Identification and cloning of nodulation genes and host specificity determinants of the broad host-range Rhizobium leguminosarum biovar phaseoli strain CIAT899

Rhizobium Ieguminosarum biovar phaseoli type II strain CIAT899 nodulates a wide range of hosts: Phaseolus vulgaris (beans), Leucaena esculenta (leucaena) and Macroptilium atropurpureum (siratro). A nodulation region from the symbiotic plasmid has been isolated and characterized. This region, which is contained in the overlapping cosmid clones pCV38 and pCV117, is able to induce nodutes in beans, leucaena and siratro roots when introduced in strains cured for the symbiotic plasmid, pSym. In addition, this cloned region extends the host range of Rhizobium meliloti and R. leguminosarum biovar (bv.) trifolii wild-type strains to nodulate beans. Analysis of constructed subclones indicates that a 6.4 kb Hin dlll fragment contains the essential genes required for nodule induction on all three hosts. Rhizobium leguminosarum bv. phaseoli type I strain CE3 nodulates only beans. However, CE3 transconjugants harbouring plasmid pCV3802 (which hybridized to a nodD heterologous probe), were capable of eliciting nodules on leucaena and siratro roots. Our results suggest that the CIAT899 DNA region hybridizing with the R. meliloti nodD detector is involved in the extension of host specificity to promote nodule formation in P. vulgaris, L. esculenta and M. atropurpureum.     

Growth promotion of maize and lettuce by phosphate-solubilizing Rhizobium leguminosarum biovar. phaseoli

Rhizobium leguminosarum bv. phaseoli strains P31 and R1, Serratia sp. strain 22b, Pseudomonas sp. strain 24 and Rhizopus sp. strain 68 were examined for their plant growth-promoting potential on lettuce and forage maize. All these phosphate solubilizing microorganisms (PSM) were isolated from Québec soils. The plants were grown in field conditions in three sites having high to low amounts of available P. In site 1 (very fertile soil), strains R1 and 22b tended to increase the dry matter yield of lettuce shoots (p≤0.10). Lettuce inoculated with rhizobia R1 had a 6% higher P concentration (p≤0.10) than the uninoculated control. In site 2 (poorly fertile soil), the dry matter of lettuce shoots was significantly increased (p≤0.05) by inoculation with strain P31 and 24 plus 35 kg ha^-1 P-superphosphate, or with strain 68 plus 70 kg ha^-1 P-superphosphate. In site 3 (moderately fertile soil), the dry matter of maize shoots was significantly increased (p≤0.05) by inoculation with strain 24 plus 17.5 kg ha^-1 P-superphosphate, or with strain P31 plus 35 kg ha^-1 P-superphosphate. Inoculation with PSM did not affect lettuce P uptake in the less fertile soil in site 2. In site 3 with the moderately fertile soil, maize plants inoculated with strain R1 had 8% higher P concentration than the uninoculated control (p≤0.01), and 6% with strains P31 and 68 (p≤0.05). The results clearly demonstrate that rhizobia specifically selected for P solubilization function as plant growth promoting rhizobacteria with the nonlegumes lettuce and maize. The P solubilization effect seems to be the most important mechanism of plant growth promotion in moderately fertile and very fertile soils when P uptake was increased with rhizobia and other PSM.     

Effect of divalent cations on succinate transport in Rhizobium tropici,R. leguminosarum bv phaseoli and R. loti

Rhizobium tropici, R. leguminosarum bv phaseoli and R. loti each have an active C₄-dicarboxylic acid transport system dependent on an energized membrane. Free thiol groups are probably involved at the active site. Since EDTA inhibited succinate transport in R. leguminosarum bv phaseoli and R. loti, divalent cations may participate in the process; the activity was reconstituted by the addition of Ca²⁺ or Mg²⁺. However, EDTA had no effect on succinate transport in R. tropici, R. meliloti or R. trifolii strains. Ca²⁺ or Mg²⁺ had a similar effect on the growth rates of R. tropici and R. leguminosarum bv phaseoli; R. tropici did not require Ca²⁺ to grow on minimal medium supplemented with succinate but R. leguminosarum bv phaseoli required either or both of the divalent cations Ca²⁺ and Mg²⁺. A R. tropici Mu-dI (lacZ) mutant defective in dicarboxylic acid transport, was isolated and found unable to form effective bean nodules.The authors are with the Division of Biochemistry, Instituto de Investigaciones Biológicas Clemente Estable, Avda, Italia 3318, 11.600 Montevideo, Uruguay     

Construction and characterization of a Rhizobium leguminosarum biovar viciae strain designed to assess horizontal gene transfer in the environment

Abstract An integration vector was developed which inserts cloned DNA in a non-essential site of the Rhizobium leguminosarum biovar viciae chromosome. The expression of integrated genes is under the control of the constitutive neomycin phosphotransferase II ( npt II) promotor of transposon Tn5. The design of the vector ensures that loss of vector sequences can be detected, enabling selection of progeny containing only the requisite DNA. The newly constructed vector was employed to insert the Escherichia coli gusA gene conferring GUS activity into R. leguminosarum bv. viciae strain LRS39401 which is cured of its symbiotic plasmid (pSym). One GUS-positive transconjugant, strain CT0370, was shown to have lost all vector sequences. Conjugal transfer of pSym2004 (a Tn5-tagged derivative of symbiotic plasmid pRL1JI, which specifies pea nodulation and symbiotic nitrogen fixation) to CT0370, restored the GUS-positive strain's symbiotic proficiency. Strain CT0370 is presently being used in a field release experiment in order to assess the extent of pSym transfer in a natural R. leguminosarum bv. viciae population under environmental conditions.     

