Modeling growth and biochemical activities of Azospirillum spp.

An unstructured mathematical model was developed and used in the evaluation of biochemical activities of four Azospirillum spp. strains grown in batch cultures in a high C/N-ratio medium. The strains were evaluated for their ability to grow on fructose and produce exo-polysaccharide, and to sustain nitrogenase activity by using fructose or polysaccharides. Quantitative expression of the regulation of polysaccharide synthesis and nitrogenase (acetylene reduction) activity from the mineral nitrogen and sugar concentration in the culture medium was achieved. It was found that, during growth, Azospirillum spp. produced significant quantities of exocellular and capsular polysaccharide, whereas after depletion of the carbon source from the culture medium polysaccharides were consumed, especially in A. lipoferum strains. Significant nitrogenase activity was detected during polysaccharide degradation. Oxygen uptake was high during assimilation of fructose and low during polysaccharide degradation.     

Fructose catabolism in Azospirillum brasilense and Azospirillum lipoferum.

The pathways for catabolism of fructose were investigated in the type strains of Azospirillum lipoferum and Azospirillum brasilense grown aerobically with (NH4)2SO4 as the nitrogen source. When grown on fructose, the former species possessed a complete Entner-Doudoroff pathway, whereas the latter species lacked activity for glucose-6-phosphate dehydrogenase. Both species possessed a complete catabolic Embden-Meyerhof-Parnas pathway. Neither species possessed the key enzyme of the hexose monophosphate pathway, 6-phosphogluconate dehydrogenase. Both species could phosphorylate fructose to fructose-1-phosphate by means of a phosphoenolpyruvate-phosphotransferase system, and high activities of 1-phosphofructokinase occurred. Both species possessed glucokinase activity, but only A. lipoferum had hexokinase activity; moreover, the cells of A. brasilense were nearly impermeable to glucose, accounting for the inability of this species to grow on glucose. Both species possessed pyruvate dehydrogenase, a complete tricarboxylic acid cycle, a glyoxylate shunt, and malic enzyme. Analysis of the acidic end products for both species indicated the formation of only small amounts of various organic acids, and most of the titratable acidity was due to utilization of the ammonium ions of the medium. Gluconic acid was not formed during growth of either species on fructose but was detected during growth of A. lipoferum on glucose; this species also possessed an NADP-linked glucose dehydrogenase and gluconokinase.     

Salinity stress responses in the plant growth promoting rhizobacteria,Azospirillum spp

In order to adapt to the fluctuations in soil salinity/osmolarity the bacteria of the genusAzospirillum accumulate compatible solutes such as glutamate, proline, glycine betaine, trehalose, etc. Proline seems to play a major role in osmoadaptation. With increase in osmotic stress the dominant osmolyte inA. brasilense shifts from glutamate to proline. Accumulation of proline inA. brasilense occurs by both uptake and synthesis. At higher osmolarityA. brasilense Sp7 accumulates high intracellular concentration of glycine betaine which is taken up via a high affinity glycine betaine transport system. A salinity stress induced, periplasmically located, glycine betaine binding protein (GBBP) of ca. 32 kDa size is involved in glycine betaine uptake inA. brasilense Sp7. Although a similar protein is also present inA. brasilense Cd it does not help in osmoprotection. It is not known ifA. brasilense Cd can also accumulate glycine betaine under salinity stress and if the GBBP-like protein plays any role in glycine betaine uptake. This strain, under salt stress, seems to have inadequate levels of ATP to support growth and glycine betaine uptake simultaneously. ExceptA. halopraeferens, all other species ofAzospirillum lack the ability to convert choline into glycine betaine. Mobilization of thebet ABT genes ofE. coli intoA. brasilense enables it to use choline for osmoprotection. Recently, aproU-like locus fromA. lipoferum showing physical homology to theproU gene region ofE. coli has been cloned. Replacement of this locus, after inactivation by the insertion of kanamycin resistance gene cassette, inA. lipoferum genome results in the recovery of mutants which fail to use glycine betaine as osmoprotectant.     

Sunflower inoculation with Azospirillum and other plant growth promoting rhizobacteria

The bacterial microflora from sunflower rhizosphere (Helianthus annus L.) and one strain ofAzospirillum lipoferum of a different origin were screened for their ability to promote sunflower growth in a 6-day germination test and in pot experiments. TwoAzospirillum lipoferum strains and oneXanthomonas maltophilia strain produced the best responses. These strains were chosen for field testing.     

