Ashok V Gomashe et al.

, IJSID, 2012, 2 (2), 310-315

ISSN:2249-5347

IJSID

International Journal of Science Innovations and Discoveries

Research Article

An International peer Review Journal for Science

Available online through www.ijsidonline.info

ISOLATION AND IDENTIFICATION OF PHOSPHATE SOLUBILIZING FUNGI FROM RHIZOSPHERE (SOIL) Shivaji Science College, Congress Nagar, Nagpur-440012, M.S. India Ashok V. Gomashe*, Sabina S. Suriya, and Preeti G. Dharmik

Received: 16.02.2012 Accepted: 30.04.2012

ABSTRACT considerable part of which accumulates as consequences of regular application of phosphate fertilizers. However a greater part of soil phosphorous, approximately 95-99% is present in the form of insoluble phosphates and hence cannot be having potential to solubilize phosphate were characterized. Two fungal isolates phosphate solubilization. utilized by plants. Aspergillus species, a soil isolate had excellent potential to solubilize phosphate in vitro. In present study fungal strains isolated from soil were identified by cultural and microscopic examination and are capable of Most agricultural soil contains large reserves of phosphorous (P), a

*Corresponding Author

were characterized as Aspergillus niger and Aspergillus flavus. Both the isolates
Address: Name: Dr. Ashok V. Gomashe Place: Shivaji Science College Congress Nagar, Nagpur, Maharastra, India E-mail: drgomashe@rediffmail.com

INTRODUCTION Key Words: Aspergillus, Phosphate, rhizosphere, fungal strain.

INTRODUCTION

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Ashok V Gomashe et al., IJSID, 2012, 2 (2), 310-315 most essential plant nutrients for maximizing crop productivity. This nutrient is limited in soils, which remain as a major Improving soil fertility is one of the most common practices in agricultural production. Phosphorus (P) is one of the INTRODUCTION

challenge to agriculturists and land managers. (1). Phosphorus is one of the major nutrients, second only to nitrogen in phosphates and cannot be utilized by the plants (2). Compared with the other major nutrients, phosphorus is by far the least forms, 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 crop mobile and available to plants in most soil conditions. Although phosphorus is abundant in soils in both organic and inorganic yields. It is important for the functioning of key enzymes that regulate the metabolic pathways (4). To increase the availability of phosphorus for plants, large amounts of fertilizer are being applied to soil. But a large proportion of fertilizer phosphorus is available to plants, making continuous application necessary (6). (PSF) (7). 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 Rhizospheric phosphate solubilizing bacteria and fungi are capable of solubilizing insoluble or inorganic phosphates after application is quickly transformed to the insoluble form (5). Therefore, very little percentage of the applied phosphorus

requirement for plants. A greater part of soil phosphorus, approximately 9599% is present in the form of insoluble

allowing a sustainable use of phosphate fertilizers. Microorganisms are involved in a range of process that effect the 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). solubilize phosphates. 1. 2. 3. MATERIAL AND METHODS

transformation of soil phosphorus (P) and thus are integral component of the soil P cycle. Many bacterial, fungal, yeast, and Owing to above facts, the current study deals with the isolation and identification of fungal strains having potential to

actinomycetes species capable of solubilizing sparingly soluble phosphorus in pure culture have been isolated and studied (8).

Phosphate solubilizing microorganisms (PSMs) play an important role in supplementing phosphorus to the plants,

Isolation of fungi from soil by serial dilution

Suspend 1gm of soil in 9ml sterile distilled water in a test tube and make serial dilutions. (Himedia, Mumbai) which is supplemented with phosphate. Incubate the plates at 25C for 4 to 5 days.

Transfer aliquots of 1ml suspension from 10 or 10th dilution tube in to agar plates containing Pikovskayas medium Identification of fungi was done on the basis of morphological characteristics by cotton blue staining.

Identification by staining technique Confirmatory test 1.

For further confirmation the colonies were inoculated in Sabourauds dextrose medium. Quantitative measurement of phosphate solubilization in culture medium incubation for 7 days.

Colonies showing clear zone in above method are inoculated in the Pikovskayas broth in two different flasks and kept for International Journal of Science Innovations and Discoveries, Volume 2, Issue 2, March-April 2012

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2. 3. 4. 5. 6.

This results in the formation of fungal matt in the Pikovskayas broth. The fungal matt was separated and the broth was filtered. taken at 660nm. Sr.no 1 2 3 4 5

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

The two filtrates of, i.e. Black and Lime green color colonies are taken as unknown sample 1and 2. Table 1: Protocol for phosphate solubilization in culture medium Blank I II III 0.2 0.4 0.6 1 0.8 0.6 0.4 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 RESULT AND DISCUSSION IV 0.8 0.2 0.5 0.5

This is further estimated quantitatively by Fiskey and subbaraos method as shown in table 1 & specific readings were By using same protocol the concentration of unknown sample i.e. solubilized phosphate is estimated Reagent Working Phosphate solution Unknown Distilled water Ammonium Molybdate ANSA V 1.0 0.5 0.5

availability has been through the application of concentrated phosphorous fertilizers, but the efficiency of this process is phosphate and cannot be utilized by plants (3).

affected by chemical immobilization of phosphorous in soil, depletion of non-renewable sources of phosphorous ore and cost non regulated cultivation techniques, deforestation, etc) results in the disturbances of soil ecosystems (11). Such degraded Conditions like irregular rainfall, long dry and hot summers and man-mediated degradative activities (overgrazing,

of fertilizer processing. A greater part of soil phosphorous approximately 95-99% is present in the form of insoluble ecosystems are usually characterized by a disturbed vegetation cover accompanied by a rapid erosion of surface soil (12). The mineral content. In fact, despite being abundant in soil, both in inorganic and organic forms, phosphorus (P) is the major plant growthlimiting nutrient after nitrogen (N). Plant mineral nutrition depends mainly on the phosphorus content of soil, which along plant roots in the rhizosphere. Phosphorus in soils is present in insoluble form complexes with cations like iron, agricultural production, a large portion of inorganic phosphates applied to soil as fertilizer is rapidly immobilized after application and becomes unavailable to plants (14). processes in soil (15). The process of microbe mediated P solubilization is generally ascribed to the production of organic acids by them (16). Still there are reasonable doubts on the exclusive role of organic acids in solubilization process (17).

