Potential Bias of Fungal 18S Rdna and Internal Transcribed Spacer Polymerase Chain Reaction Primers for Estimating Fungal Biodiversity in Soil
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Environmental Microbiology (2003) 5(1), 36–47 Potential bias of fungal 18S rDNA and internal transcribed spacer polymerase chain reaction primers for estimating fungal biodiversity in soil Ian C. Anderson,1,2* Colin D. Campbell1 and environmental samples, and it also highlights some James I. Prosser2 potential limitations of the approach with respect to 1The Macaulay Institute, Craigiebuckler, Aberdeen AB15 PCR primer specificity and bias. 8QH, UK. 2Department of Molecular and Cell Biology, University of Introduction Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK. Fungi play fundamentally important and diverse roles in terrestrial ecosystems, being involved in many of the key processes required for ecosystem functioning. They are Summary important as pathogens of plants and animals, as mycor- Four fungal 18S rDNA and internal transcribed spacer rhizal symbionts of plants (Smith and Read, 1997) and as (ITS) polymerase chain reaction (PCR) primer pairs the main agents for the decomposition of organic material. were tested for their specificity towards target fungal Fungi therefore possess the ability to control nutrient DNA in soil DNA extracts, and their ability to assess fluxes in natural ecosystems that may be aided through the diversity of fungal communities in a natural grass- extensive below-ground mycelial networks. Despite their land soil was compared. Amplified PCR products importance in terrestrial ecosystems, little is known of the were cloned, and ª 50 clones from each library were diversity of natural fungal populations. Recent estimates sequenced. Phylogenetic analysis and database suggest that 1.5 million fungal species are present in searches indicated that each of the sequenced cloned natural ecosystems, but only 5–10% have been described DNA fragments was of fungal origin for each primer formally (Hawksworth, 1991; 2001; Hawksworth and pair, with the exception of the sequences generated Rossman, 1997). The primary reason for this apparent using the 18S rDNA primers nu-SSU-0817 and nu- ignorance has been the dependence on cultivation-based SSU-1196, where 35 of the 50 sequenced clones techniques for the characterization of fungal diversity. The represented soil invertebrates. Although some of the major limitations of these techniques are the inability to primers have previously been suggested to be biased separate biomass from particulate material and a lack of towards certain fungal taxonomic groups, the ratio of growth media and cultivation conditions suitable for all sequences representing each of the four main fungal members of the community. phyla, Ascomycota, Basidiomycota, Chytridiomycota The development and application of molecular tech- and Zygomycota, was similar for each of the primer niques, particularly those based on the analysis of 16S pairs, suggesting that primer bias may be less sig- rRNA genes amplified from extracted DNA, have trans- nificant than previously thought. Collector’s curves formed studies of bacterial diversity in natural environ- were plotted to estimate the coverage obtained for ments. Similar techniques, however, have only recently each of the clone libraries after clustering the been used to characterize fungal diversity (Kowalchuk sequences into operational taxonomic units at a level et al., 1997; Kowalchuk, 1998; Smit et al., 1999; of 99% sequence similarity. The curves indicated that Borneman and Hartin, 2000; Vainio and Hantula, 2000; good coverage of diversity was achieved, with the van Elsas et al., 2000; Lowell and Klein, 2001; Möhlenhoff exception of the clone library constructed using prim- et al., 2001; Pennanen et al., 2001; Schabereiter-Gurtner ers nu-SSU-0817 and nu-SSU-1196, on account of the et al., 2001). A major challenge in applying these tech- high number of non-fungal sequences obtained. The niques to fungal communities is the design of suitable work demonstrates the usefulness of 18S rDNA and polymerase chain reaction (PCR) primers with specificity ITS PCR primers for assessing fungal diversity in for fungal DNA, while reducing co-amplification of similar Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology1462-2912Macaulay Land Use Research Institute, 20035Origi- nal ArticlePotential bias of fungal 18S rDNA and ITS primersI. C. Anderson, C. D. Campbell and J. I. Prosser target DNA from non-fungal sources. Numerous PCR Received 2 August, 2002; revised 30 September, 2002; accepted 30 primers have been described that amplify fungal rDNA September, 2002. *For correspondence at the first address. E-mail [email protected]; Tel. (+44) 1224 498 200; Fax (+44) 1224 from a wide range of taxonomic groups (White et al., 498 207. 