Content list available at http://epubs.icar.org.in, www.kiran.nic.in; ISSN: 0970-6429
Indian Journal of Hill Farming
Special Issue 2021, Volume-34, Page 268-275
Molecular characterization, microscopic characteristics, and phylogenetic analysis of
Ophiocordyceps sinensis from Sikkim, India
Vashkar Biswa1 ∙ Raju Ali1 ∙ Durga Kr. Pradhan2 ∙ Nitisha Boro3 ∙ Tikendrajit Baro1 ∙ Debajani Das1
∙ Robin Subba4 ∙ Puja Das4 ∙ Sandeep Das1
1Department of Biotechnology, Bodoland University, Assam
2Sikkim State Forest Herbarium, Deorali, Gangtok, Sikkim
3Department of Molecular Biology & Biotechnology, Tezpur University, Assam
4College of Agricultural Engineering and Post-Harvest Technology, Ranipool, Sikkim
ARTICLE INFO ABSTRACT
Received: 26th October 2021
Revision Received: 03rd Decembe 2021
Accepted: 06th December 2021
Key words: Medicinal Mushroom, Fungi,
Taxonomy, Phylogeny, Ophiocordyceps
The zombie fungi are a group of entomo
parasitic fungi comprising of 400
diverse species having tremendous pharmaceutical virtues. They have their use in
traditional practice among Himalayan highlanders and inhabitants across the globe living
in high altitude areas. Ophiocordyceps sinensis is one such representative of the entomo-
parasitic group. The current study aimed to identify Ophiocordyceps sinensis from Sikkim,
India, following classical, molecular taxonomic approaches, and culture. The classical
approach involved a microscopic study of asci, stroma, and mycelia and the macroscopic
characters of the stroma and larva. The molecular approach involved the amplification of
internal transcribed spacer (ITS) region from the stroma, cytochrome oxidase subunit-I
(COI), and cytochrome b (Cytb) from host larva for phylogenetic studies. The pure culture
was established on potato dextrose agar (PDA). The sequences were edited with Bioedit
version 7.2.5 and subjected to multiple alignments using fast fourier transform (MAFFT)
database. Model testing was performed using MegaX version 10.2.5, and the best model
was utilized to construct the maximum likelihood tree. To confirm the results of the
maximum likelihood tree, a Bayesian tree was also constructed using MrBayes 3.2.7.
Subsequently, the study confirmed that the collected specimen is O. sinensis. The main
bioactive compounds of O. sinensis are cordycepin and adenosine which has been explored
for different therapeutic applications including treatment of cancer, diabetes, anemia,
inflammation etc. Thus, such study provides the platform for their exploration for extensive
pharmaceutical and nutraceutical future studies.
Ophiordyceps sinensis (Berk.) is an entomo-
pathogenic fungus belonging to the genus Ascomycota [Zhou
et al. (2014)]. It consists of >400 different species worldwide,
which are parasitic, mainly on insects and larvae. Typically,
it exists in two stages: an asexual stage (mitosporic fungi) and
a sexual stage. The mitosporic fungi are parasitic on dead
caterpillars of the moth Hepialus spp. The spores of O.
sinensis germinate inside the caterpillars, colonizing with
hyphae and producing a stalked ascomata (sexual stage)
[Zeng, W., Yi, D.H. and Huang, T.F. (1998), Pu, Z.L. and Li,
Z.Z. (1996)]. Cordyceps are mainly found in China, Nepal,
and India at 3500 m above sea level. The fungus is a coveted
medicinal utility in traditional Tibetan and Chinese
medicine, which is commonly known in the West as
“Himalayan Viagra.” In India, the collection and trade of
Cordyceps by the Bhotiya community have been reported
from Garhwal, Uttarakhand [Caplins, L. B. (2016)]. It has
also been reported
*Corresponding author: email@example.com
an altitude of 3200 m from the snow meadows of Brahamkot,
Ultapara, Ghawardhappa, Chhipalakot, Najari in Dharchula,
Chetri Bugyal, and ChiplaKedar (4000 m), as well as from
Nagin Dhura, Ralam Bugyal at the base of Panchachuli
Hills, Laspa, Tolatop, Darti, Mapa top, Burfu top, and Milam
top in Johar Hills of Kumaun [Arora, R. K., (2014)]. Sikkim
is the organic state of India, comprising a vast diversity of
flora and fauna. The distribution of O. sinensis has been
reported from Lachen (ca.2750 masl;
27.7167°N;88.5577°E), Lachung (ca.2700 masl;
276891°N;88.7430°E) in North Sikkim, and Gnathang
valley (27.2986° N, 88.8173° E) in East Sikkim [Risley, H.
