Bacteriocin-like substances produced by Rhizobium japonicum and other slow-growing rhizobia

Bacteriocin-like substances were commonly produced by slow-growing Rhizobium japonicum and cowpea rhizobia on an L-arabinose medium. Antagonism between strains of R. japonicum was not detected in vitro; however, such strains were often sensitive to some bacteriocins produced by cowpea rhizobia. Inhibitory zones (2 to 8 mm from colony margins), produced by 58 of 66 R. japonicum test strains, were reproducibly detected with Corynebacterium nebraskense as an indicator. Quantitative production was not related to symbiotic properties of effective strains, since nine noninfective strains and one ineffective strain produced bacteriocin. Eight R. japonicum strains that did not produce bacteriocin nevertheless formed effective nodules on soybeans. R. japonicum strains that produced bacteriocin in vitro had no antagonistic effect on nonproducer strains during soybean nodulation. Under controlled conditions, a nonproducer (3I1b135) predominated over a bacteriocin producer (3I1b6) when inoculated at 1:1 and 1:9 ratios. Depending on the particular ratio, up to 38% of the total nodules formed were infected with mixed combinations. The bacteriocin(s) had a restricted host range and antibiotic-like properties which included the ability to be dialyzed and resistance to heat (75 to 80 degrees C, 30 min), Pronase, proteinase K, trypsin, ribonuclease, and deoxyribonuclease. R. japonicum strains representing genetic, serological, cultural, and geographic diversity were differentiated into three groups on the basis of bacteriocin production.     

Effect of soil salinity level and zinc application on growth,yield, and nutrient composition of rice

Summary Physiological and symbiotic characteristics were identified in fast-growing (FG)Rhizobium japonicum. Carbon nutritional patterns linked these rhizobia to other FG rhizobia. They were able to use hexoses, pentoses, disaccharides, trioses, and organic acids for growth, but they were unable to use dulcitol or citrate. These rhizobia produced acid with all carbon sources except intermediates of the Krebs cycle. FGR. japonicum showed no vitamin requirements and were tolerant to 1% NaCl but not to 2%. They nodulated cowpea, pigeon pea, and mung bean but not peanut. Effective, nitrogen-fixing symbioses were observed only with cowpea and pigeon pea. In addition, FGR. japonicum formed effective symbioses with Asian-type soybeans. We concluded that although the physiological characteristics of FGR. japonicum were similar to other FG rhizobia, their symbiotic properties were similar to slow-growing rhizobia of the cowpea miscellany.     

A Comparative Study of the Physiology of Symbioses Formed by Rhizobium japonicum with Glycine max, Vigna unguiculata, and Macroptilium atropurpurem

Although Rhizobium japonicum nodulates Vigna unguiculata and Macroptilium atropurpurem, little is known about the physiology of these symbioses. In this study, strains of R. japonicum of varying effectiveness on soybean were examined. The nonhomologous hosts were nodulated by all the strains tested, but effectiveness was not related to that of the homologous host. On siratro, compared to soybean, many strains reversed their relative effectiveness ranking. Both siratro and cowpea produced more dry matter with standard cowpea rhizobia CB756 and 176A22 than with the strains of R. japonicum. Strains USDA33 and USDA74 were more effective with siratro and cowpea than with soybean. The strain USDA122 expressed high rates of hydrogenase activity in symbiosis with the cowpea as well as the soybean host. The strains USDA61 and USDA74 expressed low levels of hydrogenase activity in symbiosis with cowpea, but no activity was found with soybean. Our results indicate host influence for the expression of hydrogenase activity, and suggest the possibility of host influence of nitrogenase for the allocation of electrons to N₂ and H⁺.     

Foliar chlorosis in legumes induced by cowpea rhizobia

Summary While screening cowpea rhizobia from West Africa for ability to nodulate various host species, foliar chlorosis was observed in young mung bean and soybean plants inoculated with certain strains. The chlorosis occurred in the first and sometimes the second trifoliate, but not on subsequent leaves. There was no correlation of symptoms with the presence of nodules. Where extreme chlorosis was induced in soybeans, there was stunting of the primary root. Disease symptoms were obtained with culture-broth supernatants free of rhizobia, indicating an extracellular toxin. In common with rhizobitoxine-producing strains ofR. japonicum, chlorosis-inducing cowpea strains were able to nodulate ‘non-nodulating’ soybeans of the rj₁rj₁ genotype.     

