Molecular characterization of endophytic fungi associated with Centella Asiatica linn. Inhabiting wildly in Arunachal Pradesh

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J. Indian bot. Soc.
e-ISSN:2455-7218, ISSN:0019 - 4468
MOLECULAR CHARACTERIZATION OF ENDOPHYTIC
FUNGI ASSOCIATED WITH CENTELLA ASIATICA LINN.,
INHABITING WILDLY IN ARUNACHAL PRADESH
RICHA SHARMA, SUMPAM TANGJANG AND
1AMRITESH C. SHUKLA
Department of Botany, Rajiv Gandhi University, Rono Hills, Doimukh, Itanagar- 791112 Arunachal
1
Pradesh, India, Department of Botany, University of Lucknow, Lucknow- 226007, Uttar Pradesh, India.
Email: amriteshcshukla@gmail.com
Date of online publication: 31st December 2020
DOI:10.5958/2455-7218.2020.00038.8
Microorganisms are found ubiquitously and
they are extremely flexible in all types of
ecosystems around the sphere. Studies express
that 5% of fungal species less than 1% of all
bacterial species are presently known, and at
least 11 million microbial species are
unrevealed, leftover species are hidden in
nature (Felício et al. 2016). De Barry (1866)
coined the term 'endophyte' which lives in
intercellularly and intracellularly in plant
tissues without causing any damage to the plant
(Jeewon et al. 2017). Endophytic fungi were
reported in approximately all the plants spp viz.
alg ae, mos s es, fer ns, and mainly in
angiosperms, gymnosperms reported from
various parts of the world (Doilom et al. 2017).
Numerous researchers have demonstrated that
endophyte is the potential source of novel
natural products for exploitation in modern
agriculture, medicine, and industry (Kaul et al.
2013). To date, it has been found that taxol can
be produced by endophytic fungi Metarhizium
anisopliae and C. cladosporioides (El-Maali et
al. 2018). Fungal endophytes are capable of
producing compounds similar to their host
plants and are able in the preservation of
world's diminishing biodiversity (Bender et al.
2016, Mane et al. 2017).
C. asiatica Linn has been considered as an
important medicinal plant and also been used
traditionally in Ayurvedic medicine for central
nervous system ailments including failing
memory, insomnia, depression, memory
enhancement stress, epilepsy, leprosy, wounds,
cancer, fever and syphilis antidepressant
activity, inflammations, diarrhea, asthma,
tuberculosis and various skin lesions and
ailments like leprosy, lupus and psoriasis
(Zheng and Qin 2007, Zainol et al. 2008,
Umbeliferae et al. 2009, Joshi and Chaturvedi
2013 ).
Studies on endophytic fungi determined on
medicinal plants have uncovered that the
curative property of the medicinal plant is not
only because of the chemicals present in the
plant but also because of the fungal endophytes
that present in the plant (Verma 2011,
Suryanarayanan 2013). Thus there is a need to
We describe the first time report on the diversity of endophytic fungi from the medicinal plant Centella asiatica Linn (Apiaceae) wildly
growing in the forest of Papum Pare, Arunachal Pradesh, India. The fungal endophytes were identified based on their morphological,
cultural and reproductive structures. Further, the phylogenetic analysis of the isolated species was identified, using the sequences of
5.8S and 28S rDNA internal transcribed spacer sequence 1 and 4. The largest number of fungal endophytes (39%) was isolated from
the leaves, followed by the roots (31%) and stems (30%). Overall twenty-eight fungal species have been isolated, out of which, 11
belongs to the class Dothideomycetes; 02 belongs to the Eurotiomycetes; 14 belongs to the class Sordariomycetes and 01 fungal
endophyte couldn't identify properly. The maximum species richness and frequency of colonization were recorded in leaf. The
observations show that Lasiodiplodia viticola was the dominant endophytic species followed by Colletotrichum gloeosporioides,
Bipolaris bicolour, Pithomyces chartarum, and Preussia sp. and others. However, Pestalotiopsis longiseta, Aureobasidium sp., C.
gloeosporioides and Fusarium merismoides were the common fungal endophytes recorded from all the plant parts viz., leaf, stem, and
roots from C. asiatica. The density of colonization (rD %) was recorded in the chronology of L. viticola 8.66% < C. gloeosporioides
(4.66%) < B. bicolour, Pithomyces chartarum and Preussia sp. (3.33%) and 0.66% to 1.33% for the remaining endophytes.
