Effects of low root zone temperatures on the early stages of symbiosis establishment between soybean [Glycine max (L.) Merr.] and Bradyrhizobium japonicum

It is known that low root zone temperatures (RZT) have moreeffect on infection and early nodule development than on nitrogenfixation by soybean [Glycine max (L.) Merr.]. However, therehave been no studies regarding how the low RZT inhibit the infectionstages of soybean. Two controlled environment experiments wereconducted to examine the effect of low RZT on bacterial attachmentto, and infection thread penetration of, soybean root hairs.The experimental designs were (1) plants maintained at 25, 17.5or 15C RZT, or transferred from 25 or 17.5 to 15C RZT at either0.5, 1, 2, or 7d after inoculation (DAI), (2) early symbioticestablishment between soybean and Bradyrhizobium japonicum wasexamined microscopically under three RZT (15, 17.5 and 25C).These results indicated that (1) keeping plants at 25C only0.5 DAI prior to transfer to a 15C RZT accelerates the onsetof N₂ fixation at 15C RZT by 6 d, (2) at RZT between 25 and17.5C the infection processes were progressively delayed astemperature declined, (3) RZT less than 17C strongly inhibitedinfection steps, such that when RZT dropped 8.5C from 25 to17.5C infection initiation was delayed 1 d, while when RZTdropped only 2.5C from 17.5 to 15C, infection initiation wasdelayed another 2 d. Key words: Bradyrhizobium japonicum, low temperature, nodulation, soybean     

Preincubation of Bradyrhizobium japonicum with Genistein Accelerates Nodule Development of Soybean at Suboptimal Root Zone Temperatures

In the soybean (Glycine max [L.] Merr.) N2-fixing symbiosis, suboptimal root zone temperatures (RZTs) slow nodule development, especially at temperatures below 17[deg]C. A step in the infection process that occurs within the first 24 h is particularly sensitive to suboptimal RZT. The first phase in the establishment of the soybean-Bradyrhizobium japonicum symbiosis is the exchange of recognition molecules. The most effective plant-to-bacterium signal is genistein. Binding of genistein to B. japonicum activates many of the B. japonicum nod genes. To our knowledge, the potential of sub-optimal RZT to disrupt this interorganismal signaling has not previously been investigated. Controlled environment experiments were conducted to determine whether the preincubation of B. japonicum with genistein increases soybean nodulation and N2 fixation at suboptimal RZT and whether the time between inoculation and root-hair curling is shortened by genistein application. The results of these experiments indicated that (a) genistein application increased soybean nodulation at suboptimal RZTs (17.5 and 15[deg]C) but not at the optimal RZT (25[deg]C); (b) the period between inoculation and root-hair curling was shortened by inoculation with bradyrhizobia preincubated with genistein; (c) at 17.5 and 15[deg]C RZT, the onset of N2 fixation occurred earlier in plants that received genistein-treated bradyrhizobia than in plants inoculated with untreated bradyrhizobia; (d) over the tested concentration range, genistein application at 15 to 20 [mu]M was the most effective in stimulating nodulation; and (e) between 25 and 15[deg]C, as RZT decreased, there was an increase in the nodulation-stimulating potential of genistein.     

Effects of root zone temperature on aluminium toxicity in two cultivars of spring wheat with different resistance to aluminium

Manifestations of aluminium (Al) toxicity in two cultivars of wheat ( Triticum aestivum L. cvs Kadett [relatively Al-resistant] and WW 20299 [relatively Al-sensitive]) were investigated at two root zone temperatures (RZT) that may occur in the field. The plants were grown for 9 days at 10 or 25°C RZT. Mineral nutrients other than CaSO₄ were supplied daily in exponentially increasing amounts to meet the demand of the plants. Al was added as Al₂(SO₄)₃ at the beginning of the culture period at concentrations ranging from 0 to 100 μ M . pH was kept constant at 4.1. Experimental data were analysed for interactions between Al and RZT on a fresh weight basis by the nonlinear Weibull function. Cultivar Kadett, when grown at 25°C RZT, was more resistant to Al than when grown at 10°C RZT. Cultivar WW 20299 was equally sensitive to Al at 10 and 25°C RZT but generally more sensitive to Al than cv. Kadett. It is suggested that cv. Kadett, in contrast to cv. WW 20299, possesses a mechanism for Al resistance that is less effective at 10°C than at 25°C RZT and therefore may be metabolically dependent. In roots, the concentrations of K, P, Mg and Ca were not negatively affected by Al or by RZT. In shoots of both cultivars the concentrations of Ca and Mg became comparatively low when the plants were treated with Al or at low RZT, the effect being larger for Ca than for Mg. At 10°C RZT under Al stress, the Ca concentrations in shoots approached the critical concentration where growth may be inhibited. As no Al was detected in the shoots, it is suggested that Al in the roots inhibits shoot growth by reducing transport of Ca from roots to shoots.     

