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1 Departamento de Producción Vegetal, Instituto de Recursos Naturales y Agrobiología, Consejo Superior de Investigaciones Científicas, Apartado 257, 37071 Salamanca, Spain
2 Departamento de Microbiología y Genética, Universidad de Salamanca, 37007 Salamanca, Spain
3 Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain
Correspondence
José-Mariano Igual
igual{at}usal.es
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ABSTRACT |
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-Proteobacteria and that the closest related genus is Herbaspirillum. The DNA G+C content ranged from 57·9 to 61·9 mol%. Growth was observed with many different carbohydrates and organic acids including caprate, malate, citrate and phenylacetate. No growth was observed with maltose, meso-inositol, meso-erythritol or adipate as sole carbon source. According to the phenotypic and genotypic data obtained in this work, the bacterium represents a novel species of the genus Herbaspirillum, and the name Herbaspirillum lusitanum sp. nov. is proposed. The type strain is P6-12T (=LMG 21710T=CECT 5661T).
Published online ahead of print on 30 May 2003 as DOI 10.1099/ijs.0.02677-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Herbaspirillum lusitanum P6-12T is AF543312.
Details of DNA G+C contents, levels of DNA–DNA relatedness and pellicle formation by the novel isolates, transmission electron micrographs of cells, a 16S rDNA-based phylogenetic tree and RFLP and TP-RAPD patterns of the novel isolates are available as supplementary material in IJSEM Online.
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). Three other species have since been included: Herbaspirillum rubrisubalbicans (Baldani et al., 1996), a mild pathogen of sugarcane formerly named Pseudomonas rubrisubalbicans, Herbaspirillum species 3 (Baldani et al., 1996; Gillis et al., 1990), which contains mainly strains of clinical origin, and, more recently, Herbaspirillum frisingense (Kirchhof et al., 2001), which occurs in C4-fibre plants. With the exception of Herbaspirillum species 3, all species of Herbaspirillum are nitrogen-fixing bacteria able to establish close associations with plants (Reinhold-Hurek & Hurek, 1998), even as endophytes in apoplastic (Elbeltagy et al., 2001; Olivares et al., 1997) or intracellular (James et al., 1997; Olivares et al., 1997) locations. When associated with plants, either as a causal agent of mild disease or as an asymptomatic bacterium, Herbaspirillum species have been found predominantly in species of the family Gramineae (Baldani et al., 1996; Kirchhof et al., 2001), and only exceptionally in other plants (Baldani et al., 1996).
Although it has been suggested for several years that the species of Herbaspirillum form a type of endophytic association in gramineous plants, in which they liberate fixed nitrogen and supply it to the plant (Döbereiner et al., 1993), direct experimental evidence for this observation has been recently obtained in rice (Oryza officinalis) inoculated with Herbaspirillum sp. strain B501 by using acetylene reduction and 15N2 gas incorporation assays (Elbeltagy et al., 2001).
Olivares et al. (1996) described the isolation of H. seropedicae not only from gramineae but also from roots of a legume species (Cajanus cajan); however, as these authors pointed out, the exact origin of this isolate is uncertain because small pieces of maize root may have been included in the sample. In the course of isolating bacteria from nodules of Phaseolus vulgaris plants growing in a soil from Portugal, we have isolated several bacteria that, based on their genotypic and phenotypic characterization, should be classified as a novel species of the genus Herbaspirillum, for which we propose the name Herbaspirillum lusitanum sp. nov. To our knowledge, this is the first report of the undoubted association of a bacterium of the genus Herbaspirillum with a leguminous plant.
The reference strains and novel isolates used in this study are listed in Table 1. A total of six novel herbaspirillum isolates was obtained from young nodules of five Phaseolus vulgaris plants growing in a soil from Sierra da Peneda in the north-east of Portugal. Isolations were made according to Vincent (1970) using YMA (Bergersen, 1961). The cultures used in further studies were purified from single colonies after 10 days of incubation at 28 °C. On YMA, colonies were mucoid, circular convex, white, slightly translucent and usually 1–2 mm in diameter within 2 days at 28 °C.
