IJERD – International Journal of Environmental and Rural Development (2014) 5-2

Research article
erd

Potential of Native Phosphate Solubilizing Bacteria Isolated
from the Rhizosphere of Economic Crops and Vermicast in
Northeast Thailand to Solubilize Insoluble Phosphates
under in vitro Conditions
KIRIYA SUNGTHONGWISES*
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand
Email: skiriy@kku.ac.th

CHULEEMAS BOONTHAI IWAI
Integrated Water Resource Management Research and Development Center in Northeast
Thailand, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand

ANAN WONGCHAROEN
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand

ARUNEE PROMKHAMBUT
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand

DIDIER LESUEUR
CIRAD, UMR Eco&Sols (CIRAD-IRD-INRA-SupAgro), Land Development Department,
Office of Science for Land Development, Chatuchak, Bangkok 10900, Thailand
Deakin University, School of Life and Environmental Sciences, Faculty of Science,
Engineering and Built Environment, Melbourne, Australia
Received 3 November 2013

Accepted 11 August 2014(*Corresponding Author)

Abstract Although soils generally contain a large amount of total P, only a small
proportion is immediately available for plant uptake making it a major constraint on crop
production in many tropical countries. Free-living bacteria and fungi can mobilize
orthophosphate from either organic or inorganic P sources such as Phosphate Rock (PR).
These phosphate-solubilizing microorganisms (PSM) are characterized by their capacity tosolubilize precipitated forms of P, the main P ingredient in PR and could be good biofertilizers for improving phosphorus plant nutrition. The present study examined
phosphate solubilising bacteria (PSB) isolated from cassava, groundnut, rubber tree,
sunchoke, rice, rice-soybean, rice-soybean-corn and rice-chili fields and vermicast ofearthworm varieties in Northeast of Thailand, where soils are mainly sandy and Pdeficient. PSB isolates were tested by using different P sources [Tri-calcium Phosphate(Ca3(PO4)2), Ferric Phosphate (FePO4) and Aluminium Phosphate (AlPO4)] on specificculture media (National Botanical Research Institute Phosphate Growth Medium, NBRIP).
Our results showed that five of the PSB isolates from economic crops and vermicast ofearthworm varieties solubilised a significantly (P  0.01) higher amount of AlPO4 andFePO4 over the uninoculated control. The highest activity of solubilization was achievedfor AlPO4 followed by FePO4 which are the main forms of insoluble phosphates in acidic
sandy soils. We found that PSB isolated from vermicast of earthworm varieties Pheretima
posthuma and Eudrilus eugeniae were able to solubilize both AlPO4 and FePO4 at
relatively high rates (up to 1,918.49 mgP/l), in contrast, PSB isolated from cassava, ricesoybean and groundnut field soil tended to have lower solubilisation rates for FePO4. Noneof the isolates tested were able to solubilize CaPO4. Finally, IAA production was observed
only in PSB isolated of E. eugeniae vermicast and rice-soybean field soil. These results
highlight variability of specific PSB isolates from different rhizospheres and vermicast ofearthworm varieties and provide essential information for the management of soil fertility.

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Keywords crop productions, phosphate solubilization, phosphate-solubilizing
bacteria, vermicast

INTRODUCTION
Phosphorus (P) is an essential element in plant development. Photosynthesis, changing sugar to
starch, genetic inheritance, nitrogen fixation, flowering, fruiting seed production (Mehrvarz et. al.,2008) and resistance to plant diseases are the attributes associated with phosphorus nutrition (Khanet al., 2009). Although soils generally contain a large amount of total P, only a small proportion isimmediately available for plant uptake. Phosphorus deficiency is the main problem to plant growthin tropical and subtropical areas (Bonser et al., 1996). Especially, in the Northeast Thailand the soil
are acidic with highly reactive of Fe3+ and Al3+ (Gyaneshwar et al., 2002), low phosphorus contents
in the parent material and low soil moisture affects the availability of phosphorus (Karmakar et al.,
1997; Raychaudhury et al., 2003). To meet the crop demand, farmers apply up to 3-4 times therequired amount of P to crops, causing a substantial increase in production costs. There are strong
evidences showed soil bacteria are capable of transforming soil P to the forms available to plant.
Recent studies reported that plants and microorganisms play a key role in soil P dynamics
which catalyze the hydrolysis of organic phosphate esters to orthophosphate anions by using
phosphatases. Free-living bacteria and fungi can mobilize orthophosphate (predominantly as
HPO42- and H2PO4-) from either organic or inorganic P sources for improving P availability.
Phosphate solubilizing bacteria (PSB) become a source of P to plants upon its release from theircells. The phosphatase efficiency is related to the microbial fauna, the soil temperature andhumidity as well as the associated bacteria communities (Zahran, 1999). Other factors that can playa significant role are the physiological state of the plant, the type of rooting system, the age of theplant and the location of ectomycorrhiza on the root (Antibus et al., 1997).
Vermicast, also called worm manure originates from the breakdown of organic matter by
earthworms. Its microbial activity is 10 to 20 times higher than in the soil (Chaoui, 2010).
Vermicast contains reduced levels of contaminants and a higher saturation of nutrients than organic
materials do before vermi composting (Ndegwa et al., 1998). Moreover, vermicast is also believed
to contain hormones and enzymes which it acquires during the passage of the organic matter
through the earthworm gut. The hormones and enzymes are believed to stimulate plant growth anddiscourage plant pathogens (Gajalakshmi and Abbasi, 2004). The analysis of the ability of bacterial
isolates to solubilize insoluble P form to available P form will enable to create a highly valuablebasis for the use of these isolates as bio-inoculums or bio-fertilizer to increase crops yield and forsustaining crop production. The objectives of this research were to isolate phosphate-solubilizing
bacteria (PSB) from cassava, groundnut, rubber tree, sunchoke, rice, rice-soybean, rice-soybeancorn and rice-chili fields and vermicast of earthworm varieties in the Northeast of Thailand wheresoils are mainly sandy and P-deprive.
MATERIALS AND METHODS
Sampling Site and Soil Sample Collection
The experiment was conducted within Khon Kaen, Mahasarakham and Roi Et provinces, Northeast
Thailand. Northeast Thailand is characterized by a tropical climate with an acid sandy soil. Soilsamples (20 points) were collected from cassava, ground nut, rubber tree, sunchoke, rice, ricesoybean, rice-soybean-corn and rice-chili fields while those plants were growing from the soil
surface at a depth of 10 cm. Collected soil samples were preserved in plastic bag at 4 C forphosphate solubilizing bacteria isolation.
Isolation of Phosphate Solubilizing Bacteria by Enrichment CultureⒸ ISERD

