A lyophilized microbial composition of beauveria bassiana and metarhizium anisopliae for pest control

Abstract

The present disclosure provides a microbial composition comprising, a first component comprising Beauveria bassiana, or spores thereof, and a first stabilizing agent selected from trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof; and a second component comprising Metarhizium anisopliae, or spores thereof, and a second stabilizing agent selected from pectin, trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof, and a method of preparation thereof. The present disclosure also provides a lyophilized product of the microbial composition disclosed herein and a corresponding biopesticide formulation.

Description

A LYOPHILIZED MICROBIAL COMPOSITION OF BEAUVERIA BASSIANA AND METARHIZIUM ANISOPLIAE FOR PEST CONTROL FIELD OF INVENTION

[0001] The present disclosure broadly relates to the field of biopesticides and, particularly to, a microbial composition comprising a combination of microbial biomass for pest control. It relates to a microbial consortium or strains for use as biopesticides. It further relates to a method of preparing the microbial composition, a method for preparing a biopesticide formulation comprising the composition, and a method of controlling pests using the composition or the formulation. BACKGROUND OF THE INVENTION

[0002] A range of insects are responsible for the huge economic losses in agriculture sector. Currently, the main available solution to this problem is the use of chemical pesticides. The major limitations of chemical pesticides are that they are expensive and generally hazardous to the environment. There are reports suggesting that insect strains develop resistance against these pesticides and sometimes, require large quantities and different chemicals to treat. The action of these pesticides is often not very specific to the pests as they may act as non-target biological control agents.

[0003] Biopesticides, comprising insect pathogens are a possible alternative to the common use of highly toxic chemical pesticides for the control of insects. Entomopathogenic fungi are one of the promising groups of insect pathogens suitable as biological agents for the control of insects.

[0004] Wettable powder of entomopathogenic fungi is the most basic kind of biopesticide formulation, which can be made into suspension liquid, and sprays. Similarly, liquid formulations of entomopathogenic fungi are being widely used as biopesticides for various foliage pest and soil borne insect pests. Powder formulations are more convenient for subsequent processing and transportation and have longer shelf lives compared to those of liquid formulations. However, both liquid, and powder formulations have the limitation of low viability of the fungal strains and poor ambient stability.

[0005] Thus, there is a need to develop a biopesticide formulation of entomopathogenic fungi with improved viability and with extended ambient stability. SUMMARY OF INVENTION

[0006] In an aspect of the present disclosure, there is provided a microbial composition comprising, a first component comprising Beauveria bassiana, or spores thereof, and a first stabilizing agent selected from trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof; and a second component comprising Metarhizium anisopliae, or spores thereof, and a second stabilizing agent selected from pectin, trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof.

[0007] In an aspect of the present disclosure, there is provided a lyophilized product, comprising the composition having a first component comprising a Beauveria bassiana, or spores thereof, and a first stabilizing agent selected from trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof; and a second component comprising Metarhizium anisopliae, or spores thereof, and a second stabilizing agent selected from pectin, trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof.

[0008] In an aspect of the present disclosure, there is provided a biopesticide formulation, comprising: the composition or the lyophilized product as disclosed herein; and a carrier, wherein the composition is in a weight range of 1% to 80% with respect to the total weight of the formulation; and the carrier is in a weight range of 20% to 99% with respect to the total weight of the formulation.

[0009] In an aspect of the present disclosure, there is provided a method of preparing the composition as disclosed herein, the method comprising: (i) mixing Beauveria bassiana biomass, or spores thereof, and a solution comprising a first stabilizing agent selected from trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof to obtain the first component; (ii) mixing Metarhizium anisopliae biomass, or spores thereof, and a solution comprising a second stabilizing agent selected from pectin, trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof to obtain secondcomponent; and (iii) mixing said first component and said second component to obtain the composition.

[0010] In an aspect of the present disclosure, there is provided a method of preparing the biopesticide formulation as disclosed herein, the method comprising: diluting the composition as disclosed herein, or a composition obtained by the method as disclosed herein, or the lyophilized product as disclosed herein, with a carrier to obtain the formulation.

[0011] In an aspect of the present disclosure, there is provided a kit comprising: the microbial composition or the formulation as disclosed herein; and optionally an instruction manual.

[0012] In an aspect of the present disclosure, there is provided a method of controlling pests, the method comprising: contacting the pests with the composition as disclosed herein or the formulation as disclosed herein.

[0013] In another aspect of the present disclosure, there is provided a microbial consortium comprising a strain of Beauveria bassiana; and a strain of Metarhizium anisopliae, wherein concentration of the strain of Beauveria bassiana is in a range of 108to 1010cfu / g dry matter of the consortium, and concentration of the strain of Metarhizium anisopliae is in a range of 108to 1010cfu / g dry matter of the consortium.

[0014] In another aspect of the present disclosure, there is provided a strain of Beauveria bassiana deposited under the accession number MTCC25687.

[0015] In another aspect of the present disclosure, there is provided a strain of Metarhizium anisopliae deposited under the accession number MTCC25686.

[0016] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

[0017] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0018] Figure 1 depicts the colony forming unit (CFU) result of storage stability study at accelerated conditions (37°C) for the microbial composition comprising Beauveria bassiana and Metarhizium anisopliae formulated in dextrose, in accordance with the embodiments herein.

[0019] Figure 2 depicts the CFU result of storage stability study at ambient conditions (25°C) for the microbial composition comprising Beauveria bassiana and Metarhizium anisopliae formulated in dextrose, in accordance with the embodiments herein.

[0020] Figure 3 depicts the CFU result of storage stability study at refrigerated conditions (4°C) for the microbial composition comprising Beauveria bassiana and Metarhizium anisopliae formulated in dextrose, in accordance with the embodiments herein. DETAILED DESCRIPTION OF THE INVENTION

[0021] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features. Definitions

[0022] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, forconvenience and completeness, particular terms and their meanings are set forth below.

[0023] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0024] The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

[0025] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0026] The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.

[0027] The term “entomopathogenic fungi”, as used herein, refers to fungi that are capable of attacking, infecting, killing, disabling, causing disease, and / or causing injury to an insect. According to the embodiments herein, the entomopathogenic fungi include strains of Beauveria bassiana, and Metarhizium anisopliae.

[0028] The term “biopesticide”, as used herein, refers to a biologically derived substance capable of killing; or inhibiting or retarding growth, reproduction or spread of plant pests like insects. According to the present disclosure, the biopesticides comprise entomopathogenic fungi, namely Beauveria bassiana, and / or Metarhizium anisoplia. It has the ability of killing or inhibiting pests; inhibiting or retarding growth of pests; affecting reproduction or spread of pests, particularly plant pests.

[0029] The term “colony forming unit (cfu)”, as used herein, is a measure of the viable fungal cells, in which a colony represents an aggregate of cells derived from a single progenitor fungal cell. Further, the term “cfu / g” refers to the cfu obtained per gram of the microbial composition or the formulation of the present disclosure.

[0030] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not onlythe numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0032] Embodiments herein provide a microbial composition for pest control, having enhanced storage stability. The disclosed embodiments provide a microbial composition, a lyophilized product, a biopesticide formulation, a kit, a method of preparing the composition, a method of preparing the biopesticide formulation and a method of controlling pests.

[0033] Further, the embodiments herein are capable of addressing the challenges associated with the development of microbial biopesticide with enhanced storage stability and activity. The microbial composition, according to embodiments herein, is suitable for preparing a biopesticide formulation that exhibits extended shelf life, along with stable microbial viability. Further, the embodiments herein also include a method for the preparation of the composition and the formulation. Furthermore, the embodiments herein provide a method of controlling the pests using said composition or the formulation. In addition, the embodiments herein include a kit comprising said formulation and / or composition. The embodiments herein also provide a microbial consortium comprising a strain of Beauveria bassiana; and a strain of Metarhizium anisopliae. The embodiments herein also provide microbial strains of Beauveria bassiana; and Metarhizium anisopliae.

