Carmen Lluch

Effects of salt stress on growth and nitrogen fixation by pea, faba-bean, common bean and soybean plants

The effects of salinity on growth, nodulation, acetylene reduction activity (ARA), nodule leghemoglobin (Lb) content and respiratory capacity of bacteroids from pea (Pisum sativwn cv. Lincoln), faba-bean (Vicia faba L. var. minor cv. Alborea), bean (Phaseolus vulgaris cv. Contender) and soybean (Glycine max L. var. Williams) were determined. The depressive effect of saline stress (50 and 100 mM NaCl) on dry weight and ARA of nodules was directly related to the salt-induced decline in dry weight and N content in shoots. The results indicate that P. savitum was the legume most severely affected by salinity, whereas G. max was the most salt-tolerant species. Saline stress was also responsible for a decrease in cytosolic protein of nodules, specifically Lb; this effect was more pronounced in pea and bean nodules than in soybean and faba-bean nodules. Bacteroids isolated from soybean and bean nodules exhibited higher respiratory activity in the absence of energy-yielding substrates than those isolated from pea and faba-bean plants. Salt added directly to the incubation mixture of bacteroids inhibited O2 uptake of bacteroids isolated from nodules of each legume. However, salt treatment of plants decreased respiratory capacity only in pea and faba-bean bacteroids. Inhibition of ARA under moderate saline stress may be related to the drop in bacteroid respiration. Under severe stress both the reduction in Lb content and inhibition of bacteroid respiration may be involved in salt-induced inhibition of nitrogen fixation.

Rhizobium tropici Genes Involved in Free-Living Salt Tolerance are Required for the Establishment of Efficient Nitrogen-Fixing Symbiosis with Phaseolus vulgaris

Characterization of nine transposon-induced mutants of Rhizobium tropici with decreased salt tolerance (DST) allowed the identification of eight gene loci required for adaptation to high external NaCl. Most of the genes also were involved in adaptation to hyperosmotic media and were required to overcome the toxicity of LiCl. According to their possible functions, genes identified could be classified into three groups. The first group included two genes involved in regulation of gene expression, such as ntrY, the sensor element of the bacterial ntrY/ntrX two-component regulatory system involved in regulation of nitrogen metabolism, and greA, which encodes a transcription elongation factor. The second group included genes related to synthesis, assembly, or maturation of proteins, such as alaS coding for alanine-tRNA synthetase, dnaJ, which encodes a molecular chaperone, and a nifS homolog probably encoding a cysteine desulfurase involved in the maturation of Fe-S proteins. Genes related with cellular build-up and maintenance were in the third group, such as a noeJ-homolog, encoding a mannose-1-phosphate guanylyltransferase likely involved in lipopolysaccharide biosynthesis, and kup, specifying an inner-membrane protein involved in potassium uptake. Another gene was identified that had no homology to known genes but that could be conserved in other rhizobia. When inoculated on Phaseolus vulgaris growing under nonsaline conditions, all DST mutants displayed severe symbiotic defects: ntrY and noeJ mutants were impaired in nodulation, and the remaining mutants formed symbiosis with very reduced nitrogenase activity. The results suggest that bacterial ability to adapt to hyperosmotic and salt stress is important for the bacteroid nitrogen-fixing function inside the legume nodule and provide genetic evidence supporting the suggestion that rhizobia face severe environmental changes after their release into plant cells.

Evolution of ethylene from roots of medicago sativa plants inoculated with rhizobium meliloti

Summary The evolution of ethylene from roots of Medicago sativa plants uninoculated and inoculated with Rhizobium meliloti was measured for plants grown in test tubes under axenic conditions.Three maxima of ethylene evolved were always observed.These appeared 9-11, 19-23, and 27-30 days after sowing.Ethylene released from roots of inoculated plants showed a considerable increase compared with uninoculated plants.Results showed that ethylene is an important factor in the control of nodule development, maintenance, and senescence.

Phenotypic and molecular characterization of chickpea rhizobia isolated from different areas of Morocco

Aims: To determine the biodiversity of rhizobial strains nodulating Cicer arietinum L. in representative soils from various areas of Morocco. 
Methods and Results: Symbiotic traits, utilization of 49 carbohydrate sources, resistance to antibiotics and heavy metals, tolerance to salinity, to extreme temperatures and pH were studied as phenotypic markers. In addition, restriction fragment length polymorphism (RFLP) of PCR‐amplified 16S rDNAs were compared with those of reference strains. Numerical analysis of the phenotypic characteristics showed that the 48 strains studied fell into three distinct groups. RFLP analysis of 16S rRNA genes revealed an additional heterogeneity and four ribotypes were identified. 
Conclusions: Chickpea rhizobia isolated from Moroccan soils are both phenotypically and genetically diverse. Most of these rhizobia belong to the Mesorhizobium genus. However, some strains originating from a particular soil appeared to have 16S rRNA genes similar to Sinorhizobium as well as very distinct auxanographic characteristics compared with Mesorhizo‐ bium isolates. 
Significance and Impact of the Study: A well characterized collection of chickpea‐nodulating rhizobia in representative soils of Morocco has been generated, which can be used to develop efficient inoculants for this crop. This is the first report evidencing that chickpeas may be nodulated by bacteria from the Sinorhizobium genus.

