Rhizobial position as a main determinant in the problem of competition for nodulation in soybean

Selected Bradyrhizobium japonicum strains inoculated on soybean seeds often fail to occupy a significant proportion of nodules when a competitor rhizobial population is established in the soil. This competition problem could result from a genetic/ physiological advantage of the adapted soil population over the introduced inoculant or from a positional advantage, as the soil population already occupies the soil profile where the roots will penetrate, whereas the inoculant remains concentrated around the seeds. Here, we have assessed the contribution of these factors with a laboratory model in which a rhizobial population is established in sterile vermiculite. We observed that the wild-type strain B. japonicum LP 3004 was able to grow in pots with N-free plant nutrient solution-watered vermiculite for six or seven generations with a duplication rate of at least 0.7 day(-1). In addition, the rhizobial population persisted for 3 months with 10(6)-10(7) colony-forming units ml(-1) of the vermiculite-retained solution. N-starved, young rhizobial cultures are more efficient in performing several steps along their early association with soybean roots. However, N starvation during growth of rhizobia used for seed inoculation did not enhance their competitiveness against a 1 month vermiculite-established rhizobial population, which occupied more than 72% of the nodules. When a similarly established rhizobial population was recovered from the vermiculite and homogeneously suspended in plant nutrient solution, these cells were significantly less competitive (29% of nodules occupied) than rhizobia obtained from a fresh, logarithmic culture in a N-poor minimal medium, thus indicating that cell position rather than intrinsic competitiveness was the determinant for nodule occupation.     

Appearance of a novel form of plant glutamine synthetase during nodule development in Phaseolus vulgaris L.

The activities of glutamine synthetase (GS), nitrogenase and leghaemoglobin were measured during nodule development in Phaseolus vulgaris infected with wild-type or two non-fixing (Fix^-) mutants of Rhizobium phaseoli. The large increase in GS activity which was observed during nodulation with the wild-type rhizobial strain occurred concomitantly with the detection and increase in activity of nitrogenase and the amount of leghaemoglobin. Moreover, this increase in GS was found to be due entirely to the appearance of a novel form of the enzyme (GS_n1) in the nodule. The activity of the form (GS_n2) similar to the root enzyme (GS_r) remained constant throughout the experiment. In nodules produced by infection with the two mutant strains of Rhizobium phaseoli (JL15 and JL19) only trace amounts of GS_n1 and leghaemoglobin were detected.Abbreviations DEAE-Sephacel diethylaminoethyl-Sephacel - GS glutamine synthetase     

超慢生大豆根瘤菌DNA G+C含量和DNA同源性测定

用热变性温度法和液相复性速率法分别测定了超慢生大豆根瘤菌(ESG,extra-slow-growing soybean rhizobia)DNA G+C mol%及与其它根瘤菌间的DNA同源性.结果表明,ESG的DNA G+C mol含量在59.2—63.5%之间,且不同地区不同血清型的ESG代表菌株DNA同源率在70%以上,说明它们是遗传型一致的类群.ESG与在大豆上结瘤的快生大豆根瘤菌(Rhizobium fredii USDA205)同源率为14.8%,与慢生大豆根瘤菌(Bradyrhizobiumjaponicum)三个DNA同源组的同源率分别为20.5%,30.0%,19.4%.测定结果还表明,ESG与其它根瘤菌遗传学的亲缘关系也很远.     

