Genetic characterization of fast-growing rhizobia able to nodulate Prosopis alba in North Spain

Prosopis is a Mimosaceae legume tree indigenous to South America and not naturalized in Europe. In this work 18 rhizobial strains nodulating Prosopis alba roots were isolated from a soil in North Spain that belong to eight different randomly amplified polymorphic DNA groups phylogenetically related to Sinorhizobium medicae, Sinorhizobium meliloti and Rhizobium giardinii according to their intergenic spacer and 16S rRNA gene sequences. The nodC genes of isolates close to S. medicae and S. meliloti were identical to those of S. medicae USDA 1,037(T) and S. meliloti LMG 6,133(T) and accordingly all these strains were able to nodulate both alfalfa and Prosopis. These nodC genes were phylogenetically divergent from those of the isolates close to R. giardinii that were identical to that of R. giardinii H152(T) and therefore all these strains formed nodules in common beans and Prosopis. The nodC genes of the strains isolated in Spain were phylogenetically divergent from that carried by Mesorhizobium chacoense Pr-5(T) and Sinorhizobium arboris LMG 1,4919(T) nodulating Prosopis in America and Africa, respectively. Therefore, Prosopis is a promiscuous host which can establish symbiosis with strains carrying very divergent nodC genes and this promiscuity may be an important advantage for this legume tree to be used in reforestation.     

Characterisation and differentiation of indigenous rhizobia isolated from Canarian shrub legumes of agricultural and ecological interest

In the course of a study on rhizobia nodulating six indigenous legume shrubs from the Canary Islands, one Rhizobium and 27 Bradyrhizobium Canarian isolates were characterised. It was found that those ascribed to Bradyrhizobium were promiscuous and formed effective nodules not only in their original host but on Chamecytisus proliferus subsp. proliferus (Tagasaste) as well. However, Rhizobium isolate RES-1 was more specific and only nodulated on its host (Teline canariensis). The serotyping of these isolates required a broad antisera panel due to the great antigenic diversity of these rhizobia, that appeared to be due to differences in their lipopolysaccharides, the main antigenic determinants, that showed great structural diversity. The 28 isolates studied produced 22 easily distinguishable electrophoretic profiles of lipopolysaccharides. Protein or plasmid electrophoretic profiles were equally or less discriminating than the lipopolysaccharides profiles and were more difficult to compare. The comparison of the lipopolysaccharide electrophoretic patterns is a more reliable and discriminating method than serotyping or electrophoretic protein and plasmid profile analysis for the identification of Bradyrhizobium strains. No correlation between the lipopolysaccharide profiles of the isolates and the plant from which they were obtained or their geographical origin was observed.     

The physiological and genetic diversity of bovine Streptococcus bovis strains(1)

Laboratory Streptococcus bovis strains and isolates obtained from a steer fed increasing amounts of grain had similar growth characteristics, but they differed in their sensitivity to 2-deoxyglucose (2DG), a non-metabolizable glucose analog. The addition of 2DG decreased both growth rate (0.92+/-0.34 h(-1)) and growth yield (ranging from 25 to 63%), but these differences could not be correlated with diet. However, isolates from a steer fed a 90% grain diet were more prone to 2DG-dependent lysis than those from a hay diet (P<0.001). All S. bovis laboratory strains and isolates had an identical restriction fragment length polymorphism pattern, when their 16S rDNA was digested with HaeIII and HhaI. However, when genomic BOX elements were amplified, 5-12 bands were observed, and the S. bovis isolates and laboratory strains could be grouped into 13 different BOX types. Strains 26 and 581AXY2 had the same BOX type, but the remaining laboratory strains did not form closely related clusters. Strains JB1 and K27FF4 were most closely related to each other. Most of the fresh isolates (24 out of 30) could be grouped into a single cluster (>90% Dice similarity). This cluster contained isolates from all three diets, but it did not have any of the laboratory strains. The majority (90%) of the isolates obtained from the hay-fed steer exhibited the same BOX type. Because more BOX types were observed if grain was added to the diet, it appears that ruminal S. bovis diversity may be a diet-dependent phenomenon.     

