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ORIGINAL PAPER
Plant growth promotion traits of phosphobacteria isolated
from Puna, Argentina
Emilce Viruel •Marı
´a E. Lucca •Faustino Sin
˜eriz
Received: 28 June 2010 / Revised: 18 November 2010 / Accepted: 28 February 2011
ÓSpringer-Verlag 2011
Abstract The ability of soil microorganisms to solubilize
phosphate is an important trait of plant growth-promoting
bacteria leading to increased yields and smaller use of
fertilizers. This study presents the isolation and character-
ization of phosphobacteria from Puna, northwestern
Argentina and the ability to produce phosphate solubili-
zation, alkaline phosphatase, siderophores, and indole
acetic acid. The P-solubilizing activity was coincidental
with a decrease in pH values of the tricalcium phosphate
medium for all strains after 72 h of incubation. All the
isolates showed the capacity to produce siderophores and
indoles. Identification by 16S rDNA sequencing and phy-
logenetic analysis revealed that these strains belong to the
genera Pantoea,Serratia,Enterobacter, and Pseudomonas.
These isolates appear attractive for exploring their plant
growth-promoting activity and potential field application.
Keywords Phosphobacteria Phosphate solubilization
Indol acetic acid Siderophore 16S rDNA
Introduction
Phosphorus (P) is one of the major plant growth-limiting
nutrients despite being abundant in soils. The free phos-
phorus concentration available to plants is very low even in
fertile soils due to the fact that soluble P reacts with cal-
cium (Ca), iron (Fe), or aluminum (Al) and organic com-
pounds that lead to P precipitation (Gyaneshwar et al.
2002). This essential plant nutrient is added to soil as
chemical fertilizers, but becomes insoluble and, therefore,
unavailable to plants. Moreover, this practice is reaching
the theoretical maximum use beyond which there will be
no further increase in yields (Son et al.2006).
Phosphobacteria have the ability to convert insoluble
compounds of phosphorus into available phosphates that
enhance nutrient availability to plants (Barea et al.2005;
Lugo et al.2008; Rodrı
´guez and Fraga 1999; Son et al.
2006; Souchie et al.2006). Strains from the genera
Pseudomonas,Bacillus,Rhizobium,Burkholderia,Achro-
mobacter,Agrobacterium,Microccocus,Aereobacter,
Flavobacterium, and Erwinia are known phosphate solu-
bilizers (Rodrı
´guez and Fraga 1999). Several studies have
shown that the release of organic acids, like gluconic and
2-ketogluconic acids, is one of the mechanisms responsi-
ble for solubilizing insoluble phosphate (Lin et al.2006)
since the organic acids produced by the microorganisms
may reduce pH and act as chelating agents, forming
complexes with Ca, Fe, or Al, and thereby releasing the
phosphates to solution. Other mechanisms of solubiliza-
tion comprise the release of other chelating substances and
inorganic acids such as sulphideric, nitric, and carbonic
acids. Secretion of phosphatase enzymes (acid and alka-
line phosphatase, phytase, phosphohydrolase) by phos-
phobacteria is also a common mode of facilitating the
conversion of insoluble forms of P to plant-available
Communicated by ursula priefer.
E. Viruel (&)M. E. Lucca F. Sin
˜eriz
Planta Piloto de Procesos Industriales Microbiolo
´gicos
(PROIMI), CONICET, Av. Belgrano y Pasaje Caseros,
4000 Tucuma
´n, Argentina
e-mail: eviruel@proimi.org.ar
M. E. Lucca F. Sin
˜eriz
Ca
´tedra de Microbiologı
´a Superior, Facultad de Bioquı
´mica,
Quı
´mica y Farmacia, Universidad Nacional de Tucuma
´n,
4000 Tucuma
´n, Argentina
123
Arch Microbiol
DOI 10.1007/s00203-011-0692-y
forms and thus enhance plant P uptake and growth (Kohler
et al.2007).
