EXERCISE NO.

EQUIPMENT, MACHINERY AND TOOLS USED FOR BIOFERTILIZERS,

BIOPESTICIDES AND BIOAGENTS 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:

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

o
C
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 approximately
90 linear feet per minute physically sweeps the work area and prevents the entry
of contaminated air.
o The space between the HEPA filter and sterile product being prepared is referred
to as the critical work surface.
o HEPA filter - removes 99.97 % of all air particles which have the diameter of 0.3
mm or larger.
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 sterile

products 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 used
for 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 for

organisms which grow at their maximum growth rate.
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 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.

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
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 testing
biochemical 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.

EXCERISE NO. 2
 MEDIA USED FOR BIOFERTILIZERS, BIOPESTICIDES AND
BIOAGENTS 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

2. CONGO RED YEAST EXTRACT MANNITOL (CRYEM)

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

3. 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

4. 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
 5. 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

6. 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 0.5g
Agar - 1.0g
Distilled water - 1000 ml 7. LGIP MEDIUM

PH - 6.6-7.0
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
8. BECKING’S MEDIUM

Sucrose - 20 gm
K2HPO4 - 0.2 gm
KH2PO4 - 0.2 gm
FeCl3 - 0.005 gm
MgSO4 7H2O - 0.5 gm
Na2MoO4 - 0.0050 gm
Agar - 20 gm
Distilled water - 1000 ml

9. 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

10. 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

 EXERCISE NO. 3

ISOLATION OF RHIZOBIUM FROM ROOT NODULES. ISOLATION OF

AZOTOBACTER , ACETOBACTOR ,BEIJERNICKIA, AZOSPIRILLIUM.

I. BY DILUTION POUR PLATE TECHNIQUE AND II. BY ENRICHMENT

CULTURE TECHNIQUE

A. 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. From
such 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 and

bring the root samples with nodules to the laboratory.

2. Wash the root system gently under running tap water, remove adhering soil with
camel’s hair brush.

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

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.
 B. 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.
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 an
incubator.
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 culture
in a slant or plate.

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.
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 is
formed 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 on
incubation 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.
7. Draw a schematic diagram showing enrichment culture technique for isolating
Azotobacter form rhizosphere of a cereal crop
8. Write the composition of Jensen's and Ashby's nutrient media.
 C. 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 of
grass 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

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 or
scalpel.
3. Rinse root bits in 0.1% mercuric chloride solution for 1 minute in sterile Petri
dish.
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 small
test 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 two
vials 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 nitrogen
free malate agar medium (1.5% agar) in sterile petri dishes.
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 rods
and 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 containing
NFb 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 on
subsequent 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 form
rhizosphere of weed Cynadon dactylon .
7. Write the composition of Sodium malate or NFb medium.
 D. 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 washed
thoroughly in the running tap water
2. Surface sterilized with 70% ethanol and subsequently washed in changes of
sterilized distilled water
3. Surface sterilized samples are to be macerated in sterile blender and serial
dilutions 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 LGIP
semisolid 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.

E. 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 non symbiotic
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 crops
and 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 with
1 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.
Observations:
The colonies of the Beijerinckia are gummy with profuse polysaccharides. They are
round in shape, wrinkled flat and raised
 EXERCISE NO. 4

ISOLATION OF BGA, PSB, SULPHUR OXIDIZING MICROORGANISMS, IRON

CHELATOR, POTASH MOBILIZERS, ORGANIC MATTER DECOMPOSERS

I. BY DILUTION POUR PLATE TECHNIQUE AND II. BY ENRICHMENT CULTURE

TECHNIQUE

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

Sodium nitrate - 0.5 g

Magnesium sulphate- 0.15 g

Calcium 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 ml

capacity), 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.

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

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 quadruplicate
plates 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 soil
sample 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 solubilizing

capacity may be estimated by the colourimetric method.

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 C
on 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 and
10-6 dilutions
Ingredients of Aleksandrov medium
Glucose - 10 gm
MgSO4.7H2O - 0.005 gm
FeCl3 - 0.1 gm
CaCO3 - 0.1 gm
CaCO4 - 2 gm
Potassium aluminium
silicate - 5 gm
Yeast extract - 5 gm
Agar - 15 gm

ISOLATION OF SULPHUR OXIDIZING MICROORGANISMS FROM SOIL

BACTERIA

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 a
wooden pestle and screened though 2 mm sieve.

