scholarly journals Problems, Limitations, and Challenges in Species Identification of Ascomycota Members on the Basis of ITS Regions

2020 ◽  
Vol 55 (1) ◽  
Author(s):  
Anna Baturo-Cieśniewska ◽  
Wojciech Pusz ◽  
Katarzyna Patejuk
Keyword(s):  
Species Identification ◽  
Its Region ◽  
Fungal Species ◽  
Plant Diseases ◽  
Its Sequences ◽  
Local Alignment ◽  
Correct Identification ◽  
Rdna Its ◽  
Its Sequence Analysis ◽  
Species Specific

The internal transcribed spacer (ITS) region is regarded as a formal fungal primary barcode with a high probability of the correct identification for a broad group of fungi. ITS sequences have been widely used to determine many fungal species and analysis of rDNA ITS is still one of the most popular tools used in mycology. However, this region is not equally variable in all groups of fungi; therefore, identification may be problematic and result in ambiguous data, especially in some species-rich genera of Ascomycota. For these reasons, identification based on rDNA ITS is usually complemented by morphological observations and analysis of additional genes. Reliable species identification of Ascomycota members is essential in diagnosing plant diseases, verifying air quality and the effectiveness of agronomic practices, or analyzing relationships between microorganisms. Therefore, the present study aimed to verify, using specific examples, the extent to which ITS sequence analysis is useful in species identification of pathogens and saprobionts from Ascomycota and demonstrate problems related to such identification in practice. We analyzed 105 ITS sequences of isolates originating from air and plant material. Basic local alignment search tool (BLASTn) significantly contributed to the reliable species identification of nearly 80% of isolates such as <em>Arthrinium arundinis</em>, <em>Beauveria bassiana</em>, <em>Boeremia exigua</em>, <em>Cladosporium cladosporioides</em>, <em>Epicoccum nigrum</em>, <em>Nigrospora oryzae</em>, <em>Sclerotinia sclerotiorum</em>, or <em>Sordaria fimicola </em>and members of the genera <em>Alternaria </em>and <em>Trichoderma</em>. However, for most isolates, additional morphological observations, information regarding the isolate origin and, where possible, a PCR with species-specific primers were helpful and complementary. Using our practical approach, we determined that ITS-based species identification and comparative analysis with GenBank sequences significantly helps identifying Ascomycota members. However, in many cases, this should be regarded as suggestive of a taxon because the data usually require the use of additional tools to verify the results of such analysis.

scholarly journals Multiplex PCR Based Strategy for Detection of Fungal Pathogen DNA in Patients with Suspected Invasive Fungal Infections

2020 ◽  
Vol 6 (4) ◽  
pp. 308
Author(s):  
Joana Carvalho-Pereira ◽  
Filipa Fernandes ◽  
Ricardo Araújo ◽  
Jan Springer ◽  
Juergen Loeffler ◽  
...  
Keyword(s):  
Fungal Infections ◽  
Fungal Species ◽  
Cross Reactivity ◽  
Invasive Fungal Infections ◽  
Clinical Samples ◽  
Correct Identification ◽  
Colony Pcr ◽  
Pcr Multiplex ◽  
Pathogen Dna ◽  
Species Specific

A new and easy polymerase chain reaction (PCR) multiplex strategy, for the identification of the most common fungal species involved in invasive fungal infections (IFI) was developed in this work. Two panels with species-specific markers were designed, the Candida Panel for the identification of Candida species, and the Filamentous Fungi Panel for the identification of Aspergillus species and Rhizopusarrhizus. The method allowed the correct identification of all targeted pathogens using extracted DNA or by colony PCR, showed no cross-reactivity with nontargeted species and allowed identification of different species in mixed infections. Sensitivity reached 10 to 1 pg of DNA and was suitable for clinical samples from sterile sites, with a sensitivity of 89% and specificity of 100%. Overall, the study showed that the new method is suitable for the identification of the ten most important fungal species involved in IFI, not only from positive blood cultures but also from clinical samples from sterile sites. The method provides a unique characteristic, of seeing the peak in the specific region of the panel with the correct fluorescence dye, that aids the ruling out of unspecific amplifications. Furthermore, the panels can be further customized, selecting markers for different species and/or resistance genes.

