Abstract
Three bacterial strains, 1AS11T, 1AS12 and 1AS13, members of the new symbiovar salignae and isolated from root nodules of Acacia saligna grown in Tunisia, were characterized using a polyphasic approach. All three strains were assigned to the Rhizobium leguminosarum complex on the basis of rrs gene analysis. Phylogenetic analysis based on 1734 nucleotides of four concatenated housekeeping genes (recA, atpD, glnII and gyrB) showed that the three strains were distinct from known rhizobia species of the R. leguminosarum complex and clustered as a separate clade within this complex. Phylogenomic analysis of 92 up-to-date bacterial core genes confirmed the unique clade. The digital DNA–DNA hybridization and blast-based average nucleotide identity values for the three strains and phylogenetically related Rhizobium species ranged from 35.9 to 60.0% and 87.16 to 94.58 %, which were lower than the 70 and 96% species delineation thresholds, respectively. The G+C contents of the strains were 60.82–60.92 mol% and the major fatty acids (>4 %) were summed feature 8 (57.81 %; C18 : 1 ω7c) and C18 : 1 ω7c 11-methyl (13.24%). Strains 1AS11T, 1AS12 and 1AS13 could also be differentiated from their closest described species (Rhizobium indicum, Rhizobium laguerreae and Rhizobium changzhiense) by phenotypic and physiological properties as well as fatty acid content. Based on the phylogenetic, genomic, physiological, genotypic and chemotaxonomic data presented in this study, strains 1AS11T, 1AS12 and 1AS13 represent a new species within the genus Rhizobium and we propose the name Rhizobium acaciae sp. nov. The type strain is 1AS11T (=DSM 113913T=ACCC 62388T).
Keywords: Acacia saligna, nodules, Rhizobium acaciae, Rhizobium leguminosarum complex
Introduction
Acacia is a multipurpose, fast-growing tree species of the family Fabaceae. This genus comprises over 1350 species that are distributed in the warm regions of the world, including Australia, the Americas, Africa and Asia [1]. Acacia saligna (Labill.) Wendl is an invasive, fast-growing woody tree that has been introduced to the Mediterranean coast in the Maghreb area including Tunisia for many different purposes, including revegetation of arid regions [2]. These plants enrich soil nitrogen in symbiotic association with various genera of rhizobia such as Mesorhizobium, Rhizobium, Ensifer and preferentially Bradyrhizobium [3]. In a recent study, Hsouna et al. [4] reported that Acacia saligna plants grown in Tunisian soils are promiscuous and were nodulated by fast growing organisms that could constitute putative new species within the genus Rhizobium. The 100 bacterial strains obtained in the study of Hsouna et al. [4] were characterized into three distinct ribotypes based on rrs PCR-RFLP analysis. Sequence analyses of rrs and four housekeeping genes (recA, atpD, gyrB and glnII) assigned 30 isolates to four putative new lineages of the genera Rhizobium and Bradyrhizobium and a single isolate to Sinorhizobium meliloti. One of these new lineages consisted of 10 isolates, including the representative strains 1AS11T, 1AS12 and 1AS13. Phylogenetic analysis of 1734-bp concatenated sequences of recA, atpD, glnII and gyrB clustered strains 1AS11T, 1AS12 and 1AS13 in a distinct clade within the R. leguminosarum complex (Rlc). The closest described species were R. laguerreae and R. indicum [4]. Interestingly, based on the divergence of nodA/nodC sequences and host range nodulation patterns used in previous study for symbiovar delineation [5,7], strains 1AS11T, 1AS12 and 1AS13 were classified as a new symbiovar named salignae [4] . The three strains effectively nodulated A. saligna, A. salicina and Leucaena leucocephala, while they failed to nodulate Glycine max, Phaseolus vulgaris and Retama raetam.
In the present study, the taxonomic status of strains 1AS11T, 1AS12 and 1AS13 was investigated using a polyphasic approach including genome-based analyses.
