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Chiang Mai J. Sci. 2013; 40(1) 11
Chiang Mai J. Sci. 2013; 40(1) : 11-25
http://it.science.cmu.ac.th/ejournal/
Contributed Paper
Characteristics of Phosphate Solubilization by
Phosphate-Solubilizing Bacteria Isolated from
Agricultural Chili Soil and Their Efficiency on the
Growth of Chili (Capsicum frutescens L. cv. Hua Rua)
Worapon Surapat [a], Charida Pukahuta [a], Pongsak Rattanachaikunsopon [a],
Tadanori Aimi [b], and Sophon Boonlue*[c]
[a] Department of Biological Sciences, Faculty of Science, Ubon Ratchathani University,
Ubon Ratchathani 34190, Thailand.
[b] Department of Biochemistry and Biotechnology, Faculty of Agriculture, Tottori University,
Tottori 680-8553, Japan.
[c] Department of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand.
*Author for correspondence; e-mail: bsopho@kku.ac.th.
Received: 10 April 2012
Accepted: 21 May 2012
ABSTRACT
No previous studies have been carried out on the isolation and characterization of
phosphate-solubilizing bacteria (PSB) in organic and conventionally grown chili (Capsicum frutescens
L. cv. Hua Rua) in Ubon Ratchathani province, Thailand. This study aimed to isolate PSB from
rhizosphere soil of chili cultivated on organic and conventional farms. Characterization of
organic acid and ability of mineral phosphate solubilization were also investigated. The effects
of PSB on the growth of chili were observed in a pot trial. The most PSB (over 68%) could
be obtained from organically managed soil. Ten isolates of PSB were selected based on
tricalcium phosphate solubilizing ability, and further testing was carried out for characterization
of organic acids. The selected PSB exhibited phosphate-solubilizing ability ranging between
126.36-488.55 μg/ml of phosphate. Additionally, the 16S rRNA gene and phylogenetic analysis
showed maximum similarity to Burkholderia ambifaria and B. tropica. HPLC analysis of organic
acids produced from these isolates in a culture medium found eight different kinds of organic
acids: acetic acid, citric acid, gluconic acid, lactic acid, succinic acid, propionic acid, and two
unknown organic acids. Among the isolates in this study, multiple organic acids could be
produced from eight isolates, and a single organic acid was found from two isolates. Multiple
organic acids did not correspond with an ability to solubilize mineral phosphate. Isolate KS04
was obtained from a conventional chili farm, and showed the greatest efficiency in promotion
of chili growth cultivated in sterile organically managed soil; it significantly increased the growth,
flowering and P uptake, compared to uninoculated plants. Thus, this isolate may be useful for
use as a bio-inoculant for chili production in organically managed soil.
Keywords: Burkholderia tropica KS04, Chili growth, Organic acid, Organically managed soil,
Phosphate-solubilizing bacteria
12 Chiang Mai J. Sci. 2013; 40(1)
1. INTRODUCTION
Chili (Capsicum frutescens L.) is an
economically important vegetable of
Thailand that can be grown all year
round [1]. The biggest production of chili is
in the northeast of Thailand [2], particularly in
the southern part of the northeast, namely
Ubon Ratchathani and Sisaket provinces [3].
Generally, agricultural practices of Thai
farmers for cultivation of chili have been
performed using two systems: conventional
and organic. High chemical inputs, such as
synthetic fertilizers and synthetic pesticides, are
typical in a conventional system, which is
practiced by most farmers. In an organic
system, green manure, animal manures,
liquid fertilizer (effective microorganism,
called EM) and compost are applied. As a
result, the quality and price of chili products
is higher than those produced in a
conventional system [4].
