MALDI-TOF mass spectrometry as a tool for differentiation of Bradyrhizobium species: Application to the identification of Lupinus nodulating strains

Systematic and Applied Microbiology 36 (2013) 565–571

Contents lists available at ScienceDirect

Systematic and Applied Microbiology journal homepage: www.elsevier.de/syapm

MALDI-TOF mass spectrometry as a tool for differentiation of Bradyrhizobium species: Application to the identification of Lupinus nodulating strains Fernando Sánchez-Juanes a,c,1 , Laura Ferreira a,1 , Pablo Alonso de la Vega d,f , Angel Valverde e,f,2 , Milagros León Barrios g , Raúl Rivas d,f , Pedro F. Mateos d,f , Eustoquio Martínez-Molina d,f , José Manuel González-Buitrago a,b,c , Martha E. Trujillo d,f , Encarna Velázquez d,f,∗ a

Unidad de Investigación, Hospital Universitario de Salamanca, Spain Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca, Spain Instituto de Investigación Biomédica, Salamanca, Spain d Departamento de Microbiología y Genética, Universidad de Salamanca, Spain e IRNASA Instituto de Recursos Naturales y Agrobiología, Consejo Superior de Investigaciones Científicas, Salamanca, Spain f Unidad Asociada Grupo de Interacción planta-microorganismo, Universidad de Salamanca, Consejo Superior de Investigaciones Científicas, Salamanca, Spain g Departamento de Microbiología y Biología Celular, Universidad de La Laguna, Spain b c

a r t i c l e

i n f o

Article history: Received 27 October 2012 Received in revised form 14 September 2013 Accepted 23 September 2013 Keywords: MALDI-TOF MS Bradyrhizobium Lupinus Rhizobia Identification

a b s t r a c t Genus Bradyrhizobium includes slow growing bacteria able to nodulate different legumes as well as species isolated from plant tumours. The slow growth presented by the members of this genus and the phylogenetic closeness of most of its species difficults their identification. In the present work we applied for the first time Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) to the analysis of Bradyrhizobium species after the extension of MALDI Biotyper 2.0 database with the currently valid species of this genus. With this methodology it was possible to identify strains belonging to phylogenetically closely related species of genus Bradyrhizobium allowing the discrimination among species with rrs gene identities higher than 99%. The application of MALDI-TOF MS to strains isolated from nodules of different Lupinus species in diverse geographical locations allowed their correct identification when comparing with the results of rrs gene and ITS analyses. The nodulation of Lupinus gredensis, an endemic species of the west of Spain, by B. canariense supports the European origin of this species. © 2013 Published by Elsevier GmbH.

Introduction The genus Bradyrhizobium comprises bacterial species of slow growth isolated from different sources that include legume nodules, plant tumours, water and soil (Table 1). The difficulties inherent to the handling of slow growing bacteria have resulted in a slow rhythm in the description of the species within genus Bradyrhizobium, although its numbers are currently increasing.

∗ Corresponding author at: Departamento de Microbiología y Genética, Lab. 209, Edificio Departamental de Biología, Campus Miguel de Unamuno, 37007 Salamanca, Spain. Tel.: +34 923 294532; fax: +34 923 224876. E-mail address: [email protected] (E. Velázquez). 1 These authors contributed equally to this work. 2 Present address: Centre for Microbial Ecology and Genomics (CMEG), Department of Genetics, University of Pretoria, Pretoria 0002, South Africa. 0723-2020/$ – see front matter © 2013 Published by Elsevier GmbH. http://dx.doi.org/10.1016/j.syapm.2013.09.003

From the three species described in 2000, B. japonicum [13], B. elkanii [17] and B. liaoningense [41] have gone to 16 effectively described species in year 2012 (see Table 1). We can expect that this number will increase in the next years because only in the past year 2012, in addition to soybean [1,36,43], several works showed that bradyrhizobia are the predominant endosymbiont of many herbaceous legumes such as peanut [12,18], cowpea [2,20,24], lupine [27,29] and several others [6,8,19]. Also Bradyrhizobium species are endosymbionts of tree legumes [1,14,16,32] and shrub legumes [1,5,10]. The slow growth and the phylogenetic closeness of most Bradyrhizobium species make their identification difficult and considering the frequency of the isolation of bradyrhizobial strains, rapid and reliable methods allowing the rapid identification of slow growing isolates will be very useful in the study of symbioses of many legumes. MALDI-TOF MS (Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry) based on the characteristic

566

F. Sánchez-Juanes et al. / Systematic and Applied Microbiology 36 (2013) 565–571

Table 1 Type and reference strains of genus Bradyrhizobium included in the extended database for MALDI-TOF MS-based species identification. Species

Strains included in database

Source of isolation

Geographical origin

Reference

B. japonicum B. elkanii B. liaoningense B. yuanmingense B. betae B. canariense B. pachyrhizi B. jicamae B. iriomotense B. lablabi B. denitrificans B. cytisi B. rifense B. huanghuaihaiense B. oligotrophicum Bradyrhizobium genosp. alpha Bradyrhizobium genosp. beta B. japonicum sv genistearum

LMG 6138T LMG 6134T LMG 18230T LMG 21827T pl7Hg1T LMG 22265T PAC48T PAC68T LMG 24129T LMG 25572T DSM 1113T CTAW11T CTAW71T LMG 26136T LMG 10732T BC-C1 BRE-1 BGA-1

Glycine max nodules Glycine max nodules Glycine max nodules Lespedeza cuneata nodules Beta vulgaris tumours Chamaecytisus proliferus nodules Pachyrhizus erosus nodules Pachyrhizus erosus nodules Entada koshunensis tumours Lablab purpureus nodules Water Cytisus villosus nodules Cytisus villosus nodules Glycine max nodules Rice paddy soil Chamaecytisus proliferus nodules Teline canariensis nodules Teline stenopetala nodules

Japan USA China China Spain Spain Costa Rica Honduras Japan China Germany Morocco Morocco China Japan Spain (Canary Islands) Spain (Canary Islands) Spain (Canary Islands)

