Compartmentalization of bacterial and fungal microbiomes in the gut of adult honeybees.

The core gut microbiome of adult honeybee comprises a set of recurring bacterial phylotypes, accompanied by lineage-specific, variable, and less abundant environmental bacterial phylotypes. Several mutual interactions and functional services to the host, including the support provided for growth, hormonal signaling, and behavior, are attributed to the core and lineage-specific taxa. By contrast, the diversity and distribution of the minor environmental phylotypes and fungal members in the gut remain overlooked. In the present study, we hypothesized that the microbial components of forager honeybees (i.e., core bacteria, minor environmental phylotypes, and fungal members) are compartmentalized along the gut portions. The diversity and distribution of such three microbial components were investigated in the context of the physico-chemical conditions of different gut compartments. We observed that changes in the distribution and abundance of microbial components in the gut are consistently compartment-specific for all the three microbial components, indicating that the ecological and physiological interactions among the host and microbiome vary with changing physico-chemical and metabolic conditions of the gut.

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.

Fungal mycelial mats used as textile by indigenous people of North America.

The indigenous people of the United States and Canada long have used forest fungi for food, tinder, medicine, paint, and many other cultural uses. New information about historical uses of fungi continues to be discovered from museums as accessions of fungi and objects made from fungi collected over the last 150+ years are examined and identified. Two textiles thought to be made from fungal mats are located in the Hood Museum of Art, Dartmouth College, and the Oakland Museum of California. Scanning electron microscopy and DNA sequencing were used to attempt to identify the fungus that produced the mats. Although DNA sequencing failed to yield a taxonomic identification, microscopy and characteristics of the mycelial mats suggest that the mats were produced by Laricifomes officinalis. This first report of fungal mats used for textile by indigenous people of North America will help to alert museum curators and conservators as well as mycological researchers to their existence and hopefully lead to more items being discovered that have been made from fungal fabric.

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.

Nitrogen Fixation and Biological Behavior of Nanodiamond Colloidal Solutions.

Detonation-produced nanodiamond, both as a powder (with adsorbed water) and especially when suspended in an aqueous colloid, can support the growth (both aerobic and anaerobic) of bacteria and fungi. These were isolated and identified by microbiological methods, optical and electron microscopy, as species of Penicillium, Purpureocillium, Beaveria, Trichoderma and Aspergillus genera. The C : N molar ratio of the developing fibers (comprising fungal mycelia with attached bacteria and entrapped nanodiamond) decreased from 25 to 11 between the 1st and 10th week of incubation (cf. 40 in initial nanodiamond, 4.6 typical for bacteria and 8.3 for fungi), and from 4 to <1 after the 12th week, as the lysis of microorganisms releases carbon as CO2 and nitrogen as NH4+ or NO3- . The nitrogen content of the colloid increased by an order of magnitude and more, due to fixation of N2 by nanodiamond under ambient conditions.The process requires water but not necessarily oxygen present.

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.

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.

Molecular identification of fungi microfossils in a Neoproterozoic shale rock.

Precambrian fossils of fungi are sparse, and the knowledge of their early evolution and the role they played in the colonization of land surface are limited. Here, we report the discovery of fungi fossils in a 810 to 715 million year old dolomitic shale from the Mbuji-Mayi Supergroup, Democratic Republic of Congo. Syngenetically preserved in a transitional, subaerially exposed paleoenvironment, these carbonaceous filaments of ~5 µm in width exhibit low-frequency septation (pseudosepta) and high-angle branching that can form dense interconnected mycelium-like structures. Using an array of microscopic (SEM, TEM, and confocal laser scanning fluorescence microscopy) and spectroscopic techniques (Raman, FTIR, and XANES), we demonstrated the presence of vestigial chitin in these fossil filaments and document the eukaryotic nature of their precursor. Based on those combined evidences, these fossil filaments and mycelium-like structures are identified as remnants of fungal networks and represent the oldest, molecularly identified remains of Fungi.

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.

Rapid discrimination of fungal species by the colony fingerprinting.

The contamination of foods and beverages by fungi is a severe health hazard. The rapid identification of fungi species in contaminated goods is important to avoid further contamination. To this end, we developed a fungal discrimination method based on the bioimage informatics approach of colony fingerprinting. This method involves imaging and visualizing microbial colonies (referred to as colony fingerprints) using a lens-less imaging system. Subsequently, the quantitative image features were extracted as discriminative parameters and subjected to analysis using machine learning approaches. Colony fingerprinting has been previously found to be a promising approach to discriminate bacteria. In the present proof-of-concept study, we tested whether this method is also useful for fungal discrimination. As a result, 5 fungi belonging to the Aspergillus, Penicilium, Eurotium, Alternaria, and Fusarium genera were successfully discriminated based on the extracted parameters, including the number of hyphae and their branches, and their intensity distributions on the images. The discrimination of 6 closely-related Aspergillus spp. was also demonstrated using additional parameters. The cultivation time required to generate the fungal colonies with a sufficient size for colony fingerprinting was less than 48 h, shorter than those for other discrimination methods, including MALDI-TOF-MS. In addition, colony fingerprinting did not require any cumbersome pre-treatment steps prior to discrimination. Colony fingerprinting is promising for the rapid and easy discrimination of fungi for use in the ensuring the safety of food manufacturing.

Unveiling the bioactive potential of the essential oil of a Portuguese endemism, Santolina impressa.

