IJERD – International Journal of Environmental and Rural Development (2012) 3-1

Research article
erd

Isolation of Phosphate-Solubilizing Bacteria from Different
Fields Crop Productions
KIRIYA SUNGTHONGWISES*
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand
E-mail: skiriy@kku.ac.th

CHULEEMAS BOONTHAI IWAI
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand

MASAYUKI MATSUOKA
Research and Education Faculty, Kochi University, Kochi, JapanKOUHEI OHNISHI
Research and Education Faculty, Kochi University, Kochi, Japan

SOTA TANAKA
Research and Education Faculty, Kochi University, Kochi, Japan
Received 14 December 2011

Accepted 18 April 2012(*: Corresponding Author)

Abstract There are many factors involved in the availability of P through phosphatases
efficiency such as the microbial fauna, soil temperature, bacteria communities, plant
physiological state, type of rooting system, age of the plant and the location ofectomycorrhiza on the root. To assess the effects of different crop productions on thediversity and the efficiency of phosphate-solubilizing bacteria (PSB), this study isolated
phosphate-solubilizing bacteria from paddy fields and eggplant fields before and afterharvest in Kochi prefecture in Japan. The results showed that a total of 9 heterotrophic
bacterial isolates present different degrees of mineral tri-calcium phosphate (Ca3(PO4)2)solubilizing activities. Klebsiella pneumonia in the paddy field with water and in theeggplant fields which solubilize Ca3(PO4)2 better than FePO4 and AlPO4. Especially,
Klebsiella pneumoniae strain M-AI-2 and Gluconacetobacter sp. isolate code Ek01 in theeggplant fields seem to have the capacity to solubilize insoluble forms of AlPO4 andFePO4 which are the main forms of insoluble phosphates in acid sandy soils.
Keywords phosphate solubilization, phosphate-solubilizing bacteria, field crop production

INTRODUCTION
Phosphorus is considered as the principal yield-limiting nutrient along with nitrogen (Zahran,1999). Phosphorus deficiency is a primary constraint to plant growth in many terrestrial ecosystems(Bonser et al., 1996) especially, in acid sandy soils with high levels of P fixation by Fe and Aloxides. Plants use phosphorus for adenosine triphosphate (ATP) synthesis. ATP is an essential
energy-provider molecule for the metabolism of organic compounds containing P such as sugar
phosphates, phospholipids, nucleic acids, nucleotides and coenzymes which are key molecules forbiological metabolisms (Schachtman et al., 1998). Plants dependent on symbiotic N2 fixation havetherefore high ATP requirements for nodule development and function (Ribet and Drevon, 1996)
and need additional P for signal transduction and membrane biosynthesis.
Normally, plants and microorganisms produce phosphatases which are released in therhizosphere and catalyze the hydrolysis of organic phosphate esters to orthophosphate anions.
Radersma and Grierson (2004) concluded that root exudation of acid phosphatases and organicacids increase the P solubility in the rhizosphere. The phosphatases efficiency is related to variousfactors such as the microbial fauna, the soil temperature and humidity and more particularly theⒸ ISERD150