Nitrogenase and antioxidant enzyme activities in Phaseolus vulgaris nodules formed by Rhizobium tropici isogenic strains with varying tolerance to salt stress

Common bean plants inoculated with salt-tolerant Rhizobium tropici wild-type strain CIAT899 formed a more active symbiosis than did its decreased salt-tolerance (DST) mutant derivatives (HB8, HB10, HB12 and HB13). The mutants formed partially effective (HB10, HB12) or almost ineffective (HB8, HB13) nodules (Fix(d)) under non-saline conditions. The DST mutant formed nodules that accumulated more proline than did the wild-type nodules, while soluble sugars were accumulated mainly in ineffective nodules. Under salt stress, plant growth, nitrogen fixation, and the activities of the antioxidant defense enzymes of nodules were affected in all symbioses tested. Overall, mutant nodules showed lower antioxidant enzyme activities than wild-type nodules. Levels of nodule catalase appeared to correlate with symbiotic nitrogen-fixing efficiency. Superoxide dismutase and dehydroascorbate reductase seem to function in the molecular mechanisms underlying the tolerance of nodules to salinity.

Isolation and characterization of Azotobacter and Azospirillum strains from the sugarcane rhizosphere

Bacteria with the ability to grow on nitrogen-free media and with nitrogenase activity under aerobic or microaerobic conditions were isolated from sugarcane roots collected from four different agricultural locations in Granada (Spain). Isolates were Gram negative rods and were identified as Azotobacter chroococcum and Azospirillum brasilense. Our results suggest that Azotobacter isolates do not have a particular affinity for sugarcane rhizospheres and that, on the contrary, Azospirillum isolates show specific association and perhaps endophytic colonization of sugarcane. However, obligate endophytes (Gluconacetobacter diazotrophicus) were not found in the apoplastic fluid of the stems and macerates extracts of sugarcane tissues with the procedure applied. Population of this microorganism might be in low number in the Spanish sugarcane varieties studied which is also discussed.

Salinity effects on growth analysis and nutrient composition in four grain legumes. Rhizobium symbiosis

Abstract The effect of salinity on growth response, nitrogen (N) fixation and tissue mineral content was investigated for four legumes: faba bean (Vicia faba L), pea (Pisum sativum L), soybean (Glycine max L), and common bean (Phaseolus vulgaris L). Plants were grown in a vermiculite culture system supplied with a N‐free nutrient solution with the addition of 0, 50, and 100 mM NaCl. Plants were harvested at the beginning of the flowering period and the dry weights of shoots and roots and acetylene reduction activity (ARA) were evaluated at the same time plant tissues were analysed for N, potassium (K), calcium (Ca), magnesium (Mg), and sodium (Na) contents. The depressive effect of saline stress on ARA of nodules was directely related to the salt induced decline in dry weight and N content in shoots. Growth inhibition by NaCl treatments was greater for the pea than for other legumes, whereas the soybean was the most salt‐tolerant Saline stress also affected the N content in shoots and roots. In general th...

Alleviation of Salt Stress in Common Bean (Phaseolus vulgaris) by Exogenous Abscisic Acid Supply

In this work the effect of abscisic acid (ABA) and 100 mM NaCl on common bean (Phaseolus vulgaris var. Coco) growth, nitrogenase activity, and nodule metabolism was studied. Experiments were carried out in a controlled environmental chamber and plants, at the vegetative growth stage (16 days old), were treated with ABA (1 μM and 10 μM) and 48 h later were exposed to saline treatment. Results revealed that plant dry weight, nodule dry weight, nitrogen fixation (acetylene reduction activity and ureides content), and most enzymes of ammonium and ureides metabolism were affected by both ABA and NaCl. The addition of 1 μM ABA to the nutrient solution before the exposure to salt stress reduced the negative effect of NaCl. Based on our results, we suggest that ABA application improves the response of Phaseolus vulgaris symbiosis under saline stress conditions, including the nitrogen fixation process and enzymes of ammonium assimilation and purine catabolism.

Effects of salinity on protein and lipopolysaccharide pattern in a salt-tolerant strain of Mesorhizobium ciceri

Aims: To characterize the physiological and metabolic responses of Mesorhizobium ciceri strain ch‐191 to salt stress, investigating the changes induced by salinity in protein and lipopolysaccharide profiles, as well as determining the accumulation of amino acids, glutamate and proline.

Influence of host genotypes on growth, symbiotic performance and nitrogen assimilation in faba bean (Vicia faba L.) under salt stress

Fifteen genotypes of faba bean (Vicia faba L.) were inoculated with salt-tolerant Rhizobium leguminosarum biovar. viciae strain GRA 19 in solution culture with 0 (control) and 75 mM NaCl added immediately after transplanting. Genotypes varied in their tolerance of high levels of NaCl. Physiological parameters (dry weight of shoot and root, number and dry weight of nodules) were not affected by salinity in lines VF46, VF64 and VF112. Faba bean line VF60 was sensitive to salt stress. Host tolearance appeared to be a major requisite for nodulation and N2 fixation under salt stress. Tolerant line VF112 sustained nitrogen fixation under saline conditions. Activity of the ammonium assimilation enzymes glutamine synthetase and glutamate synthase, and soluble protein content, were reduced by salinity in all genotypes tested. Evidence presented here suggests a need to select faba bean genotypes that are tolerant to salt stress.