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Mesorhizobium intechi sp. nov. isolated from nodules of Lotus tenuis in soils of the Flooding Pampa, Argentina
Journal Pre-proof Mesorhizobium intechi sp. nov. isolated from nodules of Lotus tenuis in soils of the Flooding Pampa, Argentina Mar´ıa Julia Estrella, Mar´ıa Florencia Fontana, Liz Marjory Cumpa ´ Velasquez, Gonzalo Arturo Torres Tejerizo, Luis Diambra, Lars ´ Sannazzaro Hestbjerg Hansen, Mariano Pistorio, Anal´ıa Ines
PII:
S0723-2020(19)30339-X
DOI:
https://doi.org/10.1016/j.syapm.2019.126044
Reference:
SYAPM 126044
To appear in:
Systematic and Applied Microbiology
Received Date:
29 July 2019
Revised Date:
21 October 2019
Accepted Date:
4 November 2019
´ Please cite this article as: Estrella MJ, Fontana MF, Cumpa Velasquez LM, Torres Tejerizo GA, Diambra L, Hansen LH, Pistorio M, Sannazzaro AI, Mesorhizobium intechi sp. nov. isolated from nodules of Lotus tenuis in soils of the Flooding Pampa, Argentina, Systematic and Applied Microbiology (2019), doi: https://doi.org/10.1016/j.syapm.2019.126044
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Mesorhizobium intechi sp. nov. isolated from nodules of Lotus tenuis in soils of the Flooding Pampa, Argentina
María Julia Estrella1, María Florencia Fontana1, Liz Marjory Cumpa Velásquez1, Gonzalo Arturo Torres Tejerizo2, Luis Diambra3, Lars Hestbjerg Hansen4, Mariano Pistorio2, Analía Inés Sannazzaro1,#
1
INTECH (Instituto Tecnológico Chascomús), CCT-La Plata, CONICET - Universidad
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Nacional de San Martín - Avenida Intendente Marino, Km 8.2, (7130) Chascomús, Argentina. 2
IBBM (Instituto de Biotecnología y Biología Molecular), CCT-La Plata, CONICET,
Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad
3
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Nacional de La Plata, (1900) La Plata, Argentina.
CREG (Centro Regional de Estudios Genómicos), CONICET - Universidad Nacional
4
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de La Plata, (1900) La Plata, Argentina.
Section of Microbial Ecology and Biotechnology, Department of Plant and
Corresponding author: Analía Inés Sannazzaro
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#
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Environmental Sciences, University of Copenhagen, 1871 Frederiksberg, Denmark
Phone: +54-2241-430323 ext. 108 Fax: +54-2241-424048
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E-mail: [email protected]
Abstract
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. opportunistum WSM
2975T, M. jarvisii ATCC 33669T and M. huakuii LMG 14107T, with sequence identities of 99.78% - 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
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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).
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Keywords: Mesorhizobium sp.nov.; Lotus tenuis; rhizobia; phylogeny
Abbreviations: nt, nucleotides; rep-PCR, repetitive element palindromic PCR; MLSA,
hybridization
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multilocus sequence analysis; ANI, average nucleotide identity; DDH, DNA-DNA
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The draft genome of Mesorhizobium intechi sp. nov. strain BD68T has been deposited at DDBJ/ENA/GenBank under accession number PNOT00000000. The version described in this paper is PNOT02000000. 16S rRNA, recA, rpoB, glnII, dnaK and nodC gene accession numbers for the type strain of Mesorhizobium intechi (BD68T) are EU748908, MH265064, MH265067, MH265070, MH265073 and MH265076, respectively. BOX-
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PCR analysis is available as a supplementary figure.
Introduction
The genus Lotus includes more than 180 legume species found worldwide, although only a few species have been domesticated and improved by selection and plant breeding for use as livestock forage. In particular, species such as L. tenuis, L.
corniculatus, L. uliginosus, L. subbiflorus and L. ornithopoides are currently used to improve pasture and hay quality where other forage legume species are not suitable [24]. Lotus species are mainly sown in South America, North America and Europe, with 1.85, 1.39 and 1.38 million hectares, respectively [5]. Like most legumes, Lotus species establish highly specific symbiotic relationships with N-fixing soil bacteria collectively known as rhizobia. The rhizobial species nodulating plants of the genus Lotus are divided into meso- and slow-growing strains [23]. The slow-growing strains, named Bradyrhizobium sp. (Lotus), are typical symbionts for L. uliginosus and L. subbiflorus, while the meso-growing strains, classified as Mesorhizobium loti [9,10], have long been
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considered to be the typical rhizobial symbionts of L. corniculatus, L. tenuis and the model L. japonicus [7,10,31]. However, recent revisions have found that strains formerly described as Mesorhizobium loti (type strain NZP 2213T) actually correspond
to the phylogenetically divergent species M. jarvisii (ATCC 33669T strain), M. erdmanii
(USDA 3471T strain) and M. japonicum (MAFF 303099T and R7A strains isolated from
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L. japonicum and L. corniculatus, respectively) [18,19]. Notwithstanding the above-
mentioned efforts to revise the taxonomic status of the Lotus-nodulating bacteria, many
strain for this species [6,16,27].
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strains are still reported as M. loti despite being phylogenetically distant from the type
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Lotus tenuis is an outstanding legume species under soil-stress conditions, but there are relatively few reports describing the identity of its associated rhizobial symbionts [6,7]. In a previous study, symbiotic nitrogen-fixing bacteria associated with L. tenuis in the
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Flooding Pampa (Argentina) have been classified mainly as members of the Mesorhizobium genus [6,33]. In the present work, three bacterial strains from the abovementioned study (BD68T, BD66 and BD73) were characterized phylogenetically as members of a distinct group within the genus Mesorhizobium, based on an analysis of their housekeeping genes (16S rRNA, recA, dnaK, glnII and rpoB). A polyphasic
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approach was employed to describe the taxonomic status of one representative strain (BD68T) compared with the type strains of related Mesorhizobium species. As a result, a novel L. tenuis nodulating species, Mesorhizobium intechi sp. nov., is proposed. Isolates BD68T, BD66 and BD73 were cultured in tryptone-yeast extract (TY) medium and stored in 30% (w/v) glycerol at -80 °C. Total DNAs used for amplification of the genes or fragments from the rhizobial strains were extracted using an AccuPrep® Genomic DNA Extraction Kit (Bioneer) according to the manufacturer’s guidelines.
BOX-PCR fingerprint profiling was carried out as previously described [32] and profile comparison revealed that the three isolates showed different patterns (Supplementary Figure S1), demonstrating that they were not clonal varieties. The 16S rRNA gene from the three strains was PCR-amplified using the primer pairs 41f/1488r [6]. Strains BD68T, BD66 and BD73 had identical sequences for a 1,270 bp internal region of the 16S rRNA gene. A 16S rRNA-based phylogenetic tree with the 49 hitherto validly published species of Mesorhizobium was performed: 16S rRNA gene sequences of mesorhizobial type strains obtained through the Ribosomal Database Project (RDP, 8) were aligned with the corresponding sequences of the novel isolates using the Clustal
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module implemented by the MEGA7 software [11]. Distances were calculated in a complete deletion procedure and the maximum-likelihood method was used to reconstruct the corresponding phylogenetic tree by means of the MEGA7 software. The
robustness of the tree topology was evaluated by bootstrap analysis (1,000 replicates).
The three novel isolates clustered together with M. japonicum MAFF 303099T, M.
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erdmanii USDA 3471T, M. carmichaelinearum ICMP 18942T, M. opportunistum WSM 2975T and M. jarvisii ATCC 33669T (Figure 1), with sequence identities of 100%,
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100%, 99.86%, 99.79% and 99.72%, respectively. In turn, the newly isolated strains did not group with the canonical rhizobial species for L. corniculatus, M. loti NZP 2213T
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(98.12% identity). It has been shown that species within the Mesorhizobium genus usually share high 16S rRNA sequence similarity values, making it difficult to delineate species entirely on the basis of this phylogenetic marker [25]. For this reason, the
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analysis of additional taxonomical markers is the recommended procedure for elucidating systematic relationships within mesorhizobial species. The selection and use of housekeeping genes for taxonomic purposes has been a critical step forward; therefore, the four protein-coding genes, recA, glnII, dnaK and rpoB, were further studied since they have been previously shown to produce a robust phylogeny of the
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genus Mesorhizobium [26], and their sequences are available for most type species. Partial recA, glnII, dnaK and rpoB sequences of strains BD68T, BD66 and BD73 were obtained according to previously described methodology [8,17,37,39]. Sequence similarities for the four housekeeping genes between the three new isolates were calculated and values between 99-100% were observed, supporting the fact that the three isolates corresponded to the same species. Additionally, phylogenetic trees for each housekeeping gene were performed in order to establish the relative taxonomic position of the three isolates within the Mesorhizobium genus. Reference sequences for
the Mesorhizobium type strains were recovered from the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov) GenBank database. Sequences for each gene in a given species were aligned and trimmed and 24 nucleotide substitution models were tested using the MEGA7 software. The lowest Bayesian information criterion scores were considered in order to choose the best substitution pattern for phylogenetic inferences. In the four phylogenies, the L. tenuis isolates grouped in a separate cluster, close to M. japonicum MAFF 303099T, M. jarvisii ATCC 33669T, M. carmichaelinearum ICMP 18942T and M. huakuii LMG 14107T (Supplementary Figure S2).
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Taken together, the results of this study suggested that the novel strains represented a species distinct from all the previously recognized species within the Mesorhizobium genus. To identify the three newly isolated strains accurately, BD68T was designated as
a group representative and its genome was fully sequenced in order to perform in silico
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whole genome comparisons. Additionally, morphological/phenotypic features and major fatty acid profiles were determined, according to the suggested recommendations
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regarding the description of new species [12,36].