The effect of potassium deficiency on growth and N2-fixation in Trifolium repens

The effects of potassium (K) deficiency on growth, N₂-fixation and photosynthesis in white clover ( Trifolium repens L.) were investigated using natural occurring gas fluxes on the nodules in real time of plants under three contrasting relative addition rates of K causing mild K deficiency, or following abrupt withdrawal of the K supply causing strong K deficiency of less than 0.65% in dry matter. A steady-state below-optimum K supply rate led to an increase in CO₂-fixation per unit leaf surface area as well as per plant leaf surface. However, nitrogenase activity per unit root weight and per unit nodule weight was maintained, as was the efficiency with which electrons were allocated to the reduction of N₂ in the nodules. Abrupt K removals stimulated nodule growth strongly without delay, but as K concentrations decreased in the plant tissue a significant decline in nitrogenase activity per unit root weight as well as per unit nodule mass occurred. Further, the rate of photosynthesis per unit leaf area was unaffected, while the CO₂ acquisition for the plant as a whole increased due to an expansion of total leaf area whereas the leaf area per unit leaf weight was unaffected. The ratio between CO₂-fixation and N₂-fixation increased, although not statistically significant, under short-term K deprivation as well as under long-term low K supply indicating a downregulation of nodule activity following morphological and growth adjustments. This downregulation took place despite a partly substitution of the K by Na. It is concluded that N₂-fixation does not limit the growth of K-deprived clover plants. K deprivation induces changes in the relative growth of roots, nodules, and shoots rather than changes in N and/or carbon uptake rates per unit mass or area of these organs.     

Genistein accumulation in soybean (Glycine max [L.] Merr.) root systems under suboptimal root zone temperatures

Genistein, as a plant-to-bacteria signal, plays an importantrole in the establishment of the soybean (Glycine max [L.] Merr.)-Bradyrhizobiumjaponicum nitrogen-fixing symbiosis. It is essential to thedevelopment of effective root nodules and responsible for inducingthe nod genes of B. japonicum. Because sub-optimal root zonetemperature (RZT) delays infection and early nodule development,and decreases plant nodule number, and genistein addition overcomessome of this, it is reasonable to hypothesize that suboptimalRZT disrupts the inter-organismal signal exchange by inhibitinggenistein synthesis. Four experiments were conducted to testthese hypotheses. The results of these studies indicated that:(1) when soybean plants were germinated and maintained at RZTsranging from 13 to 17C, root genistein concentration and contentper plant were lower than those of plants with roots maintainedat RZTs above 17C; (2) when plants were germinated at an optimalRZT (25 C) then transferred to RZTs below 17C, and acclimatedfor a few days, root genistein concentration and content perplant were higher than those of plants with roots maintainedeither at optimal RZT, or transferred to RZT above 17 C, althoughby the end of the experiment, the genistein concentration ofroot systems at below 17C RZT appeared to be declining to valuesbelow those of plants with above 17 C RZT; (3) the root genisteinconcentration increased before the onset of nitrogen fixationand decreased thereafter; and (4) part of the effect of RZTson genistein content per plant root system was from reductionsin genistein concentration at lower RZT5, and part was due todecreased plant root growth. Key words: Genistein, Glycine max, suboptimal temperature     

Thermophilic denitrifying bacteria: A survey of hot springs in Southwestern Iceland

