Rhizobium Leguminosarum Biovar Phaseoli Research Articles

Rhizobium nod Gene Inducers Exuded Naturally from Roots of Common Bean (Phaseolus vulgaris L.)

Four compounds exuded from young roots of a black-seeded bean (Phaseolus vulgaris L., cv PI165426CS) induce transcription of nod genes in Rhizobium leguminosarum biovar phaseoli. The three most active nod gene inducers were identified by spectroscopic methods (ultraviolet/visible absorbance, proton nuclear magnetic resonance, and mass spectrometry) as being eriodictyol (5,7,3',4' -tetrahydroxyflavanone), naringenin (5,7,4' -trihydroxyflavanone), and a 7-O-glycoside of genistein (5,7,4' -trihydroxyisoflavone). Comparisons with authentic standards verified the chemical structures of the aglycones and their capacity to induce beta-galactosidase activity in R. leguminosarum strains containing nodA-lacZ or nodC-lacZ fusions controlled by R. leguminosarum biovar phaseoli nodD genes. Roots of 9-day-old seedlings released 42, 281, and 337 nanomoles per plant per day of genistein, eriodictyol, and naringenin, respectively. Genistein and naringenin induced higher maximum beta-galactosidase activities and required lower concentrations for half-maximum induction than eriodictyol. Comparing the nod gene-inducing activity of seed rinses with root exudate from PI165426CS bean showed that root flavonoids were released at about 6% the rate of those from seeds on a molar basis, but on average the individual compounds from roots were approximately three times more active than nod gene inducers from seeds.

Formation and structure of root nodules induced on Macroptilium atropurpureum inoculated with various species of Rhizobium

Fifteen strains of Rhizobium leguminosarum biovar trifolii formed ineffective nodules and (or) nodule-like structures (rhizobia were re-isolated from both structures) on Macroptilium atropurpureum grown in enclosed glass tubes. Bacteria were observed among the parenchyma cells surrounding the nodule-like structures. One variant of R. leguminosarum biovar trifolii (NGR66/ST) isolated from M. atropurpureum formed nodules on this host that exhibited abnormal intercellular and intracellular infection. The bacteria (NGR66/ST) were contained within threadlike structures, surrounded by matrix material. The identities of the Rhizobium strains were confirmed serologically after reisolation and in sections of nodule tissue using immunogold labelling. Rhizobium leguminosarum biovar phaseoli strain NGR76 isolated from Phaseolus vulgaris formed nodules on M. atropurpureum resembling those formed by effective Bradyrhizobium strains. The association was partially effective in nitrogen fixation, and this was reflected in the nodule structure. The percentage of cells infected was lower than that in fully effective nodules. There was a high frequency of infected cells showing degeneration; these were located throughout the nodule tissue and were often adjacent to healthy infected cells. The rhizobia appeared to infect new nodule cells via infection threads, which were abundant both intercellularly and intracellularly in young, mature, and degenerating host nodule cells. Strains of R. leguminosarum biovar viceae and Rhizobium meliloti were unable to induce nodule-like structures on M. atropurpureum. Key words: Macroptilium, Bradyrhizobium, Rhizobium, microscopy, nodule, structure.

Competitiveness of KIM 5 and VIKING 1 bean rhizobia: Strain by cultivar interactions

Competitiveness, which in rhizobia refers to the relative ability of a strain to inject a legume and cause nodule formation in the presence of other strains, is critical to biological N 2-fixation. The mechanisms for strain competitiveness are unknown, but are probably affected by biotic and abiotic factors. The competitiveness of two strains of rhizobia ( Rhizobium leguminosarum biovar phaseoli) KIM 5 and VIKING 1 that are known to be highly competitive were evaluated when added together as inoculant to 12 bean ( Phaseolus vulgaris L.) host cultivars grown in Leonard jars in sterilized sand. Strain identification of bacteria from harvested nodules was determined by fluorescent antibodies. KIM 5 was generally more competitive than VIKING 1, but competitiveness of both strains was highly dependent on strain by cultivar interactions. With Tendergreen, no nodules contained VIKING 1 alone, whereas with Jampa, 46.3 of the nodules contained only VIKING 1. Comparisons of parent cultivar and sib progeny lines showed that even small differences in the host genotype affected the competitiveness of both strains. All symbioses were effective in fixing N 2. Based on these data, studies evaluating the competitiveness of rhizobia should utilize several host genotypes to avoid misinterpretations of the competitiveness of a given strain.

