Description:TECHNICAL FIELD OF THE INVENTION

The present disclosure pertains to the agrochemical free composition or formulation. The present disclosure relates to an agrochemical free composition comprising chitosan nano particles derived from fungal chitosan, wherein the fungal chitosan is obtained from fungus cell wall, and additives or agrochemical excipients, and a process of preparation thereof. The agrochemical free composition is useful against different plant pathogens including fungal, hence, can act as antimicrobial composition, fungicidal composition, weedicide composition, etc.

BACKGROUND OF THE INVENTION

The agriculture sector has a significant economic role and serves as a direct asset for many countries. However, the productivity of cash crops such as sugarcane, cotton, rice, tea, coffee, tobacco, cocoa, fruits, citrus fruits, rubber, medicinal, oil-producers, and so on is constantly hampered by environmental degradation, climate change, and diseases caused by various pathogens. Sugarcane (Saccharum officinarum), a well-known cash crop, produces approximately 80% of the sugar. This commercial crop has gained global acceptance due to its enormous biomass potential. Sugarcane has provided numerous commercial benefits over several decades, not only for sucrose production but also for the ethanol, paper, and bio fuel industries. Globally, India accounts for approximately 16.77% of sugarcane production and consumption. The sugar industry, a major player in India's economy, has faced numerous challenges. Initially, sugarcane production appeared to be significantly higher than consumption. However, the productivity of sugarcane is currently declining (by approximately 40%) due to the threat posed by fungal pathogens (such as Fusarium moniliforme, F. graminearum, Colletotrichum falcatum, Sporisoriumscitamineum,etc.). Aside from reducing sugar yield, the disease has a cascading effect on food safety due to the secretion of mycotoxins such as Fusaric acid (FA) by Fusarium spp. Mycotoxins are low-weight metabolites that cause harm known as mycotoxicoses in livestock, domestic animals, and humans, and thus have public health significance. The pathogens' exudation of these secondary metabolites disrupts the entire ecological balance of the food chain.
To combat pathogens, farmers use a wide range of agrochemicals. Massive population growth necessitates an increase in food supply, which is met through the use of harmful agrochemicals. However, overuse of agrochemicals or chemicals poses significant risks because they pollute the air and water and emit greenhouse gases. As a result, this scenario generates a wide range of environmental and public health risks. These agrochemicals contain harmful chemicals that can be detrimental to the environment, humans, and long-term plant health. Besides, their limited application over the surface, these fungicides can lead to pathogen resistance over time. Lastly, because of the requirement of frequent applications, the entire plant health management becomes costly too.
Thus, there is a need to make the alternative composition or formulation which avoids agrochemicals, and still provide protection to plants from different pathogens specifically to said cash crop plants with better or at par anti-pathogenic effects.
The present disclosure satisfies the existing needs, as well as others, and generally overcomes the deficiencies found in the prior and general art.

OBJECTIVES OF THE INVENTION

It is an objective of the present disclosure to provide an agrochemical composition comprising fungal chitosan nanoparticles, wherein the fungal chitosan is obtained from fungus cell wall.
It is another objective of the present disclosure to provide a process of preparation of said agrochemical free composition.
It is yet another objective of the present disclosure to provide a composition which can act as antimicrobial composition, fungicidal composition, weedicide composition, etc.

SUMMARYOF THE INVENTION

The present disclosure provides a new and improved composition which can act as antimicrobial composition, fungicidal composition, weedicide composition, etc., and that to without the presence of any chemical or agrochemical.
In an aspect, the present invention relates to an agrochemical free composition comprising:
a) fungal chitosan nanoparticles, wherein the fungal chitosan is obtained from fungus cell wall;
b) additives or excipients; and
c) a solvent.
In an embodiment, the additives or excipients comprises at least one or more non active materials or components selected from but not limited to biosurfactant, non-ionic surfactant, ionic surfactant, amphiphilic surfactant, humectant, hygroscopic agent, and solvent or any of combinations thereof.
In another aspect, the present invention relates to a process of preparation of said agrochemical free composition, comprising:
i. mixing fungal chitosan nanoparticles obtained from fungus cell wall in a solvent followed by sonicating the mixture at temperature in the range of 25 to 35 ℃ for a time period in the range of 25 to 50 minutes to obtain a solution of fungal chitosan nanoparticles; and
ii. adding said additives or excipients to said solution of step i) to obtain said agrochemical free composition.
Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

