Plant Growth-promoting Bacterium Azospirillum Brasilense Research Articles

Elemental composition of strawberry plants inoculated with the plant growth‐promoting bacteriumAzospirillum brasilenseREC3, assessed with scanning electron microscopy and energy dispersiveX‐ray analysis

The elemental composition of strawberry plants (Fragaria ananassa cv. Macarena) inoculated with the plant growth-promoting bacterium Azospirillum brasilense REC3, and non-inoculated controls, was studied using scanning electron microscopy (SEM) and energy dispersive X-ray (EDS) analysis. This allowed simultaneous semi-quantification of different elements in a small, solid sample. Plants were inoculated and grown hydroponically in 50% or 100% Hoagland solution, corresponding to limited or optimum nutrient medium, respectively. Bacteria-inoculated plants increased the growth index 45% and 80% compared to controls when grown in 100% and 50% Hoagland solution, respectively. Thus, inoculation with A.brasilense REC3 in a nutrient-limited medium had the strongest effect in terms of increasing both shoot and root biomass and growth index, as already described for Azospirillum inoculated into nutrient-poor soils. SEM-EDS spectra and maps showed the elemental composition and relative distribution of nutrients in strawberry tissues. Leaves contained C, O, N, Na, P, K, Ca and Cu, while roots also had Si and Cl. The organic fraction (C, O and N) accounted for over 96.3% of the total chemical composition; of the mineral fraction, Na had higher accumulation in both leaves and roots. Azospirillum-inoculated and control plants had similar elemental quantities; however, in bacteria-inoculated roots, P was significantly increased (34.33%), which constitutes a major benefit for plant nutrition, while Cu content decreased (35.16%).

Scale-up from shake flasks to pilot-scale production of the plant growth-promoting bacterium Azospirillum brasilense for preparing a liquid inoculant formulation

Azospirillum brasilense has industrial significance as a growth promoter in plants of commercial interest. However, there is no report in the literature disclosing a liquid product produced in pilot-scale bioreactors and is able to be stored at room temperature for more than 2years. The aim of this work was to scale up a process from a shake flask to a 10-L lab-scale and 1,000-L pilot-scale bioreactor for the production of plant growth-promoting bacterium A. brasilense for a liquid inoculant formulation. Furthermore, this work aimed to determine the shelf life of the liquid formulation stored at room temperature and to increase maize crops yield in greenhouses. Under a constant oxygen mass transfer coefficient (K L a), a fermentation process was successfully scaled up from shake flasks to 10- and 1,000-L bioreactors. A concentration ranging from 3.5 to 7.5 × 10(8)CFU/mL was obtained in shake flasks and bioreactors, and after 2years stored at room temperature, the liquid formulation showed one order of magnitude decrease. Applications of the cultured bacteria in maize yields resulted in increases of up to 95% in corncobs and 70% in aboveground biomass.

Amendment of degraded desert soil with wastewater debris containing immobilized Chlorella sorokiniana and Azospirillum brasilense significantly modifies soil bacterial community structure, diversity, and richness

The main goal of this study was to expand our knowledge of what happens to the soil bacterial community in an eroded desert soil when improvement of soil fertility is derived from the application of debris of tertiary wastewater treatment containing immobilized microalgae Chlorella sor- okiniana and the plant growth-promoting bacterium (PGPB) Azospirillum brasilense. We hypothesized that an "im- proved" non-agricultural desert soil will exhibit substantial changes in the structure of the bacterial community in a relatively short time after amendment. To assess the effect of the amendments, microalgae and PGPB alone or com- bined, on the structure of the rhizosphere bacterial commu- nity, changes in species richness and bacterial diversity over time were based on sequence differences in the 16S rRNA gene, performed with PCR-denaturing gradient gel electro- phoresis (DGGE) and then analyzed by similarity test and non-metric multidimensional scaling analysis. Root surface colonization and persistence in the rhizosphere of A. brasi- lense was monitored by fluorescent in situ hybridization and sequencing of DGGE bands. Application of waste debris significantly changed the rhizosphere bacterial population structure, whether comparisons were made over time, be- tween inoculated and non-inoculated soil, and among dif- ferent inoculated microorganisms. Species richness and diversity increased when the waste debris contained the microalgae-bacteria association and also over time. Even as its secondary role as an inoculant after wastewater treat- ment, A. brasilense colonized the root surface profusely and persisted within the rhizosphere bacterial community. This study demonstrated that small organic amendment to desert soil significantly changed soil bacterial community com- pared to the original soil and also 2 months after amend- ments were added.