Microbial phosphate solubilization may be then a solu-
tion, not only to compensates for the ever increasing costs
of manufacturing fertilizers in industry but also as a way to
mobilize the fertilizers added to soil. Phosphobacteria not
only play a significant role in supplying P to plants, but
also increase plant growth and development through other
plant growth promotion activities, like nitrogen fixation,
siderophores, and phytohormones production (Vassilev
et al.2006). Certain cooperative microbial activities can be
exploited as a low-input biotechnology and form a basis for
a strategy to help sustainable, environmentally friendly
practices fundamental to the stability and productivity of
both agricultural systems and natural ecosystems (Barea
et al.2005). The aim of this study was to isolate phos-
phobacteria from rhizospheric soil of Puna-native grasses,
to identify them using molecular tools and to evaluate their
phosphate solubilization activity, indol acetic acid, and
siderophores production. Puna is a South American harsh
biogeographical region with unique features (Lugo et al.
2008), although it is considered an alpine environment by
some authors, and is a source of microorganisms with
interesting characteristics.
Materials and methods
Isolation of phosphate-solubilizing bacteria
Rhizospheric soil samples were taken along an altitudinal
gradient (3,200–3,800 masl) from Puna (northwestern
Argentina), a South American stressed biogeographic
region with unique features, where climate is of a desert
type. The soils are superficial and immature, very poor in
organic matter, sandy and rocky (Lugo et al.2008). The
samples were stored at 4°C before processing. Batch cul-
ture of soil samples with a phosphorous limited culture
medium (1 lM of phosphorus) was performed, and con-
tinuous feeding at a dilution rate of 0.05 h
-1
allowed the
isolation of different bacterial morphotypes.
Serial dilutions of batch culture samples were then
individually inoculated on NBRIP agar plate supplemented
with 1.5% (w/v) agar (Nautiyal 1999). Phosphobacteria
were identified after 7 days of incubation at 30°C because
they developed clear zones around their colonies. They
were picked up and further purified by replating on agar
plates for qualitative estimation of phosphate solubilization
index (SI). The spot inoculation was carried out using a
sterile needle, and the Petri dishes were incubated at 30°C.
The halo (zone of solubilization) around the bacterial
colony and colony diameter were measured after incuba-
tion for 7 days. Solubilization index was evaluated
according to the ratio of the total diameter (colony ?halo
zone) and the colony diameter (Premono et al.1996). The
test was carried out on duplicate in each case.
Genomic DNA isolation and 16S rDNA sequencing
For phylogenetic characterization, single colonies of each
strain were removed from LB plates and transferred to 2-ml
microcentrifuge tubes for subsequent DNA extraction.
DNA was extracted by the cetyltrimethylammonium bro-
mide (CTAB) method as like described by Lugo et al.
(2008). Almost full-length 16S ribosomal ribonucleic acid
(rRNA) gene sequences were amplified from DNA extrac-
ted with oligonucleotide primers 27f (Escherichia coli 16S
rDNA positions 8–27) and 1492r (E.coli 16S rDNA posi-
tions 1,492–1,512) (Lane 1991). The PCR amplification
mixture contained 0.2 mM (each) dNTP (Promega), 400
nM (each) primer (Promega), GoTaq Green Master Mix
with Mg 7.5 mM 19(Promega), and 1U GoTaq
Ò
DNA
Polymerase (Promega) in a final volume of 25 ll. After a
hot start at 94°C for 3 min, 30 cycles PCR reaction were run
as follows: denaturation at 94°C for 1 min, annealing at
57°C for 30 s, and extension at 72°C for 1 min. In addition,
a final extension at 72°C for 7 min was added. Negative
controls, without DNA, were included in each experiment.
PCR products were analyzed by horizontal agarose (1%) gel
electrophoresis (4 V cm
-1
) in Tris–borate-EDTA (TBE)
running buffer (Sambrook et al.1989). Gels were stained
with ethidium bromide (0.5 mg ml
-1
) for 30 min, washed
three times with distilled water, and visualized on UV
analyzer. The determined partial 16S rDNA sequences have
been deposited in EMBL Nucleotide Sequence Database
(http://www.ncbi.nlm.nih.gov), and numbers access were
obtained for each strain.
Phylogenetic analysis
Phylogenetic and molecular evolutionary analyses were
conducted using the program MEGA version 4 (Tamura
et al.2007). The phylogenetic trees were constructed by the
neighbor-joining method (Saitou and Nei 1987) using the
distance matrix from the alignment. Distances were cal-
culated using the Kimura method (1980).