H. (1894)]. Lachenpas and Lachungpas are primary dwellers
of North Sikkim with a population of 3200 (Lachen) and
2495 (Lachung), according to the panchayat register of 2017
[Pradhan, B. K. et al. (2020)]. The Himalayan rural
population relies on herbal medicinal plants and fungi for 3–
58 percent of total yearly household income and 78 percent
of cash revenue [Pradhan, B. K. et al. (2020)]. Cordyceps
spp. has long been used to promote longevity, relieve
exhaustion, and treat numerous diseases in Chinese
traditional medicines [Russell, R.; Paterson, M. (2008)].
Recent studies have demonstrated that various species in this
genus possess multiple pharmacological properties,
including anti-tumor, anti-microbial, anti-inflammatory, and
immunomodulatory effects [Holliday, J.; Cleaver, M.
(2008), Agrawal, D. G., & Sandhu, S. S. (2020)].
The present study defines the taxonomic virtues of
the collected sample, identified as O. sinensis by both
classical and molecular taxonomy and characterized by
mycelial culture. Also, the host larva of the Cordyceps spp.
was characterized. Thus, this study could be significant for
distinguishing O. Sinensis from-related Cordyceps spp.
These features could be a measure for their systematic use as
nutraceuticals for treatments and beneficial effects. The
study can also be a tool to identify counterfeits to minimize
or regulate the trade of O. sinensis. Therefore, the current
study claims to be the first elaborate taxonomic study on O.
sinensis from Sikkim.
2. Materials and Methods
Samples were collected from Yumesamdong
(27°51'05.4"N 88°41'04.8"E), North Sikkim, in July 2021
and transported on an icebox to Bodoland University.
Photographs were taken before and after cleaning the
samples. DNeasy plant genomic, PCR purification, and gel
extraction kits were purchased from Qiagen, Germany.
Wizard SV Genomic purification system (Promega,USA)
was obtained from the USA. Taq Polymerase was procured
from Thermo Fischer, USA, and 100-bp ladder was
purchased from TaKaRa, Japan.
Isolation and Culture
The samples were cleaned with a sterile art brush and detached
from the larva, followed by washing in sterile distilled water.
0.1% mercuric chloride was used for surface sterilization.
Subsequently, the samples were cut vertically using a sterilized
scalpel. The tissue from the center of the fruiting body was
inoculated on potato dextrose agar (PDA) with 0.05% MgCl2
[Barseghyan, G. S. et al. (2011)]. The culture plates were
incubated at 18 °C in a Bio-oxygen Demand (BOD) incubator.
Mycelial growth was observed after 4-5 days. In order to obtain
pure culture, small chunks of mycelia were sub-cultured on PDA
plates containing MgCl2 and 500 mg/L ampicillin. The mycelia
were then transferred to liquid media for DNA extraction to
confirm the culture.
The microscopic characteristics, such as the total diameter of the
transverse section of stroma and the length and breadth of the
asci and mycelia, were studied [Liu, H et al. (2011)].
DNA Barcoding Analysis
The genomic DNA of the sample was extracted from stroma
(fruiting body), mycelia, and host (larva), separately. The DNA
isolation from stroma and mycelia was carried out using the
DNeasy plant genomic kit (Qiagen), and the isolation from the
host was carried out with the Wizard SV Genomic purification
kit (Promega) with some minor modifications. The DNA
samples were quantified on Qubit4 fluorometer (Thermo Fisher
Scientific). The genomic DNA was visualized on 0.8% agarose
gel electrophoresis. The primers used for rDNA internal
transcribed region (ITS) amplification were as follows: forward
primer ITS-5 (5’-GGAAGTAAAGTCGTAACAAGG-3’) and
reverse primer ITS-4 (5’-TCCTCCGCTTATTGATATGC-3’)
[Wu, D. et al. (2016)]. The amplification of the cytochrome
region from the genomic DNA of host larva was carried using
forward primer Cytb-1 (5’-
TATGTACTACCATGAGGACAAATATC-3’) and reverse
primer Cytb-2 (5’-ATTACACCTCCTAATTTATTAGGAAT-
3’) [Quan, X., & Zhou, S. L. (2011) and Wu, D. et al. (2016)].
