Identification of Prosopis juliflora and Prosopis pallida Accessions Using Molecular Markers

There has been much taxonomic confusion over the identification of Prosopis species, especially where introduced. Prosopis juliflora is the most widespread species in the arid and semi-arid tropics, although it has been confused with other species, particularly the closely related Prosopis pallida. In this study, RAPDs markers were used for the first time to distinguish between these species. Eighteen primers were used in amplification reactions, which yielded an average of 120 bands per accession. A dendrogram showing genetic similarities among accessions was constructed using UPGMA cluster analysis and the Nei and Li similarity coefficient. The genetic similarity observed between P. juliflora and P. pallida is similar to the value in sympatric Prosopis species in North America, and reconsideration of the series rank in section Algarobia is suggested. Species-specific markers confirmed that material in Burkina Faso is P. juliflora, but suggested that material collected in Brazil, Cape Verde and Senegal is P. pallida, whereas this has previously been identified as P. juliflora.     

Diversity of bacteria that nodulate Prosopis juliflora in the eastern area of Morocco

A total of 274 bacterial strains were isolated from the root nodules of Prosopis juliflora, growing in two arid soils of the eastern area of Morocco. A physiological plate screening allowed the selection of 15 strains that could tolerate NaCl concentrations between 175 and 500 mM. These were compared with 15 strains chosen from among the ones which did not tolerate high salinity. The diversity of strains was first assessed by rep-PCR amplification fingerprinting using BOXA1R and ERIC primers. An analysis of the PCR-amplified 16S rDNA gene digestion profiles using five endonucleases indicated the presence of different lineages among the taxa associated with P. juliflora nodules in the soils studied. Nucleotide sequencing of the small subunit rRNA gene and BLAST analysis showed that P. juliflora could host at least six bacterial species in this region and that the identity of those associated with high salt tolerance was clearly distinct from that of the salt-sensitive ones. Among the former, the first type displayed 99% similarity with different members of the genus Sinorhizobium, the second 97% similarity with species within the genus Rhizobium, while the third ribosomal type had 100% homology to Achromobacter xylosoxidans. Within the salt-sensitive isolates the prevailing type observed showed 98% similarity with Rhizobium multihospitium and R. tropici, a second type had 98% similarity to R. giardinii, and a further case displayed 97% colinearity with the Ensifer group including E. maghrebium and E. xericitae. All of the thirty strains encompassing these types re-nodulated P. juliflora in microbiologically controlled conditions and all of them were shown to possess a copy of the nodC gene. This is the first report detecting the betaproteobacterial genus Achromobacter as nodule-forming species for legumes. The observed variability in symbiont species and the abundance of nodulation-proficient strains is in line with the observation that the plant always appears to be nodulated and efficiently fixing nitrogen in spite of a wide range of soil and environmental conditions.     

Plasmid-mediated control of nodulation in Rhizobium trifolii

The nodulation ability was effectively eliminated from different Rhizobium trifolii strains incubated at elevated temperature (urkowski and Lorkiewicz, 1978). Non-nodulating (Nod^-) mutants were stable and no reversion of Nod^- to Nod⁺ phenotype was observed. Strains R. trifolii 24 and T12 which showed a high percentage of elimination of nodulation ability were examined in detail. Two plasmids were detected in strain 24 using neutral and alkaline sucrose gradient centrifugation of plasmid preparations. Molecular weights of the plasmids pWZ1 and pWZ2 were 460 Mdal and 190 Mdal, respectively. Rhizobium lysates labeled with ³H-thymidine and ultracentrifuged in caesium chloride — ethidium bromide gradients demonstrated a 40% reduction of the plasmid DNA content in R. trifolii 24 Nod^- mutants in comparison with the nodulating wild type strain 24. It was found further that non-nodulation of mutants 24 Nod^- was due to the absence of plasmid pWZ2. Sucrose gradient data also demonstrated that strain T12 contained two plasmids with molecular weights corresponding to those of pWZ1 and pWZ2, respectively. In Nod^- mutant clones derived from strain T12, pWZ2 plasmid was missing.Non Standard Abbreviations CCC covalently closed circular - OC open cirucular - Sarkosyl sodium N-lauroylsarcosinate     

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.     

A growth-inhibitory substance exuded from freeze-dried mesquite (Prosopis juliflora (Sw.) DC.) leaves

When lettuce seeds were incubated in a concentric circle aroundfreeze-dried mesquite leaves in a dish containing agar culture medium, thegrowth, especially radicle growth, of the seedlings was inhibited, the morestrongly the closer they were to the leaves. This result indicates thatallelopathic substance(s) inhibiting the lettuce growth are exuded from themesquite leaves. A potent substance was isolated from the exudates of thefreeze-dried mesquite leaves and identified as L-tryptophan by spectralanalyses. L-Tryptophan inhibited the radicle growth of lettuce and barnyardgrass at concentrations greater than1.5×10^–3 . The content ofL-tryptophan in the exudates of freeze-dried mesquite leaves (1 eq.) was 4.8×10^–3 . Theseresults suggest that L-tryptophan may play an important role in the allelopathyof mesquite leaves.     

