Bioaerosol studies aim to describe the microbial content and increase understanding of the aerosolization processes linked to diseases. Air samplers are used to collect, identify, and quantify ...bioaerosols. Studies comparing the performances of air samplers have typically used a culture approach or have targeted a specific microorganism in laboratory settings. The objective of this study was to use environmental field samples to compare the efficiencies of 3 high-airflow-rate samplers for describing bioaerosol diversity using a next-generation sequencing approach. Two liquid cyclonic impactors and one electrostatic filter dry sampler were used in four wastewater treatment plants to target bacterial diversity and in five dairy farms to target fungal diversity. The dry electrostatic sampler was consistently more powerful in collecting more fungal and bacterial operational taxonomic units (OTUs). Substantial differences in OTU abundances between liquid and dry sampling were revealed. The majority of the diversity revealed by dry electrostatic sampling was not identified using the cyclonic liquid impactors. The findings from this work suggest that the choice of a bioaerosol sampler should include information about the efficiency and ability of samplers to cover microbial diversity. Although these results suggest that electrostatic filters result in better coverage of the microbial diversity among the tested air samplers, further studies are needed to confirm this hypothesis. While it is difficult to determine a single universally optimal air sampler, this work provides an in-depth look at some of the considerations that are essential when choosing an air sampler for studying the microbial ecology of bioaerosols.
Associating bioaerosol exposure and health problems is challenging, and adequate exposure monitoring is a priority for scientists in the field. Conclusions that can be drawn from bioaerosol exposure studies are highly dependent on the design of the study and the methodologies used. The air sampling strategy is the first methodological step leading to an accurate interpretation of what is present in the air. Applying new molecular approaches to evaluate the efficiencies of the different types of samplers used in the field is necessary in order to circumvent traditional approaches and the biases they introduce to such studies. The results and conclusions provided in this paper should be taken in consideration when conducting a bioaerosol study.
Occupational exposure to harmful bioaerosols in industrial environments is a real threat to the workers. In particular, dairy-farm workers are exposed to high levels of fungal bioaerosols on a daily ...basis. Associating bioaerosol exposure and health problems is challenging and adequate exposure monitoring is a top priority for aerosol scientists. Using only culture-based tools does not express the overall microbial diversity and underestimate the large spectrum of microbes in bioaerosols and therefore the extended fungal profile that farmers are exposed to. The aim of this study was to provide an in-depth characterization of fungal exposure at Eastern Canadian dairy farms using qPCR and high-throughput sequencing methods. Specific primers were used for the quantification of Penicillium/Aspergillus and Aspergillus fumigatus in dairy farms air samples. Illumina Miseq sequencing of the ITS1 region provided sequences for the diversity analyses. The minimum and maximum concentration of Penicillium/Aspergillus ranged from 4.6 × 106 to 9.4 × 106 gene copies/m3 and from 1 × 104 gene copies/m3 to 4.8 × 105 gene copies/m3 for Aspergillus fumigatus, respectively. Differences in the diversity profiles of the five dairy farms support the idea that the novel approach identifies a large number of fungal taxa. The most striking differences include Microascus, Piptoporus, Parastagonospora, Dissoconium, Microdochium, Tubilicrinis, Ganoderma, Ustilago, Phlebia and Wickerhamomyces. The presence of a diverse portrait of fungi in air may represent a health risk for workers who are exposed on a daily basis. The broad spectrum of fungi detected in this study includes many known pathogens like Aspergillus, Acremonium, Alternaria and Fusarium. Adequate monitoring of bioaerosol exposure is necessary to evaluate and minimize risks.
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•Molecular and culture techniques enable the study of fungal diversity in aerosols from dairy farms•A practical bioinformatics workflow is described for the treatment of raw sequencing reads and diversity analyses•The type of cattle feed and animal confinement influence fungal composition of aerosols•Culture techniques underestimate fungal diversity compared to high-throughput sequencing•Molecular techniques allow the description of fungal diversity in aerosols for a better assessment of occupational exposure
Christmas trees are an economically and culturally important ornamental plant in North America. Many microorganisms are pathogens of firs cultivated as Christmas trees. Among those,
causes millions ...of dollars in damage to plantations annually. In Canada, it is unknown which species are responsible for Phytophthora root rot (PRR) of cultivated
species. Between 2019 and 2021, soil and root samples were collected from 40 Christmas tree plantations in Québec province. We used soil baiting and direct isolation from unidentified root fragments to assess the diversity of culturable
spp. The obtained isolates were identified using a multi-locus sequencing and phylogenetic approach. A total of 44 isolates were identified, including eight
, eight
, seven
, three
, six
, and two
isolates, plus 10 isolates belonging to a previously unknown taxon that is phylogenetically close to
and
. Among the known species,
was the most prevalent isolated species associated with trees showing aboveground PRR-like symptoms. Pathogenicity trials confirmed the pathogenicity potential of
on both Fraser fir and balsam fir seedlings. Our study provides a first snapshot of the
diversity in Québec's Christmas tree productions and describe multiple potential first associations between
species and
and
.
