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Prepared by
Mr. Mahesh Vikram Sable
Assistant Professor
Department of Plant Pathology
College of Agriculture, Ashti
Mass Production of
HaNPV and EPN
Contents
ProductionProcedureofNuclear Polyhedrosis
virusof Helicoverpa armigera
• Material Requirement
1. Nucleus culture of HaNPV
2. Culture of H. armigera
3. Natural food or gram flour based artificial diet for H. armigera
4. Small plastic bottles or tube with lid (50 ml)
5. Muslin cloth
6. Cotton
7. Honey solution
8. Haemocytometer
9. Research microscope
10. Disinfectant – Formalin, Sodium hypochloride, teepol
11. Centrifuge machine
Mass culture of Host insect H. armigera
• Collect the larva of H. armigera from chickpea and pigeon pea
plot at pod formation stage.
 Rear individual in plastic tubes and fed with natural food or
artificial diet.
 Full grown larvae undergo pupation.
Collect such pupae and place them on sawdust / fine soil with
blotting paper in glass jar or rearing cage for moth emergence.
After a week, adult start emerging, collect them and introduce
in an oviposition cage.
• The adult provided with 10 % honey solution during oviposition period.
• The black cloth is wrapped from the inner side of the oviposition cage to
facilitate the egg laying.
• The cage is raised in water filled aluminium tray dipping lower side of cloth
I water to avoid the climbing ant.
• After two days unwrap the black cloth having H. armigera egg (which are on
inner side of cloth). Take care that the moths in the case shall not fly away.
• The black cloth should be kept in 10 % formalin solution for disinfection for
10 minute and then in running tap water to wash the trace of sodium
hypochloride.
• Put the cloth with egg in 2 lit sized plastic jar for incubation.
• The newly hatched larvae are collected in plastic tube and
could be reared in mass up to 2 – 3 days on gram flour based
artificial diet.
• Thereafter, separate and keep them in individual plastic
container providing piece of artificial diet.
Preparation of Artificial Diet
Part I
• Gram flour 140 g, formaldehyde 40 % 1 ml, Yeast powder,
Ascorbic acid 4.3 g, Sorbic acid 1.6 g, Streptomycin sulphate
0.3 g, Methyl Para hydroxyl benzoate 2.6 g, Multi vit. capsules
0.3 g, distilled water 400 ml
Part II
• Agar agar 17 g, Distilled water 450 ml.
Procedure
Blend all the ingredient of part I in a mixture pot.
Boil agar agar in 2 lit. sized container and cool up to 600 C,
pour the ingredient of part I and at last the vitamin capsules.
Stir well the whole mixture.
Hurriedly pour the semisolid diet 40 * 25 * 5 cm sized
aluminium tray.
 Allow the diet contamination smear 10 ml formulated HaNPV
on the diet surface with the help of brush. Store unused diet in
refrigerator.
 Grow the larvae on un contaminated diet up to 9 days age.
Keep 20 % larval stock for further breeding and use 80 %
larvae for HaNPV production.
Propagation of NPV
• Select the 3rd or early 4th instar larvae of H. armigera for virus
inoculation and keep individually in plastic tubes. The virus
could be inoculated through different ways into larval body.
• Leaf – dip method
• Head dip method
• Diet contamination method
Diet surface contamination method
Starve these larvae for 6-8 hrs.
Prepare artificial diet and contaminate it smearing HaNPV
formulated suspension@ 10 ml/litter.
Then introduce a piece of the virus – contaminated diet in each
plastic container with starved larva.
The larva feeding on the diet facilitates ingestion of virus.
Replace uneaten part of diet after 2-3 day and simultaneously clean
or change the container of larvae.
the larvae begin to show symptom of virus infection after about 5
days and start dying between 6 to 7 days
Dead larvae become loose and emit brownish black viral ooze on
slight pressing.
Collect the dead diseased larvae in distilled water and purity for 10
days period
Filter this suspension through double layered muslin cloth and
centrifuge for 3 to 5 min at 200 rpm speed.
Pellet settled at the bottom may be discarded and the supernant is
again centrifuged at 2500 rpm for 10 to 15 min.
Hence supernant is discarded and the pellets settled at the bottom
may be responded in little quantity of distilled water.
