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.     

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.     

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     

Nitrogen fixation,biomass production,and nutrient uptake by annual Sesbania species in an alkaline soil

Summary In three field trials conducted during the summer season of 1986, 1987 and 1989 in an alkaline soil, 17 accessions of annual Sesbania spp. were evaluated for nodulation, N₂ fixation (acetylene reduction assay), dry weight of roots and shoots, woody biomass production, and nutrient uptake. At 50 days after sowing all the accessions were effectively nodulated (average 36.4 root nodules plant^-1) with a high nodule score (3.4). There was a lot of variation in nodule volume and mass and in acetylene reduction activity but not in N content (5.2%). N uptake in shoots, roots and nodules averaged 639, 31, and 13 mg plant^-1, respectively, and much of the fixed N remained in shoots. Accessions of S. cannabina complex performed better than others. S. rostrata had poor root nodulation but exhibited excellent stem nodulation (300 nodules plant^-1) even though not inoculated with Azorhizobium sp. Average concentrations of N, P, K, S, Ca, and Mg in the shoots were high, at 3.2, 0.28, 1.5, 0.28, 1.5, and 0.4% respectively, and Na was low (0.15%), reflecting the usefulness of Sesbania spp. as an integrated biofertilizer source. Green matter production was 26.0 Mg ha^-1 (5.9 Mg dry matter) and N uptake was 158 kg ha^-1, 54 days after sowing. Average woody biomass of six accessions at maturity, 200 days after sowing, was high (19.9 Mg ha^-1), showing its potential for shortterm firewood production. Total nutrient uptake for production of woody biomass (200 days of growth) was no more demanding than growing the plant to the green-manuring stage of 50–60 days' growth.     

Asymbiotic dinitrogen fixation by tundra in the Imnavait Creek drainage,Alaska, USA

Summary N₂ fixation by free-living microorganisms was investigated at an intensively studied low Arctic site near Toolik Lake in the northern foothills of the Brooks Range, Alaska, during July 1987. Four characteristic vegetation associations along an elevational gradient were assayed using minimally disruptive in situ acetylene reduction assay methods. The acetylene reduction rates did not differ significantly among vegetation associations. The mean rate for the site was 9.60 mol m^–2 h^–1 or 90 g N m^–2 day^–1, which is within the range of values given for other Arctic and alpine tundra studies. The complex microtopography and resulting patchy distribution of free-living and phycobiont diazotrophs is the most likely cause of the high spatial variability in acetylene reduction activity. Rates were most variable among samples from the lowest position, a riparian site. The potential contribution of heterotrophic diazotrophs was examined through a laboratory enrichment study. Soils from the two lower slope positions showed dramatic responses to added C, suggesting that heterotrophs may contribute fixed N₂ to this system.     

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.     

Effects of phosphate fertilizer on biomass production and N2(C2H2) fixation by pot-grown Ulex gallii Planchon in a forest soil

Summary N₂(C₂H₂) fixation by Ulex gallii Planchon (dwarf or autumn flowering gorse/furze) seedlings was determined following 8 months of growth (December-August) in the glasshouse in a very acid, N- and P-deficient forest soil. Application of Na₂HPO₄·12H₂O or North African ground rock phosphate fertilizer was essential for growth, nodulation and C₂H₂ reduction activity. Overall, both the sodium phosphate and the rock phosphate were equally effective P sources and the maximum acetylene reduction by intact roots was measured as 4.09 and 4.69 mol C₂H₄g^-1 fresh weight nodule h^-1, respectively. Applied NH₄Cl severely inhibited nodulation and restricted acetylene reduction activity but not seedling growth. The results are discussed in relation to the spread of U. gallii in the south of Ireland and its potential as a leguminous nurse crop for Sitka spruce on the very impoverished forest soils of the region.     

Effect of urea,blue green algae and<Emphasis Type="Italic"> Azolla</Emphasis> on nitrogen fixation and chlorophyll accumulation in soil under rice

Nitrogen fixing potential in terms of acetylene reducing activity (ARA) and biomass accumulation (in terms of chlorophyll) were investigated using surface and below-surface soil cores, collected from rice fields 45 and 90 days after transplanting (DAT). Treatments included different levels of urea (30, 60, 90 and 120 kg N ha^–1) in combination with inoculation using blue green algae (BGA) and Azolla biofertilizers. Application of biofertilizers brought about a significant enhancement in chlorophyll accumulation and nitrogenase activity, when measured 45 DAT. Positive effects in below-surface soil cores, on both these parameters as a result of application of biofertilizers further emphasized their contribution to the N economy of rice fields. Plots treated with 30 and 60 kg N ha^–1 along with biofertilizers exhibited the highest percentage increase in terms of algal biomass and ARA, both in surface and below-surface soil cores at 45 DAT. A definite need to examine critically the nature and metabolic activities of below-surface microflora is highlighted through our investigation.     

