Abstract
Low-molecular-weight (LMW) RNA molecules were analyzed to characterize rhizobial isolates that nodulate the common bean growing in Spain. Since LMW RNA profiles, determined by staircase electrophoresis, varied across the rhizobial species nodulating beans, we demonstrated that bean isolates recovered from Spanish soils presumptively could be characterized as Rhizobium etli, Rhizobium gallicum, Rhizobium giardinii, Rhizobium leguminosarum bv. viciae and bv. trifolii, and Sinorhizobium fredii.
The common bean (Phaseolus vulgaris), an important leguminous crop, is promiscuous because it forms nitrogen-fixing symbioses with a wide diversity of rhizobia, including six proposed species of Rhizobium and one of Sinorhizobium (1, 2, 6, 8, 10, 12, 14, 17). Because of this diversity, it would be useful to have a rapid method for the presumptive identification of newly isolated cultures that would also provide additional criteria to evaluate the taxonomic position of bean-nodulating rhizobia. While 16S rRNA gene sequencing might be considered the method of choice, this method is not always readily available to all laboratories and the expense involved would make the analysis of many isolates prohibitive. Analysis of LMW RNA cell content by staircase electrophoresis (20) is an alternative approach that may permit the characterization of numerous bean isolates by a method that is both more readily available and cheaper than sequencing analysis.
To explore the potential of this method, we first obtained the LMW RNA profiles of the type strain and other strains belonging to the different proposed rhizobial species that nodulate beans (Table 1). LMW RNA, extracted as described by Höfle (9), was separated according to molecular size using staircase electrophoresis (5) and the gels were silver-stained (7). Based on results with the reference strains, we concluded that the analysis of LMW RNA was useful for the presumptive identification of unknown bean isolates because the banding patterns within species were consistent, although they varied across species (data not shown). The banding patterns across the different biovars of Rhizobium etli (22) and Rhizobium gallicum (2) were identical with those of the type strains. However, the results obtained with ATCC 14482 (presumed to be Rhizobium leguminosarum bv. phaseoli) were different from those obtained with the type strain for R. leguminosarum and were identical with R. etli (data not shown). The reason for this may well be the misclassification of ATCC 14482 as R. leguminosarum bv. phaseoli since this is a bean isolate originally obtained by the U.S. Department of Agriculture in 1959 (3I6c15, USDA 2668), and a systematic analysis of this strain by other methods has not been done. We also were able to distinguish R. leguminosarum bv. viciae and bv. trifolii, which were included in our analysis to complement the data obtained with ATCC 14482. Analyses of other presumptive strains of R. leguminosarum bv. phaseoli were not included and, therefore, it is not known whether the banding pattern would have been different from one or both of the other two biovars of this rhizobial species.
TABLE 1.
Strains used in this study
| Strain | Host | Geographical origin | Source or reference |
|---|---|---|---|
| RPVR06, RPVR07, RPVR09, RPVR32 | Phaseolus vulgaris var. “redonda” | León, Spain | This study |
| RPVN02, RPVN03, RPVN05 | Phaseolus vulgaris var. “negra” | León, Spain | This study |
| 14-C1, 14-C3, 8-C3, 4C-4, 15C-4 | Phaseolus vulgaris var. “canellini” | Andalucía, Spain | 14 |
| 5NJ-1, 5NJ-2, 16NJ-2 | Phaseolus vulgaris var. “negra” | Andalucía, Spain | 14 |
| 4PR-2, 21PR-1, 18PR-2 | Phaseolus vulgaris var. “presenta” | Andalucía, Spain | 14 |
| R. etli bv. mimosae | |||
| Mim 1-4, Mim 1, Mim 1-2, Mim 4-2, Mim 7-5, Mim 2, Mim 11 | Mimosa affinis | Huautla, Mexico | 22 |
| R. etli | |||
| F6, F8 | Phaseolus vulgaris | Jalisco, Mexico | 11 |
| Viking 1 | Phaseolus vulgaris | Belize | 11 |
| Bra 1 | Leucaena leucocephala | Brazil | 11 |
| Rhizobium tropici IIA | |||
| Br 832, Br 833, Br 835, Br 836 | Leucaena leucocephala | Brazil | 11 |
| Br 10043 | Phaseolus vulgaris | 11 | |
| R. tropici IIB | |||
| Br 850, Br 864, Br 859, Br 852, Br 857 | Leucaena leucocephala | Brazil | 11 |
| R. gallicum bv. phaseoli | |||
| PhI21, PhD12 | Phaseolus vulgaris | Gers, France | 2 |
| R. gallicum bv. gallicum | |||
