scholarly journals Comparison of qPCR and Metabarcoding Methods as Tools for the Detection of Airborne Inoculum of Forest Fungal Pathogens

2021 ◽  
Vol 111 (3) ◽  
pp. 570-581
Author(s):  
Anne Chandelier ◽  
Julie Hulin ◽  
Gilles San Martin ◽  
Frédéric Debode ◽  
Sébastien Massart
Keyword(s):  
Fungal Pathogens ◽  
High Throughput Sequencing ◽  
Disease Outbreaks ◽  
Rapid Identification ◽  
Fungal Species ◽  
Reference Database ◽  
Airborne Inoculum ◽  
Important Pathway ◽  
Spore Trapping ◽  
The Impact

Forest diseases caused by invasive fungal pathogens are becoming more common, sometimes with dramatic consequences to forest ecosystems. The development of early detection systems is necessary for efficient surveillance and to mitigate the impact of invasive pathogens. Windborne spores are an important pathway for introduction of fungal pathogens into new areas; the design of spore trapping devices adapted to forests, capable of collecting different types of spores, and aligned with development of efficient molecular methods for detection of the pathogen, should help forest managers anticipate new disease outbreaks. Two types of Rotorod samplers were evaluated for the collection of airborne inoculum of forest fungal pathogens with a range of spore sizes in five forest types. Detection was by specific quantitative PCR (qPCR) and by high-throughput sequencing (HTS) of amplified internal transcribed spacer sequences using a new bioinformatic pipeline, FungiSearch, developed for diagnostic purposes. Validation of the pipeline was conducted on mock communities of 10 fungal species belonging to different taxa. Although the sensitivity of the new HTS pipeline was lower than the specific qPCR, it was able to detect a wide variety of fungal pathogens. FungiSearch is easy to use, and the reference database is updatable, making the tool suitable for rapid identification of new pathogens. This new approach combining spore trapping and HTS detection is promising as a diagnostic tool for invasive fungal pathogens.

scholarly journals Early Stage Root-Associated Fungi Show a High Temporal Turnover, but Are Independent of Beech Progeny

2020 ◽  
Vol 8 (2) ◽  
pp. 210 ◽  
Author(s):  
Kezia Goldmann ◽  
Silke Ammerschubert ◽  
Rodica Pena ◽  
Andrea Polle ◽  
Bin-Wei Wu ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Early Stage ◽  
European Beech ◽  
Fungal Species ◽  
Fungal Communities ◽  
Transplant Experiment ◽  
Fungal Community Structure ◽  
Temporal Turnover ◽  
One Year ◽  
The Impact

The relationship between trees and root-associated fungal communities is complex. By specific root deposits and other signal cues, different tree species are able to attract divergent sets of fungal species. Plant intraspecific differences can lead to variable fungal patterns in the root’s proximity. Therefore, within the Beech Transplant Experiment, we analyzed the impact of three different European beech ecotypes on the fungal communities in roots and the surrounding rhizosphere soil at two time points. Beech nuts were collected in three German sites in 2011. After one year, seedlings of the different progenies were out-planted on one site and eventually re-sampled in 2014 and 2017. We applied high-throughput sequencing of the fungal ITS2 to determine the correlation between tree progeny, a possible home-field advantage, plant development and root-associated fungal guilds under field conditions. Our result showed no effect of beech progeny on either fungal OTU richness or fungal community structure. However, over time the fungal OTU richness in roots increased and the fungal communities changed significantly, also in rhizosphere. In both plant compartments, the fungal communities displayed a high temporal turnover, indicating a permanent development and functional adaption of the root mycobiome of young beeches.

scholarly journals MiCoP: Microbial Community Profiling method for detecting viral and fungal organisms in metagenomic samples

2018 ◽  
Author(s):  
Nathan LaPierre ◽  
Serghei Mangul ◽  
Mohammed Alser ◽  
Igor Mandric ◽  
Nicholas C. Wu ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Human Microbiome ◽  
Real Data ◽  
Human Microbiome Project ◽  
Direct Analysis ◽  
Fungal Species ◽  
Reference Database ◽  
Marker Genes ◽  
Full Diversity ◽  
Community Profiling

