Analysis of Fungal Diversity in the Wheat Rhizosphere by Sequencing of Cloned PCR-Amplified Genes Encoding 18S rRNA and Temperature Gradient Gel Electrophoresis

Abstract

Like bacteria, fungi play an important role in the soil ecosystem. As only a small fraction of the fungi present in soil can be cultured, conventional microbiological techniques yield only limited information on the composition and dynamics of fungal communities in soil. DNA-based methods do not depend on the culturability of microorganisms, and therefore they offer an attractive alternative for the study of complex fungal community structures. For this purpose, we designed various PCR primers that allow the specific amplification of fungal 18S-ribosomal-DNA (rDNA) sequences, even in the presence of nonfungal 18S rDNA. DNA was extracted from the wheat rhizosphere, and 18S rDNA gene banks were constructed in Escherichia coli by cloning PCR products generated with primer pairs EF4-EF3 (1.4 kb) and EF4-fung5 (0.5 kb). Fragments of 0.5 kb from the cloned inserts were sequenced and compared to known rDNA sequences. Sequences from all major fungal taxa were amplified by using both primer pairs. As predicted by computer analysis, primer pair EF4-EF3 appeared slightly biased to amplify Basidiomycota and Zygomycota, whereas EF4-fung5 amplified mainly Ascomycota. The 61 clones that were sequenced matched the sequences of 24 different species in the Ribosomal Database Project (RDP) database. Similarity values ranged from 0.676 to 1. Temperature gradient gel electrophoresis (TGGE) analysis of the fungal community in the wheat rhizosphere of a microcosm experiment was carried out after amplification of total DNA with both primer pairs. This resulted in reproducible, distinctive fingerprints, confirming the difference in amplification specificity. Clear banding patterns were obtained with soil and rhizosphere samples by using both primer sets in combination. By comparing the electrophoretic mobility of community fingerprint bands to that of the bands obtained with separate clones, some could be tentatively identified. While 18S-rDNA sequences do not always provide the taxonomic resolution to identify fungal species and strains, they do provide information on the diversity and dynamics of groups of related species in environmental samples with sufficient resolution to produce discrete bands which can be separated by TGGE. This combination of 18S-rDNA PCR amplification and TGGE community analysis should allow study of the diversity, composition, and dynamics of the fungal community in bulk soil and in the rhizosphere.

Diversity of fungi in organic soils under a moorland – Scots pine (Pinus sylvestris L.) gradient

“…This compares with cultivation-based estimates by Dickinson and Dooley (1967) for uncut bogs in which 77% of isolated fungi were identified as ascomycetes. Although ITS-DGGE data may actually reflect the relative abundance of ascomycete and basidiomycete taxa present in the transect soil samples, the possibility that DNA extraction and PCR conditions might introduce bias cannot be ignored (Smit et al ., 1999). The fungal ITS region was targeted because it is taxonomically more informative than other genomic regions (e.g.…”

“…These approaches have been facilitated by the design of fungal-specific 18S rDNA (Smit et al ., 1999;Borneman and Hartin, 2000) and internal transcribed spacer (ITS) (White et al ., 1990;Gardes and Bruns, 1993) primers for the direct amplification of fungal DNA from environmental DNA extracts and subsequent identification in some cases by DNA sequencing. However, some concerns have been raised regarding potential bias of some primers to preferentially amplify certain fungal taxonomic groups (Smit et al ., 1999;Anderson et al ., 2003) and the potential lack of specificity for target fungal DNA in some instances (Borneman and Hartin, 2000;Anderson et al ., 2003). Such limitations should be taken into consideration when formulating conclusions on data generated using these techniques.…”

Dynamics of bacterial and fungal communities associated with eggshells during incubation

“…Fungal communities were assessed by amplification of the internal transcribed spacer (ITS) region for fungi, using a nested approach. The first run was performed using EF4 (5 0 -GGAAGGGRTGTATTTATTAG) and ITS4 (5 0 -TCCTC CGCTTATTGATATGC) primers (White et al 1990;Smit et al 1999). Each PCR was carried out in 25 lL.…”

Assessment of fungal diversity using terminal restriction fragment (TRF) pattern analysis: comparison of 18S and ITS ribosomal regions

“…Primers that amplify a V500-bp fragment of the 18S rDNA, Fun18S1 5P-CCATGCATGTCTAAGTWTAA-3P and Fun18S2 5P-GCTGGCACCAGACTTGCCCTCC-3P, or a V650-bp region of the ITS rDNA, EF3RCNL 5P-CAAACTTGGTCATTTAGAGGA-3P (reverse complement of EF3 from [12]) and ITS4 5P-TCCTCCGCTTAT-TGATATGC-3P [22], were used for PCR. The forward primers, Fun18S1 and EF3RCNL, were 5P-labeled with FAM 0 , a £uorescent sequencing dye (Perkin-Elmer) for use when analyzing samples on the ABI 310 Genetic Analyzer (Applied Biosystems); EF3RCNL was 5P-labeled with D4-PA, a di¡erent £uorescent dye for analyzing samples using the CEQ 2000XL DNA analysis system (Beckman-Coulter, Fullerton, CA, USA).…”