876 DOI: 10.1002/jpln.201300116 J. Plant Nutr. Soil Sci.

2013, 176, 876–882

Use of Rhizobium leguminosarum as a potential biofertilizer for Lactuca

sativa and Daucus carota crops
José D. Flores-Félix1, Esther Menéndez1, Lina P. Rivera1, Marta Marcos-García1, Pilar Martínez-Hidalgo1,
Pedro F. Mateos1,2, Eustoquio Martínez-Molina1,2, Ma de la Encarnación Velázquez1,2, Paula García-Fraile1,
and Raúl Rivas1,2*
1 Departamento de Microbiología y Genética, Edificio Departamental de Biología. Plaza Doctores de la Reina s/n, 37007 Salamanca, Spain
2 Unidad Asociada de I+D Universidad de Salamanca (USAL)-CSIC (IRNASA), Spain

Abstract
Microbial biofertilizers are becoming an effective tool for sustainable agriculture by means of the
reduction of the use of chemical fertilizers. However, the knowledge of each specific plant–mi-
croorganism interaction is essential for a correct application. In this study, we analyzed the in
vitro plant-growth-promotion mechanisms of a Rhizobium leguminosarum strain named
PEPV16 isolated from Phaseolus vulgaris nodules. This strain was able to produce sidero-
phores and indole acetic acid and to solubilize phosphate. Confocal microscopy showed that
this strain was able to colonize the roots of two horticultural crops, Lactuca sativa L. (lettuce)
and Daucus carota L. (carrot). Strain PEPV16 was also able to promote the plant growth of both
plant species increasing the dry matter of shoots and roots of lettuce and carrots, respectively,
as well as to increase the uptake of N and P in the edible parts of both plant species. These data
confirmed the suitability of Rhizobium as biofertilizer for nonlegumes.

Key words: PGPR / lettuce / carrot / GFP / rhizobia / endophyte

Accepted September 24, 2013

1 Introduction
After the Green Revolution, agriculture has been based on
the extensive use of chemical fertilizers and pesticides.
Throughout the time, the effects of these practices have
become hazardous for animal and human health and water
ecosystems have been impaired by eutrophication. More-
over, soil quality and rhizosphere diversity have been com-
promised. For all of these reasons, the reduction in the use of
chemical fertilizers is now regarded as an important neces-
sity, due to its expensiveness in production and its impact on
the environment. This reduction may help to minimize green-
house-gas emissions and to avoid contamination of ecosys-
tems (Snyder et al., 2009). However, conventional agriculture
is unable to provide a solution to this problem without increas-
ing costs or decreasing crop production (Singh et al., 2011).
Therefore, the use of biofertilizers may be an efficient and
low-cost alternative, able to face the problems described
above. Plant growth–promoting rhizobacteria, commonly
called PGPR, exhibit several mechanisms influencing the
availability of plant nutrients and enhancing plant resistance
to stress and pathogen invasion and infection (Berg, 2009;
Tikhonovich and Provorov, 2011). Among their benefits, biolo-
gical nitrogen fixation (BNF), both free-living or in symbiosis,
phosphate solubilization, siderophore production, and phyto-
hormone synthesis are some of the most valuable features of
these bacteria (Bhattacharryya and Jha, 2011).
Plant–microorganism interactions are efficient due to the
direct effect of PGPR in the plant, specifically colonizing and
occupying intercellular spaces in leafs, roots, and stems, as
endophytes (Hardoim et al., 2008; Bhattacharryya and Jha,
2011). These relationships are based on a complex and well-
regulated molecular dialogue between plant and microorgan-
isms. Concurrently, an essential requirement for biofertilizer
design is the use of innocuous bacteria for human and animal
health, given that some human pathogenic bacteria, such as
Klebsiella pneumoniae, Burkholderia cepacia, Pseudomonas
aeruginosa, or Acinetobacter show plant growth–promoting
features but cannot be used due to their pathogenicity (Gar-
cía-Fraile et al., 2012). On the other hand, symbiotic rhizobia
are safe microorganisms with a well-known ability to establish
nitrogen-fixing endosymbiosis with legumes and present in
vitro mechanisms of plant growth promotion and have some
interesting features, such as siderophore production, phos-
phate solubilization, and phytohormone production such as
indole acetic acid, gibberellins, and cytokinins (Mehboob
et al., 2009; García-Fraile et al., 2012). Although the effects
of rhizobia in legumes are the best studied, these microor-
ganisms also colonize the roots of nonlegumes such as rice,
maize, lettuce, pepper, and tomato and therefore they are
good candidates for biofertilization of these plants (Chabot
et al., 1996a; Peña and Reyes, 2007; Baset and Shamsud-
din, 2010; García-Fraile et al., 2012).
Nevertheless, there are few studies about the ability of Rhizo-
bium to promote the growth of vegetables with high agro-
nomic interest and to allow the substitution of chemical fertili-

