The Vicia sativa spp. nigra - Rhizobium leguminosarum bv. viciae symbiotic interaction is improved by Azospirillum brasilense

This study aimed to examine the effects of inoculation with wild type (Sp7) and mutant strains of Azospirillum brasilense on the Vicia sativa spp. nigra (vetch)-Rhizobium leguminosarum bv. viciae (Rlv) symbiosis. The A. brasilense mutants were ipdC ^- and napA ^-, impaired in indole pyruvate decarboxylase and periplasmic nitrate reductase, respectively; and acdS ⁺ , carrying the ACC deaminase gene. Inoculations were done in pots, pouches and hydroponics and we measured shoot and root weight parameters as well as effects on root morphology and nod gene induction ability by roots. In pots, wild type Sp7 and the acdS ⁺ strain, but ipdC ^- and napA ^- mutants, lead to an increase in root hair density, 3–4?cm above the root tip. In pouches, combined inoculation with Rlv and strains Sp7, acdS ⁺ or ipdC ^-, but napA ^-, increased shoot dry matter and nodulation relative to Rlv alone. In a hydroponic system, co-inoculation with strains Sp7 or acdS ⁺ , but with ipdC ^- and napA ^- mutants, enhanced root secretion of nod gene-inducing flavonoids in comparison with Rlv-inoculated plants. These results support that auxin production by A. brasilense has a positive effect on root secretion of nod gene-inducing flavonoids and auxin absorption activity by the plant.     

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.     

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.     

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.     

Immunoelectron microscopy investigation of the cell surface of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strains

The genus-specific surface protein antigens of Azospirillum brasilense strains were visualized immunochemically. The procedure used for cell sample preparation was optimized to ensure that the surface protein structures were detected on cells in situ. Gold and gold-silver nanoparticles were conjugated to antibodies raised against the flagellin of A. brasilense type strain Sp7, against the lipopolysaccharide of A. brasilense Sp245, and against the genus-specific protein determinants of A. brasilense Sp7. Electron microscopic analysis using nanoparticle-labeled antibodies revealed antigenic determinants of the polar flagellum on the A. brasilense Sp245 cell surface, which in these bacteria are normally screened from the surroundings by a lipopolysaccharide sheath. Pili-like structures were detected on the Sp245 wild-type strain and on its Fla^– Swa^– Omegon-Km mutant SK048, which are presumably involved in microcolonial spreading in these bacteria.     

Azospirillum sp. Promotes Root Hair Development in Tomato Plants through a Mechanism that Involves Ethylene

Tomato seeds were inoculated with the plant growth–promoting rhizobacteria Azospirillum brasilense FT326, and changes in parameters associated with plant growth were evaluated 15 days after inoculation. Azospirilla were localized on roots and within xylematic tissue. An increase in shoot and root fresh weight, main root hair length, and root surface indicated that inoculation with A. brasilense FT 326 resulted in plant growth improvement. The levels of indole-3-acetic acid (IAA) and ethylene, two of the phytohormones related to plant growth, were higher in inoculated plants. Exogenously supplied ethylene mimicked the effect of inoculation, and the addition of an inhibitor of its synthesis or of its physiological activity completely blocked A. brasilense growth promotion. Based on our results, we propose that the process of growth promotion triggered by A. brasilense inoculation involves a signaling pathway that has ethylene as a central, positive regulator.     

INVOLVEMENT OF INDOLE‐3‐ACETIC ACID PRODUCED BY THE GROWTH‐PROMOTING BACTERIUM AZOSPIRILLUM SPP. IN PROMOTING GROWTH OF CHLORELLA VULGARIS1