Inorganic fertilizer is an economical way
to supply enough nutrients to increase chili
production. Among the macronutrients used
for chili production, phosphorus (P) is one
of the most essential for plant growth, the
second being nitrogen. However, a large
amount of inorganic phosphate applied to
the soil as chemical fertilizer is immobilized
rapidly, causing unavailability and a limitation
for plant use [2, 5, 6]. Recent reports have
documented that approximately 95-99% of
phosphorus in soil is present in the form of
insoluble phosphates [7], but only 0.1% of
the total phosphorus exists in plant-accessible
form [8]. This problem may be alleviated by
releasing P from an immobilized form to a
soluble form through the activities of
solubilization and mineralization by soil
microorganisms [9].
Recently, plant growth-promoting
rhizobacteria (PGPR) have become attractive
for use as soil inoculums due to their
improvement of plant growth and yield by
various direct or indirect mechanisms [10, 11].
Among PGPR, phosphate-solubilizing
bacteria (PSB) are one of the most interesting
microorganisms concerned with plant
P nutrition. The principal mechanism for
mineral phosphate solubilization by these
bacteria is the production of low-molecular-
weight organic acids, which have high
potential as cations bound to phosphate,
as a result of their conversion into soluble
forms [9]. PSB has the ability to dissolve
tricalcium phosphate from an insoluble
form into a soluble form, as has been
reported by many researchers [12, 13, 14, 15].
Additionally, the use of PSB as inoculants
simultaneously increases P uptake by the
plant, resulting in higher crop yields - as has
been documented in maize [16], greengram
[17], sorghum [18], wheat, potato, bean [19]
and tomato [20]. There have been few reports
concerning growth promotion of chili by
co-inoculation of arbuscular mycorrhizal
fungi and PSB [21, 22]. However, there have
been no reports on the isolation of PSB
from organically managed soil; most
results have been documented from other
agricultural and rangeland soils [12, 23, 24].
In order to investigate the potential of
PSB as inoculants for chili cultivation in
organically managed soil, the present
experiment was designed to characterize the
phosphate solubilization by PSB isolated
from conventional and organic soil and to
determine the effects of PSB on the growth
and P uptake of chili (cv. Hua Rua) planted in
sterile, organically managed soil under
greenhouse condition.
2. MATERIALS AND METHODS
2.1 Soil Sampling and Isolation of PSB
Soil samples were collected from one
organic farm, Ratchathani Asok (15° 13’
34.48" N, 104° 54’ 11.05" E), and from three
conventional chili farms, Ban Hua Rua
Chiang Mai J. Sci. 2013; 40(1) 13
(15° 22’ 4.82" N, 104° 49’ 49.74" E), Ban
Kaset Nua (15° 18’ 9.97’’ N, 104° 89’ 84.91’’E)
and Ban Pho Yai (15° 21’ 16.44’’ N, 104° 92’
0.12’’ E), in Ubon Ratchathani province.
Approximately 50 g of chili root zone soil
(30 cm depth) was taken aseptically from the
central areas (4 × 4 m) of three agricultural
chili plots at each of the four sites, with
duplicates from each plot. Each soil sample
was thoroughly mixed and further enriched
on Pikovskaya’s medium, containing 0.5%
tricalcium phosphate as a sole P source, at
30° C for 24 h. Subsequently, PSB were
isolated by dilution plate technique on
Pikovskaya’s agar medium [25]. After 3 d of
incubation at 30° C, colonies with hollow
clear zones were obtained, and thereafter
purified on Pikovskaya’s agar.
2.2 Morphological and Biochemical
Characteristics
Gram staining is used to classify bacteria
on the basis of their forms, sizes, cellular
morphologies, and Gram reactions [26].
Biochemical characterization of PSB isolates
was carried out according to Coenye et al.
[27], Garrity et al. [28] and Reis et al. [19].
Utilization of sugar and fermentation were
determined by starch hydrolysis, gelatin
hydrolysis and arginine hydrolysis, as well as
tests for oxidase, catalase, urease, nitrate
reduction, glucose, D-mannitol, lactose,
dulcitol, maltose, fructose, D-raffinose, and
sucrose.
