DESC:FORM 2
The Patent Act 1970
(39 of 1970)
&
The Patent Rules, 2005

COMPLETE SPECIFICATION
(SEE SECTION 10 AND RULE 13)

TITLE OF THE INVENTION

A bio-pesticidal formulation and a process of preparation thereof

APPLICANT:

Name : Camson Biotechnologies Ltd

Nationality : India

Address : C-7, 7th Floor, Corporate Block, Golden
Enclave, Airport Road, Bangalore 560 017

The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed:-
FIELD OF INVENTION

[001] This invention relates to a bio-pesticidal formulation, and more particularly to a bio-pesticidal formulation comprising an extract from a group of bacterial species, which when cultured under appropriate conditions is capable of producing at least one pesticidally active substance.

BACKGROUND OF INVENTION
[002] For a number of years, it has been known that various microorganisms exhibit biological activity so as to be useful to control plant diseases. Although progress has been made in the field of identifying and developing biological pesticides for controlling various plant diseases of agronomic and horticultural importance, most of the pesticides in use are still synthetic compounds. Many of these chemical pesticides are classified as carcinogens and are toxic to wildlife and other non-target species. In addition, pathogens may develop resistance to chemical pesticides.
[003] Biological control offers an attractive alternative to synthetic chemical pesticides. Bio-pesticides can be safer, more biodegradable, and less expensive to develop. According to United States Environmental Protection Agency (EPA), bio-pesticides include naturally occurring substances that control pests (biochemical pesticides), microorganisms that control pests (microbial pesticides), and pesticidal substances produced by plants containing added genetic material (plant-incorporated protectants) or PIPs.
[004] In the recent years, the use of methods of biological control of pests has emerged as an attractive alternative to synthetic chemical pesticides. Bio-herbicidal agents are considered safer, eco-friendly and less expensive to develop. Various bio-pesticides involving the use of various strains of bacteria have been provided earlier. However, the use of a unique combination of various strains of different bacteria in order to provide for an effective, eco-friendly, non-toxic, biodegradable and cost effective method for controlling pests is desirable.

OBJECT OF INVENTION
[005] The principal object of this invention is to provide a method and a formulation for the control of pests from a group of bacterial species, which when cultured under appropriate conditions is capable of producing at least one pesticidally active substance.

DETAILED DESCRIPTION OF INVENTION
[006] The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.
[007] The embodiments disclosed herein encompass a bio-pesticidal formulation comprising the extract of Serratia marcescens (ATCC 13880), Bacillus subtilis (ATCC 6633), Bacillus subtilis (ATCC 6051) and Streptomyces sp. (MTCC 6531) which when cultured under appropriate conditions is capable of producing at least one insecticidally active substance. In an embodiment, the extract of Serratia marcescens (ATCC 13880), Bacillus subtilis (ATCC 6633), Bacillus subtilis (ATCC 6051) and Streptomyces sp. (MTCC 6531) disclosed herein includes a variant or mutant thereof.
[008] In an embodiment, the use of metabolites, metabolite-containing supernatant, whole broth culture from the aforementioned strains for use as a pesticide is provided.
[009] In another embodiment, the bio-pesticidal formulation is used as an insecticide. In an embodiment, the insecticide is active against Soil born insects such as those belong to the families Termitidae, Scarabaeidae and Trichostrongylidae. In one embodiment, the insects selected from the families Termitidae, Scarabaeidae and Trichostrongylidae include without limitation insects like Termites, Root grubs and Nematodes.
[0010] In another embodiment, the bio-pesticidal formulation further showed plant growth promoting response leading to high biomass production and yield.

Isolation of Microbes
[0011] In an embodiment, the microbes Serratia marcescens (ATCC 13880), Bacillus subtilis (ATCC 6633), Bacillus subtilis (ATCC 6051) and Streptomyces sp. (MTCC 6531) are isolated from various soil samples or dead insects by procedures that are generally known in the art.

