FIELD OF INVENTION

[001] This invention relates to the field of agrochemicals, and more
particularly to a biopesticidal formulation obtained from a group of microbial species.

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-friendiy and less expensive to develop. Various bio-pesticides involving the use of various strains of microbes and more specifically 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 formulation for the control of pests obtained from a group of microbes and a process for its preparation

[006] Another object of the invention is to provide a method for controlling or killing pests.

[007] Yet another object of the invention is to provide a method for protecting or treating plants from pests.

DETAILED DESCRIPTION OF INVENTION

[008] 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 / or 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.

[009] The embodiments disclosed herein encompass a formulation for controlling or killing pests which is eco-friendly, non-toxic, stable and cost effective. The biopesticidal formulation encompasses an extract 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 The embodiments further disclose a process for the preparation of the biopesticidal formulation. Furthermore, the embodiments disclose a method for controlling or killing pests and a method for protecting or treating plants from pests.

[0010] In an embodiment, the biopesticidal formulation is used as a larvicide / insecticide. In an embodiment, the insecticide is active against larva or insects belonging to the groups Lepidoptera, Coleoptera and Diptera, Hemiptera. In one embodiment, the activity is against the larva or insects selected from the order Lepidoptera including without limitation Spodoptera, Heliothis, Leaf minors, Diamondback moth, Cut worms, Corn borer, and Stem borrer.

[0011] In another embodiment, the biopesticidal formulation further showed plant growth promoting response leading to high biomass production and yield.

FORMULATION

[0012] 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); and suitable carriers. In alternate embodiments, the formulation may comprise of microbial extracts of variants, strains or mutants of Serratia marcescens (ATCC 13880), Bacillus subtilis (ATCC 6633), Bacillus subtilis (ATCC 6051) and Streptomyces sp. (MTCC 6531); and suitable carriers. The list of suitable carriers that may be used includes at least one of wetting agents, surfactants, spreading agents, dispersing agents, stabilizers, sun protection agents, effectors, solvents, diluents, and neutralizing agents.

[0013] The surfactant may be selected from the list of surfactants which include: 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.; Siiicone-Based Surfactants: Syigard® 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®, Pathfmder'^'^ II, Improved JLB Oil Pius, 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.

[0014] 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; Ethyihexyl Dimethyl PABA; Glyceryl PABA; PEG-25 PABA; Salicylic Derivatives such as: Homosalate ; Ethyihexyl Salicylate; Dipropyleneglycol Salicylate; TEA Salicylate; Cinnamic Derivatives such as: Ethyihexyl Methoxycinnamate; Isopropyl Methoxy cinnamate, Isoamyl Methoxy cinnamate; Cinoxate; DEA Methoxycinnamate; Diisopropyl Methylcinnamate; Glyceryl Ethylhexanoate Dimethoxycinnamate p,p-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 I Sulfonic Acid, Polyacrylamidomethyl Benzylidene Camphor.

[0015] Solvents, diluents and neutralizing agents used may be any solvent, diluents and neutralizing agents generally used in the art.

[0016] 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. Other suitable formulations will be known to those skilled in the art.

[0017] In an embodiment the formulation comprises of microbial extracts of Serratia marcescens (ATCC 13880) in the range of 5%-50%, Bacillus subtilis (ATCC 6633) in the range of 5%-45%, Bacillus subtilis (ATCC 6051) in the range of 5%-75%, Streptomyces sp. (MTCC 6531) in the range 0.01%-5%. In a preferred embodiment, the formulation comprises of Serratia marcescens (ATCC 13880) in the range of 30%-50%, Bacillus subtilis (ATCC 6633) in the range of 15%-45%, Bacillus subtilis (ATCC 6051) in the range of 15%-45%, Streptomyces sp. (MTCC 6531) in the range 0.1%-1%. The formulation further comprises of wetting and spreading agent in the range of 2.5%-6.4%, surfactants in the range of 2.5%-5%, and effectors, protectants and stabilizers in the range of 0-1.5%.

[0018] The details of the biopesticidal 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 1

[0019] The microbial extracts when cultured under appropriate conditions are capable of producing secondary metabolites capable of pesticidal activity. The appropriate conditions can be such that it facilitates secondary metabolism. In an embodiment, the use of metabolites, metabolite-containing supernatant, whole broth culture from the aforementioned strains for use as a formulation is provided.

[0020] The biopesticidal formulation has plant growth promoting response leading to high biomass production and yield. An increase of 15-20% in the biomass production was observed for the crops sprayed with the aforementioned formulation.

PROCESS

[0021] The process for the preparation of the disclosed formulation involves conditioning of microbes to yield an extract; and combining the extract with suitable carriers. The conditioning of microbes involves subjecting the microbes to appropriate conditions, wherein appropriate conditions can be such that it facilitates secondary metabolism. The extract may be such that it includes secondary metabolites, secondary metabolite-containing supernatant and whole broth culture obtained from the aforementioned microbes.

