ISSN 0852-257X
Jurnal
TA NAH TR OPlKA
(Journal a/Tropical Soils)
Volume
18,
No. 1
January 2013
JURNAL TANAH TROPIKA'
1_
Listed in:Journal of
OOAJ
DIRECTORY OFTropical Soils
OPEN ACCESSVol.
18,
No.1, January 2013
JOURNALShttp://www. crossref.org/ http://www.doaj .org/
TABLE OF CONTENTS
I. Effectiveness of Direct Application of Phosphate Rock in Upland Acid Inceptisols 1-9 Soils on Available-P and Maize Yield ... ... Nurjaya and Dedi Nursyamsi
2. Ameliorant Application on Variation of Carbon Stock and Ash Content on Peatland 11-16 South Kalimantan ... Siti Nurzakiah, Fahmuddin Agusand Haris Syahbuddin
3. Soil Chemical and Biological Characteristics for Diagnostic the Potency of Acid Dry 17-24 Land for Soybean Extensification ... .... ... Prihastuti and Sudaryono
4. Growth and Yield of Rice Plant by the Applications of River Sand, Coconut and 25-32 Banana Coir in Ustic Endoaquert ... Nurdin and Fauzan Zakaria
5. Fresh Organic Matter Application to Improve Aggregate Stability of Ultisols under 33-44 . Wet Tropical Region ... .. ... fulnaJatmawita, Adrinal, and Febrian Anggriani
6. Relationship between Water Content and Mineralization of Carbon and Nitrogen in 45-52 Soils Varying in Physical and Chemical Characteristics ... Akhmad Rizalli Saidy
7. The Role of Indigenous Mycorrhiza in Combination with Cattle Manure in 53-58 Improving Maize Yield (Zea Mays L) on Sandy Loam of Northern Lombok, Eastern
of Indonesia ... .. ... ... .. ... .. .... .. ... Wahyu Astiko, Ika Rochdjatun Sastrahidayat, Syamsuddin Djauhari and Anton Muhibuddin
8: Coal Waste Powder Amendment and Arbuscular Mycorrhizal Fungi Enhance the 59-66 Growth of Jabon (Anthocephalus cadamba Miq) Seedling in Ultisol Soil Medium
.. .. ... Sri Wilarso Budi and Fiona Christina
9, Isolation, Characterization, and ' Molecular Identification of Phosphate Solubilizing 67-74 Bacteria from Several Tropical Soils ... ... Fahrizal Hazra and Etty Pratiwi
10. Inoculation Effect of NrFixer and P-Solubilizer in a Mixture of Fresh Manure aiid 75-80 . Phosphate Rock Formulated as . Organonitrofos Fertilizer on Bacterial and Fungal
Population ... Sutopo Ghani Nugroho, Dermiyati, lamalam Lumbanraja, Sugeng Triyono, Hanung Ismono, Missy Kurnia Ningsih and Fitri fani Saputri
11. Assessment Erosion 3 D Hazard with USLE and Surfer Tool: A Case Study of 81-92 Sumani Watershed in West Sumatra Indonesia ... :
... Ajlizar, Roni AJrizal and Tsugiyuki Masunaga
Jurnal
TANAH
TROPlKA
(Journalo[Tropical Soils)
ISSN 0852-257X, established in 1995, accredited by Indonesian Directorate General of Higher Education (DGHE) since
1998, last accredited No. 5 I IDIKTIlKEP/20 1 0, 5 July20 I 0, valid until 5 July 20 13.
Granted for Internationalization Program of Scientific Journal by DGHE No. 017 ISP.SIPIDP2M/20 10 .
Jurnal TANAH TROPIKA (Journal a/Tropical Soils) published three times a year (January, May and September),
covers wide range of research article in which using tropical soils. Review article does not allow except it was invited by editor.
All contributors should be subscribed this journal at least one year. The cost for subscriber journal per year (3 issues) is Rp 150 .000 for individual (Indonesian) and 20 USD for Overseas, and Rp200.000 for Indonesian institution or library and 25 USD for Overseas. The cost of shipping and handling for each issue is Rp25 .000 for Indonesian and 5 USD for overseas . All cost can transfer to Bank Account of Sri Yusnaini, Rekening BNI Cabang Pembantu Universitas Lampung number 02111 20716.
