Orwik

Effect of on-field inoculation of Phaseolus vulgaris with rhizobia on soil bacterial communities.

The aim of this study was to assess the impact of inoculation of Phaseolus vulgaris with two indigenous rhizobia strains on plant growth promotion, nitrogen turnover processes, richness and structure of the Rhizobiaceae and total bacterial communitie... expand abstracts in the bulk soil. Both strains used induced a significant increase in nodulation and grain yield. Analysis of bulk soil fertility showed positive, negative and strain-dependent effects of inoculation on nitrate, phosphorus and ammonium, respectively. Terminal-restriction fragment length polymorphism profiling demonstrated that inoculation significantly increased the phylotype richness of the bacterial communities. No significant difference in richness between the strains used and no additive effect of co-inoculation were observed. However, differences between both inoculants and a clear additive effect of co-inoculation on heterogeneity were found. This work gives original insights into the effect of rhizobial inoculation outside the restricted rhizospheric area. Effects on bacterial structure and diversity are clearly sensed in the neighbourhood of 25 cm and in a limited time course. Both Alpha- and Gammaproteobacteria, together with Firmicutes and Actinobacteria, were enhanced by inoculation, No evidence of terminal-restriction fragment inhibition was found. However, it remains to be answered how the impact on taxonomic groups can be related to effects on functional capabilities of soil microbial communities. collapse abstract

Diversity of rhizobia nodulating sulla (Hedysarum coronarium L.) and selection of inoculant strains for semi-arid Tunisia

A total of 45 bacteria were isolated from root nodules of sulla (Hedysarum coronarium L.) in Tunisia and tested for their tolerance to NaCl, alkaline pH and PEG-induced water stress. Tolerance limits varied from 150 to 700 mM for NaCl, from 9 to 10.5... expand abstract for alkaline pH and from −0.5 to −0.95 MPa for PEG-induced stress. Three effective isolates with variable tolerance were retained to study the effect of water deficiency. The 16S rRNA gene sequencing showed that the closest relative to the three isolates was Rhizobium sullae strain IS 123T. Plants inoculated with the moderately tolerant strain Hc14 showed the highest shoot dry yield at 75% FC; however, for lower soil moisture levels, strain Hc14 was quite similar to the most tolerant strain Hc5. By contrast, plants inoculated with the most sensitive strain Hc1 showed the lowest nodule number and shoot dry yield. At high water deficiency (20% FC), strains Hc5 and Hc14 were still able to induce nodules; however, strain Hc1 failed. Strains Hc5 and Hc14 were then assayed in a non-irrigated field trial. The results confirmed the usefulness of these strains as inoculants under water deficiency. However, other trials should be conducted in other sites to further confirm these results. collapse abstract

Genetic diversity and salt tolerance of bacterial communities from two Tunisian soils

Microbial ecology studies on arid soils are particularly important for the analysis of biological functions during desertification. Although much is known about the arid saline flora, few researches have directly compared the bacterial communities of... expand abstract saline arid soils with cultivated soils in Northern Africa. Bacterial communities present in two soils from Soliman (north of Tunisia), one salty and neglected, and the other cultivated, were investigated by using both cultivation dependent and independent approaches. The first approach was used to assess the presence of salt tolerant bacteria and the relationships among salt (NaCl) resistance phenotype, soil characteristics and phylogenetic assignment of strains. Total community analysis, performed by T-RFLP on total DNA, was carried out to investigate the relationships between total community fingerprinting with cultivated isolates diversity. The cultivated isolates from salty soil were more genetically diverse, harbouring strains that can grow at high salt concentration. Moreover, the salt resistance of isolates was found not to be related to any particular phylogenetic group, being widespread among isolates belonging to different bacterial subdivisions. Ribotype richness, evaluated as number of different T-RFLP bands (TRFs), was shown to be higher in the agricultural soil than in the salty soil and several agricultural soil-specific TRFs were detected. Key words  bacterial communities - T-RFLP - salt tolerance - arid soil collapse abstract

Symbiotic diversity of Ensifer meliloti strains recovered from various legume species in Tunisia.

