Phylogenetic and symbiotic characterization of rhizobial bacteria nodulating Argyrolobium uniflorum in Tunisian arid soils

Forty-two bacterial isolates from root nodules of Argyrolobium uniflorum growing in the arid areas of Tunisia were characterized by phenotypic features, RFLP, and sequencing of PCR-amplified 16S rRNA genes. The isolates were found to be phenotypically diverse. The majority of the isolates tolerated 3% NaCl and grew at temperatures up to 40 degrees C. Phylogenetically, the new isolates were grouped in the genera Sinorhizobium (27), Rhizobium (13), and Agrobacterium (2). Except for the 2 Agrobacterium isolates, all strains induced nodulation on Argyrolobium uniflorum, but the number of nodules and nitrogen fixation efficiency varied among them. Sinorhizobium sp. strains STM 4034, STM 4036, and STM 4039, forming the most effective symbiosis, are potential candidates for inoculants in revegetalisation programs.     

Characterization of rhizobia nodulating Lotus subbiflorus from Uruguayan soils

Generation times, acid production, carbon utilization, immunological properties, plasmid content, protein profile and symbiotic properties of 15 isolates of rhizobia nodulating Lotus subbiflorus were studied. Based on specific growth rates, carbon source utilization and acid production, 13 out of the 15 isolates could be assigned to the slow-growing group of rhizobia (bradyrhizobia). Using antisera against whole cells of three isolates, we separated the 15 isolates into three serogroups. Only the slow-growing isolate Ls4 and the fast-growers Ls5 and Ls552 lacked cross-reactivity with any of the sera tested. Electrophoretic mobilities of whole cell protein from seven out of the eight isolates included in the serogroup represented by strain Ls31 were identical. Similarly, isolates Ls1B3 and Ls1B4, both in serogroup Ls1B3, had the same pattern of cell proteins. In contrast, isolates Ls3 and Ls7, belonging to serogroup Ls7, differed in protein profile. Plant growth experiments carried out under bacteriologically controlled conditions revealed that all of the isolates effectively nodulated L. subbiflorus and L. pedunculatus, but were unable to form effective nodules on L. tenuis and L. corniculatus. All isolates showed similar effectiveness in symbiosis with L. subbiflorus, except isolate Ls7, which gave significantly higher plant dry weight.Abbreviations ELISA enzyme linked immuno-sorbent assay - kDa kiloDalton - MM mineral medium - PBS phosphate-buffered saline - RE relative efficiency - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - YEM deyeast extract mannitol     

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

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, nifH 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.     

Genetic diversity,symbiotic efficiency,stress tolerance,and plant growth promotion traits of rhizobia nodulating Vachellia tortilis subsp. raddiana growing in dryland soils in southern Morocco

In the present study, we analyzed the genetic diversity, phylogenetic relationships, stress tolerance, phytobeneficial traits, and symbiotic characteristics of rhizobial strains isolated from root nodules of Vachellia tortilis subsp. raddiana grown in soils collected in the extreme Southwest of the Anti-Atlas Mountains in Morocco. Subsequent to Rep-PCR fingerprinting, 16S rDNA gene sequencing of 15 representative strains showed that all of them belong to the genus Ensifer. Phylogenetic analysis and concatenation of the housekeeping genes gyrB, rpoB, recA, and dnaK revealed that the entire collection (except strain LMR678) shared 99.08 % to 99.92% similarity with Ensifer sp. USDA 257 and 96.92% to 98.79% with Sinorhizobium BJ1. Phylogenetic analysis of nodC and nodA sequences showed that all strains but one (LMR678) formed a phylogenetic group with the type strain “E. aridi” LMR001^T (similarity over 98%). Moreover, it was relevant that most strains belong to the symbiovar vachelliae. In vitro tests revealed that five strains produced IAA, four solubilized inorganic phosphate, and one produced siderophores. All strains showed tolerance to NaCl concentrations ranging from 2 to 12% and grew at up to 10% of PEG6000. A greenhouse plant inoculation test conducted during five months demonstrated that most rhizobial strains were infective and efficient. Strains LMR688, LMR692, and LMR687 exhibited high relative symbiotic efficiency values (respectively 231.6 %, 171.96 %, and 140.84 %). These strains could be considered as the most suitable candidates for inoculation of V. t. subsp. raddiana, to be used as a pioneer plant for restoring arid soils threatened with desertification.     

