Nested PCR Amplification of Arbuscular Mycorrhizal Fungal 18S rRNA Genes from Field-collected Roots

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  • SAITO Katsuharu
    Laboratory of Land Ecology, Graduate School of Agricultural Science, Tohoku University
  • NISHIWAKI Aya
    Faculty of Agriculture, Miyazaki University
  • SUGAWARA Kazuo
    Laboratory of Land Ecology, Graduate School of Agricultural Science, Tohoku University

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  • 野外採取植物根からのアーバスキュラー菌根菌18SrRNA遺伝子のnested PCR増幅

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Abstract

Arbuscular mycorrhizal (AM) fungi in the order of Glomales (Zygomycetes) maintain a symbiotic association with plant roots of several species. We compared the nested PCR technique utilizing published primers to detect AM fungal DNA from field-collected mycorrhizal roots to the direct PCR technique which has been used for the evaluation of laboratory-grown plant roots or AM fungal spores. The first reaction of nested PCR was performed with a universal primer pair SS38-NS21, and the subsequent reaction was performed with the Glomales-specific primer VANS 1 in conjunction with taxon-specific primer VAGLO (Glomaceae), VAACAU (Acaulosporaceae) or VAGIGA (Gigasporaceae). The nested PCR method was capable of detecting amplified products from the DNA of four plant species roots obtained in the field, while direct PCR utilizing primers VANS 1-VAGLO, VANS 1-VAACAU and VANS 1-VAGIGA did not. To ascertain whether nested PCR end-products were derived from corresponding families of AM fungi, the PCR products were sequenced. The amplified sequences utilizing the VANS 1-VAGLO and the VANS 1-VAACAU were the most similar to sequences representative of the Glomaceae species and the Acaulosporaceae species found in the DDBJ database, respectively. However, most sequences amplified with the VANS 1-VAGIGA were not similar to the Gigasporaceae species, but appeared more similar to some Ascomycetes species. These results indicate that use of these specific primers have limitation in the detection of AM fungi in the field, though the nested PCR method itself was an effective and labor-saving technique when amplifying low amounts of fungal DNA from field-collected roots.

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