2.3 Genomic DNA Isolation, 16S rRNA
Sequencing and Phylogeny
Template DNA for 16S rRNA gene
amplification was prepared by a method
modified from Nilsson et al. [29]. To produce
the fragments of the 16S rRNA gene,
genomic DNA of PSB was amplified by
pairs of a universal primer: UFUL (5’-
GCCTAACACATGCAAGTCGA-3’) and
URUL (5’ CGTATTACCGCGGCTGCT
GG-3’). The reaction conditions included
an initial denaturation of 5 min at 94° C,
followed by 30 cycles of denaturation at
94 ° C for 30 s, annealing at 55° C for 30 s,
and extension at 72° C for 30 s. A final
extension at 72° C for 5 min was done at the
end of the amplification. PCR products
were electrophoresed using 1.5% agarose,
then stained with ethidium bromide (0.5 μg/
ml), and visualized using a GelDoc-It®
(Upland, CA) UV transilluminator [30].
Sequence data were compared visually
and aligned with available standard
sequences of bacterial lineages from the
GenBank of the National Center for
Biotechnology Information (NCBI), using
a BLASTN analysis tool [31]. Mega4
molecular software, version 4.0, was used
for multiple sequence alignment and
phylogenetic tree analysis. Multiple sequence
alignment used the ClustalW software
package. Phylogenetic trees were built using
neighbor-joining. Bootstrap analysis was
based on 1,000 resamplings [32]. Distances
were calculated according to the Kimura
2-parameter [33]. The isolates were also
compared with those present in the Ribosomal
Database Project (http://rdp.cme.msu.edu/
), comprising reference profiles of closely
typed species strains.
2.4 Determination for Growth, Phosphate
Solubilizing Abilities, pH and Analysis
of Organic acid
PSB were inoculated into Pikovskaya’s
medium containing 0.5% tricalcium
phosphate, as described previously, by shaking
at 160 rpm at 30° C for 72 h. To estimate
bacterial growth yield, optical density (OD)
of bacterial culture was determined at
560 nm (OD560); then cell numbers
were examined by standard plate count
method [34], and later centrifuged for 5 min
14 Chiang Mai J. Sci. 2013; 40(1)
at 13,081 × g (10,000 rpm) at 4° C in a
Hettich Rotina 35R to remove bacterial
biomass. The supernatant was measured for
total amount of released soluble phosphate
contents by the Molybdenum blue method
[35]. The pH of the medium was also
recorded from the cultured supernatant by a
pH meter (Sartorius PP-20, Edgewood, NY)
in order to establish a relationship between
this parameter and the soluble phosphate [36].
For analysis of organic acids, cultured
media were filtered though a 0.2 μm filter
(GTBP; Millipore, Billerica, MA); 20 μl of
filtrates were then injected into an HPLC
column (LC-20AD; Shimadzu, Kyoto,
Japan) equipped with a SPD-M20A diode
array detector. Organic acid separation was
carried out on an Inertsil ODS-3 column
(GL Sciences, Tokyo, Japan) with 0.1 M
NH4H2PO4 in phosphoric acid (pH 2.6) as
mobile phase. The retention time of each
signal was recorded at a wavelength of
210 nm.
2.5 Effects of PSB on growth of chili in
pot experiment
PSB suspensions were prepared by
culturing on NB and incubating on an
orbital shaker at 160 rpm, 30° C, for 48 h.
Then bacterial cells were harvested by
centrifugation for 5 min at 13,081 × g (10,000
rpm), 4° C, in a Hettich Rotina 35R
centrifuge, and washed with sterile distilled
water. The cell pellet was resuspended with
sterile distilled water, and then further adjusted
to be 1 at OD560 (2.0 × 108 CFU/ml).
