International Journal of Agriculture The
International Journal of Agriculture. Photon 125 (2014) 285-289
https://sites.google.com/site/photonfoundationorganization/home/international-journal-of-agriculture
Original Research Article. ISJN: 7758-2463
International Journal of Agriculture
Ph ton
The phospate soluble capability of Pseudomonas sp. and Bacillus sp.
as Superior Solubilizing Phospate Exogenous Bacteria (SPB) on
Andisols as tea planting area
Mieke Rochimi Setiawatia*, Eko Pranotob
a
b
Agricultural Faculty, Padjadjaran University (UNPAD), Bandung-West Java, Indonesia
Soil and Plant Nutrition Division Research Institute for Tea and Cinchona (RITC), Bandung, Indonesia
The authors receive Thomas Edison Award-2014 in
Agriculture for Inspiration and Knowledge Distribution
among young research scholars.
Article history:
Received: 03 January, 2014
Accepted: 10 January, 2014
Available online: 12 February, 2014
Keywords:
Andisols, exogenous, solubilizing phosphate bacteria,
soluble-P, tea planting area
Corresponding Author:
Setiawati M.R.*
Email: [email protected]
Phone: +628122147664 2
Pranoto E.
Email: [email protected]
Phone: +62225928780
Abstract
In Indonesia, tea was planted dominant on uplands
area with 52-55% from the total area on Andisols as
soil of tea planting area. The range of pH was 4,5 5,6 made high retention of phosphate was the main
problem on tea plantation. The used of Superior
Solubilizing Phosphate Exogenous Bacteria (SPB)
was one of the problem solving. Laboratory-scale
experiments have been conducted to test the
phosphate soluble capability from SPB on Andisols.
The inoculant source of SPB from agricultural food
plants with elevation 600 - 800 m above sea level.
The strain that used were: Pseudomonas
cepaceae, P. malei, Bacillus mycoides, and B.
subtilis. The experiment was using a Completely
Randomized Design with six treatments and four
repeated. The ANOVA’s analysis was not significant
in increase the soluble-P. Even it, the treatment D
(Bacillus mycoides) was the highest soluble-P. The
capability from solubilizing phosphate bacteria to
increase P-soluble depend on the pH of soil. Even
Pseudomonas cepaceae, P. malei, Bacillus
mycoides, and B. subtilis were the superior
solubilizing phosphate bacteria on agricultural food
plants soil, but they have not significantly different
on Andisols. So, for the better results, may use the
indigenous isolate.
Citation:
Setiawati M.R., Pranoto E., 2014. The phospate soluble
capability of Pseudomonas sp. and Bacillus sp. as
Superior Solubilizing Phospate Exogenous Bacteria (SPB)
on Andisols as tea planting area. International Journal of
Agriculture. Photon 125, 285-289.
1. Introduction
Andisols are soils formed in volcanic ash and
defined as soils containing high proportions of
glass and amorphous colloidal materials,
including allophane, imogolite and ferrihydrite.
In the FAO soil classification, Andisols are
known as Andosols. Because they are
generally quite young, Andisols typically are
very fertile except in cases where phosphorus
is easily fixed. They can usually support
intensive cropping areas such as tea, coffee or
tobacco.
Landforms in volcanic regions are strongly
influenced by the chemical and mineralogical
composition of the materials that were
deposited during eruptive phases. Volcanic
rocks and magmas are grouped according to
Ph ton
their silica contents in three main categories
that are Rhyolite (65-75% SiO2), Andesite (6555% SiO2) and Basalt (55-45% SiO2). The
mineralogical
properties
and
chemical
composition (notably the contents of K2O,
Na2O and CaO) distinguish individual rock
types. The mineral contents from Andisols
such are Allophane, Ferrihydrite, and
Imogolite. The hydrolisis of this mineral
released Fe and Al ionic that can protect from
organic matter biodegradation and fixation
phosphor (Kapur, 2010). Many volcanic soils
have excellent physical properties that make
them highly desirable for a wide range of uses.
Chemically, they suffer from high phosphate
retention, and they may be limiting in K and
some micronutrients. Nevertheless, these soils
285
are amongst the most fertile lands in the world
and are, therefore, very intensively cultivated,
even if the users are aware of the risks of
volcanic outbursts (Neall, 2009).
In Indonesia, tea was planted dominant on
uplands area with 52-55% from the total area
on Andisols as soil of tea planting area. Tea is
one of the kind of beverage with 37% market
segment
(Notohadiprawiro,
2006)
and
contribute around Rp. 1.2 trillion on Gross
Domestic Product (0.3% from total Gross
Domestic Product non oil and natural gas on
2010 (Indonesia Agricultural Ministry, 2010).