* Correspondence: R. Rivas; e-mail: raulrg@usal.es

 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.plant-soil.com

J. Plant Nutr. Soil Sci. 2013, 176, 876–882
zers by biofertilizers. Lettuce (Lactuca sativa) is widely con-
sumed in the world and constitutes one of the major fresh
vegetables produced in Spain, which is also the major produ-
cer of carrots (Dacus carota) in Europe. In both cases the
edible parts, shoots and roots, respectively, can be con-
sumed rawly and carrot is one of the main components in
purees for children. Therefore, it is essential that they are
free of hazardous compounds as well as pathogenic. The
objective of this study was to investigate whether Rhizobium
is able to promote the growth of lettuce and carrot and
whether it can be considered as a reliable biofertilizer for
these two horticultural crops.
2 Material and methods
2.1 Bacterial strains
Rhizobium leguminosarum L. strain PEPV16 was isolated
from an effective nodule of Phaseolus vulgaris growing in a
soil from Salamanca (Spain) using the standard method of
Vincent (1970) on YMA plates at 28°C (Flores-Félix et al.,
2011). The ability of this strain to nodulate P. vulgaris was
checked according to Mulas et al. (2011). For GFP-tagged
experiments, Escherichia coli was grown in Luria Bertani (LB)
broth at 37°C and Rhizobium strains in YMA medium. Anti-
biotics were added when required (tetracycline 10 lg mL–1).
GFP-tagged derivative Rhizobium was obtained by biparental
mating with E. coli S17.1, carrying plasmid pHC60 (Cheng
and Walker, 1998) as described in García-Fraile et al. (2012).
2.2 Phylogenetic analysis of 16S rRNA gene
The amplification and sequencing of rrs gene was carried out
according to Rivas et al. (2007). The sequences obtained
were compared with those from EzTaxon-e server (Kim et al.,
2012). Sequences were aligned using the Clustal X software
(Thompson et al., 1997). The distances were calculated
according to Kimura’s two-parameter model (Kimura, 1980).
A phylogenetic tree was inferred using the neighbor-joining
analysis (Saitou and Nei, 1987). MEGA5 software (Tamura
et al., 2011) was used for all analyses.
2.3 Analysis of in vitro PGPR mechanisms
The ability to solubilize phosphate by the strain PEPV16 was
tested in YED-P media according to Peix et al. (2004). Side-
rophore production was evaluated using M9-CAS-AGAR
media (Schwyn and Neilands, 1987) and modified by Alexan-
der and Zuberer (1991). Indole acetic acid production was
measured in JMM media (O’Hara et al., 1989) supplemented
with tryptophan (0.17g L–1) as described Khalid et al. (2004).
2.4 Colonization assays
Lettuce (Lactuca sativa L. var. romain) and carrot (Daucus
carota L. var. nantes) seeds were surface-sterilized with 70%
ethanol during 30 s and 5% sodium hypochlorite for 5 min.
Several washes were performed and seeds were spread on
agar plates. Three days after germination, seedlings were
 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Rhizobium leguminosarum as a biofertilizer 877
inoculated with 250 lL plant–1 of a bacterial suspension with
a turbidity of 5 in McFarland standards (1.5 × 109 CFU mL–1)
and incubated in a growth chamber. Uninoculated controls
were also included in the study.
Seedlings were viewed under a laser scanning confocal
microscope (Leica SP2) 15 d after inoculation. Also, a NIKON
Eclipse 80i epifluorescence microscope was used to monitor
lettuce and carrot colonization 6 and 7 d after inoculation, re-
spectively, using excitation at 472 nm in an argon laser for
green fluorescence and 510 nm for root cells which were
stained with 10 lM of propidium iodide (Sigma). Images were
processed using Leica confocal software.
2.5 Plant assays
Axenic seedlings were transferred to pots containing a mix of
sterilized soil and vermiculite (3 : 1). Eighteen plants per
treatment were inoculated with 1 mL of R. leguminosarum
PEPV16 suspension with a turbidity of 5 in McFarland stand-
ard (1.5 × 109 CFU mL–1) and watered when needed. An
uninoculated group was left under the same conditions.
Plants were harvested 45 d after inoculation to determine dry
weight and nutrient concentrations. The analyses of nitrogen,
phosphorous, potassium, magnesium, calcium, sulfur, iron,
manganese, zinc, and copper was performed using ICP-OES
ICAP6500 DUO spectrometer (Thermo Scientific) in the
Ionomic Service from CEBAS-CSIC (Spain).
2.6 Statistics
Data were analyzed with one-way ANOVA using Statview 5.0
software (SAS Institute Inc.), with a post-hoc test using
Fisher’s protected least significant difference (P ≥ 5%).
3 Results
3.1 Identification of strain PEPV16 and analysis of
in vitro PGPR mechanisms
The strain PEPV16 was classified as genus Rhizobium on
the basis of its rrs gene sequence showing 99.9% identity
with respect to Rhizobium leguminosarum USDA 2370T. Fig-
ure 1 shows the phylogenetic position of this strain in the
group formed by R. leguminosarum and R. indigoferae, two
species that are probably synonyms because they have
nearly identical rrs gene as was already pointed out by
Ferreira et al. (2011). The strain PEPV16 was able to nodu-
late P. vulgaris in agreement with other strains from the same
species isolated in North Spain where common beans are
commonly nodulated by strains of the species R. legumino-
sarum (Mulas et al., 2011).
Colonies of strain PEPV16 in M9-CAS-AGAR medium were
surrounded by a yellow-orange halo (2 mm periphery around
colonies) indicative of siderophore production. In YED-P
medium, solubilization of insoluble phosphate was detected
since strain PEPV16 showed a transparent halo of about
2 cm radius around its colonies. The strain also produced
indole-3-acetic acid (IAA) at final concentration 77 mg L–1.
www.plant-soil.com
878 Flores-Félix et al. J. Plant Nutr. Soil Sci. 2013, 176, 876–882

