Ascogenous Hyphae Research Articles

FgBUD14 is important for ascosporogenesis and involves both stage-specific alternative splicing and RNA editing during sexual reproduction.

In wheat head blight fungus Fusarium graminearum, A-to-I RNA editing occurs specifically during sexual reproduction. Among the genes with premature stop codons (PSCs) that require RNA editing to encode full-length proteins, FgBUD14 also had alternative splicing events in perithecia. In this study, we characterized the functions of FgBUD14 and its post-transcriptional modifications during sexual reproduction. The Fgbud14 deletion mutant was slightly reduced in growth, conidiation and virulence. Although deletion of FgBUD14 had no effect on perithecium morphology, the Fgbud14 mutant was defective in crozier formation and ascus development. The FgBud14-GFP localized to the apex of ascogenous hyphae and croziers, which may be related to its functions during early sexual development. During vegetative growth and asexual reproduction, FgBud14-GFP localized to hyphal tips and both ends of conidia. Furthermore, mutations blocking the splicing of intron 2 that has the PSC site had no effect on the function of FgBUD14 during sexual reproduction but caused a similar defect in growth with Fgbud14 mutant. Expression of the non-editable FgBUD14Intron2-TAA mutant allele also failed to complement the Fgbud14 mutant. Taken together, FgBUD14 plays important roles in ascus development, and both alternative splicing and RNA editing occur specifically to its transcripts during sexual reproduction in F. graminearum.

Sexual specific functions of Tub1 beta-tubulins require stage-specific RNA processing and expression in Fusarium graminearum.

The wheat head blight fungus Fusarium graminearum has two highly similar beta-tubulin genes with overlapping functions during vegetative growth but only TUB1 is important for sexual reproduction. To better understand their functional divergence during ascosporogenesis, in this study we characterized the sequence elements important for stage-specific functions of TUB1. Deletion of TUB1 blocked the late but not initial stages of perithecium formation. Perithecia formed by tub1 mutant had limited ascogenous hyphae and failed to develop asci. Silencing of TUB1 by MSUD also resulted in defects in ascospore formation. Interestingly, the 3'-UTR of TUB1 was dispensable for growth but essential for its function during sexual reproduction. RIP mutations that specifically affected Tub1 functions during sexual reproduction also were identified in two ascospore progeny. Furthermore, site-directed mutagenesis showed that whereas the non-editable mutations at three A-to-I RNA editing sites had no effects, the N347D (not T362D or I368V) edited mutation affected ascospore development. In addition, the F167Y, but not E198K or F200Y, mutation in TUB1 conferred tolerance to carbendazim and caused a minor defect in sexual reproduction. Taken together, our data indicate TUB1 plays an essential role in ascosporogenesis and sexual-specific functions of TUB1 require stage-specific RNA processing and Tub1 expression.

In Vitro Propagation of Morchella esculenta and Study of its Life Cycle

Among the edible mushrooms, Morchella esculenta (Pers.) is a highly prized mushroom which consists of a short stalk and a conical, pitted, hollow pileus. It is commonly known as a morel or sponge mushroom, or gucchi, etc. It belongs to the class Ascomycetes, order Pezizales and family Morchellaceae. It is found throughout the world. In Pakistan, it is found in northern areas from Murree, Patriata, and Gilgit under pine vegetation. It is therapeutic in nature; therefore its medicinal and biochemical properties are under investigation throughout the world, especially in regard to treating the hazardous diseases such as cancer, tumors, etc. The present in vitro study was conducted for the first time in Pakistan. The three media soil and soil extracts Agar and Luria Agar were used. Mycellial growth was obtained on all these media, but the LA medium showed better growth. Microscopic studies had shown pigmentation is most likely due to the secondary mycelium. Among these mycelium ascogenous hyphae are produced that may lead to the formation of ascogonium and antheridium with the dilated tips. The compact mass of prosenchymatous and pseudoparenchyma tissues was observed.

