Int. J. Biosci.
2015
International Journal of Biosciences | IJB |
ISSN: 2220-6655 (Print), 2222-5234 (Online)
http://www.innspub.net
Vol. 6, No. 10, p. 116-124, 2015
RESEARCH PAPER
OPEN ACCESS
Solubilization of inorganic phosphate by Pseudomonas strains
isolated from rice rhizosphere
Saida Aarab, Ferdaouss El Habil-Addas, Amin Laglaoui, Mohammed Bakkali,
Abdelhay Arakrak*
Equipe de Recherche de Biotechnologies et Génie des Biomolécules, Faculté des
Sciences et
Techniques de Tanger, B.P. 416, Tangier, Morocco
Key words: Inorganic phosphate solubilization, Pseudomonas, Rice, Carbon source, Sustainable agriculture.
http://dx.doi.org/10.12692/ijb/6.10.116-124
Article published on May 30, 2015
Abstract
The use of phosphate solubilizing bacteria to mobilize large reserves of insoluble P in soils instead of phosphate
fertilizers is an ecologically safe and economically reasonable alternative to assure a sustainable agriculture. In
this work, the mineral phosphate solubilization activity was evaluated for 4 Pseudomonas strains isolated from
rice rhizosphere. For qualitative tests, different media were used; NBRIP, YED and Pikovskaya (PVK) amended
with 0.5% Ca3(PO4)2 and modified PVK medium containing CaHPO4, Ca5HO13P3, FePO4 and AlPO4 as P source.
All bacteria were able to dissolve calcium phosphates, while none was surrounded by transparent halos when
FePO4 and AlPO4 were the P source. Quantitative test was assessed in PVK substituting glucose by sucrose and
galactose. The highest levels of soluble P were recorded in the presence of glucose, while it didn’t exceed 43.64 μg
/ ml and 19.19 µg / ml in the presence of galactose and sucrose, respectively. Therefore, glucose has been chosen
as a carbon source to assay the effect of carbon concentration on P solubilization ability. The level of dissolved P
was enhanced with increasing amount of glucose (1%, 2% and 3% (w/v)). In all cases, the P biosolubilisation was
accompanied by a significant pH decrease of the liquid media. A significant inverse correlation was found
between P concentrations and pH reduction. Thus, acidification of the media seems to be the main mechanism
for calcium phosphates solubilization in this study, and these test strains could serve as efficient biofertilizer
candidates for improving crop production in low P soils.
* Corresponding
Author: Abdelhay Arakrak  [email protected]
116 Aarab et al.
Int. J. Biosci.
2015
Introduction
simultaneously P uptake by the plant and crop yields
Phosphorus (P) is one of the essential macronutrients
(Hameeda et al., 2008; Shahab et al., 2009).
for plant growth and development. Plants acquire this
element from soil solution as phosphate anions,
Given that numerous studies reported that strains
H2PO-4
(Bhattacharyya and Jha, 2012).
from genus Pseudomonas are among the most
However, P is extremely reactive and may be fixed
efficient PSB (Hayat et al., 2010; Nico et al, 2012;
and it then becomes unavailable to plants. The
Muleta et al., 2013), the present study was designed
phenomenon of P fixation and precipitation in soil is
to evaluate inorganic phosphate solubilizing bacteria
generally highly dependent on pH and soil type. Thus,
belonging to Pseudomonas genus isolated from
in alkaline soils P is fixed by calcium, while in acid
rhizosphere of rice in the presence of some modified
soils, phosphorus is fixed by free oxides and
parameters.
and
HPO2-4
hydroxides of aluminum and iron, causing a low
efficiency of soluble inorganic P used as chemical
Materials and methods
fertilizers to overcome the deficiency of this nutrient
Isolation and screening of PSB from rice rhizosphere
(Gyaneshwar et al., 2002; Hao et al., 2002). Besides,
Seeds of rice (Oryza sativa, Puntal and Elio varieties)
regular application of chemical phosphate fertilizers
were surface sterilized with 95% ethanol for 1min and
is not only costly but is also environmentally
1.2% sodium hypochlorite for 20 min, then washed
undesirable. All these issues have led to search for an
with sterile distilled water repeatedly up to 10 min.
ecologically
Sterilized seeds were put onto Petri plates of 1%
safe
and
economically
reasonable
alternative to assure a sustainable agriculture.
