Biol Fertil Soils (2010) 46:755–763
DOI 10.1007/s00374-010-0482-8
ORIGINAL PAPER
Soil fungal isolates produce different organic acid patterns
involved in phosphate salts solubilization
Jose Martin Scervino & Milton Prieto Mesa &
Ivana Della Mónica & Marina Recchi &
Nubia Sarmiento Moreno & Alicia Godeas
Received: 22 April 2010 / Revised: 16 June 2010 / Accepted: 30 June 2010 / Published online: 14 July 2010
# Springer-Verlag 2010
Abstract Phosphorus availability is a major limiting factor
for yield of most crop species. The objective of this study
was to compare the solubilization of three sources of
phosphorus (P) by different fungal isolates and to determine
the possible mechanisms involved in the process. Talaromyces flavus (S73), T. flavus var flavus (TM), Talaromyces
helicus (L7b) and T. helicus (N24), Penicillium janthinellum (PJ), and Penicillium purpurogenum (POP), fungal
strains isolated from the rhizosphere of crops, are known to
be biocontrol agents against pathogenic fungi. The P
solubilization efficiency of these fungal strains in liquid
media supplemented either with tricalcium phosphate
(Ca3(PO4)2; PC), aluminum phosphate (AlPO4; AP), or
phosphorite (PP) depended on the source of P and the
fungal species. The type and concentration of organic acids
produced by each species varied according to the source of
available P. In the medium supplemented with PC, the
highest proportion was that of gluconic acid, whereas in the
media supplemented with the other P sources, the highest
proportion was that of citric and valeric acids. This suggests
that the release of these organic compounds in the
rhizosphere by these microorganisms may be important in
the solubilization of various inorganic P compounds.
Results also support the hypothesis that the simultaneous
J. M. Scervino (*) : I. Della Mónica : M. Recchi : A. Godeas
Departamento de Biodiversidad y Biología Experimental,
Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires,
Pabellón II, 4P Ciudad Universitaria,
1428 Buenos Aires, Argentina
e-mail: [email protected]
M. P. Mesa : N. Sarmiento Moreno
Depto de Ingeniería Química, Facultad de Ingeniería,
Universidad Nacional de Colombia,
Bogotá, Colombia
production of different organic acids by fungi may enhance
their potential for solubilizing insoluble phosphate.
Keywords Talaromyces . Penicillum . Phosphorus .
Phosphate-solubilizing microorganisms . Organic acids
Introduction
Most cultivated soils are deficient in available forms of
phosphorus (P). The average amount of P in soil is
approximately 0.05% (w/w), but only 0.1% present in the
soil solution as orthophosphate ions is available to plants.
The free inorganic P in the soil solution thus plays an
important role in the biogeochemical cycle of this element
as well as in plant nutrition (Scheffer and Schachtschabel
1992).
The rhizosphere supports a large number of organisms
that contribute to the global composition of metabolites in
the soil solution by releasing exudates. In 1948, Gerretsen
showed that the activity of these rhizosphere populations
contributes to dissolving insoluble P, thus promoting plant
growth (Gerretsen 1948). Some fungi produce and release
organic acids that allow the formation of organic mineral
complexes, and thus play an important role in the economy
of P (Richardson et al. 2001; Ryan et al. 2001). Together
with the release of organic acids, protons are produced that
contribute to the acidification of the soil solution and, as a
consequence, to the solubilization of some forms of
inorganic P. Therefore, a method to estimate the solubilizing ability of a microorganism is to determine the amount
and nature of organic acids produced (Vassilev et al. 2006).
However, the synthesis of these compounds is not the only
mechanism by which P is solubilized; other processes, such
as the assimilation of ammonium where protons that acidify
This copy belongs to 'tribhuwan'
756
Biol Fertil Soils (2010) 46:755–763
the rhizosphere are released, can also take place (Pradhan
and Sukla 2005).
Different soil microorganisms have been reported to
solubilize P in vitro. These microorganisms, therefore, have
the potential to promote plant growth by increasing soil
fertility (Richardson et al. 2001). Recently, organisms that
have a role both as biocontrol agents and as P solubilizers
have gained importance in the field of biogeochemistry and
in maintaining soil quality (Vassilev et al. 2006). Among
these organisms are species of Aspergillus, Penicillium,
Talaromyces, and Eupenicillium, which are considered “key
organisms” in the P cycle (Whitelaw 2000). Most of them
solubilize inorganic calcium phosphates and have a limited
capacity of solubilizing aluminum or iron phosphates (Illmer
and Schinner 1995). In the soil, the efficiency of organisms
to use P is very low because the P applied as fertilizer is
fixed by aluminum or iron in acid soils and by calcium in
alkaline soils (McLaughlin et al. 1988). Although a large
number of organisms have been studied in relation to their
capacity to solubilize insoluble forms of calcium phosphate
(Wakelin et al. 2004; Stephen and Jisha 2009), there are few
in vitro studies concerning the solubilization of other
phosphates or P salts (Barroso et al. 2006).
