Diversity in symbiotic specificity of cowpea rhizobia indigenous to Zimbabwean soils

Tropical cowpea rhizobia are often presumed to be generally promiscuous but poor N fixers. This study was conducted to evaluate symbiotic interactions of 59 indigenous rhizobia isolates (49 of them from cowpea (Vigna unguiculata)), with up to 13 other (mostly tropical) legume species. Host ranges averaged 2.4 and 2.3 legume species each for fast- and slow-growing isolates respectively compared to 4.3 for slow-growing reference cowpea strains. An average of 22% and 19% of fast- and slow-growing cowpea isolates respectively were effective on each of 12 legume species tested. We conclude that the indigenous cowpea rhizobia studied have relatively narrow host ranges. The ready nodulation of different legumes in tropical soils appears due to the diversity of indigenous symbiotic genotypes, each consisting of subgroups compatible with a limited number of legume species.     

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.     

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.     

Growth and nutritional characteristics of cowpea rhizobia

Summary Twenty-five slow-growing strains of cowpea rhizobia were examined for growth and nutritional characteristics. Growth and nutritional data of these isolates were surprisingly homogeneous given their proposed genetic diversity. Most strains tested were capable of anaerobic growth in the presence of nitrate and all were found capable of autotrophic growth in a defined atmosphere of CO₂ and H₂ with oxygen or nitrate as terminal electron acceptors. These isolates grew heterotrophically with various carbohydrates and organic acids. Nitrogen utilization was consistent with that of other slow-growing rhizobia. Medium composition strongly affected the final pH of the culture. Cowpea rhizobia generally did not require vitamins; those requiring vitamins exhibited good growth when biotin was supplemented to the medium.     

Research evaluation and impact analysis of biological nitrogen fixation

Although viable Rhizobium inoculation technology for cultivated legumes has long been available, there has been little sustained adoption of this technology in tropical regions. Reasons contributing to this include inadequate demonstration of the technology, presence of adequate native rhizobia, high soil mineral nitrogen levels which suppress nitrogen fixation, inadequate quality control of Rhizobium inoculum and difficulties of inoculating under tropical conditions. In order to ensure a better adoption rate of existing or emerging biological nitrogen fixation (BNF) technologies, it is proposed that future research and development efforts better focus on the research-adoption-impact continuum. The salient features of this approach are described in this paper, using the example of recently developed nodulation variants in chickpea as a potential means of increasing BNF in this crop. It is suggested that previous experience with Rhizobium inoculation technology is amenable to ex-post impact analysis to analyze bottlenecks, and that ex-ante impact analysis should be built into on-going or planned BNF research, to better ensure that technology adoption occurs.     

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.     

一氧化氮对豆科植物结瘤及固氮的影响机制

豆科植物-根瘤菌共生过程受双方基因复杂且精细的调控, 能够产生特异的根瘤结构并可将大气中的惰性氮气(N₂)转化为可被植物直接利用的氨态氮。结瘤与固氮受多种因素影响, 其中, 一氧化氮(NO)作为一种自由基反应性气体信号分子, 可参与调节植物的许多生长发育过程, 如植物的呼吸、光形态建成、种子萌发、组织和器官发育、衰老以及响应各种生物及非生物胁迫。在豆科植物中, NO不仅影响寄主与菌共生关系的建立, 还参与调控根瘤菌对氮气的固定并提高植株氮素营养利用效率。该文主要从豆科植物及共生菌内NO的产生、降解及其对结瘤、共生固氮的影响和对环境胁迫的响应, 阐述了NO调控豆科植物共生体系中根瘤形成和共生固氮过程的作用机制, 展望了NO信号分子在豆科植物共生固氮体系中的研究前景。     

Mixed infections may promote diversification of mutualistic symbionts: why are there ineffective rhizobia?

