MALDI-TOF mass spectrometry is a fast and reliable platform for identification and ecological studies of species from family Rhizobiaceae.

Family Rhizobiaceae includes fast growing bacteria currently arranged into three genera, Rhizobium, Ensifer and Shinella, that contain pathogenic, symbiotic and saprophytic species. The identification of these species is not possible on the basis of physiological or biochemical traits and should be based on sequencing of several genes. Therefore alternative methods are necessary for rapid and reliable identification of members from family Rhizobiaceae. In this work we evaluated the suitability of Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) for this purpose. Firstly, we evaluated the capability of this methodology to differentiate among species of family Rhizobiaceae including those closely related and then we extended the database of MALDI Biotyper 2.0 including the type strains of 56 species from genera Rhizobium, Ensifer and Shinella. Secondly, we evaluated the identification potential of this methodology by using several strains isolated from different sources previously identified on the basis of their rrs, recA and atpD gene sequences. The 100% of these strains were correctly identified showing that MALDI-TOF MS is an excellent tool for identification of fast growing rhizobia applicable to large populations of isolates in ecological and taxonomic studies.

Introduction

The family Rhizobiaceae currently contains fast growing species of bacteria that may be saprophytic or able to establish beneficial or deleterious plant interactions. These species are currently arranged into three genera, Rhizobium, Ensifer and Shinella [1], [2]. The former genera Agrobacterium and Allorhizobium are now included in genus Rhizobium [3] and Sinorhizobium is currently named Ensifer [4]. The identification of members of the family Rhizobiaceae is necessarily based on gene sequencing since there is not phenotypic information that allows the differentiation and identification of rhizobial species [3]. Therefore, although gene sequencing is the most reliable method for identification of rhizobia, it is still a tedious and time-consuming method to be applied to wide populations and therefore alternative methods are necessary for reliable identification of these bacteria shortening the time needed to achieve this process.

Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) has been suggested as a fast and reliable method for bacterial identification, based on the characteristic protein profiles for each microorganism. Using this technology it has been estimated that up to 99% of strains tested are correctly identified when comparing with commercial phenotypic identification panels or rrs gene sequencing [5]–[8]. However MALDI-TOF MS has been basically applied to the identification of clinical isolates [9]–[16] so most of the species currently included on available databases are those of clinical interest. For example in the case of family Rhizobiaceae only the type strains of three species, Rhizobium tropici, Rhizobium radiobacter and Rhizobium rubi, and eight pathogenic non-type strains of R. radiobacter, R. rhizogenes and Agrobacterium tumefaciens (currently R. radiobacter) are included in Biotyper 2.0 database (Bruker Daltonics) used in this study.

Therefore the objectives of this work were: (i) the evaluation of MALDI-TOF MS technology for species differentiation within family Rhizobiaceae, (ii) the construction of a database that includes the type strains of currently accepted species within family Rhizobiaceae and (iii) the validation of the MALDI-TOF MS technology to identify rhizobial strains isolated from nodules and tumours previously identified by gene sequencing.

Materials and Methods

Bacterial strains and culture conditions

To build a reference database for MALDI-TOF MS-based rhizobial species identification, the type strains of 56 species belonging to the family Rhizobiaceae were used (Table 1). In addition 35 strains isolated from legume nodules or plant tumours previously characterized by gene sequencing were used to validate MALDI-TOF MS as an identification tool for this group of bacteria (Table 2).

Table 1

Type strains of family Rhizobiaceae included in the extended database for MALDI-TOF MS- based species identification.