Numerical Taxonomic Analysis of Some Strains of Rhizobium spp. That Uses a Qualitative Coding of Immunodiffusion Reactions

Antigenic relationships among seven strains of Bradyrhizobium japonicum were examined by immunodiffusion reactions, in which cells of each strain were reacted against each of the seven corresponding antisera. Similar analyses were performed with Rhizobium trifolii (28 strains), Rhizobium meliloti (9 strains), and rhizobia of the cowpea miscellany (13 strains). Antigens and antisera were reacted within each species only; serological interspecies cross-reactions were not performed. The results, scored qualitatively as reactions of identity, cross-reactions, or no reaction, were formed into datum matrices and used to analyze the relationships between strains by applying the association measure of Bray and Curtis (J. R. Bray and J. T. Curtis, Ecol. Monogr. 27:325-349, 1957) and the UPGMA clustering algorithm (P. H. A. Sneath and R. R. Sokal, Numerical Taxonomy, 1973). No two strains were regarded as being serologically identical unless each gave the same results as the other in each immunodiffusion reaction against every antiserum. Despite the high level of cross-reactions and reactions of identity (totalling 93% of all cell-antiserum combinations) among strains of R. trifolii and R. meliloti, no strains were identical by the criterion described above; however, the strains of these species clustered rapidly and fused at the 70% similarity level. The B. japonicum strains and the rhizobia of the cowpea miscellany were much less cross-reactive (67 and 86% of all combinations were negative, respectively), and they clustered more slowly. The strains of B. japonicum fused completely only at the 4% similarity level, whereas of the 13 cowpea-nodulating strains, 4 reacted as two pairs of identical strains and 6 remained unfused.     

Utilization of carbon and nitrogen sources and acid/alkali production by cowpea rhizobia

Summary Sixteen slow-growing strains of rhizobia (15 cowpea rhizobia and oneR. japonicum) were examined to determine the effects of carbon and nitrogen sources on acid/alkali production in culture media. We found that the pH changes of the medium were more influenced by nitrogen sources than carbon sources (with the exception of ribose). When ammonium sulphate was used as a nitrogen source, all the cowpea rhizobia strains produced acid. When yeast-extract was used as a nitrogen source, however, a heterogenous pattern for acid/alkali production was found. The majority of the strains produced alkali from nitrate, glutamate and urea irrespective of carbon sources and acid from ribose irrespective of nitrogen sources.     

Mineral Soils as Carriers for Rhizobium Inoculants

Mineral soil-based inoculants of Rhizobium meliloti and Rhizobium phaseoli survived better at 4°C than at higher temperatures, but ca. 15% of the cells were viable at 37°C after 27 days. Soil-based inoculants of R. meliloti, R. phaseoli, Rhizobium japonicum, and a cowpea Rhizobium sp. applied to seeds of their host legumes also survived better at low temperatures, but the percent survival of such inoculants was higher than peat-based inoculants at 35°C. Survival of R. phaseoli, R. japonicum, and cowpea rhizobia was not markedly improved when the cells were suspended in sugar solutions before drying them in soil. Nodulation was abundant on Phaseolus vulgaris derived from seeds that had been coated with a soil-based inoculant and stored for 165 days at 25°C. The increase in yield and nitrogen content of Phaseolus angularis grown in the greenhouse was the same with soil-and peat-based inoculants. We suggest that certain mineral soils can be useful and readily available carriers for legume inoculants containing desiccation-resistant Rhizobium strains.     

Competition between inoculum and native rhizobia for nodulation of cowpea (Vigna unguiculata (L) Walp): Use of a dark-nodule strain

Summary Cowpea rhizobia strains were examined with indigenous populations in nodulating cowpea (Vigna unguiculata (L) Walp) cv. Laura B. strain IRC256 formed dark nodules on cowpea, and were used as the standard against orthodox pink-nodule strains in evaluating nodulating competitiveness. The dark nodule phenotype and intrinsic antibiotic resistance pattern were used to identify the strains in the nodules. Our results showed the usefulness of the dark-nodule strain in evaluating nodulating competitiveness of cowpea rhizobia in soils where dark-nodule strains were not indigenous.     

