Allan R. J. Eaglesham

Physiological and symbiotic characteristics of fast-growingRhizobium japonicum

SummaryPhysiological 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.

The first photosynthetic N2-fixing Rhizobium: Characteristics

Biological N2 fixation requires large amounts of energy based on either in vitro biochemical studies of nitrogenase or in vivo studies of root nodulated legume plants. For example, calorimetric experiments with effectively root nodulated soybeans suggest that the aerobic metabolism of the equivalent of about 12 g of carbohydrate to CO2 and H2O is required to fix 1 g of N2 by the rhizobial-legume process (11). This carbohydrate is provided through foliar photosynthesis by the legume macrosymbiont and is used by the rhizobial microsymbiont. A photosynthetic rhizobium that could meet directly its energy needs for biological N2 fixation could be advantageous since the legume plant might use the saved fixed carbon to produce additional harvestable plant product.

Intrinsic antibiotic resistance in relation to colony morphology in three populations of West African cowpea rhizobia

Abstract Rhizobia isolated from cowpeas ( Vigna unguiculata (L.) Walp.) grown in three west African soils were examined for intrinsic resistance to five antibiotics and were scored for one of two colony morphologies. Half of the strains tested had a “wet”, slimy colony morphology and half had small discrete “dry”, non-slimy colonies. The populations as a whole were resistant to gentamicin (87%) but varied in their resistance to streptomycin, rifampicin, kanamycin and penicillin. Thirteen patterns of resistance were found for the 128 strains screened and strains within the same pattern usually had the same colony type. The most common pattern (32%) was resistance to all five antibiotics. Associations between resistances were random within populations and colony type. Each population was diverse, expressing from 6 to 8 patterns of resistance, but one population was relatively homogeneous, with 68% of its members exhibiting the same pattern. Correlations between intrinsic antibiotic resistance and colony type were demonstrated both for the west African cowpea rhizobia and for a broader group of cowpea miscellany rhizobia. The method was practical, rapid and reliable for identification of groups within populations.

Rhizobitoxine: a phytotoxin of unknown function which is commonly produced by bradyrhizobia

SummaryFifty-six percent of 93 strains ofBradyrhizobium japonicum andBradyrhizobium sp. (various hosts) from diverse geographical areas were found to produce a chlorosis-inducing toxin. Toxin production was common among bradyrhizobia originating from the USA, Africa, Central America, and South America.Toxin produced by West African strains was compared with rhizobitoxine by cation exchange chromatography, paper chromatography, and soybean (Glycine max (L.) Merr.) bioassay. The comparison suggested that the chlorosis-inducing toxin produced by West African bradyrhizobia is rhizobitoxine.Purified toxin from a West AfricanBradyrhizobium sp. (Vigna) strain inhibited the growth ofBacillus subtilis on minimal medium. The growth inhibition was reduced by addition of yeast-extract or casamino acids but not by any of 21 individual amino acids, including methionine. The same toxin did not inhibit the growth of 14 Bradyrhizobium strains, including eight strains that did not produce toxin.Mixed inoculum experiments revealed that a toxin-producing West African strain could not assist toxin non-producingB. japonicum strains in nodulating non-nodulating (rj1 rj1) soybeans.

A stem-nodulating Rhizobium with physiological characteristics of both fast and slow growers

Summary: Rhizobium strain BTAi 1 which was isolated from a stem nodule on Aeschynomene indica was a fast grower with mean generation times of 3.2 and 4.0 h with glucose and mannitol, respectively. Its ability to utilize sucrose and lactose confirmed the similarity of BTAi 1 to other fast growers. However, its inability to utilize rhamnose, dulcitol, raffinose, trehalose, citrate, malate and fumarate linked it to the slow-growing rhizobia. The absence of the pentose phosphate pathway also placed BTAi 1 with the slow-growers as did the presence of 2-keto-3-deoxy-L-arabonate aldolase, its small colony morphology and the production of alkali with most carbon sources. Strain BTAi 1 is thus an intermediate type of Rhizobium, sharing characteristics with both fast and slow growers.