[13] [17] [41] [42] [26] [40] [23] [23] [11] [6] [9,37] [4] [5] [43] [22] [40] [40] [40]

protein profiles for each microorganism is a promising method for taxonomic purpose that was already proven to be useful in the identification of fast growing rhizobia [7]. Although this methodology has been used to identify a known strain of B. japonicum inoculated in legumes directly from nodules [44], to identify unknown isolates it is necessary to extend the MALDI Biotyper 2.0 database that currently does not contain Bradyrhizobium species. Therefore the first aim of this work was to extend this database with the currently validly described species of genus Bradyrhizobium. The following aims were to evaluate the reliability of MALDI-TOF MS to identify slow growing reference strains isolated from different hosts and the application of this methodology to the identification of Lupinus endosymbionts isolated from the wild species Lupinus angustifolius and Lupinus gredensis, the latter, an endemic species of the West Spain. The results of this work showed that MALDI-TOF MS allows the differentiation of Bradyrhizobium species, even those closely related, being a good tool for slow growing rhizobia identification. Material and methods Bacterial strains and culture conditions To build a reference database for MALDI-TOF MS-based rhizobial species identification, the type strains of the 16 Bradyrhizobium species currently described and reference strains from genospecies alpha and beta and from symbiovar genistearum of B. japonicum were used (Table 1). To validate MALDI-TOF MS as an identification tool for genus Bradyrhizobium 26 strains isolated from different hosts and previously identified by DNA-DNA hybridization and gene sequencing were used (see Table 2). Finally MALDI-TOF MS was used to identify 12 strains isolated in this study from effective nodules of the wild species L. angustifolius and L. gredensis spontaneously growing in a soil from Cabrerizos located at 40◦ 58 43 North, 5◦ 36 46 West in Salamanca province (Spain) according to Vincent [39] on YMA medium. The nodulation of these strains in Lupinus albus plants was checked as was previously published [38].

and the MALDI-TOF MS performing was carried out as previously published [7] using a matrix of saturated solution of ␣-HCCA (Bruker Daltonics, Germany) in 50% acetonitrile and 2.5% trifluoracetic acid. As indicate the manufacturer we used amounts of biomass between 5 and 100 mg to obtain the spectra and the calibration masses were the Bruker Bacterial Test Standards (BTS) which were as follows (masses as averages): RL36, 4365.3 Da; RS22, 5096.8 Da; RL34, 5381.4 Da; RL33meth, 6255.4 Da; RL29, 7274.5 Da; RS19, 10,300.1 Da; RNase A, 13,683.2 Da and myoglobin, 16,952.3 Da. MALDI-TOF MS identifications were classified using the score values proposed by the manufacturer: a score value between 2.3 and 3.00 indicated species identification; a score value between 2.0 and 2.299 indicated genus identification and possible species identification, a score value between 1.7 and 1.999 indicated genus identification, and a score value < 1.7 indicated no identification. Cluster analysis was performed based on comparison of strainspecific main spectra created as described above. The dendrogram was constructed by the statistical toolbox of Matlab 7.1 (MathWorks Inc., USA) integrated in the MALDI Biotyper 2.0 software. The parameter settings were: ‘Distance Measure = Euclidian’ and ‘Linkage = complete’. The linkage function is normalized according to the distance between 0 (perfect match) and 1000 (no match). Phylogenetic analyses The results of MALDI-TOF MS analysis of the type strains from Bradyrhizobium species were compared with those obtained after the analyses of the rrs gene sequences available in databanks. For the identification of the new Lupinus isolates we obtained the16S23S intergenic fragment sequences (ITS) by using the primers and PCR conditions described by Peix et al. [21]. The sequences were aligned using the Clustal W software [35]. The distances were calculated according to Kimura’s two-parameter model [15]. Phylogenetic trees were inferred using the neighbour-joining method [28] and the MEGA 5 package [34]. Results and discussion

MALDI-TOF MS performing and data analysis

Spectra analysis and database setting

For MALDI-TOF MS analysis we used YMA and TY media [3,38]. Due to the slow growth of Bradyrhizobium strains we tested different incubation times, 24, 48 and 72 h, at 28 ◦ C in the type strains of three species from Bradyrhizobium with different grow rates, B. japonicum, B. elkanii and B. liaoningense. The sample preparation

In a previous work we showed that for fast-growing rhizobia the most adequate medium to perform MALDI-TOF MS analysis is solid TY because on YMA plates the abundant exopolysaccharide formed by these bacteria hampered the acquisition of reliable spectra [7]. Although in the case of Bradyrhizobium the spectra were nearly

F. Sánchez-Juanes et al. / Systematic and Applied Microbiology 36 (2013) 565–571

567

Table 2 Results of the identification using MALDI-TOF MS system. Plant source

Geographical origin

References

Best match

Score values

Second match

Score values

Reference strains isolated from different hosts Beta vulgaris Salamanca, Spain TTR1

[26]

B. betae pl7Hg1T

2.739

2.152

LMG 18231 LMG 6135 PAC51

Glycine max Glycine max Pachyrhizus erosus

China USA Honduras

[41] [23] [23]

B. liaoningense LMG 18230T B. elkanii LMG 6134T (USDA 76T ) B. pachyrhizi PAC48T

2.544 2.714 2.503

PAC683 MCOC04 MCOC05 MCAH03 MCAH12 MCAH13

Pachyrhizus erosus Ornithopus compressus Ornithopus compressus Arachis hypogaea Arachis hypogaea Arachis hypogaea

Honduras Salamanca, Spain Salamanca, Spain Salamanca, Spain Salamanca, Spain Salamanca, Spain

[23] [25] [25] [25] [25] [25]

B. jicamae PAC68T B. canariense LMG 22265T B. canariense LMG 22265T B. canariense LMG 22265T B. canariense LMG 22265T B. canariense LMG 22265T

