Role of Azospirillum brasilense nitrate reductase in nitrate assimilation by wheat plants

Summary The nitrogen metabolism of wheat plants inoculated with various Azospirillum brasilense strains and nitrate reductase negative (NR^–) mutants was studied in two monoxenic test tube experiments. The spontaneous mutants selected with chlorate under anaerobic conditions with nitrite as terminal electron acceptor fixed N₂ in the presence of 10 mM NO₃ ^– and were stable after the plant passage. One strain (Sp 245) isolated from surface-sterilized wheat roots produced significant increases in plant weight at both NO₃ levels (1 and 10 mM) which were not observed with the NR^– mutants or with the two other strains. Similar effects were observed in a pot experiment with soil on dry weight and total N incorporation but only at the higher N fertilizer level. In the monoxenic test tube experiments plants inoculated with the mutants showed lower nitrogenase activities than NR⁺ strains at the low NO₃ ^– level (1 = mM) but maintained the same level of activity with 10 mM NO₃ ^– where the activity of all NR⁺ strains was completely repressed. The nitrate reductase activity of roots increased with the inoculation of the homologous strains and with the mutants at both NO₃ ^– levels. At the low NO₃ ^– level this also resulted in increased activity in the shoots, but at the high NO₃ ^– level the two homologous strains produced significantly lower nitrate reductase activity in shoots while the mutants more than doubled it. The possible role of the bacterial nitrate reductase in NO₃ ^– assimilation by the wheat plant is discussed.     

Effect of nitrogen on the yield response of Pennisetum americanum,Triticum aestivum and Zea mays to inoculation with Azospirillum brasilense under temperate conditions

Summary A nitrate-respiring strain, a denitrifying strain, and a non-nitrogen-fixing strain of Azospirillum brasilense were compared for their effect on the growth of pearl millet (Pennisetum americanum), wheat (Triticum aestivum) and maize (Zea mays) under temperate conditions in nitrogen-limited pot cultures. Increases in yield of Z. mays shoots occurred with all three strains when inoculation coincided with the addition of low levels of combined nitrogen. The inoculation of A. brasilense did not show any effect on the yield of P. americanum and T. aestivum. Increased numbers of A. brasilense became associated with Z. mays roots following the addition of low levels of combined nitrogen. Low and very variable rates of acetylene reduction activity were observed from excised roots of inoculated Z. mays plants without preincubation. Results indicate that inoculation of cereals with A. brasilense under temperate conditions has only a limited effect on plant growth.     

Yield response of wheat and barley to inoculation of plant growth promoting rhizobacteria at various levels of nitrogen fertilization

The yield response of a wheat (Kirik) and a barley (Tokak 157/37) cultivar to inoculation with Azospirillum brasilense Sp246 and Bacillus sp. OSU‐142 was studied in relation to three levels of N fertilization (0, 40, and 80 kg ha^–1) under field conditions in Erzurum, Turkey, in 1999 and 2000. Seed inoculation with A. brasilense Sp246 significantly affected yield and yield components, both in wheat and barley. On average of years and N doses, inoculation with A. brasilense Sp246 increased spike number per m², grain number per spike, grain yield, and crude protein content by 7.2, 5.9, 14.7, and 4.1 % in wheat and by 6.6, 8.1, 17.5, and 5.1 % in barley, respectively, as compared to control. Inoculation with Bacillus sp. OSU‐142 significantly increased kernel number per spike in wheat, but no significant effect was determined in the other characteristics. Grain yields and yield components were also higher at all levels of nitrogen fertilizer in the inoculated plots as compared to the control. However, these increases diminished at high fertilizer levels. These results suggest that application of the growth promoting bacteria A. brasilense Sp246 may have the potential to be used as a biofertilizer for spring wheat and barley cultivation in organic and low‐N input agriculture.     

