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The sequences highlighted in blue illustrate examples of sequences removed from PHYMYCO-DB. The compromised nature can stem from erroneous sequencing (e.g. repeated gaps), wrong annotation (e.g. sequence corresponding to another clade), high numbers of undetermined nucleotides, homopolymers insertions, erroneous alignment or reverse complementary sequences and presence of long insertions and introns or presence of deletions.
Source publication
Background: In environmental sequencing studies, fungi can be identified based on nucleic acid sequences, using either highly variable sequences as species barcodes or conserved sequences containing a high-quality phylogenetic signal. For the latter, identification relies on phylogenetic analyses and the adoption of the phylogenetic species concept...
Citations
... For the fungal taxonomic studies, a lot of databases are available such as FungiDB (Stajich et al., 2012), PHYMYCO-DB (Mahé et al., 2012), UNITE (Kõljalg et al., 2005), ITSone DB (Santamaria et al., 2012), and ITS2 Database (Schultz et al., 2006;Selig et al., 2008;Koetschan et al., 2010Koetschan et al., , 2012. FungiDB (Stajich et al., 2012) is a database for functional and genomic data. ...
... FungiDB (Stajich et al., 2012) is a database for functional and genomic data. PHYMYCO-DB (Mahé et al., 2012) is a manually curated bank used for fungal phylogenetic analyzes from TEF1-α and SSU rRNA markers derived from GenBank. For Fungi, UNITE database (Nilsson et al., 2018) is the most widely used, as it contains 2,480,043 ITS sequences from the International Nucleotide Sequence Databases (NCBI, DDBJ, EMBL) and is a curated database, and lacks any cryptic sequences (Vargas-Gastélum & Riquelme, 2020) thus helps in improved identification from the environmental samples (Kõljalg et al., 2005;Abarenkov et al., 2010). ...
... Further filtering enabled removal of all sequence clusters identified in fewer than three samples and with a threshold of 0.005% of reads. After taxonomic affiliations using Silva138 16S rRNA for bacteria (Quast et al. 2012) and Phymyco-DB databases for fungi (Mahé et al. 2012), a final filtering step was applied to affiliated sequence clusters (95% coverage and 95% identity (BLAST)). The amplicon sequences did not contain any plastidial or plant mitochondrial DNA sequence. ...
Considering the plant microbiota, temporal changes are expected depending on plant development stages and environmental pressures because of modifications in plant requirements and available soil microbial reservoir.
Herein, we analyzed the composition of root endosphere microbiota of grafted vine plants using two grapevine cultivars (Merlot and Cabernet-Sauvignon as scion grafted on rootstocks of different clones) as models both sampled in a single vineyard at three dates over a period of two growing years.
Highly conserved temporal patterns were found in the two cultivars. Intra-annual changes in microbial community composition were recorded whereas convergent microbial communities were observed on the two September dates. In particular, the increase in Actinobacteria and decrease in Glomeromycota in September were interpreted as shifts in the microbiota community patterns related to plant physiological requirements (e.g. water supply). A high proportion of non-random assembly of the root endospheric bacterial community confirmed the deterministic influence of the plant or/and the environment in microbial recruitment over time. The modified normalized stochasticity ratio (MST) showed that deterministic processes of assembly (MST < 50%) were commonplace despite the changes in the root microbiota composition observed among sampling dates.
Our study suggests an intra-annual rhythm of microbiota shifts, marginally random, with a succession within the root-microbiota endosphere likely governed by active plant filtering. A better knowledge of microbial-recruitment at work, seems important for both fundamental and applied perspectives.
... The most used ones are: The National Center for Biotechnology Information (NCBI) database (Robbertse & Tatusova, 2011), the Userfriendly Nordic ITS Ectomycorrhiza Database -UNITE database (Kõljalg et al., 2013, Nilsson et al., 2019, a Global fungi (Větrovský et al., 2020), the Barcode of Life Data Systems (Ratnasingham & Heber, 2007). It is highly possible that some other derivative databases such as PHYMYCO (Mahe et al., 2012) or specialised database would appear in the future along with those that are currently under development such as EPPO-Q-bank Fungi database (https://qbank.eppo.int/fungi/). ...
