Estimation of host-strain compatibility for symbiotic N-fixation between Rhizobium meliloti,several annual species of Medicago and Medicago sativa

Standard methods for estimating host-strain compatibility for N-fixation in vitro proved inadequate for examination of the symbiosis between Rhizobium meliloti and Medicago sativa, M. littoralis, M. tornataas well as for hybrids between M. littoralisand M. truncatula. When screening procedures in soil free of background R. melilotiwere adopted, nitrogen fixation appeared to be unrestricted by the screening environment. During the experiments it became clear that the single commercial inoculant strain available in Australia (CC169) was poorly effective at N-fixation with many of the lucerne and annual medic cultivars examined. Interspecific hybridisation between M. truncatulaand M. littoralis also produced genotypes that were particularly difficult to satisfy in a symbiotic sense. Despite the existence of substantial host-strain genotype interactions between the symbiotic partners, several alternative strains of R. meliloti proved to be broadly effective at N-fixation. One of these, WSM826, has since become commercially available in Australia.     

钙离子对紫花苜蓿及苜蓿根瘤菌耐酸能力的影响

土壤酸性是阻碍苜蓿根瘤菌与其宿主紫花苜蓿之间高效共生固氮的重要环境因子.本文研究了Ca2 对紫花苜蓿及苜蓿根瘤菌耐酸能力的影响.结果表明:加入一定浓度的Ca2 (5和10mmol·L-1)能提高苜蓿根瘤菌的生长速率,使苜蓿根瘤菌提前进入对数生长期.中性pH条件下,Ca2 的加入对苜蓿根毛变形率无显著影响;低pH条件下,加入2、5和10mmol·L-1的Ca2 均可提高根毛变形率,Ca2 浓度越高,其影响越显著,说明低pH下Ca2 可能会促进苜蓿根瘤菌与其宿主之间的识别.低pH条件下加入Ca2 可以使苜蓿结瘤提前,结瘤率提高;结瘤动力学检测结果表明,加入一定浓度的Ca2 可以使同期结瘤数增加,越是结瘤后期,环境pH越低,这种表现越明显.     

Ecological genetics of Rhizobium meliloti: symbiotic plasmid transfer in the Medicago sativa rhizosphere

A potentially important aspect of the ecological genetics of fast-growing rhizobia is lateral transfer of symbiosis-related genes between strains in the host-legume rhizosphere. We have therefore characterized transfer of the Rhizobium leguminosarum symbiotic (Sym) plasmids pJB5JI and pSymR1897 to strain WL113, a non-attaching, non-nodulating Rhizobium meliloti recipient, in the rhizosphere of its legume host Medicago sativa. Interspecific transfer of pJB5JI generated symbiotically proficient transconjugants of WL113, demonstrating that a mechanism exists for genetic flux in indigenous R. meliloti populations in response to selection pressure provided by the host legume. This could generate fluid groupings of R. meliloti rather than discrete and stable strains.     

Genetic diversity of an Italian Rhizobium meliloti population from different Medicago sativa varieties.

We investigated the genetic diversity of 96 Rhizobium meliloti strains isolated from nodules of four Medicago sativa varieties from distinct geographic areas and planted in two different northern Italian soils. The 96 isolates, which were phenotypically indistinguishable, were analyzed for DNA polymorphism with the following three methods: (i) a randomly amplified polymorphic DNA (RAPD) method, (ii) a restriction fragment length polymorphism (RFLP) analysis of the 16S-23S ribosomal operon spacer region, and (iii) an RFLP analysis of a 25-kb region of the pSym plasmid containing nod genes. Although the bacteria which were studied constituted a unique genetic population, a considerable level of genetic diversity was found. The new analysis of molecular variance (AMOVA) method was used to estimate the variance among the RAPD patterns. The results indicated that there was significant genetic diversity among strains nodulating different varieties. The AMOVA method was confirmed to be a useful tool for investigating the genetic variation in an intraspecific population. Moreover, the data obtained with the two RFLP methods were consistent with the RAPD results. The genetic diversity of the population was found to reside on the whole bacterial genome, as suggested by the RAPD analysis results, and seemed to be distributed on both the chromosome and plasmid pSym.     