Secondary metabolite production
[0012] In an embodiment, the at least one active substance present in the formulation is an effective amount of a secondary metabolite that is produced by the microbes. In another embodiment, the active substance present in the formulation is an effective amount of a whole broth extract of the microbes. In yet another embodiment, the active substance present in the formulation comprises of an effective amount of supernatant obtained from a whole broth culture.
[0013] In an embodiment the secondary metabolites, whole broth culture or the supernatant obtained from the whole broth culture were cultured under appropriate conditions. An optimized secondary metabolite production was tried by varying the media composition, duration of culturing, temperature, etc.

[0014] Media composition: In an embodiment the media composition may be selected from a list of media compositions which includes: Luria Broth, Nutrient Broth, SY (soyapowder-1%, Sodium chloride- 0.5% and Yeast extract-1%), SN (soya powader-2% and Sodium chloride-0.5%), and Potato Dextrose Broth (PDB; Potato-20%, Dextrose-2%) for ATCC 13880, ATCC 6633 and ATCC 6051; and YMDB (Yeast extract-0.4%, Malt extract-1%, Glucose-0.4%), SCA (Starch-1%, Casein-0.03%, Potassium nitrate-0.2%, Sodium chloride-0.2%, Dipotassium hydrogen phosphate-0.2%, Magnesium sulphate-0.005%, Calsium nitrate-0.002%, Ferrous sulphate-0.001%), Actiono broth and RF (Rice flakes-10%, Humic acid -5%) for MTCC 6531. In an embodiment, the media includes: SN media for ATCC 13880, SY media for ATCC 6051 and ATCC 6633, and RF media for MTCC 6531; that provided maximum insect mortality and secondary metabolite production.
[0015] The details of the media composition for the secondary metabolite production are provided in the following example by way of illustration only and should not be construed to limit the scope of the present invention.
[0016] Example 1: The details on media and percentage killing for each of these microbes are provided in Table 1.
Table 1: Media optimization
Cultures Media tried Insecticidal effect (%) (3g per lt dilution, 3 days)
ATCC 13880 LB 30
NB 20
SN 80
SY 55
PDB 45
ATCC 6051 LB 60
NB 40
SN 50
SY 75
PDB 60
ATCC 6633 LB 65
NB 40
SN 50
SY 70
PDB 50
MTCC 6531 YMDB 40
SCA 30
Actino broth 55
RF 80
Control 0.00

[0017] Growth conditions: The growth conditions such as duration of culture and temperature are crucial in the production of secondary metabolites. In an embodiment, the starter cultures were grown for 24-48 hours in SN media for ATCC 13880, SY media for ATCC 6051 and ATCC 6633, and RF media for MTCC 6531. In another embodiment, the growth condition for the culture of microbes include 50-300 rpm of shaking under light for 5 – 10 hours and under dark condition for 10-18 hours, at a temperature of 25-40°C for 3-10 days for all cultures.
[0018] The details of the growth conditions for the secondary metabolite production are provided in the following example by way of illustration only and should not be construed to limit the scope of the present invention.
[0019] Example 2: The details on growth conditions and percentage killing for each of these microbes are provided in Table 2.
[0020] In order to arrive at the optimum growth conditions, the starter cultures were grown in SN media for ATCC 13880, SY media for ATCC 6051 and ATCC 6633, and RF media for MTCC 6531 for 24-48 hours. These were used for further inoculation to SN media for ATCC 13880, SY media for ATCC 6051 and ATCC 6633, and RF media for MTCC 6531 and incubated for different periods with shaking of 150-200 rpm with 8 hr light and 14 hr dark condition at 30°C. The highest insect killing was observed by the microbial extract cultured in the growth condition with 150-200 rpm shaking under light for 8 hours and under dark condition for 16 hours, at a temperature of 30°C at 5th day for ATCC 13880, ATCC 6633 and ATCC 6051 cultures. MTCC 6531 showed highest insect mortality at 28°C at 7th day.

