Isolation and characterization of the major nod factor of Bradyrhizobium japonicum strain 532C

Bradyrhizobium japonicum 532C nodulates soybean effectively under cool Canadian spring conditions and is used in Canadian commercial inoculants. The major lipo-chitooligosaccharide (LCO), bacteria-to-plant signal was characterized by HPLC, FAB-mass spectroscopy MALDI-TOF mass spectroscopy and revealed to be LCO Nod Bj-V (C18:1, MeFuc). This LCO is produced by type I strains of B. japonicum and is therefore unlikely to account for this strains superior ability to nodulate soybean under Canadian conditions. We also found that use of yeast extract mannitol medium gave similar results to that of Bergerson minimal medium.     

A host-specific bacteria-to-plant signal molecule (Nod factor) enhances germination and early growth of diverse crop plants

Lipo-chitooligosaccharides (LCOs), or Nod factors, are host-specific bacteria-to-plant signal molecules essential for the establishment of a successful N(2)-fixing legume-rhizobia symbiosis. At submicromolar concentrations Nod factors induce physiological changes in host and non-host plants. Here we show that the Nod factor Nod Bj V(C18:1,MeFuc) of Bradyrhizobium japonicum 532C enhances germination of a variety of economically important plants belonging to diverse botanical families: Zea mays, Oryza sativa (Poaceae), Beta vulgaris (Chenopodaceae), Glycine max, Phaseolus vulgaris (Fabaceae), and Gossypium hirsutum (Malvaceae), under laboratory, greenhouse and field conditions. Similar increases in germination were observed for filtrates of genistein-induced cultures of B. japonicum 532C, while non-induced B. japonicum, induced Bj 168 (a nodC mutant of B. japonicum deficient in Nod factor synthesis) or the pentamer of chitin did not invoke such responses, demonstrating the role of Nod factor in the observed effects. In addition, three out of four synthetic LCOs evaluated also promoted germination of corn, soybean and Arabidopsis thaliana seeds. LCO also enhanced the early growth of corn seedlings under greenhouse conditions. These findings suggest the possible use of LCOs for improved crop production.     

Perception of Bradyrhizobium japonicum Nod factor by soybean [Glycine max (L.) Merr.] root hairs under abiotic stress conditions

Suboptimal growth conditions, such as low rhizosphere temperature, high salinity, and low pH can negatively affect the rhizobia-legume symbioses, resulting in poor nodulation and lower amounts of nitrogen fixed. Early stages of the Bradyrhizobium japonicum-soybean [Glycine max (L.) Merr.] symbiosis, such as excretion of genistein (the plant-to-bacteria signal) and infection initiation can be inhibited by abiotic stresses; however, the effect on early events modulated by Nod factors (bacteria-to-plant signalling), particularly root hair deformations is unknown. Thus, the objective of this study was to evaluate the perception of Nod factor by soybean root hairs under three stress conditions: low temperature, low pH, and high salinity. Three experiments were conducted using a 1:1 ratio of Nod Bj-V (C(18:1), MeFuc) and Nod Bj-V (Ac, C(16:0), MeFuc). Nod factor induced four types of root hair deformation (HAD), wiggling, bulging, curling, and branching. Under optimal experimental conditions root hair response to the three levels of Nod factor tested (10(-6), 10(-8), and 10(-10) M) was dose-dependent. The highest frequency of root hair deformations was elicited by the 10(-6) M level. Root hair deformation decreased with temperature (25, 17, and 15 degrees C), low pH, and high salinity. Nod factor concentration did not interact with either low temperature or pH. However, salinity strongly inhibited HAD responses to increases in Nod factor concentration. Thus, the addition of higher levels of Nod factor is able to overcome the effects of low pH and temperature stress, but not salinity.     

