Description:FORM-2
THE PATENT ACT, 1970
(39 OF 1970)
AND
THE PATENT RULES, 2003
(As Amended)

COMPLETE SPECIFICATION
(See section 10; rule 13)

“Biocontrol agents, compositions comprising such biocontrol agents and method for preparing these compositions”.

ECSO Global Pvt. Ltd., an Indian Cooperation, of Plot no. 68 5th, Absolute, Anant Raj Builders, Sector 44, Gurugram, Haryana 122002

The following specification particularly describes the invention and the manner in which it is to be performed:
Field of invention

The present invention relates to bio-control agents, composition comprising said bio-control agents, and a method of preparation of the composition. Furthermore, the invention also provides the bio-control agents (BCA) in an encapsulated form. The bio-control agents of the invention, compositions comprising said bio-control agents are specifically effective against wilt causing pathogens.

Background

As the global population grows, there is a pressing need to increase both the quality and quantity of agricultural products. This demand has traditionally been met by the extensive use of chemical pesticides to combat crop diseases. However, concerns over the adverse effects of these chemicals on soil and human health and the environment have driven research towards eco-friendly pest management solutions such as biological control agents and biopesticides. Bio-Control Agents (BCAs) have emerged as a promising alternative, offering an environmentally friendly way to protect crops. BCAs are beneficial microorganisms that protect plants against various biotic and abiotic stresses, such as fungi, bacteria, nematodes, insects, viruses, and viroids. These agents are integral to sustainable agriculture, as they inhibit the infection of host plants by pathogens, thus enhancing plant resistance and growth. These agents are crucial for sustainable agriculture, providing targeted pest control with minimal environmental impact. Biopesticides, derived from natural sources such as bacteria, fungi, and plant extracts, offer targeted pest control with minimal environmental impact.

However, the efficacy and stability of BCAs and biopesticides face significant challenges in practical application. They are sensitive to environmental factors like pH, extreme temperatures, moisture variations, and ultraviolet radiation, leading to rapid degradation and reduced effectiveness in field conditions. This sensitivity, along with issues like microbial competition and desiccation, limits their practical utility and requires frequent applications, increasing costs and labor.

To address these challenges, various technologies are being developed to address these challenges. Compositions and methods are being developed that can help protect them, increase efficacy, provide controlled release of the active agents at the optimal time and location, maximizing their effectiveness against pests while minimizing the frequency of application.

Despite advancements in technologies, there remains a need for improved bio-control agents (BCAs), and methods that can further enhance the performance of biopesticides and BCAs, extend their shelf life and ensure compatibility with various agriculture practices.

Therefore, the present invention addresses the above needs by providing bio-control agents that can be used alone or in combination and can be formulated in microcapsules. The invention also provides a stable product, enhancing the BCA’s ability to function under various environmental conditions and improving its overall efficacy in pest control and plant growth promotion.

Summary of the invention

It is an object of the present invention to provide bio-control agents (BCAs) that are effective, and stable.

It is also an object of the present invention to provide a process that can help preserve the BCAs' metabolic activity during storage and after application while shielding them from biotic and abiotic challenges.

It is an object of the present invention to provide a process/ composition that can provide a controlled release of the BCA’s.

It is yet another object of the present invention to prepare a composition of bio control agents BCAs to combat diseases in plants.

The object of the present invention is achieved by bio-control agents and the composition of the invention. The present invention also relates to composition comprising at least one biological control agent. In preferred embodiments, said composition comprises a consortium of biological control agents.

In preferred embodiments of the invention, the biological control agents (BCAs) are microencapsulated in one, two or more layers of a microencapsulating agent, and the microencapsulating agent is selected from the group comprising of gelatin, cellulose, starch, maltodextrin, gum arabic, xanthan gum, sodium alginate, methylcellulose.

Microencapsulation process of the invention involves entrapping microbial cells and/or spores of one or more of BCA’s within a polymeric material, creating beads permeable to nutrients, gases, and metabolites to maintain cell viability.

The encapsulation of biological control agents with multiple layers offers significant advantages, extending their effective field life and reducing application frequency and dosage, thereby minimizing environmental contamination. Encapsulated BCAs can be directly applied to plants or soil, improving plant tolerance and reducing biotic stresses. Said one or more encapsulated BCAs may also be formulated into a composition.

The present invention is also directed to a method of forming a composition for use as a bio-pesticide, the method comprising:-
a. culturing one or more bio-control agent by solid substrate fermentation and/or submerged fermentation;
b. concentrating the cells/spores of bio-control agent at least 10-15 times via continuous centrifugation and/or micro-filtration to obtain concentrated cells/spores of bio-control agent;
c. optionally mixing concentrated cells/spores of bio-control agent with a slurry comprising microencapsulation agents selected from one or more of gelatin, cellulose, starch, maltodextrin, gum arabic, xanthan gum, sodium alginate, methylcellulose and interlinking agents selected from one or more of CaCl2, NaCl and optionally buffers such as phosphate buffer to form microcapsules;
d. spray drying the microcapsules obtained in step c) or concentrated cells/spores of bio-control agent obtained in step b) to obtain spray dried microcapsules or spray dried or concentrated cells/spores of bio-control agent or;
e. optionally repeating steps c) and d) to form further encapsulation layers;
f. mixing 3-20%, preferably 5-10% of spray dried micro-capsules obtained in step (d) or mixing 3-20%, preferably 5-10% of spray dried concentrated cells/spores of bio-control agent obtained in step d) with 55-65% base carrier, 15-25% cell protectant, 1-5 % a dispersion agent, 2-4% stabilizers, 1-3% of anti-fungicidal agent such as chitinase, protease, lipase and 1-3% of UV protectant molecule.
The present invention is also directed to a method of reducing overall damage caused to plants and plant parts by pathogens, comprising the step of applying the composition as of the invention to the plants, plant parts, seeds, fruits, vegetables, and soil. In the preferred embodiment, the plants plant parts, seeds, fruits, vegetables are either infected by pathogens or are susceptible to infection by pathogens.

Utilizing a microencapsulation process for forming a stable and effective biopesticide that significantly alleviates the abiotic stress on plants against various pathogens. Each strain used in a consortium is selected so that it contributes distinct properties that, when combined, synergistically, in a highly efficient manner offers a comprehensive and potent defence mechanism against these pathogens.

The composition is meticulously designed to ensure maximum stability and efficacy. In the present invention, the most preferred percentage of spray dried micro-capsules of BCA and concentrated each cells/spores of bio-control agent in the composition is 8% and 6% respectively. The composition further comprises the base carrier/filler, primarily dextrose monohydrate which constitutes 55-65% of the composition. This carrier supports the microencapsulated BCA’ and maintains compatibility with other components. The inclusion of a cell protectant, comprising 15-25% of the composition, is critical for preserving the BCAs. Additionally, the composition incorporates a dispersion agent (1-5%) to ensure an even distribution of microcapsules, preventing clumping and facilitating uniform encapsulation. Moreover, the composition has 1-3% of fungicidal agent and 1-3% of UV protectant molecule. Stabilizers, which make up 2-4% of the composition, enhance the shelf-life and stability of the encapsulated BCAs, maintaining the integrity of the microcapsules during storage and application.

