Hypersensitivity pneumonitis associated with inhalation of Lycoperdon spores (lycoperdonosis) in a 3-month-old English setter dog in Quebec.

A 3-month-old female English setter dog was presented to the Faculty of Veterinary Medicine of the Université de Montréal (Quebec) with acute respiratory distress. The dog had moderately increased C-reactive protein concentrations, and thoracic radiographs revealed a moderate, caudodorsal, nodular-to-miliary alveolo-interstitial pulmonary pattern that was worse in the perihilar region. Initial differential diagnoses included a fungal pneumonia (e.g., blastomycosis or histoplasmosis). Cytology of the bronchoalveolar lavage revealed several round, green structures ~2 µm in diameter, consistent with fungal spores. The dog was hospitalized, but within 24 h the respiratory condition deteriorated and euthanasia was elected. Post-mortem panfungal PCR and sequencing tests identified the spores as Lycoperdon sp. Retrospectively, the owners recalled that the dog had played in a wood pile with mushrooms and had sneezed in a cloud of spores, implying inhalation of Lycoperdon spores. This is the first report of a confirmed case of canine lycoperdonosis in eastern Canada (Quebec), and the radiographic features in this case differed slightly from previous reports. Diagnosis before bronchoalveolar lavage analysis was challenging, as spore inhalation was not initially reported. Although the disease is infrequently reported in dogs, this case report reminds veterinarians to consider lycoperdonosis as a differential diagnosis when addressing animals presented with acute dyspnea with similar radiographic lesions, and highlights the importance of history and cytology in diagnosing this condition. Key clinical message: Hypersensitivity pneumonitis secondary to inhalation of Lycoperdon spores must be included in differential diagnoses for a dog with acute onset of respiratory signs and a nodular-to-miliary interstitial pulmonary pattern coalescing in patchy perihilar alveolar pulmonary lesions, and should prompt clinicians to question owners regarding inhalation of mushroom spores.Although cytological examination of a bronchoalveolar lavage reveals the presence of fungal spores, panfungal PCR and sequencing tests are needed to pinpoint the species involved.

Pneumopathie d'hypersensibilité associée à l'inhalation de spores de Lycoperdon (lycoperdonose) chez un chien setter anglais de 3 mois au Québec. Une chienne setter anglais âgée de 3 mois a été présentée à la Faculté de médecine vétérinaire de l'Université de Montréal (Québec) avec une détresse respiratoire aiguë. Le chien présentait des concentrations de protéine C-réactive modérément augmentées et les radiographies thoraciques ont révélé un schéma pulmonaire alvéolo-interstitiel modéré, caudodorsal, nodulaire à miliaire, pire dans la région périhilaire. Les diagnostics différentiels initiaux incluaient une pneumonie fongique (par exemple, blastomycose ou histoplasmose). La cytologie du lavage broncho-alvéolaire a révélé plusieurs structures rondes et vertes d'environ 2 µm de diamètre, compatibles avec des spores fongiques. Le chien a été hospitalisé, mais en 24 heures, l'état respiratoire s'est détérioré et l'euthanasie a été décidée. Les tests panfongiques PCR et de séquençage post-mortem ont identifié les spores comme étant Lycoperdon sp. Rétrospectivement, les propriétaires ont mentionné que le chien avait joué dans un tas de bois avec des champignons et avait éternué dans un nuage de spores, ce qui implique une inhalation de spores de Lycoperdon. Il s'agit du premier rapport d'un cas confirmé de lycoperdonose canine dans l'est du Canada (Québec), et les caractéristiques radiographiques de ce cas différaient légèrement des rapports précédents. Le diagnostic avant l'analyse du lavage broncho-alvéolaire était difficile, car l'inhalation de spores n'avait pas été initialement signalée. Bien que la maladie soit rarement rapportée chez les chiens, ce rapport de cas rappelle aux vétérinaires de considérer la lycoperdonose comme un diagnostic différentiel lorsqu'ils traitent des animaux présentant une dyspnée aiguë avec des lésions radiographiques similaires, et souligne l'importance de l'anamnèse et de la cytologie dans le diagnostic de cette affection.Message clinique clé : La pneumopathie d'hypersensibilité secondaire à l'inhalation de spores de Lycoperdon doit être incluse dans les diagnostics différentiels chez un chien présentant un début aigu de signes respiratoires et un schéma pulmonaire interstitiel nodulaire à miliaire fusionnant dans des lésions pulmonaires alvéolaires périhilaires inégales, et devrait inciter les cliniciens à interroger les propriétaires concernant l'inhalation de spores de champignons.Bien que l'examen cytologique d'un lavage broncho-alvéolaire révèle la présence de spores fongiques, des tests panfongiques PCR et de séquençage sont nécessaires pour identifier les espèces impliquées.(Traduit par Dr Serge Messier).

