylipins in the ascomycetous yeast Saturnispora saitoi

The distribution of 3-hydroxy oxylipins in Saturnispora saitoi was mapped using immunofluorescence microscopy. Fluorescence was observed on aggregating ascospores, indicating the presence of 3-hydroxy oxylipins on the surface or between ascospores. The oxylipin was identified as 3-hydroxy 9:1 using gas chromatography mass spectrometry. Furthermore, ultrastructural studies using scanning and transmission electron microscopy on ascospores revealed a clear equatorial ledge surrounding oval-shaped ascospores.     

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.     

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.     

Oxylipin covered ascospores of Eremothecium coryli

Eremothecium coryli is known to produce intriguing spindle-shaped ascospores with long and thin whip-like appendages. Here, ultra structural studies using scanning electron microscopy, indicate that these appendages serve to coil around themselves and around ascospores causing spore aggregation. Furthermore, using immunofluorescence confocal laser scanning microscopy it was found that hydrophobic 3-hydroxy oxylipins cover the surfaces of these ascospores. Using gas chromatography–mass spectrometry, only the oxylipin 3-hydroxy 9:1 (a monounsaturated fatty acid containing a hydroxyl group on carbon 3) could be identified. Sequential digital imaging suggests that oxylipin-coated spindle-shaped ascospores are released from enclosed asci probably by protruding through an already disintegrating ascus wall.     

Acetylsalicylic acid as antifungal in <Emphasis Type="Italic">Eremothecium</Emphasis> and other yeasts

Interesting distribution patterns of acetylsalicylic acid (ASA, aspirin) sensitive 3-hydroxy (OH) oxylipins were previously reported in some representatives of the yeast genus Eremothecium—an important group of plant pathogens. Using immunofluorescence microscopy and 3-OH oxylipin specific antibodies in this study, we were able to map the presence of these compounds also in other Eremothecium species. In Eremothecium cymbalariae, these oxylipins were found to cover mostly the spiky tips of narrowly triangular ascospores while in Eremothecium gossypii, oxylipins covered the whole spindle-shaped ascospore with terminal appendages. The presence of these oxylipins was confirmed by chemical analysis. When ASA, a 3-OH oxylipin inhibitor, was added to these yeasts in increasing concentrations, the sexual stage was found to be the most sensitive. Our results suggest that 3-OH oxylipins, produced by mitochondria through incomplete β-oxidation, are associated with the development of the sexual stages in both yeasts. Strikingly, preliminary studies on yeast growth suggest that yeasts, characterized by mainly an aerobic respiration rather than a fermentative pathway, are more sensitive to ASA than yeasts characterized by both pathways. These data further support the role of mitochondria in sexual as well as asexual reproduction of yeasts and its role to serve as a target for ASA antifungal action.     

Bioprospecting for novel oxylipins in fungi: The presence of 3-hydroxy oxylipins in Pilobolus

As previously found in various members of the Mucorales, 3-hydroxy oxylipins in Mucor genevensis are associated with the sporangia, i.e. mainly the columella structure and between aggregating sporangiospores. To determine if this phenomenon is also true in distantly related members, the mucoralean fungus Pilobolus was examined. This fungus is characterized by relatively large sub sporangial-columella structures which actively eject sporangia in a sticky liquid for attachment onto herbage surrounding its growth medium – in this case horse dung. Strikingly, this fungus produced a novel oxylipin i.e. a 3-hydroxy monounsaturated fatty acid, possibly a nonenoic acid, which is mainly associated with the sub sporangial-columella structure and aggregating sporangiospores. The specificity of the antibody against 3-hydroxy oxylipins used in immunofluorescence mapping of the mucoralean fungi, was further confirmed in the yeast, Saccharomycopsis malanga which produces 3-hydroxy palmitate in crystal form. These crystals occur between aggregating yeast cells. On the basis of the available data, we hypothesize that 3-hydroxy oxylipins probably function as adhesives, attaching fungal cells to each other or to other surfaces through entropic based hydrophobic forces and/or hydrogen bonds.     

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.     

The presence of 3-hydroxy oxylipins on the ascospore surfaces of some species representing Saccharomycopsis Schiönning

Through gas chromatography - mass spectrometry, the presence of oxylipins, mainly 3-hydroxy 9:1 and 3-hydroxy 10:1, was detected in Saccharomycopsis fermentans, Saccharomycopsis javanensis, and Saccharomycopsis vini. The distribution of these compounds was mapped using immunofluorescence microscopy, and they were found to be closely associated with the surfaces of aggregating ascospores.     

The presence of novel 3-hydroxy oxylipins on surfaces of hat-shaped ascospores of Ascoidea africana Batra & Francke-Grosmann

Immunofluorescence microscopy exposed the presence of novel 3-hydroxy oxylipins on the surfaces of aggregated hat-shaped ascospores of Ascoidea africana. These compounds were confirmed by gas chromatography - mass spectrometry analysis. Only the complete structure of a novel 3-hydroxy 10:1 could be determined.     