Application of immunodiffusion to the identification of Rhizobium meliloti strains competing for nodulation on Medicago sativa

The immunodiffusion technique was successfully used to unambiguously recognize four strains of Rhizobium meliloti in a study of competition for nodulation with Medicago sativa cv. Apollo inoculated with two-, three- and fourstrain mixtures. The serological reactions of all R. meliloti strains revealed no significant changes following plant passage indicating that the antigens involved in immunodiffusion were stable. R. meliloti 102F70 formed 50% or more of the nodules on M. sativa inoculated with two-, three- and four-strain mixtures. The remaining three strains were less competitive and produced similar proportions of nodules (14–20%) on plants inoculated with three- and four-strain mixtures. Cases of mixed-strain occupancy of nodules involving either two of three strains were detected in a sub-sample of nodules. The data also indicated considerable variation in the proportions of strains in the nodules of individual plants.     

A note on evidence for involvement of pectolytic enzymes in the infection process of Medicago sativa by Rhizobium meliloti

Evidence has been obtained of the implication of pectolytic enzymes in the infection of alfalfa roots by Rhizobium meliloti.     

Regulation of symbiotic nitrogen fixation in root nodules of alfalfa (Medicago sativa) infected with Rhizobium meliloti

Symbiotic nitrogen fixation of Rhizobium meliloti bacteroids in Medicago sativa root nodules was suppressed by several inorganic nitrogen sources. Amino acids like glutamine, glutamic acid and aspartic acid, which can serve as sole nitrogen sources for the unnodulated plant did not influence nitrogenase activity of effective nodules, even at high concentrations.Ammonia and nitrate suppressed symbiotic nitrogen fixation in vivo only at concentrations much higher than those needed for suppression of nitrogenase activity in free living nitrogen fixing bacteria. The kinetics of suppression were slow compared with that of free living nitrogen fixing bacteria. On the other hand, nitrite, which acts as a direct inhibitor of nitrogenase, suppressed very quickly and at low concentrations. Glutamic acid and glutamine enhanced the effect of ammonia dramatically, while the suppression by nitrate was enhanced only slightly.     

Motility and Chemotaxis of Rhizobium meliloti in Soil

The spreading of Rhizobium meliloti strains in various autoclaved soils was measured by using simple modifications of established techniques. Behavioral mutants were used to demonstrate that efficient spreading requires active motility and chemotaxis. The rate of spreading was affected by physical or chemical differences among soils and by changes in water content.     

Enhanced competitiveness for nodulation of Medicago sativa by Rhizobium meliloti transconjugants harbouring the root-inducing plasmids of Agrobacterium rhizogenes strain 15834

Abstract The root-inducing plasmid of Agrobacterium rhizogenes strain 15834 marked with transposon Tn5-mob with a helper plasmid RP4-4 was mobilized into nitrogen-fixing Rhizobium meliloti strain CIAM 1759. The resulting transconjugants did not induce ‘hairy’ root syndrome but developed nitrogen-fixing nodules on alfalfa. Among the 9 transconjugants tested, 6 strains had increased nodulation rates. The competitiveness of 2 of these R. meliloti (pRi) strains was significantly enhanced as compared with the parent strain CIAM 1759; this was confirmed both in tube and in pot tests.     

Enhanced competitiveness for nodulation of Medicago sativa by Rhizobium meliloti transconjugants harbouring the root-inducing plasmids of Agrobacterium rhizogenes strain 15834

Abstract The root-inducing plasmid of Agrobacterium rhizogenes strain 15834 marked with transposon Tn 5 -mob RP4-4 with a helper plasmid was mobilized into nitrogen-fixing Rhizobium meliloti strain CIAM 1759. The resulting transconjugants did not induce ‘hairy’ root syndrome but developed nitrogen-fixing nodules on alfalfa. Among the 9 transconjugants tested, 6 strains had increased modulation rates. The competitiveness of 2 of these R. meliloti (pRi) strains was significantly enhanced as compared with the parent strain CIAM 1759; this was confirmed both in tube and in pot tests.     

Rhizobium meliloti Mutants Defective in Symbiotic Nitrogen Fixation Affect the Oxygen Gradient in Alfalfa (Medicago sativa) Root Nodules