The whole genomic DNA of strain BD68T used for genome sequencing was extracted using the AccuPrep® Genomic DNA Extraction Kit (Bioneer). 2x250bp paired-end
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reads were obtained from sequencing on an Illumina MiSeq platform using a Nextera XT DNA library preparation kit. The resulting reads were quality trimmed [2] and the
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draft genome was assembled using the MIRA (version 4.9.6, [3]) and AbySS (version 1.9.0, [35]) assemblers. The Contig Integrator for Sequence Assembly (CISA, [14]) was used to integrate the sets of contigs from the two assemblers. In this way, the reads from BD68T were assembled into 393 contigs (N50 28,806 bp) with a 19.66-fold coverage. The genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline
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(PGAP). The sequences have been deposited in the NCBI GenBank under the accession number PNOT00000000. The reference genome sequences used for whole genome comparisons were retrieved from NCBI GenBank. The genome size of strain BD68T was approximately 6.6 Mbp, comprised of 6,590 predicted genes. Taking advantage of whole genome sequencing for the BD68T strain, and in order to identify precisely the closest species to be used in the subsequent analysis, a multilocus sequence analysis (MLSA) with seven housekeeping genes, recA, glnII, dnaK, rpoB, gyrB, truA and thrA, which were previously shown to produce a robust phylogeny for
the genus Mesorhizobium [26], was performed. The seven housekeeping genes were concatenated and the resulting alignment in the length of 2,951 bp was analyzed by the maximum-likelihood method based on a matrix with the distance correction calculated by the general time reversible model with 1,000 resamplings in the bootstrap analysis (Figure 2). According to the MLSA tree, the BD68T most-related type strains were M. japonicum MAFF 303099T and M. jarvisii ATCC 33669T. The average nucleotide identity (ANI) calculation is an alternative approach to the traditional DNA-DNA hybridization method [28]. ANI calculations between BD68T and its phylogenetically closest type strains were performed using the JSpeciesWS web
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server (http://jspecies.ribohost.com/jspeciesws) [29] based on pairwise alignment of the genomes using the BLAST+ (ANIb) or MUMmer (ANIm) tools. ANIb and ANIm
values (Table 1) were consistent and clearly below the 95-96% proposed cutoff for discriminating different species [28]. Additionally, in silico DNA-DNA hybridization
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(isDDH) values were calculated using the genome-to-genome distance calculation
(GGDC 2.1) online service (http://ggdc.dsmz.de/distcalc2.php), as described by MeierKolthoff and co-workers [20]. The results of the GGDC were based on the
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recommended BLAST+ method and formula 2 (sum of all identities found in highscoring segment pairs (HSPs) divided by overall HSP length), which is independent of
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the genome length and is thus robust against the use of incomplete draft genomes [1,20]. The isDDH percentage values resulting from the comparison of BD68T with closely related type strain genomes (Table 1) were well below the threshold of 70 % for
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species delineation, further supporting that the newly isolated strains belonged to a new species in the genus Mesorhizobium. Recently, a novel approach for prokaryote taxonomy using a comprehensive database of the genomes of type strains and truly whole-genome-based methods was proposed as an
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alternative to standard techniques such as DDH and G+C content, and the 16S rRNA gene and MLSA [21], and a web interface for such analysis was developed (Type Strain Genome Server, https://tygs.dsmz.de). The BD68T type strain was tested by this analysis and the resulting whole-genome phylogram also supported the aforementioned findings (Supplementary Figure S3). Phenotypic features of strains BD66, BD68T and BD73 were determined using the API 20NE (bioMérieux) and BIOLOG GENIII (Biolog Inc.) kits following the manufacturers' instructions. Distinctive features of our isolates compared with
representative strains of the closest phylogenetically related Mesorhizobium species are depicted in Table 2. The fatty acid profile of the type strain, BD68T, was determined under the growth conditions, harvesting and extraction procedures described by Sasser [34]. Briefly, BD68T was grown aerobically on tryptone-yeast extract agar plates at 28 °C for 48 h. The fatty acids were extracted and analyzed according to the recommendations of the commercial Microbial Identification System (Microbial ID, MIDI) and whole-cell fatty acid composition was determined by gas chromatography (Agilent Technologies 6890N) using the peak naming table MIDI TSBA 5.0. The fatty acid composition of
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BD68T comprised C19:0 CYCLO 8c (27.23%), C16:0 (21.49%), C18:17c (16.03%), C18:17c 11-methyl (7.87%), C12:0 (6.76%), C17:0 ISO (5.95%), C16:1 ISO H (5.39%),
C16:0 N OH (5.35%) and C18:0 (3.92%). These results were in accordance with the main fatty acid compounds detected in all species of the genus Mesorhizobium [38].
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Although housekeeping genes are useful for the establishment of rhizobial taxonomic
status, they do not offer information regarding bacterial symbiotic behavior in terms of
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the legume hosts. The analysis of the symbiotic gene nodC, located in interchangeable elements (plasmids or symbiotic islands), has long been used for the identification of
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strains at the symbiovar level [15,25,30]. The nodC sequences of the new isolates from L. tenuis nodules were compared with other Mesorhizobium spp. nodC genes in a maximum-likelihood tree using the Tamura 3 (G+I) model (Figure 3). The nodC genes
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of L. tenuis strains BD68T, BD66 and BD73 were phylogenetically related to those of M. loti and other type strains representing the symbiovar loti [13,15,22], although they clustered together on a separate branch with 100% bootstrap support. The similarity in the nodC sequences of strains BD68T, BD66 and BD73 with respect to the closest sv. loti strains (M. olivaresii CPS 13T, M. helmanticense CSLC 115NT, M. japonicum
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MAFF 303099T, M. loti NZP 2213T, M. tarimense CCBAU 83306T and M. erdmanii USDA 3471T) ranged between 96.91%-97.74%. In turn, the less related nodC sequence in this symbiovar, belonging to M. jarvisii ATCC 33669T, shared a similarity of 93.28% with the three strains. Description of Mesorhizobium intechi sp. nov.
Mesorhizobium intechi (in.te'chi N.L. gen. n. intechi, pertaining to INTECH (Technological Institute of Chascomús) where the strains of this work were isolated and characterized. Gram-negative, aerobic, non-spore-forming rods. Colonies appearing on yeast extractmannitol-agar within 2-4 days incubation at 28 ºC are circular, convex and creamcolored. The optimum temperature for aerobic growth is 28 ºC, growth was observed at 10 ºC but not at 37 ºC; all strains are sensitive to 1% NaCl and grow over a pH range of 7-10. Strains are resistant to ampicillin (10 g mL-1), gentamicin (10 g mL-1), neomycin (5 g mL-1) and kanamycin (50 g mL-1); susceptible to chloramphenicol (10
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g mL-1). The complete description of the new species can be found at the Digital
Protologue website (http://imedea.uib-csic.es/dprotologue/) under the taxonumber TA00564 (Table 3). The type strain BD68T, isolated from nodules of Lotus tenuis, has been deposited as CECT 9304T and LMG 30179T. The whole-genome shotgun project
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sequences PNOT02000001-PNOT02000393.
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has been deposited in GenBank under the accession no. PNOT00000000 and consists of
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Conflict of interest There are no conflicts of interest.
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Acknowledgements
The authors wish to thank Dr. Gustavo Somoza for helping with the Latin for the species name. This study was supported by the Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC, Argentina); Consejo Nacional de Investigaciones
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Científicas y Técnicas (CONICET, Argentina) and Agencia Nacional de Promoción Científica y Tecnológica (Argentina) by grants PICT-2013-0963, PICT-2015-3789 and PICT-2012-0518, to M.J.E, A.I.S and M.P., respectively. A.I.S., G.T.T., and M.P. are members of the Research Career of CONICET. M.J.E. is a member of the Research Career of CIC. L.M.C.V. and M.F.F. are doctoral CONICET fellows.
References [1]
Auch, A.F., von Jan, M., Klenk, H.-P., Göker, M. (2010) Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand. Genomic Sci. 2(1), 117–134, Doi: 10.4056/sigs.531120.
[2]
Bolger, A.M., Lohse, M., Usadel, B. (2014) Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30(15), 2114–2120, Doi: 10.1093/bioinformatics/btu170.
[3]
Chevreux, B., Pfisterer, T., Drescher, B., Driesel, A.J., Muller, W.E.G., Wetter,
ro of
T., Suhai, S. (2004) Using the miraEST assembler for reliable and automated
mRNA transcript assembly and SNP detection in sequenced ESTs. Genome Res. 14(6), 1147–1159, Doi: 10.1101/gr.1917404. [4]
Cole, J.R., Wang, Q., Fish, J.A., Chai, B., McGarrell, D.M., Sun, Y., Brown,
-p
C.T., Porras-Alfaro, A., Kuske, C.R., Tiedje, J.M. (2014) Ribosomal Database
Project: Data and tools for high throughput rRNA analysis. Nucleic Acids Res.
[5]
re
42(D1), 633–642, Doi: 10.1093/nar/gkt1244.
Díaz, P., Borsani, O., Monza, J. (2005) Lotus-related species and their agronomic
lP
importance. In: Márquez, A.J., Stougaard, J., Udvardi, M., Parniske, M., Spaink, H., Saalbach, G., Webb, J., Chiurazzi, M., (Eds.), Lotus japonicus Handbook,
[6]
ur na
Springer, pp. 25–37.
