Michał Kalita

Phylogeny of nodulation genes and symbiotic properties of Genista tinctoria bradyrhizobia

Pairwise comparisons of Genista tinctoria (dyer’s weed) rhizobium nodA, nodC, and nodZ gene sequences to those available in databanks revealed their highest sequence identities to nodulation loci of Bradyrhizobium sp. (Lupinus) strains and rhizobia from other genistoid legumes. On phylogenetic trees, genistoid microsymbionts were grouped together in monophyletic clusters, which suggested that their nodulation genes evolved from a common ancestor. G. tinctoria nodulators formed symbioses not only with the native host, but also with other plants of Genisteae tribe such as: Lupinus luteus, Sarothamnus scoparius, and Chamaecytisus ratisbonensis, and they were classified as the genistoid cross-inoculation group. The dyer’s weed root nodules were designated as indeterminate with apical meristem consisting of infected and uninfected cells.

Phenotypic and genomic characteristics of rhizobia isolated from Genista tinctoria root nodules.

Forty three rhizobial strains isolated from root nodules of Genista tinctoria growing in England, Ukraine, and Poland were compared with 21 representatives of the recognized rhizobial species and two unclassified Bradyrhizobium sp. (Lupinus) strains by performing a numerical analysis of 102 phenotypic features and with the reference bradyrhizobia by simplified AFLP analysis with one restriction enzyme PstI and one selective primer PstI-A. All Genista tinctoria microsymbionts were slow-growing bradyrhizobia with generation time of 10-14 h, acid tolerant, salt sensitive, and antibiotic resistant. Cluster analysis based on the phenotypic properties of all bacteria included, grouped dyers broom rhizobia together with Bradyrhizobium strains, and classified them into three major phena according to their geographic origin. Genista tinctoria nodule isolates were separated into three clusters with the strain composition as in a phenogrouping by AFLP patterns. The presented results, suggest the relationship of G. tincoria microsymbionts to Bradyrhizobium species and show the usefulness of AFLP analysis for differentiation and classification of the studied rhizobia.

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.

New taxonomic markers for identification of Rhizobium leguminosarum and discrimination between closely related species

Rhizobia, producing species-specific exopolysaccharides (EPSs), comprise a very diverse group of soil bacteria that are able to establish nitrogen-fixing symbioses with legumes. Based on the sequences of R. leguminosarum EPS synthesis genes, a sensitive and reliable PCR-based method for identification and subsequent discrimination between Rhizobium species has been developed and tested. For identification of R. leguminosarum, primer sets I–III complementary to sequences of rosR, pssA and pssY genes were proposed. Further sets of primers (IV–VII) were designed for discrimination between R. leguminosarum biovars. The usefulness of the method was examined using a wide range of R. leguminosarum strains isolated from different host plants nodules originating from different regions of Poland. We demonstrate a high discriminating power of primer sets I–III that allow distinguishing R. leguminosarum and two closely related species, R. etli and R. gallicum. This new approach is applicable to identification of R. leguminosarum strains, originating from nodules or soil, where many other closely related bacteria are expected to be present. Based on the nucleotide sequence of rosR and pssA genes, phylogenetic relationships of selected R. leguminosarum isolates were determined. Our results indicate that both rosR and pssA might be useful markers to differentiate and define relationships within a group of R. leguminosarum strains.

Analysis of genetic relationship of Sarothamnus scoparius microsymbionts and Bradyrhizobium sp. by hybridization in microdilution wells

DNA-DNA hybridization in microdilution wells was successfully used to determine the overall genomic similarity among Sarothamnus scoparius microsymbionts isolated from Poland and Japan and representatives of Bradyrhizobium species including Bradyrhizobium sp. (Lupinus) USDA3045. Geographically different S. scoparius rhizobia diverged into two genomospecies at the DNA-DNA similarity level of approximately 49%. Polish isolates exhibited high DNA similarity levels to the DNA of Bradyrhizobium japonicum strains (approximately 78%) and formed a common genomospecies. Japanese S. scoparius rhizobia were allocated into a new genomic species due to the low similarity levels of their DNA to the DNA of representative strains of the Bradyrhizobium genus (from 19% to 52%).

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.

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.

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.

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.

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.