Siderophore production byBradyrhizobium spp. strains nodulating groundnut

EighteenBradyrhizobium spp. strains, fourRhizobium spp. strains and oneAzorhizobium caulinodans strain were grown under Fe limitation and assayed for siderophore production. It was further assessed if Fe accumulation in two groundnut cultivars was influenced by inoculant strain or nitrate fertilisation. Growth ofBradyrhizobium spp. strains nodulating groundnut was slow with mean generation times from 11–24 h. All strains, except MAR 967, showed a reduced growth rate when deprived of Fe; none of the strains showed starvation at 1 M Fe. In the CAS (chrome azurol S)-agar assay, all strains, which formed colonies, produced siderophores as visualised by orange halos around the colonies on blue plates.Bradyrhizobium strains produced much smaller halos than the referenceRhizobium meliloti strain. In the CAS-supernatant assay, all strains, except MAR 967, gave positive responses (measured as absorbance at 630 nm) when supernatants of Fe-depleted cultures were assayed with CAS-indicator complex in comparison with Fe-supplemented cultures. Responses of all fourRhizobium spp. strains were large, while responses of allBradyrhizobium strains, exceptB. japonicum MAR 1491 (USDA 110), were small and mostly insignificant. A small response, i.e. a low Fe-scavenging ability, implies either the production of small quantities of siderophores or the production of low affinity siderophores. Among theBradyrhizobium strains, MAR 1574 and MAR 1587 gave the largest responses taken over the two assays. Fe accumulation in groundnut cultivar Falcon was seven times larger than in cultivar Natal Common. No correlation was found between the quantity of nodule tissue and Fe accumulation, making it unlikely that bacteroids are involved in Fe acquisition by groundnuts. Nitrate-fertilised plants accumulated significantly more Fe, suggesting involvement of nitrate reductase in Fe assimilation in groundnut. The two most successful Fe-scavengingBradyrhizobium spp. strains were also the most effective in nodulating groundnut, the reverse also being true. Strain MAR 967, with the lowest Fe requirement, produced the largest nodule dry weight. These data indicate that improved Fe scavenging properties and/or reduced Fe requirement improve rhizospheric growth and with that nodulation effectiveness.     

Soil acidity in relation to groundnut-Bradyrhizobium symbiotic performance

Effects of soil acidity on groundnut-Bradyrhizobium symbiotic performance were studied in a potted, sandy soil in a glasshouse in Zimbabwe. The soil was limed to soil-pH levels of 5.0 and 6.5. Soil acidity negatively affected plant development, measured as leaf area and plant dry weight, while nodulation was enhanced. This acidity-enhanced nodulation was most evident when nodulation was caused by the indigenousBradyrhizobium population. Effects of soil acidity differed between groundnut cultivars andBradyrhizobium spp. strains, the former having greater importance. TwoArachis hypogaea L. Spanish-type cultivars, Falcon and Plover, performed equally well at neutral soil pH, but Falcon was more acid tolerant. Comparison of the symbiotic performance in neutral versus acid soil of twoBradyrhizobium spp. strains, MAR 411 (3G4b20) and MAR 1510 (CB 756), showed that MAR 411 performed superiorly in neutral soil, but MAR 1510 in acid soil. The indigenousBradyrhizobium population was more effective than was inoculation with strains MAR 411 or MAR 1510. Comparison of twelveBradyrhizobium spp. strains for their symbiotic performance in acid soil showed that some strains were totally ineffective under acidity stress (MAR 253, MAR 967 and MAR 1506), while others performed well.Bradyrhizobium spp. strain MAR 1576 (32 H1) ranked highest for nitrogen accumulation, plant dry weight and leaf area, with strains MAR 1555 (TAL 11) and MAR 1510 following closely. Nitrate fertilisation of groundnut plants led to soil alkalinisation, while nitrogen fixation resulted in soil acidification. Soil acidity in combination with soil sterilisation gave rise to symptoms associated with Al and Mn toxicity.     

