Fungal Fragments Research Articles

Integrated eDNA metabarcoding and morphological analyses assess spatio-temporal patterns of airborne fungal spores

Fungi represent relevant allergens and plant pathogens that can disperse on long ranges, potentially producing severe consequences on public health and agriculture. Up to 11% of the bioaerosol particles are fungal spores and mycelium fragments. Estimation of fungal species diversity in time and space is decisive but may be biased by abiotic conditions and sampling methods. Traditional morphological analyses of fungal spores have been widely applied in aerobiology in the past, while recently eDNA metabarcoding can complement these studies. Here, we used both morphological analysis (spore count and taxon identification) and high-throughput sequencing to disentangle spatio-temporal variation of fungi across Northern and Central Italy and to evaluate the detection efficiency of the two approaches. Our results showed that eDNA metabarcoding detects about three times more genera and has a higher detection efficiency than the morphological analyses. However, the efficiency is high in both spore count and eDNA metabarcoding methods when the most abundant or the rarest genera are considered but it can substantially vary between the two approaches when moderately abundant genera are analyzed. Furthermore, morphological spore determination resulted in higher variance explained by PERMANOVA analysis with respect to eDNA metabarcoding (26% and 13%, respectively), which leads to a better spatio-temporal characterization of the fungal genera. As both morphological analyses and eDNA metabarcoding methods capture significant interactions between seasons and sites, they could be preferably used as complementing approaches to reliably study airborne fungal diversity and variation.

Bark decomposition in white oak soil outperforms eastern hemlock soil, while bark type leads to consistent changes in soil microbial composition

Bark decomposition is an underexamined component of soil carbon cycling and soil community assembly. Numerous studies have shown faster decomposition of leaf litter in “home” environments (i.e. within soil adjacent to the plant that produced the leaves), suggesting potential legacy effects from previous deposition of similar litter. This is expected to occur through, in part, accumulation of microorganisms that metabolize substrates the litter provides. Whether a similar “home-field advantage” (HFA) exists for bark decomposition is unknown, but this dynamic may differ because annual bark deposits to soil are minimal relative to leaf deposits. We hypothesized that (1) as with leaf litter, bark will be better decomposed near to the tree from which it was collected, and (2) that decomposing bark can initiate change in soil microbial composition. To test these hypotheses, we used a full factorial design that included two bark types (collected from eastern hemlock, Tsuga canadensis, and white oak, Quercus alba) and two soil types (‘home’ and ‘away’) within a temperate mixed hardwood forest at the Shale Hills Catchment in central Pennsylvania, USA. Bark was excised from 25 replicates of each tree type, buried in either home or away soil, and incubated belowground from July 2017 to June 2018. Decomposition was assessed through proportionate mass loss over time, while microbial composition in the bark and adjacent soil was assessed through high-throughput sequencing of 16S rRNA gene and fungal ITS fragments. Overall, bark degraded faster in white oak soils, and there was also an effect of bark type on decomposition. Although white oak bark decomposed more quickly in its home environment, this could be due to either soil conditioning or inherent differences in the soils in which each species grows. Soil microbial assemblages also sorted according to bark type rather than soil type, suggesting that bark strongly influences the composition of nearby microorganisms during decomposition. Our results suggest that both bark type and soil type are important factors during bark decomposition, but our findings suggest no clear evidence for HFA.

First Report of Southern Blight on Cynanchum stauntonii Caused by Athelia rolfsii in China

