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     

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.     

Activation of a calcium-dependent protein kinase involved in the <Emphasis Type="Italic">Azospirillum</Emphasis> growth promotion in rice

Rice seedlings (Oryza sativa) inoculated with the plant growth-promoting rhizobacteria Azospirillum brasilense FT326 showed an enhanced development of the root system 3 days after inoculation. Later on, a remarkable enlargement of shoots was also evident. An increase in the Ca²⁺-dependent histone kinase activity was also detected as a result of inoculation. The biochemical characterization and Western-blot analysis of the kinase strongly supports the hypothesis that it belongs to a member of the rice CDPK family. The fact that the amount of the protein did not change upon inoculation seems to indicate that a posttranslational activation is responsible for the change in the enzymatic activity. An in-gel kinase experiment identified a 46 kDa CDPK like protein kinase as a putative component of the signal transduction pathway triggered by Azospirillum inoculation. To our knowledge, this is the first report on the possible involvement of a Ca²⁺-dependent protein kinase in promotion of rice plants growth by A. brasilense.     

Physiological,structural and molecular traits activated in strawberry plants after inoculation with the plant growth‐promoting bacterium Azospirillum brasilense REC3

The plant growth‐promoting strain REC3 of Azospirillum brasilense, isolated from strawberry roots, prompts growth promotion and systemic protection against anthracnose disease in this crop. Hence, we hypothesised that A. brasilense REC3 can induce different physiological, structural and molecular responses in strawberry plants. Therefore, the aim of this work was to study these traits activated in Azospirillum‐colonised strawberry plants, which have not been assessed until now. Healthy, in vitro micropropagated plants were root‐inoculated with REC3 under hydroponic conditions; root and leaf tissues were sampled at different times, and oxidative burst, phenolic compound content, malondialdehyde (MDA) concentration, callose deposition, cell wall fortification and gene expression were evaluated. Azospirillum inoculation enhanced levels of soluble phenolic compounds after 12 h post‐inoculation (hpi), while amounts of cell wall bound phenolics were similar in inoculated and control plants. Other early responses activated by REC3 (at 24 hpi) were a decline of lipid peroxidation and up‐regulation of strawberry genes involved in defence (FaPR1), bacterial recognition (FaFLS2) and H₂O₂ depuration (FaCAT and FaAPXc). The last may explain the apparent absence of oxidative burst in leaves after bacterial inoculation. Also, REC3 inoculation induced delayed structural responses such as callose deposition and cell wall fortification (at 72 hpi). Results showed that A. brasilense REC3 is capable of exerting beneficial effects on strawberry plants, reinforcing their physiological and cellular characteristics, which in turns contribute to improve plant performance.     

Alterations in membrane potential and in proton efflux in plant roots induced byAzospirillum brasilense

Inoculation of soybean seedlings withAzospirillum brasilense Cd significantly reduced the membrane potential in every root part and was being maximal in the root elongation zone. Monitoring the proton efflux pattern of inoculated wheat roots by severalA. brasilense strains and byPseudomonas sp. for prolonged periods (up to 200h) revealed a change from the bimodal pattern of proton efflux of non inoculated roots. This change was not related to root colonization ability but to bacterial capacity to induce changes in root surface area. Continuous perfusion of the plant nutrient solution with a fresh solution (from inoculation time), eliminated the enhancing effect of inoculation on proton efflux. We propose thatA. brasilense inoculation influences membrane activity and subsequently proton efflux in roots, probably through the release of an as yet unidentified bacterial signal.     

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

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

Protection of Tomato Seedlings against Infection by Pseudomonas syringae pv. Tomato by Using the Plant Growth-Promoting Bacterium Azospirillum brasilense

