Induced genetic variability in Rhizobium leguminosarum for nitrogen fixation parameters in Vicia faba L.

Sumary The objective of this work was to know the behaviour and variability of Rhizobium leguminosarum after irradiation. The induced variation was tested under greenhouse conditions on the variety JV 3 of broad beans (Vicia faba) in six replications. Induced genetic variabilty was observed for strain, parent and mutant versus parent. Out of 24 irradiated strains, strain 93-32 performed better with a greater number of nodules and higher dry weight of nodules per plant and biological yield. Environment played an important role in the expression of characters observed. High heritability and genetic advance of these traits indicated that the nitrogen fixation ability of Rhizobium can easily be improved by selection.     

Correlation between infection by Rhizobium leguminosarum and lectin on the surface of Pisum sativum L. roots

The lectin on the surface of 4- and 5-dold pea roots was located by the use of indirect immunofluorescence. Specific antibodies raised in rabbits against pea seed isolectin 2, which crossreact with root lectins, were used as primary immunoglobulins and were visualized with fluorescein- or tetramethylrhodamine-isothiocyanate-labeled goat antirabbit immunoglobulin G. Lectin was observed on the tips of newly formed, growing root hairs and on epidermal cells located just below the young hairs. On both types of cells, lectin was concentrated in dense small patches rather than uniformly distributed. Lectin-positive young hairs were grouped opposite the (proto)xylematic poles. Older but still-elongating root hairs presented only traces of lectin or none at all. A similar pattern of distribution was found in different pea cultivars, as well as in a supernodulating and a non-nodulating pea mutant. Growth in a nitrate concentration which inhibits nodulation did not affect lectin distribution on the surface of pea roots of this age. We tested whether or not the root zones where lectin was observed were susceptible to infection by Rhizobium leguminosarum. When low inoculum doses (consisting of less than 10⁶ bacteria·ml^-1) were placed next to lectin-positive epidermal cells and on newly formed root hairs, nodules on the primary roots were formed in 73% and 90% of the plants, respectively. Only a few plants showed primary root nodulation when the inoculum was placed on the root zone where lectin was scarce or absent. These results show that lectin is present at those sites on the pea root that are susceptible to infection by the bacterial symbiont.Abbreviations FITC fluorescein isothiocyanate - TRIC tetramethylrhodamine isothiocyanate     

Microscopic analysis of the effect ofRhizobium leguminosarum biovartrifolii host specific nodulation genes in the infection of white clovers

Summary A microscopic assessment is presented of the comparative infection capacity of wild-type and hybrid strains ofRhizobium leguminosarum bv.viciae withR. l. bv.trifolii strain ANU 843 on white clover seedlings. TheR. l. bv.viciae hybrid strains contained defined DNA segments coding for different combinations ofR. l. bv.trifolii host-specific nodulation genes. White clover plants were examined over a 72 h period to assessRhizobium infectivity, the morphological changes in root hair growth; colonisation ability of rhizobia; infection thread initiation and the ability to induce cortical cell division.R. l. bv.viciae strain 300 induced root hair curling more slowly than strain ANU 843 or any of the hybrid strain 300 bacteria, and when curling had taken place, there was poorer colonization by strain 300 within the folded hair cell, no evidence of infection thread formation and only limited cortical cell division 72 h after inoculation. The addition of the host-specific nodulation genes ofR. l. bv.trifolii to strain 300 was necessary to induce infection threads and establish a normal pattern of nodulation of the roots of white clovers.     

