Drought tolerance ofRhizobium leguminosarum andR. meliloti

Cell suspension ofRhizobium leguminosarum bv.viciae D-253,R. leguminosarum bv.viciae D-560 andR. meliloti D-557 were incorporated into sterile diatomaceous earth (DE) and dried at room temperature. Initial numbers of colony-forming units (CFU), expressed as log₁₀, were 8.27, 8.36 and 8.51, respectively. After 5 months of storage the CFU numbers were 0.00, 5.99 and 7.43, respectively.R. meliloti D-557 showed only minor lowering of the CFU number even after 16 months of storage (log₁₀=7.07). After 7 months of storage in DE some single-colony isolates of D-253 produced 10–100 times higher CFU numbers than the original strain. The isolates of D-560 were much more drought-tolerant. The cells of the original strain died after 7 months of storage, log₁₀ of CFU was 6–7 in the isolates. In both strains some of their drought-tolerant isolates had the same specific acetylene-reducing activity of nodule tissue as the original strains. Diatomaceous earth seems to be a prospective carrier for the formulation of bacterization preparations.     

Tolerance ofRhizobium leguminosarum bv.phaseoli to acidity and drought

Ten strains ofRhizobium leguminosarum bv.phaseoli isolated from soils of Morocco were more tolerant than three culture collection strains to acid conditions in culture media or in sterile soil. The survival rate of a tolerant strain in a sandy acid soil was greater than a sensitive strain at different humidity levels. These properties should give locally selected strains an advantage in nodulatingPhaseolus vulgaris roots in soils similar to those used here.     

Habitable pore space and survival ofRhizobium leguminosarum biovartrifolii introduced into soil

The hypothesis that the population size of introduced bacteria is affected by habitable pore space was studied by varying moisture content and bulk density in sterilized, as well as in natural loamy sand and silt loam. The soils were inoculated withRhizobium leguminosarum biovartrifolii and established and maintained at soil water potentials between –5 and –20 kPa (pF 1.7 and 2.3). Rhizobial cells were enumerated when population sizes were expected to be more or less stable. In sterilized soils, the rhizobial numbers were not affected or decreased only slightly when water potentials increased from –20 to –5 kPa. In natural soils, the decrease in rhizobial numbers with increasing water potentials was more pronounced. Bulk density had only minor effects on the population sizes of rhizobia or total bacteria. Soil water retention curves of both soils were used to calculate volume and surface area of pores from different diameter classes, and an estimation of the habitable pore space was made. Combining these values of the theoretical habitable pore space with the measured rhizobial numbers showed that only 0.37 and 0.44% of the habitable pore space was occupied in the sterilized loamy sand and silt loam, respectively. The situation in natural soil is more complicated, since a whole variety of microorganisms is present. Nevertheless, it was suggested that, in general, pore space does not limit proliferation and growth of soil microorganisms.     

Identification of inoculant strains and naturalized populations ofRhizobium leguminosarum bvtrifolii using complementary methodologies

Introduced clovers (Trifolium repens andTrifolium subterraneum) fall to persist more than two years in Uruguayan improved pastures. Three strains of naturalizedRhizobium leguminosarum bvtrifolii, isolated from persistent clover pastures inoculated several years ago, and two commercial inoculant strains have been identified. Serological techniques (enzyme-linked immunosorbent assay—ELISA) and total cell protein pattern have been selected as sensitive and reliable methods for strain identification. Indirect ELISA was suitable for detecting rhizobia in cultures or in nodules. Two of the naturalized strains had distinct protein profiles but they could not be differentiated by the immunological method with a polyclonal antibody.

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Résumé Des trèfies (Trifolium repens etTrifolium subterraneum) introduits dans des pâturages améliorés d'Uruguay, n'ont pu s'y maintenir plus de deux années. On a identifié 3 souches naturalisées deRhizobium leguminoserum bvtrifolii de pâturages aux trèfies persistants, inoculés il y a plusleurs années, ainsi que 2 souches d'inoculation industrielles. Des techniques sérologiques (ELISA: enzyme-linked immunosorbent assay) et l'histogramme des protéines cellulaires totales ont été sélectionnées comme méthodes sensibles et fiables pour l'identification des souches. Le test ELISA indirect est vaiable pour détecter les rhizobia en culture ou dans les nodules. Deux des souches naturalisées avaient leur propre profil protéique mais elles n'ont pu être différenclées par la méthode immunologique utilisant un anticorps polyclonal.

