Pest problem is one of the major constraints for achieving higher production in agriculture crops. India loses about 30% of its crops due to pests and diseases each year. The damage due to these is estimated to be Rs.60,000 crores annually. The use of pesticides in crop protection has certainly contributed for minimising yield losses. The pesticides, which are needed to be applied carefully, only when the threshold limits of the pest population is exceeded. However, quite often the indiscriminate and unscientific use of pesticides has led to many problems, such as pests developing resistance, resurgence of once minor pest into a major problem besides environmental and food safety hazards.
The problem of insect-pest is acute in case of all the crops and especially so in case of commercial crops. The use of insecticides and pesticides have increased manifolds during the past 3 - 4 decades with the introduction of intensive cropping. The average consumption of pesticides in India is about 570 gms per ha. as compared to developed countries like Japan, Thailand and Germany where the consumption rate is 11 kg, 17 kg and 3 kg per ha, respectively. Though the average quantum of pesticides usage in India is low, the damage caused due to their indiscriminate usage and poor quality maintenance is alarming. Interms of value, much of the pesticide application is accounted for by a few crops. For example, cotton, paddy and vegetable crops account for 80% of the value of pesticides applied in India.
Pesticides or chemicals are meant to control harmful pests such as insects, nematodes, diseases, weeds etc. However, excessive use of pesticides not only leave residues in soil, water and air but also have adverse effects on the non target organisms such as pollinators, parasitoids, predators and wild animals. This has adversely affected the ecological balance resulting in pest resurgence, development of resistance in the pest species and environmental pollution. Development of pest resurgence and resistance has resulted in high cost of production and low income especially to cotton farmers in AP, Maharashtra.
In view of the several disadvantages associated with the unscientific use of pesticides in agriculture, there is an urgent need for minimising the use of chemical pesticides in the management of insect pests. Growing public concern over potential health hazards of synthetic pesticides and also steep increase in cost of cultivation/low profit making by farmers has led to the exploration of eco-friendly pest management tactics such as Integrated Pest Management (IPM). IPM aims at suppressing the pest species by combining more than one method of pest control in a harmonious way with least emphasis on the use of insecticides. In simple terms "IPM is the right combination of cultural, biological and chemical measures which provides the most effective, environmentally sound and socially acceptable methods of managing diseases, pests and weeds". The major components of IPM are prevention, observation and intervention. The IPM seems to be the only answer to counter some of the major pests of crops, which have become unmanageable in recent years. The success of IPM largely depends upon conservation of naturally occuring bio control agents.
Importance of Bio-pesticides
In nature every ecosystem exists in a balance. Growth and multiplication of each organism depends on the food-chain, its predetors, parasites, etc. In biological control system, these interrelations are exploited. The natural enemy of a pest, disease or weed is selected, its biology is studied for mass multiplication and utilize the same to check the target pest. They are also specific in their action and perish once their feed (i.e. the pest) is exhausted. Thus they are based on natural principles, do not leave any residue, safe and economical.
Among the alternatives, biological control of pests is one of the important means for checking pest problems in almost all agro-ecological situations.
Bio pesticides are living organisms which can intervene the life cycle of insect pests in such a way that the crop damage is minimized. The agents employed as biopesticides, include parasites, predetors and disease causing fungi, bacteria and viruses, which are the natural enemies of pests. Further, they complement and supplement other methods of pest control. Utilisation of naturally occurring parasites, predators and pathogens for pest control is a classical biological control. On the other hand, these bio agents can be conserved, preserved and multiplied under Laboratory condition for field release. Once these bio-agents are introduced in the field to build their population considerably, they are capable of bringing down the targeted pest' population below economic threshold level (ETL). However, the crux lies in their mass production and application at the appropriate time.
Major advantages of bio pesticides
Bio-pesticides are preferred over chemical pesticides for the following reasons:
- no harmful residues;
- target specific and safe to beneficial organisms like pollinators, predetors, parasites etc.;
- growth of natural enemies of pests is not affected, thus reducing the pesticide application;
- environmental friendly;
- cost effective;
- important component of IPM as 1st line and 2nd line of defence, chemicals being the last resort.