Pseudomonas syringae pv. tomato, the causal agent of bacterial speck of tomato, and the plant growth-promoting bacterium Azospirillum brasilense were inoculated onto tomato plants, either alone, as a mixed culture, or consecutively. The population dynamics in the rhizosphere and foliage, the development of bacterial speck disease, and their effects on plant growth were monitored. When inoculated onto separate plants, the A. brasilense population in the rhizosphere of tomato plants was 2 orders of magnitude greater than the population of P. syringae pv. tomato (10⁷ versus 10⁵ CFU/g [dry weight] of root). Under mist chamber conditions, the leaf population of P. syringae pv. tomato was 1 order of magnitude greater than that of A. brasilense (10⁷ versus 10⁶ CFU/g [dry weight] of leaf). Inoculation of seeds with a mixed culture of the two bacterial strains resulted in a reduction of the pathogen population in the rhizosphere, an increase in the A. brasilense population, the prevention of bacterial speck disease development, and improved plant growth. Inoculation of leaves with the mixed bacterial culture under mist conditions significantly reduced the P. syringae pv. tomato population and significantly decreased disease severity. Challenge with P. syringae pv. tomato after A. brasilense was established in the leaves further reduced both the population of P. syringae pv. tomato and disease severity and significantly enhanced plant development. Both bacteria maintained a large population in the rhizosphere for 45 days when each was inoculated separately onto tomato seeds (10⁵ to 10⁶ CFU/g [dry weight] of root). However, P. syringae pv. tomato did not survive in the rhizosphere in the presence of A. brasilense. Foliar inoculation of A. brasilense after P. syringae pv. tomato was established on the leaves did not alleviate bacterial speck disease, and A. brasilense did not survive well in the phyllosphere under these conditions, even in a mist chamber. Several applications of a low concentration of buffered malic acid significantly enhanced the leaf population of A. brasilense (>10⁸ CFU/g [dry weight] of leaf), decreased the population of P. syringae pv. tomato to almost undetectable levels, almost eliminated disease development, and improved plant growth to the level of uninoculated healthy control plants. Based on our results, we propose that A. brasilense be used in prevention programs to combat the foliar bacterial speck disease caused by P. syringae pv. tomato.     

Root-to-Root Travel of the Beneficial Bacterium Azospirillum brasilense

The root-to-root travel of the beneficial bacterium Azospirillum brasilense on wheat and soybean roots in agar, sand, and light-textured soil was monitored. We used a motile wild-type (Mot⁺) strain and a motility-deficient (Mot^-) strain which was derived from the wild-type strain. The colonization levels of inoculated roots were similar for the two strains. Mot⁺ cells moved from inoculated roots (either natural or artificial roots in agar, sand, or light-textured soil) to noninoculated roots, where they formed a band-type colonization composed of bacterial aggregates encircling a limited part of the root, regardless of the plant species. The Mot^- strain did not move toward noninoculated roots of either plant species and usually stayed at the inoculation site and root tips. The effect of attractants and repellents was the primary factor governing the motility of Mot⁺ cells in the presence of adequate water. We propose that interroot travel of A. brasilense is an essential preliminary step in the root-bacterium recognition mechanism. Bacterial motility might have a general role in getting Azospirillum cells to the site where firmer attachment favors colonization of the root system. Azospirillum travel toward plants is a nonspecific active process which is not directly dependent on nutrient deficiency but is a consequence of a nonspecific bacterial chemotaxis, influenced by the balance between attractants and possibly repellents leaked by the root.     

Enhanced Mineral Uptake by Zea mays and Sorghum bicolor Roots Inoculated with Azospirillum brasilense

Inoculation of corn (Zea mays) seeds with Azospirillum brasilense strain Cd or Sp 7 significantly enhanced (30 to 50% over controls) the uptake of NO₃⁻, K⁺, and H₂PO₄⁻ into 3- to 4-day- and 2-week-old root segments. No gross changes in root morphology were observed; altered cell arrangement in the outer four or five layers of the cortex was seen in photomicrographs of cross sections of inoculated corn roots. The surface activity involved in ion uptake probably increased, as shown by the darker staining by methylene blue of the affected area. Shoot dry weight increased 20 to 30% in inoculated plants after 3 weeks, presumably by enhancement of mineral uptake. Corn and sorghum plants grown to maturity on limiting nutrients in the greenhouse showed improved growth from inoculation approaching that of plants grown on normal nutrient concentrations. Enhanced ion uptake may be a significant factor in the crop yield enhancement reported for Azospirillum inoculation.     

Plant Growth Substances Produced by Azospirillum brasilense and Their Effect on the Growth of Pearl Millet (Pennisetum americanum L.)