While strategy variation is a key feature of symbiotic mutualisms, little work focuses on the origin of this diversity. Rhizobia strategies range from mutualistic nitrogen fixers to parasitic nonfixers that hoard plant resources to increase their own survival in soil. Host plants reward beneficial rhizobia with higher nodule growth rates, generating a trade‐off between reproduction in nodules and subsequent survival in soil. However, hosts might not discriminate between strains in mixed infections, allowing nonfixing strains to escape sanctions. We construct an adaptive dynamics model of symbiotic nitrogen‐fixation and find general situations where symbionts undergo adaptive diversification, but in most situations complete nonfixers do not evolve. Social conflict in mixed infections when symbionts face a survival–reproduction trade‐off can drive the origin of some coexisting symbiont strategies, where less mutualistic strains exploit benefits generated by better mutualists.     

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

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.     

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.     

Bacterial RuBisCO is required for efficient Bradyrhizobium/Aeschynomene symbiosis

Rhizobia and legume plants establish symbiotic associations resulting in the formation of organs specialized in nitrogen fixation. In such organs, termed nodules, bacteria differentiate into bacteroids which convert atmospheric nitrogen and supply the plant with organic nitrogen. As a counterpart, bacteroids receive carbon substrates from the plant. This rather simple model of metabolite exchange underlies symbiosis but does not describe the complexity of bacteroids' central metabolism. A previous study using the tropical symbiotic model Aeschynomene indica/photosynthetic Bradyrhizobium sp. ORS278 suggested a role of the bacterial Calvin cycle during the symbiotic process. Herein we investigated the role of two RuBisCO gene clusters of Bradyrhizobium sp. ORS278 during symbiosis. Using gene reporter fusion strains, we showed that cbbL1 but not the paralogous cbbL2 is expressed during symbiosis. Congruently, CbbL1 was detected in bacteroids by proteome analysis. The importance of CbbL1 for symbiotic nitrogen fixation was proven by a reverse genetic approach. Interestingly, despite its symbiotic nitrogen fixation defect, the cbbL1 mutant was not affected in nitrogen fixation activity under free living state. This study demonstrates a critical role for bacterial RuBisCO during a rhizobia/legume symbiotic interaction.     

Nodulation and nitrogen fixation in extreme environments

Biological nitrogen fixation is a phenomenon occurring in all known ecosystems. Symbiotic nitrogen fixation is dependent on the host plant genotype, theRhizobium strain, and the interaction of these symbionts with the pedoclimatic factors and the environmental conditions. Extremes of pH affect nodulation by reducing the colonization of soil and the legume rhizosphere by rhizobia. Highly acidic soils (pH<4.0) frequently have low levels of phosphorus, calcium, and molybdenum and high concentrations of aluminium and manganese which are often toxic for both partners; nodulation is more affected than host-plant growth and nitrogen fixation. Highly alkaline soils (pH>8.0) tend to be high in sodium chloride, bicarbonate, and borate, and are often associated with high salinity which reduce nitrogen fixation. Nodulation and N-fixation are observed under a wide range of temperatures with optima between 20–30°C. Elevated temperatures may delay nodule initiation and development, and interfere with nodule structure and functioning in temperate Iegumes, whereas in tropical legumes nitrogen fixation efficiency is mainly affected. Furthermore, temperature changes affect the competitive ability ofRhizobium strains. Low temperatures reduce nodule formation and nitrogen fixation in temperate legumes; however, in the extreme environment of the high arctic, native legumes can nodulate and fix nitrogen at rates comparable to those observed with legumes in temperate climates, indicating that both the plants and their rhizobia have successfully adapted to arctic conditions. In addition to low temperatures, arctic legumes are exposed to a short growing season, a long photoperiod, low precipitation and low soil nitrogen levels. In this review, we present results on a number of structural and physiological characteristics which allow arctic legumes to function in extreme environments.     