Status of bio pesticide use in India
Last decade has witnessed a tremendous breakthrough in this aspect, especially on standardization of production techniques of Trichoderma, Gliocladium, Paecilomyces, Pseudomonas, Trichogramma, NPV and Bacillus to use them against many insect pests and diseases.
There are a number of instances where bio control agents have been successfully employed in India. Some examples of these are given below :
- Growth of lantana weed was controlled by using the bug Telonemia scrupulosa
- Sugarcane pyrilla has been successfully controlled in a number of States by the introduction of its natural enemy Epiricania melanoleuca and Tetrastictus pyrillae.
- Trichogramma, which feeds on the eggs of sugarcane borers, has been used against the borers in the states of Tamil Nadu, Rajasthan, UP, Bihar and Haryana.
- Similarly Trichogramma, Bracon, Chelonus and Chrysopa spp. are being used for the control of cotton bollworms. Trichogramma has also been used against rice stem borer and leaf folder.
- The sugarcane scale insect has been controlled with the help of predatory coccinellid beetles in UP, West Bengal, Gujarat and Karnataka.
The popularity of biopesticides has increased in recent years, as extensive and systematic research has greatly enhanced their effectiveness. Also, techniques for the mass production, storage, transport and application of biopesticides have been improved in recent years.
Scope for Commercial Production of Biopesticides
Though there are about 140 biopesticide production units existing in the country as on today, they are able to meet the demand of only less than 1% of cropped area. There exists a wide gap, which can only be bridged by setting up of more and more units for production of biopesticides. This requires large scale investment and private participation.
Some of the local small scale industries have already started production and marketing of Trichoderma viride (against few fungal diseases) and Trichogramma (against sugarcane early shoot borer). There is a scope to enhance production and use of biological control agents in the days to come as the demand is on the increase every year.
Location of Biopesticide Units
In order to achieve optimum results, care needs to be taken to set up biopesticide facilities in areas which have appropriate climatic conditions. The production of Biopesticides requires controlled climatic conditions. Temperature control is less costly in locations where there is no extreme conditions. Besides the climatic conditions, the proximity of the location to the market is also important. However, care must be taken that the production facilities are set up at least a quarter of a mile away from farming areas, so as to prevent the contamination of production facilities by insecticides from the farming areas. Also, as air pollution can damage biopesticides, the production should be located away from industrial and urban areas.
||Production Process in brief
||i.Trichogramma spp. (egg parasite)
||Mass multiplied by using stored grain pest as a host. The production involves the multiplication of host insect on sorghum grains, allowed to be parasitized by trichogramma. Then egg are clued in cards as "tricho cards".
||Used for control of sugarcane early shoot borer, bollworms of cotton, sorghum stem borer.
|ii. Crysoperla carnea (Chrysopid predetor)
||Mass multiplied in laboratory on the eggs of stored grain pest.
||Controls larval pests in pulses, vegetables /fruits
|iii. Cryptolaemus montrouzieri (Ladybird beetle)
||Mass multiplied on already mass multiplied mealy bugs with the help of pumpkin as under laboratory conditions..
||to control mealy bugs especially on fruits.
||i. NPV of Helocoverpa armigera & Spodoptera litura
||The production starts with raising of pod borer and tobacco caterpillar larvae (host culture) on semi-synthetic diet. NP Virus is smeared on cultured larvae. Then the diseased larvae are collected to obtain virus suspension after blending, filtration, centrifugation.
||Used against boll worms in cotton and pod borers.
|ii. Trichoderma Fungal spp.
||Multiplied in laboratory and formulated in powder form with the help of carrier material (talc powder).
||To control root rot and wilt diseases especially on pulses.
|iii. Pheromone lures for Helicoverpa armigera & Spodoptera litura
||Sex pheromones are filled into plastic lures at required concentration with the help of micro pippets and placed into rubber septa. The septa is fixed to the trap.
||To trap reproductive males of gram pod borer and tobacco caterpillar.
The technology used were indigenous and the scientific aspects of production were standardised by ICAR Research Institutes and State Agricultural Universities. Machinaries and laboratory equipments are available from various manufacturers and are of BIS standards.
