Several strains of Burkholderia vietnamiensis, isolated from the rhizosphere of rice plants, and four strains formerly known as Pseudomonas cepacia including two collection strains and two clinical isolates were compared for siderophore production and iron uptake. The B. vietnamiensis (TVV strains) as well as the B. cepacia strains (ATCC 25416 and ATCC 17759) and the clinical isolates K132 and LMG 6999 were all found to produce ornibactins under iron starvation. The two ATCC strains of B. cepacia additionally produced the previously described siderophores, pyochelin and cepabactin. Analysis of the ratio of isolated ornibactins (C4, C6 and C8) by HPLC revealed nearly identical profiles. Supplementation of the production medium with ornithine (20 mM) resulted in a 2.5-fold increase in ornibactin synthesis. Ornibactin-mediated iron uptake was independent of the length of the acyl side chain and was observed with all strains of B. vietnamiensis and B. cepacia, but was absent with strains of Pseudomonas aeruginosa, Pseudomonas fluorescens and Pseudomonas stutzeri, known to produce pyoverdines or desferriferrioxamines as siderophores. These results suggest that ornibactin production is a common feature of all Burkholderia strains and that these strains develop an ornibactin-specific iron transport system which is distinct from the pyoverdine-specific transport in Pseudomonas strains.
A new, simple method for isolating Azospirillum strains from the roots and the rhizosphere of rice is described. The method is based on the capacity of Azospirillum, a nitrogen-fixing bacterial genus, to grow in nutrient-deficient liquid media such as distilled water, KCl (8.5 g/L), or soil extract medium. The enrichment efficiency of the deficient media was compared with classical N-free malate medium. Serial dilutions from 10−1 to 10−10 of rice root macerates and rhizosphere soil were incubated at 28 °C in these media without agitation for 15 days. Pellicle formation was regularly examined under a microscope to detect the spiral motility of Azospirillum cells. Tubes with putative azospirilla were plated on nutrient agar. Most probable numbers of Azospirillum in root macerates were 2 × 103/g dry root weight in KCl, 2 × 104/g dry root weight in H2O, 2 × 106/g dry root weight in malate, and 3 × 106/g dry root weight in the soil extract medium. Soil extract medium repressed the most abundant bacterial populations and facilitated isolation of azospirilla from a population representing less than 0.001% of the total microflora. The isolates had vibrioid to S-shaped cells with one polar flagellum when grown in liquid medium and fixed nitrogen according to the acetylene reduction assay. Their identification as Azospirillum lipoferum was based on biochemical tests and hybridization with a 16S rRNA probe specific for this species and was confirmed by the detection of a 150-MDa plasmid.Key words: rice, rhizosphere, soil extract, isolation, identification, Azospirillum.
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