Resin cast impressions as a tool for microscopic observations of fungal epiphytes on leaves.

A simple method for fungal epiphyte microscopic observations and preservation is described. A two-part clear casting resin, cotton leaves and two species of fungi were used to validate this protocol. We obtained very detailed images of fungal structures using this approach in addition to retaining the impressions for future reference.

New fungal cephalothecoid-like fructifications from central European Neogene deposits.

Fragments of cephalothecoid fructifications (peridia) were encountered during palynological investigations of Neogene deposits in Mizerna-Nowa/Poland and Adendorf/Germany. Isolated plates of cephalothecoid ascoma in shape and cellular structure similar to the extant members of the family Cephalothecaceae are described as Cephalothecoidomyces neogenicus fossil gen. et sp. nov. while remnants of fungal sporocarps with cephalothecoid walls with indistinct lines of dehiscence, similar in structure to peridia with cephalothecoid morphology of extant representatives the family Chaetomiaceae (mainly genus Chaetomidium) are assigned to Adendorfia miocenica fossil gen. et sp. nov. We also propose a new interpretation of some previously described fossil fungal taxa that we consider to be remnants of cephalothecoid ascomata.

New insights from an old mutant: SPADIX4 governs fruiting body development but not hyphal fusion in Sordaria macrospora.

During the sexual life cycle of filamentous fungi, multicellular fruiting bodies are generated for the dispersal of spores. The filamentous ascomycete Sordaria macrospora has a long history as a model system for studying fruiting body formation, and two collections of sterile mutants have been generated. However, for most of these mutants, the underlying genetic defect remains unknown. Here, we investigated the mutant spadix (spd) that was generated by X-ray mutagenesis in the 1950s and terminates sexual development after the formation of pre-fruiting bodies (protoperithecia). We sequenced the spd genome and found a 22 kb deletion affecting four genes, which we termed spd1-4. Generation of deletion strains revealed that only spd4 is required for fruiting body formation. Although sterility in S. macrospora is often coupled with a vegetative hyphal fusion defect, Δspd4 was still capable of fusion. This feature distinguishes SPD4 from many other regulators of sexual development. Remarkably, GFP-tagged SPD4 accumulated in the nuclei of vegetative hyphae and fruiting body initials, the ascogonial coils, but not in sterile tissue from the developing protoperithecium. Our results point to SPD4 as a specific determinant of fruiting body formation. Research on SPD4 will, therefore, contribute to understanding cellular reprogramming during initiation of sexual development in fungi.

Cephalotheca sulfurea (Ascomycota, Sordariomycetes), a new fungal pathogen of the farmed rainbow trout Oncorhynchus mykiss.

The first case of visceral mycotic infection due to Cephalotheca sulfurea (Cephalothecaceae, Ascomycota) is documented in farmed rainbow trout from a raceway culture system. The disease clinically manifested as a hyperaemic area in the liver of the fish, and histological examination using silver and PAS staining showed the presence of numerous foci of hyphae and spores. The causative agent was first isolated in pure culture from the liver and identified using morphological characteristics. Sequence data from ITS and LSU rDNA also clearly confirmed C. sulfurea as the causal agent. The pathogenicity of related species belonging to the family Cephalothecaceae has been well-documented in humans and dogs (superficial as well as systemic infections). However, C. sulfurea has never been reported as a pathogen of humans or animals, including marine and freshwater fishes. The morphological identification of C. sulfurea is difficult due to its similarity to several different fungal genera, and molecular methods are strongly recommended for reliable identification.

A fungal sarcolemmal membrane-associated protein (SLMAP) homolog plays a fundamental role in development and localizes to the nuclear envelope, endoplasmic reticulum, and mitochondria.