Long-term effects of daily grazing orbits on nutrient availability in Sahelian West Africa: I. Gradients in the chemical composition of rangeland soils and vegetation

Abstract. An investigation of the long-term effects of livestock grazing on the spatial distribution of plant nutrients was conducted in the Sahelian region of West Africa. The study area was located along the transhumance corridor used for at least the past century by a long-established population of agropastoralists in central Mali. Spatial variation of plant and soil chemistry across eighteen sites was investigated with respect to a grid, whose axes are defined by distance from a pan from which livestock disperse to graze primarily during the early dry season (D1) and by distance from the nearest of three pans used primarily during the rainy season (D2). Particle size distribution, pH, total C, N, and P of surface soil (0–10 cm) was determined for four composite samples collected at eleven of the eighteen ecological sites. Elemental analysis (N, P, Ca, Mg, K, Mn, Fe, Zn, Cu) was performed on above-ground tissue samples (excluding seeds) of the three most common grasses and the most common legume collected at all eighteen sites at the end of the rainy season. Results demonstrate that long-term grazing movements can, over the long-term, affect nutrient availability over wider spatial scales (5 km radii) than previously observed by shorter-term studies. Soil and plant chemistry were found in general to be most affected by the historic gradient of cattle presence during the rainy season (D2). Controlling for soil C and coarse sand fraction, soil pH was found to decline with D2 (?0.5 pH units per km D2) and to a lesser extent D1 (?0.15pH units per km D1), a finding that cannot be explained solely by a consideration of local geomorphology. The pH gradient was found to significantly affect micronutrient concentrations in plant tissues. Phosphorus (P) concentrations of plant tissue were found to decrease with D2. The increase in P-availability near rainy-season encampments could result from P-release from Al-complexes at higher pH and/or by the redistribution of P from outlying zones towards rainy-season encampments.     

Response of common bean lines to inoculation: comparison between the <Emphasis Type="Italic">Rhizobium tropici</Emphasis> CIAT899 and the native <Emphasis Type="Italic">Rhizobium etli</Emphasis> 12a3 and their persistence in Tunisian soils

Since Phaseolus vulgaris (L) is poorly nodulated in all regions of Tunisia where this crop is grown, the response of common-bean lines CocoT and Flamingo to inoculation with reference Rhizobium tropici CIAT 899 or native rhizobia, namely Sinorhizobium fredii 1a6, Rhizobium etli 12a3, and Rhizobium gallicum 8a3, was studied in a field station. Since R. etli 12a3 was found to be the most effective native rhizobium, it was subsequently compared with R. tropici CIAT 899 in a broader study in two stations over 3 years. A significant interaction between bean and rhizobia was observed for nodule number, shoot dry weight, grain yield, and contents of nitrogen and chlorophyll. The native rhizobia was more efficient than CIAT899 for Flamingo, though not for CocoT. The Enzyme-linked immunosorbent assay technique was used with polyclonal antibody to assess the occupancy in nodule and persistence in soil of the inoculated rhizobia. For both stations the nodule occupancy was 100% during the first year for each rhizobium, but during the next 2 years, between 7 and 15% of nodules were formed by the rhizobia inoculated in the neighboring plot. It is concluded that the first-year inoculation is sufficient to maintain an adequate rate of nodulation during three growth cycles, and that the native R etli can be recommended for the common-bean inoculation in similar soils of Tunisia.     

Influence of hydrological fluctuations on the growth and nutrient dynamics ofPhalaris arundinacea L. in a riparian environment

Seasonal changes in aboveground and belowground tissues ofPhalaris arundinacea L. were studied in a population colonizing an ancient meander of the Garonne river (France) submitted to important fluctuations of the permanent water table. Waterlogged conditions in spring stopped the growth of rhizomes and promoted the translocation of nutrient to the shoots. The early senescence of plants after flowering could be related to the withdrawal of the water table. It was characterized by a distribution of nutrients in belowground tissues and a release in litter and soil. Aerated conditions in late summer permitted the growth of belowground tissues. At this time a partition of resources between aboveground and belowground biomass of a new generation of plants was observed. Rising water and decreasing temperatures in winter induced the death of aboveground parts. Reconstitution of nutrient stocks in rhizomes and losses by leaching then occured. Beside a very high primary production this strategy confers toPhalaris arundinacea a great interest in different uses, especially in the removal of nutrients from water in riparian zones as in artificial sites.     

Study of the contribution of the shoot and/or root ofAlnus sp. in the compatibility between the host and a Sp+ Frankia strain using a grafting technique

Two alder species,Alnus glutinosa (L.) Gaertn. andAlnus incana (L) Moench, were inoculated with a Sp⁺ Frankia homogenate obtained fromA. incana root nodules. This inoculum formed effective nodules on the original host plant and ineffective nodules onA. glutinosa. Grafts between the two alder species were made to determine which part of the plant is involved in this phenomenon. The results obtained indicate that the compatibility between Alnus andFrankia is restricted to the root system.