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 inoculation with different strains of Azospirillum brasilense on cotton (Gossipium barbadense)

Summary The response of the cotton plant to inoculation with six strains of Azospirillum brasilense was investigated under subtropical conditions in Egypt. Azospirilla populations and activities were increased as a result of root inoculation with liquid inoculum of Azospirillum sp. Highest C₂H₂ — reduction activities on roots were obtained with strains S631 and Sp Br 14 (means of 216.85 and 209.50 nmol C₂H₄g^–1 root h^–1 respectively) while strain M4 gave the lowest activity (mean of 100.8 nmol C₂H₄g^–1 root h^–1). Statistical analysis showed that Azospirillum strains 5631, Sp Br 14, E15 and SC22 significantly increased the plant dry weight and nitrogen uptake while inoculation with strains M4 and SE had no significant effect in that respect.     

Peculiar effect of Azospirillum inoculation on growth and nitrogen balance of winter wheat (Triticum aestivum)

Summary Wheat plants (Triticum aestivum) grown in pots and in the field under the Mediterranean climate of the south of France were inoculated with a strain of Azospirillum brasilense. Comparisons with non-inoculated plants grown under the same conditions showed significant responses to inoculation with an increase in the number of fertile tillers, shoot and root dry weight, and root to shoot biomass ratio. The roots of inoculated plants attracted relatively more assimilates than those of the control plants until a late stage of growth (heading stage) but the rhizosphere respiration expressed per unit of root growth was not increased by inoculation. Nitrogen yield, both total and in grains, was also enhanced; however, N percentages of all aerial parts of the plants grown in pots were always statistically lower after inoculation than in the control. At maturity, the N % in seeds was 1.81 and 2.45, respectively. The possible mechanisms of this effect of inoculation under the experimental conditions of this study are discussed.     

Single and double inoculation with Azospirillum/Trichoderma: the effects on dry bean and wheat

A plant growth-promoting rhizobacterium (Azospirillum brasilense Sp7) and a bio-control fungus, which can solubilize insoluble phosphorus (Trichoderma harzianum Rifai 1295-22), were evaluated for their single and combined effects on dry bean (Phaseolus vulgaris) and wheat (Triticum aestivum L.) grown in soil. A pot experiment with bean and a field experiment with both bean and wheat were established. In contrast to single inoculation of Trichoderma, the single inoculation of Azospirillum and the double inoculation did not significantly (P >0.05) increase nodule numbers and nodule mass at 45 days after planting in pot grown beans. However, the Azospirillum inoculation with supplementary phosphorus significantly (P <0.05) increased nodule mass. There were no significant (P >0.05) differences among the inoculation treatments for plant dry weight, total plant nitrogen, and total plant phosphorus at 45 days after planting in both pot and field experiments with bean. However, the combined inoculation and rock phosphate application at 1 Mg ha^–1 significantly (P <0.05) increased bean seed yield, total seed nitrogen and phosphorus in the bean field trial. This treatment more than doubled the mentioned properties compared to the control. The microbial inoculations, with the exception of the combined inoculation, significantly (P <0.05) increased total seed nitrogen, but never affected seed yield in the wheat field trial (P >0.05). The combined inoculation improves many plant and yield parameters and, therefore, has some advantages over single inoculation provided that rock phosphate was supplied at an amount not exceeding 1 Mg ha^–1. Higher rock phosphate application rates decreased many plant and yield parameters in our study.This work was carried out at Gaziosmanpaa University, Tokat, Turkey.     

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.     

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.     

Non-isotopic method for the quantification of biological nitrogen fixation and wheat production under field conditions

 A field experiment was conducted in the Inshas area (Sharkeia governorate) to study the potential of biofertilizers, when the quantities of commercial fertilizers were reduced, for optimal wheat production. The different treatments were arranged in a completely randomized block design with seven replicates. N fertilizer was applied in three treatments with one control, i.e. zero, full, half and one-fourth rates, in the presence or absence of inocula. Azospirillum brasilense strain Sp245 was used as a biofertilizer. Generally, inoculation increased the accumulation of shoot dry matter and grain yield by about 35%, relative to the control treatment. Similar trends were observed in the case of N and P uptake by shoots and grains, as well as the efficient use of both, where inoculation increased the acquisition of the two elements as compared with the uninoculated plants. The obtained data showed that N₂ fixed by shoots and grains ranged from 2 to 10 kg N ha^–1 and from 8 to 19 kg N ha^–1, respectively. With respect to fixed N₂, the best treatment was inoculation combined with the one-fourth dose of N, followed by inoculation combined with half of the recommended N dose. Most of the fixed N was utilized by grains and the results clearly reflected the negative effect of high N fertilizer rates on biological N fixation. It is obvious that inoculation, in general, enhanced the N fertilizer utilized by both shoots and grains of wheat plants. In conclusion, the application of biofertilization technology to a light-textured soil with low fertility had a positive effect on plant growth, N gained from the air and enhancement of fertilizer N uptake (apparent recovery fraction). Received: 22 April 1999     

