DR. D. Y.

PATIL COLLEGE OF AGRICULTURE

TALSANDE

(Affiliated to M. P. K. V. Rahuri)
Talsande, Dist: Kolhapur- 4161112

Practical Journal
SEC- 111

Biofertilizers and Biopesticide Production

Credit- 2(0+2)

Course Teacher
Ms. A. R. Jadhav

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 INDEX

Sr. Particulars
No.

1 Equipment, machinery and tools used for biofertilizers &Biopesticides

production.

2 Methods of sterilization

3 Nutritional media and their preparations

4 Preparation of nutrient agar & PDA

5 Methods of isolation and purification of microbial cultures

6 Different media used for biofertilizers and biopesticides production

7 Isolation of rhizobium from root nodule of leguminous crop

8 Isolation of azotobacter from soil

9 Isolation of Azospirillum from soil and roots of hariyali (cynadon dactylon)

10 Isolation of Acetobacter from sugarcane

11 Isolation of Beijerinckia from sugarcane

12 Isolation of blue green algae from soil

13 Isolation of phosphate solubilizing microorganisms from soil

14 Isolation of potash solubilizing microbes

15 Isolation of sulphur oxidizing microorganisms from soil

16 Isolation of organic decomposer culture

17
Production of commercial biofertilizers of Rhizobium Azotobacter,
Azospirillum, Acetobacter, Organic matter decomposers

18 production of carrier biofertilizers of sulphur oxidizing microorganisms,

iron chealator, potash mobilizers

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19
study of va-mycorrhiza: growth on guinea grass roots and observations
for root colonization. methods of preparationand application of va-
mycorrhizal inoculums

20 Mass multiplication of BGA and azolla and its application in paddy field

21 Introduction to Biopesticides

22 classification of biopesticides

23 Importance & mass production of verticillium

24 Importance and mass production of beauveria

25
Importance and mass production of metarhzium

26
Importance and mass production of Paecilomyces

27 Importance and mass production of Trichoderma

28 Importance and mass production of Nomuraea

29 Importance and mass production of Hirsutella thompsoni

30 Quality control parameters of Biopesticides (as per CIB specifications)

31 Labeling and packing of Biopestide

Products

32
methods of application of Biofertilizers and Biopesticides

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 EXERCISE NO. 1

EQUIPMENT, MACHINERY AND TOOLS USED FOR

BIOFERTILIZERS &BIOPESTICIDES PRODUCTION

In biofertilizer production industry, equipment’s are the major

infrastructure, which involves 70 percent of capital investment. Any
compromise on the usage of the following mentioned equipments may finally
decline in the quality of biofertilizer. After studying the principle behind the
usage of all instruments; some of the instruments can be replaced with a culture
room fitted with a U.V.Lamp. Autoclaves, Hot Air Oven, Incubators and sealing
machines are indigenously made with proper technical specifications. The
correct use of equipments will give uninterrupted introduction with quality
inoculum.

Autoclave
Culture media like Potato Dextrose Agar (P.D.A.), Nutrient Broth (N.B.),
Nutrient Agar (N.A.) and soil, water, sand, manure, etc. which are not damaged
by high temperature of the steam under pressure are sterilized by autoclave. The
Autoclave consists of a thick walled vessel made of alloy (metal), with a close
fitting, which can be tightened with fly-nuts. The lid is provided with air or
steam cock, safety valve and pressure gauge. Water is to be filled at the bottom
and a separator stand or frame is provided at the bottom. It is operated by
electric current or in some cases gas burners can also be used.
Principle: Works on the principle of Steam under pressure.
Water is heated to generate a steam, which is collected in a closed chamber that
raises the temperature as follows:

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 Sr. No. Pressure in lbs/sq. inch. Temperature in

oC

1 5 lbs. 107.7

2 10 lbs. 115.5

3 15 lbs. 121.6

4 30 lbs. 134.4

Normally it takes 11-12 minutes at 121oC (moist heat) to kill thermophilic

bacteria. Culture media are usually sterilized at 15 lbs. pressure maintained for 15
to 20 minutes and soil at 25 to 30 lbs. pressure for 30 to 60 minutes depending
upon the volume of materials in autoclave. This is sufficient to destroy all
vegetative cells. Normally all growth medium are sterilized in the autoclave.

LAMINAR AIR FLOW CHAMBER:

Principle :

o The underlying principle of a liminar air flow hood is that a constant

flow of HEPA (High Efficiency Particulate Air Filter) filtered air at a

rate of approximately90 linear feet per minute physically sweeps the
work area and prevents the entryof contaminated air.
o The space between the HEPA filter and sterile product being prepared is

referredto as the critical work surface.

o HEPA filter - removes 99.97 % of all air particles which have the

diameter of 0.3mm or larger.

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 o The nominal filter surface air speed of 0.45 m/s (90 fpm) ensures a

continuous change of air in the enclosed area and therefore ensures the
required purity of the air.

Uses :

o The hood workspace is used to prevent the contamination of compounded

sterileproducts and parenteral preparations.

o It is used for maintaining aseptic or microorganism free environment for

performing various activities such as pouring of sterilized media in

petridishes, test tubes, isolation and sub culturing of pathogens etc.

INCUBATOR :

* In 1857, Jean Louis Paul Denuce invented the first incubator which

was usedfor birds like chicken to hatch eggs for a very long time.
* In 1891, French doctor Alexander Loin invented the modern incubator.

* It is similar to hot air oven in construction and operation.

Principle :

* It is based on the principle to provide controlled temperature and

humidity fororganisms which grow at their maximum growth rate.

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Uses :

* The range of temperature varies from room temperature to about

50-60oC.It is used for incubation of microorganisms at suitable

temperature

HOT AIR OVEN

Materials like dry glassware such as test tubes, flasks, Petri-dishes, pipettes,
funnels, etc. which get damaged by direct flame heating and which do not
contain any moisture of matter likely to be burnt or charred can be sterilized by
this method. The apparatusis known as Hot Air Oven.

Principle: The air is heated to 160oC to 180o C by electric heaters, the material
is exposed to such hot air for 1 to 2 hrs. The Hot Air Oven is made up of double
walled chamber (Inner copper and outer asbestos) provided with electric coils at
the bottom, dial with regulator switch, ventilator, pilot lamp and thermometer.
Before opening the oven, allow the temperature to lower to room temperature to
avoid cracking of glassware.

pH METER :
* pH meter was invented by Arnold O. Beckman for Testing Acidity.

* A pH meter is an electronic device used for measuring the pH (acidity or

alkalinity) of a liquid

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 Uses :

* It is used to determine the pH of solutions of unknown pH.

* It is used for setting the pH of various media used for the cultivation and

testingbiochemical activities of micro organisms.

FERMENTOR
* An apparatus that maintains optimal conditions for the growth of
microorganisms, used in large scale fermentation and in the commercial
production of antibiotics and hormones

ROTARY SHAKER

It is used for agitating culture flasks by circular motion under variable speed
control. Shaking provides aeration for growth of cultures. Shakers holding upto
20-50 flasks are generally used. The capacity of the shaker may be increased if
it is a double- decker type.

REFRIGERATOR

This equipment is used preserving all mother cultures used for biofertilizer
production. The mother culture is periodically sub-cultured and stored in the
refrigerator for long- term usage.

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Instruments used in Laboratory of Plant Pathology and their uses
1. Inoculating Needle Used in isolation procedure to transfer the fungal/ bacterial
colony from one Petri plate to another.
2. Petri Plate: Used for isolation, multiplication and maintenance of fungal
or bacterial cultures. It was named after German bacteriologist
Julius Richard Petri, who invented it when working as an assistant to Robert Koch
3. Test Tube: Used for preparing slants of media. Also used in serial
dilution method for isolation of fungi or bacteria
4. A Bunsen burner: It produces a single open gas flame, which is used for
heating, sterilization, and combustion.
5. Microscope: To observe and examine the microorganisms
6. Agar Agar powder: Used as solidification agent in different media
7. Dextrose: Used as a source of sugar in different media
8. Conical Flask: Used in preparation of media.
9. Sprit Lamp: To sterilize the needle, cork borer etc
10. Cork Borer: To prepare the holes in fungal colony in Petri Plate
11. Cotton: To plug the conical flask and test tubes
12. Glass Slide: To mount the culture for examination under microscope
13. Cover slip: To cover the culture on glass slide
14. Forceps: To transfer the bits of samples on media during isolation procedure
15. Spreader: To spread the cultures of pathogen on media.
16. Digital Colony Counter: Used to count the number of colonies of bacteria on a
medium.
17. Micro pipette To take the exact quantity of solution in different procedures
like isolation, serial dilution etc.
18. Water Distillation Unit: To obtain the distilled water for different studies.

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 EXERCISE NO. 2

METHODS OF STERILIZATION
Objective: To keep the material free from microorganisms

Sterilization: It is a process by means of which material is made sterile or free

from living microorganisms either by killing or by separating them.
It is essential to sterilize cultural media, glassware & other laboratory
instruments before their use to avoid contamination. In pure culture studies,
different physical & chemical means are used for this purpose, which are as
follows:

1. Physical agents:
1) Heats
i) Dry: (a) Direct, e.g. flame, (b) Indirect, i.e. Hot Air Oven.
ii) Moist: (a) Stem without pressure – Arnold Steam Sterilizer, (b)
Steam with pressure – autoclave.
2) Filters
For liquids: Porcelain or glass filters like
(a) Chamber land, (b) Berkefeld, (c) Seitz.
For Gases: Cotton fibers, etc.
3) Light: I) Ultraviolet rays, ii) Germicidal lamps.

2. Chemical agents: a) Inorganic KMNO4, Iodine, etc. b) Organic –

Phenol, formaldehyde, ethyl alcohol, etc.

Heat

Direct dry heating/flame sterilization: It is useful for the material made

up of metal like inoculating needles, scalpels, forceps etc. In this method, the
temperature reaches above 200o C and material is exposed for few seconds.

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 Indirect dry heating by hot air oven: Materials like dry glassware such as
test tubes, flasks, Petri-dishes, pipettes, funnels, etc. which get damaged by
direct flame heating and which do not contain any moisture of matter likely
to be burnt or charred can be sterilized by this method. The apparatus is
known as Hot Air Oven.

Principle: The air is heated to 160oC to 180o C by electric heaters, the material
is exposed to such hot air for 1 to 2 hrs. The Hot Air Oven is made up of
double walled chamber (Inner copper and outer asbestos) provided with
electric coils at the bottom, dial with regulator switch, ventilator, pilot lamp
and thermometer. Before opening the oven, allow the temperature to lower to
room temperature to avoid cracking of glassware.

Moist heating by steam without pressure (Arnold steam sterilizer):

Materials like media which contain moisture or mater likely to be affected or
spoiled by dry heating and which requires moist heating by using the apparatus
known as Arnold Steam Sterilizer. For example, certain media containing
carbohydrates, potato cylinders, gelatin, milk media, etc. are sterilized by
ordinary flowing steam or streaming steam, as it cannot be subjected to steam
pressure, which affects the carbohydrate contents.

Principle: The material is exposed to ordinary steam i.e. 100 to 110 o C for 20
to 30 minutes and the process is repeated for successive 2 more days with an
interval of 24 hrs. This repeated treatment kills the resistant spore formers that
germinate during the intermittent period. The process is also known as
‘fractional or intermittent sterilization or Tyndallization’.
The apparatus ‘Arnold steam sterilizer’ consists of double walled copper
chamber with a water holding trays at the bottom. The material to be sterilized
is placed on perforated sieves. Steam rising from bottom, heats the material,
gets condensed at the top and returns to be boiling trays through the space in
between two walls. Heating can be done by either electric heaters or gas
burners.

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Moist heating by steam under pressure (Autoclave)
Culture media like Potato Dextrose Agar (P.D.A.), Nutrient Broth
(N.B.), Nutrient Agar (N.A.) and soil, water, sand, manure, etc. which are not
damaged by high temperature of the steam under pressure are sterilized by this
method. Steam under pressure has high temperature and more power that is
penetrating and hence it is the quickest method. Materials usually sterilized by
direct heating or hot air can also be sterilized by this method. The apparatus
used in this method is called as ‘Autoclave’.

