Isolation and indentification of phosphate solubilizing fungi from rhizosphere (soil)

Ashok V Gomashe et al., IJSID, 2012, 2 (2), 310-315

International Journal of Science Innovations and Discoveries, Volume 2, Issue 2, March-April 2012

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ISOLATION AND IDENTIFICATION OF PHOSPHATE SOLUBILIZING FUNGI FROM RHIZOSPHERE (SOIL)

Ashok V. Gomashe*, Sabina S. Suriya, and Preeti G. DharmikShivaji Science College, Congress Nagar, Nagpur-440012, M.S. India

INTRODUCTION

INTRODUCTION

ISSN:2249-5347IJSID

International Journal of Science Innovations and Discoveries An International peerReview Journal for Science

Research Article Available online through www.ijsidonline.info

Received: 16.02.2012

Accepted: 30.04.2012

*Corresponding Author

Address:

Name:Dr. Ashok V. GomashePlace:Shivaji Science CollegeCongress Nagar, Nagpur,Maharastra, IndiaE-mail:[email protected]

ABSTRACT

Most agricultural soil contains large reserves of phosphorous (P), aconsiderable part of which accumulates as consequences of regular application ofphosphate fertilizers. However a greater part of soil phosphorous, approximately95-99% is present in the form of insoluble phosphates and hence cannot beutilized by plants. Aspergillus species, a soil isolate had excellent potential tosolubilize phosphate in vitro. In present study fungal strains isolated from soilhaving potential to solubilize phosphate were characterized. Two fungal isolateswere characterized as Aspergillus niger and Aspergillus flavus. Both the isolateswere identified by cultural and microscopic examination and are capable ofphosphate solubilization.Key Words: Aspergillus, Phosphate, rhizosphere, fungal strain.



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INTRODUCTIONImproving soil fertility is one of the most common practices in agricultural production. Phosphorus (P) is one of themost essential plant nutrients for maximizing crop productivity. This nutrient is limited in soils, which remain as a majorchallenge to agriculturists and land managers. (1). Phosphorus is one of the major nutrients, second only to nitrogen inrequirement for plants. A greater part of soil phosphorus, approximately 95–99% is present in the form of insolublephosphates and cannot be utilized by the plants (2). Compared with the other major nutrients, phosphorus is by far the leastmobile and available to plants in most soil conditions. Although phosphorus is abundant in soils in both organic and inorganicforms, it is frequently a major or even the prime limiting factor for plant growth (3).Phosphorus is a plant macronutrient that plays a significant role in plant metabolism, ultimately reflected on cropyields. It is important for the functioning of key enzymes that regulate the metabolic pathways (4). To increase the availabilityof phosphorus for plants, large amounts of fertilizer are being applied to soil. But a large proportion of fertilizer phosphorusafter application is quickly transformed to the insoluble form (5). Therefore, very little percentage of the applied phosphorusis available to plants, making continuous application necessary (6).Rhizospheric phosphate solubilizing bacteria and fungi are capable of solubilizing insoluble or inorganic phosphatesinto soluble organic forms. Such Phosphate solubilizing Microbes (PSMs) are known to be abundant in the rhizospheric soils ofvarious plants. They can be divided into 2 groups: phosphate solubilizing bacteria (PSB) and phosphate solubilizing fungi(PSF) (7).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 thetransformation of soil phosphorus (P) and thus are integral component of the soil ‘P’ cycle. Many bacterial, fungal, yeast, andactinomycetes species capable of solubilizing sparingly soluble phosphorus in pure culture have been isolated and studied (8).Fungi have been reported to possess greater ability to solubilize rock-phosphate than bacteria (9).Species of Aspergillus,

Penicillium and yeast have been widely reported solubilizing various forms of inorganic phosphates (10).Owing to above facts, the current study deals with the isolation and identification of fungal strains having potential tosolubilize phosphates.MATERIAL AND METHODS

Isolation of fungi from soil by serial dilution1. Suspend 1gm of soil in 9ml sterile distilled water in a test tube and make serial dilutions.2. 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.3. Incubate the plates at 25°C for 4 to 5 days.Identification by staining techniqueIdentification of fungi was done on the basis of morphological characteristics by cotton blue staining.Confirmatory testFor further confirmation the colonies were inoculated in Sabouraud’s dextrose medium.Quantitative measurement of phosphate solubilization in culture medium1. Colonies showing clear zone in above method are inoculated in the Pikovskaya’s broth in two different flasks and kept forincubation for 7 days.



