Comparative symbiotic performance of native rhizobia of the Flooding

Antonie van Leeuwenhoek (2011) 99:371–379 DOI 10.1007/s10482-010-9502-9

ORIGINAL PAPER

Comparative symbiotic performance of native rhizobia of the Flooding Pampa and strains currently used for inoculating Lotus tenuis in this region Analıá Ine´s Sannazzaro • Verońica Mariel Bergottini • Rosalıá Cristina Paz • Luis Nazareno Castagno • Ana Bernardina Meneńdez • Oscar Adolfo Ruiz • Fernando Luis Pieckenstain • Marıá Julia Estrella

Received: 14 April 2010 / Accepted: 23 August 2010 / Published online: 2 September 2010 Ó Springer Science+Business Media B.V. 2010

Abstract The Flooding Pampa (FP) is the most important area for cattle breeding in Argentina. In this region, persistence and yield of typical forage legumes are strongly limited by soil salinity and alkalinity, which affect around 30% of the total area. Instead, naturalized Lotus tenuis is the main forage legume in this region. Rhizobial strains currently used for inoculating L. tenuis in the FP are exotic or native from non-saline soils of this region, their taxonomic identity being unknown. Assuming that rhizobia native from the most restrictive environments are well adapted to adverse conditions, the use of such isolates could improve the productivity of L. tenuis in the FP. Hence, the goal of this study was to evaluate the symbiotic efficiency of selected L. tenuis rhizobia native from the FP, as compared with strains currently used for field inoculation of this legume. Under non-stressing conditions, the symbiotic performance of native strains of FP exceeded those

Analıá Ine´s Sannazzaro and Verońica Mariel Bergottini contributed equally to this study. A. I. Sannazzaro V. M. Bergottini R. C. Paz L. N. Castagno A. B. Meneńdez O. A. Ruiz F. L. Pieckenstain M. J. Estrella (&) Unidad de Biotecnologıá 1, Instituto de Investigaciones Biotecnolo´gicas- Instituto Tecnolo´gico de Chascomu´s (IIB-INTECH), UNSAM-CONICET, Camino de Circunvalacioń Km 6, CC 164 (7130), Chascomu´s, Argentina e-mail: [email protected]

ones currently used for L. tenuis. Moreover, the symbiotic performance of the native strain ML103 was considerably high under salt stress, compared with strains currently used as inoculants. Analysis of 16S rRNA gene sequencing revealed that unclassified rhizobia currently used for field inoculation of L. tenuis and native strains grouped with the genus Mesorhizobium. As a whole, results obtained demonstrate that soils of the FP are a source of efficient and diverse rhizobia that could be used as a sustainable agronomic tool to formulate inoculants that improve forage yield of L. tenuis in this region. Keywords Symbiotic performance Lotus tenuis Soil salinity Rhizobia

Introduction The Flooding Pampa (FP) is located in the east-center of Buenos Aires Province, in Argentina. In general, soils in this region are poorly drained, with low nutrient contents, high levels of sodic salts, and alkaline pH (Lavado and Alconada 1994; Salazar Lea Plaza and Moscatelli 1989). In addition, a high degree of soil heterogeneity is typical of this region, and patches with different physico-chemical properties such as salt content can be found in relatively small areas. The features of soils in the FP significantly decrease the persistence and yield of traditional legumes such as red and white clover (Trifolium