Species	Strains included in database	Source of isolation	References
Ensifer adhaerens	LMG 20216T	soil	[50]
Ensifer americanum	DSM 15007T	Acacia nodules	[36], [51]
Ensifer arboris	LMG 14919T	Prosopis chilensis nodules	[36], [52]
Ensifer fredii	USDA 205T, LMG 6217T	Glycine max nodules	[36], [53], [54]
Ensifer garamanticus	LMG 24692T	Agryrolobium uniflorum nodules	[55]
Ensifer kostiense	LMG 19227T	Acacia senegal nodules	[36], [52]
Ensifer kummerowiae	CCBAU 71714T	Kummerowia stipulacea nodules	[36], [56]
Ensifer medicae	USDA 1037T	Medicago truncatula nodules	[36], [57]
Ensifer meliloti	ATCC 9930T	Medicago sativa nodules	[36], [58]
Ensifer morelense	Lc04T	Leucaena leucocephala nodules	[36], [59]
Ensifer numidicus	LMG 24690T	Agryrolobium uniflorum nodules	[55]
Ensifer saheli	LMG 7837T	Sesbania cannabina nodules	[36], [58]
Ensifer terangae	LMG 7834T	Acacia laeta nodules	[36], [58]
Rhizobium aggregatum	DSM 1111T	Surface lake water	[30], [60]
Rhizobium alamii	LMG 24466T	Plant rhizosphere	[61]
Rhizobium alkalisoli	DSM 21826T	Caragana microphylla nodules	[62]
Rhizobium borbori	LMG 23925T	Activated sludge	[63]
Rhizobium cellulosilyticum	ALA10B2T	Populus alba sawdust	[64]
Rhizobium daejeonense	DSM 17795T	Cyanide treatment bioreactor	[65]
Rhizobium etli	CFN42T	Phaseolus vulgaris nodules	[32]
Rhizobium fabae	LMG 23997T	Vicia faba nodules	[66]
Rhizobium galegae	HAMBI 540T	Galega orientalis nodules	[67]
Rhizobium gallicum	R602spT	Phaseolus vulgaris nodules	[45]
Rhizobium giardinii	H152T	Phaseolus vulgaris nodules	[45]
Rhizobium hainanense	I66T	Desmodium sinuatum nodules	[68]
Rhizobium huautlense	S02T	Sesbania herbacea nodules	[69]
Rhizobium indigoferae	CCBAU 71042T	Indigofera amblyantha nodules	[56]
Rhizobium larrymoorei	LMG 21410T	Ficus benjamina aerial tumours	[3], [70], [71]
Rhizobium leguminosarum	USDA 2370T	Pisum sativum nodules	[29]
Rhizobium loessense	CCBAU 7190BT, LMG23187T	Astragalus complanatus nodules	[72]
Rhizobium lusitanum	p1-7T	Phaseolus vulgaris nodules	[48]
Rhizobium mesosinicum	LMG 24135T	Albizia julibrissin	[73]
Rhizobium miluonense	LMG 24208T	Lespedeza chinensis nodules	[74]
Rhizobium mongolense	USDA 1844T	Medicago ruthenica nodules	[75]
Rhizobium multihospitium	LMG 23946T	Halimodendron halodendron nodules	[76]
Rhizobium oryzae	LMG 24253T	Wild rice surface sterilized roots	[77]
Rhizobium phaseoli	ATCC 14482T	Phaseolus vulgaris nodules	[29], [32]
Rhizobium pisi	DSM 30132T	Pisum sativum nodules	[29]
Rhizobium radiobacter (formerly Agrobacterium radiobacter)	ATCC 19358T, NCBI 9042T, CIP 104325T	Soil	[3], [28]
Rhizobium rhizogenes	ATCC 11325T	Hairy roots	[3], [28]
Rhizobium rosettiformans	CCM 7583T	hexachlorocyclohexane (HCH) dump site	[30]
Rhizobium rubi	ATCC 13335T, DSM 6772T	Rubus tumours	[3], [78]
Rhizobium selenitireducens	LMG 24075T	Cyanide treatment bioreactor	[79]
Rhizobium sullae	IS 123T	Hedysarum coronarium nodules	[80]
Rhizobium tibeticum	DSM 21102T	Trigonella archiducis-nicolai	[81]
Rhizobium tropici	CIAT 899T	Leucaena leucocephala nodules	[82]
Rhizobium tubonense	LMG 25225T	Oxytropis glabra nodules	[83]
Rhizobium undicola	LMG 11875T	Neptunia natans nodules	[3], [84]
Rhizobium vitis	CECT 4799T	Vitis vinifera tumours	[3], [85]
Rhizobium vignae	LMG 25447T	Vigna nodules	[86]
Rhizobium yanglingense	CCBAU 71623T	Gueldenstaedtia nodules	[87]
Shinella granuli	DSM 18401T	Upflow anaerobic sludge blanket reactor	[88]
Shinella fusca	LMG 24714T	Domestic waste compost	[89]
Shinella kummerowiae	LMG 24136T	Kummerowia stipulacea nodules	[2]
Shinella yambaruensis	DSM 18801T	Soil	[90]
Shinella zoogloeoides	DSM 287T	Activated sludge	[88], [91]

Table 2

MALDI BioTyper identification results for family Rhizobiaceae strains.

