evaluation of substrates for quality spawn production … · certificate-ii we the members of...

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


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


supplements on the mycelial growth of Pleurotus florida

17


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


related parameters of Pleurotus florida

19


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


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.


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.


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


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



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


(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



*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


(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


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



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



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



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



Bacillus spp. 2.33

Total 12.00

Sorghum Aspergillus spp 3.00

2.00

12.66a



Bacillus spp. 2.66

Total 10.66

Barley Aspergillus spp 5.33

4.00

19.00



Bacillus spp. 3.33

Total 15.00

Oat Aspergillus spp 5.00

3.33

16.66



Bacillus spp. 3.33

Total 13.33



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


Bacillus spp. 3.33

Total 14.33

Maize+GH Asppergillus spp. 5.00

3.00

15.00ab



Bacillus spp. 2.66

Total 12.00

Sorghum+GH Asppergillus spp. 3.00

2.66

13.33a



Bacillus spp. 4.33

Total 10.66

Barley+GH Asppergillus spp. 6.00

3.33

18.33b



Bacillus spp. 5.00

Total 15.00

Wheat+PH Asppergillus spp. 5.00

3.00

18.66b



Bacillus spp. 5.66

Total 15.66

Maize+PH Asppergillus spp. 5.00

2.66

16.00ab



Bacillus spp. 5.00

Total 13.33

Sorghum+PH Asppergillus spp. 5.33

2.00

15.00ab



Bacillus spp. 4.66

Total 13.00

Barley+PH Asppergillus spp. 4.33

2.66

16.66ab



Bacillus spp. 6.66

Total 14.00



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



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



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



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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