Chief Editor: Dermiyati Managing Editor: Ainin Niswati
Editorial Boards
Abdul Kadir Salam (Soil Chemistry - The University of Lamp ung)
Ded i Nursyamsi (Soil Chemistry - Indonesian Agricultural Environment Research Institutem, Pati)
Sri Djuniwati (Soil Fertility - Bogor Agricultural University, Bogor)
So ni lsnaini (Soil Fertility- DARMAWACANAMetro)
Erry Purn o mo (Soil Fertility - Lambung Mangkurat University, Banjarbaru)
Eko Hanuddin (Soil Chemistry and Mineralogy- Gadjah Mada University, Yogjakarta)
lin Purwati Handayani (Soil Biology and Microbiology - Murray State University, USA)
Nobihiro Kaneko (Soil Ecology- Yokohama National University, Japan)
I
I·
lrwan Sukri Banuwa (Soil Conservation and Environmental Sciences - The University of Lampung)Naik Sinukaban (Soil Conservation and Watershed Management - Bogor Agricultural University, Bogor)
I
Afandi (Soil Physics - The University of Lampung)
Priyono Prawito (Soil Genesis and Classification - The University ofBengkulu, Bengkulu)
I
Tamaludin Syam (Land Evaluation and Development- The University of Lampung)
I
Treasure: Sri Yusnaini Technical Editors: Astrid Novia 'Diningrum
!
Supono
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I
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Publisher
Department of Soil Science Faculty ofAgriculture, the University ofLampung and Indonesian Society
for Soil Science (ISSS) Region Commissariat Lampung
Website: http://journal.unila.ac.idl/index.php/tropicalsoil
I
Available online at:
http;/ljournal.unila.ac.id/index.php/tropicalsoil
DOl: 1O.5400/jts.20 13.18.1.67
Isolation, Characterization,
and
Molecular Identification of
Phosphate Solubilizing Bacteria from Several Tropical Soils
Haztal and Etty
Science and Land Resource, ofAgriculture
IPB Bogar 16680, Inanm!Sla
e-mail address:[email protected]
Jalan Tentara Pe/ajar
<7,.,,,,,JlHW.m University,
Received 30 April20f2! accepted 7 January2013
ABSTRACT
ofthe research were: (i) to isolate and characterize ofphosphate bacteria
to identifY PSB based on molecular ofl6S rRNA gene. Soil were collected from in and East Nusa Several in this research were:
0)
isolation PSB inagar, (ii) a,nd biOChemical characterization (iii) measurement of
and (iv) measurement indole acetic acid As many as 29 isolates ofPSB have been collected and three isolates namely: P 3.5 Nusa P 6.2 Nusa and P 10.1 (Citeureup, were chosen for further There were many characteristics of isolate PI 0.1: it had capable to solubilize P with the value solubilization index (1.80), (ii) it had the highest phosphatase enzyme (120.40 mg kgl), and (iii) it had the pH decrease at each observation fOf six Isolates P 3.5 P 10.1 were the Gram-negative bacteria with coccus and isolate P 6.2 was a Gram-negative ｢｡｣ｴ･ｾｩ｡＠ with bacil!u$ shape. De<;3xiribonucieatAcid (DNA) amplification ofthese employing 16S rRNA primers gener-ated the 1,300bp-PCR product. The results ofth!;l analysis ofl6S rRNA gene sequences showed that isolates P 3.5 and P 10,1 has 98% similarity with Gluconacetobacter sp. strains Rg1-MS-CO and isolate P 6.2 has 97% similarity with Enterobacter strains.
"Research Institute UrJ;""7J1.'lIr,m ｊｪｬｏｴＯｾｃｦＱＬｲＯｏｬ［ｏｬＡｖ＠ and Genetic
West Nusa
P..,7'VTTI .."
16S rRNA, indole acetic acid, '""V''''''VU, phosphatase enzymes, phosphate solubilizing bacteria
INTRODUCTION
Phosphor (P) is one of the macro element
which is very for However,
content of P in plant is lower than nitrogen (N), Potassium (K) and (Ca) et at.
2005). often not
｢ｾ｡ｵｳ･＠ is bonded in unavailable form,
so it is not available to be plant. The
problem in on farmland is low
in efficiency, only 10-30% that can use by plant. This happenend because bounding process or fixation of P is by To increase efficiency of fertilizing, it
to use phosphate solubilizing microorganism, such as phosphate
(PSB).
Olievera (2009) showed that PSB produced the greatest solubilization in medium
pho:;pha:te [(Ca,(P04
)J.