Phosphate solubilization
Solubilization of tricalcium phosphate in broth culture
Phosphobacteria were grown in LB broth overnight at 30°C
with 200 rpm agitation. Flasks with 50 ml of NBRIP media
were inoculated with 100 ll of LB culture (10
2
UFC ml
-1
)
and incubated on a rotary shaker at 200 rpm and 30°C. A
5-ml sample of each culture was taken at 24, 48, and 72 h
Arch Microbiol
123
of incubation and centrifuged for 15 min at 10,000 rpm.
Soluble phosphorus (P) concentrations (lgml
-1
) (Murphy
and Riley 1962) and pH were measured in the supernatant
for each strain. Sterile uninoculated medium was used as
control. The test was carried out on triplicate in each case.
Alkaline phosphatase production
To determine alkaline phosphatase (AP) activity, synthetic
medium (in g l
-1
: Glycerol, 10; (NH
4
)
2
SO
4
, 5; NaCl, 5;
yeast extract, 0.5) was used with either 10 mM K
2
HPO
4
(Pi) (excess phosphate) or 1 lM Pi (limiting phosphate).
Cultures were grown overnight at 30°C, harvested, and the
pellets were washed with 0.1 M MgSO
4
and suspended in
0.1 M Tris-hydrochloride (pH 7.4) containing 0.001 M
MgSO
4
. AP activity was determined using the method
described by Ludtke et al.(1984) with p-nitrophenyl
phosphate (Sigma) as substrate. One unit of alkaline
phosphatase is defined as the nanomoles of p-nitrophenol
produced per min per mg of cell protein. The test was
carried out on triplicate in each case. Protein concentration
was determined by the method of Bradford (1976) using
bovine serum albumin as standard.
Other PGPR traits
IAA production
Total Indole production was determined by a colorimetric
method (Glickmann and Dessaux 1995) in M9 medium
with or without tryptophan (Trp) (0.1 mg ml
-1
). 100 llof
LB culture (DO
600nm
0.2) of each bacterial isolate was
transferred to M9 medium. Cultures were incubated at
30°C during 48 h and then centrifuged (10,000 rpm,
15 min) to obtain the supernatant. Indoles production was
determined with the Salkowski reagent. Reactions was
carried out with a ratio 1:1 of supernatant to Salkowski
reagent, followed by incubation in the dark at room tem-
perature during 30 min. Total indoles were determined
spectrophotometrically at 540 nm. Values were directly
converted to equivalent IAA concentrations (lg per ml),
using the calibration curve of Indole acetic acid. The test
was carried out on triplicate in each case.
Siderophores production
The chrome azurol sulfonate (CAS) assay was used to
determine production of siderophores (Schwyn and Neilands
1987). Ten ll of iron-free M9 pre-culture (DO
600nm
0.2) was
inoculated by triplicate in Petri dishes containing M9 solid
medium supplemented with CAS. They were incubated at
30°C for 24 h, and the siderophore halo (mm) was deter-
mined by substrate colony diameter to the total diameter.
Statistical analysis
All statistical analyses were performed using the least
significant difference (LSD) test (P=0.05) to determine
significant differences of the mean values between treat-
ments. The Infostat Analytical Software (2008) for Win-
dows was used.
Results
Isolation of phosphate-solubilizing bacteria
After evaluating 86 isolates, only 33 showed clear halos of
phosphate solubilization in NBRIP agar plate after 7 days
of incubation at 30°C (Fig. 1a). PSB were further purified,
and solubilization index in agar plate based on colony
diameter and clear halo zone for each isolate is presented in
Fig. 1b. Results showed that among PSB, EV4 was the
most efficient phosphate solubilizer on NBRIP plates with
SI =6.8 ±0.4. EV4 with other higher solubilizer isolates,
EV1 (5.0 ±1.0), EV2 (4.9 ±0.6), EV3 (6.0 ±0.6), IEXb
(4.7 ±0.4), and IEY (4.2 ±0.3), were selected for further
studies based on phosphate solubilization capacity and
different colony characteristics.
