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BBIIOOLLOOGGYY AANNDD MMOOLLEECCUULLAARR CCHHAARRAACCTTEERRIIZZAATTIIOONN OOFF VVOOLLVVAARRIIEELLLLAA SSPPPP..
By
IMRAN UL HAQ
M.Sc. (Hons.) Agriculture
A thesis submitted in partial fulfillment of the requirement for the
Degree of
DOCTOR OF PHILOSOPHY
In
Plant Pathology
Department of plant pathology FACULTY OF AGRICULTURE UNIVERSITY OF AGRICULTURE
FAISALABAD 2009
To
The Controller of Examinations,
University of Agriculture,
Faisalabad.
“We, the Supervisory Committee, certify that the contents and form of
thesis submitted by Imran ul Haq, 96-ag-1614, have been found
satisfactory and recommend that it be processed for evaluation, by the
External Examiner(s) for the award of degree.”
Supervisory Committee
1. Chairman ---------------------------------------
(Prof. Dr. M. Aslam Khan)
2. Member -------------------------------------------
(Dr. M Inam Ul Haq)
3. Member -------------------------------------------
(Dr. B. Fatima Usman)
II DDEEDDIICCAATTEE TTHHIISS HHUUMMBBLLEE EEFFFFOORRTT
TTOO
My beloved
PPaarreennttss
&&
AAffffeeccttiioonnaattee TTeeaacchheerrss
CONTENTS
CHAPTER
NO. TITLE PAGE
1 INTRODUCTION 1
2
REVIEW OF LITERATURE
2.1. METHODS OF CULTIVATION
2.1.1. FUNGUS CULTURING
2.1.2. CULTIVATION FORMS
2.2. S OLID STATE FERMENTATION
2.3. PHYSIOLOGY
2.3.1. EFFECT OF TEMPERATURE
2.3.2. EFFECT OF LIGHT
2.4. LIQUID FERMENTATION
2.4.1. BACKGROUND
2.4.2. LIQUID MEDIA USED FOR FERMENTATION
2.5. BIOCHEMICAL ANALYSIS
2.5.1. NUTRIENTS CONTENTS
2.5.2. MEDICAL EFFECTS
2.5.3. BIOCONVERSION OF LIGNOCELLULOSIC WASTES
2.5.4. UPTAKE OF HEAVY METALS
2.6. GENETICS
6
6
6
7
10
16
16
18
21
21
24
25
26
33
36
38
38
3
MATERIALS AND METHODS
3.1. STRAIN COLLECTION
3.2. CULTURE MULTIPLICATION
3.3. SELECTION OF CULTURE MEDIUM
3.4. SPAWN PREPARATION
3.5. PREPARATION OF SUBSTRATE
3.6. SPAWNING
3.7. REGULATION OF TEMPERATURE,
HUMIDITY AND LIGHT
41
41
41
42
43
43
44
44
45
3.8. WATERING
3.9. EVALUATION OF PRODUCTIVITY OF FIVE
STRAINS OF CHINESE MUSHROOM
3.10.COMPARISON OF THE MUSHROOM
GROWTH ON DIFFERENT SUBSTRATES
3.11. DATA RECORDED
i) DAYS FOR COMPLETION OF SPAWN RUNNING
ii) DAYS FOR THE PINHEAD FORMATION.
III) FRUITING BODY FORMATION
3.12. YIELD DATA
3.13. pH OF THE SUBSTRATE
3.14. BIOCHEMICAL ANALYSIS OF DIFFERENT
SUBSTRATES USED FOR THE CULTIVATION
OF VOLVARIELLA SPP. AND FRUITING
BODIES OF MUSHROOM.
3.14.1. DETERMINATION OF MOISTURE
3.14.2. ESTIMATION OF CRUDE FAT (ETHER
EXTRACT)
3.14.3. ESTIMATION OF CRUDE PROTEIN
3.14.4. DETERMINATION OF CRUDE FIBER
3.14.5. ESTIMATION OF ASH
3.15. LIQUID STATE FERMENTATION
3.15.1. PREPARATION OF THE GROWT MEDIUM
3.15.2. EFFECT OF pH ON THE MYCELIAL
GROWTH OF V. VOLVACEA
3.15.3. EFFECT OF TEMPERATURE ON THE
MYCELIAL GROWTH OF V. VOLVACEA.
3.16. MOLECULAR CHARACTERIZATION OF
VOVARIELLA VOLVACEA
3.16.1. DNA EXTRACTION
3.16.2. DNA QUANTIFICATION
3.16.3. RAPD ANALYSIS
3.16.5. CONDITION OPTIMIZATION FOR RAPD ANALYSES
3.16.6. RAPD (PCR) PROFILE
3.16.7. AGAROSE GEL ELECTROPHORESIS
45
45
46
46
46
46
46
47
47
47
48
48
49
50
50
51
51
52
52
52
53
54
56
56
56
56
58
59
3.16.8. AGAROSE GEL ELECTROPHORESIS
3.16.9. SCORING OF THE RAPD DATA
3.17. STATISTICAL ANALYSIS
4
RESULTS
4.1. SOLID STATE FERMENTATION
4.1.1. SPAWN RUNNING
4.1.2. PINHEAD FORMATION
4.1.3. FRUITING BODY FORMATION
4.1.4. YIELD
4.2 pH OF THE SUBSTRATES AT
DIFFERENT GROWT STAGES
4.3. BIOCHEMICAL ANALYSIS OF THE SUBSTRATES
4.4. BIOCHEMICAL ANALYSIS OF THE
MUSHROOM FRUITING BODIES
4.5. LIQUID FERMENTAION
4.5.1. EFFECT OF INCUBATION TIME ON
MYCELLIAL BIOMASS PRODUCTION
4.5.2. EFFECT OF TEMPERATURE ON MYCELLIAL
BIOMASS PRODUCTION
4.5.3. EFFECT OF pH ON THE MYCELLIAL
BIOMASS PRODUCTION
4.6. MOLECULAR CHARACTERIZATION
60
60
61
66
69
71
74
76
83
85
86
87
88
90
5 DISCUSSION 92
SUMMARY 104
RECOMMENDATIONS 107
LITERATURE CITED 108
LIST OF TABLES & FIGURES
Table Title Page
3.16.3. PCR Amplification 54
3.16.4. Random sequences used for amplification
55
3.16.6. RAPD (PCR) Profile 56
1 Analysis of variance table of growth of different strains of
Volvriella volvacea on different substrates (mean squares).
61
2 Effect of substrates on 50% spawn running of the strains. 37
3 Comparison of 50% spawn running of strains between two crops. 63
4 Effect of substrates on 100% spawn running of the strains 65
.5 Comparison of spawn running of strains between two crops 66
6 Effect of substrates on the pinhead formation of strains of V.volvacea 68
7 Comparison between the two crops for pinhead formation 69
8 Effect of substrates on fruiting body formation 70
9 Comparison for the fruiting body formation between two crops. 71
10 Effect of substrates on the yield of the strain. 72
11 : Comparison of the yield of the strains between two crops 73
12 Analysis of variance for pH of substrates at different growth stages. 74
13 Effect of strains on the pH at different growth stages on cotton waste 75
14 pH of the substrates at different growth stages of the strain V. v pk. 76
15 Analysis of variance table for ash contents 77
16 Ash contents in the substrates at different growth stages of the
Mushroom.
78
17 Analysis of variance table for crude fiber percentage. 79
18 Crude fiber percentage in the substrates at different growth stages of
the mushroom.
80
19 Analysis of variance table for crude protein percentage. 80
20 Crude protein percentage contents in the substrates at different growth
stages of the mushroom
81
21 Analysis of variance table for nitrogen percentage 82
22 Nitrogen percentage in the substrates at different growth stages of the mushroom
82
23 Analysis of variance table (mean squares). 83
24 Nitrogen, Crude protein, Ash, Moisture and Crude fiber percentage in
the fruiting bodies of mushroom harvested from all substrates.
84
25 Analysis of variance for protein production in Liquid medium at
different temperatures
85
26 Effect of time on mycelial production in three liquid media 86
27 Effect of temperature on mycelial production in three liquid media 87
28 Analysis of variance for protein production in liquid medium at different pH levels.
88
29 Effect of pH on mycelial production in three liquid media 89
30 Similarity matrix table Nie and Li‘s co-efficient of five fungal strains. 90
Gel elctrophorasis pictures 1&2 91
AAcckknnoowwlleeddggeemmeennttss
All oceans turn into ink and all the woods become pens, even then, the
praise of ALLAH ALMIGHTY can not expressed. He, who created the universe
and bestowed the mankind with knowledge and wisdom to search for the secrets, I
set my unfeigned and humble thanks giving before Him-Who favored and
reinforced me with the fortitude and capability to aptly complete my research
work. Trembling lips and wet eyes praise for one whom Allah as sent as a mercy
to the world HAZRAT MUHAMMAD (SAW), the illuminating torch and
rescuer of humanity from going astray. Our PROPHET (SAW), the city of
knowledge, who pursues his “Ummah” to seek knowledge for cradle to grave.
With profound gratitude and deep sense of devotion, I wish to thank my
sincere and honorable supervisor Prof. Dr. M. Aslam Khan, Professor,
Department of Plant Pathology, University of Agriculture, Faisalabad, for dynamic
supervision, constructive guidance and affectionate behavior throughout my
studies. It was his confidence in my capabilities and his appreciation of my work
that encouraged me to keep on fighting against all odds till the success was
ensured.
I am really indebted to Prof. Dr Sultan Mehmood Khan professor (Rtd)
Department of Plant Pathology, University of Agriculture, Faisalabad Prof. Dr.
Muhammad Mumtaz Khan, Professor, Institute of Horticultural Sciences,
University of Agriculture, Faisalabad and Dr. S. M. Mughal, National Professor,
Department of Plant Pathology, PMAS University of Arid Agriculture,
Rawalpindi, Dr. Abul Naveed, Assistant Professor (Assistant Director Research),
University of Agriculture, Faisalabad for their full cooperation and
encouragement, during study period. Heartiest thanks and indebtedness of mine
are due, to Dr. M Inam Ul Haq Associate Professor Department of Plant
Pathology, PMAS University of Arid Agriculture, and Dr. B. Fatima Usman,
Assistant Professor, Institute of Horticultural Sciences, University of Agriculture,
Faisalabad, for their positive criticism, co-operative behaviour, inspiring guidance
and valuable suggestions for improvement for this manuscript.
I would like to extend my sincere thanks to Dr. Nazir Javed, Chairman,
Department of Plant Pathology, University of Agriculture, Faisalabad. I wish to
thank my all seniors in the department of Plant Pathology, also heartful thanks are
due, to all my friends especially, Dr Yasir iftikar, Shahid Warsi Dr Abdul
Rehman, Dr Wasim Babar, Dr Khizar Abbas,, Imran Chisti, Dr Naveed, Qari
Shahid Azhar Mustaf, Farhan Azhar, Makshoof Ahmad, Zulfi, Tassadaq Shehbaz
and all those who are not in my mind right now. They shared their toughest hours
in conducting this piece of work and their excellent cooperation.
Last but no means least, I am highly indebted to my parents, sisters,
brothers and wife for their encouragement, raising their hands for pray, moral and
financial support. Their concern in me can never be returned but will always be
remembered.
Imran Ul Haq
1
Chapter 1 INTRODUCTION
Although fungi cause enormous losses to man, animals and crops, yet,
some fungi are beneficial. The fleshy fungi of class Ascomycetes and
Basidiomycetes are generally termed as mushroom. There are about 69 thousand
known mushroom species of which 2000 species from more than 30 genera are
regarded as prime edible mushrooms but 80 of them are grown experimentally and
around 20 are cultivated commercially (Chang, 1991). Mushrooms have been used
as food and medicine by the ancient Egyptian, Greek, Roman and Chinese
civilizations. These fungi had attained the status of a regular crop in France and
China by 17th and 19th centuries, respectively, spreading gradually to the other
countries in few years.
Today, mushrooms are liked all over the world due to their delicate taste,
flavor and health giving properties. Mushrooms contain good quality protein,
unsaturated fatty acids, minerals and vitamins (Wahid et al., 1988). These are low in
fat, carbohydrates, salts and rich source of dietary fiber (Genders, 1990). Moreover,
nucleic acid content in mushrooms is very low and hence, these are considered an
ideal food for patients suffering from hypertension, diabetes and obesity
(Anonymous, 2003 b). In addition to nutritional value, edible mushrooms possess
2
unique characteristics in terms of color, taste, aroma and texture, which make them
attractive for human consumption (Chang and Miles, 1991).
The world production of cultivated edible mushrooms was 3,772 thousand
tons during 1989-90; became higher than 72.5% in the year 1986, corresponding to
an annual increase of 24.5% in this period (Chang and Miles, 1991). The total world
mushroom crop value for the financial year 1989-90 was estimated to be US$ 7.5
billion (Chang, 1991). The world market for mushroom is estimated at 3.7 million
tons per annum against an output of 2 million tons (Anonymous, 2003). Pakistan
exported 97.0 thousand kg mushrooms during the year 1999-2000 having value of
US $ 0.69 million (GOP, 2000).
Straw mushroom (Volvariella volvacea), also known as paddy straw or
Chinese mushroom, also called Tributary mushroom or “Nanhua mushroom” and
can be consumed fresh as well as dry. Only three species of the straw mushroom,
Volvariella volvacea, Volvariella esculanta and Volvariella diplasia are under
artificial cultivation. This is not surprising as rice straw has been used for the indoor
cultivation of V. volvacea since the beginning of the 19th century, a practice from
which the mushroom has been given the common name straw mushroom, and has
been cultivated under natural conditions in many countries.
Pakistan is an agricultural country and its economy is mainly dependent on
agriculture. In the country mushroom cultivation has not been given due
importance because of many reasons, whereas the nature has gifted favorable
environmental conditions with a huge quantity of waste material required for
3
obtaining beneficial food and efficient medicine through artificial cultivation of
straw mushroom. No doubt the most easy and economical mushroom cultivation
technology is also developed by the scientists, but still the nation is consuming or
depending only upon the mushroom grown naturally.
Edible fungi are nature’s recycler, which can convert lignocellulosic wastes
in to protein rich food. According to the Food and Agriculture Organization,
(1992) about 20 million tons of usable agricultural wastes are discarded each year.
In Pakistan there are certain cheap and easily available agricultural wastes like rice
straw, wheat straw, cotton waste etc. which can be utilized for the production of
Volvariella. The most areas of the country are tropical and sub tropical and
therefore more suitable for the cultivation of Chinese mushroom. In Pakistan the
awareness of mushroom has increased to great extent for the last few years.
Among the various mushrooms cultivated in the country, Volvariella volvacea
holds a good promise and its cultivation has been introduced as a cottage industry.
This mushroom can be successfully cultivated on several crop residues like paddy
straw cotton waste wheat straw and sugarcane industrial waste (Khan et al., 1982).
Although mechanized cultivation is providing adequate amount of food, yet
the actual problem is to provide sufficient amount of protein for constantly
increasing world population. So the production of mushroom is going to narrow
down this protein gap.
In order to make the mushroom cultivation an environmentally friendly
industry the basic biology of mushrooms and the cultivation technology must be
4
researched and developed. So the present studies would help in selection of
genetically high yielding strains of Volvariella volvacea and improvement in
cultivation methods.
During this research different spp of Volvariella were collected from
different areas of the country and Volvariella volvacea was identified on
morphological basis. Exotic cultures were collected from different countries and
these were then cultivated on six different agricultural wastes, to evaluate the
productivity of all strains and substrates used for their cultivation. Biochemical
analysis of the substrates for the cultivation of the Chinese mushroom at different
growth stages and the biochemical analysis of the fruiting bodies of the mushroom
harvested from all the substrates was carried out. The effect of pH and temperature
variation on the mycelial production of mushroom was also studied during the
liquid fermentation of the mushroom.
Molecular characterization of five fungal strains was carried out by PCR
technique, to study the genetic variability among the strains used for the
cultivation of mushroom. Molecular markers reveal information concerning the
genetic structures of genotypes. DNA finger printing has evolved as a major tool
in fungal characterization. The development of Random Amplified Polymorphic
DNA (RAPD) Welsh and Clelland, (1990) has allowed the rapid generation of
reliable reproducible DNA fragments or finger prints in a wide variety of species
including several fungi.
5
The objectives of the study were to identify the genetically high yielding
strains of Volvariella volvacea and suitable substrates for its cultivation.
These objectives were accomplished by adopting the following line of work.
LINE OF WORK
1) Collection of wild and exotic cultures of Volvariella volvacea.
2) Study of spore germination in different isolates of Volvariella volvacea.
3) Physiological studies of different strains of Volvariella volvacea.
4) Production of filamentous protein in liquid medium from different crop
residues.
5) Production of mushroom crop on different farm wastes.
6) Chemical analysis of substrates, filamentous protein and fruiting bodies of
mushroom.
7) Molecular characterization of different stains of Volvariella volvacea.
6
Chapter 2 REVIEW OF LITERATURE
The Chinese mushroom has very old history. This mushroom has been used
as food for human beings since Cho dynasty about 3000 years ago in China.. It
was introduced in southeast Asian countries by overseas Chinese, (Baker, 1934
and Benemertio, 1936), since then, its cultivation has been conducted in various
countries outside the China, like Philippines, (Clora, 1937; Go, 1959), Malaysia
(Baker 1934; Sands 1935), Burma (Seth, 1944) and Thailand (Jalavicharama,
1950;Hashioka, 1962). The history of the Chinese mushroom cultivation is very
old. As far as its artificial cultivation is concerned it is believed that, it was begun
in Nanhua temple of Chaohsi, Kwantung province in southern China, almost 200
years ago, (Chang, 1977)
2.1. METHODS OF CULTIVATION
2.1.1. Fungus culturing
Munjal, (1973) reported that the productivity of spawn culture was related
to the formation of chlamydospores. He described that culture beds with dens
chlamydospores always produced a high yield of mushroom. Hence paddy straw
pieces mixed with 4% chalk powder were best suited for spawn production of
Chinese mushroom.
Delmas and Sun, (1984) reported the traditional method of Chinese
mushroom culture in humid tropical climate. For modern culture, a high cellulose
7
substrate having C/N ratio was required. It was sterilized after fermentation at
60°C for 24 hours. After spawning the temperature was required to be maintained
at 35°C-38°C for about 4 days, then reduced 30°C to encourage flushing and
raised again after flush but casing is not necessary.
Qumio, (1988) made attempts to recycle the spent rice straw residues both
from Volvariella spp. and Pleurotus spp. either producing another crop of the
same mushroom or producing another mushroom from same substrate, thus fully
utilizing the straw for production of edible mushrooms before using the spent
compost for feed.
Luh, (1996) described the cultural methods developed in Taiwan. He
reported that rice straw covered by compost was used as the medium for beds and
production started 10-12 days after inoculation.
