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Applied and Environmental Microbiology, November 2001, p. 5285-5293, Vol. 67, No. 11
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.11.5285-5293.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Endophytic Colonization and In Planta Nitrogen Fixation by a Herbaspirillum sp. Isolated from Wild Rice Species

Adel Elbeltagy,1,dagger Kiyo Nishioka,1 Tadashi Sato,1 Hisa Suzuki,1 Bin Ye,1 Toru Hamada,2,Dagger Tsuyoshi Isawa,1 Hisayuki Mitsui,1 and Kiwamu Minamisawa1,*

Institute of Genetic Ecology, Tohoku University, Katahira, Aoba-ku, Sendai 980-8577,1 and Marine Biotechnology Institute, Kamaishi Laboratories, Kamaishi City, Iwate 026-0001,2 Japan

Received 4 May 2001/Accepted 20 August 2001

Nitrogen-fixing bacteria were isolated from the stems of wild and cultivated rice on a modified Rennie medium. Based on 16S ribosomal DNA (rDNA) sequences, the diazotrophic isolates were phylogenetically close to four genera: Herbaspirillum, Ideonella, Enterobacter, and Azospirillum. Phenotypic properties and signature sequences of 16S rDNA indicated that three isolates (B65, 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 fluorescent protein (GFP) was introduced into the bacteria. Observations by fluorescence stereomicroscopy showed that the GFP-tagged bacteria colonized shoots and seeds of aseptically grown seedlings of the original wild rice after inoculation of the seeds. Conversely, for cultivated rice Oryza sativa, no GFP fluorescence was observed for shoots and only weak signals were observed for seeds. Observations by fluorescence and electron microscopy revealed that Herbaspirillum sp. strain B501 colonized mainly intercellular spaces in the leaves of wild rice. Colony counts of surface-sterilized rice seedlings inoculated with the GFP-tagged bacteria indicated significantly more bacterial populations inside the original wild rice than in cultivated rice varieties. Moreover, after bacterial inoculation, in planta nitrogen fixation in young seedlings of wild rice, O. officinalis, was detected by the acetylene reduction and 15N2 gas incorporation assays. Therefore, we conclude that Herbaspirillum sp. strain B501 is a diazotrophic endophyte compatible with wild rice, particularly O. officinalis.

* Corresponding author. Mailing address: Institute of Genetic Ecology, Tohoku University, Katahira, Aoba-ku, Sendai 980-8577, Japan. Phone: 81-22-217-5684. Fax: 81-22-263-9845. E-mail: kiwamu{at}ige.tohoku.ac.jp.

dagger Present address: Faculty of Agriculture, Minufiya University, Minufiya, Shebin Elkom, Egypt.

Dagger Present address: Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan.

Applied and Environmental Microbiology, November 2001, p. 5285-5293, Vol. 67, No. 11
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.11.5285-5293.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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