Estrella, M.J., Muñoz, S., Soto, M.J., Ruiz, O., Sanjuan, J. (2009) Genetic diversity and host range of rhizobia nodulating Lotus tenuis in typical soils of the Salado River Basin (Argentina). Appl. Environ. Microbiol. 75(4), 1088–1098, Doi: 10.1128/AEM.02405-08. Fulchieri, M.M., Estrella, M.J., Iglesias, A.A. (2001) Characterization of
Jo
[7]
Rhizobium loti strains from the Salado River Basin. Antonie Van Leeuwenhoek 79(2), 119–125.
[8]
Gaunt, M.W., Turner, S.L., Rigottier-Gois, L., Lloyd-Macgilp, S.A., Young, J.P. (2001) Phylogenies of atpD and recA support the small subunit rRNA-based classification of rhizobia. Int. J. Syst. Evol. Microbiol. 51(Pt 6), 2037–2048.
[9]
Jarvis, B.D.W., Van Berkum, P., Chen, W.X., Nour, S.M., Fernandez, M.P.,
Cleyet-Marel, J.C., Gillis, M. (1997) Transfer of Rhizobium loti, Rhizobium huakuii, Rhizobium ciceri, Rhizobium mediterraneum, and Rhizobium tianshanense to Mesorhizobium gen. nov. Int. J. Syst. Bacteriol. 47(3), 895–898, Doi: 10.1099/00207713-47-3-895. [10] Jarvis, B.D.W., Pankhurst, C.E., Patel, J.J. (1982) Rhizobium loti, a new species of legume root nodule bacteria. Int. J. Syst. Bacteriol. 32(3), 378–380, Doi: 10.1099/00207713-32-3-378. [11] Kumar, S., Stecher, G., Tamura, K. (2016) MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33(7), 1870–
ro of
1874, Doi: 10.1093/molbev/msw054.
[12] de Lajudie, P.M., Andrews, M., Ardley, J., Eardly, B., Jumas-Bilak, E.,
Kuzmanović, N., Lassalle, F., Lindström, K., Mhamdi, R., Martínez-Romero, E., Moulin, L., Mousavi, S.A., Nesme, X., Peix, A., Puławska, J., Steenkamp, E.,
-p
Stępkowski, T., Tian, C.-F., Vinuesa, P., Wei, G., Willems, A., Zilli, J., Young, P. (2019) Minimal standards for the description of new genera and species of
10.1099/ijsem.0.003426.
re
rhizobia and agrobacteria. Int. J. Syst. Evol. Microbiol. 69(7), 1852–1863, Doi:
lP
[13] Laranjo, M., Young, J.P.W., Oliveira, S. (2012) Multilocus sequence analysis reveals multiple symbiovars within Mesorhizobium species. Syst. Appl. Microbiol. 35(6), 359–367, Doi: 10.1016/j.syapm.2012.06.002.
ur na
[14] Lin, S.-H., Liao, Y.-C. (2013) CISA: Contig integrator for sequence assembly of bacterial genomes. PLoS One 8(3), e60843. [15] Lorite, M.J., Flores-Felix, J.D., Peix, A., Sanjuan, J., Velazquez, E. (2016) Mesorhizobium olivaresii sp. nov. isolated from Lotus corniculatus nodules.
Jo
Syst. Appl. Microbiol. 39(8), 557–561, Doi: 10.1016/j.syapm.2016.09.003. [16] Marcos-García, M., Menéndez, E., Cruz-González, X., Velázquez, E., Mateos, P.F., Rivas, R. (2015) The high diversity of Lotus corniculatus endosymbionts in soils of northwest Spain. Symbiosis 67(1–3), 11–20, Doi: 10.1007/s13199-0150368-5. [17] Martens, M., Dawyndt, P., Coopman, R., Gillis, M., De Vos, P., Willems, A. (2008) Advantages of multilocus sequence analysis for taxonomic studies: A case
study using 10 housekeeping genes in the genus Ensifer (including former Sinorhizobium). Int. J. Syst. Evol. Microbiol. 58(1), 200–214, Doi: 10.1099/ijs.0.65392-0. [18] Martínez-Hidalgo, P., Ramírez-Bahena, M.H., Flores-Félix, J.D., Igual, J.M., Sanjuán, J., León-Barrios, M., Peix, A., Velázquez, E. (2016) Reclassification of strains MAFF 303099T and R7A into Mesorhizobium japonicum sp. nov. Int. J. Syst. Evol. Microbiol. 66(12), 4936–4941, Doi: 10.1099/ijsem.0.001448. [19] Martínez-Hidalgo, P., Ramírez-Bahena, M.H., Flores-Félix, J.D., Rivas, R., Igual, J.M., Mateos, P.F., Martínez-Molina, E., León-Barrios, M., Peix, À.,
ro of
Velàzquez, E. (2015) Revision of the taxonomic status of type strains of
Mesorhizobium loti and reclassification of strain USDA 3471T as the type strain of Mesorhizobium erdmanii sp. nov. and ATCC 33669T as the type strain of
Mesorhizobium jarvisii sp. nov. Int. J. Syst. Evol. Microbiol. 65(6), 1703–1708,
-p
Doi: 10.1099/ijs.0.000164.
[20] Meier-Kolthoff, J.P., Auch, A.F., Klenk, H.-P., Göker, M. (2013) Genome
re
sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 14(1), 60, Doi: 10.1186/1471-2105-14-
lP
60.
[21] Meier-Kolthoff, J.P., Göker, M. (2019) TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat. Commun. 10(1), Doi:
ur na
10.1038/s41467-019-10210-3.
[22] Mohamad, R., Willems, A., Le Quéré, A., Maynaud, G., Pervent, M., Bonabaud, M., Dubois, E., Cleyet-Marel, J.C., Brunel, B. (2017) Mesorhizobium delmotii and Mesorhizobium prunaredense are two new species containing rhizobial
Jo
strains within the symbiovar anthyllidis. Syst. Appl. Microbiol. 40(3), 135–143, Doi: 10.1016/j.syapm.2017.01.004.
[23] Pankhurst, C.E. (1977) Symbiotic effectiveness of antibiotic-resistant mutants of fast- and slow-growing strains of Rhizobium nodulating Lotus species. Can. J. Microbiol. 23(8), 1026–1033, Doi: 10.1139/m77-152. [24] Papadopoulos, Y.A., Kelman, W.M. (1999) Traditional breeding of Lotus species. Trefoil: the science and technology of Lotus, Crop Science Society of America and American Society of Agronomy, Madison, WI, pp. 187–198.
[25] Peix, A., Ramírez-Bahena, M.H., Velázquez, E., Bedmar, E.J. (2015) Bacterial associations with legumes. CRC. Crit. Rev. Plant Sci. 34(1–3), 17–42, Doi: 10.1080/07352689.2014.897899. [26] Pérez-Yépez, J., Armas-Capote, N., Velázquez, E., Pérez-Galdona, R., Rivas, R., León-Barrios, M. (2014) Evaluation of seven housekeeping genes for multilocus sequence analysis of the genus Mesorhizobium: Resolving the taxonomic affiliation of the Cicer canariense rhizobia. Syst. Appl. Microbiol. 37(8), 553– 559, Doi: 10.1016/j.syapm.2014.10.003. [27] Reeve, W., Ardley, J., Tian, R., Eshragi, L., Yoon, J.W., Ngamwisetkun, P.,
ro of
Seshadri, R., Ivanova, N.N., Kyrpides, N.C. (2015) A genomic encyclopedia of the root nodule bacteria: assessing genetic diversity through a systematic
biogeographic survey. Stand. Genomic Sci. 10(1), 14, Doi: 10.1186/1944-327710-14.
-p
[28] Richter, M., Rossello-Mora, R. (2009) Shifting the genomic gold standard for the prokaryotic species definition. Proc. Natl. Acad. Sci. 106(45), 19126–19131,
re
Doi: 10.1073/pnas.0906412106.
[29] Richter, M., Rosselló-Móra, R., Oliver Glöckner, F., Peplies, J. (2016)
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JSpeciesWS: a web server for prokaryotic species circumscription based on pairwise genome comparison. Bioinformatics 32(6), 929–931. [30] Rogel, M.A., Ormeño-Orrillo, E., Romero, E.M. (2011) Symbiovars in rhizobia
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reflect bacterial adaptation to legumes. Syst. Appl. Microbiol. 34, 96–104. [31] Saeki, K., Kouchi, H. (2000) The Lotus symbiont, Mesorhizobium loti: molecular genetic techniques and application. J. Plant Res. 113(4), 457–465. [32] Sannazzaro, A.I., Bergottini, V.M., Paz, R.C., Castagno, L.N., Menéndez, A.B.,
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Ruiz, O.A., Pieckenstain, F.L., Estrella, M.J. (2011) Comparative symbiotic performance of native rhizobia of the Flooding Pampa and strains currently used for inoculating Lotus tenuis in this region. Antonie Van Leeuwenhoek 99, 371– 379, Doi: 10.1007/s10482-010-9502-9.
[33] Sannazzaro, A.I., Tejerizo, G.T., Fontana, M.F., Cumpa Velásquez, L.M., Hansen, L.H., Pistorio, M., Estrella, M.J. (2018) Mesorhizobium sanjuanii sp. nov., isolated from nodules of Lotus tenuis in the saline-alkaline lowlands of
Flooding Pampa, Argentina. Int. J. Syst. Evol. Microbiol. 68(9), 2936–2942, Doi: 10.1099/ijsem.0.002924. [34] Sasser, M. (1990) Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Tech. Note 101Newark,DEMicrobial ID, Inc. [35] Simpson, J.T., Wong, K., Jackman, S.D., Schein, J.E., Jones, S.J.M., Birol, I. (2009) ABySS: a parallel assembler for short read sequence data. Genome Res. 19(6), 1117–1123, Doi: 10.1101/gr.089532.108. [36] Stackebrandt, E., Frederiksen, W., Garrity, G.M., Grimont, P.A.D., Kampfer, P., Maiden, M.C.J., Nesme, X., Rossello-Mora, R., Swings, J., Truper, H.G.,
ro of
Vauterin, L., Ward, A.C., Whitman, W.B. (2002) Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int. J. Syst. Evol. Microbiol. 52(March 2002), 1043–1047, Doi: 10.1099/ijs.0.02360-0.02360.