Assessment of fungal spores and spore-like diversity in environmental samples by targeted lysis.

At particular stages during their life cycles, fungi use multiple strategies to form specialized structures to survive unfavorable environmental conditions. These strategies encompass sporulation, as well as cell-wall melanization, multicellular tissue formation or even dimorphism. The resulting structures are not only used to disperse to other environments, but also to survive long periods of time awaiting favorable growth conditions. As a result, these specialized fungal structures are part of the microbial seed bank, which is known to influence the microbial community composition and contribute to the maintenance of diversity. Despite the importance of the microbial seed bank in the environment, methods to study the diversity of fungal structures with improved resistance only target spores dispersing in the air, omitting the high diversity of these structures in terms of morphology and environmental distribution. In this study, we applied a separation method based on cell lysis to enrich lysis-resistant fungal structures (for instance, spores, sclerotia, melanized yeast) to obtain a proxy of the composition of the fungal seed bank. This approach was first evaluated in-vitro in selected species. The results obtained showed that DNA from fungal spores and from yeast was only obtained after the application of the enrichment method, while mycelium was always lysed. After validation, we compared the diversity of the total and lysis-resistant fractions in the polyextreme environment of the Salar de Huasco, a high-altitude athalassohaline wetland in the Chilean Altiplano. Environmental samples were collected from the salt flat and from microbial mats in small surrounding ponds. Both the lake sediments and microbial mats were dominated by Ascomycota and Basidiomycota, however, the diversity and composition of each environment differed at lower taxonomic ranks. Members of the phylum Chytridiomycota were enriched in the lysis-resistant fraction, while members of the phylum Rozellomycota were never detected in this fraction. Moreover, we show that the community composition of the lysis-resistant fraction reflects the diversity of life cycles and survival strategies developed by fungi in the environment. To the best of our knowledge this is the first time that the fungal diversity is explored in the Salar de Huasco. In addition, the method presented here provides a simple and culture independent approach to assess the diversity of fungal lysis-resistant cells in the environment.

UV-B tolerances of conidia, blastospores, and microsclerotia of Metarhizium spp. entomopathogenic fungi.

The aim of the present study was to analyze ten native Metarhizium spp. isolates as to their UV-B tolerances. Comparisons included: different fungal propagules (conidia, blastospores, or microsclerotia [MS]); conidia in aqueous suspensions or in 10% mineral oil-in-water emulsions; and conidia mixed with different types of soil. The UV-B effect was expressed as the germination of conidia or culturability of blastospores and MS relative to nongerminated propagules. Metarhizium anisopliae LCM S05 exhibited high tolerance as blastospores and/or MS, but not as conidia; LCM S10 and LCM S08 had positive results with MS or conidia but not blastospores. The formulations with 10% mineral oil did not always protect Metarhizium conidia against UV-B. Conidia of LCM S07, LCM S08, and LCM S10 exhibited the best results when in aqueous suspensions, 24 h after UV-B exposure. In general, conidia mixed with soil and exposed to UV-B yielded similar number of colony forming units as conidia from unexposed soil, regardless the soil type. It was not possible to predict which type of propagule would be the most UV-B tolerant for each fungal isolate; in conclusion, many formulations and propagule types should be investigated early in the development of new fungal biocontrol products.

