Claims:We Claim:
1. A polymeric nano composite formulation comprising Poly-(D) Glucosamine (chitosan) in combination with 2-Bromo-2-nitro-1,3-propanediol, brassinolides, 2-O-alpha-L-rhamnosyl-alpha-L-rhamnosyl-3-hydroxydecanoyl-3- hydroxy decanoic acid (rhamnolipid), along with one or more agrochemical excipients.

2. The polymeric nano composite formulation as claimed in claim 1, wherein Poly- (D) Glucosamine and a cross-linker is a nano polymer matrix and 2-Bromo-2-nitro-1,3-propanediol, brassinolides, 2-O-alpha-L-rhamnosyl-alpha-L-rhamnosyl-3-hydroxydecanoyl-3- hydroxy decanoic acid is aqueous nanofiller medium.

3. The polymeric nano composite formulation as claimed in claim 1, wherein the agrochemical excipient is selected from capsule forming agent, emulsifier, dispersing agent, anti-freeze, cross-linker, biocide, stabilizing agent, wetting agent, defoamer, adjuvant, viscosity modifier or combination thereof.

4. The polymeric nano composite formulation as claimed in claim 1, wherein Poly-(D) Glucosamine is present at a concentration in the range of 0.5% to 2% (w/v), 2-Bromo-2-nitro-1,3-propanediol is present at a concentration in the range of 0.5% to 1% (w/v), brassinolides is present at a concentration between 0.5% to 1% and 2-O-alpha-L-rhamnosyl-alpha-L-rhamnosyl-3-hydroxydecanoyl-3- hydroxy decanoic acid is present 0.5%-1% based on total volume of the formulation.

5. The polymeric nano composite formulation as claimed in claim 1, wherein the cross-linker is poly ethylene glycol selected from polypropylene glycol, poly ethylene glycol or a combination thereof, in an amount from 0.1% to 1.0% (w/v) based on total volume of the formulation.
6. The polymeric nano composite formulation as claimed in claim 1, wherein concentrations of the Poly-(D) Glucosamine, Bronopol, brassinolides and rhamnolipid in the formulation in ppm are 20000, 10000, 10000 and 5000 respectively.

7. The polymeric nano composite formulation as claimed in claim 1, wherein the particle size of the formulation is in range between 150-750 nm.

8. A method of preparing the polymeric nano composite formulation, comprising the steps of:
i. Preparation of nano polymer matrix(700ml) :
a. Weighing 20gm of (2% for final volume) of Poly- (D) Glucosamine and dissolving it in 0.5% (5 ml in 690 ml of distilled water) of CH3COOH solution using a high shear homogenizer at 800 rpm until it is dissolved completely leaving no residues to provide dissolved poly-glucosamine (solution A);
b. Adding 1% (7 ml) of poly ethylene glycol (1% of polymer matrix – 700 ml) to the dissolved poly-glucosamine (solution A), wherein the poly ethylene glycol acts as a cross-linker in producing the polymeric matrix;
ii. Preparation of nano active fillers (300 ml):
a. Dissolving 1% (of final volume -10gm) of 2-Bromo-2-nitro-1,3-propanediol (Bronopol), in 200 ml of double distilled water at 600 rpm until a clear solution is obtained;
b. Adding 0.5% (of final volume- 5 gm) of 2-O-alpha-L-rhamnosyl-alpha-L-rhamnosyl-3-hydroxydecanoyl-3- hydroxy decanoic acid to the above solution A and dissolving it at 300 rpm for 15 min or until a translucent solution C is obtained;
c. Separately dissolving 1% (of final volume -10gm) of Brassinolides in 100 ml of double distilled water at 600 rpm until homogenous solution D is obtained;
d. Blending solution C and D to form a homogenous mixture; and
e. Adding 0.5% (to final volume- 5ml) of glycerol to the homogenous mixture, wherein the glycerol acts a plasticizer for the polymer matrix in order to boost film formation;
iii. Impregnating the nano active fillers prepared from step (ii) in the nano polymer matrix of step (i), wherein the impregnation of nano active filler is done with continuous blending at 600 rpm followed by ultra-sonication with 40% amplitude at room temperature for 30 min; and
iv. Processing of polymeric nanocomposite from step iii. using wet-grinding in a nano miller for 45 min at room temperature that allows further size reduction.

9. The method of preparing the polymeric nano composite formulation as claimed in claim 8, wherein the room temperature is a temperature ranging from 20°C to 40°C.

