Nodulation of Hydroponically Grown Soybean Plants and Inhibition of Nodule Development by Nitrate

A procedure is described for obtaining a large number of nodules(approximately 1000) on a hydroponically grown soybean plantafter a brief exposure to Rhizobium japonicum. This procedurewas used to study the effect of medium nitrate upon nodulationof the soybean plant. It was found that nitrate, at concentrationsequal to or greater than 2.0 mM, restricted nodule developmentbut did not inhibit appreciably the activity or synthesis ofnitrogenase in those few nodules that were formed. Key words: Soybean, Rhizobium japonicum, Nodulation     

Synergetic Cultures of Glycine max Root Cells and Rhizobia Separated by Membrane Filters

When suspension cultures of actively growing soybean (Glycine max L.) root cells were separated by two or three membrane filters from suspension cultures of the bacteria, a synergetic (cooperative) activation of nitrogenase was observed in the Rhizobium japonicum used in the bacterial side. Either plant cells or plant cell-conditioned medium was needed for this activation to take place. Both acetylene reduction and hydrogen evolution by the activated R. japonicum persisted for several days after removal from the apparatus when (a) a suitable carbon source was provided, (b) oxygen supply was limited, and (c) growth of bacteria was suppressed by lowering of ammonia and nitrate concentrations. Activation could also take place when the bacteria were placed in media to which plant cell-conditioned medium was added. The advantages of this method for studies on symbiosis are discussed.     

Agglutination ofRhizobium japonicum 3I1b110 by soybean lectin

Summary Conditions leading to agglutination ofRhizobium japonicum 3I1b110 with soybean seed lectin were examined. Ability of cells to be agglutinated was transient and was optimal for cultures grown for 4–5 days on yeast extract mannitol plates. Similar lectin-binding results were obtained with cells from the same cultures using fluorescence microscopy with fluorescein isothiocyanate-labelled lectin. These results revise the previous model for soybean lectin-R. japonicum interactions, since it was based on the inability of soybean lectin to agglutinate these bacteria.     

Hydrogen (h(2)) evolution by rhizobia after synergetic culture with soybean cell suspensions

Rhizobium japonicum cells were grown in liquid suspension cultures and separated from soybean plant cells by two to three bacterial membrane filters. Under these conditions, the plant cells elaborated materials into the medium which aided in the expression of a major rhizobial phenotype, namely, nitrogenase activity (acetylene reduction). The evolution of H₂ was also measured and this activity relative to acetylene reduction, was influenced by: (a) O₂; (b) the quantity of conditioned plant medium; and (c) ammonia. It is concluded that plant substances are of major importance in the H₂ evolution and nitrogenase activities of free-living rhizobia in suspension cultures.     

The effect of growth medium on lectin binding byRhizobium japonicum

Summary A comparative study of soybean lectin binding byRhizobium japonicum 61A76 grown in yeast extract mannitol medium supplemented with soil extract showed that it produced cells with greater lectin binding capacity, larger cell size and a higher percentage of coccoid forms than those produced in yeast extract mannitol medium alone.     

Role of Pili (Fimbriae) in Attachment of Bradyrhizobium japonicum to Soybean Roots

Pili (fimbriae) were observed on cells of each of the five strains of Bradyrhizobium japonicum and the one strain of Rhizobium trifolii examined. Pili on B. japonicum were about 4 nm in diameter and polarly expressed. Piliated cells were estimated by transmission electron microscopy and hydrophobic attachment to polystyrene to constitute only a small percentage of the total population. The proportion of piliated cells in these populations was dependent on culture age in some strains. Piliated B. japonicum cells were selectively and quantitatively removed from suspension when cultures were incubated with either soybean roots or hydrophobic plastic surfaces, indicating that pili were involved in the attachment of the bacteria to these surfaces. Pili from B. japonicum 110 ARS were purified and found to have a subunit molecular weight of approximately 21,000. Treatment of B. japonicum suspensions with antiserum against the isolated pili reduced attachment to soybean roots by about 90% and nodulation by about 80%. Pili appear to be important mediators of attachment of B. japonicum to soybean roots under the conditions examined.     

