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Soil Biology and Biochemistry xxx (2008) 1–6
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Soil Biology and Biochemistry
journal homepage: www.elsevier.com/locate/soilbio
Phosphate solubilizing microorganisms isolated from rhizosphere
of maize cultivated in an oxisol of the Brazilian Cerrado Biome
C.A. Oliveira a, V.M.C. Alves b, I.E. Marriel b, E.A. Gomes b, M.R. Scotti a,
N.P. Carneiro b, C.T. Guimarães b, R.E. Schaffert b, N.M.H. Sá a, *
a
b
Federal University of Minas Gerais, Botany Department, CP 486, 31270-901 Belo Horizonte, MG, Brazil
Embrapa Maize and Sorghum, CP 151, 35701-970 Sete Lagoas, MG, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 September 2007
Received in revised form 21 December 2007
Accepted 4 January 2008
Available online xxx
Many soil microorganisms are able to transform insoluble forms of phosphorus to an accessible soluble
form, contributing to plant nutrition as plant growth-promoting microorganisms (PGPM). The objective
of this work was to isolate, screen and evaluate the phosphate solubilization activity of microorganisms
in maize rhizosphere soil to manage soil microbial communities and to select potential microbial
inoculants. Forty-five of the best isolates from 371 colonies were isolated from rhizosphere soil of maize
grown in an oxisol of the Cerrado Biome with P deficiency. These microorganisms were selected based on
the solubilization efficiency of inorganic and organic phosphate sources in a modified Pikovskaya’s liquid
medium culture containing sodium phytate (phytic acid), soybean lecithin, aluminum phosphate (AlPO4),
and tricalcium phosphate (Ca3(PO4)2). The isolates were identified based on nucleotide sequence data
from the 16S ribosomal DNA (rDNA) for bacteria and actinobacteria and internal transcribed spacer (ITS)
rDNA for fungi. Bacteria produced the greatest solubilization in medium containing tricalcium phosphate. Strains B17 and B5, identified as Bacillus sp. and Burkholderia sp., respectively, were the most
effective, mobilizing 67% and 58.5% of the total P (Ca3(PO4)2) after 10 days, and were isolated from the
rhizosphere of the P efficient L3 maize genotype, under P stress. The fungal population was the most
effective in solubilizing P sources of aluminum, phytate, and lecithin. A greater diversity of P-solubilizing
microorganisms was observed in the rhizosphere of the P efficient maize genotypes suggesting that the P
efficiency in these cultivars may be related to the potential to enhance microbial interactions of P-solubilizing microorganisms.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
P-solubilizing microorganisms (PSM)
Phosphorus mineralization and
solubilization
Rhizosphere
Zea mays
1. Introduction
The Cerrado Biome is a large Brazilian ecosystem characterized
by a mosaic of savanna, cropping and forest, where the soils are
characterized by low pH, low phosphorus (P) content and high P
fixation capacity (Marschner, 1995; Novais and Smyth, 1999; Hinsinger, 2001; Wakelin et al., 2004). The accumulated insoluble P, like
total soil P, occurs in either organic or inorganic forms, unavailable
to plants. Phosphate anions may be immobilized through precipitation with cations such as Ca2þ, Mg2þ, Fe3þ and Al3þ. Organic P
in the soil generally accounts for around 50% of total insoluble soil P
in soils with high organic matter content, such as the no-tillage
management systems (Bayer et al., 2001; Gyaneshwar et al., 2002).
A large proportion of the organic P is represented by inositol
phosphates and lesser amounts of other phosphate esters as
phospholipids (Richardson, 2001; Wakelin et al., 2004; Richardson
* Corresponding author. Tel.: þ55 31 3499 2688; fax: þ55 31 3499 2673.
E-mail address: [email protected] (N.M.H. Sá).
et al., 2005). The strategy of ameliorating the low P fertility
production constraint in acid soils with corrective applications of P
is limited economically and environmentally due to the high
amounts of P fertilizer required and the high P fixing capacity of
these soils (Hinsinger, 2001).
Microbial populations are key components of the soil–plant
continuum where they are immersed in a framework of interactions
affecting plant development (Wakelin et al., 2004; Barea et al., 2005;
Vassilev et al., 2006). P-solubilizing microorganisms (PSM) can
solubilise and mineralize P from inorganic and organic pools of total
soil P, and may be used as inoculants to increase P-availability to plants
(Kucey et al., 1989; Richardson, 1994, 2001; Illmer et al., 1995;
Whitelaw et al., 1999). This work may contribute in developing soil
microbial community management schemes based on specific functions (P solubilization and mineralization), and selection of microorganisms as potential microbial inoculants (biofertilizers). These results
could have significant impact on the more than six million hectares of
maize under no-till cultivation in Brazil today. There is also a potential
use of PSM in industrial bioprocessing of rock phosphate.
