Diversity analysis and characterization of antagonistic
endophytic population from
Stevia rebaudiana
Ankita Verma
*, Sandeep Saini
, B. N. Johri
and Anil Prakash
Department of Biotechnology, Barkatullah University, Bhopal-462026
Department of Biotechnology, Faculty of Science, RKDF University, Bhopal-462036
Antagonistic endophytic fungal community resident in medicinal plant Stevia rebaudiana Bertoni was studied at two
sites within Bhopal, M.P. Among 339 recovered endophytic isolates from foliar tissues, 40 fungal isolates were found
antagonistic against Sclerotinia sclerotiorum, casual agent of disease stem rot in stevia and soybean (Glycine max.).
Antagonistic fungal population (40 isolates) consisted of 52.5% Hyphomycetes and 2.5% each of Coleomycetes, Basidi-
omycetes, Ascomycetes and Sterile mycelia. The percent colonization frequency of antagonistic endophytic community
in foliar tissues ranged from 0.3%-5.3% whereas percent dominance was of the order, ranged from 2.31%-40.8%. Diver-
sity analysis of the antagonistic endophytic population was determined in terms of Shanon index, Simpson index, Spe-
cies eveness, Menhinick and Margalef richmess index .Antagonistic endophytic population was also evaluated for IAA
production, siderophore and phosphorus solubilisation, considered as plant growth promotory attributes. Identi cation
of the antagonistic endophytes was carried out by rDNAsequencing of the ITS region.
Biosci. Biotech. Res. Comm. 11(4): 754-765 (2018)
Fungal endophytes possess huge diversity morpho-
logically and biochemically (Strobel and Daisy, 2003).
Endophytic fungi are known to reside in the tissues of
plants above ground as well as below ground, parts of
the plant (Zhang et al. 2006; Kusari et al. 2012). Endo-
phytic fungi are an assemblage of microorganisms that
chie y belong to class Ascomycetes of kingdom fungi.
A signi cant literature is available so far to show that
these microorganisms, under laboratory culture condi-
tions, produce numerous structurally diverse biologi-
cally active secondary metabolites that include antimi-
crobial substances. Different ecological factors such as
seasonality, nearby vegetation and humidity in uence
the distribution of endophytic fungi in the host (Taylor
Corresponding Authors: ankita.verma1234@gmail.com
Received 19
Sep, 2018
Accepted after revision 23
Dec, 2018
BBRC Print ISSN: 0974-6455
Online ISSN: 2321-4007 CODEN: USA BBRCBA
Thomson Reuters ISI ESC / Clarivate Analytics USA
Mono of Clarivate Analytics and Crossref Indexed
Journal Mono of CR
NAAS Journal Score 2018: 4.31 SJIF 2017: 4.196
© A Society of Science and Nature Publication, Bhopal India
2018. All rights reserved.
Online Contents Available at: http//www.bbrc.in/
DOI: 10.21786/bbrc/11.4/28
Ankita Verma et al.
et al; 1999; Toofanee and Dulymamode; 2002 Lumyong
et al 2009, Dissanayake et al., 2016; Ratnaweera et al.,
2017, Ratnaweera et al., 2018).
Stevia rebaudiana Bertoniis an herbaceous polyan-
nual plant of the sun ower family (Fam. Asteraceae),
generally known as candy leaf,
sweet leaf, sweet leaf,
orsugar leaf. The Stevia leaves also contain variety of
glycosides compounds viz.,  avonoid glycosides, Stevi-
oside, Rebaudioside A, Rebaudioside C, coumarins, cin-
namic acids, phenylpropanoids and some essential oils,
(Midmore and Rank, 2002 Lavini et al., 2008).
Stevia rebaudiana Bert. Is a good source of sweeten-
ers and is about 300 times sweeter than sucrose owing
to presence of steviosides in its leaves. Previous stud-
ies have reported clearly the diversity of these fungal
fungal endpphytes from Stevia rebaudiana which also
indicates the presence of Alternaria,, Aspergillus, Mono-
dictys, and Curvularia fungal genus from leaf of Ste-
via rebaudiana (Bert.). (Prakash et al., 2008; Madhumita
and Chandra, 2013) Furthermore, these fungal isolates
have been reported from almost all climatic regions of
the globe viz., tropical, temperate and alpine (Arnold,
2007; Halmschlager et al 1993; Higgins et al 2007). The
application of biocontrol agents has become one of the
most promising tools for reducing the use of chemical
pesticides in agriculture. The antagonism of biocontrol
agent is based on different mechanisms i.e. nutrients,
mycoparasitism, plant growth promotion and induction
of the defense responses in plants (Howell, 2003, Sen
et al, 2012; Hamzah et al, 2018).
