11. MASS PRODUCTION OF BIOCONTROL AGENTS
11. MASS
PRODUCTION OF BIOCONTROL AGENTS
A.
Egg parasitoid- Trichogramma sp
Mass production of egg
parasitoid Trichigramma sp consists of two steps.
a. Mass culturing of the
facticious host Rice moth, Corcyra
cephalonica
b. Trichogramma egg card production.
a) Mass culturing of Rice
moth, Corcyra cephalonica
v Diet mixture: 2.5 Kg of
Cumbu(Broken) + 100 g Ground nut+ 5 g yeast+ 5 g Sulphur+ 0.05% Streptomycin sulphate.
v
Transfer
to a sterilized plastic container.
v Inoculate 1cc of Corcyra
eggs and cover with gaddha cloth.
v Life
historyC.cephalonica : Egg stage- 3 to 4 days;
Larval stage- 25 to 30 days; Pupal stage- 8 to 11 days;
Adult life span 7 to 10 days.
v Collect adults 35-40 days
after inoculation by using vacuum collector.
v Transfer the adults to the
mating cage and provide adult food.
v Collect eggs and clean
with sieve or egg separator.
b) Trichogramma egg card production
v Sterilize the Corcyra eggs under UV radiation.
v Smear 50% gum solution on
pre-punctured cards.
v Sprinkle sterilized rice
moth eggs on the card.
v Place it in a polythene
cover after drying.
v Inoculate nucleus card of Trichogramma sp with adult food (Honey
solution+Vitamine E).
v Adult emerges out from the
nucleus card and parasitize the sterilized rice moth eggs.
v Egg card may be tied in
the field or stored at 10°C for 20 days.
B) Mass production of Predator: Green lacewing- Chrysoperla cornea
v Place 250 chrysoperla eggs
in a plastic container with 1 CC of Corcyra eggs.
v Provide 2.5 CC of Corcyra eggs once in 2 days after hatching.
v Collect white cocoons and transfer to wooden box with black cloth cover.
v Provide adult food 50% honey solution.
v Change black cloth from 5th day; continue upto 30-40 days.
v Collect eggs and store at 10° C for field release or recycling.
v Provide 2.5 CC of Corcyra eggs once in 2 days after hatching.
v Collect white cocoons and transfer to wooden box with black cloth cover.
v Provide adult food 50% honey solution.
v Change black cloth from 5th day; continue upto 30-40 days.
v Collect eggs and store at 10° C for field release or recycling.
C) ENTOMOPATHOGENS
Fungi
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Green muscardine
fungi
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:
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Metarhizium
anisopliae
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White muscardine
fungi
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:
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Beauvaria
bassiana
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White halo
fungi
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:
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Verticillium
lecanii
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MASS
PRODUCTION OF GREEN MUSCARDINE FUNGUS, METARHIZIUM
ANISOPLIAE
Introduction
Fungi
represent a diverse group of insect pathogens.
The insects attacked by the fungus die shortly after the fungus begins
to develop in the haemocoel. The
rhinoceros beetle, Oryctes rhinoceros is
one of the serious and important pests of coconut and has a wide distribution
and persistent occurrence in all the coconut growing areas in the country. The
colony of M. anisopliae appears white
when young, but as the conidia mature, the colour turns to dark green. The conidiophores are branched, and the initial
conidium is produced at the distal end of the conidiophores.
Production procedure
The
fungus can be mass produced in conventional laboratory media as well as on
crushed maize grains, etc. The cheapest
media till date known are (1) Cassava chips mixed with rice bran supplemented
with urea or fish meal extract, and (2) Coconut water wasted from copra making
industry or (3) carrot broth.