Ensifer meliloti (formerly Sinorhizobium meliloti) was first considered as a specific microsymbiont of Medicago, Melilotus and Trigonella. However, strains of E. meliloti were recovered from root nodules of various legume species and their symbiotic ... expand abstractstatus still remains unclear. Here, we further investigate the specificity of these strains. A collection of 47 E. meliloti strains isolated in Tunisia from root nodules of Medicago truncatula, Medicago sativa, Medicago ciliaris, Medicago laciniata, Medicago marina, Medicago scutellata, Phaseolus vulgaris, Cicer arietinum, Argyrolobium uniflorum, Lotus creticus, Lotus roudairei, Ononis natrix, Retama raetam, Genista saharae, Acacia tortilis, Hedysarum carnosum and Hippocrepis bicontorta were examined by REP-PCR fingerprinting, PCR-RFLPs of the 16S-23S rDNA IGS, the nifH gene and nifD-K intergenic spacer, and sequencing of 16S rRNA and nodA genes. Their nodulation range was also assessed by cross-inoculation experiments. No clear correlation was found between chromosomal backgrounds and host plants of origin. The nodulation polyvalence of the species E. meliloti was associated with a high symbiotic heterogeneity. On the basis of PCR-RFLP data from the nifH gene and nifD-K intergenic spacer, E. meliloti strains isolated from non-Medicago legumes harboured distinct genes and possessed wider host ranges. Some strains did not nodulate Medicago species. On the basis of nodA phylogeny, the majority of the Tunisian strains, including strains from Medicago, harboured distinct nodA alleles more related to those found in E. medicae than those found in E. meliloti. However, more work is still needed to characterize this group further. The diversity observed among M. laciniata isolates, which was supported by nodA phylogeny, nifH typing and the efficiency profile on M. ciliaris, indicated that what was thought to be bv. medicaginis is certainly heterogeneous. collapse abstract

Selection of High NitrogenFixing Rhizobia Nodulating Chickpea (Cicer arietinum) for SemiArid Tunisia

Inoculation of grain legumes with rhizobia may improve biological N2 fixation and crop yield. However, drought, high temperature and soil salinity constrain legume root-nodule formation and function. Here, two rhizobial strains nodulating Tunisian ch... expand abstractickpea, Mesorhizobium ciceri strain CMG 6 and Mesorhizobium mediterraneum strain CTM 226 originating from semi-arid regions, were selected for their symbiotic performance and their salt stress tolerance (3 % NaCl). Both strains were then examined as inoculants in different soils and field conditions. Field experiments were conducted in four sites using four chickpea cultivars. Rhizobia occupying nodules in non-inoculated plots were isolated and characterized using 16S rDNA typing; to examine nodule occupancy by the inoculant strains we used polymerase chain reaction (PCR)-restriction fragment length polymorphism of 16S rDNA gene and repetitive extragenic palindromic PCR. The inoculant strains gave a significant increase in nodule number, shoot dry weight and grain yield in all the experimented fields for the four cultivars used, even in the non-irrigated soils. The improvement in plant production was equal to or better than nitrogen fertilization. Moreover, the monitoring of the nodule occupancy showed that inoculant strains competed well in the native populations of rhizobia. These results suggest that nodulation and yield of chickpea can be improved by inoculation with competitive and salt-tolerant rhizobia and is economically promising to increase chickpea production in water-limited regions. collapse abstract

Salt-tolerant rhizobia isolated from a Tunisian oasis that are highly effective for symbiotic N2-fixation with Phaseolus vulgaris constitute a novel biovar (bv. mediterranense) of Sinorhizobium meliloti.

