Rhizobium tropici nodulates field-grown Phaseolus vulgaris in France

Two hundred and eighty seven isolates of Rhizobium nodulating Phaseolus vulgaris L. were sampled in France from four geographically distant field populations. They were characterized by their colony morphology and by plasmid profiles. A representative sample was further characterized: a) by the ability of each isolate to nodulate a potential alternative host Leucaena leucocephala and to grow on specific media, and b) by RFLP analysis of PCR amplified 16S rRNA genes. On the basis of their phenotypic and genetic characteristics the isolates could be assigned either to Rhizobium leguminosarum bv phaseoli, or to R. tropici. The two species co-occurred at three sites. R. leguminosarum bv phaseoli represented 2%, 4%, 72% and 100% of the population at the four different sites. Eighteen and 22 different plasmid profiles were identified within R. tropici and R. leguminosarum bv phaseoli, respectively. Some of them were conserved between distant geographical regions. The fact that R. tropici was found in France shows that this species is not limited to tropical regions and gives additional evidence of the multi-specific nature of the Phaseolus microsymbiont, even over a geographically limited area.     

Identification and cloning of nodulation genes and host specificity determinants of the broad host-range Rhizobium leguminosarum biovar phaseoli strain CIAT899

Rhizobium Ieguminosarum biovar phaseoli type II strain CIAT899 nodulates a wide range of hosts: Phaseolus vulgaris (beans), Leucaena esculenta (leucaena) and Macroptilium atropurpureum (siratro). A nodulation region from the symbiotic plasmid has been isolated and characterized. This region, which is contained in the overlapping cosmid clones pCV38 and pCV117, is able to induce nodutes in beans, leucaena and siratro roots when introduced in strains cured for the symbiotic plasmid, pSym. In addition, this cloned region extends the host range of Rhizobium meliloti and R. leguminosarum biovar (bv.) trifolii wild-type strains to nodulate beans. Analysis of constructed subclones indicates that a 6.4 kb Hin dlll fragment contains the essential genes required for nodule induction on all three hosts. Rhizobium leguminosarum bv. phaseoli type I strain CE3 nodulates only beans. However, CE3 transconjugants harbouring plasmid pCV3802 (which hybridized to a nodD heterologous probe), were capable of eliciting nodules on leucaena and siratro roots. Our results suggest that the CIAT899 DNA region hybridizing with the R. meliloti nodD detector is involved in the extension of host specificity to promote nodule formation in P. vulgaris, L. esculenta and M. atropurpureum.     

Novel genes related to nodulation, secretion systems, and surface structures revealed by a genome draft of Rhizobium tropici strain PRF 81

Rhizobium tropici is representative of the diversity of tropical rhizobia, besides comprising strains very effective in fixing N₂ in symbiosis with the common bean (Phaseolus vulgaris L.). The genome of a Brazilian commercial inoculant R. tropici strain (PRF 81, =SEMIA 4088), estimated at 7.85 Mb, was analyzed through a total of 9,026 shotgun reads, assembled in 1,668 phrap contigs, and covering ≈30% of the genome. Annotation identified 2,135 coding DNA sequences (CDS), and only 57.2% have possible functions. The genome comprises a mosaic of genes, with CDS showing the highest similarities with 134 microorganisms, none of which represents more than 19% of the CDS with putative known functions. The high saprophytic capacity of PRF 81 may reside in a variety of genes related to transport, biodegradation of xenobiotics, defense, and secretion proteins, many of which were reported for the first time in the present study. Novelty was also found in nodulation (nodG, a double nodIJ system, nodT, nolF, nolG) and capsular polysaccharide genes, showing stronger similarities with Sinorhizobium (=Ensifer) than with the main symbionts of the common bean—R. etli and R. leguminosarum—suggesting that the original host of R. tropici might be another tropical legume or emphasizing the highly promiscuous nature of this rhizobial species.     