J Soils, VoL IS, No. 1, 2013: 67-74
is suitable by et aI, 12)
showed that 90% of PSB isolates had pot'entlal for solubilization ofsimple superphosphate
(SSP) and mono-ammonium (MAP) on the seventh day evaluation, With average values respectively 23% and 22% higher control.
ParketaL (2011) that ofPSB
rock phosphate (RP) in soil can be as an alternative technique to soluble P compounds. Research by et al. (2012) that had abilities to
phosphate, with the CO[lcerltratlOilS of solubilized P
some
in bacterial cultures from 33.48 to 69,63 mg by Montanez et ai.
are Rhanella,
Rhizobium, Pseudomona, Herbaspirillum,
Enterobacter, and Burkholderia.
All produced IAA vitro only P.
(EMA68) produced siderophores. Phosphate solubilizing bacteria is and it can
be very potential to
the
68
4
F Haua and E Pratiwi: Jdenfijicatitm oj Phosphate Solubilizing Bacteria
were isolate, and indentifiy PSB from some locations in Bogor, West Nusa
that ability
kind of bacteria molecular identification according to amplification of gen 16S
MATERIALS AND METIIODS
Soil Sampling and Media
Tropical soil samples were collected from Experiment Station Bogor Agriculture
｜ｾＧｾＢＧＧＧＧＧＧ＠ West Java), Citeureup
West Java), West Nusa (WNT), and East Nusa Tenggara Indonesia
and ofPSB were
out by Pikovskaya Medium, 2 different sources of P, Ca/P04)2 or tricalsium phosphate and CaiP04)sOH or hidroxy apatit), universal buffer (MOB) 1x, 0.5 NNaOH,
0.115 M p-nitro phenil phosphate (p-NPP), 1 mg
mL-1 p-nitrophenol (p-NP), indole
acetic acid (IAA), reagent, cristal
iodium safranin, alcohol 95%, sterile water.
Isolation of from Rhizosphere Soils
A total of 10 ofsoil each
was added into a tube containing 90 mL Pikovskaya liquid medium at pH 6.8, then it was made serial dilutions up to 1Q4. A mL 10.3 and 10'
was poured over of solid Pikovskaya medium, then spread evenly the spreader bars, and incubated for 2-3 days at room presence ofPSB
was by a clear wne ｯｦｬｩｧｨｴＭｾｯｲｯｲ･､＠
or zone around colonies that showed a dissolution of phosphate.
Characterization and Ability of in Dissolving P
some soil they were as morphology by observation on colony of PSB which included shape, elevation, and as suitable with
ｮｲＨＩｾｦＧｦｬｬｬｬｲｦＧＧＧ＠ (1993). On hand,
capabilities of in dissolving P in solid medium was also conducted by measurement diameter of Pikovskaya zone dissolution index of phosphate.
DI=
light zone
Observations made during of incubation observing the elevation, and color as well as measuring the diameter of clear
zone DI PSB colonies that
clear zone around colony.
Phosphatase Enzyme Measurement
Phosphatase enzyme measurements were out in accordance with procedures Tabatabai and Bremner A total of 10 mL
PSB culture was grown for 3 at room temperature in a liquid Pikovskaya medium (with two sources of P that were Ca (PO) and3 4 2
and was (10,000 rpm, to the bacterial colonies from
ＢＧＧＧ［ＧＧＧＧｾｵｵＮ＠ The supernatant was into a
clean test tube 1 mL, it was added 4 mL of Modified Buffer (MOB) 1x, and 1 mL of 0115 M p-nitro phenyl
(p-NPP), incubated 60 min at in a waterbath
at 37 incubation 60 a 5 mL
0.5 N NaOH was added to stop the
solution was yellow, that the bacterium the enzyme phosphatase. Absorbance measurements were perfonued at a
410 run using a 150-20 type.
