Towards improved detection and identification of rust fungal pathogens in environmental samples using a metabarcoding approach

2021 ◽  
Author(s):  
Wen Chen ◽  
Devon R. Radford ◽  
Sarah Hambleton
Keyword(s):  
In Silico ◽  
Environmental Samples ◽  
Rust Fungi ◽  
Reference Database ◽  
Its2 Region ◽  
Amplification Efficiency ◽  
Rrna Gene ◽  
Bioinformatics Pipeline ◽  
Airborne Spores ◽  
Accurate Identification

The dispersion of fungal inocula such as the airborne spores of rust fungi (Pucciniales) can be monitored by metabarcoding the internal transcribed spacer 2 (ITS2) of the rRNA gene in environmental DNAs. This is largely dependent upon a high-quality reference database (refDB) and primers with proper taxonomic coverage and specificity. For this study, a curated ITS2 reference database (named CR-ITS2-refDB) comprising representatives of the major cereal rust fungi and phylogenetically related species was compiled. Inter- and intra-specific variation analyses suggested that the ITS2 region had reasonable discriminating power for the majority of the Puccinia species or species complexes in the database. In silico evaluation of nine forward and seven reverse ITS2 primers, including three newly designed, revealed marked variation in DNA amplification efficiency for the rusts. The theoretical assessment of rust-enhanced (Rust2inv/ITS4var_H) and universal fungal (ITS9F/ITS4) ITS2 primer pairs was validated by profiling the airborne rust fungal communities from environmental samples using a metabarcoding approach. Species or subspecific level identification of the rusts was improved by using CR-ITS2-refDB, and the Automated Oligonucleotide Design Pipeline (AODP), which identified all mutations distinguishing highly conserved DNA markers amongst close relatives. A generic bioinformatics pipeline was developed, including all steps employed in this study from in silico evaluation of primers to accurate identification of short metabarcodes at the level of interest for defining phytopathogens. The results highlighted the importance of primer selection, refDBs that are resolved to reflect phylogenetic relationships, and the use of AODP for improving the reliability of metabarcoding in phytopathogen biosurveillance.

scholarly journals The Impact of Primer Design on Amplicon-Based Metagenomic Profiling Accuracy: Detailed Insights into Bifidobacterial Community Structure

2020 ◽  
Vol 8 (1) ◽  
pp. 131 ◽  
Author(s):  
Leonardo Mancabelli ◽  
Christian Milani ◽  
Gabriele Andrea Lugli ◽  
Federico Fontana ◽  
Francesca Turroni ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
In Silico ◽  
In Silico Analysis ◽  
Amplification Efficiency ◽  
Rrna Gene ◽  
Test Case ◽  
Bacterial Populations ◽  
Taxonomic Marker ◽  
The Impact

Next Generation Sequencing (NGS) technologies have overcome the limitations of cultivation-dependent approaches and allowed detailed study of bacterial populations that inhabit the human body. The consortium of bacteria residing in the human intestinal tract, also known as the gut microbiota, impacts several physiological processes important for preservation of the health status of the host. The most widespread microbiota profiling method is based on amplification and sequencing of a variable portion of the 16S rRNA gene as a universal taxonomic marker among members of the Bacteria domain. Despite its popularity and obvious advantages, this 16S rRNA gene-based approach comes with some important limitations. In particular, the choice of the primer pair for amplification plays a major role in defining the accuracy of the reconstructed bacterial profiles. In the current study, we performed an in silico PCR using all currently described 16S rRNA gene-targeting primer pairs (PP) in order to assess their efficiency. Our results show that V3, V4, V5, and V6 were the optimal regions on which to design 16S rRNA metagenomic primers. In detail, PP39 (Probio_Uni/Probio_Rev), PP41 (341F/534R), and PP72 (970F/1050R) were the most suitable primer pairs with an amplification efficiency of >98.5%. Furthermore, the Bifidobacterium genus was examined as a test case for accurate evaluation of intra-genus performances at subspecies level. Intriguingly, the in silico analysis revealed that primer pair PP55 (527f/1406r) was unable to amplify the targeted region of any member of this bacterial genus, while several other primer pairs seem to rather inefficiently amplify the target region of the main bifidobacterial taxa. These results highlight that selection of a 16S rRNA gene-based PP should be done with utmost care in order to avoid biases in microbiota profiling results.

scholarly journals Development of a Universal Microarray Based on the Ligation Detection Reaction and 16S rRNA Gene Polymorphism To Target Diversity of Cyanobacteria

