Characterization of Phosphate solubilizing Microorganism isolated from soil

 

Pervez Ahmed Khan1* , Dr. Amia Ekka2

1School of Studies in Life sciences, Pt. Ravishankar Shukla University, Raipur – 492010, Chhattisgarh, India

 2School of Studies in Life sciences, Pt. Ravishankar Shukla University, Raipur – 492010, Chhattisgarh, India

*Corresponding Author E-mail: vikkykhan@rediffmail.com, amia_tirkey@rediffmail.com

  

ABSTRACT:

Phosphorus (P) is one of the most essential macro-elements required for growth and development of plants (including photosynthesis, energy and sugar production) .  A greater part of P is present in the insoluble form and therefore, cannot be taken up by plants. The deficiency of P in turn severely restricts growth and yields in plants.   Aspergillus species, a soil isolate had excellent potential to solubilize phosphate in vitro. In present study fungal strains isolated from soil having potential to solubilize phosphate were characterized. Fungal isolates were characterized as Aspergillus Sp. and Penicillium Sp. Both the isolates were identified by cultural and microscopic examination and are capable of phosphate solubilization.

 

KEYWORDS: Aspergillus, Biofertilizer, Penicillium, Phosphate solubilizing fungi and Solubilization .

 

 


1. INTRODUCTION:

Phosphorus (P) is an essential element for plant growth and development. Plants utilize little amounts of applied P fertilizers and the rest is rapidly converted into insoluble complexes in the soil .(1)Phosphorus is a plant macronutrient that plays a significant role in plant metabolism, ultimately reflected on crop yields. It is important for the functioning of key enzymes that regulate the metabolic pathways.(2)This leads to the need of frequent application of P fertilizers, but its use on a regular basis is expensive and environmentally undesirable. Natural phosphate rocks have been recognized as a valuable alternative for P fertilizers.(3)

Rhizospheric phosphate solubilizing bacteria and fungi are capable of solubilizing insoluble or inorganic phosphates into soluble organic forms. Such Phosphate solubilizing Microbes (PSMs) are known to be abundant in the rhizospheric soils of various plants. They can be divided into 2 groups: phosphate solubilizing bacteria (PSB) and phosphate solubilizing fungi (PSF)(4)

 

Phosphate solubilizing microorganisms (PSMs) play an important role in supplementing phosphorus to the plants, allowing a sustainable use of phosphate fertilizers. Microorganisms are involved in a range of process that effect the transformation of soil phosphorus (P) and thus are integral component of the soil ‘P’ cycle. Many bacterial, fungal, yeast, and actinomycetes species capable of solubilizing sparingly soluble phosphorus in pure culture have been isolated and studied.(5)

 

Fungi are the important components of soil microbes typically constituting more of the soil biomass than bacteria, depending on soil depth and nutrient conditions. Fungi have been reported to have greater ability to solubilize insoluble phosphate than bacteria.(6) Species of Aspergillus, Penicillium and yeast have been widely reported solubilizing various forms of inorganic phosphates.(7)

 

Our aim for the present study was therefore to isolate and characterize fungi for greater phosphate solubilization efficiency

 

2. MATERIAL AND METHODS:

The present investigation was carried out in the SoS Life Sciences, Pt. Ravishankar Shukla University, Raipur. Chhattisgarh.

A-Study Site

Raipur is situated in East Central part of Chhattisgarh at latitude of 21016 N, longitude 81036 E and altitude 289.5 m above mean sea level.

 

B-Collection of soil samples

Soil samples were collected from rhizosphere of Chickpea plantation from 3 different villages of Raipur city of Chhattisgarh state. Samples were collected in polythene bags, transported to laboratory and stored in refrigerator for further processing. Soil samples were separated from roots, air dried at room temperature, crushed, sieved and collected in separate polythene bags. pH of the samples was recorded using pH meter (Elico made).

 

C-Culture media for isolation

Pikovskaya’s (8) agar medium (HIMEDIA) was used for the isolation and maintenance of phosphate solubilizing fungi. It contained (g litre-1) Dextrose 10; Calcium phosphate 5; Ammonium sulphate 0.5; Potasium chloride 0.2; Magnesium sulphate 0.1; Manganease suplhate 0.0001; Yeast extract 0.5; Ferrous sulphate 0.0001, Agar 15. The pH of medium was 7.0 (± 0.2).