The present invention represents a significant advancement in the field of sustainable agriculture. It offers a highly effective and environmentally friendly alternative to chemical pesticides, providing superior protection against fungal diseases while enhancing the stability and shelf-life of the biopesticide. The specific strains result in efficient inhibition of wilt, making this composition an important tool for protecting economically important crops. The combined action of the specific strains in the consortium results in the highest inhibition of wilt, making this composition an invaluable tool for protecting economically important crops. This innovative method further ensures the stability, efficacy, and prolonged shelf-life of the biopesticide, contributing to more sustainable and environmentally responsible agricultural practices.

Brief Description of the Drawings

The present invention is described by way of embodiments illustrated in the accompanying drawings wherein:

Figure 1: Illustrates a flowchart depicting the process flow of microencapsulated microbial composition by using spray dryer techniques with microencapsulating biopolymers according to an embodiment of the present invention.

Figure 2: Illustrates a flowchart depicting the process flow of microencapsulated microbial composition by using spray dryer techniques with microencapsulating biopolymers according to another embodiment of the present invention.

Figure 3: Illustrates a flowchart depicting the process flow of microencapsulated microbial composition by using spray dryer techniques with microencapsulating biopolymers according to another embodiment of the present invention.

Detailed description of the Invention

The present invention relates to a composition comprising at least one biological control agent.

In an embodiment, the present invention also provides a process of preparation of multi-layered microencapsulation on said BCAs through spray drying technique and its application for making a biopesticide composition.

The production of a dry microencapsulated biopesticide involves a multi-step process designed to ensure the stability and efficacy of biocontrol agents. This process not only isolates and cultivates potent microbes but also encapsulates them in protective layers, ultimately producing a durable powder form suitable for agricultural use.

In an embodiment, the preparation method for single, double or multi-layered microencapsulation of agriculturally important microbial strains like Bacillus amyloliquefaciens XenAg-B001 (Accession No. MTCC 25764), Bacillus pumilus XenAg-B009 (Accession No. MTCC 25768), Bacillus polyxma XenAg-B010 (Accession No. MTCC 25769), Trichoderma viride XenAg-F008 (Accession No. MTCC 25773) etc and/ or consortium of strains involves advanced techniques to ensure the microbes' effective protection and delivery in agricultural settings.

The consortium/combination, is designed to work synergistically to combat wilt causing organisms. Each strain contributes unique properties that enhance the overall efficacy of the biopesticide. The combination of these strains ensures comprehensive protection against wilt development, with each strain complementing the others to maximize effectiveness.

In an embodiment of the present invention, the bio-control agent of the invention is Bacillus amyloliquefaciens XenAg-B001 (Accession No. MTCC 25764), wherein the Bacillus amyloliquefaciens XenAg-B001 (Accession No. MTCC 25764) is grown in a media by submerged fermentation. The media comprising 10-15 g/L D-Mannitol or dextrose, 2-7 g/L yeast extract, 3-5 g/L peptone, 5-7 g/L Sodium Chloride and 3-6 g/L Calcium Chloride (CaCl2), and optionally 1-2 g/L soyabean meal, and having a pH 6.5.

In an embodiment of the present invention, the bio-control agent of the invention is Bacillus pumilus XenAg-B009 (Accession No. MTCC 25768), wherein the Bacillus pumilus XenAg-B009 (Accession No. MTCC 25768) is grown in a media by submerged fermentation. The media comprising 10-15 g/L D-Mannitol or dextrose, 2-7 g/L yeast extract, 3-5 g/L peptone, 5-7 g/L Sodium Chloride and 3-6 g/L Calcium Chloride (CaCl2) and optionally 1-2 g/L soyabean meal and having a pH 6.5.

In an embodiment of the present invention, the bio-control agent of the invention is Bacillus polyxma XenAg-B010 (Accession No. MTCC 25769), wherein the Bacillus polyxma XenAg-B010 (Accession No. MTCC 25769), is grown in a media by submerged fermentation. The media comprising dextrose 10-15 g/L, yeast extract 2-7 g/L, ammonium sulphate 1-3 g/L, K2HPO4 1-3 g/L, KH2PO4 1-3 g/L, and optionally comprising around 0.5 g/L MgSO4.7H2O, around 0.06 g/L CaCl2.2H2O, around 0.05 g-1L MnSO4.H2O, 0.2 g/L antifoam agent, and having a pH 6.5-7.

In an embodiment of the present invention, the bio-control agent of the invention is comprising Trichoderma viride XenAg-F008 (Accession No. MTCC 25773), wherein the Trichoderma viride XenAg-F008 (Accession No. MTCC 25773) is grown in a media by solid state fermentation. The media comprising dextrose 5-7 g/L, yeast extract 1.5-2 g/L, CaCl2 0.05-0.08 g/L, KH2PO4 1.2-1.5 g/L, MgSO4 2-4 g/L, KNO3 4.6-5.6 g/L, and having a pH 6.8.

In an embodiment of the present invention, the bio-control agent of the invention are microencapsulated in one, two or more layers of a microencapsulating agent, and wherein the microencapsulating agent is selected from the group comprising of gelatin, cellulose, starch, maltodextrin, gum arabic, xanthan gum, sodium alginate, methylcellulose.

The composition of the invention comprises concentrated cells/spores of bio-control agent in the range of 5-20%, preferably 5-10% by weight. In an embodiment, the most preferred percentage of concentrated cells/spores of bio-control agent in the composition is 6%.

The composition of the invention comprises microencapsulated bio-control agent in the range of 5-20%, preferably 5-10% by weight, more preferably 8%.

The composition further comprises a base carrier, a cell protectant, a dispersion agent, optionally a fungicide active molecule, a stabilizer or adjuvant or sticking agent and a UV protectant molecule. The base carrier is preferably dextrose monohydrate and present in an amount of 55-65% of the composition. The cell protectant is present in an amount of 15-25% of the composition. The dispersion agent is silicon dioxide or calcium silicate present in an amount of 1-5% of the composition. The composition can include any fungicidal active molecule. Preferably, such fungicidal active is selected from the group consisting of chitinase, protease, lipase and are present in an amount of 1-3% of the composition. The stabilizer or adjuvant or sticking agent is selected from CMC (carboxymethyl cellulose), guar gum, casein, alginic acid 1-2% of the composition. The UV protectant molecule is lignosulfonates and is present in an amount of 1-2% of the composition.