N2 fixation and growth of legumes as affected by sulphur fertilization

The influence of three sulphur application rates in combination with two nitrogen application rates on N₂ fixation and growth of different legumes was investigated. N was applied as N-labelled ¹⁵NH₄ ¹⁵NO₃. The ¹⁵N isotope dilution technique was used to estimate N₂ fixation. At both N increments dry matter yield was highest with high S supply. Independently of the N supply, the high S application rate resulted in a significantly higher N accumulation, which was mainly caused by a higher N₂ fixation rate. With the grain legumes the weight of nodules was increased by the high S application rate. The higher number of nodules per pot with optimum S supply was the result of a better root growth. Rates of acetylene reduction correlated significantly with S supply.     

Variation in nitrogen fixation among populations ofFrankia sp. andCeanothus sp. in actinorhizal association

Summary Wildland shrub improvement is needed for sound range and disturbed land revegetation practice. The possibility of selecting superior N₂-fixingFrankia-Ceanothus spp. actinorhizal associations was examined. Greenhouse tests were used to expose various soil-borne microsymbiont andCeanothus sp. population accessions in reciprocal combination. The acetylene reduction rate was used as a measure of N₂-fixation capacity. There was no significant interaction between host and microsymbiont regardless of source for all variables measured. The acetylene reduction rate, nodule number and mass, plant biomass, and root: shoot ratio were significantly different among soil sources. The acetylene reduction rate was not significantly different amongCeanothus sp. accessions. Neither was it strongly correlated with other variables. It was concluded that the N₂-fixation rate is more a function ofFrankia sp. than the hostCeanothus sp. in actinorhizal associations. It appears possible to select soil sources with superior N₂-fixing microsymbiont populations.The use of trade or firm names in this paper is for reader information and does not imply endorsement by the U.S. Department of Agriculture of any product or service.This article was produced by government employees and is in the public domain and not subject to copyright. It may be freely reprinted with customary crediting of the source.     

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.     

Effects of inoculation with N2-fixing cyanobacteria on the nitrogenase activity in soil and rhizosphere of wetland rice (Oryza sativa L.)

Summary A greenhouse experiment was conducted with wetland rice (Oryza sativa cv. IR-50) in a clay-loam soil (Fluventic Eutrochrept) to study the effect of cyanobacterial inoculation a mixed culture of Aulosira fertilissima, Nostoc muscorum, Nostoc spp., and Anabaena spp., applied at the rate of 0.15 g (dry weight pot^-1 or 43 kg ha^-1) on acetylene reduction activity in soil and the root system (excised root), and the grain and straw yield. The effects of applying P (40 kg ha^-1), N (60 kg ha^-1), and P+N to the soil were also evaluated. Cyanbacterial inoculation significantly increased (more than 200% on average) photo-dependent acetylene reduction activity in soils, particularly where the indigenous activity was considerably low, i.e. under unfertilized and N-fertilized conditions. The effect of inoculation was prominent at the maximum tillering and grain formation stages of the crop. This inoculation benefit was, however, marginal in P-applied soils (P and P+N), where the indigenous activity was stimulated more than threefold. The inoculation led to a remarkable increase in root-associative acetylene reduction activity after the maximum tillering stage of the crop, particularly with applied N but for other treatments this inoculation effect was not significant. Cyanobacterial inoculation also increased the grain and straw yield of the crop when N was not applied. The grain and straw yield was significantly correlated with the acetylene reduction activity in flooded soils and in the root system during the tillering and maximum tillering stages of rice growth, respectively.     

Effect of bulk density and soil water tension on denitrification in the rhizosphere of spring wheat (Triticum vulgare

Summary Pot experiments were carried out to study the influence of bulk density (D _b), soil water tension (pF) and presence of plants (spring wheat) on denitrification in a low-humus B_t-horizon of a udalf. Pots of only 5-cm depth were found to be most suitable for the experiments when using the acetylene inhibition method. Almost homogeneous soil compaction between 1.1 and 1.6g soil cm^–3 was achieved by a Proctor tamper. Water tensions were adjusted by means of ceramic plates on which negative pressure was applied. No denitrification was detected in unplanted pots. With planted pots and increasing bulk density denitrification increased more in pots with 14-day-old plants than in pots with 7-day-old plants. With 14-day-old plants N₂O emission pot^–1 increased steadily from 2 mol at D _b 1.1 to 8 mol at D _b 1.6, when soil moisture was adjusted to pF 1.5, although root growth was impaired by higher bulk density. From an experiment with different bulk densities and water tensions it could be deduced that the air-filled porosity ultimately determined the rate of denitrification. When low water tension was applied for a longer period, water tension had an overriding effect on total denitrification. Denitrification intensity, however, i.e. the amount of N₂O g^–1 root fresh weight, was highest when low water tension was accompanied by high bulk density. The results suggest that the increase in denitrification intensity at oxygen stress is partly due to higher root exudation.     

