Fungal ITS Region Research Articles

Fungal Diversity in Gut Microbiota in Patients with Cancer

Patients who have cancer are more prone to get infections than healthy individuals. In this study, we applied cloning sequencing technique to identify the mycobiome and other transient fungal components in the digestive tract of patients with acute myeloid leukemia (AML), non- Hodgkin’s lymphoma (NHL), Leucopenia and anemia and compare it with the fungal components of healthy individuals. Fungal universal primer pair targeting ITS region of the fungal genome was used to assess the fungal components in fecal samples. Approximately more than 1,481 clones were selected and subjected to sequence analysis resulted in detection of a total of 44 and 43 fungal species from both fecal samples of cancer patients and healthy control individuals respectively. The majority of fungi were assigned to the 3 taxa Ascomycota (n = 39/63, 61.9 %), Basidiomycota (n = 23/63, 36.5 %) and Zygomycota (n= 1/63, 1.6 %). Fungal species such as Candida parapsilosis , Candida dubliniensis , Malassezia restricta were represented only in the feces of cancer patients. Moreover, the results from analyzing ITS clone library revealed that the most frequently detected fungi of the Malassezia genus ( Malassezia globosa , Malassezia pachydermatis , Malassezia restricta ) were retrieved in high amount in the fecal samples of cancer patients compared with those of healthy volunteers. Further efforts and studies are needed to shed light on the complex relationships between the fungal populations and the host immune system in cancer patients.

Novel ITS1 Fungal Primers for Characterization of the Mycobiome.

Studies of the human microbiome frequently omit characterization of fungal communities (the mycobiome), which limits our ability to investigate how fungal communities influence human health. The internal transcribed spacer 1 (ITS1) region of the eukaryotic ribosomal cluster has features allowing for wide taxonomic coverage and has been recognized as a suitable barcode region for species-level identification of fungal organisms. We developed custom ITS1 primer sets using iterative alignment refinement. Primer performance was evaluated using in silico testing and experimental testing of fungal cultures and human samples. Using an expanded novel reference database, SIS (18S-ITS1-5.8S), the newly designed primers showed an average in silico taxonomic coverage of 79.9% ± 7.1% compared to a coverage of 44.6% ± 13.2% using previously published primers (P = 0.05). The newly described primer sets recovered an average of 21,830 ± 225 fungal reads from fungal isolate culture samples, whereas the previously published primers had an average of 3,305 ± 1,621 reads (P = 0.03). Of note was an increase in the taxonomic coverage of the Candida genus, which went from a mean coverage of 59.5% ± 13% to 100.0% ± 0.0% (P = 0.0015) comparing the previously described primers to the new primers, respectively. The newly developed ITS1 primer sets significantly improve general taxonomic coverage of fungal communities infecting humans and increased read depth by an order of magnitude over the best-performing published primer set tested. The overall best-performing primer pair in terms of taxonomic coverage and read recovery, ITS1-30F/ITS1-217R, will aid in advancing research in the area of the human mycobiome. IMPORTANCE The mycobiome constitutes all the fungal organisms within an environment or biological niche. The fungi are eukaryotes, are extremely heterogeneous, and include yeasts and molds that colonize humans as part of the microbiome. In addition, fungi can also infect humans and cause disease. Characterization of the bacterial component of the microbiome was revolutionized by 16S rRNA gene fragment amplification, next-generation sequencing technologies, and bioinformatics pipelines. Characterization of the mycobiome has often not been included in microbiome studies because of limitations in amplification systems. This report revisited the selection of PCR primers that amplify the fungal ITS1 region. We have identified primers with superior identification of fungi present in the database. We have compared the new primer sets against those previously used in the literature and show a significant improvement in read count and taxon identification. These primers should facilitate the study of fungi in human physiology and disease states.

Detection of fungal pathogens by a new broad range real-time PCR assay targeting the fungal ITS2 region.

