Early fungi from the Proterozoic era in Arctic Canada.

Fungi are crucial components of modern ecosystems. They may have had an important role in the colonization of land by eukaryotes, and in the appearance and success of land plants and metazoans1-3. Nevertheless, fossils that can unambiguously be identified as fungi are absent from the fossil record until the middle of the Palaeozoic era4,5. Here we show, using morphological, ultrastructural and spectroscopic analyses, that multicellular organic-walled microfossils preserved in shale of the Grassy Bay Formation (Shaler Supergroup, Arctic Canada), which dates to approximately 1,010-890 million years ago, have a fungal affinity. These microfossils are more than half a billion years older than previously reported unambiguous occurrences of fungi, a date which is consistent with data from molecular clocks for the emergence of this clade6,7. In extending the fossil record of the fungi, this finding also pushes back the minimum date for the appearance of eukaryotic crown group Opisthokonta, which comprises metazoans, fungi and their protist relatives8,9.

Links between evolutionary processes and phenotypic robustness in microbes.

The costs and benefits of random phenotypic heterogeneity in microbes have been vigorously debated and experimental tested for decades; yet, this conversation is largely independent from discussion of phenotypic robustness in other disciplines. In this review I connect microbial examples of stochasticity with studies on the ecological and population-genetic consequences of phenotypic variability. These topics illustrate the complexity of selection pressures on phenotypic robustness and provide inspiration that this complexity can be parsed with theoretical advances and the experimental power of microbial systems.

Membrane traffic related to endosome dynamics and protein secretion in filamentous fungi.

In eukaryotic cells, membrane-surrounded organelles are orchestrally organized spatiotemporally under environmental situations. Among such organelles, vesicular transports and membrane contacts occur to communicate each other, so-called membrane traffic. Filamentous fungal cells are highly polarized and thus membrane traffic is developed to have versatile functions. Early endosome (EE) is an endocytic organelle that dynamically exhibits constant long-range motility through the hyphal cell, which is proven to have physiological roles, such as other organelle distribution and signal transduction. Since filamentous fungal cells are also considered as cell factories, to produce valuable proteins extracellularly, molecular mechanisms of secretory pathway including protein glycosylation have been well investigated. In this review, molecular and physiological aspects of membrane traffic especially related to EE dynamics and protein secretion in filamentous fungi are summarized, and perspectives for application are also described.

The inhibitory effect of volatile organic compounds produced by Bacillus subtilis CL2 on pathogenic fungi of wolfberry.

Bacillus subtilis strain CL2 is antagonistic to wolfberry postharvest pathogenic fungi. In this study, we isolated and screened this strain for in vitro experiments. The result of the two-sealed-base-plates method revealed that volatile organic compounds (VOCs) emitted from the strain CL2 inhibited the hyphal growth of four pathogenic fungi Mucor circinelloides LB1, Fusarium arcuatisporum LB5, Alternaria iridiaustralis LB7, and Colletotrichum fioriniae LB8. After exposure to VOCs for 5 days, the hyphal growth of the pathogen C. fioriniae LB8 was inhibited by 73%. Scanning electron microscopy revealed that the VOCs produced by B. subtilis CL2 caused the mycelium morphology of the pathogenic fungi to deform, twist, fold, and shrink. In the in vivo experiments, we noticed that VOCs could significantly reduce the weight loss rate of wolfberry fruits caused by the pathogenic fungus M. circinelloides LB1 and that the decay incidence rate were caused by the pathogenic fungi F. arcuatisporum LB5, A. iridiaustralis LB7, and C. fioriniae LB8. On the basis of the headspace-gas chromatography-ion mobility spectrometry analysis, seven VOCs produced by strain CL2 were identified. Among them, 2,3-butanedione and 3-methylbutyric acid are the main antifungal active substances. This study investigated the antifungal properties of VOCs produced by the strain CL2 on postharvest pathogenic fungi isolated from wolfberry fruits both in vivo and in vitro, thereby providing the theoretical basis for its future applications.

