Rhizobium Sullae Research Articles

Promoting the growth of Sulla flexuosa L. by endophytic root nodule bacteria authors and affiliations.

Legume plants rely upon multipartite interactions between rhizobia and bacterial endophytes within root nodules to facilitate plant growth. This study aimed to isolate and identify indigenous endophytic bacteria from root nodules of Sulla aculeolata L. in Northeast Morocco. Based on their tri-calcium phosphate (TCP) solubilization capacity, five endophytes were chosen for further evaluation of their plant growth traits. All isolates were hydrogen cyanide (HCN) and siderophore producers, while only BCH24 tested positive for ACC deaminase activity. Indole-3-acetic acid (IAA) synthesis ranged from 1.27 mgL- 1 to 2.89 mgL- 1, while soluble phosphate concentrations was between 7.99mg L- 1 and 110.58mg L- 1. Additionally, all the endophytes were able to produce more than two lytic enzymes. Based on the analysis of 16S rRNA gene sequences five isolates were identified as Enterobacter sp (BCH13, BCH2), Pseudomonas sp (BCH16, BCH24), and Serratia sp (BCH10). The strains inhibited the growth of three phytopathogenic fungi, with BCH13 exhibiting the highest rate against Aspergillus ochraceus (45%), followed by BCH24 against Fusarium oxysporum (40%) and Botrytis cinerea (35%), respectively. In vivo inoculation of halotolerant strains Enterobacter hormaechei (BCH13) and Pseudomonas moraviensis (BCH16) under gnotobiotic conditions revealed that co-inoculation with Rhizobium sullae KS6 improved plant development compared to single inoculation, making it a promising eco-friendly bio-inoculant for legume Sulla flexuosa L. production.

Sorption–Desorption of Imazamox and 2,4-DB in Acidic Mediterranean Agricultural Soils and Herbicide Impact on Culturable Bacterial Populations and Functional Diversity

In this study, we investigated the sorption–desorption behavior of imazamox (IMZ) and 2,4-DB (DB) in two typical acidic Mediterranean agricultural soils and the impact of these herbicides on culturable soil bacterial populations, enzyme activities and functional diversity when applied at concentrations higher than recommended doses (10×, 50×, 500×). Herbicide sorption was similar in both soils and IMZ was less retained compared to DB (~0.5 vs. 40 µg g−1 soil, respectively). IMZ desorption was remarkable (70–100%) while that of DB was more limited, i.e., ~40%. Three days after spiking (DAS), IMZ and DB significantly increased the number of soil-culturable heterotrophic bacteria, actinomycetes and Pseudomonas spp., soil respiration and the potential catabolic capacity of soil microbial communities. Soil dehydrogenase activity increased by ~56–70% in IMZ-treated soils while being reduced by ~33–41% in DB-treated ones. β-glucosidase activity showed a soil-dependent behavior, while the pattern of C source utilization suggested a change of soil microbial community structure after herbicide (especially DB) spiking. At 30 DAS, the herbicides’ impact on soil microorganisms, enzyme activity and functional diversity was still visible. Moreover, a toxic effect of DB (at 50× and 500×), but not IMZ, was recorded vs. Rhizobium sullae, the bacterial symbiont of Hedysarum coronarium. The obtained results indicated that IMZ and DB are poorly sorbed and highly desorbed by both soils. Moreover, at the tested concentrations, IMZ and DB can have short- and medium-term impacts on the microbial component and the related activity of the investigated soils, likely affecting a range of ecosystem services provided by soil microorganisms.

Potential of Rhizobium sullae–Sulla coronaria Symbiotic Biological Nitrogen Fixation to Supplement Synthetic Mineral Nitrogen in Olive Tree Fertilization

The aim of the present work is to compare olive tree nitrogen fertilization over two years of trials, using synthetic chemical fertilizers along with organic fertilizers composed of the green manure of sulla (Sulla coronaria) inoculated with the symbiont Rhizobium sullae or left uninoculated. The tests indicated that symbiotic nitrogen fixation promoted by the sulla–R. sullae symbiosis represents an important source of nitrogen that can replace or supplement synthetic nitrogen fertilizers for olive tree cultivation when sulla is inoculated with R. sullae in a soil already populated by the symbiont. Integration of the indigenous population of R. sullae via sulla inoculation with a selected strain yielded nodule formation in 100% of plants and produced a sufficient amount of biomass rich in nitrogen with a low C/N ratio. On the contrary, olive tree fertilization using the green manure of sulla that was not inoculated with the symbiont supplied significantly less organic nitrogen in 2017 and 2018, respectively, compared to the control. Optimal management of the multi-factorial approaches involved in green manure olive fertilization are also reported.