Therefore, the objective of this study was to evaluate
new isolates of P-solubilizing and biocontrol fungi in order
to obtain further information about the mechanisms
involved in the solubilization of P salts. We tested the
capacity of these fungal species to produce organic acids, as
well as their efficiency in the solubilization of P sources.
(Pitt 1979) Fungi were identified as Talaromyces flavus
(S73), T. flavus var flavus (TM), Talaromyces helicus (L7b)
and T. helicus (N24). In addition, two strains of Penicillium
previously studied in this laboratory (Penicillium purpurogenum and Penicillium janthinellum) were selected from
the preliminary screening based on the size of the halo
of solubilization. The strain P. purpurogenum (POP) was
provided by the strain collection of the School of
Sciences of the University of Buenos Aires (BAFC
3303) and P. janthinellum (PJ) by the fermentation
laboratory of the Biotechnology Institute of the National
University of Colombia. The cultures routinely maintained on Malta agar in 90-mm diameter Petri plates at
28°C were grown for 5 days in a medium containing malt
extract 10.0 g, glucose 4.0 g, and distilled water 1,000 ml.
The mycelium was macerated to obtain a homogeneous
mycelial suspension.
Liquid NBRIP (100 ml) at pH 7 was added to 500-ml
conical flasks and the strains inoculated in each flask with a
uniform inoculum of 50 μl of mycelial suspension.
The NBRIP medium contained different commercial
insoluble inorganic forms of P: Ca3(PO4)2 (PC; Sigma
C5267) 5 g/L, AlPO4 (AP; Aldrich 34.145-2) 1 g/L, and
phosphorite (PP; Neiva, P2O5 30%, CaO 35–40%, F 2%)
5 g/L. The cultures were incubated for 7 days under
controlled shaking conditions (30°C in the dark) and
samples (2 ml) were taken every 24 h. These samples were
stored at −70°C until chemical analysis.
Determination of nitrogen and phosphorus
Materials and methods
Selection of microorganisms
Sixty fungal isolates isolated by Rodríguez (2004) and reported
to be biocontrol agents against Sclerotinia sclerotiorum were
inoculated in Petri dishes containing National Botanical
Research Institute's phosphate growth medium (NBRIP;
Nautiyal 1999), with glucose 10 g L−1, MgSO4 12 g L−1,
KCl 0.2 g L−1, MgCl2 5 g L−1, (NH4)2SO4 0.1 g L−1, agar
15 g L−1, and Ca3(PO4)2 5 g L−1 and supplemented with
ampicillin 0.1 g L−1. The Petri plates were incubated at 25°C
in the dark and cultures were examined every 2 days for the
presence of a halo around the fungal colony (Wakelin et al.
2004); cultures forming an obvious halo around the colony
were selected and identified.
Media and growth conditions: phosphate
solubilization experiment
The four fungal isolates showing pronounced halos were
identified using morphologic and physiologic characteristics
Cultures were harvested after 0, 24, 48, 72, 120, and
144 h, centrifuged at 10,500×g for 20 min and the
biomass was measured at 144 h. The samples taken at
different times were used to measure N, P, and pH. The
release of P from mineralogical pure substances and rock
powders was analyzed by the Merck test kit (14842) for
the determination of orthophosphate and the N consumption was determined by measuring the residual ammonium
in the supernatant by the Berthelot method (Weatherburn
1967).
Determination of organic acids
The organic acids isolated at 144 h from NBRIP liquid media
supplemented with three different sources of inorganic
phosphate were analyzed by high-performance liquid chromatography using an Aminex column HPX-87-H (Cat. No.
125-0140) and a UV detector set to 210 nm at 50°C. The
supernatants were filtered through a 0.22-μm nylon filter and
the pH was recorded. The solvent as mobile phase consisted
of 5 mM H2SO4 run at a flow rate of 0.6 ml min−1. Peaks
were identified against a set of standards from known
This copy belongs to 'tribhuwan'
Biol Fertil Soils (2010) 46:755–763
757
organic acids (gluconic, citric, glucuronic, tartaric, pyruvic,
succinic, fumaric, propionic, butyric, valeric, lactic, acetic,
malic, and oxalic acids).
