Mesorhizobium retamae sp. nov., a novel non-nodulating and non-nitrogen-fixing species isolated from the root nodules of Retama raetam sampled in Tunisia.

A comprehensive polyphasic taxonomic investigation integrating taxongenomic criteria was conducted on strain IRAMC:0171T isolated from the root nodules of Retama raetam in Tunisia. This Gram-stain-negative and aerobic bacterium thrived within a temperature range of 5-45 °C, optimal at 28 °C, and tolerated salt concentrations from 0-6 % NaCl, with an optimal range of 0-3 %. It displayed pH tolerance from pH 4 to 10, thriving best at pH 6.8-7.5. Chemotaxonomically, strain IRAMC:0171T was characterized by diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, and phosphatidylethanolamine as polar lipids. Its predominant fatty acid composition was C18 : 1 ω7c (61.2 %), and the primary ubiquinone was Q10 (97 %). Analysis of the 16S rRNA gene of strain IRAMC:0171T showed 99.08 % similarity to Mesorhizobium waimense ICMP 19557T, Mesorhizobium amorphae ACCC 19665T, and Mesorhizobium huakuii IAM 14158. However, digital DNA-DNA hybridization and average nucleotide identity analyses revealed values ranging from 21.1 to 25.2 % and 77.05 to 82.24 %, respectively, signifying significant deviation from established species demarcation thresholds. Phylogenetic studies, encompassing 16S rRNA, whole-genome-based tree reconstruction, and core protein analysis, positioned strain IRAMC:0171T closest to Mesorhizobium terrae KCTC 72278T and 'Mesorhizobium hungaricum' UASWS1009T, forming together a distinct branch within the genus Mesorhizobium. In consideration of this comprehensive data, we propose strain IRAMC:0171T (=DSM 112841T=CECT 30767T) as the type strain of a new species named Mesorhizobium retamae sp. nov.

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).

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.

Mesorhizobium atlanticum sp. nov., a new nitrogen-fixing species from soils of the Brazilian Atlantic Forest biome.

Biological nitrogen fixation performed by diazotrophic bacteria is a vital process for agricultural and environmental sustainability. In recent years, bacterial classification has been based on genomic data, accelerating our understanding about the diversity, and resulting in the description of several new species. In this study, four strains (CNPSo 3140T, CNPSo 3235, CNPSo 3236 and CNPSo 3237) trapped by Phaseolus vulgaris and Mimosa pudica from soil samples of the Brazilian Atlantic Forest biome (Mata Atlântica) were submitted to polyphasic analysis to investigate their proper classification within the genus Mesorhizobium. The 16S rRNA gene phylogram showed that the strains present sequences identical to those of Mesorhizobium acaciaeand Mesorhizobium plurifarium, not allowing a clear taxonomic classification; however, when using multilocus sequence analysis methodology, the strains were grouped into a well-supported distinct clade, with <94.5 % nucleotide identity with the other species of the genus. The average nucleotide identity of CNPSo 3140T genome showed values below the threshold in relation to the closest species, of 89.75 % with Mesorhizobium plurifariumand of 88.83 % with Mesorhizobium hawassense; the digital DNA-DNA hybridization values were 39 and 37.70 % with the same species, respectively. Nodulation gene (nodC) phylogeny positioned the strains in an isolated cluster, showing greater similarity to Mesorhizobiumshonense. All data obtained in this study support the description of the novel species Mesorhizobiumatlanticum sp. nov. The type strain is CNPSo 3140T (=ABIP 206T=LMG 30305T=U1602T), isolated from a nodule of Phaseolus vulgaris.

Mesorhizobium denitrificans sp. nov., a novel denitrifying bacterium isolated from sludge.

