The effects of low root-zone temperature stress on two soybean (Glycine max) genotypes when combined with Bradyrhizobium strains of varying geographic origin

In areas with short growing seasons, poor early vegetative growth of soybean (Glycine max [L.] Merr.) is often attributed to the restrictive effect of cool soil conditions on nodulation and N₂-fixation by this subtropical grain legume. However, there are few studies regarding potential genetic variability of soybean and Bradyrhizobium japonicum genotypes for nodulation at cool root-zone temperatures (RZT). Experiments were conducted to (1) test for a threshold temperature for low RZT inhibition of soybean nodulation and (2) ascertain whether this threshold temperature response depends mainly on the micro- or macrosymbiont. In experiment 1 soybean seedlings (Glycine max [L.] Merr. cv. Maple Arrow) were inoculated with 1 ml of a log phase culture of B. japonicum strain 532C, H8 or H15 (the latter two strains were isolated from cold soils of Hokkaido, northern Japan) and maintained at either 16, 17.5, 19 or 25°C RZT. In experiment 2 seedlings of cv. Maple Arrow and a cold-tolerant Evans isoline were combined with strain 532C and two Hokkaido strains (H5, H30) at both 19 and 25°C RZT. Results indicated that N₂-fixation at 44 days after inoculation was substantially reduced (30–40%) by RZT as high as 19°C, due to development of less nodule mass and to a delay in the onset of N₂-fixation and a small decrease in the number of nodules formed. However, the number of nodules formed was sharply reduced and the time required for the first appearance of nodules was significantly delayed below an RZT of 17.5°C. Differences between cultivars for nodulation and N accumulation were apparent at 25°C, but were abolished by growth at 19°C, indicating that, in spite of differences in growth potential between the cultivars under optimum RZT, both cultivars were equally limited by low RZT. Differences between B. japonicum strains were consistent across temperatures and were largely attributable to higher rates of specific nodule activity recorded for strain 532C, which seemed well adapted to low RZT. These results suggest that the host plant mediates the sensitivity of N₂-fixation under low RZT and that inoculation with B. japonicum strains from cold environments is unlikely to enhance soybean N₂-fixation under cool soil conditions.     

Comparison between parental and variant soybeanBradyrhizobium strains with regard to the production of lipo-chitin nodulation signals,early stages of root infection,nodule occupancy,and N2 fixation rates

Soybean is the most important leguminous crop in Brazil and the nitrogen required for plant growth is supplied byBradyrhizobium bacteria through the symbiotic relation established by the inoculation process. Since 1992, two new strains, CPAC 7 and CPAC 15, which have been shown to increase yields in several field experiments, have been recommended in Brazilian commercial inoculants. CPAC 15 is a natural variant of theB. elkanii SEMIA 566 strain, and was isolated after several years of adaptation to a Brazilian Cerrado soil, while CPAC 7 is a variant ofB. japonicum strain CB 1809, selected under laboratory conditions for higher nodulation and yield. The comparison between parental and variant strains, under greenhouse conditions, showed that both CPAC 15 and CPAC 7 increased N₂ fixation rates in relation to the parental strains. The better performance of CPAC 15 was related to an increase in nodule efficiency (mg N₂ fixed mg^-1 nodule) while with CPAC 7 the higher N₂ fixation rates were due to increased nodulation. Both CPAC 15 and CPAC 7 increased nodule occupancy, when co-inoculated at a ratio of 1:1 withB. elkanii 29w, in relation to their parental strains. Variant strains also differed from parental in their ability to increase numbers of root hairs (Hai phenotype) either when inoculated onto plants, or when supernatants of bacteria exposed to seed exudates were used as inoculants. This results lead to the hypothesis that a modification in some of the “common” nodulation genes had occurred. However, the increase in Hai phenotype with CPAC 7 was dependent on the soybean cultivar, indicating a possible alteration in some genotypic specific nodulation gene. Apparently, there were no differences in Nod metabolites produced by strains CPAC 15 and SEMIA 566, but a more detailed chemical analysis would be required to rule out subtle differences. On the contrary, significant differences were found between CPAC 7 and the parental strain CP 1809, in the profile of Nod metabolites. Consequently, it may be possible that diffusable molecules, responsible for Hai phenotype, would be related to nodulation ability, competiviveness, and N₂ fixation, resulting in the higher yields that have been associated with CPAC 7 and CPAC 15. For the CPAC 7 strain, the increase in Hai phenotype could be atributed to the differences found in the Nod molecules. Consequently, a high degree of physiological and genetic variability can result from the adaptation of rhizobial strains to the soil. Also, this variability can be found under laboratory conditions, when searching single colonies with specific properties. ei]Section editor: R O D Dixon     