The rise in the incidence of fungal infections and the expanding spectrum of fungal pathogens make early and broad detection of fungal pathogens essential. In the present study, a panfungal real-time PCR assay for the broad-range detection of fungal DNA (Fungi assay) in a wide variety of clinical specimens was developed. Our in-house, HybProbe real-time PCR assay targets the ITS2 region of fungal DNA. The applicability was evaluated by testing 105 clinical samples from 98 patients with suspected fungal infection. Samples included tissue biopsies, paraffin embedded tissues, aspirates, EDTA-anticoagulated blood, cerebrospinal fluids and bronchoalveolar lavages. Fungal pathogens were identified by the Fungi assay in 47 samples. In all of these cases, conventional methods and clinical data were also indicative for a fungal infection. Five samples were interpreted false negative. blast analyses of the amplicons derived from 11 samples revealed the presence of environmental fungal species while other tests and clinical data did not suggest a fungal infection. This fact might indicate contaminated samples. The remaining 42 samples were negative by the Fungi assay as well as the conventional methods and were therefore regarded as true negatives. Thus, sensitivity was 90.4 % and specificity 79.2 %. The Fungi assay improved the targeted diagnosis of fungal infections allowing pathogen identification in samples that were histologically positive but culture negative. For reliable diagnosis, results have to be interpreted in context with conventional methods and clinical data.

Insect frass in stored cereal products as a potential source of Lactobacillus sanfranciscensis for sourdough ecosystem.

Insect frass samples were collected from Drosophila melanogaster, Plodia interpunctella, Rhyzopertha dominica, Sitophilus granarius, Sitophilus oryzae, Sitophilus zeamais, Tribolium confusum and Tribolium castaneum to elucidate if they can be the origin of Type I sourdough micro-organisms (Lactobacillus sanfranciscensis and Candida milleri). Selective enrichments were carried out to isolate lactic acid bacteria (LBA) and yeast. A metagenetic analysis, targeted on bacterial 16S rRNA gene and fungal ITS region, was performed by using Illumina MiSeq protocol. In cultivation conditions, Lactococcus garvieae and Rhodotorula mucilaginosa were the most frequently species among LAB and yeasts respectively. The Next Generation Sequencing approach revealed that Enterobacteriaceae, Pseudomonadacae and Bacillaceae were the dominating taxa, accounting for 61% of the bacterial community. Lactobacillus genus showed a relative abundance of only 0·36%, but L. sanfranciscensis proved to be the species most frequent between lactobacilli and predominant in faecal samples of T. castaneum and T. confusum larvae. The core fungal microbiota was constituted by Saccharomycetales, Pleosporaceae and Nectriaceae that attained the 51% of recognized OTUs. While the most abundant yeast genus was Candida (17·1%), sequences belonging to C. milleri were not found. Frass released by the insects of stored cereal products can be the natural reservoir of L. sanfranciscensis. Insect dejections are potential sources of novel strains for controlled bakery productions.

Spatiotemporal Variation and Networks in the Mycobiome of the Wheat Canopy.

The phyllosphere is an important habitat for a diverse microbiome and an important entry point for many pathogens. Factors that shape the phyllosphere microbiome and also the co-existence among members and how they affect disease development are largely understudied. In this study we examined the wheat mycobiome by using metabarcoding of the fungal ITS1 region. Leaf samples were taken from four cultivars grown at two locations in Denmark. Samples were taken from the three uppermost leaves and at three growth stages to better understand spatiotemporal variation of the mycobiome. Analysis of read abundances showed that geographical location had a major effect in shaping the mycobiome in the total dataset, but also leaf position, growth stage and cultivar were important drivers of fungal communities. Cultivar was most important in explaining variation in older leaves whereas location better explained the variation in younger leaves, suggesting that communities are shaped over time by the leaf environment. Network analysis revealed negative co-existence between Zymoseptoria tritici and the yeasts Sporobolomyces, Dioszegia, and Cystofilobasidiaceae. The relative abundance of Z. tritici and the yeasts was relatively constant between individual samples, suggesting that fast growing fungi rapidly occupy empty space in the phyllosphere.