Principle: Water is heated to generate a steam, which is collected in a closed

chamber that raises the temperature as follows:

Sr. No. Pressure in lbs/sq. Temperature in

inch. oC

1 5 lbs. 107.7
2 10 lbs. 115.5
3 15 lbs. 121.6
4 30 lbs. 134.4

Culture media, water, etc. are sterilized at 15 lbs. Pressure for 15

minutes, while bulky materials like soil, sand, manure etc. required 30 lbs.
Pressure for 30 minutes.
The Autoclave consists of a thick walled vessel made of alloy (metal),
with a close fitting, which can be tightened with fly-nuts. The lid is provided
with air or steam cock, safety valve and pressure gauge. Water is to be filled
at the bottom and a separator stand or frame is provided at the bottom. It is
operated by electric current or in some cases gas burners can also be used.

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 Normally it takes 11-12 minutes at 121 oC (moist heat) to kill
thermophilic bacteria. Culture media are usually sterilized at 15 lbs. pressure
maintained for 15 to 20 minutes and soil at 25 to 30 lbs. pressure for 30 to 60
minutes depending upon the volume of materials in autoclave. The materials
like oils, fats and gasses cannot be sterilized in autoclave.

Precautions to be taken while working with Autoclave:

1. Equipment must be in working order.

2. Sufficient water should be filled before heating.
3. Pressure is allowed to rise only when all air from in side is removed
through air cock.
4. When desired pressure is obtained, it is maintained for specific time.
5. After sterilization, the apparatus is allowed to cool down and the
pressure is slowly released before opening.

Filtration
1) Liquids: Heat sensitive materials like sugars, blood serum, enzymes,
hormones, vitamins, antibiotics, vaccines, etc. require sterilization by
filtration. Bacterial filters are made up of porcelain (diatomaceous earth)
sintered glass etc. e.g. Chamber land, Berkefeld, Seitz filter. The pores of
the filters are so small that the bacteria cannot pass through and are,
therefore, hold back. Actually, this is not the sterilization but
microorganisms are separated from the media. Filtration occurs only when
vacuum is crated in receiving flask. The filters are of various designs and
grades (pore size).

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2) Gaseous sterilization: Ethylene oxide vapours under pressure in special
equipment is becoming common method of cold sterilization. Ethylene
oxide is highly toxic to viruses, bacteria, fungi as well as to theheat resistant
bacterial endospore. It is easy to handle, inexpensive, non-corrosive and
non- deleterious to materials being sterilized. Ethylene gas is inflammable
but in minute with 90% carbon dioxide it becomes flammable and more
effective, sterilizing agent.

Irradiation: High-energy ionizing radiations with gamma rays from a cobalt

60 or caseium 139 source and cathode rays from electron generators and
accelerators are used in sterilization of certainpharmaceuticals.

Irradiation with ultraviolet light is used in operation theaters and isolation

chambers, e.g. laminar airflow cabinet and germicidal lamp.

Chemicals: Chemicals used for sterilization are called disinfectants.

I) Organic chemicals
1. Phenol (Carbolic acid): 2 to 4% is used for dis-infection of wounds. It is
a standard disinfectant against which other disinfectants are compared.
Alcohol 50 to 70% is used for plant material and skin.
2. Lysol (Cresol + linseed oil) 1 to 5% in water used for pots etc.
3. Formaldehyde 5% is used for preserving plant materials and soil
sterilization.

II) Inorganic chemicals

1. Mercuric chloride 0.1% is used for plant materials, working surfaces
etc.
2. Chloride 1 to 5% is used for skin, plants, drinking water etc.
3. Silver nitrate 1 to 5% is used for skin.

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4. Iodine 2 to 5% alcoholic solution (tincture) is used for skin.
5. Potassium permanganate is used for skin at 1% and in drinking
water.
6. Soaps and detergents are used for skin sterilization.

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 EXERCISE NO. 3

NUTRITIONAL MEDIA AND THEIR PREPARATIONS

Objective:
Since the bacteria are without chlorophyll they cannot manufacture their
own food material. They have, therefore, to depend upon outside sources for
their food requirement. Hence, it is necessary to grow them on artificial
medium of certain kind of food so that large number of one kind of bacteria i.e.
pure culture will be available at a time for study.

Culture medium: Nutrient or combination of nutrients used for the growth of

microorganisms is called as a culture medium.

Culture:
It is the growth of microorganisms on a medium. If a culture contains
the growth of only one type of microorganisms it is called as pure culture, on
the other hand when it contains growth of more than one type of
microorganisms it is called as ‘Mixed’ or ‘Contaminated culture’
The main elements required for bacterial growth are carbon and nitrogen
with certain amount of minerals and vitamins with water. Carbon is usually
supplied as carbohydrates, water supplies the hydrogen, and nitrogen can be
supplied in organic or inorganic form. (Plant or animal tissue extract supplying
organic nitrogen with carbon are usually more favorable. In the laboratory,
peptone is commonly employed in the form of organic nitrogen, it also
supplies carbon. Since different organisms require various nutrients in different
proportions, general media supplying all the essential nutrients are commonly
used e.g. Nutrient broth for bacteria and
P.D.A. (Potato Dextrose Agar) for fungi.
The medium is prepared by mixing or dissolving nutrients in water that
makes a liquid medium. If a solidifying agent (Agar or gelatin) is added, a
liquefiable solid medium can be prepared.

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 Requirement of a good culture medium
1) It should supply required nutrients in proper proportion.
2) It should have a proper pH
3) It should be free from all living organisms and unwanted
materials.
4) It should have proper moisture content.
5) It should have considerable physical properties e.g. solid, liquid.

Nutrient substances commonly used to prepare bacteriological

media
1. Gelatin: It is an incomplete protein compound prepared from animal
bones, horns, hoofs, etc. It is used by certain gelatinese producing bacteria
called gelatin-liquefying organisms. It melts at 37º C and solidifies a 20º
C. The medium-containing gelatin is transparent and water of
condensation is not formed on its surface. It is used @ 10 to 20 % in the
medium for solidification.
2. Agar-agar: It is a complex carbohydrates prepared from seaweed or
marine algae. It is used as solidifying agent in culture media @ 1.5 to

3. 2% concentration. It melts at 98º C and solidifies at 37º C. The medium is

opaque when solid and covered by water of condensation. It does not
supply any nutrient to the organisms.
4. Agar agar is preferred as a solidifying agent because less quantity is
required. It remains solid at room temperature and is not acted upon or
used by micro-organisms. Gelatin is used to study the liquefying ability of
bacteria (Liquiefication).

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 Classification of media
1) According to composition:
a) Synthetic media: Exact chemical composition of ingredients is known
e.g. Richard’s medium, Ashby’s medium, Coon’s medium.
b) Non-synthetic media: Exact chemical composition is not known e.g.
P.D.A, N.A., Malt extract etc.
2) According to use:
a) General or common use e.g. P.D.A, N.B., and N. A.
b) Selective or special medium: Used for specific organisms. Ashby’s
medium for Azotobacter and yeast Manitol Agar medium for
Rhizobium.
3) Differential media:
These are used to detect the production of certain characteristic growth of
the various species, e.g. eosine methylene blue agar for testing coliform
bacteria.
Solid media are used for studying cultural characters, for maintenance and
obtaining pure cultures by streaking, plating, etc. while liquid media are
commonly used for large-scale multiplication of the organisms.

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 Adjustment of pH of culture medium
pH is the measurement of acidity or alkalinity of a solution or it is the
hydrogen-ion concentration of medium. Different organisms grow well in the
culture medium having specific pH, Bacteria require slightly alkaline medium
(pH 7 to 7.5), while fungi require slightly acidic media (pH 6 to 7).
pH range 0 to 7 - Acidic (Distilled water)
pH range 8 to 14 - Alkali.

Methods of pH Adjustment
1. Colorimetric method
2. Electrolytic method

Colorimetric method: This method is based on the use of certain indicators or

regents that exhibits different colours at different pH values, when added to a
medium.

Colour
Sr. Colour Concentration
Indicator pH range in
No. in acid (%)
Alkali
Bromo cresol
1 5.2 to 6.8 Yellow Purple 0.04
Purple (B.C.P.)
Bromothymol
2. 6.0 to 7.6 Yellow Blue 0.04
Blue (B.T.B.)
Phenol Red
3. 6.8 to 8.4 Yellow Red 0.02
(P.R.)
Methyl Red
4. 4.4 to 6.0 Red Yellow -
(M.R.)

B.T.B. is commonly used for bacteriological media. It exhibitsyellow,

grass –green and blue colours at pH 6,7 and 7.6 respectively.

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 Procedure
1) Add 0.5 ml of B.T.B. to 5 ml of medium. It will show the colour
according to original pH.
2) Place the tube alongwith another tube, containing distilled water in the
comparator block.
3) Compare the colour with the help of indicator disc and note theoriginal
pH.
4) Adjust the pH is to alkaline or acidic range by adding 0.5 N NaOH or
0.5 N HCl solution as required. Calculate the quantity of 1 N. NaOH or
HCL solutions required to be added to the total volume of medium.
5) Recheck the pH of the medium after adjustment.

Electrolytic method: This method is based on the principle that solutions of

different reactions (pH values) exhibit different degrees of resistance to an
electric current. The resistance is directly measured on the scale reading from 1
to 14. The apparatus is known as ‘Beckman’s pH meter. It gives accurate and
quick pH observations and adjustment.
Buffers: Buffers are the substances, which prevent rapid change in pH of a
medium. Due to the activities and respiration of micro-organisms, the pH of a
medium is changed and hence it is prevented by buffer e.g. Peptone, Carbonate
protein, Phosphates, etc.

Requirement of a good plug

1. It should be tight enough to bear the weight of the container.
2. It should be 1” to 1 ½” inside the mouth
3. It should be easily removable or replaceable.

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 EXERCISE NO 4
Preparation of common media

Preparation of Nutrient Agar

1) Nutrient Agar Composition
1. Beef extract - 3g
2. Peptone - 5g
3. Agar-agar - 20 g
4. Water - 1000 ml

MediumProcedure:
1. Weigh the constituents (chemicals) according to the
requiredquantity of medium.
2. Pour the required quantity of water and ingredients in conical flask.
3. Use the conical flask having double the capacity of
requiredmedium.
4. Dissolve the constituents with agitation.
5. Adjust the pH of the medium with acid or alkali.
6. Cotton plug is applied to the conical flask.
7. Autoclaved it to 121ºC, 15 lbs pressure for 15 minutes.
8. After sterilization the medium is cooled down upto 45ºC and then
poured in Petri plates.

2) P.D.A. (Potato (peeled) Dextrose Agar): Composition

1. Potato (peeled) - 200 g
2. Dextrose - 20 g
3. Agar agar - 20 g
4. Water - 1000 ml

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 Method
The boiled and filtered extract of potatoes is mixed with dextrose and
Agar-agar, dissolved by indirect heating (In boiling water).
The medium is distributed in tubes or flasks, plugged and sterilized in
autoclave at 15 lbs. pressure for 15 minutes.
Tubes are slanted duringsolidification to form slants.
This is general medium for fungi.
9.

Class work:
1) Observe the material used in the preparation of culture media.
2) Prepare NA, PDA and Ashby’s media

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 EXERCISE NO. 5

METHODS OF ISOLATION AND PURIFICATION OF MICROBIAL

CULTURES

Object: To isolate the bacteria from liquid sample.

Materials: Nutrient agar, Petriplates, inoculating needle, sprit lamp and water
sample.
Isolation: This is the process of separating the microorganisms from their
natural substrates and growing them on culture medium in pure form.

Procedure:
1) Prepare the nutrient agar medium.
2) Pour the Petriplates with N. A. medium at 45o C temperature.
3) Cool down the Petriplates so that media could be solidified.
4) Raise the lid of medium plate only far enough to permit the inoculating
needle to enter.
5) Dip the tip of inoculating needle in liquid sample (water) so that
bacterial cells stickup the tip of needle.
6) Insert the inoculating needle in nutrient agar plate and streak out the
needle on medium by making zigzag or straight and cross lines gently.
The bacterial cells present on the needle could enter in the medium and
grow.
7) Incubate the Petriplates for 3-4 days at 30oC.