Data analysis
The experiments were carried out at least twice with similar
results in five replicates per treatment. One treatment
without inoculum was used as control. Statistical analysis,
two-way analysis of variance (ANOVA), one-way ANOVA,
and plot graphics were performed with the computer
software Statistica 7.0. The differences obtained by
ANOVA were determined by post hoc Tukey's analysis.
All assumptions were tested for normality and variances.
Results
Consumption of nitrogen (ammonium sulfate)
and solubilization of P
The strains grew in media supplemented with three P
sources. The amount of P in the medium, estimated as
milligrams per liter of phosphate (PO42−), showed variations according to the fungal strain and the P source. In the
supernatant, the consumption of ammonium and the P
solubilization were inversely related. When the P added
was in the form of PC, maximum solubilization was
detected after exhaustion of the N source during the
exponential phase of growth, accompanying the fall of pH
(Fig. 1a, d, g). In contrast, in the media with AP and PP,
maximum solubilization was detected 48 h after the
exhaustion of N in the stationary phase, after reaching the
minimum pH (Fig. 1b, e, h and c, f, i, respectively).
efficiency and solubilized 0.4±0.02 and 0.36±0.08 mg L−1
(PO42−) g−1, respectively, whereas TM, L7b, N24, and PJ
(b) solubilized 0.188±0.015, 0.19±0.03, 0.14±0.015, and
0.1±0.02 mg L−1 (PO42−) g−1, respectively (Fig. 2b).
When grown in PP, the strain TM (a) showed a higher
solubilization efficiency (1.61±0.02 mg L−1 (PO42−) g−1),
whereas S73, L7b, N24, POP, and PJ (b) solubilized 0.61±
0.02, 0.68±0.03, 0.9±0.06, 0.98±0.1, and 0.82±0.01 mg L−1
(PO42−) g−1, respectively (Fig. 2c).
Growth of the strains in different sources of P
The growth of the strains differed significantly independently of the P present in the medium, according to the
ANOVA (two factors: strains×sources of P, p≤0.05).
Besides, the growth of the organisms belonging to one
strain differed according to the P source (p≤0.05).
Since the interaction between factors did not show
significant differences, we analyzed the main effects of
the P source×strain on the biomass (Fig. 3a, b) and found
that, independently of the source of available P, the growth
of the strain TM (b) was significantly lower than that of the
other strains, reaching a biomass development (dry weight)
of 222.35±55 mg. The other strains, i.e., S73, L7b, N24,
POP, and PJ (a), produced 330.34±63, 379.1±62, 353.72±
66, 291.58±65, and 322.6±58 mg of dry weight, respectively (Fig. 3a).
On the other hand, independently of the strain studied,
the growth of the organisms was significantly lower in the
presence of PP. The biomass produced in the medium with
PP was of 258.22±52 mg (b), whereas that in the media
with PC and AP (a) was of 351.02±61 and 340.70±45 mg,
respectively (Fig. 3b).
Production of organic acids
Solubilization efficiency
The efficiency of P solubilization of the strains in the different
media was determined 144 h after the inoculation as milligrams per liter of phosphate (PO42−) ions per gram of
biomass produced. The values obtained were compared for
each P source by one-factor ANOVA (p≤0.05), which
indicated that there were significant differences in the
capacity of P solubilization between some of the treatments (Fig. 2). According to the post hoc analysis
(Tukey's test), the strains that grew in PC were grouped
as follows: on one hand, TM and POP (a), which showed a
higher solubilization efficiency of 9 ± 0.22 and 8.1 ±
0.25 mg L−1 (PO42−) g−1, respectively, and on the other
hand, the remaining strains, S73, L7b, N24, and PJ (b),
which solubilized 4.5±0.23, 2.9±0.25, 5.1±0.5, and 4.2±
0.6 mg L−1 (PO42−) g−1, respectively (Fig. 2a). When
grown in AP, the strains S73 and POP (a) showed a higher
The production of organic acids in the media with different
sources of P was different: we detected 11 different organic
acids in the medium with PC (Table 1), seven in the
medium with AP (Table 2), and six in the medium with PP
(Table 3). Butyric, citric, gluconic, fumaric, lactic, and
succinic acids were produced in the three media.
In the medium with PC, 67% of the production
corresponded to gluconic acid (133.53±3.47 mg L−1),
19.2% to glucuronic acid (38.61±6.8 mg L−1), 2.85% to
citric acid (5.63±0.18 mg L−1), and 8.5% to valeric acid
(16.78±3.59 mg L−1). The remaining 2.43% corresponded
to succinic acid, propionic acid, butyric acid, lactic acid,
fumaric acid, and oxalic acid (Table 1).
