Evaluation of Antifungal and Enzymatic Potential of Endophytic Fungi Isolated from Cupressus

17 Pages·2017·0.61 MB·English

Int.J.Curr.Microbiol.App.Sci (2017) 6(7): 4084-4100
International Journal of Current Microbiology and Applied Sciences
ISSN: 2319-7706 Volume 6 Number 7 (2017) pp. 4084-4100

Journal homepage: http://www.ijcmas.com

Original Research Article https://doi.org/10.20546/ijcmas.2017.607.424

Evaluation of Antifungal and Enzymatic Potential of Endophytic Fungi
Isolated from Cupressus torulosa D. Don

Mohd Mahfooz1, Sushmita Dwedi1, Arun Bhatt1, Sharad Raghuvanshi1,
Manoj Bhatt2 and Pavan Kumar Agrawal1*

1Department of Biotechnology, G.B. Pant Engineering College, Ghurdauri, Pauri, Garhwal,
Uttarakhand, India
2Guru Gobind Singh Indraprastha University, Delhi, India
*Corresponding author

A B S T R A C T

Today, endophytic fungi have become new sources of industrially useful
Key words
enzymes such as cellulases, chitinases, lipases, amylases and proteases.

Extra cellular When endophytes reside on the plant surface, they produce enzymes to
enzym es, hydrolyze pl ant cell walls. Therefore, these enzymes can also suppress
Bioco ntrol, PGP,
Endop hytic fungi. plant pathoge n activities directly and are capable of degrading the cell walls
of fungi. In d ual plate assay fungal endophytes inhibited Fusarium solani;
A rtic le Info few isolates were inhibitory towards Ganoderma lividense and

Accepted: Colletotrichum demati. Microbial enzymes are also biotechnologically

29 June 2017
important products employed in biotechnology, agriculture, and the
Available Online:
1 0 July 2017 pharmaceutical, photographic an d food industries.

Introduction

The term endophyte is applied to fungi or laccase) with various industrial applications
bacteria which live within plant tissues, for all are also of major interest.
or part of their life cycle and causes no
apparent infections (Wilson, 2000). Like other Biological control through microorganisms
organisms invading plant tissues, endophytic which inhibit or antagonize plant pathogens
fungi produce extracellular hydrolases as a and pests reduces or eliminates the use of
resistance mechanism against pathogenic chemical products. Fungal endophytes are
invasion and to obtain nutrition from host. effective antagonists (Azevedo et al., 2000)
Such enzymes include pectinases, cellulases, and constitute a taxonomically and
lipases, laccase from the endophytic fungus metabolically diverse group of organisms that
Monotospora sp., xylanase, -1, 4- colonize internal plant tissues without causing
glucanlyase, phosphotases and proteinase apparent harm to the host plant. Indeed,
(Sunitha et al., 2013). Hydrolytic enzymes endophyte-mediated biological control has
(amylase, proteases, cellulases, chitansae and been investigated both in vivo and in vitro
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through screening experiments to verify the using a plate assay and different growth
activity of endophytes against phytopathogens substrates.
and pests (Flores et al., 2013).
Materials and Methods
Endophytic and phytopathogenic fungi
compete and interact within the same Isolation of endophytic fungi
ecological niche through the action of
hydrolytic enzymes such as proteases and The sampling procedure was designed with
chitinases, which degrade the hyphal cell the intention of isolating as many endophytic
walls of pathogenic microorganisms (Almeida fungal species as possible from the different
et al., 2007). This enzymatic activity is tissues samples. Tissues of the leaves of
closely associated with the fungus-host Cupressus torulosa D. Don were cut into 5
specificity. To facilitate the entry of mm long segments. Surface sterilization
endophytes into host tissues through natural followed the method of (Arnold et al., 2007)
or artificial openings, hydrolytic enzymes with minor modification.
including pectinases, cellulases and lipases
are secreted (Polizeli et al., 1991). Segments were surface sterilized by
consecutive immersion for 1 min in 75 %
It has been observed that some strains of Ethanol, treated for 1 min in 0.1 % mercuric
bacteria and fungi shows beneficial effects on chloride, followed by several washing in
plants and this type of evaluation is based on sterile distilled water (Sharma et al., 2016).
better emergence of seedling’s fast growth. The time of the dilution and immersion in
The mechanism of plant growth promotion ethanol and Mercuric chloride varies with
include, i] solubilization of insoluble tissues and host (At least three washing
phosphates (ii) ability to produce require).
phytoharmone auxin iii) production of HCN
iv) siderophore production. The Plant growth Under sterile conditions, tissue segments were
promoting activity of microorganism showed allowed to surface-dry before plating. Five
beneficial association with the plants by segments were then evenly placed in each 90
increasing uptake of N, P and Fe (Kloepper et mm Petri dish containing Potato dextrose agar
al., 2009). There are many reports on the use and water agar Medium. The dishes were
of micro-organisms as biocontrol agents as an sealed with parafilm and incubated at 27ºC ±
alternate to agricultural chemical fungicides 2ºC for 2-4 weeks in incubator.
(Kaur et al., 2013).
Fungal growth was observed from the tissues
In present investigation, considering the segments inoculated on the different media
shortage of information concerning the plates as colored cottony outgrowth. Hyphal
antifungal and enzymatic activities of the tips, from germinating fungi, were isolated,
endophytes from this plant, the aim of the sub cultured onto PDA and brought into pure
present study was to evaluate the antagonism culture by incubating at 28ºC for 5-7 days.
interactions between endophytic fungi and
phytopathogenic fungi in dual culture The purified fungal isolates were maintained
experiments and to detect the extracellular at 4ºC. The cultures were also submitted to
enzyme activity of these fungal endophytes the Gujarat State Biotechnology Mission
isolated from Cupressus torulosa D. Don microbial repository for preservation.