Propagation and characterization of viable arbuscular mycorrhizal fungal spores within maize plant (Zea mays L.).

BACKGROUND: The harmful effect of chemical fertilizer application on human health and the environment as a modern method of meeting the food demand of the increasing world population demands an urgent alternative that is environmentally friendly, which will pose no harm to human health and the environment. Arbuscular mycorrhizal fungi (AMF) are beneficial soil microorganisms that provide various ecological functions in increasing soil fertility and enhancing plant growth. This present study aimed to propagate, characterize and examine the effect of viable arbuscular mycorrhizal fungal spores on maize (Zea mays L) hosts using molecular methods. The propagation of AMF in the host plant using sterile soil and vermiculite was conducted in the greenhouse. RESULT: The effect of AMF inoculation revealed a significant difference (P > 0.05) in maize growth, root colonization and AMF spore count when compared with the control. In all the parameters measured in this study, all the AMF spores propagated had a positive effect on the maize plant over the control, with the highest value mostly recorded in Rhizophagus irregularis AOB1. The molecular characterization of the spore using a specific universal primer for Glomeromycota established the success of the propagation process, which enhanced the classification of the AMF species into Rhizophagus irregularis OAB1, Glomus mosseae OAB2 and Paraglomus occultum OAB3. CONCLUSION: This finding will be a starting point in producing arbuscular mycorrhizal inoculum as a biofertilizer to enhance plant growth promotion. © 2021 Society of Chemical Industry.

Extract of Ganoderma sinensis spores induces cell cycle arrest of hepatoma cell via endoplasmic reticulum stress.

CONTEXT: Ganoderma sinensis Zhao, Xu et Zhang (Ganodermataceae) has been used for the prevention or treatment of a variety of diseases, including cancer. OBJECTIVE: We investigated the antitumor activity and mechanism of an extract from G. sinensis against hepatocellular carcinoma. MATERIALS AND METHODS: A G. sinensis extract (GSE) was obtained from sporoderm-broken G. sinensis spores by supercritical fluid carbon dioxide extraction. Hepatoma cells, HepG2 cells, were treated with emulsified sample of GSE at 12.5, 25, 50, 100 and 150 µg/mL for 24 h. The Alamar Blue assay was used to examine growth inhibitory effects. Changes in cell structure and morphology were assessed via transmission electron microscopy and confocal laser scanning microscope. Cell cycle distribution was analysed by flow cytometry. RESULTS: GSE suppressed the proliferation of HepG2 cells (IC50=70.14 µg/mL). Extensive cytoplasmic vacuolation originating from dilation of the endoplasmic reticulum (ER) was shown in GSE-treated HepG2 cells. GSE treatment also upregulated the expression of ER stress-related proteins in HepG2 cells. Cells tended to be arrested at the G2/M cell cycle stage after GSE treatment (30.8 ± 1.4% and 42.2 ± 2.6% at GSE with 50 µg/mL and 100 µg/mL vs. 21.03 ± 1.10%, control). Pre-treatment with salubrinal, an inhibitor of ER stress, effectively attenuated cell cycle arrest induced by GSE. DISCUSSION AND CONCLUSIONS: Our findings provide new evidence that GSE suppresses growth of cancer cells in vitro through activating the ER stress pathway. The GSE may be clinically applied in the prevention and/or treatment of cancer.

An integrated microbiome and metabolomic analysis identifies immunoenhancing features of Ganoderma lucidum spores oil in mice.