Physiological and biochemical aspects of diversity of Bradyrhizobium sp. (Vigna) from three West African soils

Abstract Bradyrhizobial isolates from the nodules of cowpeas [ Vigna unguiculata (L.) Walp.] grown at three climatically-distinct West African locations (Ibadan and Onne in Nigeria and Maradi in Niger) were examined for physiological and biochemical characteristics to gain an understanding of the ranges of diversity of types within and between soil populations. Patterns of utilization of 20 carbohydrates and three vitamins showed similarities between locations and to other bradyrhizobia reported previously. Strains from Maradi, in the sahel-savanna, had the greatest tolerance to high temperature. Different proportions of two colony types, designed “wet” and “dry”, were found at each location. Strains with the “wet” colony morphology were tolerant of 0.5% NaCl whereas “dry” strains were not. Production of a chlorosis-inducing toxin in broth culture was more prevalent among “dry” strains. Protein-band patterns on SDS-polyacrylamide gels indicated that the “wet” and “dry” types are fundamentally different.

Increased nodulation of “non-nodulating” (rj1 rj1) soybeans by high dose inoculation

SummaryA significant increase in nodulation of “non-nodulating” (rj1 rj1) soybeans was obtained by inoculating with very high numbers (approx. 1011 cells/pot) of certain rhizobia when compared with inoculation at a moderate dose (approx. 109 cells/pot). Nodulating ability of rhizobial strains was not correlated with their ability to produce a detectable level of chlorosis-inducing toxin in culture.

Physiological comparisons of root and stem nodules of Aeschynomene scabra and Sesbania rostrata

A few legume species possess the ability to form N2-fixing nodules on stems as well as on roots. Little is known of the functional characteristics of stem nodules, or to what extent they differ from root nodules. Stem and root nodules of greenhouse-grown plants of Aeschynomene scabra (inoculated with the photosynthetic rhizobial strain BTAi 1) and Sesbania rostrata (inoculated with Azorhizobium caulinodans strain BTSr 3) were examined for assimilation of 14CO2 in the light and dark, soluble carbohydrate and starch contents, acetylene reduction activity, relative efficiency of nitrogenase in terms of uptake-hydrogenase activity, glutamine synthetase and glutamate synthase, and reduced N and ureide contents. In general, stem nodules possessed higher enzyme activities and metabolite contents than did root nodules, suggesting that they fix N2 with greater energy efficiency. This greater efficiency correlated with photosynthesis in the cortex of stem nodules. Differences in enzyme activities and metabolite contents between the stem nodules on A. scabra and those on S. rostrata probably result either from legume-species characteristics or from the photosynthetic capability of strain BTAi 1.

Light-stimulated 14CO2 uptake and acetylene reduction by bacteriochlorophyll containing stem nodule isolate BTAi 1

SummaryBradyrhizobial strain BTAi 1 nodulates both stems and roots of Aeschynomene spp. Previous work has shown that it contains bacteriochlorophyll a and forms photosynthetic reaction centers, and has provided indirect evidence of photosynthesis by bacteroids within stem nodules. Here we report physiological and biochemical characteristics of BTAi 1 ex planta, which also suggest the presence of photosynthetic activity. Light-stimulated uptake of 14CO2 by BTAi 1 was detected at all stages of growth. Inhibitors of photosynthesis, 1,10-orthophenanthroline and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and the uncoupler NH4Cl, immediately suppressed light-driven 14CO2 uptake and increased O2 uptake. BTAi 1 is strictly aerobic and was unable to grow without organic C even in the light; also, it was unable to grow chemoautotrophically in an atmosphere enriched with H2 and CO2. In micro-aerobic conditions, strain BTAi 1 expressed acetylene reducing activity ex planta in an N-free medium. The highest rates of light-stimulated 14CO2 uptake and acetylene-reducing activity occurred during the exponential and early stationary phases of growth. Acetylene-reducing rates at a low glucose concentration were increased following a light-dark cycle in comparison with continuous dark conditions.

Foliar chlorosis in legumes induced by cowpea rhizobia

SummaryWhile 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.