The Diversity of Phaseolus-Nodulating Rhizobial Populations Is Altered by Liming of Acid Soils Planted with Phaseolus vulgaris L. in Brazil

PCR-mediated restriction fragment length polymorphism (RFLP) analysis of the 16S-23S rRNA internally transcribed spacer (ITS) region and the 16S rRNA gene indicated that the rhizobial populations isolated from common bean (Phaseolus vulgaris L.) nodules in the unlimed soil from a series of five lime rates applied 6 years previously to plots of an acidic oxisol had less diversity than those from plots with higher rates of liming. Isolates affiliated with Rhizobium tropici IIB and Rhizobium leguminosarum bv. phaseoli were predominant independent of lime application. An index of richness based on the number of ITS groups increased from 2.2 to 5.7 along the soil liming gradient, and the richness index based on “species” types determined by RFLP analysis of the 16S rRNA gene varied from 0.5 to 1.4. The Shannon index of diversity, based on the number of ITS groups, increased from 1.8 in unlimed soil to 2.8 in limed soil, and, based on RFLP analysis of the 16S rRNA gene, ranged from 0.9 to 1.4. In the limed soil, the subpopulation of R. tropici IIB pattern types contained the largest number of ITS groups. In contrast, there were more R. leguminosarum bv. phaseoli types in the unlimed soil with the lowest pH than in soils with the highest pH. The number of ITS (“strain”) groups within R. leguminosarum bv. phaseoli did not change with increased abundance of rhizobia in the soil, while with R. tropici IIB, the number of strain groups increased significantly. Some cultural and biochemical characteristics of Phaseolus-nodulating isolates were significantly related to changes in soil properties caused by liming, largely due to changes in the predominance of the rhizobial species groups.     

Effect of divalent cations on succinate transport in Rhizobium tropici,R. leguminosarum bv phaseoli and R. loti

Rhizobium tropici, R. leguminosarum bv phaseoli and R. loti each have an active C₄-dicarboxylic acid transport system dependent on an energized membrane. Free thiol groups are probably involved at the active site. Since EDTA inhibited succinate transport in R. leguminosarum bv phaseoli and R. loti, divalent cations may participate in the process; the activity was reconstituted by the addition of Ca²⁺ or Mg²⁺. However, EDTA had no effect on succinate transport in R. tropici, R. meliloti or R. trifolii strains. Ca²⁺ or Mg²⁺ had a similar effect on the growth rates of R. tropici and R. leguminosarum bv phaseoli; R. tropici did not require Ca²⁺ to grow on minimal medium supplemented with succinate but R. leguminosarum bv phaseoli required either or both of the divalent cations Ca²⁺ and Mg²⁺. A R. tropici Mu-dI (lacZ) mutant defective in dicarboxylic acid transport, was isolated and found unable to form effective bean nodules.The authors are with the Division of Biochemistry, Instituto de Investigaciones Biológicas Clemente Estable, Avda, Italia 3318, 11.600 Montevideo, Uruguay     

<Emphasis Type="Italic">Rhizobium etli</Emphasis> maize populations and their competitiveness for root colonization

Rhizobium etli, which normally forms nitrogen-fixing nodules on Phaseolus vulgaris (common bean), is a natural maize endophyte. The genetic diversity of R. etli strains from bulk soil, bean nodules, the maize rhizosphere, the maize root, and inside stem tissue in traditional fields where maize is intercropped with P. vulgaris-beans was analyzed. Based on plasmid profiles and alloenzymes, it was determined that several R. etli types were preferentially encountered as putative maize endophytes. Some of these strains from maize were more competitive maize-root colonizers than other R. etli strains from the rhizosphere or from bean nodules. The dominant and highly competitive strain Ch24-10 was the most tolerant to 6-methoxy-2-benzoxazolinone (MBOA), a maize antimicrobial compound that is inhibitory to some bacteria and fungi. The R. tropici strain CIAT899, successfully used as inoculant of P. vulgaris, was also found to be a competitive maize endophyte in inoculation experiments.     

