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High taxonomic diversity of Micromonospora strains isolated from Medicago sativa nodules in Western Spain and Australia
Journal Pre-proof High taxonomic diversity of Micromonospora strains isolated from Medicago sativa nodules in Western Spain and Australia ´ Pilar Mart´ınez-Hidalgo, Jose´ David Flores-Felix, Encarna ´ Velazquez, Lambert Brau, Martha E. Trujillo, Eustoquio Mart´ınez-Molina
PII:
S0723-2020(19)30338-8
DOI:
https://doi.org/10.1016/j.syapm.2019.126043
Reference:
SYAPM 126043
To appear in:
Systematic and Applied Microbiology
Received Date:
16 May 2019
Revised Date:
8 November 2019
Accepted Date:
15 November 2019
´ JD, Velazquez ´ Please cite this article as: Mart´ınez-Hidalgo P, Flores-Felix E, Brau L, Trujillo ME, Mart´ınez-Molina E, High taxonomic diversity of Micromonospora strains isolated from Medicago sativa nodules in Western Spain and Australia, Systematic and Applied Microbiology (2019), doi: https://doi.org/10.1016/j.syapm.2019.126043
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
High taxonomic diversity of Micromonospora strains isolated from Medicago sativa nodules in Western Spain and Australia
Short Title: Micromonospora species inhabiting Medicago sativa nodules
Pilar Martínez-Hidalgo1, José David Flores-Félix2, Encarna Velázquez2,3, Lambert Brau4, Martha E. Trujillo2, Eustoquio Martínez-Molina2,3 1
Departamento de Biología, Geología, Física y Química inorgánica. Universidad Rey
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Juan Carlos. Departamental II despacho 248. Av. Tulipán s/n, 28933 Móstoles, Madrid Departamento de Microbiología y Genética and Instituto Hispanoluso de Investigaciones
Agrarias (CIALE). Universidad de Salamanca. Edificio Departamental de Biología. Lab 209. Av. Doctores de la Reina S/N. 37007 Salamanca.
Unidad Asociada Grupo de Interacción Planta-Microorganismo Universidad de
Salamanca-IRNASA-CSIC, Salamanca, Spain.
Deakin University, Geelong, Australia, Centre for Regional and Rural Futures, School
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of Life and Environmental Sciences
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3
Abstract
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*Corresponding author: Pilar Martínez-Hidalgo
The genus Micromonospora has been found in nodules of several legumes and some new
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species of this genus were isolated from these plant organs. In this study we analysed the taxonomic diversity of Micromonospora strains isolated from alfalfa nodules in Spain
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and Australia on the basis of three phylogenetic markers, the rrs and gyrB genes and 16S23S intergenic spacer (ITS). The genome analysis of selected strains representative of different clusters or lineages found after rrs, gyrB and ITS analyses confirmed the results obtained with these phylogenetic markers. They showed that the analysed strains belong to at least 18 Micromonospora species including previously described ones, such as Micromonospora noduli, Micromonospora ureilytica, Micromonospora taraxaci, Micromonospora zamorensis, Micromonospora aurantiaca and Micromonospora tulbaghiae. Most of these strains belong to undescribed species of Micromonospora
showing the high taxonomic diversity of strains from this genus inhabiting alfalfa nodules. Although Micromonospora strains are not able to induce the formation of these nodules, and it seems that they do not contribute to fix atmospheric nitrogen, they could play a role related with the mechanisms of plant growth promotion and pathogen protection presented by Micromonospora strains isolated from legume nodules.
Keywords: Micromonospora, Medicago sativa, diversity, taxonomy, Spain, Australia
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Introduction
Legume nodules are induced by soil bacteria known as rhizobia [43] but also contain phylogenetically diverse endophytic bacteria including species from the genus
Micromonospora [29, 44, 45]. Phylogenetically diverse strains from this genus have been
found in nodules of Lupinus angustifolius [40], Pisum sativum [8,11] and Medicago
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sativa [27]. Several of these strains belong to new species, such as Micromonospora
lupini isolated from nodules of L. angustifolius [41], and Micromonospora pisi,
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Micromonospora ureilytica, Micromonospora noduli, Micromonospora vinacea, Micromonospora luteifusca, Micromonospora phytophila and Micromonospora
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luteiviridis isolated from nodules of P. sativum [9, 10, 12, 15]. Strains isolated from M. sativa nodules also belong to several clusters of genus Micromonospora but to date only the 16S rRNA gene (rrs gene) has been analysed [27].
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Although the rrs gene is the universal marker used for bacterial classification, it has limitations to differentiate among closely related species [16] and additional phylogenetic
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markers are used to analyse intrageneric diversity [17, 19, 33, 37]. This occurs within genus Micromonospora [20] and the housekeeping gene gyrB has been commonly used for species differentiation within this genus [8, 20, 46]. Different studies proposed cut-
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off similarity values around 98.5% in the gyrB gene for differentiation of Micromonospora species [11, 20]. For species whit rrs and gyrB genes closely related, the analysis of the complete genomes allows the assignment of Micromonospora strains to a species [4, 6, 36], taking into account that cut-off values of ANI (Average Nucleotide Identity) and dDDH (Digital DNA-DNA hybridization) have been recently proposed for bacterial species differentiation [13].
Other genomic regions are also used for this purpose, such as the 16S-23S intergenic spacer (ITS), which is useful to analyse bacterial diversity because it allows to differentiate among different species and even among strains from the same species [31]. Although the ITS region has not been used in the case of genus Micromonospora, it has been used to differentiate between species of genus Salinispora, a closely related genus of the family Micromonosporaceae [23], having established a cut-off similarity value of 95% for Salinispora species differentiation [23]. Therefore, the objective of this work was to analyze the taxonomic diversity of Micromonospora strains previously isolated from M. sativa nodules in different
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geographical locations in Spain and Australia. To achieve this objective three phylogenetic single markers, rrs and gyrB genes and ITS region were analysed for all strains. In addition, we analysed the ANI (Average Nucleotide Identity) and dDDH
(Digital DNA-DNA hybridization) values obtained for selected strains belonging to
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different gyrB gene clusters.