Lead soaps formation and biodiversity in a XVIII Century wax seal coloured with minium.

A multidisciplinary approach was carried out in order to study the biodeterioration and the associated microbiome of a XVIII Century wax seal coloured with minium. A small wax seal fragment was observed by scanning electron microscopy combined with energy dispersive spectroscopy in non-destructive mode. The same object was analysed by Raman and Fourier-transform infrared spectroscopy. The identification of the microbiota growing on the seal was performed with both a culture-dependent strategy, combined with hydrolytic assays, and high-throughput sequencing using the MinION platform. The whole bacterial 16S rRNA gene and the fungal markers ITS and 28S rRNA were targeted. It was observed that the carnauba wax coloured with lead tetroxide (minium) was covered by a biofilm consisting of a network of filaments and other structures of microbial origin. The culture-dependent and culture-independent investigations showed the presence of a complex microbiota composed mainly by fungal members, which demonstrated interesting properties related to lipids and lead processing. The formation of lead soaps and secondary biogenic minerals was also described.

Skeleton bones in museum indoor environments offer niches for fungi and are affected by weathering and deposition of secondary minerals.

Large skeleton specimens are often featured as iconic open displays in Natural History Museums, for example, the blue whale 'Hope' at the Natural History Museum, London. A study on Hope's bone surface was performed to assess the biodeterioration potential of fungi. Fungi were isolated, and a fungal internal transcribed spacer (ITS) clone library survey was performed on dust and bone material. Mineral particles derived from bone and dust were analysed using energy dispersive X-ray spectroscopy, variable pressure scanning electron microscopy (SEM) and high vacuum SEM. Results showed that bone material, although mainly mineral in nature, and therefore less susceptible than organic materials to biodeterioration phenomena in the indoor environments, offers niches for specialized fungi and is affected by unusual and yet not so well-documented mechanisms of alteration. Areas of bone surface were covered with a dense biofilm mostly composed of fungal hyphae, which produced tunnelling and extensive deposition of calcium and iron-containing secondary minerals. Airborne halophilic and xerophilic fungi including taxa grouping into Ascomycota and Basidiomycota, capable of displacing salts and overcome little water availability, were found to dominate the microbiome of the bone surface.

Alternative therapy to manage otitis media caused by multidrug-resistant fungi.

During the last few decades, the increase in the incidence of multidrug-resistant (MDR) fungal infections has become an emerging threat to public health. Therefore, it is important to illuminate the usage of alternative therapy to treat MDR fungal infection. This study was carried out to elucidate the usage of plant extract and essential oil, either alone or with other antifungal drugs to treat otitis media caused by MDR fungi. Medicinal plant is a safe and cheap source when compared with chemical antifungal drugs. Twenty-one fungal isolates out of 104 ear swabs from patients suffering from otitis media were characterized using both phenotypic and genotypic methods. The antibiogram typing was used to determine the MDR isolates. The sensitivity of MDR fungal isolates was tested against several plant extracts and essential oils, either alone or with other antifungal drugs. Thyme oil and clove extracts proved to have synergistic effects suggesting their use in the treatment of fungal infections, especially otitis media caused by MDR fungi. The ultrastructure of MDR fungal isolates exhibited a complete destruction post exposure to the used materials when observed under the transmission microscope (TEM). Thyme oil and clove extract were found to be the most effective agents against MDR fungal isolates and they constitute a promising tool for the management of fungal infection causing the otitis media.

Subcellular structure and behaviour in fungal hyphae.