Jasmonates induce Nod factor production by Bradyrhizobium japonicum

Jasmonates are signaling molecules involved in induced systemic resistance, wounding and stress responses of plants. We have previously demonstrated that jasmonates can induce nod genes of Bradyrhizobium japonicum when measured by beta-galactosidase activity. In order to test whether jasmonates can effectively induce the production and secretion of Nod factors (lipo-chitooligosaccharides, LCOs) from B. japonicum, we induced two B. japonicum strains, 532C and USDA3, with jasmonic acid (JA), methyl jasmonate (MeJA) and genistein (Ge). As genistein is well characterized as an inducer of nod genes it was used a positive control. The high-performance liquid chromatography (HPLC) profile of LCOs isolated following treatment with jasmonates or genistein showed that both JA and MeJA effectively induced nod genes and caused production of LCOs from bacterial cultures. JA and MeJA are more efficacious inducers of LCO production than genistein. Genistein plus JA or MeJA resulted in greater LCO production than either alone. A soybean root hair deformation assay showed that jasmonate induced LCOs were as effective as those induced by genistein. This is the first report that jasmonates induce Nod factor production by B. japonicum. This report establishes the role of jasmonates as a new class of signaling molecules in the Bradyrhizobium-soybean symbiosis.     

Differential response of soybean (Glycine max (L.) Merr.) genotypes to lipo-chito-oligosaccharide Nod Bj V (C(18:1) MeFuc)

Lipo-chito-oligosaccharides (LCOs) are bacteria-to-plant signal molecules essential for the establishment of rhizobia-legume symbioses. LCOs invoke a number of physiological changes in the host plants, such as root hair deformation, cortical cell division and ontogeny of complete nodule structures. The responses of five soybean cultivars to Nod BJ: V (C(18:1) MeFuc) isolated from Bradyrhizobium japonicum strain 532C were studied with a new technique. Two distinct types of root hair deformation were evident (i) bulging, in which root hairs were swollen at the tip or at the base depending on the cultivars and (ii) curling. The nodulating capacity of B. japonicum 532C varied among cultivars. Cultivars that produced a bulging reaction when treated with LCO had fewer nodules and the roots had low phenol contents. Cultivars that produced curling had higher numbers of nodules and the roots had higher amounts of phenol. Further, the roots of cultivars that showed root hair bulging were able to degrade LCO much faster than cultivars that manifested curling. The results of the present study establish relationships among the type of LCO-induced root hair deformation, root system LCO-degrading ability and nodulation capacity of soybean cultivars.     

Evidence for the production of chemical compounds analogous to nod factor by the silicate bacterium Bacillus circulans GY92

Silicate bacteria are generally placed in the species Bacillus circulans and are widely used in biological fertilisers and biological leaching. The bacteria can form conspicuous amounts of extracellular polysaccharides in nitrogen-free media or in the presence of substrates with large C/N ratios. Using high performance liquid chromatography, we have shown that B. circulans produced a new peak/compound when induced with the plant-to-bacteria signal molecule genistein. This material co-eluted with the lipo-chitooligosaccharide (Nod Bj-V (C18:1, MeFuc)) of Bradyrhizobium japonicum. This compound exhibited root hair deformation activity on soybean, which is characteristic of lipo-chitooligosaccharides (LCOs). We propose that this might be an LCO or closely related compound with similar biological activity.     

Nod factor induces soybean resistance to powdery mildew.

