Effect of root exudates on the exopolysaccharide composition and the lipopolysaccharide profile of Azospirillum brasilense Cd under saline stress

The effect of wheat root exudates on the exopolysaccharide (EPS) composition and the lipopolysaccharide (LPS) profile of Azospirillum brasilense Cd under saline stress was studied. EPS of A. brasilense Cd was composed of glucose (47%), mannose (3%), xylose (4%), fucose (28%), rhamnose (6%), arabinose (1%) and galactose (11%). Under saline stress, A. brasilense produced a totally different EPS, composed mainly of galactose. Root exudates induced changes in A. brasilense EPS composition only under normal conditions, consisting of higher amounts of arabinose and xylose compared with EPS of bacteria grown without root exudates. No changes were induced by root exudates when A. brasilense was grown under saline stress. Additionally, root exudates induced changes in the LPS profile, both under normal and stress conditions.     

Influence of substrate composition and flow rate on growth of Azospirillum brasilense Cd in a co-culture with 3 sorghum rhizobacteria

The ability of Azospirillum brasilense Cd to colonize the niche occupied by 3 bacterial strains previously isolated from sorghum rhizosphere was studied by means of the Biolog system. The isolates were identified by different methods as strains belonging to Pseudomonas putida, Stenotrophomonas maltophilia, and Klebsiella terrigena species. Several C sources, also chosen among the constituents of sorghum root exudates, were used to evaluate the metabolic profiles of Azospirillum and the sorghum rhizobacteria. Azospirillum brasilense Cd exploited the same class of C compounds as the sorghum rhizobacteria and overlapped in their niche requirements. Since structure and functioning of a microbial community are largely affected by the flow rate of nutrient supply, the competitive behavior of A. brasilense Cd was studied in a chemostat mixed culture under C-limited conditions using disodium succinate as C source. Only at high growth rates, i.e., when the C source was highly supplied, A. brasilense Cd appeared to be a good competitor and it became the dominant species, whereas at low growth rates, it was outnumbered by the other species. However, the coexistence of all the strains was always maintained, thus suggesting that interactions other than competition or a potential cross-feeding might occur within the mixed culture.     

The effect of native and ACC deaminase-containing Azospirillum brasilense Cd1843 on the rooting of carnation cuttings

Carnation cuttings treated with non-transformed and 1-aminocyclopropane (ACC) deaminase-containing Azospirillum brasilense Cd1843 produced significantly more roots than untreated controls and fewer roots than cuttings treated with 0.1% indolebutyric acid (IBA). The roots produced by cuttings treated with ACC deaminase-containing Azospirillum brasilense Cd1843 were the longest roots resulting from any of the treatments, followed by non-transformed Azospirillum brasilense Cd1843, 0.1% IBA, and treatment with water. The results are interpreted in terms of a previously proposed model of bacterial promotion of plant growth by ACC deaminase and indoleacetic acid, and may have implications for the use of plant growth-promoting bacteria in the flower industry.     

Chemical composition and immunochemical characteristics of the lipopolysaccharide of nitrogen-fixing rhizobacterium Azospirillum brasilense CD

The chemical composition of the lipopolysaccharide of the associative diazotrophic rhizobacterium Azospirillum brasilense Cd has been studied. Among the main components of the hydrophobic part of the lipopolysaccharide, we identified 3-hydroxytetradecanoic, hexadecenoic, 3-hydroxyhexadecanoic, hexadecanoic, octadecenoic, and nanodecanoic fatty acids; the carbohydrate part contained rhamnose, galactose, and mannose. Polyclonal antibodies against the preparation under study were raised in rabbits. Serological relations between A. brasilense Cd and other strains of Azospirillum spp. were studied using double radial immunodiffusion and enzyme-linked immunosorbent assay.     

Antigenic identity of the capsule lipopolysaccharides,exopolysaccharides, and O-specific polysaccharides in Azospirillum brasilense

The antigenic identity (and close values of electrophoretic mobility) of capsular polysaccharides, exopolysaccharides, and O-specific polysaccharides was revealed in the Azospirillum brasilense strains Sp7 and Sp245 by the immunodiffusion and immunoelectrophoretic methods. Together with the literature data on the identity of the monosaccharides composition of these polymers, this gives evidence of the absence of a specific capsular antigen in the bacteria studied. Thus, extracellular Azospirillum brasilense polysaccharides are likely to represent O-antigenic lipopolysaccharide fragments excreted by the bacteria into the culture medium, and their identification as a capsule or as an exopolysaccharide depends on the strength of the attachment of these polysaccharides to the cell surface.     