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

medium.

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 and

the 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 three
successive days.

7. The flasks are then inoculated with 1 gm of soil and incubate on to-and-fro
shaker (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 color
change of the bromocresol purple indicator from purple to colorless.

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

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 clearing

around the colony of fungi and actinomycetes.

Observations :

Record the colony morphology of suphur oxidising microorganisms

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, Cellulose
agar medium.

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 mix
thoroughly 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 soil
sample in test tubes containing sterile water.

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

5. 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.

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 cultures
by 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 of
one gram of soil i.e.

Average plate count x Dilution factor

No. of cellulolytic microbes/ = -------------------------------------------------

oven dry soil Oven dry weight of 1 g of soil sample

Observations:

Record the colony morphology of cellulolytic microorganisms

 EXERCISE NO. 6

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 dilution
method to get pure colonies of desired microbe

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 organism
till 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
o High organic matter (above 60%)
o Low soluble salt content (less than 1%)
o 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

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.
 EXERCISE NO. 6

PRODUCTION OF CARRIER BIOFERTILIZERS OF SULPHUR OXIDIZING

MICROORGANISMS, 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, sealing
machine

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 dilution
method 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
 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 organism
till 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
o High organic matter (above 60%)
o Low soluble salt content (less than 1%)
o 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
 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.
 EXERCISE NO. 8

STUDY OF VA-MYCORRHIZA: GROWTH ON GUINEA GRASS ROOTS AND

OBSERVATIONS FOR ROOT COLONIZATION. METHODS OF PREPARATION
AND 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 40
microns).
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 and
observe the spores under the stereoscope (majority of the spores could be seen
in 90 and 40 micron sieves).
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 destaining
solution.
8. Take the root make small sections and observe under microscope.
9. Record presence of arbuscles, vesicles, mycelium within and out side the root
section.

B) Methods of preparation and application of VA-mycorrhizal inoculums

Methods 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

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 30 th and 45th

Day

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 mixture
of 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 1000
kg 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 time
of planting

For existing trees:

Two hundred gram of VAM inoculums is required for inoculating one tree. Apply
inoculums near the root surface at the time fertilizer application.
 EXERCISE NO. 9

MASS PRODUCTION OF TRICHOGRAMMA, CRYPTOLAEMUS AND

CRYSOPERLA

A) Mass production of Trichogramma

Introduction

 The genus Trichogramma is cosmopolitan in distribution and present in all

terrestrial habitats and is one of 80 genera in the
family Trichogrammatidae. Trichogramma are primary parasitoids eggs of
Lepidoptera, but parasitism also occurs in eggs of other orders such as
Coleoptera, Diptera, Hemiptera, Hymenoptera and Neuroptera.
 It is important for plant protection because of its wide spread natural occurrence
and its success as biological control agent by mass releasing.
 Since this parasitoid kills the pest in the egg stage itself before the pest could
cause any damage to the crop and also that it is quite amenable to mass
production in the laboratories, it has the distinction of being the highest
produced and most utilized biological control agent in the
world Trichogrammatidae includes the smallest of insects, ranging in size from
0.2 to 1.5 mm.
 Trichogramma are difficult to identify because they are so small and have
generally uniform morphological characters. Also, certain physical characteristics
such as body color and the number and length of body hairs can vary with body
size, season, rearing temperature and the host on which the adult was reared.
 A major advance in the systematics of Trichogramma was the discovery that
characteristics of male genitalia can be used to identify species. This is the
primary means of identification today, but body color, wing venation and
features of the antennae serve as supporting characteristics.
 Females cannot be identified with the same level of confidence, so collections
submitted for identification must include males in addition to physical
 characteristics; studies of reproductive compatibility and mode of reproduction
also have been especially valuable in identifying species.