PURIFICATION OF MICROBIAL CULTURES

Objective: To grow the bacterial culture in pure form.

Principles: The microorganisms are required to be grown in pure form,

developed from single cell on culture media for their detail study of
morphology, physiology and cultural characters.

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 Culture: It is the growth of microorganisms on culture medium.
Colony: Growth of microorganisms usually round in appearance on a solid
medium resulting from a single cell or group of similar cells.
Contamination: Growth of desired microorganism along with other undesired
microorganisms on culture medium

Procedure: During the course of handling the bacterial cultures may get
contaminated with undesirable micro-organisms. Such cultures can be purified
by following three methods. (A) Dilution and plating, (B) Streaking method
and (C) Spread plate method.
(A) Dilution and Plating
1. Prepare 5 to 7 water blank by pouring 9 ml sterile water in steriletest tubes.
2. Prepare nutrient agar medium
3. Lift loopful quantity of contaminated bacterial culture with inoculating needle
under aseptic conditions using Laminar AirFlow Cabinet.
4. Dispense the culture in first water blank and this will be 10-1dilution.
5. Take one ml. suspension from 1st water blank and pour in 2nd water blank and
this will be 10-2 dilution. Like wise prepare 10-5 to 10-7 serial dilutions.
6. Take 1 ml. from 10-5 to 10-7 dilution each and pour in Petriplates.
7. Pour 15 ml nutrient agar medium in the plates containing bacterial
suspensions.
8. Incubate the plates at 30oC for 3-4 days.

Observation: Observe the colonies of desired bacteria in the plates and

transfer on agar slants.

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 (B) Streak Plate Method:
1. Prepare nutrient agar plates.
2. Lift loopful bacterial contaminated culture and dispense in 1ml sterile
water in test tubes under aseptic conditions using Laminar Air Flow
Cabinet.
3. Dip the inoculating needle in bacterial suspension and streak on agar
medium by making zigzag or straight and cross lines in Petriplates.
4. Incubate the plates at 30oC for 3 to 4 days

Observation: Select well isolated colony and transfer on nutrient agar slants.

A) Spread Plate Method:

1) Prepare nutrient agar Petriplates.
2) Prepare suspension of contaminated bacterial culture in sterilewater.
3) Deep ‘L’ shaped bent glass rod in suspension and spread on agar
plates.
4) Incubate the plates at 30oC for 3 - 4 day.

Observation: Observe well isolated bacterial colonies and transfer on nutrient

agar slants.

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 EXCERISE NO.6

MEDIA USED FOR BIOFERTILIZERS ANDBIOPESTICIDES

PRODUCTION

Different organism required specific media for their growth is called as

selective media. Following are selective media for different organisms.
1. Rhizobium: : 1. Yeast Extract Mannitol Agar (YEMA)
2. Congo Red Yeast Extract Mannitol Agar
(CRYEMA)
2. Azotobacter: : Ashby’s medium /Jensen’s medium

3. Azospirrilum: : N-free semisolid malic acid medium

4. PSB: : Pikovskaya’s medium

5. Acetobacter: : LGIP medium

6. Beijerinckia : Beckings medium

7. Blue Green Algae : Fogg’s medium

Compositions of Different Medium:

1. YEAST EXTRACT MANNITOL AGAR (YEMA)

Mannitol - 10.0 g

K2HPO4 - 0.5 g
MgSO4 7H2O - 0.2 g
NaCl - 0.1 g

Yeast extract - 0.5 g

Agar - 20.0 g

Distilled water - 1000.0 ml

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 2. CONGO RED YEAST EXTRACT MANNITOL (CRYEMA)

YEMA + 10 ml of sterile 1:400 aqueous solution of Congo Red /lit or 2.5ml of

1% solution of congo red.

JENSAN’S MEDIUM

Sucrose - 20.0 g

K2HPO4 - 1.0 g

MgSO4 - 0.5 g

NaCl - 0.5 g

FeSO4 - 0.1 g
Na2MoO4 -
0.005g

CaCO3 - 2.0 g

Distilled water - 1000 ml

ASHBY’S MEDIUM
Mannitol - 20.0 g

K2HPO4 - 0.2 g

MgSO4.7H2O - 0.5 g

NaCl - 0.2 g

K2SO4 - 0.1 g

CaCO3 - 5.0 g

Agar - 15.0 g

Distilled water - 1000 ml

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PIKOVSKAYA’S BROTH

Glucose - 10.0 g

Ca3(PO4)2 - 5.0 g

(NH4)2SO4 - 0.5 g

KCl - 0.2 g

MgSO4.7H2O - 0.1 g

MnSO4 - Trace

FeSO4 - Trace

Yeast Extract - 0.5 g

Distilled Water 1000 ml

N-FREE SEMISOLID MALIC ACID MEDIUM

Malic acid - 5.0g

FeSO4.7H2O - 0.05g

K2HPO4 - 4.0g

Na2MoO4.2H2O - 0.002g

MnSO4.7H2O - 0.01g

MgSO.7H2O - 0.1g

NaCl - 0.02g

CaCl2.2H2O - 0.01g

BTB (0.5% - 5.0 ml

alcoholic solution)

Biotin - 0.001g

Yeast extract Agar 0.5g

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 1.0g

Distilled water - 1000 ml

PH - 6.6-7.0

LGIP MEDIUM

K2HPO4 - 0.6 gm

KH2PO4 - 0.2 gm

MgSO47H2O - 0.2 gm

CaCl2 - 0.02 gm

Na2MoO4 - 0.002 gm

FeCl3 - 0.01 gm

Bromo thymol - 0.5%

blue

Cane Sugar - 1000 ml

Agar - 1.8 gm

Distilled Water - 1000 ml

BECKING’S MEDIUM

Sucrose - 20 gm

K2HPO4 - 0.2 gm

KH2PO4 - 0.2 gm

FeCl3 - 0.005 gm

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MgSO4 7H2O - 0.5 gm

Na2MoO4 - 0.0050 gm

Agar -
20 gm
Distilled -
water 1000 ml

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 FOGG’S MEDIUM

KH2PO4 - 0.2 g

MgSO4 - 0.2 g

CaCl2 - 0.1 g

Na2MoO4 - 0.1 mg

MgCl2 - 0.1 mg

H3BO3 - 0.1 mg

CuSO4 - 0.1 mg

ZnSO4 - 0.1 mg

Fe-EDTA - 0.1 ml

Distilled water - 1000 ml

pH - 7.5-8

Sulphur enriched medium

(NH4)2SO4 - 0.2 g

KH2PO4 - 3.0 g

MgSO4.7H20 - 0.5 g

FeSO4.7H2O - trace

CaCl2 - 0.2 g

Elemental sulphur - 10.0 g

Distilled water - 1000 ml

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 EXERCISE NO. 7
ISOLATION OF RHIZOBIUM FROM ROOT NODULE OF
LEGUMINOUS CROP

Object: To isolate Rhizobium from root nodule of groundnut / pigeon-pea/red-

gram.

Rhizobium is a bacterium which fixes atmospheric N2

symbiotically in legume root nodules. It was first discovered in 1888 by
Beijerinck Symbiosis is a phenomenon of living together with mutual benefits.
The legume crop is benefited by the supply of NH3 fixed by becteroids (forms of
Rhizobium) in nodules, while Rhizobium is benefited by shelter in root nodules
and receipt of carbohydrates from legume plant. The capacity of rhizobial
isolate is to invade roots of a restricted number of plant species in addition to
the legume from which it is isolated. This indicates host specificity of
Rhizobium and taken as a basis for different cross-inoculation groups of
Rhizobium. For isolation generally, bigger nodules with pink colour are
effective. Fromsuch nodules it is possible to isolate pure cultures of Rhizobium.

Material required

Any leguminous crop with vigorous nodules (Groundnut/pigeon-

pea/red gram), 1:1000 HgCl2 solution, 75% alcohol, sterile petri dishes, test
tubes, glass rod, scalpel, forceps, razor-blade, sterile water, nutrient media viz.
Yeast extact mannitol agar containing congo red.

Procedure

1. Uproot carefully vigoroulsy growing legume crop preferably at

flowering andbring the root samples with nodules to the laboratory.

2. Wash the root system gently under running tap water, remove adhering

soil withcamel’s hair brush.

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3. Detach carefully the nodules from the root system with the help of

sterile sharprazor blade leaving a small portion of root attached to

nodules.

4. Place the nodules in a petri dish containing 1:1000 HgCl2 solution and

agitate thenodules using sterile forceps for 3 to 4 minutes

5. Take care that sufficient HgCl2 solution is taken in petri dish so as to

achieve complete dipping of nodules.

6. Transfer the nodules with sterile forceps to a sterile petri dish containing

75% alcohol and agitate for 2 minutes.

7. Transfer the nodules to a sterile petri dish containing sterile water. Rinse

the nodules in sterile water. Two to three more transfers of nodules in

succession are done to sterile petri dishes containing sterile water and by
rinsing the traces of surface sterilizers are removed.

8. Collect one or two surface sterilized nodules in a sterile test tube

containing 1 ml of sterile water. Crush the nodule with sterile blunt ended
glass rod or sterile forcep. Mix the nodule exudate and water. From this
transfer one or two loopful of exudate to other sterile test tube. Add 1 ml
of sterile water and dilute the nodule exudate.

9. Add 1 ml of diluted nodule exudate to a sterile petri dish.

10. Pour petri dish with solidifiable Congo red yeast extract mannitol agar

(45°C).Mix the nodule exudate and medium by rotating the plates gently.

11. Allow the medium to solidify and incubate the plates at 28°C or at room

temperature for 4 to 5 days.

12. Transfer growth from rhizobial colony to the slants of yeast mannitol agar.

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Observations

Observe for the development of rhizobial colonies after 4 to 5 days of incubation.

Note down the colony characters of Rhizobium and common
contaminant Agrobacterium.

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 EXERCISE NO. 8

ISOLATION OF AZOTOBACTER FROM SOIL

Object:

To isolate Azotobacter from rhizosphere soil of cereal crop either by

dilution plating or by enrichment culture technique and to know the selective
enrichment of Azotobacter species.

Preamble:

Amongst various nitrogen-fixing bacteria Azotobacter is one of the

important non-symbiotic free-living nitrogen fixing bacterium. It is an aerobic,
Gram-ve, short rod and heterotrophic bacterium. Its isolation from the
rhizosphere soil of any of the cereal crops can be made by two methods viz, (1)
Soil dilution and pour plate, (2) Enrichment culture technique. For isolation of
Azotobacter either Jensen’s or
Ashby’s medium can be used. These media are devoid of nitrogen source.

Method –I:

Isolation of Azotobacter by soil dilution and pour plate technique.

Principle:

Azotobacter population is abundant in the rhizosphere of cereal crops. On

dilution of rhizosphere soil, Azotobacter cells get separated and subsequently on
plating with nitrogen free nutrient medium individual cells develop into colony.
Such colonies developed from individual cells yield pure culture of Azotobacter
which can be maintained on Jensen’s agar slants.

Material required:
Rhizosphere soil of jowar / bajra / wheat / maize, petri plates, Water
blanks,pipettes, Jensen’s agar or Ashby’s agar, slants of these media, incubator,
etc.
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PROCEDURE:

1. Suspend 10 g of rhizosphere soil sample in 90 ml. sterile water and mix

thoroughly.
2. Prepare 10 fold dilutions of the above suspension from 10-1 to 10-6, using
separate water blanks.
3. Transfer 1ml aliquot of the appropriate dilution to sterile petri dishes
separately.
4. Pour petri dishes with solidifiable Jensen’s agar or Ashby’s agar having
temperature of 45°C.
5. Mix the contents of the plates carefully to avoid contact of medium to the lid.
6. Allow the medium to solidify and incubate the plates at 28°C ( 2°C) in
anincubator.
7. Observe for the development of soft, flat, milky and mucoid colonies
of Azotobacter after 3 days of incubation.
8. Transfer typical Azotobacter colonies on the slants of Ashby’s or Jensen’s agar.