0038-0717/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.soilbio.2008.01.012
Please cite this article in press as: Oliveira, C.A. et al., Phosphate solubilizing microorganisms isolated from rhizosphere of maize cultivated in an
oxisol of the Brazilian Cerrado Biome, Soil Biology and Biochemistry (2008), doi:10.1016/j.soilbio.2008.01.012
ARTICLE IN PRESS
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C.A. Oliveira et al. / Soil Biology and Biochemistry xxx (2008) 1–6
The objective of this research was to isolate phosphate solubilization and mineralization microorganisms in maize rhizosphere
cultivated under conventional tillage and no-tillage management
systems in the Brazilian Cerrado. Additionally, a new method to
screening PSM using a P-Mehlich1 phosphorus extractor was also
tested in this work. The knowledge of P-organic mineralization
processes is expected to be useful in understanding seasonal Pcycling in the Brazilian oxisols under the no-till production system.
2. Materials and methods
were inoculated on modified Pikovskaya’s agar medium containing
organic forms of P, phytic acid or sodium phytate (P-phytate) and
soybean lecithin (P-lecithin). P-Ca, P-Al, P-phytate and P-lecithin
were added in Pikovskaya’agar medium at 5 g l1, 3.5 g l1, 10 g l1
and 15 g l1, respectively. Petri plates were incubated at room
temperature for 10 days. Morphologically distinct colonies, both
with and without halos were purified by repeated subculturing,
maintained on potato dextrose agar (PDA) and incubated at room
temperature. The isolates were grouped into filamentous fungi,
actinobacteria or bacteria based on macro and microscopic
observations.
2.1. Soil samples
2.3. Screening and phosphate evaluation
Soil samples were taken from the rhizosphere of four maize
cultivars contrasting in P efficiency; BRS3060 – P efficient hybrid,
HS26 1113 – P inefficient hybrid, L3 – P efficient inbred line, and
L22 – P inefficient inbred line developed by the Maize Breeding
Program of Embrapa Maize and Sorghum (Parentoni et al., 2000) at
Sete Lagoas, Minas Gerais, Brazil. These genotypes were growing in
a conventional and no-tillage management system in an oxisol. The
soils of conventional crop system were characterized by low P
(3 mg P dm3), clay texture (55% clay, 11% silt, 34% sand) with pH
5.2 (soil/water ratio, 1:2.5 [w/v]) and 3% organic matter and 0.25,
2.29, and 0.36 cmolc kg1 of Al, Ca, and Mg, respectively, in a 1 N KCl
extraction and 0.16 mg K dm3, in a Mehlich1 extraction (Mehlich,
1978). Samples of no-tillage maize rhizosphere were collected from
oxisols at eight locations with 13–15 years of no-tillage management. Descriptions of each sample are provided in Table 1,
including the location and the crops in rotation with maize.
Samples were taken for each genotype and tillage system 60 days
after planting, during the flowering stage. Each of three replicated
samples was composed of the soil adhering to the roots of five
maize plants. The roots were shaken carefully inside plastic bags in
order to separate the soil from the roots.
2.2. Isolation of P-solubilizing microorganisms
The rhizosphere soil samples from the maize plants in a conventionally managed low P environment were serially diluted and
inoculated on modified Pikovskaya’s agar medium (Pikovskaya,
1948) containing insoluble inorganic forms of P, Ca3(PO4)2 or
tricalcium phosphate (P-Ca), AlPO4 or aluminum phosphate (P-Al).
The maize rhizosphere soil samples from the no-till management
A preliminary experiment was conducted to test the ability to
produce soluble phosphate in liquid cultures by the isolates. Three
replicate flasks containing modified Pikovskaya’s liquid medium
were inoculated with each isolate using 8 mm mycelia disks of
fungal and actinobacteria cultures, and bacterial suspension
(108 cell ml1) taken from 10 days old cultures. Insoluble sources of
P, Ca3(PO4)2, AlPO4, sodium phytate, and soybean lecithin, were
added to the liquid medium at 1.5 g l1 (300 mg P l1), 1 g l1
(250 mg P l1), 1 g l1 (280 mg P l1), 15 g l1 (101.1 mg P l1), respectively. Three replicates of a control treatment were included in
the experiment, each containing one P-insoluble source. The initial
pH was adjusted to 6.0. The cultures were centrifuged (7000g,
10 min) after 10 days of incubation, at 27 C, with gentle shaking
and 5 ml supernatant aliquots were filtered through Whatman n
42 filter paper to remove thick polysaccharide-like exudates. The
filtrates were assayed for soluble P, using the Murphy and Riley’s
(1962) colorimetric method. The amount of P solubilized was
obtained by subtracting the soluble P of the inoculated sample from
the corresponding sample uninoculated control (i.e. P released by
autoclaving of the P suspension).
