FIELD OF INVENTION

[001] The present invention generally relates to the field of agrochemicals, and more particularly to a weedicidal formulation which includes microbial extracts.

BACKGROUND OF INVENTION

[002] Weeds are unwanted plants which compete with crops for space, nutrients, light and water. They have the capacity to germinate under varied conditions. They mainly belong to the class Angiospermae, having two subclasses Monocotyledoneae (monocots) and Dicotyledoneae (dicots). On the basis of the habitat, they may be of two varieties; terrestrial and aquatic. On the basis of the duration of life they may be categorized as armuals, biennials and perennials.

[003] The ability of weeds to generate formidable competition to crops has always been a matter of botheration to cultivators. Additionally, weeds harbor pests and insects which have imdesirable, deleterious effects on crops. Many measures to control and inhibit the growth of weeds have been undertaken in the past. The mechanical methods used for control of weeds include hand-weeding, hoeing, tilling, mowing, burning, flooding, smothering, etc. These mechanical methods are time consuming, inefficient and labour intensive. Further, the chemical methods for the control of weeds include the use of chemicals, including Aryloxyphenoxy-propionate'FOPs', Cyclohexanedione (DIMs), Sulfonylurea, Triazine, etc as herbicides.

[004] Chemical herbicides, however, have widely variable toxicity. High exposure to chemical herbicides may even be carcinogenic and may contribute to long term problems. These herbicides cause a variety of health problems ranging from skin rashes to death.

[005] In the recent years, the use of methods of biological control of weeds has emerged to as an attractive alternative to synthetic chemical pesticides. Bio-herbicidal agents are considered safer, eco-friendly and less expensive to develop. Various bio-herbicides involving the use of specific strains of Streptomyces have been provided earlier. However, the use of a combination of various strains of Streptomyces in order to arrive at a single effective bio herbicidal agent has never been implemented earlier.

[006] There exists a need for an effective, eco-friendly, non-toxic, biodegradable and cost effective method for controlling weeds.

OBJECT OF INVENTION

[007] The principal object of this invention is to provide a method for controlling and/or inhibiting the growth of weeds.

[008] Another object of the invention is to provide a formulation for controlling and/or inhibiting the growth of weeds and a process for its preparation.

DETAILED DESCRIPTION OF INVENTION

[009] 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.

[0010] The embodiments herein disclose formulation for controlling and/or inhibiting weeds, and a process for the preparation of the formulation. The weedicide formulation (also referred to as bio-weedicidal formulation) is a non-toxic and eco-friendly formulation which may be used to control and/or inhibit the growth of weeds.

[0011] The disclosed embodiments of the weedicide formulation have weedicidal activity towards a wide range of weeds (or herbs), and can effectively control and/or inhibit the growth of various weeds (or herbs). In an embodiment the weedicide formulation controls and/or inhibits the growth of weeds by suppressing photosynthesis in weeds. The wide range of weeds that can be controlled by the formulations disclosed herein are as follows: Polygonaceae weeds: Polygonum convolvulus, Polygonum lapathiolium, Polygonum pennsylvanicum, Polygonum persicaria. Polygonum longisetum, Rumex japoniucs, Rumex crispus, Rumex obtusifolius, Polygonum cuspidatum;