Ganoderma lucidum (Leyss. ex Fr.) Karst. is a valuable dietary supplement used worldwide for promoting health as well as a medicinal fungus for handling fatigue, immunological disorders, and cancer. Previous studies have revealed the immunoenhancing effect of G. lucidum and the polysaccharide extract, with potential involvement of gut microbiome. The oil of G. lucidum spores (GLSO)is one of the well-known G. lucidum-related products. However, there is little evidence supporting the immune promotion activity and the underlying mechanisms. The present study aims to investigate the immunoenhancing effect of GLSO in mice. GLSO enhanced macrophage phagocytosis and NK cell cytotoxicity of mice. Further microbiome and metabolomics studies showed that GLSO induced structural rearrangement of gut microbiota, mediating alterations in a wide range of metabolites. By clustering, multivariate and correlation analysis, the immunoenhancing effect of GLSO was found to be highly correlated with elevated abundance of several bacterial genera (Lactobacillus, Turicibacter and Romboutsia) and species (Lactobacillus_intestinalis and Lactobacillus_reuteri), and decreased level of Staphylococcus and Helicobacter, which resulted in the regulation of a range of key metabolites such as dopamine, prolyl-glutamine, pentahomomethionine, leucyl-glutamine, l-threonine, stearoylcarnitine, dolichyl ß-d-glucosyl phosphate, etc. These results provide new insights into the understanding of the modulatory effect of GLSO on immune system.

An aero mycological analysis of Mucormycetes in indoor and outdoor environments of northern India.

Mucormycosis is an angio-invasive infection, predominantly acquired by inhalation of sporangiospores from the environment. However, the burden of Mucormycetes sporangiospores in the air is not well studied. We aimed to estimate the burden of Mucormycetes spores in the outdoor and indoor (hospital) environment across different seasons in north India. A total of 380 air samples from outdoor (n = 180) and indoor (n = 200) environment were included in the study. Air samples were suctioned using air sampler (100 l/min) and cultured on Dichloran Rose Bengal Chloramphenicol (DRBC) with benomyl for selective isolation of Mucormycetes. The isolates were identified by phenotypic and genotypic methods. The mean spore count (±SD) of Mucormycetes (cfu/m3) in outdoor samples varied from 0.73 (±0.96) to 8.60 (±5.70) across different seasons. In hospital, the mean spore count varied from 0.68 (±1.07) to 1.12 (±1.07) and 0.88 (±1.01) to 1.72 (±2.17) for air-conditioned wards and non-air-conditioned wards, respectively. Rhizopus arrhizus was the predominant agent isolated from both indoor and outdoor environment followed by Cunninghamella species. We also report a single isolate of the rare mucormycete agent, Apophysomyces variabilis from outdoor environment. The present study highlights the presence of low spore burden of Mucormycetes in outdoor and hospital settings in north India. This study also reports the first isolation of A. variabilis from air samples in the Indian subcontinent.

Inoculum quantification of canker-causing pathogens in prune and walnut orchards using real-time PCR.

AIMS: A real-time quantitative PCR (qPCR) assay was established to quantify the inoculum densities in the air and rainwater for six canker-causing pathogen groups in prune and walnut orchards in California. METHODS AND RESULTS: The previously published DNA primers to target six pathogen groups including Botryosphaeria dothidea, Cytospora spp., Diplodia spp., Lasiodiplodia spp., Neofusicoccum spp. and Phomopsis spp. were used in a qPCR assay. Air samples from Burkard spore traps and rain samples from special rain collector devices were collected periodically from various prune and walnut orchards. Using the qPCR approach, we were able to quantify the concentrations of these pathogen groups in rainwater and air samples and study the dynamics of pathogen inoculum in orchards showing severe canker potential. Phomopsis spp. and Diplodia spp. were not found in all rain samples in prune orchards, although they were detected in the 2016 in the walnut orchard. The other four pathogen groups were quantified at varying concentrations in the prune and walnut orchards. Cytospora spp. in some cases showed higher concentrations in the rainwater in prune orchards. CONCLUSIONS: The rainy season during winter and early spring is a highly risky period of time for infection by the pathogens when the inoculum of these pathogens can easily spread by air and rain water, thus serving as an important inoculum source for disease initiation. The different studied pathogen groups showed different concentrations during the growing season, indicating the complexity of the components of canker-causing species in various tree crops. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed the applicability of the qPCR assay in the quantification of inoculum in tree orchards to help reveal the mechanisms of canker disease epidemics and to help design disease management strategies.