The interaction of rockphosphate,Bradyrhizobium, vesicular-arbuscular mycorrhizae and phosphate-solubilizing microbes on soybean grown in a sub-Himalayan mollisol

A factorial design 2³ × 4 with two levels of Mussorie rockphosphate (RP) with or without vesicular-arbuscular mycorrhizal (VAM) fungi and Bradyrhizobium japonicum, and four treatments of phosphate-solubilizing microbes (PSM) Pseudomonas striata, Bacillus polymyxa, Aspergillus awamori was employed using Patharchatta sandy loam soil (Typic Hapludoll). The observations included mycorrhization, nodulation, grain and straw yield, N and P uptake, available soil P and the PSM population in the soil after crop harvest. Inoculation with endophytes alone caused about 70% root colonization. Addition of rockphosphate or inoculation with PSM, except B. polymyxa, stimulated root infection of native as well as introduced VAM endophytes. Application of RP or inoculation with Bradyrhizobium japonicum, mycorrhizal fungi or phosphate-solubilizing microorganisms significantly increased nodulation, N uptake, available soil P and the PSM population in the soil after the crop harvest. The grain and straw yields did not increase following RP addition or mycorrhizal inoculation but increased significantly after inoculation wit Bradyrhizobium or PSM. In general, the application of RP, Bradyrhizobium, VAM and PSM in combinations of any two or three resulted in significant increases in nodulation, plant growth, grain yield and uptake of N and P. Among the four factor interactions, rockphosphate, Bradyrhizobium and P. striata in the absence of VAM resulted in maximal nodulation, grain and straw yields and N uptake by soybean. The highest P uptake by soybean grain was recorded with Bradyrhizobium and A. awamori in the absence of rockphosphate and VAM. Generally, available soil P and PSM population after crop harvest were not significantly increased by the treatment combinations giving the maximal uptake of nutrients. However, they increased significantly in response to PSM, which produced no significant increase in total uptake of nutrients.Research paper no. 7498     

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.     

N2 fixation in Thai soybeans: Effect of tillage and inoculation on15N-determined N2 fixation in recommended cultivars and advanced breeding lines

The practice of seeding soybeans following paddy rice in Thailand has encountered difficulties in seedling germination, nodulation and crop establishment. This research project evaluated the choice of a non-fixing control to quantify N₂ fixation by¹⁵N isotope dilution, and the effect of tillage regime, soybean cultivar, strain ofBradyrhizobium japonicum and P fertilization on yield and N₂ fixation after paddy rice in northern and central Thailand.Japanese non-nodulating lines Tol-0 and A62-2 were the most appropriatecontrol plants for¹⁵N isotope dilution for Thai soybeans in these soils which contained indigenous rhizobia. Cereals such as maize, sorghum and barley were also appropriate controls at some sites. The choice of the appropriate non-fixing control plant for the¹⁵N isotope dilution technique remains a dilemma and no alternative exists other than to use several possible controls with each experiment. Acetylene reduction assay (ARA) proved of little value for screening varieties on their N₂ fixing capacity.The recommended Thai soybean cultivars (SJ1, 2, 4, 5) and an advanced line 16–4 differed little in their ability to support N₂ fixation or yield, possibly due to similar breeding ancestry. The ten AVRDC (ASET) lines showed considerable genotypic control in their ability to utilize their three available N sources (soil, fertilizer, atmosphere) and to translate them into yields. None of these lines were consistently superior to Thai cultivars SJ4 or SJ5 although ASET lines 129, 209 and 217 showed considerable promise.Neither recommended Thai or ASET cultivars were affected by tillage regime. Zero tillage resulted in superior N₂ fixation and yield at two sites but conventional tillage was superior at another site. Soybean cultivars grown in Thailand were well adapted to zero tillage. Levels of N₂ fixation were similar to world figures, averaging more than 100 kg N ha^–1 and supplying over 50% of the plant's N yield. However, seed yields seldom exceeded 2 t ha^–1, well below yields for temperately-grown soybeans. It is not clear why Thai soybeans support N₂ fixation, but do not translate this into higher seed yields.     