Abstract Samples of water, sediment and bacterial mat from hot springs in Grændalur and Hveragerdi areas in southwestern Iceland were screened at 70°C and 80°C for thermophilic denitrifying bacteria by culturing in anaerobic media containing nitrate or N₂O as the terminal oxidant. The springs ranged in temperature from 65–100°C and included both neutral (pH 7–8.5) and acidic (pH 2.5–4) types. Nitrate reducing bacteria (nitrate → nitrite) and denitrifiers (nitrate → N₂) were found that grew at 70°C but not at 80°C in nutrient media at pH 8. Samples from neutral springs that were cultured at pH 8 failed to yield a chemolithotrophic, sulfur-oxidizing and nitrate-reducing bacterium, and samples from acidic springs that were cultured at pH 3.5 seemed entirely to lack dissimilatory, nitrate-utilizing bacteria. No sample yielded an organism capable of growth solely by N₂O respiration. The denitrifiers appeared to be Bacillus . Two such Bacillus strains were examined in pure culture and found to exhibit the unusual denitrification phenotype described previously for the mesophile, Pseudomonas aeruginosa , and one other strain of thermophilic Bacillus . The phenotype is characterized by the ability to grow by reduction of nitrate to N₂ with N₂O as an intermediate but a virtual inability to reduce N₂O when N₂O was the sole oxidant.     

Thermophilic denitrifying bacteria: A survey of hot springs in Southwestern Iceland

Abstract Samples of water, sediment and bacterial mat from hot springs in Grændalur and Hveragerdi areas in southwestern Iceland were screened at 70°C and 80°C for thermophilic denitrifying bacteria by culturing in anaerobic media containing nitrate or N₂O as the terminal oxidant. The s springs ranged in temperature from 65–100°C and included both neutral (pH 7–8.5) and acidic (pH 2.5–4) types. Nitrate reducing bacteria (nitrate → nitrite) and denitrifiers (nitrate → N₂) were found that grew at 70°C but not at 80°C in nutrient media at pH 8. Samples from neutral springs that were cultured at pH 8 failed to yield a chemolithotrophic, sulfur-oxidizing and nitrate-reducing bacterium, and samples from acidic springs that were cultured at pH 3.5 seemed entirely to lack dissimilatory, nitrate-utilizing bacteria. No sample yielded an organism capable of growth solely by N₂O respiration. The denitrifiers appeared to be Bacillus . Two such Bacillus strains were examined in pure culture and found to exhibit the unusual denitrification phenotype described previously for the mesophile, Pseudomonas aeruginosa , and one other strain of thermophilic Bacillus . The phenotype is characterized by the ability to grow by reduction of nitrate to N₂ with N₂O as an intermediate but a virtual inability to reduce N₂O when N₂O was the sole oxidant.     

Oxygen-induced recovery from short-term nitrate inhibition of N2 fixation in white clover plants from spaced and dense stands

Nitrogenase (N₂ase; EC 1.18.6.1) activity (H₂ evolution) and root respiration (CO₂ evolution) were measured under either N₂:O₂ or Ar:O₂ gas mixtures in intact nodulated roots from white clover ( Trifolium repens L.) plants grown either as spaced or as dense stands. The short-term nitrate (5 m M ) inhibition of N₂-fixation was promoted by competition for light between clover shoots, which reduced CO₂ net assimilation rate. Oxygen-diffusion permeability of the nodule declined during nitrate treatment but after nitrate removal from the liquid medium its recovery parallelled that of nitrogenase activity. Rhizosphere pO₂ was increased from 20 to 80 kPa under N₂:O₂. A simple mono-exponential model, fitted to the nodule permeability response to pO₂, indicated NO⁻₃ induced changes in minimum and maximum nodule O₂-diffusion permeability. Peak H₂ production rates at 80 kPa O₂ and in Ar:O₂ were close to the pre-decline rates at 20 kPa O₂. At the end of the nitrate treatment, this O₂-induced recovery in nitrogenase activity reached 71 and 82%; for clover plants from spaced and dense stands, respectively. The respective roles of oxygen diffusion and phloem supply for the short-term inhibition of nitrogenase activity in nitrate-treated clovers are discussed.     