2.4. Evaluation for phosphate solubilization,
pH and phosphatase activity
The best 45 isolates from the preliminary screening were
selected based on the amount of P solubilized in each medium and
were again assayed for P solubilization as described above. The pH,
phosphatase production and P-Mehlich1 solubilizing activity were
also evaluated. The solution pH was measured after 10 days and
Table 1
Description of rhizosphere samples of maize cultivated in oxisols, production system, number of microorganisms (fungi, bacteria, actinobacteria) isolated from soil samples,
and source of insoluble P; tricalcium phosphate (P-Ca), aluminum phosphate (P-Al), sodium phytate (P-phytate), and soybean lecithin (P-lecithin)
Sample
City/statea
Crop management systemb
Total P inorganicsolubilizing isolatesc
P-Ca
P-Al
P-phytate
P-lecithin
L3
L22
BRS3060
HS26 1113
CNPMS
JSP
CNPS
MGO
PGO
GRGO
SCRGO
SMRGO
Sete Lagoas/MG
Sete Lagoas/MG
Sete Lagoas/MG
Sete Lagoas/MG
Sete Lagoas/MG
Jardinópolis/SP
Londrina/PR
Morrinhos/GO
Planaltina/GO
Rio Verde/GO
Rio Verde/GO
Rio Verde/GO
CT/maize
CT/maize
CT/maize
CT/maize
NT/maize BRS3060
NT/maize
NT/maize
NT/maize (soybean)
NT/maize (grassland/soybean)
NT/maize (grassland)
NT/maize (sugar cane)
SM/NT/maize
34
23
28
21
–
–
–
–
13
18
8
17
–
–
–
–
–
–
–
–
21
25
10
22
20
15
11
22
Total
(14)
(6)
(9)
(7)
Total P organicsolubilizing isolatesc
(7)
(4)
(4)
(6)
–
–
–
–
–
–
–
–
–
–
2
6
3
9
9
13
14
7
106
56
63
(1)
(0)
(1)
(3)
(1)
(3)
(10)
(0)
(6)
(3)
(0)
(7)
(0)
(0)
(3)
(5)
146
a
State’s acronym: MG, Minas Gerais; SP, São Paulo; GO, Goiás.
b
Production system: CT, conventional tillage; NT, no-tillage; SM, swine manure. In parentheses, the crop grown in rotation with maize.
c
Total number of isolates from each specific solid medium (number in parentheses represents the number of isolates showing P solubilization within 10 days in liquid
culture).
Please cite this article in press as: Oliveira, C.A. et al., Phosphate solubilizing microorganisms isolated from rhizosphere of maize cultivated in an
oxisol of the Brazilian Cerrado Biome, Soil Biology and Biochemistry (2008), doi:10.1016/j.soilbio.2008.01.012
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C.A. Oliveira et al. / Soil Biology and Biochemistry xxx (2008) 1–6
150 ml centrifuged and filtrated aliquots were assayed for phosphatase activity of the isolates according to Freitas et al. (1997)
modified protocol. Aliquots of each sample were added to 0.48 ml
universal buffer 0.1 M, pH 6.5 or pH 11 for acid or alkaline phosphatase activity, respectively, and 0.12 ml of 0.05 M p-nitrophenyl
phosphate (pNPP) solution, followed by 1 h incubation at 37 C.
Control treatments containing only liquid medium were included
in each experiment with pNPP added after incubation. The yellow
color was measured at 410 nm (Tabatai and Bremmer, 1969).
The effects of the treatments were analyzed by ANOVA and
means were compared using Tukey’s test, at 5% significant level.
2.5. P-Mehlich1 solubilizing activity – phosphorus incorporated
into the organic matrix of polysaccharides
A simultaneous test was carried out using the same samples
from the four experiments cited above to determine the amount
of phosphorus incorporated into the organic matrix of polysaccharides formed during culture growth. After 10 days of
incubation 10 ml aliquots were incubated for 24 h with 100 ml of
P-Mehlich1 extractor solution (HCl 0.05 M þ H2SO4 0.0125 M),
referred to as ‘‘double acid’’ extracting (Mehlich, 1978). After incubation, 5 ml supernatant aliquots were centrifuged, filtered, and
assayed for soluble P using the same Murphy and Riley’s (1962)
conventional method. The amount of P solubilized (P-Mehlich1)
was obtained by subtracting the soluble P concentration of the
inoculated from that of the corresponding uninoculated control
culture.
Factorial ANOVA was used to analyze the effects of the treatments and the phosphorus determination methods, whose means
were compared using the Tukey test at 5% significance.