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Material and methods
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Micromonospora strains
The Micromonospora strains analysed in this study were isolated from surface sterilized root nodules of alfalfa in previous studies [25,27]. The nodules were collected from alfalfa
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plants growing in five locations from Spain and in two locations from Western Australia (Table S1). The locations in Spain were Aldearrubia (40º 59' 57,08''N - 5º 29' 18,78''W), Babilafuente (40º 58' 22,22''N - 5º 25' 20,50''W), Palaciosrubios (41º 2' 36,34N- 5º 11'
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49,55''W), Tormes riverbank (40º 57' 42,88''N - 5º 40' 31,66''W) and Salamanca (40º 56' 29,13N - 5º 39' 21,21W) and in Australia were Binningup, Shire of Harvey (33°07'56.2"S
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115°43'22.0"E) and Murdoch University Campus, Perth (32°03'57.6"S 115°50'06.7"E).
Amplification and sequencing of the rrs and gyrB genes and 16S-23S ITS region The rrs gene and the 16S-23S ITS region were amplified and sequenced as previously reported by Trujillo et al. [40] and by Peix et al. [32], respectively. The amplification and sequencing of gyrB gene were performed using two primers designed in this study on the basis of conserved regions in this gene from Micromonospora species, GyrBF: 5’-
TCGACGGCAAGGCGTACG-3’ and GyrBR: 5’-CGCAGCTTCTCSATGTCG-3’. Amplification and sequencing conditions were as follows: 9 min at 94 ºC, 35 cycles of 1 min at 94 ºC, 1 min at 62 ºC and 2 min at 72 ºC, followed by 7 min final extension at 72 ºC. PCR products were electrophoresed in 1% agarose gels containing ethidium bromide (0.5 µg/ml) using modified Tris-Acetate EDTA buffer (Millipore, Cork, Ireland). The bands were excised and purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer instructions. The sequencing reactions were
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performed on an ABI377 sequencer (Applied Biosystems, Foster City, CA, USA) using a BigDye Terminator v3.0 Cycle Sequencing Kit as supplied by the manufacturer. The rrs gene sequences were compared with those from EzTaxon-e [48] and the sequences of
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the gyrB and ITS regions with those from GenBank using BLASTn [1]. Sequences were
aligned using the Clustal_X software [39]. The distances were calculated according to the
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Kimura two-parameter model [21]. The phylogenetic trees were inferred using the
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neighbour-joining [38] method. Bootstrap analysis was based on 1,000 resamplings. MEGA7 [22] was used for all analysis.
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Genome similarity estimation
The genomic DNA was extracted and purified using the DNeasy UltraClean Microbial DNA Isolation Kit (Qiagen) following manufacturer´s protocol. Sequencing, upon
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preparation of pair-end libraries, was performed on Illumina MiSeq sequencing platform (2x250bp). Sequencing data was assembled using Velvet 1.2.10 [49]. The genome
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characteristics are in Table S2. Average nucleotide identity blast (ANIb) was calculated using the JSpecies service [34, 35] (http://imedea.uib-csic.es/jspecies/). Digital DNA– DNA hybridization (dDDH) values were calculated using the genome-to-genome distance
calculator
website
service
from
DSMZ
(GGDC
2.1)
[30]
(http://ggdc.dsmz.de/ggdc.php/). These values were calculated using the formula two at the GGDC website [2] because it is the only function appropriate to analyse draft genomes [14].
Results and discussion
Analysis of the rrs gene The rrs gene is the main phylogenetic marker used in classification and identification of Prokaryotes, including Actinobacteria [18] and therefore it has been used in the analysis of the Micromonospora strains isolated in Spain and Australia [24, 25, 27]. The results obtained showed that two strains isolated in Australia, AMSO11 and AMSO12.1.2, have rrs gene sequences identical to those of the type strains of Micromonospora zamorensis and Micromonospora tulbaghiae, respectively, and the remaining strains presented
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similarity values (> 99%) with the type strains of different Micromonospora species. The strains AL6, AL20, ALFb4, AMSO3.1 and AMSO13, had the same similarity values with
respect to the type strains of two different Micromonospora species (Table 1). The phylogenetic analysis of the rrs genes showed that the strains from this study were
distributed into five clusters with similarity values among them lower than 99% in the rrs
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genes (Fig. 1). These clusters also included already described species of
Micromonospora, with some of them having rrs genes very closely related (similarity >
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99%), as occurred with the species included in the cluster I (Fig.1). This cluster contains, in addition to ten strains isolated from alfalfa nodules in Spain and Australia, the type
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strains of five Micromonospora species previously isolated in Spain, M. zamorensis isolated from rhizosphere of pea [8], Micromonospora saelicesensis isolated from lupine nodules [41] and M. ureilytica, M. noduli and M. vinacea isolated from pea nodules [9].