Growth requirements of Rhizobium leguminosarum,strain PRE

Summary Rhizobium leguminosarum, strain PRE, is unable to use sulphate as the sulphur source. Sulfhydryl compounds must be added to achieve growth.Omission of FeCl₃ from the synthetic growth medium resulted in a sharp decrease in growth of this Rhizobium strain as contrasted to other strains of R. leguminosarum. The pyrimidine bases uracil and cytosine could replace FeCl₃. Thymine almost completely inhibited bacterial growth. Adenine and guanine showed no effect. re]19760809     

Selection and symbiotic properties ofRhizobium leguminosarum biovarphaseoli strains harboring pRtr5a

TheRhizobium leguminosarum biovartrifolii symbiotic plasmid pRtr5a has been transferred toR. leguminosarum biovarphaseoli RCR 3644-S1. The transconjugant selection had been done byTrifolium pratense plants. All transconjugants lacked the resident pSym, but had complete pRtr5a, and were Fix⁺ onT. repens andT. alexandrinum, Fix^– onT. subterraneum, and formed a few small white and Fix^– nodules onPhaseolus vulgaris. It is shown that this nodulation onP. vulgaris is due to pRtr5a. The presence of pRtr5a and/or the passage throughTrifolium pratense nodules provoke(s) the recipient strain symbiotic plasmid loss.     

Different species and symbiotic genotypes of field rhizobia can nodulate Phaseolus vulgaris in Tunisian soils

A collection of 160 isolates of rhizobia nodulating Phaseolus vulgaris in three geographical regions in Tunisia was characterized by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified 16S rDNA, nifH and nodC genes. Nine groups of rhizobia were delineated: Rhizobium gallicum biovar (bv.) gallicum, Rhizobium leguminosarum bv. phaseoli and bv. viciae, Rhizobium etli bv. phaseoli, Rhizobium giardinii bv. giardinii, and four groups related to species of the genus Sinorhizobium, Sinorhizobium meliloti, Sinorhizobium medicae and Sinorhizobium fredii. The most abundant rhizobial species were R. gallicum, R. etli, and R. leguminosarum encompassing 29–20% of the isolates each. Among the isolates assigned to R. leguminosarum, two-thirds were ineffective in nitrogen fixation with P. vulgaris and harbored a symbiotic gene typical of the biovar viciae. The S. fredii-like isolates did not nodulate soybean plants but formed numerous effective nodules on P. vulgaris. Comparison of nodC gene sequences showed that their symbiotic genotype was not related to that of S. fredii, but to that of the S. fredii-like reference strain GR-06, which was isolated from a bean plant grown in a Spanish soil. An additional genotype including 16% of isolates was found to be closely related to species of the genus Agrobacterium. However, when re-examined, these isolates did not nodulate their original host.     

Discrete differences between strains of different Rhizobium spp. for competitive nodule occupancy on beans

Rhizobium etli strain TAL182 and R. leguminosarum bv phaseoli strain 8002, both of which produce melanin pigment, were tested for their nodulation competitiveness on beans by paired inoculation with two strains which do not produce melanin: R. tropici strain CIAT899 and Rhizobium sp. strain TAL1145. An assay was developed to distinguish nodules formed by the melanin-producing and non-producing strains. Strain TAL182 had discrete competitive superiority over CIAT899 and TAL1145 for nodulation of beans. Nodulation competitiveness was not correlated with the ability to produce melanin pigment or the host range of the Rhizobium strains tested.The authors are with the Department of Plant Molecular Physiology, University of Hawaii, 3050 Maile Way, Gillmore 402, Honolulu, HI 96822, USA     

The adaptive acid tolerance response in root nodule bacteria and Escherichia coli

