Utilizing ITS1 and ITS2 to study environmental fungal diversity using pyrosequencing

Monard, Cécile; Gantner, Stephan; Stenlid, Jan

doi:10.1111/1574-6941.12046

Cited by 78 publications

(70 citation statements)

References 35 publications

(58 reference statements)

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“…The community identified by the three tested primer pairs still differed significantly, however. These differences confirm the finding that targeting either the ITS1 or the ITS2 region may result in different pictures of the fungal communities at the OTU level, as was previously assessed by both in silico [43] and sequencing studies [40], [44]. In addition, it was found that primers targeting the same ITS region do not necessarily result in the same OTU composition (Fig.…”

Section: Discussionsupporting

confidence: 89%

Comparison and Validation of Some ITS Primer Pairs Useful for Fungal Metabarcoding Studies

Beeck

Lievens²,

Busschaert³

et al. 2014

PLoS ONE

360

214

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Current metabarcoding studies aiming to characterize microbial communities generally rely on the amplification and sequencing of relatively short DNA regions. For fungi, the internal transcribed spacer (ITS) region in the ribosomal RNA (rRNA) operon has been accepted as the formal fungal barcode. Despite an increasing number of fungal metabarcoding studies, the amplification efficiency of primers is generally not tested prior to their application in metabarcoding studies. Some of the challenges that metabarcoding primers should overcome efficiently are the amplification of target DNA strands in samples rich in non-target DNA and environmental pollutants, such as humic acids, that may have been co-extracted with DNA. In the current study, three selected primer pairs were tested for their suitability as fungal metabarcoding primers. The selected primer pairs include two primer pairs that have been frequently used in fungal metabarcoding studies (ITS1F/ITS2 and ITS3/ITS4) and a primer pair (ITS86F/ITS4) that has been shown to efficiently amplify the ITS2 region of a broad range of fungal taxa in environmental soil samples. The selected primer pairs were evaluated in a 454 amplicon pyrosequencing experiment, real-time PCR (qPCR) experiments and in silico analyses. Results indicate that experimental evaluation of primers provides valuable information that could aid in the selection of suitable primers for fungal metabarcoding studies. Furthermore, we show that the ITS86F/ITS4 primer pair outperforms other primer pairs tested in terms of in silico primer efficiency, PCR efficiency, coverage, number of reads and number of species-level operational taxonomic units (OTUs) obtained. These traits push the ITS86F/ITS4 primer pair forward as highly suitable for studying fungal diversity and community structures using DNA metabarcoding.

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Section: Discussionsupporting

confidence: 89%

Comparison and Validation of Some ITS Primer Pairs Useful for Fungal Metabarcoding Studies

Beeck

Lievens²,

Busschaert³

et al. 2014

PLoS ONE

360

214

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“…Although ITS has been widely used in fungal systematics to delimit species and to understand evolutionary relationships, there are several known issues with the effectiveness of this region including the overestimating and underestimating fungal diversity (Schoch et al 2012. On average the variability of the ITS1 exceeds that of ITS2, while the 5.8S fragment embedded between these two regions is highly conserved, and results of phylogenetic analysis of the complete sequence may differ from the analysis of the individual sub-loci (Nilsson et al 2008;Monard et al 2013).…”

Section: Discussionmentioning

confidence: 99%

Insights into the genus Diaporthe: phylogenetic species delimitation in the D. eres species complex

et al. 2014

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The genus Diaporthe comprises pathogenic, endophytic and saprobic species with both temperate and tropical distributions. Cryptic diversification, phenotypic plasticity and extensive host associations have long complicated accurate identifications of species in this genus. The delimitation of the generic type species Diaporthe eres has been uncertain due to the lack of ex-type cultures. Species limits of D. eres and closely related species were evaluated using molecular phylogenetic analysis of eight genes including nuclear ribosomal internal transcribed spacer (ITS), partial sequences of actin (ACT), DNA-lyase (Apn2), translation elongation factor 1-α (EF1-α), beta-tubulin (TUB), calmodulin (CAL), 60s ribosomal protein L37 (FG1093) and histone-3 (HIS). The occurrence of sequence heterogeneity of ITS within D. eres is observed, which complicates the analysis and may lead to overestimation of the species diversity. The strict criteria of Genealogical Concordance Phylogenetic Species Recognition (GCPSR) were applied to resolve species boundaries based on individual and combined analyses of other seven genes except the ITS. We accept nine distinct phylogenetic species including helicis and D. pulla. Modern descriptions and illustrations are provided for these species. Newly designed primers are introduced to amplify and sequence the Apn2 (DNA-lyase) gene in Diaporthe. Based on phylogenetic informativeness profiles, EF1-α, Apn2 and HIS genes are recognised as the best markers for defining species in the D. eres complex.

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“…Therefore, next generation sequencing (NGS) has also been employed to characterize the diversity of the microbial community in our aerosol samples. The NGS method has previously been used on environmental samples (Bartram et al, 2011;Monard et al, 2013) and house dust samples (Konya et al, 2014). For the identification of culturable microorganisms the matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) method is used.…”

Section: Introductionmentioning

confidence: 99%

Microbial diversity in bioaerosol samples causing ODTS compared to reference bioaerosol samples as measured using Illumina sequencing and MALDI-TOF

Madsen

Zervas

Tendal

et al. 2015

Environmental Research

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The importance of the microbial diversity of bioaerosols in relation to occupational exposure and work related health symptoms is not known. The aim of this paper is to gain knowledge on the bacterial and fungal communities in dust causing organic dust toxic syndrome (ODTS) and in reference dust not causing ODTS. Bacterial and fungal communities were described in personal exposure samples from grass seed workers developing ODTS, in dust generated from grass seeds causing ODTS and in dust generated from reference seeds not causing ODTS. Amplicon sequencing of the bacterial 16S rRNA gene and the fungal ITS region, as well as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) were used for identification of fungi and bacteria in personal exposure samples and in dust samples from grass seeds causing ODTS and in dust from reference grass seeds. Furthermore, activities of enzymes were measured in the same samples. The sequencing data revealed more than 150 bacterial and 25 fungal genera present in each sample. Streptomyces spp., Aspergillus fumigatus and Rhizopus microsporus were dominating in the dust causing ODTS but not in the reference dust. The dustiness in terms of Mucor sp. and R. microsporus were 100-1000 times higher for problematic seeds compared to reference seeds. The bacterial species in the dust causing ODTS included pathogenic species such as Klebsiella pneumonia and Streptomyces pneumonia, and it contained increased concentrations of total protein, serine protease, chitinase, and β-glucosidase. Twenty-three bacterial genera covered more than 50% of the total reads in the personal and problematic seed dust. These 23 genera accounted for less than 7% of the total reads in the reference seed dust. The microbial community of the dust from the problematic seeds showed great similarities to that from the personal air samples from the workers. In conclusion, we have shown for the first time a shift in the microbial community in aerosol samples that caused ODTS compared to the reference samples that did not cause the ODTS. Furthermore, elevated enzyme activities were found in the dust causing ODTS.

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Utilizing ITS1 and ITS2 to study environmental fungal diversity using pyrosequencing

Cited by 78 publications

References 35 publications

Comparison and Validation of Some ITS Primer Pairs Useful for Fungal Metabarcoding Studies

Comparison and Validation of Some ITS Primer Pairs Useful for Fungal Metabarcoding Studies

Insights into the genus Diaporthe: phylogenetic species delimitation in the D. eres species complex

Microbial diversity in bioaerosol samples causing ODTS compared to reference bioaerosol samples as measured using Illumina sequencing and MALDI-TOF

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