Effect ofAzospirillum inoculation on nitrogen fixation and growth of several winter legumes

Summary Inoculation of naturally nodulatedPisum sativum L. (garden pea) withAzospirillum in the greenhouse caused a significant increase in nodule numbers above controls. Field inoculation of garden peas in the winter 1981–1982 andCicer arietinum L. (chick pea), in winter 1982–1983, withAzospirillum one week after plant emergence, produced a significant increase in seed yield, but did not affect plant dry matter yield. ForVicia sativa L. (vetch) grown in soil in the greenhouse and in the field for forage, winter 1980–1981, inoculation significantly increased dry matter yield, %N, N-content, and acetylene reduction (nitrogen fixation) activity. InHedysarum coronarium L. (sulla clover), winter 1981–1982, inoculated with both its specificRhizobium (by the slurry method) andAzospirillum, 7 days after emergence, there was an increase in acetylene reduction above controls inoculated withRhizobium alone. These results suggest that it is possible, under conditions tested in this work, to increase nodulation, nitrogen fixation, and crop yields of winter legumes by inoculation withAzospirillum.     

Yield response of a common spring wheat cultivar to inoculation withAzospirillum brasilense at various levels of nitrogen fertilization

Summary The yield response of a common spring wheat cultivar,Triticum aestivum, to inoculation withAzospirillum brasilense was studied at four levels of N fertilization. Plant yield increased due to the inoculation treatment only at medium and high levels of N fertilization, with a maximum yield increase of about 8.0 per cent at the highest level (approximately 1.0 g of pure N per plant). Yield increase was mostly due to an increase in the number of grains per spike, and at the highest level of fertilization, also due to a higher number of spikes per plant. At all N levels, the inoculation caused an increase of 0.5–1.4 per cent in the number of fertile spikelets per main spike.Grain protein percentage was unaffected by the inoculation, though significantly increase due to the fertilization treatments.The occurrence of maximum yield response at the highest N level, the response by early-determined yield components, i.e. spikelet number, and the unaffected grain protein content are in accord with the suggestion that the contribution ofAzospirillum brasilense to wheat yield is not through N₂-fixation.     

Effect of combined inoculation ofAzospirillum brasilense and vesicular-arbuscular mycorrhiza on pearl millet (Pennisetum americanum)

Summary Growth and phosphorus uptake of pearl millet (Pennisetum americanum) on an unsterile, phosphorus-deficient soil was improved by the seed inoculation withAzospirillum brasilense or soil inoculation with the vesicular-arbuscular mycorrhizal fungi (Acaulospora,Gigaspora margarita, Glomus fasciculatum). These microorganisms acted synergistically when added simultaneously and the response was significant withAzospirillum brasilense + Gigaspora margarita andAzospirillum brasilense + Glomus fasciculatum combinations over uninoculated control as far as the dry matter content of shoots, root biomass and phosphorus uptake of the millet was concerned.     

Ecophysiological aspects of growth and nitrogen fixation inAzospirillum spp.