2.658 2.502 2.570 2.447 2.612 2.505

Bradyrhizobium genosp. beta BRE-1 B. betae pl7Hg1T B. pachyrhizi PAC48T B. elkanii LMG 6134T (USDA 76T ) B. lablabi LMG 25572T B. betae pl7Hg1T B. betae pl7Hg1T B. betae pl7Hg1T B. canariense LMG 22265T B. betae pl7Hg1T

[4,25,38] [25,38] [25,38] [25,38] [25,38]

B. canariense LMG 22265T B. canariense LMG 22265T B. canariense LMG 22265T B. canariense LMG 22265T B. japonicum sv genistearum BGA-1

2.549 2.068 2.464 2.297 2.543

Reference strains isolated from Lupinus albus nodules Lupinus albus Salamanca, Spain MCLA07 Lupinus albus Salamanca, Spain MCLA12 Lupinus albus Salamanca, Spain MCLA22 Lupinus albus Salamanca, Spain MCLA23 RLA11 Lupinus albus León (Spain) RLA12

Lupinus albus

León (Spain)

[25,38]

B. japonicum sv genistearum BGA-1

2.637

ISLU 203 ISLU 207 ISLU 213 ISLU 220 BLUT1

Lupinus albus Lupinus albus Lupinus albus Lupinus albus Lupinus albus

Cautín (Chile) Cautín (Chile) Cautín (Chile) Valdivia (Chile) Canary Islands

[38] [38] [38] [38] [30,38]

B. japonicumsv genistearum BGA-1 Bradyrhizobium genosp. alpha BC-C1 B. japonicum sv genistearum BGA-1 B. japonicum sv genistearum BGA-1 B. canariense LMG 22265T

2.558 2.321 2.678 2.624 2.479

BLUT5 BLUT6

Lupinus albus Lupinus albus

Canary Islands Canary Islands

[38] [38]

B. canariense LMG 22265T B. canariense LMG 22265T

2.444 2.484

BLUT8 BLUT10

Lupinus albus Lupinus albus

Canary Islands Canary Islands

[38] [38]

B. canariense LMG 22265T B. canariense LMG 22265T

2.283 2.468

This study

B. canariense LMG 22265T

2.442

This study This study This study This study This study This study This study This study This study This study This study

B. canariense LMG 22265T B. canariense LMG 22265T B. canariense LMG 22265T B. canariense LMG 22265T B. canariense LMG 22265T B. canariense LMG 22265T B. canariense LMG 22265T B. canariense LMG 22265T B. canariense LMG 22265T B. canariense LMG 22265T B. canariense LMG 22265T

2.500 2.520 2.520 2.495 2.516 2.557 2.606 2.538 2.478 2.147 2.242

New isolates from nodules of wild Lupinus species CAR02 Lupinus angustifolius Salamanca, Spain CAR03 CAR05 CAR08 CAR10 CAR12 CAR16 CGR09 CGR10 CGR11 CGR12 CGR13

Lupinus angustifolius Lupinus angustifolius Lupinus angustifolius Lupinus angustifolius Lupinus angustifolius Lupinus angustifolius Lupinus gredensis Lupinus gredensis Lupinus gredensis Lupinus gredensis Lupinus gredensis

Salamanca, Spain Salamanca, Spain Salamanca, Spain Salamanca, Spain Salamanca, Spain Salamanca, Spain Salamanca, Spain Salamanca, Spain Salamanca, Spain Salamanca, Spain Salamanca, Spain

identical using TY and YMA media (Figs. S1 and S2), in general the TY medium was more adequate than YMA due to the production of higher amounts of exopolysaccharide in the latter medium which makes the sample preparation in some strains a difficult task. In the case of fast-growing rhizobia we used cultures of 24 h incubation [7], however taking into account that Bradyrhizobium species presented slow growth, we analyzed in the present work the spectra obtained after 24, 48 and 72 h incubation at 28 ◦ C, which is the temperature commonly used for the incubation of Bradyrhizobium strains, for the type strains of B. japonicum, B. elkanii and B. liaoningense. Since the spectra of these strains were almost identical (Fig. S3), all strains were cultivated on TY plates during 48 h at 28 ◦ C to perform MALDI-TOF MS analysis. A comparison between the spectra of different species of genus Bradyrhizobium and those of the type species of fast growing rhizobial genera Rhizobium, Ensifer and Shinella showed no relevant common peaks for discrimination among species of these genera (Fig. S4). The species from the genus Bradyrhizobium (Fig. S4D–U) have several peaks in a region located between 3200 and 3900 Da

2.042 2.282 2.245 2.126 2.193 2.091 2.077 2.003 2.130

B. betae pl7Hg1T No reliable B. betae pl7Hg1T B. liaoningense LMG 18230T Bradyrhizobium genosp. beta BRE-1 Bradyrhizobium genosp. beta BRE-1 B. canariense LMG 22265T B. canariense LMG 22265T B. canariense LMG 22265T B. canariense LMG 22265T Bradyrhizobium genosp. beta BRE-1 B. betae pl7Hg1T Bradyrhizobium genosp. beta BRE-1 B. betae pl7Hg1T B. betae pl7Hg1T

2.045 1.510 1.988 1.943 2.246

Bradyrhizobium genosp. alpha BC-C1 B. liaoningense LMG 18230T B. cytisi CTAW11T B. betae pl7Hg1T B. liaoningense LMG 18230T B. liaoningense LMG 18230T B. liaoningense LMG 18230T B. betae pl7Hg1T B. betae pl7Hg1T B. betae pl7Hg1T B. liaoningense LMG 18230T B. cytisi CTAW11T

