Stimulation of root exudation of rice seedlings by Azospirillum strains: carbon budget under gnotobiotic conditions

Summary The association of rice seedlings (cv. Delta) with different strains of Azospirillum was studied under monoxenic conditions in the dark. Axenic 3-day-old seedlings were obtained on a C- and N-free medium and inoculated with 6 · 10⁷ bacteria per plant in a closed vial. Seven days later, different components of a carbon budget were evaluated on them and on sterile controls: respired CO₂, carbon of shoot and roots, bacterial and soluble carbon in the medium. Two strains (A. lipoferum 4B and A. brasilense A95) isolated from the rhizosphere of rice caused an increase in exudation, + 36% and + 17% respectively compared with sterile control. Shoot carbon incorporation and respiration were reduced by inoculation. A third strain (A. brasilense R07) caused no significant change in exudation. A. lipoferum B7C isolated from maize did not stimulate rice exudation either. We further investigated a possible effect of nitrogen fixation on this phenomenon: inhibition of nitrogen fixation by 10% C₂H₂ did not modify the extent of C exudation by rice associated with A. lipoferum 4B or with the non-motile A. lipoferum 4T.     

Maize genotype effects on the response to Azospirillum inoculation

Two field experiments were carried out in Northern Argentina, during the 1989–1990 and 1990–1991 growing seasons, on Argentinian and Brazilian maize genotypes. The inoculant consisted of a mixture of four Azospirillum brasilense strain isolated from surface-sterilized maize roots in Argentina and three A. lipoferum strains isolated from surface-sterilized maize or sorghum roots in Brazil. Establishment of the inoculated strains was confirmed by the antibiotic resistance of the strains in the highest dilution vials. In all treatments, numbers of Azospirillum spp. were increased and the inoculated strains were found in the highest dilutions. While grain yields of the different genotypes varied between 1700 and 7300 kg ha^-1, total N accumulation was much less variable. Significant inoculation effects on total N accumulation and on grain yields were consistently negative with one Argentinian genotype and positive with four Argentinian and two Brazilian genotypes. Significant inoculation effects on leaf nitrate reductase activity at the flowering stage, observed in the range-55% to +176%, indicated the presence of various interactions between the plant NO _inf3 ^sup- metabolism and Azospirillum spp. Three Brazilian and one Argentinian maize genotype showed significant decreases in leaf nitrate reductase due to inoculation while four Argentinian genotypes showed significant increases in leaf nitrate reductase activity. The results of the present study, were consistent over the two field experiments and strongly indicate that more detailed plant genotype-Azospirillum spp. strain interaction studies, taking the entire N metabolism in the plant into account, are needed to allow better inoculation results of cereal crops.     

Potential for enhancement of root growth and nodulation of soybean co-inoculated with Azospirillum and Bradyrhizobium in laboratory systems

The potential enhancement of root growth and nodulation in vegetable soybean (AGS190) was studied with application of Azospirillum brasilense (Sp7) and A. lipoferum (CCM3863) co-inoculated with two Bradyrhizobium japonicum strains (TAL102 and UPMR48). Significant root growth stimulation and nodulation were observed in Azospirillum as well as during its co-inoculation with Bradyrhizobium. Nodule formation is linked with the initiation of new roots; nodules were almost absent even in Bradyrhizobium inoculated plant due to the absence of new roots development in clipped rooted seedlings. Total root length, root number, specific root length, root dry matter, root hair development and shoot dry matter were significantly increased by Azospirillum alone and its co-inoculum. Co-inoculated plants significantly influenced the number of nodules and its fresh weight. A. brasilense seemed to perform better in root growth and nodule development compared to A. lipoferum.     

Fluorescent antibody technique to identify Azospirillum brasilense associated with roots of grasses

Bacteria associated with roots of grasses from Florida, Ecuador and Venezuela were isolated and their N₂-fixing ability was demonstrated by C₂H₂ reduction assay. The bacterial isolates have been classified as Azospirillum brasilense (formerly Spirillum lipoferum). These N₂-fixing isolates have been compared with several Brazilian strains. Fluorescent antibody (FA) techniques were used to assist identifying isolates of N₂-fixing bacteria from grass roots. Tests with antisera prepared against four strains of Azospirillum were used to define serological groups. Antigen-antibody specificity was demonstrated using both Azotobacter and Azospirillum antisera against known species of other soil microorganisms and numerous unidentified soil bacteria. Several applications of the FA technique are suggested to identify N₂-fixing bacteria associated with grass roots.     

