ZALF Centre for Agricultural Landscape and Land Use Research, Institute of Primary Production and Microbial Ecology, Eberswalder Str. 84, D-15374 Müncheberg, Germany1
Federal Research Centre for Forestry and Forest Products, Institute for Forest Genetics and Forest Tree Breeding, Eberswalder Chaussee 3, D-15377 Waldsieversdorf, Germany2
Author for correspondence: Andreas Ulrich. Tel: +49 33432 82345. Fax: +49 33432 82344. e-mail: aulrich{at}zalf.de
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ABSTRACT |
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Keywords: Mesorhizobium, Robinia pseudoacacia, black locust, 16S rDNA sequences, PCR-RFLP
The EMBL accession numbers for the sequences reported in this paper are AJ271898AJ271902 for the Mesorhizobium strains Rob6, Rob8 and Rob23 and the Rhizobium strains Rob18 and Rob20, respectively.
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INTRODUCTION |
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Comparison of 16S rDNA nucleotide sequences can be applied for classification of isolates at species and higher levels (Woese, 1987 ; Cilia et al., 1996
). Such phylogenetic analyses of representative rhizobial strains revealed that rhizobia are polyphyletic, and resulted in a reclassification of bacteria able to nodulate legumes into five genera (Young & Haukka, 1996
; Chen et al., 1988
; de Lajudie et al., 1994
; Dreyfus et al., 1988
; Jarvis et al., 1997
; Jordan, 1982
). The data also confirm that there is no branch of the evolutionary tree exclusively consisting of rhizobial species (Young, 1996
). Variations in the 16S rRNA gene sequences have been used for differentiation and classification of numerous rhizobial strains. These studies demonstrated that some legume species were infected by more than one species of rhizobia (Amarger et al., 1994
, 1997
; Laguerre et al., 1993
; Lafay & Burdon, 1998
). However, no strains capable of nodulating black locust were included in these phylogenetic studies. Our objective was to analyse phenotypic features and variation in 16S rRNA genes of rhizobial strains promoting growth of black locust plantlets.
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METHODS |
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Nodules with pink- or red-stained tissue were taken from the root system at a soil depth of about 5 cm. The nodules were surface-sterilized with 0·05% AgNO3 for 1 min and washed several times with sterile deionized water (Fox, 1993 ). The nodule contents were streaked on a glucose medium enriched with pea extract and incubated at 28 °C. The medium contained (l-1) 10 g glucose, 0·5 g K2HPO4, 0·5 g KH2PO4, 0·2 g MgSO4, 0·2 g NaCl, 0·1 g CaSO4, 1 mg (NH4)2MoO4, 500 ml pea extract (boiled from 100 g peas) and 15 g agar adjusted to pH 6·8. Bacteria from single colonies were subcultured on a modified yeast extract-mannitol agar (YMA), which contained 0·5 g instead of 3 g yeast extract (Elkan & Bunn, 1992
).
Reference strains.
As a reference strain, LMG 4270 (ATCC 10320), isolated from black locust in central Iowa, was used (Thorne & Walker, 1936 ). Furthermore, the rhizobial strains USDA 4197, USDA 4207, USDA 4212, USDA 4214, USDA 4222, USDA 4224, USDA 4236, USDA 4242 and USDA 4255, and USDA 4262, USDA 4279, USDA 4283, USDA 4297, USDA 4313, USDA 4315, USDA 4318, USDA 4322 and USDA 4340, respectively, isolated by Batzli et al. (1992
) from black locust trees at two sites in Green Ridge State Forest in Allegany County, MD, USA, were used to characterize the phylogenetic diversity of rhizobia from a native location.
Phenotypic characterization.
The growth rates of the 17 isolates were estimated by the incubation time necessary to achieve a colony size of 23 mm on YMA at 28 °C (Jarvis et al., 1997 ). Colony colour was examined macroscopically after growth on YMA for 6 d. Carbohydrate utilization was tested in yeast extract-mannitol medium in which mannitol was substituted by inositol or D-raffinose. NaCl tolerance was determined on YMA containing 5, 7·5, 10 and 12·5 g NaCl l-1 with three replications. The growth at different pH ranges was tested in 5 ml yeast extract-mannitol broth adjusted at pH 5·09·0 in steps of 0·5 (Batzli et al., 1992
; modified). Each broth was inoculated with 200 µl of a fresh liquid culture and incubated for 24 h at 27 °C. The OD660 was measured. For assaying antibiotic resistances, single colonies from 4-d-old cultures were spread on YMA containing either 10 µg rifampicin ml-1, 25 µg nalidixic acid ml-1, 50 µg kanamycin ml-1, 25 µg ampicillin ml-1, 15 µg chloramphenicol ml-1, 30 µg streptomycin ml-1 or 15 µg oxytetracycline ml-1 (Sigma). The plates were read for growth after 7 d incubation.
Plant inoculation test.