The nitrogenase activity ofAzospirillum spp. is efficiently regulated by environmental factors. InA. brasilense andA. lipoferum a rapid switch off of nitrogenase activity occurs after the addition of ammonium chloride. As in photosynthetic bacteria, a covalent modification of nitrogenase reductase (Fe-protein) is involved. InA. amazonense, a non-covalent mechanism causes only a partial inhibition of nitrogenase activity after ammonium chloride is added. In anaerobic conditions, nitrogenase reductase is also switched off by a covalent modification inA. brasilense andA. lipoferum. Short-time exposure ofAzospirillum to increased oxygen levels causes a partially reversible inhibition of nitrogenase activity, but no covalent modification is involved.Azospirillum spp. show variations in their oxygen tolerance. High levels of carotenoids confer a slightly improved oxygen tolerance. Certain amino acids (e. g. glutamate, aspartate, histidine and serine) affect growth and nitrogen fixation differently inAzospirillum spp. Amino acids may influence growth and nitrogen fixation ofAzospirillum in the association with plants.Azospirillum brasilense andA. halopraeferens are the more osmotolerant species. They utilize most amino acids poorly and accumulate glycine betaine, which also occurs in osmotically stressed grasses as a compatible solute to counteract osmotic stress. Nitrogen fixation is stimulated by glycine betaine and choline. Efficient iron acquisition is a prerequisite for competitive and aerotoleran growth and for high nitrogenase activity.Azospirillum halopraeferens andA. amazonense assimilate iron reasonably well, whereas growth of someA. brasilense andA. lipoferum strains is severely inhibited by iron limitation and by competition with foreign microbial iron chelators. However, growth of certain iron-limitedA. brasilense strains is stimulated by the phytosiderophore mugineic acid. Thus, various plant-derived substances may stimulate growth and nitrogen fixation ofAzospirillum.     

Contribution ofAzospirillum brasilense to the nitrogen needs of grain sorghum (Sorghum bicolor (L) Moench) in humid sub-tropics

Summary Field experiments were conducted to assess the contribution ofAzospirillum brasilense to the N needs of grain sorghumcv. CSH — 5 during monsoon (June–October) seasons of 1978 and 1979.A. brasilense contributed to the N uptake by crop in the range from 5.8 to 19.6 kg N/ha. However, the contribution ofA. brasilense to the N needs of sorghum was more when sorghum was manured with farmyard manure at the rate of 10 tons/ha. Publication of G.B.P.U.A.T. Expt. Station, Pantnagar — 263145, India.     

The effect of inoculation with Azospirillum brasilense on growth and nitrogen utilization by wheat plants

Azospirillium brasilense is a rhizosphere bacteria that has been reported to improve yield when inoculated on wheat plants. However, the mechanisms through which this effect is induced is still unclear. In the present work, we have studied the effects of inoculating a highly efficient A. brasilense strain on wheat plant grown in 5 kg pots with soil in a greenhouse, under three N regimes (0, 3 or 16 mM NO₃ ^–, 50 ml/pot once or twice-a -week), and in disinfected or non-disinfected soil. At the booting stage, the inoculated roots in both soils showed a similar colonization by Azospirillum sp. that was not affected by N addition. The plants grown in the disinfected soil showed a higher biomass, N content and N concentration than those in the non-disinfected soil, and in both soils the inoculation stimulated plant growth, N accumulation, and N and NO₃ ^– concentration in the tissues.At maturity, the inoculated plants showed a higher biomass, grain yield and N content than the uninoculated ones in both soils, and a higher grain protein concentration than the uninoculated. It is concluded that in the present experiments, A. brasilenseincreased plant growth by stimulating nitrogen uptake by the roots.     

Effect of Azospirillum inoculation on some growth parameters and n-content of wheat,sorghum and panicum

The potential of the nitrogen fixing bacteriumAzospirillum brasilense to enhance development and increase growth of several gramineae was investigated. In both sterilized and non-sterilized systems heading and flowering occurred earlier in the inoculated plants as compared to the noninoculated ones. Total shoot and root weights, total-N content, plant height and leaf length were significantly increased by inoculation.From the Department of Agricultural Botany.     

Use ofAzospirillum brasilense as biofertilizer in intensive wheat cropping

Summary Three field experiments were conducted on ten cultivars of winterwheat and four cultivars of springwheat to estimate the growth promoting effect ofAzospirillum brasilense under varying nitrogen doses. Independent of cultivar selection or nitrogen dose a highly significant yield increase could be observed in winterwheat: strains S631 and SpBr14 increased the average grain yield with 9.14% and 14.82% respectively. When the yield components were studied a coinciding increase in ear density could be demonstrated of resp. 10.57% and 13.55%. Less significant results were obtained with springwheat although in one experiment strain SpBr14 significantly increased grain yield. As with winterwheat tillering of the plant was markedly affected by inoculation with both strains. In a companion greenhouse experiment it was found that inoculation with Azospirillum can cause a decrease in the root mass of wheatplants except when strain SpBr14 is used. Therefore it is suggested that the presence of a higher tillering together with an undisturbed nutrient uptake capacity can result in yield increases after inoculation withAzospirillum brasilense.     