71Rhizobium multihospitium CCBAU 83401 T (EF035074)

75
Rhizobium hainanense I66 T (U71078)
87
Rhizobium tropici CIAT 899 T (HQ 850704))
99 Rhizobium miluonens e CCBAU 41251 T (EF061096)
Rhizobium rhizogenes ATCC 11325 T (AY945955)
88
63 Rhizobium lusitanum P1-7 T (AY738130)
44 Rhizobium leuc aenae LMG 9517 T (CFN 299) (X67234)
67 Rhizobium v allis CCBAU65647 T (FJ839677)
Rhizobium etli CFN42 T (U28916)
38 Rhizobium fabae CCBAU 33202 T (DQ 835306)
78
62 Rhizobium pisi DSM 30132 T (AY509899)

49
Rhizobium phaseoli ATCC 14482 T (EF141340))
44 PEPV16
100 Rhizobium leguminosarum USDA2370 (FJ595999)
T

75 Rhizobium indigoferae CCBAU71042 T (AY034027)

89 Rhizobium grahamii CCG E502 T (JF424608.1)
Rhizobium tibetic um CCBAU85039 T (EU256404)
48
74 Rhizobium endophytic um CCG E2052 T (EU867317)
80 Rhizobium c auense CCBAU101002 T (JQ 308326.1)
85 Rhizobium mes oamericanum CCG E501 T (JF424606.1)
100 Rhizobium mes osinicum CCBAU 25010 T (DQ 100063)

Rhizobium alamii G BV016 T (AM931436)

