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.     

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.     

The effect of Azospirillum brasilense Sp7 and Azotobacter chroococcum on nitrogen balance in soil under cropping with oats (Avena sativa L.)

Summary Pot experiments with oats were carried out to study the effect of Azospirillum brasilense Sp 7 and Azotobacter chroococcum 94K on the yield of plants, the N content of soil and the ¹⁴N balance. The plants were grown on gray forest soil under irrigation with deionized water and application of ¹⁵N-labelled fertilizer at a rate of 4 mg N 100 g^-1 soil. Inoculation of plants with Azospirillum spp. and Azotobacter spp. failed to increase the plant yield. However, the increase in total N in the soil at the end of the experiment and the positive ¹⁴N balance in the soil-plant system due to increased nitrogenase activity in the rhizosphere were statistically significant. The amount of N accumulated in the soil was comparable with the rate of N applied as fertilizer.     

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.     

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.     

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.     

Association of Azospirillum brasilense with pearl millet (Pennisetum americanum (L.) Leeke)

Summary Microscopic observations of the root system of pearl millet (Pennisetum americanum (L.) Leeke) var. BJ 104 after surface sterilization and incubation in phosphate malate triphenyl tetrazolium chloride (TTC) revealed extensive colonization by Azospirillum spp. when plants were grown in sterile, partially sterile and field conditions as evidenced by the TTC-reducing property of active cells of the bacterium. Quantitative studies showed the need to standardize the techniques further to ensure more precise monitoring of the bacteria in the rhizosphere, as large numbers of soil bacteria were found capable of growth on specific media, thus interfering with the plate counts. Seed inoculation with A. brasilense increased the mean grain yield of pearl millet under different agroclimatic conditions in India. The mean increase in grain yield due to inoculation over uninoculated controls was also noticed with graded levels of fertilizer nitrogen (urea). Inoculation alone contributed to increased nitrogen uptake of plants with varying levels of fertilizer nitrogen application under sandy loam soil conditions (pH 7.3). The effects of inoculation were more prominent under lower levels of nitrogen than at the higher levels. The root biomass under field conditions was increased with Azospirillum spp. inoculation at 10 and 20 kg N/ha than their corresponding uninoculated controls.     

Ammonium-excreting Azospirillum sp. become intracellularly established in maize (Zea mays) para-nodules

Maize seedlings develop nodule-like tumour knots (para-nodules) along primary roots when treated with the auxin 2,4-dichlor-phenoxy-acetic acid (2,4-D). Inoculated NH ₄ ⁺ -excreting Azospirillum brasilense cells were shown to colonize these tumours, mostly intracellularly, promoting a high level of N₂ fixation when microaerophilic conditions were imposed. The nitrogenase activity inside the para-nodules was less sensitive to free O₂ than in non-para-nodulating roots. Both light and electron microscopy showed a dense bacterial population inside intact tumour cells, with the major part of the cell infection along a central tumour tissue. The bacteria colonized the cytoplasm with a close attachment to inner cell membranes. In an auxin-free growth medium, young 2,4-D-induced para-nodules grew further to become mature differentiated root organs in which introduced bacteria survived with a stable population. These results provide evidence that gramineous plants are potentially able to create a symbiosis with diazotrophic bacteria in which the NH ₄ ⁺ -excreting symbiont will colonize para-nodule tissue intracellularly, thus becoming well protected.     

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.     

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.     

Priming effects of 15N-labelled ammonium nitrate on uptake of soil N by wheat (Triticum aestivum L.) under field conditions

Summary The uptake of labelled and unlabelled N by wheat was measured in a field experiment using ¹⁵N-labelled ammonium nitrate fertilizer. The dry matter yield and N yields were significantly increased with fertilizer N application compared to those from unfertilized soil. The uptake of applied N by wheat ranged between 25 and 34%. Fertilizer N application increased the uptake of unlabelled soil N which was attributed to a positive priming effect or added N interaction. The added N interaction observed by applying 20, 60, and 120 kg fertilizer N was 11.4, 19.1, and 27.9 kg, corresponding to 26, 44 and 64%, respectively of the N taken up from unfertilized soil. The A values did not alter with the increase in fertilizer N application. The observed added N interaction may have been the result of pool substitution whereby added labelled fertilizer N stood proxy for unlabelled soil N. A significant correlation coefficient (r=0.996^**) between the uptake of soil N and the dry matter yield showed that soil N was more important than fertilizer N in wheat production.     

