Design of a primer for ribosomal DNA internal transcribed spacer with enhanced specificity for ascomycetes

Larena, Inmaculada; Salazar, Óscar; García, Vicente González; Julián, Marı́a C.; Rubio, Víctor J.

doi:10.1016/s0168-1656(99)00154-6

Cited by 248 publications

(137 citation statements)

References 17 publications

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“…The primers ITS1 and ITS4 (White et al 1990) or ITS4A and ITS5 (Larena et al 1999) were used to amplify and sequence the ITS region. The largest subunit of RNA polymerase II (RPB1) was amplified and sequenced with RPB1-Af and RPB1-6Rlasc (Stiller andHall 1997, Hofstetter et al 2007).…”

Section: Methodsmentioning

confidence: 99%

Sexual and asexual states of some endophyticPhialocephalaspecies ofPicea

2016

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Unidentified DNA sequences in isolationbased or culture-free studies of conifer endophytes are a persistent problem that requires a field approach to resolve. An investigation of foliar endophytes of Picea glauca, P. mariana, P. rubens and Pinus strobus in eastern Canada, using a combined field, morphological, cultural and DNA sequencing approach, resulted in the frequent isolation of Phialocephala spp. and the first verified discovery of their mollisia-like sexual states in the field. Phialocephala scopiformis and Ph. piceae were the most frequent species isolated as endophytes from healthy conifer needles. Corresponding Mollisia or mollisioid sexual states for Ph. scopiformis, Ph. piceae and several undescribed species in a clade containing Ph. dimorphospora were collected in the sampling area and characterized by analysis of the nuc internal transcribed spacer rDNA (ITS) and gene for the largest subunit of RNA polymerase II (RPB1) loci. Four novel species and one new combination in a clade containing Ph. dimorphospora, the type of Phialocephala, are presented, accompanied by descriptions of apothecia and previously undocumented synanamorphs. An epitype culture and corresponding reference sequences for Phialocephala dimorphospora are proposed. The resulting ITS barcodes linked with robust taxonomic species concepts are an important resource for future research on forest ecosystems and endophytes.

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Section: Methodsmentioning

confidence: 99%

Sexual and asexual states of some endophyticPhialocephalaspecies ofPicea

2016

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“…Ascomycete isolates were PCR-amplified with the primers ITS 1-F (5′ CTT GGT CAT TTA GAG GAA GTA A 3′; Gardes and Bruns 1993) and ITS 4-A (5′ CGC CGT TAC TGG GGC AAT CCC TG 3′; Larena et al 1999). PCR conditions were as follows: initial denaturation at 95°C for 3 min, 35 cycles of denaturation at 95°C for 1 min, annealing at 52°C for 30 s, extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min (Walker and Campbell 2010) in a Thermo Electron Px2 thermocycler (Thermo Electron Corp., Waltham, MA, USA) and a Peltier Thermal Cycler PTC-100 (Bio-Rad Laboratories, Hercules, CA, USA).…”

Section: Molecular Identification Of Fungal Isolatesmentioning

confidence: 99%

“…ITS rDNA was PCRamplified in triplicate reactions for each sample using the fungal primers ITS 1-F (Gardes and Bruns 1993) and ITS 4-A (Larena et al 1999) -as most fungi reported from marine environments belong to the phylum Ascomycota (Shearer et al 2007) -with ITS 1-F labelled on the 5′ end with the fluorescent dye FAM (6-carboxyfluorescein) and PCR conditions as previously stated. All PCR products were combined and purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA, USA).…”

Section: Optimization Of Dna Extraction From Biofilms In Preparationmentioning

confidence: 99%

Fungal diversity of marine biofilms on artificial reefs in the north-central Gulf of Mexico

2016

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Abstract:We present the first characterization of fungal community diversity of natural mixed-species biofilms on artificial marine reefs. Four artificial reefs in the Mississippi (MS) Sound, USA, representing low-profile (underwater) and high-profile (periodically air-exposed) conditions were sampled every 3 months over a 23-month period to investigate changes in fungal diversity within reef biofilms. Fungal presence was assessed via PCR amplification of the internal transcribed spacer (ITS) region of fungal ribosomal DNA, and by terminal restriction fragment length polymorphism (T-RFLP) analysis of fungal ITS regions -the latter being used to track variation in fungal community structure with respect to season, location, and reef profile type. Fungal communities were also characterized taxonomically through both morphological identification and phylogenetic comparisons of ITS gene sequences, with 36 fungal genera cultured from reef biofilms. Using a multivariate statistical approach, significant temporal and spatial differences in fungal biofilm communities were detected. High-profile reefs differed significantly in biofilm fungal community composition across the 10 sampling periods. This assessment of marine fungal biofilm communities over time provides novel insights into the fungal diversity present on artificial reefs in an understudied region, the north-central Gulf of Mexico.

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“…Samples with d a o2.1 mm were not included for the analyses owing to no or weak PCR amplification. Universal fungal primers ITS1F and ITS4 (Larena et al, 1999;Manter and Vivanco, 2007) along with adaptor, key and multiplex identifier sequences were used to amplify the ITS region of fungal rDNA (Supplementary Table 2 DNA sequence processing and analyses Sequence FASTA and QUAL files were extracted from the machine output file, trimmed and parsed by sequence tag using the Ribosomal Database Project pyrosequencing pipeline (Cole et al, 2005). Trimming removed primers, sequences with one or more undefined base, sequences below a minimum machine quality score of 20 and sequences with a trimmed read length of fewer than 300 bp.…”

Section: Dna Extractionmentioning

confidence: 99%

Particle-size distributions and seasonal diversity of allergenic and pathogenic fungi in outdoor air

et al. 2012

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Fungi are ubiquitous in outdoor air, and their concentration, aerodynamic diameters and taxonomic composition have potentially important implications for human health. Although exposure to fungal allergens is considered a strong risk factor for asthma prevalence and severity, limitations in tracking fungal diversity in air have thus far prevented a clear understanding of their human pathogenic properties. This study used a cascade impactor for sampling, and quantitative real-time PCR plus 454 pyrosequencing for analysis to investigate seasonal, size-resolved fungal communities in outdoor air in an urban setting in the northeastern United States. From the 20 libraries produced with an average of ∼800 internal transcribed spacer (ITS) sequences (total 15 326 reads), 12 864 and 11 280 sequences were determined to the genus and species levels, respectively, and 558 different genera and 1172 different species were identified, including allergens and infectious pathogens. These analyses revealed strong relationships between fungal aerodynamic diameters and features of taxonomic compositions. The relative abundance of airborne allergenic fungi ranged from 2.8% to 10.7% of total airborne fungal taxa, peaked in the fall, and increased with increasing aerodynamic diameter. Fungi that can cause invasive fungal infections peaked in the spring, comprised 0.1–1.6% of fungal taxa and typically increased in relative abundance with decreasing aerodynamic diameter. Atmospheric fungal ecology is a strong function of aerodynamic diameter, whereby through physical processes, the size influences the diversity of airborne fungi that deposit in human airways and the efficiencies with which specific groups of fungi partition from outdoor air to indoor environments.

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Design of a primer for ribosomal DNA internal transcribed spacer with enhanced specificity for ascomycetes

Cited by 248 publications

References 17 publications

Sexual and asexual states of some endophyticPhialocephalaspecies ofPicea

Sexual and asexual states of some endophyticPhialocephalaspecies ofPicea

Fungal diversity of marine biofilms on artificial reefs in the north-central Gulf of Mexico

Particle-size distributions and seasonal diversity of allergenic and pathogenic fungi in outdoor air

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