	Source of isolation	Reference	Organism (best match)	score values*
A. Strains from different collections
Ensifer fredii USDA 205T	Glycine nodules	[53]	Ensifer fredii LMG 6217T	2.585
Rhizobium loessense LMG 23187T	Astragalus nodules	[72]	Rhizobium loessense CCBAU 7190BT	2.335
Rhizobium tropici CIAT 899T	Phaseolus nodules	[82]	Rhizobium tropici DSM 11418T	2.582
Rhizobium radiobacter ATCC 19358T (NCBI 9042T, CIP 104325T)	soil	[28]	Rhizobium radiobacter DSM 30147T	2.566 (2.524, 2.488)
Rhizobium rubi ATCC 13335T	Rubus tumours	[28]	Rhizobium rubi DSM 6772T	2.505
B. Species correctly reclassified in other species
Agrobacterium tumefaciens ATCC 23308 (NCBI 13307, CIP 67.1), former type strains	Malus tumours	[28]	Rhizobium radiobacter DSM 30147T (strain included in Biotyper 2.0 database)	2.522 (2.408, 2.405)
Ensifer xinjiangense LMG 17930 (CECT 4657), former type strains	Glycine nodules	[92]	Ensifer fredii LMG 6217T	2.413 (2.151)
Rhizobium trifolii ATCC 14480, former type strain	Trifolium nodules	[29]	Rhizobium leguminosarum USDA 2370T	2.128
C. Species erroneously included in other species
Ensifer morelense Lc04T	Leucaena nodules	[36], [59]	Ensifer adhaerens LMG 20216T	1.245
Rhizobium phaseoli ATCC 14482T	Phaseolus nodules	[29], [32]	Rhizobium etli CFN42T	1.991
Rhizobium pisi DSM 30132T	Pisum nodules	[29]	Rhizobium leguminosarum USDA 2370T	1.782
D. Species with problems in their identity
Ensifer kummerowiae CCBAU 71714T	Kummerowia nodules	[56]	Ensifer meliloti ATCC 9930T	2.261
Rhizobium fabae LMG 23997T	Vicia nodules	[66]	Rhizobium pisi DSM 30132T	2.258
Rhizobium indigoferae CCBAU 71042T	Indigofera nodules	[56]	Rhizobium leguminosarum USDA 2370T	2.219
Rhizobium loessense CCBAU 7190BT (LMG23187T)	Astragalus nodules	[72]	Rhizobium gallicum R602spT	2.283 (2.354)
Rhizobium mongolense USDA 1844T	Medicago nodules	[75]	Rhizobium gallicum R602spT	2.506
Rhizobium yanglingense CCBAU 1623T	Gueldenstaedtia nodules	[87]	Rhizobium gallicum R602spT	2.314
F. Strains included in already described species
RTM17	Trigonella nodules	[49]	Ensifer meliloti ATCC 9930T	2.140
GVPV12	Phaseolus nodules	[44]	Ensifer meliloti ATCC 9930T	2.145
RPA13	Prosopis nodules	[43]	Ensifer meliloti ATCC 9930T	2.241
RMP01	Melilotus nodules	[49]	Ensifer medicae USDA 1037T	2.252
RMP05	Melilotus nodules	[49]	Ensifer medicae USDA 1037T	2.114
RPA08	Prosopis nodules	[43]	Ensifer medicae USDA 1037T	2.092
RPA11	Prosopis nodules	[43]	Ensifer medicae USDA 1037T	2.177
RPA20	Prosopis nodules	[43]	Ensifer medicae USDA 1037T	2.211
FL27	Phaseolus nodules	[45]	Rhizobium gallicum R602spT	2.405
PhD12	Phaseolus nodules	[45]	Rhizobium gallicum R602spT	2.399
RPA02	Prosopis nodules	[43]	Rhizobium giardinii H152T	2.432
RPA12	Prosopis nodules	[43]	Rhizobium giardinii H152T	2.425
RPVF18	Phaseolus nodules	[38]	Rhizobium leguminosarum USDA 2370T	2.017
RVS11	Vicia nodules	[39]	Rhizobium leguminosarum USDA 2370T	2.152
ATCC 14480	Trifolium nodules	[29]	Rhizobium leguminosarum USDA 2370T	2.128
P3-13	Phaseolus nodules	[48]	Rhizobium lusitanum P1-7T	2.314
USDA 1929	Medicago nodules	[75]	Rhizobium mongolense USDA1844T	2.474
ATCC 13332	no data	[93]	Rhizobium radiobacter DSM 30147T	2.644
163C	Prunus tumours	[47]	Rhizobium rhizogenes ATCC 11325T	2.195
IAM 13571	no data	[48]	Rhizobium rhizogenes ATCC 11325T	2.267
K84	soil	[46]	Rhizobium rhizogenes ATCC 11325T	2.185
Br859	Leucaena nodules	[82]	Rhizobium tropici CIAT 899T	2.613
G. Strains do not belonging to described species
Br816	Leucaena nodules	[34], [35]	Ensifer americanum	1.775
RPVR32	Phaseolus nodules	[38]	Rhizobium leguminosarum USDA 2370T	1.066
CVIII4	Vicia nodules	[39]	Rhizobium leguminosarum USDA 2370T	1.288
RTP05	Trifolium nodules	[94]	Rhizobium leguminosarum USDA 2370T	1.701
CFN299	Leucaena nodules	[82]	Rhizobium tropici CIAT 899T	1.191
C58	Prunus tumour	[95]	Rhizobium radiobacter DSM 30147T	1.956

*score value ≥2 indicates species identification; 1.7

In order to establish the more adequate medium and growth conditions for fast-growing rhizobia analysis, selected strains were cultivated on TY [17] and YMA plates [18] and incubated at 28°C for 24 and 48 hours.