In the present investigation the endophytes recov-
ered from Stevia rebaudiana leaves have been tested for
antagonistic abilities against Sclerotinia sclerotiorum,
which is the major phytopathogen affecting varieties of
crop plants in Central India (Prakash et al., 2008, Verma
et al 2004).
Therefore, present investigation was carried out to
understand the generic diversity of endophytic fungi in
leaves of Stevia rebaudiana Bertoni and to compare the
antagonistic endophytic assemblages in samples collected
from two different sites in the same region. Thus a speci c
rationale for the selection of stevia plant for endophyte
isolation and natural product discovery is used.
Stevia rebaudiana Bertoni: Stevia rebaudiana Bertoni
was selected as the target plant for isolation of fungal
endophytes. Sampling was carried out from two sites.
The  rst site was Misrod (23º16’N; 76º36’E), a village
situated nearly 22 km away from the capital city Bhopal
in the state of Madhya Pradesh. The second site of sam-
pling was green house grown plants in the campus of
(23º20’N; 77º45’E) Barkatullah University, Bhopal.
Samples Collection and Surface sterilization: Healthy
and mature plants were carefully chosen for sampling
and leaves were collected randomly. Plant material was
brought to the laboratory in sterile bags. Surface steri-
lization of foliar tissues were done using reagents like
70% ethanol, 4% sodium hypochlorite and sterile dis-
tilled water for different time period for effective surface
sterilization process. Leaves were thoroughly washed
several times in sterile distilled water (SDW) for 5-10 sec
then sterilized by exposing them to 70% ethanol for 2
min followed by treatment with 4% sodium hypochlorite
for 2 min. The leaves were now immersed in sterile water
(SDW) for 2-5 sec and allowed to dry on blotting paper.
Isolation of endophytic fungi from foliar tissues of
plants: The surface sterilized leaf segments of 5mm size
were placed on Potato dextrose Agar (Howksworth et al
1995), supplemented with chloramphenicol (0.2gl-1) to
avoid bacterial contamination. Plates were incubated at
28±2 ºC in for 3-5 days and were observed regularly for
fungal growth.
Analysis of data: The colonization frequency (% CF)
of endophytic fungi was calculated according to Hata
and Futai (1995) and dominance as per Kumaresan and
Suryanarayanan (2002). Utilizing the data of percent-
age colonization of fungal endophytes in leaves, for two
sites. Simpson’s Diversity indices and Shanon-Wiener
indices were calculated. Species evenness and species
richness was calculated according to Simpson (1949),
Shanon and Weaver (1949), Ludwig & Reynolds (1988)
and Margelef and Menhinick (1964)
In vitro antagonistic activity of fungal endophyte
Fungal isolates were screened for antagonism against
Sclerotinia sclerotiorum by a dual culture technique on
Potato Dextrose Medium (Szekeres et al, 2005).
Characterization of endophytes: The endophytic
fungi were identi ed by their macroscopic & micro-
scopic characteristics such as the morphology of the
fruiting bodies and spore morphology. Morphological
characterization was done on the basis of color, margin,
reverse pigmentation & texture. (Rifai 1969).Antago-
nistic endophytic fungi are characterized functionally
employing plate assays for amylase cellulase, Protease,
pectinase, lipase and xylanase (Paterson & Bridge. 1994;
Teather & Wood. 1982; Shakeri et al. 2007; Pointing.
1999; Sierra. 1957, Mishra et al, 2013, Aneja, 2003).
Plant growth promoting attributes: Plant growth
promoting attributes of antagonistic endophyte were
also studied. This included IAA (Brick et al. 1991) and
siderophore production (Schwyn & Neilands. 1987) and
phosphate solubilisation ef ciency (Pikovskaya. 1948).
Both qualitative and quantitative estimation were made.