2.1.2. Cultivation forms
Ho, (1972) suggested an economical plastic house with a bamboo frame
structure that for indoor cultivation of Chinese mushroom consisting of polythene
film of 0.1mm was lined inside this mushroom house and has a layer of sugarcane
leaves on top, to block solar radiation and minimize heat penetration during hot
summer .An electric blower with a one forth horse power and a polythene air duct
was used to provide ventilation.
8
Grahum and Yaung, (1974) observed that beds of 32 cm depth gave the
highest yield of 2.25Kg /100Kg of waste and maintained at a temperature of 35°C-
38°C slightly below the optimum from mycelial growth of Volvariella volvacaea,
throughout the cropping season .
Purkayastha et al., (1981) reported that orientation of paddy straw beds has
a profound effect on the fruiting bodies production of Volvariella volvacea helix
and tyre types of beds ere found to be more productive than the conventional criss-
cross type. Several wastes for Chinese mushroom were used as substrates. Among
them cotton and jute wastes were most and least productive, respectively,
supplementation gram flour up to 600g / bed augmented the production. Climatic
conditions during June and July in west Bengal appeared to be the most favorable
for the production of Volvariella volvacea.
Qumio, (1986) reported that water hyacinth could be used for growing not
only Volvariella mushroom but also Pleurouts and Auricularia. The mushroom
grows fast and yield more if grown in this substrate than on rice straw. It could also
be used as substrate for preparation of the spawn either alone or in combination with
other substrates such as rice straw and saw dust. All parts of the plant including roots
could be used in making beds for Volvariella mushrooms or in the preparation of
mushroom bags for Pleurotus and Auricularia.
Li, (1989) studied the cultivation of Volvariella volvacea on wheat straw in
fields after the wheat harvest. High yield of Volvariella volvacea and 20% increase
in yield of the subsequent wheat crop was achieved. He also described the techniques
9
concerning the treatment of wheat straw compost, cultivation management and pest
control.
Wang, (1990) observed the key points in the management of Volvariella
volvacea grown on rice straw in open fields in relation to the stages of spawn
production, mycelial growth and fruiting bodies formation.
Pan and Li, (1990) studied the construction and design of semi-underground
sheds. Cultivation techniques including the selection of high quality spores and
substrates, bed construction and the control of growth conditions were discussed.
Hua, (1990) grew Volvariella volvacaea strain V2 on a rice straw substrate
in an apple orchard or in shelters with conventional management. The former
method gave slightly higher yields and better quality fruiting bodies than the later.
Soil samples taken from around the trees 2 years after Volvariella volvacaea
cultivation showed improved organic matter, P and K contents. The orchard
environment which provided shade and high air humidity appeared to be
favourable for Volvariella volvacaea cultivation.
Nayak et al., (1990) reported the feasibility of growing paddy straw
mushroom Volvariella volvacea inside plastic tunnels was. Tunnels (4m x 1.7m x
0.085m) were prepared with transparent UV stabilized film (200) in 2 beds (0.9m
x 0.9m) were made one of the beds was covered with black LDPE (Low density
polyethylene) film (150) under the UV stabilized film. The average mushroom
yield at the end of 41 days after spawning was 2.7 kg/m2.
10
2.2. SOLID STATE FERMENTATION
Garo, (1964) used different substrates such as paddy straw dried banana stalks
and leaves, water hyacinth, wheat straw and sugarcane baggasse for mushroom
production. He observed only the beds made of sugarcane baggasse did not produce
mushrooms. The yield obtained from the beds of banana leaves was superior to the
yield obtained from any other substrate.
Gupta et al., (1970) tried wheat maize barley oat pearl millet, and sorghum
straw but the yield was very low as compared to that produced on paddy straw.
Before, 1970, paddy or rice straw was practically the only material used for
preparing the medium for commercial cultivation of mushroom under natural
condition.
Chen and Graham, (1973) grew Volvariella volvacea successfully on oil palm
pericarp waste. French material composed for five days before spawning gave the
highest yield of about 1.54Kg/100 Kg of waste. Bed temperature may be one of the
most important factors affecting the incubation period and duration of crop.
Chang, (1974) studied the cultivation of Volvariella volvacea on cotton waste
compost in plastic green houses. The compost was prepared by adding 4% rice or
wheat bran and 4-6% lime stone to cotton waste, after which it was soaked in water
and fermented for to 4 days .Production by this method was high and cropping was
also regular.
Graham, (1974) studied the performance of three field isolates collected from
oil palm bunch waste in Selangor and four isolates from Sarawak, Hong Kong,
11
Indonesia and Singapore, which were compared in two experiments. The Hong Kong
isolate yielded 5-6 Kg/100Kg of substrate and gave a maximum bed yield of over 3.1
Kg or 7.4 Kg/100 Kg of waste. The performance approached and surpassed yield
from paddy straw.
Madane et al., (1974) produced spawn of Volvariella volvacaea on paddy
straw mixed with 2.5% by weight of oat meal. Same results were obtained when
powered wheat or gram was substituted for the oat meal and when wheat or sorghum
straw or sugarcane bagasse was used instead of paddy straw.
Granhum, (1975) found a wide range of cultural characteristics and yielding
ability in single spore isolation from five cultures of Volvariella volvacaea. A
selected isolate from Hong Kong culture out yielded the parent culture by about
125% and one from a Sarawak culture out yielded its parent by about 199%.
Jablonsky, (1981) studied the substrates consisting of standard mushroom
compost prepared with 11 days at stage 1, and 8 days at stage II, horse manure
composed for 3 days and wheat straw treated in various ways. Half of each substrate
was treated for 4 hours at 900C (pasteurized) and the other half at 1200C for 1.5 hour
sterilized. Pasteurization of substrates produced higher yield than sterilization. The
highest yield was with chopped wheat straw. The addition of 3% CaCO3 reduced the
yield.
Qumio, (1981) reported that the following substrates supported very well
mycelial growth of Volvariella volvacea, rice straw, Ipil-Ipil leaves, sigadillas leaves,
new paper prints, coconut coir dust and banana bracts. It took 8 days for mycelium to
12
fill up the entire diameter of the Petri dish containing the substrate. This only
confirmed the fact Volvariella, unlike Agaricus species, could grow directly on un
composted substrates and therefore could be considered less specific in growth
requirements than the later mushroom.
Devi and Nair, (1987) observed that Volvariella volvacaea spawn prepared
out of 3-10 days old culture supported maximum sporocarp formation, when used for
spawning the beds. Maize and wheat grains supported good mycelial growth and
found to be suitable substrate for spawn preparation. At room temperature of
(28+4OC) the spawn remained viable with out a reduced in yield for about 20-60
days.
Alam and Khan, (1989) calculated growth percentage index of locally
crushed fresh mill bagasse with 25%, 10% and 5% molasses. The growth percentage
index in the first flush ranged between 305- 425%, in the second flush between 283-
300%, in the third flush between 220-240% and in the fourth flush it was 180%.
Adewusi et al., (1993) determined the biological value of 5 mushrooms
Chlorophyllum molybditis, Psathyrella atroumbonata, Termitomyces robustus,
Termitomyces striatus and Volvariella esculenta by using weanling rats and
observed that T. robustus and V. esculenta did not support growth at all.
Khan et al., (1994) tried dried water hyacinth for the cultivation of
Volvariella volvacaea and concluded that water hyacinth + cotton waste at the rate
1:1 gave maximum yield of Chinese mushroom followed by dried water hyacinth
alone and cotton waste alone.
13
Chiu et al., (1996) described that the cultivated strains of shiitake in China
were genetically very homogeneous, very like to the cultivated strains of Agaricus
bisporus and Volvariella volvacea. However, their collection of L. edodes, covers
an enormous geographical area, (approx. 1700 km N to S, 700 km E to W) and
results demonstrated that the shiitake industry in China depends on an extremely
small gene pool.
Salmones et al., (1996) reported the mycelial growth of two Mexican
strains of Volvariella volvacea (Bull.: Fr.) Sing., in 13 agro industrial wastes. He
used, banana leaves, bracts of pineapple crown, coconut fiber, coffee bran, coffee
pulp, corn cob, corn stover, orange peel, rice bran, rice straw, sisal bagasse,
sugarcane bagasse and wheat straw as substrate and evaluated mycelial growth,
mycelial thickness and pinhead formations. Fruiting bodies were obtained only
from one strain growing in bracts of pineapple crown, coffee pulp, rice straw and
sisal bagasse. Primordia were developed between 13 and 15 days. He recorded that
the highest biological efficiency was achieved on rice straw, 33.8%, while the
results obtained for coffee pulp, sisal bagasse and bracts of pineapple crown were
15, 7.8 and 6.2%, respectively. Chemical analyses of the substrates registered C/N
ratios of 33:1 to 80:1.
Cheung, (1997) evaluated the feasibility of using food waste, such as soya
milk residue, to produce nutritive fungal biomass. They produced edible
mushroom mycelia of Volvariella bombycina, Lyophyllum ulmarius and Pleurotus
citrinopileatus, were produced in liquid culture containing soya milk waste. They
14
observed similarities in the crude protein, lipid, ash and nucleic acid contents
between the mycelia and fruiting bodies. Differences were observed in the amount
of total dietary fiber and amino acid composition.
Reyes et al., (1998) described the nutritional and physical requirements for
the efficient mycelial colonization of Volvariella volvacea (Bull. ex. Fr.). Singer.
The investigation was limited to the evaluation of two commercial strains
(designated Vvc1 and Vvc2) and two wild strains (designated EAAC-0001 and
EAAC-0002) of V. volvacea from the Philippines. The four strains of V. volvacea
had varying preferences for carbon. Vvc1 preferred polysaccharides (starch and
cellulose), whereas Vvc2 grew luxuriantly at a relatively rapid rate in sugar
alcohol (sorbitol). The two wild strains preferred starch as a carbon source. In
terms of nitrogen utilization, soytone, peptone, and glycine supported efficient
mycelial colonization of the four strains. Efficient colonization of Vvc1, Vvc2,
and EAAC-0002 with dense mycelial growth was noted in mycological agar.
EAAC-0001, on the other hand, grew more efficiently in malt extract agar. The
Philippine strains of V. volvacea grew luxuriantly when incubated at 35°C and pH
8.0 under dark and sealed conditions. They concluded that under optimum
physiological conditions, Vvc1, Vvc2, and EAAC-0002 were fast-growing strains,
whereas EAAC-0001 was a moderately growing type.
Tonial et al., (2000) used industrial residues from cassava and potato starch
processing as substrates to produce the edible mushroom Volvariella volvacea
(Bull. Fr.) Singer. Three strains of V. volvacea (LPB 08, 59, 77) were grown in a
15
medium containing 2.4% (w/v) declassified potato flour and 1.6% (w/v) cassava
bagasse (PFCB) in petri dishes. Growth performance of cultures was evaluated by
measuring their rate of radial growth and biomass production. Strain LPB77
showed highest growth on PFCB agar medium. The liquid PFCB medium was
optimized with regard to residue composition, nitrogen source and pH The best
results were obtained after 8, days of fermentation in a medium containing 4.8%
(w/v) declassified potato flour and 1.2% (w/v) cassava bagasse, pH5,
supplemented with 0.1% (w/v) of KNO3, giving a C/N ratio of 30.
Philippoussis et al., (2001) cultivated ten selected wild and commercial
strains of Pleurotus ostreatus, Pleurotus eryngii, Pleurotus pulmonarius,
Agrocybe aegerita and Volvariella volvacea on three agricultural wastes, i.e.
wheat straw (WS), cotton waste (CW) and peanut shells (PS). They observed that
one commercial strain of V. volvacea presented higher growth rates when the
composted CW medium was used. Furthermore, earliness in the fructification of
P. ostreatus, P. pulmonarius and V. volvacea strains was promoted in CW
substrates. They detected positive correlation between cellulose content and
mushroom yield for V. volvacea strains.
Obodai et al., (2003) evaluated the biological efficiencies (yield of the
mushroom against the dry matter of the substrates) of two strains of the mushroom
Volvariella volvacea, V99 and VVO, by using banana leaves, cocoyam peelings
and oil-palm pericarp as substrates. They observed that primodia were after11-12
days on banana leaves. Both strains showed their highest production on banana
16
leaves, with biological efficiencies of 43 and 72%. V99 fruited on all the
substrates but VVO fruited only on banana leaves as it had mycogone infection on
the other substrates.
Akinyele and Akinyosoye, (2005) cultivated the mushroom, Volvariella
volvacea on various agro wastes and observed Maximum mycelia extensions in
cotton waste (98.23 ± 0.1 mm) and a combination of rice husk and cotton waste
(101.87 ± 0.4 mm). A decrease in moisture content resulted in significant increase
percentage crude protein content of mushroom-treated waste compared to the
untreated they concluded that changes in crude fiber and ash content of treated and
untreated wastes were not significant, mineral contents were observed to increase.
Phosphorus and potassium ion content also increased in mushroom-treated
samples.
Belewu and Belewu, (2005) studied the solid state fermentation of banana
leaves by lignin degrading mushroom (Volvariella volvacea) ,yield of fruiting
body and compositional changes of substrates were evaluated .The biological
efficiency was 5.21while the total weight of fruit yield was 2.5 kg.
2.3. PHYSIOLOGY
2.3.1. Effect of temperature
Alicbusan and Ela, (1967) reported that the bed should be pressed lightly
because it must reach to the temperature of 40°C to 45°C because this temperature
favourable for the mushroom production but not for the organisms present in the
bed.
17
Chang, 1972 reported the best temperature for the growth and fructification
of Chinese mushroom is 26°C to 30°C and needs bed temperature of 34 to 37°C.
Agarwala, (1973) reported that tropical mushrooms can grow at a
temperature up to 45C or even more and the minimum temperature should not go
below 25C in any case even for a short period. He concluded that at higher the
temperature faster growth and higher yield will be obtained. The cropping period
lasted 25-30 days under poor temperature conditions.
Samajpati et al., (1977) studied the cultivation of V. volvacea on paddy
straw beds where growth conditions were satisfactory from April to August but
highest yield was obtained in July when the optimum temperature was 32C and
humidity was about 85%.
Khan and Kusar, (1981) reported prospects and potential of mushroom as a
cottage industry in Pakistan. Variety of climatic conditions such as northern hilly
areas with high humidity and low temperature were suitable for temperature kind of
mushrooms where as plains of Punjab, Sindh, and Balochistan were suitable for
cultivation of tropical mushrooms in summer. Many agricultural wastes and
industrial waste in the form of straws, leaves and cotton and corn wastes were
available in large quantities.
Ramakrishnan et al., (1986) observed that use of transparent polythene
sheet to cover the bed showed higher yield than with black polythene sheet and he
also reported the optimum temperature for Chinese mushroom cultivation was 35
to 37.
18
Morris et al., (1988) observed the changes in morphology and viability of
20 species of fungi including Volvariella volvacea during freezing were examined
in relation to cooling rate and the presence of glycerol. They observed that the
morphological response of Phytophthora, Aschersonia and Volvariella differed
from other genera, with shrinkage occurring at all rates of cooling.
Frank, (1989) recommended measures to obtain high yield of V. volvacaea.
The best planting date was when both day and night air temperatures were 23°C.
Lime was sprayed on the straw to increase the pH value from 7.5 to 8.5. The spawn
was planted at an age of 18-20 days.
Wang and Li, (1989) studied the various deformities occurred in cultures of
V. volavcea including the production of empty mycelium, abnormal bodies formed
from the hyphae, deformed fruiting bodies and withering of young mycelium. They
suggested that these deformities could be avoided by the maintenance of a constant
growing temperature in the range of 22-28C.
2.3.2. Effect of light
Yau and Chang, (1970) obtained fruiting under 12 hours light. They
reported the light intensity of 50lux was optimum. Antonio and Fordyce, (1972)
reported an appreciable quantity of light (15 minutes full sunlight) was required
for the initiation of fruiting bodies of Volvariella volvacea. The average yield was
84g/Kg dry weight compost.
19
Khan, (1976) described that the light is not required for the Chinese
mushroom during the period of spawn running, it respond favorably to weak light
condition and ventilation
Chakravarty and Mallick, (1979) reported that growth of V. diplasia was
vigorous in complete darkness, intermediate in diffused light and slowest in full
light. Light passing through red or blue filters produced good growth but light
from green or yellow filters inhibited the growth.
Singh and Saksena, (1983) mentioned that in case of V. volvacea light
intensity showed no effect on mushroom yield but colour, texture and shelf life of
mushroom grown under dark and diffused light conditions was better than the
mushroom produced under bright light conditions.
Fasidi, (1996) described that Volvariella esculenta (Mass) Singer, is able to
grow at a temperature range of 20-40°C (optimum = 35°C) and pH range of 3-10
(optimum = 6.0) and a wide range of agricultural wastes for growth. Of these the
rice straw and induced the widest mycelial extension and rice bran produced the
highest mycelial density. The unfermented cotton waste compost produced the
highest fruit body yield.
Banik and Nandi, (2000) described that V. volvacea known as paddy straw
mushroom grows well in humid and tropical environment, the biological
efficiency of this was very low in comparison to oyster mushroom. Low
productivity was a big hurdle for its commercial exploitation but the traditional
substrate for this mushroom cultivation, with biogas residual slurry manure
20
increased its production by producing fruit bodies bigger in size and higher in
number and also increased the protein content significantly. The mineral nutrients
viz., P, Ca, K, Fe, Cu, Zn and Mn were also increased. So, they concluded that
supplementation of this bio manure for cultivation of tropical mushroom
Volvariella volvacea may be a step towards its successful commercial
exploitation.
Zervakis et al., (2001) determined the influence of environmental
parameters on mycelial linear growth of different mushroom crops including
Volvariella volvacea in two different nutrient media in a wide range of
temperature. V. volvacea grew faster at 35°C, Wheat straw, peanut shells and
particularly cotton gin-trash supported fast growth of V. volvacea.
Jonathan et al., (2004) studies were conducted on the effects of
temperature, pH, vitamins and plant hormones on the vegetative growth of
Volvariella esculenta (Mass) Singer. They observed that, this mushroom had its
optimum radial growth at 35°C with mycelial extension of 85.0 mm and the pH
that supported best growth was 6.0. Pyridoxine was the most utilizable vitamin
with mycelial dry weight of 123 mg/30 cm3. They also observed that among the
tested phytoharmones, 2, 4-D (10.0 ppm) stimulated the best growth of 150 mg/30
cm3 but, 0.1 ppm of GA3 supported poor growth (53.0 mg/30 cm3).
Akinyele and Adetuyi, (2005) studied the effect of temperature variations
on the growth of V. volvacea cultivated on various agricultural wastes singly and
in various combinations. A pH range of 5.5 to 8.5 recorded the maximum mycelial
21
yield and the highest mycelia weight was recorded at pH6.5 while poor mycelia
growth of the mushroom and the least mycelia weight was recorded at pH 2.0.
