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.     

Molybdenum reserves of seed,and growth and N2 fixation by Phaseolus vulgaris L.

Summary Plants grown from seed with high (1.5–7.3 g Mo seed^-1) and low (0.07–1.4 g Mo seed^-1) Mo contents were grown in the presence and absence of Mo in growth media (perlite) or in a flowing-solution culture, in a controlled environment. Neither the high (1.5 g Mo seed^-1) nor the low (0.1 g Mo seed^-1) Mo content in seed from a small-seeded genotype (BAT 1297) was able to prevent Mo deficiency (reduced shoot, root and nodule dry weight, N₂ fixation and seed production) in growth media without an external supply of Mo, whereas both the high (7.3 g Mo seed^-1) and the low (0.07 g Mo seed^-1) contents in seed were able to prevent Mo deficiency in a large-seeded genotype (Canadian Wonder). Responses to Mo treatment by the Two genotypes were inconsistent between the growth media and solution culture experiments. Seed with a large Mo content (3.5 g Mo seed^-1) from the Canadian Wonder genotype was unable to prevent Mo deficiency (reduced shoot and nodule dry weight and N₂-fixation) in a solution culture without an external source of Mo, whereas both the large (1.7 g Mo seed^-1) and the small (0.13 g Mo seed^-1) contents in seed prevented a deficiency in BAT 1297. Growing plants from seed with a small Mo content, without additional Mo, reduced the seed Mo content by 83–85% and seed production by up to 38% in both genotypes. Changes in seed size and increases in shoot, root and nodule dry weight occurred, but varied with the genotype and growth conditions. These effects were also observed in some cases where plants were grown with additional Mo, demonstrating that the amount of Mo in the seed sown can influence plant nutrition irrespective of the external Mo supply. Nodule dry weight, total N content of shoots and seed production were improved by using seed with a small Mo content (1.64–3.57 g Mo seed^-1) on acid tropical soils in Northern Zambia. Plants of both the large- and small-seeded genotypes grown from seed with a small Mo content (<1.41 g Mo seed^-1) had a smaller nodule weight, accumulated less N and produced less seed. The viability of seed with a small Mo content was lower (germination up to 50% less) than that of seed with a large Mo content.     

Comparative growth and symbiotic performance of four Acacia mangium provenances from Papua New Guinea in response to the supply of phosphorus at various concentrations

Plant growth performance, the P content in root and nodule tissues, and nodulation and N₂-fixing ability were studied in four provenances of Acacia mangium from Papua New Guinea following different levels of P fertilizer application. A. mangium did not seem to need high levels of P for growth and N₂ fixation. The response by this leguminous tree to the P supply varied significantly according to provenance and to P concentrations in the culture solution. The provenances of A. mangium were classified into three types according to their P response: (1) Growth performance, nodulation, and N₂ fixation of plants were stimulated as concentrations of P increased (provenance PH 482); (2) the maximal effect of P on plant growth was found only at P concentrations higher than 500 M (provenance PH 484); and (3) the plant response to P fertilization was low, even with nutrient solutions containing P concentratins higher than 500 M (provenances PH 483 and PH 485). Provenance PH 483 was distinguished by its low nodulating ability. However, this provenance grew well, probably because of its high N₂ fixation efficiency as expressed by specific acetylene reduction activity and its high P content in nodule tissues. Therefore, in certain cases, these two parameters may be useful criteria in selecting leguminous plants for field use. Statistical analyses of the study results showed that the effect of the factor P supply on N₂ fixation efficiency and nodule development was only significant at P concentrations lower than 250 M whereas the effect of the factor plant provenance was significant regardless of the P concentration used. This observation emphasizes the value of provenance screening in the identification of plants for use in a wide range of soil types.     

