Untitled

UGC- SAP DRS-1 SCHEME
ON
ENTERPRISING MUSHROOM BIOTECHNOLOGY FOR
FOOD, FEED AND BIOMANURE
COMPLETION REPORT (2016-2020)
DEPARTMENT OF PLANT PATHOLOGY

CENTRE FOR PLANT PROTECTION STUDIES
TAMIL NADU AGRICULTURAL UNIVERSITY
COIMBATORE-641003, TAMIL NADU
2020
Title: Enterprising Mushroom Biotechnology for Food, Feed and Biomanure
(Completion Report)
Authors: A. S. Krishnamoorthy, S. Nakkeeran, G. Thiribhuvanamala,

M. Karthikeyan, P. Latha
Component: UGC-SAP-DRS1 Scheme report
Citation: Krishnamoorthy, A. S., S. Nakkeeran, G. Thiribhuvanamala, M. Karthikeyan,

P. Latha. 2020. Enterprising Mushroom Biotechnology for Food, Feed and
Biomanure (Completion Report). ISBN: 978-93-5437-078-6. PP: 291.
First Edition: December 2020
Publisher: TNAU
Copyright: TNAU
Pages: 298
Cover page: Sree Kumaran Computers, TNAU Campus, Coimbatore -3
Photographs Courtesy: Mushroom Research and Training Center, Department of

Plant Pathology, CPPS, TNAU, Coimbatore
ISBN: 978-93-5437-078-6
Printed at
TNAU offset Press

TNAU, Coimbatore - 641 003.Phone: 0422 – 6611202
CONTENT
S. No. Title Page No.
1. Foreward I
2. Preface II
3. Abstract 1
4. UGC-Approval 4
5. UGC-ASO 9
6. Advisory Committee Members 16
7. Advisory Committee Proceedings 19
8. UGC Report (as per format) 23
9. Purchase of equipments in UGC-SAP Scheme 25
10. Morphological and molecular characterization of commercially 27
important basidiomycetes - I Year
11. Organization of SAP- Mandatory skill oriented Research / 63
Entrepreneurship training programme on fungal biotechnology
targeting young scientists, research scholars, student
entrepreneurs and youth – II Year
12. Organization of II SAP-Mandatory skill oriented 72
Research/Entrepreneurship training programme –III Year
13. National Seminar Conducted 90
14. Visit of important dignitaries 95
15. Establishment of Mushroom Knowledge Park and Mushroom 99
museum at TNAU, Coimbatore to encourage student research
and entrepreneurship programme-IV Year
16. Annexure-I- Transfer of Technology through one day and five 112
days training
17. Annexure-II - Mushroom Producer and Company 118
18. Annexure-III -Training to International Students 119
19. Annexure-IV- Extension Activities 119
20. Annexure-V – Other Courses 120
21. Annexure-VI - Product and Prototype development 121
22. Annexure-VII - Neighbouring Institutions participated in the UGC- 123
SAP Seminar
23. Annexure-VIII - Other Collaborative programmes 124
24. List of publication records in referred journals 129
25. List of M.Sc and Ph.D Students 167
26. Summary 168
27. Publications 171
28. Audit Utilization Certificate (AUC -2016-2020) 271
29. Final Report Assessment / Evaluation by Expert Committee 292
I
II
III
IV
ABSTRACT
ABSTRACT
UGC – SAP scheme on Enterprising Mushroom Biotechnology for Food, Feed and
Biomanure
Period: 2016 to 2020
A UGC - SAP scheme on “Enterprising Mushroom Biotechnology for Food, Feed

and Biomanure” was operated at the Department of Plant Pathology, Tamil Nadu
Agricultural University, Coimbatore from 2016 to 2020 with a budget out lay of Rs.62
lakhs. The objectives were set forth to explore mushroom diversity, conserve and to
domesticate the edible and medicinal mushrooms, develop innovative cultivation
techniques for mushrooms, exploration of antimicrobial molecules from medicinal
mushrooms, utilization of spent mushroom substrate in to biomanure, skill
empowerment to students from UGC affiliated colleges and impart mushroom
cultivation technology for farmers and woman self help groups. The equipments viz.,
Thermostat controlled autoclave, Rotary vaccum flask evaporator, Refrigerated
Centrifuge, Refrigerated shaker and a Deep freezer for the value of Rs.28,84,534/- were
purchased from the scheme. A separate UGC-SAP laboratory was established for the
benefit of students and research activities.
Research on biodiversity and conservation of mushrooms lead to
identification of 17 isolates of wild milky mushrooms collected from various parts of Tamil
Nadu. They were identified as Agaricus augustus, Auricularia polytricha, Amanita
phalloides, Calocybe indica, Fomes fomentarius, Ganoderma lucidum, Laccaria
amethystine, Lepiota castaneidica, Mycena flavescens, Psathyrella cernua, Pluteus
cervinus, Polyporus sp., Pycnoporus sanguineus, Russula olivacea, Trametes
versicolor, Termitomyces clypeatus, Schizophyllum commune, Pleurotus sajor caju, P.
florida, P. eous, Hypsizygus ulmaris, Lentinus sp and Tricholoma based on the
morphological keys microscopic characters and molecular characterization. Among the
wild isolates of Calocybe indica, the maximum bioefficinecy percentage was on served
with the isolate CBE-TNAU-1523. Besides cultivation technology of Ganoderma lucidum
under subtropical conditions was standardized in coconut wood log saw dust, arecanut
wood log saw dust and coconut leaf stalk (chopped).
1
An ecofriendly cost effective, innovative module for oyster mushroom
(Pleurotus spp) cultivation developed mushroom spawning in Polypropylene (PP)
container yielded 109 to 148 % bioefficiency with C;B ratio of 3.1. Apart from it,
laminated carton boxes used for oyster mushroom cultivation had 103.7 to 128.3 %
bioefficiency. The Moringa wood log (300 mm and 80 mm dia. with vertical slits)
cultivation of oyster mushrooms yielded 433.8g/500g substrate with average
bioefficiency of 123.9 per cent.
Paddy straw mushroom was cultivated as an intercrop in Maize cropping
systems with an average bioefficiency of 11.8 to 17.3 % using paddy straw and oyster
mushroom spent substrates. In sugarcane based cropping systems outdoor cultivation
of paddy straw mushroom was successful with a bioefficiency of 10.3 to 15.3 % using
paddy straw and oyster mushroom spent substrates. Augmentation of oyster mushroom
SMS (paddy straw) in maize field increased the level of soil available nutrients
viz., N (308.0 kg / ha) and P 2O5 (19.1 kg / ha).
Bioactive principles from the secondary metabolites of Ganoderma lucidum had
antinemic activity against Meloidogyne incognita extracted (4H-Pyran-4-one, 5-hydroxy-
2-(hydroxymethyl) ) fruiting body of Lentinus edodes inhibited the mycelial growth of
F. oxysporum and F. solani. The metabolites extracted from cap portion of G. lucidum
fruiting body inhibited the mycelial growth of Colletotrichum gloeosporioides. Besides,
ultramicroscopic observations made with SEM revealed conidial malformation and
disintegration of conidial wall. The biomolecule of cordycepin (3’-deoxyadenosine)
Ophiocordyceps sinensis and O. neovolkiana had antifungal property isolated from
caterpillar mushroom against F. o. f. sp. cubense and F. o. f. sp. lycopersici. The
bioactive molecules of Inky cap mushroom, Coprinus cinerea inhibited the egg hatching
(65.02 per cent) and increased juvenile (J2) mortality (67.0 per cent) of root knot
nematode. The volatile organic compounds produced by mycelia of Coprinus sp.
significantly inhibited mycelial growth of Fusarium oxysporum f.sp. lycopercisi. The
squalene, the mycelial culture filtrate of G. lucidum inhibited symptom development of
Groundnut bud necrosis virus in tomato plants and triggered the transcripts level of
defense genes PR 1 (pathogenesis related gene), PAL gene (Phenyl alanine ammonia
lyase) and LOX (Lipoxygenase).
2
Skill empowerment on mushroom research were imparted to four UG students
and twelve PG students from UGC affiliated colleges on mushroom biodiversity,
biomolecules, biodegradation, value addition and animal feed. Two hands on training
on spawn production and mushroom cultivation were imparted to international students
from Sultan Quaboos University, Oman and Agricultural officers from Maldives.
Three National seminars “Enterprising Mushroom Biotechnology”, “Fungal
Diversity Conservation and Exploitation of Macromlecules” and Rise of Mushroom –
Retreats to Humanity were organized and 1200 participants attended and benefitted
across the country. A national workshop on mushroom series-I was organized to 300
students and 25 faculty members.
As a part of technology transfer, twelve one day training on mushroom
cultivation was imparted to 1489 participants and five skill oriented tissue culture, spawn
production and mushroom cultivation were imparted to 73 participants. Seven
demonstrations were imparted to 339 participants including farmers from various parts of
Tamil Nadu. Dignitaries of both national and international visited the facility. Five radio
programmes were organized involving scientists, farmers and students to create
awareness on mushroom cultivation. Twenty two demonstrations on oyster mushroom
cultivation were conducted to 1195 school / college students. Special training on spawn
and mushroom production was organized to 140 SC beneficiaries including farmers,
students and woman self help groups. About 2180 visitors including students from
various colleges of Tamil Nadu were benefitted on the aspects of mushroom cultivation.
Publicity on mushroom cultivation was provided through All India Radio and daily news
papers. About 23 entrepreneurs registered with Agri business incubator of TNAU
through UGC- SAP to utilize the facilities for commercialization. A mushroom knowledge
park has been planned with a exhibition hall show casing various oyster mushroom,
milky mushroom and button mushroom varieties released by the Department of Plant
Pathology, TNAU, Coimbatore along with the developed mushroom technologies. A
separate training hall has been planned to offer skill oriented training on spawn and
mushroom production to educated youth, farm woman and woman self help groups. A
mushroom museum has been established by displaying the wild mushroom collections
and commercial varieties.
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UGC-APPROVAL
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ADVISORY
COMMITTEE
ADVISORY COMMITTEE MEMBERS
2018-2020
S. No. Advisory members Name and Designation
1. Dr. N. Kumar
Vice-Chancellor
Tamil Nadu Agricultural University
Coimbatore
2. Dr. A. S. Krishnamoorthy,
Registrar and Coordinator
Coimbatore
3. Dr. S. N. Nakkeran
Professor and Deputy Coordinator
Coimbatore
4. Dr. K. Prabhakar,
Director (CPPS)
Coimbatore
5. Dr. M. Muthamilan
Professor and Head
Dept. of Plant Pathology, TNAU, Coimbatore
6. Dr. G. Karthikeyan
Professor and Head
Dept. of Plant Pathology
Coimbatore
7. Dr. U. Sivakumar,
Professor and Internal member
Dept. of Agrl. Microbiology
Coimbatore
8. Dr. P. Nallathambi
Principal Scientist and Internal member
IARI, Wellington
Coonoor, Ooty
9. Dr. Samir Ranjan Sikdhar
Professor and Head and UGC Committee,
Division of Plant Pathology, Centaury Building,
Bose Institute Kolkata-700054, WB
10. Dr. G. Thiribhuvanamala
Associate Professor
11. Dr. M. Karthikeyan

Asst. Professor
12. Dr. P. Latha
Asst. Professor
16
2017-2018
1. Dr. K. Ramasamy
Vice-Chancellor
Coimbatore
Director (CPPS) and Coordinator
Coimbatore
Coimbatore
5. Dr. T. Raguchander
Professor and Head
Coimbatore
Coimbatore
Principal Scientist
IARI, Wellington
Coonoor, Ooty

Professor and Head and UGC committee,
Division of Plant Pathology, Centaury Building,
10. Dr. D. Anusuya,

Professor (Retd.) and UGC nominee
Department of Botany,
Bangalore University,
Jnanbharathi Campus, Bengaluru-560058

Associate Professor

Asst. Professor
13. Dr. P. Latha

Asst. Professor
Dept. of Plant Pathology,TNAU, Coimbatore
17
2016-2017
1. Dr. K. Ramasamy
Vice-Chancellor
Coimbatore
Professor and Head and Coordinator
Coimbatore
Coimbatore
4. Dr. K. Ramaraju
Director (CPPS) and Advisory Committee member
TNAU, Coimbatore
Coimbatore
Principal Scientist
IARI, Wellington
Coonoor, Ooty
Professor and Head and UGC nominee, Division of
Plant Pathology, Centaury Building,
8. Dr. D. Anusuya,
Professor (Retd.) and UGC nominee
Department of Botany,
Bangalore University,
Jnanbharathi Campus,
Bengaluru-560058
Associate Professor
Coimbatore
Asst. Professor
Coimbatore
11. Dr. P. Latha
Asst. Professor
Coimbatore
18
ADVISORY COMMITTEE
PROCEEDINGS
ADVISORY COMMITTEE PROCEEDINGS
1. 2016-2017
The first advisory committee meeting of the UGC sponsored SAP-DRS-1
“Enterprising scheme being operated in the Department of Plant Pathology, CPPS,
TNAU, Coimbatore was held on 20.01.2017 at Committee room, O/o of the Vice-
Chancellor, TNAU, Coimbatore under the chairmanship of Dr. K. Ramasamy,
Vice-Chancellor, TNAU, Coimbatore.
The following members attended the meeting:
1. Dr. K. Ramaraju, Director (CPPS), TNAU, Coimbatore and Advisory Committee

member.
2. Dr. Samir Rajan Sikdar, Professor and Head, Division of Plant pathology,
Centaury Building, Bose Institute Kolkata-700054, West Bengal and UGC
committee
3. Dr. D. Anusuya, Professor (Retd.), Department o9f Botany, Bangalore University,
Jnanbharathi Campus, Bengaluru-560058 and UGC nominee
4. Dr. U. Sivakumar, Professor (Microbiology), TNAU, Coimbatore and Advisory
Committee Member
5. Dr. S. Nakkeeran, Professor (Plant Pathology), TNAU, Coimbatore and Deputy
Coordinator
6. Dr. A.S. Krishnamoorthy, Professor and Head, Department of Plant Pathology,
TNAU, Coimbatore- Member Secretary and Coordinator.
Dr. K. Ramaraju, Director i/c, (CPPS), TNAU, Coimbatore welcomed the

Advisory Committee members of UGC-SAP-DRS-1, Scheme presented the major
objectives, year wise plan of work, budget details, work progress for the year 2016-2017
before the committee for favour of approval Dr. Anusuya and Dr. U. Sivakumar actively
participated and offered their valuable suggestions for the successful implementation of
the programme Dr. S. Nakkeeran, Professor (Plant Pathology) proposed vote of thanks.
19
Remarks offered by the Advisory committee for further action:
¾ The committee approved the overall objectives and year wise plan of work
(2016-2021).
¾ The Vice-Chancellor insisted the importance of conversation of
economically important fungal cultures and advised the scientists involved
to visit national repositories like Agarkar Institute to get hands on
experience. He also suggested forming a time like of activities for the
successful implementation of the scheme. In his further remarks the Vice-
Chancellor advised the scientists to involve M.Phil students of
conventional Universities in the collection and identification of wild
mushrooms.
¾ The Vice-Chancellor also suggested to send proposal for establishment of
pilot plant facility for automated spawn production under NADP scheme.
¾ It was insisted to involve students working in the Department of
Environmental Science, Agricultural Microbiology and Bio-informatics to
continue with the objectives of the scheme student project work priorities
need to be identified towards mycelia biomass conversion, enzyme
extraction, identifying pharmaceutically, important genes in mushroom
fungi, nucleotide sequencing and identification of wild fungi, separation of
colour, flavour, and fragrance profiles of mushrooms, mechanisms
formation sporocarps and basidiospores and use of mushroom fungi for
environment clean up.
¾ Dr. Samir Ranjan Sikdar suggested for collaborative research projects
with Bose Institute of protoplast fusion for strain improvement in
mushroom fungi. He further suggested to ensure supply of quality spawn
through Regional Centres.
¾ Dr. D. Anusuya suggested to send the AUC in time for further release of
grant from UGC. She also suggested to specify the substrates to be used
in bioconversion by mushroom fungi based on their large- scale availability
and economic feasibility. The scientist also insisted to develop value
added mushroom products. She further insisted the importance of
20
establishing a culture collection centre for mushrooms at TNAU,
Coimbatore.
2. 2017-2018
Major Recommendations
1. Establishment of mushroom knowledge park (Dr.K. Ramasamy, Chairman-UGC-

SAP)
2. Empowerment of rural unemployment youth on spawn and mushroom production
(Dr. Anusuya)
3. Knowlegde empowerment on UG and PG students from neighbouring colleges
on antimicrobial activity of biomolecules (Dr. Anusuya)
4. Documentation of mushroom biodiversity (Dr. Anusuya)
5. Intensification on arious thematic areas of mushroom research by involving both UG
and PG scholar from neighbouring colleges (Dr. Samir Ranjan Sikdhar)
3. 2018-2019
The Annual Advisory Committee meeting of UGC-SAP-DRS-1 scheme on

“Enterprising mushroom biotechnology on Food, Feed and biomanure” was conducted
in the chamber of Vice-Chancellor committee hall, TNAU, Coimbatore on 30.09.2019
under the chairmanship of Dr.N.Kumar, Honourable Vice-Chancellor, TNAU,
Coimbatore in the presence of the following advisory committee members.
1. Dr. A. S. Krishnamoorthy, Registrar & Coordinator (UGC-SAP).

2. Dr.K.Prabakar,Director (CPPS)
3. Dr. Samir Ranjan Sikdhar, (Rtd Professor), UGC Nominee
4. Dr. S. Nakkeeran, Professor (Plant Pathology) & Deputy Coordinator (UGC-SAP)
5. Dr. U. Sivakumar, Professor (Agrl. Microbiology) & Member (UGC-SAP)
6. Dr.P.Nallathambi, Principal Scientist & Member (UGC-SAP) Wheat Research
Station, IARI , Wellington, Ooty
7. Dr. M. Muthamilan, Professor and Head, Department of Plant Pathology, TNAU,
Coimbatore
21
The advisory committee suggested following issues to strengthen the activity of
UGC-SAP.
1. Empowerment of woman self help groups on spawn and mushroom production

(Dr.N.Kumar, Vice-Chancellor)
2. Developing innovative modules/ growing kits for mushroom production as an
alternate to polybags and exploring antimicrobial activity of mushrooms
(Dr.P.Nallathambi)
3. Documentation and conservation of the biodiversity edible and medicinal
mushroom(Dr. Samir Ranjan Sikdhar)
4. Standardizing the cultivation techniques for medicinal mushroom Ganoderma
lucidum( Dr.N.Kumar, Vice-Chancellor)
5. Strengthening on various thematic areas of mushroom research through skill
empowerment of UG and PG scholar from neighbouring colleges
(Dr. Samir Ranjan Sikdhar)
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UGC REPORT
(AS PER FORMAT)
UGC Report (as per format)
ANNEXURE-VII
UNIVERSITYGRANTSCOMMISSION - NEWDELHI
PROGRESSREPORTANNUAL / FINAL REVIEW

UNDER SAP (CAS/DSA/DRS)
Date of first approval with level at inception : January 2016

Date of implementation of current phase as : 22.08.2016
noted by the UGC
Name of the University : Tamil Nadu Agricultural University,
Coimbatore
Name of the Department : Department of Plant Pathology,
CPPS, Coimbatore
Status (CAS/DSA/DRS with phase) : DRS 1
Period of Report : From 2016 to 2020
Non Recurring: Recurring: 32.50 lakhs Total: 62 lakhs
29.50 lakhs
Amount allocated for 5 years : 62 lakhs
Amount sanctioned during the year : 6.50 lakhs
Coordinator's Name : Dr. A.S. Krishnamoorthy,
Registrar, TNAU, Coimbatore
Deputy Coordinator’s Name : Dr. S. Nakkeeran, Professor (Plant
Pathology). TNAU, Coimbatore
Address : Department of Plant Pathology,
TNAU, Coimbatore
City : Coimbatore Pin: 641003
State: Tamil Nadu
Tel.: 0422-6611226
Amount utilized during the year : -
Date of first sanction (Current phase) : 01.01.2016/-
Totalgrantsreceivedsinceinception : Rs. 43,01,107/-
23
(a).Thrust Area(s) :
Ongoing Modified to, if any,

Identifiedsince inception and when UGC
approval reference
no and date
Morphological and molecular characterization of
commercially important macrobasidiomycetes Nil
Standardization of innovative systems and Completed
modules for indoor/outdoor cultivation of edible
mushrooms utilization of spent mushrooms
wastes as biomanure and animal feed.
Organization of SAP-Mandatory skill oriented Completed Nil
Research / Entrepreneurship training programme
on fungal biotechnology targeting young
Scientists, research scholars, student
entrepreneurs and youth (One each targeting
researchers and entrepreneurs).
Organization of II SAP-Mandatory skill oriented Completed Nil
Research/Entrepreneurship training programme
(One each targeting researchers and
entrepreneurs).
Technology transfer and formation of Mushroom Completed Nil
Producer Companies in coordination with the
Directorate of Agribusiness Development, TNAU,
Coimbatore.
Organization of III-SAP Mandatory skill oriented Completed Nil
Research / Entrepreneurship training programme
(One each targeting researchers and
entrepreneurs).
Strengthening fungal bioprocess technology
programmes through self supporting and PPP
mode to sustain the R&D activities.
Documentation of SAP activities for self
evaluation targeting further area of “Centre of
Excellence in Tropical Mushroom Biotechnology”
at TNAU, Coimbatore.
24
FutureThrust Area proposed
(b).UGC nominees with Address, City, Pin, State, Tel.,Fax, e-mail (as approved by
the UGC):
I. Dr. Samir Ranjan Sikdhar, Division of Plant Pathology, Bose Institute,

93/3A/1, Aacharya Prafulla Chandi road, Raja Bazzar Kolkata WB,
Mobile:09845154456.
II. Prof. Anusuya, Department of Botany, Bangalore University, Jnan bharathi

Campus, Bengalur, Karnataka 560056 Ph:09831042569.
Majorachievements:
(i) Teaching:
a.Newcoursesintroduced:
x Certificate course on Commercial Mushroom Production

x Experiential Learning on Commercial Mushroom Production
b.Curriculumlastrevised&significantchanges:
x Advances in Mycology – including the mushroom biotechnology

c. Examinationreformslastmadewithspecialfeatures: Nil
d. Teachinglab./equip./newfacilitiescreated:
Purchase of equipments in UGC-SAP-DRS-1 Scheme
S. Date of Name of the instruments Amount (Rs.)

No. purchase
1. 31.03.2017 Refrigerated shaker 498067.00
2. 31.03.2017 Rotary vaccum flask evaporator 498960.00

with accessories
3. 31.03.2017 Refrigerated Centrifuge 470000.00
4. 27.03.2018 Deep freezer 1810400 YEN(Rs.11,18,131)
5. 31.03.2017 Thermostat controlled 299376.00

autoclave
25
PURCHASE OF EQUIPMENTS IN UGC-SAP-DRS-1 SCHEME
Refrigerated Shaker Rotary Vaccum Refrigerated

Flask Evaporator Centrifuge
Deep freezer Thermostat controlled autoclave
26
(i) Research
a. Research (high light major objectives set-forth (as proposed) and

achievements made with breakt hrough, innovation broughtin, technology
transferred, international collaboration which have created resources).
Year 1. Morphological and molecular characterization of commercially important

basidiomycetes
(i) Biodiversity, conservation and identification of milky mushroom

isolates through morphological and molecular approaches
The Indian sub continent is blessed with diverse agroclimatic zones that
harbour a treasure trove of fungal diversity with about 1,200 species of fungi
belonging to the order Agaricales, Russulales and Boletales contributing to 10
percent of the global mushroom flora. So far, in India about 1,105 to 1,208
species of mushrooms belonging to 128-130 genera have been documented
and among these, 300-315 species belonging to 75-80 genera are considered
edible. The Western Ghats are globally recognised for the biodiversity
hotspots forming a long mountainous region along the west coast of India. Till
date 750 species of mushrooms has been documented. It has an unestimated
wealth of mushroom biodiversity that needs to be tapped properly as there
are still several undescribed species yet to be identified. Efforts need to be
made to identify and exploit this mushroom flora for utility as their biodiversity
and conservation strengthen the food security of a country. These data on the
occurrence of the mushrooms reveals the richness of mycoprotein in the
country. Not only in terms of edibility, there lies enormous applications of
these mushrooms for bioremediation, biodegradation, biopesticidal and
pharmacological values that could be exploited.
Biodiversity:
Survey was conducted at various agroclimatic zones of Tamil Nadu and a total
number of 17 wild isolates viz., CBE-TNAU-1513 to 1526, CBE- TNAU-1603, 1604 and
CBE-TNAU-1701 were collected along with passport data and geographical co-
27
ordinates Among the wild isolates, four were associated with the finer roots of Cocos
nucifera (L.) and 3 had ectomycorrizal relationship with Delonix regia (L). The isolates
CBE-TNAU-1515 and 11516 were had association with the finer roots of Ficus
benghalensis and Tamarindus indicus. Other eight isolates were growing
independently as humicolous mushrooms and they have been collected from banana
field, as well as from litter decomposition sites. While collecting the passport data,
growth characters like stipe length and diameter; cap length and diameter; shape of
stipe and cap; gill colour and attachment; weight of sporophore at the time of
collection; smell and field photographs were documented.Among the wild isolates
collected, one single fruiting body of CBE-TNAU-1701 weighed 14 kg followed by
CBE-TNAU-1524, which recorded 13.4 kg per sporophore. At the same time, a
minimum weight of 20 g per sporophore was also noticed in case of CBE-TNAU-1604.
Thus, the fruiting bodies of milky mushrooms (Table.1.)
Molecular characterization of wild mushrooms understanding the genetic

relationship
PCR amplification of 5.8S and 5S rRNA regions of DNA from all the 25 isolates
was performed. ITS 1, ITS 4 and IGS primers were used in the study. The ITS region
were amplified with an amplicon size of 650 bp pertaining to isolate CBE-TNAU 1516
and 1519; while, all other isolates were found to be amplified at 700bp. In case of IGS
amplification, the DNA of all the 25 strains were amplified between 1500 and 1600 bp
(Plate.1.)
RAPD analysis with 25 isolates confirmed the presence of dimorphic bands. The
amplified DNA fragments ranged from 430 bp to700 bp. Based on the analysis made
from RAPD-PCR banding patterns, all the 25 strains of milky mushroom were
grouped into two major clusters A and B. Among the 25 isolates 23 were separated in
cluster A, which includes APK2; whereas, CBE- TNAU- 1526 and 1701 were
assembled in cluster B at approximately 67.5 per cent similarity coefficient. The
cluster A is again divided into two sub clusters A1 and A2 with 48 per cent similarity.
Cluster A1 consists of two sub clusters i.e., A1a and A1b with 67 per cent similarity
coefficient. A2 contains two sub groups A2a and A2b at 54 per cent similarity. Cluster
A1a consists of APK2, CBE TNAU 1513, 1521 and 1522 at 54 per cent similarity.
28
Whereas, A2b clade consists of CBE TNAU 1517 and CBE TNAU 1525 with 57 per
cent similarity. Remaining isolates were separated in cluster A2a subdivision with 59-
82 per cent similarity. RAPD analysis showed a minimum of 48 per cent to the
maximum of 82 per cent similarities among the strains of milky mushroom
(Plate.2-5a).
Inter Simple Sequence Repeat (ISSR) analysis was performed to establish the
molecular phylogenetic relationship with 25 milky mushroom strains by using 5
random primers, of which D3 primer, performed well. The DNA fragments amplified at
different molecular weights ranged from 300-900 bp. Similarity indices and genetic
relationship revealed from the experiment is presented as dendrogram by using
UPGMA. Among the 25 isolates, 24 were found to be separated in cluster A along
with the cultivar APK2; while, CBE-TNAU-1603 alone was found to be separated in
cluster B with approximately 36 per cent similarity coefficient. The cluster A is again
divided into two sub clusters A1 and A2 with 48 per cent similarity. Cluster A1a
consists of two sub clusters i.e., A1aa and A1ab with 52 per cent similarity coefficient.
A1aa consists of variety APK2, CBE-TNAU-1513, 1517, 1521, 1522 and 1523;
whereas, A1ab consists of CI-13-02 with 49 per cent similarity. Remaining isolates
were separated in cluster A1b and A2a, b subdivisions with 44-82 per cent similarity.
ISSR analysis showed 36 to 84 per cent similarities among the milky mushroom
strains tested. (Plate.6, Fig.1.)
29
Table.1. Comparison of sporophore characters
Growth characters
Isolates Basidisporedia
Pileus Stipe Colour Gills Spore print Basidiospores
(μm)
APK2 Campanulate Robust Pure White White Golden yellow 1.2
cylindrical white
CI-13-02 Convex Robust White Yellowish White Brown 1.5
cylindrical white
CI-13-04 Flattened Cylindrical Dull Yellowish White Brown 2.1
white white
CI-13-06 Flattened Cylindrical Dirty Yellowish Creamy Brown 1.4
white white white
CI-14-02 Convex Robust Dirty White Creamy Brown 1.8
cylindrical white white
CI-14-03 Convex Robust Pure Yellowish White Golden yellow 1.0
30
cylindrical white white
CI-14-04 Flattened Cylindrical Pure White White Light yellow 1.2
white
CI-14-06 Convex Cylindrical Creamy Yellowish Creamy Brown 1.6
white white
CBE-TNAU- Flattened Bulged, Pure White White Brown 1.5
1513 cylindrical white
CBE-TNAU- Flattened Cylindrical White White Dull white Golden yellow 2.0
1514
CBE-TNAU- Flattened Robust Dirty Brownish White Golden yellow 1.4
1515 cylindrical white white
CBE-TNAU- Convex Cylindrical White Yellowish White yellow 1.8
1516 white
CBE-TNAU- Campanulate Robust White White White Golden yellow 2.3
1517 cylindrical
CBE-TNAU- Flattened Cylindrical White White Dull white Brown 2.1
1518
CBE-TNAU- Flattened Cylindrical Dull White White Brown 1.6
1519 white
CBE-TNAU- Convex Robust White Yellowish White Golden yellow 1.8
CBE-TNAU- Campanulate Robust Dull White Dull white Yellow 1.4
CBE-TNAU- Campanulate Robust Dull White White Yellow 1.3
CBE-TNAU- Campanulate Robust Pure White White Golden yellow 1.7
CBE-TNAU- Flattened Cylindrical Creamy Yellowish Dull white Brown 1.5
1524 white
CBE-TNAU- Flattened Cylindrical White Yellowish Dull white Brown 2.4
1525 white
31
CBE-TNAU- Convex Robust Creamy White White Brown 1.7
1526 cylindrical
CBE-TNAU- Convex Robust Pure White Dull white Golden yellow 1.5
CBE-TNAU- Flattened Cylindrical White Yellowish Dull white Golden yellow 1.3
1604 white
CBE-TNAU- Flattened Cylindrical Creamy Yellowish White Brown 2.1
1701 white
Plate.1. Morphology of wild milky mushroom isolates
CBE- TNAU -1513 CBE- TNAU -1514 CBE- TNAU -1515 CBE- TNAU -1516 CBE- TNAU -1517
32
CBE- TNAU -1603 CBE- TNAU -1604 CBE- TNAU -1701
33
Plate.2. PCR amplification of ITS region of wild milky mushroom
Plate.3.PCR amplification of IGS region of wild milky mushroom
34
Plate.4. RAPD profiles of wild milky mushroom strains amplified with 20
primers- Set1
35
Plate.5. RAPD profiles of milky mushroom strains amplified with 20 primers-
Set 2 (contd.)
36
Plate.5a. RAPD profiles of milky mushroom strains amplified with 20
primers- Set 2 (contd.)
37
Plate.6. ISSR profiles of milky mushroom strains amplified with five primers
38
Fig.1. ISSR profiles of milky mushroom strains amplified with five primers
Table.2. Testing the yield performance of wild milky muhsroom strain CBE-
TNAU-1523 (GEN BANK Acc No. SUB4030762)
The milky mushroom strain CBE-TNAU 1523 was collected during 2015 from
Edayarpalayam, Coimbatore district of Tamil Nadu. The pass port details of the wild
mushroom is provided below.
Specimen no. CBE- TNAU- 1523

Date of collection 20.10.15
Locality Idayarpalayam,, Coimbatore
GPS Data
a. Longitude 76.97° E
a. Lattitude 11.01 ° N
a. Altitude (MSL) 411.2m
Single/ groups/ connate both single and group in a different location
Habitat attached with the finer roots of Cocos

nucifera
Smell (Y/N) yes, fatty smell
Spore print colour pure white
Cap
a. colour attractive white colour
a. diameter 6.5cm
a. shape highly campunate
39
a. scales/smooth smooth
Stipe straightened cylindrical
a. attachment central
a. colour pure white
a. diameter and length 3.5 cm, 7.3cm
a. base not bulged

Annular ring absent
Volva absent
Veil absent
Basal association away from the trunk
Lamellae smoothly attached running down the stem
Attachment adnate
a. Gill colour pure White
a. Gill Edges Smooth
Edibility edible
Average weight of single sporophore 243g
Name (s) of the Collector Mrs. Lakshmi
Plate.7. Wild mushroom CBE-TNAU-1523
Pure white, silk thread like, quickly White Spore print Spore print with
aggregating with 1.8g dry wt. of golden yellow basidiospores measuring
mycelium 1.7μm
40
Molecular Confirmation of Calocybe indica CBE-TNAU-1523 isolate was carried out
with 5.8S rRNA sequencing by using ITS primer and the confirmed sequences were
submitted in the NCBI-GENBANK database and obtained accession number
SUB4030762.
Table.3. Performance of CBE-TNAU-1523 in comparison with Calocybe indica

var.APK 2 at TNAU Coimbatore centre (2016-17)
Milky mushroom Yield/500g Bioefficiency

Strain DFSR DFPF DFFH substrate (% )
a a a a
CBE -TNAU -1523 13.2 8.6 7.3 795.1 159.0
Calocybe indica var. a a a a

12.9 8.3 7.3 768.8 153.8
APK2
ON FARM RESEARCH TRIAL AT TNAU: Yield performance of CBE-TNAU-1523

Calocybe indica in comparison with APK2 at different research centres of
TNAU (2019-2020)
41
Centres:
AC & RI, Coimbatore; AC & RI, Madurai; AC & RI, Killikulam; AC & RI, Trichy;
RRS, Aruppukottai and KVK, Villupurum.
Treatment :
T1: Test culture ( CBE-TNAU -1523 Milky Mushroom)
T2: APK2 (TNAU Milky Mushroom variety)
Replication: 13
Design: RBD
Substrate: Paddy straw: 500g/ half cut bed
Results
Table.4. Pooled mean analysis of yield performance of CBE-TNAU-1523 milky

mushroom
Av. wt/ Average

Calocybe sporo yield
No. of Pileus:stipe BE C:B
indica Strain / DFSR DFPF DFFH
sporophores phore (g/500g ratio (%) ratio
variety
(g) substrate)
CBE-TNAU-
1523
20.2 28.4 32.3 34.7 27.9 725.59 1:2.2 145.1 1:3.5
(test culture)
APK2
21.9 29.5 34.6 30.7 25.3 611.78 1: 2.1 122.3 1:3.3
(Control)
CD (P=0.05) 0.4 0.65 0.52 2.1 1.3 13.59
DFSR: Days for spawn run; DFPF : Days for pinhead formation; DFFH : Days
for first harvest; BE : Bioefficiency
The pooled mean analysis (Table 4) of all six centres of TNAU tested for
yield performance of CBE-TNAU-1523 milky mushroom with TNAU variety APK2
showed that the spawn running was completed on 20.2 days in CBE-TNAU-1523
compared to APK2 variety (21.9 days). Also the pinheads were formed one day
earlier in CBE-TNAU-1523 (28.5 days)) compared to APK2 (29.5 days). Also the first
42
harvest was recorded two days earlier on 32.3 days in CBE-TNAU-1523 as
compared with APK2 variety (34.7 days). The average number of sporophores and
average weight per sporophore were significantly higher in test culture (34.7 and
27.9 g, respectively) compared to APK2 (30.7; 25.3 g, respectively).There was
significant differences in yield as shown by data that CBE-TNAU-1523 yielded
725.59g when compared to APK2 with 611 .78 g per 500 g substrate. Comparatively
the bioefficiency was maximum in CBE-TNAU-1523 with 145.1% when compared to
APK2 which recorded 122.3%.
TNAU-CBE 1523 APK2
TNAU- CBE- 1523 Calocybe indica
43
(ii) Biodiversity of edible and medicinal mushrooms
Survey at Coimbatore (Siruvani, TNAU campus), Ooty, Kallar and Kodaikanal

revealed the presence of about 19 mushroom cultures. It was identified as viz.,
Agaricus augustus Auricularia polytricha Amanita phalloides Calocybe indica, Fomes
fomentarius, Ganoderma lucidum,Laccaria amethystina Lepiota castaneidica
Mycena flavescens Psathyrella cernua Pluteus cervinus Polyporus sp. Pycnoporus
sanguineus, Rusulla olivacea, Trametes versicolor, Termitomyces clypeatus,
Schizophyllum commune and Tricholoma based on the morphological keys and
microscopic characters provided by DMR,Solan.
Plate. 8. Biodiversity of edible and medicinal mushrooms
TNAU- CBE 1901 Agaricus TNAU CBE-1902 TNAU -CBE1903

augustus Auricularia polytricha Calocybe indica
TNAU -CBE-1904 TNAU -CBE-1905 Fomes TNAU- CBE-1906

Calocybe indica fomentarius Ganoderma lucidum
44
TNAU- CBE-1907 TNAU- CBE-1908 Laccaria TNAU- CBE-1909
Ganoderma lucidum amethystina Lepiota
castaneidisca
TNAU- CBE-1910 Mycena TNAU- CBE- TNAU- CBE-1912

flavescens- 1911Psyatherella cernua Pluteus cervinus
TNAU- CBE-1913 TNAU- CBE-1914 TNAU- CBE-1915

Pycnoporus sanguineus Polyporus sp
Rusulla olivacea
45
TNAU- CBE-1916 TNAU- CBE-1917Trametes TNAU- CBE-1918
Schizophyllum commune versicolor Termitomyces
clypeatus
TNAU- CBE-1919 Tricholoma terreum
Table.5. Phenotypic characterization of wild mushroom fungi
S. TNAU sample Habitat Identification

Number
No.
1 TNAU-CBE-19- Soil / humus Agaricus augustus-Mild smell, Cap 4.5 cm

01 to 6.0 cm, light creamy brownish white,
on lawn
fleshy, hemispherical in button stage, later
becoming convex, flattens on
maturity,dry,cap easily peels off, Prominent
brown scales on cap in concentric
manner.Stipe fleshy,firm, long 4.5 to 10 cm,
centrally attached, hollow in centre, colour
white till the ring. Below the ring, stipe is
covered with brown scales, Clavate at base,
ring present, whitish veil present in young
buttons, veil torn in matured ones and
remains as ring,stem base deep in soil.
46
Crowded, free gills, Initially light pink later
becoming brown on maturity, gill edges
smooth, white spore print
2 TNAU-CBE-19- Felled trees Auricularia polytricha - In Groups, No

02 smell,the spore print is creamy white to light
pink.Cap is dark purplish brown ranging
from 4.5 to 9 cm, ear shaped, surface
smooth, dry, glabrous, elastic,
gelatinous.Stipe dark purple, lateral very
short stalk not visible, 0.5 to 0.7cm, thin
base.Gilled fungi
3 TNAU-CBE-19- In soil in Calocybe indica-Single, mild smell, spore

03 association print white. Cap White, fleshy, firm, 30 to 45
with Delonix
cm diameter, convex, later becoming
regia
umbrella like at maturity, margin smooth,
easily peels off. Stipe short centrally
attached, 10 cm to 12 cm, bulbous at
base.Gills crowded, edges smooth, easily
separable, forking towards the stipe, Adnexed,
creamy white, ellipsoid spores, smooth
4 TNAU-CBE-19- On soil in Calocybe indica-Single, very mild smell,

04 association spore print white cap white, fleshy, firm,
with root of
pungam 10 cm to 15 cm, convex, later becoming
tree.
umbrella like at maturity, margin smooth,
easily peels off.Stipe white, Short, centrally
attached, 7.5 cm length,6 cm diameter,
Bulbous base.Gills crowded, edges smooth,
easily separable, forking towards the stipe,
Adnexed, creamy white, ellipsoid spores,
smooth.
5 TNAU-CBE-19- In Fomes fomentarius-Single, cap, hoof like,

05 association Pale brownish white, Smooth, glabrous
with pine
when young, hard and with folded rings or
47
tree trunk zones and grey at maturity.Spore print light
yellow. Lamellae Pores minute, numerous,
white initially, later turns brown at maturity,
spores cylindrical, smooth
6 TNAU-CBE-19- Single in Ganoderma lucidum -Smell mild, Spore

06 association print colour Reddish brown. Cap Brownish,
with Delonix
whitish at growing margins, 8.0cm to 18.0
regia tree
base cm, bracket shaped, Smooth, glabrous
when young, shiny and hard when
matured.Stipe Lateral to eccentric,
Brownish, 6.0 cm to 6.5 cm. Stipe base
broad at pileus attachment and tapering at
base. Lamellae pored, Pores minute,
numerous, white initially, later turns brown
at maturity, spores ellipsoidal to oval with
warts
7 TNAU-CBE-19- Associated Ganoderma lucidum-Single, No smell,

07 with felled Spore print colour reddish brown.Pileus
tree stump
bracket shaped 7 cm, Smooth, glabrous
when young, shiny and hard when
matured.Stipe Lateral to eccentric, Reddish,
length 6.0 cm to 6.5 cm, Broad at pileus
attachment and tapering at base. Lamellae
Pores minute, numerous, white initially,
later turns brown at maturity, spores
ellipsoidal to oval with warts
8 TNAU-CBE-19- Leaf litters of Laccaria amethystina. Scattered in Groups,

08 Delonix regia No smell, creamy whitish Pileus,Purplish lilac
tree ,
Mycorrhizal colour 2.5 cm to 5.0 cm, Convex initially, later
association depressed at centre on maturity, margins
smooth but splitting at maturity,cap surface
Smooth, dry, glabrous.Stipe equal centrally
attached, Purplish lilac later fading at full
maturity, 4.0 cm to 6.0 cm, stipe base Thick,
48
hollow at centre. Gills -distantly spaced,
Adnate, gills, unequal, forking towards stipe,
Purplish lilac colour, smooth edges
9 TNAU-CBE-19- In Groups, Lepiota castaneidisca -Strong smell, spore

09 Soil/ debris print white.Cap Creamy white, delicate ,with
on lawn
orange colour at centre which fades on
maturity, 4.5 cm to 6.0 cm, Convex cap
which flattens on maturity, Smooth,moist,
breaks off easily from stipe. Stipe, Equal,
centrally attached, White, brown at base,
4.0 cm to 5.0 cm, thin, clavate at base.Ring
present, Gills - subdistant to crowded, easily
separable, Free, forking towards stipe, Gills
Creamy white ,serrated edges
10 TNAU-CBE-19- In groups, Mycena flavescens-Strong smell, white

10 Dead woods spore print. creamy white, delicate, brown at
of pine trees
the centre which fades on maturity, 4.5 cm
to 6.0 cm, Conical to broadly with striations
on cap margins at maturity, surface
smooth, dry, glabrous, cap breaks off easily.
Stipe short, centrally attached, White initially
later turning to light yellow at full maturity,
4.6 cm to 7.0 cm, base thin, fragile.Gills -
subdistant, easily separable, Adnexed,
white to cream colour gills, Creamy white to
pale grey, amyloid spores, gill edges
smooth
11 TNAU-CBE-19- In groups, Psyatherella cernua - strong smell, spore

11 association print Purple brown .Cap Light brownish
with pine
yellow, 4.5 cm to 6.0 cm, Convex, margins
tree
appendiculate, Smooth, dry, glabrous stipe
central, equal, white, 4.5 cm to 6.0 cm,
base thin gills-close, unequal, Adnexed,
white to cream colour gills later turning
49
purplish brown on maturity, ellipsoid spores,
gill edges smooth
12 TNAU-CBE-19- On soil in Pluteus cervinus, mild smell, Brownish

12 humus rich pink spore print. Cap brownish yellow, 2.5
area under
cm to 4.0 cm when opened, conical
trees
becoming broadly convex to flat on maturity,
smooth. Stipe Central, Equal, White, 4.5 cm
to 5.5 cm, base thin, gills-close or crowded,
Free from the stem; close or crowded, Gill
colour Initially whitish becoming pink to
brownish pink on maturity with salmon
coloured spores
13 TNAU-CBE-19- Groups/ Pycnoporus sanguineus -No smell, Light

13 gregarious, orangish white.Cap Corky bright thin
Dead woods
orange sporophores, does not fade at
of felled
trees maturity, 2.5 cm to 5.0 cm, bracket shaped
with zonations, Leathery, dry, glabrous,
margins acute to wavy orange powder like
basidiospores sticking to hand.Stipe
Sessile, pores minute, orange coloured.
14 TNAU-CBE-19- On felled Polyporus sp., Connate, No smell, spore

14 tree stump print Creamy Whitish , 7 to 14 cm diameter,
Bracket shaped, rough surface, dry.Stipe
central, creamy white, 3.0 cm to 3.5 cm
length, Broad at pileus attachment and
tapering at base. Pores ate tube like,
numerous, cylindrical to ellipsoid spores
15 TNAU-CBE-19- In Groups on Rusulla olivacea - Mild smell, spore print

15 pine tree Pale yellow,Pileus Creamy white at centre,
debris
greyish brown to puplish green at margins,
peels off easily, 4.5 cm to 6.0 cm, Firm,
convex later becoming plano depressed
with decurved margins, surface dry,
50
glabrous. Stipe equal, central, fleshy, stout,
White, 4.6 cm to 7.0 cm, stout base, hollow
at centre.Gills - crowded, easily separable,
adnexed, creamy white warty spores, gill
edges smooth
16 TNAU-CBE-19- on felled Schizophyllum commune -In groups,

16 trees spore print white. Cap whitish, spongy, later
becoming leathery, 1.0 to 2.0 cm, fan
shaped, wavy margins, hairy, little rough.
Split gills, white in colour; Irregular
17 TNAU-CBE-19- On felled Trametes versicolor - Gregarious /

17 wood logs scattered, No smell, creamy white light
creamy to pinkish brown, 3.0cm to 7.0 cm,
Bracket - turkey tail, grows in layers on
stumps, Leathery/ coriaceous/ with brown
zonations. older sporophores with green
algae growing on them, minute fine hairs,
Lateral, Light creamy to pinkish brown, 0.5.0
to 1.5 cm, Stipe thin, short, pores minute,
numerous, whitish to pale brown, cylindrical
smooth spores
18 TNAU-CBE-19- Humus / soil Termitomyces clypeatus - Humus / soil,

18 No smell, Spore print Creamy white.Cap
Brownish white, 4.5 cm to 8.0 cm, Initially
conical, later Umbonate, Small warts like on
cap, Stipe Central, Equal, White, long, 6.0
cm to 6.5 cm, Bulbous base, Gills-close to
crowded, Free, white, Serrate, ellipsoidal
spores
19 TNAU-CBE-19- On soil in Tricholoma terreum Single, very mild,

19 association White spore print. Cap greyish White,
with Pungam
fleshy, 4 cm to 6 cm, convex, later
tree
becoming flattened and way, margins
51
smooth split at maturity, easily peels off,
Grey scales like. Stipe short, centrally
attached, white, 5.5 cm, bulbous, gills -
crowded, uneven, forking towards the stipe,
Adnexed. Gill colour Creamy white, oval
spores, smooth.
(ii). Standardization of innovative systems and modules for indoor/outdoor

cultivation of edible mushrooms and utilization of spent mushrooms wastes
as biomanure and animal feed.
Mushrooms have been part of human diet for their ample nutrition, medicinal
value and flavor. Nutritional value of mushroom is far better than that of any
vegetable or fruit. More than 1000 species of Pleurotus have been described
throughout the world. However, only 50 valid species have been commercially
explored. Pleurotus spp are efficient producers of protein rich food from crop
residues.In Tamil Nadu moringa is cultivated in about 7408 ha mainly as an annual
crop.After harvesting the pods, the pruned wood logs are normally discarded or burnt
as these branches do not possess economic value. Light hard wood pieces of
moringa are available in plenty throughout the year with less cost. Under secondary
agricultural cropping system such kind of value less wood logs need to be profitably
utilized.
Selection of the best locally available substrates, their treatment methods and
innovative containers for cropping will have a greater stake hold, while designing
home growing to large scale production systems for oyster mushrooms. However, in
India, over years mostly, poly bags of varied sizes are used for the cultivation of this
mushroom utilizing boiled or steam treated paddy straw or wheat straw .After the
crop cycle, worn out polypropylene or polyvinyl chloride container bags are most
often burnt or disposed indiscriminately. This practice is not environmentally benign
and suffocates soil biota. Further, these containers cannot be repeatedly used, which
may result in increased cost of production.
Proper disposal of SMS always remained as a challenging task for mushroom

growers. Sequential degradation of raw materials using different mushroom fungi will
help to enhance the effective usage of a wide variety of agro wastes. Integrating
varieties of mushrooms into a permaculture system would be an intensive design
52
process, in which natural elements of an ecosystem are replaced by food-producing
relatives, creating an edible landscape. Although, not familiar in India except in one
or two cases (Krishnamoorthy et al., 2005), outdoor system of mushroom cultivation
as practiced in other countries is very much possible. If established, a sequential
cropping system with SMS of Pleurotus spp for cultivating paddy straw mushroom
fungus (Volvariella spp), it may be possible to reap additional profit. Further, the N
content in the SMS can be increased with an ultimate reduction in C: N ratio
favouring manurial value of SMS.
I. Standardization of innovative systems for cultivation of oyster mushroom
(i) Polypropylene containers for cultivation of oyster mushroom species
To reduce the risk of environmental pollution, instead of using the

polypropylene bags, recyclable polypropylene containers were tried for cultivation of
oyster mushroom species viz., Pleurotus florida (PF), Hypsizygus ulmarius (Co2)
and Pleurotus pulmonarius. The yield increased with H. ulmarius (Co2) cultivated in
polypropylene bottle (1800ml) containers (267.17g per bottle with 148.43 per cent
bioefficiency). P.platypus (Pp) was also (Table.6) and was followed by P.
pulmonarius.However, P. floridawas notequally effective in increasing the
bioefficiency (197.26 g per bottle with 109.6 per cent bioefficiency) when cultured in
polypropylene bottle containers.
Table.6. Comparative evaluation of Pleurotus spp grown in PP bottle containers

(1800 ml) and in paddy straw substrate
Avg.
DFSR Yield
No.of Avg.weight
(g/180g BE
Species DFPF DFFF sporophores (g)/
of (%)
100% harvested sporophore
50% paddy
straw)
H. ulmarius (CO
6.3 10.0 16.8 18.3 12.6 21.2 267.17 148.43
(OM)2)
P. eous(APK1) 4.2 6.8 13.6 15.3 13.3 15.6 208.16 115.64
P. platypus(Pp) 3.1 5.3 7.6 10.6 14.6 16.3 238.33 132.41
P. florida(Pf) 7.6 11.2 20.3 22.3 11.5 17.2 197.26 109.6
P.pulmonarius
7.3 11.3 16.6 18.4 15.7 14.3 225.33 125.18
(Ppl)
CD(p=0.05) 0.35 0.55 0.95 1.06 0.78 1.02 30.05
*Mean of five replications, each with six bottles
53
DFSR- Days for spawn run
DFPF- Days for pinhead initiation
DFFF- Days for first flush
On farm testing on evaluating the performance of polypropylene container as

an alternate to polythene bags
A OFT was conducted at six research centers of TNAU to evaluate the
performance of polypropylene containers as an alternative to polythene bags
The pooled mean analysis of all six centers tested for use of PP containers for
cultivation of oyster mushroom indicated that cultivation of mushroom in PP
container yielded 367g on par with the existing polythene bag method which
produced a yield of 374.9 g per 300 g paddy straw substrate. The spawn running
was completed two days earlier in container with uniform growth compared to PP
bags (16.1 days). Also the pinheads emerged two days earlier in PP containers (17.9
days) compared to PP bags (19.2 days). The mushrooms came to harvest on 0.4
days in PP containers whereas one and half days delayed harvest was noticed in PP
bags (21.9 days). There was no significant difference in the average weight of
sporophores and the average weight of mushrooms between the treatments. The
bioefficiency of yield in PP container recorded 126.3 % on par to PP bags with 128.9
%(Table.7. Plate.9).
Table.7. Pooled mean analysis of yield performance of PP containers for

oyster mushroom cultivation
Average
No. of Av.Wt/ yield Bioeff
C:B
Treatment DFSR DFPF DFFH sporop sporoph icienc
(g/300g ratio
hores ore (g) y (%)
substrate)
T1: PP 14.8 17.9 20.4 34.6 10.69 367.0 126.3 1.3.1

container 4
T2: PP bags 16.1 19.2 21.9 36.6 10.05 374.9 128.8 1:

6 3.2
CD (P=0.05) 0.74 1.58 2.23 2.0 NS 9.5
DFSR: Days for spawn run; DFPF: Days for pinhead formation; DFFH : Days
for first harvest; BE : Bioefficiency
54
(ii) Testing the suitability of laminated carton boxes as containers
Laminated carton boxes (36x18x12 cm and 18x12x8 cm) were used for the
cultivation of four different species of Pleurotus. Significantly increased yield was
obtained with P. platypus (PP) when cultivated in laminated carton box (36x18x12
cm) container (886.5g per box with 136.4 per cent bioeffficency). However,
H.ulmarius was found to give yield on par with that of P. platypus in laminated carton
box containers. P. eous and P.florida gave comparatively less yield (694.5 and
686.6g per box with 106.8 and 105.6 per cent bio efficiency). (Table.8.,
Plate 10.)
Table.8. Testing the suitability of laminated carton boxes for the cultivation of
oyster mushroom species
36x18x12 cm 18x12x8 cm
Avg. Avg.
Yield (g Yield
Species Avg. Avg.
/ 650g (g/300g BE
DFSR DFPF DFFF NSH w eight (g) / BE (%) DFSR DFPF DFFF NSH w eight (g) /
of of (%)
sporophore sporophore
paddy paddy
straw ) straw )
H.
ulmarius
10.3 19.0 21.2 37.9 22.3 846.0 130.2 11.2 18.3 21.8 20.7 16.4 338.8 112.9
CO
(OM)2
P. ous
7.6 13.2 16.0 38.4 18.1 694.5 106.8 8.0 13.0 15.3 21.3 15.2 325.9 108.7
(APK1)
P.
platypus 5.6 8.1 10.6 49.8 17.8 886.5 136.4 6.3 9.2 11.6 26.2 14.2 371.5 123.8
(Pp)
P .florida
9.8 16.6 18.4 36.4 19.8 686.6 105.6 10.6 16.3 18.6 19.9 15.6 311.2 103.7
(Pf)
CD
0.48 0.85 1.11 2.48 1.18 168.72 0.56 0.88 1.04 1.34 0.93 0.56 12.98
(p=0.05)
*Mean of five replications, each with six boxes

NSble.H – No of sporophore harvested
55
(iv) Cultivation of oyster mushrooms in M. oleifera wood log
Wood logs moringa measuring 250x105mm, 300x80mm, 350x70mm length

with four types of spawning methods viz.,vertical slits, vertical holes, horizontal slits
and horizontal holes were used for the cultivation of P. platypus. The results showed
that, wood logs measuring 300 mm and 80 mm dia. with vertical slits were found to
be superior, which recorded an average bioefficiency of 123.9 per cent and yield of
433.8g per log. This was followed by wood log measuring 300 mm and 80 mm dia.
with vertical holes which recorded of 111.7 per cent bioefficiency (390.9g yield/ log)
Comparatively, horizontal holes method of spawning found to give less yield. Vertical
slits and spawning were recorded higher yields when compared to any other
method(Table.9., Plate.11.).
Table.9. Standardization of P. platypus cultivation in Moringa oleifera wood

logs
Spawning filling method

Log size (mm)
/ weight (g)
Vertical slits Vertical holes Horizontal slits Horizontal holes
Yield BE Yield BE Yield Yield BE

Length Dia. Wt. NSH AWOS NSH AWOS NSH AWOS BE (%) NSH AWOS
(g) (%) (g) (%) (g) (g) (%)
250 105 450 31.2 14.8 462.3 102.7 31.1 14.6 453.5 100.8 29.8 15.8 471.3 104.7 26.3 15.6 410.6 91.3
300 80 350 27.8 15.6 433.8 123.9 25.5 15.3 390.9 111.7 25.7 14.6 376.6 107.6 25.8 13.8 356.4 101.8
350 70 400 26.9 15.2 408.9 102.2 28.9 14.0 405.2 101.3 26.5 15.2 402.8 100.7 26.3 14.8 388.6 97.1
CD (p=0.05) 1.79 NS 27.17 - 1.78 NS 26.04 - 1.70 0.94 26.13 - NS 0.92 24.07 -
*Mean of five replications, each with six wood logs
NSH - Number of sporophore harvested
AWOS- Average weight of sporophore
56
II. Sequential mushroom cropping system with spent mushroom substrate
(SMS) – Outdoor cropping
(i) Intercropping of paddy straw mushroom in maize and sugarcane fields on

the yield of paddy straw mushroom
Field experiments were conducted to evaluate the suitability of paddy straw

mushroom cultivation in the inter row spaces of field crops viz., maize (var. Co MH4,
spacing 60x30 cm) and sugarcane (var.CO 86032) at the farmers field, located at
Thiyagadurugam, (Villupuram Dt), Mathampatti (Coimbatore Dt) and P.N. Palayam
(Erode Dt). For cultivation of paddy straw mushroom (Volvariella volvacea) different
substrates viz., paddy straw, oyster mushroom SMS (paddy straw), banana plant
residues and oyster mushroom SMS (banana residue) were used.
Intercropping of paddy straw mushroom in maize field revealed that, all the
substrates took 7-8 days to complete spawn run and 8-10 days for the appearance
of mustard like pinhead. Intercropping of paddy straw mushroom cultivated using
paddy straw substrate showed the highest yield of 4.34kg/bed in maize field at
Thiyagadurugam, (Villupuram dist) followed by oyster mushroom SMS (paddy straw)
which recorded an average yield of 4.21kg /bed.
Intercropping of paddy straw mushroom in sugarcane field was carried out with
the mushroom beds prepared using paddy straw, oyster mushroom SMS (paddy straw),
banana plant residues and oyster mushroom SMS (banana residue).V. volvacea
cultivated in paddy straw substrate in sugarcane field at Thiyagadurugam, (Villupuram
dist), produced the maximum yield of 3.82 kg/bed (Table.10.).
Table.10.Testing the performance of oyster mushroom SMS (paddy straw) for

sequential cropping of V. volvacea in a maize based outdoor
cropping system
Paddy straw Oyster m ushroom (paddy straw ) SMS
Yield (g)
Yield (g)
No. of per
No. of eggs per
DFSR DFPF DFFF eggs 25 kg of BE (%) DFSR DFPF DFFF BE (%)
Location harvested 25 kg of
harvested paddy
SMS
straw
Mathampatti
(Coimbatore 6.8 7.8 11.6 140.3 3143.1 12.5 7.4 8.6 12.2 138.4 2948.2 11.8
Dt.)
T.N
.Palayam 6.2 7.5 11.2 187.8 4094.0 16.3 6.8 8.2 11.8 172.2 3892.4 15.6
(Erode Dt.)
57
Thiyaga-
-durugam
5.1 6.8 10.4 184.0 4342.5 17.3 6.3 7.8 11.3 174.2 4214.8 16.8
(Villupuram
Dt.)
CD (p=0.05) NS NS NS 10.73 242.97 NS NS NS 9.72 232.35

*Mean of five replications, each with six beds
Table.11. Testing the performance of oyster mushroom SMS (paddy straw) for
sequential cropping of V. volvacea in sugarcane based outdoor
cropping system
Paddy straw Oyster m ushroom (paddy straw ) SMS
Yield (g)
Yield (g)
Location No .of per No. of BE
BE per
DFSR DFPF DFFF eggs 25 kg of DFSR DFPF DFFF eggs
(%) 25 kg of
harvested paddy harvested ( %)
SMS
straw
Mathampatti
7.1 10.4 12.6 131.1 2673.4 10.7 8.1 11.2 13.3 127.2 2569.2 10.3
(Coimbatore Dt.)
T.N Palayam
6.6 9.7 11.8 171.2 3732.9 14.9 7.4 9.8 12.4 164.9 3512.4 14.05
(Erode Dt.)
Thiyagadurugam
5.8 9.0 11.2 168.4 3823.5 15.3 6.6 8.7 11.9 160.3 3702.8 14.8
(Villupuram Dt.)
CD (p=0.05) NS NS NS 215.23 NS NS NS 205.82
Effect of available nutrients in soil as influenced by Spent mushroom

susbtrate
The level of soil available nutrients due to the integration of paddy straw
mushroom cultivation in maize and sugarcane fields were recorded periodically from
before laying out the bed, 25 days after bed inclusion and 50 days after integration of
mushroom crop. The results showed that, intercropping of paddy straw mushroom in
maize field with oyster mushroom SMS (paddy straw) recorded the highest level of
soil available nutrients viz., N (308.0 kg / ha) and P 2O5 (19.1 kg / ha) in Mathampatti,
Coimbatore district followed by T.N. Palayam Erode district, which accountedto
58
about 301.0 kg N/ha and 19.0 kg P 2O5 /ha in maize field (50 days after bed
inclusion). In sugarcane field, the oyster mushroom SMS (paddy straw) recorded soil
available nutrients viz., N (306.1 kg / ha) and P 2O5 (19.1 kg / ha) (50 days after bed
preparation). Irrespective of the crops and locations, the least soil available nutrients
were recorded with banana pseudostem SMS inclusion for growing paddy straw
mushroom as inter crop. However, compared to control an improved soil nutrient
status was observed in this treatment also. There was no significant result achieved
in soil available potassium in all the treatments(Table.11-13).
Table.12. Soil available nutrients (kg/ha) as influenced by SMS in maize field
Mathampatti T.N Palayam Thiyagadurugam