Effect of Azospirillum inoculants on arbuscular mycorrhiza establishment in wheat and maize plants

Plant growth-promoting rhizobacteria and arbuscular mycorrhizal (AM) fungi represent two main groups of beneficial microorganisms of the rhizosphere. The role of different strains of Azospirillum on AM fungi development was evaluated by measuring the percentage of AM colonisation of the root system in durum wheat and maize plants, grown under both greenhouse and field conditions. The effect of wild-type Azospirillum brasilense strain Sp245 and genetically modified (GM) derivatives overproducing indole-3-acetic acid was assessed at greenhouse level in (1) three different cultivars of durum wheat, in the presence of indigenous AM fungi and (2) maize plants artificially inoculated with Glomus mosseae and Glomus macrocarpum. In addition, the establishment of natural AM fungal symbiosis was evaluated using Azospirillum lipoferum CRT1 in maize plants at field level. Despite the stimulatory effect of the different Azospirillum inocula on root growth, no significant differences in AM colonisation were found, independently of the AM fungus involved, either in wheat or in maize plants. Similarly, GM A. brasilense, which strongly stimulates root development, did not affect AM formation. Although these results were obtained in conditions in which the mycorrhization rate was moderate (15–30%), overall considered they indicate that the use of wild-type or GM Azospirillum phytostimulators does not alter mycorrhization.     

Azospirillum strains isolated from roots and rhizosphere soil of wheat (Triticum aestivum L.) grown under different soil moisture conditions

The present study deals with the isolation and characterization of Azospirillum strains isolated from roots and rhizosphere soil of wheat (at tillering and anthesis stages) plants grown under different moisture regimes in the field and in pots. The survival of Azospirillum isolates from plants of irrigated field and those from well-watered pots was higher than that of Azospirillum strains isolated from roots and rhizosphere soils of plants grown under arid and semiarid (14–8% soil moisture) field conditions and under water-stressed (8% soil moisture) conditions in pots. On the basis of carbon/nitrogen source utilization, the Azospirillum strains isolated from wheat under field and pot conditions were grouped in three groups. The unweighted pair group method with arithmetic means cluster analysis based on random amplification of polymorphic DNA showed that two groups of Azospirillum were similar. The strains isolated from plants (at tillering stage) grown under low moisture conditions either in pots or in field were genetically similar to strains isolated from plants grown under well-watered conditions in both pots and field. Inoculation of wheat with isolates from water-stressed plants induced tolerance to water stress in inoculated plants. Isolates from water-stressed conditions exhibited lower production of indole acetic acid, gibberellic acid, and trans zeatin riboside but a higher production of abscisic acid.     

Development of a real-time PCR method to quantify the PGPR strain Azospirillum lipoferum CRT1 on maize seedlings

Azospirillum lipoferum CRT1 is a promising phytostimulatory PGPR for maize, whose effect on the plant is cell density-dependent. A nested PCR method is available for detection of the strain but does not allow quantification. The objective was to develop a real-time PCR method for quantification of A. lipoferum CRT1 in the rhizosphere of maize seedlings. Primers were designed based on a strain-specific RFLP marker, and their specificity was verified under qualitative and quantitative PCR conditions based on successful CRT1 amplification and absence of cross-reaction with genomic DNA from various rhizosphere strains. Real-time PCR conditions were then optimized using DNA from inoculated or non-inoculated maize rhizosphere samples. The detection limit was 60 fg DNA (corresponding to 19 cells) with pure cultures and 4 × 10⁴ CFU equivalents g⁻¹ lyophilized sample consisting of mixture of rhizosphere soil and roots. Inoculant quantification was effective down to 10⁴ CFU equivalents g⁻¹. Assessment of CRT1 rhizosphere levels in a field trial was in accordance with estimates from semi-quantitative PCR targeting another locus. This real-time PCR method, which is now available for direct rhizosphere monitoring of A. lipoferum CRT1 in greenhouse and field experiments, could be used as a reference for developing quantification tools for other Azospirillum inoculants.     

