(1988). Inhibition of legume nodule formation and N2 fixation by nitrate. Critical Reviews in Plant Sciences: Vol. 7, No. 1, pp. 1-23.

Inhibition of legume nodule formation and N2 fixation by nitrate

Recent data indicate that the slow-growing, non-acid-producing root nodule bacteria of leguminous plants should be separated from the fast-growing, acid-producing strains and placed in a new genus. The separation is warranted by numerical taxonomy, deoxyribonucleic acid base ratio determinations, nucleic acid hybridization, ribosomal ribonucleic acid cistron similarities, serology, composition of extracellular gum, carbohydrate utilization and metabolism, bacteriophage and antibiotic susceptibilities, protein composition, and types of intracellular inclusion bodies in the bacteroid forms. The name proposed for the new genus is Bradyrhizobium. The type species of the genus is B. japonicum (Buchanan 1980) comb. nov. (basonym: Rhizobium japonicum Buchanan 1980), the type strain of which is ATCC 10324.

NOTES: Transfer of Rhizobium japonicum Buchanan 1980 to Bradyrhizobium gen. nov., a Genus of Slow-Growing, Root Nodule Bacteria from Leguminous Plants

A total of 33 strains of fast-growing soybean rhizobia isolated from soil and soybean nodules collected in China and 25 strains belonging to the genera Rhizobium, Bradyrhizobium, and Agrobacterium were compared by numerical taxonomic techniques, using 240 different characters. Our results indicated that all of the strains of fast-growing soybean rhizobia which we examined are closely related (guanine-plus-cytosine content, 59.9 to 63.8 mol%) and are separated from Rhizobium and Bradyrhizobium at the generic level. Based on numerical taxonomy, deoxyribonucleic acid (DNA) base ratio determinations, DNA-DNA hybridization data, serological analysis data, the composition of extracellular gum, bacteriophage typing data, and soluble protein patterns, we propose that the fast-growing soybean rhizobia represent members of a new genus rather than a species of Rhizobium (Rhizobium fredii); we propose Sinorhizobium gen. nov. as an appropriate generic name. The type species of the new genus is Sinorhizobium fredii comb. nov. (basonym, Rhizobium fredii Scholla and Elkan 1984), and the type strain is strain ATCC 35423 (= USDA 205). For the other species we propose the name Sinorhizobium xinjiangensis sp. nov.; the type strain of this species is strain CCBAU 110, which has been deposited in the Beijing Agricultural University Culture Collection, Beijing, People's Republic of China.

Numerical Taxonomic Study of Fast-Growing Soybean Rhizobia and a Proposal that Rhizobium fredii Be Assigned to Sinorhizobium gen. nov.

Seventeen strains of extra-slowly growing (ESG) soybean rhizobia isolated from root nodules of Glycine soja and Glycine max growing in five provinces (Liaoning, Heilongjiang, Shanxi, Hubei, and Anhui) in the People's Republic of China were compared with 48 reference strains belonging to the genera Bradyrhizobium, Rhizobium, and Agrobacterium by performing a numerical analysis of 191 phenotypic features. Our results showed that all of the ESG strains examined clustered closely in the genus Bradyrhizobium but were separated from Bradyrhizobium japonicum at the species level and that they could be differentiated from Rhizobium and Agrobacterium species at the genus level. On the basis of the results of our numerical taxonomy analysis, a genomic DNA G+C content analysis, DNA-DNA hybridization experiments, a partial 16S rRNA sequence analysis, a serological analysis, an N and C content analysis, and an N/C ratio analysis of members of the three groups of soybean rhizobia, we propose the name Bradyrhizobium liaoningense sp. nov. for the ESG strains; the type strain of this species is strain 2281.

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Bradyrhizobium liaoningense sp. nov., isolated from the root nodules of soybeans.

Nine bacterial strains isolated from root nodules of Astragalus sinicus were compared with 41 reference strains, including the type strains of the type species of the genera Rhizobium, Bradyrhizobium, and Agrobacterium, by performing a numerical analysis of 200 phenotype features. Representative strains belonging to different clusters were further compared with similar bacteria by using data from gel electrophoresis of whole-cell proteins, DNA G+C content data, and DNA-DNA hybridization data. The rhizobial strains isolated from nodules of A. sinicus constitute a distinct homology group that is quite different from previously described Rhizobium, Bradyrhizobium, and Agrobacterium species and from strains isolated from other Astragalus species. We propose the name Rhizobium huakuii sp. nov. for the strains isolated from A. sinicus. Type strain CCBAU 2609 (= 103) has been deposited in the Culture Collection of Beijing Agricultural University, Beijing, People's Republic of China.

