Establishment of inoculatedAzospirillum spp. in the rhizosphere and in roots of field grown wheat and sorghum

Summary Four field experiments were carried out with wheat or sorghum in different regions of Brazil. The aim was to study the establishment of inoculatedAzospirillum strains, marked with resistance to various antibiotics, in the rhizosphere and in roots. The levels of the various antibiotics were chosen according to the resistance of the indigenousAzospirillum population.Azospirillum brasilense strains Sp 107 and Sp 245 could be established in all three wheat experiments and predominated within theAzospirillum population in washed, and especially in surface sterilized, roots. Strains Sp 7 and Cd established poorly in wheat roots.Azospirillum lipoferum Sp S82 represented 72% of the root isolates from sorghum inoculated with this strain. This strain and naturalAzospirillum infection became concentrated in the upper parts of the root system. Improved methods for root surface sterilization in which the absence ofAzospirillum on the root surface was established by pre-incubating roots with paraffin-capped ends in NFb medium confirmed the establishment of inoculatedAzospirillum strains within sorghum roots in the field.     

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.     

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.     

Wheat root colonization by Azospirillum brasilense strains with different motility

Migration of associative bacteria Azospirillum brasilense in semisolid media is performed mainly by swarming (Swa⁺ phenotype), which depends on the flagellar functioning and intercellular contacts. Non-swarming mutants of A. brasilense Sp245 lacking a polar flagellum migrate in semisolid media with microcolony formation using a unrevealed mechanism (Gri⁺ phenotype). The study of wheat root colonization dynamics demonstrated that A. brasilense Sp245 Gri⁺ mutants exhibited lower capacity for wheat root adsorption. However, after “anchoring” has occurred, both A. brasilense Sp245 and its Swa-Gri⁺ mutants colonized the growing roots with virtually the same efficiency. All strains under study formed microcolonies on the surface of roots, stimulated root branching, and exhibited changes in the composition of protein antigens exposed on the bacterial cell surface. Indirect evidence was obtained for enhanced production of genus-specific protein antigens in the process of A. brasilense Sp245 adaptation to growth on plant roots.     

Effect of inoculation ofAzospirillum spp. on nitrogen accumulation by field-grown wheat

Summary Two experiments were performed to examine the effects of inoculation of field grown wheat with various Azospirillum strains. In the first experiment the soil was sterilized with methyl bromide to reduce the Azospirillum population and¹⁵N labelled fertilizer was added to all treatments. Two strains ofAzospirillum brasilense isolated from surface sterilized wheat roots and theA. brasilense type strain Sp7 all produced similar increases in grain yield and N content. From the¹⁵N and acetylene reduction data it was apparent that these increases were not due to N₂ fixation. In the second experiment performed in the same (unsterilized) soil, twoA. brasilense strains (Sp245, Sp246) and oneA. amazonense strain (Am YTr), all isolated from wheat roots, produced responses of dry matter and N content while the response to the strain Sp7 was much smaller. These data confirm earlier results which indicate that if natural Azospirillum populations in the soil are high (the normal situation under Brazilian conditions), strains which are isolated from wheat roots are better able to produce inoculation responses than strains isolated from other sources. The inoculation of a nitrate reductase negative mutant of the strain Sp245 produced only a very small inoculation response in wheat. This suggests that the much greater inoculation response of the original strain was not due to N₂ fixation but to an increased nitrate assimilation due to the nitrate reductase activity of the bacteria in the roots. Consultant Inter-American Institute for Cooperation in Agriculture IICA/EMBRAPA World Bank Project.     

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.     

Determination of the Structure of the Repeated Unit of the Azospirillum brasilense SR75 O-Specific Polysaccharide and Homology of the lps Loci in the Plasmids of Azospirillum brasilense strains SR75 and Sp245

The structural identity of the repeated unit in O-specific polysaccharides (OPSs) present in the outer membrane of strain SR75 of the bacterium Azospirillum brasilense, isolated from wheat rhizosphere in Saratov oblast, and the previously studied OPSs of A. brasilense strain Sp245, isolated from surfacesterilized wheat roots in Brazil, has been demonstrated. Plasmid profiles, DNA restriction, and hybridization assays suggested that A. brasilense strains SR75 and Sp245 have different genomic structures. It was shown that homologous lps loci of both strains were localized in their plasmid DNA. This fact allows us to state that, despite their different origin, the development of the strains studied was convergent. Presumably, the habitation of these bacteria in similar ecological niches influenced this process in many respects. __________ Translated from Mikrobiologiya, Vol. 74, No. 5, 2005, pp. 626–632. Original Russian Text Copyright ? 2005 by Fedonenko, Borisov, O. Konnova, Zdorovenko, Katsy, S. Konnova, Ignatov.     