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pratensis and T. repens) and lucerne (Medicago sativa). In contrast, narrow-leaf birdsfoot trefoil (Lotus tenuis), a warm-season, perennial species from Europe that was introduced in the FP only a few decades ago (c. 1930), is the forage legume of choice for beef and dairy cattle production (Montes 1988; Langer 1990). In spite to adaptation, this plant species displays a low relative growth rate during the seedling stage, a critical phase in its development (Montes 1988). Like most legumes, L. tenuis has the ability to establish mutualistic symbiotic relationships with soil N-fixing bacteria collectively known as rhizobia, thereby rendering this plant more competitive than non-legumes in soils with low N content. Taking into account the relatively recent naturalization of this legume in the FP, it is not surprising that the selection of efficient strains and the development of high quality inoculants for L. tenuis is still incipient. However, in many cases L. tenuis seeds are inoculated before sowing, by using rhizobial formulations based on strains whose taxonomic identity has not been established. These strains were originally isolated from soils different to those of the FP and selected for their ability to symbiotically fix N in environments not necessarily similar to those typical of this region. It is well known that in the soil the strains used for inoculation are subjected to the effects of numerous biotic and abiotic factors (Bushby 1982), many of which impose stressful conditions that decrease the symbiotic efficiency (Diatloff 1977). In the particular case of the FP, the adaptation and survival of rhizobia, as well as their symbiotic efficiency can be affected by soil salinity (Quadrelli et al. 1997). Thus, when efficient strains are introduced in environments different to those from which they were isolated, they can be outcompeted by welladapted native rhizobia (Fabiano and Arias 1991). In this way, it is reasonable to hypothesize that rhizobial isolates from the FP should be better adapted to the soil conditions of this region than introduced strains. Thus, the use of inoculants based on efficient native strains could be an affordable and sustainable resource to improve the yield of L. tenuis in this region. Hence, the goal of this study was to evaluate if rhizobia with a higher symbiotic performance than those currently used for inoculation of L. tenuis can be isolated from soils in the FP, and to further

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Antonie van Leeuwenhoek (2011) 99:371–379

characterize these strains. Infective strains from a previously characterized collection of rhizobia from the FP were available for this purpose (Estrella et al. 2009), but new strains were obtained in this study from constrained saline-alkaline soils of this region. Thus, the symbiotic performance of two newly obtained highly infective strains, along with three already available strains, was compared with bacteria currently used for field inoculation of L. tenuis in the FP. The taxonomic status of the new strains and the so far unidentified rhizobia used for inoculation of L. tenuis was established.

Materials and methods Rhizobia strains and culture conditions Rhizobia were isolated from nodules of L. tenuis plants naturally growing in alkaline-saline lowlands of the FP in Chascomu´s, Province of Buenos Aires, Argentina. Nodules were separated from the root, washed in distilled water, and then surface disinfected with 70% ethanol for 2 min and 2% sodium hypochlorite for 5 min (Fulchieri et al. 2001). The nodules were crushed and exudates were streaked onto yeast extract-mannitol (YEM) agar medium (Vincent 1970). Pure cultures were obtained by repeated subculturing steps. In addition, the following strains were used in this study: BSA151, BD68 and ML103 which belong to a previously characterized collection from the FP (Estrella et al. 2009) and were isolated from different soil types (saline lowlands, non-saline lowlands and transitional plains, respectively); and B733, LL32 and NZP2213 which are currently used for the formulation of inoculants for L. tenuis. B733 was isolated from a Natraqualf alkaline hydromorphic soil in Buenos Aires Province and kindly provided by Ing. Ana Quadrelli, Laboratorio de Microbiologıá de la Unidad Integrada UNMDP-INTA, Balcarce; LL32 was isolated from an Arguidol soil in Tucumań Province, Argentina and kindly given by Ing. Alejandro Perticari from Instituto de Microbiologıá y Zoologıá Agrıćola, INTA, Castelar; Mesorhizobium loti NZP2213 strain native from New Zealand is the type species for L. tenuis (Laguerre et al. 1997), and was kindly provided by Dr. Juan Sanjuań from Departamento de Microbiologıá del Suelo y Sistemas


Simbio´ticos, Estacioń Experimental del Zaidıń, Spain. YEM medium was routinely used for rhizobial isolation, purification, and culture. All strains were stored at -80°C in the same medium with 20% (v/v) glycerol.

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Sequences of the 16S rRNA genes obtained in this study have been deposited in the GenBank database under the following accession numbers: BSA1.6: HQ013312, BSA2.8: HQ013313, B733: HQ013314 and LL32: HQ013315. Phylogenetic analysis

