RESUMEN
A collection of rhizobial strains isolated from root nodules of the narrowly endemic legume species Oxytropis erecta, O. anadyrensis, O. kamtschatica, and O. pumilio originating from the Kamchatka Peninsula (Russian Federation) was obtained. Analysis of the 16S ribosomal RNA gene sequence showed a significant diversity of isolates belonging to families Rhizobiaceae (genus Rhizobium), Phyllobacteriaceae (genera Mesorhizobium, Phyllobacterium), and Bradyrhizobiaceae (genera Bosea, Tardiphaga). A plant nodulation assay showed that only strains belonging to 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 type III and type VI secretion systems (T3SS and T6SS, respectively), 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 nonsymbiotic nodulation strategies to enhance the efficiency of plant-microbe interactions by expanding the host specificity and increasing nodulation efficiency are discussed.
Asunto(s)
Bradyrhizobiaceae , Mesorhizobium , Oxytropis/microbiología , Rhizobium , Simbiosis , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo VI/genética , Bradyrhizobiaceae/genética , Mesorhizobium/genética , Filogenia , ARN Ribosómico 16S/genética , Rhizobium/genética , Nódulos de las Raíces de las Plantas/microbiologíaRESUMEN
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.
Asunto(s)
ADN Bacteriano/genética , Evolución Molecular , Fabaceae/microbiología , Genes Bacterianos , Rhizobium leguminosarum/genética , Simbiosis/genética , Proteínas Bacterianas/genética , Rhizobium leguminosarum/aislamiento & purificación , Especificidad de la EspecieRESUMEN
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.
Asunto(s)
ADN Bacteriano/genética , Fabaceae/microbiología , Genotipo , Filogenia , Rhizobium leguminosarum/genética , Nódulos de las Raíces de las Plantas/microbiología , Simbiosis/genética , Rhizobium leguminosarum/aislamiento & purificaciónRESUMEN
Two Gram-stain-negative strains, RCAM04680T and RCAM04685, were isolated from root nodules of the relict legume Caragana jubata (Pall.) Poir. originating from the south-western shore of Lake Khuvsgul (Mongolia). The 16S rRNA gene (rrs) sequencing data showed that these novel isolates belong to the genus Bosea and are phylogenetically closest to the type strains Bosea lathyri LMG 26379T, Bosea vaviloviae LMG 28367T, Bosea massiliensis LMG 26221T and Bosea lupini LMG 26383T (the rrs-similarity levels were 98.7-98.8â%). The recA gene of strain RCAM04680T showed the highest sequence similarity to the type strain B. lupini LMG 26383T (95.4â%), while its atpD gene was closest to that of B. lathyri LMG 26379T (94.4â%). The ITS, dnaK and gyrB sequences of this isolate were most similar to the B. vaviloviae LMG 28367T (86.8â% for ITS, 90.4â% for the other genes). The most abundant fatty acid was C18â:â1ω7c (40.8â%). The whole genomes of strains RCAM04680T and RCAM04685 were identical (100â% average nucleotide identity). The highest average nucleotide identity value (82.8â%) was found between the genome of strain RCAM04680T and B. vaviloviae LMG 28367T. The common nodABC genes required for legume nodulation were absent in both strains; however, some other symbiotic nol, nod, nif and fix genes were detected. Based on the genetic study, as well as analyses of the whole-cell fatty acid compositions and phenotypic properties, a new species, Boseacaraganae sp. nov. (type strain RCAM04680T (=LMG 31125T), is proposed.
Asunto(s)
Bradyrhizobiaceae/clasificación , Caragana/microbiología , Filogenia , Nódulos de las Raíces de las Plantas/microbiología , Técnicas de Tipificación Bacteriana , Composición de Base , Bradyrhizobiaceae/aislamiento & purificación , ADN Bacteriano/genética , Ácidos Grasos/química , Genes Bacterianos , Mongolia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , SimbiosisRESUMEN
Gram-negative strains Tri-36, Tri-38, Tri-48T and Tri-53 were isolated from root nodules of the relict legume Oxytropis triphylla (Pall.) Pers. originating from Zunduk Cape (Baikal Lake region, Russia). 16S rRNA gene sequencing showed that the novel isolates were phylogenetically closest to the type strains Phyllobacterium sophorae LMG 27899T, Phyllobacterium brassicacearum LMG 22836T, Phyllobacterium endophyticum LMG 26470T and Phyllobacterium bourgognense LMG 22837T while similarity levels between the isolates and the most closely related strain P. endophyticum LMG 26470T were 98.8-99.5â%. The recA and glnII genes of the isolates showed highest sequence similarities with P. sophorae LMG 27899T (95.4 and 89.5â%, respectively) and P. brassicacearum LMG 22836T (91.4 and 85.1â%, respectively). Comparative analysis of phenotypic properties between the novel isolates and the closest reference strains P. sophorae LMG 27899T, P. brassicacearum LMG 22836T and P. endophyticum LMG 26470T was performed using a microassay system. Average nucleotide identities between the whole genome sequences of the isolates Tri-38 and Tri-48T and P. sophorae LMG 27899T, P. brassicacearum LMG 22836T and P. endophyticum LMG 26470T ranged from 79.23â% for P. endophyticum LMG 26470T to 85.74â% for P. sophorae LMG 27899T. The common nodABC genes required for legume nodulation were absent from strains Tri-38 and Tri-48T, although some other symbiotic nod and fix genes were detected. On the basis of genotypic and phenotypic analysis, a novel species, Phyllobacterium zundukense sp. nov. (type strain Tri-48T=LMG 30371T=RCAM 03910T), is proposed.
Asunto(s)
Oxytropis/microbiología , Phyllobacteriaceae/clasificación , Filogenia , Nódulos de las Raíces de las Plantas/microbiología , Técnicas de Tipificación Bacteriana , ADN Bacteriano/genética , Genes Bacterianos , Phyllobacteriaceae/genética , Phyllobacteriaceae/aislamiento & purificación , ARN Ribosómico 16S/genética , Federación de Rusia , Análisis de Secuencia de ADN , SimbiosisRESUMEN
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.
Asunto(s)
Bradyrhizobium/genética , Mesorhizobium/genética , Oxytropis/microbiología , Nodulación de la Raíz de la Planta/genética , Simbiosis/genética , Bradyrhizobium/fisiología , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Mesorhizobium/fisiología , Filogenia , Nodulación de la Raíz de la Planta/fisiología , Simbiosis/fisiologíaRESUMEN
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.