A pot experiment was carried out in a
greenhouse employing randomized complete
block design with four replications and
11 treatments. The treatments consisted of
one control (non-inoculated with PSB)
and 10 PSB species. All treatments were
conducted in pots (12-in diameter) containing
8 kg of soil. The soil used for preparation
of the seedling and pot experiment was
obtained from a plot that had been organically
managed for more than five years
(Ratchathani Asok). This organic soil was
amended by adding home made compost
(5,555.56 kg/ha), coarse rice bran (5,555.56
kg/ha), soybean press cake (2,777.78 kg/ha),
sesame press cake (2,777.78 kg/ha) and
rice husk ash (5,555.56 kg/ha). It had a
silt/loam texture, pH 4.6, 1.3% organic
matter, 0.08% total N, 66.8 ppm total P,
37.5 ppm available P (Bray II Method [37]),
and 78.3 ppm extractable K (1 N NH4OAc
[38]). Soil was sterilized by fumigation with
60g/m2 of dazomet (3,5-dimethyl-1,3,5-
thiadiazinane-2-thione). Chili (cv. Hua Rua)
seedlings were prepared from sterile
(10% v/v, NaOCl solution, for 10 min) seeds
planted in small plastic pots (top diameter,
7 cm) filled with fumigated soil. After 21 d,
individual chili seedlings were transplanted
into individual plastic pots (12-in diameter)
and then inoculated with 5 ml of PSB cell
suspension (2.0 × 108 CFU/ml). The plants
were watered with sterile distilled water
for 90 d. Five ml of PSB cell suspension
(2.0 × 108 CFU/ml) was reinoculated onto
the plant root zone by injection with a
sterilized syringe at every 2 weeks of
cultivation. At the end of the study,
plant growth parameters - such as shoot
height, stem diameter, fresh weight of
shoots and roots, dry weight of shoots and
roots, number of flowers per plant, and P
uptake - were determined.
2.6 Statistical analysis
Data were analyzed using SPSS for
Windows software, version 11.5.0 (SPSS,
Chicago, IL). All data were subjected to
analysis of variance. Comparisons of means
were made by Duncan’s multiple range test
(P≤0.05).
Chiang Mai J. Sci. 2013; 40(1) 15
3. RESULTS
3.1 Isolation of PSB and Preliminary
Screening for Phosphate Solubilization
PSB were isolated from rhizosphere
soil of chili cultivated at four sites from
agricultural farms in Ubon Ratchathani
province, Thailand. Fifty isolates of PSB
were obtained (Figure 1). A large amount of
PSB isolated strains were obtained from
the organic farm at Ratchathani Asok
(34 isolates). PSB isolates at conventional
chili plots were found to be fewer than at the
organic chili farm: at Ban Hua Rua (7 isolates),
Ban Kaset Nua (6 isolates), and Ban Pho Yai
(3 isolates). In order to screen for high
phosphate-solubilizing activity of PSB
strains for a further pot experiment, all PSB
strains were cultured on Pikovskaya’s
medium containing 0.5% tricalcium
phosphate. Ten isolates - namely, RA01,
RA02, RA12, RA28, RA32, HR06, HR07,
KS01, KS04 and PI01 - exhibited high
ability of phosphate solubilization for
releasing of soluble phosphorus from
tricalcium phosphate (Figure 1).
3.2 Morphological and Biochemical
Characterization of Bacteria
The morphological characteristics of
PSB were Gram negative, rod-shaped and
non-spore forming. All isolates were positive
for nitrate reductase, citrate, oxidase, catalase,
hydrolysis of glucose, D-mannitol, and
fructose tests, but negative for a urease test.
RA01, RA02, RA12 and KS01 presented
positive results for gelatin hydrolysis,
Figure 1. Mineral solubilizing activity of PSB isolated from organic and conventional
chili farms. Organic farm: RA, Ratchathani Asok; Conventional farm: HR, Ban Hua
Rua; KS, Ban Kaset Nua; P, Ban Pho Yai.
16 Chiang Mai J. Sci. 2013; 40(1)
arginine hydrolysis, dulcitol, D-raffinose
and sucrose; whereas isolates RA28, RA32,
HR06, HR07, KS04 and PI01 showed
negative results for gelatin and arginine
hydrolysis. In addition, four isolates
(RA32, HR06, HR07 and KS04) presented
positive results for a lactose test, while
other isolates exhibited negative results.
