Agriculture:- Model Bankable Projects in India | Land Development : Biopesticide Unit Part 3 --- www.nabard.org

Agriculture:- Model Bankable Projects in India | Land Development : Biopesticide Unit Part 3 --- www.nabard.org

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B. Chrysopid predetors 1. Importance 1.1 Chrysopid predators are important for the management of bollworms and aphids in cotton and tobacco and several sucking pests in fruit crops. They are capable of bringing down the population of the pest drastically. Chrysoperla (Chrysoperla carnea) is a potential chrysopid, which is also amenable to mass multiplication. 1.2 Chrysoperla are generally green in colour, varying in length from 1.0-1.3 cm. The pre-oviposition period lasts 3 to 7 days. Adults start laying eggs from 5th day onwards and peak egg-laying period is between 9 and 23 days after emergence. The male longevity is 30-35 days. Adult female lay eggs of 600-800 eggs/female on an average. The eggs are stalked and green in colour. The eggs are laid singly or in clusters. Egg stage lasts 3-4 days. The larva has 3 instars and after 8-10 days it will form cocoons. Adult emerges in 5-7 days from cocoons. 1.3 The green lacewing is being mass released in the field for the control of aphids, white flies, mealy bugs and eggs and young larvae of lepidepteron pests. The Chrysoperla predetors may be used on cotton, groundnut, pulses, vegetables, ornamentals and several other crops. They also feed on the eggs and freshly hatched larvae of Helicoverpa armigera and such other caterpillar pests. 1.4 It is being mass produced primarily on the eggs of rice grain moth, Corcyra cephalonica in India. For mass production of chrysoperla, an efficient rearing technique is required. 2. Mass Production