The bacteriome and mycobiome associated with oral squamous cell carcinoma: metagenomic analysis of samples from Yemeni and Sri Lankan cohorts

ABSTRACTThere is increasing interest in the possible role of the oral microbiome in oral squamous cell carcinoma (OSSC). However, studies on the possible association between the oral bacteriome and OSCC remain inconclusive, while the potential role of the “mycobiome” in oral carcinogenesis has never been explored. I hereby present results from our recent studies on the bacteriome and mycobiome associated with OSCC based on analysis of samples from Yemeni and Sri Lankan cohorts. Tissue biopsies were obtained from the cases while deep buccal swabs or fibro-epithelial polyps were collected from the controls. Sequencing of the bacterial V1-V3 region of the 16S rRNA gene and the fungal ITS2 region using Illumina’s 2x300 bp chemistry was employed to study the bacteriome and mycobiome, respectively. Merged reads were classified to species level using a BLASTN-algorithm. Downstream analyses were performed using QIIME, PICRUSt, and LEfSe. Fusobacterium nucleatum subsp. polymorphum, Pseudomonas aeruginosa, Prevotella and Campylobacter spp. were more abundant in OSCC samples while Streptococcus mitis and Rothia spp. were overrepresented in the controls. Functionally, inflammatory bacterial attributes including bacterial mobility, flagellar assembly, bacterial chemotaxis and LPS synthesis were enriched in the tumors. Mycobiome analysis revealed a dysbiotic fungal community dominated by C. albicans in association with OSCC.

Conversion from long-term cultivated wheat field to Jerusalem artichoke plantation changed soil fungal communities

Understanding soil microbial communities in agroecosystems has the potential to contribute to the improvement of agricultural productivity and sustainability. Effects of conversion from long-term wheat plantation to Jerusalem artichoke (JA) plantation on soil fungal communities were determined by amplicon sequencing of total fungal ITS regions. Quantitative PCR and PCR-denaturing gradient gel electrophoresis were also used to analyze total fungal and Trichoderma spp. ITS regions and Fusarium spp. Ef1α genes. Results showed that soil organic carbon was higher in the first cropping of JA and Olsen P was lower in the third cropping of JA. Plantation conversion changed soil total fungal and Fusarium but not Trichoderma spp. community structures and compositions. The third cropping of JA had the lowest total fungal community diversity and Fusarium spp. community abundance, but had the highest total fungal and Trichoderma spp. community abundances. The relative abundances of potential fungal pathogens of wheat were higher in the wheat field. Fungal taxa with plant growth promoting, plant pathogen or insect antagonistic potentials were enriched in the first and second cropping of JA. Overall, short-term conversion from wheat to JA plantation changed soil fungal communities, which is related to changes in soil organic carbon and Olsen P contents.

Honey Bee Gut Microbiome Is Altered by In-Hive Pesticide Exposures.

Honey bees (Apis mellifera) are the primary pollinators of major horticultural crops. Over the last few decades, a substantial decline in honey bees and their colonies have been reported. While a plethora of factors could contribute to the putative decline, pathogens, and pesticides are common concerns that draw attention. In addition to potential direct effects on honey bees, indirect pesticide effects could include alteration of essential gut microbial communities and symbionts that are important to honey bee health (e.g., immune system). The primary objective of this study was to determine the microbiome associated with honey bees exposed to commonly used in-hive pesticides: coumaphos, tau-fluvalinate, and chlorothalonil. Treatments were replicated at three independent locations near Blacksburg Virginia, and included a no-pesticide amended control at each location. The microbiome was characterized through pyrosequencing of V2–V3 regions of the bacterial 16S rRNA gene and fungal ITS region. Pesticide exposure significantly affected the structure of bacterial but not fungal communities. The bee bacteriome, similar to other studies, was dominated by sequences derived from Bacilli, Actinobacteria, α-, β-, γ-proteobacteria. The fungal community sequences were dominated by Ascomycetes and Basidiomycetes. The Multi-response permutation procedures (MRPP) and subsequent Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis indicated that chlorothalonil caused significant change to the structure and functional potential of the honey bee gut bacterial community relative to control. Putative genes for oxidative phosphorylation, for example, increased while sugar metabolism and peptidase potential declined in the microbiome of chlorothalonil exposed bees. The results of this field-based study suggest the potential for pesticide induced changes to the honey bee gut microbiome that warrant further investigation.

Soil fungal community development in a high Arctic glacier foreland follows a directional replacement model, with a mid-successional diversity maximum.