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Other species isolated from pea nodules in Spain, M. phytophila [12] is included in cluster II along with two strains isolated in Spain and Australia. The remaining clusters contain strains isolated from alfalfa nodules in Spain and/or Australia together with the type
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strains of Micromonospora species which were originally isolated from non legume sources (Fig.1). The results of the rrs gene analysis suggest that several strains analysed
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in this study could represent new species of genus Micromonospora based on comparison to recently described species of this genus [5, 9, 10], however, they also highlight the need to use other more variable phylogenetic markers in order to differentiate among these strains due the very high similarity values among rrs genes. Analysis of the gyrB gene Within the phylogenetic markers used to differentiate among species with close rrs genes, the gyrB gene is the most useful in the case of genus Micromonospora because it is currently available for most of species from this genus. Kasai et al. [15] showed that gyrB
gene similarity values of 98.6% correspond to 70% of DNA homology, which is the cutoff value currently accepted for species differentiation [47] and Carro et al. [11] concluded that different genospecies of Micromonospora could be defined at 98.5% similarity. Thus, the gyrB gene sequence similarity among different species of Micromonospora should be lower than 98.6%. Based on this, the strains ALFpr19a, ALF9, AL20, AMSO11 and AMSO12.1.2 can be identified as M. noduli, M. ureilytica, Micromonospora taraxaci, M. zamorensis and M. tulbaghiae, respectively, with similarity values from 98.9% to 99.9% in the gyrB gene (Table 1, Fig. 2). The strains AL4 and ALFb5 were closely related to the type strains of Micromonospora viridifaciens and
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M. tulbaghiae, respectively, with similarity values of 98.6% and 98.8%, respectivley, which are in the limit for species delineation. Some strains isolated in this study can be
assigned to the same species, for example, the strains ALFr4, AL6 and AMSO5.2 with similarity values in the gyrB gene ranging from 98.7% to 98.9% and the strains ALF4 and AMSO1.2, with gyrB gene similarity values of 98.7%, although these values are in the
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limit for species differentiation. From them, only the first group of strains could belong to the described species M. saelicensensis, with similarity values which also are in the
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limit for species differentiation since they ranged from 98.3 and 98.8%. The remaining strains present values lower than 98% similarity and therefore belong to species different
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to those currently described in the genus Micromonospora, including the strains ALF4 and AMSO1.2 (Table 1). The results of the gyrB and rrs genes were commonly congruent with the strains being related to the same species in both cases, nevertheless for some
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strains belonging to undescribed species, such as ALF4, ALFb4, Atrumurd12, AMSO1.2 and AMSO3.1, the closest related species in gyrB and rrs genes were not coincident (Table 1). The phylogenetic analysis of the gyrB gene showed that the strains from rrs
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gene cluster I formed a gyrB cluster A with internal similarity values higher than 93% for the latter gene. In the remaining gyrB gene clusters (B and C), applying a similarity value
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of 95% as cut-off, the strains showed a distribution different to that obtained after the rrs gene analysis. This is expected due to the higher variability of the gyrB gene and that most of strains from this study belong to several undescribed species. It is remarkable that some strains isolated in Australia and Spain are closely related as occurred in the case of ALFr4, AL6 and AMSO5.2 (cluster A) and with the strains ALF4 and AMSO1.2 (cluster B). Overall, the results of the gyrB gene analysis showed high taxonomic diversity of Micromonospora strains isolated from nodules of alfalfa from different geographical
locations, belonging to at least 18 different species within genus Micromonospora, with many of them undescribed to date.
Analysis of the 16S-23S intergenic region (ITS) The ITS region is a useful genetic marker to analyse bacterial biodiversity at species and strain levels [31], but it has not been widely used as taxonomic marker because its sequences are not available for all bacterial species, as occurs in the case of the genus Micromonospora. Nevertheless, in a related genus, Salinispora, its first described species were differentiated based on their ITS sequences [23]. These species, Salinispora tropica
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and Salinispora arenicola, showed ITS sequence similarity values lower than 95%, ranging from 98% to 100% at strain level [23].
The ITS region in the strains analysed in this study contained between 295 and 319 bp
and, as expected, as the ITS is a non-coding region, the variabilitiy of this region is higher
than that found for the gyrB gene (Table S3). The species of genus Salinispora present
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similarity values that range from 93% and 94% in their ITS sequences (excluding gaps);
these values are slightly higher to those found between closely related species of genus
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Micromonospora, such as M. zamorensis and M. noduli with 91.2%, or M. aurantiaca and M. tulbaghiae with 89.2% similarity (Fig. 3). Therefore, despite its low size, the ITS
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region is a very useful phylogenetic marker that discriminates Micromonospora species with closely related rrs and gyrB genes.
The ITS similarity values among the strains from this study were equal or lower than 95%
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and therefore all of them could be considered different species, including those where the gyrB gene sequence similarities were in the limit for species delimitation (Tables 1 and S3). The identification of strain AMSO12.1.2 as M. tulbaghiae (similarity of 98.4%) was
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confirmed, however the strains ALFpr19a and AMSO11 differed from the type strains of M. noduli and M. zamorensis, respectively. In the case of strains ALF9 and AL20 no
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conclusions can be established since the ITS for the type strains of M. ureilytica and M. taraxaci are not available (Fig. 3). The type strains of other Micromonospora species closely related with our strains in rrs and/or gyrB gene analyses and whose ITS sequences are available were phylogenetically divergent (Fig. 3). Therefore, the phylogenetic analysis of the ITS region confirmed that all strains from this study formed divergent lineages among them (Fig. 3) with similar distances to those found between S. tropica and Salinispora pacifica. This suggests that they belong to different species of genus Micromonospora inhabiting alfalfa nodules as was also found in other legumes [11, 40].
Average nucleotide identity blast (ANIb) and digital DNA–DNA hybridization (dDDH) estimation The genome comparison allows to delineate bacterial species and recently the minimal standards for this purpose have been published [13]. They include the estimation of ANI and dDDH having establishing cut-off values of 9596% and 70%, respectively, for bacterial species differentiation [13]. In this study we obtained the genome sequences of strains belonging to different gyrB clusters, which were selected taking into account two main criteria: i) They showed different gyrB gene similarities with respect their closest
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related species ii) The genomes of the type strains of these species are available in Genbank.
From gyrB cluster A we selected the strains ALFpr19a and ALFpr18c that showed, respectively, high (99.7%) and low (95.2%) similarity values in this gene with respect to
their closest relatives M. noduli GUI43T and M. chokoriensis DSM 45160T. In agreement
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with these results, the genome analysis confirmed that the strain ALFpr19a belongs to the species M. noduli with ANIb and dDDH values of 99.0% and 92.4%, respectively, and
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that the strain ALFpr18c could represent a new genospecies since it has ANIb and dDDH values of 87.3% and 35.6%, respectively, with respect to its closest relative M.
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chokoriensis DSM 45160T (Table S4).