Attachment,colonization and proliferation ofAzospirillum brasilense andEnterobacter spp. on root surface of grasses

Root colonization studies, employing immunofluorescence and using locally isolated strains, showed thatEnterbacter sp. QH7 andEnterobacter agglomerans AX12 attached more readily to the roots of most plants compared withAzospirillum brasilense JM82. Heat treatment of either root or inoculum significantly decreased the adsorption of bacteria to the root surface. Kallar grass and rice root exudates sustained the growth ofA. brasilense JM82,Enterobacter sp. QH7 andE. agglomerans AX12 in Hoagland and Fahraeus medium. All the strains colonized kallar grass and rice roots in an axenic culture system. However, in studies involving mixed cultures,A. brasilense JM82 was inhibited byEnterobacter sp. QH7 in kallar grass rhizosphere and the simultaneous presence ofEnterobacter sp. QH7 andE. agglomerans AX12 suppressed the growth ofA. brasilense JM82 in rice rhizosphere. The bacterial colonization pattern changed from dispersed to aggregated within 3 days of inoculation. The colonization sites corresponded mainly to the areas where root mucigel was present. The area around the point of emergence of lateral roots usually showed maximum colonization.     

Effect of salinity and its composition on the accumulation of selenium by alfalfa

Alfalfa (Medicago sativa L.) was grown in greenhouse sand culture to examine the effect of salinity composition and concentration on Se accumulation by plants. In a 2×2×4 factorial experiment, salinity was added as either C1⁻ or SO ₄ ²⁻ salts to the irrigating solution to achieve an electrical conductivity of 0.5, 1.5–3.0, or 6.0 dS m⁻¹. Selenium was added to the nutrient solution at a concentration of 0.25 or 1.0 mg Se(VI)I⁻¹. Following the third cutting, the roots were washed and all plant material analyzed for dry weight and Se. Plant biomass production decreased with additions of either Se or salinity, regardless of composition. In the presence of Se, the yield reduction was greater with Cl⁻ salinity than with SO ₄ ²⁻ salinity. Plant Se accumulation was reduced from 948 mg Se kg⁻¹ to 6 mg Se kg⁻¹ in the presence of SO ₄ ²⁻ salts (0.5 mmol SO ₄ ²⁻ l⁻¹ vs. 40 mmol SO ₄ ²⁻ l⁻¹) due to an apparent Se(VI) −SO ₄ ²⁻ antagonism. This Se−SO ₄ ²⁻ antagonism prevented accumulation of Se and reduced Se-induced toxicity. A lesser antagonistic effect on Se accumulation was observed between Cl⁻, and Se. A synergistic interaction between SO ₄ ²⁻ and Se(VI) increased plant S concentrations in the presence of the relatively low basal SO ₄ ²⁻ concentrations but not at the higher solution SO ₄ ²⁻ concentrations. In many areas, soil and water containing high Se concentrations also contain large amounts of SO ₄ ²⁻ . The occurrence of SO ₄ ²⁻ with Se reduces plant accumulation of Se(VI) and may lower the risk of Se overexposure to animals feeding on forage material grown in high Se−SO ₄ ²⁻ regions.     

