The symbiosis between oriental goat’s rue and the root nodule bacteria <Emphasis Type="Italic">Rhizobium galegae</Emphasis>: Specificity and competitiveness

Competitiveness and genetic variation of the Rhizobium galegae strains from the collection of the All-Russia Institute of Agricultural Microbiology, Russian Academy of Agricultural Sciences, causing nodulation of oriental goat’s rue under conditions of Bashkortostan soils (lacking this rhizobial species) were studied. It was demonstrated that of all the tested strains, the strains CIAM 0702 and CIAM 0704, each carrying two megaplasmids of 1500 and 2000 MDa, were the most competitive. RAPD (random amplified polymorphic DNA) analysis showed that R. galegae strains were able to intensively exchange the genetic material in the host plant rhizosphere. We did not succeed in detecting the local root nodule bacteria that were either initially able to infect oriental goat’s rue or had adapted to infecting this species due to various genetic rearrangements.     

Transconjugants of Agrobacterium radiobacter harbouring sym genes of Rhizobium galegae can form an effective symbiosis with Medicago sativa

It is known that the Rhizobium galegae genomes contain megaplasmids. The suicide vector pSUP2111 with nifH gene of R. meliloti was introduced into the strains CIAM 0703 and CIAM 0711 of R. galegae inducing effective nodules on Galega orientalis plants. The formation of self-transmissible megaplasmids was observed. The megaplasmid transfer into non-nodulating R. meliloti mutants resulted in partial complementation of the nodulation defect in recipient strains though only one transconjugant showed the nitrogen-fixing activity in symbiosis with alfalfa and another one in symbiosis with G. orientalis plants. Among the Agrobacterium strains harbouring R. galegae megaplasmids there were four classes of transconjugants: (1) Nod+ Fix- in symbiosis with goat's rue plants (three strains); (2) Nod+ Fix- on Medicago sativa (two strains); (3) Nod+ Fix+ on M. sativa (five strains); (4) Nod- with both plant hosts (11 strains).     

Identification of Rhizobium spp. in peat-based inoculants by DNA hybridization and PCR and its application in inoculant quality control.

Procedures based on DNA hybridization and PCR were developed for quality control of Rhizobium inoculants. Inoculants for pea and goat's rue were produced by Elomestari Ltd., Juva, Finland, in sterile dry fine peat by the standard procedure used by the company. The inoculants contained Rhizobium galegae HAMBI 1174 and HAMBI 1207 and an R. leguminosarum biovar vicia strain, 16HSA, either solely or in combinations of two or three strains. DNA was isolated from 1-g samples of each peat inoculant and analyzed by nonradioactive DNA-DNA hybridization and by PCR. The hybridization probes were total DNAs from pure cultures of R. galegae HAMBI 1207 and R. leguminosarum biovar viciae 16HSA and a 264-bp strain-specific fragment from the genome of R. galegae HAMBI 1174. The total DNA probes distinguished inoculants containing R. galegae or R. leguminosarum, and the strain-specific probe distinguished inoculants containing R. galegae HAMBI 1174. The hybridization results for R. galegae were verified in a PCR experiment by amplifying an R. galegae species-specific fragment and an R. galegae HAMBI 1174 strain-specific fragment in the same reaction. When suitable probes and primers are available, the methods described here offer promising alternatives for the quality control of peat-based inoculants.     

Description of two biovars in the Rhizobium galegae species: biovar orientalis and biovar officinalis

Twenty-six Rhizobium galegae strains, representing the center of origin of the host plants Galega orientalis and G. officinalis as well as other geographic regions, were used in a polyphasic analysis of the relationships of R. galegae strains. Phage typing, lipopolysaccharide (LPS) profiling, pulsed field gel electrophoresis (PFGE) profiling and rep-PCR (use of repetitive sequences as PCR primers for genomic fingerprinting) with REP and ERIC primers investigated nonsymbiotic properties, whereas plasmid profiling and hybridisation with a nif gene probe, and with nodB, nodD, nod box and an IS sequence from the symbiotic region as probes, were used to reveal the relationships of symbiotic genes. The results were used in pairwise calculations of distances between the strains, and the distances were visualised as a dendrogram. Indexes of association were compared for all tests pooled, and for chromosomal tests and symbiotic markers separately, to display the input of the different categories of tests on the grouping of the strains. Our study shows that symbiosis related genetic traits in R. galegae divide strains belonging to the species into two groups, which correspond to strains forming an effective symbioses with G. orientalis and G. officinalis respectively. We therefore propose that Rhizobium galegae strains forming an effective symbiosis with Galega orientalis are called R. galegae bv. orientalis and strains forming an effective symbiosis with Galega officinalis are called R. galegae bv. officinalis.     

