Production technology of bio agents

1. Trichogramma
Production Technology for Bio-agents and Bio-fertilizers
ENT-422 10(0+10)
Submitted To:-
Dr. Kanchan G. Padwal
Assistant Professor(Entomology)
Institute of Agricultural Sciences,BHU

2. What is Biocontrol ?
◦ Biological control or biocontrol is a method of controlling pests such
as insects, mites, weeds and plant diseases using other organisms.
◦ There are three basic strategies for biological pest control:
◦ Classical (importation) – Where a natural enemy of a pest is introduced in the hope of
achieving control.
◦ Inundative (augmentation) – In which a large population of natural enemies are administered
for quick pest control.
◦ Inoculative (conservation) – In which measures are taken to maintain natural enemies through
regular re-establishment.

3. TRICHOGRAMMA
◦ Trichogramma is a genus of minute polyphagous wasps that are endoparasitoids of insect
eggs.Trichogramma is genera from the family Trichogrammatidae, with over 200 species
worldwide.
◦ Trichogramma wasps are small and very uniform in structure, which causes difficulty in
identifying the separate species. As females are all relatively similar, taxonomists rely upon
examination of males to tell the different species apart, using features of their antennae
and genitalia.
◦ Trichogramma spp. have been used for control of lepidopteran pests for many years. They
can be considered the Drosophila of the parasitoid world, as they have been used for
inundative releases
◦ Play important role in pest suppression programme by destroying the early stage of the
pest.

4.  More than 150 species of Trichogrammatids are distributed throughout the world
parasitizing eggs of over 200 insect species.
 In India, 26 Trichogrammatids are recorded; out of which,
1.Trichogramma chilonis (Ishii)
2. T. japonicum (Ashmead) and
3. T. acheae
4. T. brasiliensis are of significant importance.
 Worldwide over 32 million ha area under agricultural crops and forest are treated annually
with different Trichogramma species in 19 countries.
 Mostly in China and Republic of Soviet Union (Li, 1994).
 Their levels of parasitism sometimes reach to 100 percent depending upon the
environmental conditions.

5. CLASSIFICATION
◦ Domain: Eukaryota
◦ Kingdom: Metazoa
◦ Phylum: Arthropoda
◦ Subphylum: Uniramia
◦ Class: Insecta
◦ Order: Hymenoptera
◦ Family: Trichogrammatidae
◦ Genus: Trichogramma

6. • Mainly for lepidopteran pests, in sugarcane, corn,cotton,
vegetables and fruits crops pests.
T.chilonis
• Rice stem borer
T.japonicum
• For lepidopteran pests on Chickpea and Pigeonpea.
T.brasiliensis
• For avocados, ornamentals, orchards and grape pests
T.platneri

7. • For ornamentals, orchards, grapes pests and treeswhere the protective zone is
five feet or more above ground level.
T.minutum
• For vegetable, orchards, gardens and field crops pests.
T.brassicae
• Lepidopterous pests on cruciferous crops.
T.dendrolimi
• Lepidopterous pests
T.evanescence
• For cotton pests
T.exiguam
• For citrus pests
T.papilionis

8. Biology and Life Cycle
◦ The development of all Trichogramma spp. is
very similar.
◦ The female wasp inserts its ovipositor through
the chorion and deposits its eggs within the egg of
the host.
◦ It avoids super parasitism or multiple-parasitism
under natural conditions.
◦ During the 3rd instar, dark melanin granules
are deposited on the inner surface of the egg
chorion, causing the host egg to turn black.

9. How does Trichogramma actually Parasitise on other host egg
Immediately after they hatch, female Trichogramma wasps begin their
search for the egg of a 'target species’.
 Once host egg is found – through pheromones and visual cues.The wasp
drills a hole into the egg and deposits two to three eggs of her own.
How many of their own eggs to lay, is calculated by timing walks across
moth egg surfaces.
 In a mere 24 hours, the wasps eggs hatch within the host egg and the
larvae consume the yolk and embryo.
The larvae then undergo three instars before a pupae stage.

10.  They emerging about nine days after they were initially laid
within the host egg (Knutson 1998).
 Trichogramma larvae eat out the insides of pest eggs,
pupate, and cutan exit hole in moth egg shells for winged
adults to squeeze through.
 Males emerge first, wait for females, and immediately
mate. The life cycle from egg to adult is completed in 7 to
10 days (longer in cool weather).
 Short life cycle -30 generations per season, and rapid
population increase. Hence, early season releases produce
large populations positioned to fight pest invasions.

12.  Trichogramma wasps are used worldwide as highly effective
biocontrol agents.
 Female wasps lay their eggs inside the eggs of a host insect,
usually eggs the pest lays on plants, adult wasps emerge7-10
days later.
 Pest eggs parasitized by trichogramma wasps turn black.
 No pest larvae will emerge.

