WO2018042311A1 - Chitosan derivative formulations for plant growth, and building disease resistance - Google Patents

Abstract

The invention relates to chitosan derivative comprising pre-activated chitosan and chemical moieties. The invention also relates to method for preparing the chitosan derivative. The method further relates to the formulation comprising pre-activated chitosan and biomolecules for improved disease resistance, control the plant pathogens, protect the plants from infections and growth of plants.

Description

C H IT OSA N DE RIVAT IV E FOR M U LAT IO NS FOR PLA NT G R OWT H . A ND BUIL DING DISEASE R E SISTA NC E

T echnical F ield of the Invention

T he invention relates to chitosan derivative comprising pre-activated chitosan and chemical moieties. The invention also relates to method for preparing the chitosan derivative. The method further relates to the formulation comprising pre-activated chitosan and biomolecules for improved disease resistance, control the plant pathogens, protect the plants from infections and growth of plants The efficacy of in-vitro control is illustrated for plant pathogenic bacterial strain Xanthomonas causing the leaf blight of Pomegranate; plant pathogenic fungal strai ns Alternaria alternanthera, Cylindrocarpon, F usari um oxysporum, animal pathogenic yeast strain Candida albicans and free living nematode Panagrellus redivivus as examples of the disease prevention due to the use of product.

Background of the Invention

Antimicrobial activity of chitosan has been reported against various bacteria yeasts and molds such as Aeromonas, Bacillus, Bifidobacterium, brochothrix, Clostridium, E nterococcus, Escerechia, Lactobacillus, Leuconostoc, Listeria, Micrococcus, Pediococcus, Photobacterium, Pseudomonas, Salmonella, Shigella, Staphylococcus, Vibrio, Candida, Cryptococcus, Saccharomyces, Schizosaccaromyces, Zygosaccharomyces, Aspergi llus, Botrytis, Cladosporium. Penicilliurn Rhizopus, etc. and has been reviewed [J Food Sci 72 (2007) R 87 ^" R100].

US patent no. 5,374,627 describes method of control of plant diseases and damage by certain pests i n agricultural plants by compositions containi ng 1 part by weight of a chitosan hydrolyzate with an average molecular weight of 10,000 to 50,000, obtai ned by acid hydrolysis or enzymatic hydrolysis of chitosan.

T he patent application WO 03070008 discloses antimicrobial compounds and their methods of application wherein the product for use is a liquid obtained by using organic acids of 3 30 carbons and the claimed product is useful for application only under acidic pH conditions.

Although various chemical modifications of chitosan are known i n the literature that lead to solublization of chitosan at neutral to alkaline pH, the applications of chitosan are only using solution of chitosan i n organic acid preferably acetic acid. T he solution thus obtai ned is of low pH (2.0 ^" 4.0) depending on the concentration of the acid used. Low pH of the formulation can^'t be accepted as it can cause scorching on plant leaves. Further, the net positive charge developed on the chitosan molecule because of the low pH is responsible for interaction with anionic components from the product composition [such as inorganic salts, amino acids, fatty acids], in turn making the product formulation practically insoluble in water. All these undesired reactions can be avoided by making the formulation as described in the present i nvention.

A class of water soluble chitosan as described in the literature is of complexes of chitosan with respective organic acids which are typically described as chitosan acetate, chitosan lactate etc. These are dry powders of chitosan acid complex, derived by simple mixing followed by removal of un-reacted acid and drying. Such complexes are more unstable, and still could have the problems associated with low pH like that of the chitosan solution.

T herefore, there is a need to provide a composition which can that can overcome the problems listed above and boost the plant performance.

Obj ects of the invention

It is an object of the i nvention to overcome the drawbacks of the prior art.

It is another object of the invention to provide chitosan derivatives comprising pre- activated chitosan and chemical moieties.

It is another object of the invention to provide a composition comprising the chitosan derivatives which can promote the plant performance.

It is yet another object of the invention to provide a composition comprising chitosan derivatives which can improve microbial (pathogen as well as endophyte) population, bi otic factors such as leaf surface area, photosynthesis capacity, root volume, thickness of the sell wall; and abiotic factors such as improving bio-availability of the nutrients, rate of nutrient uptake.