Key words – Biodiversity, colonization frequency, endophyte, internal transcribed spacer, medicinal plant, Arunanchal Pradesh.
Received on October 21, 2020 Accepted on October 24, 2020
www.indianbotsoc.org
Vol. 100 (3&4) 2020:160-168

isolate and identify the number of fungal
endophytes and subject them to screening to
secondary metabolites. Although, number of
studies have already been reported the
association of fungal endophytes with
medicinal plants from India such as Allium
sativum, Ashwagandha, Azadarichata indica,
Catharanthus roseus, Clerodendron serratum,
Coffea Arabica, Ficus religiosa, Mamordica
cha eanti a, Not hapody tes ni mmonia na,
Part h e n ium hysterophorus , Pongamia
gra e cum, S i l ybum m a rian u m , Taxus
brevifoliaTerminalia arjuna, Trigonella
foenum (Mane et al. 2017, Mane and
Vedamurthy 2018), but there is no such report
on fungal endophytic association with C.
asiatica.
MATERIALS & METHODS
Study area
The study region of Papum Pare is situated at
the N 26°56´11˝ to 27°35´44˝ and E 93°12´45˝
to 94°13´30˝ is one of the significant hotspots
in the world, ranked 25th (Chowdhery, 1999),
and along with the 200 internationally
important eco-regions (Olson and Dinerstein,
1998). It covers an area of 2875 sq. km, having
annual rainfall 2694 mm. the main part (75%)
of this district is covered by thick forest which
has the sub-tropical, deciduous and humid type
of vegetation (Fig.1-a).
Plant & Sample collection
The selected plant C. asiatica (Linn) belongs to
the family Apiaceae. The herb plant is well-
known as Mandookaparni in Ayurvedia,
Brahmi in Unani medicine. C. asiatica has been
used as a wound-healing agent. The current
study was mainly focused on isolation and
identification of the fungal endophytes from
the medicinal.
Samples of leaves stems and roots were
collected during (2015-2017) from the healthy
plant of C. asiatica in sterile bags and brought
to the laboratory for processing within 24 hours
after sampling (Fig. 1-a).
Surface sterilization and isolation
The collected samples were washed delicately
under running tap water to remove the soil and
debris. Isolation of fungal endophytes was
determined using protocol leaf samples which
were cut into small pieces of 0.5 cm²; and,
stems and roots samples were cut into 0.5-1.0
cm. The pieces were then surface-sterilized by
dipping them serially in 70% ethanol for 5
seconds and 4% NaOCl for 90 secondes
respectively and finally rinsed in sterile
distilled water for 10 seconds. One hundred
and fifty segments of the samples were
randomly selected and plated on Potato
Dextrose Agar (PDA) medium (supplemented
with 150 mg/L chloramphenicol) contained in
Petri dishes (7.5 cm diam). Petri dishes were
incubated (12 h dark: 12 h light cycle) for 25
days at 28°C, to observe the growth of
endophytes (Suryanarayanan et al.1998). The
hyphal tips which grew out from the segments
were isolated and sub cultured on PDA
medium. The pure cultures were maintained on
PDA slants and preserved at 4°C for further
investigation.
Further, the colonization frequency (CF) was
calculated, using the following formula
(Suryanarayanan et al. 2003):
However, the density of colonization (rD%) of
a single endophyte species was calculated,
using the following formula (Fisher and Petrini
1987).
Where,
Ncol = Number of segments colonized by each
fungus
Nt = Total number of segments inoculated
Morphological identification
The endophytes thus isolated, were identified
based on their macroscopic and microscopic
characteristics viz., the morphology of fruiting
structures and spore morphology (Sutton 1980
and Nagmani 2005).
