Applied Soil Ecology 34 (2006) 33–41

www.elsevier.com/locate/apsoil

Phosphate solubilizing bacteria from subtropical soil and

their tricalcium phosphate solubilizing abilities
Y.P. Chen, P.D. Rekha, A.B. Arun, F.T. Shen,
W.-A. Lai, C.C. Young *
Department of Soil and Environmental Sciences, National Chung Hsing University,
250, Kuo-Kuang Road, Taichung, Taiwan 402, ROC
Received 20 June 2005; received in revised form 2 November 2005; accepted 19 December 2005

Abstract
The ability of a few soil microorganisms to convert insoluble forms of phosphorus to an accessible form is an important trait in
plant growth-promoting bacteria for increasing plant yields. The use of phosphate solubilizing bacteria as inoculants increases the P
uptake by plants. In this study, isolation, screening and characterization of 36 strains of phosphate solubilizing bacteria (PSB) from
Central Taiwan were carried out. Mineral phosphate solubilizing (MPS) activities of all isolates were tested on tricalcium phosphate
medium by analyzing the soluble-P content after 72 h of incubation at 30 8C. Identification and phylogenetic analysis of 36 isolates
were carried out by 16S rDNA sequencing. Ten isolates belonged to genus Bacillus, nine to genus Rhodococcus, seven to genus
Arthrobacter, six to genus Serratia and one each to genera Chryseobacterium, Delftia, Gordonia and Phyllobacterium. In addition,
four strains namely, Arthrobacter ureafaciens, Phyllobacterium myrsinacearum, Rhodococcus erythropolis and Delftia sp. are
being reported for the first time as phosphate solubilizing bacteria (PSB) after confirming their capacity to solubilize considerable
amount of tricalcium phosphate in the medium by secreting organic acids. P-solubilizing activity of these strains was associated
with the release of organic acids and a drop in the pH of the medium. HPLC analysis detected eight different kinds of organic acids,
namely: citric acid, gluconic acid, lactic acid, succinic acid, propionic acid and three unknown organic acids from the cultures of
these isolates. An inverse relationship between pH and P solubilized was apparent from this study. Identification and characteriza-
tion of soil PSB for the effective plant growth-promotion broadens the spectrum of phosphate solubilizers available for field
application.
# 2006 Elsevier B.V. All rights reserved.

Keywords: Phosphate solubilizing bacteria (PSB); Tricalcium phosphate; Organic acid; Genetic diversity; Subtropical soil

1. Introduction chemical fertilizer is immobilized rapidly and becomes

Phosphorus (P) is one of the major essential
macronutrients for plants and is applied to soil in the
form of phosphatic fertilizers. However, a large portion of
soluble inorganic phosphate applied to the soil as
* Corresponding author. Tel.: +886 4 22840373x4304;
fax: +886 4 22861495.
E-mail address: ccyoung@mail.nchu.edu.tw (C.C. Young).
unavailable to plants (Goldstein, 1986). Microorganisms
are involved in a range of processes that affect the
transformation of soil P and are thus an integral part of the
soil P cycle. In particular, soil microorganisms are
effective in releasing P from inorganic and organic pools
of total soil P through solubilization and mineralization
(Hilda and Fraga, 1999). Currently, the main purpose in
managing soil phosphorus is to optimize crop production
and minimize P loss from soils. Recently, phosphate
solubilizing microorganisms have attracted the attention

0929-1393/$ – see front matter # 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.apsoil.2005.12.002
34 Y.P. Chen et al. / Applied Soil Ecology 34 (2006) 33–41

Fig. 1. Phylogenetic analysis based on16S rDNA sequence available from European Molecular Biology Laboratory data library (accession numbers
are given in parentheses) was constructed. Distances (distance options according to the Kimura-2 model) and clustering with the neighbor-joining
 Y.P. Chen et al. / Applied Soil Ecology 34 (2006) 33–41
of agriculturists as soil inoculums to improve the plant
growth and yield (Young, 1994; Young et al., 1998;
Goldstein et al., 1999; Fasim et al., 2002). Plant growth-
promoting bacteria (PGPB) are soil and rhizosphere
bacteria that can benefit plant growth by different
mechanisms (Glick, 1995), and P-solubilization ability
of the microorganisms is considered to be one of the most
important traits associated with plant P nutrition. Given
the negative environmental impacts of chemical fertili-
zers and their increasing costs, the use of PGPB is
advantageous in the sustainable agricultural practices.
