Symbiotic efficiency of Rhizobium leguminosarum sv. trifolii strains originating from the subpolar and temperate climate regions.

Red clover (Trifolium pratense L.) is a forage legume cultivated worldwide. This plant is capable of establishing a nitrogen-fixing symbiosis with Rhizobium leguminosarum symbiovar trifolii strains. To date, no comparative analysis of the symbiotic properties and heterogeneity of T. pratense microsymbionts derived from two distinct geographic regions has been performed. In this study, the symbiotic properties of strains originating from the subpolar and temperate climate zones in a wide range of temperatures (10-25 °C) have been characterized. Our results indicate that all the studied T. pratense microsymbionts from two geographic regions were highly efficient in host plant nodulation and nitrogen fixation in a wide range of temperatures. However, some differences between the populations and between the strains within the individual population examined were observed. Based on the nodC and nifH sequences, the symbiotic diversity of the strains was estimated. In general, 13 alleles for nodC and for nifH were identified. Moreover, 21 and 61 polymorphic sites in the nodC and nifH sequences were found, respectively, indicating that the latter gene shows higher heterogeneity than the former one. Among the nodC and nifH alleles, three genotypes (I-III) were the most frequent, whereas the other alleles (IV-XIII) proved to be unique for the individual strains. Based on the nodC and nifH allele types, 20 nodC-nifH genotypes were identified. Among them, the most frequent were three genotypes marked as A (6 strains), B (5 strains), and C (3 strains). Type A was exclusively found in the temperate strains, whereas types B and C were identified in the subpolar strains. The remaining 17 genotypes were found in single strains. In conclusion, our data indicate that R. leguminosarum sv. trifolii strains derived from two climatic zones show a high diversity with respect to the symbiotic efficiency and heterogeneity. However, some of the R. leguminosarum sv. trifolii strains exhibit very good symbiotic potential in the wide range of the temperatures tested; hence, they may be used in the future for improvement of legume crop production.

Pantoea trifolii sp. nov., a novel bacterium isolated from Trifolium rubens root nodules.

A novel bacterium, designated strain MMK2T, was isolated from a surface-sterilised root nodule of a Trifolium rubens plant growing in south-eastern Poland. Cells were Gram negative, non-spore forming and rod shaped. The strain had the highest 16S rRNA gene sequence similarity with P. endophytica (99.4%), P. leporis (99.4%) P. rwandensis (98.8%) and P. rodasii (98.45%). Phylogenomic analysis clearly showed that strain MMK2T and an additional strain, MMK3, should reside in the genus Pantoea and that they were most closely related to P. endophytica and P. leporis. Genome comparisons showed that the novel strain shared 82.96-93.50% average nucleotide identity and 26.2-53. 2% digital DNA:DNA hybridization with closely related species. Both strains produced siderophores and were able to solubilise phosphates. The MMK2T strain was also able to produce indole-3-acetic acid. The tested strains differed in their antimicrobial activity, but both were able to inhibit the growth of Sclerotinia sclerotiorum 10Ss01. Based on the results of the phenotypic, phylogenomic, genomic and chemotaxonomic analyses, strains MMK2T and MMK3 belong to a novel species in the genus Pantoea for which the name Pantoea trifolii sp. nov. is proposed with the type strain MMK2T (= DSM 115063T = LMG 33049T).

Genetic diversity of microsymbionts nodulating Trifolium pratense in subpolar and temperate climate regions.

Rhizobia are soil-borne bacteria forming symbiotic associations with legumes and fixing atmospheric dinitrogen. The nitrogen-fixation potential depends on the type of host plants and microsymbionts as well as environmental factors that affect the distribution of rhizobia. In this study, we compared genetic diversity of bacteria isolated from root nodules of Trifolium pratense grown in two geographical regions (Tromsø, Norway and Lublin, Poland) located in distinct climatic (subpolar and temperate) zones. To characterize these isolates genetically, three PCR-based techniques (ERIC, BOX, and RFLP of the 16S-23S rRNA intergenic spacer), 16S rRNA sequencing, and multi-locus sequence analysis of chromosomal house-keeping genes (atpD, recA, rpoB, gyrB, and glnII) were done. Our results indicate that a great majority of the isolates are T. pratense microsymbionts belonging to Rhizobium leguminosarum sv. trifolii. A high diversity among these strains was detected. However, a lower diversity within the population derived from the subpolar region in comparison to that of the temperate region was found. Multi-locus sequence analysis showed that a majority of the strains formed distinct clusters characteristic for the individual climatic regions. The subpolar strains belonged to two (A and B) and the temperate strains to three R. leguminosarum genospecies (B, E, and K), respectively.