Comparison of the ITS Sequences of 5 Common Potentilla Species in Jilin Province of China

2012 ◽  
Vol 554-556 ◽  
pp. 1690-1693 ◽  
Author(s):  
Shao Xuan Zhang ◽  
Xin Rui Liu ◽  
Bo Chuan Wang ◽  
Yun Hui Ling ◽  
De Jun Sun ◽  
...  
Keyword(s):  
Internal Transcribed Spacer ◽  
Sequence Data ◽  
Its Region ◽  
Scientific Data ◽  
Jilin Province ◽  
Its Sequences ◽  
Universal Primers ◽  
Its Sequence ◽  
Its Sequence Analysis ◽  
Pcr Products

To find the differences in the internal transcribed spacer(ITS) sequences and provide scientific data for the authentication of Potentilla chinensis and its related species, we extracted the genome DNA from the leaves of 5 common Potetilla species in Jilin Province, amplified the ITS region using ITS universal primers of angiosperm, and sequenced the purified PCR products directly. Polymorphism of ITS sequences was found within P. chinensis and the sequence data suggested that our samples of this species might be related to hybridization. Other 4 species showed intraspecies-stability in ITS sequence. The ITS sequences of these 5 Potentilla species are significantly different. So ITS sequence analysis and other methods derived from it can be used in authentication of Potentilla.

scholarly journals The UNITE Database for Molecular Identification and for Communicating Fungal Species

2019 ◽  
Vol 3 ◽  
Author(s):  
Urmas Kõljalg ◽  
Kessy Abarenkov ◽  
R. Henrik Nilsson ◽  
Karl-Henrik Larsson ◽  
Andy F.S. Taylor
Keyword(s):  
Molecular Identification ◽  
High Throughput Sequencing ◽  
Its Region ◽  
Fungal Species ◽  
Its Sequences ◽  
Current Version ◽  
Community Members ◽  
Global Biodiversity Information Facility ◽  
Unique System ◽  
Fungal Sequence

UNITE (https://unite.ut.ee; Nilsson et al. 2018) is an international community of scientists and citizen scientists established in 2001. The ambition of UNITE is to develop: 1) datasets and tools for robust and reproducible molecular identification; 2) Persistent Identifiers based system for the communicating fungal species. Datasets of the nuclear ribosomal internal transcribed spacer (ITS) region, form the basis for UNITE. The current version includes nearly 1 million public fungal ITS sequences. Datasets are curated and annotated by community members. During the past 15 years, they made more than 275 000 improvements. In the complete absence of Latin names for species, UNITE offers a unique system where species hypotheses (SH) are provided with Digital Object Identifiers (DOIs). The current version 8 of UNITE offers more than 800 000 DOI-based SHs. One such SH DOI page is shown in Fig. 1. These DOI identifiers are also incorporated into the taxonomic backbone, making communication of taxa seamless in both directions. DOI identifiers of species hypotheses are also used by GBIF (Global Biodiversity Information Facility) in order to publish high-throughput sequencing taxon occurrence data in their data portal. UNITE serves as a data provider for a range of metabarcoding software pipelines and regularly exchanges data with all major fungal sequence databases and other community resources. Recent improvements include ITS-based species hypotheses for all eukaryotes and aggregation of full-length, high-quality ITS sequences generated by the PacBio Sequel system (https://www.pacb.com/products-and-services/sequel-system) from diverse material samples.

scholarly journals Indigenous yeast with cellulose-degrading activity in napa cabbage (Brassica pekinensis L.) waste: Characterisation and species identification