Stimulation of α-glucosidases from fast-growing rhizobia and Agrobacterium tumefaciens by K+, NH+4, and Rb+

Extracts from cultured fast-growing rhizobia and Agrobacterium tumefaciens contain enzymes for hydrolysis of the α-glucosides maltose, sucrose, and α,α-trehalose. The hydrolysis of all three sugars was stimulated by the presence of K+, Rb+, or [Formula: see text]. This stimulation varied from less than 2-fold to more than 12-fold, depending on the bacterial species, culture conditions, and experimental conditions, such as type of enzyme, buffer, and ion concentration. Eight other ions tested, including several divalent cations, did not have any stimulatory effect. Other sources of enzyme (Escherichia coli, Saccharomyces cerevisiae, Oryza sativa, porcine kidney, and Medicago sativa and Glycine max nodule cytosol) contained α-glucosidases that differed in both substrate specificity and pH optima and were not affected by K+, Rb+ or [Formula: see text] ions. Bacteroids from G. max and Phaseolus vulgaris nodules did not have detectable α-glucosidase activity. Growth of Rhizobium leguminosarum biovar phaseoli USDA 2667 with one of the α-glucosides as carbon source increased Vm and substrate affinity for all three disaccharidase activities. The pH optimum for all three enzyme activities in R. leguminosarum bv. phaseoli USDA 2667 was 6.6. Stimulation by specific monovalent cations appears to be a novel property of α-glucosidases in the bacterial family Rhizobiaceae. Key words: maltose, sucrose, trehalose, disaccharidases, Rhizobiaceae.

Two genes that regulate exopolysaccharide production in Rhizobium sp. strain NGR234: DNA sequences and resultant phenotypes

Two closely linked genes involved in the regulation of exopolysaccharide (EPS) production in Rhizobium sp. strain NGR234, exoX and exoY, were sequenced, and their corresponding phenotypes were investigated. Inhibition of EPS synthesis occurred in wild-type strains when extra copies of exoX were introduced, but only when exoY had been deleted or mutated or was present at a lower copy number. Normal EPS synthesis occurred in Rhizobium sp. when both exoX and exoY were introduced on the same replicon. Surprisingly, the presence of multiple copies of exoY in exoY:: Tn5 mutants of NGR234 adversely affected cellular growth. This was apparent when exoY was introduced into exoY mutants on IncP1 vectors, where the copy number was approximately 10, but was not apparent when present on much larger R-prime plasmids with lower copy numbers (approximately 3 per cell). Multiple copies of exoX did not adversely affect cellular growth of any strain. The exoX gene appeared analogous, in size and phenotype, to a previously described Rhizobium leguminosarum biovar phaseoli EPS gene, psi (D. Borthakur and A.W.B. Johnston, Mol. Gen. Genet. 207:149-154, 1987), and the deduced ExoX and Psi shared strikingly similar secondary structures. Despite this, ExoX and Psi showed little homology at the primary amino acid level, except for a central region of 18 amino acids. The interaction of ExoX and ExoY could form the basis of a sensitive regulatory system for EPS acids. The interaction of ExoX and ExoY could form the basis of a sensitive regulatory system for EPS biosynthesis. The presence of a multicopy exoX in Rhizobium meliloti and R. fredii similarly abolished EPS biosynthesis in these species.

Gelation of members of a family of branched anionic heteropolysaccharides produced by certain strains of Rhizobium leguminosarum

The structure of the anionic extracellular slime produced by Rhizobium leguminosarum biovar phaseoli 8002 has been shown to be unaltered by variation of the Sym plasmid. The polysaccharide is a member of a family of branched-anionic polysaccharides. Rheological studies have demonstrated gelation of members of this family of polysaccharides produced by certain R. leguminosarum strains. Gelation was found to be dependent upon the ionic strength and salt composition of the sample. Aqueous preparations of the purified polysaccharides did not gel. Thermoversible gels were formed upon addition of salts to the aqueous preparations. Divalent cations were found to be more effective than monovalent cations in promoting gelation. Possible consequences of the gelation of the extracellular polysaccharides, relating to non-specific adhesion of the bacteria to plant roots, water retention in soils, and soil aggregation and stability, are discussed.

Analysis of Periplasmic Enzymes in Intact Cultured Bacteria and Bacteroids of Bradyrhizobium japonicum and Rhizobium leguminosarum biovar phaseoli

Analysis of periplasmic enzymes of Bradyrhizobium japonicum or Rhizobium leguminosarum biovar phaseoli was hampered by the fact that only small amounts of marker enzyme activities were released from cells by osmotic shock or by lysozyme/EDTA treatment. However, up to 95 % of total activity of certain periplasmic marker enzymes could be measured in intact cells by including 003% Triton X-100 in enzyme assays. Less than 5% of the cytoplasmic marker enzyme -hydroxybutyrate dehydrogenase could be measured under these conditions, indicating that treatment with dilute detergent does not significantly perturb the cytoplasmic membrane. Various lines of evidence indicate that the dilute detergent treatment does not significantly alter wall structure but, instead, permits the assay of periplasmic enzymes by facilitating diffusion of substrates and products to and from the periplasmic space. For pyrophosphatase and phosphodiesterase, the proportion of total enzyme activity which could be measured in intact cells was higher in bacteroids than in bacteria at all Triton concentrations, suggesting that the outer membrane of bacteroids may be more permeable than the outer membrane of cultured bacteria. The effect of detergent on the hydrolysis of four disaccharides sucrose, α,α-trehalose, maltose and lactose was studied using intact cultured R. leguminosarum biovar phaseoli. The response of lactose hydrolysis to the addition of detergent was four-fold greater than the response of the other disaccharidase activities, indicating that a rhizobial lactase may be present in the periplasmic space.