[0034] The microbial composition, as disclosed in the embodiments herein, is preferably lyophilized and comprises one or more strains of entomopathogenic fungi that are capable of exhibiting biopesticide activity. Further, the microbial composition comprises stabilizing agents. The stabilizing agent protects the fungi from damage caused due to freezing, thawing, etc. during the process oflyophilization. Thus, the specific combination of stabilizing agent, according to the embodiments herein, is crucial for protecting the structural integrity of the fungi and thereby retaining viability for extended period of time. The embodiments, according to the present disclosure, are capable of achieving viability of cells for an extended period of over 36 months. In an embodiment, the microbial composition exhibits extended shelf life, with 108to 1010cfu / g, for a period of up to 36 months. In an embodiment, the microbial composition comprising the microbial strains in a concentration of between 2x109to 1x109cfu / g exhibits extended shelf life for a period of up to 36 months. In an embodiment, the microbial composition exhibits extended shelf life, having about 2x109cfu / g, for a period of up to 24 months. The retained viability of the entomopathogenic fungi, furthermore, ensures the sustained biopesticide activity even after storing the formulation for extended periods of time. Microbial Composition

[0035] Embodiment herein include a microbial composition. The microbial composition, according to embodiments herein, comprises a first component and a second component. The first component and second component, according to the present disclosure, may be in the form of mixtures, suspensions, lyophilized cake, lyophilized powder, lyophilized granules, etc. In an embodiment, the first component is a mixture comprising Beauveria bassiana, or spores thereof; and stabilizing agents. In another embodiment, the first component is a lyophilized powder or cake comprising Beauveria bassiana, or spores thereof; and stabilizing agents. In an embodiment, the second component is a mixture comprising Metarhizium anisopliae, or spores thereof; and stabilizing agents. In another embodiment, the second component is a lyophilized powder or cake comprising Metarhizium anisopliae, or spores thereof; and stabilizing agents.

[0036] In an embodiment, the first component is a stabilized composition of Beauveria bassiana, or spores thereof. In another embodiment, the first component is lyophilized composition of Beauveria bassiana, or spores thereof. In an embodiment, the second component is a stabilized composition of Metarhizium anisopliae, or spores thereof. In another embodiment, the second component is lyophilized composition of Metarhizium anisopliae, or spores thereof. The presentinvention achieves a stabilized microbial composition. In an embodiment, the microbial composition is a stabilized microbial composition.

[0037] In an embodiment, the composition comprises a first component comprising Beauveria bassiana, or spores thereof, and a stabilizing agent. In another embodiment, the composition comprises a second component comprising Metarhizium anisopliae, or spores thereof, and a stabilizing agent. The stabilizing agent for Beauveria bassiana is referred to herein as “first stabilizing agent”, and the stabilizing agent for Metarhizium anisopliae is referred to herein as “second stabilizing agent”. In an embodiment, the microbial composition comprises biomass of Beauveria bassiana and Metarhizium anisopliae. The term “biomass”, as used herein refers to fungal material including cells, spores, etc. of fungus such as Beauveria bassiana, Metarhizium anisopliae, etc. It may be produced by culturing the strains of Beauveria bassiana or Metarhizium anisopliae, in solid culture media or liquid culture media using fermentation processes that are well known in the art. Further, optimal conditions of growth will depend on the specific strains of the fungi.

[0038] In yet another embodiment, the composition comprises a first component comprising Beauveria bassiana, or spores thereof, and a first stabilizing agent; and a second component comprising Metarhizium anisopliae, or spores thereof, and a second stabilizing agent.

[0039] In an embodiment, the composition comprises a first component comprising Beauveria bassiana, or spores thereof, and a first stabilizing agent; and a second component comprising Metarhizium anisopliae, or spores thereof, and a second stabilizing agent, wherein the first component comprises Beauveria bassiana and the first stabilizing agent in a weight ratio in the range of 1:0.1 to 1:10, 1:0.1 to 1:5, or 1:0.2 to 1:2, preferably in a weight ratio of 1:0.5; and wherein the second component comprises Metarhizium anisopliae and the second stabilizing agent in a weight ratio in the range of 1:0.1 to 1:10, 1:0.1 to 1:5, or 1:0.2 to 1:2, preferably in a weight ratio of 1:0.5.

[0040] The Beauveria bassiana and Metarhizium anisopliae, according to embodiments herein, may be of any strain. The Beauveria bassiana andMetarhizium anisopliae may be obtained commercially or may be isolated strains of Beauveria bassiana and Metarhizium anisopliae. For example, the inventors have isolated various strains of Beauveria bassiana from virgin forest soil of Jaberkhet, Dehradun, Uttarakhand, India, which may be used in various embodiments herein. In another example, the inventors have isolated various strain of Metarhizium anisopliae from farmer’s crop fields in Askote, Pithoragarh, Uttarakhand, India, which may be used in various embodiments herein. In an embodiment, the Beauveria bassiana is of a strain deposited under the accession number MTCC25687. In an embodiment, the Metarhizium anisopliae is of a strain deposited under the accession number MTCC25686. It is understood that the claimed composition may also be practised with other strains of Beauveria bassiana and Metarhizium anisopliae.

[0041] In some embodiments, the composition comprises said Beauveria bassiana in an amount in the range of 1% to 80%, with respect to the total weight of the first component.

[0042] In an embodiment, the composition comprises said Beauveria bassiana in an amount in the range of 1% to 80%, with respect to the total weight of the first component. In another embodiment, said Beauveria bassiana is in an amount in the range of 5% to 25%, with respect to the total weight of the first component. In yet another embodiment, said Beauveria bassiana is in an amount in the range of 10% to 20%, with respect to the total weight of the first component.

[0043] In some embodiments, said Metarhizium anisopliae is in an amount in the range of 1% to 80%, with respect to the total weight of the second component.

[0044] In an embodiment, said Metarhizium anisopliae is in an amount in the range of 1% to 80%, with respect to the total weight of the second component. In another embodiment, said Metarhizium anisopliae is in an amount in the range of 5% to 25%, with respect to the total weight of the second component. In yet another embodiment, said Metarhizium anisopliae is in an amount in the range of 10% to 20%, with respect to the total weight of the second component.

[0045] The term “stabilizing agent”, as used herein, refers to a substance for protecting fungal cells. It refers to a lyoprotectant. The stabilizing agent, accordingto embodiments herein, are capable of protecting the fungal cells from cell injuries caused during freeze-drying process, and thus provide not only cell viability after freeze drying but also extended shelf life. The stabilizing agent, according to embodiments herein, includes a combination of compounds and may be in the form of powders, solution, etc. In an embodiment, the stabilizing agent is a solution. The stabilizing agent, according to embodiments herein, is selected from pectin; trehalose; maltose; maltodextrin; monosodium glutamate; glycerol; carboxy methyl cellulose (CMC); polyethylene glycol (PEG); an antioxidant or derivative thereof; or combinations thereof. In an embodiment, the stabilizing agent is selected from pectin; trehalose; maltose; maltodextrin; monosodium glutamate; glycerol; carboxy methyl cellulose (CMC); polyethylene glycol (PEG); an antioxidant or derivative thereof; or combinations. In an embodiment, the stabilizing agent is selected from pectin; trehalose; maltose; maltodextrin; monosodium glutamate; glycerol; carboxy methyl cellulose (CMC); polyethylene glycol (PEG); an antioxidant or derivative thereof; or combinations thereof. The term “derivative”, refers to any substance functionalized with one or more functional groups such as methyl, ethyl, acetyl, carboxyl, aryl, etc.