It is yet another object of the invention to provide a formulation comprising chitosan derivatives and one or more biologically active substances.

It is yet another object of the invention to provide a method for formulating and stabilizing the chitosan derivatives in liquid formulations.

It is yet another object of the invention to provide an improved formulation which is stable at pH ranging from pH 7 to pH 12.

It is yet another object of the invention to provide anti- microbial activity comprising chitosan derivative.

It i s yet another obj ect of the i nventi on to provi de anti -fungal activity compri si ng chi tosan derivative.

It is still an object of the i nvention to provide a composition comprising chitosan derivative for prevention of diseases of crops along with the anti-microbial activity that can prevent i nvasiveness of the pathogen and bui Id defense of the plant.

Summary of the invention

According to one aspect of the present invention, there is provided a chitosan derivative comprising pre-activated chitosan wherein amine group is partial ly substituted, and chemical moieties.

According to another aspect of the present invention, there is provided a method of obtai ning the pre-activated chitosan comprising the steps of:

i. Pre activating reaction comprising chitosan and hydrogen peroxide in a ratio of 1 :1 to 10:1 to obtain partially substituted chitosan at a temperature rangi ng from 18 to 30 degree Celsi us;

ii. Adding Hydrogen peroxide and FeCI3;

iii. Adding water to uniformly mix the reagents obtained in step ii, and adding Chitosan powder (DA value 78%) to water with constant sti rri ng; iv. Dissolving acid crystals in de-ionized water, v. Adding the solution obtained in step iv to the chitosan with constant sti rri ng; and

vi. Termi nating the reaction of step v at time intervals ranging between 1 hour to 24 hours by precipitating the reaction mixture by adding solvents with constant stirring and obtaining pre-activated chitosan.

According to another aspect of the present invention, there is provided a method a method for preparing the chitosan derivative of claim 1 comprising steps of

Step I-obtaining the pre-activated chitosan

i. Pre activating reaction comprising chitosan and hydrogen peroxide in a ratio of 1 :1 to 10:1 to obtain partially substituted chitosan at a temperature rangi ng from 18 to 30 degree Celsi us;

ii. Adding Hydrogen peroxide and FeCI3;

iii. Adding water to uniformly mix the reagents obtained in step ii, and adding Chitosan powder (DA value 78%) to water with constant sti rri ng;

iv. Dissolving acid crystals in de-ionized water,

v. Adding the solution obtained in step iv to the chitosan with constant sti rri ng; and

vi. Termi nating the reaction of step v at time intervals ranging between 1 hour to 24 hours by precipitating the reaction mixture by adding solvents with constant stirring and obtaining pre-activated chitosan.

Step II- reacting pre-activated chitosan with chemical moieties i. Reacting the pre-activated chitosan obtained in step a with chemical moieties to form a solution at a temperature ranging from 20 to 95, degree C, pH 2 ^" 5.5 and Time 3 ^" 10 hrs. and i i. Obtai ni ng chitosan derivatives.

According to another aspect of the present invention, there is provided a formulation for disease resistance and growth of plants comprising chitosan derivatives and one or more biologically active substances.

According to yet another aspect of the present invention, there is provided use of the chitosan derivatives as defined in claim 1 for an improved rate of germination.

According to yet another aspect of the present invention, there is provided a use of chitosan derivatives as defined in claim 1 for an improved growth of leaf surface area. According to yet another aspect of the present invention, there is provided a use of chitosan derivatives for an i ncrease i n the chl orophyl I content.

According to yet another aspect of the present invention, there is provided a use of chitosan derivatives for callose bio- synthesis.

According to yet another aspect of the present invention, there is provided a use of chitosan derivatives for inhibition of microbial growth.

According to yet another aspect of the present invention, there is provided a use of chitosan derivatives as defined in claim 1 for inhibition of microbicide activity.

According to yet another aspect of the present invention, there is provided an anti -antimicrobial formulation comprising chitosan derivative and one or more biomolecules. According to yet another aspect of the present invention, there is provided an anti-fungal formulation comprising chitosan derivative and one or more biomolecules.