Availability of soil and fertilizer nitrogen to wheat (Triticum aestivum L.) following rice-straw amendment

Summary A pot experiment was conducted to study the N availability to wheat and the loss of ¹⁵N-labelled fertilizer N as affected by the rate of rice-straw applied. The availability of soil N was also studied. The straw was incorporated in the soil 2 or 4 weeks before a sowing of wheat and allowed to decompose at a moisture content of 60% or 200% of the water-holding capacity. The wheat plants were harvested at maturity and the roots, straw, and grains were analysed for total N and ¹⁵N. The soil was analysed for total N and ¹⁵N after the harvest to determine the recovery of fertilizer N in the soil-plant system and assess its loss. The dry matter and N yields of wheat were significantly retarded in the soil amended with rice straw. The availability of soil N to wheat was significantly reduced due to the straw application, particularly at high moisture levels during pre-incubation, and was assumed to cause a reduction in the dry matter and N yields of wheat. A significant correlation (r=0.89) was observed between the uptake of soil N and the dry matter yield of wheat with different treatments. In unamended soil 31.44% of the fertilizer N was taken up by the wheat plants while 41.08% of fertilizer N was lost. The plant recovery of fertilizer N from the amended soil averaged 30.78% and the losses averaged 45.55%     

Nitrogen fixation by Azospirillium brasilense in soil and the rhizosphere under controlled environmental conditions

Summary Acetylene reduction activity by Azospirillum brasilense, either free-living in soils or associated with wheat roots, was determined in a sterilised root environment at controlled levels of O₂ tension and with different concentrations of mineral N. In an unplanted, inoculated soil nitrogenase activity remained low, at approximately 40 nmol C₂H₄ h^-1 per 2kg fresh soil, increasing to 300 nmol C₂H₄ h^-1 when malic acid was added as a C source via a dialyse tubing system. The N₂ fixation by A. brasilense in the rhizosphere of an actively growing plant was much less sensitive to the repressing influence of free O₂ than the free-living bacteria were. An optimum nitrogenase activity was observed at 10 kPa O₂, with a relatively high level of activity remaining even at an O₂ concentration of 20 kPa. Both NO _inf3 ^sup- and NH _inf4 ^sup+ repressed nitrogenase activity, which was less pronounced in the presence than in the absence of plants. The highest survival rates of inoculated A. brasilense and the highest rates of acetylene reduction were found in plants treated with azospirilli immediately after seedling emergence. Plants inoculated at a later stage of growth showed a lower bacterial density in the rhizosphere and, as a consequence, a lower N₂-fixing potential. Subsequent inoculations with A. brasilense during plant development did not increase root colonisation and did not stimulate the associated acetylene reduction. By using the ¹⁵N dilution method, the affect of inoculation with A. brasilense in terms of plant N was calculated as 0.067 mg N₂ fixed per plant, i.e., 3.3% of the N in the root and 1.6% in the plant shoot were of atmospheric origin. This ¹⁵N dilution was comparable to that seen in plants inoculated with non-N₂-fixing Psudomonas fluorescens.     

Influence of soluble phosphorus fertilizer on the interaction between a vesicular-arbuscular mycorrhizal fungus and Azospirillum brasilense in barley (Herdeum vulgare L.)

Summary Pot-culture studies were carried out to examine the response of barley (Hordeum vulgare L.) to inoculation with Azospirillum brasilense and Glomus versiforme, singly and/or in combination, under varying levels of nitrogenous [(¹⁵NH₄)₂SO₄] and soluble phosphatic (single superphosphate) fertilizers. The interaction between both the endophytes led to increased growth and nutrition of the barley plants. Roots from plants inoculated with Azospirillum brasilense and Glomus versiforme exhibited very low acetylene reduction activity. N₂ fixation in the plants increased with the increase in plant growth but the mycorrhiza alone gave a low level of N₂ fixation in the plants compared to combined inoculation with both the endophytes.     