It is generally accepted that the mechanism of
mineral phosphate solubilization by PSB strains is
associated with the release of low molecular weight
organic acids (Goldstein, 1995; Kim et al., 1997a),
which through their hydroxyl and carboxyl groups
chelate the cations bound to phosphate, thereby
converting it into soluble forms (Kpomblekou and
Tabatabai, 1994). However, P-solubilization is a
complex phenomenon, which depends on many factors
such as nutritional, physiological and growth conditions
of the culture (Reyes et al., 1999). There is experimental
evidence to support the role of organic acids in mineral
phosphate solubilization (Halder et al., 1990).
With the emphasis on screening for potential PSB
from the soil for the agricultural purposes the present
experiment was designed to evaluate biochemical and
genetic characteristics of indigenous PSB from the
natural subtropical soil habitats of central Taiwan.
Attempt was also made to establish the genetic
relationship of PSB with its MPS activity, types of
organic acid secreted, pH and P-solubilization.
2. Materials and methods
2.1. Isolation of phosphate solubilizing bacteria
Soil samples were collected from a non-cropped,
undisturbed site that was covered by native vegetation
near Taichung city (Taiwan, ROC). Approximately 50 g
of soil sample was taken from the upper 30 cm of the
soil profile aseptically. The soil pH and electrical
conductivity were 6.8 and 327 mS/cm. Soil contained
59.1% of sand, 11.65% clay and 29.3% silt.
The serially diluted soil samples were plated on
standard agar medium (pH 6.8–7.0) containing 5 g of
tricalcium phosphate (TCP) as sole phosphorus source
for selectively screening the bacteria which have the
ability to release inorganic phosphate from tricalcium
method was performed by using the software packages MEGA (Molecular Evolutionary Genetics Analysis) version 2.1 (Kumar et al., 2001).
Bootstrap values based on 1000 replications are listed as percentages at the branching points.
35
phosphate (Nautiyal et al., 2000). Uninoculated plates
and E. coli inoculated plates served as controls. After
3-days of incubation at 30 8C, PSB developed clear
zones around colonies. Colonies with clear zones were
further purified by replating on agar medium
supplemented with TCP. Thirty-six phosphate solu-
bilizing bacterial strains thus screened were selected
for further analysis. Isolates were designated as CC-
BC01 to CC-BC36.
2.2. Genomic DNA isolation, 16S rDNA
sequencing, phylogeny and RAPD amplification of
16S rDNA
Total genomic DNA was extracted and purified using
the method described by Pospiech and Neumann
(1995). PSB were identified by using MicroSeq 500
16S rDNA Bacterial Sequencing Kit and DNA
sequencer (model: ABI 310, Perkin-Elmer Applied
Biosystem, CA, USA) (Watts and MacBeath, 2001).
Sequence data were aligned and compared with
available standard sequences of bacterial lineage in
the National Center for Biotechnology Information
GenBank (http://www.ncbi.nlm.nih.gov/) using
BLAST. A phylogenetic tree was constructed by using
neighbor joining method from distance matrices.
Genomic profiles of bacteria were studied using
RAPD-PCR with 80 different 10-mer random primers
(data not shown). RAPD amplification was carried out
on a RoboCycler GRADIENT96 thermocycler (STRA-
TAGENE). A 25 ml reaction mixture contained 20 ng of
bacterial DNA as template, 0.2 mM of primer and 1 unit
of Taq DNA polymerase and 800 mM dNTPs. The
reaction conditions included an initial denaturation of
5 min at 94 8C followed by 40 cycles of 1 min 30 s at
94 8C, 1 min 30 s at 37 8C and 2 min 30 s at 72 8C with
a final extension of 10 min 30 s at 72 8C. PCR products
were resolved on 1.5% agarose gel with 50 V running
voltage for 1 h. The RAPD bands were recorded as
binary data and analyzed with NTSYS software using
UPGMA method (Rohlf, 1993).
