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EVALUATION OF SUBSTRATES FOR QUALITY SPAWN PRODUCTION OF
MUSHROOMS
By
Sandeep Kumar
(J-13-M-348)
Thesis submitted to Faculty of Postgraduate Studies
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE IN AGRICULTURE
PLANT PATHOLOGY
Division of Plant Pathology
Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu
Main Campus, Chatha, Jammu 180 009
2015
CERTIFICATE – I
This is to certify that the thesis entitled "Evaluation of substrates for quality
spawn production ofmushrooms" submitted in partial fulfillment of the requirements of
the degree of Master of Science in Agriculture (Plant Pathology), to the Faculty of
Post-Graduate Studies, Sher-e-Kashmir University of Agricultural Sciences and
Technology of Jammu, is a record of bonafide research carried out by Mr.Sandeep
Kumar, Registration No. J-13-M-348, under my supervision and guidance. No part of
the thesis has been submitted for any other degree or diploma.
It is further certified that such help and information received during that course of
investigation have been duly acknowledged.
Dr. Sachin Gupta
(Major Advisor)
Place: Jammu
Date:
Endorsed
(Dr. Anil Gupta)
Prof. &Head
Division of Plant Pathology
FOA, SKUAST-Jammu
CERTIFICATE-II
We the members of Advisory Committee of Mr.Sandeep Kumar, Registration No.
J-13-M-348, a candidate of the degree of Master of Science in Agriculture (Plant
Pathology), have gone through the manuscript of the thesis entitled “Evaluation of
substrates for quality spawn production of mushrooms” and recommended that it may be
submitted by the student in partial fulfillment of the requirements for the degree.
Dr. Sachin Gupta
(Major Advisor)
Place: Jammu
Date:
Advisory Committee Members:
Member from major subject
Dr. Ranbir Singh
Assistant Professor
(Division of Plant Pathology) ___________________
Member from minor subject
Dr. Arti Sharma
Assistant Professor
Division of Fruit Sciences ___________________
Dean’s Nominee
Dr. A. K. Razdan
Professor& Head
Division of Plant Breeding and Genetics
___________________
ACKNOWLEDGEMENT
I am thankful to Mahakali for her grace and immense blessing. I offer my humble
praise to the omniscient and almighty Maa Baway Wali, Datti Maa and Naag Devta who
gave me the opportunity to complete this difficult venture. Without their unceasing mercy
and compassion, it was never possible for me to complete this research work.
I strive for words to express my deep sense of gratitude to my honorable major
advisor Dr. Sachin Gupta, Assistant Professor, Division of Plant Pathology, SKUAST-J for
his expert guidance, keen interest and constant encouragement during my entire degree
program. I simply feel myself blessed being provides with an academic advisor like him.
I am grateful to Dr Anil Gupta Professor and Head Division of Plant Pathology and
Dr. V. K. Razdan Professor for providing necessary facilities during the course of my study.
I emphatically extend my heartiest thanks to the worthy members of my advisory
committee, Dr. Ranbir Singh Assistant Professor, Division of Plant Pathology, Dr. Arti
Sharma Assistant Professor, Division of Fruit Science and Dr. A. K. Razdan Professor,
Division of Plant Breeding and Genetics, for their constant help, encouragement and valuable
suggestions during the investigation and preparation of manuscript.
I equally reiterate my gratitude and indebtedness to Dr. S. K. Singh Associate
Professor, Dr. Vishal Gupta, Dr. Deepak Kumar Assistant Professor and Dr. V.B. Singh
Assistant Professor, for their help when approached.
I am also thankful to all the non-teaching staff of Division of Plant Pathology,
SKUAST-J for their help and cooperation during the research work.
I owe my thanks to my colleagues Arun Khajuria, Rewti Raman Sharma, Arvind
Kumar, Manmohan and others for their valuable suggestions.
I unfeignedly owe my parents Sh. Nathu Ram and Smt. Sudesh Kumari, a
tremendous respect and gratitude for their unending love and affection. They have been
instrumental in every success that I have achieved hitherto. I am also thankful to my wife
Mrs. Shasna Mansotra and my kids Manasvani and Manomay for their moral support and
confidence-build up provided by them in my meek times has been pivotal during my studies.
Sandeep Kumar
Place: Jammu
Date:
ABSTRACT
Title of Thesis : “Evaluation of substrates for quality spawn
production of mushrooms”
Name of the student and : SandeepKumar
Registration number J-13-M-348
Major subject : Plant Pathology
Name and designation of : Dr. Sachin Gupta
Major advisor Assistant Professor
Degree to be awarded : Master of Science in Agriculture (Plant Pathology)
Year of award of Degree : 2016
Name of the University : Sher-e- Kashmir University of Agricultural Sciences &
Technology of Jammu (J&K)
Six different grains viz. wheat, maize, bajra, sorghum, barley and oat alone and in
combination with the supplements viz. gram husk, paddy husk and wheat bran in the 1:1 ratio
(v/v) were studied to see their impact on spawn growth and yield of three species of
mushroom viz. Agaricus bisporus, Pleurotus florida and Calocybe indica.. All the grain
substrates with and without supplements favoured the mycelial growth in spawn of different
mushrooms except when the grains were supplemented with wheat bran. However yield and
quality related parameters varied with different spawn substrates. Sorghum grains took the
minimum time for mycelial run in spawn bottles followed by maize while bajra grains took
the maximum time for mycelia run. In case of A. bisporus maximum time for spawn run was
19.00 days in bags spawned by sorghum grains supplemented with gram husk. In Pleurotus
florida, wheat straw spawned with sorghum grains took the minimum time for spawn run. For
cultivation of C. indica, sorghum grain spawn took the minimum time for spawn run.
However, maximum days for spawn run was taken by spawn prepared with maize
supplemented with gram husk. For pinhead formation, bags spawned with sorghum grain
spawn took the minimum number of days. Highest biological efficiency was obtained in
A.bisporus, P. florida and C.indica by using spawn prepared by sorghum grains, while
minimum biological efficiency in A.bisporus, P.florida and C.indica was obtained by using
maize added gram husk. Maximum B:C ratio of 1.91, 2.98, and 3.91 was obtained in
A.bisporus, P. florida and C. indica respectively by using spawn prepared by sorghum grains.
Four major types of contaminants were observed which included three fungal viz. Aspergillus
spp., Penicillium spp. and Trichoderma spp. and one bacterial viz. Bacillus spp. These
contaminants were found individually as well as in combination in all the grain substrates.
The incidence of bacterial contamination was maximum in bajra grain spawn and least in
sorghum grain spawn. The treatment comprising of three boiling as well as three autoclavings
showed maximum efficiency in management of fungal and bacterial contaminants.
Tetracycline (50µg/kg) was found best for managing bacterial contamination of spawn which
resulted in the reduction up to 98.33% of bacterial contamination.
Signature of Major advisor Signature of Student
CONTENTS
Chapter No. Topics Page
1
INTRODUCTION
1-2
2 REVIEW OF LITERATURE 3-7
3 MATERIALS AND METHODS 8-15
4 RESULTS 16-24
5 DISCUSSION 25-33
6 SUMMARY AND CONCLUSIONS 34-36
REFERENCES 37-43
LIST OF TABLES
Table
No.
Particulars After
Page no.
1 Effect of different spawning substrates on the mycelial
growth of Agaricus bisporus
16
2 Effect of different spawning substrates on the myelial growth
of Pleurotus florida
16
3 Effect of different spawning substrates on the mycelial
growth of Calocybe indica
16
4 Time taken for full growth of spawn of different mushrooms
using different grains
16
5 Effect of different spawning substrates with added
supplements on the mycelial growth of Agaricus bisporus
17
6 Effect of different spawning substrates with added
supplements on the mycelial growth of Pleurotus florida
17
7 Effect of different spawning substrates with added
supplements on the mycelial growth of Calocybe indica
17
8 Time taken for full growth of spawn of different mushrooms
using supplemented grains
17
9 Effect of different spawn substrates on yield and quality
related parameters of Agaricus bisporus
19
10 Effect of different spawn substrates on yield and quality
related parameters of Pleurotus florida
19
11 Effect of different spawn substrates on yield and quality
related parameters of Calocybe indica
20
12 Incidence of contamination of spawn prepared using different
grains
21
13 Incidence of contamination of spawn prepared using grains
along with supplements
22
14 Evaluation of boiling treatments for management of spawn
contamination
23
15 Evaluation of different autoclaving treatments for
management of spawn contamination
23
16 Evaluation of chemical treatments for the management of
spawn contamination (%)
23
17 B:C ratio of different mushrooms cultivated using spawn
prepared by different substrates
24
18 Identification of contaminants from ITCC
24
LIST OF PLATES
Plate
No.
Title After page
No.
1 Substrates for spawn production 18
2 Contamination in mushroom spawn 23
3 Mushrooms cultivated using different types of Spawn 24
CERTIFICATE – IV
Certified that all the necessary corrections as suggested by external examiner/
evaluator and the advisory committee have been duly incorporated in the thesis entitled
“Evaluation of substrates for quality spawn production of mushrooms” submitted by
Sandeep Kumar, Registration No. J-13-M-348.
Dr. Sachin Gupta
(Major Advisor)
Place:
Dated:
Head
Division of Plant Pathology
CHAPTER 1
INTRODUCTION
Mushroom farming today is being practiced in more than 100 countries and its
production is increasing at an annual rate of 6-7%. (Chang, 1999) India alone produces about
600 million tons of agricultural by-products, which can profitably be utilized for the
cultivation of mushrooms (Chadha and Sharma, 1995). The state of Jammu and Kashmir in
general and Jammu division in particular being an agrarian economy is rich in terms of agro
wastes which are not being properly utilized by the farmers of this state. Keeping in view, the
availability of abundant agro-wastes and varied agro climatic conditions prevalent in Jammu
Division, mushroom cultivation has a great potential as an economic activity and means of
societal development in the region giving good remunerative returns.
Like other crops, seeding material is needed for mushroom cultivation. Spawn is the
mushroom seed, comparable to the seed in crop plants. The term spawn is generally used for
the vegetative growth of mushroom mycelium and the substrate on which the fungus grows to
seed the compost. Spawn plays an important role in the mushroom industry because the
failure or success of mushroom cultivation depends upon the availability of pure culture
spawn. The success of mushroom cultivation and its yield depend to a large extent on the
vigour and quality of the spawn used (Bahl, 1984). The yield and quality of spawn is
governed mainly by the genetic make up of the strain and the technology including the
substrates used in spawn production. Spawn quality is counted as the most important aspect
of mushroom production (Goltapeh and Pujram, 2003). As per the current data available, the
demand of quality spawn in Jammu division is 263 quintals. Department of Agriculture,
Govt. of J&K and Division of Plant Pathology, Sher-e-Kashmir University of Agriculture
Sciences and Technology of Jammu through their respective spawn production laboratories
provide about 120 quintals of spawn while the rest is arranged by the farmers on their own.
The problems being faced by the local laboratories are high cost of spawn production and the
contamination of spawn by various fungal and bacterial contaminants.
Presently, mushroom spawn is being prepared on wheat grains. Taking into
consideration, the escalating cost of wheat grains and its role in human nutrition, it is
imperative to look for other locally available cheap substrates which could be used for spawn
preparation. There is also a need to look for strategies to reduce the contamination of spawn
so that wastage of resources used in spawn preparation is reduced. Keeping in view the above
factors, the present study was designed with the following objectives:
• To evaluate different locally available substrates for spawn production.