In PC, gluconic acid accounted for 99.8%, 98.5%,
69.1%, and 63.3% of the acid produced by strain S73,
TM, N24, and POP, respectively, whereas citric acid
accounted for 95.9% of the organic acid produced by
This copy belongs to 'tribhuwan'
758
Biol Fertil Soils (2010) 46:755–763
Fig. 1 Solubilized P (a–c), changes in ammonium concentration (d–
e), and pH (g–i) during the solubilization of PC (a–d and g), AP (b–e
and h), and PP (c–f and i) by Penicillium and Talaromyces. The curve
represents values taken at different times. Values with the same letter
are not significantly different as determined by Tukey's test (p≤0.05)
L7b. Glucuronic acid accounted for 28.3% and 96.8% of
the acid produced by N24 and PJ, respectively (Table 1)
The study of the organic acids produced by the organisms
that grew in the medium supplemented with AP showed that
the highest proportion was that of valeric acid (51.77%), being
the strain POP the one that most contributed to it (29.39±
0.763 mg L−1). The total production of citric acid was
22.22% (12.63±2.15 mg L−1), whereas that of gluconic acid
was 22.09% (12.55±1.58 mg L−1).
In AP, citric acid accounted for 99.3%, 86.1%, 72.1%,
and 8.7% of the acid produced by strains S73, L7b, N24,
and PJ, respectively, whereas gluconic acid accounted for
98.2% of the organic acid produced by TM. POP produced
46.2% and 45.1% of gluconic acid and valeric acid,
respectively (Table 2).
The production of malic, succinic, fumaric, and lactic
acids constituted a little more than 5% of the total
production of organic acids (Table 2).
In the medium supplemented with PP, the organic acid that
appeared in the highest proportion (166.98±14.02 mg L−1)
was that of gluconic acid (93.71%). About 6% corresponded to
citric acid (10.53±1.045 mg L−1) and <1% corresponded to
fumaric acid (0.01±1×10−4 mg L−1), butyric acid (5.8×10−3 ±
6.5×10−4 mg L−1), succinic acid (0.36±0.015 mg L−1), and
lactic acid (0.34±0.092 mg L−1).
In PP, gluconic acid accounted for 99.5%, 99.8%,
88.3%, 93.23%, and 95.1% of the acid produced by strains
S73, TM, N24, POP, and PJ, respectively. L7b produced
gluconic acid (77.8%) and citric acid (22.1%; Table 3).
Total production of organic acids (in milligrams per
liter of total organic acids per gram of biomass) by each
strain in the different media is presented in Tables 1, 2,
and 3.
Discussion
The species of Penicillium and Talaromyces used in this
study were isolated from the rhizosphere, the interface
between plant roots and soil in which microorganisms are
This copy belongs to 'tribhuwan'
Biol Fertil Soils (2010) 46:755–763
759
Fig. 3 Principal effects during the solubilization of PC, AP, and PP
(source of P× fungal species). Dry weight independent of P sources
(a) and dry weight independent of fungal species (b). Main effects:
F(2, 36) = 19.945, p = 0.00000. Vertical bars denote 0.95 confidence
intervals. Values with the same letter are not significantly different
as determined by Tukey's test (p ≤ 0.05) between fungal species
Fig. 2 Solubilized P by grams of fungus at 144 h during the
solubilization of PC (a), AP (b), and PP (c). Values with the same
letter are not significantly different as determined by Tukey's test (p≤
0.05) between fungal species
most important for the growth and protection of plants. The
strains used in this experiment had been previously tested
as antagonist organisms of fungal pathogens (Rodríguez
2004).
Our results support the hypothesis of Vassilev et al. (2006)
that the compounds involved in the biological control against
soil-borne phytopathogens are often combined with the
production of organic acids involved in the solubilization of
insoluble phosphates.