Portulacaceae weeds: Portulaca oleracea; Caryophyllaceae weeds: Stellaria media, Cerastium glomeratum; Chenopodiaceae weeds: Chenopodium album, Kochia scoparia; Amaranthaceae weeds: Amaranthus retroflexus, Amaranthus viridis, Amaranthus lividus, Amaranthus spinosus, Amaranthus hybridus, Amaranthus rudis; Brassicaceae weeds: Raphanus raphanistrum, Sinapis arvensis, Capsella bursa-pastor&, Brassica juncea; Leguminosae weeds: Sesbania exaltata, Cassia obtusifolia, Desmodium tortuosum, Trifolium repens, Pueraria lobata, Vicia angustifolia; Malvaceae weeds: Abutilon theophrasti, Sida spinosa;Violaceae weeds: Viola arvensis, Viola tricolor; Rubiaceae weeds: Galium aparine; Convolvulaceae weeds: Ipomoea hederacea, Ipomoea purpurea, Ipomoea hederacea var integriumscula, Ipomea lacunosa, Convolvulus arvensis; Labiatae weeds: Lamiumpurpureum, Lamium amplexicaule; Solanaceae weeds: Datura stramonium, Solarium nigrum; Scrophulariaceae weeds: Veronica hederaefolia, Veronica persica, Veronica arvensis; Compositae weeds: Xanthium pensylvanicum, Helianthus annuus, Matricaria chamomilla, Matricariaperforate, Chrysantemum segetum, Matricaria matricarioides, Artemisia princeps, Solidago altissiuma, Taraxacum oDcinale, Galinosoga ciliata, Conyza canadensis, Ambrosia artemisiaefolia, Ambrosia trifida; Boraginaceae weeds: Myosotis arvensis; Asciepiadaceae weeds: Asclepias syriaca; Euphorbiaceae weeds: Euphorbia helioscopia, Euphorbia maculata; Geraniaceae weeds: Geranium carolinense, Erodium cicutarium; Papaveraceae weeds: Papaver rhoeas; Gramineae weeds: Echinochloa crus-galli, Setaria viridis, Setaria faberi, Digitaria sanguinalis, Eleusine indica, Poa annua, Alopecurus myosuroides, Avena fatua, Sorghum halepensa, Agropyron repens, Lolium multiflorum, Lolium perenne, Bromus secalinus, Bromus tectorum, Apera spicaventi, Panicum dichotomiflorum, Panicum texanum. Sorghum vulgare; Cyperaceae weeds: Cyperus iria, Cyperus rotundus, Cyperus esculentus, Kyllinga gracillima; Commelinaceae weeds: Commelina communis,
Commelina bengharensis; and Eqnisetaceae weeds: Equisetum arvense.
Formulation

[0012] The disclosed embodiments provide a formulation having weedicidal activity which includes an effective amount of microbial extracts obtained from Streptomyces sp. (MTCC No. 3001), Streptomyces hygroscopicus (ATCC Accession No. 27438), Streptomyces sp. (MTCC No. 6531), and Streptomyces viridochromogene (ATCC Accession No. 14920); and a suitable carrier. The effective amount may be any amount such that the formulation is capable of weedicidal activity. The microbial extracts are obtained when Streptomyces sp. (MTCC No. 3001), Streptomyces hygroscopicus (ATCC Accession No. 27438), Streptomyces sp. (MTCC No. 6531), and Streptomyces viridochromogene (ATCC Accession No. 14920) are conditioned under appropriate conditions. The appropriate conditions can be such that it facilitates secondary metabolism. In an embodiment, the microbial extracts comprises of secondary metabolites produced by the microbes. In another embodiment, the microbial extract includes metabolites, metabolite-containing supernatant and whole broth culture obtained from the aforementioned microbes.

[0013] In an embodiment, the formulation comprises of microbial extracts of: Streptomyces sp. (MTCC No. 3001), Streptomyces hygroscopicus (ATCC Accession No. 27438), Streptomyces sp (MTCC No. 6531), and Streptomyces viridochromogene (ATCC Accession No. 14920); and suitable carrier. In alternate embodiments the formulation may comprise of microbial extracts of variants, strains or mutants of Streptomyces sp. (MTCC No. 3001), Streptomyces hygroscopicus (ATCC Accession No. 27438), Streptomyces sp.

(MTCC No. 6531), and Streptomyces viridochromogene (ATCC Accession No. 14920). The list of suitable carriers that may be used includes stabilizers, utility adjuvants, protectants, effectors, surfactants, solvents, diluent and neutralizing agents.

[0014] 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® IF^; 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®, PathfinderTw 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®.

[0015] The utility adjuvants may be used may be LI-700® Surfactant Penetrant Acidifier

[0016] The stabilizers, effectors and UV protectants may be selected from the list which includes: ZnO, Ti02, nanodiamond; Para-Aminobenzoic Acid Derivatives such as: PABA, Ethyl PABA; Ethyl

[0017] Dihydroxypropyl PABA; Ethylhexyl Dimethyl PABA; Glyceryl PABA; PEG-25 PABA; Salicylic Derivatives such as: Homosalate ; Ethylhexyl Salicylate; Dipropyleneglycol SaUcylate; TEA Salicylate; Cinnamic Derivatives such as: Ethylhexyl Methoxycinnamate; Isopropyl Methoxy cinnamate, Isoamyl Methoxy cinnamate; Cinoxate; DEA Methoxycinnamate; Diisopropyl Methylcinnamate; Glyceryl Ethylhexanoate Dimethoxycirmamate 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: S-Benzylidene camphor, Benzylidene Camphor Sulfonic Acid, Camphor Benzalkonium Methosulfate, Terephthalylidene Dicamphor Sulfonic Acid, Polyacrylamidomethyl Benzylidene Camphor.

[0018] The solvents, diluent and neutralizing agents used may be any solvents, diluent and neutralizing agents generally used in the art.