agar/water (w/v) for 48 h at 28 °C. Germinated seeds
were sown in plastic pots filled with 0.4 kg soil, and
Evidence
of
naturally
occurring
rhizospheric
then placed in a plant growth chamber (16 h of
phosphorus solubilizing microorganism (PSM) dates
photoperiod, 18 °C/26 °C). Soil was moistened with
back to 1903 (Khan et al., 2007). Bacteria are more
an equal volume of sterile distilled water for daily
effective in phosphorus solubilization than fungi
watering. Soil composition is the following: sable,
(Alam et al., 2002). Among the whole microbial
24%, clay, 48%, silt, 28%, organic carbon, 0.76%
population in soil, PSB constitute 1 to 50%, while
(w/w), pH 8.27, and P Olsen 17.81 ppm. After 30 days
phosphorus solubilizing fungi (PSF) are only 0.1 to
of growth, plants were harvested and rhizospheric soil
0.5% in P solubilization potential (Chen et al., 2006).
was collected. Two grams of rhizosphere were
It is well documented that a significant number of
dissolved in 18 ml of sterile physiologic water. Then
bacterial species associated with plants roots are able
100 µl of each dilution were plated on King’s B (King
to convert insoluble phosphates (both organic and
et al., 1954) medium.
inorganic) complexes into available forms to the
plants (Pérez et al., 2007; Keneni et al., 2010;
Mineral phosphate solubilizing activity was tested on
Prasanna et al., 2011; Nico et al., 2012) and termed
Pikovskaya
Phosphate
or
amended with 0.5% Ca3(PO4)2. Plates were inoculated
Phosphobacteria (Muleta et al., 2013), and hence they
with all isolates. Formation of clear halo zone around
are a viable substitute to phosphate fertilizers. Many
the colonies after 3 days of incubation indicates
mechanisms like lowering the pH by acid production,
phosphate solubilizing ability.
Solubilizing
Bacteria
(PSB)
(PVK)
medium
(Pikovskaya,
1948)
ion chelation, exchange reactions and polymeric
substances formation in the growth environment have
Qualitative assay of P solubilization
been
Effect of various media on the efficiency of P
reported
to
play
a
role
in
phosphate
solubilization by PSB (Chen et al., 2006; Pérez et al.,
solubilization
2007; Delvasto et al., 2008). Application of PSB as
Phosphorus solubilizing ability of selected bacterial
inoculants
strains was tested in 3 different types of solid media
has
been
117 Aarab et al.
reported
to
increase
Int. J. Biosci.
2015
reported in literature. We used NBRIP (Nautiyal,
source for phosphate solubilization. The conditions of
1999), YED-P (Peix et al., 2001) and PVK. All these
the
media contained 0.5% Ca3(PO4)2. Plates were spot-
concentrations of dissolved P and final pH were
inoculated with 10 µl overnight culture and incubated
carried out as described previously.
experiment
and
the
determination
of
at 28 °C for 7 days. The ability to solubilize inorganic
phosphate was described by the solubilization index
Statistical analysis
(SI) (ratio of halo diameter to colony diameter).
The data are reported as means ± SD (standard
Rests of the experiment were performed in PVK
deviation) for three replicates or more. The results
media amended with 0.5 Ca3(PO4)2.
were subjected to analysis of variance (ANOVA)
according to Fisher protected LSD test (p < 0.05)
Solubilization
of
other
sources
of
inorganic
using the Statgraphics Plus version 4.0.
phosphate
Qualitative assay of solubilization of other sources of
Results
inorganic phosphate was evaluated using modified
Qualitative assay of phosphate solubilization
PVK
by
Out of 57 bacteria isolated from the rhizosphere of
hydroxyapatite (Ca5HO13P3), dicalcium phosphate
rice (Oryza sativa), 54% (31 isolates) showed clear
(CaHPO4), aluminium phosphate (AlPO4) or iron
zones on PVK solid medium supplemented with
phosphate (FePO4). The inoculated plates were
Ca3(PO4)2. Based on the diameter of the solubilizing
incubated at 28 °C for 7 days. SI was calculated as
halos on PVK medium (SI) and other characteristic
described previously.
tests (data not shown), 4 bacteria PP7, PP17, PP31
medium,
substituting
Ca3(PO4)2
and PE15, were selected for P solubilization analysis.
Quantitative assay of P solubilization
Using different media reported in the literature to
Effect of carbon source on phosphate solubilization
select PSB, SI recorded for qualitative test of
Effect of carbon sources on phosphate solubilizing
phosphate solubilization activity is presented in the
activity was determined in PVK liquid medium
table 1. The halos obtained in NBRIP were not clear to
amended with 0.5% Ca3(PO4)2 with addition of 1% of
be measured in comparison with 2 other media. The 4
respective sugars, glucose, galactose and sucrose. Test
bacteria gave the highest SI in PVK solid medium.
bacteria were inoculated into 100 ml PVK’s broth and
Besides, isolate PP17 was not able to dissolve
incubated on a rotary shaker at 30 °C for 7 days.
