Unit 01

Culture preparation of mushrooms:-

Methods of culture preparation:-

There are several methods of culture preparation of mushrooms. Here are a few
methods:

1. Spore Culture Method: In this method, spores are collected from a mature
mushroom and then germinated on the culture media. Once the spores have
germinated, they can be transferred to new media and grown into a new mushroom.
This method is commonly used for the production of large numbers of spawn.

2. Tissue Culture Method: This method involves taking a small piece of tissue from an
existing mushroom and placing it into a sterile medium. The tissue will then grow and
develop into a new mushroom. This method is commonly used for the propagation of
rare or endangered mushroom species.

3. Agar Culture Method: This method involves transferring the spores or tissue of a
mushroom into a nutrient-rich agar medium. The medium is then poured into petri
dishes and allowed to solidify. Once the agar has solidified, small pieces of the
mushroom culture can be placed onto the surface and allowed to grow. This method is
commonly used for research and experimentation.

4. Liquid Culture Method: This method involves inoculating a liquid culture medium
with spores or tissue from a mushroom culture. The liquid medium is then agitated to
promote growth of the culture. This method is often used for the production of
mushroom mycelium, which can then be used to inoculate spawn.

5. Grain Spawn Method: This method involves sterilizing and then inoculating a
nutrient-rich grain substrate with mushroom mycelium. The mycelium colonizes the
grain, which can then be used as a spawn to inoculate mushroom substrates. This is a
common method used in commercial mushroom production.

pure culture preparation:-

The pure culture preparation of mushrooms is similar to other microorganisms. Here's

a step-by-step guide:

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1. Harvest a single mushroom and remove the stem and gills. Place the cap gill-side
down onto a sterile nutrient agar plate.

2. Incubate the plate at the ideal temperature for the specific strain of mushroom you
are working with. This usually ranges from 20-30°C.

3. Within a few days, tiny white strands will begin to radiate from the cap. These
strands are mycelium, the vegetative part of the mushroom.

4. Use a sterile scalpel or inoculation loop to cut a small piece of the mycelium and
transfer it to a new sterile agar plate. This will allow the mycelium to continue
growing and form a pure culture.

5. Incubate the new plate, and within a few days to several weeks, the mycelium will
grow into a large, pure white mass.

6. Use the pure culture to inoculate a mushroom-growing substrate, such as a jar of

sterilized grain or sawdust, and incubate until the desired mushroom fruiting body
develops.

7. Repeat the process as needed to maintain a pure culture of the specific mushroom
strain.

It is important to maintain a sterile environment throughout the process to prevent

contamination from other microorganisms. The pure culture method is the basis for
commercial production of mushroom spawn, which is then used to inoculate a
substrate and grow mushrooms.

Methods of culture preservation:-

Mushroom culture preservation is the process of maintaining fungal cultures for future
use. This is important to ensure that the genetic integrity and viability of the cultures
are maintained over extended periods. Here are some common methods of culture
preservation of mushrooms:

1. Refrigeration: One of the simplest ways of preserving mushroom cultures is to store

them in the refrigerator at a temperature of around 4°C. This method is relatively
short-term and cultures typically last for a couple of months.

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2. Subculturing: This method involves transferring a small amount of the mushroom
culture onto a new agar plate. By regularly subculturing, the culture can be maintained
and kept viable for an extended period of time.

3. Lyophilization: Lyophilization, or freeze-drying, is a method of preservation that

involves removing water from the culture by rapidly freezing it and then gradually
heating it up to evaporate the water. The culture is then stored in a vacuum-sealed
container at a low temperature.

4. Cryopreservation: This is a long-term storage method that involves freezing

cultures at an ultra-low temperature, usually in liquid nitrogen (-196°C). The cultures
are stored in sterile vials and the viability of the cultures can last for decades.

5. Mineral Oil Preservation: In this method, the mushroom culture is stored in

sterilized mineral oil. The oil acts as a barrier to prevent contamination and
oxygenation, which keeps the culture viable for extended periods.