The Lotus japonicus nucleoporin GLE1 is involved in symbiotic association with rhizobia

Nucleoporins are components of the nuclear pore complexes, channels that regulate the transport of macromolecules between the nucleus and cytoplasm. The nucleoporin GLE1 (GLFG lethal1) functions in the export of messenger RNAs containing poly(A) tails from the nucleus into the cytoplasm. Here we investigated a mutant of the model legume Lotus japonicus that was defective in GLE1, which we designated Ljgle1. The growth of Ljgle1 was retarded under symbiotic association with rhizobia, and the nitrogen-fixation activities of the nodules were around one-third of those in the wild-type plant. The growth of Ljgle1 was not substantialy recovered by supplemention of combined nitrogen. Nodules formed on the Ljgle1 were smaller than those on the wild-type and colored faint pink. The numbers of infected cells of nodules on the Ljgle1 were smaller than on the wild-type plant, and the former cells remained undeveloped. Rhizobia in the cells of the Ljgle1 exhibited disordered forms, and the symbiosome membrane was closely attached to the bacterial membrane. These results indicate that GLE1 plays a distinct role in the symbiotic association between legumes and rhizobia.     

Phenotypic and molecular characterization of indigenous rhizobia nodulating chickpea in India

In a combined approach of phenotypic and genotypic characterization, 28 indigenous rhizobial isolates obtained from different chickpea growing regions in peninsular and northern India were analyzed for diversity. The field isolates were compared to two reference strains TAL620 and UPM-Ca142 representing M. ciceri and M. mediterraneum respectively. Phenotypic markers such as resistance to antibiotics, tolerance to salinity, temperature, pH, phosphate solubilization ability, growth rate and also symbiotic efficiency showed considerable diversity among rhizobial isolates. Their phenotypic patterns showed adaptations of rhizobial isolates to abiotic stresses such as heat and salinity. Two salt tolerant strains (1.5% NaCl by T1 and T4) with relatively high symbiotic efficiency and two P-solubilising strains (66.7 and 71 microg/ml by T2 and T5) were identified as potential bioinoculants. Molecular profiling by 16S ribosomal DNA Restriction Fragment Length Polymorphism (RFLP) revealed three clusters at 67% similarity level. Further, the isolates were differentiated at intraspecific level by 16S rRNA gene phylogeny. Results assigned all the chickpea rhizobial field isolates to belong to three different species of Mesorhizobium genus. 46% of the isolates grouped with Mesorhizobium loti and the rest were identified as M. ciceri and M. mediterraneum, the two species which have been formerly described as specific chickpea symbionts. This is the first report on characterization of chickpea nodulating rhizobia covering soils of both northern and peninsular India. The collection of isolates, diverse in terms of species and symbiotic effectiveness holds a vast pool of genetic material which can be effectively used to yield superior inoculant strains.     

Phenotypic characteristics of rhizobia isolates nodulating acacia species in the arid and Saharan regions of Morocco

The phenotypic characteristics of 48 isolates obtained from root nodules of four Acacia species (Acacia cyanophylla, A. gummifera, A. horrida and A. raddiana) growing in soils collected from the arid and Saharan regions of Morocco were studied. The rhizobia were very diverse with respect to their cross-nodulation patterns, as well as their physiological and biochemical properties. Dendrograms obtained through computer numerical analysis of 52 phenotypic characteristics showed that isolates could fit into four clusters below the boundary level of 0.85 average distance and that they were very distinct from the reference strains. Some interesting isolates for inoculation trials have been identified. They were able to grow at pH ranging from 4 to 9, tolerate a high salt concentration (3% NaCl) and grew at a maximum temperature between 35 and 40 degrees C.     

Genotypic and symbiotic diversity of native rhizobia nodulating red pea (Lathyrus cicera L.) in Tunisia

Nodulation and genetic diversity of native rhizobia nodulating Lathyrus cicera plants grown in 24 cultivated and marginal soils collected from northern and central Tunisia were studied. L. cicera plants were nodulated and showed the presence of native rhizobia in 21 soils. A total of 196 bacterial strains were selected and three different ribotypes were revealed after PCR-RFLP analysis. The sequence analysis of the rrs and two housekeeping genes (recA and thrC) from 36 representative isolates identified Rhizobium laguerreae as the dominant (53%) rhizobia nodulating L. cicera. To the best of our knowledge, this is the first time that this species has been reported among wild populations of the rhizobia-nodulating Lathyrus genus. Twenty-five percent of the isolates were identified as R. leguminosarum and isolates LS11.5, LS11.7 and LS8.8 clustered with Ensifer meliloti. Interestingly, five isolates (LS20.3, LS18.3, LS19.10, LS1.2 and LS21.20) were segregated from R. laguerreae and clustered as a separate clade. These isolates possibly belong to new species. According to nodC and nodA phylogeny, strains of R. laguerreae and R. leguminosarum harbored the symbiotic genes of symbiovar viciae and clustered in three different clades showing heterogeneity within the symbiovar. Strains of E. meliloti harbored symbiotic genes of Clade V and induced inefficient nodules.     

