Interaction of pea (Pisum sativum L.) lectins with rhizobial strains

Lectins from two varieties (PG-3 and LFP-48) of pea have been purified by affinity chromatography on Sephadex G-50. The specific activity increased by 23 and 25 folds, respectively. These lectins from both the varieties were found to be specific for mannose. The purified fluorescein isothiocyanate (FITC)-labelled lectins showed binding reaction with homologous as well as heterologous strains of Rhizobium spp. The results revealed that pea lectins are not highly specific to their respective rhizobia. Moreover, these lectins showed a greater stimulatory effect on homologous Rhizobium leguminosarum strains.     

Identification of enzymes involved in indole-3-acetic acid degradation

Strains of Bradyrhizobium japonicum with the ability to catabolize indole-3-acetic acid (IAA) and strains of B. japonicum, Rhizobium loti, and Rhizobium galegae, unable to catabolize IAA, were analyzed for enzymes involved in the pathway for IAA degradation. Two enzymes having isatin as substrate were detected. An isatin amidohydrolase catalyzing the hydrolysis of isatin into isatinic acid was found in some B. japonicum strains and in two Rhizobium species, R loti and R. galegae. The enzyme was inducible (4–5-fold) by its substrate, isatin, and the partially purified enzyme from R. loti showed an apparent K_M of 11 M for isatin. A NADPH-dependent isatin reductase was measured in extracts from a strain of B. japonicum lacking the isatin amidohydrolase. The structure of the reaction product, dioxindole was verified by NMR spectroscopy. Isatin reductase activity was also detected in extracts of dry pea seeds, and present in at least two isoforms. A low K_M of 10 M for isatin was found with a partially purified preparation of the pea enzyme. The presence of such an enzyme activity in pea indicates dioxindole and isatin as possible intermediates in IAA degradation in pea.     

Role of Bacterial Polysaccharides in the Adsorption Process of the Rhizobium-Pea. Symbiosis

Phase-contrast and fluorescence microscopy observations showed that pea symbiont R. leguminosarum adsorbed to pea root hairs, but non-symbiont rhizobial strains only adsorbed to a small extent. ¹⁴C-labeled cells were used to assay the number of rhizobial cells adsorbed to a pea root. Capsular polysaccharides or lipopolysaccharides obtained from R. leguminosarum specifically inhibited the adsorption of ¹⁴C-R. leguminosarum cells to a pea root and specifically adsorbed to pea root hairs. Also, they reacted specifically with pea seed lectins. These results suggest that capsular polysaccharides or lipopolysaccharides play an important role in host-specific adsorption. The interaction between the polysaccharides and pea lectins could be the key to determining host specificity in the infection process of Rhizobium-pea symbiosis.     

Glucosylation of membrane-bound proteins by lipid-linked glucose

Particulate preparations from Pisum sativum. were able to incorporate [¹⁴C]glucose from UDP-[¹⁴C]glucose into oligosaccharide-linked lipids was formed by an oligosaccharide chain containing 7-8 glucose residues linked to dolichol, presumably via a pyrophosphate. The polymer was identified as a membrane-bound glucoprotein that could be solubilized by Triton X-100. SDS gel electrophoresis showed that a polypeptide with an apparent molecular weight of 13,000 could be glucosylated from dolichyl-phosphate-glucose. This was coincident with the electrophoretic mobility of the subunit of the pea lectin in the same system. The glucosylated protein was solubilized from the membranes by sonication and showed the same carbohydrate-binding ability as pea lectins. These results strongly suggest that pea lectins can be glucosylated by the lipid intermediate pathway.Abbreviations BSA Bovine serum albumin - Dol dolichol - SDS sodium dodecyl sulfate     

Comparative Characteristics of Carbohydrate Binding by Lectins from Broad Bean,Pea, Common Vetch,and Lentil Seeds

