INTRODUCTION

Mushrooms belong to the kingdom of Fungi, a group very distinct from

plants, animals and bacteria. Fungi lack the most important feature of plants: the

ability to use energy from the sun directly through Chlorophyll. Thus, fungi

depend on other organisms for food, absorbing nutrients from the organic

material in which they live. The living body of the fungus is mycelium made

out of a tiny web of threads (or filaments) called hyphae. Under specific

conditions, sexually compatible hyphae will fuse and start to form spores. The

larger spore producing structures (bigger than about 1 mm) are called

mushrooms. In nature this is the most striking part of the organism, but in fact it

is just the fruiting body and the major part of the living organism is found under

the ground or inside the wood.

Mushrooms are not plants, and require different conditions for optimal

growth. Plants develop through photosynthesis, a process that converts

atmospheric carbon dioxide into carbohydrates, especially cellulose. While

sunlight provides an energy source for plants, mushrooms derive all of their

energy and growth materials from their growth medium, through biochemical

decomposition processes. This does not mean that light is an unnecessary

requirement, since some fungi use light as a signal for fruiting. However, all the

materials for growth must already be present in the growth medium.

1
Mushrooms grow well at relative humidity levels of around 95-100%, and

substrate moisture levels of 50 to 75%.Instead of seeds, mushrooms reproduce

sexually during underground growth, and asexually through spores. The

biotechnological advances that will help ensure mushroom cultivation without

pollution and provide mushroom products with health enhancers and get more

reliable yields, then improve substrate utilization and control disease more

effectively. Either of these can be contaminated with airborne microorganisms,

which will interfere with mushroom growth and prevent a healthy crop.

Fungi depend on other organisms for their food. Three modes of living

can be recognised Saprophytes degrading already dead material , Symbionts

living together with other organisms (especially trees) in a close, mutually

beneficial relationship , Parasites living at the expense of other organisms.

Saprophytic fungi need organic matter to decompose. In nature they will

grow on fallen leaves, animal droppings, or stumps of dead wood. Some are

specialized in breaking down the hairs of mammals, while

others may decompose bird's feathers. Saprophytes decompose the complex

organic structures left behind by plants and animals. And in the natural run of

things, plants or animals regain access to minerals and other nutrients present in

the substrate. Oyster mushrooms degrade dead wood in nature. They can be

cultivated on a wide range of ligno-cellulose waste materials. The mode of

2
living has nothing to do with edibility both edible and poisonous mushrooms

can be found in all three groups.

Mushrooms are good cash crop; they are rather easy to grow and are

brimming with protein, B vitamins and minerals. Mushrooms have been valued

throughout the world as both food and medicine for thousands of years. They

are rich source of nutrition and form a major chunk of health foods. Fats occur

in mushrooms in minor amounts, especially compared with protein and

carbohydrate and the fatty fraction consists predominantly of unsaturated fatty

acids such as linoleic acid, they may be the perfect food for maintaining a

healthy heart and cardiovascular system

The two most commonly grown species of mushroom in India are white

button mushroom and oyster mushroom .most of the production of white button

in our country is seasonal .The cultivation is done in conventional method.

However, in recent years, yield of mushroom has increased as a result of

introduction of improved agronomic practices. At present three mushrooms are

being cultivated in India; these are Agarics bisporus ,Volvariella vovvacea and

Pleurotus sajor-caju .Of these A.bisporus is the most popular and economically

sound to grow and extensively cultivated through the world. however due to its

low temperature requirement, its cultivation is restricted to the cool climatic

areas and to the winter in the plains of northern India In summer ,the paddy

straw mushrooms suitable for growing in most part of India, as a kitchen garden

3
crop it is preferred because it is very delicious and nutritious. Oyster

mushrooms can grow at moderate temperature ranging from 22 cto 28c.In north

India, the climate conditions prevailing during different seasons can be

exploited for growing mushroom through out the year.