AbstractBackgroundHigh throughput sequencing has spurred the development of metagenomics, which involves the direct analysis of microbial communities in various environments such as soil, ocean water, and the human body. Many existing methods based on marker genes or k-mers have limited sensitivity or are too computationally demanding for many users. Additionally, most work in metagenomics has focused on bacteria and archaea, neglecting to study other key microbes such as viruses and eukaryotes.ResultsHere we present a method, MiCoP (Microbiome Community Profiling), that uses fast-mapping of reads to build a comprehensive reference database of full genomes from viruses and eukaryotes to achieve maximum read usage and enable the analysis of the virome and eukaryome in each sample. We demonstrate that mapping of metagenomic reads is feasible for the smaller viral and eukaryotic reference databases. We show that our method is accurate on simulated and mock community data and identifies many more viral and fungal species than previously-reported results on real data from the Human Microbiome Project.ConclusionsMiCoP is a mapping-based method that proves more effective than existing methods at abundance profiling of viruses and eukaryotes in metagenomic samples. MiCoP can be used to detect the full diversity of these communities. The code, data, and documentation is publicly available on GitHub at: https://github.com/smangul1/MiCoP

scholarly journals 268. Fungal NGS: Identification of Etiological Agents of Invasive Fungal Infection by High-throughput Sequencing

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S148-S149
Author(s):  
Donald J Nelsen ◽  
Rohita Sinha ◽  
Aaron J Tyler ◽  
Jordyn Westergaard ◽  
Jamie Nutt ◽  
...  
Keyword(s):  
Species Identification ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Fungal Infections ◽  
Graft Loss ◽  
Rapid Identification ◽  
Fungal Species ◽  
Individual Species ◽  
Sequence Alignments ◽  
Etiological Agents

Abstract Background Invasive fungal infections (IFI) cause severe symptoms that affect immunocompromised and transplant patient populations. Antifungal therapies vary depending on the pathogenic species, and delays in diagnosis can lead to graft loss and an increase in morbidity and mortality. Therefore, rapid identification of fungi causing IFI is critical for informing antifungal therapy. Such actionable genus/species information can be obtained quickly via Next-generation Sequencing (NGS). In this study, an NGS assay was developed to identify fungal species responsible for IFI, allowing for selection of effective antifungal therapies. Methods Internal transcribed spacer (ITS) regions 1 and 2 were used for fungal identification. Primers were taken from published research and/or designed/modified by assessment in fungal sequence alignments. A DNA sequence database was compiled and a reference-assisted assembly approach utilizing % sequence ID and % coverage was developed for species identification. End-point PCR was conducted on DNA extracted from 19 pathogenic fungal species, and mixed communities (MC) for preliminary sensitivity and inclusivity. Sensitivity was assessed using dilutions of template DNA into the PCR reaction. Results NGS data of 14 individual species and 4 MC passed quality control checks. Using only ITS1 and ITS2, species identification was expected for 10 of 14 individuals. We observed species identification in 9 individual samples, and 13 were identified within the top 5 results. All individuals were identified to genus. In MC analyses, combinations of 3, 4, 6, and 10 fungal species resolved 100% of the genera present, but failed to resolve species adequately with only 2 loci evaluated. Unexpectedly, 3 tested Aspergillus spp. were correctly identified with this limited data in both single and MC samples. The lower limit of detection was assessed at 5,000 genomic equivalents/mL of eluate. Conclusion The inclusivity and sensitivity demonstrated here of an NGS approach for identification of etiological agents of IFI support this assay’s potential utility as an aid in the treatment of IFI in at-risk patient groups. This assay allows for rapid identification (<4 days) of fungal species to aid clinicians in improving case outcomes. Disclosures All authors: No reported disclosures.

scholarly journals Fungal Diversity in the Phyllosphere of Pinus heldreichii H. Christ—An Endemic and High-Altitude Pine of the Mediterranean Region

Diversity ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 172 ◽  
Author(s):  
Jelena Lazarević ◽  
Audrius Menkis
Keyword(s):  
High Altitude ◽  
Bark Beetles ◽  
Fungal Pathogens ◽  
High Throughput Sequencing ◽  
Fungal Communities ◽  
High Quality ◽  
The Balkans ◽  
Fungal Community Structure ◽  
Growing Conditions ◽  
Coniferous Tree Species

