Serological relationships of azospirilla revealed by their motility patterns in the presence of antibodies to lipopolysaccharides

Motility of the serologically different Azospirillum brasilense strains Sp245 (serogroup I) and Sp7 (serogroup II) was studied in the presence of antibodies to their lipopolysaccharides (LPS). A procedure was proposed in order to determine the motility patterns indicating the specificity of the interaction between the anti-LPS antibodies and bacteria. Analysis of the effect of such antibodies on motility of 25 strains (A. brasilense, A. lipoferum, A. irakense, and Azospirillum sp.) revealed bacteria exhibiting antigenic cross reactions with A. brasilense Sp7 or Sp245. The effect of anti-LPS antibodies on motility of azospirilla was in agreement with the results of immune agglutination analysis of bacterial cells and of immunodiffusion analysis of the LPS preparations. According to our results, strains Azospirillum sp. SR81 and A. brasilense SR14 should be included into serogroups I and II, respectively.     

Characterization of the lipopolysaccharides of serogroup II Azospirillum strains

Lipopolysaccharides of six Azospirillum brasilense strains (SR50, SR80, SR88, SR109, SR111, SR115) and A. lipoferum SR 42 isolated from the rhizosphere of cereal plants of Saratov oblast, Russia and referred to serogroup II by serological analysis were studied. In the lipid A fatty acid composition, the lipopolysaccharides under study were similar to those of other Azospirillum strains and were characterized by a predominance of 3-hydroxytetradecanoic, 3-hydroxyhexadecanoic, and octadecenoic acids. Monosaccharide analysis of the O-specific polysaccharides (including determination of the absolute configurations, methylation analysis, and one- and two-dimensional NMR spectroscopy) revealed the presence of two types of repeating units in varying ratios. A high degree of serological similarity between the strains under study was shown to result from the presence of repeating units with identical structure of their O antigens.     

Detection of putative polysaccharide biosynthesis genes in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strains from serogroups I and II

It is known that in Azospirillum brasilense strains Sp245 and SR75 included in serogroup I, the repeat units of their O-polysaccharides consist of five residues of D-rhamnose, and in strain SR15, of four; and the heteropolymeric O-polysaccharide of A. brasilense type strain Sp7 from serogroup II contains not less than five types of repeat units. In the present work, a complex of nondegenerate primers to the genes of A. brasilense Sp245 plasmids AZOBR_p6, AZOBR_p3, and AZOBR_p2, which encode putative enzymes for the biosynthesis of core oligosaccharide and O-polysaccharide of lipopolysaccharide, capsular polysaccharides, and exopolysaccharides, was proposed. By using the designed primers, products of the expected sizes were synthesized in polymerase chain reactions on genomic DNA of A. brasilense Sp245, SR75, SR15, and Sp7 in 36, 29, 23, and 12 cases, respectively. As a result of sequencing of a number of amplicons, a high (86–99%) level of identity of the corresponding putative polysaccharide biosynthesis genes in three A. brasilense strains from serogroup I was detected. In a blotting-hybridization reaction with the biotin-labeled DNA of the A. brasilense gene AZOBR_p60122 coding for putative permease of the ABC transporter of polysaccharides, localization of the homologous gene in ~120-MDa plasmids of the bacteria A. brasilense SR15 and SR75 was revealed.     

Involvement of the Lipopolysaccharides of Azospirilla in the Interaction with Wheat Seedling Roots

The present study was undertaken to comparatively investigate the attachment capacities of Azospirillum brasilenseSp245 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 Azospirillumcell surface. The lipopolysaccharides extracted from the outer membrane of A. brasilenseSp245 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 Azospirillumcells with plants is discussed.     

Oil-oxidizing potential of associative rhizobacteria of the genus Azospirillum

The oil-oxidizing potential of associative rhizobacteria of the genus Azospirillum was studied under laboratory conditions. After screening, A. brasilense strain SR80 was chosen for further investigation. The strain was capable of degrading 56.5% of crude oil (added in a concentration of 1%) over 14 days in a medium containing malate as an additional source of carbon and energy. Studies of associative properties showed that the strain had positive chemotaxis to wheat root exudates, colonized wheat roots, and produced indole-3-acetic acid. The synthesis of indole-3-acetic acid was not inhibited by oil. Under hydroponic conditions, crude oil stimulated growth of A. brasilense SR80, which promoted development of the wheat root system in the presence of oil and enhanced the level of oil degradation by the plant-microbial association.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 248–254.Original Russian Text Copyright © 2005 by Muratova, Turkovskaya, Antonyuk, Makarov, Pozdnyakova, Ignatov.     