Plants possess highly sensitive perception systems by which microbial signal molecules are recognized. In the Bradyrhizobium-soybean (Glycine max (L.) Merr.) symbiosis, recognition is initiated through exchange of signal molecules, generally flavonoids from soybean and lipo-chitooligosaccharides (Nod factors) from the microsymbiont. Application of the Nod factor Nod Bj-V (C18:1, MeFuc) induced soybean resistance to powdery mildew caused by Microsphaera diffusa. Addition of Nod factor (concentrations ranging from 10(-6) to 10(-10) M) to soybean root systems led to reductions in disease incidence. The lowest disease incidence was caused by Nod factor treatment at 10(-6) M. The effect of Nod factor application on fungal growth and development was measured at 4, 12, 48, and 96 h after inoculation. Colony diameter and number of germ tubes per conidium were decreased by 10(-6) M Nod factor. Phenylalanine ammonia lyase (PAL, EC.4.3.1.1.) is the first enzyme of the phenyl propanoid pathway, and is commonly activated as part of plant responses to disease. Treatment of soybean seedlings with Nod factor, through stem wounds, induced PAL activity; the most rapid increase followed treatment with 10(-6) M Nod factor. These data show that soybean plants are able to detect root applied LCO and respond by increased disease resistance.     

Genistein preincubation of Bradyrhizobium japonicum cells improves strain competitiveness under greenhouse,but not field conditions

In the soybean-B. japonicum symbiosis, genistein has been identified as one of the major compounds in soybean seed and root exudates responsible for inducing expression of the B. japonicum nodYABC operon. In this study, we have tested the possibility that genistein treatment prior to inoculation can increase the competitiveness of the treated B. japonicum strain under both greenhouse and field conditions. Two mutants of the two B. japonicum strains each with a different antibiotic resistant marker were selected. They were tested with one or the other treated with genistein. The results showed genistein treated mutants had higher levels of nodule occupancy than the untreated mutant or parent strain under greenhouse conditions. Mutants from 532C had higher nodule occupancies than mutants from USDA110, especially at 15 °C. In the more complex field environment, genistein treated mutants formed fewer nodules than the untreated mutants. The contradictory results of strain competitiveness for greenhouse and field experiments are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

The major Nod factor of Bradyrhizobium japonicum promotes early growth of soybean and corn

Greenhouse experiments were conducted to evaluate the effect of Nod factor Nod Bj-V (C18:1, MeFuc) of Badyrhizobium japonicum on the growth of soybean and corn. Three-day-old seedlings of soybean and corn were grown in hydroponic solutions containing four concentrations (0, 10(-7), 10(-9) or 10(-11) M) of Nod factor. After 7 d of treatment, Nod factor enhanced soybean and corn biomass. Nod factor elicited profound effects on root growth resulting in 34-44% longer roots in soybean. More detailed analyses of the roots, using a scanner based image analysis system, revealed that Nod factor increased the total length, projected area and surface area of the roots and decreased the diameter of soybean roots, while it increased the total length of corn roots. Stem injection of soybean plants with 10(-7) M Nod factor resulted in increased dry matter accumulation. These results suggest that Nod factor, besides mediating early stages of nodulation, has more general plant growth-promoting effects.     

Rhizobial Nodulation Factors Stimulate Mycorrhizal Colonization of Nodulating and Nonnodulating Soybeans

Legumes form tripartite symbiotic associations with noduleinducing rhizobia and vesicular-arbuscular mycorrhizal fungi. Co-inoculation of soybean (Glycine max [L.] Merr.) roots with Bradyrhizobium japonicum 61-A-101 considerably enhanced colonization by the mycorrhizal fungus Glomus mosseae. A similar stimulatory effect on mycorrhizal colonization was also observed in nonnodulating soybean mutants when inoculated with Bradyrhizobium japonicum and in wild-type soybean plants when inoculated with ineffective rhizobial strains, indicating that a functional rhizobial symbiosis is not necessary for enhanced mycorrhiza formation. Inoculation with the mutant Rhizobium sp. NGR[delta]nodABC, unable to produce nodulation (Nod) factors, did not show any effect on mycorrhiza. Highly purified Nod factors also increased the degree of mycorrhizal colonization. Nod factors from Rhizobium sp. NGR234 differed in their potential to promote fungal colonization. The acetylated factor NodNGR-V (MeFuc, Ac), added at concentrations as low as 10-9 M, was active, whereas the sulfated factor, NodNGR-V (MeFuc, S), was inactive. Several soybean flavonoids known to accumulate in response to the acetylated Nod factor showed a similar promoting effect on mycorrhiza. These results suggest that plant flavonoids mediate the Nod factor-induced stimulation of mycorrhizal colonization in soybean roots.     

