Guidelines for the description of rhizobial symbiovars.

Rhizobia are bacteria that form nitrogen-fixing nodules in legume plants. The sets of genes responsible for both nodulation and nitrogen fixation are carried in plasmids or genomic islands that are often mobile. Different strains within a species sometimes have different host specificities, while very similar symbiosis genes may be found in strains of different species. These specificity variants are known as symbiovars, and many of them have been given names, but there are no established guidelines for defining or naming them. Here, we discuss the requirements for guidelines to describe symbiovars, propose a set of guidelines, provide a list of all symbiovars for which descriptions have been published so far, and offer a mechanism to maintain a list in the future.

The rhizosphere of a drought-tolerant plant species in Morocco: A refuge of high microbial diversity with no taxon preference.

Arid and semi-arid areas are facing increasingly severe water deficits that are being intensified by global climate changes. Microbes associated with plants native to arid regions provide valuable benefits to plants, especially in water-stressed environments. In this study, we used 16S rDNA metabarcoding analysis to examine the bacterial communities in the bulk soil, rhizosphere and root endosphere of the plant Malva sylvestris L. in Morocco, along a gradient of precipitation. We found that the rhizosphere of M. sylvestris did not show significant differences in beta-diversity compared to bulk soil, although, it did display an increased degree of alpha-diversity. The endosphere was largely dominated by the genus Rhizobium and displayed remarkable variation between plants, which could not be attributed to any of the variables observed in this study. Overall, the effects of precipitation level were relatively weak, which may be related to the intense drought in Morocco at the time of sampling. The dominance of Rhizobium in a non-leguminous plant is particularly noteworthy and may permit the utilization of this bacterial taxon to augment drought tolerance; additionally, the absence of any notable selection of the rhizosphere of M. sylvestris suggests that it is not significatively affecting its soil environment.

Geographical and climatic distribution of lentil-nodulating rhizobia in Iran.

Lentil is one of the most important legumes cultivated in various provinces of Iran. However, there is limited information about the symbiotic rhizobia of lentils in this country. In this study, molecular identification of lentil-nodulating rhizobia was performed based on 16S-23S rRNA intergenic spacer (IGS) and recA, atpD, glnII, and nodC gene sequencing. Using PCR-RFLP analysis of 16S-23S rRNA IGS, a total of 116 rhizobia isolates were classified into 20 groups, leaving seven strains unclustered. Phylogenetic analysis of representative isolates revealed that the rhizobia strains belonged to Rhizobium leguminosarum and Rhizobium laguerreae, and the distribution of the species is partially related to geographical location. Rhizobium leguminosarum was the dominant species in North Khorasan and Zanjan, while R. laguerreae prevailed in Ardabil and East Azerbaijan. The distribution of the species was also influenced by agroecological climates; R. leguminosarum thrived in cold semiarid climates, whereas R. laguerreae adapted to humid continental climates. Both species exhibited equal dominance in the Mediterranean climate, characterized by warm, dry summers and mild, wet winters, in Lorestan and Kohgiluyeh-Boyer Ahmad provinces.

International Committee on Systematics of Prokaryotes, Subcommittee on the taxonomy of Rhizobia and Agrobacteria, minutes of the annual meeting by videoconference, 5 July 2021, followed by online discussion until 31 December 2021.

Minutes of the closed meeting of the International Committee on Systematics of Prokaryotes Subcommittee on the Taxonomy of Rhizobia and Agrobacteria held by videoconference, 5 July 2021, followed by online discussion until 31 December 2021, and list of recent species.

Complete genomic sequence and phylogenomics analysis of Agrobacterium strain AB2/73: a new Rhizobium species with a unique mega-Ti plasmid.