All mushroom growing techniques require the correct combination of

humidity, temperature, substrate (growth medium) and inoculum (spawn or

starter culture). Wild harvests, outdoor log inoculation and indoor trays all

provide these elements.

Kerala is the leading state in the production of coconuts [Thampan,

1997]. Coir pith, a highly lignocellulosic material, is available in large

quantities as a by product of coir industry. Its high lignin concentration coupled

with wide C/N ratio doesn’t permit its direct application to soil. Biodegradation

of coir pith for converting it into a nutrient rich material for plant growth not

only reduces environmental pollution but it is also an eco friendly approach.

The material on which the mycelium of the mushrooms grows is called

substrate. Agricultural waste like wood chips/sawdust, sugar cane bagasse, and

different types of straw can be used as the main ingredients

in the substrate for oyster mushrooms. The properties of a substrate determine

which mushrooms and microbe can grow in it. The more selective it is, the

better the substrate meets the demands of a specific mushroom and the less

suitable it is for others. After mixing and adding certain supplements, the

4
substrate undergoes a heat treatment to give the desired mushroom mycelium an

environment with few competitors.

A number of fungi like Pleurotus sajor-caju were found to be potent

degraders of coir pith [Savithri and Khan, 1994]. Pleurotus spp have been

successfully cultivated on aquatic weeds such as water hyacinth [Murugesan et

al., 1995] and Elio charis punt gene [Subarhan et al., 1993]. Deka et al., (1994)

studied the feasibility of cultivation of P.florida, P.sajor caju on paddy straw

and bamboo leaves. Siva Prakasum and Kandaswamy (1981) and Geetha (1994)

have reported that the yield of sporophores was positively correlated with the

cellulose content and also with the cellulose lignin ratio.

Mushroom production converts raw natural ingredients into mushroom

tissue, most notably the carbohydrate chitin. An ideal substrate will contain

enough nitrogen and carbohydrate for rapid mushroom growth.

               

5
 REVIEW OF LITERATURE

Oyster mushrooms, Pleurotus spp, are edible fungi popularly known

as wood fungi. Pleurotus is a genus of gilled mushrooms which includes one of

the most widely eaten mushrooms, P. ostreatus. Species of Pleurotus may be

called oyster, abalone, or tree mushrooms, and are some of the most commonly

cultivated edible mushrooms in the world. Pleurotus fungi have been used in

mycoremediation of pollutants such as petroleum and polycyclic aromatic

hydrocarbons. The genus was established by Fries in 1821. There are a number

of spp under this genus. According to Pegles (1970) and Singer (1986) this

genus is known to contain 50 spp, of these about 25 spp are known to occur in

India.

It is an 'oyster mushroom'. A number of different species are grown

including, Pleurotus ostreatus, Pleurotus sajor-caju, Pleurotus cystidus,

Pleurotus cystidus, Pleurotus citrinopileatus and Pleurotus flabellatus. This

mushroom is cultivated on a wide range of plant wastes (cereal straw, sawdust,

bagasse, waste cotton) often enclosed by plastic bags. Pleurotus mushrooms are

the second most important mushrooms in production in the world, 25% of total

world production of cultivated mushrooms. Pleurotus mushrooms are world-

wide, China is the major producer.

Plate 1 , 2 and 3 shows the Photos of Pleurotus spp

6
P.sajor-caju

P.florida

P.eous

7
Spawn

The mycelium of mushroom growing in its substrate and prepared for the

purpose of mushroom production is called spawn [Chang, 1982]. Kinden

(1932, 1934) was the first to introduce grain spawn in the cultivation of

mushrooms. Different kinds of grains viz., wheat, rye, millets etc can be used

as spawn substrates [Kotwaliwale et al., (1991)]. The complete procedure of

spawn production involves preparation of the medium, filling the test tubes or

Petri dishes and sterilizing them, and the process of inoculating larger

containers with this culture.