3.3 Identification and Phylogenetic
Analysis of Bacteria
Identification of PSB isolates based on
16S rRNA gene sequence and a phylogenetic
tree are presented in Figure 2. These
sequence data have been submitted to the
DDBJ/EMBL/GenBank databases under
accession number AB673035-AB673045.
All isolates displayed close homology to
the genus Burkholderia. Our isolates were
divided into two phylogenetic clades. The
first clade (PI01, RA28, RA32, KS04,
HR06 and HR07) was maximally closely
related to Burkholderia tropica Ppe8T. In
addition, two clusters were subdivided
within this clade, as cluster I (PI01 and RA
28) and cluster II (RA32, KS04, HR06 and
Figure 2. Phylogenetic analysis based on 16S rRNA sequence, which was generated based
on pairwise nucleotide distance of the Kimura 2-parameter using the neighbor-joining method
included in the MEGA4 software package. The bar indicates a difference of 1 nucleotide per
100. The number beside the node is the statistical bootstrap value. In brackets are the GenBank
accession numbers of the16S rRNA genes.
Chiang Mai J. Sci. 2013; 40(1) 17
HR07). 16S rRNA gene sequence analysis
of RA28 and PI01 in cluster I
showed 99.1% similarity to Burkholderia
tropica Ppe8T. However, members of cluster
II - HR07, HR06, KS04 and RA32 presented
98.5%, 98.6%, 98.8% and 98.5% similarities
to Burkholderia tropica Ppe8T, respectively.
The second clade (RA02, RA12, KS01 and
RA01) was closely related to Burkholderia
ambifaria, with a genetic distance farther from
other strains and completely separate from
the other clade. The isolates in this group
(RA02, RA12, KS01 and RA01) presented
99.6%, 99.7%, 99.3% and 99.7% similarities
to Burkholderia ambifaria AMMDT, respectively.
From these results, we identified our isolates
to be B. tropica and B. ambifaria.
3.4 Phosphate Solubilization, Growth,
pH and Production of Organic acid
Ten isolates with high phosphate-
solubilizing activity PSB were investigated
for phosphate solubilization during a 72 h
period by monitoring growth, pH, available
phosphorus and organic acid production in
the culture medium (Table 1). Growth rate in
terms of cell numbers measured by standard
plate count methods was not significantly
different among observed isolates (1.86 × 108
to 2.06 × 108 CFU/ml); whereas pH in the
culture medium declined (to 3.31-5.11) from
an initial pH of 6.60 after incubation for
72 h. In addition, isolate HR07 was able to
produce significantly higher acid than other
isolates (except for RA28, RA32 and PI01),
which was detected from a pH drop
(pH 3.31). In consideration of soluble
phosphorus released from mineral-solubilizing
activity of PSB, the result showed that
bacterial isolates RA02, RA12, HR06, HR07,
KS01 and KS04 had significantly higher
potential for solubilization of tricalcium
phosphate than the other isolates. Among
these strains, KS04 was proven to be the best
(499.85 μg/ml).
An analysis of organic acid production
by HPLC is presented in the chromatogram
in Figure 3. The results revealed that 8
Table 1. Bacterial cell growth, pH, soluble-P concentration and types of organic acid observed
after 72 hrs incubationa.
Note: aIn a column, means followed by a common letter are not significantly different by
DMRT0.05
bG, gluconic acid; L, lactic acid; C, citric acid; A, acetic acid; S, succinic acid; P, propionic
acid; U1, unknown acid 1; and U2, unknown acid 2.