On coconut water
Coconut water (40 ml) contained in
375 ml side wise flat liquor bottles plugged with cotton plug are sterilized in
batches of 9-10 bottles in 12 liter pressure cooker for 20 minutes at 15
psi. The bottles are inoculated with 1
ml suspension containing spores of the fungus with the help of a sterile
injection syringe. Before inserting the
needle within the sterile bottles for drawing spore suspension for inoculation,
the needle of the syringes and collar region of the bottles are flamed (Bunsen
burner). The bottles are inoculated in a
laminar flow chamber and rested on flat surface for 2 days or till the surface
of medium is fully covered by the olive green sporulated fungus. The whole culture is crushed thoroughly in
an ordinary mixer and used in the field.
From a single average size coconut 5-6 bottles of cultures can be made.
In Carrot broth
MASS PRODUCTION OF THE FUNGUS, VERTICILLIUM LECANII
Introduction
Vertilcillium
lecani commonly called as white halo
fungus is found sprouting on the body of coffee green scale Coccus viridis. It has been extensively studied against the
pest in Lower Palany Hills in Tamil Nadu.
The fungus is known to cause epizootic when the environmental conditions
are favourable.
Production
procedure
MASS
PRODUCTION OF THE FUNGUS, BEAUVERIA
BASSIANA
Introduction
The
fungus is otherwise called as white muscardine fungus. The fungus spores and mycelia are milky white
and found sprouting on the body of lepidopterous insects like Helicoverpa armigera, Spodoptera litura.
Production procedure
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 B. bassiana spores are transferred aseptically. The flasks are incubated at room
temperature. The spores are obtained in
a fortnight.
MASS PRODUCTION OF NUCLEAR
POLYHEDROSIS VIRUS –
Sl NPV and Ha NPV
Baculovirus group have a very narrow host
range and generally infests the larvae of crop pests. The research aimed at
insect pest control is, therefore, confined to nuclear polyhedrosis viruses
(NPVs) and granular viruses (GVs). NPV
is a nucleic acid (double standard, circular DNA) enclosed in protein matrix,
hence it is called polyhedral occlusion body (POB). NPV infects the nucleus of
the cell and multiplies within the nucleus. In India, extensive research has
been conducted on the use of NPVs for tackling two major pests namely Spodoptera litura and Helicoverpa armigera.
v Nuclear Polyhedrosis viruses
like Ha NPV, SINPV are increasingly being used as alternatives to chemicals.
v These viruses are highly
specific and do not affect beneficial insects like parasitoids and predetors
and
v They are safe to fish, birds,
animals and man.
Mass production of
virus involves three steps viz., mass
culturing of host insect, virus production and virus purification.
Diet
preparation for mass production of the host insects- Tobacco caterpillar and Gram pod borer
The larvae of Tobacco caterpillar and Gram
pod borer can be multiplied by using chick pea based semi-synthetic diet. The
composition of the diet for rearing larvae is as follows.
390 ml of water is mixed with fraction 'A'
of the diet in the blender which is run for two minutes. Fraction 'A' and 'C'
are mixed and the blender is run again for 1 minute. Fraction 'B' is boiled in
the remaining 390 ml water, added to the mixture of A and B and the blender is
run for a minute. Formaldehyde solution is added at the end and the blender is
again run for a minute.
Mass
culturing of host insects:
a) Mass
production of Tobacco caterpillar (Spodoptera
litura)
The culture of
Tobacco caterpillar is initiated by collecting eggs from the fields of castor,
cauliflower, lucerne, tobacco etc. Once the pure culture is established the
mass production is commenced under laboratory conditions after the first
generation established.
Pairs of newly
emerged moths of Tobacco caterpillar are placed in well ventilated plastic
containers. The inner wall of the containers is lined with paper to enable the
adults to lay eggs. The bottom of the container is lined with sponge covered
over by blotting paper. The moths are provided with 50% honey solution and
water on two cotton swabs placed in small plastic cups.