Lectins from the seeds of broad bean (Vicia faba L.), pea (Pisum sativum L.), common vetch (V. sativa L.), and lentil (Lens culinaris Medik.) were isolated and purified by affinity chromatography. The hemagglutinating activity of lectins was most effectively inhibited by methyl--D-mannopyranoside, trehalose, and D-mannose. Other carbohydrate haptens, such as methyl--D-glucopyranoside, maltose, and alginic and D-glucuronic acids were less effective. Two lectins obtained from different lentil cultivars, unlike other lectins, had a relatively high affinity for melecitose, N-acetyl-D-glucosamine, L-sorbose, and sucrose. Furthermore, these lectins interacted with soluble starch. All the lectins examined had similar, but not identical, carbohydrate-binding properties. Because of their similar D-mannose/D-glucose specificity, these lectins interacted with lipopolysaccharides and exopolysaccharides of Rhizobium leguminosarum bv. viciae, root nodule bacteria that infect broad-bean, pea, common-vetch, and lentil plants with the formation of nitrogen-fixing symbiosis. However, owing to individual distinctions of carbohydrate-binding properties, these lectins showed a higher affinity for the polysaccharides of those microsymbionts within the R. leguminosarum bv. viciae species that were better specialized towards one or the other host plant from the cross inoculation group of legumes.     

Root nodulation studies inAeschynomene aspera

Summary Fifteen isolates of nodule bacteria were isolated from root and stem nodules ofAeschynomene aspera and they were characterized as Rhizobium by well known laboratory tests. All these isolates together with other efficient strains of known rhizobia belonging to different cross-inoculation groups were evaluated for their nodulation abilities onAeschynomene aspera, Cajanus cajan (pigeon pea),Cicer arietinum (chickpea),Pisum sativum (pea),Trifolium repens (clover),Medicago sativa (lucerne),Lens culinaris (lentil),Glycine max (soybean),Vigna sinensis (cowpea),Vigna radiata (mung bean),Vigna mungo (urd bean) andArachis hypogea (peanut). The results demonstrated that Rhizobium fromAeschynomene could form nodules only on its homologous host (Aeschynomene) but not on other legumes tested. Secondly, none of the rhizobia of other cross-inoculation groups could nodulateA. aspera.     

STUDIES ON INDUCED VARIATION IN THE RHIZOBIA. III. HOST RANGE MODIFICATION OF RHIZOBIUM TRIFOLII BY SPONTANEOUS AND RADIATION-INDUCED MUTATION

Schwinghamer , E. A. (Brookhaven Natl. Lab., Upton, New York.) Studies on induced variation in the rhizobia. III. Host range modification of Rhizobium trifolii by spontaneous and radiation-induced mutation. Amer. Jour. Bot. 49(3): 269–277. Illus. 1962.—Mutant strains capable of nodulating pea seedings ineffectively (incapable of nitrogen fixation) have been obtained from 2 antibiotic-resistant marked strains of Rhizobium trifolii which normally do not form nodules on this host. Such variant forms apparently occur spontaneously in these strains at a low frequency which can be significantly increased by irradiation with ultraviolet light, X rays, and fast neutrons. Nodulation of vetch, sweet peas and several varieties of peas by the mutant strains suggests a close parallelism of the extended host range with the range of R. leguminosarum, although nitrogen fixation by the mutants on the new hosts is negligible or absent. The mutant nodules on these hosts also differ from those of the pea strains in slightly smaller size, spherical form, and lighter pink color. Nodule morphology on the homologous host, clover. appears unaltered but a slight loss of effectiveness was noted on red clover. This loss may be attributed partly to a reduction in infective ability since the average number of nodules formed per plant of clover or pea is appreciably lower than for comparable inoculation by strains of nonmutant R. trifolii or R. leguminosarum, respectively. Cultural characteristics of mutant strains resemble those of the nonmutant R. trifolii strains.     