13. Corcyra Rearing and mass production
Step 1 – Preparation of medium:
 Heat sterilized broken grains is taken at the
rate of 2.5 kg per plastic basin to which
groundnut kernel powder is added @ 100 gm
/basin and yeast powder @ 5g/basin.
To prevent bacterial infection in the food
medium Streptomycin sulphate @ 0.05 %
spray is given at 10—20 ml/basin using a
hand-operated sprayer or atomizer.

14. Step 2 – Inoculation of Corcyra eggs:
 Nucleus culture of Corcyra eggs were added @ 0.5 CC
/ basin containing 2.5 kg of grain medium. After
uniform mixing of the contents of the basin it was
covered with khadi cloth and secured by twine.
 The young Corcyra larva that hatch out from the egg in
3-4 days feeds on the medium by constructing webs.
 The adult Corcyra starts emerging from the medium
from 30-35 days onwards and continues to emerge up
to 90 days after inoculation of the eggs due to
staggered development of larvae in the medium.

15. Step 3 – Collection of moths for mating:
 The emerged adult Corcyra moth rest on the inner surface of the
cloth cover.
 They are to be collected in the morning hours using glass specimen
tubes (15x 2.5 cm) or a specially designed modified vacuum
aspirator.
 The moth collection is effectively done by keeping the basin inside a
mosquito net so that the moth cannot escape.
 The adult moths are transferred to a specially designed mating
drum made of G.I sheets with wire mesh at the bottom.
 Adult moths are provided with honey solution (50 %) added with
vitamin E (1 capsule per 20 ml of 50% honey) to boost the vigor of
the adult and to get higher quantity of healthy eggs.
 The adult food is given by dipping cotton swab and allowed to hang
inside the drum with a thread.

16. Step 4 – Collection of Corcyra eggs:
Corcyra eggs are loosely laid and they
are collected through the wire mesh at
the bottom on a receiving container with
funnel setup on an enamel tray.
Eggs are to be collected daily for 4 days
from each drum. On the fifth day it is to
be vacated and cleaned.
The eggs are cleaned and separated from
the moth’s scales.

17. Step 5 – Cleaning of eggs:
• The eggs thus obtained are further
cleaned with the help of plastic
sieves of different meshes.
• One CC of Corcyra eggs contains
approximately 18,000 eggs. About
100 pairs of Corcyra moth (50 %
female) produce 1.5 cc of eggs
during their egg laying period of 4
days.
•
• From each Corcyra rearing basin an
average of 2,500 moths emerge.

19. Precautions while rearing Corcyra
 Bacterial disease some time attacks Corcyra culture. To control this, streptomycin
sulphate is added to the crushed sorghum @0.23gm/kg and mixed thoroughly.
 Occasionally the mite Pyemotes ventricosus (Newport) contaminate the culture
and affect egg laying and larval development of Corcyra. in case of infestation the
racks, cages, boxes etc. should be disinfected with formalin and placed in the sun
for six hours.
 In case of severe infestation, acaricide dicofol (Kelthane) can be applied. For
this,muslin sheets are dipped in a 0.05% solution of dicofol and air dried for
couple of hours.
 Control of Bracon hebetor infestation: A plastic tub filled with water near racks
containing Corcyra boxes and keeps a table lamp with 60 or100 W bulb.
 The light of the table lamp should face the surface of water

20.  The trap should be operated daily and side by side the culture should also be
replaced with fresh stock.
 The boxes should be sterilized by spraying 1% formaldehyde or sodium
hypochlorite solution and sun dried for 6 hours.
 Care should be taken to remove host/parasitoid cocoons from boxes.
 The feed material jowar should also be sterilized. Mix all the ingredients in
separate room and charge with Corcyra eggs. Close the lid and these sterilized
boxes should not be open for 40 days and kept in separate room. By this method,
Bracon can be eliminated from the culture.
 In case of labs having separate Corcyra rearing rooms, on one corner disinfect
all boxes one by one and keep them in one room. All the Bracons in previous
room can be killed by spraying low concentration of any pesticide. All the
windows and doors can be kept open for 15 days.

21.  Thereafter, sprinkle whole room with 1% formaldehyde (40%) solution. After about
10 days bring back whole culture to same room. Thereafter, regularly keep light-
water trap to monitor Bracon.
 If we found discoloured larvae and white cocoons, then they are collected and
immersed in 0.5% formaldehyde to kill the parasitoids.
 Provide windows with fine iron mesh, wire mesh and double door to avoid any entry
of parasitoids Scales of laboratory host C. cephalonica may cause the respiratory
problems; it is advised to wear a mask while rearing C. cephalonica.
 Trichocard should be properly sealed before keeping in refrigerator to avoid any
type of accident.