[37] Stȩpkowski, T., Czaplińska, M., Miedzinska, K., Moulin, L. (2003) The variable
-p
part of the dnaK gene as an alternative marker for phylogenetic studies of
rhizobia and related alpha Proteobacteria. Syst. Appl. Microbiol. 26(4), 483–
re
494, Doi: 10.1078/072320203770865765.
[38] Tighe, W., de Lajudie, P., Dipietro, K., Lindström, K., Nick, G., Jarvis, B.D.W.
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(2000) Analysis of cellular fatty acids and phenotypic reIationships of Agrobacterium and Sinorhizobium species using the Sherlock Microbial
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Identification System. Int. J. Syst. Evol. Microbiol. 50, 787–801. [39] Turner, S.L., Young, J.P.W. (2000) The glutamine synthetases of rhizobia:
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Phylogenetics and evolutionary implications. Mol. Biol. Evol. 17(2), 309–319.
Figure captions: Figure 1. Maximum-likelihood phylogenetic tree based on Mesorhizobium type strain16S rRNA gene sequences (1,270 nucleotides) showing the position of Mesorhizobium intechi sp. nov. used in this study (indicated in bold). The tree was constructed using the Kimura 2-parameter (G+I) model. Bootstrap values (above 50%) calculated for 1,000 replications are indicated at the nodes. GenBank accession numbers are given between
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curved brackets. Bar, 0.02 substitutions per nucleotide position.
Figure 2. Maximum-likelihood phylogenetic tree based on concatenated recA (298 nt), glnII (405 nt), dnaK (223 nt), rpoB (441 nt), gyrB (578 nt), truA (668 nt) and thrA (280 nt) gene sequences showing the position of Mesorhizobium intechi sp. nov. used in this study (indicated in bold) within the genus Mesorhizobium. GenBank accession numbers
are detailed in Supplementary Table S1. The tree was constructed using the general time reversible (G+I) model. Bootstrap values (above 50%) calculated for 1,000 replications
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are indicated at the nodes. Bar, 0.05 substitutions per nucleotide position.
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Figure 3. Maximum-likelihood tree based on partial sequences of the nodC gene (383 nucleotides) showing the relationships between Mesorhizobium intechi sp. nov. (in bold) and other Mesorhizobium strains, indicating the different symbiovars (sv.) defined within the genus. The tree was constructed using the Tamura 3 (G+I) model. Bootstrap values (above 50%) calculated for 1,000 replications are indicated at the nodes. Bar,
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0.05 substitutions per nucleotide position. Original host legumes are given for unknown symbiovars.
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Table 1. Whole genome sequence comparisons between M. intechi BD68T isolated from L. tenuis nodules and phylogenetically closely related Mesorhizobium species.
ANIba
ANImb
isDDHc
M. jarvisii ATCC 33669T
92.71
93.81
53.20
M. japonicum MAFF 303099T
92.19
93.29
50.80
M. carmichaelinearum ICMP 18942T
89.57
91.02
41.50
M. erdmanii USDA 3471T
88.73
90.09
38.30
M. opportunistum WSM 2075T
86.97
88.78
34.80
a
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Strain
Average nucleotide identity percentage based on BLAST searches of 1 kb genome
fragments against a target genome. bAverage nucleotide identity percentage based on the MUMmer algorithm that does not require the artificial generation of 1 kb fragments.
In silico DNA-DNA hybridization values calculated using the recommended formula 2:
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c
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identities/HSP length.
Table 2. Distinctive features of M. intechi BD68T and its closest type species. 3
4
5
+ + + + + + + +
+ + + + + + +
+ + + + + + +
+ + + -
+ + + ± -
+
±
+
+ +
+ -
+ -
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Growth conditions 1% NaCl 10 ºC 37 ºC pH 10
2
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Assimilation of: L-Gluconate L-Malate L-Alanine D-Lactose D-Maltose D-Melibiose D-Trehalose D-Cellobiose para-nitrophenyl-Dphenylgalactopyranose
1
-
-
+ +
+ + + -
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Species
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Strains: 1, BD66; 2, BD68T; 3, BD73; 4, M. japonicum MAFF 30399T; 5, M. jarvisii ATCC 33669T. +, positive; -, negative; ±, weakly positive. Data are from this study.
Table 3. Description of Mesorhizobium intechi sp. nov. according to Digital Protologue TA00564 assigned by the www.imedea.uib.es/dprotologue website. TA00564 Mesorhizobium intechi Mesorhizobium intechi sp. nov. in.te'chi N.L. gen. n. intechi, pertaining to INTECH (Technological Institute of Species etymology Chascomús) where the strains of this work were isolated and characterized Estrella MJ, Fontana MF, Cumpa Velásquez LM, Torres Tejerizo G, Diambra Authors L, Hansen LH, Pistorio M, Sannazzaro AI Mesorhizobium intechi sp. nov. isolated from nodules of Lotus tenuis in soils of Title the Flooding Pampa, Argentina Corresponding author Analía Inés Sannazzaro E-mail of the [email protected] corresponding author Analía Inés Sannazzaro Submitter E-mail of the submitter [email protected] Designation of the type BD68 strain Strain collection CECT 9304 = LMG 30179 numbers 16S rRNA gene EU748908 accession number Alternative recA [MH265064], rpoB [MH265067], glnII [MH265070], dnaK [MH265073] housekeeping genes Genome accession PNOT00000000 number [RefSeq] Draft Genome status 6,580 kbp Genome size 63.2 GC mol% Argentina Country of origin Flooding Pampa Region of origin Root nodules Source of isolation Chascomús, Buenos Aires, Argentina Geographic location 35º 58' S Latitude 58º 00' W Longitude Number of strains in 3 study Source of isolation of Root nodules non-type strains Growth medium, incubation conditions Tryptone-yeast (TY) medium, 28 ºC, pH 7 used for standard cultivation Alternative medium 1 Yeast extract-mannitol (YEM), 28 ºC, pH 7 Conditions of Liquid TY medium with 30% glycerol stored at -80 °C preservation Negative Gram stain Rod Cell shape Cell size (length or 2-4 diameter)
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Taxonumber Species name Genus name Specific epithet Species status
Motile Flagellar On TY agar after 48 h at 28 °C colonies are circular with a creamy color, entire Colony morphology margin, convex elevation, shiny, mucoid Temperature optimum 28 7 pH optimum Neutrophile pH category Non-halophile (NaCl inhibitory at 1% NaCl) Salinity category Aerobe Relationship to O2 O2 conditions for strain Aerobiosis testing Chemoorganotroph Energy metabolism Positive Oxidase Negative Catalase Oxidation of: D-salicin, inosine, alpha-D-glucose, glycerol, D-sorbitol, Dglucuronic acid, pectin, bromo-succinic acid, Tween 40, gentiobiose, dextrin, Nacetyl-D-glucosamine, D-fucose, D-mannose, D-mannitol, alpha-D-lactose, Lglutamic acid, glycyl-L-proline, alpha-ketoglutaric acid, methylpyruvate, Positive tests with gamma-aminobutyric acid, sucrose, D-maltose, N-acetyl-beta-D-mannosamine, BIOLOG L-fucose, D-fructose, D-fructose-6-PO4, D-arabitol, D-malic acid, D-lactic acid methyl ester, D-turanose, D-trehalose, beta-methyl-D-glucoside, L-rhamnose, D-galactose, myo-inositol, L-pyroglutamic acid, L-arginine, L-malic acid, Llactic acid, D-cellobiose, D-melibiose Oxidation of: D-serine, L-aspartic acid, L-serine, gelatin, phydroxyphenylacetic acid, alpha-ketobutyric acid, formic acid, L-histidine, Lalanine, alpha-hydroxybutyric acid, acetic acid. Negative tests with Growth in presence of: 1% NaCl, 1% sodium lactate, troleandomycin, BIOLOG lincomycin, vancomycin, nalidixic acid, aztreonam, fusidic acid, rifamycin SV, guanidine HCl, tetrazolium violet, lithium chloride, D-serine, minocycline, Niaproof 4, tetrazolium blue, potassium tellurite, sodium bromate Oxidation of: N-acetyl neuraminic acid, D-raffinose, 3-methylglucose, Dsaccharic acid, citric acid, D-glucose-6-PO4, glucuronamide, D-galacturonic Variable tests with acid, acetoacetic acid, L-gluconic acid, music acid, L-galactonic acid lactone, BIOLOG propionic acid, N-acetyl-D-galactosamine, D-aspartic acid, quince acid, betahydroxy-D, L-butyric acid, stachyose. Growth in presence of sodium butyrate ß-galactosidase (para-nitrophenyl-ßD-galactopyranoside), assimilation of glucose, assimilation of arabinose, assimilation of mannose, assimilation of Positive tests with API mannitol, assimilation of N-acetyl-glucosamine, assimilation of maltose, hydrolysis of aesculin, assimilation of malate, urease Reduction of nitrates to nitrites, reduction of nitrates to nitrogen, indole production (tryptophan), fermentation of glucose, arginine dihydrolase, Negative tests with API assimilation of capric acid, assimilation of adipic acid, hydrolysis of gelatin (= protease), assimilation of trisodium citrate, assimilation of phenylacetic acid
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Motility If motile
C19:0 CYCLO 8c, C16:0, C18:17c 1 Biosafety level Soil (ENVO: 00001998), root nodule (ENVO: 01000164) Habitat Free-living Biotic relationship Symbiosis with the host Nitrogen-fixing nodules Known pathogenicity None
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Major fatty acids
PII:
S0723-2020(19)30339-X
DOI:
https://doi.org/10.1016/j.syapm.2019.126044
Reference:
SYAPM 126044
To appear in:
Systematic and Applied Microbiology
Received Date:
29 July 2019
Revised Date:
21 October 2019
Accepted Date:
4 November 2019
´ Please cite this article as: Estrella MJ, Fontana MF, Cumpa Velasquez LM, Torres Tejerizo GA, Diambra L, Hansen LH, Pistorio M, Sannazzaro AI, Mesorhizobium intechi sp. nov. isolated from nodules of Lotus tenuis in soils of the Flooding Pampa, Argentina, Systematic and Applied Microbiology (2019), doi: https://doi.org/10.1016/j.syapm.2019.126044
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Mesorhizobium intechi sp. nov. isolated from nodules of Lotus tenuis in soils of the Flooding Pampa, Argentina
María Julia Estrella1, María Florencia Fontana1, Liz Marjory Cumpa Velásquez1, Gonzalo Arturo Torres Tejerizo2, Luis Diambra3, Lars Hestbjerg Hansen4, Mariano Pistorio2, Analía Inés Sannazzaro1,#
1
INTECH (Instituto Tecnológico Chascomús), CCT-La Plata, CONICET - Universidad
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Nacional de San Martín - Avenida Intendente Marino, Km 8.2, (7130) Chascomús, Argentina. 2
IBBM (Instituto de Biotecnología y Biología Molecular), CCT-La Plata, CONICET,
Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad
3
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Nacional de La Plata, (1900) La Plata, Argentina.