10. The method of preparing the polymeric nano composite formulation as claimed in claim 8, wherein particle size ranges from 150-750 nm and concentrations of the chitosan, Bronopol, brassinolides and rhamnolipid in the formulation are 20000 ppm, 10000 ppm, 10000 ppm and 5000 ppm respectively.
, Description:TECHNICAL FIELD
[001] This present disclosure relates to field of agrochemical compositions having activity against plant pathogens. More particularly, the present disclosure pertains to polymeric nano composite formulation and method of preparation thereof. The said polymeric nano composite formulation has anti-fungal and anti-bacterial activity.

BACKGROUND
[002] Agriculture is inevitable when comes to livelihood and country’s economic growth. In recent years, changing climatic conditions have led to concurrent emergence of virulent pathogenic fungal and bacterial infestations and this in turn increased the use of pesticides and insecticides to combat pathogenic microorganisms. Among microbes, the fungal genera belonging to class Ascomycetes (Verticillium, Alternaria, and Fusarium) and Basidiomycetes (Rhizoctonia, Sclerotium) and the bacterial genera belong to the family Pseudomonadaceae are known to cause deterring effects in crop plants leading to significant yield losses. To dampen these infestations a dynamic and stern approach with novel technology focusing sustainable and prolonged efficiency is required.

[003] Plants are exposed to multiple biological stresses caused by bacterial and fungal pathogens. These pathogens attack the plants in combination or sequentially creating economic loss due to yield reduction and quality deteriorations of produces. Most of the control measures currently employed target either one of the pathogens and use large quantities of synthetic chemicals.

[004] The extensive and over use of synthetic chemicals to overcome fungal and bacterial infections in crops have led to the increase of resistance by these microbial pathogens besides leaving chemical residues in the produces and in the environment. Developing combined and effective alternate methods for managing fungal and bacterial plant diseases with less usage of chemicals could be an eco-friendly long-term strategy. Exploring the use of nano systems containing multiple functional molecules can be an assuring alternative to manage the pathogens in agricultural and horticultural crops. In the current invention, nano systems comprising of natural polymers and functional molecules that are Generally recognized as safe (GRAS) have been employed to create an effective and wide spectrum nano composite to manage both bacterial and fungal infections. Because of the unique surface chemistry and particle size, the nano composite knocks down the microbial pathogens at lower concentration.

[005] The state of the art has research dedicated to provide alternative methodologies to curb plant pathogens, improve crop productivity etc. For example, US10791734B2 relates to insecticidal active mixtures comprising carboxamide compound, US20110319341A1 relates to method of controlling pests with biosurfactant penetrants as carriers for active agents, TW201639461A relates to Bacillus amyloliquefaciens RTI472 compositions and methods of use for benefiting plant growth and treating plant disease and WO2018042311A1 relates to Chitosan derivative formulations for plant growth, and building disease resistance. Currently, multiple synthetic chemicals are used at different stages of the crop to manage the fungal and bacterial diseases. These chemicals are used alone or in combination. Due to the problems associated with resistance development by the pathogens to the chemicals, farmers are forced to use higher doses that results in cost escalation and eco-toxicity. Therefore, there is a need for providing collective control measures and a uniform effort towards for efficient disease management when the microbial pathogens exert combined stress to plants. There is a need to employ a combined strategy of creating nano formulation by incorporating both anti-fungal and anti-bacterial molecules in one matrix. The process of developing nano formulation also needs to be unique such that it ensures high efficacy against pathogens at lower concentration.
[006] Specifically, there is a need for novel methodologies to produce polymeric nano composite formulations in a cost effective easy manner that exert potent biological activity at lower concentration with eco-friendly benefits in a short span of time.
BRIEF SUMMARY
[007] The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the invention or delineate the scope of the invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

[008] A more complete appreciation of the present invention and the scope thereof can be obtained from the accompanying drawings which are briefly summarized below and the following detailed description of the presently preferred embodiments.

[009] An objective of the present disclosure is directed towards polymeric nano composite formulation and method of preparation of the same.

[0010] An objective of the present disclosure is directed to overcome the problem in the state of art by providing a unique process methodology to produce a polymeric nano composite formulation having potent anti-fungal and anti-bacterial activity.

[0011] It is another object of the present disclosure to provide a method of preparation of a polymeric nano composite formulation in a cost-effective and easy manner.

[0012] It is another object of the present disclosure to provide a method of preparation of a polymeric nano composite formulation by incorporating both anti-fungal and anti-bacterial molecules in one matrix. The process of developing nano formulation is unique that ensures high efficacy against pathogens at lower concentration.

[0013] It is another object of the present disclosure to provide a polymeric nanocomposite formulation wherein at least three functional molecules having fungicidal, bactericidal and growth enhancing properties have been incorporated in a single polymer matrix and developed as a nano composite formulation.