Time of testing (Coimbatore Dt.) (Erode Dt.) (Villupuram Dt.)
N P2O5 K2O N P2O5 K2O N P2O5 K2O
Initial 232.4 15.9 212.1 234.2 15.9 212.5 231.5 15.8 213.5
Paddy straw
25d after bed preparation 280.3 18.1 259.1 274.1 17.8 259.4 264.0 17.0 247.3
50 d after bed preparation 301.2 18.6 270.4 296.4 18.4 269.3 285.0 18.2 254.1
Paddy straw SMS
Banana plant residue
Banana residue SMS
Table.13. Soil available nutrients (kg/ha) as influenced by SMS in sugarcane

field
Mathampatti T.N Palayam Thiyagadurugam

Time of testing (Coimbatore Dt.) (Erode Dt.) (Villupuram Dt.)
N P2O5 K2O N P2O5 K2O N P2O5 K2O
Initial 233.2 15.8 213.5 233.1 15.7 212.0 234.1 15.9 211.5
Paddy straw
Paddy straw SMS
59
Banana plant residue
Banana residue SMS
Plate.9. PP bottle container for Pleurotus cultivation
P. pulmonarius P. eous P. florida
P. platypus
H. ulmarius
Plate.9a.Testing the performance of polypropylene containers for cultivation of

oyster mushrooms
Hypsizygus
y yg ulmarius Co2
60
Pleurotus florida PF
Plate.10. Laminated cartonbox used for Pleurotus cultivation
36x18x12cm 18x12x8cm
Plate.11. Log cultivation of P.platypus on M.oleifera stumps
Vertical slits Vertical holes
Horizontal slits Horizontal holes
61
Cultivation of medicinal mushroom Ganoderma lucidum in locally available
substrates under subtropical condition
The medicinal mushroom,Ganoderma lucidum was cultivated in different
substrates under sub tropical conditions at TNAU Coimbatore Centre. For this
purpose different substrates viz., mixed saw dust, coconut wood log saw dust,
coconut leaf stalks/petiole, coconut coir waste, arecanut wood log saw dust were
mixed with 20% wheat bran, moistened at 65% and maintained with packed in PP
bags @ 175 g and incubated in darkness at room temperature (27-28°C) until
complete spawn run. After complete spawn run bags were observed for primordial
initiation and then kept in cropping rooms at 80% relative humidity and temperature
of 28°C for pinheads to appear.Pinheads later grew longer with yellow stipe bearing
white colour at tip. The bags were then kept at rooms with temperature of 25°C with
relative humidity of 60-65% for thickening of cap margins. After harvesting of the
mushrooms bags were kept in cropping rooms as earlier for inducing second flush.
(Table.14.).
Table.14. Cultivation of medicinal mushroom Ganoderma lucidum in locally

available substrates under subtropical condition
Av. no. Average

Average wt yield Average
of
of yield TCP BE
Substrates DFSR DFPF PE DFFH fruiting
mushrooms /bed (days) (%)
bodies/ / kg subs
(g)
bed (g)/ bag
Mixed saw
58 70.5 81.0 92.25 4.75 13.25 63.75 364.3 122.5 36.4
dust
Coconut
wood log 46.5 54 62.3 70.5 5.75 13.5 77.5 442.9 100.5 44.3
saw dust
Coconut
leaf stalk 52.75 57 67.3 73.5 5.5 13.5 68.75 392.9 103.5 39.3
(chips)
Arecanut
wood log 51.5 59 69.5 77.7 5.5 12.75 69.5 397.1 107.75 39.7
saw dust
Coir pith
80 95 103.5 115.5 3.75 11.75 44.75 255.7 145.5 25.6
waste
CD
4.92 2.59 3.91 7.8 1.13 1.95 17.87 101.5 7.9
(P= 0.05)
SEd 2.26 1.19 1.79 3.7 0.52 0.89 8.20 46.7 3.7
TCP; Total cropping period PE: Pinhead expansion DFSR: days for spawn
run DFPF: days for first harvest, DFFH; Days for first harvest
62
Among the substrates, coconut wood log saw dust, arecanut wood log saw dust,
coconut leaf stalk (chopped) and mixed sawdust were on par with each other and
had bioefficiency of 44.3%, 39.7%, 39.3% and 36.4% respectively. Among the
substrates coconut saw dust and chopped coconut leaf stalk and arecanut wood log
saw dust recorded early spawn run, pinhead emergence and fruiting when compared
to other substrates. The pinhead expansion was early coconut saw dust and leaf
stalk chips and arecanut sawdust which ranged from 5 to 7 days. The total cropping
period ranged from 110.5 to 107.75 days.
Cultivation of Ganoderma lucidum in saw dust substrate
II Year
i. Organization of SAP- Mandatory skill oriented Research / Entrepreneurship

training programme on fungal biotechnology targeting young scientists,
research scholars, student entrepreneurs and youth (One each targeting
researchers and entrepreneurs).
List of student and guides (2018-2019)
S. Name of College Categ Marks/ Theme areas Guides

No. the student ory GPA
Under Graduate
1. Aparna.J. PSG College of BC 90.4% Biomolecules Dr. A.S.
Arts & Science Krishnamoorthy
Coimbatore Registrar
2. Penisha PSG College of BC 85.0% Value Addition Dr.K. Prabakar
Nirjo. N.M Arts & Science Director(CPPS)
Coimbatore
POST Graduate
3. Aiyha Bharathiar OBC 8.6 Animal feed Dr. M. Muthamilan
Thanseem University Professor and
Head (Pl.Path.)
4. Indhumathi. Bharathiar OBC 8.5 Biomolecules Dr. S. Nakkeeran
R. University Professor
(Pl.Path.)
63
5. Shankaram Annamalai SC 8.29 Biodegradation Dr. K. Angapan
mal.C. University, Professor
Parangipettai (Pl.Path.)
6. Om Rathinavel BC 6.7 Biodiversity Dr. G.
Prakash.M Subramanian and Taxonomy Thiribhuvanamala
College of Arts Assoc. Prof.
& Science (Pl.Path.)
7. Asan Ali. Sadakathulla OBC 7.54 Biodegradation Dr. M. Karthikeyan
M.K Appa College, Asst. Professor
Tirunelvelli (Pl.Path.)
8. Petchiamma Sadakathulla SC 6.9 Value Addition Dr. P. Latha
l.P Appa College, Asst. Professor
Tirunelvelli (Pl.Path.)
List of student and guides (2019-2020)
S. Name of the College Category Marks/ Theme areas Guides

No. student GPA
Under Graduate
1. Kavishree.P Karpagam OBC 86.4% Biomolecules Dr. A.S.
Academy of Krishnamoorthy
Higher Registrar
Education
2. Radhika.J Karpagam OBC 85.2% Value Addition Dr.K. Prabakar
Academy of Director(CPPS)
Higher
Education
POST Graduate
3. Jennifer PSG OC 8.6 Biomolecules Dr. A.S.
valentine College of Krishnamoorthy
Arts and Registrar
Science
4. Kiruthiga.R Bharathiar OBC 8.5 Biomolecules Dr. G. Karthikeyan
University Professor
(Pl.Path.)
5. Indumathi.R Bharathiar OBC 8.29 Biomolecules Dr. K. Angapan
University Professor
(Pl.Path.)
6. Venkat Surya Bharathiar SC 6.7 Spent Dr. G.
Univerity Mushrooom Thiribhuvanamala
Assoc. Prof.
(Pl.Path.)
7. Geethanjali.K Kumaraguru SC 7.54 Biodegradation Dr. M. Karthikeyan
College of Asst. Professor
Technology (Pl.Path.)
8. Sujithra.V Bharathiar SC 6.9 Biodegradation Dr. P. Latha
University Asst. Professor
(Pl.Path.)
64
Students working in UGC-SAP Scheme
65
ii. Characterization of bioactive molecules of mushroom fungi and testing their
pharmaceutical, nutraceutical and biopesticidial values
Mushroom fungi possess bioactive compounds with antibacterial, antifungal,

antioxidant, antiviral antinemic, anti-tumor, immunosuppressive, antiallergic, anti-
inflammatory activities, hypolipidemic, and hepatoprotective activity .Owing to the
current emphasis on the ecofriendly approaches for plant disease management,
mushroom fungi can serve as promising source of antimicrobials against plant
pathogens as evidenced by the antimicrobial activity of the culture filtrates of
Ophiocordeyceps sinensis against soil borne pathogens of Fusarium oxysporum f.
sp. lycopersici and F. oxysporum f. sp. cubense (Sangeetha and Krishnamooorthy,
2015), Coprinus comatus against F. oxysporum f. sp. brachygibbosum, Fusarium
oxysporum f. sp. lycopersici and F. oxysporum f. sp. cubense (Jeeva and
Krishnamoorhy, 2018),
i) Antimicrobial activity of Lentinus edodes and Ganoderma lucidum

A study was conducted to test the anitimicrobial activity of Lentinus edodes
against cucumber wilt pathogens. The ethyl acetate fraction from fruiting body of
Lentinus edodesat 2000 ppm reduced mycelial growth of F. oxysporum and
F. solani (cucumber wilt athogens) upto 61 and 58 per cent over control. The
characterization of secondary metabolites of L. edodes revealed the presence of 4H-
Pyran-4-one, 5-hydroxy-2-(hydroxymethyl) possessing antimicrobial activity. The
Fourier Transform Infrared Transcopy (FTIR) analysis of ethyl acetate fraction of dry
fruiting body of L. edodes expressed the presence of alkyl halide, alcohols, aliphatic
polyenes, amides, phosphorus, silicon compounds, halogens, alkanes, triazenes and
azo compounds. The culture filtrate condensate of Ganoderma lucidum (1000 ppm)
had antinemic activity against Meloidogyne incognita with 92.3 per cent mortality at
72 h after inoculation.(Plate.13.).
66
Plate.13. Antimicrobial activity of Lentinus edodes and Ganoderma lucidum
LC analysis of antimicrobial Inhibition of mycelial growth of
compounds from Lentinus edodes Fusarium sp.
2H-Pyran-2-one,
2H Pyran 2 one 6-pentyl-antifungal,
6 pentyl antifungal
(ii). Antimicrobial metabolites of Ganoderma lucidum

The ethyl acetate fraction of secondary metabolite from crude culture filtrate of
Ganoderma lucidum showed highest mycelial inhibition percentage of 54 per cent at 2000
ppm. The cap portion of G. lucidum fruiting body exhibited maximum antimicrobial activity
of 70 per cent inhibition of mycelial growth of C. gloeosporioides. Characterisation of the
ethyl acetate fractions of cap of G.lucidum fruiting body through GC - MS revealed
the presence oftwo antimicrobial compounds viz., papaverine (mycelium extracts)
and benzothiazole (cap of G. lucidum). Benzathiazole at 50ppm recorded per cent
inhibition of mycelial growth (paper disc assay) and also cent per cent inhibition of
spore germination of C. gloeosporioides. SEM analysis of antimicrobial effect of G.
lucidum against spores of C. capsici reveals that distortion of mycelium, conidial
malformation, disintegration of cell wall of conidia and inhibits conidial germination.
67
Plate.14. Antimicrobial metabolites of Ganoderma lucidum
Ethyl acetate fractions of G.lucidum cap portion Identification of
showing 70% mycelial inhibition of Benzathiazole compound
C.gloeosporioides through GC- MS from cap
portion of G.lucidum
extracts
Distorted mycelium of Ungerminated conidia Normal mycelium of

C.gloeosporioides
g p of C.gloeosporioides C.gloeosporioides
g p
(iii) Antifungal potential of bioactive molecules of Ophiocordyceps sinensis

(Berk.) against seedling blight, root rot and wilt pathogens
Ophiocordyceps sinensis and O. neovolkiana have possessed fungicidal,

nematicidal and insecticidal activitydue to the production of proteases, chitinases
and lipases. Methanolic fraction of the cell free culture (CFC) filtrate condensate
showed the maximum inhibition against F. o. f. sp. cubense (55.28) and F. o. f. sp.
lycopersici (48.23) at 200 μL of 2000ppm concentration (Plate 1 ). Besides, the
headspace volatiles produced by O.sinensis exhibited 52% and 48% inhibition of
mycelial growth of F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. cubense,
respectively. In addition the compound of cordycepin (3’-deoxyadenosine) was also
identified in O. sinensis and O. neovolkiana which was effective against Fusarium
spp, root knot nematode and coconut root grub. The metabolites of O.sinensis and
O.neovolkiana tested at 1000ppm, had inhibited 92 per cent egg hatching ability and
exhibited 94 per cent mortality of juvenile (J 2) of root-knot nematode (Plate14.).
68
Plate. 15. Methanolic fractions of CFC filtrate of O. sinensis against
Fusarium spp
Plate. 16. Effect of cell free culture filtrate of O. sinensis onM. incognita
(iv.) Harnessing the antimicrobial and nematicidal properties of Coprinus spp
Twenty-day old cell free culture filtrate (CFC) of C. cinerea exhibited

maximum growth inhibition of F. brachygibbosum,F. o. f. sp. cubense andF. o. f. sp.
lycopersici, when tested at a concentration of 2000 ppm. Ethylacetate fractions
ofCFC filtrate (20 d old) condensate, mycelial mat (20 d old) extract and methanolic
fractions of fruiting bodies (at post capping stage) of C. cinereainhibited the growth of
F. brachygibbosum, F. o. f. sp. cubense and F. o. f. sp. lycopersiciat a 2000 ppm.
The bioactive molecules of CFC filtrate condensate, mycelail mat and fruiting bodies
(at post capping stage) of C. cinerea at 2500 ppm exhibited the maximum inhibition
of egg hatching (65.02 per cent) and juvenile (J2) mortality (67.0 per cent) of root
knot nematode, Meloidogyne incognita over a period of 72 h of incubation. The
69
methanolic fraction of fruiting bodies at post capping stage of C. cinerea had antiviral
properties against GBNV (Groundnut bud necrosis virus). Simultaneous and pre
inoculation of metabolites of C. cinerea was effective in inhibiting GBNV at 3000
ppm. GC-MS analysis revealed the presence of cubenol, n-Hexadecanoic acid and
alfa copaene.(Plate.15,16.).
(v) Antiviral compound squalene from G. lucidum against Ground nut Bud
Necrosis Virus (GBNV) on tomato
Spraying the solvent extracted from mycelial culture filtrate of G. lucidum
reduced the number of lesions per square cm on local lesion host, cowpea and did
not produce symptoms even after 12 days of inoculation of GBNV in tomato plants
The q PCR analysis of tomato plants treated with culture filtrates of G.
lucidumupregulatedthe defense gene PR 1 (pathogenesis related gene), PAL gene
(Phenyl alanine ammonia lyase) and LOX (Lipoxygenase). Further, characterization
of G. lucidum metabolites revealed the presence of antimicrobial compound
squalene. The compound squalene at 500 ppm significantly reduced the number of
lesions per sq.cm (1.67 lesions/cm2) on local lesion host, cowpea. (Plate.17.).
Plate.17. Secondary metabolites of Ganoderma lucidum against GBNV

in tomato cv. PKM1
a. GBNV control b. untreated c. Treated
70
Identification of antiviral compound
squalene from G.lucidum
Volatailomes mediated antimicrobial activity of mushroom fungi against

damping off and wilt pathogens of tomato
The volatiles produced by mycelia of Coprinus sp. significantly inhibited 70%

mycelial growth of Fusarium oxysporum f.sp. lycopercisi, followed by
G. lucidum (60%) and L. edodes (35%). The GC-MS characterization revealed the
presence of antimicrobial compounds, undecanone from Coprinus cinereusand
limonene, from G. lucidum.
Volatiles produced by mycelia of Coprinus

cinereus inhibited mycelial growth (70% ) of
Fusarium oxysporum f.sp. lycopercisi
71
III Year
x Organization of II SAP-Mandatory skill oriented Research/

Entrepreneurship training programme (One each targeting researchers
and entrepreneurs).
2018-19 activities
TRAINING PROGRAMME
TRAINING PROGRAMME
One day Five days Spawn

Month
Participants Participants
(Nos.)
( No.) ( No.)
April 2018 42 - 168
May 74 10 280
June 62 6 130
July 64 - 575
August 70 5 600
September 83 - 389
October 59 4 545
November 11 - 1164
December 39 4 443
January2019 67 - 283
February 41 - 254
March 41 - 183
Total 652 29 5,014
Gender wise participation in mushroom training programmes

Training programme & date Male Female Total
One day training
05.04.2018 32 10 42
07.05.2018 50 24 74
05.06.2018 50 12 62
05.07.2018 45 19 64
06.08.2018 29 41 70
72
20.08.2018 30 53 83
05.09.2018 49 10 59
05.10.2018 9 2 11
25.10.2018 34 5 39
07.11.2018 35 32 67
05.12.2018 27 14 41
07.01.2019 22 19 41
Total 412 241 653
Five days training programme
28.05.18-01.06.18 8 2 10
25.06.18-29.06.18 5 1 6
23.07.18-27.07.18 3 2 5
27.08.18-31.08.18 4 - 4
17.12.18 -21.12.18 4 - 4
Total 24 5 29
C.Short demo/ training class to school /college students
S.
Date Name of the school/ college No. of participants
No.
Staff Students Total
1 17.4.2018 Suguna International School, 2 80 82

Gandhipuram
2 18.4.2018 Karl Kubel Institute for Development 2 16 18

Education, German
3 12.7.2018 Vanavarayar Institute of 5 139 144

Technology, Pollachi
4 23.7.2018 St. Philomena’s College ,Mysore 5 43 48
5 24.7.2018 College of Horticulture, Bagalkot 1 14 15
6 16.8.18 G.R. Damodaran Matric Hr. Sec. 1 20 21

School,Peelamedu
7 6.9.2018 Government Boys Hr. Sec. School, 4 56 60
73
Thondamuthur
8 25.9.2018 Pasumpon Muthuramalinga Thevar 4 66 70

College, Usilampatti
Karl Kubel Institute for Development 19

Education, German
10 3.10.2018
11 15.11.2018 S.F.V. Government Hr. Sec. School, 2 50 52

Seeranaickenpalayam
12 28.11.2018 National Model Matric Hr. Sec. 3 75 78

School, Peelamedu

School, Peelamedu

School, Peelamedu
15 7.12.2018 PGP College of Agricultural 2 53 55

Sciences, Namakkal
16 4.1.2019 Department of Agricultural 1 45 46

Microbiology, TNAU
17 11.1.2019 College of Horticulture and Forestry 2 43 45
18 13.2.2019 St. Francis Xavier Hr. Sec. School, 3 83 86

Erode
19 14.2.2019 Govt. College, Tripunithura 2 19 21
20 1.3.2019 Got. Arts and Science College, 2 22 24

Coimbatore

Sciences, Namakkal
22 21.3.2019 SSA Education Dept, Perur block 5 20 25
74
D.Special training programmes to Self help groups/ farmers
S. Date Name of Self help groups/ No. of participants

farmers
No. Staff Beneficiaries Total
1 18.4.2018 Manivel, IWMP Society- 1 46 47

Dharapuram
2 28.6.2018 AHO, Tumkur 34 34
3 30.8.2018 Block Technology Manager, 5 30 35

Thirivarur
3 23.10.2018 Agricultural extension Centre, 2 35 37

Theni
4 8.1.2019 AHO, Ooty 3 100 103
5 25.1.2019 Tribal welfare, Salem 12 12
6 29.1.2019 ATMA, Nedumkandam 3 13 16
7 30.1.2019 ATMA, Thiruvallur 1 54 55
Teaching and Outreach Programmes
One day training on mushroom cultivation
Hands on Five days training and demonstration
75
Demonstrations to school and college children
UG Students - Experiential learning course on Mushroom day

Commercial Mushroom Production
Mushroom scientists from other centres visit at TNAU,

Coimbatore 29-30th Oct.2018
Visit by Dr.V.P. Sharma, Director, DMR,Solan at TNAU, Coimbatore on

23.04.2019
76
Dr.V.P Sharma Director (DMR), At Mushroom Research & Training
Director (CPPS) , Head of the Dept. of Centre, TNAU, Coimbatore
Plant Pathology) meeting with
Vice – Chancellor (TNAU)
Director observing the LE strains

Director observing the casing soil in
spawned beds
milky mushroom beds
Director- Intercation with students Dr.A.S. Krishnamoorthy ( Registrar)

research activities briefing the developmental activities
Mushroom team members with Director
77
Visit by Deputy Collector, Erode dt. to Dr.Venkatesh Balan, University of
TNAU, Coimbatore Houston, USA
2019-20 activities
Demonstrations 1. Vellamadai village, Coimbatore through NSS students on

organised 23.05.2019
2. ATMA project , Gujarat on 10.09.2019
3. Farmers from Magudanchavadi, salem -4.12.2019
4. Kerala club, coimbatore-23.09.2019
5. Farmeers Association, cuddalore.2.12.2019
6. Central Prison- 14.10.2019
7. Central prison :23.10.2019
Radio 1. Mushroom cultivation: Prospects, Problems and solutions -
Programme 13.06.2019
2.Progarmme coverage and broadcast on Training on
Mushroom cultivation to SC farmers on 30.10.2019
3.One day training programme - Interaction with farmers
broadcasted - 5.12.2019
4. Mushroom day interaction with farmers broadcasted -
23.12.2019
News paper 1. Indian Express dt. 02.10.2019 Mushrooms the way to
coverage prosperity
2. Dinamani (Tamil) dt.3.10.2019-Message on Rise of
mushroomsas retreats for humanity
3. Thozhil malar (June,2019 Tamil) Making money from home
through mushroom cultivation
4. HIndu (Tamil) 1.10.2029 on National seminar on mushrooms
published.
5.Dinamalar (Tamil) dt.4.10.2019 on establishment of
mushroom park at TNAU, Coimbatore
78
Sr. Seminar/symposia/workshop/kisan
Date Venue
No. mela, etc
1 National seminar on Rise of Mushrooms : 30.09.2019 TNAU,

Retreads to Humanity Coimbatore
2 National workshop: Mushroom Series I 1.10.2019 TNAU,

Coimbatore
Number of visitors in
Sr. group
Date Visitors
No.
Male Female Total
Imayam Institute of Agrl&&

1 28.06.2019 30 47 77
Techn.Thuraiyur
Vidhya vikasini Hr.Sec.school,

2 11.07.2019 55 65 120
Tirupur
Vanavarayar Instutute of
3 16.07.2019 55 54 109
Agriculture, Pollachi
Namazhvar college of Agrl.&

4 22.07.2019 20 24 44
Technology
5 14.08.2019 Karur velalar college, Karur 30 22 52
Blue bird Mat. Hr.Sec.school,

6 26.08.2019 40 66 106
Pallladam
7 27.08.2019 NGP School, Kalapatti 50 52 102
Bharathi Mat.Hr.Sec.School,
8 05.09.2019 - 8 8
Coimbatore
Quest International school,

9 09.09.2019 20 55 75
Avinashi
Corp. Higher Sec. school,

10 26.09.2019 - 29 29
Coimbatore
VLB Janakiammal college,

11 14.10.2019 40 63 103
Coimbatore
Vivegam Mat.Hr.sec. school,

12 08.11.2019 53 - 53
Dharapurum
Vivegam Mat.Hr.sec. school,

13 13.11.2019 49 - 49
Dharapurum
14 15.11.2019 AVP Trust Hr.Sec.School, Tirupur 36 60 96
79
GKD Mat.Hr.Sec.school,
15 22.11.2019 100 79 179
Coimbatore
SIARD, Thakkalam,Arakonam,
16 25.11.2019 40 10 50
Vellore
17 03.12.2019 Farmers Association, Cuddalore 19 - 19
Maharishi International school,

18 12.12.2019 20 44 64
Coimbatore
Telanagana Agricultural university,

19 12.12.2019 10 7 17
Telangana
National model Mat.

20 18.12.2019 45 58 103
Hr.Sec.school, Coimbatore
National model Mat.

21 19.12.2019 45 52 97
Hr.Sec.school, Coimbatore
22 20.12.2019 Chinmaya vidyalaya school,Cbe 30 49 79
23 24.01.2020 CCMA Govt. Hr.Sec.School, Cbe 30 42 72
24 30.01.2020 Govt. Boys Hr.Sec.school, Erode 30 - 30
25 12.02.2020 Alvas college, Karnataka 10 31 41
Viveknanada college of Arts &

26 19.02.2020 20 29 49
Science
Erode Arts & Science college ,

27 06.03.2020 15 46 61
Erode
28 09.03.2020 Loyola college, Manvi, Karnataka 43
Farmers
29 10.09.2019 ATMU Project, Gujarat 24 - 24
30 15.10.2019 Farmers from DWDA, Erode 102 - 102
31 17.11.2019 Farmers from Kurumathur, Kannur 33 - 33
32 21.11.2019 Farmers from Kallaserry, Kerala 52 - 52
Farmers from Magudanchavadi,

33 04.12.2019 42 - 42
salem
Date Venue No. of Male Female Remarks

Participants if any
FC&RI,
31.10.2019 Mettupalayam, SC
40 36 4
Karamadai block, Farmers
Coimbatore dt
80
21.12.2019 Dept. of Plant
Pathology, TNAU, 60 45 15 SC Youth
Coimbatore
20.02.2020 Dept. of Plant SC self

Pathology, TNAU, 65 - 65
help group
Coimbatore
One day training
No. of
Date Venue Male Female Remarks if any
Participants
Dept. of Plant
Pathology, Farmers/
5.042019 27 18 9
TNAU, entrepreneurs
Coimbatore
Dept. of Plant
Pathology, Farmers/
0.5.05.2019 31 19 12
TNAU, entrepreneurs
Coimbatore
Vellmadai
28.05.2019 village- NSS
47 16 31 Farmers
students and
farmers
Dept. of Plant
Pathology, Farmers/
05.06.2019 23 17 6
TNAU, entrepreneurs
Coimbatore
Dept. of Plant
Pathology, Farmers/entrepren
05.07 2019 47 37 10
TNAU, eurs
Coimbatore
Dept. of Plant
Pathology, Farmers/
06.08 2019 47 37 10
TNAU, entrepreneurs
Coimbatore
Dept. of Plant
Pathology, Farmers/
05.09 2019 44 36 8
TNAU, entrepreneurs
Coimbatore
Kerala club,
23.09.2019 32 - 32 Woman group
Coimbatore
Dept. of Plant
30.9.2019 Pathology, Agrl.officers,
7 7 -
TNAU, Maldives
Coimbatore
Dept. of Plant
Pathology , Farmers/
05.102019 30 19 11
TNAU, entrepreneurs
Coimbatore
81
Central
prison,
14.10.2019 Coimbatore 50 50 - Prisoners
(Oyster
mushroom)
CAG pride,
22.10.2019 60 45 15 NABARD officals
Coimbatore
Central
prison,
23.10.2019
Coimbatore 3 3 - Prisoners
(Milky
mushroom)
Dept. of Plant
Pathology , Farmers/
05.11 2019 70 49 21
TNAU, entrepreneurs
Coimbatore
Dept. of Plant
Pathology, , Farmers/
05.12 2019 49 39 10
TNAU, entrepreneurs
Coimbatore
Dept. of Plant
Pathology, Farmers/
06.01 2020 105 37 68
,TNAU, entrepreneurs
Coimbatore
Dept. of Plant
Pathology, Farmers/
05.02 2020 78 55 23
TNAU, entrepreneurs
Coimbatore
Dept. of Plant
Pathology, Farmers/
05.03 2020 87 63 24
TNAU, entrepreneurs
Coimbatore
Three day training
No. of Remarks
Date Venue Male Female
Participants if any
Five day training
No. of
Date Venue Male Female Remarks if any
Participants
to Pathology, Farmers/
9 9 -
28.06.2019 TNAU, entrepreneurs
Coimbatore
82
to Pathology, Sultan Qaboos
2 - 2
20.07.2019 TNAU, university, oman
Coimbatore
7 7 -
Coimbatore
10 8 2
Coimbatore
8 6 2
Coimbatore
8 6 2
Coimbatore
ONE DAY TRAINING ON MUSHROOM CULTIVATION
83
ENTREPRENEURSHIP ORIENTED FIVE DAYS TRAINING ON SPAWN AND
MUSHROOM PRODUCTION
Participants preparing media Trainees learning tissue culture
Cerificate distribution by Registrar, Certificate distribution by

TNAU Director (CPPS) ,TNAU
84
TRAINING TO SCHOOL / COLLEGE STUDENTS
TRAINING ON OYSTER MUSHROOM CULTIVATION TO FARMERS
Training to farm women from Training to ATMA farmers fro

Thirupathisaram Salem
Training to farmers from Erode Training to Dept. officials
85
TRAINING TO INTERNATIONAL STUDENTS- SULTAN QUABOOS
UNIVERSITY, OMAN
TRAINING ON MUSHROOM CULTIVATION TO OFFICIALS FROM MALDIVES
86
MUSHROOM DAY AT TNAU, COIMBATORE ON 23.12.2019
Intercation with growers and radio Team of scientists and farmers

recording
TRAINING ON MUSHROOM CULTIVATION SC FARMERS OF KARAMADAI

BLOCK HELD AT FC&RI, METTUPALAYAM ON 31.10.2019
Inuagural function of mushroom training Dignitaries -Registrar, Director

programme- Karamadai block (CPPS), Dean (Forestry) ADA Prof &
Head (Pl.Path.) and (Horticulture)
Farmers learning oyster mushroom bed Farmers viewing exhibition on

preparation mushroom
87
TRAINING TO SC YOUTH HELD AT TNAU, COIMBATORE ON 21.12.2019
SC youth learning tissue culture techniques SC youth learning spawn

production techniques
View of exhibition arranged for SC youth SC youth interested in value

added products
TRAINING TO SC WOMEN SELF HELP GROUP AT TNAU, COIMBATORE

20.02.2020
Addressing the SC women self help

group on nutritive values of SC women SHG involved in
mushrooms mushroom bed preparation
88
A view of SHG participants SHG's with their inputs
89
NATIONAL SEMINAR ON ENTERPRISING MUSHROOM
TECHNOLOGY16.03.2017
90
NATIONAL SEMINAR ON ENTERPRISING MUSHROOM
TECHNOLOGY16.03.2017
91
NATIONAL SEMINAR ON RISE OF MUSHROOMS: RETREATS TO HUMANITY
30.09.2019
Coordinator (SAP) presenting

Book release by dignitaries
key notes
A view of participants
Vice-Chancellor, TNAU addressing the
gathering
Speaker from PSG Medical

A view of participants
College, Coimbatore on
pharmacology of mushrooms
92
Mushroom Industrialist from Hyderabad- Medicinal mushroom
sharing experience entrepreuner from Haryana
NATIONAL WORKSHOP ON MUSHROOM SERIES I HELD AT TNAU,

COIMBATORE ON 1.10.2019
93
NEWS PAPER CLIPPINGS
94
VISIT OF IMPORTANT DIGNITARIES
Dr.V.P.Sharma, Director, DMR, Solan Director (DMR) with Vice-Chancellor

(24.04.2019) (TNAU)
Dr.Thamariselvi, Vice-Chancellor, Dr.L.Jalali, Executive Director of

Thiruvallur (University ) 02.12.2020 Research (HAU) (18.12.2019)
DEMONSTRATIONS ON MUSHROOM CULTIVATION
Demonstration to women folk at Kerala Demonstration at Vellmadai

Club, Coimbatore village to young children
95
EXPERIENTIAL LEARNING STUDENTS - MUSHROOM SPAWN PRODUCTION
Spawn preparation Students preparing mushroom

Bed
Team of Experiential learning students
96
Establishment of one model permaculture cropping system with mushroom
fungi as key component to enhance farm income through secondary
agriculture.
Paddy straw mushroom is an edible mushroom of the tropics and subtropics
and is widely cultivated owing to its excellent taste compared to any other
mushroom. One remarkable feature of this mushroom is its fast growth and shorter
cropping period under a temperature of 35-40 C and relative humidity of 80 %.
Hence, a new approach was thought to cultivate this mushroom under maize and
banana cropping systems so as to utlise the available microclimate and utilize the
unspent substrate for conversion in to biomanure.
Paddy straw mushroom (Volvariella volvacea) is an edible mushroom of

tropics and is grown widely for its flavour and taste. One remarkable feature of this
mushroom is its shorter cropping cycle of 10 to 15 days . Though work started long
back in India, commercial cultivation of this mushroom is a great challenge due to its
poor shelf life and low bioefficiency. This mushroom could be cultivated indoors with
a temperature of 35 ᵒC and relative humidity of 85 % which leads to increased
production cost. Hence an alternative method of cultivation of paddy straw
mushroom by utilizing the available microclimate under maize cropping system
where a temperature of 30 to 35ᵒ C with relative humidity is obtained. Maize crop
with a spacing of 60 x30 cm at silky stage was selected for the studies. Paddy straw
bundles were soaked in water added with 2% lime for 12hours and next day the
bundles were used to prepare mushroom beds. Beds were prepared using bundle
method like 4 layers / bed (each layer with 3 feet length accommodating 1kg paddy
straw. In each layer at the periphery of 2% horse gram powder wa sprinkled and
over which paddy straw spawn was placed at equidistance. Later second layer was
prepared and each bed was prepared with 4 layers in the interspaces of maize crop
and left undisturbed for 8 days. The spawn running was completed within 10 days
and small pinheads appeared on 11 th day which put forth egg shaped buttons on
13th day. On an average 20-22 % bioefficiency was obtained within 15 days cropping
cycle with C;B ratio of 1:2.2 After harvest the spent substrate was used for
bioconversion in to manure using TNAU composting technology. The biomanure so
produced was enriched with biocontrol agentsviz.,Trichoderma viride and
Pseudomonas fluorescens and applied to the nursery plants.
97
x Technology transfer and formation of Mushroom Producer Companies in
coordination with the Directorate of Agribusiness Development, TNAU,
Coimbatore.
S. No. Name of the Mushroom Company and Product name
Producer
1. Mr. A.R.Subramanian M/s. Maga Mushrooms
Product: Mushroom spawn and cultivation
2. Mr.M.Ajith Laxman M/s. Sri Ayyappa Vegetable Merchant
3. Mr.S.Rahulray M/s. Vardhani Corporation
4. Mr.M.Nagarajan M/s. Annavarshni Foods LLP
5. Mr.S.K.Senthil Kumar M/s. Healthy life Foods
6. Mr.V.Mayilsamy Mushroom cultivation
7. Mr.N.Arul Jothi Mushroom spawn production
8. Mr.S.Kamal Mushroom spawn production and farming
9. Mr.Vigram Kannan Mushroom production
10. Ms.B.Akila Mushroom cultivation
11. Mr. S. Rajendran Mushroom production
12. Mr. B. Chaithanyan Oyster Mushroom production
13. Mrs. J. Suganthi Oyster and Milky Mushroom production
14. Mr. Senthilkumar Mushroom production
15. Mr. Yuvaraj Deivasigamani M/s. Nilgiri Farm Produce
Button mushroom production
16. Mr. Yasuhide Ito M/s. YATS Corporation Co. Ltd.,
Shiitake mushroom production
17. Mr.A.Gandhi Mushroom production
18. Mr. S.R.Mylsamy Mushroom farming
19. Mr. A. Senthil Kumar Mushroom Production
20. Mr. A. Kandavel Mushroom spawn production
21. Mr. K. Saravana Kumar M/s. Dhana Kesaan Mushroom Farm
22. Mr. Mohammed Kachakkaren M/s. HAAD Enterprises
23. Mr. S. Thilagaraj Mushroom value added products
98
IV Year
x Establishment of Mushroom Knowledge Park and Mushroom museum at

TNAU, Coimbatore to encourage student research and entrepreneurship
programme.
As the days are passing by Indian Agriculture experiences a visible shift

from production oriented approach towards market driven export oriented agro
based industry. Mushroom growing has become one of the best paying agro biotech
industry. Practically, the field of mycotechnology is fascinating and challenging, and
good days are ahead for the synthesis of newer fungal biomolecules having
commercial importance. With the advancements in fungal biotechnology and
analytical chemistry, mushrooms for food alone concept is getting changed.
Bioactive molecules with multifaceted uses obtained from macro basidiomycetes are
commercialized worldwide
A mushroom knowledge park has been planned with a exhibition hall show
casing mushroom various oyster mushroom, milky mushroom and button mushroom
varieties released by the department along with the technologies. A separate training
hall is planned to offer skill oriented training on spawn and mushroom production to
educated youth, farm woman and woman self help groups. The knowledge park also
encompass themes and concepts on medicinal mushrooms viz., Ganoderma
lucidum, Lentinus edodes, Jews' ear mushroom and Cordyceps concepts on
biodegradation and bioremediation with white rot basidiomycetes mushrooms,
conversion of spent substrate in to biomanure and animal feed, nutra and
pharmaceuticals, antimicrobials from mushrooms for pest and disease management
A proposal has been submitted to NABARD for obtaining financial assistance. Also a
project proposal on Mushroom Business Startup Incubator has been submitted to
NADP to promote commercialization of edible and medicinal mushrooms, value
added mushroom products through Innovation, Incubation and graduation; and to
create value chain for such products in order to commercialize and popularize the
edible and medicinal mushroom varieties and strains released from TNAU by
utilizing ligno cellulosic residues.Through this establishment it is expected to enrich
young minds with knowledge on mushroom science apart from providing training to
about 5000 beneficiaries within a year. Steps have been taken to set up a mushroom
museum show casing the biodiversity conservations of the western Ghats. The
facility will intend on ex situ conservation of macrobasidiomycetes of commercial
importance to explore the biopesticidal molecules promote for plant and human
99
health improvement. These services will be made available to entrepreuners,farmers
and other stakeholders to take up biomanure and animal feed production by utilizing
spent waste, post harvest mushroom processing, product development, packaging
and quality testing.
Through this it is expected that there will be larger adoption of TNAU

technologies and mushroom varieties by other R&D Institutes and industries in
India..Further, there is great scope for promotion of entrepreneurship in mushroom
biotechnology business by hand holding with TNAU on a Public Private Partnership
mode on “Win-Win” basis to meet the global competition for mushroom biotech
products apart from creating impact on the livelihood of farmers, farm women, youth
and self help groups.
x Development and testing of small scale automation for organized

spawn production and mushroom cultivation to encourage Small
Mushroom Big Business concept.
100
V Year
Organization of III-SAP Mandatory skill oriented Research / Entrepreneurship

training programme (One each targeting researchers and entrepreneurs).
Strengthening fungal bioprocess technology programmes through self

supporting and PPP mode to sustain the R&D activities.
Documentation of SAP activities for self evaluation targeting further area of

“Centre of Excellence in Tropical Mushroom Biotechnology” at TNAU,
Coimbatore.
-
b. If the objectives set-forth could not be achieved, the specific reasons there
of.
- Nil -
c. Utilization of findings in policy formulation, development and

modification of strategies (for Social Science departments mainly)
101
(ii) HumanResourceTraining:
A Persons trained (Nos.):UG-PG : 100 nos

B Rural/Tribal : 1000/165 nos
C Industrial : 10 nos
D International : 8 nos
E From other agencies : -
1. Infrastructure Developed:
a. Name major Equipments (Rs.3lacs)

S. Date of Name of the instruments Amount (Rs.)
No. purchase
1. 31.03.2017 Thermostat controlled autoclave 299376.00
2. 31.03.2017 Rotary vaccum flask evaporator with 498960.00

accessories
3. 31.03.2017 Refrigerated Centrifuge 470000.00
4. 31.03.2017 Large Capacity Refrigerated shaker 498067.00
5. 27.03.2018 Deep freezer 1810400 YEN
(Rs.11,18,131/-)
b. Centra lSchemes/ facilities for PG, Research and Extension Activities

(Please tick the one applicable to your Department:(i) STEP (ii) IIPC(iii)
USIC /RSIC - Does not arise
(iii) Patent Promotion Cell (v) Guest house with capacity (vi)
Seminar/Conference Room with capacity (vii) Regional
/Mainframe computing facilities- Does not arise
(viii) Central Library with documentation facilities (ix) Continuing Educationn
Centre (x) Women Development Cell.- Does not arise
c. Networking (Please tick the right one): (i) Library (ii) Laboratory (iii)
University Department.
2. Knowledge disseminated to (in the thrust area identified):
(i) Other teaching institution (Name,No.offaculty involved) – Need to be added

from the participants who attended symposium
102
UGC-SAP Seminar Participant List – Other Colleges
Sl. College Name Male Female Total
No
1. Rathinam College of Arts and Science 24 39 63
2. G. R. D College of Science 17 39 56
3. Annamalai University 7 16 23
4. RVS College of Arts and Science 8 8 16
5. Bharathiyar University 5 15 20
6. AJK College of Arts and Science 3 5 8
7. K. S. G College of Arts and Science 19 26 45
8. Dr. NGP College of Arts and Science 7 6 13
9. Hindustan College of Arts and Science 9 7 16
10. PSG College of Arts and Science 3 7 10
11. Rathinavel subramaniam College of Arts and 10 27 37
Science
12. Amritha University 2 - 2
13. Sri Ramakrishna College of Arts and Science 7 6 13
14. Avinashilingham Institute for Home Science & Higher - 1 1
Education for Women
15. Sadakathullah Appa College 1 2 3
16. Government Arts College , Salem 2 - 2
Total 124 204 328
(ii) Industry(Namewithamountreceivedifany) - Nil
(iii) Rural/Tribal/Govt./NGOs(ProvideNo.withamount):
(iv) International (name organisation)

1. Sultan Quaboos University, OMAN – 2 nos
2. Agriculture field Officers – MALDIVES – 6 nos
TRAINING TO INTERNATIONAL STUDENTS- SULTAN QUABOOS

UNIVERSITY, OMAN
1. Ahad Saud Sultan
2. Hanin Abdalaiah Said
103
TRAINING ON MUSHROOM CULTIVATION TO OFFICIALS FROM MALDIVES
S. No. Name of the Trainees (from Maldives)

1. Abdulla Anwar
2. Abdul Jaleel
3. Amir Ali Didi
4. Adam Imsaaz
5. Hussain Mohamed
6. Zakariyya Ibrahim
104
(v) Others:
(vi) Innovation/excellence brought in (Please specify in the identified thrust

areas only) –
¾ Significantly increased yield was obtained with H. ulmarius var. (CO(OM)2
cultivated in polypropylene bottle containers (267.17g per bottle containing
180 g of paddy straw on dry weight basis recording 148.43 per cent
bioefficiency). However, P.platypus (Pp) was also found to give equally better
yield followed by P. pulmonarius. But, P. florida gave comparatively less yield
in polypropylene bottle containers.
¾ Increased yield was obtained with P. platypus (PP), when cultivated in

laminated carton box (36x18x12 cm) container, which accounted to 886.5g
per box containing 650 g of paddy straw on dry weight basis recording 136.4
per cent bioeffficency. However, H.ulmarius was also found to give yield on
par with that of P. platypus in laminated carton box containers. P. eous and
P.florida gave comparatively less yield.
¾ Three different substrates viz., paddy straw, maize cobs and shredded
arecanut leaf sheath have been evaluated for the cultivation of different
species of Pleurotus. Paddy straw was found to be more suitable for the
cultivation of all the species of Pleurotus tested followed by shredded
arecanut leaf sheath. Among the different species, P.platypus recorded the
maximum yield of 319.4, 478.9 and 376.4 g in paddy straw, maize cob and
shredded arecanut leaf sheath substrates, respectively. Rapid weight loss
was noticed with paddy straw, which accounted to 55.7 and 53.7 per cent
when used for the cultivation of H.ulmarius and P.platypus,respectively. The
rate of weight loss had positive correlation with yield, which was significantly
higher during the I and II flush.
¾ Different sizes of moringa wood logs were used for the cultivation of P .
platypus. Among them, stumps measuring 300 mm x 80 mm size, with 4
vertical slits for spawning were found to be superior, which recorded an
average bioefficiency of 123.9 per cent and yield of 433.8g per log.
¾ Eight different pretreatment methods of moringa wood logs were tested

for the cultivation of P.platypus. Among the pretreatment methods, steaming
(at 1.42 kg cm-2 for 1h) with surface sterilization (hydrogen peroxide 2%) was
105
found to be preferable, which recorded the highest yield of 357.33g and
accounted to 104.7 per cent bioefficiency.
¾ Different sizes of (15, 25, 30, 45 cm length and 8 cm diameter) banana

pseudostem were tested for the cultivation of Pleurotus species. Banana
pseudostem measuring 25 cm length and 8 cm diameter was found to be
suitable for the cultivation of all the Pleurotus spp tested, which recorded
higher yield compared to all other sizes.
¾ Four different pretreatment methods viz., fresh pseudostem with 80 per cent
moisture, sun drying for 48 h, hot air oven processed (80 o C) for 5h and
steaming at 1.42 kg cm-2 (30 min) were tested. Among the methods, sun
drying of pseudostem for 48 h (moisture content 70 per cent) recorded the
maximum yield of 215.6, 200.7 and 202.4 g with an average bioefiiciency of
105.2, 100.3 and 100.8 per cent in case of P.platypus, P. eous and P. florida,
respectively.
¾ After the completion of cropping cycle, the oyster mushroom SMS was used
for the sequential cultivation of paddy straw mushroom. Significantly
increased yield of paddy straw mushroom (V. volvacea) was obtained in H.
ulmarius SMS (554.4g per bed with 11.1 per cent bioefficiency).
¾ In an intercropping system in maize field, paddy straw mushroom cultivated

using paddy straw substrate showed the highest yield of 4.34kg/ bed followed
by the usage of oyster mushroom SMS (paddy straw), which also recorded an
average yield of 4.21kg / bed. However, both the results were found to be on
par.
¾ Intercropping of paddy straw mushroom in maize field with oyster mushroom

SMS (paddy straw) also recorded the highest level of soil available nutrients
viz., N (308.0 kg / ha) and P 2O5 (19.1 kg/ ha) in Mathampatti village,
Coimbatore district.
3. Break through (already recognized)
x The details of Oyster mushroom, milky mushroom and button mushroom

varieties released as breakthrough events.
106
Mushroom varieties / strains released from TNAU
Variety /
Scientific Name Year of Release Station
Strain
Oyster mushroom
P. florida Pf 1983 Dept. of Pathology,
TNAU, Coimbatore
P. citrinopileatus Co1 1986 Dept. of Plant Pathology,
TNAU, Coimbatore
P. platypus Pp 1990 Dept. of Plant
Pathology,TNAU,
Coimbatore
P. eous APK1 1995 Regional Research
Station, Aruppukottai
P. djamor MDU 1 1996 Dept. of Plant Pathology,
AC&RI Madurai
P. flabellatus MDU2 2000 Dept. of Plant Pathology,
AC&RI Madurai
P. ostreatus Ooty 1 2000 Horticultural Research
Station, Ooty
Hypsizygus ulmarius Co (OM)2 2004 Dept. of Plant
Pathology,TNAU,
Coimbatore
MIlky mushroom
Calocybe indica APK2 2000 Dept. of Plant Pathology,
TNAU, Coimbatore
Tricholoma Co (TG) 3 2012 Dept. of Plant Pathology,
giganteum TNAU, Coimbatore
Button mushroom
Agaricus bisporus AB1(Ooty1) 2000 Horticultural Research
Station, Ooty
Agaricus bisporus AB 1 (Ooty 2) 2005 Horticultural Research
Station, Ooty
107
COMMERCIALLY CULTIVATED MUSHROOM VARIETIES
1.OYSTER MUSHROOM
Pleurotus florida var.PF

x Released for commercial cultivation during 1983
from Dept. of Plant Pathology,TNAU,Coimbatore.
x Sporophores white to light creamy white with good
taste.
x Highly suitable for commercial cultivation under
subtropical, conditions
x Preferred by growers due to uniform yield throughout
the year
x Soft creamy white mushroom with good cooking
quality preferred by consumers
x Bioefficiency: 120-130 per cent with cropping cycle of
45 days.
2.OYSTER MUSHROOM
Hypsizygus ulmarius Var.Co(OM2)

x Released from Dept. of Plant Pathology, TNAU,
Coimbatore during 2004.
x Sporophores fleshy, bluish grey initially later and
turning to dull white at maturity
x Highly suitable for commercial cultivation under
subtropical conditions
x Fleshy sporophores contribute for good taste
x Incurved margins of sporophores offer less damage
during transit
x Bioefficiency:120-130 per cent with cropping cycle of
45 days.
108
3. MILKY MUSHROOM - Calocybe indica var.APK 2
x Tropical mushroom released during 1998 from

Regional Research Station, Aruppukottai.
x Worlds first commercially cultivated milky
mushroom variety
x Pure milky white sporophores
x Highly suitable for tropical conditions with
temperature of 30-35 °C and relative humidity of
80 per cent
x Bioefficiency of 140-150 per cent with cropping
cycle of 50 days
x High fibre content contribute for extended shelf
life of 6-7 days under refrigerated storage
4. Milky mushroom
Tricholoma giganteum var.CO( TG) 3
x Tropical mushroom released during 2012 from

Dept. of Plant Pathology,TNAU,Coimbatore
x Pure milky white sporophores ,Sub bulbous stipe
x Highly suitable for tropical conditions with
temperature of 30-35 °C and relative humidity of
80 per cent
x Bioefficiency of 120-130 per cent with cropping
cycle of 50 days
x Less fibre content, storage up to 4 days under
refrigerated storage
4. Emerging/Hi-tech/Priority area generated
Establishment of mushroom biodiversity park. It might added as an emerging

output out of UGC SAP.
A mushroom knowledge park has been planned with a exhibition hall

show casing mushroom various oyster mushroom, milky mushroom and button
mushroom varieties released by the department along with the technologies. A
separate training hall is planned to offer skill oriented training on spawn and
mushroom production to educated youth, farm woman and woman self help groups.
109
The knowledge park also encompass themes and concepts on medicinal
mushrooms viz., Ganoderma lucidum, Lentinus edodes, Jews' ear mushroom and
Cordyceps concepts on biodegradation and bioremediation with white rot
basidiomycetes mushrooms, conversion of spent substrate in to biomanure and
animal feed, nutra and pharmaceuticals, antimicrobials from mushrooms for pest and
disease management A proposal has been submitted to NABARD for obtaining
financial assistance. Also a project proposal on Mushroom Business Startup
Incubator has been submitted to NADP to promote commercialization of edible and
medicinal mushrooms, value added mushroom products through Innovation,
Incubation and graduation; and to create value chain for such products in order to
commercialize and popularize the edible and medicinal mushroom varieties and
strains released from TNAU by utilizing ligno cellulosic residues. Through this
establishment it is expected to enrich young minds with knowledge on mushroom
science apart from providing training to about 5000 beneficiaries within a year. Steps
have been taken to set up a mushroom museum show casing the biodiversity
conservations of the western Ghats. The facility will intend on ex situ conservation of
macrobasidiomycetes of commercial importance to explore the biopesticidal
molecules promote for plant and human health improvement. These services will be
made available to entrepreuners, farmers and other stakeholders to take up
biomanure and animal feed production by utilizing spent waste, post harvest
mushroom processing, product development, packaging and quality testing.
Through this it is expected that there will be larger adoption of TNAU

technologies and mushroom varieties by other R&D Institutes and industries in
India..Further, there is great scope for promotion of entrepreneurship in mushroom
biotechnology business by hand holding with TNAU on a Public Private Partnership
mode on “Win-Win” basis to meet the global competition for mushroom biotech
products apart from creating impact on the livelihood of farmers, farm women, youth
and self help groups.
110
5. Resource generation (specify amount Rs.in lakh): Proposals are to be
submitted for the establishment of pilot projects to use the facilities at TNAU
on a commercial scale by the stakeholders on rental basis.
1. Project proposal submitted TANII: Budget Rs. 99 lakhs
Innovative cropping system approach for recycling of paddy straw and

banana pseudostem through paddy straw mushroom (Volvariella volvacea)
cultivation for food and biomanure
2. Project submitted to NADP:Establishment of an automated pilot plant

facility for quality mushroom spawn and ready to flush kit production
– Rs.175 lakhs
3. Proposal submitted to NABARD for Infrastructure development and

mushroom knowledge park –Rs.1.5 crores
Items Amount Items Amount
Consultancy - Sponsored(agency) -
Transfer of Annexure-I R&D Projects -
technology No
Patent utilization - Product & Prototype Annexure-VI
development
Industrial - Exploitation of internal Dept. of Nematology,
collaboration facilities by user departments Dept. of Microbiology,
TNAU, Coimbatore
Human Annexure-II Neighboring institutions Annexure-VII
Resource
Training
International Annexure-III Industries -
students
Industrial - National organizations -
Extension Annexure-IV International organizations -
activities
Other courses Annexure-V Any other Annexure-VIII
Collaborativeprogrammes
111
ANNEXURE-I
Transfer of Technology through oneday and five days training
Training programme & date Male Female Total
One day training
05.04.2018 32 10 42
07.05.2018 50 24 74
05.06.2018 50 12 62
05.07.2018 45 19 64
06.08.2018 29 41 70
20.08.2018 30 53 83
05.09.2018 49 10 59
05.10.2018 9 2 11
25.10.2018 34 5 39
07.11.2018 35 32 67
05.12.2018 27 14 41
07.01.2019 22 19 41
Total 412 241 653
Five days training programme
28.05.18-01.06.18 8 2 10
25.06.18-29.06.18 5 1 6
23.07.18-27.07.18 3 2 5
27.08.18-31.08.18 4 - 4
17.12.18 -21.12.18 4 - 4
Total 24 5 29
112
C.Short demo/ training class to school students/ college students
S. Date Name of the school/ college No. of participants
No.
Staff Students Total
1 17.4.2018 Suguna International School, 2 80 82

Gandhipuram
2 18.4.2018 Karl Kubel Institute for 2 16 18

Development Education, German
3 12.7.2018 Vanavarayar Institute of 5 139 144

Technology, Pollachi
5 24.7.2018 College of Horticulture, Bagalkot 1 14 15
6 16.8.18 G.R. Damodaran Matric Hr. Sec. 1 20 21

School,Peelamedu
7 6.9.2018 Government Boys Hr. Sec. School, 4 56 60

Thondamuthur
8 25.9.2018 Pasumpon Muthuramalinga 4 66 70

Thevar College, Usilampatti
Karl Kubel Institute for 19

Development Education, German
10 3.10.2018
11 15.11.2018 S.F.V. Government Hr. Sec. 2 50 52

School, Seeranaickenpalayam

School, Peelamedu

School, Peelamedu

School, Peelamedu
113
Sciences, Namakkal
16 4.1.2019 Department of Agricultural 1 45 46

Microbiology, TNAU
17 11.1.2019 College of Horticulture and 2 43 45

Forestry
18 13.2.2019 St. Francis Xavier Hr. Sec. School, 3 83 86

Erode
19 14.2.2019 Govt. College, Tripunithura 2 19 21
20 1.3.2019 Got. Arts and Science College, 2 22 24

Coimbatore

Sciences, Namakkal
22 21.3.2019 SSA Education Dept, Perur block 5 20 25
D.Special training programmes to Self help groups/ farmers
S. Date Name of Self help groups/ No. of participants

farmers
No. Staff Beneficiaries Total
1 18.4.2018 Manivel, IWMP Society- 1 46 47

Dharapuram
2 28.6.2018 AHO, Tumkur 34 34
3 30.8.2018 Block Technology Manager, 5 30 35

Thirivarur
3 23.10.2018 Agricultural extension 2 35 37

Centre, Theni
4 8.1.2019 AHO, Ooty 3 100 103
5 25.1.2019 Tribal welfare, Salem 12 12
6 29.1.2019 ATMA, Nedumkandam 3 13 16
7 30.1.2019 ATMA, Thiruvallur 1 54 55
114
Date Venue No. of Male Female Remarks
Participants if any
31.10.2019 FC&RI, 40 36 4 SC
Mettupalayam, Farmers
Karamadai block,
Coimbatore dt
21.12.2019 Dept. of Plant 60 45 15 SC Youth

Pathology, TNAU,
Coimbatore
20.02.2020 Dept. of Plant 65 - 65 SC self

Pathology, TNAU, help group
Coimbatore
One day training
Date Venue No. of Male Female Remarks if any

Participants
05.042019 Dept. of Plant 27 18 9 Farmers /
Pathology, entrepreneurs
TNAU,
Coimbatore
0.5.05.2019 Dept. of Plant 31 19 12 Farmers /
TNAU,
Coimbatore
28.05.2019 Vellmadai 47 16 31 Farmers
village- NSS
students and
farmers
05.06.2019 Dept. of Plant 23 17 6 Farmers /
TNAU,
Coimbatore
05.07 2019 Dept. of Plant 47 37 10 Farmers/entrepren
Pathology, eurs
TNAU,
Coimbatore
06.08 2019 Dept. of Plant 47 37 10 Farmers /
TNAU,
Coimbatore
TNAU,
Coimbatore
23.09.2019 Kerala club, 32 - 32 Woman group
Coimbatore
115
30.9.2019 Dept. of Plant 7 7 - Agrl.officers,
Pathology, Maldives
TNAU,
Coimbatore
05.102019 Dept. of Plant 30 19 11 Farmers /
Pathology , entrepreneurs
TNAU,
Coimbatore
14.10.2019 Central 50 50 - Prisoners
prison,
Coimbatore
(Oyster
mushroom)
22.10.2019 CAG pride, 60 45 15 NABARD officals
Coimbatore
23.10.2019 Central 3 3 - Prisoners
prison,
Coimbatore
(Milky
mushroom)
05.11 2019 Dept. of Plant 70 49 21 Farmers/
Pathology , entrepreneurs
TNAU,
Coimbatore
Pathology, , entrepreneurs
TNAU,
Coimbatore
,TNAU,
Coimbatore
TNAU,
Coimbatore
TNAU,
Coimbatore
Five day training
Date Venue No. of Male Female Remarks if any

Participants
24.06.2019 Dept. of Plant 9 9 - Farmers/

to Pathology,
Coimbatore
116
16.07.2019 Dept. of Plant 2 - 2 Sultan Qaboos
to Pathology, university, oman
20.07.2019 TNAU,
Coimbatore
28.08.2019 Dept. of Plant 7 7 - Farmers/

to Pathology,
Coimbatore
21.10.2019 Dept. of Plant 10 8 2 Farmers/

to Pathology,
Coimbatore

to Pathology,
Coimbatore

to Pathology,
Coimbatore
ANNEXURE-II
Human Resource Training
1. Number of One day training programme

Year Number of Training Number of persons
2016-2017 13 733
2017-2018 14 672
2018-2019 15 653
2. Number of Five days training programme

Year Number of Training Number of persons
2016-2017 4 40
2017-2018 5 29
2018-2019 5 29
117
Date Category No. of Participants Male Female
31.10.2019 SC Farmers 40 36 4
21.12.2019 SC Youth 60 45 15
20.02.2020 SC self help group 65 - 65
S. No. Name of the Mushroom Company and Product name

Producer
2. Mr. A.R.Subramanian M/s. Maga Mushrooms

Product: Mushroom spawn and
cultivation
2. Mr.M.Ajith Laxman M/s. Sri Ayyappa Vegetable Merchant
3. Mr.S.Rahulray M/s. Vardhani Corporation

4. Mr.M.Nagarajan M/s. Annavarshni Foods LLP
5. Mr.S.K.Senthil Kumar M/s. Healthy life Foods

6. Mr.V.Mayilsamy Mushroom cultivation
7. Mr.N.Arul Jothi Mushroom spawn production
8. Mr.S.Kamal Mushroom spawn production and
farming
9. Mr.Vigram Kannan Mushroom production
10. Ms.B.Akila Mushroom cultivation

11. Mr. S. Rajendran Mushroom production
12. Mr. B. Chaithanyan Oyster Mushroom production
13. Mrs. J. Suganthi Oyster and Milky Mushroom production
14. Mr. Senthilkumar Mushroom production
15. Mr. Yuvaraj Deivasigamani M/s. Nilgiri Farm Produce
Button mushroom production
118
16. Mr. Yasuhide Ito M/s. YATS Corporation Co. Ltd.,
Shiitake mushroom production
17. Mr.A.Gandhi Mushroom production

18. Mr. S.R.Mylsamy Mushroom farming
19. Mr. A. Senthil Kumar Mushroom Production
20. Mr. A. Kandavel Mushroom spawn production
21. Mr. K. Saravana Kumar M/s. Dhana Kesaan Mushroom Farm
22. Mr. Mohammed Kachakkaren M/s. HAAD Enterprises
23. Mr. S. Thilagaraj Mushroom value added products
ANNEXURE-III
International Students
1. Sultan Quaboos University, OMAN – 2 nos

1. Ahad Saud Sultan
2. Hanin Abdalaiah Said
2. Agriculture field Officers – MALDIVES – 6 nos
1. Abdulla Anwar
2. Abdul Jaleel
3. Amir Ali Didi
4. Adam Imsaaz
5. Hussain Mohamed
6. Zakariyya Ibrahim
ANNEXURE-IV
Extension Activities
Demonstrations 1. Vellamadai village, Coimbatore through NSS students on

organised 23.05.2019
2. ATMA project , Gujarat on 10.09.2019
3. Farmers from Magudanchavadi, salem -4.12.2019
4. Kerala club, coimbatore-23.09.2019
119
5. Farmeers Association, cuddalore.2.12.2019
6. Central Prison- 14.10.2019
7. Central prison :23.10.2019
Radio 1. Mushroom cultivation: Prospects, Problems and solutions -

Programme 13.06.2019
2.Progarmme coverage and broadcast on Training on Mushroom

cultivation to SC farmers on 30.10.2019
3.One day training programme - Interaction with farmers broadcasted

- 5.12.2019
4. Mushroom day interaction with farmers broadcasted - 23.12.2019
News paper 1. Indian Express dt. 02.10.2019 Mushrooms the way to

coverage prosperity
2. Dinamani (Tamil) dt.3.10.2019-Message on Rise of mushrooms as

retreats for humanity
3. Thozhil malar (June,2019 Tamil) Making money from home

through mushroom cultivation
4. HIndu (Tamil) 1.10.2029 on National seminar on mushrooms

published.
5.Dinamalar (Tamil) dt.4.10.2019 on establishment of mushroom

park at TNAU, Coimbatore
ANNEXURE-V
Other Courses
¾ Certificate course on Commercial Mushroom Production – Students -52 nos

¾ Experiential Learning on Commercial Mushroom Production – 95 nos
¾ Advances in Mycology – including the mushroom biotechnology – 10 nos
120
ANNEXURE-VI
Product and Prototype development
Automated Spawn Unit
x Development and testing of small scale automation for organized spawn

production and mushroom cultivation to encourage Small Mushroom Big
Business concept.
Mushroom cultivation in polypropylene bottle
Increased yield was obtained with H. ulmarius var. (CO(OM)2

cultivated in polypropylene bottle containers (267.17g per bottle containing
180 g of paddy straw on dry weight basis recording 148.43 per cent
bioefficiency). However, P.platypus (Pp) was also found to give equally better
yield followed by P. pulmonarius. But, P. florida gave comparatively less yield
in polypropylene bottle containers.
121
Laminated carton box used for Pleurotus cultivation
Increased yield was obtained with P. platypus (PP), when cultivated in

laminated carton box (36x18x12 cm) container, which accounted to 886.5g
per box containing 650 g of paddy straw on dry weight basis recording 136.4
per cent bioeffficency. However, H.ulmarius was also found to give yield on
par with that of P. platypus in laminated carton box containers.
Log cultivation of P. platypus on M.oleifera stumps
Different sizes of moringa wood logs were used for the cultivation of P.
platypus. Among them, stumps measuring 300 mm x 80 mm size, with 4 vertical slits
for spawning were found to be superior, which recorded an average bioefficiency of
123.9 per cent and yield of 433.8g per log.
122
ANNEXURE-VII
Neighbouring Institutions participated in the UGC-SAP Seminar
Sl. No College Name
1. Rathinam College of Arts and Science
2. G. R. D College of Science
3. Annamalai University
4. RVS College of Arts and Science
5. Bharathiyar University
6. AJK College of Arts and Science
7. K. S. G College of Arts and Science
8. Dr. NGP College of Arts and Science
9. Hindustan College of Arts and Science
10. PSG College of Arts and Science
11. Rathinavel subramaniam College of Arts and Science
12. Amritha University
13. Sri Ramakrishna College of Arts and Science
14. Avinashilingham Institute for Home Science & Higher Education for
Women
15. Sadakathullah Appa College
16. Government Arts College , Salem
123
ANNEXURE-VIII
Anyother Collaborativeprogrammes:
1. Project proposal submitted TANII: Budget Rs. 99 lakhs
Innovative cropping system approach for recycling of paddy straw and banana
pseudostem through paddy straw mushroom (Volvariella volvacea) cultivation for
food and biomanure
2. Project submitted to NADP: Establishment of an automated pilot plant facility

for quality mushroom spawn and ready to flush kit production – Rs.175 lakhs
3. Proposal submitted to NABARD for Infrastructure development and mushroom

knowledge park – Rs.1.5 crores
a. Total amount of resource generated from al lsources above
b. Also mention development grant received from University in other areas of

the Department. – Nil
6. Use of output of research, teaching in (tick and fillup the rightone)
Item No. Item No.
a.Industries 10 c. Other user deptts. 3
c.National orgns. 8 d.Other organisations 1
7. Other activities
Items Numbers Time duration
Seminar 5 10
Summer Institute 2 4
Conference 1 1
Refresher Courses 1 1
124
b. AutonomousCharacter: Yes/No.
a. Financial
b. Administrative
c. Academic
d.Others
b. Advisory Committee Meeting (No.withDates)
4. 2016-2017
The first advisory committee meeting of the UGC sponsored SAP- DRS-1
“Enterprising scheme being operated in the Department of Plant Pathology, CPPS,
TNAU, Coimbatore was held on 20.01.2017 at Committee room, O/o of the Vice-
Chancellor, TNAU, Coimbatore under the chairmanship of Dr. K. Ramasamy, Vice-
Chancellor, TNAU, Coimbatore.
The following members attended the meeting:
7. Dr. K. Ramaraju, Director (CPPS), TNAU, Coimbatore and Advisory

Committee member.
8. Dr. Samir Rajan Sikdar, Professor and Head, Division of Plant pathology,
Centaury Building, Bose Institute Kolkata-700054, West Bengal and UGC
committee
9. Dr. D. Anusuya, Professor (Retd.), Department o9f Botany, Bangalore
University, Jnanbharathi Campus, Bengaluru-560058 and UGC nominee
10. Dr. U. Sivakumar, Professor (Microbiology), TNAU, Coimbatore and Advisory
Committee Member
11. Dr. S. Nakkeeran, Professor (Plant Pathology), TNAU, Coimbatore and
Deputy Coordinator
12. Dr. A.S. Krishnamoorthy, Professor and Head, Department of Plant
Pathology, TNAU, Coimbatore- Member Secretary and Coordinator.
Dr. K. Ramaraju, Director i/c, (CPPS), TNAU, Coimbatore welcomed the

Advisory Committee members of UGC-SAP-DRS-1, Scheme presented the major
objectives, year wise plan of work, budget details, work progress for the year 2016-
2017 before the committee for favour of approval Dr. Anusuya and Dr. U. Sivakumar
actively participated and offered their valuable suggestions for the successful
implementation of the programme Dr. S. Nakkeeran, Professor (Plant Pathology)
proposed vote of thanks.
125
Remarks offered by the Advisory committee for further action:
¾ The committee approved the overall objectives and year wise plan of
work (2016-2021).
¾ The Vice-Chancellor insisted the importance of conversation of
economically important fungal cultures and advised the scientists
involved to visit national repositories like Agarkar Institute to get hands
on experience. He also suggested forming a time like of activities for
the successful implementation of the scheme. In his further remarks
the Vice- Chancellor advised the scientists to involve M.Phil students of
conventional Universities in the collection and identification of wild
mushrooms.
¾ The Vice-Chancellor also suggested to send proposal for
establishment of pilot plant facility for automated spawn production
under NADP scheme.
¾ It was insisted to involve students working in the Department of
Environmental Science, Agricultural Microbiology and Bio-informatics
to continue with the objectives of the scheme student project work
priorities need to be identified towards mycelia biomass conversion,
enzyme extraction, identifying pharmaceutically, important genes in
mushroom fungi, nucleotide sequencing and identification of wild fungi,
separation of colour, flavour, and fragrance profiles of mushrooms,
mechanisms formation sporocarps and basidiospores and use of
mushroom fungi for environment clean up.
¾ Dr. Samir Ranjan Sikdar suggested for collaborative research projects
with Bose Institute of protoplast fusion for strain improvement in
mushroom fungi. He further suggested to ensure supply of quality
spawn through Regional Centres.
¾ Dr. D. Anusuya suggested to send the AUC in time for further release
of grant from UGC. She also suggested to specify the substrates to be
used in bioconversion by mushroom fungi based on their large- scale
availability and economic feasibility. The scientist also insisted to
develop value added mushroom products. She further insisted the
importance of establishing a culture collection centre for mushrooms at
TNAU, Coimbatore.
126
5. 2017-2018
Major Recommendations
6. Establishment of mushroom knowledge park (Dr.K. Ramasamy, Chairman-

UGC- SAP)
7. Empowerment of rural unemployment youth on spawn and mushroom
production (Dr. Anusuya)
8. Knowlegde empowerment on UG and PG students from neighbouring
colleges on antimicrobial activity of biomolecules (Dr. Anusuya)
9. Documentation of mushroom biodiversity (Dr. Anusuya)
10. Intensification on arious thematic areas of mushroom research by involving both
UG and PG scholar from neighbouring colleges (Dr. Samir Ranjan Sikdhar)
6. 2018-2019
The Annual Advisory Committee meeting of UGC-SAP-DRS-1 scheme on

“Enterprising mushroom biotechnology on Food, Feed and biomanure” was
conducted in the chamber of Vice-Chancellor committee hall, TNAU, Coimbatore on
30.09.2019 under the chairmanship of Dr.N.Kumar, Honourable Vice-Chancellor,
TNAU, Coimbatore in the presence of the following advisory committee members.
8. Dr. A. S. Krishnamoorthy, Registrar & Coordinator (UGC-SAP).

