Agrobacterium strains isolated from root nodules of common bean specifically reduce nodulation by Rhizobium gallicum

In a previous work, we showed that non-nodulating agrobacteria strains were able to colonize root nodules of common bean. Both rhizobia and agrobacteria co-existed in the infected nodules. No impact on symbiosis was found in laboratory conditions when using sterile gravel as a support for growth. In this study, soil samples originating from different geographic and agronomic regions in Tunisia were inoculated with a mixture of agrobacteria strains isolated previously from root nodules of common bean. A significant effect on nodulation and vegetal growth of common bean was observed. Characterization of nodulating rhizobia and comparison with non-inoculated controls showed a biased genetic structure. It seemed that Rhizobium gallicum was highly inhibited, whereas nodulation by Sinorhizobium medicae was favored. Co-inoculation of non-sterile soils with R. gallicum and agrobacteria confirmed these findings. In vitro antibiosis assays indicated that agrobacteria exercised a significant antagonism against R. gallicum.     

Fragmentations of the large-subunit rRNA in the family Rhizobiaceae.

A 130-nucleotide-long rRNA species corresponding to the 5' end of the 23S rRNA gene was found in 96 strains belonging to different Rhizobium, Bradyrhizobium, and Agrobacterium species. Additional fragmentation in the central region of the large-subunit rRNA occurred in all agrobacteria, except Agrobacterium vitis, and in most Rhizobium leguminosarum and Rhizobium etli strains but did not occur in any of the other rhizobia and bradyrhizobia studied.     

Starch Gel Electrophoresis of Catalase and Esterase Isoenzymes from Some Rhizobium and Agrobacterium spp.

S ummary : The esterase and catalase isoenzymes of Agrobacterium tumefaciens and A. radiobacter have been compared to those of the fast growing rhizobia using starch gel electrophoresis. An overall similarity was found for the agrobacteria-rhizobia group although the majority of the agrobacteria possessed only one catalase. Those agro-bacteria with 2 catalases gave patterns similar to Rhizobium meliloti and were 3-ketolactose negative. Agrobacterium pseudotsugi isoenzymes, although fitting the general agrobacteria-rhizobia pattern, were not identical to any specific isogram. The taxonomic value of these findings is discussed.     

Phylogeny and genetic diversity of native rhizobia nodulating common bean (Phaseolus vulgaris L.) in Ethiopia

The diversity and phylogeny of 32 rhizobial strains isolated from nodules of common bean plants grown on 30 sites in Ethiopia were examined using AFLP fingerprinting and MLSA. Based on cluster analysis of AFLP fingerprints, test strains were grouped into six genomic clusters and six single positions. In a tree built from concatenated sequences of recA, glnII, rpoB and partial 16S rRNA genes, the strains were distributed into seven monophyletic groups. The strains in the groups B, D, E, G1 and G2 could be classified as Rhizobium phaseoli, R. etli, R. giardinii, Agrobacterium tumefaciens complex and A. radiobacter, respectively, whereas the strains in group C appeared to represent a novel species. R. phaseoli, R. etli, and the novel group were the major bean nodulating rhizobia in Ethiopia. The strains in group A were linked to R. leguminosarum species lineages but not resolved. Based on recA, rpoB and 16S rRNA genes sequences analysis, a single test strain was assigned as R. leucaenae. In the nodC tree the strains belonging to the major nodulating groups were clustered into two closely linked clades. They also had almost identical nifH gene sequences. The phylogenies of nodC and nifH genes of the strains belonging to R. leguminosarum, R. phaseoli, R. etli and the putative new species (collectively called R. leguminosarum species complex) were not consistent with the housekeeping genes, suggesting symbiotic genes have a common origin which is different from the core genome of the species and indicative of horizontal gene transfer among these rhizobia.     

Bdellovibrio bacteriovorus Parasitizing Rhizobium in Western Australia

S ummary . Bdellovibrio bacteriovorus strains which parasitize Rhizobium meliloti, R. trifolii, Agrobacterium tumefaciens and A. radiobacter have been found in Western Australian soils. Rhizobium lupini was not lysed by any strain. Some Bdellovibrio cells pass through 0·22 μm membrane filters, whilst the 0·45 μm filters allow several saprophytic soil bacteria to pass. The strength of the medium is shown to be of great importance for the detection or counting of Bdellovibrio cells. The Bdellovibrio strains are capable of destroying large numbers of rhizobia in the laboratory.     

