Chapter 3

Isolation, screening,

morphological and

biochemical

characterization of

fungal isolates

3.1 Introduction

“Phosphorus is one of the major nutrients, second only to nitrogen in requirement for plants. A

greater part of soil phosphorus, approximately 95–99 % is present in the form of insoluble

phosphates and cannot be utilize by the plants” (Vassileva et al., 2001). To increase the

availability of phosphorus for plants, large amounts of fertilizers are being applied to soil.

However, a large proportion of fertilizer phosphorus after application is quickly transformed into

the insoluble form. Therefore, very little percentage of the applied phosphorus is available to

plants. Phosphorus deficiencies are widespread on soil throughout the world and phosphorus

fertilizers represent major cost for agricultural production. “The phosphate solubilizing fungi

(PSFs) play an important role in supplementing phosphorus to the plants, allowing a sustainable

use of phosphate fertilizers. It has been reported that fungi possess greater ability to solubilize

insoluble phosphate than bacteria” (Nahas, 1996). Species of Aspergillus, Penicillium and

Trichoderma have been widely mentioned as efficient strains of phosphate solubilizers. In the

present study fungal strains having potential to solubilize, insoluble phosphates have been

isolated, screened and characterized on their morphological and biochemical means. These PSFs

were checked for the ability to solubilize insoluble phosphates on plate assay and their

comparative analysis has been performed.

3.2 Materials and Methods

3.2.1 Collection of soil sample

The soil samples were collected from different locations of the Kukrail forest, Lucknow, Lawns

of Integral University, Lucknow and Central Institute of Medicinal and Aromatic Plants

(CIMAP), Lucknow. The soil samples were taken from two zones viz., upper zone, which is 15-

17 cm deep and lower zone, which is 25-29 cm in depth.

3.2.2 Isolation and screening of phosphate solubilizing fungi

The soil rhizospheric samples were screened for the presence of phosphate solubilizing fungi

(PSF).

3.2.2.1 Media preparation for isolation of fungi

The fungi were grown on potato dextrose agar (PDA) medium. For this PDA medium

comprising of potato 20 %, dextrose 2 % was prepared and pH adjusted to 7.0. This medium was

complemented with agar 1.5 % and autoclaved at 15 psi for 15 min. Autoclaved medium was

poured in sterile petriplates (25 ml/plate) under laminar flow hood and allowed to solidify.

3.2.2.2 Serial dilutions of soil samples

Soil rhizospheric samples from various locations were taken and serial dilutions were made. For

this 1 g sample was taken and added in tube containing distilled water and mixed thoroughly.

This represented 10-1

dilution. Under aseptic conditions, 10-2

to 10-9

dilutions of samples were

prepared.

3.2.2.3 Screening of phosphate solubilizing fungi

The isolates were screened on the basis of plate assay. Two media viz., Pikovskaya’s media

(Pikovskaya, 1948) and bromophenol blue media were used for screening. The Pikovskaya’s

medium consisted of yeast extract 0.50 (g/l), dextrose 10.00 (g/l), calcium phosphate 5.00 (g/l),

ammonium sulphate 0.50 (g/l), potassium chloride 0.20 (g/l), magnesium sulphate 0.10 (g/l),

manganese sulphate 0.0001 (g/l), ferrous sulphate 0.0001(g/l). Bromophenol media consist of

yeast extract 0.50 (g/l), dextrose 10.00 (g/l), calcium phosphate 5.00 (g/l), ammonium sulphate

0.50 (g/l), potassium chloride 0.20 (g/l), magnesium sulphate 0.10 (g/l), manganese sulphate

0.0001(g/l), ferrous sulphate 0.0001 (g/l) and 0.5 % of bromophenol blue dye. Both the media

were prepared and pH adjusted to 7.0. This medium was complemented with agar 1.5 % and

autoclaved at 15 psi for 15 min. Autoclaved medium was poured in sterile petriplates (25

ml/plate) under laminar flow hood and allowed to solidify. Fungal colonies were inoculated on

petriplates containing medium for plate assay and the plates were incubated in inverted position

in incubator for up to 72 h at 28 C. Positive cultures were screened by observing transparent

halo zones in Pikovskaya’s medium and yellow halo zone on bromophenol blue medium.

3.2.2.4 Pure cultures of phosphate solubilizing fungal species

Positive fungal colonies were subculture, on fresh petriplates containing medium for plate assay.

