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Microbial formulations V. Shilpakal a TAD/14-04

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Microbial formulations

V. Shilpakala

TAD/14-04

Intoduction

• BT products dominate the microbial pesticide market.

• other pathogens which are under development for pest control include viruses, other bacteria, fungi and nematodes.

Various formulations

• Dry formulations – wettable powder (WP), water dispersible granular (WDG), granular(G)

• Liquid formulations – aqueous flowable, aqueous suspension, emulsifiable suspension, non- emulsifiable suspension

Bt formulationscompany Product name formulation Dosage

Eupnoea technisol Pvt. Ltd, New Delhi

BACIL-EU WP 1.5-2g/L

Prathibha biotech, Hyderabad

Bacillus thuringiensis var kurstaki

WP, liquid formulation

Seed treatment 10 g/kg seed4-5 ml/kg seed500-750g/acre200ml/ha

Som phytopharma (India) Ltd, Hyderabad

Lipel WP

Junna life sciences Pvt Ltd, Hyderabad

Bacillus thuringiensis var. israelensis

WP

Procedure:Concentrating 2-3% to 15-20%

Mix with adjuvents

Dried at 1750C

Mixed with additional adjuvents

Packed and use

Bt

• The pathogen is cultured on a semi-solid medium so that it is preferable to process it as a dust or wettable powder rather than attempt to separate the spores and crystals from the medium solids.

• When grinding and mixing material containing the pathogens to obtain a sufficiently fine powder, care should be taken to avoid increase in temperature or physical damage that would harm the pathogen.

• spore-forming Bacillus thuringiensis are usually concentrated prior to drying by centrifugation or filtration.

• Centrifugation using a continuous centrifuge concentrates the product from 2-3 % suspended solids to 15-20 %.

• Centrifugation may result in some loss of suspended solid as well as loss of dissolved materials. Such losses may not be acceptable and concentration using this technique can often be omitted.

• Following concentration, one of the technique mixes the crystal/spores slurry with lactose, adjuvants such as wetting agents, spreader-stickers or dispersing agents, and the whole product is spray-dried at 175oC (Dulmage, 1981).

• The dry product is blended and/or mixed with additional formulation adjuvants before packaging and/or use.

• The lactose added may act as a cryoprotectant or it may help to prevent clumping Dulmage and Rhodes, (1971).

• Dulmage at al. (1970) (see chapter 2) developed an alternative drying technique for laboratory preparations where spray drying facilities are not available; this technique of recovery of B. thuringiensis is based on the lactose-acetone processing (see 2.1.1). Many patents exist such as a foam flotation process for separating B.t. sporulation products.

NPV formulationscompany Product name formulation Dosage

BIOTECH INTERNATTONAL LTD., NEW DELHI

Biovirus H LiquidFormulation

0.625-1 ml/L

SOM PHYTOPHARMA (INDIA) LTD, HYDERABAD

SOMSTAR-Ha Liquidformulation

Pest control (india) pvt.ltd., mumbai

Heli-CideTM Liquidformulation

100 ml per acre

Spodoptera litura

Pest control (india) pvt.ltd., mumbai

Spodo- cideTM Liquidformulation

100 ML PER ACRE

NPV Spray SOMSTAR TM -SL @ 1ml / L of water

Mass Composition • Constituent W/W % function • Spodoptera litura –NPV Content mass/mass 5% Active • Inactive ingredients (Glycerol) 50% Inactive • Moisture 45% max Inactive

Biological Composition • Constituent PIB/ml. formulation • Polyhedral Inclusion Bodies of Spodoptera litura • Nuclear Polyhedrosis Virus. 1 x 109 PIB/ml Sprayable

Liquid

• Baculoviruses may remain unchanged for many years when stored under appropriated conditions (Sireesha et al., 2010), they are rapidly inactivated under field conditions by short and long wavelengths (254 – 320 nm) UV light (Jeyarani et al., 2013), particularly in the UV-B range (280-320 nm) (Lasa et al., 2007b).