[0014] It is another object of the present disclosure to provide a polymeric nanocomposite formulation involving conversion of macro-sized functional molecules (parent molecules) in to nano size and developing a nano formulation that has unique surface chemistry with higher efficacy against plant pathogens when compared to macro sized parent molecules.

[0015] It is another object of the present disclosure to provide a polymeric nanocomposite formulation involving process using at least three functional molecules (active ingredients) in an unique manner that ensures high efficacy against pathogens at lower concentration.

[0016] It is another object of the present disclosure to provide a polymeric nanocomposite formulation in which each active ingredient is added at a definite ratio and size to achieve higher formulation stability. The processes such as preparation of nano polymer matrix, preparation of nano-filler, impregnation of nano-filler in the nano polymer matrix are unique and have specific methodology to achieve required surface charge (zeta potential) of the final formulation.

[0017] It is another object of the present disclosure to provide a polymeric nanocomposite formulation using a process that has been designed to synthesise said formulation with specific particle size, zeta potential and poly dispersion index that ultimately enhances the formulation efficiency at low concentration.

[0018] According to an exemplary embodiment of the present disclosure, a method of preparing a polymeric nanocomposite formulation comprising: (i) Preparation of nano polymer matrix(700ml) :
Weighing 20gm of (2% for final volume) of Poly- (D) Glucosamine and dissolving it in 0.5% (5 ml in 690 ml of distilled water) of CH3COOH solution using a high shear homogenizer at 800 rpm until it is dissolved completely leaving no residues to provide dissolved poly-glucosamine (solution A);
Adding 1% (7 ml) of poly ethylene glycol (1% of polymer matrix – 700 ml) to the dissolved poly-glucosamine (solution A), wherein the poly ethylene glycol acts as a cross-linker in producing the polymeric matrix;
(ii) Preparation of nano active fillers (300 ml):
Dissolving 1% (of final volume -10gm) of 2-Bromo-2-nitro-1,3-propanediol (Bronopol), in 200 ml of double distilled water at 600 rpm until a clear solution is obtained;
Adding 0.5% (of final volume- 5 gm) of 2-O-alpha-L-rhamnosyl-alpha-L-rhamnosyl-3-hydroxydecanoyl-3- hydroxy decanoic acid to the above solution A and dissolving it at 300 rpm for 15 min or until a translucent solution C is obtained;
Separately dissolving 1% (of final volume -10gm) of Brassinolides in 100 ml of double distilled water at 600 rpm until homogenous solution D is obtained;
Blending solution C and D to form a homogenous mixture; and
Adding 0.5% (to final volume- 5ml) of glycerol to the homogenous mixture, wherein the glycerol acts a plasticizer for the polymer matrix in order to boost film formation;
(iii) Impregnating the nano active fillers prepared from step (ii) in the nano polymer matrix of step (i), wherein the impregnation of nano active filler is done with continuous blending at 600 rpm followed by ultra-sonication with 40% amplitude at room temperature for 30 min; and
Processing of polymeric nanocomposite from step iii. using wet-grinding in a nano miller for 45 min at room temperature that allows further size reduction.

[0019] Furthermore, the objects and advantages of this invention will become apparent from the following description and the accompanying annexed drawings.

BRIEF DESCRIPTION OF DRAWINGS
[0020] Other objects and advantages of the present invention will become apparent to those skilled in the art upon reading the following detailed description of the preferred embodiments, in conjunction with the accompanying drawings, wherein like reference numerals have been used to designate like elements, and wherein:

[0021] FIG. 1 is a diagram 100 comprising FIGS 1A, 1B wherein Figure 1A:100A is a graph showing Size Distribution of Polymeric Nanocomposite formulation and Figure 1B:100B is graph showing Zeta Potential (Stability) of Polymeric Nanocomposite formulation, in accordance with one or more embodiments.

[0022] FIG. 2 is a diagram 200 including FIGS2A-2E where Figure 2A:200A is the graph representing FTIR Spectra of the Nanocomposite; Figure 2B:200B is the graph representing FTIR Spectra of the Nanocarrier; Figure 2C:200C is the graph representing FTIR Spectra of the Bronopol; Figure 2D:200D is the graph representing FTIR Spectra of the Brassinolides and Figure 2E:200E is the graph representing FTIR Spectra of the Rhamnolipids, in accordance with one or more embodiments.

[0023] FIG. 3 is a diagram 300 depicting bio-efficacy studies performed to investigate the anti-fungal activity of Polymeric nanocomposite formulation at incremental concentrations against Fusarium sp., in accordance with one or more embodiments.