Molecular identi cation of antagonistic endophyte:
- Morphological identi cation of the organism was car-
Ankita Verma et al.
ried out at National Fungal Culture Collection of India
(NFCCI), Agharkhar Research Institute, Pune. For molec-
ular identi cation, total genomic DNA of the endophytic
fungus was isolated directely from actively growing
mycelium growing in Potato dextrose broth (PDB), using
CTAB method (Sambrook and Russel, 2001). DNA ampli-
cation was performed by PCR using primer pair ITS1:
TATGC (White et al. 1990).PCR was carried out according
to the following protocol: initial denaturation at 95 ºC
for 5 min; denaturation at 95 ºC for 1 min; annealing at
55ºC for 45 sec; extension at 72 ºC for 10 min; steps 2-4
were repeated 35 times. Sequencing of PCR product was
carried at Xcelris Labs Ltd, Ahmedabad. The sequenced
data was subjected to BLAST algorithm and submitted to
Genebank for accession number. The potential antago-
nistic endophytes were submitted at National Agricul-
turally Important Culture Collection (NAIMCC), culture
collection facility at ICAR-NBAIM, Maunath Bhanjhan
Phylogenetic analysis:To know the phylogenetic
relationship among the isolates and also to confirm
their taxonom ical status, certain ITS rDNA sequences
were chosen from GenBank databases via BLAST search
analysis. The sequences were chosen from the top 20
database hits obtained in the blast search by querying
the obtained sequences individually. These sequences
were aligned using CLUSTAL W 1.83 (Thompson et al.,
1994). Phylogenetic trees were generated by neighbour-
hood joining method with 100 bootstrapping replicates
using MEGA version 5.
Antagonistic Action and Diversity Analysis: A total of
339 recovered endophytic fungal isolates were screened
for antagonistic ability against Sclerotinia sclerotiorum
(culture obtained from Directorate of Soybean Research,
Indore, M.P.) by using dual culture technique. The inhi-
bition zone in dual plate assay averaged between 5 to
17 mm. Misrod  eld site harboured greater number of
antagonists compared to the endophytes recovered from
green house raised plants. Percent growth reduction of
the pathogenic culture was recorded after 24 hrs. Iso-
late Aspergillus  avipes (NAIMCC-F-03153) strain 63
showed highest value of growth reduction i.e. 19% after
24 hrs followed by Alternaria alternata strain 99 and
Aspergillus niger strain 89 (NAIMCC-F-03157) showed
17% and 18% growth reduction respectively (Fig 1);
Least reduction of pathogen was recorded for isolate
Alternaria brassicae strain 17 i.e. 6.9%. The percent
growth reduction values ranged from 6.9% to 19%.
A total of 40 endophytic fungal isolates which showed
antagonistic activity against Sclerotinia sclerotiorum
consisted of 52.5% Hyphomycetes followed by 2.5%
Coleomycetes, Basidiomycetes, Ascomycetes and Sterile
mycelia each. The percent colonization in tissues sam-
ples ranged from 1% - 36.6% (site 1) whereas 1%-10.6%
(site 2) (Fig 2) and percentage dominance of antagonistic
endophytes ranged from 1.1%-40.6% (site 1) and 4.3%-
45.3% at site2 (Fig 3). Diversity analysis of the antago-
nistic population was carried out which showed signi -
cant diversity index values at site 1 as compared to site
2, whereas Margalef & Menhinick’s richness index value
was maximum at site 2 as compared to site 1 (Table 1)
Characterization of antagonistic fungal endophytes
Morphological characterization of antagonistic fungal
endophytes: Based on the morphology different antago-
nistic endophytic fungal isolates were recovered on PDA
plate. Among 40 antagonistic endophytic isolates 62.5%
showed reverse pigmentation, 15% showed velvety
appearance on PDA plate while others appeared spory
and cottony texture. Isolates Aspergillus niger strain 89
FIGURE 1. Dual culture assay shown by isolate A) Strain 63 B) Strain 89 C) Strain 99
Ankita Verma et al.
FIGURE 2. % Colonization frequency of the antagonistic endophytic population from both sites.
FIGURE 3. % Dominance of the antagonistic endophytic population from both sites.
Table 1. Diversity indices of two sites (Site
1 & Site 2)
Diversity indices Site I Site II
Simpson index (1-D) 0.821 0.7982
Shanon index (H’) 1.856 1.781
Eveness (EH/s) 0.8001 0.8478
Menhinick S/√N 0.6305 1.121
Margalef S-1/ln(n) 1.378 1.638
(NAIMCC-F-03157), Aspergillus niger strain 88, Asper-
gillus sp. strain 37 (NAIMCC-F-03147), Aspergillus  a-
vipes strain 63 (NAIMCC-F-03153) and Aspergillus niger
strain 50 (NAIMCC-F-03151), showed dispersed growth
on PDA medium.