Changes in Azospirillum brasilense motility and the effect of wheat seedling exudates

The rhizobacterium Azospirillum brasilense Sp245 swims, swarms (Swa⁺ phenotype) or, very rarely, migrates with the formation of granular macrocolonies (Gri⁺ phenotype). Our aims were (i) to identify Sp245 mutants that swarm faster than the parent strain or differ from it in the mode of spreading and (ii) to compare the mutants’ responses to wheat seedling exudates. In isotropic liquid media, the swimming speeds of all motile A. brasilense strains were not influenced by the exudates. However, the exudates significantly stimulated the swarming of Sp245. In several Sp245 mutants, the superswarming phenotype was insensitive to local colonial density and to the presence of wheat seedling exudates. An A. brasilense polar-flagellum-defective Gri⁺ mutant BK759.G gave rise to stable Swa⁺⁺ derivatives with restored flagellum production. This transition was concurrent with plasmid rearrangements and was stimulated in the presence of wheat seedling exudates. The swarming rate of the Swa⁺⁺ derivatives of BK759.G was affected by the local density of their colonies but not by the presence of the exudates.     

Yield response of various wheat genotypes to inoculation withAzospirillum brasilense

Summary Twenty different wheat genotypes representing a wide genetic variability, were tested for their yield and yield components response to inoculation withAzospirillum brasilense, at two levels of N fertilization. Only two cultivars responded by a significant yield increase of 7.4 and 8.0 per cent — both at the higher N level. The response reflected an increase in the number of grains per plant added as additional spikes. The importance of the host plant genotype for a successful wheat-bacteria association is discussed.     

Fatty acid analysis of four Azospirillum species reveals three groups

Cellular fatty acid composition of 14 strains from the four species of Azospirillum was determined by gas chromatographic analysis. All strains of Azospirillum lipoferum and Azospirillum brasilense were similar in fatty acid data, thus not revealing an expected distinction between the two long established species. Strains of both Azospirillum halopraeferens and Azospirillum amazonense, however, differed significantly from this first group of strains.     

Response ofPanicum maximum Jacq. to inoculation withAzospirillum brasilense

Summary The effects on plant dry weight and acetylene reduction activity after applyingAzospirillum brasilense (strain 13t) to guineagrass,Panicum maximum Jacq., grown in clay pots under greenhouse conditions, are reported and discussed.     

Fertilizer and soil nitrogen accumulation by irrigated barley grown on a red-brown earth in south-eastern Australia

¹⁵N labelled (NH₄)₂SO₄ was applied to barley at 5 g N m⁻² (50 kg N ha⁻¹) in microplots at sowing to study the timing of the N losses and the contribution of soil and fertilizer N to the plant. Water treatments included rainfed and irrigation at 45–50 mm deficit beginning in the spring. Recovery of¹⁵N in the plant increased to a maximum of about 20% within 91 days after sowing (DAS 91) and then remained constant. Approximately 16% (0.8 g N m⁻²) of the fertilizer was in the stem and leaves at DAS 91 and this N was subsequently redistributed to the head. At maturity, approximately 75% of the¹⁵N assimilated by the tops was recovered in the grain. Soil N contributed 3.6 g N m⁻² to the head; 2.2 g N m⁻² was remobilized from the stem and leaves, and the balance, approximately 1.4 g N m⁻², was taken up from the soil between DAS 69 to 91. Effects of irrigation treatments on N accumulation were not significant. Residual¹⁵N fertilizer in the soil decreased with time from sowing, and at maturity 40% of the applied N was recovered in the surface 0.15 m.¹⁵N movement to depth was limited and less than 5% of the fertilizer was recovered below 0.15 m. Irrigation had no effect on the¹⁵N recovery at depth. Total recovery of the¹⁵N varied between 60 and 67% and implies that 33–40% was lost from the soil-plant system. The total recovery in the soil and plant was not affected by time or irrigation in the interval DAS 39 to 134. Losses occurred before DAS 39 when crop uptake of N was small and soil mineral N content was high. There was an apparent loss of 1.9 g fertilizer N m⁻² (i.e. 38% of that applied) between DAS 1 and 15. This loss occurred before crop emergence when rainfall provided conditions suitable for denitrification.     