[0024] FIG. 4 is a diagram 400 depicting a bar chart representing the Germination percentage of Maize seeds subjected to various treatments. (T1: NC 1ml in 100ml; T2: NC 2ml in 100ml; T3: Hydropriming; Absolute Control), in accordance with one or more embodiments.

[0025] FIG. 5 is a diagram 500 depicting maize germination studies by paper towel method, in accordance with one or more embodiments.

[0026] FIG. 6 is a diagram 600 depicting a flowchart for a method of preparing a polymeric nanocomposite formulation, in accordance with one or more embodiments.

[0027] FIG. 7 is a diagram 700 depicting a flowchart for a detailed methodology of preparing a polymeric nanocomposite formulation, in accordance with one or more embodiments.

[0028] FIG. 8 is a diagram 800 depicting electron microscopy image of the polymeric nanocomposite formulation, in accordance with one or more embodiments

DETAILED DESCRIPTION
[0029] It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0030] The use of “including”, “comprising” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

[0031] All publications herein are incorporated by reference to the same extent as that clearly to be included by reference if each individual publication or patent application, and individually indicated. The following description includes information that may be useful for understanding the present invention. This does not admitted that any of the information is prior art, or to be or being specifically related to the invention claimed herein or implicit any prior art publication is referred to as provided herein.

[0032] Exemplary embodiments are described with reference drawings. Exemplary embodiments disclosed herein and the drawings are intended to be regarded in an illustrative rather than a restrictive sense.

[0033] Referring to FIG. 1 is a diagram 100 comprising FIGS 1A, 1B wherein Figure 1A:100A is a graph showing Size Distribution of Polymeric Nanocomposite formulation and Figure 1B:100B is graph showing Zeta Potential (Stability) of Polymeric Nanocomposite formulation, in accordance with one or more embodiments.

[0034] Referring to FIG. 2 is a diagram 200 including FIGS2A-2E where Figure 2A:200A is the graph representing FTIR Spectra of the Nanocomposite; Figure 2B:200B is the graph representing FTIR Spectra of the Nanocarrier; Figure 2C:200C is the graph representing FTIR Spectra of the Bronopol; Figure 2D:200D is the graph representing FTIR Spectra of the Brassinolides and Figure 2E:200E is the graph representing FTIR Spectra of the Rhamnolipids, in accordance with one or more embodiments.

[0035] Referring to FIG. 3 is a diagram 300 depicting bio-efficacy studies performed to investigate the anti-fungal activity of Polymeric nanocomposite formulation at incremental concentrations against Fusarium sp., in accordance with one or more embodiments.

[0036] Referring to FIG. 4 is a diagram 400 depicting a bar chart representing the Germination percentage of Maize seeds subjected to various treatments. (T1: NC 1ml in 100ml; T2: NC 2ml in 100ml; T3: Hydropriming; Absolute Control), in accordance with one or more embodiments.

[0037] Referring to FIG. 5 is a diagram 500 depicting maize germination studies by paper towel method, in accordance with one or more embodiments.

[0038] Referring to FIG. 6 is a diagram 600 depicting a flowchart for a method of preparing a polymeric nanocomposite formulation, in accordance with one or more embodiments. The method 600 starts at step 602 with preparation of nano-polymeric matrix using Poly- (D) Glucosamine and a cross-linker. Subsequently at 604, preparation of nanofiller as aqueous medium using 2-Bromo-2-nitro-1,3-propanediol and 2-O-alpha-L-rhamnosyl-alpha-L-rhamnosyl-3-hydroxydecanoyl-3- hydroxy decanoic acid and a stabilizer is performed and finally concludes at step 606 with impregnation of nanofiller of step (604) in the nano-polymeric matrix of step (602) in a defined ratio

[0039] Referring to FIG. 7 is a diagram 700 depicting a flowchart for a detailed methodology of preparing a polymeric nanocomposite formulation, in accordance with one or more embodiments. The method 700 starts at step 702 with preparation of nano polymer matrix by:
At Step 702a, dissolving Poly- (D) Glucosamine (2%) in CH3COOH solution until it is dissolved completely leaving no residues
At Step 702b, addition of Poly ethylene glycol (1%) as cross-linker in solution (a) to produce polymeric matrix
The second step 704 initiates with preparation of nanofiller by:
At Step 704a =Dissolving Bronopol (1%) in double distilled water until a clear solution is obtained
At Step 704b =Adding 2-O-alpha-L-rhamnosyl-alpha-L-rhamnosyl-3-hydroxydecanoyl-3- hydroxydecanoic acid to step ii.a. and mixing until a translucent solution is obtained.
The third step 706 initiates with impregnation of nanofiller prepared from step (702b) in the nano polymer matrix of step (702a) in a defined ratio to provide a polymeric nanocomposite and subjecting the polymeric nanocomposite to ultrasonication. Subsequently at step 708 processing of polymeric nanocomposite obtained above by using wet-grinding in a nano miller for further size reduction to finally provide a polymeric nanocomposite formulation having specific size ranges is performed.