Speed of nodulation and competitive ability among strains of Rhizobium leguminosarum bv phaseoli

This study examines the speed of nodulation of 20 strains of Rhizobium leguminosarum bv phaseoli, and relates this trait to the competitive performance of these strains with Phaseolus vulgaris L. At 25/20°C day/night temperature, and with 10⁷ cells applied per growth pouch, there was a strong positive correlation between the speed of nodulation and the competitiveness of strains with the nod ⁺ fix^– reference strain UMR 1116. Strains UMR 1084, 1125, 1165, 1173 and 1384 combined good competitive performance with extensive nodulation in the uppermost root regions. When inoculant levels in the RTM studies were reduced to 10³ cells per pouch no correlation between the apparent competitiveness of strains and their speed in nodulation was evident, presumably because cells had to undergo multiplication before infection. Nodulation was also delayed when growth temperatures were raised to 31/26°C, but a correlation was still evident between competitive performance and nodulation in the region 0.1 to 5.0 mm below the RTM at the time of inoculation. From these results speed of nodulation can be used to estimate the competitive potential of Rhizobium strains, but only under carefully regulated conditions. The effects of inoculation level and temperature on the relationship between speed of nodulation and strain competitiveness could explain the inconsistent results obtained in earlier studies on this topic.Journal paper No. 16962, Agricultural Experiment Station, University of Minnesota, St. Paul, MN 55108, USA     

The ethylene-inhibitor aminoethoxyvinylglycine restores normal nodulation by Rhizobium leguminosarum biovar. viciae on Vicia sativa subsp. nigra by suppressing the ‘Thick and short roots’ phenotype

Nodulation of Vicia sativa subsp. nigra L. by Rhizobium bacteria is coupled to the development of thick and short roots (Tsr). This root phenotype as well as root-hair induction (Hai) and root-hair deformation (Had) are caused by a factor(s) produced by the bacteria in response to plant flavonoids. When very low inoculum concentrations (0.5–5 bacteria·ml^-1) were used, V. sativa plants did not develop the Tsr phenotype and became nodulated earlier than plants with Tsr roots. Furthermore, the nodules of these plants were located on the primary root in contrast to nodules on Tsr roots, which were all located at sites of lateral-root emergence. The average numbers of nodules per plant were not significantly different for these two types of nodulation. Root-growth inhibition and Hai, but not Had, could be mimicked by ethephon, and inhibited by aminoethoxyvinylglycine (AVG). Addition of AVG to co-cultures of Vicia sativa and the standard inoculum concentration of 5·10⁵ bacteria·ml^-1 suppressed the development of the Tsr phenotype and restored nodulation to the pattern that was observed with very low concentrations of bacteria (0.5–5 bacteria·ml^-1). The delay in nodulation on Tsr roots appeared to be caused by the fact that nodule meristems did not develop on the primary root, but only on the emerging laterals. The relationship between Tsr, Hai, Had, and nodulation is discussed.Abbreviations AVG aminoethoxyvinylglycine - cfu colonyforming units - Had root-hair deformation - Hai root-hair induction - NB naringenin-bacteria filtrate - Tsr Thick and short roots     

Synergistic interaction of Rhizobium leguminosarum bv. viciae and arbuscular mycorrhizal fungi as a plant growth promoting biofertilizers for faba bean (Vicia faba L.) in alkaline soil

Egyptian soils are generally characterized by slightly alkaline to alkaline pH values (7.5–8.7) which are mainly due to its dry environment. In arid and semi-arid regions, salts are less concentrated and sodium dominates in carbonate and bicarbonate forms, which enhance the formation of alkaline soils. Alkaline soils have fertility problems due to poor physical properties which adversely affect the growth and the yield of crops. Therefore, this study was devoted to investigating the synergistic interaction of Rhizobium and arbuscular mycorrhizal fungi for improving growth of faba bean grown in alkaline soil. A total of 20 rhizobial isolates and 4 species of arbuscular mycorrhizal fungi (AMF) were isolated. The rhizobial isolates were investigated for their ability to grow under alkaline stress. Out of 20 isolates 3 isolates were selected as tolerant isolates. These 3 rhizobial isolates were identified on the bases of the sequences of the gene encoding 16S rRNA and designated as Rhizobium sp. Egypt 16 (HM622137), Rhizobium sp. Egypt 27 (HM622138) and Rhizobium leguminosarum bv. viciae STDF-Egypt 19 (HM587713). The best alkaline tolerant was R. leguminosarum bv. viciae STDF-Egypt 19 (HM587713). The effect of R. leguminosarum bv. viciae STDF-Egypt 19 and mixture of AMF (Acaulospora laevis, Glomus geosporum, Glomus mosseae and Scutellospora armeniaca) both individually and in combination on nodulation, nitrogen fixation and growth of Vicia faba under alkalinity stress were assessed. A significant increase over control in number and mass of nodules, nitrogenase activity, leghaemoglobin content of nodule, mycorrhizal colonization, dry mass of root and shoot was recorded in dual inoculated plants than plants with individual inoculation. The enhancement of nitrogen fixation of faba bean could be attributed to AMF facilitating the mobilization of certain elements such as P, Fe, K and other minerals that involve in synthesis of nitrogenase and leghaemoglobin. Thus it is clear that the dual inoculation with Rhizobium and AMF biofertilizer is more effective for promoting growth of faba bean grown in alkaline soils than the individual treatment, reflecting the existence of synergistic relationships among the inoculants.     