The filamentous ascomycete fungus Lachnellula willkommii is the causal agent of European Larch Canker (ELC), one of the most destructive diseases of larch in Europe and a regulated plant pathogen of ...quarantine significance in Canada and the United States. L. willkommii was first detected in Massachusetts, North America in 1927 on a larch plantation cultivated with nursery stock imported from Great Britain. Despite the decades of practices aimed at eliminating the pathogen, it has reappeared in coastal areas of Canada and the United States. There is concern ELC could spread throughout the range of eastern larch, a transcontinental species typical of the Boreal forest which spans the North American landscape. There is geographic range overlap between several non-pathogenic indigenous Lachnellula species and the reported distribution of L. willkommii in North America. Morphological and biological methods to distinguish L. willkommii are often inadequate as the fungus does not always produce the phenotypic structures that distinguish it from these other saprophytic Lachnellula species. Whole genome sequencing technologies were used to obtain the draft genome sequences of L. willkommii and six other Lachnellula species. Molecular markers identified from the genomic data may be used to discriminate L. willkommii from its non-pathogenic relatives.
ABSTRACT Verticillium dahliae is responsible for Verticillium wilt on a wide range of hosts, including strawberry, on which low soil inoculum densities can cause significant crop loss. Determination ...of inoculum density is currently done by soil plating but this can take 6 to 8 weeks to complete and delay the grower's ability to make planting decisions. To provide a faster means for estimating pathogen populations in the soil, a multiplexed TaqMan real-time polymerase chain reaction (PCR) assay based on the ribosomal DNA (rDNA) intergenic spacer (IGS) was developed for V. dahliae. The assay was specific for V. dahliae and included an internal control for evaluation of inhibition due to the presence of PCR inhibitors in DNA extracted from soil samples. An excellent correlation was observed in regression analysis (R(2) = 0.96) between real-time PCR results and inoculum densities determined by soil plating in a range of field soils with pathogen densities as low as 1 to 2 microsclerotia/g of soil. Variation in copy number of the rDNA was also evaluated among isolates by SYBR Green real-time PCR amplification of the V. dahliae-specific amplicon compared with amplification of several single-copy genes and was estimated to range from ≈24 to 73 copies per haploid genome, which translated into possible differences in results among isolates of ≈1.8 cycle thresholds. Analysis of the variation in results of V. dahliae quantification among extractions of the same soil sample indicated that assaying four replicate DNA extractions for each field sample would provide accurate results. A TaqMan assay also was developed to help identify colonies of V. tricorpus on soil plates.
Composting is used all over the world to transform different types of organic matter through the actions of complex microbial communities. Moving and handling composting material may lead to the ...emission of high concentrations of bioaerosols. High exposure levels are associated with adverse health effects among compost industry workers. Fungal spores are suspected to play a role in many respiratory illnesses. There is a paucity of information related to the detailed fungal diversity in compost as well as in the aerosols emitted through composting activities. The aim of this study was to analyze the fungal diversity of both organic matter and aerosols present in facilities that process domestic compost and facilities that process pig carcasses. This was accomplished using a next generation sequencing approach that targets the ITS1 genomic region. Multivariate analyses revealed differences in the fungal community present in samples coming from compost treating both raw materials. Furthermore, results show that the compost type affects the fungal diversity of aerosols emitted. Although 8 classes were evenly distributed in all samples, Eurotiomycetes were more dominant in carcass compost while Sordariomycetes were dominant in domestic compost. A large diversity profile was observed in bioaerosols from both compost types showing the presence of a number of pathogenic fungi newly identified in bioaerosols emitted from composting plants. Members of the family Herpotrichiellaceae and Gymnoascaceae which have been shown to cause human diseases were detected in compost and air samples. Moreover, some fungi were identified in higher proportion in air compared to compost. This is the first study to identify a high level of fungal diversity in bioaerosols present in composting plants suggesting a potential exposure risk for workers. This study suggests the need for creating guidelines that address human exposure to bioaerosols. The implementation of technical and organizational measure should be a top priority. However, skin and respiratory protection for compost workers could be used to reduce the exposure as a second resort.
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•ITS Next-generation sequencing approach to study fungal diversity in bioaerosols released from composting plants is presented•A bioinformatics workflow is described for the treatment of the raw sequencing data and for the fungal diversity analyses•Differences in the fungal community in bioaerosols from compost treating different raw materials were observed•A number of pathogenic fungi newly identified in compost bioaerosols•This study suggests the need for creating guidelines addressing respiratory protection of workers in composting plants.