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.     

O-polysaccharide structure in serogroup I azospirilla

Lipopolysaccharides (LPS) and O-specific polysaccharides (OPS) were obtained from the outer membrane of four Azospirillum strains previously assigned to serogroup I based on the serological affinity revealed by the antibodies (AB) to the LPS of A. brasilense Sp245. Investigation, including determination of monosaccharide composition, methylation analysis, and one- and two-dimensional NMR spectroscopy, was carried out to determine the OPS structure. The OPSs of A. brasilense Sp107 and S27 and of A. lipoferum RG20a were found to have an identical structure of repeating units represented by a linear penta-D-rhamnan, as was previously described for the OPSs of A. brasilense Sp245 and SR75. The OPS of A. brasilense SR15 was found to consist of tetrasaccharide repeating units of the following structure: → 2)-α-D-Rhap-(1 → 2)-β-D-Rhap-(1 → 3)-α-D-Rhap-(1 → 2)-α-D-Rhap-(1 →. An opine compound, N^δ-(1-carboxyethyl)-ornithine, closely associated with the LPS of A. brasilense SR15, was identified in azospirilla for the first time. The presence of a 6-deoxisugar (D-rhamnose) in the OPS structure was shown to be the chemical basis of the serological similarity and the reason for classification of these strains within the serogroup I.     

Effect of Azospirillum brasilense Sp245 lipopolysaccharide on the functional activity of wheat root meristematic cells

We studied changes in the physiological and biochemical parameters of wheat (Triticum aestivum L. ??Saratovskaya 29??) seedlings treated with lipopolysaccharide isolated from the outer membrane of the associative bacterium Azospirillum brasilense Sp245. The obtained data were compared with (i) the results of plant inoculation with whole Sp245 cells and (ii) the effects exerted by the lipopolysaccharide and whole cells of the enterobacterium Escherichia coli K12 and the specific legume symbiont Rhizobium leguminosarum 249. The functional activity of meristematic cells was judged by their mitotic index and by the results of immunochemical determination of the proliferative antigen of initials, a molecular marker for wheat meristem cells. Treatment of the seedling root system with 10 ??g mL^?1 of Sp245 lipopolysaccharide increased the mitotic index (1.8-fold) and the antigen content (approximately 1.4-fold). These increases were comparable to the effects produced by whole cell inoculation (2- and 1.4-fold, respectively). Our findings give grounds to consider lipopolysaccharide as an active component of the Azospirillum cell surface that not only determines bacterial contact interactions with wheat roots but also participates in the induction of plant responses to these interactions. We finally discuss the linkage between the proliferative antigen of initials and the transduction of a hormonal signal to the cell, as well as the informational value of this antigen as an indicator of effectiveness of plant?Cbacterial interactions.     

Changes in cucumber hypocotyl cell wall dynamics caused by Azospirillum brasilense inoculation

We previously reported that Azospirillum brasilense induced a more elastic cell wall and a higher apoplastic water fraction in both wheat coleoptile and flag leaf. These biophysical characteristics could permit increased growth. Knowledge of the biochemical effects the bacteria could elicit in plant cell walls and how these responses change plant physiology is still scarce. The objective of this work was to analyze whether A. brasilense Sp245 inoculation affected elongation and extensibility of growing cucumber (Cucumis sativus) hypocotyls and ionically bound cell wall peroxidase activities. Hypocotyl tip and basal segments were excised from A. brasilense Sp245-inoculated cucumber seedlings growing in darkness under hydroponic conditions. Elongation, cell wall extensibility, cell wall peroxidase activities against ferulic acid and guaiacol and NADH oxidase activities were analyzed. Azospirillum-inoculated cucumber seedlings grew bigger than non-inoculated ones. Dynamic cell wall differences were detected between inoculated and non-inoculated hypocotyls. They included greater acid-induced cell wall extension and in vivo elongation when incubated in distilled water. Although there was no difference between treatments in either region of the hypocotyl NADH oxidase and ferulic acid peroxidase activities were lower in both regions in inoculated seedlings. These lesser activities could be delaying the stiffening of cell wall in inoculated seedlings. These results showed that the cell wall is a target for A. brasilense growth promotion.     