Contamination of the urban environment with excrements of companion animals as an underestimated source of Staphylococcus species posing a threat to public health.

The aim of the study was to assess the incidence, resistance, virulence, and genotypic characteristics of Staphylococcus spp. residing in the gastrointestinal tract of dogs and cats, as a group of animals causing potential contamination of the urban space. A high percentage of strains resistant to penicillin (58%), oxacillin (9%) and tetracycline (60%) were found. All isolates resistant to penicillin, kanamycin, or chloramphenicol carried genes responsible for individual resistance (blaZ, aph(3')-IIIa, and cat (pC194)/cat (pC223), respectively. The mecA gene was detected in 45% of the oxacillin-resistant Staphylococcus pseudintermedius strains. The amplification of DNA fragments surrounding rare restriction sites analysis demonstrated high heterogeneity of genotypic profiles correlating with phenotypic resistance profiles. Multilocus sequence typing analysis classified the methicillin-resistant S. pseudintermedius strains as ST71, ST890, and the totally new ST1047. The presence of a high level of resistance among Staphylococcus strains may suggest a potential risk of transfer of these bacteria between companion animals and humans.

Putative novel Bradyrhizobium and Phyllobacterium species isolated from root nodules of Chamaecytisus ruthenicus.

In this study, the diversity and the phylogenetic relationships of bacteria isolated from root nodules of Chamaecytisus ruthenicus growing in Poland were investigated using ERIC-PCR fingerprinting and by multilocus sequence analysis (MLSA). Two major clusters comprising 13 and 3 isolates were detected which 16S rRNA gene sequencing identified as Bradyrhizobium and Phyllobacterium. The results of phylogenetic analysis of individual and concatenated atpD, gyrB and recA gene sequences showed that the studied strains may represent novel species in the genera Bradyrhizobium and Phyllobacterium. In the phylogenetic tree based on the atpD-gyrB-recA concatemers, Bradyrhizobium isolates were split into two groups closely related to Bradyrhizobium algeriense STM89T and Bradyrhizobium valentinum LmjM3T. The genus Phyllobacterium isolates formed a separate cluster close to Phyllobacterium ifriqiyense LMG27887T in the atpD-gyrB-recA phylogram. Analysis of symbiotic gene sequences (nodC, nodZ, nifD, and nifH) showed that the Bradyrhizobium isolates were most closely related to Bradyrhizobium algeriense STM89T, Bradyrhizobium valentinum LmjM3T and Bradyrhizobium retamae Ro19T belonging to symbiovar retamae. This is the first report on the occurrence of members of symbiovar retamae from outside the Mediterranean region. No symbiosis related genes were amplified from Phyllobacterium strains, which were also unable to induce nodules on C. ruthenicus roots. Based on these findings Phyllobacterium isolates can be regarded as endophytic bacteria inhabitating root nodules of C. ruthenicus.

Root nodules of Genista germanica harbor Bradyrhizobium and Rhizobium bacteria exchanging nodC and nodZ genes.