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Preservation of endophytic fungi cultures Screening for secretion of protease

PDA slants Protease assay was performed by growing the
fungi on GYP media amended with 1% skim
The PDA slants were inoculated with hyphal milk and pH was adjusted to 6.5, after 5 days
tips taken from the PDA plates. The slants incubation the clear zone appeared around the
were incubated at 28ºC for 5-7 days to fungal colony indicated the presence of
observe the growth of pure culture of fungal protease enzyme.
endophyte. This was followed by refrigeration
of the slants at 4ºC. Screening for secretion of lipase

20% glycerol For lipase activity measurement, the fungus
were grown on Peptone Agar Medium
Mycelial agar plugs were placed in sterile (Peptone 10 g, NaCl 5 g, Agar 16 g, Distilled
cryovials containing PDB with 20% glycerol water: 1000ml and pH 6) supplemented with
under sterile laminar conditions and stored at 1% w/v tween 20 (separately sterilized). A
-80C. clear zone around the colony indicated the
presence of lipase enzyme.
Identification of endophytic fungi
Screening for secretion of cellulase
Fungal growth and sporulation was facilitated
by placing the isolates onto PDA culture For Cellulase test, GYP Agar media was used
medium. The plates were continuously as the medium to grow the fungus. To GYP
monitored for spore formation. Isolates were agar media, 1% CMC (carboxy methyl
identified on the basis of cultural cellulose) was added which act as a substrate.
characteristics, colour and morphology of After 4 days of incubation at 27°C, the plates
fruiting bodies and spores. Fungal isolates were flooded with 0.2% of aqueous Congo
were stained with lactophenole cotton blue red solution for 20 minutes and distained with
and examined under light microscope 1 M NaCl for 15 min. Appearance of yellow
(Olympus, USA). areas around the fungal colony indicates the
presence of cellulase activity.
Metabolic activity of fungal endophytes
Screening for secretion of chitinase
Screening for secretion of amylase
The final chitinase detection medium
To test for the production of amylase, consisted of a basal medium comprising (all
endophytes were inoculated on glucose yeast amounts are per litre) 4.5 g of colloidal chitin,
extract peptone (GYP) agar medium (glucose 0.3 g of MgSO .7H O, 3 g of (NH ) SO , 2 g
4 2 4 4
1g, yeast extract 0.1g, peptone 0.5g, agar 15 of KH PO , 1 g of citric acid monohydrate, 15
2 4
g, and distilled water 1000 mL, pH 6) g of agar, 0.15g of bromocresol purple and
containing 1% soluble starch. After 200 μl of tween-80, pH was adjusted to 4.7
incubating for 5 days, 1% iodine in 2% and then autoclaved at 121C for 15 min.
potassium iodide was flooded in the fungal After cooling the media was poured in to petri
colony plates. A clear zone appearance plates and allowed to solidify. The fresh
surrounding the colony was considered culture plugs of the isolates to be tested for
positive for amylase enzyme (Bhardwaj et al., chitinase activity was inoculated into the
2015).
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medium and incubated at 27C for 2-3 d and Antagonistic activity of endophytic fungi
observed for colored zone formation.
Chitinase activity was identified due to the Test pathogens, Aspergillus niger Fusarium
formation purple colored zone. solanii and, Fusarium oxysporum were
obtained from department of Biotechnology,
Plant growth promoting attribute of fungal GBPEC, Pauri, Garhwal, Uttrakhand. Dual
endophytes culture technique was adopted for
antagonistic test against these pathogens on
Phosphate solubilising activity PDA plates. Six-day-old mycelia disks of
(5mm diameter) of test pathogens were placed
The fungal cultures were inoculated on centre on one corner of Petri plates containing PDA
of Pikovskaya’s medium under aseptic. medium. Fungal endophytes were inoculated
Inoculated plates were incubated for 7-10 on the other corner of PDA plates. Plates were
days at 27°C. The clear zone around the incubated at 28˚C for 6 days and antagonistic
colony showed positive phosphate index was accessed according to the
solubilisation ability (Agrawal and Agrawal, following formula:
2013).
Antagonistic Index:
Production of ammonia
RM: radius of the pathogen in the control