ETHNOPHARMACOLOGICAL RELEVANCE: Santolina species are widely used in traditional medicine in the Mediterranean region for their anti-inflammatory, antimicrobial, antispasmodic, digestive, and analgesic properties. S. impressa, a Portuguese endemism, is traditionally recognized for its beneficial anti-inflammatory properties in several gastrointestinal affections and is also used in oropharyngeal infections. AIM OF THE STUDY: The present study aims to characterize the essential oil of S. impressa growing in Portugal and validate its traditional uses by assessing the anti-inflammatory potential of its essential oil at concentrations without toxicity. The antifungal properties of the oil are also addressed, as well as, the putative mechanism of action underlying these effects. MATERIAL AND METHODS: The essential oil was obtained in accordance with the European Pharmacopoeia and characterized by GC and GC-MS. The anti-inflammatory potential of the oil was assessed on LPS-stimulated macrophages, through the production of nitric oxide (NO) using the Griess reaction. Putative mechanisms of action included the role of the oil as a NO scavenger, as well as its effect on the expression of two key pro-inflammatory enzymes, iNOS and COX-2 by Western blot analysis. The antifungal effect of the oil was evaluated according to the CLSI guidelines on several yeast and filamentous strains and on two major virulence factors in Candida albicans, namely germ tubes and biofilms. Ultrastructural modifications on dermatophytes were also unveiled by transmission electron microscopy. RESULTS: S. impressa essential oil was primarily characterized by the presence of monoterpene hydrocarbons and oxygenated monoterpenes, being the main compounds ß-pinene (22.5%), 1,8-cineole (10.0%), limonene (9.1%), camphor (8.1%) and ß-phellandrene (8.0%). A significant decrease (ca 60.0%) in nitrite levels was observed in LPS-stimulated macrophages treated with the oil without affecting cell viability. This effect could be explained by a great reduction on iNOS expression (85.0% inhibition), thus underpinning the anti-inflammatory potential of the oil. The oil also showed a fungicidal effect, being more active against Cryptococcus neoformans, Epidermophyton floccosum and Trichophytum rubrum. For these dermatophytes, significant ultrastructural modifications in cell wall structure were detected. Strikingly, for C. albicans, the oil showed a significant anti-infective potential (at 0.07 mg/mL for germ tube inhibition and 0.02 mg/mL for biofilm disruption) before fungal growth inhibition occurred. CONCLUSIONS: Our results validate the main traditional use ascribed to S. impressa, namely its anti-inflammatory effect. In addition, an antifungal potential is pointed out, thus corroborating the antimicrobial uses and adding new value to an endemic species poorly recognized by the industry.

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.

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.

Ultrastructure of early stages of Rozella allomycis (Cryptomycota) infection of its host, Allomyces macrogynus (Blastocladiomycota).

This study reconstructs early stages of Rozella allomycis endoparasitic infection of its host, Allomyces macrogynus. Young thalli of A. macrogynus were inoculated with suspensions of R. allomycis zoospores and allowed to develop for 120 h. Infected thalli at intervals were fixed for electron microscopy and observed. Zoospores were attracted to host thalli, encysted on their surfaces, and penetrated their walls with an infection tube. The parasite cyst discharged its protoplast through an infection tube, which invaginated the host plasma membrane. The host plasma membrane then surrounded the parasite protoplast and formed a compartment confining it inside host cytoplasm. The earliest host-parasite interface within host cytoplasm consisted of two membranes, the outer layer the host plasma membrane and the inner layer the parasite plasma membrane. At first a wide space separated the two membranes and no material was observed within this space. Later, as the endoparasite thallus expanded within the compartment, the two membranes became closely appressed. As the endoparasite thallus continued to enlarge, the interface developed into three membrane layers. Thus, host plasma membrane surrounded the parasite protoplast initially without the parasite having to pierce the host plasma membrane for entry. Significantly, host-derived membrane was at the interface throughout development.

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.

Assessment of mold and yeast in some bakery products of Lahore, Pakistan based on LM and SEM.

The present investigation was designed to throw light on the microbial status of bakery products available in bakeries and supermarkets of Lahore. Different bakery samples such as biscuits, pizza, patties were collected from different localities such as Anar Kali, Chauburji, Faisal Town, Iqbal Town, Model Town, Muslim Town were investigated for mold and yeast using serial dilution technique inoculated over malt extract agar and potato dextrose agar under sterilized conditions. Isolated fungi were namely Alternaria alternata, Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Aspergillus terreus, Curvularia americana, Fusarium solani, Penicillium digitatum, Saccharomyces cerevisiae, and Geotrichum candidum. Results depicted maximum fungal viable count in biscuits, collected from Model Town while minimum count was in the samples of Chauburji. In the case of pizza, the maximum fungal viable count was found in the sample of Muslim Town. In the case of patties, the maximum fungal viable count was found in the sample of Muslim Town while minimum count was found in the sample of Iqbal Town. Prevalence of microorganisms may be due to the use of contaminated raw material, use of polluted waters, human handling, and the use of contaminated containers. Contaminated food intake can lead to measurable signs of liver injury, inflammation, etc. Preventive measures like washing and drying of hands before preparing food, cleaning of food preparation areas, and the use of clean equipment can avoid microbes which cause hazards to human health. RESEARCH HIGHLIGHTS: Bakery products of Lahore, Pakistan is investigated for the first time on the basis of light microscopy (LM) and scanning electron microscopy (SEM) and found very significant to check the quality of bakery products or not.

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.