Co-inoculation Effects of Rhizobium sullae and Pseudomonas sp. on Growth, Antioxidant Status, and Expression Pattern of Genes Associated with Heavy Metal Tolerance and Accumulation of Cadmium in Sulla coronaria

Recently, phytoremediation assisted by soil bacteria has emerged as a potential tool to clean up the metal-contaminated/polluted environment. Three plant-growth-promoting bacteria (PGPBs): Rhizobium sullae, Pseudomonas fluorescens, and Pseudomonas sp. were found to tolerate cadmium (Cd) stress. Sulla coronaria inoculated with these PGPBs, and grown under different Cd concentrations (0, 100, and 200 µM), showed increases in dry biomass and proline content. Notable increases in different gas-exchange characteristics such as photosynthesis rate (A), transpiration rate (E), and water-use efficiency (WUE), as well as increases in nitrogen (N) and Cd accumulations were also recorded in inoculated plants compared to non-inoculated Cd stressed plants. The activities of antioxidant enzymes superoxide dismutase (SOD), guaiacol peroxidase (GPOX), catalase (CAT), and ascorbate peroxidase (APX) in S. coronaria roots increased under Cd stress after PGPB co-inoculation, suggesting that these PGPB species could be used for amelioration of stress tolerance in S. coronaria. The expression patterns of ScPCS, ScMT, ScF-box, ScGR, and ScGST in roots of S. coronaria indicated that these genes are differentially expressed under Cd treatments, suggesting their possible roles in Cd and heavy metal stress responses. The results indicate that co-inoculation with R. sullae and Pseudomonas sp. could alleviate Cd toxicity in S. coronaria. In the present study, the obtained data suggest that the application of PGPBs could be a promising strategy for enhancing the phytostabilization efficiency of Cd-contaminated soils.

Nodule-associated microbiome diversity in wild populations of Sulla coronaria reveals clues on the relative importance of culturable rhizobial symbionts and co-infecting endophytes

The culturable bacteria from root nodules of Sulla coronaria growing in spontaneous conditions in Sardinia were characterized. This plant’s peculiarity is to represent a legume still found in both wild and cropped statuses. We tested whether culturable bacteria would differ from those commonly isolated from its field-cropped varieties, to date exclusively represented by Rhizobium sullae. 63 isolates from 60 surface-sterilized nodules were analyzed by ARDRA and 16S rDNA sequencing. The official nitrogen-fixing symbiont Rhizobium sullae was found only in 25 nodules out of 60. The remaining nodules did not yield culturable rhizobia but a number of different endophytic genera including Pseudomonas sp. (17 nodules), Microbacterium sp. (15 nodules), Pantoea agglomerans (5 nodules). The situation appears therefore a hybrid between what is commonly observed in other Mediterranean legumes occurring only in wild status (featuring non-culturable rhizobia and arrays of culturable endophytes within nodules), as opposed to cropped legumes (endowed with fully culturable rhizobia and minimal endophytic occurrence). These findings, within a species bridging the ecology between native and cropped conditions, suggest insights on the relative importance of endophytic co-occupancy vs. true N-fixing symbiont culturability within nodules. The latter trait thus appears to accompany the domestication path of plants with a main trade-off of renouncing to interactions with a diversity of endophytic co-invaders; the relationships with those being critical in the non-cropped status. In fact, endophytes are known to promote plant growth in harsh conditions, which can be particularly stressful in the Mediterranean due to drought, highly calcareous soils, and pathogens outbreaks.

Physiological responses and antioxidant enzyme changes in Sulla coronaria inoculated by cadmium resistant bacteria.