• To study the common contaminants associated with spawn production and their
management.
CHAPTER-2
REVIEW OF LITERATURE
The relevant literature pertaining to the investigation “Evaluation of substrates for
quality spawn production of mushroom” is reviewed herewith:
The mushroom is a plant life without leaves, buds and flowers and is recognised as
fleshy macro-fungi, a group of achlorophyllous organisms. These are sometimes tough and
umbrella like sporophores with spores, naturally grown in fields, forests, on manure heaps,
water channels and hilly areas mostly during and just after rains (Jiskani et al., 2007). Wood
(1985) stated that mushroom cultivation is currently the only economically viable
biotechnology process, where in waste materials or negative value crop residues may be
converted into valuable food.
Spawn is the vegetative mycelium from a selected mushroom grown on a convenient
medium or substrate (Klingman, 1950). As per Mbogoh (2011), spawn is pure culture of
mycelium grown on a solid substrate such as cereal grain. It is the mushroom seed,
comparable to the seed of crop plants. The success of mushroom cultivation and its yield
depend to a large extent on the quality of the spawn used (Bahl, 1984). It serves as the
planting material in mushroom cultivation.
2.1 Evaluation of substrates for spawn production
Various substrates have been evaluated by different researchers across the globe for
preparation of spawn. Early methods of mushroom spawn making used either horse manure
or a mixture of this and cow manure as a substrate (Mbogoh et. al., 2011). Most significant
advancement in spawn making took place in 1932 when Sinden proved that mycelium of
Agaricus bisporus grew vigorously on cereal grains. Different researchers have reported
different grains for spawn preparation. The technology of grain spawn preparation was
further improved by Stoller in 1962 and he preferred rye to sorghum. Stoller (1968) reported
faster growth of mycelium on hulled grain and cotton seed meal while buck wheat and wheat
bran showed poor growth. Hu and Lin (1972) used shell powder, starch, compost powder and
grain hull powder for making granular spawn in Taiwan. Antonio and Hwang (1971)
recommended that cereal grain can be a common substrate for commercial spawn production.
Rangad and Jandaik (1977) screened various substrates for spawn production of Pleurotus
spp. and reported jowar and bajra grains as best substrates for spawn production followed by
wheat grains. Sivaprakasam and Kandaswami (1981) studied the effect of different grain
spawn on sporophore production of P. sajor caju and reported maximum yield with the use of
sorghum and bajra grain spawn followed by maize spawn. Jowar and bajra has also been
reported as best substrate for spawn production by Chauhan and Pant (1988) after they
studied the effect of spawn prepared on different substrates on sporophore production of P.
sajor caju. Moorthy and Mohan (1991) have grown stock spawn on sorghum grains. Mansur
et al. (1992) reported that the different spawn substrates showed the order: wheat seeds,
millet seeds, ground maize cobs for efficiency in spawn production. However, Gokulapalan
et. al., (1994) reported that mycelial run was fast in normal grains and tea leaves followed by
half filled paddy grains, tea leaves, paddy chaff and saw dust after they evaluated different
substrates for spawn production of oyster mushrooms.
Mathai and Suharban (1994) reported the growth response of P. sajor caju spawn on
sporophore production. They evaluated the use of low cost materials like half filled paddy
chaff and compared with paddy grain spawn and observed that radial growth was highest in
paddy chaff followed by half filled paddy grain spawn. Kathe et al., (1994) studied the
preparation of spawn by using cotton stalk and reported that cotton stalk spawn gave the
highest yield. Moreover, cotton stalks also proved a good substrate for cultivation of P. sajor
caju. Mathew et. al., (1996) evaluated the yield performance of Pleurotus spp. on various
substrates both for spawn preparation and cultivation and concluded that sorghum, wheat and
paddy grains were equally good for spawn production. Rathaiah and Ashok (1999) prepared
spawn of oyster mushroom on partially boiled paddy and compared it with that of wheat
grain for yield and profit and reported that partially boiled paddy grain were equally as good
as wheat for spawn preparation. Hafeez et al., (2000) reported that spawn production on
sorghum grains was significantly higher than pearl millet, maize and wheat grains. Jiskani et
al., (2000) conducted experiments on the effect of different temperatures and grain media on
spawn growth of oyster mushroom (P. florida) and reported that the optimum temperature for
best growth of spawn was 300C and sorghum grains were found to be best medium for spawn
growth followed by maize, wheat and pearl millet grain, respectively. Sharma (2003)
conducted an experiment to determine the suitability of jowar, kutki (Panicum miliare), kodo
(Paspalum scrobiculatum), maize and wheat for spawn production of P. djamor and reported
that the shortest period for spawn development of 8days was recorded with kutki grains
indicating its suitability for efficient spawn production. Jiskani et. al., (2007) evaluated two
different varieties of sorghum viz. white turio and red jaunpuri individually and in
combination (red+white) for spawn production of oyster mushroom and reported that spawn
growth on red jaunpuri grains was significantly higher than white turio grains. Elhami and
Ansari (2008) studied the response of three species of Pleurotus (P. florida, P. citrinopileatus
and P. ostreatus) to three substrate types (wheat, millet and corn) for producing spawn. They
reported the maximum diameter of colony extension after inoculation with corn followed by
wheat. Stanely and Waadu (2010) studied the effect of various grains on spawn production of
Pleurotus tuberegium and P. pulmonarius, and reported white maize to be most suitable to
mycelial extension and mycelial fresh weight of P. tuberregium and P. pulmonarius.
Senthilnambi et. al. (2011) focused on finding the suitability of different grains as spawn
substrates and their effect on the yield of Calocybe indica. The results revealed the
supremacy of sorghum grains as the most suitable substrate for early spawn run which took
only 13.7 days for complete mycelial growth as compared to wheat and millet spawn. Munser
et. al., (2012) reported that wheat substrate was the best substrate for spawn production of
oyster mushroom followed by rice and wheat bran. Kumbhar (2012) reported that mycelium
of P.eous had marked preference for cereal grains over pulses and crop residues and ragi
grains took only six days for mycelial colonization followed by maize, pearl millet, sorghum,
wheat and paddy grains while pulses did not allowed growth of the fungal mycelium. Sharma
et. al., (2013) checked different wheat varieties viz. DWR16, PBW550, DWR39, DBW17,
DPW621-50, DBW-14 and HD2967 for spawn production of button mushroom and found
that DPW621-50 resulted in maximum downward linear growth followed by DBW-17 and
HD2967. They contributed the highest protein content (12.6%) and dry as well as wet gluten
content of DPW621-50 as reason for being best substrate among all the seven varieties
analysed. Sofi et. al., (2014) evaluated wheat grains, barley maize and millets for spawn
production and reported maximum growth rate in the corn (38.60mm) and minimum in millet
(26.80mm) substrates after 12 days of inoculation.
2.2 Contamination of spawn and its management
Since the spawn which acts as ‘starter’ or seed is the most important crucial input for
successful mushroom production. Its purity and quality is essentially required to be at the
highest level (Singh et. al., 2009). Contaminants are one of the major problems in mushroom
spawn production. Various microorganisms e.g, bacteria, actinomycetes, yeast and fungi
affect spawn making, sometimes leading to total spawn failure (Biserka, 1972). The ‘wet
spot’ or rottening of spawn was first reported by Stoller (1962). Biserka (1972) studied
another problem of spawn i.e ‘sour spawn’ and also the characteristics of bacteria involved in
spawn spoilage, belonging to the genus Bacillus, which become active at 30-320C causing
sour spawn disease. Oxaly (1985) and Earranna (1991) reported various
contaminants/pathogen on spawn as well as on mushroom fruit bodies. Suman and Jandaik
(1992) while studying the microbial contaminants of spawn of Agaricus bisporus reported
that the prime sources of contaminants are unsterilized wheat grains and microbes present in
the environment of spawn laboratory. They isolated and identified twenty four species of
mould and one each of bacteria and yeast in commercial spawn of Agaricus bisporus. These
researchers also isolated three contaminants namely Cladobotryum vertillatum, Absidia
glauca and Epicoccum purpurescens for the first time from the contaminated spawn bags.
Ahlawat et. al. (1997) studied the management of bacterial contamination of spawn
by physico-chemical methods. They reported that autoclaving plus application of antibiotics
were quite effective; the best results were, however, obtained when antibiotics were added
after autoclaving. Among the antibiotics ampicillin, streptocycline, streptomycin and
tetracycline were most effective at 50µg/g of spawn substrate. Three isolates of Bacillus
subtilis from contaminated spawn bags were isolated and characterized by Ahlawat et. al.
(1999). They suggested that the higher pH of wheat grains ( 7.5) and higher temperature
(0C) are the predisposing factors for spoilage by this bacterium. Mazumder and Rathaiah
(2001) found Trichoderma harzianum, Aspergillus spp and Pencillium spp as the three most
dominant fungal contaminants during spawn production in oyster mushroom. Mazumder et.
al., (2005) isolated and identified eight fungal and one bacterial contaminant from naturally
contaminated spawn of oyster mushroom. They observed month wise variation in spawn
contamination and found that the combination was highest during the monsoon season
(28.57%) followed by pre-monsoon (21.9%). They also reported that paddy grain based
spawn recorded significantly lowest (15.00%) contamination as compared to wheat grain
based (30.00%) spawn. Samadpour et. al., (2006) isolated and identified many bacteria from
the mushroom samples. Singh et. al. (2009), studied incidence of spawn contaminants and
economic losses in spawn production of button mushroom. They isolated and identified six
types of contaminants which contributed to 7.8% spoilage of the total spawn production out
of which Pencillium spp. contributed to maximum spoilage (39.3%) followed by Mucor spp.
(25.9%). Earranna et. al. (2010), isolated and identified Bacillus pumillus as mushroom
spawn contaminating bacteria using morphological, biochemical and molecular approaches.
CHAPTER-3
MATERIAL AND METHODS
The Material and methods adopted during the course of present investigation are
described here as under:
3.1 Experimental Site:
The laboratory and cultivation studies were conducted in the Mushroom Biology
Laboratory and Mushroom Cropping Unit of Division of Plant Pathology, Faculty of
Agriculture, Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu,
Jammu.
3.2 Cleaning and sterilization of glassware and media:
Glassware used for spawn preparation and other laboratory experiments were cleaned
with a cleaning mixture (100g potassium dichromate + 400 ml hot water + 600 ml of
concentrated sulphuric acid) and subsequently washed under tap water. The glassware was
sterilized by dry heat in hot air oven at 180±20C for 90 minutes while the media was
sterilized by wet heat in an autoclave at 15 lbs psi for 20 minutes during the course of
experiment.
3.3 Cleaning and disinfection of laboratory and cropping room:
Cleaning of laboratory floor and cropping room was done with laboratory washing
detergent and water. The disinfection of floor, concrete walls and air space was done with 5%
formaldehyde (formalin 40%) once a week. The air space of growing room was disinfected
by spraying 2.5 litre of the above solution for each 100 m2 of air space.
3.4 Source of master culture and its maintenance:
Pure culture of Agaricus bisporus, Pleurotus florida and Calocybe indica was
procured from Directorate of Mushroom Research Solan, Indian Institute of Horticulture
Research Bangalore and M.P.U.A.T Udaipur respectively. The pure cultures were maintained
by repeated sub culturing on potato dextrose agar (PDA) media at 15 days interval and
cultures of Agaricus bisporus and Pleurotus florida were incubated at 22±20C, however,
culture of Calocybe indica was incubated at 28±20C. After full growth, the cultures of
Agaricus bisporus and Pleurotus florida were kept in refrigerator and Calocybe indica at
room temperature till further use.