Quantitative effects of field water deficits on N2(C2H2) fixation in selected legumes grown in the Indian desert

Summary The nitrogenase activity of irrigated and rainfed plants of mung bean, cluster bean and moth bean was studied throughout the growth period in order to estimate the reduction in the potential nitrogen fixation (C₂H₂ reduction) rate due to field water deficits. Nitrogenase activity followed a similar trend in all crops and was dependent on both plant ontogeny and soil moisture levels. The loss of activity due to water deficits varied from 13% to 100% at different growth stages and was related to the plant water potential. The specific activity was directly correlated with the plant water potential under both the treatments. The average loss of nitrogen fixation rate during the season did not differ markedly among crops. There was an accumulation of ureides in the nodules with increasing field moisture stress in mung bean and moth bean while no such effect was found in cluster bean. The significance of these results is discussed in the N-economy of these legumes grown in the drought-prone areas of the Indian desert.     

Phosphorus requirements and nitrogen accumulation by N2-fixing and non-N2-fixing leguminous trees growing in low P soils

The variation in P uptake and use efficiency and N accumulation by Gliricidia sepium (N₂-fixing tree), Senna siamea and S. spectabilis (leguminous non-N₂-fixing trees) were examined in the field at Fashola (savanna zone), southwestern Nigeria, using four P rates, 0, 20, 40 and 80 kg P ha^-1. Growth of G. sepium and S. spectabilis responded to P application at 24 weeks after planting (WAP) and average yield increases of 58% and 145% were observed by the application of 40 kg P ha^-1 for the two species, respectively. Such a P response was not found in S. siamea at 24 WAP and for any of the species at 48 WAP. G. sepium accumulated more P (on average 162%) than S. siamea and S. spectabilis at 24 WAP and had greater root length and a higher percentage of mycorrhizal infection. However, at 48 WAP S. siamea had 2.5 times more P than G. sepium. Differences in the physiological P use efficiency (PPUE) between G. sepium and the non-N₂-fixing trees were significant at the 0 P level, being higher for S. siamea (average, 0.61 g shoot mg^-1 P) than for G. sepium (0.27 g shoot mg^-1 P). G. sepium had a consistently lower atom % ¹⁵N than S. spectabilis, while that of S. siamea for most of the time did not differ from that of G. sepium. The reference plant affected N₂ fixation extimates, with negative values and a higher variability (CV 60%) associated with S. siamea than with S. spectabilis (CV<20%). Consequently, S. spectabilis was selected as a better reference plant for measuring N₂ fixation in G. sepium. G. sepium fixed on average 35% and 54% of its N at 24 and 48 WAP, respectively. Except at the lowest P rate, percentage and amount of N fixed were not generally enhanced by P application.     

Chemical composition of cultures and natural samples of N2-fixing blue-green algae from rice fields

Summary Laboratory cultures, soil cultures, and natural samples of N₂-fixing blue-green algae (BGA) from rice fields were analyzed for dry matter, ash, N, C, P, and a few other constituents.Results show a very large variability of the composition. Dry matter contents ranged from 0.28% to 13.6% (average 3.3%). Ash contents ranged from 15.6% to 71.3%. Nitrogen contents ranged from 1.9% to 11.8% on an ash-free basis (average 6%). Carbon content was less variable, ranging from 37% to 72% and averaging 43.7%.A decrease in N and pigment contents, and an increase in reducing sugars, was observed in aging laboratory cultures.Large differences in composition were observed between field samples and material grown in artificial medium. Soil-grown BGA and field samples were characterized by very high ash contents, N contents lower than those in laboratory cultures, and P deficiency.Extrapolation from (1) average dry matter, ash, and N contents and (2) records of BGA biomass in rice fields indicates that an algal bloom has a potentiality of about 15–25 kg N per hectare and that a BGA biomass of agronomic significance is visible to the naked eye.     