Early seedling and seasonal N2-fixing symbiotic activity of two soybean [Glycine max (L.) Merr.] cultivars inoculated with Bradyrhizobium strains of diverse origin

In areas with a short growing season the poor adaptability of soybean [Glycine max Meer. (L.)] to cool soil conditions is considered the primary yield limiting factor. Soybean requires temperatures in the 25 to 30°C range for optimum N₂-fixation and yield. Field studies were conducted in 1990 and 1991 at Montreal, Quebec to determine whether adaptability to cool soil conditions, with respect to earlier symbiosis establishment and function, existed among either Bradyrhizobium strains or soybean genotypes. An early maturing isoline of the soybean cultivar Evans and the cultivar Maple Arrow were inoculated with one of four strains isolated from the cold soils of Hakkaido, northern Japan, or the commercially used strains 532C or USDA110, at two planting dates. Plot biomass and nodulation were assessed at seedling (V2), and flowering(R2) growth stages and harvest maturity. Soybean genotypes did not differ for pre-flowering nodulation or N₂-fixation in the cool spring conditions of the first year. Seasonal N₂-fixation rates were also determined at the final harvest by the N-balance and ¹⁵N-isotope dilution methods. Significantly higher symbiotic activity was found for two of the four Hakkaido strains and was reflected in higher final soybean seed yield and total N₂-fixation for the growing season, as compared to the two commercial strains. Planting 14 days earlier resulted in greater early vegetative and total seasonal N₂ fixation and yield in the second year when soil temperatures were warmer, emphasizing the need for the development of soybean-Bradyrhizobium combinations superior in nodule development and function under cool soil conditions.     

Soil temperature effects on competitiveness and growth ofRhizobium japonicum and on Rhizobium-induced chlorosis of soybeans

Summary The effects of temperature on growth in broth and soil and on competition for nodule formation betweenRhizobium japonicum serotypes USDA 76 and 94 compared to 6 and 110 were studied. Increasing root temperatures of Lee soybean from 20 to 35°C increased the competitiveness of 76 and 94 relative to 6 and 110 for all inoculum ratios such that at 30 and 35°C symptoms ofRhizobium-induced chlorosis appeared. Tolerance to elevated temperatures was exhibited by 76 and 110, but not 94 and 6 in broth and soil which suggested that increased competitiveness of 76 and 94 at high soil temperatures was not dependent upon growth at elevated temperatures. Nodulation and vegetative growth of Lee soybeans were at a minimum at 20°C and optimum at 30°C. Differences in competitiveness of 6 to previous studies indicated the need to standardize temperatures of assays. Differences in growth responses of 76 and 94 to temperature from a previous study suggested a confounding effect on different carbon sources in growth media. Scientific Article No. A-3721 Contribution No. 6697 of the Maryland Agric Exp Sta, Dept of Agronomy, College Park, MD 20742 and the USDA, ARS, Beltsville, MD 20705. Part of a thesis submitted by the senior author in partial fulfillment of the requirements for the M.S. Degree.     

Incubation period, spore egestion and horizontal transmission of Nosema fumiferanae (Microsporidia: Nosematidae) in spruce budworm (Choristoneura sp., Lepidoptera: Tortricidae): the role of temperature and dose