Depend on observation at volcanic soils in
Europe, that were from Italy, Portugal
(Azores), Iceland, Spain (Tenerife) and France
concluse that the decomposition kinetics that
the proportion of C in added plant material that
would be mineralized is greater for soil
microbial communities at later stages of
development than at earlier stages, and also
the efficiency of leaf litter decomposition
increases with soil development (Hopkins and
Bartoli, 2004). The use of phosphate
solubilizing
bacteria
as
inoculants
simultaneously increases P uptake by the
plant and crop yield. Strains from the genera
Pseudomonas, Bacillus and Rhizobium are
among the most powerful phosphate
solubilizers. The principal mechanism for
mineral phosphate solubilization is the
production of organic acids, and acid
phosphatases play a major role in the
mineralization of organic phosphorous in soil
(Rodriguez and Fraga, 1999).
Solubilizing
Phosphate
Bacteria
can
increasing the phosphate solubilizing by
phosphatase enzyme and organic acid as its
secondary metabolite. 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. Arthrobacter
sp. (CC-BC03) was the highest phosphate
solubilizing strain bacteria to produce solubleP in 5 g of tricalcium phosphate medium after
72 hours incubation, and then in succession
are Serratia marcescens (CC-BC14), Delftia
sp. (CC-BC21), Arthrobacter ureafaciens (CCBC02), Chryseo-bacterium sp. (CC-BC05),
Bacillus
megaterium
(CC-BC10)
Phyllobacterium myrsinacearum (CC-BC19),
Rhodococcus erythropolis (CC-BC11), and
Gordonia sp. (CC-BC07) (Chen et al., 2006).
The solubilizing bacteria were screened for
Ph ton
their capacity in solubilizing inorganic and
mineralizing organic phosphate using modified
NBRIP media in vitro. Most isolates were able
to
solubilize
Ca3(PO4)2
with
various
solubilization index in the first screening.
Secondary screening using P sources with
lower pH, further grouped these bacteria into :
(a) solubilizing inorganic Ca3(PO4)2 with pH
4.5: Erwinia sp. CK10, Roseateles sp. CK15,
Rhizobium sp. CK19, Enterobacter sp. CK23,
and Erwinia sp. CK24.; (b) mineralizing
organic C6H6(OPO3H2)6 with pH 6.0:
Roseateles sp. CK15, Rhizobium sp. CK19,
Enterobacter sp. 23CK, Erwinia sp. CK24, NI
CK53, and NI CK54; (c) solubilizing and
mineralizing both P sources: Roseateles sp.
CK15, Rhizobium sp. CK19, Enterobacter sp.
CK23, and Erwinia sp. CK24 (Sitepu et.al,
2013).
The objective of research was to obtain
solubilizing phosphate bacteria as inoculants
that increasing the P uptake by tea plants. By
using a variety of bacteria that has been
known as phosphate solubilizing superior from
rhizosphere of various plants, it will get a good
chance to get a solubilizing phosphate
bacteria that suitable for tea plants grown in
Andisols where could solve the problem from a
high phosphate retention.
2. Experimental
2.1 Preparation of Inoculant
This experiment used Superior Solubilizing
Phospate Exogenous Bacteria (SPB) Isolates
from Padjadjaran University (UNPAD)’s
collection. The inoculant source of SPB from
agricultural food plants with elevation 600 800 m above sea level. The strain that used
was: Pseudomonas cepaceae, P. malei,
Bacillus mycoides, and B. subtilis. Each of
bacteria multiplied on Pikovskaya liquid
medium. After five days incubation, the
population of SPB was variated. Before
applied in Andisol as in-vitro test, the
population was made same each other with
dilution, shown on Table 1.
2.2 Experimental Design
The research was done on UNPAD laboratoy’s
in 06° 55’ 32” S dan 107° 46’ 16” E in March
2013. This experiment used Completely
Randomized Design with six treatments and
four repeated. The statistic analyxe used was
Analyze of Variants (ANOVA) with Duncan
different test. The dose each treatment
was10% v/w, its mean 10 ml inoculant per 100
g steril Andisols with pH (H2O) 4.9.
286
Table 1: The population of Solubillizing Phosphate Bacteria
Strain of BPF
Total Population Dilution
12
(10
cfu/ml) Dose of Inoculant
before dilution
(ml)
Pseudomonas cepaceae 1.09
1.0000
P. malei
1.23
0.8831
Bacillus mycoides
1.18
0.9189
B. subtilis
1.57
0.6939
Negative control mean no inoculant, just only
100 g steril Andisols. The Positive control
mean only gave 10 ml steril liquid Pikovskaya.
Each treatment was incubated for 5 days. The
arrangement treatments were:
A. Negative control
B. Positive control
C. Pseudomonas cepacea
D. Bacillus mycoides
E. Bacillus subtilis
F. Pseudomonas mallei
The responses are:
a. Soluble-P with Bray methode
b. The water content of Andisol
gravimetric method
with
3. Results and Discussion
3.1 Soluble-P
After 5 days incubation, the soluble-P each
treatment was measured and the result shown
Table 2: The Soluble-P (ppm) each Treatment
No Treatment
Repeated
Strain
Code I
1
Negative control
A
0.0158
2
Positive control
B
0.1458
Pseudomonas cepacea
3
C
0.0633
Bacillus mycoides
4
D
0.0212
B. subtilis
5
E
0.1947
P. mallei
6
F
0.0186
TOTAL
0.4594
Average
0.0766
Table 3: The water content (%) each treatment
No Treatment
Repeated
Strain
Code
I
1
Negative control
A
34.6900
2
Positive control
B
35.1800
Pseudomonas cepacea
3
C
37.3400
Bacillus mycoides
4
D
34.0000
B. subtilis
5
E
36.6500
P. mallei
6
F
34.2900
TOTAL
212.1500
Average
35.3583
3.2 Water Content
The average water content each treatment
was 33 - 35%. Water content was measurable
at the same times with soluble-P each
treatment, that shown on Tabel 3.