A Gram-stain-negative, non-spore-forming, facultative, rod-shaped bacterium (designated LA-28T) was isolated from a sludge sample from a wastewater treatment plant in Hanam city, Republic of Korea. On the basis of 16S rRNA gene sequencing, strain LA-28T clustered with species of the genus Mesorhizobium and appeared closely related to M. jarvisii LMG 28313T (96.8%), M. waimense ICMP 19557T (96.7%), and M. huakuii LMG 14107T (96.7%). Growth occurs at 18-40°C on R2A medium in the presence of 1-4% NaCl (w/v) and at pH 6-8. The DNA G+C content was 61.2 mol%, and the predominant quinone was ubiquinone-10 (Q-10). The major cellular fatty acids (> 5%) were C16:0, C19:0ω8c cyclo, C18:1ω7c 11-methyl, and C18:1ω7c and/or C18:1ω6c (summed feature 8). Major polar lipids were phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidyl-N-methylethanolamine (PME), and phosphatidylcholine (PC). Physiological and biochemical characteristics indicated that strain LA-28T represents a novel species of the genus Mesorhizobium, for which the name Mesorhizobium denitrificans sp. nov. is proposed. The type strain is LA-28T (= KACC 19675T = LMG 30806T).

Mesorhizobium carbonis sp. nov., isolated from coal bed water.

A Gram-stain negative, aerobic, motile, short rod-shaped bacterium, designated as B2.3T, was isolated from coal bed water collected from Jincheng, Shanxi Province, China. The strain was able to grow at 10-40 °C (optimum 28-30 °C), pH 4.0-10.0 (optimum 7.0), and in the presence of 0-5.0% NaCl (optimum 3.0%, w/v). Phylogenetic analysis based on the 16S rRNA and concatenated housekeeping gene recA, atpD and glnA sequences showed strain B2.3T belongs to the genus Mesorhizobium, with Mesorhizobium oceanicum B7T as the closely related type strain. Strain B2.3T exhibited ANI value of 77.5% and GGDC value of 21.5% to M. oceanicum B7T. The major fatty acids were identified as summed feature 8 (C18:1ω7c and/or C18:1ω6c) and 11-methyl C18:1ω7c. The major polar lipids were found to consist of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and an unidentified aminophospholipid. The predominant ubiquinone was identified as Quinone 10. Phenotypic and biochemical analysis results indicated that strain B2.3T can be distinguished from closely related type strains. On the basis of phenotypic, genotypic and chemotaxonomic characteristics, strain B2.3T is concluded to represent a novel species in the genus Mesorhizobium, for which the name Mesorhizobium carbonis sp. nov. is proposed. The type strain is B2.3T (=CGMCC 1.15730T = KCTC 52461T).

Mesorhizobium wenxiniae sp. nov., isolated from chickpea (Cicer arietinum L.) in China.

Three chickpea rhizobial strains (WYCCWR 10195T=S1-3-7, WYCCWR 10198=S1-4-3 and WYCCWR 10200=S1-5-1) isolated from Northwest China formed a group affiliated to Mesorhizobium based on 16S rRNA gene sequence comparison. To clarify their species status, multilocus sequence analysis and average nucleotide identity (ANI) values of whole genome sequences between the novel group and the type strains of the related species were further performed. Similarities of 95.7-96.6 % in the concatenated sequences of atpD-recA-glnII and 91.9-93.1 % of ANI values to the closest-related species Mesorhizobium muleiense, Mesorhizobium mediterraneum and Mesorhizobium temperatum demonstrated the novel group a unique genospecies. The most abundant fatty acid in cells of WYCCWR 10195T were C19 : 0 cyclo ω8c (51.4 %), followed by C18 : 1 ω7c 11-methyl (9.5 %) and C16 : 0 (9.3 %). Its genome size was 6.37 Mbp, comprising 6633 predicted genes with a DNA G+C content of 61.9 mol%. The similarities of 99.0-99.8 % for the nodC gene and 98.3-99.44 % for the nifH gene to those of the chickpea rhizobial species and nodulation with Cicer arietinum L. confirmed the strains of the new genospecies as symbiovar ciceri. The weak utilization of most of the tested sugars/organic acids and non-utilization of l(+)-rhamnose, l-cysteine and l-glycine as sole carbon source, tolerance to 1 % (w/v) NaCl, resistance to 5 µg ml-1 chloromycetin and non-hydrolysis of l-tyrosine distinguished the novel group from the related species and supported this group as a novel species, for which the name Mesorhizobium wenxiniae sp. nov. is proposed, with WYCCWR 10195T (=S1-3-7=HAMBI 3692T=LMG 30254T) as the type strain.