22. Trichocard preparation
Materials required
Corcyra eggs, Nucleus culture of
Trichogramma , Polythene bags,
Rubber bands, Scissors,
Gum, Brush, Mesh sieve, Tricho
cards, 50% honey solution, Stapler,
Refrigerator/fridge
and UV lamp/LED light.

23. Preparation of Trichocard
Step 1 – Preparation of “Tricho cards”:
The Corcyra eggs collected from the mating drum are cleaned and finally taken
in glass petri dishes (15-20 cm dia).
◦ The eggs were sterilized by exposing to UV light (15 W) in a closed chamber
for ½ hr. duration so that the embryo may be killed without damaging the egg
contents.
◦ The UV sterilized eggs were sprinkled on drawing board cards (30 x 20 cm)
smeared with thin layer of diluted gum, at 6.0 CC of egg/ card.
◦ The card was already drawn with lines to separate 30 rectangles (7 x 2 cm) to
accommodate 6.0 CC eggs.

24. Step 2 – Inoculation of parasitoids:
The cards pasted with Corcyra eggs were dried under fan and taken into
polythene bags (45x 30 cm) containing nucleus parasitized cards at 6: 1 (fresh eggs
to parasitized eggs) for exposure.
◦ The parasitoids emerging from the nucleus parasitoid eggs start parasitizing the
fresh Corcyra eggs.
◦ After 2 days exposure the cards were kept for another 1-2 days, during which
the parasitized cards were then stored in refrigeration at 10 0 C for 21 days.
◦ The parasitoids under normal room temperature emerge in 7 days where as they
emerge in 2 days after removing from storage for field use.

25. A
• The parasitisation of Trichogramma spp., in laboratory condition on one cc eggs of
Corcyra cephalonica, which are uniformly spread and pasted on a card measuring 15
cm x 10 cm is called as Tricho card. The card has 12 demarcations (stamps).
B
• Apply gum on the card and sprinkle the cleaned eggs uniformly in a single layer
with the aid of a tea strainer
C
• The excess eggs pasted are removed by gently passing a shoe brush over the card
after sufficient air drying under fan.
D
• Treat the eggs under UV lamp for 30 minutes to kill the embryo (at present
price of UV light in market is Rs. 1,121)

26. E
• Take polythene bag, insert UV treated “Trichocard” and nucleus
card at the ratio of 6:1 (6 Corcyra egg cards: 1 Trichogramma nucleus
card) and provide 50%honey with vitamin E in a soaked cotton swab.
F
• Remove the Tricho cards after 24 hours. On fourth day, the Corcyra
eggs changes to black in colour and indicates the parasitization of
eggs.
G
• Release the tricho cards in the fields when at least 5 % adult
emergence (pharate stage) is observed. During each release
trichocards should be cut into 12 or 16 bits and staple to the lower
side of leaf of plant during morning or evening hours.

27. ◦ Field release: Trichocards can be stapled inside plastic tea cup and the plastic tea
cup can be tied to the leaf or shoot of the crop in an inverted manner to
safeguard the eggs from adverse climatic conditions and hyperparasitism.
◦ The small rectangular cards were used in the field @ 5 cards /acre for sugar
cane and @ 12 cards / acre for cotton.
◦ The dose being 1.0 CC and 2.5 CC /acre respectively. The cards were tied in
different spots avoiding border rows.
◦ For sugarcane 6 releases are to be made at fortnight intervals commencing
from 3- 3 ½ months onwards.
◦ For cotton crop the parasitoids are released soon after noticing the adult
moths of bollworms flying in the field or trapped in pheromone trap or light
traps.
◦ Two or three releases at fortnightly intervals are needed for cotton.

29. Precautionary measure
1. Emergence date should be specified on the cards to guide the user.
2. The cards should be stapled on the inner side of the leaves to avoid direct
sunlight.
3. The cards should be stapled in morning hours and just before emergence to
avoid predation.
4. Avoid application of insecticides in the field where Trichogramma are released.
5. If need arises uses selective/safer insecticides.
6. Ensure that insecticides are used 15 days after or before Trichogramma release.

30. DOSAGE

32. Maintenance of Trichogramma quality :
The following practices may be followed to maintain the quality of
Trichogramma:
1. Production colonies should be periodically replaced with
individuals from a stock culture maintained on the natural or
target host.
2. Producers should also periodically assess the per cent host egg
parasitization, adult emergence and the sex ratio of emerged
adults to be sure they are within acceptable standards.

33. Reference
◦ https://en.m.wikipedia.org/wiki/Trichogramma
◦ https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/trichogramma
◦ https://agritech.tnau.ac.in/crop_protection/crop_prot_bio_mass_parasitoids7.html
◦ https://agritech.tnau.ac.in/crop_protection/crop_prot_bio_mass_parasitoids1.html
◦ http://www.vsisugar.com/india/agriculture
◦ http://www.slideshare.net/
◦ http://www.insectary.com/

34. THANK YOU