Phylogenomic analyses and reclassification of the Mesorhizobium complex: proposal for 9 novel genera and reclassification of 15 species.

BACKGROUD: The genus Mesorhizobium is shown by phylogenomics to be paraphyletic and forms part of a complex that includes the genera Aminobacter, Aquamicrobium, Pseudaminobacter and Tianweitania. The relationships for type strains belong to these genera need to be carefully re-evaluated. RESULTS: The relationships of Mesorhizobium complex are evaluated based on phylogenomic analyses and overall genome relatedness indices (OGRIs) of 61 type strains. According to the maximum likelihood phylogenetic tree based on concatenated sequences of 539 core proteins and the tree constructed using the bac120 bacterial marker set from Genome Taxonomy Database, 65 type strains were grouped into 9 clusters. Moreover, 10 subclusters were identified based on the OGRIs including average nucleotide identity (ANI), average amino acid identity (AAI) and core-proteome average amino acid identity (cAAI), with AAI and cAAI showing a clear intra- and inter-(sub)cluster gaps of 77.40-80.91% and 83.98-86.16%, respectively. Combined with the phylogenetic trees and OGRIs, the type strains were reclassified into 15 genera. This list includes five defined genera Mesorhizobium, Aquamicrobium, Pseudaminobacter, Aminobacterand Tianweitania, among which 40/41 Mesorhizobium species and one Aminobacter species are canonical legume microsymbionts. The other nine (sub)clusters are classified as novel genera. Cluster III, comprising symbiotic M. alhagi and M. camelthorni, is classified as Allomesorhizobium gen. nov. Cluster VI harbored a single symbiotic species M. albiziae and is classified as Neomesorhizobium gen. nov. The remaining seven non-symbiotic members were proposed as: Neoaquamicrobium gen. nov., Manganibacter gen. nov., Ollibium gen. nov., Terribium gen. nov., Kumtagia gen. nov., Borborobacter gen. nov., Aerobium gen. nov.. Furthermore, the genus Corticibacterium is restored and two species in Subcluster IX-1 are reclassified as the member of this genus. CONCLUSION: The Mesorhizobium complex are classified into 15 genera based on phylogenomic analyses and OGRIs of 65 type strains. This study resolved previously non-monophyletic genera in the Mesorhizobium complex.

Aquibium microcysteis gen. nov., sp. nov., isolated from a Microcystis aeruginosa culture and reclassification of Mesorhizobium carbonis as Aquibium carbonis comb. nov. and Mesorhizobium oceanicum as Aquibium oceanicum comb. nov.

A novel bacterial strain, NIBR3T, was isolated from a Microcystis aeruginosa culture. Strain NIBR3T was characterized as Gram-negative, rod-shaped, catalase- and oxidase-positive, and aerobic. The 16S rRNA gene sequence analysis showed that strain NIBR3T was most closely related to Mesorhizobium carbonis B2.3T (=KCTC 52461), Mesorhizobium oceanicum B7T (=KCTC 42783) and Mesorhizobium qingshengii CCBAU 33460T (=HAMBI 3277), at 98.7, 97.2 and 97.2% similarity, respectively. Our phylogenetic analyses revealed that three strains [strain NIBR3T with the previously reported two Mesorhizobium species (M. carbonis B2.3T and M. oceanicum B7T)] formed a distinct cluster from other Mesorhizobium type strains. The average nucleotide identity of strain NIBR3T relative to M. carbonis B2.3T , M. oceanicum B7T, and M. qingshengii CCBAU 33460T was found to be 84.3, 79.4 and 75.8 %, with average amino-acid identities of 85.1, 74.8 and 64.3 %, and digital DNA-DNA hybridization values of 27.6, 22.6 and 20.7 %, respectively. The genome size and genomic DNA G+C content of NIBR3T were 6.1 Mbp and 67.9 mol%, respectively. Growth of strain NIBR3T was observed at 23-45 °C (optimum, 33 °C), at pH 6-11 (optimum, 8) and in the presence of 0-4 % (w/v) NaCl (optimum, 0 %). The major polar lipids in this novel strain were phosphatidylethanolamine, phosphatidylcholine and phosphatidylmethylethanolamine. The predominant respiratory quinone was Q-10. Summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) was the most abundant cellular fatty acid in strain NIBR3T. Based on genotypic characteristics using our genomic data, strain NIBR3T was identified as a member of new genus, Aquibium gen. nov., with the two aforementioned stains. The type strain f the novel species, Aquibium microcysteis sp. nov., is NIBR3T (=KACC 22092T=HAMBI 3738T). We also reclassified Mesorhizobium carbonis and M. oceanicum as Aquibium carbonis comb. nov. and A. oceanicum comb. nov., respectively.