[0046] In an embodiment, the first stabilizing agent is selected from trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof. In an embodiment, the first stabilizing agent comprises trehalose, and maltodextrin. In an embodiment, the first stabilizing agent comprises trehalose, maltose, maltodextrin, and monosodium glutamate. In another embodiment, the first stabilizing agent comprises trehalose, maltose, maltodextrin, monosodium glutamate, and glycerol. In another embodiment, the second stabilizing agent is selected from pectin, trehalose, maltose, maltodextrin, monosodium glutamate, glycerol, or combination thereof. In an embodiment, the second stabilizing agent comprises glycerol and monosodium glutamate. In an embodiment, the second stabilizing agent comprises pectin, trehalose, maltose, maltodextrin, and monosodium glutamate. In another embodiment, the second stabilizing agent comprises pectin, trehalose, maltose, maltodextrin, monosodium glutamate, and glycerol.

[0047] In some embodiments, the maltodextrin has a dextrose equivalent in a range of 3 to 20. The term “dextrose equivalent”, as used herein, refers to a measure of the degree of hydrolysis of starch. It refers to a measure of reducing sugars expressed as dextrose and calculated as a percentage on a dry basis.

[0048] In an embodiment, the maltodextrin has a dextrose equivalent value in a range of 10 to 14, preferably in a range of 10 to 12.

[0049] In an embodiment, the first stabilizing agent and / or second stabilizing agent further comprises PEG. In some embodiments, the PEG has an average molecular weight in the range of 200 to 10000 g / mol. Examples of PEG with average molecular weight in the range of 200 to 10000 include, but not limited to, PEG200, PEG 300, PEG 400, PEG 600, PEG 1500, PEG 3350, PEG 6000, and PEG 10000. For example, PEG 1000 refers to a mixture of purified PEG molecules (about 195- 265 PEG molecules) having an average MW of 10,000 g / mol.

[0050] In an embodiment, the PEG is PEG 6000 having an average molecular weight of 6000.

[0051] In an embodiment, the first stabilizing agent and / or the second stabilizing agent further comprises an antioxidant. In some embodiments, the antioxidant is selected from vitamin C, vitamin E, glutathione, or combination thereof.

[0052] In an embodiment, the antioxidant is vitamin C (ascorbic acid) or derivatives thereof.

[0053] In some embodiments, the first stabilizing agent and / or second stabilizing agent further comprises poly vinyl and carbohydrates. In some embodiments, the first stabilizing agent and / or second stabilizing agent further comprises carbohydrates, such as sorbitol and starch; wherein starch is selected from potato starch, corn starch, wheat starch, rice starch, or combination thereof. In an embodiment, the starch is potato starch (for e.g.: by Himedia, India).

[0054] In some embodiments, the polyvinyl is selected from polyvinyl alcohol, polyvinyl acetate, polyvinyl pyrrolidone, or combinations thereof. In an embodiment, the polyvinyl is polyvinyl pyrrolidone.

[0055] In an embodiment, the first component comprises a first stabilizing agent selected from from trehalose, maltose, maltodextrin, glycerol, monosodiumglutamate or combination thereof; wherein the first stabilizing agent is in an amount in the range of 0.7% to 84%, with respect to the total weight of the first component. In another embodiment, the first component comprises a first stabilizing agent selected from trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof; wherein the first stabilizing agent is in an amount in the range of 0.7% to 84%, with respect to the total weight of the first component.

[0056] In another embodiment, the first component comprises a first stabilizing agent having trehalose in an amount in the range of 0.1% to 20%, maltose in an amount in the range of 0 to 10%, maltodextrin in an amount in the range of 0.1% to 20%, monosodium glutamate in an amount in the range of 0 to 10%, and glycerol in an amount in the range of 0 to 10%, with respect to the total weight of the first component.

[0057] In another embodiment, the first component comprises a first stabilizing agent having trehalose in an amount in the range of 1% to 20% or 1% to 17%; maltose in an amount in the range of 0 to 7%, 0 to 6%, 0.1 to 7%, 0.01 to 6%, or 0.1 to 6%; maltodextrin in an amount in the range of 1% to 20%, or 1% to 17%; monosodium glutamate in an amount in the range of 0.01 to 6%, 0 to 6%, or 0 to 5%; and glycerol in an amount in the range of 0 to 5% or 0.5% to 5%, with respect to the total weight of the first component.

[0058] In another embodiment, the first component includes a stabilizing agent further comprising starch in an amount in the range of 0.1% to 10%, with respect to the total weight of the first component, wherein said starch is selected from potato starch, corn starch, wheat starch, rice starch, or combination thereof. In another embodiment, the first component includes a stabilizing agent further comprising starch in an amount in the range of 0.5% to 5%, with respect to the total weight of the first component.

[0059] In an embodiment, the first component is a mixture comprising Beauveria bassiana, or spores thereof, and a stabilizing agent comprising, trehalose in an amount in the range of 0.1% to 20%, maltose in an amount in the range of 0.1% to 10%, maltodextrin in an amount in the range of 0.1% to 20%, and monosodium glutamate in an amount in the range of 0.1% to 10%, with respect to the total weightof the first component. In another embodiment, the first component is subjected to lyophilization to obtain lyophilized first component.

[0060] In an embodiment, the first component is a suspension comprising Beauveria bassiana, or spores thereof, and a stabilizing agent comprising, trehalose in an amount in the range of 0.1% to 20%, maltose in an amount in the range of 0 to 10%, maltodextrin in an amount in the range of 0.1% to 20%, monosodium glutamate in an amount in the range of 0 to 10%, and glycerol in an amount in the range of 0 to 10%, with respect to the total weight of the first component. In another embodiment, the first component is subjected to lyophilization to obtain lyophilized first component. In another embodiment, the first component is in lyophilized form.

[0061] In an embodiment, the second component comprises a second stabilizing agent selected from pectin, trehalose, maltose, maltodextrin, monosodium glutamate, and glycerol, or combination thereof, wherein the stabilizing agent is in a weight range of 0.7% to 84%, with respect to the total weight of the second component. In another embodiment, the second component comprises a second stabilizing agent selected from pectin, trehalose, maltose, maltodextrin, monosodium glutamate, glycerol, or combination thereof, wherein the stabilizing agent is in a weight range of 0.7% to 84%, with respect to the total weight of the second component.

[0062] In another embodiment, the second component comprises a second stabilizing agent selected from pectin in an amount in the range of 0 to 10%, trehalose in an amount in the range of 0 to 10%, maltose in an amount in the range of 0 to 10%, maltodextrin in an amount in the range of 0 to 15%, monosodium glutamate in an amount in the range of 0.1% to 10%, glycerol in an amount in the range of 0 to 10%, or combination thereof, with respect to the total weight of the second component.

[0063] In another embodiment, the second component comprises a second stabilizing agent selected from pectin in an amount in the range of 0.1% to 10%, 0 to 6%, 0 to 5%, or 0.01 to 5%; trehalose in an amount in the range of 0.01% to 10%, 0.1% to 10%, 0 to 8%, 0.01% to 8%, 0 to 7% or 0.01% to 7%; maltose in an amount in the range of 0.01% to 10%, 0.1% to 10%, 0 to 8%, 0.01% to 8%, 0 to 7% or0.01% to 7%; maltodextrin in an amount in the range of 0 to 10%, 0.01% to 10%, 0.1% to 10%, 0 to 8%, 0.01% to 8%, 0 to 7% or 0.01% to 7%; monosodium glutamate in an amount in the range of 0.1% to 6%, 0.1% to 5%, or 0.01 to 5%; glycerol in an amount in the range of 0.1% to 10%, 0.01 to 5%, 0 to 5%, or 0.5% to 5%; or combination thereof, with respect to the total weight of the second component.

[0064] In another embodiment, the second component comprises a second stabilizing agent comprising pectin in an amount in the range of 0 to 10%, trehalose in an amount in the range of 0 to 10%, maltose in an amount in the range of 0 to 10%, maltodextrin in an amount in the range of 0 to 15%, and monosodium glutamate in an amount in the range of 0.1% to 10% with respect to the total weight of the second component. In yet another embodiment, the second component comprises a second stabilizing agent comprising monosodium glutamate in an amount in the range of 0.1% to 10% and glycerol in an amount in the range of 0 to 10%, with respect to the total weight of the second component.