Genetics of promiscuous nodulation in soybean: nodule dry weight and leaf color score

The symbiotic relationship between the soybean plant and rhizobium results in fixation of atmospheric nitrogen (N(2)) in the root nodules, with the result that nitrogenous fertilization of the soybean is unnecessary. The effectiveness of nodule formation and N(2) fixation with rhizobial strains is under genetic control with two general categories identified: (1) promiscuous, which produces functional nodules with cowpea-type rhizobial strains; and (2) nonpromiscuous, which forms no or nonfunctional nodules with these strains. The segregation pattern of this promiscuity trait was studied using nodule dry weight (NDW) and leaf color score (LCS) as indicators of N(2) fixation effectiveness. Individual plants in each of six populations [P(1) = nonpromiscuous, P(2) = promiscuous, F(1) = P(1) x P(2) (and the reciprocal cross), BC(1)(P(1)) = F(1) (female) x P(1), BC(1)(P(2)) = F(1) (female) x P(2), F(2)] were scored for these characters after inoculation with a rhizobial strain that would distinguish between both types. For NDW, nonpromiscuity was found to be partially dominant (h/d = 0.37), controlled by four loci. For LCS, nonpromiscuity was shown to be almost completely dominant (h/d = 0.74), controlled by two loci. LCS was a more meaningful estimate of N(2) fixation because it represented the total effectiveness of nodulation to provide nitrogen for the plant.     

Organization and expression of leghaemoglobin genes

Leghaemoglobin genes in soybean (Glycine max) are present as a moderately reiterated family of sequences. Since there are identical restriction site patterns of these sequences in DNA isolated from leaf, root, or nodule tissue, the data suggest that no major changes in the organization or methylation of leghaemoglobin genes occur during their induction. Cloned soybean leghaemoglobin-cDNA cross hybridized with RNA from root nodules of kidney bean (Phaseolus vulgaris), and to a lesser extent, of pea (Pisum sativum) indicating sequence homology in the leghaemoglobin genes of these species. Hybridization to the genomic DNA restriction fragments of two other species, Glycine soja and Vicia faba, also indicated interspecies sequence homologies. Several restriction fragments appear to be common to all species examined. The induction of these genes occurs following infection of the plant by Rhizobium and is independent of the appearance of nitrogenase activity in the nodules. The level of expression is, however, influenced by various mutations in Rhizobium that result in the development of ineffective (nonnitrogen fixing) nodules.     

The antibiosis of nodule-endophytic agrobacteria and its potential effect on nodule functioning of Phaseolus vulgaris

The effect of the nodule-endophytic Agrobacterium strain 10C2 on nodulation, plant growth and nodule functioning of Phaseolus vulgaris was investigated using two rhizobial strains differing in their sensitivity to the in vitro antibiosis of the Agrobacterium strain. In the case of the sensitive strain, Agrobacterium sp. 10C2 induced a significant decrease in the proportion of pink nodules, probably by an antibiosis effect leading to the reduction in the number of bacteroids and thereby a decrease in total soluble proteins, leghaemoglobin content, photosynthesis and nitrogen fixation. In this case, the Agrobacterium strain behaved like a plant pathogen and the nodule reacted by increasing guaiacol peroxidase (POX) activity, which assures some physiological processes linked to pathogen control. By contrast, in the case of the resistant strain, the proportion of pink nodules increased, and thereby total soluble proteins, leghaemoglobin content, biomass production and nitrogen fixation were enhanced. The Agrobacterium strain is regarded in this case as a plant growth–promoting rhizobacterium and the POX-pathogen reaction was not observed. There was even a decrease in superoxide dismutase activity. The results suggested also that the Agrobacterium strain may be also involved in retarding nodule senescence in the case of the resistant strain.     

Comparative Response of Nodulated and Nitrogen-Supplied Cowpea (Vigna unguiculata (L.) Walp. var. Tuy) Plants to Infection by Cowpea Mosaic Virus