Extraction of DNA from rhizobia isolates for PCR amplifications All rhizobial strains were grown for two days at 28°C and a loopful of young cells from each isolate was suspended in 0.1 ml of MilliQ water. The cell suspensions were centrifuged at 13,500 9 g for 5 min and the supernatant was removed. The pellet was resuspended in 0.1 ml of sterile MilliQ water and cells were lysed by boiling for 15 min, centrifuged at 13,500 9 g for 5 min and the supernatant transferred to a clean tube and used as a source of DNA for subsequent PCR amplifications. PCR fingerprinting of rhizobial DNA with BOX primers BOX-PCR (Versalovic et al. 1994) was used to assess the genetic diversity of the isolates. PCR assays were performed using the universal BOXAIR1 primer (5-CT ACggCAAggCgACgCTgACg-3; Versalovic et al. 1994) synthesized by Ruralex Fagos, Argentina. Sequencing of 16 s rRNA fragments Nearly full-length 16S rRNA genes were amplified from native isolates (BSA16 and BSA28) and strains used for the formulation of inoculants for L. tenuis (B733 and LL32) using primers 41f (5-gCTCAAgATTgAACgC TggCg-3) and 1488r (5-ggTTACCTTgTTACgACTTCACC-3) as previously described (Estrella et al. 2009). The PCR products amplified were purified using the GFX kit (GE Healthcare, Little Chalfont Buckinghamshire, UK) and were sequenced by the Applied Biosystems ABI 377 sequencer (DNA Sequencing Service, Instituto de Investigaciones Biotecnolo´gicas, Universidad Nacional de General San Martıń -UNSAM-, Argentina). The sequences obtained were compared with the sequences of reference strains deposited in the GenBank (Table 1) by using the BLASTN program (http://www.ncbi.nlm.nih.gov/blast).

Sequence alignment was performed with the ClustalW software from the EMBL server (http://www.ebi.ac. uk/). Aligned sequences were analyzed using the MEGA software, version 4.0 (Tamura et al. 2007). Phylogenetic analyses of the 16S rRNA sequences were performed by the UPGMA method (Sneath and Sokal 1973). The phylogenetic distances were computed by the p-distance method and calculated based on the proportion of different nucleotides (p-distance), which was obtained by dividing the number of nucleotide differences by the total number of nucleotides compared (Nei and Kumar 2000). Statistical support for tree nodes was evaluated by bootstrap analysis (Felsenstein 1985). Free-living salt tolerance Serial 10-fold dilutions of cell suspensions for each isolate were spotted on YEM plates supplemented with increasing NaCl concentrations (0, 50, 100, 150 and 200 mM). Plates were incubated for 2–4 days at 28°C. For each strain, the minimal dilution at which isolated colonies were observed within the inoculation spot in the control plate (0 mM NaCl) was identified. Afterwards, growth was evaluated for the same dilution in NaCl-amended plates and the maximal NaCl concentration that allowed bacterial growth was considered as the tolerance level. Plant material, growth conditions and nodulation assays L. tenuis cv. Esmeralda (Gentos, SA) seeds were scarified and surface disinfected as previously described (Estrella et al. 2009) and allowed to germinate in the dark for 24–48 h at 25°C. Plants were grown in a growth chamber with a 16/8 h photoperiod at 25/21°C (day/night) and 55/75 ± 5% RH. Light intensity (200 lmol m-2 s-1) was provided by incandescent and cool white fluorescent lamps.

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Table 1 Reference strains used for analysis of amplified 16S rRNA genes Strain

Species

Host plant of origin

Accession number (16S rRNA)