BACKGROUND: The Agrobacterium strain AB2/73 has a unique host range for the induction of crown gall tumors, and contains an exceptionally large, over 500 kbp mega Ti plasmid. We used whole genome sequencing to fully characterize and comparatively analyze the complex genome of strain AB2/73, including its Ti plasmid and virulence factors. RESULTS: We obtained a high-quality, full genomic sequence of AB2/73 by a combination of short-read Illumina sequencing and long-read Nanopore sequencing. The AB2/73 genome has a total size of 7,266,754 bp with 59.5% GC for which 7012 genes (6948 protein coding sequences) are predicted. Phylogenetic and comparative genomics analysis revealed that strain AB2/73 does not belong to the genus Agrobacterium, but to a new species in the genus Rhizobium, which is most related to Rhizobium tropici. In addition to the chromosome, the genome consists of 6 plasmids of which the largest two, of more than 1 Mbp, have chromid-like properties. The mega Ti plasmid is 605 kbp in size and contains two, one of which is incomplete, repABC replication units and thus appears to be a cointegrate consisting of about 175 kbp derived from an unknown Ti plasmid linked to 430 kbp from another large plasmid. In pTiAB2/73 we identified a complete set of virulence genes and two T-DNAs. Besides the previously described T-DNA we found a larger, second T-DNA containing a 6b-like onc gene and the acs gene for agrocinopine synthase. Also we identified two clusters of genes responsible for opine catabolism, including an acc-operon for agrocinopine degradation, and genes putatively involved in ridéopine catabolism. The plasmid also harbours tzs, iaaM and iaaH genes for the biosynthesis of the plant growth regulators cytokinin and auxin. CONCLUSIONS: The comparative genomics analysis of the high quality genome of strain AB2/73 provided insight into the unusual phylogeny and genetic composition of the limited host range Agrobacterium strain AB2/73. The description of its unique genomic composition and of all the virulence determinants in pTiAB2/73 will be an invaluable tool for further studies into the special host range properties of this bacterium.

Diversity and distribution of Sophora davidii rhizobia in habitats with different irradiances and soil traits in Loess Plateau area of China.

To investigate the diversity and distribution of rhizobia associated with Sophora davidii in habitats with different light and soil conditions at the Loess Plateau, we isolated rhizobia from root nodules of this plant grown at 14 sites at forest edge or understory in Shaanxi Province. Based on PCR-RFLP and phylogenies of 16S rRNA gene, housekeeping genes (atpD, dnaK, recA), and symbiosis genes (nodC and nifH), a total of 271 isolates were identified as 16 Mesorhizobium genospecies, belonging to four nodC lineages, and three nifH lineages. The dominance of M. waimense in the forest edge and of M. amorphae/Mesorhizobium sp. X in the understory habitat evidenced the illumination as a possible factor to affect the diversity and biogeographic patterns of rhizobia. However, the results of Canonical Correlation Analysis (CCA) among the environmental factors and distribution of rhizobial genospecies illustrated that soil pH and contents of total phosphorus, total potassium and total organic carbon were the main determinants for the community structure of S. davidii rhizobia, while the illumination conditions and available P presented similar and minor effects. In addition, high similarity of nodC and nifH genes between Mesorhizobium robiniae and some S. davidii rhizobia under the forest of Robinia pseudoacacia might be evidence for symbiotic gene lateral transfer. These findings firstly brought an insight into the diversity and distribution of rhizobia associated with S. davidii, and revealed illumination conditions a possible factor with impacts less than the soil traits to drive the symbiosis association between rhizobia and their host legumes.

Functional and genetic diversity of native rhizobial isolates nodulating cowpea (Vigna unguiculata L. Walp.) in Mozambican soils.

Identification and symbiotic characterization of indigenous rhizobial isolates are the basis for inoculant formulations needed for sustainable grain legume production. This study screened for morpho-genetic diversity of indigenous cowpea nodulating rhizobia in farmers' fields across two contrasting agroecological zones of Northern Mozambique. The photosynthetic function induced by the isolates in their homologous cowpea was assessed. The results showed high genetic variability among the isolates based on morphology and ERIC-PCR fingerprinting. The trap cowpea genotype did not influence the diversity of isolates collected from the two different agroecologies, suggesting that the cowpea-rhizobia compatibility may be conserved at species level. Phylogenetic analysis of the 16S rRNA gene assigned representative rhizobial isolates to species in the Bradyrhizobium and Rhizobium genera, with some isolates showing high divergence from the known reference type strains. The isolates from both agroecologies highly varied in the number and biomass of nodules induced in the homologous cowpea, resulting in variable plant growth and photosynthetic activities. A total of 72% and 83% of the isolates collected from the agroecological zones 7 and 8 were respectively classified as highly effective candidates with > 80% relative effectiveness compared to plants fertilized with nitrate, indicating that elite native strains populated the studied soils. Moreover, the top 25% of high N2-fixing isolates from the two agroecologies recorded relative effectiveness ranging from 115 to 154%, values higher than the effectiveness induced by the commercial Bradyrhizobium sp. strain CB756. These strains are considered as having potential for use in inoculant formulations. However, future studies should be done to assess the ecologically adaptive traits and symbiotic performance under field conditions.