Hydrogen oxidation in soil following rhizobial H2 production due to N2 fixation by a Vicia faba-Rhizobium leguminosarum symbiosis

Summary The rate of H₂ release from broad beans (Vicia faba) infected with Rhizobium leguminosarum Hup^- was much faster than from beans infected with the Hup⁺ strain. Acetylene reduction and H₂ release were abolished by cutting the plants down, by incubation in darkness, or after the addition of ammonium, indicating that the H₂ was released by N₂-fixing bacterial symbionts. In laboratory cultures using non-sterile soil, the bean plants released H₂ until an equilibrium between H₂ production and H₂ oxidation was reached. The H₂ equilibrium concentration was higher in Hup^--infected bean cultures (about 3 ppm H₂ in the gas phase) than in Hup⁺-infected cultures (0.3 ppm H₂) because of the higher H₂ production. The H₂ release from Hup^--infected bean cultures in sterile soil did not reach equilibrium. An equilibrium occurred, if Knallgas bacteria were added. However, the equilibrium value was higher (13 ppm H₂) than in non-sterile soil, which seemed to be more efficient at H₂ oxidation. The Knallgas bacteria exhibited a relatively high K _m for H₂ (> 1300 ppmv H₂); this activity was observed in unplanted non-sterile soil, and in nonsterile soil planted with Hup⁺-infected beans or planted with Hup^--infected beans which had been cut down before being assayed. All these soils also showed a second, low-K _m (<50 ppm) level of H₂ oxidation activity, which was presumably due to abiontic soil enzymes. In contrast, only one level of activity, which had an intermediate K _m (about 200 ppm H₂), was observed when the soil was planted with Hup^--infected beans. The origin of this activity, which was only observed in the presence of intact, H₂-producing beans, is still unknown.     

Effect of nitrogen levels and Rhizobium strains on symbiotic N2 fixation and grain yield of Phaseolus vulgaris L. genotypes in normal and saline-sodic soils

Summary Following screening, selection, characterization, and symbiotic N₂ fixation with 12,5, 25.0, and 40.0 mg N kg^–1 in normal and saline-sodic soils, only two Phaseolus vulgaris genotypes (HUR 137 and VL 63) and two Rhizobium spp. strains (ND 1 and ND 2) produced maximum nodulation, nitrogenase activity, plant N contents, and grain yields in saline-sodic soil, with 12.5 mg N kg^–1, compared with the other strains. However, interactions between strains (USDA 2689, USDA 2674, and ND 5) and genotypes (PDR 14, HUR 15, and HUR 138) were significant and resulted in more nodulation, and greater plant N contents, nitrogenase activity, and grain yields in normal soils with 12.5 mg N kg^–1 compared with salt-tolerant strains. Higher levels of N inhibited nodulation and nitrogenase activity without affecting grain yields. To achieve high crop yields from saline-sodic and normal soils in the plains area, simultaneous selection of favourably interacting symbionts is necessary for N economy, so that bean yields can be increased by the application of an active symbiotic system.     

Characterization of denitrification and net N2O-reduction properties of novel aerobically N2O-reducing bacteria

Nitrous oxide, one of the earth-warming and ozone-destructing gases, is produced through either nitrification or denitrification depending on the O₂ availability in soil. Aerobically denitrifying bacteria express denitrification tract even under the gas phase containing O₂ at the ambient air level. The net reduction of exogenous N₂O by novel aerobically denitrifying bacteria were studied. We carried out two different isolation strategies in the primary screening. One was to select isolates of interest out of periplasmic nitrate reductase-dependent denitrifying bacteria in a eutrophic condition. The other was to use diluted nutrient agar to allow the formation of colonies of diverse bacteria. Among aerobically denitrifying bacteria, those which showed net aerobic N₂O reduction were only minor populations. As a result, eight isolates belonging to Proteobacteria were obtained from soil and cow manure. The denitrification and net N₂O reduction properties of the three representative isolates, Pseudomonas sp. CM1, Thauera sp. PM2 and Paracoccus denitrificans 96, were determined separately by the acetylene inhibition method after exposure to aerobic or low O₂ conditions, a 24 h starvation prior to the determination of the aerobic activity and inoculation to a cow manure-amended sterile soil. The phenotype inversion from net N₂O-reducing to N₂O-emitting, and vice versa, attested to the fact that activity of the N₂O-producing and -reducing steps changed in different intensities to each other. The activity values and the direction of activity changes varied among the isolates. When they were inoculated in a sterilized soil microcosm at 40% maximum water holding capacity, the denitrification and the N₂O-reducing activities were comparable with or, in some cases, facilitated more than those determined under the low-O₂ condition. It is possible that these isolates sensed the O₂ deficiency even in such a relatively dry condition. Pseudomonas sp. CM1 was unique because it lacked nitrate reducing activity and acted as a net aerobic N₂O reducer.     