Acetylene reduction, hydrogen evolution and nodule respiration in Phaseolus vulgaris

In an experiment performed under greenhouse conditions, four cultivars of Phaseolus vulgaris L. (Venezuela-350; Aroana; Moruna; Carioca) were inoculated with three Rhizobium leguminosarum biovar phaseoli strains (C-05; C-40 = CIAT 255; C-89 = CIAT 55) and were fertilized with an N-free mineral nutrient solution. The plants were harvested 25, 40, and 55 days after emergence and the following paramenters were evaluated: Nitrogenase activity of nodulated roots, H2 evolution by the nodules; relative efficiency of nitrogenase; respiration rates of nodulated roots and detached nodules; dry weight and total N of stems, leaves, pods, roots, and nodules. Generally the bean cultivar, Rhizobium strain, had an effect and there was an interaction effect with both symbiotic partners, on all parameters. On average, nodules represented 23% of total root respiration but the best symbiotic combinations showed lower ratios of C respired to N fixed. The maximum N-assimilation rate (between 40 and 55 days after emergence) of 11.93 mg N plant−1 day−1 occurred with the symbiotic combination of Carioca × C-05, while the poorest rate of 0.55 mg N plant−1 day−1 was recorded with Venezuela-350 × C-89. The best symbiotic combinations always showed the highest relative nitrogenase efficiency, but the differences in N2-fixation rates cannot be explained solely in terms of conservation of energy by recycling of H2. This requires further investigation.

Strain identification of highly-competitive bean rhizobia isolated from root nodules: Use of fluorescent antibodies, plasmid profiles and gene probes

Kim 5 and Viking 1 are highly-competitive N-fixing bean rhizobia ( Rhizobium leguminosarum biovar phaseoli). Leonard jar studies were used to determine which of the two isolates was the most competitive. A mixed inoculant of both strains was added to bean seed ( Phaseolus vulgar is cultivar Tendergreen) in sterile sand. Single-strain inoculants were used for seed grown in soil containing indigenous rhizobia. Nodule occupancy was determined after harvest. Plasmid profiles of nodule isolates from bean seed inoculated with equal numbers of both strains showed that Kim 5 occupied 72% of the nodules. Plasmid profiles for Kim 5 revealed three distinctive bands as compared to two bands from Viking 1. One band appeared to be common to both strains, whereas Kim 5 had two unique bands. A 32P-labelled gene probe to one of the unique Kim 5 plasmids was used to-detect Kim 5 isolated from nodules by colony hybridization. This technique showed that Kim 5 was found in 76.2% of the nodules. Fluorescent antibodies were also used to distinguish between isolates. When single-strain inoculants were used in non-sterile soil, fluorescent antibodies showed that Kim 5 was recovered in 67.5% of the nodule isolates compared to 17.6% for Viking 1, while the other isolates were indigenous rhizobia. These data demonstrate that Kim 5 is more competitive than Viking 1.

Symbiotic Plasmid Rearrangement in a Hyper-recombinant Mutant of Rhizobium leguminosarum biovar phaseoli

The symbiotic plasmid of Rhizobium leguminosarum biovar phaseoli strain CFN23 has been previously shown to undergo a series of genetic rearrangements that modify the bacterial symbiotic phenotype. A hyper-recombinant derivative of strain CFN23 was isolated. This derivative (strain CFN2300) has a similar phenotype to that reported for Escherichia coli mutants defective in DNA metabolism. The frequency of CFN23 symbiotic plasmid deletion is increased in the CFN2300 background, suggesting that homologous genetic recombination is involved in the generation of this genetic rearrangement.

TEMPERATURE-SENSITIVE NODULATION AND N2 FIXATION OF Rhizobium leguminosarum BIOVAR Phaseoli STRAINS

Two strains of Rhizobium leguminosarum biovar phaseoli, both previously Nod+Fix+ and efficient in N2 fixation, now have altered symbiotic properties presumably through spontaneous mutation. Rhizobium leguminosarum biovar phaseoli strain RCR 3605 was Nod−Fix− in a cool growth temperature regime (14–23 °C) and Nod+Fix− in a warm regime (17–23 °C) on seven of nine bean cultivars studied. Rhizobium leguminosarum biovar phaseoli strain CIAT 57 was Nod+Fix− in both temperature regimes. CIAT 57 and RCR 3605 are synonyms for strains derived from the same parent strain but housed in different culture collections. The nodulation deficiency in CIAT 57 is presumably genetically predetermined in the bacterium rather than in the plant because no Nod+Fix+ phenotype was identified on nine bean cultivars tested. A control R. leguminosarum biovar phaseoli strain, RCR 3644, was efficient on all cultivars. In the warm temperature regime, the Nod+Fix− phenotype was observed with strain RCR 3605 only on some cultivars, suggesting that the host plant genome played a role in this ineffective symbiosis. It is likely that deleterious genetic mutations during prolonged storage may be altering the N2-fixing ability of these rhizobial strains and that commercial inoculants must be continually monitored for symbiotic performance of strains with a variety of host plant cultivars. Key words: Field bean, Rhizobium leguminosarum biovar phaseoli, N2 fixation