Rhizobium meliloti bacteroids carrying mutations in either fdxNor fixX isolated from alfalfa root nodules were shown to containthe nitrogenase proteins NifH, NifD and NifK. In contrast toan in vitro system of N₂-fixation based on R. meliloti wild-typebacteroids, nitrogenase activity could not be restored in crudeextracts of these mutant bacteroids by the addition of an artificialelectron donor, indicating that the nitrogenase proteins werepresent but not functional. ESR-studies revealed that both mutantslacked the FeMo-cofactor of nitrogenase. To analyse the roleof free O₂ on the damage of the nitrogenase components and theFeMo-cofactor in these mutant bacteroids, microelectrode studiesof O₂ concentrations and gradients within alfalfa root noduleswere carried out. R. meliloti mutants defective in other genesnecessary for symbiotic N₂-fixation were also included in thisstudy. Four distinct types of O₂ gradients were defined by theapparent presence or absence of an O₂ diffusion barrier andby the minimum internal O₂ concentration. These data clearlydemonstrated the influence of the microsymbiont on the O₂ gradientswithin the nodules. Nodules induced by Rm0540, an R. melilotimutant with altered exopolysaccharide production, which is notable to infect plant cells, did not contain an O₂ diffusionbarrier. In contrast, nodules containing a mutant defectivein dicarboxylate transport (dctA-), produced an O₂ gradientsimilar to the wild-type. Microelectrode measurements revealedH₂ concentrations in alfalfa wild-type nodules comparable tosoyabean, whereas no hydrogen could be detected in nodules harbouringthe dctA mutant or any other mutant strain. Key words: Nitrogen fixation, Rhizobium meliloti bacteroids, ferredoxin-like proteins, microelectrode studies     

Sequential Analysis of the Organogenesis of Lucerne (Medicago sativa) Root Nodules Using Symbiotically-Defective Mutants of Rhizobium meliloti

Legume root-nodules are differentiated organs composed of peripheral tissue containing vascular bundles, and a central tissue in which are located the nitrogen-fixing bacteroids. The morphogenesis of these eukaryotic organs is induced by a prokaryotic organism, Rhizobium , which is amenable to genetic analysis. Inoculation of lucerne seedlings with leucine-requiring (Leu⁻) mutants of R. meliloti resulted in the formation of ineffective nodules. In these nodules, bacteria were not released from the infection threads into the host cytoplasm. When urea was provided as a nitrogen source to compensate for the defect in nitrogen fixation, the nodules became anatomically similar to those of effective nodules induced by the wild-type strain. The fact that these nodules were induced by bacteria which remained sequestered in infection threads indicates that nodule morphogenesis can be triggered from a distance. We hypothesize the existence of a bacterial nodule organogenesis-inducing principle (NOIP) which can cross the plant cell wall and plasmalemma.
In nitrogen-fixing nodules the central tissue exhibited a ploidy gradient, while in ineffective Leu⁻ nodules it was found to be monosomatic. The initiation of nodule formation is therefore independent of polyploidy. Supplying the defective plant-bacterial system with l -leucine or one of its precursors, α-ketoisovalerate or α-ketoisocaproate, caused the release of rhizobia into the plant cytoplasm and a restoration of nitrogen fixation. In the central tissue infected cells were polyploid and enlarged, and uninfected cells remained small and contained small nuclei. Therefore induction of differentiation of the central tissue requires the presence of bacteria in the cytoplasm. We hypothesize the role of a bacterial central tissue differentiation inducing principle (CTDIP) which cannot pass from cell to cell.     

Use of green fluorescent protein to visualize the early events of symbiosis between Rhizobium meliloti and alfalfa (Medicago sativa).

A gene encoding a variant of green fluorescent protein (GFP) of Aequorea victoria was put under the control of a promoter which is constitutive in Rhizobium meliloti. The heterologous GFP gene was expressed at high levels during all stages of symbiosis, allowing R. meliloti cells to be visualized as they grew in the rhizosphere, on the root surface, and inside infection threads. In addition, nodules that were infected with bacteria which were synthesizing GFP fluoresced when illuminated with blue light. GFP-tagged bacteria could be seen inside infection threads, providing the opportunity to measure the growth rate and determine the patterns of growth of R. meliloti residing inside its host plant.     

Influence of Location, Host Cultivar, and Inoculation on the Composition of Naturalized Populations of Rhizobium meliloti in Medicago sativa Nodules

A phage typing system was used to evaluate the composition of indigenous populations of Rhizobium meliloti inhabiting nodules of Medicago sativa cultivars grown with and without inoculation at two field sites during 1983 and 1984. Soil at both locations contained established populations of R. meliloti at planting. Analysis of 1,920 nodule isolates revealed 55 unique phage types of indigenous R. meliloti at one site and 65 indigenous types at the other location. The distributions of phage types differed markedly between locations. At one site, the nodule population was dominated by two phage types; seven others occurred consistently but at lower frequency, and the remainder were encountered infrequently. No indigenous types predominated at the other location, although nine occurred more frequently than the remaining types. Indigenous R. meliloti predominated in nodules from inoculated plots at both sites, with inoculant recovery varying between 10 and 38% in each of two years. The frequency of occurrence of particular phage types at one location was significantly influenced by both M. sativa cultivar and inoculation. At this location, the interaction of cultivar and inoculation on the incidence of phage types suggests that the presence of an inoculant strain differentially affected nodule occupancy of M. sativa cultivars by members of the indigenous R. meliloti population. At both sites, the frequency of specific phage types differed between years. The data emphasize the importance of understanding the ecology and characteristics of indigenous Rhizobium populations as a prerequisite for elucidating problems of inoculant establishment and persistence in competitive situations.     