Polyamines in Rhizobium, Bradyrhizobium, Azorhizobium and Argobacterium

Abstract Eighteen strains of Rhizobium including four species, R. leguminosarum, R. meliloti, R. loti and R. fredii , nine strains of Bradyrhizobium japonicum and three strains of Azorhizobium caulinodans contained putrescine and honospermidine as major polyamines. All these nodulating N₂-fixing rhizobia lack spermidine. Spermidine and cadaverine were present only in a limited number of R. meliloti and B. japonicum . Polymanine-synthetic activity was not affected by the differences in ability to produce phytoxine (rhizobitoxine and dihydrorhizobitoxine) H₂-uptake-hydrogenation in the organisms. Putrescine and homospermidine were major polyamined in a strain of Agrobacterium rhizogenes . All the eight strains of Agrobacterium tumefaciens as well as A. rubi, A. radiobacter and two other strains of A. rhizogenes contained putrescine and spermidine as major polyamines and homospermidine and spermine (and thermospermine) as minor polyamines.     

Sinorhizobium americanum symbiovar mediterranense is a predominant symbiont that nodulates and fixes nitrogen with common bean (Phaseolus vulgaris L.) in a Northern Tunisian field

A total of 40 symbiotic bacterial strains isolated from root nodules of common bean grown in a soil located in the north of Tunisia were characterized by PCR-RFLP of the 16S rRNA genes. Six different ribotypes were revealed. Nine representative isolates were submitted to phylogenetic analyses of rrs, recA, atpD, dnaK, nifH and nodA genes. The strains 23C40 and 23C95 representing the most abundant ribotype were closely related to Sinorhizobium americanum CFNEI 156(T). S. americanum was isolated from Acacia spp. in Mexico, but this is the first time that this species is reported among natural populations of rhizobia nodulating common bean. These isolates nodulated and fixed nitrogen with this crop and harbored the symbiotic genes of the symbiovar mediterranense. The strains 23C2 and 23C55 were close to Rhizobium gallicum R602sp(T) but formed a well separated clade and may probably constitute a new species. The sequence similarities with R. gallicum type strain were 98.7% (rrs), 96.6% (recA), 95.8% (atpD) and 93.4% (dnaK). The remaining isolates were, respectively, affiliated to R. gallicum, E. meliloti, Rhizobium giardinii and Rhizobium radiobacter. However, some of them failed to re-nodulate their original host but promoted root growth.     

Urea and Biuret as Nitrogen Sources for Rhizobium Spp.

Ability to utilize urea as nitrogen source proved to be universal in 80 fast and 40 slow growing strains of Rhizobium spp. from 23 genera of host plants, and also in 10 strains of Agrobacterium radiobacter. Rhizobium meliloti (32 strains) as well as A. radiobacter constantly failed to utilize biuret. Most rhizobia from other host plants (with 8 exceptions among 88) were able to use biuret as a somewhat suboptimal source of nitrogen, which was generally assimilated at a slower rate than urea and rarely resulted in the same amount of growth, particularly in the fast growing strains; in exceptional cases and mostly among the slow growing strains biuret appeared slightly superior to urea. Adaptation experiments showed that urease occurred as a constitutive enzyme in Rh. leguminosarum , while the biuret decomposing enzyme appeared to be inducible.     

Group antigens in slow-growing rhizobia

Summary Internal group antigens of several slow-growing and fast-growing Rhizobium strains were tested by gel-diffusion against antisera to three strains of Rhizobium japonicum. At least one, generally two common antigens were found in 13 strains of R. japonicum, 4 strains of R. lupini, 4 strains isolated from cowpea and two slow-growing strains isolated from Lotus. Forty-six fast-growing rhizobia (including two from Lotus and 4 from Leucaena leucocephala) were clearly distinguished from the slow-growing strains in tests with the same antisera. They were wholly negative (9) or gave a much weaker non-identical line with one antiserum (24 strains), two antisera (8) or three antisera (5). The 5 strains of agrobacteria grouped with the fast-growing rhizobia.     

Phenotypic expression of mutations in a wide-host-range R plasmid in Escherichia coli and Rhizobium meliloti.

Eight different derivatives of R plasmid RP1 with thermosensitive mutations affecting maintenance in Escherichia coli and Pseudomonas aeruginosa were introduce into Rhizobium meliloti. None of the plasmids showed a thermosensitive character in R. meliloti. On the other hand, a certain deletion mutation in RP1 was found to cause plasmid instability in rhizobia and agrobacteria, but not in E. coli.     