Mix 10 times water in the purified virus. Counting POBs under
microscopic using haemocytometer may assess the strength of
polyhedral suspending in unit quantity of water
For this fill the squares of the haemocytometer releasing few
drops of HaNPV suspension between cover slip and marked
portion.
Wipe out excess suspension with tissue or sponge piece.
Observe under research microscope
 The minimum count is required 1 * 109 POBs/ml. fill the
convenient sized bottles, label properly indicating at least
brand name of product, POBs / ml, manufacture, direction for
use, use before date etc. store in cool place. Normally HaNPV
preparation is virulent up to 4 to 5 year.
Entomopathogenic Nematodes (EPN)
 Entomopathogenic Nematodes are a group of nematode
(Thread worm), causing death to insect.
 EPN live parasitically inside the infected insect host and so
they termed as endoparasitic.
 EPN have been found in all over the world and a range of
ecologically diverse habitats.
 They are highly diverse, complex and specialized.
They infect many different type of insect living in the soil like
the larval form of moth, butter flies, and beetles as well as
adult form of beetles, grass hoppers and crickets.
Productionprocedure ofNeoplectinaspp.
(DD-136)Nematode
• Three host insects can be used for mass multiplication of EPN
1. Galleria mellonella
2. Corcyra cephalonica
3. Helicoverpa armigera
• But G. mellonella larvae has been found to be susceptible for
EPN
White trap method of mass production of EPN
• Larvae are inoculated with EPN on petri dish lined with filter
paper.
• After 2-5 days infected larvae are transferred to the white trap.
• In this method two glass dishes, one big and one small is used.
One big dish is kept in a normal position and another small is
kept in a inverted position inside it. One whatmans filter paper
No. 1 is kept on this prepared trap. A distilled water is poured
in this white trap.
• The nematode migrate into the water which is then sedimented
of 2000 million juveline per 3 kg bag be obtained at relatively
low cost.
• To harvest of nematode, the sponge culture is placed in open
tray and just covered with water
• The cadavers rest on inverted watch glass, surrounded by
water.
• The central dish containing the cadavers provide a moist
surrounding for the EPN emergences from cadaver.
• New progeny of infective juveniles that emerge from cadaver
migrate to the surrounding water where they are trapped and
subsequently harvested.
Mass producation HaNPV & EPN

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Mass producation HaNPV & EPN

  • 1. Prepared by Mr. Mahesh Vikram Sable Assistant Professor Department of Plant Pathology College of Agriculture, Ashti
  • 4. ProductionProcedureofNuclear Polyhedrosis virusof Helicoverpa armigera • Material Requirement 1. Nucleus culture of HaNPV 2. Culture of H. armigera 3. Natural food or gram flour based artificial diet for H. armigera 4. Small plastic bottles or tube with lid (50 ml) 5. Muslin cloth 6. Cotton 7. Honey solution 8. Haemocytometer 9. Research microscope 10. Disinfectant – Formalin, Sodium hypochloride, teepol 11. Centrifuge machine
  • 5. Mass culture of Host insect H. armigera • Collect the larva of H. armigera from chickpea and pigeon pea plot at pod formation stage.
  • 6.  Rear individual in plastic tubes and fed with natural food or artificial diet.  Full grown larvae undergo pupation.
  • 7. Collect such pupae and place them on sawdust / fine soil with blotting paper in glass jar or rearing cage for moth emergence. After a week, adult start emerging, collect them and introduce in an oviposition cage.
  • 8. • The adult provided with 10 % honey solution during oviposition period. • The black cloth is wrapped from the inner side of the oviposition cage to facilitate the egg laying. • The cage is raised in water filled aluminium tray dipping lower side of cloth I water to avoid the climbing ant. • After two days unwrap the black cloth having H. armigera egg (which are on inner side of cloth). Take care that the moths in the case shall not fly away. • The black cloth should be kept in 10 % formalin solution for disinfection for 10 minute and then in running tap water to wash the trace of sodium hypochloride.
  • 9. • Put the cloth with egg in 2 lit sized plastic jar for incubation. • The newly hatched larvae are collected in plastic tube and could be reared in mass up to 2 – 3 days on gram flour based artificial diet. • Thereafter, separate and keep them in individual plastic container providing piece of artificial diet.