Mesorhizobium comanense sp. nov., isolated from groundwater.

Strain 3P27G6T was isolated from an artesian well connected to the thermal water basin of Comano Terme, Province of Trento, Italy. In phylogenetic analyses based on multilocus sequence analysis, strain 3P27G6T clustered together with Mesorhizobium australicum WSM2073T. Genome sequencing produced a 99.51 % complete genome, with a length of 7 363 057 bp and G+C content of 63.53 mol%, containing 6897 coding sequences, 55 tRNA and three rRNA. Average nucleotide identity analysis revealed that all distances calculated between strain 3P27G6T and the other Mesorhizobium genomes were below 0.9, indicating that strain 3P27G6T represents a new species. Therefore, we propose the name Mesorhizobium comanense sp. nov. with the type strain 3P27G6T (=DSM 110654T=CECT 30067T). Strain 3P27G6T is a Gram-negative, rod-shaped, aerobic bacterium. Growth condition, antibiotic susceptibility, metabolic and fatty acid-methyl esters profiles of the strain were determined. Only few nodulation and nitrogen fixation genes were found in the genome, suggesting that this strain may not be specialized in nodulation or in nitrogen fixation.

Psychrotolerant Mesorhizobium sp. Isolated from Temperate and Cold Desert Regions Solubilizes Potassium and Produces Multiple Plant Growth Promoting Metabolites.

Soil potassium (K) supplement depends intensively on the application of chemical fertilizers, which have substantial harmful environmental effects. However, some bacteria can act as inoculants by converting unavailable and insoluble K forms into plant-accessible forms. Such bacteria are an eco-friendly approach for enhancing plant K absorption and consequently reducing utilization of chemical fertilization. Therefore, the present research was undertaken to isolate, screen, and characterize the K solubilizing bacteria (KSB) from the rhizosphere soils of northern India. Overall, 110 strains were isolated, but only 13 isolates showed significant K solubilizing ability by forming a halo zone on solid media. They were further screened for K solubilizing activity at 0 °C, 1 °C, 3 °C, 5 °C, 7 °C, 15 °C, and 20 °C for 5, 10, and 20 days. All the bacterial isolates showed mineral K solubilization activity at these different temperatures. However, the content of K solubilization increased with the upsurge in temperature and period of incubation. The isolate KSB (Grz) showed the highest K solubilization index of 462.28% after 48 h of incubation at 20 °C. The maximum of 23.38 µg K/mL broth was solubilized by the isolate KSB (Grz) at 20 °C after 20 days of incubation. Based on morphological, biochemical, and molecular characterization (through the 16S rDNA approach), the isolate KSB (Grz) was identified as Mesorhizobium sp. The majority of the strains produced HCN and ammonia. The maximum indole acetic acid (IAA) (31.54 µM/mL) and cellulase (390 µM/mL) were produced by the isolate KSB (Grz). In contrast, the highest protease (525.12 µM/mL) and chitinase (5.20 µM/mL) activities were shown by standard strain Bacillus mucilaginosus and KSB (Gmr) isolate, respectively.

Mesorhizobium microcysteis sp. nov., isolated from a culture of Microcystis aeruginosa.