     

Selection and symbiotic properties ofRhizobium leguminosarum biovarphaseoli strains harboring pRtr5a

TheRhizobium leguminosarum biovartrifolii symbiotic plasmid pRtr5a has been transferred toR. leguminosarum biovarphaseoli RCR 3644-S1. The transconjugant selection had been done byTrifolium pratense plants. All transconjugants lacked the resident pSym, but had complete pRtr5a, and were Fix⁺ onT. repens andT. alexandrinum, Fix^– onT. subterraneum, and formed a few small white and Fix^– nodules onPhaseolus vulgaris. It is shown that this nodulation onP. vulgaris is due to pRtr5a. The presence of pRtr5a and/or the passage throughTrifolium pratense nodules provoke(s) the recipient strain symbiotic plasmid loss.     

Isolation and partial characterization of phenolate siderophore from Rhizobium leguminosarum IARI 102

Abstract Rhizobium leguminosarum IARI 102 produced 2,3-dihydroxy benzoic acid, a type of phenolate siderophore, under iron-starved conditions. Hydroxamic acids were not detected. Maximum production of siderophore was found at 26 h of growth in a chemically defined medium at 28°C with shaking. Threonine was detected as the amino acid conjugate of the siderophore. Addition of Fe³⁺ to the culture medium increased the growth yield significantly, but depressed the production of the iron chelating compound.     

Survival ofRhizobium leguminosarum biovarviceae subjected to heat,drought and salinity in soil

Two strains (RCR 1001 and 1044) and a commercial inoculant (Okadin) ofRhizobium leguminosarum biovarviceae were tested for their ability to survive in autoclaved clay soil for up to four months under heat, salinity and drought stress. Resistance to heat was tested by incubating rhizobia in soil at 27, 37 and 42 °C. Tolerance of rhizobia to salinity was investigated by growing rhizobia in soil salinized with 1 and 2 % NaCl (m/m). Drought resistance was tested by subjecting bacteria to soil moisture contents of 20, 10 and 5%. Strain RCR 1001 was more resistant to heat and nodulated faba bean better than other tested strains. A commercial inoculant Okadin survived more (plate count method) and nodulated faba bean (plant infectivity, most probable number, MPN) at moisture content of 5% and 2% NaCl. Although, strains RCR 1001 and 1044 resisted these stress conditions (plate count) they lost their abilities to nodulate faba bean (MPN-test). There is a possibility for selection of effective rhizobia which are more tolerant to harsh conditions.     

Interaction ofPhaseolus vulgaris with thermotolerant isolates ofRhizobium leguminosarum biovarphaseoli from Kenyan soils

Summary Thermotolerant strains ofRhizobium leguminosarum biovarphaseoli were isolated from nodules ofPhaseolus vulgaris grown in Kenyan soils, where high soil temperatures often exceed 40°C during sunny days. The isolates varied in their maximum growth laboratory temperature with two strains, 17 and 29B, able to grow at 42°C on yeast extract/mannitol agar media and at 40°C in liquid medium. These strains also survived better in moist clay soil at 38°C and 42°C and on seeds at room temperature but did not grow as well as several of the other strains at low pH. The thermotolerant strains nodulated three bean cultivars in a rooting medium that attained a daily maximum temperature of 36°C to 40°C but they varied in effectiveness according to the cultivar used.

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Interaction dePhaseolus vulgaris avec des isolats thermo-tolérants deRhizobium leguminosarum biovarPhaseoli de sols du Kenya

Résumé On a isolé des souches thermo-tolérantes deRhizobium leguminosarum biovarphaseoli de nodules dePhaseolus vulgaris developpés dans des sols du Kenya où la température haute du sol dépasse souvent 40°C, les jours ensoleillés. Les températures de croissance maximum au laboratoire varient d'un isolat à l'autre, avec deux souches, 17 et 29B, capables de croître à 42°C sur milieu gélosé à l'extrait de levure et au mannitol et à 40°C sur milieu liquide. Ces souches survivent également mieux en sol argileux humide respectivement à 38 et à 42°C, et sur semences à température ambiante mais ne croissent pas aussi bien que plusieurs autres souches à pH bas. Les souches thermo-tolérantes ont nodulé trois cultivars de fèves en milieux propices à la germination qui atteignaient une température diurne maximum de 36 à 40°C mais elles varient en efficacité selon le cultivar utilisé.

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This work was carried out at University of Nairobi under NairobiRhizobium MIRCEN, P.O. Box 30197, Nairobi, Kenya.     

