Mass culturing technique of entomopathogenic nematode

RK Patidar, P Dutta, V Kadam, M Pathak and RK Tombisana
The term entomopatho-genic nematode (EPN) refers to those nematodes which parasitize and killed the insect hosts. The different species of EPNs differs in the choice of insects they attack, ecological needs and stability in commercial products. Many agriculturally important insect pests can be control by them. Entomopathogenic nematodes come under the genus Steinernematids and Heterorhabditids. EPNs are attractive, organic alternatives for controlling a wide range of insect pests and they are utilized in classical, conservation and augmentative biological control programs. These groups of nematode can be multiplied o in vivo and in vitro.
Most Steinernematids and Heterorhabditidscan easily be cultured on wax moth larvae (Galleria mellonella) for use in the laboratory and small field trials, but not for large-scale production as described below.
Production of wax moth, Galleria mellonella
Wax moths are easily reared in the laboratory by using honey comb or artificial diet.
The diet compositions are:
(1) Corn meal – 400 g  (2) Wheat flour – 200 g (3) Wheat bran – 200 g (4) Milk powder 200 g (5) Yeast tablets – 100 g (6) Honey – 350 ml (7) Glycerine – 350 ml.
Preparation of diet
Corn meal, wheat flour, wheat bran, milk powder and yeast are mixed  thoroughly. Honey and glycerine are mixed separately and then mixed with the earlier mixture  The diet is then kept in plastic container 4 or 5 days before use.
Egg collection
Plastic jars with lid can be used as oviposition cage. Folded paper strips should be put inside the jars for deposition of eggs. Ten pairs of adult wax moth are released inside a jar (35 cm height x 20 cm dia). The eggs were removed daily after removing the folded paper strips and cutting the egg deposited area of paper.. The collected eggs are disinfected with 10% formalin for 10 minutes followed by 5 minutes washing in sterilized water and dried over a filter paper.
Inoculation with eggs
The prepared diets are kept in plastic jars. Perforations should be made in the jars for aeration. The dried eggs are kept over the diets in plastic jars. After hatching the young larvae feed on the diet. Overcrowding of the larvae can be avoided by transferring some of the larvae along with diet to a freshly prepared rearing jar. Each rearing jars are checked regularly. When the food is exhausted, they are fed with fresh diets after removing them from the webs. If any dead larvae are come across, they are removed from the rearing jar.
Mass production of Steinernema and Heterorhabditis
· Evenly distributed 200 infective juveniles (IJs) in 1 ml of nematode suspension on 9 cm filter paper placed on the lid of 100 mm 15 mm-1petridish.
· Add 10 galleria larvae to the dish and cover the lid with inverted petridish.
· Store the petridish in a plastic bag containing a wet pepper towel to keep it moist, at 25oC.
· After 5-7 days, place the cadaver on to the white trap.
· Harvest nematodes from the white trap (using the outer petridish of 150 mm 20 mm-1 size), and give 2-3 washing with sterilized water, concentrate and store in culture bottles, keeping flat, at 20oC.
Steinernematids and Steinernematids can be cultured on varieties of substrates. The ingredients for nematodeculture include a source ofnutrient for the symbiotic bacterium and a sterol source for the nematodes.
1. Place sterile rabbit kidney (1 g) on 1% agar (10 ml), or a small piece of autoclaved pig kidney placed aseptically on to the peptone-glucose-agar has also been used for Steinernematidi. Another artificial medium for Steinernematids and Heterorhabditisproduction used dry dog food as the main ingredient.
II. After sterilization, cool the medium and streak the symbiotic bacteria from the species to be cultured.
III. The bacteria is allowed to grow for 1-2 days and then inoculated with sterilized IJs from another monoxenic cultured. After 2-3 weeks, IJs are harvested using white trap. Bedding (198) develop a technique whereby large number of  nematodes may be produced using chicken offal medium on a porous foam substrate such as polyether polyurethane. These substrates provide the large volume ratio while providing adequate interstitial space. Glass flask is generally used, but autoclavable bag can also be used.
Surface sterilization of IJs
The IJs can be surface sterilized by using any of the four methods- 0.1% Hyamine 10X for 15 minutes; 0.1% formaldehyde for 30 minutes; 0.1% merthiolate solution (w/v) plus streptomycin (5000 units ml-1) for 2-3 hours; or 0,1% sodium hypochlorite for 1 hour. After treating, the IJs should be given 2-3 washing with sterilized water.