2004 ◽  
Vol 70 (12) ◽  
pp. 7161-7172 ◽  
Author(s):  
Bianca Castiglioni ◽  
Ermanno Rizzi ◽  
Andrea Frosini ◽  
Kaarina Sivonen ◽  
Pirjo Rajaniemi ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Environmental Samples ◽  
Rrna Gene ◽  
Accurate Identification ◽  
Culture Collections ◽  
Reliable Identification ◽  
Ligation Detection Reaction ◽  
Universal Array ◽  
Axenic Strains

ABSTRACT The cyanobacteria are photosynthetic prokaryotes of significant ecological and biotechnological interest, since they strongly contribute to primary production and are a rich source of bioactive compounds. In eutrophic fresh and brackish waters, their mass occurrences (water blooms) are often toxic and constitute a high potential risk for human health. Therefore, rapid and reliable identification of cyanobacterial species in complex environmental samples is important. Here we describe the development and validation of a microarray for the identification of cyanobacteria in aquatic environments. Our approach is based on the use of a ligation detection reaction coupled to a universal array. Probes were designed for detecting 19 cyanobacterial groups including Anabaena/Aphanizomenon, Calothrix, Cylindrospermopsis, Cylindrospermum, Gloeothece, halotolerants, Leptolyngbya, Palau Lyngbya, Microcystis, Nodularia, Nostoc, Planktothrix, Antarctic Phormidium, Prochlorococcus, Spirulina, Synechococcus, Synechocystis, Trichodesmium, and Woronichinia. These groups were identified based on an alignment of over 300 cyanobacterial 16S rRNA sequences. For validation of the microarrays, 95 samples (24 axenic strains from culture collections, 27 isolated strains, and 44 cloned fragments recovered from environmental samples) were tested. The results demonstrated a high discriminative power and sensitivity to 1 fmol of the PCR-amplified 16S rRNA gene. Accurate identification of target strains was also achieved with unbalanced mixes of PCR amplicons from different cyanobacteria and an environmental sample. Our universal array method shows great potential for rapid and reliable identification of cyanobacteria. It can be easily adapted to future development and could thus be applied both in research and environmental monitoring.

scholarly journals The mutL Gene as a Genome-Wide Taxonomic Marker for High Resolution Discrimination of Lactiplantibacillus plantarum and Its Closely Related Taxa

2021 ◽  
Vol 9 (8) ◽  
pp. 1570
Author(s):  
Chien-Hsun Huang ◽  
Chih-Chieh Chen ◽  
Yu-Chun Lin ◽  
Chia-Hsuan Chen ◽  
Ai-Yun Lee ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Target Genes ◽  
Marker Genes ◽  
Rrna Gene ◽  
Accurate Identification ◽  
Discrimination Power ◽  
Sequence Identity ◽  
Genome Wide ◽  
A Genome

The current taxonomy of the Lactiplantibacillus plantarum group comprises of 17 closely related species that are indistinguishable from each other by using commonly used 16S rRNA gene sequencing. In this study, a whole-genome-based analysis was carried out for exploring the highly distinguished target genes whose interspecific sequence identity is significantly less than those of 16S rRNA or conventional housekeeping genes. In silico analyses of 774 core genes by the cano-wgMLST_BacCompare analytics platform indicated that csbB, morA, murI, mutL, ntpJ, rutB, trmK, ydaF, and yhhX genes were the most promising candidates. Subsequently, the mutL gene was selected, and the discrimination power was further evaluated using Sanger sequencing. Among the type strains, mutL exhibited a clearly superior sequence identity (61.6–85.6%; average: 66.6%) to the 16S rRNA gene (96.7–100%; average: 98.4%) and the conventional phylogenetic marker genes (e.g., dnaJ, dnaK, pheS, recA, and rpoA), respectively, which could be used to separat tested strains into various species clusters. Consequently, species-specific primers were developed for fast and accurate identification of L. pentosus, L. argentoratensis, L. plantarum, and L. paraplantarum. During this study, one strain (BCRC 06B0048, L. pentosus) exhibited not only relatively low mutL sequence identities (97.0%) but also a low digital DNA–DNA hybridization value (78.1%) with the type strain DSM 20314T, signifying that it exhibits potential for reclassification as a novel subspecies. Our data demonstrate that mutL can be a genome-wide target for identifying and classifying the L. plantarum group species and for differentiating novel taxa from known species.