Plant growth promoting bacteria (PGPB) may help to reduce the toxicity of heavy metals on plants growing in polluted soils. In this work, Sulla coronaria inoculated with four Cd resistant bacteria (two Pseudomonas spp. and two Rhizobium sullae) were cultivated in hydroponic conditions treated by Cd; long time treatment 50µM CdCl2 for 30days and short time treatment; 100µM CdCl2 for 7days. Results showed that inoculation with Cd resistant PGPB enhanced plant biomass, thus shoot and root dry weights of control plants were enhanced by 148 and 35% respectively after 7days. Co-inoculation of plants treated with 50 and 100µM Cd increased plant biomasses as compared to Cd-treated and uninoculated plants. Cadmium treatment induced lipid peroxidation in plant tissues measured through MDA content in short 7days 100µM treatment. Antioxidant enzyme studies showed that inoculation of control plants enhanced APX, SOD and CAT activities after 30days in shoots and SOD, APX, SOD, GPOX in roots. Application of 50µM CdCl2 stimulated all enzymes in shoots and decreased SOD and CAT activities in roots. Moreover, 100µM of CdCl2 increased SOD, APX, CAT and GPOX activities in shoots and increased significantly CAT activity in roots. Metal accumulation depended on Cd concentration, plant organ and time of treatment. Furthermore, the inoculation enhanced Cd uptake in roots by 20% in all treatments. The cultivation of this symbiosis in Cd contaminated soil or in heavy metal hydroponically treated medium, showed that inoculation improved plant biomass and increased Cd uptake especially in roots. Therefore, the present study established that co-inoculation of S. coronaria by a specific consortium of heavy metal resistant PGPB formed a symbiotic system useful for soil phytostabilization.

Draft Genome Sequence of the Nitrogen-Fixing Rhizobium sullae Type Strain IS123T Focusing on the Key Genes for Symbiosis with its Host Hedysarum coronarium L.

The prominent feature of rhizobia is their molecular dialogue with plant hosts. Such interaction is enabled by the presence of a series of symbiotic genes encoding for the synthesis and export of signals triggering organogenetic and physiological responses in the plant. The genome of the Rhizobium sullae type strain IS123T nodulating the legume Hedysarum coronarium, was sequenced and resulted in 317 scaffolds for a total assembled size of 7,889,576 bp. Its features were compared with those of genomes from rhizobia representing an increasing gradient of taxonomical distance, from a conspecific isolate (Rhizobium sullae WSM1592), to two congeneric cases (Rhizobium leguminosarum bv. viciae and Rhizobium etli) and up to different genera within the legume-nodulating taxa. The host plant is of agricultural importance, but, unlike the majority of other domesticated plant species, it is able to survive quite well in the wild. Data showed that that the type strain of R. sullae, isolated from a wild host specimen, is endowed with a richer array of symbiotic genes in comparison to other strains, species or genera of rhizobia that were rescued from domesticated plant ecotypes. The analysis revealed that the bacterium by itself is incapable of surviving in the extreme conditions that its host plant can tolerate. When exposed to drought or alkaline condition, the bacterium depends on its host to survive. Data are consistent with the view of the plant phenotype as the primary factor enabling symbiotic nitrogen fixing bacteria to survive in otherwise limiting environments.

Specificity in Legume-Rhizobia Symbioses.