Azospirillum brasilense, a nitrogen-fixing bacterium found in the rhizosphere of various grass species, was investigated to establish the effect on plant growth of growth substances produced by the bacteria. Thin-layer chromatography, high-pressure liquid chromatography, and bioassay were used to separate and identify plant growth substances produced by the bacteria in liquid culture. Indole acetic acid and indole lactic acid were produced by A. brasilense from tryptophan. Indole acetic acid production increased with increasing tryptophan concentration from 1 to 100 μg/ml. Indole acetic acid concentration also increased with the age of the culture until bacteria reached the stationary phase. Shaking favored the production of indole acetic acid, especially in a medium containing nitrogen. A small but biologically significant amount of gibberellin was detected in the culture medium. Also at least three cytokinin-like substances, equivalent to about 0.001 μg of kinetin per ml, were present. The morphology of pearl millet roots changed when plants in solution culture were inoculated. The number of lateral roots was increased, and all lateral roots were densely covered with root hairs. Experiments with pure plant hormones showed that gibberellin causes increased production of lateral roots. Cytokinin stimulated root hair formation, but reduced lateral root production and elongation of the main root. Combinations of indole acetic acid, gibberellin, and kinetin produced changes in root morphology of pearl millet similar to those produced by inoculation with A. brasilense.     

Host genotype dependency and growth enhancing ability of VA-mycorrhizal fungi for Eleusine coracana (finger millet)

Inoculation of finger millet (Eleusine coracana Gaertn.) plants with one of six different vesicular, arbuscular, mycorrhizal (VAM) fungi increased plant biomass, height, leaf area and absolute growth rate; however, effectiveness of the various VAM fungi varied significantly. Maximum root colonization and mycorrhizal efficacy was observed with plants inoculated with Glomus caledonicum. Among five host genotypes tested for mycorrhizal dependency against G. caledonicum, genotype HR-374 gave the highest plant biomass, mycorrhizal efficacy and root colonization, the inoculation resulting in increased mineral (phosphate, nitrogen, Zn²⁺ and Cu²⁺) content and uptake in shoots.     

Variation of secondary metabolite levels in maize seedling roots induced by inoculation with Azospirillum, Pseudomonas and Glomus consortium under field conditions

Background and aims

Many plant-beneficial microorganisms can influence secondary plant metabolism, but whether these effects add up when plants are co-inoculated is unclear. This issue was assessed, under field conditions, by comparing the early impacts of seed inoculation on secondary metabolite profiles of maize at current or reduced mineral fertilization levels.

Methods

Maize seeds were inoculated singly with selected strains from bacterial genera Pseudomonas and Azospirillum or mycorrhizal genus Glomus, or with these strains combined two by two or all three together. At 16?days, maize root methanolic extracts were analyzed by RP-HPLC and secondary metabolites (phenolics, flavonoids, xanthones, benzoxazionoids, etc.) identified by LC/MS.

Results

Inoculation did not impact on plant biomass but resulted in enhanced total root surface, total root volume and/or root number in certain inoculated treatments, at reduced fertilization. Inoculation led to qualitative and quantitative modifications of root secondary metabolites, particularly benzoxazinoids and diethylphthalate. These modifications depended on fertilization level and microorganism(s) inoculated. The three selected strains gave distinct results when used alone, but unexpectedly all microbial consortia gave somewhat similar results.

Conclusions

The early effects on maize secondary metabolism were not additive, as combining strains gave effects similar to those of Glomus alone. This is the first study demonstrating and analyzing inoculation effects on crop secondary metabolites in the field.     

Effects of Temperature, Nitrogen Fertilization, and Plant Age on Nitrogen Fixation by Setaria italica Inoculated with Azospirillum brasilense (strain cd)

The association between the nitrogen-fixing bacterium Azospirillum brasilense (strain cd) and the grass Setaria italica was studied under different environmental and soil conditions. Highest acetylene reduction rates in intact plants were observed at the booting stage of Setaria (2350 nmol ethylene produced hour⁻¹ plant⁻¹) at 27 C. Higher temperatures, up to 32 C, enhanced ethylene reduction. Significant increases in shoot dry weight, panicle weight, and length were obtained in inoculated plants fertilized with suboptimal NH₄NO₃ levels. The increase in nitrogen content of plants inoculated with A. brasilense was shown to be due to N₂ fixation. This was demonstrated by growing plants in washed quartz sand with no combined nitrogen. The bacteria also increased branching and development of roots. It was concluded that inoculation of Setaria with A. brasilense may lead both to increases in plant yield and saving of nitrogen fertilizer.     

