부산대학교 도서관

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.apsoil.2006.01.001 Byline: Rene A. Soares (a), Luiz Fernando W. Roesch (b), Geancarlo Zanatta (a), Flavio A. de Oliveira Camargo (b), Luciane Maria P. Passaglia (a) Keywords: nifH gene; RFLP-PCR; Diazotrophs microorganisms; Auxin production; Nitrogen fixation; Soil management Abstract: Molecular and microbiological techniques were used to evaluate the nitrogen fixing bacterial community associated with oat (Avena sativa) in different soil management systems of Rio Grande do Sul State. The conventional microbiological approach made use of semi-solid nitrogen-free and solid selective media to isolate some of these nitrogen fixing bacteria. For the molecular approach, a PCR-RFLP strategy was adopted to amplify a segment of the nifH gene, which encodes nitrogenase reductase, from DNA samples extracted from rhizosphere soils, roots and leaves of oat. The amplified nifH fragments were cleaved with TaqI and HaeIII endonucleases and the products analyzed in polyacrylamide gels. One sample was analyzed in more detail. From this site rhizosphere soil and leaf samples PCR fragments were cloned into pUC18, generating 55 nifH positive clones, from which 19 different RFLP patterns were obtained. These patterns were compared with RFLP patterns generated from DNA obtained from pure strains or by theoretical digestion data. To identify and confirm the RFLP patterns obtained, at least one clone of each was sequenced and the nucleotide sequences were compared with the GenBank nucleotide database. As the partial nifH sequences analyzed had shown homologies with several known and unknown diazotrophs, we attempted to identify the diazotrophs through the positioning of the clones in the NifH partial sequence-derived tree. Through the molecular biological analysis we were able to identify a reasonable occurrence of diazotrophs in the rhizosphere soil and leaf samples analyzed, 9 and 13 different genera, respectively. Of these, two genera were particularly abundant: Azospirillum and Herbaspirillum. These two genera were also the two most abundant genera isolated by conventional microbiological techniques. Both approaches indicated that the nitrogen-fixing communities were different in soils under cultivation and permanent pasture from these under cultivation without live stock. Our results showed that while the molecular approach gives us a general view of the variability of nitrogen fixers in the community, the conventional methodology allows us to isolate those bacteria and to select some of them that have showed the highest nitrogen fixation capacity and auxin production. Author Affiliation: (a) Department of Genetics, Federal University of Rio Grande do Sul, 9500 Bento Goncalves Avenue, 91540-000 Porto Alegre, Brazil (b) Department of Soil Science, Federal University of Rio Grande do Sul, 7712 Bento Goncalves Avenue, 91540-000 Porto Alegre, Brazil Article History: Accepted 2 December 2005