1.874

2.201 2.160 2.052 2.075 2.118 2.028 2.136 2.098 1.726 1.812

1.680 1.863 2.108 1.883 2.118 1.856 2.220 2.217 2.161 1.743 1.912

that were much more intense that in fast-growing rhizobial genera (Fig. S4A–C) and were observed in both TY and YMA media (see Fig. S2). In this region, zones with multiple peaks are typical of genus Bradyrhizobium as can be observed in an enlargement of this region (Fig. S5). This typical region is present in all classic Bradyrhizobium species and it is also present in recently reclassified ones such as Bradyrhizobium denitrificans (formerly Blastobacter denitrificans) [9,37] and Bradyrhizobium oligotrophicum (formerly Agromonas oligotrophica) [22]. The presence of the region with multiple peaks in the spectra of these two species supports their reclassification into genus Bradyrhizobium. Moreover the peaks found in this region are diagnostic for each Bradyrhizobium analyzed species having different masses in each one of them (Figs. S4 and S5). Even phylogenetically related species from different origins (Table 1) such as B. japonicum, B. liaoningense and B. canariense have important differences in this region located in the first case between 3400 and 3500 Da, in the second case between 3650 and 3700 and between 3650 and 3800 in the third case (Figs. S4 and S5D, F and L). Within these regions the main peak of B. japonicum

568

F. Sánchez-Juanes et al. / Systematic and Applied Microbiology 36 (2013) 565–571

Bradyrhizobium liaoningense LMG 18230T (AJ250813) Bradyrhizobium yuanmingense CCBAU 11071T (LMG 21827T) (AF193818) 62 Bradyrhizobium genosp. beta BRE-1 (FJ428214) 69 Bradyrhizobium japonicum sv genistearum BGA-1 (AJ558024) 68 Bradyrhizobium japonicum LMG 6138T (X66024) Bradyrhizobium canariense BTA-1T (LMG 22265T) (AJ558025) Bradyrhizobium betae pl7Hg1 T (AY372184) Bradyrhizobium rifense CTAW71T (EU561074)

83 70 91

Bradyrhizobium cytisi CTAW11T (EU561065) Bradyrhizobium genosp. alpha BC-C1 (AJ558030) Bradyrhizobium iriomotense EK05T (LMG 24129T) (AB300992)

98 Bradyrhizobium huanghuaihaiense CCBAU 23303T (LMG 26136T) (HQ231463) 100

Bradyrhizobium denitrificans LMG 8443T (DSM 1113T) (X66025 ) 99

Bradyrhizobium oligotrophicum LMG 10732 T (JQ619230) Bradyrhizobium jicamae PAC68T (AY624134)

100 87

Bradyrhizobium lablabi CCBAU 23086T (LMG 25572T) (GU433448) Bradyrhizobium elkanii USDA 76T (LMG 6134T) (U35000) 100 Bradyrhizobium pachyrhizi PAC48T (AY624135) Shinella granuli Ch06T (DSM 18401T) (AB187585) Rhizobium leguminosarum USDA2370T (U29386)

94

Ensifer adhaerens LMG 20216T (AM18173)

0.01 Fig. 1. Neighbour-joining phylogenetic rooted tree based on 16S rRNA sequences (about 1475 nt) showing the taxonomic location of the species included in this study. Bootstrap values calculated for 1000 replications are indicated. Bar, 1 nt substitution per 100 nt. Accession numbers from Genbank are given in brackets.

LMG 6138T is located at 3437.5 Da, in B. liaoningense LMG 18230T is located at 3677.0 Da and in B. canariense LMG 22265T is located at 2691.1 Da (Table S1). In the spectrum of B. elkanii LMG 6134T , phylogenetically divergent to these three previous species (see Fig. 1) the region with multiple peaks (Figs. S4 and S5) is more reduced (from 3600 to 3750 Da) and has a main peak at 3678.4 Da, but this species has a second zone with multiple peaks well defined and with a main peak at 3834.1 Da (Table S1). B. pachyrhizi PAC48T which is phylogenetically very close to B. elkanii LMG 6134T (see Fig. 1), but has different origin (Table 1) also has a region with these two zones (Figs. S6 and S7C and D), but the main peaks are located at 3734.2 Da and 3820.6 Da (Table S1). In spite of the phylogenetic closeness of B. denitrificans and B. oligotrophicum, and in agreement with the results observed for the previous mentioned species, their spectra differ in the region with multiple peaks that is located 3800 and 3950 Da in the case of B. denitrificans DSM 1113T and between 3800 and 3900 Da in the case of B. oligotrophicum DSM 12412T (Figs. S4 and S5). The main peak is located at 3846.8 Da in B. denitrificans DSM 1113T and at 3833.7 in the case of B. oligotrophicum DSM 12412T (Table S1). In both cases the second region with multiple peaks was not detected (Figs. S6 and S7E and F). The MALDI-TOF MS spectra also differ in other peaks located before and after the multiple peaks region in both, close and divergent Bradyrhizobium species, allowing their differentiation by using this methodology (Table S1). These results are congruent with those previously reported for genera of fast growing rhizobia [7] suggesting that MALDI-TOF MS can also be a useful tool for the differentiation of slow growing species of rhizobia. To achieve this purpose we extended the MALDI BioTyper 2.0 database with the type strains of the 16 described species from genus Bradyrhizobium as well as with 3 strains representing the genospecies alpha and beta and the symbiovar genistearum of B. japonicum (Table 1).

The dendrogram constructed after the mathematical analysis of spectra showed an arrangement of species not being completely congruent with that obtained after rrs gene analysis (Fig. 1), although phylogenetically close species between them such as B. denitrificans and B. oligotrophicum, B. canariense and B. betae, B. jicamae and B. lablabi or B. elkanii and B. pachyrhizi were also close in the MALDI-TOF dendrogram (Fig. S8). Although in genus Rhizobium a correlation was found between MALDI-TOF dendrograms and rrs gene analysis [7], the number of species in genus Bradyrhizobium is much lower and this can affect to the mathematical analysis as reported previously for the genera Ensifer and Shinella where species were interspersed in the MALDI-TOF dendrogram [7]. Validation of MALDI-TOF MS for identification of Bradyrhizobium species To evaluate the reliability of MALDI-TOF MS in the identification of Bradyrhizobium species we analyzed several strains belonging to different species of this genus whose identity had been previously assessed by DNA–DNA hybridization and MLST analysis. Some of these strains were isolated from the same host and in the same country (Table 2) such as the strain TTR1 isolated from Beta vulgaris tumours in Spain that presented 99% DNA–DNA relatedness with respect to the type strain of this species, pl7Hg1T , from the same origin [26] and matched with this strain with a score value of 2.739. The strain LMG 6135 isolated from Glycine max nodules in USA showed 91% DNA–DNA relatedness with respect to the strain B. elkanii LMG 6134T from the same origin [23] and matched with this strain with a score value of 2.714. Finally, the strain PAC68-3 isolated from Pachyrhizus erosus nodules in Honduras showed 94% DNA–DNA relatedness with respect to B. jicamae PAC68T from the same origin [23] and matched with this strain with a score value of 2.658. The spectra of strains from the same species but different origins (Table 2) were also almost identical (Figs. S9 and S10). In