Root nodule bacteria and Escherichia coli show an adaptive acid tolerance response when grown under mildly acidic conditions. This is defined in terms of the rate of cell death upon exposure to acid shock at pH 3.0 and expressed in terms of a decimal reduction time, D. The D values varied with the strain and the pH of the culture medium. Early exponential phase cells of three strains of Rhizobium leguminosarum (WU95, 3001 and WSM710) had D values of 1, 6 and 5 min respectively when grown at pH 7.0; and D values of 5, 20 and 12 min respectively when grown at pH 5.0. Exponential phase cells of Rhizobium tropici UMR1899, Bradyrhizobium japonicum USDA110 and peanut Bradyrhizobium sp. NC92 were more tolerant with D values of 31, 35 and 42 min when grown at pH 7.0; and 56, 86 and 68 min when grown at pH 5.0. Cells of E. coli UB1301 in early exponential phase at pH 7.0 had a D value of 16 min, whereas at pH 5.0 it was 76 min. Stationary phase cells of R. leguminosarum and E. coli were more tolerant (D values usually 2 to 5-fold higher) than those in exponential phase. Cells of R. leguminosarum bv. trifolii 3001 or E. coli UB1301 transferred from cultures at pH. 7.0 to medium at pH 5.0 grew immediately and induced the acid tolerance response within one generation. This was prevented by the addition of chloramphenicol. Acidadapted cells of Rhizobium leguminosarum bv. trifolii WU95 and 3001; or E. coli UB1301, M3503 and M3504 were as sensitive to UV light as those grown at neutral pH.     

Accumulation of ppGpp in symbiotic and free-living nitrogen-fixing bacteria following amino acid starvation

Following amino acid or ammonium starvation, ppGpp is accumulated by Rhizobium meliloti strain 1021 but not by R. meliloti strain 41 or Rhizobium tropici. Azorhizobium caulinodans ORS571 produced ppGpp following amino acid deprivation; however, the free-living nitrogen-fixing bacteria Azotobacter vinelandii and Azomonas agilis did not produce ppGpp. Western blot analysis using anti-RelA antibody demonstrated that R. meliloti strain 1021, Azotobacter vinelandii and Azorhizobium caulinodans cross-reacted under conditions that detected RelA in Escherichia coli CF1648. Cross-reaction was not observed in R. meliloti strain 41, R. tropici, or Azomonas agilis. All strains that accumulated ppGpp also produced high intracellular levels of ATP. Received: 28 August 1998 / Accepted: 11 November 1998     

Rhizobium tropiciteu genes involved in specific uptake of Phaseolus vulgaris bean-exudate compounds

Rhizobium tropici nodulates and fixes nitrogen in bean. In the R. tropici strain CFN299 we identified and characterized teu genes (tropiciexudate uptake) induced by bean root exudates, localized by insertion of a promoter-less Tn5-gusA1 transposon. teu genes are present on a plasmid of around 185 kb that is conserved in all R. tropici strains. Proteins encoded by teu genes show similarity to ABC transporters, specifically to ribose transport proteins. No induction of the teu genes was obtained by treatment with root exudates from any of several other plants tested, with the exception of Macroptilium atropurpureum, which is also a host plant for R. tropici. It appears that the inducing compound is characteristic of bean and closely related legumes. It is present in root exudates, but not in seeds. This compound is removed, presumably by metabolism, from the exudates by the majority of bean-nodulating rhizobia (such as R. etli, R. leguminosarum bv. phaseoli and R.␣giardinii). The principal inducing compound has not been identified, but some induction was obtained using trigonelline. The CFN299 strain seems to have an additional uptake system, as no phenotype is observed in two different mutants. R. tropici strain CIAT899, on the other hand, must have only one uptake system, since a mutant bearing an insertion in the teu genes could not remove the compound from the exudates as efficiently as the wild type, and it showed diminished nodulation competitiveness. Received: 21 November 1997 / Accepted: 18 March 1998     

Lipopolysaccharide core components of Rhizobium etli reacting with a panel of monoclonal antibodies