Hypergrowing Indole Acetic Acid of PSB
Indole Acetic (IAA) measurements were carried out in accordance to of Gordon
Weber (1993). A total of 10 m1 was grown for 3 days at room tf'nlnf'r$ltillt medium Pikovskaya (with two sources
was (10,000 15 to separate
the colonies from supernatant was test tube
2
mL,(ratio 1 :1). ,><...,,'''''','''
mixture and the supernatant was incubated for 60minute at room The,solutions were turned this was an indication of the content
IAA. solution was at a
wavelength (A) = run using a Hitachi 150-20
Spectrophotometer
Population Growth, and Content. of Ph,osphatese of PSB
Measurement pH in liquid
Pikovskaya medium PSB isolates were carried out using a meter. A population was measured by serial dilutions
10-1, Measurement of ph()SP:hatase ",,.,cr\lfi''''
was to the previous.
carried out for 7 of mcuO'll1on.. ｾＧｮ｟Ｇｾｾ＠
J Soils, VoL 18, No.1, 2013: 67-74
69
Gram Staining the PSB
Gram staining test were done according to Hadioetomo (1993) in which ...ｮＮｾ＠ ｾＮｾｾ＠
appeared purple, while appeared pink
Bacteria Genomic DNA Isolation
Isolation of total genomic DNA a modification ofthe method of Lazo et al. (1987).
DNA Electrophoresis Process
A total ml
mlloading buffer as Suspension ofDNA with loading buffer solution was injected into the wells on gel electrophoresis. After all wells were fllled, the power supply was with a voltage by 110 V for ± 45 minutes. Gel plp<,trl"l,,,hf''''''''';''
results were appointed and subsequently immersed solution ofethidium bromide (EtBr) 10 minutes and in distilled water for 5 minutes. Further, DNA can be viewed and photographed UV Transilluminator device equipped a digital camera.
Polymerase Chain Reaction Analysis
were used in the polymerase chain reaction process, the F-63 (5'_ CAGGCCTAACACATGCAAG and R-1387 primer (5'-GGGCGGCGTGTA
(Marchesi 1998). The process was begun by the manuiaci:11rirll! protocols in which a mixture ofPCR reaction components for PCR 75 ml with the following composition: 7.50 mIl Ox PCR buffer, 2.25 mllOmMdNTP, mMMgS041:50 0.75ml 101M F, 0.75 mIlO iM primer R, 1:00 ml DNA, 1.00 mLTaq DNA and 60.25 mL of sterile aquabidest.
Total running 30 of PCR were performed with the following conditions: the initial cycle of denaruration or 94 "C for 5 minute, followed by denaruration for the next cycle at 94°C for 30 was performed for 30 at 50 °c. Polymerization out for 2 at 72 OC last the cyc1e-30 was made an extension for 7 minute nrolvm,M""l'7,,,,ti/.n
it was performed to a tp,."np.."tt
drop of 4 OC to stop the PCR reaction. PCR ..p",..tum
were in SwiftTM Maxi Thermal Cycler Blocks-"ESCO". products were by 1.0% gel electrophoresis in 0.5x TAE buffer with a voltage of 110 volts for ± 45 minutes on Mupid-2 plus. PCR were
｣ｨ･ｾｫｴｸｩ＠ by a Scope Transilluminator (Destkop
21).
DNA Sequencing
Polymerase Chain products were sequenced an Automated DNA Sequencer tool PRISM ABI 377 (perkin EImer Biosystem. USA). DNA sequencing kit was performed using BigDye Ready Reaction Mix
USA). DNA sequences obtained were compared to sequences in the database European Bioinformatics Instimte (EBI) WU-BLASTN 2.0 tools on the HYPERLfNK ''http:// www.ebLac.uk''http://www.ebLac.uk.
RESULTS AND DISCUSSION
Isolation of PSB
Phosphate grows in
medium will dissolve P Pikovskaya which is by clear or light-colored zone SUlTOlmdml! the This is to the
､ｩｳｳｯｾｵｴｩｯｾ＠ of phosphate from Ca
3
(POJ2
which iscontamed In the medium. A total of29 isolates of the PSB which produce9 clear zone was then purified on a Pikovskaya (Table 1).
Characteristics and Ability of PSB in dissolving P
isolates were observed colony morphology characteristics included the shape,
,-","ViR'U". and the color colonies acc:ordlmQ
to the Hadioetomo (1993), as well as the measurement of ｰｨｯｳｰｨ｡ｴｾ＠ dissolution index (01). Observations and measurements are shown in
l.
on generated it could be seen that the ability ofPSB to P was suitable with by et (2011) which showed 15 PSB strains exhibited inorganic ..._coAI" ....
rooging
376.62 aid 669.56 mgto Rachmiati (1995), a qualitatively clear zone area allegedly showed the of the ability of
dissolving P from insoluble phosphate.
The Ability of PSB in Produce Phosphatase
Enzyme, IAA and Gram Staining
The ability
and IAA production as well as results are shown in Table 2.