Diversity in symbiotic specificity of cowpea rhizobia indigenous to Zimbabwean soils

Tropical cowpea rhizobia are often presumed to be generally promiscuous but poor N fixers. This study was conducted to evaluate symbiotic interactions of 59 indigenous rhizobia isolates (49 of them from cowpea (Vigna unguiculata)), with up to 13 other (mostly tropical) legume species. Host ranges averaged 2.4 and 2.3 legume species each for fast- and slow-growing isolates respectively compared to 4.3 for slow-growing reference cowpea strains. An average of 22% and 19% of fast- and slow-growing cowpea isolates respectively were effective on each of 12 legume species tested. We conclude that the indigenous cowpea rhizobia studied have relatively narrow host ranges. The ready nodulation of different legumes in tropical soils appears due to the diversity of indigenous symbiotic genotypes, each consisting of subgroups compatible with a limited number of legume species.     

Distribution and diversity of rhizobia nodulating agroforestry legumes in soils from three continents in the tropics

The natural rhizobial populations of Calliandra calothyrsus, Gliricidia sepium, Leucaena leucocephala and Sesbania sesban were assessed in soils from nine sites across tropical areas of three continents. The rhizobial population size varied from undetectable numbers to 1.8 x 104 cells/g of soil depending on the trap host and the soil. Calliandra calothyrsus was the most promiscuous legume, nodulating in eight soils, while S. sesban nodulated in only one of the soils. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analyses of the 16S rRNA gene and the internally transcribed spacer (ITS) region between the 16S and 23S rRNA genes were used to assess the diversity and relative abundance of rhizobia trapped from seven of the soils by C. calothyrsus, G. sepium and L. leucocephala. Representatives of the 16S rRNA RFLP groups were also subjected to sequence analysis of the first 950 base pairs of the 16S rRNA gene. Eighty ITS groups were obtained, with none of the ITS types being sampled in more than one soil. RFLP analysis of the 16S rRNA yielded 23 'species' groups distributed among the Rhizobium, Mesorhizobium, Sinorhizobium and Agrobacterium branches of the rhizobial phylogenetic tree. The phylogeny of the isolates was independent of the site or host of isolation, with different rhizobial groups associated with each host across the soils from widely separated geographical regions. Although rhizobial populations in soils sampled from the centre of diversity of the host legumes were the most genetically diverse, soil acidity was highly correlated with the diversity of ITS types. Our results support the hypothesis that the success of these tree legumes in soils throughout the tropics is the result of their relative promiscuity (permissiveness) allowing nodulation with diverse indigenous rhizobial types.     

Comparison of rhizobia that nodulate Medicago laciniata and Medicago truncatula present in a single Tunisian arid soil

The rhizobia present in a single arid region Tunisian soil that nodulate Medicago laciniata and Medicago truncatula were compared. All isolates, 40 from each host, were Sinorhizobium meliloti based on 16S rRNA polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) patterns and subsequent confirmation by sequence analysis of the 16S rRNA genes in four representatives from each host species. There was no apparent relationship between Medicago host species of isolation and the nodulating rhizobial genome as determined by repetitive extragenic palandromic PCR. The isolates of M. laciniata were distinguished from those of M. truncatula present in the same soil by variation in PCR-RFLP of nifDK, indicating that this dissimilarity is originally genetic and not geographic. While forming effective symbioses with their own respective isolates, both M. laciniata and M. truncatula formed ineffective true nodules, nodule-like structures, or no nodules at all in cross-inoculation tests, as confirmed by the histological observations.     