A survey of symbiotic nitrogen fixation by white clover grown on metal contaminated soils

There is conflicting evidence about toxic effects of heavy metals in soil on symbiotic nitrogen fixation. This study was set-up to assess the general occurrence of such effects. Soils with metal concentration gradients were sampled from six established field trials, where sewage sludge or metal salts have been applied, or from a transect in a sludge treated soil. Additional contaminated soils were sampled near metal smelters, in floodplains, in sludge amended arable land and in a metalliferous area. Symbiotic nitrogen fixation was measured with ¹⁵N isotope dilution in white clover (Trifolium repens L.) grown in potted soil that was not re-inoculated, and using ryegrass (Lolium perenne L.) as reference crop. The fraction nitrogen in clover derived from fixation (N_dff) varied from 0 to 88% depending on soil. Pronounced metal toxicity on N_dff was only confirmed in a sludge treated soil where nitrogen fixation was halved from the control value at soil total metal concentration of 737 mg Zn kg⁻¹, 428 mg Cu kg⁻¹ and 10 mg Cd kg⁻¹. The N_dff was significantly reduced by increasing metal concentration in soils from two other sites where N_dff was low throughout and where these effects might be attributed to confounding factors. No significant effects of metals on N_dff were identified in all other gradients even up to elevated total metal concentration (e.g. 55 mg Cd kg⁻¹). The variation of N_dff among all soils (n=48), is mainly explained by the number of rhizobia in the soil (log MPN, log (cells g⁻¹ soil)), whereas correlations with total or soil solution metal concentrations were weak (R²<0.25). The is significantly affected by the presence or absence of the host plant at the sampling site. No effects of metals were identified at even at total Zn concentrations of about 2000 mg Zn kg⁻¹, whereas metal toxicity could be identified at lower most probable number (MPN) values. This survey shows that the metal toxicity on symbiotic nitrogen fixation cannot be generalized and that survival of a healthy population of the microsymbiont is probably the critical factor.     

Influence of phosphorus or phosphorus-potassium fertilization on biomass and dinitrogen fixation of the stem-nodulating green-manure legume Sesbania rostrata in different marginally productive wetland rice soils

The performance of Sesbania rostrata varies widely from site to site. This makes it difficult to predict the N yield and biomass of this plant in marginally productive soils, and to arouse the interest of farmers in green manure technology. Three consecutive pot experiments were conducted in a greenhouse at the International Rice Research Institute (IRRI) to evaluate growth, nodulation, N₂ fixation (C₂H₂ reduction assay and ¹⁵N dilution method), and N yield of 6-week-old S. rostrata on 13 physicochemically different wetland rice soils of the Philippines and on three artificial substrates. The performance of S. rostrata on the unfertilized controls was compared with two fertilizer treatments containing either P (100 mg P kg^-1 dry soil) or P+K (100 mg P kg^-1 and 200 mg K kg^-1 dry soil). In the control soils and substrates, the N yield of S. rostrata varied between 20 and 470 mg N per pot, with the N rate from N₂ fixation ranging between 0 and 95%. In three of the nutritionally poor soils even Mn toxicity symptoms apparently occurred with S. rostrata. P application alleviated these symptoms and increased the overall N yield considerably, mainly through increased biological N₂ fixation. An additional increase in N yield was obtained by the PK treatment. Multiple regression analysis between soil characteristics and the N yield of S. rostrata showed that the original level of P (Olsen-extracted) and Mn in the soil accounted for 73% of the variance in biomass production by S. rostrata among the unfertilized soils and substrates.     

Nitrogen fixation in six forage legumes in Mediterranean central Chile

Abstract

The contribution of biological nitrogen fixation (BNF) to the N nutrition of six annual forage legumes, subterranean clover (Trifolium subterraneum), burr medic (Medicago polymorpha), balansa clover (Trifolium michelianum), Persian clover (Trifolium resupinatum), yellow serradela (Ornithopus compressus), and pink serradela (Ornithopus sativus) was evaluated by the ¹⁵N natural abundance technique, using four grass species (Briza máxima, Bromus mollis, Hordeum berteroanum, Avena barbata) and two composite species (Leontodon leysseri and Hedipnois cretica) as reference plants. An additional objective was to determine whether alternative legume species to those in common use (T. subterraneum and M. polymorpha) in the area, could improve BNF. The field studies were conducted in two edaphic conditions, granitic (Entisol) and clay (Vertisol) soil, located in Cauquenes, VII Region, in the sub-humid Mediterranean zone of Chile. In the granitic soil the percentages of N derived from fixation were high in all species (74 to 94%); yellow serradela cv. Tauro presented the greatest N content in dry matter and N fixation, equivalent to 91 kg N ha^?1. In contrast, pink serradela cv. Cádiz and subterranean clover cv. Gosse presented the lowest N fixation. In the clay soil, under periodically waterlogged conditions, balansa clover cv. Paradana and persian clover cv. Prolific had high percentage values of BNF (>95%) and fixed more N (100.2 and 82.5 kg N ha^?1, respectively) than burr medic and subterranean clover cv. Gosse. The present study allowed the identification of new germplasm of high capacity of N fixation which is an additional criterion for selecting species for infertile and waterlogged soil conditions in the Mediterranean area of Chile.     