A collection of 18 previously unstudied strains isolated from root nodules of Genista germanica (German greenweed) grown in southeast Poland was evaluated for the level of genetic diversity using the BOX-PCR technique and the phylogenetic relationship based on both core (16S rRNA, dnaK, ftsA, glnII, gyrB, recA, rpoB) and nodulation (nodC and nodZ) gene sequences. Each of the 18 G. germanica root nodule isolates displayed unique BOX-PCR patterns, indicating their high level of genomic heterogeneity. Based on the comparative 16S rDNA sequence analysis, 12 isolates were affiliated to the Bradyrhizobium genus and the other strains were most similar to Rhizobium species. Phylogenetic analysis of the core gene sequences indicated that the studied Bradyrhizobium bacteria were most closely related to Bradyrhizobium japonicum, whereas Rhizobium isolates were most closely related to Rhizobium lusitanum and R. leguminosarum. The phylogenies of nodC and nodZ for the Rhizobium strains were incongruent with each other and with the phylogenies inferred from the core gene sequences. All Rhizobium nodZ gene sequences acquired in this study were grouped with the sequences of Bradyrhizobium strains. Some of the studied Rhizobium isolates were placed in the nodC phylogenetic tree together with reference Rhizobium species, while the others were closely related to Bradyrhizobium bacteria. The results provided evidence for horizontal transfer of nodulation genes between Bradyrhizobium and Rhizobium. However, the horizontal transfer of nod genes was not sufficient for Rhizobium strains to form nodules on G. germanica roots, suggesting that symbiotic genes have to be adapted to the bacterial genome.

The ftsA gene as a molecular marker for phylogenetic studies in Bradyrhizobium and identification of Bradyrhizobium japonicum.

The use of ftsA gene sequences for taxonomic studies of the genus Bradyrhizobium bacteria was assessed. The ftsA gene codes for an actin-like protein involved in prokaryotic cell division. Up to now, this gene has not been used as a phylogenetic marker for analysis of bacteria establishing root nodule symbiosis with Fabaceae plants. In this study, the ftsA gene sequences obtained for bradyrhizobia forming N2 fixing symbiosis with four Genisteae tribe plants growing in Poland and most of the type strains of the genus Bradyrhizobium species were analyzed and evaluated as molecular markers for phylogenetic studies of these bacteria for the first time. The ftsA gene sequences of all bradyrhizobial strains with completely or partially sequenced genomes, available in the GenBank database, were also included into the analysis. The phylogeny of the ftsA gene was compared to the phylogenies of other chromosomal genes commonly used in the studies of Bradyrhizobium bacteria. The results showed that the phylogenies of ftsA and the core genes recA and glnII were congruent, making the ftsA gene useful as a phylogenetic marker. Analysis of the ftsA gene sequences revealed a single-nucleotide polymorphism unique to Bradyrhizobium japonicum strains, and the potential use of this SNP for identification of this species was discussed.

Molecular phylogeny of Bradyrhizobium bacteria isolated from root nodules of tribe Genisteae plants growing in southeast Poland.

The phylogeny of 16 isolates from root nodules of Genista germanica, Genista tinctoria, Cytisus ratisbonensis, and Cytisus scoparius growing in southeast Poland was estimated by comparative sequence analysis of core (16S rDNA, atpD, glnII, recA) and symbiosis-related (nodC, nodZ, nifH) genes. All the sequences analyzed placed the studied rhizobia in the genus Bradyrhizobium. Phylogenetic analysis of individual and concatenated housekeeping genes showed that the Genisteae microsymbionts form a homogeneous group with Bradyrhizobium japonicum strains. The phylogeny of nodulation and nitrogen fixation genes indicated a close relationship of the examined rhizobia with B. japonicum, Bradyrhizobium canariense, Bradyrhizobium cytisi, Bradyrhizobium rifense and Bradyrhizobium lupini strains infecting other plants of the tribe Genisteae. For the first time, the taxonomic position of G. germanica and C. ratisbonensis rhizobia, inferred from multigenic analysis, is described. The results of the phylogenetic analysis based on the protein-coding gene sequences presented in this study also indicate potential pitfalls concerning the choice of marker and reference strains, which may lead to conflicting conclusions in species delineation.

Host-dependent symbiotic efficiency of Rhizobium leguminosarum bv. trifolii strains isolated from nodules of Trifolium rubens.