Strain MaA-C15T, a Gram-stain-negative, non-spore-forming and strictly aerobic bacterium, was isolated from a xenic culture of Microcystis aeruginosa in the Republic of Korea. Cells were motile rods showing positive reactions in catalase and oxidase tests. Growth was observed between 15 and 37 °C (optimum, 30 °C), between pH 6.0 and pH 11.0 (optimum, pH 7.5) and in the presence of 0-2.0 % (w/v) NaCl (optimum, 0 %). Strain MaA-C15T contained C16 : 0, 11-methyl-C18 : 1 ω7c, cyclo-C19 : 0 ω8c and summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c) as the major cellular fatty acids and ubiquinone-10 as the sole respiratory quinone. Phosphatidylethanolamine, phosphatidylmonomethylethanolamine, an unidentified aminophospholipid, an unidentified glycolipid and three unidentified phospholipids were detected as the major polar lipids. The G+C content of the genomic DNA was 64.1 mol%. Phylogenetic and phylogenomic analyses based on 16S rRNA gene and genome sequences revealed that strain MaA-C15T formed a phyletic lineage with Mesorhizobium sediminum YIM M12096T within the family Phyllobacteriaceae. Strain MaA-C15T was most closely related to Mesorhizobium albiziae DSM 21822T with a 98.2 % 16S rRNA sequence similarity. Average nucleotide identity and in silico DNA-DNA hybridization values between strain MaA-C15T and M. albiziae DSM 21822T were 75.4 and 20.1 %, respectively. Based on the results of phenotypic, chemotaxonomic and molecular analyses, strain MaA-C15T represents a novel species of the genus Mesorhizobium, for which the name Mesorhizobium microcysteis sp. nov. is proposed. The type strain is MaA-C15T (=KACC 21226T=JCM 33503T).

Definition of the novel symbiovar canariense within Mesorhizobium neociceri sp. nov., a new species of genus Mesorhizobium nodulating Cicer canariense in the "Caldera de Taburiente" National Park (La Palma, Canary Islands).

Cicer canariense is a highly promiscuous wild chickpea nodulated by Mesorhizobium strains in La Palma Island located at Canary archipelago. Four of these strains, CCANP34, CCANP35T, CCANP38 and CCANP95 belong to a group phylogenetically close to Mesorhizobium caraganae with 100% similarity values in the 16S rRNA gene. However, the genomes of the strains CCANP35T and M. caraganae LMG 24397T obtained in this work showed ANIb and dDDH values of 90.02% and 44.1%, respectively. These values are lower than those currently accepted for different bacterial species showing that the Canarian strains do not belong to the species M. caraganae. The Canarian strains also differ from M. caraganae in the amounts of several fatty acids and in several phenotypic traits. Based on the obtained results the Canarian strains belong to a novel species for which we propose the name Mesorhizobium neociceri sp. nov. and whose type strain is CCANP35T. The results of the phylogenetic analyses of nodC and nifH symbiotic genes showed that the Canarian strains represent a novel symbiovar within genus Mesorhizobium phylogenetically divergent to that encompassing M. caraganae. We propose the names canariense and caraganae for the symbiovars encompassing the strains of M. neociceri and M. caraganae, respectively.

Identification of the endosymbionts from Sulla spinosissima growing in a lead mine tailings in Eastern Morocco as Mesorhizobium camelthorni sv. aridi.

AIMS: To identify the bacteria nodulating Sulla spinosissima growing profusely in a lead and zinc mine tailings in Eastern Morocco. METHODS AND RESULTS: In all, 32 rhizobial cultures, isolated from root nodules of S. spinosissima growing in soils of the mining site, were tolerant to different heavy metals. The ERIC-polymerase chain reaction (PCR) fingerprinting analysis clustered the isolates into seven different groups, and the analysis of the 16S rRNA sequences of four selected representative strains, showed they were related to different species of the genus Mesorhizobium. The atpD, glnII and recA housekeeping genes analysis confirmed the affiliation of the four representative strains to Mesorhizobium camelthorni CCNWXJ40-4T , with similarity percentages varying from 96·30 to 98·30%. The sequences of the nifH gene had 97·33-97·78% similarities with that of M. camelthorni CCNWXJ40-4T ; however, the nodC phylogeny of the four strains diverged from the type and other reference strains of M. camelthorni and formed a separated cluster. The four strains nodulate also Astragalus gombiformis and A. armatus but did not nodulate A. boeticus, Vachellia gummifera, Prosopis chilensis, Cicer arietinum, Lens culinaris, Medicago truncatula, Lupinus luteus or Phaseolus vulgaris. CONCLUSIONS: Based on similarities of the nodC symbiotic gene and differences in the host range, the strains isolated from S. spinosissima growing in soils of the Sidi Boubker mining site may form a different symbiovar within Mesorhizobium for which the name aridi is proposed. SIGNIFICANCE AND IMPACT OF THE STUDY: In this work, we show that strains of M. camelthorni species nodulating S. spinosissima in the arid area of Eastern Morocco constitute a distinct phylogenetic clade of nodulation genes; we named symbiovar aridi, which encompasses also mesorhizobia from other Mediterranean desert legumes.