Effects of plant breeding and selection on yields and nitrogen fixation in soybeans under two soil nitrogen regimes

Summary Soybeans (Glycine max (L.) Merr.) have a high N requirement which is fulfilled by soil N uptake and N₂-fixation. This study was concerned with the effects of past yield selection on N₂-fixation in soybeans.The soybean cultivars, Lincoln, Shelby, and Williams, which represent successive improvements in the Lincoln germplasm, and a non-nodulating control were planted in a soil containing¹⁵N labelled organic matter. Two replications occurred on soil previously cropped to alfalfa and two on soil previously cropped to soybeans. Plants were harvested at five growth stages and leaf area, plant weight, total N, and atom percent¹⁵N were determined. Mature grain was harvested and yield components were also determined, as well as the total N and¹⁵N content.Cultivar differences in total dry matter were only evident at physiological maturity, when Williams contained the greatest dry matter. Williams exhibited the longest period of seed formation and seed fill and also had the highest grain yield which resulted from a larger weight per seed.The N content of the cultivars did not vary until physiological maturity when Williams contained the highest percent N. The quantity of N fixed at physiological maturity was highest for Williams and lowest for Lincoln. Fixed N contained in the harvested grain was greater for Williams than for the other two cultivars. The fraction of the total plant N derived from fixation was not greatly affected by cultivar and all cultivars acquired an average of 50% of their total N through N₂-fixation.Previous cropping history greatly affected the quantity of N fixed and the fraction of the total plant N derived from fixation. Soybeans following soybeans were more dependent upon N₂-fixation than soybeans following alfalfa with the former deriving 65% of the total plant N from fixation and the latter only 32%. These soybean cultivars apparently utilized soil N first and then used N₂-fixation to satisfy their N requirement.The past selection for higher yield has resulted in soybean cultivars with improved capacities to fix atmospheric N₂ and an improved ability to take up available soil N.     

Nitrogen fixation (15N dilution) with soybeans under Thai field conditions

Controlled environment and field studies were conducted to determine relationships between various measurements of N₂ fixation using soybeans and to use these measures to evaluate a number ofBradyrhizobium japonicum strains for effectiveness in N₂ fixation in Thai soils.¹⁵N dilution measurements of N₂ fixation showed levels of fixation ranging from 32 to 161 kg N ha⁻¹ depending on bacterial strain, host cultivar and location. Midseason measures of N₂ fixation were correlated with each other, but not related measures taken at maturity. Ranking ofB. japonicum strains based on performance under controlled conditions in N-free media were highly correlated with rankings based on soybean seed yields and N₂ fixation under field conditions. This study showed that inoculation of soybeans with effectiveB. japonicum strains can result in significant increases in yield and uptake of N through fixation. The most effective strains tested for use in Thai conditions were those isolated from Thai soils; however, effective strains from other locations were also of benefit.     

