Isolation, Characterization, and Molecular Identification of Phosphate Solubilizing Bacteria from Several Tropical Soils

ISSN 0852-257X

Jurnal

TA NAH TR OPlKA
(Journal a/Tropical Soils)
Volume 18, No. 1

January 2013

JURNAL TANAH TROPIKA'

Accredited by Indonesian DGHE No. 51IDIKTIIKEPI2010

Journal of

Tropical Soils
Vol. 18, No.1, January 2013

1_
http://www. crossref.org/

Listed in:

OOAJ

DIRECTORY OF
OPEN ACCESS
JOURNALS

http://www.doaj .org/

TABLE OF CONTENTS

I.

Effectiveness of Direct Application of Phosphate Rock in Upland Acid Inceptisols
Soils on Available-P and Maize Yield .......... .............. Nurjaya and Dedi Nursyamsi

1-9

2.

Ameliorant Application on Variation of Carbon Stock and Ash Content on Peatland
South Kalimantan ............ Siti Nurzakiah, Fahmuddin Agusand Haris Syahbuddin

11-16

3.

Soil Chemical and Biological Characteristics for Diagnostic the Potency of Acid Dry
Land for Soybean Extensification .............. .... .................... Prihastuti and Sudaryono

17-24

4.

Growth and Yield of Rice Plant by the Applications of River Sand, Coconut and
Banana Coir in Ustic Endoaquert .................................. Nurdin and Fauzan Zakaria

25-32

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
Soils Varying in Physical and Chemical Characteristics .......... Akhmad Rizalli Saidy

45-52

7.

The Role of Indigenous Mycorrhiza in Combination with Cattle Manure in
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

53-58

8:

Coal Waste Powder Amendment and Arbuscular Mycorrhizal Fungi Enhance the
Growth of Jabon (Anthocephalus cadamba Miq) Seedling in Ultisol Soil Medium
.. .. ..................................................................... Sri Wilarso Budi and Fiona Christina

59-66

9,

Isolation, Characterization, and ' Molecular Identification of Phosphate Solubilizing
Bacteria from Several Tropical Soils ... ................... Fahrizal Hazra and Etty Pratiwi

67-74

10.

11.

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
Assessment Erosion 3 D Hazard with USLE and Surfer Tool: A Case Study of
Sumani Watershed in West Sumatra Indonesia ........................................................... :
.......................................................... Ajlizar, Roni AJrizal and Tsugiyuki Masunaga

Published three times a year

81-92

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 10, 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)
lrwan Sukri Banuwa (Soil Conservation and Environmental Sciences - The University of Lampung)
Naik Sinukaban (Soil Conservation and Watershed Management - Bogor Agricultural University, Bogor)
Afandi (Soil Physics - The University of Lampung)
Priyono Prawito (Soil Genesis and Classification - The University ofBengkulu, Bengkulu)
Tamaludin Syam (Land Evaluation and Development- The University of Lampung)
Technical Editors: Astrid Novia 'Diningrum
Supono

Treasure: Sri Yusnaini

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

I·

I
I
I!
II

I

I
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
Hazta l and Etty
Science and Land Resource,
ofAgriculture

University,
IPB
Bogar 16680, Inanm!Sla
e-mail address:jhazra2011@yahoQ.com
Jalan Tentara Pe/ajar
UrJ;""7J1.'lIr,m ｊｪｬｏｴＯｾｃｦＱＬｲ［Ａｖ＠
and Genetic

rn,'",
Gram TlPotn.''''
Gram negative
Gram "",o."tn/'"
Gram "p(1mtn,p
Gram negative
Gram
Gram "",..,,,fn'p
Gram negative
Gram n .......",ti.",

Gram n",o·Mh,,,,

Gram "1"0'''1''",(>
Gram npo"f"" ..,
Gram negative
gram negative
Gram """.,thu'
Gram np(],,,tn,p.