2.6. Identification of isolates
The 45 most efficient isolates solubilizing P-phytate, P-lecithin,
P-Ca, and P-Al were identified based on nucleotide sequence data
from the rDNA – ITS for fungi and 16S rDNA for bacteria, including
actinobacteria. Total DNA was extracted using BIO 101 kit protocols
(FastDNA SPIN Kit, Bio 101 Inc., Vista, CA) from cultures recovered in
a potato dextrose liquid medium (0.2 g of fresh weight of fungi and
actinobacteria mycelium filtrated and 200 ml of bacterial suspension, 108 cell ml1), after 10 days growth at room temperature
without shaking. The rDNA fragments were amplified using fungi
universal primers, ITS1 and ITS4 or ITS5 and ITS2 (White et al., 1990)
and the 16S rDNA bacteria and actinobacteria fragments were
amplified using the F968 and R1401 primers (Nubel et al., 1996).
PCR reaction was performed using 20 ng DNA, 50 mM of each
dNTP, 2.5 mM of MgCl2, 20 mM Tris–HCl (pH 8.4), 50 mM KCl,
0.2 mM of each primer and 1 unit of Taq DNA polymerase (Invitrogen, Carslbad, CA) in a final reaction volume of 50 ml. Amplifications were carried out using the temperature profiles: 94 C for
2 min, followed by 30 cycles at 94 C for 1 min, 55 C for 1 min,
72 C for 2 min, and a final extension step at 72 C for 10 min for
bacteria (including actinobacteria) and 95 C for 4 min, followed by
30 cycles at 95 C for 30 s, 46 C for 60 s, 72 C for 30 s and primer
extension at 72 C for 10 min for fungal isolates. PCR products were
separated on a 1.5% (w/v) agarose gel, stained with ethidium
bromide (1 mg ml1), and visualized under UV light using Eagle Eye
II (Stratagene, La Jolla, CA).
Amplified products were removed from the gel and purified
using QIAquick Gel Extraction kit (Qiagen, Hilden, Germany) and
sequenced using the ‘‘Big Dye Terminator v3.1. Cycle Sequencing’’
(Applied Biosystems, Foster City, CA) kit in an ABI PRISM 3100
Genetic Analyzer (Applied Biosystems). Nucleotide sequence data
were compared with GenBank data (http://www.ncbi.nlm.nih.gov/
) using the BlastN search (Altschul et al., 1997).
3
3. Results
3.1. Isolation of P-solubilizing microorganisms and solubilization
assay for inorganic and organic P sources
Three hundred seventy-one morphotypes (116 fungi and 255
bacteria of which 126 were actinobacteria) were recovered from
the rhizosphere of maize plants grown in a Cerrado Biome oxisol
soil from both conventional and no-till planting systems (Table 1). A
total of 36 isolates from P-Ca medium showed P-solubilizing in
liquid culture medium solubilizing more than 80 mg P l1 (30% of
total P), in 10 days, and of these, 14 (39%) were isolated from the P
efficient maize inbred line, L3.
In this study, 63 colonies were evaluated in P-phytate medium
and 19 colonies showed P-solubilizing activity in 10 days, and of
these colonies, 10 (53%) were from maize no-tillage rhizosphere in
consortium with sugar cane (SCRGO). The numbers of P-lecithinsolubilizing isolates varied among samples, however, the highest
number of isolates that presented P-solubilizing activity was found
in no-tillage maize–soybean samples (Table 1).
3.2. Identification and evaluation of isolates for P-solubilizing
activity, pH and phosphatase activity
A total of 45 isolates screened for phosphate solubilizing efficiency in specific liquid medium were genetically identified using
rDNA sequences in comparison with GenBank database (Tables 2–5).
Morphological identification confirmed that the majority of the
genera to the species identified by the molecular techniques.
The strains of bacteria, fungi and most of the actinobacteria
(Tables 2–5) were confirmed as PSM with high P solubilization
rates, but the actinobacteria genera, Kitasatospora (Table 5), was
identified as a new PSM. Among the bacteria isolates, B17 and B5,
identified as Bacillus sp. (B17) and Burkholderia sp. (B5), were the
most efficient P-solubilizing strains from P-Ca source culture
solution (Table 2), solubilizing 67% and 58.5% of the total P,
respectively. The B5 isolate also had the largest reduction in pH in
the growth solution to 4.46. The actinobacteria most efficient in
solubilizing P-Ca source was A4, identified as Streptomyces platensis
(Table 2), according to morphological and molecular criteria.
The most efficient isolates for aluminum phosphate solubilization were B58, F39 and F50, identified as Burkholderia cepacia and
Aspergillus terreus, which were isolated from the inefficient maize
genotypes, L22 and HS26 1113 (Table 3). No correlation was found
between pH and P-Al solubilization (R ¼ 0.001; P < 0.001).