Three new ascomycetes from freshwater in China

Three new freshwater ascomycetes, Diaporthe aquatica sp. nov. (Diaporthaceae), Ophioceras aquaticus sp. nov. (Magnaporthaceae) and Togninia aquatica sp. nov. (Togniniaceae), are described and illustrated based on morphological and molecular data (ITS, 18S, 28S rDNA sequences). Diaporthe aquatica is characterized by globose to subglobose, black ascomata with long necks, broadly cylindrical to obclavate asci, and small, ellipsoidal to fusiform, one-septate, hyaline ascospores; it is unusual among Diaporthe species in the fact that it lacks a stroma and has freshwater habitat. Ophioceras aquaticus is characterized by globose ascomata with a long beak, cylindrical, eight-spored asci with J- subapical rings and 3–5-septate filiform ascospores with slightly acute ends. Togninia aquatica is characterized by globose ascomata with long necks, clavate and truncate asci clustered on distinct ascogenous hyphae, and small, reniform, hyaline ascospores. Differences among the new taxa and similar species are discussed.

Sexual Development and Ascospore Discharge in <em>Fusarium graminearum</em>

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces fruiting bodies, called perithecia, on carrot agar. Perithecia contain numerous tissue types, produced at specific stages of perithecium development. These include (in order of appearance) formation of the perithecium initials (which give rise to the ascogenous hyphae), the outer wall, paraphyses (sterile mycelia which occupy the center of the perithecium until the asci develop), the asci, and the ascospores within the asci14. The development of each of these tissues is separated by approximately 24 hours and has been the basis of transcriptomic studies during sexual development12,8. Refer to Hallen et al. (2007) for a more thorough description of development, including photographs of each stage. Here, we present the methods for generating and harvesting synchronously developing lawns of perithecia for temporal studies of gene regulation, development, and physiological processes. Although these methods are written specifically to be used with F. graminearum, the techniques can be used for a variety of other fungi, provided that fruiting can be induced in culture and there is some synchrony to development. We have recently adapted this protocol to study the sexual development of F. verticillioides. Although individual perithecia must be hand picked in this species, because a lawn of developing perithecia could not be induced, the process worked well for studying development (Sikhakolli and Trail, unpublished).The most important function of fungal fruiting bodies is the dispersal of spores. In many of the species of Ascomycota (ascus producing fungi), spores are shot from the ascus, due to the generation of turgor pressure within the ascus, driving ejection of spores (and epiplasmic fluid) through the pore in the ascus tip2,7. Our studies of forcible ascospore discharge have resulted in development of a "spore discharge assay", which we use to screen for mutations in the process. Here we present the details of this assay.F. graminearum is homothallic, and thus can form fruiting bodies in the absence of a compatible partner. The advantage of homothallism is that crossing is not necessary to generate offspring homozygous for a particular trait, a facet that has facilitated the study of sexual development in this species14,7. However, heterothallic strains have been generated that can be used for crossing5,9. It is also possible to cross homothallic strains to obtain mutants for several genes in one strain1. This is done by coinoculating one Petri dish with 2 strains. Along the meeting point, the majority of perithecia will be recombinant (provided a mutation in one of the parent strains does not inhibit outcrossing). As perithecia age, they exude ascospores en masse instead of forcibly discharging them. The resulting spore exudate (called a cirrhus) sits at the tip of the perithecium and can easily be removed for recovery of individual spores. Here we present a protocol to facilitate the identification of recombinant perithecia and the recovery of recombinant progeny.