Ph ton
Steril Aquadest
(ml)
0.0000
0.1169
0.0811
0.3061
Total
Population
12
(10
cfu/ml) after
dilution
1.09
1.09
1.09
1.09
on Table 2. Even the ANOVA’s analysis was
not significant, the the treatment D (Bacillus
mycoides) was the highest soluble-P. Even the
strain from genera of Pseudomonas and
Bacillus were the most powerful phosphate
solubilizers, but the major source of
phosphatase activity in soil is considered to be
of microbial origin (Garcia et al., 1992; Xu and
Johnson, 1995). Its mean that even the
isolates was superior, the activity was
influential by the environmental aspect like pH
of soil. In Indonesia, tea growth well on pH 4.5
- 5.6 (Widayat et al., 2006). The isolated
strains were inoculated on tea rhizosphere in
specific media containing tryptophan produce
growth regulating substances such as indole
acetic acid (IAA) under in-vitro conditions
(Sharma et al., 2012). The phosphate
solubilizing bacteria grew rapidly in the liquid
medium at pH 5 and 7 but almost no growth
occurred at pH 3 (Islam et al., 2007).
Sum
Average
0.1720
0.2620
0.2665
0.3987
0.2982
0.1036
1.5010
0.0625
0.0430
0.0655
0.0666
0.0997
0.0746
0.0259
Sum
Average
140.1100
142.8900
143.5500
142.5900
139.4200
134.9900
843.5500
35.0275
35.7225
35.8875
35.6475
34.8550
33.7475
II
0.0212
0.0843
0.0158
0.0267
0.0422
0.0266
0,2168
0,0361
III
0.0158
0.0159
0.1320
0.0267
0.0320
0.0319
0.543
0.0424
IV
0.1192
0.0160
0.0554
0.3241
0.0293
0.0265
0.5705
0.0951
II
36.1100
34.0200
37.6500
36.1600
35.1300
35.4700
214.540
0
35.7567
III
34.8900
36.6000
37.3600
36.0600
33.2900
33.1900
211.3900
IV
34.4200
37.0900
31.2000
36.3700
34.3500
32.0400
205.4700
35.2317
34.2450
35.1479
The table shown that the water content was
stable, it means that the activity of PSB was
not disturbed by water condition. Water is
important to growth of plant and as a nutrient
287
solvent. A relatively low and uniform supply of
water soluble phosphorus was maintained in
some of the lime and avocado groves. Where
this was done the trees were in excellent
condition. While the trees showed no evidence
of injury in areas of high water soluble
phosphorus concentrations, they showed no
benefit. On the average, the water soluble
phosphorus content of these soils was the
same in all seasons. Probably phosphate
fertilizers could be applied to many lime and
avocado groves less frequently and at lower
rates than at present without any harm to the
trees (Malcolm, 1951).
Because of inoculated PSB derived from soil
of agricultural food plants with a pH near
neutral, pH seems is the environment factor of
Andisols that influence decreases the activity
of PSB in phosphate solubilizing. Therefore it
is recommended to use the PSB isolates
derived from the tea plant Andisols. We
expected the indigenous isolates not inhibited
its activity on phosphate solubilizing because it
has adapted to its environment.
4. Research Highlights
The main points in this research to know were:
a. There are many microbes which can
increase the phosphate solubilizing by
phosphatase enzyme and organic acid as its
secondary metabolite
b. Some microbes have an ideal condition to
growth well and functioned. It’s better to used
the superior microbe in each habitat
c. Bacillus mycoides was producing the
highest soluble-P than other superior
solubilizing phospate exogenous bacteria on
Andisols as tea planting area in Indonesia. It’s
arround 83.78% more than control (without
solubilizing phospate bacteria).
Limitations
The important thing on this research was to
calibrate the population of microbes. Because
we used some and different microbes, so the
growth and population different too. On Table
1 we calibrated the population of all microbes.
So, the each microbe have the same
population before inoculated on the soil, that
12
cfu/ml. Different
was around 1.09 x 10
population will be confused us in interpretation
of the data.
Recommendation
and know the capability or function, it should
be done in same population. To do it, before
application we should be to regeneration of
microbe isolate and growth it on it’s medium.
After someday incubation, please count the
population and calibrated it, so we will have
the same population.
Funding and Policy Aspects
One of the “green concept” on agricultural
aspect was used some superior microbe as a
biofertilizer. The huge population in each
biofertilizer product make it dominant on the
habitat which bio-fertilizer applied without
know the capability on that habitat. This is the
basic research to prove that the exogenous
superior microbe can not significantly on other
habitat, particulary on tea plantation area. So,
the one of next research need to produce
biofertilizer special from indigenous microbes
of tea area.