The mineralization of organic P from acid phytic (P-phytate) is
shown in Table 4. In general, no reduction in pH was observed. The
species Talaromyces rotundus (F80, F102 and F105), A. terreus (F79)
and B. cepacia (B116) were the most efficient in mineralizing P, but
without significant difference. The strain of T. rotundus, F102
produced high levels of acid phosphatase.
Among the microorganisms showing ability to mineralize
P-lecithin, Penicillium citrinum (F95) and A. terreus (F93) were the
most efficient solubilizing 44% and 42% of total P, respectively and
were among the three best acid phosphatase producers (Table 5).
3.3. Quantification of P solubilization by P-Mehlich1 method
P-solubilizing ability of microorganisms was also evaluated in
liquid medium using P-Mehlich1 extractor (Mehlich, 1978). The
amount of P solubilized varied significantly among the methods,
with and without extractor, and among the isolates (Tables 2–5). In
general, the values of soluble P detected by the method P-Mehlich1
for fungi, actinobacteria and bacteria were larger than for the
conventional method of direct reading without extraction
described by Murphy and Riley (1962). A large amount of a thick
Please cite this article in press as: Oliveira, C.A. et al., Phosphate solubilizing microorganisms isolated from rhizosphere of maize cultivated in an
oxisol of the Brazilian Cerrado Biome, Soil Biology and Biochemistry (2008), doi:10.1016/j.soilbio.2008.01.012
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Table 2
Closest relatives using rDNA fragments, P solubilized, pH and phosphatase activity by microorganisms screened and isolated from samples of maize rhizosphere grown in
liquid cultures containing P inorganic insoluble form tricalcium phosphate (P-Ca)
Isolate (sample)a
Closest relatives database reference
Amount solubilized phosphate (mg P l1)b*
Phosphatase activity
(mg pNPP ml1 h1)*
Species (accession number) – similarity index
Conventional methodc
P-Mehlich1 methodd
Acid
Alkaline
A4 (L3)
A14 (L3)
A19 (L22)
A20 (L22)
A26 (BRS3060)
A29 (HS26 1113)
B2 (L3)
B5 (L3)
B7 (L22)
B17 (L3)
B46 (BRS3060)
B48 (HS26 1113)
F14 (BRS3060)
Streptomyces platensis (AB163439.1) – 99%
S. tumescens (AF346485.1) – 99%
S. chartreusis (SCH399468) – 99%
S. griseochromogenes (SGR310923) – 98%
S. collinus (SCO306623) – 99%
S. avermitilis (AB078897.2) – 98%
Pantoea ananatis (AF364846.1) – 99%
Burkholderia sp. (AY224513.1) – 97%
Bacterium H3 (AY345547.1) – 97%
Bacillus sp. (AF507879) – 89%
Burkholderia cepacia (AY268142.1) – 90%
B. cepacia (AY741360.1) – 96%
Penicillium pinophilum (AF176660) – 98%
68.0 A abc
43.1 A abc
23.5 A abc
4.1 A ab
1.8 A a
3.7 A ab
81.8 A c
175.4 A d
77.1 A c
200.0 A d
70.6 A bc
64.3 A abc
25.9 A abc
39.8 A ab
29.6 A ab
37.3 A ab
30.5 A ab
9.4 A a
29.9 A ab
179.9 Bc
167.2 A c
63.0 A ab
211.1 A c
54.9 A ab
67.9 A b
8.9 A ab
1.88 a
0
0
0
0
0
2.62 a
0
0
0
0
0
0
0.59 a
1.89 a
0
0
0
0
7.24 a
0
0
6.15 a
18.71 a
70.98 b
0
*Means followed by the same letter did not differ significantly at 5% Tukey test.
a
Sample identification are according to Table 1.
b
Means followed by different higher case letter in each row represent the differences between the P-solubilizing isolates, and lower case letter in each column represents
differences between the conventional method and the P-Mehlich1 method.
c
Amount of P solubilized by the Murphy and Riley’s (1962) procedure after 10 days of growth in a liquid medium containing 300 mg P l1 insoluble phosphate.
d
Amount of P solubilized extracted by the P-Mehlich1 procedure (Mehlich, 1978), after 10 days growth in a liquid medium containing 300 mg P l1 insoluble phosphate.
polysaccharide-like compound was observed in some flasks of
bacteria and fungi, which could have functioned as an organic
matrix adsorbing the P liberated by the microorganisms in the
culture solution.
