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 nif H) 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. may be traced using specialised symbiotic (sym) genes representing the accessory parts of bacterial genomes, which differ in their natural histories from housekeeping genes (hkg) representing the core parts of genomes [4]. As a result of co-evolutionary processes, symbiosis is formed between tightly co-adapted cross-inoculation groups of rhizobia and legumes, and their coevolution is directed by a set of symbiosis-specific genes from each partner [5][6][7]. In some rhizobia, sym genes are more susceptible to autonomous horizontal gene transfer than hkg genes, because they are located on plasmids-mobile elements of the genome [3]. This results in an intensive recombination of host specific and chromosomal markers [8]. For example, Rhizobium leguminosarum is composed of two biovars, which have diverged based on their plasmid-encoded host ranges [9]. Biovar viciae (Rlv) nodulates legumes from the Fabeae tribe, while biovar trifolii (Rlt) nodulates clovers from the Trifolieae tribe; however, they show a conservative chromosomal arrangement of hkg markers (Figure 1).Even so, divergent evolution of rhizobia is not restricted to sym genes. Application of the average nucleotide identity (ANI) method has demonstrated that a local R. leguminosarum population could be separated into five genomic species, differing in their hkg genes, representing their cor...

Two bacterial strains Ach-343 and Opo-235 were isolated, respectively from nodules of Miocene-Pliocene relict legumes Astragalus chorinensis Bunge and Oxytropis popoviana Peschkova originated from Buryatia (Baikal Lake region, Russia). For identification of these strains the sequencing of 16S rRNA (rrs) gene was used. Strain Opo-235 belonged to the species Mesorhizobium japonicum, while the strain Ach-343 was identified as M. kowhaii (100 and 99.9% rrs similarity with the type strains MAFF 303099T and ICMP 19512T, respectively). Symbiotic genes of these strains as well as some genes that promote plant growth (acdS, gibberellin- and auxin-synthesis related genes) were searched throughout the whole genome sequences. The sets of plant growth-promoting genes found were almost identical in both strains, whereas the sets of symbiotic genes were different and complemented each other with several nod, nif, and fix genes. Effects of mono- and co-inoculation of Astragalus sericeocanus, Oxytropis caespitosa, Glycyrrhiza uralensis, Medicago sativa, and Trifolium pratense plants with the strains M. kowhaii Ach-343 and M. japonicum Opo-235 expressing fluorescent proteins mCherry (red) and EGFP (green) were studied in the gnotobiotic plant nodulation assay. It was shown that both strains had a wide range of host specificity, including species of different legume genera from two tribes (Galegeae and Trifolieae). The effects of co-microsymbionts on plants depended on the plant species and varied from decrease, no effect, to increase in the number of nodules, nitrogen-fixing activity and plant biomass. One of the reasons for this phenomenon may be the discovered complementarity in co-microsymbionts of symbiotic genes responsible for the specific modification of Nod-factors and nitrogenase activity. Localization and co-localization of the strains in nodules was confirmed by the confocal microscopy. Analysis of histological and ultrastructural organization of A. chorinensis and O. popoviana root nodules was performed. It can be concluded that the strains M. kowhaii Ach-343 and M. japonicum Opo-235 demonstrate lack of high symbiotic specificity that is characteristic for primitive legume-rhizobia systems. Further study of the root nodule bacteria having complementary sets of symbiotic genes will contribute to clarify the evolutionary paths of legume-rhizobia relationships and the mechanisms of effective integration between partners.