CadA of Mesorhizobium metallidurans isolated from a zinc-rich mining soil is a P(IB-2)-type ATPase involved in cadmium and zinc resistance.

Mesorhizobium metallidurans STM 2683(T) is a nitrogen-fixing bacterium that nodulates Anthyllis vulneraria in mine tailings highly contaminated in zinc, lead and cadmium. To study the mechanisms whereby this bacterium copes with metals, we functionally screened a cosmid genomic library of M. metallidurans for zinc or cadmium tolerance. A cosmid clone harbored a gene encoding P(IB)-type ATPase homologous to CadA that leads to cadmium and zinc resistance in Escherichia coli. The CadA protein structure presents one duplication of the two N-terminal metal binding domains (i.e. a heavy metal-associated domain followed by a histidine-rich domain) which allows specific binding to zinc and cadmium cations. A cadA-deleted strain of M. metallidurans failed to grow at high zinc concentrations (2 mM) and its growth was delayed at lower zinc concentrations. Expression studies using a transcriptional fusion of cadA promoter to gfp showed that cadA is specifically induced in a dose-dependent manner by zinc and cadmium in M. metallidurans in vitro conditions and into A. vulneraria nodules after Zn stress. Metal induction sensitivity was increased in the strain where cadA gene was deleted. This study identified cadA as a first mesorhizobial resistance determinant involved in detoxification of cadmium and zinc and which confers upon M. metallidurans greater capacity for coping with high zinc concentrations. This improves the knowledge of this bacterium for potential use as a symbiotic inoculant of Anthyllis in phytostabilization strategies of metal-rich sites.

Polyphasic characterization of rhizobia isolated from Leucaena leucocephala from Panxi, China.

Leucaena leucocephala was introduced into Panxi, Sichuan, China, in the 1980s and 1990s for afforestation and preventing water loss and soil erosion in this area. The co-introduction of rhizobial symbionts of introduced plants has drawn attention since they may influence local soil communities. We studied the phylogenetic position of the L. leucocephala isolates and assessed if the rhizobia were introduced together with the host to Panxi, Sichuan, China. The glnII and atpD genes of fifteen representative isolates were sequenced and analyzed, and applied multilocus sequence analyses in which the housekeeping genes recA, glnII and atpD were included. Furthermore, we estimated the within species diversity directly with 23S rDNA and IGS RFLP and indirectly through phenotypic analysis of forty L. leucocephala isolates. The isolates represented seven species and 38 diversified strains in the genera Ensifer, Mesorhizobium, Bradyrhizobium and Rhizobium. The within species diversity of the Ensifer isolates was large, proposing a potential to occupy novel niches. There was not conclusive evidence to show that any of the strains would have been co-introduced with L. leucocephala. On the contrary, we came to a conclusion that the possible introduction should not be inferred from sequence data alone.

Mesorhizobium sangaii sp. nov., isolated from the root nodules of Astragalus luteolus and Astragalus ernestii.

Our previous published data indicated that the two rhizobial strains SCAU7(T) and SCAU27, which were isolated from the root nodules of Astragalus luteolus and Astragalus ernestii respectively, in Sichuan Province, China, might be novel species of the genus Mesorhizobium. Their exact taxonomic position was determined in the present study by using polyphasic approaches. Comparative analysis of nearly full-length 16S rRNA gene sequences showed that these strains belonged to the genus Mesorhizobium, with Mesorhizobium ciceri USDA 3383(T), Mesorhizobium loti NZP 2213(T), Mesorhizobium shangrilense CCBAU 65327(T) and Mesorhizobium australicum WSM2073(T) as the closest neighbours (>99 % 16S rRNA gene sequence similarity). Phylogenies of the housekeeping genes atpD and recA confirmed their distinct position, showing low similarity with respect to those of M. loti LMG 6125(T) (96.5 % and 92.3 % similarity respectively), M. ciceri USDA 3383(T) (96.8 % and 93.3 % similarity, respectively), M. shangrilense CCBAU 65327(T) (96.5 % and 92.7 % similarity, respectively) and M. australicum WSM2073(T) (95.4 % and 90.6 % similarity, respectively). The DNA-DNA relatedness values between strain SCAU7(T) and strain SCAU27 were 83.0 %, showing that they belong to the same species. The DNA-DNA relatedness values of SCAU7(T) with M. loti NZP 2213(T), M. ciceri USDA 3383(T) and M. shangrilense CCBAU 65327(T) were 41.1 %, 48.8 % and 23.4 %, respectively, clearly indicating that strain SCAU7(T) represents a novel species. A series of phenotypic and genotypic tests and comparison of cellular fatty acids indicated that the novel group of isolates was distinct from previously described species. Therefore, we propose that strains SCAU7(T) and SCAU27 represent a novel species of the genus Mesorhizobium, Mesorhizobium sangaii sp. nov., with strain SCAU7(T) (= HAMBI 3318(T) = ACCC 13218(T)) as the type strain.