Invasive alien tree pathogens can cause significant economic losses as well as large-scale damage to natural ecosystems. Early detection to prevent their establishment and spread is an important ...approach used by several national plant protection organizations (NPPOs). Molecular detection tools targeting 10 of the most unwanted alien forest pathogens in Canada were developed as part of the TAIGA project (http://taigaforesthealth.com/). Forest pathogens were selected following an independent prioritization. Specific TaqMan real-time PCR detection assays were designed to function under homogeneous conditions so that they may be used in 96- or 384-well plate format arrays for high-throughput testing of large numbers of samples against multiple targets. Assays were validated for 1) specificity, 2) sensitivity, 3) precision, and 4) robustness on environmental samples. All assays were highly specific when evaluated against a panel of pure cultures of target and phylogenetically closely-related species. Sensitivity, evaluated by assessing the limit of detection (with a threshold of 95% of positive samples), was found to be between one and ten target gene region copies. Precision or repeatability of each assay revealed a mean coefficient of variation of 3.4%. All assays successfully allowed detection of target pathogen on positive environmental samples, without any non-specific amplification. These molecular detection tools will allow for rapid and reliable detection of 10 of the most unwanted alien forest pathogens in Canada.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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•Environmental monitoring is crucial for protecting human and ecosystem health.•Honey bees (Apis mellifera) can serve as global, continuous biomonitoring species.•Colonies are ...resilient and accumulate or respond to stressors without collapsing.•Long-range foragers transport pollutants, pesticides, and pathogens to their hives.•A. mellifera may serve to monitor emerging threats such as climate change and AMR.
Monitoring the environment for pollution, pesticides, and pathogens is crucial for protecting human, agriculture, and overall ecosystem health. Diverse strategies ranging from physical sensors to sentinel species have been used for environmental monitoring. The European honey bee, Apis mellifera, is a globally managed pollinator that can serve as a continuous biomonitoring species. During foraging, honey bees are exposed to contaminants and pathogens and carry them to their hives where they can be detected and quantified. Although individual bees are vulnerable to environmental stressors, the honey bee colony as a whole is more resilient and can accumulate contaminants or respond to them without collapsing. This allows for long-term monitoring of the colony to map contaminants in a geographical area and study ecotoxicology gradients over space and time. In this paper, we review demonstrated and proposed uses of honey bees for environmental monitoring. We focus our discussion on heavy metals, air pollutants, pesticides, and plant pathogens that can be detected in bees and their hive materials including honey, wax, and stored pollen. We present the use of gene expression, microbiome profiling, and other high-throughput methodologies to study dose-dependent exposure and increase detection sensitivity; for example, stored pollen analysis with next generation sequencing can reveal the presence of plant viruses, fungi, and invasive species earlier than traditional detection methods. Finally, we discuss opportunities for using honey bees to monitor emerging threats such as climate change and antimicrobial resistance. This narrative review highlights the versatility and potential utility of the European honey bee as a biomonitoring species for ecosystem health.
Apple bitter rot caused by Colletotrichum species is a growing problem worldwide. Colletotrichum spp. are economically important but taxonomically un-resolved. Identification of Colletotrichum spp. ...is critical due to potential species-level differences in pathogenicity-related characteristics. A 400-isolate collection from New York apple orchards were morphologically assorted to two groups, C. acutatum species complex (CASC) and C. gloeosporioides species complex (CGSC). A sub-sample of 44 representative isolates, spanning the geographical distribution and apple varieties, were assigned to species based on multi-locus phylogenetic analyses of nrITS, GAPDH and TUB2 for CASC, and ITS, GAPDH, CAL, ACT, TUB2, APN2, ApMat and GS genes for CGSC. The dominant species was C. fioriniae, followed by C. chrysophilum and a novel species, C. noveboracense, described in this study. This study represents the first report of C. chrysophilum and C. noveboracense as pathogens of apple. We assessed the enzyme activity and fungicide sensitivity for isolates identified in New York. All isolates showed amylolytic, cellulolytic and lipolytic, but not proteolytic activity. C. chrysophilum showed the highest cellulase and the lowest lipase activity, while C. noveboracense had the highest amylase activity. Fungicide assays showed that C. fioriniae was sensitive to benzovindiflupyr and thiabendazole, while C. chrysophilum and C. noveboracense were sensitive to fludioxonil, pyraclostrobin and difenoconazole. All species were pathogenic on apple fruit with varying lesion sizes. Our findings of differing pathogenicity-related characteristics among the three species demonstrate the importance of accurate species identification for any downstream investigations of Colletotrichum spp. in major apple growing regions.
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