Biology of Trichogramma

 The development of all Trichogramma spp. is very similar. Being an egg parasite,
the female drills a hole through the chorion and deposits its eggs within the egg of
the host.
 The internal pressure of the egg forces a small drop of yolk out of the oviposition
hole. Females feed on this yolk, which increases their longevity and under
laboratory conditions a female parasitizes from one to ten eggs per day or from ten
to 190 during her life.
 Large females parasitize more eggs than smaller females. The number of eggs laid
per host egg may vary from 1 to 20 or more depending upon the size of the host
egg. However in sugarcane, in which moth borer eggs are small, generally 1 or 2
parasites develop per egg.
 A female parasitoid can distinguish already parasitised eggs, thereby avoiding
superparasitism or multiple-parasitism under natural conditions. Fecundity varies
from 20 to 200 eggs per female according to the species, the host, and the longevity
of the adult.
 Eggs in the early stages of development are more suitable for parasite
development. Older eggs, especially those in which the head capsule of the larva is
visible, are not usually parasitized and if they are, parasite survival is much lower.
Venom injected by the female at the time of oviposition is believed to cause this
predigestion of the egg’s contents.
 During the 3rd instar (3 to 4 days after the host egg was parasitized) dark melanin
granules are deposited on the inner surface of the egg chorion, causing the host egg
to turn black. This is an invaluable diagnostic character for distinguishing them from
unparasitised eggs. Larvae then transform to the inactive pupal stage.
 The adult wasps emerge from the pupae and escape the host egg by chewing a
circular hole in the egg shell. The black layer inside the chorion and the exit hole
are evidence of parasitism by Trichogramma. The egg, larval and pupal stages
of Trichogramma at 28 ± 20C are completed in about 1 day, 3 to 4 days, and 4 to 5
days respectively.
 Thus, the life cycle is completed in 8 to 10 days, but it may be prolonged at lower
temperatures or hampered at very high temperatures. The adults are short lived (6-
8 days). Mating and oviposition take place immediately after emergence. The sex
ratio is generally 1:1.

Materials required

Sterilized sorghum, Corcyra rearing boxes/trays/jars made up of plastic or wood with lid
provided with wire mesh for aeration, Corcyra egg laying cage, Black cloth, Mosquito
net, Table Racks for placing Corcyra cages, Honey, Glycerin, Tubes for collecting
Corcyra moth, Measuring cylinder
Plastic tubs for egg laying purpose, Brush, Roasted ground nut powder - 100 grams,
Yeast - 5 grams
Wettable sulphur - 5 grams, Streptomycin sulphate - 0.05 gms

Preparation of egg laying cage of Corcyra cepholonica

Take a plastic bucket with lid. Cut the lid in circular shape leaving space for providing
/fixing wire mesh for egg laying purpose in the (circular wire mesh). Make a hole on the
centre of bottom of the plastic bucket to pour the collected adults in the bucket. Keep
bucket inverted in the plastic tub for egg laying purposes.
Steps for production of Corcyra cephalonica
 Sterilize the rearing boxes (if wooden) in hot air oven for 100 degree centigrade for
30 minutes
 If plastic trays are used, wash them before use
 Dry broken grains of jowar in sunlight properly
 Pour sterilized grain - 2.5 kg/box/tray
 Add 100 grams of roasted ground nut powder, 5 grams of yeast, 5 grams of
wettable sulphur, 0.05 gms of streptomycin sulphate in each box or tray
 Mix well all ingredients
 Sprinkle 1 cubic centimeter ofCorcyra eggs/box/tray on the top of mixture(culture
medium)
 Cover the box with lid, label the date of inoculation
 Keep these boxes in racks protected by ant pans
 Favourable temperature for rearing is 28+/-2 degree centigrade and Relative
humidity, 75% +/- 5%
 The moth starts emerging on 40th day
 Bring the boxes ready for moth emergence and collect moths inside the net by glass
tubes
 Transfer the moths to egg laying chamber
 Provide cotton soaked 20% honey+ vitamin E solution as adult food in the egg
laying chamber
 Collect the eggs daily
 Pour the eggs in a paper by tilting slightly downward so eggs come down side where
as dust particles remain in upper side
 Clean the eggs further by passing through different size sieves to 10, 15 and 40
meshes
 Discard the moth after 4 days
 Utilize the Corcyra eggs for Trichogramma production (or) host culture or store them
in refrigerator at 10 degree centigrade for 7 days, if required.