This work briefly surveys the diversity of selected subcellular characteristics in hyphal tip cells of the fungal kingdom (Mycota). Hyphae are filamentous cells that grow by tip extension. It is a highly polarised mechanism that requires a robust secretory system for the delivery of materials (e.g. membrane, proteins, cell wall materials) to sites of cell growth. These events result it the self-assembly of a Spitzenkörper (Spk), found most often in the Basidiomycota, Ascomycota, and Blastocladiomycota, or an apical vesicle crescent (AVC), present in the most Mucoromycota and Zoopagomycota. The Spk is a complex apical body composed of secretory vesicles, cytoskeletal elements, and signaling proteins. The AVC appears less complex, though little is known of its composition other than secretory vesicles. Both bodies influence hyphal growth and morphogenesis. Other factors such as cytoskeletal functions, endocytosis, cytoplasmic flow, and turgor pressure are also important in sustaining hyphal growth. Clarifying subcellular structures, functions, and behaviours through bioimagining analysis are providing a better understanding of the cell biology and phylogenetic relationships of fungi. LAY DESCRIPTION: Fungi are most familiar to the public as yeast, molds, and mushrooms. They are eukaryotic organisms that inhabit diverse ecological niches around the world and are critical to the health of ecosystems performing roles in decomposition of organic matter and nutrient recycling (Heath, 1990). Fungi are heterotrophs, unlike plants, and comprise the most successful and diverse phyla of eukaryotic microbes, interacting with all other forms of life in associations that range from beneficial (e.g., mycorrhizae) to antagonistic (e.g., pathogens). Some fungi can be parasitic or pathogenic on plants (e.g., Cryphonectria parasitica, Magnaporthe grisea), insects (e.g., Beauveria bassiana, Cordyceps sp.), invertebrates (e.g., Drechslerella anchonia), vertebrates (e.g., Coccidioides immitis, Candia albicans) and other fungi (e.g., Trichoderma viride, Ampelomyces quisqualis). The majority of fungi, however, are saprophytes, obtaining nutrition through the brake down of non-living organic matter.

Automated, continuous video microscopy tracking of hyphal growth.

The growth of filamentous fungi is a complex process that involves hyphal elongation and branching. Microscopic observations provide a wealth of information on fungal growth, although often requiring laborious manual intervention to record and analyze images. Here, we introduce a novel tool for automated tracking of growth in fungal hyphae that affords quantitative analysis of growth rate and morphology. We supplied a student-grade bright field microscope with stepper motors to enable computer-control of the microscope stage. In addition, we developed an image-processing routine that detects in real-time the tip of a hypha and tracks it as the hypha elongates. To achieve continuous observation of hyphal growth, our system automatically maintains the observed sample within field-of-view and performs periodic autofocus correction in the microscope. We demonstrate automated, continuous tracking of hyphal growth in Trichoderma atroviride with sampling rates of seconds and observation times of up to 14 h. Tracking records allowed us to determine that T. atroviride hyphae grow with characteristic elongation rates of ∼70 nm/s. Surprisingly, we found that prior to the occurrence of an apical branching event the parental hypha stopped growing during a few minutes. These arrest events presented occasionally for subapical branching as well. Finally, from tracking data we found that the persistence length (a measure of filament extension before presenting a change in direction) associated to T. atroviride hyphae is 362 µm. Altogether, these results show how integration of image analysis and computer control enable quantitative microscopic observations of fungal hyphae dynamics.

Kinetid Structure of Aphelidium and Paraphelidium (Aphelida) Suggests the Features of the Common Ancestor of Fungi and Opisthosporidia.

The aphelids (phylum Aphelida) are phagotrophic parasitoids of algae and represent the most basal branch in superphylum Opisthosporidia, which contains the Microsporidia, Rozellosporidia and Aphelida. Being the closest group to traditional fungi, the aphelids should have ancestral features of both phyla. As in chytrids and other zoosporic fungi, the structure of zoospores is the most informative and important morphological feature for the phylogeny and taxonomy of aphelids. Though a general zoospore description exists for some aphelid species, their flagellar apparatus (kinetid) structure, which contains pivotal taxonomic and phylogenetic characters, has not been studied. Here we represent the kinetid structure in two genera, Aphelidium and Paraphelidium, and demonstrate independent reduction in the kinetid in each genus. The kinetid-mitochondrion connection found in Aphelidium and Paraphelidium is rare for opisthokonts in general, but present in the most basal branches of Fungi and Opisthosporidia. We suggest, therefore, that this connection represents an ancestral character for both these phyla.