4. Discussion
The frequency of isolates varied among the maize genotypes
evaluated (Table 1). A greater number of microorganisms were
isolated from the rhizosphere of the maize genotypes L3 and
BRS3060, both considered P efficient by the Embrapa Maize and
Sorghum Breeding Program (Parentoni et al., 2000). The most
efficient P-Ca solubilizing isolates, identified as Bacillus sp. (B17),
Burkholderia sp. (B5), and Streptomyces platensis (A4) were isolated
from the rhizosphere of L3 maize. This result supports the
hypothesis that these microorganisms could contribute to the
efficiency of these P efficient maize genotypes in acquiring P from
the soil. Several studies have indicated that the presence of different plant species or genotypes influence the microbial community
due to the differential response of these organisms to different root
signaling and exudation patterns, especially when plants are under
environmental stress (Lynch and Whipps, 1990; Richardson, 1994)
for example phosphorus stress (Hinsinger, 2001).
The aluminum phosphate solubilization rates were lower than
the P-Ca mobilization (Table 3), in agreement with Narsian et al.
(1994) that found lower rates of solubilizing from P-Al than P-Ca
among eight microorganisms tested in tricalcium and aluminum
phosphate. Similar results were also found by Whitelaw et al.
(1999) and Barroso and Nahas (2005) that described this
phenomenon is probably due to the higher solubility of the Ca
phosphates in culture solutions. Some authors have pointed out
that the phosphorus liberated from clay minerals with AlPO4 can
increase the level of toxic Al3þ in solution (Illmer et al., 1995) and
could possibly explain the suppression of the P-solubilizing activity
of the PSM that was observed in the P-Al medium. A low correlation
was observed between pH reduction and an increase of soluble P in
the liquid culture after 10 days of growth indicating that the decline
of the concentration of insoluble phosphorus might have been
influenced by other factors (Whitelaw et al., 1999; Barroso and
Nahas, 2005).
In this study, the fungi were the most efficient in the liberation
of P from P-phytate. Several authors have reported similar results,
affirming that the genus Aspergillus was the most efficient in
solubilizing phytate (Yadav and Tarafdar, 2003). A strain of Talaromyces (F102) producing high levels of acid phosphatase was
isolated in this study and is the first report of this genus with the
Table 3
Closest relatives using rDNA fragments, P solubilized and phosphatase activity by microorganisms screened and isolated from samples of maize rhizosphere grown in liquid
cultures containing P inorganic insoluble form, aluminum phosphate (P-Al)
Isolate (sample)a
B52 (L3)
B53 (L3)
B56 (L22)
B58 (L22)
F39 (HS26 1113, L22)
F40 (L22)
F50 (HS26 1113)
Closest relatives database reference
Amount solubilized phosphate (mg P l1)b*
Phosphatase activity
(mg pNPP ml1 h1)*
Species (accession number) – similarity index
Conventional methodc
P-Mehlich1 methodd
Acid
Alkaline
Pantoea ananatis (AF364846.1) – 96%
Pantoea agglomerans (AY924376.1) – 89%
Paenibacillus elgii (AY090110.1) – 98%
Burkholderia cepacia (F3AY509957.1) – 95%
Aspergillus terreus (AY373871.1) – 98%
Penicillium pimiteouiense (AF037436.1) – 96%
A. terreus (AJ413985.1) – 95%
10.3 A b
13.3 A b
0.03 A a
20.3 A c
14.9 A bc
12.1 A b
13.7 A bc
10.8 A a
14.6 A ab
16.0 B ab
38.4 B c
16.3 A ab
20.8 B b
18.3 A ab
3.57 a
0.36 a
0
0
48.1 b
57.9 b
50.2 b
19.5 a
50.1 b
53.1 b
64.8 b
0
0
0
*Means followed by the same letter did not differ significantly at 5% Tukey test.
a
Sample identification are according to Table 1.
b
Means followed by different higher case letter in each row represent the differences between the P-solubilizing isolates, and lower case letter in each column represents
differences between the conventional method and the P-Mehlich1 method.
c
Amount of P solubilized by the Murphy and Riley’s (1962) procedure after 10 days of growth in a liquid medium containing 250 mg P l1 insoluble phosphate.
d
Amount of P solubilized extracted by the P-Mehlich1 procedure (Mehlich, 1978), after 10 days growth in a liquid medium containing 250 mg P l1 insoluble phosphate.
