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PRODUCTION TECHNIQUES OF BIOCONTROL AGENTS TRICHOGRAMMA TRICHODERMA

SUBMITTED TO: Proff. N.N Singh Proff. P.P Singh

SUBMITTED BY:

Sonika patel I.D= 10136 B.Sc. ( Ag) 4th

year

CONTENTS
INTRODUCTION IMPORTANCE OF BIO-PESTICIDES ADVANTAGE OF BIO-PESTICIDES STATUS OF BIO-PESTICIDES IN INDIA SCOPE FOR COMMERCIAL PRODUCTION OF BIO-PESTICIDE MARKET POTENTIAL OF BIO-PESTICIDES BASIC REQUIREMENT FOR ESTABLISHMENT OF BIOPESTICIDE UNIT CHARACTERISTICS OF EFFECTIVE BIOCONTROL AGENT PRODUCTION TECHNOLOGIES 1.TRICHOGRAMMA EGG PARASITE 2MASS PRODUCTION OF TRICHODERMA FUNGI REFERENCES

INTRODUCTION

Pest problem is one of the major constraints for achieving higher production in agriculture crops. India loses contributed for minimising yield losses. The pesticides, which are needed to be applied carefully, only when 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. Th 570 gms per ha. as compared to developed countries like Japan, Thailand and Germany where the consumpti maintenance is alarming. Interms of value, much of the pesticide application is accounted for by a few crops.

Pesticides or chemicals are meant to control harmful pests such as insects, nematodes, diseases, weeds etc. H animals. This has adversely affected the ecological balance resulting in pest resurgence, development of resis Maharashtra.

In view of the several disadvantages associated with the unscientific use of pesticides in agriculture, there is increase in cost of cultivation/low profit making by farmers has led to the exploration of eco-friendly pest ma emphasis on the use of insecticides. In simple terms "IPM is the right combination of cultural, biological and prevention, observation and intervention. The IPM seems to be the only answer to counter some of the major

Importance of Bio-pesticides

They are also specific in their action and perish once their feed (i.e. the pest) is exhausted. Thus they are base

Among the alternatives, biological control of pests is one of the important means for checking pest problems

Bio pesticides are living organisms which can intervene the life cycle of insect pests in such a way that the c they complement and supplement other methods of pest control. Utilisation of naturally occurring parasites, release. Once these bio-agents are introduced in the field to build their population considerably, they are capa

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 produc There are a number of instances where bio control agents have been successfully employed in India. Some ex

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 na

Trichogramma, which feeds on the eggs of sugarcane borers, has been used against the borers in the Similarly Trichogramma, Bracon, Chelonus and Chrysopa spp. are being used for the control of cotto The sugarcane scale insect has been controlled with the help of predatory coccinellid beetles in UP, W

The popularity of biopesticides has increased in recent years, as extensive and systematic research has greatl

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 large scale investment and private participation.

Some of the local small scale industries have already started production and marketing of Trichoderma viride demand is on the increase every year. Technology Model 1

Bio-agent i.Trichogramma spp. (egg parasite) ii. Crysoperla carnea (Chrysopid predetor) iii. Cryptolaemus montrouzieri (Ladybird beetle) i. NPV of Helocoverpa armigera & Spodoptera litura

ii. Trichoderma Fungal spp. iii. Pheromone lures for Helicoverpa armigera & Spodoptera litura

Pro Ma gra Ma Ma The sme cen Mu Sex sep

The technology used were indigenous and the scientific aspects of production were standardised by ICAR Re

Objectives of Biopesticide Project Models


The primary objective of biopesticide projects is to establish the bankability of mass multiplication of To serve as guidelines for extending financial assistance to entrepreneurs who may be interested in se 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 biop

1. Land Land is required for construction of culture and rearing rooms, processing room, laboratory, office etc. In the

2. Building and civil works Biopesticides production involves rearing of insects. Hence, the basic infrastructure to be created includes on problems. For the proposed installed capacity, an estimated built up area of about 1000 sq ft is required for m Other utilities required are power, water and vehicle. Among others, the civil structure may be designed to ha may be restricted in such a way to prevent any contamination. In other words, one should not enter host cultu

3. Plant and Machinery There is no requirement of heavy plant and machinery. Racks, trays and other facilities are required for rearin 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 produc

5. Water The water requirement is mainly for feed preparation, washing, cleeaning, drinking etc.. Water quality should

6. Power Power supply is essential for bio-pesticide units. Electricity charges under recurring cost are considered in th