OBSERVATIONS :

1. Observe the growth of Azotobacter by Gram staining under microscope.

2. Record dilution wise colony count and conclude the abundance of
Azotobacter
population in a given soil sample.
3. Note down whether there is pigmentation, produced by older Azotobacter
culturein a slant or plate.

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Method –II:

Isolation of Azotobacter by enrichment culture technique.

Principle :

In enrichment culture technique the nutrient medium used is enriched with

defined chemical components which are necessary for a particular organism so
as to cause its predominance by its ability to grow more rapidly than others.
Azotobacter being N2 fixer, it can use atmospheric N2 and so far its isolation the
nutrient medium that is free of combined nitrogen but contains other minerals
and carbon source is used. Jensen’s medium is most suitable for the isolation of
Azotobacter by enrichment culture technique.

Material required:

Rhizosphere soil of jowar / bajra / wheat / maize, 250 ml conical

flasks,pipettes, Jensen’s agar or Ashby’s agar, slants of these media, incubator,
etc.

PROCEDURE:
1. Collect rhizosphere soil of any cereal crop growing profusely.
2. Add 0.5 g of rhizosphere soil separately to 3 to 4 conical flasks containing 100
ml of sterile Jensen’s broth and shake well to mix the soil with broth.
3. Incubate the flasks at 28°C ( 2°C) for a week.
4. After incubation, transfer 1 to 2 ml of growth suspension from each of these
flasks separately to another flasks containing Jensen’s liquid medium. Incubate
newly transferred flasks at 28°C ( 2°C) for a week.

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5. Similarly make 2 to 3 more transfers and observe for the growth of
Azotobacter

obtained from the finally transferred flasks.

6. Transfer loopful of growth on the slants of Jensen’s agar.

7. Examine the growth of Azotobacter under microscope for cell shape,

motility,Gram reaction, etc.
OBSERVATIONS:

1. Record the observations of common contaminants coming up along

with
Azotobacter in first few transfers.
2. Note down whether more dense and uniform layer/pellicle of Azotobacter
isformed or otherwise in the finally transferred flasks.
3. Examine under microscope and record the observations on Gram reaction,
size,shape and motility of Azotobacter isolate.

STUDY QUESTIONS:

1. What is enrichment culture technique?

2. Name the species of Azotobacter?

3. What is the reaction mediated by Azotobacter in fixation of atmospheric N ?

4. What is the reason that the colony of Azotobacter chroococcum turns black
onincubation for a linger period ?
5. In dilution pour plate technique explain how will you count the number
of Azotobacter cells in the given rhizosphere sample?
6. Draw a schematic diagram showing technique of dilution pour plate for
estimating and isolating Azotobacter form rhizosphere of a cereal crop.

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Circulated By @Infinite_Agril(Atharv Bhalerao)
 EXERCISE NO. 9

ISOLATION OF AZOSPIRILLUM FROM SOIL AND ROOTS OF

HARIYALI (Cynadon dactylon)

Object:

To isolate Azspirillum from soil and the root tissues of haryali, a C4

type ofgrass weed which harbours Azospirillum in abundance.

Preamble:

A Dutch Microbiologist, Beijerinck in 1925, observed the occurrence

of Spirillum lipoferum (renamed as Azospirillum lipoferum) from soil.
However,diazotrophic nature of this bacterium was realised and proved only
in the year 1976 by Johan Dobereiner, a Brazilian lady scientist pionioring
in associative dinitrogen fixation. This bacterium lives in close association
with the roots of many cereal crops viz., maize, sorghum, pearlmillet, finger
millet, sugarcane, rice, foxtail millet and various types of grasses including
Cyanodon dactylon

Principle:

Azospirillum being microaerophilic in nature and inhabiting in the

upper cortex region of roots, can be easily isolated by inoculation surface
sterilized root bits into semi-solid NFb-medium. This medium supplies
malate as a carbon source preferred by Azosprirllum. Thin white pellicle of
Azospirillum developes below the surface of medium which can be used for
transferring the growth to the slants of the same medium and pure culture of
Azospirillum can be obtained

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Material required:

Freshly collected rhizosphere soil and roots of haryali, Petri dishes,

sterile water, Nitrogen free-malate medium (NFb-medium), small vials or
injection waste bottles, 1.1000 HgCl2 solution, Incubator, scalpel, blade, etc.

PROCEDURE:

A. Isolation From Roots:

1. Wash the roots in tap water to remove adhering soil particles

2. Cut the roots into small bits of 0.5 to 1.0 cm length with sterile razor blade
orscalpel.
3. Rinse root bits in 0.1% mercuric chloride solution for 1 minute in sterile
Petridish.
4. Wash root bits in sterile water in three to four petri dishes.
5. Prepare malic acid semi-solid medium and dispense 5 ml quantities in the
smalltest tubes or glass vials.
6. Transfer one or two root bits aseptically to each tube/vial containing NFb
medium.
7. Incubate the tubes at 30°C ( 2°C) for a period of 3-5 days. Keep one or
twovials containing NFb medium without root bits which will serve as control.
8. Observe for the development of pellicle in the tubes below the surface of
medium.
9. Streak a loopful of the culture from the above tube over the solidified
nitrogenfree malate agar medium (1.5% agar) in sterile petri dishes.

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10. Incubate the plates at 30°C ( 2°C) for 3.5 days. Observe for the colonies
of Azospirillum and change of the colour of medium from light yellow to blue.
11. Confirm the growth of Azospirillum by observing under microscope for spiral
rodsand spiral movement using hanging drop method.

B. Isolation From Soil

Follow the same procedure as that used for isolating Azospirillum

from roots, except soil dilution aliquot is used in place of roots for
inoculating in tubes containingNFb semisolid medium

Observations:

1. Record the site of development of Azospirillum pellicle in NFb-vials.

2. Record the change of colour of medium in vials and further in petir
plates onsubsequent transfers.
STUDY QUESTIONS:

1. What is associative symbiosis?

2. Name the species of Azospirillum?
3. What is the reason that NFB medium is most suitable for
isolation of Azospirillum?
4. What are the micoaerophilic microorganisms ?
5. Why Cynadon dactylan, a most nuisance weed, is preferred a host for
isolation of Azospuruillum?
6. Draw a schematic diagram showing technique of isolation of
Azospirillum formrhizosphere of weed Cynadon dactylon .
7. Write the composition of Sodium malate or NFb medium.

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 EXERCISE NO. 10

ISOLATION OF ACETOBACTER FROM SUGARCANE

OBJECT : To isolation Acetobacter from sugarcane crops

PREAMBLE : Louis Pasteur(1864) showed that it is acetobacter bacteria that
cause the conversion of alcohol to acetic acid. Acetobacter is also called as
acetic acid bacteria. It has the ability to convert ethanol to acetic acid in the
presence of oxygen. It fixes nitrogen non-symbiotically. It is mostly used in
sugarcane crop. It also secretes the useful growth promoting hormons such as
indole acetic cid and gibberelin. It is psycharophilic bacteria.
Procedure of Isolation:

1. Take 1 gm of sugarcane sample (root/leaf/stem/bud) are to be

washedthoroughly in the running tap water

2. Surface sterilized with 70% ethanol and subsequently washed in

changes ofsterilized distilled water

3. Surface sterilized samples are to be macerated in sterile blender

and serialdilutions are prepared up to 103 dilutions

4. 1 ml of 103 dilutions is to be inoculated into various enrichment

media viz. diluted cane juice semisolid medium, LGIP semisolid

medium and acetic LGIPsemisolid media.
5. Enrichment culture is to be sub cultured for every 2-3 days.

6. The isolated cultures grown on acetic LGIP broth are used for

further characterization.

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 EXERCISE NO. 11

ISOLATION OF BEIJERINCKIA FROM SUGARCANE

OBJECT : To isolation Beijerinckia from soil

PREAMBLE: Beijerinckia named after M.W. Beijerinck the Dutch

microbiologist (1851- 1931). Döbereiner and Ruschel (1958) discovered
Beijerinckia fluminensis species. Starkey and De (1939) discovered Beijerinckia
indica. Beijerinckia is a nonsymbiotic nitrogen fixing organism present in
rhizosphere of crops and acidic soils. These are aerobic for bacteria i.e micro
aerophilic bacteria. They have ability to form cysts at adverse conditions. This
species is rust brown.

Isolation of Beijerinckia:

Bejerinckia is non symbiotic nitrogen fixing organism present in rhizosphere

of cropsand acidic soils.
1. It is isolated by dilution plating method using beckings media.

2. As it is slow growing organism, first inoculate 100 ml beckings media

broth with1 gm of acidic soils.

3. After 15 days of incubation there will be a formation of scum.

4. Streak a loopful of the culture on beckings agar plate.

5. After 10 days of incubation pure colonies of beijerinckia will be

developed.

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 EXERCISE NO. 12

ISOLATION OF BLUE GREEN ALGAE

FROM SOIL

Object : To isolate blue green algae from soil by using N-free medium and
illuminated conditions of incubation.

Blue green algae are photoautotrophic free living nitrogen fixing organisms.
Blue green algae are generally covered with mucilage and it is easy to locate
them as colonies floating on flooded rice fields. Blue green algae being
photoautotrophic use radient energy and CO2 as carbon source to perform
photosynthetic activities.

Composition of nutrient media for isolation of Blue green

algae

Composition of Fogg’s Medium

KH2PO4 - 0.2 g

MgSO4 .7H2O - 0.2 g

CaCl2 . 2H2O - 0.1 g

FeECTA - 1.0 g

Distilled water - 1000 ml

Composition of Bristol’s Sodium Nitrate Medium

Potassium phosphate - 0.5 g

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Sodium nitrate -
0.5 g
Magnesium sulphate -
0.15 gCalcium
chloride - 0.01 g
Ferric chloride - 0.01
g Tap water -
1000 ml
Material required
Fogg’s or Bristol’s sodium nitrate liquid medium conical flasks
(250 mlcapacity), water blanks, pipettes, illuminated growth cabinet, soil
sample.

Procedure

1. Prepare serial dilutions of soil or algal sample in sterile water blanks with

sterilized pipettes.

2. Prepare several conical flasks of nitrogen free blue green algae liquid

medium (fogg’s medium).

3. Inoculate aliquots of appropriate dilutions in to liquid media in flasks

and incubate for several weeks in an illuminated growth cabinets at 28°C

±-2

4. As and when the individual colonies arise, they are transferred from the

enrichment flasks to fresh aliquots of liquid media or on agar slant.

Purification of culture is done by the dilution method. Eradication of
cyanobacterial cultures with ultraviolet rays is successful in making pure
culture from contaminants.

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 EXERCISE NO. 13
ISOLATION OF PHOSPHATE SOLUBILIZING MICROORGANISMS
FROM SOIL

Object : To isolate phosphate solubilizing microorganisms from soil.

Fixation of added phosphorus through chemical fertilizers in soil poses the

problem of availability of this essential element to crops and crops suffer from P
deficiencies. Some of the microorganisms present in soil solubilize the fixed form
of phosphate and make it available to crops. Phosphate soilubilizing
microorganisms can be isolated from soil using Pikovaskaya’s medium containing
tricalcium phosphate. On plating aliquot of soil dilution, the P solubilizing
microorganisms show clear zones of P solubilizing around the colony. Such
colonies are further subcultured and pure cultures of P solubilizing
microorganisms can be maintained on slants of Pikovaskaya’s medium.

Materials
Rhizosphere soil of legume crop, 9 ml sterile water blanks, sterile
petri plates, pipettes, inoculation needle, incubator, test tubes, Pikovaskava’s,
medium.

Composition of Pikovaskaya’s Medium

Glucose - 10.0 g
(NH4)2 SO4
- 0.5 g
MgSO4 .
7H2O - 0.1 g

Yeast Extract - 0.5 g

Ca3 (PO4)2 - 5.0 g

KCl - 0.2 g

MnSO4 - Trace

FeSO4 - Trace
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Agar - 15 g

Distilled water - 1000 ml

Procedure

1. Prepare 10-fold dilutions serially up to 10-6

2. Transfer 1 ml each of 10-1, 10-2, 10-3, 10-4 and 10-5 dilution aliquots in

quadruplicateplates separately.