The effect of infection by the Cowpea Mosaic Virus (CpMV) onseveral parameters relevant to symbiotic nitrogen fixation wasdetermined in cowpea (Vigna unguiculata (L.) Walp. var. Tuy)plants nodulated with two strains of Rhizobium cowpea: IVIC–124and IVIC–38. Plants were virus-infected at the seedlingstage before Rhizobium inoculation. The effect of CpMV infectionon plant growth was analysed in nodulated and nitrogen-suppliedplants at 18, 25 and 35 d after germination. At all developmentalstages of nodulated plants CpMV infection caused a reductionof leaf chlorophyll content, leaf area, dry weight of shootsand roots, total nodule weight and nodule number. Most of thenodules from 18- and 25-d-old CpMV-infected plants did not exhibitleghaemoglobin pigmentation. CpMV infection delayed the onsetof nitrogenase activity in nodules of the rhizobial strain IVIC–124and the enzyme activity measured on a per plant basis was reducedin both strains at the first and second harvests. Significantnitrogenase activity was detected in 35-d-old infected plants.Some of the nodules of the rhizobial strain IVIC-124 and mostof the nodules from plants nodulated with the strain IVIC-38developed leghaemoglobin; however, the nodule-specific nitrogenaseactivity, estimated on a milligram nodule dry weight basis,was always higher in virus-infected plants, particularly in18-d-old CpMV-infected plants harbouring the IVIC–124strain. CpMV-infected nodules had a larger peribacteroidal space,a reduced number of peribacteroid units, a greater number ofbacteroids per unit, a lower number of vesicles and 88% lowertotal reducing sugar content. Starch accumulation was detectedin infected leaves of nodulated plants during the first harvest,while high levels of leaf reducing sugars and protein were presentat the second harvest. In healthy nodulated plants the rhizobialstrain IVIC–124 was shown to be more efficient than IVIC–38in promoting plant growth. However, the results indicate thatnodulation by rhizobial strain IVIC–124 and growth ofplants harbouring this strain were affected to a greater extentby virus infection. The effect of CpMV infection on leaf chlorophyllcontent, leaf area, carbohydrate level, leaf proteins and growthof nitrogen-supplied plants, as well as the symptoms inducedin the leaves, were less conspicuous than in nodulated plants. Key words: Cowpea, Rhizobium, virus infection, nodule untrastructure     

DNA restriction patterns and DNA-DNA solution hybridization studies of Frankia isolates from Myrica pennsylvanica (bayberry)

Sixteen Frankia strains were isolated from Myrica pennsylvanica (bayberry) root nodules collected at diverse sites in New Jersey. Restriction pattern analysis of total genomic DNA was used to group the isolates into gel groups, and the genetic relatedness among the isolates was evaluated by DNA-DNA solution hybridization studies. Restriction pattern analysis provided a distinctive reproducible fingerprint for each isolate. Isolates fell into nine separate groups (strain types). More than one strain type was isolated from most sites. Isolates from two different gel groups were found in 3 of 10 nodules examined. Of the 16 isolates, 10 contained extrachromosomal DNA. Six different extrachromosomal DNA banding patterns were found. Genomically similar isolates carried related, but different, banding patterns. DNA hybridization studies indicated that isolates from a single plant species can be minimally related as determined by total genome homology. Homology ranged from 12 to 99%. Highly divergent strains were isolated from the same plant and found to cohabit the same nodule. Thus, this study demonstrated that Frankia strains which infect the same host plant are not only phenotypically different but also genetically diverse.     

DNA restriction patterns and DNA-DNA solution hybridization studies of Frankia isolates from Myrica pennsylvanica (bayberry).

Sixteen Frankia strains were isolated from Myrica pennsylvanica (bayberry) root nodules collected at diverse sites in New Jersey. Restriction pattern analysis of total genomic DNA was used to group the isolates into gel groups, and the genetic relatedness among the isolates was evaluated by DNA-DNA solution hybridization studies. Restriction pattern analysis provided a distinctive reproducible fingerprint for each isolate. Isolates fell into nine separate groups (strain types). More than one strain type was isolated from most sites. Isolates from two different gel groups were found in 3 of 10 nodules examined. Of the 16 isolates, 10 contained extrachromosomal DNA. Six different extrachromosomal DNA banding patterns were found. Genomically similar isolates carried related, but different, banding patterns. DNA hybridization studies indicated that isolates from a single plant species can be minimally related as determined by total genome homology. Homology ranged from 12 to 99%. Highly divergent strains were isolated from the same plant and found to cohabit the same nodule. Thus, this study demonstrated that Frankia strains which infect the same host plant are not only phenotypically different but also genetically diverse.     