Genomic DNA isolation and 16S rDNA sequencing
of selected isolates
Identification of six selected phosphate-solubilizing bacte-
rial strains based on 16S rDNA sequence and their phy-
logeny are presented in Fig. 2. Molecular analysis reveals
that the sequence of EV1 showed high similitary (98.3% of
identity) with type strain Serratia marcescens subsp. sak-
uensis (AB061685), two isolates EV2 and EV4 showed
99.4 and 98.8% identity with Pantoea eucalypti LMG
24197
T
(EF688009), respectively, IEXb was closely asso-
ciated with Pseudomonas tolaasii ATCC 33618
T
(D84028)
with 99.2% of identity, and IEY showed high similitary
with Enterobacter aerogenes NCTC 10006
T
(AJ251468)
with 98.9% of identity. Interestingly, EV3 isolate showed
only 93.3% of identity with the closest known species in
the GenBank database, in this case Pantoea agglomerans
DSM 3493
T
(AJ233423). Sequences of six selected isolates
were deposited in the GenBank nucleotide sequence data
library under their respective accession numbers (Fig. 2).
Phosphate solubilization
Solubilization of tricalcium phosphate in broth culture
In order to quantify phosphate-solubilizing activity, EV1,
EV2, EV3, EV4, IEXb, and IEY isolates were inoculated in
Arch Microbiol
123
NBRIP broth and incubated for 7 days. The values of pH
and amounts of soluble P at 24, 48, and 72 h in the medium
are presented in Table 1. The solubilization of Ca
3
(PO
4
)
2
in liquid medium by different strains was accompanied by
a decrease in pH from the initial pH 7 after 72 h. No
relevant changes were observed in the blanks. In general, a
wide range of values of phosphate solubilization among
isolates was observed. The maximum P solubilization at
24 h was recorded by isolate EV1 (470.4 lgml
-1
),
471.7 lgml
-1
at 48 h, and 469.6 lgml
-1
at 72 h with a
Fig. 1 a Clear halo of
solubilization around colony of
PSB isolate after 7 days of
incubation. bPhosphate
solubilizing activity of 33
isolates in NBRIP agar plate
(mm) after 7 days of incubation
at 30°C. Data are the means of
two determinations, and the
error bars indicate standard
deviation (SD). Different letters
indicate significant differences
at P=0.05 according to LSD
test
Pseudomonas rhizosphaerae IH5T(AY152673)
IEXb (FM202487)
Pseudomonas tolaasii ATCC 33618 (D84028)
Pseudomonas fluorescens DSMT 50090 (D84013)
Pantoea eucalypti LMG 24197T (EF688009)
EV2 (FM202484)
EV4 (FM202486)
Pantoea agglomerans DSM 3493T (AJ233423)
EV3 (FM202485)
EV1 (FM202483)
Serratia marcescens subsp. sakuensis (AB061685)
IEY (GQ165811)
Enterobacter aerogenes NCTC10006T (AJ251468)
Escherichia coli ATCC 25922 (FM207087)
93
76
100
100
100
52
74
90
65
100
51
0.02
Fig. 2 Phylogenetic tree showing the relationships among the PSB
isolates and between representatives strain of other related taxa. The
tree was constructed by using the MEGA 4 after aligning the
sequences with ClustalWand generating evolutionary distance matrix
inferred by the neighbor-joining method using Kimura parameter 2.
The numbers at the nodes indicate the levels of bootstrap support
based on data for 500 replicates. Scale bar indicates 0.02 substitutions
per nucleotide position. Escherichia coli ATCC 25922 was used as
the outgroup. Accession numbers of 16S rDNA sequences are given
in parentheses
Arch Microbiol
123
drop in the final pH to 3.3. Soluble P production was
approximately ninefold higher than uninoculated NBRIP
medium (50.0 lgml
-1
). Among the isolates, the minimum
concentration of soluble P (227.4 lgml
-1
) was observed
in the cultures of IEY at 24 h and the pH of the medium
was relatively higher (5.3). Even though a larger decrease
in the pH value was generally associated with higher levels
of P solubilization, in some cases, for example, IEXb,
where pH value decreased only to 4.7, comparatively
higher amounts of soluble P (445.3 lgml
-1
) were detected
in the medium.