Mother spawn can be used to inoculate either grain spawn or a second

generation of mother spawn. In simple laboratories, grain mother spawn should

not be used to inoculate another generation of grain mother spawn because the

risk of contamination and degeneration will be too high. The main advantage of

grain is that it is very nutritious for fungi and forms kernels easily. The kernels

can easily be dispersed in the substrate. The main disadvantage is that it

provides an optimal substrate for other organisms too. The chances of

contamination are therefore much higher compared to sawdust spawn.

8
Enzymology

Extra cellular enzymes are those enzymes produced within the cells

and then liberated into the external environment to carryout the function of

cultivation of the nutrients in the substrate. The rate of extra cellular enzymes is

the production and activity of the mushrooms. Since, only by their production

and activity can the mycelium grow and produce mushroom fruit bodies. The

extra cellular enzymes are produced to degrade the large insoluble molecules of

the substrates into small soluble molecules which the mycelium can utilize

(Wood, 1990).

Fungal laccases form an important group of enzymes, as they are

involved in the degradation of lignin and in removal of potentially toxic

compounds (Thurston, 1994).For detecting laccase, some assay methods

including HPLC method, manometry, order spectrum method and

spectrophotometry are involved (Zhu et al., 2006).The extra cellular enzymes of

Pleurotus spp play a major role in the degradation of structural elements such as

cellulose, hemi cellulose, lignin and pectin present in the natural substrates.

The enzymes most frequently associated with cellulose degradation are

cellulases and those associated with lignin degradation are laccase.

9
Laccase

Laccase enzyme typically contains 15–30% carbohydrate. It has an

acidic isoelectric point and has a molecule mass of 60–90 kDa. Laccases are the

model enzymes for multi-copper oxidases and participate in cross-linking of

monomers, degradation of polymers, and ring cleavage of aromatic compounds.

For catalyzing the oxidation of non-phenolic substrates, laccase requires the

presence of a mediator in the medium. A mediator is a small molecule that

behaves like an ‘electron shuttle’ between laccase and substrate and these small

molecular-mass compounds are converted into stable radicals by means of

enzymatic oxidation.

The most efficient lignin degrading oyster mushrooms [Zadrazil and

Dube, 1992] has been reported to produce high level of laccase and acyl alcohol

oxidase on their substrates [Kirk and Farell, 1987]. Reddy (1985) reported the

capacity of Pleurotus spp to produce laccase and degrade the part of lignin and

cellulose present in their substrates. Jabionsky (1984) cultivated P.florida on

ground maize and observed high laccase activity. Assay of laccase during

different stages of growth of Pleurotus spp on paddy straw showed that laccase

production reached the maximum after 24 days of incubation and defined

thereafter.

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Lignin degradation

Discovery of novel laccases with different substrate specificities and

improved stabilities is important for industrial applications. Microbes that

produce laccases have been screened for either on solid media containing

coloured indicator compounds that enable the visual detection of laccase

production.

Lignin degradation is important in the global recycling of carbon

because of the least abundance of lignin in the atmosphere and also because it is

an important factor delimiting the degradation of cellulose and other poly

saccharides [Krick and Farell, 1987]. All fungi capable of degrading lignin are

known as white rot fungi. Pleurotus is a white rot fungus capable of degrading

ability of Pleurotus spp [Ibrahim and Pearce, 1980 and Nizkovskaya et al.,

1984].

   

11
 METHODOLOGY

1. Isolation and maintenance of pure cultures of Pleurotus spp.

Isolation and maintenance of pure cultures of Pleurotus spp viz.,

Pleurotus florida, Pleurotus sajor-caju and Pleurotus eous were developed at

the biotechnology lab in Mar Ivanios College by adopting tissue culture

methods[Scarcye 1995].

Mycelium, or actively growing mushroom culture, is placed on growth

substrate to seed or introduce mushrooms to grow on a substrate. This is also

known as inoculation, spawning or adding spawn. Its main advantages are to

reduce chances of contamination while giving mushrooms a firm beginning.

Spores are another inoculation option, but are less developed than established

mycelium. Since they are also contaminated easily, they are only manipulated in

laboratory conditions with laminar flow cabinet.