Rhizobia and endophytic bacteria isolated from rainforest fragments within an iron ore mining site of the Eastern Brazilian Amazon.

The aim of the present study was to isolate and evaluate the diversity of rhizobial and endophytic bacterial strains from undisturbed native rainforests within an iron ore mining site of the Serra Norte de Carajás in the Eastern Brazilian Amazon region to assess their biotechnological utility in reclamation of areas. Experiments were conducted to capture strains from samples of the soil of these forests at the sites Arenito II, Noroeste II, and Sul IV using Macroptilium atropurpureum and Mimosa acutistipula var. ferrea as trap host plants. Only M. atropurpureum nodulated, and the different bacterial strains were isolated from its nodules. There was no difference in the number of nodules among the areas, but the Arenito II bacterial community was the most efficient, indicated by the aboveground biomass production and suitable shoot mass/root mass ratio. Fifty-two (52) bacterial isolates were obtained, distributed in five groups, including nodulating and endophytic bacteria: 32 from Arenito II, 12 from Noroeste II, and 8 from Sul IV. The nodulating Bradyrhizobium genus was common to the three areas, whereas Paraburkholderia was found only in Arenito II. The nodD1 gene was amplified in all the strains of both nodulating genera. Strains of the nodulating genus Methylobacterium were also isolated from the three areas; however, they did not nodulate the host of origin, and their nodD1 gene was not amplified. Endophytic strains were also isolated from the genera Paenibacillus, Pantoea, and Leifsonia in Arenito II, Leifsonia in Noroeste I, and Paenibacillus in Sul IV. The greater nodulation and rhizobial and endophytic bacterial diversity observed in Arenito II were probably due to the more suitable edaphic properties of the area. The isolated strains were incorporated in the collection of the Department of Soil Science of UFLA and will be investigated in relation to their symbiotic characteristics with native host plants, as well as their ability to perform other biological processes.

Phylogenetic study of rhizobia nodulating pea (Pisum sativum) isolated from different geographic locations in Tunisia.

Nodulated Pisum sativum plants showed the presence of native rhizobia in 16 out of 23 soil samples collected especially in northern and central Tunisia. A total of 130 bacterial strains were selected and three different ribotypes were revealed after PCR-RFLP analysis. Sequence analyses of rrs and four housekeeping genes (recA, atpD, dnaK and glnII) assigned 35 isolates to Rhizobium laguerreae, R. ruizarguesonis, Agrobacterium radiobacter, Ensifer meliloti and two putative genospecies. R. laguerreae was the most dominant species nodulating P. sativum with 63%. The isolates 21PS7 and 21PS15 were assigned to R. ruizarguesonis, and this is the first report of this species in Tunisia. Two putative new lineages were identified, since strains 25PS6, 10PS4 and 12PS15 clustered distinctly from known rhizobia species but within the R. leguminosarum complex (Rlc) with the most closely related species being R. indicum with 96.4% sequence identity. Similarly, strains 16PS2, 3PS9 and 3PS18 showed 97.4% and 97.6% similarity with R. sophorae and R. laguerreae, respectively. Based on 16S-23S intergenic spacer (IGS) fingerprinting, there was no clear association between the strains and their geographic locations. According to nodC and nodA phylogenies, strains of Rlc species and, interestingly, strain 8PS18 identified as E. meliloti, harbored the symbiotic genes of symbiovar viciae and clustered in two different clades showing heterogeneity within the symbiovar. All these strains nodulated and fixed nitrogen with pea plants. However, the strains belonging to A. radiobacter and the two remaining strains of E. meliloti were unable to nodulate P. sativum, suggesting that they were non-symbiotic strains. The results of this study further suggest that the Tunisian Rhizobium community is more diverse than previously reported.

Phylogenomics reveals the evolution of root nodulating alpha- and beta-Proteobacteria (rhizobia).