Mass production

1. Different species and strains of Trichogramma typically prefer different host eggs
and crop habitats and have different searching abilities and tolerance to weather
conditions.
2. Efficacy is improved by selecting the most effective and adapted species or strain for
the specific crop / pest situation.
3. In the laboratory the parasitoids are multiplied on Corcyra eggs. The eggs laid by
the Corcyra moths are collected and sieved to remove the moth scales etc.
4. The pure eggs thus obtained are exposed to ultra-violet light in UV chamber to kill
the host embryo but at the same time permit parasitization.
5. The quantity of the sterilized eggs is assessed in a measuring cylinder
volumetrically. The eggs in volume of six cc are then sprinkled uniformly over a 144
gsm (chart paper) card of 30 x 18 cm size
6. The card is divided into two halves of 30 x 9 cm (LxB). Lengthwise it is subdivided
into 15 grids (G1 to G15) of size 2cm. The dimension of each grid is 7x2 cm. Each
grid can accommodate 0.2 cc of the eggs.
7. Label information on the manufacturer, species of the parasitoid, date of
parasitization and expected date of emergence are given in the left over spaces of
size 30 x 1 cm on the top and bottom of each half of the card.
8. A coat of 10% gum arabic is applied on the grids (G1-30) and the eggs are sprinkled
uniformly in a single layer with the aid of a tea strainer.
9. The excess eggs pasted are removed by gently passing a shoe brush over the card
after sufficient air drying under fan.
10. The egg cards are placed into polythene bags of suitable size and the nucleus card
of Trichogramma are introduced in it. The easiest way to accomplish this is to place
a piece of ‘Tricho egg card’ containing parasitized eggs (i.e. pharate adults) that are
ready to yield the adults and to hold them in subdued light for 2 to 3 days.
11. The emerging parasites readily parasitize the fresh eggs. The parasitoid - host ratio
is adjusted accordingly to 1:6 get effective parasitism.
12. The parasitized eggs in the Tricho Card turn back in 3 or 4 days and the adult
parasitoids emerge in 8 to 10 days from the date of parasitization.
13. The parasitized eggs in which the parasitoids in the larval or pupal stage (i.e. before
or after turning black) can be stored in the refrigerator (at 50C) for about 3 weeks
without any loss in emergence.
Precautions
Poor quality of mass reared Trichogramma can result in control failures. The artificial
conditions of mass rearing can select for genetic changes that reduce the effectiveness
of the Trichogramma in the field. Such rearing conditions include rearing multiple
generations on unnatural host eggs, the absence of plants, crowding and interference,
rapid generation time, and failure to rejuvenate genetic stock. Except for obvious
problems such as lack of adult emergence or wing deformities, growers and pest
consultants cannot detect poor quality Trichogramma prior to release. Commercial
suppliers are responsible for maintaining desirable characteristics necessary for good
performance in the field. Production colonies should be periodically replaced with
individuals from a stock culture maintained on the natural or target host. Suppliers also
should assess the per cent host egg parasitization, adult emergence, and the sex ratio
of emerged adults to be sure they are within acceptable standards. Standards for
established cultures on Corcyra are 95±5 per cent egg parasitization, 90±5 per cent
adult emergence, and a sex ratio of 1 to 1.5 females per male.

Delivery
Tricho cards are delivered for use in the field. The cards in volumes of 6 cc are
assembled in aerated polythene bags and packed in paper cartons for transport. The
cards have to be transported by the most rapid method of transport to reach the
destination. During transport and holding the cartons should not be exposed to extreme
conditions like toxic fumes, open sunlight, high temperature areas as the consignment
could be damaged leading to mortality of the Trichogramma stages.

Field release
The parasitoids are released in the pharate stage or when few adults begin to emerge
from the host egg during the evening hours. The cards are cut into bits neatly along the
grids with least damage to the eggs and stapled beneath the foliage in the upper
canopy level. To maximize the field parasitization it is recommended to release the
parasitoids is as many locations as possible. Recently scientists are beginning to
advocate the release of cards @ 1/5m row length.