Phosphatase enzyme is an important erur.vrrle
soil is functioned to change a phosphate which is bonded in organic compounds into a available to Table 2 shows that PSB isolates that produced the highest phosphatase enzyme in
70 "
F Hazra and E Pratiwi: Identification ofPhosphate Solubilizing Bacteria
Table 1. Morphological characteristics and the ability ofPSB to dissolve P on Solid Pikovskaya the 7 incubation.
Isolates
DI
Code Color
Pl.l
ENT
White arising 1.20P 1.2
ENT
Yellow Smooth Wrinkled 1.50 P1.3ENT
White Like crater Smooth Oval, with the edge arising 1.10 PI.4ENT
White Smooth Round 1.70 P1.5ENT
White Like crater Smooth with the 1.08P2J
ENT
White, surrounded Wavy Complex (like 1.10by pink color
P2.2
ENT
yellow as the Like crater Smooth Oval, with the edge arising 1.09center
P 2.3
ENT
White Like crater Smooth Oval, with shellfish 1.11P2.4
ENT
White Like crater Wavy with shellfish 1.22 P 3.1ENT
Brovmish yellow Arising Irregular Oval, with shellfish 1.27 P3.2ENT
Brownish Arising Wavy Oval, with edges spread 1.50 P3.3ENT
Brownish yellow Irregular Oval, with edges 1.56 P3.4ENT
Brownish yellow and spread 1.25 P 3.5ENT
Brown Arising with shellfish 1.78 P 3.6ENT
Yellow Like crater Wavy Oval, with shellfish 1.50 P 4.1ENT
White, yellow as the Like droplet Smooth Oval 1.08center
P4.2
ENT
White Like crater Smooth Oval, with the edge 1.08P 4.3
ENT
White, as the Like crater Smooth Oval, with the edge 1.09center
P4.4
ENT
White Like droplet Smooth Oval 1.08 P5.l WNT White Like crater Wavy Oval, with the arisingLIO
P5.2 WNT White Wavy Oval, with the edge arising 1.42
P53 WNT White Smooth Oval 1.57
P 6.1 WNT White, yellow as the Like droplet Smooth Oval 1.25
center
P 6.2
WNT
White Like crater Smooth with shellfish 1.22 P 7.1 CK Yellow Arising Wavy Oval, with edges spread 1.11White Smooth Oval, with edges shellfish 1.67
P 8.1 CK
P 8.2 CK Clear white Flat Smooth Oval 1.10 P9.1 CT Brownish yellow Arising Smooth Oval 1.62
Smooth Oval 1.80
ENT East Nusa CK Cikabayan (IPB
Station), and CT =
Note: DI = Dissolution Index, WNT
While the that produced lowest phosphate source Ca/P04)2 was P 3.4, that phosphatase enzyme were P isolates, which was equal to mg kg!. While the isolates 0.17 mg P 3.3 and P 7.1, respectively, lowest IAA was isolates P 1.1 isolates were isolated from of river sand East containing 0.04 rug kgl,
and rhizosphere P andP Nusa (ENT) and rhizosphere soil 1.1 were from samples
of soybean plants in IPB Cikabayan Nusa
Station (Bogor, Java). of mahogany, East N usa (ENT),
on inTable 2, it was also respectively. At source of PSB isolates that produced in Ca (P04)pH, isolates that produced
[image:7.611.66.538.104.616.2]71
JTrop JIOI. 18, No.1, 2013: 67-74
of 97.52
. Isolates
ｓ｡ｮ｛ＱｐｬＨｾｓ＠ of river
East N usa CENT) and
soil samples of paddy fields, Nusa
liquid medium, and phosphatase enzyme content during 7 ofincubation .
The different productions of
enzyme and IAA of were ca\lsed by of that were isolated on
This is with by
Tenggara CENT), respectively. Based on the results Karagoz et (2012) which showed that of Gram stainihg, it is known that all isolates of the different geographical locations, soil and
veJ;tmmCtn types in resulted in
the different population and ....""'t"'r.,,,
According to the by Mander et
phosphatase plant growth IAA thy frequency P-solubilisation in bacterial production which were in origin area of
each NTS, and NTT), the
are shoVi-TI in Table 3. Selected isolates ofPSB were then carried out further testing,
growth, of pH in the
measurement
lAAduring 3 incubation Table2. ability ofPSB in produces
its gram 0UUJ:UUE>'