Fungal cultures were isolated by incubating the plates in inverted position in incubator for up to

72 h at 28 C. Positive cultures were screened by observing transparent halo zones in

Pikovskaya’s medium, which is due to the solubilization of insoluble tricalcium phosphate into

the soluble form and yellow halo zones appears on bromophenol blue medium which is due to

the production of organic acids leading to lowering of pH within the medium.

3.2.3 Morphological characterization of fungal isolates by lactophenol staining

The identification of the isolated phosphate solubilizing fungi was done by its staining

procedure. A fungal colony was first grown on the Sabouraud agar medium and its morphology

was studied using standard cover-slip technique and lactophenol cotton blue staining procedure.

The cover slip was inserted in tilted position in the petriplate itself and the culture was allowed to

grow for some time. Then the cover slip was taken out with the help of forceps and put inverted

on slide containing a drop of lactophenol cotton blue stain and visualized under microscope at 40

X magnification. Thin mycelia of fungal isolates were also spread on the glass slide and teased

with needles followed by addition of a drop of lactophenol stain. The stained and air-dried slides

were further examined under microscope at 40 X magnification. The fungi were identified on the

basis of mycelial and spore characteristics.

3.2.4 Biochemical characterization of phosphate solubilizing fungi

Solubilization index based on colony diameter and halo zone for each PSF indicate the efficiency

of solubilization of insoluble phosphate into the soluble one thus forming a transparent halo zone

around the colony. Hence, the positive fungal isolates were also analyzed qualitatively for their

pH change, dry weight and acid phosphatase enzyme. Beside it the starch hydrolysis test and

cellulose hydrolysis test were also performed.

3.2.4.1 Solubilization index (SI)

200 mg-500 mg ml of each PSF culture preserved in sterile distilled water was placed on

Pikovskaya’s agar (Pikovskaya, 1948) plates and incubated for seven days. Solubilization Index

was measured using following formula (Edi-Premono et al., 1996).

Colony diameter + halo zone diameter

SI =

Colony diameter

3.2.4.2 pH change

200 mg-500 mg of five days old culture of fungus was added to sterile 100 ml

Pikovskaya’s broth (PB) medium and kept on shaker for seven days at 28°C. Sterile uninoculated

medium served as control. Initial pH and change in pH was recorded on 3rd

, 5th

and 7th

day by

digital pH meter.

3.2.4.3 Dry weight determination of fungus

200 mg-500 mg of 5 days old culture of fungus was added to sterile 100 ml Pikovskaya’s

broth (PB) medium and kept on shaker for seven days at 28°C. During this period, the fungal

cultures reached the maximal level of their biomass in the medium. This was determined by

measuring dry weight of cultures for which mycelia were taken and dried at 40°C and their

respective weights were measured.

3.2.4.4 Starch hydrolysis test

Starch agar medium (starch 20.0 g/l, peptone 5.0g/l, yeast extract 3.0 g/l, agar 15.0 g/l;

pH 7.0) was inoculated with isolated fungal cultures. The plates were incubated at 25 C in

inverted position for 5 to 7 days. The surface of the plates was flooded with iodine solution for

30 sec. Examined the disappearance of starch from the starch agar media plates by observing the

disappearance of clear zones around the fungal growth.

3.2.4.5 Cellulose hydrolysis test

The Czapek-mineral salt agar medium consisted of KCl 0.5 (g/l), K2HPO4 1.0 (g/l),

NaNO3 2.0 (g/l), MgSO4.7H2O 0.5 (g/l), peptone 2.0 (g/l), carboxymethyl cellulose (CMC) 5.0

(g/l). This medium was complemented with agar 2 % and autoclaved at 15 psi for 15 min.

Autoclaved medium was poured in sterile petriplates (25 ml/plate) under laminar flow hood and

allowed to solidify. The plates were allowed to inoculate with isolated fungal cultures. The plates

were incubated at 35 C in inverted position for 5 days. The surface of the plates was flooded

with 1 % aqueous solution of hexadecyltrimethyl ammonium bromide for 30 sec. The plates

were observed for the formation of a clear zone around the fungal growth.

3.2.4.6 Analysis of acid phosphatase enzyme activity from various isolates

Medium preparation and fungal culture

.