Choosing the best method of culture preservation depends on the specific needs and
preference of the researcher.

Limitations in culture preservation:-

Despite the benefits of culture preservation of mushrooms, there are some limitations
that should be considered. Here are a few limitations of culture preservation of
mushrooms:

1. Degeneration: Over time, cultures may lose their vitality and may not produce the
same yield or quality as the original culture. This is known as degeneration and can
result in a decrease in productivity or even a loss of valuable traits.

2. Contamination: Maintaining a sterile environment is crucial for the success of

culture preservation. Any contamination can cause the culture to become
compromised and render it unusable.

3. Storage Space: The storage requirements for maintaining mushroom cultures can be
significant. Some methods, such as cryopreservation or lyophilization, require
specialized equipment and adequate storage space.

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4. Long-Term Viability: While some storage methods, such as cryopreservation, can
preserve cultures for several decades, the viability of the culture over several
generations cannot be guaranteed.

5. Cost: Depending on the amount of cultures being preserved, the cost of maintaining
a culture library can be substantial - especially with some of the more sophisticated
methods like cryopreservation.

It is important to understand the limitations and choose the best method of culture
preservation that suits the research goals. Regular monitoring and updating culture
preservation methods will help minimize the challenges that may occur during the
process.

Spawn and spawning:-

Forms of spawns (liquid and substrate/grain spawn) :-

Spawn is a material that has been inoculated with mushroom mycelium and is used to
transfer the mycelium to a substrate where it can grow and produce fruiting bodies.
There are two common forms of spawn used for cultivating mushrooms:

1. Substrate/Grain Spawn: This is the most common form of spawn used for
mushroom cultivation. Grain spawn is made by inoculating sterilized grains such as
rye, millet, or wheat with mushroom cultures and allowing the mycelium to colonize
the grain.

Once the grain has been colonized, it can be used to inoculate a substrate like sawdust,
straw, or compost. The substrate is then incubated until the mycelium has colonized
the substrate, and mushroom fruiting bodies can be harvested.

2. Liquid Spawn: Liquid spawn is a less common form of spawn, but it can be useful
in certain situations. Liquid spawn is made by adding mushroom cultures to a
sterilized solution of nutrients, such as malt extract, and allowing the mycelium to
grow in the liquid.

Once the mycelium has colonized the liquid, it can be injected into a substrate for
colonization and fruiting body production. Liquid spawn is particularly useful for
inoculating substrates that are more difficult to inoculate due to texture or
composition.

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Both types of spawn can be stored for later use, either by refrigeration or freezing.
Using high-quality spawn is essential to successful mushroom cultivation, and
selection of the appropriate form of spawn is one of the key factors to successful
mushroom cultivation.

Preparation of spawn:-

The preparation of spawn for mushrooms involves the following steps:

1. Selecting a substrate: The substrate is the material that the mushrooms will grow
on. Common substrates include straw, sawdust, and compost.

2. Sterilizing the substrate: The substrate needs to be sterilized before adding

mushroom spawn to prevent contamination. This can be done by steaming or
autoclaving the substrate.

3. Inoculating the substrate with spawn: Once the substrate has cooled, it's time to
inoculate it with mushroom spawn. This can be done by mixing the spawn with the
substrate or by inserting spawn into the substrate with a sterilized tool.

4. Incubating the spawn: The inoculated substrate needs to be kept in a warm, humid
environment for the spawn to grow and spread throughout the substrate.

5. Transferring the spawn to fruiting containers: Once the spawn has colonized the
substrate, it's time to transfer it to fruiting containers such as plastic bags or trays. The
containers need to be kept in a dark, humid environment until the mushrooms begin to
form.

6. Maintaining the fruiting environment: The fruiting containers will need to be kept
in a cool, humid environment with proper air flow and lighting to ensure healthy
mushroom growth.

By following these steps, you should be able to successfully prepare spawn for
mushroom cultivation.

Mother spawn:-

Mother spawn is the starting material used to create mushroom spawn. It is also
known as mother culture or pure culture. Mother spawn is a culture of pure mushroom

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mycelium that is grown on sterilized grain or agar. It is used to inoculate a substrate to
create mushroom spawn.