Ca3(PO4)2 present in YED-P while it was screened as
Controls consisted of uninoculated culture medium.
Ca3(PO4)2 solubilizer on PVK. These results confirm
After centrifugation at 13,000 rpm for 20 min,
the effect of media composition on P solubilizing
concentrations of soluble P released in the media
activity of bacteria.
were detected by the colorimetric method as
described by Ames (Ames, 1966). The rate of P
When Ca3(PO4)2 was substituted by other sources of
solubilization was estimated by subtracting the P
inorganic P, CaHPO4, Ca5HO13P3, FePO4 and AlPO4,
concentration of uninoculated control from the final
all selected bacteria were able to dissolve calcium
concentration of soluble P obtained in the inoculated
phosphates showing transparent halos around the
media. The final pH of media was measured with a
colonies with different SI (Table 1). Moreover, the
pH meter with a glass electrode.
highest SI value was recorded for the isolate PP7 in
plates containing CaHPO4 as sole P source. In
Effect
of
concentration
of
carbon
source
on
contrast, although all bacteria were able to be grown
phosphate solubilization
in plates containing FePO4 and AlPO4 as sole P
To estimate the effect of different concentrations of
source, none were surrounded by clear zones (Table
carbon source, the test was carried out in PVK
1).
medium containing 1%, 2% and 3% of the best carbon
118 Aarab et al.
Int. J. Biosci.
2015
Considering efficacy of P solubilization in PVK
Phosphate solubilization in presence of 3 carbon
medium, rests of the experiments were performed
sources was accompanied by a significant decrease in
using PVK amended with 0.5% Ca3(PO4)2.
pH of the media by all strains (Table 2). This
acidification was intense in PVK amended with
Quantitative assay of phosphate solubilization
glucose with pH falling to 3.89 from initial pH of 7.0.
After evaluating the phosphate solubilizing ability on
The pH results show the interference of carbon source
solid medium, the quantitative assay of this activity
in the solubilization of Ca3(PO4)2. An inverse
was carried out in PVK liquid medium. The results
correlation between the amount of soluble P and the
show that the solubilization ability varied among the
reduction of pH in the inoculated media (r = - 0.74
4 PSB and was dependent on the carbon source (Fig.
and p < 0.01) was found. Thus, the efficient isolates
1). Among them, glucose was the best carbon source
solubilized high amounts of P with strong decrease in
for the insoluble phosphate solubilization. The most
pH,
efficient P solubilizer was PP17 that solubilized 234.14
accompanied by weak decrease in final pH (Fig. 1,
µg/ml of P from 0.5% Ca3(PO4)2 (Fig. 1). Low level of
Table 2).
but
low
levels
of
P
solubilization
was
P solubilization was observed in other media,
especially in the presence of sucrose as carbon source.
Table 1. Solubilization index (SI) recorded for selected bacteria after 7 days of incubation.
Isolates
Different media
PVK with different inorganic phosphates sources
PP7
PP17
PP31
NBRIP
ND
ND
ND
YED-P
1.55 (±0.09)
2.17 (±0.29)
Ca3(PO4)2
2.35 (±0.14)
1.93 (±0.35)
2.44 (±0.36)
CaHPO4
2.77 (±0.07)
2.26 (±0.13)
2.16 (±0.05)
Ca5HO13P3
1.87 (±0.24)
1.50 (±0.25)
1.83 (±0.29)
FePO4
-
AlPO4
-
PE15
ND
2.25 (±0.31)
2.41 (±0.28)
2.22 (±0.09)
1.75 (±0.10)
-
-
Values are expressed as mean ± standard deviation of three independent readings. ND: Not determined.
Considering efficacy of P solubilization obtained in
concentrations of soluble P were released at 3%
the presence of glucose, PVK amended with glucose
glucose. The most powerful P solubilizer was PP17
was used to evaluate the P solubilization ability in
releasing 794.54 mg/l of soluble P in presence of 3%
presence of various concentrations of glucose ranging
glucose followed by PP31 (742.68 µg/ml at 3%
from 1%, to 3% (w/v). The 4 selected bacteria showed
glucose).
different abilities to dissolve Ca3(PO4)2 depending on
concentration of soluble-P was observed in the
glucose concentrations (Fig. 2). The P solubilizing
cultures of PP7 strain that dissolved 522.04 µg/ml of
ability was enhanced with increasing amounts of
soluble P at 3% glucose.
glucose
in
the
medium,
and
the
Among
the
isolates
the
minimum
maximum
Table 2. Final pH of PVK media inoculated with selected bacteria after 7 days of incubation.