Table 2: Optimization of growth conditions
Cultures Duration of culturing % insect mortality (100 dilution, 3days)
ATCC 13880 3 days 40
5 days 80
7 days 70
ATCC 6051 3 days 50
5 days 75
7 days 60
ATCC 6633 3 days 40
5 days 70
7 days 60
MTCC 6531 5 40
7 80
10 60
Control 0.00

[0021] Secondary metabolite extraction: In an embodiment, the formulation comprises of an extract comprising secondary metabolites that have been extracted from microbes: Serratia marcescens (ATCC 13880), Bacillus subtilis (ATCC 6633), Bacillus subtilis (ATCC 6051) and Streptomyces sp. (MTCC 6531), wherein the microbes have been subjected to appropriate conditions to yield secondary metabolites. The extraction of secondary metabolites may be performed by any method generally known in the art. Various solvents such as methanol, acetone, ethyl acetate, etc in different concentration may be used for extraction of secondary metabolites. In an embodiment, the extraction is performed by using Methanol at 5-50% (v/v) for all cultures.
[0022] The details of the secondary metabolite extraction are provided in the following example by way of illustration only and should not be construed to limit the scope of the present invention.
[0023] Example 3: The details on metabolite recovery and percentage killing for each of these microbes are provided in Table 2.
[0024] Various solvents in different concentration were used for extraction of secondary metabolites. The extraction of secondary metabolites performed by using ethyl acetate, methanol and acetone at various concentrations. The preferred extraction method involves the use of Methanol at 25% (v/v) for ATCC 13880 and ATCC 6051 cultures, and Methanol at 50% (v/v) for MTCC 6531 and ATCC 6633, when used as live cultures.
Table 3: Metabolites recovery
Cultures Solvents Solvent ratio (solvent : broth) % insect mortality ( 3 g per lt dilution, 3 days)
ATCC 13880 Methanol (1:9) 30
(1:3) 80
(1:1) 50
(2:1) 40
Ethyle acetate (1:9) 30
(1:3) 40
(1:1) 40
(2:1) 20
Butanol (1:9) 30
(1:3) 50
(1:1) 60
(2:1) 30
Culture filtrate - 20
ATCC 6051 Methanol (1:9) 50
(1:3) 75
(1:1) 50
(2:1) 40
Ethyle acetate (1:9) 50
(1:3) 40
(1:1) 60
(2:1) 40
Petroleum ether (1:9) 40
(1:3) 60
(1:1) 50
(2:1) 50
Culture filtrate - 30
ATCC 6633 Methanol (1:9) 20
(1:3) 15
(1:1) 20
(2:1) 30
Ethyle acetate (1:9) 40
(1:3) 30
(1:1) 30
(2:1) 10
Butanol (1:9) 30
(1:3) 00
(1:1) 20
(2:1) 30
Culture filtrate - 70
MTCC 6531 Methanol (1:9) 60
(1:3) 80
(1:1) 60
(2:1) 50
Toluene (1:9) 20
(1:3) 50
(1:1) 30
(2:1) 20
Benzene (1:9) 30
(1:3) 40
(1:1) 50
(2:1) 20
Culture filtrate - 40
Control - - 0.00