USDA 110 LMG6133

Bradyrhizobium japonicum

Glycine max

D13430.1

Ensifer meliloti

Medicago sativa

X67222.2

USDA 205 NGR234

Ensifer fredii Ensifer fredii

Glycine max Lotus purpureus

AY260147.1 AY260149.1

LMG9517

Rhizobium tropici

Phaseolus vulgaris

X67234.2

CIAT899

Rhizobium tropici

Phaseolus vulgaris

U89832.1

IFO15247

Rhizobium tropici

Phaseolus vulgaris

D11344.1

USDA2671

Rhizobium leguminosarum bv phaseoli

Phaseolus vulgaris

U29388.1

CFN 42

Rhizobium etli

Phaseolus vulgaris

U28916.1

DMS 30105

Agrobacterium tumefaciens

–

M11223.1

UPM-Ca7

Mesorhizobium ciceri

Cicer arietinum

U07934.1

A-1Bs

Mesorhizobium tianshanense

Glycyrhiza pallidiflora

AF041447.1

ORS1096

Mesorhizobium plurifarium

Acacia tortilis

AJ295079.1

UPM-Ca36

Mesorhizobium mediterraneum

Cicer arietinum

L38825.1

LMG PR5

Mesorhizobium chacoense

Prosopis alba

AJ278249.1

ACCC 19665

Mesorhizobium amorphae

Amorpha fruticosa

AF041442.1

IFO 15243

Mesorhizobium huakuii

Astragalus sinicus

D13431.1

LMG 6123

Mesorhizobium loti

Lotus divaricatus

Y14159.1

MAFF303099 NZP2213

Mesorhizobium loti Mesorhizobium loti

Lotus corniculatus Lotus tenuis

BA000012 D14514.1

BD68

Mesorhizobium sp

Lotus tenuis

EU748908

BSA151

Mesorhizobium sp

Lotus tenuis

EU748911

ML103

Mesorhizobium sp

Lotus tenuis

EU748910

In order to evaluate the infectivity of isolates, 10 seedlings were aseptically transferred to square Petri dishes containing solid Rigaud-Puppo medium (Rigaud and Puppo 1975). Seedlings were inoculated with exponentially growing rhizobial cultures (108 cells per seedling) and the time elapsed until the first nodule appearance (number of days post-inoculation, d.p.i) was recorded. Non-inoculated seedlings were used as negative controls. Nodulation assays were performed in order to evaluate the symbiotic performance of the strains. L. tenuis seedlings were transferred to Leonard jars with sand-perlite 1:1 (3–5 seedlings per jar) and inoculated with each of the isolates (108 cells per seedling) in a completely randomized design (four replicate jars per treatment). Non-inoculated plants irrigated with 20 ppm or without N (as NH4NO3) in the solution were used as growth controls. Salt stress was imposed by irrigating plants with N-free nutrient

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solution (Rigaud and Puppo 1975) with 100 mM NaCl. This NaCl concentration was selected in a previous experiment, taking into account that it was found to impose a non-lethal stress that partially reduces plant growth. Plants irrigated with N-free nutrient solution without NaCl were used as controls. Plants were harvested 30 d.p.i. and shoot and nodule dry weights were determined. Statistical analyses Data were analyzed by two-way analysis of variance (Two-way ANOVA), with salt and N source (exogenously added or derived from biological fixation) as factors, followed by all pairwise multiple comparisons (post-hoc testing), using the HolmSidak method. Pearson’s correlation coefficients between shoot and nodule dry weights were calculated.


Results Selection of isolates from L. tenuis growing in saline-alkaline lowlands from the Flooding Pampa Overall, 149 rhizobial strains were isolated from field-collected nodules of L. tenuis. In order to identify non-redundant strains, BOX-PCR fingerprinting was performed for each isolate. Among all the isolates obtained, 137 non-redundant BOX profiles were observed (Fig. 1). Strains with non-redundant BOX profiles were evaluated in infectivity assays and two of them (BSA2.8 and BSA1.6), showing the highest infectivity levels (data not shown), were chosen for all subsequent studies.

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(ML103, BD68 and BSA151) isolated and characterized in a previous study (Estrella et al. 2009), and also strains usually used for L. tenuis inoculation (B733, LL32 and NZP2213) were tested for symbiotic efficiency under salt-stress and non-stressing conditions, by measuring shoot dry weight 30 d.p.i. Under non-saline conditions, plants inoculated with four out of five isolates native from the FP (BSA2.8, BSA1.6, ML103 and BD68) exhibited the highest shoot dry weights relative to non-inoculated plants grown without N (corresponding to 100% in Fig. 2). Moreover, relative shoot dry weight of plants inoculated with strain BSA2.8 was even higher than non-inoculated plants irrigated with 20 ppm N. Interestingly, relative shoot biomass of plants inoculated with BSA2.8, BSA1.6 and ML103 was higher than plants inoculated with all the strains usually used