Isolates
Glucose
Sucrose
Galactose
PP7
3,98 ab
5,06 fg
4,59 de
PP17
3,89 a
5,66 h
4,32 cd
PP31
4,20 bc
5,27 j
4,81 ef
PE15
3,89 a
5,06 fg
4,31 c
Data followed by the same letter are not significantly different according to Fisher protected LSD test (p < 0.05).
This solubilization was accompanied as well by a
(Table 3). The maximum P solubilization was
significant drop in final pH of inoculated media when
recorded by isolate PP17 with a maximum drop in the
compared with the uninoculated control (pH 7.0)
pH to 3.5, followed by PP31. Even though maximum
119 Aarab et al.
Int. J. Biosci.
2015
drop in pH was associated with higher levels of P
Plant Growth Promoting Rhizobacteria (PGPR) may
solubilization, in some cases, for example PP7 strain,
use one or more mechanisms for plant growth
where pH was decreased to 3.47, comparatively lower
improvement
amounts of soluble P was detected in the medium.
phosphates is one of the most important traits in
Statistically a weak negative correlation (r= -0.38 and
increasing plant yields, and Khan et al. (2009) has
p< 0.05) between pH decrease and P release was
emphasized
observed.
production. Pseudomonas contains the most efficient
and
the
solubilization
importance
of
of
PSB
insoluble
in
crop
and frequently encountered P solubilizing strains
Discussion
(Keneni et al., 2010). Hence, the present work
Qualitative assays of inorganic P solubilization
focused on this bacterial genus isolated from the rice
rhizosphere.
Table 3. Final pH of PVK media inoculated with selected bacteria after 7 days of incubation.
Isolates
Glucose 1%
Glucose 2%
Glucose 3%
PP7
3,98 cde
3,72 bc
3,47 ab
PP17
3,88 cd
3,71 bc
3,5 ab
PP31
4,21 e
4,08 de
4,12 de
PE15
3,95 cde
3,45 ab
3,32 a
Data followed by the same letter are not significantly different according to Fisher protected LSD test (p < 0.05).
In the literature, a lot of media were used to evaluate
compounds added to the media as source of P, the 4
phosphate
the
selected PSB were found to be more efficient in
microorganisms. In this study, PVK, NBRIP and YED-
solubilizing calcium phosphates than aluminum and
P were chosen. The SI obtained were different
iron phosphates. This finding is in agreement with a
according to the media, even if these media were
previous report of Pérez et al. (2007) who isolated
amended with the same P source, Ca3(PO4)2.
PSB from an acidic soil that were showed transparent
Correspondingly,
(2005)
halos in plates of NBRIP amended with Ca3(PO4)2
evaluated two P solubilizing fungi isolated from rice
while they were absent around the colonies in plates
filed soil, using different media reported in literature
where FePO4 and AlPO4 were sole P source. Same
to select P solubilizing microorganisms, PVK, NBRIP
data were obtained by Chang and Yang (2009). This
(Nautiyal, 1999), AYG (Halder et al., 1991) and 2
phenomenon could be explained by the use of
others described by Kim et al. (1997) and Vassilev et
experimental approaches which are inappropriate to
al. (1998). They confirmed the impact of media type
select iron and aluminum phosphate solubilizing
as well as carbon and nitrogen sources on P
bacteria or caused by the toxicity of Fe3+ and Al3+ and
solubilization ability. Similarly, Son et al. (2006)
their inhibitor effect on the solubilization activity of
affirmed the effect of the concentrations of some
PSB (Pérez et al., 2007). Besides, bacterial growth in
elements of the media as salts and nitrogen source on
the absence of any visible sign of phosphate
solubilization of the inorganic phosphates. Recently,
solubilization in plates containing FePO4 or AlPO4
Sagervanshi et al. (2012) obtained the highest
was also observed, and may have resulted from the
concentrations of soluble P in PVK liquid medium in
release of small amounts of soluble P during
comparison with other media as AYG, NBRIY and
autoclaving the media.
solubilization
Pradhan
ability
and
Sukla
of
NBRIP.
