Growth-Related Substituent Changes in Exopolysaccharides of Fast-Growing Rhizobia

Pyruvic acid and O-acetyl groups are the major noncarbohydrate substituents in exopolysaccharides (EPS) produced by fast-growing species of Rhizobium. EPS substituent variations were observed among strains of the same species. The amounts of these substituents also varied with culture age; pyruvic acid increased in the EPS of all four species, whereas O-acetyl increased in Rhizobium trifolii and R. leguminosarum EPS, decreased in R. meliloti EPS, and remained constant in R. phaseoli EPS. The use of glycerol as a substrate for R. meliloti significantly increased EPS yields, whereas mannitol increased those of the other three Rhizobium species.     

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.     

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.     

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.     

Survival of Cowpea Rhizobia in Soil as Affected by Soil Temperature and Moisture

Successful inoculation of peanuts and cowpeas depends on the survival of rhizobia in soils which fluctuate between wide temperature and moisture extremes. Survival of two cowpea rhizobial strains (TAL309 and 3281) and two peanut rhizobial strains (T-1 and 201) was measured in two soils under three moisture conditions (air-dry, moist (−0.33 bar), and saturated soil) and at two temperatures (25 and 35°C) when soil was not sterilized and at 40°C when soil was sterilized. Populations of rhizobia were measured periodically for 45 days. The results in nonsterilized soil indicated that strain 201 survived relatively well under all environmental conditions. The 35°C temperature in conjunction with the air-dry or saturated soil was the most detrimental to survival. At this temperature, the numbers of strains T-1, TAL309, and 3281 decreased about 2 logs in dry soil and 2.5 logs in saturated soil during 45 days of incubation. In sterilized soil, the populations of all strains in moist soil increased during the first 2 weeks, but decreased rapidly when incubated under dry conditions. The populations did not decline under saturated soil conditions. From these results it appears that rhizobial strains to be used for inoculant production should be screened under simulated field conditions for enhanced survival before their selection for commercial inoculant production.     

Cowpea Rhizobia Producing Dark Nodules: Use in Competition Studies

During a program of screening rhizobia from West Africa, it was found that some strains produced nodules of unusually dark appearance on cowpeas, but not on peanuts, soybeans, pigeon peas, or mung beans. The dark pigmentation was in the bacteroid zone, was not correlated with nodule effectiveness, and was additional to the leghemoglobin pigment. Only rhizobial strains with a nongummy (“dry”) colony morphology produced dark nodules. Visually distinguishable pink and dark nodules formed on the same root when a mixture of pink and dark strains was applied as inoculum. The dark-nodule phenotype was therefore appraised as a marker and found to be useful for studying nodulation competition with strains of the orthodox pink-nodule type. The competitiveness of 10 pink-nodule strains was examined relative to a black-nodule strain, IRc 256; a range of competitiveness was obtained of less competitive than, equally competitive to, or more competitive than IRc 256. Patterns of primary (early) nodulation were generally the same as patterns of secondary (later) nodulation. Mixed infections by dark and pink strains produced piebald nodules, the frequency of occurrence of which was much greater among primary than among secondary nodules.     

Kinetics of Nitrate Utilization by Mixed Populations of Denitrifying Bacteria

Kinetics of nitrate utilization by mixed bacterial populations from two agricultural soils and a pond sediment in Kentucky were measured by using progress curves of nitrous oxide production. Nitrous oxide production from anaerobic soil and sediment slurries containing added nitrate and acetylene exhibited first-order kinetics. Nitrate affinity (K_m) for mixed populations of denitrifying bacteria in unfertilized agricultural soils and pond sediments ranged from 1.8 to 13.7 μM. The affinity of bacterial populations for nitrate did not vary with habitat, and the ability to use low concentrations of nitrate was retained by bacterial populations living in environments which received large inputs of nitrate.     