Most species in the Leguminosae (legume family) can fix atmospheric nitrogen (N2) via symbiotic bacteria (rhizobia) in root nodules. Here, the literature on legume-rhizobia symbioses in field soils was reviewed and genotypically characterised rhizobia related to the taxonomy of the legumes from which they were isolated. The Leguminosae was divided into three sub-families, the Caesalpinioideae, Mimosoideae and Papilionoideae. Bradyrhizobium spp. were the exclusive rhizobial symbionts of species in the Caesalpinioideae, but data are limited. Generally, a range of rhizobia genera nodulated legume species across the two Mimosoideae tribes Ingeae and Mimoseae, but Mimosa spp. show specificity towards Burkholderia in central and southern Brazil, Rhizobium/Ensifer in central Mexico and Cupriavidus in southern Uruguay. These specific symbioses are likely to be at least in part related to the relative occurrence of the potential symbionts in soils of the different regions. Generally, Papilionoideae species were promiscuous in relation to rhizobial symbionts, but specificity for rhizobial genus appears to hold at the tribe level for the Fabeae (Rhizobium), the genus level for Cytisus (Bradyrhizobium), Lupinus (Bradyrhizobium) and the New Zealand native Sophora spp. (Mesorhizobium) and species level for Cicer arietinum (Mesorhizobium), Listia bainesii (Methylobacterium) and Listia angolensis (Microvirga). Specificity for rhizobial species/symbiovar appears to hold for Galega officinalis (Neorhizobium galegeae sv. officinalis), Galega orientalis (Neorhizobium galegeae sv. orientalis), Hedysarum coronarium (Rhizobium sullae), Medicago laciniata (Ensifer meliloti sv. medicaginis), Medicago rigiduloides (Ensifer meliloti sv. rigiduloides) and Trifolium ambiguum (Rhizobium leguminosarum sv. trifolii). Lateral gene transfer of specific symbiosis genes within rhizobial genera is an important mechanism allowing legumes to form symbioses with rhizobia adapted to particular soils. Strain-specific legume rhizobia symbioses can develop in particular habitats.

Characterization of efficient plant-growth-promoting bacteria isolated from Sulla coronaria resistant to cadmium and to other heavy metals

The inoculation of plants with plant-growth-promoting rhizobacteria has become a priority in the phytoremediation of heavy-metal-contaminated soils. A total of 82 bacteria were isolated from Sulla coronaria root nodules cultivated on four soil samples differently contaminated by heavy metals. The phenotypic characterization of these isolates demonstrated an increased tolerance to cadmium reaching 4.1mM, and to other metals, including Zn, Cu and Ni. Polymerase Chain Reaction/Restriction Fragment Length Polymorphism (PCR/RFLP) analysis showed a large diversity represented by genera related to Agrobacterium sp., R. leguminosarum, Sinorhizobium sp., Pseudomonas sp., and Rhizobium sp. Their symbiotic effectiveness was evaluated by nodulation tests. Taking into consideration efficiency and cadmium tolerance, four isolates were chosen; their 16SrRNA gene sequence showed that they belonged to Pseudomonas sp. and the Rhizobium sullae. The selected consortium of soil bacteria had the ability to produce plant-growth-promoting substances such as indole acetic acid and siderophore. The intracellular Cd accumulation was enhanced by increasing the time of incubation of the four soil bacteria cultivated in a medium supplemented with 0.1mM Cd. The existence of a cadmium-resistant gene was confirmed by PCR. These results suggested that Sulla coronaria in symbiosis with the consortium of plant-growth-promoting rhizobacteria (PGPR) could be useful in the phytoremediation of cadmium-contaminated soils.

High-quality permanent draft genome sequence of Rhizobium sullae strain WSM1592; a Hedysarum coronarium microsymbiont from Sassari, Italy.

Rhizobium sullae strain WSM1592 is an aerobic, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen (N2) fixing root nodule formed on the short-lived perennial legume Hedysarum coronarium (also known as Sulla coronaria or Sulla). WSM1592 was isolated from a nodule recovered from H. coronarium roots located in Ottava, bordering Sassari, Sardinia in 1995. WSM1592 is highly effective at fixing nitrogen with H. coronarium, and is currently the commercial Sulla inoculant strain in Australia. Here we describe the features of R. sullae strain WSM1592, together with genome sequence information and its annotation. The 7,530,820 bp high-quality permanent draft genome is arranged into 118 scaffolds of 118 contigs containing 7.453 protein-coding genes and 73 RNA-only encoding genes. This rhizobial genome is sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.Electronic supplementary materialThe online version of this article (doi:10.1186/s40793-015-0020-2) contains supplementary material, which is available to authorized users.

