Arch. Biol. Sci., Belgrade, 67(3), 793-800, 2015
DOI:10.2298/ABS141003038M
PHOSPHATE-SOLUBILIZING BACTERIA ASSOCIATED
WITH RUNNER BEAN RHIZOSPHERE
Gabriela Mihalache1, Maria-Magdalena Zamfirache1, Marius Mihasan1, Iuliu Ivanov2,
Marius Stefan1,* and Lucian Raus3
Alexandru Ioan Cuza University, Iasi, Romania
Regional Oncology Institute, Laboratory of Molecular Biology, Iasi, Romania
3
University of Agricultural Sciences and Veterinary Medicine, Iasi, Romania
1
2
Corresponding author: [email protected]
*
Abstract: Soil microorganisms, especially rhizobacteria, play a key role in soil phosphorus (P) dynamics and the subsequent availability of phosphate to plants. Utilization of phosphate-solubilizing bacteria as biofertilizers instead of synthetic
chemicals is known to improve plant growth through the supply of plant nutrients, and may help to sustain environmental
health and soil productivity. The main purpose of this study was to identify new phosphate-solubilizing bacteria isolated
from runner bean rhizosphere. Ten out of 25 isolated bacterial strains solubilized Ca3(PO4)2 in qualitative and quantitative
P-solubilization. The strain that exhibited the highest potential to solubilize Ca3(PO4)2, was selected for further determination of the mechanisms involved in the process. The medium pH was measured, organic acids released in the culture
medium were identified by HPLC analysis, and the acid and alkaline phosphatase activities were determined. Our results
showed that strain R7 solubilized phosphorous through the production of various organic acids such as lactic, isocitric,
tartaric and pyruvic acids, and that it can be used as a potential biofertilizer.
Key words: rhizobacteria; runner bean; phosphate solubilization; biofertilizer.
Received October 3, 2014; Revised November 18, 2014; Accepted November 24, 2014
INTRODUCTION
Phosphorus is one of the major nutrients for biological growth and development, but for plants is also a
limiting one, due to its low availability of soluble forms
in soil (Vessey, 2003). The biggest reserves of P in soil
are in mineral forms such as apatite, hydroxyapatite
and oxyapatite and in organic forms such as inositol
phosphate, phosphomonoesters, phosphodiesters and
phosphotriesters, which cannot be directly assimilated by plants (Rodriguez et al., 1999, Gamalero et al.,
2011).
The runner bean (Phaseolus coccineus L.) is a climbing perennial vegetable often grown as an annual crop
for dry seeds or immature pod production; it is also
grown as an ornamental vine. Although of minor importance in the United States, the crop is of importance in some parts of Europe. In the United Kingdom, for instance, runner bean is frequently grown
in order to produce a reliable crop of green beans for
commercialization. In Romania the lack of scientific
knowledge about the biology and ecology of runner
bean has contributed to the “slower” progress of this
species (Munteanu et al., 2007).
Solubilization of insoluble and fixed forms of P is
an important issue for increasing soil P availability to
plants (Nautiyal, 1999). Microorganisms are responsible
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for this process, in particular bacteria, which constitute up to 50% of the soil population capable of P
solubilization (Khan et al., 2009). A high proportion of
phosphate-solubilizing microorganisms (PSM) belongs
to the rhizosphere where they are metabolically more
active compared to other sources due to the release of
root exudates (Vazquez et al., 2000).
identify the main mechanism used by a runner bean
rhizobacteria strain to solubilize inorganic phosphate.
The mechanisms by which PSM can solubilize inorganic P are not fully understood, but the production of organic acids seems to be a major mechanism
(Alam et al., 2002). The most common organic acids
produced by PSM are gluconic, 2-ketogluconic, malonic, oxalic, succinic and fumaric acids (Rodriguez et
al., 1999). Other mechanisms that have been involved
in the solubilization of inorganic phosphate are the
release of H+, the production of chelating substances
and inorganic acids, and the synthesis of exopolysaccharides (Gamalero et al., 2011). On the other hand,
phosphatase activity may participate in inorganic
phosphate solubilization (Park et al., 2011).
Bacteria strains were isolated from the rhizosphere
of a field-grown runner bean crop from the Experimental Farm, University of Agriculture Sciences and
Veterinary Medicine, Iasi County, Romania, using the
serial dilution method (Dunca et al., 2004). Aliquots
(0.1 ml) were plated on Bunt and Rovira nutrient
medium and incubated at 28°C for seven days. Isolates were re-streaked on the same nutrient medium,
checked for purity and stored on slants at 4°C.
