Symbiosis-stimulated chitinase isoenzymes of soybean (Glycine mas (L.) Merr.)

Isoforms of endochitinase in soybean were studied in relation to root symbiosis. Five selected cultivars differing in their nodulation potential were inoculated with two strains of Bradyrhizobium japonicum, the broad host-range Rhizobium sp. NGR234, and with the mycorrhizal fungus Glomus mosseae. Total chitinase activity in nodules was up to 7-fold higher than in uninoculated roots and in mycorrhizal roots. The chitinase activity in nodules varied depending on the strain-cultivar combination. On semi-native polyacrylamide gels, four acidic isoforms were identified. Two isoforms (CH 2 and CH 4) were constitutively present in al analysed tissues. The other two isoforms (CH 1 and CH 3) were strongly induced in nodules and were simulated in mycorrhizal roots as compared to uninoculated roots. The induction of CH 1 varied in nodules depending on the soybean cultivar. This isoform was also stimulated in uninfected roots when they were treated with tri-iodobenzoic acid, rhizobial lipochitooloigosaccharides (Nod factors) and chitotetraose. CH 3 was not affected by these stimuli indicating that this isoform could represent a marker for enzymes induced in later stages of the symbiotic interactions.Key words: (Brady)rhizobium, chitinase isoenzymes, mycorrhiza, (restricted) nodulation, Nod factors      

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.     

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.     

Sugar-binding activity of pea lectin enhances heterologous infection of transgenic alfalfa plants by Rhizobium leguminosarum biovar viciae

Transgenic alfalfa (Medicago sativa L. cv Regen) roots carrying genes encoding soybean lectin or pea (Pisum sativum) seed lectin (PSL) were inoculated with Bradyrhizobium japonicum or Rhizobium leguminosarum bv viciae, respectively, and their responses were compared with those of comparably inoculated control plants. We found that nodule-like structures formed on alfalfa roots only when the rhizobial strains produced Nod factor from the alfalfa-nodulating strain, Sinorhizobium meliloti. Uninfected nodule-like structures developed on the soybean lectin-transgenic plant roots at very low inoculum concentrations, but bona fide infection threads were not detected even when B. japonicum produced the appropriate S. meliloti Nod factor. In contrast, the PSL-transgenic plants were not only well nodulated but also exhibited infection thread formation in response to R. leguminosarum bv viciae, but only when the bacteria expressed the complete set of S. meliloti nod genes. A few nodules from the PSL-transgenic plant roots were even found to be colonized by R. leguminosarum bv viciae expressing S. meliloti nod genes, but the plants were yellow and senescent, indicating that nitrogen fixation did not take place. Exopolysaccharide appears to be absolutely required for both nodule development and infection thread formation because neither occurred in PSL-transgenic plant roots following inoculation with an Exo(-) R. leguminosarum bv viciae strain that produced S. meliloti Nod factor.     

The Rhizobium sp. strain NGR234 systemically suppresses arbuscular mycorrhizal root colonization in a split‐root system of barley (Hordeum vulgare)

Nitrogen‐fixing bacteria (rhizobia) form a nodule symbiosis with legumes, but also induce certain effects on non‐host plants. Here, we used a split‐root system of barley to examine whether inoculation with Rhizobium sp. strain NGR234 on one side of a split‐root system systemically affects arbuscular mycorrhizal (AM) root colonization on the other side. Mutant strains of NGR234 deficient in Nod factor production (strain NGRΔnodABC), perception of flavonoids (strain NGRΔnodD1) and secretion of type 3 effector proteins (strain NGRΩrhcN) were included in this study. Inoculation resulted in a systemic reduction of AM root colonization with all tested strains. However, the suppressive effect of strain NGRΩrhcN was less pronounced. Moreover, levels of salicylic acid, an endogenous molecule related to plant defense, were increased in roots challenged with rhizobia. These data indicate that barley roots perceived NGR234 and that a systemic regulatory mechanism of AM root colonization was activated. The suppressive effect appears to be Nod factor independent, but enhanced by type 3 effector proteins of NGR234.     

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.     

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.     