Bayesian Phylogenetic Analysis of Rhizobia Isolated From Root-Nodules of Three Tunisian Wild Legume Species of the Genus Sulla

We used a relaxed-clock program (Multidivtime) to estimate the phylogenetic relatedness and substitution rates by comparative analyses of 16S rRNA gene and ITS region sequences. Four rhizobial strains isolated from three Tunisian wild Sulla legumes were used together with reference strains to obtain the 16S rRNA gene and ITS region alignments. Bayesian inferred trees were congruent, and showed a clear split between Agrobacterium and Rhizobium species. Among the four Tunisian isolates, Hsc01 belonged to Rhizobium species and formed a monophyletic clade with Rhizobium sullae. The strain Hc04 was placed into the Agrobacterium group, but it belonged to Rhizobium species. Two remaining strains (Hc01 and Hcar01) belonged to typically Agrobacterium species. Divergence times between these four strains were earlier the existence of legumes. Using sequence alignments of the 16S rRNA genes and ITS regions, we inferred different rates of nucleotide substitutions across these two molecular markers. The ITS region evolutionary rate was 15-fold higher than the 16S rRNA gene rate, suggesting that the ITS region represented an appropriate molecular marker for inferring phylogenies and divergence times in bacteria.

Rhizobium halotolerans sp. nov., Isolated from Chloroethylenes Contaminated Soil

The strain designated as AB21(T) was isolated from chloroethylenes contaminated soil. Cells are gram-negative, aerobic, non-spore-forming, and motile rods. Phylogenetic analysis based on 16S rRNA gene sequence showed that it belonged to the genus Rhizobium, and was closely related to Rhizobium sullae IS 123(T) (97.4 %), Rhizobium yanglingense SH 22623(T) (97.2 %), Rhizobium gallicum R 602sp(T) (97.1 %), Rhizobium alamii GBV 016(T) (97.0 %), and Rhizobium monogolense USDA 1844(T) (97.0 %). It showed less than 97 % identity with the remaining Rhizobium species. This novel isolate grew optimally at 25-37 °C (optimum, 30 °C) and pH 6-9 (optimum, pH 8.0). It grew in the presence of 0-4 % (w/v) NaCl, tolerating a 4 % (w/v) NaCl. DNA-DNA hybridization experiment shows less than 53 % binding with closely related Rhizobium. Predominant quinone is ubiquinone (Q-10). The major fatty acids were summed feature 8 (composed of C(18:1) ω7c/C(18:1) ω6c), C(19:0) cyclo ω8c, and C(16:0). The G+C molar content is 62.5 mol%. Based on the polyphasic analysis, strain AB21(T) is referred to be a novel species of the genus Rhizobium for which the name Rhizobium halotolerans sp. nov. is proposed. The type strain is AB21(T) (=KEMC 224-056(T) = JCM 17536(T)).

Relevance of Fucose-Rich Extracellular Polysaccharides Produced by Rhizobium sullae Strains Nodulating Hedysarum coronarium L. Legumes

Specific and complex interactions between soil bacteria, known as rhizobia, and their leguminous host plants result in the development of root nodules. This process implies a complex dialogue between the partners. Rhizobia synthesize different classes of polysaccharides: exopolysaccharides (EPS), Kdo-rich capsular polysaccharides, lipopolysaccharides, and cyclic β-(1,2)-glucans. These polymers are actors of a successful symbiosis with legumes. We focus here on studying the EPS produced by Rhizobium sullae bacteria that nodulate Hedysarum coronarium L., largely distributed in Algeria. We describe the influence of the carbon source on the production and on the composition of EPS produced by R. sullae A6 and RHF strains. High-molecular-weight EPS preserve the bacteria from desiccation. The structural characterization of the EPS produced by R. sullae strains has been performed through sugar analysis by gas chromatography-mass spectrometry. The low-molecular-weight EPS of one strain (RHF) has been totally elucidated using nuclear magnetic resonance and quantitative time-of-flight tandem mass spectrometry analyses. An unusual fucose-rich EPS has been characterized. The presence of this deoxy sugar seems to be related to nodulation capacity.

Influence of carbon source on the production of exopolysacharides by Rhizobium sullae and on the nodulation of Hedysarum coronarium L. legume

The exopolysaccharides (EPS) produced by species of the Rhizobium genus are essential for establishing symbiotic nitrogen fixation with legumes. Rhizobium strains were isolated from the wild plant Hedysarum coronarium L. collected in Constantine, Algeria. The effect of carbon source on the production of EPS and the infectivity of the bacteria were studied for the different Rhizobium isolates. Strains were cultivated on YMA where mannitol was replaced by different sugars. Good bacterial growth was found for sorbitol, sucrose, glucose and maltose. High production of EPS is obtained in the presence of mannitol, sucrose and maltose. Infectivity of the isolates was influenced by the carbon source; high infectivity was recorded in the presence of mannitol and sucrose. A relationship between carbon source, EPS production and nodulation capacity was observed. Key words: Rhizobium sullae, Hedysarum coronarium, exopolysaccharides (EPS), symbiosis.