The eggs which are
generally laid in batches on the paper are cut out. The freshly laid eggs can
also be surface sterilized in 0.05 percent solution of sodium hypo chlorite for
5 minutes. These eggs are washed several times in running tap water to remove
the traces of sodium hypo chlorite. The surface sterilised eggs are kept in
plastic tubes (7.5 x 25 cm) on moist tissue paper for continuing the stock
culture.
After 3 days, the
newly hatched larvae are transferred to bouquets of castor leaves and kept in a
plastic container or can be multiplied on a chickpea based semi-synthetic diet
composition.
The pupae are collected 3 days after all the
larvae enter the sand. The pupae are sexed and kept in adult emergence cage.
After 10 days, freshly emerged males and females are collected from their
respective emergence cages.
b)
Mass production of Spodoptera
litura NPV (SI NPV).
For SlNPV
production, the synthetic diet prepared is poured at 4gm/cell in the
multi-cavity trays and the diet surface is uniformly sprayed with virus
prepared in distilled sterilised water at 18 x 106 POBs / ml. Eighty
percent of the total 5-7 day old larvae are utilised for SINPV production.
The trays are
incubated at 260 C for 7 days. In case of virus infected larval
trays, the diseased larvae dies after attaining its maximum size of 6th instar,
where the dead caterpillar will have 2-6 billion poly occlusion bodies (POB)
which is in terms of larval equivalent (LE).
1 LE of H.armiegera NPV = 6 x 109 POBs; 1 LE of S.
litura = 2 x 109 POBs.
The dead larvae have to be
harvested, macerated in distilled/sterilised water and filtered through muslin
cloth to get the crude suspension of the virus.
a)
Mass production of Gram pod borer (Helicoverpa armigera)
The culture of Gram borer is initiated
either collecting the adults with the help of light traps. It could be by
collection of larvae on a large scale from its host crops in endemic areas.
Nucleus culture can also be obtained from the established laboratories.
The
adults are kept in an oviposition cage with a cloth enclosing the frame. A
circular plastic mesh (on which cotton swabs soaked in water and honey solution
are placed in small containers) rests on a support above the base of the frame.
The cloth cover is open at both ends with a 20 cm vertical slit in the centre
which can be closed with a zip or cloth clips. The cloth cover enclosing the
frame is tied with rubber bands at both ends. It is placed on tray with a
sponge at the bottom soaked in water. The temperature inside the cage is
maintained at 260 C and humidity at 60 - 90%.
The eggs are laid all over the inner
surface of the cloth cover. The egg cloth is removed daily. This cloth is
surface sterilised in 10% formalin for 10 minutes, the eggs could also be
surface sterilised using 0.2% sodium hypchlorite solution for 5-7 minutes and
treated with 10% sodium thiosulphate solution to neutralise the effect of
sodium hypo chlorite, rinsed in distilled water. The eggs are later placed on
paper towell under laminar flow for drying. The dried cloth pieces containing
eggs are kept in 2 litre flasks containing moist cotton. Flasks are plugged
with cotton wrapped in muslin cloth and the bottom of the flask is wrapped with
aluminium foil.
The larvae are removed from the top of the
aluminium foil wrapped flasks with a brush and then transferred to the diet.
Larvae are transferred to plastic containers or sterilised glass vials or multi
cavity tray contains diet. In host culture units, larvae start pupating when
they are 18-19 days old and the pupation will be over within 2-3 days. The
harvested pupae are surface sterilised using 0.2% sodium hypo chlorite solution
followed by washing with 10% sodium thiosulphate solution to neutralize sodium
hypo chlorite and then washed thoroughly with distilled, sterilised water.
After washing, the pupae are dried by rolling over blotting paper. The male and
female pupae are separated out and placed over moist sponge in adult emergence
cages.
The egg, larval, pupal and adult stages of
gram borer last 3-4, 18-29, 7-8 and 7-9 days respectively. The oviposition
period of the females is about 5 days.
b)
Mass
production of HaNPV
1 LE of H.armigera NPV = 6 x 109 POBs;
Directions
for use of NPV
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