B) Mass production of Cryptolaemus montrouzieri

Introduction
C. montrouzieri has been introduced from Australia for the control of Coccus viridis on
coffee. But the predator has established on many species of mealybugs and green
shield scale. In the field its practical use for the suppression of mealybugs viz., pink
mealy bug, Maconellicoccus hirsutus, citrus mealy bug Planococcus citri, tailed mealy
bug Ferrisia virgata and mealy scale Pulvinaria maxima on citrus, coffee, grapes and
several other fruit crops and ornamentals has been demonstrated. Use of C.
montrouzieri is the breakthrough in applied classical biological control. The coccinellid
predator is native of Australia. In 1892, it was introduced into California by Albert
Koebele for the control of citrus mealy bugs. Following the success, the beetle was
introduced into India in 1898 by New Port. It has given effective control of mealy bugs
in fruit crops like citrus, grapes, guava, etc. C. montrouzieri is one of the outstanding
examples in the biological control history. Its importance is also evident by its growing
commercialization in India.

Production procedures of predator

In the laboratory, the life cycle is completed in approximately 30 days. The

premating and preoviposition periods are about 5 and 10 days respectively. The
oviposition is about 10 days. Eggs are laid from late evening to early morning. They
are pale yellowish white, the surface being smooth and shiny. It is oval to cylindrical,
both the ends beings smoothly rounded. Incubation period ranges from 5 to 6 days but
extended in winter months. Viability of eggs is 90 to 100 per cent. The newly hatched
grub is sluggish but becomes active after 3 to 4 hours. The tiny grub is pale greyish
with white lines across the body along intra segmental regions. These white lines
become prominent after few hours and white wax strands develop after a day. The
grub has four larval instars, and the larval stage occupies about 20 days. They feed on
all stages of mealy bugs. Duration of first, second, third and fourth instar grubs are 3-
4, 4, 4-5-7-8 days respectively. Grownup grubs are entirely covered with white wax
strands. When the grub is disturbed, it exudes a yellow fluid from the dorsal surface of
the body for defensive purpose. The prepupal period is 2 to 4 days when it suspends
feeding activities. The pupal period varies from 7 to 9 days. The adult spends about
one day in the pupal case before it emerges. It is covered with a white powder like
substance for a day. The male could be distinguished from the female by the
colouration of first pair of legs. The first pair of legs in the case of male is brown and
the latter two pairs being black, whereas in the female all the three pairs are black.
Male to female ratio is 1:1. Adults are also known to attack and feed the mealy bugs.
Longevity of adults ranges from 50 to 60 days and the fecundity is about 200-220 eggs.

Feeding behaviour

Both adults and grubs are predating almost all stages of the mealy bug.
However the grubs are voracious feeders. The coccinellid grub consumes a total of 900
to 1500 mealy bug eggs in its development. A single grub can eat as many as 30
nymphs or 30 adult mealy bugs. Fourth instar grub is the most voracious feeder of the
mealy bugs. After 15 days of infestation of pumpkins with bugs they are exposed to a
set of 100 beetles for 24 hrs. After exposing, the pumpkin is kept back in a cage as
described for under production of M. hirsutus. The beetle during the period of exposure
feed on mealy bugs as well as deposits their eggs singly or in groups of 4-12. The
grubs are visible in such cages within a week of exposure to beetles. The young grubs
feed on eggs and small mealy bugs but as they grow they become voracious and feed
on all stages of mealy bugs. For facilitating the pupation of grubs dried guava leaves or
pieces of papers are kept at the base of each of the cages. The first beetle from the
cages starts emerging on 30th day of exposure to C. montrouzieri adults. The beetles
are collected daily and kept in separate cages for about 10-15 days to facilitate
completion of mating and pre-oviposition. The beetles are also fed on diet containing
agar powder (1 g), sugar (20 g), honey (40 cc) and water (100 cc). The adult diet is
prepared by boiling sugar in 70 cc of water, adding 1 g agar, diluting 40 cc honey in 30
cc of water and adding to the sugar and agar mixture when it comes to boiling point.
The hot liquid diet is kept on small white plastic cards in the form of droplets which get
solidified on cooling. Such cards containing adult diet can be fed not only to C.
montrouzieri but also to many other species of coccinellids. From each cage about 175
beetles are obtained. The emergence of the beetles is completed within 10 days.
Beetles can also be reared on Corcyra cephalonica eggs but empty ovisacs
of Planococcus citri are to be kept for inducing egg laying by the beetles. The beetles
are also multiplied on semi synthetic diet which is still in the process of further
refinement.

