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Our study evaluated in silico the potential of 14 mitochondrial genes encoding the subunits of the respiratory chain complexes, including cytochrome c oxidase I (CO1), as Basidiomycota DNA barcode. Fifteen complete and partial mitochondrial genomes were recovered and char-acterized in this study. Mitochondrial genes showed high values of molecular...
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... total of 15 mitochondrial genomes were retrieved (Table 2), among which seven [Coprinus cinereus, Cryptococcus neofor- mans var. gattii (strain R265), Laccaria bicolor, Phanerochaete chrysosporium, Phakopsora pachyrhizi, Postia placenta and Sporo- bolomyces roseus] were recovered using the reconstruction strategies 2, 3 and 4 of our bioinformatic process (Fig. 1). ...
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... mitochondrial genome size ranged from 24.8 kb for C. neoformans var. grubii (strain H99) to more than 100 kb for Moniliophthora perniciosa and P. placenta (Table 2). Such length differences can be explained by the high vari- ation in gene and intron numbers in mitochondrial genomes. ...
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... differences in gene patterns among different mito- chondrial genomes reflected either the absence of one specific gene in the mitochondrial genome, as previously reported for the atp9 gene in Podospora anserina (Yan & Xu 2005), or the limitation of the data-mining and reconstruction methods applied to incomplete Trace Archive data sets. The mitochon- drial genomes of P. placenta (CO2 and rps3 missing), S. roseus (nad6 and rps3 missing, atp9 incomplete) and P. chrysosporium (atp8, rnl and rps3 missing, CO1, CO2, CO3, cob, nad1 and nad5 incomplete) are most likely incomplete since they resulted from the concatenation of 4, 26 and 12 non-overlapping contigs, for a total size of 114.1, 45.4 and 40.1 kb, respec- tively (Table 2). ...
Citations
... The small subunit (18S) region of the ribosomal DNA repeat was amplified with NS1 (White et al. 1990)/Rust18S-R (Aime 2006) and, for weak products, nested with RustNS2-F )/NS6 (White et al. 1990) following the protocols of Aime et al. (2018). Cytochrome c oxidase subunit 3 (CO3) of the mitochondrial DNA was amplified with CO3_F1/CO3_R1 (Vialle et al. 2009) following the protocols of Vialle et al. (2009). For select members of Coleosporium, the internal transcribed spacer region of the ribosomal DNA (ITS) repeat was amplified with primers ITS1F/ITS2R (Toome and Aime 2015) following the protocols of Toome and Aime (2015). ...
... The small subunit (18S) region of the ribosomal DNA repeat was amplified with NS1 (White et al. 1990)/Rust18S-R (Aime 2006) and, for weak products, nested with RustNS2-F )/NS6 (White et al. 1990) following the protocols of Aime et al. (2018). Cytochrome c oxidase subunit 3 (CO3) of the mitochondrial DNA was amplified with CO3_F1/CO3_R1 (Vialle et al. 2009) following the protocols of Vialle et al. (2009). For select members of Coleosporium, the internal transcribed spacer region of the ribosomal DNA (ITS) repeat was amplified with primers ITS1F/ITS2R (Toome and Aime 2015) following the protocols of Toome and Aime (2015). ...
... Genomic DNA Extraction Kit (Bioneer; Daejeon, Korea). The internal transcribed spacer (ITS) and large subunit (LSU) regions of the rDNA, and the cytochrome oxidase 3 (cox3) mtDNA region were amplified and sequenced with primers ITS5u/ ITS4rust, LRust1R/LRust3, and CO3-F1/CO3-R1, respectively(Vialle et al., 2009). The resulting sequences were deposited in GenBank (Accession. ...
... 28S ribosomal RNA was amplified with Rust2INV (Aime, 2006)/LR6 or LR7 (Vilgalys & Hester, 1990) and, for weak products, nested with Rust28SF (Aime et al., 2018)/ LR5 or LR6 (Vilgalys & Hester, 1990 (White et al., 1990). The mitochondrial CO3 was amplified with CO3_F1/CO3_R1 (Vialle et al., 2009). DNA extraction, PCR and sequencing were mainly performed by TechnoSuruga Laboratory Co. Ltd. (Shizuoka, Japan). ...