In this study, epiphytic fungi were isolated from wheat seeds (Triticum aestivum L) and a polyphasic approach for their identification was applied. The initial morphological characterisation was done on selected twenty-tree strains and continued with their identification based on the carbon utilisation pattern according to the Biolog OmniLog system protocol (Hayward, CA, USA). Throughout the isolated strains, the Biolog data indicated predominance of the genera Aspergillus, Penicillium and Fusarium. The carbon utilisation pattern of five strains assigned them to several different from aforementioned genera and due to their scarcity they were not included in the current study. The indicated by the Biolog genus designation of the strains was used as a guideline for the further molecular identification of seventeen strains from the predominant genera. Molecular identification was based on a polymerase chain reaction (PCR) with universal primers, which targeted the internal transcribed spacer (ITS) region of rDNA gene. The PCR fragments were sequenced and after applying a BLAST algorithm, a high percentage of similarity was found for eleven strains. At the species level, the Biolog and molecular technique showed a discrepancy in the identification of two strains. The procedure of identification was unsuccessful for six strains either due to insufficient quantity of the PCR product or the lack of sufficient similarity of the fragments to the GenBank database. Current study showed that the unbiased identification of epiphytic fungi requires a polyphasic approach, which applies morphological, physiological and molecular techniques.
... Merged sequences were then clustered using SWARM allowing to limit the overestimation of sequence diversity which conversely might be the case when using ASVs. As recommended in the FROGS guidelines, affiliation was done by blastn+ for one representative sequence of each sequence cluster using herein PhymycoDB (Mahé et al., 2012) as a reference database (threshold of at least 95% BLAST identity and 95% coverage applied). In the particular case of uncertainty in the taxonomic affiliation (e.g. ...
Micro‐organisms associated with plants provide essential functions to their hosts, and therefore affect ecosystem productivity. Agricultural intensification has modified microbial diversity in the soil reservoir and may affect plant–microbial recruitment. Weeds develop spontaneously in crop fields, and could influence micro‐organisms associated with crop plants through a neighbourhood effect. We explore the effect of weed species on crop plant microbiota as potentially auxiliary plants that affect agricultural productivity.
We combined field and controlled laboratory studies to analyse the neighbourhood effect of weeds on wheat root endospheric mycobiota (i.e. fungi within roots) and growth. First, we analysed the effect of weed species diversity and identity recorded in the neighbourhood of individual wheat plants on soil and wheat root mycobiota in the field. Second, we used a plant‐matrix design in laboratory conditions to test the effect of weed identity (nine weed treatments) and their ability to transmit root mycobiota to wheat roots, and the resulting impact on wheat growth.
In contrast to soil mycobiota, we demonstrated that wheat root endospheric mycobiota was influenced by the diversity and identity of weeds developing in their 1 m² neighbourhood. Wheat root endospheric microbiota strongly differs in terms of richness and composition depending on the neighbouring weed plant species. Weed species transmitted from 13% to 74% of their root microbiota to wheat roots depending on weed identity in controlled conditions.
Synthesis. Weed neighbours modified wheat plant performance, possibly as a result of competitive interactions and changes in microbiota. Our findings suggest that crop root mycobiota was variable and was modulated by their weed neighbourhood. Synergistic effects between mycobiota of crops and weeds could therefore contribute to soil biodiversity and sustainable agriculture.
... Sequence clustering was combined with a rigorous chimera removal step (Escudié et al., 2018) followed by the removal of all sequence clusters detected in less than three independent samples and with a threshold of 0.005% of reads. As recommended in the FROGS guidelines (Escudié et al., 2018), affiliation was made by BLASTN+ for one representative sequence of each sequence cluster using herein PhymycoDB (Mahé et al., 2012) as a reference database (threshold of at least 95% BLAST identity and 95% coverage applied). In the particular case of getting identical BLASTN+ scores for a given representative sequence to affiliate, the last convergent taxonomic rank was kept. ...