Expression of Rhizobium galegae common nod clones in various backgrounds.

The cosmid clone pRg30, carrying common nodulation genes of Rhizobium galegae HAMBI 1174, and pRg33, a subclone of pRg30 that contains a 5.7-kb ClaI insert carrying nodDABC were conjugated into various Rhizobium nod- mutant strains and into a Ti plasmid-cured Agrobacterium tumefaciens. Complementation and expression of the nodABC genes of R. galegae were studied by following microscopically the infection process and the nodulation on different test plants. The nodABC genes of R. galegae complemented the nod- strains of other Rhizobium species. The presence of extra copies of common nod genes in the homologous R. galegae nodABC- strain induced an increased nodulation on Galega orientalis. However, the inserts of R. galegae in pRg30 and pRg33 do not carry sufficient genetic information for normal nodulation of test plants in an Agrobacterium background, because the Agrobacterium transconjugants induced root hair deformation on Galega plants, but no infection threads were detected and nodulelike structures developed only at low frequency. The Agrobacterium carrying the nodDABC of R. galegae did not cause the root hairs of Medigo sativa to deform.     

Symbiotic and genetic diversity of Rhizobium galegae isolates collected from the Galega orientalis gene center in the Caucasus

This paper explores the relationship between the genetic diversity of rhizobia and the morphological diversity of their plant hosts. Rhizobium galegae strains were isolated from nodules of wild Galega orientalis and Galega officinalis in the Caucasus, the center of origin for G. orientalis. All 101 isolates were characterized by genomic amplified fragment length polymorphism fingerprinting and by PCR-restriction fragment length polymorphism (RFLP) of the rRNA intergenic spacer and of five parts of the symbiotic region adjacent to nod box sequences. By all criteria, the R. galegae bv. officinalis and R. galegae bv. orientalis strains form distinct clusters. The nod box regions are highly conserved among strains belonging to each of the two biovars but differ structurally to various degrees between the biovars. The findings suggest varying evolutionary pressures in different parts of the symbiotic genome of closely related R. galegae biovars. Sixteen R. galegae bv. orientalis strains harbored copies of the same insertion sequence element; all were isolated from a particular site and belonged to a limited range of chromosomal genotypes. In all analyses, the Caucasian R. galegae bv. orientalis strains were more diverse than R. galegae bv. officinalis strains, in accordance with the gene center theory.     

Comparison between Rhizobium galegae and Rhizobium meliloti plasmid contents

Plasmid profiles of two strains of a newly classified rhizobial species- Rhizobium galegae -were compared with the profiles of several strains of another fast-growing Rhizobium species- Rhizobium meliloti .
The existence of a plasmid DNA band with a lower electrophoretic mobility than the R. meliloti megaplasmid band was demonstrated in the two R. galegae strains by a modified horizontal Eckhardt method. Thus R. galegae species contain giant plasmid(s) larger than the R. meliloti 1000 MD megaplasmids, previously considered to be the largest plasmids in the Rhizobiaceae family.
In one of the R. galegae strains an additional middle-size plasmid only a little smaller than 140 MD pRme41a of R. meliloti 41 was observed.     

Lipopolysaccharide and protein patterns of Rhizobium sp. (Galega)

Abstract Strains of Rhizobium sp. (Galega) (R. galegae), R. meliloti, R. leguminosarum , and R. loti were compared for their lipopolysaccharide (LPS) and whole cell protein patterns. Antigenic properties of these LPS and proteins were tested by immunoblotting with rabbit antiserum raised against R. galegae strain HAMBI 540. The LPS and protein patterns of R. galegae strains differed from those of the other rhizobia tested. By immunoblotting, a species-specific R. galegae LPS antigen and two proteins specific for R. galegae were identified. Our results support the suggestion that R. galegae strains form a distinct taxonomic group within the genus Rhizobium .     

Assessment of competitiveness of rhizobia infecting Galega orientalis on the basis of plant yield, nodulation, and strain identification by antibiotic resistance and PCR.