plate
Fungal isolates were tested for the production
rm: radius of pathogen in the dual culture
of ammonia peptone water is used. In each
plate
tube of 10ml peptone water freshly grown

cultures were inoculated and incubated for
Result and Discussion
48–72 h at 27°C.

A systematic study about the endophytic
Nessler’s reagent 0.5 ml of was added in each
fungal biodiversity in a forest plant,
tube. Development of brown to yellow colour
Cupressus torulosa D. Don. located in
indicate positive test for ammonia production.
Govind Ballabh Pant Engineering College

Campus, Pauri Garhwal, Uttarakhand were
Production of HCN
carried out to evaluate their extracellular

enzyme production and it role in biocontrol
For HCN production, fungal isolates were
activity. A total of eight endophytic fungi
screen for the production of hydrogen cyanide
were isolated from leaves of C. torulosa
by adapting the method of Lorck (1948).
D.Don by using different culture media.

These endophytic fungi were characterized
Briefly nutrient broth was amended with 4.4g
morphotypically using lactophenole cotton
glycine/l and fungal streaked on modified
blue using scotch tape techniques (Table 1).
agar plate. Then a Whatman filter paper No.1
All fungal endophytes were preserved in PDA
soaked in 0.5% picric acid and 2% Na CO
2 3 slants at 4ºC as well -80º C in glycerol stock
was placed in the lid of the Petri dish, which
for further use.
was then sealed with paraffin film. After four

days of incubation at 27°C, yellow to brown
The Majority of the recovered endophtytes
disc coloration of the paper indicated HCN
belong to the Ascomycota. Fungal endophytes
production (Agrawal et al., 2015).
are especially common among the

Ascomycota, representing at least five classes,
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dozens of families, and large numbers of amylase productivity achieved at 30˚C of
previously unknown species (Sharma et al., incubation was 0.28μ/ml and maximum
2016). Only one species from the collected fungal biomass was 5.618 g/25 ml and at pH
isolates in this study belong to the 7.0 of the cultural media showed maximum
Dothideomycetes (Petrini, 1986). amylase activity of 0.461μ /ml and the
maximum fungal biomass production was
Functional attributes of fungal endophytes 5.511 g/25 ml.

The relationship between the endophyte and Protease activity
its host plant may range from latent
phytopathogenesis to mutualistic symbiosis Out of eight a four x endophytes exhibited
(Panutahi et al., 2012). Like other organisms protease activity (Fig. 2). The protease
invading plant tissues, endophytic fungi activity was observed in Biosporus sp.,
produce extracellular hydrolases (Table 2) as Aspergillus sp., Cladosporium sp., Curvularia
a resistance mechanism against pathogenic sp., Rhizoctonia sp., Chaetomium sp.,
invasion and to obtain nutrition from host. Cladosporium sp. indicated by formation of
Such enzymes include pectinases, cellulases, clear zone around the colony because of
lipases, laccase from the endophytic fungus degradation of gelatin, while other two
Monotospora sp., xylanase, -1, 4- isolates Colletotrichum sp., Fusarium sp.
glucanlyase, phosphotases and proteinase from the medicinal plants Citrus limon and
(Sunitha et al., 2013). Hydrolytic enzymes Gossypium hirsutum respectively indicated
(amylase, cellulase and laccase) with various negative results. Similar result was reported
industrial applications are also of major by Pavithra et al., 2012, where out fungal
interest. Hydrolases also reported from Pinus isolates from Ocimum sanctum were found
rouxburghii by Bhardwaj et al., (2015) positive for protease.

