Methods for enhancing symbiotic nitrogen fixation

Biological nitrogen fixation of leguminous crops is becoming increasingly important in attempts to develop sustainable agricultural production. However, these crops are quite variable in their effectiveness in fixing nitrogen. By the use of the ¹⁵N isotope dilution method some species have been found to fix large proportions of their nitrogen, while others like common bean have been considered rather inefficient. Methods for increasing N₂ fixation are therefore of great importance in any legume work. Attempts to enhance nitrogen fixation of grain legumes has been mainly the domain of microbiologists who have selected rhizobial strains with superior effectiveness or competitive ability. Few projects have focused on the plant symbiont with the objective of improving N₂ fixation as done in the FAO/IAEA Co-ordinated Research Programme which is being reported in this volume. The objective of the present paper is to discuss some possibilities available for scientists interested in enhancing symbiotic nitrogen fixation in grain legumes. Examples will be presented on work performed using agronomic methods, as well as work on the plant and microbial symbionts. There are several methods available to scientists working on enhancement of N₂ fixation. No one approach is better than the others; rather work on the legume/Rhizobium symbiosis combining experience from various disciplines in inter-disciplinary research programmes should be pursued.     

3种水稻土中7株固氮蓝细菌的分离与特征

【背景】蓝细菌是水生和陆地生态系统中生物固氮的主要贡献者。【目的】增加对稻田土壤固氮蓝细菌的了解,获得用于进一步研究的可培养固氮蓝细菌菌株。【方法】选择3种具有不同固氮能力的水稻土,采用BG11-N培养基分离培养固氮蓝细菌菌株,对新分离菌株进行形态特征观察,通过基因组DNA的nifH基因扩增明确其固氮潜力,进一步采用乙炔还原法和~(15)N_2示踪法定量测定其固氮能力,通过基因组DNA的16SrRNA基因序列比对进行鉴定。【结果】在光照培养条件下,采用BG11-N培养基共分离纯化得到自养菌株7株,细胞呈圆形或椭圆形、单列、无分枝、丝状和念珠状,在固体培养基上形成团垫状菌落。新分离菌株在BG11-N培养基中生长状况良好,以基因组DNA为模板可扩增出nifH基因,乙炔还原法和~(15)N_2示踪法测定结果显示具有较高固氮能力,同时具有铁载体生成能力。结合16S rRNA基因序列比对和形态特征,7株菌被初步鉴定隶属于念珠藻科(Nostocaceae)。【结论】从水稻土中分离到在稻田生物固氮中发挥重要作用的蓝细菌(念珠藻科)菌株,可培养固氮蓝细菌菌株固氮能力较高,兼具铁载体生成能力,可作为进一步深入研究的微生物资源,具有潜在的研究应用价值。     

Optimising biological N2 fixation by legumes in farming systems

Whether grown as pulses for grain, as green manure, as pastures or as the tree components of agro-forestry systems, the value of leguminous crops lies in their ability to fix atmospheric N₂, so reducing the use of expensive fertiliser-N and enhancing soil fertility. N₂ fixing legumes provide the basis for developing sustainable farming systems that incorporate integrated nutrient management. By exploiting the stable nitrogen isotope ¹⁵N, it has been possible to reliably measure rates of N₂ fixation in a wide range of agro-ecological field situations involving many leguminous species. The accumulated data demonstrate that there is a wealth of genetic diversity among legumes and their Rhizobium symbionts which can be used to enhance N₂ fixation. Practical agronomic and microbiological means to maximise N inputs by legumes have also been identified.     

Yield and biological nitrogen fixation of common bean (Phaseolus vulgaris L.) in Peru

Two field experiments were performed to evaluate the nitrogen fixation potential of twenty common bean cultivars and breeding lines during summer and winter seasons of 1986 and 1988, respectively. The ¹⁵N isotope dilution method was used to quantify N₂ fixation. The cultivars and breeding lines were variable in terms of their N₂ fixation. The cv. Caballero was very efficient, with more than 50% N derived from the atmosphere and 60–80 kg N ha^–1 fixed in both seasons. Other cultivars were less efficient, since the poorest ones derived less than 30% of their nitrogen from the atmosphere and fixed less than 20 kg N ha^–1. After additional testing the best cultivars may be used directly by the farmers for cultivation. The experiments have provided information about which genotypes may be used to breed for enhanced fixation in common bean.     

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.     