Rhizobium leguminosarum inoculation decreases damage to faba bean (Vicia faba) caused by broad bean mottle bromovirus and bean yellow mosaic potyvirus

Faba bean (Vicia faba) plants were inoculated with rhizobia and then their sap was infected with broad bean mottle bromovirus (BBMV) or bean yellow mosaic potyvirus (BYMV) in a field experiment. Both viral infections significantly decreased shoot and root dry weight, number of nodules, nodule dry weight, numbers of flowers and pods/plant, total plant N, grain yield and N₂ fixation. However, inoculation withRhizobium leguminosarum significantly increased all these parameters, both in healthy and virus-infected plants. Although BYMV was more destructive than BBMV, inoculation with rhizobia could be used, with other control measures, to limit damage by both viruses.The authors are with the Department of Biochemistry and Soil Science, Faculty of Agriculture, Shambat, Sudan.     

The effects of sodium chloride-salinity upon growth, nodulation, and root nodule structure of pea (Pisum sativum L.) plants

Pea (Pisum sativum L.) plants inoculated with Rhizobium leguminosarum bv. viciae effective strain 248 were irrigated with nitrogen-free medium supplemented with 0, 25, 50 or 75 mM NaCl. The inhibitory effect of salinity on the growth of pea plants treated with 25 mM NaCl occurred 6 weeks post inoculation. In case of 75 mM NaCl treatment, the same effect was observed 2 weeks post inoculation. In contrast to investigations described in the literature our results clearly indicated that 25 mM NaCl stimulated nodule formation, however, in contrast to control nodules (the medium without NaCl), the nodules were considerably smaller. Remaining variants of salt treatment reduced plant growth, nodulation, and total nodule volume calculated per plant. Microscopic observations showed that salinity: (1) caused the loss of turgor of the nodule peripheral cells, (2) changed nodule zonation, (3) stimulated infection thread enlargement and expansion, (4) caused disturbances in bacterial release from the infection threads, and (5) induced synthesis of electron dense material (EDM) and its deposition in vacuoles. It was also found that cisternae of RER were involved in the formation of special cytoplasmic compartments responsible for synthesis of EDM. Autofluorescence study revealed that salinity increased accumulation of phenolics in pea nodules, as well.     

Activation of flavonoid biosynthesis in roots of Vicia sativa subsp. nigra plants by inoculation with Rhizobium leguminosarum biovar viciae

Infective (nodulating) Rhizobium leguminosarum biovar viciae (R.l. viciae) bacteria release Nod factors which stimulate the release of nodulation gene-inducing flavanones and chalcones from roots of the host plant Vicia sativa subsp. nigra (K. Recourt et al., Plant Mol Biol 16: 841–852; H.P. Spaink et al., Nature 354: 125–130). The hypothesis that this release results from increased synthesis of flavonoids was tested by studying the effect of inoculation of V. sativa with infective and uninfective R.l. viciae bacteria on (i) activity of L-phenylalanine ammonia-lyase, (ii) level of chalcone synthase mRNA, and (iii) activity of (eriodictyol) methyltransferase in roots. Consistent with the hypothesis, each of these parameters was found to increase 1.5 to 2-fold upon inoculation with infective R.l. viciae bacteria relative to the situation for uninoculated roots and for roots inoculated with uninfective rhizobia.     