2.1 Chrysoperla predators are mass multiplied in laboratory at 27 ± 10C and 70% RH on the eggs of Corcyra cephalonica, a laboratory host. Three days old 120 chrysopid eggs are mixed with 0.75 ml Corcyra eggs (the embryo of Corcyra eggs are inactivated by keeping them at 2 feet distance from 30 watt ultraviolet tube light for 45 minutes) in a plastic container. On hatching, the larvae feed on the contents of eggs. The second and subsequent instars are reared individually in cells of louvers on the eggs of C. cephalonica. It is assumed that for rearing 100 larvae (1cc) C. cephalonica eggs are required. Host eggs are provided twice during the course of larval rearing. First feeding of 1.75 ml for 100 larvae and second feeding of 2 ml for 100 larvae with a gap of 3 to 4 days is provided. Cocoons formed in the cells are collected after 24 hours. The cocoons are placed in oviposition cage for adult emergence (Photograph-1). In each oviposition box roughly 20 pairs can be accommodated and inside portion of the container is covered with black paper on which adults lay eggs. The adults in the oviposition boxes are provided with castor pollen, protinex mixture (equal volume of protinex, fructose, honey and powdered yeast dissolved in small quantity of water), 50% honey and drinking water in cotton swab. Adults lay eggs on the under surface of the top lid which is removed by sliding a clean lid. After 24 hours of hardening the eggs are gently brushed with a brush to dislodge on to a paper eggs are collected and either reused for mass multiplication or sent to farmers for field release. Only first instar larvae are released on to the recommended crop plants. 3. Major equipment required Facilities like rearing room (6 x 6 m), slotted angle iron racks, work tables, plastic louvers 60 x 22 cms with 2.5 cm cubical cells, acrylic sheets to cover the louvers, glass vials, adult oviposition cages (45 x 30 x 30 cms), plastic louvers, plastic containers, scissors and brushes, cotton wool, tissue paper, sponge, fructose, protinex, honey, yeast, castor pollen etc. are required for the mass rearing of chrysopids. 4. Dosage At least 1000 eggs or larvae may be used per acre. C. Australian ladybird beetle (Cryptolaemus montrouzieri) 1. Importance 1.1 Mealybugs are serious pests on fruits, vegetables, ornamentals and plantation crops. Besides causing direct loss to the plants they also reduce market value of infested fruits. The extent of damage may go upto 70 percent in severe infestation. Lady bird beetle, Cryptolaemus montrouzieri introduced from Australia is a potential bio control agent and is being utilized on many crops in Southern India. 1.2 Mealybugs or scale insects constitute the natural food of certain ladybird beetles. The adult beetles as well as their larvae (grubs) seek the pests and feed voraciously on all stages. They often wipe out the entire pest colonies. The lady bird beetles are being used for suppression of mealy bugs in citrus, coffee, grapes, guava, ornamental and a variety of other crops. 2. Equipment needed Equipments like wooden boxes/cages, iron rack, buckets etc. are needed for mass multiplication of ladybird beetles. 3. Production Technology The production involves the following steps: After 15 days of infestation of pumpkins with mealy bugs (Planococcus citri), they are exposed to a set of 100 beetles for 24 hrs. After exposing the pumpkin is kept back in a cage. The beetles during the period of exposure feed on mealybugs as well as deposit their egg singly or in groups of 4-12. The young grubs feed on eggs and small mealybugs but as they grow they become voracious and feed on all stages of mealybugs. For facilitating the pupation of grubs, dried guava leaves or pieces of papers are kept at the base of each of the eggs. The first beetle from the cages start emerging on 30th day of exposure to beetle adults. The beetles are collected daily and kept in separate cages for about 10-15 days to facilitate completion of mating and pre-oviposition. The beetles are also fed on diet containing agar powder (1gm), sugar (20gm), honey (40cc) and water (100cc). The adult beetle diet is prepared by boiling sugar in 70cc of water, adding 1gm agar, diluting 40cc honey in 30cc of water and adding to the sugar and agar mixture when it comes to boiling point. The hot liquid diet is kept on small white plastic cards in the form of droplets which get solidified on cooling. Such cards containing diet can be fed not only to C. montroozieri but also to many other species of cocinellids. From each cage about 175 beetles are obtained. The emergence of the beetles is completed within 10 days. The Beetles can also be reared on corcyra cephalanica eggs but empty ovisacs of Planococcus citri are to be kept for inducing egg laying by the beetles. 4. Field release and application Before releasing in the field in the endemic areas, moderate to severely infested plants are marked. The plant trunks are ringed one foot away with a band of 5% diazinan granules 24 hrs before the release of the beetles; this stops the patrolling of ants on the trunk atleast 3 days. On citrus 10 beetles per infested plants are released but on other crops the releases are calculated based on infestation and crop canopy. Release of 10-15 adults / tree depending up on canopy and infestation once in a season 600 to 1000 beetles may be released per acre 5. Precautions The important precautions are given below: All due precautions should be taken to avoid scarcity of food for the grubs to avoid cannibalism by grubs. All the pumpkins showing sign of rotting should be properly incinerated. - D. Production of Ha NPV and SI NPV 1. Introduction 1.1 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). 1.2 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. 1.3 In India, extensive research has been conducted on the use of NPVs for tackling two major pests namely Spodoptera litura and Helicoverpa armigera. 1.4 Nuclear Polyhedrosis viruses like Ha NPV, SINPV are increasingly being used as alternatives to chemicals. These viruses have distinct advantages over other methods of pest control. NPVs are virulent pathogens of insect characterised by the polyhedral occlusion bodies (POB). These viruses are highly specific and do not affect beneficial insects like parasitoids and predetors and are safe to fish, birds, animals and man. Considering the usefulness of NPV's there has been a growing demand amongst the farmers for these bioagents. 2. Major equipment required The major equipments like centrifuge, laminar flow, magnetic shaker, microscopes, autoclave, coolers, refrigerators, incubator, distillation units etc. are required in addition to glassware, plastic trays, basins, iron racks for mass production of Ha NPV and SI NPV. 3. Spodoptera litura (Tobacco Caterpillar) Spodoptera litura commonly known as tobacco caterpillar, is a polyphageous pest. It is a serious pest of tobacco nurseries and also a sporadic pest of cauliflower, cabbage, castor, cotton, groundnut, potato and lucerne. It causes serious crop losses. 4. SI NPV The virus is specific and infects only Tobacco Caterpillar. NPV can be successfully multiplied on tobacco caterpillar and the viral extraction can be applied in the field to control the caterpillar. For continuous production of SI NPV, it is necessary to rear Tobacco Caterpillar larvae continuously in a lab condition. 5. Gram pod borer (Helicoverpa armigera) It is widely distributed in India and infests/damages a variety of cultivated and wild plants throughout its distribution range. It is a serious pest on commercial crop like cotton; pulses like redgram and bengalgram; vegetables like tomato, bhendi and dolichos bean; oilseeds like sunflower, soybean and safflower and cereals like sorghum and maize. 6. Ha NPV Ha NPV is a highly infective microbial biopesticide which can be used to control Gram borer. It is derived from naturally diseased or under laboratory conditions artificially infected larvae of gram borer. 7. Mass production of Ha NPV and SI NPV 7.1 The mass production of Ha NPV and Si NPV involves 3 steps Rearing of adult Gram pod borer and Tobacco caterpillar for mass production of eggs. Rearing of larvae of the above species either on the host plants like chickpea and castor under semi natural condition or on the synthetic diet in the laboratory conditions. In the model only the later is considered for large scale commercial production of NPV. Inoculation of Ha NPV and Si NPV into the larvae of Gram pod borer and Tobacco caterpillar respectively for mass multiplication of viruses and extraction of polyhedral occlusion bodies(POBs) from the diseased larvae, which are used as biopesticide on the crop plants. The schematic representation depicting production and application of NPV is presented in Exhibit-II.