Marker assisted pea breeding: <Emphasis Type="Italic">eIF4E</Emphasis> allele specific markers to <Emphasis Type="Italic">pea seed-borne mosaic virus</Emphasis> (PSbMV) resistance

Traditional plant breeding relies upon crosses and subsequent selection of genotypes to meet desirable traits. The incorporation of marker-assisted selection into breeding strategies would result in a reduction in the number of offspring to be propagated, selected and tested. In the case of pea (Pisum sativum L.), the testing of resistance to viral pathogens such as pea seed-borne mosaic virus (PSbMV) is included in the breeding process. Resistance to the common strains of PSbMV is conferred by a single recessive gene (eIF4E), localized on LG VI (sbm-1 locus). We have analyzed for variation in the eIF4E genomic sequences from 43 pea varieties and breeding lines, reported as donors of resistance. This enabled a comprehensive investigation of the eIF4E gene structure and mutations responsible for PSbMV resistance were identified. Subsequently, PCR-based and gene-specific single nucleotide polymorphism and co-dominant amplicon length polymorphism markers were developed. All together 60 accessions were analyzed using sequence data and/or allele specific DNA markers. Developed allele specific markers were reproducibly amplified across a broad spectra of pea varieties and breeding lines. These were found to be 100% accurate in detecting the presence of the respective alleles when compared to symptomology and ELISA, testing (74% reliable). Hence, these molecular markers will substantially speed-up PSbMV diagnosis and resistance breeding processes in pea.     

An Ouchterlony double diffusion study on the interaction between legume lectins and rhizobial cell surface antigens

Acidic exopolysaccharides and O-antigen containing lipopolysaccharides were isolated from Rhizobium japonicum, R. leguminosarum, R. lupini, R. meliloti, R. phaseoli, cowpea Rhizobium sp. and a non-nodulating soil bacterium. Lectins from seeds of soybean (Glycine max), garden pea (Pisum sativum), lentil (Lens culinaris), alfalfa (Medicago sativa), field bean (Phaseolus vulgaris), jackbean (Canavalia ensiformis) and from wheat germ were tested for their capacity to precipitate rhizobial exopolysaccharides and lipopolysaccharides in the Ouchterlony double diffusion test. Soybean lectin precipitated exclusively with the exopolysaccharide of R. japonicum, whereas the lectins from pea and lentil precipitated exopolysaccharides from all the fast growing strains of Rhizobium. Host range specific interactions between lipopolysaccharides and lectins were observed in the pea/lentil-R. leguminosarum and in the alfalfa-R. meliloti systems. Concanavalin A precipitated the exopolysaccharides of all fast growing strains of Rhizobium, the exopolysaccharide of the cowpea strain and several lipopolysaccharides of different Rhizobium species and thus did not show any correlation between polysaccharide binding and symbiotic specificity. Non-leguminous wheat germ agglutinin did not precipitate any of the rhizobial polysaccharides tested and the lipopolysaccharide of the soil bacterium did not precipitate with any of the lectins examined.Abbreviations Con A Concanavalin A - CPC cetylpyridinium chioride - EPS exopolysaccharide - FITC fluorescein isothiocyanate - KDO 2-keto-3-deoxyoctonic acid - LPS lipopolysaccharide - PBS phosphate-buffered saline - PS polysaccharide     

Comparative mapping between<Emphasis Type="Italic"> Medicago sativa</Emphasis> and<Emphasis Type="Italic"> Pisum sativum</Emphasis>

Comparative genome analysis has been performed between alfalfa ( Medicago sativa) and pea ( Pisum sativum), species which represent two closely related tribes of the subfamily Papilionoideae with different basic chromosome numbers. The positions of genes on the most recent linkage map of diploid alfalfa were compared to those of homologous loci on the combined genetic map of pea to analyze the degree of co-linearity between their linkage groups. In addition to using unique genes, analysis of the map positions of multicopy (homologous) genes identified syntenic homologs (characterized by similar positions on the maps) and pinpointed the positions of non-syntenic homologs. The comparison revealed extensive conservation of gene order between alfalfa and pea. However, genetic rearrangements (due to breakage and reunion) were localized which can account for the difference in chromosome number (8 for alfalfa and 7 for pea). Based on these genetic events and our increasing knowledge of the genomic structure of pea, it was concluded that the difference in genome size between the two species (the pea genome is 5- to 10-fold larger than that of alfalfa) is not a consequence of genome duplication in pea. The high degree of synteny observed between pea and Medicago loci makes further map-based cloning of pea genes based on the genome resources now available for M. truncatula a promising strategy.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by W. R. McCombie     