[0065] In a further embodiment, the first stabilizing agent and / or the second stabilizing agent further comprises polyvinyl in an amount in the range of 0.1% to 10%, and sorbitol in an amount in the range of 0.1% to 10%, with respect to the total weight of the first or second component, wherein the polyvinyl is selected from polyvinyl alcohol, polyvinyl acetate, polyvinyl pyrrolidone, or combinations thereof. In a furthermore embodiment, the second component comprises a stabilizing agent, further comprising, polyvinyl pyrrolidone in an amount in the range of 0.5% to 5%, and sorbitol in an amount in the range of 0.5% to 5%, with respect to the total weight of the second component.

[0066] In an embodiment, the second component is a mixture comprising Metarhizium anisopliae, or spores thereof, and a second stabilizing agent selected from pectin in an amount in the range of 0 to 10%, trehalose in an amount in the range of 0 to 10%, maltose in an amount in the range of 0 to 10%, maltodextrin in an amount in the range of 0 to 15%, monosodium glutamate in an amount in the range of 0.1% to 10%, glycerol in an amount in the range of 0 to 10%, or combination thereof, with respect to the total weight of the second component. Inanother embodiment, the second component is subjected to lyophilization to obtain lyophilized second component. In another embodiment, the second component is in lyophilized form.

[0067] In an embodiment, the first component and the second component are in a weight ratio in the range of 1:9 to 9:1, 1:5 to 5:1, or 1:3 to 3:1, preferably in a weight ratio of 1:1. In another embodiment, the lyophilized first component and lyophilized second component are in a weight ratio in the range of 1:9 to 9:1, 1:5 to 5:1, or 1:3 to 3:1, preferably in a weight ratio of 1:1. In an embodiment, the lyophilized Beauveria bassiana and lyophilized Metarhizium anisopliae are in a weight ratio in the range of 1:9 to 9:1, 1:5 to 5:1, or 1:3 to 3:1, preferably in a weight ratio of 1:1. In another embodiment, the Beauveria bassiana and lyophilized Metarhizium anisopliae are in a weight ratio in the range of 1:9 to 9:1, 1:5 to 5:1, or 1:3 to 3:1, preferably in a weight ratio of 1:1.

[0068] Accordingly, in an embodiment, the microbial composition comprises first component and second component in a weight ratio in the range of 1:9 to 9:1, preferably in a weight ratio of 1:1. Microbial Consortium

[0069] Embodiments herein include a microbial consortium. The microbial consortium, according to embodiments herein, comprises entomopathogenic fungi including, but not limited to, Beauveria bassiana and Metarhizium anisopliae. The microbial consortium, according to embodiment herein, have the ability of killing or inhibiting pests; inhibiting or retarding growth of pests; affecting reproduction or spread of pests, particularly plant pests.

[0070] In an embodiment, there is provided a microbial consortium comprising, a strain of Beauveria bassiana; and a strain of Metarhizium anisopliae; wherein the concentration of, each of, Beauveria bassiana; and the Metarhizium anisopliae is in a range of 108to 1010cfu / g dry matter of the consortium.

[0071] In another embodiment, there is provided a microbial consortium comprising a strain of Beauveria bassiana; and a strain of Metarhizium anisopliae; wherein the concentration of the strain of Beauveria bassiana; and the strain of Metarhizium anisopliae is in a range of 108to 1010cfu / g dry matter of theconsortium; wherein the Beauveria bassiana is of a strain deposited under the accession number MTCC 25687; and the Metarhizium anisopliae is of a strain deposited under the accession number MTCC 25686. The present inventors have deposited a strain of Beauveria bassiana at Microbial Type Culture Collection and Gene Bank (MTCC) under the accession number MTCC25687. A strain of Metarhizium anisopliae has also been deposited at Microbial Type Culture Collection and Gene Bank (MTCC) under the accession number MTCC25686.

[0072] In an embodiment, there is provided a microbial consortium comprising a strain of Beauveria bassiana; and a strain of Metarhizium anisopliae; wherein the Beauveria bassiana is of the strain deposited under the accession number MTCC 25687; and the Metarhizium anisopliae is of the strain deposited under the accession number MTCC25686.

[0073] In an embodiment of the present disclosure, there is provided a strain of Beauveria bassiana deposited under the accession number MTCC25687.

[0074] In an embodiment of the present disclosure, there is provided a strain of Metarhizium anisopliae deposited under the accession number MTCC25686.

[0075] The strain(s), according to embodiments herein, may be used as biopesticides, having the ability of killing or inhibiting pests; inhibiting or retarding growth of pests; affecting reproduction or spread of pests, particularly plant pests. Lyophilized product for reconstitution

[0076] Embodiments herein include a lyophilized product, comprising the microbial composition described herein. The lyophilized product, in various embodiments herein, may comprise the lyophilized microbial composition wherein the microbial composition comprises the first component and second component. In other embodiments, the lyophilized product comprises a mixture of the lyophilized first component and lyophilized second component. In an embodiment, the lyophilized product comprises lyophilized first component and lyophilized second component, wherein the first component comprises stabilized Beauveria bassiana, or spores thereof, and the second component comprises stabilized Metarhizium anisopliae, or spores thereof. Accordingly, in another embodiment, there is provided a lyophilized product, comprising the lyophilizedfirst component comprising Beauveria bassiana, or spores thereof, and a first stabilizing agent selected from trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof; and a second component comprising Metarhizium anisopliae, or spores thereof, and a second stabilizing agent selected from pectin, trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof.

[0077] In an embodiment, there is provided a lyophilized product, comprising the lyophilized first component comprising Beauveria bassiana, or spores thereof, and a first stabilizing agent selected from trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof; and a second component comprising Metarhizium anisopliae, or spores thereof, and a second stabilizing agent selected from pectin, trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof.

[0078] The lyophilized product may be in any suitable form including, but not limited to powder, granules, beads, cakes, etc. In some embodiments, the lyophilized product is a lyophilized powder for reconstitution. Reconstitution may be performed using a suitable carrier.

[0079] In an embodiment, there is provided a lyophilized product comprising the lyophilized microbial composition, wherein the microbial composition comprises: a first component comprising Beauveria bassiana, or spores thereof, and a first stabilizing agent selected from trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof; and a second component comprising Metarhizium anisopliae, or spores thereof, and a second stabilizing agent selected from pectin, trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof. In an embodiment, the lyophilized product comprises: a lyophilized first component comprising Beauveria bassiana, or spores thereof, and a first stabilizing agent comprising trehalose, maltose, maltodextrin, and monosodium glutamate; and a lyophilized second component comprising a biomass of Metarhizium anisopliae, or spores thereof, and a second stabilizing agent comprising pectin, trehalose, maltose, maltodextrin, and monosodium glutamate.

[0080] In an embodiment, the lyophilized product comprises: a lyophilized first component comprising Beauveria bassiana, or spores thereof, and a first stabilizing agent comprising trehalose, maltodextrin, monosodium glutamate, and glycerol; and a lyophilized second component comprising a biomass of Metarhizium anisopliae, or spores thereof, and a second stabilizing agent comprising pectin, trehalose, maltose, maltodextrin, monosodium glutamate, and glycerol. In another embodiment, the lyophilized product comprises: a lyophilized first component comprising Beauveria bassiana, or spores thereof, and a first stabilizing agent comprising trehalose, maltose, maltodextrin, monosodium glutamate, and glycerol; and a lyophilized second component comprising a biomass of Metarhizium anisopliae, or spores thereof, and a second stabilizing agent comprising pectin, trehalose, maltose, maltodextrin, monosodium glutamate, and glycerol.

[0081] In another embodiment, the lyophilized product comprises: a lyophilized first component comprising Beauveria bassiana, or spores thereof, and a first stabilizing agent comprising trehalose and maltodextrin; and a lyophilized second component comprising a biomass of Metarhizium anisopliae, or spores thereof, and a second stabilizing agent comprising monosodium glutamate, and glycerol.