Justification of Research
The objective of research was to obtain the
phospate soluble capability from the superior
solubilizing phosphate bacteria. The inoculant
from agricultural food plants with elevation 600
- 800 m above sea level in Indonesia. The
strain that used was Pseudomonas cepaceae,
P. malei, Bacillus mycoides, and B. subtilis. In
Indonesia, tea was planted dominant on
uplands area with 52-55% from the total area
on Andisols as soil of tea planting area. To
know the capability of that superior exogenous
microbe on Andisol, the research should be
done.
Conclusion
The capability from solubilizing phosphate
bacteria to increase P-soluble depend on the
pH of soil. Andisols as a tea planting area in
Indonesia had range of pH 4.5 – 5.6 that made
P retention more. Even Pseudomonas
cepaceae, P. malei, Bacillus mycoides, and B.
subtilis were the superior solubilizing
phosphate bacteria on agricultural food plants
soil, but they have not significantly diffrent on
Andisols. The Bacillus mycoides was the
highest soluble-P produced. The average
water content each treatment was 33 - 35%, it
means that the activity of PSB was not disturb
by water condition. Depend on the result, we
recommendation to used the indigeneous
isolate to each areal plantation.
In every research by used microbe to compare
Ph ton
288
Author’s
Interest
Contribution
and
Competing
Mieke Rochimi Setiawati as a main author
contributes on produce the inoculant. She was
a lecturer on UNPAD with specialization on
soil biotechnology, particulary microbe on
agricultural food plants. Eko Pranoto as coauthor
contributes
in
applied
and
experimented it on Andisol as tea planting
area. He was a researcher from Indonesia
Research Institute for Tea and Cinchona.
References
Chen Y.P., Rekha P.D., Arun A.B., Shen F.T., Lai
W.A.,Young C.C., 2006. Phosphate solubilizing
bacteria from subtropical soil and their tricalcium
phosphate solubilizing abilities. Applied Soil
Ecology, 34, 33-41.
Garcia C., Fernandez T., Costa F., Cerranti B.,
Masciandaro G., 1992. Kinetics of phosphatase
activity in organic wastes. Soil Biol Biochem
Journal, 25, 361-365.
Sharma B.C., Subba R., Saha A., 2012. In vitro
solubilization of tricalcium
phosphate and
production of IAA by phosphate solubilizing bacteria
isolated from tea rhizosphere of Darjeeling
Himalaya. Plant Sciences Feed Journal, 2(6), 9699.
Sitepu, I.R., Hashidoko V., Santoso V., Tahara V.,
2013. Potential of phosphate-solubilizing bacteria
isolated from dipterocarps grown in peat swamp
forest in central Kalimantan and their possible
utilization for biorehabilitation of degraded peatland.
[Online] Available :
http://www.geog.le.ac.uk/carbopeat/media/pdf/yogy
apapers/p17.pdf
Widayat W., Santoso V., Martosupono V., Astika
G.P.W., Dharmadi A., Kartawijaya W.S., Sukasman,
Tobroni M., Suwardi E., Topani, Samudi J.B., 2006.
rd
Tea cultivation guideline. 3 edition, Indonesia
language. Indonesia Research Institute for Tea and
Cinchona, Gambung, Indonesia.
Xu J.G., Johnson R.L., 1995. Root growth, microbial
activity and phosphatase activity in oilcontaminated, remediated and uncontaminated
soils planted to barley and field pea. Plant Soil
Journal, 173, 3-10.
Hopkins D.W., Bartoli F., 2004. Size and activity of
the soil microbial community from a range of
European volcanic soils. Volcanic soil resources in
Europe. Agricultural Research Institute. Reykjavik,
Iceland. Rala Report, 214, 35-36.
Indonesia
Agricultural
Ministry
(Welcome
Speaking). 2010. Anually meeting of Indonesia tea
board, Bandung.
Islam M.T., Deora A., Hashidoko Y., Rahman A., Ito
T., Tahara S., 2007. Isolation and Identification of
Potential Phosphate Solubilizing Bacteria from the
Rhizoplane of Oryza sativa L. cv. BR29 of
Bangladesh.
Verlag
der
Zeitschrift
für
Naturforschung Journal, Tübingen, 62c, 103-110.
Kapur S., 2010. Andosols. University of Çukurova.
Departments of Soil Science and Archaeometry,
Adana, Turkey,
Malcolm J.L., 1951. Water soluble phosphorus and
potassium in the soil of lime and avocado groves in
dade county. Proc. Fla. State Hort. Soc, 64, 285292.
Neall V. E., 2009. Volcanic soils. Land use and land
cover. UNESCO-EOLSS, VII, 23-45.
Notohadiprawiro T., 2006. The limited concept on
agricultural aspect; the serious constraint on
national
development.
Soil
Science
and
Environment Journal, 6(1), 63-70.
Rodriguez H., Fraga V., 1999. Phosphate
solubilizing bacteria and their role in plant growth
promotion. Biotechnology Advances, 17, 319-339.