Quantitative assays of Ca3(PO4)2 solubilization
As the transparent halos around the isolates on agar
To determine the effect of carbon source on P
plates amended with insoluble phosphates indicate
solubilization, 3 different carbon sources, sucrose,
the solubilization of these inorganic phosphate
galactose
120 Aarab et al.
and
glucose,
were
provided
at
the
Int. J. Biosci.
2015
concentration of 1% (w/v). The selected bacteria
works (Son et al., 2006; Keneni et al., 2010).
demonstrated diverse levels of P solubilization
According to several studies, this acidification is
activity in PVK liquid media depending on carbon
caused by production of organic acids by bacteria
sources. The PVK medium containing glucose showed
(Pérez et al., 2007; Song et al., 2008; Mardad et al.,
maximum P solubilization. Low levels of soluble P
2013).
were observed in other media, especially in the
between pH and P indicates the major role of these
presence of sucrose as carbon source. This finding
organic acids in mineral P solubilization. Most of the
may be related to the complexity of sucrose and the
previous reports state that the main mechanism of
preference of glucose. These results affirm the
mineral phosphates solubilization by PSB strains is
interference of carbon source in the solubilization of
through biosynthesis and release of a wide variety of
Ca3(PO4)2. This observation is consistent with Son et
organic acids and gluconic acid is the principal
al. (2006) and Song et al. (2008) who have shown
organic acid produced by most P solubilizing bacteria
that the dissolved P in glucose medium was at less 2
(Song
times more than that of sucrose and galactose
Consequently, production of acids is greatly affected
medium. Moreover, Sagervanshi et al. (2012) found
by the nature and the concentration of carbon sources
that PVK with glucose gave maximum P solubilization
(Pradhan
followed by galactose.
microorganism that acidifies its external medium will
The
et
statistically
al.,
and
2008;
Sukla,
significant
Mardad
2005).
et
relationship
al.,
Therefore,
2013).
any
show some level of phosphorus solubilizing activity.
Nevertheless, Hamdali et al. (2012) have reported
alkalinization during the solubilization of rock
phosphate by actinobacteria isolated from a Togolese
phosphate mine and absence of organic acids in the
growth medium. This explains the involvement of
other mechanisms in the process of inorganic P
solubilization as the production of siderophores
(Hamdali et al., 2008; 2012). Thus, microbial PFig. 1. Concentration of P dissolved by 4 selected
bacteria in presence of different carbon sources after
7 days of incubation.
solubilization is a complex phenomenon that involves
several mechanisms. It depends on many factors such
as nutritional, physiological and growth conditions of
the culture (Pradhan and Sukla, 2005; Son et al.,
As the best solubilization was obtained in the
2006; Sagervanshi et al., 2012).
presence of glucose, it has been chosen as carbon
source to assay the effect of 3 various carbon
concentrations on P solubilization ability. This
activity was enhanced with increasing amounts of
glucose in the medium, and the maximum levels of
soluble P were released at 3% glucose. Similarly, Song
et al (2008) confirmed that P solubilization was more
effective when the glucose concentration in the
medium was raised from 1 to 3%, but decreased in the
5% of glucose.
Fig. 2. Concentration of P dissolved by 4 selected
bacteria in presence of different amounts of glucose
In all cases, the P solubilization was concomitant with
after 7 days of incubation.
a significant pH decrease. A significant negative
correlation was found between dissolved P and pH of
Conclusion
the media, which has been found also by previous
The overall results highlight one of P solubilization
121 Aarab et al.
Int. J. Biosci.
2015
mechanisms, which is acidification of the medium.
JA, González F, Blázquez ML, Igual JM,
Moreover, this study makes the 4 test bacteria
García-Balboa C. 2008. Diversity and activity of
attractive as phosphate solubilizers and might be used
phosphate
as biofertilizers to increase the available P in soil, to
phosphorus iron ore. Hydrometallurgy 92, 124–129.
minimize chemical fertilization, and to reduce
http://dx.doi.org/10.1016/j.hydromet.2008.02.007
environmental pollution in order to achieve a
Gyaneshwar P, Kumar GN, Parekh LJ, Poole
sustainable agriculture. However, further studies are
PS. 2002. Role of soil microorganisms in improving
required based on the plant growth promoting effect
P nutrition of plants. Plant Soil 245, 83–93.
of these isolates under pot culture as well as field
http://dx.doi.org/10.1023/A:1020663916259
conditions
before
they
are
recommended
bioleaching
bacteria
from
a
high-
as
biofertilizers.
Halder AK, Mishra AK, Chakarbarthy PK. 1991.
Solubilization
of
inorganic
phosphate
by
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