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

• DOI: 10.1128/aem.65.6.2614-2621.1999
• Publication Date: 2019-12-19T19:52:12Z
• Authors: Eric Smit; Paula Leeflang; Boet Glandorf; Jan Dirk van Elsas; Karel Wernars
• Keywords: Primer (cosmetics)

Like bacteria, fungi play an important role in the soil ecosystem. As only a small fraction of present can be cultured, conventional microbiological techniques yield limited information on composition and dynamics fungal communities soil. DNA-based methods do not depend culturability microorganisms, therefore they offer attractive alternative for study complex community structures. For this purpose, we designed various PCR primers that allow specific amplification 18S-ribosomal-DNA (rDNA) sequences, even presence nonfungal 18S rDNA. DNA was extracted from wheat rhizosphere, rDNA gene banks were constructed Escherichia coli by cloning products generated with primer pairs EF4-EF3 (1. 4 kb) EF4-fung5 (0.5 kb). Fragments 0.5 kb cloned inserts sequenced compared to known sequences. Sequences all major taxa amplified using both pairs. predicted computer analysis, pair appeared slightly biased amplify Basidiomycota Zygomycota, whereas mainly Ascomycota. The 61 clones matched sequences 24 different species Ribosomal Database Project (RDP) database. Similarity values ranged 0.676 1. Temperature gradient gel electrophoresis (TGGE) analysis rhizosphere microcosm experiment carried out after total This resulted reproducible, distinctive fingerprints, confirming difference specificity. Clear banding patterns obtained samples sets combination. By comparing electrophoretic mobility fingerprint bands separate clones, some could tentatively identified. While 18S-rDNA always provide taxonomic resolution identify strains, diversity groups related environmental sufficient produce discrete which separated TGGE. combination TGGE should diversity, composition, bulk rhizosphere.