Plant growth-altering effects of Azospirillum brasilense and Bacillus C–11–25 on two wheat cultivars

The effect of Azospirillum brasilense Cd, Bacillus C–11–25, indole acetic acid, gibberellic acid and cytokinin on plant growth characteristics of two wheat ( Triticum aestivum L. emend Thell) cultivars was studied under laboratory and greenhouse conditions. Responses of wheat plants to bacterial inoculation were similar to those caused by the addition of gibberellic acid in growth pouches. Chester and Fielder wheat varieties differed in responses to the bacteria and hormone additions. When added to growth pouches, bacterial culture filtrates and dead bacterial cells caused plant growth responses similar to those caused by the addition of live cells. Bacteria and hormone additions resulted in increased permeability of Fielder wheat to ¹⁵Nlabelled nitrate, and decreased nitrate permeability of Chester wheat. Bacterial inoculation of soil in pots caused ¹⁵N isotope dilution in Fielder but not in Chester wheat. Hormone addition to pots caused isotope dilution in Chester wheat. It appeared that genetic differences between cultivars affected plant growth responses. The accuracy of estimates of N₂ fixation by associative bacteria based on ¹⁵N isotope dilution calculations may be reduced if control plants differ in plant response to these bacteria.     

Factors inducing transition from growth to dormancy in rhizobacteria Azospirillum brasilense

The factors suppressing division of the cells of the rhizobacterium Azospirillum brasilense and inducing their transition to a dormant state were analyzed. These included the presence of hexylresorcinol or heavy metals (Cu and Co) in the medium, oxygen stress, and transfer of the cells into the physiological saline or phosphate buffer solution. The results were used to develop a protocol for obtaining of nonculturable cells of A. brasilense Sp245, a natural symbiont of wheat. The cells lost their ability to grow on synthetic agar medium, but could revert to growth when incubated in freshly prepared liquid medium. Needle-shaped crystals differing from struvite, which has been previously reported for this strain, were found in the dormant culture of A. brasilense Sp245.     

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.     

Flocculation in Azospirillum brasilense and Azospirillum lipoferum: exopolysaccharides and cyst formation.

The phenomena of flocculation and floc formation by Azospirillum brasilense Sp7 (ATCC 29145) and Azospirillum lipoferum Sp59b (ATCC 29707) were studied in aerobic liquid cultures. Carbon sources representative of various entry pathways in combination with various nitrogen sources induced flocculation in both species of azospirilla. Noticeably, the combination of fructose and nitrate was the most effective in terms of floc yields. Phase-contrast microscopic observations revealed a transition in cell morphology from freely motile, vibrioid cells to nonmotile, highly refractile encysting forms during the formation of flocs. The nonmotile forms in flocs appeared to be entangled within a fibrillar matrix, and the cells were highly resistant to desiccation. Dried flocs kept for almost 6 months still maintained the highly refractile encysting forms, and their viability was confirmed by pellicle formation and acetylene reduction in semisolid malate medium. Electron microscopic observations of the desiccated flocs revealed the presence of cell forms containing abundant poly beta-hydroxybutyrate granules within a central body and surrounded by a thick layer of exopolysaccharides. The latter were characterized by alkali and acid digestion, crude cellulase hydrolysis, and calcofluor staining. It was concluded that the overproduction of exocellular polymers induces the flocculent growth and is associated with the concomitant transformation of vegetative cells to the desiccation-resistant encysting forms under limiting cultural conditions.     

Azospirillum amazonense inoculation: effects on growth, yield and N2 fixation of rice (Oryza sativa L.)

Bacteria of the genus Azospirillum are considered to be plant-growth promoting bacteria (PGPR) and stimulate plant growth directly either by synthesising phyto-hormones or by promoting nutrition by the process of biological nitrogen fixation (BNF). Although this genus extensively studied, the effects of inoculation and the possible BNF contribution of the Azospirillum amazonense specie are very scarce. The aim of this study was to isolate, characterise and evaluate auxin production and nitrogenase activity of this species and to select, by inoculation of rice plants, promising isolates based on their ability to improve plant growth, yield and the BNF contribution. One hundred and ten isolates obtained from rice were characterised and grouped according to colony features. Forty-two isolates, confirmed as A. amazonense by the fluorescent in situ hybridization (FISH) technique, were tested for auxin production and nitrogenase activity in vitro. Subsequently plant growth promotion related to plant nutrition effect was evaluated, in vitro and in greenhouse experiments. The BNF contribution to plant growth was evaluated using the ¹⁵N isotope dilution technique. All A. amazonense strains tested produced indoles, but only 10% of them showed high production, above 1.33 μM mg protein⁻¹. The nitrogenase activity also was variable and only 9% of isolates showed high nitrogenase activity and the majority (54%) exhibited a low potential. The inoculation of selected strains in rice under gnotobiotic conditions reduced the growth of root and aerial part when compared to the control, showing the negative effects of excess of phytohormone accumulation in the medium. However, in the greenhouse experiment, inoculation of strains of A. amazonense increased grain dry matter accumulation (7 to 11.6%), the number of panicles (3 to 18.6%) and nitrogen accumulation at grain maturation (3.5 to 18.5%). BNF contributions up to 27% were observed for A. amazonense Y2 (wild type strain). The data presented here is the first report that the PGPR effect of A. amazonense for rice plants grown under greenhouse conditions is mainly due the BNF contribution as measured by ¹⁵N isotope dilution technique, in contrast to the hormonal effect observed with other Azospirillum species studied.     