Ph ton
289
https://sites.google.com/site/photonfoundationorganization/home/international-journal-of-agriculture
Original Research Article. ISJN: 7758-2463
International Journal of Agriculture
Ph ton
The phospate soluble capability of Pseudomonas sp. and Bacillus sp.
as Superior Solubilizing Phospate Exogenous Bacteria (SPB) on
Andisols as tea planting area
Mieke Rochimi Setiawatia*, Eko Pranotob
a
b
Agricultural Faculty, Padjadjaran University (UNPAD), Bandung-West Java, Indonesia
Soil and Plant Nutrition Division Research Institute for Tea and Cinchona (RITC), Bandung, Indonesia
The authors receive Thomas Edison Award-2014 in
Agriculture for Inspiration and Knowledge Distribution
among young research scholars.
Article history:
Received: 03 January, 2014
Accepted: 10 January, 2014
Available online: 12 February, 2014
Keywords:
Andisols, exogenous, solubilizing phosphate bacteria,
soluble-P, tea planting area
Corresponding Author:
Setiawati M.R.*
Email: [email protected]
Phone: +628122147664 2
Pranoto E.
Email: [email protected]
Phone: +62225928780
Abstract
In Indonesia, tea was planted dominant on uplands
area with 52-55% from the total area on Andisols as
soil of tea planting area. The range of pH was 4,5 5,6 made high retention of phosphate was the main
problem on tea plantation. The used of Superior
Solubilizing Phosphate Exogenous Bacteria (SPB)
was one of the problem solving. Laboratory-scale
experiments have been conducted to test the
phosphate soluble capability from SPB on Andisols.
The inoculant source of SPB from agricultural food
plants with elevation 600 - 800 m above sea level.
The strain that used were: Pseudomonas
cepaceae, P. malei, Bacillus mycoides, and B.
subtilis. The experiment was using a Completely
Randomized Design with six treatments and four
repeated. The ANOVA’s analysis was not significant
in increase the soluble-P. Even it, the treatment D
(Bacillus mycoides) was the highest soluble-P. The
capability from solubilizing phosphate bacteria to
increase P-soluble depend on the pH of soil. Even
Pseudomonas cepaceae, P. malei, Bacillus
mycoides, and B. subtilis were the superior
solubilizing phosphate bacteria on agricultural food
plants soil, but they have not significantly different
on Andisols. So, for the better results, may use the
indigenous isolate.
Citation:
Setiawati M.R., Pranoto E., 2014. The phospate soluble
capability of Pseudomonas sp. and Bacillus sp. as
Superior Solubilizing Phospate Exogenous Bacteria (SPB)
on Andisols as tea planting area. International Journal of
Agriculture. Photon 125, 285-289.
1. Introduction
Andisols are soils formed in volcanic ash and
defined as soils containing high proportions of
glass and amorphous colloidal materials,
including allophane, imogolite and ferrihydrite.
In the FAO soil classification, Andisols are
known as Andosols. Because they are
generally quite young, Andisols typically are
very fertile except in cases where phosphorus
is easily fixed. They can usually support
intensive cropping areas such as tea, coffee or
tobacco.
Landforms in volcanic regions are strongly
influenced by the chemical and mineralogical
composition of the materials that were
deposited during eruptive phases. Volcanic
rocks and magmas are grouped according to
Ph ton
their silica contents in three main categories
that are Rhyolite (65-75% SiO2), Andesite (6555% SiO2) and Basalt (55-45% SiO2). The
mineralogical
properties
and
chemical
composition (notably the contents of K2O,
Na2O and CaO) distinguish individual rock
types. The mineral contents from Andisols
such are Allophane, Ferrihydrite, and
Imogolite. The hydrolisis of this mineral
released Fe and Al ionic that can protect from
organic matter biodegradation and fixation
phosphor (Kapur, 2010). Many volcanic soils
have excellent physical properties that make
them highly desirable for a wide range of uses.
Chemically, they suffer from high phosphate
retention, and they may be limiting in K and
some micronutrients. Nevertheless, these soils
285
are amongst the most fertile lands in the world
and are, therefore, very intensively cultivated,
even if the users are aware of the risks of
volcanic outbursts (Neall, 2009).
In Indonesia, tea was planted dominant on
uplands area with 52-55% from the total area
on Andisols as soil of tea planting area. Tea is
one of the kind of beverage with 37% market
segment
(Notohadiprawiro,
2006)
and
contribute around Rp. 1.2 trillion on Gross
Domestic Product (0.3% from total Gross
Domestic Product non oil and natural gas on
2010 (Indonesia Agricultural Ministry, 2010).
Depend on observation at volcanic soils in
Europe, that were from Italy, Portugal
(Azores), Iceland, Spain (Tenerife) and France
concluse that the decomposition kinetics that
the proportion of C in added plant material that
would be mineralized is greater for soil
microbial communities at later stages of
development than at earlier stages, and also
the efficiency of leaf litter decomposition
increases with soil development (Hopkins and
Bartoli, 2004). The use of phosphate
solubilizing
bacteria
as
inoculants
simultaneously increases P uptake by the
plant and crop yield. Strains from the genera
Pseudomonas, Bacillus and Rhizobium are
among the most powerful phosphate
solubilizers. The principal mechanism for
mineral phosphate solubilization is the
production of organic acids, and acid
phosphatases play a major role in the
mineralization of organic phosphorous in soil
(Rodriguez and Fraga, 1999).