Ectomycorrhizal symbiosis enhanced the efficiency of inoculation with two Bradyrhizobium strains and Acacia holosericea growth

Abstract Two strains of Bradyrhizobium sp., Aust 13C and Aust 11C, were dually or singly inoculated with an ectomycorrhizal fungus, Pisolithus albus to assess the interactions between ectomycorrhizal symbiosis and the nodulation process in glasshouse conditions. Sequencing of strains Aust 13C and Aust 11C confirmed their previous placement in the genus Bradyrhizobium. After 4 months culture, the ectomycorrhizal symbiosis promoted plant growth and the nodulation process of both Bradyrhizobium strains, singly or dually inoculated. PCR/RFLP analysis of the nodules randomly collected in each treatment with Aust 13C and/or Aust 11C: (1) showed that all the nodules exhibited the same patterns as those of the Bradyrhizobium strains, and (2) did not detect contaminant rhizobia. When both Bradyrhizobium isolates were inoculated together, but without P. albus IR100, Aust 11C was recorded in 13% of the treated nodules compared to 87% for Aust 13C, whereas Aust 11C and Aust 13C were represented in 20 and 80% of the treated nodules, respectively, in the ectomycorrhizal treatment. Therefore Aust 13C had a high competitive ability and a great persistence in soil. The presence of the fungus did not significantly influence the frequencies of each Bradyrhizobium sp. root nodules. Although the mechanisms remain unknown, these results showed that the ectomycorrhizal and biological nitrogen-fixing symbioses were very dependent on each other. From a practical point of view, the role of ectomycorrhizal symbiosis is of great importance to N₂ fixation and, consequently, these kinds of symbiosis must be associated in any controlled inoculation.     

Short communication: Screening of native and foreign Bradyrhizobium japonicum strains for high N2 fixation in soybean

Four local rhizobia isolates selected after two screening experiments and five USDA Bradyrhizobium japonicum strains were estimated for N₂ fixation in soybean using the ¹⁵N isotope dilution technique. Strain USDA 110 was superior to the local isolates in nodulation and N₂ fixation when inoculated onto soybean cv TGX 1497-ID in a Nigerian soil and could therefore be used as an inoculant for enhanced N₂ fixation in soybean in Nigeria.     

Exclusion of inefficient Bradyrhizobium japonicum serogroups by soybean genotypes

Soybean [Glycine max (L.) Merr.] forms a symbiosis with serogroups of Bradyrhizobium japonicum that differ in their dinitrogen fixing abilities. The objectives of this study were to identify soybean genotypes that would restrict nodulation by relatively inefficient serogroups indigenous to a large portion of the southeastern USA, and then characterize the nodulation responses of selected genotypes with specific bradyrhizobial strains under controlled conditions. From field screening trials followed by controlled single and competitive inoculations of serogroups USDA 31, 76 and 110, twelve soybean genotypes out of 382 tested were identified with varying levels of exclusion abilities. Soybean nodule occupancies and nodulation characteristics were influenced by plant genotype, environment (i.e. field or greenhouse), bradyrhizobial serogroup, and location of nodules (i.e. tap or lateral root). The cultivar Centennial sustains high seed yields even though it nodulates to a high degree with the inefficient serogroup USDA 31. In contrast, data from the released cultivars Braxton, Centennial and Coker 368 indicate that they may have been selected to exclude the inefficient serogroup USDA 76 from their tap root nodules, possibly contributing to high seed yield.     

Effect of plant genotype and nitrogen fertilizer on symbiotic nitrogen fixation by soybean cultivars

Summary Isotopic as well as non-isotopic methods were used to assess symbiotic nitrogen fixation within eight soybean [Glycine max (L.) Merr.] cultivars grown at 20 and 100 kg N/ha levels of nitrogen fertilizer under field conditions.The¹⁵N methodology revealed large differences between soybean cultivars in their abilities to support nitrogen fixation. In almost all cases, the application of 100 kg N/ha resulted in lower N₂ fixed in soybean than at 20 kg N/ha in the first year of the study. However, N₂ fixed in one cultivar, Dunadja, was not significantly affected by the higher rate of N fertilizer application. These results were confirmed by measurements of acetylene reduction activity, nodule dry weight and N₂ fixed as measured by the difference method. Further proof of differences in N₂ fixed within soybean cultivars and the ability of Dunadja to fix similar amounts of N₂ at 20 and 100 kg N/ha was obtained during a second year experiment. Dunadja yield was affected by N fertilizer and produced larger yield at 100 kg N/ha than at 20 kg N/ha. This type of cultivar could be particularly useful in situations where soil N levels are high or where there is need to apply high amounts of N fertilizer.The present study reveals the great variability between legume germplasms in the ability to fix N₂ at different inorganic N levels, and also the potential that exists in breeding for nitrogen fixation associative traits. The¹⁵N methodology offers a unique tool to evaluate germplasms directly in the field for their N₂ fixation abilities at different N fertilizer levels.     