3.5 Evaluation of substrates and supplements for spawn production of Agaricus
bisporus, Pleurotus florida and Calocybe indica:
Substrates viz. Wheat grain, Barley grain, Bajra grain, Sorghum grain, Oat grain and
Maize grain were evaluated for their efficacy in spawn production of different mushrooms.
3.5.1 Spawn preparation:
All the substrates viz., wheat, barley, sorghum, maize and bajra were cleaned, washed
and then boiled until they became soften but did not split apart. The excess water was drained
out and the substrates were then cooled at room temperature by spreading them on muslin
cloth under shade. Calcium carbonate and Calcium sulphate @ 0.5% and 2% respectively on
dry wt. basis of grain were mixed thoroughly with the substrates. These substrates were filled
in clean and sterilized glass bottles (empty glucose saline bottles of 500 ml) up to 2/3rd
of
their capacity, plugged tightly with non absorbent cotton and the cotton plug was wrapped
with a piece of butter paper. The grain filled bottles were sterilized in autoclave at 15 lbs psi
pressure at 1210C for 2 hours.
3.5.2 Inoculation of spawn bottles:
After sterilization and cooling, the bottles containing different substrates were shaken
to remove clumps and were aseptically inoculated with small bits of fungal mycelium taken
from pure culture of mushroom grown on Potato Dextrose Agar (PDA) medium. The bits
were placed at the top of the substrates and incubated at 25±20C except C. indica which was
incubated at 28±20C.
3.5.3 Evaluation of substrates:
The downward linear extension of the mycelium in spawn bottles containing
substrates were measured with the help of ruler. Time taken for mycelial run (linear extension
of mycelium in spawn), time taken for full spawn run in spawn bottles and incidence of
contaminants were observed on daily basis till full mycelial run was observed in wheat grain
spawn bottles which served as control treatment.
3.5.4 Evaluation of supplements:
Substrates viz. wheat, barley, sorghum and maize were combined with supplements
viz. gram husk, paddy husk and wheat bran in the ratio of 1:1 (v/v). Spawn bottles of these
substrates prepared as described in 3.5.1 were inoculated with small bits of fungal mycelium
taken from pure culture of mushroom grown on Potato Dextrose Agar medium and incubated
at 25±20C except C. indica which was incubated at 28±2
0C. Observations as described in
3.5.3 were taken.
3.6 Evaluation of spawn prepared from different substrates and supplements for their
efficacy in cultivation of Agaricus bisporus, Pleurotus florida and Calocybe indica:
Spawn prepared from different substrates and supplements were evaluated for their
efficiency in yield and quality related parameters during cultivation of Agaricus bisporus,
Pleurotus florida and Calocybe indica.
3.6.1 Cultivation of Agaricus bisporus:
3.6.1.1 Preparation of compost:
Compost was prepared by long method of composting using the formula proposed by
SKUAST-J (Gupta et. al.,2014) The ingredients of compost pile were as follows:
• Wheat straw 500 kg
• Chicken manure 200 kg
• Oil cake 25 kg
• Rice/wheat bran 50 kg
• Gypsum 50 kg
• Urea 4kg
3.6.1.1.1 Mixing of ingredients and pile preparation:
Wheat was spread over a cemented floor and wetted thoroughly (48 hrs) by sprinkling
water. All the ingredients except gypsum were mixed thoroughly in wet straw which was
finally stacked into a pile of 5 feet height and same width. The heap was compressed by
applying light pressure with the help of wooden board. The turnings were given as per the
following schedule:
Operation Day
Stacking of pile 0
1st
turning 6
2nd
turning 10
3rd
turning 13
4th
turning 16
5th
turning 19
6th
turning 22
7th
turning 25
Opening of pile 28
Gypsum was added during third turning while after 7th
turning, the compost mixture was
sprayed with 0.05% malathion. After each turning, water was sprinkled to make up the loss of
water due to evaporation.
On 28th
day, the pile was spread, cooled and noticed for the properties of ideal compost
which was dark brown in colour with inoffensive sweet smell, not greasy or sticky, free from
smell of ammonia, not contain more than 65-70% of moisture, not contain any visible growth
of undesirable fungi except fire fangs and free from insects and nematodes.
3.6.1.2 Spawning:
Before spawning the floor surface was washed with 2% formalin. Spawning i.e.
addition of spawn prepared on different substrates was done in the compost following layer
method (Gupta et. al., 2014) Spawn was used @ 0.75% of the prepared compost.
3.6.1.3 Spawn run:
The compost after spawning was filled in polythene bags of the size 18 x 24 inches.
Before shifting the bags, the cropping room was disinfected with 2% formalin. A temperature
range of 20-250C and relative humidity of 85-90% was maintained in the cropping room till
full spawn run in the compost bags. Observation for number of days taken for full spawn run
in the bags was noted. As soon as the spawn run was completed, casing was done.
3.6.1.4 Preparation and application of casing soil:
Casing soil was prepared by mixing 1.5 years old cowdung + loam soil :: 3:1 (v/v).
The casing soil was sterilized with 4% formalin. The casing soil was piled up into a heap and
was treated with formalin and covered with plastic sheet for 48 hrs. Later the soil was
uncovered and stirred frequently to remove formalin fumes. After two days when it became
free from the smell of formalin, a 2.5cm thick layer was applied uniformly on the upper
surface of compost bags and the bags were watered regularly.
3.6.1.5 Harvesting of fruit bodies:
Fruit bodies of button mushroom were harvested when the pin heads matured fully
(2.5-3.0 cm) by twisting them gently clockwise and anticlockwise so that the young
developing pinheads were not damaged. The whole experiment was conducted in CRD and
observations on number of days for spawn run, days for pinhead initiation, average weight of
fruit body and total yield were recorded. Biological efficiency was calculated as:
3.6.2 Cultivation of Pleurotus florida:
The substrate (wheat straw) was soaked in hot water (55-600C) for half an hour and
after taking it out from hot water, excess water was drained out for 2-3 hours and the straw
was completely cooled. A moisture content of about 60-70% was maintained in the substrate
prior to spawning.
3.6.2.1 Spawning:
Spawning was done @ 5% by dry weight of the substrate. Cultivation was done in
high density polythene bags (18 x 24 inch with 100gauze). Each treatment was replicated
thrice.
3.6.2.2 Spawn run and opening of bags:
These bags were placed in the mushroom cropping room maintained at 80-85% RH
and 20- 250C temperature during spawn run and 18-20
0C during fruiting. When the substrate
was fully colonized by the spawn, resulting in block formation, the polythene bags were
removed and blocks left for fructification. The colonized substrate blocks were kept on the
iron stall shelves and kept humid by spraying water on them as and when required.
3.6.2.3 Harvesting of fruit bodies:
Observations on number of days for spawn run, days for pin head formation, days of
first harvest, average weight of fruit bodies, total yield and economic cropping period were
recorded Mushrooms were harvested and biological efficiency was calculated as:
3.6.3 Cultivation of Calocybe indica:
Substrate preparation and spawning for cultivation of C.indica was performed similar
to P.florida as already described in 3.6.2 and 3.6.2.1 respectively.
3.6.3.1 Spawn run:
The spawned bags were placed in the mushroom cropping room maintained at 85-
90% RH and 28- 300C temperature during spawn run and 32-36
0C during fruiting.
Observation for number of days taken for spawn run was noted.
3.6.3.2 Preparation and application of casing soil:
On completion of spawn run in C.indica bags, casing soil was prepared and applied as
described in 3.6.1.4.
3.6.3.3 Harvesting of fruit bodies:
Fresh fruit bodies of C.indica were harvested and biological efficiency was noted as
in 3.6.2.3.
3.7 Benefit: Cost ratio:
The cost involved in preparation of spawn with different substrates and the yield of
mushrooms obtained with use of different types of spawn was taken into consideration for
calculating the Benefit: Cost ratio of using spawn prepared from different substrates.
3.8 Incidence of contaminants:
Spawn bottles prepared from different substrates and supplements and inoculated with
different mushroom fungi were constantly monitored for appearance of any contamination or
competitor moulds during the period of mycelial growth in bottles. The bottles showing any
type of contamination were removed from the incubation room for recording observation and
identification of contaminants. Identity of contaminants was established on the basis of their
morphocultural characters and further confirmed through ITCC identification services of
IARI, N.Delhi.
3.8.1 Isolation and identification of bacterial contaminants:
Bacterial contaminants were isolated by streak method. Infected grains were placed
on nutrient agar medium in petri plates and incubated at 250C for 2-3 days. These bacterial
contaminants were purified by repeated streaking and colony characters were recorded. The
bacterium was identified by consulting the Bergy’s Manual of Systematic Bacteriology
(Sneath, 1986). Shape of cells was recorded after staining the pure culture with crystal violet.
Gram reaction was observed by Gram staining. A week old culture grown on the nutrient agar
was stained for endospore using malachite green. Catalase activity was studied using
hydrogen peroxide on the grown colonies for efflorescence. Methyl Red Vogues-Proskuer
(MRVP) test was conducted using MRVP medium. Starch hydrolysis was done by growing
the bacterium on starch agar and colonies were flooded with iodine solution for clear zone.
3.8.2 Isolation and identification of fungal contaminants:
For identification of fungal contaminants, infected spawn grains were inoculated at
three points on potato dextrose agar and incubated at 250C. Isolations were made from the
hyphal tip of the growing culture. Preleminary identification of the fungi was made on the
basis of morphocultural characteristics which were further confirmed by getting the cultures
identified from ITCC identification services of IARI, N.Delhi.
3.9 Management of contaminants
Three types of treatments were adopted for management of contaminants. These
include boiling treatments, autoclaving treatments and use of antibiotics. One, two and three
boiling treatments were given with a gap of 24 hours between the two subsequent treatments.
The time period of boiling was dependent on the hardness of the grain and was adjusted in a
way that the grains should not split apart. Like boiling treatments, one, two and three
autoclaving treatments were also given with a gap of 24 hours between the two subsequent
treatments. The time period of each autoclaving was 20 minutes at 15 lbs psi. The antibiotics
were used in 30, 40 and 50 µg concentration and were added at the time of inoculation.
3.10 Statistical analysis:
The experiments were conducted in completely randomized design with three
replications of each treatment. The analysis of variance was performed using SPSS version
16.0 and means were compared by Duncan’s multiple range tests at 5% level of probability
for interpretation of results (Gomez and Gomez, 1984).
CHAPTER IV
RESULTS
The results of the present investigation “Evaluation of substrates for quality spawn
production of mushrooms” are described here under the following heads:
4.1 Evaluation of grain substrates for the mycelial growth in mushroom spawn
preparation:
The effect of different grain substrates viz., wheat, maize, sorghum, barley, oat and
bajra on the mycelial growth for the preparation of spawn of different mushrooms viz.,
Agaricus bisporus, Pleurotus florida and Calocybe indica was studied. The results depicted
in Table 1 revealed that in case of A.bisporus spawn prepared on sorghum grains after 12
days of inoculation, showed maximum mycelial growth (11.65cm @ 0.404mm/h) followed
by maize (10.61cm @ 0.368mm/h ). However, minimum growth of mycelium was observed
in spawn prepared using bajra grains (5.04 cm @ 0.175mm/h).