Large reserves of P are found in most agricultural
soils due to the regular application of chemical fertilizers (Rodriguez et al., 1999). However, 75-90% of
added P fertilizers is rapidly precipitated, soon after
application, by Fe, Al and Ca complexes present in
the soil, making it unavailable to plants (Gyaneshwar et al., 2002). Excessive use of chemicals in traditional cultivation practices has led to a reduction
in soil fertility by modifying its physical, chemical
and biological characteristics, also causing environmental degradation. As an eco-friendly alternative, P
biofertilizers in the form of rhizobacteria can help in
increasing the availability of accumulated phosphates
for plant growth by solubilization (Gyaneshwar et al.,
2002). Therefore, the main purpose of this study was
to identify new phosphate-solubilizing bacteria isolated from runner bean rhizosphere and to determine
their potential to be used as biofertilizer in organic
agriculture. In addition, there are no data concerning
the phosphate solubilization processes in runner bean
rhizosphere and the mechanisms involved in bacterial phosphate solubilization are very controversial.
Therefore, a second objective of our study was to
MATERIALS AND METHODS
Isolation of bacteria
Plate assay
The ability of rhizobacterial strain to solubilize P was
assessed following the plate method (Chaiharn et al.,
2011) using Pikovskaya (PVK) agar medium containing Ca3(PO4)2 as insoluble inorganic form of P. Bacterial strains were streaked in the center of PVK agar
plate and incubated at 28°C for seven days. Bacterial
colonies surrounded by a halo, indicating phosphate
removal, were visually observed and measured. The
solubilization index was measured according to the
formula: the sum of colony diameter and the halo
zone diameter/the colony diameter (Alam et al., 2002).
Three replicate plates were used for each isolate.
Quantitative P assay
Bacteria strains were grown in triplicate in 25 ml
Pikovskaya (PVK) agar medium with and without
Ca3(PO4)2 on a gyratory shaker (190 rpm) at 28°C
(Nautiyal, 1999). Control flasks were not inoculated
with bacteria. A 5-ml aliquot was aseptically removed
from each flask at 0, 3, 5 and 7 days, centrifuged at 4
500 rpm for 30 min and assessed for pH. A supernatant
volume of 1.5 ml from the flasks with Ca3(PO4)2 was
further centrifuged at 14 000 rpm for 30 min and the
Mechanisms of inorganic phosphate solubilization
795
amount of P released was estimated using the phosphomolybdate method (Artenie et al., 2008).
Production of organic acids during
phosphate solubilization
PCR amplification, 16S rDNA sequence
analysis and bacterial identification
For organic acid identification and quantification, 1
ml of culture from each flask was centrifuged at 14
000 rpm for 30 min and filtered through a 0.2-μm
PES syringe filter (Roth, Germany); 20-μl of culturefiltrate was injected on a PRP-x300 PSDVB-Sulfonic
acid column (Hamilton, Germany) and organic acids were monitored using a Bischoff Lambda 1010
UV detector at 220 nm. The mobile phase consisted
of 1 mN sulfuric acid with a flow rate of 1 ml/min
(Walser, 1988).
Total genomic DNA was extracted and purified according to (Ausubel et al., 2002). The 16S rDNA fragment
was amplified with the primers F27 (AGAGTTTGATCMTGGCTCAG) and R1492 (TACGGYTACCTTGTTACGACTT) (Muyzer et al., 1993). PCR reaction was
performed in 20 µl of reaction mixture containing 5 µl
PfuDNA Polymerase 10X Buffer with MgSO4 (Promega), 1µl dNTP mix (10mM each), 10 pmol of forward and reverse primers and 1U PfuDNA Polymerase
(Promega). The reaction conditions included an initial
denaturation of 2 min at 95°C followed by 30 cycles of
30 s at 94°C, 30 s at 52°C and 2 min at 72°C.
The amplification fragment was purified from gel
with Wizard® SV Gel and PCR Clean-up System (Promega Inc, Madison, WI, USA) and then sequenced in
forward and reverse reactions, using the Dye Terminator Cycle Sequencing (DTCS) Quick Start Kit (Beckman
Coulter). The sequencing cycle consisted of 30 cycles
at 96°C for 20 s, 52°C for 20 s and 60°C for 4 min. The
sequencing product was purified by ethanol precipitation according to the manufacturer’s instructions, and
sequence analysis was performed using the CEQ8000
Investigator (Beckman CoulterTM) software. The 16S
rDNA sequence was analyzed using BLAST algorithm
against the SILVA database (Pruesse et al., 2012).