Analysis of pss genes of Rhizobium leguminosarum required for exopolysaccharide synthesis and nodulation of peas: their primary structure and their interaction with psi and other nodulation genes

Summary Strains of Rhizobium leguminosarum (R. l.) biovar viciae containing pss mutations fail to make the acidic exopolysaccharides (EPS) and are unable to nodulate peas. It was found that they also failed to nodulate Vicia hirsuta, another host of this biovar. When peas were co-inoculated with pss mutant derivatives of a strain of R.l. bv viciae containing a sym plasmid plus a cured strain lacking a sym plasmid (and which is thus Nod^-, but for different reasons) but which makes the acidic EPS, normal numbers of nodules were formed, the majority of which failed to fix nitrogen (the occasional Fix⁺ nodules were pressumably induced by strains that arose as a result of genetic exchange between cells of the two inoculants in the rhizosphere). Bacteria from the Fix^- nodules contained, exclusively, the strain lacking its sym plasmid. When pss mutant strains were co-inoculated with a Nod^- strain with a mutation in the regulatory gene nodD (which is on the sym plasmid pRL1JI), normal numbers of Fix⁺ nodules were formed, all of which were occupiced solely by the nodD mutant strain. Since a mutation in nodD abolishes activation of other nod genes required for early stages of infection, these nod genes appear to be dispensable for subsequent stages in nodule development. Recombinant plasmids, containing cloned pss genes, overcame the inhibitory effects of psi, a gene which when cloned in the plasmid vector pKT230, inhibits both EPS production and nodulation ability. Determination of the sequence of the pss DNA showed that one, or perhaps two, genes are required for correcting strains that either carry pss mutations or contain multi-copy psi. The predicted polypeptide product of one of the pss genes had a hydrophobic aminoterminal region, suggesting that it may be located in the membrane. Since the psi gene product may also be associated with the bacterial membrane, the products of psi and pss may interact with each other.     

Rhizobium leguminosarum genes required for expression and transfer of host specific nodulation

The contributions of various nod genes from Rhizobium leguminosarum biovar viceae to host-specific nodulation have been assessed by transferring specific genes and groups of genes to R. leguminosarum bv. trifolii and testing the levels of nodulation on Pisum sativum (peas) and Vicia hirsuta. Many of the nod genes are important in determination of host-specificity; the nodE gene plays a key (but not essential) role and the efficiency of transfer of host specific nodulation increased with additional genes such that nodFE < nodFEL < nodFELMN. In addition the nodD gene was shown to play an important role in host-specific nodulation of peas and Vicia whilst other genes in the nodABCIJ gene region also appeared to be important. In a reciprocal series of experiments involving nod genes cloned from R. leguminosarum bv. trifolii it was found that the nodD gene enabled bv. viciae to nodulate Trifolium pratense (red clover) but the nodFEL gene region did not. The bv. trifolii nodD or nodFEL genes did significantly increase nodulation of Trifolium subterraneum (sub-clover) by R. leguminosarum bv. viciae. It is concluded that host specificity determinants are encoded by several different nod genes.     

Genetic and functional analysis of the nodulation region of the Rhizobium leguminosarum Sym plasmid pRL1JI

Thirty Tn5- or Tn1831-induced nodulation (nod) mutants of Rhizobium leguminosarum were examined for their genetic and symbiotic properties. Thirteen mutants contained a deletion in Sym plasmid pRL1JI. These deletions cover the whole nod region and are 50 kb in size. All remaining seventeen mutations are located in a 6.6 kb EcoRI nod fragment of the Sym plasmid. Mutations in a 3.5 kb part on the right hand side of this 6.6 kb fragment completely prevent nodulation on Vicia sativa. All mutants in this 3.5 kb area are unable to induce marked root hair curling and thick and short roots.Mutations in a 1.5 kb area on the left hand side of the 6.6 kb nod fragment generate other symbiotic defects in that nodules are only rarely formed and only so after a delay of several days. Moreover, infection thread formation is delayed and root hair curling is more excessive than that caused by the parental strain. Their ability to induce thick and short roots is unaltered.Mutations in this 1.5 kb region are not complemented by pRmSL26, which carries nod genes of R. meliloti, whereas mutations in the 3.5 kb region are all complemented by pRmSL26.Abbreviations Rps repression of production of small bacteriocin - Mep medium bacteriocin production - Nod nodulation - Fix fixation - Tsr thick and short roots - Flac root hair curling - Hsp host specificity - Flad root hair deformation - Tc tetracycline - Km kanamycin - Cm chloramphenicol - Sp spectinomycin - Sm streptomycin - R resistant     