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associated bacteria communities (Zahran, 1999). Phosphatases activity can be induced by low
inorganic phosphorus concentrations in the soil solution, but other factors can play a significantrole as the environmental conditions, the physiological state of the plant, the type of rooting system,
the age of the plant and the location of ectomycorrhiza on the root (Antibus et al., 1997).
Within the rhizosphere, the solubility of phosphate can be enhanced by the secretion oforganic acids in root exudates and enable phytic acid to be more available to microorganisms
responsible for its mineralization. Despite its importance in soils and particularly in different cropproductions, the isolation of phosphate-solubilizing bacteria and their P solubilization capacity inthe phosphorus cycle remains poorly studied and only few studied have aimed at exploring themicrobial diversity and its role in the regulation of the cycle of the phytic acid. Finally, the analysisof the ability of bacterial isolates to hydrolyze phytic acid will enable to create a highly valuablebasis for the use of these isolates as bio-inoculums or for the use of enzymes in the food industry.
The objectives of this research were to isolate phosphate-solubilizing bacteria (PSB) from different
field crop productions in Kochi, Japan, evaluate their P solubilization capacity and identify the PSB.
METHODOLOGYIsolation of phosphate solubilizing bacteria by enrichment culture
The experiment was conducted in Kochi prefecture, Japan. Kochi prefecture is characterized by asemiarid tropical climate with a distinct rainy season from May to September. Soil samples werecollected from paddy fields and eggplant fields from the soil surface until a depth of 10 cm. To
isolate phosphate-solubilizing bacteria, 5 g of soil samples were transferred to the National
Botanical Research Institute phosphate growth medium (NBRIP). Per liter, this growth liquidmedium contains 10 g glucose with 5 g of different insoluble forms of phosphate (AlPO4,Ca3(PO4)2 and FePO4), 5 g MgCl2·6H2O, 0.25 g MgSO4·7H2O, 0.2 g KCl and 0.1 g (NH4)2SO4.
Additionally, modified NBRIP media, containing either FePO4 or AlPO4 as the sole source of P,
were also used for the initial screening step. The pH of the agar medium was adjusted to 7.0.
Tricalcium phosphate was autoclaved separately and the other sterile ingredients were aseptically
mixed after autoclaving. Erlenmeyer flasks containing 50 mL of the medium with inoculants wereincubated for 7 days at 30 °C on a IWAKI Incubator shaker at medium speed (150 cycles min −1).
For the following week, 5 ml of this incubated medium with inoculants were transferred into 50
mL Erlenmeyer flasks again with new liquid medium for 7 more days at 30 °C on a IWAKI
Incubator shaker at medium speed (150 cycles min−1). At the end of each week in NBRIP growthliquid media, aliquots of each dilution were spread on NBRIP medium and incubated at 30 °C for
14 days. Colonies were selected from the plates on the basis of the appearance of a clear halo; theclones were further purified on minimal medium based on each insoluble phosphate forms.
Mineral phosphate solubilization assays
The phosphate solubilizing (PS) activity of each of the isolates was determined by molybdenumblue method (Murphy and Riley, 1962). The isolates were grown in NBRIP liquid medium
containing different insoluble forms of phosphate (AlPO4, Ca3(PO4)2 and FePO4) for 3 days at
30 °C on a IWAKI Incubator shaker at medium speed (150 cycles min−1). The solubilization
efficiencies were determined by reaction with ammonium molybdate for phosphorus compounds asammonium phosphomolybdate and reduced with a compound ascorbic acid to molybdenum blue.
Then, the isolates were incubated for 30 min at room temperature for color development. And
finally, the absorption of light in the wavelength range 595 nm was measured by 680 XR
Microplate Reader.

