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

ABSTRACT

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.

KEY WORDS: DIVERSITY INDEX,

S. SCLEROTIORUM

, ITS REGION, ANTAGONISTIC ENDOPHYTES, SEQUENCE PHYLOGENY

754

Biotechnological

Communication

Biosci. Biotech. Res. Comm. 11(4): 754-765 (2018)

INTRODUCTION

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

ARTICLE INFORMATION:

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

Online Contents Available at: http//www.bbrc.in/

DOI: 10.21786/bbrc/11.4/28

Ankita Verma et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS DIVERSITY ANALYSIS AND CHARACTERIZATION OF ANTAGONISTIC ENDOPHYTIC POPULATION 755

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.

MATERIALS AND METHODS

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.

756 DIVERSITY ANALYSIS AND CHARACTERIZATION OF ANTAGONISTIC ENDOPHYTIC POPULATION BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

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:

TCCGTAGGTGAACCTGCGG and ITS4: TCCTCCGCTTGA-

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

(U.P).

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.

RESULTS

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

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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-

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758 DIVERSITY ANALYSIS AND CHARACTERIZATION OF ANTAGONISTIC ENDOPHYTIC POPULATION BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Table 2. Plant growth promoting attributes of antagonistic endophytes

S.no Isolates Identity IAA (μg/ml)

Siderophore

(mg/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

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BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS DIVERSITY ANALYSIS AND CHARACTERIZATION OF ANTAGONISTIC ENDOPHYTIC POPULATION 759

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

-1

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

1.9-11μgml

-1

(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)

DISCUSSIONS

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.

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760 DIVERSITY ANALYSIS AND CHARACTERIZATION OF ANTAGONISTIC ENDOPHYTIC POPULATION BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

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

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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.

762 DIVERSITY ANALYSIS AND CHARACTERIZATION OF ANTAGONISTIC ENDOPHYTIC POPULATION BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

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

-1

, 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

-1

.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

-1

(Holz-

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.

ACKNOWLEDGEMENTS

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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