scholarly journals Dermacentor variabilis is the Predominant Dermacentor spp. (Acari: Ixodidae) Feeding on Dogs and Cats Throughout the United States

2021 ◽  
Author(s):  
Kathryn T Duncan ◽  
Meriam N Saleh ◽  
Kellee D Sundstrom ◽  
Susan E Little
Keyword(s):  
United States ◽  
Spotted Fever ◽  
Rocky Mountain ◽  
The United States ◽  
Dermacentor Variabilis ◽  
Western United States ◽  
Its2 Region ◽  
Rrna Gene ◽  
Rocky Mountain Spotted Fever ◽  
Dermacentor Albipictus

Abstract Throughout North America, Dermacentor spp. ticks are often found feeding on animals and humans, and are known to transmit pathogens, including the Rocky Mountain spotted fever agent. To better define the identity and distribution of Dermacentor spp. removed from dogs and cats in the United States, ticks submitted from 1,457 dogs (n = 2,924 ticks) and 137 cats (n = 209 ticks) from veterinary practices in 44/50 states from February 2018-January 2020 were identified morphologically (n = 3,133); the identity of ticks from regions where Dermacentor andersoni (Stiles) have been reported, and a subset of ticks from other regions, were confirmed molecularly through amplification and sequencing of the ITS2 region and a 16S rRNA gene fragment. Of the ticks submitted, 99.3% (3,112/3,133) were Dermacentor variabilis (Say), 0.4% (12/3,133) were D. andersoni, and 0.3% (9/3,133) were Dermacentor albipictus (Packard). While translocation of pets prior to tick removal cannot be discounted, the majority (106/122; 87%) of Dermacentor spp. ticks removed from dogs and cats in six Rocky Mountain states (Montana, Idaho, Wyoming, Nevada, Utah, and Colorado) were D. variabilis, suggesting this species may be more widespread in the western United States than is currently recognized, or that D. andersoni, if still common in the region, preferentially feeds on hosts other than dogs and cats. Together, these data support the interpretation that D. variabilis is the predominant Dermacentor species found on pets throughout the United States, a finding that may reflect recent shifts in tick distribution.

scholarly journals Yeast Species Associated with Orange Juice: Evaluation of Different Identification Methods

2002 ◽  
Vol 68 (4) ◽  
pp. 1955-1961 ◽  
Author(s):  
Covadonga R. Arias ◽  
Jacqueline K. Burns ◽  
Lorrie M. Friedrich ◽  
Renee M. Goodrich ◽  
Mickey E. Parish
Keyword(s):  
Orange Juice ◽  
Restriction Pattern ◽  
Partial Sequence ◽  
Rrna Gene ◽  
Correct Identification ◽  
Internal Transcribed Spacer Region ◽  
Accurate Identification ◽  
Hanseniaspora Uvarum ◽  
Classical Methodology ◽  
26S Rrna

ABSTRACT Five different methods were used to identify yeast isolates from a variety of citrus juice sources. A total of 99 strains, including reference strains, were identified using a partial sequence of the 26S rRNA gene, restriction pattern analysis of the internal transcribed spacer region (5.8S-ITS), classical methodology, the RapID Yeast Plus system, and API 20C AUX. Twenty-three different species were identified representing 11 different genera. Distribution of the species was considerably different depending on the type of sample. Fourteen different species were identified from pasteurized single-strength orange juice that had been contaminated after pasteurization (PSOJ), while only six species were isolated from fresh-squeezed, unpasteurized orange juice (FSOJ). Among PSOJ isolates, Candida intermedia and Candida parapsilosis were the predominant species. Hanseniaspora occidentalis and Hanseniaspora uvarum represented up to 73% of total FSOJ isolates. Partial sequence of the 26S rRNA gene yielded the best results in terms of correct identification, followed by classical techniques and 5.8S-ITS analysis. The commercial identification kits RapID Yeast Plus system and API 20C AUX were able to correctly identify only 35 and 13% of the isolates, respectively. Six new 5.8S-ITS profiles were described, corresponding to Clavispora lusitaniae, Geotrichum citri-aurantii, H. occidentalis, H. vineae, Pichia fermentans, and Saccharomycopsis crataegensis. With the addition of these new profiles to the existing database, the use of 5.8S-ITS sequence became the best tool for rapid and accurate identification of yeast isolates from orange juice.