Inoculation of field-grown wheat (Triticum aestivum) with Azospirillum spp. in Brazil

Summary Three field experiments with wheat were conducted in 1983, 1984, and 1985 in Terra Roxa soil in Paraná, the major Brazilian wheat-growing region, to study inoculation effects of various strains of Azospirillum brasilense and A. amazonense. In all three experiments inoculation with A. brasilense Sp 245 isolated from surface-sterilized wheat roots in Paraná produced the highest plant dry weights and highest N% in plant tops and grain. Grain yield increases with this strain were up to 31 % but were not significant. The application of 60 or 100 kg N ha^–1 to the controls increased N accumulation and produced yields less than inoculation with this strain. Another A. brasilense strain from surface-sterilized wheat roots (Sp 107st) also produced increased N assimilation at the lower N fertilizer level but reduced dry weights at the high N level, while strain Sp 7 + Cd reduced dry weights and N% in the straw at both N levels. The A. amazonense strain isolated from washed roots and a nitrate reductase negative mutant of strain Sp 245 were ineffective. Strains Sp 245 and Sp 107st showed the best establishment within roots while strain Cd established only in the soil.     

Growth and yield responses of a Nepalese spring wheat cultivar to the inoculation with Nepalese Azospirillum spp at various levels of nitrogen fertilization

The Azospirillum 10SW used in our experiments was isolated from roots of wheat growing in nitrogen-poor soil of a hilly region of Nepal, where inorganic nitrogen fertilizers were never used. The main objectives of this work were to assess the effects of inorganic nitrogen fertilization in the yield responses of wheat grown in association with the bacteria. The in vitro experiments were done in laboratory, whereas the pot experiments were performed in a greenhouse. The nitrogenase activities of in vitro grown Azospirillum were repressed by nitrate. The magnitude of repression was lower when the bacteria were growing in association with wheat. The number of roots per plant was increased significantly in inoculated plants irrespective of the nitrate concentration of the medium. Inoculation with Azospirillum 10SW also increased the yield of wheat grown in pots with medium levels of nitrogen fertilization. These data show the possibility of inoculation of this Azospirillum spp. in combination with nitrogen fertilizer to improve the yield of wheat. Azospirillum inoculation enhanced the development of roots and shoots in the early growth stages of wheat. It may be one of the factors responsible for the yield increases. Received: 11 December 1996     

Seedlings growth promotion by Azospirillum brasilense under normal and drought conditions remains unaltered in Tebuconazole-treated wheat seeds

The wide use of pesticides in modern agriculture may cause side effects on the non-target microflora. Data on the fungicide Tebuconazole effects on Azospirillum-wheat association are scarce. We analyzed the effects of Tebuconazole on: (a) Azospirillum brasilense Sp245 growth in pure culture, (b) A. brasilense Sp245 colonization of Triticum aestivum cv ProINTA Oasis roots, (c) A. brasilense Sp245-inoculated seedlings growth under normal and water stress conditions in the presence of 20% polyethylene glycol 8000. Seeds were separated in Tebuconazole-free and Tebuconazole-treated lots. Inoculated and non-inoculated seedlings were grown in hydroponics in the dark at 20 °C for 72 h. Root surface, coleoptile length, fresh and dry (DW) weights in both tissues and diazotrophic bacterial most probable number in roots were determined. Water contents and shoot-to-roots DW ratio were calculated. Neither Azospirillum growth nor root colonization was affected by Tebuconazole. Under normal growth conditions most of the growth parameters analyzed, revealed a clear positive effect of A. brasilense on wheat seedlings up to 72 h treatments. The characteristic Azospirillum enhancing effects observed on roots remained unaltered by Tebuconazole. The present study shows that Tebuconazole is compatible with A. brasilense Sp245-wheat inoculation.     