53
73
Rhizobium sullae IS 123 T (Y10170)
49 Rhizobium loes sens e CCBAU7190B T (AF364069)
70
Rhizobium mongolense USDA 1844 T (U89817)
100 Rhizobium gallicum R602 T (U86343)
Rhizobium yanglingense SH 22623 T (AF003375)
22
Rhizobium tubonens e CCBAU 85046 T (EU256434)
72 Rhizobium ps eudoryzae J3-A127 T (DQ 454123)
43
Rhizobium oryzae Alt 505 T (EU056823)
21
Rhizobium gilianshanens e CCNW Q LS01 T (JQ 728555.1)
Rhizobium phenanthrenillyticum F11 T (FJ43436.1)
100
Rhizobium petrolearum SL-1 T (EU556969.C3)
13
Rhizobium soli DS-42 T (EF363715)
17
Rhizobium kunmingense LXD30 T (FJ560597.1)
23
Rhizobium tarimense PL-41 T (HM371420.2)
Rhizobium cellulos ilytic um ALA10B2 T (DQ 855276)
59
85 Rhizobium vignae CCBAU 05176 T (G U128881)
95
Rhizobium galegae ATCC 43677 T (D11343)
100 Rhizobium alk alis oli CCBAU 01393 T (EU074168)
86 Rhizobium huautlense SO 2 T (AF025852)
89 Rhizobium subbaraonis JC85 T (FR714938.1)
20 Rhizobium halophy tocola YC6881 T (G U322905.2)
Rhizobium borbori DN316 T (EF125187)
18
Rhizobium undicola LMG 11875 T (Y17047)
100 Rhizobium helanshanense CCNW MQ 14-2 T (HQ 132341.1)
46 Rhizobium sphaerophysae CCNW G S0238 T (FJ154088.1)
23
Rhizobium herbae CCBAU83011 T (G U565534)
100 Rhizobium giardinii H152 T (U86344)
96 Rhizobium naphtalenivorans TSY03 T (AB663504.1)
47
70 Rhizobium selenitireducens B1 T (EF440185)
Rhizobium daejeonense L61 T (AY341343)
100 Rhizobium taibaishanens e CCNW SX0483 T (HM776997.1)
64
75 Rhizobium vitis NCPPB 3554 T (D14502)
Rhizobium rossettiformans W 3 T (EU781656)
80 Rhizobium aggregatum IFAM1003 T (X73041)
36 74 Rhizobium pusens e NRCPB10 T (FJ969841)
Rhizobium larry mooreii AF3.10 T (Z30542)
94 Rhizobium radiobac ter ATCC19358 T (HQ 735085)
48 Rhizobium nepotum 39/7 T (FR870231.1)
78 Rhizobium sk ierniewicense Ch 11 T (HQ 823551.1)
99 Rhizobium rubi IFO 13261 T (D14503)
Azorhizobium caulinodans O RS57 T (NC 009937)

0.01

Figure 1: Neighbor-joining tree based on nearly complete 16S rRNA gene sequences of strain PEPV16. The significance of each branch is
indicated by a bootstrap value calculated for 1000 subsets. Bar, 1 nt substitutions per 100 nt.