Response of introduced Bradyrhizobium strains infectinga promiscuous soybean cultivar

Two field experiments were established to assess the competitiveness of foreign bradyrhizobia in infecting the promiscuous soybean cultivar TGX 536-02D. Seeds were inoculated with antibiotic mutants of the bradyrhizobia strains before planting after land preparation. Soybean plants were harvested at pre-determined days after planting for estimating nodule number, nodule dry weight, nodule occupancy, shoot dry weight and seed yield. Results show that nodule number and dry weight significantly increased and showed great variability at 84 days after planting (DAP), probably due to differences in the ability of inoculant bradyrhizobia to form nodules with the soybean cultivar TGX 536-02D. Increased shoot dry weight, %N, total N and seed yield were a result of increased nodulation by the effective and competitive inoculant Bradyrhizobium strains. Strain USDA 110 occupied the highest percentage of nodule sites because it was more competitive than the other Bradyrhizobium strains. These results show that there was high potential for increasing growth and seed yield of the promiscuous soybean cultivar TGX 536-02D by inoculation with foreign Bradyrhizobium strains.     

Effect of dinitroaniline herbicides on rhizobia, nodulation and N2(C2H2) fixation of four groundnut cultivars

Field, greenhouse and laboratory investigations were conducted to determine the effect of four dinitroaniline herbicides on rhizobia, nodulation and nitrogen fixation of four groundnut cultivars. Benefin, dinitramine and nitralin used at recommended levels decreased nodule dry weight, nitrogenase activity and total nitrogen of groundnut tops and pod yield in three cultivars Kadiri 71-1, Kadiri-2, ICGS-11 and not for a fourth cultivar, Kadiri-3 of groundnut (Arachis hypogaea L.), but fluchloralin used at the recommended level increased the nodulation rate, nitrogenase activity and total nitrogen of groundnut tops and pod yield compared to untreated plants. Studies were conducted in vitro to determine the relative toxicity of the herbicides on four Rhizobium strains isolated from the nodules of four cultivars of groundnut. It was found that various strains of rhizobia differ in their sensitivity to different rates of the herbicides tested. Carbon dioxide exchange rate (CER) of all the cultivars which received herbicide treatment was measured at different time intervals to determine the relationship between photosynthesis and inhibition of nodulation. The lack of adverse effect on the CER of four cultivars when treated at recommended concentrations indicated that nitrogen fixation was affected in cultivars Kadiri 71-1, Kadiri-2 and ICGS-11 due to inhibition of nodulation.     

Soil P-status and cultivar maturity effects on pea-Rhizobium symbiosis

In a green-house experiment, five cultivars of Pisum sativum L. grown on soils from 10 different locations in Tunisia, showed significant differences in nodulation, shoot dry matter (shDM) yield and shoot nitrogen content (shNC). The effect of soil on biological nitrogen fixation, as evidenced by the number and weight of nodules, was mainly attributable to the available phosphorus content. Cate-Nelson ANOVA analysis established a critical value of soil test phosphorus (STP) of 20 mg P kg^–1 soil for nodule weight and number for the majority of cultivars. Within cultivars, nodulation varied with maturation period and was correlated with shoot NC. Thus, the overall interaction of soil-P content and cultivar-maturation period were correlated positively with nodulation and to symbiotic effectiveness of strains of Rhizobium leguminosarum bv. viceae indigenous to these soils. Based on an antibiotic susceptibility test and main variable factor analysis of the data obtained, 70 isolates of Rhizobia that nodulate pea, obtained from soils from agricultural sites throughout Tunisia, were identified as belonging to 18 distinct strains. These classes were identified on the basis of symbiotic efficiency parameters (shoot DM yield and shoot NC) as: ineffective (33 isolates), moderately effective (27 isolates), and efficient strains (10 isolates). This study shows that the Mateur site, an agricultural area for millennia in the northern region of Tunisia, harbors rhizobial strains that are highly efficient in fixing N₂ with peas. These results also indicate the importance of strain-cultivar interrelationships and specificity.     

Nitrogen-fixing potential of micropropagated clones of Acacia mangium inoculated with different Bradyrhizobium spp. strains

Five A. mangium seedlings of different shoot lengths were selected from a 600-seed screening experiment and micropropagated. Two-week-old rooted microcuttings of the 5 micropropagated clones were inoculated with 3 specific Bradyrhizobium spp. strains in 15 combinations. After 5 months of growth, nodule dry weight and shoot dry weight data showed significant effects of clone and Bradyrhizobium spp. strain. Clones RR-G₁ and IR-M₂ and Bradyrhizobium sp. Aust13c resulted in the highest dry-matter production and most efficient nodulation. No interaction was observed between clone and Bradyrhizobium spp. strain, which indicates that the Bradyrhizobium spp. strain and the host plant can be selected separately.     

Effect of seed size and plant growth on nodulation and nodule development in lima bean (Phaseolus lunatus L.)