Cynanchum stauntonii is a medicinal plant belonging to the Apocynaceae family, and its rhizome can be used as antitussive and anti-inflammatory medicine. C. stauntonii is widely cultivated in Tuanfeng, Xinzhou, Yingshan, and Qichun cities in Hubei Province. However, serious outbreaks of southern blight were observed in C. stauntonii fields in June and July in Tuanfeng County, Huanggang City (N30°48′48.943″, E115°01′15.967″) from 2018 to 2019, and the disease incidence was about 10 to 40%. The diseased C. stauntonii plants were characterized by water-soaked lesions on stems with white mycelia and white to brown sclerotia densely distributed on the infected sites and the surrounding soil. Leaf yellowing and wilting, root rot, and plant death are very common symptoms of infected C. stauntonii in warm and humid weather, especially in June and July. To isolate the fungal pathogen, mycelial fragments and sclerotia were collected from the field and cultivated directly on potato dextrose agar (PDA containing 100 µg/ml of kanamycin) medium and incubated at 27°C. The purified isolate BQ-1 showed white and fluffy aerial mycelia, which grew radially with an average growth rate of 32.3 to 37.5 mm/day (n = 10). White microsclerotia could be observed on PDA at 5 days postinoculation (dpi), and brown mature sclerotia appeared at 9 dpi. The number of mature sclerotia produced per plate ranged from 67 to 128 (92 ± 18; n = 10), and the diameter of mature sclerotia ranged from 1.00 to 4.15 mm (2.65 ± 0.83 mm; n = 50). No spores were observed under the microscope, and the fungal hyphae formed septa. For molecular identification, the 18S rDNA region of isolate BQ-1 was amplified by employing primers NS1 and NS6 (Li et al. 2014). The 18S rDNA sequence was sequenced by Sangon Biotech (Shanghai, China) and was deposited in GenBank under accession number MN243786. This sequence was 100% similar to Athelia rolfsii isolate AFTOL-ID 664 (AY665774.1). In addition, part of the elongation factor 1-alpha (TEF-1α) gene was amplified by using the primers EF595F and EF1160R (Wendland and Kothe 1997). The TEF-1α sequence was deposited in GenBank with accession number MN262526 and had 99.63% similarity to A. rolfsii strain Sr_12 (JF267794.1). The phylogenetic tree also shows that isolate BQ-1 is very close to seven A. rolfsii genes screened from GenBank. Pathogenicity tests were conducted using cleaned roots, stems, and leaves of healthy C. stauntonii. The upper, middle, and lower parts of roots (n = 3), stems (n = 3), and leaves (n = 3) were inoculated with mature sclerotia in a moist chamber for 5 days at 25 ± 2°C following the method described previously (Mahadevakumar et al. 2018). Samples inoculated with sterile water were used as a control. Sclerotia began to germinate and formed small colonies on the surface of tissues at 1 dpi. Mycelia began to infect tissues and formed black-brown spots on the tissues at 2 dpi, and the tissues were rotted at 5 dpi. In the pot experiment, stem bases of three C. stauntonii seedlings were punctured with a sterile needle and then inoculated with 5-day-old PDA-grown BQ-1 mycelia. Three seedlings treated with sterile water were used as controls. Three days later, the inoculated seedlings showed typical disease symptoms. Fungal pathogens were reisolated from the inoculated site and were reconfirmed through morphological features. This is the first report of A. rolfsii causing southern blight on C. stauntonii in China. This report will provide resources and reference for controlling of the increased incidence and economic losses of southern blight on C. stauntonii.

Allergenic fungal spores and hyphal fragments in the aerosol of Havana, Cuba

Fungal spores have a recognized allergenicity, but in recent years, fungal fragments have also been reported as inducers of Type I allergy. However, airborne hyphae are scarcely studied in tropical regions. The objective of this research was to analyze the fungal composition of the Havana aerosol during 2017 and 2018, emphasizing the presence of hyphae fragments. Air sampling was performed using a Lanzoni VPPS-2000 sampler. The fungal propagules collected were identified and counted following the recommendations of the Spanish Aerobiology Network in two longitudinal transepts of each daily slides. In 2017, the spore count and hyphae count were higher than in 2018 (108,607 spores and 3822 fungal fragments vs. 40,896 spores and 2268 fungal fragments). Cladosporium was the most abundant spore (77.2–76.6%), predominating in the month of May. Leptosphaeria ascospores were identified at lower levels (16.2–17.9%) but also abundant during May. The behavior of the hyphal fragments does not show great variations throughout the year. The contribution of this component to the aerosol ranged between 3.4 and 5.3% in 2017 and 2018, respectively. The intradiurnal periodicity showed a diurnal pattern for Cladosporium and nocturnal for Leptosphaeria, while the hyphae pattern does not show great variations. The temperature and relative humidity correlated with the concentration of the detected airborne fungi propagules. The composition of the fungal aerosol in Havana during the 2 years was characterized, including both spores and hyphal fragments, since both structures can be related to respiratory allergy pathologies.