Rhizobium huakuii sp. nov. isolated from the root nodules of astragalus sinicus

IT has been widely believed that the legume root-nodule bacteria (Rhizobium spp.; rhizobia) fix N2 only within the tissues of the host plant1. There have however been recent reports that a strain of cowpea rhizobia, 32H1, developed nitrogenase activity when grown in association with legume or non-legume plant cells2,3. The induction of nitrogenase was apparently due to a diffusible factor(s) secreted by the plant cells, since nitrogenase activity was detected when strain 32H1 was grown adjacent to, but not in contact with, tobacco cells3. Several plant metabolites, including sugars known to favour rhizobial growth4,5 and citric acid cycle intermediates6,7, were examined for their effect on this latter system and as possible direct inducers of nitrogenase activity in cultured rhizobia. This led to the formulation of a defined medium on which strain 32H1 fixed N2 in the absence of plant cells.

Nitrogen fixation by Rhizobium cultured on a defined medium

The effect of nitrate on the symbiotic properties of nitrate-reductase-deficient mutants of a strain of cowpea rhizobia (32H1), and of a strain of Rhizobium trifolii (TA1), were examined; the host species were Macroptilium atropurpureum (DC.) Urb. and Trifolium subterraneum L. Nitrate retarded initial nodulation by the mutant strains to an extent similar to that found with the parent strains. It is therefore unlikely that nitrite produced from nitrate by the rhizobia, plays a significant role in the inhibition of nodulation by nitrate. Nitrite is an inhibitor of nitrogenase, and its possible production in the nodule tissue by the action of nitrate reductase could be responsible for the observed inhibition of nitrogen fixation when nodulated plants are exposed to nitrate. However, the results of this investigation show that nitrogen fixation by the plants nodulated by parent or mutant strains was depressed by similar amounts in the presence of nitrate. No nitrite was detected in the nodules. Nodule growth, and to a lesser extent, the nitrogenase specific activity of the nodules (μmol C2H4g(-1) nodule fr. wt. h(-1)), were both affected by the added nitrate.

Nitrate effects on the nodulation of legumes inoculated with nitrate-reductase-deficient mutants of Rhizobium.

Nutritional and physical conditions affecting nitrogenase activity in the strain of “cowpea” rhizobia, 32H1, were examined using cultures grown on agar medium. Arabinose in the basic medium (CS7) could be replaced by ribose, xylose, or glycerol, but mannitol, glucose, sucrose, or galactose only supported low nitrogenase (C2H2 reduction) activity. Succinate could be replaced by pyruvate, fumarate, malate, or 2-oxoglutarate, but without any carboxylic acid, nitrogenase activity was low or undetectable unless a high level of arabinose was provided. Inositol was not essential. Several nitrogen sources could replace glutamine including glutamate, urea, (NH4)2SO4 and asparagine.

Nitrogenase activity in cultured Rhizobium sp. strain 32H1: nutritional and physical considerations.

Forty-eight mutants unable to reduce nitrate were isolated from "cowpea" Rhizobium sp. strain 32Hl and examined for nitrogenase activity in culture. All but two of the mutants had nitrogenase activity comparable with the parental sttain and two nitrogenase-defective strains showed alterations in their symbiotic properties. One strain was unable to nodulate either Macroptilium atropurpureum or Vigna uguiculata and, with the other, nodules appeared promptly, but effective nitrogen fixation was delayed. These results, and the relatively low proportion of nitrate reductase mutants with impaired nitrogenase activity, do not support the proposed commanality between nitrogenase and nitrate reductase in cowpea rhizobia. Inhibition studies of the effect of nitrate and its reduction products on the nitrogenase activity in cultured strains 32Hl and the nitrate reductase-deficient, Nif+ strains, indicated that nitrogenase activity was sensitive to nitrite rather than to nitrate. Images

Nitrogen fixation in nitrate reductase-deficient mutants of cultured rhizobia.

Nitrogen fixation was induced in a strain of “cowpea” rhizobia, 32Hl, when it was grown in association with cell cultures of the non-legume, tobacco (Nicotiana tabacum). Rhizobia grown alone on the various media examined did not show nitrogenase activity, indicating the involvement of particular plant metabolites in nitrogenase induction. Nitrogenase activity, as measured by C2H2 reduction, was maximized at an O2 concentration of 20% and at an assay temperature of 30°C, the conditions under which the plant cell-rhizobia associations developed. Glutamine, as a nitrogen source, could be replaced by other organic nitrogen sources, but NH4+ and NO3- repressed nitrogenase activity. Nitrogenase activity induced in rhizobia when cultured adjacent to, but not in contact with, the plant cells could be stimulated by providing succinate in the medium. At least 12 other strains of rhizobia also reduced C2H2 in association with tobacco cells; the highest levels of activity were found among cowpea strains.

Janet D. Pagan

Papers

Nitrogen fixation by Rhizobium cultured on a defined medium

Nitrate effects on the nodulation of legumes inoculated with nitrate-reductase-deficient mutants of Rhizobium.

Nitrogenase activity in cultured Rhizobium sp. strain 32H1: nutritional and physical considerations.

Nitrogen fixation in nitrate reductase-deficient mutants of cultured rhizobia.

The induction of nitrogenase activity in Rhizobium by non-legume plant cells.