Lima bean (Phaseolus lunatus L.) cultivars vary widely in their growth habit and seed size. Preliminary experiments indicated that a large-seeded pole cultivar (King of the Garden) formed many more nodules than a small-seeded bush cultivar (Henderson). The relative importance of seed size and shoot mass in determining nodule number and mass was assessed in five lima bean cultivars differing in seed size and growth habit. Between cultivars, significant positive correlations between initial seed mass, plant weight and nodule number and mass were observed during the first four weeks after planting. Comparisons within cultivars indicated a strong correlation between nodule mass and shoot dry weight. The influence of plant morphology on nodule formation and mass was secondary to the effects of seed and shoot mass. As plants matured, the increase in nodule mass paralleled the increase in plant mass, while nodule number was relatively stable after day 18. These results suggest that the highly regulated process of nodule formation was under the influence of seed derived factors, while the continued accumulation of nodule tissue was related to shoot growth.     

Differences between Bradyrhizobium strains NC92 and TAL1000 in their nodulation and nitrogen fixation with peanut in iron deficient soil

The effects of Bradyrhizobium (strains NC92 and TAL1000) and Fe supply on nodulation and nitrogen fixation of two peanut (Arachis hypogaea L.) cultivars (cv. Tainan 9 (Fe inefficient) and cv. 71-234 (Fe efficient)) grown under Fe deficient conditions (imposed by adding 40% CaCO₃ to a ferruginous soil) were examined in a glasshouse experiment. When inoculated with TAL1000 without Fe, both cultivars had low shoot N concentration, very low nodule numbers and weight and no measurable acetylene reduction activity per plant. Inoculation with NC92 without Fe increased all these parameters substantially; addition of Fe with NC92 had no further effect on N concentration but doubled nodule number, weight and acetylene reduction activity per plant. Addition of Fe with TAL1000 increased all parameters to the same level as Fe+NC92, indicating that the poorer nodulation and N₂ fixation of TAL1000 in the absence of Fe, resulted from a poorer ability in getting its Fe supply from the alkaline soil. The nodules from all treatments with measurable activity had the same specific acetylene reduction activity suggesting that Fe deficiency limited nodule development.The results support previous suggestions that Bradyrhizobium strains differ greatly in their ability to obtain Fe from soils and that selection of Fe efficient strains could complement plant breeding in the selection of legume crops for Fe deficient soils.     

The root-nodule symbiosis between Sarothamnus scoparius L. and its microsymbionts

When nitrogen fixing root nodules are formed, Sarothamnus scoparius (broom) is inoculated with its microsymbionts. Nodules studied under light and electron microscopy exhibited typical indeterminate nodule histology with apical, persistent meristem, age gradient of nodule tissues, and open vascular bundles, and also with some particular features such as: the presence of mitotic activity in the infected meristematic cells, lack of infection threads, distribution of bacteria by process of host cell division, and occurrence of a large bacteroid zone only with infected cells. The results of cross-inoculation tests have shown a broad host range for S. scoparius microsymbionts including not only the native host but also species such as: Lupinus luteus, Ornithopus sativa, Lotus corniculatus, Genista tinctoria, Chamaecitisus ratisbonensis, Macroptilium atropurpureum, and Phaseolus vulgaris. In addition, our data established a close symbiotic relationship of S. scoparius nodulators to Bradyrhizobium sp. (Lupinus) by comparison of the partial sequence of nodC gene of the strain CYT7, specific for the broom, to those from Bradyrhizobium sp. (Lupinus) strain D1 and others available in the public databases.     

Early growth and final yield of autumn sownVicia faba L cultivars given different forms of fertiliser N over winter