FIG. 1showscontact angle (º) data of the agrochemical free composition on to a hydrophobic leaf surface of Nelumbo nucifera, where (A) is leaf surface of Nelumbo nucifera, (B) is glass slide covered with leaf, (C) isc ontrol - no surface contact without nano-formulation, and (D) is test-improved surface contact by said nano-formulation (D).
FIG. 2 shows contact angle (º) data of the agrochemical free composition on toa hydrophilic leaf surface of sugarcane i.e., Saccharum officinarum L., where (A) isleaf surface of Saccharum officinarum L, (B) is glass slide covered with leaf, (C) is control - no surface contact without nano-formulation, and (D) is test-improved surface contact by said nano-formulation (D).
FIG. 3 shows comparative antifungal activity data of the agrochemical free composition against Fusarium moniliforme ITCC 191. It covers poisoned food technique depicting the antifungal potential of FCHNPs-based nano-formulation preparations (N1 to N11) by measuring the diameter (mm) of the spread of the fungus from the mycelial pit placed at the centre of the plate (black arrow insert).

DETAILED DESCRIPTION OF THE INVENTION

The following is a detailed description of embodiments of the present disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
Unless the context requires otherwise, throughout the specification, which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, process conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.

The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

All methods described herein can be performed in suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

Various terms are used herein. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.

The term “agrochemical free” used herein means that there is no use of agrochemical or chemical agents normally used in agro industry for crop protection, e.g. Benomyl, flutolanil, fenfuran, pyrimethanil, azoxystrobin, tridemorph, epoxiconazole, etc.

The terms “composition” “agrochemical free composition”, “formulation” and “nano formulation” used herein are used interchangeably with the same meaning or relatively same meaning.

In an embodiment, the present invention relates to an agrochemical free composition comprising:
a) fungal chitosan nanoparticles, wherein the fungal chitosan is obtained from fungus cell wall;
b) additives or excipients; and
c) a solvent.
In specific embodiment, the agrochemical free composition comprising:
a) fungal chitosan nanoparticles, wherein the fungal chitosan is obtained from fungus cell wall;
b) glycolipid biosurfactant;
c) non-ionic surfactant; and
d) humectant or hygroscopic agent.
In another embodiment, the fungus from which the fungal chitosan obtained is Cunninghamellaechinulata NCIM 691.

In another embodiment, the additives or excipients comprises at least one or more non active materials or components selected from but not limited to biosurfactant, non-ionic surfactant, ionic surfactant, amphiphilic surfactant, humectant, hygroscopic agent, and solvent or any of combinations thereof.

In another embodiment, the additives or excipients comprises three non-active materials selected from biosurfactant, non-ionic surfactant, and humectant or hygroscopic agent.

In another embodiment, the biosurfactant is selected from but not limited to glycolipid, lipopeptide, and high molecular weight bio-emulsifier, or any of combination thereof.

In another embodiment, the glycolipid is selected from but not limited to rhamnolipids, trehalolipids, sophorolipids, and mannosylerythritol, or any of combination thereof.
In another embodiment, the lipopeptideis selected from but not limited tosurfactin, iturin, lichenysin, and arthrofactin, or any of combination thereof.

In another embodiment, the high molecular weight bio-emulsifieris selected from but not limited to Emulsan, Alasan and mannoprotein, or any of combination thereof.

In another embodiment, the non-ionic surfactant is selected from but not limited to Tween, Span, crown ether, glucosyl dialkyl ether, polyglycerol alkyl ether, ester-linked surfactant, Brij, and polyoxyethylene alkyl ethers, or any of combination thereof.

In another embodiment, the Tween is selected from but not limited to Tween 80, Tween 20, Tween 40, and Tween 60 or any of combination thereof.
Here, the Tween 20 covers a polyoxyethylene sorbitan monolaurate; the Tween 40 covers apolyoxyethylene sorbitan monopalmitate; the Tween 60 covers apolyoxyethylene sorbitan monostearate; and the Tween 80 covers apolyoxyethylene sorbitan monooleate that's often used in biochemical applications. This can act as solubilizing agent, emulsifying agent and/or dispersing agent.