According to yet another aspect of the present invention, there is provided an anti -viral formulation comprising chitosan derivative and one or more biomolecules.

According to yet another aspect of the present invention, there is provided an improved composition comprising chitosan derivative and one or more biomolecules for degradation of toxic pesticide. According to yet another aspect of the present invention, there is provided an improved composition comprising chitosan derivative and one or more biomolecules having shelf life more than 2 years.

According to yet another aspect of the present invention, there is provided an in-vitro method for callose i nduction i n plants comprising the steps of treating the plant tissues with formulation comprising chitosan derivative and one or more biomolecules.

According to yet another aspect of the present invention, there is provided a method of disease resistance and growth of plants comprising contacting plants with formulation comprising chitosan derivative and one or more biomolecules.

Brief Description of the Accompanying Drawings

Figure 1a ill ustrates FTIR report of chitosan

Figure 1 b-1 e illustrates FTIR report of chitosan derivatives at different time intervals of 1 hr, 2 hrs, 7 hrs, 24 hrs respectively.

Detailed Description of the Accompanying Drawings

Figure 1a ill ustrates FTIR report of unmodified chitosan

Figure 1 b-1 e illustrates FTIR report of chitosan derivatives at different time intervals of 1 hr, 2 hrs, 7 hrs, 24 hrs respectively.

Source of biological materials:

Chitosan (commercial grade) was purchased from Mahtani Chitosan, India Sea Food, or Marine Chemicals. E nzymatically processed protein hydrolysate was procured from Food BioTech, Mumbai. Y east Extract Sugars, Inorganic and organic chemicals and solvents were all of commercial grade (95% purity) obtained from the local traders. Microbial cultures were procured from National Collection of Industrial Micro-organisms, NC L, Pune or MTCC Chandigarh and were grown and processed as described. Culture of X anthomonas was isolated from the infected plant parts of pomegranate. Dried herbs were procured from the I ocal suppl i ers and the extracts were prepared as descri bed. Detailed Description of the Invention

Not getting bound to this or any other theory the formulations/composition described in this invention promote the plant performance by causing positive effects on more than one factor such as microbial (pathogen as well as endophyte) population, biotic factors such as leaf surface area, photosynthesis capacity, root vol ume, thickness of the sell wall; and abiotic factors such as improving bio-availability of the nutrients, rate of nutrient uptake etc.

It is known in the literature that chitosan reacts with Hydrogen peroxide in presence of various cations. The reaction leads to de-acetylation, as well as oxidative degradation, leading to formation of water soluble small oligosaccharides and the product of complete hydrolysis is monosaccharide. For complete hydrolysis typically chitosan: Hydrogen peroxide ratio is about 1 :5 and the temperature of about 80 degree C (Ma Z, Wang et al., (2014) Oxidative Degradation of Chitosan to the Low Molecular Water-Sol uble Chitosan over Peroxotungstate as Chemical Scissors; PLoS ONE 9(6): e100743 ( https://doi . org/10.1371/i ournal . pone.0100743) : Ke Liang B et al.; (2001) Kinetics and Products of the Degradation of Chitosan by Hydrogen Peroxide J . Agric. Food Chem, 49 (10), pp 4845^"4851.

It has been found that when a similar reaction is carried out under controlled conditions, a derivative of chitosan (partially oxidized) which is different i n its reactivity with various reagents under the reaction conditions that have been developed further. The controlled reaction of chitosan with Hydrogen peroxide is termed as :pre-activation reaction^".

T he :pre activated^" products have been characterized using FTIR analysis. There are progressive changes in the FTIR pattern of the chitosan from : non pre- activated ^" form to 1 hr, 3hr, 7 hr, and 24 hr reactions. The FTIR scans of the chitosan in both forms ^" preci pitate suspended in isopropanol as well as dried powder are identical. T his confirms that sequential changes in the structure of chitosan can be detected in the time chase study.

In one embodiment, the ratio of chitosan: hydrogen peroxide: FeCI3 in 5 Kg: 1 L: 1 gm (5:1 :0.001). The ratio of hydrogen peroxide ranges from 3 ^" 0.5 and FeC I3 0.01 to 0.0005 for the chitosan. The chitosan as used herein ranging in the DA value is between 60 to 99%.