Ecological corridors promote species coexistence in fragmented habitats where dispersal limits species fluxes. The corridor concept was developed and investigated with macroorganisms in mind, while microorganisms, the invisible majority of biodiversity, were disregarded.
We analyzed the effect of corridors on the dynamics of endospheric fungal assemblages associated with plant roots at the scale of 1 m over 2 years (i.e. at five time points) by combining an experimental corridor‐mesocosm with high‐throughput amplicon sequencing.
We showed that plant root endospheric mycobiota were sensitive to corridor effects when the corridors were set up at a small spatial scale. The endospheric mycobiota of connected plants had higher species richness, lower beta‐diversity, and more deterministic assembly than the mycobiota of isolated plants. These effects became more pronounced with the development of host plants.
Biotic corridors composed of host plants may thus play a key role in the spatial dynamics of microbial communities and may influence microbial diversity and related ecological functions.
... Further filtering enabled removal of all sequence clusters identified in fewer than three samples and with a threshold of 0.005% of reads. After taxonomic affiliations using Silva138 16S rRNA for bacteria (57) and Phymyco-DB databases for fungi (58), a final filtering step was applied to affiliated sequence clusters (95% coverage and 95% identity (BLAST)). ...
Le concept d’holobionte considère l’unité fonctionnelle composée des plantes et de ces microorganismes. Il promeut une approche holistique à la gestion des cultures et, plus généralement, à la vision du vivant. Cependant, ce concept est sujet à débat en raison du manque de preuves expérimentales de son existence. De plus, la compréhension fine des mécanismes régissant l’assemblage des microorganismes associés aux plantes reste un des enjeux majeurs de l’écologie microbienne et de l’agriculture. Le but de cette thèse était de tester la validité du concept d’holobionte, d’étudier les facteurs qui impactent l’assemblage des communautés microbiennes de l’endosphère racinaire et d’analyser la dynamique intra- et inter-annuelle du microbiote de la vigne. Grâce à une expérimentation mise en place en association avec une pépinière viticole (greffage et plantes chimériques), nous avons pu démontrer l’existence d’un recrutement actif et déterminé de microorganismes par la plante, sous l’effet dominant du porte-greffe, ce qui nous a permis d’augmenter les preuves expérimentales à l’existence du concept d’holobionte. Dans le cadre d’une seconde partie, nous nous sommes intéressés aux dynamiques d’assemblages du microbiote de la vigne, grâce à un vaste plan d’échantillonnage mis en place au sein d’un domaine viticole. Nous avons mis en avant le rôle fondamental des facteurs environnementaux, de l’âge et du cépage dans l’assemblage du microbiote endosphérique racinaire de la vigne à petite échelle géographique et l'existence de patrons temporels intra-annuels marqués dans la structuration de ces communautés. Ces travaux fournissent un ensemble de connaissances nouvelles dans le domaine de l’écologie microbienne, soutiennent l’existence d’un terroir microbiologique et soulignent l’importance de la prise en compte de ce terroir microbien dans le cadre d’une gestion durable de la vigne.
... Besides, SILVA, as well as PR 2 (a database dedicated to protists; Guillou et al., 2013), do not include any taxonomic rank between phyla and genera (see Supporting Information 1). PHYMYCO-DB (Mahé et al., 2012), a database dedicated to Fungi, does not contain microsporidian sequences. The existing databases being inappropriate, another one based on the phylogeny rather than on morphological characters, was thus required. ...