Solubilizing
Phosphate
Bacteria
can
increasing the phosphate solubilizing by
phosphatase enzyme and organic acid as its
secondary metabolite. 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. Arthrobacter
sp. (CC-BC03) was the highest phosphate
solubilizing strain bacteria to produce solubleP in 5 g of tricalcium phosphate medium after
72 hours incubation, and then in succession
are Serratia marcescens (CC-BC14), Delftia
sp. (CC-BC21), Arthrobacter ureafaciens (CCBC02), Chryseo-bacterium sp. (CC-BC05),
Bacillus
megaterium
(CC-BC10)
Phyllobacterium myrsinacearum (CC-BC19),
Rhodococcus erythropolis (CC-BC11), and
Gordonia sp. (CC-BC07) (Chen et al., 2006).
The solubilizing bacteria were screened for
Ph ton
their capacity in solubilizing inorganic and
mineralizing organic phosphate using modified
NBRIP media in vitro. Most isolates were able
to
solubilize
Ca3(PO4)2
with
various
solubilization index in the first screening.
Secondary screening using P sources with
lower pH, further grouped these bacteria into :
(a) solubilizing inorganic Ca3(PO4)2 with pH
4.5: Erwinia sp. CK10, Roseateles sp. CK15,
Rhizobium sp. CK19, Enterobacter sp. CK23,
and Erwinia sp. CK24.; (b) mineralizing
organic C6H6(OPO3H2)6 with pH 6.0:
Roseateles sp. CK15, Rhizobium sp. CK19,
Enterobacter sp. 23CK, Erwinia sp. CK24, NI
CK53, and NI CK54; (c) solubilizing and
mineralizing both P sources: Roseateles sp.
CK15, Rhizobium sp. CK19, Enterobacter sp.
CK23, and Erwinia sp. CK24 (Sitepu et.al,
2013).
The objective of research was to obtain
solubilizing phosphate bacteria as inoculants
that increasing the P uptake by tea plants. By
using a variety of bacteria that has been
known as phosphate solubilizing superior from
rhizosphere of various plants, it will get a good
chance to get a solubilizing phosphate
bacteria that suitable for tea plants grown in
Andisols where could solve the problem from a
high phosphate retention.
2. Experimental
2.1 Preparation of Inoculant
This experiment used Superior Solubilizing
Phospate Exogenous Bacteria (SPB) Isolates
from Padjadjaran University (UNPAD)’s
collection. The inoculant source of SPB from
agricultural food plants with elevation 600 800 m above sea level. The strain that used
was: Pseudomonas cepaceae, P. malei,
Bacillus mycoides, and B. subtilis. Each of
bacteria multiplied on Pikovskaya liquid
medium. After five days incubation, the
population of SPB was variated. Before
applied in Andisol as in-vitro test, the
population was made same each other with
dilution, shown on Table 1.
2.2 Experimental Design
The research was done on UNPAD laboratoy’s
in 06° 55’ 32” S dan 107° 46’ 16” E in March
2013. This experiment used Completely
Randomized Design with six treatments and
four repeated. The statistic analyxe used was
Analyze of Variants (ANOVA) with Duncan
different test. The dose each treatment
was10% v/w, its mean 10 ml inoculant per 100
g steril Andisols with pH (H2O) 4.9.
286
Table 1: The population of Solubillizing Phosphate Bacteria
Strain of BPF
Total Population Dilution
12
(10
cfu/ml) Dose of Inoculant
before dilution
(ml)
Pseudomonas cepaceae 1.09
1.0000
P. malei
1.23
0.8831
Bacillus mycoides
1.18
0.9189
B. subtilis
1.57
0.6939
Negative control mean no inoculant, just only
100 g steril Andisols. The Positive control
mean only gave 10 ml steril liquid Pikovskaya.
Each treatment was incubated for 5 days. The
arrangement treatments were:
A. Negative control
B. Positive control
C. Pseudomonas cepacea
D. Bacillus mycoides
E. Bacillus subtilis
F. Pseudomonas mallei
The responses are:
a. Soluble-P with Bray methode
b. The water content of Andisol
gravimetric method
with
3. Results and Discussion
3.1 Soluble-P
After 5 days incubation, the soluble-P each
treatment was measured and the result shown
Table 2: The Soluble-P (ppm) each Treatment
No Treatment
Repeated
Strain
Code I
1
Negative control
A
0.0158
2
Positive control
B
0.1458
Pseudomonas cepacea
3
C
0.0633
Bacillus mycoides
4
D
0.0212
B. subtilis
5
E
0.1947
P. mallei
6
F
0.0186
TOTAL
0.4594
Average
0.0766
Table 3: The water content (%) each treatment
No Treatment
Repeated
Strain
Code
I
1
Negative control
A
34.6900
2
Positive control
B
35.1800
Pseudomonas cepacea
3
C
37.3400
Bacillus mycoides
4
D
34.0000
B. subtilis
5
E
36.6500
P. mallei
6
F
34.2900
TOTAL
212.1500
Average
35.3583
3.2 Water Content
The average water content each treatment
was 33 - 35%. Water content was measurable
at the same times with soluble-P each
treatment, that shown on Tabel 3.