Without getting bound to any theory the inventors have partially substituted the amine group of chitosan to form pre-activated chitosan. The partially reacted (: pre-activated^") chitosan has partial oxidation and partial blocking/capping/inactivating the amine/ amide group. This partial modification adds asymmetric charges, which correlates with the altered reactivity.

T he chitosan treated by : pre- activation^" reaction is terminated at different time intervals by precipitation. The precipitation reaction has been carried out using methanol, butanol, isopropanol or Sodium hydroxide. The precipitates derived by different reagents for a particular reaction and time interval show same FTIR output. This indicates that preci pitation prior to chemical modification can be carried out using any of these reagents.

In accordance with the invention, pre-activation^" reaction has been carried out with chitosan: with hydrogen peroxide and FeCI3 in presence of organic/inorganic acid in the rati o rangi ng from 8: 1 to 1 : 1 , preferably 2: 1.

T he organic acids can be selected from acetic acid, citric acid, ascorbic acid, lactic acid and inorganic acid can be hydrochloric acid.

In this preferred ratio of reagents, optimum swelling of the chitosan can take place in the stipulated time. When this swelling takes place the tertiary structure of the chitosan is disrupted.

T he chitosan : pre-activated^" for different ti me i nterval is subj ected to Sephadex÷ col umn chromatography. It was found that the retention time of the major fraction of the derivative within the column increases with increase in the reaction time. Sephadex÷ stands for a porous dextran gel used as molecular sieve wherein after loading on to gel of Sephadex÷ , molecular species having largest molecular size gets eluted first and the molecular species having least molecular size elutes last and rest of the molecular species el ute i n the order of thei r mol ecul ar si zes, I arger ones el uti ng earl i er than the smal I er ones. T herefore, the results indicate that the molecular weight of the chitosan is controlled by specifyi ng the reaction conditions for : pre-activation^". According to the invention, the : pre-activation is carried out in a single step reaction which in addition to altering the reactivity of chitosan ^" also pre-processes the poly- disperse molecules in to uniform tertiary structure, molecular weight and Degree of Acetylation (DA) %.

T he partial degradation products of chitin and chitosan like chito-oligosaccharides made by suspending or dissolving in acids are known to be water soluble. However, these molecules have a lower anti- microbial activity, and may get precipitated or show further reduction in the anti -microbial activity when diluted in water with pH above 7.0, or having dissolved salts like sodi unri magnesium, calcium etc. Patrida Martinez and Heil [Frontiers in Plant Sciences V ol. 2: 2-16 (2011)] have concl uded that ^"A plant that is completely free of microorganisms (pathogens) represents an exotic exception, rather than the ^"biologically relevant ^" rule. The net outcome of most plant ^" endophyte (microbe) interaction is highly conditional and depends on the detailed biotic and abiotic environment_,. The chitosan is soluble in water only below pH 3.0, but the derivatives of chitosan described in the present i nvention are soluble at a higher pH value ranging from pH 7 to pH 12.

In an embodiment of the invention, the chitosan derivative comprises pre-activated chitosan and chemical moieties. The chemical moieties are selected from Formaldehyde, Succinic anhydride, G lucose, Galactose, Salicylic acid, Linoleic acid or Oleic acid and Gallic acid.

T he chitosan derivatives have a molecular weight in the range of 100,000 to 300,000 Da. T he Degree of Acetylation ranges from 60 to 99%, preferably 70-80%, more preferably 78%.

A ccordi ng to another embodi ment of the present i nventi on, a method of obtai ni ng the pre- activated chitosan is provided involving pre activating chitosan and hydrogen peroxide in a ratio of 1 :1 to 10:1 to obtai n partially substituted chitosan at a temperature ranging from 18 to 30 degree Celsius, adding Hydrogen peroxide, FeCI3 and water to uniformly mix the reagents and dissolving acid crystals in de- ionized water and terminating the reaction at time intervals ranging between 1 hour to 24 hours by precipitating the reaction mixture by adding solvents and obtaining partially substituted chitosan. T he ratio of chitosan: hydrogen peroxide: FeCI3 is in 5:1 :0.001.