Tissue from the junction of pileus and stipe of sporocarp was scooped

out and surface sterilized by placing in 95% ethyl alcohol for one minute, after

that the PDA slants were prepared, autoclaved for 15 minutes at 120C. Then

inoculation of mycelium under the laminar flow in PDA slants and incubated at

room temperature for 7-18 days. Following sub culturing it is maintained on

PDA slants.

12
2. Spawn production

The mycelium will colonise the substrate and use the available nutrients.

This is commonly referred to as the spawn run. When some nutrients run out, or

when the weather changes, the mycelium will reach a different phase: the

reproductive stage. A temperature of about 25 °C is optimal for the spawn run

of most species. The environment can also enhance the growth of the desired

mycelium: a high CO2 concentration is favorable for mycelial growth (but not

for cropping). During spawn run stage the mycelium will grow through the

substrate. The spawn run time is different for each species and depends on the

size of the bag, amount of spawn, the strain used and the temperature.

Rice grain spawn of the three Pleurotus spp was prepared adopting the

method described by Siva Prakasam (1980). Rice grains were boiled in water

and are half boiled. After draining excess water, it was mixed with calcium

carbonate at the rate of 50g per kg of rice grains to prevent adhesion of grains

and for optimizing the pH for spawn. Bottles of 750ml capacity were filled with

the grains to 2/3 of its capacity, plugged with cotton and autoclaved at 1.05kg

cm-2 for 2 hours. The substrate should have cooled down (whether pasteurised

by steam or by immersion in hot water) to 30 C. The spawn (3% to 8% of the

weight of the substrate) can be mixed in with when filling the bags. Or a layer

of substrate can be topped with some spawn, layer by layer.

Inoculation of the grains with pure cultures of pleurotus spp was carried

out and incubated at room temperature. The nature of growth and time taken

13
for completing mycelium colonization of the grains was recorded. The spawn

thus prepared was utilized for laying out mushroom beds.

3. Preparation of mushroom beds

Preparation of bags : Use 60x30 cm polythene bags (both side open).Tie one

end of bag, put two holes of 1 cm diameter in the middle.Put handful of cooked

straw in the bag to a height of 5 cm; sprinkle about 25 g of spawn. Layer the

straw to 25 cm height. Repeat the process to get four layers of spawn and 5

layers of straw. Tie the mouth and arrange beds in tiers in the spawn running

room. After 15-20 days, cut and remove the polythene bag and transfer the beds

to cropping room. Keep the beds moist by periodical spraying with water.

In the bed system mushrooms are grown in wooden receptacles which are

approximately 65" wide×60 foot long. These receptacles may be wider or

narrower than 65" and/or they can be shorter or longer than 60 feet long. The

depth of these receptacles is generally from 6" to 8" deep, however, they can be

any suitable size. In what is referred to as a mushroom house these receptacles

are usually stacked in four tiers, In each of these tiers six receptacles are usually

stacked in a superimposed arrangement so that one mushroom house contains 4

tiers and 24 receptacles.

In the tray system mushrooms are also grown in wooden receptacles.

These receptacles or trays can be square or rectangular, however, the most

popular size is 48" wide×72" long×8" deep. In a farm using the tray system

14
approximately 220 of these trays are put into one growing room. The trays are

generally stacked four to five trays high and are arranged in eleven rows four

stacks to a row. A typical mushroom tray is about 175 cm. long by 120 cm.

wide with the tray height being 17.5 cm. and the corner post extending

upwardly from the tray sides a further distance of about 12.5-15 cm.

Mushrooms are grown on decaying organic material. This material can be

horse manure, hay, cereal straw or a variety of other vegetable wastes. The

breakdown of this organic material is accelerated by the mushroom grower by

composting. The purpose of composting is to convert the crude, often variable,

raw material into a medium rich in nutrition which is specific for the growth of

mushrooms. Three different substrates were tried for mushroom production viz.,

paddy straw, non retted coir pith [obtained by manually extracting the

mesocrap] and retted coir pith. Mushroom beds were prepared following the

method described by Baskaran et al., (1978) in the case of paddy straw or non

retted coir pith as the substrate and the method described by Theradinani and

Marimuthu (1991) was adopted for the preparation of beds using retted coir pith

as the substrate.