Amylase activity Lipase activity

All fungal endophytes were able to produce These all eight isolates were screened for
extracellular amylase (Fig. 1). The amylolytic lipase activity seven isolates such as
potential of these endophytes may help them WCTS21, WCTS33, PCTS13, PCTS21,
to degrade starch which is available when the PCTS25, KCTS15 and KCTS34 produce
plant senesces. The isolate KCTS34 was show lipase activity while one isolate WCTS31 was
highest amylase activity with the diameter of not produce lipase activity (Fig. 3). The
76 mm The Amylase activity was found to be isolate PCTS21 was show highest lipase
higher with Tuber aestivum than with Tuber activity with the diameter of 64 mm.
maculatum (Nadim et al., 2015). Rhizoctonia Cladosporium sp. of Phyllanthus emblica
sp. showed highest production of amylase medicinal plant was the maximum producer
enzyme i.e. 0.26U/ml (Patil et al., 2015). of lipase activity (Patil et al., 2015). Amirita
Endophytic fungi such as Penicillium et al., (2012) reported lipolytic activity of
frequentans isolated from Pinus rouxburghii Curvularia brachyspora, C. vermiformis,
were screened for amylolytic activity. Drechslera hawaiiensis, Colletotrichum
Influence of various physical and chemical falcatum and Phyllosticta sp. isolated from
factors such as pH, temperature, carbon and medicinal plants. Venkates agowda et al.,
nitrogen sources on amylase production by P. (2012) reported lipolytic endophytic fungal
frequentans in liquid media was studied isolates from the different oil-bearing seeds.
(Bhardwaz et al., 2015). The maximal
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Cellulase activity Plant growth promoting activity of
endophytic fungi
All fungal endophytes were able to produce
extracellular cellulase (Fig. 4). The isolate Many endophytes are reported to be capable
KCTS34 was show highest cellulase activity of nitrogen (N) fixation, solubilization of
with the diameter of 50 mm. Cellulolytic phosphate, enhance uptake of phosphorus (P),
activity was prominent in Talaromyces production of siderophores, ACC deaminase,
emersonii (Ci1), followed by Discosia sp. (Ci and plant hormones such as auxin, abscisins,
5), of Calophyllum inophyllum and ethylene, gibberellins, and indole acetic acid
Drechsclera sp. (Cr2) from Catharanthus (IAA), which are important for plant growth
roseus. The production of cellulase was not and development regulation (Selim et al.,
significant from isolates of other two plants. 2012).
Only 32% of the endophytes tested were able
to produce cellulose (Sunitha et al., 2013). Phosphate solubilizing activity

Similar result was reported by (Maria et al., Eight isolates were screened for Phosphate
2005) from mangrove angiosperm isolates. solubilizing activity, only six isolates such as
However, 66% cellulolytic activity was WCTS21, WCTS33, PCTS13, KCTS15,
reported by (Choi et al., 2005) from isolates PCTS25 AND KCTS34 showed the
of Brucea javanica. Bezerra et al., (2003) Phosphate solubilizing activity while two
reported 53.84% cellulolytic activity of isolates such as WCTS31 and PCTS21 were
endophytes from Opuntiaficus-indica Mill., not produce Phosphate solubilizing activity
Cladosporium cladosporioides with (Fig. 5 and Table 3).
maximum activity.
Nahas (1996) reported that among the
Chitinase activity microbial group, fungi are more efficient in
solubilizing phosphate than bacteria. The
All fungal isolates exhibited the chitinase highest range was observed in T.
activity. The isolate PCTS13 showed the peudokoningii (37.45±2.78 to 64.32±2.87)
highest chitinase activity with the diameter of µg/ml followed by C. globosum (33.62±5.92
53 mm while the isolate WCTS31 showed the to 69.32±3.21) µg/ml, F. semitectum
lowest chitinase activity with the diameter of (32.64±1.89 to 57.63±2.11) µg/ml, A.
20mm. the chitinase activity of 0.00093 U/mL versicolor (31.63±2.02 to 63.72±2.36) µg/ml
in Streptomyces sp., which corresponds to a (Chadha et al., 2015).
specific activity of 0.050 U/mg protein, was
four times lower than that in S. viridificans Production of ammonia
(0.0038 U/mL) (Gupta et al., 1995).
All fungal isolates exhibited the ammonia
As with Trichoderma sp, S. elegans is capable production (Fig. 6, Table 3). Most of the
of degrading Rhizoctonia solani cell walls ammonia produced by fungi will be
(Morissette et al., 2003) and releases the transformed to ammonium in solution.
CWDEs, β-1,3-glucanases and chitinases, into However, at higher pH a larger proportion of
culture medium amended with R. solani cell the ammonium becomes toxic unionized
wall fragments or with chitin as a carbon ammonia; the approximate ratios of NH + %
3
source (Tweddell et al., 1994). to NH will be 1800:1 at pH 6, and 9:1 at pH
3
8±3. Ammonia is very toxic because it is lipid
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soluble and raises intracellular pH, thus Radial growth of the pathogen was
inhibiting protein synthesis and enzyme considerably hindered by all the test
activity (Doyle and Butler, 1990). antagonists under the conditions of this study.
WCTS21 was the most antagonistic and
Production of HCN inhibited the radial growth of the pathogen
most while KCTS34 was the least
All eight fungal endophytes isolates screened antagonistic. The fungal isolate PCTS25 was
for the production of HCN and any isolates more active against A. niger with antagonistic
was not produce HCN. HCN is the common index 75.55 while WCTS31 more active
secondary metabolite produced by against F. oxysporum with antagonistic index
rhizosphere Pseudomonas (Schippers, 1988). 42.18 (Figs. 8, 9 and 10; Table 4) and isolate
Meena et al., (2001) compared the HCN WCTS21 is more active against F. solani with
production of several strains of P. fluorescens antagonistic index 51.66. Aspergillus flavus,
and their efficacy in controlling root rot of A. fumigatus, A. niger, A. sydowi, A.
groundnut caused by M. phaseolina. sulphureus, Penicillium sp. and three
biocontrol agents namely Trichoderma
Antagonistic activity of endophytic fungi harzianum, T. Koeningii and T. viride were
isolates tested against one plant pathogen namely
Pythium debaryanum. The percentage
The isolates were tested for antagonistic inhibition of growth of pathogen against
activity by the dual culture technique. A total Trichoderma harzianum, T. koeningii and T.
of eight endophytic fungal isolates out of viride were 66.6, 62.5, 60.4 percentage with
seventeen were showed antagonism activity respectively (Gomathi et al., 2011).
against A. niger, F. oxyspourm and F. solani.