Ph ton
Steril Aquadest
(ml)
0.0000
0.1169
0.0811
0.3061
Total
Population
12
(10
cfu/ml) after
dilution
1.09
1.09
1.09
1.09
on Table 2. Even the ANOVA’s analysis was
not significant, the the treatment D (Bacillus
mycoides) was the highest soluble-P. Even the
strain from genera of Pseudomonas and
Bacillus were the most powerful phosphate
solubilizers, but the major source of
phosphatase activity in soil is considered to be
of microbial origin (Garcia et al., 1992; Xu and
Johnson, 1995). Its mean that even the
isolates was superior, the activity was
influential by the environmental aspect like pH
of soil. In Indonesia, tea growth well on pH 4.5
- 5.6 (Widayat et al., 2006). The isolated
strains were inoculated on tea rhizosphere in
specific media containing tryptophan produce
growth regulating substances such as indole
acetic acid (IAA) under in-vitro conditions
(Sharma et al., 2012). The phosphate
solubilizing bacteria grew rapidly in the liquid
medium at pH 5 and 7 but almost no growth
occurred at pH 3 (Islam et al., 2007).
Sum
Average
0.1720
0.2620
0.2665
0.3987
0.2982
0.1036
1.5010
0.0625
0.0430
0.0655
0.0666
0.0997
0.0746
0.0259
Sum
Average
140.1100
142.8900
143.5500
142.5900
139.4200
134.9900
843.5500
35.0275
35.7225
35.8875
35.6475
34.8550
33.7475
II
0.0212
0.0843
0.0158
0.0267
0.0422
0.0266
0,2168
0,0361
III
0.0158
0.0159
0.1320
0.0267
0.0320
0.0319
0.543
0.0424
IV
0.1192
0.0160
0.0554
0.3241
0.0293
0.0265
0.5705
0.0951
II
36.1100
34.0200
37.6500
36.1600
35.1300
35.4700
214.540
0
35.7567
III
34.8900
36.6000
37.3600
36.0600
33.2900
33.1900
211.3900
IV
34.4200
37.0900
31.2000
36.3700
34.3500
32.0400
205.4700
35.2317
34.2450
35.1479
The table shown that the water content was
stable, it means that the activity of PSB was
not disturbed by water condition. Water is
important to growth of plant and as a nutrient
287
solvent. A relatively low and uniform supply of
water soluble phosphorus was maintained in
some of the lime and avocado groves. Where
this was done the trees were in excellent
condition. While the trees showed no evidence
of injury in areas of high water soluble
phosphorus concentrations, they showed no
benefit. On the average, the water soluble
phosphorus content of these soils was the
same in all seasons. Probably phosphate
fertilizers could be applied to many lime and
avocado groves less frequently and at lower
rates than at present without any harm to the
trees (Malcolm, 1951).
Because of inoculated PSB derived from soil
of agricultural food plants with a pH near
neutral, pH seems is the environment factor of
Andisols that influence decreases the activity
of PSB in phosphate solubilizing. Therefore it
is recommended to use the PSB isolates
derived from the tea plant Andisols. We
expected the indigenous isolates not inhibited
its activity on phosphate solubilizing because it
has adapted to its environment.
4. Research Highlights
The main points in this research to know were:
a. There are many microbes which can
increase the phosphate solubilizing by
phosphatase enzyme and organic acid as its
secondary metabolite
b. Some microbes have an ideal condition to
growth well and functioned. It’s better to used
the superior microbe in each habitat
c. Bacillus mycoides was producing the
highest soluble-P than other superior
solubilizing phospate exogenous bacteria on
Andisols as tea planting area in Indonesia. It’s
arround 83.78% more than control (without
solubilizing phospate bacteria).
Limitations
The important thing on this research was to
calibrate the population of microbes. Because
we used some and different microbes, so the
growth and population different too. On Table
1 we calibrated the population of all microbes.
So, the each microbe have the same
population before inoculated on the soil, that
12
cfu/ml. Different
was around 1.09 x 10
population will be confused us in interpretation
of the data.
Recommendation
and know the capability or function, it should
be done in same population. To do it, before
application we should be to regeneration of
microbe isolate and growth it on it’s medium.
After someday incubation, please count the
population and calibrated it, so we will have
the same population.
Funding and Policy Aspects
One of the “green concept” on agricultural
aspect was used some superior microbe as a
biofertilizer. The huge population in each
biofertilizer product make it dominant on the
habitat which bio-fertilizer applied without
know the capability on that habitat. This is the
basic research to prove that the exogenous
superior microbe can not significantly on other
habitat, particulary on tea plantation area. So,
the one of next research need to produce
biofertilizer special from indigenous microbes
of tea area.