Nodulation of common bean was explored in six oases in the south of Tunisia. Nineteen isolates were characterized by PCR-RFLP of 16S rDNA. Three species of rhizobia were identified, Rhizobium etli, Rhizobium gallicum and Sinorhizobium meliloti. The d... expand abstractiversity of the symbiotic genes was then assessed by PCR-RFLP of nodC and nifH genes. The majority of the symbiotic genotypes were conserved between oases and other soils of the north of the country. Sinorhizobia isolated from bean were then compared with isolates from Medicago truncatula plants grown in the oases soils. All the nodC types except for nodC type p that was specific to common bean isolates were shared by both hosts. The four isolates with nodC type p induced N(2)-fixing effective nodules on common bean but did not nodulate M. truncatula and Medicago sativa. The phylogenetic analysis of nifH and nodC genes showed that these isolates carry symbiotic genes different from those previously characterized among Medicago and bean symbionts, but closely related to those of S. fredii Spanish and Tunisian isolates effective in symbiosis with common bean but unable to nodulate soybean. The creation of a novel biovar shared by S. meliloti and S. fredii, bv. mediterranense, was proposed. collapse abstract

Agrobacterium strains isolated from root nodules of common bean specifically reduce nodulation by Rhizobium gallicum.

In a previous work, we showed that non-nodulating agrobacteria strains were able to colonize root nodules of common bean. Both rhizobia and agrobacteria co-existed in the infected nodules. No impact on symbiosis was found in laboratory conditions whe... expand abstractn using sterile gravel as a support for growth. In this study, soil samples originating from different geographic and agronomic regions in Tunisia were inoculated with a mixture of agrobacteria strains isolated previously from root nodules of common bean. A significant effect on nodulation and vegetal growth of common bean was observed. Characterization of nodulating rhizobia and comparison with non-inoculated controls showed a biased genetic structure. It seemed that Rhizobium gallicum was highly inhibited, whereas nodulation by Sinorhizobium medicae was favored. Co-inoculation of non-sterile soils with R. gallicum and agrobacteria confirmed these findings. In vitro antibiosis assays indicated that agrobacteria exercised a significant antagonism against R. gallicum. collapse abstract

Colonization of Phaseolus vulgaris nodules by Agrobacterium-like strains.

Non-nodulating Agrobacterium-like strains identified among root nodule isolates of common bean were labeled with gusA, a reporter gene encoding beta-glucuronidase (GUS). Bean plants were then co-inoculated with an infective Rhizobium strain and label... expand abstracted transconjugants of Agrobacterium-like strains. Blue staining of nodules showed that Agrobacterium-like strains were able to colonize these symbiotic organs. Isolation and characterization by restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes revealed a mixed population of Rhizobium and Agrobacterium-like strains in all nodules showing GUS activity. PCR amplification of the nifH gene and nodulation tests did not show any evidence of acquisition of symbiotic gene by lateral transfer from Rhizobium to Agrobacterium-like strains. Moreover, these strains were able to invade mature nodules. Based on sequencing of the 16S rRNA gene, one of these Agrobacterium-like strains showed 99.4% sequence similarity with Agrobacterium bv. 1 reference strains and 99% similarity with an Agrobacterium bv. 1 strain isolated from Acacia mollisima in Senegal. Agrobacterium tumefaciens C58 and the disarmed variant AT123 did not show any ability to colonize nodules. Co-inoculation of bean seeds with Agrobacterium and Rhizobium strains did not enhance nodulation and plant yield under controlled conditions. collapse abstract

Different species and symbiotic genotypes of field rhizobia can nodulate Phaseolus vulgaris in Tunisian soils.