Highest mycelia growth of mushroom was recorded between 25°C and 30°C and
the least mycelial dry weight of 0.5 mg obtained at 10°C.
2.4. LIQUID FERMENTATION
2.4.1. Background
Anon, (1954) while working in the Syracuse University, New York,
conculede that it is possible to grow the highly priced mushroom Morchella
hortensis, without any problem and at very low cost in water culture under
constant motion.
Atacador, (1967) reported that out of five edible mushrooms cultivated in
liquid medium, V. volvacea gave the highest mycelial yield. The best medium for
its production had pH 5, and contained 4% urea, 4% sucrose and best propagation
period was 5 days to get maximum mycelial yield.
Kostadinov et al., (1972) described a method of producing Pleurotus
ostreatus mycelium in sub-merged culture which may be used as inoculation
material (spawn) for production of sporophores on a substrate of crushed corn
cobs. They also reported the most suitable nutritive medium was a combination of
cane molasses with supplementation of NH4NO3, NH4SO4 and Potassium
dihydrogen phosphate.
Lin and Hu, (1972) reported that sugarcane baggase was found to be
suitable and more economical for mushroom cultivation as straw compost. The
22
dried product was amended by the wheat bran (10%), ammonium dihydrogen
phisohate (1%), calcium chloride (5%) and water (60%) followed by heat
treatment at 55°C for 72 hours.
Bukhalo, (1982) selected some species and strains of edible fungi for
biomass production in submerged culture. Similarly Khan et al., (1982) reported
that Pleurotus can be can grown in cane molasses medium and resulting biomass
can be used as a source of protein rich food.
Garo and Neelakanton, (1982) investigated the production of single cell
protein by Aspergillus terrens by using alkali treated baggase as potential
substrate. They obtained maximum biomass protein content of 20% by
continuously shaking of culture for seven days at pH 4.
Quimio, (1984) mentioned potential of using coconut water, first time as a
liquid medium for mycelial production of Lentinus sajor caju and other edible
mushroom, secondly as a routine agar medium for the growth of edible
mushrooms. He observed that coconut water both from young and mature nuts can
support the mycelial growth of edible mushrooms. After first week of incubation,
12gm of dry weight of Lentinus sajor- caju mycelia was harvested from 100ml of
sterilized coconut water. When the coconut water was incorporated with agar
medium it performed almost equally to the PDA medium. Trials with V. volvacea
and some other mushrooms showed that coconut water agar medium can be used
as a routine laboratory medium in place of potato dextrose agar medium.
23
Mahmood, (1986) optimized the concentration of inorganic growth
nutrients for the production of mycelial protein from rice polishing with
Arachniotus spp. Maximum crude protein percentage was obtained in the shake
medium containing calcium chloride 0.001%, magnesium sulphate 0.001%, and
potassium dihydrogen phosphate 0.08%, after 72 hours of incubation at pH 4 and
30°C temperature.
Dey et al., (1995) investigated biosorptions of Pb2+, Cr6+, Cd2+ and Ni2+ by
using live and dead fungal mycelia. Of the four fungi, namely Polyporus
ostreiformis, Volvariella volvacea, Pleurotus sajor-caju and Phanerochaete
chrysosporium, they observed total biosorption was effected in 6 days up to the
Pb2+ concentration of 6 mg/l, with a specific uptake of 1.33 mg Pb2+ /g dry cell
mass. The removal of other three metals varied between 28.8-73.3% from a
medium containing 4 mg/l of each of the metals.
Cai, et al., (1998) studied that the edible straw mushroom, Volvariella
volvacea (V-14), produced β-glucosidase when grown in liquid culture on a
variety of carbon sources including cellulose, cellobiose, salicin, sorbose, lactose,
esculin, cotton wool, and filter paper. They purified two cell-associated β-
glucosidases, BGL-I and BGL-II, 32-fold and 23-fold, respectively, from extracts
of cellulose-grown mycelium. The enzymes were found to be homogeneous and to
have native molecular weights of 158 kDa (BGL-I) and 256 kDa (BGL-II) by gel
filtration. Both isozymes displayed relatively broad pH optima with maximum
reaction velocities recorded at pH 7.0 for BGL-I and pH 6.2 for BGL-II, and were
24
rapidly denatured at temperatures of 60°C and above. Isozyme activities were
adversely affected by several reported β-glucosidase inhibitors, various metal ions,
and lignin-derived aromatic acids and aldehydes. Glucose production from
microcrystalline cellulose by a commercial cellulase preparation was enhanced by
9.7% in reaction mixtures supplemented with BGL-II.
Chen et al., (2004) isolated a Laccase (lac1) from culture fluid of
Volvariella volvacea, grown in a defined medium containing 150 micro m CuSO4
subscript by ion exchange and gel filtration chromatography. RT-PCR analysis of
gene transcription in fungal mycelia grown on rice straw revealed that, apart from
during the early stages of substrate colonization, lac1 was expressed at every stage
of mushroom developmental cycle defined in their study, although the levels of
transcription varied considerably depending upon the developmental phase.
2.4.2. Liquid media used for fermentation
Edwards, (1954) observed the growth of edible fungi in sub-merged
culture, consisted of a liquid medium through which air was constantly bubbled.
The rapid growth of mycelium was obtained from one cream and one white
variety of cultivated mushroom Agricus compestris. He concluded that sub-
merged culture might be much cheaper than ordinary mushroom culture. The
mycelial biomass produced could be used for soups or flavouring of food items.
Block, (1959) concluded that yields were high and no special production
problems were encountered when mushroom mycelium were grown in sub-
merged liquid culture with carbohydrates and nitrogen compounds with mineral
salts.
25
Torver, (1968) worked out a technology for the cultivation of mycelium of
different species of higher fungi in liquid nutritive medium. He assumed that
mycelium of some mushroom species produced in liquid culture would be used as
spawn in mushroom cultivation.
Anthony, (1977) reported that fungi can be successfully propagated on a
wide variety of substrates. He found that protein contents of fungi were strain
dependant and influenced by the growth conditions.
Bukhalo et al., (1978) studied that Pl was successfully grown in liquid
nutrient medium containing 10% red clover extract, sucrose, peptone and mineral
salts on commercial scale. The fungus was cultivated on complex media
containing potato waste and molasses. The rate of mycelial growth was greater on
sub-merged culture than that in surface culture.
Nagaso and Yoshikawa, (1978) observed that mycelial growth and yield
was best with shaking at 100 cycles/minute rather than shaking at 90 or 120
cycles/ minute.
2.5. BIOCHEMICAL ANALYSIS
2.5.1. Nutrients contents
Tumwasorn et al., (1980) reported 11.5% to 12% ash in the straw of different paddy
varieties and crude fat contents of paddy straw before and after mushroom
production were reported to be 0.5% and 0.2%, respectively.
26
Vijaya nad Pandaya (1981) reported proximate composition of paddy straw
mushroom. The value for moisture fat, crude protein, carbohydrates, crude fiber and
ash were 92%, 0.21%, 2.19%, 1.15% and .99% respectively.
Khowala and Sengupta, (1985) purified the enzyme, endo-alpha-
mannanase, from culture filtrate of a mushroom Volvariella volvacea by acetone
precipitation, ion-exchange chromatography (DEAE-Sephadex), and gel-
permeation chromatographies on Bio-Gel P-300 and on Sephacryl S-200 columns.
They observed single protein band on sodium dodecyl sulfate-disc gel
electrophoresis at pH 6.8 and has a molecular weight of approx. 56,000. It has no
alpha- or beta-mannosidase activity and does not act on beta-gluco-or
galactomannan. The enzyme shows maximum activity on baker's yeast alpha-
mannan at pH 5.0 and at 55°C, and is fairly stable between pH 3 and 6 and
temperatures up to 50°C. Enzyme activity is inhibited by Hg2+, sodium azide,
iodoacetic acid, EDTA, and Ag+, in decreasing order.
Kalisz et al., (1986) concluded that Agaricus bisporus was grown on defined
liquid media with protein as sole source of carbon, nitrogen or sulpher and with these
nutrients supplied in the form of glucose, ammonium or sulphate. The culture
filtrates of Agaricus were tested for extracellular laccase activity. Constitutive
laccase production was observed under all conditions tested. However, Agaricus
laccase, though constitutive, was induced by protein and repressed by ammonium.
No detectable extracellular laccase activity was found in tested cultures of Coprinus
cinereus or Volvariella volvacea.
27
Kishida et al., (1989) concluded that A (1--3)-beta-D-glucan branched by
O-6 substitution (FCAP), obtained from the cold-alkali extract of the fruiting body
of V. volvacea, exhibited potent growth-inhibitory activity against implanted
tumors in mice. They suggested that the Volvariella glucan was structurally
heterogeneous with regard to the distribution of branches, having less branched,
moderately branched, and highly branched segments.
Cheung (1996) analysed the mycelia caps and stalks of fruiting bodies of four
edible mushrooms (Lentinus edodes, Lycophyllum shimeji, Pleurotus sajor-caju,
and Volvariella volvacea) for their total dietary fiber contents.The TDF cotents of
all of the mushrooms were considerably greater than those determined by using
the Uppsala method. Mushroom mycelia had higher TDF values than did the
fruiting bodies .The TDF composition of the mycelia and the TDF composition of
the caps and stalks of the mushroom fruiting bodies were similar. Sugar
composition reflected that β-glucans were the major fiber polysaccharide with
chitin, hemicelluloses, and polyuronides as minor ones.
Phutela et al., (1996) screened two strains of Volvariella diplasia (Vd IIHR
and Vd TNAU) and three of V. volvacea (Vv IARI, Vv MU and Vv TNAU) for
the production of cellulases and xylanases. They observed Vd IIHR exhibited
maximum activity of cellulases. This strain also showed maximum biomass
production (5.8 g I- broth) and yield potential (BE 8.6%). The xylanase enzyme
showed maximum enzyme activity (9.73 U mg-1 protein) in Vd TNAU strain. All
the enzyme activities were the maximum in culture filtrates after 8 d of growth.
28
Lin et al., (1996) crystallized Volvatoxin A2; an ion channel disturbed
cardiotoxic and hemolytic protein from the edible mushroom, V. volvacea, by the
vapor diffusion method using polyethylene glycol 4000 and ammonium sulfate in
sodium acetate buffer pH 4.6.
Zhi et al., (1998) purified a novel single chained ribosome inactivating
protein (RIP) from fruiting bodies of the edible mushroom Volvariella volvacea.
The mushroom RIP, designated volvarin, exhibited a potent inhibitory action on
protein synthesis in the rabbit reticulocyte lysate system. It also exerted a
deoxyribonuclease activity on super coiled SV-40 and demonstrated a strong
abortifacient effect in mice.
Wang et al., (1998) summarized existing information about mushroom
lectins, with an emphasis on those from the species which have been most
extensively characterized including various Agaricus species, Areanita
pantherina, Boletus satanas, Coprinus cinereus, Ganoderma lucidum, Flammulina
velutipes, Grifola frondosa, Hericium erinaceum, Ischnoderma resinosum,
Lactarius delerrimus, Laetiporus sulphureus, Tricholoma mongolicum and
Volvariella volvacea. Immunomodulatory and antitumour/cytotoxic activities have
been carried out on lectins from Agaricus bisporus, Boletus satanas, Flammulina
velutipes, Ganoderma lucidum, Grifola frondosa, Tricholoma mongolicum and
Volvariella volvacea.
Chiu et al., (1998) explained that recalcitrant nature, persistence and
toxicities of chlorophenols make them priority pollutants for treatment. He
29
compared the ability of various fungi including (Armillaria gallica, A. mellea,
Ganoderma lucidum, Lentinula edodes, Phanerochaete chrysosporium, Pleurotus
pulmonarius, a Polyporus sp., Coprinus cinereus and Volvariella volvacea), and
the spent mushroom substrate of P. pulmonarius (SMS) to remove
pentachlorophenol (PCP) using a batch cultivation system. All these fungi showed
active breakdown in addition to bio sorption as their PCP removal mechanisms.
Mau, (1998) irradiated fresh common (Agaricus bisporus) and high-
temperature mushrooms (A. bitorquis) with ultraviolet-C (UV-C) for 0, 0.5, 1, and
2 h at 12 °C. Fresh common, shiitake (Lentinula edodes), and straw mushrooms
(Volvariella volvacea) were irradiated with UV-B for 0, 0.5, 1, and 2 h at 12 °C.
After UV-C irradiation for 2 h, vitamin D2 contents in common and high-
temperature mushrooms increased from 2.20 and 4.01 µg/g of dry weight to 7.30
and 5.32 µg/g, respectively. After UV-B irradiation for 2 h, vitamin D2 contents in
shiitake and straw mushrooms increased from 2.16 and 3.86µg/g to 6.58 and 7.58
µg/g, respectively. The increase rates in shiitake and straw mushrooms were not as
high as in common mushrooms.
Yao et al., (1998) purified a novel single-chained ribosome-inactivating
protein (RIP) with a molecular weight of 29,000 from fruiting bodies of the edible
mushroom V. volvacea. The mushroom RIP, designated volvarin, exhibited a
potent inhibitory action on protein synthesis in the rabbit reticulocyte lysate
system with an IC50 value of 0.5 nM. It also exerted a deoxyribonuclease activity
on supercoiled SV-40 DNA and demonstrated a strong abortifacient effect in mice.
30
She et al., (1998) purified a novel lectin from the fruiting bodies as well as
cultured mycelia of the edible mushroom V. volvacea. They observed that the
lectin, designated as VVL, was a homodimeric protein and had no carbohydrate
moiety, and its hemagglutinating activity was inhibited by thyroglobulin. The
immunomodulatory activity was demonstrated by its potent stimulatory activity
toward murine splenic lymphocytes. VVL also enhanced the transcriptional
expression of interleukin-2 and interferon-γ by reverse transcriptase-polymerase
chain reaction. VVL possessed a molecular structure distinct from other
immunomodulatory proteins previously reported in the same fungus.
Cai et al., (1999) described that the edible straw mushroom, Volvariella
volvacea produced a multi component enzyme system consisting of endo-1, 4-b-
glucanase, cellobiohydrolyse, and glucosidase for the conversion of cellulose to
glucose. Biochemical analysis of different culture fractions in cultures exhibiting
peak enzyme production revealed that most of the endoglucase was present either
in the culture filtrate (45.8%) of the total or associated with the insoluble pellet
fraction remaining after centrifugation of homogenized mycelia (32.6%).
Cellobiohydrolyse distributed with 58.9% of the total enzyme present in cultural
filtrates and 31 % associated with the pellet fraction.
Whiteford, (2000) described the types, economic significance and methods
of production of the principal cultivated mushrooms are described in outline.
These organisms are all less than ideal for conventional genetic analysis and
breeding, so molecular methods afford a particular opportunity to advance our
31
understanding of their biology and potentially give the prospect of improvement
by gene manipulation. They also described the gene sequences isolated from the
paddy straw mushroom V. volvacea and many other mushrooms.
Isiloglu et al., (2001) determined the concentrations of Cu, Cd, Co, Ni, Mn,
Pb, Zn and Fe in 66 samples of mushroom fruiting bodies, representing seven
species, mainly all edible, were determined by atomic absorption
spectrophotometry. The mushrooms were collected from near roads and inner
parts of forest and lawns in Balikesir in the north western part of Turkey. The
results indicated that the Fe level in the species Volvariella speciosa (Fr.) Sing.
from near the road was the highest with a mean of 6990 mg/kg. The Cd was
accumulated mostly by Lactarius sanguifluus (Paulet: Fr.) Fr. and V. speciosa
from near road with a mean of 1.60 mg/kg.
Liu et al., (2001) studied the antiproliferative activity of a fungal lectin
(VVL) isolated from the mushroom, V. volvacea. It was observed that VVL did
not exert ribosome-inactivating activity or induce any changes in the expression of
cyclins A, D1, and E. However, it did activate the expression of cyclin kinase
inhibitors, namely p21, p27, p53, and Rb, in a dose-dependent manner. Flow
cytometric analysis demonstrated an accumulation of cells in the G2/M phase in a
time- and dose-dependent manner, indicating that VVL arrested cell proliferation
by blocking cell cycle progression in the G2/M phase.
Fu et al., (2002) studied the antioxidative potency of commercially
available mushrooms in Taiwan. The order suggested by him of inhibitory activity
32
of mushroom extracts on oxidation in emulsion system was Agaricus bisporus >
Hypsizigus marmoreus > Volvariella volvacea > Flammulina velutipes >
Pleurotus eryngii > Pleurotus ostreatus > Hericium erinaceus > Lentinula
edodes. In the thermal oxidative stability test, using lard, the order of antioxidative
activity of test materials showed similar tendencies, except for the extract of
Lentinula edodes.
Wang and Liu, (2002) isolated the lectin from the dried fruiting bodies of
the mushroom Agrocybe cylindracea a heterodimeric lectin with a molecular
weight of 31.5 kDa and displaying high hemagglutinating activity. The larger and
the smaller subunits resembled Agaricus bisporus lectin and fungal
immunomodulatory protein from Volvariella volvacea respectively in N-terminal
sequence. The lectin exhibited potent mitogenic activity toward mouse
splenocytes. The hemagglutinating activity of the lectin was inhibited by lactose,
salicyclic acid and inulin.
Ahlawat et al.,(2005) isolated and evaluated the six parent strains and 11
monosporous isolates from two strains of V. volvacea for their enzymes induction
level, substrate colonization and yield potential. They observed that all parent
strains preferred wild grass over wheat straw and paddy straw for vegetative
growth along with highest level of induction of β-glucosidase and xylanase.
Mushroom yield in parent strains was related to the level of various
lingocellulolytic enzymes produced on a substrate. Monosporous isolates and
33
parent strains produced more cellulases at 8 and 10 d, while xylanase, laccase and
polyphenol oxidase were more at 10 or 13 d growth on paddy straw.
2.5.2. Medical effects
Lin et al., (1974) isolated the cardiotoxic protein volvatoxin from
Volvariella volvacea and another cardio toxic protein isolated from the
Flammulina velutipes (Curt. ex Fries Sing), which is widely eaten in the Orient.
This protein is called flammutoxin. Flammutoxin has three biological activities
similar to those of volvatoxin, direct hemolytic action against human group ‘O’
blood cells; ability to cause a writhing reaction with a delay before onset; and
effect on the electrocardiogram at a dose of 0.25 mg per kg body weight, causing
depression of the ST segment and inversion of the T wave.
Fassold et al., (1976) isolated Volvatoxin A, present in the mushroom
Volvariella volvacea, causes a competitive, dose and time dependent inhibition of
the Ca2+ accumulating activity of a sarcoplasmic reticulum rich microsomal
fraction isolated from guinea pig ventricular muscle. They explained why
volvatoxin A increases the diastolic resting tension in heart muscle.