Competition between effective and ineffective Rhizobium galegae strains nodulating Galega orientalis was examined on the basis of plant growth, nodulation, antibiotic resistance, and PCR results. In a preliminary experiment in Leonard's jars, ineffective R. galegae strains HAMBI 1207 and HAMBI 1209 competed in similar manners with the effective strain R. galegae HAMBI 1174. In a pot experiment, soil was inoculated with 0 to 10(5) HAMBI 1207 cells per g before G. orientalis was sown. Seeds of G. orientalis were surface inoculated with 2 x 10(4) and 2 x 10(5) cells of HAMBI 1174 per seed (which represent half and fivefold the commercially recommended amount of inoculant, respectively). Plant yield and nodulation by the effective strain were significantly reduced, with as few as 10(2) ineffective rhizobia per g of soil, and the inoculation response was not improved by the 10-fold greater dose of the inoculant. Bacteria occupying the nodules were identified by antibiotic resistance and PCR with primers specific for R. galegae HAMBI 1174, R. galegae, and genes coding for bacterial 16S rRNA (bacterial 16S rDNA). Sixty-two large nodules examined were occupied by the effective strain HAMBI 1174, as proven by antibiotic resistance and amplification of the strain-specific fragment. From 20 small nodules, only the species-specific fragment could be amplified, and isolated bacteria had the same antibiotic resistance and 16S PCR restriction pattern as strain HAMBI 1207. PCR with our strain-specific and species-specific primers provides a powerful tool for strain identification of R. galegae directly from nodules without genetic modification of the bacteria.     

Symbiotic nitrogen fixation of Rhizobium (Galega) in acid soils,and its survival in soil under acid and cold stress

Summary Goat's rue (Galega orientalis) is a potential perennial forage legume for northern temperate acid soils. Greenhouse experiments were performed to compare symbiotically nitrogen fixing goat's rue with plants receiving mineral nitrogen in five different acid soils. Soil acidity had the same effect on yields of symbiotically grown plants as on plants receiving mineral nitrogen, suggesting that the acid sensitivity of the symbiosis was not limiting plant growth, even under very acidic conditions. The survival of an antibiotic resistant Rhizobium (Galega) strain in acid soil and freezing conditions was also studied. The survival of the bacteria was not affected at 15°C, when the pH of the soil (measured in 0.01M CaCl₂) was 5.2 or 4.9. In pH 3.4, and after freezing to –5°C, the population density decreased from 3×10⁸ to 1×10⁵/g in a few weeks. It is concluded that goat's rue, its symbiotic nitrogen fixation and R. (Galega) are tolerant of moderately acid agricultural soils, but that harsh winters may reduce bacterial numbers in the soil.     

Fermentation of a high-protein plant biomass by introduction of lactic acid bacteria

Lactic acid bacteria displaying increased ability to produce lactic acid, medium proteolytic activity, and tolerance to osmotic stress were isolated under selective conditions from phyllosphere and rhizosphere of registered and raised cultivars of legumes. Lactic fermentation of poorly ensilable leguminous plants (red clover and Caucasian goat's rue) was performed by introduction of rifampin-resistant homofermenting representatives of the genus Lactobacillus (selected according to a set of technologically important characteristics). The results demonstrate that introduction of active local strains of lactobacteria, as well as the collection strain Lactobacillus plantarum BS 933, enhances activation of ensiling and increases the quality of fodder, as assessed according to the standard criteria (a decrease in pH of the medium, the ratio of lactic acid to fatty acid homologues, and the composition of silage microflora).     

Pulsed-field gel electrophoresis-fingerprinting, genome size estimation and rrn loci number of Rhizobium galegae

I. HUBER AND S. SELENSKA-POBELL. 1994. The genomes of several Rhizobium galegae (of.) strains, which effectively nodulate Galega officinalis host, were analysed by pulsed-field gel electrophoresis (PFGE). Individual PFGE-fingerprints were obtained for every particular strain when the rarely cutting restriction endonucleases Spe I and Asn I were applied. In hybridization experiments, where a DNA fragment carrying the rrnB ribosomal RNA operon of Escherichia coli was used as a probe, the number of the resulting strain-specific Spe I and Asn I bands was reduced to three for all of the strains studied. This suggests that in Rh. galegae (of.) there are at least three rrn loci. On the basis of the lengths of the Spe I fragments, separated by PFGE, the genome size of five Rh. galegae (of.) strains was estimated to be 5852 ± 198 kbp.     