Phosphatase activity assay
Acid and alkaline phosphatase activities were determined using a modified assay of Juma and Tabatabai
(De Freitas et al., 1997). One milliliter of culture supernatant was incubated at 28°C with 1 ml 25mM
p-nitrophenyl phosphate and 4 ml modified universal buffer, pH 6.5 or pH 11 (Thornton et al., 1975),
for assay of acid or alkaline phosphatase. After 3 h of
incubation, the reaction was terminated by adding
1 ml 0.5 M CaCl2 and 4 ml 0.5 M NaOH. The assay
mixtures were centrifuged for 10 min and the yellow
color measured at 410 nm using a Beckman Coulter
DU 720 spectrophotometer.
Statistical analysis
The experimental data were statistically processed
using ANOVA two-factor with replication and Stu-
Fig. 1. Changes in P solubilization (left) and pH value (right) recorded for strain R7 during 7 days of incubation in PVK broth supplemented with Ca3(PO4)2.
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Mihalache et al.
phates activities; Pearson correlation was used for
exploring the relation between solubilized P and
phosphatase activity.
RESULTS
Qualitative screening of bacterial strains
isolated from runner bean rhizosphere
Ten out twenty-five isolated bacterial strains showed
clearly visible halos around their colonies on PVK
agar medium after 7 days of incubation. The solubilization index, based on colony diameter and halo
zone, ranged from 0.33 to 3.19 (Table 1). The highest
solubilization index was recorded for R4 (3.19) followed by R1 (2.89) and R2 (2.87).
Quantitative estimation of phosphate
solubilization in PVK broth
Phosphate solubilization results recorded in liquid
media showed that all the isolates have the potential
to solubilize the inorganic form of P as indicated by a
gradual increase in the amount of soluble P in the medium. The bacterial strain that exhibited the highest Psolubilizing potential was R7 (19.5 μg P/ml), identified
by 16S DNA analysis as belonging to the Pseudomonas
genus. The strain was used for further tests.
Fig. 2. HPLC chromatograms of R7 strain culture in PVK without
Ca3(PO4)2 (left) and in PVK supplemented with Ca3(PO4)2 (right):
A – 3 days of incubation; B – 5 days of incubation; C – 7 days of
incubation.
dent t-test to calculate the differences between the
different solubilization indices and alkaline phos-
The pH value in the uninoculated control flasks
remained almost the same (Fig. 1). A gradual pH
decrease from the initial value of 7.03 to 4.92 on
the 7th day was noticed in PVK broth supplemented
with tricalcium phosphate (Fig. 1). Moreover, it was
observed that the amount of solubilized P increased
along with the medium pH decrease. The lowest pH
value was assessed on the 7th day (4.92) when the
amount of solubilized P reached the maximum value
(19.5 μg P/ml).
Mechanisms of inorganic phosphate solubilization
Table 1. Solubilization index of rhizobacteria isolates after 7 days
of incubation
Bacterial strain
Solubilization index
R1
2.89 ± 0.43
R2
2.87 ± 0.57
R3
2.26 ± 0.10
R4
3.19 ± 0.10
R5
2.7 ± 0.32
R6
0.33 ± 0.33
R7
1.5 ± 0.5
R8
2.62 ± 0.11
R9
0.33 ± 0.33
R10
1.33 ± 0.88
797
PVK supplemented with Ca3(PO4)2 was lactic acid
compared to the control, followed by tartaric and
pyruvic acids (Fig. 2).
Phosphatase activity
The acid phosphatase activity varied from 1.37 pNP/ml/h
to 4.30 pNP/ml/h in the case of R7 strain cultivated in
PVK without Ca3(PO4)2, and between 2.15 and 7.40
pNP/ml/h for the culture from PVK supplemented with
the calcium phosphate. The alkaline phosphatase activity recorded in PVK medium without Ca3(PO4)2 varied
between 1.59 and 7.76 pNP/ml/h, and from 3.33 to 7.75
pNP/ml/h for PVK supplemented with Ca3(PO4)2. The
analysis showed that there are no significant differences
between the acid and alkaline phosphatase activity recorded in PVK medium compared to PVK supplemented with the inorganic P source.
The values are means ± SEM
DISCUSSION
HPLC analysis
HPLC analysis of the culture filtrates was performed in order to identify the organic acids produced by R7 in the presence or absence of inorganic phosphate source from the PVK medium.