Effect of inoculation with wild type Azospirillum brasilense and A. irakense strains on development and nitrogen uptake of spring wheat and grain maize

Under the controlled conditions of the greenhouse and by varying some biotic and abiotic factors, we tried to identify some of the factors critical to obtain successful Azospirillum inoculation. Spring wheat and grain maize were inoculated with different concentrations of the wild type strains A. brasilense Sp245 and A. irakense KBC1, and grown in a substrate with varying concentrations of organic matter (OM) and N fertiliser. The inoculum concentration was one of the factors that influenced most the outcome of an inoculation experiment on wheat, with lower inoculum concentrations (10⁵-10⁶ cfu plant^-1) stimulating root development and plant dry weight and higher inoculum concentrations (10⁷-10⁸ cfu plant^-1) having no effect or sometimes even inhibiting root development. The effect of inoculation was most pronounced at low to intermediate N fertilisation levels, while the OM content of the substrate had no effect. Inoculation was found to affect early plant and root development, plant and root dry weight, grain yield and the N-uptake efficiency of plants. However, inoculation did not change the N concentration in plants or grains. In addition, a difference in the ability of both strains to stimulate plant growth and N uptake of wheat and maize was observed, with A. brasilense Sp245 having most effect on spring wheat and A. irakense KBC1 being more effective on grain maize. The significance of the obtained results for agriculture is discussed.     

Effect of Azospirillum brasilense inoculation on root morphology and respiration in tomato seedlings

Summary The level of Azospirillum brasilense strain Cd colonization in the rhizosphere of some vegetables was 10⁴–10⁵ colony-forming units (CFU) per root of one plant in 2-week-old plants inoculated with 5 × 10⁸ Azospirillum cells. Significant increases in root length (35%) and in top (90%) and root (50%) dry weight and total leaf area (90%) were observed in 18-day-old inoculated tomato plants compared with non-inoculated controls. An inoculum concentration of 1 × 10⁸ to 5 × 10⁸ CFU/ml stimulated the appearance of root hairs. Large numbers of bacteria (1 × 10⁹ CFU/ml) caused asymmetrical growth of the root tip. In a petri dish system, Azospirillum (1 × 10⁸ CFU/ml) increased root dry weight (150%), protein content (20%), respiration rate per root (70%) and the specific activity of malate dehydrogenase (45%–65%) over non-inoculated controls. The specific respiration rate, expressed as micromol of O₂ per minute per milligram of dry weight of roots, was significantly lower in inoculated roots, suggesting that less energy was spent for accumulation of more dry material.     

N2-fixation by ammonium-excreting Azospirillum brasilense in auxine-induced root tumours of wheat (Triticum aestivum L.)

Summary Wheat seedlings, treated with the auxine 2,4-dichlor-phenoxy acetic acid (2,4-D) during germination developed only a residual root system. Root elongation was extremely restricted and root tips were deformed to thick club-shaped tumours. When 2,4-D was added in a later stage of plant growth the plants developed additional nodule-like knots along primary roots. Root and shoot dry-matter production was slightly repressed in all 2,4-D treatments and N translocation from roots to shoots was repressed as well. When transferred to an auxine-free growth medium, the 2,4-D-affected roots were not capable of complete recovery. In plants inoculated gnotobiotically with Azospirillum brasilense, either with the wild type or with the NH ₄ ⁺ -excreting mutant strain C3, a 2,4-D addition increased rhizosphere acetylene-reduction activity at pO₂ 1.5 kPa. The O₂ sensitivity of root-associated nitrogenase activity tended to be reduced. The number of root-colonizing bacteria, at approximately 10⁸ colony-forming units (cfu) per g dry root, was similar in the 2,4-D treatments and untreated controls. Plant treatment with high concentrations of the chemical isomer 3,5-dichlor-phenoxy acetic acid (3,5-D) did not have comparable effects, either on plant development or on rhizosphere-associated nitrogenase activity. Root-tumour tissue inhabited by A. brasilense showed purple staining when subjected to a tetrazolium chloride solution, which may indicate intensive local nitrogenase activity in this tissue. Exposed to an ¹⁵N₂-enriched atmosphere, plants treated with 2,4-D and with A. brasilense incorporated significantly higher amounts of ¹⁵N than untreated controls. In all cases the highest values of ¹⁵N enrichment were found following inoculation with the NH ₄ ⁺ -excreting mutant strain C3. Present address: F. A. Janssens Laboratory of Genetics, Catholic University of Leuven, Willem de Croylan 42, B-3001 Heverlee, Belgium     