[0082] In another embodiment, there is provided a lyophilized product comprising a first component comprising Beauveria bassiana, or spores thereof, and a first stabilizing agent selected from trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof; and a second component comprising Metarhizium anisopliae, or spores thereof, and a second stabilizing agent selected from pectin, trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof; wherein the Beauveria bassiana retains viability of about 108to 1010cfu / g at a temperature in the range of -20 to 37 ℃, and Metarhizium anisopliae retains viability of about 108to 1010cfu / g at a temperature in the range of -20 to 37 ℃. Biopesticide formulation

[0083] Embodiments herein include a biopesticide formulation. In an embodiment, there is provided a biopesticide formulation comprising, (a) the microbial composition described herein; and (b) a carrier.

[0084] The term “carrier”, as used herein, refers to chemical substances that are used to maintain the stability and / or activity of the formulation. In an example, it refers to a solvent. The carriers are capable of providing an environment to support the viability of the fungal spores. Unless the formulation is prepared immediately prior to use, the carrier may be used to maintain the activity of the biopesticide during storage (e.g., in a container for the entire shelf-life of the formulation). The carrier may also be used to maintain the activity of the biopesticide formulation after the formulation described herein has been applied to the application surface.

[0085] Various carriers are known in the art and can be suitably used as part of the biopesticide formulation disclosed herein. According to the present disclosure, the carrier is water soluble. Examples of carriers include, but are not limited to, lactose, sucrose, dextrose, mannitol, sorbitol, or soluble starch, or derivatives thereof.

[0086] In an embodiment, there is provided a biopesticide formulation comprising, (a) the microbial composition described herein; and (b) a carrier; wherein the composition is in a weight range of 1% to 80% with respect to the total weight of the formulation; and the carrier is in a weight range of 20% to 99% with respect to the total weight of the formulation.

[0087] In another embodiment, there is provided a biopesticide formulation comprising, (a) the microbial composition, comprising a first component comprising Beauveria bassiana, or spores thereof, and a first stabilizing agent selected from trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof; and a second component comprising Metarhizium anisopliae, or spores thereof, and a second stabilizing agent selected from pectin, trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof; and (b) a carrier selected from lactose, sucrose, dextrose, mannitol, sorbitol, or soluble starch, or combination thereof, preferably dextrose; wherein the composition is in a weight range of 5% to 25% with respect to the total weight of the formulation; and the carrier is in a weight range of 75% to 95% with respect to the total weight of the formulation.

[0088] In another embodiment, there is provided a biopesticide formulation comprising, (a) the microbial composition, comprising a first componentcomprising Beauveria bassiana, or spores thereof, and a first stabilizing agent selected from trehalose, maltose, maltodextrin, glycerol monosodium glutamate or combination thereof; and a second component comprising Metarhizium anisopliae, or spores thereof, and a second stabilizing agent selected from pectin, trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof; and (b) a carrier selected from lactose, sucrose, dextrose, mannitol, sorbitol, soluble starch, or combinations thereof, preferably dextrose; wherein the composition is in a weight range of 5% to 25% with respect to the total weight of the formulation; and the carrier is in a weight range of 75% to 95% with respect to the total weight of the formulation.

[0089] In another embodiment, there is provided a biopesticide formulation comprising, (a) the microbial composition, comprising a first component comprising Beauveria bassiana, or spores thereof, and a first stabilizing agent selected from trehalose, maltose, maltodextrin, glycerol monosodium glutamate or combination thereof; and a second component comprising Metarhizium anisopliae, or spores thereof, and a second stabilizing agent selected from pectin, trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof; and (b) a carrier selected from lactose, sucrose, dextrose, mannitol, sorbitol, soluble starch, or combinations thereof, preferably dextrose; wherein the composition is in a weight range of 10% to 15% with respect to the total weight of the formulation; and the carrier is dextrose in a weight range of 88% to 92% with respect to the total weight of the formulation. Kit

[0090] Embodiments herein provide a kit. In an embodiment, there is provided a kit comprising the microbial composition, lyophilized product, or the biopesticide formulation; a container; and optionally an instruction manual.

[0091] In another embodiment, there is provided a kit comprising the biopesticide formulation having the microbial composition, and a carrier; a container; and optionally an instruction manual; wherein the (a) microbial composition, comprises a first component comprising Beauveria bassiana, or spores thereof, and a firststabilizing agent selected from trehalose, maltose, maltodextrin, glycerol monosodium glutamate or combination thereof; and a second component comprising Metarhizium anisopliae, or spores thereof, and a second stabilizing agent selected from pectin, trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof; and (b) the carrier is selected from lactose, sucrose, dextrose, mannitol, sorbitol, soluble starch, or combinations thereof, preferably dextrose; wherein the composition is in a weight range of 1% to 80% with respect to the total weight of the formulation; and the carrier is in a weight range of 20% to 99% with respect to the total weight of the formulation. Preparation and Methods

[0092] Embodiments herein include a method for preparing the microbial composition disclosed in various embodiments herein.

[0093] In an embodiment of the present disclosure, the method comprises, (i) mixing Beauveria bassiana biomass, or spores thereof, and a solution comprising a first stabilizing agent selected from trehalose, maltose, maltodextrin, glycerol monosodium glutamate or combination thereof, and lyophilizing to obtain a first component; (ii) mixing Metarhizium anisopliae biomass, or spores thereof, and a solution comprising a second stabilizing agent selected from pectin, trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof, and lyophilizing to obtain a second component; (iii) mixing said first component and said second component to obtain the composition.

[0094] The Beauveria bassiana and Metarhizium anisopliae, according to embodiments herein, may be cultured in vitro in suitable culture media. The culture media may have suitable amounts of carbon, nitrogen, and optionally salt sources, to enable high yields of the fungal spores. Various culture media compositions are known in the art and may be used in embodiments herein.

[0095] In an embodiment, the culture media comprises (a) a carbon source selected in a weight range of 0.1% to 5% with respect to the total weight of the medium; and (b) a nitrogen source in a weight range of 0.1% to 6% with respect to the total weight of the medium.

[0096] In another embodiment of the present disclosure, the culture media optionally comprises salt in a weight range of 0.001 to 5% with respect to the total weight of the medium.

[0097] In another embodiment, the culture media comprises (a) a carbon source selected from dextrose, sucrose, soluble starches, jaggery, glycerol, maltose, succinic acid or combinations thereof, in a weight range of 0.1% to 5% with respect to the total weight of the medium; and (b) a nitrogen source selected from fish meal, corn meal, soy flour, soyabean meal, oatmeal, soy peptone, fish peptone, casamino acids, casein (tryptone), gelatin hydrolysate, proteose peptone, bacteriological peptone, animal derived peptones, yeast extract, rice bran extract, barley extract, meat extract or combinations thereof, in a weight range of 0.1% to 6% with respect to the total weight of the medium.

[0098] In another embodiment of the present disclosure, the culture media optionally comprises a salt selected from zinc sulfate, copper sulfate, magnesium sulfate, manganese sulfate, citric acid, ferric citrate, calcium chloride, sodium chloride, magnesium chloride, calcium carbonate, ferrous sulfate, boric acid, sodium molybdate, chloroform, potassium hydrogen phosphate or combinations thereof, in a weight range of 0.001 to 5% with respect to the total weight of the medium.

[0099] In yet another embodiment, the culture media for culturing Beauveria bassiana comprises: (a) a carbon source selected from dextrose, sucrose, soluble starches, jaggery, glycerol, maltose, succinic acid or combinations thereof, in a weight range of 0.1% to 5% with respect to the total weight of the medium; (b) a first nitrogen source selected from soy peptone, fish peptone, casamino acids, casein (tryptone), gelatin hydrolysate, proteose peptone, bacteriological peptone, animal derived peptones or combinations thereof, in a weight range of 0.1% to 3% with respect to the total weight of the medium; and (c) a second nitrogen source selected from yeast extract, rice bran extract, barley extract, meat extract or combinations thereof, in a weight range of 0.1% to 3%, with respect to the total weight of the medium.