Analysis of DNA, lipopolysaccharide structure, and some cultural and morphological properties in closely related strains of Azospirillum brasilense

We studied closely related Azospirillum brasilense strains Sp7 and Cd. For probing of their genomes, the fragments of 85-MDa (p85) and 120-MDa (p120) from A. brasilense Sp245 plasmids were hybridized with 115-MDa (p115) and 90-Mda (p90) plasmids of strain Sp7, respectively. Strain Cd was found to lose the 115-Mda plasmid and one of the two EcoRI restriction fragments of the total DNA (localized within p115 and the chromosome) that was homologous to an EcoRI-generated p85 fragment of 2.4 kb. On the contrary, in the total DNA of strain Sp7-S, in spite of the previously established disappearance of the 115-Mda replicon, two fragments homologous to p85 were revealed, as with strain Sp7. It is suggested that the Sp7-S genome contains the total p115 DNA or at least a certain part of it. Strains Sp7 and Cd were found to differ in size and morphology of colonies on solid and semisolid media, in the levels of resistance to a cation surfactant cetavlon, and in the antigen structure of lipopolysaccharides.     

Structure of the O-specific polysaccharide of the lipopolysaccharide of Azospirillum brasilense Sp245

An O-specific polysaccharide was isolated from the lipopolysaccharide of a plant-growth-promoting bacterium Azospirillum brasilense Sp245 and studied by sugar analyses along with one- and two-dimensional 1H and 13C NMR spectroscopy, including NOESY. The polysaccharide was found to be a new rhamnan with a pentasaccharide repeating unit having the following structure:-->2)-beta-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->2)-alpha-D-Rhap-(1-->2)-alpha-D-Rhap-(1-->     

Evidence for the occurrence of an alternative nitrogenase system in Azospirillum brasilense

Abstract Azospirillum brasilense strain Cd, but not A. lipoferum , could grow and reduce acetylene to ethylene when grown on Mo-free Nfb medium with Fe. Purified Nfb medium lacking both Mo and Fe did not support growth of A. brasilense Cd. Supplementation of − Mo + Fe Nfb medium with vanadium or several other metal salts did not stimulate growth or acetylene reduction. In Mo-free Nfb semisolid medium containing Fe and W, the organism showed good pellicle growth and reduced acetylene to ethylene and ethane. Moreover, a significantly higher amount of H₂ was produced in Mo-free medium than in Nfb containing Mo. The data suggest the presence of an alternate nitrogenase system 3 (Fe-nitrogenase) in A. brasilense Cd besides the normal Mo-nitrogenase.     

Development and growth effects in the Sorghum–Azospirillum association

Sorghum plants were inoculated with a pigmented strain of Azospirillum brasilense (Cd) or were not inoculated and received an N-amended nutrient solution (1.0 mmol/l NH₄NO₃). A third set of plants were non-inoculated and not fertilized with N (control). Plants were grown in a steam-sterilized, low-fertility soil and harvested after growing for 2, 4, 6, 8 and 10 weeks. Dry weights for Azospirillum -inoculated plants were significantly greater than the N-fertilized sorghum at weeks 2 and 4, but A. brasilense -colonized plants weighed significantly less than the N-fertilized sorghum at weeks 8 and 10. Shoot and root N concentrations increased for N-amended plants but remained fairly steady for sorghum inoculated with strain Cd indicating enhanced N-use efficiency in plants colonized with the endophyte. In general, the concentration of Fe, Mn, Zn and Cu in Azospirillum -colonized plants was relatively less than that in N-amended sorghum early in ontogeny when the growth rate of the inoculated plants was greatest. The number of inoculated Cd cells per plant was correlated with percentage increase in dry weight ( r = 0.92*) or total N content ( r = 0.93*) relative to the N-fertilized plants. Growth enhancement relative to N-fertilized sorghum was not observed when the number of Azospirillum cells per gram of root dropped below 1.0 x 10⁵ (or 3.0 x 10⁶ cells/plant). Therefore, the persistence of Azospirillum in the endorhizosphere of sorghum had a direct impact on host growth, physiology and nutrition.     

