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BIOFERTILIZER AND MUSHROOM TECHNOLOGY

UNIT I: BIOFERTILIZERS
Definition, Advantages, Microbes used as Biofertilizers. Isolation, Characteristics,
Identification, Mass inoculum production, Field application and marketing of Rhizobium,
Azospirillum, Azotobacter.

UNIT II:
Cyanobacteria (BGA) - Isolation, Characteristics, Mass inoculum production and Field
application. Azolla – Isolation, Characteristics, Mass inoculum production and Field
application.

REFERENCE:
1. Kumarasan.B, 2001, Biotechnology, Saras Publication, Tamil Nadu.
2. Dubey, R.C., 2001, Text Book of biotechnology, S.Chand & Co., New Delhi.
3. Bagyaraj,D.J., & Rangasamy A.,2005,Agricultural Microbiology- Tata McGraw Hill.,
NewDelhi
4. Subba Rao, N.S.,1995, Biofertlizers, Oxford and IBH Publishing Co., Pvt. Ltd., New

Prepared by

DR.R.SAGAYA GIRI,
ASSISTANT
PROFESSOR,
DEPARTMENT OF
BOTANY,
KN GOVT ARTS COLLEGE FOR
WOMEN, THANJAVUR-7
UNIT I: BIOFERTILIZERS
Definition, Advantages, Microbes used as
Biofertilizers

Introduction
Biofertilizer is a substance which contains living microorganisms
which, when applied to seeds, plant surfaces, or soil and promotes
growth by increasing the supply or availability of primary nutrients to
the host plant.It adds nutrients through the natural processes of
nitrogen fixation, solubilizing phosphorus, and stimulating plant
growth through the synthesis of growth-promoting substances.

Definition

Biofertilizers are the substance that contains microbes, which

helps in promoting the growth of plants and trees by increasing the
supply of essential nutrients to the plants. It comprises living organisms
which include mycorrhizal fungi, blue-green algae, and bacteria.
Mycorrhizal fungi preferentially withdraw minerals from organic matter
for the plant whereas cyanobacteria are characterized by the property of
nitrogen fixation.

Types of Biofertilizers

Following are the important types of biofertilizers:

1. Nitrogen-fixing biofertilizers

Cultivated crop determine what type of nitrogen biofertilizer should be

used:
• Rhizobium for legume crops.
• Azotobacter/Azospirillum for non legume crops.
• Acetobacter for sugarcane only.
• Blue-Green Algae (BGA) and Azolla for low paddy land used to grow
rice.
2. Phosphorus fixing biofertilizers

Phosphorus biofertilizers are not dependent on the crops cultivated on

the soil
• Phosphatika for all crops to be applied with
• Rhizobium
• Azotobacter
• Azospirillum and Acetobacter
Biofertilizers can use to enrich your compost and the bacterial
processes breaking down the compost waste
• Phosphotika and Azotobacter culture.

MICROBES USED AS BIOFERTILIZERS

1. Symbiotic Nitrogen-Fixing Bacteria

Rhizobium is one of the vital symbiotic nitrogen-fixing bacteria. Here

bacteria seek shelter and obtain food from plants. In return, they help by
providing fixed nitrogen to the plants.

2. Loose Association of Nitrogen-Fixing Bacteria

Azospirillum is a nitrogen-fixing bacteria that live around the roots of

higher plants but do not develop an intimate relationship with plants. It is
often termed as rhizosphere association as this bacteria collect plant
exudate and the same is used as a food by them. This process is termed
as associative mutualism.

3. Symbiotic Nitrogen-Fixing Cyanobacteria

Blue-Green algae or Cyanobacteria from the symbiotic association with

several plants. Liverworts, cycad roots, fern, and lichens are some of the
Nitrogen-fixing cyanobacteria. Anabaena is found at the leaf cavities of
the fern. It is responsible for nitrogen fixation. The fern plants decay and
release the same for utilization of the rice plants. Azolla pinnate is a fern
that resides in rice fields but they do not regulate the growth of the plant.
4. Free-Living Nitrogen-Fixing Bacteria

They are free-living soil bacteria which perform nitrogen fixation. They
are saprotrophic anaerobes such as Clostridium beijerinckii, Azotobacter,
etc.

Among all the types of biofertilizers, Rhizobium and Azospirillum are most
widely used.

Importance of Biofertilizers
Biofertilizers are important for the following reasons:

1. Biofertilizers improve soil texture and yield of plants.

2. They do not allow pathogens to flourish.

3. They are eco-friendly and cost-effective.

4. Biofertilizers protect the environment from pollutants since they are

natural fertilizers.
5. They destroy many harmful substances present in the soil that can
cause plant diseases.
6. Biofertilizers are proved to be effective even under semi-arid
conditions.

Advantages of Biofertilizers

1. It helps in maintaining environmental health by reducing the

level of pollution.
2. Reduces human & animal hazards by reducing the level of
residue in the product.
3. Increases the agricultural products and makes it sustainable
4. Ensures the optimum utilization of natural resources.
5. Reduces the risk of crop failure.
6. Improves the physical and chemical properties of soil
7. Biofertilizers are cost-effective when compared to synthetic
fertilizers
8. Using biofertilizers can increase crop yield by 20 or 30 percent.
Drawback of biofertilizers

1. Slow-release
2. Crop specific
3. Strain-specific
4. Soil specific—lose effectiveness if soil too dry or hot
5. Lesser efficient than synthetic fertilizers
 6. Crops show less response to biofertilizers then chemical
fertilizers
7. Much lower nutrient density — requires large amounts to get
enough for most crops.

Applications of Biofertilizers
Following are the important applications of biofertilizers:

1. Seedling root dip

This method is applicable to rice crops. The seedlings are planted in the
bed of water for 8-10 hours.

2. Seed Treatment

The seeds are dipped in the mixture of nitrogen and phosphorus

fertilizers. These seeds are then dried and sown as soon as possible.

3. Soil Treatment

The biofertilizers along with the compost fertilizers are mixed and kept
for one night. This mixture is then spread on the soil where the seeds
have to be sown.

Frequently Asked Questions on Biofertilizers

1. What do you understand by biofertilizers?

Biofertilizers are substances that contain microorganisms, which when

added to the soil increases the crop yield and promotes plant growth.

2. What are the advantages of biofertilizers over chemical fertilizers?

• Biofertilizers are cost-effective.

• They reduce the risk of plant diseases.
• The health of the people consuming the vegetables grown by the
addition of chemical fertilizers is more at risk.
• Biofertilizers do not cause any type of pollution.
3. What is the need of using biofertilizers?

Biofertilizers are required to restore the fertility of the soil. Prolonged use
of chemical fertilizers degrades the soil and affects the crop yield.
Biofertilizers, on the other hand, enhance the water holding capacity of
the soil and add essential nutrients such as nitrogen, vitamins and
proteins to the soil. They are the natural form of fertilizers and hence,
widely used in agriculture.

4. Name a few microorganisms used as biofertilizers.

Microorganism used as biofertilizers are:

• Rhizobium
• Azotobacter
• Azospirilium
5. How do biofertilizers promote plant growth?

Biofertilizers utilise certain microorganisms. These microorganisms trap

atmospheric nitrogen and convert it into nitrates and nitrites and make it
available to the plants. They also convert insoluble phosphates into the
forms required by the plants.

6. What are the main sources of biofertilizers?

The main sources of biofertilizers include bacteria, cyanobacteria and

fungi.

7. Name an important nitrogen-fixing bacteria.

Rhizobium is an important nitrogen-fixing bacteria. Rhizobium lives in

symbiotic association with the leguminous plants, specifically in their
root nodules. It traps the atmospheric nitrogen and converts it into
usable forms that enhance the growth of the plants.

Isolation, Characteristics, Identification, Mass inoculum

production, Field application and marketing of Rhizobium

Rhizobium
 Rhizobium is a gram negative, rod shaped, aerobic, nitrogen
fixing , soil habitat bacterium which can able to colonize the legume
roots and fixes the atmospheric nitrogen symbiotically.

They have seven genera and highly specific to form nodule in

legumes, referred as cross inoculation group.They are the most
efficient biofertilizer as per the quantity of nitrogen fixed concerned.
The morphology and physiology of Rhizobium will vary from free-living
condition

The bacteria infect the legume root and form root nodules within
which they reduce molecular nitrogen to ammonia which is reality
utilized by the plant .This belongs to bacterial group and the classical
example is symbiotic nitrogen fixation. Rhizobium inoculant was first
made in USA and commercialized by private enterprise in 1930s.

Classification of Rhizobium

There are different types of Rhizobium that are categorized on the

basis of the rate of growth and the type of plant they are associated with.

Some species of Rhizobium include:

• R. leguminosarum
• R. alamii
• R. lentis
• R. japonicum
• R. metallidurans
• R. smilacinae
• R. phaseoli
• R. trifolii

Along with Bradyrhizobium, Sinorhizobium, Mesorhizobium,

Azorhizobium, and Allorhizobium, Rhizobium is a soil Rhizobia, which
means that it consists of bacteria with the ability to fix nitrogen. As
such, it presents a significant advantage to the plants it infects by
contributing to their growth and development.

In addition to this classification, Rhizobium bacteria are also

categorized based on the species of legume that they nodulate. This
type of grouping is known as cross-inoculation.
 Based on studies on a wide variety of legumes, it became
evident that not all Rhizobia are capable of nodulating all types of
legumes. This resulted in a need to group Rhizobium into appropriate
groups (cross-inoculation groups) which are simply groups of legumes
that given species of Rhizobium nodulate.

The cross-inoculation groups include:

❖ Clover groups - R. trifolii infects and nodulates plants of genus

Trifolium (clovers/trefoil)
❖ Alfalfa groups - R. meliloti infects and nodulates the roots of
medicago, melilotus and medicago
❖ Bean group - R. phaseoli infects and nodulates plants of genus
Phaseolus (e.g. beans)
❖ Lupine group - R. lupine nodulates lupines and serradella
(Ornithopus)
❖ Pea group - R. leguminosarum infects and nodulates pea,
sweet pea, lentil, and vetch
❖ Soybean group - R. japonicum nodulates Glycine such as
soybean
❖ Cowpea group - Rhizobium sp. nodulates cowpea, pegionpea,
lespedza, groundnut and kudz among a few others.

* The word Rhizobium comes from the Greek words: "rhiza" which
refers to root, and "bios" which refers to life.

Characteristics of Rhizobium

Rizobium is discovered and described in 1889, R. leguminosarum

is the type species of Rhizobium in the same way Rhizobium has been
the type genus of family Rhizobiaceae. As such, it can be viewed as a
representative of the genus (Rhizobium).

Some of the characteristics of the bacteria include:

❖ They appear as elongated rods when viewed under the

microscope
❖ Like a number of other bacteria, Rhizobium do not form spores
in their life cycle
❖ They posses several flagella on their polar end. This allows them
to move from one location to another
 ❖ They are aerobic. As such, they need oxygen for respiratory
purposes
❖ There are various strains of the bacteria some of which have
granules
❖ They are Gram-negative bacteria
❖ Although they can tolerate higher temperatures of about 38
degrees Celsius, Rhizobium ideally grow in temperatures of
between 20 and 28 degrees Celsius
❖ Apart from various types of carbohydrates, the bacteria also
uses nitrates and nitrite, ammonium salts and various amino
acids among others for development

Isolation of Rhizobium (Vincent, 1970)

The selected nodules were properly washed with running water

to remove the soil particles. Then the undamaged root nodules were
selected and again washed with distilled water. These selected
nodules were kept immersed in 0.1% acidified potassium chloride
solution for 5 minutes and washed repeatedly with sterile water. Then,
they were immersed in ethyl alcohol solution. This treatment was
followed by repeated washing with sterile distilled water. These
sterilized root nodules were crushed simply with pestle and mortar.
The homogenate was sieved through a fine sieve. The extract was
serially diluted with distilled water and inoculated into a Yeast Extract
Mannitol Agar (YEMA) medium (Vincent, 1970).