The effects on N2 fixation (C2H2 reduction), bacterial population and rice plant growth of two modes of straw application to a wetland rice field

Summary The effects of incorporation and surface application of straw to a wetland rice field on nitrogen fixation (C₂H₂ reduction), bacterial population and rice plant growth were studied. Rice straw (5 t ha^–1) was chopped (10- to 15-cm pieces) and applied to the field 2 weeks before transplanting IR42, a long-duration variety, and IR50, a short-duration variety. The acetylene-reducing activity (ARA) of IR42 and IR50 measured at heading stage for 3 consecutive days showed significantly higher ARA in IR42 as a result of the 2 straw application methods. Mostly up to 20 days after straw surface application and incorporation, the dark ARA in the soil, total and N₂-fixing heterotrophs, and photoorganotrophic purple nonsulphur bacteria (POPNS) in the soil and in association with degrading straw were stimulated. Higher bacterial populations were associated with straw on the surface than with straw incorporated. The POPNS counts, in particular, were increased hundreds fold in the surface-applied straw treatment. Straw applications also increased the root, shoot and total plant biomass at heading stage and the total dry matter yield at harvest in both varieties. The data show the potentials of straw as a source of substrate for the production of microbial biomass and for the non-symbiotic N₂ fixation to improve soil fertility and plant nutrition.     

Rapid and reversible nitrate inhibition of nodule growth and N2 fixation activity in soybean (Glycine max (L.) Merr.)

Nodulated soybean (Glycine max. (L) Merr. cv. Williams) plants were hydroponically cultured, and various combinations of 1-week culture with 5 or 0 mm nitrate were applied using 13-d-old soybean seedlings during three successive weeks. The treatments were designated as 0-0-0, 5-5-5, 5-5-0, 5-0-0, 5-0-5, 0-5-5, and 0-0-5, where the three sequential numbers denote the nitrate concentration (mm) applied in the first-second-third weeks. The size of the individual nodule was measured periodically using a slide caliper. All the plants were harvested after measurement of the acetylene reduction activity (ARA) at the end of the treatments. In the 0-0-0 treatment, the nodules grew continuously during the treatment period. Individual nodule growth was immediately suppressed after 5 mm nitrate supply. However, the nodule growth rapidly recovered by changing the 5 mm nitrate solution to a 0 mm nitrate solution in the 5-0-0 and 5-5-0 treatments. In the 5-0-5 treatment, nodule growth was completely inhibited in the first and the third weeks with 5 mm nitrate, but the nodule growth was enhanced in the second week with 0 mm nitrate. The nodule growth response to 5 mm nitrate was similar between small and large size nodules. After the 5-5-5, 5-0-5, 0-0-5, and 0-5-5 treatments, where the plants were cultured with 5 mm nitrate in the last third week, the ARA per plant was significantly lower compared with the 0-0-0 treatment. On the other hand, the ARA after the 5-0-0 and 5-5-0 treatments was relatively higher than that after the 0-0-0 treatment, possibly due to the higher photosynthate supply associated with the vigorous vegetative growth of the plants supplemented with nitrate nitrogen. It is concluded that both soybean nodule growth and N₂ fixation activity sensitively responded to the external nitrate level, and that these parameters were reversibly regulated by the current status of nitrate in the culture solution, possibly through sensing of the nitrate concentration in roots and / or nodules.     