Microsporidia are obligate intracellular eukaryotic parasites known to parasitize many species of the animal kingdom as well as some protists. However, their diversity is underestimated, in part as a consequence of the failure of ‘universal’ primers to detect them in metabarcoding studies. Besides, due to the inconsistency between taxonomy and phylogenetic data, available databases may assign incorrectly sequences obtained with high‐throughput sequencing. In this work, we developed a comprehensive reference database which positions microsporidian SSU rRNA gene sequences within a coherent ranked phylogenetic framework. We used this phylogenetic framework to study the microsporidian diversity in lacustrine ecosystems, focusing on <150 μm planktonic size fractions. Our analysis shows a high diversity of Microsporidia, with the identification of 1531 OTUs distributed within 7 clades, of which 76% were affiliated to clade IV2 and 20% to clade I (nomenclature presented hereby). About a quarter of the obtained sequences shared less than 85% identity to the closest known species, which might represent undescribed genera or families infecting small hosts. Variations in the abundance of Microsporidia were recorded between the two lakes sampled and across the sampling period, which might be explained by spatio‐temporal variations of their potential hosts such as microeukaryotes and metazooplankton. This article is protected by copyright. All rights reserved.
... This article is protected by copyright. All rights reserved DB (containing SSU and EF1-α sequences for Fungi; Mahé et al., 2012) and ITSoneDB (containing ITS1 sequences for Eukarya; Santamaria et al., 2018). ...
... All rights reserved because it does not detect putative chimeric sequences that are merged into clusters with nonchimeric centroids, thus remaining hidden. Clustering can be useful when tackling sequencing errors, as the most abundant sequences tend to have a lower probability to contain errors compared to rare sequences (Kunin et al., 2010;Smith, Heger, & Sudbery, 2017 Although gDAT provides the MaarjAM database for analysis of AM fungal SSU data, it also contains tools to build custom databases, making the pipeline applicable for other organisms and marker regions (Quast et al., 2012;Mahé et al., 2012;Santamaria et al., 2018;Nilsson et al., 2019b). Analysis of sequence data here showed that a general eukaryotic primer can pick up abundant AM fungal species and provided a general description of the organisms harboured in the soil. ...
High‐throughput sequencing (HTS) of multiple organisms in parallel (metabarcoding) has become a routine and cost‐effective method for the analysis of microbial communities in environmental samples. However, careful data treatment is required to identify potential errors in HTS data, and the large volume of data generated by HTS requires in‐house experience with command line tools for downstream analysis. This paper introduces a pipeline that incorporates the most common command line tools into an easy‐to‐use graphical interface ‐ gDAT. By using the Python scripting language, the pipeline is compatible with the latest Windows, macOS and Linux operating systems. The pipeline supports analysis of Sanger, 454, IonTorrent, Illumina and PacBio sequences, allows custom modification of quality filtering steps, and implements both open and closed‐reference operational taxonomic unit‐picking for sequence identification. Predefined parameters are optimised for analysis of SSU rRNA gene amplicons from arbuscular mycorrhizal fungi, but the pipeline is widely applicable to metabarcoding studies targeting a broad range of organisms. The pipeline was additionally tested with data using general eukaryotic primers from the SSU gene region and fungal primers from the ITS marker region. We describe the pipeline design and evaluate its performance and speed by conducting analysis of example datasets using different marker regions sequenced on Illumina platforms. The graphical interface, with the option to use the command line if needed, provides an accessible tool for rapid data analysis with repeatability and logging capabilities. Keeping the software open‐source maximises code accessibility, allowing scrutiny and bug fixes by the community.
... The 18S rRNA gene sequence from isolates CA1, CA3, CA4, CA5, CA7, and CA8 were aligned and analysed using Basic Local Alignment Search Tool, PHYMYCO-DB, and FungiDB [18,19]. An evolutionary relationship of samples was constructed using the Maximum Likelihood method based on the Tamura-Nei model in MEGA7 software package with bootstrap consensus tree from 500 replicates [20][21][22]. ...