F. Sánchez-Juanes et al. / Systematic and Applied Microbiology 36 (2013) 565–571

569

56 Bradyrhizobium canariense MCLA22 (EF694747) Bradyrhizobium canariense BTA-1T (LMG 22265T) (AY386708) Bradyrhizobium canariense MCLA07 (EF694745) Bradyrhizobium canariense BLUT1 (EU333383) Bradyrhizobium canariense BLUT5 (GQ863552)

99 63

Bradyrhizobium canariense CAR03 (HF547301)

66 Bradyrhizobium canariense MCLA12 (EF694746) Bradyrhizobium canariense MCLA23 (EF694748) Bradyrhizobium canariense CGR11 (HF547303) 53 Bradyrhizobium canariense CAR10 (HF547302) 84 Bradyrhizobium canariense CGR12 (HF547304) Bradyrhizobium japonicum sv genistearum BGA-1 (AY386714) 56 Bradyrhizobium sp. ISLU203 (GQ863555) 84 Bradyrhizobium sp. RLA08 (EF694749) Bradyrhizobium japonicum LMG 6138T (AF345255 ) 90 94 Bradyrhizobium sp. ISLU207 (EF990556) Bradyrhizobium genosp. alpha BC-C1 (AY386703) Bradyrhizobium rifense CTAW71T (KC247123)

63 74

Bradyrhizobium cytisi CTAW11T (KC247124) Bradyrhizobium betae pl7Hg1T (AJ631967)

50

Bradyrhizobium genosp. beta BRE-1 (AY386715) Bradyrhizobium liaoningense LMG 18230T (AF345256)

97

Bradyrhizobium yuanmingense CCBAU 11071T (LMG 21827T) (AJ534605) Bradyrhizobium huanghuaihaiense CCBAU 23303T (LMG 26136T) (HQ428043)

93

Bradyrhizobium iriomotense EK05T (LMG 24129T) (AB300993) 98

Bradyrhizobium pachyrhizi PAC48T (AY628092) Bradyrhizobium elkanii USDA 76T (LMG 6134T) (AJ279308 ) Bradyrhizobium jicamae PAC68T (AY628094)

99

Bradyrhizobium lablabi CCBAU 23086T (LMG 25572T) (GU433583)

99

Bradyrhizobium denitrificans LMG 8443T (DSM 1113T) (AJ279318) 100

Bradyrhizobium oligotrophicum LMG 10732 T (KF583880)

0.02 Fig. 2. Neighbour-joining phylogenetic tree based on ITS fragment sequences (about 740 nt) showing the position of species included in this study. Bootstrap values calculated for 1000 replications are indicated. Bar, 1 nt substitution per 100 nt. Accession numbers from Genbank are given in brackets. Those obtained in this study are in bold.

this way the strain MCLA07 isolated from Lupinus albus nodules in mainland Spain showed 92% DNA–DNA relatedness with respect to the type strain of B. canariense LMG 22265T (LMG 22265T ) isolated from Chamaecytisus proliferus nodules in Canary Islands [4] and matched with this strain with a score value of 2.549 in agreement with the high similarity of their spectra (Figs. S9 and S10A and B). The strain PAC51 isolated from Pachyrhizus erosus nodules showed 82% DNA–DNA relatedness with respect to B. pachyrhizi PAC48T isolated from the same host but in a different country [23] and matched with this strain with a score value of 2.478 accordingly to their nearly identical spectra (Figs. S9 and S10C and D). Finally, the strain LMG 8231 isolated from Glycine max nodules [41] showed 100% DNA–DNA relatedness and similar spectrum (Figs. S9 and S10E and F) with respect to the type strain of B. liaoningense LMG 18230T isolated from the same host but in a different region of China [26] and matched with this strain with a score value of 2.554. Other strains, such as MCOC04 and MCOC05 isolated from Ornithopus compressus and MCAH03, MCAH12 and MCAH13 isolated from Arachis hypogaea in Spain were previously identified as B. canariense on the basis of MLST analysis [25]. The results of the MALDI-TOF MS analysis showed that all these strains matched with B. canariense LMG 22265T with score values near to 2.5. Since score values near to or higher than 2.5 according to the standards of Bruker system used in this work indicate good species identification, these results indicated that MALDI-TOF MS is a reliable system for differentiation of Bradyrhizobium species. Therefore

we applied this methodology to the identification of isolates from Lupinus, a legume nodulated by B. canariense and B. japonicum in Europe [25,33,38], in order to establish the potential of MALDITOF MS to differentiate between phylogenetically closely related species of genus Bradyrhizobium. Identification of Lupinus nodulating strains by MALDI-TOF MS Lupinus is an abundant legume in Spanish soils that comprises cultivated herbaceous legumes such as L. albus and wild shrub legumes such as L. angustifolius and L. gredensis an endemism from the Northwest region of Spain that includes the province of Salamanca. Some strains isolated from L. albus have been previously investigated in this province [25,30,38], however up to date there are no data about the endosymbionts of L. angustifolius or L. gredensis. Therefore in the present work we used for the first time MALDITOF MS to identify Lupinus isolates comparing the results obtained with those of rrs and ITS analysis, since this last region allowed a better differentiation between B. canariense and B. japonicum, the two main lupine endosymbionts in Europe [33] (Figs. 1 and 2). Initially we analyzed the results of MALDI-TOF MS analysis in several strains previously isolated from L. albus nodules in different countries and secondly we applied this methodology to the identification of the new isolates from L. angustifolius and L. gredensis (Table 2).