The biological control agents (BCAs) are microencapsulated in one, two or more layers of a microencapsulating agents using linking agents, like Calcium Chloride and thereafter spray dried.

The composition of the invention is preferably used as a biopesticide for inhibition of wilt causing pathogens.

The present invention is also related to a method of forming a composition for use as a bio-pesticide, the method comprising:-
a. culturing one or more bio-control agent by solid substrate fermentation and/or submerged fermentation;
b. concentrating the cells/spores of bio-control agent at least 10-15 times via continuous centrifugation and/or micro-filtration to obtain concentrated cells/spores of bio-control agent;
c. optionally mixing concentrated cells/spores of bio-control agent with a slurry comprising microencapsulation agents selected from one or more of gelatin, cellulose, starch, maltodextrin, gum arabic, xanthan gum, sodium alginate, methylcellulose and interlinking agents selected from one or more of CaCl2, NaCl and optionally buffers such as phosphate buffer to form microcapsules;
d. spray drying the microcapsules obtained in step c) or concentrated cells/spores of bio-control agent obtained in step b) to obtain spray dried microcapsules or spray dried or concentrated cells/spores of bio-control agent or;
e. optionally repeating steps c) and d) to form further encapsulation layers;
f. mixing 5-20%, preferably 5-10% of spray dried micro-capsules obtained in step (d) or mixing 3-20%, preferably 5-10% of spray dried concentrated cells/spores of bio-control agent obtained in step d) with 55-65% base carrier, 15-25% cell protectant, 1-5 % a dispersion agent, 2-4% stabilizers, 1-3% of anti-fungicidal agent such as chitinase, protease, lipase and 1-3% of UV protectant molecule.

In preferred embodiments, of the method, the bio-control agent is a consortium of XenAg-B001, XenAg-B009, XenAg-B010 and XenAg-F008 in step f), wherein 1-3% of the microcapsules are of XenAg-B001, 1-3% of the microcapsules are of XenAg-B009, 1-3% of the microcapsules are of XenAg-B010 and 2-4% of the microcapsules are of XenAg-F008 are included in the consortium.

In a preferred embodiment of the method, in step c), the slurry comprises starch in an amount in the range of from 0.33%-0.50%, gelatin in an amount in the range of from 0.1%-0.5%, methylcellulose in an amount in the range of from 1.0%-1.5%, maltodextrin in an amount in the range of from 1.0%-1.5%, xanthan gum in an amount in the range of from 1.0%-1.5%, and/or sodium alginate in an amount in the range of from 2.0%-4.0%.

In a preferred embodiment of the method, in step c), the slurry comprises interlinking agents selected from one or more of CaCl2, NaCl in an amount ranging from 0.5% to 1.0%, and phosphate buffer in an amount ranging from 0.8%-1.5%.

In a preferred embodiment of the method, in step c) the slurry is mixed with the cell concentrated cells/spores of bio-control agent in the ratio of 1:1 to 1:5, preferably 1:3.

The present invention is also directed to a method for reducing overall damage caused to plants and plant parts by wilt causing pathogens, comprising the step of applying the composition of the invention to the plants, plant parts, seeds, fruits, vegetables, and soil. In the preferred embodiment, the plants the plants, plant parts, seeds, fruits, vegetables are either infected by fungal wilt or are susceptible to infection by wilt causing pathogens.

1. SELECTED STRAINS – BIO- CONTROL AGENTS OF THE INVENTION
For example purposes the present invention is demonstrate with the help of the following strains or a consortium of one or more of said strains: -

Table 1: The accession code, geographical culture isolation source of the selected strains.
Code MTCC No. Culture Source Address Strain Name
XenAg-B001 MTCC 25764 Isolates from Sugarcane field soil Village-Bakhari, PO. Bhatpar Block: Bankata, Distt. Deoria, UP-274702 Bacillus amyloliquefaciens
XenAg-B009 MTCC 25768 Isolates from soil, Saurashtra Village-Wanadgaon, District-Jalna, Maharashtra-431204 Bacillus pumilus
XenAg-B010 MTCC 25769 From dead caterpillar from castor crop field Village- Aghavn; Tahsil- Khargone; District-Khargone, Madhya Pradesh-477582 Bacillus polyxma

XenAg-F008 MTCC 25773 From ground nut crop field: Highly Saline soil Village- Chandroda; Tahsil- Anjar; Dist.-Anjar, Kutch Gujarat-370110 Trichoderma viride

2. ANALYSIS OF COMPATIBILITY OF THE STRAINS
The selected strains were subjected to compatibility check to evaluate the compatibility with each other. The selected BCAs were tested for compatibility by cross streak method. Two different bacterial isolates were streaked vertically and horizontally on Potato dextrose agar into petri dish. Petri dishes were incubated for 5 days and observed for lysis at the juncture of the streaks. The compatibility of the individual strain vis-à-vis each another is provided below in Table 2. It indicates that the strains would work in a synergistic manner in a consortium.

Table 2: Compatibility test of the selected strains
Culture XenAg-B001 XenAg-B009 XenAg-B010 XenAg-F008
XenAg-B001 ++++ ++++ ++++
XenAg-B009 ++++ ++++ ++++
XenAg-B010 ++++ ++++ ++++
XenAg-F008 ++++ ++++ ++++

“-“ indicates no compatibility
“+” indicates some compatibility
“++“ indicates average compatibility
“+++” indicates good compatibility
“++++” indicates excellent compatibility

3. ANALYSIS OF CULTURE INTERACTION AND EFFECTIVENESS

The strong fungicidal activity is attributed to presence of potent fungicidal enzymes. The pesticidal traits of selected strains were assessed by evaluation of fungicidal enzymes like lipase, chitinase, phospholipase, cellulose, amylase, protease, pectinase. The selected strains exhibiting exceptional and significant pesticidal traits and also resulted in higher quantity of fungicidal enzymes, for example proteases, or lipases, with the capacity to lyse pesticide cells and were compatible, synergistic with one another.