Sample preparation for MALDI-TOF MS

Cells of a whole colony were transferred from the plate to a 1.5 ml tube (Eppendorf, Germany) with a pipette tip and mixed thoroughly in 300 µl of water to resuspend the bacterial cells. Then, 900 µl of absolute ethanol was added and the mixture was centrifuged at 15,500 g for 2 min, and the supernatant was discarded. The pellet was air-dried at room temperature for 1 hour. Subsequently, 50 µl of formic acid (70% v/v) was added to the pellet and mixed thoroughly before the addition of 50 µl acetonitrile to the mixture. The mixture was centrifuged again at 15,500 g for 2 min. One microliter of the supernatant was placed onto a spot of the steel target and air-dried at room temperature. Each sample was overlaid with 1 µl of matrix solution and air-dried.

MALDI-TOF MS

Measurements were performed on an Autoflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Leipzig, Germany) equipped with a 200-Hz smartbeam laser. Spectra were recorded in the linear, positive mode at a laser frequency of 200 Hz within a mass range from 2,000 to 20,000 Da. The IS1 voltage was 20 kV, the IS2 voltage was maintained at 18.6 kV, the lens voltage was 6 kV, and the extraction delay time was 40 ns.

For each spectrum, 500 laser shots were collected and analyzed (10×50 laser shots from different positions of the target spot). The spectra were calibrated externally using the standard calibrant mixture (Escherichia coli extracts including the additional proteins RNase A and myoglobin, Bruker Daltonics). Calibration masses were as follows: RL36, 4364.3 Da; RS22, 5095.8 Da; RL34, 5380.4 Da; RL33meth, 6254.4 Da; RL32, 6315 Da; RL29, 7273.5 Da; RS19, 10299.1 Da; RNase A, 13682.2 Da; myoglobin, 16952.5 Da.

Spectrum generation and data analysis

For automated data analysis, raw spectra were processed using the MALDI Biotyper 2.0 software (Bruker Daltonics, Leipzig, Germany) at default settings. The software performs normalization, smoothing, baseline subtraction, and peak picking, creating a list of the most significant peaks of the spectrum (m/z values with a given intensity, with the threshold set to a minimum of 1% of the highest peak and a maximum of 100 peaks). To identify unknown bacteria, each peak list generated was matched directly against reference libraries (3,476 species) using the integrated patterns matching algorithm of the Biotyper 2.0 software (Bruker Daltonics, GmbH, Germany). The unknown spectra were compared with a library of reference spectra based on a pattern recognition algorithm using peak position, peak intensity distributions and peak frequencies. Once a spectrum has been generated and captured by the software, the whole identification process was performed automatically, without any user intervention. MALDI-TOF MS identifications were classified using modified score values proposed by the manufacturer: a score value ≥2 indicated species identification; a score value between 1.7 and 1.9 indicated genus identification, and a score value <1.7 indicated no identification.

For reference library construction, 36 independent spectra were recorded for each bacterial isolate (three independent measurements at twelve different spots each). Manual/visual estimation of the mass spectra was performed using Flex Analysis 3.0 (Bruker Daltonics GmbH, Germany) performing smoothing and baseline substraction. Checking existence of flatlines, outliers or single spectra with remarkable peaks differing from the other spectra was done, taking into account that mass deviation within the spectra set shall not be more than 500 ppm. Finally, 20 spectra were selected, removing questionable spectra from the collection. To create peak lists of the spectra, the BioTyper software was used as described above. The 20 independent peak lists of a strain were used for automated “main spectrum” generation with default settings of the BioTyper software. Thereby, for each library entry a reference peak list (main spectrum) which contains information about average masses, average intensities, and relative abundances in the 20 measurements for all characteristic peaks of a given strain was created, so a main spectrum displayed the most reproducible peaks typical for a certain bacterial strain.

