The effect of waterlogging on the synthesis of the nitrogenase components in bacteroids of Rhizobium leguminosarum in root nodules of Pisum sativum

The effect of waterlogging of root nodules on nitrogenase activity and synthesis was studied in Pisum sativum inoculated with Rhizobium leguminosarum (strain PRE). It was shown that: 1. nitrogenase activity of intact pea plants was decreased by waterlogging, 2. this decrease was paralleled by a decline of the amount of active nitrogenase determined in toluene EDTA treated bacteroids, 3. SDS-polyacrylamide gel electrophoresis revealed that the amount of nitrogenase component II (CII) decreased by waterlogging while the amount of component I (CI) was not markedly affected, and 4. analysis of bacteroid proteins after ³⁵SO₄ labeling of pea plants showed that CII synthesis was repressed while CI synthesis continued indicating that the synthesis of CI and CII is regulated by independent mechanisms.     

Presence of Rhizobium Etli bv . Phaseoli and Rhizobium Gallicum bv . Gallicum in Egyptian Soils

Seven bean rhizobial strains EBRI 2, 3, 21, 24, 26, 27 and 29 identified as Rhizobium etli, and EBRI 32 identified as Rhizobium gallicum, isolated from Egyptian soils and which nodulated Phaseolus vulgaris efficiently, were subjected to hybridization with a nifH probe in order to estimate the copy number of this gene. Seven strains (EBRI 2, 3, 21, 24, 26, 27 and 29) which were only able to nodulate Phaseolus vulgaris, contained three copies of the nifH gene, consistent with their identification as Rhizobium etli bv. phaseoli. Only one strain (EBRI 32) which nodulated both Phaseolus vulgaris and Leucaena leucocephala, had one copy of nifH gene. This confirmed the classification of this strain as Rhizobium gallicum bv. gallicum.     

Appearance of a novel form of plant glutamine synthetase during nodule development in Phaseolus vulgaris L.

The activities of glutamine synthetase (GS), nitrogenase and leghaemoglobin were measured during nodule development in Phaseolus vulgaris infected with wild-type or two non-fixing (Fix^-) mutants of Rhizobium phaseoli. The large increase in GS activity which was observed during nodulation with the wild-type rhizobial strain occurred concomitantly with the detection and increase in activity of nitrogenase and the amount of leghaemoglobin. Moreover, this increase in GS was found to be due entirely to the appearance of a novel form of the enzyme (GS_n1) in the nodule. The activity of the form (GS_n2) similar to the root enzyme (GS_r) remained constant throughout the experiment. In nodules produced by infection with the two mutant strains of Rhizobium phaseoli (JL15 and JL19) only trace amounts of GS_n1 and leghaemoglobin were detected.Abbreviations DEAE-Sephacel diethylaminoethyl-Sephacel - GS glutamine synthetase     

In vitro andin vivo nitrogenase activity ofRhizobium mutants and their symbiotic effectivity

A slow growing nitrogen-fixing strain ofVigna radiata var.aureus (mung bean)Rhizobium which expressed nitrogenase activity in a synthetic medium was isolated from its native population. Mutants with decreased and increased nitrogenase activity were derived from this strain by treatment with acridine orange and ethidium bromide. These mutants were tested for symbiotic effectivity invivo. The effectivity of mutants with decreased nitrogenase activity in the culture medium was lower than the parent strain; however, the effectivity of mutants with higher nitrogenase activity did not increase above that of the parent. This suggests that the plant is perhaps a limiting factor in the full expression of rhizobial nitrogenase in the nodules.     

Characterization of Rhizobial Isolates of Phaseolus vulgaris by Staircase Electrophoresis of Low-Molecular-Weight RNA

Low-molecular-weight (LMW) RNA molecules were analyzed to characterize rhizobial isolates that nodulate the common bean growing in Spain. Since LMW RNA profiles, determined by staircase electrophoresis, varied across the rhizobial species nodulating beans, we demonstrated that bean isolates recovered from Spanish soils presumptively could be characterized as Rhizobium etli, Rhizobium gallicum, Rhizobium giardinii, Rhizobium leguminosarum bv. viciae and bv. trifolii, and Sinorhizobium fredii.     