Pinus heldreichii is a high-altitude coniferous tree species naturaly occurring in small and disjuncted populations in the Balkans and southern Italy. The aim of this study was to assess diversity and composition of fungal communities in living needles of P. heldreichii specifically focusing on fungal pathogens. Sampling was carried out at six different sites in Montenegro, where 2-4 year-old living needles of P. heldreichii were collected. Following DNA isolation, it was amplified using ITS2 rDNA as a marker and subjected to high-throughput sequencing. Sequencing resulted in 31,831 high quality reads, which after assembly were found to represent 375 fungal taxa. The detected fungi were 295 (78.7%) Ascomycota, 79 (21.0%) Basidiomycota and 1 (0.2%) Mortierellomycotina. The most common fungi were Lophodermium pinastri (12.5% of all high-quality sequences), L. conigenum (10.9%), Sydowia polyspora (8.8%), Cyclaneusma niveum (5.5%), Unidentified sp. 2814_1 (5.4%) and Phaeosphaeria punctiformis (4.4%). The community composition varied among different sites, but in this respect two sites at higher altitudes (harsh growing conditions) were separated from three sites at lower altitudes (milder growing conditions), suggesting that environmental conditions were among major determinants of fungal communities associated with needles of P. heldreichii. Trees on one study site were attacked by bark beetles, leading to discolouration and frequent dieback of needles, thereby strongly affecting the fungal community structure. Among all functional groups of fungi, pathogens appeared to be an important component of fungal communities in the phyllosphere of P. heldreichii, especially in those trees under strong abiotic and biotic stress.

scholarly journals Post-translational regulation of autophagy is involved in intra-microbiome suppression of fungal pathogens

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Jing Wang ◽  
Chaoyun Xu ◽  
Qiming Sun ◽  
Jinrong Xu ◽  
Yunrong Chai ◽  
...  
Keyword(s):  
Translational Regulation ◽  
Fungal Pathogens ◽  
Fungal Growth ◽  
Fungal Species ◽  
26S Proteasome ◽  
Healthy Plant ◽  
Autophagy Induction ◽  
Histone Acetyltransferase Gcn5 ◽  
Plant Microbiota ◽  
Autophagic Process

Abstract Background Microbiome interactions are important determinants for ecosystem functioning, stability, and health. In previous studies, it was often observed that bacteria suppress potentially pathogenic fungal species that are part of the same plant microbiota; however, the underlying microbe-microbe interplay remains mostly elusive. Here, we explored antagonistic interactions of the fungus Fusarium graminearum and bacterium Streptomyces hygroscopicus at the molecular level. Both are ubiquitous members of the healthy wheat microbiota; under dysbiosis, the fungus causes devastating diseases. Results In co-cultures, we found that Streptomyces alters the fungal acetylome leading to substantial induction of fungal autophagy. The bacterium secrets rapamycin to inactivate the target of rapamycin (TOR), which subsequently promotes the degradation of the fungal histone acetyltransferase Gcn5 through the 26S proteasome. Gcn5 negatively regulates fungal autophagy by acetylating the autophagy-related protein Atg8 at the lysine site K13 and blocking cellular relocalization of Atg8. Thus, degradation of Gcn5 triggered by rapamycin was found to reduce Atg8 acetylation, resulting in autophagy induction in F. graminearum. Conclusions Autophagy homeostasis plays an essential role in fungal growth and competition, as well as for virulence. Our work reveals a novel post-translational regulation of autophagy initiated by a bacterial antibiotic. Rapamycin was shown to be a powerful modulator of bacteria–fungi interactions with potential importance in explaining microbial homeostasis in healthy plant microbiomes. The autophagic process provides novel possibilities and targets to biologically control pathogens.

Effects of pH shock on microaerobic activated sludge system with a graphene oxide/nano-magnetic powder composite

2020 ◽  
Vol 35 (3) ◽  
pp. 457-463
Author(s):  
Huixia Lan ◽  
Xiangzhi Wang ◽  
Shixin Qi ◽  
Da Yang ◽  
Hao Zhang
Keyword(s):  
Graphene Oxide ◽  
Activated Sludge ◽  
High Throughput Sequencing ◽  
Impact Resistance ◽  
Magnetic Powder ◽  
Blank Group ◽  
Activated Sludge System ◽  
Ph Shock ◽  
Dominant Bacteria ◽  
The Impact