Subchronic inhalation of Aspergillus versicolor conidia leads to sustained innate immune responses and is associated with irreversible remodeling of pulmonary arterial tissue following recovery

Abstract Aspergillus versicolor is an environmentally ubiquitous fungal species that is particularly abundant in environments with water infiltration. Respiration of fungal products, including conidia, hyphal fragments, mycotoxins, and other secondary metabolites may cause adverse pulmonary health effects including asthma and allergy. In the present study, pulmonary immune responses in B6C3F1 mice were examined following 1, 2, 4, 8, and 13 weeks of repeated respiratory challenge to 3×105A. versicolor conidia. Increased alveolar macrophages, inflammatory monocytes, neutrophils, and eosinophils, which peaked between 4 and 8 weeks, were detected in the lungs of mice exposed to viable conidia. Type 2 innate lymphoid cells (ILC2s) peaked between 8 and 13 weeks compared to total T cells which increased between 2 and 8 weeks and decreased at 13 weeks. Protein concentrations of the Th2 cytokines IL4, IL5, and IL13 were highest in bronchoaveolar lavage fluid of exposed mice following 4 weeks and exhibited a time-dependent reduction at 8 and 13 weeks. Inhalation of conidia for 13 weeks led to goblet cell hyperplasia, vascular wall thickening, and perivascular inflammation. Finally, mixed perivascular inflammation and smooth muscle hypertrophy were sustained at 4 weeks post-exposure. These data indicate that exposure to A. versicolor elicits a Th2-biased immune response that is driven by innate cell populations and is associated with the induction of remodeling of the pulmonary vasculature.

The fluctuations of airborne fungal components in western Romania

Outdoor regional concentrations of airborne fungal spores and fungal fragments were measured using the 7-day Lanzoni sampler. The daily and intradiurnal fluctuations recorded during the monitored period have been studied. The multiple regression analysis was performed in order to determine how much of the total variance in these concentrations could be explained by meteorological parameters. Daytime periodicity statistically significant was established only in a few cases. As the relative importance of the weather factors varied, they could not be ranked according to their effect.

Dynamic Surface Proteomes of Allergenic Fungal Conidia.

Fungal spores and hyphal fragments play an important role as allergens in respiratory diseases. In this study, we performed trypsin shaving and secretome analyses to identify the surface-exposed proteins and secreted/shed proteins of Aspergillus fumigatus conidia, respectively. We investigated the surface proteome under different conditions, including temperature variation and germination. We found that the surface proteome of resting A. fumigatus conidia is not static but instead unexpectedly dynamic, as evidenced by drastically different surface proteomes under different growth conditions. Knockouts of two abundant A. fumigatus surface proteins, ScwA and CweA, were found to function only in fine-tuning the cell wall stress response, implying that the conidial surface is very robust against perturbations. We then compared the surface proteome of A. fumigatus to other allergy-inducing molds, including Alternaria alternata, Penicillium rubens, and Cladosporium herbarum, and performed comparative proteomics on resting and swollen conidia, as well as secreted proteins from germinating conidia. We detected 125 protein ortholog groups, including 80 with putative catalytic activity, in the extracellular region of all four molds, and 42 nonorthologous proteins produced solely by A. fumigatus. Ultimately, this study highlights the dynamic nature of the A. fumigatus conidial surface and provides targets for future diagnostics and immunotherapy.