Biological nitrogen fixation in non-leguminous field crops: Facilitating the evolution of an effective association between Azospirillum and wheat

Recent advances towards achieving significant nitrogen fixation by diazotrophs in symbioses with cereals are reviewed, referring to the literature on the evolution of effective symbioses involving rhizobia and Frankia as microsymbionts. Data indicating that strains of Acetobacter and Herbaspirillum colonizing specific cultivars of sugarcane as endophytes make a significant contribution to the nitrogen economy of this crop improves the prospects that similar associative systems may be developed for other gramineous species such as rice and wheat. By contrast, the transfer of nodulation genes similar to those in legumes or Parasponia to achieve nodulation in crops like rice and wheat is considered to be a more ambitious and distant goal. Progress in developing an effective associative system for cereals has been materially assisted by the development of genetic tools based on the application of lacZ and gusA fusions with the promoters of genes associated with nitrogen fixation. These reporter genes have provided clear evidence that crack-entry at the points of emergence of lateral roots or of 2,4-D induced para-nodules is the most significant route of endophytic colonization. Furthermore, using the laboratory model of para-nodulated wheat, there is now evidence that the ability of azospirilla and other nitrogen fixing bacteria to colonize extensively as endophytes can be genetically controlled. The most successful strain of Azospirillum brasilense (Sp7-S) for endophytic colonization and nitrogen fixation in wheat seedlings is a mutant with reduced exopolysaccharide production. Most other strains of azospirilla do not colonize as endophytes and it is concluded that though these are poorly adapted to providing nitrogen for the host plant, they are well adapted for survival and persistence in soil. A research program combining the study of endophytic colonization by azospirilla with an examination of the factors controlling the effectiveness of association (oxygen tolerance and nitrogen transfer) is now being pursued. It is proposed that a process of facilitated evolution of para-nodulated wheat involving the stepwise genetic improvement of both the prospective microsymbionts and the cereal host will eventually lead to effective nitrogen-fixing associations. In the attempt to achieve this goal, continued study of the endophytes occurring naturally in sugar cane and other grasses (e.g. Azoarcus sp.) should be of assistance.     

Plant growth regulators and amino acids released by Azospirillum sp in chemically defined media

AIMS: To investigate the ability of Azospirillum sp., a facultative endophitic diazotrophic bacterium, to release plant growth regulators (PGR) such as polyamines, ethylene, indoleacetic acid and amino acids in both combined-N and N-free cultures. METHODS AND RESULTS: The presence of those substances was analysed by HPLC. Azospirillum sp. is capable of releasing PGR and amino acids into the culture medium. CONCLUSIONS: The type and quantity of the released substances varied, depending on the presence of combined-N in the medium. SIGNIFICANCE AND IMPACT OF THE STUDY: A better knowledge of PGR produced by Azospirillum sp. has been gained.     

Development and standardization of cyst based liquid formulation of Azospirillum bioinoculant

Azospirillum bioinoculant is well known for its high nitrogen fixing and plant growth promoting characters. The carrier based bioinoculants generally suffer from shorter shelf life, poor quality, high contamination and low field performance. Therefore, it is necessary to develop alternative new formulation of inoculants where cyst based inoculants can play significant role. The cyst based liquid formulation was developed by inoculating Azospirillum into the cyst inducing minimal salts medium (MSM). One hundred per cent conversion of vegetative cells into cyst cells was noticed in 96 h. The survival of cyst cells in the MSM was observed up to one year and two months and interestingly, the population level of 10(8) was maintained till the final observation. The cyst cells of Azospirillum accumulated poly-beta-hydroxybutyrate (PHB) granules and exhibited desiccation tolerance up to 20 days and temperature tolerance up to 40 degrees C. Thus the cyst based liquid formulation has twin advantage of longer shelf life and tolerance to harmful environmental conditions. Regeneration of cyst cells into vegetative cells in different media viz., tap water, sterile water, rice gruel and nutrient broth was studied. The changes started within 3 h and complete return of vegetative cells was observed at 24 h. Although all the media tested favoured regeneration, comparatively quicker regeneration was observed in nutrient broth and followed by rice gruel. Thus, cyst based liquid formulation of Azospirillum has all the survival advantages and can be used as a potential bioinoculant.     