Isolates OD560 Cell density
(log CFU/ml)
pH of
medium
Soluble-P
(μg/ml)
Type of organic acidb
RA01
RA02
RA12
RA28
RA32
HR06
HR07
KS01
KS04
PI01
CV%
1.120a
1.288a
1.236a
1.153a
1.228a
1.219a
1.181a
1.155a
1.199a
1.232a
16.4
1.86 × 108a
2.14 × 108a
2.06 × 108a
1.92 × 108a
2.04 × 108a
2.03 × 108a
1.96 × 108a
1.92 × 108a
2.00 × 108a
2.05 × 108a
41.34
4.25bc
5.01d
5.11d
3.76ab
3.53ab
4.77cd
3.31a
4.86cd
4.05b
3.99ab
19.95
294.26b
479.51de
403.44cd
129.28a
335.15bc
448.44de
427.54de
433.81de
499.85e
133.60a
98.76
G L A C S U1 U2
G L A C P U2
G L A C P U2
A S U1
A
A U1
A S
G L C P U2
A
G A C P U2
18 Chiang Mai J. Sci. 2013; 40(1)
different kinds of organic acid - lactic acid,
acetic acid, citric acid, succinic acid, propionic
acid, gluconic acid, and 2 unknown
acids - could be produced from all PSB
isolates. Eight isolates (RA01, RA02, RA12,
RA28, HR06, HR07, KS01 and PI01) showed
the presence of multiple organic acids
(as shown in Table 2 and Figure 3). Only
two isolates (RA32 and KS04) could
produce only one organic acid (acetic acid).
In addition, acetic acid could be produced
from all observed isolates except KS01.
The retention times of the unknown organic
acids were 2.6 and 20.4 min for unknown
1 and unknown 2, respectively.
3.5 Effects of PSB on the Growth of Chili
in a Pot Experiment
The effects of PSB on the growth of
chili in a pot trial demonstrated that chili
inoculated with PSB isolate KS04 showed
the best growth performance. All plant
growth parameters including height, fresh
weight, dry weight of shoots and roots and
number of flowerings found in plants
inoculated with KS04 were significantly
Figure 3. HPLC chromatogram of organic acids produced from PSB cultured on Pikovskaya’s
medium for 72 h. G, gluconic acid; L, lactic acid; C, citric acid; A, acetic acid; S, succinic acid;
P, propionic acid; U1, unknown acid 1; and U2, unknown acid 2.
Chiang Mai J. Sci. 2013; 40(1) 19
Table 2. Growth parameters and phosphorus uptake in chili inoculated with ten different
PSB isolates cultivated on sterile organically managed soila.
higher than in uninoculated plants
(control). Chili inoculated with RA12,
HR07, KS01, KS04 and PI01 showed
significantly higher P uptake than the
control (uninoculated plant). Among them,
the highest P uptake was evident in the
plant inoculated with KS04. Furthermore,
the height and P uptake of chili inoculated
with KS04 was significantly higher than
plants inoculated with the other PSB
isolates.
4. DISCUSSION
PSB is a phosphate-solubilizing
microorganism which can be routinely
screened by a plate assay method using
Pikovskaya medium. The bacteria will
grow on this medium and form a clear
zone around the colony [25, 39]. These
bacteria can convert tricalcium phosphate in
the medium from insoluble to soluble forms
[40]. A total of 50 PSB isolates were screened
by the halo zone presented around a colony
in Pikovskaya’s medium which were obtained
from organically managed soil (Ratchathani
Asok), higher than at conventional chili
farms (Ban Hua Rua, Ban Kaset Nua and
Ban Pho Yai). Juottonen et al. [41] reported
that environmental conditions and farm
management practices had a considerable
impact on soil microorganisms. The
numbers of microorganisms found in each
soil sample were different because the
composition and function of the microbial
community was shown to vary according to
land-use type, plant species, temperature,
nutrients, oxygen availability, pollutants and
other environmental variables. Our results
corresponded with findings by Chen et al.