Monoclonal antibodies that react with Rhizobium leguminosarum lipopolysaccharide core antigens (LPS-2) have been used to investigate LPS-2 structure in Rhizobium etli. The panel of antibodies (JIM 32 - JIM 35, JIM 37, JIM 38) specific for LPS-2 of R. leguminosarum strain 3841 and its core components displays similar reactivities towards isolated LPS-2 from R. etli CE109 (a mutant of wild-type strain R. etli CE3 that displays LPS-2 as its main LPS form on the cell surface). This result suggests the antibodies bind to similar epitopes on both strains and, hence, that R. leguminosarum and R. etli have very similar LPS core and lipid A antigen structures. More detailed analysis of the antibody binding sites with isolated LPS-2 and lipid A from R. etli suggests that some of the antibodies (JIM 32, 33, 34, and MASM-I) bind some part of the core oligosaccharides, while others (JIM 35 and JIM 38) involve lipid A. These antibodies have already proven useful in the biochemical analysis of the LPS antigen forms. For example, the loss of reactivity of certain LPS forms with antibody JIM 37 has led to the discovery of a hitherto unnoticed form of the LPS antigen in a precipitate formed during the phenol/water extraction procedure. This new form reacts with the JIM 37 antibody. Furthermore, the positive reaction of some of the antibodies with only sonicated wild-type R. etli cells suggests that either an effective way of masking the display of core antigens on whole bacterial cells is occurring or that core forms of the LPSs are never displayed on the surface of the bacterial cells. Either possibility, once confirmed, could be important for our picture of the Rhizobium cell surface and could also have some bearing on symbiotic nodule infection and development.Abbreviations LPS lipopolysaccharide     

Determination of bean nodule occupancy by <Emphasis Type="Italic">Rhizobium tropici</Emphasis> using the double <Emphasis Type="Italic">gfp</Emphasis> and <Emphasis Type="Italic">gusA</Emphasis> genetic markers constitutively expressed from a new broad-host-range vector

A new broad-host-range vector expressing constitutively the reporter genes gfp and gusA was used to evaluate nodule occupancy of Phaseolus vulgaris nodules by Rhizobium tropici. The results showed that the pHRGFPGUS plasmid was stably maintained in R. tropici over 45 generations and can therefore be used in nodule competitiveness assays. A new method for determining the nodule occupancy using the green fluorescent protein as a marker is described and is shown to be quick, inexpensive and reliable.     

Genes Essential for Nod Factor Production and Nodulation Are Located on a Symbiotic Amplicon (AMPRtrCFN299pc60) in Rhizobium tropici

Amplifiable DNA regions (amplicons) have been identified in the genome of Rhizobium etli. Here we report the isolation and molecular characterization of a symbiotic amplicon of Rhizobium tropici. To search for symbiotic amplicons, a cartridge containing a kanamycin resistance marker that responds to gene dosage and conditional origins of replication and transfer was inserted in the nodulation region of the symbiotic plasmid (pSym) of R. tropici CFN299. Derivatives harboring amplifications were selected by increasing the concentration of kanamycin in the cell culture. The amplified DNA region was mobilized into Escherichia coli and then into Agrobacterium tumefaciens. The 60-kb symbiotic amplicon, which we termed AMPRtrCFN299pc60, contains several nodulation and nitrogen fixation genes and is flanked by a novel insertion sequence ISRtr1. Amplification of AMPRtrCFN299pc60 through homologous recombination between ISRtr1 repeats increased the amount of Nod factors. Strikingly, the conjugal transfer of the amplicon into a plasmidless A. tumefaciens strain confers on the transconjugant the ability to produce R. tropici Nod factors and to nodulate Phaseolus vulgaris, indicating that R. tropici genes essential for the nodulation process are confined to an ampliable DNA region of the pSym.     

Genetic diversity and phylogeny of root nodule bacteria entering into symbiosis with bitter peavine <Emphasis Type="Italic">Lathyrus vernus</Emphasis> (L.) Bernh.