"'

17.09
3.61
15.35
l.M
8.39
2.74
1.44
94.04

Gram negative
Gram

Gram negative
Gram negative

and E Pratiwi: Identification ofPlwspitate Solubilizing Bacteria

of
Table 3. Selected
with
region oforigin soil samples.
lClVl,a.""",

I> 3.5
P 6.2
P 10.1

ENT

WNT

Dn()SDnatase enzymes and lAA production in each

76.70
66.70
30.17

144.52
43.44  
100.17 

3.06

97.52
15.35
94.04 

'" 
20.13 

*= Not detected.
10

their
provides the
the availability of soil P

9 

ｾ＠

8 

u

'0  7

.3  

6 

o 

2 

3 

4 

5 

6 

1.  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. 
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.
is one indicator
Changes ofacidity he
001 metabolic activity. Research by Perez: 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
by Yu et al.
2) which showed
with
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
of the phosphate solubilizing
detected in the
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) 

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 
2). 
decrease 
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, 
(1glicooxalate, 
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 

73

J Trop Soils, VoL 18, No.1, 2013: 67-74
7

6

ｾＧＯＢ

ｾ＠

ｾ＠

14D
OIl
E 120
c:
100
0

,..A

'-'

ｾＢＧ＠

.ｾＢＮＭ
ＢＧＵｾ

Ｎｾ＠

•• Ｇｾ＠

'\'"(::0 . - EJ.
ＬｾＮ＠

ｾＢ＠

........
c:
u
c:

.'

80

C!)

-'8

.. '....····iI......
ｾＭ

ｾ＠

"0

......-.

8
'"
«l
C!)

1;;
..c

0..
til
0

..c:

o

.1&'"

p...

234

6

Time (days)

.......

....
...,
,,-......
,
)A
.,'
" ...
.'..' ,"
.'

60
40

•••• oJ.'.

A

..,

/;

"

..../i.","" .

,,':'.,4 .. -e' . -0 . -[3' .... EJ
.... ,,'

20
0
0

234

6

Time (days)

Figure 2. The changes in pH of selected PSB in
Pikovskaya medium with a Ca 3(P0 4 )2 as
a source Of P....... = P 3.5, -0'. = P
6.2, and ...... ··· = P 10.1.

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.

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.

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:

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

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

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.

A number of29 phosphate solubilizing bacteria
isolates were obtained from soil at Experiment
Station of Cikabayan IPB (Bogor, West Java),
Citeureup (Bogor, West Java), West Nusa Tenggara
(WNT), and East Nusa Tenggara (ENT). Based
on the ability to produce enzyme phosphatase and
to obtain the highest IAA three isolates were
selected, namely isolates of P 3.5 (ENT), P 6.2
(WNT), and P 10.1 (Citeureup) and all isolates of
bacteria were a Gram-negative bacteria. Isolates
of P 10.1 had the highest ability to dissolve P with
the largest dissolution index (IP), in which 1.80, the

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.

REFERENCES
CTaule,A Valverde,JMlgual
and A Arias. 2012. Abundance, diversity and
prospecting of culturable phosphate solubilizing
bacteria on soils under crop-pasture rotaions in a noin Uruguay. App Soil Eco161: 320-326.
Coutinho 
WP  Felix,  AM  Yano­Melo.  2012. 
Solubilization 
in vitro by Aspergillus
spp. and Penicillium spp. 
42: 85­89. 
AH 
K 
2008. 
Promotion of growth of bean (Phaseolus !l7il'JfJr'"
in a calcareous soil by a phosphate­solubilizing, 
rhizosphere­competent  isolate of Micromonosporaendolithica. 
161­171. 
Gordon SA and KJ Weber.  1993. Colorimetric estimation 
of indole acetic acid.  Plant Physiol26: 192­195.. 
Hadioetomo RS. 1993. Mikrobiologi Dasar dalam Praktek. 
Gramedia. Jakarta (in Indonesian). 
Havlin JL, ill Beaton, SL Tisdale and WL Nelson. 2005. Soil

Fertility and Nutrient Management: An Introduction
to Nutrient Management. 7th  Edition.  Pearson/ 
Prentice HalL 
Saddle River, NJ,  515 p. 
.... ,,,',,orl.  R Kotan and R Cakmakci. 
2012. Characterization of plant growth­promot,ing 
traits of bacteria isolated from  the rhizosphere of 
in alkaline and acidic soils. EurJ Soil