In another embodiment, the humectant or hygroscopic agent is selected from but not limited to polyethylene glycol, glycerin, urea, hyaluronic acid, salicylic acid, alpha hydroxy acids (AHAs)such as glycolic acid or lactic acid, honey and sorbitol or any of combination thereof.

In another embodiment, the solvent is selected from but not limited to distilled water and demineralized water.

In another embodiment, the weight % of fungal chitosan nanoparticles in said composition is in the range of 0.005 to0.2 %.

In specific embodiment, the weight % of fungal chitosan nanoparticles in said composition is 0.005, 0.006, 0.007, 0.0075, 0.008, 0.009, 0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.125, 0.15, 0.175 or 0.2%.

In another embodiment, the weight % of additives or excipients in said composition is in the range of 0.5 to 2 %.

In another embodiment, the weight % of glycolipid in said composition is in the range of 0.005 to 0.2 %.

In specific embodiment, the weight % of glycolipidin said composition is 0.005, 0.006, 0.007, 0.0075, 0.008, 0.009, 0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.125, 0.15, 0.175 or 0.2%.
In another embodiment, the weight % of non-ionic surfactant in said composition is in the range of 0.05 to 0.2 %.

In specific embodiment, the weight % of non-ionic surfactant in said composition is 0.05, 0.075, 0.1, 0.125, 0.15, 0.175 or 0.2 %.

In another embodiment, the weight % of humectant or hygroscopic agent in said composition is in the range of 0.5 to 2 % or rest of the weight % of the excipients or additives apart from glycolipid and non-ionic surfactant.

In specific embodiment, the weight % of humectant or hygroscopic agent in said composition is 0.5, 0.75, 1, 1.25, 1.5, 1.75 or 2 %.

In another embodiment, the weight % of solvent in said composition is in the range of 97-99 % or rest of the weight % of the composition apart from weight percentages of said fungal chitosan nanoparticles and said additives or excipients.
In another embodiment, the composition comprises 1 part of the fungal chitosan nanoparticles, 1 part of glycolipid biosurfactant, 10 parts of non-ionic surfactant and 100 parts of humectant or hygroscopic agent of the total active components of the composition. Here, the total components are fungal chitosan nanoparticles, and additives or excipients, wherein the additives or excipients compriseglycolipid biosurfactant, non-ionic surfactant, and humectant or hygroscopic agent.

In another embodiment, the surface tension of said composition is in the range of 25 to 28mN/m.

In another embodiment, the fungal chitosan nanoparticles (FCHNPs) are in a spherical shape according to Scanning Electron Microscopy (SEM) analysis.

In another embodiment, the fungal chitosan nanoparticles (FCHNPs) are homogenous in nature with a particle size distribution in the range of 134.11 to 154.53 nm.
Specifically, the particle size distribution of said fungal chitosan nanoparticles (FCHNPs) is about 134.13, 144.33 or 154.53.

In another embodiment, the fungal chitosan nanoparticles (FCHNPs) show optical properties with higher intensity absorption peaks at 300 nm.

In another embodiment, the zeta potential for FCHNPs showed the cationic nature with 45.6 mV >24.5 mV.

The agrochemical free composition disclosed herein is useful in controlling fungus growth in crop plants hence acting as protecting agent or antimicrobial agent.

In another embodiment, the present invention relates to a process of preparation of said agrochemical free composition, comprising:
i. mixing fungal chitosan nanoparticles obtained from fungus cell wall in a solvent followed by sonicating the mixture at temperature in the range of 25 to 35 ℃ for a time period in the range of 25 to 50 minutes to obtain a solution of fungal chitosan nanoparticles; and
ii. adding said additives or excipients to said solution of step i) and optionally adding solvent to obtain said agrochemical free composition.