Evaluation of the new Id-Fungi plates from Conidia for MALDI-TOF MS identification of filamentous fungi and comparison with conventional methods as identification tool for dermatophytes from nails, hair and skin samples.

OBJECTIVES: We first compare the efficiency of mould/dermatophyte identification by MALDI-TOF MS using a new medium called Id-Fungi plates (IDFP) from Conidia® and two different databases. For the second purpose, we evaluated a new version of the medium supplemented with cycloheximide, Id-Fungi plates Plus (IDFPC) for the direct inoculation of nails, hair and skin samples and compared the efficiency of MALDI-TOF MS identification of dermatophytes to classical methods based on culture and microscopy. METHODS: A total of 71 strains have been cultured IDFP and Sabouraud gentamicin plates (SGC2) and were identified by MALDI-TOF MS. For the evaluation of the combination IDFPC/ MALDI-TOF MS as a method of identification for dermatophytes, 428 samples of hair nails and skin were cultivated in parallel on IDFPC and Sabouraud + cycloheximide medium (SAB-ACTI). RESULTS: For Aspergillus sp and non-Aspergillus moulds, the best performances were obtained on IDFP after maximum 48-h growth, following protein extraction. For dermatophytes, the best condition was using the IDFP at 72 hours, after extended direct deposit. Regarding the direct inoculation of nails, hair skin on IDFPC, 129/428 (30.1%) showed a positive culture against 150/428 (35%) on SAB-ACTI medium. Among the 129 positive strains, the identification by MALDI-TOF MS was correct for 92/129 (71.4%). CONCLUSION: The IDFP allows the generation of better spectra by MALDI-TOF MS compared to SGC2. It facilitates sampling and deposit. Regarding the use of IDFPC, this medium seems less sensitive than SAB-ACTI but among positive strains, the rate of correct identification by MALDI-TOF MS is satisfactory.

A staining method for detection of Enterocytozoon hepatopenaei (EHP) spores with calcofluor white.

A fluorescent stain, calcofluor white (CFW), was used for detection of spores of the microsporidium Enterocytozoon hepatopenaei (EHP). EHP spores in suspension, in feces, or in the infected hepatopancreas of the shrimp Penaeus vannamei, can be easily stained with this chitin and cellulose binding dye to show distinct blue-white fluorescent oval walls. The dye does not stain the host tissues. EHP spores showed orange-red spots by staining with hematoxylin and phloxine (H&P) in the section. CFW staining provides a simple and rapid method for determining the presence of EHP spores in fecal or tissue samples.

A specific molecular label for identifying mature Nosema bombycis spores.

Microsporidia are a fascinating phylum of obligate intracellular pathogens with unique infection processes and complicated life cycles. Microsporidian life cycles can be divided roughly into intracellular and extracellular stages. Currently, research on their life cycles were mainly explored by morphology because there are few molecular markers available with which to distinguish the different life stages. In this study, we generated H20, a monoclonal antibody (MAb) to label mature spores of Nosema bombycis. Immunofluorescence assays showed that the target protein of H20, which is highly stable and was barely affected by alkali and sodium dodecyl sulfate (SDS) treatments, was located on the mature spore surface. Western blot analysis showed that spore wall protein 26 (SWP26) was the likely target of H20. This MAb can specifically identify mature spores in a complex biological sample based on immunological detection of the parasite.

Human Pythiosis: Emergence of Fungal-Like Organism.