Alginate beads provide a beneficial physical barrier against native microorganisms in wastewater treated with immobilized bacteria and microalgae

When the freshwater microalga Chlorella sorokiniana and the plant growth-promoting bacterium Azospirillum brasilense were deployed as free suspensions in unsterile, municipal wastewater for tertiary wastewater treatment, their population was significantly lower compared with their populations in sterile wastewater. At the same time, the numbers of natural microfauna and wastewater bacteria increased. Immobilization of C. sorokiniana and A. brasilense in small (2-4 mm in diameter), polymer Ca-alginate beads significantly enhanced their populations when these beads were suspended in normal wastewater. All microbial populations within and on the surface of the beads were evaluated by quantitative fluorescence in situ hybridization combined with scanning electron microscopy and direct measurements. Submerging immobilizing beads in wastewater created the following sequence of events: (a) a biofilm composed of wastewater bacteria and A. brasilense was created on the surface of the beads, (b) the bead inhibited penetration of outside organisms into the beads, (c) the bead inhibited liberation of the immobilized microorganisms into the wastewater, and (d) permitted an uninterrupted reduction of ammonium and phosphorus from the wastewater. This study demonstrated that wastewater microbial populations are responsible for decreasing populations of biological agents used for wastewater treatment and immobilization in alginate beads provided a protective environment for these agents to carry out uninterrupted tertiary wastewater treatment.

CELL-CELL INTERACTION IN THE EUKARYOTE-PROKARYOTE MODEL OF THE MICROALGAE CHLORELLA VULGARIS AND THE BACTERIUM AZOSPIRILLUM BRASILENSE IMMOBILIZED IN POLYMER BEADS(1).

Cell-cell interaction in the eukaryote-prokaryote model of the unicellular, freshwater microalga Chlorella vulgaris Beij. and the plant growth-promoting bacterium Azospirillum brasilense, when jointly immobilized in small polymer alginate beads, was evaluated by quantitative fluorescence in situ hybridization (FISH) combined with SEM. This step revealed significant changes, with an increase in the populations of both partners, cluster (mixed colonies) mode of colonization of the bead by the two microorganisms, increase in the size of microalgae-bacterial clusters, movement of the motile bacteria cells toward the immotile microalgae cells within solid matrix, and formation of firm structures among the bacteria, microalgae cells, and the inert matrix that creates a biofilm. This biofilm was sufficiently strong to keep the two species attached to each other, even after eliminating the alginate support. This study showed that the common structural phenotypic interaction of Azospirillum with roots of higher plants, via fibrils and sheath material, is also formed and maintained during the interaction of this bacterium with the surface of rootless single-cell microalgae.

Recycling waste debris of immobilized microalgae and plant growth-promoting bacteria from wastewater treatment as a resource to improve fertility of eroded desert soil

This study attempted to demonstrate that biological residue from a new biological wastewater treatment is a resource for improving quality of arid soils and plant growth. After tertiary wastewater treatment, debris composed of alginate beads containing the microalgae Chlorella sorokiniana and the plant growth-promoting bacterium Azospirillum brasilense was used as an amendment for eroded, infertile desert soil having low levels of organic matter. A. brasilense survived in these used dried beads for at least one year. Three consecutive applications of the dry debris increased organic matter, organic carbon, and microbial carbon in the soil. Growth of sorghum in the amended soil was greater than plants grown in low organic matter, untreated soil or soil amended with beads containing other combinations of alginate, microalgae, or bacteria. The surface of plant roots growing in the amended soil was heavily colonized by A. brasilense, with no endophytic colonization; root tips were the preferred sites of colonization.

Activity of two catabolic enzymes of the phosphogluconate pathway in mesquite roots inoculated with Azospirillum brasilense Cd

The mesquite amargo (Prosopis articulate), one of the main nurse trees of the Sonoran Desert in Mexico, is responsible for major, natural re-vegetation processes. It exudes gluconic acid in root exudates, a favorite carbon source for the plant growth-promoting bacterium Azospirillum brasilense. Two enzymes, gluconokinase (EC 2.7.1.12) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44), participating in the phosphogluconate pathway, are active in the bacteria. Bacterial 6-phosphogluconate dehydrogenase is a constitutive enzyme, while gluconokinase is induced upon exposure to gluconic acid. Both enzymes are active in young, non-inoculated mesquite seedlings growing under hydroponic conditions. When A. brasilense Cd bacteria are inoculated on the root system, the roots exhibit much higher activity of gluconokinase, but not 6-phosphogluconate dehydrogenase. Mesquite roots exhibit high levels of root colonization by the inoculating bacteria. At the same time, and also for plants growing under sand culture conditions, the seedlings grew taller, greener, had longer leaves, and were heavier.