[0040] FIG. 8 is a diagram 800 depicting electron microscopy image of the polymeric nanocomposite formulation, in accordance with one or more embodiments

[0041] In an embodiment, the present disclosure aims to provide polymeric nanocomposite formulation comprising of four unique functional molecules such as Poly- (D) Glucosamine (chitosan), 2-Bromo-2-nitro-1,3-propanediol (bronopol), Brassinolides and 2-O-alpha-L-rhamnosyl-alpha-L-rhamnosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid (rhamnolipid) at a definite ratio and size while achieving significant size and stability of the active molecules. The present invention achieves size reduction, entrapment and enhancing the efficacy of functional molecules to potentially control fungal and bacterial diseases.

[0042] Nanocomposites involving a nanopolymer matrix and a nanofiller with specific nanometric size, surface charge and stability are described herein. The nanofillers are bioactive molecules that are entrapped within the polymer matrix as filler through cross-linking and electrostatic interactions. The concentrations of the functional molecules such as chitosan, Bronopol, brassinolides and rhamnolipid in the formulation in ppm are 20000, 10000, 10000 and 5000 respectively.

[0043] In an embodiment, the process of preparing the said formulation comprises:
I. Preparation of nano-polymeric matrix using Poly- (D) Glucosamine and a cross-linker
II. Preparation of nanofiller as aqueous medium using 2-Bromo-2-nitro-1,3-propanediol brassinolides and 2-O-alpha-L-rhamnosyl-alpha-L-rhamnosyl-3-hydroxydecanoyl-3- hydroxy decanoic acid as a stabilizer
III. Impregnation of nanofiller of step (II) in the nano-polymeric matrix of step (I) in a defined ratio.

[0044] In an embodiment, the polymeric nanocomposite formulation has four functional molecules in which chitosan acts both as functional molecule and polymer matrix, bronopol acts as anti-microbial agent, Brassinolides act as growth promotor and rhamnolipid acts as functional molecule and surfactant. Chitosan aids in the control of fungal pathogens and also acts as a bacteriostatic agent, bronopol acts as an excellent anti-microbial agent, brassinolides that is a class of steroid plant hormone plays a role in regulation of numerous developmental processes, including root and shoot growth, vascular differentiation, fertility, flowering, and seed germination and rhamnolipid acts as antifungal agent and as a stabilizer.

[0045] The brassinolides are obtained from the plants itself produced via cycloartenol and cycloartanol dependent pathways. But, more than 17 compounds were characterized as inhibitors of the BR biosynthesis. Thus, providing Brassinolides as an external source can improve the efficiency of the seeds and as the dephosphorylated BZR1 (Brassinolide resistant 1) and BES1 (BRI1-EMS suppressor 1) transcription factors enter the nucleus and function in regulating genes responsible for enhancing plant stress tolerance the seeds coated with formulation containing Brassinolides increases the capacity of antioxidant enzymes and regulating accumulation of endogenous hormones.

[0046] The formulation has been designed for the management of both bacterial and fungal pathogens besides promoting seed germination and growth. The nano ranged size of the particle in the formulation improve its efficiency against target pathogens are high as the plants absorb the formulation effectively