Effect of nitrogen supply and Azospirillum brasilense Sp-248 on the response of wheat to seawater irrigation

Response of wheat to Azospirillum brasilense Sp-248 inoculation with different N-fertilizer levels using seawater irrigation was investigated. All inoculated treatments increased plant height, shoot and root dry weight, and tiller number in compared with uninoculated treatments. Yield parameters measured were also increased due to the inoculation. In terms of the effect of saline irrigation, there were no significant differences in growth and yield parameters in plants treated with tap water and others irrigated with 8.0% seawater concentration. This would indicate a relatively high tolerance of A. brasilense to saline irrigation and its ability to reduce the deleterious effects of saline on growth by increasing the plant’s adaptation. However, increasing the seawater concentration in the irrigation water to 16.0% significantly decreased all tested parameters. Inoculation treatments generally increased NPKCa contents and decreased sodium ratio of the grains in compared with the uninoculated treatments. Overall results clearly revealed that the Azospirillum inoculation saved about 20 units of N-fertilizer and that saving was made economically feasible by decreasing the chemical fertilizers needed, improving the nitrogen content and counteracting the effects of salinity.     

Transposon Mutagenesis of Azospirillum brasilense and Azospirillum lipoferum: Physical Analysis of Tn5 and Tn5-Mob Insertion Mutants

Tn5-induced insertion mutants were generated in Azospirillum brasilense Sp7 and A. lipoferum SpBr17 by mating with Escherichia coli strains carrying suicide plasmid vectors. The sources of Tn5 were the suicide plasmids pGS9 and pSUP2021. Kanamycin-resistant Azospirillum colonies appeared from crosses with E. coli at maximum frequencies of 10⁻⁷ per recipient cell. Transposon Tn5 also conferred streptomycin resistance on Azospirillum colonies as was observed earlier for Rhizobium sp. Eight Tn5-induced Km^r Sm^rA. brasilense Sp7 mutants with reduced nitrogen-fixing capacity were isolated. The potential use of Tn5-Mob for labeling and mobilization of Azospirillum-indigenous plasmids was demonstrated by isolating Tn5-Mob insertions in the megaplasmids of A. brasilense Sp7.     

A method of acoustic analysis for detection of bacteriophage-infected microbial cells

The dependence of the changes of physical parameters of the suspension of Azospirillum brasilense Sp7 cells infected by FAb-Sp7 bacteriophage on their number and exposure time was studied using a biological sensor based on a piezoelectric resonator with a lateral electric field. The change in the value of the analytical signal was recorded at 1 minute from the beginning of the infection of the cells by bacteriophage. The selectivity of the action of the FAb-Sp7 bacteriophage was studied for Azospirillum brasilense (strains Cd, Sp107, Sp245, Jm6B2, Br14, KR77, S17, S27, SR55, and SR75), A. lipoferum (strains Sp59b, SR65, and RG20a), A. halopraeferans Au4, Nitrospirillum amazonense Am14, Niveispirillum irakense (strains KBC1 and KA3) bacteria, as well as for heterologous bacteria of the genera Escherichia coli (strains XL-1 and B-878), Pseudomonas putida (strains C-11 and BA-11), and Acinetobacter calcoaceticum A-122. The limit of the reliable determination of the concentration of microbial cells during bacteriophage infection process was found: ~10⁴ cells/mL. At the same time, the presence of heterologous cell cultures (E. coli XL-1 cells) did not complicate the detection. It was shown that the method of electroacoustical analysis of cell suspensions can be used for the detection of microbial cells of Azospirillum infected by the FAb-Sp7 bacteriophage. The results are promising for the development of methods for determining and controlling the number of soil microorganisms.     

Azospirillum brasilense colonisation of wheat roots and the role of lectin–carbohydrate interactions in bacterial adsorption and root-hair deformation

The dynamics of adsorption of the nitrogen-fixing soil bacteria Azospirillum brasilense 75 and 80 (isolated from soil samples collected in Saratov Oblast, southern Russia) and A. brasilense Sp245 to the roots of seedlings of common spring wheat was studied in relation to inoculum size, period of incubation with the roots and bacterial-growth phase. The number of root-attached cells increased with increasing size of inoculum and time of contact. The saturation of root-surface adsorption was observed by 24 h of co-incubation for A. brasilense 75, by 6 h for A. brasilense 80, and by 3 h for A. brasilense Sp245. The firmness of bacterial–root attachment increased after extended co-incubation. Differences in the adsorption kinetics of the azospirilla were found that were associated with bacterial-growth phases. Azospirilla attached to the roots of their host cultivar more actively than they did to the roots of a non-host cultivar. Adsorption was partially inhibited when the roots were treated with N-acetyl-D-glucosamine. Maximal inhibition occurred after a 3-h exposure of the roots to the bacteria. Root-hair deformation induced with polysaccharide-containing complexes from the Azospirillum capsular material was inhibited by N-acetyl-D-glucosamine and chitotriose, specific haptens of wheat germ agglutinin. A possible mechanism of the mutual influence of bacteria and plants may involve key roles of wheat germ agglutinin, present on the roots, and the polysaccharide-containing components of the Azospirillum capsule.     