Kishida et al., (1992) purified a potent antitumor-active branched (1 → 3)-
β-D-glucan (VVG) from fruiting body of Volvariella volvacea. They observed that
conversion of the glucosyl groups substituted at 0-6 atoms of the (1 → 3)-linked
D-glucose residues into the corresponding polyhydroxyl groups gave significant
enhancement of the original activities, whereas deletion of the polyhydroxyl
groups resulted in a great reduction of the activity. When D-glucose residues of
34
the branches were modified to the 3,6-anhydro D-glucose residues. They observed
that the previous findings that, besides the conformation of (1 → 3)-β-glucan
backbone, the molecular shape and the distribution pattern of the substituted
groups located outside the backbone chains, must also play an important role in
exhibiting anti tumor action.
Chiu et al., (1995) described that the straw mushroom Volvariella volvacea
was one of the common edible mushrooms cultivated in Southeast Asian
countries. It has been reported to produce a hypotensive response in animals
including humans. An aqueous extract of the mushroom (SME) was prepared and
given through intravenous injections to normotensive rats. They examined the
effects of SME on the kidney function of water-loaded rats and on isolated tissue
preparations of the tail artery and right atrium. An IV injection of SME produced a
hypotensive effect in rats with an ED50 of 25 mg dry weight/kg body weight. This
hypotensive effect of SME was attenuated or blunted in the presence of
hexamethonium, phentolamine, pyrilamine and cimetidine suggesting the
involvement of the α-adrenergic component of the autonomic system and/or
histaminergic stimulation. They also observed that SME did not increase urinary
excretion nor sodium diuresis. It produced positive chronotropic and inotropic
effects on isolated right atria and induced contraction of isolated tail artery strips.
This latter contractile response was inhibited by antagonists of serotonin and α-
adrenoceptor, ketanserin and phentolamine respectively. Partial purification using
dialysis and liquid chromatography revealed that the hypotensive active
35
substances had molecular masses between 8000 and 12000 dalton. These
substances were heat stable and resistant to trypsin digestion. In view of the
similarity in blood pressure and cardiovascular response, SME might contain
serotonin-like substances.
Fasidi and Kadiri, (1995) studied the toxicological aspects of seven
Nigerian mushrooms, namely, Chlorophyllum molybditis (Mayer ex. fr.) Masse,
Cortinarius melliolens Fries, Lentinus subnudus Berk, Pleurotus tuber-regium
(Fries) Singer, Termitomyces robustus (Beeli) Heim, Tricholoma lobayensis Heim
and Volvariella esculenta (Mass) Singer. Amatoxin spot test and chromatographic
screening of the mushrooms revealed the absence of amatoxins and phallotoxins
because none of the mushroom extracts tested killed the experimental rats.
Cheung, (1996) fed male Sprague-Dawley rats with two semisynthetic diets
supplemented with 2% cholesterol and 1% β-glucan type extracellular
polysaccharide isolated from two liquid cultures of straw mushroom (Volvariella
volvacea) mycelium containing different carbon sources. They concluded that both
mycelial extracellular polysaccharides exhibited hypocholesterolemic activity in
rats with alimentary-induced hypercholesterolemia.
Fasidi and Akwakwa, (1996) studied the growth requirements of
Volvariella speciosa (Fr. ex. Fr.) Sing. were studied. They observed all the tested
carbohydrates except cellulose significantly enhanced mycelial growth and
Mannitol was the most utilized, followed in order by fructose and maltose. All the
organic and inorganic nitrogen sources investigated significantly improved
36
growth. Calcium, magnesium, sodium, potassium and zinc significantly enhanced
growth whereas hormones and vitamins did not.
2.5.3. Bioconversion of lignocellulosic wastes
Buswell et al., (1996) described that edible mushroom cultivation was one
of the most economically-viable processes for the bioconversion of many types of
lignocellulosic wastes. According to him Lentinula edodes, Volvariella volvacea
and Pleurotus sajor-caju were three important commercially cultivated
mushrooms which exhibit varying ability to utilize different lignocellulosics as
growth substrate he explained that V. volvacea, which preferred high cellulose-,
low lignin-containing substrates produces a family of cellulolytic enzymes
including at least five endoglucanases, five cellobiohydrolases and two β-
glucosidases, but none of the recognized lignin-degrading enzymes.
Rajor, (1996) described that the Sawdust, a bulky waste generated by wood
processing industries, has very few profitable and eco friendly uses and has a
problem of proper disposal. They observed that treatment with the fungus V.
volvacea and a dilute solution of urea converted sawdust from a phytoinhibitory
material to a phytostimulatory soil conditioner. Analyses of the major biopolymers
of sawdust after fungal treatment indicated the decrease in the levels of cellulose,
hemicellulose and lignin.
Chiu et al., (2000) described that mushroom cultivation was a direct
utilization of their ecological role in the bioconversion of solid wastes generated
from industry and agriculture into edible biomass, which could also be regarded as
37
a functional food or as a source of drugs and pharmaceuticals. They concluded
that, this was very true for Lentinula edodes, Volvariella volvacea, and
Ganoderma lucidum, which were commonly consumed in Asian communities but
are now gaining popularity worldwide. Besides the conventional method, strain
improvement could also be exploited by protoplast fusion and transformation.
Biodiversity is the key contribution to the genetic resource for breeding programs
to fulfill different consumer demands. Spent mushroom compost, a bulky solid
waste generated from the mushroom industry, however, could be exploited as a
soil fertilizer and as a prospective bioremediating agent.
Datta and Chakravarty, (2001) studied comparative utilization of
lignocellulosic componnts of paddy straw by Tricholoma lobayense and
Volvariella volvacea. They observed that T. lobayense degraded the lignin more
actively till the end of spawn run phase. However, V. volvacea could utilize
cellulose and hemicellulose throughout spawn run and cropping phases, but was
unable to utilize lignin at any stage.
Yadav et al., (2002) devised an optimized protocol for the bioconversion of
eucalyptus bark. It comprised: (i) mechanical reduction in bark size to 0.5-3.0 cm,
(ii) moistening to 60-65%, (iii) fortification with ligninase-rich fungus Volvariella
sp. (S-1) and 2% urea and (iv) maintenance of this composting mix under aerobic
and ambient condition for 14-15 weeks. The resulting bark soil conditioner (BSC),
with physico-chemical and microbial properties which would enrich soil
fertility/productivity.
38
2.5.4. Uptake of heavy metals Purkayastha and Mitra, (1992) observed the uptake of a few metals by V.
volvacea during submerged growth of the organism in sub lethal concentration of
each metal salt. They concluded that the uptake of Pb2+ and Hg2+ was 5 and 5.23
micrograms g-1 respectively while that of Cu2+ was 500 micrograms g-1 under
experimental conditions. Treatment of spawned substrate separately with different
metal salts showed maximum and minimum uptake of Pb2+ (100 micrograms g-1)
and Cd2+ (2.93 micrograms g-1) respectively by sporocarps. All metal salts at test
concentrations reduced biological efficiency of sporocarp production but markedly
by Co2+. Cd2+ and Co2+ were highly toxic to mycelia and sporocarps
respectively and the uptake of Cu2+ by mycelia and Pb2+ by sporocarps were
highest among the five metals tested.
2.6. GENETICS
Boekhout and Enderle, (1996) designated a neotype for Volvariella
gloiocephala (DC. Fr.) Boekhout & Enderle, to serve as a representative collection
for the current concept of this species, that generally was considered conspecific
with V. speciosa (Fr.: Fr.) Kummer.
Chiu and Moore, (1999) determined the electrophoretic karyotype of the
Chinese straw mushroom, Volvariella volvacea. They observed that haploid strain
V34 of V. volvacea has 15 chromosomes ranging in size from 1.4 to 5.1 Mb. No
chromosomal polymorphism in terms of size and number was seen in either of two
growth stages: vegetative mycelia or fruit-body gill tissues. They also prepared
39
DNA fingerprints by the arbitrarily-primed polymerase chain reaction. Variation
in DNA fingerprints was evident in protoplast regenerants derived from the same
vegetative mycelium. Thus the haploid mycelium of strain V34 was heterokaryotic
but the bulk genotype is stable during fruit body development. They overviewed
the known mechanisms to generate genetic variation and proposed a novel
mechanism that could account for the 1:1 segregation ratio of self-fertile to self-
sterile progeny regularly obtained from selfed Volvariella volvacea fruit bodies.
Ding at el., (2001) isolated an endoglucanase, EGI, from fluid of
Volvariella volvacea grown on crystalline cellulose by ion exchange and gel
filtration chromatography and preparative PAGE .Their work was aimed at
generating improved strains of V. volvacea with enhanced growth, substrate
conversion capacity and higher production capacity.
Guo et al., (2002) used PCR technique for amplifying THP gene in an
unknown vector with primer AFP1 and AFP2. Then THP gene was ligated to
pGEM T-Vector to be the plasmid pGTHP4. Then isolated and purified big
fragment containing promoter with the Agarose Gel DNA Extraction Kit, and also
purified the small fragment containing THP gene from 1% agarose gel with the
Agarose Gel DNA Extraction Kit. The big fragment and the small fragment were
ligated at Nco I digested cohesive-end. The ligation product was re-ligated to be
cyclic plasmid by addition to a specific adapter, resulting in the pCTH823, a
expression vectorof V. volvacea.
40
Shi et al., (2002) assessed aqueous extracts of the sporophores of eight
mushroom species for their ability to prevent H2O2-induced oxidative damage to
cellular DNA using the single-cell gel electrophoresis assay. They observed the
highest genoprotective effects with cold (20°C) and hot (100°C) water extracts of
Agaricus bisporus and Ganoderma lucidum fruit bodies, respectively. No
protective effects were observed with mushroom derived preparations (MDPs)
from Lentinula edodes, Pleurotus sajor-caju, and Volvariella volvacea. They
concluded that some edible mushrooms represent a valuable source of biologically
active compounds with potential for protecting cellular DNA from oxidative
damage.
41
Chapter 3 MATERIALS AND METHODS
Experiments were carried out in the Mushroom Laboratory and growing room
of Department of Plant Pathology, University of Agriculture, Faisalabad, and private
mushroom production farm Faisalabad.
The growing room was consisted of two compartments, one meant for spawn
running and other for cropping. Each portion had iron and wooden racks. The
arrangement of racks maximizes the utility of available space for the cultivation of
mushroom. The temperature of growing room was kept constant at 30oC spawn
running and for production of fruiting bodies temperature was kept below 40oC with
spraying of water on beds and floor of the room.
3.1. STRAIN COLLECTION
Mushroom species were colleted from different areas like University of
Agriculture Faisalabad, Gutwala forest area Faisalabad, Changa manga forest area
Lahore and from different field areas. From these collected species Volvariella spp.
were identified on morphological basis.
The exotic strains i.e., Vv436 (Malaysia), Vv422 (Philippines), Vv428 (Hong
Kong) and Vv430 (Taiwan), were imported from the mushroom spawn laboratory of
Pennsylvania State University America.
3.2. CULTURE MULTIPLICATION
Culture of V.v. PK, (local strain) was prepared by tissue culture method on
potato dextrose agar (PDA) and malt extract (MEA) culture medium. Exotic cultures
42
were also multiplied on the selected culture media in Petri dishes. These Petri dishes
were incubated at 30°C for 7 days in the incubator. The procedure of culture
multiplication was repeated after every 30 days to keep the cultures viable and fresh.
From these cultures spawn was prepared for the cultivation of mushroom.
3.3. SELECTION OF CULTURE MEDIUM
The selected strains were multiplied on Malt Extract Agar (MEA) and Potato
dextrose agar medium (PDA) at 30oC. The composition of culture media used during
study is given as:
Malt Extract Agar (MEA)
Malt extract = 20 gm
Dextrose = 20 gm
Agar = 20 gm
Peptone = 1 gm
Distilled water = to make volume one liter
Potato Dextrose Agar (PDA)
Potato starch = 20 gm
Dextrose = 20 gm
Agar = 20 gm
Distilled water = to make volume one liter
The medium was sterilized in autoclave at 15 psi at 121oC for 30 minutes, and
then poured in clean test tubes. Streptomycin was added in the sterilized medium at
the rate of 1 g/L. When the medium was cooled to 40oC, the strains were inoculated
43
into above mentioned media in test tubes. Test tubes were incubated at 30oC for
seven days. From these mother cultures then spawn was prepared. For the spawn
preparation sterilized and autoclaved cotton waste was used. The prepared spawn of
each strain was then used for the cultivation of mushroom.
3.4. SPAWN PREPARATION
Spawn was prepared from the multiplied cultures of five strains of V.
volvacea. The spawn of each strain was prepared on cotton waste in clean and
autoclaved jam bottles. Along with the cotton waste supplementation of Gram flour
was also used in the spawn preparation. The bottles were covered with aluminum foil
and kept at room temperature. The cooled spawn of V. volvacea was used for the
cultivation of the mushroom.
3.5. PREPARATION OF SUBSTRATE
The following six substrates were used in the experiment:
1. Cotton waste (By-product of textile industry)
2. Paddy straw (Agricultural waste with no economic supplement use)
3. Banana leaves (Agricultural waste with no economic supplement use)
4. Sugarcane baggase (Agricultural waste with no economic supplement use)
5. Corn Stovers (Used as fodder for animals)
6. Pulses straw (Agricultural waste with no economic supplement use)
Paddy straw, lentil straw and corn stovers were collected from the field.
Cotton waste, a byproduct of textile mills was collected from Faisalabad industrial
area. Pulses (lentil) straw, paddy straw, corn stovers and sugarcane baggase were
44
collected from university research area. All these agro-wastes except cotton waste
were chopped into small pieces with the help of fodder cutter. These chopped agro-
wastes and cotton waste were soaked in to water for 24 hours and then placed on
cemented floor, to remove the excessive water from the wastes to keep the moisture
up to 70%.
Banana leaves were collected from the University of Agriculture Faisalabad.
These leaves were also chopped with help of fodder cutter. These chopped leaves
were soaked in water for 4 hours and then drained later. Lime was mixed @ 5% to
each substrate on dry weight basis. Later on these substrates were covered with
polythene for four days for fermentation. Then five beds were made of each substrate
and each bed was of 3 kg weight on dry weight basis of the substrate.
3.6. SPAWNING
The seven days old, prepared spawn of each strain was then inoculated on all
the substrates. Five replications were made of each substrate and each replication
was of 3 kg, of agro-waste on dry weight basis. Gram flour was dusted on each layer
of the beds to provide the instant energy to fungus for growth. Each bed was again
covered with polythene sheath from all sides till the completion of spawn running.
3.7. REGULATION OF TEMPERATURE, HUMIDITY AND LIGHT
For spawn running and fructification of Chinese Mushroom, the
temperature of 300C for spawn running and fruiting body formation was required.
The average relative humidity was required above 80 %. The temperature and
humidity are two critical factors for the spawn running and fructification of the
45
mushroom. Efforts were made to maintain the required temperature and humidity by
wetting the flour, thrice in a day.
For 10-15 days spawn running room was kept dark but on completion of
spawn running light 50-250 lux (White fluorescent) was provided daily for eight
hours. Moreover, fresh air wash was given to growing room by exhaust fan to
lower the CO2 below 1000 ppm after every 3-4 hours.
3.8. WATERING
Usually no watering is required during spawn running. However, when the
beds were exceptionally dry, slight watering was done before the appearance of first
fruiting bodies. After this beds were just kept moist by watering.
3.9. EVALUATION OF PRODUCTIVITY OF FIVE STRAINS OF
CHINESE MUSHROOM
Two crops were grown on six substrates during the months of June, July
and August. Five strains of V. volvacea were grown on six substrates individually
and growth was recorded in terms of spawn running time, the time taken by the
mushroom for pinhead formation, time taken for fruiting body formation and yield
in grams of the fruiting bodies of different strains on all substrates from three
flushes.
3.10. COMPARISON OF THE MUSHROOM GROWTH ON
DIFFERENT SUBSTRATES
In this experiment the growth of different strains of Chinese mushroom
(Volvariella volvacea) was compared on six substrates i.e., cotton wastes, paddy
46
straw sugarcane baggase, banana leaves, corn stovers and lentil straw. The
comparison among the substrates was made by calculating the average yield
obtained from each substrate. These substrates were sterilized and inoculated as
mentioned above and all strains were used for comparison of growth.
3.11. DATA RECORDED
The following data was recorded during the experiments:
i) Days for completion of spawn running
Time was recorded in days for completion of 50% and then 100% spawn running
on each substrate by the five strains of V. volvacea
ii) Days for the pinhead formation.
Time was recorded in day for the pinhead formation by each strains on the
substrates used in the experiments.
iii) Fruiting body formation
Time was recorded in days for the fruiting body formation by the fungal strains on
each substrate.
3.12. YIELD DATA
Mushroom harvesting was done at maturity in three flushes of the five strains.
Yield data were recorded by weighing the fresh mushroom fruiting bodies harvested
from the substrates in three flushes.
47
3.13. pH OF THE SUBSTRATE
The samples of the substrate from different treatments were taken before
inoculation of spawn, after spawn running and termination of crop. Five grams of
each sample were taken from each treatment at different growth phases of the
mushroom, in 250 ml conical flasks. Flasks were placed as such for 24 hours for
complete saturation of distilled water in each sample, pH of filtrate was determined
with the help of pH meter.
3.14. BIOCHEMICAL ANALYSIS OF DIFFERENT SUBSTRATES
USED FOR THE CULTIVATION OF VOLVARIELLA SPP. AND
FRUITING BODIES OF MUSHROOM.
The freshly harvested mushrooms harvested from six different substrates
were analyzed for moisture, crude protein (N X 6.25), crude fiber and ash
contents, (AOAC, 1990).
3.14.1. DETERMINATION OF MOISTURE
A sample of 5g fresh mushrooms was taken in an aluminum dish and
placed in an air oven at 105oC for 24 hours for drying till constant weight. The
following formula was used to calculate the percentage of moisture.
Wt. of original sample – wt of dried sample (g) Moisture (%) = x 100 Wt. of original sample (g)
48
3.14.2. ESTIMATION OF CRUDE FAT (ETHER EXTRACT)
For crude fat estimation, 3g of dried mushroom samples were taken in a
thimble and put in an extraction tube of Soxhlet apparatus. The temperature of the
heater was so adjusted that continuous drops of ether fell on the sample in the
extraction tube. The process of extraction was carried out with petroleum ether
(B.P. 40-60oC) for 16 hours. The sample was removed and the solvent was
allowed to evaporate under the fume hood. The extract was completely dried in an
air oven for 30 minutes at 105oC. The weight of the extract was recorded after
cooling in a desiccator. Crude fat was calculated with the help of following
formula:
Wt. of fat in sample (g) Crude fat (%) = ___________________________ x 100
Wt. of sample (g)
3.14.3. ESTIMATION OF CRUDE PROTEIN
The nitrogen present in the samples was determined using the Kjeldhal
method.