9. Dr.K.Prabakar,Director (CPPS)
10. Dr. Samir Ranjan Sikdhar, (Rtd Professor), UGC Nominee
11. Dr. S. Nakkeeran, Professor (Plant Pathology) & Deputy Coordinator (UGC-SAP)
12. Dr. U. Sivakumar, Professor (Agrl. Microbiology) & Member (UGC-SAP)
13. Dr.P.Nallathambi, Principal Scientist & Member (UGC-SAP) Wheat Research
Station, IARI , Wellington, Ooty
14. Dr. M. Muthamilan, Professor and Head, Department of Plant Pathology,
TNAU, Coimbatore
The advisory committee suggested following issues to strengthen the activity
of UGC-SAP.
6. Empowerment of woman self help groups on spawn and mushroom

production (Dr.N.Kumar, Vice-Chancellor)
7. Developing innovative modules/ growing kits for mushroom production as an
alternate to polybags and exploring antimicrobial activity of mushrooms
(Dr.P.Nallathambi)
127
8. Documentation and conservation of the biodiversity edible and medicinal
mushroom(Dr. Samir Ranjan Sikdhar)
9. Standardizing the cultivation techniques for medicinal mushroom Ganoderma
lucidum( Dr.N.Kumar, Vice-Chancellor)
10. Strengthening on various thematic areas of mushroom research through skill
empowerment of UG and PG scholar from neighbouring colleges
(Dr. Samir Ranjan Sikdhar)
10. Faculty Involved
a.(Put Numbers) In thrust Area(1) Other Areas(2) (1) (2) (1) (2)under SAP/ ASIST
FacultyStrength PositionsAvailable Working Vacant

Professors Dr. A. S. Krishnamoorthy Registrar -
TNAU,
Coimbatore
Dr. S. Nakkeeran Professor -
(Plant Pathology)
TNAU, Coimbatore
Associate Professor Dr. G. Thiribhuvanamala Assoc.Professor
(Plant Pathology)
TNAU, Coimbatore
Assistant Professors Dr. M. Karthikeyan Asst.Professor -
(Plant Pathology),
TNAU, Coimbatore
Dr. P. Latha Asst.Professor -
(Plant Pathology),
TNAU, Coimbatore
Others Mr. Venkatesan Lab-Assistant
Mrs. Shantha PUSM

Mrs.Kaliammal PUSM
Mr. Selvaraj PUSM
128
b. In the identified thrust area(s)*:Include the details of identified persons in the
respective thrust areas (Advisory committee members from ICAR/Other Dept. can be
added).
Membership Specialization/
(INSA/ Specific
Faculty Name
BHATNAGAR/ Areas of
BIRLA) expertise
Dr. P.Nallathambi Principal Scientist Mycology
Dr. U. Sivakumar, Professor (Agrl. Microbes and

Microbiology) enzymes
Dr. S.D. Sivakumar Associate Professor Fodder crops

(Agronomy)
Dr. M. Thirunavukkarasu Veterinary Science Animal Science
Dr. P.Kaliselvi Assistant Professor Bioremediation

(ENS)
Dr. P. Geetha Assoc. Professor Food Science

(FSN) and Nutrition
*Provide a list of publication records in referred journals (group area wise,

faculty memberwise, year-wise).
Mushroom
Krishnamoorthy, A.S. and Venkatesh Balan.2015. A Comprehensive Review of

Tropical Milky White Mushroom (Calocybe indica P&C). Mycobiology 43(3):
184-194.
SangeethaC., A. S. Krishnamoorthy and S.Nakkeeran. 2015. Evaluation of bioactive

compounds of Ophiocordyceps sinensis [berk.] sacc. againstFusarium spp.
Biochem. Cell. Arch. 15 (2), 431-435.
Sangeetha, C., Krishnamoorthy, A.S., and Ramakrishnan,S.2015. Testing bioactive

compounds of chinese caterpillar fungus, Ophiocordyceps spp against root
nematode (Meloidogyne incognita). Research Journal of Agricultural sciences.
6(6): 1129-1133.
129
Sangeetha, C., Krishnamoorthy, A.S., Nakkeeran. S., Ramakrishnan, S and
Amirtham.D. 2015. Evaluation of bioactive compounds of Ophiocordyceps
sinensis (Berk.) Sacc. Against Fusarium spp. Biochemical and cellular
Archive. 15(2): 431-435.
Senthilmurugan, S. And Krishnamoorthy, A.S. 2015. Growth promoting

Mycobacteria, Acinetobacter calcoaceticus and Agrobacterium tumifaciens
(Rhizobium radiobacter) isolated from oyster mushroom mycelium. Research
Journal of Agricultural sciences. 6(6): 1190-1193
Thiribhuvanamala. G., Prakasam V., D., Alice, Parthasarathi S and Krishnamoorthy

A.S. 2015. Utilization of Agrowaste for Production of Protein Rich Food and
Biomanure by the white Rot fungi, Pleurotus pulmonarius .Trends in
Bioscience 8:3701-3705
Senthilmurugan, S. and Krishnamoorthy, A.S. 2015. Innovative containers for oyster

mushroom cultivation. International journal of Tropical agriculture. 33(3):2107-
2111.
Thiribhuvanamala, G. G.Kalaiselvi,.S.Parthasarathy, S. Madhavan and V.

Prakasam.2017 Extracellular secretion of lignocellulolytic enzymes by diverse
white rot basidiomycetes fungi. Annals of Phytomedicine 6(1): 20-29
Kiran kumar.N, Krishnamoorthy. A. S, Kamalakannan. A and Amirtham. D, 2017.

Influence of temperature and pH on the mycelial growth and chlamydospore
production of paddy straw mushroom Volvariella volvaceae (Bull. Ex Fr.), The
Journal of research, 44(1&2),1-7
Kiran Nannapaneni, Krishnamoorthy Akkanna Subbiah and Kamalakannan. A,

2017. Formulation of liquid spawn base of paddy straw mushroom, Volvariella
volvacea (Bull. Ex Fr.) sing. Int. Journal of Chemical Studies. 5(3), 138-142
Kiran Nannapaneni, Krishnamoorthy Akkanna Subbiah and Kamalakannan.A, 2017.

Tyrosinase inhibitory potential of phytochemicals and mycomolecules.
International journal of green Pharmacy,10(4), 1-6.
Viruthambigai S, R Kannan, M Arumugam Pillai, V Ramamoorthy, R Reihana and

VK Parthiban. 2020 Studies on morphological and growth characters of new
Pleurotus isolates. Journal of Phytocognasy and Phytochemistry 8 (3): 3328-
3330.
130
Sakthivel, K., G.Thiribhuvanamala, S. Madhavan and V. Prakasam. 2019 Substrate
induced lignolytic enzyme secretion for maximising the yield of Black Poplar
mushroom, Agrocybe aegerita under subtropical condition. Journal of
Mycology and Plant Pathology, 49(2):17-176.
New Disease reports Disease Diagnosis and surveillance
Dheepa R., P. Renukadevi, S.Vinodkumar and S.Nakkeeran. 2015. First Report of

Chrysanthemum White Rust (Puccinia horiana) in India. Plant Disease, 99
(9):1279.
Aravintharaj, R., C. G. Balaji, K. Nagendran, R. Priyanka and G. Karthikeyan. 2017.

First report of Lily mottle virus on lily (Lilium sp.) in southern India. Virus
Dis.,DOI 10.1007 /s13337 -017-0381-9
Priyadharshini, S., A. Kamalakannan and V. Paranidharan. (2019). First

characterization report of Botrytis cinerea infecting Tuberose in India. Journal
of Pharmacognosy and Phytochemistry, 8 (3): 4709-4712.
Jamuna, S., L. Rajendran, Betsy Haokip, K. Nagendran, G. Karthikeyan, and S. K.

Manoranjitham. 2017. First report of natural infection of Solanum nigrum L.
with Tomato mosaic virus in India. Plant Disease, http://doi.org/10.1094/
PDIS-08-17-1264-PDN
Balamurugan, A., K. Sakthivel, A. Kumar, M. Muthamilan 2019. First reportof

Curvularia hominis inciting fruit rot of ridge gourd (Luffa acutangula) in Tamil
Nadu, India. Journal of Plant Pathology, 1-1.
Kannan Bapu J. R, A. Thanga Hemavathy, N. Nadarajan, A.R. Muthiah, D.

Packiaraj, D. Malarvizhi, K. Ganesamurthy, E.Rajeswari and D.Rajabaskar
.2017.CO 8 : A high yielding long duration new red gram variety suitable for
Tamil Nadu. Electronic Journal of Plant Breeding, 8(4): 1053-1058 . DOI:
10.5958/0975-928X.2017.00156.9
Nagendran Krishnan, Shweta Kumari, Awadhesh Bahadur Rai, Manimurugan

Chinnappa, Bijendra Singh, Karthikeyan Gandhi and Naidu Rayapati. 2017.
First report of Peanut bud necrosis virus infecting bitter gourd (Momordica
charantia L.) in India. Plant Disease, 102(3): 690
Prabhukarthikeyan, S. R., Manikandan, R., Durgadevi, D., Keerthana, U., Harish, S.,
Karthikeyan, G and Raguchander. T. 2017. Bio-suppression of turmeric
rhizome rot disease and understanding the molecular basis of tripartite
131
interaction among Curcuma longa, Pythium aphanidermatum and
Pseudomonas fluorescens.Biological Control, 111: 23-31.
Prabhukarthikeyan, S. R., Manikandan, R., Durgadevi, D., Keerthana, U., Harish, S.,
Karthikeyan, G and Raguchander. T. 2017. Antibiotic-producing
Pseudomonas fluorescens mediates rhizome rot disease resistance and
promotes plant growth in turmeric plants. Mycological Research. 210: 65-73
K.P.Smitha, E.Rajeswari, D.Alice and T.Raguchander .2015.Assessment of

Vascular Wilt and Dry Root Rot of Pigeonpea in Tamil Nadu International
Journal of Tropical Agriculture. 33(3): 2145-2151
Latha, T.K.S. and P. Lava Kumar. 2015. Preliminary characterization of Coimbatore

isolate of Pigeonpea sterility mosaic virus. Madras Agric. J.102 : 344-347.
M. Suganyadevi, S.Nakkeeran and P. Renukadevi. 2015. Molecular characterization of

plant quarantine pathogen pv. causing bacterial blight in anthurium and its
management Xanthomonas axonopodis dieffenbachiae. J. Mycology and
Plant Pathology, 45 (4): 235 -240.
Renukadevi P., K. Nagendran, S.Nakkeeran, G. Karthikeyan and M. Jawaharlal.

2015. First report of Tomato spotted wilt virus infection of chrysanthemum in
India, Plant Disease, 99 (8):1190.
VinodkumarS., P. Rajeshkumar, C.Senthilraja, S.Nakkeeran and WGD. Fernando.

2015. First report of Sclerotinia sclerotiorum causing stem rot of carnation
(Dianthus caryophyllus) in India. Plant Disease, 99 (9): 1280.
VinodkumarS., P.Rajeshkumarand S.Nakkeeran. 2015.Developmental biology and

infection cycle of Sclerotiniasclerotiorum causing stem rot of carnation in
India. African Journal of Microbiology Research 9 (49): 2328-2336.
DheepaR., S. Vinodkumar, P. Renukadevi and S.Nakkeeran. 2016. Phenotypic and

molecular characterization of chrysanthemum white rust pathogen Puccinia
horiana (Henn) and the effect of liquid based formulation of Bacillus spp. for
the management of chrysanthemum white rust under protected cultivation.
Biological Control 103: 172-186.
RageshwariS., P. Renukadevi, V.G. Malathi and S.Nakkeeran. 2016. Occurrence,

biological and serological assay of Tobacco streak virus infecting cotton in
Tamil Nadu. J.Mycol Pl. Pathol, 46 (2): 67-77.
132
RajeshkumarP., P. Adhipathi, S.Nakkeeran. 2016. RAPD–PCR technique for
molecular detection and diversity of Fusarium oxysporum f.sp. dianthi causing
vascular wilt of carnation. International Journal of Forestryand Crop
Improvement, 7 (1):85-91
SmithaK.P., E.Rajeswari,D. Alice and P.Latha.2016.Morphological and molecular

variability in Rhizoctonia bataticola Taub Butler causing root rot of pigeonpea
in Tamil Nadu. Madras Agricultural Journal 103( 1-3) : 45 -50
Parthasarathy S., M. Muthamilan, S. Harish, D. Alice and T. Raguchander 2017

.Natural Incidence and Genetic Variability of Erysiphe pisi, the Causal Agent
of Powdery Mildew on Peas in the Nilgiris District, Tamil Nadu, India Current
Journal of Applied Science and Technology, 24(5): 1-11
Sara Shomo, H. Shifa, C. Senthilraja, Madam Gurivi Reddy, V. Paranidharan and R.

Velazhahan. 2016. Transgenic Groundnut Plants Modified For Resistance
Against Tobacco Streak Virus. Biochem. Cell. Arch. Vol. 16 p. 245-250
Archana,S., Prabukarthikeyan, K., Raguchander, T. and Prabakar, K. 2017.

Comparative assessment of RAPD and ISSR markers to study genetic
polymorphism in Colletotrichum gloeosorioides isolates of Mango. Asian
Journal of Plant Pathology, 11 (3): 130-138.
Nagendran, K., S. Mohankumar, P. Mohammed Faisal, B. Bagewadi and G.

Karthikeyan 2017. Molecular evidence for the occurrence of Tomato leaf curl
New Delhi virus on chayote (Sechium edule) in southern India. Virus Dis., DOI
10.1007/s13337-017-0403-7
Nagendran, K., S. Mohankumar, R. Aravintharaj, C.G. Balaji, S. K. Manoranjitham,

A.K. Singh, A. B. Rai, B. Singh and G. Karthikeyan. 2017. The occurrence
and distribution of major viruses infecting cucurbits in Tamil Nadu state, Indi a.
Crop Protection, 99:10-16.
Senthilraja, C., Renukadevi, P., Malathi, V. G., Nakkeeran, S and Pappu, H. R. 2018.
Occurrence of Tomato spotted wilt virus infecting snapdragon (Antirrhinum
majus) in India. Plant Disease, 102 (8): 1676.
Kothandan Manivannan, Perumal Renukadevi, Varagur Ganesan Malathi, Gandhi

Karthikeyan and Natarajan Balakrishnan. 2019. A new seed-transmissible
begomovirus in bitter gourd (Momordica charantia L.). Microbial
Pathogenesis, 128 (2019) 82–89.
133
Biocontrol
Balakrishnan, S., M. Muthamilan, A. Ramanathan, D. Sudhakar, L. Mahalingam, L.

Rajendran and S. Parthasarathy. 2020. Field evaluation of fungicide, biotic
and abiotic inducers for the management of Alternaria alternata causing leaf
blight of sunflower. Journal of Pharmacognosy and Phytochemistry 9 (1): 574
– 575. NAAS: 5.21
Chaithra M., S.Vanitha, A. Ramanathan, V. Jegadeeshwari, V. Rajesh, V. Hegde

and E. Apshara, 2020. Profiling secondary metabolites of Cocoa (Theobroma
cacao L.) Endophytic fungi Lasiodiplodia pseudotheobromae PAK-7 and
Lasiodiplodia theobromaeTN-R-3 and antimicrobial activities. Current Journal
of Applied Science and Technology, 39(2):47-56, 2020.
Naveenkumar K., S.K.Manoranjitham, L. Rajendran, K. Darshan. 2020.

Morphological and molecular characterization of two devastating chili
pathogens Collectotrichum acutatum and Fusarium brachygibbosum. Journal
of Pharmacognosy and Phytochemistry, 9(2):916-921.
Dileepkumar, G., N. Natarajan and S.Nakkeeran. 2015. Synthesis and

characterization of silver (Ag) nanoparticles and its antifungal activity against
Sclerotium rolfsii in chilli (Capsicum annum L.). International Journal of
Agricultural Science and Research, 5 (3): 211-218
Durgadevi, D., S. Harish, D. Alice and T. Raguchander. 2015. Mycolytic enzymes

produced by plant growth promoting endophytic bacteria inhibits sheath blight
of rice incited by Rhizoctonia solani Kuhn. Journal of Mycology & Plant
Pathology 45 (1) : p. 101 -102
Durgadevi, D., S. Harish, D. Alice, and T. Raguchander. 2015. Bioprospecting and

antifungal potential of novel endophytic Bacillus in rice against sheath blight
disease incited by Rhizoctonia solani. Journal of Pure and Applied
Microbiology 9 (3) : 2361-2372.
Durgadevi, D., S. Harish, D. Alice, and T. Raguchander. 2015. Characterization of

novel endophytic Bacillus spp. from rice exhibiting plant growth promoting and
antifungal activities against Rhizoctonia solani.. African journal of Microbiology
research 9 (11) : 6016-6021.
Kodoth Padinhare Smitha, Rajeswari Mohan, Alice Devadason and Thiruvengadam

Raguchander 2015 Exploiting novel rhizosphere Bacillus species to suppress
134
the root rot and wilt pathogens of chickpea .African Journal of Microbiology
Research 9(15), pp. 1098-1104
Meena, B. 2015. Disease management in medicinal crops. Int. J. Noni Res, Vol. 10
p. 43-57.
Meena, B. and K.Rajamani. 2015. Biological control of leaf blight disease in Gloriosa
superba using Pseudomonas fluorescens. Journal of Pure and Applied
Microbiology, Vol. 8 p. 2579-2586.
Meena, B. and K.Rajamani. 2015. Integrated management of leaf blight disease in

Gloriosa superba. World Journal of Pharmaceutical Research, Vol. 4 p. 1545-
1551.
Raj C., S. Singh, M. Hubballi, S.Nakkeeran and G Chandrasekar. 2015. Diversity

assessment of Colletotrichum species associated with chilli anthracnose and
their characterization via species specific primers. Indian Journal of Plant
Protection 43 (3), 341-349.
Suganyadevi, M., S.Nakkeeran and P. Renukadevi. 2015. Molecular characterization of

plant quarantine pathogen pv. causing bacterial blight in anthurium and its
management Xanthomonas axonopodis dieffenbachiae. J. Mycology and
Plant Pathology, 45 (4): 235 -240.
Dileep KumarG., N. Natarajan and S.Nakkeeran.2016. Antifungal activity of

nanofungicide Trifloxystrobin 25%+ Tebuconazole 50% against
Macrophomina phaseolina. African Journal of Microbiology Research 10 (4),
100-105.
KrishnamoorthyK., A .Sankaralingam and S.Nakkeeran. 2016. Efficacy of biocontrol

agents in the management of head rot of cabbage (Brassica oleracea var.
capitata) caused by Sclerotinia sclerotiorum. African Journal of Microbiology
Research 10 (40), 1711-1714.
KrishnamoorthyK., A. Sankaralingam and S.Nakkeeran. 2016. Growth of Sclerotinia

sclerotiorum causing head rot disease of cabbage on various carbon and
nitrogen sources. World Journal of Agricultural Sciences 12 (4), 261-263.
Karthick M., C. Gopalakrishnan, E. Rajeswari and V. Karthik Pandi. 2017. In vitro

efficacy of Bacillus spp. against Fusarium oxysporum f. sp. ciceri, the causal
agent of Fusarium wilt of Chickpea, Int.J.Curr.Microbiol.App.Sci 6(11): 2751-
2756
135
Keerthana, U., K. Nagendran, T. Raguchander,K. Prabakar, L. Rajendran and
G. Karthikeyan, 2017. Deciphering the Role of Bacillus subtilis var.
amyloliquefaciens in the Management of Late blight Pathogen of Potato,
Phytophthora infestans. Proceedings of the National Academy of Sciences,
India - Section B: Biological Sciences, doi:10.1007/s40011-017-0842-3.ISSN:
0369-8211
Krishnamoorthy,K.K., Sankaralingam, A., and Nakkeeran,S. 2017. Compatibility

between fungicides and Bacillus amyloliquefaciens isolate B15 used in the
management of Sclerotinia sclerotiorum causing head rot of cabbage. Int. J.
Chemical Studies, 5 (6), 239-24.
Krishnamoorthy,K.K., Sankaralingam, A., and Nakkeeran,S. 2017. Management of

head rot of cabbage caused by Sclerotinia sclerotiorum through combined
application of fungicides and biocontrol Bacillus amyloliquefaciens. Int. J.
Chemical Studies, 5 (2): 401 -404.
LakshmiS..S.Nakkeeran and S.Sathya. 2016. Combined effect of biopriming and

polymer coating against chilli damping off. International Journal of Agriculture
Science and Research 6 (3), 45-64.
Meena, B. 2016. Integrated disease management of root rot in Coleus forskohlii.

World Journal of Pharmaceutical Research, Vol. 5 p. 1312-1317.
RajeswariE., K.P. Smitha, A. Kamalakannan, D.Alice and J.R. Kannan Bapu.2016.

Management of pigeonpea sterility mosaic disease. Legume Research.
39(4) :648 - 650
Sathya S.and S.Nakkeeran. 2016. Effect of biopriming on population of Bacillus

amyloliquefaciens VB7 in chilli seeds. The Bioscan 11 (2), 907-919
SuganyadeviM., P. Renuka devi and S.Nakkeeran. 2016. Efficacy of biocontrol

agents and bactericides for the management of bacterial blight incited by
Xanthomonas axonopodis pv. dieffenbachiae in Anthurium andraeanum.
INTERNATIONAL J. OF Plant Protection, 9(1): 292 -296.
Lincy K.B.,, Nakkeeran,S., Manoharan,T and Gunasekaran,K. 2017. Pathogenicity

of indigenous Beauveria bassiana isolates against diamond back moth,
Plutella xylostella. International Journal of Agricultural Sciences, 8 (62):3512 -
3514.
136
Lincy, K.B., Nakkeeran,S., Kennedy, J.S., and Manoharan,T. 2017. Morphoilogical
and molecular characterization of entomopathogenic fungi Beauveria
bassiana isolated from different districts in India. Green Farming. 8 (4): 940 -
944
Madhavan,s., Adhipathi pasuvaraj, R.Velazhahan and M. Karthikeyan,-2017

Management of Chilli (Capsicum annuum) Anthracnose Using Fungicides and
Biocontrol Agents Indian Phytopathology, 70(1) : 86-9.
Dheepa,R.,Vinodkumar,S., Renukadevi,P. and Nakkeeran,S. 2017. Phenotypic and

molecular characterization of chrysanthemum white rust pathogen
Pucciniahoriana (Henn) and the effect of liquid based formulation of Bacillus
spp. for the management of chrysanthemum white rust under protected
cultivation. Biological Control 103, 172
Balakrishnan.S, Gopalakrishnan. C, Kamalakannan. A, Senthil Kuppusamy. K and

Parthasarathy. S, 2017. Evaluation of antagonistic activity and plant growth
promotion by paste formulation of Trichoderma harzianum, Journal of
Pharmacognosy and Phytochemistry,6(6),355-360.
Johnson, I., R. Ramjegathesh, J. Sheela, N. Shoba and H. P. Maheshwarappa.

2017. Development of Microbial Consortia for the Management of Leaf Blight
Disease of Coconut, ActaPhytopathologica et EntomologicaHungarica, 52 (1),
pp. 1−14
Rajamanickam, S., G. Karthikeyan, M. Kavino and S. K. Manoranjitham. 2017.

Biohardening of micropropagated banana using endophytic bacteria to induce
plant growth promotion and restrain rhizome rot disease caused by
Pectobacterium carotovorum subsp. carotovorum. Scientia Horticulturae,
231:179-187
Rajamanickam, S., Rageshwari, S., Renukadevi, P. and Nakkeeran, S. 2017.

Occurrence of Plantagoasiaticamosaicvirus infecting oriental lily in India. The
Horticultural Society of India, 74 (4): 574-578
Ramyabharathi, SR., K. Sankari Meena, K., Rajendran, L., Karthikeyan, G.,

Jonathan, E. I and Raguchander. T. 2018. Biocontrol of wilt-nematode
complex infecting gerbera by Bacillus subtilis under protected cultivation.
Egyptian Journal of Biological Pest Control. 28: 21-29.
137
Sathya,S., Lakshmi,S. and Nakkeeran,S. 2017. Biopriming and polymer coating for
healthy seedling production under hydrophonic condition in chilli cv.K2. Green
Farming, 8 (1): 222 -226.
Senthil Raja, G., Anand, T., Mohankumar, S., Raguchander, T and Samiyappan, R.
2017. Analysis of variation in virulence of Beauveria bassiana against insect
pests of pigeonpea using qPCR. Journal of Basic Microbiology,1: 1-6
ParthasarathyS., M. Muthamilan, S. Harish, D. Alice and T. Raguchander

2017.Studies on Morphological Characterization of Erysiphe pisi Causing
Powdery Mildew of Pisum sativum by Environmental Scanning Electron
Microscope.Int. J. Pure App. Biosci.5 (6):1348-1355.
Manikandan, R., G. Karthikeyan, T. Raguchander. 2017. Soil proteomics for

exploitation of microbial diversity in Fusarium wilt infected and healthy
rhizosphere soils of tomato. Physiological and Molecular Plant Pathology,
100: 185- 193
Muthulakshmi,P . Ragavi.B and Parthasarathy,S.2017. Influence of Abiotic factors on

the growth of Colletotrichum musae causing post harvest anthracnose
disease in banana. Accepted for publication in the International Journal of
Agricultural Science and Research ,vol.7,issue,2,Apr.2017,121-128
Shanmugapackiam, S., Parthasarathy, S and Raguchander, T. 2017. Detection of

Phytotoxin Produced from Leaf, Neck and Finger Blast Disease Causing
Magnaporthe grisea through GC-MS Analysis, International Journal of
Biochemistry Research & Review, 19(3): 1-10
Shanmugapackiam, S., Parthasarathy, S., Nakkeeran, S. and Raguchander, T.

2017. Detection of finger millet blast pathogen Magnaporthe grisea using
FTIR spectroscopy, Chem Sci Rev Lett., 6 (22): 956-960.
Smitha K.P. , E. Rajeswari, D. Alice and T. Raguchander2017 Exploring Quorum

Sensing Loci and Biofilm Formation in Bacillus Isolates from Pigeonpea
Rhizosphere,.Int.J.Curr.Microbiol.App.Sci 6 (11) : 3252-3262
Smitha Kodoth Padinhare,Rajeswari Mohan, Alice Devadason and

ThiruvengadamRaguchander2017. Morphological and Molecular Variability in
Fusarium udum isolates causing Vascular Wilt of Pigeonpea in Tamil Nadu,
ChemSci Rev Lett 6(24), 2281-2288
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Venkataramanamma K., L. Rajendran and C. Gopalakrishnan. 2020. Sunflower

diseases and their management by integrated approach. Diseases of Crops:
Diagnosis and management, Vol 2: Pulses, Oilseeds, Narcotics and Sugar
crops (CRC Press- 978-1-77188-840-0) pp. 287-306.
Balakrishnan, S., M. Muthamilan, A. Ramanathan, D. Sudhakar, L. Mahalingam, L.

Rajendran and S. Parthasarathy. 2020. Assessment of different culture media
on the growth and sporulation of Alternaria alternata causing leaf blight
disease of sunflower. The Pharma Innovation Journal 9 (1): 408 – 411.
Pushpam R., V Ramamoorthy, N Nithya, S Fathima Nasrin, E Meena, VK Parthiban

and M Arumugam Pillai. 2020. Identification of suitable rice cultivar for sheath
rot disease resistance by artificial screening in the Germplasm. Journal of
Phytocognasy and Phytochemistry (3): 1533-1536.
149
Induced resistance
Vanthana, M., Nakkeeran, S., Malathi, V.G., Renukadevi, P., Vinodkumar, S. 2019.
Induction of in planta resistance by flagellin (Flg) and elongation factor-TU
(EF-Tu) of Bacillus amyloliquefaciens (VB7) against Groundnut bud necrosis
virus in tomato. Microbial Pathogenesis, 137, 103757.
Surega R., S. Ramakrishnan, B. Anita, K. Gunasekaran and S.Nakkeeran. 2015.

Antifungal effect of plant mediated silver nanoparticles against Fusarium
oxysporum f sp lycopersici. International Journal of Tropical Agriculture 33 (1):
157-160.
SuregaR., S. Ramakrishnan, B. Anita, K. Gunasekaran and S.Nakkeeran.

2015.Characterization of green synthesized silver nanoparticles using Tridax
procumbans. International Journal of Tropical Agriculture 33 (1): 131-134.
Prabhukarthikeyan, S.R., Keerthana, U., Archana, S. and Raguchander, T. 2017.

Induced resistance in tomato plants to Helicoverpa armigera by mixed
formulation of Bacillus subtilis and Beauveria bassiana. Research Journal of
Biotechnology, 12 (10): 53-59.
Prabukarthikeyan, S. R., Keerthana, S., Archana, S and Raguchander, T. 2017.

Analysis of genetic diversity among different isolates of Beauveria bassiana
by RAPD-PCR. Journal of Biological Control, 31 (1): 18-24
Rageshwari,S., Renukadevi,P., Malathi,V.G., Amalabalu, P and Nakkeeran,S. 2017.

DAC-ELISA and RT-PCR based confirmation of systemic and latent infection
by tobacco streak virus in cotton and parthenium. Journal of Plant Pathology,
99 (2): 17
Mohan,C., Sridharan,S, Subramanian,K.S., Natarajan,N and Nakkeeran,S. 2017.

Effect of nano emulsion of hexanal on honey bees (Hymenoptera, Aphidae).
Journal of Entomology and Zoology studies, 5 (3) – 1415 – 1418.
Murali Sankar P., T. Raguchander and Shanmugabackiam S. 2017. Seed

Bacterization a Novel Prime Tool for Growth Promotion in Tomato (Solanum
lycopersicum M.) under in vitro. Int.J.Curr.Microbiol.App.Sci, 6(11): 3076-
3080.
Jebaraj,M.D., Aiyanathan, K.E.A., and Nakkeeran,S. 2017. Virulence and genetic

diversity of Sclerotium rolfsii Sacc., infecting groundnut using nuclear (RAPD
& ISSR) markers. Journal of Environmental Biology, 38 (1), 147 – 158.
150
Krishnamoorthy,K.K., Sankaralingam, A., and Nakkeeran,S. 2017. Effect of
Temperature and Salinity on the Growth of Sclerotinia sclerotiorum Causing
Head Rot of Cabbage. Int. J. Curr. Microbiol. App. Sci., 6 (2), 950-954.
Daimei, G., Harpreet Singh, R., Pushpa Devi, P., Gunjan Kumar, S., Renukadevi,
P.,Malathi,V.G., Senthilraja, C., Mandal, B.and Rajagopal, R. 2017. Influence
of groundnut bud necrosis virus on the life history traits and feeding
preference of its vector, Thrips palmi. Phytopathology. 107 (11), 1440-1445.
Kanimoli Mathivathana, M., N. Jagadeeshselvam, B. Madhumitha, A. Karthikeyan,

M. Pandiyan, G. Karthikeyan, C. Vanniarajan, M. Raveendran, N. Senthil and
M. Sudha. 2018. Screening and identification of SSR markers for genetic
diversity for mungbean [Vigna radiate (L.) Wilczek]. Int. J. Curr. Microbiol.
App. Sci., 7(4): 789-793 (https://doi.org/10.20546/ijcmas.2018 .704.088)
Kanimoli Mathivathana, M., S.M. Samyuktha, R. Deepa Priya, I. Mariyammal, P.

Bharathi, N. Jagadeeshselvam, A. Karthikeyan, M. Sudha, M. Pandiyan, G.
Karthikeyan, C. Vanniarajan, M. Raveendran and N. Senthil. 2018.
Association and path analysis of yield and yield components in the RIL
population of Vigna radiate × Vigna umbellate. Int. J. Curr. Microbiol. App.
Sci., 7(4): 1231-123
Kumar, A.S., Aiyanathan, K.E.A., Nakkeeran, S., and Manickam,S. 2018. Documentation
of virulence and races of Xanthomonas citri pv. malvacearum in India and its
correlation with genetic diversity revealed by repetitive elements (REP ). 3 Biotech
8 (11), 479.
Manikandan Rajendran, Harish Sankarasubramanian, Karthikeyan Gandhi and

Raguchander Thiruvengadam. 2018. Comparative proteomic analysis of
different isolates of Fusarium oxysporum f.sp. lycopersici to exploit the
differentially expressed proteins responsible for virulence on tomato plants.
Front. Microbiol., doi: 10.3389/fmicb.2018.00420
Karthick, K., T. Arumugam, V. Rajasree, K.N., Ganesan and M. Karthikeyan. 2019.

Studies on correlation and path analysis of yield attributes in cucumber
(Cucumis sativus L.). Journal of Pharmacognosy and Phytochemistry. 8(6):
342-345.
Gowrisri, N, Kamalakannan, A, Malathi, V.G, Rajendran, L. and Rajesh, S. 2019.

Morphological and molecular characterization of Magnaporthe oryzae, inciting
agent of rice blast disease, Madras Agric. J., 106: 255-260.
151
Jadhav, K.P., N Senthil, P.M. Tamilarasi, K.N. Ganesan, V. Paranidharan, M.
Raveendran and J. Ramalingam. 2019. QTL mapping for sorghum downy
mildew disease resistance in maize (Zea mays L.) in recombinant inbred line
population of UMI79 X UMI936 (w). Current Plant Biology 20: 100-124.
Latha M., Raja K., Subramanian K.S., Karthikeyan M. and Lakshmanan A. 2019.
Fabrication and characterization of tebuconazole loaded PVA Nano fibre.
International Journal of Agriculture Sciences, 11(10): 8514-8517.
Mayalagu Kanimoli Mathivathana, Jayakodi Murukarthick, Adhimoolam
Karthikeyan, Woojong Jang, Manickam Dhasarathan, Nallathambi
Jagadeeshselvam, Manickam Sudha, ChocklingamVanniarajan, Gandhi
Karthikeyan, Tae-Jin Yang, Muthurajan Raveendran, Muthaiyan Pandiyan
and Natesan Senthil. 2019. Detection of QTLs associated with mungbean
yellow mosaic virus (MYMV) resistance using the interspecific cross of
Vignaradiata × Vigna umbellate. Journal of Applied Genetics.
https://doi.org/10.1007/s13353-019-00506-x.
Nivedha, P., V. Rajasree, T. Arumugam, M. Karthikeyan and V. Thiruvengadam.
2019. Evaluation of parents and hybrids of chilli (Capsicum annuum L.) for
yield and resistance to chilli leaf curl disease. Journal of Pharmacognosy and
Phytochemistry. 8(3): 4763-4766
Prasad Basavaraj Purad, H. Usha Nandhini Devi, T. Arumugam and M. Karthikeyan.
2019. Growth and performance of different chilli genotypes for yield and yield
attributing characters. Journal of Pharmacognosy and Phytochemistry, 8(4):
210-213. NAAS: 5.21
Priyanka, R. and Nakkeeran, S. 2019. Ochrobactrum ciceri mediated induction of
defence genes and antifungal metabolites enhance the biocontrol efficacy for
the management of Botrytis leaf blight of Lilium under protected conditions.
Journal of Plant Pathology 101 (2): 323-337.
Ramjegathesh, R., G. Karthikeyan, D. Balachander, K. Ramaraju, L. Rajendran, T.
Raguchander and R. Samiyappan. 2019. Nested and TaqMan® probe based
quantitative PCR for the diagnosis of Ca. Phytoplasma in coconut palms. Mol.
Biol. Rep., http://doi.org/10.1007/s11033.018-4500-5. (Impact Factor: 2.11).
Shanmugapriya. S, JeyaSundara Sharmila, D, Karthikeyan, G, and K. S.
Subramanian. 2019. Bioassay of Azadirachtin nanoformulation against
Bemisia tabaci, the vector of Mungbean yellow mosaic virus. Madras
Agricultural Journal, DOI:10.29321/MAJ.2019.000293.
152
Shiva, N., V. Paranidharan, M. Karthikeyan, D. Balachandar and K. Gunasekaran.
2019. Systemic induction of defense-related genes in tomato by
Pseudomonas fluorescens suppresses bacterial wilt disease incited by
Ralstonia solanacearum. J. Entomol. And Zool. Stud. 7(3): 813-819.
Sneha, R.P. and V. Paranidharan. 2019. Transcript analysis of five defense genes in
tomato in response to streptocycline resistant and sensitive strains of
Xanthomonas euvesicatoria. Multilogic Sci. pp. 249-253.
Sudha A. 2019. A new symptom report in mundu chilli (Rainfed Chilli)

(Capsicum Spp.) Caused by Colletotrichum capsici in Ramanathapuram
District, Tamil Nadu. International Journal of Plant Sciences. 14(2) :99-100.
Intake (Please put numbers)
Intake Identified thrust area Other than thrust

area
Ph.D. 6 50
PG 10 40
Fellows 5 41
NETscholar - -
GATEScholar: - -
Res.Asso. - -
Proj.Asstt. 2 -
Others
11. National/Nodal Character of the Department National/Nodal/All India Centre

a. Resource Persons Invited (Nos.)-
International: Nil
153
National – External Examiner
1. A. Bharani Deepan ID. No. 2015801502 - Dr. V. Shanmugam

2. RAJESH KUMAR, P. ID. No. 2015801505 - Dr. M. LOGANATHAN
3. TANUJA, N. ID. No. 2015801507 - Dr. R. SELVARAJAN
4. Chaithra, M. (2016801503) - Dr. Y.M. Somasekhara
5. Karthick, M. ID. No. 2016801505 - Dr. R. THANGAVELU
6. Praveen, N.M. ID. No. 2016801507 - Dr. P. RAJI
7. Patil Sneha Rashtrapal (2016801506) - Dr. V. Devappa
8. Vanthana, M. ID. No. 2016801510 - Dr. R. VISWANATHAN
9. Balamurugan, A. ID. No. 2014801501 - Dr. V. DEVAPPA
10 R. DHEEPA ID. No. 2014801502 - Dr. Robin Gogoi
11. KARTHIK PANDI, V. ID. No. 2014801503 - Dr. P. Nallathambi
12. MURALI SANKAR P. ID. No. 2014801504 - Dr. P. Nallathambi
13. Parthasarathy, S., ID. No. 2014801505 - Dr. S. Sriram
14. Priyadharshini. B., ID. No. 2014801511 - Dr. P. Nallathambi
15. R. PRIYANKA ID. No. 2014801512 - Dr. A. Kumar
16. SAMPATH KUMAR, A. ID. No. 2014801507 - Dr. M. Loganathan
17 SENTHILRAJA, C., ID. No. 2014801508 - Dr. Kajal K. Biswas
18. SUGANYADEVI, M. ID. No. 2014801509- Dr. N. Nagaraju
19. Vinod Kumar, S, ID. No. 2014801510 - Dr. R. Viswanathan
20. Vasumathi, S. (13-613-007) - Dr. M. Loganathan
21. Sara Hamad Shomo Sharaf Eldin ID. No. 13-613-009 - Dr. K. Umamaheswaran
22. J. Rajalakshmi, ID. No. 13-613-005 - Dr. R. Rajesh Rathod
23. Betsy Deineihoi Haokip, I. D. No. 11-613-001 - Dr. T. Makeshkumar
24. S. Rageshwari, I. D. No. 11-613-008 - Dr. Indranil Dasgupta
25. C. Anju (12-613-001) - Dr. K. Umamaheswaran
26. M. Daniel Jebaraj (12-613-002) - Dr. A. Kumar
27. D. Durgadevi (12-613-003) - Dr. A. Naseema
28. Hassen Shifa Gebeyo (12-613-011) -Dr. R. Viswanathan
29. S. R. Prabhukarthikeyan (12-613-006) - Dr. A. Nassema
30. Sangeetha, C. (12-613-007) - Dr. Lulu Das
31. Smitha, K.P (12-613-009) - Dr. A. Nassema
32. Sireesha Yeturi (12-613-008) - Dr. M. Anandaraj
33. N. Kalieswari (11-613-002) - Dr. M. K. Naik
34. P. Lakshmidevi (11-613-004) - Dr. A. Ramesh Sundar
35. SA. Ramyabharathi (11-613-007) - Dr. N. P. Eswara Reddy
36. S. Shanmugapackiam-(11-613-008) - Dr. M. Loganathan
154
1. Dr. M. Loganathan
Principal Scientist(Plant Pathology)
ICAR- National Research centre for Banana,
Thogamalai Road, Thayanur (Po)
Tiruchirapalli-620 102
2. Dr. R. Viswanathan
Principal Scientist & Head
Division of Crop Protection
ICAR-Sugarcane Breeding Institute
Coimbatore – 641 007
3. Dr. R. Thangavelu,
Principal Scientist,
National Research Centre for Banana,
Tiruchchirappalli - 620 102.
4. Dr. P. Raji
Professor (Plant Pathology)
Regional Agricultural Research Station,
Pattambi – 679 306.
Principal Scientist (Plant Pathology)
Indian Agricultural Research Institute,
Regional Station,
Wellington – 643 231.
6. Dr. N. Nagaraju
Professor
Department of Plant Pathology
University of Agricultural Sciences,
Bangalore – 560 024.
7. Dr. V. Shanmugam
Senior Scientist (Plant Pathology)
Division of Plant Pathology
ICAR –IARI, PUSA,
New Delhi – 110 012.
8. Dr. A. Kumar
Principal Scientist
Division of Plant Pathology
I.A.R.I.
155
9. Dr. Y.M. Somasekhara
Professor
College of Agriculture
UAS, GKVK, Bengaluru – 560 065
Email id: [email protected]
10. Dr. R. Selvarajan
ICAR-NRC for Banana Thayanur Post,
Tiruchirapalli – 620 102
11. Dr. K.Umamaheshwaran, Ph.D,
Associate Director of Research,
Kerala Agricultural University,
Vellayani, Thiruvananthapuram – 695522.
12. Dr. Kajal K. Biswas
Principal Scientist (Professor),
Division of Plant Pathology,
New Delhi - 110012
13. Dr. S.Sriram
Principal Scientist (Plant Pathology) &
Indian Institute of Horticultural Research (IIHR),
Hessaraghatta Lake post,
Bengaluru - 560 089.
14. Dr. A. Nassema
Professor (Plant Pathology),
College of Agriculture,
Vellayani,
Thiruvananthapuram,
Kerala-695 522
15. Dr. Lulu Das
Professor
Kerala Agricultural University
Vellayani -695 522
16. Dr. T. Makeshkumar
ICAR- Central Tuber Research Institute
Sreekariyam,Thiruvanathapuram- 695017, Kerala, India.
156
17. Dr. Indranil Dasgupta
Professor of Plant Molecular Biology
University of Delhi South Campus
18. Dr. R. Rajesh Rathod
Assistant Professor (Jr. Plant Pathology),
ACIRP on Spices, Dept. of Horticulture,
College of Agriculture, Dapoli- 415 712
19. Dr. V. Devappa
Professor & Head
College of Horticulture
UHS Campus, GKVK Post
Bengaluru – 560 065
20. Dr. Robin Gogoi
Principal Scientist,
Division of Plant Pathology,
New Delhi – 110012
21. Dr. M. Anandaraj
Director,
ICAR- Indian Institute of Spices Research,
P.B.No.1701, Marikunnu,
P.O.Kozhikode-673 012,
Kerala
22. Dr. N. P. Eswara Reddy
Associate Dean, S.V. Agricultural College,
Tirupati – 517501
23. Dr. A. Ramesh Sundar
Principal Scientist (Plant Pathology),
Sugarcane Breeding Institute,
Indian Council of Agricultural Research,
Coimbatore - 641 007.
24. Dr. M. K. Naik,
Dean, PG Studies,
University of Agricultural Sciences,
Raichur – 584 104.
157
b. Serving for outside user departments in (Nos.&hrs.)
I. Hands-on OR technical training touniversity/college teachers include

workshops
II. Collaborative (international)
III. Teaching to neighboring nstitutions – Nil
IV. Visiting Teachers to foreign university – Nil
V. Equipment facilities – From Department
Generic Name of Equipment Make

HPLC with fluorescence and ELSD detector Agilent
GC/MS- Quadruple Perkin-Elmer
Rotavapor Buchi
Gel documentation system Alpha
UV-Vis Spectrophotometer Varian
Column chromatography unit Bio-Rad
Freezer (-20 C) Sanyo
Freezer (-70 C) Panosonic
Vacuum gel dryer Hoefer
UV Trans illuminator Hoefer
Hybridization oven Hoefer
Mini gel system Hoefer
Mini submarine unit Hoefer
Trans blot SD semi-dry transfer system Hoefer
Ultra-centrifuge Hitachi
Digital Autoclave TOMMY
VI. Other major infrastructure facilities - –
DST- FIST
1. Phase Contrast Microscope

2. Real time PCR
3. Growth Chamber
12. Most critical and essential requirements that may be required to continue the
programmes if the UGC agrees to continue or extend support based on the
evaluation and final review by expert committee.
Non-recurring : Recurring: Total(Rs.in lakh) : (As per
items given in the guidelines

{Please add Annexure)
158
a. Whether the State Government will take up the liability of the faculties and
the staff approved under SAP after cessation of the tenure of the
programme i.e. five years. - Nil
b. Whether the State Government has already agreed or has taken up the
liability after five years of completion of the tenure of the programme as was
communicated along with the approval letter? - Nil
c. How the Department is going to maintain infrastructure and the status if UGC
disagrees to continue the support further. Whether the Department /University
will agree for upgradation of the status on no cost basis, if it so happens as
per the recommendation of the Committee.
13. Utilization Certificates may be provided as per the UGC format. The
accounts of the earlier phase be completed, finalised, audited and duly
authenticated by the competent authority (Registrar and Finance Officer both)
(item-wise and year-wise) for all the allocations and sanctions given to the
Department for ongoing/current phase are to be submitted by the Department
so that UGC, if provides support again, may immediately release the funds
for the phase to be approved as per the above activities.
Programme Coordinator Head of the Department
Director (CPPS) Director of Research
Registrar of the University
159
Courses to students
One ODL Certificate Course on Mushroom Cultivation was conducted from

11.03.2018 to 19.08.2018 through the Directorate of Open Distance
Learning, TNAU, Coimbatore. A total number of 17 students have registered
for the course.
One ODL Certificate Course on Mushroom Cultivation was conducted from
15.03.2019 to 25.08.2019 through the Directorate of Open Distance
Learning, TNAU, Coimbatore. A total number of 15 students have registered
for the course.
Forty one UG – IV year BSc.,(Agri.) & B.S (ABM)students (37 girls and 4
Boys) were taught EXP 401 Experiential Learning in Mushroom Production
Technology 0+5 Course during VIII Semester 2017-18.
Thirty nine UG – IV year BSc.,(Agri.) & B.S (ABM)students (29 girls and 10
Boys) were taught EXP 401 Experiential Learning in Mushroom Production
Technology 0+5 Course during VIII Semester 2018-19.
MSc(Ag.) Plant Pathology Students research work a Mushroom

Research Laboratory:
x Characterization of antimicrobial compounds from mushroom fungi and plant
extracts against chilli fruit rot pathogen, Colletotrichum capsici
x Bioactive molecules from mushroom fungi against post harvest anthracnose
fruit rot of mango caused by Colletotrichum gloeosporioides
Ph.D (Plant Pathology)students research work a Mushroom Research

Laboratory:
x Studies on genetic variability, morphogenesis and fruiting body architecturing
of milky mushroom (Calocybe indica p&c)
x Harnessing the antifungal properties of hexanal and mycomolecules against
post harvest pathogens of Mango and Banana
x Formulation of Microbial Molecules against Fusarium nematode complex in
Cucumber and Capsicum
x Bioprospecting with antiviral phyto and microbial molecules against
RNAviruses
x Volatilomes Mediated Defense against Damping off and Wilt Pathogens of
Tomato under Protected Cultivation
160
F. Important Visitors
Name of Visitor Designation Date of Visit
K.V.Rajkumar Deputy Collector,Erode 21.05.2018
C. Vidya Deputy Collector,Villupuram 09.01.2019
Dr.Venkatesh Balan Dept. of Engineering 23.12.2018

Technology, University of
Houston, USA
Dr. D.S.Rathore, Dr. R.P. QRT TEAM Visit 29.10.2018 to

Tewari ,Dr. R.K. Pal, Dr. Chairman & Members 30.10.2018
S.R. Bhat,
Dr. H.B. Singh, Dr. Shwet
Kamal
Dr. N.Krishnakumar International Biodiversity, 09.03.2019
New Delhi
Dr. V.P. Sharma Director, ICAR – DMR, 24.04.2019

Solan
Posters presented at NRCB: (21-23 December, 2018)
x Invitro screening of volatilomes of certain basidiomycetes fungus against

Fusarium oxysporum f.sp. lycopersici and Pythium sp in tomato(T. Praveen)
x Standardization for effective growth of Ophiocordyceps sinensis under invitro
condition(S. B.Akshaya)
x Inhibitory potential of bioactive molecules of Ophiocordyceps sinensis against
plant pathogens(C. Sangeetha)
x Emergence of Tomato leaf curl New Delhi virus infecting bittergourd in Tamil
Nadu(B. Sangeetha)
x Activity of metabolites from mushroom fungi against Colletotrichum capsici,
the chilli anthracnose and fruit rot pathogen of chilli (K. Priya)
x Antimicrobial Activity of Cucumber Endophytes against Fusarium Wilt
Pathogen(S. Priyanka)
161
AWARDS
x Best Poster Presentation Award was received by T.Praveen, S.B.Akshaya,

C. Sangeetha for the topics Invitro screening of volatilomes of certain
basidiomycetes fungus against Fusarium oxysporum f.sp. lycopersici and
Pythium sp in tomato (T. Praveen), Standardization for effective growth of
Ophiocordyceps sinensis under invitro condition(S. B.Akshaya), Inhibitory
potential of bioactive molecules of Ophiocordyceps sinensis against plant
pathogens (C. Sangeetha)
G. Publications
Krishnamoorthy, A. S. and B.Priyadharshini.2016. Physical, Chemical and Biological

Properties of Casing Soil Used for Milky Mushroom (Calocybe indica P&C)
Production. Madras Agric. J.,103(10-12) : 338-343.
Krishnamoorthy, A.S. and Venkatesh Balan.2015. A Comprehensive Review of

Tropical Milky White Mushroom (Calocybe indica P&C). Mycobiology43(3):
184-194.
Kirankumar, N.., A. S. Krishnamoorthy, A.Kamalakkannan and D. Amirtham. 2016.

Influence of temperature and pH on mycelial growth and chlamydospore
production of paddy straw mushroom, Volvariella volvaceae (Bull. Ex Fr.)
Sing. J.Res. ANGRAU .44(1&2) 1-7
Kiran Kumar Nannapaneni, Krishnamoorthy Akkanna Subbiah, Kamalakannan

Ayyanar, Amirtham Damodarasamy. 2016. Tyrosinase inhibitory potential of
phytochemicals and mycomolecules. International Journal of Green
Pharmacy. 10 (4): 1-7.
Kiran Kumar Nannappaneni, Akkanna Subbiah Krishnamoorthy and Ayyanar

Kamalakannan.2017. Formulation of liquid spawn base of paddy straw
mushroom, Volvariella volvacea (Bull.Ex.Fr.)sing. International Journal of
Chemical Studies5(3):138-142.
Priyadharshini, Bhupathy and Krishnamoorthy Akkanna Subbiah. 2017. Volatilomes

of milky mushroom (Calocybe indica P&C) estimated through GCMS/MS.
International Journal of Chemical Studies (IJCS) 5(3): 387-391.
162
Priyadharshini Bhupathi, Akkanna Subbiah Krishnamoorthy and Sivakumar Uthandi.
2017. Profiling of morphogenesis related enzymes of milky mushroom
Calocybe indica. (P & C). Journal of Pharmacognosy and Phytochemistry 6(5):
2537-2543
Sangeetha Chinnusamy and Akkanna Subbiah Krishnamoorthy. 2017. Identification

of 3’deoxyadenosine (Cordycepin) from the medicinal mushrooms,
Ophiocordyceps spp. Intl. Journal of Chemical Studies (IJCS) 5(3): 788-792 .
Sangeetha, C., Krishnamoorthy, A.S., Nakkeeran, S., Ramakrishnan., S. and D.