7. Manpower Production of bio-pesticides required skilled manpower. There is need for a number of labourers at each stag

Scale of production

These biopesticides can be produced on a small or large scale. Small scale production is particularly suitable simple, the local farmers/SHGs can be trained to undertake the production. Medium and large scale productio house technological expertise and marketing resources, are ideally suited for producing biopesticides on a lar

Market Potential

Considering the negative effects of indiscriminate case of pesticides, importance for organic farming and pro AP, UP, West Bengal and Karnataka, where crops such as sugarcane, pulses, cereals and vegetable crops are

The National Integrated Pest Management Workshop, 1992 estimated the gross demand for a few biopesticid

At present, in some states, state government is purchasing the product from the private parties and selling it t

of bio-pesticides in the country. As per this, all the units have to meet the Indian standards and technical spec

At present, Bacillus thuringensis, neem based formulations, microbial pesticides like fungi, NPV etc., are inc prepared and circulated to prospective entrepreneurs by MoA. Now as such, any person dealing with biopest

Major types of bio-agents available for commercial production


Parasitoids Trichogramma chilonis, T.brasiliensis and T.pretiosum (egg parasites) - for tomato fruit borer

There are different types of bio-agents which can be commercially mass produced for large scale distribution

Predato

Trichogramma chilonis - for brinjal shoot and fruit borer, shoot borers of cotton, sugarcane, rice etc.

Field efficacy of biopesticides Field efficacy trials have been conducted by State Agricultural Universities and ICAR Research Institutes/St

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 comp

While using natural enemies, it is important to have fast growing biocontrol organism in the fields which can

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 v 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

PRODUCTION TECHNOLOGIES
A. Trichogramma egg parasite

1. Introduction Trichogramma spp. belongs to the category of egg parasitoid of biological agents. Trichogramma spp., the m

itself and hence damage done by larvae is avoided. It offers a lower cost but more effective plant protection o

Trichogramma are dark coloured tiny wasps and the female wasp lays 20-40 eggs into the host's eggs. The en black in 3-4 days. The Trichogramma eggs on hatching, feed the embronic contents of host's egg, completes

2. Major equipment needed Equipments like semi-automatic corcera rearing cages, trays, iron racks, hot air oven, air conditioner, UV ch

3. Steps involved in production i) Identification of host The Trichogramma of multiplication starts with identification of a suitable host species, with the following c

easily available. easy to culture with the locally available material. should yield maximum host egg/larvae/pupae per unit cost.

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 m i.e. eggs of Corcyra cephalonica are introduced in rearing cages. In the model use of semiautomatic rearing c iii) Preparation of feed material Corcyra feed may be prepared from bold white sorghum grains without any insecticide residues. This can be grains may be decided. The requisite quantum of sorghum is milled to make 3-4 pieces of each grain. Sorghu 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

v) Collection of moths After about 40 days of charging, moths start emerging and the emergence continues for two months. 10 to 75 Collect the moths daily and transfer to the specially designed oviposition cages for egg laying. Roughly 2000

vi) Collection of eggs Eggs are collected by means of manual suction and are placed in tubes and counted with measuring cylinder. 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 summ

i) Egg preparation The eggs of Corcyra thus collected are cleaned to make it free from insect scales etc. They are sieved thrice a screen. These eggs of Corcyra are exposed to UV rays of 15 watt UV tube for 45 minutes to prevent hatching

ii) Introduction of Trichogramma After the sterilisation the egg cards are placed in plastic bottles and are introduced with nucleus culture of Tr

iii) What is a tricho card ? The parasitisation of Trichogramma spp., in laboratory condition on one cc eggs of Corcyra cephalonica, wh this card in 7-8 days after parasitisation. To delay the emergence of Trichogramma, these cards can be stored

in a refrigerator for a period of 1 month without any spoilage.

5. Dosage For controlling sugarcane early shoot borer : Start releasing 6,000 parasites per week per acre area, for a peri 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

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 p cool hours, in windward direction and there should not be any pesticide spray. Before releasing the parasite, 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 Cost of pest control is very nominal. Added to all these, environmental pollution is avoided.

8. Precautions 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

E. Technology for mass production of Trichoderma fungi

1. Introduction Crop losses due to soil borne plant pathogens worldwide are Pythium spp., Furarium oxysporum, sclerotium

an effective biocontrol agent of wilt diseases.

