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Appl. Environ. Microbiol. doi:10.1128/AEM.02532-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Cultivation-independent characterization of Methylobacterium populations in the plant phyllosphere by automated ribosomal intergenic spacer analysis (ARISA)

Laboratoire des Interactions Plantes Micro-organismes, INRA/CNRS, BP 52627, 31326 Castanet-Tolosan, France; Institute of Microbiology, ETH Zurich, 8093 Zurich, Switzerland

^* To whom correspondence should be addressed. Email: knief{at}micro.biol.ethz.ch.

	Abstract

Bacteria of the genus Methylobacterium are widespread in the environment, but their ecological role in ecosystems such as the plant phyllosphere is not very well understood. To gain a better insight into the distribution of different Methylobacterium species in diverse ecosystems, a rapid and specific cultivation-independent method for their detection and the analysis of their community structure is needed. Therefore, 16S rRNA gene-targeted primers specific for this genus were designed and evaluated. These were used in PCR in combination with a reverse primer that binds to the tRNA-Ala gene, located upstream of the 23S rRNA gene in the 16S – 23S intergenic spacer (IGS). PCR products of different length were obtained due to the length heterogeneity of the IGS of different Methylobacterium species. This length variation allowed the generation of fingerprints of Methylobacterium communities in environmental samples by automated ribosomal intergenic spacer analysis (ARISA). The Methylobacterium communities on leaves of different plant species in a natural field were compared using this method. The new method allows rapid comparisons of Methylobacterium communities, and is thus a useful tool to study Methylobacterium communities in different ecosystems.