Sexual Development and Ascospore Discharge in <em>Fusarium graminearum</em>

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces fruiting bodies, called perithecia, on carrot agar. Perithecia contain numerous tissue types, produced at specific stages of perithecium development. These include (in order of appearance) formation of the perithecium initials (which give rise to the ascogenous hyphae), the outer wall, paraphyses (sterile mycelia which occupy the center of the perithecium until the asci develop), the asci, and the ascospores within the asci14. The development of each of these tissues is separated by approximately 24 hours and has been the basis of transcriptomic studies during sexual development12,8. Refer to Hallen et al. (2007) for a more thorough description of development, including photographs of each stage. Here, we present the methods for generating and harvesting synchronously developing lawns of perithecia for temporal studies of gene regulation, development, and physiological processes. Although these methods are written specifically to be used with F. graminearum, the techniques can be used for a variety of other fungi, provided that fruiting can be induced in culture and there is some synchrony to development. We have recently adapted this protocol to study the sexual development of F. verticillioides. Although individual perithecia must be hand picked in this species, because a lawn of developing perithecia could not be induced, the process worked well for studying development (Sikhakolli and Trail, unpublished). The most important function of fungal fruiting bodies is the dispersal of spores. In many of the species of Ascomycota (ascus producing fungi), spores are shot from the ascus, due to the generation of turgor pressure within the ascus, driving ejection of spores (and epiplasmic fluid) through the pore in the ascus tip2,7. Our studies of forcible ascospore discharge have resulted in development of a "spore discharge assay", which we use to screen for mutations in the process. Here we present the details of this assay. F. graminearum is homothallic, and thus can form fruiting bodies in the absence of a compatible partner. The advantage of homothallism is that crossing is not necessary to generate offspring homozygous for a particular trait, a facet that has facilitated the study of sexual development in this species14,7. However, heterothallic strains have been generated that can be used for crossing5,9. It is also possible to cross homothallic strains to obtain mutants for several genes in one strain1. This is done by coinoculating one Petri dish with 2 strains. Along the meeting point, the majority of perithecia will be recombinant (provided a mutation in one of the parent strains does not inhibit outcrossing). As perithecia age, they exude ascospores en masse instead of forcibly discharging them. The resulting spore exudate (called a cirrhus) sits at the tip of the perithecium and can easily be removed for recovery of individual spores. Here we present a protocol to facilitate the identification of recombinant perithecia and the recovery of recombinant progeny.

Orbilia ultrastructure, character evolution and phylogeny of Pezizomycotina

Molecular phylogenetic analyses indicate that the monophyletic classes Orbiliomycetes and Pezizomycetes are among the earliest diverging branches of Pezizomycotina, the largest subphylum of the Ascomycota. Although Orbiliomycetes is resolved as the most basal lineage in some analyses, molecular support for the node resolving the relationships between the two classes is low and topologies are unstable. We provide ultrastructural evidence to inform the placement of Orbiliomycetes by studying an Orbilia, a member of the only order (Orbiliales) of the class. The truncate ascus apex in the Orbilia is thin-walled except at the margin, and an irregular wall rupture of the apex permits ascospore discharge. Ascus, ascogenous and non-ascogenous hyphae were simple septate, with septal pores plugged by unelaborated electron-dense, non-membranous occlusions. Globose Woronin bodies were located on both sides of the septum. Nuclear division was characterized by the retention of an intact nuclear envelope, and a two-layered disk-shaped spindle pole body. The less differentiated nature of the spore discharge apparatus and septal pore organization supports an earliest diverging position of Orbiliomycetes within the subphylum, while the closed nuclear division and disk-shaped spindle pole body are interpreted as ancestral state characters for Ascomycota.

New insights into the systematics and phylogeny of the genus Jattaea and similar fungi of the Calosphaeriales