Trifolium rubens L., commonly known as the red feather clover, is capable of symbiotic interactions with rhizobia. Up to now, no specific symbionts of T. rubens and their symbiotic compatibility with Trifolium spp. have been described. We characterized the genomic diversity of T. rubens symbionts by analyses of plasmid profiles and BOX-PCR. The phylogeny of T. rubens isolates was inferred based on the nucleotide sequences of 16S rRNA and two core genes (atpD, recA). The nodC phylogeny allowed classification of rhizobia nodulating T. rubens as Rhizobium leguminosarum symbiovar trifolii (Rlt). The symbiotic efficiency of the Rlt isolates was determined on four clover species: T. rubens, T. pratense, T. repens and T. resupinatum. We determined that Rlt strains formed mostly inefficient symbiosis with their native host plant T. rubens and weakly effective (sub-optimal) symbiosis with T. repens and T. pratense. The same Rlt strains were fully compatible in the symbiosis with T. resupinatum. T. rubens did not exhibit strict selectivity in regard to the symbionts and rhizobia closely related to Rhizobium grahamii, Rhizobium galegae and Agrobacterium radiobacter, which did not nodulate Trifolium spp., were found amongst T. rubens nodule isolates.

Diversity and plant growth promoting properties of rhizobia isolated from root nodules of Ononis arvensis.

This is the first report describing isolates from root nodules of Ononis arvensis (field restharrow). The aim of this investigation was to describe the diversity, phylogeny, and plant growth promoting features of microsymbionts of O. arvensis, i.e., a legume plant growing in different places of the southern part of Poland. Twenty-nine bacterial isolates were characterized in terms of their phenotypic properties, genome fingerprinting, and comparative analysis of their 16S rRNA, nodC and acdS gene sequences. Based on the nodC and 16S rRNA gene phylogenies, the O. arvensis symbionts were grouped close to bacteria of the genera Rhizobium and Mesorhizobium, which formed monophyletic clusters. The acdS gene sequences of all the isolates tested exhibited the highest similarities to the corresponding gene sequences of genus Mesorhizobium strains. The presence of the acdS genes in the genomes of rhizobia specific for O. arvensis implies that these bacteria may promote the growth and development of their host plant in stress conditions. The isolated bacteria showed a high genomic diversity and, in the BOX-PCR reaction, all of them (except three) exhibited DNA fingerprints specific only for them. Our studies showed that restharrow isolates formed effective symbiotic interactions with their native host (O. arvensis) and Ononis spinosa but not with Trifolium repens and Medicago sativa belonging to the same tribe Trifolieae as Ononis species and not with Lotus corniculatus, representing the tribe Loteae.

Fluorine-free electrolytes for all-solid sodium-ion batteries based on percyano-substituted organic salts.

A new family of fluorine-free solid-polymer electrolytes, for use in sodium-ion battery applications, is presented. Three novel sodium salts withdiffuse negative charges: sodium pentacyanopropenide (NaPCPI), sodium 2,3,4,5-tetracyanopirolate (NaTCP) and sodium 2,4,5-tricyanoimidazolate (NaTIM) were designed andtested in a poly(ethylene oxide) (PEO) matrix as polymer electrolytes for anall-solid sodium-ion battery. Due to unique, non-covalent structural configurations of anions, improved ionic conductivities were observed. As an example, "liquid-like" high conductivities (>1 mS cm-1) were obtained above 70 °C for solid-polymer electrolyte with a PEO to NaTCP molar ratio of 16:1. All presented salts showed high thermal stability and suitable windows of electrochemical stability between 3 and 5 V. These new anions open a new class of compounds with non-covalent structure for electrolytes system applications.

Multilocus sequence analysis supports the taxonomic position of Astragalus glycyphyllos symbionts based on DNA-DNA hybridization.