2019 ◽  
pp. 321-328
Author(s):  
Gemilang L. Utama ◽  
Widia D. Lestari ◽  
Indira L. Kayaputri ◽  
Roostita L. Balia
Keyword(s):  
Species Identification ◽  
Total Population ◽  
Its Region ◽  
Local Alignment ◽  
Rrna Gene ◽  
Yeast Isolate ◽  
Degrading Enzyme ◽  
Average Activity ◽  
Waste Characterisation ◽  
Indigenous Yeast

Napa cabbage waste contains an organic component, cellulose, which can be utilised as an ingredient for cellulose-degrading enzyme production with the help of indigenous yeast. The aim of the research was to identify and characterise potential indigenous yeast isolated from napa cabbage waste, which has cellulose-degrading activity. Indigenous yeast were isolated and characterised using the RapID Yeast Plus System, then turbidity was used to determine the yeast total population. Indigenous yeast was grown at napa cabbage waste at 27, 37, and 40°C for three days, and cellulose-degrading activity was determined by the Dinitrosalicylic Acid (DNS) method. The potential yeast isolate with the highest cellulose-degrading activity was identified by a sequence analysis of the rRNA gene internal transcribed spacer (ITS) region with using primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′- TCCTCCGCTTATTGATATGC-3′). The results were compared to the GenBank database using the Basic Local Alignment Search Tools/BLAST algorithm. Three species of indigenous yeast were isolated from napa cabbage waste (S2, S6, and S8). S8, incubated at 37ºC for three days, demonstrated the highest cellulose-degrading enzyme activity (1.188 U/mL), with the average activity of 0.684U/mL. Species identification results indicated that the S8 isolate had a 100% similarity to Pichia fermentans UniFGPF2 (KT029805.1).

scholarly journals Development of a Method for Detection of the Biocontrol Agent Penicillium oxalicum Strain 212 by Combining PCR and a Selective Medium

Plant Disease ◽  
2009 ◽  
Vol 93 (9) ◽  
pp. 919-928 ◽  
Author(s):  
I. Larena ◽  
P. Melgarejo
Keyword(s):  
Its Region ◽  
Fungal Species ◽  
Penicillium Oxalicum ◽  
Selective Medium ◽  
Nucleotide Sequences ◽  
Tomato Plants ◽  
Its Regions ◽  
Specific Pair ◽  
Species Specific ◽  
Biocontrol Strain

The registration of biological control agents requires the development of monitoring systems to detect and quantify the agent in the environment. Penicillium oxalicum strain 212 (PO212) is being developed for the control of tomato pathogens. In this study, we demonstrated that PO212 was more effective for controlling Fusarium oxysporum f. sp. lycopersici in tomato plants than 13 other P. oxalicum strains. A new semiselective medium was developed as a preliminary screen for P. oxalicum from soil. This semiselective medium was a modified Fusarium selective medium that contained 0.006 g of nystatin per liter. The growth of P. oxalicum strain 212 was not inhibited on this medium, but it did inhibit the growth of 11 fungal species. Specific identification of the biocontrol strain and its quantification were achieved using a polymerase chain reaction with a strain-specific pair of primers (POITS1F/POITS2R1) and dilution plating. This primer set differentiated the biocontrol strain from 13 other strains of P. oxalicum. There were differences in the nucleotide sequences of the internal transcribed spacer (ITS) regions of the ribosomal DNA of 25 strains of P. oxalicum and those of PO212. Based on the differences in the nucleotide sequences of the ITS regions in rDNA of PO212 and other P. oxalicum strains, a relationship between the nucleotide sequences in the ITS region and biocontrol efficacy is postulated.

scholarly journals First Report of Colletotrichum acutatum Associated with Stem Blight of Blueberry Plants in China