We used PC in solid medium as the source of
insoluble P. This source, which has been used before
(Shantaram and Saraswathy 1985; Kang et al. 2002; Elzouni 2008; Xiao et al. 2008), overestimates the solubilizing capacity of microorganisms. In media with PC,
some microorganisms can solubilize P by decreasing the
pH of the medium as a result of their metabolic activity
(Wakelin et al. 2004); this does not occur in media with
AP and PP. After the experiments in solid medium
supplemented with PC, the analysis of the efficiency and
capacity of production of organic acids in liquid medium
This copy belongs to 'tribhuwan'
760
Biol Fertil Soils (2010) 46:755–763
Table 1 PC solubilization and organic acid production by different fungal strains
PC
Organic acid (mg L−1)a
S73
TM
L7b
N24
POP
PJ
Acetic acid
Butyric acid
Citric acid
Fumaric acid
Gluconic acid
ND
ND
ND
1.9×10−3 ±8.1×10−5
28.66±0.42
ND
ND
ND
ND
46.78±2.39
0.17±2.6×10−3
ND
3.94±0.14
ND
ND
ND
0.083±4×10−3
ND
ND
26.15±0.21
ND
ND
1.69±0.04
0.024±3×10−3
31.94±0.45
ND
ND
ND
0.92±0.08
ND
Glucuronic acid
Lactic acid
Oxalic acid
Propionic acid
Succinic acid
Valeric acid
Organic acid productivity
(mg L−1 g OA/mg M)b
ND
0.0631±8.6×10−5
ND
ND
ND
ND
28.72±0.42
ND
ND
0.68±0.09
ND
ND
ND
47.47±2.44
ND
ND
ND
ND
ND
ND
4.11±0.14
10.73±1.31
ND
0.44±0.014
0.22±0.05
0.21±0.023
ND
37.85±2.65
ND
ND
ND
ND
ND
16.78±3.59
50.45±4.08
28.01±5.49
0.003±8.8×10−5
ND
ND
ND
ND
28.94±5.61
Values given are the mean±SD of three independent replicates per treatment
ND not detected, S73 T. flavus, TM T. flavus var flavus, L7b T. helicus, N24 T. helicus, POP P. purpurogenum, PJ P. janthinellum
a
Organic acid calculated as parts per million of organic acid (in milligrams per liter)
b
Calculated as grams of organic acid produced per gram of dry cell mass (in milligrams per liter OA per milligram M) per gram of dry cell mass
(PC, AP, and PP) were tested, showing that the solubilization of P varies according to the source of P and also
varied with the strain and species of fungi. This clearly
demonstrated that the capacity and efficiency to solubilize
P depends on the chemical property of the P source
(Mehta et al. 1979; Gadagi et al. 2007) and on the
solubilizing strain (Eckhardt 1979).
The role of the genus Penicillium as a P solubilizer has
extensively studied, whereas there are few reports on the role
of Talaromyces (Wakelin et al. 2004; Oliveira et al. 2009).
Recent studies have shown that T. flavus is able to mobilize
46.6 mg PO4−2 L−1 in the first week of the experiments
(Wakelin et al. 2004). In these studies, the media used by
Wakelin was different than that used by us and the sucrose
and both KNO3 and NH4 can affect the amount of P
solubilized. Our findings show that the Talaromyces isolates
were able to solubilize almost 10 times more inorganic P
than the isolates by Wakelin et al. (2004).
Table 2 AP solubilization and organic acid production by different fungal strains
AP
Organic acid (mg L−1)a
S73
TM
L7b
N24
POP
PJ
Citric acid
Fumaric acid
Gluconic acid
Lactic acid
Malic acid
Succinic acid
Valeric acid
Organic acid productivity
(mg L−1 OA/g M)b
10.88±1.66
1×10−4 ±2.2×10−5
ND
ND
ND
0.02±6.5×10−3
0.04±2.5×10−3
10.95±1.67
ND
4×10−4 ±1×10−4
3.97±0.81
ND
ND
0.07±5×10−4
ND
4.04±0.81
0.51±0.26
1.2×10−3 ±2×10−4
ND
0.01±4×10−3
0.07±0.021
0.31±0.056
3×10−3 ±5.×10−4
ND
ND
0.080±0.012
0.041±0.035
ND
0.43±0.11
0.57±0.118
0.01±1.5×10−3
6.67±0.69
ND
ND
0.06±0.005
29.39±0.763
36.65±1.58
0.36±0.063
ND
1.91±0.081
ND
ND
1.86±0.31
ND
4.13±0.46
ND
0.59±0.29
Values given are the mean±SD of three independent replicates per treatment
ND not detected, S73 T. flavus, TM T. flavus var flavus, L7b T. helicus, N24 T. helicus, POP P. purpurogenum, PJ P. janthinellum
a
Organic acid calculated as parts per million of organic acid (in milligrams per liter) per gram of dry cell mass
b
Calculated as grams of organic acid produced per gram of dry cell mass (in milligrams per liter per gram OA per gram M)
This copy belongs to 'tribhuwan'
Biol Fertil Soils (2010) 46:755–763
761
Table 3 PP solubilization and organic acid production by different fungal strains
PP
Organic acid
(mg L−1)a
S73
TM
L7b
N24
POP
PJ
Butyric acid
Citric acid
Fumaric acid
Gluconic acid
Lactic acid
Succinic acid
Organic acid
productivity
(mg L−1 OA/g M)b
ND
ND
4×10−4 ±1.2×10−5
25.32±0.26
ND
0.11±2.3×10−3
25.44±0.26
ND
ND
1×10−5 ±1.2×10−8
42.57±3.02
ND
0.077±1×10−3
42.65±3.02
5.8×10−3 ±6.5×10−4
4.26±0.19
4.8×10−3 ±4.7×10−5
15±0.07
ND
ND
19.27±0.26
ND
3.03±0.14
7.3×10−6 ±9.24×10−7
24.36±2.34
