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1 Departamento de Microbiología y Genética, Universidad de Salamanca, Salamanca, Spain
2 Laboratorium voor Microbiologie, Vakgroep Biochemie, Fysiologie en Microbiologie, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
3 Centro Regional de Diagnóstico de la Junta de Castilla y León, Salamanca, Spain
Correspondence
Encarna Velázquez
evp{at}gugu.usal.es
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Published online ahead of print on 9 February 2004 as DOI 10.1099/ijs.0.02971-0.
The GenBank accession number for the 16S rRNA gene sequence of Bradyrhizobium betae PL7HG1T is AY372184.
Photographs of the tumour-like deformations described here are available as supplementary material in IJSEM Online.
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). Phylogenetic analysis of the 16S rRNA molecule revealed that these strains belong to the genus Bradyrhizobium. This genus currently includes four species able to produce nodules in several legumes: Bradyrhizobium japonicum (Jordan, 1982), Bradyrhizobium elkanii (Kuykendall et al., 1992) and Bradyrhizobium liaoningense (Xu et al., 1995) establish symbioses with soybean plants. The most recently described species, Bradyrhizobium yuanmingense (Yao et al., 2002), produces nodules in Lespedeza but not in soybean. Although rhizobia are soil-inhabitants, rhizobial species are not commonly isolated from sources other than nodules. We show here that modern molecular techniques can help to identify and classify new rhizobial strains not isolated from nodules. A polyphasic study of these strains, including phenotypic and molecular taxonomic approaches, showed that these strains represent a novel species of Bradyrhizobium phylogenetically similar to B. japonicum, for which we propose the name Bradyrhizobium betae sp. nov.
For isolation of bacterial strains, several tumours present in Beta vulgaris roots were surface sterilized using a 2·5 % aqueous solution of HgCl2 for 2 min. Tumours were washed ten times in sterile water. They were then disrupted in sterile water and cultivated on YMA medium (Bergersen, 1961) at 28 °C for 7 days. Four strains (PL7HG1T, TTR1, PL7HG3 and PL7HG5) were isolated from different tumours present in two plants. These strains showed a slow rate of growth and their colonies were mucoid, similar to those of B. japonicum.
To test the ability of the isolates to produce tumours, five plants per strain were inoculated between the cotyledons and the first true leaves, injecting 10 µl of a suspension of about 108 c.f.u. ml–1 prepared from a YMA plate culture grown for 48 h. A positive control inoculated with the type strain of Agrobacterium tumefaciens, ATCC 23308T, and a negative control using just sterile water were also prepared. Plants were maintained in a greenhouse for 30 days at 18–25 °C. None of the strains was able to reproduce tumours on Nicotiana tabacum, which is commonly used as a plant model of tumour growth. The strains isolated were also unable to reproduce tumours in Beta vulgaris.
Nodulation was tested using soybean (Glycine max cv. Peking) and yam bean (Pachyrrhizus ahipa), the latter a promiscuous plant that can be nodulated by several species of Rhizobium and Bradyrhizobium (Fuentes et al., 2002). B. japonicum LMG 6138T was used as a positive control. None of the isolates was able to nodulate the two plants used in this study.
PCR amplifications of nodD, nifH and virA genes were carried out using the primer pairs 5'-CTCGTCGCGCTCGACGCATTGA-3' and 5'-TGCCCCATGGACATGTA-3' (nodD), 5'-GTCTCCTATGACGTGCTCGG-3' and 5'-GCTTCCATGGTGATCGGGGT-3' (nifH) and 5'-ATGAATGGAAGGTATTCACCG-3' and 5'-GGCTCAGGCAGCTTCGCTGCG-3' (virA) under the following conditions: pre-heating at 95 °C for 9 min, 35 cycles of denaturing at 94 °C for 1 min, annealing at 54 °C for 2 min and extension at 72 °C for 2 min and a final extension at 72 °C for 7 min. As a positive control in amplification of symbiotic genes, strain EC-550 nodulating yam bean (Fuentes et al., 2002) was used and A. tumefaciens ATCC 23308T was used as a positive control for amplification of the virA gene. None of these genes was detected in any of the strains isolated here, whereas they were amplified from the positive controls (data not shown).
Strains were analysed by two-primers random amplified polymorphic DNA (TP-RAPD) fingerprinting according to the method previously described (Rivas et al., 2002a), using the primers 849F (5'-GCCTGGGGAGTACGGCCGCA-3', Escherichia coli positions 829–849) and 1522R (5'-AAGGAGGTGATCCANCCRCA-3', E. coli positions 1509–1522). We have previously shown that TP-RAPD patterns allow differentiation among rhizobial species (Rivas et al., 2001) and, because these patterns are not strain dependent, all strains showing identical TP-RAPD pattern are considered to belong to the same species. All strains isolated in this study presented the same TP-RAPD pattern (Fig. 1) and therefore they probably belong to the same species. These results were confirmed from sequencing of 16S–23S rDNA intergenic spacer (ITS) regions (see below); based on these data, PL7HG1T was considered as the type strain. The nearly complete 16S rDNA sequence from this strain was obtained according the method previously described (Rivas et al., 2002b). The sequence obtained was compared with those from the GenBank database using the FASTA program (Pearson & Lipman, 1988), indicating that this strain is phylogenetically related to members of the genus Bradyrhizobium. Sequences of the novel isolate and related bacteria were aligned using CLUSTAL W software (Thompson et al., 1997). 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 MEGA2 package (Kumar et al., 2001) was used for all analyses. The resulting neighbour-joining tree is shown in Fig. 2. The 16S rDNA sequence of strain PL7HG1T showed 99·2 % similarity to that of B. japonicum USDA110 (genospecies Ia), 99 % to that of B. japonicum LMG 6138T (genospecies I), 99·1 % to that of B. liaoningense LMG 18230T, 98·5 % to that of B. yuanmingense CCBAU 11073T and 96·4 % to that of B. elkanii USDA 76T.