Objectives of Biopesticide Project Models
- The primary objective of biopesticide projects is to establish the bankability of mass multiplication of various bioagents discussed in the models
- To serve as guidelines for extending financial assistance to entrepreneurs who may be interested in setting up biopesticide units
- To promote setting up of more bio-control production units
- To disseminate widely the technology
Basic requirements for establishment of Biopesticide units
Based on the field visits to bio-control production units and in line with the technology and objective of biopesticides production, various facilities required for the successful implementation of such projects are indicated below:
Land is required for construction of culture and rearing rooms, processing room, laboratory, office etc. In the present models, we have assumed only rented buildings, hence no land cost has been considered except for poly house.
2. Building and civil works
Biopesticides production involves rearing of insects. Hence, the basic infrastructure to be created includes only the civil structures built in such a way as to provide environmental conditions suitable for rearing of insects. The production unit has to be located away from industrial unit to avoid pollution problems. For the proposed installed capacity, an estimated built up area of about 1000 sq ft is required for model-I (mass production of Trichogramma, Chrysoperla and Cryptolaemous beetles) & for Model-II (production of NPV, Trichoderma and pheromone lures) about 2400 sq.ft. area is required. Other utilities required are power, water and vehicle. Among others, the civil structure may be designed to have separate room for diet preparation, corcera culture, egg production, host culture etc. The host culture room for NPV production should be kept at a distance with proper hygiene and entry may be restricted in such a way to prevent any contamination. In other words, one should not enter host culture room after visiting a facility, where NPV is extracted from dead infected larvae.
3. Plant and Machinery
There is no requirement of heavy plant and machinery. Racks, trays and other facilities are required for rearing insects. Apart from this centrifuge, mixers and some fabricated equipments for insect collection and rearing are required. For production of Trichoderma fermentors, laminar flow apparatus etc. are required. All the machinery required are locally manufactured.
4. Raw material
For rearing of insects special diet is required which comprises of pulses, vitamins, antibiotics etc. For production of Trichoderma molasses-yeast medium, is required. All these materials are available locally.
The water requirement is mainly for feed preparation, washing, cleeaning, drinking etc.. Water quality should be tested to establish the suitability.
Power supply is essential for bio-pesticide units. Electricity charges under recurring cost are considered in the models.
Production of bio-pesticides required skilled manpower. There is need for a number of labourers at each stage of production. The project is labour intensive. The manpower requirement is as under:
Scale of production
These biopesticides can be produced on a small or large scale. Small scale production is particularly suitable to village or community level cooperatives, which can produce and distribute these for local use. As the production technology of some of these agents (particularly Trichogramma) is relatively simple, the local farmers/SHGs can be trained to undertake the production. Medium and large scale production can be undertaken by firms, sugar mills cooperatives engaged in the manufacture and distribution of agro-chemicals. Foe example, fertilizer companies, which already possess sufficient in-house technological expertise and marketing resources, are ideally suited for producing biopesticides on a large scale. Similarly, seed companies are particularly well placed for undertaking the production and marketing of Trichoderma.
Considering the negative effects of indiscriminate case of pesticides, importance for organic farming and promotion of sustainable farming practices it is estimated that there will be further scope for new units, particularly in the states of Maharashtra, Gujarat, Rajasthan, Madya Pradesh, Tamil Nadu, AP, UP, West Bengal and Karnataka, where crops such as sugarcane, pulses, cereals and vegetable crops are grown in large scale.
The National Integrated Pest Management Workshop, 1992 estimated the gross demand for a few biopesticides which is given below:
690 million cards
Heliothis NPV (HNPV)
5293 million LE
Spodoptera NPV (SINPV)
3729 million LE
At present, in some states, state government is purchasing the product from the private parties and selling it to the individual farmers at a subsidized rate.
As the bio-control agents are living organisms, it is very important to have effective regulatory measures. The quality control of commercial bioagents must be strictly enforced by the Government. In this connection, the Directorate of Plant Protection Quarantine and Storage, Department of Agriculture and Cooperation, Ministry of Agriculture, GOI have issued guidelines/data requirements for registration of bio-pesticides in the country. As per this, all the units have to meet the Indian standards and technical specifications to be eligible for registration under the Insecticides Act, 1968.