Abstract A collection of 160 isolates of rhizobia nodulating Phaseolus vulgaris in three geographical regions in Tunisia was characterized by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified 16S rDNA, nif... expand abstractH and nodC genes. Nine groups of rhizobia were delineated: Rhizobium gallicum biovar (bv.) gallicum, Rhizobium leguminosarum bv. phaseoli and bv. viciae, Rhizobium etli bv. phaseoli, Rhizobium giardinii bv. giardinii, and four groups related to species of the genus Sinorhizobium, Sinorhizobium meliloti, Sinorhizobium medicae and Sinorhizobium fredii. The most abundant rhizobial species were R. gallicum, R. etli, and R. leguminosarum encompassing 29-20% of the isolates each. Among the isolates assigned to R. leguminosarum, two-thirds were ineffective in nitrogen fixation with P. vulgaris and harbored a symbiotic gene typical of the biovar viciae. The S. fredii-like isolates did not nodulate soybean plants but formed numerous effective nodules on P. vulgaris. Comparison of nodC gene sequences showed that their symbiotic genotype was not related to that of S. fredii, but to that of the S. fredii-like reference strain GR-06, which was isolated from a bean plant grown in a Spanish soil. An additional genotype including 16% of isolates was found to be closely related to species of the genus Agrobacterium. However, when re-examined, these isolates did not nodulate their original host. collapse abstract

Genetic diversity of Sinorhizobium populations recovered from different medicago varieties cultivated in Tunisian soils.

A collection of 468 rhizobial isolates was obtained from different ecological areas of Tunisia by trapping them on Medicago sativa cv. Gabes, Medicago scutelleta cv. Kelson, Medicago truncatula, and Medicago ciliaris. A subsample of 134 rhizobia was ... expand abstractchosen to determine their plasmid profile, and 89 isolates were subjected to multilocus enzyme electrophoresis (MLEE) and PCR/RFLP analysis using 16S, IGS (inter genic spacer), and nifKD probes. Twenty-five representatives from these isolates were evaluated for their nodulation and nitrogen fixation capacities. MLEE studies revealed two groups with highly heterogeneous host specificity and geographical origin. The discriminatory power was found to be slightly better with the amplified ribosomal intergenic region, than the nifKD genes. Divisions detected by nifKD amplified DNA analysis matched those established by ribosomal PCR- RFLPs. The comparison between different analyses revealed that MLEE illustrated better phenotypic properties of isolates than PCR-RFLP or plasmid content analysis. Clear distinction between Sinorhizobium meliloti and Sinorhizobium medicae were observed by analysis of the IGS symbiotic regions between nifD and nifK genes. Were able to distinguish three inoculation groups; isolates trapped from M. sativa cv. Gabes and M. scutelleta cv. Kelson formed one inoculation group which was more closely related to isolates trapped from M. truncatula than those trapped from M. ciliaris. collapse abstract

Potential for inoculation of common bean in Tunisian soils

Nodulation and growth of common bean under NaCl stress

Characterization of rhizobia nodulating chickpea in Tunisia

Distribution and genetic diversity of rhizobia nodulating natural populations of Medicago truncatula in Tunisian soils

Competitiveness and symbiotic effectiveness of a R. gallicum strain isolated from root nodules of Phaseolus vulgaris

Nodulation and growth of common bean (Phaseolus vulgaris) under water deficiency

Rhizobium gallicum as an efficient symbiont for bean cultivation

The diversity of rhizobia nodulating chickpea (Cicer arietinum) under water deficiency as a source of more efficient inoculants

Diversity of S. meliloti and S. medicae nodulating Medicago truncatula according to host and soil origin

Genotypic diversity and symbiotic effectiveness of rhizobia isolated from root nodules of Phaseolus vulgaris grown in Tunisian soils

Legume rhizobia symbiotic interactionunder salt and drought constraints: generation of reactive oxygen species and protective role of antioxydant enzymzs within nodules

Effet du sel sur des isolats de Sinorhizobium de Tunisie in vitro ou en association avec Medicago

Diversité génétique et polymorphysme symbiotique de Sinorhizobium meliloti nodulant Medicago truncatula en sols Tunisiens des régions arides

Nodulation, biomass production, and diversity of rhizobia nodulating chickpea under water deficiency

The evaluation of a salt-tolerant strain nodulating common bean isolated from the oases