In another specific embodiment, the present invention relates to a process of preparation of said agrochemical free composition, comprising:
i. mixing fungal chitosan nanoparticles obtained from fungus cell wall in a solvent followed by sonicating the mixture at temperature in the range of 25 to 35 ℃ for a time period in the range of 25 to 50 minutes to obtain a solution of fungal chitosan nanoparticles; and
ii. mixing and dissolving said glycolipid to said solution of step i) followed by adding non-ionic surfactant and humectant or hygroscopic agent, and optionally adding solvent to obtain said agrochemical free composition.

In another embodiment, the solvent is selected from but not limited to distilled water and demineralized water.

In specific embodiment, the surface tension of said composition is in the range of 26.10mN/m.

Generally, in the literature, the use of chemical methods is widespread that it is hard to overlook the harm they cause to living systems. The introduction of disease-resistant cultivars and other biocontrol methods has provided protection for sugarcane or any other crops to some extent. Recent advances in the area of agriculture biotechnology are obliging to move on the roller coaster to minimize the economic losses. The present nano-formulation or composition possessing multifunctional molecules is appreciable since it has exceptional antimicrobial activity against targeted pathogens and spreads efficiently all over the surface (refer, contact angle data provided in figures 1and 2). Combinatorial studies of all said components in the composition evidenced the promising activity of foliar application. Analysis of various biochemical parameters and enzymes associated with innate immune response in the treated sugarcane plants were also supportive to prove the efficiency and the positive impact of the nanoformulation on sugarcane plants. This eco-friendly nano-formulations at defined concentration is noteworthy to defeat phytopathogens of sugarcane.

While the foregoing description discloses various embodiments of the disclosure, other and further embodiments of the invention may be devised without departing from the basic scope of the disclosure. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.

EXAMPLES

The present disclosure is further explained in the form of following examples. However, it is to be understood that the foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.

Procurement Details:

The fungal culture Cunninghamellaechinulata NCIM 691 is procured from National Collection of Industrial Microorganism (NCIM), Pune, MH, India.The Rhamnolipid biosurfactant is procured from AGAE Technologies, United States(SET Lab India,Pune, MH, India).The Poly Ethylene Glycol (PEG) and Tween 80 both are HPLC analytical grade procured from Sharad agencies, anil kunj, near gas godown, walvekarnagar, pune:- 411009, India.

Example 1: Preparation of Fungal Chitosan nanoparticles:

A: Growth and maintenance of the fungal culture:

Cunninghamellaechinulata NCIM 691 was revived by scraping fungal growth from PDA plate and growing in PDB for 6 days. The spore suspension (1.0 ×108 spores/ml) was prepared by gently scraping the culture from the PDA plate in phosphate buffer saline (PBS – pH 7.0). The spore suspension was inoculated into 250 ml Erlenmeyer flasks with 100 ml of culture medium at 29±1℃ for 16 hours on a rotary shaker (100 rpm). Further, 7.5% (v/v) of the inoculum was aseptically transferred to the 500 ml Erlenmeyer flasks containing 100 ml of fermentation broth. Flasks were incubated at 29±1℃ under 100 rpm for 6 days.

B: Extraction, purification of fungal chitosan (FCH) andsynthesis of fungal chitosan nanoparticles (FCHNPs):