Physiological Characteristics of Cowpea Rhizobia: Evaluation of Symbiotic Efficiency in Vigna unguiculata

One fast-growing and three slow-growing strains of Rhizobium (isolated from cowpeas) were evaluated for symbiotic performance on Vigna unguiculata (L.) Walp. cultivar California no. 5 blackeyes. Plants inoculated with slow-growing strains 176A22, 176A30, and 176A32 developed a maximum acetylene reduction activity of 24.6, 27.0, and 32 μmol of ethylene formed per plant per h, respectively, versus 6.4 μmol per plant per h in plants inoculated with the fast-growing strain 176A28. When inoculated with approximately equal proportions of rhizobia, the fast-growing strain 176A28 produced 95% of the nodules when challenged with the slow-growing strain 176A22, but formed only 6% of the nodules when challenged with the slow-growing strain 176A30. Consequently, there was no relation between the growth rate in vitro and the capability of rhizobia to compete for nodule-forming sites. Plants inoculated with strain 176A28 and subjected to drought during the vegetative growth period recovered to the same level of nitrogen fixation and nodulation as those that received adequate irrigation. On the other hand, plants inoculated with strains 176A22, 176A30, and 176A32 failed to achieve the same levels of nodulation and nitrogen fixation under drought as compared with irrigated conditions.     

Asymbiotic Acetylene Reduction by a Fast-Growing Cowpea Rhizobium Strain with Nitrogenase Structural Genes Located on a Symbiotic Plasmid

A procedure was designed which enabled the detection of ex planta nitrogenase activity in the fast-growing cowpea Rhizobium strain IHP100. Nitrogenase activity in agar culture under air occurred at a rate similar to that found for Bradyrhizobium strain CB756 but lower than that for Rhizobium strain ORS571. Hybridization studies showed that both nod and nif genes were located on a 410-kilobase Sym plasmid in strain IHP100.     

Differences Among Cowpea Rhizobia in Tolerance to High Temperature and Desiccation in Soil

Strains of cowpea rhizobia grew in mannitol-amended, nonsterile soil at 29 to 35°C but not at 40°C. Little decline in numbers of these bacteria occurred in dry, nonsterile soil incubated at 42°C for 7 days. Strains of cowpea rhizobia differed widely in their tolerances to drying at 30°C in nonsterile and sterile soil, and from less than 1 to 50% of the bacteria were still viable after 11 days. No relation was evident between tolerance to desiccation and the degree of aridity of the site from which the bacteria were isolated or their growth rates in culture, but strains not producing extracellular polysaccharide were often more tolerant than those producing extracellular polysaccharide. It is suggested that desiccation-tolerant rhizobia be used for the production of legume inoculants.     

Growth of Fast- and Slow-Growing Rhizobia on Ethanol

Free-living soybean rhizobia and Bradyrhizobium spp. (lupine) have the ability to catabolize ethanol. Of the 30 strains of rhizobia examined, only the fast- and slow-growing soybean rhizobia and the slow-growing Bradyrhizobium sp. (lupine) were capable of using ethanol as a sole source of carbon and energy for growth. Two strains from each of the other Rhizobium species examined (R. meliloti, R. loti, and R. leguminosarum biovars phaseoli, trifolii, and viceae) failed to grow on ethanol. One Rhizobium fredii (fast-growing) strain, USDA 191, and one (slow-growing) Bradyrhizobium japonicum strain, USDA 110, grew in ethanol up to concentrations of 3.0 and 1.0%, respectively. While three of the R. fredii strains examined (USDA 192, USDA 194, and USDA 205) utilized 0.2% acetate, only USDA 192 utilized 0.1% n-propanol. None of the three strains utilized 0.1% methanol, formate, or n-butanol as the sole carbon source.     

Effect of Aldrin on Growth and Oxidative Metabolism of Rhizobia

At 500 μg ml⁻¹ of aldrin, Rhizobium sp. Bengal gram ( Cicer arietinum ) and Rhizobium sp. Green gram (Vigna radiata ) showed a lag of about 12 h after which the growth returned to normal. The lag period was extended at concentrations above 500 μg ml⁻¹ of aldrin and it was more in the case of Rhizobium sp. Green gram than Rhizobium sp. Bengal gram. However, at high concentrations of aldrin, after the lag, the growth rate was very slow. On the seventh day, Rhizobium sp. Bengal gram showed 72, 81 and 84° inhibition while Rhizobium sp. Green gram exhibited 74, 85 and 88° inhibition of growth at 1000, 1500 and 2000 μg ml⁻¹ of aldrin, respectively. In general, oxidative activity of both of the Rhizobium spp. on pentoses, hexoses and TCA cycle intermediates was more strongly inhibited in aldrin grown cells than normal cells. The inhibitory effect of aldrin on the oxidative activity of rhizobia was partially released with a high concentration of glucose.     