Nod factors and tri-iodobenzoic acid stimulate mycorrhizal colonization and affect carbohydrate partitioning in mycorrhizal roots of Lablab purpureus

Roots of Lablab purpureus (L.) Sweet were treated with tri -iodobenzoic acid (TIBA), kinetin or with nodulation factors (Nod factors) purified from Rhizobium sp. NGR234 and grown in the presence of a mycorrhizal inoculum ( Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe. Colonization by the mycorrhizal fungus was increased from <30% to c . 65% of root length when roots were treated with these growth regulators. Moreover, treatment of mycorrhizal L. purpureus roots with Nod factors or TIBA strongly induced sporocarp formation of Glomus mosseae . In parallel, the pool size of the fungal disaccharide trehalose was significantly affected in roots treated with TIBA and Nod factors alone, and with TIBA combined with all effectors, and increased from 0·06 mg g⁻¹ d. wt in control roots to up to 1·7 mg g⁻¹ d. wt (TIBA+kinetin). Conversely, the sucrose pool decreased from 5% d. wt to less than a half in roots treated with Nod factors. Activities of trehalase were significantly enhanced in mycorrhizal roots by the treatment with Nod factors or TIBA. When Nod factors and TIBA were added in combination, these activities were strongly enhanced suggesting synergism between these growth regulators.     

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.     

Nod factors of Rhizobium are a key to the legume door

Symbiotic interactions between rhizobia and legumes are largely controlled by reciprocal signal exchange. Legume roots excrete flavonoids which induce rhizobial nodulation genes to synthesize and excrete lopo-oligosaccharide Nod factors. In turn, Nod factors provoke deformation of the root hairs and nodule primordium formation. Normally, rhizobia enter roots through infection threads in markedly curled root hairs. If Nod factors are responsible for symbiosis-specific root hair deformation, they could also be the signal for entry of rhizobia into legume roots. We tested this hypothesis by adding, at inoculation, NodNGR-factors to signal-production-deficient mutants of the broad-host-range Rhizobium sp. NGR234 and Bradyrhizobium japorticum strain USDA110. Between 10 ⁻⁷ M and 10⁻⁶ M NodNGR factors permitted these NodABC mutants to penetrate, nodulate and fix nitrogen on Vigna unguiculata and Glycine max, respectively. NodNGR factors also allowed Rhizobium fredii strain USDA257 to enter and fix nitrogen on Calopogonium caeruleum, a non-host. Detailed cytological investigations of V. unguiculata showed that the NodABC mutant UGR AnodABC, in the presence of NodNGR factors, entered roots in the same way as the wild-type bacterium. Since infection threads were also present in the resulting nodules, we conclude that Nod factors are the signals that permit rhizobia to penetrate legume roots via infection threads.     

Symbiotic conditions induce structural modifications of Sinorhizobium sp. NGR234 surface polysaccharides

When the rhizosphere is starved of nitrogen, the soil bacteria Rhizobium are able to infect legume roots and invade root nodules, where they can fix atmospheric nitrogen. Nod boxes, the nod gene promoters located on the rhizobial symbiotic plasmid, are activated by means of flavonoids present in the legume root exudates, leading to the synthesis of lipochitooligomers: the Nod factors. Several recent works pointed out the importance of rhizobial surface polysaccharides in establishing the highly specific symbiosis between rhizobia and legumes. Lipopolysaccharides (LPSs) exhibit specific active roles in the later stages of the nodulation processes, such as the penetration of the infection thread into the cortical cells or the setting up of the nitrogen-fixing phenotype. The study reported here concerns the structural modifications affecting surface (lipo)polysaccharides when Sinorhizobium sp. NGR234 strains are grown with nod gene induction under nitrogen starvation. In the absence of induction, NGR234 only produces fast-migrating LPSs. When cultured in the presence of flavonoids, the same strain produces large quantities of a high-molecular-weight rhamnose-rich lipopolysaccharide (RLPS). Because the synthesis of this compound seems to be coded by the symbiotic plasmid under direct or indirect gene induction by flavonoids, this RLPS is thought to be biologically relevant.     

Charcoal and shrubs modify soil processes in ponderosa pine forests of western Montana

In split-root systems of alfalfa (Medicago sativa L.), already existing nodules or arbuscular mycorrhizal roots suppress further establishment of symbiosis in other root parts, a phenomenon named autoregulation. Roots treated with rhizobial nodulation signals (Nod factors) induce a similar systemic suppression of symbiosis.In order to test the hypothesis that flavonoids play a role in this systemic suppression, split-root systems of alfalfa plants were inoculated on one side of the split-root system with Sinorhizobium meliloti or Glomus mosseae or were treated with Nod factor. HPLC-analysis of alfalfa root extracts from both sides of the split-root system revealed a persistent local and systemic accumulation pattern of some flavonoids associated with the different treatments. The two flavonoids, formononetin and ononin, could be identified to be similarily altered after rhizobial or mycorrhizal inoculation or when treated with Nod factor.Exogenous application of formononetin and ononin partially restored nodulation and mycorrhization pointing towards the involvement of these two secondary compounds in the autoregulation of both symbioses.     