Quantification of Azospirillum brasilense FP2 Bacteria in Wheat Roots by Strain-Specific Quantitative PCR

Azospirillum is a rhizobacterial genus containing plant growth-promoting species associated with different crops worldwide. Azospirillum brasilense strains exhibit a growth-promoting effect by means of phytohormone production and possibly by N₂ fixation. However, one of the most important factors for achieving an increase in crop yield by plant growth-promoting rhizobacteria is the survival of the inoculant in the rhizosphere, which is not always achieved. The objective of this study was to develop quantitative PCR protocols for the strain-specific quantification of A. brasilense FP2. A novel approach was applied to identify strain-specific DNA sequences based on a comparison of the genomic sequences within the same species. The draft genome sequences of A. brasilense FP2 and Sp245 were aligned, and FP2-specific regions were filtered and checked for other possible matches in public databases. Strain-specific regions were then selected to design and evaluate strain-specific primer pairs. The primer pairs AzoR2.1, AzoR2.2, AzoR5.1, AzoR5.2, and AzoR5.3 were specific for the A. brasilense FP2 strain. These primer pairs were used to monitor quantitatively the population of A. brasilense in wheat roots under sterile and nonsterile growth conditions. In addition, coinoculations with other plant growth-promoting bacteria in wheat were performed under nonsterile conditions. The results showed that A. brasilense FP2 inoculated into wheat roots is highly competitive and achieves high cell numbers (∼10⁷ CFU/g [fresh weight] of root) in the rhizosphere even under nonsterile conditions and when coinoculated with other rhizobacteria, maintaining the population at rather stable levels for at least up to 13 days after inoculation. The strategy used here can be applied to other organisms whose genome sequences are available.     

The participation of the cell wall hydrolytic enzymes in the initial colonization of Azospirillum brasilense on wheat roots

The effect of cellulase and pectinase on bacterial colonization of wheat was studied by three different experiments. In the first experiment, the root colonization of 3 wheat cultivars (Ghods, Roshan and Omid) by two A. brasilense strains (Sp7 and Dol) was compared using pre-treated roots with cellulase and pectinase, and non-treated with these enzymes (control). Although the root colonization varied greatly among strain-plant combinations in controls, the pre-treatment of roots with polysaccharide degrading enzymes significantly increased the bacterial count in roots, regardless of the strain-plant combination. This might be an indication that cell wall may act as an important factor in plant-Azospirillum interaction. In the second experiment, the root cellulase activity of the same wheat cultivars treated with and without the two Azospirillum brasilense, strains (Sp7 and Dol) was compared. The pre-treatment of wheat roots with Azospirillum enhanced the cellulase activity of wheat root extracts. Thus, the cellulase activity might participate in the initial colonization of wheat roots by Azospirillum. The comparison of the cellulase activity of root extracts within inoculated and non-inoculated seedlings showed that the inoculation had enhanced the cellulase activity in root extracts, but this effect was directly dependent on the strain-plant combination. Strain Sp7 stimulated the highest cellulase activity in cv. Roshan, but strain Dol induced the highest enzyme activity in cv. Ghods. In the third experiment, several growth parameters of those 3 wheat cultivars treated with and without those two bacterial strains (Sp7 and Dol) were compared. The highest magnitude of growth responses caused by Sp7 strain was in the cv Roshan, but Dol strain stimulated the highest growth in cv Ghods. Therefore, effective colonization may contribute to more growth responses.     

Azospirillum himalayense sp. nov., a nifH bacterium isolated from Himalayan valley soil,India

Gram-negative, free-living bacterial strain ptl-3^T was isolated from Himalayan valley soil, India. Polyphasic taxonomy was performed including morphological characterization, fatty acid analysis, biochemical tests, 16S rRNA and nifH gene sequence analyses. 16S rRNA gene sequence analysis showed that the strain ptl-3^T belonged to the genus Azospirillum and was closely related to A. brasilense (98.7 % similarity) and A. rugosum (97 % similarity). 16S rRNA gene sequence similarity (96–95 %) was shown with other members of the genus Azospirillum. Major fatty acid 18:1ω7c was also similar to the genus Azospirillum. DNA–DNA relatedness value between strain ptl-3^T and A. brasilense was found to be 47 %. Various biochemical tests showed that the strain ptl-3^T differed from its closely related species A. brasilense. On the basis of phenotypic, chemotaxonomic and molecular genetics evidence, a bacterium with the type strain ptl-3^T is proposed as a novel species of the genus Azospirillum. The name of bacterial strain ptl-3^T has been proposed as Azospirillum himalayense sp. nov. The type strain of ptl-3^T (CCUG 58760^T, KCTC 23189^T) has been submitted to two culture collection centres. The accession numbers for 16S rRNA and nifH gene are GQ 284588 and GQ 249665. respectively.     