Effect of an increasing carbon: nitrate-N ratio on the reliability of acetylene in blocking the N2O-reductase activity of denitrifying bacteria in soil

Summary In model experiments with a silty loam soil the effect of different C : NO _inf3 ^sup- -N ratios on the reliability of C₂H₂ (1% v/v) in blocking N₂O-reductase activity was examined. The soil was carefully mixed with different amounts of powdered lime leaves (Tilia vulgaris) to obtain organic C contents of about 1.8, 2.3, and 2.8%, and of NO _inf3 ^sup- solution to give C : NO _inf3 ^sup- -N ratios of 84, 107, 130, 156, 200, and 243. The soil samples were incubated in specially modified anaerobic jars (22 days, 25°C, 80% water-holding capacity, He atmosphere) and the atmosphere was analysed for N₂, N₂O, CO₂, and C₂H₂ by gas chromatography at regular intervals. Destruction jars were used to analyse soil NO _inf3 ^sup- , NH ₄ ⁺ and C. The results clearly showed that N₂O-reductase activity was completely blocked by 1% (v/v) C₂H₂ only as long as NO _inf3 ^sup- was present. In the presence of C₂H₂, NO _inf3 ^sup- was apparently entirely converted into N₂O. The C₂H₂ blockage of N₂O-reductase activity ceased earlier in soils with a wide C : NO _inf3 ^sup- -N ratio (156, 200, and 243) than in those with closer C : NO _inf3 ^sup- -N ratios (84, 107, and 130). As soon as NO _inf3 ^sup- was exhausted, N₂O was reduced to N₂ in spite of C₂H₂. The wider the C : NO _inf3 ^sup- -N ratio, the earlier the production of N₂ and the less the reliability of the C₂H₂ blockage. In the untreated control complete inhibition of N₂O-reductase activity by C₂H₂ lasted for 7–12 days. In the field, estimates of total denitrification losses by the C₂H₂ inhibition technique should be considered reliable only as long as NO _inf3 ^sup- is present. Consequently, NO _inf3 ^sup- monitoring in the field is essential, particularly in soils supplied with easily decomposable organic matter.     

Application of N2/Ar ratio method for N2 fixation studies in submerged soil

Abstract

Recently there has been developments in the measurement of N₂ fixation due mainly to the C₂H₂ reduction method (1). This method, however, has several disadvantages, especially for submerged soil, and the estimated amount of fixed N₂ on the basis of the C₂H₂ reduction activity is not very reliable. The tracer ¹⁵N₂ technique which gives a reliable estimation of the fixed N₂ is too expensive for common use. Development of an alternative method suitable for submerged soil would therefore be desirable. The present authors expected that the measurement of the ratio N₂/Ar in the soil solution might provide advantages for the estimation of the fixed N₂ in submerged soil.     

A yield-independent, 15N-isotope dilution method to estimate legume symbiotic dependence without a non-N2-fixing reference plant

The proportional contribution of atmospheric N₂ to the N nutrition of lupin (P _atm) was estimated in a field experiment following addition of NH₄Cl of KNO₃ to unconfined microplots (1.5 m²) at 2.5 g N m^-2 (10 atom% ¹⁵N). The integrated ¹⁵N enrichment, or mean pool abundance, of nitrate extracted from 0- to 15-cm samples taken under the lupin crop on eight occasion between 28 and 190 days after sowing was used as the reference criterion to estimate P _atm by the ¹⁵N-isotope dilution technique. Estimates of P _atm were similar to those obtained using canola as a non-fixing reference plant, but were higher than estimates obtained using a yield-dependent model. Use of mean pool abundance obviates the need for a non-fixing reference plant, and the frequent sampling and isotope-ratio analysis of the legume biomass required with the yield-dependent model is unnecessary. However, further work is needed to validate a sampling strategy commensurate with the growth of the legume roots.     