T he acids as used in the method can be organic or inorganic acids. Organic acids are selected from acetic acid, citric acid, ascorbic acid and lactic acid. Inorganic acids can be hydrochloric acid or phosphorus acid.

Solvents as used herein can be selected from methanol, butanol, isopropanol and Sodium hydroxide.

T he chitosan and acid can be present in a ratio ranging from 8:1 to 1 :1, preferably 2:1.

According to another embodiment of the invention, a method of preparing the chitosan derivatives is provided which comprises the steps of obtaini ng the pre-activated chitosan by the method as mentioned above and further reacting it with the chemical moieties selected from Formaldehyde, Succinic anhydride, Glucose, Galactose, Salicylic acid, L inoleic acid or Oleic acid and Gallic acid. The chitosan is reacted in a suspension form in a solvent comprising of alcohol (70 ^" 90 parts) and water (30 ^" 10 parts), at temperature between 20 and 95 degree C, pH between 2 and 6, and with constant stirring. T he invention also embodies methods for formulati ng the said chitosan derivative in liquid formulations that are stable and active for a significant time period of up to two years, so that they can be commercially prepared and used as pre-harvest products for controlling the plant pathogens, building disease resistance, or promoting plant growth.

In one of the embodiments, the formulation is stable at a pH ranging from pH 7 to pH 12. In one of the embodiments of this invention, chitosan derivatives are dissolved in water and the resultant solution(s) are stabi lized by adding water misci ble organic solvents such as methanol, ethanol, isopropanol, and butanol. The water miscible organic solvents can be present i n a range from 1 - 30%.

In yet another embodiment the chitosan derivatives are dissolved in water and the resultant solution(s) are stabilized by adding chemical preservative such as sodium azide, bronopol, QUAT 188, copper chloride etc. Said preservatives are present in a range 0.1 % to 10% w /vol, preferably 5% wt/Vol.

T he preservatives are selected from sodium azide, bronopol, QUAT 188, and copper chlori de. According to another embodiment of the i nvention, a formulation comprising chitosan derivatives and biologically active substances is provided.

T he biologically active substances can be selected from ol igosaccharides, protein hydrolyzate, oils and fatty acids, aldehydes, carboxylic acids, inorganic salts, or chemically complex plant and microbial extracts, commercial yeast extract, herbal extracts, protein hydrolysate, and extracts of micro-organisms.

T he chitosan derivatives can be present i n an amount ranging from 1 to 10% wtA ol in the composition.

T he said biologically active substances can be present in an amount ranging from 1 to 10% wt/vol.

T he inventors have surprisingly found that all the individual formulations provide enhancement in a distinct bio-activity such as rate of germination, growth of root, growth of leaf surface area, increase in the chlorophyll content, increase in the nutrient bioavailability, i nduction of disease resistance, callose bio-synthesis, induction of enzyme synthesis, thickeni ng of plant cell wall, inhibition of quorum sensing, inhibition of spore germination, inhibition of microbial growth, microbicide activity, etc. The examples given below are of some such peculiar combinations of distinct biological activities that are enhanced by the particular formulation and effect of the said formulation on the performance of the plant.

Not getting bound by any theory we observe a correlation between the biological activity and i nteraction of chitosan with other ingredients which may be leading to : self-assembly of biomolecules^" i n association with chitosan.

In a further embodiment of this invention the solution of chitosan derivative(s) is formulated with commercial yeast extract or herbal extracts, or protein hydrolysate, or extracts of micro-organisms such as Xanthomonas, Pseudomonas, Alternaria, Trichodernna, Saccharonryces etc. prepared by autolysis of the micro-organism in the fermentation broth using chitosan derivative(s).

A yet another embodiment of this i nvention discloses the in-vitro anti- microbial activity of the formulation(s) and the technical material as tested against the bacterial culture of Xanthomonas sp. A yet another embodiment of this invention discloses the plant growth promotion activity of the formulation(s) as tested in the two plot demonstrations using plant crop of potato and cauliflower.

Use of the chitosan derivatives for an improved rate of germi nation, improved growth of leaf surface area, increase i n the chlorophyll content, for callose bio- synthesis, for inhibition of microbial growth and i nhibition of microbicide activity.