Isolation and ecology
The three strains were isolated from root nodules of A. saligna grown in the locality of Borj Cedria in Tunisia [4]. Nodules were surface sterilized and bacteria were extracted following the standard procedures of Vincent [8]. Pure single colonies of the bacterial cultures were obtained by repetitive streaking on yeast–mannitol agar (YMA) as described previously [4]. Aliquots of the purified single colonies were preserved in 15 % (v/v) glycerol suspensions at −80 °C. The nodulation and nitrogen-fixation performance of the three novel strains was tested on various legume species.
rrs, recA, atpD, gyrB and glnII phylogeny
Bacterial DNA was extracted from three isolates and used as template for 50 µl PCR reactions [4]. The rrs gene were sequenced using the primers fD1 and rD1 [9]. Amplification and sequencing of recA, atpD, glnII and gyrB genes were carried out according to the methods of Hsouna et al. [4]. PCR-amplified products were purified from agarose gels using the EZ-10 spin column DNA gel extraction Minipreps Kit (Bio Basic Canada Inc.) following the manufacturer’s instructions. Purified DNA fragments were sequenced at the Centre of Biotechnology, Borj-Cédria, Tunisia. The sequence similarities in the DNA databases were examined using the blast program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The rrs gene sequences were compared with NCBI reference sequences. Sequences were aligned using the ClustalW2 software (www.ebi.ac.uk/Tools/clustalw2/). Maximum-likelihood (ML) phylogenetic trees were inferred using the mega7 [10] using the Kimura two-parameter substitution model [11] with the pairwise deletion option [12]. Bootstrap analysis was based on 1000 re-samplings and values >50 % are shown on the phylogenetic trees.
In the phylogenetic tree based on partial rrs gene sequences (1215 nt), all three new strains grouped together and showed 100 % similarity to R. laguerreae, R. changzhiense, R. anhuiense, R. leguminosarum, R. ruizarguesonis, R. acidisoli, R. hidalgonense, R. indicum, R. sophorae and R. redzepovicii type strains. Except for R. anhuiense, R. acidisoli and R. hidalgonense, these species were recently named to belong to the Rlc [13]. Fig. S1 (available in the online version of this article) shows an ML phylogenetic tree with the three strains of the proposed R. acaciae sp. nov. clustering with the type strains of the Rlc at a bootstrap value of 76 %. This suggests that the three strains of R. acaciae sp. nov. belong to the Rlc. Neighbour-joining and minimum-evolution algorithms were also used to reconstruct phylogenetic relationships and showed similar topologies (Figs S2 and S3). Given that, rrs phylogenies have been reported to show low-resolution power among closely related species [14,15], multilocus sequence analysis was performed using four housekeeping genes (recA, atpD, gyrB and glnII). Based on recA-atpD-gyrB-glnII concatenated sequences (1734 bp), the three novel strains were divergent from strains of type strains of previously described Rhizobium species of the Rlc group and the closest described species with were R. indicum, R. laguerrea and R. changzhiense. Based on the phylogenetic analysis of recA-atpD-gyrB-glnII, the three strains of R. acaciae sp. nov. clustered in a distinct clade (bootstrap of 100 %), an indication that these strains are unique (Fig. 1).
Fig. 1. Maximum-Likelihood-based phylogenetic analysis of recA-atpD-gyrB-glnII concatenated gene sequences (1734 nucleotides). The three novel strains are in bold. Bootstrap values ≥50 are indicated for each node (1000 replicates). After species name, the strain designation is given followed by the NCBI accession number of the sequence used. The scale indicates the number of substitutions per site.
Genomic relatedness and phylogenomics
Draft whole-genome sequences of the three new strains were generated using Illumina NovaSeq 6000 technology (Génome Québec, Montreal, Canada). The quality of the raw reads (2×150 bp) was checked using the FastQC algorithm [16]. De novo assembly was performed using Unicyler version 0.4.8 [17] and assessed using quast [18] as implemented in patric 3.6.12 [19,20]. Contigs of length <300 bp were discarded. Where required, assembled genome sequences were annotated using Prokka version 1.12 [21]. Table 1 shows the basic statistics of the sequenced draft genomes. For each of the strains, 33 112 583 raw reads were generated. After quality checks, the readsused in the assembling processed gave an average coverage depth of 1213.9×, 1314.8× and 1226.6× with contig counts of 84, 79 and 112, for strains 1AS11T, 1AS12 and 1AS13, respectively. The completeness of the genomes was 100 % with coarse and fine consistencies of 99.8–99.9% and 96.8–96.9 %, respectively, with a contamination rate of 0.5 % (CheckM algorithm) [22]. The largest contigs were 761366, 1 110 614 and 761 366 bases and had N50 values of 247715, 270 518 and 247 715 bases for strains 1AS11T, 1AS12 and 1AS13. The total lengths of the draft genomes were 8.01, 7.49 and 8.02 Mb, respectively, with G+C contents of 60.82–60.92 mol% (Table 1). Annotation reports of the genome sequences identified a total of 7714, 7127 and 7758 protein-coding sequences as well as 48 or 50 tRNAs, three rRNAs and one tmRNA (Table 1). The generated new draft whole-genome sequences were used to confirm the uniqueness of these strains relative to closest related Rhizobium species.