Effect of Piriformospora indica and Azospirillum strains from saline or non-saline soil on mitigation of the effects of NaCl

Salinity toxicity is a worldwide agricultural and eco-environmental problem. The intent of this study was to determine the salt tolerance of Piriformospora indica and strains of Azospirillum, isolated from non-saline and saline soil, as well as to determine their affect on the tolerance of wheat to soil salinity. In this study, an experiment was conducted to investigate the salt stress tolerance abilities of the endophytic fungi, P. indica, and Azospirillum strains, isolated from non-saline and saline soil, at five NaCl levels (0, 0.1, 0.2, 0.3, 0.4, 0.5 mol L^?1). Additionally, a greenhouse experiment was conducted to test the effects of these selected microorganisms under increasing salinity levels on seedling growth, solute accumulation (proline and sugars), and photosynthetic pigments (Chl a, b, ab) of seedling wheat. Azospirillum strains were isolated in Iran from the root of field-grown maize from non-saline soil with an EC = 0.7 dS m^?1 and from saline soil with an EC = 4.7 dS m^?1. Plants were irrigated with non-saline water–tap water with an electrical conductivity water (ECw) value of 0.2 dS m^?1, as well as low, moderate and severe saline water-irrigation with saline water with an ECw of 4 dS m^?1, 8 dS m^?1 and 12 dS m^?1, respectively. The upper threshold of P. indica salinity tolerance was 0.4 mol L^?1 NaCl in both liquid and solid broth medium. The upper thresholds of the salt adapted and non-adapted Azospirillum strains were 0.2 and 0.4 mol L^?1 NaCl, respectively. The results indicated a positive influence of the organisms on salinity tolerance, more with the saline-adapted Azospirillum strains than the non-adapted strains. P. indica was more effective than the Azospirillum strains. These results could be related to a better water status, higher photosynthetic pigment contents and proline accumulation in wheat seedlings inoculated with P. indica. The benefits of both isolates and P. indica depended on two factors: water salinity and growth stage of the host plant. Inoculation with the two isolates increased salinity tolerance of wheat plants; the saline-adapted Azospirillum strains showed better performance with respect to improved fresh and dry weights at 80 and 100 days after sowing under both non-saline and saline conditions. When compared to plants inoculated with non-saline-adapted Azospirillum strains, those inoculated with adapted Azospirillum strains had much better performance with respect to the presence of photosynthetic pigment (Chl a, b and ab) and proline accumulation. Overall, these results indicate that the symbiotic association between P. indica fungus and wheat plants improved wheat growth, regardless of the salinity. It is concluded that the mechanisms for protecting plants from the detrimental effects of salinity by P. indica fungus and Azospirillum strains may differ in their salinity tolerance and influence the uptake of water, photosynthetic pigment contents and proline accumulation in wheat seedlings.     

Growth Promotion of Maize (Zea mays L.) by Plant-Growth-Promoting Rhizobacteria under Field Conditions

Plant-growth promoting rhizobacteria (PGPR) play an important role in plant health and soil fertility. The experiment was conducted as factorial experiment with two factors of Azospirillum and Azotobacter. The bacterial strains were Azospirillum lipoferum s-21, A. brasilense DSM 1690, A. lipoferum DSM 1691, Azotobacter chroococcum s-5, and A. chroococcum DSM 2286. The results indicated that growth promotion by PGPR appears from early stages of growth, 45 days after inoculation (DAI). Beneficial effects of bacterial inoculation on ear growth were observed after 75 DAI. Inoculation with PGPR increased dry weights of leaf, stem, and grain and hence total biomass sampled at 90, 105, and 120 (harvest time) DAI. The greatest grain weight was produced by Azospirillum s-21 inoculation. Dual inoculation with Azotobacter s-5 + Azospirillum s-21 significantly increased total dry weight up to 115%. Results of this study showed that leaf area index and crop growth index were significantly affected by bacterial treatments.     

Azospirillum inoculation and nitrogen fertilization effect on grain yield and on the diversity of endophytic bacteria in the phyllosphere of rice rainfed crop

