BioMed Central Page 1 of 11 (page number not for citation purposes) BMC Microbiology Open Access Methodology article Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts Kendall J Martin*1 and Paul T Rygiewicz2 Address: 1Dynamac Corporation, National Health and Environmental Effects Research Laboratory, Corvallis, OR USA and 2USEPA National Health and Environmental Effects Research Laboratory, Corvallis, OR, USA Email: Kendall J Martin* - kendall@lifetime.oregonstate.edu Paul T Rygiewicz - rygiewicz.paul@epa.gov * Corresponding author PCRprimersbasidiomycetesascomycetesInternal Transcribed Spacer (ITS)X/T rapid extraction method Abstract Background: The 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. Results: Numerous 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. Conclusion: These 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. Published: 18 May 2005 BMC Microbiology 2005, 5:28 doi:10.1186/1471-2180-5-28 Received: 30 November 2004 Accepted: 18 May 2005 This article is available from: http://www.biomedcentral.com/1471-2180/5/28 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

BMC Microbiology 2005, 5:28 http://www.biomedcentral.com/1471-2180/5/28 Page 2 of 11 (page number not for citation purposes) Background Studies of fungi (Kingdom Eumycota) in natural environ- ments often require simultaneous analysis of a broad tax- onomic range. For instance, the fungi forming ectomycorrhizal symbioses number over 5000 species [1]. Analysis of natural ectomycorrhizal fungal communities traditionally has been a laborious, highly-skilled process with heavy reliance on gross morphological characteriza- tion of the ectomycorrhizal root-tips. The laborious nature of microscopic analysis and identification is the driving force behind developing such methods as molec- ular verification of identified morphotypes by means of PCR-RFLP [2]. Analysis of natural populations of ectomy- corrhizas requires high rates of sampling because of the high number of fungal species involved and the high spa- tial variability observed in natural systems [3,4]. Primers allowing simultaneous analysis of all the fungal phyla which are involved in ectomycorrhizal symbioses would be a useful tool in studying the ecology of these fungi. Of particular interest for such work is the division Dikaryo- mycota, which includes the subdivisions Basidiomycotina and Ascomycotina, and encompasses all ectomycorrhizal fungi [1]. The earliest PCR primers to gain wide acceptance for work with fungal Internal Transcribe Sequences (ITS) were "ITS1" and "ITS4" which amplify the highly variable ITS1 and ITS2 sequences surrounding the 5.8S-coding sequence and situated between the Small SubUnit-coding sequence (SSU) and the Large SubUnit-coding sequence (LSU) of the ribosomal operon [5]. These primers amplify a wide range of fungal targets and work well to analyze DNA isolated from individual organisms, but do not exclude effectively the plant host sequences in mixed, phytosphere DNA extracts typical of studies of plant-asso- ciated microbiota. Subsequently, the plant-excluding primers ITS1-F and ITS4-B came into wide use for analyses of fungal ITS sequences, but these primers were "intended to be specific to fungi and basidiomycetes, respectively" [6]. We present here a suite of primers designed to amplify Dikaryomycota efficiently, requiring little optimization for use in the hands of even relatively untrained operators. In addition to the increased efficiency needed for well- established methods such as PCR-RFLP analyses of single fungal species [7], robust primers are needed for newer molecular methods currently used to characterize micro- bial communities: Length Heterogeneity PCR (LH-PCR) [8], high-throughput sequencing [9], and Terminal Restriction Fragment Length Polymorphism (T-RFLP) [10]. LH-PCR is a reliable and effective approach to ana- lyze targets with high variability in overall length. In T- RFLP analyses, as with LH-PCR, a fluorescent label on the PCR primer is used for detection, but in this case only the primer-terminal fragments of restriction digested PCR products are detected. These fragments contain the labelled primer and extend to the first instance of a restric- tion site for the enzyme used. With increased access to capillary-electrophoresis instruments capable of high res- olution discrimination of oligonucleotide lengths, these methods have become both rapid and reliable. Such methods enable rapid analysis of environmental samples and can provide extensive data on microbial communities as defined or restricted by the specificity range of primers used. These data include both relative abundances of dominant microbial phylotypes and characteristic PCR- product or TRF (terminal restriction fragment) sizes for these phylotypes. For comparison of fungal communities, either method provides a relatively complete, culture- independent analysis. Additionally, where the effort is jus- tified, identified phylotypes subsequently can be taxo- nomically characterized (by applying sequence analysis to amplified targets) with the advantage that resources can be focused on phylotypes that are most important to the study at hand. Terminal restriction fragment sizes also can be compared to a database of theoretical restriction frag- ments derived from sequence information to approximate taxonomic identity [11,12]. While LH-PCR or T-RFLP gives an estimate of relative abundance of phylotypes in a community, quantitative PCR (QPCR) gives an overall quantification for the target sequence which can then be subdivided mathematically to provide an estimate of absolute population level for individual phylotypes. Higuchi et al. [13,14] developed a method for real-time detection of PCR products to quan- tify sample target sequences based on fluorescence of the intercalating dye ethidium bromide, and numerous com- panies now offer kits for such quantification using SYBR Green I (Invitrogen Life Technologies, Carlsbad, CA) as the intercalating dye. Most of these instruments now include the ability to perform a melting curve analysis on the PCR product after quantification [15], which partially ameliorates uncertainty arising from the inability of the dye to distinguish target amplicons from non-target PCR products. We describe a set of primers which performs well in all of these analytical approaches. This characteristic of wide applicability to studies of Dikaryomycota from diverse environmental samples, particularly those with signifi- cant plant tissue content, lends a degree of interoperabil- ity between analytical approaches. For instance, real-time PCR analysis of samples can guide the choice of cycle numbers for LH-PCR or T-RFLP reactions which are sensi- tive to late stage PCR. This is important for comparing data between more established approaches and newer methods coming into popular use.