Fig. 1 BOX–PCR profiles of L. tenuis rhizobia native from alkaline-saline lowlands of the Flooding Pampa examples of BOX profiles obtained for some of the bacterial strains used in this study, after analyzing PCR products by agarose gel electrophoresis. Profile analysis was carried out on a range of fragments comprised between 2027 bp and 125 bp, by using Lambda DNA/EcoRI ? Hind III as a molecular marker (lane MM). As an example, some isolates that have identical BOX profiles are indicated with arrows

b

b bc b

N

ab

a

ab

NZP2213

d ab

cd

cd

cd

LL32

ab

b

ML103

ab

bc a

B733

***

BSA151

***

BD68

a

BSA1.6

750 700 650 600 550 500 450 400 350 300 250 200 150 100 50 0

BSA2.8

Native strains BSA2.8 and BSA1.6, isolated in this study and selected on the basis of their high infectivity, along with three efficient native strains

% relative shoot dry weight

Symbiotic performance

Fig. 2 Symbiotic efficiency of rhizobia isolated from soils of the Flooding Pampa and strains currently used for inoculation of L. tenuis, under saline and control conditions. Plants were inoculated with selected strains from the Flooding Pampa (see Results). Salt stress was imposed by irrigating plants with Nfree nutrient solution (Rigaud and Puppo 1975) with 100 mM NaCl. Plants irrigated with the same solution without NaCl were used as stress controls. Non-inoculated plants irrigated with 20 ppm nitrogen (N) or without this nutrient were used as growth controls. Symbiotic performance of strains was assessed 30 dpi and expressed as % relative shoot dry weight (shoot dry weight of inoculated plants 9 100/shoot dry weight of non-inoculated plants without N). White and gray columns represent the results obtained under control and saline conditions, respectively. Statistically significant differences in symbiotic efficiency between different strains under control or saline conditions (P B 0.05) are indicated by different letters. Comparisons are only valid within a given condition (control or saline). Statistical differences in symbiotic efficiency for each strain under control and saline conditions are shown as: *** P B 0.001

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weight (r = 0.9464), both under control (r = 0.8026) and stress conditions (r = 0.9347), showing that in this system, any of these parameters could equally serve to estimate symbiotic performance of the isolates.

for L. tenuis inoculation hereby tested (B733, LL32 and NZP2213). BSA151 was the only strain that showed a poor symbiotic efficiency (Fig. 2). Under saline conditions, plants inoculated with native strain ML103 exhibited higher relative shoot biomass than those inoculated with LL32, one of the strains currently used for L. tenuis inoculation (Fig. 2). On the other hand, plants inoculated with native isolates BSA2.8 and BSA1.6, which had a good performance in control conditions, were strongly affected by salinity (denoted by asterisks in Fig. 2). In any case, under stress conditions, growth of plants inoculated with these two strains was still similar to plants inoculated with any other strain evaluated in this study. As a general rule, a highly significant correlation was found between shoot dry weight and nodule dry

Taxonomic position of the selected strains by sequencing of amplified 16S rRNA PCR amplification of nearly full-length 16S rRNA gene and further sequence analysis were performed in order to identify the taxonomic position of isolates BSA2.8 and BSA1.6, as well as two strains of unknown taxonomic identity currently used for the formulation of inoculants (B733 and LL32). Figure 3 shows a phylogenetic tree based on the similarity of the 16SrRNA gene sequences of the 65 58 67

M sp BD68 LL32 BSA28 M loti MAFF303099 M loti LMG6123 M amorphae ACCC19665 M sp ML103 M huakuii IFO15243

86

M plurifarium ORS1096 M mediterraneum UMPCa36 93 95

M. sp BSA151 M tianshanense A1Bs M chacoense LMGPR5 BSA16

99 100

99 100

100

57

M ciceri UPMCa7 M loti NZP2213 B733 E fredii USDA205 E fredii NGR234 E meliloti LMG6133 A tumefaciens DMS30105 R leguminosarum bv phaseoli USDA267 R etli CFN42 R tropici subgroupIIA LMG9517

99 100

R tropici CIAT899 R tropici IFO15247 B japonicum USDA110

0.05

0.04

0.03

0.02

0.01

Fig. 3 16S rRNA gene phylogeny of rhizobial strains isolated from nodules of L. tenuis in alkaline-saline soils of Flooding Pampa and strains currently used for inoculation of L. tenuis. The tree was constructed from the nucleotide sequence data by using the UPGMA algorithm, and phylogenetic distances were