J. Indian bot. Soc. Vol. 100 (3&4) 2020:161
Molecular characterization of endophytic fungi associated
with C. asiatica

Molecular identification
DNA extraction and PCR amplification of
rDNA
Genomic DNA was extracted from fresh fungal
mycelia (5-8 days old culture) growing on PDA
0
Plates (at 28 C), using Qiagen microbial
extraction kit. Fragments of 18S rRNA gene
were amplified by PCR, using forward ITS1 (5'
TCCG TA GGTGAA C C TGCGG- 3 ' ) a nd
r e v e r s e I T S 4 ( 5 ' -
TCCTCCGCTTATTGATATGC-3') primer
(White et al. 1990). Each PCR amplification
reaction was performed in a final volume of 25
µL containing 25 ng template DNA, 10X PCR
buffer (10 mM Tris-HCl, 50 mM KCl, pH 8.3),
1.5 mM MgCl2, 200 µM of each dNTP, 50 pM
of each primer, 1 unit of Taq polymerase (Genei,
Banglore) and distilled water (Sigma, USA).
The conditions of the PCR was initial
denaturation at 95°C for 5 minutes followed by
35 cycles for 45 sec, annealing at 56 °C for 1
minute (35 cycles) and primer extension at
72°C for 1 minute (again 35 cycles), final
extension at 72°C for 7 minutes (1 cycle) and
hold (cooling) at 4°C. PCR products were
checked on 1.2 % Agarose gel in Tris–acetate-
EDTA buffer (TAE) at pH 8.0, stained with
ethidium bromide (0.3lg/mL) and visualized
under UV light by using Gel documentation
system. Sequencing was done by GeNei Labs
Private Limited, Bengaluru using PRISM® Big
Dye TM Terminator Cycle Sequencing Kits
with AmpliTaq® DNA polymerase, as per
manufacturer's instructions.
Phylogenetic analysis procedures
The primer pairs ITS1/ITS4 were used to
amplify ITS-rDNA and partial. The sequences
thus obtained, were subjected to BLAST and
submitted to GenBank of NCBI as well as got
the accession number. Further, to get the
homology search and multiple sequence
alignments were completed by ClustalW.
Phylogenetic relationship of all fungal species;
drawn their phylogenetic relationship. Further,
it was analyzed using Neighbor-Joining
methods of MEGA 5 (Tamura et al. 2011) and
the robustness of the inferred phylogeny was
assessed using bootstrap value at 1,000
replications.
RESULTS AND DISCUSSION
The current investigation deals with the
isolation of fungal endophytes, from C.
asiatica; ethnomedicinal plants frequently
used for the treatment of different health
problems in human beings. 15 plant materials
of C. asiatica, 150 segments were processed
(including root, stem and leaves plant parts);
for the isolation of endophytic fungi. The
observations recorded a total of 104 isolates of
fungal endophytes; which belong to 28 fungal
spp. The maximum number of fungal isolates
were recorded from leaves (39%), followed by
roots (31%) and stems (30%) [Fig.1-b]
respectively. Further, the species richness and
the frequency of colonization were also
recorded, highest in the leaves.
The observations show that L. viticola was the
most dominant endophytic myco-flora
followed by various fungal endophytes (Table
1). However, P. longiseta, Aureobasidium sp.,
C. gloeosporioides were the common fungal
endophytes recorded from all the plant parts
viz., leaf, stem, and roots. The density of
colonization (rD%) was recorded in the
chronological order (Table 1; Fig. 1-c).
While observing the fungal taxonomy of all
the isolated 28 fungal endophytes; 11 belongs
to the class Dothideomycetes; 02 belongs to the
class Eurotiomycetes;14 belongs to the class
Sordariomycetes and 01 fungal endophyte
couldn't identify properly. While categorizing
up to the order level; it was observed that L.
v i t i c o l a b e l o n g s t o t h e o r d e r
Botryosphaeriales; Cladosporium sp. and C.
sphaerospermum belongs to the order
Capnodiales; Aureobasidium sp. belongs to the
order Diaporthales; D. microsperma and D. sp.
belongs to the order Dothideales; A. fischeri
and A. niger belongs to the order Eurotiale; C.
fructicola, C. gloeosporioides and C.
sublineola belongs to the order Glomerellales;
J. Indian bot. Soc. Vol. 100 (3&4) 2020:162
Richa Sharma, Sumpam Tangjang and Amritesh C. Shukla

F. incarnatum and F. merismoides belongs to
the order Hypocreales; Cylindrocladium sp.
belongs to the order Hypocreomycetidae; S.
brevicaulis belongs to the order Microascales;
C. borreriae, C. spicifera, B. bicolor, H.
dalbergiae, P. chartarum, Preussia spp. and P.
brabeji belongs to the order Pleosporales; S.
fimicola belongs to the order Sordariales; N.
oryzae belongs to the order Trichosphaeriales;
Eutypa sp., P. longiseta and Sordariomycetes
sp. belong to the order Xylariales.