Functional characterization of antagonistic fun-
gal endophytes: The antagonistic fungal endophytes
recovered from Stevia, were checked for their hydro-
lytic potential. They were screened for multiple enzyme
activity on starch, pectin, lipid, carboxy methyl cellu-
Ankita Verma et al.
Table 2. Plant growth promoting attributes of antagonistic endophytes
S.no Isolates Identity IAA (μg/ml)
% solubilising ef ciency
of Phosphate
1 SR/II/2 Alternaria porri - 8.42±0.20 75
2 SR/II/4 Alternaria alternata 6.2±0.03 1.6±0.20 52
3 SR/II/5 Alternaria brassicae - 3.79±0.05 71.4
4 SR/II/6 Alternaria porri 3±0.04 6.2±0.06 60
5 SR/II/42 Alternaria sp. 8±0.03 3.72±0.08 -
6 SR/II/45 Penicillium mallochii 6.3±0.05 4.2±0.04 32
7 SR/II/50 Aspergillus niger 4.8±0.01 - 6.25
8 SR/II/52 Phoma sp. - 6.6±0.03 10
9 SR/II/54 Phoma sp. 4.6±0.02 8.7±0.03 20
10 SR/I/76 Alternaria alternata - 3.2±0.02 5.4
11 SR/I/77 Alternaria sp. 5.5±0.03 6.9±0.02 22
12 SR/I/78 Alternaria tenuissima 6.5±0.02 8.02±0.03 11
13 SR/I/94 Alternaria alternata 3.8±0.01 2.8±0.03 8
14 SR/I/99 Alternaria alternata 5.3±0.3 - 36
15 SR/II/63 Aspergillus  avipes 4.6±0.04 11.12±0.16 22.2
16 SR/II/1 Alternaria alternata - 17.09±0.1 -
17 SR/II/9 Alternaria alternata 9.8±0.6 7.45±0.12 17.3
18 SR/II/20 Alternaria sp. 6.3±0.06 8.03±0.11 70
19 SR/II/21 Alternaria alternata - 8.03±0.11 50
20 SR/II/23 Chaetomiumglobosum 6.7±0.05 15.51±0.12 14
21 SR/II/24 Alternaria alternata - 14.63±0.14 12
22 SR/II/26 Alternaria alternata 1.9±0.04 3.15±.10 10
23 SR/II/33 Phoma sp. 4.5±0.01 1.66±0.031 -
24 SR/II/36 Alternaria brassicae 8.2±0.7 27±20.22 65
25 SR/II/55 Alternaria alternata - 4.42±0.14 14
26 SR/II/60 Ustilago tritici 5.5±0.04 9.51±0.17 -
27 SR/I/72 Alternaria brassicae - 23±0.08 34
28 SR/II/81 Alternaria alternata - 22±0.06 -
29 SR/I/85 Alternaria alternata 5.7±0.05 6.07±0.03 37
30 SR/I/95 Alternaria alternata - 4.5±0.04 -
31 SR/II/100 Alternaria alternata 5.8±0.07 5.8±0.07 19
32 SR/I/83 Alternaria alternata 4.5±0.05 6.18±0.19 -
33 SR/II/18 Phoma sp. - 1.3±0.02 85.7
34 SR/II/37
Aspergillus sp. 11±0.01 13.3±1.12 -
35 SR/II/46 Aspergillus sp. 6.8±0.06 8.06±0.07 -
36 SR/I/88 Aspergillus niger 8.1±0.2 - -
37 SR/I/89 Aspergillus niger 11.2±0.4 6.72±1.4 -
38 SR/I/90 Alternaria alternata - - 35
39 SR/II/3 Sterile mycelia - 8.42±0.20 85.7
40 SR/II/17 Alternaria brassicae 6.25±0.05 24.33±0.12 -
lose, xylan and skim milk. Endophytes exhibited good
amylolytic, cellulolytic, proteolytic, pectinolytic activi-
ties while xylanolytic and lipolytic activities were pos-
sesed by only few isolates. The con rmation of enzy-
matic activity was recorded by the presence of zone
of clearance around the culture. Alternaria alternata
strain 76 showed maximum zone of about 15 mm on
starch agar plate while 11 mm zone was recorded for
Ankita Verma et al.