All the fungal isolates tested positive in plate assay were subjected to analyses of activity

of acid phosphatase enzyme. Fungal colonies tested positive in plate assay were inoculated in

Pikovskaya’s broth, poured in test tubes (20 ml/tube) and autoclaved at 15 psi for 15 min. The

tubes were incubated in incubator shaker at 120 rpm, 28 C for 48-72 h. 10 ml of above grown

fungal culture was taken and filtered through Whatman no. 1 filter paper. This was considered as

enzyme or protein sample. The enzyme acid phosphatase was assayed using para nitrophenyl

phosphate (PNP-P) as a substrate. The reaction mixture contained 2.5 ml (0.1 M) sodium acetate

buffer (pH 5.8), 1 ml (1 mM) magnesium chloride, 0.5 ml 1 % PNP-P and 0.5 ml of a suitable

dilution of enzyme preparation. One ml of the reaction mixture was transferred to 2 ml of 0.2 M

sodium hydroxide before and after 15 min incubation at 37 C to stop the reaction. The sodium

hydroxide solution added before incubation acts as a control sample for each analysis. The

amount of para nitro phenol (PNP) liberated was measured by recording the absorbance at 420

nm using an appropriate calibration curve. Activity is expressed as nmol PNP liberated min-1

.

The blank was run in a similar manner using distilled water.

Reaction showing acid phosphatase enzyme activity with para nitrophenyl phosphate

substrate

3.2.4.7 Determination of protein content by Lowry’s method

500 l of fungal culture was taken in microfuge tube and protein was precipitated with

equal volume of ice-cold 20 % trichloroacetic acid (TCA) and kept at 4 C overnight. The pellet

was recovered by centrifuging at 12,000 rpm for 5 min at room temperature and decanting the

supernatant. The pellet was washed with 0.1 ml ice-cold 10 % TCA and ice-cold acetone.

Depending on the pellet size, it was dissolved in 0.5-1.0 ml of 0.1 N NaOH. The solution was

subjected to heating for 5 min in boiling water bath and vortexed vigorously. The protein content

was determined by Lowry’s method (Lowry et al., 1951). For protein content determination, 0.5

ml of protein solution was taken in test tube and 2.5 ml of alkaline solution [prepared by mixing

2 % Na2CO3 solution (in NaOH), 2 % sodium potassium tartrate and 1 % CuSO4.5H2O in

100:1:1. was added. The contents were mixed well and the tubes were incubated at room

temperature for 10 min. This was followed by addition of 0.25 ml of 1.0 N Folin’s reagent. The

contents in the tube were mixed thoroughly and after 10 min, absorbance at 660 nm against

reagent blank was determined spectrophotometrically using bovine serum albumin fraction V as

standard.

3.3 Results and Discussion

3.3.1 Isolation and screening of phosphate solubilizing fungi

Based on serial dilution thirty-two fungal isolates from different soil samples were

isolated, from which 20 fungal isolates having potential phosphate solubilizing ability were

screened by plate assay. Various fungi, which were isolated from soil, were preserved on the

potato dextrose medium (PDA). The shape, structure and type of colony were analyzed on the

Sabouraud agar medium and further the lactophenol staining was performed. The tricalcium

phosphate present in Pikovskaya’s medium in insoluble form was converted into the soluble

form by the phosphate solubilizing fungi (PSF), thereby giving a clear zone around a positive

colony (Fig. 3.1).

Figure 3.1 Fungi showing phosphate solubilization leading to formation of clear zone in

Pikovskaya's medium

In bromophenol blue medium (Fig. 3.2) all the positive fungal isolates were capable of

producing organic acids, which led to change in pH and thereby color change from blue to

yellow.

Figure 3.2 Fungi showing yellowing of bromophenol blue medium

Fungal isolates were characterized by analyzing their shape and the structure of colonies,

presence of amylase and cellulase/cellobiose enzyme. The isolates were examined and the results

are given in Table 3.1. Variations were observed among the isolates in their colony morphology.

3.3.2 Morphological characterization of PSFs by microscopic examination

All the twenty fungal isolates were characterized by microscopic analysis using lactophenol

cotton blue staining procedure. Wide variations were observed among the fungal isolates in their

colony morphology (Table 3.1).