Evaluation of N2 fixation by applying 15N labeled plant material and ammonium sulfate

Effect of different ¹⁵N labeled sources on the estimation of N₂ fixation was investigated. The combination of ¹⁵N labeled ammonium sulfate, ¹⁵N labeled plant material, and ¹⁵N labeled ammonium sulfate with unlabeled plant material, was examined in pot experiments. Two cultivars of soybean (Glycine max) and one of mungbean (Vigna radiata) were used. No significant difference was observed among the treatments for the estimation of N₂ fixation. This was due to the homogeneity and stability of the ¹⁵N abundance in soil which resulted in a similar N uptake from the soil by the N² fixing and reference crops. The plant yield, total N uptake and amount of N₂ fixed were higher in the Yellow Soil than in the Andosol. The amount of N₂ fixed was strongly influenced by the plant growth and consequently it affected the plant yield. The slow decomposition of plant material in the Andosol resulted in a low yield in both the N₂ fixing and reference crops. Thus, the artificial decrease of the available N content in soil, by application of plant material, did not stimulate N, fixation but suppressed plant growth and N₂ fixation.     

Enhanced denitrification in plots of N2-fixing faba beans compared to plots of a non-fixing legume and non-legumes

Denitrification rates were studied using the C₂H₂ inhibition technique in a 2-year field experiment within plots of nodulated and non-nodulated faba beans, ryegrass, and cabbage. Denitrification rates ranged from 14.40 to 0.02 ng N₂O–N g^–1 soil dry weight h^–1. Mean denitrification increased fourfold in plots of N₂–fixing Vicia faba compared to non-nodulated V. faba mutant F48, Lolium perenne, and Brassica oleracea. The results with and without C₂H₂ treatment indicate that in the field the major part of this enhanced denitrification led to the endproduct N₂ rather than to the ozone-degrading N₂O. Higher denitrification rates of plots with N₂–fixing plants in September seemed to be caused by an increase in soil NO _inf3 ^sup- of about 20 kg ha^–1 found between July and August. Soil NO _inf3 ^sup- and soil moisture explained 67% of the variation in denitrification rates of the different soil samples over the growing seasons in the 2 years. Soil moisture explained 44% of the variation for soil planted with N₂–fixing plants and 62% for soil planted with non-fixing plants. Positive exponential relationships were obtained between denitrification rates and soil nitrate (r=0.71) and soil moisture (r=0.82).     

Effect of inoculation with Azotobacter chroococcum on nitrogenase activity of Zea mays roots grown in agricultural soils under aseptic and non-sterile conditions

Summary Inoculated and non-inoculated seedlings of Zea mays were grown in agricultural soils under aseptic and non-sterile conditions. Acetylene reduction activity and microbial counts were determined after 7 and 30 days of growth. Irrespective of the soil type Azotobacter spp. were commonly isolated under maize cultivation. Inoculation of agricultural soils with a suspension of A. chroococcum led to an increase in Azotobacter numbers, although this effect diminished with time. Nitrogenase activity was detected on maize roots and increased in response to the inoculation with A. chroococcum, showing that this associative growth could be of primary importance for the plant. The results of assays for acetylene reduction activity indicated that the nitrogenase activity was associated only with the root systems.     

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.     

Nitrogen fixation and soil N level during maturation affect the contents of storage compounds of soybean seeds

We compared the concentrations and contents of protein and oil in mature seeds from nodulated and non-nodulated soybean plants grown on soils with four different N levels during maturation. We observed a positive correlation between the contents of protein and oil in seeds from nodulated plants. Seeds from nodulated plants grown on urea-treated soil showed higher protein and lower oil contents than those from plants grown on soil treated with coated slow release N fertilizer (LP-100). Contents of these compounds in seeds from nodulated plants grown on LP-100 soil were almost the same as those from non-nodulated plants on the same soil. These observations indicated that N economy in roots during seed maturation affects the contents of storage compounds. We suggested that the control of the N₂ fixation activity of soybean plants and management of soil N level during seed maturation are important to determine the contents of protein and oil in seeds.     

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.