Diversity of Rhizobia Nodulating Phaseolus vulgaris L. in Two Kenyan Soils with Contrasting pHs

Rhizobia were isolated from two Kenyan soils with pHs of 4.5 and 6.8 and characterized on the basis of their host ranges for nodulation and nitrogen fixation, colony morphologies, restriction fragment fingerprints, and hybridization with a nifH probe. The populations of rhizobia nodulating Phaseolus vulgaris in the two soils were similar in numbers and in effectiveness of N(inf2) fixation but were markedly different in composition. The population in the Naivasha soil (pH 6.8) was dominated by isolates specific in host range for nodulation to P. vulgaris; these all had multiple copies, in most cases four, of the structural nitrogenase gene nifH. Only one of the isolates from this soil formed effective nodules on Leucaena leucocephala, and this isolate had only a single copy of nifH. By contrast, the population in the acid Daka-ini soil (pH 4.5) was composed largely of broad-host-range isolates which had single copies of nifH. The isolates from the Daka-ini soil which were specific to P. vulgaris generally had three copies of nifH, although one isolate had only two copies. These rhizobial isolates are indigenous to Kenyan soils and yet have marked similarities to previously described Rhizobium species from other continents.     

Strains of Mesorhizobium amorphae and Mesorhizobium tianshanense, carrying symbiotic genes of common chickpea endosymbiotic species, constitute a novel biovar (ciceri) capable of nodulating Cicer arietinum

AIMS: To identify several strains of Mesorhizobium amorphae and Mesorhizobium tianshanense nodulating Cicer arietinum in Spain and Portugal, and to study the symbiotic genes carried by these strains. METHODS AND RESULTS: The sequences of 16S-23S intergenic spacer (ITS), 16S rRNA gene and symbiotic genes nodC and nifH were analysed. According to their 16S rRNA gene and ITS sequences, the strains from this study were identified as M. amorphae and M. tianshanense. The type strains of these species were isolated in China from Glycyrrhiza pallidiflora and Amorpha fruticosa nodules, respectively, and are not capable of nodulating chickpea. These strains carry symbiotic genes, phylogenetically divergent from those of the chickpea isolates, whose nodC and nifH genes showed more than 99% similarity with respect to those from Mesorhizobium ciceri and Mesorhizobium mediterraneum, the two common chickpea nodulating species in Spain and Portugal. CONCLUSIONS: The results from this study showed that different symbiotic genes have been acquired by strains from the same species during their coevolution with different legumes in distinct geographical locations. SIGNIFICANCE AND IMPACT OF THE STUDY: A new infrasubspecific division named biovar ciceri is proposed within M. amorphae and M. tianshanense to include the strains able to effectively nodulate Cicer arietinum.     

Strains nodulating Lupinus albus on different continents belong to several new chromosomal and symbiotic lineages within Bradyrhizobium

In this work we analysed different chromosomal and symbiotic markers in rhizobial strains nodulating Lupinus albus (white lupin) in several continents. Collectively the analysis of their rrs and atpD genes, and 16S-23S intergenic spacers (ITS), showed that they belong to at least four chromosomal lineages within the genus Bradyrhizobium. Most isolates from the Canary Islands (near to the African continent) grouped with some strains isolated on mainland Spain and were identified as Bradyrhizobium canariense. These strains are divided into two ITS subgroups coincident with those previously described from isolates nodulating Ornithopus. The remaining strains isolated on mainland Spain grouped with most isolates from Chile (American continent) forming a new lineage related to Bradyrhizobium japonicum. The strains BLUT2 and ISLU207 isolated from the Canary Islands and Chile, respectively, formed two new lineages phylogenetically close to different species of Bradyrhizobium depending on the marker analyzed. The analysis of the nodC gene showed that all strains nodulating L. albus belong to the biovar genistearum; nevertheless they form four different nodC lineages of which lineage C is at present exclusively formed by L. albus endosymbionts isolated from different continents.     