Procedure for Digestion
2.5g of dried mushroom sample were digested with 25ml concentrated
H2SO4 and 5g of digestion mixture (CuSO4 9: K2SO4 90: FeSO4 1) till transparent
digested material was obtained. The digested material was diluted and 10ml of it
were distilled with 40% NaOH in a micro-Kjeldhal apparatus. The ammonia, thus
liberated was condensed and collected in 4% boric acid solution containing methyl
49
red as an indicator. The distillate was titrated against standard N/10 H2SO4
solution taken in the burette until golden yellow end point.
Calculation
ml of N/10 H2SO4 x 0.0014 x volume/dilution Nitrogen (%) = x 100 Wt of sample x ml of digested material used
Protein (%) = N% x 6.25
3.14.4. DETERMINATION OF CRUDE FIBER
Three grams dried and fat free mushroom sample was taken in a 1000ml
capacity beaker and 200ml of 1.25% H2SO4 were added to it and the level of
beaker was marked. The contents of the beaker were boiled for 30 minutes with
constant stirring; also the level of the water was supplemented, contents were
filtered giving 2-3 washings with hot water (150ml) until it was acid free. The
residue was transferred to a 1000ml beaker again and 200ml of 1.25% NaOH were
added into it. The contents were again boiled for 30 minutes and made up volume
during boiling. The contents were filtered and 2-3 washings with hot water were
given until alkali free. The residue was carefully transferred to a tared crucible and
dried in an oven at 100oC for 3-4 hours until constant weight was obtained. The
contents were heated on oxidizing (blue) flame until the smoke ceased to come out
of the sample. Then the sample was placed in a muffle furnace at 550oC for 4 h
until a grey ash was obtained, then cooled in a desiccator and weighed. The
difference in weight was reflected as crude fiber as calculated by using the
following formula:
50
Loss of weight after ignition (g) Crude fiber (%) = x 100 Weight of sample (g)
3.14.5. ESTIMATION OF ASH
Procedure
Three grams of dried mushroom sample were taken in a crucible and heated
on oxidizing flame till smoke subsided. The crucible was transferred to muffle
furnace at 550oC for 6 hours. The sample was cooled in a desiccator and weighed.
The ash in the sample was calculated as under.
Wt of Ash in sample (g) Ash % = x 100 Wt of the sample (g)
3.15. LIQUID STATE FERMENTATION
Liquid fermentation of the mushroom was carried out and for this
experiment only one strain of Volvariella volvacea (VvPk.) was used. Three agro-
wastes, Cotton waste, paddy straw and pulses straw, in powdered form were used
to make three different liquid media. The mycelial biomass production was
calculated in each liquid medium. The effect of pH and temperature variations on
the mycelial growth of V. volvacea strain (Vvpk) in three different liquid media
was also studied.
51
3.15.1. PREPARATION OF THE GROWTH MEDIUM
The liquid medium was prepared as described by Kadiri, (1998). The basal
medium contained, FeSO4.7H2O (0.01 g), MgSO4.7H2O (0.05 g), KH2PO4 (0.05
g), KCl (0.05 g), yeast extract (2.50 g), KNO3 (1.55 g) and D-glucose (10.0g) in
1000 ml of distilled water. The 30 ml of prepared liquid medium was poured into
each of the 250 ml flasks. Powder of dried cotton waste, paddy straw and pulses
straw was prepared. Twenty grams of each powdered agro waste was added in the
basal medium to make three different liquid media. Three replications were made
of each liquid medium.
3.15.2. EFFECT OF pH ON THE MYCELIAL GROWTH OF
V. VOLVACEA
In this experiment 30 ml of the basal medium was taken in 250 ml flasks.
Three different groups of these flasks were made by adding three powdered agro
waste in each group. Each group of flasks containing basal medium with agro
waste had three replications. The medium in the flasks was adjusted at six
different pH levels of 3.0, 4.0, 5.0 6.0 7.0 and 8.0 by using 1M NaOH or 1 M HCl
that was added in drops Each pH medium was dispensed in 100 ml conical flask
and all flasks were autoclaved at 121°C for 15 min and 30mg /100ml of
streptomycin was added to each flask to suppress the bacterial growth. These
flasks were then inoculated with 5 days old culture of V. volvacea strain V.v pk.
52
These flasks were incubated for 7 days at 30°C on rotary shaker at 100 rpm.
Mycelial dry weight and pH of the culture filtrate were determined.
3.15.3. EFFECT OF TEMPERATURE ON THE MYCELIAL
GROWTH OF V. VOLVACEA.
Same experiment was repeated as described above. But in this experiment the
pH of the medium was maintained 7.0 and different temperature for the incubation of
the medium were used to determine the optimum temperature for mycelial growth.
The basal medium employed in this experiment was prepared according to Kadiri,
(1998). The basal medium contained all the components in the same proportion as
stated above. These flasks were incubated for 7 days at 20, 30, 40 and 50°C
temperatures. Each treatment was replicated three times. Means of readings from
three replicates were determined and subjected to analyses of variance.
3.16. MOLECULAR CHARACTERIZATION OF VOVARIELLA
VOLVACEA
3.16.1. DNA EXTRACTION
The following protocol was adopted for DNA extraction:
1- A few mycelial threads were multiplied in 1.5 ml eppendorf tubes
containing 500 µl of liquid potato dextrose agar medium and allowed to
grow for 72 hrs at 25 °C.
2- The mycelial mat was centrifuged for 5 min at 10000 rpm, washed with 500
µl of TE buffer peleted again.
53
3- The buffer was decanted and 300 µl of extraction buffer (200 mM Tris
HCL, pH 8.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS) was added.
4- The Mycelium of each isolate was crushed with a conical grinder for few
min following the addition of 150 µl of 3 M Sodium acetate (pH 5.2).
5- Tubes were placed at –20oC for 10 min and centrifuged.
6- The supernatant was transferred to another tube. An equal volume of
isopropanol was added.
7- After 5 min, the precipitated DNA was pelleted by centrifugation.
8- The pellet was washed with 70% ethanol, vacuum dried and dissolved in 20
µl TE buffer.
3.16.2. DNA QUANTIFICATION
Concentration of DNA
The concentration of total genomic DNA was measured by DNA Quant
flourometer (Hoefer Dyna Quant TM 200, San Francisco, USA) using Hoechst
33258 dye. This method measured the concentration of DNA based on the
fluorescence of Hoechst 33258 dye when it bound to DNA.
Quality of DNA
The quality of DNA was checked by running 5 ul DNA on % 8 agarose gel
prepared in 0.5X TBE buffer. The DNA samples giving smear in the gel were
rejected.
54
Dilution of DNA
Dilutions of 15 ng/µl were made from stock solutions. The dilutions were
also checked by comparing them with the DNA quantification standards in
agarose gel.
3.16.3. RAPD ANALYSIS
PCR Amplification
PCR was performed in 0.2 µl PCR tubes with total volumes of 25 µl
containing the following reagents:-
Reagents Concentration Volume
Template DNA (15 ng/) 2.5 µl dNTPs (2.5mM) 4.0 µl Buffer (10 X) 2.5 µl Gelatin (0.001%) 2.5 µl Mg Cl2 (25 mM) 3.0 µl Primer (15 µl) 2.0 µl Taq polymerase (5 U/µl) 8.5 µl Total Volume 25 µl
A total of 15 random primers were surveyed as detailed in table. The
Oligionculeotide primers were purchased form Operon Technologies, Inc.
Alameda, CA. USA while Taq polymerase together with buffer, MgCl2 , dNTP’s
and Gelatin were arranged form Perkin Elmer, Norwalk USA.
55
3.16.4. Random sequences used for amplification
Sr. No. Primer code Primer sequence
1 GL DecamerA-009 CTACAGCCAC
2 GL DecamerA-006 GAAACGGGTC
3 GL DecamerA-004 GTGCTGTAGG
4 GL DecamerA-13 CAGCACCCAC
5 GL DecamerA-01 AGCCATCAAA
6 GL DecamerB-02 CATGGCGATG
7 GL DecamerB-01 TCGCTGGACT
8 GL DecamerB-13 TTCCCCCGCT
9 GL DecamerB-17 GACGTGTGTT
10 GL DecamerB-10 CCACAGCAGT
11 GL DecamerC-12 TTCGAGCCAG
12 GL DecamerC-03 TCGTAGTCTT
13 GL DecamerC-06 CGATGGACTC
14 GL DecamerC-07 CTCACCGTCC
15 GL DecamerC-16 TGCGAGCTTG
56
3.16.5. Condition optimization for RAPD analyses
DNA concentration in the working solution of approximately 15 ng/ ul in
d3 H2O were confirmed by spectrophotometer. For RAPD analyses (Williams et
al., 1990), concentration of genomic DNA, 10X PCR buffer with (NH4)2SO4, Mg
Cl2, dNTPs (dATp, dCTP, dGTP, dTTP). 10 mer random primer and Taq DNA
polymerase were optimized. The 10 base oligonucleotide primers obtained from
gene link company (USA) sere used for the amplification of DNA.
3.16.6. RAPD (PCR) Profile
Denaturation at 94 oC for 5 minutes
Denaturation at 94 oC for 1 minute
Annealing at 36 oC for 1 minute
Extension at 72 oC for 2 minutes
Total cycles 40
Final extension for 10 minutes
3.16.7. Agarose gel electrophoresis
The PCR products were electrophoresed at 80 V in 1.2 % agarose gel for
approximately 2 hours using 0.5 X Tris Boric acids EDTA (TBE) buffer along
with a DNA molecular size marker.
3.16.8. Agarose gel electrophoresis involved the following steps
1. An adequate volume of electrophoresis buffer i.e. 0.5X TBE buffer was
prepared to fill electrophoresis tank and to prepare the agarose gel.
57
2. Desired amount of agarose (1.2g) was taken in a flask, containing
electrophoresis buffer 0.5X TBE (100 ml), to melt in microwave oven for
three minutes and was swirled to ensure even mixing.
3. Casting tray was prepared by wrapping mashing tape on its both sides and a
suitable comb was adjusted on it.
4. The melted agarose was cooled to 55oC to 45 oC before pouring on to the
gel casting tray and Ethidium Bromide (3 ul) was added in to it.
5. Melted agarose poured on the gel-casting tray, gels typically pour between
the thicknesses of 0.5- 1.0 cm.
6. Bubbles were removed, if any, underneath the comb or on the surface of gel
and were solidified at room temperature.
7. After solidification of gel at room temperature the comb was removed
carefully to avoid tearing of wells. The mashing tape was also removed
from the sides of casting tray.
8. The gel-casting tray containing the gel was placed in electrophoresis tank,
having 0.5X TBE buffer in to it.
9. Before loading the PCR products in to the wells of gel, 5 ul of 6X Tracking
dye (Bromophenol blue) was added to the PCR products. Only 10 ul of
RAPD products were loaded on the gel. Marker of known molecular size
58
was loaded on both sides of the gel to compare the size of different
amplified fragments of genomic DNA.
10. The wires of electrophoresis tank were connected to power supply and were
adjusted to a certain voltage (50 V). Movement of dye indicated migration
of DNA from anode to cathode through gel.
The fingerprints were examined under ultra violet Trans illuminator and
photographed using the SynGene Gel Documentation System.
The data of the primers were used to estimate genetic similarity on the basis
of number of shared amplification products (Nei and Li, 1979). The coefficients
were calculated by the following statistical equation.
F= 2Nxy/ (Nx + Ny)
Where
F is the similarity coefficient in which Nx and Ny are the number of
fragments in population x and y, respectively, where Nxy is the fragment shared
by the two populations.
3.16.9. SCORING OF THE RAPD DATA
Good quality photographs were used to read the amplification profiles. The
first and last lane of the gel was loaded with DNA markers of known molecular
weight (1kb) provided from Fermentas USA. All visible scorable fragments
amplified by primers were scored under the heading of total scorable fragments.
The DNA fragments that were repeatedly present in one individual and absent in
59
the other were scored as polymorphic fragments. The presence of fragments was
denoted by 1 and absence by 0.The RAPD data was analyzed by using PopGen
software version 10.2.
3.17. Statistical analysis All the data were subjected to analysis of variance (ANOVA) technique and
all possible interactions were calculated. Duncan’s Multiple Range (DMR) test
was applied to separate significant differences among different strains and
substrate means (Duncan, 1955).
60
Chapter 4 RESULTS 4.1. SOLID STATE FERMENTATION
The mean squares values showed the different growth stages such as spawn
running, pinhead formation, fructification and ultimate yield of the five strains of
Volvariella volvcea on six different substrates i.e., cotton waste, paddy straw,
banana leaves, sugarcane baggase, corn stovers and pulses straw.. The individual
effect of crop, strains and substrates on the yield and different growth phases of
the mushroom was significant. Two way interaction between crops and strains,
crops and substrates was non significant, the difference in the growth and yield of
all the strains between the two crops was not significant statistically. The two way
interaction between strains and substrates was significant, both they had
significant effect on the yield and growth phases of the mushroom The yield of the
strains was significantly affected by the different substrates used in the
experiment. The three way interaction among the crops, strains and the substrates
was non significant, (Table1).
61
Table 1: Analysis of variance table of growth of different strains of Volvriella volvacea on different substrates (mean squares).
Mean squares Source of variation
Degrees of
freedom Yield
(g) Pinhead
time (Days)
Fruiting time
(Days)
50% spawn (Days)
100% spawn (Days)
Crops 1 5799.2** 3.6300* 1.6133* 9.0133* 8.6700**
Stains 4 151237.9** 7.5300** 2.2783** 65.1883** 24.1750**
Treatment 5 1685599.4** 1.8513** 0.5173* 6.5733** 2.6673**
Crops x Stains
4 9.4 NS 0.0467 NS 0.0717 NS 0.0550 NS 0.2617 NS
Crops x Treatment
5 10.0 NS 0.0220 NS 0.1973 NS 0.0293 NS 0.1180 NS
Stains x Treatment
20 1890.3** 0.7880** 0.2423 NS 0.3383 NS 0.4190 NS
Crops x Stains x Treatment
20 24.1 NS 0.0287 NS 0.0357 NS 0.0410 NS 0.0897 NS
Error 240 86.0 0.2417 0.2250 0.3733 0.2767 NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01) 4.1.1. SPAWN RUNNING
The mean values indicated the effect of the substrates on 50% spawn
running of the strains, (Table2). The data showed that maximum average time was
taken for 50% spawn running both on pulses straw, (6.88 days) and sugarcane
baggase (6.68 days), substrates by all the strains. The minimum average time was
taken on paddy straw (5.92 days) followed by the cotton waste (6.0 days), banana
leaves (6.08) corn stovers (6.30 days). All the strains showed the efficient spawn
running on paddy straw and cotton waste. The difference of the time taken for
spawn running between cotton waste and paddy straw substrates was not
62
significant and difference of time for 50% spawn running was also non significant
between the sugarcane baggase and pulses straw, (Table2).
The comparison among the strains for the 50% spawn running between two crops
showed that, strain Vv pk(local) showed the fastest growth and took minimum
time (5 days), for 50% spawn running, followed by the strain Vv 430, (6.95 days),
strain Vv 428, (5.77 days) and strain Vv 436, (5.97 days), while strain Vv 422
showed the slowest growth and took maximum time (7.72 days), for 50% spawn
running.
Table 2: Effect of substrates on 50% spawn running of the strains.
Strains Days Treatment
Vv(PK) Vv436 Vv428 Vv430 Vv422
Mean (Crop x Treat)
Cotton waste 4.70±0.153 5.50±0.167 5.40±0.163
6.80±0.249
7.60±0.163
6.00±0.169 C*
Paddy straw 4.90±0.180 5.50±0.224
5.40±0.163
6.40±0.163
7.40±0.163
5.92±0.148 C
Banana Leaves
4.80±0.200 5.80±0.200
5.60±0.221
6.60±0.221
7.60±0.163
6.08±0.161 BC
Corn stovers 4.70±0.213 6.10±0.180
5.90±0.233
6.90±0.180
7.90±0.100
6.30±0.172 B
Sugarcane baggase
5.70±0.213 6.40±0.221
6.00±0.298
7.50±0.167
7.80±0.200
6.68±0.152 A
Pulses straw 5.60±0.163 6.50±0.224 6.30±0.153 7.50±0.167 8.00±0.149 6.78±0.144 A
LSD 0.24
*Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). * CW=Cotton waste, PS=Paddy straw, BL=Banana leaves, CS=Corn stover, S.Baggasse=Sugarcane baggase, PS=Pulse straw
63
The difference for the 50% spawn running among the strains Vvpk, Vv430 and Vv
422 was significant while this difference was non significant between the strain
Vv436 and Vv 428.The time taken for 50% spawn running by the crop 1 grown
during June- July was more than the crop 2 which was grown during the months of
July-August. The difference of time between two crops was significant, (Table 3).
Table 3: Comparison of 50% spawn running of strains between two crops.
Crop Days
Strains
1 2
Mean
Vv(PK) 5.20±0.139 4.93±0.117
5.07±0.092 D
Vv436
6.17±0.145
5.77±0.114
5.97±0.095 C*
Vv428
5.97±0.140
5.57±0.114
5.77±0.093 C
Vv430
7.10±0.139
6.80±0.121
6.95±0.093 B
Vv422 7.90±0.088 7.53±0.093
7.72±0.068 A
Mean 6.47±0.096 A 6.12±0.091 B
LSD = 0.21
*Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).
The data for the effect of the substrates on 100% spawn running showed
that maximum time was taken by the strains for 100% spawn running (5.50 days),
both on pulses straw and corn stovers, and slowest spawn running on these
substrates, while minimum time was taken for 100% spawn running on cotton
waste by all the strains followed by the paddy straw (5 days), sugarcane baggase
(5.18 days) and banana leaves (5.30 days).This difference of time was non
64
significant between the corn stovers and pulses straw but the difference was
significant among the other substrates. The comparison for the 100% spawn
running time taken by the strains between two crops showed that, strain Vv
pk(local) and strain Vv436 showed the fastest growth and took (4.65) and (4.72)
days respectively, for 100% spawn running, followed by the Vv 428 (5.0), Vv
430, (5.57 days). The strain Vv 422 took (6.15 days) and showed the slowest
growth and took maximum time for 100% spawn running. The difference of time
taken by the strain Vv436 and Vv (pk) was non significant but this difference was
significant among the strains Vv 430, vv428 and Vv422. Mean values showed that
time taken for 100% spawn running by the crop 1 grown during June- July was
more than the crop 2 which was grown during the months of July-August. The
difference of 100%spawn running time between the two crops was significant,
(Table 5).
