Genetic diversity of N2-fixing bacteria associated with rice roots by molecular evolutionary analysis of a nifD library

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Abstract
The rhizosphere of wetland rice has significant N2-fixing activity. It has been suggested that N2 fixation in the rice root zone is associated with the activity of various N2-fixing heterotrophic bacteria that inhabit the rice rhizosphere. Because of the generic diversity, many different isolation media and conditions are required to count and isolate these bacteria. In an attempt to overcome any bias from culture-dependent methods we amplified nifD segments from crude rice root DNA by the polymerase chain reaction. The nifD fragments were then cloned into a pT7 BlueT-vector to construct a nifD library. Sixteen cloned nifD genes chosen at random from the library were sequenced. A comparison with published sequences indicated the presence of seven novel groups of NifD proteins, which implies the existence of at least seven components in the diazotrophic community of rice roots, dominated mainly by proteobacteria. We also observed genetic variability within the clusters, which suggests the coexistence of many closely related bacterial lineages. However, we did not find Azospirillum-like nifD clones, although many reports indicated the widespread presence of Azospirillum spp. Therefore, it remains to be clarified whether Azospirillum species are the widespread N2-fixing bacteria in rice roots.Key words: N2 fixation, molecular evolution, nifD, rice rhizosphere.