ND
0.16±0.012
27.57±2.50
ND
1.77±0.64
2.8×10−3 ±4.5×10−5
24.4±4.33
ND
ND
26.17±4.97
ND
1.45±0.075
ND
35.30±4
0.33±0.092
ND
37.10±4.17
Values given are the mean±SD of three independent replicates per treatment
ND not detected, S73 T. flavus, TM T. flavus var flavus, L7b T. helicus, N24 T. helicus, POP P. purpurogenum, PJ P. janthinellum
a
Organic acid calculated as parts per million of organic acid (in milligrams per liter)
b
Calculated as grams of organic acid produced per gram of dry cell mass (in milligrams per liter per gram OA per gram M)
Importance of the N source in studies of solubilization of
inorganic P has been studied by Pradhan and Sukla (2005).
They reported experiments with two species of Penicillium
and Aspergillus that the consumption of (NH4)2SO4
decreases the pH of the medium, which in turn leads to
the solubilization of PC. In addition, these authors observed
that a decrease in pH not related to the N consumption
leads to a partial solubilization of other sources of inorganic
P. In agreement with observations of Pradhan and Sukla
(2005), our experiments support the hypothesis that the N
consumption, together with a decrease in the pH, can be
related to the solubilization of PC (Asea et al. 1988).
The mechanisms responsible for the solubilization of P are
different depending on the source of P used. AP and PP differ
from that of PC. The exponential increase of P solubilization
in the media with AP and PP takes place after the N
consumption, indicating that P solubilization depends not
only on the consumption of ammonium but also on the
production of organic acids (Illmer et al. 1995).
The results of biomass production of the strains used
with the different sources of P indicate that the solubilization of P using PP and AP as the P source is independent of
biomass production. On the other hand, the analysis of the
main effects (strains×sources of P) indicates that, independently of the fungal species used, when PP is used, there is
a lower development of microbial biomass.
Our results show that S73 and POP are the most efficient
fungal strains solubilizing AP; however, the production of
organic acids by S73 was four times lower than that
produced by POP, supporting the hypothesis that the
solubilization of the different sources of P does not only
depend on the total amount of acids produced but also on
the type of acids produced (Cunningham and Kuiack 1992).
Barroso et al. (2006) have shown that the production of
organic acids by Aspergillus niger is higher in the media
with AP than other P salts. Our data suggest that the
production of organic acids depends both on the microorganism and source of P in which the microorganism grows.
The data obtained show that, in the presence of PC and
PP, gluconic acid is produced in higher percentages than in
the source that contains aluminum. It is well known that
gluconic acid produced by bacteria (Illmer and Schinner
1992; Liu et al. 1992; Rodriguez et al. 2004), as well as that
produced by several fungi (Vassilev et al. 2004), acts by
solubilizing the calcium minerals by acidification of the
medium (Lin et al. 2006). In the PC medium, gluconic acid
is not present in as high proportion in the highest
solubilizers as it only accounted for 63% of the acid
produced by POP compared to over 95% for S73 and TM.
In PJ, glucuronic acid is the most abundant acid produced.
Our findings show that the production of this acid is
important for the solubilization of PC, independently of the
fungus studied. Other studies with bacteria have shown that
glucuronic acid is involved in the solubilization of calcium
minerals (Harrison et al. 1972). Although our results are in
agreement with this, further studies should be carried out to
determine the action of this acid.
The production of other acids present in lower concentrations is also important. Kpomblekou and Tabatabai
(1994), Gyaneshwar et al. (1998), and Vazquez et al.
(2000) have reported that bacteria produce citric, oxalic,
propionic, lactic, valeric, and succinic acids and that these
are involved in the solubilization of calcium minerals in
concentrations lower than that of gluconic acid. This could
indicate that the solubilization of calcium minerals is a
generalized mechanism in fungi and bacteria.
This copy belongs to 'tribhuwan'
762
Biol Fertil Soils (2010) 46:755–763
The fungi studied that use AP as the P source produced a
higher percentage of citric and valeric acids than of
gluconic acid. Burgstaller et al. (1992) and Illmer et al.