Response of Azospirillum brasilense Cd to Sodium Chloride Stress

Growth of Azospirillum brasilense Cd in the presence of different NaCl concentrations showed that it tolerates up to 200 mM NaCl in the medium, without appreciable decline in growth rate. At 300 mM NaCl, a decrease of 66% in growth was observed at 24 h of culture. At 48 h of culture, bacteria in the presence of 300 mM NaCl reached the maximum optical density value that was attained at 12 h by control cultures. This investigation was designed to elucidate the effect of saline stress on Azospirillum brasilense Cd and the physiologic mechanism involved in its possible salinity tolerance. For this reason, studies of other osmolytes, as well as of putrescine metabolism and protein patterns were done with bacteria grown with this NaCl concentration in the medium, at 24 and at 48 hours. A. brasilense responded to saline stress elevating the intracellular concentration of glutamate at 24 h, and of K⁺at 48 h. Glucan pattern, putrescine metabolism, and total and periplasmic protein patterns of the treated group showed several changes with respect to the control. In spite of the several cellular functions affected by saline stress, the results imply that A. brasilense Cd shows salinity tolerance in these experimental conditions.     

Structural analysis of the O-polysaccharide from the lipopolysaccharide of Azospirillum brasilense S17

A mixture of two structurally distinct neutral O-polysaccharides was obtained by mild acid degradation of the lipopolysaccharide isolated by the phenol/water extraction from the asymbiotic diazotrophic rhizobacterium Azospirillum brasilense S17. The following structures of the O-polysaccharides were established by composition and methylation analyses, Smith degradation, and 1H and 13C NMR spectroscopy, including a 2D NOESY experiment: [Formula: see text] where L-Rha2Me stands for 2-O-methyl-L-rhamnose and SHb for the (S)-3-hydroxybutanoyl group. The occurrence of two distinct polysaccharides is reported for the first time in Azospirillum spp.     

The nodPQ genes in Azospirillum brasilense Sp7 are involved in sulfation of lipopolysaccharides

Here we report on the presence of sulfated lipopolysaccharide molecules in Azospirillum brasilense, a plant growth-promoting rhizosphere bacterium. Chemical analysis provided structural data on the O-antigen composition and demonstrated the possible involvement of the nodPQ genes in O-antigen sulfation.     

Detection of chemotaxis in Azospirillum brasilense

Azospirillum brasilense strain Cd responded chemotactically to amino acids, sugars and organic acids. Chemotactic rings were observed in semisolid agar plates containing oxidizable substrates. Increasing sodium succinate concentration decreased the velocity of ring expansion. Chemotactic activity of Azospirillum was also examined by a newly developed technique using a channelled chamber. Varying the concentrations of aspartic and glutamic acids affected the chemotactic response of the bacteria. In both assays chemotaxis was obtained under conditions that prevented aerotaxis.     

Promotion of nod Gene Inducers and Nodulation in Common Bean (Phaseolus vulgaris) Roots Inoculated with Azospirillum brasilense Cd

Inoculation of Phaseolus vulgaris with Azospirillum brasilense Cd promoted root hair formation in seedling roots and significantly increased total and upper nodule numbers at different concentrations of Rhizobium inoculum. In experiments carried out in a hydroponic system, A. brasilense caused an increase in the secretion of nod gene-inducing flavonoids, as was observed by nod gene induction assays of root exudates fractionated by high-performance liquid chromatography. Possible mechanisms involved in the influence of A. brasilense on this symbiotic system are discussed.     

Wheat root colonization and nitrogenase activity by Azospirillum isolates from crop plants in Korea

Nitrogen-fixing bacteria were isolated from the rhizosphere of different crops of Korea. A total of 16 isolates were selected and characterized. Thirteen of the isolates produced characteristics similar to those of the reference strains of Azospirillum, and the remaining 3 isolates were found to be Enterobacter spp. The isolates could be categorized into 3 groups based on their ARDRA patterns, and the first 2 groups comprised Azospirillum brasilense and Azospirillum lipoferum. The acetylene reduction activity (ARA) of these isolates was determined for free cultures and in association with wheat roots. There was no correlation between pure culture and plant-associated nitrogenase activity of the different strains. The isolates that showed higher nitrogenase activities in association with wheat roots in each group were selected and sequenced. Isolates of Azospirillum brasilense CW301, Azospirillum brasilense CW903, and Azospirillum lipoferum CW1503 were selected to study colonization in association with wheat roots. We observed higher expression of beta-galactosidase activity in A. brasilense strains than in A. lipoferum strains, which could be attributed to their higher population in association with wheat roots. All strains tested colonized and exhibited the strongest beta-galactosidase activity at the sites of lateral roots emergence.     