The strains AMSO1.2 and AMSO3.1 selected from cluster B also showed different similarity values in the gyrB gene (97.9% and 95.4%, respectively) with respect to their
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closest relatives M. echinofusca DSM 43913T and M. coxensis DSM 45161T. ANIb and dDDH values of 92.5% and 50.8%, respectively, showed that the strain AMSO1.2 does not belong to M. echinofusca. Therefore this strain could represent new genospecies
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within Micromonospora despite of the gyrB gene similarity value is near to the cut-off values proposed for species differentiation within this genus [11, 20]. In agreement with
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the results of gyrB gene analysis the strain AMSO3.1 could also represent a new genospecies with ANIb and dDDH values of 83.6% and 38.7%, respectively, with respect to its closest relative M. coxensis DSM 45161T. The strains AMSO12.1.2 and ALFb5 from cluster C were selected because they presented high gyrB gene similarity values (near to or higher than 98%) with respect to the type strains of M. tulbaghiae and M. aurantiaca, two closely related species showing ANIb and dDDH values near to the limit for species differentiation (Table S4). In the case of the strain AMSO12.1.2, ANIb and dDDH values of 99.0% and 93.1%, respectively,
confirmed its identification as M. tulbaghiae. However, although in the gyrB gene analysis M. tulbaghiae also was the closest relative to the strain ALFb5 (98.8% similarity), the obtained ANIb and dDDH values (94.4% and 68.0%, respectively) showed that this strain do not belong to M. tulbaghiae. According to the results of ANIb and dDDH (98.0% and 88.9%, respectively) the strain ALFb5 should be identified as M. aurantiaca (Table S4). These results agree with those found after the rrs gene and ITS analyses, which showed that the strain AMSO12.1.2 is closely related to M. tulbaghiae and Alfb5 to M. aurantiaca (Table 1, Figs. 1 and 3). Therefore, in the case of phylogenetically close related species, as occurs with M.
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aurantiaca and M. tulbaghiae, we can found strains closely related to one or another species depending on the analysed marker, being the genome analysis the most reliable criterion for closely related species differentiation as well as for the assignment of new isolates to these species [36]. Nevertheless, the gyrB gene continues to be a good
phylogenetic marker for differentiation of most Micromonospora species and the ITS
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region will be an excellent tool for intra and interspecific differentiation when the
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sequences of this region are available for all species.
In conclusion, the results from this study showed that Micromonospora is a common
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inhabitant of alfalfa nodules in countries as geographically far apart as Spain and Australia. Our results also highlight the huge number of species that can inhabit these nodules in agreement with the results of other studies where Micromonospora were
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isolated from other legume nodules, such as lupine and peas [11, 40] and from actinorhizal nodules [7]. Although Micromonospora strains are not able to induce the formation of legume nodules and they do not appear to contribute to atmosferic nitrogen
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fixation [26], nodule isolated Micromonospora strains acts as a Rhizobia Helper Bacteria (RHB) agent and has probiotic effects, promoting plant growth and increasing nutrition
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efficiency [27, 42] and as biocontrol agents combining both direct antifungal activity against plant pathogens and the ability to prime plant immunity [28]. These roles could be related with the ability to infect the internal tissues of legume nodules when they are coinoculated with rhizobia even in the case of legumes from which they were not originally isolated [3, 24]. Taking this into account and considering the high taxonomic diversity of Micomonospora inhabiting legume nodules and the low number of legumes and geographical locations studied to date, it is necessary to carry out further studies in
order to improve the knowledge of the biodiversity of the association Micromonospora-
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legumes.
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[49] Zerbino, D.R., Birney, E. (2008) Velvet: Algorithms for de novo short read assembly
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Figure legends.
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Figure 1. Neighbour-joining phylogenetic rooted tree based on rrs gene sequences (1440 nt) of the representative strains from this study and those of closely related species of genus Micromonospora. Bootstrap values calculated for 1000 replications are indicated.
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Bar, 5 nt substitution per 1000 nt. Accession numbers from GenBank are given in
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brackets. Only bootstrap values >50% are shown.
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Figure 2. Neighbour-joining phylogenetic tree based on gyrB gene sequences (1030 nt) showing the position of representative strains from this study and those of closely related
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species of genus Micromonospora. Bootstrap values calculated for 1000 replications are indicated. Bar, 1 nt substitution per 100 nt. Accession numbers from GenBank are given
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in brackets. Only bootstrap values >50% are shown.
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Figure 3. Neighbour-joining phylogenetic tree based on the 16S-23S intergenic spacer
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(ITS) region sequences (295-319 nt) of the representative strains from this study comparing to Salinispora species. Bootstrap values calculated for 1000 replications are indicated. Bar, 5 nt substitution per 100 nt. Accession numbers from GenBank are given
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in brackets. Only bootstrap values >50% are shown.
Table 1. Strains isolated in this study and their closest related species.
ALF1 ALF2 ALF4 ALF9 ALFb4
ALFb5 ALFr4 ALFpr18c ALFpr19a Australia Atrumurd12 AMSO1.2 AMSO3.1
Closest related species (gyrB gene)
Similarity (%)
Cluster
M. yasonensis M. viridifaciens M. saelicesensis M. vinacea M. chaiyaphumensis M. taraxaci M. chokoriensis M. schwarzwaldensis M. yasonensis M. phytophila M. echinofusca M. ureilytica M. tulbaghiae M. citrea M. chalcea M. aurantiaca M. tulbaghiae M. vinacea M.saelicesensis M. chokoriensis M. noduli
99.6 99.8 99.7 99.7 99.6 99.9 99.9 99.6 99.1 99.7 98.9 99.6 99.2 99.2 98.5 99.9 98.7 99.7 99.5 99.4 99.9
V V I
M. yasonensis M. viridifaciens M. saelicesensis M. vinacea M. chaiyaphumensis M. taraxaci M. chokoriensis M. schwarzwaldensis M. yasonensis M. echinofusca M. phytophila M. ureilytica M. chalcea M. tulbaghiae M. citrea M. tulbaghiae M. aurantiaca M. saelicesensis M. vinacea M. chokoriensis M. noduli
94.8 98.6 98.3 97.4 97.3 99.2 97.8 97.6 96.1 97.9 92.8 99.9 96.2 94.8 92.7 98.8 97.9 98.4 97.2 95.2 99.7
C C A
M. chokoriensis M. coriariae M. phytophila M. echinofusca M. chaiyaphumensis M. citrea M. coxensis M. saelicesensis M. zamorensis M. tulbaghiae M. vinacea M. saelicesensis
99.8 97.7 99.7 98.9 99.7 99.7 98.8 99.9 100 100 99.7 99.7
I
M. coriariae M. chokoriensis M. echinofusca M. phytophila M. coxensis M. chaiyaphumensis M. citrea M. saelicesensis M. zamorensis M. tulbaghiae M. vinacea M. saelicesensis
95.9 92.1 97.9 92.9 95.4 94.3 92.8 98.8 98.9 99.9 98.0 97.9
A
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AMSO5.2 AMSO11 AMSO12.1.2 AMSO13
Cluster
V I IV V II I V
III I I I
II
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AL8 AL20
Similarity (%)
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Spain AL3 AL4 AL6
Closest related species (rrs gene)
V
I I V I
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Strains
C A C C B A C
C A A A
B B
A C A
PII:
S0723-2020(19)30338-8
DOI:
https://doi.org/10.1016/j.syapm.2019.126043
Reference:
SYAPM 126043
To appear in:
Systematic and Applied Microbiology
Received Date:
16 May 2019
Revised Date:
8 November 2019
Accepted Date:
15 November 2019
´ JD, Velazquez ´ Please cite this article as: Mart´ınez-Hidalgo P, Flores-Felix E, Brau L, Trujillo ME, Mart´ınez-Molina E, High taxonomic diversity of Micromonospora strains isolated from Medicago sativa nodules in Western Spain and Australia, Systematic and Applied Microbiology (2019), doi: https://doi.org/10.1016/j.syapm.2019.126043
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
High taxonomic diversity of Micromonospora strains isolated from Medicago sativa nodules in Western Spain and Australia
Short Title: Micromonospora species inhabiting Medicago sativa nodules
Pilar Martínez-Hidalgo1, José David Flores-Félix2, Encarna Velázquez2,3, Lambert Brau4, Martha E. Trujillo2, Eustoquio Martínez-Molina2,3 1
Departamento de Biología, Geología, Física y Química inorgánica. Universidad Rey
2
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Juan Carlos. Departamental II despacho 248. Av. Tulipán s/n, 28933 Móstoles, Madrid Departamento de Microbiología y Genética and Instituto Hispanoluso de Investigaciones
Agrarias (CIALE). Universidad de Salamanca. Edificio Departamental de Biología. Lab 209. Av. Doctores de la Reina S/N. 37007 Salamanca.
