Field performance of three Bradyrhizobium japonicum strains with two soybean (Glycine max. L.) cultivars

Summary A field experiment was condutced in a clay loam soil to study the performance of three Bradyrhizobium japonicum strains; USDA 110, USDA 138 and TAL 379, in relation to their N₂-fixing potential and competitiveness on two soybean cultivars (Clark and Calland). Inoculation of soybean cultivars with these strains, either singly or in combination, induced significant increases in plant dry weight, N₂ fixation and seed yields. However, no significant differences were found between the rhizobial strains and/or their mixtures in N₂ fixation and increased seed yield for both cultivars. The two soybean cultivars gave similar responses to inoculation. No significant differences in seed yield were observed between Clark and Calland cultivars. The interaction between inoculant strain and soybean cultivar was not significant. The competition between strains for nodulation was assessed. Strain USDA 110 was the most competitive, followed by USDA 138. Strain TAL 379 was always less competitive on both cultivars. The incidence of double-strain occupancy of nodules varied from 8% to 40%.     

Nitrogen fixation capacity and nodule occupancy by Bradyrhizobium japonicum and B. elkanii strains

 In a previous study soybean Bradyrhizobium strains, used in Brazilian studies and inoculants over the last 30 years, and strains adapted to the Brazilian Cerrados, a region frequently submitted to environmental and nutritional stresses, were analyzed for 32 morphological and physiological parameters in vivo and in vitro. A cluster analysis allowed the subdivision of these strains into species Bradyrhizobium japonicum, Bradyrhizobium elkanii and a mixed genotype. In this study, the bacteria were analyzed for nodulation, N₂ fixation capacity, nodule occupancy and the ability to increase yield. The goal was to find a relationship between the strain groups and the symbiotic performance. Two strains of Brazilian B. japonicum showed higher rates of N₂ fixation and nodule efficiency (mg of N mg^–1 of nodules) under axenic conditions. These strains also showed greater yield increases in field experiments when compared to B. elkanii strains. However, no differences were detected between B. japonicum and B. elkanii strains when comparing nodule occupancy capacity. The adapted strains belonging to the serogroup B. elkanii SEMIA 566, most clustered in a mixed genotype, were more competitive than the parental strain, and some showed a higher capacity of N₂ fixation. Some of the adapted strains, such as S-370 and S-372, have shown similar N₂ fixation rates and nodulation competitiveness to two Brazilian strains of B. japonicum. This similarity demonstrates the possibility of enhancing N₂ fixing ability, after local adaptation, even within B. elkanii species. Differences in the DNA profiles were also detected between the parental SEMIA 566 and the adapted strains by analyses with the ERIC and REP-PCR techniques. Consequently, genetic, morphological and physiological changes can be a result of adaptation of rhizobia to the soil. This variability can be used to select strains capable of increasing the contribution of N₂ fixation to soybean nutrition. Received: 28 May 1997     

Microbial inoculation of Rhizobium and phosphate-solubilizing bacteria along with inorganic fertilizers for sustainable yield of soybean [Glycine max (L.) Merrill]

A field experiment was conducted to assess the effect of microbial inoculants and inorganic fertilizers for sustaining the yield of soybean. Application of 100% recommended dose of fertilizer (RDF) gave significantly highest yield (2433 kg ha^?1) over 75% RDF (2317 kg ha^?1) and without RDF (2205 kg ha^?1). Seeds inoculated with Rhizobium (Bradyrhizobium japonicum) and phosphate-solubilizing bacteria (2480 kg ha^?1) gave significantly highest soybean yield over without inoculation (2191 kg ha^?1). Rhizobium and phosphate-solubilizing bacteria with 100% RDF (2674 kg ha^?1) gave significantly highest seed yield than rest of the treatment combinations. Root nodules and their dry weight were remained un-influenced due to fertilizer levels, whereas in bio-fertilizers, it was significantly higher with Rhizobium inoculation (24.3 and 408 mg, respectively) followed by dual inoculation of Rhizobium and PSB. 100% RDF and dual inoculation with Rhizobium and PSB earned Rs. 47916/- and Rs. 51182/- net returns per ha, respectively.     