Potato dextrose agar (PDA, HIMEDIA) was used for the isolation maintenance of fungal cultures. It contained (g.litre-1) potato infusion 200; Dextrose 20; Agar 15 and the pH of medium was 5.6 (± 0.2) The pH of culture media was adjusted using 1N NaOH or 1N HCl. Media were sterilized by autoclaving at 121°C for 15 min.

 

D-Isolation of fungi from soil by serial dilution

a.     Suspend 1gm of soil in 9ml sterile distilled water in a test tube and make serial dilutions.

b.     Transfer aliquots of 1ml suspension from 10 or 10th dilution tube in to agar plates containing Pikovskaya’s medium  (Himedia, Mumbai) which is supplemented with phosphate.

c.     Incubate the plates at 26°C for 4 to 5 days.

 

E-Screening

The isolates were screened by inoculating on plates containing Pikovskaya’s Agar (PKA) medium (6) amended with 0.5% tricalcium phosphate (TCP) as insoluble phosphate source and were incubated at 28±2ºC for 5 days. Fungal colonies with clear halozone around them were screened as phosphate solubilizers.  

 

 

F- Identification

The fungal cultures were identified on the basis of colony characteristics and microscopic examination (9,10 and 11). Some of the fungal isolates have been sent and deposited to NFCCI for identification.

 

G-Quantitative measurement of phosphate solubilization in culture medium

a.     Colonies showing clear zone in above method are inoculated in the Pikovskaya’s broth in two different flasks and kept for incubation for 7 days.

b.     This results in the formation of fungal matt in the Pikovskaya’s broth.

c.     The fungal matt was separated and the broth was filtered.

d.     The two filtrates of, i.e.Aspergillus Sp.  and Penicillium Sp. colonies are taken as unknown sample 1and 2.

e.     This is further estimated quantitatively by Fiskey and subbarao’s method as shown in table 1 and specific   readings were taken at 660nm.

f.      By using same protocol the concentration of unknown sample i.e. solubilized phosphate is estimated. (TABLE 1)

 

3. RESULT AND  DISCUSSION:

Phosphatases play a key role in transforming organic forms of phosphorus into plant available inorganic forms and are found to be active in microorganisms, plants and roots. A significant correlation had been noticed between the depletion of organic P and phosphatase activity in the rhizosphere soil of wheat (12).

 

 

TABLE 1.

S. No

 

Standard Phosphate Solution(ml)

Distilled Water (ml)

Ammonium Molybdate (ml)

Ammonium Nepthal Sulphonic Acid (ml)

Concentration

µgm/ml

Optical density

Blank

o

1

0.5

0.5

0

0

St.1

0.2

0.8

0.5

0.5

2

0.20

St.2

0.4

0.6

0.5

0.5

4

0.39

St.3

0.6

0.4

0.5

0.5

6

0.58

St.4

0.8

0.2

0.5

0.5

8

0.82

St.5

1

0

0.5

0.5

1

10.2

Aspergillus

1

0

0.5

0.5

4.8  µgm

0.48

Penicillium

1

0

0.5

0.5

5.1 µgm

0.51

 


Fungi that showed halo zones around the colony are good phosphate solubilizers and belong mainly to the genera of Aspergillus, and Penicillium. Aspergillus niger was found to be the dominant group followed by Penicillium sp. and other species of Aspergillus. The similar results were highlighted by Mahamuni et al. (13) and Deepa et al. (14). The higher number of the species of the fungus can be attributed to its ability to grow in diverse conditions (15). Soil fungi make a very important part of the ecosystem along with other microbes in turnover of the biomass (16). It is known that the species of Aspergillus, Trichoderma, Penicillium, Fusarium, Mucor etc. are the dominantly occurring fungi isolated from different rhizospheric soils and screened for phosphate solubilization by many workers (17 and 18).