65
Table 4: Effect of substrates on 100% spawn running of the strains Strains
Days Treatment Vv(PK) Vv436 Vv428 Vv430 Vv422
Mean (Crop x Treat)
Cotton waste 4.20±0.133 4.40±0.163 4.80±0.133 5.30±0.153 5.70±0.153 4.88±0.102 D Paddy straw 4.40±0.163 4.50±0.167 4.70±0.153 5.70±0.153 5.90±0.100 5.04±0.111 CD
Banana Leaves 4.80±0.249 4.80±0.291 4.70±0.213 5.70±0.213 6.50±0.167 5.30±0.141 AB Corn stovers 5.00±0.211 4.80±0.200 5.40±0.163 5.60±0.163 6.20±0.133 5.40±0.103 A
Sugarcane. baggase 4.70±0.153 4.60±0.163 4.80±0.133 5.50±0.167 6.30±0.153 5.18±0.113 BC Pulses straw 4.80±0.133 5.20±0.133 5.60±0.163 5.60±0.163 6.30±0.153 5.50±0.096 A LSD 0.20
Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). * CW=Cotton waste, PS=Paddy straw, BL=Banana leaves, CS=Corn stover, S.B=Sugarcane baggase, PS=Pulse straw
66
Table 5: Comparison of spawn running of strains between two crops
Crop Days Strains
1 2 Mean
Vv(PK) 4.87±0.115 4.43±0.092 4.65±0.078 D* Vv436 4.97±0.122 4.47±0.093 4.72±0.083 D
Vv428 5.17±0.108 4.83±0.108 5.00±0.079 C Vv430 5.70±0.098 5.43±0.092 5.57±0.069 B Vv422 6.23±0.092 6.07±0.095 6.15±0.066 A
Mean 5.39±0.063 A 5.05±0.066 B LSD = 0.18
*Means sharig similar letter in a row or in a column are statistically non-significant (P>0. 4.1.2. PINHEAD FORMATION The data for the effect of substrates on the pinhead formation by the strains of the
mushroom showed that strain Vv (pk) took minimum time (2.50 days) on cotton
waste and showed the fastest growth, followed by paddy straw, (3.36 days), corn
stovers (3.16 days), pulses straw (3.42 days), banana leaves (3.48 days), while it
took maximum time for pinhead formation on sugarcane baggase (3.50 days). The
strain Vv 436 took minimum time (2.70 days) on cotton waste and maximum time
on sugarcane baggase (3.50 days) for pinhead formation. The strain Vv 428 took
minimum time on cotton waste 2.80 days and maximum time on pulses straw and
banana leaves (3.70 days). The strain Vv 430 took minimum time on cotton waste
3.30 days and maximum time on sugarcane baggase, pulses straw and banana
leaves (3.70 days). The strain Vv422 took minimum time on banana leaves (3.50
days) and maximum time on paddy straw 4.0. Among all the strains, strain Vv4pk
was the fast growing strain, which took minimum time for pinhead formation on
67
cotton waste (2.50 days), while strain Vv422 was the slowest growing strain which
took maximum time for pinhead formation on paddy straw (4.00 days). The mean
values of the table showed that minim time taken for pinhead formation was taken
on cotton waste (3.02 days) and corn stovers (3.16 days) as compared to the other
substrates used for the cultivation. Among the substrates the cotton waste and corn
stovers promoted the pinhead formation of the mushroom as compared to the other
substrates. The difference of time for pinhead formation by the strains on banana
leaves, pulses straw, sugarcane baggase and paddy straw was found to be non
significant and this time difference was also non significant between the cotton
waste and corn stovers, (Table 6).
The comparison between the strains for the time taken for pinhead
formation, showed that strain Vv pk, (local strain) was more efficient and took
minimum average time (2.87 days) followed by the strain Vv 436 which took
(3.13 days), Vv 428 which took (3.28 days), Vv 430 took (3.57 days) and
maximum average time was taken by the strain Vv 422 which was (3.77 days)
with lowest efficiency among all the strains. The difference of time taken for
pinhead formation by the strain Vv436 and Vv 428 was non significant, while the
difference of time taken for pinhead formation among the strain Vv(pk), Vv430
and Vv422 was significant. The mean values fro the two crops showed that crop 2
took minimum average time (2.48 days) than crop 1, 2.63 days. The difference of
time taken for pinhead formation by the mushroom between two crops was
significant, (Table 7).
68
Table 6: Effect of substrates on the pinhead formation of strains of V.volvacea
Strains Days Treatment
Vv(PK) Vv436 Vv428 Vv430 Vv422
Mean (Crop x Treat)
Cotton waste 2.50±0.167 g
2.70±0.153 fg
2.80±0.133 fg
3.30±0.153 b-e
3.80±0.133 ab
3.02±0.093 B*
Paddy straw 2.70±0.153 fg
3.30±0.153 b-e
3.30±0.153 b-e
3.50±0.167 a-d
4.00±0.000 a
3.36±0.085 A
Banana Leaves 2.80±0.133 eg
3.70±0.153 abc
3.70±0.153 abc
3.70±0.153 abc
3.50±0.167 a-d
3.48±0.082 A
Corn stovers 2.90±0.100 efg
2.80±0.133 fg
2.70±0.213 fg
3.50±0.167 a-d
3.90±0.100 aa
3.16±0.092 B
Sugarcane baggase
3.20±0.200 c-f
3.50±0.167 a-d
3.50±0.167 a-d
3.70±0.153 abc
3.60±0.163 a-e
3.50±0.077 A
Pulses straw 3.10±0.100 def 2.80±0.200 fg 3.70±0.153 abc 3.70±0.153 abc 3.80±0.133 ab 3.42±0.086 A
LSD 0.19
*Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.
69
Table 7: Comparison between the two crops for pinhead formation
Crop Days Strains
1 2 Mean
Vv(PK)
3.00±0.083
2.73±0.095
2.87±0.065 D
Vv436
3.27±0.106
3.00±0.117 3.13±0.080 C*
Vv428
3.40±0.113
3.17±0.118
3.28±0.083 C
Vv430
3.67±0.088
3.47±0.093
3.57±0.065 B
Vv422
3.83±0.069 3.70±0.085
3.77±0.055 A
Mean 2.63±0.040 A 2.48±0.041 B LSD =0.17 *Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.
4.1.3. FRUITING BODY FORMATION
The data for the effect of substrates on the fructification time showed that
minimum average time was taken by strains for the fructification on cotton waste
(2.44) days, paddy straw and the pulses straw also took almost same time (2.48
days) and (2.50 days) respectively after cotton waste. The difference for the
fructification time between the cotton waste, paddy straw and pulses straw was not
significant. The sugarcane baggase showed minimum efficiency and took
maximum average time (2.72 days) for fructification by the strains. Banana leaves
and corn stovers took same time (2.60 days) and (2.58 days) respectively for the
fruiting bodies formation of mushroom, (Table 8).
The comparison among the strains for fructification time showed that,
strain Vv pk (local) showed best growth and took minimum time for fruiting
70
bodies formation which was (2.32 days) followed by the strain Vv 436 which took
(2.40 days), strain Vv428 took (2.57 days) and strain Vv430 took (2.72 days) for
fructification. Maximum time was taken by the strain Vv 422 which took (2.77
days) and showed lowest efficiency among all the strains. The mean values of
table for the comparison between the two crops showed the significant difference.
The data showed that less time was taken by the strains for fruiting body formation
during the crop 2, which was (2.48 days) as compared to the crop 1(2.630 days),
(Table 9).
Table 8: Effect of substrates on fruiting body formation
Strains
Days Treatment
Vv(PK) Vv436 Vv428 Vv430 Vv422
Mean (Crop x Treat)
Cotton waste 2.20±0.133 2.20±0.133 2.50±0.167 2.70±0.153 2.60±0.163 2.44±0.071 B*
Paddy straw 2.10±0.100 2.30±0.153 2.40±0.163 2.70±0.153 2.90±0.100 2.48±0.071 B
Banana Leaves
2.50±0.167 2.40±0.163 2.60±0.163 2.70±0.153 2.80±0.133 2.60±0.070 AB
Corn stovers 2.50±0.167 2.30±0.153 2.70±0.153 2.60±0.163 2.80±0.133 2.58±0.071 AB
Sugarcane baggasse
2.30±0.153 2.70±0.153 3.00±0.000 2.80±0.133 2.80±0.133 2.72±0.064 A
Pulses straw 2.30±0.153 2.50±0.167 2.20±0.133 2.80±0.133 2.70±0.153 2.50±0.071 B
LSD 0.18
*Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.
71
Table 9: Comparison for the fruiting body formation between two crops.
Crop Days Strains
1 2 Mean
Vv(PK)
2.43±0.092
2.20±0.074
2.32±0.061 D*
Vv436
2.50±0.093
2.30±0.085
2.40±0.064 CD
Vv428
2.63±0.089
2.50±0.093
2.57±0.065 BC
Vv430 2.77±0.079 2.67±0.088 2.72±0.059 AB
Vv422 2.80±0.074 2.73±0.082 2.77±0.055 A
Mean 2.63±0.040 A 2.48±0.041 B
LSD =0.17
*Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.
4.1.4. YIELD
The effect of the substrates on the yield of the strains showed that highest
average yield of the mushroom was obtained from the cotton waste (734.3g),
followed by the paddy straw (590.8g), banana leaves (483.2g), pulses straw
(385.4g), sugarcane baggase, (297.2g) and corn stover, (251.0 g), respectively.
The highest yield was obtained from the strain Vv pk (821g), on cotton waste
while lowest yield was obtained by the strain Vv 422 on the corn stovers. The
individual yield data of the strain Vv pk showed that it produced highest yield on
the cotton waste (821g), followed by the paddy straw (651.4 g), banana leaves
(547.3g), pulse straw (439.1g), sugarcane baggase, (318.3g), and lowest yield was
obtained from the corn stovers, (291.4g). The strain Vv 436 produced maximum
72
yield on cotton waste (769.4g) followed by the paddy straw (623.9g), banana
leaves, (523.5g), pulses straw, (417.3g), sugarcane baggase, (343.1g), and lowest
yield was obtained on the corn stovers, (287.2g). The strain Vv 428 produced
highest yield on cotton waste, (734g), and lowest yield was obtained from corn
stovers, (226g). The strain Vv430 produced highest yield on the cotton waste,
(969g) while the strain Vv 422 maximum yield up to (650g), on cotton waste
while lowest yield was obtained from corn stovers, (192.2 g), (Table 10).
Table10: Effect of substrates on the yield of the strains
Strains Fresh weight (g)
Treatment Vv(PK) Vv436 Vv428 Vv430 Vv422
Mean (Crop x
Treat)
Cotton waste
821.0±5.09 a 769.4±3.91 b 734.1±4.08 c 696.1±2.64 d 650.7±2.75 e 734.3±8.54 A
Paddy straw
651.4±2.93 e 623.9±3.16 f 591.7±2.20 g 567.1±3.21 h 520.1±1.99 j 590.8±6.60 B
Banana Leaves
547.3±3.07 i 523.5±2.23 j 496.2±3.12 k 443.7±3.53 l 405.1±2.34 n 483.2±7.54 C
Corn stovers
291.4±2.43 s 287.2±3.77 s 257.0±1.89 u 226.5±2.82 v
192.9±3.83 w 251.0±5.49 F
Sugarcane baggase
318.3±2.81 r 343.1±4.37 q 319.4±3.61 r
273.6±2.69 t 231.7±2.72 v 297.2±5.85 E
Pulses straw
439.1±2.23 l 417.3±2.84m 386.3±3.90 o 361.0±2.75 p 323.4±3.38 r 385.4±5.98 D
LSD 3.65
Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.
The comparison of the yield between the strains during two crops showed
that maximum average yield was produced by the strain Vv pk on cotton waste
(511.4g), while minimum yield was produced by the strain Vv422, (387g), strain
73
Vv 436, (494.1g), Vv 428 (464.1g), Vv430 (428g). The comparison between two
crops for yields showed the significant difference, (Table11).
Table 11: Comparison of the yield of the strains between two crops
Crop Fresh weight (g) Strains
1 2 Mean
Vv(PK) 506.6±34.39 516.2±34.78
511.4±24.25 A
Vv436
489.5±31.20 498.7±30.49 494.1±21.63 B
Vv428 460.1±30.18 468.1±30.57 464.1±21.30 C
Vv430
424.1±30.35
431.9±30.45
428.0±21.32 D
Vv422 382.7±29.72 391.9±29.73
387.3±20.85 E
Mean 452.6±14.25 B 461.4±14.27 A
LSD =3.33
Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.
74
4.2 pH OF THE SUBSTRATES AT DIFFERENT GROWT STAGES
The individual effect of different growth stages of the mushroom and the
strains and substrates on the pH change after inoculation and after termination of
the fruiting bodies was significant. The two way interaction between the different
growth stages and the strains was also significant. The two way interaction
between the growth stages and the substrates was also significant. The two way
interaction between the strains and the treatments and three way interaction among
the growth stages, strains and treatments for the change in pH values was non
significant, (Table 12).
Table 12: Analysis of variance for pH of substrates at different growth stages.
Source of variance Degrees of freedom Mean squares F value
Growth stage (G)
Strain
G x Strain
Treatment
G x Treatment
Strain x Treatment
G x Strain x Treatment
Error
2
4
8
5
10
20
40
180
13.1371
0.3168
0.0858
26.5351
0.2548
0.0034
0.0041
0.0032
4163.6664**
100.4052**
27.1977**
8410.0006**
80.7669**
1.0743 NS
1.2936 NS
NS = Non-significant (P>0.05); ** = highly significant (P<0.01)
75
The effect of the strains on the pH of different growth stages showed that
strain Vvpk had more effect on the pH of the substrates (6.57) as compared to the
other strains. The strain Vv422 showed little effect on the pH change among all
the strains, (Table 13)
The data showed that all the substrates had significantly different pH values
before inoculation of the spawn which decreased in all the substrates as the growth
of the mushroom proceeded. The pH value of all the substrates decreased
gradually after spawn running and became minimum after termination of the
fruiting bodies of the mushroom.
Table 13: Effect of strains on the pH at different growth stages on cotton waste
Growth Stage Strain Before
inoculation After spawn
running After
termination Mean
Vv(PK)
6.89±0.144 a
6.52±0.185 b
6.32±0.174 e
6.57±0.101 A
Vv436
6.89±0.144 a
6.41±0.194 c
6.22±0.183 g
6.51±0.106 B
Vv428
6.89±0.144 a
6.36±0.196 d
6.12±0.173 h
6.46±0.107 C
Vv430
6.89±0.144 a
6.33±0.194 de
6.08±0.172 i
6.43±0.108 D
Vv422
6.89±0.144 a 6.26±0.198 f 5.97±0.176 j 6.37±0.112 E
LSD 0.021 Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.
The mean values of the growth stages showed the gradual decrease in pH,
the average pH value of all the substrates before inoculation was (6.89), which
gradually decreased after spawn running and became (6.38) and minimized after
76
the termination of the fruiting bodies of the mushroom and became 6.14. The pH
mean values of the substrates in the Table showed that maximum change was
found in case of cotton waste (7.14), followed by the paddy straw (7.05) and
minimum or less change was observed in pulses straw (5.62), (Table 14).
Table 14: pH of the substrates at different growth stages of the strain V. v pk.
Growth Stage Treatment Before
inoculation After spawn
running After
termination Mean
Cotton waste 7.76±0.003 a
7.39±0.023 b
7.08±0.022 d
7.41±0.043 A
Paddy straw 7.37±0.007 b
7.05±0.017 d
6.73±0.041 f
7.05±0.042 B
Banana Leaves 7.19±0.004 c
6.97±0.026 e
6.68±0.041 g
6.95±0.035 C
Corn stovers 6.57±0.045 h
6.04±0.019 j
5.79±0.036 k
6.13±0.053 D
Sugarcane baggasse
6.22±0.006 i
5.44±0.028 l
5.30±0.030 n
5.65±0.063 E
Pulses straw 6.22±0.008 i 5.38±0.038 m 5.26±0.028 o 5.62±0.066 F
Mean 6.89±0.063 A 6.38±0.085 B 6.14±0.078 C LSD =0.23
Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.
4.3. BIOCHEMICAL ANALYSIS OF THE SUBSTRATES
The analysis for the ash contents in the substrates at different growth stages
showed that, different phases of mushroom growth had significant effect on the
ash contents of the substrates and individual effect of the substrates on ash
contents was also significant. The two way interaction between the growth stages
and substrates was non-significant (Table 15).
77
Table 15: Analysis of variance table for ash contents
Source of variance Degrees of freedom Mean squares F value
Growth stages
Treatment
Growth stages x treatment
Error
2
5
10
36
26.971
367.300
2.266
1.562
17.2683**
235.1679**
1.4506 NS
NS = Non-significant (P>0.05); ** = highly significant (P<0.01)
The Ash contents were high in cotton waste (22.30%) followed by the
paddy straw (20.20%) and corn stover. Banana leaves, pulses straw and sugarcane
baggase had same ash contents. The ash contents were minimum before
inoculation (12.18%) which increased after spawn running (14.59%) and
ultimately decreased after termination of the fruiting bodies of the mushroom
(12.99%). The comparison among the substrates showed that difference of ash
among all the substrates was significant. The mean values showed that ash
contents of the substrates at different growth stages were also significantly
different, (Table 16).
78
Table 16: Ash contents in the substrates at different growth stages of the mushroom.
Growth Stage Before
inoculation After spawn
running After
termination Treatment Ash contents (%)
Mean
Paddy straw 18.74±1.04 23.05±2.73 18.82±0.36 20.20±1.11 B
Cotton waste 20.82±0.15 24.34±0.31 21.73±0.33 22.30±0.55 A
Banana Leaves 7.84±0.32 8.91±0.17 8.11±0.09 8.29±0.19 D*
Corn stovers 11.34±0.25 12.62±0.15 12.02±0.06 11.99±0.20 C
Sugarcane
baggasse 7.47±0.34 9.32±0.33 8.19±0.20 8.32±0.31 D
Pulses straw 6.90±0.07 9.30±0.05 9.06±0.04 8.42±0.38 D
Mean 12.18±1.37 B 14.59±1.64 A 12.99±1.31 B
*Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.
The individual effect of different growth stages on the crude fiber
percentage was significant. The fiber percentage was significantly different at each
growth stage of the mushroom. The individual effect of the substrates on fiber
percentage was also significant. The interaction between the different growth
stages and treatments for fiber percentage was also significant, (Table 17).
79
Table 17: Analysis of variance table for crude fiber percentage.
Source of variance Degrees of freedom Mean squares F value
Growth stages.