Sarcolemmal membrane-associated protein (SLMAP) is a tail-anchored protein involved in fundamental cellular processes, such as myoblast fusion, cell cycle progression, and chromosomal inheritance. Further, SLMAP misexpression is associated with endothelial dysfunctions in diabetes and cancer. SLMAP is part of the conserved striatin-interacting phosphatase and kinase (STRIPAK) complex required for specific signaling pathways in yeasts, filamentous fungi, insects, and mammals. In filamentous fungi, STRIPAK was initially discovered in Sordaria macrospora, a model system for fungal differentiation. Here, we functionally characterize the STRIPAK subunit PRO45, a homolog of human SLMAP. We show that PRO45 is required for sexual propagation and cell-to-cell fusion and that its forkhead-associated (FHA) domain is essential for these processes. Protein-protein interaction studies revealed that PRO45 binds to STRIPAK subunits PRO11 and SmMOB3, which are also required for sexual propagation. Superresolution structured-illumination microscopy (SIM) further established that PRO45 localizes to the nuclear envelope, endoplasmic reticulum, and mitochondria. SIM also showed that localization to the nuclear envelope requires STRIPAK subunits PRO11 and PRO22, whereas for mitochondria it does not. Taken together, our study provides important insights into fundamental roles of the fungal SLMAP homolog PRO45 and suggests STRIPAK-related and STRIPAK-unrelated functions.

Autophagic kinases SmVPS34 and SmVPS15 are required for viability in the filamentous ascomycete Sordaria macrospora.

Autophagy is a tightly controlled degradation process of all eukaryotes. It includes the sequestration of cytoplasmic contents and organelles within a double-membraned autophagosome. Autophagy involves core autophagy related (atg) genes as well as genes regulating vesicle trafficking. Previously, we analyzed the impact of proteins of the core autophagic machinery SmATG7, SmATG8 and SmATG4 on the sexual and vegetative development of the filamentous ascomycete Sordaria macrospora. While deletion of Smatg8 and Smatg4 abolished fruiting-body formation and impaired vegetative growth, Smatg7 is required for viability. In yeast, the phosphatidylinositol 3-kinase vacuolar protein sorting 34 (Vps34) and its myristoylated membrane targeting unit, the protein kinase Vps15 have been shown to be important regulators of autophagy and vacuolar protein sorting. However, their exact role in filamentous ascomycetes remains elusive. To determine the function of Smvps34 and Smvps15 we isolated genes with high sequence similarity to Saccharomyces cerevisiae VPS34 and VPS15. For both genes we were not able to generate a homokaryotic knockout mutant in S. macrospora, suggesting that Smvps34 and Smvps15 are required for viability. Furthermore, we analyzed the repertoire of vps genes encoded by S. macrospora and could identify putative homologs of nearly all of the 61 VPS genes of S. cerevisiae.

Autophagy genes Smatg8 and Smatg4 are required for fruiting-body development, vegetative growth and ascospore germination in the filamentous ascomycete Sordaria macrospora.

Autophagy is a tightly controlled degradation process involved in various developmental aspects of eukaryotes. However, its involvement in developmental processes of multicellular filamentous ascomycetes is largely unknown. Here, we analyzed the impact of the autophagic proteins SmATG8 and SmATG4 on the sexual and vegetative development of the filamentous ascomycete Sordaria macrospora. A Saccharomyces cerevisiae complementation assay demonstrated that the S. macrospora Smatg8 and Smatg4 genes can functionally replace the yeast homologs. By generating homokaryotic deletion mutants, we showed that the S. macrospora SmATG8 and SmATG4 orthologs were associated with autophagy-dependent processes. Smatg8 and Smatg4 deletions abolished fruiting-body formation and impaired vegetative growth and ascospore germination, but not hyphal fusion. We demonstrated that SmATG4 was capable of processing the SmATG8 precursor. SmATG8 was localized to autophagosomes, whereas SmATG4 was distributed throughout the cytoplasm of S. macrospora. Furthermore, we could show that Smatg8 and Smatg4 are not only required for nonselective macroautophagy, but for selective macropexophagy as well. Taken together, our results suggest that in S. macrospora, autophagy seems to be an essential and constitutively active process to sustain high energy levels for filamentous growth and multicellular development even under nonstarvation conditions.

The anamorphic genus Monotosporella (Ascomycota) from Eocene amber and from modern Agathis resin.