Some USDA studies on the soybean-Rhizobium symbiosis

Summary The ecology, strain evaluation, genetics of host strain interactions and physiology of nitrogen fixation ofRhizobium japonicum in association with the soybean,Glycine max, were studied. Results of inoculation experiments with selected strains ofRhizobium japonicum indicated that indigenous strains occupied most of the nodules of soybeans grown in highRhizobium japonicum populated soils. Nodule sampling indicated that inoculation did not result in quicker nodulation or a higher incidence of root nodules (primary or secondary) than uninoculated checks. Rhizosphere studies indicated that colonization by introduced strains did occur but did not compete successfully with field strains for nodule sites. Recovery of specific serological types from nodules was influenced by planting intervals. The distribution of the serotypes varied with the time of planting and the age of the plant. Temperature studies indicated that the distribution of serotypes recovered from the nodules was influenced by temperature. Field studies showed the selectivity of soybean genotypes on strains ofRhizobium japonicum. Some strains were more common in the nodules of some varieties than in others. Closely related varieties had similar populations in their nodules. Three genes which control nodule response in soybeans are reported. Nitrogen fixation profiles were determined for some variety-strain interactions. Combinations previously classified as inefficient showed some nitrogenase activity as measured by the acetylene reduction technique. Research Microbiologist; Research Agronomist; Research Plant Physiologist, Soybean Investigations, Crops Research Division, Beltsville, Md. (USDA, ARS); and Plant Pathologist currently located at Michigan State University, East Lansing, Michigan.     

Isolation from Rhizobium for factors which transfer electrons to soybean nitrogenase

Rhizobium japonicum grown anaerobically with nitrate produced several factors capable of transferring electrons from spinach chloroplasts lacking photosystem II to soybean root nodule nitrogenase. The same organism grown aerobically produced no factors with detectable activity. It is concluded that the bacterium is potentially capable of producing these proteins in the low oxygen conditions of the leguminous root nodule.     

A new perfusion system for the measurement and characterization of potential rates of soil nitrification

The effect of two isoflavonoids, coumestrol and daidzein which are present in aseptically grown roots and root exudates of soybean, was tested on some rhizospheric microorganisms. It was found that coumestrol promotes the growth ofR. japonicum USDA 138 (about 30%) andR. leguminosarum (about 15%) whereas it inhibits the growth ofAgrobacterium tumefaciens (about 50%) andPseudomonas sp. (about 20%). The following microorganisms were unaffected by this molecule:R. japonicum W505,Agrobacterium radiobacter, Micrococcus luteus andCryptococcus laurentii. It was found that daidzein promotesR. japonicum USDA 138 growth (about 20%) and inhibitsPseudomonas sp. growth (about 20%); other microorganisms were unaffected. In addition, coumestrol favoured the formation of ‘coccoids’ cells byRhizobium japonicum USDA 138 which could be the infective state of this strain. It seems that this compound is able to help nodulation of soybean by aRhizobium strain. This result supports the work of Peterset al. (1986) and Redmondet al. (1986) who show that flavones present in plant exudates induces expression of nodulation genes in Rhizobium.     

Competitive growth of slow growingRhizobium japonicum against fast growingEnterobacter andPseudomonas species at low concentrations of succinate and other substrates in dialysis culture