Amirtham. 2015. Evaluation of Bioactive compounds of Ophiocordyceps
sinensis (Berk.) Sacc. Against Fusarium spp. Biochem.Cell.Arch.15:431-435.
Sangeetha, C., Krishnamoorthy, A.S., and S.Ramakrishnan.2015. Testing bioactive

compounds of Chinese caterpillar fungus, Ophiocordyceps spp against root
knot nematode. Research Journal of Agricultural Sciences6 (5): 1129-1133.
Senthilmurugan and A.S.Krishnamoorthy.2015. Innovative containers for Oyster

Mushroom Cultivation. 2015. International Journal of Tropical Agriculture
(IJTA)33(3): 2107-2111.
Senthilmurugan, s. and A.S.Krishnamoorthy.2015. Growth Promoting Mycobacteria,

Acinetobacter calcoaceticus and Agrobacterium tumifaciens (Rhizobium
radiobacter) Isolated from Oyster Mushroom. Research Journal of Agricultural
Sciences6 (6): 1190-1193.
Srinivasan V.M., and A.S. Krishnamoorthy. 2017. Compatibility of azoxystrobin and

chaetoglobosin biomolecules with fungal antagonists.International Journal of
Chemical Studies (IJCS)5(5): 2222-2225.
Srinivasan V.M., and A.S. Krishnamoorthy. 2017. Compatibility and phytotoxicity

nature of azoxystrobin on potato .International Journal of Chemical Studies
(IJCS)5(5): 2226-2228.
Srinivasan V.M., and A.S. Krishnamoorthy. 2017. Compatibility of azoxystrobin and

chaetoglobosin biomolecules with bacterial antagonists.International Journal
of Chemical Studies (IJCS)5(5): 2229-2231.
163
Srinivasan V.M., and A.S. Krishnamoorthy. 2017. Performance evaluation of
chaetoglobosin against fruit rot disease on chilli.International Journal of
Chemical Studies (IJCS) 5(5): 2232-2237
Srinivasan, V.M., Krishnamoothy,A.S.,Kuttalam,S., Raguchander and Chinnamuthu,

C.R.2014. Performance evaluation of azoxystrobin in the control of fruit rot
and powdery mildew diseases on chilli. Pestology. 1118 (4): 64-68.
Sumathy, R., R.Kumuthakalavalli, A.S.Krishnamoorthy and Venkatesh balan, 2015.

Phytochemical investigation and total Antioxidant Potential of the Milky
mushroom, Calocybe indica var. APK2 cultivated in Tamilnadu, India. Int J
Pharm Bio Sci.6 (2): (P) 485 – 497
Sumathy, R., R.Kumuthakalavalli, A.S.Krishnamoorthy and Venkatesh Balan, 2015.

Effect of phytochemicals and antioxidant compounds enriched extract from
Calocybe indica var. APK2 on proliferation of human MCF-7 breast carcinoma
cells. Der Pharmacia Sinica6 (2):6-11
Sumathy, R., R.Kumuthakalavalli and A.S.Krishnamoorthy. 2015. Prroximate,

vitamin, aminoacid and mineral composition of milky mushroom, Calocybe
Indica (P&C). var. APK2, commonly cultivated in Tamil Nadu.J. Nat. Prod.
Plant Resour., 5 (1):38-43.
Krishnamoorthy, A. S., S.Nakkeeran, T.Raguchander and C.Ponniah. 2013. Training

Manual on “Fascinating Microbial Biomolecules for Plant and Human Health”.
ICAR-NAIP-National Training organized between 24 th Feb. and 10th
March.2013 in the Directorate of Agribusiness Development, TNAU,
Coimbatore-3
Krishnamoorthy, A.S., K.Kalpana, G.Amutha and M.Ragul.2013. Commercial

Prospects of Milky mushroom (Calocybe indica var.APK2) and its Chitosan
Biopolymer for Plant and Human Health. In: Proc. of the 7 th Intl. Medicinal
Mushroom Conference., 26-29th Aug.2013, Beijing, China. pp: 453-459.
Krishnamoorthy, A.S., A..Kandan and S.Mohan.2013.Effect of Ganoderma lucidum

in Indian HIV carriers. In: Proc. of the 7 th Intl. Medicinal Mushroom
Conference., 26-29th Aug.2013, Beijing, China. pp: 626-631.
Krishnamoorthy, A.S. 2014. Biodiversity exploration of Milky mushroom (Calocybe

indica P&C) Concept to Commercialization. In: Proc. of 8th Int. Conf. on
164
Mushroom Biology and Mushroom Products vol.II (ed. Manjit Singh et al.),
organized by WSMBMP, ICAR-DMR and MSI, 19-22 Nov.2014, NASC
Complex, Pusa, New Delhi. pp:490-495.
Ganesh Kumar, M. and A.S.Krishnamoorthy.2014. Exploration of antifungal bio-

active compounds of Pisolithus tinctorius (Pers.) Coker against soil borne
plant pathogens. In: Abstr. of 8th Int. Conf. on Mushroom Biology and
Mushroom Products vol.II (ed. Manjit Singh et al.), organized by WSMBMP,
ICAR-DMR and MSI, 19-22 Nov.2014, NASC Complex, Pusa, New Delhi. p:
139.
Sangeetha, C., Krishnamoorthy, A.S., Nakkeeran, S., Ramakrishnan, S. and

Govindaraju, P. 2014. Efficacy of bioactive molecules of Ophiocardyceps
sinensis (Berk.) Sacc. againstFusarium Oxysporum f.sp.cubense (E.F.Smith),
the Panama wilt pathogen of banana, and its predisposing nematode,
Meloidogyne incognita (Kofold& White) Chit. In: Abstr. of 8th Int. Conf. on
Mushroom Biology and Mushroom Products vol.II (ed. Manjit Singh et al.),
organized by WSMBMP, ICAR-DMR and MSI, 19-22 Nov.2014, NASC
Complex, Pusa, New Delhi. p: 140.
Senthilmurugan, S., Prabhu Krishnan and A.S.Krishnamoorthy.2014. Performance

evaluation of Volvariella volvacea (Bull.ex Fr.) Singer strain PS 1 for outdoor
cultivation in maize field. In: Abstr. of 8th Int. Conf. on Mushroom Biology and
Mushroom Products vol.II (ed. Manjit Singh et al.), organized by
WSMBMP, ICAR-DMR and MSI, 19-22 Nov.2014, NASC Complex, Pusa,
New Delhi. p: 79.
Krishnamoorthy, A.S., Ragul, M., Srinivasan, V.M. and Ganeshkumar, P. 2015.

Mycomolecules and Blooming Biotech Business Opportunities - The
Changing Scenario of Small Mushroom to Big Industry. Lead paper presented
in the Session VII-Food Mycology during 36th Ann. Conf. and National
Symposium organized by ISMPP between 12-14th Feb.2015 at AC& RI,
Madurai.
Srinivasan, V.M., A.S.Krishnamoorthy, S. Kuttalam, T.Raguchander and

C.R.Chinnamuthu. 2014. Performance Evaluation of Azoxystrobin in the
control of Phytophthora infestans (Mont.) de Bary in potato.
PestologyXXXVIII : 52-55.
165
Sangeetha, C., A.S. Krishnamoorthy., S. Nakkeeran., S. Ramakrishnan and D.
Amirtham. 2015. Evaluation of bioactive compounds of Ophiocordyceps
sinensis (berk.) sacc. againstFusarium spp. Biochem. Cell. Arch.,15(1): 431-
435.
Thiribhuvanamala, G., M.Bharathiraja , D.Amirtham and K.Rajamani. 2018.

Exploitation of Vetiver (Chrysopogon zizanioides L.) shoot biomass for
production of oyster mushrooms Medicinal Plants International Journal of
Phytomedicines and Related Industries.10 (2), p.133-137.
Jeeva S, Krishnamoorthy. A.S, 2018, Antifungal Potential of Myco-molecules of

Coprinopsis cinerea (Schaeff) S. Gray s.lat. against Fusarium spp., Madras
Agric. J., 105(1-3) : 56-60.
Praveen T, Reihana R, Parthiban V.K, Ramamoorthy V, 2018, Molecular

Characterization and Phenotypic Study of New Pleurotus djamor Isolate
KKM1, International Journal of Current Microbiology and Applied
Sciences,7(8):3574-3582.
Sangeetha C, Krishnamoorthy. A.S, Kiran Kumar N, Arumuga Praveen I, 2018,

Effect of Headspace and trapped volatile organic compounds (VOCs) of the
Chinese Caterpillar Mushroom, Ophiocordyceps sinensis (Ascomycetes),
against Soil- borne pathogens, International Journal of Medicinal Mushrooms,
20(9):825-835.
Priyadharshini Bhupathi, Akkanna Subbiah Krishnamoorthy, Sivakumar Uthandi,

2017, Profiling of morphogenesis related enzymes of milky mushroom
Calocybe indica(P &C), Journal of Pharmacognosy and Phytochemistry,
6(5):2537-2543.
166
M.Sc. and Ph.D students guided by Dr. A.S. Krishnamoorthy, Professor
List of M.Sc Students
S.No Name Year Thesis topic
1. Nannapaneni. 2015 Studies on molecular basis of morphogenesis,
KiranKumar chlamydospores production, shelf life and yield of
Volvariellaspp
2. Akshaya S B 2016 Studies on antifungal bioactive compounds of
Ophiocordycepssinensis against selected
phytopathogens
3. Jothi R 2016 Standardization of liquid spawn for the production
of Paddy straw mushroom
4. Manukonda 2017 Exploration of environmentally benign strategies for
JalaRajini the management of bacterial blotch disease in
button mushroom, Agaricusbisporus (Lange.)
5. Jeeva S 2018 Harnessing the Antimicrobial and Nematicidal
properties of Coprinusspp
6 K. Priya 2019 Exploration of biomolecules from mushroom fungi
against chillies fruit rot
7 Priyanka S 2019 Exploitation of endophytes against damping off and
wilt pathogens infecting cucumber
List of Ph.D Students
S.No Name Year Thesis topic
1. S. SenthilMurugan 2015 Developing innovative systems and modules for

oyster mushroom cultivation
2. Sangeetha .C 2015 Exploration of the antifungal potential of bioactive

molecules of Ophiocordycepssinensis (Berk.)
against Seedling blight,Root rot and wilt
Pathogens
3. Priyadharsini. B 2017 Studies on genetic variability, morphogenesis and
fruiting body architechturing of milky mushroom
(Calocybeindica P&C).
4. ArumukaPravin. I 2018 Harnessing the antifungal property of hexanal
and mycomolecules against post-harvest
pathogens of mango and banana
5. Akshaya. S. B 2019 Formulation of consortium of microbial molecules
against Fusarium nematode complex in
cucumber and capsicum
6. Sangeetha. B 2019 Bioprospecting with antiviral phyto and microbial
molecules against begomovirus
7. Praveen. T 2020 Volatilomes mediated defense against damping

off and wilt pathogens under protected cultivation
167
SUMMARY
Summary
x An exclusive UGC-SAP laboratory was established in Department of Plant

Pathology by housing equipments viz., Thermostat controlled autoclave, Rotary
vacuum flask evaporator, Refrigerated Centrifuge, Refrigerated shaker and a
Deep freezer purchased for the value of Rs.28,84,534.
x Thirty six isolates of wild milky mushroom viz., CBE-TNAU-1513 to 1526, CBE-
TNAU-1603, 1604 and CBE-TNAU-1701 were collected from various parts of Tamil
Nadu. They were as Agaricus augustus, Auricularia polytricha, Amanita
phalloides, Calocybe indica, Fomes fomentarius, Ganoderma lucidum, Laccaria
amethystine, Lepiota castaneidica, Mycena flavescens, Psathyrella cernua,
Pluteus cervinus Polyporus sp. Pycnoporus sanguineus, Russula olivacea,
Trametes versicolor, Termitomyces clypeatus, Schizophyllum commune and
Tricholoma based on the morphological keys, microscopic characters and
molecular characterization.
x Among the wild milky mushroom collections, strain TNAU-CBE-1523 was
superior in bioefficiency. The maximum yield was 725.59g (145% bioefficiency)
compared to milky mushroom APK2 (122.3% bioefficiency).
x The substrates coconut saw dust and chopped coconut leaf stalk and arecanut
wood log saw dust were optimize as the best substrate for the cultivation of
medicinal mushroom, Ganoderma lucidum with a total cropping cycle of 107 to
110 days.
x As an alternate to polythene bags, polypropylene (PP) containers were effectively
used for oyster mushroom cultivation with bioefficiency of 109 to 148 % The on
farm trial conducted at various research centres of TNAU also confirmed 126.3 %
bioefficiency in PP containers with C:B ratio of 3.1.
x Bioactive principles from secondary metabolites of medicinal mushroom,
Ganoderma lucidum had antinemic activity against Meloidogyne incognita with
92.3 per cent mortality at 72h after inoculation. The ethyl acetate fraction from
fruiting body of Lentinus edodes at 2000 ppm inhibited mycelial growth of F.
oxysporum and F. solani (cucumber wilt pathogens) up to 61 and 58 per cent
over control. It revealed the presence of 4H-Pyran-4-one, 5-hydroxy-2-
(hydroxymethyl) which was responsible for antimicrobial activity. The Fourier
Transform Infrared Transcopy (FTIR) analysis of ethyl acetate fraction of dry
fruiting body of L. edodes confirmed the presence of alkyl halide, alcohols,
168
aliphatic polyenes, amides, phosphorus, silicon compounds, halogens, alkanes,
triazenes and azo compounds with antimicrobial activity.
x The fruiting body of G. lucidum had the maximum antimicrobial activity against
Colletotrichum gloeosporioides. Biomolecules of G. lucidum induced
malformation in mycelium, conidia and disintegration of cell wall. Characterization
of the ethyl acetate fractions from G.lucidum cap fruiting body indicated the
presence of antimicrobial compounds viz., papaverine (mycelium extracts) and
benzothiazole (cap of G. lucidum).
x Squalene from G. lucidum inhibited symptom development of Ground nut bud
necrosis virus in tomato plants apart from up regulation of defense genes PR 1
(pathogenesis related gene), PAL gene (Phenyl alanine ammonia lyase) and
LOX (Lipoxygenase). Application of squalene at 500 ppm significantly reduced
the number of lesions per sq.cm (1.67 lesions/cm2) on local lesion host, cowpea.
x Cordycepin (3’-deoxyadenosine) from caterpillar mushroom, Ophiocordyceps
sinensis and O. neovolkiana had antifungal against F. o. f. sp. cubense and F. o.
f. sp. lycopersici. It also inhibited egg hatching of juvenile (J2) of root-knot
nematode.
x The bioactive molecules of culture filtrate of Inky cap mushroom, Coprinus
cinerea at 2500 ppm inhibited egg hatching and increased the mortality of
juvenile (J2) of root knot nematode, Meloidogyne incognita The volatiles produced
by mycelia of Coprinus sp. inhibited the mycelial growth of Fusarium oxysporum
f.sp. lycopercisi.
x Hands on skill oriented training on research techniques were imparted to four
UG students from PSG college of Arts & science, Coimbtaore and Karpagam
Academy of Hifgher Education, Coimbatore and twelve PG students from UGC
affiliated Colleges, Bharathiar University, Coimbatore, Annamalai University,
Parangipettai, Rathinavel Subramanian College of Arts & Science, Sulur and
Sadakathulla Appa College, Tirunelvelli on the various research themes viz.,
mushroom biodiversity, biomolecules, biodegradation, value addition and animal
feed under the guidance of faculty members.
x Two numbers of hands on training on spawn production and mushroom
cultivation were imparted to two international students from Sultan Quaboos
university, Oman and seven Agricultural officers from Maldives.
x Three National seminars –“Enterprising Mushroom Biotechnology”, “Fungal
Diversity Conservation and Exploitation of Macromlecules” and Rise of
169
Mushroom–Retreats to Humanity was organized to about 1200 participants
across the country and a National workshop on mushroom series-I was
organized to about 300 students and 25 faculty members during the period. A
permaculture cropping based outdoor cultivation of paddy straw mushroom as
inter crop in Maize was developed which recorded bioefficiency of 18 to 22 per
cent which depict the inclusion of mushroom as a component in Integrated
Farming system.
x Twelve numbers of one day training on mushroom cultivation was imparted to
1489 participants and five numbers of skill oriented tissue culture, spawn
production and mushroom cultivation were imparted to 73 participants. Seven
numbers of demonstrations were imparted to 339 participants including farmers
from various parts of Tamil Nadu.
x Twenty two numbers of demonstrations on oyster mushroom cultivation were
conducted to 1195 school / college students. Special training on spawn and
mushroom production was organized to 140 numbers of SC benefici aries
including farmers, students and woman self help groups. About 2180 visitors
including students from various colleges of Tamil Nadu from were benefitted on
the aspects of mushroom cultivation.
x Publicity on mushroom cultivation was provided through All India Radio and daily
news papers. About 23 entrepreneurs registered with Agri business incubator of
TNAU to utilize the facilities for their enterprise. Five radio programmes were
organized involving scientists, farmers and students to create awareness on
mushroom cultivation.
x A mushroom knowledge park has been planned with a exhibition hall show
casing mushroom various oyster mushroom, milky mushroom and button
mushroom varieties released by the department along with the technologies.
Proposal has been submitted to NABARD and a separate facility for automated
spawn unit has been proposed to NADP A separate training hall is planned to
offer skill oriented training on spawn and mushroom production to educated
youth, farm woman and woman self help groups. A mushroom museum has been
established by displaying the wild mushroom collections and commercial
varieties.
x Dignitaries Dr.V.P.Sharma, Director, DMR, Solan, Dr.Jalali, Executive Director
(HAU), Deputy Collector, Erode, Dr.S.Thamaraiselvi, Vice-Chancellor, Thiruvallur
University, Dr.Venkatesh Balan, University of Houston, USA and many others
both national and international visited the facility.
170
PUBLICATIONS
Madras Agric. J., 103 (1-3): 35-40, March 2016
Detection of Variability among the Strains of Volvariella volvacea

(Bull. Ex Fr.) Sing. and Volvariella bombycina (Schaeff.) Sing.
Using RAPD analysis
Nannapaneni Kiran kumar1, A.S. Krishnamoorthy*2 and D. Amirtham3

1&2
Department of Plant Pathology,
3
Department of Food and Agricultural Process Engineering, AEC & RI,
Tamil Nadu Agricultural University, Coimbatore-641 003, India.
A total of 9 strains consisting of both V. volvacea (5 cultivated strains viz., CBE TNAU 1401 to
1405; 2 wild strains viz., CBE TNAU 1505 and 1516) and V. bombycina (one cultivated strain CBE
TNAU 1406 and one wild strain CBE TNAU 1504) were evaluated to ascertain the variation in
their morphological, cultural and molecular characters. Among strains tested, CBE TNAU 1505
was the best strain, which showed earliness in spawn run, more biomass production in unit time
and more chlamydospores production with high density. In the trials conducted with partially
decomposed paddy straw, the highest yield of 1041.2 g per 5 kg of substrate with an average
ELRORJLFDOHI¿FLHQF\RISHUFHQWZDVUHFRUGHGZLWK&%(71$80ROHFXODUGHWHFWLRQRI
9ROYDULHOODVWUDLQVWKURXJK6U'1$UHJLRQVVKRZHGWKDW,76SULPHUVKDGDPSOL¿HGDSURGXFW
size between 700 and 740 bp. RAPD - PCR phylogenetic analyses revealed 2 major clusters.
V. volvacea strains showed approximately 13 per cent genetic similarities with the strains of
V. bombycina.
Key words: V. volvacea, V. bombycina, Chlamydospore, Morphology, Strains, ITS and RAPD
Indian mushroom industry is witnessing a fabulous variations occur in the isolates of same strains and
change in recent years with respect to the types and needs efficient screening to obtain the isolates
strains cultivated (Krishnamoorthy, 2014). Volvariella with desired attributes. Keeping this background,
belonging to the family Pluteacea (Kolt and Pouz) of the present study aimed to explore the best strain
Phylum Basidiomycotina ranks sixth amongst the of Volvariella and to detect the variation in their
cultivated mushrooms accounting to about 5 per cent morphological growth characteristics, yield potential
of the total world mushroom production. China has and genetic relatedness using RAPD.
produced 3, 30,000 tons accounting for more than
0DWHULDOVDQG0HWKRGV
80 per cent of global production (Bao et al., 2013).
Volvariella volvaceae (Bull. Ex Fr.) Sing. is known as All experiments were conducted at Mushroom
paddy straw mushroom or Chinese mushroom that Research and Training Centre, Department of Plant
grows in tropical and sub-tropical regions. Although Pathology, Coimbatore during 2014-15.
V. volvacea has been cultivated for 300 years, Collection of cultures
multiple problems associated with the practice have
greatly restricted the development of paddy straw In the attempt to conduct the experiment, 6
mushroom industry. In India, Krishnamoorthy et al. strains of paddy straw mushroom fungus (Volvariella
(2005) developed a new conscionable approach by volvacea) viz., CBE TNAU 1401 to 1405 and
cultivating paddy straw mushroom as an intercrop V. bombycina strain CBE TNAU 1406 used in the
LQPDL]H¿HOGVZLWKDQDYHUDJHELRORJLFDOHI¿FLHQF\ study were collected from the germplasm bank
of about 8.75 per cent and explored the possibility of Mushroom Research Laboratory, Tamil Nadu
of introducing this mushroom as an intercrop in Agricultural University, Coimbatore, India. The wild
high temperature and high humid agro eco system. strains namely, CBE TNAU 1504, 1505 and 1516
Although, Volvariella is an aggressive colonizer of used were isolated from various habitats of Tamil
any cellulosic substrate, its competitive saprophytic Nadu such as dead wood of Ceiba pentandra (Theni),
ability, hydrolytic enzyme production potential is compost of paddy straw (Perundurai) and paddy straw
very poor. Cellulases play a critical role during waste (Coimbatore).
substrate colonization while, laccase dominates In vitro testing of growth and biomass production
during sporophore development. These enzymes
were found to be expressed differently with different The strains were maintained on Potato Dextrose
strains of V. volvacea (Ahlawat et al., 2005). However, Agar (PDA) medium in Petri dishes and 100 ml
RI 3RWDWR GH[WURVH EURWK LQ PO FRQLFDO ÀDVNV
*Corresponding author email: [email protected] The media was sterilized at 121°C for 30 min in an
171
36
DXWRFODYH(DFKÀDVNDQG3HWULGLVKZDVLQRFXODWHG paper and dried. One g of the dried mycelial mat

with 9 mm mycelial discs of strains and incubated at ZDV JURXQG WR ¿QH SRZGHU XVLQJ OLTXLG QLWURJHQ
32 + 2°C for seven days. The radial mycelial growth, Powdered mycelia were vortexed in pre-cooled
colony characters, formation of aerial hyphae and CTAB buffer (N-cetyl-N, N, N trimethyl ammonium
the intensity of chlamydospores were recorded for bromide 2 g, 0.1 M Tris Hcl (pH: 8.0), 1.4 M NaCl,
every 24 h. In order to maintain the vigour, fresh 0.5 M EDTA (pH: 8.0), 1 g polyvinyl pyrolidone (PVP),
isolations were made from the fruiting bodies every 1 ml mercaptoethanol and 1 g of sodium sulphite
time after 2 to 3 subcultures. For this purpose the and incubated at 65°C for 30 min followed by the
strains were propagated in straw spawn and grown addition of 750 μl of chloroform and isoamylalcohol
on paddy straw following the method suggested by (24:1 v/v). The contents were gently mixed to form
Thomas et al. (1943). To measure the quantity of an emulsion by inverting the tube for 4-5 times and
biomass produced, the mycelial mats that had grown then centrifuged at 10,000 rpm for 10 min. The upper
RQ SRWDWR GH[WURVH EURWK ZHUH ¿OWHUHG VHSDUDWHO\ aqueous phase (300 μl) was taken without disturbing
WKURXJK SUHZHLJKHG :KDWPDQ 1R ¿OWHU SDSHU the inter phase in a fresh 1.5 ml microfuge tube and
oven dried at 55 to 60°C for constant weight and added with half the volume of 5 M NaCl and two times
WKH ¿QDO ZHLJKW ZDV GHWHUPLQHG E\ DVVHVVLQJ WKH the volume of ice cold ethanol. The contents were
weight difference. Aerial mycelial growth, colony mixed well and incubated at -20°C for overnight. The
characters, days taken for chlamydospores formation contents were centrifuged at 13,000 rpm at 4°C for 10
and the density were recorded by visible observation. min; the ethanol fraction was decanted and the DNA
Micrometric observation on the diameter of hyphae pellet was air dried. Later, the pellet was resuspended
and chlamydospore were observed with the help of in 50 μl of Tris-EDTA buffer (10 mM Tris-HCl and 1mM
image analyzer (N-400T, Optika, Italy). EDTA, pH 8.0). The genomic DNA was checked by
Evaluation of yield potential
using 0.8 per cent agarose gel electrophoresis and
stored at -20°C for further use.
In order to evaluate the yield potential, circular
$PSOL¿FDWLRQRI6U'1$
compact bed method was followed by using paddy
straw based spawn (Sangeetha, 2002) with well The Polymerase Chain Reaction (PCR)
performing 6 strains i.e. CBE TNAU 1401, 1402, DPSOL¿FDWLRQ ZDV SHUIRUPHG E\ XVLQJ WKH SULPHUV
1404, 1406, 1505 and 1516. Yield potential of the ITS 1 (5’-TCC GTA GGT GAA CCT GCG G-3’) and
VHOHFWHGVWUDLQVZHUHFRQ¿UPHGE\FRQGXFWLQJWZR ITS 4 (5’-TCC TCC GCT TAT TGA TAT GC -3’) to
trials i.e., trial 1 during September - October, 2014 amplify the ITS region along with 5.8S rDNA. PCR
and trial 2 during December – January, 2015. Steam was undertaken in 30 μl reaction mixture containing
sterilized paddy straw twists of 2.5 m length and 5 3 μl of template DNA (50 ng), 15 μl of master mix
to 8 cm diameter; each twist weighing 1.25 kg was (Bangalore Genei, Pvt. Ltd., Bangalore, India), 3 μl
used for bed preparation. The twists were presoaked each of ITS primers (0.01 mM) and 6 μl of nuclease
in cold water for about 24 h and steam sterilized at free water (Fermentas, USA). The reactions were
1.46 kg / cm2 for 1 h. Later, they were shade dried performed in Eppendorf gradient S Master cycler
to get 65 to 75 per cent moisture. The twists were (Eppendorf, Hamburg, Germany). Cycling conditions
compactly placed clockwise in a circular fashion as were 94°C for 5 min followed by 36 cycles at 95°C for
FORVHDVSRVVLEOHRQDZRRGHQSODQNWRPDNHWKH¿UVW 1 minute, 50°C for 30 seconds, 72°C for 80 seconds
layer. Straw spawn was placed at the periphery of the DQG¿QDOH[WHQVLRQVWHSDW&IRUPLQZLWKOLG
¿UVWOD\HURIWKHEHGDQGJRIDXWRFODYHGKRUVH KHDWLQJRSWLRQDW&7KH3&5DPSOL¿HGSURGXFWV
gram powder was sprinkled over spawn. Second (10 μl) were electrophoretically separated by running
OD\HU ZDV IRUPHG RYHU WKH ¿UVW OD\HU IROORZLQJ WKH the agarose gel prepared in 1x Tris-acetate-EDTA
same procedure but the twist was placed compactly (TAE) buffer (1.2 g in 100 ml of buffer) at 80 V for 1h.
in anticlock wise direction. Similarly, the third and Staining was done with ethidium bromide and the
fourth layers of bed were formed. The size of the bed gel was visualized and photographed using Bio-rad
measured 30 cm diameter and 20 cm height. Total imaging system. Comparison of DNA amplicon length
weight of each bed was 5 kg on dry weight basis. with standard molecular weight marker (100 bp) was
The perfectly prepared bed was pressed tightly and done and conclusions were made.
placed in a poly house for cropping at 32 to 35°C and RAPD analysis
relative humidity of 80 to 85 per cent. Based on the
\LHOGGDWDELRORJLFDOHI¿FLHQF\ZDVFDOFXODWHGEDVHG Multilocus genotyping was performed by RAPD using
on the following formula: BE (%) = Fresh weight of 9 decamer primers, OPA 01(5’-CAGGCCCTTC-3’),
the mushroom yield in kg / quantity of dry substrate OPA 03 (5’-AGTCAGCCAC-3’), OPA 5 (5’-AGG
used x 100. GGTCTTG-3’), OPA 07 (5’-GAAACGGGTG-3’),
O PA 0 9 ( 5 ’ - G G G TA A C G C C - 3 ’ ) , O PA 1 8
Extraction of total genomic DNA
(5’-AGGTGACCGT-3’), OPB 03 (5’-CATCCCCCTG-3’),
Total genomic DNA was extracted following the OPB 11 (5’-GTAGACCCGT-3’) and OPB 15
method suggested by Liu et al. (2000). The mycelial ¶**$***7*77¶$PSOL¿FDWLRQZDVSHUIRUPHG
mats of the 9 Volvariella strains were harvested in 30 μl reaction mixture, containing 6 μl primer (50
E\ ¿OWUDWLRQ WKURXJK GRXEOH OD\HUV RI FRXQWU\ ¿OWHU pM), 15 μl of master mix (Bangalore Genei, Pvt. Ltd.,
172
37
Bangalore, India), 3 μl template DNA (50 ng) and 6 construct the dendrogram employing the Unweighted
μl of nuclease free water (Fermentas, USA). RAPD- Paired Group Method using Arithmetic averages
3&5 DPSOL¿FDWLRQV ZHUH SHUIRUPHG LQ (SSHQGRUI (UPGMA) and the SAHN (Sequential Agglomerative
gradient S Master cycler (Eppendorf, Hamburg, Hierarchical and Nested Clustering) using NTSYS
Germany) with initial denaturation at 94°C for three software. Four replications were maintained for each
min followed by 40 cycles of 94°C for forty seconds, strain in every experiment. The statistical analyses of
&IRUIRUW\VHFRQGVDQG&IRURQHPLQDQG¿QDO all the experiments conducted were laid out based
elongation at 72°C for 10 min with lid heating option at on Completely Randomized Block Design (CRBD)
104°C. The agarose gel was prepared as mentioned (Gomez and Gomez, 1984). Statistical software
above and electrophoresis was performed by using AGRES was used for the analyses of the data. In
standard molecular weight marker (1 Kb). The band case of zero values the data was log transformed
SUR¿OHRIHDFKJHOZDVVFRUHGDVµ¶IRUWKHSUHVHQFH (X+0.5) before statistical analysis.
DQGµ¶IRUDEVHQFHRIEDQGVDQGFRPELQHGELQDU\
Results and Discussion
data matrix for all the markers was constructed. The
data matrix was entered in the numerical taxonomy 6HOHFWLRQRI9ROYDULHOODVWUDLQVEDVHGRQ
morphological characters and yield potential
and multivariate analysis system package NTSYS-
pc programme version 2.2 (Numerical Taxonomy The nine strains of Volvariella, varied in
System, Applied Biostatistics Inc, New York) to their morphological characters such as aerial
estimate similarity indices using SIMQUAL route to mycelial growth, colony characters, earliness of
REWDLQ -DFFDUG¶V VLPLODULW\ FRHI¿FLHQWV -DFFDUGV chlamydospores production and their density on PDA
1908). The similarity coefficients were used to
Table 1. Selection of superior strains of Volvariella spp based on morphological characters
Micrometric
Radial observations#
Bio
growth
Aerial Colony mass Performance Chlamydospores
Strain in DTTCPP* DTFCP* Hyphal Chlamydo
hyphae* morphology in g in broth density*
mm dia spore dia
15 DAI*
(6 DAI)* (μm) (μm)
Depressed
in centre Floating on 16.8e
CBETNAU 1401 90.0a 5.5cd +++ 0.9c ++ 4.8 40.0
and raised surface (4.1)
in margins
Thick Floating on 14.5c

CBETNAU 1402 90.0a 5.2bc ++++ 1.1b +++ 6.4 44.4
Cottony surface (3.8)
Fried egg Floating on

CBETNAU 1403 52.1c 11.4h ++ 0.7ef - NP 3.8 -
like surface
Fried egg
like in centre
Floating on 16.6e
CBETNAU 1404 62.0bc 9.5f +++ and cottony 0.8d + 5.2 43.7
surface (4.1)
towards
margins
Depressed Floating on
CBETNAU 1405 65.7bc 8.6g - 0.7de - NP 3.3 -
and uniform surface
Slightly
yellowish 7.3a
CBETNAU 1406 90.0a 4.9ab + 0.6g Submerged ++++ 5.3 22.5
and raised (2.8)
in centre
Depressed
in centre Floating on 13.8c
CBETNAU 1505 90.0a 4.7a ++++ 1.2a +++ 7.3 24.2
and raised surface (3.7)
in margins
Irregular and 8.5b

CBETNAU 1504 81.0a 5.6d + 0.7f Submerged ++++ 7.1 28.5
raised (3.0)
Greyish and 15.5d

CBETNAU 1516 76.5ab 7.3e + 0.8d Submerged ++ 5.5 28.1
regular (4.0)
CD (P = 0.05) 15.0 0.3 0.05 0.11
Aerial hyphae, chlamydospores density “- to ++++” absent to highly densed.

DTTCPP – Days taken to cover 90 mm Petri plate. NP – not produced.
DTFCP – Days taken for chlamydospores production.
*, # Values are mean of 4 and 25 replications. Data in parenthesis were square root transformed values.
)LJXUHVLQSDUHQWKHVHVDUHORJWUDQVIRUPHGYDOXHV0HDQVIROORZHGE\DFRPPRQOHWWHUDUHQRWVLJQL¿FDQWO\GLIIHUHQWDW3 E\RQHZD\$129$
medium (Table 1 and Fig.1). The highest radial growth virulent (52.1 mm) and took 11.4 d to complete full
of 90 mm was observed in strain CBE TNAU 1505, SODWH 7KH SURGXFWLRQ RI FKODP\GRVSRUHV ZDV ¿UVW
which took 4.7 d to complete full plate, followed by observed in CBE TNAU 1406 in 7.3 d, followed by
CBE TNAU 1406, 1402 and 1401 (4.9 d, 5.2 d and CBE TNAU 1504 in 8.5 d; while, CBE TNAU 1401 took
5.5 d, respectively). The strain CBE TNAU 1403 took 16.8 d. No chlamydospore production was observed
longer time for proliferation and was found to be less in CBE TNAU 1403 and 1405. The strain CBE TNAU
173
38
Table 2. Yield performance of different isolates of Volvariella spp

Trail 1 (September – October, 2014) * Trail 2 (December, 2014 – January,2015) *
Colour No. of Avg. Avg. No. Avg. Avg.
Strain BE BE
of egg DFSR DFPF DFFH eggs weight of yield DFSR DFPF DFFH of eggs weight of yield
(%) (%)
harvested egg (g) (g) harvested egg (g) (g)
CBE TNAU 7.1bc 11.5b 39.1b 22.3ab 874.0b 17.4c 751.8c 15.0c
Greyish 6.5 7.2 8.8 13.2d 36.9c 20.3d
1401 (2.7) (3.4) (6.2) (4.7) (29.5) (25.0) (27.4) (22.8)
CBE TNAU 6.8ab 10.8a 43.3a 22.8a 991.1a 19.8b 876.3b 17.5b
Greyish 5.7 6.2 7.5 12.1b 40.3b 21.6c
1402 (2.7) (3.3) (6.6) (4.8) (31.4) (26.7) (29.6) (24.7)
CBE TNAU 7.8d 12.7c 31.4d 21.4b 674.6d 13.4e 608.4d 12.1d
Greyish 7.1 7.7 9.1 14e 24.7e 24.5a
1404 (2.8) (3.6) (5.6) (4.6) (25.9) (21.9) (24.6) (20.3)
CBE TNAU 32.6e 0.6e

White 10.4 NP NP NP NP NP NP 6.7 10.5 11.2a 2f 16.3e
1406 (5.7) (4.6)
CBE TNAU 6.6a 10.4a 44.9a 23.1a 1041.2a 20.8a 940.7a 18.8a
Greyish 5.2 5.8 7.16 11.1a 42.7a 22.0bc
1505 (2.6) (3.3) (6.7) (4.8) (32.2) (27.4) (30.6) (25.6)
CBE TNAU 7.3c 12.0b 36.1c 22.0ab 797.8c 15.9d 767.9c 15.3c
Greyish 6.8 7.1 8.3 12.8c 33.8d 22.6b
1516 (2.7) (3.5) (6.0) (4.7) (28.2) (23.9) (27.7) (23.0)
CD
NS 0.08 0.09 0.1 0.1 0.8 0.4 NS NS 0.3 1.0 0.8 0.7 0.4
(P =0.05)
DFSR - Days taken for spawn run.

DFPF - Days taken for pinhead formation.
'))+'D\VWDNHQIRU¿UVWKDUYHVW
%(%LRORJLFDOHI¿FLHQF\7KHGDWDLQSDUHQWKHVLVDUHDUFVLQHWUDQVIRUPHGYDOXHV
9DOXHVDUHPHDQRIIRXUUHSOLFDWLRQV0HDQVIROORZHGE\DFRPPRQOHWWHUDUHQRWVLJQL¿FDQWO\GLIIHUHQWDW3 E\RQHZD\$129$'DWD
in parenthesis were square root transformed values.
SURGXFHG VLJQL¿FDQWO\ PRUH ELRPDVV J In the present investigation, the yield potential
on 15 DAI under in vitro. This was followed by CBE of six strains which performed better under in vitro
TNAU 1402 and 1401 (1.0 and 0.9 g), whereas CBE studies i.e., CBE TNAU 1401, 1402, 1404, 1406,
TNAU 1406 produced less biomass of only 0.6 g. The 1505 and 1516 was evaluated by conducting two
Fig. 1. Cultural characters of Volvariella strains independent trials (one in September to October,
on PDA medium 2014 and the other in December 2014 to January,
Figure 1. Cultural characters of Volvariella strains on PDA medium
Fig. 2. Fruiting bodies of Volvariella strains grown
on paddy straw
CBE TNAU 1401 CBE TNAU 1402
CBE TNAU 1401 CBE TNAU 1402 CBE TNAU 1403


mycelial mats had grown very well over the surface CBE TNAU 1404 CBE TNAU 1516
of the broth except in case of CBE TNAU 1406, 1504
and 1516 where the mats were partially submerged
(Table 1). Days taken for chlamydospore production
and the density were recorded by the presence of
pink coloured colonies in the Petriplate however, the
procedure to accurately measure the chlamydospore
density based on optical density is lacking and future
studies in this context are warranted. Chang et al, 2015) on partially decomposed paddy straw. Among
(1981) corroborated that monosporous isolates of the strains evaluated during trial 1, CBE TNAU 1505
paddy straw mushroom varied in their growth rate, DQG&%(71$8SHUIRUPHGVLJQL¿FDQWO\VXSHULRU
abundance of mycelia, aerial hyphae and presence and found to be on par by producing the maximum
of chlamydospores. They divided isolates of paddy \LHOGVDQGELRORJLFDOHI¿FLHQFLHVRIJSHU
VWUDZPXVKURRPLQWR¿YHGLIIHUHQWJURXSVµ11¶µ1¶ cent and 991.1 g, 19.8 per cent per 5 kg of substrate,
µ$1¶ µ$¶ DQG µ$$¶ EDVHG RQ WKHLU PRUSKRORORJLFDO respectively. This was followed by CBE TNAU 1401
characteristics. (874.0 g and 17.4 per cent) and CBE TNAU 1516
174
39
(797.8 g and 15.9 per cent). Minimum yield and Fig. 4 b. Dendrogram of molecular phylogenetic
ELRORJLFDOHI¿FLHQF\ZDVUHFRUGHGZLWK&%(71$8 relationship of Volvariella strains
1404 (674.6 g and 13.4 per cent). CBE TNAU 1406
VWUDLQZDVVLJQL¿FDQWO\GLIIHUHQWIURPDOO)LJ
Fig. 3. PCR amplification of ITS regions of
Volvariella strains
700- 740
V. volvacea strains CBE TNAU 1401, 1402,

1404, 1505 and 1516 are adapted to wide varied
temperatures between 29 and 36°C and produced
better yields; whereas, V. bombycina strain produced
M - 1 kb ladder 4 - CBE TNAU 15058 - CBE TNAU 1504
a rudimentary fruiting body at the temperature
1 - CBE TNAU 1401 5 - CBE TNAU 14049 - CBE TNAU 1516 between 26 and 31°C. According to Datta and
2 - CBE TNAU 1402 6 - CBE TNAU 1405 Chakravarty (2002) V. volvacea was found to utilize
3 - CBE TNAU 1403 7 - CBE TNAU 1406 cellulose and hemicellulose throughout spawn run
3&5DPSOL¿FDWLRQRI,76UHJLRQVRI9ROYDULHOODVWUDLQV and was unable to utilize lignin at any stage due to
the strains in all parameters and recorded no yield. lack of lignolytic system. Substrates preference by
In the second trial, all the strains except CBE TNAU V. volvacea and V. bombycina, respectively could
1406 were found to be on par in terms of yield. CBE be well understood from the fact that V. volvacea
TNAU 1406 produced minimum yield of only 32.6 g collected from partially fermented straw and
per 5 kg substrate (Table 2 and Fig.2). V. bombycina from the dead wood of Ceiba pentandra.
,QFRQ¿UPLW\ZLWK6LQJKV. bombycina requires
Fig. 4 a. RAPD profile of Volvariella strains
the temperature of 26 to 30°C for mycelial growth and
DPSOL¿HGZLWKGLIIHUHQWSULPHUV
fruiting. This lucidly indicates that low temperature
OPB 03 OPA 07
M 1 2 3 4 5 6 7 8 9 M M 1 2 3 4 5 6 7 8 9 M and lignicolous substrate are suitable for extensive
growth and fruiting of V. bombycina.
Molecular variation and genetic relatedness of
Volvariella strains using RAPD
PCR amplification of 5.8S rDNA region was

performed with the nine strains of Volvariella sp.
M 1
OPA 03
2 3 4 5 6 7 8 9 M M 1 2 3
OPB 11
4 5 6 7 8 9 M by using ITS 1 and ITS 4 primers. The DNA of nine
VWUDLQVDPSOL¿HGDSSUR[LPDWHO\EHWZHHQES
(Figure 3). 2 parent and 15 monosporous isolates of
V. volvaceaDPSOL¿HGDSSUR[LPDWHO\ESSURGXFW
in all the strains (Ahlawat et al., 2010). Though
the amplification was observed at 700-740 bp,
M - 1 kb ladder 4 - CBE TNAU 1505 8 - CBE TNAU 1504
comparing the scrupulous results of earlier workers it
1 - CBE TNAU 1401
2 - CBE TNAU 1402
5 - CBE TNAU 1404
6 - CBE TNAU 1405
9 - CBE TNAU 1516
LVFRQFOXGHGWKDWDPSOL¿FDWLRQRI,76DQGUHJLRQV
3 - CBE TNAU 1403 7 - CBE TNAU 1406 alone will not provide cent per cent agreement to
The data presented in Table 1 and 2 revealed that FRQ¿UP WKH SK\ORJHQHWLF UHODWLRQVKLS EHWZHHQ WKH
the strain, CBE TNAU 1505 had exhibited the highest tested strains.
growth with plenty of aerial hyphae, chlamydospores
To study the molecular variation and genetic
density and yield performance with more biological
relatedness among the nine strains, RAPD was
HI¿FLHQF\ IROORZHG E\ FXOWLYDWHG VWUDLQ &%( 71$8
performed by using 9 random decamer primers, out
1402. Ahlawat et al. (2010) disclosed that there was
of which 4 primers produced no bands in most of the
no relationship between chlamydospores formation,
VWUDLQV 7KH '1$ IUDJPHQWV VKRZHG DPSOL¿FDWLRQ
rate of mycelial growth and yield. In a separate study,
with different molecular weights up to 2000 bp (Figure
Ahlawat et al. (2014) also screened ten strains of
D5HSURGXFLELOLW\RIWKHDPSOL¿FDWLRQSDWWHUQZDV
V. volvacea, among which the fastest growing strain
FRQ¿UPHG LQ WZR LQGHSHQGHQW H[SHULPHQWV %DVHG
OSM-1 exhibited the highest activity of exoglucanase,
on the analysis made from RAPD-PCR banding
low level of endoglucanase and increased levels
patterns, all the nine strains of Volvariella were
RI ȕJOXFRVLGDVH DQG [\ODQDVH7KLV SHUVSLFXRXVO\
grouped into 2 major clusters A and B (Figure 4b)
indicates that strain CBE TNAU 1505 may have
with V. volvacea strains CBE TNAU 1401, 1402,
exhibited more activity of hydrolytic enzymes on
1404, 1405, 1505, 1403 and 1516 in cluster A; V.
comparison to other strains.
175
40
bombycina strains CBE TNAU 1504 and 1406 in References

cluster B. The cluster A is again divided into 2 sub
$KODZDW23$KODZDW.DQG'KDU%/,QÀXHQFH
clusters A1 and A2. Sub cluster A2 consists of CBE of lignocellulolytic enzymes on substrate colonization
TNAU 1402, which was approximately 13.8 per cent and yield in monosporous isolates and parent strains
similar with the strains of A1. Cluster B consists of of Volvariella volvacea (Bull. Fr.) Sing. Indian J.
V. bombycina strains CBE TNAU 1504 and CBE Microbiol., 45(3): 205-210.
TNAU 1406 at 19 per cent similarity, which were Ahlawat, O. P., Pardeep, G., Kamal, S. and Dhar, B. L. 2010.
approximately 13 per cent similar with that of strains 9DULDELOLW\LQLQWUDVSHFL¿FDQGPRQRVSRURXV,VRODWHV
in cluster A. Analysis of the RAPD showed minimum of Volvariella volvacea based on enzyme activity, ITS
and maximum per cent similarities among the strains and RAPD. Indian J. Microbiology., 50(2): 192-198.
of Volvariella, which were in the range between 13 Ahlawat, O.P., Mohapatra, K.B., Kaur, H. and Singh, M.
and 27.5 per cent, respectively. Whereas, Ahlawat 2014. Genetic variability in strains of Volvariella
volvacea collected from the state of Odisha. In:
et al.SXUSRUWHGLQWUDVSHFL¿FYDULDWLRQRI
Proceedings of the 8th International Conference on
per cent within the 2 parent strains and their single Mushroom Biology and Mushroom Products., pp.
spore isolates of V. volvacea. Whole genome of 135-144.
V. volvacea with a total genome size of 35.7 Mb Bao, D., Gong, M., Zheng, H., Chen, M., Zhang, L., Wang,
was sequenced by Bao et al. (2013). This provided H., Jiang, J., Wu, L., Zhu, Y., Zhu, G., Zhou, Y., Li,
explicit understanding of molecular levels involved C., Wang, S., Zhao, Y., Zhao, G. and Tan, Q. 2013.
in sexual reproduction mechanism, degradation of Sequencing and Comparative Analysis of the Straw
compost and the sensitivity to low temperatures by Mushroom (Volvariella volvacea) Genome. PLoS
V. volvacea. Commercial exploitation and application ONE., (3): e58294.
of this genome information may be used to improve Chang, S.T. 1969. A cytological study of spore germination
the chlamydospores production, yield and shelf life of Volvariella volvacea. Bot. Mag. Tokyo., 82: 102-109.
of Volvariella sp by regulating the responsible genes. Chang, S.T., Miles, P.F. and Wai, C.C. 1981. A study
The pattern of distribution of genotypes into two of monosporous isolates of Volvariella volvacea.
broad clusters at random indicated that geographical International Journal for mushroom science., 11(2):
and genetic diversity were not related. This further 603-621.
suggested that forces other than geographical origin Datta, S. and Chakravarty, D. K. 2002. Comparative
VXFKDVJHQHWLFGULIWQDWXUDODQGDUWL¿FLDOVHOHFWLRQ utilisation of lignocellulosic components of paddy
exchange of cultivars might have played an important straw by Pleurotus sajor-caju and Volvariella volvacea.
Indian Phytopath., 55(3): 308-309.
role in the evolution of diversity of tested strains.
Variations in environment could also be responsible Gomez, K. A. and A. A. Gomez. 1984. Statistical procedures
for Agriculture research. John Wiley & sons. Inc., New
for such diversity. The RAPD data presented here
York, p. 680.
confirmed that, the genetic similarity among the
Jaccards, P. 1908. Nouvelles recherches sur la distribution
strains exists and the level of similarity is medium.
ÀRUDOH%XOO6RF9DXGRLVH6FL1DW44: 223.
High yielding and chlamydospores producing wild
Krishnamoorthy, A.S. 2014. Biodiversity exploration of
strain of V. volvacea CBE TNAU 1505 was found to
Milky mushroom (Calocybe indica P.) and - Concept
be genetically 27.5 per cent similar to wild strain CBE to commercialization, In: Proceedings of the 8th
TNAU 1516. Varied conscientious reports indicate International Conference on Mushroom Biology and
that the DNA fingerprints showed polymorphism Mushroom Products, pp;490-495.
between strains of V. volvacea and V. bombycina. Krishnamoorthy, A. S., Thiribhuvanamala, G., Shanthi,
Systematic investigations in this context may give K. and Marimuthu, T. 2005. Outdoor cultivation of
lead for exploration of elite Volvariella strains. SDGG\VWUDZPXVKURRPDVLQWHUFURSLQPDL]H¿HOG
Mushroom Res., 14(1): 9-12.
Conclusion Liu, D., Coloe, S., Baird, R. and Pederson, J. 2000. Rapid
From this concerted study, it was straight mini-preparation of fungal DNA for PCR. J. Clin.
Microbiol., : 471.
laced that Volvariella strains showing variation in
morphological and cultural characteristics also Sangeetha, G. 2002. Exploring the possibilities of increasing
the yield potential of paddy straw mushroom,
VKRZHG YDULDWLRQ LQ WKHLU 5$3' SUR¿OHV UHYHDOHG
Volvariella volvacea (Bull. ex Fr.) Sing. M. Sc.
WKURXJK SK\ORJHQHWLF DQDO\VLV 7KH VWUDLQ VSHFL¿F (Ag.) Thesis, Tamil Nadu Agricultural University,
RAPD bands of high yielding Volvariella strains could Coimbatore, India. p.114.
be utilized to identify the phylogenetic belongingness. Singh. M. 2014. Culture viability, commercial scale
Acknowledgement cultivation and shelf life studies on the silver-silk straw
mushroom, Volvariella bombycina. In: Annual report,
The authors express their gratitude to Department DMR, pp, 2-16.
of Plant Pathology, TNAU, Coimbatore and ICAR- Thomas, K. M., Ramakrishan, T. S. and Narsimhalu, I. L.
AICRP on mushroom for the valuable support 1943. Paddy straw Mushroom. Madras Agric. J., 31:
rendered during the course of investigation. 57-59.
Received after revision: February 12, 2016; Accepted: March 28, 2016
176
International Journal of Agriculture Sciences
ISSN: 0975-3710&E-ISSN: 0975-9107, Volume 8, Issue 37, 2016, pp.-1759-1762.
Available online at http://www.bioinfopublication.org/jouarchive.php?opt=&jouid=BPJ0000217
Research Article
MOLECULAR DYNAMICS OF MORPHOGENESIS IN Volvariella volvacea (BULL. EX FR.) SING
KIRANKUMAR N.*, KRISHNAMOORTHY A.S., KAMALAKANNAN A. AND AMIRTHAM D.

Mushroom Research Laboratory, Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore-641003, Tamil Nadu, India
*Corresponding Author: [email protected]
Received: May 16, 2016; Revised: May 23, 2016; Accepted: May 25, 2016; Published: September 18, 2016
Abstract- Mannitol dehydrogenase, tyrosinase and water activity (a w) were found to be variously expressed during morphogenesis of V. volvacea. Very less enzymatic
activity was recorded at pinhead stage, which increased with progression of growth. In different parts of mushroom maximum activities of mannitol dehydrogenase,
tyrosinase, browning and water activity were found in pileus followed by volva and stipe. Mannitol dehydrogenase was found to be involved in morphogenesis whereas
tyrosinase was found to be involved during senescence and browning of the sporophores of V. volvacea. Browning degree, water activity and tyrosinase activities are
presumably conjugated and are responsible for the senescence of the mushroom. Further studies involving the molecular interacti on of these enzymes in the
developmental stages is most warranted for commercial marketing of mushrooms.
Keywords- Browning, enzymes, mannitol dehydrogenase, tyrosinase, water activity.
Citation: Kirankumar N., et al., (2016) Molecular Dynamics of Morphogenesis in Volvariella volvacea (Bull. Ex Fr.) Sing.. International Journal of Agriculture Sciences, ISSN:
0975-3710 & E-ISSN: 0975-9107, Volume 8, Issue 37, pp.-1759-1762.
Copyright: Copyright©2016 Kirankumar N., et al., This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.
Academic Editor / Reviewer: Mishra Ved Kumar, Ansari Rizwan Ali, Singh Priyanka
Introduction volvacea.
Mushroom fungi are major chunk of nutritious healthy foods, which are eulogized
throughout the world both as food and as medicine for thousands of years. Among Materials and Methods
the edible mushrooms, gourmet species such as Volvariella volvacea (Bull. Ex Fr.) V. volvacea strain CBE TNAU 1505 obtained from the germplasm bank of
Sing, known as paddy straw mushroom belonging to the family Pluteacea (Kolt Mushroom Research Laboratory, Tamil Nadu Agricultural University, Coimbatore,
and Pouz) of Phylum Basidiomycetes, ranks sixth amongst the cultivated Tamil Nadu, India was used in this experiment. It was maintained on 90 mm petri
mushrooms; accounting to about 5 per cent of the total world mushroom dishes with potato dextrose agar medium at 32+ 20C.
production. Cellulases play a critical role during substrate colonization while,
laccase dominates during sporophore development. These enzymes were found Spawn preparation and crop rising
to be expressed differently with different strains of V. volvacea [1]. Mannitol is the Paddy straw-based spawn was prepared by following the method suggested by
most common polyol, found in large quantities in spores, fruiting bodies, sclerotia Krishnamoorthy et al. [14]. Fresh, good quality paddy straw bits (1-2 inches) were
and mycelia contributing to about 20 per cent of the mycelium. This sugar alcohol soaked in water for 6 h and dried under shade up to 65% moisture content. The
increases dramatically to 30-50 per cent in differentiating sporophores [23, 27]. paddy straw substrate was supplemented with horse gram powder @ 2% (dry
In mushroom fungi, mannitol was purported to function as an osmoregulator, weight basis) and the contents were packed in polypropylene bags and
which encouraged influx of water from the environment to develop turgor pressure autoclaved at 1.46 Kg/cm2 for 90 min. On the next day 90 mm mycelial mats of 7
thereby helped in fruiting body development [3, 12]. It also acted as a respiratory days old culture was propagated and the bags were incubated at room
substrate during the post-harvest storage and senescence of mushroom [10]. temperature. Fifteen days old spawn with well-developed chlamydospores were
Earlier, mannitol-1-phosphate dehydrogenase (M1PDH), a zinc-containing long- used to seed the beds. For bed preparation, circular compact bed method was
chain alcohol dehydrogenase was thought to be limited to Zygomycetes and followed [24]. In which, golden yellow, well-dried, good quality paddy straw was
Ascomycetes. However, their activities were reported in Basidiomycetes such as, made into small twists of 2.5 m length and 5 to 8 cm diameter; each twist weighing
Pleurotus ostreatus and Cryptococcus neoformans [4, 28]. 1.25 kg. After draining the excess water, the twists were pre-soaked in cold water
Sporocarps of Volvariella are perishable and tend to lose their appearance due to for about 24 h and steam sterilized at 1.46 kg/cm2 for 1 h. Later, the paddy straw
short shelf life, a ravaging impediment to the distribution and marketing of the twists were shade dried to get 65 to 75% moisture. The twists were compactly
fresh mushrooms. Extension of the quality and shelf life is therefore a scientific, placed clockwise in a circular fashion as close as possible on a wooden plank to
technical and economical challenge. Tyrosinase, a glycosylated cytosolic copper- make the first layer. Eight paddy straw spawn bits each weighing 25 g were placed
containing monooxygenase belonging to poly phenol oxidase family, is the at the periphery of the first layer of the bed leaving equal distance between them.
principal enzyme for the synthesis of melanin pigment from tyrosine, which is Over the spawn bits 20 g of pre sterilized horse gram powder was sprinkled.
responsible for enzymatic browning during development and postharvest storage Second layer was formed over the first layer following the same procedure but the
[6, 22]. Keeping this information, the present study is thus aimed to emphasize an twist was placed compactly in the anticlockwise direction. Similarly, the third and
arrant knowledge on enzymatic dynamics involved in the morphogenesis of V. fourth layers of bed were formed. The size of the bed measured 30 cm diameter

ISSN: 0975-3710&E-ISSN: 0975-9107, Volume 8, Issue 37, 2016
|| Bioinfo Publications || 177 1759
Molecular Dynamics of Morphogenesis in Volvariella volvacea (Bull. Ex Fr.) Sing
and 20 cm height. Total weight of each bed was 5 kg on dry weight basis. The Whereas, stage 1 recorded poor activities of MtDH (1.23) [Table-1]. The rate of
perfectly prepared bed was pressed tightly and placed in a poly house for increase in activity of MtDH. was higher from stage 1 to 4 when compared to stage
cropping. Appropriate room temperature inside the cropping room ranged from 32 5 and 6 [Fig-2]. Pileus recorded the maximum activity of the enzyme (1.80),
to 350C and the relative humidity of 80 to 85% was maintained to prevent followed by stipe (1.74) and volva (1.66) [Table-2].
desiccation of young buttons. The sporocarps of the fungus at different growth
stages were collected separately as described by Chakraborty et al. [5] and used
for morphogenesis related enzyme assay.
Assay of mannitol dehydrogenase (MtDH) (EC 1.1.1.138)

Mannitol dehydrogenase was assayed as suggested by Chakraborty et al. [4] with
some modifications. The fresh mushroom tissue was extracted with 20 mM
HEPES (Hydroxyethyl piperazin ethane sulfonic acid) KOH buffer (pH 7.5)
containing 1 mM EDTA, 2 mM 2- mercapto ethanol and 2 mM PMSF (Phenyl
Methyl Sulfonyl Fluoride). Unbroken cells, and cell debri were removed by
centrifugation at 32,000 rpm for 60 min at 4°C and the supernatant was used as
the cell extract. MtDH activity was determined by incubating the cell extract at Fig-1 Morphogenesis of Volvariella
25°C in 20 mM HEPES-KOH (pH 7.5) - 500 mM fructose - 0.25 mM NADPH. The
enzyme activity was monitored by recording the change in absorbance at 340 nm.
Assay of tyrosinase (EC 1.14.18.1)

The tissue was macerated with phosphate buffer (pH 6.8) at 1:1 (w/v) ratio. The
extract was collected and centrifuged at 15,000 rpm for 30 min at 4°C. The
supernatant was used as enzyme source. Assay was conducted with the reaction
mixture, total volume of 3.0 ml: 3 mM-L-tyrosine, 47 mM-sodium phosphate buffer
(pH 6.8) and 25 μl of enzyme source. The rate of formation of dopachrome was
measured at 475 nm. The control was kept by adding the same reaction mixture
without enzyme source. The enzyme activity was expressed as change in
absorbance at 470 nm.
Expression of Tyrosinase
Determination of water activity Tyrosinase is an enzyme that belongs to PPO family and its expression was
The harvested mushrooms were sorted into six stages of maturity viz., pinhead, significantly different at all stages of morphogenesis. It is expressed as change in
tiny button, button, egg, elongation and maturation stages. Sample of 3 g of absorbance at 475 nm. Stage 6 registered significantly high levels of tyrosinase
freshly harvested mushroom tissue was placed in cups of Aqua lab series water activity of 0.19, followed by stage 5 recording 0.156 and stage 4 (0.11). Whereas,
activity meter for 1 min at 30°C and the water activity was determined and stage 1 showed less tyrosinase activity (0.042) as shown in [Table-1] and [Fig-3].
expressed in aw. When tested with different parts of the mushroom, high tyrosinase activity was
recorded in pileus (0.13) followed by volva (0.10) and stipe (0.091) [Table-2].
Measurement of browning
The browning degree of V. volvacea was evaluated by a spectrophotometric
method described by Zhou et al. [29] with some modification. A sample containing
4 g of mushroom sample was ground with 8 ml of 0.2 M phosphate buffer solution
(pH 6.8) in an ice bath. After centrifugation at 10000 rpm for 20 min at 4oC, the
browning degree was determined by measuring the absorbance of the
supernatant at 420 nm using a UV-vis spectrophotometer (GS5703AT).
Statistical Analysis
In order to avoid experimental errors the enzyme assays were carried out for five
times and the results presented are mean values ± standard deviations. Statistical
software AGRES was used for the data analysis. In case of zero values the data
was log transformed (X+0.5) before statistical analysis.
Browning degree
Results Browning of mushroom is an important senescence related factor that causes
As the enzymes like mannitol dehydrogenase and tyrosinase are related to economical loss. In this study, degree of browning at different growth stages was
morphogenesis and shelf life at different growth stages of mushrooms viz., stage 1 evaluated by spectrophotometric method. Increased level of browning was noticed
(pin head), stage 2 (tiny button), stage 3 (button), stage 4 (egg), stage 5 at stage 6 (1.08) that was statistically different from all other stages. This was
(elongation) and stage 6 (maturity) [Fig-1], the present study was undertaken to followed by stage 5 (0.81) and stage 4 (0.63). Stage 1 recorded very less
find out their level of expression at different growth stages of V. volvacea strain browning activity (0.19). Browning is recorded at all stages of maturity and it was
CBE TNAU 1505. found to be accelerated at late stages i.e., after opening of the pileus [Table-1]
and [Fig-4]. When tested with different parts of mushroom, the degree of browning
Expression of mannitol dehydrogenase (MtDH) was found to be significantly high in pileus (0.98) followed by volva (0.49) and
Mannitol dehydrogenase activity was spectrophotometrically assayed at all the stipe (0.34), respectively [Table-2].
growth stages of mushroom. Enzyme activity was expressed as change in
absorbance at 340 nm. The results indicated that stage 6 and stage 5 are on par Water activity
and expressed more activity of MtDH (1.97, and 1.88), followed by stage 4 (1.66). The water activity was found to be significantly higher at stage 4 to 6 (0.976, 0.983

Kirankumar N., Krishnamoorthy A.S., Kamalakannan A. and Amirtham D.
and 0.997 aw), respectively. This was followed by stage 3 (0.947 a w). Minimum
water activity was recorded at stage 1 (0.881 a w) [Table-1] and [Fig-5]. There was
no significant difference between the water activity of pileus, volva and stipe
[Table-2].
Discussion
Volvariella volvacea is a large pileate fungus with dark grey basidiocarps, which
are roughly divided into six stages of growth namely, pinhead, tiny button, button,
egg, elongation and mature stages; each stage having its own morphological and
anatomical characteristics [7].
Table-1 Enzymatic dynamics during the morphogenesis of V. volvacea

Stage Mannitol Tyrosinase activity* Browning degree (OD420)* Water activity (aw) at 300C*
dehydrogenase activity*
Pinhead 1.23 ± 0.28d 0.042 ± 0.0060f 0.19 ± 0.012f 0.88 ± 0.010c
Tiny button 1.47 ± 0.54c 0.061 ± 0.0035e 0.30 ± 0.047e 0.89 ± 0.026c
Button 1.59 ± 0.33b 0.097 ±0.0024d 0.44 ± 0.039d 0.94 ± 0.022b
Egg 1.66 ± 0.25b 0.11 ± 0.063c 0.63 ± 0.080c 0.97 ± 0.014a
Elongation 1.88 ± 0.19a 0.15 ± 0.030b 0.81 ± 0.014b 0.98 ± 0.011a
Maturity 1.97 ± 0.30a 0.19 ± 0.060a 1.08 ± 0.048a 0.99 ± 0.021a
CD (P = 0.05) 0.10 0.0030 0.024 0.024
*Data are expressed as mean ± Standard deviation (n=5). Mannitol dehydrogenase and tyrosinase activity; browning degree are e xpressed as change in absorbance at 340, 470 and 420 nm,
respectively. Water activity is measured at 30 0C. Means in a column followed by the same letter are not significantly different at P = 0.05 by one way ANOVA.
Table-2 Enzymatic activities in the morphological parts of V. volvacea

Part of mushroom Mannitol Tyrosinase activity* Browning degree* Water activity (aw)*
dehydrogenase activity*
Volva 1.66 ± 0.28c 0.10 ± 0.021b 0.49 ± 0.031b 0.96 ± 0.025
Stipe 1.74 ± 0.41b 0.091 ± 0.007c 0.34 ± 0.053c 0.95 ± 0.051
Pileus 1.80 ± 0.17a 0.13 ± 0.014a 0.98 ± 0.028a 0.99 ± 0.020
CD 0.048 0.0036 0.031 NS
(P = 0.05)
*Data are expressed as mean ± Standard deviation (n=5). Mannitol dehydrogenase and tyrosinase activity; browning degree are e xpressed as change in absorbance at 340, 470 and 420 nm,
respectively. Water activity is measured at 30 0C. Means in a column followed by the same letter are not significantly different at P = 0.05 by one way ANOVA.
The characteristic feature of genus Volvariella is opening of basidiocarp before full with considerable change in carbohydrate metabolism during flushing. This
maturity (hemi-angiocarpous) with the universal veil. The aerial hyphae aggregate fascinatingly insinuates that greater participation of metabolic pathways is
into knots on mycelial cultures and are known as pinheads. Under favourable certainly responsible for enzymatic activities during the morphogenesis of V.
conditions, these primordia (pinheads) would grow into button stage and then to volvacea.
commercially marketable egg stage. Mannitol, a six carbon polyol accumulates in In mushrooms, browning is the kenspeckle quality factor and economically
growing sporophores i.e. in both pileus and stipe during and between the flushes ascertained phenomenon in which phenols are enzymatically processed into
of fruiting body development and is also responsible for salt tolerance of A. quinone’s that evolve eventually into melanins [6, 26]. Concentrations of Water
bisporus [11, 27]. In accordance with Bonner et al. [3] and Jennings [12] mannitol activity, pH, temperature, active PPO and phenolic compounds determine the
was indeed supposed to function as an osmoregulator by encouraging the influx of browning degree of the tissue [17]. Tyrosinase, a multifunctional, glycosylated
water from the environment to develop turgor pressure thereby, helps in fruiting copper containing oxidase is responsible for enzymatic browning in fresh fruits,
body development. In edible mushrooms such as A. bisporus and Lentinulla vegetables, beverages and mushrooms [16]. It catalyzes the rate limiting steps in
edodes, fructose is reduced to mannitol and the reaction is catalysed by MtDH in melanogenesis i.e. oxidation of both monophenols and o-diphenols into o-
the presence of NADPH, obtained from pentose phosphate pathway [8]. In the quinones, which finally leads to melanins [6, 13]. The results presented in the
present study, MtDH activity was recorded in pinhead stage and showed a gradual [Table-1 and 2] cogently imply that tyrosinase activity increased with subsequent
uptick in subsequent stages of maturity, which is positively, compared to stages of maturity, which showed positive correlation with browning degree and
accumulation of mannitol in developmental stages of the fruiting bodies of A. water activity. In fungi, tyrosinases are associated with formation, stability of
bisporus and L. edodes, as reported by Hammond and Nichols [11] and Kulkarni spores and also for melanin production, which constitute a mechanism of defence
[15]. In contrast, cytoplasmic non glycosylated MtDH activity was reported to be to stress such as dehydration, UV radiation, extreme temperatures and free
abundant in vegetative cells and early developmental stages of A. bisporus which radicals. Moreover, they also contribute to the fungal cell-wall resistance to
dwindled progressively during sporophore growth and expressed at very low level hydrolytic enzymes, avoiding cellular lysis [2, 20, 25]. Meng et al. [18] reported
after sporulation [19, 21]. Perhaps, the increase in MtDH activity in developmental that the polyphenoloxidase (PPO) activity of Agaricus bisporus increased
stages had been attributed to the increase in fructose-6-phosphate by Hexose gradually with storage time. The increased activities of tyrosinase and browning in
monophosphate (HMP) activity [9]. However, the depletion of mannitol ensuing developmental stages may be featured to the two distinct mechanisms of
concentration during mycelial growth might be due to unavailability of NADPH. phenol oxidation i.e., by the activation of tyrosinase that belongs to PPO family
According to Hammond and Nichols [11] the levels of enzymatic activities change and by spontaneous oxidation [16, 26].