The genetic diversity and phylogeny of root nodule bacteria entering into symbiosis with bitter peavine Lathyrus vernus (L.) Bernh. (Fabaceae) growing in various regions of the Republic of Bashkortostan were studied. RAPD analysis revealed a high degree of polymorphism of the DNA of the isolated strains giving evidence of the heterogeneity of the microorganisms in question. The study of the phylogeny of microsymbionts based on comparative analysis of the nucleotide sequences of 16S rRNA genes showed that the bacteria isolated from the plant nodules of L. vernus growing on the territory of Ufa and Beloretsk raions belonged to the species Rhizobium leguminosarum, whereas the microsymbionts of L. vernus growing on the territory of Tatyshly raion belonged to the species Rhizobium tropici,@ except for several strains of Rhizobium leguminosarum     

Rapid identification ofRhizobium species based on cellular fatty acid analysis

As understanding of the evolutionary relationships between strains and species of root nodule bacteria increases the need for a rapid identification method that correlates well with phylogenetic relationships is clear. We have examined 123 strains ofRhizobium: R. fredii (19),R. galegae (20),R. leguminosarum (22),R. loti (17),R. meliloti (21), andR. tropici (18) and six unknowns. All strains were grown on modified tryptone yeast-extract (TY) agar, as log phase cultures, scraped from the agar, lysed, and the released fatty acids derivatized to their corresponding methyl esters. The methyl esters were analysed by gas-chromatography using the MIDI/Hewlett-Packard Microbial Identification System. All species studied contained 16:0, 17:0, 18:0 and 19cyclo_w9C fatty acids but onlyR loti andR tropici produced 12:0 3 OH,13:0 iso 3 OH,18:1_w9C and 15:0 iso 3 OH,17:0 iso 3 OH and 20:2_w6,9C fatty acids respectively. Principal component analysis was used to show that strains could be divided into clusters corresponding to the six species. Fatty acid profiles for each species were developed and these correctly identified at least 95% of the strains belonging to each species. A dendrogram is presented showing the relationships betweenRhizobium species based on fatty acid composition. The data base was used to identify unknown soil isolates as strains ofRhizobium lacking a symbiotic plasmid and a bacterium capable of expressing a symbiotic plasmid fromR. leguminosarum asSphingobacterium spiritovorum.     

Occurrence of Choline and Glycine Betaine Uptake and Metabolism in the Family Rhizobiaceae and Their Roles in Osmoprotection

The role of glycine betaine and choline in osmoprotection of various Rhizobium, Sinorhizobium, Mesorhizobium, Agrobacterium, and Bradyrhizobium reference strains which display a large variation in salt tolerance was investigated. When externally provided, both compounds enhanced the growth of Rhizobium tropici, Sinorhizobium meliloti, Sinorhizobium fredii, Rhizobium galegae, Agrobacterium tumefaciens, Mesorhizobium loti, and Mesorhizobium huakuii, demonstrating their utilization as osmoprotectants. However, both compounds were inefficient for the most salt-sensitive strains, such as Rhizobium leguminosarum (all biovars), Agrobacterium rhizogenes, Rhizobium etli, and Bradyrhizobium japonicum. Except for B. japonicum, all strains exhibit transport activity for glycine betaine and choline. When the medium osmolarity was raised, choline uptake activity was inhibited, whereas glycine betaine uptake was either increased in R. leguminosarum and S. meliloti or, more surprisingly, reduced in R. tropici, S. fredii, and M. loti. The transport of glycine betaine was increased by growing the cells in the presence of the substrate. With the exception of B. japonicum, all strains were able to use glycine betaine and choline as sole carbon and nitrogen sources. This catabolic function, reported for only a few soil bacteria, could increase competitiveness of rhizobial species in the rhizosphere. Choline dehydrogenase and betaine-aldehyde dehydrogenase activities were present in the cells of all strains with the exception of M. huakuii and B. japonicum. The main physiological role of glycine betaine in the family Rhizobiaceae seems to be as an energy source, while its contribution to osmoprotection is restricted to certain strains.