Autecology of Bradyrhizobium japonicum in soybean-rice rotations

The effect of rice culture on changes in the number of a strain of soybean root-nodule bacteria, (Bradyrhizobium japonicum CB1809), already established in the soil by growing inoculated soybean crops, was investigated in transitional red-brown earth soils at two sites in south-western New South Wales. At the first site, 5.5 years elapsed between the harvest of the last of four successive crops of soybean and the sowing of the next. In this period three crops of rice and one crop of triticale were sown and in the intervals between these crops, and after the crop of triticale, the land was fallowed. Before sowing the first rice crop, the number of Bradyrhizobium japonicum was 1.32×10⁵ g^–1 soil. The respective numbers of bradyrhizobia after the first, second and third rice crops were 4.52 ×10⁴, 1.26×10⁴ and 6.40×10² g^–1 soil. In the following two years the population remained constant. Thus sufficient bradyrhizobia survived in soil to nodulate and allow N₂-fixation by the succeeding soybean crop. At the second site, numbers of bradyrhizobia declined during a rice crop, but the decline was less than when the soil was fallowed (400-fold cf. 2200-fold). Multiplication of bradyrhizobia was rapid in the rhizosphere of soybean seedlings sown without inoculation in the rice bays. At 16 days after sowing, their numbers were not significantly different (p<0.05) from those in plots where rice had not been sown. Nodulation of soybeans was greatest in plots where rice had not been grown, but yield and grain nitrogen were not significantly different (p<0.05). Our results indicate that flooding soil has a deleterious effect on the survival of bradyrhizobia but, under the conditions of the experiments, sufficient B. japonicum strain CB 1809 survived to provide good nodulation after three crops of rice covering a total period of 5.5 years between crops of soybean.     

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.     

Nodulation and nitrogen fixation by fast- and slow-growing rhizobia strains of soybean on several temperate and tropical legumes

Three slow-growingBradyrhizobium japonicum (G₃, USDA-110 and KUL-150) of diverse origins and two fast-growing strains ofRhizobium fredii (USDA-192 and USDA-193) were tested with a cropped soybean (Glycine max L. Merrill) cultivar, two cowpeas (Vigna unguiculata), one mung-bean (Phaseolus radiata), one winged-bean (Psophocarpus tetragonolobus) and one field bean (Phaseolus vulgaris) varieties.TheR. fredii strains nodulated and fixed Nitrogen as effectively as the strains ofB. japonicum in a modern european soybean cultivar, namely Fiskeby V. The other western bred soybeans tested were not nodulated by theseR. fredii strains. All of the soybean rhizobia produced nodules in both cowpeas and in mung-bean; theR. fredii strains showed effective N₂-fixation in the cowpeas, particularly USDA-193, yielding shoot dry weights greater than those from theB. japonicum. The symbiotic performance of theR. fredii strains with soybean and other legumes indicated that they should be placed in an intermediate group between the slow-growingB. japonicum and cowpearhizobium sp.The hydrogen uptake activites suggested a possible host effect on the expression of such genes in one out of theB. japonicum strains tested. Furthermore, the slow-growing rhizobia showed significantly higher nitrate-reduction than theR. fredii in the nodules.     