Table 1. Basic statistics of whole-genome sequences of the three strains of Rhizobium acaciae sp. nov, sequenced using Illumina NovaSeq 6000 technology.
| Description | 1AS11T | 1AS12 | 1AS13 |
| Number of short reads (151 bp length) | 33 112 583 | 33 112 583 | 33 112 583 |
| Average coverage depth | 1213.9× | 1314.8× | 1226.6× |
| Contig count | 84 | 79 | 112 |
| Largest contig (bp) | 761 366 | 1 110 614 | 761 366 |
| Total length (bp) | 8 011 864 | 7 494 653 | 8 015 410 |
| Contigs N50 (bp) | 247 715 | 270 518 | 247 715 |
| Contigs L50 | 12 | 9 | 12 |
| Protein-encoding genes with functional assignment | 5 037 | 4 743 | 5 036 |
| Total protein-coding sequences (CDSs) | 7 714 | 7 127 | 7 758 |
| misc_RNA | 75 | 67 | 75 |
| Number of rRNA | 3 | 3 | 3 |
| Number of tRNA | 48 | 50 | 48 |
| Number of tmRNA | 1 | 1 | 1 |
| G+C content (mol%) | 60.82 | 60.92 | 60.82 |
Genome-sequence-based digital DNA–DNA hybridization (dDDH; GGDC 3.1 [23]), blast-based average nucleotide identity (ANIb; JSpecies [24]) and ANI (Fastani [25]) values were computed and compared to 12 validly published close Rhizobium species to validate the taxonomic position of strains. The three strains (1AS11T, 1AS12 and 1AS13) exhibited dDDH, ANIb and ANI values significantly above the species-level cut-offs of 70 and 96 %, validating that these three belong to the same Rhizobium species (Table 2). For example, strain 1AS11T showed dDDH, ANI and ANIb values of 100, 99.99 and 99.97 % with strain 1AS13 (Table 2). Strain 1AS12 exhibited lower dDDH, ANb and ANI values with 1AS11T but still significantly above the species-level cut-off (Table 2). The type strain of the proposed R. acaciae sp. nov, strain 1AS11T, had lower relatedness with the 12 closest species with values significantly below the cut-off values of 70 and 96 % for dDDH and ANIb and ANI, respectively (Table 2). R. indicum JKLM 12A2T showed the highest dDDH of 60.0 % (cut-off, 70 %) and ANIb and ANI values of 94.58 and 94.95 % (cut-off, 96.00%) with strain 1AS11T followed by R. laguerreae FB206T with values of 56.10, 93.19 and 94.08 % (Table 2), all below the respective cut-off species delineation threshold. These data are consistent with the results of the recA-atpD-gyrB-glnII phylogenetic analysis, suggesting that strains 1AS11T, 1AS12 and 1AS13 constituted a novel species in the genus Rhizobium. The dDDH, ANIb and ANI results were corroborated by the phylogenomics tree [26,27] inferred using GBDP-derived intergenomic distances and computed on the Type (Strain) Genome Server (TyGS [28,29]) (Fig. S4). Also, the uniqueness of strains 1AS11T, 1AS12 and 1AS13 was validated by phylogenetic analysis of a concatenated alignment of 92 core genes as implemented in the up-to-date bacterial core gene (UBCG) tool [30] (Fig. 2). Both TyGS and UBCG pipelines generated phylogenomic trees that showed strains 1AS11T, 1AS12 and 1AS13 clustering uniquely from the closest known species within the genus Rhizobium (Fig. S4; Fig. 2). Based on these genome data and analyses, these new strains of the genus Rhizobium constitute a new species.