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. Cell morphology was observed by scanning electron microscopy under a Zeiss EM900 electron microscope, with cells grown for 3 days at 28 °C in liquid YED medium. Cells were Gram-negative and showed a short, curved rod morphology (1·6x0·5 µm). Cells were also grown in nutrient agar for 48 h to check for motility by phase-contrast microscopy. To observe flagella, cells were treated with 2 % uranyl acetate and were observed under a Zeiss EM209 transmission electron microscope. Cells were motile and showed one or two polar flagella (see Supplementary Fig. A in IJSEM Online).
The 16S rRNA gene of a representative strain, P6-12T, was sequenced as described by Rivas et al. (2002). The sequence obtained was compared with those from GenBank using the FASTA program (Pearson & Lipman, 1988). Sequences were aligned using CLUSTAL X software (Thompson et al., 1997) and distances were calculated according to Kimura's two-parameter method (Kimura, 1980). Phylogenetic trees were inferred using the neighbour-joining method (Saitou & Nei, 1987). Bootstrap analysis was based on 1000 resamplings. The MEGA 2.1 package (Kumar et al., 2001) was used for all analyses. The trees were rooted using Comamonas testosteroni ATCC 11996T as the outgroup (Supplementary Fig. B). Sequence similarity calculations after neighbour-joining analysis indicated that the organism was phylogenetically related to members of the family ‘Oxalobacteraceae’. The 16S rDNA sequence of strain P6-12T showed 97·9 % similarity to that of H. frisingense, its closest relative, indicating that strain P6-12T could constitute a novel species of the genus Herbaspirillum.
Determinations of DNA base composition and DNA–DNA hybridization analysis (Supplementary Table A) were performed as described by Arahal et al. (2001). The G+C contents of the six strains isolated in this study ranged from 57·9 to 61·9 mol%. These values are similar to those obtained for Herbaspirillum species. The results of DNA–DNA hybridization showed 92–98 % relatedness between strain P6-12T and the other five strains isolated. The relatedness of strain P6-12T to H. frisingense DSM 13128T, H. seropedicae DSM 6445T and H. rubrisubalbicans DSM 9440T was respectively 28, 10 and 29 %. These results indicate that the strains isolated in this study do not belong to any of the known species of Herbaspirillum when the recommendation of a threshold value of 70 % DNA–DNA relatedness for definition of species is considered (Wayne et al., 1987).
PCR products of 16S rDNA amplification were digested with the restriction endonucleases DdeI and CfoI (Amersham-Pharmacia Biotech) as recommended by the manufacturer and electrophoresed in 2 % agarose gels. Each endonuclease produced the same RFLP pattern in all strains isolated in this study, which was different from those obtained for Herbaspirillum species already described (Supplementary Fig. C).
TP-RAPD (two-primer randomly amplified polymorphic DNA) patterns were obtained according to Rivas et al. (2001) by using the primer 849F (5'-GCCTGGGGAGTACGGCCGCA-3'; Escherichia coli positions 829–849) and the reverse primer 1522R (5'-AAGGAGGTGATCCANCCRCA-3'; E. coli positions 1502–1522), both originally designed for amplification of 16S rDNA. Each species of the genus Herbaspirillum showed a different pattern and all strains from this study showed the same pattern that was different from those of the species of the genus Herbaspirillum (Supplementary Fig. D). According to our previous results, strains showing different TP-RAPD patterns belong to different species (Rivas et al., 2001, 2002). Therefore, the TP-RAPD and RFLP patterns confirm the results obtained by 16S rDNA sequence analyses, DNA base composition and DNA–DNA hybridization analyses and indicate that the novel isolates belong to a novel species of the genus Herbaspirillum.
The six strains isolated in this study, together with the type strain of the previously described species of Herbaspirillum, were subjected to several phenotypic tests. The ability to grow at temperatures between 20 and 40 °C and at pH values between 5 and 8 was determined on YMA medium. Catalase production was assayed by using 0·3 % hydrogen peroxide with one colony taken from YMA plates. Oxidase activity was detected by using N,N,N',N'-tetramethyl-1,4-phenylenediamine dihydrochloride. For testing antibiotic resistance, API ATB G- strips (bioMérieux) were used following the manufacturer's instructions. Other physiological and biochemical tests were carried out using API 20NE and API ZYM strips (bioMérieux) following the manufacturer's instructions.