Cluster analysis was performed based on comparison of strain-specific 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 were compared with those obtained after rrs, recA, atpD and nodC gene sequence analyses. In this work we obtained some sequences of these genes that are absent in databases according to Rivas et al. [19] for rrs gene, Gaunt et al. [20] for recA and atpD genes and Laguerre et al. [21] for nodC gene. The sequences were aligned using the Clustal W software [22]. The distances were calculated according to Kimura's two-parameter model [23]. Phylogenetic trees were inferred using the neighbour-joining method [24] and the MEGA 4.0 package [25].

Results and Discussion

Database setting

In Biotyper 2.0 database only three species of genus Rhizobium are included and none of genus Ensifer or Shinella. Therefore a database extension in order to include the species currently described in these genera is necessary before applying MALDI-TOF MS to the identification of rhizobial isolates.

Owing to the fact that in Biotyper 2.0 database the type strains of three species of genus Rhizobium are already included, R. tropici DSM 11418T, R. rubi DSM 6772T and R. radiobacter DSM 30147T, we verified the reproducibility of MALDI-TOF MS using the type strains of these species that were cultivated in two different media (YMA and TY) and incubated at 24 and 48 h.

The results obtained showed that the analysed strains matched with high score values (higher than 2.5) with each corresponding type strain already present in Biotyper 2.0 database when TY medium and 24 h incubation were used (Table 3). Lower score values were found with YMA medium incubated at 24 h and only R. rubi ATCC 13335T and R. radiobacter ATCC 19358T were correctly identified with score values higher than 2 (Table 3). This was probably due to the production of higher amounts of exopolysaccharide in YMA medium which makes the sample preparation difficult. After an incubation time of 48 h the score values were lower when both TY and YMA media were used and only R. rubi ATCC 13335T and R. radiobacter ATCC 19358T were correctly identified using YMA medium. Therefore best results for rhizobial species were obtained with TY medium and 24 h incubation, in spite of previous studies that have demonstrated high reproducibility of MALDI-TOF MS analysis in different culture media and growth phases [14], [26], [27].

Table 3

Comparison of identification results by MALDI-TOF MS with different conditions.

Media and incubation times (extraction method)
Strain	Incubated in YMA during 24 h (best match)	Score value*	Incubated in TY during 24 h (best match)	Score value*
Rhizobium radiobacter ATCC 19358T	Rhizobium radiobacter DSM 30147T	2.388	Rhizobium radiobacter DSM 30147T	2.566
Rhizobium tropici CIAT 899T	Rhizobium tropici DSM 11418T	1.897	Rhizobium tropici DSM 11418T	2.582
Rhizobium rubi ATCC 13335T	Rhizobium rubi DSM 6772T	2.500	Rhizobium rubi DSM 6772T	2.505

*score value ≥2 indicates species identification; 1.7

Before the extension of Biotyper 2.0 database we also checked the suitability of MALDI-TOF MS system to differentiate the spectra of representative species from the three genera currently accepted in Family Rhizobiaceae.

Firstly we compared the spectra of the type strains from the type species of the three genera currently included in family Rhizobiaceae. The results obtained showed that the spectra of Rhizobium leguminosarum USDA 2370T, Ensifer adhaerens LMG 20216T and Shinella granuli DSM18401T were clearly distinguishable since there were not common peaks among their spectra (figure 1).

Figure 1

MALDI-TOF MS spectra of whole-cell extracts obtained from the type strains of two close and one divergent species from each genus analysed in this study: (A) Rhizobium, (B) Ensifer and (C) Shinella.

Subsequently, we analyzed the spectra of two phylogenetically close and one phylogenetically divergent species from each genus. We selected from genus Rhizobium the close species R. leguminosarum (type species of genus Rhizobium) and R. pisi as well as the species R. cellulosilyticum, phylogenetically distant from them. From genus Ensifer we chose the close species E. meliloti and E. medicae and the species E. adhaerens, which is the type species of genus Ensifer and it is phylogenetically distant from the other two species. Finally, from genus Shinella we chose the close species S. granuli, type species of genus Shinella, and S. kummerowiae and the phylogenetically distant S. fusca.

All these spectra were quite different with almost any common peaks among those of species belonging to different genera as we previously observed for the type species of each genus. Within the same genus the spectra of close species were more similar than those from divergent species. For example, considering the mass tolerance ±2 m/z for each peak as we have previously described [13], in genus Rhizobium, R. leguminosarum USDA 2370T and R. pisi DSM 30132T shared peaks at 3126, 4689, 6773, 7298 and 9380 Da that are not in R. cellulosilyticum ALAB2T (figure 1A). In genus Ensifer, peaks at 2502, 2605, 4652, 5005, 5211 and 9304 Da were present in E. meliloti ATCC 9930T and E. medicae USDA 1037T and not in E. adherens LMG 20216T, although there are two common peaks in the three species compared (3741 and 7484 Da). (figure 1B). In genus Shinella, we found peaks at 5006, 4613 and 2504 Da in S. granuli and S. kummerowiae that were not present in S. fusca and, although S. fusca shared few peaks with S. granuli (3615, 3723, 7229, 7444 Da), this phylogenetically distant species had many specific peaks that were not in the other two species (2070, 2237, 2326, 2497, 3254, 5096, 6351, 6507 Da) (figure 1C).