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.     

Sinorhizobium americanum symbiovar mediterranense is a predominant symbiont that nodulates and fixes nitrogen with common bean (Phaseolus vulgaris L.) in a Northern Tunisian field

A total of 40 symbiotic bacterial strains isolated from root nodules of common bean grown in a soil located in the north of Tunisia were characterized by PCR-RFLP of the 16S rRNA genes. Six different ribotypes were revealed. Nine representative isolates were submitted to phylogenetic analyses of rrs, recA, atpD, dnaK, nifH and nodA genes. The strains 23C40 and 23C95 representing the most abundant ribotype were closely related to Sinorhizobium americanum CFNEI 156(T). S. americanum was isolated from Acacia spp. in Mexico, but this is the first time that this species is reported among natural populations of rhizobia nodulating common bean. These isolates nodulated and fixed nitrogen with this crop and harbored the symbiotic genes of the symbiovar mediterranense. The strains 23C2 and 23C55 were close to Rhizobium gallicum R602sp(T) but formed a well separated clade and may probably constitute a new species. The sequence similarities with R. gallicum type strain were 98.7% (rrs), 96.6% (recA), 95.8% (atpD) and 93.4% (dnaK). The remaining isolates were, respectively, affiliated to R. gallicum, E. meliloti, Rhizobium giardinii and Rhizobium radiobacter. However, some of them failed to re-nodulate their original host but promoted root growth.     

Trichinella spiralis infections of inbred mice: genetics of the host response following infection with different Trichinella isolates

The immune response of inbred strains of mice was studied following infection with isolates of Trichinella from a pig (P1), an arctic fox (AF1), and T. spiralis var. pseudospiralis (TP). Strains of mice previously characterized as highly resistant to a separate pig isolate of T. spiralis responded to the P1 and AF1 isolates by expelling over 80% of the worms by day 10 postinfection (PI), and by suppressing the in vitro release of newborn larvae by female worms. However, the response induced by AF1 worms was expressed more quickly when compared to responses induced by the P1 and TP isolates. The host response to TP was less as recovery was always higher at day 10 PI and antifecundity effects were not induced in TP worms even in highly resistant strains of mice. Strains of mice previously characterized as susceptible to T. spiralis infection were slow to develop resistance when compared to the resistant mouse strains, but even among the susceptible strains, infection with AF1 induced a more rapid response. The mouse strains used in these experiments allowed us to assess the role of the major histocompatibility complex (MHC) and/or non-MHC genes in influencing the responses observed. As previously reported for a pig isolate of T. spiralis, both MHC and non-MHC genes influenced the rate at which worms were expelled from the gut and the host response that limits the fecundity of adult female worms.     

Genotypic and symbiotic diversity of Rhizobium populations associated with cultivated lentil and pea in sub-humid and semi-arid regions of Eastern Algeria

The genetic structure of rhizobia nodulating pea and lentil in Algeria, Northern Africa was determined. A total of 237 isolates were obtained from root nodules collected on lentil (Lens culinaris), proteaginous and forage pea (Pisum sativum) growing in two eco-climatic zones, sub-humid and semi-arid, in Eastern Algeria. They were characterised by PCR-restriction fragment length polymorphism (RFLP) of the 16S–23S rRNA intergenic region (IGS), and the nodD-F symbiotic region. The combination of these haplotypes allowed the isolates to be clustered into 26 distinct genotypes, and all isolates were classified as Rhizobium leguminosarum. Symbiotic marker variation (nodD-F) was low but with the predominance of one nod haplotype (g), which had been recovered previously at a high frequency in Europe. Sequence analysis of the IGS further confirmed its high variability in the studied strains. An AMOVA analysis showed highly significant differentiation in the IGS haplotype distribution between populations from both eco-climatic zones. This differentiation was reflected by differences in dominant genotype frequencies. Conversely, no host plant effect was detected. The nodD gene sequence-based phylogeny suggested that symbiotic gene diversity in pea and lentil nodulating rhizobial populations in Algeria was low compared to that reported elsewhere in the world.     