Table 2. Digital DNA–DNA hybridiziation (dDDH), average nucleotide identity (ANI) and blast-based average nucleotide identity (ANIb) values among Rhizobium acaciae sp. nov. AS11T (type strain) and related Rhizobium species.
| Rhizobium species | dDDH | ANI | ANIb |
| Rhizobium acaciae sp. nov. 1AS11T (JAPCZO01) | 100 | 100 | 100 |
| Rhizobium acaciae sp. nov. 1AS13 (JAPCZM01) | 100 (0) | 99.99 | 99.97 |
| Rhizobium acaciae sp. nov. 1AS12 (JAPCZN01) | 90.7 (0.0114) | 98.84 | 98.72 |
| Rhizobium indicum JKLM 12A2T (CP054021) | 60.0 (0.0515) | 94.95 | 94.58 |
| Rhizobium laguerreae FB 206T (MRDM01) | 56.1 (0.0588) | 94.08 | 93.19 |
| Rhizobium changzhiense WYCCWR 11279T(JABFCQ01) | 53.8 (0.0634) | 93.68 | 93.08 |
| Rhizobium sophorae CCBAU 03386T (JABFCN01) | 53.2 (0.0645) | 93.59 | 92.50 |
| Rhizobium ruizarguesonis UPM 1133T (PQIG01) | 52.7 (0.0656) | 93,52 | 92,27 |
| Rhizobium leguminosarum DSM 106839T (JACHFX01) | 49.1 (0.0737) | 92.67 | 91.34 |
| Rhizobium indigoferae CCBAU 71042 (JABFCO01) | 49.0 (0.0740) | 92.66 | 91.57 |
| Rhizobium trifolii DSM 30141T (JACJHV01) | 49.2 (0.0736) | 92.60 | 91.48 |
| Rhizobium anhuiense CGMCC 112621T (BMFI01) | 42.5 (0.0915) | 91.07 | 89.97 |
| Rhizobium acidisoli FH23T (CP034998) | 37.3 (0.1090) | 89.50 | 88.79 |
| Rhizobium redzepovicii 18TT (JAILYH01) | 36.5 (0.1123) | 88.91 | 87.60 |
| Rhizobium hidalgenense FH14T (LODW01) | 35.9 (0.1146) | 88.68 | 87.16 |
Fig. 2. Unrooted maximum-likelihood phylogenetic tree based on UBCGs (concatenated alignment of 92 core genes) inferred using RAxML-ng with the GTR +CAT model. Percentage bootstrap values are given at branching points. Bar, 0.005 substitution per position.
In addition, the whole genome sequences generated in this study were used to locate the proposed new species within the 18 genomospecies (gsp) groupings according to Young et al. [13] . The UBCG tool was used to implement an ML phylogenomics tree of strains 1AS11T, 1AS12 and 1AS13 and the proposed representatives of the genomospecies of Young et al. [13]. Fig. 3 shows an unrooted tree of the R. acaciae strains clustering closely with the representative strain of gsp J. The gsp J was represented by two strains WSM1455 (AJUF01) and WSM1481 (AQUM01) and these strains were classified as unique [13] but deposited in GenBank as Rhizobium leguminosarum bv. viciae. Interestingly, a taxonomy check using GenBank’s algorithm flagged these two strains as “inconclusive “ to be classified as R. leguminosarum. These two unique strains, WSM1455 (AJUF01) and WSM1481 (AQUM01), exhibited dDDH and ANI values of 83.10 and 98.07 % with the type strain of the proposed R. acaciae 1AS11T. These values are significantly above the species-level cut-off of 70 and 96 %, respectively, suggesting that strains WSM1455 and WSM1481 are genetically valid members of the proposed new species, R. acaciae sp. nov. Both WSM1481 and WSM1455 were isolated from roots of Vicia faba plants in the Greek island of Mykonos (GOLD database; https://gold.jgi.doe.gov/organism?id=Go0007354). This suggests that members of the proposed new species, R. acaciae sp. nov., might have preference for plant species of the family Fabaceae, from cultivated legumes (broad beans) to small wild trees, e.g. golden wreath wattle (Acacia saligna).