7.2 Details of mass production 7.2.1 Diet preparation The larvae ofGram pod borer and Tobacco caterpillar can be multiplied by using chick pea based semi-synthetic diet. The composition of the diet for rearing larvae is as follows:-

Item Quantity 'A' fraction: Chickpea (Kabuli chenna) flour 105.00 gm   Methyl para-hydroxt benzoate 2.00 gm   Sorbic acid 1.00 gm   Streptomycin sulphate 0.25 gm   10% formaldehyde solution 2.00 ml 'B' fraction: Agar-agar 12.75 gm 'C' fraction: Ascorbic acid 3.25 gm   Yeast tablets 25 tablets   Multivitaplex 2 capsules   Vitamin E 2 capsules   Distilled water 780.00 ml

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. 7.2.2 Mass production of eggs Tobacco caterpillar The culture of Tobacco caterpillar is initiated by collecting eggs from the fields of castor, cauliflower, lucerne, tobacco etc. These field collected eggs are reared in isolation to eliminate the emerging parasitoids and diseases, if any. The culture can also be established by collecting the gravid females with the help of light traps. 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 cottons swabs placed in small plastic cups. The eggs which are generally laid in batches on the paper are cut out. Freshly laid egg masses are sterilised by dipping in 10% formalin for 30 minutes, washed in running water for 30 minutes, dried on blotting paper and kept for hatching in sterilised glass vials. The freshly laid eggs can also be surface sterilised 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 traces of sodium hypo chlorite could be neutralized by dipping the eggs in 10% sodium thiosulphase solution and again the eggs are washed thoroughly under running tap water. 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 with stand for pupation. The pupae are collected 3 days after all the larvae enter the sand. The pupae are sexed and kept on a lid over a wet sponge in adult emergence cage. After 10 days, freshly emerged males and females are collected from their respective emergence cages. Tobacco caterpillar larvae can be multiplied on a chickpea based semi-synthetic diet composition and preparation of which is detailed under point 7.2.1. 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 material thus obtained is reared in the laboratory in aseptic conditions and the healthy progeny is selected and established. The production starts with the availability of 250 pairs of adults every day, which will yield 10,500 eggs daily. The adults are kept @ 100 pairs in each 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.

7.2.3 Rearing of larvae on semi-synthetic diet Tobacco caterpillar Stage - I (rearing of early instar larvae): The rearing unit is prepared by placing a sponge piece on a glass sheet. The sponge is covered with a single layer of soft tissue paper. A small plastic container containing 200 surface sterilised eggs of Tobacco caterpillar is placed in the centre over the tissue paper. A petri dish containing about 200 ml of diet is placed inverted over the tissue paper. The eggs hatch within 25 hr and neonate larvae crawl and spread out on the diet. Stage - II (rearing of late instar larvae): Late instar larvae are reared in a modified plastic boxes. One window each on the four sides of the box is cut and covered with a fine plastic mesh to provide sufficient ventilation and to prevent moisture accumulation inside the box. A thick layer of sterilised sand is spread at the bottom of the box. A small piece of tissue paper is kept at the centre over the sand. The diet in the petri dish (containing 200 larvae) is divided into five equal pieces. One piece of diet bearing 40 larvae is kept in plastic box over the tissue paper so that the sand does not soil the diet. In this way, 5 boxes are charged with larvae from 1 petri dish. A plastic grill is fitted into the box in such a manner so that it forms a crest higher than the brim of the box. Thick cake of diet (about 500 gm) in a petri dish is divided into two equal pieces. One such piece is kept on the top of the crest and the lid of the box is then fixed so that the diet and grill crest are opposed to each other just beneath the lid. After consuming the small quantity of diet on tissue paper the larvae crawl and perch on the grill and feed from the ceiling of the box. The boxes are stacked and left intact for 3 days. During this time the diet is almost completely consumed. Now another piece of fresh diet (about 250 gm) is kept on the crest in each box and the boxes are closed and stacked again. During the last 3/4 days of larval stage the food consumption is maximum and so is the fecal matter accumulation on the sand layer. After 20 days from hatching the larvae move into the sand and start pupating. In a period of 25 days, all the larvae, pupate and the chitinisation of pupae is also completed. The boxes are now ready for the pupal harvest. The pupae are collected, cleaned, sterilised and placed in adult emergence cages. The freshly emerged moths are then placed in oviposition cages. Gram borer The larvae of gram borer can also be reared on a chickpea based semisynthetic diet as detailed under point 7.2.1. The diet is poured as per the requirement either on the nylon mesh for rearing 5-7 day old larvae or in tray cells for rearing the older larvae or poured into sterilised petri plates and allowed to solidify. The diet could be stored in the refrigerators for upto 2 weeks. For preparing large quantities of diet, the quantity of diet ingredients to be used should be calculated accordingly and industrial type waring blenders could be used. The larvae are removed from the top of the aluminium foil wrapped flasks with a brush and then transferred to the diet. 220 larvae are transferred to diet impregnated on nylon mesh and placed in plastic containers or sterilised glass vials. 100 such containers are maintained daily for 5-7 days. Multi-cellular trays with semi-synthetic diet is advantageous for rearing a large number of larvae. Starting with 10,500 eggs, the total number of larvae available is 10,000 considering an estimated 5% mortality in initial 5 days of emerging and 10% mortality upto first 5 - 7 days. The total number of larvae available for virus production is 8000 (80%). The rest of 20% will be utilized for maintenance of host culture continuously. The diet requirements at various stages of production of larva are: for the young larvae upto 5-7 days will be 2 gms / larva. for 5-7 day old larvae for Ha NPV production will be 4gms/larva for five to seven day old larvae for continuation of host culture will be 6 gms/larvae. for rearing the field collected larvae for augmenting the nucleus stock will be about 1 kg 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 chloride and then washed thoroughly with distilled, sterilised water. After washing, the eggs 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. 7.2.4 Production of Helicoverpa armigera NPV (Ha NPV) and Spodoptera litura NPV (SI NPV). For Ha NPV and SINPV 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 Ha NPV and 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. The extraction is centrifuged to further clarify the solution. 8. Other Important Aspects 8.1 General precautions to be followed while maintaining host cultures