The symbiosis between legumes and nodulating Proteobacteria (so-called rhizobia) contributes greatly to nitrogen fixation in terrestrial ecosystems. Root nodulating Proteobacteria produce nodulation (Nod) factors during the initiation of rhizobial nodule organogenesis on the roots of legumes. Here, we screened the Nod factor production capacity of the previously reported nodule inducing Proteobacteria genera using their genome sequences and assessed the evolutionary history of symbiosis based on phylogenomics. Our analysis revealed 12 genera as potentially Nod factor producing taxa exclusively from alpha- and beta-Proteobacteria. Based on molecular clock analysis, we estimate that rhizobial nitrogen-fixing symbiosis appeared for the first time about 51 Mya (Eocene epoch) in Rhizobiaceae, and it was laterally transferred to multiple symbiotic taxa in alpha- and beta-Proteobacteria. Coevolutionary tests conducted for measuring the phylogenetic congruence between hosts and symbionts revealed only weak topological similarity between legumes and their bacterial symbionts. We conclude that frequent lateral transfer of symbiotic genes, facultative symbiotic nature of rhizobia, differential evolutionary processes of chromosome versus plasmids, and complex multispecies coevolutionary processes have shaped the rhizobia-host associations.

Peteryoungia gen. nov. with four new species combinations and description of Peteryoungia desertarenae sp. nov., and taxonomic revision of the genus Ciceribacter based on phylogenomics of Rhizobiaceae.

A novel bacterial strain designated as ADMK78T was isolated from the saline desert soil. The cells were rod-shaped, Gram-stain-negative, and non-motile. The strain ADMK78T grows best at 28 °C. Phylogeny of 16S rRNA gene placed the strain ADMK78T with the members of genera Ciceribacter and Rhizobium, while the highest sequence similarity was with Rhizobium wuzhouense W44T (98.7%) and Rhizobium ipomoeae shin9-1 T (97.9%). Phylogenetic analysis based on 92 core-genes extracted from the genome sequences and average amino acid identity (AAI) revealed that the strain ADMK78T forms a distinct cluster including five species of Rhizobium, which is separate from the cluster of the genera Rhizobium and Ciceribacter. We propose re-classification of Rhizobium ipomoeae, R. wuzhouense, R. rosettiformans and R. rhizophilum into the novel genus Peteryoungia. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values of ADMK78T were less than 82 and 81%, respectively, among all type strains included in the genus Peteryoungia. The strain ADMK78T showed differences in physiological, phenotypic, and protein profiles estimated by MALDI-TOF MS to its closest relatives. Based on the phenotypic, chemotaxonomic properties, and phylogenetic analyses, the strain ADMK78T represents a novel species, Peteryoungia desertarenae sp. nov. The type strain is ADMK78T (= MCC 3400T; KACC 21383T; JCM 33657T). We also proposed the reclassification of Rhizobium daejeonense, R. naphthalenivorans and R. selenitireducens, into the genus Ciceribacter, based on core gene phylogeny and AAI values.

Diversity of rhizobia and non-rhizobia endophytes isolated from root nodules of Trifolium sp. growing in lead and zinc mine site Guelma, Algeria.

High concentrations of heavy metals in mine soil disturb the interactions between legumes and microorganisms leading to select strains adapted to these specific conditions. In this work, we analyzed the diversity of fifty strains isolated from Trifolium sp. nodules growing on Pb-Zn mine soil, in the Northeastern of Algeria and highlighted their potential symbiotic traits. The phylogeny of the 16S rRNA gene sequences revealed a high bacterial diversity with a predominance of non-rhizobial endophytes. The identified isolates belong to the thirteen following genera Cupriavidus, Pseudomonas, Bacillus, Acinetobacter, Enterobacter, Roseomonas, Paracoccus, Frondihabitans, Microbacterium, Kocuria, Providencia, Micrococcus and Staphylococcus. Regarding rhizobial strains, only isolates affiliated to Rhizobium genus were obtained. The symbiotic gene nodC and the nitrogen fixation gene nifH present showed that Rhizobium isolates belonged to the symbiovar trifolii. In addition to bacterial, one yeast strain was isolated and identified as Rhodotorula mucilaginosa by sequencing the internal transcribed spacer (ITS) region.

Diversity of rhizobial and non-rhizobial bacteria nodulating wild ancestors of grain legume crop plants.