Response of soil microbial communities to continuously mono-cropped cucumber under greenhouse conditions in a calcareous soil of north China

Microbes are the engines for nutrient cycling in the soil, playing an important role in maintaining soil quality and agricultural production, but it is unclear how soil microbial communities respond to continuously mono-cropped cucumber under greenhouse conditions. High-throughput sequencing of the bacterial 16S rRNA and fungal 18S rRNA gene fragments was conducted to compare the variations of microbial communities within greenhouse mono-cropped cucumber systems across 8, 15, and 22 cultivation years with new-build greenhouse of cucumber cultivated just for one season (1 year). Results showed that soil pH and fungal diversity significantly decreased, whereas soil EC, organic matter (OM), and nutrient concentrations (total nitrogen (N), nitrate N (NO3−-N), total and available phosphorus, and potassium) significantly increased when cucumber was mono-cropped for 8 or more years. The mono-cropped cucumber for 8 or more years reduced the relative abundance of the dominant bacterial phyla Actinobacteria, but increased the abundances of Acidobacteria and Firmicutes significantly compared with the cucumber planted for one season. Moreover, the predominant fungal phylum Ascomycota increased significantly with increasing cultivation years of cucumber. Soil OM and NO3−-N are most important factors to drive the variation of soil bacterial community, while soil pH, OM, and NO3−-N markedly responded to the changes of the fungal community in the greenhouse cucumber mono-cropped system. This research provides insights into the response of microbial communities to continuous mono-cropping cucumber, and this has implications on the sustainability of such intensive production systems.

Characterization and pro-inflammatory potential of indoor mold particles.

A number of epidemiological studies find an association between indoor air dampness and respiratory health effects. This is often suggested to be linked to enhanced mold growth. However, the role of mold is obviously difficult to disentangle from other dampness-related exposure including microbes as well as non-biological particles and chemical pollutants. The association may partly be due to visible mycelial growth and a characteristic musty smell of mold. Thus, the potential role of mold exposure should be further explored by evaluating information from experimental studies elucidating possible mechanistic links. Such studies show that exposure to spores and hyphal fragments may act as allergens and pro-inflammatory mediators and that they may damage airways by the production of toxins, enzymes, and volatile organic compounds. In the present review, we hypothesize that continuous exposure to mold particles may result in chronic low-grade pro-inflammatory responses contributing to respiratory diseases. We summarize some of the main methods for detection and characterization of fungal aerosols and highlight in vitro research elucidating how molds may induce toxicity and pro-inflammatory reactions in human cell models relevant for airway exposure. Data suggest that the fraction of fungal hyphal fragments in indoor air is much higher than that of airborne spores, and the hyphal fragments often have a higher pro-inflammatory potential. Thus, hyphal fragments of prevalent mold species with strong pro-inflammatory potential may be particularly relevant candidates for respiratory diseases associated with damp/mold-contaminated indoor air. Future studies linking of indoor air dampness with health effects should assess the toxicity and pro-inflammatory potential of indoor air particulate matter and combined this information with a better characterization of biological components including hyphal fragments from both pathogenic and non-pathogenic mold species. Such studies may increase our understanding of the potential role of mold exposure.

Silencing of the Ornithine Decarboxylase Gene of Fusarium oxysporum f. sp. lycopersici by Host-Induced RNAi Confers Resistance to Fusarium Wilt in Tomato

Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici, a hemibiotrophic filamentous fungal pathogen is one of the important diseases of tomato. Recently, RNA interference (RNAi) has emerged as a novel alternative strategy for the control of plant diseases caused by fungal pathogens. Therefore, we tested the potential of RNAi for the control of Fusarium wilt in tomato by targeting a key polyamine (PA) biosynthesis gene, ornithine decarboxylase (ODC) of the pathogen as PAs (putrescine, spermidine, and spermine) are absolutely essential for normal growth and development. The target fungal ODC gene fragment was cloned in a hairpin RNA construct, which was used to develop several transgenic tomato plants. These RNAi transgenic lines expressed the intended small interfering RNAs (siRNAs) and exhibited moderate to high resistance when challenged with the spores of F. oxysporum. Interestingly, the silencing effect was confined only to the fungal pathogen and had no influence on the plant ODC gene expression, polyamine levels, and morphology. These results confirm that the ODC gene is vital for fungal growth and is a suitable target for disease control through host-induced gene silencing (HIGS) and also implicates the transfer of dsRNA/siRNAs from host plant cells to the fungal cells. To further validate the uptake of plant-derived siRNAs by fungal cells in a visual manner, silencing of the GFP transgene was observed in F. oxysporum GFP transformants upon infecting the dsGFP-expressing transgenic tomato plants. These experiments demonstrate the applicability of RNAi-based approach for crop protection from the filamentous fungal pathogens.