Pleiotropic physiological effects in the plant growth-promoting bacterium Azospirillum brasilense following chromosomal labeling in the clpX gene

Azospirillum brasilense 8-I was chromosomally labeled with green fluorescent protein (gfp) genes, using either the native promoterless gfp gene or the mutant gfpmut2 gene under the transcriptional control of the neomycin phosphate transferase (npt2) promoter inserted into Tn5 suicide plasmid vectors. One A. brasilense exconjugant, showing a steady and strong fluorescence following irradiation with 365-nm UV light was characterized in detail. This strain, A. brasilense 8-I-gfp showed increased N(2)-fixation of approximately threefold, up to a twofold increase in exopolysaccharide production, and a significant decrease in indole-3-acetic acid and poly-beta-hydroxybutyrate production over the parental strain. Sequence analysis showed that the Tn5 carrying the gfp gene was inserted in the clpX gene encoding a heat-shock protein. This data is consistent with a model in which the observed physiological changes are a consequence of pleiotropic changes that occur as a consequence of impaired heat shock (stress) protein synthesis. In summary, (i) chromosomally labelled Azospirillum brasilense was obtained carrying either native or mutant gfp genes, (ii) Pleiotropic physiological effects were caused by disruption of the clpX gene as the consequence of the insertion, (iii) a new indole-3-acetic acid-attenuated mutant of A. brasilense producing only 0.25% of the indole-3-acetic acid produced by the wild-type is presented.

Increase in auxiliary photoprotective photosynthetic pigments in wheat seedlings induced by Azospirillum brasilense

Inoculation of wheat seedlings with the plant growth-promoting bacterium Azospirillum brasilense Cd was immobilized in alginate microbeads and, without applying any stress, significantly increased the quantity of several photosynthetic pigments, such as chlorophyll a, chlorophyll b, and the auxiliary photoprotective pigments violaxanthin, zeaxanthin, antheroxanthin, lutein, neoxanthin, and β-carotene. This resulted in greener plants with no apparent visible stress. After monitoring the quantity of photosynthetic pigments for 4 weeks, we observed that inoculated plants had higher quantities of pigments in shoot and stem. The greatest difference in the quantity of all pigments between inoculated and noninoculated plants occurred in the first week of growth. Regardless of treatment, the quantity of pigments in stems was three to four times less than the quantity of these pigments in shoots. Application of Azospirillum, either as liquid inoculant or as alginate microbeads, did not alter the positive effect of the bacteria on pigment production or the positive response of the plants towards A. brasilense Cd inoculation.

Annotation of the pRhico plasmid of Azospirillum brasilense reveals its role in determining the outer surface composition

The plant growth-promoting soil bacterium Azospirillum brasilense enhances growth of economically important crops, such as wheat, corn and rice. In order to improve plant growth, a close bacterial association with the plant roots is needed. Genes encoded on a 90-MDa plasmid, denoted pRhico plasmid, present in A. brasilense Sp7, play an important role in plant root interaction. Sequencing, annotation and in silico analysis of this 90-MDa plasmid revealed the presence of a large collection of genes encoding enzymes involved in surface polysaccharide biosynthesis. Analysis of the 90-MDa plasmid genome provided evidence for its essential role in the viability of the bacterial cell.

Alleviation of noxious effects of cattle ranch composts on wheat seed germination by inoculation with Azospirillum spp.

Two commonly-used composts from dairy cow manure that are used to improve poor structure and fertility of desert soils have inhibitory effects on wheat seed germination, probably as a result of their high levels of humic acids. Inoculation of wheat seeds with two species of the plant growth-promoting bacteria Azospirillum brasilense Cd and A. lipoferum JA4 (separately) prior to sowing in these amended soils improved germination, similar to the natural level of germination of seeds in desert soil without compost amendment. Both compost amendments increased height of wheat seedlings in the range of 20–25%, increased shoot dry weight by 15–19%, but severely decreased (51–54% less) root dry weight. Inoculation of wheat seeds with A. brasilense Cd, but not with A. lipoferum JA4, significantly increased plant growth parameters (height, shoot and root dry weight) over control plants grown in soil-compost mixtures. This bacterial species could survive for a period of 20 days in compost humic acid solution, could increase its population when the humic acids served as the sole carbon source, and may change the composition of humic acids in which it grows. We suggest that inoculation with A. brasilense may alleviate noxious effects on germinating seeds caused by compost application by possibly transforming the composition of humic acids in the compost.

Increased acidification in the rhizosphere of cactus seedlings induced by Azospirillum brasilense.

Acidification of the rhizosphere of cactus seedlings (giant cardon, Pachycereus pringlei) after inoculation with the plant growth-promoting bacterium Azospirillum brasilense Cd, in the presence or absence of ammonium and nitrate, was studied to understand how to increase growth of cardon seedlings in poor desert soils. While ammonium enhanced rhizosphere and liquid culture acidification, inoculation with the bacteria enhanced it further. On the other hand, nitrate increased pH of the rhizosphere, but combined with the bacterial inoculation, increase in pH was significantly smaller. Bacterial inoculation with ammonium enhanced plant growth.