[0019] The weedicidal activity of the formulation may be enhanced by combining it with other weedicidally active compound(s). In an embodiment, the formulation may be mixed with ingredients such as insecticides, bactericides, plant growth regulating agents, fertilizers, phytotoxicity reducing agents (safeners), and soil conditioners.

[0020] In an embodiment the formulation comprises of microbial extract in the range of 1% to 30% of Streptomyces hygroscopicus (ATCC Accession No. 27438), 1% to 30% of Streptomyces viridochromogene (ATCC Accession No. 14920), 1% to 30% of Streptomyces sp. (MTCC No. 6531), 1% to 30% of Streptomyces sp. (MTCC No. 3001). In another embodiment the formulation comprising the microbial extracts further comprises of carriers in the range of 1% to 20 % benzoic acid ammonium salt, 1% to 10 % ammonium sulphate, 0.1% to 5 % modified silicon wetting agent (MSW), 0.5% to 8 % TweenSO and 0.5% to 5 % glycerol.

[0021] The details of weedicidal 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

[0022] The process for the preparation of the disclosed formulation involves conditioning of microbes to yield an extract; and combining the extract with an acceptable carrier. 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.

Microbes

[0023] 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 and Gene Bank (MTCC), etc. The biological resource center's reference/ accession number for the microbes are as follows: Streptomyces hygroscopicus: ATCC Accession No. 27438, Streptomyces viridochromogene: ATCC Accession No. 14920, Streptomyces sp.: MTCC No. 6531, Streptomyces sp.: MTCC No. 3001. 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

Secondary metabolite production

[0024] The secondary metabolites produced are such that it has weedicidal activity and are instrumental in controlling or inhibiting weeds. The secondary metabolites control the growth of weeds by suppressing photosynthesis by acting as analogues to molecules involved in photosynthesis.

[0025] In an embodiment the secondary metabolites are produced when the microbes; ATCC 27438 {Streptomyces hygroscopicus), MTCC 6531 {Streptomyces sp.), ATCC 14920 {Streptomyces viridochromogene) and MTCC 3001 {Streptomyces sp.) 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.

[0026] Media composition: In an embodiment the media composition may be selected from the list of media compositions which includes: 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 %).

[0027] 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.

[0028] Examples 4: The details on media and percentage killing for each of these microbes are provided in Table 1.

[0029] The list of media compositions that were used to culture the microbes include: 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%, Magnesiimi 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 %). The microbes were cultured in RP media to achieve maximum metabolite production and maximum weedicidal activity within 15 days.

[0030] Table 1: media optimization

[0031] Growth conditions: The growth conditions such as duration of culture and temperature are crucial in the production of secondary metabolites. In an embodiment, the growth condition for the culture of microbes include; 50-300rpm of shaking under light for 5 - lOhrs and under dark condition for 10-18hrs, at a temperature of 25-40°C for 3-10 days for all cultures. Killing was observed within 10 to 15 days of spraying. In another embodiment, the highest weed killing was observed by the microbial extract cultured in the growth condition of; 150-200rpm shaking under light for 8hrs and under dark condition for 14hrs, at a temperature of 30°C for 5 days for all cultures.

[0032] 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.

[0033] Example 5: The details on growth conditions and percentage killing for each of these microbes are provided in Table 2.

[0034] In order to arrive at the optimum growth conditions, the starter cultures were inoculated to the RF media 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 weed killing was observed by the microbial extract cultured in the growth condition of; 150-200rpm shaking under light for 8hrs and under dark condition for 14hrs, at a temperature of 30°C for 5 days for all cultures at. Killing was observed on the 12*'' day of spraying.

[0035] Table 2: Optimization of growtli conditions

[0036] Secondary metabolite extraction: In an embodiment, the formulation comprises of an extract comprising secondary metabolites that have been extracted from microbes: Streptomyces sp.; Streptomyces hygroscopicus; Streptomyces sp.; and Streptomyces viridochromogene, 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, chloroform, ethyl acetate, etc in different concentration may be used for extraction of secondary metabolites. In an embodiment, the extraction is
5 performed by using Ethyl acetate at 20-30% (v/v) for Streptomyces hygroscopicus (ATCC 27438) and Streptomyces viridochromogene (ATCC 14920), and chloroform:methanol (wherein chloroform is in the range of 10-30%, and methanol is in the range of around 90-70%) at 20-30% for Streptomyces sp. (MTCC No. 6531) and Streptomyces sp. (MTCC 3001).

[0037] 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.

[0038] Example 6: The details on metabolite recovery and percentage killing for each of these microbes are provided in Table 3.