The phylogenetic relationship of Jattaea and Wegelina, two morphologically related genera of the Calosphaeriales, is analyzed in two molecular analyses using the Internal transcribed spacer region of the DNA operon, and the nuclear large and small subunits of ribosomal DNA combined with the second largest subunit of RNA polymerase for a multigene analysis. For each data set maximum likelihood, maximum parsimony and Bayesian analyses were performed. Type and herbarium material of two lectotype species Jattaea algeriensis and Wegelina discreta were examined and correlated with results from in vitro studies. The phylogenetic analyses show that both species are congeneric. The name Jattaea is accepted to include species with hyaline, allantoid to suballantoid ascospores in clavate, stipitate asci without apical annulus borne on individual cells on ascogenous hyphae and with phialophora-like anamorphs produced in axenic culture. Twelve species are accepted in Jattaea, the concept of the genus is outlined and a key to its species is provided. The genus Wegelina is reduced to its synonymy. Molecular data also show that septation of ascospores, a diagnostic feature used to separate calosphaeria-like fungi into the genus Phragmocalosphaeria, does not appear to be relevant in distinguishing genera in the Calosphaeriales. Therefore, Jattaea includes species with both one-celled and septate ascospores and Phragmocalosphaeria is accepted as its generic synonym. The genus Jattaea and its phialophora-like anamorphs is compared with other genera of the Calosphaeriales and the phylogenetically related Togniniaceae. The revision of four species of Ceratostomella, i.e., C. echinella, C. leptorrhyncha, C. mali and C. microspora, revealed their striking morphological similarities with the Calosphaeriaceae and the Togniniaceae. These are identified as species of Jattaea, Togninia and Togniniella and the respective new combinations are proposed.

Ultrastructural observations on the ascomata and ascospores of the truffle Mattirolomyces terfezioides

The septal pore ultrastructure of non-ascogenous and reproductive hyphae, as well as development of the spore wall were examined in the truffle Mattirolomyces terfezioides (Mattir.) E. Fisch. (Ascomycota, Pezizales, Pezizaceae) and compared with other pezizalean taxa. The septal pore in ascogenous hyphae of M. terfezioides is a uni- or bi-convex band of the type found in the Pezizaceae. Septal pore ultrastructure in other hyphae included a uniconvex band with lamellate structure. Woronin bodies were globose to hexagonal, and infrequently ellipsoid in shape. Hexagonal Woronin bodies have not been previously reported in the Pezizaceae. Ascospore delimitation starts with two delimiting membranes and the spore-wall development is typical of the Pezizales. The heterogeneously staining spines of the secondary cell wall differ from the structures of Terfezia species. The ultrastructural characteristics of M. terfezioides show the general characteristics of the Pezizaceae, except that the hexagonal Woronin body form is new to this family. The spore delimitation, spine-cap structure, and Woronin body shape of M. terfezioides presented here are in agreement with molecular taxonomic results, which separate the genus from Terfezia and call into question the generic position of Mattirolomyces tiffanyae Healy.

Crypthonia, a new genus of byssoid Arthoniaceae (lichenised Ascomycota)

Crypthonia with 11 species is described as belonging to the Arthoniaceae. The new genus is characterised by (1) white to pale carneous, maculiform ascomata lacking a distinct exciple, (2) a weakly gelatinised, hydrophobic hymenium of paraphysoids, (3) a discontinuous layer of branched ascogenous hyphae in the hypothecium, (4) small, clavate to broadly clavate asci of the Arthonia-type lacking a hemiamyloid ring structure in the tholus, (5) eight small, hyaline, 1–3-septate, thin-walled ascospores per ascus, (6) loosely attached thalli with a byssoid, coloured hypothallus, and (7) a chemistry of mainly β-orcinol depsidones and tridepsides including psoromic, norstictic, and gyrophoric acids. Rugulosin occurs in C. bella and C. citrina. Crypthonia athertoniensis Frisch & G. Thor, C. bella Frisch & G. Thor, C. brevispora Frisch & G. Thor, C. citrina Frisch & G. Thor, C. olivacea Frisch & G. Thor, C. palaeotropica Frisch & G. Thor, and C. vandenboomii Frisch & G. Thor are described. C. albida (Fee) Frisch & G. Thor, C. biseptata (Aptroot & Wolseley) Frisch & G. Thor, C. mycelioides (Vain.) Frisch & G. Thor, and C. polillensis (Vain.) Frisch & G. Thor are new combinations. The delimitation from similar genera and the position within the Arthoniaceae is discussed.