Samples of the fungal endophytes thus
isolated (28 samples) were used for extraction
of the DNA and its molecular characterization
as well as submitted to the NCBI and got the
ac c es s io n n um b er ( Fi g .2 ) . Fu r th e r,
phylogenetic analyses based on the ITS1-ITS4
sequencing data of the isolated fungal
endo p h y t es were a l s o recorded. The
observations of the neighbor-joining tree as
constructed based on the sequence-structure
alignment, and recorded in figure two; shows
the structure alignment clearly in six well-
separated groups. Group 'A' consisted
L.viticola, H. dalbergiae, F. incarnatum, F.
merismoides, C.gloeosporioides and C.
fructicola. Similarly, group 'B' .C. sublineola,
C. sphaerospermum, Sordariomycetes spp. and
Preussia spp; group 'C' contains P.longiseta, P.
brabeji, S. brevicaulis, S. fimicola, A. niger,
and A. fischeri; group 'D' contains Fungal sp.,
Eutypa sp., B. bicolor, P. chartarum,
Cylindrocladium sp. and Cladosporium sp.;
gr o u p 'E' c o nsi s ted N . o ryza e a nd
Aureobasidium sp. Further,Group 'F' D.
microsperma, C. spicifera and C. borreriae.
However, Dothidea sp. was recorded as an out-
group relationship (Fig. 2).
S.N. Isolated fungal endophytes Plant parts
(C. asiatica)
CF
( %)
rD (%)
Leaf Stem Root
1 Pestalotiopsis longiseta Leaf Stem Root 4 1.33
2 Colletotrichum gloeosporioides Leaf Stem Root 14 4.66
3 Lasiodiplodia viticola Leaf Stem Root 26 8.66
4 Sordaria fimicola Leaf --- --- 2 0.66
5 Curvularia borreriae --- Stem Root 8 2.00
6 C. fructicola Leaf --- --- 8 2.66
7 Diplodina microsperma --- --- Root 4 1.33
8 C. sublineola Leaf --- Root 8 2.66
9 Cylindrocladium sp. --- Stem --- 4 1.33
10 Aspergillus niger Leaf --- --- 4 1.33
11 Nigrospora oryzae Leaf Root 2 0.66
12 Dothidea sp. Leaf Stem Root 6 2.00
13 Cladosporium sp. Leaf Root 8 2.66
14 C. sphaerospermum Leaf Stem --- 8 2.66
15 Fusarium merismoides Leaf Stem Root 4 1.33
16 Sordariomycete sp. Leaf --- Root 8 2.66
17 Bipolaris bicolor Leaf Stem --- 10 3.33
18 Helminthosporium dalbergiae --- Stem --- 4 1.33
19 Curvularia spicifera Leaf --- --- 2 0.66
20 Pithomyces chartarum --- Stem --- 10 3.33
21 Scopulariopsis brevicaulis Leaf Root 2 0.66
22 Eutypa sp. Leaf --- --- 2 0.66
23 Fungal sp. Leaf Root 6 2.00
24 F. incarnatum --- Stem --- 8 2.66
25 A. fischeri Leaf --- --- 4 1.33
26 Preussia spp. Leaf Root 10 3.33
27 Pseudocamarosporium brabeji Stem Root 8 2.66
28 Aureobasidium sp. Leaf Stem Root 4 1.33
Table 1: Fungal endophytes isolated from C. asiatica, its frequency (CF %) and density (rD %)
--- indicates absences of the fungal endophytes.
J. Indian bot. Soc. Vol. 100 (3&4) 2020:163
Molecular characterization of endophytic fungi associated
with C. asiatica

Figure 1 (a) Sample collection site of C. asiatica L., in Arunachal Pradesh, India (satellite image), where, red circle
indicates collection site. (b) Fungal endophytes isolated from different parts of C. asiatica (c) Isolated fungal
endophytes and its rD (%)
J. Indian bot. Soc. Vol. 100 (3&4) 2020:164
Richa Sharma, Sumpam Tangjang and Amritesh C. Shukla

MK398249 Lasiodiplodia viticola
MK332495 Helminthosporium dalbergiae
MK332494 Fusarium incarnatum
MK299158 Fusarium merismoides
MK299152 Colletotrichum gloeosporioides
MK299144 Colletotrichum fructicola
MK379589 Colletotrichum sublineola
MK332486 Cladosporium sphaerospermum
MK379583 Sordariomycetes sp.