Alder and lupine enhance nitrogen cycling in a degraded forest soil in Northern Sweden

Positive effects of legumes and actinorhizal plants on N-poor soils have been observed in many studies but few have been done at high latitudes, which was the location of our study. We measured N₂ fixation and several indices of soil N at a site near the Arctic Circle in northern Sweden. More than 20 years ago lupine (Lupinus nootkatensis Donn) and gray alder (Alnus incana L. Moench) were planted on this degraded forest site. We measured total soil N, net N mineralization and nitrification with a buried bag technique, and fluxes of NH⁺ ₄ and NO^– ₃ as collected on ion exchange membranes. We also estimated N₂ fixation activity of the N₂-fixing plants by the natural abundance of ¹⁵N of leaves with Betula pendula Roth. as reference species. Foliar nitrogen in the N₂-fixing plants was almost totally derived from N₂ fixation. Plots containing N₂-fixing species generally had significantly higher soil N and N availability than a control plot without N₂-fixing plants. Taken together, all measurements indicated that N₂-fixing plants can be used to effectively improve soil fertility at high latitudes in northern Sweden.     

A field evaluation using the 15N isotope dilution method of lines of Phaseolus vulgaris L. bred for increased nitrogen fixation

N₂ fixation in lines of Phaseolus vulgaris was measured by ¹⁵N-isotope dilution to determine whether a programme of crossing and recurrent selection had resulted in enhanced nitrogen fixation. In field experiments on an isohyperthermic Aquic Hapludoll soil the amount of N₂ fixed by the different lines ranged from 18 to 36 kg ha^–1 (32 to 47% of plant N) in 56 days. The quantity of N₂ fixed and the proportion of plant N derived from fixation was not significantly greater in the lines selected for N₂ fixation (RIZ lines) than parental lines. Total shoot N ranged from 53 to 77 kg ha^–1 and partitioning of N to pods differed from 28 to 52% among the lines which all had similar growth habit and duration. Nodulation patterns were also distinct. Nodules formed early (10 to 15 plant^–1 at 13 days) in many lines, and smallest amounts of fixation were observed in those lines which nodulated slowly and did not form substantial nodule mass until after 40 days. The screening criteria used in the selection of the RIZ lines had been largely indirect with other factors such as disease resistance also being included. Progress for increasing N₂ fixation over good-fixing parental lines such as BAT76 was not significant and it is recommended that more attention be paid to early nodulation, to the use of soils with lower available N and to inter-crossing of lines having different good N₂ fixation traits in order to further enhance the potential for N₂ fixation in beans.     

Does carbon isotope discrimination correlate with biological nitrogen fixation?

It has recently been reported that N₂ fixation and carbon isotope discrimination (Δ) are negatively correlated. To further test this hypothesis, a greenhouse experiment was conducted to investigate if Δ is correlated with the efficiency of lentil (Lens culinaris cv Laird) in fixing atmospheric nitrogen. Lentil seed was inoculated with one of 10 Rhizobium leguminosarum strains that varied in their effectiveness in symbiotic N₂ fixation. Carbon-13 discrimination was positively correlated with N₂ fixation (r²=0.60^*). Although the amount of N₂ fixed ranged from 1.5 mg N to 13.5 mg N shoot⁻¹, the range of Δ values was only 25.8 to 26.6%.. It is unlikely that variability of such small magnitude could be of any practical use in selecting for N₂-fixing efficiency.     

Nitrogen fixation by white clover in pastures grazed by dairy cows: Temporal variation and effects of nitrogen fertilization

Effects of rate of nitrogen (N) fertilizer and stocking rate on production and N₂ fixation by white clover (Trifolium repens L.) grown with perennial ryegrass (Lolium perenne L.) were determined over 5 years in farmlets near Hamilton, New Zealand. Three farmlets carried 3.3 dairy cows ha^–1 and received urea at 0, 200 or 400 kg N ha^–1 yr^–1 in 8–10 split applications. A fourth farmlet received 400 kg N ha^–1 yr^–1 and had 4.4 cows ha^–1.There was large variation in annual clover production and total N₂ fixation, which in the 0 N treatment ranged from 9 to 20% clover content in pasture and from 79 to 212 kg N fixed ha^–1 yr^–1. Despite this variation, total pasture production in the 0 N treatment remained at 75–85% of that in the 400 N treatments in all years, due in part to the moderating effect of carry-over of fixed N between years.Fertilizer N application decreased the average proportion of clover N derived from N₂ fixation (P_N; estimated by ¹⁵N dilution) from 77% in the 0 N treatment to 43–48% in the 400 N treatments. The corresponding average total N₂ fixation decreased from 154 kg N ha^–1 yr^–1 to 39–53 kg N ha^–1 yr^–1. This includes N₂ fixation in clover tissue below grazing height estimated at 70% of N₂ fixation in above grazing height tissue, based on associated measurements, and confirmed by field N balance calculations. Effects of N fertilizer on clover growth and N₂ fixation were greatest in spring and summer. In autumn, the 200 N treatment grew more clover than the 0 N treatment and N₂ fixation was the same. This was attributed to more severe grazing during summer in the 0 N treatment, resulting in higher surface soil temperatures and a deleterious effect on clover stolons.In the 400 N treatments, a 33% increase in cow stocking rate tended to decrease P_N from 48 to 43% due to more N cycling in excreta, but resulted in up to 2-fold more clover dry matter and N₂ fixation because lower pasture mass reduced grass competition, particularly during spring.     