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PCR amplification of 16S rRNA and sequencing
The gene-encoding 16S rRNA was amplified from selected strains by the polymerase chain
reaction (PCR) using bacterial universal   primers   proR2   (5′-AGAGTTTGATCMTGGCTCAG-3′)  and 907R  (5′-CCGTCAATTCCTTTRAGTTT-3′)  (Weisburg et al., 1992). The PCR mix consistedof  0.25  μM  of  each  primer,  1X  PCR  buffer  and  0.2  U  of  Taq  DNA  polymerase.  A  suspension  of  cells on MilliQ water, coming from a fresh colony grown on Nutrient Agar, was used as target
DNA. The following cycle conditions were used: 85 °C for 5 min, followed by 35 cycles of 95 °Cfor 1 min, 55 °C for 1 min and 72 °C for 2 min, and a final extension step at 72 °C for 3 min (Lane,1991).
The PCR products were purified from agarose gels with the PCR Clean-up Gel Extraction Kit(Macherey-Nagel, Germany) and sequenced. The nucleotide sequences were compared using theBlastN program (Altschul et al., 1997), and the closest match of known phylogenetic affiliation
was used to assign the isolated strains to specific taxonomic groups.
RESULTS AND DISCUSSION
Isolation of PSB from soil samples
The screening strategy employed during this research enabled the identification of PSB colonies onNBRIP medium containing different insoluble forms of phosphate (AlPO4, Ca3(PO4)2 and FePO4)
as sole P source. No colonies exhibiting a clear halo were observed on agar plates supplemented
with either FePO4 or AlPO4. Approximately 9 bacterial isolates showed clear halos of Ca3(PO4)2solubilization. Some obvious differences in the size of the halos of different isolates were observed(not shown). This preliminary observation suggested the existence of bacterial isolates exhibiting
different degrees of PS efficiencies in the soil samples collected. To confirm this observation, the 9purified isolates were tested following the protocol of Murphy and Riley (1962), a method previously
shown to be a reliable and qualitative indicator of the PS activity of different bacterial isolates.
Table 1 shows the OD595 nm shift of the culture supernatants of each of the 9 PSB isolates after a
3-day cultivation period in NBRIP medium. Indeed, some isolates did not show any significant
change in the absorbance of the supernatant while others exhibited OD595 nm changes in theabsorbance. Furthermore, we noticed that the most dramatic changes in the color of the supernatantcorrelated with a total solubilization of Ca3(PO4)2 in the medium. Based on these results weselected 9 isolates exhibiting the highest PS activities for further studies. The solubilization
efficiencies of these isolates were calculated and are shown in Table 1. After evaluating their P
solubilization capacity, we concluded that all of the 9 isolates can solubilize Ca 3(PO4)2   better thanFePO4   and AlPO4   especially the isolates with the codes Rk02, Rk03 and Ek04. Moreover, isolate
codes Ek01 and Ek04 look interesting for solubilizing FePO4   and AlPO4, which are the main forms
of insoluble phosphates in acid sandy soils.
Table 1 Phosphate solubilizing effectiveness of tested bacteria, 3 days after inoculation
Isolate Code
Rk01
Rk02
Rk03
Ek01
Ek02
Ek03
Ek04
Ek05
Ek06

Solubilized Phosphate (mgP/l) fromCa3(PO4)2
FePO4
AlPO4
1,015b0c0b1,534a0c0b1,580a0c0b805d
93a0b941bc
0c0b943bc
0c15b1,548a47b161a770d0c0b912c0c0bⒸ ISERD152IJERD – International Journal of Environmental and Rural Development (2012) 3-1

Identification of PSB isolates
Nucleotide sequencing of PCR-amplified 16S rRNA genes and sequence comparison withavailable data in the GenBank using the BLAST algorithm (Altschul et al., 1997) allowed us toidentify the majority of the PSB isolates (Table 2). Based on a sequence identification of 94% orgreater (Van Waasbergen, 2004),   they   were   all   affiliated   to   the   β- or   γ- sub-divisions of theProteobacteria: three isolates were similar to species of the Klebsiella genus, another three weresimilar to Gluconacetobacter sp. and one was closely related to Sphingobacterium sp. Klebsiellapneumonia in the paddy field with water and in the eggplant fields which solubilize Ca 3(PO4)2  better than FePO4   and AlPO4. Klebsiella pneumonia strain M-AI-2 seems to be more interesting tosolubilize AlPO4   and FePO4. Moreover, Gluconacetobacter sp. isolate code Ek01 in the eggplant
field in Kochi University can solubilize FePO4 as well.
Table 2 Identification of PSB isolates from soil samples of paddy fields and eggplant fields in
Kochi by 16S rRNA sequencing after inoculation

Rk01
Rk02
Rk03
Ek01
Ek02
Ek03
Ek04

Length of 16SrRNA genesequenced  503
814
488
546
710
990
938

GenBank
accessionno.
lcl/23633
lcl/45645
lcl/20369
lcl/19025
lcl/39135
lcl/26231
lcl/51159

Ek05

966

lcl/30399

Ek06

983

lcl/27533

IsolateCode

Most closely related organism/
Species (Strain)

Accession no.  