Sources and Quantities of Yeast Extract for Growth of Rhizobia

S ummary : The growth of 4 strains of rhizobia representing 4 inoculation groups (clover, medic, cowpea-type and lupin) in liquid medium containing one of 4 commercially available sources of yeast extract (Vegemite, Oxoid powder, Oxoid paste and BBL yeast) was compared at 4 levels of each extract (1, 2, 4 and 8 g/l). Total cell numbers increased with increasing levels of yeast extract, but viable cell numbers were significantly depressed by some yeast sources. For the clover, cowpea-type and lupin strains strong depressive effects of BBL yeast were recorded at the highest levels, but satisfactory growth was obtained at the lower levels. Oxoid powder depressed viable numbers slightly but Vegemite did not. All yeast sources increased viable cell numbers of the medic strain with increasing levels in the medium. Reasons for these depressive effects and their practical implication are discussed in relation to legume inoculant preparation.     

Anaerobic Growth and Denitrification among Different Serogroups of Soybean Rhizobia

We screened soybean rhizobia originating from three germplasm collections for the ability to grow anaerobically in the presence of NO₃⁻ and for differences in final product formation from anaerobic NO₃⁻ metabolism. Denitrification abilities of selected strains as free-living bacteria and as bacteroids were compared. Anaerobic growth in the presence of NO₃⁻ was observed in 270 of 321 strains of soybean rhizobia. All strains belonging to the 135 serogroup did not grow anaerobically in the presence of NO₃⁻. An investigation with several strains indicated that bacteria not growing anaerobically in the presence of NO₃⁻ also did not utilize NO₃⁻ as the sole N source aerobically. An exception was strain USDA 33, which grew on NO₃⁻ but failed to denitrify. Dissimilation of NO₃⁻ by the free-living cultures proceeded without the significant release of intermediate products. Nitrous oxide reductase was inhibited by C₂H₂, but preceding steps of denitrification were not affected. Final products of denitrification were NO₂⁻, N₂O, or N₂; serogroups 31, 46, 76, and 94 predominantly liberated NO₂⁻, whereas evolution of N₂ was prevalent in serogroups 110 and 122, and all three were formed as final products by strains belonging to serogroups 6 and 123. Anaerobic metabolism of NO₃⁻ by bacteroid preparations of Bradyrhizobium japonicum proceeded without delay and was evident by NO₂⁻ accumulation irrespective of which final product was formed by the strain as free-living bacteria. Anaerobic C₂H₂ reduction in the presence of NO₃⁻ was observed in bacteroid preparations capable of NO₃⁻ respiration but was absent in bacteria that were determined to be deficient in dissimilatory nitrate reductase.     

Bromate Reduction by Denitrifying Bacteria

In the presence of bromide, ozonation as applied in water treatment results in the formation of bromate, an ion with carcinogenic properties. The reduction of bromate by mixed bacterial populations as well as pure cultures was studied under laboratory conditions. Bromate was reduced to bromide by a mixed bacterial population with and without a preceding nitrate reduction step in an anaerobically incubated medium with ethanol as the energy and carbon source at 20 and 25 deg C. The predominating bacteria isolated from the batches showing bromate reduction were identified as Pseudomonas spp. Strains of Pseudomonas fluorescens reduced BrO(inf3)(sup-) to Br(sup-) but at a much lower rate than the mixed bacterial population did. Nitrate is a preferred electron acceptor for the bromate-reducing bacteria. Bromate reduction did not occur in the presence of NO(inf3)(sup-), and the rate of bromate reduction was at least 100 times lower than the rate of nitrate reduction. Bromate was completely converted to Br(sup-), indicating that intermediates, e.g., BrO(inf2)(sup-), did not accumulate during bromate reduction.