Hypoxylon cohaerens: Cytology of the ascus

The haploid chromosome number ofHypoxylon cohaerens apparently is 5, based on counts made at meiotic prophase and meiotic and mitotic metaphases. Newly formed ascospores are at first uninucleate, becoming binucleate following mitosis in the ascospore. Subsequently, one of the two nuclei disappears. Maturing ascospores are uninucleate.Scientific Paper No. 3732 Washington State University. College of Agriculture, Project 1767. This study was supported in part by National Science Foundation Grant GB-19924.     

MORPHOLOGY OF CHAETOMIUM ERRATICUM

Development of perithecia from single, uninucleate ascospores disclosed a homothallic condition for Chaetomium erraticum. This species was found to produce sessile ascogonial coil initials from uninucleate vegetative cells that become enveloped by hyphae formed at the base of the ascogonium. The ascogonium consists of several cells that are uninucleate or binucleate. A perithecium forms from numerous divisions and enlargement of the surrounding uninucleate cells. Differentiation of the perithecial cells results in the formation of a carbonaceous wall, perithecial hairs, and an ostiole lined with periphyses. A convex hymenial cluster of ascogenous cells forms in the lower half of the centrum from which typical croziers develop. Asci push up into the pseudoparenchyma cells of the centrum. The growth of the ascogenous system is in part responsible for increase in perithecial size. The breakdown of the pseudoparenchyma cells around the developing asci results in the formation of a central cavity in which ascospores are released when the asci deliquesce. No paraphyses are present. The type of development and features of the centrum of C. erraticum and other species of Chaetomium indicate a distinct Xylaria-type centrum.     

Hypoxylon rubiginosum: Cytology of the ascus and surface morphology of the ascospore

Summary The haploid chromosome number ofHypoxylon rubiginosum is 5. The ascospore is uninucleate when formed, becoming binucleate following a mitosis. One of the nuclei subsequently disintegrates. Maturing ascospores are uninucleate.The morphology of the ascospore, as revealed by the scanning electron microscope, is described. The outer wall layer — the perisporium — shows heretofore undescribed surface fibrils. The possible significance of the fibrils is discussed.Paper No. 3205. Washington State University College of Agriculture Project 1767. This study was supported in part by National Science Foundation Grants GB-5219 and GB-8004.     

ULTRASTRUCTURE OF ASCOCARP DEVELOPMENT IN SPORORMIA AUSTRALIS

Thin sections taken from intact ascocarps were examined to trace the developmental sequence of ascocarp formation in Sporormia australis Speg. The ascocarp originated from a uninucleate vegetative hyphal cell which underwent repeated divisions and formed an ascostroma. In the center of the young ascostroma a cavity formed, apparently from cell disintegrations, and enlarged as the ascocarp enlarged. Within the cavity pseudoparaphyses developed from undifferentiated pseudoparenchymatous cells at the apex of the cavity and extended downward. Ascogenous hyphae arose from proliferating uninucleate cells at the base of the cavity. As the ascocarp matured, the pseudoparenchymatous cells differentiated into three layers, none of which were considered homologous to the perithecial wall lining the cavity of pyrenomycetes. The cells of the apex were not differentiated into layers and light microscopy revealed the presence of an ostiole through which bitunicate asci discharged their eight 4-celled ascospores.     

The meiosis‐specific APC activator FgAMA1 is dispensable for meiosis but important for ascosporogenesis in Fusarium graminearum