3. Pour about 15 ml molten and cooled Pikovaskaya’s medium into the

above plates and mix the soil suspension by gently rotating the plates and
allow the medium to solidify.

4. Incubate the plates at 30°C for 3 to 4 days.

Observations

Observe for the growth of microbial colonies showing clear zone around them.

Count such colonies and calculate the number of P solubilizing microbes in

original soilsample by following formula.

No. of P solubilizer/g soil = Av. No. of colonies x Dilution Factor with clear
zones.

Maintain the P solubilizing isolates on Pikovaskaya’s medium and their P

solubilizingcapacity may be estimated by the colourimetric method.

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 EXERCISE NO. 14
ISOLATION OF POTASH SOLUBILIZING MICROBES

Objective : To isolate potash solubilizing bacteria from soil.

Materials and Methods : Soil sample, test tubes etc.

Procedure :
a) Adaptation and Enrichment :

Mix the soil samples with insoluble potassium (Feldspar). Incubate it for
1 week at room temperature. After adaptation inoculate 1 gm of soil in 100 ml
liquid medium containing 1 % glucose, 0.05 % yeast extract and 0.5% feldspar
and incubate at 37 o Con 120 rpm for 1 week.

b) Isolation and screening of Potassium solubilizing Microbes :

1. Take this 1 gm soil and dilute it 10-4, 10-5 and 10-6 by serial dilutions.

2. Inoculate this on Aleksandrov Agar medium at 37O C for one week.

3. Pick the colonies exhibiting clear zone of potasium solublization from 10-4,

10-5 and10-6 dilutions

Ingredients of Aleksandrov medium

Glucose - 10 gm
MgSO4.7H2O - 0.005 gm
FeCl3 - 0.1 gm
CaCO3 - 0.1 gm
CaCO4 - 2gm
Potassium aluminium
silicate - 5 gm
Yeast extract -5gm
Agar - 15 gm

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 EXERCISE NO. 15

ISOLATION OF SULPHUR OXIDIZING MICROORGANISMS FROM

SOILBACTERIA
1. Collect the sulphur enriched rhizosphere soil from a depth of 22.5 cm or

soil from suphur hot-spring and air-dry under shed.

2. The stones, pebbles etc. should be separated and the soil should be

grinded in awooden pestle and screened though 2 mm sieve.

3. Isolation is carried out by an enrichment techniques using sulphur

enrichedmedium.

4. The pH of the medium is adjusted to 5 – 8.0

5. 5 ml of bromocresol purple (0.25%) indicator is added to per litre

medium andthe medium is then distributed in each of the 250 ml

Erlenmeyer flasks..

6. The flasks are sterilised in autoclave at 1 kg/cm3 pressure for 20 min

for threesuccessive days.

7. The flasks are then inoculated with 1 gm of soil and incubate on to-

and-froshaker (150 to-and-fro actions) at room temperature for 8 day.

8. The flasks showing the fall in the pH of the medium is evidenced by

the colorchange of the bromocresol purple indicator from purple to

colorless.

9. Such flasks are presumed to contain the growth of sulphur oxidising

bacteria

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FUNGI AND ACTIONOMYCETES

1. For isolation purpose, an enrichment technique is used

2. The medium consists of sodium thiosuphate as sulphur source

3. The pH is adjusted to 5.0 to 8.0 by using 0.1 N NaOH and as required

bromocresol purple (0.25%) indicator is added for detection of colonies

4. Presumptive sulphur oxidation is indicated by the formation of zone of

clearingaround the colony of fungi and actinomycetes.

Observations :

Record the colony morphology of suphur oxidising microorganisms

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 EXERCISE NO. 16

ISOLATION OF ORGANIC DECOMPOSER CULTURE

Cellulose is a major constituent of higher plant and plant residues

following in soil is therefore difficult to decompose. Nature has provided the
microrganisms responsible for decomposition of cellulolytic residues in the soil.
Aerobic, anaerobic and mesophilic bacteria viz., (Cytophaga sp., Paracytophaga
sp., Clostridium sp., Bacillus sp.,) mesophillic and thermophillic actinomycetes
viz., Streptomyces sp., Micromonosporus sp., Thermomonosporus sp.,
Thermoactinomyces sp., and numerous fungi viz., Trichoderma sp., Trichurus
sp., Aspergillus sp. Penicillium sp. and Fusarium sp. actively degrade the
cellulolytic plant residues and make the nutrients available in soil.

ISOLATION AND MAINTENANCE OF ORGANIC DECOMPOSER

CULTURES

Cellulose is the most abundant biomass on the earth. It is a crystalline

polymer of D-glucose residues connected by β-1, 4 glucosidic linkages, which
is the primary structural material present in cell of plants.
The microorganism producing cellulase enzymes are playing very
important role cellulose degradation. The cellulase enzyme is produced mostly
by fungi, bacteria and protozoans. Bacteria has high growth rate of reproduction
as compared to fungi and hence are high potential to degrade cellulose.
The species of bacterial genera of Cellulomonas, Pseudomonas,
Bacillus and Micrococus are very important in degradation of cellulose.
Material required :

Soil sample, sterile water test tubs (water blanks), petri plates,
Celluloseagar medium.

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Composition of Cellulose agar medium

KH2PO4 - 3.0 g

K2HPO4 - 2.0 g

Cellulose powder - 10.0

g

MgSO4 - 0.5 g

Asparagine - 2.0 g

Agar-agar - 15 g
Distilled water -
1000 ml

Add BTB (Bromo Thymol Blue) till you get grass green colour indicating neutral
pH.
Procedure

1. Add 1 g soil sample to 9 ml sterile water in test tube. It will give 10-1
dilution.

2. Transfer 1 ml suspension of 10-1 dilution to next 9 ml sterile water blank

and mixthoroughly by shaking for 1 minute. This will give 10-2 dilution of
original soil sample.

3. Repeat the above steps and prepare 10-3, 10-4, 10-6, 10-7, 10-8 dilutions of
the soilsample in test tubes containing sterile water.

4. 1 ml suspension from each dilution into sterile Petri plates separately.

5. Transfer Pour the plates with molten cellulose agar medium. Mix the soil
dilution aliquot and medium by gentle rotating and allow the medium in
plates to solidify.

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 6. Incubate the plates at 30°C for 8-10 days.

7. Count the number of colonies of microorganisms showing a change in

colour (from grass green to yellow) around the colonies, which is an
evidence for the production of acid from degradation of the cellulose into
simple carbon compound

8. Pick up colonies of cellulolytic microorgnisms and transfer them to the

slants of cellulose agar medium or potato dextrose agar medium. Maintain
the culturesby periodic transfers to the fresh slants of these nutrient media
within six months.

Observations

Count the number of cellulolytic microbial colonies in each plates and compute
the average number per gram of soil.

The number of cellulolytic microorganisms of oven dry soil are calculated by

multiplying the average plate count by the dilution factor and divided by the
oven dry weight ofone gram of soil i.e.

Average plate count x Dilution

No. of cellulolytic microbes/ =
oven dry soil1 g of soil sample

Observations:

Record the colony morphology of cellulolytic microorganisms

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 EXERCISE NO. 17

PRODUCTION OF COMMERCIAL BIOFERTILIZERS OF

RHIZOBIUM AZOTOBACTER, AZOSPIRILLUM, ACETOBACTER,
ORGANIC MATTER DECOMPOSERS

Materials

Nutrient liquid media required by specific N2 fixing organisms (Yeast extract

mannitol broth for Rhizobium, Jensen’s liquid medium for Azotobacter,
nitrogen free broth for Azospirillum, LGIP broth for Acetobacter, cellulolytic
media for organic matter decomposers, Efficient cultures of diazotrophs,
Suitable carrier material (e.g. peat, lignite etc), Autoclave. Shaker, Polythene
bags, Flasks, Inoculating needle, sealing machine

Procedure :

Production of carrier biofertilizers involves the following steps

1. Strain Selection ,maintenance of pure culture

2. Preparation of starter culture

3. Preparation of broth culture

4. Preparation of carriers

5. Preparation of inoculants in powder form

1. Strain Selection, maintenance of pure culture:

Isolation of bacterial culture performed under inoculation chamber using serial

dilutionmethod to get pure colonies of desired microbe

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 2. Preparation of starter culture

Pure culture of efficient strain of organism is grown on respective agar medium A

loopful of inoculum from it is transferred in a 300 ml of liquid medium
The flask is kept on shaker (260 rpm) for 72-96 hrs
If shaker is not available incubate it 28 °C for 5-6 days

3. Preparation of broth culture

Each flask containing suitable broth is inoculated with the starter culture 1:2
proportion, aseptically.

Incubate the flasks at 28 C for 2-5 days, depending upon the type of organismtill
the count per ml reaches to 109 cells.
Broth culture with population of 109cell/ml should be used for preparation of
carrier based biofertilizers.

4. Preparation of carrier

Finely powdered peat, lignite or soil + compost or cellulose powder or soil +

wood charcoals may be used as carrier
The carrier should have the following characteristics
High organic matter (above 60%)
Low soluble salt content (less than 1%)
High moisture holding capacity (150 -200% by weight)
Carriers provide a nutritive medium for growth of the bacteria and prolong their
survival in culture as well as on inoculated seed
The carriers are powdered to 230-300 mesh (about 75 micron pore size)
Carrier should be neutralized with 1% calcium carbonate
Sterilized at 15 pounds psi for 4 hrs in autoclave

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 5. Preparation of inoculants in powder form

Usually about one part (by weight) of broth is required for two parts of dry
carrier
Final moisture content varies from 30-50% depending on quality of carriers
After adding the broth culture to carrier powder in 1:2 proportion by weight, it is
kept for curing at room temperature 28 °C for 5 – 10 days in 10 cm deep trays
After curing, it is sieved to disperse the concentrated packets of growth and to
break lumps
Then packed in polythene bags of 0.5 mm thickness, leaving 2/3 space open for
aeration of bacteria

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 EXERCISE NO. 18

PRODUCTION OF CARRIER BIOFERTILIZERS OF SULPHUR

OXIDIZINGMICROORGANISMS, IRON CHEALATOR,
POTASH MOBILIZERS

Materials

Nutrient liquid media required for specific sulphur oxidizing microorganisms,

iron chealator, potash mobilizers, Efficient cultures, Suitable carrier material
(e.g. peat, lignite etc), Autoclave. Shaker, Polythene bags, flasks, inoculating
needle, sealingmachine

Procedure

Production of biofertilizers involves the following steps

6. Strain Selection ,maintenance of pure culture

7. Preparation of starter culture

8. Preparation of broth culture

9. Preparation of carriers

10.Preparation of inoculants in powder form

Strain Selection, maintenance of pure culture:

Isolation of bacterial culture performed under inoculation chamber using serial

dilutionmethod to get pure colonies of desired microbe

Preparation of starter culture

Pure culture of efficient strain of organism is grown on respective agar medium

A loopful of inoculum from it is transferred in a 300 ml of liquid medium

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The flask is kept on shaker (260 rpm) for 72-96 hrs
If shaker is not available incubate it 28 °C for 5-6 days

Preparation of broth culture

Each flask containing suitable broth is inoculated with the starter culture 1:20
proportion, aseptically.
Incubate the flasks at 28 C for 2-5 days, depending upon the type of organismtill
the count per ml reaches to 109 cells.

Broth culture with population of 109cell/ml should be used for preparation of

carrier based biofertilizers.

Preparation of carrier

Finely powdered peat, lignite or soil + compost or cellulose powder or soil +

wood charcoals may be used as carrier
The carrier should have the following characteristics
High organic matter (above 60%)
Low soluble salt content (less than 1%)
High moisture holding capacity (150 -200% by weight)
Carriers provide a nutritive medium for growth of the bacteria and prolong their
survival in culture as well as on inoculated seed
The carriers are powdered to 230-300 mesh (about 75 micron pore size)
Carrier should be neutralized with 1% calcium carbonate
Sterilized at 15 pounds psi for 4 hrs in autoclave

Preparation of inoculants in powder form

Usually about one part (by weight) of broth is required for two parts of dry
carrier
Final moisture content varies from 30-50% depending on quality of carriers

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After adding the broth culture to carrier powder in 1:2 proportion by weight, it is
kept for curing at room temperature 28 °C for 5 – 10 days in 10 cm deep trays
After curing, it is sieved to disperse the concentrated packets of growth and to
break lumps
Then packed in polythene bags of 0.5 mm thickness, leaving 2/3 space open for
aeration of bacteria.