Summary Between 3 Nov. 1983 and 9 Apr. 1984, six applications of fertiliser N (ammonium, nitrate or urea) were given to four autumn sown (26 Oct. 1983)Vicia faba L cultivars, Banner Winter (BW) and Maris Beagle (MBg), cold tolerant cultivars normally sown in the autumn, and Herz Freya (HF) and Maris Bead (MBd), cold sensitive cultivars more commonly sown in the spring. The effects of additional N were determined by comparison with plants given zero-N (controls). Application of N, regardless of form, had no effect on % emergence at the first sampling (15 Dec. 1983); >90% for BW, MBg and HF, but only 40–60% for MBd. At this time the dry weight, carbon content and nitrogen content of all cultivars was approximately 20% less than that of the seed on planting. No more plants emerged after 15 Dec. 1983. Between 15 Dec. 1983 and 20 Feb. 1984, all cultivars, regardless of N treatment, showed little change in dry weight, carbon content and nitrogen content but the proportion of total plant dry weight, carbon content and nitrogen content in the cotyledons decreased while the proportions in root, stem and leaf tissue increased. On 20 Feb. 1984 there were no N effects. All cultivars but especially BW and MBg, showed progressive increases in dry weight, carbon content and nitrogen content during the period 20 Feb. 1984 to 8 May 1984. Pooled results for all four cultivars indicated that on 8 May 1984, plants given ammonium and urea had a greater dry weight, carbon content and nitrogen content than controls. At harvest (1–3 Sep. 1984), BW and MBg outyielded (g dw seed m⁻²) HF and MBd. Pooled results for all cultivars indicated that application of N regardless of form gave increased yield and an increased N concentration (mg N g⁻¹ dw) in the seed.     

Biology of theParasponia-Bradyrhizobium symbiosis

Parasponia remains the only non-legume known to nodulate withRhizobium/Bradyrhizobium. It is a pioneer plant that is capable of rapid growth and fixing large quantities of nitrogen. In addition to its high agronomic potential, the symbiosis offers the scientist the unique opportunity of studying differences at the molecular level of both partners, and to investigate any possible extension of the symbiosis to other non-legumes of importance. Haemoglobin has been found in the nodule tissue ofParasponia and other nodulated non-legumes and the gene for it has been found and expressed in non-nodulating plants such asTrema tomentosa andCeltis australis. Bradyrhizobium strains isolated from species ofParasponia growing in Papua New Guinea form a group that are more specific in their host requirements thanBradyrhizobium strains from tropical legumes from the same area. They do not effectively nodulate (except CP283) tropical legumes, andParasponia is not readily nodulated withRhizobium andBradyrhizobium strains from legumes. The effectiveness of the symbiosis is influenced by host species, theBradyrhizobium strain and the environment.Parasponia andersonii forms a more effective symbiosis than the other species tested. In competition studies with strains from legumes, isolates fromParasponia always dominate in nodules onParasponia.     

Peanut (Arachis hypogaea) response to inoculation with Bradyrhizobium sp. in soils of Argentina

Soil bacteria (rhizobia) of the genus Bradyrhizobium form symbiotic relationships with peanut root cells and fix atmospheric nitrogen by converting it to nitrogenous compounds. Inoculation of peanut with rhizobia can enhance the plant’s ability to fix nitrogen from the air and thereby reduce the requirement for nitrogen fertiliser. We evaluated three Bradyrhizobium sp. strains for effect on root nodulation and on pod yield of peanut in Argentina soils, using laboratory and field experiments. Of these, strain C‐145 was the most effective in laboratory studies. In‐furrow inoculation with this strain produced increased nodule number, relative to seed inoculation. However, pod yield was not increased significantly by either type of inoculation. In view of the inconsistent response of peanut to inoculation, we examined the effect of indigenous strains of bradyrhizobia. The high degree of nodulation and nitrogen fixation produced by indigenous rhizobia were sufficient for maximal yield under the field and inoculation conditions used in this study. The data are important for future investigation of alternative inoculant strains and conditions for improving peanut production.     

Inoculation of pea by application of Rhizobium in the planting furrow

Summary Selected streptomycin resistant strains ofRhizobium leguminosarum suspended in nutrient broth were added to the planting furrow immediately before the sowing of pea. The nodule occupancy by a strain isolated from Risø soil (Risø la) was increased from 74 to 90%, when the inoculum rate was increased from 3.7×10⁶ to 3.7×10⁸ cells per cm row. The experimental soil contained 10³ to 10⁴ cells ofR. leguminosarum per gram. An almost inefficient strain isolated from Risø soil (SV10) was less competitive with respect to nodulation on two pea cultivars than an efficient Risø strain (SV15) and an efficient non-Risø strain (R1045). The nodule occupancy by the introduced strains varied between pea cultivars.Irrespective of the generally high nodulation by the efficient strains introduced to the soil, the pea seed yield, compared to pea nodulated by the indigenous population, was not significantly increased. Neither were two commercial inoculants, applied in rates corresponding to 3 times the recommended rate, able to increase the yield. This suggests that the indigenous populations ofR. leguminosarum were sufficient in number and nitrogen fixing capacity to ensure an optimal pea crop. However, some inoculation treatments slightly increased the seed N concentration and total N accumulation, indicating that it may be possible to select or develop bacterial strains that may increase the yield.     