Characterisation of rhizobia isolated from nodules on locally-adapted Glycine max grown in Nigeria

Strains of Rhizobium spp. (cowpea miscellany) and R. japonicum are characterised by their symbiotic performance on two hosts, Vigna unguiculata cv. Ife Brown and Glycine rnax cv. Bossier. The results indicate that isolates from G. max cv. Malayan, a promiscuously nodulating cultivar adapted to Nigerian conditions, form a group intermediate between R. japonicum and R. spp. but are more closely related to the latter. We tentatively suggest on the evidence of symbiotic performance that R. japonicum is a specialised group within R. spp.     

Intestinal colonization potential of turbot (Scophthalmus maximus)- and dab (Limanda limanda)-associated bacteria with inhibitory effects against Vibrio anguillarum.

Of more than 400 bacteria isolated from turbot (Scophthalmus maximus), 89 have previously been shown to inhibit the in vitro growth of the fish pathogen Vibrio anguillarum. The aim of the present study was to investigate the potential of seven of these strains, as well as of intestinal isolates (four strains) from a closely related fish, dab (Limanda limanda), for colonizing farmed turbot as a means of protecting the host from infection by V. anguillarum. In addition, the inhibitory effect of these strains on the pathogen was further studied. Colonization potential was measured by the capacity of the strains to adhere to and grow in turbot intestinal mucus. These parameters were also used to investigate the potential of V. anguillarum to amplify in the turbot intestinal tract. Because of the observed rapid growth of V. anguillarum in intestinal mucus, it can be proposed that the intestinal tract is a site for V. anguillarum multiplication. Strains isolated from the intestine showed greater capacity for adhesion to and growth in fish intestinal mucus than did the pathogen and the skin mucus isolates. All of the isolates released metabolites into the culture medium that had inhibitory effects against V. anguillarum. The results are discussed with emphasis on administering bacteria of host origin to farmed turbot in order to control V. anguillarum-induced disease.     

Origins of cheating and loss of symbiosis in wild Bradyrhizobium

Rhizobial bacteria nodulate legume roots and fix nitrogen in exchange for photosynthates. These symbionts are infectiously acquired from the environment and in such cases selection models predict evolutionary spread of uncooperative mutants. Uncooperative rhizobia – including nonfixing and non‐nodulating strains – appear common in agriculture, yet their population biology and origins remain unknown in natural soils. Here, a phylogenetically broad sample of 62 wild‐collected rhizobial isolates was experimentally inoculated onto Lotus strigosus to assess their nodulation ability and effects on host growth. A cheater strain was discovered that proliferated in host tissue while offering no benefit; its fitness was superior to that of beneficial strains. Phylogenetic reconstruction of Bradyrhizobium rDNA and transmissible symbiosis‐island loci suggest that the cheater evolved via symbiotic gene transfer. Many strains were also identified that failed to nodulate L. strigosus, and it appears that nodulation ability on this host has been recurrently lost in the symbiont population. This is the first study to reveal the adaptive nature of rhizobial cheating and to trace the evolutionary origins of uncooperative rhizobial mutants.     

Intestinal colonization potential of turbot (Scophthalmus maximus)- and dab (Limanda limanda)-associated bacteria with inhibitory effects against Vibrio anguillarum.