cellulolytic activity by isolate Alternaria alternata strain
26.Maximum pectinolytic activity was recorded by iso-
late Alternaria alternata strain 99, whereas isolate Alter-
naria alternata strain 4 (NAIMCC-F-03138), showed
maximum zone (11 mm) of clearance on CMC plate. The
isolates Aspergillus sp. strain 46, Phoma sp. strain18,
Phoma sp. strain 33 and Alternaria alternata strain 94,
Aspergillus niger strain 50, Alternaria porri strain 6
(NAIMCC-F-03139), and Alternaria brassicae strain 5
(NAIMCC-F-03140), showed minimum inhibition (2mm)
on same substrate.
Plant growth promotiong attributes of antagonistic
endophytes:The antagonistic endophytes were studied
for plant growth promoting traits such as siderophore
production, phosphate solubilisation, Indole acetic acid
production. A comprehensive overview of the PGP
traits of antagonistic endophytes is given in the Table
2. About 40% of the antagonistic endophytes were
positive for siderophore production and the amount of
siderophore produced ranged between 1.3-27 mgml
On Pikovyaskya’s agar, 31% of the antagonistic endo-
phytes showed phosphate solubilisation; ef ciency of P
solubilisation ranged between 5.4%-85.7percent. Based
on the results isolates Phoma sp. strain 18 and Sterile
mycelia strain 3 were found to be most ef cient P solu-
bilizer. A total of 29% of the antagonistic endophytes
were positive for IAA production which ranged between
(Table 2).
Sequence analysis of ITS region of rDNA gene fragments
All the selected isolates produced a single PCR prod-
uct with approximately 600 bp. Puri cation of the PCR
product was performed using Banglore Genei puri-
cation kit and sequencing was performed by Xcelris
Genomics, Ahemdabad using the same set of primers as
mentioned earlier. The full length sequences of the iso-
lates were compared with the related fungal sequences
in the GenBank databases and sequence similarities were
determined using BLAST sequence similarity search tool
(Altschul et al., 1990). The sequences of ITS region of
rDNA gene of the fungal endophytes were deposited in
the GenBank and given accession numbers (Table 3).
Phylogenetic analysis
To know the phylogenetic relationship among the iso-
lates and also to confirm their taxonom ical status, cer-
tain ITS rDNA sequences were chosen from GenBank
databases via BLAST search analysis. The sequences
were chosen from the top 20 database hits obtained in
the blast search by querying the obtained sequences indi-
vidually. These sequences were aligned using CLUSTAL
W 1.83 (Thompson et al., 1994). Phylogenetic trees were
generated by neighbourhood joining method with 100
bootstrapping replicates using MEGA version 5.( Fig:4)
Characterization of fungal endophytes from Stevia
rebaudiana Bertoni was considered important because
only few attempts have been made earlier to character-
ize fungal endophytes from this useful plant, (Begum
et al 2008; Kumari and Chandra 2013). Various fungal
diseases have been reported to pose serious problems
to S.rebaudiana Bertoni commonly known as Stevia, a
popular non calori c sweetener. These include Verticil-
lium dahlia on leaves (Farrar et al., 2000), S. sclerotio-
rum reported in Canada (Chang et al., 1997), S. rolfsii in
India (Kamalakannan et al., 2007) and Botryis cinerea in
Itlay (Garibaldi et al., 2009). Sclerotinia stem rot (white
mold) of soybean was  rst reported in Hungary in 1924
and since has been reported in Argentina, Brazil, Canada,
India, Nepal, South Africa and United States. A great
economic loss to crop plants by different phytopatho-
gens results in low yields. The species composition of
the endophytic assemblage and frequency of infection
varies according to host species and site characteristic
such as elevation, exposure, associated vegetation, tis-
sue type (Fisher et al., 1994) and tissue age (Fisher et al.,
1986; Rodrigues, 1994).