Survival and symbiotic properties of Bradyrhizobium japonicum in the presence of thiram: isolation of fungicide resistant strains

The fungicide thiram, widely used as a chemical seed protectant, induces a strong inhibition of primary nodulation in the crown zone of soybean roots. The present work reports on the isolation of Bradyrhizobium japonicum strains resistant to thiram, some of which (T3B, A86 and A2) maintained their capacity for nodulation and were still efficient symbionts, but some (A1, C1 and C6) lost the ability to stimulate nodulation. Characterization tests such as growth at different pH, denitrifying ability, salt tolerance, production of siderophores and phosphate solubilization were performed on the resistant strains. Inoculants produced from these strains could be appropriate for use with thiram-treated seeds, without causing a loss of bacteria viability. Received: 16 September 1996     

Hydrogen metabolism and nitrogen fixation of soybean (Glycine max. L.) inoculated with different strains ofRhizobium spp.

Summary Hydrogenase activities and N₂-fixing capacities of soybean nodules (Glycine max. cv. Hodgson), inoculated with strains ofBradyrhizobium japonicum andRhizobium fredii from different geographical regions, were measured after 35 days of culture under controlled conditions. Of the strains tested, 47% induced nodules with bacteroids which recycled H₂. The data obtained suggest that H₂-recycling ability is not a major factor influencing early N₂-fixation which depends essentially on the precocity and intensity of the initial nodulation.     

Effects of Rhizobium inoculation and magnesium application on growth and nodulation of soybean (Glycine max L.)

Abstract

Magnesium (Mg) deficiency is one of the major nutritional problems in tropic and subtropic areas, where the most soils are acidic. In this study, the effects of Mg application and Bradyrhizobium inoculation on growth, nodulation, symbiotic nitrogen (N) fixation as well as N nutrition status in soybean (Glycine max L.) were investigated in hydroponics under greenhouse conditions. With the increase of Mg up to 0.75?mM at low N and up to 0.5?mM at high N solutions, the dry weights of shoots, roots, and pod grain yield in soybean were increased, while further increase in Mg supply inhibited soybean growth. The availability of Mg was found to entail an improved uptake of N by plants and nodulation process in the root by Bradyrhizobium. Inoculation with rhizobial inoculants not only formed many nodules, but also increased soybean shoot, root biomass and yield, as well as plant N nutrient status.     

Variation of nitrogen accumulation and yield in response to phosphorus nutrition of soybean (Glycine max L. Merr.)

Phosphorus (P) is essential macronutrient for soybean [Glycine max (L.) Merr.] growth and function. The objective of this study was to determine effect of phosphorus nutrition (including phosphorus nutrition level and interruption of phosphorus supply) on nitrogen accumulation, nodule nitrogen fixation and yield of soybean plants by ¹⁵N labeling with sand culture. The results showed that they all presented a single peak curve with improvement of phosphorus nutrition level, when phosphorus concentration of nutrient solution was about 31 mg/L, they all reached the maximum and effect of phosphorus nutrition level on nodule nitrogen fixation was lower than that on yield formation level. Interruption of phosphorus supply during soybean growth period, nitrogen accumulation and nodule nitrogen fixation were seriously inhibited, and yield was decreased significantly when interruption of phosphorus supply during V₃-R₁ and R₁-R₅ period, while interruption of phosphorus supply during R₅-R₇ period had no significant effect on nitrogen accumulation, nodule nitrogen fixation and yield. So soybean nitrogen metabolism and yield were sensitive to phosphorus nutrition in the V₃-R₅ period, those were not sensitive to phosphorus nutrition after R₅ period.     

Enhanced soybean (Glycine max L.) plant growth and nodulation by Bradyrhizobium japonicum-SB1 in presence of Bacillus thuringiensis-KR1

Abstract

Nodulation and subsequent nitrogen fixation are important factors that determine the productivity of soybean (Glycine max L.). The beneficial effects of nodulation can be enhanced when rhizobial inoculation is combined with plant-growth-promoting bacteria (PGPB). The PGPB strain Bacillus thuringiensis-KR1, originally isolated from the nodules of Kudzu vine (Pueraria thunbergiana), was found to promote growth of soybean plants (variety VL Soya 2) under Jensen's tube and growth pouch conditions, when co-inoculated with Bradyrhizobium japonicum-SB1. Co-inoculation with Bacillus thuringiensis-KR1 (at a cell density of 10 cfu) provided the highest and most consistent increase in nodule number, shoot weight, root weight, root volume, and total biomass, over rhizobial inoculation and control, under both conditions. The results demonstrate the potential benefits of using nonrhizobial nodule occupants of wild legumes for the co-inoculation of soybean, with Bradyrhizobium japonicum-SB1, in order to achieve plant-growth promotion and increased nodulation.     

Azospirillum brasilense Az39 and Bradyrhizobium japonicum E109, inoculated singly or in combination,promote seed germination and early seedling growth in corn (Zea mays L.) and soybean (Glycine max L.)