The FCH was extracted and purified successfully by following an alkali insoluble method (Pochanavanich, P. and Suntornsuk, W. (2002) Fungal Chitosan Production and Its Characterization. Letters in Applied Microbiology, 35, 17-21. http://dx.doi.org/10.1046/j.1472-765X.2002.01118.x) and was preserved at 4℃ and used further for synthesis of NPs. This part has been published previously (Karamchandani et al., Front. Bioeng. Biotechnol, Sec. Industrial Biotechnology, Volume 10, 09 August 2022,| https://doi.org/10.3389/fbioe.2022.917105). Ionotropic gelation method was followed for synthesis of fungal chitosan nanoparticles (FCHNPs) using FCH extracted from C. echinulata. Successful synthesis of FCHNPs followed has been reported in the publication (Karamchandani et al. 2022). The ionic gelation protocol described by Carvalho et al. 2019 (Synthesis and characterization of TPP/chitosan nanoparticles: Colloidal mechanism of reaction and antifungal effect on C. albicans biofilm formation. Mater. Sci. Eng., 104, 109885, 2019) was employed to synthesize FCHNPs. Briefly, the FCH was dissolved in acetic acid (1% v/v) and pH was adjusted to acidic condition (5.0). The solution of sodium tripolyphosphate (TPP) was prepared in distilled water (0.1% w/v). About 5 ml of TPP solution was added to 15 ml of the FCH preparation resulting in a ratio of 3:1 (FCH:TPP). The TPP solution was filtered through a 0.2 µm membrane and was then added to FCH solution dropwise followed by stirring at 700-800 rpm (room temperature) to achieve a clear solution. The mixture was allowed to dissolve completely for 1 hour under previously discussed stirring conditions. After TPP addition, the solution was left under the stirring process for another 30 min. The positively charged amino acid group of FCH interacts with the negative charge of TPP resulting in an ionotropic gelation reaction. The ionic gelation method of nano-FCH synthesis was initiated with meshwork formation upon TPP addition and enabled the formation of FCHNPs. Afterward, the solution was centrifuged at 10,000 rpm for 20 min. The FCHNPs were obtained in the form of precipitate and subsequently washed (aqueous) twice to remove the un reacted substances. The purified FCHNPs were then freeze-dried and stored at 4°C until further use. A similar protocol was used to synthesize CHNPs from commercial CH (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). The physico-chemical characterization along with other detailed analysis of FCH and FCHNPs has already been publishedin Karamchandani et al. 2022 and hereby referred fully for any support or other details reference. Some of them are provided below for ready reference:

Scanning Electron Microscopy (SEM) analysis showed the spherical shape of the FCHNPs. The Nanoparticle Tracking Analysis (NTA) suggested the superior homogenous nature of FCHNPs with a particle size distribution 144.33 ± 10.20 nm.

The UV-Visible Spectroscopy technique displayed optical properties of FCHNPs with higher intensity absorption peaks at 300 nm. The zeta potential measurements for FCHNPs showed the cationic nature (45.6 mV > 24.5 mV). Fourier Transform Infra-Red Spectroscopy (FTIR) successfully characterized functional groups of FCHNPs.

Example 2: General procedure for the preparation of agrochemical free composition

The said composition is prepared by adding fungal chitosan nanoparticles synthesize using chitosan extracted from fungus from Cunninghamellaechinulata NCIM 691. The nanoparticles were dissolved in distilled water and homogenized using sonicator machine for 30 minutes (Department of Microbiology, SPPU). The mixing was carried out in Falcon tubes using micropipettes at room temperature with no further purification steps. Further Rhamnolipid biosurfactant was added simultaneously. The additives Polyethylene glycol and Tween 80 were finally added using micropipettes to achieve required concentration of the formulation for biological assays.
The composition containing fungal chitosan nanoparticles, rhamnolipid as glycolipid, Tween 80 as non-ionic surfactant, and polyethylene glycol (PEG) as humectant or hygroscopic agent is prepared according to above general procedure, and tested further. Details of the same are given below:

Table 1: Composition of Formulation A

Components Concentration μg/ml In percentage
Fungal chitosan nanoparticles synthesized usingchitosan extracted from Cunninghamellaechinulata NCIM 691 100
0.010%
Rhamnolipid biosurfactant 100 0.010%
Poly ethylene glycol 10 1%
Tween 80 1 0.1%
Distilled water To make up 98.88%
Total 100%

Table 2: Composition of Formulation B

Components Concentration μg/ml In percentage
Fungal chitosan nanoparticles synthesized using chitosan extracted from Cunninghamellaechinulata NCIM 691 200
0.020 %
Rhamnolipid biosurfactant 200 0.020%
Poly ethylene glycol 10 1%
Tween 80 1 0.1%
Distilled water To make up 98.86%
Total 100

Measurement of Contact Angle of Formulation A:

The measurement of contact angle (º) on hydrophobic leaf surface - Nelumbo nuciferawas performed in the lab. The following results of table 3 explained the efficacy of nanoformulation. The images obtained are shown in Figure 1.

Table 3
S.NO COMPOUND CONTACT ANGLE
A Water on Lotus (Nelumbo nucifera) 135.4⁰
B Nanoformulation on Lotus surface 38.8⁰

Similarly, the measurement of contact angle (º) on hydrophobic leaf surface S. officinarum was performed in the lab. The following results in table 4 explained the efficacy of nanoformulation. The images obtained are shown in Figure 2.