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To extract PSB from soil, 5 g of soil samples and vermicast of earthworm varieties were transferredto the NBRIP growth medium. Per liter, this growth liquid medium contains 10 g glucose with 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 or Ca3(PO4)2 as the sole source of insoluble P, were also
used for the initial screening step. The pH of the agar medium was adjusted to 7.0. The sources of
insoluble P were 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 incubator shaker at medium speed (150 cycles min −1). For thefollowing 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 incubator shaker at
medium speed (150 cycles min−1). At the end of each week in NBRIP growth liquid media, aliquotsof each dilution were spread on NBRIP medium and incubated at 30 °C for 14 days. After PSB
isolation for 6 weeks, colonies were selected from the plates on the basis of the appearance of aclear halo; the clones were further purified on minimal medium based on each insoluble phosphateforms. Once purified, each isolate was stored as a glycerol stock at -20 °C.
Mineral Phosphate Solubilization Assays
The phosphate solubilizing (PS) activity of each of the isolates was determined by molybdenumblue method (Land Development Department, 2005). The isolates were grown in NBRIP liquidmedium containing different insoluble forms of phosphate (AlPO4, Ca3(PO4)2 and FePO4) for 3-7
days at 30 °C on 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. Finally,
the absorption of light in the wavelength range 880 nm was measured by Shimadzu UV-120-01
Spectrophotometer.
Indole Acetic Acid Production
Selected PSB strains based on their ability to solubilize P were analyzed for IAA production(Nuntagij, 1997). The selective bacterial strains were grown in 45 ml of Lysogeny Broth medium(LB) contains 10 g/l Tryptone with 5 g/l NaCl and 5 g/l yeast extract at 30 °C for 2 days. The 5 l
PSB solution were determined by reaction with 1 ml of Tris-TMRT reagent contains 10 g/l Dmanital, 0.2 g/l yeast extract, 0.2/l CaCl2. 2H2O, 0.2 g/l MgSO4.7H2O, 1.21 g/l Tris-base and 0.061
g/l L-Tryptophane 28 °C for 10 days. The 2 ml mixture was determined by 0.01 M FeCl 3 in 35%
HClO4 for 30 min at 25 °C in the dark. The positive isolated were showed red color for indole
acetic acid production.
Statistical Analysis
An analysis of variance was done on data obtained from each parameter in each treatment. Allanalyses were carried out using Statistical analysis version 8.0. Least significant differences(L.S.D.) were calculated at p < 0.05 and Duncan’s multiple-range test was used to test significant
differences between treatments. Standard deviation was also calculated for the variance.
RESULTS AND DISCUSSION
Isolation of PSB from the Rhizosphere and Vermicast of Earthworm Varieties
The screening strategy employed during this research enabled the identification of PSB colonies on
NBRIP 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Ⓒ ISERD112