After ten days of inoculation, mycelium of P.florida on sorghum grains attained the
maximum growth (11.93cm @ 0.497mm/h) which was statistically at par with maize
(10.90cm @ 0.454mm/h) (Table 2). However, mycelium on bajra grains showed minimum
growth (5.51cm @0.229mm/h).
Mycelial growth of spawn of Calocybe indica has been presented in Table No. 3. The results
reveal that after 10 days of inoculation, maximum mycelial extension (11.36cm @
0.473mm/h) was observed in sorghum grains followed by maize (10.62cm 0.442mm/h ). Like
other mushrooms, minimum growth of mycelium was observed in bajra (4.79cm
@0.199mm/h) after 10 days of inoculation.
4.2 Effect of different grain substrates on the time taken for full mycelial growth
during spawn preparation of different mushrooms:
The time (number of days) taken for full growth of spawn of different mushrooms viz.
A.bisporus, P.florida and C.indica prepared using different substrates viz. sorghum, maize,
Table 1 : Effect of different spawning substrates on the mycelial growth of Agaricus
bisporus
Substrate
Mycelial growth (cm)
Growth rate
( mm/ h)
2 *DAI 4 *DAI 6 *DAI
8*DAI
10 *DAI 12 *DAI
Wheat 1.17
b
2.55b
4.48c
6.26c
7.50bc
8.86c
0.307
c
Maize 2.35
a
5.65a
8.00a
8.91a
9.78a
10.61b
0.368
b
Sorghum 2.49
a
5.69a
8.08a
9.06a
10.13a
11.65a
0.404
a
Barley 1.18
b
2.65b
5.33b
6.74b
8.11b
8.84c
0.306
c
Oat 1.17
b
2.53b
5.15b
6.48bc
7.26c
8.36d
0.290
d
Bajra 0.50
c
0.92c
1.93d
3.00d
4.03d
5.04e
0.175
e
Table 2: Effect of different spawning substrates on the myelial growth of Pleurotu florida
Substrate
Mycelial growth (cm) Growth rate
(mm/h) 2 *DAI 4 *DAI
6 *DAI 8 *DAI 10 *DAI
Wheat 2.24
bc
4.45b
7.66a
8.76bc
9.76b
0.406
b
Maize 2.62
ab
6.03a
8.45a
9.06ab
10.90ab
0.454
ab
Sorghum 2.92
a
6.33a
8.64a
10.40a
11.93
a 0.497
a
Barley 1.69
c
3.56b
6.05b
7.45c
8.73bc
0.363
bc
Oat 1.68
c
3.51b
6.00b
7.32c
8.56c
0.356
c
Bajra 0.91
d
1.53c
2.98c
4.29d
5.51d
0.229
d
Means followed by the same letter(s) within the same column in a treatment group are not
significantly different statistically at 5% level of probability using DMRT.
*DAI- Days after inoculation
Table 3: Effect of different spawning substrates on the mycelial growth of Calocybe
indica
Substrate Mycelial growth (cm) Growth rate
(mm/h) 2 *DAI 4 *DAI
6*DAI 8 *DAI
10 *DAI
Wheat 1.37
c
2.78b
5.54b
6.99c
8.43c
0.351
c
Maize 2.50
b
5.92a
8.07a
9.65b
10.62b
0.442
b
Sorghum 2.80
a
5.98a
8.08a
9.93a
11.36a
0.473
a
Barley 1.36
c
2.74bc
5.45b
6.89cd
8.27c
0.344
c
Oat 1.25
d
2.65c
5.36b
6.77d
8.10c
0.337
c
Bajra 0.76
e
1.08d
2.27c
3.29e
4.79d
0.199
d
*DAI- Days after inoculation
Table 4: Time taken for full growth of spawn of different mushrooms using different
grains
Substrate Days taken for full growth
Agaricus bisporus Pleurotus florida Calocybe indica
Wheat 18.66
c
14.66c
17.00c
Maize 15.66
b
12.33b
13.66b
Sorghum 12.66
a
10.33a
10.66a
Barley 18.66
c
16.66d
17.66cd
Oat 19.66
c
17.00d
18.66d
Bajra 25.33
d
22.00e
23.33e
Means followed by the same letter(s) within the same column in a treatment group are not
significantly different statistically at 5% level of probability using DMRT.
wheat, barley, oat and bajra has been presented in Table 4. The results reveal that in case of
A.bisporus spawn prepared using sorghum, maize, wheat, barley, oat and bajra, the time
taken for full growth of spawn was 12.66, 15.66, 18.66, 19.66 and 25.33 days, respectively.
In case of P. florida, the number of days for full growth was 10.33, 12.33, 14.66, 16.66 17.00
and 22.00 days using sorghum, maize, wheat, barley, oat and bajra respectively. However, for
C. indica, sorghum grain spawn took 10.66 days, maize grains 13.66 days, wheat grains
17.00 days, barley grains 17.66 days, oat grains 18.66 days and bajra grains 23.33 days for
full growth.
4.3 Evaluation of grain substrates in combination with supplements for mycelial growth
of spawn:
The effect of four grain substrates viz., sorghum, maize, wheat and barley in combination
with supplements viz. gram husk, paddy husk and wheat bran in 1:1 ratio (v/v) on the
mycelial growth of spawn of A.bisporus, P.florida and C.indica was studied and the results
have been presented in Table 5, 6 and 7.
In case of spawn of A.bisporus, after 14th day of inoculation, the maximum mycelial
extension (11.31cm @ 0.336mm/h) was observed in sorghum supplemented with gram husk
which was statistically at par with maize grain supplemented with gram husk (10.98cm @
0.326mm/h). However, spawn prepared by supplementing barley with paddy husk showed
minimum mycelia growth of 7.11cm @ 0.211mm/h (Table 5). Results presented in Table 6
revealed that after 12 days of inoculation with Pleurotus florida culture, sorghum
supplemented with gram husk attained the maximum mycelial extension (11.52cm @
0.400mm/h) which was statistically at par with maize added gram husk which showed
mycelia growth of 10.90 cm @0.378mm/h mycelia growth in bottles. However, minimum
mycelial extension was observed in barley supplemented with paddy husk (7.43cm @
0.257mm/h). After 12 days of inoculation, in case of mycelial growth of spawn of Calocybe
indica (Table 7) prepared by sorghum grains supplemented with gram husk attained the
maximum mycelial extension (10.98cm @ 0.381mm/h ) which was statistically at par with
maize supplemented with gram husk which showed mycelia growth of 10.92cm @
0.379mm/h. However, barley supplemented with paddy husk attained the minimum growth
(6.89cm @ 0.239mm/h).
Table 5: Effect of different spawning substrates with added supplements on the mycelial
growth of Agaricus bisporus
Substrate
Mycelial growth (cm) Growth rate
(mm/h) 2
DAI*
4
DAI*
6
DAI*
8
DAI*
10
DAI*
12
DAI*
14
DAI*
Wheat+GH 1.20c
2.49bc
5.16b
6.51b
7.81b
8.33b
9.30b
0.276b
Maize+GH 2.36a
5.06a
7.22a
8.34a
9.41a
10.10a
10.98a
0.326a
Sorghum+GH 2.33a
5.63a
7.95a
8.93a
9.80a
10.60a
11.31a
0.336a
Barley+GH 1.07cd
2.32cd
4.99b
6.21b
7.46b
8.01b
8.91b
0.247b
Wheat+PH 0.95de
1.57de
3.24c
4.32c
5.77cd
6.89c
7.80c
0.232c
Maize+PH 1.16c
2.47bc
5.15b
6.49b
7.80b
8.39b
9.33b
0.277b
Sorghum+PH 1.99b
3.30b
4.71b
5.61b
6.79bc
8.09b
8.95b
0.266b
Barley+PH 0.83e
1.13e
2.32c
3.36c
4.91d
6.25c
7.11c
0.211c
Table 6: Effect of different spawning substrates with added supplements on the
mycelial growth of Pleurotus florida
Substrate Mycelial growth (cm) Growth rate
(mm/h) 2
DAI*
4
DAI*
6
DAI*
8
DAI*
10
DAI*
12
DAI*
Wheat+GH 1.83c
3.29b
5.09b
6.27bc
7.47bc
8.38b
0.290b
Maize+GH 2.39ab
5.80a
8.36a
9.36a
10.31a
10.90a
0.378a
Sorghum+GH 2.52a
5.95a
8.57a
9.86a
10.76a
11.52a
0.400a
Barley+GH 1.35d
2.74c
5.44b
6.91b
8.30b
8.99b
0.312b
Wheat+PH 1.18de
2.56c
5.22b
6.60b
7.95b
8.66b
0.300b
Maize+PH 1.37d
2.75c
5.49b
6.93b
8.34b
8.99b
0.312b
Sorghum+PH 2.17b
3.73b
5.18b
6.40b
7.76b
8.72b
0.302b
Barley+PH 1.00e
1.94d
4.10c
5.24c
6.59c
7.43c
0.257c
Means followed by the same letter(s) within the same column in a treatment group are not
significantly different statistically at 5% level of probability using DMRT.
*DAI- Days after Inoculation,
GH-Gram husk
PH-Paddy husk
Table 7: Effect of different spawning substrates with added supplements on the
mycelial growth of Calocybe indica
Substrate
Mycelial growth (cm) Growth rate
(mm/h) 2*DAI
4 *DAI
6 *DAI
8 *DAI
10*DAI 12*DAI
Wheat+GH 1.31
c
2.70c
5.37b
6.82b
8.18b
8.78b
0.304
b
Maize+GH 2.42
a
5.82a
8.39a
9.38a
10.34a
10.92a
0.379
a
Sorghum+GH 2.42
a
5.81a
8.39a
9.46a
10.41a
10.98a
0.381
a
Barley+GH 1.16
c
2.47c
5.15b
6.49b
7.80b
8.39bc
0.291
bc
Wheat+PH 1.07
d
2.33c
4.77b
6.22b
7.47b
8.00c
0.277
c
Maize+PH 1.31
c
2.70c
5.43b
6.88b
8.27b
8.97b
0.311
b
Sorghum+PH 2.17
b
3.64b
4.99b
6.07b
7.31b
8.38c
0.290
c
Barley+PH 0.95
d
1.57c
3.24c
4.31c
5.77c
6.89d
0.239
d
*DAI- Days after inoculation
Table 8: Time taken for full growth of spawn of different mushrooms using
supplemented grains
Substrates Days taken for full growth
Calocybe indica Agaricus bisporus Pleurotus florida
Wheat+GH 18.66
bc
19.66bc
17.66c
Maize+GH 14.66
a
16.00a
14.66b
Sorghum+GH 14.33
a
15.66a
13.33a
Barley+GH 19.66
cd
20.66cd
17.66c
Wheat+PH 20.66
de
21.66de
19.33d
Maize+PH 18.00
b
19.66bc
17.66c
Sorghum+PH 17.66
b
18.66b
17.33c
Barley+PH 21.66
e
22.66e
21.33e
Means followed by the same letter(s) within the same column in a treatment group are not
significantly different statistically at 5% level of probability using DMRT.