The Ultrastructure of Sanchytrium tribonematis (Sanchytriaceae, Fungi incertae sedis) Confirms its Close Relationship to Amoeboradix.

Fungi encompass, in addition to classically well-studied lineages, an ever-expanding diversity of poorly known lineages that include, among others, zoosporic chytrid-like parasites. According to recent phylogenetic analysis based on 18S + 28S rRNA concatenated genes two unusual chytrid-like fungi Amoeboradix gromovi and Sanchytrium tribonematis form a monophyletic group, the family Sanchytriaceae, which represents a new divergent taxon that remains incertae sedis within Fungi. Zoospores of Amoeboradix gromovi contain one of the longest kinetosomes known in eukaryotic cells, which are composed of microtubular singlets or doublets. However, the ultrastructure of S. tribonematis, the type species of the genus had not been yet studied. Here, we provide the results of TEM investigations of zoospores and sporangia from two strains of S. tribonematis. The two strains are endowed with unusual features. Like in A. gromovi, amoeboid zoospores of S. tribonematis contain a long kinetosome composed of microtubular singlets, and the two orthogonal centrioles in their sporangia have nine microtubular singlets with an internal ring. The morphological and ultrastructural features of S. tribonematis are now included in the improved taxonomic diagnosis for this species.

Autophagy in plant pathogenic fungi.

Autophagy is a conserved cellular process that degrades cytoplasmic constituents in vacuoles. Plant pathogenic fungi develop special infection structures and/or secrete a range of enzymes to invade their plant hosts. It has been demonstrated that monitoring autophagy processes can be extremely useful in visualizing the sequence of events leading to pathogenicity of plant pathogenic fungi. In this review, we introduce the molecular mechanisms involved in autophagy. In addition, we explore the relationship between autophagy and pathogenicity in plant pathogenic fungi. Finally, we discuss the various experimental strategies available for use in the study of autophagy in plant pathogenic fungi.

Physiology of biodeterioration on canvas paintings.

The study of the physiological action of microorganisms in artistic materials is one of the most interesting topics in biodeterioration nowadays. Pathologies and illnesses of organic and inorganic materials provoked by microorganisms can be treated by experts by a variety of preventive interventions. Artistic medicine encompasses the monitoring of the exhibition and storage of art, as well as proper environmental conditions and the regular cleaning of museums. Biodeterioration control is essential in order to prevent fungal and bacterial contamination in artwork. Biodeterioration of canvas paintings is a complex phenomenon, not well-known at the moment. Canvas paintings are created by several artistic techniques on textile supports that are not always kept in the best conditions, and the best parameters of preventive conservation are often not applied. Therefore, we need to research the agents and the main causes that provoke canvas painting biodeterioration. By applying new methodologies, we can identify the alterations and the treatments needed in order to manage the diverse materials employed in artwork correctly. Herein, we review the causes of biodeterioration that affect artwork, especially art created on textile supports. We also study the alterations of the natural filmogenic materials employed in traditional pictorial techniques, such as agglutinants and protection layers, and the biodeterioration agents that impact them. Additionally, we review current scientific methods employed for the identification of microbial species, and the types of alterations of the materials where the organisms grow. Finally, we summarize the different biocides and preventive conservation treatments that are currently employed.

Unity in diversity: structural and functional insights into the ancient partnerships between plants and fungi.