[12] which reported that several isolates of
PSB could be isolated from fertile soil in
Taichung, Taiwan. Kumar et al. [42] also
found different numbers of PSB isolates in
various agricultural farms in eastern Uttar
Note: aIn a column, means followed by a common letter are not significantly different by
DMRT0.05
Treatments
Shoot
Height
(cm)
Diameter
(cm)
Fresh
weight
(g/pot)
Dry
weight
(g/pot)
Root
Fresh
weight
(g/pot)
Dry
weight
(g/pot)
No. of
flowerings
per pot
P uptake
(mg/pot)
Control
RA01
RA02
RA12
RA28
RA32
HR06
HR07
KS01
KS04
PI01
CV%
23.88a
32.50b
29.75b
42.00b
29.50b
43.00b
33.50b
42.50b
41.50b
49.00c
39.00b
35.7
0. 26a
0.30ab
0.26a
0.32ab
0.26a
0.40b
0.33ab
0.40b
0.37ab
0.41b
0.35ab
26.5
2.72a
5.18a
4.23a
8.45ab
4.57ab
9.17ab
6.88ab
10.81ab
9.36ab
14.50b
8.32ab
73.4
0.38a
0.82a
0.65a
1.39ab
0.58ab
1.52ab
1.09ab
1.70ab
1.72ab
2.66b
1.38ab
91.3
2.40ab
2.02ab
2.72bc
2.00ab
2.27abc
1.61ab
2.40abc
1.76ab
1.20a
3.25c
1.97ab
92.8
0.06a
0.28a
0.26a
0.53ab
0.54ab
0.61ab
0.26a
0.70ab
0.54ab
1.17b
0.51ab
84.9
1.00ab
4.50cd
0.00a
1.50ab
3.00bc
1.50ab
2.75bc
4.25cd
1.50ab
5.50d
0.25a
190.28
0.002a
0.006abc
0.005ab
0.014bc
0.006abc
0.011abc
0.006abc
0.017bc
0.017bc
0.028d
0.015bc
84.85
20 Chiang Mai J. Sci. 2013; 40(1)
Pradesh, India.
Based on the ability of solubilization of
tricalcium phosphate, PSB isolated from
conventional farm had higher solubilizing
activity than those isolated from organic
farm. This finding was similar to the report
of Bhakta et al. [43] who found that
phosphate solubilizing activity of bacteria
isolated from sediment soil exposed to
various doses of chemical fertilizer
increased correlating to the amount of
fertilizer in the soil.
In addition, the general characteristics
of the isolates in this study corresponded
with the genus Burkholderia as described by
Linu et al. [44]: Gram negative, rod-shaped,
motile, non-sporing, and containing catalase
and citrate. Analysis of 16S rRNA and
the phylogeny of PSB found that all
isolates belonged to Burkholderia spp.
(Figure 2), which are completely separated
from Enterobacter sp. by 100 bootstrap values.
In addition, all isolates in this study were
maximally related to Burkholderia tropica and
B. ambifaria. Our results contrasted with the
report of Kumar et al. [42], which could only
isolate genera belonging to members of
Enterobacteriaceae (Enterobacter spp. and
Exiguobacterium spp.) from agricultural farms.
However, our results corresponded with the
findings of Balandreau et al. [45], which
documented that Burkholderia was a common
plant-associated bacterium which could be
found naturally in wet soil and decaying
plants [46].
Generally, available phosphorus in soil
can be increased by low-molecular-mass
organic acids produced from PSB [12].
The results of this study demonstrated
that various types of organic acids were
produced by PSB. This was a significant
characteristic of these bacteria, including a
drop in pH and soluble phosphorus which
was obtained after culturing in mineral
phosphate for a few days. The results in
Table 1 and Figure 3. demonstrate that most
PSB isolates (8 isolates) produced multiple
organic acids, while a few (2 isolates)
produced only one type of organic acid
(acetic acid). However, multiple organic
acid producers did not show phosphate-
solubilizing activity or a pH drop greater
than those from one organic acid producer.
This result contrasting with the findings of
Chen et al. [12], who reported that a
combination of multiple organic acids could
accomplish mineral solubilization and
pH drop better than a single organic acid.