To create mother spawn, the following steps are typically taken:

1. Prepare the substrate: Sterilize a grain such as rye or wheat berries and mix it with
water. Then fill mason jars or bags with the sterilized grain and let it cool.

2. Add mushroom spores: Inoculate the sterilized grain with mushroom spores or a
small piece of mushroom tissue. Cover with a sterile lid and let the mycelium grow.

3. Incubate the mother culture: Keep the jars or bags at a specific temperature and
humidity for 2-4 weeks to allow the mycelium to colonize the grain.

4. Harvest the mother spawn: Once the mycelium has colonized the grain, the mother
spawn can be used to inoculate more grain or agar to create mushroom spawn.

Mother spawn is used to ensure that the spawn created is identical to the original
culture and free of any contaminants. It serves as a reliable starting point for
mushroom cultivation.

Spawn formulations and commercial spawn:-

Spawn formulations and commercial spawn are both used in mushroom cultivation for
the purpose of inoculating substrates with mushroom mycelium.

Spawn formulations:- typically refer to the materials used to create the spawn. This
can include using organic grains, sawdust, or other substrates that have been sterilized
and inoculated with mushroom spores or mycelium.

Commercial spawn:- on the other hand, refers to pre-made spawn that is purchased
from a supplier. These suppliers specialize in producing high-quality spawn that is
free of contamination and optimized for specific mushroom varieties. Commercial
spawn can be purchased in various forms, including grain spawn, sawdust spawn, and
plug spawn for specific mushrooms.

The choice between using a spawn formulation or commercial spawn often depends
on the resources available to the mushroom grower. For small-scale growers, creating
their own spawn formulation using organic grains or sawdust and inoculating it with
mushroom spores can be more cost-effective. However, commercial spawn can be

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advantageous for larger-scale growers or those looking for consistent quality and
productivity, as commercial suppliers have the equipment and expertise to produce
high-quality spawn consistently.

Regardless of the choice between spawn formulation or commercial spawn, it is

important to ensure that all materials used are well-sanitized and that the appropriate
measures are taken to minimize contamination. Proper inoculation with clean spawn is
critical to successful mushroom cultivation.

Problems in spawn production, diagnostics and solution:-

When problems arise in spawn production, diagnosing the issue and determining the
appropriate solution is crucial. Here are some common problems, diagnostics and
solutions in spawn production for mushrooms:

1. Contamination: Indicators of contamination include the appearance of mold,

bacteria or yeast on the spawn, or an off-colour appearance. Strict hygiene protocols
should be instituted to avoid contamination. Once contamination is detected, the
infected material should be isolated and discarded. Measures should be taken to
ensure that the remaining materials are sterilized and not contaminated.

2. Low yield: If mushroom yield is low, there are a number of potential causes that
should be considered. The quality of the substrate, spawn production, and incubation
conditions should all be evaluated. A lower inoculation density or using poor-quality
substrate could reduce yield. In addition, environmental factors such as temperature,
humidity and light can all have an impact on yield. Proper management of these
factors should help to address the problem.

3. Abnormal mushroom development: If mushroom caps are small or malformed, it is

possible that the environmental conditions were unsuitable during growing.
Everything from light levels, temperature, humidity, and elevated carbon dioxide
levels can all potentially contribute to abnormal mushroom development. To address
this, the environmental conditions should be adjusted to optimize growth conditions.

4. Insufficient colonization: If spawn is used to inoculate a substrate, and colonization

takes an excessive amount of time or fails entirely, there may be an issue with the
quality of the spores or mycelium introduced. Contamination may also be a factor.
Sterilization protocols should be inspected and optimized to ensure proper penetration
and safe, hygienic conditions are maintained throughout the process.

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In conclusion, spawning production for mushroom cultivation can be sensitive, and
problems may occur. The best way to address issues that may arise is through
diagnosing the cause and implementing corrective actions to optimize the spawning
process. A thorough understanding of potential problems and techniques for reducing
their risks (or addressing them as they happen), is important for successful mushroom
cultivation.