Role of bacterial polysaccharides in the derepression of ex-planta nitrogenase activity with rhizobia

Abstract Since bacterial polysaccharides may limit the availability of oxygen to the cells, we have investigated the role of rhizobial extracellular polysaccharides (EPS) and the non-rhizobial polyscharide, xanthan, in the depression of ex-planta nitrogenase activity with rhizobia in liquid medium. Two rhizobial strains known to exhibit ex-planta nitrogenase activity on solid media were used; the slow-growing Bradyrhizobium japonicum USDA 110 and the arctic Rhizobium strain N31, both being prolific EPS producers. In low nitrogen mannitol (LNM) liquid medium strain N31 exhibited nitrogenase activity only after 15 days, when sufficient EPS had accumulated in the medium, and activity was correlated with EPS production. When rhizobial EPS from an old culture was added to the LNM medium, nitrogenase activity was detected after 48 h incubation, indicating that EPS of the medium decreased oxygen diffusion to cells to a level that depressed nitrogenase activity. In modified LNM medium with xanthan nitrogenase activity was readily depressed. In both strains activity increased with increased xanthan concentration, but decreased sharply at higher concentrations. Strain N31 exhibited a narrower range of polysaccharide concentration for nitrogenase activity than the slow strain USDA 110. Thus, the condition for derepression of nitrogenase might be a careful balancing of the oxygen concentration surrounding the cells, and this condition is met when a balancing of polsaccharide, either synthesized by the rhizobia or added to the medium, can permit oxygen diffusion to within the narrow range required for the depression and expression of nitrogenase.     

Hydrogen (h(2)) evolution by rhizobia after synergetic culture with soybean cell suspensions

Rhizobium japonicum cells were grown in liquid suspension cultures and separated from soybean plant cells by two to three bacterial membrane filters. Under these conditions, the plant cells elaborated materials into the medium which aided in the expression of a major rhizobial phenotype, namely, nitrogenase activity (acetylene reduction). The evolution of H₂ was also measured and this activity relative to acetylene reduction, was influenced by: (a) O₂; (b) the quantity of conditioned plant medium; and (c) ammonia. It is concluded that plant substances are of major importance in the H₂ evolution and nitrogenase activities of free-living rhizobia in suspension cultures.     

Characterization of the fixABC region of Azorhizobium caulinodans ORS571 and identification of a new nitrogen fixation gene

Summary The fast growing strain, Azorhizobium caulinodans ORS571, isolated from stem nodules of the tropical legume Sesbania rostrata, can grow in the free-living state at the expense of molecular nitrogen. Five point mutants impaired in nitrogen fixation in the free-living state have been complemented by a plasmid containing the cloned fix-ABC region of strain ORS571. Genetic analysis of the mutants showed that one was impaired in fixC, one in fixA and the three others in a new gene, located upstream from fixA and designated nifO. Site-directed Tn5 mutagenesis was performed to obtain Tn5 insertions in fixB and fixC. The four genes are required for nitrogen fixation both in the free-living state and under symbiotic conditions. The nucleotide sequence of nifO was established. The gene is transcribed independently of fixA and does not correspond to fixX, recently identified in Rhizobium meliloti and R. leguminosarum. Biochemical analysis of the five point mutants showed that they synthesized normal amounts of nitrogenase components. It is unlikely that fixA, fixC and nifO are involved in electron transport to nitrogenase. FixC could be required for the formation of a functional nitrogenase component 2.     

Effect of plant nutrient supply on nodule effectiveness and rhizobium strain competition for nodulation ofLotus pedunculatus

Summary The effect of nutrient supply on nodule formation and competition between Rhizobium strains for nodulation ofLotus pedunculatus was studied. Limiting plant growth by decreasing the supply of nutrients in an otherwise nitrogen-free medium, increased the size but decreased the number and the nitrogenase activity of nodules formed by a fast-growing strain of Lotus Rhizobium (NZP2037). In contrast decreasing nutrient supply caused only a small decline in the size, number and nitrogenase activity of nodules formed by a slow-growing strain (CC814s). Providing small quantities of NH₄NO₃ (50 to 250 g N) to plants grown with a normal supply of other nutrients stimulated nodule development by both Rhizobium strains and increased the nitrogenase activity of the NZP2037 nodules. Differences in the level of effectiveness (nitrogen-fixing ability) of nodules formed by different Rhizobium strains on plants grown with a normal supply of nutrients were less apparent when the plants were grown with decreased nutrient supply or when the plants were supplied with low levels of inorganic N.Inter-strain competition for nodulation ofL. pedunculatus between the highly effective slow-growing strain CC814s and 7 other fast- and slow-growing strains, showed CC814s to form 42 to 100% of the nodules in all associations. The greater nodulating competitiveness of strain CC814s prevailed despite changes in the nutrient supply to the host plant. A tendency was observed for partially effective Lotus Rhizobium strains to become more competitive in nodule formation when plant growth was supplemented with low levels of inorganic nitrogen.     