CREG (Centro Regional de Estudios Genómicos), CONICET - Universidad Nacional
4
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de La Plata, (1900) La Plata, Argentina.
Section of Microbial Ecology and Biotechnology, Department of Plant and
Corresponding author: Analía Inés Sannazzaro
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#
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Environmental Sciences, University of Copenhagen, 1871 Frederiksberg, Denmark
Phone: +54-2241-430323 ext. 108 Fax: +54-2241-424048
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E-mail: [email protected]
Abstract
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. opportunistum WSM
2975T, M. jarvisii ATCC 33669T and M. huakuii LMG 14107T, with sequence identities of 99.78% - 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
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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).
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Keywords: Mesorhizobium sp.nov.; Lotus tenuis; rhizobia; phylogeny
Abbreviations: nt, nucleotides; rep-PCR, repetitive element palindromic PCR; MLSA,
hybridization
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multilocus sequence analysis; ANI, average nucleotide identity; DDH, DNA-DNA
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The draft genome of Mesorhizobium intechi sp. nov. strain BD68T has been deposited at DDBJ/ENA/GenBank under accession number PNOT00000000. The version described in this paper is PNOT02000000. 16S rRNA, recA, rpoB, glnII, dnaK and nodC gene accession numbers for the type strain of Mesorhizobium intechi (BD68T) are EU748908, MH265064, MH265067, MH265070, MH265073 and MH265076, respectively. BOX-
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PCR analysis is available as a supplementary figure.
Introduction
The genus Lotus includes more than 180 legume species found worldwide, although only a few species have been domesticated and improved by selection and plant breeding for use as livestock forage. In particular, species such as L. tenuis, L.
corniculatus, L. uliginosus, L. subbiflorus and L. ornithopoides are currently used to improve pasture and hay quality where other forage legume species are not suitable [24]. Lotus species are mainly sown in South America, North America and Europe, with 1.85, 1.39 and 1.38 million hectares, respectively [5]. Like most legumes, Lotus species establish highly specific symbiotic relationships with N-fixing soil bacteria collectively known as rhizobia. The rhizobial species nodulating plants of the genus Lotus are divided into meso- and slow-growing strains [23]. The slow-growing strains, named Bradyrhizobium sp. (Lotus), are typical symbionts for L. uliginosus and L. subbiflorus, while the meso-growing strains, classified as Mesorhizobium loti [9,10], have long been
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considered to be the typical rhizobial symbionts of L. corniculatus, L. tenuis and the model L. japonicus [7,10,31]. However, recent revisions have found that strains formerly described as Mesorhizobium loti (type strain NZP 2213T) actually correspond
to the phylogenetically divergent species M. jarvisii (ATCC 33669T strain), M. erdmanii
(USDA 3471T strain) and M. japonicum (MAFF 303099T and R7A strains isolated from
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L. japonicum and L. corniculatus, respectively) [18,19]. Notwithstanding the above-
mentioned efforts to revise the taxonomic status of the Lotus-nodulating bacteria, many
strain for this species [6,16,27].
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strains are still reported as M. loti despite being phylogenetically distant from the type
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Lotus tenuis is an outstanding legume species under soil-stress conditions, but there are relatively few reports describing the identity of its associated rhizobial symbionts [6,7]. In a previous study, symbiotic nitrogen-fixing bacteria associated with L. tenuis in the
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Flooding Pampa (Argentina) have been classified mainly as members of the Mesorhizobium genus [6,33]. In the present work, three bacterial strains from the abovementioned study (BD68T, BD66 and BD73) were characterized phylogenetically as members of a distinct group within the genus Mesorhizobium, based on an analysis of their housekeeping genes (16S rRNA, recA, dnaK, glnII and rpoB). A polyphasic
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approach was employed to describe the taxonomic status of one representative strain (BD68T) compared with the type strains of related Mesorhizobium species. As a result, a novel L. tenuis nodulating species, Mesorhizobium intechi sp. nov., is proposed. Isolates BD68T, BD66 and BD73 were cultured in tryptone-yeast extract (TY) medium and stored in 30% (w/v) glycerol at -80 °C. Total DNAs used for amplification of the genes or fragments from the rhizobial strains were extracted using an AccuPrep® Genomic DNA Extraction Kit (Bioneer) according to the manufacturer’s guidelines.
BOX-PCR fingerprint profiling was carried out as previously described [32] and profile comparison revealed that the three isolates showed different patterns (Supplementary Figure S1), demonstrating that they were not clonal varieties. The 16S rRNA gene from the three strains was PCR-amplified using the primer pairs 41f/1488r [6]. Strains BD68T, BD66 and BD73 had identical sequences for a 1,270 bp internal region of the 16S rRNA gene. A 16S rRNA-based phylogenetic tree with the 49 hitherto validly published species of Mesorhizobium was performed: 16S rRNA gene sequences of mesorhizobial type strains obtained through the Ribosomal Database Project (RDP, 8) were aligned with the corresponding sequences of the novel isolates using the Clustal
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module implemented by the MEGA7 software [11]. Distances were calculated in a complete deletion procedure and the maximum-likelihood method was used to reconstruct the corresponding phylogenetic tree by means of the MEGA7 software. The
robustness of the tree topology was evaluated by bootstrap analysis (1,000 replicates).
The three novel isolates clustered together with M. japonicum MAFF 303099T, M.
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erdmanii USDA 3471T, M. carmichaelinearum ICMP 18942T, M. opportunistum WSM 2975T and M. jarvisii ATCC 33669T (Figure 1), with sequence identities of 100%,
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100%, 99.86%, 99.79% and 99.72%, respectively. In turn, the newly isolated strains did not group with the canonical rhizobial species for L. corniculatus, M. loti NZP 2213T
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(98.12% identity). It has been shown that species within the Mesorhizobium genus usually share high 16S rRNA sequence similarity values, making it difficult to delineate species entirely on the basis of this phylogenetic marker [25]. For this reason, the
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analysis of additional taxonomical markers is the recommended procedure for elucidating systematic relationships within mesorhizobial species. The selection and use of housekeeping genes for taxonomic purposes has been a critical step forward; therefore, the four protein-coding genes, recA, glnII, dnaK and rpoB, were further studied since they have been previously shown to produce a robust phylogeny of the
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genus Mesorhizobium [26], and their sequences are available for most type species. Partial recA, glnII, dnaK and rpoB sequences of strains BD68T, BD66 and BD73 were obtained according to previously described methodology [8,17,37,39]. Sequence similarities for the four housekeeping genes between the three new isolates were calculated and values between 99-100% were observed, supporting the fact that the three isolates corresponded to the same species. Additionally, phylogenetic trees for each housekeeping gene were performed in order to establish the relative taxonomic position of the three isolates within the Mesorhizobium genus. Reference sequences for
the Mesorhizobium type strains were recovered from the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov) GenBank database. Sequences for each gene in a given species were aligned and trimmed and 24 nucleotide substitution models were tested using the MEGA7 software. The lowest Bayesian information criterion scores were considered in order to choose the best substitution pattern for phylogenetic inferences. In the four phylogenies, the L. tenuis isolates grouped in a separate cluster, close to M. japonicum MAFF 303099T, M. jarvisii ATCC 33669T, M. carmichaelinearum ICMP 18942T and M. huakuii LMG 14107T (Supplementary Figure S2).
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Taken together, the results of this study suggested that the novel strains represented a species distinct from all the previously recognized species within the Mesorhizobium genus. To identify the three newly isolated strains accurately, BD68T was designated as
a group representative and its genome was fully sequenced in order to perform in silico
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whole genome comparisons. Additionally, morphological/phenotypic features and major fatty acid profiles were determined, according to the suggested recommendations
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regarding the description of new species [12,36].