Treatment
Growth stages x treatment
Error
2
5
10
36
8.386
1120.413
1.670
0.054
156.4051**
20896.0167**
31.1392**
** = Highly significant (P<0.01)
The mean values of crude fiber percentage in the substrates at different
growth stages showed significant difference.The fiber percentage increased after
spawn running of the mushroom (30.89%) which gradually decreased after the
termination of the fruiting bodies (29.98%) which was comparatively higher than
the fiber percentage before inoculation. The mean values of the fiber percentage of
different substrates showed the significant difference among the substrates. The
highest crude fiber was found in cotton waste (42.36%), followed by the sugarcane
baggase (39.49%), rice straw (33.1%), corn stovers (30.88%), pulses straw
(22.76%) and lowest percentage of the fiber was found in the banana leaves,
(12.20%),(Table 18).
80
Table 18: Crude fiber percentage in the substrates at different growth stages of the mushroom.
Growth Stage Before
inoculation After spawn
running After
termination Treatment
Crude fibre (%)
Mean
Paddy straw 33.00±0.06j 33.31±0.06f
33.10±0.05g 33.14±0.05D
Cotton waste 41.62±0.17c
43.62±0.22a 41.86±0.05b
42.36±0.33A
Banana Leaves 12.45±0.17hij
12.87±0.03e
11.30±0.05e
12.20±0.24C
Corn stovers 28.90±0.37e
32.47±0.16d
31.28±0.09e
30.88±0.54B
Sugarcane baggasse
39.05±0.08k
39.99±0.05h
39.43±0.04hi
39.49±0.14E*
Pulses straw 22.3±0.11ij 23.08±0.03hi 22.91±0.04hij 22.76v0.12E
Mean 29.55±2.41C 30.89±2.50A 29.98±2.51B
*Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean. The data for the crude protein analysis showed that individual effect of
different growth stages and the treatments on the protein percentage was
significant. The two way interaction between the different growth stages and
substrates was also significant. (Table19).
Table 19: Analysis of variance table for crude protein percentage. Source of variance Degrees of freedom Mean squares F value
Storage days
Treatment
Storage days x treatment
Error
2
5
10
36
12.692
222.622
0.932
0.035
364.1828**
6388.0018**
26.7535**
** = Highly significant (P<0.01)
81
The data for the crude protein percentage showed that protein percentage
increased after the spawn running of the mushroom (10.94%) which was (9.28%)
before inoculation of spawn, but that percentage was decreased after the
termination of the fruiting bodies and became (10.35%). The mean values of the
protein percentage of the substrates showed that highest protein was present in the
cotton waste (20.00%) followed by the corn Stover (10.28%). Banana leaves
(8.96%), Paddy straw (7.98%), pulses straw (7.05%) and sugarcane baggase
(6.86%), (Table20).
Table 20: Crude protein percentage contents in the substrates at different growth stages of the mushroom
Growth Stage Before
inoculation After spawn
running After termination Treatment
Crude Protein (%)
Mean
Paddy straw 6.71±0.05k
9.19±0.05g
8.03±0.10h
7.98±0.36D
Cotton waste 19.17±0.06c
20.67±0.20a
20.17±0.05b
20.00±0.23A
Banana Leaves 7.09±0.09ij
10.09±0.06e
9.69±0.04f
8.96±0.47C
Corn stovers 9.85±0.08ef
11.02±0.07d
9.97±0.03ef
10.28±0.19B
Sugarcane baggasse
6.03±0.03l
7.40±0.10i
7.17±0.33i
6.86±0.23F
Pulses straw 6.83±0.05jk 7.24±0.04i 7.06±0.03ij 7.05±0.06E
Mean 9.28±1.11C 10.94±1.11A 10.35±1.10B
*Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.
82
The mean square values showed that individual effect of the growth stages
and the treatments on the nitrogen percentage of the substrates was significant.
The interaction between the different growth stages and treatments was also
significant, (Table 21).
Table 21: Analysis of variance table for nitrogen percentage. Source of variance Degrees of freedom Mean squares F value
Growth stages
Treatment
Growth stages x treatment
Error
2
5
10
36
0.367
4.282
0.081
0.023
15.6997**
183.4397**
3.4908**
** = Highly significant (P<0.01)
Table 22: Nitrogen percentage in the substrates at different growth stages of
the mushroom.
Growth Stage Before
inoculation After spawn
running After termination Treatment
N (%)
Mean
Paddy straw 1.59±0.19f
1.96±0.03de
1.85±0.04def
1.80±0.08C*
Cotton waste 3.22±0.10a
2.92±0.06b
3.09±0.04ab
3.08±0.06A
Banana Leaves 1.03±0.04h
1.26±0.05gh
1.02±0.02h
1.11±0.04E
Corn stovers 1.69±0.15ef
2.00±0.06d
1.71±0.15ef
1.80±0.08C
Sugarcane baggasse
1.05±0.05h
1.68±0.15ef
1.32±0.06g
1.35±0.10D
Pulses straw 1.90±0.07de 2.33±0.03c 2.06±0.04d 2.10±0.07B
Mean 1.75±0.18B 2.03±0.13A 1.84±0.16B
*Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.
83
The mean values of the nitrogen percentage of the substrates showed that it
was high in the cotton waste (3.80) followed by the pulses straw, paddy straw and
sugarcane baggase have same nitrogen percentage and minimum nitrogen was
found in the banana leaves, (Table 22).
4.4. BIOCHEMICAL ANALYSIS OF THE MUSHROOM FRUITING
BODIES
The biochemical analysis of the fruiting bodies obtained showed that, the
fruiting bodies harvested from different substrates had different nitrogen, crude
protein, ash and moisture percentage. The individual effect of the treatments on
these contents of the fruiting bodies was significant, (Table 23).
Table 23: Analysis of variance table (mean squares).
Mean squares Source of variation
Degrees of freedom N% Crude protein Ash % Moisture
% Crude fibre
Treatment
Error
5
12
12.657**
0.140
88.410**
0.290
43.348**
0.094
5.293*
1.569
10.129**
0.209
* = Significant (P<0.05); ** = Highly significant (P<0.01)
The nitrogen percentage of the fruiting bodies harvested from cotton waste
was higher (5.56%) followed by the paddy straw, (4.87%) sugarcane baggase
(4.45%).The fruiting bodies harvested from corn stovers and banana leaves had
almost same nitrogen percentage. The lowest percentage of nitrogen was found in
pulses straw (3.22%).The biochemical analyses of the fruiting body showed that
fruiting body harvested from cotton waste contained maximum protein (34.17%)
84
followed by the sugarcane baggase (30.51%), paddy straw (28.57%), banana
leaves (23.92%), corn srover (21.77%) and pulses straw (20.25%). The protein
percentage was significantly different in the fruiting bodies harvested from
different substrates.The ash %age was maximum in the fruiting bodies harvested
from cotton waste (10.8%), followed by the banana leaves(5.93%) paddy straw
(2.43%) corn stover (1.53%) pulses straw (2.05%) sugarcane baggase (1.96%).The
crude fiber %age was maximum in the fruiting bodies harvested from cotton waste
(11.9%) followed by the pulses straw (8.14%), sugarcane baggase and corn Stover
(7.22%) paddy straw and lowest fiber percentage was found in the fruiting bodies
harvested from banana leaves (7.88%). The difference in the moisture contents of
the fruiting bodies from all the substrates was not significant, (Table 24).
Table 24: Nitrogen, Crude protein, Ash, Moisture and Crude fiber percentage in the fruiting bodies of mushroom harvested from all substrates. Treatments N
(%) Crude protein
(%)
Ash (%)
Moisture (%)
Crude fiber (%)
Paddy straw 4.87±0.03 B 28.57±0.24 C 2.43±0.19 C 89.9±0.12 A 7.88±0.63 BC*
Cotton waste 5.65±0.04 A 34.17±0.29 A 10.8±0.37 A 89.3±0.22 A 11.9±0.11 A
Banana Leaves 3.72±0.13 D
23.92±0.12 D
5.93±0.12 B
88.6±0.21 A
7.34±0.07 BC
Corn stovers 3.54±0.04 D
21.77±0.07 E
1.53±0.03 D
88.2±0.54 AB
7.04±0.05 C
Sugarcane baggasse
4.45±0.04 C
30.51±0.64 B
0.96±0.03 E
86.0±1.59 B
7.22±0.04 C
Pulses straw 3.22±0.05 E
20.25±0.04 F 2.05±0.04 CD 88.1±0.46 AB 8.14±0.04 B
LSD 0.19 0.95 0.54 2.22 0.81 *Means sharing similar letters in a column are statistically non significant (P>0.05)
85
4.5. LIQUID FERMENTAION
The data for the filamentous protein production in the three different liquid
media at different temperatures showed that individual effect of liquid media, time
and different temperatures on myceilal production was highly significant. The two
way interaction between the liquid media and time, and two way interaction
between the time and temperature was also significant .Three way interaction
among the liquid media, time and temperature was also significant, (Table 25).
Table 25: Analysis of variance for protein production in Liquid medium at ` different temperatures.
Source of variance Degrees of freedom Mean squares F value
Medium
Days
Temperature
Medium x Days
Medium x Temperature
Days x Temperature
Medium x Days x Temperature
Error
2
5
3
10
6
15
30
144
9507.03
1729.39
867.76
507.28
216.29
35.36
10.29
1.07
8874.3082**
1614.2901**
473.5179**
810.0041**
201.8962**
33.0048**
9.6042**
** = Highly significant (P<0.01)
86
4.5.1. EFFECT OF INCUBATION TIME ON MYCELLIAL BIOMASS
PRODUCTION
The effect of time on the mycelial production in the three different liquid
media, the pH of which was kept at 7 and temperature at 30°C, the maximum
mycelial production was obtained at the seventh day of fermentation on the rotary
shaker at 100rpm. The difference in the yield of the sixth and seventh days was not
significant. On the other hand the data also showed that mycelial production was
high in the liquid medium containing cotton waste (38.62mg/30ml at the seventh
day of incubation), followed by the paddy straw (15.03mg/30ml) and lowest yield
was obtained from the pulses straw liquid medium (3.10mg/30ml). The yield
difference of three liquid media was significant. (Table26).
Table26: Effect of time on mycelial production in three liquid media Medium
Days Cotton waste (mg)
Paddy straw (mg)
Pulses straw (mg)
Mean
2 5.39±0.82 g
1.15±0.13 jk
0.51±0.11 k
2.35±0.46 E
3
11.68±1.22 e 4.16±0.47 h
1.15±0.14 jk
5.66±0.86 D
4
21.98±1.89 c 7.69±1.12 f
1.76±0.24 j
10.48±1.60 C
5
31.47±2.29 b
12.46±1.31 e
2.73±0.32 i
15.55±2.19 B
6
38.17±2.95 a
14.64±1.45 d
3.13±0.37 i
18.64±2.69 A*
7 38.62±3.05 a 15.03±1.51 d 3.10±0.40 i 18.92±2.73 A
*Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.
87
4.5.2. EFFECT OF TEMPERATURE ON MYCELLIAL BIOMASS
PRODUCTION
The data for the effect of different temperatures on the mycelial production
of the fungus showed that maximum production was obtained at 30°C from all the
liquid media, which was (33.82mg) from cotton waste liquid medium, (13.20mg)
from paddy straw liquid medium and (43mg) from pulses straw liquid medium.
Table27: Effect of temperature on mycelial production in three liquid media
Medium Temperature °C Cotton waste
(mg) Paddy straw
(mg) Pulses straw
(mg) Mean
20
15.80±2.04 d 6.00±0.88 g 1.30±0.18 j 7.70±1.10 D
30
33.82±3.90 a 13.20±1.72 e 3.43±0.39 h 16.82±2.23 A
40
26.39±3.41 b 11.57±1.68 f 2.17±0.24 i 13.38±1.85 B
50 22.19±3.09 c 5.98±0.88 g 1.36±0.18 j 9.84±1.62 C
Mean 24.55±1.74 A 9.19±0.77 B 2.07±0.16 C LSD =0.48
Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.
The difference between the average mycelial productions in all the liquid
media, different temperature was significant. The lowest average yield was
obtained both at the temperature 20°C and 50°C which was (7.70mg) and
(9.48mg) respectively. The mean values of the yield in three liquid media showed
that maximum average yield was obtained from the liquid media containing cotton
waste at all the temperatures (24.55mg), followed by the paddy straw liquid
88
medium which was (9.19mg) and lowest average yield was obtained from the
pulses straw liquid medium (2.07mg), (Table 27).
4.5.3. EFFECT OF pH ON THE MYCELLIAL BIOMASS PRODUCTION
The mean square values showed that individual effect of liquid media, time
and different pH levels had highly significant effect on the mycelial production in
the liquid media. The two way interaction between the liquid media and time, two
way interaction between the liquid media, different pH levels and two way
interaction between the time and different pH levels had significant effect on the
mycelial production in the liquid media. The data also showed that three way
interactions among the time, different liquid media and different pH levels were
highly significant, (Table 28).
Table 28: Analysis of variance for protein production in liquid medium at different pH levels.
Source of variance Degrees of freedom Mean squares F value
Medium
Days
pH level
Medium x Days
Medium x pH level
Days x pH level
Medium x Days x pH level
Error
2
5
5
10
10
25
50
216
4823.76
1069.46
2348.12
229.76
662.06
119.46
32.13
1.21
3994.9289**
885.7027**
190.2833**
1944.6586**
548.3067**
98.9369**
26.6113**
** = Highly significant (P<0.01)
The mean values of the table showed that highest mycelial production was
obtained at pH 7, from all the liquid media used in the experiment. The liquid
89
media containing cotton waste produced maximum mycelial production at pH 7
i.e., (33.99mg/30ml), followed by the paddy straw liquid medium production
(13.20mg) and pulses straw liquid medium production (3.43mg).The mycelial
production at 6 pH was (11.76mg) which was higher than the production at other
pH levels but less than the production at pH 7. The average yield mycelial
production at pH 3 and pH 4 was not significantly different. The mean values of
the mycelial production in the liquid media showed that the liquid medium
containing cotton waste produced maximum average yield (15.0mg/30ml)
followed by the average yield obtained from the paddy straw liquid medium with
(6.25mg/30ml) and lowest average yield was obtained from pulse straw liquid
medium (2.49mg/30ml.), (Table 29).
Table29: Effect of pH on mycelial production in three liquid media. Medium
pH Cotton waste (mg)
Paddy straw
Pulses straw (mg)
Mean
3
1.15±0.10 klm
0.45±0.09 mn
0.13±0.02 n
0.58±0.07 E*
4
1.39±0.14 kl
0.84±0.13 lmn
0.08±0.01 n
0.77±0.10 E
5
9.74±1.42 f
4.56±0.76 g
1.88±0.25 jk
5.39±0.70 D
6
23.74±3.11 b
9.15±1.45 f
2.41±0.30 ij
11.76±1.66 B
7
33.99±3.97 a
13.20±1.72 d
3.43±0.39 h
16.87±2.25 A
8 19.98±2.19 c 10.90±1.58 e 2.94±0.36 hi 11.27±1.31 C
Mean 15.00±1.49 A 6.52±0.66 B 1.81±0.17 C LSD = 0.41
*Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.
90
4.6. MOLECULAR CHARACTERIZATION Table.30: Similarity matrix table Nie and Li ‘s co-efficient of five fungal strains. Pop ID 1 2 3 4 5 1 0.6456 0.5823 0.7342 0.7342 2 0.5063 0.5570 0.6835 3 0.5949 0.6456 4 0.7722
1= Vv PK, 2= Vv 436, 3= Vv 428, 4= Vv 430, 5= Vv 422
The similarity matrix table showed the genetic similarity between the
strains. With the help of the table similarity between the strains was calculated.
The strain 1,(Vv pk) was 64% similar with the strain 2, (Vv 436), 58% similar
with the strain3, (Vv428),73% similar with the strain 4, (Vv430) and strain 5,
(Vv422).The strain 2, (Vv436) was 50 % similar with the strain 3, (Vv428), 55%
similar with the strain 4, (Vv430) and 68% similar with the strain5, (Vv422). The
strain3, (Vv428) was 59% similar with the strain4, (Vv430), 64% similar with the
strain 5, (Vv422).The strain4 was 77% similar with the strarin5, (Vv422).
The picture1 and 2 showed the bands for the diversity among the five strain
of the fungus. In these pictures at 0, position was the band of primer (DNA of
known sequence) and at position 1, was the band of strain Vvpk, at position 2,
strain Vv436, at position 3, Vv428, at position 4, Vv430 and at position 5, band of
Vv422 was shown. The bands which showed the same sequence had the genetic
similarity, while where the bands showed different sequence, the strain differ
genetically at that position from each other.
91
Primer = Genetic Diversity among the five Strain of V. volvacea
Lane 1= Vv PK, Lane 2= Vv 436, Lane 3= Vv 428, Lane 4= Vv 430, Lane 5= Vv 422
1 2 3 4 5 1 2 3 4 5
1 2 3 4 5 1 2 3 4 5
92
Chapter 5 DISCUSSION
Mushrooms are good source of sugar, fiber, protein and minerals (Senatore,
1990; Adewusi et al., 1993), with comparable amino acid with animal protein
(Aletor, 1995). Shortage of supply and high cost of animal protein necessitates the
search for and cultivation of alternative and cheap protein sources. The
lignocellulose bioconversion, such as cultivation of edible fungi, could be an
alternative source of food production for human and upgrading fiber rich by-
products for animal feeding (Smith et al., 1988, Rajarathnam and Bano 1991). The
Chinese mushroom (V. volvacea) has the ability to grow well on a wide range of
cellulosic wastes. The rice straw has been used for the indoor cultivation of V.
volvacea, since the beginning of the 19th century, a practice from which the
mushroom has been given the common name straw mushroom. The Chinese
mushroom (V. volvacea) is appropriate for cultivation in tropical regions.
Moreover this species is the most common edible mushroom in south-east Asia
and produced almost all over the world (Chang and Miles, 1991).
In the present study six different agricultural wastes, cotton waste, rice
straw, banana leaves, corn stovers, sugarcane baggase and pulses straw were used
to find out the most cheap and high yielding substrates for the commercial
cultivation of the Chinese mushroom. These above mentioned agro-wastes were
selected, because all these were abundantly and cheaply available in the fields of
Pakistan, round the year. Each agro- waste was used as a single substrate, no
combination of the agro-wastes was used in order to make the mushroom
93
cultivation more easy and to calculate the potential of each agro-waste for
mushroom production. These wastes were also selected, by keeping in view the
previous experimentation of the different scientists which used variety of agro-
wastes for cultivation of V. volvacea in the past. Garo, (1964) used paddy straw,
dried banana stalks and leaves, water hyacinth, wheat straw and sugarcane
baggasse for Chinese mushroom cultivation. Gupta et al., (1970) tried wheat
maize barley oat pearl millet, and sorghum straw. Chang, (1974) cultivated
Volvariella volvacea on cotton waste compost in plastic green houses. Qumio,
(1981) reported the mycelial growth of Volvariella volvacea, on rice straw, Ipil-
Ipil leaves, sigadillas leaves, new paper prints, coconut coir dust and banana
bracts. Khan et al., (1994) tried dried water hyacinth for the cultivation of
Volvariella volvacaea. Salmones, (1996) used 13 agro industrial wastes, banana
leaves, bracts of pineapple crown, coconut fiber, coffee bran, coffee pulp, corn
cob, corn stover, orange peel, rice bran, rice straw, sisal bagasse, sugarcane
bagasse and wheat straw as substrate. Tonial, (2000) used industrial residues from
cassava and potato starch processing as substrates to produce the edible mushroom
Volvariella volvacea. Philippoussis et al., (2001) used three agricultural wastes,
i.e. wheat straw, cotton waste and peanut shells. Zervakis et al., 2001, evaluated
seven mushroom cultivation substrates: wheat straw, cotton gin-trash, peanut
shells, poplar sawdust, oak sawdust, corn cobs and olive press-cake. Obodai et al.,
(2003) tried banana leaves, cocoyam peelings and oil-palm pericarp. Belewu and
Belewu, (2005) studied the solid state fermentation of banana leaves.