570

F. Sánchez-Juanes et al. / Systematic and Applied Microbiology 36 (2013) 565–571

The previously mentioned strain MACL07 and the strain MCLA22 were isolated from L. albus in Salamanca (Spain) and identified as B. canariense on the basis of MLST [25] and ITS sequence analysis [38]. Accordingly, the MALDI-TOF MS analysis showed they matched with B. canariense LMG 22265T with score values higher than 2.3. The strains MCLA12 and MCLA23 showed values slightly lower than 2.3 and accordingly belong to a different ITS subgroup than the type strain of B. canariense (Fig. 2). The strains BLUT1, BLUT5, BLUT6, BLUT8, BLUT10, isolated from L. albus in Canary Islands, were also identified as B. canariense on the basis of housekeeping gene analysis [38] and also matched with the type strain of this species with score values higher than 2.3 with the exception of strain BLUT8 which showed a value slightly lower. Strains RLA11 and RLA12 isolated in León (Spain) and ISLU203, ISLU213, ISLU220 isolated in Chile from L. albus nodules are related to B. japonicum BGA-1 isolated from Teline stenopetala in Canary Islands on the basis of ITS analysis [38] (Fig. 2). Accordingly, these strains showed similar spectra (Figs. S11 and S12A–C) and matched with score values higher than 2.3 with this last strain after MALDITOF MS identification (Table 2). Also the results of MALDI-TOF MS identification coincide with those of ITS analysis (Fig. 2) in the case of the strain ISLU207 isolated from Lupinus albus in Chile that was related to Bradyrhizobium genosp. alpha BC-C1 isolated in Canary Islands from Chamaecytisus proliferus nodules [38], showing similar spectra (Figs. S11 and S12D and E) and matching with this strain with a score value of 2.321 (Table 2). The dendrogram resulting from MALDI-TOF MS analysis (Fig. S8) showed that this technique allows the differentiation between B. canariense and the group of B. japonicum BGA-1 to which belong the two main endosymbionts of Genisteae including Lupinus [33]. The results of MALDI-TOF MS analysis for the new isolates from L. angustifolius and L. gredensis showed that all of them belong to the species B. canariense having almost identical spectra (Figs. S13 and S14) and matching with the type strain of this species with score values higher than 2.3 with the exception of strains CGR12 and CGR13 that showed values slightly lower (Table 2). These results agree with those obtained after ITS analysis, since the strain CGR12 belongs to the same subgroup than strains MCLA12 and MCLA23 which is phylogenetically divergent to that containing the type strain of B. canariense (Fig. 2). The results of this work confirm that B. canariense is the most frequent endosymbiont of Lupinus in Salamanca province in both cultivated species, L. albus, and wild species, L. angustifolius and L. gredensis. Although more strains nodulating this host in Spain should be analyzed to establish definitive conclusions, the identification of B. canariense as endosymbiont of L. angustifolius and particularly of L. gredensis, endemic of Spain, supports the coevolution of Lupinus species with B. canariense in Spanish soils and the European origin of this rhizobial species suggested by Stepkowski et al. [31] (2005). In summary, since the second matching species in all cases belonged to genus Bradyrhizobium with score values lower than 2.3 that according to the specifications of Biotyper 2.0 correspond to different species from the same genus (Table 2), the results of this work showed that MALDI-TOF MS is a good tool for rapid and reliable identification of slow-growing strains from genus Bradyrhizobium allowing the differentiation of all currently described species of this genus even those phylogenetically close such as B. canariense and B. japonicum, the most frequent endosymbionts of Lupinus species worldwide. Acknowledgements This work was supported by MICINN to EV, RR and MET; JCYL to EMM and RR and by the Instituto de Salud Carlos III (Ministerio de Ciencia e Innovación, SPAIN) (Ayuda de Infraestructura) to JMGB.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.syapm. 2013.09.003.

