basic information on preparation and availability of various microbial formulations

1. Microbial formulations
V. Shilpakala
TAD/14-04

2. Intoduction
• BT products dominate the microbial pesticide
market.
• other pathogens which are under development
for pest control include viruses, other bacteria,
fungi and nematodes.

3. Various formulations
• Dry formulations – wettable powder (WP),
water dispersible granular (WDG), granular(G)
• Liquid formulations – aqueous flowable,
aqueous suspension, emulsifiable suspension,
non- emulsifiable suspension

5. Bt formulations
company 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 seed
4-5 ml/kg seed
500-750g/acre
200ml/ha
Som phytopharma
(India) Ltd,
Hyderabad
Lipel WP
Junna life sciences
Pvt Ltd, Hyderabad
Bacillus
thuringiensis var.
israelensis
WP

6. Procedure:
Concentrating 2-3% to 15-20%
Mix with adjuvents
Dried at 1750C
Mixed with additional adjuvents
Packed and use

7. 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.

8. • 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.

11. NPV formulations
company Product name formulation Dosage
BIOTECH
INTERNATTONA
L LTD., NEW
DELHI
Biovirus H Liquid
Formulation
0.625-1 ml/L
SOM
PHYTOPHARMA
(INDIA) LTD,
HYDERABAD
SOMSTAR-Ha Liquid
formulation
Pest control (india)
pvt.ltd., mumbai
Heli-CideTM Liquid
formulation
100 ml per acre
Spodoptera litura
Pest control (india)
pvt.ltd., mumbai
Spodo- cideTM Liquid
formulation
100 ML PER ACRE

12. 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

13. • 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

14. 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

15. 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.

17. 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.

19. 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.

20. 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 .

21. • 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.

22. 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.

24. 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.

25. 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).

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

27. Thank you