(1995) have previously reported that the production of citric
acid plays a crucial role in the solubilization of AP. In our
experiments, the higher release of valeric acid in the
medium with AP than in those with other sources of P
may indicate the important role of this acid. The simultaneous production of different organic acids by the fungi
isolated in this study may enhance their potential for
solubilizing insoluble phosphate.
The present study confirms that the species of Penicillium and Talaromyces, which are biocontrol agents of
rhizosphere fungal pathogens, have more than one mode of
action. These species clearly demonstrated their efficiency
to release P using different mechanisms of solubilization,
such as release of organic acid to the medium or
assimilation of ammonium, according to the P available.
The efficiency of solubilization depended on the kind of
organic acids released to the medium and their concentration.
This study thus supports the hypothesis that the quality of the
acid is more important than the total amount of acids.
There are other microorganisms in the rhizosphere with
more than one known function. All of these polyfunctional
groups establish a challenge for further research and an
attractive field of application.
Acknowledgements We thank Dr. Larry Petersen for the critical
review of this manuscript. This work was supported by the following
institutions: Universidad de Buenos Aires (UBA), Consejo Nacional
de Investigaciones Científicas y Técnicas (CONICET), Agencia
Nacional de Promoción Científica y Tecnológicas (ANCYPT), and
Red BIOFAG CYTED.
References
Asea PEA, Kucey RMN, Stewart JWB (1988) Inorganic phosphate
solubilization by two Penicillium species in solution culture and
soil. Soil Biol Biochem 20:459–464
Barroso CB, Pereira GT, Nahas E (2006) Solubilization of CaHPO4
and AlPO4 by Aspergillus niger in culture media with different
carbon a nitrogen source. Braz J Microbiol 37:434–438
Burgstaller W, Strasser H, Schinner F (1992) Solubilization of zinc
oxide from filter dust with Penicillium simplicissimum;
bioreactor, leaching and stoichiometry. Environ Sci Technol
26:340–346
Cunningham J, Kuiack C (1992) Production of citric and oxalic acids
and solubilization of calcium phosphate by Penicillium bilaii.
Appl Environ Microbiol 85:1451–1458
Eckhardt FEW (1979) Über die Einwirkung heterotropher Mikroorganismen auf die Zersetzung silikatischer Minerale. Z Pflanzenerntihr Bodenkund 142:434–445
El-zouni IM (2008) Effect of phosphate solubilizing fungi on growth
and nutrient uptake of soybean (Glycine max L.) plants. J Appl
Sci Res 4:592–598
Gadagi RS, Shin WS, Sa TM (2007) Malic acid mediated
aluminum phosphate solubilization by Penicillium oxalicum
CBPS-3F-Tsa isolated from Korean paddy rhizosphere soil. In:
Velasuez E, Rodriguez-Barrueco C (eds) First International
Meeting on Microbial Phosphate Solubilization. Developments
in plant and soil sciences, vol 102. Springer, Berlin, pp 286–
290
Gerretsen FC (1948) The influence of microorganisms on the
phosphorus uptake by the plants. Plant Soil 1:51–81
Gyaneshwar G, Kumar N, Parekh LJ (1998) Effect of buffering on the
phosphate-solubilizing ability of microorganisms P. J Microbiol
Biotechnol 14:669–673
Harrison MJ, Pacha RE, Morita RY (1972) Solubilization of inorganic
phosphates by bacteria isolated from Upper Klamath Lake
Sediment. Limnol Oceanogr 17:50–57
Illmer P, Schinner F (1992) Solubilization of inorganic phosphates by
microorganisms isolated from forest soils. Soil Biol Biochem
24:389–395
Illmer P, Schinner F (1995) Solubilization of inorganic calcium
phosphates—solubilization mechanisms. Soil Biol Biochem
27:257–263
Illmer P, Barbato A, Schinner F (1995) Solubilization of hardlysoluble AlPO4, with P-solubilizing microorganisms. Soil Biol
Biochem 27:265–270
Kang S, Chul GH, Lee TG, Maheshwari DK (2002) Solubilization of
insoluble inorganic phosphates by a soil-inhabiting fungus
Fomitopsis sp. PS 102. Curr Sci 82:439–442