Mesorhizobium jarvisii is a dominant and widespread species symbiotically efficient on Astragalus sinicus L. in the Southwest of China.

In order to identify rhizobia of Astragalus sinicus L. and estimate their geographic distribution in the Southwest China, native rhizobia nodulating A. sinicus were isolated and their genetic diversity were studied at 13 sites cultivated in four Chinese provinces. A total of 451 rhizobial isolates were trapped with A. sinicus plants from soils and classified into 8 different genotypes defined by PCR-based restriction fragment length polymorphism (RFLP) of 16S-23S rRNA intergenic spacer (IGS). Twenty-one representative strains were further identified into three defined Mesorhizobium species by phylogenetic analyses of 16S rRNA genes and housekeeping genes (glnII and atpD). M. jarvisii was dominant accounting for 76.3% of the total isolates, 22.8% of the isolates were identified as M. huakuii and five strains belonged to M. qingshengii. All representatives were assigned to the symbiovar astragali by sharing high nodC sequence similarities of more than 99%. Furthermore, the biogeography distribution of these rhizobial genotypes and species was mainly affected by contents of available phosphorus, available potassium, total salts and pH in soils. The most remarkable point was the identification of M. jarvisii as a widespread and predominant species of A. sinicus in southwest of China. These results revealed a novel geographic pattern of rhizobia associated with A. sinicus in China.

Symbiosis islands of Loteae-nodulating Mesorhizobium comprise three radiating lineages with concordant nod gene complements and nodulation host-range groupings.

Mesorhizobium is a genus of soil bacteria, some isolates of which form an endosymbiotic relationship with diverse legumes of the Loteae tribe. The symbiotic genes of these mesorhizobia are generally carried on integrative and conjugative elements termed symbiosis islands (ICESyms). Mesorhizobium strains that nodulate Lotus spp. have been divided into host-range groupings. Group I (GI) strains nodulate L. corniculatus and L. japonicus ecotype Gifu, while group II (GII) strains have a broader host range, which includes L. pedunculatus. To identify the basis of this extended host range, and better understand Mesorhizobium and ICESym genomics, the genomes of eight Mesorhizobium strains were completed using hybrid long- and short-read assembly. Bioinformatic comparison with previously sequenced mesorhizobia genomes indicated host range was not predicted by Mesorhizobium genospecies but rather by the evolutionary relationship between ICESym symbiotic regions. Three radiating lineages of Loteae ICESyms were identified on this basis, which correlate with Lotus spp. host-range grouping and have lineage-specific nod gene complements. Pangenomic analysis of the completed GI and GII ICESyms identified 155 core genes (on average 30.1 % of a given ICESym). Individual GI or GII ICESyms carried diverse accessory genes with an average of 34.6 % of genes unique to a given ICESym. Identification and comparative analysis of NodD symbiotic regulatory motifs - nod boxes - identified 21 branches across the NodD regulons. Four of these branches were associated with seven genes unique to the five GII ICESyms. The nod boxes preceding the host-range gene nodZ in GI and GII ICESyms were disparate, suggesting regulation of nodZ may differ between GI and GII ICESyms. The broad host-range determinant(s) of GII ICESyms that confer nodulation of L. pedunculatus are likely present amongst the 53 GII-unique genes identified.

Genomic diversity of chickpea-nodulating rhizobia in Ningxia (north central China) and gene flow within symbiotic Mesorhizobium muleiense populations.