We assessed the Azospirillum inoculation and N-fertilization effect on grain yield and on the phyllosphere endophytic diversity of nitrogen-fixing bacteria in a rice rainfed crop. We used cultivation-based techniques and cultivation-independent methods involving PCR-16S rRNA and denaturing gradient gel electrophoresis (DGGE). In general, we observed that grain yield was improved when inoculated with Azospirillum (depending on the genotype) and/or fertilized with urea. A similar behavior was observed in total N-content in grain and the MPN determination, as the highest values occurred when seeds were inoculated with A. brasilense REC3 (S1) than with A brasilense 13-2C (S2). A positive nitrogenase activity and PCR-nifH amplification suggests that the bacteria associated to inner tissues of rice phyllosphere could have contributed to the different N-contents detected. The bacterial diversity, observed in the number and intensity of DGGE profiles, showed a higher number of bands when total DNA was obtained using only CTAB than with CTAB + PVP. The DGGE profiles revealed great stability in the dominating bands, which presumably represent numerically dominant species. Application of A. brasilense strains as inoculants did not influence the dominant members of the endophytic microbial communities in the phyllosphere, but improved N-content and production of rainfed rice crop.     

A spontaneous chlorate-resistant mutant of Azospirillum brasilense Sp245 displays defects in nitrate reduction and plant root colonization

Azospirillum, a soil bacterium capable of colonizing plant roots, can reduce NO₃^-. In this work, a spontaneous chlorate-resistant mutant of Azospirillum brasilense Sp245, named Sp245chl1, was phenotypically characterized. The mutant is defective in both assimilatory and periplasmic dissimilatory nitrate reductase activity. Using the gusA reporter gene methodology, Sp245chl1 was found to be significantly affected in its ability to colonize roots of wheat and rice seedlings.     

Interactions between azospirillum and va mycorrhiza and their effects on growth and nutrition of maize and ryegrass

Interactions between the N₂-fixing bacterium Azospirillum brasilense and the mycorrhizal fungus Glomus mosseae were studied in relation to their effects on the growth and nutrition of Zea mays (C₄) and Lolium perenne (C₃) plants. Although roots from plants inoculated with Azospirillum exhibited C₂H₂ reduction activity no significant effect of inoculation on N concentration in the plant shoots was found. With non-mycorrhizal plants, inoculation with Azospirillum resulted in increased dry matter production at the first harvest compared to the effect achieved by supplying N as fertilizer, but this trend was reversed at the last harvest. However, with mycorrhizal maize plants, Azospirillum, which stimulated the development of VA mycorrhiza, was still effective in improving plant growth and nutrient uptake at the last harvest. Azospirillum and N behaved similarly in enhancing the growth and nutrition of mycorrhizal maize. The dual inoculation of maize by Azospirillum and Glomus produced plants of a similar size, N content, and a higher P content, than those supplied with N and P.     

Inoculation of wheat with Azospirillum brasilense and Pseudomonas fluorescens: Impact on the production and culturable rhizosphere microflora

Scientific evidence recognizes that the operation of a terrestrial ecosystem depends on soil microbial activity. Some Azospirillum strains produce plant growth regulators, increase the development of roots, and fix atmospheric nitrogen (N₂). Some Pseudomonas strains are capable of producing cytokinins and solubilizing organic phosphorus. A sustainability analysis requires a detailed knowledge of the interrelationships between the microorganisms added to the system and those present in the soil. This study examines the effect of three commercial inoculants Azospirillum brasilense Az1 and Az2 as well as Pseudomonas fluorescens Pf on biomass production, grain yield and rhizosphere colonization of wheat, combined with two levels of N-addition. Plate counts of rhizosphere soil showed that the inoculation and N-addition did not affect the number of P. fluorescens, whereas it significantly affected the number of Azospirillum. N-addition and inoculation did not change the communities of actinomycetes and bacteria but they changed the number of fungi at the rhizosphere of wheat plants. Community-level physiological profiles of carbon-source utilization of rhizosphere soil microbial communities were changed after inoculation with Az1, Az2 and Pf depending on the phenological stage of the crop. Although no significant responses were observed, in average, PGPB inoculation increased aerial biomass by 12%, root biomass by 40% and grain yield by 16%. These increases represent important earnings for the farmer and they may help to obtain a greater sustainability of the agroecosystems.     