In the present study, foliar endophytic microorgan-
isms were studied using various diversity indices viz
Simpson index (Simpson, 1949), Shannon index (Shan-
non and Weaver, 1949), evenness index (Ludwig and
Reynolds, 1988) and richness index (Margalef, 1958;
Menhinick, 1964). Higher value of Shannon index and
evenness index with lower values of Simpson index
indicated greater diversity. Bills et al. (2002) described
a signi cant difference between tropical and temperate
endophytes, in terms of their ability to produce num-
ber of bioactive natural compounds isolated from endo-
phytes. This observation suggests the importance of
the host plant in in uencing the general metabolism of
endophytic microbes.
Among 339 recovered endophytic fungal isolates,
40 isolates were screened out as potential antagonistic
endophytes against broad spectrum plant pathogen S.
sclerotiorum following using dual culture technique. It
was found that maximum numbers of antagonistic endo-
phytes were recovered from site1 as compared to site 2.
This may be explained as site 1 is open agricultural  eld
which was exposed to wide variety of phytopathogens
so in order to overcome these pathogens several bioac-
tive compounds are produced by them whereas site 2
is closed area which was exposed to limited number of
phytopathogens. Sadrati et al. (2013) screened 20 endo-
phytic fungi from wheat which showed antimicrobial
activities against 12 pathogenic bacteria, yeast and two
phytopathogenic fungi. Percentage growth inhibition
ranged between 6.9%-19% after 24 hr of incubation.
Ankita Verma et al.
Table 3. Genetic relatedness of GenBank to antagonistic fungal endophytes recovered from
Stevia rebaudiana Bertoni using ITS rDNA gene sequence analysis
Isolate No. Site Accession no. % Similarity Organism
SR/II/2 1 KJ592050 99% Alternaria porri
SR/II/4 1 KJ592051 100% Alternaria alternata
SR/II/5 1 KJ592052 99% Alternaria brassicae
SR/II/6 1 KJ603463 98% Alternaria porri
SR/II/42 1 KJ713969 100% Alternaria sp.
SR/II/45 1 KJ713970 99% Penicillium mallochii
SR/II/50 1 KJ648618 100% Aspergillus niger
SR/II/52 1 KJ713971 99% Phoma sp.
SR/II/54 1 KJ648619 99% Phoma sp.
SR/I/76 1 KJ713972 99% Alternaria alternate
SR/I/77 2 KJ713973 100% Alternaria sp.
SR/I/78 1 KJ728832 100% Alternaria tenuissima
SR/I/94 1 KJ728833 100% Alternaria alternate
SR/I/99 1 KJ728834 99% Alternaria alternate
SR/II/63 1 KF671231 94% Aspergillus  avipes
SR/II/1 1 KJ728835 100% Alternaria alternate
SR/II/9 1 KJ735925 99% Alternaria alternate
SR/II/20 1 KJ728836 98% Alternaria sp.
SR/II/21 1 KJ728837 99% Alternaria alternata
SR/II/23 1 KJ728838 100% Chaetomiumglobosum
SR/II/24 1 KJ728839 99% Alternaria alternate
SR/II/26 1 KJ728840 100% Alternaria alternate
SR/II/33 1 KJ728841 100% Phoma sp.
SR/II/36 1 KJ728842 99% Alternaria brassicae
SR/II/55 1 KJ728843 99% Alternaria alternata
SR/II/60 1 KJ735919 100% Ustilago tritici
SR/I/72 1 KJ735920 99% Alternaria brassicae
SR/II/81 1 KJ735921 100% Alternaria alternata
SR/I/85 1 KJ735922 100% Alternaria alternata
SR/I/95 1 KJ735923 100% Alternaria alternata
SR/II/100 1 KJ735924 100% Alternaria alternata
SR/I/83 2 KJ748009 100% Alternaria alternata
SR/II/18 1 KJ748010 100% Phoma sp.
SR/II/37 1 KJ767528 100% Aspergillus sp.
SR/II/46 1 KJ767529 100% Aspergillus sp.
SR/I/88 1 KJ767530 100% Aspergillus niger
SR/I/89 1 KJ767531 100% Aspergillus niger
SR/I/90 1 KJ767532 100% Alternaria alternata
SR/II/3 1 NS NS Sterile mycelia
SR/II/17 1 KJ767533 100% Alternaria brassicae
1= Misrod Agriculture Field 2= Green House NS= Not Sequenced
Ankita Verma et al.