Composition of YEMA medium

Agar - 20 g
Mannitol - 10 g
Yeast Extract - 1.0 g
K2HPO4 - 0.5 g
MgS04.7H20 - 0.2 g
NaCl - 0.1 g
Distilled water - 1000 ml
Congo red (1%) - 2.5 ml (only for solid medium during isolation)

The pH of the medium was adjusted to 6.8 before adding agar.

The plates were incubated at 28 + 2°C for 2 to 3 days. This

medium allowed Agrobacterium and Rhizobium to grow and develop
into colonies. The rhizobial colonies appeared as white translucent,
elevated colonies on YEMA medium. They were removed and purified
by repeated streaking. Pure rhizobial cultures were maintained on
YEMA slant

Identification of Rhizobium

The bacterium was identified by the following tests.

Growth on media: The pure culture from the slants was spread
on peptone glucose agar and on YEMA media and the growth was
observed.

Gram staining: The bacterial cultures were subjected to gram

staining procedures and observed.

Congo red test (Hahn, 1966): An aliquot of 2.5 ml of 1 per cent

solution of the dye in water was added to a litre of YEMA. The isolated
bacterial culture was inoculated on to the plated YEMA and observed.

Hofer's alkaline broth test (Hofer, 1941): The isolated bacterial

cultures were spread on YEMA with high pH 11 and observed for
grovi^h.

Lactose Agar Test (Bemaerts and Delay, 1963): Isolated nodule

bacteria were grown on lactose plates for 4-10 days and the plates
were flooded with Benedict's reagent. Finally the plates were
observed for colour development.

Staining of poly P-hydroxy butyrate (PHB) (Mc Kinney, 1953): A

loopM of selected rhizobia were spread out on a microscope slide in a
drop of water and allowed to dry in air. The cooled smear was flooded
with 5 times diluted carbol fiichsin for 30 seconds. The slide was
washed in running water and allowed to air dry. The pinkish PHB
granules were observed under the oil immersion microscope.
Methods of Mass Cultivation

Following are the steps of mass cultivation of Rhizobium.

(a) sterilize the growth medium and inoculate with broth of mother
culture prepared in advance,

(b) incubate for 3-4 days at 30 - 32°C,

(c) test the cultures for its purity and transfer to a large fermenter,
wait for 4-9 days for bacterial growth (for good bacterial growth make
the device for its aeration),

(d) allow to grow the bacteria either in a large fermenter containing

broth or in small flasks as per demand,

(e) check the quality of broth,

(f) blend the broth with sterile carrier e.g. peat, lignite, farmyard
manure and charcoal powder,

(g) pack the culture in polyethylene bags and keep at 25°C,

(h) check the quality of carrier culture,

(i) store at 4°C in a controlled-temperature room, and

(j) supply to farmers.

During the blending of broth a variety of carriers are used, for

example, peat, lignite, farmyard manure, charcoal powder, etc. In
India powdered farmyard manure and charcoal powder are good
carrier and an alternative to peat and lignite. Good quality of carrier
culture is that which contains sufficient amount of rhizobial
cells i.e. 1000 x 106 to 4000 x 106 rhizobia/g carrier. Seed inoculation
with aqueous suspension of carrier culture during sowing has revealed
the luxuriant nodulation and good yield of crops.

Methods of seed inoculation with rhizobial culture

 The steps of seed inoculation with rhizobial culture are given
Dissolve 10 per cent sugar or gur (jaggery) in water by boiling it for
some time. Leave the content to cool down. Gum arabic solution (10%
) may also be added to the solution. This serves as sticker
for Rhizobium cells to seeds. Mix this carrier based culture
of Rhizobium to form the inoculum slurry. For one hectare, 400 g
charcoal based culture would be sufficient for mixing the seeds.
Transfer the inoculum slurry on seeds and mix properly. The number of
rhizobial cells/ seed should be between 105 to 106. Spread the seeds in
shade for drying on cement floor or plastic sheets.

While using rhizobial cultures, certain precautions are taken into

account. For example, use of culture before expiry date, use of small
amount of pesticides when required, immediate sowing of seeds after
mixing, etc. Seeds must be stored at 4°C when not used immediately
to protect the rhizobial cells.

Pelleting

When soil has the adverse conditions such as dryness, acidity,

excess fertilizers and pesticides, etc., the rhizobial cells are protected
by adopting special method of inoculation. One of these methods is
pelleting i.e. preparation of pelleted seeds. This method involves the
procedure as described earlier. High amount of gum arabic (40%) or
carboxymethylcellulose (20%) is added to the inoculum slurry before
mixing with seeds. Finally, pelleting agent is mixed when inoclated
seeds are moist (before seed drying ) to get the seeds evenly coated.

The commonly used pelleting agents are calcium carbonate, rock

phoshate, charcoal powder, gypsum and betonite.

Effects of rhizobial inoculants on crop yield

Effect of rhizobial culture on yield of different pulses, and

subsequent crops grown after harvesting the pulse crops are given in
Table 12.1 based on the study made under All India Co-ordinated Pulse
Improvement Research Programme of I.C.A.R., New Delhi. Yield of
pulse crops can be substantially increased by rhizobial inoculation.
Legume crops get benefit from rhizobial symbiosis. In addition, certain
amount of N is left over in soil which is taken by the other plants also.
 Subba Rao and Tilak (1977) studied the effect of residual
nitrogen of many legumes on the yield of subsequent crops of wheat
or rice. They always found more yield of subsequent crops
in Rhizobium inoculated fields than in uninoculated control.

Application of biofertilizers to crops

1.Seed treatment
Each packet (200g) of inoculant is mixed with 200 ml of rice gruel or
jaggery solution. The seeds required for one hectre are mixed in the
slurry so as to have uniform coating of the inoculants over the seeds
and then shade dried for 30 minutes. The treated seeds should be
used within 24 hous. One packet of inoculant is sufficient to treat to
10 kg seeds. Rhizobium, Azospirillum, Azotobacter and
Phosphobacteria are applied as seed treatment.

2. Seedling root dip

This method is used for transplanted crops. Five packets (1.0 kg) of
the inoculants are required for one ha and mixed with 40 litres of
water. The root portion of the seedlings is dipped in the solutions for 5
to 10 minutes and then transplanted. Azospirillum is used for seedling
root dip particularly for rice.

3. Soil treatment
4 kg each of the recommended biofertilizers are mixed in 200 kg of
compost and kept overnight. This mixture is incorporated in the soil at
the time of sowing or planting.
Isolation, Characteristics, Identification, Mass inoculum
production, Field application and marketing of Azospirillum,

Azospirillum, a free-living nitrogen-fixing bacteria closely

associated with grasses. Azospirillum Bacteria is a Gram negative
motile bacteria belonging to the order Rhodospirillales, associated
with roots of monocots, including important crops, such as wheat,
corn and rice.
Azospirillum contains 109/gm spores of Azospirillum species.
This Azospirillum bacterium fixes the atmospheric nitrogen and makes
it available to plants in non-symbiotic manner that can replace 50-90%
of the nitrogen fertilizer required by plants.
 Azospirillum biofertilizer also secretes some fungicides, enzymes
but in minute amount. Use of Azospirillum biofertilizer increases the
crop production in large scale. We are engaged in manufacturing and
marketing of Bio control agents comprise of different types of
beneficial Bacterial, Fungal and Viral cultures.
Azospirillum is mainly useful for monocot vegetables.It is an eco-
friendly liquid biological fertilizer formulation containing bacteria, It
contains large amount of lipid granules, which enters the cortical cells
of the root and fix up atmospheric nitrogen and also produces
biologically active substances like vitamins, nicotinic acid, in dole
acetic acid, gibberellins etc and helps in better retention of flowers
and enhances the plant growth.

Azospirillum Biofertilizer is a nitrogen fixing biofertilizer.

Nitrogen is a major nutrient for all plants. Azospirillum lipofereum is a
very useful soil and root bacterium. It is an associative symbiotic
nitrogen fixing bacteria. It is found in the soil around plant roots and
root surface.

It also produces growth-promoting substances like indole acetic

acid (IAA), gibberellins, pantothenic acid, thiamine and niacin and it
promotes root proliferation and it improves the plant growth yield. It
increases the rootlet density and root branching resulting in the
increased uptake of mineral and water.
Suitable for:

Millets, oilseeds, fruits & vegetables, sugarcane, banana,

coconut, oil palm, cotton, chilly, lime, coffee & tea, arecanut & rubber,
flower, spices, & contiments, herbs, lawns & ornaments, trees etc.

It is suitable for all crops and applications including field crops,

potting soil, vegetable and flower gardens, orchards and turf grass.
Promotes balanced growth of crops, boosts the capacity of immunity
and Greater nutritional value: Rapidly complement the nutrients, im-
prove the quality of product.

Characteristics:
Azospirillum is a rod to spirillum-shaped nitrogen fixing
bacterium and freely lives in soil forming nonspecific symbiotic
associations with various plants in particular, corn. This genus
consists of species, namely, A. lipoferum, A. brasilense, A.
amazonense, A. halopraeferans, A. nitrocaptans, and A. seropedica.
Azospirillum lipoferum was originally described and named Spirillum
lipoferum by Beijerinck in 1922.

Azospirillum characteristically develops white, dense, and

undulating pellicles on a semi-solid malate containing enrichment
medium. The pellicle is formed 2 mm below the surface of the medium
indicating the microaerophilic nature of the bacterium. Azospirillum in
gram-negative contains poly-β-hydroxy butyrate (PHB) granules, and
shows polymorphism and spirillar movement.

It fixes atmospheric nitrogen (dinitrogen) in microaerophilic

surroundings (low oxygen conditions) but possesses ability to grow
profusely in ammonium- rich environment without fixing nitrogen. The
bacterium also produces growth substances such as indole acetic
acid (IAA), kinetins, and gibberellins.

Criteria for Strain Selection:

The efficient nitrogen fixing strain is evolved or selected in

laboratory, maintained and multiplied on nutritionally rich artificial
medium before inoculating the seed or soil. In soil, the strain has to
survive and multiply to compete for infection site on roots against
hostile environment in soil.

Steps for Preparing Bio-Fertilizer:

The isolated strain is inoculated in small flasks containing

suitable medium for inoculum production. The volume of the starter
culture should be a minimum of 1% to obtain atleast 1×109 cells/ml.
Now the culture obtained is added to the carrier for inoculant (bio-
fertilizer) preparation.
Carriers carry the nitrogen fixing organisms to the fields. In some
cases carrier is first sterilised and then inoculated, while in other
cases it is first inoculated and then sterilised by UV irradiation. The
inoculum is now packed with 109-1010 viable cells per gram. Final
moisture content should be around 40-60%. For large scale production
of inoculum, culture fermenters are used.
Application of biofertilizers to crops

1.Seed treatment
Each packet (200g) of inoculant is mixed with 200 ml of rice gruel or
jaggery solution. The seeds required for one hectre are mixed in the
slurry so as to have uniform coating of the inoculants over the seeds
and then shade dried for 30 minutes. The treated seeds should be
used within 24 hous. One packet of inoculant is sufficient to treat to
10 kg seeds. Rhizobium, Azospirillum, Azotobacter and
Phosphobacteria are applied as seed treatment.

2. Seedling root dip

This method is used for transplanted crops. Five packets (1.0 kg) of
the inoculants are required for one ha and mixed with 40 litres of
water. The root portion of the seedlings is dipped in the solutions for 5
to 10 minutes and then transplanted. Azospirillum is used for seedling
root dip particularly for rice.
3. Soil treatment
4 kg each of the recommended biofertilizers are mixed in 200 kg of
compost and kept overnight. This mixture is incorporated in the soil at
the time of sowing or planting.