Gram-stain-negative strains V5/3MT, V5/5K, V5/5M and V5/13 were isolated from root nodules of Vicia alpestris plants growing in the North Ossetia region (Caucasus). Sequencing of the partial 16S rRNA gene (rrs) and four housekeeping genes (dnaK, gyrB, recA and rpoB) showed that the isolates from V. alpestris were most closely related to the species Microvirga zambiensis (order Rhizobiales, family Methylobacteriaceae) which was described for the single isolate from root nodule of Listia angolensis growing in Zambia. Sequence similarities between the Microvirga-related isolates and M. zambiensis WSM3693T ranged from 98.5 to 98.7 % for rrs and from 79.7 to 95.8 % for housekeeping genes. Cellular fatty acids of the isolates V5/3MT, V5/5K, V5/5M and V5/13 included important amounts of C18 : 1ω7c (54.0-67.2 %), C16 : 0 (6.0-7.8 %), C19 : 0 cyclo ω8c (3.1-10.2 %), summed feature 2 (comprising one or more of iso-C16 : 1 I, C14 : 0 3-OH and unknown ECL 10.938, 5.8-22.5 %) and summed feature 3 (comprising C16 : 1ω7c and/or iso-C15 : 02-OH, 2.9-4.0 %). DNA-DNA hybridization between the isolate V5/3MT and M. zambiensis WSM3693T revealed DNA-DNA relatedness of 35.3 %. Analysis of morphological and physiological features of the novel isolates demonstrated their unique phenotypic profile in comparison with reference strains from closely related species of the genus Microvirga. On the basis of genotypic and phenotypic analysis, a novel species named Microvirga ossetica sp. nov. is proposed. The type strain is V5/3MT (=LMG 29787T=RCAM 02728T). Three additional strains of the species are V5/5K, V5/5M and V5/13.

Ten rhizobial strains were isolated from root nodules of a relict legume Oxytropis popoviana Peschkova. For identification of the isolates, sequencing of rrs, the internal transcribed spacer region, and housekeeping genes recA, glnII, and rpoB was used. Nine fast-growing isolates were Mesorhizobium-related; eight strains were identified as M. japonicum and one isolate belonged to M. kowhaii. The only slow-growing isolate was identified as a Bradyrhizobium sp. Two strains, M. japonicum Opo-242 and Bradyrhizobium sp. strain Opo-243, were isolated from the same nodule. Symbiotic genes of these isolates were searched throughout the whole-genome sequences. The common nodABC genes and other symbiotic genes required for plant nodulation and nitrogen fixation were present in the isolate Opo-242. Strain Opo-243 did not contain the principal nod, nif, and fix genes; however, five genes (nodP, nodQ, nifL, nolK, and noeL) affecting the specificity of plant-rhizobia interactions but absent in isolate Opo-242 were detected. Strain Opo-243 could not induce nodules but significantly accelerated the root nodule formation after coinoculation with isolate Opo-242. Thus, we demonstrated that taxonomically different strains of the archaic symbiotic system can be co-microsymbionts infecting the same nodule and promoting the nodulation process due to complementary sets of symbiotic genes.

A collection of rhizobial strains isolated from root nodules of the narrowly endemic legume species Oxytropis erecta, O. anadyrensis, O. kamtschatica and O. pumilio originated from the Kamchatka Peninsula (Russian Federation) was obtained. Analysis of the 16S rRNA gene sequence showed a significant diversity of isolates belonging to families Rhizobiaceae (genus Rhizobium), Phyllobacteriaceae (genera Mesorhizobium, Phyllobacterium) and Bradyrhizobiaceae (genera Bosea, Tardiphaga). Plant nodulation assay showed that only strains belonging to the genus Mesorhizobium could form nitrogen-fixing nodules on Oxytropis plants. The strains M. loti 582 and M. huakuii 583, in addition to symbiotic clusters, possessed genes of the T3SS and T6SS secretion systems, which can influence the host specificity of strains. These strains formed nodules of two types (elongated and rounded) on O. kamtschatica roots. We suggest this phenomenon may result from Nod-factor dependent and independent nodulation strategies. The obtained strains are of interest for further study of the T3SS and T6SS gene function and their role in the development of rhizobium-legume symbiosis. The prospects of using rhizobia having both gene systems related to symbiotic and non-symbiotic nodulation strategies to enhance the efficiency of plant-microbe interactions by expanding the host specificity and increasing nodulation efficiency are discussed.