Mesorhizobium qingshengii sp. nov., isolated from effective nodules of Astragalus sinicus.

In a study on the diversity of rhizobia isolated from root nodules of Astragalus sinicus, five strains showed identical 16S rRNA gene sequences. They were related most closely to the type strains of Mesorhizobium loti, Mesorhizobium shangrilense, Mesorhizobium ciceri and Mesorhizobium australicum, with sequence similarities of 99.6-99.8%. A polyphasic approach, including 16S-23S intergenic spacer (IGS) RFLP, comparative sequence analysis of 16S rRNA, atpD, glnII and recA genes, DNA-DNA hybridization and phenotypic tests, clustered the five isolates into a coherent group distinct from all recognized Mesorhizobium species. Except for strain CCBAU 33446, from which no symbiotic gene was detected, the four remaining strains shared identical nifH and nodC gene sequences and nodulated with Astragalus sinicus. In addition, these five strains showed similar but different fingerprints in IGS-RFLP and BOX-repeat-based PCR, indicating that they were not clones of the same strain. They were also distinguished from recognized Mesorhizobium species by several phenotypic features and fatty acid profiles. Based upon all the results, we suggest that the five strains represent a novel species for which the name Mesorhizobium qingshengii sp. nov. is proposed. The type strain is CCBAU 33460(T) (=CGMCC 1.12097(T)=LMG 26793(T)=HAMBI 3277(T)). The DNA G+C content of the type strain is 59.52 mol% (Tm).

Mesorhizobium shonense sp. nov., Mesorhizobium hawassense sp. nov. and Mesorhizobium abyssinicae sp. nov., isolated from root nodules of different agroforestry legume trees.

A total of 18 strains, representing members of the genus Mesorhizobium, obtained from root nodules of woody legumes growing in Ethiopia, have been previously shown, by multilocus sequence analysis (MLSA) of five housekeeping genes, to form three novel genospecies. In the present study, the phylogenetic relationship between representative strains of these three genospecies and the type strains of their closest phylogenetic neighbours Mesorhizobium plurifarium, Mesorhizobium amorphae, Mesorhizobium septentrionale and Mesorhizobium huakuii was further evaluated using a polyphasic taxonomic approach. In line with our earlier MLSA of other housekeeping genes, the phylogenetic trees derived from the atpD and glnII genes grouped the test strains into three well-supported, distinct lineages that exclude all defined species of the genus Mesorhizobium. The DNA-DNA relatedness between the representative strains of genospecies I-III and the type strains of their closest phylogenetic neighbours was low (≤59 %). They differed from each other and from their closest phylogenetic neighbours by the presence/absence of several fatty acids, or by large differences in the relative amounts of particular fatty acids. While showing distinctive features, they were generally able to utilize a wide range of substrates as sole carbon and nitrogen sources. The strains belonging to genospecies I, II and III therefore represent novel species for which we propose the names Mesorhizobium shonense sp. nov., Mesorhizobium hawassense sp. nov. and Mesorhizobium abyssinicae sp. nov. The isolates AC39a(T) ( = LMG 26966(T) = HAMBI 3295(T)), AC99b(T) ( = LMG 26968(T) = HAMBI 3301(T)) and AC98c(T) ( = LMG 26967(T) = HAMBI 3306(T)) are proposed as type strains for the respective novel species.

Polymorphic infection and organogenesis patterns induced by a Rhizobium leguminosarum isolate from Lotus root nodules are determined by the host genotype.