Molecular Dynamics of Morphogenesis in Volvariella volvacea (Bull. Ex Fr.) Sing
The compelling results of present investigation [Tables-1 and 2] envisaged that 1233-1240.
the browning degree gradually accreted with the developmental stages of V. [26] Singh P., Langowski H.C., Wanib A.A. and Saengerlaub S. (2010) J. Sci.
volvacea i.e., from pin head to flattening and final maturity. Noticeably, the water Food Agric., 90, 1393-1402.
activity (aw) showed positive correlation with the browning degree; a w increased [27] Stoop J.M.H. and Mooibroek H. (1998) Applied and Environmental
significantly with morphogenesis of fungi towards final maturity. Similar results Microbiology, 64(12), 4689-4696.
were obtained in A. bisporus in which, browning degree showed negative and [28] Suvarna K., Bartiss A. and Wong B. (2000) Microbiology, 146, 2705-2713.
positive correlation with firmness and respiration rate, respectively due to its thin [29] Zhou L., Wang Y., Hu X., Wu J. and Liao X. (2009) Innovat Food Sci Emerg
and porous epidermal structure [16]. This explicitly indicates that browning and Technol., 10, 321-327.
water activity are partly responsible for firmness and respiration rate of V.
volvacea. Further, this information univocally explains that browning degree, water
activity and tyrosinase activities are concatenated thereby, amenable for
accelerating senescence in fruiting bodies of V. volvacea. Further studies in this
context are very much warranted for commercial marketing of Volvariella.
Acknowledgement
The authors express their gratitude to Department of Plant Pathology, TNAU,
Coimbatore for the valuable support rendered during the course of investigation
Conflict of Interest: None declared
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International Journal of Chemical Studies 2017; 5(3): 138-142

P-ISSN: 2349–8528
E-ISSN: 2321–4902
IJCS 2017; 5(3): 138-142 Formulation of liquid spawn base of paddy straw
© 2017 JEZS
Received: 01-03-2017 mushroom, Volvariella volvacea (Bull. Ex Fr.) sing
Accepted: 02-04-2017

Nannapaneni Kiran Kumar Nannapaneni Kiran Kumar, Akkanna Subbiah Krishnamoorthy and
Mushroom Research Laboratory,
Ayyanar Kamalakannan
Tamil Nadu Agricultural
University, Coimbatore, India Abstract
Mushroom spawn in liquid form could be more handily employed and its dispersion would be greatly
Akkanna Subbiah Krishnamoorthy superior to solid spawn. Underpinning this, an isotonic liquid spawn base is formulated with gum acacia,
Mushroom Research Laboratory, trehalose, carboxymethyl cellulose (CMC), glycerol, polyethylene glycol (PEG) and aloe gel, based on
their water activity for uniform suspension and preservation of chlamydospores and basidiospores. The
University, Coimbatore, India
spores were suspended in the formulation and viability counts were recorded at 15 days interval. The
outcome perspicuously indicated that the chlamydospores suspended in PEG were found to be the best
Ayyanar Kamalakannan even after 45 days (1.5×104 spores ml-1) followed by aloe gel based formulation (1.2×104 spores ml-1). In
Mushroom Research Laboratory, the case of basidiospores, the isotonic formulation with glycerol as preservative unveiled more number of
Department of Plant Pathology, viable spore counts (8.7×104 spores ml-1) followed by aloe gel (7.5×104 spores ml-1). The efficiency of
Tamil Nadu Agricultural liquid formulation tested in terms of laccase activity indicated that the substrate seeded with
University, Coimbatore, India chlamydospore based formulation had exhibited comparatively more laccase activity than that of the
basidiospore based formulation.
Keywords: Basidiospore, chlamydospore, mycelium, spore, Volvariella volvacea, water activity
Introduction
Species of Volvariella are perishable and tend to lose their appearance due to short shelf life,
an impediment to the distribution and marketing of fresh mushrooms. Extension of the quality
and shelf life is therefore, a scientific, technical and economical challenge. Despite of
multifarious approaches for extending the shelf life of mushrooms, an alternative safe, cheaper
and benign advent is of utmost importance (Fernandes et al., 2013) [8]. The production of
liquid spawn is much more rapid than with a solid base material. Liquid spawn is widely used
in the cultivation of many mushrooms, including button mushroom, Agaricus bisporus (Friel
and McLoughlin, 2000) [9]; Pleurotus ostreatus (Laniece, 1966 and Silveira et al., 2008) [13, 23];
Lentinus edodes (Kirchhoff and Lelley, 1991 and Pellinen et al., 1987) [12, 19]; Brazilian oyster
mushroom, Pleurotus ostreatus (Rosado et al., 2002) [21]; Paris mushroom (A. bisporus var.
hortensis), wood blewit (Tricholoma nudum), garden morel (Morchella hortensis), yellow
morel (Morchella esculenta) and Chanterelle, Cantharellus cibarius (Laniece, 1966) [13].
Monosporous and vegetative hyphae of Volvariella volvacea (Bull.Ex Fr.) Sing. is known to
produce two kinds of spores namely, brown, thick walled and multinucleate asexual
chlamydospores and brown, sexual basidiospores (Chang, 1969) [3]. Chlamydospores represent
enlarged, highly refractile, thick-walled vegetative cells with varied forms and condensed
cytoplasm that form within hyphae or at hyphal tips (Odds, 1988) [17]. They are not formed by
direct transformation of hyphal cells but are borne on specialized spore-bearing branches
consisting several swollen cells and at maturity, these spores are detached. Commonly,
chlamydospores are spherical with an average diameter of 58.8μ. Chlamydospore is thickened
uniformly; the germ tube or tubes can emerge out of the spore at any point by means of
rupturing the wall (Chang and Yau, 1971) [4]. Mycelium borne chlamydospores rapidly lose
their viability in water when subjected to stress, suggesting that they are unlikely to act as
long-term storage structures (Teixido et al., 1998) [25]. Basidiospores tend to be in egg shape
Correspondence
Nannapaneni Kiran Kumar with an average length of 7-9μ; the widest part 5-6μ and the narrowest part 3-4μ across. The
Mushroom Research Laboratory, spore is generally uninucleate, but occasionally multinucleated. Spore wall is relatively thick
Department of Plant Pathology, and brown in color when spores shed. Protrusion of the germ tube is always at the hilum
TamilNadu, Agricultural region. Germination of the basidiospore commences with the appearance of a small clear
University, Coimbatore, India round vesicle, which emerges from a minute pore on the cell wall.
~138~
181
International Journal of Chemical Studies

The temperature of 20 oC and above is imperative for spore observations were recorded with the help of image analyzer
discharge by the fruiting body and the process continues for (N-400T, Optika, Italy).
18 hours at 25 oC (Chang, 1969) [3].
The spores may be collected and formulated in a liquid Collection and Purification of Chlamydospores and
suspension for more uniform distribution of inoculum during Basidiospores
the process of seeding the substrate (Eyal, 1991) [7]. Highly A Nine mm mycelial disc of seven days old actively growing
concentrated biomass in a liquid formulation is a worthwhile culture was inoculated into potato dextrose broth. Flasks were
prerequisite in order to curtail the cost of transit (Abadias et incubated at 30-35oC for 3 days without shaking and were
al., 2003) [1]. In the course of developing spore based liquid kept in an orbital shaker at 150 rpm with incubation at 30-
spawn, the viability of the spores needs to be given sheer 35oC for 6 days in the stationary condition. After 15 days
priority. Lowering water activity (aw) was an efficient way to formation of red to pinkish colored dots (chlamydospores) on
control bacterial growth in liquid formulations (Mugnier and the lower surface of the mycelial mat was observed. The
Jung, 1985) [16]. The fermentation process was scaled up using mycelial mat was then collected and macerated in a pestle and
a low-cost culture medium for the mass production of yeast, mortar for 5 minutes to ensure the detachment of spores from
Rhodotorula minuta (109 CFU ml-1) by adding glycerol (20%) the hyphae as suggested by Chang (1969) [3]. The final
and xanthan (5g l-l) to avert contamination and sedimentation macerated product was filtered through glass wool in the
(Patino-Vera et al., 2005) [18]. funnel where most of the hyphae were barred by the glass
The liquid spawn of oyster mushroom (P. ostreatus) was wool while chlamydospores passed through it easily. The
immobilized by using a mixture of cottonseed hull, corn core filtrate was kept in sonicator at 25 kHz for 10 min for
and wheat bran with a ratio of 4.5:4.5:1 by weight to prolong complete separation of chlamydospores, following the method
the storage time and also to provide accessible transportation suggested by Citiulo et al. (2009) [6] from hyphae. The filtrate
of liquid spawn (Wang et al., 2011) [27]. Compared to solid was once again filtered through glass wool kept in the funnel
spawn, there are ample advantages of adapting the liquid to harvest the chlamydospores (completely separated from
spawn which entail improved automation, more uniform hyphae). Fruiting bodies were produced by following the
distribution of inocula in substrate, quick and year-round method suggested by Sangeetha, 2002 [22]. Basidiospores were
production of large mycelial chunks, more homogeneous collected by placing the freshly harvested pileus at elongation
fungal growth, lower cost, early fruiting and elevated yield stage on a white sheet and keeping it undisturbed for 7 hours
(Eyal, 1991; Kirchhoff and Lelley, 1991; Kawai et al., 1995 in aseptic condition. The basidiospores were further collected
and Rosado et al., 2002) [7, 12, 10, 21]. Besides, it is strenuous to by scrapping the spore print on the white sheet using the
store and transport liquid spawn. Sometimes the residual sterile blade. The collected basidiospores were suspended in
nutrients may cause contamination. In addition, liquid spawn 10 ml sterile water and serially diluted to get 1:10 dilution,
of several mushrooms does not colonize on all substrates which is further used for formulation. The chlamydospores
(Friel and McLoughlin, 2000 and Leatham and Griffin, 1984) and basidiospores obtained were counted by using
[9, 14]
. The liquid spawn without nourishment, could not hemocytometer.
survive in pasteurized compost and that the biomass levels Standardization of Buffer for the Storage of
were significantly lower than that of conventional grain spawn Chlamydospores and Basidiospores
with the entrapment of both mycelium and nutrients (Friel and Viable chlamydospores were stored at 30-35 oC in an isotonic
McLoughlin, 2000) [9]. Consistent, more homogenous spawn buffer prepared based on the water activity of the spores as
production of V. volvacea is a laborious and more sensitive suggested by Patino-Vera et al., 2005 [18]. The buffer
process and the requirement of mushrooms is proliferating preservative solution contained NaCl, 8.0 g; KH2PO4, 2.0 g;
constantly. Alternate spawn base to replace the widely used KCl, 2.0 g and Na2HPO4, 2.9 g l-1 (pH 6.9). The water activity
straw spawn assents. Hence, the present inquest has been of chlamydospores and basidiospores was measured by water
fascinatingly conducted to identify the ideal liquid activity meter at 30 oC (aw = 0.89 and 0.60, respectively)
formulation for maintaining the spore viability. (Aqua LAB Model series TE8056). The water activity of
phosphate buffer was adjusted to aw = 0.89 and 0.60 by
Materials and Methods adding several protective agents like gum arabica, trehalose,
Volvariella volvacea strain CBE TNAU 1505, which is found carboxymethyl cellulose (CMC), glycerol, polyethylene
to show superior characteristics in terms of growth and glycol (PEG) and Aloe vera extract. Each preservation
chlamydospore production in our previous work (Kumar et medium was added with L-ascorbic acid (0.02 g w/v) as an
al., 2016) [11] was used in this study. This was maintained on antioxidant. The formulations to preserve chlamydospores
Potato Dextrose Agar (PDA) medium by incubating at were named as VVCF1 to VVCF6 and those used to preserve
32+2oC. Chlamydospore production is proportional to vigor, basidiospores were named as VVBF1 to VVBF6. Each
in order to maintain vigor; fresh isolations were made from formulation contained 100 ml of isotonic buffer with 0.02 g of
the fruiting bodies every time after 2 to 3 subcultures. For this L- Ascorbic acid. In addition, protective agents viz., 1.78 g of
purpose, the strains were propagated in straw spawn and gum Arabica (VVCF1), 1.33 g of trehalose (VVCF2), 1.22 g
grown on paddy straw following the method suggested by of CMC (VVCF3), 1.44 ml of glycerol (VVCF4), 1.78 g of
Thomas et al. (1943) [26]. Freshly harvested sporophores were PEG (VVCF5), 1.78 g of Aloe vera extract (VVCF6), 1.09 g
swabbed with 70 percent ethanol. At the junction of the pileus of gum Arabica (VVBF1), 0.81 g of trehalose (VVBF2), 1.75
and stipe, tissue bits were removed aseptically, surface g of CMC (VVBF3), 0.88 ml of glycerol (VVBF4), 1.09 g of
sterilized with 70% ethanol for 30 seconds and repeatedly PEG (VVBF5), 1.09 g of Aloe vera extract (VVBF6) were
washed in sterile water and placed on PDA medium taken in added. The spores suspended in the buffer solution with L-
sterile Petri dishes. The dishes were incubated at 30 to 35 oC ascorbic acid (VVCF7 and VVBF7) and in sterile water
for seven days. Following single hyphal tip method (VVCF8 and VVBF8) served as controls. All liquid
(Rangasamy, 1972) [20] pure culture was made and stored in formulation media were inoculated with 10 ml of
PDA slants to carry out further studies. Micrometric chlamydospore and basidiospore suspensions. The initial
~139~
182

concentration of viable cells was determined by counting the Table 1: Sustainability of chlamydospores in isotonic solution
spores (ml-1) by using hemocytometer. The samples were Formulation 15 days* 30 days* 45 days*
stored at 30oC in order to maintain the water activity and shelf 4.7 × 104 c 7.6 × 103 e 3.9 × 103 e
life. The observations were recorded at 15 days interval. The VVCF1
(4.67) (3.88) (3.59)
spore viability was confirmed by spraying 1ml of spore 3.3 × 104 e 8.7 × 103 d 5.5 × 103 d
VVCF2
suspension on 1-3 cm pounded paddy straw bits. (4.52) (3.94) (3.74)
2.9 × 104 f 6.2 × 103 f 2.7 × 103 f
VVCF3
Assay of Laccase (EC 1.10.3.2) (4.46) (3.79) (3.43)
As laccase is essential for colonization of substrate its activity 3.6 × 104 d 1 × 104 c 8 × 103 c
VVCF4
is determined. The conventional, paddy straw based spawn (4.56) (4.00) (3.90)
(Sangeetha, 2002) [22] and spore suspension inoculated fresh 5.7 × 104 a 2 × 104 a 1.5× 104 a
VVCF5
straw bits of 1 g were grounded in one ml of 0.1 M sodium (4.76) (4.30) (4.18)
phosphate buffer (pH 6.8). The homogenate was centrifuged 5.1 × 104 b 1.7 × 104 b 1.2 × 104 b
VVCF6
(4.71) (4.23) (4.08)
at 15,000 rpm for 15 min at 4oC and the supernatant was used
2.5 × 104 g 6 × 103 f 2.3 × 103 g
as the enzyme source. Laccase was assayed by adding 0.3 mL VVCF7
(4.40) (3.78) (3.36)
enzyme source to 2.5 mL of 30 μM Guaiacol in phosphate 2 × 103 h 5.9 × 102 g 1.7 × 102 h
buffer (0.1 M) at pH 6.0. Absorbance was read at 470 nm VVCF8
(3.30) (2.78) (2.23)
after incubating the reaction mixture for 30 min at 25oC CD (P = 0.05) 0.030 0.034 0.031
against zero time control. One unit of laccase activity was
calculated as the change in absorbance by 0.001 min-1 mL-1 of VVCF1 - gum acacia, VVCF2 - trehalose, VVCF3 - carboxy
enzyme source at 25oC. methyl cellulose, VVCF4 - glycerol, VVCF5 - poly ethylene
glycol, VVCF6 - aloe gel, VVCF7 - buffer and VVCF8 -
Statistical Analysis water. Viability counts were taken at 15 days interval for 45
In order to avoid errors, the experiment has been carried out days by using haemocytometer, with initial count of 7.5 × 104
in 4 replicas and the results presented are mean values. spore’s ml-1 at 1:10 dilution. *The data presented are mean of
Statistical software AGRES was used for the data analysis. In 4 replications and the data in parenthesis are square root
the case of zero values, the data was square-foot transformed transformed values. Means in a column followed by the same
(X+0.5) before statistical analysis. letter are not significantly different at P = 0.05 by one way
ANOVA.
Results
Viability of basidiospores in isotonic formulation Viability of basidiospores in isotonic formulation
Chlamydospores collected were tested for their total count Basidiospores were collected and diluted to 1:10 dilution with
and viability by using different substances in the isotonic the isotonic buffer designed based on the water activity (0.60
buffer designed based on the water activity (0.89 aw at 30oC) aw at 30oC). The spores were diffused in various substances
and the viability counts were taken at 15 days interval for 45 and the viable spore counts were taken by using
days by using haemocytometer, with initial count of 7.5 × 104 hemocytometer up to 45 days at 15 days interval, with the
spores ml-1 at 1:10 dilution. The results are presented in table initial count of 8.2 × 106 spores ml-1. The results obtained are
1. Among the substances tested the spores diffused in PEG presented in table 2. The basidiospores diffused in glycerol
performed significantly better in maintaining spore viability at were significantly different in terms of spore viability at 15,
15, 30 and 45 DAI (5.7 × 104, 2 × 104 and 1.5× 104 spores ml- 30 and 45 DAI (1.4 × 106, 1.02 × 106 and 8.7 × 104 spores ml-
1
1
). This was followed by aloe gel (5.1 × 104, 1.7 × 104 and 1.2 ). This was followed by aloe gel (1.35 × 106, 9.25 × 105 and
× 104 spores ml-1), gum acacia on 15 DAI (4.7 × 104 spores 7.5 × 104 spores ml-1) and gum acacia (1.1 × 106, 7.4 × 105
ml-1) and glycerol at 30 and 45 DAI (1 × 104, 8 × 103 spores and 5.3 × 104 spores ml-1), respectively. Comparatively less
ml-1), respectively. Among the substances evaluated, gum number of viable spores was recorded in PEG at 15, 30 and
acacia suspension had registered the minimum number of 45 DAI (9.7 × 105, 7.3 × 105 and 4.6 × 104 spores ml-1). Less
spores at 45 DAI (3.9 × 103) ml-1. Very less number of viable number of spores was observed in water i.e., 8.6 × 105, 4 ×
spores were observed in water at 45 DAI (1.7 × 102 spores ml- 105 and 3.5 × 104 spores ml-1 at 15, 30 and 45 DAI,
1
) when used as a control. respectively.
Table 2: Sustainability of basidiospores in isotonic formulation
Formulation 15 days* 30 days* 40 days*
1.1 × 106 c 7.4 × 105 c 5.3 × 104 c
VVBF1
(6.04) (5.86) (4.72)
1.02 × 106 d 6.65 × 105 d 5.1 × 104 c
VVBF2
(6.01) (5.82) (4.70)
1.25 × 106 b 8.8 × 105 b 7.3 × 104 b
VVBF3
(6.10) (5.94) (4.86)
1.4 × 106 a 1.02 × 106 a 8.7 × 104 a
VVBF4
(6.15) (6.00) (4.93)
9.7 × 105 de 7.3 × 105 c 4.6 × 104 e
VVBF5
(5.99) (5.86) (4.66)
1.35 × 106 a 9.25 × 105 b 7.5 × 104 b
VVBF6
(6.13) (5.96) (4.87)
9.5 × 105 e 5.8 × 105 e 5.08× 104 c
VVBF7
(5.98) (5.76) (4.54)
8.6 × 105 f 4 × 105 f 3.5 × 104 f
VVBF8
(5.93) (5.60) (4.54)
CD (P = 0.05) 0.030 0.037 0.037
~140~
183

VVBF1 - gum acacia, VVBF2 - trehalose, VVBF3 - carboxy liquid spawn and sometimes, residual nutrients may cause
methyl cellulose, VVBF4 - glycerol, VVBF5 - poly ethylene contamination (Friel and Mcloughlin, 2000) [9]. During the
glycol, VVBF6 - aloe gel, VVBF7 - buffer and VVBF8 - course of the investigation, bacterial contamination of
water. Viable spore counts were taken by using chlamydospore and basidiospore based isotonic suspension
haemocytometer up to 45 days at 15 days interval, with the was observed. This is surmounted by the addition of 0.3 ml of
initial count of 8.2 × 106 spore’s ml-1 at 1:10 dilution. *The 500 ppm streptomycin sulfate to the suspensions. For
data presented are mean of 4 replications and the data in avoiding damage during storage and transit, the spores may be
parenthesis are square root transformed values. Means in a immobilized by entrapment in alginate beads or polymer gel
column followed by the same letter are not significantly matrix to protect the cells from fluctuations in the macro
different at P = 0.05 by one way ANOVA. environments.
Laccase activity in colonized substrate

The chlamydospores and basidiospores formulated in the
isotonic buffers containing glycerol and PEG were sprayed on
steam sterilized paddy straw bits keeping conventional straw
spawn as a control. The activity of laccase was determined
and expressed as change in absorbance min-1 ml-1 of enzyme
source in the colonized paddy straw bits after 6 days of
inoculation. Laccase activity was found to be more in
conventional spawn colonized substrate prepared from tissue
culture (0.4). This was followed by chlamydospores colonized
substrate (0.19). Basidiospores colonized substrate recorded
the minimum laccase activity of 0.12 as shown in figure 1.
Fig 2: Chlamydospores suspended in isotonic buffer

A - Viable chlamydospore, B - Partially broken chlamydospore,
C - Fully broken chlamydospore (empty spore).
Fig 1: Laccase Activity in Spore Suspension Colonized Substrate
Discussion
The addition of chemical amendments to the formulation is
capable of enhancing cell tolerance to desiccation, osmotic
pressure and temperature stress (Streeter, 1985). In the present
study, chlamydospores and basidiospores were collected and
formulated in an isotonic buffer designed with disparate
preservatives for perpetuating the viability of spores. It was
observed that chlamydospores suspended in PEG and
basidiospores suspended in glycerol significantly performed
better over a period of 45 days when stored at 30 to 35oC. The
outcome obtained by chlamydospores based isotonic
suspension pertained to PEG, a polymer commercially used in
the manufacturing of medicines, in commensuration to
immobilization by entrapment in polymer gels, allows for a Fig 3: Chlamydospores and Basidiospores of Volvariella volvacea
A - Thick double walled chlamydospore on swollen hyphal cells (20 X)
greater level of microenvironmental control (McLoughlin, B - Double walled chlamydospore with half-filled protoplasm (20 X)
1994). The results were cognate to other immobilization C - Oval shaped Basidiospores (plasmamembrane (a), outer wall (b),
techniques. Patino-Vera et al. (2005) [18] developed a liquid hilum (c)
formulation of yeast, Rhodotorula minuta by adding glycerol D - Basidium (a) with basidospores (b) on sterigmata (c)
(20%) which helped to uphold the viability of cells @ 107 cfu
ml-1 up to 6 months at 4oC. During the current investigation, In mushroom fungi, laccases play a dynamic role in lignin
oval shaped basidiospores and three types of chlamydospores degradation and sporophore development (Ardon et al., 1998
were observed viz., spores with full, partial and no protoplasm and Wood, 1980). According to Chen et al. (2003), laccase is
(Figure 2 and 3). In consonance with Chang (1969) [3], the imperative in the mushroom developmental cycle involving
spores with full protoplasm are considered as viable spores. fruit body morphogenesis and also indicated that low levels of
The breaking down of chlamydospores might be due to laccase are detected all during the vegetative growth phase
osmotic imbalance and change in the environment around but enzyme activity increases sharply during sporophore
spore walls. Accordingly, it is difficult to store and transport development. Laccase activity was determined in paddy straw
~141~
184

substrates colonized by conventional straw spawn, and 11. Kumar NK, Krishnamoorthy AS, Amirtham D. Detection
isotonic suspensions of chlamydospore and basidiospore on of variability among the strains of Volvariella volvacea
the sixth day after inoculation. It is divulged that, (Bull. Ex Fr.) Sing. And Volvariella bombycina
conventional straw spawn colonized substrate had shown (Schaeff.) Sing. Using RAPD analysis. Madras
more laccase activity, followed by chlamydospore based Agricultural Journal 2016; 103(1-3):35-40.
liquid formulation. The upshot also highlighted the 12. Kirchhoff B, Lelley J. Investigations of shiitake [Lentinus
discrepancy in adoption period of both the inocula on edodes (Berk.) Sing. Bag-log cultivation to increase the
sterilized and partially fermented paddy straw. Friel and yield in Germany. In: Science and cultivation of edible
McLoughlin (2000) [9] tested mycelia based liquid spawn of fungi, Netherlands. 1991, 509-516.
A. bisporus for laccase activity and disclosed its superiority. 13. Laniece A. Production and use of liquid mushroom
In conformity with Chang (1969) [3], basidiospores desire spawn. U.S. patent. 1966; 3:286-399.
40oC for 48 hours for effective germination. The 14. Leatham GF, Griffin TJ. Adapting liquid spawn Lentinus
basidiospores suspended in liquid formulation might not have edodes to oak wood. Appl Microbiol Biotechnol. 1984;
experienced 40oC at bed surface for effective colonization. 20:360-363.
Henceforth, it is propounded that the basidiospore inoculated 15. McLoughlin AJ. Controlled release of immobilized cells
substrate should be incubated at 40oC at least for 48 hours to as a strategy to regulate ecological competence of
promote spore germination and subsequent colonization. inocula. Advances in Biocontrol
Future research in this context is warranted by augmenting the Engineering/Biotechnology 1994; 51:1-45.
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protective agents for maintaining the spore viability. water in biopolymer gels. Appl Environ Microbiol. 1985;
50:108-114.
Acknowledgement 17. Odds FC. Candida and Candidosis. Journal of Basic
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mushroom for the valuable support rendered during the course R, Carrillo A, Galindo E. Pilot-scale production and
of the investigation. liquid formulation of Rhodotorula minuta, a potential
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Table 1: Tyrosinase inhibitory potential of phytochemicals

Medicinal Plant Tyrosinase inhibition (%)*
plants part Methanolic extract Acetonic extract Water extract
used
100 zg/ml 250 zg/ml 500 zg/ml 100 zg/ml 250 zg/ml 500 zg/ml 100 zg/ml 250 zg/ml 500 zg/ml
A. vasica Leaves 29.80c 40.46bc 53.42c 19.34d 25.70d 34.01f 4.46d 11.69d 19.75e
(33.08) (39.50) (46.96) (26.08) (30.46) (35.67) (12.19) (19.99) (26.38)
M. citrifolia Fruits 21.26f 35.23d 54.28c 16.19e 30.46c 43.65cd 10.36bc 22.11b 35.30b
(27.45) (36.40) (47.45) (23.72) (33.49) (41.35) (18.77) (28.04) (36.45)
A. vera Leaves 23.76e 39.17cd 49.05c 16.43e 28.93c 32.56f 9.33c 17.30c 25.48d
(29.17) (38.74) (44.45) (23.91) (32.53) (34.79) (17.78) (24.57) (30.31)
G. glabra Bark 31.91b 52.19a 65.67ab 26.63b 39.08b 53.59b 11.00b 21.44b 29.71c
(34.39) (46.25) (54.13) (31.06) (38.69) (47.05) (19.36) (27.58) (33.02)
P. betle Leaves 14.34g 27.04e 35.01d 19.45d 30.41c 41.84de 3.86de 6.50f 14.12f
(22.25) (31.33) (36.27) (26.16) (33.46) (40.30) (11.33) (14.77) (22.07)
C. limon Peel 4.10j 9.15h 16.36f 1.41g 5.79f 9.17i 9.88c 17.92c 28.34cd
(11.68) (17.60) (23.85) (6.81) (13.92) (17.62) (18.32) (25.04) (32.16)
A. indica Leaves 14.60g 22.86f 28.23e 9.65f 14.97e 21.83g 2.26f 5.93f 11.52g
(22.46) (28.56) (32.09) (18.09) (22.76) (27.85) (8.64) (14.09) (19.84)
M. alba Leaves 28.40d 43.83b 60.80b 23.66c 35.94b 46.94c 3.41e 8.91e 14.14f
(32.20) (41.45) (51.23) (29.10) (36.83) (43.24) (10.64) (17.36) (22.08)
V. negundo Roots 12.26h 21.37f 37.66d 9.26f 13.52e 17.23h 4.51d 10.48d 15.76f
(20.49) (27.53) (37.85) (17.71) (21.57) (24.52) (12.26) (18.88) (23.39)
C. longa Rhizome 9.50i 14.54g 23.86e 8.04f 23.91d 39.15e 1.22g 3.58g 7.91h
(17.95) (22.41) (29.23) (16.47) (29.27) (38.73) (6.34) (10.90) (16.33)
L- ascorbic - 41.62a 54.02a 67.58a 41.62a 54.02a 67.58a 41.62a 54.02a 67.58a
acid (40.17) (47.30) (55.29) (40.17) (47.30) (55.29) (40.17) (47.30) (55.29)
CD (P=0.05) 1.57 2.53 3.38 1.64 1.90 2.46 1.02 1.43 1.91
*Data in parenthesis are arcsine transformed values. Means in a column followed by the same letter are not significantly different at P=0.05
by one way ANOVA. C. longa: Curcuma longa, V. negundo: Vitex negundo, M. alba: Morus alba, A. indica: Azadirachta indica, C. limon:
Citrus limon, P. betle: Piper betle, G. glabra: Glycyrrhiza glabra, A. vera: Aloe vera, M. citrifolia: Morinda citrifolia, A. vasica: Adathoda
vasica
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189
Table 2: Tyrosinase inhibitory potential of mycomolecules

Fungi Tyrosinase inhibition (%)*
Methanolic extract Acetonic extract Water extract
100 zg/ml 250 zg/ml 500 zg/ml 100 zg/ml 250 zg/ml 500 zg/ml 100 zg/ml 250 zg/ml 500 zg/ml
G. lucidum 21.15c 41.98b 53.3b 19.34c 31.95b 40.44b 11.97b 20.5b 29.3b
(27.38) (40.38) (46.89) (26.08) (34.41) (39.48) (20.24) (26.92) (32.77)
C. versicolor 21.56c 33.12cd 45.68c 17.62c 28.97b 36.07c 7.71c 13.6bc 23.72c
(27.66) (35.13) (42.52) (24.81) (32.56) (36.91) (16.12) (21.64) (29.14)
P. albus 18.88c 28.9d 33.43d 12.4d 23.44c 29.33e 6.5d 16.71bc 22.94c
(25.75) (32.51) (35.32) (20.61) (28.95) (32.79) (14.77) (24.12) (28.61)
O. neovolkiana 28.84b 36.14c 47.71bc 22.69b 29.57b 33.99cd 12.11b 18.99b 26.45bc
(32.48) (36.95) (43.68) (28.44) (32.94) (35.66) (20.36) (25.83) (30.95)
O. sinensis 19.39c 28.9d 32.72d 10.47de 17.59d 25.38f 5.83d 11.24cd 15.14d
(56.12) (32.51) (34.89) (18.87) (24.79) (30.25) (13.97) (19.58) (22.89)
L. edodes 25.61b 33.12cd 41.85c 17.62c 25.19c 31.27de 12.41b 20.84b 26.07bc
(30.40) (35.13) (40.30) (24.81) (30.12) (34.00) (20.62) (27.16) (30.70)
S. commune 12d 18.42e 24.65e 9.66e 14.21e 19.22g 2.07f 7.64d 11.57e
(20.26) (25.41) (29.76) (18.10) (22.14) (26.00) (8.27) (16.04) (19.88)
L. esculentum 19.76c 22.53e 29.49de 11.88d 18.9d 24.39f 4.69e 10.43cd 15.04d
(26.39) (28.33) (32.89) (20.16) (25.76) (29.59) (12.50) (18.84) (22.81)
L- Ascorbic 41.62a 54.02a 67.58a 41.62a 54.02a 67.58a 41.62a 54.02a 67.58a
acid (40.17) (47.30) (55.29) (40.17) (47.30) (55.29) (40.17) (47.30) (55.29)
CD (P=0.05) 2.21 3.22 3.57 1.93 2.21 2.05 1.16 5.79 2.72
*Data in parenthesis are arcsine transformed values. Means in a column followed by the same letter are not significantly different at P=0.05
by one way ANOVA. L. esculentum: Lycoperdon esculentum, O. sinensis: Ophiocordyceps sinensis, O. neovolkiana: Ophiocordyceps
neovolkiana, S. commune: Schizophyllum commune, L. edodes: Lentinus edodes, P. albus: Pisolithus albus, C. versicolor: Coriolus
versicolor, G. lucidum: Ganoderma lucidum
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(41.29) (30.32) (26.28)
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L. esculentum 32.85d 24.58e 16.49e PHFKDQLVPV7KHW\URVLQDVHLQKLELWLRQDELOLW\PLJKWGHSHQG
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O. neovolkiana 56.67b 34.96cd 23.77bc IRUPDK\GURJHQERQGWRWKHDFWLYHVLWHRIHQ]\PHOHDGLQJ
(48.83) (36.24) (29.17) WRORZHUHQ]\PDWLFDFWLYLW\>@*DOOLFDFLGHSLFDWHFKLQ
O. sinensis 33.47d 29.18de 19.97cde SURF\DQLGLQ % DQG HSLFDWHFKLQJDOODWH IODYRQRLGV
(35.34) (32.69) (26.54) LVRIODYRQRLGVDQGSKHQROVLQPXVKURRPVDUHFRJHQWO\SURYHG
L. edodes 54.21b 46.02b 21.50bcd WRKDYHHIIHFWLYHW\URVLQDVHLQKLELWRU\FRQGLWLRQLQJ >@,W
(47.41) (42.71) (27.62) ZDVDOLNHUHFRUGHGWKDWWKHW\URVLQDVHLQKLELWRU\DFWLYLW\RI
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192


P-ISSN: 2349–8528
E-ISSN: 2321–4902
IJCS 2017; 5(3): 788-792 Identification of 3’deoxyadenosine (Cordycepin)
© 2017 JEZS
Received: 06-03-2017 from the medicinal mushrooms, Ophiocordyceps spp

Sangeetha Chinnusamy Sangeetha Chinnusamy and Akkanna Subbiah Krishnamoorthy
Abstract
University, Coimbatore, Tamil
Nadu, India
The antifungal metabolite cordycepin was extracted and purified from cell free culture filtrate condensate
and mycelial mat extract of Ophiocordyceps sinensis and Ophiocordyceps neovolkiana by TLC. The
Akkanna Subbiah Krishnamoorthy compound exhibited the band with the Rf value of 0.63. Further confirmation by HPLC indicated only
Department of Plant Pathology, from the culture filtrate and mycelial mat extract of O. sinensis and the retention time had ranged
Tamil Nadu Agricultural between 5.8 - 5.9 min as against 5.8 min with the standard cordycepin molecule (Sigma Aldrich. Inc.,
University, Coimbatore, Tamil USA). Inopportunely, the native isolate of O. neovolkiana not had the similar peak at the same retention
Nadu, India time. But the proton NMR spectral analysis of cordycepin produced by both, O. sinensis and O.
neovolkiana (CFC filtrate condensate and mycelial mat extract) showed anomeric carbon peaks at 3.4
ppm, which were comparable with proton NMR spectrum of standard cordycepin already done.
Keywords: O. sinensis, O. neovolkiana, cordycepin, Thin Layer Chromatography (TLC), High

performance liquid chromatography (HPLC) and NMR
1. Introduction
The Chinese caterpillar fungi, Ophiocordyceps spp are usually identified based on the fungal
growth observed on insect cadavers and they are formulated as inundative biocontrol agents of
Lepidopteran insects, mites and ticks (Goettel et al., 2005 and Vincent et al., 2007) [6, 27].
Ophiocordyceps sinensis is known to parasitize the larvae of ghost moth (Hepialus
armoricanus Oberthur) belonging to the family Hepialidae. Adherence of either ascospores or
conidia of the fungus with the cuticle of the larva is very common. The fungus colonizes the
caterpillars and multiplies inside the host, filling the entire caterpillar with thread like hyphae.
The fungus Ophiocordyceps spp are known to produce several secondary metabolites
including a nucleoside antibiotic, cordycepin (Isaka et al., 2000) [11]. Ophiocordyceps sinensis
is well known for its invigorating and immunological effects on human body (Jiang et al.,
2002) [12]. Medicinal properties of O. sinensis are attributed to cordycepin, cordycepic acid,
triterpenoids and other active compounds (Rana, 2004) [22]. Investigations have proved that, O.
sinensis had possessed anti tumor, free radical scavenging and anticancer principles. It was
further suggested that, polysaccharide and sterol fractions of the fungus were responsible for
such activities (Zhang et al., 2004 and Wang et al., 2005) [31, 28]. Analytical studies proved that
low-molecular weight fractions had showed antimicrobial effects (Kuo et al., 1996) [17]. A
number of bioactive compounds from Ophiocordyceps spp have been reported to possess
multiple benefits including anti-tumor, anti-microbial, anti-inflammatory and
immunomodulatory activities (Schuffler and Anke, 2009) [24]. Ophiocordyceps spp extract has
been known to contain cordycepin, cordycepic acid, N-acetylgalactosamine, adenosine,
ergosterol, ȕ-glucans, exo-polysaccharides and cordymin (Cunningham et al., 1950; Holliday
and Cleaver, 2008; Yan et al., 2010 and Wonga et al., 2011) [5, 7, 29, 30].
Among these molecules cordycepin (3’-deoxyadenosine) was the first compound isolated from
C. militaris (Cunningham et al., 1950) [5]. Bentley et al. (1951) [1] reported that the cordycepin
would be an adenine nucleoside containing a 3’-deoxypentose with a branched carbon chain.
The structure of cordycepin is very much similar with cellular nucleoside(adenosine),but lacks
3’ hydroxyl group facilitating the compound to act as anucleoside antibiotic. The cordycepin is
Correspondence
known to have many biological activities including the inhibition of cell proliferation, platelet
Sangeetha Chinnusamy
Department of Plant Pathology, aggregation, cell migration and inflammation (Kim et al., 2006 and Cho et al., 2007) [15, 3].
Tamil Nadu Agricultural Apparently, a large part of the metabolism of C. militaris is directed towards the production of
University, Coimbatore, Tamil cordycepin (25-100 mg per L of medium). But, the biosynthesis depends upon the method of
Nadu, India isolation, which may require around 20 days of incubation (Phillips, 1960) [21].
~788~
193

Hsu et al. (2002) [8] indicated that the content of 3’- and the cell free culture (CFC) filtrate was extracted with
deoxyadenosine in the mycelial fermentation process would methanol. Liquid-liquid extraction was carried out three to
be more (40.8 mg /g) as compared to recovery from fruiting four times. Each fungal extract of methanolic solvent was
bodies (less than 5.4 mg /g). Therefore, the present evaporated separately under reduced pressure using a rotary
investigation conducted for the isolation of most important evaporator to obtain the residues. The residue was dried and
pharmaceutical molecule of cordycepin from the culture dissolved in methanol (1mg/mL) and filtered with membrane
filtrates as well as from the mycelium of Ophiocordyceps filter (0.2 ȝm), stored at 4 °C for further studies.
sinensis and the native isolates of Ophiocordyceps
neovolkiana. Further, it was confirmed through the studies of Extraction of cordycepin from the mycelia
HPLC and NMR. Five gram of freeze dried mycelia obtained from the
submerged cultures of O. sinensis and O. neovolkiana
Materials and Methods maintained at 25 °C for 15 days in MC broth (pH 5.5), was
Ophiocordyceps sinensis isolate no.1220 was obtained from powdered separately with liquid nitrogen and extracted three
Forest Research Institute (FRI), Dehradun, India and a local times with equal volume of methanol at the rate of one ml
isolate of Ophiocordyceps sp. was collected from the cadavers each time. The extracted sample taken in the Eppendorf tubes
of coconut root grub, Leucopholis coneophora Burm at was centrifuged at 10,000 rpm for 15 min in a bench
College of Agriculture, Padannakkad (Latitude 12o26”55’N, centrifuge (at 4 °C) and the supernatants were evaporated to
Longitude 75 o11”41’ E) Kasaragod, Kerala during March dryness, dissolved in methanol and stored at 4 °C for further
2014. Morphological identification was done by visual and studies.
microscopic examination. The specimens were stored at 4 ͼC
prior to the isolation and purification of the fungal culture. Detection of Bioactive Molecules by Thin Layer
The purified cultures were further identified by molecular Chromatography (TLC)
techniques. The cordycepin extract from culture filtrate and mycelial mat
For the purpose of tissue isolation, the fruiting bodies of wild of O. sinensis and O. neovolkiana were spotted separately on
isolate were cut into small pieces, surface sterilized with one to silica gel 60 TLC plates (60 F 254, 0.12 mm thick, 5×20
per cent (w/v) sodium hypochlorite for 60 sec. The tissue bits cm, Merck, Germany) at the rate of 5 μL /spot along with
were washed twice with sterile distilled water for 60 sec and standard cordycepin was purchased from Sigma- Aldrich,
placed in sterile Petri plates containing 20 mL of potato Inc., USA (Product Number C 3394). After drying,
dextrose agar. The plates were incubated at 25 ͼC for 15 days. Chromatographs were developed using solvent system
butanol: water (86:14). The developed TLC plates were air
Extraction of cordycepin from culture filtrate dried overnight to remove the remaining solvents. TLC plates
Four mycelial discs measuring 6 mm diameter each, cut from were viewed under UV light at 250 nm. The retention factor
the margin of a 5days old colony of O. sinensis and O. (Rf) values of various compounds resolved on TLC plates
neovolkiana were inoculated separately in 250 mL conical were calculated using the formula given by Sadasivam and
flasks containing 100 mL of sterilized mushroom complete Manickam (1992) [23].
(MC) broth in each flask (adjusted to pH 5.5). The flasks were
placed on a rotary shaker maintained at 120 rpm and Detection of cordycepin through HPLC
incubated at 25 °C in diffused day light (600-800 lux) for 15 The extracted cordycepin was further confirmed by High
days. After incubation, the culture filtrate and the mycelial Performance Liquid Chromatography (HPLC). The
mat were separated by filtration through Whatman No.40 instrumental parameters and details for HPLC are presented
filter paper. The filtrate was further centrifuged at 10,000 rpm below:
Table 1
Instrumental parameters Details
Name of equipment Agilent 1200 HPLC system and AB Sciex API 4000
Column Atlantis –dC18 (100mm x 2.0 mm,5μm)
Guard column C18 (10mm x 2.1 mm, 5 μm)
Detector Photo-Diode Array (PDA)
Mobile phase A and B Methanol and water (20:80)
Injection volume 10 μl
Column temperature 30 ºC
Flow rate 1.0 mL /min
Detection wavelength 254 nm
Cordycepin standard was separately prepared for HPLC, injected in to HPLC column at the rate of 10 μL. The
dissolved in methanol and made to 1000 ppm (one mg /mL) retention time and peak area were recorded. The similarity
and diluted serially with methanol to get 0.5, 5 and 10 ppm. between cordycepin standard and extracted samples was
These dilutions were injected in to HPLC column at the rate analyzed by comparing the data.
of 10 μL and observations on retention time and peak area
was recorded. The dry form of extracted cordycepin Proton Nuclear Magnetic Resonance (NMR) Spectrum of
molecules from culture filtrate and powdered mycelial mat of cordycepin
O. sinensis and O. neovolkiana were collected after The bioactive compound present in CFC filtrate and mycelial
condensation and dissolved in HPLC grade methanol at the mat of O. sinensis and O. neovolkiana was purified by TLC to
rate of 1 mg /mL to get 1000 ppm concentration. The diluted obtained 50 mg for each sample. The TLC purified bioactive
extract was passed through the membrane filter (0.2 μm) and compounds were sent for NMR analysis at the Sophisticated
~789~
194

Analytical Instrument Facility (SAIF), Central Drug Research H NMR spectrum of bioactive molecules of O. sinensis
Institute (CSIR), Lucknow. The proton NMR spectral analysis of cordycepin produced by
O. sinensis and O. neovolkiana (CFC filtrate condensate and
Results mycelial mat extract) showed anomeric carbon peaks at 3.4
Purification of cordycepin by TLC ppm (Fig 4, 5, 6 and7) which was comparable with 1H NMR
The methanolic fraction of culture filtrate condensate and spectrum of standard cordycepin (Fig 8).
mycelial mat extract collected on 15 days after inoculation
and it was purified by stepwise gradient with butanol: water
(86:14) and three fractions were obtained (F1-F3). Presence of
cordycepin was found in fraction F2 (Rf value 0.63). This was
sub-fractionated into four fractions (S1-S4) using elution
gradient with butanol: water (86:14). Sub fraction S3 was
found to carry cordycepin, which was further vacuum dried
and crystallized in methanol, which resulted in a creamy
white powdery product. Spectral analysis of purified
cordycepin was performed using HPLC and proton NMR.
Detection of cordycepin through HPLC

High performance liquid chromatography analysis for the
standard (cordycepin) at 254nm recorded a retention time of
5.92 min (Fig 1). Regression analysis data showed a linear
response at three different concentrations (0.1, 0.5 and 1.0
ppm). Similarly, cordycepin was detected in the culture
filtrate condensate and mycelial mat extract of O. sinensis at
254nm with a retention time of 5.84 (Fig 2) at a retention time Fig 4: H NMR spectral analysis of culture filtrate condensate of O.
of 5.9 min (Fig 3). But, O. neovolkiana did not show any peak sinensis
at the same retention time.
Fig 1: HPLC analysis of cordycepin standard (0.1ppm)
Fig 5: H NMR spectral analysis of mycelial mat extract of O.

sinensis
Fig 2: Culture filtrate condensate contained cordycepin produced by

O. sinensis
Fig 6: H NMR spectral analysis of culture filtrates of O. neovolkiana

Fig 3: Mycelial mat extract contained cordycepin produced by O.
sinensis
~790~
195

comparing the HPLC results of cordycepin standard and the
extracted metabolite, final confirmation of cordycepin was
made only O. sinensis. In a similar study, Huang et al. (2009),
Varshney et al. (2011) and Kumar and Spandana (2013) [10, 24,
15]
have also extracted cordycepin from the sporophores of O.
sinensis and conformed the identity by TLC and HPTLC.
The proton NMR spectral analysis of cordycepin purified
from the CFC filtrate condensate and mycelial mat extract of
O. sinensis and O. neovolkiana demonstrated the presence of
anomeric carbon peaks at 3.4 ppm (Fig 4, 5, 6, 7 and 8). This
observation has been further compared with 1H NMR
spectrum of standard cordycepin reported by Tuli et al. (2014)
[23]
. Further, the NMR analytical data obtained during the
current investigation was found to be consistent with the
earlier reports (Cunningham et al., 1950 and Chatterjee et al.,
1957) [5, 2].
Fig 7: H NMR spectral analysis of mycelial mat extract of O.
neovolkiana Acknowledgement
The authorssincere thanks to department of plant pathology,
Tamil Nadu Agricultural University, Coimbatore and also
thanks to funding agent ICAR AICRP.
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197
8430 Advances8430-8436,
Advances in Life Sciences 5(19), Print : ISSN 2278-3849, in Life Sciences
20165(19), 2016
Studies on the Influence of Carbon and Nitrogen Nutrition on the Chlamydospores

Production of Volvariella volvaceae (Bull. Ex Fr.) Sing. and Volvariella bombycina
(Schaeff.) Singer.
KIRAN KUMAR NANNAPANENI1*, KRISHNAMOORTHY AKKANNA SUBBIAH2 AND AMIRTHAM
DAMODARASAMY3
1
Department of Plant Pathology, Acharya N.G. Ranga Agricultural University, Andhra Pradesh.
2
Department of Plant Pathology;
3
Department of Food and Agricultural Process Engineering, AEC&RI, Tamil Nadu Agricultural University, Coimbatore
email*: [email protected]
ABSTRACT (Stoop and Mooibroek, 1998). Complex organic nitrogen

sources like yeast extract, peptone, tryptophan, aspartic
Carbon sources viz., glucose, fructose, sucrose, mannitol
acid, serine and casein hydrolysate; inorganic nitrogen
and sorbitol; Nitrogen sources viz., ammonium nitrate,
sources such as ammonium di-hydrogen phosphate were
sodium nitrate, potassium nitrate, glycine and calcium
found to be the best for protein production, germination
nitrate were supplemented in Czapek’s dox medium to and germ tube elongation of the spores of V. diplasia
study their influence on chlamydospores production of (Banerjee and Samajpati, 1989; Banerjee et al., 1990).
Volvariella. Supplementation of sucrose and potassium Keeping this bounded facts in hark, the present
nitrate to the growth medium each at 3 per cent level investigation has been made for selecting the best
encouraged the mycelial growth and chlamydospores nutritional parameters for enhancing chlamydospores
production of both V. volvacea strain CBE TNAU 1505 production of Volvariella.
and V. bombycina strain CBE TNAU 1406.
MATERIALS AND METHODS
Key words Chlamydospores, Fungi, Hyphae, Isolate, V. volvacea strain CBE TNAU 1505 and V. bombycina
Ions, Nutrition, Volvariella strain CBE TNAU 1406 were obtained from the germplasm
bank of Mushroom Research Laboratory, Tamil Nadu
Agricultural University, Coimbatore, Tamil Nadu, India was
Mushrooms are myriad of nutritious healthy foods
used in this experiment. It was maintained on 90 mm
that are valued throughout the world as luscious medicine
petridishes with potato dextrose agar medium at 32+ 20C.
for thousands of years. Among the edible mushrooms,
Volvariella volvaceae (Bull. Ex Fr.) Sing. is known as paddy Maintenance of Pure Cultures
straw mushroom or Chinese mushroom that grows in tropical The sub-cultures of paddy straw mushroom strains
and sub-tropical regions at a temperature range of 28-350C. used in the study were maintained on Potato Dextrose Agar
V. bombycina (Schaeff.) Singer is popularized as silver silk (PDA) medium. In order to maintain the vigour fresh
straw mushroom or tree mushroom grows in temperate isolations were made from the fruiting bodies every time
conditions at a temperature range of 23-250C (Ahlawat and after 2 to 3 subcultures. For this purpose the strains were
Singh, 2014). Poor shelf life, hydrolytic enzyme potential propagated in straw spawn and grown on paddy straw
and productivity are the major hindrances for this praise following the method suggested by Thomas et al. (1943).
worthy mushroom for not gaining commercial importance Freshly harvested sporophores were swabbed with 70 per
in India. Genome sequencing of Volvariella suggests that it cent ethanol. At the junction of the pileus and stipe, tissue
is a secondarily homothallic fungus that produces brown, bits were removed aseptically, surface sterilized with 70 per
thick walled and multinucleated asexual chlamydospores cent ethanol for 30 sec and repeatedly washed in sterile
and sexual basidiospores. (Chang, 1969 and Bao et al., 2013). water and placed on PDA medium taken in sterile Petri
Chlamydospores are produced by many mushroom fungi dishes. The dishes were incubated at 30 to 35ºC for 7 d.
and represent thick walled vegetative cells with varied forms Following single hyphal tip method (Rangasamy, 1972) pure
of condensed cytoplasm normally formed at hyphal tips cultures were made and stored in PDA slants to carry out
(on apical cells) or within hyphae. Commonly, further studies.
chlamydospores of Volvariella are uniformly thickened
spherical with an average diameter of 58.8 μ (Chang and Effect of Carbon (C) and Nitrogen (N) Nutrition
Yau, 1971). Monosporous isolates of paddy straw To select the best source of carbon and nitrogen
mushroom vary in their growth rate, abundance of mycelia, nutrition that would induce the growth and chlamydospores
aerial hyphae and presence of chlamydospores. Chang et formation of V. volvacea strain CBE TNAU 1505 and V.
al., 1981. The selection of strains is normally done based bombycina strain CBE TNAU 1406, some of the C sources
on the earliness in mycelial growth and chlamydospores like glucose, fructose, sucrose, mannitol and sorbitol and
production. A positive correlation has been established N sources like ammonium nitrate, sodium nitrate, potassium
with the yield potential and chlamydospores production of nitrate, glycine and calcium nitrate were substituted
Volvariella (Chang, 1972). Mannitol contributes up to 20 separately in the basal Czapek’s dox medium (Thom and
per cent of the mycelial dry weight which further increases Raper, 1945). Conical flasks containing the nutrient
dramatically to 30-50 per cent in differentiating sporophores substituted medium were steam sterilized at 1.46 kg / cm2
198
NANNAPANENI et al., Studies on the Influence of Carbon and Nitrogen Nutrition on the Chlamydospores Production 8431
Table 1. Carbon nutrition and chlamydospores production by V. volvacea strain (CBE TNAU 1505)
C source Radial DTTCPP* Aerial Colony DTFCP* Chlamydospores Micrometric observations#

growth hyphae* morphology* density*
in Hyphal Chlamydospore
mm* diameter diameter (μm)
(6 (μm)
DAI)
Mannitol 42.1c 12.6c ++ Thin 15.8b ++ 6.0 26.4
transparent
radiating
Sorbitol 81.7b 8.9b +++ Thick 15.2b ++ 5.2 24.0
projecting
Dextrose 35.0d 14.5d + Thin strandy 19.9c + 4.7 22.5
Fructose 28.3e 22.6e - Thin 24.8d + 4.3 23.8

transparent
Sucrose 88.2a 7.5a ++++ Thick fluffy 14.5a +++ 5.8 25.3
CD (P = 4.8 0.4 0.6

0.05)
Aerial hyphae, chlamydospores density “- to ++++” absent to highly densed,

DTTCPP - Days taken to cover 90 mm Petri plate.
*, # Values are mean of 4 and 25 replications.
Means followed by a common letter are not significantly different at P = 0.05 by one way ANOVA.
for 20 min. After cooling, 0.3 ml of streptomycin sulphate incubated at 30 - 35°C. Visual observations on the radial
(500 ppm) was incorporated to 100 ml of the medium to mycelial growth, colony characters, and formation of
avoid bacterial contamination. The medium was aseptically chlamydospores were recorded.
poured in 90 mm Petri dishes. A 9 mm mycelial disc of Statistical Analysis
specific strain was separately inoculated aseptically in
Statistical software AGRES (Developed by Dept. of
replicates on medium amended with respective carbon and
Physical Science, TNAU, Coimbatore) was used for analysis
nitrogen sources. The inoculated Petri dishes were
Table 2. Carbon nutrition and chlamydospores production by V. bombycina strain (CBE TNAU 1406)
C source Radial DTTCPP* Aerial Colony DTFCP* Chlamydospores Micrometric observations#
Hyphal Chlamydospore
in mm*
diameter diameter (μm)
(8 DAI) (μm)
Mannitol 77.0b 9.7c + Thin 8.7c +++ 5.7 24.2
transparent
irregularly
projecting
Sorbitol 88.0a 9.1b + Thin 8.3b +++ 5.0 24.0
uniformly
projecting
Dextrose 46.7c 12.9e - Thin wavy 11.4e ++ 4.6 26.4
b d
Fructose 71.7 12.4 - Thin 10.2d ++ 4.9 25.1
Sucrose 89.5a 8.6a ++ Thin fluffy 7.8a ++++ 5.9 26.7
CD (P = 0.05) 8.1 0.3 0.3

DTTCPP – Days taken to cover 90 mm Petri plate.
*, #Values are mean of 4 and 25 replications.
199
8432 Advances in Life Sciences 5(19), 2016
Table 3. Nitrogen nutrition and chlamydospores production by V. volvacea strain (CBE TNAU 1505)
N source Radial DTTCPP* Aerial Colony DTFCP* Chlamydospores Micrometric observations#
in
mm*
(μm)
(6
DAI)
Ammonium 86.2a 7.3b ++ Thick strandy 14.3b +++ 5.6 25.4
nitrate
Calcium 83.7a 8.4c - Thin 15.2c ++ 5.1 25.8
nitrate transparent
Potassium 89.5a 6.6a ++++ Thick fluffy 13.6a ++++ 6.0 26.2
nitrate
Sodium 73.7b 9.8d - Thin 15.9d ++ 4.9 21.2
nitrate radiating
Glycine 57.2c 11.4e + Highly thin 17.2e + 4.7 23.8
transparent
CD (P = 8.2 0.3 0.6
0.05)

of data obtained in the experiment. All the visual RESULTS AND DISCUSSION
observation parameters were carried out in 4 replications
Five carbon and nitrogen sources were used to check
whereas; micrometric observation parameters were carried
the growth and chlamydospores production of V. volvacea
out in 25 replications. In case of zero values the data was
strain CBE TNAU 1505 and V. bombycina strain CBE TNAU
log transformed (X+0.5) before statistical analysis.
1406. The readings were taken at sixth day after inoculation
Table 4. Nitrogen nutrition and chlamydospores production by V. bombycina strain (CBE TNAU 1406)
Nitrogen Radial DTTCPP* Aerial Colony DTFCP* Chlamydospores Micrometric observations#

source growth hyphae* morphology* density*
in
mm*
(μm)
(8
DAI)
Ammonium 89.5a 10.5b +++ Thick 9.2b +++ 5.7 26.1
nitrate
Calcium 89.2a 11.2c + Thin 9.9c ++ 5.4 24.5
nitrate transparent
Potassium 90.0a 9.4a ++ Thick strandy 8.2a ++++ 6.6 24.8
nitrate
Sodium 79.5b 12.7d - Thin 11.7d ++ 6.3 25.2
nitrate transparent
Glycine 75.5c 13.5e - Thin irregular 13.7e + 4.8 23.4
transparent
CD (P = 4.2 0.4 0.3
0.05)
Aerial hyphae, chlamydospores intensity “- to ++++” absent to highly densed.