Subnanomolar concentrations of membrane chitolipooligosaccharides from Rhizobium leguminosarum biovar trifolii are fully capable of eliciting symbiosis-related responses on white clover

Axenic seedling bioassays were performed on white clover, vetch, and alfalfa to assess the variety and dose responses of biological activities exhibited by membrane chitolipooligosaccharides (CLOSs) from wild type Rhizobium leguminosarum bv. trifolii ANU843. Subnanomolar concentrations of CLOSs induced deformation of root hairs (Had) and increased the number of foci of cortical cell divisions (Ccd) in white clover, some of which developed into nodule meristems. In contrast, ANU843 CLOSs were unable to induce Had in alfalfa and required a 10⁴-fold higher threshold concentration to induce this response in vetch. Also, ANU843 CLOSs were not mitogenic on either of these non-host legumes. In addition, CLOS action also increased chitinase activity in white clover root exudate. Thus, the membrane CLOSs from wild type R. leguminosarum bv. trifolii are fully capable of eliciting various symbiosis-related responses in white clover in the same concentration range as extracellular CLOSs of other rhizobia on their respective legume hosts. These results and our earlier studies indicate that membrane CLOSs represent one of many different classes of bioactive metabolites made by R. leguminosarum bv. trifolii which elicit more intense symbiosis-related responses in white clover than in other legumes. Therefore, CLOSs evidently play an important role in symbiotic development, but they may not be the sole determinant of host-range in the Rhizobium-clover symbiosis.Abbreviations Ccd cortical cell division - CLOS chitolipooligosaccharide - Had root hair deformation     

The role of motility in the efficiency of nodulation by Rhizobium meliloti

Tn5 mutants of Rhizobium meliloti L5.30 defective in motility (Mot^-) were isolated and compared to the parent with respect to the nodulation activity. Each of the mutants was able to generate normal nodules on the alfalfa (Medicago sativa) but had slightly delayed nodule formation. Coinoculation of lucerne with wild type Mot⁺ and Mot^- cells in the wide range of ratios resulted in nodules occupied in the majority by a motile strain suggesting that motility is a factor involved in the competition for nodule formation.     

Ultrastructure of infection-thread development during the infection of soybean by Rhizobium japonicum

The location and topography of infection sites in soybean (Glycine max (L.) Merr.) root hairs spot-inoculated with Rhizobium japonicum have been studied at the ultrastructural level. Infections commonly developed at sites created when the induced deformation of an emerging root hair caused a portion of the root-hair cell wall to press against an adjacent epidermal cell, entrapping rhizobia within the pocket between the two host cells. Infections were initiated by bacteria which became embedded in the mucigel in the enclosed groove. Infection-thread formation in soybean appears to involve degradation of mucigel material and localized disruption of the outer layer of the folded hair cell wall by one or more entrapped rhizobia. Rhizobia at the site of penetration are separated from the host cytoplasm by the host plasmalemma and by a layer of wall material that appears similar or identical to the normal inner layer of the hair cell wall. Proliferation of the bacteria results in an irregular, wall-bound sac near the site of penetration. Tubular infection threads, bounded by wall material of the same appearance as that surrounding the sac, emerge from the sac to carry rhizobia roughly single-file into the hair cell. Growing regions of the infection sac or thread are surrounded by host cytoplasm with high concentrations of organelles associated with synthesis and deposition of membrane and cell-wall material. The threads follow a highly irregular path toward the base of the hair cell. Threads commonly run along the base of the hair cell for some distance, and may branch and penetrate into subjacent cortical cells at several points in a manner analagous to the initial penetration of the root hair.     