The growth of the R7 strain on the PVK medium
supplemented with tricalcium phosphate during
the 7-day incubation resulted in the accumulation
of a major organic acid, namely lactic acid, compared to the control. Other organic acids accumulated during incubation were isocitric, tartaric and
pyruvic acids. The organic acids accumulated in
PVK supplemented with Ca3(PO4)2 on day 3 were
isocitric and lactic (Fig. 2). Day-5 chromatographic
analysis showed an important accumulation of lactic and tartaric acids compared to the previous day
when an increase of solubilized P was also observed
(Fig. 2). On day 7, when the amount of solubilized
P was maximum, the major acid accumulated in
Bacteria isolated from runner bean rhizosphere
were first assayed for their ability to solubilize insoluble phosphate using a plate screening method.
The production of clearing zones around the microbial colonies on PVK medium containing tricalcium
phosphate as the sole P source indicated the presence
of phosphate-solubilizing traits. The solubilization
index results were similar with those recorded by
Alam et al. (2002) for phosphate-solubilizing bacteria
(PBS) isolated from maize.
Because of the contradictory results between
plate halo detection and phosphate solubilization in
liquid media (Fankem et al., 2006), bacterial strains
exhibiting a clear halo on PVK plates were tested for
P solubilization in PVK broth. The results showed
that the best strain that solubilized the same phosphate source in liquid media was one of the strains
that did not have the greatest solubilization index.
This confirms the fact that the plate method is reli-
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Mihalache et al.
able only for isolation and preliminary characterization of phosphate-solubilizing microorganisms
The tricalcium phosphate solubilization during the
7 days of incubation was accompanied by a gradual
pH decrease. The decrease in pH value is in agreement with the findings of Sharma et al. (2012) and
Chaiharn et al. (2011), who observed that the solubility of Ca-phosphate increased exponentially with
pH decrease. The medium pH drop indicates the
production of organic acids as suggested by Mehta
et al. (2001). The production of organic acids seems
to be the main mechanism used by rhizobacteria
for mineral phosphate solubilization (De Freitas
et al., 1997, Rodriguez et al., 1999). The organic
acid produced by phosphate-solubilizing bacteria
most frequently is gluconic acid (Rodriguez et al.,
1999, Vyas et al., 2009, Gulati et al., 2010). Other major organic acids that have been identified
among phosphate solubilizers in high amounts are
oxalic and citric acid (Alam et al., 2002; Rashid et
al., 2004), 2-ketogluconic acid (Muleta et al., 2013)
and succinic acid (Panhwar et al., 2012). Organic
acids found in small quantities during phosphate
solubilization are lactic, formic (Vyas et al., 2009),
malic, propionic (Panhwar et al., 2012), acetic and
fumaric (Alam et al., 2002). However, our results
indicate that lactic, isocitric, tartaric and pyruvic
acids are the main acids produced by the strain R7
(isolated from runner bean rhizosphere). Moreover,
it has been found that during the 7 days of incuba-
tion the amount of solubilized P gradually increased
with the accumulation of lactic acid in combination with isocitric, tartaric and pyruvic acids. This
indicates that organic acid production may have
a central role in the solubilization of an insoluble
phosphate source.
Several authors suggested that phosphatase activity underlies the mechanism of mineral phosphate
solubilization (Tarafdar et al., 1987; Ponmurugan et
al., 2006; Pantujit et al., 2010; Park et al., 2011). Our
results indicate that there is no significant positive
correlation between the amount of solubilized P and
acid and alkaline phosphatase activity (Fig. 3). This
can be explained by the fact that acid and alkaline
phosphatases catalyze the hydrolysis of organic P
compounds such as esters and anhydrides of orthophosphoric acid, and do not solubilize rock phosphates (De Freitas et al., 1997). However, the synthesis of these phosphatases is enhanced when the
level of inorganic P in the growth medium is limiting
(Kaleeswari, 2007; Nannipieri et al., 2011). Hence, an
apparent relationship between P solubilization and
phosphatase activity can be considered coincidental.
CONCLUSION
Our results confirm the potential use of strain R7,
a rhizospheric isolate from runner bean roots, for
the development of a biofertilizer formula based on
Fig. 3. Correlation between phosphorous solubilization and acid (left) and alkaline (right) activity.
Mechanisms of inorganic phosphate solubilization
phosphorous solubilizing bacteria for organic agriculture. The main mechanism used by R7 to solubilize inorganic P consisted in lowering the medium
pH due to the production of organic acids such as
lactic and tartaric acids. The relationship between
phosphatase activity and phosphate solubilization
can be considered coincidental.
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