Directional replacement and directional non-replacement models are two alternative paradigms for community development in primary successional environments. The first model emphasizes turnover in species between early and late successional niches. The second emphasizes accumulation of additional diversity over time. To test whether the development of soil fungal communities in the foreland of an Arctic glacier conforms to either of these models, we collected samples from the Midtre Lovénbreen Glacier, Svalbard, along a soil successional series spanning >80 years. Soil DNA was extracted, and fungal ITS1 region was amplified and sequenced on an Illumina Miseq. There was a progressive change in community composition in the soil fungal community, with greatest fungal OTU richness in the Mid Stage (50–80 years). A nestedness analysis showed that the Early Stage (20–50 years) and the Late Stage (>80 years) fungal communities were nested within the Mid Stage communities. These results imply that fungal community development in this glacier succession follows a directional replacement model. Soil development processes may initially be important in facilitating arrival of additional fungal species, to give a mid-successional diversity maximum that contains both early- and late-successional fungi. Competition may then decrease the overall diversity due to the loss of early successional species.

Responses of microbial community from tropical pristine coastal soil to crude oil contamination.

Brazilian offshore crude oil exploration has increased after the discovery of new reservoirs in the region known as pré-sal, in a depth of 7.000 m under the water surface. Oceanic islands near these areas represent sensitive environments, where changes in microbial communities due oil contamination could stand for the loss of metabolic functions, with catastrophic effects to the soil services provided from these locations. This work aimed to evaluate the effect of petroleum contamination on microbial community shifts (Archaea, Bacteria and Fungi) from Trindade Island coastal soils. Microcosms were assembled and divided in two treatments, control and contaminated (weathered crude oil at the concentration of 30 g kg−1), in triplicate. Soils were incubated for 38 days, with CO2 measurements every four hours. After incubation, the total DNA was extracted, purified and submitted for target sequencing of 16S rDNA, for Bacteria and Archaea domains and Fungal ITS1 region, using the Illumina MiSeq platform. Three days after contamination, the CO2 emission rate peaked at more than 20 × the control and the emissions remained higher during the whole incubation period. Microbial alpha-diversity was reduced for contaminated-samples. Fungal relative abundance of contaminated samples was reduced to almost 40% of the total observed species. Taxonomy comparisons showed rise of the Actinobacteria phylum, shifts in several Proteobacteria classes and reduction of the Archaea class Nitrososphaerales. This is the first effort in acquiring knowledge concerning the effect of crude oil contamination in soils of a Brazilian oceanic island. This information is important to guide any future bioremediation strategy that can be required.

Transplanting Soil Microbiomes Leads to Lasting Effects on Willow Growth, but not on the Rhizosphere Microbiome.

Plants interact closely with microbes, which are partly responsible for plant growth, health, and adaptation to stressful environments. Engineering the plant-associated microbiome could improve plant survival and performance in stressful environments such as contaminated soils. Here, willow cuttings were planted into highly petroleum-contaminated soils that had been gamma-irradiated and subjected to one of four treatments: inoculation with rhizosphere soil from a willow that grew well (LA) or sub-optimally (SM) in highly contaminated soils or with bulk soil in which the planted willow had died (DE) or no inoculation (CO). Samples were taken from the starting inoculum, at the beginning of the experiment (T0) and after 100 days of growth (TF). Short hypervariable regions of archaeal/bacterial 16S rRNA genes and the fungal ITS region were amplified from soil DNA extracts and sequenced on the Illumina MiSeq. Willow growth was monitored throughout the experiment, and plant biomass was measured at TF. CO willows were significantly smaller throughout the experiment, while DE willows were the largest at TF. Microbiomes of different treatments were divergent at T0, but for most samples, had converged on highly similar communities by TF. Willow biomass was more strongly linked to overall microbial community structure at T0 than to microbial community structure at TF, and the relative abundance of many genera at T0 was significantly correlated to final willow root and shoot biomass. Although microbial communities had mostly converged at TF, lasting differences in willow growth were observed, probably linked to differences in T0 microbial communities.

Microbiome composition and geochemical characteristics of deep subsurface high-pressure environment, Pyhäsalmi mine Finland.