At present, Bacillus thuringensis, neem based formulations, microbial pesticides like fungi, NPV etc., are included in the schedule of Insecticides Act, 1968. This ensures the quality of bio-pesticides at farmers level. The standard parameters, protocols for data generation, guidelines for registration are prepared and circulated to prospective entrepreneurs by MoA. Now as such, any person dealing with biopesticides without registration is ill-legal.
Technical Aspects of Biopesticides
Bio pesticides are living organisms which can intervene the life cycle of insect pests in such a way that the crop damage is minimized. The agents employed as biopesticides are parasites, predetors, fungi, bacteria and viruses which are natural enemies of pests. These bio agents can be conserved, preserved and multiplied under laboratory condition for field release.
Major types of bio-agents available for commercial production
There are different types of bio-agents which can be commercially mass produced for large scale distribution among the farmers for control of insect pests. They are:
- Trichogramma chilonis, T.brasiliensis and T.pretiosum (egg parasites) - for tomato fruit borer
- Trichogramma chilonis - for brinjal shoot and fruit borer, shoot borers of cotton, sugarcane, rice etc.
- Cryptolaemus montrouzieri (Austrtralian ladybird beetle) for control of several species of mealy bugs and soft scales
- Chrysopa spp. (green lacewing bug) - for the control of aphids, white flies etc.
- Virus: Nuclear Polyhedrosis Virus (NPV) - for major polyphagous pest like Helicoverpa armigera (gram pod borer) and Spodoptera litura (Tobacco caterpillar)
- Bacteria: Bacillus thuringiences (B.t) - for control of lepidopterous pests
- Fungi: Trichoderma viride and Trichoderma harziarum against soil borne fungal diseases
- Namatodes : for control of soil-borne grubs, lepidopterans and some foliar pests
Field efficacy of biopesticides
Field efficacy trials have been conducted by State Agricultural Universities and ICAR Research Institutes/Stations to know the extent of pest control by application of biopesticides. The percentage of pest control achieved for selected bio-control agents is as under:
||Efficacy of pest control
Essential characteristics of effective biocontrol agents
- Speed/Mobility to prevent pathogen to develop resistant structures.
- Longevity, enough to protect plant during its vulnerable period, whatever that may be
- Environmental tolerance, to sustain activity under different soil and climatic conditions.
- Mode of Action, varies from pathogen to pathogen, physical contact, chemical nature of killing component.
While using natural enemies, it is important to have fast growing biocontrol organism in the fields which can eventually make the conditions unfavourable for the pathogens proliferation
Technical consultancy for setting up of mass production of bioagents
Setting up of unit for mass production of bio agents especially those which are based on fungi, bacteria and virus is highly technical in nature. The skill required to handle the mass production process is also higher. For scientific and successful setting up of a unit, the entrepreneurs can take consultancy services from the following agencies:
- Project Directorate, Biological control, ICAR, Bangalore
- Indian Institute of Horticulture Research, Hesaragatta, Bangalore
- Central Integrated Pest Management Centre (CIPMC), White field, Bangalore
- Central Institute for Cotton Research (CICR), Nagpur
A. Trichogramma egg parasite
Trichogramma spp. belongs to the category of egg parasitoid of biological agents. Trichogramma spp., the most widely used bio-control agent in the world and is effective against bollworms of cotton, stem borers of sugarcane, fruit borers of fruits and vegetables. It attacks the pest at the egg stage itself and hence damage done by larvae is avoided. It offers a lower cost but more effective plant protection option in comparison to insecticides. Two species i.e., T. chilonis and T. japonicum are predominantly used in India.
Trichogramma are dark coloured tiny wasps and the female wasp lays 20-40 eggs into the host's eggs. The entire cycle is completed within 8-12 days. The tiny adult wasps search for the host (pest) eggs in the field and lay their eggs into the eggs of the pests. The parasitised host's eggs turn uniformly black in 3-4 days. The Trichogramma eggs on hatching, feed the embronic contents of host's egg, completes its development and adult comes out of the host egg by chewing a circular hole. A single Trichogramma, while multiplying itself, can thus destroy over 100 eggs of the pest.