  • 10. Preparation of Artificial Diet Part I • Gram flour 140 g, formaldehyde 40 % 1 ml, Yeast powder, Ascorbic acid 4.3 g, Sorbic acid 1.6 g, Streptomycin sulphate 0.3 g, Methyl Para hydroxyl benzoate 2.6 g, Multi vit. capsules 0.3 g, distilled water 400 ml Part II • Agar agar 17 g, Distilled water 450 ml.
  • 11. Procedure Blend all the ingredient of part I in a mixture pot. Boil agar agar in 2 lit. sized container and cool up to 600 C, pour the ingredient of part I and at last the vitamin capsules. Stir well the whole mixture. Hurriedly pour the semisolid diet 40 * 25 * 5 cm sized aluminium tray.
  • 12.  Allow the diet contamination smear 10 ml formulated HaNPV on the diet surface with the help of brush. Store unused diet in refrigerator.  Grow the larvae on un contaminated diet up to 9 days age. Keep 20 % larval stock for further breeding and use 80 % larvae for HaNPV production.
  • 13. Propagation of NPV • Select the 3rd or early 4th instar larvae of H. armigera for virus inoculation and keep individually in plastic tubes. The virus could be inoculated through different ways into larval body. • Leaf – dip method • Head dip method • Diet contamination method
  • 14. Diet surface contamination method Starve these larvae for 6-8 hrs. Prepare artificial diet and contaminate it smearing HaNPV formulated suspension@ 10 ml/litter. Then introduce a piece of the virus – contaminated diet in each plastic container with starved larva. The larva feeding on the diet facilitates ingestion of virus.
  • 15. Replace uneaten part of diet after 2-3 day and simultaneously clean or change the container of larvae. the larvae begin to show symptom of virus infection after about 5 days and start dying between 6 to 7 days Dead larvae become loose and emit brownish black viral ooze on slight pressing. Collect the dead diseased larvae in distilled water and purity for 10 days period
  • 16. Filter this suspension through double layered muslin cloth and centrifuge for 3 to 5 min at 200 rpm speed. Pellet settled at the bottom may be discarded and the supernant is again centrifuged at 2500 rpm for 10 to 15 min. Hence supernant is discarded and the pellets settled at the bottom may be responded in little quantity of distilled water. Mix 10 times water in the purified virus. Counting POBs under microscopic using haemocytometer may assess the strength of polyhedral suspending in unit quantity of water
  • 17. For this fill the squares of the haemocytometer releasing few drops of HaNPV suspension between cover slip and marked portion. Wipe out excess suspension with tissue or sponge piece. Observe under research microscope
  • 18.  The minimum count is required 1 * 109 POBs/ml. fill the convenient sized bottles, label properly indicating at least brand name of product, POBs / ml, manufacture, direction for use, use before date etc. store in cool place. Normally HaNPV preparation is virulent up to 4 to 5 year.
  • 20.  Entomopathogenic Nematodes are a group of nematode (Thread worm), causing death to insect.  EPN live parasitically inside the infected insect host and so they termed as endoparasitic.  EPN have been found in all over the world and a range of ecologically diverse habitats.  They are highly diverse, complex and specialized.
  • 21. They infect many different type of insect living in the soil like the larval form of moth, butter flies, and beetles as well as adult form of beetles, grass hoppers and crickets.
  • 22. Productionprocedure ofNeoplectinaspp. (DD-136)Nematode • Three host insects can be used for mass multiplication of EPN 1. Galleria mellonella 2. Corcyra cephalonica 3. Helicoverpa armigera • But G. mellonella larvae has been found to be susceptible for EPN
  • 23. White trap method of mass production of EPN • Larvae are inoculated with EPN on petri dish lined with filter paper. • After 2-5 days infected larvae are transferred to the white trap. • In this method two glass dishes, one big and one small is used. One big dish is kept in a normal position and another small is kept in a inverted position inside it. One whatmans filter paper No. 1 is kept on this prepared trap. A distilled water is poured in this white trap.
  • 24. • The nematode migrate into the water which is then sedimented of 2000 million juveline per 3 kg bag be obtained at relatively low cost. • To harvest of nematode, the sponge culture is placed in open tray and just covered with water
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  • 27. • The cadavers rest on inverted watch glass, surrounded by water. • The central dish containing the cadavers provide a moist surrounding for the EPN emergences from cadaver. • New progeny of infective juveniles that emerge from cadaver migrate to the surrounding water where they are trapped and subsequently harvested.