Please cite this article in press as: Oliveira, C.A. et al., Phosphate solubilizing microorganisms isolated from rhizosphere of maize cultivated in an
oxisol of the Brazilian Cerrado Biome, Soil Biology and Biochemistry (2008), doi:10.1016/j.soilbio.2008.01.012
ARTICLE IN PRESS
C.A. Oliveira et al. / Soil Biology and Biochemistry xxx (2008) 1–6
5
Table 4
Closest relatives, P solubilized and phosphatase activity by microorganisms screened and isolated from samples of maize rhizosphere grown in liquid cultures containing P
organic insoluble form, sodium phytate (P-phytate)
Isolate (sample)a
Closest relatives database reference
Amount solubilized phosphate (mg P l1)b
Species (accession number) – Similarity index
Conventional methodc
P-Mehlich1 method
Acid
Alkaline
B116 (MGO)
B118 (MGO)
B119 (PGO)
B121 (GRGO)
Burkholderia cepacia (AY741355.1) – 98%
Arthrobacter sp. (AF408967.1) – 99%
Bacillus megaterium (AY167865.1) – 98%
Uncultured gamma
prokaryote (AJ575715.1) – 94%
Pantoea agglomerans (AY924376.1) – 90%
Arthrobacter sp. (AF408952.1) – 99%
Aspergillus terreus (ATE413985) – 96%
Talaromyces rotundus (AF285115) – 96%
T. rotundus (AF285115) – 97%
T. rotundus (AF285115) – 96%
52.7 A de
28.0 A abc
19.5 A ab
4.9 A a
92.8 B f
28.2 A ab
37.3 B bc
1.5 A a
0
0
46.4 bc
0
9.7 a
23.6 de
21.6 cd
20.9 cd
25.5 A abc
30.5 A bcd
46.5 A cde
57.5 A e
48.6 A cde
47.4 A cde
24.9 A ab
31.4 A b
9.1 A cd
67.6 A e
54.2 A cde
60.3 B de
0
0
26.4 ab
12.4 a
62.6 c
45.4 bc
31.9 e
20.7 bcd
10.8 a
13.4 abc
12.0 a
12.2 ab
B122 (GRGO)
B124 (GRGO)
F79 (SCRGO)
F80 (SCRGO)
F102 (SCRGO)
F105 (SCRGO)
Phosphatase activity
(mg pNPP ml1 h1)*
d
*Means followed by the same letter did not differ significantly at 5% Tukey test.
a
Sample identification are according to Table 1.
b
Means followed by different higher case letter in each row represent the differences between the P-solubilizing isolates, and lower case letter in each column represents
differences between the Conventional method and the P-Mehlich1 method.
c
Amount of P solubilized by the Murphy and Riley’s (1962) procedure after 10 days of growth in a liquid medium containing 280 mg P l1 insoluble phosphate.
d
Amount of P solubilized extracted by the P-Melich1 procedure (Mehlich, 1978), after 10 days growth in a liquid medium containing 280 mg P l1 insoluble phosphate.
potential to mineralize P-phytate. The ability of soil microorganisms to solubilise various forms of inorganic P is well documented
(Kucey et al., 1989; Richardson, 1994; Whitelaw et al., 1999;
Goldstein et al., 2003); however, there have few reports regarding
the potential of soil microorganisms to increase the availability of P
from phytin. The results of this work clearly demonstrate the
efficiency of newly identified microorganisms able to release P from
phytate (P-phytin) and other organic P sources.
The fungus A. terreus has been cited in the literature as an
important phytase producing fungus (Mitchell et al., 1997). In this
study, A. terreus was also able to solubilise P from aluminum
phosphate, phytate and lecithin (Tables 3-5). T. rotundus was one of
the highest phosphatase producers in the phytate (Table 4) and
lecithin (Table 5) media, indicating that this fungus represents an
important species for producing phytase and phospholipases.
The bacteria Pantoea agglomerans and Burkholderia sp. identified
in this study produced phosphatase and mineralized phytate
(Table 4). P. agglomerans also solubilized P-Al (Table 3) and
Burkholderia sp. solubilized P-Ca (Table 2). According to Nacamulli
et al. (1997), B. cepacia represents one of the predominant bacterial
species among the microorganisms occurring in maize rhizosphere.
Even though this is the first report of B. cepacia being capable of
solubilizing unavailable sources of phosphorus in maize, several
studies have shown that B. cepacia is able to compete with the indigenous microflora, survive, colonize roots of various maize cultivars (Nacamulli et al., 1997; Bevivino et al., 2000), enhance the
yield of several crop plants (Rodrigues and Fraga, 1999; Baldani
et al., 2001), antagonize and repress the major soil-borne fungal
pathogens of maize (Bevivino et al., 2000). The mechanism or
mechanisms involved in enhanced phosphorus solubilization by
this bacteria need to be further investigated to utilize it as a biofertilizer in maize production systems.