2. Major equipment needed
Equipments like semi-automatic corcera rearing cages, trays, iron racks, hot air oven, air conditioner, UV chamber, incubator, moth breeding tins, grinder, mating chambers, parasitization jars, refrigerator, wire mesh, netlon etc. are required for mass rearing of corcera and Trichogramma production.
3. Steps involved in production
i) Identification of host
The Trichogramma of multiplication starts with identification of a suitable host species, with the following characteristics :
In India Corcera cephalonica, a stored grain pest has been used for mass multiplication of targetted species.
ii) Rearing of host insect
The host rearing containers are made of materials which are non-toxic, cheap and optimum sized to permit mating and host searching and amenable to easy cleaning. Most commonly used cages are wooden cages, which are now replaced with semi automatic corcyra rearing cages. The nuclear culture, i.e. eggs of Corcyra cephalonica are introduced in rearing cages. In the model use of semiautomatic rearing cages of 30 no. is considered.
iii) Preparation of feed material
Corcyra feed may be prepared from bold white sorghum grains without any insecticide residues. This can be tested by taking a sample of 100 g from each bag. The crushed sample is fed to 20 number of 1st/2nd instar Corcyra larvae for 2-3 days. Based on the mortality of the larvae, suitability of grains may be decided. The requisite quantum of sorghum is milled to make 3-4 pieces of each grain. Sorghum grains are heat sterilised in oven at 1000C for 30 minutes and the grains are sprayed with 0.1% formalin. This treatment helps in preventing the growth of moulds as well as to increase the grain moisture to the optimum (15-16%), which was lost due to heat sterilisation. Then grains are air dried.
iv) Corcyra charging
In each rearing cage, 7.5 kg of sorghum grains are filled and charged with 0.5cc eggs (1cc = 20,000 eggs) of mother culture. Yeast, groundnut kernel and streptomycin is added to enhance egg laying capacity of the adult moths and for enriching the diet.
v) Collection of moths
After about 40 days of charging, moths start emerging and the emergence continues for two months. 10 to 75 moths emerge daily with the peak emergence being between 65th and 75th day.
Collect the moths daily and transfer to the specially designed oviposition cages for egg laying. Roughly 2000-3000 pairs of moth can be placed in one chamber. Moth emergence reduces after 100 days of initial infestation and cages are released for cleaning.
vi) Collection of eggs
Eggs are collected by means of manual suction and are placed in tubes and counted with measuring cylinder. Approximately one cc of eggs of Corcyra counts about 20,000 at the fresh harvest. After that due to shrinkage of eggs the count may be increased. The present model assumes 20,000 eggs per one cc for calculation purpose. The final output of Corcyra eggs from one cage has been assumed at 7.5 cc.
4. Production of Trichocards
The demand for Trichocards will start from the onset of kharif season and extends to rabi season. The summer season vegetables offer an extra demand.
i) Egg preparation
The eggs of Corcyra thus collected are cleaned to make it free from insect scales etc. They are sieved thrice and then poured on a plain paper. By slowly tapping eggs come downward stick on to gummed card. Thus, the cleaned eggs are spread on the gummed cards (15 cm x 10 cm) with the help of screen. These eggs of Corcyra are exposed to UV rays of 15 watt UV tube for 45 minutes to prevent hatching. While UV exposure, egg card should be kept about 12-15 cm away from tube.
ii) Introduction of Trichogramma
After the sterilisation the egg cards are placed in plastic bottles and are introduced with nucleus culture of Trichogramma species of egg or pupal stage. The ratio of host egg and parasite adult should be maintained at 1:5.
iii) What is a tricho card ?
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). About 12,000 Trichogramma adults emerge out from this card in 7-8 days after parasitisation. To delay the emergence of Trichogramma, these cards can be stored in refrigerator at 5-100C for 10-15 days. On removing the cards to room temperature, the parasitoids emerge normally. Trichocards have a shelf life of 2-3 days. However, these can be stored in a refrigerator for a period of 1 month without any spoilage.