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2. This results in the formation of fungal matt in the Pikovskaya’s broth.3. The fungal matt was separated and the broth was filtered.4. The two filtrates of, i.e. Black and Lime green color colonies are taken as unknown sample 1and 2.5. This is further estimated quantitatively by Fiskey and subbarao’s method as shown in table 1 & specific readings weretaken at 660nm.6. By using same protocol the concentration of unknown sample i.e. solubilized phosphate is estimatedTable 1: Protocol for phosphate solubilization in culture medium

Sr.no Reagent Blank I II III IV V Unknown1 Working Phosphate solution - 0.2 0.4 0.6 0.8 1.0 -2 Unknown - - - - - - 1ml3 Distilled water 1 0.8 0.6 0.4 0.2 - -4 Ammonium Molybdate 0.5 0.5 0.5 0.5 0.5 0.5 0.55 ANSA 0.5 0.5 0.5 0.5 0.5 0.5 0.5RESULT AND DISCUSSIONThe phosphorous is essential for the general health and vigor off all plants. In agricultural system, low phosphorousavailability has been through the application of concentrated phosphorous fertilizers, but the efficiency of this process isaffected by chemical immobilization of phosphorous in soil, depletion of non-renewable sources of phosphorous ore and costof fertilizer processing. A greater part of soil phosphorous approximately 95-99% is present in the form of insolublephosphate and cannot be utilized by plants (3).Conditions like irregular rainfall, long dry and hot summers and man-mediated degradative activities (overgrazing,non regulated cultivation techniques, deforestation, etc) results in the disturbances of soil ecosystems (11). Such degradedecosystems are usually characterized by a disturbed vegetation cover accompanied by a rapid erosion of surface soil (12). Thedesertification process involves a loss or reduction of major physicochemical and biological soil properties (13), including itsmineral content. In fact, despite being abundant in soil, both in inorganic and organic forms, phosphorus (P) is the major plantgrowth–limiting nutrient after nitrogen (N). Plant mineral nutrition depends mainly on the phosphorus content of soil, whichcan be assimilated only as soluble phosphate. Physical and chemical weathering of mineral phosphates is mainly realizedalong plant roots in the rhizosphere. Phosphorus in soils is present in insoluble form complexes with cations like iron,aluminum and calcium. Although, use of chemical fertilizers for improving soil fertility is the common approach of increasingagricultural production, a large portion of inorganic phosphates applied to soil as fertilizer is rapidly immobilized afterapplication and becomes unavailable to plants (14).Microorganisms substantially influence the soil productivity by solubilizing this insoluble P through their metabolicprocesses in soil (15). The process of microbe mediated P solubilization is generally ascribed to the production of organicacids by them (16). Still there are reasonable doubts on the exclusive role of organic acids in solubilization process (17).The present study aimed to isolate and identify the phosphate solubilizing fungi from rhizosphere. For isolation ofphosphate solubilizing fungi, soil was serially diluted and plated on Pikovskaya’s medium and incubated at 25°C for 4-5days.The colonies on the plate were identified by cotton blue staining technique and later on confirmed as Aspergillus flavus and

Aspergillus niger.The colonies showing clear transparent zone which indicate the phosphate in the medium has been solubilized wereselected and further identified by lactophenol cotton blue staining technique and characterized by inoculating them on to



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Sabouraud’s dextrose medium. After staining Aspergillus flavus showed septate hyphae with rough colorless conidiophoresand same colony on Sabouraud’s dextrose medium after 5 days incubation at 25°C appear yellow to green powdery in nature.

Figure 1: Black colony of Aspergillus niger on Sabouraud’s dextrose agar. The colony showing typical“salt and pepperappearance”.

Figure 2: yellow-green colony of Aspergillus fl avus on Sabouraud’s dextrose agar.On the contrary Aspergillus niger were identified as septate hyphae with smooth walled containing simpleconidiophores and on Sabouraud’s dextrose medium showed black color colonies.Similar types of findings were also shown by pradhan and sukla. These workers isolated aspergillus species andpenicillium species from soil (18).After confirmation of phosphate solubilizing fungi as Aspergillus flavus and Aspergillus niger, they were used forquantitative measurement of phosphate solubilization given by Fiskey and subbarao’s. The results showed that the amount ofphosphate solubilized by Aspergillus niger was 5.2µg/ml and Aspergillus flavus 5.5 µg/ml as shown in table 2. The result



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coincides with the findings of Vazquez et.al that Aspergillus niger solubilize insoluble phosphate well in liquid mediumsupplemented with tricalcium phosphate (19).Table 2: Quantitative measurement of phosphate solubilization in culture medium

Test tube no. Concentration in μg/ml O.D1 2 μg 0.212 4 μg 0.233 6 μg 0.234 8 μg 0.3055 10 μg 0.38Unknown sample 1 5.2 μg 0.20Unknown sample 2 5.5 μg 0.21According to Noppart et.al findings, three isolates of A. tubingensis and two isolates of A. niger isolated fromrhizospheric soils were tested on solubilization of different rock phosphates. All isolates of Aspergillus were capable ofsolubilizing all natural rock phosphates. A. tubingensis AT1 showed maximum percent solubilization in all rock phosphatestested when compared to other isolates. (20)Reddy et.al showed that isolates of Aspergillus tubingensis showed highest phosphate solubilization when grown inpresence of 2% rock phosphate. (21) Similarly kavita Sharma had isolated two fungi having phosphate solubilizing efficiencyin both solid and liquid medium. Isolates were identified as Aspergillus sp. and Penicillium sp. (22). This finding was inagreement with data obtained in earlier reports.