Fig. 3. Maximum-likelihood phylogenetic tree based on UBCGs (concatenated alignment of 92 core genes) of strains of Rhizobium acaciae sp. nov. and representatives of the 18 genomospecies proposed by Young et al. [13]. Tree was inferred using RAxML-ng with the GTR +CAT model. Percentage bootstrap values are given at branching points. Strains of R. acaciae sp. nov. are affiliated with genomospecies j. Letters in bold denote the genomospecies proposed by Young et al. [13] .
Physiology and chemotaxonomy
Colonies of strains 1AS11T, 1AS12 and 1AS13, as examined on YMA, were typical rhizobial-like translucent, cream-white and convex. The morphology of the bacterial species was examined with an atomic force microscope (FlexAFM Nanosurf AG) operated in the tapping imaging mode in phase contrast, at room temperature, using a CoreAFM Microscope, Nanosurf Isostage 300. Cells were 0.4–0.5×1–2 µm in size (Fig. S5). The phenotypic and physiological features of the novel isolates were determined and compared with their most related type strains. The characteristics tested included the use of sole carbon and nitrogen sources, tolerance to NaCl, pH, resistance to antibiotics and fatty methyl ester content. Biochemical characteristics, enzyme activities and utilization of carbon sources were performed using the API ZYM systems (07584D and 25 200, bioMérieux) and the Biolog GEN III system following the manufacturers’ instructions. Antibiotic resistance testing was carried out on Yeast extract Mannitol (YEM) plates supplemented with five concentrations of each antibiotic (5, 10, 50 80 and 100 µg ml−1). The antibiotics used were streptomycin, kanamycin, ampicillin, neomycin, erythromycin, chloramphenicol and tetracycline. Tolerance to NaCl, pH and temperature was checked in liquid YEM as previously reported [31] .The optimum pH for growth was assessed on YMA medium plates adjusted to pH 4–10 using 1 M HCl (pH 4.0 and 5.0) or 1M NaOH (pH 7–10 prior to autoclaving).
Carbon substrate characteristics (Table 3) shared by all three strains of R. acaciae sp. nov. that differed from at least one of their closest related type strains were the ability to utilize trehalose, turanose, raffinose, lactose, melibiose, methyl β-d-glucoside, N-acetyl-d-glucosamine, N-acetyl-β-d-mannosamine, α-d-glucose, d-mannose, d-galactose, d-fucose, l-rhamnose, arabitol and myo-inositol. In addition, strains of R. acaciae were able to use l-glutamic acid, l-histidine and l-pyroglutamic acid. Other characteristics shown by the strains of the novel species that differed from at least one of these closest type strains were their weak positive enzymatic reaction for cystinol-arylamidase, α-mannosidase and β-fucosidase and negative reactions for N-acetyl-β-glucosaminase. Rhizobium acaciae sp. nov. strains were resistant to kanamycin (10 µg ml−1) and streptomycin (10 µg ml−1) but sensitive to lincomycin and rifamycin. Fatty acid methyl esters were analysed using the Microbial Identification System (midi) and the Microbial Identification software package (Sherlock version 6.1; midi database, TSBA6). The fatty acid composition and abundance varied between 1AS11T and closely related type strains of Rhizobium species (Table 4). Indeed, the amount of C18 : 1 ω7c 11-methyl in strain 1AS11T was higher than that in strains FB206T of R. laguerreae and JKLM12A2T of R. indicum. Also, we detected small amounts of C15 : 0 3OH (0.27 %), C17 : 0 (0.78 %), C17 : 1 anteiso ω9c (0.32 %), C17 : 1 ω8c (0.26 %), C17 : 1 ω6c (0,1 %), C17 : 0 3OH (0.16 %), C20 : 1 ω7c (0.45 %) and C20 : 2 ω6,9c (0.17 %) that were absent in FB206T and JKLM12A2T.
Table 3. Phenotypic results on Biolog GENIII MicroPlates.
