Minor Research Project:

Title: “ISOLATION AND IDENTIFICATION OF

PHOSPHATE SOLUBILIZING FUNGI”

Submitted by

DR. MANDAKINI INGLE

Assistant Professor,

Department of Botany

Financial Assistance: Dnyanasadhan Trust

Satish Pradhan Dnyanasadhana College of Arts Commerce, Thane

INDEX

SR. NO CONTENT PAGE NO.

01 Introduction 01-02

02 Review of Literature 03-04

03 Material and Method 05-06

04 Results 07-12

05 Discussion 13

06 Conclusion 14

07 Bibligraphy 15-16

CHAPTER 1

INTRODUCTION

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Microorganisms are important component of soil. Soil bacteria and fungi mediate soil processes

such as decomposition, nutrient mobilization and mineralization, storage release of nutrients and

water, nitrogen fixation and denitrification. In the frame of agriculture, the micro flora is of great

significance because it has both beneficial and detrimental influence upon mans ability to feed

himself (Gaur, 1990; Motsara etal., 1995; White law, 2000). Phosphate Soluble Microorganisms

include different types of microorganisms that convert insoluble phosphatic compounds into

soluble forms (Prerena-Akhaury etal., 1997; Raju and Reddy, 1999).

Phosphorus is one of the major plant nutrients required in optimum amount for proper plant

growth. Is also known to involve many functions in the plant growth and metabolism. Several

important cellular, metabolic and reproductive functions rely on sufficient phosphorus supply.

Only about 25 percent of the phosphorus applied to the soil is available for the crops and the rest

become unavailable due to chemical fixation with aluminum and iron in acidic soils. Indian soils

are characterized by poor and medium status with respect to available phosphorus (Baby 2002;

Li etal. 2003; Ramanathan etal. 2004; Gerretsen, 1948). Phosphorus ranks next to Nitrogen in

importance for living plants.

Many types of microorganisms are known to inhabit soil, especially rhizosphere and play an

important role in plant growth and development, further Gerretsen, 1948 initially demonstrated

that microbiological activity in the rhizosphere could dissolve sparingly soluble inorganic P and

increase plant growth. Phosphorus in different forms might be taken up by the plants, but by far,

the major form absorbed either is HPO4 2- or H2 PO4

- (Beeverand Burns, 1981).

A greater part of soil phosphorus, approximately 95 99%, is present in insoluble form complexed

with cations like iron, aluminum, and calcium that cannot be utilized by the plants (Firew Elias;

2016).Only 1 to 5% of the total soil P is in a soluble plant available form (Arcand& Schneider,

2006).

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According to LOuahmaneetal., utilization of microbial mediated RP (Rock phosphate) has

several advantages over conventional chemical fertilizers for agricultural purposes. These

advantages are as follows:

(1) Microbial products are considered safer than many of the chemical fertilizers now in use;

(2) Neither toxic substances nor microbes themselves will be accumulated in the food chain; and

(3) Self-replication of microbes circumvents the need for repeated application. Thus, inoculation

with phosphate solubilizing microorganisms along with rock phosphate could be another strategy

to improve the physicochemical and biological properties of soil and help in improving crop

production.

Fungi have been reported to pose greater ability to solubilize insoluble phosphate than bacteria

(Nahas, 1996)

CHAPTER 2

REVIEW OF LITERATURE

Phosphate solubilizing microorganisms refer to a group of soil microorganisms that as

components of phosphorus cycle, can release it from insoluble sources by different mechanisms.

Phosphate solubilizing fungi and bacteria are known as effective organisms in this process. The

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research on Mycorrhiza fungus and its role in soil and plant has been an interesting scientific

subject since 1800. The presence of this fungus in rhizosphere provides with an advantageous

and interactive symbiosis relationship between a higher plant root and a nonpathogenic fungus.

Singh et.al., (2011) isolated many filamentous fungi from the soils of Ny-Alesund, Spitsber-gen,

Svalbard, and screened in vitro for their phosphate solubilizing ability. Two strains of Aspergillus

niger showed good tricalcium phosphate (TCP) solubilizing ability in Pikovskaya’s medium. The

TCP solubilization index was calculated at varying levels of pH and tempretures. The ability of

Aspergillus niger strain-2 solubilized 262µg ml-1 from 0.5% TCP after seven days. This is the

first reported of TCP solubilization by Arctic strains that may serve as very good phosphate

solubilizers in the form of biofertilizer.

Phosphate solubilizing fungi were found in most soils tested; however, they constituted a small

percentage of the total soil microbial population. As a group, the fungi were superior to bacteria

in solubilizing both freshly precipitated calcium phosphate and Idaho rock phosphate. Fungi also

retained this ability over many sub culturing transfers.

A novel defined microbiological growth medium, National Botanical Research Institute’s

phosphate growth medium, which is more efficient than Pikovskaya medium (PVK), was

developed for screening phosphate solubilizing microorganism’s. In plate assay the efficiency of

NBRIP was comparable to PVK; however,, in broth assay NBRIP consistently demonstrated

about 3-fold higher efficiency compared to PVK. The result indicated that the criterion for

isolation of phosphate solubilizers based on the formation of visible halo/zone on agar plates not

a reliable technique, as many isolates which did not show any clear zone on agar plates

solubilized insolubilized inorganic phosphates in liquid medium. It may be concluded that soil

microbes should be screened in NBRIP broth assay for the identification of most efficient

phosphate solubilizers. (C. Shekhar Nautiyal, 1998).