Plant Disease ◽  
2013 ◽  
Vol 97 (3) ◽  
pp. 422-422 ◽  
Author(s):  
C.-N. Xu ◽  
Z.-S. Zhou ◽  
Y.-X. Wu ◽  
F.-M. Chi ◽  
Z.-R. Ji ◽  
...  
Keyword(s):  
Vaccinium Corymbosum ◽  
Colletotrichum Acutatum ◽  
Post Inoculation ◽  
Pathogenicity Test ◽  
Distilled Water ◽  
Conidial Suspension ◽  
First Report ◽  
Rdna Its ◽  
Its Sequence Analysis ◽  
Species Specific

An anthracnose disease was observed on stems of high-bush blueberry plants (Vaccinium corymbosum L.) in Liaoning Province, China in 2012. The typical symptoms consist of sudden wilting and dieback of stems during the growing season. Dark brown lesions originate from infected buds and kill portions of the stems. Lesions have grayish white centers, with the necrotic areas becoming 6 to 8 cm in length. Disinfected stem pieces were placed on potato dextrose agar (PDA) and incubated at 28°C for 5 to 7 days, after which the emerging colonies were transferred to fresh PDA. All isolates initially produced white growth, but turned pink after 7 days before becoming blackish green. The average colony diameter was 65.5 to 75.0 mm after 7 days. Conidia were aseptate, hyaline, fusiform to ellipsoid, 8.5 to 16.5 × 2.5 to 4.0 μm in size and single celled with two to seven oil globules. Setae were not found on the acervuli. These characteristics matched published descriptions of Colletotrichum acutatum (1) (teleomorph Glomerella acutata). Pathogenicity test was confirmed in 15 2-year-old healthy potted plants of cv. Berkeley. Stems of 10 plants were punctured with flamed needles and sprayed with 5 ml of conidial suspension (106 conidia per ml in sterile distilled water) of isolate LNSW1. Five control plants were inoculated with sterile distilled water. Seven days after inoculation, eight of the 10 blueberry plants exhibited stem lesions, leaf chlorosis, followed by branch dieback 15 days post-inoculation. The symptoms were similar to those observed on diseased plants in the field, and no lesions were observed on control plants. The pathogen was reisolated from the margin of lesions and identified by colony growth characteristics on PDA. PCR amplification of one isolate (LNSW1) was carried out by utilizing the universal rDNA-ITS primer pair ITS1/ITS4. The sequence (557 bp) of isolate LNSW1 (GenBank Accession No. JX392857) showed 99% identity to G. acutata (AB443950) and C. acutatum (AJ749672) in a BLAST search. An approximately 490-bp fragment was amplified from LNSW1 by the species-specific primer pair CaInt2/ITS4 (2). The pathogen was identified as G. acutata (asexual stage: C. acutatum J.H. Simmonds) on the basis of morphological characters, rDNA-ITS sequence analysis, and a PCR product with species-specific primers. To our knowledge, this is the first report of C. acutatum in high-bush blueberry plants in China. References: (1) C. Lei et al. Fungal Diversity 12:183, 2009. (2) S. Sreenivasaprasad et al. Plant Pathol. 45:650, 1996

scholarly journals rDNA based analysis of autochtonous fungal species from Serbia

Genetika ◽  
2014 ◽  
Vol 46 (1) ◽  
pp. 33-42
Author(s):  
Eleonora Capelja ◽  
Nevena Stevic ◽  
Vladislava Galovic ◽  
Milana Novakovic ◽  
Maja Karaman
Keyword(s):  
Phylogenetic Tree ◽  
Molecular Data ◽  
Fungal Species ◽  
Its Sequences ◽  
Local Alignment ◽  
Phylogenetic Species Concept ◽  
Morphological Approach ◽  
Search Tool ◽  
Morphological And Molecular Data

