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Characterization of potential phosphate solubilizing bacteria from the local paddy fields in Perlis.

INTRODUCTION

Phosphorus solubilizing bacteria (PSB) play an important role in phosphorus (P) nutrition by enhancing plant P availability. These PSB have been shown to improve growth and yield of rice [1]. Phosphorus is taken up by plant mostly as an anion of phosphate. However large proportion of phosphorus fertilizer applied to the soil is quickly converted into the insoluble forms by forming complexes with Al or Fe in acid soils or Ca in acidic soil [2] and become unavailable to the roots. It is established that PSB have the capability to transform the insoluble phosphorus to soluble forms by secreting organic acids from their metabolitic activities [3]. Several soil bacteria, particularly those belonging to the genera Pseudomonas and Bacillus were reported to be involved in solubilizing phosphorus by secreting organic acids [4]. These acids will lower the pH and bring about the dissolution of bound forms of phosphate.

Isolation of potent PSB from the local paddy ecosystem would enhance bio-fertilizer efficacywhich can complement the application of commercial in organic fertilizer. It is known that continuous application of chemical fertilizers can affect soil chemical and physical characteristic as well as crops yield. Hence, biofertilizer has been accepted as a complement to chemical fertilizer to increase soil fertility and crop production such as rice. The aim of this research is to isolate, identify and characterize local PSB strains from rhizosphere of rice plant grown in soils of Perlis paddy fields which subsequently be used for bio fertilizer formulation.

MATERIALS AND METHODS

Soils Sampling:

Soil samples were collected from wetland paddy fields, namely Simpang Empat and Beseri, Perlis. Simpang Empat is located in MADA irrigation project area while Beseri is located outside MADA area. The samples were taken based on two soil types (Marine Alluvium and Riverine Alluvium) and high yield profiles.

Isolation of PSB from Rhizospheric Soil:

PSB were isolated from each sample by serial dilution and spread plate method. 10 g of soil sample was placed in an Erlenmeyer flask (250 ml) that containing 90 ml of autoclaved 0.9% saline solution and was thoroughly shaken. 1 ml of the above solution was again transferred to 9 ml of sterile saline solution to form 10-2 dilution. Similarly, serial dilution was made until 10-8 for each soil sample. 0.1 ml of each dilution was spread on National Botanical Research Institute Phosphate (NBRIP) agar growth medium [5] containing: (Glucose 5 g, Mg[Cl.sub.2].6[H.sub.2]O 5 g, MgS[O.sub.4].7[H.sub.2]O 0.25 g, KCl 0.1 g, [(N[H.sub.4]).sub.2]S[O.sub.4] 0.1 g, agar 15 g, dissolved in 1 L of distilled water supplemented by 5 g of Tricalcium Phosphate (TCP) as sole of phosphorus source that have the ability to releases soluble inorganic phosphate from TCP. The pH was adjusted to 7.0 and incubated at 30[degrees]C for 2 days.

Morphological Characterization and Gram Staining:

Morphological characterization of isolates including; shape, margin, elevation, size, texture, appearance, pigmentation (color), and optical property were observed for their characterization. The isolates were gram stained and examined for cellular morphology and arrangement according to the standard procedure [6]. The red colonies will show the gram negative bacteria and the purple colonies will show the gram positive bacteria.

Screening of the Isolates for Phosphate Solubilization:

Each bacterial isolate were aseptically streaked onto NBRIP agar that supplemented with TCP and incubated at 30[degrees]C for 7 days. The solubilization of phosphate was observed as a zone of clearance with a diameter that was measured in millimeters. The phosphate solubilizing ability of the isolates was analyzed by determining the phosphate solubilization efficiency [7]. The formula was expressed as below.