Kpomblekou AK, Tabatabai MA (1994) Effect of organic acids on the
release of phosphorus from phosphate rocks. Soil Sci 158:442–
448
Lin T, Huang H, Shen F, Young C (2006) The protons of gluconic acid
are the major factor responsible for the dissolution of tricalcium
phosphate by Burkholderia cepacia CC-Al74. Bioresour Technol
97:957–960
Liu ST, Lee LY, Tai CY, Hung CH, Chang YS, Wolfram JH,
Rogers R, Goldstein AH (1992) Cloning of an Erwinia
herbicola gene necessary for gluconic acid production and
enhanced mineral phosphate solubilisation in E. coli HB101:
nucleotide sequence and probable involvement in biosynthesis
of the coenzyme pyrroloquinoline quinone. J Bacteriol
174:5814–5819
McLaughlin MJ, Alston AM, Martin JK (1988) Phosphorus cycling in
wheat-pasture rotations. II. The role of the microbial biomass in
phosphorus cycling. Aust J Soil Res 26:333–342
Mehta A, Torma AE, Murr LE (1979) Effect of environmental
parameters on the efficiency of biodegradation of basalt rock by
fungi. Biotechnol Bioeng 21:875–885
Nautiyal CS (1999) An efficient microbiological growth medium for
screening phosphate solubilizing microorganisms. FEMS Microbiol Lett 170:265–270
Oliveira CA, Alves VMC, Marriel IE, Gomes EA, Scotti MR,
Carneiro NP, Guimarães CT, Schaffert RE, Sa NMH (2009)
Phosphate solubilizing microorganisms isolated from rhizosphere
of maize cultivated in an oxisol of the Brazilian Cerrado Biome.
Soil Biol Biochem 41:1782–1787
Pitt JI (1979) The genus Penicillium and its teleomorphic states
Eupenicillium and Talaromyces. Academic, London, p 634
Pradhan N, Sukla LB (2005) Solubilization of inorganic phosphates
by fungi isolated from agriculture soil. Afr J Biotechnol 5:850–
854
Richardson AE, Hadobas PA, Hayes JE, O'Hara CP, Simpson RJ
(2001) Utilization of phosphorus by pasture plants supplied with
myo-inositol hexaphosphate is enhanced by the presence of soil
microorganisms. Plant Soil 229:47–56
Rodríguez A (2004) Hongos del suelo antagonistas de Sclerotinia
sclerotiorum. Selección y estudio de potenciales agentes de
biocontrol. Thesis, Universidad de Buenos Aires, Argentina pp
280
This copy belongs to 'tribhuwan'
Biol Fertil Soils (2010) 46:755–763
763
Rodriguez H, Gonzalez T, Goire I, Bashan Y (2004) Gluconic acid
production and phosphate solubilization by the plant growthpromoting bacterium Azospirillum spp. Naturwissenschaften
91:552–555
Ryan PR, Delhaize E, Jones DL (2001) Function and mechanism of
organic anion exudation from plant roots. Annu Rev Plant
Physiol 52:527–560
Scheffer F, Schachtschabel P (1992) Lehrbuch der Bodenkunde.
Ferdinand Enke Verlag, Stuttgart, p 46
Shantaram MV, Saraswathy N (1985) Occurrence and activity of
phosphate-solubilizing fungi from coconut plantation soils. Plant
Soil 87:357–364
Stephen J, Jisha MS (2009) Buffering reduces phosphate solubilizing
ability of selected strains of bacteria. World J Agric Sci 5:135–137
Vassilev N, Fenice MI, Federici F (2004) Rock phosphate solubilization with gluconic acid produced by immobilized Penicillium
variabile P16. Biotechnol Tech 10:585–588
Vassilev N, Vassileva M, Nikolaeva I (2006) Simultaneous Psolubilizing and biocontrol activity of microorganisms: potentials
and future trends. Appl Microbiol Biotechnol 71:137–144
Vazquez P, Holguin G, Puente ME, Lopez-Cortes A, Bashan Y (2000)
Phosphate-solubilizing microorganisms associated with the rhizosphere of mangroves in a semiarid coastal lagoon. Biol Fertil
Soils 30:460–468
Wakelin S, Warren R, Harvey P, Ryder M (2004) Phosphate
solubilization by Penicillium spp. closely associated with wheat
roots. Biol Fertil Soils 40:36–43
Weatherburn MW (1967) Phenol-hypochlorite reaction for determination of ammonia. Anal Chem 39:971–974
Whitelaw MA (2000) Growth promotion of plants inoculated with
phosphate-solubilizing fungi. Adv Agron 69:99–151
Xiao C, Chi R, Huang X, Zhang W, Qiu G, Wang D (2008) Optimization
for rock phosphate solubilization by phosphate-solubilizing fungi
isolated from phosphate mines. Ecol Eng 33:187–193
This copy belongs to 'tribhuwan'