Pythiosis is an emerging infectious disease caused by the aquatic oomycete Pythium insidiosum, a fungal-like organism. It is believed that P. insidiosum's zoospores, its infected form, play major role in pathogenesis. Vascular and ocular infections are the most common clinical manifestation in humans. It is difficult to establish the diagnosis given its relatively rarity and difficulty to distinguish P. insidiosum from other molds. Delay in diagnosis and treatment has been associated with poor outcomes. High index of suspicion is the key, particularly in thalassemia patients with arterial insufficiency and patients with fungal keratitis/endophthalmitis without improvement on antifungal therapy. Tissue culture and zoospore induction remain gold standard for diagnosis; however, DNA-based method should be performed simultaneously. The combination of radical surgery, antifungal agents, and immunotherapy has been recommended. It was previously believed that surgery with negative surgical margins was the essential to survive in vascular pythiosis; however, it was recently found that patients could have residual disease despite documented negative surgical margins as infected clot may be dislodged to proximal arterial sites prior to surgery. Serum ß-D-glucan (BG) has been used to monitor disease response after treatment initiation in vascular pythiosis. A significant decrease in BG levels within 2 weeks after surgery is indicative of the absence of residual infection. Unfortunately, monitoring tools for ocular pythiosis are not yet available. Itraconazole plus terbinafine have generally been used in P. insidiosum-infected patients; however, antibacterial agents, including azithromycin and linezolid, have also been used with favorable outcomes in ocular disease. Recently, azithromycin or clarithromycin plus doxycyclin were used in two relapsed vascular pythiosis patients with good outcomes.

Conventional detection and quantification real-time PCR of the pks-1 gene of Chaetomium globosum.

Chaetomium globosum is known as a potential biocontrol indicator against various soil and seedborne pathogens. Precise data are necessary for population monitoring of C. globosum for its effective use in agriculture. A sequence-characterized amplified region marker has been applied for the detection of this biocontrol agent, which will help to detect C. globosum at the site of its application. Out of 17 isolates of C. globosum, only 8 isolates of C. globosum amplified a monomorphic band of 1,900 bp. C. globosum is known for producing chaetoglobosin A. The pks-1 gene is unique in C. globosum in that it is involved in chaetoglobosin A production, melanin formation, and sporulation. Real-time PCR of pks-1 was used to compare the expressions of the pks-1 gene and chaetoglobosin A biosynthesis and sporulation. It was found that the sporulation of C. globosum was associated with the levels of pks-1 gene expression; Cg2 isolate showed high expression of the pks-1 gene, 41.21%, and also produced a great number of spores and perithecia. The association between the pks-1 gene expression and chaetoglobosin A production was estimated. The Pks-1 gene was expressed by all C. globosum isolates except one isolate, C1, which is another species of Chaetomium. In addition, all C. globosum isolates produced chaetoglobosin A with different concentrations and did not express the same levels of pks-1. This finding may be a result of the solvent type used in the extraction.

Fungal spores and pollen are correlated with meteorological variables: effects in human health at Hermosillo, Sonora, Mexico.

We conducted the first study of air pollen and fungal spores for Hermosillo, Sonora, where the human population is exposed to high temperatures and high levels of dust and suffers from diseases related to air quality. We sampled pollen and fungal spores daily in the air during 2016 using a volumetric spore trap Hirst-type sampler. We used simple linear correlation to investigate the association between pollen and spore counts and daily weather conditions. We found an Annual Pollen Integral of 16,243 pollen day/m3 and an Annual Spore Integral higher 222,365 spore day/m3. We identified 32 pollen taxa and 15 different spores. We found two periods of higher pollen and spore concentrations: March to May and August to October, the latter was the most severe. Spore and pollen concentrations in the air increased at higher temperature and higher relative humidity but decreased at higher precipitation. We detected negative impacts during summer and fall on population health, with 13,454 cases of people who presented diseases related to allergies. A peak in allergies is centered during October and correlates well with our peaks in pollen and spore concentrations; it seems that pollen of Poaceae is the one that generates most effects in allergic people.

Recovery of Fungal Cells from Air Samples: a Tale of Loss and Gain.

