Mass producation HaNPV & EPN

Helicoverpa armigera nucleo poyhydrosis virus
Entopathogenic Nematod

1. Prepared by Mr. Mahesh Vikram Sable Assistant Professor Department of Plant Pathology College of Agriculture, Ashti

2. Mass Production of HaNPV and EPN

3. Contents

4. Production Procedure of Nuclear Polyhedrosis virus of Helicoverpa armigera Material Requirement Nucleus culture of HaNPV Culture of H. armigera Natural food or gram flour based artificial diet for H. armigera Small plastic bottles or tube with lid (50 ml) Muslin cloth Cotton Honey solution Haemocytometer Research microscope Disinfectant – Formalin, Sodium hypochloride , teepol 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 60 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 3 rd or early 4 th 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 * 10 9 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.

19. Entomopathogenic Nematodes (EPN)

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. Production procedure of Neoplectina spp. (DD-136) Nematode Three host insects can be used for mass multiplication of EPN Galleria mellonella Corcyra cephalonica 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

25. 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.