Purification and properties of pea (Pisum sativum L.) thioredoxin f,a plant thioredoxin with unique features in the activation of chloroplast fructose-1,6-bisphosphatase

Thioredoxin (Td) f from pea (Pisum sativum L.) leaves was purified by a simple method, which provided a high yield of homogeneous Td f. Purified Td f had an isoelectric point of 5.4 and a relative molecular mass (M_r) of 12 kilodaltons (kDa) when determined by filtration through Superose 12, but an M_r of 15.8 kDa when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein remained fully active for several months when conserved frozen at — 20° C. The pea protein was able to activate fructose1,6-bisphosphatase (FBPase; EC 3.1.3.11), but in contrast to other higher-plant Td f proteins, was not functional in the modulation of NADP⁺-malate dehydrogenase activity. In spite of the absence of immunological cross-reactions of pea and spinach Td f proteins with the corresponding antibodies, pea Td f activated not only the homologous FBPase, but also the spinach enzyme. The saturation curves for pea FBPase, either with fructose-1,6-bisphosphate in the presence of different concentrations of homologous Td f, or with pea Td f in the presence of excess substrate, showed sigmoid kinetics; this can be explained on the basis of a random distribution of fructose-1,6-bisphosphate, and of the oxidized and reduced forms of the activator, among the four Td f- and substrate-binding sites of this tetrameric enzyme. From the saturation curves of pea and spinach Td f proteins against pea FBPase, a 4:1 stoichiometry was determined for the Td f-enzyme binding. This is in contrast to the 2:1 stoichiometry found for the spinach FBPase. The UV spectrum of pea Td f had a maximum at 277 nm, which shifted to 281 nm after reduction with dithiothreitol (s at 280 nm for 15.8-kDa M_r = 6324 M^–1 · cm^–1). The fluorescence emission spectrum after 280-nm excitation had a maximum at 334 nm, related to tyrosine residues; after denaturation with guanidine isothiocyanate an additional maximum appeared at 350 nm, which is concerned with tryptophan groups. Neither the native nor the denatured form showed a significant increase in fluorescence after reduction by dithiothreitol, which means that the tyrosine and tryptophan groups in the reduced Td f are similarly exposed. Pea Td f appears to have one cysteine residue more than the three cysteines earlier described for spinach and Scenedesmus Td f proteins.Abbreviations DDT dithiothreitol - ELISA enzyme-linked immunosorbent assay - FBPase fructose- 1,6-bisphosphatase - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Td thioredoxin The authors are grateful to Mrs. Francisca Castro and Mr. Narciso Algaba for skilful technical assistance. This work was supported by grant PB87-0431 of Dirección General de Investigación Cientifica y Técnica (DGICYT, Spain).     

Lectin Analysis of Surface Saccharides in Two Trichomonas vaginalis Strains Differing in Pathogenicity*