[0022] Microbes - The microbes instrumental in the disclosed formulation may be obtained from any source generally known in the art. The microbes may be obtained from a biological resource center such as American Type Culture Collection (ATCC), Microbial Type Culture Collection (MTCC), etc. The biological resource center's reference / accession number for the microbes are as follows: Serratia marcescens - ATCC Accession number 13880, Bacillus subtilis - ATCC Accession number 6633, Bacillus subtilis - ATCC Accession number 6051 and Streptomyces sp. - MTCC
Accession number 6531. Alternatively, the microbes may also be isolated from various soil samples by procedures that are generally known in the art. The isolated microbes may further be cultured separately to obtain starter cultures.

Conditioning of microbes to yield an extract

[0023] Secondary metabolite production - The secondary metabolites produced have biopesticidal activity and play an important role in controlling or killing pests. In an embodiment, the secondary metabolites are produced when the microbes; Serratia marcescens (ATCC 13880), Bacillus subtilis (ATCC 6633), Bacillus subtilis (ATCC 6051) and Streptomyces sp. (MTCC 6531) are conditioned under appropriate conditions.
The appropriate conditions can be such that it facilitates secondary metabolism. An
optimized secondary metabolite production was tried by varying the media composition, duration of culturing, temperature, etc.

[0024] Media composition: In an embodiment the media composition may be selected from a list of media compositions which includes: Luria Broth (LB), Nutrient Broth (NB), SN (soya powader-2% and Sodium chIoride-0.5%), SY (soyapowder-1%. Sodium chloride- 0.5% and Yeast extract-1%) and Potato Dextrose Broth (Potato-20%, Dextrose-2%) (PDB) 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%, Calcium nitrate-0.002%, Ferrous sulphate-0.001%), Actiono broth and RF (Rice flakes in the range of 5%-15%, Humic acid in the range of 2%-8 %) for MTCC 6531. In an embodiment, the media includes: SN media for ATCC 13880, SY media for ATCC 6051 and ATCC 6633, RF media for MTCC 6531; which provided maximum larvae / insect mortality and secondary metabolite production.

[0025] 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.

[0026] Examples 5: Media optimization - The details on media and percentage killing for each of these microbes is provided in table below.

[0027] 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 hours in SN media for ATCC 13880, SY media for ATCC 6051 and ATCC 6633, 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.

[0028] 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.

[0029] Example 6: Optimization of growth conditions – The details on growth conditions and percentage killing for each of these 5 microbes are orovided in table below;

[0030] 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, RF media for MTCC 6531 for 24 hours 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 larvae / 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 14 hours, at a temperature of 30°C for 5 days for ATCC 13880, ATCC 6633 and ATCC 6051 cultures and 7 days for MTCC 6531 culture.

[0031] Secondary metabolite extraction: In an embodiment, the formulation comprises of an extract comprising secondary metabolites that have been extracted from microbes: ATCC 13880 {Serratia marcescens), ATCC 6633 (Bacillus subtilis), ATCC 6051 {Bacillus subtilis), MTCC 6531 {Streptomyces sp.), 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 20-30% (v/v) for ATCC 13880, ATCC 6051 and ATCC 6633, and Methanol at 40-60% (v/v) for MTCC 6531.

[0032] 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.

[0033] Example 7: Metabolites recovery - The details on metabolite recovery and percentage killing for each of these microbes are provided below.

[0034] 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, ATCC 6051 and ATCC 6633; and 50%forMTCC6531.

Combining the extract with suitable carrier

[0035] The microbial extract obtained from conditioning may further be combined with suitable carriers such as stabilizers, utility adjuvants, protectants, effectors, surfactants, solvents, diluents and neutralizing agents, to yield the formulation, according to the various embodiments disclosed herein. In an embodiment, the microbial extract that may be combined with the carriers includes secondary metabolites, metabolite-containing supernatant and/or whole broth culture obtained from the aforementioned microbes. The supernatant may be obtained by well known processes in the art including centrifugation, filtration, sedimentation and the like.

Method of controlling pests and treating plants

[0036] Any of the aforementioned strains, metabolites, fractions, supematants and formulations may be used to provide a method of treating or protecting plants from pest infections or provide a method of controlling pests.

[0037] 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 metabolite-containing supernatant or purified metabolites are provided.

[0038] 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 biopesticidal formulation comprising the extract of Serratia marcescens (ATCC 13880), Bacillus subtilis (ATCC 6633), Bacillus subtilis (ATCC 6051) and Streptomyces sp. (MTCC 6531), or metabolite-containing supernatant or purified metabolites.

[0039] In another embodiment, the method comprises of treatment of plants, soil, seeds, fruits and other products obtained from plants to prevent infestation by pests.

[0040] 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.

[0041] 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

We Claim,

1. A biopesticidal 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 biopesticidal formulation as claimed in claim 1, wherein the biopesticidal formulation has larvicidal or insecticidal activity.

3. The biopesticidal formulation as claimed in claim 2, wherein the larvicidal or insecticidal activity is against larva or insects of the order Lepidoptera.

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 5%-50%, ATCC 6633 extract is in the range of 5%-45%, ATCC 6051 extract is in the range of 5%-75% and MTCC 6531 extract is in the range 0.01%-5%.

6. The biopesticidal formulation as claimed in claim 1, wherein the suitable carriers comprise at least one of wetting agents, dispersing agents, stabilizers, sun protection agents, effectors, 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%, and effectors, protectants and stabilizers in the range of 0-1.5%

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 agents, dispersing agents, stabilizers, sun protection agents, effectors, 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.