Invalidity of the Concept of Slow Growth and Alkali Production in Cowpea Rhizobia

A total of 103 rhizobial strains representing the cowpea miscellany and Rhizobium japonicum were studied with regard to growth rate, glucose metabolic pathways, and pH change in culture medium. Doubling times ranged from 1.4 ± 0.04 to 44.1 ± 5.2 h; although two populations of “fast-growing” and “slow-growing” rhizobia were noted, they overlapped and were not distinctly separated. Twenty-four strains which had doubling times of less than 8 h all showed NADP-linked 6-phosphogluconate dehydrogenase (6-PGD) activity, whereas only one slow-growing strain (doubling time, 10.8 ± 0.9 h) of all those tested showed 6-PGD activity. Doubling times among fast growers could not be explained solely by the presence or absence of 6-PGD activity (r² = 0.14) because the tricarboxylic acid cycle and the Emden-Meyerhoff-Parnas pathway were operative in both 6-PGD-positive and 6-PGD-negative strains. Growth rate and pH change were unrelated to each other. Fast- or slow-growing strains were not associated with any particular legume species or group of species from which they were originally isolated, with the exception of Stylosanthes spp., all nine isolates of which were slow growers. We conclude that 6-PGD activity is a more distinctive characteristic among physiologically different groups of rhizobia than doubling times and that characterization of the cowpea rhizobia as slow-growing alkali producers is an invalid concept.     

Group antigens in slow-growing rhizobia

Summary Internal group antigens of several slow-growing and fast-growing Rhizobium strains were tested by gel-diffusion against antisera to three strains of Rhizobium japonicum. At least one, generally two common antigens were found in 13 strains of R. japonicum, 4 strains of R. lupini, 4 strains isolated from cowpea and two slow-growing strains isolated from Lotus. Forty-six fast-growing rhizobia (including two from Lotus and 4 from Leucaena leucocephala) were clearly distinguished from the slow-growing strains in tests with the same antisera. They were wholly negative (9) or gave a much weaker non-identical line with one antiserum (24 strains), two antisera (8) or three antisera (5). The 5 strains of agrobacteria grouped with the fast-growing rhizobia.     

Nodulation and nitrogen fixation by fast- and slow-growing rhizobia strains of soybean on several temperate and tropical legumes

Three slow-growingBradyrhizobium japonicum (G₃, USDA-110 and KUL-150) of diverse origins and two fast-growing strains ofRhizobium fredii (USDA-192 and USDA-193) were tested with a cropped soybean (Glycine max L. Merrill) cultivar, two cowpeas (Vigna unguiculata), one mung-bean (Phaseolus radiata), one winged-bean (Psophocarpus tetragonolobus) and one field bean (Phaseolus vulgaris) varieties.TheR. fredii strains nodulated and fixed Nitrogen as effectively as the strains ofB. japonicum in a modern european soybean cultivar, namely Fiskeby V. The other western bred soybeans tested were not nodulated by theseR. fredii strains. All of the soybean rhizobia produced nodules in both cowpeas and in mung-bean; theR. fredii strains showed effective N₂-fixation in the cowpeas, particularly USDA-193, yielding shoot dry weights greater than those from theB. japonicum. The symbiotic performance of theR. fredii strains with soybean and other legumes indicated that they should be placed in an intermediate group between the slow-growingB. japonicum and cowpearhizobium sp.The hydrogen uptake activites suggested a possible host effect on the expression of such genes in one out of theB. japonicum strains tested. Furthermore, the slow-growing rhizobia showed significantly higher nitrate-reduction than theR. fredii in the nodules.     

Role of Lectins in Plant-Microorganism Interactions: III. Influence of Rhizosphere/Rhizoplane Culture Conditions on the Soybean Lectin-binding Properties of Rhizobia

The influence of rhizosphere/rhizoplane culture conditions on the ability of various rhizobia to bind soybean seed lectin (SBL) was examined. Eleven strains of the soybean symbiont, Rhizobium japonicum, and six strains of various heterologous Rhizobium species were cultured in root exudate of soybean (Glycine max [L.] Merr.) and in association with roots of soybean seedlings which were growing either hydroponically or in montmorillonite clay soil amendment (Turface). All 11 of the R. japonicum strains developed biochemically specific receptors for the lectin when cultured under these conditions, whereas six of the 11 did not develop such receptors when cultured in synthetic salts medium. Two cowpea strains also developed receptors for SBL. The other four heterologous strains of rhizobia gave no evidence of biochemically specific SBL binding in either synthetic salts media or rhizosphere/rhizoplane cultures. These results demonstrate that the environment provided by plant roots is an important factor in the development of specific lectin receptors on the cell surface of R. japonicum.     