Isolation and developmental characterization of temperature-sensitive carrot cell variants

Summary Carrot cell lines multiply indefinitely in the presence of the auxin 2,4-D. If auxin is removed, the cells regenerate plantlets in a process that closely resembles embryogenesis in vivo. We isolated a temperature-sensitive variant, ts 2, which is unable to regenerate at 31 °C (non-permissive temperature), but does form embryos and plants at 24 °C (permissive temperature). The temperature treatment had no effect on fully differentiated ts 2 plantlets. In other variants (ts 5 and ts 11) cell proliferation was inhibited at the restrictive temperature. These lines were leaky with respect to the inhibition of embryogenesis at 31 °C.Abbreviations EMS ethylmethanesulfonate - EU embryogenic unit (see Materials and methods) - ts temperature-sensitive - 2,4-D 2,4-dichlorophenoxyacetic acid     

Efficiency of isolates ofRhizobium leguminosarum (Pea) in relation to their dimensions

A study of the efficiencies and dimensions of forty isolates ofRhizobium leguminosarum isolated from pea nodules revealed that efficiencies of the isolates were related positively and significantly with the surface areas of the rod forms and less strongly with the volumes. No such correlation was observed in the case of coccal forms of the isolates.     

Comparison of peas nodulated with a hydrogen-uptake positive or negative Strain ofRhizobium leguminosarum

During a 49-d growth period peas nodulated with a Hup⁺ strain ofR. leguminosarum (17-to 66-d-old plants) had a significantly higher total (by 80 %) and specific (by 155 %) C₂H₂-reducing nitrogenase activity a lower dihydrogen production by the root nodules (by 53 %), and a higher value of the relative efficiency coefficient of nitrogen fixation (by 45 %) in comparison with the plants nodulated with a Hup^- strain. The nodules induced by the Hup⁺ strain evolved less CO₂ per unit of the acetylene-reducing activity than those induced by the Hup^- strain (by 30 %). However, the accumulation of the nodule dry mass was significantly lower in the plants nodulated with the Hup⁺ strain.     

Effect of iron on siderophore production and on outer membrane proteins ofRhizobium leguminosarum IARI 102

Rhizobium leguminosarum IARI 102 produced a phenolate type siderophore (a derivative of 2,3-DHBA) under iron-limited conditions. Addition of Fe³⁺ to the culture medium increased the growth yield significantly, but repressed the production of the iron-chelating compound. Iron level of culture medium also had a significant role in the composition of outer membrane proteins ofR. leguminosarum IARI 102. Maximum iron uptake was observed only in the presence of its own siderophore.     

Influence ofRhizobium leguminosarum biovartrifolii host specific nodulation genes on the ontogeny of clover nodulation

Summary The infection of white clover seedlings byRhizobium strains with different host range properties was assessed using various microscopic techniques. Several wild-type andRhizobium leguminosarum biovarvicias hybrid strains containing definedR. l. bv.trifolii host range genes were used. The morphological changes in the root tissue of uninoculated and rhizobia inoculated white clovers were identified and compared. In particular, changes were observed in the induction of inner cortical cell division, alterations to nodule development and lateral root formation. The responses of the infected roots and the types of structures formed support the hypothesis that lateral roots and nodules may be physiologically homologous structures. To establish a normal pattern of nodulation on white clover roots, both sets of known host specific nodulation genes (operonsnod FERL andnod MNX) ofR. l. bv.trifolii were required. However, some nodule development occurred when only thenod FERL genes were present in the hybrid strain.     

Isolation and characterization of a virus (RL 1) infective on Rhizobium leguminosarum

A virus (RL 1) that infects Rhizobium leguminosarum was isolated and studied. The virus has phagelike morphology; it has a hexagonal head and a long, flexible, noncontractile tail with a baseplate. The edgeto-edge diameter of head is 760 Å. The tail is 1515 Å long and 115 Å wide. RL 1 is stable at 4° C in distilled water, with only 20% loss in the titer after one month storage. It does not require any ion for stability, and is stable between pH 6.0 and 8.0. The virus is composed of two components; one is thermal sensitive and the other is relatively thermal resistant. Adsorption and one step growth experiments under normal growth conditions showed a slow adsorption rate (0.82×10^-9 cm³ min^-1) followed by a 90 min latent period. The burst size was approximately 100 virus particles per cell.     