Caeoma mori (≡ Aecidium mori), known as the mulberry rust which is an anamorphic rust fungus forming only aecidioid uredinia, were found on Morus alba in Ibaraki and Saitama Prefectures, Japan. Molecular phylogenetic analyses using the combined dataset of sequences from 28S and 18S of the nuclear ribosomal RNA gene and Cytochrome-c-oxidase subunit 3 of the mitochondrial DNA revealed that this anamorphic rust fungus was a member of the clade composed of the genus Gymnosporangium. Therefore, a new combination, Gymnosporangium mori is proposed for this species. Additionally, a new combination, G. brucense for Roestelia brucensis is proposed by phylogenetic evidence.
... The intraspecific heterogeneity among copies of ITS can result in inaccurate sequences [44]. Length variation of ITS occurred because of multiple introns, which are not consistently present in intraspecies [50]. However, despite these drawbacks, based on a combined assessment of primer universality, PCR success, and discriminating power, the ITS region emerged as the consensus primary fungal barcode among the four initially assessed markers [44]. ...
A functional identification system is the core and basis of fungal taxonomy, which provides sufficient diagnostic characteristics for species delimitation. Phenotype-based identification systems have exhibited significant drawbacks, such as being laborious and time-consuming. Thus, a molecular-based identification system (rDNA, DNA fingerprint, etc.) is proposed for application to fungi that lack reliable morphological characteristics. High Throughput Sequencing also makes great contributions to fungal taxonomy. However, the formal naming of nonculturable fungi from environmental sequencing is a significant challenge. Biochemical profile-based identification systems have outstanding value in fungal taxonomy and can occasionally be indispensable. This method utilizes biomarker metabolites and proteins that are expected to be unequivocal and stable. Of these, Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry has become the method of choice for chemotaxonomy. In sum, these described identification systems cannot solve all problems of species delimitation, and considerable attention to the updating of fungal nomenclature, standardization of techniques, knowledge sharing, and dissemination will be necessary.
... Genomic DNA was extracted from rust pustules on dried herbarium specimens using a MagListo 5M plant Genomic DNA Extraction Kit (Bioneer, Daejeon, Republic of Korea). The internal transcribed spacer (ITS), large subunit (LSU) rDNA regions, and the cytochrome oxidase 3 (cox3) gene were amplified using primer pairs ITS5-u/ITS4rust [16,17], LRust1R/LRust3 [16] and CO3_F1/CO3_R1 [18], respectively. The PCR products were purified using an AccuPrep ® PCR/Gel Purification Kit (Bioneer, Daejeon, Republic of Korea) and were sequenced in both directions by a DNA sequencing service (Macrogen, Seoul, Republic of Korea) with the same primers used for the amplification. ...
The present study performed an in-depth investigation of rust diseases affecting Meliosma myriantha and Meliosma oldhamii trees (Sabiaceae) in Korea. The analysis identified four distinct species of the genus Neophysopella (Pucciniales) as the causal agents. Among these, N. hornotina was found to infect only M. oldhamii, whereas three Neophysopella species (N. meliosmae, N. meliosmae-myrianthae, and N. vitis) were parasitic on M. myriantha. To our knowledge, this is the first report of the former two species (N. hornotina and N. meliosmae) in Korea. In addition, we specified their alternate host plants for two heteroecious species (N. meliosmae-myrianthae and N. vitis) in Korea, completing the life cycles of the four rust species, and provided detailed morphological descriptions at each stage of their life cycles. Phylogenetic relationships of these rust species were uncovered using a comprehensive sample size, and we have constructed a phylogenetic tree for Neophysopella using the cytochrome c oxidase subunit III (cox3) gene sequences, demonstrating an effective approach for species delineation within this genus. The findings contribute to identifying and managing rust diseases affecting Meliosma species.
... The subsequent DNA extraction was done using the Qiagen DNeasy Blood & Tissue Kit (Valencia, California) following the manufacturer's protocol. Polymerase chain reactions (PCRs) to amplify fragments of the 28S, 18S, and CO3 gene regions were performed using primer pairs LR0R/LR6 (Moncalvo et al. 1995;Vilgalys and Hester 1990), NS1/Rust18SR (Aime 2006;White et al. 1990), and CO3-R1/CO3-F1 (Vialle et al. 2009), respectively, following conditions described in Ebinghaus et al. (2020), Ebinghaus et al. (2022)). PCR products were sequenced at Macrogen (Seoul, South Korea) using the PCR primers. ...