200
Fig. 1. Carbon and Nitrogen nutrition and chlamydospore production of V. volvacea strain CBE TNAU 1505
in case of CBE TNAU 1505 and at eighth day after influence on chlamydospores production of V. volvacea
inoculation in case of CBE TNAU 1406. and V. bombycina strain CBE TNAU 1505 and CBE TNAU
Carbon nutrition and chlamydospores production 1406. Among which, sucrose amended medium had shown
the maximum radial growth of 88.2 mm and complete growth
Various kinds of monosaccharides, disaccharides and of 90 mm was reached in 7.5 d, followed by sorbitol (81.7
sugar alcohols were used as carbon sources to test their mm and 8.9 d) and mannitol (42.1 mm and 12.6 d) amendments.
201
Fig. 2. Carbon and Nitrogen nutrition and chlamydospore production of V. volvacea strain CBE TNAU 1406
Fructose supplemented medium showed minimum radial In case of V. bombycina strain CBE TNAU 1406,
growth of 28.3 mm, which had taken 22.6 d for completing sucrose amended medium showed the maximum radial
90 mm growth in Petri plates. Sucrose amended medium growth of 89.5 mm and reached the growth of 90 mm in Petri
took 14.5 d for chlamydospores formation followed by plates in 8.6 d, followed by sorbitol (88 mm and 9.1 d) and
sorbitol (15.2 d), mannitol (15.8 d), dextrose (19.9 d) and mannitol (77 mm and 9.7 d). Dextrose amended medium
fructose (24.2 d), respectively. showed minimum radial growth of 46.7 mm, which took 12.9
202
d for completing 90 mm growth. Sucrose amended medium sources like asparagine and glutamic acid were the best
took 7.8 d for chlamydospores formation followed by sources for the growth of V. diplasia. Normally, some fungus
sorbitol (8.3 d), mannitol (8.7 d), fructose (10.2 d) and dextrose prefers nitrate form of nitrogen while, others be partial to
(11.4 d), respectively. The results of the experiment are ammonical form still, others can utilize neither nitrate nor
presented in Table 1 and 2 and the microscopic observations ammonia and may require an organic nitrogen containing
were presented in Fig 1 and 2. In the present inquest sucrose compound. This requisite for organic nitrogen may actually
was found to be best carbon source for chlamydospores be a requirement for a specific aminoacid. A fungus that
production and growth of both the strains, followed by can utilize nitrate will also harness ammonium and organic
sorbitol. The results partially support the findings of nitrogen compounds for its nitrogen requirements (Chang
Rangasamy (1956), who had tested various carbon sources and Miles, 1982). Fascinatingly, in the present investigation,
to induce the growth and morphogenesis of V. diplasia V. volvacea strain CBE TNAU 1505 and V. bombycina strain
and revealed that the highest biomass production was from CBE TNAU 1406 flaunted preference for both nitrate and
starch supplemented broth followed by sucrose. Besides, ammonical form of nitrogen (potassium nitrate followed by
Prabhu (2006) found that sugar alcohols such as sorbitol ammonium nitrate). When nitrate nitrogen in the substrate
and mannitol greatly encouraged the biomass and was preferred by the organism, it resulted in alkaline pH of
chlamydospores production by V. volvacea strain PS1. the medium by the release of excess cations. In contrast,
Jyothi and Anitha (2014) accustomed sucrose rich substrates preference of ammonical nitrogen resulted in acidic medium
such as sugar cane baggase along with paddy straw (1:1) by the increased release of anions (Kurtzman and Chang,
to increase the yield of Volvariella sp. This work further 1982). Hereby, further study by involving different carbon
substantiates the superiority of sucrose as C source for the and nitrogen sources on the chlamydospores production
growth and chlamydospores production of Volvariella spp. is much warranted for commercial exploitation of Volvariella.
Nitrogen nutrition and chlamydospores production ACKNOWLEDGEMENT
Among the different N sources evaluated with V. The authors express their gratitude to Department of
volvacea strain CBE TNAU 1505, potassium nitrate (89.5 Plant Pathology, TNAU, Coimbatore for the valuable
mm, 6.6 d), ammonium nitrate (86.2 mm, 7.3 d) and calcium support rendered during the course of investigation.
nitrate (83.7 mm, 8.4 d) were found to be on par in terms of LITERATURE CITED
radial growth and days taken to cover 90 mm in Petri plates,
respectively, followed by sodium nitrate (73.7 mm, 9.8 d); Ahlawat, O.P. and Singh, M. 2014. Cultivation of Volvariella
bombycina, a temperate mushroom species. ICAR NEWS,
whereas, minimum growth was observed in glycine
20(4): 1-3.
amended medium (57.2 mm, 11.4 d). KNO3 amended medium
Banerjee, M., Banerjee, P. and Samajpati, N. 1990. Environmental
showed the chlamydospores formation in 13.6 d, followed
factors and nutritional requirements on spore germination and
by NH4NO3 (14.3 d) and CaNO3 (15.2 d). Glycine amended germ tube growth of Volvariella diplasia. Mush. J. Tropics, 10:
medium had shown chlamydospores after 17.2 DAI. 40-46.
In case of V. bombycina strain CBE TNAU 1406, KNO3 Banerjee, M.M. and Samajpati, N. 1989. Effect of some
(90mm, 9.4 d), NH4NO3 (89.5 mm, 10.5 d) and CaNO3 (89.2 environmental factors and exogenous nutritional source on the
mm, 11.2 d) were found to be on par in terms of radial growth protein content of Volvariella diplasia in submerged culture.
and days taken to complete 90 mm in Petri plates. This was Mushroom J. tropics, 9: 139-146.
followed by NaNO3 (79.5 mm, 13.5 d). The least mycelial Bao, D., Gong, M., Zheng, H., Chen, M., Zhang, L., Wang, H., Jiang,
growth was observed in the medium amended with glycine J., Wu, L., Zhu, Y., Zhu, G., Zhou, Y., Li, C., Wang, S., Zhao, Y.,
(75.5, 12.7 d). KNO 3 amended medium showed Zhao, G. and Tan, Q. 2013. Sequencing and Comparative Analysis
of the Straw Mushroom (Volvariella volvacea) Genome. PLoS
chlamydospores formation in 8.22 d followed by NH4NO3
ONE, 8(3): e58294.
(9.2 d) and CaNO3 (9.9 d). Glycine amended medium showed
chlamydospores after 13.7 DAI. The results of the Chang, S.T. 1969. A cytological study of spore germination of
Volvariella volvacea. Bot. Mag. Tokyo, 82: 102-109.
experiment are presented in Table 3 and 4 and the
microscopic observations were presented in Fig 1 and 2. Chang, S.T. 1972. The cineaste mushroom (V. volvacea). In:
Morphology, cytology, genetics, nutrition and cultivation. The
Nitrogen is the prime constituent of the amino acids chinese university press, Hong Kong, pp. 14-35.
which make up the proteins and polysaccharide chitin, a Chang, S.T. and Yau, C.K. 1971. Volvariella volvacea and its life
cell wall component of many fungi (Chang and Quimio, history. Amer J. Bot. 58: 552-561.
1982). Darlington and Scazzocchio (1967) chronicled that if Chang, S. T., and Miles, P.G. 1982. Introduction to mushroom science.
one nitrogen source was utilized by a fungus for a long In: Tropical mushrooms: biological nature and cultivation
time, the pH of the medium would rise. Later, Sprent (1987) methods. The Chinese Univ. Press, Hong Kong, Pp. 3-6.
first reported the association between pH and nitrogen Chang, S.T. and Quimio, T.H. 1982. Biological nature and cultivation
metabolism. Amidst the different nitrogen sources used the methods.
strains CBE TNAU 1505 and CBE TNAU 1406 preferred In: Tropical Mushrooms, Chinese University Press.
potassium nitrate followed by ammonium nitrate for mycelial Chang, S.T., Miles, P.F. and Wai, C.C. 1981. A study of monosporous
growth and chlamydospores formation. But, glycine was isolates of
found to be the best nitrogen source for biomass production
Volvariella volvacea. International society for mushroom science,
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(1997) perspicuously indicated that organic nitrogen
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substrate-sensitivity of mutants in the analysis of purine uptake Nadu Agricultural University, Coimbatore, India. p. 87.
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Received on 14-09-2016 Accepted on 18-09-2016
204
International Journal of Medicinal Mushrooms, 20(9):825–835 (2018)
Effect of Headspace and Trapped Volatile Organic

Compounds (VOCs) of the Chinese Caterpillar
Mushroom, Ophiocordyceps sinensis (Ascomycetes),
against Soil-Borne Plant Pathogens
C. Sangeetha, A.S. Krishnamoorthy,* N. Kiran Kumar, & I. Arumuka Pravin
Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India
*Address all correspondence to: A.S. Krishnamoorthy, Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, Tamil
Nadu 641003, India; [email protected] or [email protected]
ABSTRACT: Headspace volatile metabolites produced by Ophiocordyceps sinensis were tested against soil-borne
plant pathogens (namely, Fusarium oxysporum f. sp. lycopersici, F. oxysporum f. sp. cubense, Thanatephorus cuc-
umeris, Athelia rolfsii, and Macrophomina phaseolina). Diffusible volatile metabolites produced by O. sinensis
inhibited 52% and 48% of the mycelial growth of F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. cubense,
respectively. In addition to inhibiting mycelial growth, the headspace volatile metabolites also induced several mor-
phological changes in the culture characteristics and mycelia of the tested fungi. Stunted and depressed colony growth
was observed for F. oxysporum f. sp. cubense and F. oxysporum f. sp. lycopersici. The headspace volatile compounds
produced by O. sinensis were trapped in a glass cartridge (Porapak Q). The trapped compounds were eluted from
WKHFROXPQE\XVLQJKH[DQHDQGWKHQE\XVLQJJDVFKURPDWRJUDSK\±PDVVVSHFWURPHWU\ZHUHLGHQWL¿HGDVWHWUDWHW-
racontane, 1(2H)-naphthalenone, 3, 4-dihydro-3-methyl, 3-hexenoic acid, 1-methyl-3-ethyladamantane, and phenol,
3-ethyl.
KEY WORDS: air entrainment technique, GC-MS, medicinal mushrooms, mushroom complete medium,
Ophiocordyceps sinensis, volatile organic compounds
ABBREVIATIONS: GC-MS, gas chromatography–mass spectrometry; MC, Mushroom Complete; PDA, potato dextrose agar;
PI, percentage inhibition; RT, retention time; VOC, volatile organic compound
I. INTRODUCTION
Soil-borne plant pathogens are the causal agents of seedling blight, root rot, and vascular wilt in almost all
plants and can result in heavy crop losses. These organisms persist in soil for long periods and produce resilient
survival structures such as chlamydospores and sclerotia.1 Complete control of soil-borne fungal pathogens
LVGLI¿FXOWWRDFKLHYHZLWKFRQYHQWLRQDOPHWKRGV5HOLDEOHFKHPLFDOVDUHODFNLQJIXQJLFLGDOUHVLVWDQFH
occurs, and pathogens can circumvent host resistance—these are some of the critical constraints underlying
efforts to develop new molecules for managing crop diseases. Stories of success with strobilurins, a group
of environmentally safe natural products with a wide spectrum of fungicidal activity and low mammalian
toxicity, were the inspiration for the current investigation. Strobilurins have been obtained from the wood-
inhabiting mushrooms Strobilurus tenacellus and Oudemansiella mucida and are commercially marketed
DVD]R[\VWURELQȕPHWKR[\DFU\ODWHVHOHFWHGIURPDPRQJFRPSRXQGV2,3
The Chinese caterpillar mushroom, Ophiocordyceps sinensis (Berk.) G.H. Sung et al. (=Cordyceps
sinensis; Ophiocordycipitaceae, Ascomycetes), was selected for use in this research; the mushroom parasit-
izes the larvae of ghost moths (Hepialus armoricanus).4,5 The anamorphic state(s) of the genus Cordyceps
(namely, Beauveria spp., Metarhizium spp., and Paecilomyces spp.) have been reported as biocontrol
agents against insect pests.6 In addition, the fungus produces volatile compounds including aldehydes
1521-9437/18/$35.00 © 2018 Begell House, Inc. www.begellhouse.com 825
205
826 Sangeetha et al.
(benzaldehyde, benzene acetaldehyde, nonanal, and decanal), aromatic alcohols (phenylethyl alcohol,
2-[methylthio]-3-pyridinol, 7-octadien-1-ol, azulene, 2-6,dimethylnaphthalene, and 1,6-dimethylnaphtha-
lene), and phenols (2-methyl-phenol, butylated hydroxyl toluene).7 Exploration of such volatile compounds
shows their promise for use in the management of soil-borne plant pathogens, nematodes, and harmful
VRLOLQVHFWV7KHUHIRUHWKLVLQYHVWLJDWLRQH[DPLQHGWKHHI¿FDF\WHVWHGXQGHUin vitro conditions, of volatile
organic compounds (VOCs) produced by O. sinensis against soil-borne plant pathogens.
II. MATERIALS AND METHODS
A. Culture Collection
O. sinensis LVRODWHZDVREWDLQHGIURPWKH)RUHVW5HVHDUFK,QVWLWXWH'HKUDGXQ8WWDUDNKDQG,QGLD
Pure cultures of Fusarium oxysporum f. sp. lycopersici (Sacc.) Synder & Hansen, F. oxysporum f. sp.
cubense (E.F. Smith) Synder & Hansen, Macrophomina phaseolina (Tassi) Goid., Thanatephorus cuc-
umeris (A.B. Frank) Donk, and Athelia rolfsii (Curzi) C.C. Tu & Kimbr. were obtained from the culture
collection facility at the Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore,
Tamil Nadu, India.
B. Effect of Headspace Volatile Metabolites of O. sinensis against Plant

Pathogenic Fungi
O. sinensis was grown on mushroom complete medium and incubated at 25°C under alternate light
(600–800 lux) and dark conditions. The volatile metabolites produced by O. sinensis were tested by
using the inverted plate technique.8 The center of each Petri plate containing potato dextrose agar (PDA)
was inoculated with 6-mm mycelial discs of F. oxysporum f. sp. lycopersici, F. oxysporum f. sp. cubense,
M. phaseolina, T. cucumeris, and A. rolfsii. Over these plates were inverted bottom plates of the same
size that contained actively growing 5-day-old culture of O. sinensis; the cultures were facing. Then the
SODWHVZHUHVHDOHGZLWKFOLQJ¿OP7KH3HWULSODWHVZLWKRXWO. sinensis at the top served as the controls,
and each treatment was replicated 3 times. The radial growth of the pathogen was measured constantly
until the control plate showed 90 mm of growth. The percentage inhibition (PI) was calculated by using
the following formula9:
C T
Growth inhibition (%) = u 100
C
where C is the growth of the pathogen (in millimeters) in the control plates and T is the growth of pathogen
(in millimeters) in the treated plates.
C. Trapping of Volatile Metabolites Produced by O. sinensis
The volatile metabolites produced by O. sinensis were trapped through the use of the air-entrainment
technique.10 Headspace volatile compounds were collected by using a special setup comprising a glass
container closed tight with a lid. The lid had 2 holes, one acting as an inlet and the other as an outlet.
%HIRUHWKHVWDUWRIWKHH[SHULPHQWWKHP/JODVVFKDPEHUFPORQJDQGFPZLGHZDV¿OOHG
with 100 mL sterile Mushroom Complete (MC) broth and inoculated with discs of O. sinensis mycelia.
7KHOLGZDVFORVHGZLWKDUXEEHUFRUNWKDWFRQWDLQHGKROHVDQGZDVVHDOHGWLJKWO\ZLWKSDUD¿OP7KH
MC broth without fungus in the enclosed container served as the control. An aquarium pump was used to
SXPSSXUL¿HGDLULQWRWKHFRQWDLQHU)LJ7KHDLUZDVSXUL¿HGWKURXJKDJODVVFDUWULGJHFRQWDLQLQJ
International Journal of Medicinal Mushrooms
206
Effect of Ophiocordyceps sinensis against Soil-Borne Plant Pathogens 827
FIG. 1: Air-entrainment technique used to collect volatile compounds produced by Ophiocordyceps sinensis:
aquarium pump (a), Porapak Q (b), air inlet (c), O. sinensis culture in mushroom complete medium (d), and the
glass cartridges containing 50 mg Porapak Q (e)
mg activated charcoal, then passed through the inlet hole in the cork. A glass cartridge containing 50 mg
Porapak Q adsorbent was connected to the outlet hole. The headspace volatile compounds pumped out
by the air pressure were collected in the glass cartridge containing the Porapak Q. The experiment was
run for 15 days, and the column containing the collected volatiles was washed 4 times with 2 mL high-
performance liquid chromatography–grade hexane and the washed volatiles were collected in an airtight
glass tube.
D. Bioassay of Trapped Volatiles against Plant Pathogenic Fungi
The hexane fraction of trapped volatiles was tested against F. oxysporum f. sp. lycopersici, F. oxysporum
f. sp. cubense, M. phaseolina, T. cucumeris, and A. rolfsii by using poisoned food. Sterilized molten PDA
medium (100 mL) was mixed separately with the hexane fraction at 1000 ppm, poured into sterile Petri plates,
and allowed to solidify. A 6-mm mycelial disc of the test pathogen was introduced at the center of the poi-
soned medium, and the Petri plates were incubated at 28 ± 2°C. The plates were observed constantly, and
the PI of mycelial growth was recorded and compared with that of the control. Hexane without trapped
volatiles served as the negative control.
E. Detection of Bioactive Molecules through Gas Chromatography–Mass Spectrometry

Analysis
The trapped volatile compounds produced by 15-day-old cultures of O. sinensisZHUHLGHQWL¿HGE\JDV

chromatography–mass spectrometry (GC-MS) analysis through the use of a Hewlett-Packard Agilent 6890
gas chromatograph coupled to a Hewlett-Packard 5973 quadrupole mass spectrometer. We used an Elite-
06FDSLOODU\FROXPQGLPHWK\OSRO\VLOR[DQHîPPîȝPGIHTXLSSHGZLWKD&ODUXV
Volume 20, Issue 9, 2018
207
GC 500 Gas Chromatograph/Mass Spectrometer and a TurboMass Gold detector (both from PerkinElmer).
+HOLXPZDVWKHFDUULHUJDVÀRZUDWHP/PLQDQGWKHLQMHFWLRQYROXPHZDV/PLQ7KHLQLWLDO
column temperature was maintained at 110°C, attained at a rate of 10°C/min, then the temperature was
increased up to 280°C at a rate of 5°C/min; this temperature was held for 9 minutes. The electron impact
energy was 70 eV and the ion source and quadrupole temperatures were set at 230°C and 150°C, respec-
tively. Electron impact mass spectra, recorded at 1-second intervals, were in the range of 40–600 amu.
:HLGHQWL¿HGWKHFRPSRXQGVSUHVHQWLQWKHVDPSOHE\VHDUFKLQJWKH1,676WDQGDUG5HIHUHQFH'DWDEDVH
mass spectrometry data library (version 2011) and comparing the spectra obtained through GC-MS.
F. Statistical Analysis
The design of experiments (completely randomized block design) and statistical analyses were followed
as suggested by Gomez and Gomez.11:HXVHGWKHVWDWLVWLFDO$*5(6DSSOLFDWLRQGHYHORSHGDWWKH
Department of Physical Science, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India)
to analyze the data.
III. RESULTS
$(IÀFDF\RI+HDGVSDFH9RODWLOHV
The effects of headspace volatiles produced by O. sinensis against plant pathogens over a period of 10 days
are presented in Table 1 and Fig. 2. The volatile compounds produced by O. sinensis inhibited mycelial
growth of F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. cubense by 52% and 48%, respectively;
the PI of mycelial growth was 38.44% for T. cucumeris, 22.22% for M. phaseolina, and 11.77% for
A. rolfsii. In addition to inhibiting mycelial growth, the headspace volatiles also induced several morpho-
logical changes in the culture characteristics and the mycelia of the tested fungi. We observed stunted and
depressed colony growth for F. oxysporum f. sp. cubense and F. oxysporum f. sp. lycopersici.
%(IÀFDF\RI7UDSSHG9RODWLOH&RPSRXQGV
The trapped volatile compounds in hexane were aseptically mixed with PDA at a rate of 1000 ppm.
They periodically inhibited the growth and caused malformations of fungal mycelia and spores; these
data are presented in Table 1. The results revealed that the growth of F. oxysporum f. sp. lycopersici and
F. oxysporum f. sp. cubense was inhibited by 41.1% and 29.11%, respectively. T. cucumeris growth was
inhibited by 16% and that of M. phaseolina was inhibited by 2.44%.
&,GHQWLÀFDWLRQWKURXJK*&06RI92&V3URGXFHGE\O. sinensis
The volatile metabolites trapped from both the medium inoculated with the fungus (treatment) and the
noninoculated control were analyzed separately. A total of 37 VOCs were detected in the medium inocu-
lated with O. sinensis, whereas 22 compounds were detected in the control (Table 2, Figs. 3 and 4). Hence
we can conclude that the additional 15 volatile compounds found in the treated medium were produced by
the fungus, O. sinensis (Table 3). These compounds include ethyl chloride; p-cresol; phenylethyl alcohol;
phenol, 2,4-dimethyl; phenol, 3-ethyl; naphthalene; naphthalene, 1-methyl; carbamo(dithioperoxoic) acid,
WULÀXRURPHWK\OHVWHUQRUERUQDQRQHHSLWKLRHWK\OGLPHWK\O66GLR[LGHPHWK\OHWK\ODGD-
mantane; 3-hexenoic acid; 1-dodecanol, 2-octyl; dodecane; tetratetracontane; and 1(2H)-naphthalenone,
3,4-dihydro-3-methyl. Among these volatile compounds, tetratetracontane showed the largest peak area
208
TABLE 1: Inhibitory Potential of Headspace Volatiles and Trapped Volatiles from Ophiocordyceps sinensis against
Plant Pathogens
Treatment Fusarium F. oxysporum Macrophomina Thanatephorus Athelia rolfsii

oxysporum f. sp. f. sp. cubense phaseolina cucumeris
lycopersici
Growth PI Growth PI Growth PI Growth PI Growth PI
(mm) (mm) (mm) (mm) (mm)
Headspace volatiles 43.20a 52.00 46.80a 48.00 70.00a 22.22 55.40a 38.44 79.40a 11.77
Control 90.00c 0.00 90.00c 0.00 90.00b 0.00 90.00c 0.00 90.00b 0.00
Trapped volatiles 53.00b 41.11 63.80b 29.11 87.80b 2.44 75.60b 16.00 90.00b 0.00
Control (volatile from 90.00c 0.00 90.00c 0.00 90.00b 0.00 90.00c 0.00 90.00b 0.00
growth medium)
Control 90.00c 0.00 90.00c 0.00 90.00b 0.00 90.00c 0.00 90.00b 0.00
CD (P = 0.05) 1.409 — 1.165 — 1.421 — 8.189 — 6.779 —
'DWDDUHWKHPHDQRIUHSOLFDWLRQV9DOXHVVHWZLWKWKHVDPHVXSHUVFULSWOHWWHULQWKHVDPHFROXPQDUHQRWVLJQL¿FDQWO\GLI-
ferent (1-way analysis of variance). CD, critical difference; PI, percentage inhibition.
FIG. 2: Effect of headspace volatile compounds produced by Ophiocordyceps sinensis against various fungal
pathogens: Fusarium oxysporum f. sp. lycopersici (a), F. oxysporum f. sp. cubense (b), Macrophomina phaseolina
(c), Thanatephorus cucumeris (d), Athelia rolfsii (e). The 3 dishes shown for each pathogen represent different
conditions: control without O. sinensis in the bottom lid (1), growth of the pathogen on the top lid (2), and growth
of O. sinensis on the bottom lid (3).
209
TABLE 2: Organic Compounds Present in Trapped Volatiles from Ophiocordyceps sinensis Inoculated with
Medium and Not Inoculated with Medium (after 15 Days)
RT Headspace Volatile Compounds

From O. sinensis From Medium (Control)
Compound Peak Compound Peak Area
Area (%) (%)
2.101 Pentane, 2-methyl 30.67 Pentane, 2-methyl 29.07
2.527 Cyclopentane, methyl 48.93 Cyclopentane, methyl 45.98
3.627 3-Pentanol, 3-methyl 0.12 3-Pentanol, 3-methyl 0.16
4.137 2-Hexanone 0.78 2-Hexanone 0.91
5.044 Cyclopentanol, 3-methyl 0.08 Cyclopentanol, 3-methyl 0.09
5.558 Ethyl chloride 0.11 ND —
6.682 Methane sulfonyl acetic acid 0.05 Methane sulfonyl acetic acid 0.09
7.544 (N-[-2-acetamido])-2-aminoethanesu 0.05 (N-[-2-Acetamido])-2-aminoethanesu 0.13
9.133 1-Hexanol, 2-ethyl 0.34 1-Hexanol, 2-ethyl 0.25
9.792 Undecane, 5-methyl 0.09 ND —
10.339 p-Cresol 0.12 ND —
10.952 Nonanal 0.26 Nonanal 0.36
11.289 Phenylethyl alcohol 0.27 ND —
12.111 Phenol, 2,4-dimethyl 0.17 ND —
12.565 Phenol, 3-ethyl 0.23 ND —
12.986 Naphthalene 0.23 ND —
13.740 Benzaldehyde, 3,4-dimethyl 0.22 Benzaldehyde, 3,4-dimethyl 0.39
14.00 Benzothiazole 0.57 Benzothiazole 0.32
14.579 4,5-Dihydro-5-oxo-3-(p-tolyl)isoxazole 0.20 4,5-Dihydro-5-oxo-3-(p-tolyl)isoxazole 0.19
15.571 Naphthalene, 1-methyl 0.11 ND —

16.968 &DUEDPRGLWKLRSHUR[RLFDFLGWULÀXR- 0.16 ND —
romethyl ester
17.361 2-Norbornanone, 1-(epithioethyl)-7,7- 0.03 ND —
dimethyl-,
S,S-dioxide
17.838 1,2,3-Benzenetriol 0.95 1,2,3-Benzenetriol 1.83
18.784 1-Methyl-3-ethyladamantane 0.28 ND —
19.073 Nonane, 4,5-dimethyl 0.14 ND —
19.383 Cyclododecane 1.07 Cyclododecane 1.06
19.858 Nonadecane 1.17 Nonadecane 1.05
20.186 3-Hexenoic acid 0.52 ND —
20.598 Trichloroacetic acid, undec-10- 0.93 Trichloroacetic acid, undec-10- 0.11
enyl ester enyl ester
210
TABLE 2: (continued)
21.269 1-Octadecanesulphonyl chloride 1.00 1-Octadecanesulphonyl chloride 0.17

21.845 Heneicosane 1.69 Heneicosane 0.26
22.340 1-Dodecanol, 2-octyl 0.04 ND —
22.775 Dodecane 0.90 ND —
23.745 Tetratetracontane 2.94 ND —
24.253 n-Decanoic acid 2.45 n-Decanoic acid 6.65
24.891 1(2H)-Naphthalenone, 3,4-dihydro- 0.69 ND —
3-methyl
25.214 Pyrrolo[1,2-a]pyrazine-1,4-dione 0.51 Pyrrolo[1,2-a]pyrazine-1,4-dione 1.25
ND, not detected; RT, retention time.
TABLE 3: Gas Chromatography–Mass Spectrometry Analysis of Trapped Volatiles from Ophiocordyceps sinensis
RT Compound Molecular Formula MW Peak Area (%)

5.55 Ethyl chloride C2H5Cl 64.00 0.11
10.33 p-Cresol C 7H 8O 108.00 0.12
11.28 Phenylethyl alcohol C8H10O 122.00 0.27
12.11 Phenol, 2,4-dimethyl C8H10O 122.16 0.17
12.56 Phenol, 3-ethyl C8H10O 122.00 0.23
12.98 Naphthalene C10H8 28.17 0.23
15.57 Naphthalene, 1-methyl C11H10 142.20 0.11
16.96 &DUEDPRGLWKLRSHUR[RLFDFLGWULÀXRURPHWK\OHVWHU C17H13ClF3N5O2 411.70 0.16
17.36 2-Norbornanone, 1-(epithioethyl)-7,7-dimethyl-, C13H21NO 207.30 0.03
S,S-dioxide
18.78 1-Methyl-3-ethyladamantane C13H22 178.00 0.28
20.18 3-Hexenoic acid C6H10O2 114.00 0.52
22.34 1-Dodecanol, 2-octyl C20H42O 298.00 0.04
22.77 Dodecane C12H26 170.00 0.09
23.74 Tetratetracontane C44H90 619.00 2.94
24.89 1(2H)-Naphthalenone, 3,4-dihydro-3 –methyl C14H24O — 0.69
MW, molecular weight; 57UHWHQWLRQWLPH
DWDUHWHQWLRQWLPH57RI)LJIROORZHGE\+QDSKWKDOHQRQHGLK\GURPHWK\O

DWDQ57RI
IV. DISCUSSION
0DQ\VFLHQWLVWVKDYHH[WHQVLYHO\SUR¿OHGDQGUHYLHZHGYRODWLOHPHWDEROLWHVSURGXFHGE\IXQJLDQGEDF-
teria.12,13 O. sinensis has been reported to produce volatile metabolites including aldehydes, alcohols, and
211
FIG. 3: Gas chromatography–mass spectrometry analysis of volatile organic compounds from mushroom com-
plete medium broth (control)
FIG. 4: Gas chromatography–mass spectrometry analysis of volatile organic compounds produced by

Ophiocordyceps sinensis inoculated with the medium
212
FIG. 5: Gas chromatography–mass spectrometry analysis of tetratetracontane
phenols.7 Mercier and Jimenez14 found that VOCs produced by the fungus Muscodor albus inhibited the
growth of Botrytis cinerea, Colletotrichum acutatum, C. coccodes, Geotrichum candidum, Monilinia
fructicola, Penicillium digitatum, and Rhizopus spp. Dennis and Webster8 recommended an inverted
plate technique for laboratory testing of acetaldehyde-like inhibitory volatile metabolites produced by
Trichoderma viride. Our results indicate the inhibitory, mainly fungistatic action of the volatiles produced
by O. sinensis against selected plant pathogenic fungi, which ranged from 11.77% to 52% (Table 1). The
highest PI was observed for F. oxysporum f. sp. lycopersici (52%), followed by F. oxysporum f. sp. cubense
(48%); the lowest inhibition was recorded for A. rolfsii (11.77%). The headspace volatile compounds also
caused several morphological derangements in the culture characteristics and mycelia of tested fungi.
Stunted and depressed colony growth was noticed for F. oxysporum f. sp. cubense and F. oxysporum f.
sp. lycopersici. In addition to the testing of headspace volatile compounds, diffused metabolites were
trapped with the use of Porapak Q dissolved with hexane and then tested against the plant pathogenic
fungi. The trapped volatiles also inhibited the tested plant pathogens. From the results of this investiga-
tion we conclude that the volatile compounds produced by O. sinensis exhibit a pronounced fungistatic
effect. In a similar study, the biofumigant fungus M. albus was shown to produce 20 volatile compounds
with fungistatic or fungicidal properties.15–17
During this investigation, the headspace volatile compounds that accumulated across the surface of the
O. sinensis mycelial mat over a period of 15 days were trapped by a special air-entrainment technique with
WKHXVHRI3RUDSDN4WKHVHFRPSRXQGVZHUHVXEMHFWHGWR*&06DQDO\VLV,QWRWDORUJDQLFFRPSRXQGV
ZHUHWUDSSHGDQGLGHQWL¿HG7DEOH+RZHYHUZKHQFRPSDUHGZLWKWKHFRQWUROKHDGVSDFHYRODWLOH
compounds collected from the growth medium without the inoculation with the fungus over the same per-
iod), 15 volatile compounds—namely ethyl chloride; p-cresol; phenylethyl alcohol; phenol, 2,4-dimethyl;
SKHQROHWK\OQDSKWKDOHQHQDSKWKDOHQHPHWK\OFDUEDPRGLWKLRSHUR[RLFDFLGWULÀXRURPHWK\OHVWHU
2-norbornanone, 1-(epithioethyl)-7,7-dimethyl-, S,S-dioxide; 1-methyl-3-ethyladamantane; 3-hexenoic acid;
1-dodecanol, 2-octyl; dodecane; tetratetracontane; and 1(2H)-naphthalenone, 3,4-dihydro-3-methyl—were
found to be additional compounds produced by the fungus. Among these 15 compounds, p-cresol; pheny-
OHWK\ODOFRKROSKHQROGLPHWK\ODQGSKHQROHWK\ODUHVSHFL¿FSKHQROLFFRPSRXQGVWKDWDUHNQRZQ
to play important roles in protecting plants against insect pathogens and predators.18 Middleton et al.19
and Manach et al.20 DOVRLOOXVWUDWHGWKHVHFRPSRXQGV¶DQWLDOOHUJHQLFDQWLLQÀDPPDWRU\DQWLPLFURELDODQG
antioxidant activities. Entrapped compounds such as naphthalene (0.23%) and naphthalene derivatives
(naphthalene, 1-methyl [0.11%]; 1[2H]-naphthalenone, 3,4-dihydro-3-methyl [0.69%]) are known to repel
insects.21 Daisy et al.22 reported that volatile antimicrobial compounds such as naphthalene produced by
Muscodor vitigenusHIIHFWLYHO\UHSHOOHGDGXOWZKHDWVWHPVDZÀLHVCephus cinctus). In a similar way, the
FRPSRXQGKH[HQRLFDFLGGHWHFWHGDWDQ57RIKDVEHHQXVHGDVDSUHVHUYDWLYHLQEULQH
213
solution to inhibit the growth of yeast.23 The highest percentage recovery was achieved with tetratetracon-
WDQHZKLFKLVDQDONDQHH[SUHVVHGDWDQ57RI)LJ2QHVXFKFRPSRXQGH[WUDFWHGIURP
essential oil of Teucrium polium possessed antimicrobial and antioxidant activities.24 Thus, the results of
this investigation pave the way for exploring hitherto unused biomolecules of O. sinensis for management
of fungal plant pathogens. Further systematic insight into individual molecules and developing step-up
protocols to enhance their level of production and innovative formulation will give these a greater stake
in the agrochemical industry.
ACKNOWLEDGMENTS
The authors thank the Department of Plant Pathology and the Department of Agricultural Entomology for
providing the laboratory support necessary to conduct this study. The authors also thank the Department of
Science and Technology, Government of India (through the Fund for Improvement of S&T Infrastructure),
and Special Assistance Programme of the University Grant Commission for providing laboratory support
facilities.
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216


P-ISSN: 2349–8528
E-ISSN: 2321–4902
IJCS 2017; 5(3): 387-381 Volatilomes of milky mushroom (Calocybe indica
© 2017 JEZS
Received: 15-03-2017 P&C) estimated through GCMS/MS

Priyadharshini Bhupathi Priyadharshini Bhupathi and Krishnamoorthy Akkanna Subbiah
PhD Scholar, Department of
Plant Pathology, Tamil Nadu
Agricultural University, Abstract
Coimbatore, India The volatilomes of both fresh and dried samples of milky mushroom (Calocybe indica P&C var. APK2)
were characterized with GCMS/MS. The gas chromatogram was performed with the ethanolic extract of
Krishnamoorthy Akkanna the samples. The results revealed the presence of increased levels of 1, 4:3, 6-Dianhydro-Į-d-
Subbiah glucopyranose (57.77%) in the fresh and oleic acid (56.58%) in the dried fruiting bodies. The other
Professor and Head, Department important fatty acid components identified both in fresh and dried milky mushroom samples were
of Plant Pathology, Tamil Nadu octadecenoic acid and hexadecanoic acid, which are known for their specific fatty or cucumber like
Agricultural University, aroma and flavour. The aroma quality of dried samples differed from that of fresh ones with increased
Coimbatore, India levels of n- hexadecenoic acid (peak area - 8.46 %) compared to 0.38% in fresh samples. In addition, Į-
D-Glucopyranose (18.91%) and ergosterol (5.5%) have been identified in fresh and dried samples
respectively. The presence of increased levels of ergosterol indicates the availability of antioxidants and
anticancer biomolecules in milky mushroom, which needs further exploration. The presence of Į-D-
Glucopyranose (trehalose) components reveals the chemo attractive nature of the biopolymers of milky
mushroom, which can be utilized to enhance the bioavailability of pharmaceutical or nutraceutical
preparations.
Keywords: GCMS/MS, Milky mushroom (Calocybe indica P&C var. APK2), Octa and hexadecanoic
acid, Trehalose, Volatilomes
1. Introduction
Mushrooms are known to produce a wide range of volatile and flavour compounds with
distinct profiles that may vary according to the species, variety and sometimes due to cultural
conditions (Rapior et al. 1997) [21]. The flavour profile also changes when mushrooms are
dried, primarily due to high level of oxidation (Morath et al. 2012) [17]. Solvent extraction,
vacuum distillation, nitrogen flow conveyance and capillary gas chromatography are normally
followed for the concentration of flavour compounds. Depending upon the extraction method,
nature of sample and sample preparation procedure significant changes in flavour profiles of
mushrooms have been reported (Beltran-Garcia et al. 1997) [2]. Analyzing the complete
volatilomes of freshly harvested samples of Calocybe indica sporophores, (Chandravadana et
al. 2005) [4] confirmed the presence of an eight carbon volatile compound, 1-octen-3-ol
(58.3%) and n-octanol (17.9%). They also concluded that the concentration of these
compounds decreased up to 10.6% and 2.4%, respectively after drying. Noticeably, benzyl
alcohol and n-Hexanal present in traces in the fresh mushroom samples increased to about
10.2% and 15.3%, respectively after drying.
Linoleic and palmitic acids were the predominant fatty acid fractions reported in oyster
mushroom (Pleurotus florida) samples by Kwon and Uhm (1984) [12]. The involvement of
these compounds in fungal aroma, interactions with pests, pathogens and reproductive events
have been elaborately reviewed by Combet and his co-workers. In most the fungi, it is
understood that linoleic acid is oxidized to form a 10-hydroperoxide intermediate, which is
then cleaved to form an eight-carbon volatile (1-octen-3-ol) and a ten-carbon oxoacid (10-
ODA) by Wurzenberger and Grosch (1984c) [26]. The 10-Oxodecanoic acid (10-ODA)
possesses hormone-like properties accelerating the growth of mushroom stipe and
Correspondence development of fungal structures. Hence, it is suggested that both 1-octen-3-ol and 10-ODA
Priyadharshini Bhupathi could work together to regulate the transition between vegetative and reproductive growth in
PhD Scholar, Department of fungi (Champavier et al. 2000) [3]. Trehalose released by the mycelium of Laccaria bicolor
Plant Pathology, Tamil Nadu
Agricultural University,
was shown to be a chemo attractant for pseudomonads (Frey-Klett et al. 2007) [8].
Coimbatore, India
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217

The aroma compounds of fresh and dried samples of C. ratio of 10:1). The column oven temperature program is given
gambosa were extracted by an aroma extract dilution analysis below: 110 °C hold for 2 min; up to 200 °C @ 10 °C per min-
(AEDA) system to identify the key flavour compounds and no hold; up to 280 °C @ 5 °C per 10 min hold. The injector
the total volatilomes were quantified by GC flame ionization temperature was maintained at 280 °C and the total GC
detection by Kleofas et al. (2015) [11]. The key odour running time was 38.50 min. This last increase was to clean
compound detected in the fresh sample was (E)-non-2-enal, the column from any residues. The mass spectrometer was
which, together with (E)-non-2-en-1-ol was found to be operated in the positive electron ionization (EI) mode with
responsible for the characteristic flavour and cucumber-like ionization energy of 70eV. The solvent delay was 0-3.5 min.
odour. However, in the dried fruiting bodies, odour compound A scan interval of 0.5 sec. and fragments from m/z 50 to 500
like 3- methylbutanoic acid were dominating and (E)-non-2- Da was programmed. The inlet temperature was set at 290 °C,
enal was not at all detected. Hence, the present study was source temperature at 250 °C and total running time was
undertaken to investigate on the volatilomes of milky 37.50 min. The relative percentage of each component was
mushroom, Calocybe indica P&C var. APK2 through GC- calculated by comparing its average peak area to the total
MS/MS flavour spectrum. areas. Software adopted to handle mass spectra and
chromatograms was MS Work station 8. The NIST Version
2. Materials and Methods 2.0 library database of National Institute Standard and
Samples of fresh milky mushroom sporophores, C. indica Technology (NIST) having more than 62,000 patterns was
P&C var. APK2 grown on paddy straw substrate were used for identifying the chemical components. The spectrum
collected from the Mushroom Research and Training Centre, of the unknown component was compared with the spectrum
Department of Plant Pathology, Tamil Nadu Agricultural of the known components stored in the NIST library. The
University, Coimbatore, India. The fresh experimental name, molecular weight and structure of the components of
samples included mixed preparation of both pileus and stipe, the test materials were ascertained.
collected at harvest stage from five randomly selected
mushrooms. For making dried mushroom samples, freshly cut 3. Results and Discussions
sporophores were oven dried at 55o C for 6 h, until the The major volatilomes identified through GC-MS/MS in fresh
moisture content of the sample reached 12 per cent. The dried and matured fruiting bodies at harvest stage of milky
samples were macerated with liquid nitrogen to obtain mushroom was 1,4:3,6-Dianhydro-Į-d-glucopyranose with
powdered form for the extraction of flavour compounds. the highest peak area of 57.77% followed by Į,ȕ-Gluco-
octonic acid lactone recording a peak area of 22.16% (Table 1
2.1 Extraction of volatilomes and Fig.2). European Food Safety Authority (2010)
Volatile compounds from the samples were extracted determined that the 1,4:3,6-Dianhydro-Į-d-glucopyranose is
following the method suggested by Srinivasan and Kumaravel one of the smoke flavouring primary product, which was
(2015) [23]. A sample size of 30 g of fresh or dried and obtained from beech wood sawdust (Fagus grandifolia) used
powdered milky mushroom tissue was soaked in 30 ml of for preservation purposes and they demonstrated that the
ethanol overnight and then filtered through a rough filter smoke flavour is produced by controlled thermal degradation
paper. The filtrate was then concentrated to one ml by of wood in a limited supply of oxygen (pyrolysis). It has also
flushing nitrogen gas into the solution. The concentrate along been sufficiently demonstrated that, it does not cause risk to
with 2g sodium sulfate (used to remove the sediments and human health. The most abundant compound that was found
traces of water in the filtrate) was once again filtered through to be present in dry milky mushroom was oleic acid, which
Whatman No.41 filter paper. The final sample was subjected showed the highest peak area of 56.58% followed by 9-
to volatilomes analysis. Octadecenoic acid, methyl ester, (E)- with the peak area of
16.94 % (Table 2 and Fig 2). In addition to these compounds
2.2 GC-MS/MS analysis (both in fresh and dried milky mushroom samples) different
GCMS/MS analysis of the samples through electron types of terpenes, alcohols, fatty acids and sterols. The most
ionization (GC-MS/EI) mode was performed at Food Safety abundant compound was present in dry milky mushroom was
and Quality Testing Laboratory, Indian Institute of Crop oleic acid with the highest peak area of 56.58% followed by
Processing Technology, Thanjavur, Tamil Nadu. The GC- 9-Octadecenoic acid, methyl ester, (E)- with the peak area of
MS/MS (Scion 436-GC Bruker model) coupled with a triple- 16.94 % (Table 2 and Fig. 2). Pedneault et al. (2006) [19]
quadruple mass spectrophotometer with fused silica capillary reported that oleic acid is a major component available in
column BR-5MS (5% diphenyl / 95% dimethyl polysiloxane); some mushrooms that belong to the genus Boletus. This
length-30m; Internal diameter-0.25 mm; thickness- 0.25ȝm bioactive compound is known to strongly inhibit the activity
was used during the experimentation. Helium gas (99.99%) of human telomerase in a cell-free enzyme assay (Masako et
was used as the carrier gas at a constant flow rate of one ml al. 2002) [14]. Further, Won et al. (2007) [25] proved that, it is
per min and an injection volume of 2 ȝl was employed (split an efficient inhibitor of glucosyl transferase.
Table 1: Volatile compounds identified in the freshly harvested milky mushroom
Molecular Molecular Peak
No. RT Name of the compound
Formulae Weight Area %
1. 6.55 1,4:3,6-Dianhydro-Į-d-glucopyranose C6H8O4 144 57.77
2. 10.25 Į-D-Glucopyranose, 4-O-ȕ-D-galactopyranosyl- C12H22O11 342 18.91
3. 12.33 Į,ȕ-Gluco-octonic acid lactone C8H14O8 238 22.16
4. 13.11 Undecanoic acid, 11-mercapto- C11H22O2 S 218 0.04
5. 13.92 Pyrano[4,3-b]benzopyran-1,9-dione, 5a-methoxy-9a-methyl-3-(1-propenyl)perhydro- C17H2O5 308 0.04
6 14.29 10-Undecynoic acid, trimethyl ester C14H26O2 254 0.20
7. 15.53 n-Hexadecanoic acid C16H32O2 256 0.38
8. 23.79 Glycerol 1-palmitate C19H38O4 330 0.36
9. 26.83 d-Galactose, 1,2:3,4-di-O-isopropylidene-, 6-decanoate C22H38O7 414 0.13
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218

Table 2: Volatile compounds identified in the dried milky mushroom in powder form
Molecular Molecular Peak Area
No. RT Name of the compound
Formulae Weight %
1. 6.08 3-Hydroxymethyl-2-trimethyl pentane C9H22O2Si 190 0.54
2. 9.39 Allyl (2tetrahydrofurylmethoxy) dimethyl C10H20O2 200 0.78
3. 11.72 ȕ-D-Glucopyranose, 4-O-ȕ-D-galactopyranosyl- C12H22O11 342 0.22
4. 14.30 10-Undecynoic acid, trimethyl ester C14H26O2 254 0.15
5. 15.10 Hexadecanoic acid, methyl ester C17H34O2 270 0.55
6. 15.68 n-Hexadecanoic acid C16H32O2 256 8.46
7. 17.50 9-Octadecenoic acid (Z)-, methyl ester C19H36O2 296 1.23
8. 18.28 Oleic Acid C18H34O2 282 56.58
9. 18.47 9-Octadecenoic acid, methyl ester, (E)- C19H36O2 296 16.94
10. 22.68 cis-13-Eicosenoic acid C20H38O2 310 1.08
11. 23.54 Glycerol 1-palmitate C19H38O4 330 1.20
12. 24.64 9,10-Secocholesta-5,7,10(19)-triene-3,24,25-triol, (3ȕ,5Z,7E)- C27H44O3 416 1.06
13. 26.15 9-Octadecenoic acid (Z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester C21H40O4 356 2.99
14. 29.80 9(11)-Dehydroergosteryl benzoate C35H46O2 498 1.33
15. 33.51 9(11)-Dehydroergosterol tosylate C35H48O3S 548 0.68
16. 34.38 Ergosterol C28H44O 396 5.51
17. 34.76 Ergosta-14,22-dien-3-ol, (3ȕ,5Į,22E)- C28H46O 398 0.71
The other important compounds found both in dry and fresh formed by two molecules of Į-D-glucopyranose bound by 1-1
mushrooms were Ergosterol (5.51%) and sugar related glycosidic bond) in those mushrooms. These water soluble
compounds like Į-D-Glucopyranose (18.91%), respectively. sugars partly contributed to the taste of such mushrooms. He
Ergosterol is the principal sterol of the cell membrane of fungi also concluded that these sugars could participate to supply a
by Czub and Baginski (2006) [5] is known to activate considerable proportion of C for the growth and firmness of
expression of a number of defense genes and increase the fruiting bodies. Trehalose was found to have growth-
resistance of plants against the pathogens by Lochman and promoting effects on the mycorrhization helper bacteria
Mikes (2006) [13]. The ergosterol is also an important dietary (MHB), Pseudomonas monteilii, when inoculated with the
source of vitamin D. Novaes et al. (2011) [18] reviewed that ECM fungus Pisolithus albus (Duponnois and Kisa, 2006) [6].
the ergosterol or provitamin D2 is the precursor of The chromatogram of the ethanolic extract of fresh and dry
ergocalciferol, an important substrate in vitamin D fruting bodies of milky mushroom is given in Fig. 1 and 2.
biosynthesis frequently found in the lipid fraction of The results indicated that among the compounds identified in
Agaricales extracts. The pharmacological effects of ergosterol Calocybe indica var. APK2, the notable ones were
rich mushrooms have been reported in several clinical studies polyunsaturated fatty acids like Octadecenoic acid and
with promising results in the treatment of breast cancer Hexadecanoic acid (present in both dry and fresh samples),
mainly mediated through the improvements in immunological which are known for their fatty or cucumber like flavour. The
and hematological parameters, ultimately enhancing the aroma quality of dried fruiting bodies differed from that of
quality of life in cancer prone patients. Afieroho and Ugoeze fresh samples by the presence of n- hexadecenoic acid (peak
(2014) [1] performed GCMS and reported about the presence area- 8.46 % compared to 0.38% in fresh samples).
of Į-ergosterol in Lentinus tuber regium with a peak area Moliszewska (2014) [16] reported that the most characteristic
percentage of 2.16. This steroid component predominantly flavour compound is defined mainly by C8 volatiles, which
possessed anti-cancer, antioxidant, hypoglycemic, are known to exhibit fruit-like, cucumber, potato, garlic,
hypocholesterolemic and thyroid inhibiting properties. The cheese-garlic, and even flour-like smell in mushrooms.
results of present study obtained through GCMS analysis
obviously indicated increased levels of ergosterol (peak area -
5.51%) in milky mushroom samples. This may be a good
indication relating vitamin D synthesis by the milky
mushroom fungus, Calocybe indica (P&C) var. APK2.
Jedinak and Sliva, (2008) [9] suggested that P.ostreatus,
known to contain ergosterol could significantly inhibit the
proliferation of human breast and colon cancer cells by the
means of cell cycle arrest.
Raina et al. (2014) [20] quantified the ergosterol content in four
different mushrooms using HPLC and indicated that Calocybe
indica contained 243 ȝg/g while, other commonly cultivated
mushrooms like Pleurotus florida and Volvariella volvacea
contained 113ȝg and159 ȝg/g of samples, respectively. They
further reported that the well known medicinal mushroom,
Ganoderma lucidum contained comparatively increased levels
of ergosterol (403 ȝg/g). Working with Agaricus, Boletus,
Amanita, Canthatrellus and Coprinus, Kalac (2016) [10]
reported the presence of mannitol and trehalose (Į,Į, trehalose Fig 1: GCMS-MS Chromotogram for fresh milky mushroom
~389~
219

compounds in dry and 9 different compounds in fresh
mushroom samples. The results also indicated that milky
mushroom samples are rich in polyunsaturated and essential
fatty acids. The presence of specific compounds like
Octacecenoic and Hexadecenoic acid in both fresh and dry
mushroom samples could be responsible for the cucumber
like extra mushroom flavour of the samples. The increased
level of polyunsaturated fatty acids like n-Hexadecenoic acid
and Octadecenoic acid is more related to the growth and
development of mushroom fungi (Mau and Beelman, 1996)
[15]
. The presence of increased levels of ergosterol (5.5%) and
Į-D-Glucopyranose (trehalose) (18.91%) in milky mushroom
could be useful in anti-cancer therapy, which needs further
exploration. The presence of trehalose component reveals the
chemo attractive nature of the biopolymers of milky
mushroom, which can be utilized to enhance the
bioavailability of pharmaceutical or nutraceutical
Fig 2: GCMS-MS Chromotogram for dry milky mushroom preparations.
Mau and Beelman, (1996) [15] have identified that 10-oxo- Acknowledgement
trans-8 decenoic acid (ODA) is a major mushroom aroma The authors are thankful to University Grants Commission for
component, which is a product formed coincidentally with 1- financial support through Rajiv Gandhi National Fellowship.
octen-3ol through two enzyme catalyzed reaction and also he The support and facilities provided for this research through
concluded that the ODA was found to stimulate mycelial Indian Council of Agricultural Research-All India
growth, post harvest development and stipe elongation in Coordinated Research Project on Mushroom Scheme,
Agaricus bisporus under in vitro condition at a concentration University Grants Commission-Special Assistance
of 900 ppm. Also, it might be involved in the initiation of Programme-Departmental Research Support-1 “Enterprising
fruiting bodies, which could be proved by means of ODA Mushroom Biotechnology for Food, Feed and Biomanure”
supplementation (in the form of mushroom powder) to and Department of Science and Technology-Fund for
compost at spawning. It can be considered as a mushroom Improvement of Science and Technology Infrastructure,
growth hormone. Venkateshwarlu et al., (1999) [24] identified Government of India operated in the Department of Plant
the volatile flavour compounds from three different types of Pathology, Tamil Nadu Agricultural University, Coimbatore
mushrooms by simultaneous distillation and extraction and are greatly acknowledged.
concluded that, 1-octen-3-ol was the major constituent, and its
relative percentage was found to be the highest in Pleurotus References
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48.7% in Calocybe indica correlating with the strong spectroscopic (GC-MS) analysis of n-Hexane extract of
mushroom flavour of P. florida. The aliphatic aldehydes, i.e. Lentinus tuber-regium (Fr) Fr (Polyporaceae) syn
pentanal, hexanal, octanal and 2-octenal, were recorded Pleurotus tuber regium Fr sclerotia. Tropical Journal of
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J.Res. ANGRAU 44(1&2) 1-7, 2016
INFLUENCE OF TEMPERATURE AND pH ON MYCELIAL GROWTH AND

CHLAMYDOSPORE PRODUCTION OF PADDY STRAW MUSHROOM
Volvariella volvaceae (Bull. Ex Fr.) Sing
N. KIRAN KUMAR, A. S. KRISHNAMOORTHY, A. KAMALAKANNAN AND D. AMIRTHAM
Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore-641003
Date of Receipt : 27.7.2016 Date of Acceptance : 03.9.2016

ABSTRACT
The present study was conducted to identify the optimum temperature and pH for both mycelial growth and
chlamydospore production of Volvariella volvacea strain CBE TNAU 1505. The mycelium was subjected to varied
temperatures (150C-400C) and pH (5-10). The optimum temperature that favoured the maximum mycelial growth and
chlamydospore production of V. volvacea was 350C and 300C, respectively and the optimum pH range was 6 to 8. The
temperatures below 200C and above 400C and the pH of below 6.0 and above 8.0 are not favourable for the growth
and chlamydospore production of V. volvacea. Strain specific preference for temperature and pH optima to induce
chlamydospores and stress metabolism needs future attention.
INTRODUCTION on luscious morphological characters, mycelial

growth rate, type of mycelia, aerial hyphae formation
Volvariella volvaceae (Bull.Ex Fr.) Sing is the
and chlamydospore production (Chang and Yau,
tropical as well as subtropical mushroom that prefers
1971). The asexual chlamydospore is a thick walled
to grow at high temperature and humidity. Optimum
vegetative cell normally formed at hyphal tips and
temperature for the production of paddy straw
are visually represented as pink coloured dots in
mushroom is 300C-350C (Chang, 1969). Amidst the
culture. Factors such as pH, low temperature and
cultivated mushrooms, it is the fastest growing one
oxygen limitation are also likely to be important
and the fruiting bodies are formed within 10-12 days
inducers of the morphogenetic pathways required for
of spawning. Regulation of temperature between 35
chlamydospore formation (Citiulo et al., 2009).
to 370C by covering the beds with polybag gives the
Keeping this acquirable literature as background, the
maximum sporophore production of V. diplasia
present disquisition has been intended for selecting
(Ramakrishnan et al., 1968). Temperatures below
the best suitable temperature and pH for enhancing
150C cause chilling damage to the fruiting body and
chlamydospore production of V. volvacea.
adversely affect the viability of fungal mycelia.
Storage at low temperatures (40C) causes autolysis MATERIAL AND METHODS
of fruiting body (Chang, 1978), thereby shortening
V. volvacea strain CBE TNAU 1505 obtained
mushroom shelf life (Bao et al., 2013). V. diplasia
from the germplasm repository of Mushroom
prefers 280C to 350C for mycelial growth whereas,
Research Laboratory, Tamil Nadu Agricultural
optimum temperature for spore germination was 400C
University, Coimbatore was used in this experiment.
(Singh and Saxena, 1983 and Banerjee et al., 1990).
Hydrogen ion concentration of around 6.0 was most Maintenance of Pure Cultures
favourable for the growth, protein production and The sub-culture of paddy straw mushroom
cellulase activity of V. diplasia (Banerjee and strain used in the study was maintained on Potato
Samajpati, 1989 and Gupta et al., 1996). A potential Dextrose Agar (PDA) medium. In order to maintain
high yielding strain of Volvariella is apparent based the vigour fresh isolations were made from the fruiting
E-mail: [email protected]
222
KIRAN KUMAR et al.
bodies every time after 2 to 3 subcultures. For this and incubated at 350C. The radial growth of mycelia,
purpose the strains were propagated in straw spawn colony characters and production of aerial hyphae
and grown on paddy straw following the method were recorded periodically at 2 days interval for every
suggested by Thomas et al. (1943). Freshly harvested 20 days. Chlamydospores production was visually
sporophores were swabbed with 70 per cent ethanol. observed and the conclusions were drawn out.
At the junction of the pileus and stipe, tissue bits Statistical Analysis
were removed aseptically, surface sterilized with 70
Statistical software AGRES (Developed by
per cent ethanol for 30 sec and repeatedly washed in
Department of Physical Science, TNAU, Coimbatore)
sterile water and placed on PDA medium taken in
was used for analysis of data obtained in the
sterile Petri dishes. The dishes were incubated at 30
experiment. All the visual and micrometric
to 350C for seven days. Following single hyphal tip
observation parameters were carried out in 4 and 25
method (Rangasamy, 1972) pure cultures were made
replicas, respectively. In case of zero values the data
and stored in PDA slants to carry out further studies.
was log transformed (X+0.5) before statistical
Micrometric observations on the diameter of hyphae
analysis.
and chlamydospores were observed with the help of
image analyzer (N-400T, Optika, Italy). RESULTS AND DISCUSSION
Effect of Temperature Influence of Temperature
To assess the best temperature range which Optimum temperature that favoured the
would favour the growth and chlamydospores maximum growth of the V. volvacea strain CBE
production, a 9 mm mycelial disc of CBE TNAU 1505 TNAU 1505 was 300C-350C. At temperature below
strain was inoculated aseptically on PDA medium in 200C, the fungus did not grow well. Growth of the
sterile Petri dishes. The plates in 4 replicates were fungus increased positively with the number of days
incubated in B.O.D. incubators set at different of incubation. At 300C and 350C, 89.2 and 90 mm
temperature optima viz., 15 C, 20 C, 25 C, 30 C, 35 C
0 0 0 0 0
radial growth of the fungus, respectively was recorded
and 400C. Linear hyphal growth, colony characters, on sixth day. This was followed by 250C (84.7
production of aerial hyphae were recorded periodically mm).However, in lower temperature i.e., at 150C very
at 2 days interval for every 20 days. Chlamydospores poor growth was observed. At 350C, the 90 mm Petri
production was also visually observed and the dish was covered within 6.10 days followed by
conclusions were drawn out. incubation at 300C (6.4 days) and 250C (7.3 days).
Effect of pH Whereas, it had taken 12.2 days at 150C. More number
of chlamydospores was observed at 250C and 300C
To check the best pH for the growth and
than at lower and higher temperatures. No
chlamydospores production of CBE TNAU 1505
chlamydospore production was noticed at 15 0C and
isolate, PDA medium was prepared by adjusting the
400C (Table 1 and Fig. 1).
pH with 0.1 N hydrochloric acid or 0.1 N sodium
hydroxide at different pH levels viz., 5.0, 6.0, 7.0, Optimum temperature for mycelial growth and
8.0, 9.0 and 10.0. A 9 mm mycelial disc of CBE TNAU fruiting bodies differs with the stage of mushrooms
1505 isolate was inoculated aseptically on PDA (Zadrazil and Grabbe, 1983). In accordance to Chang
medium maintained at respective pH in 4 replicates (1978), temperature below 150C affects the viability
223
Table 1. Influence of temperature on mycelial growth and chlamydospores production of V. volvacea strain (CBE TNAU
1505)
224
Aerial hyphae, chlamydospores density “- to ++++” absent to highly densed; DTTCPP (Days taken to cover 90 mm Petri plate); DTFCP (Days taken for
chlamydospores production). Data in parenthesis are square root transformed values. *, #Values are mean of 4 and 25 replications. Means followed by
a common letter are not significantly different at P = 0.05 by one-way ANOVA
INFLUENCE OF TEMPERATURE AND pH ON MYCELIAL GROWTH AND CHLAMYDOSPORE PRODUCTION
Table 2. Influence of pH on mycelial growth and chlamydospores production of V. volvacea strain (CBE TNAU 1505)
225
KIRAN KUMAR et al.
Aerial hyphae, chlamydospores density “- to ++++” absent to highly densed; DTTCPP (Days taken to cover 90 mm Petri plate); DTFCP (Days taken for
chlamydospores production). Data in parenthesis are square root transformed values. *, #Values are mean of 4 and 25 replications. Means followed by
a common letter are not significantly different at P = 0.05 by one-way ANOVA
226
KIRAN KUMAR et al.
of fungal mycelia and also invokes chilling injury to lack of initiation of biosynthesis of unsaturated fatty
the fruiting bodies of Volvariella. The present findings acids, trehalose and glycogen that are incredibly
indicated that the optimum temperature that favoured essential for the formation of chlamydospores (Bao
the maximum growth of V. volvacea strain CBE TNAU et al., 2013).
1505 was 350C. However, the mycelium does not grow Influence of pH
well below 200C and above 400C. At these extreme Hydrogen ion concentration of 7.0 and 8.0 were
temperatures the hypha was found to be very thin. statistically on par favouring radial mycelial growth
Cognate results were reported by Sangeetha (2002) followed by pH of 6.0 (Table 2). The mycelial growth
and Prabhu (2006). Slightly deviating from these was minimum at a pH 10.0 (74.1 mm). At pH 7.0,
results the maximum mycelial growth of the V. the fungus took a minimum duration of 6.3 days to
volvacea was observed between 25 and 300C and the cover the entire Petri plate of 90 mm diameter,
highest mycelial dry weight of 80 mg was obtained at followed by pH 8.0 (6.9 days). However, at pH 10.0,
300C by Akinyele and Adetuyi (2005). it had taken 9.7 days. The mushroom fungus grown
In the present disquisition, chlamydospores at pH 6.0, 7.0 and 8.0 produced more number of
were produced with more density at 30 C. 0
chlamydospores at 15.9, 14.4 and 15.2 days with
This may be attributed to the induction of mild high density (Fig. 2 and Fig. 3).
stress at 30 C resulting in more number of
0
CONCLUSION
chlamydospores on aerial hyphae. However, with The present study indicated that V. volvacea
increased stress well below 250C, no chlamydospore produced the maximum radial growth and
production was observed which may be attributed to chlamydospores at pH 6.0 to 8.0. However,
Fig. 3. Chlamydospores produced terminally on apical cells (20 X)
227
Sangeetha (2002) reported that slightly acidic to Candida albicans and Candida dubliniensis
neutral pH of 6.5 to 7.0 was quite suitable for chlamydospores cultured in liquid media.
chlamydospores production of V. volvacea. Banerjee FEMS Yeast Research. 9: 1051-1060.
and Samajpati (1989) and Kalra et al. (1997) reported Gupta, U., Kalra, R and Phutela, R. P. 1996. Factors
that pH 6.0 favoured the growth and protein production affecting cellulase production in Volvariella, the
of V. diplasia. straw mushroom. Mushroom Research. 5: 29-32.
REFERENCES Kalra, R., Phutela, R. P and Sodhi, H. S. 1997.
Akinyele, B. J and Adetuyi, F. C. 2005. Effect of Studies on growth of Volvariella diplasia.
agrowastes, pH and temperature variation on Mushroom Research. 6: 47-50.
the growth of Volvariella volvacea. African
Prabhu, K. 2006. Biodegradation of agrowastes with
Journal of Biotechnology. 4 (12): 1390-1395.
Volvariella volvacea (Bull. ex Fr.) Sing and
Banerjee, M. M and Samajpati, N. 1989. Effect of
subsequent introduction of Trichoderma viride
some environmental factors and exogenous
Pres. ex Gray for the management of Damping-
nutritional source on the protein content of
off disease in Tomato. M.Sc. Thesis submitted
Volvariella diplasia in submerged culture.
to Tamil Nadu Agricultural University,
Mushroom Journal for the Tropics. 9: 139-146.
Coimbatore.
Banerjee, M., Banerjee, P and Samajpati, N. 1990.
Environmental factors and nutritional Ramakrishnan, K., Lalithakumari, D., Shanmugam,
requirements on spore germination and germ N and Krishnamoorthy, C. S. 1968. A simple
tube growth of Volvariella diplasia. Mushroom technique for increasing the yield of straw
Journal for the Tropics. 10: 40-46. mushroom (Volvariella diplasia). Madras
Bao, D., Gong, M., Zheng, H., Chen, M., Zhang, L., Agricultural Journal. 55: 194-195.
Wang, H., Jiang, J., Wu, L., Zhu, Y., Zhu, G., Rangasamy, G. 1972. Diseases of crop plants in India.
Zhou, Y., Li, C., Wang, S., Zhao, Y., Zhao, G Prentice Hall of India Pvt. Ltd. New Delhi. pp. 520.
and Tan, Q. 2013. Sequencing and Sangeetha, G. 2002. Exploring the possibilities of
comparative analysis of the straw mushroom increasing the yield potential of paddy straw
(Volvariella volvacea) genome. PLoS ONE. 8 mushroom, Volvariella volvacea (Bull. ex Fr.)
(3): e58294. Sing. M.Sc. Thesis submitted to Tamil Nadu
Chang, S. T. 1969. A cytological study of spore Agricultural University, Coimbatore.
germination of Volvariella volvacea. Botanical
Singh, R. P and Saxsena, H. K. 1983. Effect of
Magazine, Tokyo. 82: 102-109.
temperature, humidity, light intensity and NPK
Chang, S. T and Yau, C. K. 1971. Volvariella volvacea
fertilization on the yield of V. diplasia (Berk &
and its life history. American Journal of Botany.
Curt) Sing. Mushroom Newsletter for Tropics.
58: 552-561.
3: 10-13.
Chang, S. T. 1978. Volvariella volvacea. In: The
biology and cultivation of edible mushrooms. Thomas, K. M., Ramakrishan, T. S and Narsimhulu,
Chang, S.T., Hayes, W.A. (Eds). Academic I. L. 1943. Paddy straw Mushroom. Madras
Press, New York. pp. 573-603. Agricultural Journal. 31: 57-59.