In the case of paddy straw and non retted coir pith, substrates were

kept in water overnight, boiled for half an hour, drained and dried. In

perforated polythene bags of size 60×30cm, 3 layers of substrate [each of 5 to

8cm thickness] was placed with spawning of each layer. 100gm spawn per

15
mushroom beds was used. The bags were then tied and incubated in the dark

for 15 days, after which they were opened and transferred to cropping room

were adequate ventilation and moisture was maintained.

In the case of retted coir pith, well perforated polythene bags of size

30cm was first filled with 0.5kg of the substrate and inoculated with pleurotus

spawn [at the rate of 100g per kg of the substrate] uniformly over surface and

covered with another layer of 0.5kg of coir pith. This process was repeated till

the bag was filled. The bag was tied and incubated in a room where adequate

moisture and ventilation were present.

The nature and rate of growth and yield characteristics in all the

substrates were recorded. Samples were drawn at 10, 20 and 30 days interval to

study laccase production and also to estimate the laccase content.

4. Cultivation of oyster mushroom

Cultivation of Pleurotus spp on their natural habitat was first described at

the beginning of 20th century [ Falck, 1917] and on a saw dust-cereal mixture by

Kaufert (1935). The foundation for the industrial production of Pleurotus on

different substrates was laid by several workers [Kalberes and Vogel, 1974;

Zadrazil, 1974 and Kurtz man, 1979].

Pleurotus spp have been successfully cultivated of different agricultural

wastes such as mixture of coconut fiber and coffee pulp [Bernabe et al., 1993],

kidney bean stubbles [Sobal et al., 1993], sugarcane bagasse [Shi, 1994] and

cotton wastes [Haq et al., 1994].

16
5. Enzyme Assay
Mycelium is used as the source of extra cellular enzyme. To detect intra

cellular laccase, grind the mycelium in 4ml of cold 0.1M phosphate buffer p H in

a pre-chilled mortar with pestle. Centrifuge the homogenate at 9000rpm for

24min to remove cell debris. Use supernatant as enzyme source.

Pipette 4ml of sodium phosphate buffer, pH 6 containing guaiacol into test

tubes and equilibrate at 25C. Add 0.1ml of the enzyme source. After 5min,

determine the absorbance at 470nm. Express the results as enzyme units.

Maintain control with heat killed enzymes.

6. Assay of laccase

Laccase production by Pleurotus spp in different substrates at different

time intervals was assayed using the method described by Frochner and

Eriksson (1974).

4ml sodium phosphate butter, pH 6 containing 10mM guaiacal was added

to the test tubes. Enzyme source of 0.1ml was added to this and the mixture

incubated for 5 min. The absorbance was obtained at 470nm in

spectrophotometer. Sodium phosphate buffer served as control. The activity of

laccase was expressed in terms of enzyme units. [1 unit the change in

absorbance of 0.01 per minute

17
 RESULT AND DISCUSSIONS

1. Isolation and maintenance of pure cultures of Pleurotus spp:

Pure cultures of three Pleurotus spp viz., P.sajor-caju, P.florida and P.

eous were obtained from the sporocarp by tissue culture method. The cultures

were maintained on PDA slant.

2. Spawn preparation:
Wheat grain spawns of the three Pleurotus spp was prepared and their rate

of growth on wheat grain was evaluated. The three species showed statistically

significant differences in the growth if spawn bottles. (Table 1)

P.sajor-caju required the maximum period of 18 days for completion of

mycelium run, P.florida 14 days and P.eous 15 days.