The amplification of COI gene used forward primer COI-F (5’-
GGTCAACAAATCATAAAGATATTG-3’) and reverse primer
COI-R (5’-TAAACTTCAGGGTGACCAAAAAAT3’). The
polymerase chain reaction (PCR) for the ITS region of the
genomic DNA from stroma (fruiting body) was as follows: an
initial denaturation step of 95 °C for 5 min, followed by 35
cycles of 95 °C for 30 s, 51 °C for 2 min, and 72°C for 1 min,
and a final extension step of 72 °C for 10 min. The PCR
conditions for the amplification of Cytb gene of host larva
consisted of an initial denaturation step of 95 °C for 5 min,
followed by 35 cycles of 95 °C for 30 s, 50 °C for 2 min, and 72
°C for 1 min, and a final extension step of 72 °C for 10 min.
The PCR amplification of
gene of host larva consisted of an
initial denaturation step of 95 °C for 5 min, followed by 35 cycles
of 95 °C for 30 s, 48 °C for 2 min, and 72°C for 1 min, and a
final extension step of 72 °C for 10 min [Wu, D et al. (2016)].
The amplification products were purified using the Qiaquick
PCR purification kit (Qiagen, Germany) and confirmed on 1.5%
agarose gel electrophoresis with a 100-bp ladder. The fragments
were excised and purified using QIAEX gel extraction kit
(Qiagen), and the concentration was measured on a Qubit 4
Fluorometer (Invitrogen, USA). Subsequently, the DNA was
used for sequencing in a AB13730XL, Applied Biosystem
Sequencer following sanger sequencing method. The ITS, Cytb,
and COI sequences were subjected to homology search by
BLAST tool in the NCBI nucleotide blast portal
=BlastSearch). Finally, the sequences were submitted, and the
accession number was obtained from the NCBI database
Sequences were edited using BioEdit version 7.2.5. Phylogenetic
analysis was performed using MegaXversion 10.2.5, and
reference sequences were downloaded from NCBI. The
evolutionary history was obtained using the maximum
likelihood method and Kimura 2-parameter model [Kimura, M.
(1980)]. The tree with the highest log likelihood (-3549.73) is
shown in figure-2. The percentage of trees in which the
associated taxa are clustered together is shown next to the
branches. The initial tree(s) for the heuristic search were
obtained by applying the neighbor-joining (NJ) method to a
matrix of pairwise distances estimated using the maximum
composite likelihood (MCL) approach. A discrete gamma
distribution was used to model the evolutionary rate differences
among the sites (5 categories (+G, parameter = 1.1526)). This
analysis involved 23 nucleotide sequences and a total of 880
positions in the final dataset. The evolutionary analysis was
conducted in MEGA X10.2.5 [Kumar S et al, 2018].
3. Results and Discussion
The collected samples were submitted to Sikkim State
Forest Herbarium (SSFH), Deorali, Sikkim vide (SSFH
SK005007) and cultured in Petri plates containing PDA media
supplemented with 0.5 g/L and 50 mg thiamine hydrochloride.
The mycelia started to grow after 5–7 days, followed by a
subculture to obtain a pure culture of the sample. Then, the
collected samples were cleaned, and DNA was extracted
(DNeasy Plant Genomic kit, Qiagen). The DNA from stroma,
pure-cultured mycelia, and host larva was isolated independently
and amplified on a Thermal Cycler (2720, Applied Biosystems).
The PCR product of ITS, COI,
respectively. These were then sequenced (AB13730XL, Applied
Biosystem Sequencer) following sanger sequencing method.
The sequencing data of stroma, mycelia, and larva were blasted
in the NCBI database
rch). The ITS amplicon of sample Cordyceps Bodoland
University Sikkim-3 (CBUS3 fruiting body and culture) covered
a query of 97% and 99%, which is identical with O. sinensis. The
sequencing data of COI gene had query cover of 100% and 93%
identity to Thitarodes spp, while Cytb gene had a 92% query
coverage and 96% identity to Thitarodes spp. The presence of
grouped, cylindrical, and embedded asci confirmed O. sinensis.
The phylogenetic analysis of the ITS region of stroma and COI
gene of the host was performed separately. A total of 22
sequences of Cordyceps spp. were retrieved from NCBI for the
phylogenetic analysis of the ITS region of the stroma, and 19
sequences were retrieved for the phylogenetic analysis of the
COI region of the host. The sequences were subject to multiple
alignment using fast fourier transform (MAFFT) online tool.
The alignment was optimized visually, and ambiguous regions
were excluded from subsequent phylogenetic analyses. The best
model was calculated by the model testing in Mega (version
10.2.5), and the K2+G model was chosen [Kimura, M. (1980)].