Table 3.1 Morphological characterization of phosphate solubilizing fungal isolates by

colony morphology and microscopic analysis

Fungal

isolates

Colony morphology

analysis on

Sabouraud agar medium

Microscopic analysis Fungus

identified

FNP 1

Aspergillus

niger

FUK 29

Bipolaris

tetramera

FNC 27

Alternaria

brassicae

FUC 5

Verticillium

FSK 12

Rhizoctonia

FNK 18

Fusarium

oxysporum

FSI 33

Gliocladium

FNK 2

Schizophyllum

commune

FNI 10

Alternaria

SPWF166

FUI 16

Trichoderma

FUK 17

Phoma

FNC 11

Aspergillus

flavus

FNU 4

Aspergillus

paecelomyces

FSC 15

Ascobolus

FUK 6

Alternaria

azaubiae

FSK 14

Gliocladium

FNK 20

Amauroderma

FNI 7

Helminthosporium

FUC 13

Botrytis

FNK 19

Aspergillus

sulphuracea

3.3.3 Biochemical characterization of fungal isolates

3.3.3.1 Solubilization index (SI)

All the 20 isolates were able to solubilize tricalcium phosphate (TCP) in Pikovskaya’s agar

medium and the diameter of the zones of solubilization indicated wide variations among the

isolates. The results are shown in Table 3.2. Fungal isolates FUK 29, FNK 18, FNK 2, FNI 10,

FNK 20 and FNC 11 have shown higher solubilization zones (Fig. 3.3) indicating a high level of

phosphate solubilization while others have showed less solubilization index.

Table 3.2 Comparative analysis of solubilization indices of fungal isolates

Fungal isolates Solubilization Index (SI)

FNP 1 2±0.15

FUK 29 3.8±0.12

FNC 27 2.8±0.13

FUC 5 2.7±0.09

FSK 12 3.2±0.11

FNK 18 3.5±0.12

FSI 33 2.5±0.13

FNK 2 3.7±0.129

FNI 10 3.6±0.1

FUI 16 2.1±0.13

FUK 17 3.3±0.14

FNC 11 3.9±0.1

FNU 4 2.4±0.137

FSC 15 1.9±0.12

FUK 6 2.5±0.15

FSK 14 2.1±0.12

FNK 20 3.6±0.12

FNI 7 2.2±0.13

FUC 13 1.9±0.18

FNK 19 1.2±0.19

Figure 3.3 Histogram showing solubilization indices of fungal isolates

3.3.3.2 pH change

All the 20 isolates were allowed to grow on Pikovskaya’s broth supplemented with TCP (0.5 %

w/v). Decrease in pH was observed among fungal isolates with time up to 7 days. Minimum pH

was observed on the 7th

day. The pH lowered down due to the liberation of organic acids in

broth media. The maximum decrease of pH was exhibited by FUK 29 i.e., 2.9 after 7th

day of

growth. Even the pH of FNK 20, FNI 10, FNK 18, FNC 11 and FNK 2 showed decreasing trend

in pH, which was observed as 3, 3.1, 3.2, 3.3 and 3, respectively (Table 3.3) thus confirmed of

exhibiting the phosphate solubilizing ability. However, no significant decrease in pH were found

in two isolates FSK 14 and FSC 15 viz., even after 7th day which was observed as 5.9 and 5.4,

respectively (Fig. 3.4).

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

FNP 1 FUK 29 FNC 27 FUC 5 FSK 12 FNK 18 FSI 33 FNK 2 FNI 10 FUI 16 FUK 17 FNC 11 FNU 4 FSC 15 FUK 6 FSK 14 FNK 20 FNI 7 FUC 13 FNK 19

SI(

cm

)

Fungal isolates

Table 3.3 Comparative analysis of pH change on 3rd

, 5th

and 7th

day

Fungal isolates pH

3rd

day

pH

5th day

pH

7th day

FNP 1 6.8 5.9 3.9

FUK 29 6.2 5.2 2.9

FNC 27 6.2 5.7 4.9

FUC 5 6.3 5.6 4.7

FSK 12 6.5 5.5 4.7

FNK 18 6.5 5.2 3.2

FSI 33 6.6 6 5.5

FNK 2 6.6 5.4 3.4

FNI 10 6.5 5.9 3.1

FUI 16 6.8 5.7 4.5

FUK 17 6.8 5.9 4.9

FNC 11 5.9 4.8 3.3

FNU 4 6.2 5.8 5.2

FSC 15 6.3 5.9 5.3

FUK 6 6.7 5.8 5.1

FSK 14 6.8 6.2 5.9

FNK 20 6.7 5.4 3

FNI 7 6.8 6.2 4.9

FUC 13 6.8 5.9 5.2

FNK 19 6.7 6.1 5.4

Figure 3.4 Histogram showing comparative analysis of pH on different days of growth

3.3.3.3 Dry weight determination weight of fungus

After 1 week of incubation, the fungal mycelia were filtered and dried at 45 C followed by

measuring their dry weight. Thus, fungal isolate FNP 1, FUI 16, FNC 11, FNU 4 and FNU 4

exhibited an increase in the dry weight, which was 2.031, 2.494, 2.102, 1.904 and 2.234,

respectively (Table 3.4).