A cultivation system with simultaneous growth of six bacterial cultures in separate bags in dialysis culture was developed. In a medium with no added carbon source (one half concentrated Hoagland solution, water deionized and distilled), cell number ofRhizobium japonicum increased during a 7 day period by a factor of 35, whereas the number ofEnterobacter aerogenes cells decreased to one half. With a concentration of 100 nM succinate as an additional carbon source in the inflow,Rhizobium japonicum 61-A-101 cell number increased by a factor of 50 during an 8 day period, whereas cell number ofEnterobacter cloacae NCTC 10005 only doubled and ofEnterobacter aerogenes NCTC 10006 decreased. At 10 mM concentration of succinate in the inflow, doubling time the twoEnterobacter strains was about 12 h, compared to about 24 h for theRhizobium japonicum strain. Varying the succinate concentration from 10 mM to 100 nM in the inflow,Rhizobium japonicum 61-A-101 surpassed theEnterobacter aerogenes strains in the growth rate between 1 mM and 100 M succinate in the inflowing medium. Three otherRhizobium japonicum strains (fix⁺ and fix^-) did grow with a similar rate as strain 61-A-101 at very low concentrations of substrate. Growth rates for the strains were confirmed by protein data per culture. Growing in competition with twoPseudomonas strains,Rhizobium japonicum RH 31 Marburg (fix^-) did overgrow alsoPseudomonas fluorescens, was however outgrown byPseudomonas putida. In utilizing low concentrations of a¹⁴C labelled organic acid (malonate), three strains ofRhizobium japonicum left 2–4 times smaller amounts of¹⁴C in the medium than two species ofPseudomonas and two species ofArthrobacter.On sabbatical leave at ANU     

Influence of Glycine spp. on Competitiveness of Bradyrhizobium japonicum and Rhizobium fredii

The displacement of indigenous Bradyrhizobium japonicum in soybean nodules with more effective strains offers the possibility of enhanced N₂ fixation in soybean (Glycine max (L.) Merr.). Our objective was to determine whether the wild soybean (G. soja Sieb. & Zucc.) genotype PI 468397 would cause reduced competitiveness of important indigenous B. japonicum strains USDA 31, 76, and 123 and thereby permit nodulation by Rhizobium fredii, the fast-growing microsymbiont of soybean. In an initial experiment, PI 468397 nodulated and fixed moderate amounts of N₂ with USDA 31 and 76 but, despite the formation of nodules, fixed essentially no N₂ with USDA 123. In contrast, PI 468397 formed a highly effective symbiosis with R. fredii strain USDA 193. In two subsequent experiments, Williams soybean and PI 468397 were grown in a pasteurized soil mixture or in soybean rhizobium-free soil and inoculated with both USDA 123 and USDA 193. In each experiment, more than 90% of the nodules of Williams contained USDA 123, while only a maximum of 2% were occupied with USDA 193. In contrast, in the two experiments, 16 and 11%, respectively, of the nodules produced on PI 468397 were occupied by USDA 123, while in both experiments 87% contained USDA 193. Thus, in relation to the cultivar Williams, which is commonly grown and used as a parent in soybean breeding programs in the United States, PI 468397 substantially reduced the competitive ability of B. japonicum strain USDA 123 in relation to R. fredii strain USDA 193.     

The adaptive acid tolerance response in root nodule bacteria and Escherichia coli

Root nodule bacteria and Escherichia coli show an adaptive acid tolerance response when grown under mildly acidic conditions. This is defined in terms of the rate of cell death upon exposure to acid shock at pH 3.0 and expressed in terms of a decimal reduction time, D. The D values varied with the strain and the pH of the culture medium. Early exponential phase cells of three strains of Rhizobium leguminosarum (WU95, 3001 and WSM710) had D values of 1, 6 and 5 min respectively when grown at pH 7.0; and D values of 5, 20 and 12 min respectively when grown at pH 5.0. Exponential phase cells of Rhizobium tropici UMR1899, Bradyrhizobium japonicum USDA110 and peanut Bradyrhizobium sp. NC92 were more tolerant with D values of 31, 35 and 42 min when grown at pH 7.0; and 56, 86 and 68 min when grown at pH 5.0. Cells of E. coli UB1301 in early exponential phase at pH 7.0 had a D value of 16 min, whereas at pH 5.0 it was 76 min. Stationary phase cells of R. leguminosarum and E. coli were more tolerant (D values usually 2 to 5-fold higher) than those in exponential phase. Cells of R. leguminosarum bv. trifolii 3001 or E. coli UB1301 transferred from cultures at pH. 7.0 to medium at pH 5.0 grew immediately and induced the acid tolerance response within one generation. This was prevented by the addition of chloramphenicol. Acidadapted cells of Rhizobium leguminosarum bv. trifolii WU95 and 3001; or E. coli UB1301, M3503 and M3504 were as sensitive to UV light as those grown at neutral pH.     