Unidad Asociada Grupo de Interacción Planta-Microorganismo Universidad de
Salamanca-IRNASA-CSIC, Salamanca, Spain.
Deakin University, Geelong, Australia, Centre for Regional and Rural Futures, School
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of Life and Environmental Sciences
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3
Abstract
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*Corresponding author: Pilar Martínez-Hidalgo
The genus Micromonospora has been found in nodules of several legumes and some new
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species of this genus were isolated from these plant organs. In this study we analysed the taxonomic diversity of Micromonospora strains isolated from alfalfa nodules in Spain
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and Australia on the basis of three phylogenetic markers, the rrs and gyrB genes and 16S23S intergenic spacer (ITS). The genome analysis of selected strains representative of different clusters or lineages found after rrs, gyrB and ITS analyses confirmed the results obtained with these phylogenetic markers. They showed that the analysed strains belong to at least 18 Micromonospora species including previously described ones, such as Micromonospora noduli, Micromonospora ureilytica, Micromonospora taraxaci, Micromonospora zamorensis, Micromonospora aurantiaca and Micromonospora tulbaghiae. Most of these strains belong to undescribed species of Micromonospora
showing the high taxonomic diversity of strains from this genus inhabiting alfalfa nodules. Although Micromonospora strains are not able to induce the formation of these nodules, and it seems that they do not contribute to fix atmospheric nitrogen, they could play a role related with the mechanisms of plant growth promotion and pathogen protection presented by Micromonospora strains isolated from legume nodules.
Keywords: Micromonospora, Medicago sativa, diversity, taxonomy, Spain, Australia
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Introduction
Legume nodules are induced by soil bacteria known as rhizobia [43] but also contain phylogenetically diverse endophytic bacteria including species from the genus
Micromonospora [29, 44, 45]. Phylogenetically diverse strains from this genus have been
found in nodules of Lupinus angustifolius [40], Pisum sativum [8,11] and Medicago
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sativa [27]. Several of these strains belong to new species, such as Micromonospora
lupini isolated from nodules of L. angustifolius [41], and Micromonospora pisi,
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Micromonospora ureilytica, Micromonospora noduli, Micromonospora vinacea, Micromonospora luteifusca, Micromonospora phytophila and Micromonospora
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luteiviridis isolated from nodules of P. sativum [9, 10, 12, 15]. Strains isolated from M. sativa nodules also belong to several clusters of genus Micromonospora but to date only the 16S rRNA gene (rrs gene) has been analysed [27].
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Although the rrs gene is the universal marker used for bacterial classification, it has limitations to differentiate among closely related species [16] and additional phylogenetic
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markers are used to analyse intrageneric diversity [17, 19, 33, 37]. This occurs within genus Micromonospora [20] and the housekeeping gene gyrB has been commonly used for species differentiation within this genus [8, 20, 46]. Different studies proposed cut-
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off similarity values around 98.5% in the gyrB gene for differentiation of Micromonospora species [11, 20]. For species whit rrs and gyrB genes closely related, the analysis of the complete genomes allows the assignment of Micromonospora strains to a species [4, 6, 36], taking into account that cut-off values of ANI (Average Nucleotide Identity) and dDDH (Digital DNA-DNA hybridization) have been recently proposed for bacterial species differentiation [13].
Other genomic regions are also used for this purpose, such as the 16S-23S intergenic spacer (ITS), which is useful to analyse bacterial diversity because it allows to differentiate among different species and even among strains from the same species [31]. Although the ITS region has not been used in the case of genus Micromonospora, it has been used to differentiate between species of genus Salinispora, a closely related genus of the family Micromonosporaceae [23], having established a cut-off similarity value of 95% for Salinispora species differentiation [23]. Therefore, the objective of this work was to analyze the taxonomic diversity of Micromonospora strains previously isolated from M. sativa nodules in different
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geographical locations in Spain and Australia. To achieve this objective three phylogenetic single markers, rrs and gyrB genes and ITS region were analysed for all strains. In addition, we analysed the ANI (Average Nucleotide Identity) and dDDH
(Digital DNA-DNA hybridization) values obtained for selected strains belonging to
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different gyrB gene clusters.