94
The growth of the mushroom on the substrates was measured in terms of
spawn running, pinhead formation and fructification and ultimate yield of three
flushes from each substrate. In Pakistan the performance of these five strains was
measured for the first time. The other data regarding the yield performance of
V.volvacea in general also confirmed the results obtained by the other scientists.
The results for the spawn running showed that all the strains took minimum time
for spawn running, both on the cotton waste and paddy straw as compared to the
other substrates. The slow rate of spawn running was observed on the sugarcane
baggase and pulses straw. The time taken by the strains for pinhead formation was
minimum on the cotton waste and corn stovers but on the other substrates all the
strains took almost same time for pinhead formation and did not showed the
significant difference. The strain Vv pk took shortest time for pinhead formation
on cotton waste and longest time was taken by the strain Vv422 on corn stover.
The efficiency for pinhead formation by the strain Vvpk was high on all the
substrates followed by the strain Vv436, while, the strain Vv422 showed lowest
efficiency and took more time for pinhead formation as compared to the other
strains on all the substrates. All the strains showed fructification in minimum time
on cotton waste, paddy straw and pulses straw respectively, maximum time for the
fructification of the mushroom was observed at sugarcane baggase. All the five
strains used for the cultivation produced highest yield on the cotton waste
followed by the paddy straw. The average yield of the five strains on cotton waste
was (734.3g) and on paddy straw it was (590g) and (483.2g).The lowest average
95
yield was obtained on the corn stovers which was (251g). These result reflected
that although the rice straw is the traditional substrate used for the cultivation of
the Chinese mushroom but cotton waste gave the high yield as compared to the
paddy straw. The banana leaves also produced good yield less than the cotton
waste and paddy straw but more than the other substrates. These three substrates,
cotton waste, pulses straw and banana leaves produced high yield than the other
three substrates; sugarcane baggase, corn stovers and pulses straw. The results
showed that difference in the yield of the six substrates was significant.
Different scientists had calculated the yield of Volvariella volvacea, on
different agricultural wastes. Most of them obtained the appreciable yield on
cotton waste, paddy straw and banana leaves. Garo, (1964) obtained highest yield
from the beds of banana leaves as compared to any other substrate, dried banana
stalks and leaves, water hyacinth, wheat straw and sugarcane baggase. Gupta et
al., (1970) observed low yield on wheat maize barley oat pearl millet, and
sorghum straw as compared to that produced on paddy straw. Khan et al., (1994)
concluded that water hyacinth + cotton waste at the rate 1:1 gave maximum yield.
Salmones et al., (1996) recorded that, the highest biological efficiency was
achieved on rice straw. Philippoussis et al., (2001) observed that V. volvacea
presented higher growth rates when the composted cotton waste medium was
used. Akinyele and Akinyosoye, (2005) reported maximum fungal growth on
cotton waste (98.23 ± 0.1 mm) and a mixture of rice husk and cotton waste.
Zervakis et al., (2001) found the highest mycelium extension rates (linear growth
96
and colonization rates) on cotton gin-trash. Obodai et al., (2003) highest
production on banana leaves banana leaves, cocoyam peelings and oil-palm
pericarp as substrates. Belewu and Belewu, (2005) recorded the total number of
fruits and the total weight of the fruits 2.5 kg, with biological value put at 15.21%.
The comparison among the strains for the yield showed that, the strain
Vvpk was proved to be the high yielding among the five strains, by producing
(821gm) mushroom on the cotton waste substrate. Similarly it produced high yield
on all other five substrates, followed by the strain Vv 436, which produced
(769.4gm) on cotton waste. The strain Vv422 was low yielding strain and
produced lowest yield on all the substrates as compared to the other substrates. Its
lowest yield was recorded on the corn stovers which was (192.9gm). These
differences in strain yield on different substrates may depend on the physical
conditions and chemical composition of the substrates. It is conclusive that V.
volvacea is a cellulolitic mushroom (Kwang and Chang 1981, Ho1985), because it
required high C/N ratios, (Chang-Ho and Yee, 1978).
The comparison between the two crops for spawn running, pinhead
formation, fruiting bodies formation and yield showed that the difference for these
parameters between two crops was not significant. The crop 1 which was grown
during the month of June- July showed low yield as compared to the crop 2 which
was grown during the months of July-August. Samajpati et al., (1977) reported
satisfactory growth conditions for the cultivation of V. volvacea from April to
97
August, but highest yield was obtained in July when the optimum temperature was
32C and humidity was about 85%.
The pH of substrates has also a great effect on the mushroom growth. The
results showed significant difference in the of the pH values of the substrates at
different growth stages of the mushroom. The pH of the substrates before
inoculation was higher than the pH after spawn running and termination of the
fruiting bodies of the mushroom. It was obvious from the results that as mushroom
growth proceeded in terms of, spawn running, pinhead formation and fruiting
body formation resulted in lowering the pH level of the substrates compared to the
level before inoculation of spawn, the reason may be the degradation of lignin
which is considered to contribute substantially to the formation of humus. During
cropping the change in pH was significant. The strain (Vv pk), had maximum
effect on the pH change of the substrates while strain Vv422, has minimum effect
on the pH of the substrates Maximum change in pH of cotton waste was observed
while minimum change was recorded in case of Pulses straw. Frank, (1989)
recommended the pH value from 7.5 to 8.5 to obtain high yield of V. volvacaea.
Fasidi et al., (1996) described that Volvariella esculenta (Mass) Singer, has the
ability to grow at a temperature range of 20-40°C (optimum = 35°C) and pH range
of 3-10 (optimum = 6.0). Jonathan et al., (2004) concluded the pH 6.0, for the
best mushroom growth.
The results of proximate composition of the agricultural wastes used for the
cultivation of mushroom showed high protein content in substrates after the
98
treatment with mushroom spawn as compared to the protein content before
treatment. Similarly crude fiber and ash contents were also high in treated agro
wastes as compared to untreated. Among all the substrates cotton waste was rich
in ash, fiber and protein contents before inoculation of the spawn, 20.82%,
41.62%, 19.17%, respectively. After spawn running these contents became,
24.34% ash, 43.62% fiber and 20.67% protein, which showed the significant
increase in the percentage of these contents. Different scientists reported that, the
fungal growth on different wastes enhance their nutritional values (Zadrazil, 1993;
Belewu and Okhawere, 1998). The increase in the protein content of the substrates
after the treatment with mushroom spawn may be due to secretion of certain
extracellular enzymes which are involved the breakdown and subsequent
metabolism of the substrates. (Kadiri, 1999). Hydrolysis of starch to glucose,
which is sued by the fungus for it growth could also be another reason for increase
in protein of the substrates. (Bender, 1970; Hammond and Wood, 1985). Belewu
and Belewue, (2005), observed that the banana leaves treated with V. volvacea
decreased in the polysaccharide components and with a greater percentage of the
fiber components being degraded. The crude protein content was enhanced by the
incubation of the mushroom. Biological treatment of agricultural wastes with
fungi reduces the environmental pollution and these treated substrates are also
used as animals feed. (Bisaria et al., 1987; Belewu and Banjo, 1990, 2000). The
rams fed with the fungal treated wastes, showed the increase in digestibility of
these wastes and better growth rate. (Bano and Rajatham, 1989). Rice husks
99
treated with Trichoderma harzianum, consumed by animals resulted in, increase in
nutrient composition of rice straw and hay in the tropical environment (Belewu,
1999; Belewu and Banjo, 2000).
The biochemical analyses of the fruiting bodies harvested from different
substrates showed that cotton waste fruiting body harvested from cotton waste
contained maximum protein (34.17%) followed by the sugarcane baggase
(30.51%), paddy straw (28.57%), banana leaves (23.92%), corn stover (21.77%)
and pulses straw (20.25%).The ash %age was maximum in cotton waste (10.8%),
followed by the banana leaves (5.93%) paddy straw (2.43%) %corn stover
(1.53%) pulses straw (2.05%) sugarcane baggase (1.96%). The crude fiber %age
was maximum in cotton waste (11.9%) followed by the pulses straw (8.14%),
sugarcane baggase and corn Stover (7.22%) paddy straw and banana leaves
(7.88%). Cheng, 1979, reported the proximate analysis of Volvariella volvaceae,
which showed that mushrooms contain crude protein 30-43%, crude fat 1-6%,
carbohydrates 12-48%, crude fiber 4-10% and ash varied between 5-13%, on dry
weight basis.
Liquid fermentation of the Volvariella volvace strain pk, was carried out in
the study. Three agro wastes were used i.e., cotton waste, paddy straw and pulses
straw, to make three different liquid cultures. With help of liquid fermentation,
fungal mycelium can be obtained within short period of time and that mycelium
can be used for the spawn preparation, in the food industry for flavouring and
aroma and in animal feed. Some scientists have explored the possibility of
100
growing mushroom mycelia in a variety of submerged liquid media, as a potential
source of fungal mycelium and aromatic compounds, which may be used in
different food items. (Berger et al., 1987; Sastry et al.1980). The effect of time on
the mycelial production was significant during the liquid fermentation of the
mushroom. Maximum mycelial growth was recorded at 7th day of the fermentation
in all the three media used for fermentation at 100rpm. The temperature of 30°C
was best to get the maximum mycelial production in all media used. Liquid
medium contained cotton waste produced maximum mycelial growth followed by
the paddy straw and pulses straw. Anon, (1954) concluded that it is possible to
grow the highly priced mushroom Morchella hortensis, at very low cost in water
culture under constant motion. Edwards, (1954) observed the growth of edible
fungi in sub-merged culture, consisted of a liquid medium. Block, (1959)
produced mushroom mycelium in sub-merged liquid culture with carbohydrates
and nitrogen compounds with mineral salts. Atacador, (1967) reported that out of
five edible mushrooms cultivated in liquid medium, V. volvacea gave the highest
mycelial yield. Troev, (1968) worked out a technology for the cultivation of
mycelium of different species of higher fungi in liquid nutritive medium.
Kostadinov et al., (1972) described a method of producing Pleurotus ostreatus
mycelium in sub-merged culture which may be used as (spawn) Bukhalo et al.,
(1978) studied that Pleurotus was successfully grown in liquid nutrient medium
containing 10% red clover extract, sucrose, peptone and mineral salts on
commercial scale. Nagaso and yoshikawa, (1978) observed that mycelial growth
101
and yield was best with shaking at 100 cycles/minute rather than shaking at 90 or
120 cycles/ minute. Quimio, (1984) mentioned potential of using coconut water as
a liquid medium for mycelial production of Lentinus sajor caju. Cai, et al., (1998)
studied that Volvariella volvacea (V-14), produced β-glucosidase when grown in
liquid culture on a variety of carbon sources
Temperature and pH, both have the significant effect on the growth of most
microorganisms. The effect pH and temperature variation on mycelial growth was
studied. Each medium had different potential to produce the mycelia at different
pH levels. The pH 7 was best to get the maximum mycelial production in all the
liquid media. The optimum pH of 7 and optimum temperature of 30ºC reported for
V. volvacea agreed with the report of Chang and Yau (1977),which was 30°C –
35ºC for the same mushroom. Fasidi, (1996), reported that Volvariella esculenta
was able to tolerate temperature range of 20°C –40ºC and pH range of 3 – 10.
Kuforiji and Fasidi (1998) obtained an optimal temperature of 35ºC for Pleurotus
tuberregium and pH range of 5 – 7 for same mushroom. Chang et al., (1981)
reported a temperature range of 10 – 20ºC for Pleurotus sajor-caju while Jonathan
and Fasidi, (2000) reported the growth of Psathyrella atroumbonata at 30ºC and
pH 6.5. This work had shown that significant improvement in the mycelia growth
of V. volvacea can be attained through liquid fermentation with agro wastes i.e.,
cotton wastes, rice straw, which gave the best mycelial growth, while pH 7 and
temperature of 30ºC were the most suitable for mycelial production.
DNA polymorphism may be detected in a variety of way. Recent
102
developments in a molecular biology enables us to find out species-specific DNA
markerswhich are indepandant of environmental vaiations.With the advent of PCR
technology it has become possible to study genetic differences in plants. RAPD is
a technique based on PCR. It prodced consistent results with optimized conditions
and has potential to be employed for phylogenetic relationships and taxonomic
classification. Polymorphism revealed by RAPD could be a result of nucletoid
changes t the primer annealing site or due to addition or deletion between two
priming sites which results in different lengths of the amplification product.(
Williams et al, 1990). The genetic study is alsohelpful in plant pathology in a
variety of way.
The analysis of genetic variation in a population is an important
prerequisite to understand the co-evolution of plant pathogens. Chiu and Moore,
(1999) determined the electrophoretic karyotype of the Chinese straw mushroom,
Volvariella volvacea. Ding at el., (2001) isolated an endoglucanase, EGI, from
fluid of Volvariella volvacea by PAGE, to generate improved strains of V.
volvacea with better growth, with higher potential of production and substrate
conversion. Guo et al., (2002), used PCR technique for amplifying THP gene in an
unknown vector with primer AFP1 and AFP2. Jia et al., (1999) obtained the
regenerating protoplast from mycelial culture of the mushroom Volvariella
volvacea by the action of the lytic enzyme.
In this study the bands of same size amplified from different varieties are
considered as the same loci and band from different sizes are considered as
103
different loci. Amplification products of the same size from different strains might
have homologous sequences. The faint bands were also observed during the study
but were not scored. The objective of the genetic study of the five different strains
was to know either these strains which are producing different yields on the
different substrates are genetically different from each other or they have the same
genetic makeup. PCR of five strains V. v.pk, Vv436, Vv430, Vv428 and Vv422
showed that all these strains had genetic diversity. In coming time further research
could be done, in order to identify the genetically high yielding strain. That could
be helpful to select the strain for the commercial production of the Chinese
mushroom in Pakistan. The genetic study during this study was the preliminary
study just to ensure that these five strains were genetically different.
104
SUMMARY
The cultivation of mushroom is a way to overcome the problem of
pollution, caused by the different crops residues, because the fungi have the ability
to recycle these wastes and convert them in the edible protein. So the cultivation
of the mushroom can also be helpful to overcome the shortage of food.
The objectives of this study were to identify the genetically highly yielding
strains of the Chinese mushroom, (Volvarieela volvacea), and selection of some
suitable substrates for the cultivation of the Chinese mushroom. For this purpose
six different substrates, cotton wastes, paddy straw, banana leaves, corn stovers,
sugarcane baggase and pulses straw were evaluated for the cultivation of the
mushroom.
Among these substrates cotton waste, paddy straw and banana leaves were
proved to be the best for mushroom cultivation. Cotton waste was best among all
the substrates by producing maximum yield of the mushroom, followed by the
paddy straw. Six different wild and exotic strains, the strain Vvpk which was local
strain and the strain Vv436, Vv428, Vv430 and strain Vv422 were exotic strains
imported from different foreign countries. These strains were evaluated for their
productivity on the different substrates. The comparison between the strains for
the yield showed that strain pk, which was the local strain produced high yield as
compared to the other strains. The strains Vv436 also produced good yield after
Vvpk, while strain Vv422 was the lowest yield producing strain among all the
strains. The temperature and relative humidity requirements were easily available
105
during the summer months, from April to September. The Chinese mushroom is a
mushroom of hot summer days, its temperature requirement is above 30ºC and
relative humidity above 80%, which was easily available in these months.
The liquid fermentation was also done during the study, in which three
liquid media were used, containing cotton waste, paddy straw and pulses straw.
The effect of temperature and pH was studied on the filamentous protein
production in these liquid media. The results showed that 30ºC temperature and
pH 7 was the best for maximum mycelial production. The liquid media containing
cotton waste produced highest mycelial weight as compared to the paddy straw
and pulses straw containing liquid medium. The time of incubation for liquid
fermentation also had significant effect on the mycelial production. The maximum
production was recorded at the 7th day of the incubation of the mushroom.
The biochemical analysis of the substrates showed that protein contents,
fiber and ash contents of all the substrates enhanced after the treatment with the
mushroom as compared the untreated substrates. Among these substrates had high
percentage of protein, fiber and ash contents as compared to all other strains.
The biochemical analysis of the fruiting bodies showed that the crude
protein, crude fiber and ash percentage was high in the fruiting body harvested
from cotton waste, the moisture percentage of the fruiting bodies were almost
same from all the substrates. The pH data of the substrates at different growth
stages showed that it decreased as the growth phases of the mushroom proceeded
and become less than the pH before the inoculation of the substrates.
106
Molecular characterization of the strains revealed the genetically high
yielding strain and there was genetic diversity among the strains.
107
SUGGESTIONS AND RECOMMENDATIONS
• The Chinese mushroom is the mushroom of tropical areas; most of the area
of Pakistan is of same character. The environmental conditions required for
its cultivation are easily available in the country, therefore more suitable for
the cultivation of the Chinese mushroom.
• This mushroom can be grown on the wide variety of agricultural wastes.
These waste materials are also abundantly and cheaply available in the
country. Cotton waste, paddy straw and banana leaves are the good
substrates for the mushroom cultivation.
• The local strain Vvpk and exotic strain Vv436 are the best strains for the
cultivation.
• Liquid fermentation is also a good source to get the filamentous protein in a
short period of time, which can be used for spawn preparation, in human
feed like soups and in other food products to produce aroma, and in animal
feed also.
• The best pH is 7 and 30ºC temperature is best to get maximum yield of the
mushroom.
• The treatment of the substrates with the mushroom enhanced certain
contents in the substrates like protein, fiber and ash, which can be used as
animal feed.
• The mushroom can be used directly as a balance diet, as health food and
can also be used in the canned form.
• Commercially it could be a multi million business in future. Mushroom
cultivation can also be adopted as cottage industry easily because it does
not require high skills and investment, but it only requires some knowledge
and some training regarding its cultivation.
108
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