Study of theRhizobium japonicum-soybean symbiosis

Summary Capsular polysaccharides were isolated fromRhizobium japonicum (61A76NS) and conjugated to a fluorescent dye to determine if the specificity in theRhizobium japonicum-soybean symbiosis is expressed by a component (lectin) located on soybean roots which binds to the sugars of the bacterial capsules.The conjugated Fraction A capsular polysaccharides ofR. japonicum bound only to the root hair tips of soybean seedlings. The polysaccharide would not bind specifically to the roots of clover or alfalfa seedlings. Rhodamine conjugated polysaccharides ofR. japonicum could be inhibited from binding to soybean root hairs by the addition of N-acetylgalactosamine or galactose, effective hapten inhibitors of this type of binding. This is the first report of hapten-reversible binding of an isolated rhizobial component to soybean root hairs, the differentiated epidermal cells which are subsequently infected by this nitrogen-fixing symbiont.Paper number6046 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina.     

Root hair cell enhancement in tissue cultures from soybean roots: a useful model system: in vitro Rhizobium symbiosis

A technique for obtaining large numbers of root hair cells in cell cultures from soybeans is described. The cells were grown on agar containing the Prairie Regional Laboratory B5 (PRL-B5) medium for periods longer than 60 days. Mixed populations of cultured root hair cells and cortical cells were used to study the in vitro association between soybean cells and Rhizobium japonicum. The advantages of these types of root cell cultures in studies of symbiosis are discussed.     

Spatial relationships between uninfected and infected cells in root nodules of soybean

In soybean (Glycine max (L.) Merr.) the uninfected cells of the root nodule are responsible for the final steps in ureide production from recently fixed nitrogen. Stereological methods and an original quantitative method were used to investigate the organization of these cells and their spatial relationships to infected cells in the central region of nodules of soybean inoculated with Rhizobium japonicum strain USDA 3I1B110 and grown with and without nitrogen (as nitrate) in the nutrient medium. The volume occupied by the uninfected tissue was 21% of the total volume of the central infected region for nodules of plants grown without nitrate, and 31% for nodules of plants grown with nitrate. Despite their low relative volume, the uninfected cells outnumbered the much larger infected cells in nodules of plants grown both without and with nitrate. The surface density of the interface between the ininfected and infected tissue in the infected region was similar for nodules in both cases also, the total range being from 24 to 26 mm²/mm³. In nodules of plants grown without nitrate, all sampled infected cells were found to be in contact with at least one uninfected cell. The study demonstrates that although the uninfected tissue in soybean nodules occupies a relatively small volume, it is organized so as to produce a large surface area for interaction with the infected tissue.     

Binding and penetration ofRhizobium japonicum to cultured soybean cells

A cultured soybean cell line, SB-1 was used to evaluate the initial interaction between the soybean cells andRhizobium japonicum. Co-culturing ofR. japonicum with SB-1 cells in suspension resulted in strain-specific polar attachment. This attachment can be inhibited by galactose and antibodies raised against seed soybean agglutinin (SBA). A lectin was purified from SB-1 cells which shares properties with SBA in terms of immunological reactivity, sugar binding activity, polypeptide molecular weight and peptide maps. When the SB-1 cells were co-cultured withR. japonicum for three weeks in solid agar medium, histological staining revealed bacterial penetration into certain SB-1 cells. Furthermore, there were focal regions of cells with prominent nuclei representing actively proliferating regions. These observations are analogous to that ofin vivo nodule initiation in soybean roots.     

Subcellular localization of enzymes involved in the assimilation of Ammonia by soybean root nodules

Ammonia, the primary product of nitrogen fixation is rapidly incorporated into a number of amino acids such as glutamate and aspartate. A novel enzyme system glutamine: 2-oxoglutarate aminotransferase oxidoreductase, which probably has an important role in ammonia assimilation has been detected, in the present studies, in the rhizobial fraction of soybean root nodules and in Rhizobium japonicum grown in culture. The role of this latter enzyme and other enzymes such as glutamate dehydrogenase, aspartate aminotransferase and alanine aminotransferase in ammonia assimilation by soybean nodules is discussed.     