The phosphorous is essential for the general health and vigor off all plants. In agricultural system, low phosphorous

Unknown 1ml 0.5 0.5

desertification process involves a loss or reduction of major physicochemical and biological soil properties (13), including its can be assimilated only as soluble phosphate. Physical and chemical weathering of mineral phosphates is mainly realized aluminum and calcium. Although, use of chemical fertilizers for improving soil fertility is the common approach of increasing Microorganisms substantially influence the soil productivity by solubilizing this insoluble P through their metabolic The present study aimed to isolate and identify the phosphate solubilizing fungi from rhizosphere. For isolation of

phosphate solubilizing fungi, soil was serially diluted and plated on Pikovskayas medium and incubated at 25C for 4-5days. Aspergillus niger. selected and further identified by lactophenol cotton blue staining technique and characterized by inoculating them on to International Journal of Science Innovations and Discoveries, Volume 2, Issue 2, March-April 2012 The colonies showing clear transparent zone which indicate the phosphate in the medium has been solubilized were

The colonies on the plate were identified by cotton blue staining technique and later on confirmed as Aspergillus flavus and

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Sabourauds dextrose medium. After staining Aspergillus flavus showed septate hyphae with rough colorless conidiophores and same colony on Sabourauds dextrose medium after 5 days incubation at 25C appear yellow to green powdery in nature.

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

Figure 1: Black colony of Aspergillus niger on Sabourauds dextrose agar. The colony showing typicalsalt and pepper appearance.

conidiophores and on Sabourauds dextrose medium showed black color colonies. penicillium species from soil (18).

On the contrary Aspergillus niger were identified as septate hyphae with smooth walled containing simple Similar types of findings were also shown by pradhan and sukla. These workers isolated aspergillus species and After confirmation of phosphate solubilizing fungi as Aspergillus flavus and Aspergillus niger, they were used for

Figure 2: yellow-green colony of Aspergillus fl avus on Sabourauds dextrose agar.

quantitative measurement of phosphate solubilization given by Fiskey and subbaraos. The results showed that the amount of International Journal of Science Innovations and Discoveries, Volume 2, Issue 2, March-April 2012

phosphate solubilized by Aspergillus niger was 5.2g/ml and Aspergillus flavus 5.5 g/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 medium supplemented with tricalcium phosphate (19). Table 2: Quantitative measurement of phosphate solubilization in culture medium Test tube no. 1 2 3 4 5 Unknown sample 1 Unknown sample 2 Concentration in g/ml 2 g 4 g 6 g 8 g 10 g 5.2 g 5.5 g O.D 0.21 0.23 0.23 0.305 0.38 0.20 0.21

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rhizospheric soils were tested on solubilization of different rock phosphates. All isolates of Aspergillus were capable of tested when compared to other isolates. (20) presence of 2% rock phosphate. (21) Similarly kavita Sharma had isolated two fungi having phosphate solubilizing efficiency in both solid and liquid medium. Isolates were identified as Aspergillus sp. and Penicillium sp. (22). This finding was in agreement with data obtained in earlier reports. CONCLUSION Reddy et.al showed that isolates of Aspergillus tubingensis showed highest phosphate solubilization when grown in

solubilizing all natural rock phosphates. A. tubingensis AT1 showed maximum percent solubilization in all rock phosphates

According to Noppart et.al findings, three isolates of A. tubingensis and two isolates of A. niger isolated from

using these two species (Aspergillus flavus and Aspergillus niger) of phosphate solubilizing fungi, one can improve the physiochemical, biochemical and biological properties of rock phosphate amended soil. Beyond phosphate solubilization these organism also increase the mycorrhizal root colonization by production of specific metabolites as vitamins, amino acids and microorganisms aiming at their co-inoculation to increase the nutrition and growth of plant species. 1. 2. 3. 4. 5. 6. REFERENCES Programme, Gurgaon, Hariyana, India. hormones. Therefore, one can recommend these species to improve the functional knowledge of compatibility of those

From the current investigation one may conclude that Aspergillus flavus and Aspergillus niger solubilize phosphate. By

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10. Whitelaw MA (2000). Growth promotion of plants inoculated with phosphate solubilizing fungi. Edited by Donald L. 12. Herrera, M.A., C.P. Salamanca and J.M. Barea: Inoculation of woody legumes with selected arbuscular mycorrhizal fungi 14. Yadav, K.S. and K.R. Dadarwal: Biotechnological approaches in soil microorganisms for Scientific Publishers, Jodhpur, India. pp. 293-308 (1997). Environ. Biol., 28, 109-114 (2007). and rhizobia to recover desertified Mediterranean ecosystems. Appl. Environ. Microb., 59, 129-133 (1993). aids restoration of desertified ecosystems. Appl. Environ. Microb., 67, 495-498 (2001). Sparks. Advances in Agronomy, Academic press 69 : 99-151. Environ. Biol., 28, 185-191 (2007).

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