in Table 2, three isolates Were based on the content of the enzyme
(P < 0.001) in
Gram Isolates
Pl.1 42.78 17.13 8.83 Gram negative
P 1.2
*
17.57*
5.78 Gram npo',>rn,'",P1.3 28.65 43.44 * 16.22 Gram TlPo<>tn.''''
PIA 30.61 8.44 2.24 3.61 Gram negative
P 1.5 30.39 14.96
*
11.87 Gram "",o."tn/'"Gram "p(1mtn,p
P 2.1 5.39 21.26 * 8.39
P2.2 24.74 17.35
*
14.04 Gram negativeP 2.3 24.09 11l.44
*
1.00 GramP2.4 4257 24.96
*
10.57 Gram "",..,,,fn'pP3.1 63.00 9.30 7.59 45.35 Gram negative
Gram n ...",ti.",
P3.2 34.74 242.13 4.04
2.13 38.99 1.87 Gram n",o·Mh,,,,
P3.3 98.22
P3.4 92.35 3.44 74.45 18.83 Gram "1"0'''1''",(>
P 3.5 76.70 144.52 3.06 97.52 Gram npo"f"" ..,
P
3.6 50.83 6.04 37.59 13.6] Gram negativeP 4.1 36.10 31.48 *
*
gram negative9.52 21.48 1.87 Gram """.,thu'
P4.2
*
P4.3 39.30 18.M
*
12.30P4.4 41.48 16.26
*
2.30P 5.1 21.70 47.13
"'
2.74P 5.2 41.10 17.78
'"
"'
P5.3 28.44 20.61
*
17.09P 6.1 31.26 10.61
*
3.61P 6.2 66.70 43.44
*
15.35P 7.1 0.17 14.09
'"
l.M Gram negativeP 8.1 21.70 14.52
*
8.39 GramP8.2 29.52 21.48
'"
2.74 Gram negativeP 9.1 31.26 70.39 7.96 1.44 Gram negative
p 30.17 100,17 20.13 94.04
Gram np(],,,tn,p.
and E Pratiwi: Identification ofPlwspitate Solubilizing Bacteria
Table 3. Selected lClVl,a.""", of with Dn()SDnatase enzymes and lAA production in each
region oforigin soil samples.
I> 3.5 ENT 76.70 144.52 3.06 97.52
P 6.2 WNT 66.70
P 10.1 30.17
*= Not detected.
their provides the the availability of soil P
isolates had a lag or beginning phase on day-O to day-I. The stationary phase was occured on day-3 to day-4.
While the phase was from 5 to day-6.
Changes ofacidity he is one indicator
001
metabolic activity.Research
byPerez:
et al.(2007) sowed that 10 of 130 the isblates were able to mediate almost solubilization of in liquid Acidification of culture supernatants were likely to be the main mechanism for P solubilization. was suitable with by Yu et al. 2) which showed was found between pH and soluble P concentration, as well as total organic
acid production P PSB. This
was suitable with by EI-Tarabily et al.
(2008) which showed that a significant (P < 0.05) a concurrent (P <
"'1'-0":>'-'U P concentration were
detected in the of the phosphate solubilizing isolates in PRP-amended broth compared to the
non-"""PO;'rf'h by Muleta
et (201 showed that solubilisation of hydroxyapatite (HAP)ltricalcium phosphate (TCP)
43.44
'"
15.35100.17 20.13 94.04
10
9
8
ｾ＠
u
'0
7.3
6
o
2 3 4 51. The population growth of selected PSB in Pikovskaya medium with a Ca,(P04)2
as a source
--+-.
P 3.5, -0'-= P 6.2, and·· ....··• = P 10.1.all were a in
medium pH and production of organic acids kind phosphobacteria that could considered
as involved solubilisation
of insoluble HAPITCP. Value on Pikovskaya liquid medium for all isolates were to
decrease 2).
ｔｨｾ＠ in medium was caused by
secreting organic acids that was acquitted by a number ofPSB in the activity. Phosphate solubilizing bacteria will deliver a number of orgamc acids such as citric acid, glutamic, lactic,
glicooxalate,
(1-ketobutirat. acids are usually
followed by a sharp decrease in pH, thus resulting
in the dissolution (SupbaRao 1982).
Rachmiati (1995) that each has
ability to produce genetically different in the
number organic acids that a role
of dilution P.