Genotypic and phenotypic characterization of rhizobia that nodulate snap bean (Phaseolus vulgaris L.) in Egyptian soils

Snap bean fields in 12 of the 25 governorates of Egypt were surveyed to determine the distribution and taxonomy of snap bean-nodulating rhizobia. Nodulation rates in the field were very low, indicating that Egyptian soils do not have sufficient numbers of snap bean-compatible Rhizobium spp. A total of 87 rhizobial isolates were assayed on the most commonly grown cultivars in order to identify the most effective strains. The five most effective isolates (R11, R13, R28, R49 and R52) were fast-growing and utilized a wide range of carbon and nitrogen sources. A phylogenetic assignment of these strains by analysis of the 16S ribosomal RNA gene suggested that all fell within the Rhizobium etli–Rhizobium leguminosarum group. Strains R11, R49 and R52 all clustered with other identified R. etli strains, while strains R13 and R28 were more distinct. The distinctness of R13 and R28 was supported by physiological characteristics, such as their ability to utilize citrate, erythritol, dulcitol and lactate. Strains R13 and R28 also yielded the highest plant nitrogen content of all isolates.The highly effective strains isolated in this study, in particular strains R13 and R28, are promising candidates for improving crop yields. The data also suggested that these two strains represented a novel sub-group within the R. etli–R. leguminosarum group. As snap bean is a crop of great economic value to Egypt, the identification of highly effective rhizobial strains adapted to Egyptian soils, such as strains R13 and R28, is of great interest.     

Molecular and phylogenetic characterization of potentially toxic cyanobacteria in Tunisian freshwaters

This study presents a genetic characterization of 27 potentially toxic cyanobacterial strains isolated from seven reservoirs located in the north and centre of Tunisia. These strains belonged mainly to Microcystis aeruginosa, Cylindrospermopsis raciborskii and Planktothrix agardhii species. Their toxicological potential was evaluated by molecular biology tools, which showed that none of the isolated strains carried segments of the gene cluster responsible for the production of cylindrospermopsin and saxitoxin. The majority of Microcystis isolates were able to synthesize microcystin, since they presented the six characteristic segments of the microcystin synthetase mcy cluster (mcyA, -B, -C, -D, -E and -G). This was further confirmed by MALDI-TOF analysis that showed the presence of eight microcystin variants, including microcystin-LR. The taxonomic identification of the strains was assessed based on the variability of the 16S rRNA gene sequences. Furthermore, the 16S-23S rRNA ITS sequences of Microcystis isolates and rpoC1 sequences of Cylindrospermopsis strains were also used in the phylogenetic analysis.     

Metabolic properties, stress tolerance and macromolecular profiles of rhizobia nodulating Hedysarum coronarium

The drought-tolerant legume Hedysarum coronarium is a Mediterranean species valued as a forage crop for its high performance in stressful conditions. The plant shows peculiar capabilities of nodulating above pH 9 and thriving in highly calcareous soils. With the aim of providing an adequate characterization of its bacterial symbiotic partner, a study was undertaken, approaching from several viewpoints the physiology and structural features of bacteria isolated from nodules of H. coronarium . Tests involved trophic capabilities on different carbon and nitrogen sources, vitamin requirements, and resistance to factors including antibiotics, heavy metals, salinity, pH, and temperature. Enzyme activities, including those of cellulase, pectinase, urease, β-galactosidase, nitrate and nitrite reductase, were evaluated. The DNA G + C percentage content was determined. Species-specific bacteriophages were isolated and a strain-typing grid established. In order to characterize further and fingerprint the different Rhizobium 'hedysari' isolates, electrophoretic pattern of proteins, plasmid DNA, and digested genomic DNA (in pulsed-field gel separation) were compared. Adansonian taxonomy yielded similarity clusters of the different isolates.     

Genotypic and symbiotic diversity studies of rhizobia nodulating Acacia saligna in Tunisia reveal two novel symbiovars within the Rhizobium leguminosarum complex and Bradyrhizobium