Implications of Azospirillum brasilense inoculation and nutrient addition on maize in soils of the Brazilian Cerrado under greenhouse and field conditions

The combination of nitrogen and Azospirillum can ensure greater nutrient absorption and crop yield in agricultural areas using high technology. Thus, the objective was to evaluate maize response to Azopirillum brasilense (AZ) inoculation and nutrient (macronutrients and micronutrients) application under greenhouse and field conditions in clay and sandy soils of the Brazilian Cerrado. In the greenhouse assays, the following parameters were measured: shoot dry weight (SDW), root dry weight (RDW), and root volume (RV). In the field experiments, the maize yield was determined after drying the grains at 60 °C for 48 h. In clay soil, there was a significant increase in the SDW, RDW and RV in the treatment with AZ concentrated (10¹¹ cells ml⁻¹ of inoculum) when compared with the control treatment and the treatment with AZ diluted (10⁶ cells ml⁻¹ of inoculum). In this soil, adding micronutrients did not affect the maize response under greenhouse conditions. In sandy soil, there was no difference between the AZ treatment and the control, except for treatments where nutrients and AZ were both added leading to a significant increase in the maize response. In both soils, the RV:RDW ratio was higher in the treatment with AZ concentrated compared to that in the treatment with AZ diluted, but the yield response depended on the addition of nutrients. Inoculation with A. brasilense gave comparable yield to the nitrogen treatment. The grain production was increased by 29% in the treatment with A. brasilense and nitrogen compared to nitrogen fertilization alone. In this study, the yield response was affected significantly when maize was inoculated with A. brasilense, but this response was dependent on the soil type under greenhouse conditions.     

Effect of Azospirillum brasilense inoculation on rhizobacterial communities analyzed by denaturing gradient gel electrophoresis and automated ribosomal intergenic spacer analysis

Nucleic acid-based techniques allow the exploration of microbial communities in the environments such as the rhizosphere. Azospirillumbrasilense, a plant growth promoting rhizobacterium (PGPR), causes morphological changes in the plant root system. These changes in root physiology may indirectly affect the microbial diversity of the rhizosphere. In this study, the changes in the rhizobacterial structure following A. brasilense inoculation of maize (Zea mays) plants was examined by PCR-denaturating gradient gel electrophoresis (DGGE) and automated ribosomal intergenic spacer analysis (ARISA), using two universal primers sets for the 16S rRNA gene, and an intergenic 16S-23S rDNA primer set, respectively. Similar results were obtained when using either ARISA or DGGE performed with these different primer sets, and analyzed by different statistical methods: no prominent effect of A. brasilense inoculation was observed on the bacterial communities of plant roots grown in two different soils and in different growth systems. In contrast, plant age caused significant shifts in the bacterial populations.     

Biological nitrogen fixation at elevated temperature in different Azospirillum species and strains

Nitrogenase activity, nitrogenase synthesis, and the growth of four species of Azospirillum were examined at 30°C and 42°C. Azospirillum brasilense Sp 7 did not synthesize nitrogenase at 42°C nor was the enzyme stable at this temperature. In A. lipoferum 708 and A. brasilense 12S the nitrogenase activity was stable at 42°C but the synthesis of nitrogenase was reduced. In A. brasilense Sp 9 the nitrogenase activity was stable and showed the highest C₂H₂ reduction activity at 42°C.     

Effect of calcium carbonate, sand, and organic matter levels on mortality of five species of Azospirillum in natural and artificial bulk soils

 Five bacterial strains, one from each of the five known species of the plant growth-promoting bacteria (PGPB) Azospirillum (A. brasilense, A. lipoferum, A. amazonense, A. halopraeference, and A. irakense) were inoculated into two natural, semiarid soils (terra rosa and loessial sandy) from Israel, and two artificial soils constructed to simulate the native soils. Within 60 days, the populations of all five Azospirillum species declined significantly in a linear fashion, in both the native soils and in the homologous artificial soils. Increased levels of CaCO₃, and fine and rough sand, had significant detrimental effects on the survival of the five Azospirillum species, whereas increased organic matter content improved survival. In contrast, when the bacterial strains were incubated in the rhizosphere of tomato seedlings grown in the artificial soils, manipulation of these soil variables had only a marginal effect on bacterial survival; all Azospirillum species survived well in the tomato rhizosphere under conditions that are otherwise detrimental. This study indicates that most cells of the strains of five known species of Azospirillum died out linearly over time in two semiarid soils, and that only the major soil components affected Azospirillum survival in soil. Because mortality was similar in native soils and in artificial homologous soils, artificial soils can be used to study the soil behavior of Azospirillum. Received: 9 April 1999