[0100] In yet another embodiment, the culture media for culturing Metarhizium anisopliae comprises: (a) a carbon source selected from dextrose, sucrose, soluble starches, jaggery, glycerol, maltose, succinic acid or combinations thereof, in a weight range of 0.1% to 5% with respect to the total weight of the medium; (b) a first nitrogen source selected from fish meal, corn meal, soyabean meal, soy flour, oatmeal, or combinations thereof, in a weight range of 0.1% to 3% with respect to the total weight of the medium; (c) a second nitrogen source selected from soy peptone, fish peptone, casamino acids, casein (tryptone), gelatin hydrolysate, proteose peptone, bacteriological peptone, animal derived peptones or combinations thereof, in a weight range of 0.1% to 3% with respect to the total weight of the medium; and (d) a salt selected from zinc sulfate, copper sulfate, magnesium sulfate, manganese sulfate, citric acid, ferric citrate, calcium chloride, sodium chloride, magnesium chloride, calcium carbonate, ferrous sulfate, boric acid, sodium molybdate, chloroform, potassium hydrogen phosphate or combinations thereof, in a weight range of 0.001 to 5%, with respect to the total weight of the medium.

[0101] The culturing of fungi, according to embodiments herein, may be performed in a bioreactor. The bioreactor may be a lab scale or an industrial scale fermenter and may be operated in batch mode or continuous mode. Based on the needs and preferred conditions, a person skilled in the art will be able to choose an appropriate size of bioreactor and as well as a mode of operating the bioreactor.

[0102] The fungi grown in a bioreactor may be suspended or immobilized.

[0103] In an embodiment, the strain of Beauveria bassiana or the strain of Metarhizium anisopliae is grown in a suspended state in a culture medium having a pH in the range of 3.5 to 7; at a temperature in the range of 20°C to 35°C for a time period in the range of 18 to 96 hours.

[0104] Further, the biomass of Beauveria bassiana, or spores thereof, or Metarhizium anisopliae, or spores thereof, may be harvested and / or concentrated using any suitable method(s) known in the art. Examples include, but not limited to, centrifugation, coagulation, decanting, filtration, or flocculation. In an embodiment, the grown cultures of Beauveria bassiana or spores thereof, orMetarhizium anisopliae or spores thereof is harvested by centrifugation under aseptic conditions.

[0105] In further embodiments, the harvested biomass is subjected to a drying process. Various drying processes are known in the art and may be used in embodiments herein. Examples of drying processes include, but are not limited to, air-drying, freeze drying, or spray drying to a low moisture level, and storing at a suitable temperature, e.g., room temperature. In an embodiment, the drying process comprises freeze-drying.

[0106] In an embodiment, the harvested biomass of Beauveria bassiana or spores thereof, or Metarhizium anisopliae or spores thereof, is subjected to freeze drying process. The term “lyophilization”, as used herein, refers to a process of freezing microbial cells for preservation of the microbial cells. Accordingly, the term “lyophilized”, as used herein, refers to frozen state of a substance. Lyophilization, also referred to as freeze-drying, is a drying method for microbial preservation in order to achieve higher viability and shelf-life. The freeze-drying process involves freezing at low temperature (below eutectic temperature) followed by primary drying (sublimation) and secondary drying under low vacuum. During primary drying, any unbound solution is removed through sublimation. Secondary drying involves removal of water that did not freeze. The underlying physical phenomenon of sublimation means a direct transition from the solid to the vapor state, bypassing the liquid aggregate state. The frozen product is thus dried under vacuum without thawing. In this process, microbes / product do not undergo thermal stress during unbound and bound water removal.

[0107] In an embodiment, said lyophilization comprises freezing and drying under a vacuum pressure to remove water, preferably to remove 85 to 90% of water, and to obtain the lyophilized cake or powder of the first component or the second component.

[0108] In another embodiment, the lyophilization in step (i) or (ii) comprises, (a) freezing the first component or the second component to obtain a first solid mass; (b) drying the first solid mass under a vacuum pressure in the range of 0.5 to 4 millibar (mbar), at a temperature in the range of -35 °C to +40°C for a duration inthe range of 10 to 20 hour or until 85% to 90% of water is removed to obtain a second solid mass, preferably until 90% of water is removed to obtain a second solid mass; and (c) drying the second solid mass under a vacuum pressure in the range of 0.01 to 0.5 millibar (mbar), at a temperature in the range of 4°C to 40°C for a period of time in the range of 10 to 25 hour or until 85% to 90% of water is removed, to obtain the composition.

[0109] In an embodiment, the first component or the second component are frozen at a temperature in the range of -45°C to -80°C to obtain a first solid mass. In another embodiment, the first component or the second component are frozen at a temperature in the range of -45°C to -60°C to obtain a first solid mass. In yet another embodiment, the first component or the second component are frozen at a temperature in the range of -45°C to -50°C to obtain a first solid mass.

[0110] In an embodiment, drying the first solid mass under a vacuum pressure in the range of 0.5 to 4 millibar (mbar), at a temperature in the range of -35°C to +40°C for a duration in the range of 10 to 20 hour or until 85% to 90% of water is removed to obtain a second solid mass. In another embodiment, drying the first solid mass under a vacuum pressure in the range of 0.5 to 4 millibar (mbar), at a temperature in the range of -35°C to +40°C for a duration in the range of 10 to 20 hour, until 90% of water is removed to obtain a second solid mass.

[0111] In an embodiment, drying the second solid mass under a vacuum pressure in the range of 0.01 to 0.5 millibar (mbar), at a temperature in the range of 4°C to 40°C for a period of time in the range of 10 to 25 hour or until 85% to 90% of water is removed, to obtain the lyophilized cake or powder of the first or second component.

[0112] In some embodiments, the composition prepared using the method disclosed herein is a solid. In an embodiment, the composition prepared using the method disclosed herein is a powder.

[0113] Various methods are known in the art to produce a powder form of the composition with desired particle size, disclosed in various embodiments herein, such as milling, sieving etc. In an embodiment the method further comprises millingand passing the composition through a sieve with a mesh size in a range of 100 micron to 1000 micron to obtain a dry lyophilized powder of the composition.

[0114] In an embodiment, there is provided a lyophilized product for reconstitution. In another embodiment, lyophilized product for reconstitution is a mixture comprising, a first component having Beauveria bassiana, or spores thereof, and a first stabilizing agent selected from trehalose, maltose, maltodextrin, glycerol monosodium glutamate or combination thereof; and a second component comprising Metarhizium anisopliae, or spores thereof, and a second stabilizing agent selected from pectin, trehalose, maltose, maltodextrin, glycerol, monosodium glutamate or combination thereof; wherein the Beauveria bassiana and Metarhizium anisopliae retains viability of about 108to 1010cfu / g for a period in the range of 1 to 3 months at a temperature in the range of -20 to +37℃.

[0115] In some embodiments, there is provided a method of preparing the consortium. In an embodiment, the method of preparing the consortium comprises, (i) culturing the strain of Beauveria bassiana or the strain of Metarhizium anisopliae by a method described herein above; and harvesting the culture from step (i) aseptically, to obtain the consortium; wherein the concentration of the strain of Beauveria bassiana; and the strain of Metarhizium anisopliae is in a range of 108to 1010cfu / g dry matter of the consortium.

[0116] In some embodiments, there is provided a method of preparing a biopesticide formulation comprising diluting the microbial composition in a carrier.

[0117] In an embodiment, there is provided a method of preparing a biopesticide formulation comprising diluting the microbial composition using a carrier, preferably to contain a minimum of 1 × 109cfu / g of Beauveria bassiana or Metarhizium anisopliae. Application and Use

[0118] Embodiments herein include a method for controlling pests, preferably plant infesting insects. In embodiment, there is provided a method for controlling the pests comprising, contacting the pests with the microbial composition as described herein or the biopesticide formulation as described herein.

[0119] In some embodiments, the microbial composition or the biopesticide formulation is capable of controlling the pests at the egg stage, the nymph stage, the instar stage, or the adult stage.