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Material and methods
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Micromonospora strains
The Micromonospora strains analysed in this study were isolated from surface sterilized root nodules of alfalfa in previous studies [25,27]. The nodules were collected from alfalfa
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plants growing in five locations from Spain and in two locations from Western Australia (Table S1). The locations in Spain were Aldearrubia (40º 59' 57,08''N - 5º 29' 18,78''W), Babilafuente (40º 58' 22,22''N - 5º 25' 20,50''W), Palaciosrubios (41º 2' 36,34N- 5º 11'
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49,55''W), Tormes riverbank (40º 57' 42,88''N - 5º 40' 31,66''W) and Salamanca (40º 56' 29,13N - 5º 39' 21,21W) and in Australia were Binningup, Shire of Harvey (33°07'56.2"S
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115°43'22.0"E) and Murdoch University Campus, Perth (32°03'57.6"S 115°50'06.7"E).
Amplification and sequencing of the rrs and gyrB genes and 16S-23S ITS region The rrs gene and the 16S-23S ITS region were amplified and sequenced as previously reported by Trujillo et al. [40] and by Peix et al. [32], respectively. The amplification and sequencing of gyrB gene were performed using two primers designed in this study on the basis of conserved regions in this gene from Micromonospora species, GyrBF: 5’-
TCGACGGCAAGGCGTACG-3’ and GyrBR: 5’-CGCAGCTTCTCSATGTCG-3’. Amplification and sequencing conditions were as follows: 9 min at 94 ºC, 35 cycles of 1 min at 94 ºC, 1 min at 62 ºC and 2 min at 72 ºC, followed by 7 min final extension at 72 ºC. PCR products were electrophoresed in 1% agarose gels containing ethidium bromide (0.5 µg/ml) using modified Tris-Acetate EDTA buffer (Millipore, Cork, Ireland). The bands were excised and purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer instructions. The sequencing reactions were
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performed on an ABI377 sequencer (Applied Biosystems, Foster City, CA, USA) using a BigDye Terminator v3.0 Cycle Sequencing Kit as supplied by the manufacturer. The rrs gene sequences were compared with those from EzTaxon-e [48] and the sequences of
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the gyrB and ITS regions with those from GenBank using BLASTn [1]. Sequences were
aligned using the Clustal_X software [39]. The distances were calculated according to the
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Kimura two-parameter model [21]. The phylogenetic trees were inferred using the
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neighbour-joining [38] method. Bootstrap analysis was based on 1,000 resamplings. MEGA7 [22] was used for all analysis.
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Genome similarity estimation
The genomic DNA was extracted and purified using the DNeasy UltraClean Microbial DNA Isolation Kit (Qiagen) following manufacturer´s protocol. Sequencing, upon
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preparation of pair-end libraries, was performed on Illumina MiSeq sequencing platform (2x250bp). Sequencing data was assembled using Velvet 1.2.10 [49]. The genome
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characteristics are in Table S2. Average nucleotide identity blast (ANIb) was calculated using the JSpecies service [34, 35] (http://imedea.uib-csic.es/jspecies/). Digital DNA– DNA hybridization (dDDH) values were calculated using the genome-to-genome distance
calculator
website
service
from
DSMZ
(GGDC
2.1)
[30]
(http://ggdc.dsmz.de/ggdc.php/). These values were calculated using the formula two at the GGDC website [2] because it is the only function appropriate to analyse draft genomes [14].
Results and discussion
Analysis of the rrs gene The rrs gene is the main phylogenetic marker used in classification and identification of Prokaryotes, including Actinobacteria [18] and therefore it has been used in the analysis of the Micromonospora strains isolated in Spain and Australia [24, 25, 27]. The results obtained showed that two strains isolated in Australia, AMSO11 and AMSO12.1.2, have rrs gene sequences identical to those of the type strains of Micromonospora zamorensis and Micromonospora tulbaghiae, respectively, and the remaining strains presented
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similarity values (> 99%) with the type strains of different Micromonospora species. The strains AL6, AL20, ALFb4, AMSO3.1 and AMSO13, had the same similarity values with
respect to the type strains of two different Micromonospora species (Table 1). The phylogenetic analysis of the rrs genes showed that the strains from this study were
distributed into five clusters with similarity values among them lower than 99% in the rrs
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genes (Fig. 1). These clusters also included already described species of
Micromonospora, with some of them having rrs genes very closely related (similarity >
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99%), as occurred with the species included in the cluster I (Fig.1). This cluster contains, in addition to ten strains isolated from alfalfa nodules in Spain and Australia, the type
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strains of five Micromonospora species previously isolated in Spain, M. zamorensis isolated from rhizosphere of pea [8], Micromonospora saelicesensis isolated from lupine nodules [41] and M. ureilytica, M. noduli and M. vinacea isolated from pea nodules [9].