These results showed that the spectra of both phylogenetically close and distant species from the same genus, as well as those of species of different genera within family Rhizobiaceae can be differentiated by MALDI-TOF MS. Therefore we extended the database MALDI BioTyper 2.0 with 56 type strains of species from genera Rhizobium, Ensifer and Shinella belonging to Family Rhizobiaceae (Table 1).

Comparison between MALDI-TOF MS and phylogenetic analyses

To compare the data obtained by MALDI-TOF MS analysis with those based on gene sequence analysis (figures 2, 3 and 4), a cluster analysis was performed based on a correlation matrix using the integrated tools of the MALDI Biotyper 2.0 software package. Figure 5 showed that the genus Rhizobium was divided into several clusters whose distribution basically coincided with that observed after rrs, recA and atpD gene analyses. The results evidenced that some reclassifications performed within genus Rhizobium are correct as occurs in the case of the former species Agrobacterium tumefaciens reclassified into A. radiobacter [28]. MALDI-TOF MS results confirmed that they are the same species since their type strains held in different collections matched with score values higher than 2 (Table 2A and 2B). These results are congruent with those obtained from recA and atpD gene analyses since these strains presented nearly identical sequences (figures 3 and 4). After reclassification of the complete genus Agrobacterium into Rhizobium, the current valid name for these species is Rhizobium radiobacter [3].

Figure 2

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. Accesion numbers from Genbank are given in brackets.

Figure 3

Neighbour-joining phylogenetic tree based on recA gene sequences (about 520 nt) showing the position of species included in this study.

Bootstrap values calculated for 1000 replications are indicated. Bar, 2 nt substitution per 100 nt. Accesion numbers from Genbank are given in brackets.

Figure 4

Neighbour-joining phylogenetic tree based on atpD gene sequences (about 500 nt) showing the position of species included in this study.

Bootstrap values calculated for 1000 replications are indicated. Bar, 2 nt substitution per 100 nt. Accesion numbers from Genbank are given in brackets.

Figure 5

Cluster analysis of MALDI-TOF MS spectra of different species and strains from the genus Rhizobium.

Distance is displayed in relative units.

MALDI-TOF MS analysis also confirmed the R. trifolii ATCC 14480 reclassification into R. leguminosarum [29], since the strain ATCC 14480 matched with R. leguminosarum USDA 2370T with a score value higher than 2 (Table 2B), and Blastobacter aggregatus DSM 1111T into R. aggregatum [30] since strain DSM 1111T clustered with species of genus Rhizobium (Figure 5).

On the contrary, some species of genus Rhizobium were erroneously reclassified. For example, R. phaseoli type strain was reclassified into R. leguminosarum [31]. Later the biovar phaseoli type I of this species was reclassified into R. etli [32], so it was not clear the location of the R. phaseoli type strain. A revision based on rrs, recA and atpD analysis showed that R. phaseoli is a valid species distinguishable from both R. leguminosarum and R. etli [29]. The results of the MALDI-TOF MS confirmed these results since R. phaseoli ATCC 14482T matched with R. etli CFN42T with score values lower than 2 (Table 2C).

Moreover, the MALDI-TOF MS cluster analysis showed, in agreement with rrs, recA and atpD gene analyses, that some current Rhizobium species are indistinguishable (figures 2, 3, 4 and 5). For example, the type strains of R. mongolense, R. loessense and R. yanglingense matched with R. gallicum R602spT with score values higher than 2 (Table 2D). In addition, R. indigoferae CCBAU 71042T matched with R. leguminosarum USDA 2370T with a score value of 2.219 and R. fabae LMG 23997T matched with R. pisi DSM 30132T with a score value of 2.258 (Table 2D). Therefore the taxonomic status of all these species should be revised according to the current rules of bacterial taxonomy.

The genera Shinella and Ensifer MALDI-TOF cluster analysis was performed together (figure 6) since they are closely related on the basis of recA and atpD gene analyses (see figures 3 and 4). This closeness was confirmed after MALDI-TOF cluster analysis although the distribution of Shinella species was slightly different (figure 6). The species S. yambaruensis was the closest related species to S. granuli on the basis of MALDI-TOF MS analysis, whereas these two species were distant according to their rrs gene sequences (figure 2). However, S. yambaruensis DSM 18801T matched with S. granuli DSM 18401T with a score value lower than 2 corresponding to different species from the same genus.