Trichoderma spp. are commonly found in almost any soil and other natural habitats consisting of organic ma which limits the growth and activity of plant pathogenic fungi. In addition, they produce toxic metabolites w

Trichoderma spp. are saprophytic fungi that grow best in neutral and acid soils and thrive well in moist cond 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, wat 2. Major steps in production process Inoculation | Fermenter run | Harvesting | Blending | Drying and Packing

MATERIALS AND METHODS

Isolation and Identification of T. Viride


Fungal species Trichoderma viride was isolated from soil samples by using potato dextrose agar (PDA) medium. Samples were inoculated over plates by multiple tube dilution technique (MTDT) and the plates were incubated at 26C for 4 days. The fungal colonies which were picked up and purified by streaking and incubated at 26C for 7-8 days. Green conidia forming fungal bodies were selected and microscopic observation was identified to be Trichoderma viride. The culture was maintained on PDA slant METHODOLOGY Collection of soil (100g)-2g is necessary Dissolve 2g soil in 10ml sterilized water Took 1ml solution and diluted in 9ml water Repeat dilution till 10 times Incubated in ROSE BENGAL MEDIUM

PREPARATION OF ROSE BENGAL MEDIUM

Formula / Liter Enzymatic Digest of Soybean meal Dextrose .

5g 10 g

Monopotassium Phosphate

1g

Magnesium Sulfate

0.5 g

Rose Bengal

0.05 g

Chloramphenicol

0.1 g

Agar

15.5 g

AUTOCLAVING OF MEDIUM
Rose Bengal medium should be autoclave at 121.6 before incubation

Maintenance of culture
A loopful of inoculum from sub cultured plates of Trichoderma viride were transferred to Potato Dextrose Agar (PDA) slants and maintained as pure culture.

PDA CULTURE FOR 1 LIT. 200 g 18-20 g 18-20 g 0.05 mg potato dextrose Agar Streptomycin

the fungus was cultured on PDA medium. The medium was sterilized at 15 psi for 30 min in autoclave, poured to sterilized plates, cooled and inoculated with pure culture of the fungus under aseptic con The plates were then incubated at room temperature (262C) for ten days. After complete sporulation, conid the medium were harvested by washing them thoroughly with sterilized water containing Tween-20 (0.2%) f immediate use. Otherwise, spores were harvested with the help of a small sterile metal spatula. Harvested co were air dried under laminar air flow and stored in a small air tight screw cap vials (10 cm with 2.5 cm diam refrigerator at 4C before using for further studies. Colony forming units (cfu) were estimated by plating tech Suspension of spores was made using distilled water with Tween-20 (0.2%) and filtered through a double lay muslin cloth.

Grain Medium

Three Grains viz rice, wheat and pulses were used for estimating the biomass of Trichoderma viride at 26 C of each grain was washed well and boiled in distilled water for 1 hr. and then mesh properly and filter it, now makeup 1 liter with distilled water. Now these grain mediums were packed separately in individual 500 ml c flask for Trichoderma viride. They were plugged with cotton wool and autoclaved at 15 psi for 1hr at 121C cooling, 1 gm of the fungal culture was inoculated into each flask, separately. All these action were done und laminar air flow chamber. They were incubated in BOD incubator at 26C for 3 weeks. To avoid clumping, a

days of inoculation, the flasks were shaken vigorously to separate the culture and to break the mycelial mat. After 14 days of incubation, the mycelial mat appeared in flasks. Now it was grow well for 21 days. The flas shaken in mechanical shaker for 10 min. The suspension was filtered through double layered muslin cloth an taken biomass in each grain medium.

Outlines for production of Trichoderma


The pure mother culture of Trichoderma fungi is being maintained in Agri. Universities, IARI production by adopting standard techniques under the supervision of trained microbiologist / p The culture has to be mass multiplied in two levels namely (i) at primary level using shakers i 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 cu The molasis based culture media is continuously aerated by passing sterile air from compresso While the inoculated culture is gathering ready in the fermenters, the carrier material is sterili The cultured (fungi) and sterilised carriers are mixed mechanically in a blender and the mater

Dosage Advantages of Trichoderma applicatiom


Talc based formulations of the fungal antagonists are applied at the rate of 4gm per kg of seed for controlling

Ecofriendly 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

Application

Soil application Trichoderma spp. suppress the activity of soil borne fungal pathogens, especially Rhizoctania solani and Pyth