An anti -anti -mi crobial/anti -fungal /anti -viral formulation comprising chitosan derivative and one or more biomolecules is provided.

An improved composition comprising chitosan derivative and one or more biomolecules for degradati on of toxi c pesti ci de i s provi ded.

T he composition comprising chitosan derivative and one or more biomolecules having shelf I if e more than 2 years.

An in-vitro method for callose induction in plants comprising the steps of treati ng the plant tissues with formulation comprising chitosan derivative and one or more biomolecules.

A method of disease resistance and growth of plants comprising contacting plants with formulation comprising chitosan derivative and one or more biomolecules.

In the following are described experiments conducted that serve as non-limiting illustrations of how the i nvention is performed. Any modifications or variations in the parameters, including but not limited to process for producing hydrolysates and further modifications, the method of use of the products and their various steps used are merely illustrative and any equivalents of them that are obvious to a person skilled in the art and capable of achieving the same objective may be used in their place and yet they shall be considered as i ncluded in the scope of this specification.

E xample 1 : P re-activation reaction

T he reaction is carried out at room temperature, where the temperature of the reaction mixture is between 18 and 30 degree Celsius. De- ionized water 30 L is taken in a Constantly Stirred Tank Reactor (Stainless steel) of 300 L capacity is added with Hydrogen peroxide (80%) 1 L and FeCI3 1 gm. T he water is stirred (100 rpm for 10 min) to uniformly mix the reagents. Chitosan powder (DA value 78%) 5 Kg is added to water with constant stirring. Chitosan gets uniformly dispersed and soaked typically in about 30 min time. Phosphorus acid crystals 2.5 kg are dissolved in 5 L de-ionized water, and the solution is added to the soaked chitosan with constant stirri ng. The chitosan starts getting dissolved and the viscous solution starts forming. The reaction is terminated at different time intervals from 1 hr to 24 hr by precipitating this reaction mixture by adding 150 L isopropanol with constant stirring.

T he phosphorus acid used in this example may be replaced with various inorganic acids (except sulfuric acid) such as hydrochloric acid, boric acid; or organic acids such as acetic acid, citric acid, lactic acid ascorbic acid.

E xample 2 Precipitation of Pre-activated chitosan and stability

Pre-activated chitosan processed as described in Example 1 was precipitated using Isopropanol, and the precipitate was recovered by filtration. The precipitate was dried under vacuum at temperature ranging between 20 and 40 degree C. The dried precipitate was in the form of lumps, and was powdered mechanically to obtain 60 mesh size material. The powdered pre-activated chitosan was stored in air tight glass bottles. The stored pre-activated chitosan has been tested suitable for further reaction, and remained unchanged in its FTIR pattern up to 190 days. This was not tested beyond 190 days for storage shelf life.

Chitosan from the same batch, having exactly identical physico-chemical characteristics was processed exactly in the same way without :pre-activation^" and termed as : corresponding control

E xample 2a

T he reaction for makin : corresponding control ^"to the process described in Example 1 is as follows:

The reaction is carried out at room temperature, where the temperature of the reaction mixture is between 18 and 30 degree Celsius. De- ionized water 30 L is taken in a Constantly Stirred Tank Reactor (Stainless steel) of 300 L capacity. The water is stirred (100 rpm for 10 min). Chitosan powder (DA value 78%) 5 Kg is added to water with constant stirring. Chitosan gets uniformly dispersed and soaked typically in about 30 min time. Phosphorus acid crystals 2.5 kg are dissolved in 5 L de-ionized water, and the solution is added to the soaked chitosan with constant stirri ng. The chitosan starts getting dissolved and the viscous solution starts forming. The reaction is terminated at different time intervals from 1 hr to 24 hr by precipitating this reaction mixture by adding 150 L i sopropanol with constant sti rri ng.

T he : corresponding control ^" products have been characterized using FTIR analysis. The FTIR pattern of the chitosan of corresponding control ^" form at 1 hr, 3hr, 7 hr, and 24 hr reactions are identical. The FTIR scan of : corresponding control ^" drawn after 24hr reacti on ti me i s shown i n F igure 1 a to 1 e.