[0047] EXAMPLE 1: METHOD OF PREPARING THE SYNTHESIS OF POLYMERIC NANOCOMPOSITE FORMULATION (1 LITER) OF PRESENT APPLICATION INVOLVES FOLLOWING STEPS.
[0048] Source and Biological Origin of the components used in the nanomeric polymeric formulation: The formulation ingredients such as rhamnolipids and poly-D-glucosamine (chitosan) have been isolated from Pseudomonas sp. and Mucor sp respectively.
i. Preparation of nano polymer matrix (700 ml):
a. Weigh 20gm of (2% for final volume) of Poly- (D) Glucosamine and dissolve it in 0.5% (5 ml in 690 ml of distilled water) of CH3COOH solution using high shear homogenizer at 800 rpm until it is dissolved completely leaving no residues.
b. Then, to the dissolved poly-glucosamine (solution a) 1% (7 ml) of Poly ethylene glycol (1% of polymer matrix – 700 ml) is added that acts as a cross-linker in producing the polymeric matrix
ii. Preparation of nano active fillers (300 ml):
a. Dissolve 1% (of final volume -10gm) of Bronopol in 200 ml of double distilled water at 600 rpm until a clear solution is obtained.
b. Then, to the above solution (i- a.) add 0.5% (of final volume- 5 gm) of 2-O-alpha-L-rhamnosyl-alpha-L-rhamnosyl-3-hydroxydecanoyl-3- hydroxy decanoic acid and dissolve it at 300 rpm for 15 min or until a translucent solution is obtained. Consider it as solution C.
c. Then separately dissolve 1% (of final volume -10gm) of Brassinolides in 100 ml of double distilled water at 600 rpm until homogenous solution is obtained. Consider this as Solution D
d. Now, blend solution C and D to form a homogenous mixture. To this 0.5% (to final volume- 5ml) of glycerol is added that acts a plasticizer for the polymer matrix in order to boost film formation.
iii. Impregnation of nano active fillers prepared from step (ii) in the nano polymer matrix of step (i). This is done with continuous blending at 600 rpm followed by ultrasonication with 40% amplitude at room temperature for 30 min
iv. Processing of polymeric nanocomposite from step iii. using wet-grinding in a nano miller for 45 min at room temperature that allows further size reduction.

[0049] EXAMPLE 2: CHARACTERIZATION OF CURCUMIN AND THE MULTICOMPONENT NANOPARTICLES OF THE PRESENT APPLICATION
Example 2A: Particle size Analysis
Fig.100A represents the Size Distribution of Polymeric Nanocomposite formulation. The particle size of the Nano formulation is measured based on the principle of dynamic light scattering in Nano Particle Size Analyzer and found to be in the size regime of 150-750 nm while the encapsulant cum functional molecule, chitosan alone was measured between 200-300 nm. The poly-dispersion index (PI) of the nano formulation is 0.492 while the PI of chitosan is 0.9.

[0050] Example 2B. FIG. 8: 800 represents the electron microscopy image of the polymeric nanocomposite. The particle size and morphology of the Nanocomposite formulation is confirmed with High resolution transmission electron microscopy that clearly depicts the size distribution of composite nanoparticle between 100-300 nm.

[0051] Example 2C: Surface Charge
Zeta Potential is the potential difference between the dispersion medium and the stationary layer of fluid attached to the particles. It is a characterization technique for estimating the surface charge of the particles and is used for attaining the right surface charge that can ensure physical stability of the nanosuspensions. FIG.1B: 100B represents the Zeta Potential (Stability) of Polymeric Nanocomposite formulation.
The chitosan nanoparticle was stable with a zeta potential of above +60mV. The positive and negative charge of the zeta potential is directly proportional to the surface charge of the sample. The chitosan is positively charged and the same has been confirmed with obtained result. Bronopol is neutrally charged that is encapsulated into the chitosan matrix. The nano formulation has the zeta potential of +60mV and the positive charge obtained indicates that the active ingredient is loaded into the cationic nanocarrier.

[0052] Example 2D: Fourier Transform Infra-Red Spectroscopy (FTIR Spectroscopy)
The main compounds contributing to the activities of nanocomposites are Poly- (D) Glucosamine, 2-Bromo-2-nitro-1,3-propanediol, 1S,2R,4R,5S,7S,11S,12S,15R,16S)-15-[(2S,3R,4R,5S)-3,4-dihydroxy-5,6-dimethylheptan-2-yl]-4,5-dihydroxy-2,16-dimethyl-9-oxatetracyclo octadecan-8-one and 2-O-alpha-L-rhamnosyl-alpha-L-rhamnosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid.
The infrared spectroscopy is measured between 4000 cm-1 to 400 cm-1. Infra-red active molecules have unique IR absorption spectra that may be used as a “fingerprint” for identification. Since each functional group absorbs a specific wavelength no two unique molecules have the same absorption spectra. Thus, this technique is utilized to identify the functional groups. FIG.2A: FTIR spectra of nano formulation. FIG.2B: FTIR spectra of nano Carrier (Nano chitosan). FIG.2C: FTIR spectra of Bronopol. FIG.2D: FTIR spectra of Brassinolides and FIG.2E FTIR spectra of Rhamnolipids.

[0053] The FTIR Spectra (FIG 2A) confirms the presence of all four functional molecules in the formulation. The FTIR spectra of nanocarrier, Bronopol and rhamnolipids of the present application are provided in FIGS 2B AND 2C and 2E. The series of bands between 3000–2800 cm–1 belong to stretching vibrations of the hydrocarbon groups shown in FIG 2D.