| PhP222, PhF29 | Phaseolus vulgaris | France | 2 |
| R. leguminosarum bv. phaseoli | |||
| 8002 | Phaseolus vulgaris | Norwich | |
| Rhizobium mongolense | |||
| USDA 1884 | Medicago ruthenica | Hutan Lao, China | 18 |
| USDA 1929 | Medicago ruthenica | Xilinhot, China | 18 |
| R. giardinii H152T | Phaseolus vulgaris | Côte d'Or, France | 2 |
| R. gallicum R602T | Phaseolus vulgaris | Maine et Loire, France | 2 |
| R. tropici IIA CFN 299T | Phaseolus vulgaris | Brazil | 16 |
| R. tropici IIB CIAT899T | Phaseolus vulgaris | Colombia | 16 |
| R. etli CFN42T | Phaseolus vulgaris | Guanajuato, Mexico | 16 |
| R. leguminosarum bv. viceae ATCC 10004T | Pisum sativum | Illinois, United States | |
| R. leguminosarum bv. trifolii ATCC 14480 | Trifolium repens | ||
| R. leguminosarum bv. phaseoli ATCC14482 | Phaseolus vulgaris | ||
| R. mongolense USDA 1844T | Medicago ruthenica | Tongliao, China | 18 |
The staircase electrophoresis method was used to characterize bean isolates obtained from two regions of Spain, León and Andalucía. We used the bean isolates originating from Andalucía that were characterized by Rodríguez-Navarro et al. (14). Nodules from bean plants growing in a field in León were used to isolate rhizobia according to the method of Vincent (21). Purified isolates were confirmed to nodulate beans by standard plant tests replicated five times using NaClO3-surface-sterilized seeds and with plants grown in pots containing sterilized vermiculite moistened with N-free Rigaud and Puppo nutrient solution (13) and inoculated with approximately 109 cells. Plants were grown for 30 days in a growth chamber with a 16-h photoperiod at 26°C and 50 to 60% rH. The isolates from León were characterized by the production of 10 exocellular glucosidases using 1:1 mixtures of bacterial suspensions (6 × 109 CFU/ml) grown for 4 days in Bergensen minimal medium (3) at 28°C and para-nitrophenyl substrates at 0.4% concentrations (wt/vol) in 50 mM phosphate buffer, pH 7.0. Chromophores were developed by adding equal volumes of 4% NaCO3. From these results we placed the isolates RPVR07, RPVR32, and RPVN03 within R. leguminosarum bv. viciae and the isolates RPVR06, RPVR09, RPVN02, and RPVN03 within R. leguminosarum bv. trifolii (Table 2). Our presumptive placement of these isolates by using exocellular enzyme profiles was supported by the results of LMW RNA profiles (Fig. 1A). The profiles of RPVR07, RPVR32, RPVN03, and R. leguminosarum bv. viciae (Fig. 1A, lanes 2, 6, 7, and 8, respectively) were identical. Similarly, the profiles of RPVR06, RPVR09, RPVN02, RPVN03, and R. leguminosarum bv. trifolii (Fig. 1A, lanes 1, 3, 4, 5, and 9, respectively) were identical. These results indicate that in some cases R. leguminosarum bv. viciae and bv. trifolii may cross-nodulate onto beans or that within these two biovars variants exist that are able to nodulate beans. Although nodulation of clover and peas by these isolates was not determined, this unexpected result would have been missed using sequencing analysis of the 16S rRNA gene since this locus is conserved among the three biovars belonging to this species.
TABLE 2.
Physiological and biochemical characteristics of rhizobia used in this studya
| Substrateb | R. leguminosarum bv. viceae | R. leguminosarum bv. trifolii | R. etli | R. tropici IIB | R. tropici IIA | R. gallicum | R. mongolense | R. giardinii | S. fredii | RPVR07 | RPVR32 | RPVN03 | RPVR06 | RPVR09 | RPVN02 | RPVN05 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| PNP-N-acglc | +c | − | − | + | − | + | + | + | + | + | + | + | − | − | − | − |
| PNP-α-d-ara | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
| PNP-β-d-ara | − | − | + | + | − | − | − | + | + | − | − | − | − | − | − | − |
| PNP-α-l-fuco | + | + | + | − | − | − | − | − | − | + | + | + | + | + | + | + |
| PNP-β-d-fuco | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
| PNP-α-d-gal | + | + | + | − | − | + | − | + | + | + | + | + | + | + | + | + |
| PNP-β-d-gal | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
| PNP-N-lact | + | − | + | − | + | − | − | − | − | + | + | + | − | − | − | − |
| PNP-α-d-mal | + | − | − | − | − | − | − | − | − | + | + | + | − | − | − | − |
| PNP-α-d-xyl | + | − | − | + | − | − | − | + | − | + | + | + | − | − | − | − |
| PNP-β-d-xyl | + | + | + | − | + | + | + | + | − | + | + | + | + | + | + | + |
Type strain was used except for strains isolated in this study.