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, 2003). Therefore, we determined the ITS sequence of the four novel isolates as described by Willems et al. (2001a). The fragment obtained was 789 bp in length for all strains. Strains TTR1, PL7HG1T, PL7HG3 and PL7HG5 had identical sequences (accession no. AJ631967). A comparison with ITS sequences of other bradyrhizobia showed that the novel strains are most closely related to B. japonicum genospecies Ia and I (as described by Hollis et al., 1981), B. liaoningense and B. yuanmingense. ITS sequence similarities (as calculated with BIONUMERICS version 3.0, after multiple alignment with a gap penalty of 800 % and without gap penalties in the calculation of sequence similarities) of PL7HG1T with each of these genospecies or species were 94·2–94·3, 90·7–91·0, 93·2 and 86·8 %, respectively. Willems et al. (2003) have shown that for those bradyrhizobia closely related to B. japonicum, ITS sequence similarities of more than 95·5 % indicate a genospecies-level relatedness.
Based on this information, we selected the taxa to perform DNA–DNA hybridizations using a protocol described by Willems et al. (2001b), differing only in that we used white instead of black polystyrene microplates. White plates gave higher fluorescence readings and therefore resulted in better agreement of reciprocal hybridizations. Strains PL7HG1 and TTR1 were hybridized with B. japonicum genospecies I and Ia (Willems et al., 2003) and with B. liaoningense. Each hybridization experiment (using four replicate vials) was carried out twice, and the values reported in Table 1 are means of the two experiments. Standard deviation ranged from 0 to 11 % with a mean of 4 %. Values for combinations hybridized previously are slightly higher than those reported by Willems et al. (2001b) and this was observed consistently. Our data indicate that strains PL7HG1 and TTR1, as expected, showed almost 100 % DNA–DNA relatedness to each other. They showed about 60 % DNA–DNA hybridization with representatives of B. japonicum genospecies Ia and I and with B. liaoningense. In view of the relatively high hybridization values found here and in Willems et al. (2001b, 2003) between all Bradyrhizobium species (except B. elkanii), we propose that the novel group represented by PL7HG1T represents a novel genospecies close to B. japonicum.
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), was 63·7 and 64·0 mol%, respectively.
Phenotypic characterization of strains from this study was based on growth with different carbon sources, as described by Velázquez et al. (2001), Yao et al. (2002) and Xu et al. (1995) and using the commercial system API 20NE according to the manufacturer's instructions (bioMérieux). The type strains of B. japonicum, B. liaoningense, B. yuanmingense and B. elkanii were used as references. The following antibiotics were used to test for resistance: ampicillin (2 µg), erythromycin (2 µg), ciprofloxacin (5 µg), penicillin (10 IU), polymyxin (300 IU), cloxacillin (1 µg), oxytetracycline (30 µg), gentamicin (10 µg), cefuroxime (30 µg) and neomicin (5 µg) (all from Becton Dickinson). The basal medium was YMB (Vincent, 1970) supplemented with 0·5 % yeast extract. Each antibiotic disc was added under sterile conditions to 5 ml basal medium. All newly isolated strains had identical phenotypic characteristics but several differences from the recognized species of Bradyrhizobium (Table 2). The main differences observed between the novel species and B. japonicum are in nitrate reduction, growth in lactose and sucrose as the carbon source and resistance to polymyxin B.
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) and we therefore propose to name the new group Bradyrhizobium betae sp. nov.
Description of Bradyrhizobium betae sp. nov.
Bradyrhizobium betae (bet'ae. N.L. gen. n. betae of Beta, a plant genus, because the organism was isolated from Beta vulgaris, sugar beet).
Gram-negative rods as for the other species of the genus. Colonies are small, pearl white in YMA at 28 °C, the optimal growth temperature. Optimum pH for growth is 7–7·5. Isolated from sugar beet tumours; they are not able to reproduce these symptoms in Beta vulgaris or Nicotiana tabacum and strains are not able to nodulate soybean or yam bean. Nitrate reduction is negative. The strains produce 
-galactosidase and urease and hydrolyse aesculin. They use glucose, L-arabinose, galactose, mannose, mannitol, N-acetylglucosamine, maltose and L-sorbose as carbon sources. Strains do not grow on lactose, L-rhamnose, trehalose, raffinose, sucrose or adonitol. All strains are resistant to cloxacillin, polymyxin B, penicillin, gentamicin, oxytetracycline and amoxicillin. They do not grow in the presence of ciprofloxacin, cefuroxime or neomicin. Growth is weak in the presence of erythromycin. The G+C content of the type strain is 63·7 mol%.
The type strain, PL7HG1T (=LMG 21987T=CECT 5829T), was isolated from a tumour on sugar beet.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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