An alternative explanation for plant growth promotion by bacteria of the genus Azospirillum

Experiments were performed to identify the substances that are excreted by the soil bacterium Azospirillum brasilense Sp7 and that were reported to stimulate the formation of lateral roots and of root hairs of grasses. Azospirillum forms indole-3 acetic acid (IAA) but only in the late stationary growth phase or when tryptophan is present in the medium, but not in continuous cultures or in the logarithmic growth phase of batch cultures. Formation of IAA by Azospirillum requires aerobic conditions. Nitrite can replace IAA in several phytohormone assay, and is even more active than IAA in a test with wheat root segments in which the increase of wet weight is determined. Higher amounts of nitrite are necessary for activity in other classical auxin assays. Nitrite shows 40–60% of the activity of IAA in the straight-growth test of Avena coleoptiles and in the formation of C₂H₄ by pea epicotyl segments. Like IAA, nitrite is inactive in promoting C₂H₄ formation by ripe apple tissues. Since nitrite alone can hardly exert phytohormonal effects, it is postulated that nitrite reacts with a substance in the cells and that a product formed by this reaction functions as auxin. Such a substance could be ascorbate. Exogenously added ascorbate enhances the rate of nitrite-dependent C₂H₄ formation by pea epicotyl sections and the nitrite-dependent increase in the wet weight of wheat root segments. Nitrite is formed by nitrate respiration of Azospirillum. The findings that nitrite can have phytohormonal effects offers an alternative explanation of the promotion of the growth of roots and the enhancement of mineral uptake of grasses by Azospirillum. Indole-acetic acid completely and nitrite partly substitute for an inoculation with Azospirillum in an assay where the increase of the dry weight of intact wheat roots is determined after an incubation for 10 d. Nitrite and IAA are, therefore, possibly the only factors causing an enhancement of the growth of roots of grasses.Abbreviations HPLC high-performance liquid chromatography - IAA indole-3-acetic acid     

Search for conjugative plasmids in Azospirillum brasilense and Azospirillum lipoferum associated with plants

The conjugative plasmids in Azospirillum brasilense strains S17. Sp107, Sp245, SpBr14, JM6B2, JM82Al, UQ1794, UQI796 and in Azospirillum lipoferum strain RG20 were prove to exist for the first time in connection with their potency to mobilize a non-conjugative IncQ-plasmid pVZ361 from IncQ-group (ori RSF1010, KmR, SuR. 11.4 kb) for conjugated transfer to aplasmid strains Agrobacterium tumefaciens and Pseudomonas putida at high frequencies.     

Regulation of nitrogenase activity by oxygen in Azospirillum brasilense and Azospirillum lipoferum.

The nitrogenase activity of the microaerophilic bacteria Azospirillum brasilense and A. lipoferum was completely inhibited by 2.0 kPa of oxygen (approximately 0.02 atm of O2) in equilibrium with the solution. The activity could be partially recovered at optimal oxygen concentrations of 0.2 kPa. In contrast to the NH4+ switch off, no covalent modification of the nitrogenase reductase (Fe protein) was involved, as demonstrated by Western-blotting and 32P-labeling experiments. However, the inhibition of the nitrogenase activity under anaerobic conditions was correlated with covalent modification of the Fe protein. In contrast to the NH4+ switch off, no increase in the cellular glutamine pool and no modification of the glutamine synthetase occurred under anaerobic switch-off conditions. Therefore, a redox signal, independent of the nitrogen control of the cell, may trigger the covalent modification of the nitrogenase reductase of A. brasilense and A. lipoferum.     