Chickpeas, lentils, and peas are the oldest grain legume species that spread to other regions after their first domestication in Fertile Crescent, and they could reveal the rhizobial evolution in relation to the microsymbionts of wild species in this region. This study investigated the phenotypic and genotypic diversity of the nodule-forming rhizobial bacteria recovered from Pisum sativum subsp., Cicer pinnatifidum, and Lens culinaris subsp. orientalis exhibiting natural distribution in the Gaziantep province of Turkey. PCA analyses of rhizobial isolates, which were tested to be highly resistant to stress conditions, showed that especially pH and salt concentrations had an important effect on these bacteria. Phylogenetic analysis based on 16S rRNA determined that these wild species were nodulated by at least 7 groups including Rhizobium and non-Rhizobium. The largest group comprised of Rhizobium leguminosarum and Rhizobium sp. while R. pusense, which was previously determined as non-symbiotic species, was found to nodulate C. pinnatifidum and L. culinaris subsp. orientalis. In recent studies, Klebsiella sp., which is stated to be able to nodulate different species, strong evidences have been obtained in present study exhibiting that Klebsiella sp. can nodulate C. pinnatifidum and Pseudomonas sp. was able to nodulate C. pinnatifidum and P. sativum subsp. Additionally, L. culinaris subsp. orientalis unlike other plant species, was nodulated by Burkholderia sp. and Serratia sp. associated isolates. Some isolates could not be characterized at the species level since the 16S rRNA sequence similarity rate was low and the fact that they were in a separate group supported with high bootstrap values in the phylogenetic tree may indicate that these isolates could be new species. The REP-PCR fingerprinting provided results supporting the existence of new species nodulating wild ancestors.

Diverse Rhizobium strains isolated from root nodules of Trifolium alexandrinum in Egypt and symbiovars.

Berseem clover (T. alexandrinum) is the main forage legume crop used as animal feed in Egypt. Here, eighty rhizobial isolates were isolated from root nodules of berseem clover grown in different regions in Egypt and were grouped by RFLP-16S rRNA ribotyping. Representative isolates were characterized using phylogenetic analyses of the 16S rRNA, rpoB, glnA, pgi, and nodC genes. We also investigated the performance of these isolates using phenotypic tests and nitrogen fixation efficiency assays. The majority of strains (<90%) were closely related to Rhizobium aegyptiacum and Rhizobium aethiopicum and of the remaining strains, six belonged to the Rhizobium leguminosarum genospecies complex and only one strain was assigned to Agrobacterium fabacearum. Despite their heterogeneous chromosomal background, most of the strains shared nodC gene alleles corresponding to symbiovar trifolii. Some of the strains closely affiliated to R. aegyptiacum and R. aethiopicum had superior nodulation and nitrogen fixation capabilities in berseem clover, compared to the commercial inoculant (Okadein®) and N-added treatments. R. leguminosarum strain NGB-CR 17 that harbored a nodC allele typical of symbiovar viciae, was also able to form an effective symbiosis with clover. Two strains with nodC alleles of symbiovar trifolii, R. aegyptiacum strains NGB-CR 129 and 136, were capable of forming effective nodules in Phaseolus vulgaris in axenic greenhouse conditions. This adds the symbiovar trifolii which is well-established in the Egyptian soils to the list of symbiovars that form nodules in P. vulgaris.

Genetic diversity and phylogeny of indigenous rhizobia nodulating faba bean (Vicia faba L.) in Greece.

The genetic diversity and phylogeny of fast-growing rhizobia isolated from root nodules of Vicia faba grown in different geographical regions of Greece were assessed. Although Rhizobium leguminosarum sv. viciae is the most common symbiont of Vicia spp. in European soils, there is no available information on native rhizobia nodulating faba bean in Greece. Seventy bacterial strains were isolated and grouped into sixteen distinct profiles based on BOX-PCR fingerprinting. The phylogenetic affiliation was further defined by sequence analysis of the rrs and multilocus sequence analysis (MLSA) of three housekeeping genes (recA, atpD and gyrB). Fifty-eight isolates were affiliated with recently described genospecies gsF-2, represented by R. laguerreae FB206T, whereas six isolates were closely related to gsB and two isolates might belong to gsA. Two isolates assigned to R. hidalgonense and another two non-nodulating strains could not be assigned to any validly defined species and possibly belong to a new rhizobial lineage. Interestingly, R. laguerreae strains were commonly found at all sampling sites, suggesting that they could be the main symbionts of faba beans in Greek soils. According to the phylogenies of two symbiosis-related genes (nodC and nifH), all nodulating isolates belonged to symbiovar (sv.) viciae harboring four distinct nodC gene haplotypes and they were grouped into two clades together with strains assigned to R. laguerreae and genospecies of R. leguminosarum isolated from other countries and continents. This is the first report that R. hidalgonense strains belong to sv. viciae. No correlation was observed between the nodC haplotypes, geographic origin and chromosomal background of the isolates in the study.