Effects:

➢ Better root systems: Promoting the development of roots

➢ Healthier foliage and fruit appearance: Thicken, enlarge and

balance the leaf growth, supply well-balanced crop nutrients,
stimulating cell division, improves the fruit set, improves
blossom and fruit set

➢ Greater resistance to disease and pests: Containing antitoxins to

fend off bacteria and viruses, and to repel insects. Helps plants
to endure environmental stress

➢ Improved seed germination: Promoting the development of

shoots

➢ As formulation: Seaweed Extract can be used not only on crops,

but also to formulate types of fertilizers. A little addition of
seaweed extract on common fertilizer will height the quality
greatly resistance, improves crop quality and increase yield

Isolation, Characteristics, Identification, Mass inoculum

production, Field application and marketing of Azotobacter:

Azotobacter Structure and Characteristics:

Azotobacter is a soil-inhabiting bacterium and comprises large,

gram-negative, obligately aerobic rods .This bacterium freely lives in
soil and fixes atmospheric nitrogen nonsymbiotically. The first species
of Azotobacter was discovered by the Dutch microbiologist M.
Beijerinck in the beginning of 20th century, and was named by him
Azotobacter chroococcum.
 While growing, Azotobacter produces flat, slimy, paste-like
colonies with a diameter of 5–10 mm, which may form films in liquid
nutrient media. The colonies can be dark-brown, green, or other
colors, or may be colorless, depending on the species. The growth is
favored at a temperature of 20–30°C.

Subsequently, many other species of Azotobacter were isolated

from different soils of the world and some important ones are: A.
agilis, A. vinelandii, A. beinjerinckii, A. insignis, A. macrocytogenes, A.
paspali, etc. Azotobacter cells are large, many isolates being almost
the size of yeasts, with diameters of 2-4 μm or more. Pleomorphism is
common and a variety of cell shapes and sizes have been described.
Some strains possess peritrichous flagella.

Although the Azotobacter is an obligate aerobe, its enzyme

callcd nitrogenasc that catalyzes atmospheric nitrogen fixation is
oxygen-sensitive. It has been studied that the high respiratory rate
characteristic of Azotobacter and the abundant capsular slime help
protect nitrogenase from oxygen.

This bacterium grows on a wide variety of carbohydrates,

alcohols, organic acids, amonia, urea, and nitrate. Azotobacter forms
cysts the resting structures, which are resistant to desiccation,
mechanical disintegration, and ultraviolet and ionizing radiation. Each
cyst measures about 3 μm in diameter.

Mechanism of N2 Fixation:

Azotobacter (A. chroococcum and A. vinelandii ) is one of the

most extensively investigated member amongst free-living nitrogen
fixing bacteria. The use of 15N tracer and acetylene reduction method
have however enriched our knowledge regarding the biochemical
pathway between atmospheric nitrogen (dinitrogen; N2) and ammonia
(NH3) but the exact nature of intermediate products have eluded even
critical investigators.
Nevertheless, the overall reaction in the enzymic reaction of N 2 to
NH3 can be postulated as under:
Nitrogenase of Azotobacter:

Nitrogenase, the enzyme that catalyzes atmospheric nitrogen

fixation, consists of two protein fractions:
(i) The Mo-Fe containing protein (molecular weight 220,000-2,70,000)
and

(ii) Fe containing protein (molecular weight 55,000-66,800).

In Azotobacter (A. vinelandii), two additional nitrogenases have been

investigated. One of these possesses vanadium (V) instead of
molybdenum (Mo) and the other has neither molybdenum nor
vanadium.

The characterization of these nitrogenases has generated fresh

problems in pinpointing evidences to demonstrate the essentiality of
molybdenum for N2-fixation and characterization of the site at which
nitrogen binds to nitrogenase.

Production of azotobacter:
i. Mother culture:

A pure growth of any organism on a small scale is called as a

mother culture. Mother culture is always prepared in a conical flask of
500 or 1000 ml. Capacity and then this mother culture is used for
furtherproduction.
For this purpose, one liter conical flasks are taken to which 500
ml of broth of nitrogen free medium is added and these flasks are then
plugged with non-absorbent cotton, sterilized in an auto slave for 15-
20 minutes at 75 lbs pressure for 15 minutes. Flasks are then
inoculated with mother culture with the help of inoculating needle
aseptically. The flasks are transferred to shaker and shaking is done
for 72-90 hours so as to get optimum growth of bacteria in broth.
Bacteria are multiplied by binary method i.e. cell division. After about
90 days, the number of per milliliters comes to about 100 crores. Total
growth of bacteria in this broth means starter culture or mother
culture, which should carefully be done, since further purity of
biofertilizer or quality of biofertilizer depends upon how mother culture
isprepared.

ii. Production on a large scale:

Azotobacter is multiplied on a large scale by two ways viz.

Fermenter and Shaker. The fermenter is most automatic and accurate
method of multiplication of any micro-organism. In this method, the
medium is taken in a fermenter and then sterilized. After this pH of the
medium is adjusted and 1% mother culture is added. In order to get an
optimum growth of the Azotobacter required temperature and oxygen
supply is adjusted so that concentrated broth is made. This
concentrated broth of the culture is then mixed with a carrier
previously sterilized and bio-fertilizers are prepared. Depending upon
the demand and supply suitable fermenter is selected.

In the 2nd method i.e. shake method, a suitable medium is

prepared transferred to conical flask of suitable capacity. These
flasks are then sterilized in an autoclave at 15 lbs pressure for 15
minutes. Each flask is inoclulated with 10 ml mother culture and they
are transferred to shaker for multiplication where they are kept for 72-
90 hours. This broth is mixed with a suitable carrier previously
sterilized. Thus biofertilizer is prepared, filled in plastic bags and
stored in cool place.

iii.Selection of carrier:
A carrier is nothing but a substance which has high organic
matter, higher water holding capacity and supports the growth of
organism. In order to transport the biofertilizer and becomes easy to
use the suitable carrier is selected. Generally Lignite cool, compost
and peat soil are suitable carriers for Azotobacter. Out of these
carriers lignite is most suitable for this organism, since it is cheaper,
keeps organism living for longer period and does not lower the quality
ofbio-fertilizers.

The lignite comes in clouds and hence it is ground in fine powder

by grinding machine. Its finesses should be 250-300 mesh. The pH of
the carrier is adjusted to neutral by adding CaCO3. The lignite
naturally has a variety of micro-organism and hence it is sterilized in
autoclave at 30 lbs. Pressure for 30 minutes. After this the broth is
mixed with lignite 1:2 proportion by following method.

Galvanized trays are sterilized and used. To these trays,

previously sterilized lignite is transferred and broth is then added
(lignite2: broth 1) and mixed properly. Trays are then kept one above
the other for 10-12 hours for allowing the organism to multiply in the
carrier. This mixture is then filled in plastic bags of 250 g or 500 g
capacity. Plastic bags are properly. Trays are then kept one above the
other for 10-12 hours for allowing the organism to multiply in the
carrier. This mixture is then filled in plastic bags of 250 g or 500 g
capacity. Plastic bags are properly sealed. All the required information
such as name of biofertilizer, method of use expiry date, etc. is printed
on plastic bags. In this way biofertilizer is ready to sell or use. If
biofertilizer is used immediately then bags are stored in cool place
otherwise they should be stored in cold storage in order to keep
biofertilizeringoodquality.

As per ISI standards, one gram of biofertilizer immediately after it is

prepared should have one crore cells of bacteria and 15 days before
expiry date one gram of biofertilizer should have 10 lakh bacteria. If
biofertilizer is stored at 15-20 0C then it will remain effective for 6
months. However, at 0 to 4 0C (cold storage) the bacteria will remain
active for 2 years. The storage periods are decided after testing the
biofertilizer for that particular storage conditions, such temperature
and humidity.

How to apply Azotobacter bio-fertilizer?

1. Seed inoculation:

On the basis of efficiency of Azotobacter, other micro-

organisms present in the soil, benefits obtained from biofertilizer and
expenditure it has been fixed to use Azotobacter - bio-fertilizer at the
rate of 250 g biofertilizer for 10-15 kg. If one knows this proportion
then take a definite quantity of seed to be inoculated. The required
quantity of fresh biofertilizer is secured and slurry is made by adding
adequate, quantity of water. This slurry is uniformly applied to seed,
seed is then dried in shed and sown. Some stickers are used in order
to adher biofertilizer to seeds. Viz. Jaggery or gumarabica.

2. Seedling inoculation:

This method of inoculation is used where seedlings are used to

grow the crop. In this method, seedlings required for one acre are
inoculated using 4-5 packets (2-2.5 kg). For this, in a bucket adequate
quantity of water is taken and biofertilizer from these packets is
added to bucket and mixed properly. Roots or seedlings are then
dipped in this mixture so as to enable roots to get inoculums. These
seedlings are then transplanted e.g. Tomato,
Rice,Onion,Cole,Crops,flowers.

c.Self inoculation or tube inoculation:

In this method 50 litres of water is taken in a drum and 4-5 kg of

Azotobacter biofertilizer is added and mixed properly. Sets are
required for one acre of land are dipped in this mixture. Potato tubers
are dipped in the mixture of biofertilizer and planting is done.

d.Soil application:
This method is mostly used for fruit crops, sugarcane, and trees. At
the time of planting fruit tree 20 g of biofertilizer mixed with compost
is to be added per sappling, when trees became matured the same
quantity of biofertilizer is applied.
In sugarcane after two to three months of planting i.e. before
earthing up 5-6 kg of biofertilizer per acre is applied by mixing with
compost or soil. Although, Azotobacter fixes nitrogen non-
symbiotically, it also fixes atmospheric nitrogen in the rhizospere
region i.e. soil around the seedlings or trees. Biofertilizer applied to
seed or seedlings bacteria remain around seeds or seedlings and use
organic carbon for their metabolism. When seeds are germinated or
seedlings set in soil they leave or exude root exudates which become
food of these bacteria. They grow on these substances which include
sugars, organic acids, and amino acids and fix atmospheric nitrogen
most efficiently. Nitrogen so fixed by these bacteria becomes
available to plants after dead and degradation of bacterial cells.

Use of Azotobacter as Biofertilizer:

Plant needs nitrogen for its growth and Azotobacter fixes
atmospheric nitrogen non-symbiotically. Therefore, all plants, trees,
vegetables, get benefited. However, especially cereals, vegetables,
fruits, trees, sugarcane, cotton, grapes, banana, etc. are known to get
addition nitrogen requirements from Azotobacter. Azotobacter also
increases germination of seeds. Seeds having less germinating
percent if inoculated can increase germination by 20-30%.

Advantages of Azotobacter:

1. Azotobacter contributes moderate benefits

2. Azotobacter is heaviest breathing organism and requires a large

amount of organic carbon for its growth.

3. It is poor competitor for nutrients in soil and hence its growth

promoting substances, fungistatic substances.

4. It can benefit crops by Nitrogen fixation, growth promoting

substances, fungi static substances.

5. Azotobacter is less effective in soils with poor organic matter

content.

6. It improves seed germination and plant growth

7. It thrives even in alkaline soils.

8. Azotobacter is tolerant to high salts.

Conclusion:
 Azotobacter is a broad spectrum biofertilizer and can be used as
inoculant for most agricultural crops. Earlier, its utility as a
biofertilizer was not a priority due to its relatively low population in
the plant rhizosphere. However, seeding treatment with Azotobacter
of several crops brought about an increase in yield. Besides, because
of its well known N2 nutritional function, it is now recognized to play a
multiple role in helping crop plants to improve their growth potential,
yield and maintenance of soil health for sustainable agriculture. Hence
there is renewed interest in this rhizobacterium. However, quantitative
understanding of the ecological factors that control the performance
of biological N2fixation systems of the bacterium in crop fields is
essential for promotion and successful adoption of the bio-fertilizer
production technology.
UNIT II:
Cyanobacteria (BGA) - Isolation, Characteristics, Mass inoculum production
and Field application. Azolla – Isolation, Characteristics, Mass inoculum
production and Field application
Cyanobacteria (BGA) - Isolation, Characteristics, Mass
inoculum production and Field application.
The blue-green algae are photoautotrophic,prokaryotic algae.
They are free living. They are called as cyanobacteria and are fixing
atmospheric nitrogen and release it into the surroundings in the form
of amino acids, proteins and other growth promoting substances . e.g.
Aulosira, Anabaena, Cylindrospermum, Ocillatoria, Nostoc,
Plectonema, Tolypothrix , and Gleocapsa.