Isolation and characterization of peanut agglutinin-resistant embryonal carcinoma cell-surface variants

The isolation and characterization of variant embryonal carcinoma (EC) cells possessing altered cell-surface structures is described. The lectin peanut agglutinin (PNA), which binds to EC cells but not their differentiated derivatives, was used to select the variants. Clones resistant to the cytotoxic effect of PNA were isolated at a frequency of 4 × 10^–5 following mutagenesis. The resistant phenotype was stable in the absence of selection in all eight clones tested. The increased frequency of resistant clones following mutagenesis and the stability of the phenotype suggests a mutational origin. Somatic cell hybrids constructed between wild-type cells and two different PNA-resistant cell lines were sensitive to PNA; this suggests that the resistant phenotype is recessive. Binding assays demonstrated that resistant cells exhibited a twofold to fourfold reduction in the total amount of PNA bound. Together with the recessive behavior of the phenotype, this suggests that resistant cells are deficient for PNA receptors. The PNA-resistant cells also showed reduced binding of monoclonal antibody against stage-specific embryonic antigen 1 (SSEA–1) in indirect cytotoxicity tests. All eight PNA-resistant lines isolated were tumorigenic in syngeneic mice and gave rise to well-differentiated teratocarcinomas. The PNA-resistant cells behaved like their wild-type parents in a cell recognition assay; when incubated in suspension with endodermal cells, they sorted out to form simple embryoid bodies (a core of EC cells surrounded by an endodermal rind). Thus, EC cells can form tumors, differentiate, and recognize differentiated cells in a sorting assay despite a reduction in expression of the embryo-specific cell surface structures (s) that bind PNA and anti-SSEA-1 antibody.     

Release of periplasmic enzymes fromRhizobium leguminosarum bvphaseoli bacteroids by lysozyme is enhanced by pretreatment of cells at low pH

Treatment ofRhizobium leguminosarum bvphaseoli bacteroids with 25 mM citrate buffer, pH 4.0, prior to treatment with 50 mM Tris, pH 8.0, containing 1 mM EDTA, 20% (wt/vol) sucrose, and 1 mg lysozyme/mL increased by two- to three-fold the release of the periplasmic marker enzymes phosphodiesterase and pyrophosphatase. Release of malate dehydrogenase, a cytoplasmic marker, was largely unaffected by the citrate pretreatment. The pretreatment was shown to be a pH effect, with a narrow optimum pH range. Above pH 4.0 the enhancement of release of periplasmic enzymes decreased sharply to almost no effect at pH 5.5, and below pH 4.0 release of malate dehydrogenase increased sharply. The effectiveness of pretreatment with low pH did not appear to be owing to increased entry of lysozyme into the periplasmic space, and an effect of low pH on the suceptibility of the peptidoglycan layer to hydrolysis is suggested.     

Isolation and characterization of interferon-resistant variants from S49 mouse lymphoma

S49 mouse lymphoma cells were found to be extremely sensitive to the antiproliferative activity of interferon. These characteristics were studied to select for IFN-resistant cell variants. Some 0.6% of the parental S49 cell population were resistant to the antiproliferative and cytotoxic activities of IFN. The resistant cells were cloned and analyzed for their responses to several of the activities of IFN, namely, inhibition of encephalomyocarditis (EMC) virus, murine leukemia virus (MuLV) replications, and the induction of (2'-5') oligoadenylate synthetase. Among the clones selected some were highly resistant while others demonstrated only partial responsiveness to IFN. S49 cells demonstrate tubular structures in the cytoplasm. These structures were previously reported to be antigenically related to mouse mammary tumor virus (MMTV). We report here that IFN treatment decreases the expression of these cytoplasmic viral structures as revealed by electron microscopy. To correlate this novel antiviral activity to the more established functions of IFN we utilized the above mentioned S49 IFN-resistant variants. The anti-MMTV activity of IFN correlated with the other effects of IFN in both the highly resistant and partially responsive S49 clones. Our findings indicate that a relatively high proportion of S49 cells vary in their response to IFN. The defect in the resistant cells appears to affect a primary response to IFN which is common to its diverse activities. Furthermore, the effect of IFN on MMTV-related structures involves the usual pathway of IFN action.     

Isolation and characterization of the histone variants in chicken erythrocytes.

Chicken erythrocyte histones 2A, 2B, and 3 can be resolved into nonallelic primary structure variants by polyacrylamide gel electrophoresis in the presence of Triton X-100. These variants were isolated and characterized by analysis of their tryptic and thermolytic peptides. The major variants of chicken H2A and H2B differ from the analogous component of calf thymus by a small number of conservative amino acid substitutions in the basic terminal regions, which interact with DNA. This moderate rate of allelic evolution of the slightly lysine-rich histones contrasts with the complete conservatism found in the arginine-rich histones. Chicken H4 and both chicken H3 variants are identical with their corresponding components in mammals. The amino acid substitutions distinguishing histone variants are located within the highly conserved hydrophobic regions, which are involved in histone--histone interactions.