Rhizobium nepotum sp. nov. isolated from tumors on different plant species

Five Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from galls on different plant species in Hungary: strain 39/7(T) from Prunus cerasifera Myrobalan, strain 0 from grapevine var. Ezerjó, strain 7/1 from raspberry var. Findus and in Poland, strain C3.4.1 from Colt rootstock (Prunus avium × Prunus pseudocerasus) and strain CP17.2.2 from Prunus avium. Only one of these isolates, strain 0, is able to cause crown gall on different plant species. On the basis of 16S rRNA gene sequence similarity, the strains cluster together and belong to the genus Rhizobium and their closest relative is Rhizobium radiobacter (99.1%). Phylogenetic analysis of the novel strains using housekeeping genes atpD, glnA, gyrB, recA and rpoB revealed their distinct position separate from other known Rhizobium species and confirmed their relation to Rhizobium radiobacter. The major cellular fatty acids are 18:1 w7c, 16:0, 16:0 3OH, summed feature 2 (comprising 12:0 aldehyde, 16:1 iso I and/or 14:0 3OH) and summed feature 3 (comprising 16:1 w7c and/or 15 iso 2OH). DNA-DNA hybridization of strain 39/7(T) with the type strain of R. radiobacter LMG 140(T) revealed 45% DNA-DNA hybridization. Phenotypic and physiological properties differentiate the novel isolates from other closely related species. On the basis of the results obtained, the five isolates are considered to represent a novel species of the genus Rhizobium, for which the name Rhizobium nepotum sp. nov. (type strain 39/7(T)=LMG 26435(T)=CFBP 7436(T)) is proposed.     

Utilization of octopine and nopaline by Agrobacterium

Tests for utilization of d-octopine and nopaline in defined media containing a carbon and nitrogen source were made on 60 strains of Agrobacterium representing four species and on a representative of each of five species of Rhizobium. Among 46 virulent strains of Agrobacterium, only two strains were found which utilized neither compound, while three strains were found which could utilize both. Of the remaining virulent strains, 27 utilized octopine and 14 utilized nopaline. Each of six strains of A. rhizogenes tested utilized only octopine but at a slower rate relative to growth than most A. tumefaciens. All eight of the A. radiobacter strains failed to utilize either compound, as did four of six nonvirulent strains of A. tumefaciens. The rhizobia did not utilize octopine or, with the possible exception of R. japonicum, nopaline. Virulence in the genus Agrobacterium is concluded to be highly correlated with the ability to utilize one or both of these compounds.     

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.     

Nodulation of Sesbania species by Rhizobium (Agrobacterium) strain IRBG74 and other rhizobia

Concatenated sequence analysis with 16S rRNA, rpoB and fusA genes identified a bacterial strain (IRBG74) isolated from root nodules of the aquatic legume Sesbania cannabina as a close relative of the plant pathogen Rhizobium radiobacter (syn. Agrobacterium tumefaciens ). However, DNA:DNA hybridization with R. radiobacter , R. rubi , R. vitis and R. huautlense gave only 44%, 5%, 8% and 8% similarity respectively, suggesting that IRBG74 is potentially a new species. Additionally, it contained no vir genes and lacked tumour-forming ability, but harboured a sym -plasmid containing nifH and nodA genes similar to those in other Sesbania symbionts. Indeed, IRBG74 effectively nodulated S. cannabina and seven other Sesbania spp. that nodulate with Ensifer ( Sinorhizobium )/ Rhizobium strains with similar nodA genes to IRBG74, but not species that nodulate with Azorhizobium or Mesorhizobium . Light and electron microscopy revealed that IRBG74 infected Sesbania spp. via lateral root junctions under flooded conditions, but via root hairs under non-flooded conditions. Thus, IRBG74 is the first confirmed legume-nodulating symbiont from the Rhizobium ( Agrobacterium ) clade. Cross-inoculation studies with various Sesbania symbionts showed that S. cannabina could form fully effective symbioses with strains in the genera Rhizobium and Ensifer , only ineffective ones with Azorhizobium strains, and either partially effective ( Mesorhizobium huakii ) or ineffective ( Mesorhizobium plurifarium ) symbioses with Mesorhizobium . These data are discussed in terms of the molecular phylogeny of Sesbania and its symbionts.     

Phylogeny of fast-growing soybean-nodulating rhizobia support synonymy of Sinorhizobium and Rhizobium and assignment to Rhizobium fredii.

