MSc

Cordyceps which is an entomo-pathogenic fungus got the name from two
Latin words, cord and ceps, meaning ‘club’and ‘head’. This mysterious fungus has a
long reputation of being the single-most expensive raw material used in traditional
Chinese medicine due to its dearth in availability. As a result of its high efficacy and
potency in curing various diseases, this fungus has been recommended by medicinal
practioners as a ‘panacea of all ills’. Distribution of Cordyceps sp. is cosmopolitan,
but mostly confined to high Himalayan Mountains in China, Tibet, Nepal and India,
at an altitude of 3000 to 5000 metres or in Asian high altitude grass land ecosystems.
Cordyceps sp. which is renamed as Ophiocordyceps sp. attacks on the coconut root
grub (Leucopholis coneophora Burm.) in coastal sandy areas of Kasargod district of
Kerala. In this context, in the present study, ‘Characterization of medicinal
mushroom, Cordyceps sp. from Kasargod district’ an attempt was made to untangle
many of the mysteries of Cordyceps sp. by isolation, identification, characterization,
cultivation and assessment of the proximate composition with special attention to
Cordyceps sp.
Purposive sampling surveys were conducted in three locations of coastal
sandy tracts of Kasargod district during June to September months of 2017 and 2018.
The fruiting bodies of Cordyceps sp. were collected from three locations viz.,
Instructional farm of College of Agriculture, Padannakkad, Valiyaparamba area of
Nileshwar and Regional Agricultural Research Station, Pilicode. Maximum number
of fruiting bodies were obtained from the soil under cashew trees in the Instructional
farm of College of Agriculture, Padannakkad (Average of 13.86/m2) due to the
richness in organic carbon (1.39%), and shaded condition in the soil under cashew
orchard compared to Valiaparamba and Pilicode. Macro minerals viz., potassium,
calcium and micro mineral like, manganese were in higher quantity under soil of
cashew trees compared to other two regions, which may be helping in the survival
and germination of spores.
The fungus was isolated on PDA medium from the stroma portion of the
fruiting body and the isolate obtained after pure culturing was numbered as CD-1.
The isolate completed the full growth in a 9 cm Petri dish by 55 days after inoculation
in PDA medium. The anamorph produced similar type of conidia as produced in the
field.
Characterization of Cordyceps sp. based on cultural, morphological and
physiological characters were carried out. The cultural characters like colour, shape,
texture and the growth rate of the mycelium were studied in different media. Among
the different media tested yeast potato dextrose agar medium (YPDA) was found to
be the best medium followed by potato dextrose agar media (PDA), which were
significantly superior from other medium.
Various macroscopic characters like shape, size and colour of sclerotia, stipe
and stroma and branching pattern of the stroma in three locations were observed.
Microscopic studies of both anamorph (hyphae, synnemata, conidiophores and
conidia) and teleomorph (perithetia, asci and ascospores) were conducted.
The molecular characterization of the fungus was carried out at Rajiv Gandhi
Centre for Biotechnology (RGCB), Thiruvananthapuram and showed homology with
Ophiocordyceps neovolkiana (Kobayasi) strain KC1 having 98 per cent identity. The
sequence of the fungus was deposited at the Gen Bank of NCBI and the accession
number obtained was MH 668282. The culture was deposited in National Fungal
Culture Collection of India at MACS Agharkar Institute, Pune with culture no.
NFCCI 4331.
Physiological characterization of O. neovolkiana was carried out. Among eight
different temperatures tested, 30oC was found to be the optimum temperature in
which the growth was completed within 38 days after inoculation. The neutral pH
seven was found to be optimum for mycelial growth. Among the different light
conditions, 24 hrs of darkness under incubator (30oC) was preferred by this fungus.
Fructose was the best source of carbon in which maximum mycelial growth was
obtained within 36 days after inoculation. The best source of nitrogen was yeast
extract in which maximum mycelial growth was obtained within 35 days after
inoculation. KH2PO4 was the best source of macro mineral for the growth, in which
the fungus completed the growth within 38 days after inoculation. Among micro
minerals, ZnCl2 was the best for the growth in which the fungus completed the
growth within 35 days after inoculation. Folic acid was the best source of vitamin for
the growth in which the fungus completed the growth within 37 days after
inoculation.
One optimum media for the growth of O. neovolkiana was standardized as
Yeast Potato Fructose medium supplemented with one gram of KH2PO4, 500 mg of
ZnCl2, 10 mg of folic acid in one litre of distilled water which was named as Yeast
Extract Potato Fructose Agar (YEPFA).
The growth of O. neovolkiana in the YEPFA medium was significantly higher
than the best media YPDA and PDA, so that the time required for the mycelial
growth could be considerably reduced from 55.15 days to 30.85 days. The mycelial
dry weight was also significantly higher in YEPFB medium than YPDB and PDB and
growth completed within 32.57 days after inoculation.
Different cereal grains viz., rice, wheat and sorghum were tried for artificial
culturing of O. neovolkiana under in vitro conditions. The number of days taken for
coverage of the mycelium in 50g of grains was 96.4 days for sorghum which was
significantly different from rice (115.2 days) and wheat (142.2 days). Hence sorghum
was selected as the best substrate for artificial culturing.
Analysis of proximate constituents was estimated using the collected sporocarps
from the field and mycelial growth obtained on the broth of YEPF medium. The
carbohydrate was more in artificially cultivated mycelia than fruiting bodies
(5 and 9.7% respectively).
But protein, (2.9 and 1.75%) and vitamin C
(3.27 and 2.5) were more in fruiting bodies than mycelia. The crude fibre was more in
mycelia than the fruiting bodies (16.8 and 25%). The total minerals content was
almost similar in fruiting bodies and mycelia.
Present work is the first attempt in India, which resulted in a detailed systematic
study on O. neovolkiana parasitizing on coconut root grub. Future line of work
should be concentrated on mass production of this fungus, the estimation of its anti-
cancerous components and the antifungal and antibacterial effect of this fungus on
pathogens in plant disease management and its potential as a biocontrol agent in pest
management.