Structural peculiarities of the O-specific polysaccharides of <Emphasis Type="Italic">Azospirillum</Emphasis> bacteria of serogroup III

Lipopolysaccharides and O-specific polysaccharides were isolated from the outer membrane of bacterial cells of three strains belonging to two Azospirillum species, and their structures were established by monosaccharide analysis including determination of the absolute configurations, methylation analysis, and one- and two-dimensional NMR spectroscopy. It was shown that while having the identical composition, the O-polysaccharides have different branched tetrasaccharide repeating units. Two neutral polysaccharides were found in the lipopolysaccharide of A. brasilense 54, and the structure for the predominant O-polysaccharide was determined. The structural data, together with results of serological studies, enabled assignment of strains examined to a novel serogroup, III. The chemical basis for the serological relatedness among the azospirilla of this serogroup is presumably the presence of a common →3)-α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-α-L-Rhap-(1→oligosaccharide motif in their O-polysaccharides.     

Inoculation with selected strains of Azospirillum brasilense and A. lipoferum improves yields of maize and wheat in Brazil

Interest in the use of inoculants containing bacteria that promote plant growth is likely to increase in the coming years, due to higher costs of fertilizers, concerns over pollution and emphasis on sustainable agriculture. Although Brazil has a long tradition in research on nitrogen fixation in Azospirillum-grass associations, it has not led to recommendations of strains for use in commercial inoculants. In this study, we report the selection and evaluation of Azospirillum strains for the maize (Zea mays L.) and wheat (Triticum aestivum L.) crops, following protocols established by the Brazilian legislature, i.e. field experiments have to be performed in at least two different localities representing the crop growing regions, and for at least two seasons. In a first set of nine trials performed at Londrina and Ponta Grossa, southern Brazil, nine Azospirillum strains were evaluated after application to seeds as peat-based inoculants. A. brasilense strains Ab-V4, Ab-V5, Ab-V6 and Ab-V7 increased grain yields of maize by 662–823 kg ha^?1, or 24–30%, in relation to non-inoculated controls. Two A. lipoferum strains were tested in two of these experiments and promising results were also obtained. With wheat, A. brasilense strains Ab-V1, Ab-V5, Ab-V6 and Ab-V8 were the most effective, increasing yields by 312–423 kg ha^?1, or 13–18%. In a second trial set with eight field experiments at Londrina an Ponta Grossa, liquid and peat-based inoculants carrying a combination of A. brasilense strains Ab-V5 and Ab-V6 increased maize and wheat yields by 27% and 31%, respectively. Effects of inoculation were attributed to general increases in uptake of several macro and micronutrients and not specifically to biological nitrogen fixation. All experiments received only a low N-fertilizer starter at sowing (24 kg and 20 kg of N ha^?1 for the maize and wheat, respectively) and although yields can be globally considered low, they were compatible with Brazilian mean yields. This study resulted in the identification of the first Azospirillum strains authorized for the production of commercial inoculants in Brazil.     

Chemotaxis of Azospirillum Species to Aromatic Compounds

Chemotaxis of Azospirillum lipoferum Sp 59b and Azospirillum brasilense Sp 7 and Sp CD to malate and to the aromatic substrates benzoate, protocatechuate, 4-hydroxybenzoate, and catechol was assayed by the capillary method and direct cell counts. A. lipoferum required induction by growth on 4-hydroxybenzoate for positive chemotaxis to this compound. Chemotaxis of Azospirillum spp. to all other substrates did not require induction. Maximum chemotactic responses for most aromatic compounds occurred at concentrations of 1 to 10 mM for A. lipoferum and 100 μM to 1 mM for A. brasilense. Threshold levels of these chemoattractants ranged from nanomolar to micromolar, with A. brasilense Sp CD showing the lowest threshold levels for the substrates tested. Benzoate was the strongest chemoattractant tested, with threshold concentrations in the nanomolar to picomolar range for all strains. Azospirillum spp. clearly have more sensitive chemosensory mechanisms for certain aromatic substrates than previously reported in some other soil bacteria. This sensitivity allows Azospirillum spp. to detect and respond to aromatic substrates at concentrations relevant to the soil and rhizosphere environments. The ability to detect such low concentrations of aromatic compounds in soils may confer advantages in survival and colonization of the rhizosphere by Azospirillum species.