Uptake hydrogenase system in urd bean (Vigna mungo) Rhizobium in relation to nitrogen fixation

Twenty-five isolates of urd bean ( Vigna mungo ) Rhizobium were tested for the presence of an H₂-uptake system using triphenyl tetrazolium chloride reduction as the screening procedure. The isolates which reduced the dye rapidly at early stages of growth were found to recycle H₂ both in culture as well as in nodules. H₂-uptake positive, H₂-uptake negative strains and H₂-uptake negative mutants were compared on the basis of their effect on dry matter accumulation and N₂-fixation. Greenhouse experiments have shown the beneficial effect of the H₂-uptake positive system in nodules on N₂-fixation. Plants inoculated with H₂-uptake positive strains produced higher N content and dry matter over the plants inoculated with H₂-uptake negative strains.     

Plant regulated aspects of nodulation and N2 fixation

Abstract. Root nodule organogenesis is described. Plant regulated aspects of nodulation and N₂ fixation are reviewed and discussed. Since the effective N₂ fixing symbiosis requires the interaction of the host plant and bacterium in an appropriate environment (the rhizosphere and the root nodule) it is essential that research aimed at improving N₂ fixation involve a knowledge and understanding of the plant genes that affect nodule development, growth, and function. Current knowledge of host plant genes involved in N₂ fixation is summarized. Various experimental approaches to the study of the host plant's contribution to nodulation are noted. The functions of nodule specific proteins (nodulins) in symbiosis are delineated. Future areas of research are suggested.     

Effects of mycorrhizal colonization on biomass production and nitrogen fixation of black locust (Robinia pseudoacacia) seedlings grown under elevated atmospheric carbon dioxide

Interactive effects of elevated atmospheric CO₂ and arbuscular mycorrhizal (AM) fungi on biomass production and N₂ fixation were investigated using black locust ( Robinia pseudoacacia ). Seedlings were grown in growth chambers maintained at either 350 μmol mol⁻¹ or 710 μmol mol⁻¹ CO₂. Seedlings were inoculated with Rhizobium spp. and were grown with or without AM fungi. The ¹⁵N isotope dilution method was used to determine N source partitioning between N₂ fixation and inorganic fertilizer uptake. Elevated atmospheric CO₂ significantly increased the percentage of fine roots that were colonized by AM fungi. Mycorrhizal seedlings grown under elevated CO₂ had the greatest overall plant biomass production, nodulation, N and P content, and root N absorption. Additionally, elevated CO₂ levels enhanced nodule and root mass production, as well as N₂ fixation rates, of non- mycorrhizal seedlings. However, the relative response of biomass production to CO₂ enrichment was greater in non-mycorrhizal seedlings than in mycorrhizal seedlings. This study provides strong evidence that arbuscular mycorrhizal fungi play an important role in the extent to which plant nutrition of symbiotic N₂-fixing tree species is affected by enriched atmospheric CO₂.     

Effects of gaseous environment and temperature on the storage behaviour of Listeria monocytogenes on chicken breast meat

Portions of skinless chicken breast meat (pH 5·8) were inoculated with a strain of Listeria monocytogenes and stored at 1, 6 or 15°C in (1) aerobic conditions; (2) 30% CO₂+ air; (3) 30% CO₂+ N₂; and (4) 100% CO₂. When samples were held at 1°C the organism failed to grow under any of the test conditions, despite marked differences between treatments in spoilage rate and ultimate microflora. At 6°C counts of L. monocytogenes increased ca 10-fold in aerobic conditions before spoilage of the meat, but only when the inoculum culture was incubated at 1°C rather than 37°C. In CO₂ atmospheres growth of L. monocytogenes was inhibited on meat held at 6°C, especially under 100% CO₂. By contrast, storage at 15°C led to spoilage of the meat within 2 d, in all gaseous environments, and listeria levels increased up to 100-fold. Differences in the behaviour of L. monocytogenes on poultry and red meats are discussed.     