TheSaccharomyces cerevisiae PRM1Homolog inNeurospora crassaIs Involved in Vegetative and Sexual Cell Fusion Events but Also Has Postfertilization Functions

Cell-cell fusion is essential for a variety of developmental steps in many eukaryotic organisms, during both fertilization and vegetative cell growth. Although the molecular mechanisms associated with intracellular membrane fusion are well characterized, the molecular mechanisms of plasma membrane merger between cells are poorly understood. In the filamentous fungus Neurospora crassa, cell fusion events occur during both vegetative and sexual stages of its life cycle, thus making it an attractive model for studying the molecular basis of cell fusion during vegetative growth vs. sexual reproduction. In the unicellular yeast Saccharomyces cerevisiae, one of the few proteins implicated in plasma membrane merger during mating is Prm1p; prm1Delta mutants show an approximately 50% reduction in mating cell fusion. Here we report on the role of the PRM1 homolog in N. crassa. N. crassa strains with deletions of a Prm1-like gene (Prm1) showed an approximately 50% reduction in both vegetative and sexual cell fusion events, suggesting that PRM1 is part of the general cell fusion machinery. However, unlike S. cerevisiae, N. crassa strains carrying a Prm1 deletion exhibited complete sterility as either a male or female mating partner, a phenotype that was not complemented in a heterokaryon with wild type (WT). Crosses with DeltaPrm1 strains were blocked early in sexual development, well before development of ascogenous hyphae. The DeltaPrm1 sexual defect in N. crassa was not suppressed by mutations in Sad-1, which is required for meiotic silencing of unpaired DNA (MSUD). However, mutations in Sad-1 increased the number of progeny obtained in crosses with a DeltaPrm1 (Prm1-gfp) complemented strain. These data indicate multiple roles for PRM1 during sexual development.

Unresolved Problems in the Life Cycle of Truffles

The mating system and ploidy level of truffles, both mycorrhizal and sterile fruit-body hyphae are the subject of conflicting hypotheses. The current dogma concerning Ascomycetes, supported by recent studies on truffles, maintains that the mycorrhizing mycelium is homokaryotic and that the short-lived ascogenous heterokaryotic hyphae resulting from the fertilization process occur only in truffle primordia. Therefore the gleba and sterile veins are also homokaryotic. Other studies show heterokaryons both in gleba and mantle hyphae of some truffle species, leading to the conclusion that long- term heterokaryons do exist in truffles. The matter is discussed. Some fundamental aspects concerning the life cycle of truffles (Pezizales) (1) have yet to be fully elucidated. Among these are the mating system and the ploidy level of both mycorrhizal and sterile fruit-body hyphae. Both are the subject of conflicting hypotheses. Tuber and Terfezia hyphal cells, like those of other As- comycetes, are multinucleate (e.g. (2)). Ascertaining whether a mycelial culture is a homo or heterokaryon is, therefore, not straightforward. Nowadays, however, the prevailing per- ception, which is based on in-depth research, casts doubt on the long-term subsistence of natural vegetative heterokary- ons in most filamentous Ascomycetes (e.g. (3)). The hypothesis that tends to be accepted today - a hy- pothesis that has been thoroughly investigated - maintains that the vegetative mycelium of such fungi is homokaryotic and that plasmogamy occurs only in the primordia of the fruit bodies. Plasmogamy in the developing fruit body leads to the formation of heterokaryotic ascogenous hyphae, from which, after karyogamy and meiosis, haploid ascospores develop (e.g. (2)). As a result, the heterokaryotic phase is brief and is restricted to the developing ascocarps. Yet, it had long been known that long-term heterokary- ons exist in Neurospora crassa, Pezizomycotina (4). This was later shown to be dependent on the existence of identical het alleles in the individual strains forming the heterokaryon (5). Wild type Neurospora tetrasperma isolates are invaria- bly heterokaryons, mostly selfing, but maintain some level of outcrossing in nature, as well as in the laboratory (6). This phenomenon is also dependent on het allele identity in both nuclei forming the long-term heterokaryion (ibid). Thus con- ditions under which long term ascomyceteous heterokaryons may be maintained - do exist. No het genes were, so far, identified or demonstrated in truffles. But Terfezia boudieri and Kalaharituber pfeilii het- erokaryon mycelial cultures originating from glebal out- growth seem to be the rule rather than exception: In glebal K. pfeilii and T. boudieri cultures, the hyphal cells invariably