Acacia saligna is an invasive alien species that has the ability to establish symbiotic relationships with rhizobia. In the present study, genotypic and symbiotic diversity of native rhizobia associated with A. saligna in Tunisia were studied. A total of 100 bacterial strains were selected and three different ribotypes were identified based on rrs PCR-RFLP analysis. Sequence analyses of rrs and four housekeeping genes (recA, atpD, gyrB and glnII) assigned 30 isolates to four putative new lineages and a single strain to Sinorhizobium meliloti. Thirteen slow-growing isolates representing the most dominant IGS (intergenic spacer) profile clustered distinctly from known rhizobia species within Bradyrhizobium with the closest related species being Bradyrhizobium shewense and Bradyrhizobium niftali, which had 95.17% and 95.1% sequence identity, respectively. Two slow-growing isolates, 1AS28L and 5AS6L, had B. frederekii as their closest species with a sequence identity of 95.2%, an indication that these strains could constitute a new lineage. Strains 1AS14I, 1AS12I and 6AS6 clustered distinctly from known rhizobia species but within the Rhizobium leguminosarum complex (Rlc) with the most closely related species being Rhizobium indicum with 96.3% sequence identity. Similarly, the remaining 11 strains showed 96.9 % and 97.2% similarity values with R. changzhiense and R. indicum, respectively. Based on nodC and nodA phylogenies and cross inoculation tests, these 14 strains of Rlc species clearly diverged from strains of Sinorhizobium and Rlc symbiovars, and formed a new symbiovar for which the name sv. “salignae” is proposed. Bacterial strains isolated in this study that were taxonomically assigned to Bradyrhizobium harbored different symbiotic genes and the data suggested a new symbiovar, for which sv. “cyanophyllae” is proposed. Isolates formed effective nodules on A. saligna.     

Metabolic properties, stress tolerance and macromolecular profiles of rhizobia nodulating Hedysarum coronarium

The drought-tolerant legume Hedysarum coronarium is a Mediterranean species valued as a forage crop for its high performance in stressful conditions. The plant shows peculiar capabilities of nodulating above pH 9 and thriving in highly calcareous soils. With the aim of providing an adequate characterization of its bacterial symbiotic partner, a study was undertaken, approaching from several viewpoints the physiology and structural features of bacteria isolated from nodules of H. coronarium. Tests involved trophic capabilities on different carbon and nitrogen sources, vitamin requirements, and resistance to factors including antibiotics, heavy metals, salinity, pH, and temperature. Enzyme activities, including those of cellulase, pectinase, urease, beta-galactosidase, nitrate and nitrite reductase, were evaluated. The DNA G + C percentage content was determined. Species-specific bacteriophages were isolated and a strain-typing grid established. In order to characterize further and fingerprint the different Rhizobium 'hedysari' isolates, electrophoretic pattern of proteins, plasmid DNA, and digested genomic DNA (in pulsed-field gel separation) were compared. Adansonian taxonomy yielded similarity clusters of the different isolates.     

The novel symbiotic phenotype of enhanced-nodulating mutant of Lotus japonicus: astray mutant is an early nodulating mutant with wider nodulation zone

We have isolated a novel enhanced-nodulating mutant astray (Ljsym77) from Lotus japonicus. The name astray derives from the non-symbiotic phenotype of this mutant, agravitropic lateral roots that go "astray" against gravity. In this report we evaluated the symbiotic aspects of this mutant in detail. The astray mutant developed approximately twice the number of nodules on a wider area of roots compared with the wild type. Furthermore, the astray mutant demonstrated early initiation of nodule development, which is an unprecedented symbiotic phenotype. The astray seedlings showed normal sensitivity to the general inhibitors of nodulation such as ethylene and nitrate. These results indicate that the astray mutant is distinct from the hypernodulating mutants reported previously, and that the ASTRAY gene acts as an early and negative regulator in the cascade of nodule development.     

Molecular and phenotypic characterization of strains nodulating Anthyllis vulneraria in mine tailings, and proposal of Aminobacter anthyllidis sp. nov., the first definition of Aminobacter as legume-nodulating bacteria

Bacterial strains from Zn-Pb mine tailings were isolated by trapping with Anthyllis vulneraria, a legume-host suitable for mine substratum phytostabilisation. Sequence analysis of the 16S rRNA gene and three housekeeping genes (atpD, dnaK and recA) showed that they were related to those of the genus Aminobacter. DNA-DNA relatedness of representative isolates supported the placement of novel strains in Aminobacter as a new species. Phenotypic data emphasize their differentiation from the other related species of Aminobacter and Mesorhizobium. Aminobacter isolates exhibited nodA sequences tightly related with M. loti as the closest nodA relative. By contrast, their nodA sequences were highly divergent from those of M. metallidurans, another species associated with A. vulneraria that carries two complete copies of nodA. Therefore, the novel bacterial strains efficient on A. vulneraria represented the first occurrence of legume symbionts in the genus Aminobacter. They represent a new species for which the name Aminobacter anthyllidis sp. nov. is proposed (type strain STM4645(T)=LMG26462(T)=CFBP7437(T)).