C) Mass production of Chrysoperla carnea

Introduction

In India, 65 species of chrysopids belonging to 21 genera have been recorded from

various crop ecosystems. Some species are distributed widely and are important
natural enemies for aphids and other soft bodied insects. Amongst them, Chrysoperla
carnea is the most common. It has been used in cotton ecosystem for protection from
aphids and other soft bodied insects. C. carnea is now used extensively all over the
country.

Morphology and biology

The eggs are stalked and green in colour. The length of the egg in various species
ranges between 0.7 to 2.3 mm and that of the stalk between 2 to 26 mm. The eggs
are laid singly or in clusters. Eggs turn pale whitish and then black before hatching.
Egg period lasts 3-4 days. The larva is white in colour on hatching. The larva has 3
instars which are completed in 8-10 days. The larva spins a cocoon from which the
adult emerges in 5-7 days. Adults on emergence mate repeatedly. Generally, pre-
oviposition period lasts for 3-7 days. Adult females start laying eggs from 5th day
onwards and peak egg-laying period is between 9-23 days after emergence. The male
longevity is 30-35 days and female can even live up to 60 days. Fecundity is 600-800
eggs/female. The sex ratio Male: Female is 1: 0.85. The adult males and females live
41 and 53 days, respectively.

Production procedure

In mass production, the adults are fed on various types of diets. The larvae are either
reared in plastic tubes or empty injection vials or in groups in large containers or in
individual cells. The adults are collected daily and transferred to pneumatic glass
troughs or G.I. round troughs (30 cm x 12 cm). Before allowing the adults, the rearing
troughs are wrapped inside with brown sheet which act as egg receiving card. About
250 adults (60% females) are allowed into each trough and covered with white nylon or
georgette cloth secured by rubber band. On the cloth outside three bits of foam
sponge (2 sq.in) dripped in water is kept. Besides an artificial protein rich diet is
provided in semisolid paste form in three spots on the cloth outside. This diet consists
of one part of yeast, fructose, honey, Proteinex R and water in the ratio 1:1:1:1. The
adults lay eggs on the brown sheet. The adults are collected daily and allowed into
fresh rearing troughs with fresh food. From the old troughs, the brown paper sheets
along with Chrysopa eggs are removed.

Storage and destalking of eggs

The brown paper sheet kept inside the adult rearing troughs contain large
number of eggs each laid on a stalk or pedicel. As such the sheets are stored at 10oC
in B.O.D. incubator or refrigerator for about 21 days. When the eggs are required for
culturing or for field release the egg sheets are kept at room temperature for a day and
the eggs during this period turn brown and hatch on 3 days later. The first larvae are
either taken for culture or for recycling or for field release.

Group rearing of grubs

It is done in GI round basins (28 cm dia) @ 250 larvae/basin, covered

with khada cloth. The eggs of Corcyra cephalonica is given as feeding material for the
larvae in the laboratory. For rearing 500 Chrysopa larvae the total quantity of Corcyra
eggs required is 22 cc @ 5 cc/feeding for five feedings in alternate days.
The Chrysopa larvae pupate into round white coloured silken cocoons in 10 days. The
cocoons are collected with fine brush and transferred into 1 lit plastic containers with
wire mesh window for emergence of adults. From the cocoons, pale green coloured
adults with transparent lace like wings emerge in 9-10 days.