Nitrogen fixation efficiency,interspecies N transfer,and root growth in barley-field pea intercrop on a Black Chernozemic soil

Summary Barley-field pea intercrops have been shown to increase N yield when grown under cryoboreal subhumid conditions. In this study, we extended previous research by testing the hypotheses that (1) the intercropped field pea fixes a greater proportion of its shoot and root N than does sole-cropped field pea; (2) N is transferred from the annual legume to the cereal during the growing season; and (3) root production is greater under intercropped than sole-cropped conditions. Unconfined microplots seeded to barley, field peas, or a barley-field pea intercrop were fertilized with N at 10 kg ha^-1 as (NH₄)₂SO₄ (5.21 atom % ¹⁵N excess). Both the intercropped and sole-cropped barley derived more than 93% of their N from the soil. In contrast, 40% of N in the intercropped field pea was derived from soil. This study provided no evidence for transfer of N from the legume to the cereal. On average, the proportion of N derived from air by both pea intercrops was 39% higher than that derived by the sole-cropped pea. Root length determined by a grid intersection method following digitization using an image analyzer tended to be higher under intercropping than in sole crops. We conclude that even on fertile soils benefits may accrue from annual intercropping that includes a legume. The benefits arise from (1) increased N production, (2) greater N-fixation efficiency, and/or (3) more shoot and root residue-N mineralization for subsequent crops.     

Nitrogen fixation and balance studies of rice soil

Summary A nitrogen balance study conducted in ceramic pots under net house conditions for four seasons showed that flooded rice soil leaves a positive nitrogen balance (N increase) in soil after rice cropping in both fertilized and unfertilized soil. Recovery of nitrogen from rice soil was more than its input in unfertilized soil, but it was reverse in fertilized soil. Incorporation of Azolla or BGA twice as basal and 20 days after transplanting (DAT) alone or in combination showed higher nitrogen balance and N₂-fixation (N gain) in soil than in that where it was applied once either as basal or 20 DAT. Planted soil showed more N₂-fixation than that of fallow rice, and flooded soil fixed more nitrogen in comparison to non-flooded soil in light but less in dark. Soil exposed to light fixed more nitrogen than that of unexposed soil in both flooded and non-flooded conditions.     

Nitrogen fixation in soybeans as influenced by cultivar and Rhizobium strain differences

Summary The effect of soybean variety and Rhizobium strain, as well as the level of nitrogen fertilizer, were tested in a field experiment in Greece. Three nodulating varieties were used and one non-nodulating variety as control, with two Rhizobium strains, one commercially available and one isolated from the soil of the experimental site. Nitrogen fertilizer labeled with ¹⁵N enabled the nitrogen derived from the atmosphere (Ndfa), from the soil (Ndfs), and from the fertilizer (Ndff) to be estimated. Statistical analyses showed that the combined variety-strain effect was responsible for most of the variation observed in all parameters, either measured directly or calculated, while the nitrogen level had no effect. The locally isolated strain generally performed equally as well as the commercial one. With the highest yielding variety in particular, the nitrogen-fixing efficiency of soybean nodules, expressed as plant total nitrogen in excess of that in the non-nodulated control in relation to nodule dry weight, was even superior. Taking into account the low fertilizer recovery by plants, it is suggested that adapted cultivars properly inoculated can fix most of the nitrogen they need for high yields. This is of great economic importance for crops with high nitrogen-fixing efficiency, like the soybeans used in this work, which can fix more than 200 kg N/ha, as ¹⁵N dilution and nitrogen difference evaluations have confirmed. The local cultivars in this connection were superior to the United States cultivar, while the difference between cultivars in symbiotic performance were not due to the ability to nodulate, but rather to a much higher nodule-specific efficiency. The commercially available strain was found to produce generally more nodules and the locally isolated strain produced less but larger and more efficient nodules. Finally, the grains from the nodulated plants contained significantly smaller quantities of Fe, Ni, Cu, Zn, and especially Mo compared with non-nodulated plants, as determined by X-ray fluorescence techniques.     

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.