 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.plant-soil.com

J. Plant Nutr. Soil Sci. 2013, 176, 876–882
3. 2 Colonization assays
GFP-tagged bacteria allow confocal and fluorescence micro-
scopy assays. With this technology, the interaction between
L. sativa seedlings and PEPV16 strai L. sativa seedlings was
tested. This strain is able to colonize the root surface, occu-
pying intercellular spaces in cortical cells. PEPV16 was dis-
tributed ubiquitously on the root, forming a smooth layer
(Fig. 2A). When checked under confocal microscope,
PEPV16 strain was located at root surface and clearly shows
invasion of root epidermis intercellular spaces. These results
confirm its behavior as an endophyte in this plant (Fig. 2B).
Daucus carota seedlings inoculated with GFP-tagged
PEPV16 strain showed uniform colonization along root cell
depressions (Fig. 2C), as observed for L. sativa seedlings.
Also D. carota seedlings showed similar bacterial distribution
(Fig. 2D). Therefore, PEPV16 strain shows the ability to colo-
nize and adhere to root hairs in lettuce and carrots with a sim-
ilar distribution and to penetrate intercellular spaces during
early stages of development in lettuce. The capacity to
become an endophyte is expected to be the same as in let-
tuce and carrot because the colonization of the root surface
followed a similar pattern.
3. 3 Plant assays
We have analyzed the ability of strain PEPV16 to promote
the plant growth of L. sativa and D. carota plants under micro-
cosmos conditions, evaluating effects on edible parts in both
cases (Tab. 1).
 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Rhizobium leguminosarum as a biofertilizer 879
Figure 2: A: Fluorescent microscopy
showing Lactuca sativa roots with Rhi-
zobium leguminosarum strain PEPV16
colonizing the surface 6 d after inocula-
tion. B: Confocal fluorescent microscopy
shows where Rhizobium leguminosarum
strain PEPV16 appears to colonize
intercellular spaces of the Lactuca sativa
roots. Rhizobium leguminosarum
PEPV16 bacteria appear in green due
to the GFP transformation, and plant
cells were stained red with a propidium
iodide solution. C: Daucus carota root
surface colonized with Rhizobium legu-
minosarum strain PEPV16 visualized
with fluorescent microscopy. D: Fluor-
escent confocal microscopy showing
Rhizobium leguminosarum strain
PEPV16 colonizing the surface of a
Daucus carota root.
Concerning L. sativa, the dry weight of shoots was signifi-
cantly increased for plants inoculated with PEPV16, compar-
ed to uninoculated ones. Also the concentrations of N, P, and
Ca were significantly higher in inoculated plants indicating
that they had higher potential for nutrient uptake than control
plants (Tab. 1). The remaining macronutrients were not signif-
icantly different between inoculated and uninoculated plants
except in the case of K and S whose percentages were signif-
icantly lower in inoculated plants. In the case of micronutri-
ents, only the percentage of Fe was significantly higher in the
inoculated plants while the rest of micronutrients showed a
nonsiginificant increase.
The results obtained for D. carota confirmed those for lettuce,
since the root dry weight of inoculated plants was significantly
higher (40%) than that of the control plants (Tab. 1). As in the
case of lettuce, the concentrations of N, P, and Ca were sig-
nificantly higher in the inoculated plants. In addition, the con-
centrations of K and Mg were also higher in inoculated plants.
The percentage of S was slightly but not significantly higher
in the inoculated plants. As in the case of lettuce, the Fe con-
centration was significantly higher in inoculated plants but no
significant differences were found in the remaining micronutri-
ents.
4 Discussion
In the current worldwide scenario of increasing food prices
and constant environment impoverishment, the fertilizer
application should be optimized by the use of low-cost and
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880 Flores-Félix et al. J. Plant Nutr. Soil Sci. 2013, 176, 876–882

Table 1: Effect of R. leguminosarum strain PEPV16 on dry weights and nutrient concentrations of lettuce shoots and carrot roots.

Treatment Dry weight Macronutrients Micronutrients

per edible part
/g N/% P/% K/% Mg / % Ca / % S/% Fe / mg kg–1 Mn / mg kg–1 Zn / mg kg–1 Cu / mg kg–1
(± SE) (± SE$) (± SE) (± SE) (± SE) (± SE) (± SE) (± SE) (± S.E.) (± S.E.) (± S.E.)
Lettuce
Control 4.53 3.44 0.62 7.4 0.28 0.68 0.26 67.66 58.82 39.34 3.80
(± 0.19)a§ (± 0.01)a (± 0.01)a (± 0.01)a (± 0.01)a (± 0.01)a (± 0.01)a (± 2.11)a (± 4.00)a (± 2.67)a (± 0.17)a
PEPV16 5.05 3.72 0.73 6.7 0.27 0.72 0.23 77.21 59.72 41.26 3.74
(± 0.16)b (± 0.03)b (± 0.02)b (± 0.01)b (± 0.01)a (± 0.01)b (± 0.01)a (± 3.00)b (± 0.81)a (± 1.73)a (± 0.31)a
Carrot
Control 0.90 2.87 0.99 4.60 0.22 0.44 0.09 8.13 16.64 26.94 3.66
(± 0.08)c (± 0.03)c (± 0.02)c (± 0.09)c (± 0.01)c (± 0.01)c (± 0.00)a (± 0.04)a (± 0.33)a (± 0.52)a (± 0.35)a
PEPV16 1.53 5.31 1.66 7.03 0.36 0.81 0.10 10.10 15.32 25.47 4.25
(± 0.9)d (± 0.08)d (± 0.06)d (± 0.44)d (± 0.01)d (± 0.03)d (± 0.01)a (± 0.01)b (± 0.51)a (± 0.96)a (± 0.64)a

$SE = standard error.