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0.00

calculated by the p-distance method. The numbers at branch points are the significant bootstrap values (expressed as percentages based on 1,000 replicates; only values greater than 50% are shown). The horizontal branch lines are proportional and indicate the p-distances


above mentioned rhizobia and reference strains (Table 1). On the basis of 16S rRNA gene sequences, isolates BSA2.8 and BSA1.6 and the above mentioned inoculant strains B733 and LL32 grouped with species belonging to the genus Mesorhizobium (Fig. 3). Strains LL32 and BSA2.8 were closely related with reference strains M. loti MAFF303099, M. loti LMG6123, and BD68 previously characterized by Estrella et al. (2009), showing 98–99% identity with sequences of this cluster (Fig. 3). On the contrary, 16SrRNA gene of strains BSA1.6 and B733 only shared 94–97% sequence identity with reference strains of the genus Mesorhizobium (Fig. 3). Free-living salt tolerance All native and foreign strains were able to grow in YEM with 100 mM NaCl, whereas BSA1.6, BSA2.8 and BSA151 strains, isolated from saline lowlands; ML103 strain isolated from a transitional plain and the reference strain NZP2213 tolerated NaCl concentrations up to 200 mM.

Discussion The general aim of this study was to evaluate and characterize L. tenuis rhizobial strains from the FP, particularly from the more restrictive environments, the alkaline-saline lowlands and to compare their efficiency with that of strains currently used for inoculation of this forage legume. Strains usually employed for inoculation of L. tenuis have been isolated from other geographic locations, different to the FP in terms of soil types and environmental conditions. It is well known that survival and symbiotic efficiency of the inoculant can be affected by the environmental conditions (Quadrelli et al. 1997). Consequently, inoculation may be unsuccessful in the presence of native rhizobial populations that outcompete the foreign rhizobia regardless of their efficiency (Lie et al. 1978; Denton et al. 2002). Native rhizobia are generally well adapted to the properties of the soils in which they interact with their symbiotic partners, thus being highly competitive in these environments (Jardim-Freire 1997; Denton et al. 2002). In this study, a previous collection of native rhizobia obtained from the FP (Estrella et al. 2009) was expanded by isolating and characterizing new

377

strains from the most constrained saline-alkaline lowlands of this region, where it would be advantageous to increase L. tenuis productivity. The existence of a high percentage of non-redundant profiles between the isolates indicated a high genetic diversity among them. These results are in agreement with previous studies, in different soils of the same area, where genetically diverse symbionts of L. tenuis were found (Estrella et al. 2009). In addition, a considerable variation in the infective ability of the isolates evaluated in this study was also evident. Among the novel isolates obtained, highly infective strains were selected for further taxonomic characterization together with the strains currently used for L. tenuis inoculation, whose taxonomic position was also unknown up to date. Although all of them grouped with species of the genus Mesorhizobium, the 16S rRNA gene sequences of strains B733 and BSA1.6 were rather distinct from those of reference strains tested, showing a great diversity at the species level. On the other hand LL32, B733, BSA28 and BSA16 were phyllogenetically more related to bacteria other than M. loti NZP 2213, the type species for L. tenuis and L. corniculatus symbionts (Sullivan et al. 1996; Saeki and Kouchi 2000). In this way, the phylogenetic analysis uncovered a taxonomic diversity among novel isolates and strains currently used for inoculation of L. tenuis, in agreement with previous findings (Estrella et al. 2009). Interestingly, the evaluation of the symbiotic performance of the different strains analysed in this study revealed that under control conditions, some native strains (BSA2.8, BSA1.6 and ML103) are more efficient than those currently used for inoculating L. tenuis in the field. This observation suggests that using native strains for the formulation of rhizobial inoculants for L. tenuis could significantly improve forage yield, at least in those soils of the FP in which salinity is not a major constrain. However, it should be kept in mind that soil salinity imposes a serious limitation for agriculture worldwide (Munns and Tester 2008) and particularly in a significant proportion of the soils of the FP, where high salt levels strongly affect the implantation and development of forage legumes. Thus, selection of salt tolerant legumes such as L. tenuis, able to symbiotically fix atmospheric nitrogen under these restrictive conditions could improve forage production. As occurred under control conditions, rhizobial strains currently used for