Table 3: Pesticidal traits assessment of selected strains
Microorganisms Pesticidal traits
Lipase Chitinase Phospholipase Cellulase Amylase Protease Pectinase
XenAg-B001 ++ +++ +++ - +++ +++ ++
XenAg-B009 ++ ++ +++ - +++ +++ ++
XenAg-B010 +++ +++ +++ - +++ +++ ++
XenAg-F008 +++ +++ +++ + +++ + ++

+ = Low; ++ = Medium; +++ = High; - = No Activity

The interaction of selected strains or BCAs was tested alone and in combination for their effectiveness against wilt development. The table below summarizes the percentage mortality of wilt causing organism by different BCAs strains and their combinations:

Table 4: Antagonistic efficacy of selected strains or BCAs, alone and in combinations under dual culture technique against wilt causing organism

Treatments Percentage Inhibition (%)
Fusarium oxysporum Macrophomina phaseolina Rhizoctonia solani
XenAg-B001 42.00±0.00 57.33±0.38 38.33±0.58
XenAg-B009 34.33±0.48 56.33±0.51 54.33±0.58
XenAg-B010 48.66±0.38 44.66±0.29 45.00±0.00
XenAg-F008 39.00±0.27 57.33±0.51 78.33±0.54
XenAg-B001+ XenAg-B009 50.33±0.58 56.33±0.28 54.33±0.38
XenAg-B009+XenAg-B010 60.00±0.00 57.33±0.32 78.33±0.53
XenAg-B010+XenAg-F008 56.66±0.58 74.66±0.42 65.00±0.27
XenAg-B001 +XenAg-F008 61.00±0.23 57.33±0.34 78.33±0. 58
XenAg-B009+ XenAg-F008 70.33±0.58 56.33±0.56 54.33±0.28
XenAg-B001+XenAg-B009+XenAg-B010 88.66±0.58 78.66±0.36 81.00±0.23
XenAg-B009+XenAg-B010+XenAg-F008 73.33±0.58 86.33±0.52 84.33±0.38

The data obtained clearly shows that XenAg-B001, XenAg-B009, XenAg-B010 and XenAg-F008 when tested alone were effective, with highest inhibition achieved by XenAg-F008 against Rhizoctonia solani when tested. On the other hand, the combination or consortium of XenAg-B001, XenAg-B009, XenAg-B010 exhibited highest inhibition against the infection caused by Fusarium oxysporum at 88.66%. The combination or consortium of XenAg-B009, XenAg-B010 and XenAg-F008 achieved the highest percentage inhibition against infections caused by Macrophomina phaseolina and Rhizoctonia solani at 86.33% and 84.33% respectively. This superior performance demonstrates the synergistic effect of the combined strains, which significantly outperforms any single strain. The high percentage inhibition indicates the consortium's potent biocontrol capabilities, making it a highly effective solution for wilt management.

4. PROCESSING OF BCA’s

In an embodiment, the preparation method for single layered, double layered or multi layered microencapsulation of agriculturally important microbial strains and or consortium of multiple strains involves advanced techniques to ensure the microbes' effective protection and delivery in agricultural settings. This method employs a variety of biopolymers in specific combinations, including starch (0.33-0.50%), gelatin (0.1-0.5%), methylcellulose (1.0-1.5%), maltodextrin (1.0-1.5%), xanthan gum (1.0-1.5%), and sodium alginate (2.0-4.0%). Additionally, the method integrates binding agents such as CaCl2 and/or NaCl at concentrations ranging from 0.5% to 1.0%, along with phosphate buffer (0.8-1.5%), to enhance the structural integrity and efficacy of the encapsulated microbes. These carefully selected biopolymers and binding agents provide an optimal delivery system, ensuring the encapsulated microbes remain viable and effective in agricultural applications.

Referring to Figures 1, 2 and 3, flowcharts depicting a process flow of microencapsulated microbial composition by using spray dryer techniques with microencapsulating biopolymers is illustrated according to one or more embodiments of the present invention. The process to obtain a dry microencapsulated biopesticide includes a step of maintaining microbes according to steps 200, 210 of the Figure 3.

Potent microbes are assured by passing a strain through bioassay by exposing PGPR (plant growth promoting), antifungal and pesticidal traits of selected strains. For microbial culture maintenance, one or more strains are maintained on 25% glycerol stock and stored at 2-80C in refrigerator. Culture is also stored in deep freezer at around -800C for long time storage. The purity of culture is always monitored using the microscopic analysis of strains and their colony appearance on respective medium. The contaminated plates/slants are discarded by proper autoclaving. Before starting every batch, the culture should be checked for its purity.

The abovementioned step is followed by the step of inoculation and incubation of seed according to step 220, 230 of Figure 3. In an embodiment, for seed inoculum development, the pure culture of the strains is inoculated in sterile respective modified production media composition with set pH. Inoculated media incubated at 320C ±20C. After incubation and before proceeding for inoculation, the purity of culture is again confirmed using microscopic observation and respective pH and OD (optical density). Only pure culture inoculum is used for further inoculation and contaminated inoculum is discarded after proper autoclaving.

The preferred media composition for each individual strain of the invention is as follows:

1. XenAg-B001
Table 5: Media Composition of XenAg-B001
Ingredient g L-1
D-Mannitol 10-15
Yeast Extract 2-7
Peptone 3-5
Sodium chloride (NaCl) 5-7
Calcium chloride (CaCl2) 3-6
pH 6.5
In even more preferred embodiment, the media composition of XenAg-B001 includes D-Mannitol 10 gL-1 , yeast extract 5 gL-1, peptone 3 gL-1, sodium chloride (NaCl) 5 gL-1, calcium chloride (CaCl2) 3 gL-1 and pH 6.5±0.2. Each ingredient is selected within the ranges specified in Table 5 to achieve the desired performance, such as enhanced growth and stability.

2. XenAg-B009
Table 6: Media Composition of XenAg-B009
Ingredient g L-1
Dextrose 10-15
Yeast Extract 2-7
Peptone 3-5
Sodium chloride (NaCl) 5-7
Calcium chloride (CaCl2) 3-6
Soyabean meal 1-2
pH 6.5

In even more preferred embodiment, the media composition of XenAg-B009 includes dextrose 10g L-1, yeast extract 5 gL-1, peptone 3 gL-1, sodium chloride (NaCl) 5 gL-1, calcium chloride (CaCl2) 3 gL-1, soyabean meal 1gL-1 and pH 6.5±0.2. Each ingredient is selected within the ranges specified in Table 6 to achieve the desired performance, such as enhanced growth and stability.

3. XenAg-B010
Table 7: Media Composition of XenAg-B010
Ingredient g L-1
Dextrose 10-15
Yeast extract 2-7
Ammonium Sulphate 1.5
K2HPO4 1.5
KH2PO4 1.5
MgSO4.7H2O 0.5
CaCl2.2H2O 0.06
MnSO4.H2O 0.05
Antifoam 0.2
pH 6.5-7

In even a more preferred embodiment, the media composition of XenAg-B010 includes dextrose 15g L-1, yeast extract 5 gL-1, ammonium sulphate 1.5 gL-1, K2HPO4 1.5 gL-1, KH2PO4 1.5 gL-1, MgSO4.7H2O 0.5 gL-1, CaCl2.2H2O 0.06 gL-1 , Antifoam 0.2 gL-1 and pH 7.0±0.2. Each ingredient is selected within the ranges specified in Table 7 to achieve the desired performance, such as enhanced growth and stability.