The anamorphic fungal genus Monotosporella (Ascomycota, Sordariomycetes) has been reco-vered from a piece of Early Eocene Indian amber, as well as from the surface of extant resin flows in New Caledonia. The fossil fungus was obtained from the Tarkeshwar Lignite Mine of Gujarat State, western India, and was part of the biota of an early tropical angiosperm rainforest. The amber inclusion represents the second fossil record of Sordariomycetes, as well as the first fossil of its particular order (either Savoryellales or Chaetosphaeriales). The fossil fungus is distinguished from extant representatives by possessing both short conidiophores and small two-septate pyriform conidia, and is described as Monotosporella doerfeltii sp. nov. Inside the amber, the anamorph is attached to its substrate, which is likely the degraded thallus of a cladoniform lichen. The extant New Caledonian species is assigned to Monotosporella setosa. It was found growing on semi-solidified resin flows of Agathis ovata (Araucariaceae), and is the first record of Monotosporella from modern resin substrates.

Diplogelasinospora grovesii IMI 171018 immobilized in polyurethane foam. An efficient biocatalyst for stereoselective reduction of ketones.

Diplogelasinospora grovesii has been reported as a very active biocatalyst in the reduction of ketones. Along the text, the properties of this filamentous fungus as an immobilized catalyst are described. For this purpose, several immobilization supports as agar and polyurethane foam were tested. Experimental assays were also performed to test different co-substrates for the regeneration of the required enzyme cofactor. The fungus immobilized in polyurethane foam lead to the most stable and active catalyst. This derivative, using i-PrOH as co-substrate, could be reused at least 18 times without appreciable activity loss (>90% activity remains). Kinetic runs experiments shown that the reduction of cyclohexanone, selected as model substrate, followed a pseudo-first kinetic order and that the rate controlling step was the mass transfer through the cell wall. The deactivation kinetic constants were also determined. The reduction of different chiral ketones showed that the ketone reductase activity followed the Prelog's rule.

Sme4 coiled-coil protein mediates synaptonemal complex assembly, recombinosome relocalization, and spindle pole body morphogenesis.

We identify a large coiled-coil protein, Sme4/PaMe4, that is highly conserved among the large group of Sordariales and plays central roles in two temporally and functionally distinct aspects of the fungal sexual cycle: first as a component of the meiotic synaptonemal complex (SC) and then, after disappearing and reappearing, as a component of the spindle pole body (SPB). In both cases, the protein mediates spatial juxtaposition of two major structures: linkage of homolog axes through the SC and a change in the SPB from a planar to a bent conformation. Corresponding mutants exhibit defects, respectively, in SC and SPB morphogenesis, with downstream consequences for recombination and astral-microtubule nucleation plus postmeiotic nuclear migration. Sme4 is also required for reorganization of recombination complexes in which Rad51, Mer3, and Msh4 foci relocalize from an on-axis position to a between-axis (on-SC) position concomitant with SC installation. Because involved recombinosome foci represent total recombinational interactions, these dynamics are irrespective of their designation for maturation into cross-overs or noncross-overs. The defined dual roles for Sme4 in two different structures that function at distinct phases of the sexual cycle also provide more functional links and evolutionary dynamics among the nuclear envelope, SPB, and SC.

Invasive Myceliophthora thermophila infection mimicking invasive aspergillosis in a neutropenic patient: a new cause of cross-reactivity with the Aspergillus galactomannan serum antigen assay.

Myceliophthora thermophila is a thermophilic mould widely found in the environment but rarely responsible for human infections. We describe a case of invasive Myceliophthora thermophila infection mimicking invasive aspergillosis in a neutropenic patient with haematological malignancy. Cross-reactivity with Aspergillus galactomannan assay (GM) was demonstrated by repeated positive results and confirmed by cross-reaction between the fungal isolate and the GM assay. The patient was successfully treated with voriconazole. Potential GM cross-reactivity must be considered in future studies including patients categorized as having probable invasive aspergillosis using the GM as the only mycological criterion.

A fatal case with disseminated Myceliophthora thermophila infection in a lymphoma patient.

We report a lethal Myceliophthora thermophila infection in an immunocompromised patient. Based upon the clinical and mycological presentation, an initial diagnosis of invasive aspergillosis was made, possibly delaying optimal management in the patient. Melanized fungi are emerging pathogens that require early identification to improve their unfavorable prognosis.