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inoculation of L. tenuis (B733, LL32 and NZP2213) exhibited a low symbiotic performance under salinity. Noteworthy, one of the native rhizobial strains analyzed in this study (ML103) showed a considerable high symbiotic performance under salt stress. Moreover, in terms of symbiotic performance under salinity, strain ML103 exceeded LL32, one of the strains currently used for inoculation. Thus, it is evident that native strains able to perform well both under restrictive and non-restrictive conditions can be obtained from soils in the FP, and could be tested as inoculants for L. tenuis cultivation in different types of soils of this region. Other native strains, such as BSA2.8 and BSA1.6 showed a strong decrease of their symbiotic performance under salinity, resembling the symbiotic performance of strains currently used for inoculating L. tenuis (B733, LL32 and NZP2213). Thus, this observation does not necessarily imply that the formulation of inoculants based on native strains such as BSA2.8 and BSA1.6 should be dismissed, since these strains showed a high symbiotic performance under control conditions. Nevertheless, further identification of strains that alike ML103 show a high performance both under control and salt-stress conditions would probably contribute to improve the versatility of inoculants. The Rhizobium-legume symbiosis, and particularly nodule formation, has been shown to be more sensitive to soil salinity than the microsymbiont itself (Zahran and Sprent 1986; El-Shinnawi et al. 1989; Velagaleti et al. 1990; Zahran 1991). In this way, salt concentrations that decrease symbiotic performance are usually different to those that affect growth of each symbiotic partner (Bordeleau and Pre´svost 1994; El-Hamdaoui et al. 2003). In agreement with these observations and in spite that all the isolates hereby studied tolerated 200 mM NaCl in free-living conditions, the symbiotic performance of two strains (BSA2.8 and BSA1.6) from saline soils was considerably impaired under 100 mM NaCl. In addition, a relation between free-living salt tolerance and the type of soil from which bacteria were isolated could not be established, in that strains with different tolerance levels were isolated either from saline and non-saline soils. The results of this study suggest that rhizobial adaptation to saline environments may not necessarily translate into a better symbiotic performance under saltstress conditions. Therefore, evaluation of the symbiotic performance of rhizobial strains under salt stress, rather

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than their salt tolerance in the free-living state, seems to be a good criterion for selecting rhizobial strains for inoculation of L. tenuis in saline soils. As a whole, the results hereby presented demonstrate that soils of the FP are a source of efficient rhizobial strains that could be used as an ecologically sustainable agronomic tool to improve forage yield of L. tenuis in this region. Thus, the formulation of inoculants for the production of L. tenuis in the FP would greatly benefit from the selection of native strains that exhibit good symbiotic behavior in both normal and stress conditions. Current study is in progress to evaluate under different field conditions the symbiotic performance of some of the native strains hereby studied. Preliminar tests performed by our group detected that the density of native rhizobial populations are highly variable in soils of the FP. In the most restrictive areas such as the saline-alkaline lowlands, the number of native rhizobia is usually low (between 0 to 102 rhizobia/g of soil). Thus, strains to be used in the future as inoculants are not expected to be exposed to a strong competition by native rhizobial populations in such environments. On the other hand, in soils less restrictive than saline alkaline lowlands, native rhizobia are more abundant (between 1 9 103 and 1 9 106 rhizobia/g of soil). Therefore, introduced strains would face a stronger competition by native populations in these cases, and inoculum density should be adjusted accordingly in order to favor inoculant competitiveness. Acknowledgments This study was supported by: Comisioń de Investigaciones Cientı´ficas de la Provincia de Buenos Aires (CIC, Argentina); Consejo Nacional de Investigaciones Cientı´ficas y Tećnicas (CONICET, Argentina, PIP 112-2008 01-00734 and PIP 04-5740); Agencia Nacional de Promocioń Cientı´fica y Tecnolo´gica (Argentina, PICT 2007-02034); European Union (INCO Project PL 517617), Universidad Nacional de General San Martıń (Argentina, Project SA08/ 001) and the Iberoamerican Network for Biofertilizers (BIOFAG-CYTED). LNC and RCP are doctoral fellows of CONICET. AIS, ABM, OAR and FLP are researchers of CONICET. MJE is a member of the Research Career of CIC.

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