For controlling sugarcane early shoot borer : Start releasing 6,000 parasites per week per acre area, for a period of 5 weeks, starting from 4th week of planting i.e., as soon as the adult male moths of early shoot borer are noticed in the field. Totally 30,000 parasites are to be released per acre. More parasites may also be released depending upon the crop and pest density.
In cotton- The Trichocards are released in the field at 45 days after sowing @ 5 cards / ha (one lakh eggs). In total three release are necessary.
6. How to use 'Tricho card'
The cards are to be used before the emergence of the adult parasite. Cut or tear each Tricho card into small pieces and distribute them all over the field. The pieces may be stapled to sugarcane leaf at 7-8 m distance. Care is to be taken to release the parasites either in morning or evening i.e., during cool hours, in windward direction and there should not be any pesticide spray. Before releasing the parasite, the infected shoots are to be cut to ground level and buried inside the soil so as to avoid secondary infestation.
7. Advantages of using Tricho cards
- Less cost, more effective.
- field application (releases) is very simple as compared to other methods.
- Records show higher yield in sugarcane (about 4-5 tonnes), as secondary infestation is avoided while using Tricho cards.
- Cost of pest control is very nominal.
- Added to all these, environmental pollution is avoided.
The following precautions are required to be taken while using Trichocards :
- Trichocards should be packed in such a way that the parasitised surface is on the inner side.
- Emergence date should be specified on cards for the guidance of the users.
- Trichocards should be stapled on the inner-side of the leaf to avoid direct sunlight.
- Card should be stapled in morning hours and just before emergence to avoid predation.
- Farmers should refrain from using pesticides in the field where Trichogramma are released. If need arises selective / safer pesticides can be used and it is to be ensured that pesticides are used 15 days before or after release of Trichogramma
B. Chrysopid predetors
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.
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.
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.
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
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.
At least 1000 eggs or larvae may be used per acre.
C. Ladybird beetle
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.
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
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
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).
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.
In India, extensive research has been conducted on the use of NPVs for tackling two major pests namely Spodoptera litura and Helicoverpa armigera.
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
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.
7.2 Details of mass production
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:-
||Chickpea (Kabuli chenna) flour
||Methyl para-hydroxt benzoate
||10% formaldehyde solution
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.
Mass production of eggs
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.
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.
Rearing of larvae on semi-synthetic diet
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.
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.
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
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.
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.
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.
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.
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.
E. Technology for mass production of Trichoderma fungi
Crop losses due to soil borne plant pathogens worldwide are Pythium spp., Furarium oxysporum, sclerotium rolfsii, Rhizoctonia solani and Phytophthora spp. These fungi pathogens generally cause wilt disease in many crops. Trichoderma, a fungi, which grow saprophytically in soils have proved as an effective biocontrol agent of wilt diseases.
Trichoderma spp. are commonly found in almost any soil and other natural habitats consisting of organic matter such as decaying bark, plant material, etc. They grow trophically towards hyphae of other pathogenic fungi, coil them and degrade their cell walls. This process is called "mycoparasitism", which limits the growth and activity of plant pathogenic fungi. In addition, they produce toxic metabolites which protect the seeds from soil borne pathogenic fungi, by forming a protective coating on them.
Trichoderma spp. are saprophytic fungi that grow best in neutral and acid soils and thrive well in moist conditions.
The important species available for mass production are Trichoderma viride and Trichoderma harzianum
Equipments required: Equipments like fermentor, rotary mixer, auto packer, rotary shaker, laminar flow, water distillation unit, refrigerator, haemo cytometer etc. are required for the production of Trichoderma fungi.
2. Major steps in production process
Inoculation | Fermenter run | Harvesting | Blending | Drying and Packing
3. Outlines for production of Trichoderma
- The pure mother culture of Trichoderma fungi is being maintained in Agri. Universities, IARI, some ICAR institutions (like PDBC, Bangalore) etc. The mother culture can be purchased from the identified sources. They have to be further sub-cultured and maintained purely for mass production by adopting standard techniques under the supervision of trained microbiologist / pathologist.