Ascospores are the primary inoculum in Fusarium graminearum. Interestingly, 70 of its genes have premature stop codons (PSC) and require A‐to‐I editing during sexual reproduction to encode full‐length proteins, including the ortholog of yeast Ama1, a meiosis‐specific activator of APC/C. In this study, we characterized the function of FgAMA1 and its PSC editing. FgAMA1 was specifically expressed during sexual reproduction. The Fgama1 mutant was normal in growth and perithecium formation but defective in ascospogenesis. Instead of forming four‐celled, uninucleate ascospores, Fgama1 mutant produced oval, single‐celled, binucleated ascospores by selfing. Some mutant ascospores began to bud and underwent additional mitosis inside asci. Expression of the wild‐type or edited FgAMA1 but not the uneditable allele complemented Fgama1. In the Fgama1 x mat‐1‐1 outcross, over 60% of the asci had eight Fgama1 or intermediate (elongated but single‐celled) ascospores, suggesting efficient meiotic silencing of unpaired FgAMA1. Deletion of FgPAL1, one of the genes upregulated in Fgama1 also resulted in defects in ascospore morphology and budding. Overall, our results showed that FgAMA1 is dispensable for meiosis but important for ascospore formation and discharge. In F. graminearum, whereas some of its targets are functional during meiosis, FgAma1 may target other proteins that function after spore delimitation.     

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.     

Uninucleate spores of Phycomyces

Summary Under most culture conditions only 0.3% of the vegetative spores of Phycomyces blakesleeanus are uninucleate. On an acidified minimal medium, the uninucleate fraction can be raised up to 4.5% of the spores. The spore population can be fractionated in a gradient under gravity (1xg) yielding fractions that contain over 80% uninucleate spores. These uninucleate spores are fully viable. When the spores to be fractionated are obtained from a heterokaryotic mycelium, the uninucleate fraction produces homokaryotic mycelia.     

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.     

Four new species and a new Chinese record of the nectrioid fungi

Four new species belonging to Bionectria, Calonectria, Haematonectria and Neonectria on plant substrates collected from nature reserves in southern and central China are described. Bionectria truncata has smooth perithecia of a flattened to shallow discoid apex, clavate asci with an apical ring, and ellipsoid, smooth to spinulose ascospores. Calonectria dicephalospora is characterized by pyriform perithecia with a warted surface, clavate asci with a simple apex and long, narrow stalk, and fusoid ascospores with a cap-like appendage at each end. Haematonectria lushanensis possesses warted perithecia which are laterally collapsing when dry, cylindrical asci with a simple apex, and ellipsoid, spinulose ascospores. Neonectria dinghushanica is distinguishable by subglobose perithecia with a warted surface, clavate asci, and striate ascospores. Morphological features of these new species are described comprehensively and compared with their related fungi. Neonectria castaneicola is recorded as new to China.     

Development of microsporesin vivo andin vitro inBrassica napus L.

Summary Populations of highly homogeneous uninucleate and binucleate microspores ofBrassica napus cv. Topas were obtained by bud selection and percoll fractionation. The development of the uninucleate and the binucleate microspores in culture was compared to thosein vivo using the fluorochrome DAPI to stain DNA. The major developmental pathway of the uninucleate microsporesin vitro resulted in embryo formation. The characteristic of this pathway was that the first division produced two diffusely stained nuclei and subsequent divisions gave rise to a multinucleate embryoid. The second pathway which occurred in a small number of the uninucleate microspores led to callus formation. The majority of the binucleate microsporesin vitro followed the developmental pattern of their counterpartsin vivo and were not embryogenic. The embryogenic binucleate microspores produced embryos through the divisions of the vegetative nucleus.Plant Research Centre Contribution # 1147     

Immunodetection of Venturia inaequalis Ascospores with Phage Antibodies

We describe the bacterial expression of single chain variable fragment (scFv) antibodies that bind specifically to the ascospores of Venturia inaequalis (Cooke). A scFv phage display library was prepared from the expressed V‐gene repertoire of a mouse immunized with whole V. inaequalis ascospores. Affinity selection was then carried out using intact, non‐germinated ascospores. The binding of selected phage antibodies was monitored by enzyme‐linked immunosorbent assay, flow cytometry and fluorescence microscopy. Several scFv antibodies were found to bind specifically to V. inaequalis ascospores. No cross‐reactivity was detected with spores from other phytopathogenic fungi, such as Plectosphaerella cucumerina, Cladosporium spp. and Alternaria brassicicola. Moreover, the scFvs did not bind to V. inaequalis conidiospores or mycelia. This is the first report describing the immunodetection of V. inaequalis ascospores by phage‐derived scFv antibody fragments. The degree of specificity of the antibodies is sufficiently high to allow rapid detection of ascospores within environmental probes such as those from particle samplers.     