Contents Summary 996 I. Introduction 996 II. An ancient, and diverse, symbiosis 998 III. Structural diversity in ancient plant-fungal partnerships 1000 IV. Mycorrhizal unity in host plant nutrition 1002 V. Plant-to-fungus carbon transfer 1003 VI. From individuals to networks 1003 VII. Diverse responses of mycorrhizal functioning to dynamic environments 1006 VIII. Summary of future research direction 1007 Acknowledgements 1006 References 1006 SUMMARY: Mycorrhizal symbiosis is an ancient and widespread mutualism between plants and fungi that facilitated plant terrestrialisation > 500 million years ago, with key roles in ecosystem functioning at multiple scales. Central to the symbiosis is the bidirectional exchange of plant-fixed carbon for fungal-acquired nutrients. Within this unifying role of mycorrhizas, considerable diversity in structure and function reflects the diversity of the partners involved. Early diverging plants form mutualisms not only with arbuscular mycorrhizal Glomeromycotina fungi, but also with poorly characterised Mucoromycotina, which may also colonise the roots of 'higher' plants as fine root endophytes. Functional diversity in these symbioses depends on both fungal and plant life histories and is influenced by the environment. Recent studies have highlighted the roles of lipids/fatty acids in plant-to-fungus carbon transport and potential contributions of Glomeromycotina fungi to plant nitrogen nutrition. Together with emerging appreciation of mycorrhizal networks as multi-species resource-sharing systems, these insights are broadening our views on mycorrhizas and their roles in nutrient cycling. It is crucial that the diverse array of biotic and abiotic factors that together shape the dynamics of carbon-for-nutrient exchange between plants and fungi are integrated, in addition to embracing the unfolding and potentially key role of Mucoromycotina fungi in these processes.

Morphology, Ultrastructure, and Molecular Phylogeny of Rozella multimorpha, a New Species in Cryptomycota.

Increasing numbers of sequences of basal fungi from environmental DNA studies are being deposited in public databases. Many of these sequences remain unclassified below the phylum level because sequence information from identified species is sparse. Lack of basic biological knowledge due to a dearth of identified species is extreme in Cryptomycota, a new phylum widespread in the environment and phylogenetically basal within the fungal lineage. Consequently, we are attempting to fill gaps in the knowledge of Rozella, the best-known genus in this lineage. Rozella is a genus of unwalled, holocarpic, endobiotic parasites of hosts including Chytridiomycota, Blastocladiomycota, Oomycota, Basidiomycota, and a green alga, with most species descriptions based on morphology and host specificity. We found a Rozella parasitizing a Pythium host that was a saprobe on spruce pollen bait placed with an aquatic sample. We characterized the parasite with light microscopy, TEM of its zoospores and sporangia, and its 18S/28S rDNA. Comparison with other Rozella species indicates that the new isolate differs morphologically, ultrastructurally, and genetically from Rozella species for which we have data. Features of the zoospore also differ from those of previously studied species. Herein we describe the Rozella as a new species, R. multimorpha.

Ultrastructural and microbial analyses of cellulose degradation in leaf-cutter ant colonies.

Leaf-cutter ants (Atta and Acromyrmex) use fresh leaves to cultivate a mutualistic fungus (Leucoagaricus gongylophorus) for food in underground gardens. A new ant queen propagates the cultivar by taking a small fragment of fungus from her parent colony on her nuptial flight and uses it to begin her own colony. Recent research has shown that the ants' fungus gardens are colonized by symbiotic bacteria that perform important functions related to nitrogen fixation and have been implicated in contributing to plant biomass degradation. Here, we combine bacterial culturing in several media for counts and identification using the 16S rRNA gene with electron microscopy to investigate the process of cellulose degradation in the fungus garden and refuse dumps, and to assess the potential role of symbiotic bacteria. We show through electron microscopy that plant cell walls are visibly degraded in the bottom section of fungus gardens and refuse dumps, and that bacteria are more abundant in these sections. We also consistently isolated cellulolytic bacteria from all sections of fungus gardens. Finally, we show by culture-dependent and electron microscopy analysis that the fungus garden pellets carried by recently mated queens are colonized by fungus garden-associated bacteria. Taken together, our results indicate that cellulose is degraded in fungus gardens, and that fungus garden bacteria that may contribute to this deconstruction are vertically transmitted by new queens.