- The culture has to be mass multiplied in two levels namely (i) at primary level using shakers in flasks and (ii) secondary stage multiplication in fermenters. The important factor in this is the preparation of growing medium in which the culture is mass multiplied. For Trichoderma Fungi, the growing media used in the model is molasis and protein material.
- After the growing media is formulated and sterilised in fermenter, it is inoculated using the culture multiplied in the flasks.
- The molasis based culture media is continuously aerated by passing sterile air from compressors. After about 3-4 days fermentation period, the culture will be ready for packing in a carrier material.
- While the inoculated culture is gathering ready in the fermenters, the carrier material is sterilised in autoclaves and kept ready for mixing the culture. Talk powder is reported to be the commonly used carrier material for Trichoderma Fungi.
- The cultured (fungi) and sterilised carriers are mixed mechanically in a blender and the material is packed using semi automatic packing and sealing machine.
Talc based formulations of the fungal antagonists are applied at the rate of 4gm per kg of seed for controlling soilborne plant diseases. Mix the powder with sufficient quantity of water to make slurry for treating seed before sowing.
5. Advantages of Trichoderma applicatiom
- Can be used along with organic manure
- Trichoderma spp. are also known to suppress plant parasitic nematodes (roo-knot nematodes).
- Lower cost and longer efficacy than fungicides
- Does not lead to development of resistance in plant pathogens
- No physotoxic effects
- Minimises losses and cost of production and increases yield & profit.
- Promoter plant growth
Trichoderma spp. suppress the activity of soil borne fungal pathogens, especially Rhizoctania solani and Pythium spp. and protect transplanted seedlings by colonizing their roots.
Seed treatment is an alternative approach to introduce Trichoderma spp. into the soil. This method requires smaller amounts of biological material than soil treatment. Unlike chemical fungicides, Trichoderma spp. provide long term protection without any adverse side effects.
F. Sex pheromone traps of Helicoverpa armigera and Spodoptera litura
Sex pheromones are single or complex blend of different chemicals released by one insect to attract the opposite sex of the same species. In general, females (especially the moths) emit sex attractants to attract males for mating. Sex pheromones are artificially synthesized in the laboratories and supplied as sex pheromone lures. Such pheromones are placed in the field to attract trap and kill the males, thus matting is not allowed. Hence, sex pheromone traps can be considered as a key component in Integrated Pest Management (IPM).
Ready-to-use Sex pheromone lures and traps are available for Helicoverpa armigera (attacking crops like cotton, redgram, tomato, okra, sunflower, chillies, maize, sorghum etc.) and spodoptera litura (attacking crops like tobacco, groundnut, sunflower, cotton, cabbage, beetroot, cauliflower, etc.)
2. Advantages of pheromone lures
- No harmful effects to beneficial insects, non-target organism or an environment.
- Helps in monitoring & early detection of pests (at moth stage only)
- Helps in scheduling pest control measures
- Reduction in usage of insecticides
- Much simpler
3. Equipment needed
Only micropippets are required in addition to rubber septas, traps and pouches.
4. Production of Pheromone Traps
Sex pheromones are insect specific, produced artificially in laboratories and they are generally imported. In India, it is available from National Chemical Laboratory (NCL), Pune. Chemicals obtained from laboratory is diluted to the required dosage and filled into plastic lures with the help of micro pippets and closed with rubber septa. Lures are individually sachet packed and should be stored under refrigerated conditions when not in use.
5. Field application
Lures containing sex pheromones are placed into insect trap and erected in the field at a recommended spacing. The lure will release the sex pheromane at a constant rate over a period of 2-4 weeks. Male months are attracted and while attempting for matting, fall into a container having pesticide. Thus the female moths in the field are deprived of successful mates and fail to reproduce or lay viable eggs.
Timely use of sex pheromone helps in early detection and prompt action against pests. In general, 2-3 traps / acre are recommended for 'monitoring' or more for 'mass-trapping'. These are arranged such that the trap is 1-2 feet above the crop canopy. On the field each lure is effective for atleast 15 days. Change the lures once in two weeks.