Figure 6

Cluster analysis of MALDI-TOF MS spectra of different species and strains from the genera Ensifer and Shinella.

Distance is displayed in relative units.

The distribution of species in the genus Ensifer was coherent with those found after rrs analysis with E. medicae and E. meliloti forming the same group, E. morelense close to E. adhaerens and E. americanum (a not yet validated species) close to E. fredii (figure 6).

In the genus Ensifer also MALDI-TOF MS analysis confirmed some reclassifications as that of species E. xinjiangense into E. fredii [33] since the former type strains E. xinjiangense LMG 17930 and CECT 4657 matched with E. fredii LMG 6217T with score values of 2.413 and 2.151, respectively (Table 2B). Also was confirmed the reclassification of the strain Rhizobium sp. Br816 as Ensifer sp. [34], [35] since it clustered with E. americanum (figure 6). However, in agreement with rrs, recA and atpD gene analyses (figures 2, 3 and 4), strain Br816 does not belong to this species since it matched with E. americanum DSM 15007T with score values lower than 2 (Table 2G).

However, other reclassifications were not correct as occurs with E. morelense reclassified into E. adhaerens [36] since E. morelense Lc04T matched with E. adhaerens LMG 20216T with a score value of only 1.245 (Table 2C) in agreement with rrs, recA and atpD gene analyses (figures 2, 3 and 4).

In the genus Ensifer, also some species were indistinguishable, for example, E. kummerowiae CCBAU 71714T matched with E. meliloti ATCC 9930T with a score value of 2.261 suggesting that they belong to the same species (Table 2D). Since this result coincides with the analysis of rrs, recA and atpD genes, the taxonomic status of E. kummerowiae should be revised.

All these findings showed that MALDI-TOF MS results are comparable to those obtained after the phylogenetic analysis of core genes from members of family Rhizobiaceae including that of rrs gene in which is currently based the classification within this family [1]. These results are in agreement with those previously reported for other bacterial groups [37] and therefore we analysed the potential of MALDI-TOF MS for identification of fast-growing rhizobia isolates.

Identification of rhizobial strains by MALDI-TOF MS

To prove the suitability of the extended MALDI Biotyper 2.0 database for routine identification and discrimination of fast-growing rhizobial species we analysed several strains previously identified by rrs and housekeeping gene sequencing belonging to different species and genera of family Rhizobiaceae (Table 2F and 2G).

The species R. leguminosarum contains some strains with identical rrs gene and divergent recA and atpD genes [29], [38], [39]. For example, the strains RPVF18, RVS11 and ATCC 14480 have housekeeping genes closely related to the type strain of this species USDA 2370T and other strains have phylogenetically distant ones, such as RPVR31, CVIII4 and RTP05 (figures 3 and 4). Although all these strains clustered with R. leguminosarum USDA 2370T after MALDI-TOF MS cluster analysis (figure 5), only when the housekeeping genes were almost identical the score values were higher than 2 with respect to R. leguminosarum USDA 2370T (Table 2F). These results were congruent with those from recA and atpD gene analyses showing that, in spite of the complete identity of rrs gene, R. leguminosarum could contain several subspecies perfectly distinguishable by MALDI-TOF MS analysis as it has already been observed in other bacterial species [26], [40], [41].

Although housekeeping gene sequences present higher variability than those of rrs genes, the ITS fragment located between 16S and 23S gene in fast growing rhizobia is the most hypervariable chromosomic region and has been proposed as a tool for species differentiation [42]. However, MALDI-TOF MS showed that strains with housekeeping genes nearly identical but different ITS sequences belong to the same species. For example, the strains RPA12 and RPA02 shared only 73% identity in their ITS sequences with respect to R. giardinii H152T suggesting they can represent different species [43]. However, in agreement with rrs, recA and atpD gene analyses, MALDI-TOF MS showed that they belong to R. giardinii since they matched with the type strain of this species with score values higher than 2 (Table 2F).

The same was found for the genus Ensifer strains RTM17 and GVPV12 that matched with E. meliloti ATCC 9930T with score values higher than 2 (Table 2F) in spite of the differences in the ITS region (95% identity) [44] and in agreement with the results of the housekeeping gene analyses (figures 3 and 4).

Intraspecific variability in species of family Rhizobiaceae could be also due to the presence of large plasmids codifying for symbiotic or virulence factors. Nodulating species may contain different biovars that carry different nodC genes [21], [38], [44], [45] and pathogenic species contain strains that carry plasmids involved in tumour (pTi) or hairy roots induction (pRi) [46], [47]. Therefore we analysed strains with different combinations of chromosomal backgrounds and symbiotic or virulence plasmids by MALDI-TOF MS.