Nodule-specific host proteins in effective and ineffective root nodules of Pisum sativum

Nodule-specific root proteins – so called nodulins – were identified in root nodules of pea plants by an immunological assay. Nodulin patterns were examined at different stages of nodule development. About 30 nodulins were detectable during development. Some were preferentially synthesized before nitrogen fixation started, whereas the majority were synthesized concomitantly with leghaemoglobin. Some of the nodulins were located within the peribacteroid membrane. Ineffective Rhizobium strains (a natural nod⁺fix^- and a pop ^-fix^-) appeared to be useful in studying the expression of nodulin genes. Synthesis of some nodulins was repressed in ineffective root nodules, indicating that nodulins are essential for the establishment of nitrogen fixation. In both types of ineffective root nodules, leghaemoglobin synthesis was not completely repressed. Low amounts of leghaemoglobin were always detected in young ineffective root nodules whereas in old nodules no leghaemoglobin was present.     

Preparation of a complementary DNA for leghaemoglobin and direct demonstration that leghaemoglobin is encoded by the soybean genome

In soybean root nodules, leghaemoglobin (Lb) accounts for 25--30% of the total soluble protein but is not detected in other tissues. In order to determine whether the Lb genes are plant or bacterial in origin a cDNA probe for Lb was prepared from 9S poly (A) containing mRNA of root nodules. Although this 9S mRNA directed synthesis of predominantly three forms of Lb in vitro, the kinetics of hybridisation of cDNA and the 9S mRNA showed a transition at about 30% hybridisation which suggested that the 9S-cDNA was not pure Lb-cDNA. The abundant, Lb-cDNA was prepared by two cycles of hybridising 9S mRNA and cDNA to a Rot of 3 X 10(-3) and isolation of the hybridised cDNA on hydroxyapatite. The Lb-cDNA was homogeneous in hybridisation analysis with 9S mRNA and electrophoresis in 98% formamide gels. This cDNA hybridised with soybean DNA and not with Rhizobium DNA, thus directly demonstrating that Lb genes are of plant origin. Titration of Lb-cDNA with soybean DNA showed that Lb genes are reiterated about forty-fold per haploid genome.     

Genetic characterization of soybean rhizobia in Paraguay

The soybean is an exotic plant introduced in Paraguay in this century; commercial cropping expanded after the 1970s. Inoculation is practiced in just 15 to 20% of the cropping areas, but root nodulation occurs in most sites where soybeans grow. Little is known about rhizobial diversity in South America, and no study has been performed in Paraguay until this time. Therefore, in this study, the molecular characterization of 78 rhizobial isolates from soybean root nodules, collected under field conditions in 16 sites located in the two main producing states, Alto Paraná and Itapúa, was undertaken. A high level of genetic diversity was detected by an ERIC-REP-PCR analysis, with the majority of the isolates representing unique strains. Most of the 58 isolates characterized by slow growth and alkaline reactions in a medium containing mannitol as a carbon source were clustered with strains representative of the Bradyrhizobium japonicum and Bradyrhizobium elkanii species, and the 16S ribosomal DNA (rDNA) sequences of 5 of those isolates confirmed the species identities. However, slow growers were highly polymorphic in relation to the reference strains, including five carried in commercial inoculants in neighboring countries, thus indicating that the Paraguayan isolates might represent native bradyrhizobia. Twenty isolates highly polymorphic in the ERIC-REP-PCR profiles were characterized by fast growth and acid reactions in vitro, and two of them showed high 16S rDNA identities with Rhizobium genomic species Q. However, two other fast growers showed high 16S rDNA identity with Agrobacterium spp., and both of these strains established efficient symbioses with soybean plants.     