Individual rearing of grubs

1. In the first step of larval rearing, 120 three day old chrysopid eggs are mixed
with 0.75 ml of Corcyra eggs (the embryo of Corcyra eggs are inactivated by
keeping them at 2 feet distance from 15 watt ultraviolet tube light for 45
minutes) in a plastic container (27x18x6 cms).
2. On hatching, the larvae start feeding. On 3rd day the larvae are transferred to
2.5 cm cubical cells of plastic louvers @ one per cell. Each louver can hold 192
larvae.
3. Corcyra eggs are provided in all the cells of each louver by sprinkling through the
modified salt shaker. Feeding is provided in two doses.
4. First feeding of 1.5 ml Corcyra eggs for 100 larvae and second feeding of 2 ml
for 100 larvae with a gap of 3-4 days is done.
5. Total quantity of Corcyra eggs required for rearing 100 chrysopid larvae is 4.25
ml.
6. The louvers are secured on one side by orgami or brown paper sheet and after
transfer of larvae covered with acrylic sheet and clamped.
7. Orgami or brown paper is used for facilitating pupation and clear visibility of
eggs. The louvers are stacked in racks.
8. One 2m x 1m x 45 cms angle iron rack can hold 100 louvers containing 19,200
larvae.
9. Cocoons are collected after 24 hours of formation (when they get hardened) by
removing orgami or paper from one side.
10. The cocoons are placed in adult oviposition cages for emergence (Adults are
sometimes allowed to emerge in louvers and released on glass window panes
from where they are collected using suction pumps).
 EXERCISE NO. 10

MASS PRODUCTION OF HANPV, SLNPV AND EPN

Introduction

In India, Helicoverpa armigera is of major importance damaging a wide variety of food,

fibre, oilseed, fodder and horticultural crops. The nuclear polyhedrosis virus of H.
armigera (HaNPV) is currently used for the management of H. armigera on chickpea,
cotton, pigeon pea, tomato and sunflower. Mass production of Nuclear Polyhedrosis
Virus (NPV) on commercial scale is restricted to in vivo procedures in host larvae which
are obtained by

 Field collection from cotton, pigeon pea and chickpea – H. armigera

 Mass culturing in the laboratory in semisynthetic diet – H. armiger

Some small scale producers use field – collected larvae for mass production of NPV in
spite of the following constraints.

 Collection of a large number of larvae in optimum stage (late IV / early V instars)

is time-consuming and can be expensive in terms of labour and transportation
costs.
 Wild populations of insects may carry disease causing organisms like
microsporidians, cytoplasmic polyhedrosis virus, stunt virus and fungal pathogens
which will affect both virus production and quality.
 Introduction of wild strains of NPV resulting in quality control problems.
 Transportation of a large number of larvae with cannibalistic behaviour will be a
difficult task.
 Parasitized larvae collected from the field will die prematurely yielding little virus.

Rearing of larvae in the natural host plant will involve frequent change of food at least
once a day during the incubation period of 5-9 days increasing the handling time and
hence the cost. In order to reduce the cost, field collected larvae are released into semi
synthetic diet treated with virus inoculum. Mass culturing of insects in semi synthetic
diet involves high level of expertise, hygiene and cleanliness.

Production procedure
The NPV of H. armigera is propagated in early fifth instar larvae. The virus is multiplied
in a facility away from the host culture laboratory. The dose of the inoculum used is 5 x
105 polyhedral occlusion bodies (POB) in 10 ml suspension. The virus is applied on to
the semisynthetic diet (lacking formaldehyde) dispensed previously in 5 ml glass vials.
A blunt end polished glass rod (6 mm) is used to distribute the suspension containing
the virus uniformly over the diet surface. Early fifth instar stage of larvae are released
singly into the glass vials after inoculation and plugged with cotton and incubated at a
constant temperature of 25oC in a laboratory incubator. When the larvae exhausted
the feed, fresh untreated diet is provided. The larvae are observed for the
development of virosis and the cadavers collected carefully from individual bottles
starting from fifth day. Approximately, 200 cadavers are collected per sterile cheese
cup (300 ml) and the contents are frozen immediately. Depending upon need,
cadavers are removed from the refrigerator and thawed very rapidly by agitation in
water.
Processing of NPV
The method of processing of NPV requires greater care to avoid losses during
processing. The cadavers are brought to normal room temperature by repeatedly
thawing the container with cadaver under running tap water. The cadavers are
homogenized in sterile ice cold distilled water at the ratio 1: 2.5 (w/v) in a blender or
precooled all glass pestle and mortar. The homogenate is filtered through double
layered muslin and repeatedly washed with distilled water. The ratio of water to be
used for this purpose is 1: 7.5-12.5 (w/v) for the original weight of the cadaver
processed. The left over mat on the muslin is discarded and the filtrate can be semi-
purified by differential centrifugation. The filtrate is centrifuged for 30-60 sec. at 500
rpm to remove debris. The supernatant is next centrifuged for 20 min at 5,000 rpm.
Then the pellet containing the polyhedral occlusion bodies (POB) is suspended in sterile
distilled water and washed three times by centrifuging the pellet in distilled water at low
rpm followed by centrifugation at high rpm. The pellet finally collected is suspended in
distilled water and made up to a known volume, which is necessary to calculate the
strength of the POB in the purified suspension.