[0120] Various methods are known in the art for contacting the pests with the microbial composition or the biopesticide formulation disclosed in various embodiments herein. Examples of contacting methods include but are not limited to spraying, coating, or applying the composition or the formulation described herein to the one or more pests or surfaces which may come into contact with the one or more pests.

[0121] In an embodiment, there is provided a method for controlling plant infesting pests, comprising administering the microbial composition or the biopesticide formulation to plants or soil.

[0122] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art upon reference to the description of the subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present subject matter as defined. EXAMPLES

[0123] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.Materials

[0124] Specific isolated strains of Beauveria bassiana and Metarhizium anisopliae were used in the present disclosure. The Beauveria bassiana strain deposited under the accession number MTCC25687 (reference id: IPL / BB / 201) was used. The Metarhizium anisopliae strain deposited under the accession number MTCC25686 (reference id: IPL / KC / 88) was used.

[0125] The reagents used in the following examples were procured from Hi Media laboratories Limited, Sisco Research laboratories Pvt Ltd and Thermo Fisher Scientific Inc. EXAMPLE 1 Example 1: Preparation of microbial composition

[0126] The objective of this example is to describe the method of preparing the microbial composition, comprising: (i) a first component comprising a biomass of Beauveria bassiana, or spores thereof, and a first stabilizing agent; and (ii) a second component comprising a biomass of Metarhizium anisopliae, or spores thereof, and a second stabilizing agent. Preparation of the biomass of Beauveria bassiana and Metarhizium anisopliae

[0127] Individual cultures of Beauveria bassiana and Metarhizium anisopliae were grown in an aqueous medium (Table 1a and 1b, respectively) at the optimal temperature (20°C to 35°C) and pH (3.5 to 7.0) for a period of time to achieve above 90% sporulation. The fermented broths of Beauveria bassiana and Metarhizium anisopliae had a bacterial count ranging from 1 × 107to 1 × 109colony forming units (cfu / ml). The grown cultures were harvested using centrifugation at 8000 rpm for 20 min at 4°C, aseptically, to obtain the biomass of Beauveria bassiana and Metarhizium anisopliae. Table 1a: Shows the optimized nutrient media composition for the fermentation of selected strain of Beauveria bassiana.Table 1b: Shows the optimized nutrient media composition for the fermentation of selected strain of Metarhizium anisopliae.Example 2: Preparation of the first stabilizing agent composition

[0128] The stabilizing agent composition for Beauveria bassiana was prepared using the ingredients as depicted in the Table 2a. To prepare composition 9 depicted in Table 2a, accurately weighed amounts of trehalose (3.5 g), maltose (5.0 g), maltodextrin (10.0 g), monosodium glutamate (1.5 g), and glycerol (0.8 g) were dissolved in 100 ml of water. The solution obtained was then sterilized by autoclaving at 121°C and 15 lbs pressure for 15 minutes. After autoclaving, the solution was allowed to cool down to room temperature (25 to 30°C). Similarly, thestabilizing agent compositions 1 to 8, and 10 depicted in Table 2a were also prepared. Table 2a: List of various stabilizing agent compositions used for Beauveria bassiana, according to embodiments herein.Example 3: Preparation of the second stabilizing agent composition

[0129] The stabilizing agent compositions Metarhizium anisopliae was prepared using the ingredients as depicted in the Table 2b. To prepare composition 8 depicted in Table 2b, accurately weighed amounts of pectin (0.5 g), trehalose (4.0g), maltose (2.0g), monosodium glutamate (2.0g), maltodextrin (4.5g), and glycerol (0.2g) were dissolved in 100 ml of water. The solution was sterilized by autoclaving at 121°C and 15 lbs pressure for 15 minutes. After autoclaving, the solution was allowed to cool down to room temperature (25 to 30°C). Similarly, the stabilizing agent compositions 1 to 7, 9, and 10 depicted in Table 2b were also prepared. Table 2b: The stabilizing agent compositions used for Metarhizium anisopliae.Example 4: Preparation of the first component and second component.

[0130] 10 grams of Beauveria bassiana biomass was mixed with 5 ml of first stabilizing agent composition (refer Example 2) to obtain a mixture having Beauveria bassiana biomass and first stabilizing agent in a ratio of 1:0.5, also referred to herein as the first component.

[0131] 10 grams of Metarhizium anisopliae biomass was mixed with 5ml of second stabilizing agent composition (refer Example 3) to obtain a mixture having Metarhizium anisopliae biomass and second stabilizing agent in a ratio of 1:0.5, also referred to herein as the second component. Example 5: Preparation of lyophilized Beauveria bassiana (lyophilized first component)

[0132] 10 grams of Beauveria bassiana biomass was mixed with 5 ml of first stabilizing agent composition (refer Example 2) to obtain a mixture having Beauveria bassiana biomass and first stabilizing agent in a ratio of 1:0.5. The mixture was then loaded onto trays and frozen at -45°C. These trays containing frozen mass were loaded onto the freeze-drying instrument. Primary drying of solid frozen mass on the tray was carried out under a vacuum pressure ranging from 0.6 millibar (mbar), at -35°C for the duration until 90% of water is evaporated / removed. Secondary drying was carried out under a vacuum pressure of 0.01 millibar (mbar), at a temperature of +30°C for a period of time sufficient to reduce the moisture content to less than 2% thereby obtaining a dry composition of the invention. Example 6: Preparation of lyophilized Metarhizium anisopliae (lyophilized second component)

[0133] 10 g of Metarhizium anisopliae biomass was mixed with 5 ml of second stabilizing agent composition (refer Example 3) to obtain a mixture having Metarhizium anisopliae biomass and first stabilizing agent in a ratio of 1:0.5. Themixture was then loaded onto trays and frozen at -45°C. These trays containing frozen mass were loaded onto the freeze-drying instrument. Primary drying of solid frozen mass on the tray was carried out under a vacuum pressure ranging from 0.6 millibar (mbar), at -35°C for the duration until 90% of water is evaporated / removed. Secondary drying was carried out under a vacuum pressure of 0.01 millibar (mbar), at a temperature of +30°C for a period of time sufficient to reduce the moisture content to less than 2% thereby obtaining a dry composition of the invention. Example 7: Preparation of the microbial composition

[0134] The lyophilized Beauveria bassiana and Metarhizium anisopliae (Examples 5 and 6) was milled and sieved individually to obtain fine free flowing powder. The lyophilized Beauveria bassiana and lyophilized Metarhizium anisopliae were mixed at 1:1 ratio to obtain the microbial composition. Example 8: Preparation of the biopesticide formulation

[0135] The microbial composition (Example 7) was then standardized or diluted with a carrier, such as dextrose to contain a minimum viability of Beauveria bassiana and Metarhizium anisopliae at 1 × 109cfu / g of the formulation. Example 9: Determination of storage stability of the microbial composition

[0136] The stability studies of the composition of the present disclosure were conducted at three different conditions i.e., accelerated (37°C), real time (25°C), and refrigerated conditions (4°C). The microbial compositions prepared in Example 7 was diluted with dextrose (carrier) in order to obtain the fungal strains viability of ≥ 1 × 109cfu / g in the formulation. The standardized soluble powder samples of lyophilized formulation were packed and sealed in aluminium bags. These samples were tested for the colony-forming units (CFU) per gram, using the growth assay. Viable cell counts (cfu) were determined in freeze-dried powder immediately after freeze-drying and also at selected time points during the stability studies. A standard spread plating method was used to determine cfu. The samples were suspended in sterile saline and mixed well by vortexing. After 30 minutes of revitalization, thecell suspension was serially diluted in sterile saline. The dilutions were plated in duplicates for the determination of the cfu of Beauveria bassiana and Metarhizium anisopliae. The agar plates were incubated aerobically for three days at 37°C. Plates with 3 - 30 colonies were chosen for counting of colony forming units. The result was reported as average cfu / g of Beauveria bassiana and Metarhizium anisopliae, which was calculated from the duplicates. Observation and Results:

[0137] From Figure 1, it is evident that at accelerated conditions (37°C) the microbial composition comprising the stabilizing agent exhibited a viability of 1 × 108cfu / g to 1 × 109cfu / g for up to 4 months when compared to the microbial composition prepared without any stabilizing agent.