The whole genomic DNA of strain BD68T used for genome sequencing was extracted using the AccuPrep® Genomic DNA Extraction Kit (Bioneer). 2x250bp paired-end
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reads were obtained from sequencing on an Illumina MiSeq platform using a Nextera XT DNA library preparation kit. The resulting reads were quality trimmed [2] and the
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draft genome was assembled using the MIRA (version 4.9.6, [3]) and AbySS (version 1.9.0, [35]) assemblers. The Contig Integrator for Sequence Assembly (CISA, [14]) was used to integrate the sets of contigs from the two assemblers. In this way, the reads from BD68T were assembled into 393 contigs (N50 28,806 bp) with a 19.66-fold coverage. The genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline
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(PGAP). The sequences have been deposited in the NCBI GenBank under the accession number PNOT00000000. The reference genome sequences used for whole genome comparisons were retrieved from NCBI GenBank. The genome size of strain BD68T was approximately 6.6 Mbp, comprised of 6,590 predicted genes. Taking advantage of whole genome sequencing for the BD68T strain, and in order to identify precisely the closest species to be used in the subsequent analysis, a multilocus sequence analysis (MLSA) with seven housekeeping genes, recA, glnII, dnaK, rpoB, gyrB, truA and thrA, which were previously shown to produce a robust phylogeny for
the genus Mesorhizobium [26], was performed. The seven housekeeping genes were concatenated and the resulting alignment in the length of 2,951 bp was analyzed by the maximum-likelihood method based on a matrix with the distance correction calculated by the general time reversible model with 1,000 resamplings in the bootstrap analysis (Figure 2). According to the MLSA tree, the BD68T most-related type strains were M. japonicum MAFF 303099T and M. jarvisii ATCC 33669T. The average nucleotide identity (ANI) calculation is an alternative approach to the traditional DNA-DNA hybridization method [28]. ANI calculations between BD68T and its phylogenetically closest type strains were performed using the JSpeciesWS web
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server (http://jspecies.ribohost.com/jspeciesws) [29] based on pairwise alignment of the genomes using the BLAST+ (ANIb) or MUMmer (ANIm) tools. ANIb and ANIm
values (Table 1) were consistent and clearly below the 95-96% proposed cutoff for discriminating different species [28]. Additionally, in silico DNA-DNA hybridization
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(isDDH) values were calculated using the genome-to-genome distance calculation
(GGDC 2.1) online service (http://ggdc.dsmz.de/distcalc2.php), as described by MeierKolthoff and co-workers [20]. The results of the GGDC were based on the
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recommended BLAST+ method and formula 2 (sum of all identities found in highscoring segment pairs (HSPs) divided by overall HSP length), which is independent of
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the genome length and is thus robust against the use of incomplete draft genomes [1,20]. The isDDH percentage values resulting from the comparison of BD68T with closely related type strain genomes (Table 1) were well below the threshold of 70 % for
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species delineation, further supporting that the newly isolated strains belonged to a new species in the genus Mesorhizobium. Recently, a novel approach for prokaryote taxonomy using a comprehensive database of the genomes of type strains and truly whole-genome-based methods was proposed as an
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alternative to standard techniques such as DDH and G+C content, and the 16S rRNA gene and MLSA [21], and a web interface for such analysis was developed (Type Strain Genome Server, https://tygs.dsmz.de). The BD68T type strain was tested by this analysis and the resulting whole-genome phylogram also supported the aforementioned findings (Supplementary Figure S3). Phenotypic features of strains BD66, BD68T and BD73 were determined using the API 20NE (bioMérieux) and BIOLOG GENIII (Biolog Inc.) kits following the manufacturers' instructions. Distinctive features of our isolates compared with
representative strains of the closest phylogenetically related Mesorhizobium species are depicted in Table 2. The fatty acid profile of the type strain, BD68T, was determined under the growth conditions, harvesting and extraction procedures described by Sasser [34]. Briefly, BD68T was grown aerobically on tryptone-yeast extract agar plates at 28 °C for 48 h. The fatty acids were extracted and analyzed according to the recommendations of the commercial Microbial Identification System (Microbial ID, MIDI) and whole-cell fatty acid composition was determined by gas chromatography (Agilent Technologies 6890N) using the peak naming table MIDI TSBA 5.0. The fatty acid composition of
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BD68T comprised C19:0 CYCLO 8c (27.23%), C16:0 (21.49%), C18:17c (16.03%), C18:17c 11-methyl (7.87%), C12:0 (6.76%), C17:0 ISO (5.95%), C16:1 ISO H (5.39%),
C16:0 N OH (5.35%) and C18:0 (3.92%). These results were in accordance with the main fatty acid compounds detected in all species of the genus Mesorhizobium [38].
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Although housekeeping genes are useful for the establishment of rhizobial taxonomic
status, they do not offer information regarding bacterial symbiotic behavior in terms of
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the legume hosts. The analysis of the symbiotic gene nodC, located in interchangeable elements (plasmids or symbiotic islands), has long been used for the identification of
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strains at the symbiovar level [15,25,30]. The nodC sequences of the new isolates from L. tenuis nodules were compared with other Mesorhizobium spp. nodC genes in a maximum-likelihood tree using the Tamura 3 (G+I) model (Figure 3). The nodC genes
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of L. tenuis strains BD68T, BD66 and BD73 were phylogenetically related to those of M. loti and other type strains representing the symbiovar loti [13,15,22], although they clustered together on a separate branch with 100% bootstrap support. The similarity in the nodC sequences of strains BD68T, BD66 and BD73 with respect to the closest sv. loti strains (M. olivaresii CPS 13T, M. helmanticense CSLC 115NT, M. japonicum
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MAFF 303099T, M. loti NZP 2213T, M. tarimense CCBAU 83306T and M. erdmanii USDA 3471T) ranged between 96.91%-97.74%. In turn, the less related nodC sequence in this symbiovar, belonging to M. jarvisii ATCC 33669T, shared a similarity of 93.28% with the three strains. Description of Mesorhizobium intechi sp. nov.
Mesorhizobium intechi (in.te'chi N.L. gen. n. intechi, pertaining to INTECH (Technological Institute of Chascomús) where the strains of this work were isolated and characterized. Gram-negative, aerobic, non-spore-forming rods. Colonies appearing on yeast extractmannitol-agar within 2-4 days incubation at 28 ºC are circular, convex and creamcolored. The optimum temperature for aerobic growth is 28 ºC, growth was observed at 10 ºC but not at 37 ºC; all strains are sensitive to 1% NaCl and grow over a pH range of 7-10. Strains are resistant to ampicillin (10 g mL-1), gentamicin (10 g mL-1), neomycin (5 g mL-1) and kanamycin (50 g mL-1); susceptible to chloramphenicol (10
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g mL-1). The complete description of the new species can be found at the Digital
Protologue website (http://imedea.uib-csic.es/dprotologue/) under the taxonumber TA00564 (Table 3). The type strain BD68T, isolated from nodules of Lotus tenuis, has been deposited as CECT 9304T and LMG 30179T. The whole-genome shotgun project
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sequences PNOT02000001-PNOT02000393.
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has been deposited in GenBank under the accession no. PNOT00000000 and consists of
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Conflict of interest There are no conflicts of interest.
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Acknowledgements
The authors wish to thank Dr. Gustavo Somoza for helping with the Latin for the species name. This study was supported by the Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC, Argentina); Consejo Nacional de Investigaciones
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Científicas y Técnicas (CONICET, Argentina) and Agencia Nacional de Promoción Científica y Tecnológica (Argentina) by grants PICT-2013-0963, PICT-2015-3789 and PICT-2012-0518, to M.J.E, A.I.S and M.P., respectively. A.I.S., G.T.T., and M.P. are members of the Research Career of CONICET. M.J.E. is a member of the Research Career of CIC. L.M.C.V. and M.F.F. are doctoral CONICET fellows.
References [1]
Auch, A.F., von Jan, M., Klenk, H.-P., Göker, M. (2010) Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand. Genomic Sci. 2(1), 117–134, Doi: 10.4056/sigs.531120.
[2]
Bolger, A.M., Lohse, M., Usadel, B. (2014) Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30(15), 2114–2120, Doi: 10.1093/bioinformatics/btu170.
[3]
Chevreux, B., Pfisterer, T., Drescher, B., Driesel, A.J., Muller, W.E.G., Wetter,
ro of
T., Suhai, S. (2004) Using the miraEST assembler for reliable and automated
mRNA transcript assembly and SNP detection in sequenced ESTs. Genome Res. 14(6), 1147–1159, Doi: 10.1101/gr.1917404. [4]
Cole, J.R., Wang, Q., Fish, J.A., Chai, B., McGarrell, D.M., Sun, Y., Brown,
-p
C.T., Porras-Alfaro, A., Kuske, C.R., Tiedje, J.M. (2014) Ribosomal Database
Project: Data and tools for high throughput rRNA analysis. Nucleic Acids Res.
[5]
re
42(D1), 633–642, Doi: 10.1093/nar/gkt1244.
Díaz, P., Borsani, O., Monza, J. (2005) Lotus-related species and their agronomic
lP
importance. In: Márquez, A.J., Stougaard, J., Udvardi, M., Parniske, M., Spaink, H., Saalbach, G., Webb, J., Chiurazzi, M., (Eds.), Lotus japonicus Handbook,
[6]
ur na
Springer, pp. 25–37.
Estrella, M.J., Muñoz, S., Soto, M.J., Ruiz, O., Sanjuan, J. (2009) Genetic diversity and host range of rhizobia nodulating Lotus tenuis in typical soils of the Salado River Basin (Argentina). Appl. Environ. Microbiol. 75(4), 1088–1098, Doi: 10.1128/AEM.02405-08. Fulchieri, M.M., Estrella, M.J., Iglesias, A.A. (2001) Characterization of
Jo
[7]
Rhizobium loti strains from the Salado River Basin. Antonie Van Leeuwenhoek 79(2), 119–125.