The six strains isolated in this work showed the same physiological and biochemical characteristics. Their range of temperature for growth was 20–35 °C. However, the known species of Herbaspirillum were able to grow at up to 40 °C, which is in agreement with previous reports (Baldani et al., 1996; Kirchhof et al., 2001). All isolates were able to grow at pH 5–8. The strains isolated in this study can be distinguished from previously described Herbaspirillum species on the basis of phenotypic properties such as nitrate reduction, -galactosidase production, assimilation of N-acetyl D-glucosamine, meso-inositol, meso-erythritol, L-rhamnose and arabinose and resistance to gentamicin, cefotaxime, ceftazidime, tobramicin, netilmicin and amikacin (Table 2).
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), as described for H. frisingense (Kirchhof et al., 2001). All strains isolated in this study were able to grow, forming a pellicle, in such medium supplemented with malate, D-glucose, N-acetyl D-glucosamine, L-arabinose, mannitol, D-fructose, L-tartrate and L-rhamnose, but not when supplemented with meso-inositol or meso-erythritol (Supplementary Table B). Furthermore, the presence of the nifD gene was confirmed by using a PCR approach with universal nifD primers (Stoltzfus et al., 1997). After electrophoresis in a 1·5 % agarose gel, a band of 390 bp was observed (data not shown), coinciding with the results obtained in H. frisingense (Kirchhof et al., 2001) and in other endophytic bacteria (Stoltzfus et al., 1997).
The infectivity of strain P6-12T was assayed in Phaseolus vulgaris plants. Seeds of Phaseolus vulgaris were surface-sterilized for 10 min using 5 % sodium hypochlorite and then washed repeatedly with sterile, distilled water. After sterilization, the seeds were sown in pots containing autoclaved vermiculite. One week after germination, roots were inoculated with 1 ml (105 cells) of a 48 h culture in YMB medium or with sterile water as a negative control. Two weeks after inoculation, plants were withdrawn from the pots and bacteria were reisolated from the roots as described by Elbeltagy et al. (2001). Finally, decimal dilutions were inoculated on YMA plates and incubated for 48 h at 28 °C. From these plates, several colonies were chosen at random and the identity of these isolates was checked by TP-RAPD fingerprinting (Rivas et al., 2001). Strain P6-12T was recovered from root tissues at concentrations of 2·3–3·7x103 c.f.u. (g fresh weight)-1, which is in accordance with results reported for the species of Herbaspirillum known to be endophytes (Elbeltagy et al., 2000).
Therefore, on the basis of phylogenetic, genotypic and phenotypic data, we propose that the isolates from this study should be classified as the novel species Herbaspirillum lusitanum sp. nov.
Description of Herbaspirillum lusitanum sp. nov.
Herbaspirillum lusitanum (lu.si.ta'num. L. neut. adj. lusitanum of Lusitania, the Roman name of Portugal, where the strains reported in this study were isolated).
Gram-negative, aerobic, non-spore-forming curved cells, 1·6 µm long and 0·5 µm in diameter. Motile by polar flagella. Colonies on YMA are circular convex, white, slightly translucent and usually 1–2 mm in diameter within 2 days at 28 °C. Carbon source utilization, hydrolytic enzyme production and resistance to antibiotics (including differentiating characters for all Herbaspirillum species) are indicated in Table 2
. The G+C content of the DNA is 59·9±2 mol%.
The type strain is P6-12T (=LMG 21710T=CECT 5661T), isolated from root nodules of Phaseolus vulgaris plants grown in a soil from Sierra da Peneda (Portugal). Its DNA G+C content is 57·9 mol%.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Baldani, J. I., Baldani, V. L. D., Seldin, L. & Döbereiner, J. (1986). Characterization of Herbaspirillum seropedicae gen. nov., sp. nov., a root-associated nitrogen-fixing bacterium. Int J Syst Bacteriol 36, 86–93.
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, esterase (C4)
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