[0054] The peak at 2871cm-1 in nanocomposite corresponds to chitosan which serves as nano polymer matrix or carrier and antifungal molecule of the present application represents the C-H Symmetric stretching vibration.

[0055] The bands at 1636 cm-1 observed in the polymeric nano composite of the present application correspond to Residual N-acetyl group in chitosan (– C-N Stretching of amide I)

[0056] The peak at 1559 cm-1 in polymeric nano composite corresponds to Residual N-acetyl group in chitosan (– C-N Stretching of amide II) in the multicomponent nanocomposite of the present application.

[0057] The Residual N-acetyl group in chitosan (– C-N Stretching of amide III) in nanocomposite is at 1376 cm-1 of the present application. The peak, 1042 cm-1 in nanocomposite corresponds to the C-O-C stretching vibrations of rhamnose in 2-O-alpha-L-rhamnosyl-alpha-L-rhamnosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid

[0058] 615-500 cm-1, 1100-1200 cm-1 and 1428 cm-1 correspond to Alkyl halides that are compounds, having a C–X bond, where X is a halogen: bromine, chlorine, fluorene. In general, C–X vibration frequencies appear in the region 850-515 cm-1 while C–Br stretches appear at slightly lower wavenumbers from 690-515 cm-1.

[0059] The formulation has been designed for the management of both bacterial and fungal pathogens besides promoting seed germination and growth. The nano range size of the particle in the formulation improves its efficiency against target pathogens are high as the plants absorb the formulation effectively. The product was tested against Fusarium sp. for bio efficacy at incremental concentrations of 300, 500, 1000 and 2000 ppm compared to the control plate using poison food technique.

[0060] FIGS.3 (A-D) Anti-fungal activity of Polymeric nanocomposite formulation against Fusarium sp. The results clearly indicated that at 1000 and 2000 ppm no growth was observed after 14th day and at 300 and 500 ppm 1.45 and 1.12 cm growth was observed after 5th day where as in control 3.6 cm growth was observed.

[0061] The formulation can be an ideal candidate for the integrated control of wide spectrum of bacterial and fungal pathogens in agriculture by overcoming the issue of resistance against chemical pesticides. As all the ingredients are eco-safe molecules, the formulation does not leave any chemical residues in the produces and the environment and be promoted in sustainable agriculture.

[0062] EXAMPLE 3: Field trials for testing the bio-efficacy of the polymeric nanocomposite formulation as seed priming agent in maize and green gram to overcome seed borne pathogens and to further boost the germination percentage and vigor index.
The FIG.4 shows the graph indicating the germination percentage of maize seedlings (mean value of three replications) treated with polymeric nanocomposite at different dilutions such as 1ml in 100ml (T1) and 2 ml in 100 ml (T2) compared with hydro primed seeds (T3) and absolute control. The result clearly indicates that T1 has recorded higher germination percentage followed by T2 when compared to control. Table 1 represents the vigor index and other growth parameters of treatments as mentioned above. The vigor index of T1 is 4747 whereas it is 3959 and 3554 for T3 and control respectively. This trial clearly proves the efficacy of nano-polymeric composite as seed priming agent. The same study has performed with poor quality green gram seeds and the results are presented in Table 2.

Table1: Growth parameters of maize seedling
Treatment Avg. Shoot Length
(cm) Avg. Root Length (cm) Vigour Index Root Biomass (g) Fresh weight
(g)
T1 18.9 32.74 4747 2.48 70.82
T2 17.8 33.8 4627.5 2.34 73.43
T3 17.5 29.6 3959 1.56 66.36
Control 16.45 29.15 3544 1.3 67.46

Table 2: Growth parameters of Green gram seedling
Treatment Germination Percentage
(%) Average shoot length (cm) Average Root length
(cm) Vigour Index Fresh Weight
(gm) Dry Weight
(gm)
T1 100 16.9 23.2 4010 2.46 0.26
T2 96 14.5 21 3408 2.40 0.17
T3 92 12.5 22.5 3220 2.90 0.18
Control 84 12 17.5 2478 2.30 0.11
[0063] The polymeric nano composite formulation treated seeds recorded higher germination percentage and vigour index when compared to control and the seeds were free from any pathogen infections.

[0064] In an embodiment, a polymeric nano composite formulation comprising Poly-(D) Glucosamine (chitosan) in combination with 2-Bromo-2-nitro-1,3-propanediol brassinolides, 2-O-alpha-L-rhamnosyl-alpha-L-rhamnosyl-3-hydroxydecanoyl-3- hydroxy decanoic acid (rhamnolipid), along with one or more agrochemical excipients.