CONCLUSIONFrom the current investigation one may conclude that Aspergillus flavus and Aspergillus niger solubilize phosphate. Byusing these two species (Aspergillus flavus and Aspergillus niger) of phosphate solubilizing fungi, one can improve the physio-chemical, biochemical and biological properties of rock phosphate amended soil. Beyond phosphate solubilization theseorganism also increase the mycorrhizal root colonization by production of specific metabolites as vitamins, amino acids andhormones. Therefore, one can recommend these species to improve the functional knowledge of compatibility of thosemicroorganisms aiming at their co-inoculation to increase the nutrition and growth of plant species.REFERENCES1. Sarkar, M.C. and Uppal, K.S. (1994) Phosphorus research in India. Potash and Phosphate Institute of Canada. IndiaProgramme, Gurgaon, Hariyana, India.2. Khan, M.S., A. Zaidi and P.A. Wani, 2007. Role of phosphate-solubilizing microorganisms in sustainable agriculture – Areview. Agron. Sustain. Dev. 27: 29-43.3. Vassileva M, Vassilev N, Fenice M, Federici F (2001). Immobilized cell technology applied in solubilization of insolubleinorganic (rock) phosphate and P plant acquisition. Bioresource Technol. 79: 263-271.4. Th eodorou ME, Plaxton WC. Metabolic adaptations of plant respiration to nutritional phosphate deprivation. Plant Physiol101: 339-344, 1993.5. Omar SA (1998). The role of rock-phosphate-solubilizing fungi and vesicular-arbusular-mycorrhiza (VAM) in growth ofwheat plants fertilized with rock phosphate. World J. Microbiol. Biotech. 14 : 211–218.6. Abd Alla MH (1994). Phosphatases and the utilization of organic phosphorus by Rhizobium leguminosarum biovarviceae. Lett. Appl. Microbiol. 18 : 294–296.



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7. Khiari L, Parent LE. Phosphorus transformations in acid lighttexturedsoils treated with dry swine manure. Can J Soil Sci85:75-87, 2005.8. Halder AK, Mishra AK, Chakarbarthy PK (1991), Solubilization ofinorganic phosphate by Bradyrhizobium. Ind. J. Exp. Biol.29: 28-31.9. Nahas, E. (1999). In: Inter-relacao fertilidale, biologia de solo e nutricae de plantas. Vicosa:SECS, Lairas: UFLA/DCS, pp.467-468.10. Whitelaw MA (2000). Growth promotion of plants inoculated with phosphate solubilizing fungi. Edited by Donald L.Sparks. Advances in Agronomy, Academic press 69 : 99-151.11. Misir, N., M. Misir, U. Karahalil and H. Yavuz: Characterization of soil erosion and its implication to forest management. J.Environ. Biol., 28, 185-191 (2007).12. Herrera, M.A., C.P. Salamanca and J.M. Barea: Inoculation of woody legumes with selected arbuscular mycorrhizal fungiand rhizobia to recover desertified Mediterranean ecosystems. Appl. Environ. Microb., 59, 129-133 (1993).13. Requena, N., E. Perez Solis, C. Azcon Aguilar, P. Jeffries and J.M Barea: Management of indigenous plant-microbe symbiosesaids restoration of desertified ecosystems. Appl. Environ. Microb., 67, 495-498 (2001).14. Yadav, K.S. and K.R. Dadarwal: Biotechnological approaches in soil microorganisms for sustainable crop production.Scientific Publishers, Jodhpur, India. pp. 293-308 (1997).15. Ravikumar, S., 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).16. Fleischer, S., M. Bengtsson and G. Johansson: Mechanism of aerobic FE (III) phosphate solubilization at the sediment waterinterface. Verh. Int. Verein. Limnol., 23, 1825-1829 (1988).17. Asea, P.E.A., R.M.N. Kucey and J.W.B. Stewart: Inorganic phosphate solubilization by two Penicillium species in solutionculture and soil. Soil Biol. Biochem., 20, 459-464 (1988).18. N Pradhan*, and LB Sukla Solubilization of inorganic phosphates by fungi isolated19. from agriculture soil. African Journal of Biotechnology Vol. 5 (10), pp. 850-854, 16 May 200520. Vazquez, P., Holguin, G., Puente, E.M., Lopez-Cortes, A. and Bashan, Y. 2000 Phosphate- Solubilizing MicroorganismsAssociated with the Rhizosphere of Mangroves in a Semiarid Coastal Lagoon, Biology and Fertility of Soils, 30, 5 - 6.21. Nopparat,C., Jatupornpipat M., and Rittiboon A. Isolation of phosphate solubilizing fungi in soil from Kanchanaburi,Thailand. KMITL Sci. Tech. J. Vol. 7 No. S2 Nov. 200722. Reddy, S. M., Kumar, S., Babita, K. and Reddy, M.S. 2002 Bio solubilization of Poorly Soluble Rock Phosphates byAspergillus tubingensis and Aspergillus niger, Bioresource Technology, 84, 187 - 189.23. Kavita Sharma* Inorganic Phosphate Solubilization by Fungi Isolated from Agriculture Soil Journal of Phytology 2011, 3(4):

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Isolation and indentification of phosphate solubilizing fungi from rhizosphere (soil)

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