Justification of Research
The objective of research was to obtain the
phospate soluble capability from the superior
solubilizing phosphate bacteria. The inoculant
from agricultural food plants with elevation 600
- 800 m above sea level in Indonesia. The
strain that used was Pseudomonas cepaceae,
P. malei, Bacillus mycoides, and B. subtilis. In
Indonesia, tea was planted dominant on
uplands area with 52-55% from the total area
on Andisols as soil of tea planting area. To
know the capability of that superior exogenous
microbe on Andisol, the research should be
done.
Conclusion
The capability from solubilizing phosphate
bacteria to increase P-soluble depend on the
pH of soil. Andisols as a tea planting area in
Indonesia had range of pH 4.5 – 5.6 that made
P retention more. Even Pseudomonas
cepaceae, P. malei, Bacillus mycoides, and B.
subtilis were the superior solubilizing
phosphate bacteria on agricultural food plants
soil, but they have not significantly diffrent on
Andisols. The Bacillus mycoides was the
highest soluble-P produced. The average
water content each treatment was 33 - 35%, it
means that the activity of PSB was not disturb
by water condition. Depend on the result, we
recommendation to used the indigeneous
isolate to each areal plantation.
In every research by used microbe to compare
Ph ton
288
Author’s
Interest
Contribution
and
Competing
Mieke Rochimi Setiawati as a main author
contributes on produce the inoculant. She was
a lecturer on UNPAD with specialization on
soil biotechnology, particulary microbe on
agricultural food plants. Eko Pranoto as coauthor
contributes
in
applied
and
experimented it on Andisol as tea planting
area. He was a researcher from Indonesia
Research Institute for Tea and Cinchona.
References
Chen Y.P., Rekha P.D., Arun A.B., Shen F.T., Lai
W.A.,Young C.C., 2006. Phosphate solubilizing
bacteria from subtropical soil and their tricalcium
phosphate solubilizing abilities. Applied Soil
Ecology, 34, 33-41.
Garcia C., Fernandez T., Costa F., Cerranti B.,
Masciandaro G., 1992. Kinetics of phosphatase
activity in organic wastes. Soil Biol Biochem
Journal, 25, 361-365.
Sharma B.C., Subba R., Saha A., 2012. In vitro
solubilization of tricalcium
phosphate and
production of IAA by phosphate solubilizing bacteria
isolated from tea rhizosphere of Darjeeling
Himalaya. Plant Sciences Feed Journal, 2(6), 9699.
Sitepu, I.R., Hashidoko V., Santoso V., Tahara V.,
2013. Potential of phosphate-solubilizing bacteria
isolated from dipterocarps grown in peat swamp
forest in central Kalimantan and their possible
utilization for biorehabilitation of degraded peatland.
[Online] Available :
http://www.geog.le.ac.uk/carbopeat/media/pdf/yogy
apapers/p17.pdf
Widayat W., Santoso V., Martosupono V., Astika
G.P.W., Dharmadi A., Kartawijaya W.S., Sukasman,
Tobroni M., Suwardi E., Topani, Samudi J.B., 2006.
rd
Tea cultivation guideline. 3 edition, Indonesia
language. Indonesia Research Institute for Tea and
Cinchona, Gambung, Indonesia.
Xu J.G., Johnson R.L., 1995. Root growth, microbial
activity and phosphatase activity in oilcontaminated, remediated and uncontaminated
soils planted to barley and field pea. Plant Soil
Journal, 173, 3-10.
Hopkins D.W., Bartoli F., 2004. Size and activity of
the soil microbial community from a range of
European volcanic soils. Volcanic soil resources in
Europe. Agricultural Research Institute. Reykjavik,
Iceland. Rala Report, 214, 35-36.
Indonesia
Agricultural
Ministry
(Welcome
Speaking). 2010. Anually meeting of Indonesia tea
board, Bandung.
Islam M.T., Deora A., Hashidoko Y., Rahman A., Ito
T., Tahara S., 2007. Isolation and Identification of
Potential Phosphate Solubilizing Bacteria from the
Rhizoplane of Oryza sativa L. cv. BR29 of
Bangladesh.
Verlag
der
Zeitschrift
für
Naturforschung Journal, Tübingen, 62c, 103-110.
Kapur S., 2010. Andosols. University of Çukurova.
Departments of Soil Science and Archaeometry,
Adana, Turkey,
Malcolm J.L., 1951. Water soluble phosphorus and
potassium in the soil of lime and avocado groves in
dade county. Proc. Fla. State Hort. Soc, 64, 285292.
Neall V. E., 2009. Volcanic soils. Land use and land
cover. UNESCO-EOLSS, VII, 23-45.
Notohadiprawiro T., 2006. The limited concept on
agricultural aspect; the serious constraint on
national
development.
Soil
Science
and
Environment Journal, 6(1), 63-70.
Rodriguez H., Fraga V., 1999. Phosphate
solubilizing bacteria and their role in plant growth
promotion. Biotechnology Advances, 17, 319-339.
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