Oxylipin and mitochondrion probes to track yeast sexual cells

When oxylipin and mitochondrion probes, i.e., fluorescing antibodies specific for 3-hydroxy fatty acids (3-OH oxylipins) and rhodamine 123 (Rh123), were added to yeast cells, these probes accumulated mainly in the sexual cells (i.e., both associated with ascospores) and not in the vegetative cells. This suggests increased mitochondrial activity in asci, since 3-OH oxylipins are mitochondrially produced and it is known that Rh123 accumulates selectively in functional mitochondria that maintain a high transmembrane potential (Delta Psi m). This increased activity may be necessary for the production and effective release of the many spores found in single-celled asci. These results may be useful in the rapid identification of asci and in yeast sexual spore mechanics, which may find application in yeast systematics as well as hydro-, aero-, and nano-technologies.     

Airborne Ascomycotina on the island of Crete: Seasonal patternsbased on an 8-year volumetric survey

An 8-year study was conducted on the island of Crete in order to identify airborne ascospores and to determine their seasonal pattern. A Burkard 7-day, volumetric spore-trap was continuously operated in the city of Irakleion – located in the center of the island – from 1994 through 2001. Relatively „high” ascospore counts (20 – 48 spores/m ³) were obtained from mid-spring through summer, while the rest of the year exhibited lower activity (8–16 spores/m³). The predominant ascospores identified were those of Leptosphaeria and Chaetomium; their concentrations varied from 1 or 2 spores up to a few dozens of spores/m³. Other spores encountered sporadically were: Ascobolus, Endophragmiella, Didymella, Diatrypaceae, Leptosphaerulina, Massaria, Pleospora, Sporormiella, Xylaria. The mean daily concentration of all identified ascospores was 30/m³ for the entire study period, corresponding to 13.9% of the total fungal load. Ascospores have been recognized as important inhalant allergens and have been implicated as contributing to symptoms of both rhinitis and asthma.     

Three new <Emphasis Type="Italic">Ophiostoma</Emphasis> species isolated from Japanese red pine

Three new species of Ophiostoma found on Japanese red pine are described as Ophiostoma pusillum sp. nov., O. botuliforme sp. nov., and O. nigrogranum sp. nov. Ophiostoma pusillum is characterized by oblong ascospores and a Hyalorhinocladiella anamorph. Ophiostoma botuliforme has ostioles covered with a hyaline gelatinous cap, allantoid ascospores, and a Pesotum anamorph with hyaline to pale brown stipes. Ophiostoma nigrogranum has hyaline ostiolar hyphae with rounded tips, allantoid ascospores, and sclerotium-like structures.Contribution no. 172, Laboratory of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba     

Three new <Emphasis Type="Italic">Ophiostoma</Emphasis> species with <Emphasis Type="Italic">Pesotum</Emphasis> anamorphs associated with bark beetles infesting <Emphasis Type="Italic">Abies</Emphasis> species in Nikko,Japan

Three species of Ophiostoma possessing Pesotum anamorphs isolated from bark beetles and their galleries infesting Abies species in Nikko, Japan, are described as new species. Ophiostoma nikkoense is characterized by brush-shaped synnemata producing long septate clavate conidia, perithecia with neck, and allantoid ascospores. Ophiostoma microcarpum has smaller perithecia with hyphoid ostiolar hyphae on the neck, and the ascospores are cylindrical or ossiform in side and face views. Ophiostoma abieticola has perithecia without ostiolar hyphae on the neck and produces orange-section-shaped or reniform ascospores.Contribution no. 187, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba     

Airborne fungal spores and the thunderstorm of 24 June 1994

On the evening of 24 June 1994 there were thunderstorms which passed from the south to the north over large areas of England, followed by an extensive and initially alarming episode of 'thunderstorm asthma'. It was eventually concluded that the probable cause of this episode was the release of allergenic particles from wetted grass pollen. As part of the investigation of this episode changes in the fungal air spora were investigated at several sites. The fungal spores present varied from site to site. At London, Addlestone and Leicester there were very marked transient peaks of smut ustilospores (and to a much smaller extent at Cambridge), particularly those of Ustilago segetum (Bull.) Roussel. At many sites there were transient peaks of Cladosporium conidia. The possible causes of the transient peaks are discussed. At most of the other sites except London there were high concentrations of ascospores after the rain. Typically, Didymella ascospores occur after rain, but on this occasion they occurred at low concentrations at all sites, because of dry weather in the previous month. Instead, there were high concentrations of ascospores which normally occur in far lower numbers, viz. ascospores of Phaeosphaeria nigrans and Diatrypaceous ascospores. The reasons for this were related to increased rainfall in the previous thirteen months. There were site to site differences in the ascospore composition of the air spora, and this was related to habitat differences, where this was known. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mapping the distribution of 3-hydroxylipins in the Mucorales using immunofluorescence microscopy

The distribution of endogenous 3-hydroxylipins (3-OH oxylipins) in representatives of the Mucorales was mapped using immunofluorescence microscopy. Strains of each of the following genera were examined: Absidia, Actinomucor, Cunninghamella, Mortierella (subgenus Micromucor), Mortierella (subgenus Mortierella), Mucor and Rhizomucor. Immunofluorescence microscopy was carried out using an antibody that was raised against 3R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid (3R-HETE), which cross-reacts with other 3-OH oxylipins. Subsequently, the occurrence and distribution of the antibody on the various reproductive stages of each fungus was noted. In Absidia, Actinomucor, Mortierella (subgenus Micromucor), Mucor and Rhizomucor, 3-OH oxylipins were found to be associated with the columellae and/or wall of the sporangium. In the representative of Cunninghamella, the 3-OH oxylipins were associated with the single-spored sporangiola. No 3-OH oxylipins were detected in the strains representing Mortierella (subgenus Mortierella).     