The contribution of associative and symbiotic nitrogen fixation to the nitrogen nutrition of non-legumes

During the past 10 years estimates of N₂ fixation associated with sugar cane, forage grasses, cereals and actinorhizal plants grown in soil with and without addition of inoculum have been obtained using the ¹⁵N isotope dilution technique. These experiments are reviewed in this paper with the aim of determining the proportional and absolute contribution of N₂ fixation to the N nutrition of non-legumes, and its role as a source of N in agriculture. The review also identifies deficiencies in both the totality of data which are currently available and the experimental approaches used to quantify N₂ fixation associated with non-legumes.Field data indicate that associative N₂ fixation can potentially contribute agronomically-significant amounts of N (>30–40 kg N ha^-1 y^-1) to the N nutrition of plants of importance in tropical agriculture, including sugar cane (Saccharum sp.) and forage grasses (Panicum maximum, Brachiaria sp. and Leptochloa fusca) when grown in uninoculated, N-deficient soils. Marked variations in proportions of plant N derived from the atmosphere have been measured between species or cultivars within species.Limited pot-culture data indicate that rice can benefit naturally from associative N₂ fixation, and that inoculation responses due to N₂ fixation can occur. Wheat can also respond to inoculation but responses do not appear to be due to associative N₂ fixation. ¹⁵N dilution studies confirm that substantial amounts of N₂ can be fixed by actinorhizal plants.     

Nitrogen fixation in breeding lines of Phaseolus vulgaris L.

Nitrogen fixation in nine common bean (Phaseolus vulgaris L.) lines was estimated using the ¹⁵N isotope dilution method at two locations in two seasons. In the first season at one location no N₂ fixation was detected while in the second season up to 51 kg N ha^–1 were estimated. There were significant differences between lines and correlations between trials were significant for the amounts of N₂ fixed, but not for total shoot nitrogen. The plants that fixed the most nitrogen nodulated rapidly after germination. Differences in maximum nodule mass, but not specific nodule activity, were detected also.     

Breding common bean for improved biological nitrogen fixation

Common bean (Phaseolus vulgaris L.), which is an important food crop in the Americas, Africa and Asia, usually is thought to fix only small amounts of atmospheric nitrogen. However, field data indicate considerable genetic variability for total N₂ fixation and traits associated with fixation. Studies have shown that selection to increase N₂ fixation will be successful if: (1) discriminating traits (selection criteria) are measured precisely, (2) variability in germplasm is heritable, (3) selected parents are also agronomically suitable, (4) units of selection facilitate quantification of selection criteria, and (5) a breeding procedure that allows maximum genetic gain for N₂ fixation and recombination with essential agronomic traits is chosen. Breeding lines capable of fixing enough atmospheric N₂ to support seed yields of 1000–2000 kg ha^–1 have been identified and new cultivars with high N₂ fixation potential are being released.     

Response to inoculation and N fertilization for increased yield and biological nitrogen fixation of common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris L.) is able to fix 20–60 kg N ha^–1 under tropical environments in Brazil, but these amounts are inadequate to meet the N requirement for economically attractive seed yields. When the plant is supplemented with N fertilizer, N₂ fixation by Rhizobium can be suppressed even at low rates of N. Using the ¹⁵N enriched method, two field experiments were conducted to compare the effect of foliar and soil applications of N-urea on N₂ fixation traits and seed yield. All treatments received a similar fertilization including 10 kg N ha^–1 at sowing. Increasing rates of N (10, 30 and 50 kg N ha^–1) were applied for both methods. Foliar application significantly enhanced nodulation, N₂ fixation (acetylene reduction activity) and yield at low N level (10 kg N ha^–1). Foliar nitrogen was less suppressive to nodulation, even at higher N levels, than soil N treatments. In the site where established Rhizobium was in low numbers, inoculation contributed substantially to increased N₂ fixation traits and yield. Both foliar and soil methods inhibited nodulation at high N rates and did not significantly increase bean yield, when comparing low (10 kg N ha^–1) and high (50 kg N ha^–1) rates applied after emergence. In both experiments, up to 30 kg N ha^–1 of biologically fixed N₂ were obtained when low rates of N were applied onto the leaves.     