The effect of silicon on the growth of Prosopis juliflora growing in saline soil

A decrease in whole plant dry weight was observed when Prosopis juliflora (Swartz) DC. was treated with saline irrigation water for 24 days which was partially alleviated by the addition of 0.47 mM SiO₂ to the irrigation water. The plants treated with high salinity and SiO₂ showed a greater distribution of dry material to the leaves at the expense of the stems and roots compared to control plants. The possible use of SiO₂ to grow plants may be beneficial in areas of high soil salinities.     

Leaching of the allelopathic substance, -tryptophan from the foliage of mesquite (Prosopis juliflora (Sw.) DC.) plants by water spraying

The allelopathic potential of -tryptophan in the leachate from the foliage of mesquite (Prosopis juliflora (Sw.) DC.) plants was investigated. Distilled water (500 ml) was sprayed on mesquite plants from above for 50 min and water passing through the foliage was collected. The content of -tryptophan in the sample (30 ml eq.) was analyzed using a physicochemical method and the concentration was shown to be 17.9 µM. The sample (30 ml eq.) caused 69.8% inhibition of the root growth of barnyard grass (Echinochloa crus-galli L.). The concentration of authentic -tryptophan required for 69.8% inhibition of the root growth of barnyard grass was estimated at 20.0 µM from the dose-response curve. Moreover, to establish the origin of -tryptophan in the leachate, the amount of -tryptophan in both the leaves and the leachates was determined. The amount of -tryptophan containing in the foliage significantly decreased by soaking with time, whereas in the leachates the amount increased. After 60 min, its content in the leachates was nearly equivalent to that of the leaves. These results suggest that -tryptophan leached from the foliage may play an important role in the allelopathy imposed by mesquite.     

Involvement of Rhizobium leguminosarum nodulation genes in gene expression in pea root hairs

The mRNA population in pea root hairs was characterized by means of in vitro translation of total root hair RNA followed by 2-dimensional gel electrophoresis of the translation products. Root hairs contain several mRNAs not detectable in total RNA preparations from roots. Most of these root hair-specific mRNAs occur in elongating root hairs at higher levels than in mature root hairs. The expression of some genes in pea root hairs is typically affected by inoculation with Rhizobium leguminosarum. One gene, encoding RH-42, is specifically induced while the expression of another gene, encoding RH-44, is markedly enhanced. Using R. leguminosarum mutants it was shown that the nodC gene is required for the induction and enhancement of expression of the RH-42 and RH-44 genes, respectively, while the Rhizobium chromosomal gene pss1, involved in exopolysaccharide synthesis, is not essential. After induction of the nod genes with apigenin the bacteria excrete into the culture medium a factor that causes root hair deformation. This deformation factor stimulates the expression of the RH-44 gene but does not induce the expression of the gene encoding RH-42.     

Isolation of adenylate cyclase mutants from Rhizobium meliloti deficient in nodulation

Six Rhizobium meliloti mutants were isolated after Tn5-mediated mutagenesis as resistant to inhibition by a mixture of amino acids (serine, methionine, glycine and leucine). All were defective in adenylate cyclase activity and failed to form nodules in infected roots of Medicago sativa. Furthermore, like other nodulation mutants, they showed altered motility and increased secretion of exopolysaccharides; addition of cAMP to the growth medium abolished some of these phenotypic defects. The possibility that adenylate cyclase participates in the transduction of signals inducing nodulation is discussed.     

Host-specific nodulation is encoded on a 14kb DNA fragment in Rhizobium trifolii

Summary The Rhizobium trifolii genes necessary for nodule induction and development have been isolated on a 14.0kb fragment of symbiotic (Sym) plasmid DNA. When cloned into a broad-host-range plasmid vector, these sequences confer a clover nodulation phenotype on a derivative of R. trifolii which has been cured of its endogenous Sym plasmid. Furthermore, these sequences encode both host specificity and nodulation functions since they confer the ability to recognize and nodulate clover plants on Agrobacterium and a fast-growing cowpea Rhizobium. This indicates that the bacterial genes essential for the initial, highly-specific interaction with plants are closely linked.     