GH-Gram husk
PH-Paddy husk
4.4 Effect of grain substrates in combination with supplements on the time taken for
full growth of spawn of different mushrooms:
The results depicted in the Table 8 reveal that in case of A.bisporus, the spawn
prepared by supplementing sorghum grains with gram husk took the minimum time (15.66
days) which was at par with maize supplemented with gram husk supplement in which full
growth of spawn took 16.00 days. However, barley grains supplemented with paddy husk
took the maximum time (22.66 days) for full growth. In case of P.florida, spawn prepared by
supplementing sorghum grains with gram husk took the minimum time (13.33 days) which
was statistically at par with maize grains supplemented with gram husk (14.66 days) for the
full growth of mycelium in spawn bottles. Barley grains supplemented with paddy husk was
the slowest and took the maximum time (21.33days) for full growth of spawn. For C. indica,
the results reveal that sorghum grains supplemented with gram husk, took the minimum time
for full growth of spawn (14.33 days) which was statistically at par with maize grains
supplemented with gram husk (14.66 days) followed by wheat grains supplemented with
gram husk, maize grains supplemented with paddy husk and sorghum grains supplemented
with paddy husk which took 18.66, 18.00 and 17.66 days respectively, for the full growth of
mycelium in spawn bottles. However, barley supplemented with paddy husk took the
maximum time for full mycelial growth in spawn bottles (21.66 days).
4.5 Evaluation of spawn substrates on yield and quality related parameters of different
mushrooms:
Spawn of different mushrooms (A.bisporus, P.florida and C.indica) prepared using
grain substrates viz. sorghum, maize, wheat, sorghum supplemented with gram husk and
maize grains supplemented with gram husk were evaluated for their effect on the yield and
quality related parameters such as spawn run time, days for pin head formation, average
weight of fruiting body, number of fruiting bodies, biological efficiency and economic
cropping period. The results have been presented in Table 9, 10 and 11.
Plate I Substrates for spawn production
Barley grains Maize grains
Oat grains Full mycelial growth on sorghum grains
Sorghum supplemented with gram husk Sorghum supplemented with paddy husk
4.5.1 Effect of spawn substrates on yield and quality related parameters of Agaricus
bisporus:
Results presented in Table 9 revealed that in case of A. bisporus, spawn run time for
bags spawned using sorghum, maize and wheat grains based spawn was 16.33, 17.00 and
17.33 days respectively and were statistically at par with each other. The maximum time for
spawn run was 19.00 and 19.66 days recorded in bags spawned by sorghum grains
supplemented with gram husk and maize grains supplemented with gram husk respectively.
Similar results were obtained in case of days taken for pin head formation where sorghum,
maize and wheat grains took 35.33, 36.00 and 36.33 days respectively and were statistically
at par with each other. However, maize grains supplemented with gram husk took the
maximum time (41.00 days) for pinhead formation.
The data regarding average fruit body weight and number of fruit bodies of
A.bisporus reveals that the fruit bodies grown using sorghum grain spawn and maize grain
spawn recorded highest fruit body weight of 8.33g and 8.00g respectively followed by wheat
grain spawn (7.66g). However, the weight of the fruit body of maize supplemented with gram
husk spawn was lowest 6.66g. Sorghum grain based spawn recorded highest number of
fruiting bodies (22.00) followed by maize (21.00) while maize supplemented with gram husk
had minimum number of fruiting bodies (19.00).
In case of biological efficiency data, sorghum spawn recoded the highest biological
efficiency of 18.40% which was statistically at par with maize and wheat grain spawn giving
18.30% and 16.80% respectively. However, spawn prepared by using maize supplemented
with gram husk gave the lowest biological efficiency (12.70%). In terms of economic
cropping period, sorghum, maize and wheat spawn were reported to be statistically at par
having the economic cropping period of 81.00, 82.00 and 82.66 days respectively. Maize
supplemented with gram husk had the longest economic cropping period (91.00 days).
Table 9: Effect of different spawn substrates on yield and quality related parameters of
Agaricus bisporus
Spawn
substrates
Days
for
spawn
run
Days for
pinhead
formation
Economic
cropping
period
Avg.
Weight
(g/Frui
t body)
No. of Fruit
body
harvested
Yield
(g/kg)
Biological
efficiency
(%)
Sorghum 16.33a
35.33a
81.00a
8.33a
22.0a
184.0a
18.40a
Maize 17.00a
36.00a
82.00a
8.00a
21.0b
183.0a
18.30a
Wheat 17.33a
36.33a
82.66a
7.66ab
20.0c
168.0ab
16.80ab
Sorghum+GH 19.00b
38.66b
87.33b
7.00b
19.33cd
145.0bc
14.50bc
Maize+GH 19.66b
41.00c
91.00c
6.66b
19.0d
127.0c
12.70c
Table 10: Effect of different spawn substrates on yield and quality related parameters
of Pleurotus florida
Spawn
substrate
Days
for
spawn
run
Days for
pinhead
formation
Economic
Cropping
Period
Avg.
Weight
(g/fruit
body)
No. of
fruit body
harvested
(per kg)
Yield
(g/kg)
Biological
efficiency
(%)
Sorghum 19.66a
30.66a
71.66a
8.83a
63.33a
559.5a
55.95a
Maize 20.66ab
31.66ab
72.66ab
8.66ab
62.00ab
537.5ab
53.75ab
Wheat 21.00b
31.66ab
73.33b
8.50ab
62.00ab
526.0b
52.6b
Sorghum+GH 22.66c
32.66bc
74.66c
8.16b
60.66ab
495.3c
49.53c
Maize+GH
22.66c
33.33c
75.33c
8.00c
58.33b
466.6c
46.66c
Means followed by the same letter(s) within the same column in a treatment group are not
significantly different statistically at 5% level of probability using DMRT.
GH-Gram husk
4.5.2 Effect of spawn substrates on yield and quality related parameters of Pleurotus
florida:
Results presented in Table 10 reveal that the substrate (wheat straw) bags spawned
with sorghum grains took the minimum time (19.66 days) for spawn run which was
statistically at par with maize spawn (20.66 days). However, bags spawned with spawn
prepared from sorghum supplemented with gram husk and maize supplemented with gram
husk took the maximum time (22.66 days) for full spawn run of P.florida bags. Similar trend
was observed for pin head formation, where sorghum, maize and wheat took the minimum
time of 30.66, 31.66 and 31.66 days, respectively. However, sorghum supplemented with
gram husk and maize supplemented with gram husk took the maximum time of 45.66 and
46.33 days, respectively for pinhead formation. In terms of average fruit body weight of
P.florida, the results revealed that the fruit body weight from bags spawned with spawn
prepared from sorghum, maize and wheat grains were statistically at par with each other with
average weight of 8.83, 8.66 and 8.50g, respectively. However, the average fruit body weight
was observed to be 8.16g in case of fruit bodies yielded from sorghum modified with gram
husk spawned bags and 8.00g in case of fruit bodies harvested from bags spawned with maize
supplemented with gram husk. In P. florida, sorghum spawn had the maximum number of
fruiting bodies (63.33) which was statistically at par with maize (62.00), wheat (62.00) and
sorghum supplemented with gram husk (60.66). Maize supplemented with gram husk has the
minimum number of fruiting bodies (58.33).
In terms of biological efficiency of different spawn substrates, sorghum spawn gave
the highest biological efficiency (55.95%) which was statistically at par with maize grain
based spawn (53.75%) and wheat grain based spawn (52.6%). However, spawn prepared with
maize supplemented with gram husk had the lowest biological efficiency (46.66%) when
used for spawning wheat straw substrate bags. Similar trend was observed in case of
economic cropping period, where sorghum spawn has the minimum economic cropping
period (71.66 days) which was statistically at par with maize (72.66 days). However, maize
supplemented with gram husk spawn has the longest economic cropping period (75.33 days).
Table 11: Effect of different spawn substrates on yield and quality related parameters
of Calocybe indica
Spawn
substrates
Days
for
spawn
run
Days for
pinhead
formation
Economic
Cropping
period
Avg.
Weight
(g/fruit
body)
No. of
Fruit body
harvested
Yield
(g/kg)
Biological
efficiency
(%)
Sorghum 14.66a
25.00a
70.33a
61.00a
11.00a
670.6a
67.06a
Maize 17.33b
29.00b
76.66b
60.66a
10.66a
662.6a
66.26b
Wheat 17.66bc
30.00b
77.66bc
58.66b
10.66ab
625.6ab
62.56ab
Sorghum+GH 18.33bc
31.00b
79.33c
58.33bc
9.66ab
564.0bc
56.40bc
Maize+GH 18.66c
31.33b
80.00c
57.00c
9.33b
531.6c
53.16c
Means followed by the same letter(s) within the same column in a treatment group are not
significantly different statistically at 5% level of probability using DMRT.
GH-Gram husk
4.5.3 Effect of spawn substrates on yield and quality related parameters of Calocybe
indica:
Results presented in Table 11 reveal that during the cultivation of C. indica, sorghum
grain spawn took the minimum time for spawn run (14.66 days) followed by maize (17.33
days). However, maximum (18.66) days for spawn run was taken by spawn prepared with
maize supplemented with gram husk. For pinhead formation, bags spawned with sorghum
grain spawn took the minimum number of days (25.00 days). However, all other substrates
viz. maize, wheat, sorghum supplemented with gram husk and maize supplemented with
gram husk were statistically at par with each other taking 29.00, 30.00, 31.00 and 31.33 days
respectively. Average fruit body weight of C. indica harvested from bags spawned with
sorghum and maize spawn had the maximum average fruit body weight of 61.00g and 60.66g
respectively followed by wheat (58.66g). However, the average weight of fruit bodies
harvested from maize added gram husk spawned was the lowest (57.00g). Similar trend was
observed in case of number of fruit bodies harvested when spawn prepared from sorghum and
maize grains had highest number of fruiting bodies i.e 11.00 and 10.33 respectively).
However, spawn from maize supplemented with gram husk yielded the minimum number
(9.33) of fruiting bodies.
In terms of biological efficiency, spawn prepared from sorghum and maize grains
showed the highest biological efficiency of 67.06% and 66.26% respectively. However,
biological efficiency in C.indica bags spawned with maize supplemented with gram husk
recorded the lowest biological efficiency (53.16%). In terms of economic cropping period,
sorghum had the shortest economic cropping period (70.33 days) followed by maize (76.66
days). However, the longest economic cropping period of 80 days was observed in case of
C.indica bags spawned with maize added gram husk.
4.6 Effect of grain substrates on incidence of contamination:
Incidence of contamination was observed during spawn production in all the six grain
substrates and the results have been presented in Table 12. The data reveals that four types of
contaminants were observed in the grain spawn substrates. These
Table 12: Incidence of contamination of spawn prepared using different grains
Spawn
substrate
Contaminants Contamination %
Individual Combined
2.33
G.Total
Wheat Aspergillus spp. 6.00
18.33b Penicillium spp. 3.33
Trichoderma spp. 2.66
Bacillus spp. 4.00
Total 16.00
Bajra Aspergillus spp 4.00
3.00
20.66c
Penicillium spp 2.66
Trichoderma spp 1.66
Bacillus spp. 9.33
Total 17.66
Maize Aspergillus spp 4.66
2.33
14.33a
Penicillium spp 2.33
Trichoderma spp 2.66
Bacillus spp. 2.33
Total 12.00
Sorghum Aspergillus spp 3.00
2.00
12.66a
Penicillium spp 2.66
Trichoderma spp 2.33
Bacillus spp. 2.66
Total 10.66
Barley Aspergillus spp 5.33
4.00
19.00
Penicillium spp 4.00
Trichoderma spp 2.33
Bacillus spp. 3.33
Total 15.00
Oat Aspergillus spp 5.00
3.33
16.66
Penicillium spp 2.66
Trichoderma spp 2.33
Bacillus spp. 3.33
Total 13.33
Means followed by the same letter(s) within the same column in a treatment group are not
significantly different statistically at 5% level of probability using DMRT.
include three fungal viz., Aspergillus spp.,Penicillium spp. and Trichoderma spp. and
one bacterial contaminant viz. Bacillus spp. The containment were sent to ITCC for
identification and the results are presented in table 18. These contaminants were found
individually as well as collectively in the spawn substrates. Percentage contamination varied
significantly among different spawn substrates. Sorghum grain spawn showed the minimum
contamination (12.66%) which is statistically at par with maize grain spawn (14.33%).