Genetic variability in nodulation and root growth affects nitrogen fixation and accumulation in pea

BACKGROUND AND AIMS: Legume nitrogen is derived from two different sources, symbiotically fixed atmospheric N(2) and soil N. The effect of genetic variability of root and nodule establishment on N acquisition and seed protein yield was investigated under field conditions in pea (Pisum sativum). In addition, these parameters were related to the variability in preference for rhizobial genotypes. METHODS: Five different spring pea lines (two hypernodulating mutants and three cultivars), previously identified in artificial conditions as contrasted for both root and nodule development, were characterized under field conditions. Root and nodule establishment was examined from the four-leaf stage up to the beginning of seed filling and was related to the patterns of shoot dry matter and nitrogen accumulation. The genetic structure of rhizobial populations associated with the pea lines was obtained by analysis of nodule samples. The fraction of nitrogen derived from symbiotic fixation was estimated at the beginning of seed filling and at physiological maturity, when seed protein content and yield were determined. KEY RESULTS: The hypernodulating mutants established nodules earlier and maintained them longer than was the case for the three cultivars, whereas their root development and nitrogen accumulation were lower. The seed protein yield was higher in 'Athos' and 'Austin', the two cultivars with increased root development, consistent with their higher N absorption during seed filling. CONCLUSION: The hypernodulating mutants did not accumulate more nitrogen, probably due to the C cost for nodulation being higher than for root development. Enhancing exogenous nitrogen supply at the end of the growth cycle, by increasing the potential for root N uptake from soil, seems a good option for improving pea seed filling.     

Nonparametric approach to multitrait selection for yield in groundnut (Arachis hypogaea L.)

Summary Eight characters related to nitrogen fixation and pod development measured 30 days after flowering were evaluated for their correct grading of the relative yield performance of 17 genetically diverse lines of groundnut (Arachis hypogaea L.). Each line was assigned a high or low yield status based on its pod yield, shelling percentage, and 100-kernel weight. Seventeen character combinations were examined for their relative merit in correct identification of the yield status of lines. The character sets, nitrogenase activity alone or in combination with nitrogen percent or shoot weight identified the status of 77% of lines correctly. The extent to which various characters accounted for the variation in pod yield was also checked by multiple regression analysis. While the character combination, nitrogen percent plus leaf area explained 75% of variation in pod yield, nodule mass, nitrogenase activity, and leaf area occurred in some other combinations that explained yield variation to a lesser extent. These analyses point to the profitability of involving crop physiological traits such as leaf area and nitrogen percent in selecting for relative yield performance in groundnut.IARI Regional Station, Tutikandi, Simla 171004, IndiaNational Research Center for Groundnut, Timbawadi, Junagadh 362015, India     

Breeding for increased nitrogen fixing ability among wild and cultivated species of chickpea

Two chickpea species, one wild (Cicer reticulatum JM2106) and one cultivated (C. arietinum ICC8923) were selected as the parents for this study. C. reticulatum showed high nodule number, nodule dry weight and nitrogen content/plant as compared to C. arietinum. In lines derived from the crosses H208 × (ICC8923 × JM2106) and BG274 × (ICC8923 × JM2106), increase in nodule dry weight, nitrogen content/plant, plant dry weight and grain yield was observed over the parent ICC8923. Similarly F₆ lines also showed improvement in these traits over the cultivated parent. It is concluded that the increase in grain yield and dry matter is the result of improvement in nitrogen utilisation together with an increase in the available fixed nitrogen.     