Nitric Oxide is Involved in the <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>-induced Lateral Root Formation in Tomato

Azospirillum spp. is a well known plant-growth-promoting rhizobacterium. Azospirillum-inoculated plants have shown to display enhanced lateral root and root hair development. These promoting effects have been attributed mainly to the production of hormone-like substances. Nitric oxide (NO) has recently been described to act as a signal molecule in the hormonal cascade leading to root formation. However, data on the possible role of NO in free-living diazotrophs associated to plant roots, is unavailable. In this work, NO production by Azospirillum brasilense Sp245 was detected by electron paramagnetic resonance (6.4 nmol. g^–1 of bacteria) and confirmed by the NO-specific fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA). The observed green fluorescence was significantly diminished by the addition of the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). Azospirillum-inoculated and noninoculated tomato (Lycopersicon esculentum L.) roots were incubated with DAF-2 DA and examined by epifluorescence microscopy. Azospirillum-inoculated roots displayed higher fluorescence intensity which was located mainly at the vascular tissues and subepidermal cells of roots. The Azospirillum-mediated induction of lateral root formation (LRF) appears to be NO-dependent since it was completely blocked by treatment with cPTIO, whereas the addition of the NO donor sodium nitroprusside partially reverted the inhibitory effect of cPTIO. Overall, the results strongly support the participation of NO in the Azospirillum-promoted LRF in tomato seedlings.     

Field inoculation of sorghum and rice withAzospirillum spp. andHerbaspirillum seropedicae

Two field experiments were carried out at the UAPNPBS experimental station, Seropédica, with two sorghum and one rice cultivars. The establishment, and inoculation effects, ofAzospirillum spp. andHerbaspirillum strains marked with antibiotic resistance were investigated. One grain sorghum (BR 300) and one sugar sorghum (Br 505) cultivar were used.Azospirillum lipoferum strain S82 (isolated from surface sterilized roots of sorghum) established in both cultivars and comprised 40 to 80% of theAzospirillum spp. population in roots and stems 60 days after plant emergence (DAE).Azospirillum amazonense strain AmS91 (isolated from surface-sterilized roots of sorghum) reached only 50%. At 90 DAE, S82 almost disappeared (less than 30% of establishment) while the establishment of AmS91 remained constant in roots and stems. No establishment ofH. seropedicae strain H25 (isolated from surface-sterilized roots of sorghum) orA. lipoferum strain S65 (isolated from the root surface of sorghum) could be observed on inoculated roots. Inoculation with S82, AmS91 or S65 but not withH. seropedicae H25, increased plant dry weight of both cultivars and total N in grain of the grain sorghum. In rice,A. lipoferum Al 121 andA. brasilense Sp 245 (isolated from surface sterilized rice and wheat roots respectively) established in the roots but there was no increase inAzospirillum spp. numbers due to inoculation. None of the strains affected plant growth or rice grain yield.Azospirillum amazonense, A82 andH. seropedicae Z95, which did not establish in roots, significantly enhanced seed germination.     

Field release of genetically marked Azospirillum brasilense in association with Sorghum bicolor L.

The agronomic impact of genetically tagged azospirilla (Azospirillum brasilense)was assessed in open field and their fluctuation were monitored in the soil/rhizosphere. Strain performance, upon inoculation of sorghum, was evaluated over a two-years period; agronomic treatments included nitrogen application (0, 80, 160 kg ha^–1), and types of inoculant (Sp245 lacZ, Sp6 gusA, Sp6 IAA⁺⁺ gusA). Grain yield was higher for inoculated seed plots than in non-inoculated ones, whereas nitrogen content, biomass of plant residues and nitrogen in plant residues gave values that were not statistically different. Root length density (RLD) of sorghum at the end of the stem elongation stage was affected only by the indole-3-acetic acid (IAA) overproducer Azospirillum strain (A. brasilense Sp6 IAA⁺⁺ gusA) with respect to the normal IAA producer (A. brasilense Sp6 gusA), being higher in the first 40 cm of depth, notwithstanding the level of nitrogen fertilization. The traceability of the released genetically modified strains enabled to monitor their ability to colonise soil and roots. Moreover, the genetic modification per se vs. the non-modified counterpart, did not affect the culturable aerobic population in soil, microfungi, streptomycetes, fluorescent pseudomonads, soil microbial biomass, or some microbial activities, all selected as important indicators.     