Compatibility of rhizobial genotypes within natural populations of Rhizobium leguminosarum biovar viciae for nodulation of host legumes

Populations of Rhizobium leguminosarum biovar viciae were sampled from two bulk soils, rhizosphere, and nodules of host legumes, fava bean (Vicia faba) and pea (Pisum sativum) grown in the same soils. Additional populations nodulating peas, fava beans, and vetches (Vicia sativa) grown in other soils and fava bean-nodulating strains from various geographic sites were also analyzed. The rhizobia were characterized by repetitive extragenomic palindromic-PCR fingerprinting and/or PCR-restriction fragment length polymorphism (RFLP) of 16S-23S ribosomal DNA intergenic spacers as markers of the genomic background and PCR-RFLP of a nodulation gene region, nodD, as a marker of the symbiotic component of the genome. Pairwise comparisons showed differences among the genetic structures of the bulk soil, rhizosphere, and nodule populations and in the degree of host specificity within the Vicieae cross-inoculation group. With fava bean, the symbiotic genotype appeared to be the preponderant determinant of the success in nodule occupancy of rhizobial genotypes independently of the associated genomic background, the plant genotype, and the soil sampled. The interaction between one particular rhizobial symbiotic genotype and fava bean seems to be highly specific for nodulation and linked to the efficiency of nitrogen fixation. By contrast with bulk soil and fava bean-nodulating populations, the analysis of pea-nodulating populations showed preferential associations between genomic backgrounds and symbiotic genotypes. Both components of the rhizobial genome may influence competitiveness for nodulation of pea, and rhizosphere colonization may be a decisive step in competition for nodule occupancy.     

Genetic Characterization of Soybean Rhizobia in Paraguay

The soybean is an exotic plant introduced in Paraguay in this century; commercial cropping expanded after the 1970s. Inoculation is practiced in just 15 to 20% of the cropping areas, but root nodulation occurs in most sites where soybeans grow. Little is known about rhizobial diversity in South America, and no study has been performed in Paraguay until this time. Therefore, in this study, the molecular characterization of 78 rhizobial isolates from soybean root nodules, collected under field conditions in 16 sites located in the two main producing states, Alto Paraná and Itapúa, was undertaken. A high level of genetic diversity was detected by an ERIC-REP-PCR analysis, with the majority of the isolates representing unique strains. Most of the 58 isolates characterized by slow growth and alkaline reactions in a medium containing mannitol as a carbon source were clustered with strains representative of the Bradyrhizobium japonicum and Bradyrhizobium elkanii species, and the 16S ribosomal DNA (rDNA) sequences of 5 of those isolates confirmed the species identities. However, slow growers were highly polymorphic in relation to the reference strains, including five carried in commercial inoculants in neighboring countries, thus indicating that the Paraguayan isolates might represent native bradyrhizobia. Twenty isolates highly polymorphic in the ERIC-REP-PCR profiles were characterized by fast growth and acid reactions in vitro, and two of them showed high 16S rDNA identities with Rhizobium genomic species Q. However, two other fast growers showed high 16S rDNA identity with Agrobacterium spp., and both of these strains established efficient symbioses with soybean plants.     

Assessing host range,symbiotic effectiveness,and photosynthetic rates induced by native soybean rhizobia isolated from Mozambican and South African soils