Table 4
S.NO COMPOUND CONTACT ANGLE
A Water on Sugarcane (Saccharum officinarum) leaf 73.4⁰
B Nanoformulation on Sugarcane leaf 11.4⁰

Example 3: Antimicrobial testing of prepared formulation B:

A: FCHNPs-based foliar nano-formulation with additives:

CunnighamellaechinulataNCIM 691 originated Chitosan was utilized for synthesis of nanoparticles. The multifunctional chitosan nanoparticles from fungus were further explored for designing foliar nano-formulation against fungal pathogens associated with sugarcane plants. The nano-formulation was incorporated with few other surface active and wetting or spreading agents. A well-known biosurfactant namely Rhamnolipid was incorporated as one of the additives in the preparation. In addition to these two components the formulation was also incorporated with polyethylene glycol (PEG, HPLC grade) and Tween 80 (HPLC grade). PEG is a non-ionic polyether which has been used widely due to its exceptional properties like solvent, plasticizer, lubricant conferring homogeneity to the solutions. Additives were incorporated to improve the spreading abilities of foliar spray. Tween 80, an amphiphilic, chemical surfactant that reduces surface tension (ST) and interfacial tension (IFT) effectively thereby possesses substantial spreading abilities over different surfaces. The formulation was tested in vitro by poisoned food technique as described below-

B: Antifungal potential of nano-formulation by poisoned food technique

Poisoned food assay was performed to evaluate the antifungal efficiency of the bio-vaccine my conanosprayagainst Fusariummoniliforme ITCC 191. Around 200 μg/ml amount of each component (refer, table 5) wasadded in Potato dextrose agar medium before being individually placed in a polystyrene sterile petri dish (90 × 15 mm, HiMedia, Mumbai, India). The fungal mycelial bit of uniform size (diameter, 5.0 mm) was taken from seven-day-old culture and placed at the center of petri dishes impregnated with the test compounds individually. After that, these petri plates were incubated for seven days at 29 ± 1ºC to observe the growth of radial mycelial cells. Further, the percentage inhibition rate of the mycelia for the corresponding pathogen was determined. The inoculated plates (test) were compared with positive (commercial fungicide Carbendazim, CBZ) and negative control (without test compounds).

Where, Mc is the mycelial growth in the control plate, Mt is the mycelial growth treated with the test compounds.

Table 5: Assessment of antimicrobial effect of various components of bio vaccine-myconanosprayby poisoned food technique.

Treat. code Compound Conc. (%)
N1 Control DW
N2 Carbendazim 0.005
N3 FCHNPs 0.010
N4 Rhamnolipid 0.010
N5 Tween 80 0.10
N6 FCHNPs +Tween 80 0.020 + 0.10
N7 Rhamnolipid+ Tween 80 0.010 + 0.10
N8 FCHNPs + BS Rhamnolipid 0.010 +0.010
N9 FCHNPs + BS Rhamnolipid +Tween 80 0.020 +0.010 + 0.10
N10 FCHNPs + BS Rhamnolipid+ Poly Ethylene Glycol + Tween 80 0.010 +0.010 + 1 + 0.1
N11 FCHNPs + BS Rhamnolipid+ Poly Ethylene Glycol + Tween 80 0.020+0.020 + 1+0.1

Results-
Inhibition in growth of Fusarium spp. by nanoformulation

In-vitro poisoned food technique enabled to assess the effect of each component individually and in combination againstF. moniliforme ITCC 191.The antifungal activity of the test compounds was manifested from the reduced mycelial mat in poisoned plates as compared to control plates (refer, FIG. 3). The control plates (absence of the test compounds) denoted luxuriant growth of all fungi on PDA. Antifungal potential of different preparations (codes: N1 to N11) of nano-formulation are represented in FIG. 3. This data and FIG. 3 proves that the formulation or composition containing FCHNPs with additives or excipients are inhibiting or killing the fungal growth specifically refer, N6, N8, N9 and N10 formulations with maximum inhibition % of around 92.6±0.53%.