SE = Total diameter of colony + clear zone (D)

Colony diameter (d)

Quantification of Phosphate Solubilization:

The isolates that showing large clear zone in solid media were further examined for their ability to released soluble phosphate in NBRIP broth media. B. megaterium was used as a controlled. The PSB cultures were grown in NBRIP broth for eight days with continuous shaking 150 rpm at 30[degrees]C. A 10 ml of each culture was taken in centrifuge tube and centrifuge for 15 minutes at 10,000 rpm. Supernatant was decanted and 5 ml of supernatant was added to 20 ml of AB-DTPA extracting solution. P in solution was extracted with Ammonium bicarbonate diethylene -triamine penta acetic acid (AB-DTPA) [8]. Broth P was determined by Molybdenum blue method [9]. 1 ml of broth sample extract was taken in 100 ml of conical flask and 9 ml of distilled water + 2.5 ml of freshly prepared color reagent. The optical density of the blue color developed after 15 minutes was measured at 880 nm by UV-VIS spectrophotometer and the concentration of available P (ppm) was measured. Initial pH and pH changes were measured at last day of incubation by digital pH meter

RESULTS AND DISCUSSIONS

Isolation of PSB from Rhizospheric Soil and Gram Staining:

About 16 isolates were found to be able to solubilize phosphate. This isolates had the morphological characteristics like circular and irregular shape of colonies. These colonies mostly appeared in cream, yellow and colourless pigmentation. Upon gram staining, 4 isolates were identified as gram positive bacteria, while the 12 isolates shown as gram negative bacteria. Based on the results of the colouring gram, the gram negative rods bacteria is expected to be from Pseudomonas spp. and while the gram-positive rods bacteria is expected from Bacillus spp, due to the presence of endosphere. Soil bacteria are in cocci (sphere, 0.5[micro]m), bacilli (rod, 0.50.3[micro]m) and spiral (1-100[micro]m) shapes [10]. Bacilli are common in soil, whereas spirili are very rare in natural environment. The result was expressed in the Table 1.

Screening of the Isolates for Phosphate Solubilization:

The solubilization efficiency of 16 isolates was measured at the end of 7 days incubation. The result was expressed in the Table 1. Among these isolates, PL 13 from Padang Lati, Perlis showed the highest phosphate solubilization efficiency index (1.96), followed by the isolate PL 16 (1.87) whereas all the other isolates showed lower efficiency which range from 1.16 to 1.69. This result proved that PL 13 was very efficient isolate in solubilizing TCP. In screening process, the PL 13 was showed the highest clearing zone because it released low molecular weight acid and it produced high amount of soluble P as compared to other isolates. However, all the selected isolates were able to solubilize TCP in NBRIP agar media. Generally, halo zone increased with increased in colony diameter [7]. The screened isolates were able to solubilize TCP on solid culture state by forming clear zone also depending on the type of organism involved.

Quantification of Phosphate Solubilization:

Test for available P in NBRIP broth were also conducted to quantify the phosphate solubilizing activity of the isolates. The ability of PSB to measured inorganic P and pH of the broth media was also measured up to 8 days incubation. The amount of soluble P released in the NBRIP broth by each of the 16 isolates was quantitatively measured by Molybdenum Blue method describes by Watanabe and Olsen in 1965. In quantitative estimation, range of soluble P released between 7.23 mg/L to 87.36 mg/L which is shown in Figure 1. SE9 (25.35 mg/L) is not significantly different to PL12 (22.73 mg/L) but significantly different to B. megaterium and SE1 (19.16 mg/L). B. megaterium used as control was solubilized TCP only 18.18 mg/L. Result showed the maximum soluble P produced by PL 13 (87.36 mg/L), PL 16 (78.52 mg/L), PL 11 (44.26 mg/L), SE 6 (39.89 mg/L) and SE 10 (36.55 mg/L) after 8 days incubation. 8 isolates were better than commercially B. megaterium. P solubilization is a complex phenomenon, which depend on many factors such as nutritional like carbon and nitrogen source for their metabolic activity, physiological and growth condition of the culture like temperature and time of incubation [11]. Measurement of the pH was measured at the end of the incubation day. After 8 days, pH was decreased 5.84 to 4.18. 5 bacteria isolates which were SE 3, SE 6, SE 10, PL 12 and PL 16 have significantly lower than B. megaterium. It was revealed that pH was gradually decreased with the increased incubation time. Generally, bacteria secreting low molecular weight of organic acid from their metabolic process [12] and may due to acidic condition in NBRIP broth. Thus, in present study PL 11, PL 13 and PL 16 were found to be more efficient bacterial isolates to solubilize TCP.