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 EXERCISE NO. 19

STUDY OF VA-MYCORRHIZA: GROWTH ON GUINEA GRASS

ROOTS AND OBSERVATIONS FOR ROOT COLONIZATION.
METHODS OF PREPARATIONAND APPLICATION OF VA-
MYCORRHIZAL INOCULUMS

A) Study of VA-mycorrhiza: growth on Guinea grass roots and

observations for root colonization.
Objective :
To isolate VA mycorrhizal spores and to observe VA mycorhizal
colonization in plant roots.
Material required :
Soil samples, Root samples, Distilled water, Sieves (425, 250, 110, 90, 45
and 40) micron, Nylon mesh, Stereo microscope, screw cap bottles, 10% KOH
solution, 2% HCl Tryptophan blue solution, lactic acid, destaining solution.

Procedure :
A) Isolation of VA mycorrhizal spores:

1. Take 50 g of soil and suspend in 500 ml of water.

2. Mix thoroughly and allow the soil particles to settle down for one
minute.
3. Decant the soil solution through a set of sieves (425, 250, 110, 90, 45
and 40microns).
4. Repeat the same four to five times.
5. Wash the residue in each sieve with more water and collect them
separately.
6. Take little quantity of the solution collected from each sieve on a nylon
mesh andobserve the spores under the stereoscope (majority of the spores
could be seen in 90 and 40 micron sieves).

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 Express the number of spores per gram of soil sample
B) Observation of VA mycorrhizal colonization in roots:
1. Take one gram or roots and cut into small pieces (approx. 1 cm.)
2. Keep the root in 15ml screw cap bottles.
3. Add 10 ml of 10% KOH solution and heat at 900C for one hr.
4. Cool the vials and decant the KOH solution.
5. Wash root twice carefully with distilled water and once with 2% HCl.
6. Add 5 ml of staining solution to the vials.
7. Decant the stain after one hr. and remove the excess stain using
destainingsolution.

8. Take the root make small sections and observe under microscope.
9. Record presence of arbuscles, vesicles, mycelium within and out side
the rootsection

B) Methods of preparation and application of VA-mycorrhizal

inoculumsMethods of preparation
Vermiculite contained raised AM infected Maize plants

1. A trench (1m x 1m x 0.3m) is formed and lined with black polythene

sheet to be used as plant growth tub.

2. Mixed 50 kg of vermiculite and 5 kg sterilized soil and packed in the

trench up to a height of 20 cm

3. Spread 1 kg of AM inoculums (mother culture) 2-5 cm below the surface

of vermiculite

4. Maize seeds surface sterilized with 5 % sodium hypochlorite for 2

minutes are sown

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 5. Applied 2g urea, 2 g super phosphate and 1g muriate of potash for each

trench at the time of sowing seeds. Further 10 g of urea is applied twice

on 30 and 45 days after sowing for each trench

6. Quality test on AM colonization in root samples is carried out on 30th

and 45thDay

7. Stock plants are grown for 60 days (8 weeks). The inoculum is obtained

by cutting all the roots of stock plants. The inoculum produced consists of
a mixtureof vermiculite spores, pieces of hyphae and infected root pieces.

8. Thus within 60 days 55 kg of AM inoculums could be produced from 1

sq mter area. Thus inoculums will be sufficient to treat 550 sqmtr nursery
area having 11,000 seedlings

Application of VA-mycorrhizal inoculums Nursery application:

100 g bulk inoculums is sufficient for one meter square. The inoculums should
be applied at 2-3 cm below the soil at the time of sowing. The seeds/cutting
should be sown/planted above the VAM inoculum to cause infection.

For polythene bag raised crops :

5 to 10 g bulk inoculum is sufficient for each packet. Mix 10 kg of inoculums

with 1000kg of sand potting mixture in polythene bag before sowing

For out-planting

Twenty grams of VAM inoculums is required per seedling. Apply inoculums

at the timeof planting

For existing trees:

Two hundred gram of VAM inoculums is required for inoculating one tree.
Applyinoculums near the root surface at the time fertilizer application.

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 EXERCISE No. 20

MASS MULTIPLICATION OF BGA AND AZOLLA AND ITS

APPLICATION IN PADDYFIELD

Object

1. To know the method for large scale production of blue green algae as a
biofertilizer foruse in paddy fields.
2. To know the method of mass-multiplication of Azolla.

1) Mass multiplication of Blue Green Algae

Open air shallow culture tanks made up of G I sheets or bricks and cement can
be used. The tanks should preferably of (2m x 1 m x 23m) size. About 5 to 6 kg
sieved soil is put in these tanks along with 250 g of super phosphate and 2.0 g of
sodium molybdate. Water is filled upto ( 10-15 cm) height depending upon the
sun-shine and rate of evaporation. If the soil is acidic this may be corrected by
addition of lime. The contents of the tank are thoroughly mixed and allowed to
stand for a day and when the water becomes clear, sprinkle the starter culture of
algae on the surface. The algae are allowed to grow for 15-20 days in sunlight.
To prevent mosquito breeding, the tank is covered with wire mosquito net or
insecticides like parathion (1 ml per tank), carbofuran (25 g per tank) may be
added in the tanks. Watering of tank is done if necessary. After the algae are
fully grown forming a thick scum on the surface of the water, watering of tank
is stopped and the contents are allowed to dry. The dried flakes formed in the
tanks are collected and used for immediate application or stored in polythene
bags for further use.

For application, the powdered flakes obtained from the culture tanks are
broadcasted on standing water, one week after paddy transplantation at the rate
of 7-10 kg/ha (soil based).

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2) Mass production of Azolla biofertilizer

The field selected for Azolla nursery should be thoroughly prepared and leveled
uniformly.
The field is divided into plots of size 20x2 m by providing suitable bunds and
irrigation channels.
Water is maintained to a height of 10 cm.
Ten kg of cattle dung is mixed in 20 liters of water and sprinkled per plot.
The Azolla inoculum of 8 kg is introduced in each plot.
Super phosphate is applied in 3 split doses @ of 100 g with 4 days interval as
top dressing fertilizer for Azolla.
Furadan granules @ of 100 g per plot is also applied on 7th day after inoculation
of Azolla to control insect pests.
The beds are filled in with water periodically to maintain a height of 10 cm.
In about 20-25 days a thick mat of Azolla is formed which floats on the surface
of the water.
It is harvested and introduced into the main field as a source of primary
inoculum.
About 40 to 55 kg of fresh Azolla is harvested in one harvest from each plot.

Azolla nursery plot may be prepared, while rice nursery is raised. Three per cent
of Azolla nursery will provide inoculum for one acre of transplanted paddy main
field. Azolla is inoculated @ of 300 g/m2 after one week of planting rice. In
about 3 week, Azolla growth will cover the entire surface of the plot. It is
incorporated on 25th day after planting.

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METHODS OF APPLICATION IN PADDY FIELD

Blue green algae application

In case of blue green algae application methods involve spreading to 10 kg blue

green algal culture (powdered flakes obtained from culture tanks) /ha in
standing water one week after paddy transplantation.

Azolla

The two methods of Azolla application have been recommended in India as a

green manure by incorporating in fields prior to rice planting and secondly by
dual cropping with rice when the fern grows side by side with the main crop for
sometime.

In the first method, Azolla is grown on the fallow field for 2 to 3 weeks, the
water drained and the green manure incorporated in soil by ploughing followed
by rice planting within a week time.

In the dual culture 1 to 4 kg/sq. m. (fresh weight) is inoculated a week after

planting rice seedlings sooner or later Azolla mat is formed. At this stage water
is drained and Azolla is incorporated in soil.

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 EXERCISE NO. 21

Introduction to Biopesticides
Biopesticides:
Biopesticides are the substances derived from nature, such as microorganisms or
botanical or semiochemical, that may be formulated and applied in a mannner
similar to a conventional chemical pesticide used to control the pests.
Bio means involving life or living organisms.
Pesticides includes substances or mixture of substances intended for preventing,
destroying or controlling any pests.
Biopesticides refers introduction of any living microorganisms including bacteria,
fungi, nematodes, viruses, protozoa and parasitoids and predators that controls
pests by biological non toxic means.
e.g. Trichoderma sp., Bacillus thuringiensis, Beauveria etc.

The various disadvantages of conventional chemical pesticides over biopesticides.

Conventional Chemical Pesticides Biopesticides
Synthesised or produced from Use naturally occurring compounds derived
artificial/chemicals from living organisms for the production
They cause environmental pollution and They do not cause environmental harm
are not eco-friendly
Harmful to nontarget organisms Do not cause harm to nontarget organisms
Cost ineffective Cost efficient and cheaper, compared to
chemical fertilisers
Microorganisms develop resistance Pests do not develop resistance
gradually as the application increases
High market value Not preferred in the market
Contaminate water and soil Cannot contaminate water sources
Lead to bioaccumulation Do not lead to bioaccumulation

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 EXERCISE NO. 22

Classification of Biopesticides

Based on origin Based on use

1. Microbial pesticides
1.Bioinsectisides
2.PIP (Plant incorporated protectants
2.Bionematicides
3.Biochemical pesticides
3.Bioherbicides
4.Botanical pesticides
5.Biotic agents ( Parasitiods, predators)

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 Bio I s ti i s

i Bacterial:
etc.
ii Fungal: ( ntomogenous fungi :
a Aseronija ( hitefly of many crops.

b Boverin and Boverol ( Pine caterpillar, reen leaf

hipper, Colorado potato beetle etc.

c Mycotal and ertalec ( hitefly and Aphids of

glasshouse crop.

iii iral:
a uclear polyhedrosis viruses ( P ,

(b ranulosis viruses ( of Baculoviridae, and

(c Cytoplasmic polyhedrosis viruses (CP of Reoviridae.

i. NPV E CA Cotto oll or

ii NPV GYPCHEK Gy sy ot a l s ars

iii NPV V O E ro a sa ly

iv CPV A S KE IN Pi at r illar

v GV A E I s ts o i r t r it ro s lik Co li g ot s

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 Bio N ati i s
ematode trapping fungi ematophaeguos fungi

ifferent fungi are known to act as nematicide. Fungi of different genera like
Arthrobotrys , actylella, actylaria and Monacrosporium are used to control
different members of genera like Heterodera , Meloidogyne and Rotylenchulus ,
cause diseases of different crop plants.

Bio H r i i s

Fungus like is used to control uropean blackberry

in Chile and to control rush skeleton weed in Australia.
thers, like , has been developed as herbicide at
commercial level.
Commercial products:
i. I ( Milk weed vine in USA,

ii. LUB A ( odders in

China,

iii. BI MAL ( Round leaved

mallow in Canada and USA.

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 EXERCISE NO. 23

Importance and Mass Production of Verticillium lecanii

A group of fungi that kill an insect by attacking and infecting its insect host is
called Entomopathogenic fungi
Entomopathogenic fungi (EPF) are considered as potential biocontrol agents against
various insect pests since late nineteenth century.
Since then more than 170 mycoinsecticides have been produced with at least 12
species, out of over 800 fungal species identified as pathogenic to insects.
Importance of Verticillium lecanii
1. Verticillium lecanii fungus inserts its mycelium through the body wall of all
stages of insects
and kills the insects by infecting it.
2. The death of the insect occurs because of release of certain toxic elements like
destraksin
desmethyl, destraksin etc. which kill the hardy stage-like pupae of the insects.
3. Verticillium lecanii (formerly known as Cephalosporium lecanii) was first
described in 1861
and is a cosmopolitan fungus found on insects.
4. It is a common pathogen of scale insects in tropical and sub tropical climates.
5. Verticillium lecanii is known as a “white-halo” fungus because of the white
mycelia growth
on the edges of infected scale insects.
6. The conidia of Verticillium lecanii are slimy and attach to the cuticle of insects.
7. The fungus infects insects by producing hyphae from germinating spores that
penetrate the
insect’s integument; the fungus destroys the internal contents and the insect dies.
8. The fungus eventually grows out through the cuticle and sporulates on the
outside of the
body.