Evidence of apothecial ancestry in the cleistothecial ascomata of Pleuroascus nicholsonii

Ascomata of Pleuroascus nicholsonii, a rarely reported cleistothecial ascomycete, show little overt evidence of a putative affiliation with the Leotiomycetes. However, close examination of the plectomycetous centrum reveals a distorted hymenium arising from a system of branched ascogenous hyphae, and twisted or coiled uniseriate ascospores enclosed within what appears to be the remains of the spore investing membrane of a clavate ascus precursor. Abundant sterile elements arising from the inner wall layer of the peridium and interspersed throughout the centrum are interpreted as representing vestiges of apically branched paraphyses. Whole ascomata show limited signs of polarity, although the characteristic, tightly coiled appendages generally arise along or below the equatorial region and there is a marked thinning of subicular hyphae over the crown of the cleistothecium. The mature peridium, which consists of a thin, melanized outer layer of squamulose cells, splits irregularly along intercellular grooves when disturbed. The adaptive significance of these characteristics is unknown, but the persistent paraphyses, the easily fractured and darkly pigmented membranous peridium, an ascospore mass that is dry at maturity, and the tendency for ascomata to cling together in clumps can be rationalized in the context of a coprophilous life-style. Collection data for P. nicholsonii provide further support for this supposition because five of the six reported specimens are from rodent dung.

Ascoma development in the marine ascomycete Corollospora gracilis (Halosphaeriales, Hypocreomycetidae, Sordariomycetes)

Abstract Ascoma initials in the marine ascomycete Corollospora gracilis (Halosphaeriales) are formed from a coiled ascogonium following antheridium-ascogonium conjugation. Dikaryotic ascogenous initial cells derived from the distal end of the ascogonial coil produce ascogenous hyphae that then form the hymenium. Basal regions of the ascogonial coil and the initial investing hyphae form the sterile centrum tissue including the plug, separation layer, and centrum pseudoparenchyma. Pit-connections of the centrum pseudoparenchyma are normal ascomycetous septa. Thick-walled, non-melanized plug cells and a thin melanized separation layer form at the base of the ostiolar canal to delimit the centrum from the external environment. The subiculum that attaches the ascomata to sand grains is comprised of aggregated hyphopodia and mucilage.

Anatomy and cytology of Taphrina entomospora during infection of Nothofagus

Taphrina entomospora is one of the few species of the genus described on native plants of the Southern Hemisphere and also one of the few leaf pathogens known on Nothofagus species. The anatomical changes it produces on N. pumilio leaves, and its morphology, cytology, and sporogenesis were studied. The fungus is a perennial species that overwinters as mycelium in the foliar buds and infects the developing leaves, so the whole blade develops the disease symptoms. Interveinal areas of the leaves become chlorotic, thickened and rounded. Palisade parenchyma fails to develop, with spongy parenchyma developing as packed, rounded, isodiametric cells with little intercellular space. The mycelium is subcuticular, dikaryotic, and produces ascogenous hyphae, asci, and ascospores as described for other species in the genus. Before ascus discharge, ascospores bud in a regular, unique way. The life-cycle of T. entomospora is compared with other representative taxa in the genus and the distribution of this pathogen is discussed.