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Other species isolated from pea nodules in Spain, M. phytophila [12] is included in cluster II along with two strains isolated in Spain and Australia. The remaining clusters contain strains isolated from alfalfa nodules in Spain and/or Australia together with the type
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strains of Micromonospora species which were originally isolated from non legume sources (Fig.1). The results of the rrs gene analysis suggest that several strains analysed
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in this study could represent new species of genus Micromonospora based on comparison to recently described species of this genus [5, 9, 10], however, they also highlight the need to use other more variable phylogenetic markers in order to differentiate among these strains due the very high similarity values among rrs genes. Analysis of the gyrB gene Within the phylogenetic markers used to differentiate among species with close rrs genes, the gyrB gene is the most useful in the case of genus Micromonospora because it is currently available for most of species from this genus. Kasai et al. [15] showed that gyrB
gene similarity values of 98.6% correspond to 70% of DNA homology, which is the cutoff value currently accepted for species differentiation [47] and Carro et al. [11] concluded that different genospecies of Micromonospora could be defined at 98.5% similarity. Thus, the gyrB gene sequence similarity among different species of Micromonospora should be lower than 98.6%. Based on this, the strains ALFpr19a, ALF9, AL20, AMSO11 and AMSO12.1.2 can be identified as M. noduli, M. ureilytica, Micromonospora taraxaci, M. zamorensis and M. tulbaghiae, respectively, with similarity values from 98.9% to 99.9% in the gyrB gene (Table 1, Fig. 2). The strains AL4 and ALFb5 were closely related to the type strains of Micromonospora viridifaciens and
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M. tulbaghiae, respectively, with similarity values of 98.6% and 98.8%, respectivley, which are in the limit for species delineation. Some strains isolated in this study can be
assigned to the same species, for example, the strains ALFr4, AL6 and AMSO5.2 with similarity values in the gyrB gene ranging from 98.7% to 98.9% and the strains ALF4 and AMSO1.2, with gyrB gene similarity values of 98.7%, although these values are in the
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limit for species differentiation. From them, only the first group of strains could belong to the described species M. saelicensensis, with similarity values which also are in the
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limit for species differentiation since they ranged from 98.3 and 98.8%. The remaining strains present values lower than 98% similarity and therefore belong to species different
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to those currently described in the genus Micromonospora, including the strains ALF4 and AMSO1.2 (Table 1). The results of the gyrB and rrs genes were commonly congruent with the strains being related to the same species in both cases, nevertheless for some
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strains belonging to undescribed species, such as ALF4, ALFb4, Atrumurd12, AMSO1.2 and AMSO3.1, the closest related species in gyrB and rrs genes were not coincident (Table 1). The phylogenetic analysis of the gyrB gene showed that the strains from rrs
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gene cluster I formed a gyrB cluster A with internal similarity values higher than 93% for the latter gene. In the remaining gyrB gene clusters (B and C), applying a similarity value
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of 95% as cut-off, the strains showed a distribution different to that obtained after the rrs gene analysis. This is expected due to the higher variability of the gyrB gene and that most of strains from this study belong to several undescribed species. It is remarkable that some strains isolated in Australia and Spain are closely related as occurred in the case of ALFr4, AL6 and AMSO5.2 (cluster A) and with the strains ALF4 and AMSO1.2 (cluster B). Overall, the results of the gyrB gene analysis showed high taxonomic diversity of Micromonospora strains isolated from nodules of alfalfa from different geographical
locations, belonging to at least 18 different species within genus Micromonospora, with many of them undescribed to date.
Analysis of the 16S-23S intergenic region (ITS) The ITS region is a useful genetic marker to analyse bacterial biodiversity at species and strain levels [31], but it has not been widely used as taxonomic marker because its sequences are not available for all bacterial species, as occurs in the case of the genus Micromonospora. Nevertheless, in a related genus, Salinispora, its first described species were differentiated based on their ITS sequences [23]. These species, Salinispora tropica
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and Salinispora arenicola, showed ITS sequence similarity values lower than 95%, ranging from 98% to 100% at strain level [23].
The ITS region in the strains analysed in this study contained between 295 and 319 bp
and, as expected, as the ITS is a non-coding region, the variabilitiy of this region is higher
than that found for the gyrB gene (Table S3). The species of genus Salinispora present
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similarity values that range from 93% and 94% in their ITS sequences (excluding gaps);
these values are slightly higher to those found between closely related species of genus
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Micromonospora, such as M. zamorensis and M. noduli with 91.2%, or M. aurantiaca and M. tulbaghiae with 89.2% similarity (Fig. 3). Therefore, despite its low size, the ITS
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region is a very useful phylogenetic marker that discriminates Micromonospora species with closely related rrs and gyrB genes.
The ITS similarity values among the strains from this study were equal or lower than 95%
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and therefore all of them could be considered different species, including those where the gyrB gene sequence similarities were in the limit for species delimitation (Tables 1 and S3). The identification of strain AMSO12.1.2 as M. tulbaghiae (similarity of 98.4%) was
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confirmed, however the strains ALFpr19a and AMSO11 differed from the type strains of M. noduli and M. zamorensis, respectively. In the case of strains ALF9 and AL20 no
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conclusions can be established since the ITS for the type strains of M. ureilytica and M. taraxaci are not available (Fig. 3). The type strains of other Micromonospora species closely related with our strains in rrs and/or gyrB gene analyses and whose ITS sequences are available were phylogenetically divergent (Fig. 3). Therefore, the phylogenetic analysis of the ITS region confirmed that all strains from this study formed divergent lineages among them (Fig. 3) with similar distances to those found between S. tropica and Salinispora pacifica. This suggests that they belong to different species of genus Micromonospora inhabiting alfalfa nodules as was also found in other legumes [11, 40].
Average nucleotide identity blast (ANIb) and digital DNA–DNA hybridization (dDDH) estimation The genome comparison allows to delineate bacterial species and recently the minimal standards for this purpose have been published [13]. They include the estimation of ANI and dDDH having establishing cut-off values of 9596% and 70%, respectively, for bacterial species differentiation [13]. In this study we obtained the genome sequences of strains belonging to different gyrB clusters, which were selected taking into account two main criteria: i) They showed different gyrB gene similarities with respect their closest
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related species ii) The genomes of the type strains of these species are available in Genbank.
From gyrB cluster A we selected the strains ALFpr19a and ALFpr18c that showed, respectively, high (99.7%) and low (95.2%) similarity values in this gene with respect to
their closest relatives M. noduli GUI43T and M. chokoriensis DSM 45160T. In agreement
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with these results, the genome analysis confirmed that the strain ALFpr19a belongs to the species M. noduli with ANIb and dDDH values of 99.0% and 92.4%, respectively, and
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that the strain ALFpr18c could represent a new genospecies since it has ANIb and dDDH values of 87.3% and 35.6%, respectively, with respect to its closest relative M.
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chokoriensis DSM 45160T (Table S4).
The strains AMSO1.2 and AMSO3.1 selected from cluster B also showed different similarity values in the gyrB gene (97.9% and 95.4%, respectively) with respect to their
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closest relatives M. echinofusca DSM 43913T and M. coxensis DSM 45161T. ANIb and dDDH values of 92.5% and 50.8%, respectively, showed that the strain AMSO1.2 does not belong to M. echinofusca. Therefore this strain could represent new genospecies
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within Micromonospora despite of the gyrB gene similarity value is near to the cut-off values proposed for species differentiation within this genus [11, 20]. In agreement with
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the results of gyrB gene analysis the strain AMSO3.1 could also represent a new genospecies with ANIb and dDDH values of 83.6% and 38.7%, respectively, with respect to its closest relative M. coxensis DSM 45161T. The strains AMSO12.1.2 and ALFb5 from cluster C were selected because they presented high gyrB gene similarity values (near to or higher than 98%) with respect to the type strains of M. tulbaghiae and M. aurantiaca, two closely related species showing ANIb and dDDH values near to the limit for species differentiation (Table S4). In the case of the strain AMSO12.1.2, ANIb and dDDH values of 99.0% and 93.1%, respectively,
confirmed its identification as M. tulbaghiae. However, although in the gyrB gene analysis M. tulbaghiae also was the closest relative to the strain ALFb5 (98.8% similarity), the obtained ANIb and dDDH values (94.4% and 68.0%, respectively) showed that this strain do not belong to M. tulbaghiae. According to the results of ANIb and dDDH (98.0% and 88.9%, respectively) the strain ALFb5 should be identified as M. aurantiaca (Table S4). These results agree with those found after the rrs gene and ITS analyses, which showed that the strain AMSO12.1.2 is closely related to M. tulbaghiae and Alfb5 to M. aurantiaca (Table 1, Figs. 1 and 3). Therefore, in the case of phylogenetically close related species, as occurs with M.