The multicellular discoid convex teliospore heads represent a prominent generic feature of the genus Ravenelia. However, recent molecular phylogenetic work has shown that this is a convergent trait, and that this genus does not represent a natural group. In 2000, a rust fungus infecting the Caesalpinioid species Cenostigma macrophyllum (= C. gardnerianum) was described as Ravenelia cenostigmatis. This species shows some rare features, such as an extra layer of sterile cells between the cysts and the fertile teliospores, spirally ornamented urediniospores, as well as strongly incurved paraphyses giving the telia and uredinia a basket-like appearance. Using freshly collected specimens of Rav. cenostigmatis and Rav. spiralis on C. macrophyllum, our phylogenetic analyses based on the nuc 28S, nuc 18S, and mt CO3 (cytochrome c oxidase subunit 3) gene sequences demonstrated that these two rust fungi belong in a lineage within the Raveneliineae that is distinct from Ravenelia s. str. Besides proposing their recombination into the new genus Raveneliopsis (type species R. cenostigmatis) and briefly discussing their potentially close phylogenetic affiliations, we suggest that five other Ravenelia species that are morphologically and ecologically close to the type species of Raveneliopsis, i.e., Rav. corbula, Rav. corbuloides, Rav. parahybana, Rav. pileolarioides, and Rav. Striatiformis, may be recombined pending new collections and confirmation through molecular phylogenetic analyses.
... Primers in red were generated for the current study. Primers in black were not generated for the current study but are commonly used for sequencing rust (Vilgalys & Hester, 1990;White et al., 1990;Scholin et al., 1994;Aime, 2006;Vialle et al., 2009;Aime et al., 2018;Chen et al., 2022). Black arrows signify primer direction (primers on the top are in the forward direction and primers on the bottom are in the reverse direction). ...
Sequencing herbarium specimens can be instrumental in answering ecological, evolutionary, and taxonomic inquiries. We developed a protocol for sequencing herbarium specimens of rust fungi (Pucciniales) and proceeded to sequence specimens ranging from 4 to 211 yr old from five different genera.
We then obtained sequences from an economically important biological control agent, Puccinia suaveolens, to highlight the potential of sequencing herbarium specimens in an ecological sense and to evaluate the following hypotheses: (1) The population structure of a plant pathogen changes over time, and (2) introduced pathogens are more diverse in their native range.
Our efforts resulted in sequences from 87 herbarium specimens that revealed a high level of diversity with a population structure that exhibited spatial–temporal patterns. The specimens sequenced from Europe showed more diversity than the ones from North America, uncovering an invasion pattern likely related to its European native host in North America. Additionally, to the best of our knowledge, the specimen from France collected in c. 1811 is the oldest herbarium specimen sequenced from kingdom Fungi.
In conclusion, sequencing old herbarium specimens is an important tool that can be extrapolated to better understand plant–microbe evolution and to evaluate old type specimens to solidify the taxonomy of plant pathogenic fungi.
... Spores were crushed in three consecutive freeze-thaw cycles as described in , and DNA was isolated using the Qiagen DNeasy Blood & Tissue Kit (Valencia, CA, USA) following the manufacturer's protocol. Polymerase chain reactions (PCRs) of segments of LSU (large subunit rDNA, SSU (small subunit rDNA), and CO3 (cytochrome c oxidase subunit III) genes were performed using the GoTaq G2 Hot Start Polymerase kit (Promega, Mannheim, Germany) and the primer pairs LR0R/LR6 (Vilgalys and Hester 1990;Moncalvo et al. 1995), NS1/Rust18SR (White et al. 1990;Aime 2006), and CO3-R1/CO3-F1 (Vialle et al. 2009) for the LSU, SSU, and CO3 gene regions, respectively. For the LSU and CO3 regions, PCR conditions were as described in Ebinghaus et al. (2020). ...
The genus Cerradoa (type species Cerradoa palmaea) was established in 1978 by Hennen and Ono and named after the Brazilian Cerrado biome. The holotype collected in Planaltina, Federal District, Brazil, belonged to the first rust fungus reported on palms (Arecaceae). For decades, the status of Cerradoa as a distinct genus has been regarded as doubtful, representing a synonym of Edythea (Uropyxidaceae) starting with the second edition of the Illustrated Genera of Rust Fungi in 1983. Our molecular phylogenetic analyses, as well as our morphological investigations, allowed us to reject this synonymy, leading to the reinstatement of Cerradoa within the Pucciniaceae. Cerradoa, together with morphologically similar genera such as the newly established Pseudocerradoa with two species (Ps. paullula and Ps. rhaphidophorae) infecting araceous hosts, the fern rust Desmella, and also P. engleriana, could not be assigned to any of the seven identified major lineages within the Pucciniaceae. Edythea, instead of being maintained as a member of the Uropyxidaceae, was herein placed in Pucciniaceae, shown phylogenetically in close relationship to Cumminsiella mirabilissima, both infecting the Berberidaceae. Additionally, our extensive phylogenetic analyses add guidance for future taxonomic revisions in the highly polyphyletic genus Puccinia and other established taxa within the family Pucciniaceae.
