Endophytic colonization and in planta nitrogen fixation by a Herbaspirillum sp isolated from wild rice species

Nitrogen-fixing bacteria were isolated from the stems of wild and cultivate d rice on a modified Rennie medium. Based on 16S ribosomal DNA (rDNA) seque nces, the diazotrophic isolates were phylogenetically close to four genera: Herbaspirillum, Ideonella, Enterobacter, and Azospirillum. Phenotypic prop erties and signature sequences of 16S rDNA indicated that three isolates (B 65, B501, and B512) belong to the Herbaspirillum genus. To examine whether Herbaspirillum sp. strain B501 isolated from wild rice, Oryza officinalis, endophytically colonizes rice plants, the gfp gene encoding green fluoresce nt protein (GFP) was introduced into the bacteria. Observations by fluoresc ence stereomicroscopy showed that the GFP-tagged bacteria colonized shoots and seeds of aseptically grown seedlings of the original Mld rice after ino culation of the seeds. Conversely, for cultivated rice Oryza sativa, no GFP fluorescence was observed for shoots and only weak signals were observed f or seeds. Observations by fluorescence and electron microscopy revealed tha t Herbaspirillum sp. strain B501 colonized mainly intercellular spaces in t he leaves of wild rice. Colony counts of surface-sterilized rice seedlings inoculated with the GFP-tagged bacteria indicated significantly more bacter ial populations inside the original wild rice than in cultivated rice varie ties. Moreover, after bacterial inoculation, in planta nitrogen fixation in young seedlings of wild rice, O. officinalis, was detected by the acetylen e reduction and N-15(2) gas incorporation assays. Therefore, we conclude th at Herbaspirillum sp. strain B501 is a diazotrophic endophyte compatible wi th wild rice, particularly O. officinalis.