Determination of fungal species by traditional morphological approach can often be problematic. In the phylum Basidiomycota, sporocarps of different species can share very similar morphoanatomical characteristics. Using molecular markers and phylogenetic species concept this problem can be reduced. In this study identification of six autochtonous fungal species, collected from several locations in Serbia (Tara, Kopaonik, Stara planina) was done by comparison between morphological and molecular data of fungal species, as well as information obtained from phylogenetic tree. ITS sequences amplified from 11 specimens of two genera of ph. Basidiomycota: Marasmius and Ganoderma, were compared with ITS sequences from database using basic local alignment search tool (BLAST). Phylogenetic tree was constructed using Neighbor joining method based on differences between analyzed ITS sequences. Our results showed that within genera Marasmius and Ganoderma morphological and molecular determinations are usually in accordance, but for proper species delimitation both approaches should be used.

scholarly journals First Report of Leaf Spot Caused by Alternaria alternata on Chinese Dwarf Banana in China

Plant Disease ◽  
2014 ◽  
Vol 98 (5) ◽  
pp. 691-691 ◽  
Author(s):  
B. Z. Fu ◽  
Z. H. Zhang ◽  
L. H. Wang ◽  
G. Y. Li ◽  
J. Z. Zhang ◽  
...  
Keyword(s):  
Alternaria Alternata ◽  
Leaf Spot ◽  
Morphological Characteristics ◽  
Pcr Amplification ◽  
Its Region ◽  
Leaf Spot Disease ◽  
First Report ◽  
Spot Disease ◽  
Rdna Its ◽  
Its Sequence Analysis

The Chinese dwarf banana (Ensete lasiocarpum) is one of the ornamental bananas that belongs to Musaceae family. The plant is native to the southwestern China, where it grows semi-wild in the mountains between 1,500 and 2,500 m above sea level. During July 2011, a leaf spot disease on this plant was observed in the campus and parks in Kunming, Yunnan Province. The incidence level was about 22%, mainly on the old leaves. The leaf symptoms were irregular spots with gray to off-white centers surrounded by dark brown margins, and usually also surrounded by chlorotic halos. Leaf tissues (3 × 5 mm), cut from the margins of lesions, were surface-disinfected (95% ethanol for 3 min, 0.1% HgCl2 for 2 min, rinsed three times with sterile water), plated on potato sucrose agar (PSA), and incubated at 26°C under natural lights. The same fungus was consistently isolated from the diseased leaves. Colonies of white-to-dark gray mycelia formed on PSA that were black on the underside. The colonies were further identified as Alternaria sp. based on the dark brown, obclavate to obpyriform catenulate conidia with longitudinal and transverse septa tapering to a prominent beak attached in chains on a simple and short conidiophore (2). Conidia were 5.26 to 30.26 μm long and 3.95 to 15.79 μm wide, averaging 10.21 (±3.17) × 20.02 (±5.75) μm (n = 50), with a beak length of 0 to 7.89 μm, and had 3 to 8 transverse and 0 to 3 longitudinal septa. PCR amplification was carried out by utilizing universal rDNA-ITS primer pair ITS4/ITS5 (1). The ITS region of isolate DY1 (GenBank Accession No. KF516556) was 572 bp in length. BLAST search revealed 99% identity with two Alternaria alternata isolates (JF440581.1 and GQ121322.2). Phylogenetic analysis (MEGA 5.1) using the neighbor-joining algorithm placed the isolate in a well-supported cluster with other A. alternata isolates. The pathogen was identified as A. alternate (Fr.:Fr.) Keissler based on the morphological characteristics and rDNA-ITS sequence analysis. To confirm pathogenicity, Koch's postulates were performed on detached leaves of E. lasiocarpum inoculated with mycelial plugs with ddH2O and agar plugs as a control. Leaf spots identical to those observed in the field developed in 9 days on the inoculated leaves but not on the control. The inoculation assay used three leaves, totaling 72 spots for control and 36 spots for inoculation. The experiments were repeated once. A. alternata was consistently re-isolated from the inoculated leaves. The symptom developed easier with wounds. To our knowledge, this is the first report of E. lasiocarpum leaf spot disease caused by A. alternata in China and the world. References: (1) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990. (2) T. Y. Zhang. Flora Fungorum Sinicorum, Vol. 16: Alternaria. Science Press, Beijing, China, 2003.