15N-Determined effect of inoculation with N2 fixing bacteria on nitrogen assimilation in Western Canadian wheats

Summary A two-year field study was undertaken using¹⁵N isotope techniques to differentiate between stimulation of N uptake and N₂ fixation in Western Canadian cultivars of spring wheat (Triticum aestivum L. emend Thell) and durum (T. turgidum L. emend Bowden) in response to inoculation with N₂-fixing bacteria. Bacterial inoculation either had no effect or lowered the % N derived from the fertilizer and the fertilizer use efficiency. Despite the depression of fertilizer uptake, inoculants did not alter the relative uptake from soil and fertilizer-N pools indicating that bacterial inoculation did not alter rooting patterns. Nitrogen-15 isotope dilution indicated that N₂ fixation did occur. In 1984, % plant N derived from the atmosphere (% Ndfa) due to inoculation with Bacillus C-11-25 averaged 23.9% while that withAzospirillum brasilense ATCC 29729 (Cd) averaged 15.5%. In 1985, higher soil N levels reduced these values by approximately one-half. Cultivar x inoculant interactions, while significant, were not consistent across years. However, these interactions did not affect cultivars ‘Cadet’ and ‘Rescue’. In agreement with previous results, ‘Cadet’ performed well with all inoculants in both years while ‘Rescue’ performed poorly. Among 1984 treatments, the N increament in inoculated plants was positively correlated with % Ndfa but no such correlation existed in 1985. N₂ fixation averaged over all cultivars and strains was 17.9 and 6.7 kg N fixed ha⁻¹ in 1984 and 1985, respectively. Highest rates of N₂ fixation were estimated at 52.4 kg N ha⁻¹ for ‘Cadet’ in 1984 and 31.3 kg N ha⁻¹ for ‘Owens’ in 1985, both inoculated with Bacillus C-11-25, an isolate from southern Alberta soils. Inoculation with either ofAzospirillum brasilense strain Cd (ATCC29729) or 245 did not result in as consistent or as high N₂ fixation, suggesting that these wheats had not evolved genetic compatability with this exogenous microorganism. These agronomically significant amounts of N₂ fixation occurred under optimally controlled experimental conditions in the field. It is yet to be determined if N₂ fixation would occur in response to bacterial inoculation under dryland conditions commonly occurring in Western Canada. Contribution from Agriculture Canada Research Station, Lethbridge, Alberta, Canada.     

Uptake of nitrogen by flue-cured tobacco during maturation and senescence

A field experiment with flue-cured tobacco,Nicotiana tabacum L., was conducted to estimate the uptake and partitioning of nitrogen during maturation and senescence. On the 83rd day after transplanting (crop day 83), nitrate which had been leached from the plow layer was replaced with an equivalent amount of¹⁵N-labeled nitrate. Plants were harvested at crop day 83, 90, 96, 106, 113, and 127, and each of 11 plant parts was analyzed for nitrogen derived from the soil (NDS) and from the applied¹⁵N-labeled fertilizer (NDF). Equivalent quantities of NDF and NDS were taken up during the initial week after¹⁵N-fertilizer application; in the subsequent 5 weeks, ten times more NDS than NDF were taken up. It appears likely that the leached nitrate (NDS) accumulated below the hard pan where it became available to plants as their roots penetrated this layer via fractures originating from prior deep chiseling. Of the NDF taken up during the initial week, 20% was partitioned to the root and 42%, 24%, 14% respectively, to the upper, middle and bottom node positions (leaves plus stems). The partitioning reflected the respective growth rates of the tissues. Little change in partitioning was evident during the subsequent 5-week period, indicating that little remobilization of NDF from older to younger tissues occurred. In contrast, some remobilization of NDS was apparent between crop day 96 and 106 when the uptake of both NDF and NDS was negligible. During this period root growth was sustained by the apparent transfer of NDS from the root stump and from the adjacent lower leaf and stem tissues. These responses occurred in tobacco grown under higher nitrogen fertility levels than those usually considered optimal for the growth of flue-cured tobacco, but under conditions which are sometimes encountered. Paper no. 11640 of the Journal series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. Part of a thesis submitted by the senior author in partial fulfilment of the requirements of the Ph.D degree.     