Diversity and taxonomic affiliation of chickpea rhizobia were investigated from Ningxia in north central China and their genomic relationships were compared with those from northwestern adjacent regions (Gansu and Xinjiang). Rhizobia were isolated from root-nodules after trapping by chickpea grown in soils from a single site of Ningxia and typed by IGS PCR-RFLP. Representative strains were phylogenetically analyzed on the basis of the 16S rRNA, housekeeping (atpD, recA and glnII) and symbiosis (nodC and nifH) genes. Genetic differentiation and gene flow were estimated among the chickpea microsymbionts from Ningxia, Gansu and Xinjiang. Fifty chickpea rhizobial isolates were obtained and identified as Mesorhizobium muleiense. Their symbiosis genes nodC and nifH were highly similar (98.4 to 100%) to those of other chickpea microsymbionts, except for one representative strain (NG24) that showed low nifH similarities with all the defined Mesorhizobium species. The rhizobial population from Ningxia was genetically similar to that from Gansu, but different from that in Xinjiang as shown by high chromosomal gene flow/low differentiation with the Gansu population but the reverse with the Xinjiang population. This reveals a biogeographic pattern with two main populations in M. muleiense, the Xinjiang population being chromosomally differentiated from Ningxia-Gansu one. M. muleiense was found as the sole main chickpea-nodulating rhizobial symbiont of Ningxia and it was also found in Gansu sharing alkaline-saline soils with Ningxia. Introduction of chickpea in recently cultivated areas in China seems to select from alkaline-saline soils of M. muleiense that acquired symbiotic genes from symbiovar ciceri.

Mesorhizobium terrae sp. nov., a novel species isolated from soil in Jangsu, Korea.

A gram-negative, white-pigmented, aerobic, rod-shaped bacterium, designated as strain NIBRBAC000500504T, was isolated from soil in Jangsu, Korea. Optimal growth of this strain was observed at 25 °C, pH 7.0, and in the presence of 0% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain NIBRBAC000500504T belonged to the genus Mesorhizobium and was closely related to Mesorhizobium shangrilense LMG 24762T (98.3% sequence similarity), Mesorhizobium australicum LMG 24608T (98.2%), Mesorhizobium qingshengii LMG 26793T (98.1%), Mesorhizobium ciceri ATCC 51585T (98.0%), Mesorhizobium loti DSM 2626T (98.0%), Mesorhizobium sophorae LMG 28223T (97.9%), Mesorhizobium waitakense LMG 28227T (97.8%), and Mesorhizobium cantuariense LMG 28225T (97.8%). Next-generation sequencing analysis indicated that the genome of strain NIBRBAC000500504T comprised a circular chromosome (5,731,152 bp, G+C content: 63.26%) and a plasmid (293,638 bp, G+C content: 61.39%) with 5672 coding sequences, 50 tRNAs, and 6 rRNAs. The major respiratory isoprenoid quinone was Q10; the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine; the major fatty acids were summed feature 8 (comprising C18:1 ω7c/C18:1 ω6c), C19:0 cyclo ω8c, C16:0, and C18:1 ω7c 11-methyl; and the G+C content of the genomic DNA was 62.9 mol%. The DNA-DNA relatedness values between NIBRBAC000500504T and its closest type strains were low. On the basis of these polyphasic taxonomic data, it is proposed that strain NIBRBAC000500504T represents a novel species of the genus Mesorhizobium, with the type strain being NIBRBAC000500504T (= KCTC 72278T = JCM 33432T).

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.

Mesorhizobium rhizophilum sp. nov., a 1-aminocyclopropane-1-carboxylate deaminase producing bacterium isolated from rhizosphere of maize in Northeast China.

