A series of Tn5-induced mutants which showed enhanced symbiotic effectiveness (Eff ) was isolated from Rhizobiurn rneliloti strains CXMI-105 and CXMI-188. Alfalfa plants inoculated by the Eff + + mutants had significantly higher shoot dry mass than plants inoculated by the parental strains. In the greenhouse, the most effective mutants increased the shoot dry mass of the host plants by 23-26 O/ O and plant total nitrogen by 23-27 %. Interestingly, the frequency of the E f f -* mutants in strain CXMI-188 was higher than in the strain CXMI-105 (1.1 O/ O versus 0.4%); this was also the case for the auxotrophic mutants (1.2O/0 in CXMI-188 versus 0.3% in CXMI-105). Genetic analysis of the mutants showed that the enhanced symbiotic effectiveness was cotransducible with Tn5. By the use of Southern hybridization and plasmid transfer, it was found that ten Tn5 insertions were located in the chromosome, five in megaplasmid 1, and six in megaplasmid 2.

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...

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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.

242(1) The third sentence of the abstract should read: It was found that wild species of Medicago , Melilotus , and Trigonella preferably formed symbioses with salttolerant root-nodule bacteria in both salinized and nonsalinized soils.(2) On p. 78, left column, the last sentence of the first paragraph should read: Species identification of the natural isolates was performed by amplified 16S ribosomal DNA restriction analysis (ARDRA) as in [13].(3) On p. 80, right column, second paragraph, the first sentence should read: Another problem was the correlation between the salt tolerance of the N and T isolates and their symbiotic efficiency in the presence of salt.

Two Tn5-mutants, T37 and T107, with decreased nodulation competitiveness (Cmp-) were isolated in Rhizobium meliloti strain CXM1-105. Their Cmp-phenotype was repeatedly displayed in sterile plant tests, where competitiveness was measured against a Fix-reference strain CXM1-48 and the parental strain CXMI-105. In addition mutant T37 showed a decreased nodulation rate (Nra-) at the early stages of alfalfa development (9-18 days after germination). Its mutant phenotype was demonstrated also under greenhouse conditions when it was evaluated against reference strain CXM1-48, and against indigenous rhizobial strains persisting in the soil.Tn5 insertions from mutants T37 and T107 were transduced using phage ~bM12 to the parental strain CXM1-105. All tested transductants showed Cmp-and Nra-phenotype in the case of T37 and Cmp-phenotype in the case of T107. By means of conjugations with avirulent R. meliloti mutant ZB 121 and plasmidless Agrobacterium tumefaciens UBAPF2 we located both Tn5-insertions on the R. meliloti Sym-plasmid.

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