ADVANTAGES OF THE INVENTION:

• Avoids use of chemicals or agrochemicals in the composition for crop protection against fungus and other pathogens.
• Simple and economic.
• Provides up to 93% inhibition of fungus growth which is confirmed by in-vitro testing.
• It is best alternative to chemical based agro formulations which are not safe, environment friendly and avoids resistance related issues. , Claims:We Claim:

1. An agrochemical free composition comprising:
a) fungal chitosan nanoparticles, wherein the fungal chitosan is obtained from fungus cell wall;
b) additives or excipients; and
c) a solvent.

2. The composition as claimed in claim 1, wherein the fungus from which the fungal chitosan obtained is Cunninghamellaechinulata NCIM 691.

3. The composition as claimed in claim 1, wherein the additives or excipients comprises at least one or more non active materials or components selected from biosurfactant, non-ionic surfactant, ionic surfactant, amphiphilic surfactant, humectant, hygroscopic agent, and solvent or any of combinations thereof.

4. The composition as claimed in claim 1, wherein the additives or excipients comprises three non-active materials selected from biosurfactant, non-ionic surfactant, and humectant or hygroscopic agent.

5. The composition as claimed in claim 4, whereinthe biosurfactant is selected from glycolipid, lipopeptide, and high molecular weight bio-emulsifier, or any of combination thereof.

6. The composition as claimed in claim 5, whereinthe glycolipid is selected rhamnolipids, trehalolipids, sophorolipids, and mannosylerythritol, or any of combination thereof; wherein the lipopeptide is selected from surfactin, iturin, lichenysin, and arthrofactin, or any of combination thereof; and wherein the high molecular weight bio-emulsifier is selected from Emulsan, Alasan and mannoprotein, or any of combination thereof.

7. The composition as claimed in claim 4, wherein the non-ionic surfactant is selected from Tween, Span, crown ether, glucosyl dialkyl ether, polyglycerol alkyl ether, ester-linked surfactant, Brij, and polyoxyethylene alkyl ethers, or any of combination thereof.

8. The composition as claimed in claim 7,the Tween is selected from Tween 80, Tween 20, Tween 40, and Tween 60 or any of combination thereof.

9. The composition as claimed in claim 1, whereinthe humectant or hygroscopic agent is selected from polyethylene glycol, glycerin, urea, hyaluronic acid, salicylic acid, alpha hydroxy acids (AHAs) selected from glycolic acid or lactic acid, honey and sorbitol or any of combination thereof.

10. The composition as claimed in claim 1, wherein weight % of the fungal chitosan nanoparticles in said composition is in the range of 0.005 to 0.2 %.

11. The composition as claimed in claim 1, wherein weight % of the additives or excipients in said composition is in the range of 0.5 to 2 %; and weight % of the solvent in said composition is in the range of 97-99 % or rest of the weight % of the composition to make it 100%.

12. The composition as claimed in claim 1,wherein the agrochemical free composition preferably comprises:
a. fungal chitosan nanoparticles, wherein the fungal chitosan is obtained from fungus cell wall;
b. glycolipid biosurfactant;
c. non-ionic surfactant;
d. humectant or hygroscopic agent; and
e. solvent.

13. The composition as claimed in claim 5 or 12, wherein weight % of glycolipid in said composition is in the range of 0.005 to 0.2 %.

14. The composition as claimed in claim 4 or 12,wherein weight % of non-ionic surfactant in said composition is in the range of 0.05 to 0.2 %.

15. The composition as claimed in claim 4 or 12,wherein the weight % of humectant or hygroscopic agent in said composition is in the range of 0.5 to 2 %.

16. A process for the preparation of the agrochemical free composition as claimed in claim 1, comprising steps of:
a. mixing fungal chitosan nanoparticles obtained from fungus cell wall in a solvent followed by sonicating the mixture at temperature in the range of 25 to 35 ℃ for a time period in the range of 25 to 50 minutes to obtain a solution of fungal chitosan nanoparticles; and
b. adding said additives or excipients to said solution of step i) and optionally adding solvent to obtain said agrochemical free composition.

17. The process as claimed in claim 16, wherein said solvent is distilled water or demineralized water.