Involvement of indole‐3‐acetic acid (IAA), produced by the microalgae‐growth‐promoting bacteria Azospirillum brasilens and A. lipoferum, in promoting growth of the microalga Chlorella vulgaris Beij. was studied. Four wildtype strains of Azospirillum and their IAA‐deficient mutants were co‐immobilized with C. vulgaris in alginate beads. Cultures were grown in synthetic growth medium supplemented with tryptophan. Growth promotion of microalgae and production of exogenous IAA by Azospirillum spp. were monitored. All wildtype Azospirillum spp. produced significant but varying amounts of IAA, while their mutant forms produced significantly less. The results demonstrated a significant growth promotion in Chlorella cultures when immobilized with the four wildtype strains of Azospirillum, while very low or no enhanced growth was induced by the four IAA‐deficient mutants, compared to when C. vulgaris is immobilized alone. A complementation experiment, where an IAA‐attenuated mutant (A. brasilense SpM7918) was supplemented with IAA produced by its parental wildtype strain (A. brasilense Sp6), restored growth promotion in the microalgae‐mutant culture.     

An <Emphasis Type="Italic">ipdC</Emphasis> gene knock-out of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strain SM and its implications on indole-3-acetic acid biosynthesis and plant growth promotion

The indole-3-pyruvate decarboxylase gene (ipdC), coding for a key enzyme of the indole-3-pyruvic acid pathway of IAA biosynthesis in Azospirillum brasilense SM was functionally disrupted in a site-specific manner. This disruption was brought about by group II intron-based Targetron gene knock-out system as other conventional methods were unsuccessful in generating an IAA-attenuated mutant. Intron insertion was targeted to position 568 on the sense strand of ipdC, resulting in the knock-out strain, SMIT568s10 which showed a significant (∼50%) decrease in the levels of indole-3-acetic acid, indole-3-acetaldehyde and tryptophol compared to the wild type strain SM. In addition, a significant decrease in indole-3-pyruvate decarboxylase enzyme activity by ∼50% was identified confirming a functional knock-out. Consequently, a reduction in the plant growth promoting response of strain SMIT568s10 was observed in terms of root length and lateral root proliferation as well as the total dry weight of the treated plants. Residual indole-3-pyruvate decarboxylase enzyme activity, and indole-3-acetic acid, tryptophol and indole-3-acetaldehyde formed along with the plant growth promoting response by strain SMIT568s10 in comparison with an untreated set suggest the presence of more than one copy of ipdC in the A. brasilense SM genome.     

The benefits of foliar inoculation with <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> in soybean are explained by an auxin signaling model

Azospirillum sp. is one of the most studied genera of plant growth-promoting rhizobacteria (PGPR). The ability of Azospirillum sp. to promote plant growth has been associated with its ability to produce several phytohormones, such as auxins, gibberellins and cytokinins, but mainly indole-3-acetic acid (IAA). It has been propoosed that the production of IAA explains the positive effects of co-inoculation with Azospirillum sp. on the rhizobia-legume symbiosis. In this study, we constructed an IAA-deficient mutant of A. brasilense Az39 (ipdC ^? ) by using a restriction-free cloning method. We inoculated soybean seeds with 1·10⁶ cfu·seed^?1 of Bradyrhizobium japonicum E109 and co-inoculating leaves at the V3 stage with 1·10⁸ cfu.plant^?1 of A. brasilense Az39 wt or ipdC ^? or inoculated leaves with 20 μg.plant^?1 synthetic IAA. The results confirmed soybean growth promotion as there was increased total plant and root length, aerial and root dry weight, number of nodules on the primary root, and an increase in the symbiosis established with B. japonicum E109. Nodule weight also increased after foliar co-inoculation with the IAA- producer A. brasilense Az39. The exogenous application of IAA decreased aerial and root length, as well as the number of nodules on primary roots in comparison with the Az39 wt strain. These results allow us to propose a biological model of response to foliar co-inoculation of soybean with IAA-producing rhizobacteria. This model clearly shows that both the presence of microorganism as part of the colonization process and the production of IAA in situ are co-responsible, via plant signaling molecules, for the positive effects on plant growth and symbiosis establishment.     