The utilization in this study of the P-Mehlich1 extractor to
measure the quantity of P released indicates that the P solubilization estimated in part of this work and in other studies may have
been underestimated and may have resulted in discarding isolates
Table 5
Closest relatives, P solubilized and phosphatase activity by microorganisms screened and isolated from samples of maize rhizosphere grown in liquid cultures containing P
organic insoluble form, soybean lecithin (P-lecithin)
Isolate (sample)a
A62 (CNPMS)
A65 (CNPMS)
A68 (JSP)
A80 (MGO)
A83 (MGO)
B62 (CNPMS)
B65 (JSP)
B70 (MGO)
B76 (SMRGO)
B86 (JSP)
B104 (SCRGO)
F87 (MGO)
F93 (SCRGO)
F94 (SCRGO)
F95 (SCRGO)
Closest relatives database reference
Amount solubilized phosphate (mg P l1)b*
Species (accession number) – similarity index
Conventional methodc
P-Mehlich1 methodd
Acid
Alkaline
Kitasatospora putterlickiae (AY189976.1) – 98%
K. paracochleatus (U93328) – 99%
Streptomyces ipomoeae (AY207593.1) – 98%
S. hygroscopicus (SH16SRR) – 98%
S. hygroscopicus (SH16SRR) – 98%
Arthrobacter sp. (AF408967.1) – 99%
Methylobacterium sp. (D32232.1) – 98%
Arthrobacter sp. (AF408967.1) – 99%
Uncultured bacterium (P reactor) (AF527601.1) – 96%
Arthrobacter sp. (AY177350.3) – 97%
Uncultured Bacillus sp. (AY242537.1) – 93%
Acremonium strictum (AY138846.1) – 93%
Aspergillus terreus (AF516138.1) – 98%
Talaromyces rotundus (AF285115) – 95%
Penicillium citrinum (AY373904.1) – 99%
14.9 A ab
17.4 A ab
19.0 A abc
2.1 A a
3.4 A a
2.1 a
0
0.6 A a
0
0.9 A a
6.7 A ab
29.8A bcd
42.9 B cd
24.0 A abcd
44.0 B d
13.2 A ab
24.9 A bc
22.4 A bc
24.4 B bc
34.3 B c
0
36.5 c
15.5 B ab
24.8 bc
24.4 B bc
7.1 A ab
23.8 A bc
24.7 A bc
20.3 A bc
20.2 A bc
76.1 cde
67.1 cde
53.9 abcd
66.1 bcde
64.6 bcde
5.1 ab
1.1 a
16.4 abc
0
26.0 abc
4.9 ab
77.5 cde
0
117.6 e
95.8 de
0
0
0
0
0
0.7 a
0.7 a
8.2 b
8.1 b
4.2 ab
7.9 b
19.4 c
0
0.8 a
0
Phosphatase activity
(mg pNPP ml1 h1)*
*Means followed by the same letter did not differ significantly at 5% Tukey test.
a
Sample identification are according to Table 1.
b
Means followed by different higher case letter in each row represent the differences between the P-solubilizing isolates, and lower case letter in each column represents
differences between the Conventional method and the P-Mehlich1 method.
c
Amount of P solubilized by the Murphy and Riley’s (1962) procedure after 10 days of growth in a liquid medium containing 101.1 mg P l1 insoluble phosphate.
d
Amount of P solubilized extracted by the P-Mehlich1 procedure (Mehlich, 1978), after 10 days growth in a liquid medium containing 101.1 mg P l1 insoluble phosphate.
Please cite this article in press as: Oliveira, C.A. et al., Phosphate solubilizing microorganisms isolated from rhizosphere of maize cultivated in an
oxisol of the Brazilian Cerrado Biome, Soil Biology and Biochemistry (2008), doi:10.1016/j.soilbio.2008.01.012
ARTICLE IN PRESS
6
C.A. Oliveira et al. / Soil Biology and Biochemistry xxx (2008) 1–6
with potential for P solubilization. Wakelin et al. (2004), studying
strains of Penicillium isolated from wheat roots, reported a lower P
level in the solution when the production of the polysaccharide was
high. Further adjustments in P extraction methodology should be
investigated to improve the accuracy in measuring the P mobilized
by P-solubilizing microorganisms.
We observed in these experiments, comparing the relative
efficiency of phosphate solubilizing microorganisms using different
insoluble P sources for plant absorption, that P solubilization and P
mobilizing mechanisms depended on the nature of the P source
and the organisms involved in the process. These microorganisms
are being maintained in a genetic bank for future research and for
potential biotechnological applications in the development of more
sustainable production systems. These microorganisms are of
interest as potential for inoculants to improve the efficiency of P
acquisition from the soil and fertilizer sources, especially rock
phosphates.
Acknowledgements
This work was supported by Embrapa Maize and Sorghum and
the McKnight Foundation. C.A. Oliveira acknowledges the Brazilian
Council for Scientific and Technological Development (CNPq) for
supporting her PhD studies.
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oxisol of the Brazilian Cerrado Biome, Soil Biology and Biochemistry (2008), doi:10.1016/j.soilbio.2008.01.012