0

1

2

3

4

5

6

7

8

FNP 1 FUK 29 FNC 27 FUC 5 FSK 12 FNK 18 FSI 33 FNK 2 FNI 10 FUI 16 FUK 17 FNC 11 FNU 4 FSC 15 FUK 6 FSK 14 FNK 20 FNI 7 FUC 13 FNK 19

pH

Fungal isloates

Table 3.4 Dry weight determination of fungal isolates

Fungal isolates Dry weight (g)

FNP 1 2.031±0.05

FUK 29 1.999±0.06

FNC 27 1.002±0.07

FUC 5 1.262±0.09

FSK 12 1.564±0.16

FNK 18 1.765±0.09

FSI 33 1.212±0.06

FNK 2 1.223±0.06

FNI 10 2.435±0.04

FUI 16 1.604±0.07

FUK 17 1.31±0.04

FNC 11 2.494±0.03

FNU 4 1.402±0.01

FSC 15 1.462±0.012

FUK 6 1.564±0.092

FSK 14 1.684±0.07

FNK 20 1.321±0.09

FNI 7 1.023±0.07

FUC 13 0.999±0.09

FNK 19 1.234±0.08

Figure 3.5 Histogram representing the dry weight of fungal isolates

3.3.3.4 Starch hydrolysis test

This test was used to determine the ability of an organism to hydrolyze starch, by enzymatic

action. Starch is a mixture of two polyglucose polysaccharide molecules: linear amylase and

branched amylopectin. Iodine combines with the amylase starch fraction to form an intense, deep

blue colour complex. If starch is hydrolyzed, this network disintegrates and test fails to maintain

the blue colour. Here some PSF gave positive results and some were tested negative. The colony,

which showed the disappearance of blue color from the starch agar media plates due to

utilization of starch, was a positive colony for starch test (Table 3.5).

3.3.3.5 Cellulose hydrolysis test

Cellulose is a polysaccharide comprising of long linear chain of glucose units linked by β-1, 4

glycosidic bonds. Fungi, bacteria and actinomycetes bring about degradation of cellulose by the

secretion of extracellular enzyme, cellulose. It is a complex enzyme composed of at least three

components viz., endoglucanase, exoglucanase and β-glucosidase. The cooperative action of

these three enzymes is required for the complete hydrolysis of cellulose to glucose. Evidence for

0

0.5

1

1.5

2

2.5

3

FNP

1

FUK

29

FNC

27

FUC

5

FSK

12

FNK

18

FSI 3

3

FNK

2

FNI 1

0

FUI 1

6

FUK

17

FNC

11

FNU

4

FSC

15

FUK

6

FSK

14

FNK

20

FNI 7

FUC

13

FNK

19

Dry

weig

ht

(g)

Fungal isolates

the microbial utilization of cellulose can be detected using hexadecyltrimethyl ammonium

bromide. This reagent precipitates intact carboxymethyl cellulose (CMC) in the medium and thus

clear zones around the colony in an otherwise opaque medium indicating degradation of CMC.

In the cellulose hydrolysis test, all fungal isolates gave positive results (Table 3.5).

Table 3.5 Starch hydrolysis and cellulose hydrolysis tests of fungal isolates

Fungal isolates Starch hydrolysis test Cellulose hydrolysis test

FNP 1 Positive Positive

FUK 29 Negative Positive

FNC 27 Negative Negative

FUC 5 Positive Positive

FSK 12 Positive Positive

FNK 18 Positive Negative

FSI 33 Negative Positive

FNK 2 Positive Positive

FNI 10 Negative Negative

FUI 16 Negative Negative

FUK 17 Positive Positive

FNC 11 Positive Positive

FNU 4 Positive Positive

FSC 15 Positive Positive

FUK 6 Positive Negative

FSK 14 Negative Positive

FNK 20 Positive Positive

FNI 7 Positive Positive

FUC 13 Negative Positive

FNK 19 Positive Positive

3.3.3.6 Quantitative analysis of acid phosphatase enzyme activities

The selected PSFs were grown on Pikovskaya’s broth containing 0.5 % TCP and their acid

phosphatase activities were measured. The phosphatase activity was estimated in the supernatant

of broth taken after centrifugation at 10,000 X g at 4°C. The main mechanism for the

solubilization of insoluble organic and inorganic phosphate was due to production of an enzyme

acid phosphatase, which catalyzes hydrolysis of phosphate to liberate inorganic phosphorus (Pi).