PSIX-3 Comparison of 16S rRNA gene profiles of rumen microbiome from Raramuri Criollo, European and Criollo x European lineages

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 441-442
Author(s):  
Adrian Maynez-Perez ◽  
Francisco Jahuey-Martinez ◽  
Jose A Martinez-Quintana ◽  
Michael E Hume ◽  
Robin C Anderson ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Beta Diversity ◽  
Chihuahuan Desert ◽  
Reference Database ◽  
Rrna Gene ◽  
Linear Discriminant ◽  
Microbiome Composition ◽  
Northern Mexico ◽  
Rumen Microbiome

Abstract Raramuri Criollo cattle from the Chihuahuan desert in northern Mexico have been described as an ecological ecotype due to their enormous advantage in land grass utilization and their capacity to diversify their diet with cacti, forbs and woody plants. This diversification in diet utilization, could reflect upon their microbiome composition. The aim of this study was to characterize the rumen microbiome of Raramuri criollo cattle and to compare it to other lineages that graze in the same area. A total of 28 cows representing three linages [Criollo (n = 13), European (n = 9) and Criollo x European Crossbred (n = 6)] were grazed without supplementation for 45 days. DNA was extracted from ruminal samples and the V4 region of the 16S rRNA gene was sequenced on an Illumina platform. Data were analyzed with the QIIME2 software package and DADA2 plugin and the amplicon sequence variants were taxonomically classified with naïve Bayesian using the SILVA 16S rRNA gene reference database (version 132). Statistical analysis was performed by ANOVA and PERMANOVA for alpha and beta diversity indexes, respectively, and the non-strict version of linear discriminant analysis effect size (LEfSe) was used to determine significantly different taxa among lineages. Differences in beta diversity indexes (P < 0.05) were found in ruminal microbiome composition between Criollo and European groups, whereas the Crossbred showed intermediate values when compared to the pure breeds (Table 1). LEfSe analysis identified a total of 20 bacterial groups that explained differences between lineages, including one for Crossbreed, ten for European and nine for Criollo. These results show ruminal microbiome differences between Raramuri criollo cattle and the mainstream European breeds used in the northern Mexico Chihuahuan desert and reflect that those differences could be a consequence of dissimilar grazing behavior.

scholarly journals Bioconductor workflow for microbiome data analysis: from raw reads to community analyses

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 1492 ◽  
Author(s):  
Ben J. Callahan ◽  
Kris Sankaran ◽  
Julia A. Fukuyama ◽  
Paul J. McMurdie ◽  
Susan P. Holmes
Keyword(s):  
High Throughput Sequencing ◽  
Linear Models ◽  
Reference Database ◽  
Rrna Gene ◽  
Microbial Composition ◽  
Nonparametric Testing ◽  
Abundance Estimates ◽  
Taxonomic Markers ◽  
Microbiome Data ◽  
Microbiome Data Analysis

High-throughput sequencing of PCR-amplified taxonomic markers (like the 16S rRNA gene) has enabled a new level of analysis of complex bacterial communities known as microbiomes. Many tools exist to quantify and compare abundance levels or microbial composition of communities in different conditions. The sequencing reads have to be denoised and assigned to the closest taxa from a reference database. Common approaches use a notion of 97% similarity and normalize the data by subsampling to equalize library sizes. In this paper, we show that statistical models allow more accurate abundance estimates. By providing a complete workflow in R, we enable the user to do sophisticated downstream statistical analyses, including both parameteric and nonparametric methods. We provide examples of using the R packages dada2, phyloseq, DESeq2, ggplot2 and vegan to filter, visualize and test microbiome data. We also provide examples of supervised analyses using random forests, partial least squares and linear models as well as nonparametric testing using community networks and the ggnetwork package.

scholarly journals An 18S V4 rDNA metabarcoding dataset of protist diversity in the Atlantic inflow to the Arctic Ocean, through the year and down to 1000 m depth

2021 ◽  
Author(s):  
Elianne Egge ◽  
Stephanie Elferink ◽  
Daniel Vaulot ◽  
Uwe John ◽  
Gunnar Bratbak ◽  
...  
Keyword(s):  
Arctic Ocean ◽  
High Throughput Sequencing ◽  
Sea Water ◽  
Size Fraction ◽  
The Arctic ◽  
Reference Database ◽  
Rrna Gene ◽  
Svalbard Archipelago ◽  
The Arctic Ocean ◽  
Epipelagic Zone