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9. Infected insects appear as white to yellowish cottony particles. Diseased insects
usually
appear in 7 days.
10. However, due to environmental conditions, there may be some considerable lag
time from
infection to death of insects.
11. Verticillium lecanii works best at temperatures of 15-25 0C and a relative
humidity of 85-
90%.
12. The fungus needs high humidity for at least 10-12 h. This can be a problem as
many plant
pathogenic fungi favour the environmental conditions.
13. Verticillium lecanii spores are damaged by ultraviolet radiation.
14. The fungal mycelium of Verticillium lecanii produces a cyclodepsipeptide toxin
called
bassianolide, which has been shown to kill silkworm.
15. The fungus produces other insecticidal toxins such as dipicolinic acid.
Verticillium lecanii
strains with small spores infect aphids, where as fungal strains with large spores
infect white
flies.
16. Certain strains have also been reported to be pathogenic on rust fungi.
17. It is effective against hardy insects like scales, aphids, thrips, hoppers etc.
Mode of action
The spores of this fungus when come in contact with the cuticle of target insects
germinate and
grow directly through the cuticle to the inner body of their host. The fungus
proliferates throughout
the insect’s body, draining the insect of nutrients and eventually killing it in around
a week’s time.

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Crops: Ornamentals and vegetables in green houses, nurseries, lawns, landscape
perimeters.
Vegetables, field crops and other agricultural crops.
Target pests: Whiteflies, thrips, aphids, scales, mites and mealy bugs.

Application methods: Foliar spray: 400g/ acre in 200 L water.

MASS PRODUCTION OF VERTICILLIUM

1. Multiplied on cheap media for large scale production

2. Sorghum grains devoid of pesticide residue (40 g) is washed in potable

water

3. Transferred to conical flask (250 ml) and 15 ml of distilled water is added

4. Plugged conical flasks with cotton and autoclaved for 20 min at 15 psi

5. Flasks are allowed to cool and taken to laminar flow chamber for
inoculation

6. From a clean uncontaminated mother culture in slant loopful quantities

of V. lecanii spores are transferred aseptically

7. Incubated at room temperature

8. Spores are obtained in a fortnight

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 EXERCISE NO. 24

Importance and Mass Production of Beauveria bassiana

Importance of Beauveria bassiana

1. It is an Entomopathogenic fungus for protecting root and stem from destroying
insect pests.
2. It directly invades the insect body, when conidia become attached to the insect
cuticle and after germination the hyphae penetrate the cuticle and proliferate in the
insect body.
3. High humidity or free water is essential for conidial germination and infection
establishes between 24 and 48h.
4. The infected insect may live for 3-5 days after hyphal penetration, and after
death, the conidiophores bearing conidia are produced on cadaver.
Crops: cotton, Helicoverpa; cabbage, DBM; Sugarcane, root grub; coffee, berry
borer; black pepper, pollubeetle; palms, red weevils; and paddy, BPH
Target pests: caterpillars, weevils, leaf hoppers, bugs, grubs and leaf-feeding
insects.
Method of application: Foliar application (borer and cutworm): The product
should be sprayed (400
g/acre in 200 L of water) on growing plants using hand, ground or aerial equipment.
Soil application: 4- 5 kg/acre can be applied around the root zone and incorporated
into the soil
along with FYM (500kg) during June-July.

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 Mass Production:

• Carrot cut into small pieces (40 g) is washed in potable water and transferred
to conical flask (250 ml) and 15 ml of distilled water is added.
• The conical flasks are plugged with cotton and autoclaved for 20 min at 15
psi. The flasks are allowed to cool and taken to laminar flow chamber for i
inoculation.

From a clean uncontaminated mother culture in slant loopful quantities of B.

bassiana spores are transferred aseptically.

• The flasks are incubated at room temperature. The spores are obtained in a
fortnight

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 EXERCISE NO. 25

Importance And Mass Production of Metarhzium

Importance of Metarhizium anisopliae (Green muscardine fungus)
1. Metarhizium anisopliae is categorized as a green muscardine fungus due to the
green color of
the sporulating colonies.
2. It has been reported to infect approximately 200 species of insects and other
arthropods.
3. Metarhizium anisopliae generally enters insects through spiracles and pores in
the sense
organs.
4. Once inside the insect, the fungus produces a lateral extension of hyphae, which
eventually
proliferate and consume the internal contents of the insect. Hyphal growth
continues until the
insect is filled with mycelia.
5. When the internal contents have been consumed, the fungus breaks through the
cuticle and
sporulates, which makes the insect appear "fuzzy."
6. Metarhizium anisopliae can release spores (conidia) under low humidity
conditions (<50%).
7. In addition, Metarhizium anisopliae can obtain nutrition from the lipids on the
cuticle.
8. The fungus can also produce secondary metabolites, such as destruxin, which
have
insecticidal properties on moth and fly larvae.
9. The colony of M. anisopliae appears white when young, but as the conidia
mature, the colour turns to dark green.

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10. Host covered by weft of mycelium, hyphae septate, hyaline, 1.8-4.0 μm wide.
11. The conidiophores are branched and the initial conidium is produced at the
distal end of the conidiophores.
12. A chain of conidia is formed on each conidiophore with the youngest conidium
being adjacent to the conidiophore.
13. The mass of spore chains becomes so dense and coheres with each other to
produce prismatic masses of columns of spore chains.
14. It infects insects that come in contact with it. Once the fungus spores attach to
the surface of the insect, germinate and begin to grow, they then penetrate the
exoskeleton of the insect and grow very rapidly inside the insect causing the insect
to die. Other insects that come in contact with infected insects also become infected
with the fungus.
15. Several commercial formulations are developed based on Metarhizium
anisopliae (biogreen, biopath, bioblast, bio 1020) for control of cockroaches, termits
and vine weevil.
16. This pathogenic fungus is used to control mainly coconut rhinoceros beetle,
groundnut cut worm, rice brown plant hopper, diamond back moth and early shoot
borer, top shoot borer
and internode borer of sugarcane.
17. It is based on formulation of a friendly fungus Metarhizium anisopliae which
effectively
controls: white grubs, brown leafhoppers of paddy, pyrilla of sugarcane, green
semilooper, termites, spotted pod borer of pigeonpea, coconut rhinoceros beetle,
mango hopper, rhizomes weevil of banana and DBM of cauliflower.
Mode of action
The spores of this fungus when come in contact with the cuticle of susceptible
insects germinate and grow directly through the cuticle to the inner body of their
host.

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The fungus proliferates throughout the insect body and drains the insect of
nutrients, eventually killing it.
Crops: White grubs, brown leafhoppers of paddy, pyrilla of sugarcane, green
semilooper, termites, spotted pod borer of pigeonpea, coconut rhinoceros beetle,
mango hopper, rhizome weevil of banana and DBM of cauliflower.
Target pests: White grubs, brown leafhoppers, pyrilla, semilooper, termites,
spotted pod borer, rhinoceros beetle, mango hopper, rhizome weevil, root weevils
and DBM.
Application methods
Foliar spray: The product should be sprayed on the growing plants using hand
spraying equipments.
Soil application (root grubs and weevils): It (4-5kg/acre) can be applied around the
root zone and incorporated into the soil along with FYM (500 kg) during June-July.

Mass Production:
In Carrot broth

• Carrot cut into small pieces (40 g) is washed in potable water and transferred
to conical flask (250 ml) and 15 ml of distilled water is added.
• The conical flasks are plugged with cotton and autoclaved for 20 min at 15
psi.
• The flasks are allowed to cool and taken to laminar flow chamber for
inoculation.
• From a clean uncontaminated mother culture in slant loopful quantities of M.
anisopliae spores are transferred aseptically.
• The flasks are incubated at room temperature. The spores can be harvested in
a fortnight.

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 EXERCISE NO. 26

Importance and Mass Production of Paecilomyces lilacinus

Importance of Paecilomyces lilacinus

It is an opportunistic fungus which is a very good egg parasite of nematodes.
Mode of action
1. Then fungus has been reported to parasitize eggs of many sedentary
endoparasitic nematodes.
2. The infection process starts with growth off ungal hyphae in the gelatinous
matrix and eventually the eggs of nematodes are engulfed by the mycelia hyhae.
3. The proliferated hyphal branches penetrate the eggs. In cyst nematodes, the
fungus penetrates through vulva or the broken and exposed neck region.
4. After entering the cyst, the fungus grows saprophytically on the body content
surrounding the eggs during or before its parasitism of the eggs
5. In all cases, eggs in the early embryonic developmental stages are more
vulnerable to infection.
6. Once the hypha is in contact with the eggs a series of ultra structural changes
occurs in the eggs due to effects of exogenous metabolites and chitinolyic activities
of the fungus.
7. Once inside, the fungal mycelium radiated profusely in the eggs of early
embryonic development and the entire embryo is replaced by the mycelia biomass.
8. Occasionally, P. lilacinum may penetrate the egg lying female through the anus
or vulva.
9. In such cases, the infected female body cavity filled with the fungal biomass and
the nematodedie
10. P. lilacinumis compatible with organic amendments like neem cake, castor cake
and greenmanures.

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11. It can also be combined with Trichoderma viride, Verticilium chlamydosporium
against root knot nematode and best as seed treatment.
12. It can also be applied in soil. In that case, its culture filtrate should be used to
enrich the vermi-compost and this should be used before sowing or transplanting.
13. Its liquid and powder formulations are available in different names.

MASS MULTIPLICATION
100 ml of the different liquid media like P B, 10%Molasses, Richard’s medium
and Semi selective medium were taken in a 250 ml conical flask.
They were autoclaved at 15 psi for 20 minutes.
Each flask was inoculated aseptically with 8mm disc of the Paecilomyces lilacinus,
maintained on Potato dextrose agar (PDA) as a pureculture.
The flasks were incubated at room temperature for 30 days.
The fungus inoculated in semi selective medium served as control.
All the treatments were replicated five times.
Fungal biomass was recorded in each treatment.
Spore load was enumerated by serial dilution and plating method on Rose Bengal
Agar.
FORMULATION
The culture flasks in the above treatments were used for formulation with different
carrier materials viz., talc, fly ash, rice hull ash and vermiculite.
The mycelial mat along with the broth was homogenized and mixed with carrier
material in the ratios 1:2. Carboxyl methyl cellulose was added@5g/kg of the
product.
The acidity of the medium was neutralized by adding 20g of chalk /kg of product.
Then the product was shade dried to reduce the moisture content to 12% and packed
in opaque polythene bags and stored at room temperature for further studies.
The spore load at the time of packing was 20 x10 cfu/g in the product.
The formulated product was tested for the viability by serial dilution and plating at
15 days interval for120 days.

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 EXERCISE NO. 27

Importance and Mass production of Trichoderma viride

Trichoderma
• Trichoderma spp. is one of the most versatile biocontrol agents which have
long been used for managing plant pathogenic fungi.
• The role of Trichoderma spp. is not only to control growth of pathogenic
microbes, but there are various other uses for Trichoderma such as,
Stimulate colonization of rhizosphores,
Stimulates plant growth, root growth, and
Enhance plant defence responses
• They are the common inhabitants of soil and other natural habitats containing
organic matter.
• Trichoderma sp. is common inhabitants of almost every soil and is
antagonistic to other fungi.
• Through the action/phenomenon of antibiosis (production of volatile end
non-volatile antibiotics) and mycoparasitism (parasitizing other fungi),
Trichoderma sp. suppress/destroy/lyse the phytopathogenic soil borne
(rhizosphere) as well as foliage (phyllosphere) fungi.
• Trichoderma spp. has been reported as most potent antagonists and
mycoparasites of the pathogenic fungi, such as: Pythium, Phytophthora,
Rhizoctonia, Fusarium, Sclerotium, Sclerotinia, Macrophomina, Botrytis etc.
• Trichoderma species are economically important for their production of
industrial enzymes (cellulases and hemicellulases), antibiotics and their
action as biocontrol agents against plant pathogens based on various
mechanisms such as the production of antifungal metabolites, competition for
space and nutrients and mycoparasitism.