4. XenAg-F008
Table 8: Media Composition of XenAg-F008

Ingredient g L-1
Dextrose 5-7
Yeast Extract 1.5-2.0
CaCl2 0.05-0.08
KH2PO4 1.2-1.5
MgSO4 2-4
KNO3 4.6-5.6
pH 6.8

In even more preferred embodiment, the media composition of XenAg-F008 includes dextrose 7g L-1, yeast extract 1.5 gL-1, calcium chloride (CaCl2) 0.06 gL-1, KH2PO4 1.2 gL-1 , MgSO4 3 gL-1, KNO3 4.8 gL-1 and pH 6.8±0.2. Each ingredient is selected within the ranges specified in Table 4 to achieve the desired performance, such as enhanced growth and stability.

In an embodiment of the present invention, the appropriate production medium specific for each strain is used for mass production. The media is sterilized at 1210C±20C for 30 minutes. After sterilization, cool the media at their respective running temperature. Inoculated the seed inoculum 5-10% of seed inoculum in batch aseptically.

Inoculation and incubation are followed by mass production of microbes through solid substrate fermentation and/or submerged fermentation. The step of mass production according to, for example, step 400 of Figure 1, step 100 of Figure 2 and step 240 of Figure 3. The preferred fermentation parameter for each strain are provided below:

1.XenAg-B001
Parameter for submerged fermentation
Temperature– 320C
Aeration – 1 VVM
Back pressure – 0.5 bar
Log Hours – 6 to 9 hrs
Agitation – 150 to 200 rpm
Incubation hrs- 48 hrs
Harvesting criteria-85±10% sporulation
Collect samples every 3 hrs, checked under microscopy, pH, OD

2. XenAg-B009
Batch Parameter for submerged Fermentation
Temperature– 370C
Aeration – 1-2 VVM
Back pressure – 0.5 bar
Log Hours – 6 to 9 hrs
Agitation – 150 to 180 rpm
Incubation hrs- 48 hrs
Harvesting criteria – 85 ± 10% sporulation

3. XenAg-B010
Batch Parameter for submerged Fermentation
Temperature– 370C
Aeration – 1-1.5 VVM
Back pressure – 0.5 bar
Log Hours – 6 to 9 hrs
Agitation – 200 to 250 rpm
Incubation hrs- 48 hrs
Harvesting criteria – 85 ± 10% sporulation

4. XenAg-F008
Batch Parameter for solid-state fermentation
Incubation Temperature: 27±20 C
Relative Humidity: 80±5%
Incubation time: 7-15 days

For the solid-state fermentation (SSF), 200 grams of rice flakes are used along with 80-100 ml of a liquid medium. The liquid medium consists of dextrose (5-7 g L-1), CaCl2 (0.05-0.08 g L-1), KH2PO4 (1.2-1.5 g L-1), MgSO4 (2.0-4.0 g L-1), KNO3 (4.5-6.5g g L-1), yeast extract (1.5-2.0 g L-1), and an antibiotic (0.5-0.7 g L-1). Alternatively, in some cases, a simpler solution of 5.0-7.0 g L-1 dextrose and 0.5-0. g L-1antibiotic is used as the liquid medium, which is added to 200 grams of rice flakes in autoclavable polybags. These bags are then sterilized. Once cooled, the bags are inoculated with 10-15% of the culture inoculum and incubated at a temperature of 27°C ± 2°C. For aeration, a pinning procedure is applied to the inoculated polybags using sterile forceps, and incubation continues for 7-8 days, or in some cases up to 15 days, in temperature-controlled air handling unit (AHU) supported growth rooms. Following the formation of conidia, the harvesting procedure is carried out.

Following mass production, the step of cells/spore’s concentration is carried out according to step 410 of Figure 1, step 110 of Figure 2 and step 250 of Figure 3.

The cells/spores are concentrated at least 10-15 times of the ready batch via continuous centrifuge/micro-filtration unit. Check the quality physical and biological parameters of concentration i.e., spore percentage, purity, viscosity, spin test. If the quality is good, then proceed for encapsulation step. Cell concentrate must be stored at 40C during the whole process of microencapsulation and spray drying.

Processing of XenAg-B001, XenAg-B009, XenAg-B010 - when used in concentrated form alone:
By using the submerged fermentation of the strains XenAg-B001 (Accession No. MTCC 25764), XenAg-B010 (Accession No. MTCC 25769), XenAg-B010 (Accession No. MTCC 25769) were processed through a similar procedure described above. The cell mass was concentrated by centrifugation at 4500 RPM for 20 minutes, after which the supernatant was removed. The collected cell mass was then utilized for subsequent drying processes.

All strains of cell concentrate were sprayed using a 0.7 mm single fluid nozzle within the heat spray dryer chamber. Prior to the spraying process, it was essential to ensure the fluidity of the slurry material. The solution was atomized in a co-current flow with hot air serving as the drying medium. The inlet air temperature for the spray drying of the cultures was maintained between 180°C and 185°C, while the outlet temperature was regulated to remain between 90°C and 95°C for all cultures examined in this study, following the initial standardization of the optimal outlet temperature to achieve improved yield and minimal cell loss. After the spray drying process, the entire powdered material was passed through a 200-micron sieve.

Processing of XenAg-F008 - when used in concentrated form alone
The spores of strain XenAg-F008 (Accession No. MTCC 25773) were collected following a 10-day incubation period utilizing solid-state fermentation. The fermented substrate was subjected to a drying process until the moisture content was reduced to 8%. Subsequently, the dried substrate was processed through a vibro sifter to obtain pure spores. These pure spores were utilized for formulation without any additional processing steps. It is important to note that no protectants or coating agents were employed in any of the strains, resulting in an uncoated or non-encapsulated product. The collected spores were then utilized for subsequent drying processes.

All strains of cell concentrate were sprayed using a 0.7 mm single fluid nozzle within the heat spray dryer chamber. Prior to the spraying process, it was essential to ensure the fluidity of the slurry material. The solution was atomized in a co-current flow with hot air serving as the drying medium. The inlet air temperature for the spray drying of the cultures was maintained between 180°C and 185°C, while the outlet temperature was regulated to remain between 90°C and 95°C for all cultures examined in this study, following the initial standardization of the optimal outlet temperature to achieve improved yield and minimal cell loss. After the spray drying process, the entire powdered material was passed through a 200-micron sieve.

Thereafter, the microencapsulation of concentrated cell biomass is processed according to step 420 of Figure 1, steps 120, 130 of Figure 2 and steps 260-300 of Figure 3.

Process details of producing encapsulated forms of BCAs
In an embodiment of the present invention, before spray drying encapsulation of concentrated cell biomass is conducted by coating with mixed microencapsulation agents such as gelatin, cellulose, starch, maltodextrin, gum arabic, xanthan gum, sodium alginate, methylcellulose and added the interlinking agents (CaCl2, NaCl) and buffers (phosphate buffer) as stabilizer. Properly mixed the encapsulating material and filter with muslin cloth in stainless steel vessel. Sterilized this slurry at 1210C±20 C for 15 minutes. Cool the mixture before adding in cell concentrate and mix properly to ensure the microencapsulation finished and observed microscopically to ensure the cell uniform wrapped with mixture and compare with non-encapsulated cells.