In addition, our results revealed that acetic
acid could be produced from all isolates
in this study, except isolate KS01. This
indicated that acetic acid may be a major
acid concerned with the solubilization
activity of PSB [47]. However, the
phosphate solubilization efficiency may
depend on other factors such as substrate,
medium, temperature, time, and other
mechanisms in addition to organic acid
production. However, the acidification of
culture supernatants seemed to be generally
the main mechanism for phosphate
solubilization [48]. When they released
organic acid, the medium pH dropped to
a low level rapidly. Valverde et al. [49]
reported that Burkholderia spp. produced
gluconic acid, acetic acid and citric acid,
and showed that these bacterial genera had
high efficiency in solubilizing insoluble
phosphate. Other organic acids could be
produced by them as well, such as butyric acid,
lactic acid, succinic acid, malic acid, glycolic
acid, fumaric acid [50], propionic acid,
and unknown organic acids [51].
The effects of PSB on the growth
of chili in a pot trial found that KS04
significantly increased shoot height and P
uptake, to a greater extent than the other
PSB isolates observed in this study.
Chiang Mai J. Sci. 2013; 40(1) 21
Furthermore, KS04 was the best isolate for
significantly enhancing all plant growth
parameters (higher than uninoculated
plants), including fresh and dry weight of
shoots, fresh and dry weight of roots,
number of flowerings per plant, and P
uptake (Table 3). Regarding the ability of
mineral phosphate solubilization and types
of organic acids of KS04 (Table 2), we
found that this isolate had high efficiency
for solubilization of tricalcium phosphate,
and produced only acetic acid. This indicated
that acetic acid may be related to the
mechanism of PSB in promoting chili
growth. Generally, soil pH was induced
to decline to pH 4.0-5.0 by 99% pure acetic
acid. At these pH values, phosphorus in the
soil could be combined with aluminum
or ferrous metals and converted into
hydroxyapatites (insoluble phosphorus) [52].
The subsequent activity of acidification by
organic acids produced from PSB was high
under these conditions.
This study showed that KS04
significantly enhanced P uptake and growth
of chili. Our findings correspond to results
reported by Afzal and Bano [53] who
reported that wheat (Triticum aestivum)
inoculated with PSB (Pseudomonas sp. strain
54RB) significantly increased root and
shoot weight, plant height, spike length,
grain yield and P uptake higher than the
control. Similar results were also found in
cowpea (Vigna unguiculata (L.) Walp.) which
revealed the enhancement of nodulation,
root and shoot biomass, straw and grain
yield and P and N uptake of plants inoculated
with Gluconacetobacter sp. and Burkholderia sp.
[44]. The growth parameters and P uptake
of chili plants in the present study suggested
that bacterial isolate KS04 had the highest
potential for promoting chili growth, as a
result of its highest potential for enhancing
P uptake of the plant. In addition, KS04
showed the strongest activity for
solubilization of insoluble tricalcium
phosphate as well. Thus, this indicated that
P-solubilization was the main mechanism
responsible for positive growth response.
Based on the results found in present study,
we concluded that KS04 had the great
potential for use as soil inoculants for
organic chili production in Thailand.
However, before using of this bacterial
isolates as a biofertilizer, its effect on the
growth of chili in the actual organic chili
cropping should be investigated.
5. CONCLUSIONS
The results of this study represented
that the number of PSB obtained from
organic chili farms was higher than from
conventional chili farms. A high ability of
organic acid producer was identified using
conventional biochemical characteristics of
Burkholderia genera. The results of 16S rRNA
gene and phylogenetic analysis of these
isolates showed a close relationship to
Burkholderia ambifaria and B. tropica. Eight
different kinds of organic acid were
detected from the culture medium by
HPLC analysis after 72 h. The multiple
organic acids and the single one which were
found did not correspond with the ability
of mineral phosphate solubilization and
pH drop. Chili inoculated with KS04 which
was planted in organic soil showed
significantly higher values for all plant
growth parameters and P uptake than
uninoculated plants (control). These findings
suggest the potential of PSB isolate KS04
for use as a bio-inoculant for production of
chili by organic farming in Thailand.
ACKNOWLEDGMENTS
This research work was kindly
supported by grants from the Commission
on Higher Education of Thailand, the
22 Chiang Mai J. Sci. 2013; 40(1)
Royal Thai government, the Thailand
Research Fund (TRF), and Microbial
Resources and Applications group.
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