Pyhäsalmi mine in central Finland provides an excellent opportunity to study microbial and geochemical processes in a deep subsurface crystalline rock environment through near-vertical drill holes that reach to a depth of more than two kilometers below the surface. However, microbial sampling was challenging in this high-pressure environment. Nucleic acid yields obtained were extremely low when compared to the cell counts detected (1.4 × 104 cells mL−1) in water. The water for nucleic acid analysis went through high decompression (60–130 bar) during sampling, whereas water samples for detection of cell counts by microscopy could be collected with slow decompression. No clear cells could be identified in water that went through high decompression. The high-pressure decompression may have damaged part of the cells and the nucleic acids escaped through the filter. The microbial diversity was analyzed from two drill holes by pyrosequencing amplicons of the bacterial and archaeal 16S rRNA genes and from the fungal ITS regions from both DNA and RNA fractions. The identified prokaryotic diversity was low, dominated by Firmicute, Beta- and Gammaproteobacteria species that are common in deep subsurface environments. The archaeal diversity consisted mainly of Methanobacteriales. Ascomycota dominated the fungal diversity and fungi were discovered to be active and to produce ribosomes in the deep oligotrophic biosphere. The deep fluids from the Pyhäsalmi mine shared several features with other deep Precambrian continental subsurface environments including saline, Ca-dominated water and stable isotope compositions positioning left from the meteoric water line. The dissolved gas phase was dominated by nitrogen but the gas composition clearly differed from that of atmospheric air. Despite carbon-poor conditions indicated by the lack of carbon-rich fracture fillings and only minor amounts of dissolved carbon detected in formation waters, some methane was found in the drill holes. No dramatic differences in gas compositions were observed between different gas sampling methods tested. For simple characterization of gas composition the most convenient way to collect samples is from free flowing fluid. However, compared to a pressurized method a relative decrease in the least soluble gases may appear.

Influence of salinity on fungal communities in a submerged fixed bed bioreactor for wastewater treatment

Salinity is known to influence the performance of biological wastewater treatment plants. While its impact on bacterial communities has been thoroughly studied, its influence on fungal communities has been largely overlooked. To address this knowledge gap, we assessed the effect of saline influents (0, 3.7, 24.1 and 44.1gNaCl/L) on the community structure and diversity of fungi in a submerged fixed bed bioreactor (SFBBR). For this purpose, denaturing gradient gel electrophoresis (DGGE) and 454-pyrosequencing of PCR-amplified fungal 18S rRNA gene fragments and ITS regions, respectively, were used. Significant differences in the fungal community structure were found in relation to the NaCl concentration. Fungal diversity increased as salinity increased to a concentration up to 24.1gNaCl/L, but was significantly reduced at 44.1gNaCl/L. Basidiomycota dominated the fungal community in the absence of NaCl but decreased in relative abundance with increasing salinity, being replaced progressively by Ascomycota.

Root-associated fungal community response to drought-associated changes in vegetation community

Recent droughts in southwestern USA have led to large-scale mortality of piñon (Pinus edulis) in piñon-juniper woodlands. Piñon mortality alters soil moisture, nutrient and carbon availability, which could affect the root-associated fungal (RAF) communities and therefore the fitness of the remaining plants. We collected fine root samples at a piñon-juniper woodland and a juniper savannah site in central New Mexico. Roots were collected from piñon and juniper (Juniperus monosperma) trees whose nearest neighbors were live piñon, live juniper or dead piñon. RAF communities were analyzed by 454 pyrosequencing of the universal fungal ITS region. The most common taxa were Hypocreales and Chaetothyriales. More than 10% of ITS sequences could not be assigned taxonomy at the phylum level. Two of the unclassified OTUs significantly differed between savanna and woodland, had few like sequences in GenBank and formed new fungal clades with other unclassified RAF from arid plants, highlighting how little study has been done on the RAF of arid ecosystems. Plant host or neighbor did not affect RAF community composition. However, there was a significant difference between RAF communities from woodland vs. savanna, indicating that abiotic factors such as temperature and aridity might be more important in structuring these RAF communities than biotic factors such as plant host or neighbor identity. Ectomycorrhizal fungi (EM) were present in juniper as well as piñon in the woodland site, in contrast with previous research, but did not occur in juniper savanna, suggesting a potential shared EM network with juniper. RAF richness was lower in hosts that were neighbors of the opposite host. This may indicate competitive exclusion between fungi from different hosts. Characterizing these communities and their responses to environment and plant neighborhood is a step toward understanding the effects of drought on a biome that spans 19 000 000 ha of southwestern USA.