Particle size distribution of the major Alternaria alternata allergen, Alt a 1, derived from airborne spores and subspore fragments

Fungal fragments are abundant immunoreactive bioaerosols that may outnumber the concentrations of intact spores in the air. To investigate the importance of Alternaria fragments as sources of allergens compared to Alternaria spores, we determined the levels of Alternaria spores and Alt a 1 (the major allergen in Alternaria alternata spores) collected on filters within three fractions of particulate matter (PM) of different aerodynamic diameter: (1) PM>10, (diameter>10 μm); (2) PM2.5-10 (2.5–10μm); (3) PM2.5 (0.12–2.5 μm). The airborne particles were collected using a three stage high-volume ChemVol cascade impactor during the Alternaria sporulation season in Poznań, Poland (30 d between 6 July and 22 September 2016). The quantification of Alt a 1 was performed using the enzyme-linked immunosorbent assay. High concentrations of Alt a 1 were recorded during warm and dry d characterized by high sunshine duration, lack of clouds and high dew point values. Atmospheric concentrations of Alternaria spores correlated significantly (r = 0.930, p < 0.001) with Alt a 1 levels. The highest Alt a 1 was recorded in PM2.5-10 (66.8 % of total Alt a 1), while the lowest in PM2.5 (<1.0 %). Significantly more Alt a 1 per spore (>30 %) was observed in PM2.5-10 than in PM>10. This Alt a 1 excess may be derived from sources other than spores, e.g. hyphal fragments. Overall, in outdoor air the major source of Alt a 1 are intact Alternaria spores, but the impact of other fungal fragments (hyphal parts, broken spores, conidiophores) cannot be neglected, as they may increase the total atmospheric Alt a 1 concentration.

Sonicated extracts from the Quorn fermentation co-product as oil-lowering emulsifiers and foaming agents

This study assessed the impact of sonication on the structure and properties of a functional extract (retentate 100 or R100) from the Quorn fermentation co-product (centrate). In a previous study, we reported that the R100 fraction displayed good foaming, emulsifying and rheological properties. Sonication of a R100 solution led to the breakdown of the large hyphal structures characteristic of this extract into smaller fragments. Foams prepared with sonicated R100 displayed a higher foaming ability than with untreated R100 and a high foam stability but lower than untreated R100 ones. Oil-in-water emulsions prepared with sonicated R100 displayed smaller oil droplet size distributions than with untreated R100. Confocal micrographs suggested that small fungal fragments contributed to the stabilisation of oil droplets. 50% oil-reduced R100 emulsions were prepared by mixing R100 emulsions (untreated or sonicated) with a sonicated R100 solution at a 1:1 ratio. Smaller oil droplet size distributions were reported for the oil-reduced emulsions. These results showed that the addition of small hyphal fragments or surface-active molecules and molecular aggregates released during sonication contributed to the formation and stabilisation of smaller oil droplets. This study highlighted the potential to modulate the structure, emulsifying and foaming properties of functional extracts from the Quorn fermentation co-product by sonication and the potential of these extracts as oil-lowering agents in emulsion-based products through the reduction of oil droplet size and their stabilisation.Graphical abstract

Atmospheric fungal nanoparticle bursts.

Aerosol nanoparticles play an important role in the climate system by affecting cloud formation and properties, as well as in human health because of their deep reach into lungs and the circulatory system. Determining nanoparticle sources and composition is a major challenge in assessing their impacts in these areas. The sudden appearance of large numbers of atmospheric nanoparticles is commonly attributed to secondary formation from gas-phase precursors, but in many cases, the evidence for this is equivocal. We report the detection of a mode of fungal fragments with a mobility diameter of roughly 30 nm released in episodic bursts in ambient air over an agricultural area in northern Oklahoma. These events reached concentrations orders of magnitude higher than other reports of biological particles and show similarities to unclarified events reported previously in the Amazon. These particles potentially represent a large source of both cloud-forming ice nuclei and respirable allergens in a variety of ecosystems.