Strains: 1AS11T; 2, 1AS12; 3, 1AS13; 4, Rhizobium indicum JKLM 12A2T; 5, Rhizobium laguerreae FB206T; 6, Rhizobium changzhiense WYCCWR 11279T. +, Positive; −, negative; w, weakly positive.
| Rhizobium acaciae sp. nov. | ||||||
| Biolog substrate | 1 | 2 | 3 | 4 | 5 | 6 |
| Trehalose | + | + | + | w | − | − |
| Sucrose | − | − | − | + | − | − |
| Turanose | + | + | + | w | − | − |
| Raffinose | + | + | + | − | − | − |
| Lactose | + | + | + | − | − | − |
| Melibiose | + | + | + | − | + | − |
| Methyl β-d-glucoside | + | + | + | − | + | + |
| N-Acetyl-d-glucosamine | + | + | + | − | + | + |
| N-Acetyl-β-d-mannosamine | + | + | + | − | + | − |
| N-Acetyl-d-galactosamine | − | − | − | − | + | − |
| N-Acetyl neuraminic acid | − | − | − | − | − | + |
| α-d-Glucose | + | + | + | − | − | − |
| d-Mannose | + | + | + | − | − | − |
| d-Galactose | + | + | + | − | + | + |
| d-Fucose | + | + | + | − | + | + |
| l-Rhamnose | + | + | + | − | − | + |
| dArabitol | + | + | + | − | − | + |
| myo-Inositol | + | + | + | − | − | + |
| Glycerol | + | + | + | − | + | + |
| d-Fructose-6-PO4 | − | − | − | + | + | − |
| l-Glutamic acid | + | + | + | − | + | + |
| l-Histidine | + | + | + | − | − | + |
| l-Pyroglutamic acid | + | + | + | − | + | − |
| d-Galacturonic acid | − | − | − | + | + | − |
| d-Gluconic acid | + | + | + | − | + | + |
| d-Glucuronic acid | − | − | − | + | + | − |
| Mucic acid | − | − | − | − | − | + |
| Quinic acid | − | − | − | − | − | + |
| Methyl pyruvate | + | + | + | − | − | − |
| l-Lactic acid | − | − | − | − | − | + |
| d-Malic acid | − | − | − | − | − | + |
| l-Malic acid | − | − | − | − | + | w |
| Tween 40 | − | − | − | − | w | + |
| α-Hydroxy-butyric acid | − | − | − | − | − | + |
| β-Hydroxy-d,l butyric acid | − | − | − | − | + | + |
| α-Keto-butyric acid | − | − | − | − | − | + |
| Acetoacetic acid | − | − | − | − | − | + |
| Propionic acid | − | − | − | − | − | + |
| Acetic acid | − | − | − | w | − | + |
| Formic acid | − | − | − | − | − | + |
| Chemical sensitivity | ||||||
| pH 6 | + | + | + | − | − | − |
| pH 5 | + | + | + | − | − | − |
| NaCl 1% | − | − | − | − | + | − |
| Antbiotic resistance | ||||||
| Rifamycin | − | − | − | − | − | + |
| Lincomycin | − | − | − | − | − | + |
| Kanamycin | + | + | + | NA | − | − |
| Streptomycin | + | + | + | NA | − | − |
| API ZYM | ||||||
| Alkaline phosphomonoesterase | − | − | − | − | − | w |
| Esterase | − | − | − | NA | − | w |
| Cystinol-arylamidase | w | w | w | NA | + | + |
| N-Acetyl-β-glucosaminase | − | − | − | NA | − | + |
| α-Mannosidase | w | w | w | NA | + | + |
| β-Fucosidase | w | w | w | NA | + | + |
Table 4. Fatty acid composition (%) of 1AS11T and its related Rhizobium type strains.
Strains: 1, 1AS11T; 2, Rhizobium indicum JKLM 12A2T; 3, Rhizobium laguerreae FB206T; 4, Rhizobium changzhiense WYCCWR 11279T. –, Not detected.
| Fatty acid | 1 | 2 | 4 | 4 |
| C15 : 0 3OH | 0.27 | – | – | – |
| C17 : 0 | 0.78 | – | – | – |
| C17 : 1 ω8c | 0.26 | – | – | – |
| C17 : 1 ω6c | 0.1 | – | – | – |
| C17 : 0 3OH | 0.16 | – | – | – |
| C17 : 1 anteisoω9c | 0.32 | – | – | – |
| C18 : 1 ω9c | 0.6 | – | – | 0.5 |
| C18 : 1 ω7c 11-methyl | 13.24 | 1.8 | 1.9 | – |
| C20 : 1 ω7c | 0.45 | – | – | – |
| C20 : 2 ω6,9c | 0.17 | – | – | – |
| Summed feature 8* | 57.81 | 68 | 69.3 | 74.9 |
*Summed feature 8: C18 : 1 ω7c.