2.3. Phosphate solubilization, pH and identification
of organic acids
Phosphate solubilizing bacteria were inoculated
into 100 ml tricalcium phosphate medium as described
earlier without agar. Sterile water inoculated media
was treated as blank and Escherichia coli served as
36 Y.P. Chen et al. / Applied Soil Ecology 34 (2006) 33–41
control. After incubation for 3-days, pH of the medium
was recorded with a pH meter equipped with glass
electrode; cell numbers were estimated by standard
plate count method and amount of soluble phosphate
was measured by Mo-blue method (Watanabe and
Olsen, 1965).
For the analysis of organic acids, bacterial cultures
were filtrated through 0.2 mm filter (Millipore, GTBP)
and 20 ml of filtrates were injected to HPLC (Model:
Hitachi L-5000) equipped with a Hitachi L-3000 Poto
Diode Array detector. The organic acid separation was
carried out on Aminex HPX-87C column (Bio-Rad
Laboratories, Inc.) with 10.8% acetonitrile in 0.0035 M
H2SO4 as mobile phase. Retention time of each signal
was recorded at a wavelength of 210 nm.
2.4. Statistical analysis
Differences in the results were statistically analyzed
using Student’s t-test and comparison between treat-
Fig. 2. The dendrogram of 36 bacterial isolates obtained based on the similarity of RAPD markers.
ment means were calculated using Duncan’s multiple
range test (CoStat version 6.3, CoHort Software,
Berkeley, CA, USA) at 5% probability level ( p < 0.05).
3. Results
3.1. Isolation of phosphate solubilizing bacteria,
phylogeny and RAPD analysis
The identification of phosphate solubilizing bacterial
strains based on 16S rDNA sequence and their phylogeny
are presented in Fig. 1. Nine isolates of Rhodococcus
erythropolis, 6 isolates of Serratia marcescens, 10
isolates of Bacillus megaterium and 5 isolates of
Arthrobacter ureafaciens, 2 isolates of Arthrobacter
sp. and single isolate each of Gordonia sp., Chryseo-
bacterium sp., Phyllobacterium myrsinacearum and
Delftia sp. were confirmed as phosphate solubilizing
bacteria. Interestingly, two isolates Chryseobacterium
sp. CC-BC05 and Gordonia sp. CC-BC07 showed only
 Y.P. Chen et al. / Applied Soil Ecology 34 (2006) 33–41
96–97% similarities with the closest known species in the
GenBank.
The RAPD technique was used to elucidate the
polymorphism among the 36 isolates and to ascertain
their possible relationship with MPS activities. Out of
the 80 random primers used only 20 were effective in
resolving the difference. The similarity coefficient was
calculated by NTSYS software based on the RAPD
binary data showed that similarity among 36 isolates
ranged between 0.15 and 0.82 and formed 4 distinct
clusters (Fig. 2).
3.2. P solubilization and pH
The details of the cell densities, pH and amounts of
soluble-P in the medium after 72 h of incubation are
presented in Table 1. The solubilization of TCP in the
liquid medium by different strains was accompanied by a
significant drop in pH (to 4.9 and 6.0) from an initial pH
of 6.8–7.0 after 72 h. The soluble-P concentration in the
medium ranged between 31.5 and 519.7 mg/l with
variations among different isolates. In the blank
treatment no soluble-P was detected as well no drop in
pH was observed while, in the E. coli cultures (control)
the pH dropped to 6.3 with 20.2 mg/l of soluble-P in the
medium. The maximum P solubilization was recorded by
Arthrobacter sp. (CC-BC03) followed by S. marcescens
(CC-BC14) (421.8 mg/l) with a maximum drop in the pH
to 4.9. Among the isolates the minimum concentration of
soluble-P (31.5 mg/l) was observed in the cultures of
Gordonia sp. (CC-BC07) and the pH of the medium was
relatively higher (6.0). Eventhough maximum drop in pH
was associated with higher levels of P solubilization, in
some cases, for example, Chryseobacterium sp. (CC-
BC05), where pH was decreased only to 6.0, compara-
tively higher amounts of soluble-P (289.8 mg/l) was
detected in the medium. Statistically a strong negative
correlation (r = 0.80; p < 0.01) between pH and soluble-
P concentration was observed. In addition, the cell
densities and the P solubilized were also positively
correlated (r = 0.417; p < 0.01).