Formulation
[0025] The disclosed embodiments provide a formulation comprising the microbial extracts of: Serratia marcescens (ATCC 13880), Bacillus subtilis (ATCC 6633), Bacillus subtilis (ATCC 6051) and Streptomyces sp. (MTCC 6531) which when cultured under appropriate conditions is capable of producing at least one active substance capable of pesticidal activity. In an embodiment, the active substance is secondary metabolite produced by the microbes.
[0026] In an embodiment, the formulation comprises of microbial extracts of: ATCC 13880, ATCC 6633, ATCC 6051 and MTCC 6531. In alternate embodiments the formulation may comprise of microbial extracts of variants, strains or mutants of ATCC 13880, ATCC 6633, ATCC 6051 and MTCC 6531.
[0027] In another embodiment disclosed herein, the formulation comprises at least one of a carrier material, surfactant, stabilizer, protectors, effectors, solvent, diluent, and a neutralizing agent.
[0028] The surfactant may be selected from the list of surfactant which includes: Ethoxylated fatty amines (Cationic): Entry™ II, POEA - Roundup®; Alkylphenol ethoxylate-based surfactants (non-ionic): R-11® Spreader Activator, Activator 90, X-77®, Latron AG-98™, Latron AG-98™, Cide-kick®, Cide-kick® II™; Alcohol ethoxylate-based surfactants (non-ionic): Activator N.F.; Silicone-Based Surfactants: Sylgard® 309–silicones, Freeway®–silicone blend, Dyne-Amic® - silicone blend, Silwet L-77®- silicones: Oils: Vegetable Oils:- The methylated seed oils are formed from common seed oils, such as canola, soybean, or cotton, MSO® Concentrate Methylated Seed Oil, Hasten®, Pathfinder™ II, Improved JLB Oil Plus, Cide-Kick and Cide-Kick II, Blends of vegetable oils and silicone-based surfactants, Syl-tac™, Phase™; Crop Oils and Crop Oil Concentrates: kerosene (found in the triclopyr formulation Garlon 4)Agri-dex®, Red-Top Mor-Act®. The formulation may further comprise of utility adjuvants which includes LI-700® Surfactant Penetrant Acidifier.
[0029] The stabilizers, effectors and UV protectants may be selected from a list which includes: ZnO, Ti02, nanodiamond; Para-Aminobenzoic Acid Derivatives such as: PABA, Ethyl PABA; Ethyl Dihydroxypropyl PABA; Ethylhexyl Dimethyl PABA; Glyceryl PABA; PEG-25 PABA; Salicylic Derivatives such as: Homosalate ; Ethylhexyl Salicylate; Dipropyleneglycol Salicylate; TEA Salicylate; Cinnamic Derivatives such as: Ethylhexyl Methoxycinnamate; Isopropyl Methoxy cinnamate, Isoamyl Methoxy cinnamate; Cinoxate; DEA Methoxycinnamate; Diisopropyl Methylcinnamate; Glyceryl Ethylhexanoate Dimethoxycinnamate ß,ß-Diphenylacrylate Derivatives such as: Octocrylene, Etocrylene; Benzophenone Derivatives such as: Benzophenone-1, Benzophenone-2, Benzophenone-3 or Oxybenzone, Benzophenone-4, Benzophenone-5, Benzophenone-6, Benzophenone-8, Benzophenone-9, Benzophenone- 12; Benzylidenecamphor Derivatives such as: 3-Benzylidene camphor, Benzylidene Camphor Sulfonic Acid, Camphor Benzalkonium Methosulfate, Terephthalylidene Dicamphor Sulfonic Acid, Polyacrylamidomethyl Benzylidene Camphor.
[0030] In another embodiment, the formulation comprising the microbial extracts may further comprise at least one of carrier material, solvent, diluents and neutralizing agents. The carrier material, solvent, diluents and neutralizing agents used may be carrier materials, solvents, diluents and neutralizing agents generally used in the industry.
[0031] In order to achieve good dispersion and adhesion of formulations within the scope of the present invention, it may be advantageous to formulate the metabolites, supernatant and/or whole broth culture with components that aid dispersion and adhesion. Suitable formulations will be known to those skilled in the art. In an embodiment, the formulations can be formulated as wettable powders, granules and the like, or can be microencapsulated in a suitable medium and the like. Examples of other formulations include, but are not limited to soluble powders, wettable granules, dry flowables, aqueous flowables, wettable dispersible granules, emulsifiable concentrates and aqueous suspensions. In an embodiment, the formulation is in the form of a powder. Other suitable formulations will be known to those skilled in the art.
[0032] In an embodiment, the formulation comprises of microbial extracts of ATCC 13880 in the range of 15-45%, ATCC 6633 in the range of 25-60%, ATCC 6051 in the range of 10-35%, MTCC 6531 in the range of 1-10%. The formulation further comprises of carrier materials around 20%, wetting and spreading agent in the range of 2.5-6.4%, surfactants in the range of 2.5-5%, and effectors, protectants, stabilizers in the range of 0-1.5%, and attractants such as Carboxy methyl cellulose (CMC) in the range of 0.5-5%.
[0033] The details of the pesticidally active formulations are provided in the following examples by way of illustration only and should not be construed to limit the scope of the present invention.
Example 4
Sample Cultures Proportion
% % insect mortality (3 g per lt dilution)
Formulation ATCC 13880 extract 30 70
ATCC 6051 extract 30
ATCC 6633 culture 30
MTCC 6531 extract 2
Camwet (Prorietary) 5
Surfactant (Tween 80) 2.5
CMC 0.5
effectors, protectant and stabilizers (Glycerol) 1.5
Control - - 0.00