Several protectants have been successfully used, such as

1.reflectants (metallic aluminium, aluminium oxide or titanium dioxide),2.general absorbents (carbon and naphthalene black), 3.Selective absorbents (amelozan and paminobenzoic acid), 4.optical brighteners (derivatives of stilbene, oxazole, pyrazole, naphthalic acid, lactone and coumarin), and

EPF formulations

company Entomopathogenic fungus

Product name formulation

Varsha bioscience and technology India Pvt Ltd Hyderabad

Metarhizium anisopliae

Biostorm TM POWDER

Manidharma Biotech Private Ltd, Chennai

Metarhizium anisopliae

BIOPROTECTOR talc

Varsha bioscience and technology India Pvt Ltd Hyderabad

Verticillium lecanii shock talc

Ruchi biochemicals Maharashtra

Verticillium lecanii spider WP

Eupnoea technisol Pvt Ltd, Gujarat

Verticillium lecanii Lecan- EU WP

Entomopathogenic fungus

• For preparing the formulations viz.• Carrier based powder formulation (CBPF)

using talcum powder, glycerine and gum, • Oil-based liquid formulation (OBLF) using

corn oil, gum and glycerin,• Bentonite oil-based liquid formulation

(BOBLF) using corn oil, gum, glycerin and bentonite.

Preparing of WP formulation of conidia and storage conditions

• At first, we selected the Petri dishes with well grown and sporulated fungus on SDAY medium,

• then collected the pure conidia of each Petri dish by scraping off the sporulated fungus using sterile scalpels and maintained at a low temperature (4°C).

• For accession of proportion of carries that should be used with conidia, we carried out experiments based on adding some proportions of supplements to conidia and stored them for 30 days (unpublished data).

• In 2.5 g of fresh conidial powder, the concentration was equal and amounted to 2×1010 conidia ml–1. Conidia were mixed with silica gel powder in 2.5:1 weight ratio.

• Then, other selected materials or carriers such as stickers, stabilizers, UV-protectant and wetting agents were added to the mixture of conidia and silica gel into the sterile cabinet.

• Approximately 12 g of dry powder was obtained for each case and poured in to 50-ml falcon plastic tubes. These tubes were covered with aluminum foil and stored under different conditions: in laboratory at a temperature ranging from 23–26°C and in a refrigerator at a temperature of 4±2°C for 30 days.

EPN formulations

• Nematodes can be stored and formulated in different ways including the use of polyurethane sponge, water-dispersible granules, vermiculite, alginate gels and baits.

PREPARING ENTOMOPATHOGENIC NEMATODE FORMUTATIONS

A) Talc formulation: 25 ml of distilled water added in 250 g of talc powder in a 500 ml beaker, they were mixed thoroughly with help of glass rod. 50 ml of freshly harvested lJs of H. indica @ 1 lakh/ml were added in the moisten talc then thoroughly mixed till the nematodes suspension spread over evenly into the talc. packed in a polythene envelop and sealed .

• b) Coir pith and saw dust formulation: A readymade coconut coir brick and saw dust/ powder were taken. Both the raw material was grinded separately to get fine dust with help of domestic mixer and they were sieved with fine mesh, sterilized under sun light (1 hr).100 g of each was moistened adding 50 ml of distilled water separately. lJs suspension of 50 ml @ l lakh/ml were added evenly and mixed them gently till nematodes spread over into the coir and the same procedure was followed to saw dust powder too and sealed them individually in a plastic container, stored for further observation.

c) Sponge formulation: 2 g sponge pieces (4 cm. length X 2 cm.height X 2 cm. width) were washed thoroughly in tap water and autoclaved for 10 min. IJs of H. indica impregnated by squeezing in 50 ml nematode suspension (1 lakh/ml) and they packed in a suitable polythene cover, sealed them smoothly.

d) Gel formulation :1 g of hydrogel granules were directly mixed by adding 50 ml lJs suspensions and distribution of lJs evenly in hydrogel content were done with help of a glass rod and sealed them finally in a polythene paper.

e) Water: Freshly harvested lJs were washed twice in distilled water and 50 ml @ 1 lakh /ml were stored in 250 ml conical flask. Flask was closed with non absorbent cotton which was used in the experiment as control treatment.

Tested Formulae: Three materials were selected to test their viability as carriers for the nematodes as follows:

1) Hydrogel (Horie et al., 2002).

2) kaolinite or kaolin with the chemical composition Al2Si2O5(OH)4.

3) Calcium alginate.

Preparation of the nematode formulae• A matrix of nematode gel was prepared as per Kaya and Nelsen

(1985).• The matrix was prepared by dissolving 2g of hydrogel, kaoline

and calcium alginate in 100 ml water and blended for 4-5 minutes.• Drops of nematode suspension (2000 IJs/ml) were mixed with the

carrying material (hydrogel, kaoline and calcium alginate) in 4 x 9 cm plastic bags.

• An antifungal agent (0.05mg Streptomycin sulphate) was added to prevent the growth of microbes and pH was adjusted to 7.0.

• The bags were sealed with a plastic sealing machine and left at room temperature (25±2 oC).

• The pathogenicity of IJs which were formulated in Kaolinite was more virulent against G. mellonella than the other two carriers

Thank you