'Strengthening the fungal cell wall through chitin-glucan cross-links: effects on morphogenesis and cell integrity'.

The cross-linking of polysaccharides to assemble new cell wall in fungi requires transglycosylation mechanisms by which preexisting glycosidic linkages are broken and new linkages are created between the polysaccharides. The molecular mechanisms for these processes, which are essential for fungal cell biology, are only now beginning to be elucidated. Recent development of in vivo and in vitro biochemical approaches has allowed characterization of important aspects about the formation of chitin-glucan covalent cell wall cross-links by cell wall transglycosylases of the CRH family and their biological function. Covalent linkages between chitin and glucan mediated by Crh proteins control morphogenesis and also play important roles in the remodeling of the fungal cell wall as part of the compensatory responses necessary to counterbalance cell wall stress. These enzymes are encoded by multigene families of redundant proteins very well conserved in fungal genomes but absent in mammalian cells. Understanding the molecular basis of fungal adaptation to cell wall stress through these and other cell wall remodeling enzymatic activities offers an opportunity to explore novel antifungal treatments and to identify potential fungal virulence factors.

Growth of indoor fungi on gypsum.

AIMS: To have a better understanding of fungal growth on gypsum building materials to prevent indoor fungal growth. METHODS AND RESULTS: Gypsum is acquired by mining or as a by-product of flue-gas desulphurization or treatment of phosphate ore for the production of fertilizer. Natural gypsum, flue-gas gypsum and phosphogypsum therefore have different mineral compositions. Here, growth of fungi on these types of gypsum was assessed. Conidia of the indoor fungi Aspergillus niger, Cladosporium halotolerans and Penicillium rubens were inoculated and observed using microscopic techniques including low-temperature scanning electron microscopy. Elemental analysis of gypsum was done using inductively coupled plasma atomic emission spectroscopy and segmented flow analysis. Moisture content of the gypsum was determined using a dynamic vapour sorption apparatus. Aspergillus niger, C. halotolerans and P. rubens hardly germinated on natural gypsum and flue-gas gypsum. The latter two fungi did show germination, outgrowth, and conidiation on phosphogypsum, while A. niger hardly germinated on this substrate. Other experiments show that C. halotolerans and P. rubens can develop in pure water, but A. niger does not. CONCLUSIONS: The observations show that the lack of germination of three indoor fungi is explained by the low amount of phosphor in natural, flue-gas and laboratory-grade gypsum. Additionally, C. halotolerans and P. rubens can develop in pure water, while conidia of A. niger do not show any germination, which is explained by the need for organic molecules of this species to induce germination. SIGNIFICANCE AND IMPACT OF THE STUDY: Indoor fungal growth is a potential threat to human health and causes damage to building materials. This study possibly helps in the application of the right type of gypsum in buildings.

In Vitro Antifungal Activity of Hexahydropyrimidine Derivatives against the Causative Agents of Dermatomycosis.

Nitrogenated heterocyclic compounds are present in both natural and synthetic drugs, and hexahydropyrimidine derivatives may prove to be efficient in treating dermatomycosis causing fungi. This study evaluated the antifungal activity of four hexahydropyrimidine derivatives against the dermatomycosis causing fungi. These derivatives were synthesized, characterized, and assessed in terms of their activity against Trichophyton mentagrophytes, Microsporum canis, Microsporum gypseum, Trichophyton rubrum, Fusarium oxysporum, and Epidermophyton floccosum between concentrations 7.8 and 1,000 µg mL-1. Scanning electron micrographs were assessed for the active derivatives and reference drugs, and these micrographs revealed that new agents cause morphological changes in fungi. The derivatives HHP1, HHP3, and HHP4 revealed poor activity against the four fungal strains (MICs range 500-1000 µg mL-1). Compound HHP3 was found to be the best potential antifungal agent among those tested and was the most effective among all the active derivatives that caused morphological changes in the susceptible strains.