Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts

By K. Martin, P. Rygiewicz. BMC Microbiology, 2005

716 citations recorded by the source corpus.

BackgroundThe Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) are highly variable sequences of great importance in distinguishing fungal species by PCR analysis. Previously published PCR primers available for amplifying these sequences from environmental samples provide varying degrees of success at discriminating against plant DNA while maintaining a broad range of compatibility. Typically, it has been necessary to use multiple primer sets to accommodate the range of fungi under study, potentially creating artificial distinctions for fungal sequences that amplify with more than one primer set.ResultsNumerous sequences for PCR primers were tested to develop PCR assays with a wide range of fungal compatibility and high discrimination from plant DNA. A nested set of 4 primers was developed that reflected these criteria and performed well amplifying ITS regions of fungal rDNA. Primers in the 5.8S sequence were also developed that would permit separate amplifications of ITS1 and ITS2. A range of basidiomycete fruiting bodies and ascomycete cultures were analyzed with the nested set of primers and Restriction Fragment Length Polymorphism (RFLP) fingerprinting to demonstrate the specificity of the assay. Single ectomycorrhizal root tips were similarly analyzed. These primers have also been successfully applied to Quantitative PCR (QPCR), Length Heterogeneity PCR (LH-PCR) and Terminal Restriction Fragment Length Polymorphism (T-RFLP) analyses of fungi. A set of wide-range plant-specific primers were developed at positions corresponding to one pair of the fungal primers. These were used to verify that the host plant DNA was not being amplified with the fungal primers.ConclusionThese plant primers have been successfully applied to PCR-RFLP analyses of forest plant tissues from above- and below-ground samples and work well at distinguishing a selection of plants to the species level. The complete set of primers was developed with an emphasis on discrimination between plant and fungal sequences and should be particularly useful for studies of fungi where samples also contain high levels of background plant DNA, such as verifying ectomycorrhizal morphotypes or characterizing phylosphere communities.

Subfields: Ecology, Evolution, Behavior and Systematics, Insect Science, Plant Science

View Paper: https://www.semanticscholar.org/paper/28552f788382dd5dc2fe0f173f2f0bde86ee7da9

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Citations in the corpus

Sequences, the environment and fungi (2006, background): “Phyla-favouring primers have been designed for the ITS region allowing selective amplification from species of the Basidiomycota or Ascomycota (Gardes & Bruns 1993; Larena et al. 1999).”
Effects of clearcut logging and tree species composition on the diversity and community composition of epigeous fruit bodies formed by ectomycorrhizal fungi (2006, neutral): “and Bruns 1993) and NLB4 (Kendall and Rygiewicz 2005).”
Plant compartment and biogeography affect microbiome composition in cultivated and native Agave species (2015, background): “This apparent lack of Glomeromycota may in part be attributable to primer bias, as it is well known that ‘universal fungal’ primers often fail to amplify these basal organisms (Martin & Rygiewicz, 2005).”
Biochar enhances seedling growth and alters root symbioses and properties of sub-boreal forest soils (2012, neutral): “1990) and the fungalspecific primer NLB4 (Martin and Rygiewicz 2005).”
ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases (2010, methodology): “We are currently performing further bioinformatics analyses using the tool 'ecoPrimer' (http://www.grenoble.prabi.fr/trac/ecoPrimers; Riaz et al. unpublished) to identify the most appropriate barcoding primers within the ITS region and other regions, with the intent of determining whether new ITS pr”
High-Coverage ITS Primers for the DNA-Based Identification of Ascomycetes and Basidiomycetes in Environmental Samples (2012, methodology): “The coverage of the designed ITS primers was evaluated and compared with those of existing fungal ITS primers developed in previous studies [40–43] based on in silico PCR using the ecoPCR v0.”
Botryosphaeriaceae as endophytes and latent pathogens of woody plants: diversity, ecology and impact (2007, background): “…molecular and phylogenetic tools have been developed for in vivo detection of DNA of specific species or representing communities of organisms (as examples, see Higgins et al. 2007 and Martin & Rygiewicz 2005 for fungi in general, and Moon et al. 1999 for specific groups of grass endophytes).”
Biodiversity of fungal endophyte communities inhabiting switchgrass (Panicum virgatum L.) growing in the native tallgrass prairie of northern Oklahoma (2011, methodology): “These primer sets have been used widely (Gardes and Bruns 1993; Martin and Rygiewicz 2005) and are thus well represented in the NCBI nucleotide database.”