Pairwise distance matrices were generated using Kimura models
of nucleotide substitutions [Kimura, M. (1980); Kumar S. et al.
(2018); Swofford, D. L. (1998)], and the phylogenetic analysis
was performed in the Mega version 10.2.5. An NJ tree [Jukes,
T.H. and Cantor, C.R. (1969)] with bootstrapping was
constructed with distance measured by the Jukes Cantor distance
[Jukes, T.H. and Cantor, C.R. (1969)] model and Kimura’s two-
parameter distance. To assess the confidence of phylogenetic
relationships, the bootstrap test [Felsenstein, J. (1985)] was
conducted with 1000 resampling for NJ analysis. The
phylogenetic relationships of O. sinensis were analyzed using
the Bayesian method [Ronquist, F. et al. (2012)]. Volvariella
volvacea is used as an outgroup for phylogenetic analysis of the
ITS region and Anthera assama as an outgroup for COI region
of host larva. The comparative phylogenetics results using ITS
sequence indicated that the presence of the sample collected
from Sikkim clubbed with the sequences of O. Sinensis
sequences (China) retrieved from NCBI. The presence in the
same clade diverging from the other related species confirmed
that the sample belong to O. Sinensis, which is an entomo-
parasitic fungus infecting larva of various genera (Thitarodes
spp., Endoclita spp., Napialus spp.). The phylogenetic analysis
was performed to confirm the larva. The sequence of COI of
larva from the sample was compared to that of the common larva
of O. Sinensis. The sequences were retrieved from NCBI, and
phylogenetic analysis was carried out. Subsequently, the sample
clubbed and grouped
with the sequences from
Thitarodes spp., which are synonyms, confirmed that the larva
belongs to Thitarodes spp. The tree from Bayesian inference
showed an identical tree topology. The recent studies have
shown cordycepin from cordyceps spp. are capable of reducing
pain, inflammation and joint pathology in rodent model
[Ashraf, S, et al. (2019)] also cordycepin have been known to
be involve in activation of AMPK and induction of apoptosis
H. et al. (2013); Zhang, Y.et al. (2018); Li, S. Z. et al. (2019)].
Although O. sinensis has been reported, a detailed microscopic
and phylogenetics analysis of O. sinensis from Sikkim is reported
in this study. Thus, this study will be helpful in properly
characterizing and differentiating different species of cordyceps
which have diverse therapeutic potential.
Table 1. Details of macro-morphological characters of O. sinensis
Thitarodes sp. (CBUS3-
and brown when dried, 38–40 mm in length, and
about 2.5–3 mm in diameter. The total diameter of
the transverse section is 298 µm, length and
breadth of the asci were 84 µm and 33 µm,
silkworm, 29–32 mm in length
and 4–5 mm in breadth.
Yellowish in color with 8 pairs of
with 3-µm diameter.
larva with 8 pairs of the legs on the abdomen and 4 pairs at the center. (D)
Transverse section of the stroma. (E) Perithecia at 20x (F) Perithecia at 40x (G) Mycelial culture of O. sinensis
grown on PDA media. (H)
Mycelia of O. sinensis. (I) Central portion of the stroma.
elongated, and grouped at the fertile portion of
stroma. Mycelia embedded with the asci. The
outer layer of the stroma stained dark with Congo
The collection and trade of O. sinensis in Lachung and
Lachen valley of North Sikkim is a common practice for
livelihood management [Pradhan, B. K. et al. (2020)].
Traditional use of O. sinensis as aphrodisiac, treating fatigue,
anti-inflammation, immune booster and treatment of
respiratory ailments is a practice of choice by the traditional
healers in Himalayan region [Panda, A. K., & Swain, K. C.
(2011)]. A detailed scientific characterization and
authentication of the collected sample of O.sinensis shall
facilitate differentiation between other counterfeit species
and establish the collected Cordyceps spp. from Sikkim to be
We are thankful to DBT Sponsored Advanced Level
Institutional Biotech Hub & Technology Incubation Centre,
Bodoland University for providing us the research facility.
We are grateful to Sikkim Biodiversity Board & Sikkim State
Forest Herbarium, Deorali, Sikkim for granting us the
research permission. We sincerely thank Bidur Biswa, Sajesh
Chhetri, Sonam Wangyal Bhutia, Aruna Rai, Dhanmaya
Poudyal and Sewantika Sharma for their assistance.
6. Conflict of Interest
The authors declare no conflict of interest.
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