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aurantiaca and M. tulbaghiae, we can found strains closely related to one or another species depending on the analysed marker, being the genome analysis the most reliable criterion for closely related species differentiation as well as for the assignment of new isolates to these species [36]. Nevertheless, the gyrB gene continues to be a good
phylogenetic marker for differentiation of most Micromonospora species and the ITS
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region will be an excellent tool for intra and interspecific differentiation when the
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sequences of this region are available for all species.
In conclusion, the results from this study showed that Micromonospora is a common
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inhabitant of alfalfa nodules in countries as geographically far apart as Spain and Australia. Our results also highlight the huge number of species that can inhabit these nodules in agreement with the results of other studies where Micromonospora were
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isolated from other legume nodules, such as lupine and peas [11, 40] and from actinorhizal nodules [7]. Although Micromonospora strains are not able to induce the formation of legume nodules and they do not appear to contribute to atmosferic nitrogen
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fixation [26], nodule isolated Micromonospora strains acts as a Rhizobia Helper Bacteria (RHB) agent and has probiotic effects, promoting plant growth and increasing nutrition
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efficiency [27, 42] and as biocontrol agents combining both direct antifungal activity against plant pathogens and the ability to prime plant immunity [28]. These roles could be related with the ability to infect the internal tissues of legume nodules when they are coinoculated with rhizobia even in the case of legumes from which they were not originally isolated [3, 24]. Taking this into account and considering the high taxonomic diversity of Micomonospora inhabiting legume nodules and the low number of legumes and geographical locations studied to date, it is necessary to carry out further studies in
order to improve the knowledge of the biodiversity of the association Micromonospora-
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legumes.
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Figure legends.
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Figure 1. Neighbour-joining phylogenetic rooted tree based on rrs gene sequences (1440 nt) of the representative strains from this study and those of closely related species of genus Micromonospora. Bootstrap values calculated for 1000 replications are indicated.
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Bar, 5 nt substitution per 1000 nt. Accession numbers from GenBank are given in
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brackets. Only bootstrap values >50% are shown.
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Figure 2. Neighbour-joining phylogenetic tree based on gyrB gene sequences (1030 nt) showing the position of representative strains from this study and those of closely related
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species of genus Micromonospora. Bootstrap values calculated for 1000 replications are indicated. Bar, 1 nt substitution per 100 nt. Accession numbers from GenBank are given
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in brackets. Only bootstrap values >50% are shown.
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Figure 3. Neighbour-joining phylogenetic tree based on the 16S-23S intergenic spacer
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(ITS) region sequences (295-319 nt) of the representative strains from this study comparing to Salinispora species. Bootstrap values calculated for 1000 replications are indicated. Bar, 5 nt substitution per 100 nt. Accession numbers from GenBank are given
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in brackets. Only bootstrap values >50% are shown.
Table 1. Strains isolated in this study and their closest related species.
ALF1 ALF2 ALF4 ALF9 ALFb4
ALFb5 ALFr4 ALFpr18c ALFpr19a Australia Atrumurd12 AMSO1.2 AMSO3.1
Closest related species (gyrB gene)
Similarity (%)
Cluster
M. yasonensis M. viridifaciens M. saelicesensis M. vinacea M. chaiyaphumensis M. taraxaci M. chokoriensis M. schwarzwaldensis M. yasonensis M. phytophila M. echinofusca M. ureilytica M. tulbaghiae M. citrea M. chalcea M. aurantiaca M. tulbaghiae M. vinacea M.saelicesensis M. chokoriensis M. noduli
99.6 99.8 99.7 99.7 99.6 99.9 99.9 99.6 99.1 99.7 98.9 99.6 99.2 99.2 98.5 99.9 98.7 99.7 99.5 99.4 99.9
V V I
M. yasonensis M. viridifaciens M. saelicesensis M. vinacea M. chaiyaphumensis M. taraxaci M. chokoriensis M. schwarzwaldensis M. yasonensis M. echinofusca M. phytophila M. ureilytica M. chalcea M. tulbaghiae M. citrea M. tulbaghiae M. aurantiaca M. saelicesensis M. vinacea M. chokoriensis M. noduli
94.8 98.6 98.3 97.4 97.3 99.2 97.8 97.6 96.1 97.9 92.8 99.9 96.2 94.8 92.7 98.8 97.9 98.4 97.2 95.2 99.7
C C A
M. chokoriensis M. coriariae M. phytophila M. echinofusca M. chaiyaphumensis M. citrea M. coxensis M. saelicesensis M. zamorensis M. tulbaghiae M. vinacea M. saelicesensis
99.8 97.7 99.7 98.9 99.7 99.7 98.8 99.9 100 100 99.7 99.7
I
M. coriariae M. chokoriensis M. echinofusca M. phytophila M. coxensis M. chaiyaphumensis M. citrea M. saelicesensis M. zamorensis M. tulbaghiae M. vinacea M. saelicesensis
95.9 92.1 97.9 92.9 95.4 94.3 92.8 98.8 98.9 99.9 98.0 97.9
A
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AMSO5.2 AMSO11 AMSO12.1.2 AMSO13
Cluster
V I IV V II I V
III I I I
II
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AL8 AL20
Similarity (%)
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Spain AL3 AL4 AL6
Closest related species (rrs gene)
V
I I V I
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Strains
C A C C B A C
C A A A
B B
A C A