Production of the nuclear polyhedrosis virus Spodoptera litura

Introduction

 The virus is effective against the pest on several crops.

 The virus is generally propagated in early fifth instar by surface contamination of
semi-synthetic diet.

Production procedure

 The mass production of the NPV is carried out in early fifth instar stage of S.
litura, which yields maximum amount of the NPV. Therefore, in the host culture
laboratory a continuous culture of the insects is maintained with proper handling
procedures.
 The larvae are grown in diet held in 5 ml glass vials. When the larvae reach the
appropriate stage they are transferred to the virus production facility.
 The NPV is multiplied by feeding the semi synthetic diet coated with a clean
inoculum of the NPV that has previously been standardized.
 This is accomplished by placing aliquots of 10 ml of the viral suspension of
concentration 1 x 108 Polyhedral Occlusion Bodies (POB) in the centre over the
diet surface either in glass vials and spreading the suspension uniformly all over
the surface with a polished glass rod.
 Larvae are released singly after 15 min. into each glass vial/cell and incubated at
25oC for 10 days.
  The larvae begin to die from 5th day onward. The cadavers are collected
individually and transferred to 500 ml plastic containers and frozen immediately
until processing.
 The processing method is similar to that of H. armigera.

Mass production of entomopathogenic nematodes (EPN)

Entomopathogenic nematodes of the genera Heterorhabditis and Steinernema are

commercially used to control pest insects. They are symbiotically associated with
bacteria of the genera Photorhabdus and Xenorhabdus, respectively, which are the
major food source for the nematodes.

Life cycle:

• Bacteria colonizes inside the infective juveniles (IJs)

• IJs live in the soil until they invade a susceptible insect host, seeking the
hemolymph

• IJs release the bacteria into the insect’s hemolymph

• The bacteria participate in overcoming the insect’s defense system and killing
the host

• Nematode growth and reproduction take place

• Nutrient limitation in the host insect cadaver

• Non-feeding IJ stage emerges into the soil to forage for a new host
 EXERCISE NO. 11

MASS PRODUCTION OF VERTICILLIUM /BEAUVERIA/ METARHZIUM/NOMURAEA/

PAECILOMYCES /HIRSUTELLA THOMPSONI/TRICHODERMA

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

MASS PRODUCTION OF BEAUVERIA

MASS PRODUCTION OF METARHZIUM

Mass production of Trichoderma viride

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.
 EXERCISE No. 12

MASS MULTIPLICATION OF BGA AND AZOLLA AND ITS APPLICATION IN PADDY

FIELD

Object

1. To know the method for large scale production of blue green algae as a biofertilizer for
use 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).

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.

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.
 EXCERCISE NO. 13

METHODS OF APPLICATION OF BIOFERTILIZERS, BIOPESTICIDES AND

BIOAGENTS

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 one
acre land.

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

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

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

follows

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

3. Soil treatment: when biofertilizer application to seeds or seedlings is not

possible, 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 24
hours 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, preferably
during morning and evening hours

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

2. Sett inoculation:

i. The setts or tubers are immersed in liquid biofertilizers for 20 minutes and
are planted in the field

3. Seedling treatment:

i. Roots of seedlings are dipped in biofertilizer solution for 8-10 min and
transplanted immediately

ii. The liquid bioinoculant (500 ml) is sufficient for seedling treatment for 1
acre area.

4. Soil treatment:

i. Seed treatment with liquid bioinoculant is not possible then 2 lit liquid
bioinoculant 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 with
50 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

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 this
solution on sugarcane plant preferably in the evening

8. Drip irrigation :

i. Two liters of liquid biofertilizer is given through drip irrigation for 1 acre
area

METHODS OF APPLICATION OF BIOPESTICIDES

METHODS OF APPLICATION OF BIOCONTROL AGENTS