Rhizobium indicum sp. nov., isolated from root nodules of pea (Pisum sativum) cultivated in the Indian trans-Himalayas.

Three strains of rhizobia isolated from effective root nodules of pea (Pisum sativum L.) collected from the Indian trans-Himalayas were characterized using 16S rRNA, atpD and recA genes. Phylogeny of the 16S rRNA genes revealed that the newly isolated strains were members of the genus Rhizobium with ≥99.9% sequence similarity to the members within the "Rhizobium leguminosarum" group. Phylogenetic analyses based on the concatenated sequences of atpD and recA gene, and 92 core genes extracted from the genome sequences indicated that strains JKLM 12A2T and JKLM 13E are grouped as a separate clade closely related to R. laguerreae FB206T. In contrast, the strain JKLM 19E was placed with "R. hidalgonense" FH14T. Whole-genome average nucleotide identity (ANI) values were 97.6% within strains JKLM 12A2T and JKLM 13E, and less than 94% with closely related species. The digital DNA-DNA hybridization (dDDH) values were 81.45 within the two strains and less than 54.8% to closely related species. The major cellular fatty acids were C18:1w7c in summed feature 8, C14:0 3OH/C16:1 iso I in summed feature 2, and C18:0. The DNA G+C content of JKLM 12A2T and JKLM 13E was 60.8mol%. The data on genomic, chemotaxonomic, and phenotypic characteristics indicates that the strains JKLM 12A2T and JKLM 13E represent a novel species, Rhizobium indicum sp. nov. The type strain is JKLM 12A2T (= MCC 3961T=KACC 21380T=JCM 33658T). However, the strain JKLM 19E represents a member of "R. hidalgonense" and the symbiovar viciae.

Rhizobium rhizophilum sp. nov., an indole acetic acid-producing bacterium isolated from rape (Brassica napus L.) rhizosphere soil.

A novel Gram-stain-negative, aerobic, rod-shaped and indole acetic acid-producing strain, designated 7209-2T, was isolated from rhizosphere of rape (Brassica napus L.) grown in the Yakeshi City, Inner Mongolia, PR China. The 16S rRNA gene sequence analysis indicated that strain 7209-2T belongs to the genus Rhizobium and is closely related to Rhizobium rosettiformans W3T, Rhizobium ipomoeae shin9-1T and Rhizobium wuzhouense W44T with sequence similarities of 98.2, 98.1 and 97.9 %, respectively. Phylogenetic analysis based on concatenated housekeeping recA and atpD gene sequences showed that strain 7209-2T formed a group together with R. wuzhouense W44T and R. rosettiformans W3T, with sequences similarities of 92.6 and 91.1 %, respectively. The genome size of strain 7209-2T was 5.25 Mb, comprising 5027 predicted genes with a DNA G+C content of 61.2 mol%. The average nucleotide identity and digital DNA-DNA hybridization comparisons among 7209-2T and reference strains for the most closely related species showed values below the accepted threshold for species discrimination. The major fatty acids of strain 7209-2T were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and summed feature 2 (C12 : 0 aldehyde and/or unknown 10.953) . The major polar lipids were found to consist of phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and an unidentified aminophospholipid. The predominant ubiquinone was identified as quinone 10. Based on all the above results, strain 7209-2T represents a novel species of the genus Rhizobium, for which the name Rhizobium rhizophilum sp. nov. is proposed with 7209-2T (=CGMCC 1.15691T=DSM 103161T) as the type strain.

Rhizobium dioscoreae sp. nov., a plant growth-promoting bacterium isolated from yam (Dioscorea species).