3.3. Organic acid production in broth culture
HPLC analysis showed the presence of organic acids
in the culture mediums of by all the strains except three
(CC-BC07, CC-BC31 and CC-BC34). Eight different
organic acids were produced by 33 isolates, among
them 3 were unknown acids (citric acid, lactic acid,
gluconic acid, succinic acid, propionic acid, unknown1,
unknown2 and unknown3). Many of the isolates showed
the presence of multiple organic acids. The details are
37
given in Table 1 and Fig. 3. The retention times of the
three unknown organic acids were 12.5, 25.1 and 22.4
for unknown1, unknown2 and unknown3, respectively.
Three isolates (CC-BC07, CC-BC31 and CC-BC34) did
not produce any organic acid in detectable amounts and
the phosphate solubilization was minimum (Table 1).
Bacterial strains such as Arthrobacter sp. (CC-BC03)
and S. marcescens (CC-BC12, CC-BC14 and CC-
BC16) which synthesized maximum number of organic
acids showed maximum decline in pH (4.9) with higher
levels of P solubilization. It was also evident that the
PSB isolates, which secreted citric acid either singly or
in mixtures showed higher MPS activities with rare
exceptions such as A. ureafaciens (CC-BC32), and B.
megaterium (CC-BC35 and CC-BC36). Further, inter-
estingly, all the five isolates of S. marcescens (CC-
BC12-CC-BC16) synthesized citric acid in combination
with other acids resulting in a maximum drop in the pH
and higher levels of soluble phosphorus in the culture
medium. Among the 33 isolates that secreted organic
acids in the medium, propionic acid was produced by
only four B. megaterium isolates (CC-BC26 to CC-
BC30). There was a significant negative correlation
(r = 0.68, p < 0.01) between number of acids produced
and the pH.
4. Discussion
Identification of individual isolates by 16S rDNA
and subsequent assignment of isolates to their
respective genera and genetic variations among the
isolates of same species was successfully achieved by
adopting RAPD method. But, these variations were not
well depicted with phosphate solubilizing trait. The
RAPD technique was developed as an efficient tool to
analyze phylogenetic relationships among and within
closely related species (Williams et al., 1990). Intra-
specific variations have been described in the RAPD
both in bacteria and fungi; therefore this technique is of
great use in biodiversity studies, but at an intra-specific
level (Peix and Martinez-Molina, 2002). Interestingly,
for the first time certain difference was observed in the
type of organic acid produced by the isolates that are
identified by 16S rDNA sequencing as the strains
representing same species. Examples include B.
megaterium (CC-BC31), which was unable to produce
any type of organic acids, while all the other nine
isolates of the B. megaterium produced organic acids,
signifying that each isolate is independent of its MPS
activity, amount and type of acid secreted with its
genetic identity even when they were provided with the
similar nutrient conditions.
38 Y.P. Chen et al. / Applied Soil Ecology 34 (2006) 33–41

Table 1
Variations in cell densities, pH, and soluble-P concentration observed after 72 h incubation
Isolates Cell density (log CFU/ml) pH of medium Soluble-P (mg/l) Organic acids*
Rhodococcus erythropolis
CC-BC11 8.0 b 5.3 a 186.9e G + U1
CC-BC09 8.4 b 5.3 a 170.7ef G + U1
CC-BC20 8.0 b 5.3 a 169.3ef G + U1
CC-BC22 8.1 b 5.3 a 162.9ef G + U1
CC-BC17 8.3 b 5.4 ab 151.2ef G + U1
CC-BC04 8.2 b 5.4 ab 135.0f C + U1
CC-BC08 8.2 b 5.5 ab 130.7f U1
CC-BC01 8.1 b 5.5 ab 118.1f U1
CC-BC25 8.5 ab 5.6 ab 87.0g U1
Bacillus megaterium
CC-BC10 7.7 b 5.1 a 270.2d C + L + U1
CC-BC26 7.5 bc 5.4 ab 211.7de L+P
CC-BC35 8.1 b 5.1 a 183.9e C + U1
CC-BC36 6.0 c 5.4 ab 151.4ef C + U1
CC-BC30 6.8 c 5.9 b 140.6ef L+P
CC-BC27 8.0 b 6.0 b 132.2f L+P
CC-BC31 6.8 c 5.6 ab 129.2f None
CC-BC28 6.0 c 6.0 b 126.8f L+P
CC-BC29 6.6 c 5.5 ab 113.8f P
CC-BC06 8.0 b 5.8 b 72.2g U1
Arthrobacter sp.