Of more than 400 bacteria isolated from turbot (Scophthalmus maximus), 89 have previously been shown to inhibit the in vitro growth of the fish pathogen Vibrio anguillarum. The aim of the present study was to investigate the potential of seven of these strains, as well as of intestinal isolates (four strains) from a closely related fish, dab (Limanda limanda), for colonizing farmed turbot as a means of protecting the host from infection by V. anguillarum. In addition, the inhibitory effect of these strains on the pathogen was further studied. Colonization potential was measured by the capacity of the strains to adhere to and grow in turbot intestinal mucus. These parameters were also used to investigate the potential of V. anguillarum to amplify in the turbot intestinal tract. Because of the observed rapid growth of V. anguillarum in intestinal mucus, it can be proposed that the intestinal tract is a site for V. anguillarum multiplication. Strains isolated from the intestine showed greater capacity for adhesion to and growth in fish intestinal mucus than did the pathogen and the skin mucus isolates. All of the isolates released metabolites into the culture medium that had inhibitory effects against V. anguillarum. The results are discussed with emphasis on administering bacteria of host origin to farmed turbot in order to control V. anguillarum-induced disease.     

Partial 16S rRNA gene sequence diversity and numerical taxonomy of slow growing pigeonpea (Cajanus cajan L Millsp) nodulating rhizobia

An investigation was carried out to determine the diversity of 30 isolates of slow growing pigeonpea nodulating rhizobia based on variations in partial sequences of the 16S rRNA gene and numerical analysis of 80 phenotypic traits. Phylogenetic analysis using molecular sequences of 23 isolates showed that ARPE1 separated from the other isolates at an average distance of >14% divergence level. The other isolates were all within 5% divergence from each other but separated into four main groups, with group 1 containing 16 of the 23 isolates. Comparisons to sequences of reference strains revealed that the group 1 isolates were phylogenetically closely related to the slow growing soybean nodulating rhizobia belonging to Bradyrhizobium elkanii, although only three of these isolates were able to nodulate soybean. Numerical analysis of phenotypic data of 19 isolates showed that 14 isolates clustered together in one branch of the phenogram, which included the group 1, group 2 and group 4 isolates from the phylogenetic analysis. The group 3 isolates were highly variable in the phenogram with similarity levels lower than 50% among these isolates.     

Characterization of root-nodule bacteria isolated from Vicia faba and selection of plant growth promoting isolates

A collection of 104 isolates from root-nodules of Vicia faba was submitted to 16S rRNA PCR–RFLP typing. A representative sample was further submitted to sequence analysis of 16S rRNA. Isolates were assigned to 12 genera. All the nodulating isolates (45 %) were closely related to Rhizobium leguminosarum USDA2370^T (99.34 %). The remaining isolates, including potential human pathogens, failed to nodulate their original host. They were checked for presence of symbiotic genes, P-solubilization, phytohormone and siderophore production, and then tested for their growth promoting abilities. Results indicated that 9 strains could induce significant increase (41–71 %) in shoot dry yield of faba bean. A Pseudomonas strain was further assessed in on-farm trial in combination with a selected rhizobial strain. This work indicated that nodule-associated bacteria could be a valuable pool for selection of effective plant growth promoting isolates. Nevertheless, the possible involvement of nodules in increasing risks related to pathogenic bacteria should not be neglected and needs to be investigated further.     

Phylogenetic analysis of Bacillus cereus isolates from severe systemic infections using multilocus sequence typing scheme

Bacillus cereus strains from cases of severe or lethal systemic infections, including respiratory symptoms cases, were analyzed using multilocus sequence typing scheme of B. cereus MLST database. The isolates were evenly distributed between the two main clades, and 60% of them had allele profiles new to the database. Half of the collection's strains clustered in a lineage neighboring Bacillus anthracis phylogenetic origin. Strains from lethal cases with respiratory symptoms were allocated in both main clades. This is the first report of strains causing respiratory symptoms to be identified as genetically distant from B. anthracis. The phylogenetic location of the presented here strains was compared with all previously submitted to the database isolates from systemic infections, and were found to appear in the same clusters where clinical isolates from other studies had been assigned. It seems that the pathogenic strains are forming clusters on the phylogenetic tree.     