Rhizobial communication with rice roots: Induction of phenotypic changes,mode of invasion and extent of colonization

Legume-rhizobial interactions culminate in the formation of structures known as nodules. In this specialized niche, rhizobia are insulated from microbial competition and fix nitrogen which becomes directly available to the legume plant. It has been a long-standing goal in the field of biological nitrogen fixation to extend the nitrogen-fixing symbiosis to non-nodulated cereal plants, such as rice. To achieve this goal, extensive knowledge of the legume-rhizobia symbioses should help in formulating strategies for developing potential rice-rhizobia symbioses or endophytic interactions. As a first step to assess opportunities for developing a rice-rhizobia symbiosis, we evaluated certain aspects of rice-rhizobia associations to determine the extent of predisposition of rice roots for forming an intimate association with rhizobia. Our studies indicate that: a. Rice root exudates do not activate the expression of nodulation genes such as nodY of Bradyrhizobium japonicum USDA110, nodA of R. leguminosarum bv. trifolii, or nodSU of Rhizobium. sp. NGR234; b. Neither viable wild-type rhizobia, nor purified chitolipooligosaccharide (CLOS) Nod factors elicit root hair deformation or true nodule formation in rice; c. Rhizobia-produced indole-3-acetic acid, but neither trans-zeatin nor CLOS Nod factors, seem to promote the formation of thick, short lateral roots in rice; d. Rhizobia develop neither the symbiont-specific pattern of root hair attachment nor extensive cellulose microfibril production on the rice root epidermis; e. A primary mode of rhizobial invasion of rice roots is through cracks in the epidermis and fissures created during emergence of lateral roots; f. This infection process is nod-gene independent, nonspecific, and does not involve the formation of infection threads; g. Endophytic colonization observed so far is restricted to intercellular spaces or within host cells undergoing lysis. h. The cortical sclerenchymatous layer containing tightly packed, thick walled fibers appears to be a significant barrier that restricts rhizobial invasion into deeper layers of the root cortex. Therefore, we conclude that the molecular and cell biology of the Rhizobium-rice association differs in many respects from the biology underlying the development of root nodules in the Rhizobium-legume symbiosis.     

Rhizobial lipo-oligosaccharide nodulation factors: multidimensional chromatographic analysis of symbiotic signals involved in the development of legume root nodules

Nod factors are a group of biologically active oligosaccharidesignals that are secreted by symbiotically competent bacteriaof the family Rhizobiaceae. Their biosynthesis is determinedby rhizobial nodulation (nod) genes, and is specifically inducedin response to flavonoids secreted from the roots of host leguminousplants. The biological activity of Nod factors on these hostlegumes dramatically mimics the early developmental symptomsof the Rhizobium-legame symbiosis including, amongst other effects,root hair deformations and nodule initiation. Structurally,all Nod factors are short oligomers of ß-1,4-linkedN-acetylglucos-amine residues [usually degree of polymerization(dp) 4 or 5] that are N-acylated on the distal glucosarnine.This common ‘core’ structure may be modified bya number of species-specific substituents on the distal or reducingsugars. These modifications are governed by rhizobial host specificitynod genes. The biological activity of purified Nod factors mirrorsthis host specificity, indicating that the symbiotic host rangeof individual Rhizobium species is, at least partially, determinedby the variety of Nod factors they are able to produce. Herewe describe techniques that are universally applicable to theextraction, chromatographic separation and identification ofNod factors. We have applied these techniques to Nod factorsfrom the broad-host-range species Rhizobium fredii USDA257 andRhizobium spp. NGR234, and the more narrow-host-range Bradyrhizobiumjaponicum USDA110, and have identified a group of novel, relativelyhydrophilic Nod factors from the NGR234 species that may haveimplications for Nod factor biosynthesis. lipo-oligosaccharide Nod factor rhibozobia singals TLC     

Phylogeny of fast-growing soybean-nodulating rhizobia support synonymy of Sinorhizobium and Rhizobium and assignment to Rhizobium fredii.

We determined the sequences for a 260-base segment amplified by the polymerase chain reaction (corresponding to positions 44 to 337 in the Escherichia coli 16S rRNA sequence) from seven strains of fast-growing soybean-nodulating rhizobia (including the type strains of Rhizobium fredii chemovar fredii, Rhizobium fredii chemovar siensis, Sinorhizobium fredii, and Sinorhizobium xinjiangensis) and broad-host-range Rhizobium sp. strain NGR 234. These sequences were compared with the corresponding previously published sequences of Rhizobium leguminosarum, Rhizobium meliloti, Agrobacterium tumefaciens, Azorhizobium caulinodans, and Bradyrhizobium japonicum. All of the sequences of the fast-growing soybean rhizobia, including strain NGR 234, were identical to the sequence of R. meliloti and similar to the sequence of R. leguminosarum. These results are discussed in relation to previous findings; we concluded that the fast-growing soybean-nodulating rhizobia belong in the genus Rhizobium and should be called Rhizobium fredii.     

