Rhizobial isolates in active layer samples of permafrost soil of Spitsbergen, Arctic

Twenty-nine strains were isolated from two samples of the permafrost active layer of the Spitsbergen archipelago. The estimated number of bacteria ranged from 4.0⋅104 to 1.7⋅107 CFU∙g-1. As a result of sequencing of the 16S rRNA (rrs) genes, the isolates were assigned to 13 genera belonging to the phyla Actinobacteria, Proteobacteria (classes α, β, and γ), Bacteroidetes, and Firmicutes. Six isolates belonged to the rhizobial genus Mesorhizobium (order Rhizobiales). A plant nodulation assay with seedlings of legume plants Astragalus norvegicus, A. frigidus, A. subpolaris and Oxytropis sordida, originated from Khibiny (Murmansk region, Russia) and inoculated with Mesorhizobium isolates, showed the inability of these strains to form nodules on plant roots. Symbiotic (sym) genes nodC and nodD were not detected in Mesorhizobium strains either.

Vavilovia formosa rhizobia symbionts belong to Rhizobium leguminosarum bv. viciae species, but form a separate group within it

Ecological isolation, group separation of hkg and sym genes, along with the results of the sterile tube test demonstrate that symbionts of V. formosa belong to R. leguminosarum bv. viciae species, but form a separate group within it.

Search for Ancestral Features in Genomes of Rhizobium leguminosarum bv. viciae Strains Isolated from the Relict Legume Vavilovia formosa

Vavilovia formosa is a relict leguminous plant growing in hard-to-reach habitats in the rocky highlands of the Caucasus and Middle East, and it is considered as the putative closest living relative of the last common ancestor (LCA) of the Fabeae tribe. Symbionts of Vavilovia belonging to Rhizobium leguminosarum bv. viciae compose a discrete group that differs from the other strains, especially in the nucleotide sequences of the symbiotically specialised (sym) genes. Comparison of the genomes of Vavilovia strains with the reference group composed of R. leguminosarum bv. viciae strains isolated from Pisum and Vicia demonstrated that the vavilovia strains have a set of genomic features, probably indicating the important stages of microevolution of the symbiotic system. Specifically, symbionts of Vavilovia (considered as an ancestral group) demonstrated a scattered arrangement of sym genes (>90 kb cluster on pSym), with the location of nodT gene outside of the other nod operons, the presence of nodX and fixW, and the absence of chromosomal fixNOPQ copies. In contrast, the reference (derived) group harboured sym genes as a compact cluster (<60 kb) on a single pSym, lacking nodX and fixW, with nodT between nodN and nodO, and possessing chromosomal fixNOPQ copies. The TOM strain, obtained from nodules of the primitive “Afghan” peas, occupied an intermediate position because it has the chromosomal fixNOPQ copy, while the other features, the most important of which is presence of nodX and fixW, were similar to the Vavilovia strains. We suggest that genome evolution from the ancestral to the derived R. leguminosarum bv. viciae groups follows the “gain-and-loss of sym genes” and the “compaction of sym cluster” strategies, which are common for the macro-evolutionary and micro-evolutionary processes. The revealed genomic features are in concordance with a relict status of the vavilovia strains, indicating that V. formosa coexists with ancestral microsymbionts, which are presumably close to the LCA of R. leguminosarum bv. viciae.

Rhizobia Isolated from the Relict Legume Vavilovia formosa Represent a Genetically Specific Group within Rhizobium leguminosarum biovar viciae

Twenty-two rhizobia strains isolated from three distinct populations (North Ossetia, Dagestan, and Armenia) of a relict legume Vavilovia formosa were analysed to determine their position within Rhizobium leguminosarum biovar viciae (Rlv). These bacteria are described as symbionts of four plant genera Pisum, Vicia, Lathyrus, and Lens from the Fabeae tribe, of which Vavilovia is considered to be closest to its last common ancestor (LCA). In contrast to biovar viciae, bacteria from Rhizobium leguminosarum biovar trifolii (Rlt) inoculate plants from the Trifolieae tribe. Comparison of house-keeping (hkg: 16S rRNA, glnII, gltA, and dnaK) and symbiotic (sym: nodA, nodC, nodD, and nifH) genes of the symbionts of V. formosa with those of other Rlv and Rlt strains reveals a significant group separation, which was most pronounced for sym genes. A remarkable feature of the strains isolated from V. formosa was the presence of the nodX gene, which was commonly found in Rlv strains isolated from Afghanistan pea genotypes. Tube testing of different strains on nine plant species, including all genera from the Fabeae tribe, demonstrated that the strains from V. formosa nodulated the same cross inoculation group as the other Rlv strains. Comparison of nucleotide similarity in sym genes suggested that their diversification within sym-biotypes of Rlv was elicited by host plants. Contrariwise, that of hkg genes could be caused by either local adaptation to soil niches or by genetic drift. Long-term ecological isolation, genetic separation, and the ancestral position of V. formosa suggested that symbionts of V. formosa could be responsible for preserving ancestral genotypes of the Rlv biovar.

Phenotypic characterization of sym8 and sym9, two genes conditioning non-nodulation in Pisum sativum 'Sparkle'

Two non-nodulating and non-allelic mutants of P. sativum (L.) 'Sparkle', R25 (sym8) and R72 (sym9), were obtained by γ-radiation. Non-nodulation in each mutant was conditioned by a single recessive gene. Growth analysis indicated that if the mutants were provided with adequate fixed nitrogen, they both had the same growth rate as their nodulating parent, 'Sparkle'. Reciprocal grafts between mutants and parent plants indicated that non-nodulation was controlled by the root genotype. Both mutants induced the common nod genes in rhizobia, and had either higher or similar numbers of rhizobia attached to their roots as had 'Sparkle'. Microscopic studies indicated that root hairs of R72 deformed after inoculation, whereas root hairs of R25 did not show such deformation. In both mutants hair curling, infection thread formation, and nodule meristem initiation did not occur. Key words: mutants, sym genes, nodulation stages, symbiosis.