The phocein homologue SmMOB3 is essential for vegetative cell fusion and sexual development in the filamentous ascomycete Sordaria macrospora.

Members of the striatin family and their highly conserved interacting protein phocein/Mob3 are key components in the regulation of cell differentiation in multicellular eukaryotes. The striatin homologue PRO11 of the filamentous ascomycete Sordaria macrospora has a crucial role in fruiting body development. Here, we functionally characterized the phocein/Mob3 orthologue SmMOB3 of S. macrospora. We isolated the gene and showed that both, pro11 and Smmob3 are expressed during early and late developmental stages. Deletion of Smmob3 resulted in a sexually sterile strain, similar to the previously characterized pro11 mutant. Fusion assays revealed that ∆Smmob3 was unable to undergo self-fusion and fusion with the pro11 strain. The essential function of the SmMOB3 N-terminus containing the conserved mob domain was demonstrated by complementation analysis of the sterile S. macrospora ∆Smmob3 strain. Downregulation of either pro11 in ∆Smmob3, or Smmob3 in pro11 mutants by means of RNA interference (RNAi) resulted in synthetic sexual defects, demonstrating for the first time the importance of a putative PRO11/SmMOB3 complex in fruiting body development.

Optimal culture conditions for keratinase production by a novel thermophilic Myceliophthora thermophila strain GZUIFR-H49-1.

AIMS: To investigate the effect of medium compositions and culture conditions on keratinase production by a novel thermophilic fungus Myceliophthora thermophila (Apinis) Oorschot strain GZUIFR-H49-1. METHODS AND RESULTS: The thermophilic strain GZUIFR-H49-1 with keratinolytic ability was characterized and identified as a strain of M. thermophila on the basis of its morphological characters and molecular analysis of ITS1-5.8S-ITS2 rDNA sequence. Among the medium compositions tested, the soluble starch (SS), urea, sodium thiosulfate and CaCl2 were the most effective C-source, N-source, S-source and mineral ion, respectively, by employing the single-factor experiment. The urea and pH value were the significant factors (P < 0·05) for the keratinase production in this experiment condition using Plackett­Burman factorial design. The conditions of keratinase production were further optimized by Box­Behnken design. Consequently, there was a 6·4-fold increase (5100 U l−1) in the keratinase activity than the initial value (800 U l−1) by this optimal process. CONCLUSIONS: This study indicated that the optimization design proved a useful and powerful tool for the development of optimal medium compositions and culture conditions. Myceliophthora thermophila strain GZUIFR-H49-1 was a promising fungus strain for keratinase production.

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.

Bioprocess strategies for enhanced production of xylanase by Melanocarpus albomyces IITD3A on agro-residual extract.

The production of high titer xylanase without cellulase is required for prebleaching of pulps in pulp and paper industry. The mutant IITD3A of Melanocarpus albomyces developed from the spores of the wild type organism was used in this study. The statistical optimization of the process parameters by response surface methodology revealed that the production of xylanase was most affected by changes in the pH of the production medium which contained a soluble extract of wheat straw as the sole carbon source. When the pH of the production medium in a 14 L bioreactor was controlled on-line at 7.8, xylanase activity of 415 IU mL⁻¹ was obtained after 36 h fermentation. On cycling the pH between 7.8 and 8.2, the same activity could be attained in 24 h with an overall productivity of 16,670 IU L⁻¹ h⁻¹. The production of xylanase was also influenced by the fungus morphology; the activity being maximum when it exhibited pellet form at an agitation speed of 600 rpm. On optimization of aeration rate to 0.25 vvm, the xylanase activity further increased to 550 IU mL⁻¹ with a very high overall volumetric productivity of 22,000 IU L⁻¹ h⁻¹. Thus, a 5.2-fold enhancement in overall volumetric productivity of xylanase could be obtained by the mutant in comparison to that obtained on insoluble wheat straw.

Sordaria macrospora, a model organism to study fungal cellular development.