Reduction of bacterial speck (Pseudomonas syringae pv. tomato) of tomato by combined treatments of plant growth-promoting bacterium, Azospirillum brasilense, streptomycin sulfate, and chemo-thermal seed treatment

Inoculation of tomato seeds with the plant growth-promoting bacterium Azospirillum brasilense, or spraying tomato foliage with A. brasilense, streptomycin sulfate, or commercial copper bactericides, separately, before or after inoculation with Pseudomonas syringae pv. tomato, the casual agent of bacterial speck of tomato, had no lasting effect on disease severity or on plant height and dry weight. Seed inoculation with A. brasilense combined with a single streptomycin foliar treatment and two foliar bactericide applications at 5-day intervals (a third or less of the recommended commercial dose) reduced disease severity in tomato seedlings by over 90% after 4 weeks, and significantly slowed disease development under mist conditions. A. brasilense did not induce significant systemic resistance against the pathogen although the level of salicylic acid increased in inoculated plants. Treatment of tomato seeds that were artificially inoculated with P. syringae pv. tomato, with a combination of mild chemo-thermal treatment, A. brasilense seed inoculation, and later, a single foliar application of a copper bactericide, nearly eliminated bacterial leaf speck even when the plants were grown under mist for 6 weeks. This study shows that a combination of otherwise ineffective disease management tactics, when applied in concert, can reduce bacterial speck intensity in tomatoes under mist conditions.

Ultrastructure of interaction in alginate beads between the microalgaChlorella vulgariswith its natural associative bacteriumPhyllobacterium myrsinacearumand with the plant growth-promoting bacteriumAzospirillum brasilense

Chlorella vulgaris, a microalga often used in wastewater treatment, was coimmobilized and coincubated either with the plant growth-promoting bacterium Azospirillum brasilense, or with its natural associative bacterium Phyllobacterium myrsinacearum, in alginate beads designed for advanced wastewater treatment. Interactions between the microalga and each of the bacterial species were followed using transmission electron microscopy for 10 days. Initially, most of the small cavities within the beads were colonized by microcolonies of only one microorganism, regardless of the bacterial species cocultured with the microalga. Subsequently, the bacterial and microalgal microcolonies merged to form large, mixed colonies within the cavities. At this stage, the effect of bacterial association with the microalga differed depending on the bacterium present. Though the microalga entered a senescence phase in the presence of P. myrsinacearum, it remained in a growth phase in the presence of A. brasilense. This study suggests that there are commensal interactions between the microalga and the two plant associative bacteria, and that with time the bacterial species determined whether the outcome for the microalga is senescence or continuous multiplication.

Ultrastructure of interaction in alginate beads between the microalga <i>Chlorella vulgaris</i> with its natural associative bacterium <i>Phyllobacterium myrsinacearum</i> and with the plant growth-promoting bacterium <i>Azospirillum brasilense</i>

Chlorella vulgaris, a microalga often used in wastewater treatment, was coimmobilized and coincubated either with the plant growth-promoting bacterium Azospirillum brasilense, or with its natural associative bacterium Phyllobacterium myrsinacearum, in alginate beads designed for advanced wastewater treatment. Interactions between the microalga and each of the bacterial species were followed using transmission electron microscopy for 10 days. Initially, most of the small cavities within the beads were colonized by microcolonies of only one microorganism, regardless of the bacterial species cocultured with the microalga. Subsequently, the bacterial and microalgal microcolonies merged to form large, mixed colonies within the cavities. At this stage, the effect of bacterial association with the microalga differed depending on the bacterium present. Though the microalga entered a senescence phase in the presence of P. myrsinacearum, it remained in a growth phase in the presence of A. brasilense. This study suggests that there are commensal interactions between the microalga and the two plant associative bacteria, and that with time the bacterial species determined whether the outcome for the microalga is senescence or continuous multiplication.

Physiological and cytological studies on the inhibition of Striga seed germination by the plant growth-promoting bacterium Azospirillum brasilense.

Striga hermonthica (Del.) Benth. is an obligate parasitic weed of tropical cereals whose rhizosphere can also be colonised by bacteria of the genus Azospirillum. A previous study demonstrated that the two organisms (Azospirillum and striga) interacted during cerealroot colonisation. Two strains of A. brasilense isolated from an African sorghum rhizosphere prevented the germination of striga seeds although they were stimulated to germinate by the presence of sorghum roots. Azospirillum cells suspended in a synthetic germination stimulant (GR24) did not inhibit striga seed germination, but did block radicle elongation. Those radicles had an abnormal morphology, and contained no vacuolated cells in the root elongation zone. Lipophilic compounds extracted from the medium of bacteria in the log and stationary growth phases prevented the germination of striga seeds.