N2 Fixation by Azospirillum brasilense and Its Incorporation into Host Setaria italica

Growth and nitrogen fixation were followed during the life cycle of Setaria italica (foxtail millet) inoculated with Azospirillum brasilense in controlled-environment growth chambers. The plants were fertilized at seeding with a limiting amount of combined nitrogen and maintained with an N-free mineral solution. During maturation of the plants, substantial nitrogenase activity, measured by acetylene reduction, developed in the rhizosphere, with total fixation estimated to be equivalent to 20% of the N in the inoculated plants. The peak of this activity coincided with depletion of soluble nitrogen from the system, which in turn was reflected by a sharp decrease in the nitrate reductase activity of the leaves. A. brasilense was found in association with the root populations at 8 × 10⁷ cells per gram of dry weight. An increase in shoot growth occurred at this time, but no significant increase in total plant nitrogen could be demonstrated. ¹⁵N₂ enrichment experiments confirmed that fixation was occurring, but only about 5% of the nitrogen fixed by A. brasilense was incorporated into the plants within 3 weeks. There was thus no evidence of direct bacterium-to-plant transport of fixed nitrogen, but rather a slow transfer suggesting the gradual death of bacteria and subsequent mineralization of their nitrogen, at least under growth-room conditions.     

A Novel Interaction between Plant-Beneficial Rhizobacteria and Roots: Colonization Induces Corn Resistance against the Root Herbivore Diabrotica speciosa

A number of soil-borne microorganisms, such as mycorrhizal fungi and rhizobacteria, establish mutualistic interactions with plants, which can indirectly affect other organisms. Knowledge of the plant-mediated effects of mutualistic microorganisms is limited to aboveground insects, whereas there is little understanding of what role beneficial soil bacteria may play in plant defense against root herbivory. Here, we establish that colonization by the beneficial rhizobacterium Azospirillum brasilense affects the host selection and performance of the insect Diabrotica speciosa. Root larvae preferentially orient toward the roots of non-inoculated plants versus inoculated roots and gain less weight when feeding on inoculated plants. As inoculation by A. brasilense induces higher emissions of (E)-β-caryophyllene compared with non-inoculated plants, it is plausible that the non-preference of D. speciosa for inoculated plants is related to this sesquiterpene, which is well known to mediate belowground insect-plant interactions. To the best of our knowledge, this is the first study showing that a beneficial rhizobacterium inoculant indirectly alters belowground plant-insect interactions. The role of A. brasilense as part of an integrative pest management (IPM) program for the protection of corn against the South American corn rootworm, D. speciosa, is considered.     

Enhancement of Wheat Root Colonization and Plant Development by Azospirillum brasilense Cd. Following Temporary Depression of Rhizosphere Microflora

Inoculation of wheat with Azospirillum brasilense, combined with the application of four fungal and bacterium-inhibiting substances to which A. brasilense is resistant in the soil, decreased the rhizosphere population, while it increased wheat root colonization by A. brasilense, even in cases of poor inoculation. The inoculation significantly increased the following wheat plant parameters as well: plant dry weight, number of tillers per plant, spikelet fertility, harvest index, and grain yield. This model may provide a new approach to improve control of root colonization by beneficial bacteria.     

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.     

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.     

Tomato genotype and Azospirillum inoculation modulate the changes in bacterial communities associated with roots and leaves

AIMS: To evaluate the effect of plant variety and Azospirillum brasilense inoculation on the microbial communities colonizing roots and leaves of tomato (Lycopersicon esculentum Mill.) plants. METHODS AND RESULTS: Seeds of cherry and fresh-market tomato were inoculated with A. brasilense BNM65. Sixty days after planting, plants were harvested and the microbial communities of the rhizoplane and phyllosphere were analysed by community-level physiological profiles (CLPP) using BIOLOG EcoPlates and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Differences on the rhizoplane and phyllosphere bacterial communities between the two tomato types were detected by principal component analysis of the CLPP; DGGE fingerprints also showed differences at the phyllosphere level. Fresh-market tomato had a more complex phyllosphere bacterial community than cherry tomato, as determined by DGGE profiles. Physiological and genetic changes on phyllosphere and rhizoplane bacterial communities by Azospirillum seed inoculation were evident only on cherry tomato. CONCLUSIONS: Tomato genotype affects the response of native bacterial communities associated with the roots and leaves to A. brasilense seed inoculation. SIGNIFICANCE AND IMPACT OF THE STUDY: The successful implementation of Azospirillum inoculation requires not only the consideration of the interactions between A. brasilense strains and plant genotypes, but also the plant-associated microflora.