1990), although few were designed for use with DNA © 2003 Macaulay Land Use Research Institute Potential bias of fungal 18S rDNA and ITS primers 37 extracted from mixed communities, and their lack of selec- 800 bp (nu-SSU-0817 and nu-SSU-1536); 1.4–1.5 kb tivity may lead to inaccurate estimates of fungal diversity (EF4 and EF3); 500–800 bp (ITS1-F and ITS4). PCRs when applied to complex environmental samples. Gardes were performed in both the presence and the absence of and Bruns (1993) designed ITS1-F and ITS4-B primers bovine serum albumin (BSA) for each primer pair. In each specifically to amplify fungal internal transcribed spacer case, the PCR product yield was considerably increased (ITS) regions, without the co-amplification of plant or other in the presence of BSA. eukaryotic DNA. Furthermore, several PCR primers have been designed recently to amplify partial 18S rRNA gene Identification and phylogenetic analysis of sequences from species belonging to the four major fun- nu-SSU-0817 and nu-SSU-1196 (A) clones gal phyla (Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota) from DNA extracted directly from soil A phylogenetic analysis and FASTA database search were (Smit et al., 1999; Borneman and Hartin, 2000). Although conducted for the sequences of 50 random clones from a critical factor in designing PCR primers to fungal 18S the nu-SSU-0817 and nu-SSU-1196 (A) clone library. Of rRNA genes is their specificity towards the target fungal the 50 clones sequenced, 35 were found to have 89–95% DNA, achieving that specificity may ultimately bias the sequence similarity to Crossodonthina koreana or Hypo- view that we obtain by attempting to avoid amplification of gastrura dolsana, both of which are species of inverte- non-fungal DNA. This is because some regions of fungal brates that are commonly found in soil. The only 18S rRNA gene sequences share high similarity with exceptions were clone A30, which is most closely related other eukaryotes. This has indeed been suggested to be to Rhinosporidium seeberi (89% sequence similarity), an the case with recently designed fungal 18S rDNA primer aquatic protistan parasite, and clone A1, which is most pairs EF4/EF3 and EF4/fung5 (Smit et al., 1999). There closely related to Diplolaimelloides meyli (86% sequence have also been conflicting reports about the specificity of similarity), a soil arthropod. All these sequences had newly designed fungal 18S rDNA primers (Smit et al., 93.1–100% sequence similarity to each other over the 1999; Borneman and Hartin, 2000). For example, entire sequence length, and they clustered together in the although the PCR primer pairs EF4/EF3 and EF4/fung5 phylogenetic tree (Fig. 1). The remaining 15 clones clus- were shown in one study to amplify only fungal 18S rDNA tered in a single clade of the phylogenetic tree and were sequences from wheat rhizosphere soil (Smit et al., 1999), separated from the non-fungal sequences by a strongly other investigators have shown that the same primers can supported branch (92% bootstrap support). Each of these also amplify some non-fungal template (Borneman and cloned sequences had a high sequence identity to fungal Hartin, 2000). In addition, 18S rRNA gene sequences are 18S rDNA sequences in the GenBank database (Fig. 1). generally only able to resolve taxonomic groups to the A further phylogenetic analysis was carried out using the level of genus, and the taxonomic resolution of fungal 18S 15 fungal sequences to determine more accurately the rDNA and ITS sequences is limited by the current avail- relationships between sequences within this group ability of information held within databases. The aim of the (Fig. 2). All four major fungal phyla (Ascomycota, Basidi- current study was to assess the specificity of previously omycota, Chytridiomycota and Zygomycota) were repre- published fungal 18S rDNA and ITS primers for estimating sented among the 15 fungal clones. fungal diversity in environmental samples and to use them to characterize the diversity of fungal communities in a Identification and phylogenetic analysis of natural grassland soil. This was achieved by the construc- nu-SSU-0817 and nu-SSU-1536 (B) clones tion of clone libraries from PCR products amplified using published primer sets and comparison with respect to the In contrast to the nu-SSU-0817 and nu-SSU-1196 (A) relative abundances of sequences falling within the major clone library, the FASTA results for the sequences of 48 fungal groups. clones analysed from the nu-SSU-0817 and nu-SSU-1536 (B) clone library revealed that all sequences were of fun- gal origin (Fig. 2). Thirty-two were most closely related to Results fungal basidiomycetes, whereas 15 were most closely related to species of ascomycetes. All 32 basidiomycete Amplification of fungal 18S rDNA and ITS fragments clones had 94.2–99.0% sequence similarity to basidio- PCR