During the development of multicellular eukaryotes, the processes of cellular growth and organogenesis are tightly coordinated. Since the 1940s, filamentous fungi have served as genetic model organisms to decipher basic mechanisms underlying eukaryotic cell differentiation. Here, we focus on Sordaria macrospora, a homothallic ascomycete and important model organism for developmental biology. During its sexual life cycle, S. macrospora forms three-dimensional fruiting bodies, a complex process involving the formation of different cell types. S. macrospora can be used for genetic, biochemical and cellular experimental approaches since diverse tools, including fluorescence microscopy, a marker recycling system and gene libraries, are available. Moreover, the genome of S. macrospora has been sequenced and allows functional genomics analyses. Over the past years, our group has generated and analysed a number of developmental mutants which has greatly enhanced our fundamental understanding about fungal morphogenesis. In addition, our recent research activities have established a link between developmental proteins and conserved signalling cascades, ultimately leading to a regulatory network controlling differentiation processes in a eukaryotic model organism. This review summarizes the results of our recent findings, thus advancing current knowledge of the general principles and paradigms underpinning eukaryotic cell differentiation and development.

Recombination proteins mediate meiotic spatial chromosome organization and pairing.

Meiotic chromosome pairing involves not only recognition of homology but also juxtaposition of entire chromosomes in a topologically regular way. Analysis of filamentous fungus Sordaria macrospora reveals that recombination proteins Mer3, Msh4, and Mlh1 play direct roles in all of these aspects, in advance of their known roles in recombination. Absence of Mer3 helicase results in interwoven chromosomes, thereby revealing the existence of features that specifically ensure "entanglement avoidance." Entanglements that remain at zygotene, i.e., "interlockings," require Mlh1 for resolution, likely to eliminate constraining recombinational connections. Patterns of Mer3 and Msh4 foci along aligned chromosomes show that the double-strand breaks mediating homologous alignment have spatially separated ends, one localized to each partner axis, and that pairing involves interference among developing interhomolog interactions. We propose that Mer3, Msh4, and Mlh1 execute all of these roles during pairing by modulating the state of nascent double-strand break/partner DNA contacts within axis-associated recombination complexes.

Observing meiosis in filamentous fungi: Sordaria and Neurospora.

The filamentous fungi Neurospora crassa and Sordaria macrospora are materials of choice for recombination studies because each of the DNA strands involved in meiosis can be visually analyzed using spore-color mutants. Well-advanced molecular genetic methodologies have been developed for each of these fungi, and several mutants defective in recombination and/or pairing are available. Moreover, the complete genome sequence of N. crassa has made it possible to clone virtually any gene involved in their life cycle. Both fungi provide also a particularly attractive experimental system for cytological analysis of meiosis: stages can be determined independently of chromosomal morphology and their seven chromosomes are easily identified. The techniques for light, immunofluorescence and electron microscopy presented here have been used, with success, for monitoring of chromosome behavior during both meiotic and sporulation processes. They have also proved useful for the analysis of mitochondria and peroxisomes as well as cytoskeleton and spindle pole-body components. Moreover, all techniques of this chapter can be easily applied to other filamentous ascomycetes, including other Sordaria and Neurospora species as well as Podospora, Ascobolus, Ascophanus, Fusarium, Neotiella, and Aspergillus species.

A re-evaluation of genus Chaetomidium based on molecular and morphological characters.

Chaetomidium, a genus in the Chaetomiaceae, comprises 12 species that produce similar cleistothecial ascomata with a membranous, mostly pilose, peridium. Approximately six species of this genus produce some type of modified peridium composed of cephalothecoid plates that previous authors have hypothesized to be a homologous character within the genus. To better understand the phylogenetic affiliations of Chaetomidium and distribution of the cephalothecoid peridium within this genus we performed phylogenetic analyses with LSU, beta-tubulin and rpb2 sequence data. The results of these analyses showed that Chaetomidium is polyphyletic and should be restricted to its type, C. fimeti, and C. subfimeti. The remaining cephalothecoid and non-cephalothecoid species were scattered throughout the Chaetomiaceae and Lasiosphaeriaceae. The cephalothecoid species of Chaetomidium were distributed in three unrelated clades, suggesting that the morphological similarity amo'ng these particular species resulted from convergence instead of ancestry.