Isolation and Partial Characterization of Bacterial Strains on Low Organic Carbon Medium from Soils Fertilized with Different Organic Amendments

A total of 720 bacterial strains were isolated from soils with four different organic amendment regimes on a low organic carbon (low-C) agar medium (10 µg C ml(-1)) traditionally used for isolation of oligotrophs. Organic amendments in combination with field history resulted in differences in dissolved organic carbon contents in these soils. There were negative correlations between total and dissolved organic carbon content and the number of isolates on low-C agar medium, whereas these correlations were absent for bacterial strains isolated from the same soil on high-C agar medium (1,000 µg C ml(-1)). Repeated transfers (up to ten times) of the isolates from low-C agar medium to fresh low- and high-C agar media were done to test for exclusive growth under oligotrophic conditions. The number of isolates exclusively growing under oligotrophic conditions dropped after each subsequent transfer from 241 after the first to 98 after the third transfer step. Identification on the basis of partial 16S rRNA gene sequences revealed that most of the 241 isolates (as well as the subset of 98 isolates) belong to widespread genera such as Streptomyces, Rhizobium, Bradyrhizobium, and Mesorhizobium, and the taxonomic composition of dominant genera changed from the first transfer step to the third. A selected subset of 17 isolates were further identified and characterized for exclusive growth on low-C agar medium. Two isolates continued to grow only on low-C agar medium up to the tenth transfer step and matched most closely with Rhizobium sullae and an uncultured bacterium on the basis of the almost full-length 16S rRNA gene. It was concluded that the vast majority of strains which are isolated on low-C agar media belong to the trophic group of microorganisms adapted to a "broad range" of carbon concentrations, including well-known and widespread bacterial genera. Oligotrophy is a physiological, not a taxonomic property, and can only be identified by cultural means so far. We showed that true oligotrophs that are unable to grow on high carbon media are rare and belong to genera that also contain broad-range and copiotrophic strains.

Rhizobium mesosinicum sp. nov., isolated from root nodules of three different legumes

Thirteen novel strains were isolated from root nodules of three different leguminous plants of the genera Albizia, Kummerowia and Dalbergia grown in China. Cells of these strains were Gram-negative, strictly aerobic, non-spore-forming, motile rods. Phylogenetic analysis of 16S rRNA gene sequences revealed that they belong to the genus Rhizobium. A representative strain, CCBAU 25010T, showed 98.8% 16S rRNA gene sequence similarity to its closest phylogenetic relative, Rhizobium sullae IS123T. The 16S-23S intergenic spacer (ITS) sequence of CCBAU 25010T shared 91.5 and 87.2% sequence similarity, respectively, with those of Rhizobium etli CFN 42T and Rhizobium leguminosarum LMG 14904T. Analysis of the sequences of the housekeeping genes atpD and recA was in agreement with the results of ITS sequence analysis. The nodC gene sequence of CCBAU 25010T was identical to that of Rhizobium tropici CFN 299. DNA-DNA hybridization values for strain CCBAU 25010T ranged from 20.7% (with Rhizobium mongolense USDA 1844T) to 34.4% (with R. leguminosarum USDA 2370T). Cell protein SDS-PAGE, BOX-PCR and several phenotypic characteristics, such as use of sole carbon sources and antibiotic resistance, could differentiate the novel strains from defined Rhizobium species. We therefore propose that the novel strains reported in this study form a novel species, Rhizobium mesosinicum sp. nov., with the type strain CCBAU 25010T (=LMG 24135T =JCM 14777T).