花生根瘤菌Bradyrhizobium sp.MM6 Ⅲ型分泌系统的结构和功能

【目的】探究花生根瘤菌Bradyrhizobium sp.MM6的Ⅲ型分泌系统(T3SS)的结构及其在根瘤菌与不同宿主建立共生关系中的作用。【方法】同源比对分析菌株MM6的T3SS基因簇的结构特征,并采用三亲本接合转移的方法构建T3SS调节基因ttsI突变菌株;通过蛭石结瘤和石蜡切片实验,比较突变体与野生型的共生固氮表型差异。【结果】经预测,MM6的T3SS基因簇编码区长约34.1 kb,可分为3个区域,包含10个保守结构基因和8个效应蛋白基因,与B.diazoefficiens USDA110相应基因的序列相似性为83%–93%;成功构建了MM6的ttsI突变株;ttsI突变株与野生型分别与花生(S523和Y45)、野大豆和大豆中黄57结瘤,ttsI突变体在花生中的总瘤数显著增加(P<0.05),根瘤中含菌细胞更多;ttsI突变体在野大豆中平均每株植物增加4个根瘤,根瘤中含菌细胞更多,地上部干重相比野生型MM6显著增加(P<0.05);在大豆中黄57中,野生型MM6能形成红色的有效根瘤,ttsI突变体不结瘤,且植株叶片发黄,地上部干重相比野生型MM6显著降低(P<0.05)。【结论】MM6的T3SS在花生和野大豆共生体系中起着有害的作用,而在大豆中黄57的共生体系中起着有利的作用。     

Effects of water deficit on N2(C2H2) fixation in cowpea and groundnut

The effect of water deficit on nodulation, N₂ fixation, photosynthesis, and total soluble sugars and leghemoglobin in nodules was investigated in cowpea and groundnut. Nitrogenase activity completely ceased in cowpea with a decrease in leaf water potential ( _leaf) from –0.4 MPa to –0.9 MPa, while in groundnut it continued down to –1.7 MPa. With increasing water stress, the acetylene reduction activity (ARA) declined very sharply in cowpea, but ARA gradually decreased in groundnut. Even with mild water stress ( _leaf of 0.2 MPa), nodule fresh weight declined 50% in cowpea partly due to a severe nodule shedding whereas nodule fresh weight declined in groundnut only when _leaf decreased by 1.0 MPa. No nodule shedding was noticed even at a higher stress level in groundnut. Photosynthesis and stomatal conductance were also more stable in groundnut than in cowpea under water stress. There was a sharp increase in total soluble sugars and leghemoglobin in the nodules of groundut with water stress, but no definite trend could be found in cowpea.     

Research on the aflatoxin problem in groundnut at ICRISAT

Summary Aflatoxin contamination of groundnut is a serious problem in most groundnut producing countries and as such is given high research priority by the Groundnut Improvement Program of ICRISAT. Since 1979 we have concentrated on selecting cultivars resistant to seed invasion and colonization by toxigenicAspergillus flavus, and/or to aflatoxin production following invasion by the fungus. Resistance to invasion and colonization byA. flavus of rehydrated, mature seed has been found, and confirmed, in some cultivars. We have also screened several groundnut cultivars for seed resistance in the field, both under natural conditions and with the inoculum of the fungus added to the soil in the pod zone. Some cultivars with resistance to seed colonization also showed resistance to seed invasion byA. flavus. None of the cultivars tested has shown complete resistance to aflatoxin production but significant cultivar differences occurred in the amounts of aflatoxin produced in seeds inoculated with a toxigenic strain ofA. flavus.ICRISAT Journal Article No. JA-316     

Nitrogen fixation and growth of soybean as influenced by varying the methods of inoculation with Bradyrhizobium japonicum

The effects of inoculating soil with a water suspension of Bradyrhizobium japonicum (i) at seeding, (ii) 7, or (iii) 14 days after planting (DAP), (iv) seed slurry inoculation and (v) seed slurry supplemented with postemergence inoculation of a water suspension of Bradyrhizobium at 7 or (vi) 14 DAP, on nodulation, N₂ fixation and yield of soybean (Glycine max. [L.] Merrill) were compared in the greenhouse. The ¹⁵N isotope dilution technique was used to quantify N₂ fixed at flowering, early pod filling and physiological maturity stages (36, 52 and 70 DAP, respectively). On average, the water suspension inoculation formed the greatest number of nodules, and seed plus postemergence inoculation formed slightly more nodules than the seed-only inoculated plants (27, 19 and 12 nodules/plant respectively at physiological maturity). Seed slurry inoculation followed by postemergence inoculation at 14 DAP gave the highest nodule weight, with the plants fixing significantly more (P<0.05) N₂ (125 mg N plant⁻¹ or 56% N) than any other treatment (mean, 75 mg plant⁻¹ or 35% N). However, the higher N₂ fixation was not translated into higher N or dry matter yields. Estimates of N₂ fixed by the ostemergence Bradyrhizobium inoculations as well as plant yield were not significantly different from those of the seed slurry inoculation. Thus, delaying inoculation (e.g., by two weeks as in this study) did not reduce the symbiotic ability of soybean plants.