Comparative in situ analysis of ipdC-gfpmut3 promoter fusions of Azospirillum brasilense strains Sp7 and Sp245

Inoculation of wheat roots with Azospirillum brasilense results in an increase of plant growth and yield, which is proposed to be mainly due to the bacterial production of indole-3-acetic acid in the rhizosphere. Field inoculation experiments had revealed more consistent plant growth stimulation using A. brasilense strain Sp245 as compared with the strain Sp7. Therefore, the in situ expression of the key gene ipdC (indole-3-pyruvate decarboxylase) was examined in these two strains. Within the ipdC promoter of strain Sp245 a region of 150 bases was identified, which was missing in strain Sp7. Thus, three different translational ipdC promoter fusions with gfpmut3 were constructed on plasmid level: the first contained the part of the Sp245 promoter region homologous to strain Sp7, the second was bearing the complete promoter region of Sp245 including the specific insertion and the third comprised the Sp7 promoter region. By comparing the fluorescence levels of these constructs after growth on mineral medium with and without inducing amino acids, it could be demonstrated that ipdC expression in A. brasilense Sp245 was subject to a stricter control compared with strain Sp7. Microscopic detection of these reporter strains colonizing the rhizoplane documented for the first time an in situ expression of ipdC.     

Effect of co-inoculation of methylotrophic Methylobacterium oryzae with Azospirillum brasilense and Burkholderia pyrrocinia on the growth and nutrient uptake of tomato, red pepper and rice

The present greenhouse study was undertaken to evaluate the effects of co-inoculating methylotrophic Methylobacterium oryzae CBMB20 along with nitrogen-fixing Azospirillum brasilense CW903 or a phosphate solubilizing bacterium Burkholderia pyrrocinia CBPB-HOD on the growth and nutrient uptake of tomato, red pepper and rice. Seed inoculation and soil/foliar application of the bacterial strains alone or under dual inoculation increased the plant growth in terms of shoot or root length and increased the nutrient uptake in the plants studied compared to uninoculated control plants. Co-inoculation of M. oryzae CBMB20 with A. brasilense CW903 or B. pyrrocinia CBPB-HOD improved the N and P concentration of plants, while the results varied among the plant species tested. Also, co-inoculation of the bacterial strains increased the activity of nitrogenase, urease and phosphatase enzymes in soil when compared to uninoculated control or individual inoculations. Though the inoculation effects were analyzed at an early stage of plant growth, the results conclusively suggest that M. oryzae being compatible with other microorganisms in the rhizosphere can potentially be used as individual inoculant or co-inoculated with other plant growth promoting bacteria to increase the production in sustainable agricultural systems.     

Promotion of Peanut Growth by Co-inoculation with Selected Strains of <Emphasis Type="Italic">Bradyrhizobium</Emphasis> and <Emphasis Type="Italic">Azospirillum</Emphasis>

The ability of inoculated rhizobial strains to increase root nodulation of host legumes often depends on their competitiveness with existing native soil strains. Results of studies to date on rhizobial inoculation for improvement of peanut (Arachis hypogaea L.) production in Argentina have been inconsistent and controversial. In many cases, nodulation and yield of peanut crops have been increased by inoculation of specific rhizobial strains. Native peanut-nodulating strains are generally present in soils of agricultural areas, but their growth-promoting effect is often lower than that of inoculated strains. Many species of the genus Bradyrhizobium interact in a host-specific manner with legume species and form nitrogen-fixing root nodules. Other free-living rhizobacteria such as species of the genus Azospirillum are facultatively capable of interacting with legume roots and promoting plant growth. We evaluated and compared the effects of various single inoculation and co-inoculation treatments on peanut growth parameters in greenhouse and field experiments. In the greenhouse studies, co-inoculation with various Bradyrhizobium strains (native 15A and PC34, and recommended peanut inoculant C145), and Azospirillum brasilense strain Az39 generally resulted in increases in the measured parameters. The growth-promoting effect of 15A was similar to or higher than that of C145. In the field studies, 15A-Az39 co-inoculation had a greater promoting effect on measured growth parameters than did C145-Az39 co-inoculation. Our findings indicate that careful selection of native rhizobacterial strains adapted to peanut soils is useful in strategies for growth promotion, and that 15A in particular is a promising candidate for future inoculant formulation.     