AbstractArctic marine protist communities have been understudied due to challenging sampling conditions, in particular during winter and in deep waters. The aim of this study was to improve our knowledge on Arctic protist diversity through the year, both in the epipelagic (< 200 m depth) and mesopelagic zones (200-1000 m depth). Sampling campaigns were performed in 2014, during five different months, to capture the various phases of the Arctic primary production: January (winter), March (pre-bloom), May (spring bloom), August (post-bloom) and November (early winter). The cruises were undertaken west and north of the Svalbard archipelago, where warmer Atlantic waters from the West Spitsbergen Current meets cold Arctic waters from the Arctic Ocean. From each cruise, station, and depth, 50 L of sea water were collected and the plankton was size-fractionated by serial filtration into four size fractions between 0.45-200 µm, representing the picoplankton, nanoplankton and microplankton. In addition vertical net hauls were taken from 50 m depth to the surface at selected stations. From the plankton samples DNA was extracted, the V4 region of the 18S rRNA-gene was amplified by PCR with universal eukaryote primers and the amplicons were sequenced by Illumina high-throughput sequencing. Sequences were clustered into Amplicon Sequence Variants (ASVs), representing protist genotypes, with the dada2 pipeline. Taxonomic classification was made against the curated Protist Ribosomal Reference database (PR2). Altogether 6,536 protist ASVs were obtained (including 54 fungal ASVs). Both ASV richness and taxonomic composition were strongly dependent on size-fraction, season, and depth. ASV richness was generally higher in the smaller fractions, and higher in winter and the mesopelagic samples than in samples from the well-lit epipelagic zone during summer. During spring and summer, the phytoplankton groups diatoms, chlorophytes and haptophytes dominated in the epipelagic zone. Parasitic and heterotrophic groups such as Syndiniales and certain dinoflagel-lates dominated in the mesopelagic zone all year, as well as in the epipelagic zone during the winter. The dataset is available at https://doi.org/10.17882/79823, (Egge et al., 2014).

scholarly journals Influence of Agaricus Bisporus Establishment and Fungicidal Treatments on Casing Soil Metataxonomy During Mushroom Cultivation

2021 ◽  
Author(s):  
Maria Luisa Tello ◽  
Rebeca Lavega ◽  
Margarita Pérez ◽  
Antonio J. Pérez ◽  
Michael Thon ◽  
...  
Keyword(s):  
Agaricus Bisporus ◽  
Illumina Miseq ◽  
Biological Efficiency ◽  
Its2 Region ◽  
Rrna Gene ◽  
Mushroom Cultivation ◽  
Microbial Composition ◽  
Mycelium Growth ◽  
Bacterial Phyla ◽  
The Impact

Abstract The cultivation of edible mushroom is an emerging sector with a potential yet to be discovered. Unlike plants, it is a less developed agriculture where many studies are lacking to optimize the cultivation. Mushrooms are a source of resources still to be revealed, which have applications not only in food, but in many other sectors such as health, industry and biotechnology. Mushroom cultivation consists of the development of selective substrates through composting where the mushroom grows via solid fermentation process. In case of Agaricus bisporus, the compost fully colonized by mycelium hardly produces mushrooms and it is necessary to apply a casing layer with certain physical, chemical and biological characteristics to shift from the vegetative mycelium to the reproductive one, where the native microbiota plays crucial roles. Currently, the industry faces a challenge to substitute the actual peat based casing materials due to the limited natural resources and the impact on the peatlands where peat is extracted.In this work we have employed high-throughput techniques by next generation sequencing to screen the microbial structure of casing soil employed in mushroom cultivation while sequencing V3-V4 of the 16S rRNA gene for bacteria and the ITS2 region of rRNA for fungi in an Illumina MiSeq. In addition, the microbiome dynamics and evolution (bacterial and fungal communities) in peat based casing along the process of incubation of Agaricus bisporus have been studied, while comparing the effect of fungicidal treatment (Chlorothalonil and Metrafenone). Statistically significant changes in populations of bacteria and fungi were observed. Microbial composition differed significantly based on incubation day, changing radically from the original communities to a specific microbial composition adapted to enhance the A. bisporus mycelium growth. Chlorothalonil treatment seems to delay casing colonization by A. bisporus. Proteobacteria and Bacteroidota appeared as the most dominant bacterial phyla. We observed a great change in the structure of the bacteria populations between day 0 and the following days. Fungi populations changed more gradually, A. bisporus displacing the rest of the species as the cultivation cycle progresses. A better understanding of the microbial communities in the casing will hopefully allow us to increase the biological efficiency during production as well as possibly help us to have a clearer view of the microbial community-pathogen relationships as they are directly related to disease development.

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