Effect of lectins from <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> to peroxidase and oxalate oxidase activity regulation in wheat roots

Lectins were extracted from the surface of nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 and from its mutant A. brasilense Sp7.2.3 defective in lectin activity. The ability of lectins to stimulate the rapid formation of hydrogen peroxide related to increase of oxalate oxidase and peroxidase activity in the roots of wheat seedlings has been demonstrated. The most rapid induced pathway of hydrogen peroxide formation in the roots of wheat seedlings was the oxalic acid oxidation by oxalate oxidase which is the effect of lectin in under 10 min in a concentration of 10 μg/ml. The obtained results show that lectins from Azospirillum are capable of inducing the adaptation processes in the roots of wheat seedlings.     

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.     

Changes in Azospirillum brasilense motility and the effect of wheat seedling exudates

The rhizobacterium Azospirillum brasilense Sp245 swims, swarms (Swa⁺ phenotype) or, very rarely, migrates with the formation of granular macrocolonies (Gri⁺ phenotype). Our aims were (i) to identify Sp245 mutants that swarm faster than the parent strain or differ from it in the mode of spreading and (ii) to compare the mutants’ responses to wheat seedling exudates. In isotropic liquid media, the swimming speeds of all motile A. brasilense strains were not influenced by the exudates. However, the exudates significantly stimulated the swarming of Sp245. In several Sp245 mutants, the superswarming phenotype was insensitive to local colonial density and to the presence of wheat seedling exudates. An A. brasilense polar-flagellum-defective Gri⁺ mutant BK759.G gave rise to stable Swa⁺⁺ derivatives with restored flagellum production. This transition was concurrent with plasmid rearrangements and was stimulated in the presence of wheat seedling exudates. The swarming rate of the Swa⁺⁺ derivatives of BK759.G was affected by the local density of their colonies but not by the presence of the exudates.     

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.     

Attachment,colonization and proliferation ofAzospirillum brasilense andEnterobacter spp. on root surface of grasses

Root colonization studies, employing immunofluorescence and using locally isolated strains, showed thatEnterbacter sp. QH7 andEnterobacter agglomerans AX12 attached more readily to the roots of most plants compared withAzospirillum brasilense JM82. Heat treatment of either root or inoculum significantly decreased the adsorption of bacteria to the root surface. Kallar grass and rice root exudates sustained the growth ofA. brasilense JM82,Enterobacter sp. QH7 andE. agglomerans AX12 in Hoagland and Fahraeus medium. All the strains colonized kallar grass and rice roots in an axenic culture system. However, in studies involving mixed cultures,A. brasilense JM82 was inhibited byEnterobacter sp. QH7 in kallar grass rhizosphere and the simultaneous presence ofEnterobacter sp. QH7 andE. agglomerans AX12 suppressed the growth ofA. brasilense JM82 in rice rhizosphere. The bacterial colonization pattern changed from dispersed to aggregated within 3 days of inoculation. The colonization sites corresponded mainly to the areas where root mucigel was present. The area around the point of emergence of lateral roots usually showed maximum colonization.     

Structural and functional peculiarities of the lipopolysaccharide of Azospirillum brasilense SR55, isolated from the roots of Triticum durum

Azospirillum brasilense SR55, isolated from the rhizosphere of Triticum durum, was classified as serogroup II on the basis of serological tests. Such serogroup affiliation is uncharacteristic of wheat-associated Azospirillum species. The lipid A of A. brasilense SR55 lipopolysaccharide contained 3-hydroxytetradecanoic, 3-hydroxyhexadecanoic, hexadecanoic and octadecenoic fatty acids. The structure of the lipopolysaccharide's O polysaccharide was established, with the branched octasaccharide repeating unit being represented by l-rhamnose, l-3-O-Me-rhamnose, d-galactose and d-glucuronic acid. The SR55 lipopolysaccharide induced deformations of wheat root hairs. The lipopolysaccharide was not involved in bacterial cell aggregation, but its use to pretreat wheat roots was conducive to cell adsorption. This study shows that Azospirillum bacteria can utilise their own lipopolysaccharide as a carbon source, which may give them an advantage in competitive natural environments.     