Development and dehiscence of the cephalothecoid peridium in Aporothielavia leptoderma shows it belongs in Chaetomidium

The development of the cephalothecoid peridium of Aporothielavia leptoderma was examined using light and electron microscopy. Early stages in ascoma initiation were consistent with previous reports for other species in the Chaetomiaceae. However, as young cleistothecia increased in size, clusters of peridial cells in the outer textura angularis elongated in a radial pattern around a central cell or cell cluster to form rosettes of relatively thick-walled segments that marked the central areas of incipient cephalothecoid plates. The external flank along median portions of the radial cells became thin walled and swelled outwards so that each plate became concave and was separated from adjacent plates by a more or less circular to polygonal ridge of knuckle-shaped swellings. When dry, mature peridia split apart along some of the ridges demarcating individual plates but an internal mechanism for liberating ascospores from the confines of the ascoma was not observed. Physical disturbance of mature cleistothecia by beetles, when enclosed together in a Petri dish, shattered the peridia, liberating the ascospores. Smaller insects were unable to cause disarticulation of the cephalothecoid plates. Because of the presence of an apical germ pore in the ascospores and morphological similarity to Chaetomidium arxii, the new combination Chaetomidium leptoderma (syn. Thielavia leptoderma) comb. nov. is proposed.     

Chemotaxonomic discrimination among the fungal genera Tolypocladium, beauveria and Paecilomyces

Fungi of the genera Beauveria and Paecilomyces were found to produce cyclotetradepsipeptides, beauverolides. Production of beauverolides was not detected at the genus Tolypocladium. Analysis of beauverolides therefore provides a very simple chemotaxonomic test which seems to be suitable for fast discrimination between the genera Beauveria vs Tolypocladium and complementing morphological examination. A GC-MS study of β-hydroxy acid distribution in the beauverolide hydrolyzates revealed that all strains prdouce γ-methyl-β-hydroxy acids only. Their occurrence thus cannot be used as a taxonomic marker of different species within the genera Beauveria and Paecilomyces.     

Two new species of <Emphasis Type="Italic">Neosartorya</Emphasis> from Amazonian soil,Brazil

Neosartorya indohii and N. tsurutae, two new Neosartorya species isolated from tropical rainforest soil in the Amazonian area, Brazil, are described and illustrated. Neosartorya indohii is characterized by its spreading growth on Czapeks and malt extract agars, light yellow cleistothecia, broadly lenticular ascospores with two conspicuously serrate-incised equatorial crests and tuberculate convex surfaces, and globose to subglobose conidia with a smooth wall. Neosartorya tsurutae is characterized by its spreading growth on Czapeks and malt extract agars, white cleistothecia, broadly lenticular ascospores with four equatorial crests and rugulose-ruminate convex surfaces, and ovoid to broadly ellipsoidal conidia with a smooth wall.     

Japanese species of Hypomyces and its anamorph III

Three new species of Hypomyces with KOH-negative subiculi occurring on the Aphyllophorales are described. Hypomyces pseudocorticiicola is characterized by a grayish-yellow to brown subiculum, fusiform ascospores, always with one septum, and its Cladobotryum-anamorph. Hypomyces laeticolor is characterized by a bright-colored subiculum, small apiculi on the ascospores, and well-developed verticillate conidiogenous cells. Hypomyces penicillatus forms a Trichothecium-like anamorph with well-developed verticils of conidiogenous cells.     

Homothallism in Sclerotinia minor

Sclerotinia species are sexually reproducing ascomycetes. In the past S. minor and S. sclerotiorum, have been assumed to be homothallic because of the self-fertility of colonies derived from single ascospores. S. trifoliorum has previously been shown to be bipolar heterothallic due to the presence of four self-fertile and four self-sterile ascospores within a single ascus [Uhm, J.Y., Fujii, H., 1983a. Ascospore dimorphism in Sclerotinia trifoliorum and cultural characters of strains from different-sized spores. Phytopathology 73: 565–569]. However, isolates of S. minor and S. sclerotiorum were proven to be homothallic ascomycetes, by self-fertility of all eight ascospores within an ascus. Apothecia were raised from all eight ascospores of a single tetrad from four isolates of S. minor and from an isolate of S. sclerotiorum, indicating that inbreeding may be the predominant breeding mechanism of S. minor. Ascospores from asci of S. minor and S. sclerotiorum were predominantly monomorphic, but rare examples of ascospore dimorphism similar to S. trifoliorum were found.