 

Microorganisms substantially influence the soil productivity by solubilizing this insoluble P through their metabolic processes in soil (19). The process of microbe mediated P solubilization is generally ascribed to the production of organic acids by them (20).

 

After confirmation of phosphate solubilizing fungi as Aspergillus  Sp. and Penecillium Sp. they were used for quantitative measurement of phosphate solubilization given by Fiskey and Subbarao’s. The results showed that the amount of phosphate solubilized by Aspergillus niger was 4.8μg/ml and Penecillium, 5.1 μg/ml. The result coincides with the findings of Vazquez et.al that Aspergillus niger solubilize insoluble phosphate well in liquid medium supplemented with tricalcium phosphate (18).

 

According to Noppart et.al findings, three isolates of A. tubingensis and two isolates of A. niger isolated from rhizospheric soils were tested on solubilization of different rock phosphates. All isolates of Aspergillus were capable of solubilizing all natural rock phosphates. A. tubingensis AT1 showed maximum percent  solubilization in all rock phosphates tested when compared to other isolates. (21)

 

Reddy et.al showed that isolates of Aspergillus tubingensis showed highest phosphate solubilization when grown in presence of 2% rock phosphate. (22) A number of different filamentous fungi have been reported as potential P solubilizers by many researchers and among these fungi, Aspergillus and Penicillium are predominant (23) Penicillium pupurogenum has been reported as phosphate solubilizer by earlier researchers (24). The appearance of clear halozone around the colony indicated phosphate solubilization by the fungus which is in accordance with the findings of Gupta et al. (2007).(25)

 

Fig. 1 Photographs of Clear Zone formation. 

  Fig. 2 Staining- Aspergillus sp.

 

 

4. CONCLUSION:

It is concluded  from this study that  rhizosphere contains various types of phosphate solubilizing fungi; Aspergillus  and Penicillium are found as dominant strains. Penicillium species showed maximum phosphate solubilization and it can be used as potential phosphate biofertilizer for the cultivation of  different crop plants. Further nursery and field trials are required to confirm its inoculation effects on different crop plants.

 

5. ACKNOWLEDGEMENT:

The author would like to thank Head, Department of Life Science, Pt. Ravishankar Shukla, University, Raipur, his constant support and laboratory facilities

 

6. REFERENCES:

1.          V. Narsian, H.H. Patel, “Aspergillus aculeatus as rock phosphate solubilizers”, Soil Biol. Biochem. 32, pp. 559-565,2000.

2.          Th eodorou ME, Plaxton WC. “Metabolic adaptations of plant respiration to nutritional phosphate deprivation”, Plant Physiol 101: 339-344, 1993.

3.          M.S. Reddy, S. Kumar, B. Khosla, “Biosolubilization of poorly soluble rock phosphates by Aspergillus tubingensis and Aspergillus niger”, Bioresour. Technol. 84: pp. 187-189, 2002.

4.          L. Khiari, L.E. Parent, “Phosphorus transformations in acid light textured soils treated with dry swine manure”. Can J Soil Sci 85:75-87, 2005.

5.          A.K. Halder, A.K. Mishra, and P.K. Chakarbarthy, “Solubilization of inorganic phosphate by Bradyrhizobium”, Ind. J. Exp. Biol. 29: 28-31, 1991.

6.          E. Nahas, “Factors determining rock phosphate solubilization by microorganisms isolated from soil” World J Microbiol Biotech 12: 567-572, 1996.

7.          M.A. Whitelaw, “Growth promotion of plants inoculated with phosphate solubilizing fungi”  Edited by Donald L. Sparks. Advances in Agronomy, Academic press 69 : 99-151, 2000.

8.          R.I. Pikovskaya, “Mobilization of phosphorus in soil in connection with vital activity of some microbial species”, Microbiologiya, 17: 362-370, 1948.

9.          M.B. Ellis, “Dematiceous hyphomycetes”, Commonwealth Mycological Institute, Kew, Surrey, England,1971.

10.        H.L.Barnett, and B.B. Hunter, “Illustrated Genera of Imperfect Fungi”, 4th ed., St. Paul Minnesota, APS   Press, 1998.