... Coupling phenotypic taxonomy with DNA-based technologies, such as DNA barcoding, enables a more detailed view in the phylogeny of said cryptic species. DNA barcoding analyses using the ribosomal ITS rDNA region, which is the universal barcode for fungi (Schoch et al. 2012), enabled species identification and phylogenetic resolutions in a variety of fungal lineages, such as macromycetes (Zhang et al. 2004, Frøslev et al. 2007, Zhang et al. 2010, smuts (Vialle et al. 2009), even for pin molds and lichenized fungi (Kelly et al. 2011), and the Mucorales of Mucoromycotina (Schwarz et al. 2006). Several groups even adopted a multi-marker barcoding approach in order to facilitate fungal species identification either for specific clades or for megaphylogenies. ...
Lagiotis G, Topalidou E, Bosmali I, Osathanunkul M, Madesis P 2021-DNA-based species identification of Greek macromycetes. Abstract Fungi comprise one of the largest and diverse groups of eukaryotes. Macromycetes, which are commonly known as mushrooms, include species in Basidiomycota and Ascomycota. Macromycetes are essential for ecosystem functioning and have high commercial value owing to their nutritional and medicinal properties. Despite the importance of macrofungi for the ecosystem and human welfare, macromycete diversity and phylogeny are poorly characterized, owing to the lack of molecular-based biodiversity descriptors supporting phenotypic classifications, especially for biodiversity rich countries such as Greece. In this study, we implemented a multi-marker DNA barcoding approach, utilizing the Internal Transcribed Spacer 1 (ITS1) and part of the 28S nuclear ribosomal Large Subunit (nrLSU) rDNA regions, for the molecular identification of representative Greek macromycetes. Our analysis involved 103 Greek macromycetes covering seven genera of Basidiomycota (Agaricus, Amanita, Boletus, Cantharellus, Lactarius, Pleurotus, and Russula) and one genus of Ascomycota (Morchella). Phylogenetic inference based on the generated rDNA sequences, revealed high DNA divergence among most of the examined macromycete genera, which formed discrete monophyletic groups. Our phylogenetic analysis, in accordance with previous studies in the field, further supports the early divergence of the Cantharellus clade, followed by the subsequent split of the Russulaceae from a sister clade formed by the Agaricus, Amanita, Boletus and Pleurotus genera.
... Two barcodes based on mitochondrial DNA are available for rust fungus (Chrysomyxa ledi (Alb. and Schwein.) de Bary): one of cytochrome c oxidase subunit III (CO3) coding gene, and the second of dehydrogenase subunit 6 (nad6) gene [147]. The vast majority of sequences belong to genomic DNA and RNA sequences. ...
... Four other genes, i.e., Mcm7 (DNA replication licensing factor), tub2 (beta-tubulin), EF1-α (translation elongation factor 1-α), and RPB2 (DNA-dependent RNA polymerase second largest subunit), were sequenced less frequently. In total, 41 species are represented in the GenBank dataset [59], including 25 basidiomycetes, ten ascomycetes, and six FLOs species from the Phytophthora genus [122,130,[147][148][149][150]. The detailed information on molecular data is given as a Supplementary Material (Table S1). ...
... The detailed information on molecular data is given as a Supplementary Material (Table S1). The search in the iBOL (BOLD) [62] database retrieved only two sequences of Chrysomyxa ledi, i.e., 28S rRNA and ITS2 [147]. ...
The history of mycological research and current activities in the Polish part of the Białowieża Primeval Forest are presented. The review of literature-derived and unpublished data on species of non-lichenized fungi and protozoan and chromistan fungal analogues indicates a min-imum of 3504 species observed in this area. The gaps in the exploration of fungi: unstudied taxa, plant communities, habitats, hosts, and substrates, as well as the limitations of former studies, are discussed. Our estimates show that a total of 8000 fungal species possibly occur in the Białowieża National Park alone, and more than 10,000 are expected to be found in the Polish part of the Białowieża Primeval Forest. Despite more than a centennial history of mycological research, the majority of data come from only a few older scientific projects and several more recent citizen-sci-ence-related activities, emphasizing the need for a modern, interdisciplinary study on the diversity and ecology of fungi in this area.