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.     

The flavonoid naringenin enhances intercellular colonization of rice roots by<Emphasis Type="Italic"> Azorhizobium caulinodans</Emphasis>

The intercellular colonization of rice roots by Azorhizobium caulinodans and other diazotrophic bacteria has been studied using strains marked with the lacZ reporter gene. A. caulinodans were able to enter the roots of rice at emerging lateral roots (lateral root cracks) by crack entry and this was observed by light microscopy. After colonization of lateral roots, bacteria moved into intercellular space within the cortical cell layer of roots. Naringenin at 1×10^-5 and 5×10^-5 M concentration significantly enhanced root colonization. The role of nodABC and regulatory nodD genes was also studied; lateral root crack (LRC) colonization of rice was shown to be Nod factor and NodD independent. Lateral root crack colonization of rice was also observed with similar frequency following inoculation with Azospirillum brasilense and the colonization by A. brasilense was stimulated by naringenin and other flavonoid molecules.     

Yield response of spring wheat cultivars (Triticum aestivum and T. turgidum) to inoculation with Azospirillum brasilense under field conditions

Summary Eight commercial Israeli spring wheat cultivars (six Triticum aestivum and two T. turgidum) grown with 40 and 120 kg N/ha were tested for responses to inoculation with Azospirillum brasilense. At the low level of N fertilization (40 kg/ha), five cultivars showed significant increases in plant dry weight measured at the milky ripe stage; however, by maturation only the cultivar Miriam showed a significant increase in grain yield. Two cultivars, which had shown a positive inoculation effect at the earlier stages, had a significant decrease in grain yield. No significant effect of inoculation was found at the high N level. To confirm those results, four wheat (T. aestivum) cultivars were tested separately over 4 years in 4 different locations under varying N levels. Only Miriam showed a consistently positive effect of Azospirillum inoculation on grain yield. Inoculation increased the number of roots per plant on Miriam compared with uninoculated plants. This effect was found at all N levels. Nutrient (N, P and K) accumulation and number of fertile tillers per unit area were also enhanced by Azospirillum, but these parameters were greatly affected by the level of applied N. It is suggested that the positive response of the spring wheat cultivar Miriam to Azospirillum inoculation is due to its capacity to escape water stresses at the end of the growth season.     

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.     

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.     

Using the spermosphere model technique to describe the dominant nitrogen-fixing microflora associated with wetland rice in two Egyptian soils

Summary This study is an attempt to describe the dominant N₂-fixing microflora associated with the roots of wetland rice. Rice cultivar Giza 171 was grown in a phytotron on two alluvial Egyptian soils for 8 days, a stage when the nitrogenase activity of undisturbed plants reached a level of 245 × 10^–6 mol C₂H₄ h^–1 g^–1 dry weight of leaf. The roots and rhizosphere soils were then used for counting and isolating dominant diazotrophs. Counts and initial enrichment steps were carried out on a selective medium made of an axenic rice plantlet, the spermosphere model, incubated under 1 % acetylene. The counts were very high, exceeding 10⁸ bacteria g^–1 dry weight of rhizosphere soil. Enterobacteriaceae were dominant; most isolates were Enterobacter cloacae belonging to different biotypes in the two soils. Enterobacter agglomerans, Citrobacter freundii and Klebsiella planticola were also present as members of the dominant microflora. Azospirillum brasilense and Azospirillum lipoferum were present as well, but less abundant.     

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.