Introduction

5/26/2018 Introduction

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PRODUCTION TECHNIQUES OF BIOCONTROL AGENTS

TRICHOGRAMMA TRICHODERMA

SUBMITTED TO: SUBMITTED BY:

Proff. N.N Singh

Proff. P.P Singh Sonika patel

I.D= 10136

B.Sc. ( Ag) 4th

year


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P


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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


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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 consumptmaintenance 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.

animals. This has adversely affected the ecological balance resulting in pest resurgence, development of resi

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 m

emphasis on the use of insecticides. In simple terms "IPM is the right combination of cultural, biological an

prevention, observation and intervention. The IPM seems to be the only answer to counter some of the majo


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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 bas

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

Bio pesticidesare living organisms which can intervene the life cycle of insect pests in such a way that the

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 cap

Major advantages of bio pesticidesBio-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.


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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 e

Growth of lantana weed was controlled by using the bug Telonemia scrupulosa Sugarcane pyrillahas been successfully controlled in a number of States by the introduction of its n


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Trichogramma, which feeds on the eggs of sugarcane borers, has been used against the borers in th

Similarly Trichogramma, Bracon, Chelonus and Chrysopa spp. are being used for the control of cott

The sugarcane scale insect has been controlled with the help of predatory coccinellidbeetles in UP,

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 t

large scale investment and private participation.

Some of the local small scale industries have already started production and marketing of Trichoderma virid

demand is on the increase every year.

TechnologyModel Bio-agent Pr

1 i.Trichogramma spp. (egg parasite) M

gra

ii. Crysoperla carnea (Chrysopid predetor) M

iii. Cryptolaemus montrouzieri (Ladybird beetle) M

2 i. NPV of Helocoverpa armigera & Spodoptera litura Th

sm

ce

ii. Trichoderma Fungal spp. M

iii. Pheromone lures for Helicoverpa armigera & Spodoptera litura Sesep

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

Objectives of Biopesticide Project Models

The primary objective of biopesticide projects is to establish the bankability of mass multiplication o

To serve as guidelines for extending financial assistance to entrepreneurs who may be interested in s

To promote setting up of more bio-control production units

To disseminate widely the technology

Basic requirements for establishment of Biopesticide unitsBased on the field visits to bio-control production units and in line with the technology and objective of bio

1. Land

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


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2. Building and civil worksBiopesticides production involves rearing of insects. Hence, the basic infrastructure to be created includes o

problems. For the proposed installed capacity, an estimated built up area of about 1000 sq ft is required for

Other utilities required are power, water and vehicle. Among others, the civil structure may be designed to h

may be restricted in such a way to prevent any contamination. In other words, one should not enter host cult

3. Plant and MachineryThere is no requirement of heavy plant and machinery. Racks, trays and other facilities are required for reari

etc. are required. All the machinery required are locally manufactured.

4. Raw materialFor rearing of insects special diet is required which comprises of pulses, vitamins, antibiotics etc. For produ

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

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

7. Manpower

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

Scale of productionThese 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 producti

house technological expertise and marketing resources, are ideally suited for producing biopesticides on a la

Market PotentialConsidering the negative effects of indiscriminate case of pesticides, importance for organic farming and pr

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 biopestici

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

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

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


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Major types of bio-agents available for commercial productionThere are different types of bio-agents which can be commercially mass produced for large scale distributio

Parasitoids Predat

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.

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

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 com

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

Technical consultancy for setting up of mass production of bioagentSetting up of unit for mass production of bio agents especially those which are based on fungi, bacteria andservices 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


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PRODUCTION TECHNOLOGIES

A. Trichogramma egg parasite

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


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

Trichogramma are dark coloured tiny wasps and the female wasp lays 20-40 eggs into the host's eggs. The e

black in 3-4 days. The Trichogramma eggs on hatching, feed the embronic contents of host's egg, completes

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

3. Steps involved in production

i) Identification of hostThe 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 insectThe host rearing containers are made of materials which are non-toxic, cheap and optimum sized to permit

i.e. eggs of Corcyra cephalonica are introduced in rearing cages. In the model use of semiautomatic rearing

iii) Preparation of feed materialCorcyra 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. Sorgh

grain moisture to the optimum (15-16%), which was lost due to heat sterilisation. Then grains are air dried.

iv) Corcyra chargingIn each rearing cage, 7.5 kg of sorghum grains are filled and charged with 0.5cc eggs (1cc = 20,000 eggs) o

v) Collection of mothsAfter about 40 days of charging, moths start emerging and the emergence continues for two months. 10 to 7

Collect the moths daily and transfer to the specially designed oviposition cages for egg laying. Roughly 200

vi) Collection of eggsEggs 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 TrichocardsThe demand for Trichocards will start from the onset of kharif season and extends to rabi season. The summ

i) Egg preparationThe eggs of Corcyra thus collected are cleaned to make it free from insect scales etc. They are sieved thrice

screen. These eggs of Corcyra are exposed to UV rays of 15 watt UV tube for 45 minutes to prevent hatchin

ii) Introduction of TrichogrammaAfter the sterilisation the egg cards are placed in plastic bottles and are introduced with nucleus culture of T

iii)What is a tricho card ?