Selection of Rhizobium leguminosarum bv. viceae strains for inoculation of Pisum sativum L. cultivars: Analysis of symbiotic efficiency and nodulation competitiveness

From an analysis of 481 Rhizobium leguminosarum bv. viceae strains with 7 pea cultivars in pot and field experiments, we demonstrated that effective strains could be isolated from a rich medium-acid grey forest soil of the Oröl area (Central region of the European part of Russia) but not from a poor acid podzolic soil of the St. Petersburg area (North-West Russia). The proportion of the isolates significantly increasing N accumulation in pea plants (10.2%) is higher than that of strains increasing the shoot dry mass (4.6%) in the pot experiments. The mean values of the increase for N accumulation (33.8%) upon inoculation are also higher than for shoot mass (27.0%) in these experiments. N accumulation in the inoculated pea plants in the pot experiments was significantly correlated with seed yield and seed N accumulation in field experiments, while for shoot dry mass these correlations were either weak or not significant. Two-factor analysis of variance demonstrated that the contribution of plant cultivars to the variation of the major symbiotic efficiency parameters is higher (30.8–31.6%) and contributions of cultivar-strain specificity is lower (5.4–8.8%) than the contributions of strain genotypes (13.4–14.9%). We identified an ineffective R. leguminosarum bv. viceae strain 50 which can be used as a tester for assessing the nodulation competitiveness of the effective strains by an indirect method (analysis of dry mass and N accumulation in pea plants inoculated with the mixture of the tested effective strains and the tester strain). The relative competitive ability (RCA) determined by this method was 75.7–82.8% for strain 52 but only 10.5–13.8% for strain 250a; this difference was confirmed by a direct method (use of the streptomycin-resistant mutants). Results of screening of the diverse collection of 53 effective R. leguminosarum bv. viceae strains by the indirect method permits us to divide them into 3 groups (32 high-competitive, 10 medium-competitive and 11 low-competitive strains) but reveals no correlation between the competitiveness and symbiotic efficiency. N accumulation in the pea shoots is demonstrated to be a much more suitable criterion than the shoot mass for selection either of the highly-effective or of highly-competitive (by the indirect estimation) R. leguminosarum bv. viceae strains in the pot experiments.     

Effects of mycorrhizae and chitin-hydrolysing microbes on Vicia faba

The effect of Streptomyces albovinaceus (S-22) and Bacillus sp. (B1) on the growth response, nodulation, nutrition and nitrogenase activities of faba bean (Vicia faba) varieties infected with Glomus mosseae under pot conditions in sterile soil amended with chitin was studied. The growth, nodulation, nutrients content and nitrogenase activity of mycorrhiza-treated plants of Giza-667 were significantly increased compared to untreated ones. Such increases were related to the increase in mycorrhizal root infection. Amendment of soil with chitin alone reduced the growth, nodulation, total nitrogen contents and nitrogenase activities of mycorrhiza-treated faba bean plants (Giza-667) compared to untreated plants. Inoculation of plants with S. albovinaceus or Bacillus sp. significantly increased the level of mycorrhizal roots infection, but addition of chitin to the soil in combination with Bacillus sp. reduced the mycorrhizal infection of faba bean roots. Highest phosphorus contents of faba bean Giza-667 were recorded after G. mosseae inoculation in the presence of all treatments. Similar results were observed for the other varieties. The microbial populations were significantly increased in rhizospheres amended with chitin. Such increases were not in response to the mycorrhizal inoculation. Generally, the microflora of faba bean rhizospheres was increased after treatment with G. mosseae in the absence of chitin amendment alone compared with non-mycorrhizal rhizospheres.     