Interaction of 15N-labelled ammonium nitrate,urea and ammonium sulphate with soil N during growth of wheat (Triticum aestivum L.) under field conditions

The effects of ¹⁵N-labelled ammonium nitrate, urea and ammonium sulphate on yield and uptake of labelled and unlabelled N by wheat (Triticum aestivum L. cv. Mexi-Pak-65) were studied in a field experiment. The dry matter and N yields were significantly increased with fertilizer N application compared to those from unfertilized soil. The wheat crop used 64.0–74.8%, 61.5–64.7% and 61.7–63.4% of the N from ammonium nitrate, urea and ammonium sulphate, respectively. The fertilizer N uptake showed that ammonium nitrate was a more available source of N for wheat than urea and ammonium sulphate. The effective use of fertilizer N (ratio of fertilizer N in grain to fertilizer N in whole plant) was statistically similar for the three N fertilizers. The application of fertilizer N increased the uptake of unlabelled soil N by wheat, a result attributed to a positive added N interaction, which varied with the method of application of fertilizer N. Ammonium nitrate, urea and ammonium sulphate gave 59.3%, 42.8% and 26.3% more added N interaction, respectively, when applied by the broadcast/worked-in method than with band placement. A highly significant correlation between soil N and grain yield, dry matter and added N interaction showed that soil N was more important than fertilizer N in wheat production. A values were not significantly correlated with added N interaction (r=0.719). The observed added N interaction may have been the result of pool substitution, whereby added labelled fertilizer N stood proxy for unlabelled soil N.     

Azospirillum — plant root associations: A review

Summary Bacteria of the genus Azospirillum are extensively studied for their plant-growth promoting effect following inoculation. Physiological and biochemical studies of these diazotrophic bacteria are now benefiting from recent breakthroughs in the development of genetic tools for Azospirilum. Moreover, the identification and cloning of Azospirillum genes involved in N₂ fixation, plant interaction, and phytohormone production have given new life to many research projects on Azospirillum. The finding that Azospirillum genes can complement specific mutations in other intensively studied rhizosphere bacteria like Rhizobia will certainly trigger the exploration of new areas in rhizosphere biology. Therefore a review of the Azospirillum-plant interactions is particularly timely.     

Macronutrient accumulation in wheat crop (Triticum aestivum L.) with Azospirillum brasilense associated with nitrogen doses and sources

Abstract

New studies are needed to optimize the nitrogen (N) amount that can be applied to utilize the Azospirillum brasilense benefits. In addition, information regarding the interaction between the urease inhibitor and biological nitrogen fixation (BNF) and how they affect the macronutrients accumulation are also needed. We evaluate the effect of N sources and doses associated with A. brasilense regarding the macronutrients accumulation in straw and grains and wheat grain yield in tropical conditions. A randomized block experimental design was used with four replications in a 2?×?5?×?2 factorial arrangement as follows: two N sources (urea and urea with urease enzyme inhibitor NBPT; five N doses (0, 50, 100, 150, and 200?kg ha^?1) applied in topdressing; with and without A. brasilense inoculation. We found that an increase in N doses positively influenced the accumulation of macronutrients in straw and grains and the wheat grain yield. N sources have similar effects. Inoculation with A. brasilense increased accumulation of Mg and S in straw and P, Ca, and Mg in grains, regardless of the N dose. The inoculation with A. brasilense associated with 140?kg ha^?1 of N increased wheat grain yield. The inoculation can contribute in a more sustainable way to wheat nutrition and optimizing N fertilization.     