Various instars of Choristoneura occidentalis were fed with a range of doses of Nosema fumiferanae and reared at 20, 24 and 28 degrees C to determine the influence of temperature and dose on the time to spore egestion and the number of spores egested in the frass. When larvae were fed in the third stadium, as few as 10(2) spores per larva initiated infection, and both onset of spore egestion and the number of spores egested were affected by a complex relationship between temperature and inoculation dose. Onset of spore egestion varied from 11 to 15 days postinoculation. At 20 degrees C, the onset was delayed and spore production decreased with increasing inoculation dose whereas at higher temperatures spores were first egested at the lowest dose and spore production increased with dose. When larvae were fed spores in the fifth and sixth stadium, no spores were egested because pupation occurred before completion of the incubation period. To assess the effect of temperature on horizontal transmission, Choristoneura fumiferana larvae fed with 10(4) N. fumiferanae spores per larva were reared with uninfected larvae at 15, 20 and 25 degrees C. At 15 degrees C, we observed the highest degree of horizontal transmission, defined by the largest change in N. fumiferanae prevalence, even though the density of spores available for horizontal transmission was the lowest. Infected adults eclosed later than uninfected adults and the time to eclosion was also dependent on sex and temperature. We relate our experimental findings to consequences for horizontal and vertical transmission of N. fumiferanae in spruce budworm populations.     

Nod factor enhances calcium uptake by soybean

Inoculation with rhizobia or application of Nod factors (lipo-chitooligosaccharides, LCOs) causes transient increases in cytosolic calcium concentration in root hairs of legume plants. We conducted experiments to evaluate whether application of LCO and inoculation with rhizobia improved (45)CaCl(2) uptake into soybean (Glycine max [L.] Merr.) leaves. Roots of soybean seedlings with one developing trifoliolate were immersed in Murashige and Skoog (MS) basal liquid medium containing treatment solutions and (45)CaCl(2), and the plants were incubated under continuous light. After 24 h, leaf samples were taken, and their radioactivity levels were determined. Addition of NodBj-V (C18:1 MeFuc) at a concentration of 10(-7) M increased (45)Ca(2+) uptake. Inoculation with genistein-induced Bradyrhizobium japonicum strain 532C and USDA3 also increased (45)Ca(2+) uptake; whereas, inoculation with strain Bj-168, a nodC-mutant incapable of producing LCO, did not. Rhizobia that do not normally nodulate soybean, i.e. Rhizobium leguminosarum, and Sinorhizobium meliloti did not affect calcium uptake, nor did the tetramer or pentamer of chitosan, or lumichrome. Surprisingly, Rhizobium sp. NGR234, which can nodulate some types of soybean, although without effective N(2)-fixation, also did not affect calcium uptake. This work suggests that the rhizobial symbiosis, in addition to its known role in provision of nitrogen fixation, also improves early calcium uptake into soybean plants.     

Chitinases produced by Paenibacillus illinoisensis and Bacillus thuringiensis subsp. pakistani degrade Nod factor from Bradyrhizobium japonicum

Chitinases are enzymes that hydrolyze internal β-1,4-N-acetyl-d-glucosamine linkages of chitin. Since the backbone of Nod factors is a chitin oligomer, we investigated whether chitinases produced by soil bacteria Paenibacillus illinoisensis KJA-424 and Bacillus thuringiensis subsp. Pakistani HD 395 are able to degrade Nod factor produced by Bradyrhizobium japonicum, a phenomenon that could disrupt B. japonicum-soybean signaling and nodule establishment when chitinases are present. Purified Nod factor [LCO Nod Bj-V (C_18:1, MeFuc)] was isolated from Bradyrhizobium japonicum and incubated with crude chitinases isolated from KJA-424 and HD395, with or without acetate buffer.

After 15 h of incubation, Nod factor in the resulting solution was quantified by HPLC. Degradation was greatest following treatment with KJA-424 (91.9%) and HD395 (86.5%) chitinases in acetate buffer. Treatments that included acetate buffer had higher levels of degradation than those without. For all treatments degradation was greater than 77%.     