References [1] Aserse, A.A., Räsänen, L.A., Aseffa, F., Hailemariam, A., Lindström, K. (2012) Phylogenetically diverse groups of Bradyrhizobium isolated from nodules of Crotalaria spp., Indigofera spp., Erythrina brucei and Glycine max growing in Ethiopia. Mol. Phylogenet. Evol. 65, 595–609. [2] Azarias-Guimaraes, A., Duque-Jaramillo, P.A., Abrahao-Nófrega, R.S., Aparecida-Florentino, L., Barroso-Silva, K., Souza-Moreira, F.M. (2012) Genetic and symbiotic diversity of nitrogen-fixing bacteria isolated from agricultural soils in the Western Amazon by using cowpea as the plant tram. Appl. Environ. Microbiol. 78, 6726–6733. [3] Beringer, J.E. (1974) R factors transfer in Rhizobium leguminosarum. J. Gen. Microbiol. 84, 188–198. [4] Chahboune, R., Carro, L., Peix, A., Barrijal, S., Velázquez, E., Bedmar, E.J. (2011) Bradyrhizobium cytisi sp. nov., isolated from effective nodules of Cytisus villosus. Int. J. Syst. Evol. Microbiol. 61, 2922–2927. [5] Chahboune, R., Carro, L., Peix, A., Ramírez-Bahena, M.H., Barrijal, S., Velázquez, E., Bedmar, E.J. (2012) Bradyrhizobium rifense sp. nov. isolated from effective nodules of Cytisus villosus grown in the Moroccan Rif. Syst. Appl. Microbiol. 35, 302–305. [6] Chang, Y.L., Wang, J.Y., Wang, T., Liu, H.C., Sui, X.H., Chen, W.X. (2011) Bradyrhizobium lablabi sp. nov., isolated from effective nodules of Lablab purpureus and Arachis hypogaea. Int. J. Syst. Evol. Microbiol. 61, 2496–2502. [7] Ferreira, L., Sánchez-Juanes, F., García-Fraile, P., Rivas, R., Mateos, P.F., MartínezMolina, E., González-Buitrago, J.M., Velázquez, E. (2011) MALDI-TOF mass spectrometry is a fast and reliable platform for identification and ecological studies of species from family Rhizobiaceae. PLoS ONE 6, e20223. [8] Gehlot, H.S., Panwar, D., Tak, N., Tak, A., Sankhla, I.S., Poonar, N., Parihar, R., Shekhawat, M.S., Kumar, M., Tiwari, R., Ardley, J., James, E.K., Sprent, J.I. (2012) Nodulation of legumes from the Thar desert of India and molecular characterization of their rhizobia. Plant Soil 357, 227–243. [9] Hirsch, P., Müller, M. (1985) Blastobacter aggregatus sp. nov., Blastobacter capsulatus sp. nov., and Blastobacter denitrificans sp. nov., new budding bacteria from freshwater habitats. Syst. Appl. Microbiol. 6, 281–286. [10] Hossain, A.K.M.M., Mian, M.H., Hakim, M.A., Islam, M.M., Ferdous, J. (2012) Isolation and selection of Bradyrhizobium from the root nodules of indigo plants (Indigofera tinctoria L.). Afr. J. Biotechnol. 111, 2183–12191. [11] Islam, M.S., Kawasaki, H., Muramatsu, Y., Nakagawa, Y., Seki, T. (2008) Bradyrhizobium iriomotense sp. nov., isolated from a tumor-like root of the legume Entada koshunensis from Iriomote Island in Japan. Biosci. Biotechnol. Biochem. 72, 1416–1429. [12] Jaiswal, S.K., Anand, A., Dhar, B., Vaishampayan, A. (2012) Genotypic characterization of phage-typed indigenous soybean Bradyrhizobia and their host range symbiotic effectiveness. Microb. Ecol. 63, 116–126. [13] Jordan, D.C. (1982) Transfer of Rhizobium japonicum Buchanan 1980 to Bradyrhizobium gen. nov., a genus of slow-growing, root nodule bacteria from leguminous plants. Int. J. Syst. Bacteriol. 32, 136–139. [14] Kang, J.W., Song, J., Doty, S.L., Lee, D.K. (2012) Diversity of rhizobia associated with leguminous trees growing in South Korea. J. Basic Microbiol. 53, 291–298. [15] Kimura, M. (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111–120. [16] Koppell, J.H., Parker, M.A. (2012) Phylogenetic clustering of Bradyrhizobium symbionts on legumes indigenous to North America. Microbiology 158, 2050–2059. [17] Kuykendall, L.D., Saxena, B., Devine, T.E., Udell, S.E. (1992) Genetic diversity in Bradyrhizobium japonicum Jordan 1982 and a proposal for Bradyrhizobium elkanii sp. nov. Can. J. Microbiol. 38, 501–505. [18] Nievas, F., Bogino, P., Nocelli, N., Giordano, W. (2012) Genotypic analysis of isolated peanut-nodulating rhizobial strains reveals differences among populations obtained from soils with different cropping histories. Appl. Soil Ecol. 53, 74–82. [19] Noisangiam, R., Teamtisong, K., Tittabutr, P., Boonkerd, N., Toshiki, U., Minamisawa, K., Teaumroong, N. (2012) Genetic diversity, symbiotic evolution, and proposed infection process of Bradyrhizobium strains isolated from root nodules of Aeschynomene americana L. in Thailand. Appl. Environ. Microbiol. 78, 6236–6250. ˜ E., Rogel-Hernández, M.A., Lloret, L., López-López, A., Martínez, [20] Ormeno-Orrillo, J., Barois, I., Martínez-Romero, E. (2012) Change in land use alters the diversity and composition of Bradyrhizobium communities and led to the introduction of Rhizobium etli into the tropical rain forest of Los Tuxtlas (Mexico). Microb. Ecol. 63, 822–834. ˜ [21] Peix, A., Rivas, R., Velázquez, E., Mateos, P.F., Martínez-Molina, E., MunozHerrera, A., Gómez-Alonso, A., Velázquez, E. (2005) Application of horizontal staircase electrophoresis in agarose minigels to the random intergenic spacer analysis of clinical samples. Electrophoresis 26, 4402–4410.