For example, within genus Rhizobium, R. leguminosarum contains three biovars: viciae, trifolii and phaseoli [31], [38], perfectly distinguishable on the basis of their nodC gene sequences (figure 7). However MALDI-TOF MS analysis showed that strains with housekeeping genes close to R. leguminosarum USDA 2370T (RVS11, RPVF18 and ATCC 14480) [29], [38], [39] matched with score values higher than 2 with this strain with independence to the biovar they belong to (figure 7). Likewise, the strains FL27 from biovar gallicum [45] and PhD12 from biovar phaseoli [21] carrying divergent nodC genes (figure 7) matched with R. gallicum R602spT with score values higher than 2 (Table 2F). The same was found in R. lusitanum whose strains P1-7T and P3-13 have phylogenetically distant nodC genes (figure 7) but they matched with a score value of 2.314 (Table 2F).

Figure 7

Neighbour-joining phylogenetic tree based on nodC gene sequences (about 860 nt) showing the position of representative strains from each biovar analysed in this study.

Bootstrap values calculated for 1000 replications are indicated. Bar, 5 nt substitution per 100 nt. Accesion numbers from Genbank are given in brackets.

In genus Ensifer, E. meliloti also contains different biovars with divergent nodC genes (figure 7). However, the strains RPA13 and RTM17 from biovar meliloti and the strain GVPV12 from biovar mediterranense [44] were matched with E. meliloti ATCC 9930T with score values higher than 2 by MALDI-TOF MS (Table 2F).

Conversely, strains from the same biovar but divergent housekeeping genes were perfectly distinguished by MALDI-TOF MS in genus Rhizobium. For example, the strain CVIII14 matched with a score value lower than 2 with R. leguminosarum USDA 2370T, although both strains belong to the biovar viciae (Table 2G). To this biovar also belongs R. pisi DSM 30132T that was correctly distinguished by MALDI-TOF MS from R. leguminosarum USDA 2370T (figure 7 and Table 2C). Moreover, strains CFN299 and CIAT 899T, whose rrs and housekeeping genes showed they belong to different species [48], matched with score values lower than 2 (Table 2F) in spite of the complete identity of their nodC genes (figure 7).

In genus Ensifer, E. meliloti RPA13 and RTM17 and E. medicae RMP01, RMP05, RPA08, RPA11 and RPA20 belong to the same biovar (meliloti) [49]. However, in agreement with rrs and housekeeping gene analyses (figures 2, 3 and 4), the strains of these both species were clearly distinguished by MALDI-TOF MS (Table 2F).

Finally, two species of genus Rhizobium, R. rhizogenes and R. radiobacter, contain non-pathogenic strains, tumourigenic strains and hairy roots inducing strains (Table 2F). In both cases their strains were correctly identified by MALDI-TOF MS in agreement with the rrs and housekeeping gene analyses (figures 2, 3 and 4) in spite of the plasmidic content. In this way the non-pathogenic strain K84 [46], the tumourigenic strain 163C and the root inducing strain IAM 13571, matched with R. rhizogenes ATCC 11325T, a root inducing strain, with high score values (2.185, 2.195 and 2.158, respectively). The tumourigenic strain ATCC 23308 (type strain of the former species A. tumefaciens) and the root inducing strain ATCC 13332 (erroneously named R. rhizogenes) also matched with the non-pathogenic strain R. radiobacter DSM 30147T with score values higher than 2 (Table 2F).

Conversely, although the pTi plasmids of the tumourigenic strains 163C and C58 are closely related [47], they belong to different species according to MALDI-TOF MS results (Table 2G) in agreement with the rrs and housekeeping gene analyses (figures 2, 3 and 4).

All these results showed that plasmids carried by fast growing rhizobial strains do not affect their identification by MALDI-TOF MS since strains of the same species carrying very different plasmids and strains from different species carrying similar plasmids were correctly identified by MALDI-TOF MS.

In conclusion, the results presented in this work clearly showed that MALDI-TOF MS is a reliable and rapid method for rhizobial identification comparable to housekeeping gene sequence analysis since it is able to discriminate between strains with identical rrs gene sequences but divergent recA and atpD. This feature represents important advantages based on the rapidity and cost per sample with respect to gene sequencing. With this methodology, if the databases include all rhizobial species described in each moment, it will be possible to identify all isolates belonging to species already described as well as the detection of new species. Therefore, MALDI-TOF MS open a new and very useful way for diversity and ecological studies applicable to analysis of large populations of isolates allowing the differentiation of strains, species and genera of fast-growing rhizobia with an effectiveness of 100% in the identification at species level.