11.        J.C. Gilman, “A manual of soil fungi”. Biotech books, Delhi, India. 2008.

12.        J. Tarafdar, and  A. “Jungk Phosphatase activity in the rhizosphere and its relation to the   depletion of soil organic phosphorus”,  J. Biol. Fert. Soils 3: 199-204,1987.

13.        S.V.  Mahamuni, P.V. Wani, and A.S. Patil, “Isolation of phosphate solubilizing fungi from rhizosphere of sugarcane and sugar beet using TCP and RP solubilization” Asian Journal of Biochemical and Pharmaceutical Research, 2: 237-244, 2012.

14.        V. Deepa, A. Prasanna, B.P. Murthy, and R. Sridhar, “Efficient phosphate solubilization by fungal strains isolated from rice-rhizosphere soils for the phosphorus release”, Research Journal of Agriculture and Biological Sciences, 6: 487-492, 2010.

15.        R. Saikia,  K. Das,  S. Deka, and P. Azad, “Status and Prospects of Soil Microbial Diversity of Dibru- Saikhowa Biosphere Reserve”, Himalayan Biosphere Reserves 6(1-2):61-63, 2004.

16.        E.B.G. James, and K.D. Hyde, “Methods for the study of Mangrove Fungi, In: Mangrove Microbiology. Role of Microorganisms in Nutrient Cycling of Mangrove Soils and Waters”, Ed by A.D. Agate, C.V. Subramanian, H. Vannuccie. UNDP 9-27,1998.

17.        P. Illmer, and F. Schinner, “Solubilization of inorganic phosphates by microorganisms isolated from forest soils”, Soil Biol Biochem.24: 89-395, 1992.

18.        P. Vazquez, G. Holguin, M.E. Puente, A. Lopez Cortes, and Y. Bashan, “Phosphate solubilizing Microorganisms Associated with the Rhizosphere of Mangroves in a Semi-arid coastal Lagoon”, Biol. Fertil. Soils (30): 460-468, 2000.

19.        S. Ravikumar,  P. Williams, S. Shanthy, N. Anitha, A. Gracelin, S. Babu, and P.S. Parimala, “Effect of heavy metals (Hg and Zn) on the growth and phosphate solubilising activity in halophilic phosphobacteria isolated from Manakudi mangrove”, J. Environ. Biol., 28:109-114, 2007.

20.        S. Fleischer, M. Bengtsson, and G. Johansson, “Mechanism of aerobic FE (III) phosphate solubilization at the sediment water interface” Verh. Int. Verein. Limnol., 23:1825-1829, 1988.

21.        C. Nopparat,  M. Jatupornpipat, and A. Rittiboon, “Isolation of phosphate solubilizing fungi in soil from Kanchanaburi”, Thailand. KMITL Sci. Tech. J. Vol. 7 No. S2 Nov. 2007.

22.        S. M., Reddy, S. Kumar,  K. Babita, and M.S. Reddy, “Bio solubilization of Poorly Soluble Rock Phosphates by Aspergillus tubingensis and Aspergillus niger”, Bioresource Technology, 84:187 – 189, 2002.

23.        S.V. Mahamuni, P.V. Wani, and A.S. Patil,  “Isolation of phosphate solubilizing fungi from rhizosphere of sugarcane and sugar TCP and RP solubilization”,  Asian J. Biochem. Pharm. Res. 1:237-244, 2012.

24.        J.M. Scervino, M.P. Mesa, I.D. Monica, M. Recchi, N.S. Moreno, A. Godeas, “Soil fungi isolates produced different organic acid patterns involved in phosphate salts solubilization”,  Biol. Fertil. Soils 46:755- 763, 2010.

25.        N. Gupta, J. Sabat, R Parida, D, Kerkatta, “Solubilization of tricalcium phosphate and rock phosphate by microbes isolated from chromite, iron and manganese mines”. Acta Bot. Croat. 66:197-204, 2007.

 



 

 

 

Received on 30.01.2019           Modified on 25.02.2019

Accepted on 02.03.2019         © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(3): 1353-1356.

DOI: 10.5958/0974-360X.2019.00227.0