[0138] From the Figure 2, it is evident that at ambient conditions (25°C) the microbial composition comprising the stabilizing agent exhibited a viability of 1 × 108cfu / g to 1 × 109cfu / g for up to 5 months when compared to the microbial composition prepared without any stabilizing agent.

[0139] From the Figure 3, it is evident that at refrigerated conditions (4°C) the microbial composition comprising the stabilizing agent exhibited a viability of 1 × 108cfu / g to 1 × 109cfu / g for up to 5 months when compared to the microbial composition prepared without any stabilizing agent. Example 10: Shelf-life stability study

[0140] The lyophilized product comprising Beauveria bassiana and Metarhizium anisopliae were tested to understand shelf life. The lyophilized product was shelved for a period of 0 to 36 months and stability of the tested at the end of 3, 6, 9,12, 15, 16, 18, 21, 24, 27, 30, 33, and 36 months was determined using known methods. It was observed that the lyophilized product is stable even after 36 months of storage, exhibiting about 2x10⁹ cfu / g for upto about 24 months and about 1x10⁹ cfu / g for upto 36 months. Hence, the product is stable for an extended period. Table 3 depicts the shelf-life data for the lyophilized microbial consortium of Beauveria bassiana and Metarhizium anisopliae, according to embodiment herein.Table 3: Shelf-life data

[0141] Overall, the microbial composition of the present disclosure comprising the selected strains of Beauveria bassiana and Metarhizium anisopliae along with the stabilizing agent resulted in a stable and viable biopesticide formulation with extended shelf life, at varied temperature. Advantages of the present disclosure

[0142] The present disclosure provides a microbial composition for pest control with the following advantages: a. The microbial composition is a water-soluble lyophilized powder comprising the spores of Beauveria bassiana and Metarhizium anisopliae. b. The stabilizing agent used as a part of the microbial composition protects the fungal spores from injuries caused due to freezing and therefore maintains their structural integrity. Also, the stabilizing agent protects the spores from mechanical injuries during down steam processing, such as milling. Therefore, the use of specific stabilizing agents increases thecellular integrity of the fungal spores, reduces process loss, and even protects the spores against oxidation during storage. c. The microbial composition has reduced moisture content, which ensures low or almost nil reactivation of the spores during storage. d. The lyophilized spores of Beauveria bassiana and Metarhizium anisopliae are stable at a temperature in the range of -20 to +37℃ and exhibits viability in the range of 108to 1010cfu / gm of the composition. e. Th composition is convenient for storage and transportation, owing to the extended shelf life. The microbial composition of the present invention exhibits an extended shelf life with about 2x109cfu / g for a period of up to 24 months.

Claims

I / We claim:

1. A microbial composition, comprising: (i) a first component comprising Beauveria bassiana, or spores thereof, and a first stabilizing agent selected from trehalose, maltose, maltodextrin, monosodium glutamate, glycerol, or combination thereof; and (ii) a second component comprising Metarhizium anisopliae, or spores thereof, and a second stabilizing agent selected from pectin, trehalose, maltose, maltodextrin, monosodium glutamate, glycerol, or combination thereof.

2. The composition as claimed in claim 1, wherein the first component comprises Beauveria bassiana and the first stabilizing agent in a weight ratio range of 1:0.1 to 1:10, preferably in a weight ratio of 1:0.5; and wherein the second component comprises Metarhizium anisopliae and the second stabilizing agent in a weight ratio in the range of 1:0.1 to 1:10, preferably in a weight ratio of 1:0.

5.

3. The composition as claimed in claim 1, wherein Beauveria bassiana is of a strain deposited under the accession number MTCC25687.

4. The composition as claimed in claim 1, wherein Metarhizium anisopliae is of a strain deposited under the accession number MTCC25686.

5. The microbial composition as claimed in claim 1, wherein the first component and the second component are in a weight ratio in the range of 1:9 to 9:1, preferably in a weight ratio of 1:

1.

6. The composition as claimed in claim 1, wherein the first stabilizing agent in said first component comprises trehalose in an amount in the range of 0.1% to 20%, maltose in an amount in the range of 0 to 10%, maltodextrin in an amount in the range of 0.1% to 20%, monosodium glutamate in an amount in the range of 0 to 10%, and glycerol in an amount in the range of 0 to 10%, with respect to the total weight of the first component.

7. The composition as claimed in claim 1, wherein the second stabilizing agent in said second component comprises pectin in an amount in the range of 0to 10%, trehalose in an amount in the range of 0 to 10%, maltose in an amount in the range of 0 to 10%, maltodextrin in an amount in the range of 0 to 15%, monosodium glutamate in an amount in the range of 0.1% to 10%, and glycerol in an amount in the range of 0 to 10%, with respect to the total weight of the second component.

8. The composition as claimed in claim 1, wherein said first component and second component are in lyophilized form.

9. A lyophilized product, comprising the composition as claimed in claim 1.

10. The lyophilized product as claimed in claim 9, wherein the first component and the second component are in a weight ratio in the range of 1:9 to 9:1, preferably in a weight ratio of 1:

1.

11. A method of preparing the composition as claimed in claim 1, the method comprising: (i) mixing Beauveria bassiana biomass, or spores thereof, and a solution comprising the first stabilizing agent selected from trehalose, maltose, maltodextrin, glycerol, monosodium glutamate, or combination thereof, to obtain a first component; (ii) mixing Metarhizium anisopliae biomass, or spores thereof, and a solution comprising the second stabilizing agent selected from pectin, trehalose, maltose, maltodextrin, monosodium glutamate, glycerol, or combination thereof, to obtain a second component; and (iii) mixing said first component and said second component to obtain the composition.

12. The method as claimed in claim 11, wherein said step(i) further comprises lyophilizing said first component and said step(ii) further comprises lyophilizing said second component.

13. The method as claimed in claim 12, wherein said lyophilizing is performed by freezing at a temperature in the range of -45°C to -60°C to obtain a first solid mass, and drying under vacuum pressure to remove water, preferably to remove 85 to 90% of water to obtain a lyophilized cake or powder.

14. A biopesticide formulation, comprising:(a) the composition as claimed in claim 1 or the lyophilized product as claimed in claim 9; and (b) a carrier.

15. The formulation as claimed in claim 14, wherein the carrier is selected from lactose, sucrose, dextrose, mannitol, sorbitol, soluble starch, or derivatives thereof.

16. A method of preparing the formulation as claimed in claim 14, the method comprising: diluting the composition as claimed in claim 1 or the lyophilized product as claimed in claim 9, with a carrier to obtain the formulation.

17. A kit comprising the formulation as claimed in claim 14, the lyophilized product as claimed in claim 9, or the composition as claimed in claim 1; and optionally an instruction manual.

18. A method of controlling pests, the method comprising: contacting the pests with the composition as claimed in claim 1 or the lyophilized product as claimed in claim 9 or the formulation as claimed in claim 14.

19. Use of the composition as claimed in claim 1 or the or the lyophilized product as claimed in claim 9 or the formulation as claimed in claim 14, for controlling pests.

20. A microbial consortium comprising, a strain of Beauveria bassiana; and a strain of Metarhizium anisopliae.

21. The microbial consortium as claimed in claim 20, wherein the Beauveria bassiana is of a strain deposited under the accession number MTCC25687; and the Metarhizium anisopliae is of a strain deposited under the accession number MTCC25686, wherein concentration of the Beauveria bassiana is in a range of 108to 1010cfu / g dry matter of the consortium, and concentration of the Metarhizium anisopliae is in a range of 108to 1010cfu / g dry matter of the consortium.

22. A strain of Beauveria bassiana deposited under the accession number MTCC25687.

23. A strain of Metarhizium anisopliae deposited under the accession number MTCC25686.

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