In this study, the phylogenetic relationship and taxonomic status of six strains, representing different phenons and genomic groups of Astragalus glycyphyllos symbionts, originating from Poland, were established by comparative analysis of five concatenated housekeeping gene sequences (atpD, dnaK, glnA, recA and rpoB), DNA-DNA hybridization and total DNA G+C content. Maximum-likelihood phylogenetic analysis of combined atpD, dnaK, glnA, recA and rpoB sequence data placed the studied bacteria into the clade comprising the genus Mesorhizobium. In the core gene phylograms, four A. glycyphyllos nodule isolates (AG1, AG7, AG15 and AG27) formed a cluster common with Mesorhizobium ciceri, whereas the two other A. glycyphyllos symbionts (AG17 and AG22) were grouped together with Mesorhizobium amorphae and M. septentrionale. The species position of the studied bacteria was clarified by DNA-DNA hybridization. The DNA-DNA relatedness between isolates AG1, AG7, AG15 and AG27 and reference strain M. ciceri USDA 3383T was 76.4-84.2%, and all these A. glycyphyllos nodulators were defined as members of the genomospecies M. ciceri. DNA-DNA relatedness for isolates AG17 and AG22 and the reference strain M. amorphae ICMP 15022T was 77.5 and 80.1%, respectively. We propose that the nodule isolates AG17 and AG22 belong to the genomic species M. amorphae. Additionally, it was found that the total DNA G+C content of the six test A. glycyphyllos symbionts was 59.4-62.1 mol%, within the range for species of the genus Mesorhizobium.

Phylogeny of Symbiotic Genes and the Symbiotic Properties of Rhizobia Specific to Astragalus glycyphyllos L.

The phylogeny of symbiotic genes of Astragalus glycyphyllos L. (liquorice milkvetch) nodule isolates was studied by comparative sequence analysis of nodA, nodC, nodH and nifH loci. In all these genes phylograms, liquorice milkvetch rhizobia (closely related to bacteria of three species, i.e. Mesorhizobium amorphae, Mesorhizobium septentrionale and Mesorhizobium ciceri) formed one clearly separate cluster suggesting the horizontal transfer of symbiotic genes from a single ancestor to the bacteria being studied. The high sequence similarity of the symbiotic genes of A. glycyphyllos rhizobia (99-100% in the case of nodAC and nifH genes, and 98-99% in the case of nodH one) points to the relatively recent (in evolutionary scale) lateral transfer of these genes. In the nodACH and nifH phylograms, A. glycyphyllos nodule isolates were grouped together with the genus Mesorhizobium species in one monophyletic clade, close to M. ciceri, Mesorhizobium opportunistum and Mesorhizobium australicum symbiovar biserrulae bacteria, which correlates with the close relationship of these rhizobia host plants. Plant tests revealed the narrow host range of A. glycyphyllos rhizobia. They formed effective symbiotic interactions with their native host (A. glycyphyllos) and Amorpha fruticosa but not with 11 other fabacean species. The nodules induced on A. glycyphyllos roots were indeterminate with apical, persistent meristem, an age gradient of nodule tissues and cortical vascular bundles. To reflect the symbiosis-adaptive phenotype of rhizobia, specific for A. glycyphyllos, we propose for these bacteria the new symbiovar "glycyphyllae", based on nodA and nodC genes sequences.

Insight into the genomic diversity and relationship of Astragalus glycyphyllos symbionts by RAPD, ERIC-PCR, and AFLP fingerprinting.

We assessed the genomic diversity and genomic relationship of 28 Astragalus glycyphyllos symbionts by three methodologies based on PCR reaction, i.e., RAPD, ERIC-PCR, and AFLP. The AFLP method with one PstI restriction enzyme and selective PstI-GC primer pair had a comparable discriminatory power as ERIC-PCR one and these fingerprinting techniques distinguished among the studied 28 A. glycyphyllos symbionts 18 and 17 genomotypes, respectively. RAPD method was less discriminatory in the genomotyping of rhizobia analyzed and it efficiently resolved nine genomotypes. The cluster analysis of RAPD, ERIC-PCR, and AFLP profiles resulted in a generally similar grouping of the test strains on generated dendrograms supporting a great potential of these DNA fingerprinting techniques for study of genomic polymorphism and evolutionary relationship of A. glycyphyllos nodulators. The RAPD, ERIC-PCR, and AFLP pattern similarity coefficients between A. glycyphyllos symbionts studied was in the ranges 8-100, 18-100, and 23-100%, respectively.

Phenotypic characterization of Astragalus glycyphyllos symbionts and their phylogeny based on the 16S rDNA sequences and RFLP of 16S rRNA gene.