SYNOPSIS. Surface saccharides in 2 Trichomonas vaginalis strains, the moderately pathogenic, JH34A, and the mild, JH162A, were analyzed with the aid of plant lectins. Concanavalin A (Con A), wheat germ agglutinin (WGA), soybean agglutinin (SBA), castor bean agglutinin (CBA), and lectin from the garden pea (GPA) were employed in agglutination tests and in treatment of ultrathin sections for electron microscopy according to the horseradish peroxidase-3,3′-diaminobenzidine method. With Con A and WGA, small quantitative differences were noted between the 2 strains in the results of agglutination and in the reaction-product deposits observed by electron microscopy. Distribution of the binding sites for the 2 lectins was also somewhat different in the JH34A and JH162A trichomonads. In general, the reactions with the more pathogenic strain were slightly stronger. Although the reactions with SBA and CBA lectins were weaker than those with Con A or WGA, they provided the means for qualitative differentiation between the 2 trichomonad strains. SBA alone agglutinated the JH34A strain and formed demonstrable deposits on the cell surfaces. On the other hand, only CBA reacted with JH162A flagellates. The garden pea lectin failed to bind to the surface of either strain. On the basis of results obtained with the control preparations incubated in the presence of specific inhibitors, it was concluded that both strains had α-methyl-D-mannoside and/or α-methyl-D-mannoside-like as well as N-acetyl-D-glucosamine residues on their surfaces. In addition, JH34A strain had D-lactose-containing residues while JH162A trichomonads had residues with D-galactose. Neither strain appeared to possess residues containing N-acetyl-D-galactosamine.     

Isolation and properties of a lectin from the roots of Pisum sativum (Garden pea)

Abstract The roots of pea (Pisum sativum L. ev. Feltham First) seedlings contained haemagglutinating activity and a protein which reacted with antibodies directed against pea seed lectin. This protein was shown to be present on the surface of root hairs and in the root cortical cells by immunofluorescence. Lectin (haemagglutinin) was purified from pea seedling roots by both immunoaffinity chromatography and affinity chromatography on Sephadex G-100. The pea root lectin was similar to the seed lectin when analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and was antigenically identical: however, the isoelectric focussing band patterns of the proteins differed. The sugar specificity of the root lectin differed from that of the seed lectin, and the haemagglutinating activity of the root lectin was less than the seed lectin. These results are discussed with reference to the hypothesis that lectins mediate in the symbiotic association of legume and Rhizobium through their carbohydrate-binding properties.     

Suppression of important pea diseases by bacterial antagonists

The potential for widespreadand severe infection makes ascochyta blight,seedling blight, and root rots major hindrancesto pea production in Alberta, Canada. Over 300bacterial strains were isolated from pea seedand soil samples taken from pea fields. Thesestrains were investigated for their biologicalcontrol potential against four fungal pathogens(Pythium ultimum, Rhizoctoniasolani, Fusarium avenaceum and Ascochyta pisi) of field pea in vitro. Selected bacterial strains were furtherevaluated in vivo. In an initial agarplate bioassay, 30 strains exhibitedantagonistic properties against the fourpathogens, with inhibition zones ranging from 5to 25 mm. Thirteen of these strains, allisolated from soil, inhibited only one or twoof the pathogens, while the remainingseventeen, including nine strains isolated frompea seeds, inhibited either three or all fourpathogens. In a more stringent bioassay, eightof the thirty strains failed to demonstrate theantagonistic features shown in the initialbioassay. Eight strains inhibited only onepathogen, six inhibited two, four inhibitedthree, and four strains inhibited all fourpathogens tested. Two strains ofPseudomonas fluorescens, five strains of Serratia spp. and two strains of Bacillus spp. were further evaluated ingreenhouse experiments. Five of the isolatesreduced the severity of diseases caused byPythium or Ascochyta, two isolatesreduced the severity of Rhizoctonia andone reduced the severity of Fusarium.     

Co-evolution of the legume-Rhizobium association

Summary A number of examples is given demonstrating the co-existence of pea genotypes and their specific Rhizobium, strains isolated within the same region.R. leguminosarum strains compatible with the cultivated pea have a narrow symbiotic range and they are widely distributed in European soils. This is presumably due to the narrow genetic base of the cultivated pea and its wide-spread cultivation in European soils. Rhizobium strains capable of nodulating a primitive pea line from Afghanistan were only found in soils of the Middle East and Central Asia. A more restricted distribution of specific Rhizobium strains was found for fulvum peas from Israel. Rhizobium strains effective with the fulvum pea were found in Israeli soils. A good example of co-evolution due to geographical isolation was found in south Turkey. Here a pea line was found which can form an effective symbiosis with local Rhizobium strains but not with strains from other parts of Turkey.     