A novel 1-aminocyclopropane-1-carboxylate deaminase producing bacterium, Gram- stain-negative, aerobic, motile, rod-shaped strain designated YM1C-6-2T was isolated from rhizosphere of maize grown in Northeast China. The 16S rRNA gene sequence analysis indicated that strain YM1C-6-2T belongs to the genus Mesorhizobium and is closely related to Mesorhizobium alhagi CCNWXJ12-2T and M. camelthorni CCNWXJ40-4T with sequence similarities of 98.4% and 97.9%, respectively. Multilocus sequence analysis of other housekeeping genes revealed that the new isolates YM1C-6-2T forms a phylogenetically group with some species in the genus Mesorhizobium. The genome size of strain YM1C-6-2T was 5.51 Mb, comprising 5378 predicted genes with a DNA G+C content of 64.5%. The average nucleotide identity and digital DNA-DNA hybridization comparisons between YM1C-6-2T and the most related type strains showed values below the accepted threshold for species discrimination. The major fatty acids of strain YM1C-6-2T were C19:0 cyclo ω8c (47.5%), summed feature 8 (C18:1ω7c and/or C18:1ω6c) (19.5%) and C16:0 (15.1%), which differed from the closely related reference strains in their relative abundance. The major polar lipids consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and an unidentified aminophospholipid. The predominant ubiquinone was identified as Quinone 10. Phenotypic and biochemical analysis results indicated that strain YM1C-6-2T can be distinguished from closely related type strains. Based on the above results, strain YM1C-6-2T represents a novel species of the genus Mesorhizobium, for which the name Mesorhizobium rhizophilum sp. nov. is proposed with YM1C-6-2T (= CGMCC 1.15487T = DSM 101712T) as the type strain.

Mesorhizobium alexandrii sp. nov., isolated from phycosphere microbiota of PSTs-producing marine dinoflagellate Alexandrium minutum amtk4.

An aerobic, Gram-stain-negative, motile and rod-shaped bacterial strain, designated as Z1-4T, was isolated from the phycosphere microbiota of marine dinoflagellate Alexandrium minutum that produces paralytic shellfish poisoning toxins. Phylogenetic analysis based on 16S rRNA gene sequences showed that the new isolate belongs to the genus Mesorhizobium, and it was closely related to Mesorhizobium waimense LMG 28228T and Mesorhizobium amorphae LMG 18977T with both 16S rRNA gene sequence similarities of 97.3%. The values of average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) relatedness between strain Z1-4T and its relatives are both well below the thresholds used for the delineation of a new species. A genome-based phylogenetic tree constructed by up-to-date bacterial core gene set (UBCG) indicates that strain Z1-4T forms an independent branch within the genus Mesorhizobium. The respiratory quinone of strain Z1-4T was Q-10. The major fatty acids were similar to other members of the genus Mesorhizobium containing the summed feature 8, C16:0, C19:0cycloω8c, C17:0 and summed feature 3. The polar lipids are phosphatidylmonomethylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, an unidentified aminophospholipid, five glycolipids and seven unknown polar lipids. The DNA G + C content was determined to be 62.1 mol % based on its genomic sequence. Combined evidences based on the genotypic, chemotaxonomic and phenotypic characteristics clearly indicates that strain Z1-4T represents a novel species of the genus Mesorhizobium, for which the name Mesorhizobium alexandrii sp. nov. is proposed. The type strain is Z1-4T (= KCTC 72512T = CCTCC AB 2019101T).

Novel putative Mesorhizobium and Ensifer genomospecies together with a novel symbiovar psoraleae nodulate legumes of agronomic interest grown in Tunisia.

Forty rhizobial strains were isolated from Lotus creticus, L. pusillus and Bituminaria bituminosa endemic to Tunisia, and they belonged to the Mesorhizobium and Ensifer genera based on 16S rDNA sequence phylogeny. According to the concatenated recA and glnII sequence-based phylogeny, four Bituminaria isolates Pb5, Pb12, Pb8 and Pb17 formed a monophyletic group with Mesorhizobium chacoense ICMP14587T, whereas four other strains Pb1, Pb6, Pb13 and Pb15 formed two separate lineages within the Ensifer genus. Among the L. pusillus strains, Lpus9 and Lpus10 showed a 96% identical nucleotide with Ensifer meliloti CCBAU83493T; whereas six other strains could belong to previously undescribed Mesorhizobium and Ensifer species. For L. creticus strains, Lcus37, Lcus39 and Lcus44 showed 98% sequence identity with Ensifer aridi JNVU TP6, and Lcus42 shared a 96% identical nucleotide with Ensifer meliloti CCBAU83493T; whereas another four strains were divergent from all the described Ensifer and Mesorhizobium species. The analysis of the nodC gene-based phylogeny identified four symbiovar groups; Mesorhizobium sp. sv. anthyllidis (Lpus3 and Lpus11 from L. pusillus, Lcus43 from L. creticus), Ensifer medicae sv. meliloti (four strains from L. creticus and two strains from L. pusillus), E. meliloti sv. meliloti (four from L. creticus, four from L. pusillus and four from B. bituminosa). In addition, four B. bituminosa strains (Pb5, Pb8, Pb12, and Pb17) displayed a distinctive nodC sequence distant from those of other symbiovars described to date. According to their symbiotic gene sequences and host range, the B. bituminosa symbionts (Pb5, Pb8, Pb12 and Pb17) would represent a new symbiovar of M. chacoense for which sv. psoraleae is proposed.