Effect of various gas atmospheres on the growth and survival of Campylobacter jejuni on beef

Pieces of fresh beef were inoculated with three strains of Campylobacter jejuni . The meat was then allocated to three treatments: (a) vacuum packaged, (b) packaged in an atmosphere of 20% CO₂+ 80% N₂, and (c) packaged into sterile Petri dishes in anaerobic cultivation boxes, which were filled with a gas mixture of 5% O₂+ 10% CO₂+ 85% N₂. The packaging material in the first two treatments was PA 80/PE 100–PE 100/PA 80/PE 100. The survival of Campylobacter cells was followed at 37°C, 20°C and 4°C for 48 h, 4 days and 25 days, respectively. At 37°C the counts of two Campylobacter strains increased in each package treatment for 48 h. At 20°C and at 4°C the counts of the same two strains decreased by 1 to 2 log units and 0.5 to 1 log unit, respectively, during storage. The survival of the two strains was about the same in all package treatments. The third strain was the most sensitive of the strains studied. At 37°C its numbers increased only in the optimal gas atmosphere; at 20°C the strain was not detectable after 24 to 48 h storage and at 4°C after 4 days storage. The aerobic plate counts were determined for all samples at the same time as Campylobacter counts. The high indigenous bacterial numbers of the meat samples did not appear to have a great effect on the survival or growth of campylobacters.     

C4 photosynthesis in Cyperus longus L., a species occurring in temperate climates

Abstract. Cyperus longus L. , which has a widespread but disjunct distribution throughout Europe and extends northwards into Britain, was found to be a C₄ species based upon its Kranz leaf anatomy, low CO₂ compensation point and the labelling of malate as an early product of ¹⁴CO₂ fixation. The photosynthetic characteristics of C. longus are similar to many other C₄ species with a high maximum rate of photosynthesis (> 1.5 mg CO₂ m ⁻² s ⁻¹) and a relatively high temperature optimum (30–35°C), but unlike many C₄ species the rate of photosynthesis does not decline rapidly below the optimum temperature and a substantial rate (0.6 mgCO₂ m⁻²s⁻¹)occursat 15°C. Leaf extension is very slow at 15°C and shows a curvilinear response to temperatures between 15 and 25°C. Leaves extend at a rate of almost 4 cm d⁻¹ at 25°C.     

Field determination of denitrification in water-logged forest soils

Abstract Denitrification rates were measured as N₂O production in two water-logged forest soils at monthly intervals. The effect of acetylene inhibition and the addition of nitrate, glucose, acetate and celloboise in field incubations was examined.
N₂O release from the two soils was very low, 26 mg N₂m⁻²y⁻¹ in ash and 178 mg N₂ in alder. In acetylene inhibited incubations N₂O production was higher, 296 and 486 mg N₂m⁻² y⁻¹ in ash and alder respectively. After addition of nitrate and C-sources to a 10 mM concentration, denitrification rates increased to 5–15 times higher values.
The denitrification rates below 4°C were low and most N₂O was produced in late spring and summer.
The highest rate of denitrification during a 50 h incubation experiment occurred between 3 and 23 h.     

Gas exchange and carbon isotope composition of Ananas comosus in response to elevated CO2 and temperature

Ananas comosus L. (Merr.) (pineapple) was grown at three day/night temperatures and 350 (ambient) and 700 (elevated) μ mol mol^–1 CO₂ to examine the interactive effects of these factors on leaf gas exchange and stable carbon isotope discrimination ( Δ ,‰). All data were collected on the youngest mature leaf for 24 h every 6 weeks. CO₂ uptake (mmol m^–2 d^–1) at ambient and elevated CO₂, respectively, were 306 and 352 at 30/20 °C, 175 and 346 at 30/25 °C and 187 and 343 at 35/25 °C. CO₂ enrichment enhanced CO₂ uptake substantially in the day in all environments. Uptake at night at elevated CO₂, relative to that at ambient CO₂, was unchanged at 30/20 °C, but was 80% higher at 30/25 °C and 44% higher at 35/25 °C suggesting that phosphoenolpyruvate carboxylase was not CO₂-saturated at ambient CO₂ levels and a 25 °C night temperature. Photosynthetic water use efficiency (WUE) was higher at elevated than at ambient CO₂. Leaf Δ -values were higher at elevated than at ambient CO₂ due to relatively higher assimilation in the light. Leaf Δ was significantly and linearly related to the fraction of total CO₂ assimilated at night. The data suggest that a simultaneous increase in CO₂ level and temperature associated with global warming would enhance carbon assimilation, increase WUE, and reduce the temperature dependence of CO₂ uptake by A. comosus .     