Example 5
Sample Cultures Proportion
% % insect mortality (3 g per lt dilution)
Formulation ATCC 13880 extract 30 80
ATCC 6051 extract 20
ATCC 6633 culture 40
MTCC 6531 extract 5
Camwet (Prorietary) 2.5
Surfactant (Tween 80) 2.5
CMC 1.0
effectors, protectant and stabilizers (Glycerol) 0
Control - - 0.00

Example 6
Sample Cultures Proportion
% % insect mortality (3 g per lt dilution)
Formulation ATCC 13880 extract 30 100
ATCC 6051 extract 20
ATCC 6633 culture 35
MTCC 6531 extract 5
Camwet (Prorietary) 2.5
Surfactant (Tween 80) 5.0
CMC 2.0
effectors, protectant and stabilizers (Glycerol) 2.5
Control - - 0.00

[0034] The bio-pesticidal formulation further showed plant growth promoting response leading to high biomass production and yield. An increase of 10-20% in the biomass production was observed for the crops sprayed with the aforementioned formulation.

Method of controlling pests and treating plants
[0035] Any of the aforementioned strains, metabolites, fractions, supernatants and formulations containing active ingredients may be used to provide a method of treating or protecting plants from pest infections or provide a method of controlling pests.
[0036] In another embodiment, the method comprises of treating plants or soil to control pest infestations with an extract of Serratia marcescens (ATCC 13880), Bacillus subtilis (ATCC 6633), Bacillus subtilis (ATCC 6051) and Streptomyces sp. (MTCC 6531), or a metabolite-containing supernatant or purified metabolites is provided; which when cultured under appropriate conditions is capable of producing at least one insecticidally active substance.
[0037] In another embodiment, the method comprises of controlling pests which comprises applying to the pests or to the locus of the pests a pesticidally effective amount of a bio-pesticidal formulation comprising the extract of Serratia marcescens (ATCC 13880), Bacillus subtilis (ATCC 6633), Bacillus subtilis (ATCC 6051) and Streptomyces sp. (MTCC 6531), characterized that ATCC 13880, ATCC 6633, ATCC 6051 and MTCC 6531 when cultured under appropriate conditions is capable of producing at least one insecticidally active substance.
[0038] In an embodiment, the invention encompasses a method of treating or protecting plants from insect infections and comprising applying an effective amount of the whole broth of the extract of ATCC 13880, ATCC 6633, ATCC 6051 and MTCC 6531, which when cultured under appropriate conditions is capable of producing at least one insecticidally active substance. In another embodiment, the invention comprises applying an effective amount of the metabolites produced by ATCC 13880, ATCC 6633, ATCC 6051 and MTCC 6531. In another embodiment, the invention comprises applying an effective amount of a supernatant obtained from a whole broth culture of a group of bacteria and its strains, variants or mutants thereof. The supernatant may be obtained well known in the art including centrifugation, filtration, sedimentation and the like. In another embodiment, the method comprises of treatment of plants, soil, seeds, fruits and other products obtained from plants to prevent infestation by pests.
[0039] In an embodiment, method of treating plants to control pesticidal infestations using the claimed strains, either alone, or in combination with other chemical or biological pesticides is provided.
[0040] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.
,CLAIMS:CLAIMS
We claim,
1. A bio-pesticidal formulation comprising an effective amount of an extract obtained from a group of microbes, wherein the group comprises of Serratia marcescens (ATCC 13880), Bacillus subtilis (ATCC 6633), Bacillus subtilis (ATCC 6051) and Streptomyces sp. (MTCC 6531), or their strains, variants or mutants thereof; and suitable carriers.