[0039] Various solvents in different concentration were used for extraction of secondary metabolites. The extraction of secondary metabolites performed by using ethyl acetate, methanol and chloroform at various concentrations. The preferred extraction method involves the use of Ethyl acetate at 25% (v/v) for 5". hygroscopicus (ATCC 27438) and S. viridochromogene fATCC 14920), and chloroform:methanol (20:80) at 25% for Streptomyces sp. (MTCC 6531) and Streptomyces sp. (MTCC 3001).

[0040]

Table 3: metabolites recovery

Combining the extract with suitable carrier

[0041] The microbial extract obtained from conditioning may further be combined with suitable carriers such as stabilizers, utility adjuvants, protectants, effectors, surfactants, solvents, diluent 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 well known in the art including centrifugation, filtration, sedimentation and the like.

Method for controlling and/or inhibiting weeds

[0042] Further, the growth of weeds may be controlled and/or inhibited by applying an effective amount of the disclosed formulation to the soil or weeds. The effective amoimt may be any amount such that the formulation is capable of weedicidal activity

[0043] In an embodiment, a method for controlling weeds is provided, wherein the method comprises of applying the disclosed formulation to the soil or to the weeds. In another embodiment, the formulation comprising microbial extracts is applied in an amount sufficient to control the growth of weeds. The formulation may be applied by well-known methods of application such as spraying or as a soil drench. The application may be pre-emergent, post-emergent or as a nonselective contact herbicide.

[0044] The formulation may be formulated in numerous forms, including flowables, dry flowables, water dispersible granules or emulsified concentrates. Other suitable formulations will be known to those skilled in the art.

[0045] The foregoing description of the specific embodiments will so flilly 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 formulation having weedicidal activity comprising an effective amount of extracts obtained from a group of microbes, wherein the group comprises of Streptomyces sp (MTCC No. 3001), Streptomyces hygroscopicus (ATCC No. 27438), Streptomyces sp. (MTCC No. 6531) and Streptomyces viridochromogene (ATCC No. 14920) or their strains, variants or mutants thereof; and suitable carriers.

2. The formulation having weedicidal activity as claimed in claim 1, wherein the extracts comprise of at least one secondary metabolite.

3. The formulation having weedicidal activity as claimed in claim 1, wherein the extract from each microbe is present in the range of 1% to 30%.

4. The formulation having weedicidal activity as claimed in claim 1, wherein the suitable carriers comprise at least one of stabilizers, utility adjuvants, protectants, effectors, surfactants, solvents, diluent and neutralizing agents.

5. The formulation having weedicidal activity as claimed in claim 1, wherein the suitable carriers include at least one of benzoic acid ammonium salt, ammonium sulphate, Modified silicon wetting agent, TweenSO and glycerol.

6. The formulation having weedicidal activity as claimed in claim 5, wherein benzoic acid ammonium salt is present in the range of 1% to 20 %, ammonium sulphate is present in the range of 1% to 10%, Modified silicon wetting agent is present in the range of 0.1% to 5 %, TweenSO is present in the range of 0.5% to 8 % and glycerol is present in the range of 0.5% to 5 %.

7. The formulation having weedicidal activity as claimed in claim 1, wherein the formulation is formulated as a wettable powder, a granule, an aqueous suspension, emulsifiable concentrate or a microencapsulated formulation.

8. A process for the preparation of a formulation having weedicidal activity, the process comprising:

conditioning of microbes to yield an extract, wherein the microbes include Streptomyces sp (MTCC No. 3001), Streptomyces hygroscopicus (ATCC No. 27438), Streptomyces sp. (MTCC No. 6531) and Streptomyces viridochromogene (ATCC No. 14920), or their strains, variants or mutants thereof; and combining the extract with suitable carriers.

9. The process for the preparation of a formulation having weedicidal activity as claimed in claim 8, wherein the conditioning involves subjecting the cultured microbes to secondary metabolism.

10. The process for the preparation of a formulation having weedicidal activity as claimed in claim 8, wherein the suitable carriers comprise at least one of stabilizers, utility adjuvants, protectants, effectors, surfactants, solvents, diluent and neutralizing agents.

11. A method for controlling or inhibiting weeds comprising:

applying, to soil or weed, an effective amount of a formulation having weedicidal activity comprising an extract obtained from a group of microbes, wherein the group comprises of Streptomyces sp (MTCC No. 3001), Streptomyces hygroscopicus (ATCC No. 27438), Streptomyces sp. (MTCC No. 6531) and Streptomyces viridochromogene (ATCC No. 14920), or their strains, variants or mutants thereof; and suitable carriers.