Mass cultivation of cyanobacterial biofertilizers

For outdoor mass cultivation of cyanobacterial biofertilizers, the

regional specific strains should be used. However, many germplasm
collection laboratories have been established by the D.B.T. in different
parts of the country for the development of starter inoculum. Mixture
of 5 or 6 regional acclimatized strains of cyanobacteria, e.g. species
of Anabaena, Aulosira, Cylindrospermum, Gloeotrichia, Nostoc,
Plectonema, Tolypothrix are generally used for starter inoculum.

The following four methods are used for mass cultivation

(i) cemented tank method.,

(ii) shallow metal troughs method,

(iii) polythene lined pit method, and

(iv) field method.

The polythene lined pit method is most suitable for small and
marginal farmers to prepared algal biofertilizer. In this method, small
pits are prepared in field and lined with thick polythene sheets.

Mass cultivation of cyanobacteria is done by using any of the

four methods under the following steps:

1. Prepare the cemented tanks, shallow trays of iron sheets or

polythene lined pits in an open area. Width of tanks or pits should not
be more than 1.5 m. This will facilitate the proper handling of culture.
2. Transfer 2 -3 Kg soil (collected from open place for lm 2
area of the
tank) and add 100 g of superphosphate. Water the pit to about 10 cm
height. Mix lime to adjust the pH 7. Add 2 ml of
insecticide e.g. malathion to protect the culture from mosquitoes. Mix
well and allow to settle down soil particles.

3. When water becomes clear, sprinkle 100 g of starter inoculum on

the surface of water.

4. When temperature remains between 35-40° during summer,

optimum growth of cyanobacteria is achieved. Always maintain the
water level to about 10 cm during this period,

5. After drying, the algal mat will get separated from the soil and
forms flakes. During summer about 1 kg pure algal mat per m2 area is
produced. These are collected, powdered, kept in sealed polythene
bags and supplied to the farmers.

6. The algal flakes can be used as starter inoculum if the same

process is repeated.

PRODUCTION OF ALGAE FOR FIELD APPLICATION

The introduction of algal biofertilizer as a package of practices

in rice cultivation will depend largely upon an efficient and economical
system of large-scale production of blue-green algae, its preservation
and transport. The method is credited with simplicity of operation and
ad- aptability by small and marginal farmers. The algae used in this
system is a mixture of species of Tolypothrix, Aulosira, Anabaena,
Nostoc and Plectonema. The same system is reported to be working
well in Burma. The procedure, starting in the laboratory and ending in
the field, is described below. It may be noted that other efficient
strains of algae may be selected and used, depending on the regions
and loca- tions.

1. Laboratory Culture

Maintain stock cultures of different nitrogen-fixing blue-green algae on

1 to 1.5% agar slants.
❖ Maintain the same cultures also in a soil extract medium (1 g of
soil + 10 cm3 of Fogg’s medium, sterilized together in test-
tubes). For Fogg’s medium and soil extract medium
❖ Grow the algae in 250 cm3 flasks containing 100 cm3 of Fogg’s
medium in the light.
❖ Scale up the cultures in aspirator bottles or carboys.
❖ Transfer the algae to troughs to prepare soil-based starter
material as described later under ‘Trough Method’. Different algal
forms are cultured and maintained separately before transferring
them to the troughs. The pH of all the cultures should be between
7.0 and 7.5 unless acid or alkali-tolerant forms are used. All the
cultures are grown in light.

2 .Trough Method

➢ Prepare shallow trays (2 m x 23 cm) of galvanized iron sheet or

permanent tanks The size can be increased if more material is to
be produced.
➢ Introduce 8 - 10 kg of soil and mix well with 200 g of super-
phosphate
➢ Place from 5 to 15 cm of water in the trays depending on the
local conditions and rate of evaporation. The reaction of the soil
should be about neutral; if acidic correct by adding lime .
➢ To prevent insects, add Carbofuron (3% granules) at the rate of
25 g per tray or BHC or other suitable insecticide.
➢ After the soil has settled, sprinkle the algal culture on the
surface of the standing water Keep the units in the open air and
completely exposed to the sun.
➢ In hot summer months, the growth of the algae will be rapid and
in about seven to ten days they form a thick mat . If the daily
rate of evaporation is high, add water inter- mittently. When the
algal growth becomes sufficiently thick, stop watering.
➢ Allow the water to evaporate completely in the sun the dry algae
cracks into flakes.
➢ Collect the dry algal flakes from the trays and store in bags for
use in the fields
➢ Fill the troughs again with water and add a small amount of the
dry algal flakes as further inoculum. Continue the process as
above. Once the soil in the troughs is exhausted (usually after
three or four harvests) replace it with fresh soil mixed with
superphosphate and continue as above. A single harvest of sur-
face algae from one trough of the given dimensions will give
about 1.5 to 2.0 kg of material.
2. Pit Method
This method does not differ from the trough method except in
magnitude. Instead of troughs or tanks, shallow pits are dug in
the ground and layered with a thick polythene sheet to hold the
water . Other procedures are the same as in the trough method.
This method is easy and less expensive to operate by small
farmers. Also the accidental collection of excess soil along with
the algae during harvesting is avoided. Sometimes the addition
of 200 g of superphosphate as a single dose results in problems
of acidity. To avoid this, split the dose into two or three smaller
doses; similarly the amount of soil can be split.
3. Field Production

The field production of algae is really a scaled up operation of

the trough or pit methods to produce the material on a
commercial scale: it is being adopted by a number of farmers in
south India
a. Demarcate the area in the field for algal production; the
suggested area is 40 m2. No special preparation is
necessary although if algal production is envisaged
immediately after crop harvest, the stubble is to be
removed and if the soil is loamy it should be well puddle to
facilitate water logging.

b. Bund the area with strong 15 cm earth bunds. Flood the

area with water to a depth of about 2.5 cm . In the trough
and pit methods, flooding is done only in the beginning; in
the field, flooding is repeatedly needed to keep the water
standing.
 c. Apply superphosphate at 12 kg 40 m-2
d. If the field has previously received algal application for at
least two consecutive cropping seasons, no fresh algal
appli- cation is required. Otherwise, apply the composite
algal culture at 5 kg 40 m-2
e. To control predators like daphnids, snails and mosquitoes,
apply Carbofuron (3% granules) or Ekalux (5% granules) at
250 g 40 m-2 or BHC or Furadon
f. In clayey soils, good growth of algae takes olace in about
two weeks in clear, sunny weather, while in loamy soils its
takes about three to four weeks.
g. Once the algae have grown and form floating mats, they are
allowed to dry in the sun in the field and the dried algal
flakes are then collected and stored in sacks for further
use . One can continually harvest algae from the same area
by re- flooding the plot and applying superphosphate and
pesticides.
h. Addition of algal inoculum for subsequent production is not
necessary.
i. During summer months (April-June), the average yield of
algae per harvest ranges from 16 - 30 kg per 40 m2 .
Adopting this method, a record production of 15.6
tonnes/ha of wet blue- green algae has been obtained by
farmers within three weeks

4. Nursery-cum-algal Production

Farmers can produce algae along with seedlings in their

nurseries. If 320 m2 of land are allotted to prepare a nursery, an
additional 40 m2 alongside can be prepared for algal production as
described in the pre- ceding section. By the time the rice seedlings are
ready for trans- plantation, an amount of 15 to 20 kg of algal material
will be available and sufficient to inoculate about one and a half
hectares. Transplan- tation is made in the nursery and algal plots and
in this way land is neither wasted nor locked up exclusively for algal
production during the growing season. The algae are first grown in
flasks containing sterile medium and are then transferred to large
glass bowls under non-sterile conditions.

Nursery plots of 5-7 m x 1 m x 20 cm, containing 6-7 cm of water

are inoculated at the rate of 150 g algae per m2. After about seven
days the algae attain a biomass of 500 - 1 000 g/m2. The nursery plots
are covered with transparent plastic sheets to protect them from low
temperatures .The field inoculation is done by spreading the algae at
the rate of 750 kg/ha, which grows to attain a biomass of 7.5 tonnes/
ha within 10 - 15 days and if the temperature exceeds 303 kelvin (30
°C) 15 tonnes/ha can be reached.

RECOMMENDATIONS FOR FIELD APPLICATION OF BLUE-GREEN

ALGAE

1. If mineral nitrogen fertilizers are not being used, apply

blue-green algae in order to gain the benefit of from 20 - 30
kg N/ha.

2. Broadcast the dry algal material over the standing water

in the rice field at a rate of 10 - 15 kg/ha one week after
transplanting the seedlings. Addition of excess algal
material is not harmful and will accelerate the
multiplication and establishment in the field. Providing that
the field is not being used and that water facilities are
available, algal application can be done well in advance of
transplanting the rice.

3. When mineral nitrogen fertilizer is used reduce its dose

by 25 kg/ha and supplement with algal application.

4. If so desired, algae can also be used along with high

levels of nitrogen fertilizer.

5. The sun-dried algal material can be stored for a long time

in a dry state without any loss in viability.

6. Do not store the algal material in direct contact with

fertilizers or other chemicals.
 7. Apply algae for at least three consecutive seasons.
8. Recommended pest control measures and other
management practices do not interfere with the
establishment and activity of the algae in the field.

Azolla - Isolation, Characteristics, Mass inoculum production

and Field application.

Azolla is an aquatic heterosporous fern which contains an

endophytic cyanobacterium, Anabaena azollae, in its leaf cavity. The
significance of Azolla as biofertilizer in rice field was realized in
Vietnam. Recently, it has become very popular in China, Indonesia,
Philippines, India and Bangladesh.A total of six species of Azolla are
known so far viz., A. caroliniana, A. filiculoides, A. mexicana, A.
microphylla, A. nilotica, A. pinnata and A. rubra. Out of these A.
pinnate is commonly found in India. The global collections of several
species of Azolla are maintained at CRRI (Cuttack). Within the leaf
cavity filaments of Anabaena azollae are present. Dr. P.K. Singh, at
CRRI has done an outstanding work on mass cultivation of Azolla and
its use as biofertilizer in rice and other crop fields.
Azolla cultivation procedure

1. Create an artificial pond for growing Azolla.

2. For creating Azolla cultivation pond, select partially shaded
area because Azolla needs 30% sunlight too much sunlight will
destroy the plant. The area under the tree is preferable.
3. If you decide to grow an Azolla for the large scale you can make
small concrete tanks otherwise you can make the pond any size
you want.
4. Dig out the soil for pond and level the soil after that spread the
plastic sheet around the ground to prevent water loss. Make sure
the pond is at least 20 CM Deep.
5. Add some soil uniformly on the plastic sheet in the pond. For 2M
X 2M size pond add 10-15 kg soil.
6. Azolla needs Phosphorus to grow well you can use Super
Phosphate along with cow dung slurry. Cow dung increases the
available nutrients. Use, cow dung 4-5 days old.
7. Next, fill the pond with water to a level of about 10 cm this will
allow the short route of the Azolla Plant to float freely then
leave the pond for 2 to 3 days so the ingredients can settle.
8. After 2-3 days add Azolla culture in the pond by gently rubbing
Azolla in hands. It helps break Azolla into smaller pieces for
faster multiplication.
9. After two-week start harvesting. form pond of 2M X 2M size, you
can harvest 1kg Azolla each day.
Important tips to grow Azolla

1. Azolla rapidly grows so maintain Azolla biomass 300 gms – 350

gms /sq.meter hence harvest daily to avoid overcrowding.
2. Add once in 5 days mixture of Super Phosphate, and cow dung
also add mixture containing magnesium, iron, copper,
Sulfur etc. at weekly intervals to enhance the mineral content of
Azolla.
3. Replace 25 to 30% old water with fresh water, once in 10 days; it
helps to prevent nitrogen build up in the pond.
4. Replace complete water and soil, at least once in six months and
then add Fresh Azolla seeds.
5. Maintain the water level of at least 10 cm, so Azolla root doesn’t
grow in the soil by keeping the roots floating it becomes easy to
harvest.
6. Harvested Azolla wash thoroughly, so it removes dirt and smell
of cow dung and then feeds them to animals.