FIGURE 4. Phylogenetic tree generated on the basis of ITS rDNAsequences of antagonistic endophytic fungal
isolates aligned with sequences in GenBank databases
Diversity analysis of the antagonistic endophytic popu-
lation showed signi cant diversity index values. Maxi-
mum antifungal activity against S. sclerotiorum was
recorded by Aspergillus  avipes strain 63 followed by
strain Aspergillus niger strain 89 and Alternaria alter-
nata strain 99 after 24 hrs of incubation.
For instance, site1 is open agricultural  eld with sur-
rounding vegetation like wheat and soybean, which
favours the establishment of endophytic colonization
whereas site 2 is devoid of natural open conditions of
environment which seems to be the major factor for tis-
sue speci c uctuations in the recovery of endophytes
The increased species richness in foliar tissues may be a
result of super infection of the leaves overtime by air-
borne inocula (Carroll et al., 1977; Suryanarayanan and
Vijaykrishna, 2001).
The 40 antagonistic endophytic fungal isolates were
studied for morphological, functional and genotypic
characterization. In the present study, signi cant func-
tional diversity was observed among the antagonistic
endophytes with respect to their hydrolytic potential
viz. amylolytic, cellulolytic, lipolytic, proteolytic, pec-
tinolytic and xylanolytic activities. 19% of the antago-
nistic endophytic fungal population showed amylase,
cellulase, pectinase and protease production whereas
17% were xylanase producing and rest 7% were lipase
producers. Fifty fungal strains isolated from medici-
nal plants (Alpinia calcarata, Bixa orellana, Calophyl-
lum inophyllum and Catharanthus roseus) showed 64%
were lipase producers, 62% were amylase and pectinase
whereas 32% were cellulase and 30% were laccase pro-
ducers (Sunitha et al 2013). Begum et al. (2008) reported
Ankita Verma et al.
that majority of the endophytes from Stevia leaves were
cellulolytic in nature. Suganthi et al. (2011) isolated and
characterized Aspergillus niger (BAN 3E) out of  ve fun-
gal isolates as the most potent -amylase producer. Sid-
key (2011) found endophytic strain F2Mbb to produce
extracellular amylase.
These potential antagonistic endophytes were screened
for plant growth promoting attributes like siderophore
production, Indole acetic acid production and phos-
phate solubilisation. In present study, about 40% of the
antagonistic endophytes were positive for siderophore
production and amount of siderophore produced ranged
between 1.3-27 mg ml
, 31% of the antagonistic endo-
phytes showed positive results for phosphate solubilisa-
tion. Phosphate solubilisation ef ciency ranged between
5.4-85.7% whereas 29% of the antagonistic endophytes
were recorded positive for IAA production between the
range 1.9-11 μg ml
.Certain endophytes were observed
to improve the ecological adaptability of host enhanc-
ing their tolerance to environmental stress and resist-
ance to phytopathogens (Kimmon et al., 1990; Struz et
al., 1999). In a study performed on Absidia corymbifera,
fungi isolated from rhizospheric soil, were found to pro-
duce siderophore in the range of 4-4.55 μg ml
berg and Artis, 1983). Maliha (2004) found Aspergillus
avus, Aspergillus niger and P.canescens as the most
potent phosphate solubilizers, (Bilal et al, 2018).
Sequence analysis revealed that majority of fungal
endophytes belonged to Alternaria alternata followed
by Aspergillus niger, Phoma, Chaetomium globosum,
and Ustilago tritici. Mandyam et al. (2010) employed
sequencing of ITS region for studying Dark septate
endophytes (DSE) in annually burned tallgrass prairie. In
a nut shell, present investigation has shown that Stevia
rebaudiana Bertoni harbours a good deal of antagonistic
fungal endophytic community. These endophytes have
exhibited various characteristics features which may
pose better  tness to Stevia plant and reveal ecological
signi cance of endophyte- host relationship.
First author is supported as Junior Research Fellowship
from M.P.Biotechnology Council, Bhopal. Authors are
grateful to Dr S.K. Singh, Coordinator, National Facility
(NFCCI), Pune for identi cation of fungi. Financial sup-
port from NASI for sequencing of the samples is highly
acknowledged. First author is extremely thankful to Dr.
S.K. Sharma, Incharge NAIMCC at ICAR-NBAIM, Mau-
nath Bhanjan (U.P) for culture deposition at his facil-
ity and providing accessions numbers. Authors are also
thankful to Dr. Nidhi Gujar for their help in conduct of
this research.
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