§Values followed by the same letter in each treatment are not significantly different from each other at P = 5% according to Fisher’s Protected
LSD (least significant differences).

less harmful technologies including the use of microorgan-
isms (Dawson and Hilton, 2011; Van Vuuren et al., 2010).
Some PGPR bacteria may act as endophytes, but some of
them could be harmful for human and animal health (Rosen-
blueth and Martínez-Romero, 2006) and their use must be
avoided in biofertilization schemes (García-Fraile et al.,
2012). The genus Rhizobium is an important PGPR whose
safety for humans, animals, and plants has been widely
shown after decades of inoculation of legumes (Bhattachar-
jee et al., 2012; Glick, 2012). Moreover, the ability of Rhizo-
bium strains to promote the growth of some nonlegumes has
been reported as was pointed out in the introduction of this
paper. In a recent study, we analyzed the ability of a strain of
Rhizobium leguminosarum isolated from P. vulgaris nodules
to produce siderophores and IAA and to promote plant
growth increasing the fruit production of tomato and pepper
(García-Fraile et al., 2012). Nevertheless, there are many
species and varieties of vegetables in which the effect of Rhi-
zobium inoculation remains unexplored as occurs in the case
of carrots and the variety of lettuce “Romana” used in this
study. Moreover, the strain PEPV16, in addition to the pro-
duction of siderophores and indole acetic acid, was able to
solubilize phosphate (not shown).
The ability of Rhizobium to colonize lettuce roots previously
reported by Chabot et al. (1996b) has been confirmed in this
study using GFP-labeled strains and confocal microscopy.
Moreover the strain PEPV16 was able to colonize the inner
part of roots showing that this strain is an endophyte in let-
tuce. In carrots, although the colonization of intercellular
spaces was not clear, the colonization of root surfaces was
abundant as observed using confocal microscopy. These
results confirm the need of performing colonization assays on
different plant species, since the ability of different strains to
establish effective molecular interactions depends on the
host plant and interaction efficiency (Bais et al., 2006).
Both lettuce and carrot seedlings, 6 d after inoculation,
showed more higher root-hair density on their roots and an in-
creased root length in comparison to uninoculated seedlings
(data not shown). An increase in root-hair number and, con-
sequently, larger absorption surface is closely related to the
improved ability for nutrient uptake, compared to uninocu-
lated seedings. These data match perfectly with the ability of
PEPV16 strain to produce indole-3-acetic acid since Meh-
boob et al. (2009) described a relationship between inocula-
tion with indole acetic acid–producing bacteria and an in-
creased development and root-hair number in early stages.
The increase in the ability to take up nutrients was clearly
shown since N and P concentrations were significantly in-
creased in shoots (lettuce) and roots (carrot) inoculated with
the strain PEPV16. Although Rhizobium strains are unable to
fix nitrogen with nonlegumes, an increased level of this ele-
ment in pepper has been also reported after inoculation with
a Rhizobium strain isolated from P. vulgaris (García-Fraile
et al., 2012). The P uptake was increased by 15% in the let-
tuce shoots, in concordance with the results obtained by Cha-
bot et al. (1996a) when lettuce was inoculated with a R. legu-
minosarum strain isolated from P. vulgaris. In carrots, the
increase of P concentration in roots of inoculated plants
compared to the control ones went up by 40%, with the same
increase in dry weight. This increase was higher than that
found by Antoun et al. (1998) for radish which was inoculated
with a R. leguminosarum strain also isolated from P. vulgaris.
With regard to the micronutrients, it is remarkable that Fe in-
creased in inoculated plants of both crops. This can be
related to the ability of the strain PEPV16 to produce sidero-
phores since it has been reported that plants inoculated with
PGPR are able to produce siderophores and are capable of
absorbing Fe from these compounds (Glick, 2012). Magne-
sium also plays an important role as cofactor in many
enzymes (Mengel and Kirkby, 2001). The increase of this ele-
ment in carrot root leading to enhanced metabolic activity
could be related with the higher dry weight of these plants.
5 Conclusion
The results show that Rhizobium leguminosarum PEPV16, a
strain that actively colonizes the rhizosphere of L. sativa and
D. carota, increased plant growth as well as the content of N

 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.plant-soil.com

J. Plant Nutr. Soil Sci. 2013, 176, 876–882
and P of the edible parts in both vegetables being a good
potential biofertilizer for these nonlegume crops.
Acknowledgment
This work was funded and supported by Junta de Castilla y
León project SA183A11-2 and MICINN grant AGL2011-
29227. E. M. acknowlegdes a FPI-MICINN PhD fellowship. L.
P. R. and P. M. H. acknowledge JAE-PREDOC PhD fellow-
ships given by CSIC. M. M. G. acknowledges a grant from
The Miguel Casado San José Foundation.
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