The Rhizobial hemA Gene Is Required for Symbiosis in Species with Deficient [delta]-Aminolevulinic Acid Uptake Activity

Most rhizobial hemA mutants induce root nodules on their respective legume hosts that lack nitrogen fixation activity and leghemoglobin expression. However, a Bradyrhizobium japonicum hemA mutant elicits effective nodules on soybean, and we proposed previously that synthesis and uptake of the heme precursor [delta]-aminolevulinic acid (ALA) by the plant and bacterial symbiont, respectively, allow mutant rescue (I. Sangwan, M.R. O'Brian [1991] Science 251: 1220-1222). In the present work, the B. japonicum hemA mutant MLG1 elicited normal nodules on three hosts, including cowpea, a plant that is not effectively nodulated by a hemA mutant of Rhizobium sp. These data indicate that B. japonicum rather than soybean possesses the unique trait that allows normal nodule development by a hemA mutant. Cowpea expressed glutamate-dependent ALA formation activity in nodules induced by B. japonicum strains I110 or MLG1 and by Rhizobium sp. ANU240. Exogenous ALA was taken up by B. japonicum bacteroids isolated from soybean or cowpea nodules, and the kinetics of uptake were biphasic. By comparison, Rhizobium sp. ANU240 had very low ALA uptake activity. In addition, ALA uptake was observed in cultured cells of B. japonicum but not in cultured cells of three other rhizobial species tested. We suggest that the differential success of legume-rhizobial hemA symbioses is due to an ALA uptake activity in B. japonicum that is deficient in other rhizobia, thereby further validating the ALA rescue hypothesis.     

Endogenous isoflavones are essential for the establishment of symbiosis between soybean and Bradyrhizobium japonicum

Legume iso/flavonoids have been implicated in the nodulation process, but questions remain as to their specific role(s), and no unequivocal evidence exists showing that these compounds are essential for nodulation. Two hypotheses suggest that the primary role of iso/flavonoids is their ability to induce rhizobial nod gene expression and/or their ability to modulate internal root auxin concentrations. The present work provides direct, genetic evidence that isoflavones are essential for nodulation of soybean roots because of their ability to induce the nodulation genes of Bradyrhizobium japonicum. Expression of isoflavone synthase (IFS), a key enzyme in the biosynthesis of isoflavones, is specifically induced by B. japonicum. When IFS was silenced using RNA interference in soybean hairy root composite plants, these plants had severely reduced nodulation. Surprisingly, pre-treatment of B. japonicum or exogenous application to the root system of either of the major soybean isoflavones, daidzein or genistein, failed to restore normal nodulation. Silencing of chalcone reductase led to very low levels of daidzein and increased levels of genistein, but did not affect nodulation, suggesting that the endogenous production of genistein was sufficient to support nodulation. Consistent with a role for isoflavones as endogenous regulators of auxin transport in soybean roots, silencing of IFS resulted in altered auxin-inducible gene expression and auxin transport. However, use of a genistein-hypersensitive B. japonicum strain or purified B. japonicum Nod signals rescued normal nodulation in IFS-silenced roots, indicating that the ability of isoflavones to modulate auxin transport is not essential to nodulation.     

Nod Bj-V (C18:1, MeFuc) production by Bradyrhizobium japonicum (USDA110, 532C) at suboptimal growth temperatures

Nod factors (Lipo-chitooligosaccharides, or LCOs) act as bacteria-to-plant signal molecules that modulate early events of the Bradyrhizobium-soybean symbiosis. It is known that low root zone temperature inhibits the early stages of this symbiosis; however, the effect of low soil temperature on bacteria-to-plant signaling is largely uninvestigated. We evaluated the effect of low growth temperatures on the production kinetics of Nod factor (LCO) by B. japonicum. Two strains of B. japonicum, 532C and USDA110, were tested for ability to synthesize Nod Bj-V (C(18:1), MeFuc) at three growth temperatures (15, 17 and 28 degrees C). The greatest amounts of the major Nod factor, Nod Bj-V (C(18:1), MeFuc), were produced at 28 degrees C for both strains. At 17 and 15 degrees C, the Nod factor production efficiency, per cell, of B. japonicum 532C and USDA110 was markedly decreased with the lowest Nod factor concentration per cell occurring at 15 degrees C. Strain 532C was more efficient at Nod factor production per cell than strain USDA 110 at all growth temperatures. The biological activity of the extracted Nod factor was unaffected by culture temperature. This study constitutes the first demonstration of reduced Nod factor production efficiency (per cell production) under reduced temperatures, suggesting another way that lower temperatures inhibit establishment of the soybean N(2) fixing symbiosis.     

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.