Use of intrinsic antibiotic resistance for characterisation and identification of rhizobia from nodules of Vigna unguiculata (L) Walp. and Phaseolus vulgaris (L)

Intrinsic resistance to low concentrations of antibiotics was used to characterise 83 isolates from nodules of cowpea (Vigna unguiculata) and field bean (Phaseolus vulgaris). Characterisation and differentiation of isolates from cowpea was made difficult by associated fast-growing bacteria inside the nodule tissue. Thus, reliable pure culture was difficult to secure without repeated isolation and even via nodulation of the appropriate homologous host. Although the technique may be satisfactory for differentiation and identification of fast-growing rhizobia, it is rated inferior to serology on aspects of facility, time and accuracy where rhizobia from cowpea nodules are concerned. Fingerprint patterns of isolates revealed considerable heterogeneity amongst the populations even where there was commonality of location and/or host plant. Pure cultures of slow-growing rhizobia from V. unguiculata nodules were generally more resistant to the concentrations of antibiotics used than fast-growing nodule bacteria from P. vulgaris.     

Hydrogen sulfide and rhizobia synergistically regulate nitrogen (N) assimilation and remobilization during N deficiency-induced senescence in soybean

Hydrogen sulfide (H₂S) is emerging as an important signalling molecule that regulates plant growth and abiotic stress responses. However, the roles of H₂S in symbiotic nitrogen (N) assimilation and remobilization have not been characterized. Therefore, we examined how H₂S influences the soybean (Glycine max)/rhizobia interaction in terms of symbiotic N fixation and mobilization during N deficiency-induced senescence. H₂S enhanced biomass accumulation and delayed leaf senescence through effects on nodule numbers, leaf chlorophyll contents, leaf N resorption efficiency, and the N contents in different tissues. Moreover, grain numbers and yield were regulated by H₂S and rhizobia, together with N accumulation in the organs, and N use efficiency. The synergistic effects of H₂S and rhizobia were also demonstrated by effects on the enzyme activities, protein abundances, and gene expressions associated with N metabolism, and senescence-associated genes (SAGs) expression in soybeans grown under conditions of N deficiency. Taken together, these results show that H₂S and rhizobia accelerate N assimilation and remobilization by regulation of the expression of SAGs during N deficiency-induced senescence. Thus, H₂S enhances the vegetative and reproductive growth of soybean, presumably through interactions with rhizobia under conditions of N deficiency.     

Genetic and symbiotic diversity of nitrogen-fixing bacteria isolated from agricultural soils in the Western Amazon by using cowpea as the trap plant

Cowpea is a legume of great agronomic importance that establishes symbiotic relationships with nitrogen-fixing bacteria. However, little is known about the genetic and symbiotic diversity of these bacteria in distinct ecosystems. Our study evaluated the genetic diversity and symbiotic efficiencies of 119 bacterial strains isolated from agriculture soils in the western Amazon using cowpea as a trap plant. These strains were clustered into 11 cultural groups according to growth rate and pH. The 57 nonnodulating strains were predominantly fast growing and acidifying, indicating a high incidence of endophytic strains in the nodules. The other 62 strains, authenticated as nodulating bacteria, exhibited various symbiotic efficiencies, with 68% of strains promoting a significant increase in shoot dry matter of cowpea compared with the control with no inoculation and low levels of mineral nitrogen. Fifty genotypes with 70% similarity and 21 genotypes with 30% similarity were obtained through repetitive DNA sequence (BOX element)-based PCR (BOX-PCR) clustering. The 16S rRNA gene sequencing of strains representative of BOX-PCR clusters showed a predominance of bacteria from the genus Bradyrhizobium but with high species diversity. Rhizobium, Burkholderia, and Achromobacter species were also identified. These results support observations of cowpea promiscuity and demonstrate the high symbiotic and genetic diversity of rhizobia species in areas under cultivation in the western Amazon.     