2. The bio-pesticidal formulation as claimed in claim 1, wherein the biopesticidal formulation has insecticidal activity.

3. The bio-pesticidal formulation as claimed in claim 2, wherein the insecticidal activity is against insects of family Termitidae, Scarabaeidae and Trichostrongylidae.

4. The biopesticidal formulation as claimed in claim 1, wherein the extract comprises of at least one secondary metabolite.

5. The biopesticidal formulation as claimed in claim 1, wherein ATCC 13880 extract is in the range of 15-45%, ATCC 6633 extract is in the range of 25-60%, ATCC 6051 extract is in the range of 10-35% and MTCC 6531 extract is in the range of 1-10%.

6. The biopesticidal formulation as claimed in claim 1, wherein the suitable carriers comprise at least one of wetting and spreading agents, surfactants, protectors, effectors, stabilizers, attractants, solvents, diluents and neutralizing agents.

7. The biopesticidal formulation of claim 6, wherein the suitable carriers comprises of wetting and spreading agent in the range of 2.5-6.4%, surfactants in the range of 2.5-5%, effectors, protectants, stabilizers in the range of 0-1.5%, and attractants in the range of 0.5-5%

8. The biopesticidal formulation of claim 1, wherein the formulation is formulated as a wettable powder, a granule, an aqueous suspension, emulsifiable concentrate and a microencapsulated formulation.

9. A process for the preparation of a biopesticidal formulation, the process comprising:
conditioning of microbes to yield an extract, wherein the microbes include Serratia marcescens (ATCC 13880), Bacillus subtilis (ATCC 6633), Bacillus subtilis (ATCC 6051) and Streptomyces sp. (MTCC 6531), or their strains, variants or mutants thereof; and
combining the extract with suitable carriers.

10. The process for the preparation of a biopesticidal formulation as claimed in claim 9, wherein the conditioning involves subjecting the cultured microbes to secondary metabolism.

11. The process for the preparation of a biopesticidal formulation as claimed in claim 9, wherein the suitable carriers comprise at least one of wetting and spreading agents, surfactants, protectors, effectors, stabilizers, attractants, solvents, diluents and neutralizing agents.

12. A method for controlling or killing pests comprising:
applying to the pests, or to the locus of the pests an insecticidally effective amount of a biopesticidal formulation, wherein the biopesticidal formulation comprises of an extract obtained from a group of microbes, wherein the group comprises of Serratia marcescens (ATCC 13880), Bacillus subtilis (ATCC 6633), Bacillus subtilis (ATCC 6051) and Streptomyces sp. (MTCC 6531), or their strains, variants or mutants thereof; and suitable carriers.
13. A method for protecting or treating plants from pests comprising:
applying to the plants, soil, roots of plants an effective amount of a biopesticidal formulation, wherein the biopesticidal formulation comprises of an extract obtained from a group of microbes, wherein the group comprises of Serratia marcescens (ATCC 13880), Bacillus subtilis (ATCC 6633), Bacillus subtilis (ATCC 6051) and Streptomyces sp. (MTCC 6531), or their strains, variants or mutants thereof; and suitable carriers.

Date 22nd May, 2012 Signature
Mr. Vikram Pratap Singh Thakur
Patent Agent