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with AlPO4, Ca3(PO4)2 and FePO4 for rubber and cassava from Roi Et, rubber tree, cassava and ricefrom Mahasarakham, sonchoke, rice-soybean-corn and rice-chili from Khon Kaen provinces andfrom vermicast of P. peguana and E. foetida (not shown). Five bacterial isolates from cassava andrice-soybean field at Khon Kaen province, ground nut field from Mahasarakham province and fromvermicast of earthworm varieties showed clear halos of AlPO4 and FePO4 solubilization (Table 1
and Fig. 1). Some obvious differences in the size of the halos of different isolates were observed(not shown). This preliminary observation suggests the existence of bacterial isolates exhibiting
different degrees of PS efficiencies in the soil samples and vermicast of earthworm varieties
collected. This preliminary observation suggested the existence of bacterial isolates exhibiting
different degrees of PS efficiencies in the soil samples collected (not shown). Yahya and Azawi(1998) reported that in general high abundances of PSB are found in agricultural and rangelandsoils. Furthermore, Kim et al. (1989) showed that the cultural activities and different soil properties
such as soil physical and chemical properties, organic matter and soil phosphorus content all playroles in determining the abundance of PSB in soils.
Table 1 Phosphate solubilizing effectiveness of tested bacteria, 10 days after inoculation
IsolateCassava (Khon Kaen)
Rice-Soybean (Khon Kaen)
Groundnut (Mahasarakham)
Eudrilus eugeniae
Pheretima posthuma

Solubilized phosphate (mgP/l) from
FePO4
AlPO4
959.53b1,860.00 a
682.13b1,789.71 a
741.17b1,605.09 a
1,582.46a
1,834.96 a
1,405.00a
1,918.49 a

Ca3(PO4)2
0
0
0
0
0

Efficiency of Phosphate Solubilization by NBRIP
To confirm this observation, the 5 purified isolates were tested following the protocol of Land
Development Department (2005), a method previously shown to be a reliable and qualitative
indicator of the PS activity of different bacterial isolates. Table 1 shows the OD880 nm shift of theculture supernatants of each of the 5 PSB isolates after a 3-7 day cultivation period in NBRIP
medium. Indeed, some isolates did not show any significant change in the absorbance of thesupernatant while others exhibited OD880 nm changes in the absorbance. After evaluating their P
solubilization capacity, the results showed that all of the 5 isolates can solubilize AlPO4 and FePO4
better than Ca3(PO4)2 especially the PSB isolates from the vermicast of earthworm varieties (Table1). Moreover, PSB isolate from the cassava and rice-soybean at Khon Kaen province andgroundnut at Mahasarakham province, look interesting for solubilizing FePO4 and AlPO4, which
are the main forms of insoluble phosphates in acid sandy soils.

Fig. 1 Appearance of a clear halo from PSB isolates
Indole Acetic Acid Production of PSB Isolates
The plant growth promoting effect of selected PSB was evaluated by analyzing IAA production(Table 2). The IAA production was observed from the isolates of rice-Soybean field and vermicastⒸ ISERD

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of E. eugeniae (Fig. 2), indicating that these strains could utilize l-tryptophan as a precursor for
growth. Phosphate solubilizing bacteria isolated from rice-Soybean field and vermicast of E.
eugeniae showed the highest IAA production (111.30-111.34 mg/L). Generally, bacteria canenhance plant growth directly or indirectly by increasing available P, fixing nitrogen, sequestering
iron by siderophores, producing antibiotics and plant hormones (Glick et al., 1998 and Mantelin
and Touraine, 2004). This group of bacteria is known as plant growth promoting bacteria (PGPB).
Moreover, PSB have been widely used as inoculants to increase P uptake and crop yield (Chen etal., 2008). IAA production contributes to phytostabilization by increasing root and shoots biomass(Liphadzi et al., 2006). In the context of sustainable agriculture with bio-fertilizers (phosphatesolubilizing bacteria) to supply phosphorus at a favorable level for enhance plant growth byproviding nutrients in a readily absorbable form. The application of inoculants provided from thesemicroorganisms seem to increase an abundant population of active and effective microorganisms to
the root activity zone which increases plant ability to uptake more nutrients.
Table 2 Plant growth promoting properties of isolates
IsolateCassava (Khon Kaen)
Rice-Soybean (Khon Kaen)
Groundnut (Mahasarakham)
Eudrilus eugeniae
Pheretima posthuma

Phosphate solubilization
+
+
+
+
+

IAA production++-

Fig. 2 IAA production from PSB isolates
CONCLUSION
The native PSB isolated from cassava, rice-soybean and groundnut field soil, vermicast of P.
posthuma and E. eugeniae seems to have the capacity to solubilize insoluble forms of AlPO4 andFePO4 which are the main forms of insoluble phosphates in acid sandy soils. The PSB isolates fromvermicast of earthworm varieties P. posthuma and E. eugeniae show the highest P solubilization
capacity in vitro conditions follow by the PSB isolated from cassava and rice-soybean field soil
from Khon Kaen province and groundnut field soil from Mahasarakham province. However, the
PSB isolates from vermicast of earthworm variety E. eugeniae and rice-soybean field soil from
Khon Kaen province look interested for IAA production. This work provides a first step towards byusing bio-fertilizers (phosphate solubilizing bacteria) for improving plant nutrient uptake, soil
fertility and sustainable crop production in nutrient poor systems.

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ACKNOWLEDGEMENTS
This research was supported by Khon Kaen University under Incubation Researcher Project andKnowledge Development of Rubber Tree in Northeast Research Group. The Research Institute of
Molecular Genetics and the North East Agriculture Research and Development Center at Faculty ofAgriculture Khon Kaen University (Thailand) and the partnership Land Use changes and Soil
Ecosystem Services (LUSES) are also thanked.
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