Each value is expresses as mean (n = 3)

Bars graph not connected by same letters are significantly different (P < 0.05)

Conclusion:

This result showed that diverse types PSB exist in rice ecosystem in MADA area and this PSB isolates exhibited wide range of phosphate solubilizing capabilities and few of them were better than commercially available B. megaterium. It can be conclude that, all selected PSB has the capacity to solubilized Tricalcium phosphate (TCP) that suplemented in NBRIP medium. P released by the PSB was associated with reduction in pH of the medium. PL 11, PL 13 and PL 16 were found more efficient bacterial isolates to solubilize TCP. PL 13 is the most efficient strains on the basis on their phosphate solubilizing activity. Based on morphological characteristics, PL 13 was confirmed as a bacteria because it have coccus shaped and gram negative characteristic. These PSB isolate have the potential for development more effective bio-fertilizer.

ARTICLE INFO

Article history:

Received 25 September 2014

Received in revised form 26 October 2014

Accepted 25 November 2014

Available online 31 December 2014

ACKNOWLEDGEMENT

The authors wish to express their gratitude to the Ministry of Higher Education (MOHE) for the financial support given under LRGS - Food Security project (9012-00002), MyBrain15 (MyMaster) and School of Bioprocess Engineering for the realization of this work.

REFERENCES

[1] Hira, G.S. and K.L. Khera, 2000. Water Resource Management in Punjab under Rice -Wheat Production System. Research Bulletin, Department of Soils, Punjab Agricultural University, Ludhiana.

[2] Lindsay, W.L., P.L.G. Vlek and S.H. Chien, 1989. Phosphate minerals. In Minerals in soil environment, 2nd edn, in: J. B. Dixon and S. B. Weed (Eds.), Soil Science Society of America, Madison, WI, USA., pp: 1089-1130.

[3] Chang, C.H. and S.S. Yang, 2009. Thermo-tolerant phosphate-solubilizing microbes for multi-functional biofertilizer preparation. Bioresource Technology, 100: 1648-1658.

[4] Afzal, A., M. Ashraf, S.A. Asad and M. Farooq, 2005. Effect of phosphate solubilizing microorganisms on phosphorus uptake, yield and yield traits of wheat (Triticum aestivum L.) in rainfed area. International Journal Agricultural Biology, 7: 207-209.

[5] Geonadi, D.H., Y. Siswanto and Y. Sugiarto, 2000. Journal of Soil Science Society of America, 5(4): 384350.

[6] Blazevic, D.J. and M.E. Grace, 1975. Principle of Biochemical Test in Diagnostic Microbiology (Techniques in Pure and Applied Microbiology). New York: John Wiley & Sons Inc.

[7] Premono, E.M., A.M. Moawad and P.L.G. Vleck, 1996. Effect of phosphate solubilizing Pseudomonas putida on the growth of maize and its survival in the rhizohsphre. Journal of Indonesia Crop Science, 11: 13-23.

[8] Soltanpour, P.N. and S.M. Workman, 1979. Soil testing methods used at Colorado State University soil testing laboratory for the evaluation of fertility, salininty, and trace element tociticy. Colorado State University Experiment Statikn. Food Colin, Colorado Technological Bulletin, pp: 14-22.

[9] Watanabe, F.S. and S.R. Olsen, 1965. Test of an ascorbic acid method for determining phosphorus in water and NaHC[O.sub.3] extract from soil. Soil Science Society American Proceeding, pp: 273-700.