Identification and Isolation of the Indole-3-Pyruvate Decarboxylase Gene from Azospirillum brasilense Sp7: Sequencing and Functional Analysis of the Gene Locus

The root-associated bacterium Azospirillum brasilense Sp7 produces the growth-stimulating phytohormone indole-3-acetic acid (=IAA) via the indole-3-pyruvate pathway. The DNA region containing ipdC, the structural gene for indole-3-pyruvate decarboxylase, was identified in a cosmid gene library of strain Sp7 by hybridization and has been sequenced. Upstream of the gene, two other ORF homologous to gltX and cysS were sequenced that are transcribed in the opposite direction. A functional analysis of the cloned ipdC region has been performed. To test the expression of the gene, a lacZ-Km cartridge was introduced into the gene. By this construct, tryptophan-dependent stimulation of gene expression in A. brasilense Sp7 was observed. Evidences for the existence of another copy of the ipdC gene in the Azospirillum genome are also reported. Received: 31 October 1997 / Accepted: 8 December 1997     

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.     

Molecular cloning and sequence analysis of an Azospirilium brasilense indole-3-pyruvate decarboxylase gene

Azospirillum brasilense isolated from the rhizosphere of different plants has the ability to excrete indole-3-acetic acid (IAA) into the culture media. Cosmid p0.2, isolated from an A. brasilense Sp245 genome library in pLAFR1, complements the Tn5-induced mutant SpM7918 of A. brasilense Sp6 which excretes reduced amounts of IAA. Restriction mapping and gene expression studies identified a BglII-EcoRI 4.3 kb fragment of p0.2 sufficient for the restoration of high levels of IAA production in mutant SpM7918. Tn5 mutagenesis localized the gene responsible on a 1.8 kb SmaI fragment. Nucleotide sequence analysis revealed that this fragment contains one complete open reading grame. The predicted protein sequence shows extensive homology with the indole-3-pyruvate decarboxylase of Enterobacter cloacae and the pyruvate decarboxylases of Saccharomyces cerevisiae and Zymomonas mobilis. The A. brasilense mutant Sp245a, constructed by homogenotization of a Tn5 insertion derivative of the 1.8 kb SmaI fragment, also displayed reduced IAA production. Introduction of the cloned wild-type gene into Rhizobium meliloti 1021 resulted in increased IAA production. Cell-free extracts prepared from R. meliloti and A. brasilense transconjugants harboring this gene could convert indole-3-pyruvic acid to indole-3-acetaldehyde and tryptophol. These results clearly demonstrate that IAA production in A. brasilense is mediated by indole-3-pyruvate decarboxylase.     

Physiological and genetic analysis of root responsiveness to auxin-producing plant growth-promoting bacteria in common bean (Phaseolus vulgaris L.)

Plant root development can be largely affected through the association of roots with plant growth-promoting rhizobacteria (PGPR). However, little is known about the identity of plant genes enabling such PGPR-plant root associations. Differences in the responsiveness to PGPR among cultivars suggest genetic variation for this trait within germplasm. In this study, two genotypes of common bean (Phaseolus vulgaris L.), BAT477 and DOR364, were identified showing contrasting responsiveness in root development to inoculation with the PGPR Azospirillum brasilense Sp245. Inoculation with an A. brasilense Sp245 mutant strain strongly reduced in auxin biosynthesis or addition of increasing concentrations of exogenous auxin to the plant growth medium, indicated that the differential response to A. brasilense Sp245 among the bean genotypes is related to a differential response to the bacterial produced auxin. To further assess the role of the plant host in root responsiveness, a population of Recombinant Inbred Lines (RILs) of the DOR364×BAT477 cross was used to evaluate the efficacy of exogenous auxin on root development. We detected significant phenotypic variation among the RILs for basal root formation during germination upon addition of auxin to the growth medium. Genetic analysis revealed two quantitative trait loci (QTLs) associated with basal root responsiveness to auxin of which one explained 36% of the phenotypic variation among the RILs. This latter QTL mapped to the same location as a QTL for root tip formation at low P, suggesting that the host effect on root responsiveness to IAA interacts with specific root development. Also, significant correlations between basal root responsiveness to auxin and growth, root tips and root dry weight at low P were identified. To our knowledge, this is the first report on QTL detection for root responsiveness to auxin.     