A putative high affinity hexose transporter, hxtA, of Aspergillus nidulans is induced in vegetative hyphae upon starvation and in ascogenous hyphae during cleistothecium formation

Fungi employ different carbohydrate uptake systems to adapt to certain environmental conditions and to different carbon source concentrations. The hydrolysis of polymeric carbohydrates and the subsequent uptake of monomeric forms may also play a role in development. Aspergillus nidulans accumulates cell wall components during vegetative growth and degrades them during sexual development. We have identified the hxtA (high affinity hexose transporter) gene in a differential library, which was enriched for sexual-specific genes. The hxtA gene is disrupted by 6 introns and predicted to encode a 531 amino acid protein with high similarity to major facilitator superfamily members including the high affinity hexose transporter Gtt1 from Trichoderma harzianum. A. nidulans HxtA contains the 12 predicted transmembrane domains characteristic for this family. Deletion of hxtA did not impair growth of A. nidulans on a variety of carbon sources nor did it inhibit sexual development suggesting redundant sugar uptake systems. We found at least 17 putative hexose transporters in the genome of A. nidulans. Despite the high similarity of HxtA to fungal high affinity glucose transporters, the hxtA gene did not restore growth on glucose of a Saccharomyces cerevisiae mutant, in which all hexose transporters were deleted. Northern blot analysis revealed that the A. nidulans hxtA gene was repressed under high glucose conditions and expressed in vegetative hyphae upon carbon starvation and during sexual development. We found hxtA(p)::sgfp expression in developing cleistothecia specifically in ascogenous hyphae and propose that HxtA is a high affinity glucose transporter involved in sugar metabolism during sexual development.

Morphology and ultrastructure of Neolecta species

Several independent molecular phylogenetic analyses have indicated that the genus Neolecta has a unique position within the Ascomycota. It is the only taxon outside the core-group of filamentous, ascoma-forming ascomycetes that also has the ability to form ascomata. Light and electron microscope studies indicate that hymenial structure and development in Neolecta spp. are unique. Ascogenous hyphae in N. vitellina branched repeatedly and successively to produce asci. Non-ascogenous hyphae were multinucleate, often with nuclei in pairs. Nuclear pairing was particularily prominent in the ascogenous hyphae. A basal septum delimited the dikaryotic ascus. Ascosporogenesis was initiated by nuclear fusion followed by a meiotic and mitotic division to form eight nuclei. The ascus apex was thin with an annular subapical thickening. Ascospores were forcibly released through a 'split' in the ascus apex. Woronin bodies were frequently associated with hyphal septa. Attempts to culture N. vitellina and to obtain molecular information from the type species, N. flarovirescens, were unsuccessful. However, N. flavovirescens showed several microscopic characters that indicated close relationships with the two other species in the genus, N. vitellina and N. irregularis. The position of Neolecta spp. within the Ascomycota is discussed.

Ascogenous hyphae in foliicolous species of Arthonia and allied genera

Ascogenous hyphae of foliicolous members of the Arthoniaceae , including the genera Arthonia, Eremothecella, Amazonomyces , and Cryptothecia , were studied by epifluorescence microscopy. Two additional representatives of Opegrapha were included. The ascogenous hyphae in Arthonia, Eremothecella, Amazonomyces , and Opegrapha arise from a central region of the ascoma and form a dense and branched tree-like structure. The asci of Cryptothecia are not connected by such structures and arise singly. Differences of measurements, structures and branching patterns of ascogenous hyphae indicate that these are species-specific characters. Typical ascogones were not observed. The fusion of tips or croziers of ascogenous hyphae and haploid mycelium were seen in several species. The systematic significance of the observations is discussed.

Ascogenous hyphae in foliicolous species of Arthonia and allied genera

Ascogenous hyphae of foliicolous members of the Arthoniaceae, including the genera Arthonia, Eremothecella, Amazonomyces, and Cryptothecia, were studied by epifluorescence microscopy. Two additional representatives of Opegrapha were included. The ascogenous hyphae in Arthonia, Eremothecella, Amazonomyces, and Opegrapha arise from a central region of the ascoma and form a dense and branched tree-like structure. The asci of Cryptothecia are not connected by such structures and arise singly. Differences of measurements, structures and branching patterns of ascogenous hyphae indicate that these are species-specific characters. Typical ascogones were not observed. The fusion of tips or croziers of ascogenous hyphae and haploid mycelium were seen in several species. The systematic significance of the observations is discussed.

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.