Phosphatase enzyme content tended to increase until day then decreased on day5 but
again on (for P 3.5 and P 10.1). As
the P the "...o'vrr'p
phosphatase content did not so sharp as compared
[image:9.611.304.520.107.377.2]73
J Trop Soils, VoL 18, No.1, 2013: 67-74
7
ｾＢＧＧＧＧ＠
6
ｾＧＯＢＧｾ
.
ｾＢＢＢＮＭＮｾ
ＢＧＵｾ •• Ｇｾ＠'\'"(::0 . -
EJ.ＬｾＮ＠ - ' 8
ｾＢ＠
.. ····iI
ｾ＠"0
'....
...
ｾＭＭＭ
...
-.
o 2 3 4 6
[image:10.611.87.298.66.243.2] [image:10.611.323.536.73.240.2]Time (days)
Figure 2. The changes in pH of selected PSB in Pikovskaya medium with a Ca3(P04)2 as
a source Of P... = P 3.5, -0'. = P 6.2, and ... ··· = P 10.1.
to isolates of P 3.5 and P 10.1. isolates of P 1.1 0 had the highest content of a phosphatase enzyme followed by isolates ofP 3.5 and P 6.2.
Molecular Identification of Phosphate Solubilizing Bacteria
Deoxiribonucleat Acid has been isolated and tested to detect the presence of DNA using agarose gel electrophoresis. Having obtained the DNA. the peR was then performed. The result of PCR amplification PSB isolates using 16S rRNA primers that produced ｾ＠ PCR amplicon or product was abeM 1,300 bp (Figure 4). Amplicon was subsequently sequenced to determine the nucleotide sequence of . the 16S rRNA gene of each isolate.
Based on the results of 16S rRNA gene, sequence analysis of the program WU-BLASTN
2 3 4
Figure 4. The result ofamplification 16s r RNA gene from PSB. 1 = 1 kb DNA ladder marker, 2
=
isolate P 3.5, 3=
isolate P 6.2 and 4 = == isolate P 10.1.ｾ＠ 14D
ｾ＠
OIl 120
E
' - '
c:
0 100
Ｎｾ＠
....
....
c: 80C!)
u
c: 60
8
C!)
«l'" 40
1;; ..c
0.. 20
til
0
..c:
p... 0
,..A
.1&'" ...
.' •••• oJ.'. / ;
....
...,
)A.' ,,-... A ,"
.,'
"
...
..,
..
'"
.'
,
/i." " .
....
,",,':'.,4 ..
-e' . - 0 . - [ 3 ' .... EJ....
,,'0 2 3 4
Time (days)
Figure 2. Phosphatase concentration on selected PSB in Pikovskaya medium with a Ca3(P04)2 as a source of P.•••• = P 3.5, -0'- = P 6.2, and ... = P 10.1.
2.0 homology was done to know species from three isolates ofPSB. Isolates of P 3.5 (ENT) and isolates ofP 10.1 (Citeureup) had a similarity of98% with
Glucortacetobacter sp. strain RH I-MS-CO, whereas isolates ofP 6.2 (WNT) had a similarity of 97% with Enterobacter sp. pp9c strain. ｂｾｳ･､＠ on resea{ch by Ribeiro et al. (2012), analysis of fatty acid profiles on PGPR by partial sequencing ofthe 16S rRNA gene produced approximately 550--650 base pairs and the evaluation ofthe similarity of the bacteria also showed the same three families:
Bacillaceae, Enterobacteriaceae and
Pseudomonadaceae. The results of research by Azzis et al. (2012) showed that two hundred and fifty PSB were isolated in 2007 arid they were classified according to their phosphate solubilization activity in vitro. Twelve of these isolates showing the greatest solubilization activity were selected for 16S rDNAsequencing. Ten isolates were presumably belong to the genus Pseudomonas and two isolates showed high similarity with members of the genera
Burkholderia and Acinetobac(er,.
CONCLUSIONS
[image:10.611.82.300.539.719.2]74 F Hazra and E Pratiwi: Identification ofPhosphate Solubilizing Bacteria
highest phosphatase enzyme content (120.40 mg kg·i ),
and the highest decrease. The results of 168 rRNA
gene sequence analysis showed that isolates of P 3.5
had 98% to Gluconacetobacter
sp. strain RHI·M8·CO on the GenBank data, while the
isolates P6.2 had a 97% to Enterobacter sp.
pp9c strain on the GenBank data.
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