Methods of spawning:-

There are several methods of spawning for mushroom cultivation, including:

1. Spawn to substrate transfer: In this method, the mushroom spawn is transferred

directly onto the substrate. This can be done by scattering or broadcasting the spawn
or by placing it in a pattern on the substrate. This method can be effective for outdoor
cultivation or for substrates that are already in a container.

2. Inoculation by grain spawn: In this method, the mushroom mycelium is grown on

sterilized grain, such as rye berries, and then added to the substrate. The grain spawn
can be mixed with the substrate or layered on top of it.

3. Inoculation by sawdust spawn: In this method, the mushroom mycelium is grown

on sterilized sawdust. This sawdust can then be mixed with the substrate or placed on
top of it.

4. Plug spawn: This method involves drilling small holes into the substrate and
inserting small plugs that are spawned with mushroom mycelium. This method is
commonly used for log cultivation.

5. Liquid inoculation: In this method, the mushroom spawn is mixed with water or a
nutrient-rich liquid and then used to inoculate the substrate. This can be a quicker
method of inoculation, and the liquid spawn can be easily distributed throughout the
substrate.

Regardless of the specific method chosen, it is important to ensure proper sterilization

and hygienic practices throughout the spawning process to avoid contamination. It is
also important to select the most appropriate method for the substrate and type of
mushroom being cultivated.

Unit 02

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Compost and composting:-

Methods of composting:-

Composting is an important process for preparing the substrate for mushroom

cultivation. There are several methods of composting used in mushroom cultivation,
including:

1. Phase 1 composting: In this method, the compost is prepared by mixing straw or

other organic materials with nitrogen-rich supplements. The compost is then
pasteurized or sterilized to eliminate any harmful bacteria or fungi. The compost is
then cooled and inoculated with mushroom spawn.

2. Phase 2 composting: After phase 1 composting, the compost is further broken down
by microorganisms, producing a highly nitrogen-rich material preferred by mushroom
mycelium. The temperature is carefully monitored and managed during this process.

3. Self-heating compost: This method involves creating a compost pile that undergoes
natural thermophilic heating processes. The pile is created by mixing organic
materials such as straw or manure with nitrogen supplements. The pile will typically
reach temperatures above 55°C, which will eliminate harmful bacteria and fungi that
may compete with mushroom mycelium. Careful management of moisture and
oxygen levels is crucial for success.

4. Aerated static pile (ASP) composting: This method involves creating aerated piles
of compost that enhance microbial activity and promote the breakdown of organic
matter. Compost is mixed with supplements, is moistened, and aerated structures are
added to promote airflow and microorganism activity.

5. In-vessel composting: In this method, the composting process occurs in an enclosed

system with highly controlled environmental factors. The compost is mixed with a
supplement and stored in a container where it undergoes various stages of composting
over several weeks. This method is ideal for larger scale mushroom production.

The type of composting method used will depend on the resources available, scale of
production, and type of mushroom being cultivated. Regardless of the method of
choice, maintaining optimal hygiene and composting conditions ensures a more
desirable substrate for mushroom cultivation.

Role of composting in mushroom cultivation:-

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Composting plays a crucial role in mushroom cultivation as it is the process by which
various nutrients and microorganisms are introduced to create the ideal growing
medium for oyster mushrooms, shiitake mushrooms, and other types of edible fungi.

Mushrooms require a specific mix of nutrients, primarily carbon, nitrogen, and

microbial organisms, to grow optimally. Composting is the process of decomposing
organic materials into a nutrient-rich soil amendment that contains high levels of these
necessary nutrients and microorganisms.

The composting process is initiated by combining raw materials such as straw,

sawdust, manure, and agricultural waste, which are then mixed thoroughly and
composted for several weeks. During this period, microorganisms such as bacteria and
fungi break down the organic matter, transforming it into humus, a dark, crumbly,
nutrient-rich material that can be used as a growing medium.