Cassava as a Cheap Source of Carbon for Rhizobial Inoculant Production using an Amylase-producing Fungus and a Glycerol-producing Yeast

Summary The aim of this research was to develop methods to use low-cost carbon compounds for rhizobial inoculant production. Five raw starch materials; steamed cassava, sticky rice, fresh corn, dry corn and sorghum were tested for sugar production by an amylase-producing fungus. Streamed cassava produced the highest amount of reducing sugar after fermentation. Bradyrhizobium japonicum USDA110, Azorhizobium caulinodans IRBG23, Rhizobium phaseoli TAL1383, Sinorhizobium fredii HH103, and Mesorhizobium ciceri USDA2429 were tested on minimal medium supplemented with reducing sugar obtained from cassava fermentation. All strains, except B. japonicum USDA110, could grow in medium containing cassava sugar derived from 100 g steamed cassava per litre, and the growth rates for these strains were similar to those in medium containing 0.5 (w/v) mannitol. The sugar derived from steamed cassava was further used for production of glycerol using yeast. After 1 day of yeast fermentation, the culture containing glycerol and heat-killed yeast cells, was used to formulate media for culturing bradyrhizobia. A formulation medium, FM4, with a glycerol concentration of 0.6 g/l and yeast cells (OD₆₀₀ = 0.1) supported growth of B. japonicum USDA110 up to 3.61 × 10⁹ c.f.u./ml in 7 days. These results demonstrate that steamed cassava could be used to provide cheap and effective carbon sources for rhizobial inoculant production.     

A lipochito-oligosaccharide, Nod factor, induces transient calcium influx in soybean suspension-cultured cells

Lipochito-oligosaccharides (Nod factors) produced by Rhizobium or Bradyrhizobium are the key signal molecules for eliciting nodulation in their corresponding host legumes. To elucidate the signal transduction events mediated by Nod factors, we investigated the effects of Nod factors on the cytosolic [Ca2+] of protoplasts prepared from roots and suspension-cultured cells of soybean (Glycine max and G. soja) using a fluorescent Ca2+ indicator, Fura-PE3. NodBj-V (C18:1, MeFuc), which is a major component of Nod factors produced by Bradyrhizobium japonicum, induces transient elevation of cytosolic [Ca2+] in the cells of soybean within a few minutes. This effect is specific to soybean cells and was not observed in the tobacco BY-2 cells. Furthermore, NodBj-V without MeFuc did not induce any cytosolic [Ca2+] elevation in soybean cells. Exclusion of Ca2+ from the medium, as well as pre-treatment of the cells with an external Ca2+ chelator or with a plasma membrane voltage-dependent Ca2+ channel inhibitor, suppressed the Nod factor-dependent cytosolic [Ca2+] elevation. These results indicate that transient Ca2+ influx from extracellular fluid is one of the earliest responses of soybean cells to NodBj-V (C18:1, MeFuc) in a host-specific manner.     

Photosynthetic Bradyrhizobium Sp. strain ORS285 synthesizes 2-O-methylfucosylated lipochitooligosaccharides for nod gene? Dependent interaction with Aeschynomene plants

Bradyrhizobium sp. strain ORS285 is a photosynthetic bacterium that forms nitrogen-fixing nodules on the roots and stems of tropical aquatic legumes of the Aeschynomene genus. The symbiotic interaction of Bradyrhizobium sp. strain ORS285 with certain Aeschynomene spp. depends on the presence of nodulation (nod) genes whereas the interaction with other species is nod gene independent. To study the nod gene-dependent molecular dialogue between Bradyrhizobium sp. strain ORS285 and Aeschynomene spp., we used a nodB-lacZ reporter strain to monitor the nod gene expression with various flavonoids. The flavanones liquiritigenin and naringenin were found to be the strongest inducers of nod gene expression. Chemical analysis of the culture supernatant of cells grown in the presence of naringenin showed that the major Nod factor produced by Bradyrhizobium sp. strain ORS285 is a modified chitin pentasaccharide molecule with a terminal N-C(18:1)-glucosamine and with a 2-O-methyl fucose linked to C-6 of the reducing glucosamine. In this respect, the Bradyrhizobium sp. strain ORS285 Nod factor is the same as the major Nod factor produced by the nonphotosynthetic Bradyrhizobium japonicum USDA110 that nodulates the roots of soybean. This suggests a classic nod gene-dependent molecular dialogue between Bradyrhizobium sp. strain ORS285 and certain Aeschynomene spp. This is supported by the fact that B. japonicum USDA110 is able to form N(2)-fixing nodules on both the roots and stems of Aeschynomene afraspera.     