Rashid et.al.,(2004) reported that, After screening a large number of phosphate solubilizing

microorganisms (PSM) isolated from rice rhizosphere on the basis of solubilization index (SI)

and pH drop ten best bacterial strains and three best fungal strains {( Aspergillusflavus (1RF),

Penicilliumcanescens (4RF), A. niger (8RF)}. SI and change of pH of these isolates showed

abrupt changes during seven days of incubation in Pikovskayas agar and broth medium

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respectively. This drop of pH clearly indicated the production of organic acids. Among bacterial

strains 10RB produced largest amount of organic acids. While among fungal isolates

Aspergillusflavus produced greatest amount of acids than the other two fungal strains.

Aspergillus niger immobilizes highest amount of available phosphorus in culture. Overall fungal

strains immobilize greater amounts of phosphorus.

Kundu and Gaur (1984) reported that the grain and straw yields of rice increased significantly

due to inoculations. They futher reported that the phosphate solubilizing microorganisms

improved phosphorus uotake over control with and without chemical fertilizers. Phosphorus

solubilizing fungi enable P to become available for plant uptake solubilization (Afzal.,

et.al.,2005).

Praveen M. Patilet. et.al.,(2012) reported, Several soil fungi, particularly those belonging to the

genera Penicillium species and Aspergills species Possess ability to bring insoluble soil

phosphotes into soluble Forms by sceting weak Organic acids such as Formic, acetic, propionic

lactic etc. Plant growth, plant height differed significantly among different doses of phosphorus

application and phosphorus solubilizing fungi inoculation at 30 and 60 DAS at harvest of the

crop.

The present study was conducted with the following objectives:

To Isolate fungal species from soil sample collected from Pali, District Raigad. Sub culturing of fungal species for pure strains. Identification of fungi by macroscopic and microscopic characters. To study the phosphate solubilizing fungi.

CHAPTER 3

MATERIALS AND METHODS

3.1 Collection of Soil samples:

The Soil samples required for the experiment were collected from a farm located at Pali, Raigad-

District in Maharashtra. Soil sample was sterilized in oven at 60⁰C for 20 minutes to eliminate

contamination. The soil has been stored in air tight container for the further analysis.

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3.2 Isolation of Phosphate solubilizing microorganisms:

The collected soil samples were used for the isolation of phosphate solubilizing fungi on suitable

culturing media.

3.3 Preparation of the Culture media:

Nutritional requirement of every microorganisms may get vary. Hence the media were selected

according to the growth and requirement of culturing microbes. Pikovskyas agar is

recommended for detection of phosphate solubilizing soil microorganisms. (pikovskaya 1948).

Initially, the 500ml media were prepared according to the standard protocol( HiMidea ).

The prepared media were autoclaved at 12 C for 15 minutes. 11

3.4 Inoculation of Soil Culture:

Ten grams of the soil sample were weighed aseptically and transferred to 250mL flask containing

100 mL of sterile water. The serial dilution method was applied for inoculation. Aliquots of 1mL

of the supernatant from the sample was transferred to 9mL of sterile water solution dispensed

into test tubes and serially diluted to 10ˉ¹º, 10 ˉ9, 10 ˉ8, 10 ˉ7, and 10 ˉ6.

The serially diluted aliquots of soil sample were mixed with culture media. The Pour plate

technique were performed. Each dilution plate was prepared in triplicates. The plates were kept

in incubator at37o C for 3 days.

3.6. Identification and Characterized of P- solubilizing fungi

PDA was used for accelerate the growth rate and the production of enough conidia as reported byDiba et.al., (2007). The characteristics of fresh cultures were compared with mycologicalidentification keys and taxonomic description to identify the isolated fungi. Identification wasbased on colonial characteristics and microscopic features, among the colonial characteristicssuch as surface appearance, and colour of the colonies both from upper and lower side. Inaddition, conidia, conidiophores, arrangement of spores, and vegetative structures weredetermined with microscopy.

Phosphate solubilization index was calculated using the following formula (Lal Sahab Yadavet.al. 2011)

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CHAPTER 4

OBSERVATIONS AND RESULTS

4.1 Isolation and identification of fungi: Various fungal colonies were obtained with serial

dilution method.To get the pure culture regular sub culturing were perform on Potato Dextrose

Agar media (PDA) by using the fungal colonies obtained on preliminary inoculum plates. The

obtained isolates were maintained on potato dextrose agar (PDA) and incubated at 27ºC.

The isolates were grouped into filamentous fungi. (Figure 1). Fungal isolates examined and were

observed under light microscope using lacto phenol cotton blue staining according to Stevens.

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4.2 Isolation of phosphate solubilizing fungi :

The screening of phosphate solubilizing fungi on PKV media was done.. After incubation, the

observed colonies were morphologically distinct, both with and without halos. Those fungal

colonies showing clear zone around the colonies were selected.