Influence of Azospirillum inoculation on the mineral uptake and growth of rice under hydroponic conditions

Seedlings of rice (Oryza sativa L. var. IR42) were inoculated with nitrogen-fixingAzospirillum lipoferum (strain 34H) by immersing the roots in the inoculum for 6 h. The plants were grown in the prescence of NH₄ ⁺-N for 47 days in a hydroponic system under greenhouse conditions. Inoculation significantly enhanced PO₄-ion uptake of the plants in 4 of the 7 samplings tested while the uptake of NH₄-ion was significantly increased in two samplings and was decreased in one sampling. Inoculation reduced root length significantly and caused significant increases in shoot fresh and dry weights. Root surface area was not affected by inoculation. Bacterial population counts suggested thatA. lipoferum survived on the roots till the end of the experiment.     

Azospirillum lipoferum and Azospirillum brasilense surface polysaccharide mutants that are affected in flocculation

M ichiels , K., V erreth , C. & V anderleyden , J. 1990. Azospirillum lipoferum and Azospirillum brasilense surface polysaccharide mutants that are affected in flocculation. Journal of Applied Bacteriology 69 , 705–711.
Surface polysaccharide production by Azospirillum is demonstrated by fluorescence of colonies grown on media containing the fluorescent dye Calcofluor, which binds to β-linked polysaccharides. Mutants showing decreased and increased levels of fluorescence are obtained from Azospirillum lipoferum strain Sp59b by chemical mutagenesis, and from A. brasilense strain 7030 by Tn5 mutagenesis.
The A. brasilense 7030 fluorescence mutants produce wild-type levels of exo-polysaccharide in their culture supernatant fluids, but are affected in flocculation in liquid culture. On the basis of these observations, we postulate that an A. brasilense surface polysaccharide, different from the exopolysaccharide, is involved in both Calcofluor staining and flocculation.
It is shown by DNA hybridization that the genetic loci affected in the A. brasilense 7030 fluorescence mutants are different from the A. brasilense exoB and exoC loci, which are involved in exopolysaccharide production.     

Influence of amino acids on nitrogen fixation ability and growth of Azospirillum spp

The utilization of amino acids for growth and their effects on nitrogen fixation differ greatly among the several strains of each species of Azospirillum spp. that were examined. A. brasiliense grew poorly or not at all on glutamate, aspartate, serine, or histidine as the sole nitrogen and carbon sources. Nitrogen fixation by most A. brasiliense strains was inhibited only slightly even by 10 mM concentrations of these amino acids. In contrast, A. lipoferum and A. amazonense grew very well on glutamate, aspartate, serine, or histidine as the sole nitrogen and carbon sources; nitrogen fixation, which was measured in the presence of malate or sucrose, was severely inhibited by these amino acids. It was concluded that growth on histidine as the sole source of nitrogen, carbon, and energy may be used for the taxonomic characterization of Azospirillum spp. and for the selective isolation of A. lipoferum. The different utilization of various amino acids by Azospirillum spp. may be important for their establishment in the rhizosphere and for their associative nitrogen fixation with plants. The physiological basis for the different utilization of glutamate by Azospirillum spp. was investigated further. A. brasiliense and A. lipoferum exhibited a high affinity for glutamate uptake (Km values for uptake were 8 and 40 microM, respectively); the Vmax was 6 times higher in A. lipoferum than in A. brasiliense. At high substrate concentrations (10 mM), the nonsaturable component of glutamate uptake was most active in A. lipoferum and A. amazonense.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of plant growth regulators on acetylene-reducing associations between Azospirillum brasilense and wheat

Treatment of wheat seedlings with the synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-d), induced nodule-like structures or tumours (termed para-nodules) where lateral roots would normally emerge. The formation of these structures promoted increased rates of acetylene reduction at reduced oxygen pressure (0.02–0.04 atm) in seedling inoculated with Azospirillum brasilense, compared to seedlings inoculated without auxin treatment. Fluorescent microscopy, laser scanning confocal microscopy and direct bacterial counts all showed that the 2,4-d treatment stimulated internal colonization of the root system with azospirilla, particularly in the basal region of the nodular structures. Both colonization with azospirilla and acetylene-reducing activity were further stimulated by simultaneous treatment with another synthetic auxin, naphthaleneacetic acid (NAA) and, less reliably, with indoleacetic acid (IAA) and indolebutyric acid (IBA). These auxins produced shortening of many initiated lateral roots, although 20 times the concentration of NAA was required to achieve rounded structures similar to those obtained with 2,4-d. Treatment with NAA, IAA or IBA alone also stimulated colonization with azospirilla and acetylene reduction rates at 0.02 atm oxygen, but less effectively than by treatment with 2,4-d. Such exogenous treatments of wheat seedlings with synthetic growth regulators provide an effective laboratory model for studies on the development of a N₂-fixing system in cereals.