Rooted plantlets of five different genotypes of Rob. pseudoacacia var. rectissima were placed on sterile filter paper saturated with 2 ml sterile water and 1 ml Herridges plant nutrient solution (Qian et al., 1996 ) and inoculated with 250 µl bacterial suspension of approximately 106 cells ml-1. The root system of the plants was covered with moist filter paper, and the dishes were closed with Parafilm strips and cultivated for 30 d at 23 °C and 2000 lx (Philips TLD58W/93). During the first week, the dishes were covered with a thin layer of cellulose to protect green parts of plants against light. The test was performed in triplicate.
PCR amplification.
For template preparation, DNA was isolated from bacteria grown on YMA for 24 d at 28 °C. Single colonies were washed with 0·3% NaCl, resuspended in 20 µl 25 mM NaOH/0·25% SDS and heated for 15 min at 95 °C. Aliquots (0·2 µl) of the resulting lysate were directly used for PCR without further purification. The primers fD1 and rD1 (Weisburg et al., 1991 ) used in this study are capable of amplifying almost the entire 16S rRNA gene of most eubacteria. For routine assays, a 50 µl reaction mixture containing 10 pmol of each primer in a standard buffer (Gibco-BRL) was used. The amplifications were performed in a GeneAmp PCR System 2400 (PE Biosystems) with the following protocol: initial denaturation at 95 °C for 2 min; 30 cycles of 40 s at 94 °C, 40 s at 52 °C, 1 min at 72 °C; followed by a single final extension at 72 °C for 3 min and a final soak at 4 °C (Ulrich & Müller, 1998
; modified). After the reaction, aliquots (3 µl) of the PCR products were examined electrophoretically in a 1% agarose gel.
RFLP analysis.
PCR products (28 µl) were digested with each of the following enzymes: CfoI, HaeIII, AluI, HinfI, MspI, Sau3A and ScrFI (Roche Molecular Biochemicals, New England Biolabs). To detect minor differences between bands, the DNA fragments were separated in 23·5% Metaphor agarose gels (FMC Bioproducts) depending on the fragment sizes to be distinguished. The DNA molecular mass markers V and VI were used as size standards (Roche Molecular Biochemicals). The gels were stained with ethidium bromide and documented with a video camera image system (EasyImage plus, Herolab).
Cloning and sequencing.
The isolates Rob20, Rob18, Rob6, Rob8 and Rob23 representing genotypes III, IV, V, VI and IX, respectively, were used in 16S rDNA sequence determinations. The PCR products obtained after amplification with Pfu DNA polymerase (Promega) were purified using the QIAquick PCR purification kit (Qiagen), digested at the HindIII and SalI restriction endonuclease sites within the linker sequence of the primers and subsequently cloned into the vector pSVB30 (Arnold & Pühler, 1988 ), which had been digested with the same enzymes. A cycle-sequencing protocol was applied for sequencing both DNA strands with a Li-Cor Sequencer, model 4200 (MWG Biotech), using standard M13 primers. The resulting rDNA sequences of 1436 or 1440 nucleotides, corresponding to the genotypes III and IV, and V, VI and IX, respectively, were analysed for homologies to sequences deposited in the GenBank and EMBL databases. The similarity values were based on a pair-wise comparison of sequences. The determined DNA sequences and various 16S rDNA sequences from selected rhizobial species were aligned using the CLUSTAL W algorithm (program version 1.74; Thompson et al., 1994
). The phylogenetic analyses were performed with the PHYLIP computer program package, version 3.55c (Felsenstein, 1993
). The neighbour-joining algorithm and the parsimony method were used to generate phylogenetic trees. The neighbour-joining algorithm (Saitou & Nei, 1987
; NEIGHBOR using Bradyrhizobium japonicum as outgroup) was based on a matrix of pair-wise distances corrected for multiple base substitutions by the method of Kimura (1980
) (DNADIST with transition/transversion ratio of 2·0). The parsimony method (DNAPARS) was applied with ten jumbles of the data set. Both trees were constructed using the original data set and 100 bootstrap data sets.
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RESULTS |
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DNA sequence analysis of isolates with unknown RFLP patterns
16S rDNA sequencing was performed on a representative isolate of the genotypes that could not be classified by restriction analysis. All sequences showed a high homology to sequences from rhizobial species. The sequence of Rob6 (genotype V) was identical to that of Mesorhizobium amorphae. Best conformities of Rob8 (VI) and Rob23 (IX) were found with rDNA sequences of M. amorphae (99·9%) and the Mesorhizobium strain R88b (99·8%), respectively. The 16S rDNA sequences of Rob6 and Rob8 differed only in two nucleotides, but both genotypes were differentiated by RFLP analysis, illustrating the high resolution of this technique.
With respect to their restriction patterns, genotypes III and IV were more distinct in comparison to genotypes VIX (Tables 2 and 3
). These differences were also demonstrated by the similarity of 16S rDNA sequences; thus genotypes III and IV (Rob20 and Rob18) showed a similarity of 91·1% and 91·9%, respectively, to genotype V (Rob6). The search for homologous sequences for Rob20 and for Rob18 resulted in a similarity index of 98·1% to a Rhizobium strain nodulating Phaseolus vulgaris (RCR 3618D) and of 96·4% to Rhizobium gallicum, respectively.