4.3 Solubilizing index: It was calculated by the following formula:

Solubilizing index = colony diameter + clearing zone / colony diameter.

It was observed that Penicillium sp. and Aspergillus sp. had a greater solubilizing index, ascompared to other fungi.

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10ˉ¹° 10ˉ9 10ˉ8

10-7 10⁻6

Figure.1 : Fungal isolates obtained by serial dilution

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A1 A2

Figure.2 . Phosphate solubilizing fungi on Potato Dextrose Agar media

A1 – Penicillium sp. A2 – Aspergillus niger A3 - A. flavus A4 - A. fumigatus

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A3 A4

A5 A66

Figure.2 . Phosphate solubilizing fungi on Potato Dextrose Agar media

A5 –Aspergillus sp. A6 – Aspergillus sp A7 – Aspergillus sp

Table 1: Morphological characters of Phosphate solubilize fungi:

FungalIsolates

GrowthPattern

Configuration Margins Colour ofMycelium

A1 Slow Regular andspreading

Smooth Aerial-Greyishgreen Reverse-Ruby

A2 Fast Irregular andspreading

Smooth Aerial-CarbonblackReverse-ColourLess topale yellow

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A7

A3 Rapidly Irregular andradiating margin

Smooth toroughened

Aerial-GreenishyellowReverse-colourless

A4 Median Spreading Filamentous Aerial-white atyoung Reverse-Colourless tovarying inshades.

A5 Median Cottony Filamentous Aerial-GreyishredReverse-Brownish orange

A6 Fast Powdery Smooth Aerial-Greyishyellow Reverse-Yellow

A7 Medium Cottony Filamentous Aerial-Yellowishgrey Reverse-Darkyellow

Table 2: Phosphate solubilizing fungi

Sr no Fungal isolate Species name

1 A1 Penicillium species

2 A2 Aspergillus niger

3 A3 Aspergillus flavus

4 A4 Aspergilus fumigatus

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5 A5 Aspergillus species

6 A6 Aspergillus species

7 A7 Aspergillus species

Table.3: Solubilizing index of fungal isolates

Sr. no. Fungal isolate Solubilizing indexmm

SI

1 A1 0.25+ 0.02 / 0.25 1.05

2 A2 0.72 + 0.03 / 0.72 0.76

3 A3 0.35 + 0.01 / 0.35 0.38

4 A4 0.9 + 0 / 0.9 0.90

5 A5 0.6 + 0.05 / 0.6 0.68

6 A6 0.86 + 0.04 / 0.86 0.90

7 A7 0.45 + 0.03 / 0.45 0.52

CHAPTER 5

CONCLUSION

Identified pure colonies having similar morphology. Result obtained with the present studyshows that Aspergillus species and Penicillium species are the fungi which are Phosphatesolubilizing fungi. From filamentous fungi, Aspergillus species are dominant (32%), Penicilliumspecies (30%). The phosphate solubilizer fungi isolates were also identified based on their

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colony morphology that is pigmentation, shape, size, texture, elevation and margin, the followingtable will summarize (Table 1).

Representation filamentous fungal isolates from clustered group were stained using lacto phenol

to confirm to which genera filamentous fungi belonged to. The result revealed that 6 filamentous

fungi species associated okra soil. Identified the phosphate solubilization, they produced the

halos around their colony. Yaseer et.al., (2014) reported that, ten fungal isolates from thirty soil

samples showed clear inhibition zones formed around the colonies after 5-7 days of incubation of

solidified PKV medium.

CHAPTER 6

DISCUSSION

Phosphorus deficiencies are wide spread on soil throughout the world and one of the limitating

factors for crop productivity. Phosphorus fertilizers represent major cost for agricultural

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production. Many bacteria, fungi and few actinomycetes are potential solubilizes of bound

phosphates in soil thus playing an important role making it available to plants in the soluble

form. Solubilization of insoluble phosphorus by microorganisms was reported by Pikovskaya.

From the present report it is evident that Penicillium and Aspergillus species are more potent in

solubilizing phosphate and thus can be put into further use for agro- industrial applications.

CHAPTER 7

BIBLIOGRAPHY

1. Alexopoulous C. J., W. Mims, Introductory Mycology. Third edition.

2. Arcand, M. M., Schneider, K. D. (2006). Plant- and microbial-based mechanisms toimprove the agronomic effectiveness of phosphate rock: A review. Ann. Brazil Sci., 78,791-807.

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3. Asea, P.E.A. and Kucey R.M.N. (1988). Inorganic phosphate solubilization by twoPenicilliumspecies in solution culture and soil. Soil Biology and Biochemistry, 20,459_464.

4. Beever, R. E. and Burns, D. J. W., (1981). Phosphorus uptake, storage and utilization byfungi. Adv. Bot. Res., 8: 127-219.

5. Earl, K., Syers J. and McLaughlin R.M. 1979. Origin of the effect of citrate, tartrate,and acetate on phosphate sorption by soils and synthetic gels. Soil Science Society ofAmerica Journal43, 674_678.

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