Pesticidal effect on soybean-rhizobia symbiosis

Summary Relative compatibility of selected pesticides at two levels of application (recommended rate and 5× or 10 ×) with soybean-rhizobia symbiosis was tested in pot culture experiments using a prepared peat inoculant.PCNB, carboxin and carboxin+captan at recommended level were innocuous to growth, nodulation, N₂-fixation and total N content of shoot. Carboxin and carboxin+captan but not PCNB at 10 times recommended level proved detrimental to nodulation and N₂-fixation. Carbaryl and malathion at recommended level had no adverse effect but at 10 times recommended level severely reduced N₂-fixation but not other parameters. Acephate, diazinon and toxaphene at both levels reduced N₂-fixation and total N content but not growth and nodulation. All five herbicides used at recommended and 5 times recommended level adversely affected nodulation and N₂-fixation. Glyphosate proved least toxic to all parameters. 2,4-DB at recommended level was less harmful to nodulation and N₂-fixation than trifluralin, alachlor and metribuzin.     

Row spacing effects of N2-fixation,N-yield and soil N uptake of intercropped cowpea and maize

In the tropics, cowpea is often intercropped with maize. Little is known about the effect of the intercropped maize on N₂-fixation by cowpea or how intercropping affects nitrogen fertilizer use effiency or soil N-uptake of both crops. Cowpea and maize were grown as a monocrop at row spacings of 40, 50, 60, 80, and 120 cm and intercropped at row spacing of 40, 50, and 60 cm. Plots were fertilized with 50 kg N as (NH₄)₂SO₄; microplots within each plot received the same amount of¹⁵N-depleted (NH₄)₂SO₄. Using the¹⁵N-dilution method, the percentage of N derived from N₂-fixation by cowpea and the recovery of N-fertilizer and soil N-uptake was measured for both crops at 50 and 80 days after planting.Significant differences in yield and total N for cowpea and maize at both harvest periods were dependent on row spacing and cropping systems. Maize grown at the closer row spacing accumulated most of its N during the first 50 days after planting, whereas maize grown at the widest row spacing accumulated a significant portion of its N during the last 30 days before the final harvest, 80 days after planting.Overall, no significant differences in the percentage of N derived from N₂-fixation for monocropped or intercropped cowpea was observed and between 30 and 50% of its N was derived from N₂.At 50 DAP, fertilizer and soil N uptake was dependent on row spacing with maize grown at the narrowest row spacing having a higher fertilizer and soil N recovery than maize grown at wider spacings. At 50 and 80 DAP, intercropped maize/cowpea did not have a higher fertilizer and soil N uptake than monocropped cowpea or maize at the same row spacing. Monocropped maize and cowpea at the same row spacing took up about the same amount of fertilizer or soil N. When intercropped, maize took up twice as much soil and fertilizer N as cowpea. Apparently intercropped cowpea was not able to maintain its yield potential.Whereas significant differences in total N for maize was observed at 50 and 80 DAP, no significant differences in the atom %¹⁴N excess were observed. Therefore, in this study, the atom %¹⁴N excess of the reference crop was yield independent. Furthermore, the similarity in the atom %¹⁴N excess for intercropped and monocropped maize indicated that transfer of N from the legume to the non-legume was small or not detectable.     

Field evaluation of N2-fixation and N-utilization by phaseolus bean varieties determined by15N isotope dilution*

Summary Differences in N₂-fixation byPhaseolus vulgaris bean cultivars were successfully evaluated in the field using¹⁵N isotope dilution technique with a non-fixing test crop of a different species (wheat). The Phaseolus cultivars could have been similarly ranked for N₂-fixation capacity from either seed yield or total nitrogen yield, but the isotope method provided a direct measure of N₂-fixation and made it possible to estimate the proportion of fixed to total nitrogen in the crop and in plant parts. Amounts of nitrogen fixed varied between 24.59 kg N/ha for the 60-day cultivar Goiano precoce to 64.91 kg N/ha for the 90-day cultivar Carioca. The per cent of plant nitrogen due to fixation was 57–68% for the 90-day cultivars and 37% for Goiano precoce (60-day cultivar). Fertilizer utilization was 17–30% of a 20 kg N/ha fertilizer application. 100 kg N/ha fertilizer application decreased N₂-fixation without suppressing it totally. Differences in yield between the highest yielding (Carioca) and the lowest (Moruna) 90-day cultivars were also due apparently to varietal differences in efficiency of conversion of nitrogen to economic matteri.e. seed, as well as to differences in capacity of genotypes for N₂-fixation. The work described here was in part supported by IAEA Research Contract No. RC/2084 UNDP/IAEA Project BRA/78/006     