L-Canavanine made by Medicago sativa interferes with quorum sensing in Sinorhizobium meliloti

Sinorhizobium meliloti is a gram-negative soil bacterium, capable of establishing a nitrogen-fixing symbiosis with its legume host, alfalfa (Medicago sativa). Quorum sensing plays a crucial role in this symbiosis, where it influences the nodulation process and the synthesis of the symbiotically important exopolysaccharide II (EPS II). S. meliloti has three quorum-sensing systems (Sin, Tra, and Mel) that use N-acyl homoserine lactones as their quorum-sensing signal molecule. Increasing evidence indicates that certain eukaryotic hosts involved in symbiotic or pathogenic relationships with gram-negative bacteria produce quorum-sensing-interfering (QSI) compounds that can cross-communicate with the bacterial quorum-sensing system. Our studies of alfalfa seed exudates suggested the presence of multiple signal molecules capable of interfering with quorum-sensing-regulated gene expression in different bacterial strains. In this work, we choose one of these QSI molecules (SWI) for further characterization. SWI inhibited violacein production, a phenotype that is regulated by quorum sensing in Chromobacterium violaceum. In addition, this signal molecule also inhibits the expression of the S. meliloti exp genes, responsible for the production of EPS II, a quorum-sensing-regulated phenotype. We identified this molecule as l-canavanine, an arginine analog, produced in large quantities by alfalfa and other legumes.     

Role of the Differentiation of Root Epidermal Cells in Nod Factor (from Rhizobium meliloti)-Induced Root-Hair Depolarization of Medicago sativa

The stage of differentiation of epidermal cells and the development of root hairs was found to be important for the induction of depolarization in root hairs of Medicago sativa by Nod factor [NodRm-IV(S)] isolated from the bacterium Rhizobium meliloti. The electrical membrane response was concentration dependent, having its major effect (amplitude of the depolarization and number of root hairs that responded) at 10-8 and 10-7 M Nod factor. This response was correlated with a morphological effect of Nod factor in the root-hair-deformation bioassay at similar concentrations. The effect of Nod factor on depolarization and root-hair deformation showed specificity with respect to the structure, since unsulfated Nod molecules were inactive, as was the synthetic N,N',N",N"'- tetraacetylchitotetraose. The Nod factor that is O-acetylated at the nonreducing sugar was as efficient in root-hair deformation and membrane depolarization as the sulfated Nod factor.     

人参不同栽培群体遗传关系的RAPD分析

用随机扩增多态ＤＮＡ（ＲＡＰＤ）标记法对４个人参（Ｐａｎａｘ ｇｉｎｓｅｇ Ｃ．Ａ．Ｍｅｙｅｒ）栽培群体中存在较丰富的遗传多样性。分析一路参、三路参、系选品系５９号、北京参等４个人参栽培群体和１个西洋参（Ｐ．ｑｕｉｎｑｕｅｆｏｌｉｕｍ Ｌ．）群体的遗传分化指数值表明,三路参变异量最大（０．４１６９）,一路参降为０．２５６５,边条参系选品系５９号最低为０．１８８１,表明选择方式和选择代数的纯化作用十分     

Conserved Nodulation Genes in Rhizobium meliloti and Rhizobium trifolii

Plasmids which contained wild-type or mutated Rhizobium meliloti nodulation (nod) genes were introduced into Nod⁻R. trifolii mutants ANU453 and ANU851 and tested for their ability to nodulate clover. Cloned wild-type and mutated R. meliloti nod gene segments restored ANU851 to Nod⁺, with the exception of nodD mutants. Similarly, wild-type and mutant R. meliloti nod genes complemented ANU453 to Nod⁺, except for nodCII mutants. Thus, ANU851 identifies the equivalent of the R. meliloti nodD genes, and ANU453 specifies the equivalent of the R. meliloti nodCII genes. In addition, cloned wild-type R. trifolii nod genes were introduced into seven R. meliloti Nod⁻ mutants. All seven mutants were restored to Nod⁺ on alfalfa. Our results indicate that these genes represent common nodulation functions and argue for an allelic relationship between nod genes in R. meliloti and R. trifolii.