Effects of root-associated N2-fixing cyanobacteria on the growth and nitrogen content of wheat (Triticum vulgare L.) seedlings

Summary Following co-cultivation of wheat with N₂-fixing cyanobacterial isolates capable of forming associations, Nostoc 2S6B, 2S9B or Anabaena C5, for 15 days in the presence or absence of combined N a large stimulation of root length was observed without any increase in root dry weight. Increases in the N concentrations of both roots and shoots occurred following co-cultivation with most cyanobacteria tested. The increase in plant N concentrations appeared to be dependent on the wheat cultivar and the cyanobacterial isolate used. Nostoc isolates had similar nitrogenase activities when associated with roots and when grown in shake-flask cultures. The nitrogenase activity of roots colonized by Anabaena C5 or Nostoc 2S6B was higher following removal of loosely associated cyanobacteria.     

Estimating N2 fixation by field-grown lupins (Lupinus angustifolius L.) using soil and plant 15N enrichment

Summary Biological N₂ fixation was estimated in a field experiment following the addition of NH₄Cl or KNO₃ to unconfined microplots (1.5 m²) at 2.5 g N m^-2 (10 atom% ¹⁵N). A model of total N and ¹⁵N accumulation in lupins and decreasing ¹⁵N enrichment in the KCl-extractable soil-N pool (0–0.15 m depth) was used to estimate the proportion of N in lupins derived from biological N₂ fixation. Estimates of N₂ fixation derived from the model were compared with ¹⁵N isotope-dilution estimates obtained using canola, annual ryegrass, and wheat as nonfixing reference plants. Biomass, total N accumulation, or ¹⁵N enrichment in the lupin and reference crops did not differ whether NH _inf4 ^sup+ or NO _inf3 ^sup- was added as the labelled inorganic-N source. The decrease in soil ¹⁵N enrichment was described by first-order kinetics, whereas total N and ¹⁵N accumulation in the lupins were described by logistical equations. Using these equations, the uptake of soil N by lupins was estimated and was then used to calculate fixed N₂. Estimates of N₂ fixation derived from the model increased from 0 at 50 days after sowing to a maximum of 0.79 at 190 days after sowing. Those based on the ¹⁵N enrichment of the NO _inf3 ^sup- pool were 10% higher than those based on the mineral-N pool. ¹⁵N isotope-dilution estimates of N₂ fixation ranged from 0.37 to 0.55 at 68 days after sowing and from 0.71 to 0.77 at 190 days after sowing. Reference plant-derived values of N₂ fixation were all higher than modelled estimates during the early states of growth, but were similar to modelled estimates at physiological maturity. The use of the model to estimate N₂ derived from the atmosphere has the intrinsic advantage that the need for a non-fixing reference plant is avoided.     

The fate of markerAzospirillum lipoferum inoculated into rice and its effect on growth,yield and N2 fixation of plants studied by acetylene reduction,15N2 feeding and15N dilution techniques

Summary A spontaneous mutant ofAzospirillum lipoferum, resistant to streptomycin and rifampicin, was inoculated into the soil immediately before and 10 days after transplanting of rice (Oryza sativa L.). Two rice varieties with high and low nitrogen-fixing supporting traits, Hua-chou-chi-mo-mor (Hua) and OS4, were used for the plant bacterial interaction study. The effect of inoculation on growth and grain and dry matter yields was evaluated in relation to nitrogen fixation, by in situ acetylene reduction assay,¹⁵N₂ feeding and¹⁵N dilution techniques. A survey of the population of marker bacteria at maximum tillering, booting and heading revealed poor effectivety. The population of nativeAzospirillum followed no definite pattern. Acetylene-reducing activity (ARA) did not differ due to inoculation at two early stages but decreased in the inoculated plants at heading. In contrast, inoculation increased tiller number, plant height of Hua and early reproductive growth of both varieties. Grain yield of both varieties significantly increased along with the dry matter. Total N also increased in inoculated plants, which was less compared with dry matter increase.¹⁵N₂ feeding of OS4 at heading showed more¹⁵N₂ incorporation in the control than in the inoculated plants. The ARA,¹⁵N and N balance studies did not provide clear evidence that the promotion of growth and nitrogen uptake was due to higher N₂ fixation.