Symbiotic N2-fixation by the cover crop Pueraria phaseoloides as influenced by litter mineralization

The perennial legume Pueraria phaseoloides is widely used as a cover crop in rubber and oil palm plantations. However, very little knowledge exists on the effect of litter mineralization from P. phaseoloides on its symbiotic N₂-fixation. The contribution from symbiotic N₂-fixation (Ndfa) and litter N (Ndfl) to total plant N in P. phaseoloides was determined in a pot experiment using a ¹⁵N cross-labeling technique. For determination of N₂-fixation the non-fixing plant Axonopus compressus was used as a reference. The experiment was carried out in a growth chamber during 9 weeks with a sandy soil and 4 rates of ground litter (C/N=16,2.8% N). P. phaseoloides plants supplied with the highest amount of litter produced 26% more dry matter and fixed 23% more N than plants grown in soil with no litter application, but the percentage of Ndfa decreased slightly, but significantly, from 87 to 84%. The litter N uptake was directly proportional to the rate of application and constituted 10% of total plant N at the highest application rate. Additionally, a positive correlation was found between litter N uptake and the amount of fixed N₂. The total recovery of litter N in plants averaged 26% at harvest (shoot + root) and was not affected by the quantity added. A parallel incubation experiment also showed that, as an average of all litter levels, 26% of the litter N was present in the inorganic N pool. The amounts of fertilizer and soil N taken up by plants decreased with litter application, probably due to microbial immobilization and denitrification. It is concluded that, within the litter levels studied, litter mineralization will result in a higher amount of N₂-fixed by P. phaseoloides.     

Assessment of potential sources of error in nitrogen fixation measurements by the nitrogen-15 isotope dilution technique

Although the use of ¹⁵N fertilizers to measure nitrogen (N₂) fixed in crops has increased substantially in recent years, some methodological uncertainties still remain unresolved. The results obtained from a greenhouse study of soybean [Glycine max. (L.) Merrill] inoculated by six different methods have been examined for potential errors arising from incorporating ¹⁵N labelled fertilizer into soil to estimate N₂ fixed in pods or shoots or the whole plant at three growth stages (50% flowering, pod-initiation and physiological maturity) using as reference crops, an uninoculated soybean cultivar and a non-nodulating soybean isoline. At the first harvest when N₂ fixed was very low, the estimates of N₂ fixed by the two reference crops did not match. At this stage the uninoculated soybean estimated about four times as much N₂ fixed in the symbiotic soybean as that measured using the non-nodulating soybean. For the second and third harvests, there were substantial increases in N₂ fixed, and both the non-nodulating and uninoculated soybean were equally suitable as reference crops for assessing N₂ fixed in the symbiotic soybean. These results indicate how critical and difficult the choice of the reference crop could be at early harvests, or when N₂ fixed is low. Even though there were significant differences in ¹⁵N enrichments in different organs (generally nodules < pods < roots < shoots), the estimates of N₂ fixed in soybean plants obtained by excluding roots and nodules did not differ much from those based on the whole plant. Of the above-ground organs, % N₂ fixed in pods (containing seeds) was closest to that of the whole plant (similar at P<0.05 at physiological maturity). However, the total N₂ fixed in pods or shoots was substantially lower than that fixed by the whole plant (P<0.05), although that for the pods and enclosed seeds once again was closer to N₂ fixed in the whole plant than that in the shoots.     

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.