Thus, the isolates were evaluated for their acid phosphatase producing ability by measuring Pi

liberated. Among all the positive isolates, six fungi exhibited significantly higher amount of acid

phosphatase enzyme activity, including fungi FUK 29, FNK 20, FNI 10, FNK 18, FNC 11 and

FNK 2 (Fig. 3.6). Moreover, these isolates also showed relatively higher solubilization index.

Thus, the solubilization index and the acid phosphatase enzyme activity are directly proportional

to each other indicating that high enzymatic activity results in the formation of large halo zone.

The acid phosphates activities of all the isolates are presented in Table 3.6.

Table 3.6 Acid phosphatase enzyme activity of fungal isolates

Fungal isolates Acid phosphatase enzyme

activity (nmole/ml)

Acid phosphatase enzyme

specific activity (Activity/mg

protein)

FNP 1 1.760 ±0.065 0.243±0.015

FUK 29 2.806 ±0.091 0.873±0.035

FNC 27 1.014 ±0.018 0.545±0.023

FUC 5 1.424 ±0.046 0.132±0.043

FSK 12 1.036 ±0.034 0.435±0.032

FNK 18 2.335 ±0.028 1.057±0.029

FSI 33 1.062 ±0.017 0.584±0.017

FNK 2 2.432 ±0.1009 0.879±0.023

FNI 10 2.832 ±0.090 0.787±0.015

FUI 16 1.007 ±0.114 0.683±0.025

FUK 17 1.364 ±0.119 0.363±0.018

FNC 11 2.323 ±0.049 0.845±0.034

Figure 3.6 Histogram indicating acid phosphatase activity of fungal isolates

3.4 Conclusion

The results indicated existence of variation among the fungal isolates in their morphology. Some

of fungi have black conidiophores, sickle shaped spores, some of them having brown colony

while others formed light green, brown and orange colonies. All the fungal isolates showed halo

zones on Pikovskaya’s agar media indicating there phosphate solubilizing ability. Based on

diameter of transparent halo zone, the solubilization index of the isolates was measured, Further,

dry weight determination, change in pH of media at different days and acid phosphatase

activities were also determined. The isolated strains were identified on the basis of microscopic

analysis using lactophenol cotton blue stain which includes Schizophyllum commune,

0

0.5

1

1.5

2

2.5

3

3.5

FNP 1 FUK 29

FNC 27

FUC 5

FSK 12

FNK 18

FSI 33

FNK 2 FNI 10

FUI 16

FUK 17

FNC 11

FNU 4

FSC 15

FUK 6 FSK 14

FNK 20

FNI 7 FUC 13

FNK 19n

mole

s of

Pi/

ml

Fungal isolates

FNU 4 1.534 ±0.023 0.342±0.075

FSC 15 2.001 ±0.066 0.698±0.051

FUK 6 2.031 ±0.133 0.765±0.025

FSK 14 1.032±0.073 0.721±0.037

FNK 20 2.098±0.056 0.693±0.015

FNI 7 1.043±0.131 0.649±0.035

FUC 13 1.544±0.102 0.542±0.025

FNK 19 1.001±0.045 0.432±0.032

Trichoderma, Aspergillus sulphuracea, Botrytis, Helminthosporium, Amauroderma,

Gliocladium, Alternaria azaubiae, Ascobolus, Aspergillus paecelomyces, Aspergillus flavus,

Alternaria sp., Gliocladium, Fusarium oxysporum, Aspergillus niger, Bipolaris tetramera,

Alternaria brassicae, Verticillium, Rhizoctonia and Phoma.

Prominent halo zones were found in case of positive PSF isolates on Pikovskaya’s agar.

Based on transparent halo zone the isolates that exhibited higher SI also exhibited higher acid

phosphatase activity. Beside it, the starch and cellulose hydrolysis test were also studied.

However, morphological and biochemical characterization of isolates did not give complete

information about isolates. Hence, polyphasic approach involving molecular marker analyses

along with phenotypic evaluation is essential for identification of fungal isolates and

determination of genetic variation. Thus, all fungal isolates were further subjected to diversity

analysis at molecular level.