Abbreviations: PNP, para-nitrophenyl; PNP-N-acglc, PNP-N-acetylglucosaminide; PNP-α-d-ara, PNP-α-d-arabinopyranoside; PNP-β-d-ara, PNP-β-d-arabinopyranoside; PNP-α-l-fuco, PNP-α-l-fucopyranoside; PNP-β-l-fuco, PNP-β-l-fucopyranoside; PNP-α-d-gal, PNP-α-d-galactopyranoside; PNP-β-d-gal, PNP-β-d-galactopyranoside; PNP-α-d-mal, PNP-α-d-maltopyranoside; PNP-lact, PNP-lactopyranoside; PNP-α-d-xyl, PNP-α-d-xylopyranoside; PNP-β-d-xyl, PNP-β-d-xylopyranoside.
Plus indicates that compound is hydrolyzed; minus indicates that compound is not hydrolyzed.
FIG. 1.
LMW RNA profiles of rhizobial isolates from Spanish soils. Analysis of isolates (A) from León: lane 1, RPVR06; lane 2, RPVR07; lane 3, RPVR09; lane 4, RPVN02; lane 5, RPVN05; lane 6, RPVR32; lane 7, RPVN03; lane 8, R. leguminosarum bv. viciae ATCC 10004T; lane 9, R. leguminosarum bv. trifolii ATCC 14480; and (B) from Andalucía: lane 1, 15C-4; lane 2, 8C-3; lane 3, 14C-3; lane 4, 4C-4; lane 5, 16NJ-2; lane 6, 8002; lane 7, 4PR-2; lane 8, 18PR-2; lane 9, 5NJ-1; lane 10, 5NJ-2; lane 11, 21PR-1; lane 12, 14C-1; lane 13, R. leguminosarum bv. trifolii ATCC 14480; lane 14, R. etli CFN42T; lane 15, R. giardinii H152T; lane 16, R. gallicum R602T; and lane 17, S. fredii ATCC 35423T.
The isolates from Andalucía were more heterogeneous than those from León, since we distinguished five different LMW RNA profiles (Fig. 1B). Isolates 4C-4, 8C-3, 16NJ-2, 4PR-2, 21PR-1, and 14C-1 (Fig. 1B, lanes 2, 5, 7, 11, and 12) were identified with R. etli (lane 14), 5NJ-2 and 14C-3 (lanes 3 and 10) with R. gallicum (lane 16), 5NJ-1 and 18PR-2 (lanes 8 and 9) with Rhizobium giardinii (lane 15), and 8002 (lane 6) with R. leguminosarum bv. trifolii (lane 13). The profile of isolate 15C-4 (lane 1) was identical with that of Sinorhizobium fredii (lane 17). The placement of isolate 15C-4 within Sinorhizobium was confirmed by full-length sequencing of the 16S rRNA gene according to the method of van Berkum et al. (19) since the sequence was identical with that of S. fredii (data not shown). There is a precedence for the nodulation of bean by S. fredii (15), and Herrera-Cervera et al. (8) reported the isolation of three bean-nodulating rhizobia from a soil in Granada that had 16S rRNA genes similar to those of S. fredii, based on PCR restriction fragment length polymorphism and partial sequencing analysis. Therefore, we conclude that staircase electrophoresis of LMW RNA is a rapid and cost effective method for the presumptive characterization of bean isolates and that in the case of the biovars of R. leguminosarum the approach has more resolution than sequencing analysis of the 16S rRNA gene.
Acknowledgments
We thank Patrick Elia for technical assistance and N. Skinner for an initial review of the manuscript. We thank N. Amarger for providing strains.
This work was supported by the Junta de Castilla y León of Spain, grant numbers ES18/98 and SA35/99.
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