Long-term effects of timber harvesting on hemicellulolytic microbial populations in coniferous forest soils.

Forest ecosystems need to be sustainably managed, as they are major reservoirs of biodiversity, provide important economic resources and modulate global climate. We have a poor knowledge of populations responsible for key biomass degradation processes in forest soils and the effects of forest harvesting on these populations. Here, we investigated the effects of three timber-harvesting methods, varying in the degree of organic matter removal, on putatively hemicellulolytic bacterial and fungal populations 10 or more years after harvesting and replanting. We used stable-isotope probing to identify populations that incorporated (13)C from labeled hemicellulose, analyzing (13)C-enriched phospholipid fatty acids, bacterial 16 S rRNA genes and fungal ITS regions. In soil microcosms, we identified 104 bacterial and 52 fungal hemicellulolytic operational taxonomic units (OTUs). Several of these OTUs are affiliated with taxa not previously reported to degrade hemicellulose, including the bacterial genera Methylibium, Pelomonas and Rhodoferax, and the fungal genera Cladosporium, Pseudeurotiaceae, Capronia, Xenopolyscytalum and Venturia. The effect of harvesting on hemicellulolytic populations was evaluated based on in situ bacterial and fungal OTUs. Harvesting treatments had significant but modest long-term effects on relative abundances of hemicellulolytic populations, which differed in strength between two ecozones and between soil layers. For soils incubated in microcosms, prior harvesting treatments did not affect the rate of incorporation of hemicellulose carbon into microbial biomass. In six ecozones across North America, distributions of the bacterial hemicellulolytic OTUs were similar, whereas distributions of fungal ones differed. Our work demonstrates that diverse taxa in soil are hemicellulolytic, many of which are differentially affected by the impact of harvesting on environmental conditions. However, the hemicellulolytic capacity of soil communities appears resilient.

A revision of the history of the Colletotrichum acutatum species complex in the Nordic countries based on herbarium specimens.

Herbaria collections containing plants with disease symptoms are highly valuable, and they are often the only way to investigate outbreaks and epidemics from the past as the number of viable isolates in culture collections is often limited. Species belonging to the Colletotrichum acutatum complex infect a range of important crops. As members of the C. acutatum complex are easily confused with other Colletotrichum species, molecular methods are central for the correct identification. We performed molecular analyses on 21 herbaria specimens, displaying anthracnose symptoms, collected in Norway and Denmark before the first confirmed findings of C. acutatum complex members in this region. Sequencing parts of the fungal ITS regions showed that members of the species complex were present in 13 of the 21 specimens collected in different parts of Norway and Denmark between 1948 and 1991, representing seven plant hosts (three cherry species, apple, raspberry and rhododendron). This is the first time herbarium specimens have been used to study these pathogens under Nordic conditions. Differences in the ITS sequences suggest the presence of different genotypes within the complex, indicating a well-established population.

Metabarcoding Analysis of Fungal Diversity in the Phyllosphere and Carposphere of Olive (Olea europaea).

The fungal diversity associated with leaves, flowers and fruits of olive (Olea europaea) was investigated in different phenological stages (May, June, October and December) using an implemented metabarcoding approach. It consisted of the 454 pyrosequencing of the fungal ITS2 region and the subsequent phylogenetic analysis of relevant genera along with validated reference sequences. Most sequences were identified up to the species level or were associated with a restricted number of related taxa enabling supported speculations regarding their biological role. Analyses revealed a rich fungal community with 195 different OTUs. Ascomycota was the dominating phyla representing 93.6% of the total number of detected sequences followed by unidentified fungi (3.6%) and Basidiomycota (2.8%). A higher level of diversity was revealed for leaves compared to flowers and fruits. Among plant pathogens the genus Colletotrichum represented by three species (C. godetiae syn. C. clavatum, C. acutatum s.s and C. karstii) was the most abundant on ripe fruits but it was also detected in other organs. Pseudocercospora cladosporioides was detected with a high frequency in all leaf samples and to a less extent in ripe fruits. A much lower relative frequency was revealed for Spilocaea oleagina and for other putative pathogens including Fusarium spp., Neofusicoccum spp., and Alternaria spp. Among non-pathogen taxa, Aureobasidium pullulans, the species complex of Cladosporium cladosporioides and Devriesia spp. were the most represented. This study highlights the existence of a complex fungal consortium including both phytopathogenic and potentially antagonistic microorganisms that can have a significant impact on olive productions.