We determined the sequences for a 260-base segment amplified by the polymerase chain reaction (corresponding to positions 44 to 337 in the Escherichia coli 16S rRNA sequence) from seven strains of fast-growing soybean-nodulating rhizobia (including the type strains of Rhizobium fredii chemovar fredii, Rhizobium fredii chemovar siensis, Sinorhizobium fredii, and Sinorhizobium xinjiangensis) and broad-host-range Rhizobium sp. strain NGR 234. These sequences were compared with the corresponding previously published sequences of Rhizobium leguminosarum, Rhizobium meliloti, Agrobacterium tumefaciens, Azorhizobium caulinodans, and Bradyrhizobium japonicum. All of the sequences of the fast-growing soybean rhizobia, including strain NGR 234, were identical to the sequence of R. meliloti and similar to the sequence of R. leguminosarum. These results are discussed in relation to previous findings; we concluded that the fast-growing soybean-nodulating rhizobia belong in the genus Rhizobium and should be called Rhizobium fredii.     

The variable part of the dnaK gene as an alternative marker for phylogenetic studies of rhizobia and related alpha Proteobacteria

DnaK is the 70 kDa chaperone that prevents protein aggregation and supports the refolding of damaged proteins. Due to sequence conservation and its ubiquity this chaperone has been widely used in phylogenetic studies. In this study, we applied the less conserved part that encodes the so-called alpha-subdomain of the substrate-binding domain of DnaK for phylogenetic analysis of rhizobia and related non-symbiotic alpha-Proteobacteria. A single 330 bp DNA fragment was routinely amplified from DNA templates isolated from the species of the genera, Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium, but also from some non-symbiotic alpha Proteobacteria such as Blastochloris, Chelatobacter and Chelatococcus. Phylogenetic analyses revealed high congruence between dnaK sequences and 16S rDNA trees, but they were not identical. In contrast, the partition homogeneity tests revealed that dnaK sequence data could be combined with other housekeeping genes such as recA, atpD or glnA. The dnaK trees exhibited good resolution in the cases of the genera Mesorhizobium, Sinorhizobium and Rhizobium, even better than usually shown by 16S rDNA phylogeny. The dnaK phylogeny supported the close phylogenetic relationship of Rhizobium galegae and Agrobacterium tumefaciens (R. radiobacter) C58, which together formed a separate branch within the fast-growing rhizobia, albeit closer to the genus Sinorhizobium. The Rhizobium and Sinorhizobium genera carried an insertion composed of two amino acids, which additionally supported the phylogenetic affinity of these two genera, as well as their distinctness from the Mesorhizobium genus. Consistently with the phylogeny shown by 16S-23S rDNA intergenic region sequences, the dnaK trees divided the genus Bradyrhizobium into three main lineages, corresponding to B. japonicum, B. elkanii, and photosynthetic Bradyrhizobium strains that infect Aeschynomene plants. Our results suggest that the 330 bp dnaK sequences could be used as an additional taxonomic marker for rhizobia and related species (alternatively to the 16S rRNA gene phylogeny).     

Phylogenetic assignment and mechanism of action of a crop growth promoting <Emphasis Type="Italic">Rhizobium radiobacter</Emphasis> strain used as a biofertiliser on graminaceous crops in Russia

The taxonomic position of “Agrobacterium radiobacter strain 204,” used in Russia as a cereal crop growth promoting inoculant, was derived by a polyphasic approach. The phenotypic analyses gave very similar biochemical profiles for strain 204, Rhizobium radiobacter NCIMB 9042 (formerly the A. radiobacter type strain) and R. radiobacter NCIMB 13307 (formerly the Agrobacterium␣tumefaciens type strain). High percentage similarities, above the species separation level, were observed between the 16S rRNA, fusA and rpoB housekeeping gene sequences of these three strains, and the genomic DNA–DNA hybridisation of strain 204 against the type strain of R. radiobacter NCIMB 9042 was over 70%. Strain 204 is not phytopathogenic and it does not fix atmospheric N₂ or form a physical association with the roots of barley. Strain 204 culture and culture supernatant stimulated the rate of mobilisation of seed reserves of barley in darkness and promoted its shoot growth in the light. Gibberellic acid (GA) concentration was 1.3 μM but indole acetic acid was undetectable (<50 nM) in cultures of strain 204. It is concluded that strain 204 is phenotypically and genotypically very similar to the current R. radiobacter type strain and that the mechanism of its effect on growth of cereals is via the production of plant growth promoting substances. GA is likely to play an important role in the strain 204 stimulation of early growth of barley.     