Table 9: Examples of composition of various microencapsulation polymers with binding agent
(a)
Ingredients - encapsulation slurry Percentage Preferred Percentage - for XenAg-B001 (Accession No. MTCC 25764)
Remarks
Gelatin 0.1-0.5% 0.5% First prepared the slurry and maintained the pH and then autoclaved at 1210C for 20 minute and cool down this slurry till 250C. Mixed properly according to ratio 1:3 by using mixer; that is - 1 part of slurry material, mixed in three parts of concentrated cell that have 20% cell concentrate w/w.
Maltodextrin 0.5-1.0% 1%
PSB (Phosphate saline buffer) 0.8-1.5% 1% 7.4 pH; 0.01M
Calcium chloride (CaCl2) 0.5-1.0% 0.5%
(b)
Ingredients - encapsulation slurry Percentage Preferred Percentage - for XenAg-B009 (Accession No. MTCC 25768) Remarks
Starch 0.33-0.50% 0.50% First prepared the slurry and autoclaved at 1210C for 20 minute and cool down this slurry till 250C. Mixed properly according to ratio 1:3 by using mixer; that is - 1 part of slurry material, mixed in three parts of concentrated cell that have 40% cell concentrate w/w.
Maltodextrin 0.5-1.0% 1.0%
PSB (Phosphate saline buffer) 0.8-1.5% 1.0% 7.4 pH; 0.01M
Calcium chloride (CaCl2) 0.5-1% 0.5%

(c)
Ingredients - encapsulation slurry Percentage Preferred Percentage - for XenAg-B009 (Accession No. MTCC 25768) Remarks
Xanthan gum 1-1.5% 1.5% First prepared the slurry and autoclaved at 1210C for 20 minute and cool down this slurry till 250C. Mixed properly according to ratio 1:3 by using mixer; that is - 1 part of slurry material, mixed in three parts of concentrated cell that have 20% cell concentrate w/w.
Maltodextrin 0.5-1.5% 1%
PSB (Phosphate saline buffer) 0.8-1.5% 1% 7.4 pH; 0.01M
Calcium chloride (CaCl2) 0.5-1.0% 1%
(d)
Ingredients - encapsulation slurry Percentage Preferred Percentage - for XenAg-B010 Remarks
Methylcellulose 1-1.5% 1.5% First prepared the slurry and autoclaved at 1210C for 20 minute and cool down this slurry till 250C. Mixed properly according to ratio 1:3 by using mixer; that is - 1 part of slurry material, mixed in three parts of concentrated cell that have 40% cell concentrate w/w.
Maltodextrin 0.5-1.5% 1%
Sodium Chloride (NaCl) 1.0-2.0% 1%
PSB (Phosphate saline buffer) 0.8-1.5% 1%

(e)
Ingredients - encapsulation slurry Percentage Preferred Percentage - for Trichoderma viride XenAg-F008 (Accession No. MTCC 25773) Remarks
Sodium alginate 2-4% 3.0% First prepare the slurry and autoclaved at 1210C for 20 minute and cool down this slurry till 250C. Mixed properly according to ratio 1:1.5 by using mixer; that is - 1 part of slurry material, mixed in 1.5 parts of concentrated cell that have 20% cell concentrate w/w.
Calcium chloride (CaCl2) 0.5-2% 1.0%
Sodium Chloride (NaCl) 1-2% 1.0%
PSB (Phosphate saline buffer) 0.8-1.5% 1.0%

Spray Drying Process Conditions:
The encapsulation of the one or more strains of microbes is followed by spray drying of microcapsules so formed according to step 430 of Figure 1, step 140 of Figure 2 and step 310 of Figure 3. The ready encapsulated slurry material (slurry material mixed with cells /spores concentrated) is sprayed using a 0.7 mm single fluid nozzle in heat spray dryer chamber. Before the spray, it is ensured that the fluidity of slurry material is appropriate according to predefined parameters. The solution is sprayed in a co-current flow with hot air as drying medium. The inlet air temperature used for spray drying of culture ranged from 180°C- 185°C and outlet temperatures was adjusted to 90-95°C for all the cultures used in this study, after initial standardization of optimal outlet temperature for a high yield and minimal loss of cells. After spray drying the whole powder material passed through 250-micron sieve, spray dried powder was collected as fine particles and transferred into pouches, sealed, and stored at 4°C.

The yield (%) was calculated as follows:
Yield (%) = (Total solid – recovered solids) / Total solids X 100

According to an embodiment of the present invention, the aforementioned process flow steps of Figures 1, 2 and 3 are repeated for a second coating of a double microencapsulated microbial composition.

After the spray drying, stability of the microencapsulated bacterial cells/fungal spores is assessed and the microcapsules are characterized according to, for example, step 440 of Figure 1 and step 150 of Figure 2.

The numbers of viable bacteria before and after microencapsulation during storage were enumerated by a standard poured-plate method. Microencapsulated microbes were suspended in sterile saline and serial dilutions of each sample were prepared. All dilutions were poured into Nutrient-broth for bacterial cell and Potato dextrose agar plates were incubated at 37±2°C for 48 h for bacterial cell and 28±2°C for fungal spores. After the incubation time, colonies were enumerated, and the mean number of bacteria was expressed as CFU g-1 or Spore g-1. All experiments were repeated in triplicate.

In an embodiment of the present invention, the biological control agents (BCAs) may be encapsulated in single layer, double, layer or multiple layers. The dried microencapsulated product undergoes in vitro characterization to evaluate key parameters such as particle size, encapsulation efficiency, and the viability of the encapsulated microbes. This step ensures the quality and performance of the biopesticide meet the required standards.

5. COMPOSITION AND BLENDING OF MICROCAPSULES

Steps 160, 320, 330, 340 and 450 relates to blending of either the concentrated cells/spores or microcapsules of one or more BCA’s. and forming composition. This blending process tailors the biopesticide to specific agricultural needs, ensuring it is effective against targeted pests or diseases.

When XenAg-B001, XenAg-B009, XenAg-B010, XenAg-F008 are used in concentrated form;
Method of blending of consortia:
All concentrated strain (without encapsulated) sXenAg-B001 (Accession No. MTCC 25764), XenAg-B009 (Accession No. MTCC 25768), and XenAg-B010 (Accession No. MTCC 25769) and XenAg-F008 (Accession No. MTCC 25773) were collected according to their respective composition percentages. Initially, the base carrier was introduced into a sterile v-cone blender. Subsequently, each ingredient was added individually, ensuring thorough mixing by rotating the v-cone blender at a speed of 25 RPM for a duration of 30 minutes. V-cone blender used for gentle and effective mixing.