Microbial diversity in bioaerosol samples causing ODTS compared to reference bioaerosol samples as measured using Illumina sequencing and MALDI-TOF

The importance of the microbial diversity of bioaerosols in relation to occupational exposure and work related health symptoms is not known. The aim of this paper is to gain knowledge on the bacterial and fungal communities in dust causing organic dust toxic syndrome (ODTS) and in reference dust not causing ODTS. Bacterial and fungal communities were described in personal exposure samples from grass seed workers developing ODTS, in dust generated from grass seeds causing ODTS and in dust generated from reference seeds not causing ODTS. Amplicon sequencing of the bacterial 16S rRNA gene and the fungal ITS region, as well as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) were used for identification of fungi and bacteria in personal exposure samples and in dust samples from grass seeds causing ODTS and in dust from reference grass seeds. Furthermore, activities of enzymes were measured in the same samples.The sequencing data revealed more than 150 bacterial and 25 fungal genera present in each sample. Streptomyces spp., Aspergillus fumigatus and Rhizopus microsporus were dominating in the dust causing ODTS but not in the reference dust. The dustiness in terms of Mucor sp. and R. microsporus were 100–1000 times higher for problematic seeds compared to reference seeds. The bacterial species in the dust causing ODTS included pathogenic species such as Klebsiella pneumonia and Streptomyces pneumonia, and it contained increased concentrations of total protein, serine protease, chitinase, and β-glucosidase. Twenty-three bacterial genera covered more than 50% of the total reads in the personal and problematic seed dust. These 23 genera accounted for less than 7% of the total reads in the reference seed dust. The microbial community of the dust from the problematic seeds showed great similarities to that from the personal air samples from the workers. In conclusion, we have shown for the first time a shift in the microbial community in aerosol samples that caused ODTS compared to the reference samples that did not cause the ODTS. Furthermore, elevated enzyme activities were found in the dust causing ODTS.

Impacts of Flood Damage on Airborne Bacteria and Fungi in Homes after the 2013 Colorado Front Range Flood

Flood-damaged homes typically have elevated microbial loads, and their occupants have an increased incidence of allergies, asthma, and other respiratory ailments, yet the microbial communities in these homes remain under-studied. Using culture-independent approaches, we characterized bacterial and fungal communities in homes in Boulder, CO, USA 2-3 months after the historic September, 2013 flooding event. We collected passive air samples from basements in 50 homes (36 flood-damaged, 14 non-flooded), and we sequenced the bacterial 16S rRNA gene (V4-V5 region) and the fungal ITS1 region from these samples for community analyses. Quantitative PCR was used to estimate the abundances of bacteria and fungi in the passive air samples. Results indicate significant differences in bacterial and fungal community composition between flooded and non-flooded homes. Fungal abundances were estimated to be three times higher in flooded, relative to non-flooded homes, but there were no significant differences in bacterial abundances. Penicillium (fungi) and Pseudomonadaceae and Enterobacteriaceae (bacteria) were among the most abundant taxa in flooded homes. Our results suggest that bacterial and fungal communities continue to be affected by flooding, even after relative humidity has returned to baseline levels and remediation has removed any visible evidence of flood damage.

Molecular analysis of fungi associated with the Mediterranean orchid Ophrys bertolonii Mor

Mycorrhizal fungi are fundamental in orchid growth and metabolism and influence the distribution and rarity of these delicate plants. Fungal molecular systematics has been instrumental for the identification of orchid mycorrhizal symbionts because it overcomes the limits associated with in vitro isolation and morphological characterization of orchid endophytes. In this paper, fungal diversity in the Mediterranean orchid Ophrys bertolonii was analyzed using molecular methods. Total DNA from the roots of 12 adult orchid individuals, collected in Central Italy, was extracted and fungal ITS regions were PCR amplified using the primer pair ITS1F/ITS4, cloned and sequenced. Several fungal taxa belonging to Basidiomycota and Ascomycota were found in the analyzed orchid root samples. Results suggest that the studied orchid species may establish symbiotic relationships with a dominant fungal partner belonging to Tulasnellaceae.