To sample the natural variation in genes controlling compatibility in the legume-rhizobium symbiosis, we isolated rhizobia from nodules of endemic Lotus species from 21 sites across Europe. The majority of isolates were identified as Mesorhizobium- or Bradyrhizobium-related and formed nitrogen-fixing root nodules on Lotus corniculatus and L. pendunculatus, respectively, thus confirming previously defined cross-inoculation groups. Rhizobium leguminosarum (Rl) strain Norway, isolated from L. corniculatus nodules, displayed an exceptional phenotypic variation on different Lotus genotypes. On L. burttii, Rl Norway formed infected nodules, whereas tumors and elongated infected swellings were induced on L. glaber and L. japonicus ecotype Nepal, respectively. A symbiosis- and Nod-factor-responsive promoter:uidA fusion was strongly and rapidly induced in L. japonicus Gifu, but infection threads or signs of nodule organogenesis were absent. This complex phenotypic pattern was not mimicked by either of three engineered R. leguminosarum bv viciae strains producing different Nod-factor variants. Intriguingly, Rl Norway formed infection threads on Pisum sativum cv Sparkle, but failed to induce organogenesis. Rl Norway thus uncovered variation in symbiotic capabilities among diploid Lotus species and ecotypes that are obscured by optimally adapted M. loti strains. These contrasting infection and organogenesis phenotypes reveal recent diversification of recognition determinants in Lotus.

Mesorhizobial strains nodulating Anagyris latifolia and Lotus berthelotii in Tamadaya ravine (Tenerife, Canary Islands) are two symbiovars of the same species, Mesorhizobium tamadayense sp. nov.

Barranco de Tamadaya is a deep ravine located in southern Tenerife, which is included within a protected area where several endemic plants grow. Among them, two legumes are catalogued as critically endangered, Anagyris latifolia and Lotus berthelotii. Rhizobial strains isolated from their root nodules grown in soil samples from this ravine harboured symbiotic genes belonging to two distant symbiovars, but they shared identical 16S rRNA gene sequences (rrs). The phylogeny based on the rrs sequences placed these isolates in a separate subbranch that did not include any of the currently recognised Mesorhizobium species, but the resolution of the ribosomal tree did not permit further taxonomic conclusions. Nevertheless, multilocus sequence analysis (MLSA) of four housekeeping genes (atpD, recA, glnII and dnaK) and the rrs gene generated a highly supported Bayesian phylogeny, identifying these isolates as a new Mesorhizobium lineage. DNA-DNA hybridisation homology percentages were lower than 30% compared to type strains of the closest related species, and supported the phylogenetic data. Phenotypic characterisation also distinguished this lineage from the other closest Mesorhizobium species. The polyphasic approach thus confirmed that the isolates represented a novel species for which we propose the name Mesorhizobium tamadayense sp. nov. The type strain is Ala-3(T) (CECT 8040(T), LMG 26736(T)).

Mesorhizobium silamurunense sp. nov., isolated from root nodules of Astragalus species.

Four rhizobial strains representing a previously defined novel group in the genus Mesorhizobium and isolated from Astragalus species in China were further characterized using a polyphasic approach. Phylogenetic analysis of 16S rRNA gene sequences showed that these Gram-negative bacteria belonged to the genus Mesorhizobium, with Mesorhizobium plurifarium LMG 11892(T) as the closest neighbour sharing a sequence similarity of 99.8 %. Comparative sequence analysis of the atpD, recA, glnII, rpoB, nodC and nifH genes, SDS-PAGE of whole-cell soluble proteins, DNA-DNA hybridization, fatty acid profiles and a series of phenotypic and physiological tests differentiated the novel group from all recognized species of the genus Mesorhizobium. Based on the data obtained in the present and previous studies, this group represents a novel species within the genus Mesorhizobium, for which the name Mesorhizobium silamurunense sp. nov. is proposed. The type strain is CCBAU 01550(T) (= HAMBI 3029(T) = LMG 24822(T)), and could form effective nodules on Astragalus membranaceus, Astragalus adsurgens and Caragana intermedia, and ineffective nodules on Phaseolus vulgaris in cross-nodulation tests.