Citiulo, F., Moran, G. P., Coleman, D. C and Sullivan, Zadrazil, F and Grabbe, K. 1983. Edible mushroom.
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228
Madras Agric. J., 103 (10-12): 338-343, December 2016
Physical, Chemical and Biological Properties of Casing Soil Used

for Milky Mushroom (Calocybe indica P&C) Production
A.S. Krishnamoorthy* and B. Priyadharshini

Tamil Nadu Agricultural University, Coimbatore - 641 003.
([periments Zere conducted to ¿nd out the effect of different casing soils viz., peat, clay loam
soil, sandy soil, biogas slurry, FYM, cocopeat on the yield and yield attributing parameters of
milky mushroom (Calocybe indica var.APK2). Among the different casing media, clay loam soil
has given the maximum yield (390 g / bed containing 250 g of paddy straw substrate on dry
weight basis recording 5 bioef¿ciency) and more number of buttons (. /bed) than other
casing media. Analysis of physical, chemical and biological properties of clay loam soil revealed
that it contained high p+ .0. Cation (xchange Capacity (C(C) was high in clay loam (2.20
milliequivalents / 100 g of soil). Bulk density and particle density were comparatively higher in
sand (1.67 g/cc) and clay loam soils (1.33 g/cc). The maximum pore space was recorded in clay
loam soil (55.01 per cent) followed by peat (39.57 per cent) and sand (20.6 per cent). The :+C
revelaed high per cent in peat soil (6.27) followed by clay loam soil (39.0). +ence, peat and clay
loam soils required less water to maintain surface moisture and a delay in spraying did not lead
to the total drying of bed surface. In addition, the population of Pseudomonas spp were found
to be high in clay loam soil (3.0 cfu/ g of soil).
Key words: Milky mushroom, Calocybe indica, Casing soil properties
Calocybe indica P&C, a tropical edible mushroom properties of a good casing should be high porosity
popular in India loves warm humid climate. It is and water holding capacity (WHC), 7.2–8.2 pH, low
mainly a grassland species, saprophytic in nature content of soluble inorganic and organic nutrients,
and sometimes ectomycorrhizal with Cocos nucifera, and free of disease and pests (Taherzadeh and
Borossus flabellifer, Tamarindus indicus and Jafarpour, 2013). Trehalose was found to have
Peltaphorum ferruginum. Casing is an important growth-promoting effects on the mycorrhization-
agronomic practice followed in the cultivation of any helper bacteria (MHB), Pseudomonas monteilii, when
humicolous mushroom (Krishnamoorthy and Balan, inoculated with the ECM fungus Pisolithus albus
2015) and milky mushroom is not an exception. (Duponnois and Kisa, 2006). Trehalose released by
Casing triggers off the change from vegetative to the mycelium of Laccaria bicolor in soil was shown to
generative phase. Compact casing interfaces impede attract Pseudomonads (Frey-Klett et al., 2007). With
the diffusion of harmful metabolic gases on mushroom this background, the current investigation was made
bed surface (Mac Canna, 1983). The casing layer to screen the best casing medium for growing milky
must be sufficiently loose to facilitate primordia mushroom and to determine its physical, chemical
emergence (Sassine et al., 2005). Casing soil also and biological properties.
protects the compost against desiccation and support
the growth of Agaricus blazei and provide anchorage Materials and Methods
for the developing sporophores (Colauto et al., 2011). Mushroom Bed Preparation
Purkayastha (1984-1985) used loam soil and sand
Cylindrical polythene bag mushroom beds (60 x
(1:1), mixed with calcium carbonate at 12 per cent
30 cm size and 100 gauge thickness) were prepared
level (pH 7.0) for casing C.indica beds. Purkayastha
by following the methods suggested by Baskaran et
(1984-1985) used loam soil and sand (1:1), mixed
al., (1978) and Sivaprakasam, (1980) using paddy
with calcium carbonate at 12 per cent level (pH 7.0)
straw substrate. A total quantity of 0.5 kg of paddy
for casing C.indica beds. However, Krishnamoorthy et
straw (dry weight basis) was used for making each
alUHSRUWHGLQFUHDVHG\LHOGDQGELRHI¿FLHQF\
bed and layer spawning @ 5% to the wet weight of
when clay loam soil was used for the cultivation of
the substrate was followed (Sivaprakasam,1980) .
milky mushroom. In addition to physical and chemical
Substrate moisture at the time of bed preparation was
properties, the biological properties of the casing
65 %. After preparation, the mushroom beds were
VRLODUHNQRZQWRLQÀXHQFHWKHLQGXFWLRQRIIUXLWLQJ
incubated at 30 ± 2°C for spawn running. Relative
bodies in case of Agaricus bisporus (Grewal and
humidity ranged from 65 to 70 per cent in the spawn
Rainey, 1991). The required physical and chemical
running rooms. After 15 days, when the beds were
*Corresponding author email:[email protected]
229
339
found fully colonized by the mushroom fungus, Estimation of microbial population in casing soil
each bed was cut into two equal halves (each bed The population of fungi, bacteria and actinomycetes
contained 250g of paddy straw substrate (dry weight in the casing soil was estimated by serial dilution
basisi) colonized by the milky mushroom fungus) and technique using Rose Bengal agar (Martin, 1950)
applied with casing soil. After casing the beds were for fungi; soil extract agar (Allen, 1957) for bacteria;
shifted to cropping room where, the temperature was King’s B medium (King et al., 1954) for pseudomonads
maintained at 33±2oC and RH at 85-90 per cent. The and Kuster’s agar (Kuster and Williams, 1964) for
roof of cropping room was lined with transparent HDP actinobacteria. Ten g sample of casing medium was
blue polythene sheet (120 G thick Silpaulin sheet). WUDQVIHUUHGWRPOFRQLFDOÀDVNFRQWDLQLQJPO
Preparation of casing soils and casing RIVWHULOHGLVWLOOHGZDWHU7KHÀDVNZDVVKDNHQRQD
rotary shaker for 10 min. Serial dilutions up to 106
Peat soil (pH 6.0), clay loam soil (pH 8.4), sand
were prepared with sterile pipette and dilutions of
(pH 6.3), biogass slurry (pH 7.3), well decomposed
103, 105 and 106 were used for actinomycetes, fungi
farm yard manure (pH 7.6) and cocopeat (pH 6.8)
and bacterial population count, respectively. One ml
were used in this experiment. The casing medium
of the aliquot was aseptically transferred to sterile
was taken in mud pots and the moisture content
Petri dishes and agar medium after thawing and
was adjusted to 20 per cent following the method
cooling was added. The plates were gently shacked
RI 'HYDGRVV 7KH SRWV ¿OOHG ZLWK VRLO ZHUH
to mix the medium and aliquot. Later,the Petri dishes
steamed in an autoclave at 1.2 kg/cm2 for 60 min
were incubated at room temperature (30± 20°C). The
and used for casing after 24 h. The casing soil was
colonies appearing on the plates were counted and
uniformly spread over the spawn run beds to a height
expressed as number of colony farming units (cfu)
of 2 cm. Regular spraying with water on the casing
per g of air-dried substrate or casing soil.
surface was done to maintain the required moisture
level. Result and Discussion
Determination of physical and chemical properties of Effect of different casing soils on yield
casing soil Among the different soils used for casing, clay
Physical characters of casing soil namely, loam soil (pH 8.4) gave the maximum yield of
electrical conductivity, cation exchange capacity 388.0 g of mushrooms per bed with more number
(CEC), texture, bulk density, particle density, water of buttons (8.0). Peat soil (pH 6.0) was the second
holding capacity (WHC), pore space and percolation best medium for casing (Table 1). In sandy soil, the
rate were estimated following the methods of De fungus took more number of days for the production
(1965) and the pH was estimated by digital pH of pinheads (10.2 d) and they attained harvesting
meter. Chemical analysis of each sample was done maturity only after 10.6 days. Again, the average
for essential nutrients namely, nitrogen, phosphorus, weight of individual mushroom, pileus weight and
potash and organic carbon following standard stipe weight were much less in mushrooms harvested
methods (AOAC., 1970). from sandy soil (Table 1). Casing is generally done
to provide anchorage and essential reserves for the
Effect of calcium carbonate supplementation to casing
developing sporophores over an uniform surface
soil
(Shandilya, 2002). Amin et al., (2010) indicated that
Steamed clay loam soil was mixed with calcium the cow dung and loamy soil (3 cm thick) was the
carbonate at the rate of 2.5, 5.0 and 10.0 per cent best casing medium for growing Calocybe indica.
(W/W) level and used for casing. Soil, not mixed with But, compared to the results obtained in the present
calcium carbonate served as control. VWXG\WKHELRHI¿FLHQF\
Table 1. Effect of different casing soils on the yield of C. indica
Size of mushroom
Bio- No. of Average
Yield (g/ Pileus Stipe
Casing soil pH DFPF DFFH HI¿FLHQF\ buttons weight (g/
bed)
(%) harvested button) Breadth Weight Length
Weight
(cm) (g) (cm)
Peat soil 6.0 8.0 8.0 341.0 136.4 7.0 49.1 5.5 26.7 7.5 21.8
Clay loam soil 8.4 8.2 8.6 390.0 156.0 8.1 51.1 5.8 26.2 6.9 23.2
Sandy soil 6.3 10.2 10.6 114.0 45.6 3.1 36.5 6.0 16.9 8.0 19.6
Bio-gass slurry 7.3 8.8 9.0 145.0 58.0 2.9 49.7 4.8 25.7 7.6 24.0
Farm yard manure 7.6 10.0 9.2 181.0 72.4 4.4 40.8 5.2 20.6 6.6 20.2
Coco peat 6.8 8.4 8.6 138.4 55.4 3.2 43.8 6.6 23.6 8.0 20.2
CD (P=0.05) 1.20 1.41 34.86 0.88 7.62 NS 8.35 1.08 NS
')3) 'D\VIRUSLQKHDGIRUPDWLRQ '))+'D\VIRU¿UVWKDUYHVW
230
340
reported was much less. Krishnamoorthy et al., cocopeat were subjected to different physical and
(1998) and Pani (2012) concluded that application of chemical analyses and the results are presented
casing soil up to 2 cm depth could be ideal for quick in Table 2. Among the casing media, clay loam soil
primordial induction, increased number of buttons and exhibited alkaline pH (8.4) and the pH in peat soil
maximum mushroom yield in case of Calocybe indica. was 6.0. Electrical conductivity was almost equal in
Physical and chemical properties of casing soils and
both clay loam and peat soils (0.43 and 0.42 mmhos/
WKHLULQÀXHQFHRQ\LHOG cm). Higher levels of nitrogen and phosphrous were
noticed in clay loam than in peat. However, Potash
The different casing media namely, clay loam soil, content was much high in peat. Sandy soil exhibited
peat soil, sand, farm yard manure, biogass slurry and less of N and P as compared to clay loam and peat
Table 2. Physical and chemical properties of casing soils
pH Clay loam soil Peat soil Sandy soil Biogass slurry FYM Coco peat
PH 8.40 5.40 6.30 7.30 7.60 6.80
EC (mmhos/cm) 0.43 0.42 0.34 - - 0.80
N (per cent) 0.69 0.35 0.22 0.43 0.52 0.44
P (per cent) 0.30 0.21 0.25 1.36 1.52 1.52
K (per cent) 1.55 3.15 2.50 2.34 2.53 2.59
C (per cent) 0.37 0.84 0.12 9.80 10.05 14.10
CEC (*) 28.20 12.20 0.80 - - -
Ca++ 16.00 7.50 0.80 - - -
Mg++ 5.00 2.50 7.00 - - -
Na++ 0.87 0.54 T - - -
K+ 0.77 0.03 0.06 - - -
Clay (per cent) 38.26 - 0.12 - - -
Silt (per cent) 25.83 - 0.30 - - -
Coarse sand (per cent) 33.87 - 82.17 - - -
Fine sand (per cent) 15.60 - 17.31 - - -
Bulk density (g/cc) 1.33 0.99 1.67 - - -
Particle density (g/cc) 2.37 1.63 2.21 - - -
WHC (per cent) 39.04 46.27 19.91 27.0 37.0 70.0
Pore space (per cent) 55.01 39.56 20.46 - - -
Water percolation (ml/h) 1240 377 1960 1118 1260 235
(*) ECE expressed as milliequivalents per 100 g of soil
and increased level of K when compared to clay loam exceptionally high in clay loam (28.20 milliequivalents
soil. Among all the media, cocopeat contained more / 100 g of soil). All the cations namely, Ca++, Mg++,
of organic carbon (14.10 per cent). When the three Na+ and K+ were also found to be high in clay
soils alone were considered, C content was higher loam soil. When the texture of the casing soils was
in peat soil. Cation Exchange Capacity (CEC) was examined, the percentage of clay
Table 3. Effect of mixing calcium carbonate to casing soil on yield

Size of mushroom
Level of CaCo3 Bio- No. of Average
in casing soil DFPF DFFH Yield (g/ HI¿FLHQF\ buttons weight (g/ Pileus Stipe
bed)
(%) (%) harvested button)
Breadth (cm) Weight (g) Length (cm) Weight
2.5 8.4 9.0 334.0 133.6 7.8 42.9 10.3 26.39 13.0 16.0
5.0 7.2 7.8 328.0 131.2 6.4 50.9 7.9 24.5 12.7 26.4
10.0 7.6 8.0 320.0 128.0 7.0 45.8 8.8 24.1 12.2 21.7
Control 8.8 9.0 318.5 127.5 7.3 43.6 9.0 22.9 10.4 20.7
CD (P=0.05) 0.78 0.79 NS NS NS NS NS NS NS
')3) 'D\VIRUSLQKHDGIRUPDWLRQ '))+'D\VIRU¿UVWKDUYHVW
231
341
and silt were maximum in clay loam soil (38.26 and availability of soluble inorganic ions and low electrical
&RDUVHDQG¿QHVDQGSHUFHQWZDVPRUHLQ conductivity (Singh et al., 1992). In the present
sandy soil. Bulk density and particle density were study, clay loam soil alone possessed all these
comparatively higher in sand and clay loam soils. desirable characters. Visscher (1980) reported that
The maximum pore space was recorded in clay loam soils containing higher levels of magnesium delayed
soil (55.01 per cent) followed by peat (39.57 per cent) sporophore production and reduced the yield upto
and sand (20.46 per cent). Among all the media water 50 per cent in A. bisporus. But in the present study,
holding capacity (WHC) was exceptionally high in clay loam soil contained more of magnesium (Table
cocopeat followed by peat soil. WHC in clay loam 2) than any other media tested. Yet, this soil gave
soil and farm yard manure was almost equal. the highest yield. Zied et al ., (2012) indicated that
high amount of organic matter in casing medium
Noble (1999) studied the properties of peat based
could reduce the yield (1.67%) and biological
FDVLQJVRLOVDQGWKHLULQÀXHQFHRQWKHZDWHUUHODWLRQV
HI¿FLHQF\GXULQJWKHFXOWLYDWLRQRIAgaricus
and growth of button mushroom.
subrufescens due to the continuous proliferation
7KH YHUPLFRPSRVW ZDV DOVR EHFDXVH RI ¿QH of the vegetative mycelium and a nutrient stress is
texture and compact nature; high viscosity and always needed to induce fruiting. Water percolation
electrical conductivity (EC) not suitable for use as was poor in cocopeat and peat soil, while it was much
a casing soil for Agaricus blazei (Taherzadeh and higher in sand. Farm yard manure and clay loam soil
Jafarpour, 2013) and there is the possibility of mixing were almost on par showing moderate levels of water
it with other substrates having low EC to increase percolation (Table 2). Lambert and Humfeld (1939)
the re-use potential. Besides, alkaline pH, electrical and Flegg (1991) indicated that heavy clay soils
conductivity and cation exchange capacity were also yielded better than others. This could be correlated to
considered important in the sporophore formation the slow release of water to the growing sporocarps
of A.bisporus. A good casing soil should have low from such casing soils.
Table . Casing soil microÀora (cfu / g of soil)

before steaming after steaming at the time of pinning
casing soil
F B A P F B A P F B A P
Peat soil 18.3 41.3 33.7 23.3 2.3 18.7 31.3 16.0 7.3 24.3 33.0 26.3
Clay loam soil 19.0 48.0 38.3 29.3 1.3 25.7 21.0 21.0 3.0 32.7 28.3 34.0
Sandy soil 8.7 21.0 28.7 6.7 1.3 12.7 19.3 1.7 2.0 18.3 19.0 4.3
Biogass slurry 12.4 28.0 17.7 8.3 3.7 19.0 10.3 3.0 8.0 25.7 12.0 8.3
Farm Yard Manure 20.7 64.7 63.7 15.7 5.7 18.0 49.7 6.0 9.7 25.0 56.3 13.0
Cocopeat 15.3 51.7 27.0 4.3 8.7 14.7 17.3 0.6 10.0 18.0 22.3 2.3
CD (P = 0.05) 2.85 6.62 7.56 5.44 2.18 4.43 9.35 3.96 1.54 5.64 10.30 6.80
5 6 4 6
F - Total fungi (x 10 cfu); B - Bacteria (x 10 cfu); A - Actinobacteria (x 10 cfu); P - Pseudomonads (x10 cfu)
Effect of addition of calcium carbonate to casing soil ,QÀXHQFHRIFDVLQJVRLOPLFURÀRUD
In a separate experiment clay loam soil (pH 8.4) The results obtained in the experiment are given
used for casing was mixed with 2.5, 5.0 and 10.0 per in Table 4. Before steaming, farm yard manure, clay
FHQWOHYHORIFDOFLXPFDUERQDWHLQRUGHUWR¿QGRXWLWV loam soil and peat soil contained large population
effect on the yield of C. indica. The maximum yield of total fungi. Bacterial and actinobacterial colonies
of 334.0 g per beds was harvested when the casing ZHUHVLJQL¿FDQWO\KLJKHULQIDUP\DUGPDQXUHIROORZHG
soil was mixed with calcium carbonate at 2.5 per cent by clay loam and peat soil. Cocopeat also contained
level. However, this observation was not statistically more population of bacteria as comparable to other
different from the yield obtained in the control or casing media.. When the pseduomonads alone were
other treatments (Table 3). When days for pin head counted, it was observed that clay loam soil contained
IRUPDWLRQ')3)DQGGD\VIRU¿UVWKDUYHVW'))+ more number of cfu followed by peat soil and farm
were compared, pinheads appeared earlier and also yard manure (Table 4).
attained the harvesting maturity well in advance in the
After steaming, the cfu of total fungi in cocopeat
treatments that involved mixing of calcium carbonate
was found to be more followed by steamed farm
at the rate of 5.0 and 10.0 per cent level. Oei (2003)
yard manure and biogass slurry. Total bacterial
reported that calcium carbonate (CaCO3) could be
count was maximum in clay loam soil followed by
used to correct casing soil acidity, as it could elevate
farm yard manure and others. The actinobacterial
the pH and keep low Mg values in casing medium
colonies were found to be higher in farm yard manure
during button mushroom cultivation.
followed by peat soil and clay loam soils. However,
232
342
the cfu of pseudomonads were continued to be casing amendments used in cultivation of Agaricus
higher in clay loam soil even after steaming followed bisporus. Indian Journal of Biotechnology. : 97-109.
by peat soil (Table 4). Compost or casing soil borne Colauto, N.B., Silveira, A.R.D., Eira, A.F.D. and Linde, G.A.
Pseudomonas putida is known to have a positive 3URGXFWLRQÀXVKRIAgaricus blazei on Brazilian
relationship in the primordial induction and fruiting casing layers. Brazilian Journal of Microbiology,
body formation of button mushroom (Choudhary et 2(2):616-623.
al., 2009).
De, S.K. 1965. Practical Agricultural Chemistry. Narayan
When the casing soil samples were analyzed at Publishing home, Allahabad, India.1:110.
the time of pinhead formation, it was noted that the Devadoss, R. 1971. Studies on damping-off of vegetables.
FIXRIWRWDOIXQJLZHUHVLJQL¿FDQWO\PRUHLQFRFRSHDW M.Sc.(Ag.)., Thesis, Tamil Nadu Agricultural University,
and farm yard manure (Table 4). Clay loam soil Coimbatore. India. 1:123.
and sand contained lesser number of total fungal
Duponnois, R., and Kisa,M.2006.The possible roles of
population. Saprophytic bacterial count was much
trehalose in the mycorrhiza helper effect. Can.J.Bot.
higher in biogass slurry, farm yard manure and peat
: 1005–1008.
soil. However, when pseudomonads alone were
counted in King’s B medium, it was observed that clay Flegg, P.B. 1991. The casing circle. Mushroom J., 223:
loam soil contained more cfu than others. Counts of 24-32.
actinobacteria continued to be higher in farm yard. Frey-Klett,P., Garbaye,J. and Tarkka,M.2007.The mycorrhiza
helper bacteria revisited. New Phyt. 176: 22–36.
From the results of the study, it is concluded that
clay loam soil with pH 8.4; CEC 28.2 milli equivalents Grewal, S.I.S. and Rainey, P.B. 1991.Phenotypic variation
/ 100 g of soil; bulk density and particle density at of Pseudomonas putida and P. tolaasii a!ects the
1.33 g/cc; pore space 55.0 % and WHC 39.04% chemotactic response to Agaricus bisporus mycelial
could be the best casing soil medium for growing exudate. J. Gen. Microbiol., 137:2761-2768.
milky mushroom. Further, clay loam soil required King, E.O., Ward M.K.and Raney,D.E. 1954. Two simple
less water to maintain the required moisture level media for the demonstration of pyocyanin and
on bed surface. Because of good water retention ÀXRUHVFLQJ. Lab. Clin. Med., : 301-307.
and slow release, even a delay in spraying over the Krishnamoorthy A.S and Balan, V., 2015. A Comprehensive
casing surface did not lead to the total drying of bed Review of Tropical Milky White Mushroom (Calocybe
moisture. In addition, increased populations of soil indica P&C). Mycobiology, 3(3): 184-194.
pseudomonads (34.0 cfu/ g of soil) recorded in clay
Krishnamoorthy,A.S.,Muthusamy,M.,Marimuthu,T.,Narasim
loam casing soil favoured the appearance of more
han,V., Muthusankaranarayanan, A. 1998. APK2 milky
number of fruiting bodies of milky mushroom.
mushroom-Extn. Bulletin, RRS, TNAU, Aruppukottai.
Acknowledgement P:18
Kuster, E. and Williams,S.T. 1964. Selection of media for
The authors greatly acknowledge the support
isolation of Streptomyces. Nature. 202: 928-929.
provided through ICAR-AICRP on Mushroom Scheme
and UGC-SAP-DRS 1 “Enterprising Mushroom Lambert, E.B. and H. Humfeld. 1939. Mushroom casing soil
Biotechnology for Food, Feed and Biomanure” in relation to yield. U.S. Dept. Agric. Circ. 509.
operated in the Department of Plant Pathology, Mac Canna C. 1983.Spawned casing. Mushroom J.129:329-
TNAU, Coimbatore. 333.
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Visscher, H.R. 1974. Structure of mushroom casing soil and
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LWV LQÀXHQFH RQ \LHOG DQG PLFURÀRUD Neth. J. agric.
1992. Physico chemical parameters of casing soil
Sci., 23: 36-47.
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Sivaprakasam, K. 1980. Studies on oyster mushroom
of Agaricus subrufescens growth based on chemical
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sciences, 19(3):343-347.
Received after revision : December 28, 2016; Accepted : December 31, 2016
234
Madras Agric. J., 105 (1-3): 56-60, March 2018
Antifungal Potential of Myco-molecules of Coprinopsis cinerea

6FKDHႇ6*UD\VODWDJDLQVWFusarium spp
6-HHYD DQG$6.ULVKQDPRRUWK\
Tamil Nadu Agricultural University, Coimbatore - 641 003
,QN\FDSPXVKURRPCoprinusVSSRVVHVVHVYLWDODQWLIXQJDOSURSHUWLHVDJDLQVWWKHPDMRUZLOW

FDXVLQJSDWKRJHQVRIEDQDQDWRPDWRDQGFKLOOL:LOWGLVHDVHFDXVHGE\Fusarium oxysporum IVS
cubense in banana, F. oIVS. lycopersici LQWRPDWRDQGF.brachygibbosum LQFKLOOLZHUHREWDLQHG
DQGWKHDQWLIXQJDODFWLYLWLHVRICoprinus cinereaZDVDVVHVVHG0HWKDQROLFH[WUDFWIURPWKHIUXLWLQJ
ERGLHVRILQN\FDSC. cinereaFRQVLVWHGRIPROHFXOHVZKLFKFRXOGLQKLELWWKHJURZWKRIFusarium
VSSDWSSP5HVXOWVVKRZHGDQGSHUFHQWRIP\FHOLDLQKLELWLRQRIF. oxysporum
IVS cubense, F. oIVS. lycopersiciDQGF. brachygibbosum,UHVSHFWLYHO\ZKHQWKHPHWKDQROLF
H[WUDFWRIC. cinereaZDVXVHGLQDJDUZHOOVLQSHWULGLVKHV*&06DQDO\VLVRIWKHPHWKDQROLF
H[WUDFWRIWKHIUXLWLQJERGLHVRILQN\FDSLQGLFDWHGWKHSUHVHQFHRIGLႇHUHQWFRPSRXQGVviz
IRUPLFDFLGSURSHQ\OHVWHUDFHWLFDFLG'DODQLQH1SURSDUJ\OR[\FDUERQ\OLVRKH[\OHVWHU
+3\UDQRQH GLK\GURGLK\GUR[\PHWK\O QKH[DGHFDQRLF DFLG RFWDVLOR[DQH
KH[DGHFDPHWK\O GLSKHQ\OGLR[RS\ULGD]LQR
>¶¶@S\UUROR>¶¶G@S\ULGD]LQHVTXDOHQHDQG1RQDGHFDQRQHGLQLWURSKHQ\OK\GUD]LQH
$PRQJ WKHVH FRPSRXQGV IRUPLF DFLG SURSHQ\O HVWHU DFHWLF DFLG QKH[DGHFDQRLF DFLG
GLSKHQ\OGLR[RS\ULGD]LQR>¶¶@S\UUROR>¶¶G@S\ULGD]LQHVTXDOHQHQRQDGHFDQRQH
GLQLWURSKHQ\OK\GUD]LQHDUHKDYHEHHQUHSRUWHGWRSRVVHVVDQWLIXQJDODFWLYLWLHV
.H\ZRUGV Inky cap, Coprinopsis cinerea, GC-MS, Antifungal biomolecules and Fusarium spp
Coprinopsis cinerea6FKDHႇ6*UD\VODW “Ink 0DWHULDODQG0HWKRGV

cap mushroom” belongs to the family Psathyrellaceae &ROOHFWLRQDQGLGHQWL¿FDWLRQRI&RSULQRSVLVFLQHUHD
(Badalyan et al., SK\OXP %DVLGLRP\FRWD
is one of that most valued mushroom fungus by Coprinopsis cinerea fruiting bodies were collected
pharmaceutical industry. The inky caps are common from the contaminated oyster mushroom beds at
saprophytic fungi growing on road side. They can also 0XVKURRP 5HVHDUFK /DERUDWRU\ 'HSDUWPHQW RI
be sighted in places where high levels of ammonia and plant pathology, TNAU, Coimbatore. The study area
nitrogen, cellulose and xenobiotic waste materials are fall under the geographical co - ordinates of 11.0 °N,
dumped. Many times they grow on mushroom beds as 76.9°E and 411.2 MSL For the purpose of tissue
weed moulds, especially in oyster, paddy straw and isolation, the fruiting bodies of wild isolate was cut
milky mushroom beds when the substrate moisture into small pieces, surface sterilized with one per cent
LV H[FHVV .ULVKQDPRRUWK\ DQG %DODQ 5LFH ZYVRGLXPK\SRFKORULWHIRUVHF7KHWLVVXHELWV
straw enriched with more of nitrogenous supplements were washed twice with sterile distilled water for 60
increase Coprinus VSS 3DUN DQG /HH $ sec and placed in sterile Petri dishes containing 20
number of bioactive compounds obtained from mL of PDA. The plates were incubated at 30϶C for 6
Coprinus spp have been reported to possess several G)ROORZLQJVLQJOHK\SKDOWLSPHWKRG5DQJDVZDP\
antimicrobial, antinemic, antitumor, anticancer, anti- SXUHFXOWXUHVZHUHWUDQVIHUUHGWR3'$VODQWV
hypoglycemic, antioxidant and immunomodulatory and stored at refrigerated condition.
activities. Yuanzhen et al UHSRUWHG WKDW WKH 0ROHFXODUFKDUDFWHUL]DWLRQRI&FLQHUHD
Coprinus spp produced several bioactive molecules
viz., coprinol, coprinolone, coprinastatin, coprinacins, Genomic DNA was extracted from suspension
xanthothone, lagopodins, illudins and armillol. In cultures of C. cinerea by using Cetyl Trimethyl
the present study, the bioactive compounds of $PPRQLXP%URPLGH&7$%PHWKRG0DUWLQV
fruiting bodies of C. cinerea at post capping stage Guimaraes et al 7KH LQWHUQDO WUDQVFULEHG
were characterized by GCMS in methanol and the VSDFHUV,76UHJLRQFRQWDLQLQJSDUWLDOSRUWLRQVRIWKH
concoction of the biomolecules was tested against the VPDOOVXEXQLWU51$6ERWKLQWHUQDOWUDQVFULEHG
wilt disease causing pathogens F. brachygibbosum, VSDFHUV,76DQGDQGWKH6RIWKHU'1$UHSHDW
F. o. f. sp. cubense and F. o. f. sp. lycopersici under XQLWZDVDPSOL¿HGXVLQJWKHROLJRQXFOHRWLGHVSULPHUV
in vitro condition. ,76 ¶**$$*7$$$$*7&*7$$&$$**¶ DQG
,76 ¶7&&7&&*&77$77*$7$7*&¶ *DUGHV
*Corresponding author’s email: [email protected] DQG %UXQV 3&5 UHDFWLRQV ZHUH FDUULHG RXW
235
57
RQ D 7KHUPDO &\FOHU %,25$' DQG FRQVLVWHG RI )LVKHU6FLHQWL¿F/LPLWHGZDVHQJDJHGIRUDQDO\VLV

initial denaturing at 94ºC for 2 min followed by 37 The instrument was set as follows Injector port
F\FOHVDW&IRUVHF&IRUVHFDQG& temperature set to 250oC, Interface temperature
for one min. The reaction was completed by a 4 min set as 250 oC, source kept at 200 oC. The oven
H[WHQVLRQDW&7KH3&5DPSOL¿HGSURGXFWV temperature was programmed as available, 70oC
/ZHUHVHSDUDWHGE\HOHFWURSKRUHVLV*HQHL0D[L for 2 min, 150o&#oC /min. up to 260oC @ 10oC /
6XE6\VWHP*HQHL%DQJDORUH,QGLDRQ min. Split ratio was set as 1:50 and the injector used
SHUFHQWDJDURVHJHOXVLQJ7$(EXႇHUDW9FRQVWDQW was in splitless mode. The DB-35 MS non polar
current for 1h. The gel was stained with ethidium column was used, whose dimensions were 0.25 mm
bromide and visualized with gel documentation 2'[ȝP,'[PHWHUVOHQJWKSURFXUHGIURP
V\VWHP89,7(&&DPEULGJH8.7KHVL]HVRIWKH Agilent Co., USA. Helium was used as the carrier gas
3&5SURGXFWVZHUHGHWHUPLQHGLQFRPSDULVRQZLWK at one mL /min. The MS was set to scan from 50 to
standard 100 bp molecular marker (Genei Pvt. Ltd., 650 Da. The source was maintained at 200oC and <
%DQJDORUH,QGLD 40 motor vacuum pressure. The ionization energy
([WUDFWLRQDQGWHVWLQJRIDQWLIXQJDOELRPROHFXOHV
ZDVH97KH06ZDVDOVRKDYLQJLQEXLOWSUH¿OWHU
against Fusarium spp which reduced the neutral particles. The data system
had two inbuilt libraries for searching and matching
The bioactive compounds were extracted from the spectrum. NIST4 and WILEY9 each contain more
the fruiting bodies at post capping stage, utilizing WKDQ¿YHPLOOLRQUHIHUHQFHV7KRVHFRPSRXQGVZLWK
GLႇHUHQWVROYHQWVviz., methanol, ethanol, chloroform, VSHFWUDO¿WYDOXHVHTXDOWRRUJUHDWHUWKDQZHUH
petroleum ether and aqueous phase. Ten grams of FRQVLGHUHGSRVLWLYHLGHQWL¿FDWLRQ%DVHGRQ06GDWD
freshly picked mushrooms were crushed in a pestle library and comparing the spectrum obtained through
DQGPRWRUDQGVRDNHGLQP/RIVSHFL¿FVROYHQWV GC – MS chromatogram, the compounds present in
used and kept in a shaker at 120 rpm for 12 hours. WKHVDPSOHZHUHLGHQWL¿HG
([WUDFWZDV¿OWHUHGWKURXJKDGRXEOHOD\HUHGPXVOLQ
FORWK IROORZHG E\ :KDWPDQ 1R ¿OWHU SDSHU 7KH 5HVXOWVDQG'LVFXVVLRQ
extracts, collected were evaporated under reduced ,GHQWL¿FDWLRQRIWKHIXQJXV0ROHFXODU
pressure, using a rotary evaporator to obtain characterization
concentrated fraction. This was further dried in an
already weighed sterile Petri plate in the controlled The rDNA fragment of locally collected C. cinerea
environment and the per cent recovery of substance ZDVVXEMHFWHGWR3&5DPSOL¿FDWLRQ,76U'1$UHJLRQ
was calculated in terms of w/v and dissolved in of the fungus comprising two non-coding internal
PHWKDQROPJP/7KHFRQWHQWVZHUH¿OWHUHGWKURXJK WUDQVFULEHGVSDFHUV,76DQG,76DQG6U'1$
D PHPEUDQH ¿OWHU P DQG VWRUHG DW & IRU gene was DPSOL¿HGXVLQJJHQHUDOSULPHUV,76DQG
further study. Antimicrobial activity of this concoction ,767KHDPSOL¿HGSURGXFWVL]HPHDVXUHG
was evaluated against F. o. f. sp. brachygibbosum, F. ES3ODWH%)XUWKHUWKHSXUL¿HG3&5SURGXFWZDV
o. f. sp. cubense and F. o. f. sp. lycopersici by agar subjected to partial sequence and nucleotide BLAST
ZHOOGLႇXVLRQPHWKRG6WRNHDQG5LGJZD\ %ODVWQ DQDO\VLV ZDV SHUIRUPHG LQ WKH 1&%, 7KH
results revealed that, partial sequence of the DNA
$IWHUVROLGL¿FDWLRQRIWKHVWHULOH3'$PHGLXPLQ region had exhibited 96 per cent homology with
Petri dishes, wells of 5 mm in diameter were made on Coprinopsis cinerea 1DVHKL DQG -DYDQ1LNNKDK
each of the plate using sterile cork borer on all four LVRODWHV LQ WKH *HQ%DQN GDWDEDVH 0)
sides, giving equal distance and also by leaving one Based on the nucleotide BLAST results of ITS-rDNA
cm space from the periphery. Fruiting body fractions VHTXHQFHVWKHLGHQWLW\RIORFDOLVRODWHZDVFRQ¿UPHG
were poured separately into the wells at the rate of as Coprinopsis cinerea. The DNA sequences were
1000 ppm per well using a micro pipette. Actively registered in NCBI GenBank database with an accession
growing ten days old mycelial discs of pathogenic no. MH444367.
fungi measuring 5 mm in diameter were inoculated
'HWHFWLRQRIELRDFWLYHFRPSRXQGVE\*&06
separately, at the centre of each of the Petri dish analysis
DQGLQFXEDWHGDWÛ&IRUVHYHQGD\V&RQVWDQW
observations were made and the per cent growth Bioactive compounds extracted from the
inhibition of F. o. f. sp. lycopersici, F. o. f. sp. cubense fruiting bodies at post capping stage in methanolic
and F. brachygibbosum were recorded. Based on fraction are listed in Table 1. GC – MS analysis of
WKH UHVXOW WKH HႇHFWLYH VROYHQWV ZHUH XVHG DW IRXU methanolic fraction of fruiting body indicated the
GLႇHUHQWFRQFHQWUDWLRQVDQG presence of 9 different compounds viz., formic
SSPDQGWHVWHGDJDLQVWFusarium spp. DFLG SURSHQ\O HVWHU 57 DFHWLF DFLG
57 'DODQLQH 1SURSDUJ\OR[\FDUERQ\O LVRKH[\O
'HWHFWLRQRIELRDFWLYHPROHFXOHVRIIUXLWLQJERGLHVRI
C. cinerea by GC - MS analysis
HVWHU 57 +S\UDQRQH GLK\GUR
GLK\GUR[\PHWK\O 57 QKH[DGHFDQRLF
Characterization of biomolecules of fruiting DFLG57RFWDVLOR[DQH
body condensate of C. cinerea0HWKDQROLFIUDFWLRQ K H [ D G H F D P H W K \ O
was done by GC – MS analysis. In this study, the 2,7-Diphenyl-1,6-d ioxopyridazino[4,5:2’,3’]
trace GC Ultra and DSQII model MS from Thermo S\UUROR>¶¶G@S\ULGD]LQH57VTXDOHQH
236

7DEOH*&06DQDO\VLVRIPHWKDQROLFIUDFWLRQRIIUXLWLQJERGLHVDWSRVWFDSSLQJH[WUDFWRIC. cinerea
57 Compounds Molecular formula Molecular weight Structure Area %
4.06 Formic acid, 2-propenyl ester C4H6O2 JPRO 1.51
Acitic acid CH3COOH 60.05 g/mol 1.54
D-Alanine, N-propargyloxycarbonyl-,
C13H21NO4 222.37 g/mol
isohexyl ester
4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-
6.35 &+2 144.12 g/mol 1.17
6-methyl-
n-Hexadecanoic acid C16H32O2 257.42 g/mol 2.17
Octasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,1
31.39 &+26L 577.23 g/mol
3,15,15- hexadecamethyl-
2,7-Diphenyl-1,6-dioxopyridazino[4,5:2’,3’]
33.01 C20H13N5O2 355.34 g/mol 1.50
pyrrolo[4’,5’-d]pyridazine
34.24 Squalene C30H50 410.73 g/mol
35.03 2-Nonadecanone 2,4-dinitrophenylhydrazine C25H42N4O4 462.63 g/mol 0.75
57±5HWHQWLRQWLPH
QRQDGHFDQRQHGLQLWURSKHQ\OK\GUD]LQH acetic acid (Sercan et al DQG VTXDOHQH

$PRQJ WKH GLႇHUHQW FRPSRXQGV IRUPLF (Godio et al,. DUH NQRZQ WR SRVVHVV
DFLG ùHKLUOL et al SURSHQ\O HVWHU antifungal activities. In the present experiment
7DEOH2SWLPL]DWLRQRILQKLELWRU\FRQFHQWUDWLRQRIPHWKDQROLFIUDFWLRQVRIIUXLWLQJERGLHVDWSRVWFDSSLQJ
VWDJHFRQGHQVDWHRIC. cinerea against Fusarium spp
F. brachygibbosum F. o. f. sp. cubense F. o. f. sp. lycopersici

Concentration
Solvent
SSP Growth
*URZWKPP PI PI *URZWKPP PI
PP
70.00d 69.00d 72.00d

500 22.22 23.33 20.00

62.00c 60.00c 63.00c

1000 31.11 33.33 30.00
Methanolic
fraction
53.00b 50.00b 56.00b
1500 41.11 44.44

44.00a 41.00a 43.00a

2000 51.11 54.44 52.22

Control 90.00e 90.00e 90.00e

0.00 0.00 0.00
:DWHU
&'S - - - 1.26 -
PI - Per cent inhibition
9DOXHVDUHPHDQRIWKUHHUHSOLFDWLRQV
237
59
F. brachygibbosum F. o. f. sp. cubense F. o. f. sp. lycopersici

A) Treated plates
B
B B B B

B) Untreated plates
C) Note : a, b, c, d – 500, 1000, 1500 and 2000ppm
3ODWH$,QKLELWRU\HႇHFWRIPHWKDQROLFIUDFWLRQVRIIUXLWLQJERGLHVDWSRVWFDSSLQJ
stage of C. cinerea against plant pathogens
600 – 650bp
%3&5DPSOL¿FDWLRQDQG,76UHJLRQRICoprinus spp
Means followed by a common letter is not cubenseSHUFHQWDQG)RIVSlycopersici

VLJQLILFDQWO\ GLIIHUHQW E\ RQH ZD\ $129$ WKH SHU FHQW )LJ 3ODWH$ DQG 7DEOH
concoction containing the biomolecules of fruiting Formic acid, 2-propenyl ester and acetic acid
bodies of C. cinerea at 2000 ppm showed inhibition are the organic compounds as well as a type of
of F. brachygibbosum SHU FHQW ) R I VS organic acids. These formic and acetic acids were
238
60
)LJ*&06DQDO\VLVRIPHWKDQROLFH[WUDFWRIIUXWLQJERGLHVRIC. cinerea
UHSRUWHGDVDQDQWLPLFURELDODFWLYLW\DQGFDQDႇHFW JM, Foulongne-Oriol M., Largeteau M. and Barroso

the survival of soil borne pathogens like Fusarium G. Eds, France. pp: 140-154.
oxysporum, Fusarium solani, F. o.f. sp. lycopersici *RGLR53)RXFHV5DQG-)0DUWLQ$VTXDOHQH
and bacteria like Ralstonia solanacearum. Squalene epoxidase is involved in biosynthesis of both the
DOVR LGHQWL¿HG IURP WKH IUXLWLQJ ERGLHV H[WUDFW RI antitumor compound clavaric acid and sterols in the
basidiomycete H. sublateritium. Chem. & Biol., :
C. cinerea ZLWK WKH SHDN DUHD SHU FHQW RI
1334-1346.
Godio et alZRUNLQJZLWKAgaricus bisporus
*XLPDUDHV-%3HUHLUD3&KDPEHO/DQG57HQUHLUR
squalene epoxidase indicated that clavaric acid
$VVHVVPHQW RI ¿ODPHQWRXV IXQJDO GLYHUVLW\
formation during the biosynthesis in the ergosterol using classic and molecular approaches: case study–
biosynthesis of Apergillus fumigates which exhibited Mediterranean ecosystem. F. Ecol., : 309-321.
antifungal activity. Liu, Y., Li, Y., Ou, Y., Xiao, S., Lu, C., Zheng, Z. and Y. Shen.
2012. Guanacastane-type diterpenoids with cytotoxic
&RQFOXVLRQ
activity from Coprinus plicatilis. Bioorg. Med. Chem.
The results of the current study indicated the Lett., : 5059-5062.
possibility of extraction of antifungal compounds from Martins, A. 2004. Micorrizacao controlada de Castanea
the inky cap fungus C. cinerea against wilt disease sativa Mill.: Aspectos fisiologicos da micorrizacao
causing Fusarium spp. The methanolic extract of the in vitro. Dissertacao de doutoramento em Biologia/
%LRWHFQRORJLD 9HJHWDO )DFXOGDGH GH &LHQFLDV GH
fungus could inhibit the F. brachygibbosum, F. o. f. sp.
Lisboa. Universidade Classica de Lisboa, p: 506.
cubense and F. o. f. sp. lycopersici upto 54 per cent.
Mshandete, A.M., Lyantagaye, S. L. and T. Ndyetabura.
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from edible tanzanian Coprinus cinereus VFKDHႇ
The authors express their thanks and gratitude s. Gray s. lat. Cultivated on grasses supplemented
WR8*&6$3'56DQG'67±),67*276FKHPHV with cow dung manure. ARPN J. Agric. Biol. Sci.,
operated in the Department of Plant Pathology,
TNAU, Coimbatore for the instrumentation and Park, W.H. and H.D. Lee. 1996. Wild fungi of Korea. Published
laboratory support. (6th edition) in Kyo-Hak Publishing Co., Ltd.S
5DQJDVZDPL*'LVHDVHVRI&URS3ODQWVLQ,QGLD
5HIHUHQFHV Prentice Hall of India Pvt. Ltd., New Delhi, p: 520.
$NNDQQD6XEELDK.DQG9%DODQ$&RPSUHKHQVLYH Sangeetha, C., Krishnamoorthy, A.S., Nakkeeran, S.,
5HYLHZRIWURSLFDOPLON\ZKLWHPXVKURRPCalocybe 5DPDNULVKQDQ6DQG'$PLUWKDP(YDOXDWLRQ
indica3 &Microbiol., of bioactive compounds of Ophiocordyceps sinensis
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resistance. Front. Microbiol., : 439. and Propionic Acids on Plant Pathogenic Fungi. J. of
Badalyan, S. M., Navarro-Gonzalez, M. and U. Kues. 2011. Biol. & Environ. Sci., : 129-137.
7D[RQRPLFVLJQL¿FDQFHRIDQDPRUSKLFFKDUDFWHULVWLFVLQ 6WRNH-(DQG*/5LGJZD\&OLQLFDOEDFWHULRORJ\
the life cycle of Coprinoid mushrooms. In: Proceedings Edward Arnold Ltd. London, p. 143.
of VII Int. Conf. Mushr. Biol. & Mushr. Prod. Savoie
5HFHLYHG0DUFK5HYLVHG0DUFK$FFHSWHG0DUFK
239
Journal of Pharmacognosy and Phytochemistry 2017; 6(5): 2537-2543
E-ISSN: 2278-4136
P-ISSN: 2349-8234 Profiling of morphogenesis related enzymes of milky
JPP 2017; 6(5): 2537-2543
Received: 05-07-2017 mushroom Calocybe indica (P & C)
Priyadharshini Bhupathi
PhD Research Scholar, Tamil
Priyadharshini Bhupathi, Akkanna Subbiah Krishnamoorthy and
Nadu Agricultural University, Sivakumar Uthandi
Akkanna Subbiah
Abstract
Krishnamoorthy Milky mushroom (Calocybe indica P&C) is one among the prevalent edible tropical mushrooms
Professor and Head, Mushroom cultivated in India. It is well known that secretion of lignocellulolytic enzymes in mushrooms play an
Research and Training Centre, important role in degradation of agroresidues but it is understood that certain enzymes are also related to
Centre for Plant Protection morphogenesis of mushrooms. In this regard, the present study was undertaken to study the function of
Studies, Department of Plant morphogenesis related enzymes viz., mannitol dehydrogenase (MtDH), xylanase, laccase, tyrosinase and
Pathology and Tamil Nadu lipoxygenase (LOX) activities at seven different growth stages viz., pinhead, tiny button, button, I
Agricultural University, elongation, II elongation, maturation, complete maturation both in pileus and stipe of Calocybe indica
Coimbatore, Tamil Nadu, India var. APK2 and CBE-TNAU-1523 wild isolate. The results indicated that activity of MtDH, xylanase,
laccase, tyrosinase and LOX in the mushroom is found to be maximum at stage five (II elongation)
Sivakumar Uthandi followed by stage four (I elongation) and the rate of increase in activity was higher from stage one to
Professor, Department of stage five in pileus when compared to stipe in both APK2 variety and CBE-TNAU-1523 wild strain of
Agricultural Microbiology, Tamil
Calocybe indica. Among, all the enzymes assayed, the activity of xylanase was recorded maximum at all
Nadu Agricultural University,
the seven stages of mushroom growth followed by lipoxygenase activity. Maximum activity of xylanase
was recorded in pileus of APK2 variety and CBE-TNAU-1523 wild strain (3.514μmoles/min/g and
3.551μmoles/min/g, respectively) when compared to stipe.
Keywords: Calocybe indica (P&C) var. APK2, CBE-TNAU-1523, morphogenesis related enzymes,
mannitol dehydrogenase, laccase, xylanase, tyrosinase and lipoxygenase
Introduction
Huge amount of lignocellulosic agricultural crop residues and agro-industrial by-products
generated annually which are rich in organic compounds that are worthy of being recovered
and transformed. However, their handling and disposal are often problematic, due to their
chemical structure and decomposition properties and there by burnt or incorporated into the
soil. But these residues are of particular interest for the agricultural economy of temperate and
subtropical countries, since they are produced in large quantities. Philippoussis (2009)
accomplished that the cultivation of mushroom is a prominent biotechnological process to
enhance the value of agro-industrial production. Previous research work indicates that the
lignocellulose degrading mushroom species are used in various solid state fermentation
applications for bioremediation and biodegradation of hazardous compounds (Perez et al.,
2007) [32], biological detoxification of toxic agro-industrial residues (Soccoland Vandenberghe,
2003) [38], biotransformation of agro industrial residues to mushroom food and animal feed
(Okano et al., 2006) [30], compost and product developments such as biologically active
metabolites, enzymes, and food flavour compounds (Nikitina et al., 2007) [29].
Among applications of solid state fermentation (SSF), mushroom cultivation has proved its
economic strength and ecological importance for efficient utilization, value-addition and
biotransformation of agro-industrial residues (Zervakis and Philippoussis 2000) [45].
Commercial mushroom productionis yet another efficient and relatively short biological
process of food protein recovery from unutilized lignocellulosic materials through enzymatic
degrading capabilities of mushroom fungi (Chiu and Moore, 2001) [8].
The use of crop residues depends on the capacity of the fungus to produce a lignocellulolytic
enzyme complex (Buswell et al., 1996) [5]. This complex includes the oxidative enzymes
laccase and manganese peroxidase (MnP), which are involved in lignin degradation (Shah and
Nerud, 2002) [36], and the hydrolytic enzymes xylanase and cellulase (Eira, 2004), which are
Correspondence involved in hemicellulose and cellulose degradation, respectively. In nature, mostly all the
Priyadharshini Bhupathi
basidiomyceteous fungi are able to degrade lignin efficiently by the means of solubilization
PhD Research Scholar, Tamil
Nadu Agricultural University, and mineralization (Kirk and Cullen 1998) [19]. In mushrooms mannitol functions as an
Coimbatore, Tamil Nadu, India osmoregulator by encouraging the influx of water from the environment to develop turgor
~ 2537 ~
240
Journal of Pharmacognosy and Phytochemistry
pressure thereby, helps in fruiting body development rpm for 60 min at 4°C and the supernatant was used as the
(Jennings, 1985) [16]. The extracellular secretion of laccases by cell extract. MtDH activity was determined by incubating the
basidiomycete fungi are capable of mineralizing lignin that is cell extract at 25°C in 20 mM HEPES-KOH (pH 7.5) - 500
apparently unique to this group of organisms (Thurston, 1994) mM fructose - 0.25 mM NADPH. The enzyme activity was
[41]
. The formation of rhizomorphs (mycelial strands formed monitored by recording the change in absorbance at 340 nm.
from large numbers of tightly adpressed hyphae) is a different The enzyme assay was repeated three times to avoid
developmental change that is associated with laccase experimental errors.
synthesis in Armillaria mellea (Worrall et al., 1986) [44] and is
responsible for making a polyphenolic glue that sticks the Assay of Laccase
hyphae together. Xylanases are similar to cellulases and it can Laccase activity was determined using guaiacol as the
act synergistically to achieve hydrolysis; predominant substrate according to the method of Sandhu and Arora
enzymes within this system are endoxylanases which attack (1985) [35]. One gram of tissue was extracted with phosphate
the polysaccharide backbone, and b-xylosidases, which buffer. Cell debris were removed by centrifugation at 32,000
hydrolyze short xylooligosaccharidesto xylose (Montoya et rpm for 60 min at 4°C and the supernatant was used as the
al., 2012) [26]. Tyrosinase is a multifunctional, glycosylated cell extract. Laccase was assayed by adding 0.3 mL enzyme
and copper containing oxidase, which catalyzes the first two source to 2.5 mL of 30 μM Guaiacol in phosphate buffer (0.1
steps in mammalian melanogenesis and is responsible for M) at pH 6.0. Absorbance was read at 470 nm after
enzymatic browning in fresh fruits, vegetables, beverages and incubating the reaction mixture for 30 min at 25oC against
mushrooms (Liu et al., 2013) [22]. Lipoxygenase are involved zero time control. One unit of laccase activity was calculated
in the biosynthesis of leukotrienes and lipoxins, which are as change in absorbance by 0.001 min–1 mL–1 of enzyme
potent mediators of inflammatory responses (Nicolaou et al., source at 250C.
1989) [28]. Also they play role in the production of volatile
molecules that can positively or negatively influence the Assay of Xylanase
flavor and aroma (Tasaki et al., 2013) [40]. Xylanase activity assay was performed according to Miller
Milky mushroom (Calocybe indica) var.APK2 is one of such (1959) [25]. One gram of tissue was macerate and extracted
mushroom varieties, where complete commercial production with sodium citrate buffer (pH 5.3) and centrifugation at
techniques have been standardized for the first time of Tamil 32,000 rpm for 60 min at 4°C and the supernatant was used as
Nadu Agricultural University (Krishnamoorthy, 1998) [20]. the cell extract. The reaction mixture consisting of 1.8 mL of
The milky mushroom Calocybe indica is highly suitable for a 1.0% (w/v) suspension of birch-wood xylan in
hot humid climate and can be cultivated almost throughout 50mMsodiumcitrate and 0.2 mL of enzyme dilution (in 50
the year in India except few places (Pani, 2010). Since this mM sodium citrate at pH 5.3) was incubated at 50oC for 5
mushrooms is morphologically similar to Agaricus bisporus min. Released reducing sugars were determined by
(button mushroom), it has been quite popular in southern dinitrosalicylic acid reagent (DNS) method, by adding 3 mL
Indian states and slowly getting popular in other countries of DNS solution and then incubating the mixture at 95oC for5
(Krishnamoorthy and Balan, 2015). In this regard, the present min. Absorbance was measured at 540 nm. One unit of
study focuses the role of the assay of Calocybe indica var. enzyme is defined as the amount of enzyme catalyzing the
APK2 and CBE-TNAU-1523 wild strain morphogenesis release of 1 mmol of xylose equivalent per minute.
related enzymes at different growth stages of milky Assay of Tyrosinase
mushroom. The tyrosinase activity assay was performed according to
Boiret et al., (1985) [3] with slight modifications. The one
Materials and methods gram of tissue was macerated with phosphate buffer (pH 6.8)
Preparation of milky mushroom bed at 1:1 (w/v) ratio. The extract was collected and centrifuged at
Beds were prepared following “polybag method” described by 15,000 rpm for 30 min at 4°C. The supernatant was used as
Baskaran et al., (1978) [2] using paddy straw. enzyme source. Assay was conducted with the reaction
mixture, total volume of 3.0 ml: 3 mM-L-tyrosine, 47 mM-
Preparation of Casing soil sodium phosphate buffer (pH 6.8) and 25 μl of enzyme
Garden soil (clay loam; pH 8.4) was used for casing source. The rate of formation of dopachrome was measured at
throughout the study. The soil moisture content was adjusted 475 nm. Control was maintained without adding enzyme
to 20 per cent following the method of Devadoss (1971) [9]. mixture. The enzyme activity was expressed as changes in
absorbance at 470 nm
Collection of sample
Fresh fruiting bodies of Calocybe indica (P&C) var. APK2 Assay of Lipoxygenase
and wild strain CBE-TNAU-1523 at seven different growth Lipoxygenase was assayed according to Axelrod et al., (1981)
stages were collected from separately as described by [1]
. The cells were homogenized in an ice bath with 0.1 M
Chakraborthy et al., (2000) and used for morphogenesis TRIS-HCl buffer (pH 8.5) containing 1% PVP (w/v), 1 mM
related enzyme assay. CaCl2, 5 mM DTT, and 10% (v/v) glycerol. The homogenate
was centrifuged at 11 000 rpm for 20 min at 4 °C, and the
Assay of Mannitol dehydrogenase (MtDH) supernatant was used as the enzyme extract. 50 mg of linoleic
Mannitol dehydrogenase was assayed as suggested by acid was added to 50 mg Tween 20 and mixed with 10 ml of
Chakraborthy et al. (2004) with some modifications. One Na2HPO4 buffer (0.1 M, pH 8.7) by stirring. The solution was
gram of tissue was extracted with 20 mM HEPES cleared by addition of 250 ml of 1 M NaOH and diluted to 25
(Hydroxyethylpiperazinethanesulfonic acid) KOH buffer (pH ml with the buffer. One ml of the enzyme reaction mixture
7.5) containing 1 mM EDTA, 2 mM 2- mercaptoethanol and 2 contained 50 ml enzyme extract, 0.95 mlNa2HPO4 buffer and
mM PMSF (Phenyl Methyl Sulfonyl Fluoride). Unbroken 5 ml of substrate solution. The increase in absorbance was
cells, and cell debri were removed by centrifugation at 32,000 monitored at 234 nm.
~ 2538 ~
241
Result and Discussion Pleurotus ostreatus, with the minimum of activity occurring
The enzymes activities were spectrophotometrically assayed when the mushrooms were mature.
in all seven growth stages of the milky mushrooms viz.,
Calocybe indica (P&C) APK2 variety and CBE-TNAU-1523 Xylanase activity
wild strain at 24 hours interval. The results revealed that the Among all the enzymes tested xylanase shows maximum
morphogenesis related enzymes viz., mannitol dehydrogenase, activity at all the growth stages of both Calocybe indica
xylanase, laccase, tyrosinase and lipoxygenase were secreted (P&C) var. APK2 and wild strain CBE-TNAU-1523 but the
in higher levels in pileus when compared to stipe. Among all level of enzyme induction in stage six and seven was
the enzymes tested, xylanase showed maximum activity decreased when compared to other stages. The results
followed by lipoxygenase in both the mushrooms. Tyrosinase indicated that stage five is having more activity of
enzyme recorded lesser activity in all the stages of APK2 xylanase3.514μmoles/min/g, followed by stage four (3.443
variety and CBE-TNAU-1523 wild strain. μmoles/min/g) in APK2variety (Table 1) similar trend was
observed in CBE-TNAU-1523 wild strain with higher
Morphological stages of milky mushroom xylanase activity in stage five (3.551μmoles/min/g) and stage
The different growth stages of Calocybe indica is refers to four (3.452 μmoles/min/g) (Table 2). Whereas, stage1 and
pinhead (stage 1), tiny button (stage 2), button (stage 3), I stage2 recorded less activity of xylanase in both mushrooms.
elongation (stage 4), II elongation (stage 5), maturation (stage However the activity of xylanase was maximum in pileus
6), complete maturation (stage 7) (Plate 1 and 2). when compared to stipe in both mushrooms. Elisashvili et al.,
(2008) [11] reported that the P. ostreatus xylanase activities
Mannitol dehydrogenase activity (Mtdh) gradually increased during second fruiting stage of mushroom
In the present study, mannitol dehydrogenase activity was and then decreased. Matsumoto (1998) found that the
maximum in stage five (0.635 and 0.711 μmoles/min/g in cellulase and xylanase activities increased during the
APK2 and CBE-TNAU-1523, respectively) followed by stage development of the fruiting bodies of Lentinula edodes, with
four (0.617 and 0.623 μmoles/min/g in both mushrooms). highest levels during mushroom maturation which may be due
Among the parts of mushroom, pileus recorded maximum to the fungus need to mobilize large amounts of carbon for
(0.635 and 0.711 μmoles/min/g) activity of the enzyme mushroom formation.
followed by stipe (0.221 and 0.133 μmoles/min/g) in both
APK2 and CBE-TNAU-1523 milky mushrooms (Tables 1 Tyrosinase activity
and 2). The rate of increase in activity was higher from stage Tyrosinase is an enzyme that belongs to Poly phenol oxidase
one to stage five when compared to stage six and stage seven. family. The present study indicates that the presence of
During the mannitol synthesis NADPH is obtained from tyrosinase recorded minimum activity when compared to all
pentose phosphate pathway (Dutsch and Rast, 1972) [10]. the enzymes assayed. Stage five registered significantly high
Mannitol, a six carbon polyol that accumulates in growing tyrosinase activity (0.043μmoles/min/g)followed by stage
sporophores i.e. in both pileus and stipe during and between four (0.033μmoles/min/g) in APK2 variety and 0.142 and
the flushes of fruiting body development (Hammond & 0.112 μmoles/min/g in CBE-TNAU-1523 (Table 1 and 2).
Nichols, 1976) [13]. The increase in MtDH. activity in The tyrosinase activity increased with subsequent stages of
developmental stages had been attributed to the increase in maturity, which was correlated with senescence of fruiting
Hexose monophosphate (HMP) activity (Hammond, 1981) bodies of V. volvacea (Kiran Kumar, 2015) [18] and browning
[14]
. In contrast, mannitol dehydrogenase activity was reported effect in Agaricus mushrooms (Singh et al., 2010) [37].
to be high during the developmental stage 1 of A. bisporus
and decreased progressively in subsequent stages (Morton et Lipoxygenase
al., 1985) [27]. In this present study, lipoxygenase is recorded as a second
highest enzyme activity present in all the stages of mushroom
Laccase activity next to xylanase. Among the parts of the mushroom high
The laccase activity was found to be significantly higher in lipoxygenase activity was recorded in pileus (3.435
stage 5 (1.519 and 1.681μmoles/min/g) in Calocybeindica μmoles/min/g) in stage five followed by stage four (3.159
(P&C) var. APK2 and wild strain CBE-TNAU-1523, μmoles/min/g) in variety APK2 (Table 1). In CBE-TNAU-
respectively. Minimum laccase activity was recorded at stage 1523, 3.453and 3.132 μmoles/min/g recorded in pileus of
1 (0.333μmoles/min/g in APK2 variety and stage 5 and stage 4, respectively (Table 2). Hiroi (1988) [15]
0.411μmoles/min/g CBE-TNAU-1523).Widiastuti et al., and Kuribayashi et al., (2002) [21] reportedthat the purified
(2008) described that the activity of laccase was very high form of lipoxygenase (LOX) showed that free linoleic acid,
(1.762 U/ml) during the mycelial colonization of P. ostreatus which is the most abundant fatty acid (72.4% of total fatty
and which declined after two weeks and further increased in acid) present in the pileus of P. ostreatus converts linoleic
three weeks, but during fruit body formation the laccase acid to 13-Z,E-HPOD (hydroperoxy-(Z, E)-11, 13-
activity reduced sharply. In the cultivated button mushroom eicosadienoic acid). In the other hand Mau et al., (1992) [24]
Agaricus bisporus, laccase activity accumulates during reported that 10-oxo-trans-8-decenic acid produced
vegetative growth but undergoes rapid inactivation shortly concurrently with 1-octen-3-ol might be involved in the
after the onset of fruit body formation (Wood, 1980) [43]. development of fruiting bodies of Agaricus bisporus. Because
Kalmis and Sargi (2004) [17] reported that there existed an it has been assumed that the formation of 1-octen-3-ol
inverted correlation between laccase and manganese requires actions of lipoxygenase and hydroperoxidelyase-like
peroxidase activities and fruiting body maturation of enzyme, involved in the fruiting formation of mushroom.
~ 2539 ~
242
Plate 1: Different growth stages of Calocybe indica var. APK2
S1-Pinhead S5-II Elongation