An unrooted phylogenetic tree including further rhizobial species was constructed to illustrate the phylogenetic position of the identified genotypes (Fig. 1). As a result, isolates of groups V, VI, VII, VIII and IX were assigned to the genus Mesorhizobium (bootstrap values of 100%). These genotypes represent 76% of the isolates. The isolates Rob6 and Rob8 were unequivocally clustered with M. amorphae. Furthermore, the phylogenetic tree showed the strong similarity of isolate Rob23 and Mesorhizobium strain R88b. Like the M. loti type strain, this strain was isolated from Lotus corniculatus. The isolate Rob18 branched with the Rh. gallicum/Rhizobium mongolense grouping with a relatively low similarity and weak bootstrap support (58%). Therefore, phylogenetic assignment of this isolate remains open. There is a good support (97%) for the branching of Rob20 with the Rhizobium sp. strain RCR 3618D. Both isolates form a branch together with Rh. leguminosarum/Rhizobium etli (72% bootstrap support).
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DISCUSSION |
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Rob. pseudoacacia was introduced into middle Europe approximately 300 years ago. Although native only to the eastern USA, the tree species is now naturalized and widely planted throughout the world from temperate to subtropical areas. The rhizobia nodulating Rob. pseudoacacia are usually effective for nitrogen fixation as indicated by the red colour of the nodules or shown by their symbiotic host response (Batzli et al., 1992 ). It may be possible that some rhizobia were seed-borne and introduced together with the neophytic plant, as has been suggested for the transmission of Rh. etli on seeds of P. vulgaris (Pérez-Ramirez et al., 1998
). However, both the high phylogenetic diversity of isolates effective with Rob. pseudoacacia and its broad nodulation capacity support the view that indigenous rhizobia may be associated with this tree species. On the other hand, about half of the 16S rDNA genotypes were identical at both locations and, as shown for L. corniculatus, symbiotic gene transfer could also cause diversity of nodulating strains (Sullivan et al., 1996
). The highest proportion of the isolates obtained from the German location (47%, genotypes V and VI) showed identical or closely related (99·9%) 16S rDNA sequences to M. amorphae. In general, we found a parallel picture of phylogenetic diversity of isolates nodulating Rob. pseudoacacia to those nodulating Amorpha fruticosa (Wang et al., 1999
). Wang et al. (1999
) analysed 55 isolates and identified five 16S rDNA genotypes. The highest proportion of the isolates were M. amorphae, whilst two other genotypes belonging to the genus Mesorhizobium as well as some isolates related to Rh. leguminosarum and Bradyrhizobium elkanii were also identified. Similar to Rob. pseudoacacia, A. fruticosa is a neophytic plant native to south-eastern and mid-Western USA and was introduced into Asia more than 50 years ago. We suggest that neophytic and other legumes, especially when widely distributed, are less specific and form nodules with several phylogenetically different rhizobia, whereas archaeophytic plants could preferably host a singular or few specialist micro-symbionts. A narrow host range of rhizobial strains is considered a specialization which developed for certain plants in restricted niches (Perret et al., 2000
). However, further studies are necessary to evaluate this hypothesis.
The phylogenetic classification was mostly supported by phenotypic characteristics. Thus isolates classified in the genus Mesorhizobium generally showed a longer incubation time than the Rhizobium isolates (Rob17Rob20). The growth rates of the reference strains used in the study are in the same range, although there were some differences from the incubation time given by Jarvis et al. (1997 ). These authors showed an incubation time of 7 d for M. loti and of 56 d for M. huakuii. The markedly low salt tolerance of the isolates differed from the salt tolerance of the reference strains. However, our isolates were similar to all rhizobial groups isolated from A. fruticosa in that they did not grow at salt concentrations above 1% NaCl (Wang et al., 1999
). The phenotypic characters tested indicate a diversity of black-locust-nodulating rhizobia as already described by Batzli et al. (1992
) for the rhizobial strains used as reference strains in this study. These strains demonstrated a high heterogeneity of several phenotypic characters, including carbohydrate utilization, nitrogen fixation, generation times between 3 and 9 h and different protein profiles. As shown for the phylogenetic diversity, there was a similar phenotypic heterogeneity of strains to those reported for rhizobia nodulating the leguminous shrub A. fruticosa.
Our results revealed a high phylogenetic diversity with seven genotypes among 17 isolates analysed, which suggests the existence of further taxa nodulating Rob. pseudoacacia. The high phenotypic and phylogenetic diversity of rhizobial strains obtained from black locust seems to be characteristic for widely distributed legumes. This could enhance their ability to spread into new areas. Thus rhizobial diversity is considered to be a reason for the success of black locust as a pioneer tree species outside its native range and the suitability of the tree for natural succession on former arable land and recultivation areas under temperate conditions.
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ACKNOWLEDGEMENTS |
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We thank Peter van Berkum and Douglas K. Jones for kindly providing rhizobial strains isolated from black locust in Maryland. We are grateful to Mrs Eva Riedel, Mrs Renate Rietz and Mrs Sigune Weinert for technical assistance.
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Received 28 February 2000;
revised 8 June 2000;
accepted 9 August 2000.
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