[10] Shornam, S., K. Vijay and B.T. Ram, 2011. Isolation of Phosphate Solubilizing Microorganism (PSMs) From Soil. Journal of Microbiology and Biotechnology Research, 1(2): 90-95.

[11] Reyes, I., L. Bernier, R.R. Simard and H. Antoun, 1999. Effect of nitrogen source on the solubilization of different inorganic phosphates by an isolate of Penicillium rugulosum and two UV-induced mutants. FEMS Microbiology Ecology, 28(3): 281-290.

[12] Park, J., N. Bolen, M. Megharaj and R. Naidu, 2010. Isolation of phosphate solubilizing bacteria and characterization of their effects on lead immobilization. J. Hazard. Mater, 185(2-3): 829-836.

(1) Emey Suziana Ahmad Mukri, (2) Abdul Razak Shaari and (3) Khadijah Hanim Abdul Rahman

(1) School of Bioprocess Engineering Universiti Malaysia Perlis (UniMAP), Kompleks Pusat Pengajian Jejawi 3, 02600 Arau, Perlis, Malaysia

(2,3) Faculty of Engineering Technology, Universiti Malaysia Perlis, Pauh Putra Main Campus, 02600 Arau, Perlis, Malaysia

Corresponding Author: Emey Suziana Ahmad Mukri, School of Bioprocess Engineering Universiti Malaysia Perlis (UniMAP), Kompleks Pusat Pengajian Jejawi 3, 02600 Arau, Perlis, Malaysia.

E-mail: emeyrina_86@yahoo.com

Table 1: Gram staining and solubilization index of potential phosphate
solubilizing bacteria in National Botanical Research Institute's
phosphate (NBRIP) growth medium.

Isolates        Gram staining          Cell         Colony
                                    morphology   diameter (d)
                                                      mm

SE 1         Red, Gram negative        Rods          17.0
SE 2         Red, Gram negative        Rods          7.0
SE 3         Red, Gram negative       Coccus         13.0
SE 4        Purple, Gram positive      Rods          7.0
SE 5         Red, Gram negative        Rods          8.0
SE 6         Red, Gram negative        Rods          20.0
SE 7         Red, Gram negative        Rods          7.5
SE 8         Red, Gram negative        Rods          17.0
SE 9         Red, Gram negative        Rods          20.0
SE 10        Red, Gram negative       Coccus         21.0
PL 11        Red, Gram negative        Rods          6.5
PL 12       Purple, Gram positive      Rods          19.0
PL 13        Red, Gram negative       Coccus         15.0
PL 14       Purple, Gram positive      Rods          20.0
PL 15       Purple, Gram positive      Rods          25.0
PL 16        Red, Gram negative       Coccus         16.0

Isolates     Total diameter of    Solubilization Index/
            colony + clear zone        efficiency
                    (D)                    D/d
                    mm

SE 1               25.0                   1.47
SE 2               10.0                   1.42
SE 3               19.0                   1.46
SE 4               10.0                   1.42
SE 5               11.0                   1.37
SE 6               31.0                   1.55
SE 7               11.0                   1.46
SE 8               22.0                   1.29
SE 9               30.0                   1.50
SE 10              32.0                   1.52
PL 11              11.0                   1.69
PL 12              28.0                   1.47
PL 13              29.5                   1.96
PL 14              25.0                   1.25
PL 15              29.0                   1.16
PL 16              30.5                   1.87

The experiment was done in triplicate

* SE = Simpang Empat Phosphate Solubilizing Bacteria

* PL = Padang Lati Phosphate Solubilizing Bacteria
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Title Annotation:Malaysia
Author:Mukri, Emey Suziana Ahmad; Shaari, Abdul Razak; Rahman, Khadijah Hanim Abdul
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:9MALA
Date:Nov 1, 2014
Words:2433
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