Neurospora sitophila in indoor environment of buildings

Neurospora sitophila is a microscopic filamentous fungus of the Ascomycete taxon that rapidly expands in the environment. Although the fungus has been recorded primarily as a known laboratory contaminant, this contribution presents evidence of its frequent occurrence in indoor environment of offices at the university, especially in places where a coffee maker was part of the equipment. A total of 50 smears were made from the environment of the University of VŠB - TU Ostrava from various parts of the fixtures. It was found that spores and fragments of N. sitophila mycelia are an active part of the building air using the BIOLOG identification system. Although no direct evidence has yet been obtained that N. sitophila is the causative agent of human disease or infection, the presence of this fungus in the environment should be regarded as a risk factor for human health. Exposure to fungal spores and fragments of mycelia in the indoor environment of buildings can cause hypersensitivity known to be an influencing factor in allergic reactions and asthma.

Cultivation and aerosolization of Stachybotrys chartarum for modeling pulmonary inhalation exposure

Objective: Stachybotrys chartarum is a hydrophilic fungal species commonly found as a contaminant in water-damaged building materials. Although several studies have suggested that S. chartarum exposure elicits a variety of adverse health effects, the ability to characterize the pulmonary immune responses to exposure is limited by delivery methods that do not replicate environmental exposure. This study aimed to develop a method of S. chartarum aerosolization to better model inhalation exposures. Materials and methods: An acoustical generator system (AGS) was previously developed and utilized to aerosolize and deliver fungal spores to mice housed in a multi-animal nose-only exposure chamber. In this study, methods for cultivating, heat-inactivating, and aerosolizing two macrocyclic trichothecene-producing strains of S. chartartum using the AGS are described. Results and discussion: In addition to conidia, acoustical generation of one strain of S. chartarum resulted in the aerosolization of fungal fragments (<2 µm aerodynamic diameter) derived from conidia, phialides, and hyphae that initially comprised 50% of the total fungal particle count but was reduced to less than 10% over the duration of aerosolization. Acoustical generation of heat-inactivated S. chartarum did not result in a similar level of fragmentation. Delivery of dry, unextracted S. chartarum using these aerosolization methods resulted in pulmonary inflammation and immune cell infiltration in mice inhaling viable, but not heat-inactivated S. chartarum. Conclusions: These methods of S. chartarum growth and aerosolization allow for the delivery of fungal bioaerosols to rodents that may better simulate natural exposure within water-damaged indoor environments.

Gut microbiome composition in the Hispanic Community Health Study/Study of Latinos is shaped by geographic relocation, environmental factors, and obesity

BackgroundHispanics living in the USA may have unrecognized potential birthplace and lifestyle influences on the gut microbiome. We report a cross-sectional analysis of 1674 participants from four centers of the Hispanic Community Health Study/Study of Latinos (HCHS/SOL), aged 18 to 74 years old at recruitment.ResultsAmplicon sequencing of 16S rRNA gene V4 and fungal ITS1 fragments from self-collected stool samples indicate that the host microbiome is determined by sociodemographic and migration-related variables. Those who relocate from Latin America to the USA at an early age have reductions in Prevotella to Bacteroides ratios that persist across the life course. Shannon index of alpha diversity in fungi and bacteria is low in those who relocate to the USA in early life. In contrast, those who relocate to the USA during adulthood, over 45 years old, have high bacterial and fungal diversity and high Prevotella to Bacteroides ratios, compared to USA-born and childhood arrivals. Low bacterial diversity is associated in turn with obesity. Contrasting with prior studies, our study of the Latino population shows increasing Prevotella to Bacteroides ratio with greater obesity. Taxa within Acidaminococcus, Megasphaera, Ruminococcaceae, Coriobacteriaceae, Clostridiales, Christensenellaceae, YS2 (Cyanobacteria), and Victivallaceae are significantly associated with both obesity and earlier exposure to the USA, while Oscillospira and Anaerotruncus show paradoxical associations with both obesity and late-life introduction to the USA.ConclusionsOur analysis of the gut microbiome of Latinos demonstrates unique features that might be responsible for health disparities affecting Hispanics living in the USA.

