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Culture-Independent Assessment of Rhizobiales-Related Alphaproteobacteria and the Diversity of Methylobacterium in the Rhizosphere and Rhizoplane of Transgenic Eucalyptus

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Abstract

The rhizosphere is an ecosystem exploited by a variety of organisms involved in plant health and environmental sustainability. Abiotic factors influence microorganism–plant interactions, but the microbial community is also affected by expression of heterologous genes from host plants. In the present work, we assessed the community shifts of Alphaproteobacteria phylogenetically related to the Rhizobiales order (Rhizobiales-like community) in rhizoplane and rhizosphere soils of wild-type and transgenic eucalyptus. A greenhouse experiment was performed and the bacterial communities associated with two wild-type (WT17 and WT18) and four transgenic (TR-9, TR-15, TR-22, and TR-23) eucalyptus plant lines were evaluated. The culture-independent approach consisted of the quantification, by real-time polymerase chain reaction (PCR), of a targeted subset of Alphaproteobacteria and the assessment of its diversity using PCR–denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Real-time quantification revealed a lesser density of the targeted community in TR-9 and TR-15 plants and diversity analysis by principal components analysis, based on PCR–DGGE, revealed differences between bacterial communities, not only between transgenic and nontransgenic plants, but also among wild-type plants. The comparison between clone libraries obtained from the transgenic plant TR-15 and wild-type WT17 revealed distinct bacterial communities associated with these plants. In addition, a culturable approach was used to quantify the Methylobacterium spp. in the samples where the identification of isolates, based on 16S rRNA gene sequences, showed similarities to the species Methylobacterium nodulans, Methylobacterium isbiliense, Methylobacterium variable, Methylobacterium fujisawaense, and Methylobacterium radiotolerans. Colonies classified into this genus were not isolated from the rhizosphere but brought in culture from rhizoplane samples, except for one line of the transgenic plants (TR-15). In general, the data suggested that, in most cases, shifts in bacterial communities due to cultivation of transgenic plants are similar to those observed when different wild-type cultivars are compared, although shifts directly correlated to transgenic plant cultivation may be found.

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Acknowledgements

This work was supported by a grant from FAPESP (Foundation for Research Assistance, São Paulo State, Brazil, Grant 02/14143-3) and CNPq (National Council of Research, Brazil). We thank FAPESP for the fellowship to W.L.A. (Proc. n° 03/10527-4). We also thank Leo van Overbeek and Arjen Speksnijder for critical reading of the paper.

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Authors and Affiliations

  1. Departamento de Genética, Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, P.O. Box 83, 13400-970, Piracicaba, Brazil

    Fernando Dini Andreote, Raphael Tozelli Carneiro, Joelma Marcon, Carlos Alberto Labate, João Lúcio Azevedo & Welington Luiz Araújo

  2. Ecologie Microbienne UMR 5557 (CNRS/UCBL/USC INRA 1193), Université Lyon 1, Villeurbanne, France

    Joana Falcão Salles

  3. Núcleo Integrado em Biotecnologia, Universidade de Mogi das Cruzes, Mogi das Cruzes, Brazil

    Welington Luiz Araújo

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  1. Fernando Dini Andreote
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  2. Raphael Tozelli Carneiro
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  7. Welington Luiz Araújo
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Correspondence to Fernando Dini Andreote.

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Andreote, F.D., Carneiro, R.T., Salles, J.F. et al. Culture-Independent Assessment of Rhizobiales-Related Alphaproteobacteria and the Diversity of Methylobacterium in the Rhizosphere and Rhizoplane of Transgenic Eucalyptus. Microb Ecol 57, 82–93 (2009). https://doi.org/10.1007/s00248-008-9405-8

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