[0065] In an embodiment, Poly- (D) Glucosamine and a cross-linker is a nano polymer matrix and 2-Bromo-2-nitro-1,3-propanediol brassinolides, 2-O-alpha-L-rhamnosyl-alpha-L-rhamnosyl-3-hydroxydecanoyl-3- hydroxy decanoic acid is aqueous nanofiller medium.

[0066] In an embodiment, the agrochemical excipient is selected from capsule forming agent, emulsifier, dispersing agent, anti-freeze, cross-linker, biocide, stabilizing agent, wetting agent, defoamer, adjuvant, viscosity modifier or combination thereof.

[0067] In an embodiment, Poly-(D) Glucosamine is present at a concentration in the range of 0.5% to 2% (w/v), 2-Bromo-2-nitro-1,3-propanediol brassinolides is present at a concentration in the range of 0.5% to 1% (w/v) and 2-O-alpha-L-rhamnosyl-alpha-L-rhamnosyl-3-hydroxydecanoyl-3- hydroxy decanoic acid is present 0.5%-1% based on total volume of the formulation.

[0068] In an embodiment, the cross-linker is poly ethylene glycol selected from polypropylene glycol, poly ethylene glycol or a combination thereof, in an amount from 0.1% to 1.0% (w/v) based on total volume of the formulation.

[0069] In an embodiment, concentrations of the Poly-(D) Glucosamine, Bronopol, brassinolides and rhamnolipid in the formulation in ppm are 20000, 10000, 10000 and 5000 respectively.

[0070] In an embodiment, the particle size of the formulation is in range between 150-750 nm.

[0071] In an embodiment, a method of preparing the polymeric nano composite formulation, comprising the steps of:
i. Preparation of nano polymer matrix(700ml) :
a. Weighing 20gm of (2% for final volume) of Poly- (D) Glucosamine and dissolving it in 0.5% (5 ml in 690 ml of distilled water) of CH3COOH solution using a high shear homogenizer at 800 rpm until it is dissolved completely leaving no residues to provide dissolved poly-glucosamine (solution A);
b. Adding 1% (7 ml) of poly ethylene glycol (1% of polymer matrix – 700 ml) to the dissolved poly-glucosamine (solution A), wherein the poly ethylene glycol acts as a cross-linker in producing the polymeric matrix;
ii. Preparation of nano active fillers (300 ml):
a. Dissolving 1% (of final volume -10gm) of 2-Bromo-2-nitro-1,3-propanediol (Bronopol), in 200 ml of double distilled water at 600 rpm until a clear solution is obtained;
b. Adding 0.5% (of final volume- 5 gm) of 2-O-alpha-L-rhamnosyl-alpha-L-rhamnosyl-3-hydroxydecanoyl-3- hydroxy decanoic acid to the above solution A and dissolving it at 300 rpm for 15 min or until a translucent solution C is obtained;
c. Separately dissolving 1% (of final volume -10gm) of Brassinolides in 100 ml of double distilled water at 600 rpm until homogenous solution D is obtained;
d. Blending solution C and D to form a homogenous mixture; and
e. Adding 0.5% (to final volume- 5ml) of glycerol to the homogenous mixture, wherein the glycerol acts a plasticizer for the polymer matrix in order to boost film formation;
iii. Impregnating the nano active fillers prepared from step (ii) in the nano polymer matrix of step (i), wherein the impregnation of nano active filler is done with continuous blending at 600 rpm followed by ultra-sonication with 40% amplitude at room temperature for 30 min; and
iv. Processing of polymeric nanocomposite from step iii. using wet-grinding in a nano miller for 45 min at room temperature that allows further size reduction.
[0072] In an embodiment, the room temperature is a temperature ranging from 20°C to 40°C.
[0073] In an embodiment, the particle size ranges from 150-750 nm and concentrations of the chitosan, Bronopol, brassinolides and rhamnolipid in the formulation are 20000 ppm, 10000 ppm, 10000 ppm and 5000 ppm respectively.

[0074] The present disclosure has been described in terms of certain preferred embodiments and illustrations thereof, other embodiments and modifications to preferred embodiments may be possible that are within the principles and spirit of the invention. The above descriptions and figures are therefore to be regarded as illustrative and not restrictive.

[0075] Thus the scope of the present disclosure is defined by the appended claims and includes both combinations and sub combinations of the various features described herein above as well as variations and modifications thereof, which would occur to persons skilled in the art upon reading the foregoing description.