AFLP fingerprinting as a tool to study the genetic diversity of Rhizobium galegae isolated from Galega orientalis and Galega officinalis

AFLP fingerprints of Rhizobium galegae strains that infect Galega orientalis and Galega officinalis obtained from different geographical sources, and of taxonomically diverse rhizobia representing the recognized species, were generated. Comparisons of the fingerprints from fluorescent labeled AFLP products using capillary electrophoresis on ABI prism 310, slab gel electrophoresis on ABI prism 377 genetic analyzers and silver staining were in good agreement. All methods delineated the G. orientalis strains from G. officinalis strains, the G. orientalis strains formed a tight cluster whereas the G. officinalis strains seem to show a greater level of genetic diversity. Comparison of fluorescent AFLP with other detection methods revealed that fluorescent labeling is more sensitive and practical, in addition, the deleterious effect of radioactivity associated with 32P-labeling, the delicate process of blotting polyacrylamide gels or the tedious procedure of silver staining can be avoided. The automated system facilitated a large number of runs at a time and the subsequent analysis of the data by generating exportable raw data. The congruency of the experiments was analyzed using the Bionumerics software.     

Comparison of different methods for plant regeneration and transformation of the legume Galega orientalis Lam. (goat's rue)

 For the first time, regeneration and transformation have been achieved from the legume Galega orientalis Lam. (goat's rue). Two different regeneration protocols are described, one based on direct shoot induction from meristems and the other involving callus induction and shoot induction from callus with the plant growth regulator thidiazuron (TDZ). Different media and explants were evaluated. Three different transformation methods were compared: cocultivation with four different Agrobacterium tumefaciens strains, electroporation of embryos and apical meristems and particle bombardment of embryos. TDZ-promoted shoot induction on calli from immature embryos gave the best results. Transformation using this regeneration protocol was most successful with particle bombardment. Stable transformation has yet to be proven. Received: 11August 1997 / Revision received: 6 April 1998 / Accepted: 1 March 1999     

Polyamine-induced changes in symbiotic parameters of the Galega orientalis–Rhizobium galegae nitrogen-fixing system

Plants of goat's rue (Galega orientalis) inoculated with Rhizobium galegae strain HAMBI 540 were grown in the presence of putrescine (Put), spermidine (Spd) or spermine (Spm), and several symbiotic characteristics were investigated to delineate the influence of polyamines (PA) on this nitrogen-fixing system. All three PA exerted a concentration-dependent effect on the nodule parameters tested. The increment of nodulation ability and nodule biomass accumulation was extreme (from 2.4- to 4.0-fold) when plants were subjected to 10 and 50 M of various PA. However, at 100 M a negative effect was observed. The acetylene-reduction activity of nodulated roots was increased also in response to treatment with the lower PA concentrations. The level of nitrogenase activity supported by succinate was significantly higher in bacteroids isolated from PA-treated nodules than in bacteroids from control nodules. The symbiotic parameters were also dependent on the type of PA used; the most effective being the diamine Put, while Spm showed a smaller physiological effect with respect to the others. Polyamines altered the ultrastructure of Galega nodule infected cells. After treatment with these substances, pronounced changes in the relative volume of the main components of infected cells and their compartments were observed. The significance of the structural observations and morphometric analyses, their relationship to differences in nitrogen fixation and possible modes of action are discussed.     

Rhizobial 16S rRNA and dnaK genes: mosaicism and the uncertain phylogenetic placement of Rhizobium galegae

The phylogenetic relatedness among 12 agriculturally important species in the order Rhizobiales was estimated by comparative 16S rRNA and dnaK sequence analyses. Two groups of related species were identified by neighbor-joining and maximum-parsimony analysis. One group consisted of Mesorhizobium loti and Mesorhizobium ciceri, and the other group consisted of Agrobacterium rhizogenes, Rhizobium tropici, Rhizobium etli, and Rhizobium leguminosarum. Although bootstrap support for the placement of the remaining six species varied, A. tumefaciens, Agrobacterium rubi, and Agrobacterium vitis were consistently associated in the same subcluster. The three other species included Rhizobium galegae, Sinorhizobium meliloti, and Brucella ovis. Among these, the placement of R. galegae was the least consistent, in that it was placed flanking the A. rhizogenes-Rhizobium cluster in the dnaK nucleotide sequence trees, while it was placed with the other three Agrobacterium species in the 16S rRNA and the DnaK amino acid trees. In an effort to explain the inconsistent placement of R. galegae, we examined polymorphic site distribution patterns among the various species. Localized runs of nucleotide sequence similarity were evident between R. galegae and certain other species, suggesting that the R. galegae genes are chimeric. These results provide a tenable explanation for the weak statistical support often associated with the phylogenetic placement of R. galegae, and they also illustrate a potential pitfall in the use of partial sequences for species identification.     