When all XenAg-B001, XenAg-B009, XenAg-B010, XenAg-F008 are used in encapsulated form
Method of blending of consortia:
All encapsulated strain XenAg-B001 (Accession No. MTCC 25764), XenAg-B009 (Accession No. MTCC 25768), and XenAg-B010 (Accession No. MTCC 25769) and XenAg-F008 (Accession No. MTCC 25773) were collected according to their respective composition percentages. Initially, the base carrier was introduced into a sterile v-cone blender. Subsequently, each ingredient was added individually, ensuring thorough mixing by rotating the v-cone blender at a speed of 25 RPM for a duration of 30 minutes. V-cone blender used for gentle and effective mixing.

The production of microencapsulated biopesticide involves making a carefully balanced composition to ensure stability and efficacy of the microencapsulated BCA’s. The primary component of the composition is the base carrier, which constitutes 55-65% of the composition. Dextrose monohydrate is the most preferred base carrier due to its compatibility with the other components and the microencapsulated BCA’s. Additionally, a cell protectant, making up 15-25% of the composition, is included to further safeguard the BCAs from thermal and other stresses. The composition also includes a dispersion agent (1-5%) such as silicon dioxide, calcium silicate to ensure even distribution of the microencapsulated BCAs within the mixture, preventing clumping and ensuring uniform distribution. Stabilizers, such as CMC (Carboxymethyl cellulose), guar gum, casein, alginic acid which make up 2-4% of the composition, are added as adjuvant and to enhance the shelf-life and stability of the encapsulated BCAs, maintaining the integrity of the microcapsules during storage and application. Additionally, the composition has 1-3% of fungicide agent such as chitinase, protease, lipase and 1-3% of UV protectant molecule. The main component is the active biocontrol agents, which are the strains XenAg-B001, XenAg-B009, XenAg-B010 and XenAg-F008, or a consortium thereof. The preferred composition involves using individual strains, with XenAg-B001 at 1-3%, XenAg-B009 at 1-3%, XenAg-B010 at 1-3% and XenAg-F008 2-4%.

The table below shows the composition as described above.

Table 10: Showing the constituents of the composition - comprising encapsulated forms of the BCA

Components Percentage
Base Carrier (Dextrose, Monohydrate) 55-65%
Cell protectant 15-25%
Dispersion agent 1-5%
Fungicidal Active molecule (optionally added to further increase composition activity) 1-3%
Stabilizer/Adjuvant/Sticking agent 2-4%
UV protectant molecule (Lignosulfonates) 1-3%
XenAg-B001 2-3%
XenAg-B009 2-3%
XenAg-B010 2-3%
XenAg-F008 3-4%

In most preferred embodiments, the composition comprises 60% base carrier, 20% cell protectant, 4 % dispersion agent, 3% stabilizers, 2% of anti-fungicidal agent such as chitinase, protease, lipase and 2% of UV protectant molecule ( as also used for experimental purposes as disclosed herein). The composition further comprises encapsulated form of BCAs having 2% XenAg-B001, 2% XenAg-B0092%, 2% XenAg-B010 and 3% XenAg-F008.
If non-encapsulated concentrated forms are used, the composition further comprises encapsulated form of BCAs having 1% XenAg-B001, 1% XenAg-B0092%, 1% XenAg-B010 and 3% XenAg-F008.

6. SHELF-LIFE IMPROVEMENT ANALYSIS

The biocontrol agents of the invention can also be used as a bio pesticide, however, the same can also be encapsulated by the method as described above and this significantly improves the shelf-life of the BCAs compared to non-encapsulated cultures.

The composition comprising 8% spray dried micro-capsules of BCA or 6% concentrated cells/spores of bio-control agent and components as described in table 10 was analysed for stability and shelf life.

The numbers of viable bacteria before and after microencapsulation during storage were enumerated by a standard poured-plate method. Microencapsulated microbes were suspended in sterile saline and serial dilutions of each sample were prepared. All dilutions were poured into Nutrient-broth for bacterial cell and Potato dextrose agar plates were incubated at 37±2°C for 48 h for bacterial cell and 28±2°C for fungal spores. After the incubation time, colonies were enumerated, and the mean number of bacteria was expressed as CFU g-1 or Spore g-1. All experiments were repeated in triplicate. The following table compares the colony-forming units (CFU) per gram of encapsulated BCAs versus non-encapsulated BCA cultures over time:

Table 11: Stability of non-encapsulated microbes during long time storage
Non-encapsulated Microbes Time Intervals (CFU g-1)
Initial (0 Days) 30 days 60 days 90 days
XenAg-B001 3.5±0.34×1010 2.7±0.24×1010 1.7±0.35×1010 6.2±0.32×109
XenAg-B009 4.2±0.04×1010 3.2±0.05×1010 2.1±0.06×1010 7.1±0.04×109
XenAg-B010 3.7±0.08×1011 2.7±0.08×1011 1.5±0.08×1011 4.4±0.07×1010
XenAg-F008 4.0±0.06×108 3.0±0.05×108 1.9±0.03×108 5.9±0.09×107

Table 12: Stability of single layered microencapsulated microbes during long time storage
Encapsulated Microbes Time Intervals (CFU g-1)
Initial (0 Days) 30 days 60 days 90 days
XenAg-B001 4.5±0.30×1010 2.8±0.24×1010 2.7±0.30×1010 2.2±0.29×1010
XenAg-B009 4.2±0.06×1010 3.9±0.06×1010 2.9±0.09×1010 2.1±0.09×1010
XenAg-B010 3.7±0.07×1010 2.6±0.09×1010 2.5±0.08×1010 2.4±0.07×1010
XenAg-F008 4.5±0.06×108 3.9±0.05×108 3.2±0.03×108 2.9±0.09×108

Table 13: Stability of double layered microencapsulated microbes during long time storage
Encapsulated Microbes Time Intervals (CFU g-1)
Initial (0 Days) 30 days 60 days 90 days
XenAg-B001 4.8±0.37×1010 4.8±0.31×1010 4.7±0.33×1010 4.7±0.31×1010
XenAg-B009 3.2±0.05×1010 3.2±0.06×1010 3.1±0.04×1010 3.1±0.08×1010
XenAg-B010 2.7±0.04×1010 2.7±0.07×1010 2.±0.03×1010 2.4±0.12x1010
XenAg-F008 4.1±0.03×108 4.0±0.07×108 3.9±0.06×108 3.9±0.08×108

The encapsulated cultures maintained a significantly higher CFU count over 90 days, demonstrating enhanced stability and prolonged shelf-life compared to non-encapsulated cultures. For instance, the initial cfu amount in case of XenAg-B001 in non-encapsulated, single layer-microencapsulated and double layer-microencapsulated was 3.5±0.34×1010, 4.5±0.30×1010 and 4.8±0.37×1010 respectively and after 90 days, it was 6.2±0.32×109, 2.2±0.29×1010 and 4.7±0.31×1010 in non-encapsulated, single layer-microencapsulated and double layer-microencapsulated form. Thus, non-encapsulated form of the BCAs decreased significantly. This indicates that the encapsulation process effectively protects the BCAs from environmental degradation, ensuring their viability and effectiveness over a longer period.