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 • The ability to promote growth and induce resistance in plants is a mechanism
which has also been described for members of this genus.
• The advantage of using Trichoderma in managing soil borne plant pathogens
are ecofriendly,
• effective, ease of mass culturing with less cost of production and growth
promoting effect.
Benefits of Trichoderma
Disease Control: Trichoderma is a potent biocontrol agent and used extensively for
soil born diseases. It has been used successfully against pathogenic fungi belonging
to various genera,
viz. Fusarium, Phytopthara, Scelerotia etc
Plant Growth Promoter: Trichoderma strains solubilize phosphates and
micronutrients. The
application of Trichoderma strains with plants increases the number of deep roots,
thereby
increasing the plant's ability to resist drought
Biochemical Elicitors of Disease: Trichoderma strains are known to induce
resistance in plants. Three classes of compounds that are produced by Trichoderma
and induce resistance in plants are now known. These compounds induce ethylene
production, hypersensitive responses and other defense related reactions in plant
cultivars.
Transgenic Plants: Introduction of endochitinase gene from Trichoderma into
plants such as tobacco and potato plants has increased their resistance to fungal
growth. Selected transgenic lines are highly tolerant to foliar pathogens such as
Alternaria alternata, A. solani, and Botrytis cirerea as well as to the soil-borne
pathogen, Rhizectonia spp.
Bioremediation: Trichoderma strains play an important role in the bioremediation
of soil that are contaminated with pesticides and herbicides. They have the ability to
degrade a wide range of insecticides: organochlorines, organophosphates and
carbonates.

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Mass production
Preparation of mother culture
Molasses yeast medium is prepared as
detailed below.
Molasses : 30 g
Yeast :5g
Distiller water: 1000 ml
The medium is prepared and dispensed into conical flasks and sterilized at 15 lb
pressure for 15 minutes in an autoclave.
After the medium is cooled it is in inoculated with 10 days old fungal disc of T.
viride and then incubated for 10 days for fungal growth.
This serves as mother culture.

Mass multiplication:

Molasses yeast medium is prepared in fermentor and sterilized as

described earlier. Then after the medium is cooled, the mother culture is added
to the fermentor @ 1.5 lit / 50 lit of the medium and incubated at room
temperature for 10 days. Then the incubated broth containing the fungal culture
is used for commercial formulation preparation using talc powder.

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 EXERCISE NO. 28
Importance and Mass Production of Nomuraea rileyi
Importance of Nomuraea rileyi
1. This fungus is an Entomopathogenic fungus found in several countries, including
India.
2. This fungus attacks important caterpillar pests of soybean, groundnut and maize
such as
Spodoptera and Helicoverpa causing epizootics.
3. It targets larvae and immature stages of leaf rollers, cut worms, leaf-eating
caterpillars and
other lepidopteran pests.
4. Environmental conditions (solar radiation, temperature, humidity etc.) greatly
affect this
microorganism and bring down their field viability and persistence.
5. It acts on harmful caterpillars by gradually paralysing and thus killing them.
Mode of action
The spores of this fungus when come in contact with the cuticle of susceptible
insects
germinate and grow directly through the cuticle to the inner body of their host. The
fungus
proliferates throughout the insect’s body and drains the insect of nutrients,
eventually killing it.
Crops: Soybean, groundnut, chilli, tomato, potato, cauliflower, paddy, lucerne and
corn
Target pests: Leaf eating caterpillars, borers,

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 EXCERCISE NO. 29
Importance and Mass Production of Hirsutella thompsonii

Importance of Hirsutella thompsonii

Hirsutella is one of the most abundant and important entomogenous fungi and
might play an important role in the control of pest insects in nature.
Hirsutella includes three important species, Hirsutella thompsonii, Hirsutella
gigantea, and Hirsutella citriformis.
Green Hirsutella is a bio-miticide containing the fungus Hirsutella thompsomii.
It is effective in the management of red spider mites, yellow mites and eriophid
mites on a variety of crops such as coconut, tea, vegetables, fruit trees etc
When Hirsutella spores come into contact with the body of the mite host, they
germinate, penetrate the cuticle by enzymatic action and grow inside the mite
body killing the mites within few days. After the death of the mite the fungal
hyphae emerges from the cadaver who produces abundant of spores.
Hirsutella sp has been found efficient against nymph and adults of red spider mite,
adults. Varroa mite, (Varroa destructor) of Honey bees, Coconut eriophyid
mite (Aceria gueneronis). Hirsutella sp. penetrates into the mites mainly through
the legs, which later on forms hyphal bodies in chains in the hemolymph.
Virulence of the Hirsutella strains against target pests is an important characteristic
and its ability for mass production.
One of the most important advantage is they have restricted host range and are
harmless to non-target organisms, Therefore, it might be used within Integrated Pest
Management programs, the disadvantages of biopesticides is that it has a relatively
short shelf life that can be for a few weeks and is highly sensitive to the
environmental conditions.

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 Procedure
1. For mass production on solid media, we can use cereal grains like rice, wheat,
maize, sorghum and ragi.
2. First take 1000 gm/1 kg raw grains and soaked for 12 hours in tap water and
excess water drained out completely.
3. Soaked raw grains 100 gm placed in autoclavable polypropylene bags.
4. In each bag add 2 gm of calcium sulphate and mixed thoroughly out to get
uniform coating of these over grains.
5. This process helped in preventing the grain particles sticking together and there
by providing more surface area for the growth of fungus.
6. Then bags are sterilized twice at 121°C for 20 minutes in autoclave.
7. Then grain media was inoculated with 1 ml of fungal suspension- Beauveria spp/
Verticillium/ Nomuraea/ Paecilomyces/ Hirsutella/Metarhizium (1 X 107 spores per
ml) under laminar air flow cabinet (aseptic condition).
8. The bags are seeded manually and incubated for 14 days at 25° C and 90%
relative humidity in growth chamber.
9. After 14 days of incubation, grains with fungal growth dried under aseptic
condition at 30° c until the moisture content is reduced to 8%.
10. After drying spore production is estimated by eubauer’s haemocytometer.
11. Then grains with fungal growth are sieved with vigorous agitation.
12. Then coarse dust thus collected further sieved through a sterile 105 mm sieve to
get fine spore dust and packed in polythene bags and stored at room temperature for
further studies.

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 EXCERCISE NO. 30

QUALITY CONTROL PARAMETERS OF BIOPESTICIDES (AS PER CIB

SPECIFICATIONS)
___________________________________________________________________
________
1. Inoculation should contain a minimum of 108 viable cells/gm of the carrier on
dry weight
basis at the time of manufacture and 107 cells on expiry date marked on the packet.
2. No contaminations at 106 dilution.
3. PH of inoculants should be 6.0 to 7.5.
4. Carrier materials should pass through 50 micron sieve size.
5. Packing low density polythene bags of 50 to 75 micron.
6. Each packet should be marked with the details name of the product, crop for
which recommended, strain number, date of manufacture, date of expiry, method of
application and

storage.

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 EXCERCISE NO. 31

LABELING AND PACKING OF BIOAGENTS

AND BIOPESTIDES PRODUCT
The fungal biomass collected from fermentor is mixed with talc powder at
1:2 ratio.
The mixture is air dried in shade and mixed with carboxy methyl cellulose (CMC)
@ 5g/kg the products
It is packed in polythene bags and should be used within 4 months.
Biological control agents have characteristics that make them different from
chemical pesticides and the product labels should reflect following points:
Description and quantification of the active ingredient (a.i.) in the formulation:
The label should provide a description and quantification of the active substance in
the formulated
product as follows:
Microbial: The content of the active substance can be expressed, for example, as
colony forming unit (CFU) per kilogram or litre and or amount of relevant
secondary compound (metabolite) or in terms of biopotency (e.g. Bacillus
thuringiensis is expressed in terms of Billions of International Units, or BIU).
The label may also give information on the amount of other materials such as spent
fermentation media.
2. Botanicals: The active substance content can be expressed as the amount of
botanical source materials, the lead component, or biopotency.
3. Formulation: Details should be provided on the type of formulation.
4. Safety advice: The label should contain safety advice (and pictograms) for
humans and the environment, as well as appropriate personal protection equipment
(PPE).
5. Effectiveness: The label should indicate the expected effect (e.g. pest kill, yield
improvement, post-harvest protection) and the expected level of this effect (e.g. pest
control, pest reduction, pest suppression, increased yield).

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6. Directions for use: The label should provide advice on how to mix, prepare and
apply the product for each target pest and situation, as well as advice on application
equipment, water volumes, adjuvant, and conditions under which the product
should not be used.
7. Directions for storage: The label should specify storage conditions that align
with the test conditions for the (real-time) storage study and any specific conditions
that are required (e.g. refrigeration).
8. Integrated pest or vector management: It is good practice for the label to
recommend use of the product as part of IPM and to indicate compatibility with
other control measures, if known.

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 EXERCISE NO. 32

METHODS OF APPLICATION OF BIOFERTILIZERS

Carrier-based Biofertilizer

1. Seed treatment

2. Seedlings treatment

3. Soil treatment

1. Seed treatment :

i. Prepare the slurry of 250 g of biofertlizer in 200-500 ml water

ii. Pour this slurry slowly on 10-15 kg of seeds, or seeds required

for oneacre land.

iii. Mix the seeds with hands evenly to get uniform coating of
biofertilizers onall of them

iv. Dry the treated seeds in shade and then sow immediately

2. Seedlings treatment: For seedling-transplanted crops, treatment is

asfollows

i. Prepare the suspension of 1-2 kg of Biofertilizer in 10-15 liters of

water

ii. Dip the roots of seedlings obtained from 250-500 seeds into the
suspension for 20-30 minutes

iii. Transplant the seedlings immediately

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 3. Soil treatment: when biofertilizer application to seeds or seedlings
is notpossible, the soil method of application is followed

i. Prepare the mixture of 2-4 kg of biofertilizer in 40-60 kg of soil

compost

ii. Broadcast the mixture in one acre of land either at sowing time
or 24hours before sowing

iii. For fruit crops, 5kg FYM + 25 gm Azotobacter + 25 gm PSB +

25 gm
Trichoderma is applied

Liquid Biofertilizer

1. Seed treatment:

i. For 1 kg seed material, 25 ml liquid bioinoculant is used

ii. Seeds are dipped in this suspension for 10 mins

iii. Dry the seeds in shade and then sow them as early as possible,
preferablyduring morning and evening hours

iv. Seeds with hard seed coat like cotton, dipping should be
carried outovernight

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2. Sett inoculation:

i. The setts or tubers are immersed in liquid biofertilizers for 20

minutes andare planted in the field

3. Seedling treatment:

i. Roots of seedlings are dipped in biofertilizer solution for 8-10

min andtransplanted immediately

ii. The liquid bioinoculant (500 ml) is sufficient for seedling

treatment for 1acre area.

4. Soil treatment:

i. Seed treatment with liquid bioinoculant is not possible then 2

lit liquidbioinoculant is mixed with 50 kg of FYM or compost

ii. Mixture is spread uniformly on the field before irrigation

iii. Also be supplied through drip irrigation system

5. Soil broadcasting :

i. Dilute 100 ml of liquid bioinoculants in 5 lit of water and then

mixing with50 kg of cowdung and 5 kg rock phosphate

ii. Keep this mixture overnight

iii. Next day apply the mixture over one acre of land at root zone and
irrigate

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6. Seed pelleting:

i. Take 2 kg of sieved soil

ii. Sprinkle 25 ml of liquid bioinoculant solution on sieved soil

iii. Keep this mixture overnight

iv. Mix 7-10 kg of seeds in this mixture

v. Allow the seeds to dry in shade before sowing

7. Foliar spray :

i. Dilute 3 liter of liquid bioinoculants in 200 liter water and

spray thissolution on sugarcane plant preferably in the evening

8. Drip irrigation :

i. Two liters of liquid biofertilizer is given through drip irrigation

for 1 acrearea

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METHODS OF APPLICATION OF BIOPESTICIDES

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