The comparative effects of nitrogen and oxygen on the microflora of beef steaks in carbon dioxide-containing modified atmosphere vacuum skin-packaging (MA-VSP) systems

The effects of 80% oxygen–20% carbon dioxide (O₂–CO₂) and 80% nitrogen–20% carbon dioxide (N₂–CO₂) atmospheres were compared with respect to the microbial and sensory characteristics of vacuum skin-packaged grain-fed beef steaks stored at −1 and 4 °C. In both N₂–CO₂ and O₂–CO₂ atmospheres, lactobacilli were predominant over Brochothrix , pseudomonads, enterobacteria and yeasts and moulds. The results of the current investigation showed that the O₂–CO₂ atmospheres did not yield total viable counts in excess of 10⁵ cfu cm⁻² on beef steaks after 4 weeks of storage. However, the sensory analysis and thiobarbituric acid (TBA) values (as a measure of oxidative rancidity) of the products were unacceptable at this time. In contrast, the N₂–CO₂ atmospheres yielded maximum total viable counts of approximately 10⁷ cfu cm⁻² and the sensory analysis and TBA values of the product were judged to be acceptable after 4 weeks of storage at −1 °C. These results indicate that sensory effects of the product were influenced to a greater extent by the chemical effects of high concentration of O₂ on rancidity than by the high levels of lactobacilli.     

Efficacy of Beauveria bassiana (Bals.) Vuill. against the spruce bark beetle, Ips typographus L., in the laboratory under various conditions

Abstract:  In laboratory bioassays, the efficacy of the entomopathogenic fungus Beauveria bassiana against the spruce bark beetle, Ips typographus , was tested under various conditions. Four of the tested isolates and the commercial product Boverol^® caused 99–100% mortality when tested at a concentration of 1.0 × 10⁷ conidia/ml at 25°C. Using B. bassiana isolate 138 at a concentration of 1.0 × 10⁶, the median survival time (MST) was 6.1 d and significantly longer compared with the MST of 4.2 and 4.0 d at 1.0 × 10⁷ and 1.0 × 10⁸ conidia/ml, respectively. In the next experiment, the beetles were maintained on spruce bark, filter paper or artificial diet during the bioassay with Boverol^®, and significant differences in the MST of 3.6, 2.5 and 5.3 d, respectively, were noticed. The experiment with Boverol^® at different temperatures showed that the beetles lived significantly longer at 15°C (MST 8.7 d) than at 20, 25, 30 and 35°C. At 25°C, the beetles died most rapidly (MST 3.5 d). At different relative humidities (RH) of 40, 70 and 100%, nearly all beetles were dead after treatment with a suspension of Boverol^® at 1.0 × 10⁷ conidia/ml. At 40% RH, 49% of the untreated beetles died after 7 d. The best effects were achieved with the following bioassay: beetles were fed for three days on artificial diet, then dipped into a solution of 1.0 × 10⁷ conidia/ml and transferred on a piece of spruce bark in Petri dishes at 25°C and 70% RH.     

Growth in elevated CO2 enhances temperature response of photosynthesis in wheat

The temperature dependence of C₃ photosynthesis may be altered by the growth environment. The effects of long-term growth in elevated CO₂ on photosynthesis temperature response have been investigated in wheat ( Triticum aestivum L.) grown in controlled chambers with 370 or 700 μmol mol⁻¹ CO₂ from sowing through to anthesis. Gas exchange was measured in flag leaves at ear emergence, and the parameters of a biochemical photosynthesis model were determined along with their temperature responses. Elevated CO₂ slightly decreased the CO₂ compensation point and increased the rate of respiration in the light and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) V_cmax, although the latter effect was reversed at 15°C. With elevated CO₂, J_max decreased in the 15–25°C temperature range and increased at 30 and 35°C. The temperature response (activation energy) of V_cmax and J_max increased with growth in elevated CO₂. CO₂ enrichment decreased the ribulose 1,5-bisphosphate (RuBP)-limited photosynthesis rates at lower temperatures and increased Rubisco- and RuBP-limited rates at higher temperatures. The results show that the photosynthesis temperature response is enhanced by growth in elevated CO₂. We conclude that if temperature acclimation and factors such as nutrients or water availability do not modify or negate this enhancement, the effects of future increases in air CO₂ on photosynthetic electron transport and Rubisco kinetics may improve the photosynthetic response of wheat to global warming.     

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.