CC-BC03 7.2 c 4.9 a 519.7a C+L
CC-BC34 8.8 a 5.5 ab 60.1g None
Arthrobacter ureafaciens
CC-BC02 8.2 b 5.0 a 316.1cd C + U2
CC-BC23 8.7 a 5.0 a 247.5d C + U1
CC-BC32 8.7 a 5.4 ab 92.3g C + U1
CC-BC33 7.2 c 5.5 ab 89.9g U1
CC-BC24 7.3 c 5.9 b 36.8h U1
Serratia marcescens
CC-BC14 9.0 a 4.9 a 421.8b C + G + S + L + U1
CC-BC12 9.3 a 4.9 a 392.8bc C + G + S + L + U1
CC-BC16 8.7 a 4.9 a 391.6bc C + S + L + U1
CC-BC13 9.1 a 4.9 a 355.3c C + G + S + L + U1
CC-BC18 8.9 a 5.0 a 307.8cd C + G + S + L + U1
CC-BC 15 8.9 a 5.1 a 232.1d C + S + L + U1
Delftia sp.
CC-BC21 9.0 a 4.9 a 346.1c S + U1
Chryseobacterium sp.
CC-BC05 8.5 ab 6.0 b 289.8d C + U3
Phyllobacterium myrsinacearum
CC-BC19 9.0 a 5.2 a 201.2e G + U1
Gordonia sp. CC-BC07
CC-BC07 8.6 a 6.0 b 31.5h None
(a–h) Data following comment letter in superscripts in each column are not significantly different at 5% probability level as per Duncan’s multiple
range test.
*
C, citric acid; G, gluconic acid; S, succinic acid; L, lactic acid; P, propionic acid; U1, U2 and U3, unknown acid 1, 2 and 3

Members of genus Serretia displayed higher MPS beneficial to plant P nutrition and growth. Among the 36
activities compared to the members of the genera isolates Arthrobacter sp. (CC-BC03) was an excellent
Bacillus, Rhodococcus and Arthrobacter. Hence, pre- solubilizer of tricalcium phosphate and four bacterial
sence of Serretial members in the rhizosphere might be isolates namely, A. ureafaciens, Delftia sp., P. myrsi-
 Y.P. Chen et al. / Applied Soil Ecology 34 (2006) 33–41 39

Fig. 3. HPLC analysis of the known and unknown organic acids detected in the exudates of some representative isolates: Arthrobacter sp. (CC-BC03
and CC-BC34), Rhodococcus erythropolis (CC-BC01 and CC-BC09), Gordonia sp. (CC-BC07), Arthrobacter ureafaciens (CC-BC02), Bacillus
megaterium (CC-BC10 and CC-BC26), Serratia marcescens (CC-BC12), Chryseobacterium sp. (CC-BC05).
40 Y.P. Chen et al. / Applied Soil Ecology 34 (2006) 33–41
nacearum and R. erythropolis, are being reported for the
first time as phosphate solubilizers.
The P-solubilizing activity is determined by the
microbial biochemical ability to produce and release
organic acids, which through their carboxylic groups
chelate the cations (mainly Ca) bound to phosphate
converting them into the soluble forms (Kpomblekou
and Tabatabai, 1994). From the results of this study, it is
being reaffirmed that the phosphate solubilization by
different PSBs is involved with the production of
organic acids (Halder et al., 1990; Goldstein, 1995; Kim
et al., 1998; Rashid et al., 2004). The inverse
relationship observed between the pH and soluble-P-
concentration indicates that organic acid production by
these PSB strains plays a significant role in the
acidification of the medium facilitating the P solubi-
lization. Similar inverse relationship between pH and
soluble phosphate was reported earlier by Illmer and
Schinner (1995) and Hwangbo et al. (2003).
Although the HPLC system that was used here to
analyze the organic acids was able to detect 2-keto
gluconic acid, none of our isolates showed the release of
this acid instead, three unknown acids were detected
and interestingly, unknown1 was secreted by 26/36
isolates which implies for the first time on the role of
major and previously unknown acid, which could be
involved in MPS activity by PSBs. Major acid
unknown1 in combination with other acids was
successful in decreasing pH and induced higher MPS
activity, while, it failed when it was secreted alone (CC-
BC01). Three isolates, which did not produce any kinds
of organic acids but showed P solubilization, could be
ascribed to the participation of an unknown substance or
mechanism, other than acid production. Similar
interesting observations were recorded by Kim et al.