Table.1 Morphotypic characterization of endophytic fungi

Code of Source of Colony Probable Class
isolate Endophytic charecterstics on endophytic
Fungi PDA media fungus
PCTS13 Leaves Appears olivaceous Penicillium Ascomycetous
brown in colour oxalicum
PCTS21 Leaves Appears grayish Alternaria Dothideomycetes
green in colour alternata
KCTS34 Leaves Appears cottony Daldinia sp. Zygomycetes
white in colour
WCTS31 Leaves Whitish Fusarium Sordariomycetes
appearance, rapid circinatum
growth horizontally
WCTS33 Leaves Whitish Pestalotiopsis Sordariomycetes
appearance, cotton versicolor
growth
WCTS21 Leaves Brown Penicillium Sordariomycetes
megasporum
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Table.2 Extracellular enzymatic activity of endophytic fungi

S.NO. Isolates Diameter of clear zone (in mm)

Amylase Protease Lipase Cellulase Chitinase
activity activity activity activity activity
1 WCTS21 30 50 48 48 36
2 WCTS31 65 40 - 42 20
3 WCTS33 20 63 60 20 30
4 PCTS13 40 - 48 34 53
5 PCTS21 60 - 64 50 25
6 PCTS25 54 - 52 36 34
7 KCTS15 68 46 30 30 30
8 KCTS34 76 - 54 50 26

Table.3 Plant growth promoting activity of endophytic fungi

Strain/test Phosphate solubilizing Production of ammonia Production of HCN
activity

WCTS21 + + -
WCTS31 + + -
WCTS33 + + -
PCTS13 + + -
PCTS21 + + -
PCTS25 + + -
KCTS15 + + -
KCTS34 + + -

Table.4 Antagonistic activity evaluation of endophytic fungi against fungal pathogens

S. No. Isolates Antagonistic index (%)

Aspergillus Fusarium Fusarium solani

niger oxyspourm

1. WCTS21 44.44 34.37 51.66

2. WCTS31 55.55 42.18 28.33

3. WCTS33 55.55 21.87 15.00

4. PCTS13 60.00 37.50 43.33

5 PCTS21 48.88 37.50 15.00

6. PCTS25 75.55 34.37 23.33

7. KCTS15 44.44 37.50 46.66

8. KCTS34 44.44 34.37 28.33

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Fig.1 Amylase activity of endophytic fungi

Fig.2 Protease activity of endophytic fungi

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Fig.3 Lipase activity of Endophytic fungi

Fig.4 Cellulase activity of endophytic fungi

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