[8]
Gaunt, M.W., Turner, S.L., Rigottier-Gois, L., Lloyd-Macgilp, S.A., Young, J.P. (2001) Phylogenies of atpD and recA support the small subunit rRNA-based classification of rhizobia. Int. J. Syst. Evol. Microbiol. 51(Pt 6), 2037–2048.
[9]
Jarvis, B.D.W., Van Berkum, P., Chen, W.X., Nour, S.M., Fernandez, M.P.,
Cleyet-Marel, J.C., Gillis, M. (1997) Transfer of Rhizobium loti, Rhizobium huakuii, Rhizobium ciceri, Rhizobium mediterraneum, and Rhizobium tianshanense to Mesorhizobium gen. nov. Int. J. Syst. Bacteriol. 47(3), 895–898, Doi: 10.1099/00207713-47-3-895. [10] Jarvis, B.D.W., Pankhurst, C.E., Patel, J.J. (1982) Rhizobium loti, a new species of legume root nodule bacteria. Int. J. Syst. Bacteriol. 32(3), 378–380, Doi: 10.1099/00207713-32-3-378. [11] Kumar, S., Stecher, G., Tamura, K. (2016) MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33(7), 1870–
ro of
1874, Doi: 10.1093/molbev/msw054.
[12] de Lajudie, P.M., Andrews, M., Ardley, J., Eardly, B., Jumas-Bilak, E.,
Kuzmanović, N., Lassalle, F., Lindström, K., Mhamdi, R., Martínez-Romero, E., Moulin, L., Mousavi, S.A., Nesme, X., Peix, A., Puławska, J., Steenkamp, E.,
-p
Stępkowski, T., Tian, C.-F., Vinuesa, P., Wei, G., Willems, A., Zilli, J., Young, P. (2019) Minimal standards for the description of new genera and species of
10.1099/ijsem.0.003426.
re
rhizobia and agrobacteria. Int. J. Syst. Evol. Microbiol. 69(7), 1852–1863, Doi:
lP
[13] Laranjo, M., Young, J.P.W., Oliveira, S. (2012) Multilocus sequence analysis reveals multiple symbiovars within Mesorhizobium species. Syst. Appl. Microbiol. 35(6), 359–367, Doi: 10.1016/j.syapm.2012.06.002.
ur na
[14] Lin, S.-H., Liao, Y.-C. (2013) CISA: Contig integrator for sequence assembly of bacterial genomes. PLoS One 8(3), e60843. [15] Lorite, M.J., Flores-Felix, J.D., Peix, A., Sanjuan, J., Velazquez, E. (2016) Mesorhizobium olivaresii sp. nov. isolated from Lotus corniculatus nodules.
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Syst. Appl. Microbiol. 39(8), 557–561, Doi: 10.1016/j.syapm.2016.09.003. [16] Marcos-García, M., Menéndez, E., Cruz-González, X., Velázquez, E., Mateos, P.F., Rivas, R. (2015) The high diversity of Lotus corniculatus endosymbionts in soils of northwest Spain. Symbiosis 67(1–3), 11–20, Doi: 10.1007/s13199-0150368-5. [17] Martens, M., Dawyndt, P., Coopman, R., Gillis, M., De Vos, P., Willems, A. (2008) Advantages of multilocus sequence analysis for taxonomic studies: A case
study using 10 housekeeping genes in the genus Ensifer (including former Sinorhizobium). Int. J. Syst. Evol. Microbiol. 58(1), 200–214, Doi: 10.1099/ijs.0.65392-0. [18] Martínez-Hidalgo, P., Ramírez-Bahena, M.H., Flores-Félix, J.D., Igual, J.M., Sanjuán, J., León-Barrios, M., Peix, A., Velázquez, E. (2016) Reclassification of strains MAFF 303099T and R7A into Mesorhizobium japonicum sp. nov. Int. J. Syst. Evol. Microbiol. 66(12), 4936–4941, Doi: 10.1099/ijsem.0.001448. [19] Martínez-Hidalgo, P., Ramírez-Bahena, M.H., Flores-Félix, J.D., Rivas, R., Igual, J.M., Mateos, P.F., Martínez-Molina, E., León-Barrios, M., Peix, À.,
ro of
Velàzquez, E. (2015) Revision of the taxonomic status of type strains of
Mesorhizobium loti and reclassification of strain USDA 3471T as the type strain of Mesorhizobium erdmanii sp. nov. and ATCC 33669T as the type strain of
Mesorhizobium jarvisii sp. nov. Int. J. Syst. Evol. Microbiol. 65(6), 1703–1708,
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Doi: 10.1099/ijs.0.000164.
[20] Meier-Kolthoff, J.P., Auch, A.F., Klenk, H.-P., Göker, M. (2013) Genome
re
sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 14(1), 60, Doi: 10.1186/1471-2105-14-
lP
60.
[21] Meier-Kolthoff, J.P., Göker, M. (2019) TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat. Commun. 10(1), Doi:
ur na
10.1038/s41467-019-10210-3.
[22] Mohamad, R., Willems, A., Le Quéré, A., Maynaud, G., Pervent, M., Bonabaud, M., Dubois, E., Cleyet-Marel, J.C., Brunel, B. (2017) Mesorhizobium delmotii and Mesorhizobium prunaredense are two new species containing rhizobial
Jo
strains within the symbiovar anthyllidis. Syst. Appl. Microbiol. 40(3), 135–143, Doi: 10.1016/j.syapm.2017.01.004.
[23] Pankhurst, C.E. (1977) Symbiotic effectiveness of antibiotic-resistant mutants of fast- and slow-growing strains of Rhizobium nodulating Lotus species. Can. J. Microbiol. 23(8), 1026–1033, Doi: 10.1139/m77-152. [24] Papadopoulos, Y.A., Kelman, W.M. (1999) Traditional breeding of Lotus species. Trefoil: the science and technology of Lotus, Crop Science Society of America and American Society of Agronomy, Madison, WI, pp. 187–198.
[25] Peix, A., Ramírez-Bahena, M.H., Velázquez, E., Bedmar, E.J. (2015) Bacterial associations with legumes. CRC. Crit. Rev. Plant Sci. 34(1–3), 17–42, Doi: 10.1080/07352689.2014.897899. [26] Pérez-Yépez, J., Armas-Capote, N., Velázquez, E., Pérez-Galdona, R., Rivas, R., León-Barrios, M. (2014) Evaluation of seven housekeeping genes for multilocus sequence analysis of the genus Mesorhizobium: Resolving the taxonomic affiliation of the Cicer canariense rhizobia. Syst. Appl. Microbiol. 37(8), 553– 559, Doi: 10.1016/j.syapm.2014.10.003. [27] Reeve, W., Ardley, J., Tian, R., Eshragi, L., Yoon, J.W., Ngamwisetkun, P.,
ro of
Seshadri, R., Ivanova, N.N., Kyrpides, N.C. (2015) A genomic encyclopedia of the root nodule bacteria: assessing genetic diversity through a systematic
biogeographic survey. Stand. Genomic Sci. 10(1), 14, Doi: 10.1186/1944-327710-14.
-p
[28] Richter, M., Rossello-Mora, R. (2009) Shifting the genomic gold standard for the prokaryotic species definition. Proc. Natl. Acad. Sci. 106(45), 19126–19131,
re
Doi: 10.1073/pnas.0906412106.
[29] Richter, M., Rosselló-Móra, R., Oliver Glöckner, F., Peplies, J. (2016)
lP
JSpeciesWS: a web server for prokaryotic species circumscription based on pairwise genome comparison. Bioinformatics 32(6), 929–931. [30] Rogel, M.A., Ormeño-Orrillo, E., Romero, E.M. (2011) Symbiovars in rhizobia
ur na
reflect bacterial adaptation to legumes. Syst. Appl. Microbiol. 34, 96–104. [31] Saeki, K., Kouchi, H. (2000) The Lotus symbiont, Mesorhizobium loti: molecular genetic techniques and application. J. Plant Res. 113(4), 457–465. [32] Sannazzaro, A.I., Bergottini, V.M., Paz, R.C., Castagno, L.N., Menéndez, A.B.,
Jo
Ruiz, O.A., Pieckenstain, F.L., Estrella, M.J. (2011) Comparative symbiotic performance of native rhizobia of the Flooding Pampa and strains currently used for inoculating Lotus tenuis in this region. Antonie Van Leeuwenhoek 99, 371– 379, Doi: 10.1007/s10482-010-9502-9.
[33] Sannazzaro, A.I., Tejerizo, G.T., Fontana, M.F., Cumpa Velásquez, L.M., Hansen, L.H., Pistorio, M., Estrella, M.J. (2018) Mesorhizobium sanjuanii sp. nov., isolated from nodules of Lotus tenuis in the saline-alkaline lowlands of
Flooding Pampa, Argentina. Int. J. Syst. Evol. Microbiol. 68(9), 2936–2942, Doi: 10.1099/ijsem.0.002924. [34] Sasser, M. (1990) Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Tech. Note 101Newark,DEMicrobial ID, Inc. [35] Simpson, J.T., Wong, K., Jackman, S.D., Schein, J.E., Jones, S.J.M., Birol, I. (2009) ABySS: a parallel assembler for short read sequence data. Genome Res. 19(6), 1117–1123, Doi: 10.1101/gr.089532.108. [36] Stackebrandt, E., Frederiksen, W., Garrity, G.M., Grimont, P.A.D., Kampfer, P., Maiden, M.C.J., Nesme, X., Rossello-Mora, R., Swings, J., Truper, H.G.,
ro of
Vauterin, L., Ward, A.C., Whitman, W.B. (2002) Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int. J. Syst. Evol. Microbiol. 52(March 2002), 1043–1047, Doi: 10.1099/ijs.0.02360-0.02360.