Genetic diversity of rhizobia isolated from Caragana intermedia in Maowusu sandland,north of China

AIMS: Thirty-three rhizobial strains isolated from nodules of Caragana intermedia in Maowusu sandland were examined for their genetic diversity and putative phylogenetic position. METHODS AND RESULTS: Isolates from Caragana intermedia were classified into 12 genotypes by 16S rDNA polymerase chain reaction-restriction fragment length polymorphism (RFLP), which showed no distinct relationships with those of the reference strains. The genotypes of rhizobia were not related to geographical location. Thr 16S rDNA sequence of representative strain GH2001 from dominant genotype 2 shared high homologuey with some Rhizobium species: Rh. giardinii (96.4%), Rh. huautlense (95.3%), Rh. galegae (95.7%), Rh. yanglingense (95.2%), Rh. mongolense (95.6%), Rh. radiobacter (99%) and Rh. rubi (98.3%). CONCLUSIONS: A high degree of genetic diversity existed among rhizobia nodulating Caragana intermedia in Maowusu sandland. Most of the new isolates might belong to Rhizobium. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that the rich diversity of rhizobia might have contributed to the adaptation of the arid region. These strains could be valuable at the economic and ecosystem level.     

Enhanced competitiveness for nodulation of Medicago sativa by Rhizobium meliloti transconjugants harbouring the root-inducing plasmids of Agrobacterium rhizogenes strain 15834

Abstract The root-inducing plasmid of Agrobacterium rhizogenes strain 15834 marked with transposon Tn 5 -mob RP4-4 with a helper plasmid was mobilized into nitrogen-fixing Rhizobium meliloti strain CIAM 1759. The resulting transconjugants did not induce ‘hairy’ root syndrome but developed nitrogen-fixing nodules on alfalfa. Among the 9 transconjugants tested, 6 strains had increased modulation rates. The competitiveness of 2 of these R. meliloti (pRi) strains was significantly enhanced as compared with the parent strain CIAM 1759; this was confirmed both in tube and in pot tests.     

Identification and structure of the Rhizobium galegae common nodulation genes: evidence for horizontal gene transfer

Rhizobia are soil bacteria able to fix atmospheric nitrogen in symbiosis with leguminous plants. In response to a signal cascade coded by genes of both symbiotic partners, a specific plant organ, the nodule, is formed. Rhizobial nodulation (nod) genes trigger nodule formation through the synthesis of Nod factors, a family of chitolipooligosaccharides that are specifically recognized by the host plant at the first stages of the nodulation process. Here, we present the organization and sequence of the common nod genes from Rhizobium galegae, a symbiotic member of the RHIZOBIACEAE: This species has an intriguing phylogenetic position, being symbiotic among pathogenic agrobacteria, which induce tumors instead of nodules in plant shoots or roots. This apparent incongruence raises special interest in the origin of the symbiotic apparatus of R. galegae. Our analysis of DNA sequence data indicated that the organization of the common nod gene region of R. galegae was similar to that of Sinorhizobium meliloti and Rhizobium leguminosarum, with nodIJ downstream of nodABC and the regulatory nodD gene closely linked to the common nod operon. Moreover, phylogenetic analyses of the nod gene sequences showed a close relationship especially between the common nodA sequences of R. galegae, S. meliloti, and R. leguminosarum biovars viciae and trifolii. This relationship in structure and sequence contrasts with the phylogeny based on 16S rRNA, which groups R. galegae close to agrobacteria and separate from most other rhizobia. The topology of the nodA tree was similar to that of the corresponding host plant tree. Taken together, these observations indicate that lateral nod gene transfer occurred from fast-growing rhizobia toward agrobacteria, after which the symbiotic apparatus evolved under host plant constraint.     

Enhanced competitiveness for nodulation of Medicago sativa by Rhizobium meliloti transconjugants harbouring the root-inducing plasmids of Agrobacterium rhizogenes strain 15834

Abstract The root-inducing plasmid of Agrobacterium rhizogenes strain 15834 marked with transposon Tn5-mob with a helper plasmid RP4-4 was mobilized into nitrogen-fixing Rhizobium meliloti strain CIAM 1759. The resulting transconjugants did not induce ‘hairy’ root syndrome but developed nitrogen-fixing nodules on alfalfa. Among the 9 transconjugants tested, 6 strains had increased nodulation rates. The competitiveness of 2 of these R. meliloti (pRi) strains was significantly enhanced as compared with the parent strain CIAM 1759; this was confirmed both in tube and in pot tests.