Azospirillum brasilense Sp 245 produces ABA in chemically-defined culture medium and increases ABA content in arabidopsis plants

Azospirillum sp. are plant growth promoting bacteria (PGPB) that increase grain yield in cereals and other species via growth promotion and/or stress alleviation. The PGPB beneficial effects have been partially attributed to bacterial production of plant hormones, especially growth promoters like auxins, gibberellins and cytokinins. This paper reports the characterization of the stress-like plant hormone abscisic acid (ABA) by GC-EIMS in cultures of A. brasilense Sp 245 after 120 h of incubation in chemically-defined media, and chemically-defined media with moderate stress (100 mM NaCl). Chemical characterization of ABA was done by gas chromatography-electron impact mass spectrometry (GC-EIMS) and quantification by selected ion monitoring (SIM) with a stable isotope of the hormone as internal standard in the media. A. brasilense cultures produced higher amounts of ABA per ml of culture when NaCl was incorporated in the culture medium. Inoculation of Arabidopsis thaliana with A. brasilense Sp 245 enhanced two-fold the plant’s ABA content. These results contribute to explain, at least to some extent, the beneficial effects of Azospirillum sp. previously found in inoculated plants placed under adverse environmental conditions.     

Alternative mechanism for the evaluation of indole-3-acetic acid (IAA) production by Azospirillum brasilense strains and its effects on the germination and growth of maize seedlings

We evaluated the production of indole-3-acetic acid (IAA) by Azospirillum brasilense strains in vitro (cell culture supernatants) and in vivo (stems and roots of maize seedlings) to clarify the role of this phytohormone as a signaling and effector molecule in the symbiotic interaction between maize and A. brasilense. The three strains all showed IAA production when cultured in NFb medium supplemented with 100 μg/ml L-tryptophan. The level of IAA production was 41.5 μg/ml for Yu62, 12.9 μg/ml for Az39, and 0.15 μg/ml for ipdC-. The release of IAA into culture medium by the bacteria appeared to be the main activator of the early growth promotion observed in the inoculated maize seedlings. The application of supernatants with different IAA contents caused significant differences in the seedling growth. This observation provides the basis for novel technological tools for effective quality control procedures on inoculants. The approach described can be incorporated into different inoculation methods, including line sowing, downspout, and foliar techniques, and increase the sustainability of symbiotic plant-bacteria systems.     

Inter-root movement of Azospirillum brasilense and subsequent root colonization of crop and weed seedlings growing in soil

Inter-root movement and dispersion of the beneficial bacterium Azospirillum brasilense were monitored in root systems of wheat seedlings growing in the field and in growth chamber soil trays. Two strains were used, a motile wild-type strain (Cd, mot⁺) and a motility deficient strain (mot^–), which was derived from the Cd strain. Root colonization by two wild-type strains (Cd and Sp-245) was studied in 64 plant species growing in pots in the greenhouse. The two wild-type strains of A. brasilense were capable of colonizing all tested plant species. In soil trays and in the field, mot⁺ cells moved from inoculated roots to non-inoculated roots of either wheat plants or weeds growing in the same field plot, but the mot^– strain did not move toward non-inoculated roots of either plant species. In the field, both mot⁺ and mot^– strains of A. brasilense survived well in the rhizosphere of wheat for 30 days, but only mot⁺ moved between different weeds, regardless of the species, botanical family, or whether they were annuals or perennials. In plant-free, water-saturated soils, either in columns or in the field, both strains remained at the inoculation site and did not move.It is proposed (a) that A. brasilense is not a plant-specific bacterium and that (b) colonization of the entire root system in soil is an active process determined by bacterial motility; it is not plant specific, but depends on the presence of plants. Correspondence to: Y. Bashan