In this study, the nitrogen fixing Astragalus glycyphyllos symbionts were characterized by phenotypic properties, restriction fragment length polymorphism (RFLP), and sequences of 16S rDNA. The generation time of A. glycyphyllos rhizobia in yeast extract mannitol medium was in the range 4-6 h. The studied isolates exhibited a low resistance to antibiotics, a moderate tolerance to NaCl, assimilated di- and trisaccharides, and produced acid in medium containing mannitol as a sole carbon source. In the cluster analysis, based on 86 phenotypic properties of A. glycyphyllos symbionts and the reference rhizobia, examined isolates and the genus Mesorhizobium strains were placed on a single branch, clearly distinct from other lineages of rhizobial genera. By the comparative analysis of 16S rRNA gene sequences and 16S rDNA-RFLP, A. glycyphyllos nodulators were also identified as the members of the genus Mesorhizobium. On the 16S rDNA sequence phylogram, the representatives of A. glycyphyllos nodule isolates formed a robust, monophyletic cluster together with the Mesorhizobium species at 16S rDNA sequence similarity of these bacteria between 95 and 99 %. Similarly, the cluster analysis of the combined RFLP-16S rDNA patterns, obtained with seven restriction endonucleases, showed that A. glycyphyllos rhizobia are closely related to the genus Mesorhizobium bacteria. The taxonomic approaches used in this paper allowed us to classify the studied bacteria into the genus Mesorhizobium.

Isolation of cultivable microorganisms from Polish notes and coins.

The potential role of currency in the spread of pathogenic microflora has been evaluated in many countries. In this study Polish paper notes and the coins in general circulation were assayed for the presence of cultivable bacteria and fungi. Bacterial isolates identification was based on cultural and biochemical characters and by comparison of the 16S rRNA gene sequence. Fungal isolates were recognized with biochemical and morphological criteria. Coagulase-negative staphylococci, (43.6% of the total bacterial count) including Staphylococcus saprophyticus, S. epidermidis, and S. hominis, and Enteroccus spp. (30.8% of the total bacterial count), i.e. E.faecalis, E.faecium and E. durans, were the most numerous bacterial contamination. Penicillium spp., and Aspergillus spp. were the most frequently detected moulds whereas Candida spp. was the most frequent yeast isolated from currency. A visible dependence between the banknote denomination, the physical condition of paper currency, and the number of bacteria and fungi was found. The overall count of bacteria isolated from currency was thousand-fold higher than that of fungal isolates. The total amount of bacteria and fungi recovered from the coins was approximately 2.7-fold lower than that isolated from the notes. In summary, the Polish currency notes were found to be contaminated mainly with commensal bacteria and fungi while the opportunistic pathogenic microorganisms Escherichia coli, Pseudomonas stutzeri and C. albicans were detected at a low frequency.

Protein profiles from intact cells as a tool in Bifidobacterium characteristics.

In this study sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) profiles were analysed and differences were confirmed by a unweighted pair group method with arithmetic average (UPGMA) analysis between bifidobacterial species, such as B. infanis ATCC1567, B. bifidum Bb-12, B. longum KN29, B. catenulatum KD14, and B. animalis BI30. Two dimensional electrophoresis separation profiles were compared, and the most characteristic spots were characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We propose proteins extracted from intact cells as an additional trait for bifidobacteria characterization, together with molecular techniques, which can be used to analyze bacterial protein polymorphism and to distinguish among species.

Phylogenetic diversity of rhizobia associated with horsegram [Macrotyloma uniflorum (Lam.) Verdc.] grown in South India based on glnII, recA and 16S-23S intergenic sequence analyses.