(1997b), wherein genomic DNA fragment from
Enterobacter agglomerans (Pantoea agglomerans)
expressed MPS activity in E. coli JM109 without any
noticeable alteration in pH of the medium. These results
indicate that, though the production of organic acid by
the microorganisms is one of the major factors but not
the sole factor responsible for phosphate solubilization
by bacteria.
P is abundant in several soils and is one of the major
nutrients limiting the plant growth. The overall P use
efficiency following phosphate fertilizer application is
low because of the formation of insoluble complexes
(Vassilev and Vassileva, 2003). Hence, frequent
application of soluble forms of inorganic P is necessary
for crop production and which leaches to the ground
water and results in eutrophication of aquatic systems
(Del Campillo et al., 1999). In view of environmental
concerns and current developments in sustainability,
research efforts are concentrated on elaboration of
techniques that involve the use of less expensive, though
less bio-available sources of plant nutrients such as rock
phosphate and by application of PSB the agronomic
effectiveness can be enhanced (Whitelaw, 2000).
From the present study, we demonstrate that the
natural subtropical soil supports a diverse group of
potential phosphate solubilizing bacteria. These P
solubilizing soil bacteria could serve as efficient
biofertilizer candidates for improving the P-nutrition
of crop plants. The advantage of using natural soil
isolates over the genetically manipulated or the one
which has been isolated from a different environmental
set up is the easier adaptation and succession when
inoculated into the plant rhizosphere.
It is concluded from the present study that all the
PSB isolates except three produced multiple organic
acids followed by a decrease in the pH of the culture
medium there by solubilizing the insoluble tricalcium
phosphate. More studies are warranted to understand
the significance and mechanism used by an unknown
acid1 in MPS activity. Use of these PSB as bio-
inoculants will increase the available P in soil, helps to
minimize the P-fertilizer application, reduces environ-
mental pollution and promotes sustainable agriculture.
Acknowledgements
This research work was kindly supported by grants
from the National Science Council of Taiwan, ROC and
Council of Agriculture, Executive Yuan, Taiwan, ROC.
References
Del Campillo, S.E., Van der Zee, S.E.A.T.M., Torrent, J., 1999.
Modelling long-term phosphorous leaching and changes in phos-
phorous fertility in selectively fertilized acid sandy soils. Eur. J.
Soil Sci. 50, 391–399.
Fasim, F., Ahmed, N., Parson, R., Gadd, G.M., 2002. Solubilization of
zinc salts by a bacterium isolated from air environment of a
tannery. FEMS Microbiol. Lett. 213, 1–6.
Glick, B.R., 1995. The enhancement of plant growth by free-living
bacteria. Can. J. Microbiol. 41, 109–117.
Goldstein, A.H., 1986. Bacterial solubilization of mineral phosphates:
historical perspectives and future prospects. Am. J. Altern. Agri-
cult. 1, 57–65.
Goldstein, A.H., 1995. Recent progress in understanding the mole-
cular genetics and biochemistry of calcium phosphate solubiliza-
tion by Gram negative bacteria. Biol. Agric. Hort. 12, 185–193.
Goldstein, A.H., Braverman, K., Osorio, N., 1999. Evidence for
mutualism between a plant growing in a phosphate-limited desert
environment and a mineral phosphate solubilizing (MPS) bacter-
ium. FEMS Microbiol. Ecol. 3, 295–300.
 Y.P. Chen et al. / Applied Soil Ecology 34 (2006) 33–41
Halder, A.K., Mishra, A.K., Bhattacharya, P., Chakrabarthy, P.K.,
1990. Solubilization of rock phosphate by Rhizobium and Bra-
dyrhizobium. J. Gen. Appl. Microbiol. 36, 81–92.
Hilda, R., Fraga, R., 1999. Phosphate solubilizing bacteria and their
role in plant growth promotion. Biotechnol. Adv. 17, 319–359.
Hwangbo, H., Park, R.D., Kim, Y.W., Rim, Y.S., Park, K.H., Kim,
T.H., Suh, J.S., Kim, K.Y., 2003. 2-Ketogluconic production and
phosphate solubilization by Enterobacter intermedium. Curr.