Alkaline phosphatase production
The AP production by six selected isolates in limiting and
excess of phosphate was measured in cell extracts by
incubation with p-nitrophenyl phosphate as substrate. It is
known that synthesis of periplasmic AP is repressed at high
phosphate concentrations and derepressed in limiting con-
ditions of Pi (Yashphe et al.1990). Table 2shows specific
activities of AP of our isolates in both conditions. It was
evident the inhibitory effect of high concentration of
phosphate (10 mM) respect to the P-limiting condition
(1 lM) in enzyme production. With 1 lM of phosphate,
EV1 and IEXb isolates produced higher levels of AP (82.1
and 77.3 U/mg protein, respectively), EV4 and IEY were
lower producers with 4.1 and 6.0 U/mg of protein,
respectively, while EV2 and EV3 produced values under
detection limit of AP. On the other hand, values of the
enzyme with 10 mM of phosphate were undetectable for
all isolates assessed here, which should be consequence of
the inhibitory effect at high levels of phosphate in enzyme
production.
Other PGPR traits
IAA production
Considering the phytohormone production as one of the
features for bacteria as PGPR, biosynthesis of total indoles
in selected isolates in M9 medium supplemented or not
with Trp was studied. Figure 3shows the values of total
indoles detected in each case. It can be seen that the
presence of Trp has a significant incidence in the biosyn-
thesis of total indoles in all the isolates. In general, a var-
iation of indolic compounds values among isolates was
observed. The strain IEXb was the higher producer in
absence (5.9 ±0.3 lg IAA ml
-1
) or presence of Trp
(16.8 ±2.9 lg IAA ml
-1
) (Fig. 3).
Table 1 Solubilization of inorganic phosphate and pH by selected
isolates in broth using National Botanical Research Institute’s phos-
phate growth medium (NBRIP) medium with 5 g l
-1
of Ca
3
(PO
4
)
2
at
24, 48 and 72 h
Isolates P conc. (lgml
-1
)pH
24 h 48 h 72 h 24 h 48 h 72 h
Control 50.0
a
49.8
a
49.7
a
7.0 6.9 6.9
EV1 470.4
e
471.7
f
469.6
e
4.1 3.9 3.3
EV2 317.2
d
438.1
e
445.4
d
4.6 4.5 3.3
EV3 351.6
d
370.5
c
387.1
c
4.9 3.8 3.3
EV4 273.9
c
451.2
e
458.1
e
4.5 4.5 3.1
IEXb 330.8
d
391.5
d
445.3
d
4.6 4.6 4.7
IEY 227.4
b
311.5
b
350.0
b
5.3 5.0 5.0
Values within same column followed by the same letter are not sig-
nificantly different according to Fisher’s protected LSD (least sig-
nificant differences)
Table 2 Alkaline phosphatase activities of six selected isolates in
M9 medium with limiting (1 lM) or excess (10 mM) concentrations
of phosphate
Isolates Alkaline phosphatase activity
a
Limiting phosphate Excess phosphate
EV1 82.1 ±1.0 N.D.
EV2 N.D. N.D.
EV3 N.D. N.D.
EV4 4.1 ±0.2 N.D.
IEXb 77.3 ±1.0 N.D.
IEY 6.0 ±0.8 N.D.
a
Activity is expressed in units per mg of protein cell
N.D. not detectable
Fig. 3 Total indoles production of selected isolates grown in M9
medium supplemented (black bar) or not (gray bar) with tryptophan
(Trp) (0.1 mg ml
-1
) after 48 h of incubation at 30°C in rotatory
shaker. Data are the means of three determinations, and the error bars
indicate SD. Different letters indicate significant differences accord-
ing to LSD test (P=0.05)
Arch Microbiol
123
Siderophores production
Additionally, the production of siderophores by selected
strains was tested in CAS agar. After 24 h of incubation,
considering the halo size, EV4 was the larger siderophore
producer, with 9.4 ±0.4, followed by EV2 (7.0 ±0.3),
IEXb (6.7 ±0.3), EV3 (5.4 ±0.2), EV1 (4.8 ±0.2), and
IEY (4.8 ±0.2) mm (Fig. 4). All the bacterial strains
employed in our study produced the same change in color
(from blue to orange), as reported by the literature (Mila-
gres et al.1999; Rosas et al.2006).