Characteristics of gas exchange and morphology of a spring ephemeral, Erythronium japonicum, in comparison with a sun plant, Glycine max

Morphological characteristics and responses of gas exchanges to light intensity were examined in a typical vernal species, Erythronium japonicum Decne (E. japonicum), grown (i) on the floor of a deciduous broad-leaved Quercus mongolica forest (one of its native habitats, the Q. mongolica stand); (ii) bare land left undisturbed for 9 years after forest clearing (the bare stand); and (iii) in a sun crop, soybean, grown for 110 days in an experimental field and for 17 days in pots, in order to evaluate the adaptability of the photosynthetic process of this vernal species to its shady native habitats. The daytime solar radiation, ai and leaf temperatures and leaf–air vapor pressure difference (VPD) were significantly higher at the bare stand than at the Q. mongolica stand. When environmental factors observed at the Q. mongolica and bare stands were reproduced in an assimilation chamber, leaf temperatures of E. japonicum plants increased markedly with increased radiation, whereas those of soybean plants differed little from the respective air temperatures. The photosynthetic and transpiration rates and stomatal conductance in the former plants placed under conditions at the Q. mongolica stand increased with radiation and reached respective steady state values at maximum radiation at the site; whereas, under the conditions at the bare stand, they also increased and reached respective steady state values, but then continuously decreased to be lower than the respective value at the Q. mongolica stand. However, both rates and the conductance in the soybean plants under both conditions increased significantly with radiation and reached much higher respective values at the respective maximum radiations. Water use efficiency for E. japonicum plants was much higher under conditions at the Q. mongolica stand than at the bare stand and was practically equal to those for soybean plants under both conditions. Water potential in the leaves of E. japonicum at maximum radiation at the bare stand was one-third that of those at the Q. mongolica stand. The potential in soybean leaves differed little between both conditions and was roughly equal to the low value in E. japonicum leaves at the bare stand conditions. The stomatal densities on upper and lower leaf surfaces and the ratio of root weight to leaf area (R : L) differed little between E. japonicum plants grown at both stands as well as between young and adult soybean plants. However, the densities on the upper and lower surfaces of E. japonicum were 25% and 60% of the respective values of both soybean plants. The ratios of R : L of the E. japonicum plants were only one-quarter that of the young and adult soybean plants. The cooperation between these morphological and gas exchange characteristics in E. japonicum plants is discussed in relation to adaptation to the environment in native habitats.     

Enhancing Soybean Rhizosphere Colonization by Rhizobium japonicum

A study was conducted to seek means to increase the colonization of the rhizosphere of soybeans (Glycine max L. Merrill) by Rhizobium japonicum. For this purpose, a strain of R. japonicum that was resistant to benomyl, streptomycin, and erythromycin was used. The numbers of R. japonicum rose quickly in the first 2 days after soybean seeds were planted in soil and then rapidly fell. The decline was slower if the seeds were coated with benomyl. This fungicide reduced the numbers of bacteria and protozoa in the rhizosphere, but the effect became less or disappeared as the plants grew. In sterile soil inoculated with R. japonicum and a mixture of microorganisms, the numbers of R. japonicum were usually lower if protozoa were present than if they were absent. Nodulation and plant yield were increased by the addition of benomyl to soybean seeds sown in sterile soil inoculated with R. japonicum and a mixture of microorganisms. The addition of streptomycin and erythromycin to soil stimulated the growth of R. japonicum but inhibited other bacteria in the presence or absence of soybeans. The data indicate that colonization can be increased by the use of antimicrobial agents and R. japonicum strains resistant to those inhibitors.     

Modified Fluorescent Technique, Using Rhodamine, for Studies of Rhizobium japonicum-Soybean Symbiosis

Rhodamine-conjugated capsular polysaccharides isolated from Rhizobium japonicum 61A76NS were used to examine binding between the labeled polysaccharides and soybean roots. Fluorescein-labeled polysaccharides were not satisfactory because soybean root hairs autofluoresce in the fluorescein region.