Regulation of nitrogen partitioning in field-grown almond trees: Effects of fruit load and foliar nitrogen applications

Two treatments were employed to influence the amount of amino nitrogen (N) transport in phloem. In walnut trees (Juglans regia L.), developing fruit significantly reduced the efflux of foliar-applied ¹⁵N-enriched urea from treated spurs over a 33-day period in comparison with similarly-treated defruited spurs. Those data suggest that local aboveground demand for N influences vascular transport of amino N. In another experiment, a 1% urea solution was applied foliarly to 5-year old `Mission' almond trees [Prunus dulcis (Mill.) D. A. Webb] to increase the concentration of amino N in the phloem. The effect of foliar N treatments on a) the transport and distribution of labelled urea N within the trees over the experimental period and b) the uptake of soil-applied labelled N were determined by replicated whole tree excavation, fractionation into various tree components and mass spectrometric analyses of the ¹⁴N/¹⁵N ratios. Concentrations and composition of amino acids in the phloem and xylem saps of control trees and trees receiving foliar-applied urea were also determined. In foliar urea-treated trees, the amino acid concentrations increased significantly in leaf and bark phloem exudate, within 24 and 96 h, respectively. Foliar-applied urea N was translocated to the roots of almond trees over the experimental period and decreased soil N uptake. The results of these experiments are consistent with the hypothesis that aboveground N demand affects the amount of amino N cycling between shoots and roots, and may be involved in the regulation of soil N uptake. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of Azospirillum Lectins on the Activities of Wheat-root Hydrolytic Enzymes

This work studied the effect of two cell-surface lectins isolated from the nitrogen-fixing soil bacterium Azospirillum brasilense Sp7 and from its mutant defective in hemagglutinating activity, A. brasilense Sp7.2.3, on the activities of α-glucosidase, β-glucosidase and β-galactosidase in the exocomponent, membrane and apoplast fractions of wheat-seedling roots. Lectin (40 μg mL⁻¹) incubation for 1 h of the plant fractions increased the enzymes’ activities; both wild-type and mutant lectins were most stimulatory to the activities of all the exocomponent-fraction enzymes studied and to the apoplast-fraction β-glucosidase. Pretreatment of the lectins with their carbohydrate hapten, L-fucose, lowered the effect. The observed differences in the lectins’ ability to influence enzyme catalytic activity are explained by change in the antigenic properties of the mutant lectin.     

Colonization of wheat by Azospirillum brasilense Cd is impaired by saline stress

The effect of saline stress on the colonization of wheat was analyzed by using Azospirillum brasilense Cd carrying the fusion of the reporter gene lacZ (β-galactosidase) with the N₂ fixation gene promoter nifA. Colonization was also studied by inducing para-nodules on wheat roots using 2,4-D, establishing that these structures acted as bacterium protected niches. Bacteria grown under standard conditions were distributed along the whole root system, except the elongation zone, and colonized the para-nodules. Bacteria experiencing saline stress were mainly localized at the root tips and the lateral roots. In 2,4-D treated plants, most of the bacteria were present around the basal surface of the modified lateral root structures. Using the MPN method, there were not statistical differences between the numbers of control and stressed bacteria. As this method estimates endophytic colonization in contrast with the one using X-gal, which emphasizes colonization on the root surface, both procedures demonstrated to be necessary, concluding that salt treatment reduced surface colonization (X-gal) but not colonization inside the root. The bacterial counts made on inoculated wheat roots indicated higher numbers of both control and stressed bacteria in roots treated with 2,4-D compared with untreated roots. This revised version was published online in June 2006 with corrections to the Cover Date.