Measurements of N2 fixation by common bean in Central Brazil as affected by different reference crops

An experiment was conducted at EMBRAPA/CNPAF, Goiânia, Goias, Brazil, on a typic haplustox soil to evaluate growth and N₂ fixation-related parameters of Phaseolus vulgaris L. Bean lines, which had been selected for N₂ fixation at CNPAF, including production cultivars, germplasm bank entries, and parents and progenies of a cross made to improve this characteristic. Wheat (Triticum aestivum L.) and dwarf sorghum (Sorghum bicolor (L.) Moench) were evaluated as non-N₂-fixing reference crops for difference method (DM) and ¹⁵N isotope dilution technique (IDT) estimates of N₂ fixation. IDT estimates ranges from 4 to 18 kg N₂ fixed ha^-1. High variability associated with low levels of N₂ fixation precluded definitive identification of the best N₂ fixing bean lines. Due to differences in growth cycle and in patterns and amounts of soil N uptake during the season, neither of the reference crops tested appears to be an adequate control for either DM or IDT estimates of N₂ fixation. However, ranking of lines for effectiveness in N₂ fixation could be performed without the use of any reference crops.     

N2-Fixation, nodule efficiency and biomass accumulation after two years in three Chilean legume trees and Tagasaste Chamaecytisus proliferus subsp. palmensis

Initial results of a long-term field experiment are presented for the above and below-ground biomass accumulation after two years, as well as root nodulation, nodule efficiency (g N fixed/g nodules) and biological N₂ fixation (using the ¹⁵N isotope dilution method) of four N₂-fixing tree species (NFTs) grown in the subhumid mediterranean-climate zone of central Chile. Two non-legume tree species, Fraxinus excelsior and Schinus polygamus, were used as reference plants for the isotope dilution calculations.Over two years, Tagasaste (Chamaecytisus proliferus subsp. palmensis, a Papilionoideae from the Canary Islands), produced 10 to 20 times more biomass than the other three NFTS (Acacia caven, Prosopis alba and P. chilensis); all Mimosoideae native to Chile, and nodulation and nitrogen fixed were an order of magnitude higher as well. At the end of the second year, the percentage of N derived from N₂ fixation (%Ndfa) in Tagasate averaged 85.6, equivalent to ca. 49.1 g N fixed per tree. For all four NFTs, however, %Ndfa, nodule efficiency, and total N accumulation varied from one year to the next; caution is thus required in interpreting or predicting NFT performance over the long term, even if Tagasaste can already be considered a highly promising NFT for central Chile.     

Biological nitrogen fixation: An efficient source of nitrogen for sustainable agricultural production?

A fundamental shift has taken place in agricultural research and world food production. In the past, the principal driving force was to increase the yield potential of food crops and to maximize productivity. Today, the drive for productivity is increasingly combined with a desire for sustainability. For farming systems to remain productive, and to be sustainable in the long-term, it will be necessary to replenish the reserves of nutrients which are removed or lost from the soil. In the case of nitrogen (N), inputs into agricultural systems may be in the form of N-fertilizer, or be derived from atmospheric N₂ via biological N₂ fixation (BNF).Although BNF has long been a component of many farming systems throughout the world, its importance as a primary source of N for agriculture has diminished in recent decades as increasing amounts of fertilizer-N are used for the production of food and cash crops. However, international emphasis on environmentally sustainable development with the use of renewable resources is likely to focus attention on the potential role of BNF in supplying N for agriculture. This paper documents inputs of N via symbiotic N₂ fixation measured in experimental plots and in farmers' fields in tropical and temperate regions. It considers contributions of fixed N from legumes (crop, pasture, green manures and trees), Casuarina, and Azolla, and compares the relative utilization of N derived from these sources with fertilizer N.     