Large genotypic variation but small variation in N2 fixation among rhizobia nodulating red clover in soils of northern Scandinavia

AIMS: To analyse the symbiotic variations within indigenous populations of rhizobia nodulating red clover (Trifolium pratense L.) in soils of northern Norway and Sweden at different times of the growing season. METHODS AND RESULTS: A total of 431 nodule isolates sampled under field conditions in summer and autumn, were characterized genetically by targeting both chromosomal and symbiotic genes. The Enterobacterial Repetitive Intergenic Consensus polymerase chain reaction (PCR) fingerprinting of chromosomal DNA revealed considerable variation within the isolated populations that was more influenced by geographical origin than sampling time. Analysis of PCR amplified nodEF gene on the symbiotic plasmid by restriction fragment length polymorphism revealed a high proportion of nod types common to the two studied sites. The symbiotic efficiency of the isolates, representing both dominating and rare nodEF genotypes, showed high N(2) fixation rates in symbiosis with the host plant in a greenhouse experiment using the (15)N isotope dilution method. CONCLUSIONS: Effective N(2)-fixing strains of Rhizobium leguminosarum bv. trifolii nodulating red clover are common and genetically diverse in these northern Scandinavia soils. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on the variability, stability and dynamics of resident populations of rhizobia nodulating red clover in Scandinavian soils which has practical implications for applying biological nitrogen fixation in subarctic plant production.     

Characterization of root-nodulating bacteria associated to <Emphasis Type="Italic">Prosopis farcta</Emphasis> growing in the arid regions of Tunisia

Diversity of 50 bacterial isolates recovered from root nodules of Prosopis farcta grown in different arid soils in Tunisia, was investigated. Characterization of isolates was assessed using a polyphasic approach including phenotypic characteristics, 16S rRNA gene PCR–RFLP and sequencing, nodA gene sequencing and MLSA. It was found that most of isolates are tolerant to high temperature (40°C) and salinity (3%). Genetic characterization emphasizes that isolates were assigned to the genus Ensifer (80%), Mesorhizobium (4%) and non-nodulating endophytic bacteria (16%). Forty isolates belonging to the genus Ensifer were affiliated to Ensifer meliloti, Ensifer xinjiangense/Ensifer fredii and Ensifer numidicus species. Two isolates belonged to the genus Mesorhizobium. Eight isolates failing to renodulate their host plant were endophytic bacteria and belonged to Bacillus, Paenibacillus and Acinetobacter genera. Symbiotic properties of nodulating isolates showed a diversity in their capacity to infect their host plant and fix atmospheric nitrogen. Isolate PG29 identified as Ensifer meliloti was the most effective one. Ability of Prosopis farcta to establish symbiosis with rhizobial species confers an important advantage for this species to be used in reforestation programs. This study offered the first systematic information about the diversity of microsymbionts nodulating Prosopis farcta in the arid regions of Tunisia.     

The diversity of rhizobia nodulating beans in Northwest Argentina as a source of more efficient inoculant strains

The common bean (Phaseolus vulgaris L.) is cultivated widely in Central and South America and particularly in the Northwest of Argentina. In order to describe the diversity of the common bean nodulating rhizobial population from the bean producing area in Northwest Argentina (NWA), a collection of about 400 isolates of common beans recovered from nodules and soil samples from NWA were characterized by using nifH-PCR, analysis of genes coding for 16S rRNA and nodC, and REP-fingerprinting, respectively. It was found that species Rhizobium etli is predominant in common bean nodules although a high degree of diversity was found within the species. Other bean nodulating genotypes recovered from soils by using Leucaena sp. as the trapping host was found to have the 16S rDNA alleles of species such as Sinorhizobium fredii, Sinorhizobium saheli, Sinorhizobium teranga, Mesorhizobium loti, and Rhizobium tropici. Some of the bean genotypes that were found to be more efficient in green house experiments were selected and assayed in two successive bean-cropping seasons in the field environment in NWA, and an increase in yields with inoculation was found. The performance of strains isolated from the region indicates potential for exploiting the diversity.