Inoculants are biological formulations that combine a stable microorganism population and various types of compounds produced and released during fermentation, such as phytohormones and plant growth regulators. Azospirillum brasilense strain Az39 and Brayrhizobium japonicum strain E109 were previously shown to produce indole 3-acetic acid (IAA), gibberellic acid (GA₃) and zeatin (Z). We tested the hypothesis that such compounds are responsible for early growth promotion in inoculated corn (Zea mays L.) and soybean (Glycine max L.) seedlings. Seeds were inoculated with Az39, E109, or both, and kept in a chamber at 20–30 °C under a controlled photoperiod to evaluate seed germination. To evaluate root and shoot length and dry weight, and number of nodules and percentage of nodulated seedlings, in soybean, seedlings were kept in a growth chamber for 14 days under similar photoperiod and temperature conditions. Az39 and E109, singly or in combination, showed the capacity to promote seed germination, nodule formation, and early development of corn and soybean seedlings. Both strains were able to excrete IAA, GA₃ and Z into the culture medium, at a concentration sufficient to produce morphological and physiological changes in young seed tissues.     

Iron requirement for soybean [Glycine max (L.) Merrill] inoculated with selected exotic and native isolates of Bradyrhizobium sp. under irrigated conditions

A field and greenhouse experiments were conducted to determine the requirement of Fe nutrient supplied through foliar and soil application in soybean inoculated with different selected isolates of exotic and native Bradyrhizobium spp. in saline soils. Six soybean genotypes and three Bradyrhizobium spp. were used for the greenhouse experiments, whereas only two soybean genotypes, namely TGx-1336424 and GIZA, were selected for further study under field conditions. Two levels of FeSO₄ (0 and 4 mg Fe kg^?1 soil) directly supplied to the soil and three levels of Fe-ethylenediaminetetraacetic acid (0–2% of Fe) through foliar application were used for greenhouse and field experiments, respectively. The results of the greenhouse experiment indicated a non-significant effect of Fe application on nodulation and shoot biomass in soybean. Fe application did not improve the grain yield and total biomass yield in soybean inoculated with UK isolate and local isolate but showed remarkable improvement with TAL-379. High soil native N might be the cause for insignificant effect of Fe applied at 2% in highly effective inoculated plants. Therefore, it can be concluded that the symbiotic effectiveness of Bradyrhizobium sp. and the native soil N would affect the soybean Fe requirement supplied through foliar application.     

Rhizobial inoculation improves drought tolerance,biomass and grain yields of common bean (Phaseolus vulgaris L.) and soybean (Glycine max L.) at Halaba and Boricha in Southern Ethiopia

ABSTRACT

While pulses are staple food-legumes in Ethiopia, their productivity is low due to low soil fertility. Elite rhizobial strains that significantly increased shoot dry weight and nitrogen (N) contents of common beans and soybeans in greenhouse were selected for two-year field trials to evaluate their effect on yields of the pulses in the field. Each pulse had six treatments, namely four rhizobial inoculants, uninoculated control, and synthetic N fertilizer. In the drought-affected year 2015, inoculated pulses tolerated moisture stress better than non-inoculated controls. Inoculation was conducive to higher or equivalent yields compared to synthetic N fertilizer. At Halaba, bean inoculated with strain HAMBI3562 gave the highest grain yield (1500 ± 81 kg ha^?1; mean±SE) while the control yielded only 653 ± 22 kg ha^?1. At Boricha, HAMBI3570 gave a grain yield (640 ± 35 kg ha^?1) comparable to synthetic N. When rainfall was optimal in 2016, inoculation with HAMBI3562 and HAMBI3570 gave grain yields (around 4300 kg ha^?1) equivalent to synthetic N. With soybean, strain HAMBI3513 produced consistently higher or comparable biomass and grain yields compared to synthetic N. In conclusion, HAMBI3562 and HAMBI3570 for beans and HAMBI3513 for soybeans can serve as inoculants for areas having similar conditions as the test areas.     