Discussion
Phosphate is one of the major nutrients limiting plant
growth, and frequent application of high amounts of
P-fertilizer leads to fixation of phosphate in soil. Phos-
phate-solubilizing microorganisms produce a major con-
tribution to overall plant P nutrition and growth and have
increased yields of many crops. They help to minimize the
application of P-fertilizer, reduce environmental pollution,
and promote sustainable agriculture (Vikram and Ham-
zehzarghani 2008). In this study, 33 rhizobacteria were
isolated based on their inorganic phosphate-solubilizing
capability. Several isolates were stronger solubilizers, and
values of solubilization index in agar plate were higher
than those observed by other authors (Fig. 1b). The
Ca
3
(PO
4
)
2
solubilization by these isolates in agar plate
ranged from 2.2 to 6.8 of SI values compared with 1.5–6.0
of isolates of peat soils from Indonesia (Sitepu et al.2007).
Alam et al.(2002) have reported SI of 3.3 by the best
bacterial strain isolate from maize rhizosphere of Pakistan
soils. Six isolates, EV1, EV2, EV3, EV4, IEXb, and IEY,
were selected due to their high solubilization activity. In
NBRIP broth culture, an inverse correlation between the
pH value of the culture and the released P was observed in
all cases (Table 1). This fact may indicate that phosphate
solubility was directly correlated with the organic acids
produced. There are reports in the literature indicating that
PSB released many kinds of organic acids, but the type of
acid produced is dependent on the microorganism (Chen
et al.2006; Kohler et al.2007; Lin et al.2006; Pandey et al.
2006; Rodrı
´guez and Fraga 1999; Son et al.2006). In fact,
an inverse relationship between pH and soluble phosphate
was reported by the same authors.
Production of enzyme like phosphatases is other mech-
anism of phosphate solubilization (Rodrı
´guez and Fraga
1999). Two isolates, IEXb and EV1, showed high activity
of AP at 1 lM of P; however, the production of this enzyme
was under detection limit in excess of phosphate (10 mM)
compared to limiting condition, which could explained that
the synthesis of alkaline phosphatase by these bacteria was
inducible in low Pi, while it was repressed in high con-
centration. These results are in concordance with solubili-
zation activity in NBRIP broth, where both isolates were
strong P solubilizers. Interestingly, IEXb strain produced a
smaller drop in pH value compared to others isolates. This
might suggest that this strain is capable to solubilize
phosphate by other ways than the production of organic
acid. Therefore, we found a positive correlation between
phosphate-solubilizing capacity and phosphatase enzyme
activity.
Phosphobacteria are capable of producing physiologi-
cally active auxins that may have pronounced effects on
plant growth (Vassilev et al.2006). Bacterial strains were
able to produce IAA in broth culture, and this production
increased in the presence of a physiological precursor, L-
tryptophan. IEXb was the isolate that showed the highest
level of IAA, both in presence or absence of Trp (Fig. 3),
and these values were higher to those recorded by other
researchers. Recently, the IAA production in presence of
the amino acid in the culture media of 9.88 lgml
-1
by
Pseudomonas fragi CS11RH1 and 7.4 lgml
-1
by Pantoea
dispersa strain 1A isolated from a high-altitude Himalayan
rhizosphere has been reported (Selvakumar et al.2008b,
2009). Ali et al.(2010) report auxin production ranged
from 0.05 to 0.84 and 1.16 to 8.22 lgml
-1
in the absence
and presence of L-tryptophan, respectively. On the other
hand, production of siderophores in agar CAS was found in
all isolates, being EV4 isolate the highest producer (Fig. 4).
Siderophore production is beneficial to plants by solubi-
lizing iron formerly unavailable to the plant and has also
some biocontrol properties because it helps a particular
microorganism to compete effectively against other
organisms for available iron, especially pathogenic fungi
Fig. 4 Siderophore production (halo size in mm) by selected isolates
after 24 h of incubation at 30°C in CAS agar plate. Data are the
means of three determinations, and the error bars indicate SD.