There are limitations in establishing a direct link between fungal exposure and health effects due to the methodology used, among other reasons. Culture methods ignore the nonviable/uncultivable fraction of airborne fungi. Molecular methods allow for a better understanding of the environmental health impacts of microbial communities. However, there are challenges when applying these techniques to bioaerosols, particularly to fungal cells. This study reveals that there is a loss of fungal cells when samples are recovered from air using wet samplers and aimed to create and test an improved protocol for concentrating mold spores via filtration prior to DNA extraction. Results obtained using the new technique showed that up to 3 orders of magnitude more fungal DNA was retrieved from the samples using quantitative PCR. A sequencing approach with MiSeq revealed a different diversity profile depending on the methodology used. Specifically, 8 fungal families out of 19 families tested were highlighted to be differentially abundant in centrifuged and filtered samples. An experiment using laboratory settings showed the same spore loss during centrifugation for Aspergillus niger and Penicillium roquefortii strains. We believe that this work helped identify and address fungal cell loss during processing of air samples, including centrifugation steps, and propose an alternative method for a more accurate evaluation of fungal exposure and diversity.IMPORTANCE This work shed light on a significant issue regarding the loss of fungal spores when recovered from air samples using liquid medium and centrifugation to concentrate air particles before DNA extraction. We provide proof that the loss affects the overall fungal diversity of aerosols and that some taxa are differentially more affected than others. Furthermore, a laboratory experiment confirmed the environmental results obtained during field sampling. The filtration protocol described in this work offers a better description of the fungal diversity of aerosols and should be used in fungal aerosol studies.

Carbohydrate, glutathione, and polyamine metabolism are central to Aspergillus flavus oxidative stress responses over time.

BACKGROUND: The primary and secondary metabolites of fungi are critical for adaptation to environmental stresses, host pathogenicity, competition with other microbes, and reproductive fitness. Drought-derived reactive oxygen species (ROS) have been shown to stimulate aflatoxin production and regulate in Aspergillus flavus, and may function in signaling with host plants. Here, we have performed global, untargeted metabolomics to better understand the role of aflatoxin production in oxidative stress responses, and also explore isolate-specific oxidative stress responses over time. RESULTS: Two field isolates of A. flavus, AF13 and NRRL3357, possessing high and moderate aflatoxin production, respectively, were cultured in medium with and without supplementation with 15 mM H2O2, and mycelia were collected following 4 and 7 days in culture for global metabolomics. Overall, 389 compounds were described in the analysis which encompassed 9 biological super-pathways and 47 sub-pathways. These metabolites were examined for differential accumulation. Significant differences were observed in both isolates in response to oxidative stress and when comparing sampling time points. CONCLUSIONS: The moderately high aflatoxin-producing isolate, NRRL3357, showed extensive stimulation of antioxidant mechanisms and pathways including polyamines metabolism, glutathione metabolism, TCA cycle, and lipid metabolism while the highly aflatoxigenic isolate, AF13, showed a less vigorous response to stress. Carbohydrate pathway levels also imply that carbohydrate repression and starvation may influence metabolite accumulation at the later timepoint. Higher conidial oxidative stress tolerance and antioxidant capacity in AF13 compared to NRRL3357, inferred from their metabolomic profiles and growth curves over time, may be connected to aflatoxin production capability and aflatoxin-related antioxidant accumulation. The coincidence of several of the detected metabolites in H2O2-stressed A. flavus and drought-stressed hosts also suggests their potential role in the interaction between these organisms and their use as markers/targets to enhance host resistance through biomarker selection or genetic engineering.

Continuous separation of fungal spores in a microfluidic flow focusing device.

The research of fungi is of great importance in a number of fields, such as environmental and healthcare studies. While there are a large number of optical and molecular methods available for characterization and identification of fungi and their spores, their isolation is still conducted using slow and labor-intensive methods. Here, we develop a microfluidic device for the continuous separation of fungal spores from other eukaryotic cells. The spores were separated through the microfluidic device by expanding pinched flow fractionation (PFF) containing the spores, achieving a spatial separation perpendicular to the flow direction according to the spore size. Further branch flow fractionation (BFF) and co-flow of a Newtonian and viscoelastic fluid were used to enhance the separation performance. Using this microfluidic device, we demonstrated the separation of two different types of fungal spores and further separation of fungal spores from eukaryotic cells with a separation efficiency of above 90%. Compared to the existing conventional methods, our microfluidic flow focusing device requires little manual handling and uses small amounts of samples without any pre-treatment steps of the samples.