T he pre-activated chitosan is reacted with chemical moieties selected from Formaldehyde, Succinic anhydride, G lucose, Galactose, Salicylic acid, Linoleic acid or Oleic acid and G al I i c aci d. T he f ol I owi ng exampl es show the reacti on steps.

E xample 3: R eaction of : pre-activated ^"chitosan with Formaldehyde:

Pre-activated Chitosan [60 mesh powder recovered after 1 to 3 hrs of reaction] 5 gm is taken in a round bottom flask and 100 ml methanol (99%) is added to it The suspension is mixed well by stirring at 100 rpm for 30 min. The mixture is heated to 70 degree C.

Formaldehyde (37%) in different proportions ranging from 0.5 g to 0.1 gm is diluted with water to make 10 ml final volume. The dilute formaldehyde solution thus formed is added drop-wise over one hr whi le the chitosan suspension is held at temperature of 65 degree C (+/- 5 degree C) with constant stirring. The stirring and heating is continued for another

0.5 to 3hour reaction period. The temperature is maintained between 60 ^" 70 degree C and preferably at 65 degree C.

T he same reaction is carried out by using corresponding control of chitosan instead of the pre-activated chitosan.

T he suspension from the reaction mixture using pre-activated chitosan is allowed to cool and filtered and the solid is air dried. The free flowing powder is formed by breaking the loose lumps mechanically and sieved using 60 mesh. The free flowing powder is then further dried in hot air oven at 70degree C for 3 ^" 6 hours. Solubility of the free flowing particles is checked by adding 5 gm powder slowly to 100 ml distil led water (pH 6.5) over 5 min. The mixture is stirred continuously during addition and further for 10 more min. and then the solubility is checked visibly. T he powder is soluble i n water when 0.1 to 0.3 gm formaldehyde is added to 5 gm of pre-activated chitosan in the said reaction. T he powder derived using corresponding control of chitosan forms lumps. These lumps are dried and converted to 60 mesh powder mechanically. In the solubility test, the powder only swel Is to produce gel I i ke parti cl es suspended i n water.

T he powder does not dissolve compl etely even when the mixi ng is conti nued for 3 hrs.

E xample 4: R eaction of : pre-activated ^"chitosan with Succinic anhydride

Pre-activated Chitosan [60 mesh powder recovered after 1 to 3 hrs of reaction] 5 gm is taken in a round bottom flask and 100 ml methanol (99%) is added to it The suspension is mixed well by stirring at 100 rpm for 30 min. The mixture is heated to 70degree C. Succinic anhydride is added in different proportions ranging from 0.5 g to 0.1 gm to this mixture and stirring is continued for 1 hour. The temperature is maintained between 60 ^" 70degree C and preferably at 65 degree C.

T he same reaction is carried out by using corresponding control of chitosan instead of the pre-activated chitosan.

T he suspension is allowed to cool and fi ltered and the solid is air dried. T he free flowing powder is formed by breaking the loose lumps mechanically and sieved using 60 mesh. T he free flowing powder is then further dried in hot air oven at 70 degree C for 3 ^" 6 hours. Solubility of the free flowing particles is checked by adding 5 gm powder slowly to 100 ml distilled water (pH 6.5) over 5 mi n. The mixture is stirred continuously during addition and further for 10 more min. and then the solubility is checked visibly. The powder is soluble in water when 0.3 to 0.5 gm succinic acid is added to 5 gm of pre- activated chitosan in the said reaction.

T he powder derived using corresponding control of chitosan does not dissolve completely even when the mixi ng is conti nued for 3 hrs.

E xample 5: R eaction of : pre-activated ^"chitosan with G lucose:

Pre-activated Chitosan [60 mesh powder recovered after 7 to 16 hrs of reaction] 5 gm is taken i n a round bottom flask and 80 ml methanol (99%) is added to it. The suspension is mixed well by stirring at 100 rpm for 30 min. Acetic acid (Glacial) is added (0.5 ^" 5 ml) and the mixture is heated to 70 degree C. D G lucose (1 ^" 5 gm) was dissolved in water to make 15 ml solution. The glucose solution was added to the reaction drop wise over 30 min with constant stirring. The temperature of the reaction was increased to 90 degree C. T he reacti on i s conti nued at 90 degree C wi th constant sti rri ng for 1 to 5 hrs.