Genotypic characterization of indigenous Sinorhizobium meliloti and Rhizobium sullae by rep- PCR, RAPD and ARDRA analyses

The rhizobia, Sinorhizobium meliloti and Rhizobium sullae, which fix nitrogen in root nodules of alfalfa (Medicago sativa L.) and sulla (Hedysarum sp.) forage legumes, respectively, were isolated from root nodules and soils from Morocco. We used three PCR-based techniques namely, rep-PCR, RAPD and ARDRA techniques for genotypic characterization of 10 isolates of S. meliloti and R. sullae, in order to identify rapid and reliable techniques for applications in population genetic analysis of these species. The analysis revealed characteristic banding patterns for S. meliloti and R. sullae isolates by all the three techniques, even though the isolates are from a narrow geographic region in Morocco. Furthermore, the results showed that the rep-PCR with REP and ERIC primers was more efficient than RAPD and ARDRA technique for genotyping S. meliloti isolates; and rep-PCR with REP primers and the ARDRA technique with restriction enzyme HinfI, were more efficient than the other rep-PCR and RAPD-PCR techniques for genotyping R. sullae isolates.

Rhizobium alamii sp. nov., an exopolysaccharide-producing species isolated from legume and non-legume rhizospheres

A group of exopolysaccharide-producing bacteria was isolated from the root environment of Arabidopsis thaliana. The genetic diversity revealed by REP-PCR fingerprinting indicated that the isolates correspond to different strains. 16S rRNA gene sequence analysis showed that the isolates are closely related to the strains Rhizobium sp. YAS34 and USDA 1920, respectively isolated from sunflower roots and Medicago ruthenica nodules. These bacteria belong to the Rhizobium lineage of the Alphaproteobacteria, and the closest known species was Rhizobium sullae. DNA-DNA hybridization experiments and biochemical analysis demonstrated that the nine strains isolated from A. thaliana and Rhizobium strains YAS34 and USDA 1920 constitute a novel species within the genus Rhizobium, for which the name Rhizobium alamii sp. nov. is proposed. The type strain is GBV016(T) (=CFBP 7146(T) =LMG 24466(T)).

Genotypic characterization of indigenous Sinorhizobium meliloti and Rhizobium sullae by rep- PCR, RAPD and ARDRA analyses

The rhizobia, Sinorhizobium meliloti and Rhizobium sullae, which fix nitrogen in root nodules of alfalfa (Medicago sativa L.) and sulla (Hedysarum sp.) forage legumes, respectively, were isolated from root nodules and soils from Morocco. We used three PCR-based techniques namely, rep-PCR, RAPD and ARDRA techniques for genotypic characterization of 10 isolates of S. meliloti and R. sullae, in order to identify rapid and reliable techniques for applications in population genetic analysis of these species. The analysis revealed characteristic banding patterns for S. meliloti and R. sullae isolates by all the three techniques, even though the isolates are from a narrow geographic region in Morocco. Furthermore, the results showed that the rep-PCR with REP and ERIC primers was more efficient than RAPD and ARDRA technique for genotyping S. meliloti isolates; and rep-PCR with REP primers and the ARDRA technique with restriction enzyme HinfI, were more efficient than the other rep-PCR and RAPD-PCR techniques for genotyping R. sullae isolates.

Selenite-reducing capacity of the copper-containing nitrite reductase ofRhizobium sullae

Rhizobium sullae strain HCNT1 contains a nitric oxide-producing nitrite reductase of unknown function due to the absence of a complementary nitric oxide reductase. HCNT1 had the ability to grow on selenite concentrations as high as 50 mM, and during growth, selenite was reduced to the less toxic elemental selenium. An HCNT1 mutant lacking nitrite reductase grew poorly in the presence of 5 mM selenite, was unable to grow in the presence of 25 or 50 mM selenite and also showed no evidence of selenite reduction. A naturally occurring nitrite reductase-deficient R. sullae strain, CC1335, also showed little growth on the higher concentrations of selenite. Mobilization of a plasmid containing the HCNT1 gene encoding nitrite reductase into CC1335 increased its resistance to selenite. To confirm that this ability to grow in the presence of high concentrations of selenite correlated with nitrite reductase activity, a new nitrite reductase-containing strain was isolated from the same location where HCNT1 was isolated. This strain was also resistant to high concentrations of selenite. Inactivation of the gene encoding nitrite reductase in this strain increased selenite sensitivity. These data suggest that the nitrite reductase of R. sullae provides resistance to selenite and offers an explanation for the radically truncated denitrification found uniquely in this bacterium.