In production units, keep the host culture in a separate room and the virus production and storage facility should be located in a different facility. In the NPV production units, inspite of best care, 100% larvae are not infected, the larvae which do not turn inactive after 4 - 5 days and keep consuming the normal diet should be culled out regularly from the NPV production unit. Utmost care should be taken to prevent the break in the chain of the production system. This could be achieved only if highly dedicated and disciplined workers are engaged for such production units. Strict hygiene should be maintained in different facilities. The equipments used should be either heat sterilised or sterilised using steam or chemicals. The work place should be thoroughly disinfected with sodium hypo chlorite solution. The host culture should be initiated from a batch of healthy adults. Microbial infection could be avoided if good insect husbandry practices are followed. If infection is detected, the culture or infected part should be destroyed immediately. Besides hygienic conditions, optimum temperature (24o C- 26o C) and humidy (65 - 70%) should also be maintained. The texture and quality of the natural/semisynthetic diet should be good. entry to host culture unit after visiting virus production unit should be avoided. 8.2 Mode of action NPV acts as a stomach poison only to the target host (pest) and hence beneficial insects are not affected. The infected larvae become pale and glossy and tissue get disintegrated and liquified. Most of the body tissues and organs (except gut) get infected by polyhedral occlusion bodies (POBs), which contains the virions. The liquid which oozes out of the infected larvae (which hang upside down) contains millions of POBs. Each POB measuring about one micron in diameter and possessing a characteristic movement can be identified under the microscope. 8.3 Field application and dosage Ha NPV is used for controlling H.armigera attacking cotton, redgram, bengalgram, tomato, okra, sunflower, groundnut, chillies, maize, sorgram etc., whereas, SI NPV is used for controlling tobacco caterpillar attacking tobacco, groundnut, soyabean, sunflower, cotton, cabbage, beetroot, cauliflower etc. 8.4 Directions for use of NPV The recommended dosage is 200 ml of NPV/acre or 500 ml/ha containing 100 and 250 larval equivalent (LE) of NPV respectively as active infective material (one LE = 6 x 109 POBs). 100 ml of NPV could be diluted in 200-400 litres of water when high volume sprayer is used and in 50-70 litres of water in case of power sprayers. Preferable to spray using high volume knap-sack sprayer. Virus should be sprayed during evening hours. Spray should be initiated as soon as some newly hatched larvae are observed or three to five days after a trap catch of 5 moths per pheromone trap. Subsequent sprays should be made at 7-10 days intervals depending upon the pest population. 8.5 Compatibility with other insecticides The viral pathogens seems to be less sensitive to chemical pesticides. When the combination of pathogen and pesticide is used, sometimes synergistic action is noticed. But is recent years mixing of NPV with insecticides is not advisable due to cross resistance problem.

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