S2-Tiny button S6-Maturation
S3-Button S7-Complete maturation
S4-I Elongation
~ 2540 ~
243
Plate 2: Different growth stages of Calocybe indica CBE-TNAU-1523wild strain
S1-Pinhead S5-II Elongation

S2-Tiny button S6-Maturation
S3-Button S7-Complete maturation
S4-I Elongation
Fig 1: Assay of morhogenesis related enzymes of Calocybe indica Var. APK2
Conclusion through genome editing techniques to architect the mushroom

The morphogenesis related enzymes viz., mannitol size and to improve the flavour of milky mushroom.
dehydrogenase, laccase, xylanase, tyrosinase and
lipoxygenase highly mediate the growth and development of Acknowledgements
milky mushroom Calocybe indica (P&C) var. APK2 and The authors are thankful to UGC for financial support through
CBE-TNAU-1523 wild strain. The production of Rajiv Gandhi National Fellowship. The support and facilities
morphogenesis related enzymes were higher in vegetative provided for this research through ICAR-AICRP on
growth phases of C. indica and dropped at the harvest of the Mushroom Scheme, UGC-SAP-DRS1“Enterprising
mature mushrooms. The drastic changes of enzyme activities Mushroom Biotechnology for Food, Feed and Biomanure”
as observed in milky mushroom Calocybe indica from stage and DST-FIST, Government of India operated in the
one to stage seven was assumed to be due to the influence of Department of Plant Pathology, TNAU, Coimbatore are
internal physiological conditions of the mushrooms which is greatly acknowledged.
related to synchronization and oxidation as reported by
Bonnen et al., (1994) [4] and Ruhl et al., (2008) [34] in Agaricus References
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Int.J.Curr.Microbiol.App.Sci (2019) 8(6): 1172-1186
International Journal of Current Microbiology and Applied Sciences

ISSN: 2319-7706 Volume 8 Number 06 (2019)
Journal homepage: http://www.ijcmas.com
Original Research Article https://doi.org/10.20546/ijcmas.2019.806.145
Antimicrobial Activity of Biomolecules from Mushroom Fungi

against Colletotrichum capsici (Syd.) Butler and Bisby,
the Fruit Rot Pathogen of Chilli
K. Priya, G. Thiribhuvanamala*, A. Kamalakannan and A.S. Krishnamoorthy
Department of Plant Pathology, Tamil Nadu Agricultural University,

Coimbatore- 641 003, India
*Corresponding author
ABSTRACT
Mushroom fungi secrete antifungal, antibacterial and antiviral bioactive compounds of

therapeutic and pharmacological value. Very limited work has been done on the
exploration of antimicrobial principles from macrobasidiomycetes against plant pathogens.
In this view, a study was proposed to screen eight mushroom fungi viz., Auricularia
polytricha, Coprinus comatus, Ganoderma lucidum, Volvariella volvaceae, Lentinus
Keywords
edodes, Pycnoporus sanguineus, Schizophyllum commune, Trametes versicolor against
Chilli, spore germination and mycelial growth of Colletotrichum capsici , the fruit rot pathogen of
Colletotrichum Chilli and to extract antimicrobial molecules from the selected mushroom fungi using
capsici, Ganoderma different solvents viz., Chloroform, Diethyl ether and Ethyl acetate. Results from dual
lucidum, Solvents, culture technique revealed that Ganoderma lucidum, Auricularia polytricha and Lentinus
Mushroom fungi, edodes showed maximum antifungal activity by inhibiting the mycelial growth of C.
Antimicrobial capsici (54.81%, 53.70 % and 45.55% respectively) with maximum inhibition zone of
activity, Inhibition
(4.86 mm, 2.86 mm and 4.86 mm respectively). Though the Chloroform, Diethyl ether and
per cent
Ethyl acetate fractions of G. lucidum cell free extracts inhibited spore germination of C.
Article Info capsici both at 12 and 24 hours, maximum inhibition of spore germination was observed at
24 hours. Among the mushroom fungi, the chloroform extracted fractions followed by
Accepted:
Diethyl ether and Ethyl acetate fractions of G. lucidum cell free culture filtrates exhibited
10 May 2019
Available Online:
maximum inhibition of spore germination of C. capsici (inhibition of 88 %, 79% and 78 %
10 June 2019 respectively) at 24 hours. Similarly, maximum inhibition of mycelial growth of C. capsici
with 40 %, 34.07% and 29.25 % inhibition respectively was recorded in the chloroform
extracted fractions followed by Diethyl ether and Ethyl acetate fractions of G. lucidum cell
free culture filtrates when compared to solvent extracted fractions of L. edodes and A.
polytricha by agar well diffusion technique. The chloroform extracted metabolite of G.
lucidum followed by Ethyl acetate and Diethyl ether fractions at 2000 ppm concentration
inhibited maximum mycelial growth of C. capsici (60.55 %, 58.88 % and 55.47 %
respectively). It is well proven that chloroform extracted fractions of G. lucidum possess
antimicrobial activities against the growth of C. capsici. Hence, further studies towards the
identification of these compounds will pave for development of fungicides against C
.capsici.
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Introduction antibacterial, antifungal, antioxidant, antiviral

antinemic, antitumor, immunosuppressive,
Chilli (Capsicum annum L.) is an important antiallergic, anti-inflammatory activities,
commercial spice crop grown in almost all the hypolipidemic, and hepatoprotective activity
states of India with 1.49 million tonnes of dry (Hatvani, 2001; Wasser, 2002; Lindequist et
chilli produced annually (FAOSTAT, 2013). al., 2005; Reis et al., 2011; Rouhana-Toubi et
Such an important crop is affected by many al., 2015). Owing to the current emphasis on
diseases including fungi, bacteria and viruses, the ecofriendly approaches for plant disease
the most important being anthracnose (fruit management, mushroom fungi can serve as
rot) disease caused by a complex of promising source of antimicrobials against
Colletotrichum species that cause latent plant pathogens as evidenced by the
infection and affects ripe fruits (Jeffries et al., antimicrobial activity of the culture filtrates of
1990) and resulting in both pre and post- Ophiocordeyceps sinensis against soil borne
harvest fruit decay with yield losses of up to pathogens of Fusarium oxysporum f. sp.
50% (Liu et al., 2016), about 25% fruit loss at lycopersici and F. oxysporum f. sp. cubense
pre-harvest stage and 25-40% loss at post- (Sangeetha and Krishnamooorthy, 2015),
harvest stage (Sharma and Shenoy, 2014) and Coprinus comatus against F. oxysporum f. sp.
severe losses of 10-60% both in yield and brachygibbosum, Fusarium oxysporum f. sp.
quality of the chilli (Bansal and Grover, lycopersici and F. oxysporum f. sp. cubense
1969). Among these, Colletotrichum capsici (Jeeva and Krishnamoorhy, 2018), ethanolic
(Syd.) Butler and Bisby is reported to survive extracts of Leucopaxillus gignatea against
in plant debris, spreads through seeds from Aspergillus niger, Fusarium solani,
infected fruits and causes both fruit rot and Collectotrichum graminicolum and
die back in chillies thereby leading to severe Helminthosporium maydis, Xanthomonas
yield reduction. Moreover, secondary spread axanopodis pv. punicae, Pseudomonas
in the field is favoured at a temperature syringa and Bacillus subtilis, (Feleke and
around 27-28°C with relative humidity of 80 Anila Doshi, 2017). Perusal of literature
per cent through wind borne conidia that aids showed that no work has been attempted on
in fast spread of the disease (Roberts et al., the exploitation of antimicrobials from
2001). The intensive use of fungicides has mushroom fungi against Colletotrichum
resulted in the accumulation of toxic capsici and this kindled interest to undertake
compounds potentially hazardous to humans the present investigation with an aim to
and the environment, and also in the build-up identify a potential mushroom fungus with
of resistance of the pathogens. antimicrobial activity against C. capsici, the
chilli fruit rot pathogen.
As chilli is an edible crop and large quantity
of pesticides was being used, there is a Materials and Methods
growing demand for chemical pesticide free
organic chilli world over. Current research is The chilli fruit rot pathogen Colletotrichum
focused on search of antimicrobials from capsici (Acc. No. MK758061) and the
green channels such as plants, fungi and mushroom fungal cultures viz., Ganoderma
bacteria in order to identify biopesticidal lucidum, Auricularia polytricha, Lentinus
compounds. Perusal of literature showed that edodes, Coprinus sinensis, Schizophyllum
apart from food, mushroom fungi are commune, Trametes versicolor, Volvariella
important as natural sources of medicines and volvaceae and Pycnoporus sanguineus
possess number of bioactive compounds viz., obtained from the Department of Plant
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Pathology, Tamil Nadu Agricultural Mycelial discs (measuring 9 mm dia.) was cut
University, Coimbatore were used for the from margin of a 10 day old culture of G.
studies. lucidum, L. edodes, A. polytricha grown in
PDA medium in petridishes and inoculated in
In vitro screening of mushroom fungi 250 ml conical flasks containing 100 ml of
against C. capsici sterilized PD broth. The flasks were placed on
a rotary shaker maintained at 120 rpm and
Mushroom fungi viz. Ganoderma lucidum, incubated at 25ºC for 20 days. After
Auricularia polytricha, Lentinus edodes, incubation, the culture filtrate and the
Coprinus sinensis, Schizophyllum commune, mycelial mat were separated by filtration
Trametes versicolor, Volvariella volvaceae through Whatman No. 40 filter paper. The
and Pycnoporus sanguineus were tested for filtrate was further centrifuged at 10,000 rpm
its antagonistic activity against C. capsici by and the Cell Free Culture filtrate (CFC) of G.
following dual culture technique (Dennis and lucidum, L. edodes, A. polytricha was
Webster, 1971). A 9 mm mycelial disc of extracted separately with three different
mushroom fungi was placed at the edge of the solvents viz., ethyl acetate, chloroform and
Petri plates containing PDA medium on one diethyl ether. Liquid-liquid extraction was
side. Similarly, on the opposite side a 9 mm carried out three to four times for each
mycelial disc of C. capsici was placed. The solvent. The ethyl acetate, chloroform and
dual culture plates were incubated at 28±2ºC diethyl ether solvent extracts from CFC of G.
for 7 days. Three replications were lucidum, L. edodes, A. polytricha was
maintained for each treatment. Plates with C. evaporated separately under reduced pressure
capsici only and respective mushroom fungi using a rotary evaporator to obtain the
served as control. The plates were examined residues. The condensate or residue so
periodically and measurements on the radial obtained from solvent was dried and
mycelial growth of C. capsici and mushroom dissolved in methanol (1mg/ml) and filtered
fungi were recorded till the control plate with membrane filter (0.48 μm), stored at 4ºC
attained full growth (90mm). The percent used for further studies.
inhibition of mycelial growth of C. capsici
was calculated by using the formula proposed Effect of different solvent extracted
by Vincent (1947). metabolites from selected mushroom fungi
on spore germination and mycelial growth
Percent inhibition of growth (PI) = C- of C. capsici
T/C×100
Spore germination test
Where, C is the growth of pathogen in control
(mm) and T is the growth of pathogen in The different solvent (chloroform, diethyl
treatment (mm). ether, ethyl acetate) extracted metabolites of
G. lucidum, L. edodes, A. polytricha were
Preparation of solvent extracted tested separately against spore germination of
metabolites from selected mushroom fungi C. capsici using cavity slides (Anonymous,
1943).
Based on the above studies, the mushroom
fungi that showed maximum inhibition of A drop of chloroform, diethyl ether and ethyl
mycelial growth of C. capsici was selected acetate solvent extracted metabolites of G.
and used for further studies. lucidum, L. edodes, A. polytricha were placed
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separately in a cavity slide and a drop of spore Effect of different concentrations of solvent
suspension (1×106 spores/ml) of C. capsici extracted metabolites of G. lucidum on
prepared in sterile distilled water was added mycelial growth of C. capsici by Agar well
to each of the solvent extracted metabolite diffusion assay
and thoroughly mixed. The cavity slide was
placed in the Petri dish moistened with cotton Based on the above studies, Ganoderma
and incubated at room temperature (28 ± lucidum was identified to possess maximum
2ºC). Three replications were maintained for antimicrobial activity against C. capsici and
each treatment. The spore suspension in used for the studies. Different solvents viz.,
sterile water alone served as control. The chloroform, diethyl ether and ethyl acetate
spore germination was observed and recorded extracted metabolites from culture filtrate of
after 6, 12 and 24 hours under phase contrast Ganoderma lucidum were prepared as
microscope and the percent inhibition of mentioned earlier. The solvent extracted
spore germination was calculated using the metabolites of G. lucidum were made up to
formula (Akhter et al., 2006). 1000, 1500 and 2000 ppm and used for testing
the Minimum Inhibitory Concentration (MIC)
Inhibition of conidial germination (%) = of the metabolites that could inhibit the
mycelial growth of C. capsici by agar well
Total number of - Number of germinated
conidia conidia diffusion assay.
x 100
Total number of conidia Results and Discussion
Mycelial growth inhibition test Though it is well proven that mushrooms are
used as food and in pharmaceuticals since
The different solvent (chloroform, diethyl ancient times, the recent research has proved
ether, ethyl acetate) extracted metabolites of that the mushroom fungi possess secondary
G. lucidum, L. edodes, A. polytricha were metabolites of antimicrobial nature to be
tested separately against mycelial growth of effective against many plant pathogens. There
C. capsici by agar well diffusion method is great scope for developing biopesticidal
(Stroke and Ridgway, 1980). After molecules from mushroom fungi that can be
solidification of PDA medium in Petri dishes, used for development of fungicides in plant
four wells (5mm in diameter) were made on disease management. The best evidence is the
the plate using sterile cork borer on all four fungicide Azoxystrobin derived from
sides, giving equal distance and also by mushroom fungi Strobilurus tenacellus
leaving one cm space from the periphery. The effective against downy mildew and powdery
concentration of solvent extracted mildew of grapes. Since the last decade, lot of
(chloroform, diethyl ether, ethyl acetate) work has been initiated on the identification
metabolites of G. lucidum, A. polytricha and of antimicrobial metabolites from mushroom
L. edodes were made up to 500 ppm and fungi against clinical pathogens especially
poured into agar wells at the rate of 100μl per bacteria. Very limited work has been done on
well using micro pipette. Then, mycelial disc the fungal plant pathogens. The easiest and
of C. capsici (5mm diameter) taken from ten most reliable way to assess the antagonistic
days old culture was placed at the centre of potential of mushroom fungi has to be done
each Petri dish and incubated at 28±2ºC for by dual culture test (Dennis and Webster,
seven days. Observations on the per cent 1971) where the growth nature of the
inhibition of mycelial growth of C. capsici mushroom fungi and test pathogen will give
were recorded (Vincent, 1947).
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an indication of the presence or absence of mushroom fungi, Lentinus edodes, Trametes

antimicrobial activity of the mushroom fungi. versicolor, Volvariella volvaceae and
Coprinus sinensis also showed mycelial
In vitro screening of mushroom fungi growth inhibition of C. capsici with 45.55 per
against C. capsici by dual culture technique cent, 43.63 per cent, 42.92 per cent and 40 per
cent respectively. However, inhibition zone
In our study, among the mushroom fungi was maximum (4.86 mm and 4.86 mm) in G.
tested, Pycnoporus sanguineus, followed by lucidum and L. edodes respectively followed
Ganoderma lucidum and Auricularia by A. polytricha (2.76 mm), V. volavceae
polytricha showed reduced mycelial growth (2.83 mm) and T. versicolor (1.1mm) (Table
of C. capsici (49 mm, 40.67mm and 41. 67 1; Plate 1). The interactions between
mm respectively) when compared to control respective mushroom fungi and C.capsici in
(90 mm) with inhibition per cent of 57.41, dual culture technique is furnished below.
54.81 and 53.70 respectively. Other
Interactions between mushroom fungi and C. capsici
Mushroom fungi and Pathogen Nature of Interaction

Ganoderma lucidum and C. capsici Clear inhibition zone of 4.86mm; both mushroom
fungi and pathogen did not grow over each other
even after 10 days
Auricularia polytricha and C. capsici Initially clear inhibition zone of 2.76mm; later
mushroom fungi hyperparasitised over the pathogen
Lentinus edodes and C. capsici Pathogen growth was retarded and pushed back with
an inhibition zone of 4.86mm
Trametes versicolor and C. capsici Inhibition zone of 1.10 mm; both pathogen and
mushroom fungi did not grow over each other
Pycnoporus sanguineus and C. capsici No inhibition zone, but thick mat was formed at the
contact of both fungi; later mushroom fungi
hyperparasited over the pathogen
Schizophyllum commune and C. capsici No inhibition zone; but hyperparasitization of
mushroom fungi over pathogen
Coprinus sinensis and C. capsici No inhibition zone, thick mat formed between both
fungi
Volvariella volvaceae and C. capsici No inhibition zone, both fungi did not grow over
each other
Similar to our study, Badalyan et al., (2014) aegerita was found to reducing local lesions
reported the antagonistic activity of Pleurotus of Ground nut bud necrosis virus in cowpea
ostreatus, Hypholoma fasciculare, (Sajeena and Marimuthu, 2013) and Tobacco
Ganoderma lucidum, Lentinus tigrinus and mosaic virus infection (Sun et al., 2003). This
Schizophyllum commune Cochliobolus could be due to the effect of Ganoderma
sativus, Fusarium culmorum, constituents in inhibiting the viral replication
Gaeumannomyces graminis and Rhizoctonia by interfering with their adsorption, viral
cerealis by dual culture technique. integration, assembly and release (Gao et al.,
Constituents of Ganoderma and Agrocybe 2003).
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germination inhibition at 24 hours was

Based on the inhibition zone and maximum observed to be 66.25% in Ethyl acetate
inhibition per cent of mycelial growth of the fraction followed by Chloroform and Diethyl
pathogen, the mushroom fungi G. lucidum, A. ether (59.5 % and 59% respectively). The
polytricha and L. edodes possessing chloroform fraction of L. edodes inhibited
antagonistic activity was assumed to secrete spore germination of C. capsici with
antimicrobial compounds and hence selected inhibition of 57.75 % and 67.25% at 12 and
for further studies. 24 hours respectively. Similarly, the Ethyl
acetate fractions and Diethyl ether fractions
Testing the solvent extracted metabolites of recorded inhibition of spore germination
selected mushroom fungi (G. lucidum, A. (56.5% and 65.25% respectively) at 12 hours
polytricha and L. edodes) against spore and (53.25 % and 64.25% respectively) at 24
germination and mycelial growth of C. hours (Table 2a). Chen and Hyuang (2010)
capsici reported that the culture filtrates of Lentinula
edodes and Clitocybe nuda completely
The use of extraction solvents is important to inhibited the spore germination of
extract antimicrobial components of interest Colletotrichum higginsianum. Also, culture
as many of the macro fungi extracted with filtrates of Ganoderma lucidum inhibited
polar and non polar solvents contained spore germination of Alternaria brassicicola
bioactive compounds with antifungal, and culture filtrates of Coprinus comatus, L.
antibacterial and antiviral activities against edodes, Tremella aurantialba and C. nuda
human pathogens (Wasser, 2002). In our study suppressed the germination of Phytophthora
different solvents viz., Chloroform, Diethyl capsici.
ether and Ethyl acetate were used to extract
the antimicrobial compounds from 20 day old The agar well diffusion of solvent extracted
crude cell free culture filtrates of G. lucidum, constituents of cell free culture filtrates of G.
L. edodes and A. polytricha. All three extracts lucidum, L. edodes and A. polytricha (Table
used in the study had antimicrobial activity of 2b) showed that all the metabolites extracted
C. capsici. The results obtained in the present from all the three solvents (Chloroform,
study showed that the Chloroform, Diethyl Diethyl ether and Ethyl acetate) at 500 ppm
ether and Ethyl acetate fractions of G. lucidum exhibited inhibition of mycelial growth of C.
cell free extracts inhibited spore germination capsici. Variations in inhibition of mycelial
of C. capsici both at 12 and 24 hours; with growth were observed among different
maximum inhibition of spore germination at solvent extracted metabolites against C.
24 hours. The Chloroform, Diethyl ether and capsici. In the present study, among the
Ethyl acetate extracted constituents of G. different solvents, Chloroform extracted
lucidum showed spore germination inhibition metabolites of G. lucidum recorded 40 per
of 85 %, 73.75% and 70.25 % respectively at cent inhibition of mycelial growth of C.
12 hours and inhibition of 88 %, 79% and 78 capsici followed by Diethyl ether (34.07 %
% respectively at 24 hours. In the case of A. inhibition) and Ethyl acetate fractions
polytricha, ethyl acetate fractions of (29.25% inhibition). In the case of L. edodes,
metabolites exhibited inhibition of spore Chloroform fractions exhibited 36 per cent
germination of C. capsici with inhibition of inhibition of mycelial growth followed by
55.75 % followed by Chloroform and Diethyl Ethyl acetate (32.96%) and Diethyl ether
ether fractions at 52.75 and 52.5 per cent fractions (31 per cent). The Ethyl acetate
inhibition respectively at 12 hours. The spore fractions followed by Diethyl ether fractions
1177
252
of A. polytricha recorded 32.96 and 30 per Komemushi et al., 1996; Quereshi et al.,
cent inhibition of mycelial growth 2010). In some other studies, crude
respectively followed by Chloroform methanolic extract (100 ppm) from Clitocybe
fractions (22 per cent inhibition). From the sp, Boletus affinis var. maculosus would
study, it is observed that chloroform extracted exhibited maximum inhibition against
fractions followed by Diethyl ether and Ethyl Colletotrichum coffaenum (89.08 per cent and
acetate fractions of G. lucidum showed 76.69 per cent) followed by Leucocoprinus
maximum inhibition of mycelial growth of C. fragilissimus, Collybia strictipes and
capsici with 40 %, 34.07% and 29.25 % Lactarius sp( Shahid et al., 2016)
respectively when compared to other solvent
extracted fractions of L. edodes and A. Based on the above studies, G. lucidum
polytricha which clearly shows the possessing maximum antimicrobial activity
antimicrobial nature of metabolites from G. with respect of inhibition of spore
lucidum. Widest inhibitory zone (33mm) was germination and mycelial growth inhibition
obtained with acetone extract from mycelium was taken for further studies.
of Ganoderma lucidum against Pseudomonas
aeruginosa (Sheetal Mehta and Savitha Testing different concentrations of solvent
Jandaik, 2012). extracted metabolites of G. lucidum against
C. capsici
Variations in antimicrobial activity of G.
lucidum extracts was observed in different The antimicrobial metabolites of G. lucidum
solvent fractions and it is explained that the were extracted using different solvents
reason for the differences in their (chloroform, diethyl ether, ethyl acetate) were
antimicrobial effectiveness may be due to the made up to different concentrations of
differences in the molecular weight of the 1000,1500 and 2000 ppm to test the desired
compounds or due to the genetic makeup of concentration that could inhibit maximum
the test organisms (Uma Gowrie et al.,2014; mycelial growth of C. capsici. From the
Gebreyohannes et.al., 2019). Moreover the results (Table 3; Plate 2) it is observed that all
Ganoderma compounds identified are mostly the solvent extracted antimicrobial
Triterpenes (lanostanoid-type triterpene and metabolites of G. lucidum at 1000 ppm, 1500
polyketides (Farnesyl quinone), small ppm and 2000 ppm exhibited mycelial growth
peptides (ganodermin) and polysaccharides inhibition of C. capsici ranging (43.75 per
with antimicrobial properties (Wang and Ng, cent to 60.55 per cent). The Chloroform
2006; Zhang et al., 2015; Basnet et al., 2017). extracted metabolite of G. lucidum (60.55 %)
The antifungal agent phellinsin A from followed by Ethyl acetate and Diethyl ether
Phellinus sp. inhibited the growth of (58.88 % and 55.47 % respectively) fractions
Colletotrichum lagenarium, Pyricularia at 2000 ppm inhibited maximum mycelial
grisea, Rhizoctonia solani, Aspergillus growth of C. capsici. The chloroform extract
fumigatus and Trichophyton mentagrophytes of Hygrophorus agathosmus and the
(Hwang et al., 2000; Chowdhary et al., 2015). dichloromethane extract of Suillus collitinus
Antibacterial activity of L. edodes against were the most active extracts against both
Gram negative and Gram positive bacteria yeast and bacteria (Yamac and Fatma Bilgili,
viz., Staphylococcus aureus, Bacillus subtilis, 2006). The chloroform extract (100 μl
Escherichia coli, Klebsiella pneumoniae, concentration) of G. lucidum basidiocarp
Pseudomonas aeruginosa, and Salmonella showed antibacterial activity against S. typhi
typhi has been reported (Ishikawa et al., 2001; with inhibition zone of 18mm and antifungal
1178
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activity against C. albicans with inhibition Fusarium oxysporum. f. sp. lycopersici,

zone of 17mm (Uma Gowrie et al., 2014). Macrophomina phaseolina and Rhizoconia
Ethyl acetate was found to be the best solvent solani (Ganeshkumar and Krishnamoorthy,
for extracting antimicrobial substances from 2014). The fruiting body, mycelia and spores
L. edodes, A. polytricha and V. volvaceae of G. lucidum contain ganoderic acid,
which showed inhibition of mycelial growth polysaccharides, triterpenoids, fatty acids,
of Alternaria solani, Colletotrichum capsici, nucleotides, protein, peptides, sterols (Yoon et
Phytopthora and Rhizoctonia solani al., 1994; Mizuno et al., 1995; Kim et al.,
(Radhajeyalakshmi et al., 2011). Also ethyl 1999; Uma Gowrie et al., 2014) which
acetate fractions from fruiting body and CFC account for more than 400 bioactive
filtrate condensate of Pisolithus albus compounds.
exhibited antifungal activities against
Table.1 Screening the antagonistic activity of mushroom fungi against C. capsici by dual culture
technique
Treatment Mushroom Inhibition

Colletotrichum capsici fungal growth zone (mm) %inhibition over
Average radial mycelial (mm) control
growth (mm)
Ganoderma lucidum 40.67ab 44.40c 4.86 54.81ab
(39.58) (41.78) (47.76)
Auricularia 41.67b 45.33c 2.76 53.70b
polytricha (40.16) (42.32) (47.12)
Lentinus edodes 49.00cd 36.00a 4.86 45.55cd
(44.43) (36.87) (42.45)
Pycnoporus 38.33a 51.70d 0 57.41a
sanguineus (38.23) (45.97) (49.26)
Coprinus sinensis 54.00e 36.00a 0 40.00e
(47.29) (36.87) (39.23)
Schizophyllum 47.00c 39.00b 0 47.78c
commune (43.28) (38.65) (43.73)
Volvariella volvaceae 51.37de 35.70a 2.83 42.92de
(45.74) (36.69) (40.93)
Trametes versicolor 50.73de 38.30b 1.10 43.63de
(45.40) (38.23) (41.34)
Control 90.00f - - 0.00f
(71.57) (0.57)
SEd 2.9511 0.5156
CD (p=0.05) 1.4047 1.0930

Values are the mean of three replications. Means followed by a common letter are not significantly different at
5%level by DMRT.Values in parenthesis are arcsine transformed values
1179
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Table.2a Effect of solvent fractions of culture filtrates of selected mushroom fungi on spore germination of C. capsici
Solvent extracted metabolites

Ethyl acetate Chloroform Diethyl ether
Mushroom 6 hours 12 hours 24 hours 6 hours 12 hours 24 hours 6hours 12 hours 24 hours
fungi SG PI SG PI SG PI S PI SG PI SG PI S PI SG PI SG PI
G G
a a a a a a a a a a
Ganoderma - 100 29.75 70.25 22.00 78.0 - 100 15.00 85.0 12.0 88.0 - 100a 26.25a 73.75a 21.25a 79.0a
lucidum (89.19) (33.05) (56.95) (27.97) (62.03) (89.19) (22.79) (67.21) (20.27) (69.73) (89.19) (30.82) (59.18) (27.45) (62.73)
a b b b b a c c c c a b b c
Auricularia - 100 44.25 55.75 33.75 66.25 - 100 47.25 52.75 40.5 59.5 - 100 47.5 52.5 41.00 59.0c
polytricha (89.19) (41.70) (48.30) (35.52) (54.48) (89.19) (43.42) (46.58) (39.52) (50.48) (89.19) (43.57) (46.43) (39.82) (50.18)
a b b b b a b b b b a b b b
Lentinus - 100 43.5 56.5 34.75 65.25 - 100 42.25 57.75 32.75 67.25 - 100 46.75 53.25 35.75 64.25b
edodes (89.19) (41.27) (48.73) (36.12) (53.88) (89.19) (40.54) (49.46) (34.91) (55.09) (89.19) (43.14) (46.86) (36.72) (53.28)
a c c d d a d d e e a c c e
Control - 100 54.0 46.0 83.0 17.0 - 100 54.0 46.0 83.0 17.0 - 100 54.0 46.0 83.0 17.0e
255
(89.19) (47.29) (42.71) (65.65) (24.35) (89.19) (47.29) (42.71) (65.65) (24.35) (89.19) (47.29) (42.71) (65.65) (24.35)
a b b c c a c c d d a b b d
Control - 100 47.0 53.0 77.0 23.0 - 100 47.0 53.0 77.0 23.0 - 100 47.0 53.0 77.0 23.0d
(without (89.19) (43.28) (46.72) (61.34) (28.66) (89.19) (43.28) (46.72) (61.34) (28.66) (89.19) (43.28) (46.72) (61.34) (28.66)
metabolite)
SEd - 1.7369 1.4720 - 6.5256 1.0288 - 1.2179 1.0878
CD (p=0.05) - 3.7021 3.1374 - 13.909 2.1928 - 2.5960 2.3186
1
Where, SG- No. of spores germinated, PI- Percent inhibition of spore germination. Values are the mean of four replications. Means followed by a common letter
are not significantly different at 5%level by DMRT. Values in parenthesis are arcsine transformed values
1180
Table.2b Antimicrobial activity of solvent extracted metabolites of selected mushroom fungi

against C. capsici by agar well diffusion technique
Mushroom fungi Solvents used Average mycelial growth (mm) %inhibition over control
ef
Ethyl acetate 63.67 29.25ef
(52.93) (32.74)
Ganoderma lucidum Chloroform 54.00a 40.00a
(47.29) (39.23)
c
Diethyl ether 59.33 34.07c
(50.38) (35.71)
Ethyl acetate 61.00cde 32.00cde
(51.35) (34.45)
b
Chloroform 57.00 36.00b
Lentinus edodes (49.02) (36.87)
Diethyl ether 62.00def 31.00def
(51.94) (33.83)
cd
Ethyl acetate 60.33 32.96cd
(50.96) (35.04)
Chloroform 70.00g 22.00g
Auricularia polytricha (56.79) (27.97)
Diethyl ether 63.00ef 30.00ef
(52.54) (33.21)
Control 90.00h 0.00h
(71.57) (0.51)
SEd 0.9661
CD (p=0.05) 2.0152
Values are the mean of three replications
Means followed by a common letter are not significantly different at 5%level by DMRT
Values in parenthesis are arcsine transformed values
1181
256
Table.3 Testing the different concentrations of solvent extracted metabolites of G. lucidum

against C. capsici
Treatment Solvent Concentation (ppm) Average mycelial growth(mm) %inhibition over control
Ganoderma lucidum Ethyl acetate 1000 50.62f(45.36) 43.75f (41.41)
e
1500 48.07 (43.89) 46.58e (43.04)
b
2000 37.00 (37.46) 58.88b (50.11)
Chloroform 1000 45.10d (42.19) 49.88d (44.93)
c
1500 40.17 (39.33) 55.36c (48.08)
a
2000 35.52 (36.58) 60.55a (51.09)
f
Diethyl ether 1000 50.15 (45.09) 44.27f (41.71)
d
1500 44.50 (41.84) 50.55d (45.32)
2000 40.07c (39.27) 55.47c (48.14)
g
Control 90.00 (71.57) 0.00g (0.44)
SEd 0.5104
CD (p=0.05) 1.0425
Values are the mean of four replications. Means followed by a common letter are not significantly different at 5%level by
DMRT. Values in parenthesis are arcsine transformed values
Plate.1 Invitro effect of antagonistic activity of mushroom fungi against C. capsici by dual
culture technique
G. lucidum A. polytricha L. edodes T. versicolor P. sanguineus
S. commune C. sinensis V. volvaceae Control

Fungus on left: C.capsici
Fungus on right: Mushroom fungus
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257
Plate.2 Antimicrobial activity of different solvent extracted fractions of Cell free culture filtrates
of G. lucidum against C. capsici
Ethyl acetate
1000 ppm 1500 ppm 2000 ppm
Chloroform
1000 ppm 1500 ppm 2000 ppm
Diethyl ether
1000 ppm 1500 ppm 2000 ppm
Control
1183
258
Since, the chloroform extracted antimicrobial Fungi and Their Antagonists. 8th
metabolites of G. lucidum at 2000 ppm internation conference on mushroom
showed maximum antimicrobial activity as biology and mushroom, 4.
evidenced by the above results, further work Bansal, R.D. and Grover, R.K. 1969. Reaction
proceeded with higher concentrations using of chilli (Capsicum frutescens) varieties
chloroform as solvent that could show to Colletotrichum capsici. J.Res.
maximum mycelial growth inhibition. Ludhiana 6 (2): 345-348.
Basnet, B.B., Li Liua, Li Bao and Hongwei Liu.
Globally research is focused on the 2017. Current and future perspective on
identification of antimicrobial molecules from antimicrobial and anti-parasitic
activities of Ganoderma sp.: an update.
fungi, bacteria and plants to manage plant
Mycology, vol. 8, No. 2, 111–124.
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Dennis, C and Webster, J. 1971. Antagonistic
the environmental hazards and pollution by
properties of species-groups of
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How to cite this article:
Priya, K., G. Thiribhuvanamala, A. Kamalakannan and Krishnamoorthy, A.S. 2019.

Antimicrobial Activity of Biomolecules from Mushroom Fungi against Colletotrichum capsici
(Syd.) Butler and Bisby, the Fruit Rot Pathogen of Chilli. Int.J.Curr.Microbiol.App.Sci. 8(06):
1172-1186. doi: https://doi.org/10.20546/ijcmas.2019.806.145
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International Journal of Current Microbiology and Applied Sciences

ISSN: 2319-7706 Volume 9 Number 7 (2020)
Journal homepage: http://www.ijcmas.com
Original Research Article https://doi.org/10.20546/ijcmas.2020.907.030
Harnessing the Antimicrobial Prospects of Mushroom Fungi against

Colletotrichum gloeosporioides Penz. Causing Post-Harvest Anthracnose
Disease of Mango
M. Gayathri, G. Thiribhuvanamala* and A. S. Krishnamoorthy
Department of Plant Pathology, Tamil Nadu Agricultural University,

Coimbatore-641 003, India
*Corresponding author
ABSTRACT
Mushrooms contain various bio active potential including antifungal, antiviral and antibacterial
property. But very limited work has been done on the productive utilization of the antimicrobial
Keywords properties for plant disease management practices. The current study is formulated to harness the
antimicrobial potential of Ganoderma lucidum, Auricularia polytricha, Lentinus edodes, Coprinus
Mango anthracnose, sinensis, Schizophyllum commune, Pleurotus florida, Trichloma mastukae, Calocybe
Mushroom fungi, indica,Volvariella volvacea, Fomes spp and Pycnoporus sanguineus against Colletotrichum
Inhibition percent, gloeosporioides causing post-harvest anthracnose disease of mango.Results from dual culture test
Cell free culture revealed that the Ganoderma lucidum, Auricularia polytricha, Pycnoporus sanguineus and Pleurotus
filtrate florida showed inhibition of radial mycelial growth with inhibition percentage of 56.3 to 70.77
%.Although the mushrooms screened exhibited varied degree of inhibition of mycelial growth of
Article Info
pathogen, A. polytricha and G.lucidum performed well and the crude cell free culture filtrates from
Accepted: G. lucidum and A. polytricha tested at 20th day showed maximum inhibition of 36.33% and 47.11%
05 June 2020 of mycelial growth of C.gloeosporioides by G. lucidum and A. polytricha respectively.These results
Available Online: predicted that the cell free culture filtrate collected contained some antimicrobial compounds that
10 July 2020 would have been responsible for the antimicrobial effect and offers better scope for development of
mycomolecules based fungicide against plant diseases.
Introduction et al., 2010; Arauz, 2000). Anthracnose

disease causes 30-60% yield losses on mango
Mango is one the well-known ancient fruit in across different countries of the world causing
the world known for its high nutritive value, both qualitative and quantitative losses (Shad
superb flavour, delicious taste.Though this et al., 2002; Akem, 2006; Chowdhury and
fruit crop is affected by fungi, bacteria and Rahim, 2009, Lakshmi et al., 2011). Under
phytoplasma, Mango anthracnose caused by the current scenario, continuous and judicious
Colletotrichum gloeosporioides Penz being application of fungicide against the pathogen
the most important disease leads to severe has resulted in loss of effectiveness of
yield loss due to the pre harvest and post fungicide due to buildup of fungicide resistant
harvest infection even up to 100 per cent ( Jha pathogen apart from creating environmental
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262
pollution and health problem.As an commune, Fomes spp, Trichloma mastukae,

alternative, scientists have started probing for Calocybe indica,Volvariella volvaceae,
antimicrobial bio active compounds from Pleurotus florida and Pycnoporus sanguineus
natural source to overcome the current were obtained from the Department of Plant
situation. In that context, mushrooms are in Pathology, Tamil Nadu Agricultural
the lime light and evoked interest globally for University,Coimbatore.
their bioactive compounds that finds
application in pharmaceutical and therapeutic Screening of antagonistic potential of
values apart from their nutritive value mushroom fungi under in vitro
(Poucheret et al., 2006). Mushroom contain
various natural compounds with bio active Mycelial cultures of mushrooms viz.,
potentials as antifungal, antibacterial, Volvariella volvacea,Pleurotus florida,
antiviral, antitumor, antinemic, anti- Ganoderma lucidum, Auricularia polytricha,
inflammatory, anti-allergic, anti-antherogenic, Lentinus edodes, Coprinus sinensis,
antidiabetic properties( Hatvani,2001;Wasser Schizophyllum commune, Calocybe indica,
2002; Lindequist et al., 2005; Quang et al., Trichloma mastukae, Fomes spp and
2006). Research is focused towards Pycnoporus sanguineus were screened for
exploitation of antimicrobial compounds from their antagonistic potential against
mushrooms against plant pathogens as is Colletotrichum gloeosporioides causing post
evidenced by the effect of culture filtrates of harvest anthracnose disease of mango by dual
Lentinula edodes and Clitocybe nuda against culture technique (Dennis and Webster,
Colletotrichum higginsianum (Chen and 1971).
Huang, 2010), Ganoderma lucidum against
Colletotrichum capsici (Priya and The PDA medium was prepared and poured
Thiribhuvanamala, 2019) and Coprinus into Petri dish.A 9 mm mycelial disc of the
comatus against Fusarium oxysporum f.sp. mushroom fungi were placed at one side of
lycopersici and F.oxysporum f.sp.cubense Petri dish and similarly test pathogen was
(Jeeva and Krishnamoorthy, 2018). Perusal of placed at opposite side of the same Petri dish.
literature shows no reports on the The pathogen were inoculated separately
antimicrobials from mushrooms against post- which served as control.Three replications
harvest pathogens. Currently there is were maintained for each treatment.The Petri
increasing attention to derive safe dishes were incubated at 28 ± 2°C. Periodical
antimicrobials for post-harvest treatments to observations on the mycelial growth, pattern
mitigate the residual issues in fruits and and their antagonistic behaviour were done.
vegetables. In that context, the present study The percentage inhibition of mycelial growth
was attempted to test different mushrooms in pathogen over control was calculated by
against mango post-harvest anthracnose using the formula (Vincent, 1947).
pathogen Colletotrichum gloeosporioides.
Materials and Methods
The mango anthracnose pathogen

Colletotrichum gloeosporioides and the pure Where, C is the radial mycelial growth of the
cultures of mushrooms viz Ganoderma pathogen (mm) in control
lucidum, Auricularia polytricha, Lentinus T is the radial mycelial growth of the
edodes, Coprinus sinensis, Schizophyllum pathogen (mm) in dual culture plate.
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263
Testing the efficacy of crude culture distance leaving 1cm space from edge of the
filtrates of mushroom fungi against plate using sterilized corkborer. A 100 μl of
colletotrichum gloeosporioides crude culture filtrate of G.lucidum and
A.polytricha (separately) was pipetted and
Extraction of crude culture filtrates from poured into the well. Using sterilized cork
mycelium of mushroom fungi borer, a 9 mm mycelial disc of C.
gloeosporioides from 10 days old culture was
Based on results obtained from the placed at the centre of the plates and
preliminary antagonistic screening of incubated at room temperature (28 ± 2°C).
mushroom fungi against C.gloeopsorioides, Three replications maintained for each
the mushrooms Auricularia polytricha and treatment. Sterile water served as control
Ganoderma lucidum were shown to be instead of using culture filtrate. The
effective against Colletotrichum percentage inhibition was calculated by using
gloeosporioides as shown by the inhibition of the formula (Vincent 1947).
mycelial growth of pathogen. The mycelial
disc (9mm) from 10 days old culture of Results and Discussion
Auricularia polytricha and Ganoderma
lucidum were placed into 250 ml conical flask The results obtained from this study indicated
containing sterilized Potato Dextrose (PD) that mushrooms screened showed various
broth. The flasks were kept in incubator cum degrees of antagonistic activity against
shaker at 25°C with agitation at 150 rpm. C.gloeopsorioides and it proved the earlier
After incubation, the mycelial mat was success of derivation of Azoxystrobin from
separated from broth using Whatman filter Strobilurus tenacellus against downy mildew
paper No 1. Then the culture filtrate was and powdery mildew diseases of grapes. In
centrifuged at 10,000 rpm for 10 mins. The our study, among the mushrooms screened,
supernatant was then filtered through the the mycelium of Auricularia polytricha,
membrane filter (0.2 μm) to avoid bacterial Ganoderma lucidum, Pycnoporus sanguineus
contamination. This extract is used as cell free and Pleurotus florida inhibited the mycelial
crude culture filtrates. The crude culture growth of Colletotrichum gloeosporioides
filtrate were collected from Auricularia (26.3mm,36.6mm,39.6mm and 39.3mm
polytricha and Ganoderma lucidum at various respectively) as observed by inhibition per
periodical intervals viz., 10th,15th,20th and 25th cent of 70.77, 59.33, 56.00 and 56.33
days of inoculation and tested for mycelial respectively. Other mushrooms, Fomes spp,
inhibition studies. Volvariella volvacea, Schizophyllum
commune, Coprinus sinensis, Calocybe indica
Mycelial inhibition test and Tricholoma matsukae also showed
mycelial growth inhibition of 50.44%,
The crude culture filtrate of G. lucidum and A. 41.11%, 51.88%, 50.00%, 22.11%, 22.66%
polytricha extracted at various periodical respectively against the test pathogen
intervals (10th,15th,20th,25 days) were tested Colletotrichum gloeosporioides (Fig.1).
against the mycelial growth of
C.gloeosporioides by agar well diffusion As in our study, Priya et al., (2018) reported
technique (Stokes and Ridgway,1980).The that the antagonistic activity of Ganoderma
PDA medium was poured into the sterilized lucidum followed by Auricularia polytricha
plates and allowed to solidify. After against Colletotrichum capsici. Similarly,
solidification, four wells were made at equal Badalyan et al., (2014) reported that the
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mushrooms Ganoderma lucidum, Pleurotus competitive interactions viz., over growth of

ostreatus,Hypholoma fasiculare, Lentinus pathogen, inhibition at mycelial contact,
tigrinus exhibited various antimicrobial inhibition of mycelia at distance and partial or
activity against Cochliobolus sativus, complete replacement of plant pathogenic
Fusarium culmorum, Gaeumannomyces fungi were reported by Badalyan,2002; Jeeva
graminis and Rhizoctonia cerealis. The and Krishnamoorthy, 2018; Priya et.al.,
interactions between the pathogen 2019). This clearly shows that the interactions
Colletotrichum gloeosporioides and the could be due to certain metabolites that would
mushroom were documented from the dual be responsible for the retardation of C.
culture assay. Plates are given in Table-1. gloeosporioides. These results paved way to
Antagonistic interactions between different exploit antimicrobial metabolites from
mushroom fungi and plant pathogenic fungi potential mushrooms G.lucidum and
in dual cultures and various types of A.polytricha against C. gloeosporioides.
Table.1 Interaction between mushroom fungi and C.gloeosporioides
Mushroom fungi with Nature of interaction

C.gloeosporioides
Ganoderma lucidum Pathogen growth was retarded and pushed back.
Auricularia polytricha Formation of light yellowish green pigment at the interaction
zone
Lentinus edodes Clear zone of inhibition were formed ;both the mushroom and
pathogen did not grow each other
Fomes spp Thick mat of hyphae were formed at zonal point and later
pathogen gets hyper parasitized by the mushroom fungi
Volvariella volvacea The mushroom fungi hyperparasitised over the pathogen but no
inhibition zone
Pycnoporus sanguineus No inhibition zone.A very thin zone were formed at contact
point
Pleurotus florida No inhibition zone ;no hyperparasitization;both the mushroom
and the pathogen growth ceases at the point of interaction
Schizophyllum commune Thick mat of mycelial hyphae were formed at the zonal point
and further growth of pathogen gets retarded
Coprinus sinensis No inhibition zone.Both fungi did not grow each other
Calocybe indica Linear mycelial growth of the pathogen near the mushroom
fungi
Tricholoma matsukae Pathogen grow over the mushroom fungi
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Table.2 Testing the efficacy of crude culture filtrate of

G.lucidum and A.polytricha against C.gloeosporioides
Days G.lucidum A.polytricha

interval
Mean mycelial of the % inhibition Mean mycelial of the % inhibition over
pathogen Growth over control pathogen Growth control
10th day b
90.00 (71.61) 0.00 c
90.00 (71.61) 0.00
a ab b b
15th day 58.66 (49.96) 34.88 (36.18) 52.33 (46.31) 41.88 (40.31)
20th day a
57.33 (49.19)
a
36.33 (37.05)
a
47.60 (43.60)
a
47.11 (43.32)
25th day a
59.33 (50.35)
b
34.11 (35.71)
b
51.33 (4.74)
b
43.00 (40.95)
d
Control 90.00 (71.61) 00 90.00 (71.61) 0.00
SEd 1.265 - 1.235 -
CD 2.854 - 2.787 -
(p=0.05)
Values are the mean of three replications. Means followed by a common letter are not significantly different at
5%level by DMRT. Values in parenthesis are arcsine transformed values.
1. Ganoderma lucidum 7. Pleurotus florida

2. Auricularia polytricha 8. Schizophyllum commune
3. Lentinus edodes 9. Coprinus sinensis
4. Fomes spp 10. Calocybe indica
5. Volvariella volvacea 11. Tricholoma mastukae
6. Pycnoporus sanguineus 12. Control
Fig.1 Screening the antagonisitic activity of mushroom fungi against C.gloeosporioides
by dual culture assay
285
266
Ganoderma lucidum Auricularia polytricha Lentinus edodes
Fomes spp Volvariella volvacea Pycnoporus sanguineus
Pleurotus florida Schizophyllum commune Coprinus sinensis
Calocybe indica Tricholoma mastukae Control
Plate.1 In vitro effect of antagonistic activity of mushroom fungi against

Colletotrichum gloesporioides by dual culture
286
267
10th day 15th day 20th day
25th day Control
Plate.2a Antimicrobial activity crude culture filtrate of Ganoderma lucidum and against
Colletotrichum gloeosporioides
10th day 15th day 20th day
25th day Control
Plate.2b Antimicrobial activity crude culture filtrate of Auricularia polytricha against

Colletotrichum gloeosporioides
287
268
Testing the efficacy of crude culture filtrate fungicide against broad spectrum activity.
from potential mushroom fungi The study paves way and kindles interest for
Ganoderma lucidum and Auricularia identification of the antimicrobial compound
polytricha against C.gloeosporioides in A. polytricha and G. lucidum responsible
for inhibition of pathogen growth.
Based on the maximun inhibition per cent
and interaction studies the culture filtrates of Acknowledgement
G.lucidum and A. polytricha collected at
various periodical interval 10th, 15th, 20th and The author thanks the University for
25th days were tested to exploit the providing facilities through the scheme UGC-
antimicrobial metabolites against SAP DRS1 on Mushroom biotechnology to
C.gloeosporioides, Results indicated that the carry out the research work.
crude culture filtrate collected on 10th day did
not exhibit any antifungal effect against any References
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collected from 20th day showed maximum Akem, C.N., 2006. Mango anthracnose
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2 a and b). Arauz, L.F. 2000.Mango anthracnose:
economic impact and current options
From the findings, it is concluded that for integrated management. Plant
antagonistic effect of mushroom fungi by dual Disease, 84, p. 600-611
culture assay may offer an indication for the Badalyan, S. M., Gharibyan, N. G., Innocenti,
presence of antimicrobial metabolites and G. 2014. Antifungal/Antagonistic
such tests will be useful to screen large Activity of Different Ganoderma
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mushroom G. lucidum and A.polytricha Fungi and Their Antagonists. 8th
secreted maximal production of antifungal international conference on mushroom
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mitigate the pesticide residues in food (3):220-225.
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Hatvani, N. 2001. Antibacterial effect of the Kamalakannan and Krishnamoorthy,
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How to cite this article:
Gayathri. M., G. Thiribhuvanamala and A. S. Krishnamoorthy. 2020. Harnessing the

Antimicrobial Prospects of Mushroom and Fungi against Colletotrichum gloeosporioides Penz.
Causing Post-Harvest Anthracnose Disease of Mango. Int.J.Curr.Microbiol.App.Sci. 9(07):
281-289. doi: https://doi.org/10.20546/ijcmas.2020.907.030
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UGC- SAP DRS-1 SCHEME

COMPLETION REPORT (2016-2020)

DEPARTMENT OF PLANT PATHOLOGY

Authors: A. S. Krishnamoorthy, S. Nakkeeran, G. Thiribhuvanamala,

Component: UGC-SAP-DRS1 Scheme report

Citation: Krishnamoorthy, A. S., S. Nakkeeran, G. Thiribhuvanamala, M. Karthikeyan,

First Edition: December 2020

Cover page: Sree Kumaran Computers, TNAU Campus, Coimbatore -3

Photographs Courtesy: Mushroom Research and Training Center, Department of

TNAU offset Press

Period: 2016 to 2020

A UGC - SAP scheme on “Enterprising Mushroom Biotechnology for Food, Feed

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11. Dr. M. Karthikeyan

9. Dr. Samir Ranjan Sikdhar

10. Dr. D. Anusuya,

11. Dr. G. Thiribhuvanamala

12. Dr. M. Karthikeyan

13. Dr. P. Latha

The following members attended the meeting:

1. Dr. K. Ramaraju, Director (CPPS), TNAU, Coimbatore and Advisory Committee

Dr. K. Ramaraju, Director i/c, (CPPS), TNAU, Coimbatore welcomed the

1. Establishment of mushroom knowledge park (Dr.K. Ramasamy, Chairman-UGC-

The Annual Advisory Committee meeting of UGC-SAP-DRS-1 scheme on

1. Dr. A. S. Krishnamoorthy, Registrar & Coordinator (UGC-SAP).

1. Empowerment of woman self help groups on spawn and mushroom production

PROGRESSREPORTANNUAL / FINAL REVIEW

Date of first approval with level at inception : January 2016

Date of first sanction (Current phase) : 01.01.2016/-

Totalgrantsreceivedsinceinception : Rs. 43,01,107/-

Ongoing Modified to, if any,

I. Dr. Samir Ranjan Sikdhar, Division of Plant Pathology, Bose Institute,

II. Prof. Anusuya, Department of Botany, Bangalore University, Jnan bharathi

x Certificate course on Commercial Mushroom Production

x Advances in Mycology – including the mushroom biotechnology

Purchase of equipments in UGC-SAP-DRS-1 Scheme

S. Date of Name of the instruments Amount (Rs.)

1. 31.03.2017 Refrigerated shaker 498067.00

2. 31.03.2017 Rotary vaccum flask evaporator 498960.00

3. 31.03.2017 Refrigerated Centrifuge 470000.00

4. 27.03.2018 Deep freezer 1810400 YEN(Rs.11,18,131)

5. 31.03.2017 Thermostat controlled 299376.00

Refrigerated Shaker Rotary Vaccum Refrigerated

Deep freezer Thermostat controlled autoclave

a. Research (high light major objectives set-forth (as proposed) and

Year 1. Morphological and molecular characterization of commercially important

(i) Biodiversity, conservation and identification of milky mushroom

Molecular characterization of wild mushrooms understanding the genetic

CBE- TNAU -1603 CBE- TNAU -1604 CBE- TNAU -1701

Plate.3.PCR amplification of IGS region of wild milky mushroom

Specimen no. CBE- TNAU- 1523

Habitat attached with the finer roots of Cocos

a. base not bulged

Name (s) of the Collector Mrs. Lakshmi

Plate.7. Wild mushroom CBE-TNAU-1523

Table.3. Performance of CBE-TNAU-1523 in comparison with Calocybe indica

Milky mushroom Yield/500g Bioefficiency

Calocybe indica var. a a a a

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