Inoculation of pea by application of Rhizobium in the planting furrow

Summary Selected streptomycin resistant strains ofRhizobium leguminosarum suspended in nutrient broth were added to the planting furrow immediately before the sowing of pea. The nodule occupancy by a strain isolated from Risø soil (Risø la) was increased from 74 to 90%, when the inoculum rate was increased from 3.7×10⁶ to 3.7×10⁸ cells per cm row. The experimental soil contained 10³ to 10⁴ cells ofR. leguminosarum per gram. An almost inefficient strain isolated from Risø soil (SV10) was less competitive with respect to nodulation on two pea cultivars than an efficient Risø strain (SV15) and an efficient non-Risø strain (R1045). The nodule occupancy by the introduced strains varied between pea cultivars.Irrespective of the generally high nodulation by the efficient strains introduced to the soil, the pea seed yield, compared to pea nodulated by the indigenous population, was not significantly increased. Neither were two commercial inoculants, applied in rates corresponding to 3 times the recommended rate, able to increase the yield. This suggests that the indigenous populations ofR. leguminosarum were sufficient in number and nitrogen fixing capacity to ensure an optimal pea crop. However, some inoculation treatments slightly increased the seed N concentration and total N accumulation, indicating that it may be possible to select or develop bacterial strains that may increase the yield.     

Pea chloroplast DNA primase: characterization and role in initiation of replication

A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19_+ 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of ³H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115–120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19+2.1. Primers synthesized using M13mp19+2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.     

HMG protein binding to an A/T-rich positive regulatory region of the pea plastocyanin gene promoter

Gel retardation assays using pea nuclear extracts have detected specific binding to regions of the promoter of the pea plastocyanin gene (petE). Several complexes which differ in sensitivity to competition with unlabelled promoter fragments and various DNA alternating copolymers, to heat treatment and to digestion with proteinase K have been detected. A protein factor, PCF1, forming one of these complexes was heat-stable and most sensitive to competition with poly(dAdT).poly(dAdT) compared to other alternating copolymers. DNase I footprinting assays showed that tracts of A/T-rich sequence within the -444 to -177 positive regulatory region of the petE promoter were protected in the presence of the pea nuclear extract. The factor PCF1 copurified with a high-mobility-group (HMG) protein preparation from pea chromatin. DNase I footprinting with the HMG protein preparation demonstrated that similar tracts of A/T-rich sequences within the promoter were protected. Southwestern-blot analysis of pea HMG proteins purified by gel filtration through Superose 12 detected a single DNA-binding species of 21 kDa. The properties of the factor PCF1 suggest that it is likely to be an HMG I protein.     

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.     

The potential of <Emphasis Type="Italic">Rhizobium</Emphasis> strains for biological control of <Emphasis Type="Italic">Orobanche crenata</Emphasis>

Orobanche crenata Forsk is a chlorophyll lacking holoparasite that subsists on the roots of plants and causes significant damage to the culture of leguminous plants and, in particular, to peas (Pisum sativum L.). Here, we investigated the potential of Rhizobium strains for biological control of Orobanche crenata using a commercial pea cultivar (Douce de province) and different Rhizobium strains. Firstly, benefit of bacterial inoculation on plant growth and efficiency in N-incorporation were demonstrated with four isolates, P.SOM, P.1001, P.Mat.95 and P.1236. After five Rhizobium strains (three efficient: P.SOM, P.1236, P.Mat.95 and two not efficient: P.OM1.92, P.MleTem.92) were investigated for their ability to control Orobanche crenata using pot and Petri dish experiments. Inoculation of peas with two (P.SOM and P.1236) of the five strains induced a significant decrease in O. crenata seed germination and in the number of tubercles on pea roots. Furthermore, other symptoms, including the non-penetration of the germinated seeds into pea roots followed by radicle browning and death of the parasites, were observed in the presence of these inoculated pea plants. The hypothesis that roots secrete toxic compounds related to Rhizobium inoculation is discussed.