Once the compost is ready, it is pasteurized, which destroys any harmful bacteria or
pathogens and triggers the growth of beneficial microbes that are essential to a healthy
mushroom crop. The pasteurized compost is then inoculated with mushroom spores or
spawn, which initiates the growth of mycelium, the vegetative part of the mushroom,
which decomposes the compost and absorbs the nutrients.

In summary, composting is a critical step in the mushroom cultivation process, as it

provides a nutrient-rich medium that is essential for the growth and development of
mushrooms.

Composting for white button and oyster mushroom cultivation:-

Composting is an essential part of mushroom cultivation, and making sure you have
the right compost can help create a perfect environment for white button and oyster
mushrooms to grow. Here are some tips for composting for white button and oyster
mushroom cultivation:

1. Select the right substrate: For white button and oyster mushrooms, the best
substrate to use is a combination of straw and manure. You want to aim for a mixture
that is approximately 70% straw and 30% manure.

2. Add nitrogen sources: For the compost to be effective, it is important to add

nitrogen-rich materials. These can include chicken manure, soybean meal, or

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cottonseed meal. You should aim for a ratio of 1 part nitrogen to 20 parts of the straw
and manure mixture.

3. Wet the compost: You want to keep the compost moist, but not soaking wet. You
can add water to the compost as needed, and you should aim for a moisture content of
around 65%.

4. Monitor temperature: During the composting process, it is important to monitor the

temperature. You want to aim for a temperature range of 140-160°F (60-71°C). This
will help to kill off any pathogens or bacteria in the compost.

5. Let the compost mature: Once the compost has reached the desired temperature
range, it is important to let it mature for a few weeks. This will help to break down
any remaining organic matter and create a more stable compost.

These tips should help you create a great compost for white button and oyster
mushroom cultivation. Remember to follow proper sanitation practices and keep your
growing area clean to help prevent contamination.

Qualities of good compost:-

Casing and casing material used in mushroom cultivation:-

Casing is a layer of material that is added on top of the mushroom substrate during the
mushroom cultivation process. It serves several functions, including retaining
moisture, providing nutrients to the mycelium, and creating a microclimate that is
conducive to mushroom growth. Here are some common casing materials used in
mushroom cultivation:

1. Peat moss: Peat moss is a popular casing material due to its ability to retain
moisture and its high carbon content. It is also relatively inexpensive and widely
available.

2. Vermiculite: Vermiculite is a lightweight, mineral-based material that is often used

for casing. It is known for its ability to retain moisture and its good insulation
properties.

3. Coir: Coir is a byproduct of coconut processing and is often used as a substitute for
peat moss in mushroom cultivation. It is a sustainable and renewable resource and has
good water retention properties.

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4. Soil: Soil can also be used as a casing material, although it is not as commonly used
as other materials. It should be sterilized before use to prevent contamination.

5. Compost: Compost can also be used as a casing material, especially for oyster
mushrooms. It is nutrient-rich and can help to promote mycelial growth and fruiting.

When choosing a casing material, it is important to consider factors such as water

retention, nutrient content, and availability. You should also ensure that the casing
material is sterilized before use to prevent contamination.

Production and crop management of Button mushroom and Oyster mushroom:-

Harvesting of mushroom:-

Harvesting is an important step in mushroom cultivation, as it determines the final

yield and quality of the mushrooms. Here are some tips for harvesting button and
oyster mushrooms:

1. Observe the mushrooms: Observe the mushrooms as they grow, and harvest them
when they are mature. For button mushrooms, this is typically when the cap is fully
rounded and has not yet started to flatten out. For oyster mushrooms, the caps should
be fully formed and the edges should begin to curl.

2. Use a clean, sharp knife: Use a clean, sharp knife to harvest the mushrooms. Cut the
mushroom at the base of the stem, being careful not to damage the other mushrooms
or the substrate.

3. Harvest in batches: Harvest the mushrooms in batches, rather than all at once. This
allows the other mushrooms to continue to grow and mature.