Transfer of plasmids RP4 and R68.45 and chromosomal mobilization in cowpea rhizobia

R-plasmids RP4 and its derivatives R68.45 were transferred from Escherichia coli to two cowpea rhizobia strains. The frequency of RP4 transfer in cowpea rhizobia strains JRC23-SM20 and IRC256-HA409 was 1,000-fold higher than transfer frequency of R68.45. The transconjugants were further used to transfer R-plasmids within (isogenic) and between (non-isogenic) cowpea rhizobia strains. The plasmid transfer frequency was higher in isogenic than non-isogenic strains. The ability of R-plasmids to mobilize chromosomal genes in cowpea rhizobia was also examined. R-plasmids mediated the chromosomal transfer; however, mobilization of chromosomal markers Sm^R and Met⁺ by RP4 in isogenic strains was more efficient than by R68.45. Chromosomal mobilization has not previously been reported in cowpea rhizobia.Abbreviations Ap ampicillin - Km kanamycin - Tc tetracycline - Rif rifampicin - TYS tryptone yeast-extract sodium chloride - YEMA yeast-extract mannitol agar - YEMB yeast-extract mannitol broth Part of the work was presented in 6th International Symposium on Nitrogen Fixation at Oregon State University, Corvallis, August 4–10, 1985     

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.     

Determination of nitrogen fixation effectiveness in selected <Emphasis Type="Italic">Medicago truncatula</Emphasis> isolates by measuring nitrogen isotope incorporation into pheophytin

Effectiveness is a term used to describe the input that a bacterial nitrogen-fixing symbiosis makes to plant nitrogen metabolism. In legumes, effectiveness is considered a polymorphic trait where specific interactions between the plant and symbiotic rhizobia contribute to the success of the interaction. Evaluation of effectiveness using model legumes like Medicago truncatula may open new avenues for genetic studies. In previous work, an isotope dilution mass spectrometry method, which uses the effect of nitrogen fixation on the nitrogen isotope composition of chlorophyll in plants grown on ¹⁵N fertilizer as a measure of effectiveness, was developed for estimating the contribution of symbiotic nitrogen fixation to plant nitrogen content. This ¹⁵N-dilution assay was used to evaluate the level of nitrogen fixation effectiveness in three Medicago truncatula lines that have been used as parents in generating recombinant inbred lines. Three Sinorhizobium meliloti strains, USDA 1600, 102F51 and MK506, differ in this measure of effectiveness on three lines of M. truncatula: Jemalong A17, DZA315.16 and F83005.5. Plant–rhizobia combinations grown in two different conditions showed comparable differences in effectiveness.     

Microbiological strategies for enhancing biological nitrogen fixation in nonlegumes

In an agro-ecosystem, industrially produced nitrogenous fertilizers are the principal sources of nitrogen for plant growth; unfortunately these also serve as the leading sources of pollution. Hence, it becomes imperative to find pollution-free methods of providing nitrogen to crop plants. A diverse group of free-living, plant associative and symbiotic prokaryotes are able to perform biological nitrogen fixation (BNF). BNF is a two component process involving the nitrogen fixing diazotrophs and the host plant. Symbiotic nitrogen fixation is most efficient as it can fix nitrogen inside the nodule formed on the roots of the plant; delivering nitrogen directly to the host. However, most of the important crop plants are nonleguminous and are unable to form symbiotic associations. In this context, the plant associative and endophytic diazotrophs assume importance. BNF in nonlegumes can be encouraged either through the transfer of BNF traits from legumes or by elevating the nitrogen fixing capacity of the associative and endophytic diazotrophs. In this review we discuss mainly the microbiological strategies which may be used in nonleguminous crops for enhancement of BNF.     

A rhizobia strain isolated from root nodule of gymnosperm Podocarpus macrophyllus

Symbiotic nitrogen fixation of rhizobia and leguminous plants is considered as the most important biologic nitrogen fixation system on earth. Symbiotic nodulafion of gymnosperm Podocarpus macrophyllus and rhizobia has never been reported. In this study, 11 endophytic bacteria strains were isolated from root nodules of P. macrophyllus and its variation P. macrophyllus var. maki. The plant infection tests on these strains indicated that the isolated strains could be nodulated on P. macrophyllus plants, and weak nitrogenase activity of nodules was found in acetylene reduction method. According to the physiological and biochemical characteristics of the 11 strains, GXLO 02 was selected as the representative strain. 16S rDNA full-length sequence analysis of GXLO 02 confirmed that the representative strain GXLO 02 belongs to Rhizobium sp.