Airborne ascospores in Mérida (SW Spain) and the effect of rain and other meteorological parameters on their concentration

Airborne ascospores have been reported to be allergenic or plant pathogens, and their presence has traditionally been associated with rainfall events. The aim of the present study was to analyze the presence of airborne ascospores in relation to weather parameters in a town in SW Spain. A seven-day recording spore trap (Burkard) was used to sample the air over 2 years at 15 m above ground level on the terrace roof of the hospital in Mérida (SW Spain). Fungal spores were identified and counted by means of two longitudinal scans over the slides under ×1000 microscopy. A correlation analysis was made of the daily meteorological data and the airborne ascospore concentrations, and t-tests were used to compare data between the 2 years. Nineteen ascospore types were defined, including one-cell ascospores (Chaetomium, Diatrype, Helvella, Xylaria), tow-cell ascospores (Diaporthe, Mycosphaerella, Nectria, Venturia), transversally septate ascospores (Melanomma, Leptosphaeria, Paraphaeosphaeria, Sporormiella, Massaria), transversally and longitudinally septate ascospores (Pleospora), and ascospores within asci (Sordaria). Leptosphaeria consisted of a group of four types described according to the number of cells, hyaline grade, wall thickness, and ornamentation, and other ascospores comprised one last additional type. The average airborne ascospore concentration was 153 ascospores/m3. One-third each of this total were from the Leptosphaeria group, with an average 54 ascospores/m3, and the two-cell ascospores or Venturia-like group (Diaporthe, Mycosphaerella, Nectria, Venturia) with 51 ascospores/m3 on average. In third position was Pleospora with 27 ascospores/m3 on average. The month with highest concentration was September, with 238 ascospores/m3, and the lowest March, with 56 ascospores/m3. By seasons, autumn had the highest concentrations, followed by winter, spring, and summer. The maximum daily concentration reached was 3,371 ascospores/m3. Daily rainfall was significantly correlated with the ascospore types Diatrype, Mycosphaerella, Nectria, two subtypes of Leptosphaeria, and Pleospora. Relative humidity was positively correlated with those ascospore types and also with Diaporthe and Paraphaeosphaeria, and negatively with Chaetomium and Melanomma. The concentration was higher on rainy days than on days without rain for Pleospora, Leptosphaeria (3 subtypes), Diatrype, Diaporthe, Nectria, Mycosphaerella, and Paraphaeosphaeria. The daily temperatures were in general correlated with the same types as the relative humidity, but with the opposite sign. For the monthly data, there were no statistically significant differences between the 2 years studied.     

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     

Norlevinea n. g., a New Genus for Glugea daphniae (Protozoa: Microspore), a Parasite of Daphnia longispina (Crustacea: Phyllopoda)1

ABSTRACT. Norlevinea n. g. is established for microsporidia in which a uninucleate meront changes into a sporont by secreting a thin, membranous, sporontogcnetic and fragile sporophorous vesicle (pansporoblast membrane) in which four uninucleate sporoblasts are formed. In contrast to the genus Gurleya, the sporoblasts and later the spores are permanently joined into doublets, being laterally cemented by an electron-dense substance structurally identical to and continuous with the exospore layer. The polar filament is of the anisofilar type. The type species is Norlevinea daphniae (Weiser, 1947) n. comb., a parasite of the ovaries of Daphnia longispina occurring in several carp ponds in Czechoslovakia.     