Seed treatment Seed treatment is an alternative approach to introduce Trichoderma spp. into the soil. This method requires s

LIST OF REGISTERED BIOPESTICIDES & THEIR FORMULATIONS FOR

Microbial biopesticides 1. Bacillus thuringiensis var.israelensis (a Serotype H-14, Strain 164, WP (b)Serotype H-14, Strain VCRC B-17, slow release granules (c)Serotype H-14, Strain VCRC B-17, WP (d)Serotype H-14, 12 AS (e)Serotype H14, Strain VCRC B-17, 5 AS (f) Serotype H-14, 5% WP 2. Bacillus thuringiensis var.kurstaki (a)Strain A-97, serotype H-3a, 35 WP (b)Serotype-(3a, 3b 3c), Strain DOR-bt-1, 0.5% WP (c) Serotype 3a, 3b, Strain HD-1 3.5% ES (d) Serotype 3a, 3b, Strain Z-52 3. 4. 5. Bacillus thuringiensis var. galleriae, Serotype 3a, 3b, Strain R 1593m 1.3% FC Bacillus sphaericus Beauveria bassiana (Strain Umiam) (Strain AAI) Stain T-stains 6. Trichoderma viride (Strain TNAU) (Strain KAU) (Strain T-4 (MP)) 1% WP, 0.5% WS, 1.5% WP, 0.5%WP 1% WP 5%WP Serotype 5a, 5b, Strain 1593m 1.3% EC 1% WP, 1.15% WP, 2.15% WP, 10% SC, 1.5% WP, 5% WP 1.15% WP 1.15%WP

7.

Verticillum lecanii (Strain: Umiam) 1.15% WP

8.

NPV of Helicoverpa armigera (Strain:GBS/HNPV-01) 0.43% AS, 0.5% AS, 0.64% AS, 1.0% AS, 2% AS

9. 10.

NPV of Spodoptera litura Trichoderma harzianum (Strain: IIHR-Th-2)

0.5% AS

1% WP, 2% WP, 0.5% WS 1.0% WP, 1.15% WP 0.5% WP, 1.0% WP, 1.25% WP, 1.5% WP, 1.25% WP 0.5% WP

11. 12.

Metarrhizium anisopliae Pseudomonas fluorescence (Strain TNAU) (Strain KAU)

13. 14.

Verticillum chlamydosporium Paecilomyces lilacinus (Strain: IIHR)

1% WP 0.5% WP, 1.15% WP,1.0 % WP

15. 16. 17.

Ampelomyces quisqalis Bacillus subtilis

2 % WP 1.5 % AS

Photorhabdous luminescens akhurstii strain K-1 52% (w/v) EC

Botanical biopesticides

18. 19.

Azadirachtin Pyrethrum/Pyrethrin

0.03% EC, 0.3% , 0.1% EC, 0.15% EC, 1% EC, 1%, 2.5%, 25% 0.2% Aerosol, 0.4% Mat, 0.3% Agarbatti, 0.2% Coil, 0.05% Spray, 25% Extract, 0.2% DP, 0.2% PH, 2% EC & 2.5% EC

RESULT AND DISCUSSION

The conventional chemical pesticides has not only enhanced the food production, but also adversely affected the environment and non-target organisms. Due to the side-effects of chemical pesticides, the sustainable crop production through eco-friendly pest management is essentially required in recent scenario. Among the several micro-organisms viz., bacteria, fungi, virus, protozoans and entomopathogenic nematodes, a few have been systematically studied for their effective beneficial characteristics.Trichogramma and Trichoderma viride is very promising method against soil borne plant parasitic fungi and various diseases and pest. The fungal pathogens play a major role in the development of disea many important field and horticultural crops; resulting in severe plant yield losses. Intensified used of fungicides has resulted in accumulation of toxic compound potentially hazardous to human and environment an also in the buildup of resistance of the pathogens. In order to tackle these national and global problems, effective alternatives to chemical control are being employed. Biological control by an antagonism is a potential, no chemical and ecofriendly approach for managing plant diseases Therefore considering the cost of chemical pesticides and hazardous involves, biological control of plant diseases appears to be an effective and ecofriendly approach being practice world over. Further biological control strategy is highly compatible with sustainable agriculture and has a major role to play as a component of integrated pest management (IPM) programme.

. REFERENCES: www.nabard.org www.insectariumvirtual.com www.bugsforbugs.com.au/images/Heliothis.JPG www.geocities.com www.treknature.com www.richard-seaman.com

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