This research was funded by Cargill, Ltd. and the AAFC/NSERC Research Partnership Program.


A. Elbeltagy1, K. Nishioka1, T. Sato1, H. Suzuki1, B. Ye1, T. Hamada2, T. Isawa1,

H. Mitsui1, K. Minamisawa institute of Genetic Ecology, Tohoku University, Sendai, Japan

2Marine Biotechnology Institute, Kamaishi, Japan

From surface-sterilized culms of wild and cultivated rice, we obtained 11 gram-negative isolates of nitrogen-fixing bacteria by a modified Rennie medium. Based on 16S rDNA sequences, those diazotrophic isolates were phylogenetically close to Herbaspirillum, Ideonella, Enterobacter and Azospirillum. Isolates B65, B501 and B512 from wild rice phylogenetically belong within Herbaspirillum, however, the result of basic properties, carbon source utilization and the diagnostic probe sequences indicated that those three isolates cannot be classified into any known species of this genus. The isolates B65, B501 and B512 are possibly new species of Herbaspirillum.

To examine endophytic behavior of Herbaspirillum sp. B501 isolated from Oryza officinalis WOO 12 in the original wild rice and cultivated 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 seed inoculation. Conversely, no GFP fluorescence was observed for shoots, and only weak signals for seeds, of cultivated rice O. sativa.

Transmission electron microscopy demonstrated that Herbaspirillum sp. B501 colonized intercellular spaces in young leaves and in coleoptiles that were easily detected in the young seeding stage. Bacteria were often found outside the exodermis of roots, but rarely seen within the tissues. According to our observations, the majority of cells of Herbaspirillum sp. B501 invaded and colonized intercellular spaces and also found in intracellular spaces of rice shoots. 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 with the acetylene reduction and 15N2 gas incorporation assays. Therefore, we conclude that Herbaspirillum sp. B501 is a diazotrophic endophyte compatible with wild rice, particularly O. officinalis.


M. Rojas, J. Martinez, M. Heydrich

Dept of Microbiology, Faculty of Biology, Univ. Havana, CP 10400, La Habana, Cuba

1. Introduction

Polyhydroxyalkanoates (PHA) are biocompatible, biodegradable and thermoplastic polymers that can be applied in multiple biotechnological branches, industry and agriculture. Some microbial genera are able to produce PHAs, in recent studies it was demonstrated that diazotrophic microorganisms are also strong producers of PHA. In this study the capacity to produce PHA of Gluconacetobacter diazotrophicus, a nitrogen-fixing endophyte of sugarcane, in different conditions was demonstrated.

2. Materials and Methods

Type strain PA15 (ATCC 49037) of Gluconacetobacter diazotrophicus and two indigenous strains isolated from sugarcane in Cuba (4-02 and 1-05) were tested. Screening of colonies was carried out in LGI-P (Reis et al. 1994) and SYP media (Caballero and Martinez 1994) supplemented with Nile Red. PHA extraction-quantification was tested by our modification of Lan and Slepecky (1960) spectrophotometric method.

3. Results and Discussion

These results demonstrated that G. diazotrophicus can produce the polymer. Some strains were selected using a plate method with Nile Red as fluorogenic indicator of PHA accumulation. Microscopic examination of strains grown in media plus Nile Red showed fluorescence in some cells. G. diazotrophicus accumulate efficiently PHA, in nitrogen-fixing and not nitrogen-fixing conditions, establishing the capacity for production of high levels of PHA (Table 1).

Table 1. Comparison of PHA accumulation, yield (product/biomass), carbon:nitrogen and carbon:phosphorous ratio among strains of G. diazotrophicus at 48 h of culture in different media.




Not nitrogen fixation

Nitrogen fixation SYP

Pal 5 4-02 1-05

Pal 5 4-02 1-05 Pal 5 4-02 1-05


0.235 0.063 0.226

0.138 0.014 .0.101 0.982 1.265 0.998

Yield (P/B)

3.95 15.36 3.58

2.89 0.80 2.60 48.37 36.12 35.01



5264:1 13.58:1



243.4:1 4.38:1

Bergensen et al. (1991) propose that PHA reserves can support N2 fixation during prolonged periods ofN2 fixation. Bacteria can also use PHA as a source of energy and reductive power.

4. References

Bergensen et al. (1991) Proc. R. Soc. London B 245, 59-64 Caballero M, Romero M (1994) Appl. Environ. Microbiol. 60, 1532-1537 Reis et al. (1994) World J. Microbiol. Biotechnol. 10, 101-104

5. Acknowledgements

This work was partially supported by the Canadian International Development Agency (CIDA).

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