Microbiol. 47, 87–92.
Illmer, P., Schinner, F., 1995. Solubilization of inorganic calcium
phosphates-solubilization mechanisms. Soil Biol. Biochem. 27,
257–263.
Kim, K.Y., Jordan, D., Krishnan, H.B., 1997a. Rahnella aqualitis, a
bacterim isolated from soybean rhizosphere, can solubilize hydro-
xyapatite. FEMS Microbiol. Lett. 153, 273–277.
Kim, K.Y., McDonald, G.A., Jordan, D., 1997b. Solubilization of
hydroxypatite by Enterobacter agglomerans and cloned Escher-
ichia coli in culture medium. Biol. Fertil. Soils 24, 347–352.
Kim, K.Y., Jordan, D., McDonald, G.A., 1998. Enterobacter agglom-
erans, phosphate solubilizing bacteria, and microbial activity in
soil: effect of carbon sources. Soil Biol. Biochem. 30, 995–1003.
Kpomblekou, K., Tabatabai, M.A., 1994. Effect of organic acids on
release of phosphorus from phosphate rocks. Soil Sci. 158, 442–
453.
Kumar, S., Tamura, K., Jakobsen, I.B., Nei, M., 2001. MEGA2:
molecular evolutionary genetics analysis software. Bioinformatics
17, 1244–1245.
Nautiyal, C.S., Bhadauria, S., Kumar, P., Lal, H., Verma, M.D., 2000.
Stress induced phosphate solubilization in bacteria isolated from
alkaline soils. FEMS Microbiol. Lett. 182, 291–296.
Peix, A., Martinez-Molina, E., 2002. Molecular methods for biodi-
versity analysis of PSB. In: First Meeting on Microbial Phosphate
Solubilization. Salamanca, Spain, 16–19 July (http://webcd.u-
sal.es/web/psm/abstracts/Alvaro.htm).
41
Pospiech, A., Neumann, B., 1995. A versatile quick prep of genomic
DNA from Gram-positive bacteria. Trends Gen. 11, 217–218.
Rashid, M., Khalil, S., Ayub, N., Alam, S., Latif, F., 2004. Organic
acids production and phosphate solubilization by phosphate solu-
bilizing microorganisms (PSM) under in vitro conditions. Pak. J.
Biol. Sci. 7, 187–196.
Reyes, I., Bernier, L., Simard, R., Antoun, H., 1999. Effect of nitrogen
source on solubilization of different inorganic phosphates by an
isolate of Pencillium rugulosum and two UV-induced mutants.
FEMS Microbiol. Ecol. 28, 281–290.
Rohlf, F.J., 1993. NTSYS-pc: Numerical Taxonomy and Multivariate
Analysis System Version 1.80. Department of Ecology and Evolu-
tion, State University of New York, Stony Brook, NY.
Vassilev, N., Vassileva, M., 2003. Biotechnological solubilization of
rock phosphate on media containing agro-industrial wastes. Appl.
Microbiol. Biotechnol. 61, 435–440.
Watanabe, F.S., Olsen, S.R., 1965. Test of an ascorbic acid method for
determining phosphorous in water and NaHCO3 extracts from soil.
Soil Sci. Soc. Am. Proc. 29, 677–678.
Watts, D., MacBeath, J.R., 2001. Automated fluorescent DNA sequen-
cing on the ABI PRISM 310 Genetic Analyzer. Meth. Mol. Biol.
167, 153–157.
Whitelaw, M.A., 2000. Growth promotion of plants inoculated with
phosphate solubilizing fungi. Adv. Agron. 69, 99–151.
Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A., Tingey,
S.V., 1990. DNA polymorphisms amplified by arbitrary primers
are as genetic markers. Nucl. Acids Res. 18, 6531–6535.
Young, C.C., Chang, C.H., Chen, L.F., Chao, C.C., 1998. Character-
ization of the nitrogen fixing and ferric phosphate solubilizing
bacteria isolated from Taiwan soil. J. Chin. Agricult. Chem. Soc.
36, 201–210 (in Chinese).
Young, C.C., 1994. Selection and application of biofertilizers in
Taiwan. Food and Fertilizer Technology Center. Tech. Bull.
141, 1–9.