Benefits of Using Azolla:

Azolla as a Nitrogen Fertilizer for expanding Crop Yields:

➢ Azolla plants are described through the Chinese and Vietnamese as

being miniature nitrogen fertilizer factories.
 ➢ The Vietnamese use them as nitrogen fertilizer since Azolla
endured to supply nitrogen fertilizer for Vietnamese rice paddies.

➢ The nitrogen fertilizer mounted by way of Azolla turns into to be had

to the rice after the Azolla mat is included into the soil and its
nitrogen starts to be released through decomposition. For Azolla, it
takes 25 to 35 days to supply enough nitrogen for a four to six
ton/ha rice crop right through the wet season, or a five to eight
ton/ha crop underneath irrigation all over the dry season.

Maintaining Soil Fertility:

➢ As a inexperienced manure, Azolla’s affect on soil fertility is
because of its natural subject and nitrogen.

➢ Incorporation and decomposition of Azolla will shape a humus

compound. Humus will increase the water keeping capability of soil
and promotes aeration, drainage, and the aggregation crucial for
extremely productive soils, Organic matter can bind together soil
debris and makes clayey soils extra friable.

➢ Apart from its influence on soil physical homes, Azolla is vital within
the biking of vitamins, whilst Azolla is rising in the paddy, it fixes
nitrogen and absorbs vitamins out of the water that might in a
different way be washed away. When the Azolla is composed with
the soil and humus is shaped, and these nutrients are slowly
launched into the soil as decomposition progresses.

Controlling the Growth of Aquatic Weeds:

➢ Agricultural economists have calculated that the Asian farmers,
particularly women, pay longer time on weeding than on another
activity required for rice manufacturing. Although analysis is
inadequate, it’s frequently believed that Azolla suppresses the
expansion of certain aquatic weeds.

➢ Weed enlargement is suppressed as soon as Azolla forms a thick,

light-proof mat. There are mainly two mechanisms for this
 suppression, the most effective mechanism is the light-starvation of
young weed seedlings by way of the blockage of daylight.

➢ The opposite is the bodily resistance to weed seedling is exposed

in a heavy, interlocking Azolla mat. In the case of weed-infested
rice fields, the benefit from Azolla weed suppression may also
surpass its get advantages as a nitrogen supply. Rice seedlings
aren’t affected by Azolla’s weed suppression impact as a result of,
when transplanted, they stand above the Azolla mat.

Azolla Is Used as a Green Manure:

➢ Azolla can be utilized as a inexperienced manure by way of growing

it as a monocrop after which incorporating it as a basal manure
earlier than the rice is transplanted; or transported to any other
website for use on upland plants; growing it as an intercrop and
incorporating it as a most sensible dressing manure after the rice is
transplanted; or by way of rising it both as a monocrop and an
intercrop. All those programs may also be a hit, however, and is not
unusual in agriculture, use of the green manure crop requires some
changes in the control of both the golf green manure and the main
crop.

➢ Monocrop Azolla is used in China and Vietnam all the way through
the iciness and spring to produce nitrogen for the spring rice crop.
The identical methodology is used to provide nitrogen for the early
summer season rice crop, but that is much less not unusual for the
reason that growth of Azolla pinnata is affected by top temperature
and heavy pest attack during mid to late summer.

➢ Azolla is most often grown with the rice in places the place there is
no time available within the cropping gadget for the monocropping
of Azolla.

➢ Azolla as an intercrop Azolla shall be to start with presented via

hand or rotary rice weeder and then later killed by way of heavy
shading and/or top temperatures, along side next decomposition
and free up of nitrogen to the crop, which maximizes the grain
production.
Unit:III

Mycorrhiza:A mycorrhiza (from Greek μύκης mýkēs, "fungus", and ῥίζα

rhiza, "root"; pl. mycorrhizae, mycorrhiza or mycorrhizas[1]) is a mutual
symbiotic association between a fungus and a plant.[2] The term
mycorrhiza refers to the role of the fungus in the plant's
rhizosphere, its root system. Mycorrhizae play important roles in plant
nutrition, soil biology and soil chemistry.

In a mycorrhizal association, the fungus colonizes the

host plant's root tissues, either intracellularly as
in arbuscularmycorrhizal fungi (AMF or AM), or
extracellularly as in ectomycorrhizal fungi. The association is sometimes
mutualistic. In particular species or in particular circumstances
mycorrhizae may have a parasitic association with host plants.[3]
Definition
Typs:
Mycorrhizas are commonly divided into ectomycorrhizasand
endomycorrhizas. The two types are differentiated by the fact that the
hyphae of ectomycorrhizal fungi do not penetrate individual cells within
the root, while the hyphae of endomycorrhizal fungi penetrate the cell wall
and invaginate the cell membrane.[7][8]
Endomycorrhiza includes arbuscular, ericoid,and orchidmycorrhiza.
Ectomycorrhizae

Leccinumaurantiacum, an ectomycorrhizal fungus

Ectomycorrhiza
Ectomycorrhizas, or EcM, are symbiotic associations between the
roots of around 10% of plant families, mostly woody plants
including the birch, dipterocarp, eucalyptus, oak, pine, and rose[9]
families, orchids,[10] and fungi belonging to the Basidiomycota,
Ascomycota, and Zygomycota. Some EcM fungi, such as many
Leccinumand Suillus, are symbiotic with only one particular genus of
plant, while other fungi, such as the Amanita, are generalists that form
mycorrhizas with many different plants.[11] An individual tree may
have 15 or more different fungal EcM partners at one time.[12]
Thousands of ectomycorrhizal fungal species exist, hosted in over 200
genera. A recent study has conservatively estimated global
ectomycorrhizal fungal
species richness at approximately 7750 species, although, on the basis
of estimates of knowns and unknowns in macromycete diversity, a final
estimate of ECM species richness would probably be between 20,000
and 25,000.[13]
Ectomycorrhizas consist of a hyphal sheath, or mantle, covering the
root tip and a Hartig net of hyphae surrounding the plant cells within
the root cortex. In some cases the hyphae may also penetrate the
plant cells, in which case the mycorrhiza is called an
ectendomycorrhiza. Outside the root, ectomycorrhizal extramatrical
mycelium forms an extensive network within the soil and leaf litter.
Wheat is arbuscularmycorrhizal
Arbuscularmycorrhiza:
Arbuscular mycorrhizas, or AM (formerly known as vesicular-arbuscular
mycorrhizas, or VAM), are mycorrhizas whose hyphae penetrate plant
cells, producing structures that are either balloon-like (vesicles) or
dichotomously branching invaginations (arbuscules) as a means of
nutrient exchange. The fungal hyphae do not in fact penetrate the
protoplast (i.e. the interior of the cell), but invaginate the cell
membrane. The structure of the arbuscules greatly increases the
contact surface area between the hypha and the cell cytoplasm to
facilitate the transfer of nutrients between them.

Ericoid mycorrhiza:Ericoid mycorrhizas are the third of the three more

ecologically important types. They have a simple intraradical (grow in
cells) phase, consisting of dense coils of hyphae in the outermost
layer of root cells. There is no periradical phase and the extraradical
phase consists of sparse hyphae that don't extend very far into the
surrounding soil. They might form sporocarps (probably in the form of
Vesicular Arbuscular
small cups), Mycorrhiza
but their reproductive biology(VAM
is little understood.[8]

IsolationofVAMSporesfromSoil:
 1. Wet Sieving and Decanting:
Principle:
When soil suspension is passed through sieves having smaller mesh
size than VAM spores, they will be retained in the sieve and can be
collected.

Requirements:
a. Rhizosphere soil sample.
b. Beaker, glass rod, water, slides, etc.
 c. Set of sieves (710-45 µm mesh size).
d. Binocular and microscope.
e. Lactophen
ol.
Procedure:
1. Place approximately 250 g of rhizosphere soil in a 500 ml Erlenmeyer
beaker and add 400 ml
of water.
2. Stir well in order to disperse the soil par¬ticles and allow heavier particles
to settle down.
3. Decant the suspension through the set of sieves, 710-45 µm placed one
below the other with 710 µm one on top and 45 µm and one at bottom.
4. Collect the residue of sieves 75 µm and 45 µm after removing them and
wash¬ing them on a filter paper.
5. Examine the filter paper under a bin¬ocular and remove spores gently with
the help of a flat needle to a drop of water on a slide and observe.
6. Blot water, place a drop of lactophenol on the slide, place a coverslip
gently and observe under a microscope.
7. Count the number of spores and calcu¬late for one gram of soil.
8. Permanent slides can be made by mount¬ing spores in Hoyer s medium
which pre¬serves all characters of the spores in origi¬nal form. Staining
is not essential since most of the spores are brightly coloured.

2. Sucrose Centrifugation:
Principle:Spore suspension when placed in 2(M) su¬crose solution and
centrifuged, they easily float in the supernatant and these are filtered and
collected.

Requirements:
1. Suspension of VAM spores from sieves.
2. Centrifuge.
3. 2(M) sucrose solution.
4. Slides and coverslips.
5. Distilled
water.
Procedure:
1. Place the suspension from 75 and 45 µm sieves in a 50 ml centrifuge tube
and make up the volume to 35 ml with dis¬tilled water.
2. Centrifuge at 2000 rpm for 10 minutes and filter supernatant.
3. Suspend the pellet in the centrifuge tube in 2(M) sucrose solution and
make up the volume to 35 ml.
4. Stir well and centrifuge at 2000 rpm for 10 minutes.
5. Filter the supernatant and collect the spores.
 6. Mount the spores and examine them.

3. Mass Collection VAM Spores:

Principle:Larger spores like Gigaspora also can be col¬lected by taking the
fractions from 425 and 250 µm sieves besides 75 and 45 µm.

Requirements:
a. Rhizosphere soil sample.
b. Beakers glass rod, etc.
c. Set of sieves 710-45 µm.
d. 30% (W/V) aqueous sucrose gradients.
e. Aspirator.
f. Centrifuge.
g. Distilled water.
h. Sucrose
(2M).
Procedure:
1. Subject the soil sample to wet sieving and decanting with cold water.
2. Collect spore fraction from 425 and 250 µm sieve with debris for
Gigaspora margarita and for other genera from sieves 75 and 45 µm.
3. Decant and remove debris or vacuum aspirate the surface of the
suspension in a 4 litre beaker which has the debris and spores from 250
µm sieve.
4. For the remaining portion from 75 and 45 µm sieve separate spores by
using dis¬continuous 30% (W/V) aqueous sucrose gradients.
5. Take a one litre beaker add 600 ml of water over 200 ml sucrose.
6. Gently place the sieved material in lay¬ers on this large gradient.
7. Spores and debris get collected at the interface due to gravity.
8. Remove this layer by vacuum aspiration and rinse with cold water.
9. Centrifuge at 1600 rpm for 2 to 5 min¬utes on 2nd gradient (15 ml water
and 20 ml sucrose).
10. Aspirate spore fraction from gradient in¬terface and store in cold water.

4. Modification in Our Laboratory:

 A. Wax Coated Plates:
Principle:Spores floating on the surface will move and stick to paraffin from
.where it can easily be collected.