4. Store the mushrooms properly: After harvesting, immediately store the mushrooms
in a cool, dark place. They can be stored in a paper bag or wrapped in a damp towel.

5. Dispose of the substrate: After harvesting the mushrooms, the substrate can be
disposed of or used for composting.

It is important to maintain good sanitation practices during harvesting to prevent

contamination. Always wash your hands before harvesting and use clean equipment to
minimize the risk of introducing harmful bacteria or other pathogens.

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Identification of right stage of mushroom harvest:-

Button mushrooms:- are typically harvested when the caps have fully expanded and
the veil beneath the cap has just started to break. This usually occurs around 2-3 days
after the mushrooms first appear. The caps should be firm and tightly closed before
harvest. If the caps start to flatten out and the gills become visible, the mushrooms are
overripe and should not be harvested.

Oyster mushrooms:- should be harvested when the caps have fully expanded, but are
not yet starting to curl upwards. This usually occurs around 1-2 days after the
mushrooms first appear. The caps should be firm and not slimy to the touch. Overripe
oyster mushrooms will have curled caps and sometimes a slimy texture.

Methods of harvesting:-

The method of harvesting button and oyster mushrooms is quite different from each
other. Here are the methods for both:

Harvesting Button Mushrooms:

1. Firmly grasp the stem of the button mushroom and gently twist it at the base until it
comes loose from the substrate.
2. Be careful not to damage the surrounding mushrooms or the substrate as you
harvest.
3. Use a clean and sharp knife to trim any remaining stem from the cap.
4. Discard any mushrooms that show signs of damage or overripeness.

Harvesting Oyster Mushrooms:

1. Use a clean and sharp knife to cut the stem of the oyster mushroom at the base
where it connects to the growing substrate.
2. Be careful not to damage the mushroom or the substrate as you harvest.
3. Oyster mushrooms grow in clusters, so try to harvest the entire cluster at once to
avoid damaging the growth of future mushrooms.
4. Discard any mushrooms that show signs of damage or overripeness.

It's important to handle both button and oyster mushrooms with care during harvest, as
rough handling can damage the delicate caps and lead to bruising or rotting. Also,
keep in mind that the harvesting methods may vary depending on the growing
conditions and the preferences of the grower.

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Diseases and environmental stress management in mushroom cultivation:-

Diseases and environmental stress can affect the growth and yield of button and oyster
mushrooms. Here are some management techniques for both:

Button Mushrooms:

1. Crop hygiene: It is important to maintain good crop hygiene to prevent the spread
of diseases. Keep the growing environment clean and free of debris and dispose of any
infected mushrooms or substrate as soon as possible.

2. Proper ventilation: Button mushrooms require a high humidity environment, but too
much moisture can lead to mould and other fungal growth. Proper ventilation of the
growing area can help maintain optimal humidity levels and reduce stress on the crop.

3. Temperature control: Button mushrooms grow best at temperatures between

18-25°C. Any significant fluctuations in temperature can cause stress and lead to
reduced yield.

4. Disease-resistant strains: Use disease-resistant strains of button mushrooms to

prevent or reduce the impact of common diseases.

Oyster Mushrooms:

1. Proper substrate preparation: Oyster mushrooms grow best on substrates that are
properly prepared and pasteurized. Any contamination in the substrate can lead to the
growth of harmful fungi and reduce the yield of healthy mushrooms.

2. Proper fruiting conditions: Oyster mushrooms require cooler temperatures and

lower humidity levels during fruiting. High humidity can lead to bacterial and fungal
growth, causing less yield.

3. Biological controls: Using natural predators and beneficial microorganisms to

control pests and disease can be an effective method of management in oyster
mushroom cultivation.

4. Monitoring and timely intervention: Regular monitoring for signs of disease or pest
infestation can help catch problems early and minimize their impact on the crop.
Timely intervention with appropriate measures, such as selective picking or pruning,
can help keep the crop healthy and maintain the yield over time.

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It is also important to keep in mind that environmental stress and disease management
techniques may vary depending on the specific growing conditions and location of the
cultivation.

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