Possible Involvement of Phage-Like Structures in Antagonism of Cowpea Rhizobia by Rhizobium trifolii

A reduction in the viability of cowpea rhizobia was observed when Rhizobium trifolii IARI and cowpea Rhizobium strain 3824 were inoculated together in soil. The reduction in number of cowpea rhizobia in soil was found to be associated with the reduction in number of nodules per plant and retardation in plant growth. An antimicrobial substance was isolated from R. trifolii which, on electron microscopic investigation, demonstrated the presence of several phage-like structures.     

Uptake Hydrogenase (Hup) in Common Bean (Phaseolus vulgaris) Symbioses

Strains of Rhizobium forming nitrogen-fixing symbioses with common bean were systematically examined for the presence of the uptake hydrogenase (hup) structural genes and expression of uptake hydrogenase (Hup) activity. DNA with homology to the hup structural genes of Bradyrhizobium japonicum was present in 100 of 248 strains examined. EcoRI fragments with molecular sizes of approximately 20.0 and 2.2 kb hybridized with an internal SacI fragment, which contains part of both bradyrhizobial hup structural genes. The DNA with homology to the hup genes was located on pSym of one of the bean rhizobia. Hup activity was observed in bean symbioses with 13 of 30 strains containing DNA homologous with the hup structural genes. However, the Hup activity was not sufficient to eliminate hydrogen evolution from the nodules. Varying the host plant with two of the Hup⁺ strains indicated that expression of Hup activity was host regulated, as has been reported with soybean, pea, and cowpea strains.     

Possible Involvement of a Megaplasmid in Nodulation of Soybeans by Fast-Growing Rhizobia from China

Several isolates from a newly described group of fast-growing acid-producing soybean rhizobia, Rhizobium japonicum, were analyzed for plasmid content. All contained from one to four plasmids with molecular weights of 100 × 10⁶ or larger. Although most of the isolates shared plasmids of similar size, the restriction endonuclease (BamHI, EcoRI, and HindIII) patterns of the plasmids from three of the isolates were vastly different. Growth in the presence of acridine orange was effective in producing mutants cured of the largest plasmid in one of the strains. These mutants also lost the ability to form nodules on soybeans. High-temperature curing of a smaller plasmid in another strain did not lead to loss of nodulating ability or alteration of symbiotic effectiveness on soybean cultivars. The identities of all of the isolates and mutants were ascertained by immunofluoresence and immunodiffusion. The new fast-growing strains of R. japonicum may provide a better genetic system for the study of the soybean symbiosis than the slow-growing R. japonicum, not all of which can be shown to contain plasmids.     

Nitrate Inhibition of Legume Nodule Growth and Activity : II. Short Term Studies with High Nitrate Supply

Soybean plants (Glycine max [L.] Merr) were grown in sand culture with 2 millimolar nitrate for 37 days and then supplied with 15 millimolar nitrate for 7 days. Control plants received 2 millimolar nitrate and 13 millimolar chloride and, after the 7-day treatment period, all plants were supplied with nil nitrate. The temporary treatment with high nitrate inhibited nitrogenase (acetylene reduction) activity by 80% whether or not Rhizobium japonicum bacteroids had nitrate reductase (NR) activity. The pattern of nitrite accumulation in nodules formed by NR⁺ rhizobia was inversely related to the decrease and recovery of nitrogenase activity. However, nitrite concentration in nodules formed by NR⁻ rhizobia appeared to be too low to explain the inhibition of nitrogenase. Carbohydrate composition was similar in control nodules and nodules receiving 15 millimolar nitrate suggesting that the inhibition of nitrogenase by nitrate was not related to the availability of carbohydrate.

Nodules on plants treated with 15 millimolar nitrate contained higher concentrations of amino N and, especially, ureide N than control nodules and, after withdrawal of nitrate, reduced N content of treated and control nodules returned to similar levels. The accumulation of N₂ fixation products in nodules in response to high nitrate treatment was observed with three R. japonicum strains, two NR⁺ and one NR⁻. The high nitrate treatment did not affect the allantoate/allantoin ratio or the proportion of amino N or ureide N in bacteroids (4%) and cytosol (96%).