Requirements:
1. Debris from sieves.
2. Petri dishes coated on the sides (inside)
with paraffin. Procedure:
 1. Suspend debris and spores in Petri plates coated with paraffin wax.
2. Due to gravity debris gets settled and spores float on the surface and
gets stuck to the paraffin coating.
3. Decant water and collect spores in cold water.

B. Funnel Assembly:
Principle:As water is let out, little by little, the air bub¬ble that comes up
push the spores through waves to the periphery.

Requirements:
1. Washing from sieves.
2. Funnel fitted to a suction pump with
closed stopcock. Procedure:
1. Place water in the funnel fined to a suc¬tion pump and with the stopcock
closed.
2. Suspend washings with water from sieve in funnel. Most of the spores
move to the periphery and stick to the sides of the funnel.
3. Open the cock gently and allow a bit of water to go.
4. Spores present in the central region also now move to the periphery.
5. Repeat this process to reduce the quan¬tity of water in the funnel and
ultimately drain off the whole water by opening the cock.
6. Rings of spores get stuck to the sides of the funnel. Collect
them in cold water. Assessment of Mycorrhizal infection in roots.

Principle:
Using KOH the tissues of roots are macerated and fungal element
which remains intact is stained and observed.

Requirements:
a. Feeder roots of plants with VAM infec¬tion.
b. 10% KOH.
c. 1(N) HCl.
d. 0.5% trypan blue/cotton blue in lacto¬phenol.
e. H2O2
f. Slides, cover slips.
g. Needles and forceps.
h. Distilled water.
i. Petri
dish.
Procedure:
1. Wash infected roots thoroughly in wa¬ter.
2. Add 10% KOH and place them covered in a Petri dish in an oven at 90°c
for 30 minutes to 1
hour.
3. Wash the root segments gently two to three times with distilled water.
4. Add H2O2 and leave aside for one to two hours.
5. Acidify by immersing in dilute in HCl for a few minutes.
6. Stain the roots with cotton blue in lactophenol and remove excess of
stain with lactophenol. Mount on slides in lactophenol or acetic acid:
glycerol (l:lv/v)
7. Place a cover slip seal and
observe. For Pigmented Roots:

Principle:
Pigmented roots are decolourised by treating it with stronger solution
of KOH and heat¬ing it for a longer time.

Requirements:
1. 10-20% KOH.
2. VAM infected feeder
roots. 3. 1(n) (HCI).
4. 0.5% cotton blue in
lactophenol. 5. H2O2.
6. Slides and coverslips.
7. Needles, forceps.
8. Distilled water.
9. Petri
plates.
Procedure:
Same as above but depending on the colour of roots 10 or 20% KOH is
used and this is kept for a longer period of at least one hour.

MeasurementofRootInfection:

Requirements:
1. Stained segments of infected root.
2. Calibrated microscope.
3. Slides and cover slips.
4. Needle
s.
Procedure:
1. Select at random, stained segments of roots and mount them on
microscopic slides in groups of ten.
2. Examine under the microscope and as¬sess length of cortical colonization
of VAM in µm for each segment.
 IdentificationofVAMSpores:

i. ELISA
Method
(EISA)
Requiremen
ts:
a. Freund’s complete adjuvant.
b. P04 buffer.
c. Carbonate coating buffer.
d. 0.05% Tween 20 in phosphate buffered saline (PBST).
e. Sterile saline.
f. lg G-Horse radish peroxidase-labellcdimmunoconjugate.
g. Orthophenylenediamine.
h. H2S04.4N
i. Cultures of VAM in sterile sand grown host plant maintained in glass house
with 16 hour light per day.
j. Incubator.
k. Polyvinyl microlitre trays.
l. Rabbits.
m. Needle and syringe.
n. Centrifu
ge.
Procedure:
1. Place roots of infected plant on a 250 µm sieve and wash with a strong
jet of water (to remove external hyphae) into the 250 µm sieve.
2. Place sieving in a Petri plate and remove soil particles by observing under
a sterioscan binocular.
3. Take hyphal mass, lyophilize, grind and store in desiccator at room
temperature.
This will be approximately 20 mg (dry weight) of hyphae from a single
heavily infected host.
4. Antigen when required for immunisa¬tion is prepared by resuspending 5 mg
of powder in 1ml sterile saline and emulsifying it with equal volume of
Freund’s complete adjuvant.
5. Inject antigen into white rabbits.
6. A booster dose of 2 mg is given after 3 weeks in sterile saline
intravenously.
7. Take 5 ml of blood after seven days by cardiac puncture, allow the blood
to clot and draw off the serum.
8. 5 ml of pre-immune serum serves as con¬trol.
9. For ELISA suspend 10 mg of powdered antigen in sterile distilled water,
centri¬fuge at 15000 rpm for 20 minutes, dis¬card surface lipids and
lyophilize supernatant, suspend the powder in car¬bonate coating buffer (pH
9.6) at a con¬centration of 5 µg-ml-1.
10. Disperse 50 µl aliquots of the antigen suspension into polyvinyl
microlitre trays. Allow it to
dry at 37°C for 12 hours.
11. Wash tray three times with phosphate buffered saline containing 0.05%
Tween 20 (PBST).
12. Make serial dilutions of serum in phos¬phate buffered saline containing
0.05% Tween 20 (PB$T) (50µl) and add in quadruplicates to the wells.
13. Seal the plates with tape, incubate for one hour at 37°C.
14. Repeat washing with PBST.
15. Add 50 µl of goat anti-rabbits Ig G-horse raddish peroxidase labelled
immuno conjugate diluted to 1:1000 in PBST in each well and incubate at
37°C for 30 minutes.
16. Give a final wash to the trays and add 50 µl of orthophenylene diamine
substrate.
17. Using NH2SO4 stop the reaction after 15 minutes.
18. Read wells on a spectrophotometer at 490 nm using normal rabbit
serum as control.
19. From the absorbance of the test serum, the absorbance of control
serum is to be deducted. This gives specific reading.
20. Select a positive serum as reference to give an absorbance of 1.0.
Compare other antisera to this reference.
PureInoculumProductionofVAM:

Principle:
Since VAM cannot be grown in vitro, they are multiplied on the root system
of host plants.

Requirements:
1. Seeds of host plant-Jowar.
2. Plastic pots 7 X 10 X 10 cm.
3. Sand (sterile).
4. Inoculum obtained by wet sieving and decanting.
5. Inorganic nutrient solution—Hoglands.
6. Starter inoculum.
7. Growth
chamber.
Procedure:
1. Wash Jowar seeds under running tap wa¬ter to remove pesticides.
2. Soak them over night and germinate.
3. Add a 3 cm layer of sand (sterile) to the clean plastic pots.
4. Then place 1 cm depth of substrate with VAM inoculum (starter).
5. Place maximum 5-6 seeds on top of the inoculum per pot.
6. Cover them with sand.

7. Irrigate pots with Hogland’s solution (composition given under N2

fixation) at the rate of 20 ml
per pot.
8. Keep the pots at 28°-30°C (room tem¬perature) with a photo period of 14
hours light and 10 hours dark with a light intensity of 100 lux supplied with
fluorescent tubes for 3 weeks. Now pre¬pare second phase with expanded
clay.

MassProductionofVAM:

Principle:
The pure inoculum produced must be trans¬ferred to expanded clay along
with roots to be supplied as VAM inoculum.

Requirements:
1. Plants grown for pure inoculum.
2. Ethanol 70%.
3. Na hypochlorite-2%.
4. Sterile expanded clay.
5. 5 litre pots (clay ones).
6. Growth room with photoperiod of 14 hours light and 10 hours dark.
7. P free medium (liquid) / Hogland’s
so¬lution. Procedure:
There are several steps involved in the produc¬tion of VAM on a large
scale.

a. Separation of host plants:

Plants grown with pure inoculum in pots in the above experiment should be
uprooted carefully.

b. Sterilisation of host plants:

Wash thor¬oughly the uprooted host plants and sur¬face sterilise the root
system of these up¬rooted plants to prevent the transfer of pathogenic
organisms to the expanded clay. For this, dip the root system in 70%
ethanol for 2 minutes or 2% Na hypochlo¬rite solution. (This is optional
for plants grown under controlled conditions in growth chamber or the
ones that are upto three weeks old).

c. Preparation of expanded clay:

Either pasteurize or expose the clay to steam for 4 hours and cool to 20°C.

Place this in 5 litre or larger pots and plant two surface sterilised
plants (Jowar) hav¬ing maximum VAM colonisation in each pot
carefully without damaging the root system and water the plants.
 d. Parameters:
Five parameters are to be fol¬lowed:
i. Optional illumination of host plant.
ii. Optional temperature range for fungi in the root.
iii. No water logging or drought.
iv. Plant nutrition should be optional for symbiosis.
v. Plants should be treated for
pathogen. Illumination:
In temperate countries mass production should be done in green
houses with an intensity of 5000 Lux for 16 hours per day with sodium
vapour high pressure lamps; 10,000 Lux or more by mercury vapour
lamps for 14 hours. In tropi¬cal countries 1000 Lux supplied by
fluores¬cent mercury vapour tubes for 14 hours per day is sufficient.

Temperature:
Pots should be shaded by placing them in wide well-aerated trays.

Irrigation:
Since expanded clay has larger particles on top and smaller ones
below, more moisture will be present in the lower layers. To avoid this,
use clay pots instead of plastic ones and cover them with thin plastic
sheets with holes to allow exchange of gases.

Plant nutrition:
Modified Hogland and Arnon (1938)Treatment against pathogens Water,
plants once a month. Treat with fun¬gicide (Previeur 0.15%) to prevent
pathogens like Pythium.

e. Growth period:
It takes about 3—4 months for mass production of inoculum in
ex¬panded clay. Stop irrigation, after check¬ing if growth and
colonisation is found good. A drought stress caused by stopping
irrigation will induce formation of VAM spores.
f. Hence expose plants to drought for a week and then cut the plants.
g. After three weeks spread the substrate in a layer and air dry. Test for
contamination.
h. Store the inoculum under cool dry condi¬tions. They retain infectivity
for several years. Vermicompost:

Vermicompost (vermi-compost) is the product of the decomposition process

using various species of worms, usually red wigglers, white worms, and
other earthworms, to create a mixture of decomposing vegetable or
food waste, bedding materials, and vermicast. This process is called
vermicomposting, while the rearing of worms for this purpose is called
vermiculture.
Vermicomposting uses worms to decompose waste and make nutrient-
rich "worm manure".