Quantitative evaluation of bioaerosols in different particle size fractions in dust collected on the International Space Station (ISS).

Exposure to bioaerosols can adversely influence human health through respiratory tract, eye, and skin irritation. Bioaerosol composition is unique on the International Space Station (ISS), where the size distribution of particles in the air differs from those on Earth. This is due to the lack of gravitational settling and sources of biological particles. However, we do not understand how microbes are influenced by particle size in this environment. We analyzed two types of samples from the ISS: (1) vacuum bag debris which had been sieved into five different size fractions and (2) passively collected particles on a tape substrate with a passive aerosol sampler. Using quantitative polymerase chain reaction (qPCR), the highest concentration of fungal spores was found in the 106-150 µm-sized sieved dust particles, while the highest concentration of bacterial cells was found in the 150-250 µm-sized sieved dust particles. Illumina MiSeq DNA sequencing revealed that particle size was associated with bacterial and fungal communities and statistically significant (p = 0.035, p = 0.036 respectively). Similar fungal and bacterial species were found within the passive aerosol sample and the sieved dust samples. The most abundant fungal species identified in the aerosol and sieved samples are commonly found in food and plant material. Abundant bacterial species were most associated with the oral microbiome and human upper respiratory tract. One limitation to this study was the suboptimal storage conditions of the sieved samples prior to analysis. Overall, our results indicate that microbial exposure in space may depend on particle size. This has implications for ventilation and filtration system design for future space vehicles and habitats.

Studies in the Stypella vermiformis group (Auriculariales, Basidiomycota).

Stypella vermiformis is a heterobasidiomycete producing minute gelatinous basidiocarps on rotten wood of conifers in the Northern Hemisphere. In the current literature, Stypella papillata, the genus type of Stypella (described from Brazil), is treated as a taxonomic synonym of S. vermiformis. In the present paper, we revise the type material of S. papillata and a number of specimens addressed to S. vermiformis. As a result, the presumed synonymy of S. papillata and S. vermiformis is rejected and the genus Stypella is restricted to the single species S. papillata. Morphological and molecular phylogenetic studies of specimens from the Northern Hemisphere corresponding to the current concept of S. vermiformis uncovered three species from two newly described genera. S. vermiformis s.str. is distributed in temperate Europe and has small-sized basidia and basidiospores, and it is placed in a new genus, Mycostilla. Another genus, Stypellopsis, is created for two other species, the North American Stypellopsis farlowii, comb. nov., and the North European Stypellopsis hyperborea, sp. nov. Basidia and basidiospores of Stypellopsis spp. are larger than in Mycostilla vermiformis but other morphological characters are very similar. In addition, Spiculogloea minuta (Spiculogloeomycetes, Pucciniomycotina) is reported as new to Norway, parasitising basidiocarps of M. vermiformis and Tulasnella spp.

Fungal aerosol composition in moldy basements.

Experimental aerosolization studies revealed that fungal fragments including small fragments in the submicrometer size are released from fungal cultures and have been suggested to represent an important fraction of overall fungal aerosols in indoor environments. However, their prevalence indoors and outdoors remains poorly characterized. Moldy basements were investigated for airborne fungal particles including spores, submicron fragments, and larger fragments. Particles were collected onto poly-L-lysine-coated polycarbonate filters and qualitatively and quantitatively analyzed using immunogold labeling combined with field emission scanning electron microscopy. We found that the total fungal aerosol levels including spores, submicrometer, and larger fragments in the moldy basements (median: 80 × 103  m-3 ) were not different from that estimated in control basements (63 × 103  m-3 ) and outdoor (90 × 103  m-3 ). However, mixed effect modeling of the fungal aerosol composition revealed that the fraction of fragments increased significantly in moldy basements, versus the spore fraction that increased significantly in outdoor air. These findings provide new insight on the compositional variation of mixed fungal aerosols in indoor as compared to outdoor air. Our results also suggest that further studies, aiming to investigate the role of fungal aerosols in the fungal exposure-disease relationships, should consider the mixed composition of various types of fungal particles.