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
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