Characteristics of dissociation in cultures of Aspergillus brasilense Sp7

Peculiarities of dissociation in the cultures of nitrogen-fixating soil microorganism Azospirillum brasilense have been studied. The possible transfer among colony-morphology variants is established. The relations between variants are described by the following scheme: R in equilibrium with SR----S Electrophoretic analysis of plasmid contents in different variants of Azospirillum brasilense supposes the possible participation of plasmid DNA in the dissociation process in this microorganism.     

Participation of azospirillium polysaccharides in interaction with wheat root surface]

The present study was undertaken to comparatively investigate the attachment capacities of Azospirillum brasilense Sp245 and its lipopolysaccharide-defective Omegon-Km mutants KM018 and KM252, as well as their activities with respect to the alteration of the morphology of wheat seedling root hairs. The adsorption dynamics of the parent Sp245 and mutant KM252 strains of azospirilla on the seedling roots of the soft spring wheat cv. Saratovskaya 29 were similar; however, the attachment capacity of the mutant KM252 was lower than that of the parent strain throughout the incubation period (15 min to 48 h). The mutation led to a considerable decrease in the hydrophobicity of the Azospirillum cell surface. The lipopolysaccharides extracted from the outer membrane of A. brasilense Sp245 and mutant cells with hot phenol and purified by chromatographic methods were found to induce the deformation of the wheat seedling root hairs, the lipopolysaccharide of the parent strain being the most active in this respect. The role of the carbohydrate moiety of lipopolysaccharides in the interaction of Azospirillum cells with plants is discussed.     

The role of chemotaxis genes in establishing the associative relations between Azospirillum brasilense and wheat

The chemotactic properties of a number of Azospirillum brasilense natural strains have been studied. Azospirillum demonstrate the positive chemotactic reaction towards the organic acids salts but a poor reaction towards the presence of the attractants like hydrocarbons and aminoacids except for arabinose and glutamic acid. The series of Che- mutants deficient in general chemotaxis has been selected by introducing the transposon Tn5 into the cells of rifampicinresistant mutant strain Azospirillum brasilense 5T-2. The ability of the mutant cells to fast and solid adsorption on the roots of the sterile wheat sprouts is shown to be decreased 2-3 folds as compared with the one of the wild type strain. Chemotaxis is suggested to affect the adsorbtion ability of azospirillum and their associative properties.     

Enhancement of polygalacturonase activity during auxin induced para nodulation and endorhizosphere colonization of Azospirillum in rice roots

Effect of different auxins, namely, 2,4-dichlorophenoxyacetic acid (2,4-D), naphthalene acetic acid (NAA) and indole acetic acid (IAA) and Azospirillum brasilense bioinoculation on the enhancement of polygalacturonase (PG) activity in rice roots during para nodulation and endorhizosphere colonization of Azospirillum was studied under in vitro condition. It was observed that Azospirillum bioinoculation could augment PG activity of rice roots to a lesser extent without any root morphogenesis whereas auxin application together with Azospirillum bioinoculation enhanced PG activity of rice roots to a higher level which resulted in better root morphogenesis (para nodule) and endorhizosphere colonisation of A. brasilense. Among the three auxins tested, 2,4-D, even at lower concentration (0.5 ppm) enhanced the rice root PG activity, root morphogenesis and endorhizosphere colonization of Azospirillum while it was 2.0 ppm with NAA and variable with IAA. It is concluded that there is a positive correlation existing among PG activity, degree of root morphogenesis and endorhizosphere colonization of Azospirillum brasilense in rice roots and the degree of correlation is determined by the chemical composition, concentration and mode of action of the auxin utilised.     

Azospirillum brasilense and Azospirillum lipoferum hydrolyze conjugates of GA20 and metabolize the resultant aglycones to GA1 in seedlings of rice dwarf mutants

Azospirillum species are plant growth-promotive bacteria whose beneficial effects have been postulated to be partially due to production of phytohormones, including gibberellins (GAs). In this work, Azospirillum brasilense strain Cd and Azospirillum lipoferum strain USA 5b promoted sheath elongation growth of two single gene GA-deficient dwarf rice (Oryza sativa) mutants, dy and dx, when the inoculated seedlings were supplied with [17,17-2H2]GA20-glucosyl ester or [17,17- 2H2]GA20-glucosyl ether. Results of capillary gas chromatography-mass spectrometry analysis show that this growth was due primarily to release of the aglycone [17,17-2H2]GA20 and its subsequent 3beta-hydroxylation to [17,17-2H2]GA1 by the microorganism for the dy mutant, and by both the rice plant and microorganism for the dx mutant.