Kiyo Nishioka

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

ABSTRACT 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, andAzospirillum. 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 15N2gas incorporation assays. Therefore, we conclude thatHerbaspirillum sp. strain B501 is a diazotrophic endophyte compatible with wild rice, particularly O. officinalis.

Isolation and Characterization of Endophytic Bacteria from Wild and Traditionally Cultivated Rice Varieties

Abstract Endophytic bacteria were isolated from surface-sterilized stems, seeds, and leaf sheaths of wild and traditionally cultivated rice varieties. Phylogenetic analyses based on 16S rDNA revealed a wide divergence among the isolates. However, the most frequently isolated groups were Methylobacterium sp. in the α-subdivision of Proteobacteria, and Curtobacterium sp. in the high G+ C Gram-positive group. Various phenotypic traits that are expected to be involved in the persistence and functions of the bacteria were analyzed: Most of the isolates from rice excreted pectinase, were motile, and showed an osmotic resistance to 0.6 M sucrose. These traits may be involved in endophytic characteristics in rice. About 50% of the isolates showed a cellulase activity. A few isolates fixed nitrogen, produced indole-3-acetic acid, and formed capsules. These activities were partially correlated with the phylogenetic group.

Anaerobic Nitrogen-Fixing Consortia Consisting of Clostridia Isolated from Gramineous Plants

ABSTRACT We report here the existence of anaerobic nitrogen-fixing consortia (ANFICOs) consisting of N2-fixing clostridia and diverse nondiazotrophic bacteria in nonleguminous plants; we found these ANFICOs while attempting to overcome a problem with culturing nitrogen-fixing microbes from various gramineous plants. A major feature of ANFICOs is that N2 fixation by the anaerobic clostridia is supported by the elimination of oxygen by the accompanying bacteria in the culture. In a few ANFICOs, nondiazotrophic bacteria specifically induced nitrogen fixation of the clostridia in culture. ANFICOs are widespread in wild rice species and pioneer plants, which are able to grow in unfavorable locations. These results indicate that clostridia are naturally occurring endophytes in gramineous plants and that clostridial N2 fixation arises in association with nondiazotrophic endophytes.

Erythromycin Inhibits Adhesion of Pseudomonas Aeruginosa and Branhamella Catarrhalis to Human Nasal Epithelial Cells

Ciliated epithelial cells were obtained from nasal polyps. Bacterial adherence to these cells was compared for the ability to bind Hemophilus influenzae, Pseudomonas aeruginosa, and Branhamella catarrhalis in the presence of 10–5 M erythromycin, which was comparable with a physiologically attainable concentration in the nasal secretion and the maxillary sinus mucosa. Quantification of bacterial adherence showed the strongest ability of P. aeruginosa to the cells. Erythromycin has an inhibitory effect on adherence of P. aeruginose and B. catarrhalis to the nasal epithelial cell. Our findings suggest that the reduced adherence to the host cell is one of the underlying mechanisms to account for efficacy of erythromycin treatment in respiratory disorders.