title: Combining nested PCR and restriction digest of the internal transcribed spacer region to characterize arbuscular mycorrhizal fungi on roots from the field.
abstract: Identification of arbuscular mycorrhizal fungi (AMF) on roots is almost impossible with morphological methods and, due to the presence of contaminating fungi, it is also difficult with molecular biological techniques. To allow broad investigation of the population structure of AMF in the field, we have established a new method to selectively amplify the internal transcribed spacer (ITS) region of most AMF with a unique primer set. Based on available sequences of the rDNA, one primer pair specific for AMF and a few other fungal groups was designed and combined in a nested PCR with the already established primer pair ITS5/ITS4. Amplification from contaminating organisms was reduced by an AluI restriction after the first reaction of the nested PCR. The method was assessed at five different field sites representing different types of habitats. Members of all major groups within the Glomeromycota (except Archaeosporaceae) were detected at the different sites. Gigasporaceae also proved detectable with the method based on cultivated strains.
authors.affiliation: Department of Environmental Sciences, Institute of Ecology, University of Jena, Dornburger Str. 159, 07743, Jena, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Renker
authors.forename: Jochen
authors.initials: J
authors.lastname: Heinrichs
authors.forename: Michael
authors.initials: M
authors.lastname: Kaldorf
authors.forename: François
authors.initials: F
authors.lastname: Buscot
mesh_terms: D001203:Ascomycota / Q000235:genetics; D001483:Base Sequence; D001487:Basidiomycota / Q000235:genetics; D044822:Biodiversity; D008411:Chytridiomycota / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific; D005658:Fungi / Q000235:genetics; D008969:Molecular Sequence Data; D038821:Mycorrhizae / Q000235:genetics*; D010802:Phylogeny; D018517:Plant Roots / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment
chemical_list: D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer; C024669:endodeoxyribonuclease AluI; D015252:Deoxyribonucleases, Type II Site-Specific