Table 1: mycelium growth in spawn bottles

Pleurotus species Nature of growth Period required
completion of growth

P.florida Cottony growth 5-8 days

P.sajor-caju Cottony growth 18 days
P.eous Cottony growth 14-18 days

3. Laccase Production:

18
 There was significant difference in the level of laccase production on

three different substrates by Pleurotus spp. The maximum level of laccase

production was recorded by P.sajor-caju on paddy straw.

Among the different Pleurotus spp tested, P.sajor-caju was the most

efficient lignin degrader. Maximum lignin content was found in retted coir pith

followed by non retted coir pith and paddy straw.

The control used in laccase was buffer. The quantification of laccase

in the enzyme source using spectrophotometer in P.florida, P.sajor-caju and

P.eous were 0.127, 0.210, and 0.08 respectively.

Plate 4 and 5 shows the tissue culture of Purotus Mushroom and

its spawn.
Plate 6 and 7 showing Mother spawn having the growth of mushroom and
bed preparation

19
Plate 4

Plate 5

20
Plate 6

Plate 7

SUMMARY

21
 Pleurotus spp are edible fungi popularly known as ‘wood fungi’.

Pleurotus spp have been successfully cultivated in different agricultural wastes

such as mixture of coconut fiber and coffee pulp, kidney bean and broad bean

stubbles, sugar cane bagasse and cotton wastes. Three Pleurotus spp viz.,

P.florida, P.sajor-caju, P.eous were used here in the laccase assay. The extra

cellular enzymes are produced to degrade the large molecules of the substrate

into small soluble molecules which the mycelium can utilize.

The importance of this report was, Kerala is the leading state in the

production of coconut coir pith, a highly lingo-cellulosic material which is

available in large quantities as a by product of coir industries. In conclusion,

this study evaluated the maximum level of laccase production was recorded by

P.sajor-caju on paddy straw. Among the different Pleurotus spp tested,

P.sajor-caju was the most efficient lignin degrader. Maximum lignin content

was found in retted coir pith followed by non retted coir pith and paddy straw.

The importance of this report was Mushrooms had long been used for

medicinal and food purposes .It is now increasingly recognized that correct diet,

controls and modulates many functions of human body and consequently

participates in the maintenance of state of good health, necessary to reduce the

risk of many diseases. Modern pharmacological research confirms large parts of

traditional knowledge regarding the medicinal effects of mushrooms due to their

antifungal, antibacterial, antioxidant and antiviral properties, besides being used

as functional foods.

22
 In conclusion, this study evaluated Mushroom cultivation very well with

sustainable farming and has several advantages , It uses agricultural waste

products , A high production per surface area can be obtained , After picking

the spent substrate is still a good soil conditioner . These mushrooms are rather

easy to grow on a small scale.

CONCLUSION

ASSAY OF LACCASE ENZYME IN PLEUROTUS SPECIES

23
Assay of Laccase
Trial Volume Volume Volume Volum OD at Difference
No. of of H2O2 of e 450 nm in OD
PO4 with (ml) enzyme of

Incubation for 5’ at room temperature

Guaicol enzyme
(ml)
B - 1.6 - - - -

C1 1.5 - - 0.1 0.066 0.061

T1 1.5 - 0.1 - 0.127 -

C2 1.5 - - 0.1 0.0112 0.098

T2 1.5 - 0.1 - 0.210 -

C3 1.5 - - 0.1 0.092 0.074

T3 1.5 - 0.1 - 0.161 -

Pleurotus florida (T1)

OD units per 5’ = 0.061
OD units per minutes = 0.0122

Pleurotus sajor-caju (T2)

OD units per 5’ = 0.098
OD units per minutes = 0.0196

Pleurotus eous (T3)

OD units per 5’ = 0.07

24
OD units per minutes =0.014

Specific activity of Laccase in Pleurotus florida = 1.045 units/mg of protein

Specific activity of Laccase in Pleurotus sajor-caju = 1.78 units/mg of protein
Specific activity of Laccase in Pleurotus eous = 1.233 units/mg of protein

RESULT
The Specific activity of laccase enzyme from pleurotus species lies
between 1 and 2 units /mg of proteins.

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