Pleistophora finisterrensis n. sp., a microsporidian parasite of blue whiting Micromesistius poutassou

Pleistophora finisterrensis n. sp. is a microsporidian parasite of the hypoaxial musculature of the blue whiting Micromesistius poutassou (Risso). Foci of infection are between 3 and 6 mm in length and have no evident effects on adjacent muscle fibres. We found only a single type of spore (uninucleate, with mean dimensions of 4×2 µm in fresh preparations), contained within sporophorous vesicles (mean diameter 19 µm in fresh preparations; 150–250 spores per vesicle). All of the development stages of this microsporidian are monokaryotic. The meronts are initially uninucleate and bounded by a plasmalemma. Towards the end of merogony, meronts are multinucleate plasmodia with a well-defined surface coat. Sporogony is polysporous, with multinucleate sporonts, which likewise have a well-defined surface coat (about 130 nm thick), dividing by plasmotomy to give rise to uninucleate sporoblasts. The polar tube is isofilar and consists of 8–9 turns in the posterior half of spore. The polaroplast is made up of an anterior lamellar part and a posterior vesicular part.     

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.     

Two new species of Neosartorya isolated from soil in Xinjiang, China

Two new species, Neosartorya shendaweii and N. tsunodae, isolated from soil in Xinjing, China and in Pernambuco, Brazil, are described and illustrated. The first species is characterized by its ascospores with two widely separated equatorial crests and tuberculate to verrucose convex surfaces. This species has affinities with several known species of the genus, bearing ascospores with a similar ornamentation, but can be distinguished from these species by other morphological characteristics such as smaller cleistothecia and conidiophores, spathulate vesicles and rather ellipsoidal conidia. The second species is characterized by its unique ascospores with two low equatorial crests, an evident furrow as a deep depression, and finely reticulate convex surfaces. The validation of these new species is supported further by analyses of the β-tubulin, calmodulin and actin gene sequences.     

A new species ofClohiesia from Hong Kong

Clohiesia lignicola sp. nov. (freshwater ascomycetes) is introduced based on a specimen collected on submerged wood in the Tung Chung River, Hong Kong. Ascomata are clypeate, asci are cylindric-clavate with a relatively massive apical apparatus and ascospores are fusoid-ellipsoidal.Clohiesia lignicola differs fromC. corticola in having wider asci and wider fusoid-ellipsoidal ascospores, and larger ascomata.Clohiesia lignicola is described and illustrated with light micrographs and is also compared with species in the genus,Annulatascus.     

Importance of ascospore ornamentation in the taxonomy of Daldinia

Representative specimens of fifteen Daldinia spp. were studied for ultrastructural characteristics of their ascospores by scanning electron microscopy (SEM). The ornamentation of their outermost spore layers was found to be species-consistent, confirming the results of concurrent studies on the morphology of their teleomorphs and anamorphs, secondary metabolite profiles and PCR-based genetic fingerprints. Daldinia spp. may either show smooth or transversally striated ascospores. The spores of the species within the latter group are always ellipsoid-equilateral to ellipsoid-inequilateral with narrowly rounded ends. Smooth, broadly ellipsoid to cylindrical ascospores were observed in all species (D. caldariorum, D. fissa and D. loculata) that are known to produce their stromata on substrates damaged by fire. The ascospores of D. concentrica differed from those of D. childiae (i.e., the cosmopolitan taxon previously regarded as D. concentrica ss. auct.) and other Daldinia spp. in showing a very faint ornamentation, which only became visible at 10000× magnification by SEM. A specimen collected on the isle of Jersey (Channel Islands, UK) showed morphological similarities to the pantropical D. eschscholzii, but its ascospores appeared smooth by SEM, and it may therefore represent a previously undescribed species. Dedicated to Professor Yoshinori Asakawa, Tokushima, Japan, on the occasion of his 60^th birthday PH-R Life Science Center Natural Products