Effect of Agglutinins from Rhizobium leguminosarum Strain 252 on the Activity of Hydrolytic Enzymes

Cells of the nitrogen-fixing soil bacterium Rhizobium leguminosarum 252 and its hemagglutination-deficient mutant strain 252/7 were found to possess the activities of a variety of hydrolytic enzymes. The agglutinin proteins of rhizobia diminished β-glucosidase activity, pectinolytic activity, and acid and alkaline phosphatase activities while completely inhibiting proteolytic enzyme activity in the bacterial cell. The results here show that rhizobial agglutinins are capable of affecting enzyme functioning in Rhizobium. Received: 18 November 1999 / Accepted: 10 February 2000     

Gene centres,a source for genetic variants in symbiotic nitrogen fixation: The symbiotic response of the cultivated pea toRhizobium leguminosarum strains from Europe and the Middle East

Summary Soil samples from several European countries; Sweden, the Netherlands, Spain, Italy and Greece, contained rhizobial populations capable of forming an effective symbiosis with the cultivated pea cv. Rondo from the Netherlands. The range of variation among the European Rhizobium strains, as expressed on pea cv. Rondo, was not so large and almost the same variation could be found within the rhizobial population within each country. Superior Rhizobium strains for the Dutch pea were not restricted to soils from the Netherlands but were also found in those from Sweden and Italy.Soils from Turkey and Israel also contained Rhizobium strains capable of nodulating pea cv. Rondo. However, the genetic variation among these Middle East Rhizobium strains was much larger than that of the European strains. When tested on pea cv. Rondo the majority of the Middle East strains belonged to the medium or low effective classes and only a few strains were comparable with European Rhizobium strains.Dutch Rhizobium strains induced effective nodules on both the Dutch pea cv. Rondo and the Swedish cv. L 110. However, in association with a Turkish Rhizobium strain effective nodules were formed on pea cv. Rondo and ineffective nodules on cv. L 110.We suggest that the genetic uniformity of EuropeanR. leguminosarum strains is the result of selection and domestication of Rhizobium strains originally derived from the gene centres of the pea plant.     

Effect of the VAM fungus Glomus sp. on the growth and yield of soybean inoculated with Bradyrhizobium japonicum

The effect of two Bradyrhizobium japonicum strains (D344 and Urbana), on the frequency and intensity of infection by a VAM fungal Glomus sp. and the effect of VAM on biomass production by nodulating plants were tested in soybean growing in a soil containing low levels of accessible P and N. During the initial stage of vegetative growth, mycorrhiza frequency in roots inoculated with the two rhizobial strains did not differ. However, during flowering it was 178% higher in roots with the strain D344 than in the presence of the strain Ubrana. At final harvest (green pods) the VAM frequency did not differ in the presence of either strain. VAM positively affected biomass production, foliar concentrations of P, Zn and Cu, and number and dry matter yield of pods, but did not increase concentrations of total N and K. In nonmycorrhizal plants total nitrogenase activity (not nodule mass) and growth were higher with the rhizobial strain Urbana. The greatest nitrogenase activity, growth and yield occurred in the presence of the VAM fungus, and did not differ for plants with different strains of rhizobia.     

Identification ofRhizobium strains and evaluation of their competitiveness

Six strains ofRhizobium leguminosarum bv.viciœ, three strains ofBradyrhizobium japonicum and three strains ofRhizobium fredii were evaluated by the polymerase chain reaction (PCR). The possibility of identification of individual rhizobial strains and the way of product analysis were verified. The result of amplifications proved rich spectra along the whole length scale. Numerous identical bands could be found in related strains. Verification of the expected identity of some strains confirmed the applicability of this method for identification of individual bacterial strains of generaRhizobium andBradyrhizobium. Furthermore, competitiveness of two strains ofR. leguminosarum bv.viciœ against the native rhizobial population was evaluated in a pot experiment. When using PCR as the identification method, the presence of the strains in host plant's nodules was ascertained after inoculation by different rates of inoculum strains. With increasing the inoculum rate, the presence of inoculum strains in pea nodules also increased. On the basis of mathematical models by Amarger and Lobreu the competitiveness of the mentioned strains was estimated at certain inoculum rates. Both tested strains displayed a higher competitiveness than native rhizobia in the soil used. As they are also effective N₂ fixators (one strain being HUP⁺), one may expect successful field inoculations with them.     