Nitrogen fixation in soybean as influenced by cultivar and Rhizobium strain

Summary The¹⁵N-substratum labeling technique and other indirect methods were used to compare nitrogen (N₂) fixation in soybean varieties grown in the field in Greece and Romania. Significant variation in the amount (Ndfa) and proportion of N derived from fixation (% Ndfa) was found in different varieties. With 20 kg N/ha applied to soil, N₂ fixed ranged from 22 to 236 kg N/ha in Greece and from 17 to 132 kg N/ha in Romania. In general, varieties or treatments with higher dry matter yield supported greater fixation. Also, varieties with high Ndfa had high % Ndfa andvice versa. Breeding N₂-fixing legumes for high yields at low soil N levels therefore appears to be a reasonable strategy for enhancing N₂ fixation. Heavy applications of inorganic N fertilizer severely depressed N₂ fixation in two out of the three varieties used in Romania. One variety, F 74–412, however, derived slightly higher amounts of N₂ from fixation at 100 kg N/ha rate than when fertilized with 20 kg N/ha. In Greece, Chippewa, Williams and Amsoy-71 inoculated with a Nitragin inoculant fixed similar amounts of N₂ at both 20 and 100 kg N/ha fertilizer rates. However, when Chippewa and Williams were inoculated with amother, locally-isolated Rhizobium strain, N₂ fixation was substantially depressed at the higher N rate.     

Misconceptions and practical problems in the use of 15N soil enrichment techniques for estimating N2 fixation

The ¹⁵N methods are potentially accurate for measuring N₂ fixation in plants. The only problem with those methods is, how to ensure that the ¹⁵N/¹⁴N ratio in the plant accurately reflects the integrated ¹⁵N/¹⁴N ratio (R) in soil which is variable in time and with soil depth. However, the consequences of using an inappropriate reference plant vary with the level of N₂ fixation and the conditions under which the study was made. For example, the errors introduced into the values of N₂ fixation are higher at low levels of fixation, and decrease with increasing rates of fixation. At very high N₂ fixation rates, the errors are often insignificant. Also, the magnitude of error is proportional to the rate of decline of the ¹⁵N/¹⁴N ratio with time. Since N₂ fixation in most plants would be expected to below 60%, the question of how to select a good reference plant is still pertinent. In this paper, we have discussed some of the criteria to adopt in selecting reference plants, e.g. how to ensure that the reference plant is not fixing N₂, is absorbing most of its N from the same zone as the fixing plant, and in the same pattern with time, etc. In addition, we have discussed ¹⁵N labelling materials and methods that are likely to minimize any errors even when the fixing and reference plants don't match well in certain important criteria. The use of slow release ¹⁵N fertilizer or ¹⁵N labelled plant materials results in slow changes in the ¹⁵N/¹⁴N ratio of soil, and is strongly recommended. Where ¹⁵N inorganic fertilizers are used, the application of the fertilizer in small splits at various intervals is recommended over a one-time application. The problem with the reference crop, which has sometimes discouraged potential users of the ¹⁵N methods, is surmountable, as discussed in this paper.     

Measurement of N2 fixation in maize (Zea mays L.)—ricebean (Vigna umbellata [Thunb.] Ohwi and Ohashi) intercrops

The yield of N in maize (Zea mays L.) and ricebean (Vigna umbellata [Thumb.] Ohwi and Ohashi) were compared on a Tropoqualf soil in North Thailand in 1984 and 1985. Both species were grown in field plots in monoculture or as intercrops at a constant planting density equivalent to 8 maize or 16 ricebean plants per m². The contribution of symbiotic N₂ fixation to ricebean growth was estimated from measurements of the natural abundance of¹⁵N (δ¹⁵N) in shoot nitrogen and from analysis of ureides in xylem sap vacuumextracted from detached stems. The natural abundance of¹⁵N in the intercropped ricebean was found to be considerably less than that in monoculture in both growing seasons. Using maize and a weed (Ageratum conyzoides L.) as non-fixing¹⁵N reference plants the proportions (P ¹⁵N) of ricebean shoot N derived from N₂ fixation ranged from 0.27 to 0.36 in monoculture ricebean up to 0.86 when grown in a 75% maize: 25% ricebean intercrop. When glasshouse-derived calibration curves were used to calculate plant proportional N₂ fixation (Pur) from the relative ureide contents of field collected xylem exudates, the contribution of N₂ fixation to ricebean N yields throughout the 1985 growing season were greater in intercrop than in monocrop even at the lowest maize:legume ratio (25∶75). Seasonal patterns of sap ureide abundance indicated that N₂ fixation was greatest at the time of ricebean podset. The averagePur andP ¹⁵N in ricebean during the first 90 days of growth showed identical rankings of monocrop and intercrop treatments in terms of N₂ fixation, although the two sets ofP values were different. Nonetheless, seasonal estimates of N₂ fixation during the entire 147 days of legume growth determined from ureide analyses indicated that equivalent amounts of N could be fixed by ricebean in a 75∶25 intercrop and in monoculture despite the former being planted at one-quarter the density.     

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.