[37] Stȩpkowski, T., Czaplińska, M., Miedzinska, K., Moulin, L. (2003) The variable
-p
part of the dnaK gene as an alternative marker for phylogenetic studies of
rhizobia and related alpha Proteobacteria. Syst. Appl. Microbiol. 26(4), 483–
re
494, Doi: 10.1078/072320203770865765.
[38] Tighe, W., de Lajudie, P., Dipietro, K., Lindström, K., Nick, G., Jarvis, B.D.W.
lP
(2000) Analysis of cellular fatty acids and phenotypic reIationships of Agrobacterium and Sinorhizobium species using the Sherlock Microbial
ur na
Identification System. Int. J. Syst. Evol. Microbiol. 50, 787–801. [39] Turner, S.L., Young, J.P.W. (2000) The glutamine synthetases of rhizobia:
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Phylogenetics and evolutionary implications. Mol. Biol. Evol. 17(2), 309–319.
Figure captions: Figure 1. Maximum-likelihood phylogenetic tree based on Mesorhizobium type strain16S rRNA gene sequences (1,270 nucleotides) showing the position of Mesorhizobium intechi sp. nov. used in this study (indicated in bold). The tree was constructed using the Kimura 2-parameter (G+I) model. Bootstrap values (above 50%) calculated for 1,000 replications are indicated at the nodes. GenBank accession numbers are given between
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curved brackets. Bar, 0.02 substitutions per nucleotide position.
Figure 2. Maximum-likelihood phylogenetic tree based on concatenated recA (298 nt), glnII (405 nt), dnaK (223 nt), rpoB (441 nt), gyrB (578 nt), truA (668 nt) and thrA (280 nt) gene sequences showing the position of Mesorhizobium intechi sp. nov. used in this study (indicated in bold) within the genus Mesorhizobium. GenBank accession numbers
are detailed in Supplementary Table S1. The tree was constructed using the general time reversible (G+I) model. Bootstrap values (above 50%) calculated for 1,000 replications
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are indicated at the nodes. Bar, 0.05 substitutions per nucleotide position.
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Figure 3. Maximum-likelihood tree based on partial sequences of the nodC gene (383 nucleotides) showing the relationships between Mesorhizobium intechi sp. nov. (in bold) and other Mesorhizobium strains, indicating the different symbiovars (sv.) defined within the genus. The tree was constructed using the Tamura 3 (G+I) model. Bootstrap values (above 50%) calculated for 1,000 replications are indicated at the nodes. Bar,
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0.05 substitutions per nucleotide position. Original host legumes are given for unknown symbiovars.
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Table 1. Whole genome sequence comparisons between M. intechi BD68T isolated from L. tenuis nodules and phylogenetically closely related Mesorhizobium species.
ANIba
ANImb
isDDHc
M. jarvisii ATCC 33669T
92.71
93.81
53.20
M. japonicum MAFF 303099T
92.19
93.29
50.80
M. carmichaelinearum ICMP 18942T
89.57
91.02
41.50
M. erdmanii USDA 3471T
88.73
90.09
38.30
M. opportunistum WSM 2075T
86.97
88.78
34.80
a
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Strain
Average nucleotide identity percentage based on BLAST searches of 1 kb genome
fragments against a target genome. bAverage nucleotide identity percentage based on the MUMmer algorithm that does not require the artificial generation of 1 kb fragments.
In silico DNA-DNA hybridization values calculated using the recommended formula 2:
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c
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identities/HSP length.
Table 2. Distinctive features of M. intechi BD68T and its closest type species. 3
4
5
+ + + + + + + +
+ + + + + + +
+ + + + + + +
+ + + -
+ + + ± -
+
±
+
+ +
+ -
+ -
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Growth conditions 1% NaCl 10 ºC 37 ºC pH 10
2
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Assimilation of: L-Gluconate L-Malate L-Alanine D-Lactose D-Maltose D-Melibiose D-Trehalose D-Cellobiose para-nitrophenyl-Dphenylgalactopyranose
1
-
-
+ +
+ + + -
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Species
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Strains: 1, BD66; 2, BD68T; 3, BD73; 4, M. japonicum MAFF 30399T; 5, M. jarvisii ATCC 33669T. +, positive; -, negative; ±, weakly positive. Data are from this study.
Table 3. Description of Mesorhizobium intechi sp. nov. according to Digital Protologue TA00564 assigned by the www.imedea.uib.es/dprotologue website. TA00564 Mesorhizobium intechi Mesorhizobium intechi sp. nov. in.te'chi N.L. gen. n. intechi, pertaining to INTECH (Technological Institute of Species etymology Chascomús) where the strains of this work were isolated and characterized Estrella MJ, Fontana MF, Cumpa Velásquez LM, Torres Tejerizo G, Diambra Authors L, Hansen LH, Pistorio M, Sannazzaro AI Mesorhizobium intechi sp. nov. isolated from nodules of Lotus tenuis in soils of Title the Flooding Pampa, Argentina Corresponding author Analía Inés Sannazzaro E-mail of the [email protected] corresponding author Analía Inés Sannazzaro Submitter E-mail of the submitter [email protected] Designation of the type BD68 strain Strain collection CECT 9304 = LMG 30179 numbers 16S rRNA gene EU748908 accession number Alternative recA [MH265064], rpoB [MH265067], glnII [MH265070], dnaK [MH265073] housekeeping genes Genome accession PNOT00000000 number [RefSeq] Draft Genome status 6,580 kbp Genome size 63.2 GC mol% Argentina Country of origin Flooding Pampa Region of origin Root nodules Source of isolation Chascomús, Buenos Aires, Argentina Geographic location 35º 58' S Latitude 58º 00' W Longitude Number of strains in 3 study Source of isolation of Root nodules non-type strains Growth medium, incubation conditions Tryptone-yeast (TY) medium, 28 ºC, pH 7 used for standard cultivation Alternative medium 1 Yeast extract-mannitol (YEM), 28 ºC, pH 7 Conditions of Liquid TY medium with 30% glycerol stored at -80 °C preservation Negative Gram stain Rod Cell shape Cell size (length or 2-4 diameter)
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Taxonumber Species name Genus name Specific epithet Species status
Motile Flagellar On TY agar after 48 h at 28 °C colonies are circular with a creamy color, entire Colony morphology margin, convex elevation, shiny, mucoid Temperature optimum 28 7 pH optimum Neutrophile pH category Non-halophile (NaCl inhibitory at 1% NaCl) Salinity category Aerobe Relationship to O2 O2 conditions for strain Aerobiosis testing Chemoorganotroph Energy metabolism Positive Oxidase Negative Catalase Oxidation of: D-salicin, inosine, alpha-D-glucose, glycerol, D-sorbitol, Dglucuronic acid, pectin, bromo-succinic acid, Tween 40, gentiobiose, dextrin, Nacetyl-D-glucosamine, D-fucose, D-mannose, D-mannitol, alpha-D-lactose, Lglutamic acid, glycyl-L-proline, alpha-ketoglutaric acid, methylpyruvate, Positive tests with gamma-aminobutyric acid, sucrose, D-maltose, N-acetyl-beta-D-mannosamine, BIOLOG L-fucose, D-fructose, D-fructose-6-PO4, D-arabitol, D-malic acid, D-lactic acid methyl ester, D-turanose, D-trehalose, beta-methyl-D-glucoside, L-rhamnose, D-galactose, myo-inositol, L-pyroglutamic acid, L-arginine, L-malic acid, Llactic acid, D-cellobiose, D-melibiose Oxidation of: D-serine, L-aspartic acid, L-serine, gelatin, phydroxyphenylacetic acid, alpha-ketobutyric acid, formic acid, L-histidine, Lalanine, alpha-hydroxybutyric acid, acetic acid. Negative tests with Growth in presence of: 1% NaCl, 1% sodium lactate, troleandomycin, BIOLOG lincomycin, vancomycin, nalidixic acid, aztreonam, fusidic acid, rifamycin SV, guanidine HCl, tetrazolium violet, lithium chloride, D-serine, minocycline, Niaproof 4, tetrazolium blue, potassium tellurite, sodium bromate Oxidation of: N-acetyl neuraminic acid, D-raffinose, 3-methylglucose, Dsaccharic acid, citric acid, D-glucose-6-PO4, glucuronamide, D-galacturonic Variable tests with acid, acetoacetic acid, L-gluconic acid, music acid, L-galactonic acid lactone, BIOLOG propionic acid, N-acetyl-D-galactosamine, D-aspartic acid, quince acid, betahydroxy-D, L-butyric acid, stachyose. Growth in presence of sodium butyrate ß-galactosidase (para-nitrophenyl-ßD-galactopyranoside), assimilation of glucose, assimilation of arabinose, assimilation of mannose, assimilation of Positive tests with API mannitol, assimilation of N-acetyl-glucosamine, assimilation of maltose, hydrolysis of aesculin, assimilation of malate, urease Reduction of nitrates to nitrites, reduction of nitrates to nitrogen, indole production (tryptophan), fermentation of glucose, arginine dihydrolase, Negative tests with API assimilation of capric acid, assimilation of adipic acid, hydrolysis of gelatin (= protease), assimilation of trisodium citrate, assimilation of phenylacetic acid
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Motility If motile
C19:0 CYCLO 8c, C16:0, C18:17c 1 Biosafety level Soil (ENVO: 00001998), root nodule (ENVO: 01000164) Habitat Free-living Biotic relationship Symbiosis with the host Nitrogen-fixing nodules Known pathogenicity None
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Major fatty acids