Impact of pyrochar and hydrochar on soybean (Glycine max L.) root nodulation and biological nitrogen fixation

The aim of this study was to identify effects of carbonized organic material (“biochar”) on soybean growth, root nodulation and biological nitrogen fixation, and to elucidate possible underlying mechanisms. Soybean (Glycine max L.) was grown in four arable soils amended with carbonized organic material produced from wood or maize as feedstocks, by pyrolysis (“pyrochar”) or hydrothermal carbonization (“hydrochar”). Nodulation by Bradyrhizobium , biological nitrogen fixation (BNF) assessed by ¹⁵N techniques, plant growth, nutrient uptake and changes in chemical soil properties after soil amendment were determined. Data were analyzed by means of a three way ANOVA on the factors soil, carbonization technique and feedstock. It turned out that soybean root nodulation and BNF was influenced by the carbonization technique used to prepare the soil amendment. Hydrochar, in average and across all soils, increased nodule dry matter and BNF by factors of 3.4 and 2.3, respectively, considerably more than pyrochar, which led to 1.8 and 1.2 fold increases, respectively. Nodule dry matter and BNF correlated positively with available soil sulfur and negatively with available soil nitrogen. Hydrochars provided more available sulfur than pyrochars, and hydrochars caused a decrease in nitrogen availability in the soil solution, thereby exerting a positive influence on nodulation and BNF. Pyrochar amendment increased soil pH but had no effect on nodulation and BNF. Plant growth was affected by the soil and by the feedstock used for the “biochar”, and increased slightly more in treatments with pyrochar and hydrochar made from maize, which was richer in nitrogen and potassium. The results show that carbonized organic materials, and specifically hydrochar, have the capacity to increase BNF in soils. We suggest that this enhancement in BNF in response to soil amendments with carbonized organic materials is due to an increase in available sulfur and a reduction of available soil nitrogen.     

Evaluation of effective Myanmar Bradyrhizobium strains isolated from Myanmar soybean and effects of coinoculation with Streptomyces griseoflavus P4 for nitrogen fixation

In order to substitute the use of chemical fertilizers in legume production, there is a need for the production of rhizobial inoculants which are capable of being used as biofertilizers. To achieve this, an effective symbiotic nitrogen (N) fixation between legumes and root nodule bacteria will be essential. Evaluation of effective Myanmar Bradyrhizobium (Jordan 1982) strains isolated from Myanmar soybean (Glycine max L. Merr.) and effects of coinoculation with Streptomyces griseoflavus Krainsky 1914 P4 for N fixation were studied in pot experiments using sterilized vermiculite and Hoagland solution in the Phytotron (25°C and 70% relative humidity) with completely randomized design and three replicates. N fixation ability of soybean was evaluated by acetylene reduction activity (ARA) by gas chromatography. It was found that MAS23 showed a relatively high degree of stability and a high level of ARA per plant on both Yezin-3 and Yezin-6 soybean varieties. In the symbiotic relationship between Bradyrhizobium strains and P4 experiments, the treatments consisted of six Bradyrhizobium strains (MAS23, MAS33, MAS34, MAS43, MAS48 and USDA110) and Streptomyces griseoflavus P4 were evaluated with four Myanmar soybean varieties (Yezin-3, Yezin-6, Hinthada and Shan Sein). In the Yezin-3 soybean variety, the best treatment for ARA per plant was found in the dual inoculation of P4 and MAS34. In the Yezin-6 soybean variety, the highest nodule dry weight was found in dual inoculation of P4 with MAS34 but the highest ARA per plant was observed in the dual inoculation of P4 and MAS23. On the other hand, single inoculation of MAS43 and coinoculation of P4 with MAS48 were significantly higher in N fixation of Hinthada, and coinoculation of P4 with MAS33 was significant improvement of ARA per plant (P < 0.05) in Shan Sein soybean.     

Effect of CO2-free air treatment on nitrogen fixation of soybeans inoculated with Bradyrhizobium japonicum and Rhizobium fredii

Soybean (Glycine max L. Merr.) cultivars Akisirome and Peking were inoculated with Bradyrhizobium japonicum Is-21 and Rhizobium fredii USDA 194, respectively, and were grown in cylindrical pots containing sterilized vermiculite which were aerated with CO₂-free air or ambient air to study the effects of CO₂ deficiency in the rhizosphere on plant growth, nodulation, and nitrogen fixation. The repressive effects of CO₂-free air treatment were more conspicuous in Peking than Akisirome, and nodule number, nodule mass, amount of biologically fixed N and plant growth of Peking were reduced remarkably by the CO₂-free air treatment.

Acetylene reduction activity (ARA) of Peking inoculated with USDA 194 and Akisirome inoculated Is-21 was assayed in the absence and presence of CO₂, ARA of Akisirome was not affected by the absence of CO₂, while that of Peking decreased drastically.

Based on these results, it was concluded that nitrogen fixation by Akisirome inoculated with B. japonicum Is-21 was not appreciably influenced by CO₂ deficiency in the rhizosphere, while that of Peking inoculated with R. fredii USDA 194 was severely repressed, and the decrease was estimated to be due to both the reduction of the nodule mass and specific nitrogen-fixing activity.     