Mesorhizobium intechi sp. nov. isolated from nodules of Lotus tenuis in soils of the Flooding Pampa, Argentina.

Three symbiotic nitrogen-fixing bacteria (BD68T, BD66 and BD73) isolated from root nodules of Lotus tenuis in lowland soils of the Flooding Pampa (Argentina), previously classified as members of the Mesorhizobium genus, were characterized in this study. Phylogenetic analysis of their 16S rRNA gene sequences showed a close relationship to M. japonicum MAFF 303099T, M. erdmanii USDA 3471T, M. carmichaelinearum ICMP 18942T, M. opportunistum WSM 2975T and M. jarvisii ATCC 33699T, with sequence identities of 99.72%-100%. Multilocus sequence analysis of other housekeeping genes revealed that the three isolates belonged to a phylogenetically distinct clade within the genus Mesorhizobium. Strain BD68T was designated as the group representative and its genome was fully sequenced. The average nucleotide identity and in silico DNA-DNA hybridization comparisons between BD68T and the most related type strains showed values below the accepted threshold for species discrimination. Phenotypic and chemotaxonomic features were also studied. Based on these results, BD68T, BD66 and BD73 could be considered to represent a novel species of the genus Mesorhizobium, for which the name Mesorhizobium intechi sp. nov. is hereby proposed. The type strain of this species is BD68T (=CECT 9304T=LMG 30179T).

Mesorhizobium norvegicum sp. nov., a rhizobium isolated from a Lotus corniculatus root nodule in Norway.

Strain 10.2.2T was isolated from a root nodule of a Lotus corniculatus plant growing near Skammestein (Norway). Phenotypic and chemotaxonomic characterization revealed that colonies grown on yeast-mannitol broth agar were circular, convex and slimy. Growth occurred at 28 °C in 0-1 % NaCl and in a pH range from above 4 to 10. Cells were resistant to kanamycin and phosphomycin. They could assimilate carbon sources such as l-lysine, d-mannose, d-mannitol, and l-alanine. Major fatty acids found in the organism were 11-methyl C18  :  1ω7c, C16  :  0, C18  :  1ω7c, C18  :  0 and C19  :  0 cyclo ω8c. Genome sequencing and characterization of the genome revealed its size to be 8.27 Mbp with a G+C content of 62.4 mol%. Phylogenetic analyses based on the 16S rRNA gene and housekeeping gene alignments placed this strain within the genus Mesorhizobium. Pairwise genome-wide average nucleotide identity values supported that strain 10.2.2T represents a new species, for which we propose the name Mesorhizobium norvegicum sp. nov. with the type strain 10.2.2T (=DSM 108834T=LMG 31153T).

Global-level population genomics reveals differential effects of geography and phylogeny on horizontal gene transfer in soil bacteria.