Description of Rhizobium acaciae sp. nov.
Rhizobium acaciae (a.ca'ci.ae. L. gen. n. acaciae, of the plant genus Acacia the host plant from which the novel species was isolated).
Cells of R. acaciae are Gram-stain-negative, non-spore forming rods. Colony morphology, as examined on YMA, was typical rhizobial-like translucent, cream-white and convex after 2–3 days of incubation at 28 °C. All strains grew at 16–37 °C (optimum, 28 °C) and pH 6–10 (optimum, pH 7). Strains 1AS11T, 1AS12 and 1AS13 were not able to grow at NaCl concentrations higher than 0.6 %. Detailed phenotypic and biochemical data for the R. acaciae strains are given in Table 3. The strains can be distinguished from their most closely related Rhizobium species by their ability to use trehalose, turanose, raffinose, lactose, melibiose, methyl β-d-glucoside, N-acetyl-d-glucosamine, N-acetyl-β-d-mannosamine, α-d-glucose, d-mannose, d-galactose, fucose, l-rhamnose, d-arabitol and myo-inositol as sole carbon sources; the strains showed weak positive enzymatic reactions for cystinol-arylamidase, α-mannosidase and β-fucosidase and negative reactions for N-acetyl-β-glucosaminase. Strains are resistant to 10 µg ml−1 kanamycin and 10 µg ml−1 streptomycin, but sensitive to lincomycin and rifamycin. The major fatty acid of the type strain 1AS11T is C18 : 1 ω7c with 57.81 % content.
The type strain of Rhizobium acaciae sp. nov. is 1AS11T (=DSM 113913T=ACCC 62388T), which was isolated from root nodules of Acacia saligna grown in Borj Cedria, a city in Ben Arous governorate, Tunisia. The G+C content of 1AS11T is 60.82 mol% and the genome size is approximately 8.01 Mb.
supplementary material
Acknowledgements
All contributions made by members of the Centre of biotechnology of Borj Cedria are sincerely appreciated.
Abbreviations
- ANI
Average Nucleotide Identity
- dDDH
Digital DNA-DNA hybridization
- YMA
Yeast Mannitol Agar
Footnotes
Funding: This study was funded by the PHC Maghreb project (34MAG21) and the Tunisian Ministry of Higher Education and Scientific Research. The contribution of James T. Tambong was funded by Agriculture and Agri-Food Canada through projects J-002272 and J-002295.
Accession no: The 16S rRNA, gyrB, atpD, recA and glnII sequences of strains 1AS11T, 1AS12 and 1AS13 have been deposited in the GenBank under the accession numbers OM392037, OP782378, OP782379 (16 rRNA), OM429456, OM429455, OM429457 (gyrB), OM429388, OM429386, OM429385 (atpD), OM429391, OM429392, OM429393 (recA), OM429438, OM429440 and OM429442 (glnII). The draft genome sequences of 1AS11T, 1AS12 and 1AS13 were deposited under the accession numbers JAPCZM000000000, JAPCZN000000000 and JAPCZO000000000, respectively.The Illumina NovaSeq 6000 raw reads are deposited in the Sequence Read Archive under bioproject number PRJNA893020.
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Walid Ellouze, Email: oualid.ellouz@agr.gc.ca.
Xiao Xia Zhang, Email: zhangxiaoxia@caas.cn.
Maroua Mansouri, Email: mar.waa98@yahoo.com.
Praveen Rahi, Email: praveen_rahi22@yahoo.co.in.
Mustapha Missbah El Idrissi, Email: missbah49@yahoo.fr.
Mouad Lamrabet, Email: mouadlamrabet16@gmail.com.
M'hamed Oubla, Email: m.oubla@um5r.ac.ma.
Pierre Emmanuel Courty, Email: pierre-emmanuel.courty@inrae.fr.
Daniel Wipf, Email: daniel.wipf@u-bourgogne.fr.
James T. Tambong, Email: james.tambong@agr.gc.ca.
Bacem Mnasri, Email: mnbacemm@yahoo.com.
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