Isolation of symbiotic bacteria from IJs
The symbiotic bacteria can be isolated either from IJs or from haemolymph of the infected host (Akhurst, 1980).
· Suspend the surface sterilized IJs in yeast salt or nutrient broth.
· Allow the bacteria to multiply in the medium kept on shaker for 24-48 h.
Isolation of Bacteria from haemolymph
· Infect 10 larvae of G.mellonella with 100 IJs.
· After 48 h, surface sterilized the dead larvae by 1% sodium hypochlorite.
· Open the cadaver without rupturing the gut. Streak a drop of haemolymph on to nutrient agar. Transfer the colony into YS broth and incubate for 1-3 days at 25oC. Fill a 2.2 ml cap with 0.3 ml sterile glycerol. Add 1.7 ml bacterial suspension.
· Store caps at -20oC, till use.
The insect killed by nematode are flaccid and do not have putrid odour because the mutualistic bacteria produce antibiotics which prevent the growth of secondary microorganisms. Insect killed by Steinernematids turn yellow-brown, pale or black whereas those killed by Heterorhabditids turn red, brick red, purple, orange or some time green. In the dark, the Heterorhabditids infected insects produce luminescence due to presence of bacteria, Photorhabdus luminescence.
Production of EPN on a commercial scale has been accomplished by using inexpensive protein and sterol source (Bedding, 1981; 1984). This method is applicable for cottage industries or biotechnology companies. The EPN has also been produced monoxenically in large liquid fermenters using a combination of plant and quite suitable for companies with large capital investment because of economic of scale. Friedman et al. (1989) reported up to 110,000 IJs ml-1 in 8 days using a medium containing soy flour, corn oil and egg yolk.
A major constraint on commercializing of EPN is their poor storage capability. It is extremely important that IJs and their mutualistic bacterium must process a high and stable level of pathogenicity from production to application (Georgis, 1990). Active IJs must be immobilized to prevent depletion of their lipids and glycogen reserved. Immobilisation has been accomplished by storing them in refrigerator (5-15oC in oxygenated water where these can be stored up to 5 years (Friedman, 1990). However, this is not commercial feasible. Diminishing the activity of nematodes through immobilization and or partial desiccation or on a substrate increased their viability. Therefore, nematodes Formulated in various carriers such as alginate, clay, vermiculate and gel forming polyacrylamides store for at least 6 month under refrigeration and at least 3 month at room temperature.
Heterorhabditidbacteriophora is quite sensitive to physical stresses but was found to be tolerant to 3 days of laboratory immersion in 58 out of 75 fungicides, herbicides, insecticides and nematicides assayed (Rovesti et al., 1988). Das and Divakar (1987) found that S.carpocapsae was compatible with carbaryl, dimethoate, malathion, zineb at normal recommended doses. The compatibility of S.glaseri was found with urea, murate of potash and super phosphate.
Nematodes cab be applied with common agrochemical equipment including small pressurized sprayer, mist blower, electrostatic sprayer, fan sprayer and helicopter. They stand pressure of lb sq.-1 inch and can be delivered with all common nozzle type sprayers with opening as small as 50 microns diameter. Other methods of delivering nematodes to the target are injection, baits and alginate capsules. Nematodes can also be applied by drip and sprinkler irrigation. This is attractive since it is labour saving, automatically provide sufficient moisture for the nematodes; provide flexibility of timing of application. Early morning and late evening application are recommended. Field concentration exceeding 2.5 billion nematodes ha-1are usually applied to ensure, that sufficient nematode population will come in contact with the target insects
Practically EPNs can stand against the pressure of 300 lb sq-1 and cab be delivered with general agrochemical tools like hand guns, small pressurized sprayers, mist blowers, electrostatic sprayers, fan sprayers and helicopters which can be delivered through a nozzle with openings as small as 50 microns diameter. The following methods can be used for application of entomopathogenicnematodes:
· Spraying: A dose of about 100,000 IJs plant-1 or 2.5-2.7 billion IJs ha-1 is sufficient in the field for pest control results. About 50 IJs/flying insect are sufficient.
· Trickle irrigation: Button type of trickle irrigation outlet is used to deliver the inoculums to the area on one side of the plants.
(To be contd)