However, maximum contamination of 20.66% was observed in spawn prepared using bajra
grains. Differences were observed in incidence of fungal and bacterial contaminants among
the different types of grain spawn substrate. Bacterial contamination was maximum (9.33%)
in case of bajra spawn and minimum in case of maize (2.33%). Among the fungal
contaminants, Aspergillus spp. was the most prevalent ranging from 3.00 to 6.00% followed
by Penicillium spp. (2.33 to 4.00%). The percentage of mixed contamination varied from
2.00 to 4.00% in different grain substrates.
4.7 Effect of grain substrates in combination with supplements on incidence of
contamination:
The effect of four grain substrates viz. wheat, maize, sorghum and barley in
combination with gram husk and paddy husk on incidence of contamination was observed
during spawn preparation and the results are presented in Table. 13. The results reveal that
four types of contaminants as described in 4.6 appeared individually as well as collectively in
the substrates. Barley supplemented with gram husk, wheat supplemented with gram husk as
well as wheat supplemented with paddy husk had the highest contamination of 18.33, 18.00
and 18.66% respectively. The lowest incidence of contamination was observed in sorghum
supplemented with gram husk (13.33%) which was statistically at par with maize
supplemented with gram husk (15.00%), sorghum supplemented with paddy husk (15.00%),
maize supplemented with paddy husk (16.00%) and barley supplemented with paddy husk
16.66%). Moreover, sorghum supplemented with paddy husk (15.00%), maize supplemented
with paddy husk 16.00 % and barley supplemented with paddy husk 16.66%) are also
statistically at par with barley supplemented with gram husk (18.33%), wheat supplemented
with gram husk (18.00%) and wheat supplemented with paddy husk(18.66%). The incidence
Table 13: Incidence of contamination of spawn prepared using different grains
Sppawn
Substrate
Contaminants Contamination (%)
Individual Combined Total
Wheat+GH Asppergillus spp. 6.33
3.66
18.00b
Penicillium spp. 2.33
Trichoderma spp. 2.33
Bacillus spp. 3.33
Total 14.33
Maize+GH Asppergillus spp. 5.00
3.00
15.00ab
Penicillium spp. 2.33
Trichoderma spp. 2.00
Bacillus spp. 2.66
Total 12.00
Sorghum+GH Asppergillus spp. 3.00
2.66
13.33a
Penicillium spp. 2.00
Trichoderma spp. 1.33
Bacillus spp. 4.33
Total 10.66
Barley+GH Asppergillus spp. 6.00
3.33
18.33b
Penicillium spp. 2.33
Trichoderma spp. 1.66
Bacillus spp. 5.00
Total 15.00
Wheat+PH Asppergillus spp. 5.00
3.00
18.66b
Penicillium spp. 2.66
Trichoderma spp. 2.33
Bacillus spp. 5.66
Total 15.66
Maize+PH Asppergillus spp. 5.00
2.66
16.00ab
Penicillium spp. 1.66
Trichoderma spp. 1.66
Bacillus spp. 5.00
Total 13.33
Sorghum+PH Asppergillus spp. 5.33
2.00
15.00ab
Penicillium spp. 1.66
Trichoderma spp. 1.33
Bacillus spp. 4.66
Total 13.00
Barley+PH Asppergillus spp. 4.33
2.66
16.66ab
Penicillium spp. 1.66
Trichoderma spp. 1.33
Bacillus spp. 6.66
Total 14.00
Means followed by the same letter(s) within the same column in a treatment group are not
significantly different statistically at 5% level of probability using DMRT.
GH-Gram husk
PH-Paddy husk
of fungal and bacterial contaminants also varied with the type of spawn substrates.
The Bacterial contamination was highest (6.66%) in case of spawn prepared with barley
supplemented with paddy husk and lowest (2.66%) in case of maize supplemented with gram
husk. Among the fungal contaminants, Aspergillus spp. was the most prevalent (3.00-6.33%)
whereas the Trichoderma spp. was the lowest (1.66-2.33%) in almost all the substrates.
Incidence of contamination showing presence of more than one contaminant ranged from
2.00-3.66% in various spawn substrates.
4.8 Management of contamination:
Various physical and chemical treatments for management of contamination of spawn
including boiling treatments, autoclaving treatments and use of antibiotics were evaluated and
the results are presented in Table 14 and 15.
4.8.1 Effect of different boiling treatments for management of contaminants:
The effect of one, two and three boiling treatments was tested for the management of
contamination and significant variation was found in the results for both fungal and bacterial
contaminants (Table 14). The results reveal that the treatment comprising of three boiling
showed maximum efficiency in management of fungal and bacterial contaminants and
reduced the fungal contamination up to 78.33%. However, the reduction in fungal
contamination was only up to 36.66% in the treatment comprising of one boiling. Similarly,
the treatment of three boiling reduced Bacterial contamination up to 88.33% whereas it was
up to 23.33% in case of treatment comprising of one boiling only.
4.8.2 Effect of different autoclaving treatments for management of contaminants:
Like boiling treatments, the effect of one, two and three autoclaving treatments was
tested for the management of contamination and significant variation was observed in
management of fungal and bacterial contamination and the results are represented in table 15.
Up to 98.66% reduction in fungal as well as bacterial contamination of spawn was observed
with three autoclavings where as it was up to 54.00% in case of fungal and 23.33% in case of
bacterial contamination after one autoclaving treatment.
Table 14: Evaluation of boiling treatments for management of spawn contamination
No. of Boiling Reduction in fungal
spoilage symptoms (%)
Reduction in bacterial spoilage
symptoms (%)
I 36.66
c
23.33c
II 58.33
b
68.33b
III 78.33
a
88.33a
Table 15: Evaluation of different autoclaving treatments for management of spawn
contamination
No. of Boiling Reduction in fungal spoilage
symptoms (%)
Reduction in bacterial
spoilage symptoms (%)
I 54.00
c
23.33c
II 89.33
b
83.33b
III 98.66
a
98.66a
Means followed by the same letter(s) within the same column in a treatment group are not
significantly different statistically at 5% level of probability using DMRT.
Table 16: Evaluation of chemical treatments for the management of spawn
contamination (%)
Treatment Concentration.
(µg/kg of seed)
Reduction in spoilage
percentage
Streptocycline 30 79.33c
40 93.33ab
50 96.66a
Streptomycin 30 88.33b
40 93.33ab
50 96.66a
Tetracycline 30 73.33c
40 88.33b
50 98.33a
Vancomycin 30 18.33de
40 23.33d
50 25.00d
Bleaching Powder 30 11.66e
40 11.66e
50 11.66e
Neomycin 30 18.33de
40 18.33de
50 18.33de
Means followed by the same letter(s) within the same column in a treatment group are not
significantly different statistically at 5% level of probability using DMRT.
Plate II Contamination in mushroom spawn
Contaminated spawn
Bacterial contamination Contamination at later stages of spawn maturity
Pure culture of of fungal contaminants Pure culture of bacterial contaminants
4.8.3 Effect of chemical treatments for management of bacterial contaminants:
The results of efficacy of different antibiotics in the management of bacterial
contaminants of spawn have been presented in Table 14. The results reveal significant
variation among these antibiotics as well as their concentration in managing the bacterial
contaminants. Tetracycline (50µg/kg) was found best for managing bacterial contamination
of spawn which resulted in the reduction up to 98.33%. However, it was statistically at par
with streptocycline and streptomycin which resulted in the reduction up to 96.66% when used
as 50 µg/kg concentration. The effect of bleaching powder was minimum in management of
spawn spoilage (11.66%).
4.9 Benefit: Cost ratio:
The cost of production and income from the sale of all the three mushrooms viz.
Agaricus bisporus, Pleurotus florida and Calocybe indica was calculated and the results of
Benefit: Cost ratio obtained by cultivating mushrooms using different types of spawn have
been presented in Table 15. The results reveal that both sorghum and maize spawn recorded
highest B: C ratio followed by wheat spawn in all the mushrooms. In case of A.bisporus,
sorghum and maize spawn exhibited highest B: C ratio of 1.91 and 1.90, respectively.
However, minimum B: C ratio (1.31) was observed when the compost was spawned with
maize supplemented with gram husk spawn. In case of Pleurotus florida, sorghum and maize
spawn recorded highest B: C ratio of 2.98 and 2.96 respectively. Minimum B: C ratio was
observed in spawn prepared from maize supplemented with gram husk substrate (2.70). In
case of C.indica, similar results were observed where sorghum spawn exhibited
highest B:C ratio of 3.91 followed by maize 3.90. However, minimum B: C ratio (3.29) was
recorded in bags spawned with maize supplemented with gram husk based spawn.
Table 17: B:C ratio of different mushrooms cultivated using spawn prepared by
different substrates
Substrate Calocybe indica Agaricus bisporus Pleurotus florida
Sorghum 3.91
a
1.91a
2.98a
Maize 3.90
a
1.90a
2.96a
Wheat 3.50
b
1.75b
2.83b
Sorghum +GH 3.32
c
1.50c
2.79c
Maize+GH 3.29
c
1.31d
2.70d
Means followed by the same letter(s) within the same column in a treatment group are not
significantly different statistically at 5% level of probability using DMRT.
GH-Gram husk
Table 18: Identification of contaminants from ITCC
I.D. No. Source Fungus
9583.14 Mushroom spawn Aspergillus niger
9584.14 Mushroom spawn Aspergillus flavus
9585.14 Mushroom spawn Penicillium+Aspergillus
Plate III Mushrooms cultivated using different types of Spawn
Pleurotus florida
Agaricus bisporus
Calocybe indica
CHAPTER-5
DISCUSSION
The results of the present investigation “Evaluation of substrates for quality spawn
production of mushrooms” are discussed here under:
5.1 Evaluation of grain substrates for the mycelial growth in mushroom spawn:
The effect of different grain substrates (wheat, maize, sorghum, barley, oat, bajra,
sorghum supplemented with gram husk, maize supplemented with gram husk, wheat
supplemented with gram husk, barley supplemented with gram husk, sorghum supplemented
with paddy husk, maize supplemented with paddy husk, wheat supplemented with paddy
husk and barley supplemented with paddy husk) on the mycelial growth promotion in spawn
of Agaricus bisporus, Pleurotus florida and Calocybe indica was evaluated. The number of
days taken for full growth of spawn was also studied and the results have been presented in
Table 1 to 8. Observations for maximum growth were taken when the spawn bottles prepared
using any of the substrates showed full mycelial growth and the mycelial growth rate was
calculated as mycelial extension (mm/h).