Deoxyribonucleic acid homology and taxonomy of Agrobacterium, Rhizobium, and Chromobacterium

Hybridization experiments were carried out between high molecular weight, denatured, agar-embedded deoxyribonucleic acid (DNA) and homologous, nonembedded, sheared, denatured (14)C-labeled DNA from a strain of Agrobacterium tumefaciens and Rhizobium leguminosarum (the reference strains) in the presence of sheared, nonembedded, nonlabeled DNA (competing DNA) from the same or different nomen-species of Agrobacterium, Rhizobium, Chromobacterium, and several other organisms. Percentage of DNA homology was calculated from the results. The findings are discussed in relation to previous taximetric studies, present classification schemes, and guanine-cytosine content of the DNA. Strains of A. tumefaciens, A. radiobacter, A. rubi, A. rhizogenes, R. leguminosarum, and R. meliloti exhibited a mean percentage of DNA homology greater than 50 with the two reference strains. A. tumefaciens, A. radiobacter, and A. rubi were indistinguishable on the basis of DNA homology, with strain variations for this group involving up to 30% of their base sequences. The remainder of the organisms studied fall into at least six distinct genetic groups: (i) R. (Agrobacterium) rhizogenes, which is more homologous to R. leguminosarum than to the A. tumefaciens-A. radiobacter group; (ii) R. leguminosarum; (iii) R. meliloti; (iv) R. japonicum, which has a mean DNA homology of some 38 to 45% with the reference strains; (v) Chromobacterium, which is as genetically remote from the reference strains as, for example, Pseudomonas; and (vi) A. pseudotsugae strain 180, which has a DNA homology with A. tumefaciens and R. leguminosarum of only about 10%. Since this latter homology value is similar to what was found after hybridizations between the reference strains and organisms such as Escherichia coli and Bacillus subtilis, A. pseudotsugae should definitely be removed from the genus.     

Acquisition of an Agrobacterium Ri plasmid and pathogenicity by other alpha-Proteobacteria in cucumber and tomato crops affected by root mat

Root mat of cucumbers and tomatoes has previously been shown to be caused by Agrobacterium radiobacter strains harboring a root-inducing Ri plasmid (pRi). Nine other pRi-harboring alpha-Proteobacteria have subsequently been isolated from root mat-infected crops. Fatty acid profiling and partial 16S rRNA sequence analysis identified three of these strains as being in the genus Ochrobactrum, five as being in the genus Rhizobium, and one as being in the genus Sinorhizobium: An in vitro pathogenicity test involving inoculation of cucumber cotyledons was developed. All pRi-harboring alpha-Proteobacteria induced typical root mat symptoms from the cotyledons. Average transformation rates for rhizogenic Ochrobactrum (46%) and Rhizobium (44%) strains were lower than those observed for rhizogenic A. radiobacter strains (64%). However, individual strains from these three genera all had transformation rates comparable to those observed from cotyledons inoculated with a rhizogenic Sinorhizobium strain (75%).     

Rhizobium selenireducens sp. nov.: A Selenite-Reducing α-Proteobacteria Isolated From a Bioreactor

A Gram-negative, nonpigmented bacterium designated strain B1 was isolated from a laboratory bioreactor that reduced selenate to elemental red selenium (Se(0)). 16S rRNA gene-sequence alignment identified the isolate as a Rhizobium sp. belonging to the Rhizobium clade, which includes R. daejeonense, R. giardinii, R. undicola, R. larrymoorei, R. radiobacter, R. rubi, and R. vitis. R. radiobacter and R. rubi are its closest relatives as indicated by 16S rRNA gene-sequence alignments, which differ from strain B1 by 2.6% and 2.8%, respectively. Within this group, strains that show variances > 0.8% to 2.2% have been classified as different species. The major cellular fatty acids present in the B1 strain were C16:0 (1.8%), C18:0 (3.38%), 18:0 3-OH (1.6%), 18:1 omega7c (86.8%), 19:0 cycloomega8c (1.5%), and summed features 2 (3.8%) and 3 (1.2%). The large amount of 18:1 omega7c present is constant with members of this group of bacteria, but the small amounts of 16:0, 19:0 cycloomega8c, and summed feature 3 shows variance from R. radiobacter and R. rubi. The strain's phenotypic and biochemical characteristics are consistent with its placement in this genus.