Host range and cross-infectivity studies are important for identifying rhizobial strains with potential for use as inoculants. In this study, 10 native soybean rhizobia isolated from Mozambican and South African soils were evaluated for host range, symbiotic effectiveness and ability to induce high rates of photosynthesis leading to enhanced plant growth in cowpea (Vigna unguiculata L. Walp.), Bambara groundnut (Vigna subterranean L. Verdc.), Kersting’s groundnut (Macrotyloma geocarpum Harm) and soybean (Glycine max L. Merr). The test isolates had different growth rates and colony sizes. Molecular analysis based on enterobacterial repetitive intergenic consensus (ERIC)-PCR revealed high genetic diversity among the test isolates. The results further showed that isolate TUTLBC2B failed to elicit nodulation in all test plants, just as TUTNSN2A and TUTDAIAP3B were also unable to nodulate cowpea, Kersting’s bean and Bambara groundnut. Although the remaining strains formed ineffective nodules on cowpea and Kersting’s bean, they induced effective nodules on Bambara groundnut and the two soybean genotypes. Bacterial stimulation of nodule numbers, nodule dry weights and photosynthetic rates was generally greater with isolates TUTRSRH3A, TUTM19373A, TUTMCJ7B, TUTRLR3B and TUTRJN5A. As a result, these isolates elicited significantly increased accumulation of biomass in shoots and whole plants of Bambara groundnut and the two soybean genotypes. Whole-plant symbiotic nitrogen (N) of soybean and Bambara groundnut was highest for the commercial strains CB756 and WB74, as well as for TUTRLR3B, TUTMCJ7B and TUTRSRH3A, suggesting that the three native rhizobial isolates have potential for use as inoculants.     

A search for a leghaemoglobin-like compound in root nodules of Trema cannabina Lour.

Root nodules collected from the non-leguminous plant Trema cannabina Lour. grown under natural field conditions were examined for the presence of leghaemoglobin. No trace of any soluble haemoglobin-like compound could be detected by spectrophotometric analysis or molecular weight comparison studies, although a small amount of soluble haemoprotein with peroxidase activity was isolated. Possible alternatives to the possession of a leghaemoglobin in non-legume/Rhizobium N2-fixing associations are discussed.     

The symbiosis between oriental goat’s rue and the root nodule bacteria <Emphasis Type="Italic">Rhizobium galegae</Emphasis>: Specificity and competitiveness

Competitiveness and genetic variation of the Rhizobium galegae strains from the collection of the All-Russia Institute of Agricultural Microbiology, Russian Academy of Agricultural Sciences, causing nodulation of oriental goat’s rue under conditions of Bashkortostan soils (lacking this rhizobial species) were studied. It was demonstrated that of all the tested strains, the strains CIAM 0702 and CIAM 0704, each carrying two megaplasmids of 1500 and 2000 MDa, were the most competitive. RAPD (random amplified polymorphic DNA) analysis showed that R. galegae strains were able to intensively exchange the genetic material in the host plant rhizosphere. We did not succeed in detecting the local root nodule bacteria that were either initially able to infect oriental goat’s rue or had adapted to infecting this species due to various genetic rearrangements.     

Phenotypic and genotypic analysis of rhizobia isolated from pasture legumes native of Sardinia and Asinara Island

Thirty-five rhizobial strains were isolated from nodules of Lotus edulis, L. ornithopodioides, L. cytisoides, Hedysarum coronarium, Ornithopus compressus and Scorpiurus muricatus growing in Sardinia and Asinara Island. Basic characteristics applied to identification of rhizobia such as symbiotic properties, antibiotic- and salt-resistance, temperate-sensitivities, utilization of different sources of carbon and nitrogen were studied. The results from the 74 metabolic tests were used for cluster analysis of the new rhizobial isolates and 28 reference strains, belonging to previously classified and unclassified fast-, intermediate- and slow-growing rhizobia. All strains examined were divided into two large groups at a linkage distance of 0.58. None of the reference strains clustered with the new rhizobial isolates, which formed five subgroups almost respective of their plant origin. RFLP analysis of PCR-amplified 16S-23S rDNA IGS showed that the levels of similarity between rhizobial isolates from Ornithopus, Hedysarum and Scorpiurus, and the type strains of Rhizobium leguminosarum, Mesorhizobium loti, M. ciceri, M. mediterraneum, Sinorhizobium meliloti and Bradyrhizobium japonicum were not more than 30%. Thus, it can be assumed that these groups of new rhizobial isolates are not closely related to the validly described rhizobial species.