Vermicast (also called worm castings, worm humus, worm manure, or

worm faeces) is the end- product of the breakdown of organic matter by
earthworms.[1] These castings have been shown to contain reduced
levels of contaminants and a higher saturation of nutrients than the
organic materials before vermicomposting.[2]
Vermicompost contains water-soluble nutrients and is an excellent,
nutrient-rich organic fertilizer and soil conditioner.[3] It is used in farming
and small scale sustainable, organic farming.
Vermicomposting can also be applied for treatment of sewage. A
variation of the process is vermifiltration (or vermidigestion) which
is used to remove organic matter, pathogens and oxygen demand
from wastewater or directly from blackwater of flush toilets.[4][5]
HarvestingEdit

Worms in a bin being harvested

Vermicompost is ready for harvest when it contains few-to-no scraps

of uneaten food or bedding.[25] There are several methods of
harvesting from small-scale systems: "dump and hand sort", "let the
worms do the sorting", "alternate containers" and "divide and
dump."[30] These differ on the amount of time and labor involved and
whether the vermicomposter wants to save as many worms as
possible from being trapped in the harvested compost.
The pyramid method of harvesting worm compost is commonly used
in small-scale vermiculture, and is considered the simplest method for
single layer bins.[31] In this process, compost is separated into large
clumps, which is placed back into composting for further breakdown, and
lighter compost, with which the rest of the process continues. This
lighter mix is placed into small piles on a tarp under the sunlight. The
worms instinctively burrow to the bottom of the pile. After a few
minutes, the top of the pyramid is removed repeatedly, until the worms
are again visible. This repeats until the mound is composed mostly of
worms.
When harvesting the compost, it is possible to separate eggs and
cocoons and return them to the bin, thereby ensuring new worms are
hatched. Cocoons are small, lemon-shaped yellowish objects that can
usually be seen with the naked eye.[32] The cocoons can hold up to 20
worms (though 2-3 is most common). Cocoons can lay dormant for as
long as two years if conditions
are not conducive for
hatching.[33] Soil
Improves soil aeration
Enriches soil with micro-organisms (adding enzymes such as phosphatase
and cellulase)
Microbial activity in worm castings is 10 to 20 times higher than in the soil
and organic matter that the worm ingests[41]
Attracts deep-burrowing earthworms already present in the soil
Improves water holding capacity[42]

Plant growth
Enhances germination, plant growth, and crop yield
Improves root growth and structure
Enriches soil with micro-organisms (adding plant hormones such as
auxins and gibberellic acid)[citationneeded]

Economic
Biowastes conversion reduces waste flow to landfills
Elimination of biowastes from the waste stream reduces contamination of
other recyclables collected in a single bin (a common problem in
communities practicing single-stream recycling)
Creates low-skill jobs at local level
Low capital investment and relatively simple technologies make
vermicomposting practical for less-developed agricultural regions

Environmental
Helps to close the "metabolic gap" through recycling waste on-site
Large systems often use temperature control and mechanized harvesting,
however other equipment is relatively simple and does not wear out
quickly[citationneeded]
Production reduces greenhouse gas emissions such as methane and nitric
oxide (produced in landfills or incinerators when not composted).
 Marks
Semester Course Hours Credit Sub.Code
Internal External Total
I MBE1 6 4 18KP1BELBl 25 75 100

BIOFERTILIZER AND MUSHROOM TECHNOLOGY

UNIT – IV & UNIT - V

Unit - IV
Introduction - History and Scope of edible and medicinal mushroom – Types of
mushroom and Economic imporatance. Cultivation method – Isolation, Spawn preparation,
Growth media, Spawn running and harvesting and Marketing of mushroom
Unit – V
Cultivation technology – Agaricus sp., Pleurotus sp., Vovariella sp., Storage – Short term
storge and Long term storge. Nutritional value and Food preparation – Types of food prepared
from mushroom (Cutlet, Omelet, Pickels, Curry, Soup & Biriyani
UNIT - IV & UNIT - V
REFERENCE
1. Kumaresan. B,2001, Biotechnology, Saras Publication, Tamil nadu
2. Dubey, R.C., 2001, Text Book of biotechnology, S. Chand & Co., New Delhi.

Prepared by
Dr. V. Latha,
Assistant professor,
KN. GOVT ARTS COLLEGE FOR WOMEN,
THANJAVUR -7
 MUSHROOM TECHNOLOGY
INTRODUCTION
A mushroom or toadstool is the fleshy, spore-bearing fruiting body of a fungus,
typically produced above ground, on soil, or on its food source. The standard for the name
"mushroom" is the cultivated white button mushroom, Agaricus bisporus; hence the word
"mushroom" is most often applied to those fungi (Basidiomycota, Agaricomycetes) that
have a stem (stipe), a cap (pileus), and gills (lamellae, sing. lamella) on the underside of the
cap. "Mushroom" also describes a variety of other gilled fungi, with or without stems,
therefore the term is used to describe the fleshy fruiting bodies of some Ascomycota. These
gills produce microscopic spores that help the fungus spread across the ground or its
occupant surface. Forms deviating from the standard morphology usually have more
specific names, such as bolete, puffball, stinkhorn, and morel, and gilled mushrooms
themselves are often called agarics in reference to their similarity to Agaricus or their order
Agaricales. By extension, the term mushroom can also refer to either the entire fungus
when in culture, the thallus (called a mycelium) of species forming the fruiting bodies
called mushrooms, or the species itself.
HISTORY
Mycophagy the act of consuming mushrooms, dates back to ancient times. Edible
mushroom species have been found in association with 13,000-year-old archaeological sites
in Chile. Ötzi, the mummy of a man who lived between 3400 and 3100 BCE in Europe, was
found with two types of mushroom. The Chinese value mushrooms for supposed medicinal
properties as well as for food. Ancient Romans and Greeks, particularly the upper classes,
used mushrooms for culinary purposes. Food tasters were employed by Roman
emperors to ensure that mushrooms were safe to eat
Edible mushrooms are the fleshy and edible fruit bodies of several species of
macrofungi (fungi which bear fruiting structures that are large enough to be seen with the
naked eye). They can appear either below ground (hypogeous) or above ground (epigeous)
where they may be picked by hand. Edibility may be defined by criteria that include
absence of poisonous effects on humans and desirable taste and aroma. Edible mushrooms
are consumed for their nutritional and culinary value. Mushrooms, especially dried
shiitake, are sources of umami flavor from guanylate. Mushrooms consumed by those
practicing folk medicine are known as medicinal mushrooms. While psychedelic
mushrooms are occasionally consumed for recreational or entheogenic purposes, they can
produce psychological effects, and are therefore not commonly used as food. There is no
evidence from high-quality clinical research that "medicinal" mushrooms have any effect
on human diseases.
Edible mushrooms include many fungal species that are either harvested
wild or cultivated. Easily cultivated and common wild mushrooms are often available
in markets, and those that are more difficult to obtain (such as the
 prized truffle, matsutake and morel) may be collected on a smaller scale by private
gatherers. Some preparations may render certain poisonous mushrooms fit for
consumption.
SCOPE
Mushroom production has tremendous scope in Tamil Nadu. Mushroom has
excellent medicinal properties. It is rich in protein, fibre, and amino acids. Mushroom is a
100 per cent vegetarian food and is good for diabetes and joint pains
HISTORY OF MEDICINAL MUSHROOM
The ancestors have used mushrooms as medicine for thousands of years. The Greek
physician Hippocrates, circa 450 bce, classified the amadou mushroom (Fomes
fomentarius) as a potent anti-inflammatory and for cauterizing wounds.
TYPES OF MUSHROOMS
1. Button Mushrooms. Button mushrooms are also called baby mushrooms or white
mushrooms.
2. Cremini Mushrooms.
3. Portobello Mushrooms.
4. Oyster Mushrooms.
5. King Oyster Mushrooms.
6. Chanterelle Mushrooms.
7. Porcini Mushrooms.
8. Hedgehog Mushrooms.

ECNOMIC IMPORTANCE
Nowadays, mushrooms are popular valuable foods because they are low in calories,
carbohydrates, fat, and sodium: also, they are cholesterol-free. Besides, mushrooms
provide important nutrients, including selenium, potassium, riboflavin, niacin, vitamin D,
proteins, and fiber. General populace is less aware about the economic value of
mushrooms. Mushroom is a saprophytic organism and hence it utilizes organic and
agricultural waste. This reduces the burden of farmers to dispose his farm wastes.
Additional income is obtained through quality mushrooms production by utilizing these
residues. Mushroom cultivation both seasonal and commercial nature is giving handsome
income to the growers. The employment generation through cultivation and associated
allied activities is so immense. The value addition to mushrooms in terms of quality
products is another economic avenue. The positive use of spent mushroom substrate viz.,
biofuel, biogas production, manures, potting medium, etc also generates additional revenue
to the farmer.
CULTAVATION METHOD
Cultivation is the process of tilling or loosening soil to prepare it for planting. It is
often an essential method for maintaining soil health, preventing weed development, and
encouraging crop growth. Medicinal plants can be cultivated by two methods (applicable to
non-medicinal plants). Sexual and Asexual method
Sexual Method (seed propagation).
In this method, the plants are raised from seeds. Such plants are known as seedlings.
Seeds are sown in the fields by methods like broadcast, dibbling, or placing them in drills
or holes. The seeds must be of good quality, capable of high germination rate, and free
from diseases.
ADVANTAGES:
Seedlings are comparatively much cheaper and easy to raise. Seedlings are long-
lived, bear more heavy fruits and plants obtained are more sturdy. In those plants where
other methods of cultivation cannot be utilized, seed propagation becomes the only method
of choice. There are chances of production of some chance-seedlings of very high
superiority which may be of great importance for e.g., orange, papaya, etc.
DISADVANTAGES:
The seedlings obtained from this method require more time to bear and are not
uniform in their growth and yielding capacity as compared to other methods like grafting.
Also, the cost involved in harvesting and protection from pests is more.
Asexual Methods (vegetative propagation):
In this method, any of the vegetative part of the plant like root or stem is provided
such an environment that it develops into a new plant. The environment is provided by
setting various parts of the plant in well prepared soil.
Bulbs: A bulb is originally and structurally a bud, which possesses the capability of
perennation. It consists of a very short stem ending in an apical meristem and enclosed by
closely set leaves, which are thick and fleshy , being stored with reserves of food. Each of
the leaves has of course its axillary bud .
Corms: In a corm, the storage organ is swollen base of the stem and this is wrapped in thin
scale-leaves, each of which, of course, has an axillary bud e.g. colchicum, saffron.
Tubers: It is a swelling on an underground stem branch. The stem grows axillary buds
formed low down on the aerial stem and push through the soil, swelling at their ends to
form the tubers. The of the tuber are very small scale-leaves, each with three axillary buds
e.g., jalap, aconite, potato.
Rhizomes: In underground stems, the older parts of rhizome die off. The buds borne on the
detached younger portions thus become separate new plants e.g., ginger, turmeric.
Runners: The stem grows along the ground (horizontally over the surface of the soil), and
produces roots and erect flowering shoots from lateral buds at many of its nodes. The
growth of the creeping stem is continued by the terminal bud. Some of the older internodes
die, and the detached rooted and shoot bearing parts become independent plants e.g.,
peppermint, strawberry.
Suckers: A shoot arising from a root of a woody plant e.g., mint, pineapple, banana.
Offsets: These originate from the axil of the leaf as short thick horizontal branches and also
characterized by the presence of rosette type of leaves and a cluster of roots at their bottom
e.g., aloe, valerian.
Stolons: A creeping stem that roots at nodes e.g., arrow-root, liquorice. Other methods,
Cutting: A clear cut is made preferably below the node and the lower leaves are removed.
It is then placed in a suitable medium and provided with suitable conditions of moist
atmosphere, temperature which favouring the development of roots e.g., mint, vanilla.
Layering: A layer is a branch or a shoot which is induced to develop roots before it is
completely severed from the parent plant. It is done by a cut or ligature and embedding the
part so treated in the soil e.g., cascara.
Grafting and Budding: Grafting is a process in which two cut surfaces of different but
closely related plants are placed so as to unite and grow together. The rooted portion is
called the stock and the cut off is the scion or graft e.g., female scion of Myristica fragrans
on male stock to increase fruit bearing proportion. Budding consists of the introduction of
a piece of bark bearing a bud into a suitable cavity or shaped slit made in the bark of the
stock e.g., citrus species, sweet and sour oranges. Aseptic methods of propagation: In this
method, the plants are developed in an artificial medium under aseptic conditions from
very fine pieces of plants like single cells, callus, seeds, embryos, root tips, shoot tips, pollen
grains, etc. They are provided with nutritional and hormonal requirements.
Advantages: (Asexual method): There is no variation between the plant grown and plant
from which it is grown. As such, the plants are uniform in growth and yielding capacity.
Seedless varieties of fruits can only be propagated vegetatively e.g., pomegranates, grapes,
lemon. Plants start bearing earlier as compared to seedlings. Budding or grafting
encourages disease-resistant varieties of plants.
Disadvantages: In comparison to seedling trees, these are not vigorous in growth and are
not long lived. No new varieties can be evolved by this method.
ISOLATION
In microbiology, the term isolation refers to the separation of a strain from a
natural, mixed population of living microbes, as present in the environment, for example in
water or soil flora, or from living beings with skin flora, oral flora or gut flora, in order to
identify the microbes of interest.
SPAWN PREPARATION
In the spawn-production process, mycelium from a mushroom culture is placed onto
steam-sterilized grain, and in time the mycelium completely grows through the grain. This
grain/mycelium mixture is called spawn, and spawn is used to seed mushroom compost.
250 ml Flasks, 50 ml beaker level full of rye grain, 1/2 tsp. Calcium Carbonate,
powder (lime), 1/4 tsp. Calcium Sulfate (gypsum), 60 ml warm water Autoclave 35 minutes
at 121°C - Fast Exhaust