Root exuded nod-gene inducing signals limit the nodulation capacity of different alfalfa varieties with Rhizobium meliloti

Summary Different nodulation capacities were found among nine different varieties of alfalfa, cultivated in the Central region of Mexico, by Rhizobium meliloti 2011. A correlation between nodulation capacity and foliar dry weight was observed, which points to a genotype dependance on these parameters. A correlation between the nodulation capacity and the R. meliloti nod-gene inducing activity of the root exudates from the different varieties, as measured by -galactosidase induction in a test system consisting of a R. meliloti nodC-lacZ strain incubated with each root exudate, was established. When the root exudate from the best nodulating variety was added to the four poorest nodulating varieties, an increase in nodule formation was observed. We conclude that root exuded nod-gene inducing signals are a symbiotically-limiting component in natural populations of the poorest nodulating varieties of alfalfa.     

Effect of glutamate transport and catabolism on symbiotic effectiveness in Rhizobium leguminosarum bv. phaseoli

Seven Tn5 induced mutants unable to use glutamate as sole carbon and nitrogen source were isolated from the effective Rhizobium leguminosarum bv. phaseoli strain P121-R. As indicated by restriction and hybridisation analysis, all the mutants arose from a single Tn5 insertion in the chromosome. The ¹⁴C-glutamate uptake rate of the mutants was 76 to 88% lower than that of strain P121-R. Inoculation of Phaseolus vulgaris cv. Labrador with these mutants significantly decreased shoot dry matter yield and the total nitrogen content respectively, as compared to inoculation with the parental strain P121-R. All the mutants formed nodules, however they were smaller, white to greenish and approximately 30% less numerous than those formed by strain P121-R. These observations suggest that glutamate transport and catabolism in R. leguminosarum bv. phaseoli P121-R may play an important role in the establishment of an effective symbiosis in field bean. None of the mutants isolated was an auxotroph. All mutants were unable to grow on aspartate suggesting that glutamate and aspartate, probably have the same transporter as indicated in Rhizobium meliloti and in Bacillus subtilis. All mutants readily used glutamine, proline, arginine as sole carbon and nitrogen source, but grew more slowly than the wild type strain. On the other hand, all the mutants were impaired in growth on histidine and -aminobutyrate as sole carbon and nitrogen source. As the catabolism of these amino acids occurs predominantly through glutamate, our results indicate that mutants are also impaired in their ability to use histidine and -aminobutyrate as a nitrogen source. Our results also suggest that other amino acids catabolized through the glutamate pathways may be an additional important carbon source for bacteroids in nodules.     

Studies on the nodulation of strain-specific resistant pea lines using single,mixed and delayed inoculation by Rhizobium leguminosarum

Symbiotic interactions between peas and Rhizobium leguminosarum were investigated by inoculating four pea lines, three of which are strain-specific resistant to the European strain 311d, with various combinations of two strains of Rhizobium, 311d and Tom⁺⁺. The strains were almost equally good to infect the susceptible European cultivar Hero when added singly inoculated. After mixed inoculation (1:1 proportion) strain analysis by ELISA revealed that the nodules were preferentially formed by 311d, although some Tom⁺⁺ nodules were also found mainly on the upper part of the root. Our conclusion is that Tom⁺⁺ is less compatible in comparison with 311d. In addition, we found that as the Hero plants emerged, they were becoming more resistant towards infection with not adapted bacteria. The strain-specific resistant lines from Afghanistan belong to two different systems: Afgh. I, completely resistant to 311d and highly nodulating with Tom⁺⁺, and Afgh. III, incompletely resistant to 311d and poorly nodulating with Tom⁺⁺. Mixed inoculations resulted in nodule depressions, as compared to single inoculations with Tom⁺⁺ ranging from 87% to 14%. The ability of 311d to block infection sites on the roots were found to depend on the degree of symbiotic adaptation between Afgh. I and Tom⁺⁺, respectively Afgh. III and Tom⁺⁺. Strain analysis after double strain inoculation of Afgh. I plants revealed that some nodules were induced by strain 311d. Thus, the presence of Tom⁺⁺ in this case influences the degree of host resistance. However, in Afgh. III plants the resistance towards nodulation were unaffected by the presence of Tom⁺⁺. We suggest that the degree of symbiotic adaptation may change the barrier of resistance towards infection.