Gluconacetobacter sp.
Klebsiella pneumoniae/ BRp_2A
Klebsiella pneumoniae/ BRp_2A
Gluconacetobacter sp.
Gluconacetobacter sp.
Gluconacetobacter sp.
Klebsiella pneumoniae/M-AI-2
Uncultured bacterium
clone MS-115
Sphingobacterium sp. 21

EF493039.1
JN644536.1
JN644536.1
EF493039.1
EF493039.1
EF493039.1
FJ828890.2

Gene
identity  (%)
97
100
99
99
99
99
99

GQ477848.1

99CP002584.1

94

CONCLUSION
Klebsiella pneumoniae showed the highest P solubilization capacity in the paddy field with waterand eggplant fields. Klebsiella pneumoniae strain M-AI-2 and Gluconacetobacter sp. isolate code
Ek01 in the eggplant field in Kochi University seem to have the capacity to solubilize insoluble
forms of AlPO4 and FePO4.
ACKNOWLEDGEMENTS
This research was supported thanks to a cooperation between the Japan Society for the Promotionof Science and Kochi University under the Invitation Program for East Asian Young Researchers
on Field science towards the resolution of environmental and food problems in Southeast Asia.
Finally, thanks to the kind support of the research institute of molecular genetics at Kochi
University (Japan) and Khon Kaen University (Thailand).
REFERENCES
Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. 1997.
Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. NucleicAcids Research. 25(33), 89-3402.
Antibus, R.K., Bower, D. and Dighton, J. 1997. Root surface phosphatase activities and uptake of 32Plabelled inositol phosphate in field-collected gray birch and red maple roots. Mycorrhiza, 7, 39-46.
Bonser, A.M.Y.M., Lynch, J. and Snapp, S. 1996. Effect of phosphorus deficiency on growth angle of basalroots in Phaseolus vulgaris. New Phytol. 132, 281-288.
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IJERD – International Journal of Environmental and Rural Development (2012) 3-1

Lane, D.J. 1991. 16S/23S rRNA sequencing, E. Stackebrandt, M. Goodfellow, Editors, Nucleic AcidTechniques in Bacterial Systematics, Wiley, Chichester. 130-141.
Murphy, J. and Riley, J.P. 1962. A modified single solution method for the determination of phosphate innatural waters. Anal. Chem. Acta. 27, 31-36.
Radersma, S. and Grierson, P.F. 2004. Phosphorus mobilization in agroforestry: Organic anions, phosphataseactivity and phosphorus fractions in the rhizosphere. Plant Soil. 259, 209-219.
Ribet, J. and Drevon, J.J. 1996. The phosphorus requirement of N2-fixing and urea-fed Acacia mangium.
New Phytol, 132, 383-390.
Schachtman, D.P., Reid, R.J. and Ayling, S.M. 1998. Phosphorus uptake by plants: from soil to cell. Plant
Physiol, 116, 447-453.
Van Waasbergen, L.G. 2004. What makes a bacterial species? When molecular sequence data are used, isrRNA enough?, In R.V. Miller, M.J. Day, Editors, Microbial Evolution: Gene Establishment, Survival
and Exchange, ASM Press, Washington, DC, 339-356.
Weisburg, W.G., Barns, S.M., Pelletier, D.A. and Lane, D.J. 1992. 16S ribosomal DNA amplification forphylogenetic studies. Journal of Bacteriology, 173, 697-703.
Zahran, H.H. 1999. Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in anarid climate. Microbiol. Molecu. Bio. Rev. 63, 968-989.

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