The results revealed that sorghum grain spawn was the best in terms of mycelial
growth in all the three mushrooms viz. A.bisporus, P.florida and C.indica. Out of fourteen
substrates evaluated, maximum growth was observed in sorghum grains in all three
mushrooms i.e. 11.65cm @ 0.404mm/h in A.bisporus spawn after 12 days of inoculation,
11.93cm @ 0.497mm/h in P.florida after 10 days of inoculation and 11.36cm @ 0.473mm/h
in case of C.indica after 10 days of inoculation. However, in spawn bottles prepared using
bajra grains, the growth was minimum, 5.04cm @ 0.175mm/h in A.bisporus, 4.29cm @
5.51mm/h in P.florida and in case of C.indica, it was 4.79cm @ 0.199mm/h. It was found
that grain substrates without supplements were better than when used with supplements for
spawn preparation.
The trend in number of days taken for full growth of spawn prepared using different
substrates was just reverse to the trend of different substrates promoting the mycelial growth
of spawn. In case of A.bisporus spawn, sorghum grains took the minimum time (12.66 days)
for full growth (12.00cm) followed by maize (15.66 days). The maximum time for full
growth was taken by bajra (25.33 days). In case of P.florida, sorghum grains took the
minimum time for full growth of spawn in spawn bottles (10.33 days) followed by maize
(12.33days). The maximum time for full growth of spawn in spawn bottles was taken by
bajra (22.00 days). In case of C.indica, the minimum time for full growth of spawn was taken
by sorghum grains (10.66 days) followed by maize (13.66 days). Bajra grains took the
maximum time (23.33 days) for full growth of spawn in spawn bottles.
Suitability of different substrates for preparation of spawn has been evaluated by
different workers (Solangi, 1988; Mathew, 1996; Jiskani et.al.2000). In our results, sorghum
grains promoted maximum spawn growth of different mushrooms. Our results are in
corroboration with findings of Solangi (1988) who evaluated sorghum, maize, wheat and
pearl millet grains for spawn preparation of Pleurotus ostreatus and reported that sorghum
grains were the best followed by maize, wheat and pearl millet grains. Mathew et al., (1996),
Hafeez et al.,(2000) and Jiskani et al., (2000) also reported sorghum grains to be the best
substrate for spawn preparation of different mushrooms. Senthilnambi et al (2011) also
reported the supremacy of sorghum grains for spawn preparation. It may be due to the reason
that either sorghum may have soft aleuronic layer compared to other substrates or
composition of the aleuronic layer containing protein and starch may be different.
In our results, performance of maize seeds for spawn preparation of P.florida was at
par with sorghum while it was second to sorghum in case of A.bisporus and C.indica. Our
results are in agreement with Mbogoh et al. (2011) who observed clear differences in
mycelial growth in maize, wheat and millet substrates and found that mycelia penetrated the
maize grain faster as compared to wheat grain. The rate of mycelia penetration in the millet
grain was slowest. This is because larger grains like maize have more nutrients for mycelia
growth than smaller grains (Mottaghi, 2004). Tinoco et. al. (2001) however found that larger
the surface area and pore of substrates, more is the mycelial growth rate. For the reason that
corn seeds size are larger than wheat and millet seeds size, consequently, corn seeds pore is
also larger. As a result, it has a fast mycelial growth rate.
The larger grains carry a greater reserve of food material per grain for mushroom
mycelium so the spawn prepared with larger grains can withstand adverse condition such as
poor composting (Bahl, 1984). The results differ from the observations of Hu and Lin (1972)
who found that small grains like millet and grain powder provided more points of inoculums
per gram of spawn, so spawn prepared with smaller or powdered grains will cover the
compost faster and hence yield greater mycelia growth. Rangad and Jandiak (1977) screened
various substrates for spawn production of Pleurotus spp. and reported jowar and bajra grains
as best substrates for spawn production followed by wheat grains. The findings of other
scientists in conformity with present results are Jandaik and Kapoor (1974), Garcha (1981),
Kumar (1997), Sharma (2003) and Narh et al.(2011). All the research findings clearly
established the suitability of grain substrates for spawn preparation of mushrooms.
Sofi et al. (2014) determined that maximum and minimum growth rate was seen in
the corn and millet substrates respectively. Probably the increased mycelium growth in corn
is due to increased ventilation and oxygen concentration in corn substrate as oxygen is one of
the most important environmental factors. It was found that the grains alone were
significantly superior in terms mycelial extension and full spawn growth compared to the
grain spawn in combination with the supplements. Our results are in corroboration with
findings of Sivaprakasan and Kandaswamy (1983) who reported that grain substrates were
better than straw substrates for the production of P. sajor caju spawn. Sharma and Pultoo
(2004) studied the grain substrates like corn, crushed corn, pearl millet, oat, barley, sorghum
paddy and straw substrates like wheat bran, paddy husk, coconut husk, paddy straw, wheat
straw, spent tea leaves, saw dust for spawn production. They reported that sorghum, barley,
pearl millet and corn grains were more efficient than wheat grains. This may be due to the
production of adaptive enzymes with straw which may immediately start utilizing the straw
substrate and by eliminating any lag period which may occur in case of grain spawn. In
practice, cereal grains proved better than other substrates because this may be due to the fact
that unlike millet grains or straw they do not clump up and thus afford better aeration to the
fungus to grow quickly around each grain.
5.2 Evaluation of spawn substrates on yield and quality related parameters of different
mushrooms:
Spawn of different mushrooms (A.bisporus, P.florida and C.indica) prepared using
substrates viz. sorghum, maize, wheat, sorghum supplemented with gram husk and maize
grains supplemented with gram husk were evaluated for their effect on the yield and quality
related parameters associated with mushroom cultivation such as spawn run time, days for
pin head formation, average weight of fruit body, number of fruit bodies, biological
efficiency and economic cropping period. The results have been presented in Table 9, 10 and
11 which reveal that spawn run time for bags spawned using sorghum was minimum (16.33
days in A.bisporus,19.66 days in P.florida and 14.66 days in C.indica). However, in case of
A.bisporus, sorghum, maize and wheat were statistically at par and in P.florida sorghum and
maize were statistically at par in terms of spawn run time. The maximum time for spawn run
was 19.00 and 19.66 days reported in bags spawned with sorghum grains supplemented with
gram husk and maize grains supplemented with gram husk respectively in A.bisporus, and in
P.florida bags spawned with spawn prepared of sorghum supplemented with gram husk and
maize supplemented with gram husk took the maximum time (22.66 days) for full spawn run.
However, in C. indica, maximum (18.66) days for spawn run was taken by spawn prepared
with maize grains supplemented with gram husk. Similar results were obtained in case of
A.bisporus in respect of days for pin head formation which were 35.33, 36.00 and 36.33 days
for sorghum, maize and wheat grains respectively and were found statistically at par while
maize grains supplemented with gram husk took the maximum time (41.00 days) for pinhead
formation. In P. florida, sorghum, maize and wheat took the minimum time of 30.66, 31.66
and 31.66 days respectively, for pin head formation. However, maize supplemented with
gram husk took the maximum time for pinhead formation (33.33 days). In C. indica, for
pinhead formation, bags spawned with sorghum grain spawn took the minimum number of
days (25.00 days). However, all other substrates viz. maize, wheat, sorghum supplemented
with gram husk and maize supplemented with gram husk were statistically at par with 29.00,
30.00, 31.00 and 31.33 days respectively, for pin head formation. The data regarding average
weight of fruit body and number of fruiting bodies of A.bisporus reveals that the fruit bodies
grown using sorghum and maize grain spawn have highest average weight (8.33g 82.00g)
followed by fruit body from wheat grain spawn (7.66gms). However, the weight of the fruit
body of maize supplemented with gram husk spawn was least (6.66g). In terms of average
fruit body weight of P.florida, the results revealed that the average fruit body weight from
bags spawned with spawn prepared from sorghum, maize and wheat grains was 8.83, 8.66
and 8.50g respectively and were statistically at par with each other. However, the average
fruit body weight was observed to be 8.16g in case of fruit bodies yielded from sorghum
supplemented with gram husk spawned bags and 8.00g in case of fruit bodies harvested from
bags spawned with maize supplemented with gram husk. Sorghum spawn recorded highest
number of fruiting bodies (22.00) while maize supplemented with gram husk gave minimum
number of fruiting bodies (19.00) in case of A. bisporus. In P. florida, sorghum spawn had
the highest number of fruiting bodies (63.33) which was statistically at par with maize and
wheat (62.00), and sorghum supplemented with gram husk (60.66). Maize supplemented with
gram husk had the least number of fruiting bodies (58.33) in P. florida. In C. indica,
sorghum recorded highest number of fruiting bodies (11.00) which was statistically at par
with maize (10.66). However, spawn from maize supplemented with gram husk gave the least
number (9.33) of fruiting bodies.
In terms of biological efficiency, similar results were obtained in all the three
mushrooms viz. A.bisporus, P.florida and C.indica where sorghum spawn had the highest
biological efficiency which was statistically at par with biological efficiency of maize i.e.
18.4% and 18.3%, respectively, in A. bisporus, 55.95% and 53.75% respectively in P. florida
and 67.06% and 66.26% respectively in C. indica. However, spawn prepared by using maize
supplemented with gram husk has the lowest biological efficiency of 12.7%, 46.66% and
53.16% in A. bisporus, P. florida and C. indica respectively. In terms of economic cropping
period, sorghum, maize and wheat spawn were reported to be statistically at par having the
economic cropping period of 81.00, 82.00 and 82.66 days respectively in A. bisporus whereas
maize supplemented with gram husk has the longest economic cropping period (91.00 days).
In case of economic cropping period of P.florida,sorghum spawn had the minimum economic
cropping period (71.66 days) which was statistically at par with maize (72.66 days).
However, maize supplemented with gram husk spawn had the longest economic cropping
period (75.33 days). For C. indica, sorghum had the smallest economic cropping period
(70.33 days) followed by maize (76.66 days). However, the longest economic cropping
period of 80 days was observed in case of C.indica bags spawned with maize added gram
husk.
Different workers across the globe have investigated the various yield and quality
related parameters of mushrooms cultivated using spawn prepared from different substrates.
Sharma (2003) reported that different grain spawn viz jowar, kutki, kodo, maize and wheat
influenced the duration of spawn run and pinhead formation as well as the biological
efficiency of cultivated mushrooms. The grain substrates were better than straw substrates for
the production of Pleurotus sajor caju (Sivaprakasan and Kandaswamy, 1983). Our results
are in corroboration with findings of Senthilnambi et al. (2011) who reported the supremacy
of sorghum grain as the most suitable substrate for early spawn run in C. indica, which took
only 13.7 days for complete mycelial growth. The yield and number of buttons harvested
were maximum in the spawn prepared from sorghum grain, which recorded 390.6g/bed and
5.7 buttons, respectively. The days for pinhead formation and first harvest of the crop were
earlier in the case of spawn prepared using sorghum grain followed by ragi. Similar results
were reported by Doshi et al. (1989) who observed that early fruit body production was
noticed on sorghum grain spawn. The variation in the colonization of different substrates
could be due to the variation in the amount of moisture observed during boiling, which is one
of the critical factors responsible for mycelial growth (Mehta, 1985). Rangad and Jandaik
(1977) observed maximum yield with sorghum spawn in different Pleurotus spp.
Sivaprakasam and Kandaswamy (1981) obtained good yield of P. sajor-caju with sorghum
and pearl millet. However, our results are in contradiction to Senthilnambi et al. (2011) as
they recorded low yield from maize grains spawn as compared to other spawn substrates.