Compatibility and Coselection of Vesicular-Arbuscular Mycorrhizal Fungi and Rhizobia for Tropical Legumes

Abstract

The effect of dual inoculation on three local cultivars (Miss Kelly, Portland Red, Round Red) of red kidney bean (Phaseolus vulgaris, L.) with four strains of Rhizobium phaseoli (B36, B17, T2, and CIAT652) and three species of vesicular-arbuscular mycorrhizal (VAM) fungi (Glomus pallidum, Glomus aggregatum, and Sclerocystis microcarpa) was examined in sterilized and nonsterilized soil. Symbiotic efficiency (improved plant growth response and enhanced nitrogen (N) and phosphorus (P) nutrition) was dependent on the particular combination of Rhizobium strain, VAM fungus, and cultivar of kidney bean. Whereas rhizobial strains B36 and B17 paired with G. pallidum or G. aggregatum showed increased growth response of cv. Miss Kelly and Portland Red, rhizobial strain T2 paired with any of the three VAM fungi was the best compatible pairing for the cv. Round Red. It is suggested that even though dual inoculation significantly improves the growth response of the bean, the best pairings of VAM fungus and rhizobia for legumes is a question to be carefully examined.     

Usage of strain-producers of adhesin RapA1 from <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> for the creation of binary biofertilizers

Binary biofertilizers consisting of a rhizobial strain specific to the plant Rhizobium leguminosarum and a strain-producer of the RapA1 adhesin protein, which enhances the adhesion of rhizobacteria to the plant roots, have been designed. A system consisting of highly productive bacteria strains and additional strains that enhance the action of the main component have been obtained.     

Promotion of Infection Thread Formation by Substances from Rhizobium

An extrinsic substance (ES-6000) was isolated from the periplasmic space of Rhizobium trifolii (strain 4S) cells by osmotic shock, using a high-density sucrose solution. This substance promoted infection thread formation in root hairs of white clover when inoculated together with the infectious strain (4S). However, ES-6000 obtained from another rhizobial species and from strain A1, which is a noninfectious mutant strain obtained from strain 4S, did not have this effect. The promoter in the ES-6000 from strain 4S is a relatively small molecule since it passed through a hollow-fiber membrane (molecular weight, 6,000). This substance was also recognized as an R_f 0.1 fraction by paper chromatography. Sucrose was effective in promoting nodulation and root elongation.     

Substances from cultured soybean cells which stimulate or inhibit acetylene reduction by free-living Rhizobium japonicum

The effect of cultured soybean ( Glycine max (L.) Merr. cv. Acme) cells and extracts thereof on acetylene reduction by Rhizobium japanicum 61A76 was determined under two culture conditions. In the first (submerged culture) Rhizobium growing on solid medium were submerged in an aqueous layer containing soybean cell suspension or extract; both soybean cells and crude cell extract inhibited free living rhizobial nitrogenase measured by acetylene reduction. In the second culture condition (surface culture) Rhizobium was grown on the surface of an agar medium which contained expressed soybean cell sap (7.4% v/v), which caused an increase in free living rhizobial nitrogenase activity. Larger concentrations (12.9 to 19.4% v/v) of soybean cell extract inhibited acetylene reduction also in surface culture, inhibition in submerged culture or surface culture was found after autoclaving of cells or extract, or treating the extract with pH extremes or with Streptomyces protease. Each of these treatments destroyed stimulatory activity of cell extract in surface cultures. Fractionation of the soybean cell extract on a Biogel P-6 column showed that there are three inhibitory fractions and at least one stimulatory fraction. Using column data and the fact that stimulatory and inhibitory factors were dialysable against 0.1 M phosphate buffer, we estimated the molecular weights of the factors. The stimulatory factor has a molecular mass ( M _r) between 6,000 and 14,000. The inhibitory factors have molecular masses less than 6,000. Crude extracts from cultured carrot ( Daucus carota (L.) cv. Danvers) cells gave results similar to those seen with soybean cell extract Extracts from cultured winged bean ( Psophocarpus tetragonolobus (L.) DS. cv. TPt2) cells gave inhibition but no stimulation.     

The physiology and biochemistry of cultivar-strain interactions in the white clover-Rhizobium symbiosis

Two white clover cultivars were inoculated with two Rhizobium leguminosarum bv. trifolii strains in a factorial series of experiments. Plants were grown in axenic conditions in nitrogen free nutrient solution in a controlled environment room. Variations in nitrogen fixation were dependent partly upon general strain effects, partly upon general cultivar effects but there were also substantial differences attributable to precise interactions between specific combinations. The physiological and biochemical basis of these differences was examined. There were variations in the onset of nodulation and nitrogenase (acetylene reduction) activity. The rate at which nitrogenase activity developed also differed between associations as did the average size and number of nodules but none of these effects correlated well with differences in plant dry matter accumulation. Studies on nodule biochemistry revealed that the major nitrogen fixation enzymes were present in all four associations. Nodule protein content and enzyme activity (on a g nodule fresh weight basis) were substantially greater in associations formed by the more effective strain but cannot explain the interactive effect on dry matter accumulation. The relevance of these data to our understanding of factors regulating variations in nitrogen fixation is discussed.