Bio150; 144-150.
ｵｾＬ
.....Ｌｾ｟
....  DW Gabriel. 1987. Conservation 
of  plasmid  DNA  sequences  and  pathovar 
identification  of strains  )(atlthIJmllnQ'JiCt:lm
Phytopathology 77: 448­453. 
Liu  XQ Wu, JH Ren and JR Yeo  2011. Isolation and 
identification  of phosphobacteria  in  poplar 
rhizosphere  from  different  regions  of China. 
Pedosphere 21: 90­97. 
Mander 
S  Wakelin,  S 
L  Condron  and  M 
O'Callaghan.  2012.  Incidence  and  diversity of 
bacteria  are  linked  to 
phosphorus  status  in  grassland  soils.  Soil Biol

Biochem44: 93-101.
Marchesi  JR.  1998. 
and  evaluation of useful 
bacterium spesific PCR primers that amplifY genes 
coding  for  bacterial  16S  rRNA. 
Environ

Microbiol64: 795-799.

Montanez A, ARBlanco, C 
M Beracochea and 
M  Sicardi.  2012.  Characterization  of cultivable 
endophytic  plant 
promoting 
bacteria associated with maize cultivars 
L.) and their inoculation effects  in vitro.

Eco158: 21-28.
Muleta D, F Assefa, E 
and U Granhall. 2013. 
Phosphate­solubilising  rhizobacteria  associated 
with  coffea Arabica  L. in  natural coffee forest  of 
southwestern Ethiopia. J Saudi Soc Agr Sci 12: 73-

84. 
OliveiraCA, VMCAlves, 
BV"flU"''';;  solubilzing 

IIZGisol1tere of maize cultivated in an 
oxisol  of the Brazilian  Cerado  Biome.  Soil Bioi

Biochem41: 17&2-1787.
Park JH,  N  Bolan,  M 
and R  Naidu.  2011. 
Concomitant rock phosphate dissolution and lead 
immobilization by phosphate solubilizing bacteria 
(Enterobacter sp.).J Environ Manage 92:  11151120. 
Perez E, M Sulbaran, MM Ball and L A Yarzabal. 2007. 
Isolation and characterization of mineral phosphatesolubilizing bacteria naturally colonizing a Iimotic 
crust in  the south­eastern Venezuelan region. Soil
Bioi Biochem 39: 2905­2914. 
RRchmiati  Y.  1995.  Bakteripelarut  fosfa  dari  rizosfer 
tanaman  dan  kemampuannya  dalam  melarutkan 
fOsfat.  Prosiding Kongres Nasional VI IDTI Jakarta, 
12­15 Desember 1995 (in Indonesian). 
Ribeiro CM and EJBN Cardoso. 2012. Isolation, selection 
and  characterization  of root­associated 
bacteria  in  Brazil  Pine  (Araucaria
Microbiol Resch 167: 69­7&. 
Subba­RRo NS. 1982. Biofertiiizer in Agriculture. OxfOrd 
and 
Co: New Delhi. 
Tabatabai  MA  and  JM  Bremner.  1969.  Use  of pni trophenyl 
for  assay  of  soi I
phosphatase activity.  Soil Bioi Biochem 1:  301307. 
Yang  
LMa, MH 
JQ 
F 
CQ 
MH 
Mo,  DH 
YQ  Duan  and FX  Yang.  2012. 
Phosphate  solubilizing  ability and  phylogenetic 
diversity ofbacteria from P­rich 
around Dianchi 
lake drainage area of China.  Pedosphere 22: 707716. 
Yu  X,  X Liu, TH Zhu,  GH Liu and C Mao. 2012. Coinoculation  with  phosphate­solubilizing  and 
V",­,.,U­,lI.A"Ue:  bacteria on  solubilization on rock 
phosphate  and  their effect  on  growth  promotion 
and nutrient uptake 
Eur J Soil Bio/50:
112­112.