MK398264 Preussia spp
MK299129 Pestalotiopsis longiseta
MK398252 Pseudocamarosporium brabeji
MK299149 Scopulariopsis brevicaulis
MK299157 Sordaria fimicola
MK248608 Aspergillus niger
MH748593 Aspergillus fischeri
MH686149 Fungal sp.
MK299142 Eutypa sp.
MK332477 Bipolaris bicolor
MK332479 Pithomyces chartarum
MK299153 Cylindrocladium sp.
MK332485 Cladosporium sp.
MK299154 Nigrospora oryzae
MK398283 Aureobasidium sp.
MK332492 Diplodina microsperma
MK332478 Curvularia spicifera
MK299132 Curvularia borreriae
MK299156 Dothidea sp.
99
52
40
39
39
31
29
27
25
20
20
19
17
13
10
5
14
2
1
1
0
0
0
0
3
Group A
Group BGroup C
Group D
Group EGroup F
Figure 2 Phylogenetic analysis based on neighbor-joining tree based on ITS rDNA sequences of the isolated 28 fungal
endophytes. Numerical values indicate bootstrap percentile from 1000 iterations.
J. Indian bot. Soc. Vol. 100 (3&4) 2020:165
Molecular characterization of endophytic fungi associated
with C. asiatica

Literature reveals that diversity of fungal
endophytes has previously been reported from
numerous medicinal plants viz., Arthrinium sp.
Diaporthe sp., Fomitopsis sp. Penicillium sp.,
Phomopsis sp. Schizophyllum sp. have been
reported from Garcinia parvifolia and G.
mangostana (Sim et al., 2010); A. strictum, A.
tuberculatum, H. fuscoatra, H. Grisea,
Mortierella hyaline, M. sphaerica, N.
minutissima, Oidiodendron griseum and O.
echinulatum reported from Elaeocarpus
sphaericus (Shukla et al., 2012); P. citrinum, A.
alternate, A. niger, Cladosporium sp., Rhizopus
sp., C. vermiformis reported from Helicteres
isora (Gayathri and Chandra 2017); Periconia
hispidula, A. arbuscula, N. sphaerica, A.
falciforme, P. chrysogenum Aureobasidium sp.
Chaet o m i u m sp. from Litsea c u b e ba
(Dee panwit a and D hruva 2 018); and
Aspergillus sp., B. maydis, Chaetomium sp., D.
phaseolorum, F. solani, Fusarium spp,
Macrophomina phaseolina, Penicillium sp., R.
bataticola and Setosphaeria rostrata, reported
from Chlorophytum borivilianum (de Carvalho
et al., 2019: Chowdhary et al., 2019). Similarly,
in current study, 28 fungal spp have been
isolated from the plant parts, we observed that
L.viticola was the most dominant endophytic
fu n gi a nd A u re ob a sid i um s p., C .
gl oeos porioides, Do thid ea sp. and F.
merismoides were the common fungal
endophytes from all the plant parts viz., leaf,
stem and roots. L. viticola, P. longiseta,
Aureobasidium sp., C. gloeosporioides,
Dothidea sp. and F. merismoides were the
common fungal endophytes recorded from all
the plant parts viz., leaf, stem and roots.
Maximum density of colonization (rD %) was
recorded in L. viticola (Table 1; Fig. 1-c).
Besides this, extraction of the DNA and its
molec ular ch aracterizatio n as well as
phylogenetic analyses based on the ITS1-ITS4
sequencing data of the isolated twenty-eight
fungal endophytes, have also been recorded
(Fig. 2).
The authors are thankful to the tribal peoples of
Ar u na n ch a l P r ad e sh , f or p rov idi ng
et hnomedicinal informatio n about the
particular plant and its uses for various
ailments. Further, authors are also thankful to
the Head Department of Botany, Rajiv Gandhi
University, Arunanchal Pradesh as well as to
Head Department of Botany, University of
Lucknow for providing various facilities
during the course of the present study. Besides
this, one of the authors is thankful to the
MHRD, Govt of India; for providing financial
support.
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