GROWTH MEDIA
A growth medium or culture medium is a solid, liquid or semi-solid designed to
support the growth of a population of microorganisms or cells via the process of cell
proliferation, or small plants like the moss Physcomitrella patens. Different types of media
are used for growing different types of cells. The two major types of growth media are
those used for cell culture, which use specific cell types derived from plants or animals, and
microbiological culture, which are used for growing microorganisms, such as bacteria or
fungi. The most common growth media for microorganisms are nutrient broths and agar
plates; specialized media are sometimes required for microorganism and cell culture
growth. Some organisms, termed fastidious organisms, require specialized environments
due to complex nutritional requirements. Viruses, for example, are obligate intracellular
parasites and require a growth medium containing living cells.
SPAWN RUNNING
After spawning, comes spawn-run or the stage where the spawn or the mycelium is
allowed to grow through the substrate. At this stage, the mushroom mycelium starts to
produce enzymes that will further break down the macromolecules of the substrates and
absorb simple molcules into the mycelium for further growth and development. Thus, as
the mycelium develops, the substrate starts to break down and becomes available to the
growing mycelium. This period is also called the vegetative growth or incubation period.
The length of the time for spawn running depends upon the mushroom being cultivated:
10-15 days for Volvariella volvacea, 2-3 months for Lentnula edodes and Auricularia and 2-
4 weeks for Pleurotus and Agaricus.
As soon as spawning has been completed, the farmer has been advised to maintain
the growing conditions at the optimum level and as uniform as possible inside the spawning
room. For while button mushroom which is most common in India. The room should be
maintained at about 25°C temperature. The humidity should be built up by frequently
watering the floor and walls. The room may be kept closed as only a small amount of fresh
air is needed for spawn growing and mushroom mycelium is quite tolerant to carbon-
dioxide. The temperature of the bed is more important than the temperature of the
atmosphere. The bed temperature must not be allowed to exceed 24°C. The temperature
rises particularly when compost beds are thick, and this leads to the development of fruit
bodies later only at the edges of the bed. This problem can be dealt with in two ways: by
providing thinner compost beds, and by lowering the room temperature.
HARVESTING
Harvesting is the process of gathering a ripe crop from the fields. Reaping is the
cutting of grain or pulse for harvest, typically using a scythe, sickle, or reaper. On smaller
farms with minimal mechanization, harvesting is the most labor-intensive activity of the
growing season. On large mechanized farms, harvesting utilizes the most expensive and
sophisticated farm machinery, such as the combine harvester. Process automation has
increased the efficiency of both the seeding and harvesting process. Specialized harvesting
equipment utilizing conveyor belts to mimic gentle gripping and mass transport replaces
the manual task of removing each seedling by hand. The term harvesting in general usage
may include immediate postharvest handling, including cleaning, sorting, packing, and
cooling. The completion of harvesting marks the end of the growing season, or the growing
cycle for a particular crop, and the social importance of this event makes it the focus of
seasonal celebrations such as harvest festivals, found in many religions.
MARKETING OF MUSHROOM
The mushroom market, in terms of value, is projected to reach $50,034.12 million
by 2019, at a CAGR of 9.5% from 2014. The growth of this market is primarily triggered
by factors such as a rise in the consumption of processed food and growing awareness
about health and wellness.
UNIT - V
 CULTIVATION TECHNOLOGY

Mushroom cultivation is a process utilizing waste materials such as horse manure,

chicken manure, pig manure, wheat straw, rice straw, corn cobs, wood bark, sawdust, and
cottonseed hulls to produce a delicious and nutritious food. Therefore, it can be considered
as a twofold beneficial operation
Agaricus sp.,
Agaricus is a genus of mushrooms containing both edible and poisonous species,
with possibly over 300 members worldwide. The genus includes the common (button)
mushroom (Agaricus bisporus) and the field mushroom (A. campestris), the dominant
cultivated mushrooms of the West. Members of Agaricus are characterized by having a
fleshy cap or pileus, from the underside of which grow a number of radiating plates or gills
on which are produced the naked spores. They are distinguished from other members of
their family, Agaricaceae, by their chocolate-brown spores. Members of Agaricus also have
a stem or stipe, which elevates it above the object on which the mushroom grows, or
substrate, and a partial veil, which protects the developing gills and later forms a ring
or annulus on the stalk.
Pleurotus sp.,
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. Oyster mushrooms contain lovastatin, a form of
cholesterol lowering statin.
Vovariella sp.,
Volvariella volvacea (also known as paddy straw mushroom or straw mushroom) is a
species of edible mushroom cultivated throughout East and Southeast Asia and used
extensively in Asian cuisines. They are often available fresh in Asia, but are more
frequently found in canned or dried forms outside their nations of cultivation. Worldwide,
straw mushrooms rank third in consumption, although their use in the West is somewhat
uncommon and usually confined to use in Oriental cooking.
Storage
Storing: Store mushrooms in their original packaging or in a porous paper bag for
prolonged shelf-life. Some mushrooms may keep for up to one week in the refrigerator.
Fresh mushrooms should never be frozen, but frozen sautéed mushrooms will keep for up
to one month.
Short term storage
Short term storage is generally defined as storage for three months or less, but it's
really just storage where you know you want to be able to access your belongings sooner
rather than later
 Long term storage
With long term, you are looking at prolonged periods of time of storage, which goes
well with seasonal storing of clothes, furniture, or storing during the winter. When
comparing short-term vs. long-term storage, it is good to know just how much of a
difference
Natural value
In mathematics, the natural numbers are those used for counting (as in there are six
coins on the table) and ordering (as in this is the third largest city in the country). In
common mathematical terminology, words colloquially used for counting are cardinal
numbers, and words used for ordering are ordinal numbers. The natural numbers can, at
times, appear as a convenient set of codes (labels or names) that is, as
what linguists call nominal numbers, forgoing many or all of the properties of being a
number in a mathematical sense. The set of natural numbers is often denoted by the
symbol. Some definitions, including the standard begin the natural numbers with,
corresponding to the non-negative integers 0, 1, 2, 3, (often collectively denoted by the
symbol for emphasizing that zero is included), whereas others start with 1, corresponding
to the positive integers (sometimes collectively denoted by the symbol for emphasizing that
zero is excluded).
Food preparation
Mushrooms can easily be regarded as nature's hidden treasure. It was once
considered an exotic ingredient but is now rated as a new superfood. What's so super about
them? Mushrooms are one of the few natural sources of Vitamin D. They contain no fat
and are a valuable source of fiber. They are also packed with selenium which you don't
find in most fruits and vegetables. According to a latest study, lentinan found in shiitake
mushrooms may increase the survival rate in cancer patients. Slowly but surely
mushrooms are gaining prominence on an Indian platter. All the goodness along with its
unique earthy texture is reason enough for you to let them adorn your dinner table more
often. The versatility of mushrooms can work its magic in almost any dish. Not only are
there a number of varieties to cook with, but the culinary possibilities are almost
inexhaustible. You can choose to grill, bake, stir-fry, sauté, stuff, roast or experiment with
something new. With more than 90 percent water content, the best way to cook mushrooms
is to allow them to release all their moisture. Pick a pan with a wide surface area because
you should be able to spread them out which will make the moisture evaporate quickly.

Types of food prepared from mushroom

• Wild Mushroom Ragout. A ragout is a stew like preparation. ...
• Mushroom Risotto. ...
• Mushroom Khichda. ...
• Mushroom Samosa. ...
• Mushroom Chettinad. ...
• Whole Wheat Pasta in Mushroom Sauce. ...
• Herb Coated Mushrooms with Cheese Sauce. ...
• Stuffed Masala Mushrooms.

Cutlet

1. thin slice of meat from the leg or ribs of veal, pork, chicken, or mutton
2. dish made of such slice, often breaded (also known in various languages as
a cotoletta, Kotelett, kotlet or kotleta)
3. croquette or cutlet-shaped patty made of ground meat
4. a kind of fish cut where the fish is sliced perpendicular to the spine, rather than
parallel (as with fillets); often synonymous with steak
5. prawn or shrimp with its head and outer shell removed, leaving only the flesh and
tail
6. mash of vegetables (usually potatoes) fried with bread
Omelet
In cuisine, an omelette or omelet is a dish made from beaten eggs, fried with butter
or oil in a frying pan (without stirring as in scrambled egg). It is quite common for the
omelette to be folded around fillings such as cheese, chives, vegetables, mushrooms, meat
(often ham or bacon), or some combination of the above. Whole eggs or egg whites are
often beaten with a small amount of milk, cream, or water.
Pickeles
Pickling is the process of preserving or extending the shelf life of food by
either anaerobic fermentation in brine or immersion in vinegar. The pickling procedure
typically affects the food's texture, taste and flavor. The resulting food is called a pickle, or,
to prevent ambiguity, prefaced with pickled. Foods that are pickled include vegetables,
fruits, meats, fish, dairy and eggs. A distinguishing characteristic is a pH of 4.6 or lower,
which is sufficient to kill most bacteria. Pickling can preserve perishable foods for
months. Antimicrobial herbs and spices, such as mustard seed, garlic, cinnamon or cloves,
are often added. If the food contains sufficient moisture, a pickling brine may be produced
simply by adding dry salt. For example, sauerkraut and Korean kimchi are produced by
salting the vegetables to draw out excess water. Natural fermentation at room temperature,
by lactic acid bacteria, produces the required acidity. Other pickles are made by placing
vegetables in vinegar. Like the canning process, pickling (which includes fermentation)
does not require that the food be completely sterile before it is sealed. The acidity or
salinity of the solution, the temperature of fermentation, and the exclusion of oxygen
determine which microorganisms dominate, and determine the flavor of the end product
Curry
Mushroom gravy is a simple, usually red sauce that can be composed from stock
(beef is typical, but chicken may be used), roux (a mixture of equal parts butter and flour
to thicken), and mushroom base.
Soup
Soup is a primarily liquid food, generally served warm or hot (but may be cool or
cold), that is made by combining ingredients of meat or vegetables with stock, or water.
Hot soups are additionally characterized by boiling solid ingredients in liquids in a pot
until the flavors are extracted, forming a broth. Soups are similar to stews, and in some
cases there may not be a clear distinction between the two; however, soups generally have
more liquid (broth) than stews. In traditional French cuisine, soups are classified into two
main groups: clear soups and thick soups. The established French classifications of clear
soups are bouillon and consommé. Thick soups are classified depending upon the type of
thickening agent used: purées are vegetable soups thickened with starch; bisques are made
from puréed shellfish or vegetables thickened with cream; cream soups may be thickened
with béchamel sauce; and veloutés are thickened with eggs, butter, and cream. Other
ingredients commonly used to thicken soups and broths include rice, lentils, flour, and
grains; many popular soups also include pumpkin, carrots, potatoes, pig's trotters and
bird's nests.
Briyani
I have removed the paragraph in the etymology section talking about vegetarian
biryani and pulao as it does not belong there. There is already an extensive section talking
about the difference between biryani and pulao, which led to inconsistency in this article
with that extra paragraph in the etymology section. I also had it's only source as an opinion
piece done only just a few months ago with no actual factual evidence for the claim. I'm
perfectly fine with people saying veg biryani is not biryani, but if you're going to put that in
the article you need a factual source rather than a source that is just an opinion. It also
needs to be in the proper section. The etymology section is there to explain the origin of the
word, not to advance personal opinions.