Uji Efektivitas Inokulum Fungi Mikoriza Arbuskula Terhadap Pertumbuhan Bibit Jati (Tectona Grandis Linn. F)

Arbuscular mycorrhizal fungi is a phylum of Glomeromycota. Arbuscular mycorrhizal fungi (AMF) propagule are spores, mycor-rhizal fungal hyphae and infected root fragments. The aims of this research were to analyze the effectivity of root inoculum of AMF to enhance teak (Tectona grandis Linn F.) seedling growth. The research was used complete randomized design (CRD)-split plot design. The main plot was root inoculum of AMF, sub plot is a media sterilization and media is not sterilized. The results showed that root inoculum of AMF and media effectively improved teak growth, especially in height, diameter, and shoot dry weight. Root inoculum of AMF is able to be used as the source of inoculum for the growth teak seedling. Fresh inoculum was found to be better than root inoculum stored at room temperature and root inoculum stored at refrigerator temperature (5°C). Storage of root inocu-lum at room temperature and refrigerator temperature (5°C) for two weeks decreased the effectiveness of inoculum. Type of mixed inoculum and inoculum of Acaulospora sp. root resulted in better growth compared with G. clarum root inoculum.

Molecular detection of fungal and bacterial DNA from pustules in patients with palmoplantar pustulosis: special focus on Malassezia species.

Palmoplantar pustulosis (PPP) is a distinct, chronic skin disorder characterized by intraepidermal pustules on the palms and soles. It is hypothesized that microorganisms on the skin might induce the symptoms of PPP via inflammatory cell activation. However, the microbiota has not been studied in detail because of the assumption that the pustules in PPP are sterile. To elucidate the role of microorganisms in pathogenesis of PPP. PCR analysis was performed of microbial DNA fragments in the pustules of patients with PPP. The sequence of the D1/D2 LSU 26s rRNA gene and that of the 16S rRNA gene was used for fungal and bacterial DNA detection, respectively. In total, 71 samples were carefully collected from the pustules of patients with PPP. Fungal DNA bands were detected in 68 samples, and fungi including Malassezia spp. were identified in 30 of 71 samples (42.3%). Malassezia restricta was the most frequently encountered fungus (14/71; 19.7%). However, bacterial DNA was not detected by the methods used. Furthermore, identical fungal DNA was not detected in the outer lid of the pustules, suggesting that the fungi detected within the pustule did not derive from contamination via the skin surface. In the present study, we demonstrated for the first time that certain pustules in patients with PPP contain fungal DNA fragments, especially those of Malassezia spp. Our findings provide new insights on the role of skin microbiota in the pathogenesis of PPP.

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.

Ectomycorrhizal and Saprotrophic Fungal Communities Vary Across mm-Scale Soil Microsites Differing in Phosphatase Activity

To understand nutrient cycling in soils, soil processes and microorganisms need be better characterized. To determine whether specific trophic groups of fungi are associated with soil enzyme activity, we used soil imprinting to guide mm-scale sampling from microsites with high and low phosphatase activities in birch/Douglas-fir stands. Study 1 involved sampling one root window per site at 12 sites of different ages (stands); study 2 was conducted at one of the stem-exclusion stands, at which 5 root windows had been installed. Total fungal and ectomycorrhizal (EM) fungal terminal-restriction fragment length polymorphism (TRFLP) fingerprints differed between high- and low-phosphatase activity microsites at 8 of 12 root windows across 12 sites. Where differences were detected, fewer EM fungi were detected in high- than low-phosphatase activity microsites. Using 5 root windows at one site, next-generation sequencing detected similar fungal communities across microsites, but the ratio of saprotrophic to EM fungal reads was higher in high-phosphatase activity microsites in the two windows that had low EM fungal richness. In windows with differences in fungal communities, both studies indicated that EM fungi were less successful than saprotrophic fungi in colonizing fine-scale, organic matter-rich microsites. Fine-scale sampling linked with in situ detection of enzyme activity revealed relationships between soil fungal communities and phosphatase activity that could not be observed at the scales employed by conventional approaches, thereby contributing to the understanding of fine-scale phosphorus cycling in forest soils.