Involvement of Bradyrhizobium japonicum denitrification in symbiotic nitrogen fixation by soybean plants subjected to flooding

Denitrification by Bradyrhizobium japonicum bacteroids contributes to nitric oxide (NO) production within soybean nodules in response to flooding conditions. However, the physiological relevance of NO production by denitrification in B. japonicum-Glycine max symbiosis is still unclear. In this work, soybean plants were inoculated with B. japonicum strains lacking the nirK or norC genes which encode the copper-containing nitrite reductase and the c-type nitric oxide reductase enzymes, respectively. 14 days flooding increased nodule number of plants inoculated with the WT and norC strains, but not of plants inoculated with the nirK mutant. However, nodule dry weight was not affected by 14 days flooding regardless of the strain used for inoculation. Supporting this observation, individual nodule growth was significantly higher in plants inoculated with nirK than those inoculated with WT or norC after 14 days flooding. Nodule functioning was strongly inhibited by flooding since leghemoglobin content of the nodules induced by any of the strains was significantly decreased after 7 or 14 days flooding compared to control plants. However, this effect was more relevant in nodules of plants inoculated with the WT or norC mutant than in those inoculated with the nirK mutant. Nitrogen fixation was also estimated by analyzing nitrogen content derived from biological nitrogen fixation in shoots, using the ¹⁵N isotope dilution technique. By using this approach, we observed that the negative effect of 14 days flooding on nitrogen fixation was more pronounced in plants inoculated with the norC mutant. However, nitrogen fixation of plants inoculated with nirK showed the highest tolerance to 14 days flooding. These findings allowed us to demonstrate the previously proposed hypothesis which suggests that NO formed by copper-containing nitrite reductase in soybean nodules, in response to flooding, has a negative effect on nitrogenase activity. We propose that inoculation of soybeans with a B. japonicum nirK mutant, which does not produce NO from nitrate, increases the tolerance of symbiotic nitrogen fixation to flooding.     

Biological nitrogen fixation in faba bean (Vicia faba L.) in the Ethiopian highlands as affected by P fertilization and inoculation

 N₂ fixation by leguminous crops is a relatively low-cost alternative to N fertilizer for small-holder farmers in developing countries. N₂ fixation in faba bean (Vicia faba L.) as affected by P fertilization (0 and 20 kg P ha^–1) and inoculation (uninoculated and inoculated) with Rhizobium leguminosarium biovar viciae (strain S-18) was studied using the ¹⁵N isotope dilution method in the southeastern Ethiopian highlands at three sites differing in soil conditions and length of growing period. Nodulation at the late flowering stage was significantly influenced by P and inoculation only at the location exhibiting the lowest soil P and pH levels. The percentage of N derived from the atmosphere ranged from 66 to 74%, 58 to 74% and 62 to 73% with a corresponding total amount of N₂ fixed ranging from 169 to 210 kg N ha^–1, 139 to 184 kg N ha^–1 and 147 to 174 kg N ha^–1 at Bekoji, Kulumsa and Asasa, respectively. The total N₂ fixed was not significantly affected by P fertilizer or inoculation across all locations, and there was no interaction between the factors. However, at all three locations, N₂ fixation was highly positively correlated with the dry matter production and total N yield of faba bean. Soil N balances after faba bean were positive (12–58 kg N ha^–1) relative to the highly negative N balances (–9–44 kg N ha^–1) following wheat (Triticum aestivum L.), highlighting the importance of rotation with faba bean in the cereal-based cropping systems of Ethiopia. Received: 13 January 2000     

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 some fungicide seed treatments on the survival of Bradyrhizobium japonicum and on the nodulation and yield of soybean [Glycine max. (L) Merr.]

Several commercial fungicide seed treatments were evaluated for their possible effect on the survival of Bradyrhizobium japonicum on seeds and on the nodulation and yield of soybeans in a greenhouse and a field experiment. quinolate Pro (carbendazim and oxine copper), Vitavax 200FF (carboxin and thiram), and Monceren (pencycuron) had a small effect or no effect on the survival of B. japonicum and on the nodulation and yield of soybeans. They can thus be considered compatible with soybean seed inoculation. Germipro UFB (carbendazim and iprodione), Apron 35J (metalaxyl), and Tachigaren (hymexazol) decreased B. japonicum survival and the nodulation and yield of soybeans and thus cannot be considered compatible with soybean seed inoculation.     

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