     

Bradyrhizobium japonicum-Environment Interactions: Nodulation and Interstrain Competition in Soils along an Elevational Transect

The effects of temperature and soil type on interstrain competition of Bradyrhizobium japonicum and on nodulation and nitrogen accumulation in five soybean varieties belonging to four maturity groups were investigated at three sites devoid of soybean rhizobia along an elevational transect in Hawaii. Competition patterns of the three B. japonicum strains were unaffected by soil type or soil temperature. Strain USDA 110 was the best competitor, occupying on the average 81 and 64% of the nodules in the field and greenhouse experiments, respectively. Strain USDA 138 was the least successful in the field (4%), although it formed 34% of the nodules in the greenhouse. Nodule occupancy by B. japonicum strains was found to be related to soybean maturity group. Strain USDA 110 formed 61, 71, 88, 88, and 98% of the nodules in the field on Clay (00), Clark (IV), D68-0099 (VI), N77-4262 (VI), and Hardee (VIII), respectively. Strain USDA 136b formed few nodules on Hardee, an Rj2 soybean variety incompatible with that strain, in both experiments. Nodule number and weight at the 1,050-m site were reduced to 41 and 27%, respectively, of those at the 320-m site because of the decrease in temperature. Nodule number increased with increasing maturity group number at each site; however, there was not a corresponding increase in nodule weight. Nitrogen accumulation decreased from 246 mg of N per plant at the lowest elevation site to 26 mg of N per plant at the highest elevation. While soil type and temperature had no effect on strain competition, temperature had a profound influence on nodule parameters and plant growth.     

Comparison of the host range of fast-growingR. japonicum strains with a fast-growing isolate from Lablab

Summary Fast-growingRhizobium japnicum strains derived from the People's Republic of China were compared with a fast-growingRhizobium isolate from Lablab for their ability to nodulate tropical legumes grown in Leonard-jars and test tube culture. Fast-growingR. japonicum strains were all effective to varying degrees in their symbiosis withVigna unguiculata. Two strains USDA 192 and USDA 201, effectively nodulatedGlycine whightii and one strain, USDA 193, effectively nodulatedMacroptilium atropurpureum. Other nodulation responses in tropical legumes were ineffective. The fast-growing isolate from Lablab was more promiscuous, effectively nodulating with a larger host range. The fast-growing Lablab strain was considered more akin, on a symbiotic basis, to the slow-growing cowpea type rhizobia than the fast-growing China strains ofR. japonicum whilst maintaining physiological characteristics of other fast-growing rhizobia.     

Use of Two-Dimensional Polyacrylamide Gel Electrophoresis to Identify and Classify Rhizobium Strains

Fifty-seven strains of various Rhizobium species were analyzed by two-dimensional gel electrophoresis. Since the protein pattern on such gels is a reflection of the genetic background of the tested strains, similarities in pattern allowed us to estimate the relatedness between these strains. All group II rhizobia (slow growing) were closely related and were very distinct from group I rhizobia (fast growing). Rhizobium meliloti strains formed a distinct group. The collection of R. leguminosarum and R. trifolii strains together formed another distinct group. Although there were some similarities within the R. phaseoli, sesbania rhizobia, and lotus rhizobia, the members within these seemed much more diverse than the members of the above groups. The technique also is useful to determine whether two unknown strains are identical.     

Efficiency of nodule initiation in cowpea and soybean

When serial dilutions of a suspension of Bradyrhizobium japonicum strain 138 were inoculated onto both soybean and cowpea roots, the formation of nodules in the initially susceptible region of the roots of both hosts was found to be linearly dependent on the log of the inoculum dosage until an optimum dosage was reached. Approximately 30- to 100-fold higher dosages were required to elicit half-maximal nodulation on cowpea than on soybean in the initially susceptible zone of the root. However, at optimal dosages, about six times as many nodules formed in this region on cowpea roots than on soybean roots. There was no appreciable difference in the apparent rate of nodule initiation on these two hosts nor in the number of inoculum bacteria in contact with the root. These results are consistent with the possibility that cowpea roots have a substantially higher threshold of response to symbiotic signals from the bacteria than do soybean roots. Storage of B. japonicum cells in distilled water for several weeks did not affect their viability or efficiency of nodule initiation on soybean. However, the nodulation efficiency of these same cells on cowpea diminished markedly over a 2 week period. These differential effects of water storage indicate that at least some aspects of signal production by the bacteria during nodule initiation are different on the two hosts. Mutants of B. japonicum 138 defective in synthesis of soybean lectin binding polysaccharide were defective in their efficiency of nodule initiation on soybean but not on cowpea. These results also suggest that B. japonicum may produce different substances to initiate nodules on these two hosts.