Although microorganisms are known to dominate Earth's biospheres and drive biogeochemical cycling, little is known about the geographic distributions of microbial populations or the environmental factors that pattern those distributions. We used a global-level hierarchical sampling scheme to comprehensively characterize the evolutionary relationships and distributional limitations of the nitrogen-fixing bacterial symbionts of the crop chickpea, generating 1,027 draft whole-genome sequences at the level of bacterial populations, including 14 high-quality PacBio genomes from a phylogenetically representative subset. We find that diverse Mesorhizobium taxa perform symbiosis with chickpea and have largely overlapping global distributions. However, sampled locations cluster based on the phylogenetic diversity of Mesorhizobium populations, and diversity clusters correspond to edaphic and environmental factors, primarily soil type and latitude. Despite long-standing evolutionary divergence and geographic isolation, the diverse taxa observed to nodulate chickpea share a set of integrative conjugative elements (ICEs) that encode the major functions of the symbiosis. This symbiosis ICE takes 2 forms in the bacterial chromosome-tripartite and monopartite-with tripartite ICEs confined to a broadly distributed superspecies clade. The pairwise evolutionary relatedness of these elements is controlled as much by geographic distance as by the evolutionary relatedness of the background genome. In contrast, diversity in the broader gene content of Mesorhizobium genomes follows a tight linear relationship with core genome phylogenetic distance, with little detectable effect of geography. These results illustrate how geography and demography can operate differentially on the evolution of bacterial genomes and offer useful insights for the development of improved technologies for sustainable agriculture.

Mesorhizobium composti sp. nov., isolated from compost.

A polyphasic taxonomic approach was used to characterize a presumptively novel diazotrophic bacterium, designated strain CC-YTH430T, isolated from a compost sample in Taiwan. Cells of strain CC-YTH430T were found to be Gram-stain negative, facultative anaerobic rods that formed yellow-colored colonies on nutrient agar. Cell growth occurred at 15-40 °C, pH 5.0-9.0 and in the presence of 0-2% NaCl. Strain CC-YTH430T resembled Mesorhizobium species while sharing high pair-wise 16S rRNA gene sequence similarities with Mesorhizobium silamurunense, Mesorhizobium thiogangeticum, Mesorhizobium plurifarium, Mesorhizobium tamadayense, Mesorhizobium amorphae (96.9% each), Mesorhizobium sediminum (96.8%), and Mesorhizobium soli (96.5%) and < 96.5% similarity to other species. Strain CC-YTH430T showed 78.8-79.7% average nucleotide identity compared to the type strains of M. amorphae, M. plurifarium, M. soli, M. tamadayense and M. wenxiniae. The N2-fixing activity of strain CC-YTH430T was 0.2 nmol ethylene h-1 at 30 °C. The respiratory system was ubiquinone 10 (Q-10) and the DNA G+C content was 62.0 ± 0.2 mol%. The major fatty acids (> 5%) were C16:0, C17:0 cyclo, C19:0 cyclo ω8c, C14:0 3OH/C16:1 iso I and C18:1ω7c/C18:1ω6c. The polar lipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylmonomethylethanolamine and an unidentified aminolipid in major amounts. In addition, phosphatidylethanolamine, an unidentified lipid and several unidentified polar lipids were also found in moderate-to-trace amounts. Based on the phylogenetic, phenotypic and chemotaxonomic features, strain CC-YTH430T is proposed to represent a novel Mesorhizobium species, for which the name Mesorhizobium composti sp. nov. (type strain CC-YTH430T = BCRC 81024T = JCM 31762T) is proposed.

Biodiversity of rhizobia present in plant nodules of Biserrula pelecinus across Southwest Spain.

Biodiversity studies of native Mesorhizobium spp. strains able to nodulate the annual herbaceous legume Biserrula pelecinus L. in soils from Southwest Spain have been carried out. One or two isolates per plant, 30 in total, were randomly selected for further characterization. There was no association between the presence of mesorhizobia nodulating-B. pelecinus and the chemical or textural properties of the soils. The isolates were tested for their symbiotic effectiveness on this forage legume under greenhouse conditions and characterized on the basis of physiological parameters: carbon source utilisation (API 50CH), 16S rRNA sequencing and ERIC-PCR, lipopolysaccharide, protein and plasmid profiles. Our results show that in spite of the great diversity found among the native isolates, most of them belong to the genus Mesorhizobium, the exception being strain B24 which sequence matches 97.52% with Neorhizobium huautlense; this is the first description of a Neorhizobium strain effectively nodulating-biserrula plants. Results of a field trial indicated that some of these isolates could be recommended as inoculants for this legume. B24=DSM 28743=CECT 8815; ENA (HF955513) 16S rRNA sequences of isolates B13, B18, B26, B30 and B1 are deposited at ENA under numbers LS999402 to LS999406, respectively.