Low sequence similarity and gene content of symbiotic clusters of Bradyrhizobium sp. WM9 (Lupinus) indicate early divergence of “lupin” lineage in the genus Bradyrhizobium

Two sequenced nodulation regions of lupin Bradyrhizobium sp. WM9 carried the majority of genes involved in the Nod factor production. The nod region I harbored: nolA, nodD, nodA, nodB, nodC, nodS, nodI, nodJ, nolO, nodZ, fixR, nifA, fixA, nodM, nolK and noeL. This gene arrangement resembled that found in the nodulation region of Bradyrhizobium japonicum USDA110, however strain WM9 harbored only one nodD gene copy, while the nodM, nolK and noeL genes had no counterparts in the 410 kb symbiotic region of strain USDA110. Region II harbored nolL and nodW, but lacked an nodV gene. Both regions carried ORFs that lacked similarity to the published USDA110 sequences, though they had homologues in symbiotic regions of Rhizobium etli, Sinorhizobium sp. NGR234 and Mesorhizobium loti. These differences in gene content, as well as a low average sequence identity (70%) of symbiotic genes with respect to B. japonicum USDA110 were in contrast with the phylogenetic relationship of USDA110 and WM9 revealed by the analysis of 16S rDNA and dnaK sequences. This most likely reflected an early divergence of symbiotic loci, and possible co-speciation with distinct legumes. During this process the loss of a noeI gene and the acquisition of a nolL gene could be regarded as an adaptation towards these legumes that responded to Nod factors carrying 4-O-acetylfucose rather than 2-O-methylfucose. This explained various responses of lupins and serradella plants to infection by mutants in nodZ and nolL genes, knowing that serradella is a stringent legume while lupins are more promiscuous legumes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Growth of Rhizobium japonicum Strains at Temperatures Above 27 degrees C

A study was conducted to examine the growth responses of different Rhizobium japonicum strains to increasing temperatures, determine the degree of variability among strains in those responses, and identify temperature-related growth characteristics that could be used to select temperature-tolerant strains. Each of 42 strains was grown in liquid culture for 96 h at 19 incubation temperatures ranging from 27.4 to 54.1 degrees C in a temperature gradient apparatus. Growth was estimated by measuring the change in optical density over time. Strains differed in their responses to increasing temperatures. Three characteristic temperatures were determined for each strain: the temperature giving the maximum optical density at 96 h (optimum temperature), the maximum temperature allowing a continuous increase in optical density during the 96-h period (maximum permissive temperature), and the maximum temperature allowing growth of the cultures after they were transferred to a uniform incubation temperature of 28 degrees C (maximum survival temperature). The three characteristic temperatures varied among strains and had the following ranges: optimum temperature, from 27.4 to 35.2 degrees C; maximum permissive temperature, from 29.8 to 38.0 degrees C; and maximum survival temperature, from 33.7 to 48.7 degrees C. Significant positive correlations were found between maximum permissive temperature and optimum temperature and between maximum permissive temperature and maximum survival temperature. Eight strains which had the highest maximum permissive temperature, optimum temperature, and maximum survival temperature were considered tolerant of high temperatures and were able to grow at temperatures higher than those previously reported for the most tolerant R. japonicum strains. The strains were of diverse geographical origin, but the response to high temperatures was not related to their origin. Evaluation of the temperature responses in pure culture may be useful in the search for R. japonicum strains better suited to environments in which high soil temperature is a limiting factor.