AbstractUsing the acclimated activated sludge from the pulping middle-stage effluent, the effect of pH shock on the micro-oxygen activated sludge system with a nano-magnetic powder/graphene oxide composite was studied. The results showed that the removal rates of chemical oxygen demand (CODCr) and ultraviolet adsorption at 254 nm (UV254) decreased. Also, the sludge settling performance was poor due to the impact of pH, but the impact resistance of nano-magnetic powder/graphene oxide group (MGO group) was higher and the recovery was faster. Results of high throughput sequencing indicated that the diversity of microbial community was reduced by the impact of pH, but it was significantly higher in MGO group than in the blank group. The dominant bacteria after pH shock or recovery in both of the system had a large difference. The percentage of the dominant bacteria in the MGO group was higher than that in the blank group. The MGO group had higher electron transfer system (ETS) activity which made the system having a strong pH impact resistance.

scholarly journals Digital proximity tracing on empirical contact networks for pandemic control

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
G. Cencetti ◽  
G. Santin ◽  
A. Longa ◽  
E. Pigani ◽  
A. Barrat ◽  
...  
Keyword(s):  
Large Scale ◽  
Disease Outbreaks ◽  
Physical Distance ◽  
Contact Tracing ◽  
Reproductive Number ◽  
Modeling Framework ◽  
Infectious Disease Outbreaks ◽  
Contact Patterns ◽  
Contact Data ◽  
The Impact

AbstractDigital contact tracing is a relevant tool to control infectious disease outbreaks, including the COVID-19 epidemic. Early work evaluating digital contact tracing omitted important features and heterogeneities of real-world contact patterns influencing contagion dynamics. We fill this gap with a modeling framework informed by empirical high-resolution contact data to analyze the impact of digital contact tracing in the COVID-19 pandemic. We investigate how well contact tracing apps, coupled with the quarantine of identified contacts, can mitigate the spread in real environments. We find that restrictive policies are more effective in containing the epidemic but come at the cost of unnecessary large-scale quarantines. Policy evaluation through their efficiency and cost results in optimized solutions which only consider contacts longer than 15–20 minutes and closer than 2–3 meters to be at risk. Our results show that isolation and tracing can help control re-emerging outbreaks when some conditions are met: (i) a reduction of the reproductive number through masks and physical distance; (ii) a low-delay isolation of infected individuals; (iii) a high compliance. Finally, we observe the inefficacy of a less privacy-preserving tracing involving second order contacts. Our results may inform digital contact tracing efforts currently being implemented across several countries worldwide.

scholarly journals Mitotic Recombination and Adaptive Genomic Changes in Human Pathogenic Fungi

Genes ◽  
2019 ◽  
Vol 10 (11) ◽  
pp. 901 ◽  
Author(s):  
Asiya Gusa ◽  
Sue Jinks-Robertson
Keyword(s):  
Fungal Pathogens ◽  
Repetitive Sequences ◽  
Pathogenic Fungi ◽  
Genetic Alterations ◽  
Fungal Species ◽  
Chronic Infections ◽  
Yeast Saccharomyces Cerevisiae ◽  
Genomic Changes ◽  
Break Site ◽  
Repair Mechanisms

Genome rearrangements and ploidy alterations are important for adaptive change in the pathogenic fungal species Candida and Cryptococcus, which propagate primarily through clonal, asexual reproduction. These changes can occur during mitotic growth and lead to enhanced virulence, drug resistance, and persistence in chronic infections. Examples of microevolution during the course of infection were described in both human infections and mouse models. Recent discoveries defining the role of sexual, parasexual, and unisexual cycles in the evolution of these pathogenic fungi further expanded our understanding of the diversity found in and between species. During mitotic growth, damage to DNA in the form of double-strand breaks (DSBs) is repaired, and genome integrity is restored by the homologous recombination and non-homologous end-joining pathways. In addition to faithful repair, these pathways can introduce minor sequence alterations at the break site or lead to more extensive genetic alterations that include loss of heterozygosity, inversions, duplications, deletions, and translocations. In particular, the prevalence of repetitive sequences in fungal genomes provides opportunities for structural rearrangements to be generated by non-allelic (ectopic) recombination. In this review, we describe DSB repair mechanisms and the types of resulting genome alterations that were documented in the model yeast Saccharomyces cerevisiae. The relevance of similar recombination events to stress- and drug-related adaptations and in generating species diversity are discussed for the human fungal pathogens Candida albicans and Cryptococcus neoformans.

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