The BCA consortium (XenAg-B001, XenAg-B009, XenAg-B010 and XenAg-F008) through microencapsulation presents a further effective solution for combating fungal wilt diseases such as Fusarium oxysporum, Macrophomina phaseolina, and Rhizoctonia solani. This consortium composition leverages the synergistic effects of its components to provide superior protection against the said disease. The microencapsulation process not only enhances the stability and shelf-life of the biopesticide but also ensures its efficacy under field conditions. The combination of these specific strains achieves the highest inhibition of wilt causing organism, making this composition a valuable tool in sustainable agriculture. This method ensures the stability, efficacy, and prolonged shelf-life of the biopesticide, offering a sustainable and environmentally friendly alternative to chemical pesticides. The combination of these specific strains has proven to provide the highest inhibition of wilt causing pathogen, making this composition highly effective in protecting economically important crops.

Steps 170, 180, 350 and 360 relate to packing and storage of microencapsulated biopesticide powder. The microencapsulated biopesticide powder is packed into suitable containers such as bottles, pouches, or vials. Each container is labelled and sealed, ready for distribution and use. Proper storage conditions are maintained to preserve the efficacy and shelf life of the biopesticide.

This present invention encompasses several uses, applications, and advantages including:

1. The strains of the invention XenAg-B001, XenAg-B009, XenAg-B010 and XenAg-F008, and the consortiums can be used as a bio-fungicide to fungicide to alleviate the abiotic stress on plants against damping off and fungal wilt diseases. The synergistic effect of these strains ensures comprehensive and effective protection against these common plant pathogens.
2. This consortium composition can be applied directly to soil or plants, enhancing plant tolerance to various environmental stresses while reducing biotic stresses. Its use extends to rice crops, providing a sustainable and environmentally friendly alternative to chemical pesticides, thereby improving crop yield and overall plant health.
3. Double-layered microencapsulation significantly extends the shelf life of BCAs by protecting them from environmental degradation during storage and use. This ensures long-term viability and effectiveness of the biopesticide in field conditions.
4. The double-layer encapsulation enables BCAs to withstand harsh climatic conditions, including extreme temperatures and variable moisture levels, which would otherwise degrade unprotected agents.
5. Encapsulated BCAs exhibit greater tolerance to abiotic stresses such as UV radiation, desiccation, and extreme pH levels, maintaining their functionality and potency over extended periods.
6. The technology facilitates the gradual and controlled release of BCAs, ensuring a sustained and effective response against plant diseases and pests over time, without immediate loss of viability.
7. With the enhanced stability and controlled release, the encapsulated BCAs can be applied less frequently and at lower dosages, reducing overall chemical input and associated costs.
8. By lowering the necessary application doses and frequency, double-layered microencapsulation minimizes chemical runoff and soil contamination, promoting a cleaner and safer environment.
9. The versatility of the double-layer encapsulation allows BCAs to remain effective across various ecological settings, adapting to diverse environmental conditions and soil types.
10. Scientific evidence supports that double-layer microencapsulation maintains the stability of BCAs, preserving their colony-forming ability over time, which is crucial for their long-term effectiveness.
11. Encapsulated BCAs can be directly applied to plants or soil, where they enhance plant tolerance to biotic stresses and improve overall plant health. This direct application method simplifies the integration of BCAs into existing agricultural practices.
12. Double-layered microencapsulated BCAs help control diseases in key crops such as cereals, vegetables, and fruits, thereby safeguarding yield and quality, which is essential for food security and economic stability.
13. The technology allows for effective composition at lower dosages per acre, which is cost-efficient and reduces the environmental footprint of agricultural practices.
14. Microencapsulation addresses problems associated with free-form BCAs by providing a stable and manageable composition, which simplifies storage, handling, and application.
, Claims:We claim:
1. A method of forming a composition for use as a bio-pesticide, the method comprising: -
a. culturing one or more bio-control agent by solid substrate fermentation and/or submerged fermentation;
b. concentrating the cells/spores of bio-control agent at least 10-15 times via continuous centrifugation and/or micro-filtration to obtain concentrated cells/spores of bio-control agent;
c. optionally mixing concentrated cells/spores of bio-control agent with a slurry comprising microencapsulation agents selected from one or more of gelatin, cellulose, starch, maltodextrin, gum arabic, xanthan gum, sodium alginate, methylcellulose and interlinking agents selected from one or more of CaCl2, NaCl and optionally buffers such as phosphate buffer to form microcapsules;
d. spray drying the microcapsules obtained in step c) or concentrated cells/spores of bio-control agent obtained in step b) to obtain spray dried microcapsules or spray dried or concentrated cells/spores of bio-control agent or;
e. optionally repeating steps c) and d) to form further encapsulation layers;
f. mixing 5-20%, preferably 5-10% of spray dried micro-capsules obtained in step (d) or mixing 3-20%, preferably 5-10% of spray dried concentrated cells/spores of bio-control agent obtained in step d) with 55-65% base carrier, 15-25% cell protectant, 1-5 % a dispersion agent, 2-4% stabilizers, 1-3% of anti-fungicidal agent such as chitinase, protease, lipase and 1-3% of UV protectant molecule.

2. The method of forming a composition for use as a bio-pesticide, as claimed in claim 1, wherein in step c), the slurry comprises starch in an amount in the range of from 0.33%-0.50%, gelatin in an amount in the range of from 0.1%-0.5%, methylcellulose in an amount in the range of from 1.0%-1.5%, maltodextrin in an amount in the range of from 1.0%-1.5%, xanthan gum in an amount in the range of from 1.0%-1.5%, and/or sodium alginate in an amount in the range of from 2.0%-4.0%.

3. The method of forming a composition for use as a bio-pesticide, as claimed in claim 1, wherein in step c), the slurry comprises interlinking agents selected from one or more of CaCl2, NaCl in an amount ranging from 0.5% to 1.0%, and phosphate buffer in an amount ranging from 0.8%-1.5%.

4. The method of forming a composition for use as a bio-pesticide, as claimed in any of claims 1 to 3, wherein in step c) the slurry is mixed with the cell concentrated cells/spores of bio-control agent in the ratio of 1:1 to 1:5, preferably 1:3.

5. A method for reducing overall damage caused to plants and plant parts by wilt causing pathogens, comprising the step of applying the composition as produced by the method claimed in any of the claims provided above to the plants, plant parts, seeds, fruits, vegetables and soil.

Dated this 25th day of June 2025

(Digitally Signed)
Gitika Suri
IN/PA 1788
Of Cyril Amarchand Mangaldas
Agent for the Applicant