scholarly journals Phylogenetic analysis of Phoma species

2007 ◽  
pp. 100-107
Author(s):  
László Irinyi ◽  
György Kövics ◽  
Erzsébet Sándor
Keyword(s):  
Molecular Markers ◽  
Phylogenetic Relationships ◽  
Phylogenetic Trees ◽  
Elongation Factor ◽  
Its Region ◽  
Morphological Characterization ◽  
Fungal Species ◽  
Its Sequences ◽  
Translation Elongation ◽  
5.8S Rdna

The cosmopolitan Phoma genus contains mainly phytopathogenic, opportunistic parasites, and saprophyte fungal species. Up to now, the characterization of Phoma species and other taxa of Phoma has been determined on the basis of morphology on standardized media, and gene sequence analysis was only used as a confirmative or distinctive complement.In this study, we tried to find molecular markers which can be used as phylogenetics markers in the molecular based classification in the Phoma genus.We employed a part of the translation elongation factor 1 subunit alpha (EF-1α=tef1) containing both introns and exons and ITS region containing the internal transcribed spacer regions 1 and 2 and the 5.8S rDNA, as potential genetic markers to infer phylogenetic relationships among different Phoma taxa. Twelve different Phoma species sequences were analysed together with the closely related Ascochyta ones. The constructed phylogenetic trees, based on tef1 and ITS sequences, do not support the traditional Phoma sections based on morphological characterization. However, we managed to distinguish between the Phoma strains and Ascochyta species by comparing their tef1 sequences through parsimony analysis. We proved that a tef1 can be a useful phylogenetic marker to resolve phylogenetic relationships at species level in Phoma genus.Both parsimony sequence analyses confirmed that the Phyllosticta sojicola species is identical to the Phoma exigua var. exigua species as Kövics et al. (1999) claimed. However, the evolutionary distance by ITS sequences within Phoma species is too small to get well based consequences for the phylogenetic relationships of Phoma genus.Further investigations would be necessary to clarify whether the tef1 and ITS sequences as phylogenetic molecular markers are well suited for the classification of Phoma species.

scholarly journals IDENTIFICATION OF SUNFLOWER PATHOGENIC FUNGUS PLENODOMUS LINDQUISTII USING PCR WITH SPECIES-SPECIFIC OLIGONUCLEOTIDE PRIMERS

2020 ◽  
Vol 103 (3) ◽  
pp. 207-210
Author(s):  
М. М. Gomzhina ◽  
Ph. B. Gannibal
Keyword(s):  
Causative Agent ◽  
Pathogenic Fungus ◽  
Its Region ◽  
Fungal Species ◽  
Cross Reaction ◽  
Pcr Assay ◽  
Selective Amplification ◽  
Oligonucleotide Primers ◽  
Pure Cultures ◽  
Species Specific

Plenodomus lindquistii causes Phoma black stem of sunflower which is the most common stem disease of this crop in Russia. The diagnostics of both field specimens and pure cultures of P. lindquistii is troublesome. Molecular methods involving the use of the PCR are rapid diagnostic express tests that can precisely identify and detect fungal species. The aim of this study was to develop species-specific oligonucleotide primers for selective amplification of P. lindquistii DNA. The primers LepliF2/LepliR2 were designed on the basis of ITS region analysis and showed stable amplification of the target fungus DNA with no cross-reaction with other fungal species. The primers are recommended for express detection of the causative agent of Phoma black stem of sunflower. This is the first PCR assay that could be used to rapidly reveal and identify this pathogen.

Sign in / Sign up

Export Citation Format

Share Document