The parasitisation of Trichogramma spp., in laboratory condition on one cc eggs of Corcyra cephalonica, wthis card in 7-8 days after parasitisation. To delay the emergence of Trichogramma, these cards can be store


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

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

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). I

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. Advantagesof 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. PrecautionsThe 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. I

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


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an effective biocontrol agent of wilt diseases.

Trichoderma spp. are commonly found in almost any soil and other natural habitats consisting of organic m

which limits the growth and activity of plant pathogenic fungi. In addition, they produce toxic metabolites

Trichoderma spp. are saprophytic fungi that grow best in neutral and acid soils and thrive well in moist condThe 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, wa

2. Major steps in production processInoculation | Fermenter run | Harvesting | Blending | Drying and Packing

MATERIALS AND METHODS

Isolation and Identification ofT. Vir ide

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 at26C 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 / LiterEnzymatic Digest of Soybean meal 5 g

Dextrose . 10 g

Monopotassium Phosphate 1 g

Magnesium Sulfate 0.5 g

Rose Bengal 0.05 g


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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 potato

18-20 g dextrose

18-20 g Agar

0.05 mg 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 co

The plates were then incubated at room temperature (262C) for ten days. After complete sporulation, coni

the medium were harvested by washing them thoroughly with sterilized water containing Tween-20 (0.2%)

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 tec

Suspension of spores was made using distilled water with Tween-20 (0.2%) and filtered through a double la

muslin cloth.

Grain Medium

Three Grains viz rice, wheat and pulses were used for estimating the biomass of Trichoderma viride at 26

of each grain was washed well and boiled in distilled water for 1 hr. and then mesh properly and filter it, no

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 unlaminar air flow chamber. They were incubated in BOD incubator at 26C for 3 weeks. To avoid clumping,


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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 a

taken biomass in each grain medium.

.

Outlines for production of Trichoderma

The pure mother culture of Trichoderma fungi is being maintained in Agri. Universities, IAR

production by adopting standard techniques under the supervision of trained microbiologist /

The culture has to be mass multiplied in two levels namely (i) at primary level using shakers

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 c

The molasis based culture media is continuously aerated by passing sterile air from compress 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

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

Advantages of Trichoderma applicatiom

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 applicationTrichoderma spp. suppress the activity of soil borne fungal pathogens, especially Rhizoctania solani and Pyt

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

LIST OF REGISTERED BIOPESTICIDES & THEIR FORMULATIONS FO


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Microbial biopesticides

1. Bacillus thuringiensisvar.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 thuringiensisvar.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. Bacillus thuringiensis var. galleriae, Serotype 3a, 3b, Strain R 1593m 1.3% FC

4. Bacillus sphaericus Serotype 5a, 5b, Strain 1593m 1.3% EC

5. Beauveria bassiana 1% WP, 1.15% WP, 2.15% WP, 10% SC, 1.5% WP,

(Strain Umiam) 5% WP

(Strain AAI) 1.15% WP

Stain T-stains 1.15%WP

6. Trichoderma viride

(Strain TNAU) 1% WP, 0.5% WS, 1.5% WP, 0.5%WP

(Strain KAU) 1% WP

(Strain T-4 (MP)) 5%WP

7. Verticillum lecanii

(Strain: Umiam) 1.15% WP


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8. NPV of Helicoverpa armigera

(Strain:GBS/HNPV-01) 0.43% AS, 0.5% AS, 0.64% AS,

1.0% AS, 2% AS

9. NPV of Spodoptera litura 0.5% AS

10. Trichoderma harzianum

(Strain: IIHR-Th-2) 1% WP, 2% WP, 0.5% WS

11. Metarrhizium anisopliae 1.0% WP, 1.15% WP

12. Pseudomonas fluorescence 0.5% WP, 1.0% WP, 1.25% WP,

(Strain TNAU) 1.5% WP, 1.25% WP

(Strain KAU) 0.5% WP

13. Verticillum chlamydosporium 1% WP

14. Paecilomyces lilacinus 0.5% WP, 1.15% WP,1.0 % WP

(Strain: IIHR)

15. Ampelomyces quisqalis 2 % WP

16. Bacillus subtilis 1.5 % AS

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

Botanical biopesticides

18. Azadirachtin 0.03% EC, 0.3% , 0.1% EC, 0.15% EC, 1% EC, 1%, 2.5%, 25%

19. Pyrethrum/Pyrethrin 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


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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 severalmicro-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 dise

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 buildupof 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 diseasesTherefore 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.


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.

REFERENCES:

www.nabard.org

www.insectariumvirtual.com

www.bugsforbugs.com.au/images/Heliothis.JPG

www.geocities.com

www.treknature.com

www.richard-seaman.com

Documents

Introduction