Growth regulators,Rhizobium and nodulation in peas

The cytokinin content of roots and nodules of pea and the culture supernatants from two strains of Rhizobium leguminosarum has been examined. Roots, nodules and wild-type Rhizobium culture medium contained very little cytokinin as indicated by bioassay. Chemical ionisation gas chromatography-mass spectrometric analysis of the isopentenyladenine content of the culture medium from the Rhizobium strains confirmed that the content of the wild-type was low (approx. 1 ng dm^-3) but that it was increased by the introduction of the Agrobacterium Ti plasmid into the Rhizobium strain.Abbreviations CI chemical ionisation - GCMS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - iPAde isopentenyladenine - MIM multiple ion monitoring     

NifA-NtrA regulatory system activates transcription of nfe,a gene locus involved in nodulation competitiveness of Rhizobium meliloti

We have previously demonstrated that the Rhizobium meliloti large plasmid pRmeGR4b carries the gene locus nodule formation efficiency (nfe) which is responsible for nodulation efficiency and competitive ability of strain GR4 on alfalfa roots. In this study we report that expression of nfe-lacZ fusions in Escherichia coli is activated in the presence of the cloned nifA gene of R. meliloti. This activation was found to be oxygen sensitive and to require the E. coli ntrA gene product. In contrast to the R. meliloti nifA, the cloned nifA gene of Klebsiella pneumoniae was able to activate expression of nfe in aerobically grown cells of both E. coli and R. meliloti. Hybridization experiments did not show homology to nfe in four R. meliloti wild-type strains tested. These strains were uncompetitive when coinoculated with a GR4 derivative carrying plasmid pRmeGR4b, but were competitive when coinoculated with a GR4 derivative carrying a single transposon mutation into the nfe region. When nfe DNA was introduced into the four wild-type strains, a significant increase in the competitive ability of two of them was observed, as deduced from their respective percentages of alfalfa root nodule occupancy in two-strains coinoculation experiments.     

Organisation of nodulation and nitrogen fixation genes on a Rhizobium trifolii symbiotic plasmid

A Rhizobium trifolii symbiotic plasmid specific gene library was constructed and the physical organisation of regions homologous to nifHDK, nifA and nod genes was determined. These symbiotic gene regions were localised to u 25 kb region on the sym-plasmid, pPN1. In addition four copies of a reiterated sequence were identified on this plasmid, with one copy adjacent to nifH. No rearrangement of these reiterated sequences was observed between R. trifolii bacterial and bacteroid DNA. Analysis of a deletion derivative of pPN1 showed that these sequences were spread over a 110 kb region to the left of nifA.     

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.     

Competition between strains ofBradyrhizobium japonicum for nodulation of soybeans at different nitrogen fertilizer levels

Competitive abilities of 3 strains ofBradyrhizobium japonicum (E104, E109, E110) for nodulation of soybean (Glycine max) at increasing nitrogen fertilizer levels were studied. Dry weight of plants nodulated by strain E110 were depressed at 10 g N·m^–2, the highest fertilizer level, even when mixed with strain E109. Strain E104 alone or mixed with E109 increased dry matter production. Strain E110 formed many dually infected nodules with strain E104 present but not with strain E109. However, strain E104 formed nodules containing strain E109. Neither strain E110 or E109 produced bacteriocin, so the incompatibility of these two strains had to be due to another reason. Strain E104 successfully competes with strain E109 but not with E110 at 10 g N·m^–2. It is concluded that strain E110 dominates the symbiotic relationships even if other strains are also present in the nodules. However, at a high N-fertilizer level strain E110 decreases the plant yield in contrast to E104, which could be recommended as inoculant at increased levels of soluble soil-N.     

Effects of Rhizobium inoculation on growth and nodulation ofAlbizia falcataria (L.) Fosh. andAcacia mangium Willd. in the nursery

Three separate experiments were conducted in the nursery using grassland soil as a growing medium. The first experiment was conducted to assess the nodulation of the two legume trees grown in unamended soil, the second was done to determine the effects of N-fertilizer application on the interaction of the five Rhizobium isolates withA. falcataria and the third experiment was conducted to determine the effects of liming on theRhizobium × A. mangium interaction. Three local Rhizobium isolates, A₁₆, A₁₈, and A₁₄ were effective forA. falcataria with A₁₆ as the most promising strain under the conditions described. In general, application of combined nitrogen suppressed nodulation ofA. falcataria. Nodulation in the absence of combined N exceeded those fertilized with 30 kg N ha⁻¹ by 114.0%, 60 kg N ha⁻¹ by 209.6% and 100 kg N ha⁻¹ by 237.1%. Two local isolates, Am₁₀ and Am₂, and an introduced strain, NA 1533 from Australia were promising forA. mangium. Unlike inA. falcataria, the application of combined nitrogen at the rate of 100 kg N ha⁻¹ did not suppress nodulation inA. mangium. Liming the soil to pH 6.5 regardless of nitrogen fertilizer application improved the performance of the Rhizobium—A. mangium symbiosis.