Different letters indicate significant differences according to LSD test
(P=0.05)
Arch Microbiol
123
(Deepa et al.2010). Indole-3-acetic acid (IAA) and sid-
erophores, which are among the most frequently studied
metabolites with plant growth promotion capability, are
found to be released by microorganisms that express
P-solubilizing activity (Selvakumar et al.2008a,2009).
These characteristics could give an added value as PGPR to
the bacterial strains assessed here.
The 16S rDNA partial sequence analysis placed EV1,
EV2, EV3, EV4, and IEY isolates at the genus level in
Enterobacter,Serratia, and Pantoea, which are grouped
under one family, Enterobacteriaceae. Only one isolate
IEXb belong to Pseudomonaceae family. Pseudomonas
species are known P-solubilizer (Nautiyal 1999; Peix et al.
2003; Selvakumar et al.2009; Rosas et al.2006), while
several reports have described strains of Serratia marces-
cens (Chen et al.2006; Farhat et al.2009; Selvakumar et al.
2008a), Pantoea agglomerans (Chung et al.2005; Son
et al.2006; Sulbara
´netal.2009), and Enterobacter aer-
ogenes (Deepa et al.2010; Fernandez et al.2007; Wu and
Zhou 2005) as good phosphate solubilizers. Thus, the EV1,
EV3, and IEY strains are similar to other organisms already
related to phosphate solubilizing. However, EV2 and EV4
isolates were identified as Pantoea eucalypti strains, a
novel species recently described by Brady et al.(2008)
isolated from soils of Argentina, Colombia, Uruguay,
South Africa, and Uganda, and IEXb was identified as
Pseudomonas tolaasii strain, none described previously as
P solubilizers.
The properties of these six strains which include solu-
bilization of insoluble phosphate, siderophore, and phyto-
hormones (IAA) production could be useful for
technological applications in the area of plant production.
The growth promoter effect in plants gives extra value to
the isolates, since they may not only be increasing the
bioavailability of one of the most important plant nutrient,
like phosphorus, but also would release substances that
have an antibiotic activity and improve iron nutrition for
the plant, in case of siderophores, and increased lateral and
root hair growth, in the case of IAA production.
The exogenous introduction of phosphobacteria in
agriculture soils would help to decrease the use of chemical
fertilizer while increasing yields. This would mean that a
low-cost ecotechnology engineered through specific bac-
teria responsible for solubilization of rock phosphate could
be of considerable economic importance in the developing
countries. The good results obtained in vitro cannot always
be dependably reproduced under field conditions. It is
expected that inoculation with rhizobacteria containing
PGP characteristics consequently promotes root and shoot
growth. Further, evaluation of the isolates exhibiting mul-
tiple plant growth-promoting (PGP) traits on soil–plant
system is needed to uncover their efficacy as effective
PGPR. The present study is important in view of
identification of native bacterial strains with strong poten-
tial for development as bioinoculants.
Conclusion
In conclusion, the strains Serratia marcencens EV1,
Enterobacter aerogenes IEY, Pantoea agglomerans EV3,
Pantoea eucalypti EV2, Pantoea eucalypti EV4, and
Pseudomonas tolaasii IEXb solubilized larger amounts of
P in NBRIP liquid medium, compared with other native
isolates. All the selected isolates showed to produce sid-
erophores and indoles, two important characteristics within
the PGPR group. Organic acid production was, perhaps,
not the only possible reason for phosphate solubilization in
case of IEXb. These isolates with high phosphate-solubi-
lizing ability appear attractive for exploring their plant
growth-promoting activity toward the development of
microbial inoculants.
Acknowledgments This work was supported by Consejo de In-
vestigacio
´n de la Universidad Nacional de Tucuma
´n. (CIUNT) Pro-
gram 26/D434. Faustino Sin
˜eriz is researcher of CONICET and Maria
Ester Lucca of CIUNT. The authors are thankful to the CONICET for
providing a fellowship to Emilce Viruel. We also thank to Marcela A.
Ferrero for her support and providing laboratory facilities.
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