Horsegram [Macrotyloma uniflorum (Lam.) Verdc.) is an important grain legume and fodder crop in India. Information on root nodule endosymbionts of this legume in India is limited. In the present study, 69 isolates from naturally occurring root nodules of horsegram collected from two agro-eco-climatic regions of South India was analyzed by generation rate, acid/alkali reaction on YMA medium, restriction fragment length polymorphism analysis of 16S-23S rDNA intergenic spacer region (IGS), and sequence analyses of IGS and housekeeping genes glnII and recA. Based on the rDNA IGS RFLP by means of three restriction enzymes rhizobia were grouped in five clusters (I-V). By sequence analysis of 16S-23S rDNA IGS identified genotypes of horsegram rhizobia were distributed into five divergent lineages of Bradyrhizobium genus which comprised (I) the IGS type IV rhizobia and valid species B. yuanmingense, (II) the strains of IGS type I and Bradyrhizobium sp. ORS 3257 isolated from Vigna sp., (III) the strains of the IGS type II and Bradyrhizobium sp. CIRADAc12 from Acacia sp., (IV) the IGS type V strains and Bradyrhizobium sp. genospecies IV, and (V) comprising genetically distinct IGS type III strains which probably represent an uncharacterized new genomic species. Nearly, 87% of indigenous horsegram isolates (IGS types I, II, III, and V) could not be related to any other species within the genus Bradyrhizobium. Phylogeny based on housekeeping glnII and recA genes confirmed those results found by the analysis of the IGS sequence. All the isolated rhizobia nodulated Macrotyloma sp. and Vigna spp., and only some of them formed nodules on Arachis hypogeae. The isolates within each IGS type varied in their ability to fix nitrogen. Selection for high symbiotic effective strains could reward horsegram production in poor soils of South India where this legume is largely cultivated.

Genista tinctoria microsymbionts from Poland are new members of Bradyrhizobium japonicum bv. genistearum.

The phylogeny and taxonomic position of slow-growing Genista tinctoria rhizobia from Poland, Ukraine and England were estimated by comparative 16S rDNA, atpD, and dnaK sequence analyses, PCR-RFLP of 16S rDNA, DNA G+C content, and DNA-DNA hybridization. Each core gene studied placed the G. tinctoria rhizobia in the genus Bradyrhizobium cluster with unequivocal bootstrap support. G. tinctoria symbionts and bradyrhizobial strains shared 96-99% similarity in 16S rDNA sequences. Their similarity for atpD and dnaK sequences was 93-99% and 89-99%, respectively. These data clearly showed that G. tinctoria rhizobia belonged to the genus Bradyrhizobium. 16S rDNA sequence analysis was in good agreement with the results of the PCR-RFLP of the 16S rRNA gene. Although the tested strains formed separate lineages to the reference bradyrhizobia their RFLP 16S rDNA patterns were quite similar. The genomic DNA G+C content of three G. tinctoria rhizobia was in the range from 60.64 to 62.83 mol%. Data for species identification were obtained from DNA-DNA hybridization experiments. G. tinctoria microsymbionts from Poland were classified within Bradyrhizobium japonicum genomospecies based on 56-82% DNA-DNA similarity.

Insight into the evolutionary history of symbiotic genes of Robinia pseudoacacia rhizobia deriving from Poland and Japan.

The phylogeny of symbiotic genes of Robinia pseudoacacia (black locust) rhizobia derived from Poland and Japan was studied by comparative sequence analysis of nodA, nodC, nodH, and nifH loci. In phylogenetic trees, black locust symbionts formed a branch of their own suggesting that the spread and maintenance of symbiotic genes within Robinia pseudoacacia rhizobia occurred through vertical transmission. There was 99-100% sequence similarity for nodA genes of Robinia pseudoacacia nodulators, 97-98% for nodC, and 97-100% for nodH and nifH loci. A considerable sequence conservation of sym genes shows that the symbiotic apparatus of Robinia pseudoacacia rhizobia might have evolved under strong host plant constraints. In the nodA and nodC gene phylograms, Robinia pseudoacacia rhizobia grouped with Phaseolus sp. symbionts, although they were not closely related to our isolates based on 16S rRNA genes, and with Mesorhizobium amorphae. nifH gene phylogeny of our isolates followed the evolutionary history of 16S rDNA and Robinia pseudoacacia rhizobia grouped with Mesorhizobium genus species. Nodulation assays revealed that Robinia pseudoacacia rhizobia effectively nodulated their native host and also Amorpha fruticosa and Amorpha californica resulting in a significant enhancement of plant growth. The black locust root nodules are shown to be of indeterminate type.