Candida auris is an emerging multi-drug resistant pathogen with high mortality rate; nosocomial infections have been reported worldwide, causing a major challenge for clinicians and microbiological laboratories. The study aims to describe new cases of C. auris and detect drug resistance-associated mutations of C. auris by the sequencing of ERG11 and FKS1 genes. A total of six specimens were collected from blood, urine, ear swab, and groin screening samples. Isolates were incubated for 48 h on Sabouraud Dextrose agar (SDA) at 42 °C, then confirmed by MALDI-TOF MS. Furthermore, antifungal susceptibility testing was performed using the Vitek 2 system to detect Minimum Inhibitory Concentrations (MICs) of six antifungals. Sequences of 18S rRNA gene and ITS regions from isolates and phylogenetic analysis were performed. Gene sequencing was analysed to detect drug resistance-associated mutations by FKS1 and ERG11 genes sequencing. All C. auris isolates were confirmed by MALDI-TOF MS, and evolutionary analyses using sequences of 18S rRNA gene and ITS region. Antifungal susceptibility testing showed that all isolates were resistant to fluconazole. Sequencing of ERG11 and FKS1 genes from the isolates revealed the presence of two (F132Y and K143R) drug resistance-associated mutations in ERG11, however, FKS1 gene was devoid of mutations. The study sheds light on a public health threat of an emerging pathogen, and the hospital implemented strict contact screening and infection control precautions to prevent C. auris infection. Finally, there is a critical need to monitor the antifungal resistance in different geographical areas and implementation of efficient guidelines for treatment.
Citation: AlJindan, R.; AlEraky, D.M.; Mahmoud, N.; Abdalhamid, B.; Almustafa, M.; AbdulAzeez, S.; Borgio, J.F. Drug Resistance-Associated Mutations in ERG11 of Multidrug-Resistant Candida auris in a Tertiary Care Hospital of Eastern Saudi Arabia. J. Fungi 2021, 7, 18. https://doi.
... Following the removal of genes encoding protein sequences < 300 amino acids long, 150 CYP450s and 92 UGTs were analyzed further. Multiple sequence alignment was conducted using ClustalX 2.1 software [52]. A neighborjoining algorithm with a Poisson model and pairwise deletion was used to generate a phylogenetic tree with MEGAX software [53], with 1000 replicates being used for bootstrap testing to validate this tree. ...
Background:
Members of the cytochrome P450 (CYP450) and UDP-glycosyltransferase (UGT) gene superfamily have been shown to play essential roles in regulating secondary metabolite biosynthesis. However, the systematic identification of CYP450s and UGTs has not been reported in Aralia elata (Miq.) Seem, a highly valued medicinal plant.
Results:
In the present study, we conducted the RNA-sequencing (RNA-seq) analysis of the leaves, stems, and roots of A. elata, yielding 66,713 total unigenes. Following annotation and KEGG pathway analysis, we were able to identify 64 unigenes related to triterpenoid skeleton biosynthesis, 254 CYP450s and 122 UGTs, respectively. A total of 150 CYP450s and 92 UGTs encoding > 300 amino acid proteins were utilized for phylogenetic and tissue-specific expression analyses. This allowed us to cluster 150 CYP450s into 9 clans and 40 families, and then these CYP450 proteins were further grouped into two primary branches: A-type (53%) and non-A-type (47%). A phylogenetic analysis of 92 UGTs and other plant UGTs led to clustering into 16 groups (A-P). We further assessed the expression patterns of these CYP450 and UGT genes across A. elata tissues, with 23 CYP450 and 16 UGT members being selected for qRT-PCR validation, respectively. From these data, we identified CYP716A295 and CYP716A296 as the candidate genes most likely to be associated with oleanolic acid synthesis, while CYP72A763 and CYP72A776 were identified as being the most likely to play roles in hederagenin biosynthesis. We also selected five unigenes as the best candidates for oleanolic acid 3-O-glucosyltransferase. Finally, we assessed the subcellular localization of three CYP450 proteins within Arabidopsis protoplasts, highlighting the fact that they localize to the endoplasmic reticulum.
Conclusions:
This study presents a systematic analysis of the CYP450 and UGT gene family in A. elata and provides a foundation for further functional characterization of these two multigene families.