Natural occurrence of Azospirillum brasilense in strawberry plants

Azospirillum species are free-living nitrogen-fixing bacteria commonly found in soil and in association with roots of different plant species. For their capacity to stimulate growth they are known as plant growth-promoting bacteria (PGPB). In this work, we demonstrate the natural occurrence and colonization of different parts of strawberry plants by Azospirillum brasilense in the cropping area of Tucumán, Argentina. Although bacteria isolations were carried out from two strawberry cultivars, e.g., Camarosa and Pájaro, attempts were successful only with the cultivar Camarosa. Whereas different strains of Azospirillum were isolated from the root surface and inner tissues of roots and stolons of the cultivar Camarosa, we have not obtained Azospirillum isolates from the cultivar Pájaro. After microbiological and molecular characterization (ARDRA) we determined that the isolates belonged to the species A. brasilense. All isolates showed to have the capacity to fix nitrogen, to produce siderophores and indoles. Local isolates exhibited different yields of indoles production when growing in N-free NFb semisolid media supplemented or not with tryptophan (0.1 mg ml⁻¹). This is the first report on the natural occurrence of A. brasilense in strawberry plants, especially colonizing inner tissues of stolons, as well as roots. The local isolates showed three important characteristics within the PGPB group: N₂-fixation, siderophores, and indoles production.     

Arsenic Transformation by Azospirillum Brasilense Sp245 in Association with Wheat (Triticum Aestivum L.) Roots

The transformation of sodium arsenite and sodium arsenate by the rhizospheric nitrogen-fixing bacterium Azospirillum brasilense Sp245 in association with wheat (Triticum aestivum L. ‘Saratovskaya 29’) was studied. The effect produced by the A. brasilense strain on the morphological parameters of wheat in an As-polluted environment was examined. The plants were cultivated in a hydroponic system, with glass beads serving as a support for root growth. The plant-growth medium (an artificial soil solution) was deficient in P and Fe. The total initial As concentrations used were 75, 750, and 7500 μg l⁻¹. The As compounds used contained sodium arsenate and sodium arsenite at an As(V):As(III) ratio of 1:3.6 (in terms of As) in all experiments. Inoculation of A. brasilense Sp245 led to a decrease in the overall root length and to the formation of lateral roots; both effects are possibly related to the bacteria’s ability to synthesize auxins. Inoculation also changed the As(V): As(III) ratio of the plant-growth medium. In all experiments, the concentration of As(V) in the nutrient medium increased relative to the initial one and was approximately 1.5-fold higher than that in the medium of uninoculated plants. This value slightly decreased (1.6 > 1.5 > 1.4) with increasing concentration of As in the medium. Azospirillum-inoculated plants accumulated less As than did the surface-sterilized uninoculated plants. This study shows that A. brasilense Sp245 in association with wheat changes the speciation, bioavailability, and plant uptake of As.     

Root Hair Deformation, Bacterial Attachment, and Plant Growth in Wheat-Azospirillum Associations

Seven Azospirillum strains induced more deformation of root hairs of wheat than did strains of Rhizobium leguminosarum, Azotobacter chroococcum, or Escherichia coli. Azospirillum sp. strain Sp245 caused the most deformation. Strain Sp245 (isolated from surface sterile roots of wheat) and strain Sp7 (isolated from the rhizosphere of a forage grass) were compared with regard to their effects on root hair deformation, their attachment to roots, and their effects on the growth of four wheat cultivars. The amount of deformation caused by the two strains in the four cultivars increased in the following order: cv. Tobari, cv. Tonari, cv. BH1146, cv. Lagoa. Strain Sp245 attached to the roots of all cultivars in low numbers, and attachment did not increase with time (up to 48 h). Strain Sp7 attached in higher numbers, and attachment increased with time. Inoculation of the four cultivars of wheat had pronounced effects on root mass measured at maturity. The magnitude of the effects in the four cultivars increased in the following order: Tobari, Tonari, BH1146, Lagoa; these effects were progressively more positive for strain Sp245 and progressively more negative for strain Sp7. Concentrations of N in wheat did not vary substantially between cultivars or strains. Concentrations of K and P did not vary substantially between cultivars but did vary between strains, Sp245 effecting increases and Sp7 effecting decreases.     

Aspects of nitrogen fixation and denitrification byAzospirillum

Summary Model experiments were performed to investigate the nitrogen fixation (C₂H₂ reduction) and denitrification (N₂O formation) capabilities ofAzospirillum spp. in association with wheat. Plants and bacteria were grown together for a week and then assayed for activities. This association performed C₂H₂ reduction or N₂O formation, depending on the concentrations of nitrate and oxygen in the vessels. Both activities depended on theAzospirillum strains used. The newly isolatedAzospirillum amazonense strains Y1 and Y6 showed significant C₂H₂ reduction and low N₂O formation in association with wheat under the conditions employed and are possibly useful in practice. A cell-free preparation fromAzospirillum brasilense Sp 7 possessed a cytochrome cd type dissimilatory nitrite reductase.