F. Sánchez-Juanes et al. / Systematic and Applied Microbiology 36 (2013) 565–571 [22] Ramírez-Bahena, M.H., Chahboune, R., Peix, A., Velázquez, E. (2012) Reclassification of Agromonas oligotrophica into genus Bradyrhizobium as Bradyrhizobium oligotrophicum comb. nov. Int. J. Syst. Evol. Microbiol. 63, 1013–1016. [23] Ramírez-Bahena, M.H., Peix, A., Rivas, R., Camacho, M., Rodríguez-Navarro, D.N., Mateos, P.F., Martínez-Molina, E., Willems, A., Velázquez, E. (2009) Bradyrhizobium pachyrhizi sp. nov. and Bradyrhizobium jicamae sp. nov., isolated from effective nodules of Pachyrhizus erosus. Int. J. Syst. Evol. Microbiol. 59, 1929–1934. [24] Risal, C.P., Djedidi, S., Dhakal, D., Ohkama-Ohtsu, N., Sekimito, H., Yokoyama, T. (2012) Phylogenetic diversity and symbiotic funtioning in mungbean (Vigna radiata L. Wilczek) bradyrhizobia from contrast agro-ecological regions of Nepal. Syst. Appl. Microbiol. 35, 45–53. [25] Rivas, R., Martens, M., de Lajudie, P., Willems, A. (2009) Multilocus sequence analysis of the genus Bradyrhizobium. Syst. Appl. Microbiol. 32, 101–110. [26] Rivas, R., Willems, A., Palomo, J.L., García-Benavides, P., Mateos, P.F., MartínezMolina, E., Gillis, M., Velázquez, E. (2004) Bradyrhizobium betae sp. nov., isolated from roots of Beta vulgaris affected by tumour-like deformations. Int. J. Syst. Evol. Microbiol. 54, 1271–1275. [27] Ruiz-Díez, B., Fajardo, S., Felipe, M.R., Fernández-Pascual, M. (2012) Characterization of rhizobia from legumes of agronomic interest grown in semi-arid areas of Central Spain relates genetic differences to soil properties. J. Basic Microbiol. 52, 66–78. [28] Saitou, N., Nei, M. (1987) A neighbour-joining method: a new method for reconstructing phylogenetics trees. Mol. Biol. Evol. 44, 406–425. ˜ [29] Sánchez-Canizares, C., Rey, L., Durán, D., Temprano, F., Sánchez-Jiménez, P., Navarro, A., Polajnar, M., Imperial, J., Ruiz-Argüeso, T. (2011) Endosymbiotic bacteria nodulating a new endemic lupine Lupinus mariae-josephi from alkaline soils in Eastern Spain represent a new lineage within the Bradyrhizobium genus. Syst. Appl. Microbiol. 34, 207–215. ˛ [30] Stepkowski, T., Hughes, C.E., Law, I.J., Markiewicz, Ł., Gurda, D., Chlebicka, A., Moulin, L. (2007) Diversification of lupine Bradyrhizobium strains: evidence from nodulation gene trees. Appl. Environ. Microbiol. 73, 3254–3264. ˛ ´ [31] Stepkowski, T., Moulin, L., Krzyzanska, A., McInnes, A., Law, I.J., Howieson, J. (2005) European origin of Bradyrhizobium populations infecting lupins and serradella in soils of Western Australia and South Africa. Appl. Environ. Microbiol. 71, 7041–7052. ˛ [32] Stepkowski, T., Watkin, E., McInnes, A., Gurda, D., Gracz, J., Steenkamp, E.T. (2012) Distinct Bradyrhizobium communities nodulate legumes native to temperate and tropical monsoon Australia. Mol. Phylogenet. Evol. 63, 265–277. ˛ ´ ˙ [33] Stepkowski, T., Zak, M., Moulin, L., Króliczak, J., Golinska, B., Narozna, D., ˛ C.J. (2011) Bradyrhizobium canariense and BradyrhiSafronova, V.I., Madrzak, zobium japonicum are the two dominant rhizobium species in root nodules of lupin and serradella plants growing in Europe. Syst. Appl. Microbiol. 34, 368–375.

571

[34] Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S. (2011) MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. [35] Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G. (1997) The clustalX windows interface: flexible strategies for multiple sequence alignement aided by quality analysis tools. Nucleic Acids Res. 24, 4876–4882. [36] van Berkum, P., Elia, P., Song, Q., Eardly, B.D. (2012) Development and application of a multilocus sequence analysis method for the identification of genotypes within genus Bradyrhizobium and for establishing nodule occupancy of soybean (Glycine max L. Merr). Mol. Plant Microbe Interact. 25, 321–330. [37] van Berkum, P., Leibold, J.M., Eardly, B.D. (2006) Proposal for combining Bradyrhizobium spp. (Aeschynomene indica) with Blastobacter denitrificans and to transfer Blastobacter denitrificans (Hirsch and Muller, 1985) to the genus Bradyrhizobium as Bradyrhizobium denitrificans (comb. nov.). Syst. Appl. Microbiol. 29, 207–215. [38] Velázquez, E., Valverde, A., Rivas, R., Gomis, V., Peix, A., Gantois, I., Igual, J.M., León-Barrios, M., Willems, A., Mateos, P.F., Martínez-Molina, E. (2010) Strains nodulating Lupinus albus on different continents belong to several new chromosomal and symbiotic lineages within Bradyrhizobium. Antonie Van Leeuwenhoek 97, 363–376. [39] Vincent, J. 1970 A Manual for the Practical Study of the Root Nodule Bacteria, International Biological Programme Handbook, vol. 15, Blackwell, Oxford. [40] Vinuesa, P., León-Barrios, M., Silva, C., Willems, A., Jarabo-Lorenzo, A., PérezGaldona, R., Werner, D., Martínez-Romero, E. (2005) Bradyrhizobium canariense sp. nov., an acid-tolerant endosymbiont that nodulates endemic genistoid legumes (Papilionoideae: Genisteae) from the Canary Islands, along with Bradyrhizobium japonicum bv. genistearum, Bradyrhizobium genospecies alpha and Bradyrhizobium genospecies beta. Int. J. Syst. Evol. Microbiol. 55, 569–575. [41] Xu, L.M., Ge, C., Cui, Z., Li, J., Fan, H. (1995) Bradyrhizobium liaoningense sp. nov., isolated from the root nodules of soybeans. Int. J. Syst. Bacteriol. 45, 706–711. [42] Yao, Z.Y., Kan, F.L., Wang, E.T., Chen, W.X. (2002) Characterization of rhizobia that nodulate legume species within the genus Lespedeza and description of Bradyrhizobium yuanmingense sp. nov. Int. J. Syst. Evol. Microbiol. 52, 2219–2230. [43] Zhang, Y.M., Li, Y., Jr., Chen, W.F., Wang, E.T., Sui, X.H., Li, Q.Q., Zhang, Y.Z., Zhou, Y.G., Chen, W.X. (2012) Bradyrhizobium huanghuaihaiense sp. nov., an effective symbiotic bacterium isolated from soybean (Glycine max L.) nodules. Int. J. Syst. Evol. Microbiol. 62, 1951–1957. [44] Ziegler, D., Mariotti, A., Pflüger, V., Saad, M., Vogel, G., Tonolla, M., Perret, X. (2012) In situ identification of plant-invasive bacteria with MALDI-TOF mass spectrometry. PLoS ONE 7, e37189.