This study investigated endophytic nitrogen-fixing bacteria isolated from two species of yam (water yam, Dioscorea alata L.; lesser yam, Dioscorea esculenta L.) grown in nutrient-poor alkaline soil conditions on Miyako Island, Okinawa, Japan. Two bacterial strains of the genus Rhizobium, S-93T and S-62, were isolated. The phylogenetic tree, based on the almost-complete 16S rRNA gene sequences (1476 bp for each strain), placed them in a distinct clade, with Rhizobium miluonense CCBAU 41251T, Rhizobium hainanense I66T, Rhizobium multihospitium HAMBI 2975T, Rhizobium freirei PRF 81T and Rhizobium tropici CIAT 899T being their closest species. Their bacterial fatty acid profile, with major components of C19 : 0 cyclo ω8c and summed feature 8, as well as other phenotypic characteristics and DNA G+C content (59.65 mol%) indicated that the novel strains belong to the genus Rhizobium. Pairwise average nucleotide identity analyses separated the novel strains from their most closely related species with similarity values of 90.5, 88.9, 88.5, 84.5 and 84.4 % for R. multihospitium HAMBI 2975T, R. tropici CIAT 899T, R. hainanense CCBAU 57015T, R. miluonense HAMBI 2971T and R. freirei PRF 81T, respectively; digital DNA-DNA hybridization values were in the range of 26-42 %. Considering the phenotypic characteristics as well as the genomic data, it is suggested that strains S-93T and S-62 represent a new species, for which the name Rhizobium dioscoreae is proposed. The type strain is S-93T (=NRIC 0988T=NBRC 114257T=DSM 110498T).

Rhizobium ruizarguesonis sp. nov., isolated from nodules of Pisum sativum L.

Four strains, coded as UPM1132, UPM1133T, UPM1134 and UPM1135, and isolated from nodules of Pisum sativum plants grown on Ni-rich soils were characterised through a polyphasic taxonomy approach. Their 16S rRNA gene sequences were identical and showed 100% similarity with their closest phylogenetic neighbors, the species included in the 'R. leguminosarum group': R. laguerreae FB206T, R. leguminosarum USDA 2370T, R. anhuiense CCBAU 23252T, R. sophoreae CCBAU 03386T, R. acidisoli FH13T and R. hidalgonense FH14T, and 99.6% sequence similarity with R. esperanzae CNPSo 668T. The analysis of combined housekeeping genes recA, atpD and glnII sequences showed similarities of 92-95% with the closest relatives. Whole genome average nucleotide identity (ANI) values were 97.5-99.7% ANIb similarity among the four strains, and less than 92.4% with closely related species, while digital DNA-DNA hybridization average values (dDDH) were 82-85% within our strains and 34-52% with closely related species. Major fatty acids in strain UPM1133T were C18:1 ω7c / C18:1 ω6c in summed feature 8, C14:0 3OH/ C16:1 iso I in summed feature 2 and C18:0. Colonies were small to medium, pearl-white coloured in YMA at 28°C and growth was observed in the ranges 8-34°C, pH 5.5-7.5 and 0-0.7% (w/v) NaCl. The DNA G+C content was 60.8mol %. The combined genotypic, phenotypic and chemotaxonomic data support the classification of strains UPM1132, UPM1133T, UPM1134 and UPM1135 into a novel species of Rhizobium, for which the name Rhizobium ruizarguesonis sp. nov. is proposed. The type strain is UPM1133T (=CECT 9542T=LMG 30526T).

Biochemical, functional and molecular characterization of pigeon pea rhizobia isolated from semi-arid regions of India.

Pigeon pea (Cajanus cajan (L.) Millspaugh) is among the top ten legumes grown globally not only having high tolerance to environmental stresses along, but also has the high biomass and productivity with optimal nutritional profiles. In the present study, 55 isolates of rhizobia were identified from 22 nodule samples of pigeon pea collected from semi-arid regions of India on the basis of morphological, biochemical, plant growth promoting activities and their ability to tolerate the stress conditions viz. pH, salt, temperature and drought stress. Amongst all the 55 isolates, 37 isolates showed effective nodulation under in vitro conditions in pigeon pea. Further, five isolates having multiple PGP activities and high in vitro symbiotic efficiency were subjected to 16S rRNA sequencing and confirmed their identities as Rhizobium, Mesorhizobium, Sinorhizobium sp. Further these 37 isolates were characterized at molecular level using ARDRA and revealed significant molecular diversity. Based on UPGMA clustering analysis, these isolates showed significant molecular diversity. The high degree of molecular diversity is due to mixed cropping of legumes in the region. The assessment of genetic diversity and molecular characterization of novel strains is a very important tool for the replacement of ineffective rhizobial strains with the efficient strains for the improvement in the nodulation and pigeon pea quality. The pigeon pea isolates with multiple PGPR activities could be further used for commercial production.