5.3 Effect of spawn substrates on incidence of contamination and management of
contamination:
Incidence of contamination was observed during spawn production in all the six grain
substrates and eight grain added supplement substrates and the results have been presented in
Table 12 and 13. The data reveals that four types of contaminants were observed in all spawn
substrates. These included three fungal viz. Aspergillus spp., Penicillium spp. and
Trichoderma spp. and one bacterial contaminant viz. Bacillus spp. These contaminants were
found individually as well as collectively in the spawn substrates. Percentage contamination
varied significantly among different spawn substrates. Sorghum grain spawn showed the least
contamination (12.66%) and the maximum contamination was observed in spawn prepared
by using bajra grains (20.66%). Differences were observed in incidence of fungal and
bacterial contaminants among the type of spawn substrate. Bacterial contamination was
maximum (9.33%) in case of bajra spawn and least in case of maize (2.33%). Among the
fungal contaminants, Aspergillus spp. was the most prevalent ranging from 3.00 to 6.33%
followed by Penicillium spp.(1.66 to 4.00%). The percentage of mixed contamination was
2.00 to 4.00% in different substrates.
Various physical and chemical treatments for management of contamination of spawn
including boiling treatments, autoclaving treatments and use of antibiotics were evaluated and
the results are presented in Table 14, 15 and 16. The effect of one, two and three boiling
treatments was tested for the management of contamination and significant variation was
found in the results for both fungal and bacterial contaminants (Table 14). The results reveal
that the treatment comprising of three boiling showed maximum efficiency in management of
fungal and bacterial contaminants and reduced the fungal contamination up to 78.33%.
However, the reduction in fungal contamination was up to 36.66% in the treatment
comprising of one boiling. Similarly, the treatment of three boiling reduced bacterial
contamination up to 88.33% whereas reduction was up to 23.33% in case of treatment
comprising of one boiling. Like boiling treatments, the effect of one, two and three
autoclaving treatments was tested for the management of contamination and significant
variation was observed in management of fungal and bacterial contamination through the
treatments comprising of varying number of autoclavings (Table 15). Up to 98.66% reduction
in fungal as well as bacterial contamination of spawn was observed with three autoclavings
where as it was up to 54.00% in case of fungal and 23.33% in case of bacterial contamination
after one autoclaving treatment.
The results of efficacy of different antibiotics in the management of bacterial
contaminants of spawn have been presented in Table 16. The results reveal significant
variation in the effect of these antibiotics as well as their concentration in managing the
bacterial contaminants. Tetracycline (50µg/kg) was found best for managing bacterial
contamination of spawn which results in the reduction up to 98.33% of bacterial
contamination. However, it was statistically at par with streptocycline and streptomycin
which resulted in the reduction up to 96.66% when used at 50 µg/kg concentration and up to
93.30% when used at 40 µg/kg concentration respectively. The effect of bleaching powder
was minimum in management of spawn spoilage (up to 11.66%). Contamination of spawn,
identification of major contaminants and comparison of different methods for the
management of these contaminants has been worked out by Mazumder et al. (2005), Mishra
and Shukla (2007) and Earanna et al. (2010). Mazumder et al., (2005) isolated and identified
eight fungal contaminants and one bacterial contaminant (Bacillus brevis) from severly
contaminated spawn bearing wet spot symptom from the naturally contaminated paddy grain
base spawn and reported that contamination on paddy was significantly lower (15.00%) than
the wheat grain (30.00%). High incidence of contamination in wheat grains as compared to
paddy grains might be due to soft texture of wheat seeds with very thin seed coat creating
portal for easy entry of any bacteria including B.brevis into the wheat seeds. Moreover,
Suman and Jandaik stated that wheat grain itself was considered as the primary source of
contamination. Similar studies have also been made on isolation and identification of various
microorganisms from contaminated spawn (Bitner, 1971; Biserka 1972, Suman and Jandaik,
1992 and Singh et al., 2002). Earlier many species of Bacillus were reported from
contaminated spawn of oyster and button mushroom (Biserka, 1972; Suman and Jandaik,
1992; Pattanaik, 1998; Ahlawat et al., 1999; Singh et al., 2002).
Wheat itself has been considered as primary source of contamination (Suman and
Jandaik, 1992 ). About 34 species of bacteria, mostly Bacillus, Pseudomonas and
Xanthomonas spp. have been reported to be associated with cereal grains (Pepper and
Kiesling,1963). Singh et al. (2009) isolated and identified various moulds (Penicillium,
Aspergillus, Rhizopus, Mucor, Dehliomyces) and one bacteria (Bacillus spp.) from the spawn
of Agaricus bisporus. They reported that the percentage of contaminants in spawn normally
ranged from 2.0 to 15.0 percent but in certain cases it may increase up to 24.0 to 63.0%. The
maximum spoilage was caused by Penicillium spp.(39.3%) followed by Mucor spp. (25.9%)
and Aspergillus spp. (14.7%). However, bacterium Bacillus spp. caused least contamination
(2.6%) in the spawn bags of button mushroom. Suman (1993) also reported that the spawn
spoilage by various contaminants varied from 1.0% to 6.3% but in certain cases it was as high
as 20.2%. Aspergillus spp. contributed to the maximum spoilage (28.1%) followed by
Penicillium spp. (11.7%). Suman and Jandaik (1992) while studying the microbial
contaminants of spawn of A. bisporus reported that the sources of contaminants are both the
un-sterilized wheat grains and microbes present in spawn laboratory environment. Mazumder
and Rathaiah (2001) found Trichoderma harzianum, Aspergillus spp. and Penicillium spp. as
the three most dominant fungal contaminants during spawn production in oyster mushroom.
Three isolates of Bacillus subtilis from contaminated spawn bags were isolated and
characterized by Ahlawat et al. (1999).
Our results are in corroboration with Ahlawat et al. (1997), who indicated that in the event of
high incidence of bacterial contamination due to seasonal or any other condition, a
combination of chemical and physical methods may become indispensable. They reported
under in vitro conditions all the bacterial isolates were found sensitive to neomycin,
streptomycin and streptocycline. They also reported that ‘wet spot’ symptom development
decreased with increasing number of boiling and autoclaving treatments, and negligible
symptoms appeared in substrate bags after 3rd
boiling treatment followed by single
autoclaving. Moreover, no symptom appeared after 3rd
autoclaving treatment.
5.4 Benefit: cost ratio:
In our studies, production of spawn with different substrates resulted in varying
benefit: cost ratios. Highest benefit: cost ratio was recorded when spawn prepared using
sorghum and maize followed by wheat was used in all three mushrooms. In case of
A.bisporus, sorghum and maize spawn exhibited highest B: C ratio of 1.91 and 1.92
respectively. However, minimum B: C ratio (1.31) was observed when the compost was
spawned with maize supplemented with gram husk spawn. In case of P. florida, sorghum
and maize spawn recorded highest B: C ratio of 2.98 and 2.99 respectively. Minimum B: C
ratio was observed in spawn prepared from maize supplemented with gram husk substrate
(2.70). In case of C.indica, similar results were reported where sorghum spawn exhibited
highest B: C ratio of 3.91 followed by maize 3.93. However, minimum B: C ratio (3.29) was
recorded in bags spawned with maize supplemented with gram husk based spawn.
CHAPTER-6
SUMMARY AND CONCLUSION
The findings of the present investigation “Evaluation of substrates for quality spawn
production of mushrooms” are summarized and concluded as under:
• All the grain substrates with and without supplements favoured the mycelial growth in
spawn of different mushrooms except when the grains were supplemented with wheat
bran. However, rate of mycelial growth in spawn, number of days taken for full
mycelial growth in spawn bottles, spawn run in substrate bags, days taken for initial
pinhead formation, average weight of sporocarps at maturity, economic cropping
period, incidence of contamination, biological efficiency and benefit: cost ratio varied
with different spawn substrates
• Sorghum grains took the minimum time for mycelial run in spawn bottles (12.66 days
for Agaricus bisporus, 9.66 days for Pleurotus florida and 10.66 days in case of
Calcybe indica) followed by maize (15.66 days for Agaricus bisporus, 12.33 days for
Pleurotus florida, and 13.66 days for Calocybe indica) while bajra grains took the
maximum time for mycelia run and consequent full mycelial colonization of spawn in
spawn bottles (25.33, 22.00 and 23.33 days in case of Agaricus bisporus, Pleurotus
florida and Calocybe indica respectively).
• In case of A. bisporus, spawn run time in compost bags spawned using sorghum,
maize and wheat grains spawn was 16.33, 17.00 and 17.33 days respectively and were
statistically at par with each other. The maximum time for spawn run was 19.00 and
19.66 days reported in bags spawned by sorghum grains supplemented with gram
husk and maize grains supplemented with gram husk spawn respectively.
• Similar results were obtained in case of initial pin head formation where compost
bags spawned using sorghum, maize and wheat grain took 35.33, 36.00 and 36.33
days for pin head formation. The maximum time for pin head formation was 41.00
days reported in bags spawned by maize grains supplemented with gram husk.
• In Pleurotus florida, the substrate (wheat straw) bags spawned with sorghum grains
took the minimum time (19.66 days) for spawn run which was statistically at par with
maize grains (20.66 days). However, bags spawned with spawn prepared of sorghum
supplemented with gram husk and maize supplemented with gram husk took the
maximum time (22.66 days) for full spawn run of P.florida bags.
• Similar results were obtained for pin head formation where the substrate (wheat
straw) bags spawned with sorghum grains took the minimum time for pin head
formation (30.66days). However, bags spawned with spawn prepared with maize
supplemented with gram husk took the maximum time (33.33 days).
• For cultivation of C. indica, sorghum grain spawn took the minimum time for spawn
run (14.66 days) followed by maize (17.33 days). However, maximum (18.66) days
for spawn run was taken by spawn prepared with maize supplemented with gram
husk. For pinhead formation, bags spawned with sorghum grain spawn took the
minimum number of days (25.00 days).
• Highest biological efficiency of 18.40%, 55.95% and 67.06% was obtained in
A.bisporus, P. florida and C.indica respectively by using spawn prepared by sorghum
grains, while minimum biological efficiency of 12.70%, 46.66% and 53.16% in
A.bisporus, P.florida and C.indica was obtained by using maize added gram husk.
• Maximum B:C ratio of 1.91, 2.98, and3.91 was obtained in A.bisporus, P. florida and
C. indica, respectively by using spawn prepared by sorghum grains. However,
sorghum and maize were found at par in all the three species of mushroom.
• The contamination in spawn ranged from12.66% (sorghum) to 20.66% (bajra). Four
major types of contaminants were observed which included three fungal viz.,
Aspergillus spp., Penicillium spp. and Trichderma spp. and one bacterial viz., Bacillus
spp. These contaminants were found individually as well as in combination in all the
grain substrates. The incidence of bacterial contamination was maximum in bajra
grain spawn and least in sorghum grain spawn.
• The treatment comprising of three boiling showed maximum efficiency in
management of fungal and bacterial contaminants that reduced the fungal
contamination up to 78.33%. Up to 98.66% reduction in fungal as well as bacterial
contamination of spawn was obtained with three autoclavings.
• Tetracycline (50µg/kg) was found best for managing bacterial contamination of
spawn which resulted in the reduction up to 98.33% of bacterial contamination.
However, it was found statistically at par with streptocycline and streptomycin.
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