T he suspension is allowed to cool and fi ltered and the solid is air dried. T he free flowing powder is formed by breaking the loose lumps mechanically and sieved using 60 mesh. T he free flowing powder is then further dried in hot air oven at 70 degree C for 3 ^" 6 hours. Solubility of the free flowing particles is checked by adding 5 gm powder slowly to 100 ml distilled water (pH 6.5) over 5 mi n. The mixture is stirred continuously during addition and further for 10 more min. and then the solubility is checked visibly. The powder is freely soluble in water.

T he powder derived using corresponding control of chitosan does not dissolve completely even when the mixi ng is conti nued for 3 hrs.

E xample 6: R eaction of :pre-activated~ chitosan with Galactose

Pre-activated Chitosan [60 mesh powder recovered after 7 to 16 hrs of reaction] 5 gm is taken i n a round bottom flask and 80 ml methanol (99%) is added to it. The suspension is mixed well by stirring at 100 rpm for 30 min. Acetic acid (Glacial) is added (0.5 ^" 5 ml) and the mixture is heated to 70 degree C. D Galactose (1 ^" 5 gm) was dissolved in water to make 15 ml solution. Acetic acid (5 gm) is added to the solution. The acidic galactose solution was added to the reaction with constant stirring. The temperature of the reaction was increased to 95 degree C. The reaction is continued at 95 degree C with constant sti rri ng for 1 to 5 hrs.

T he suspension is allowed to cool and fi ltered and the solid is air dried. T he free flowing powder is formed by breaking the loose lumps mechanically and sieved using 60 mesh. T he free flowing powder is then further dried in hot air oven at 70 degree C for 3 ^" 6 hours. Solubility of the free flowing particles is checked by adding 5 gm powder slowly to 100 ml distilled water (pH 6.5) over 5 mi n. The mixture is stirred continuously during addition and further for 10 more min. and then the solubility is checked visibly. The powder is freely soluble in water. T he powder derived using corresponding control of chitosan does not dissolve completely even when the mixi ng is conti nued for 3 hrs.

E xample 7: R eaction of : pre-activated ^"chitosan with Salicylic acid

Pre-activated Chitosan [60 mesh powder recovered after 7 to 16 hrs of reaction] 5 gm is taken i n a round bottom flask and 80 ml methanol (99%) is added to it. The suspension is mixed well by stirring at 100 rpm for 30 min. Salicylic acid (0.3 ^" 1.5 gm) was dissolved in water to make 20 ml solution. The salicylic solution was added to the reaction drop wise over 30 min with constant stirring. The temperature of the reaction was increased to 65 degree C, and the reaction is continued with constant stirring for 1 to 5 hrs.

T he suspension is allowed to cool and fi ltered and the solid is air dried. T he free flowing powder is formed by breaking the loose lumps mechanically and sieved using 60 mesh. T he free flowing powder is then further dried in hot air oven at 70 degree C for 3 ^" 6 hours. Solubility of the free flowing particles is checked by adding 5 gm powder slowly to 100 ml distilled water (pH 6.5) over 5 mi n. The mixture is stirred continuously during addition and further for 10 more min. and then the solubility is checked visibly. The powder is freely soluble in water.

T he powder derived using corresponding control of chitosan does not dissolve completely even when the mixi ng is conti nued for 3 hrs.

E xample 8: R eaction of : pre-activated ^"chitosan with L inoleic acid or Oleic acid: Pre-activated Chitosan [60 mesh powder recovered after 3 to 16 hrs of reaction] 5 gm is taken in a round bottom flask and 50 ml methanol (99%) is added to it T he suspension is mixed well by stirring at 100 rpm for 30 min. The solution of linoleic (LA) or oleic (OA) acid (2.5 gm) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (E DCXHCI) (1.8 gm) was made separately by adding methanol (99%) to make 50 ml volume. The methanolic mixture of fatty acid and E DC.HCI was added drop wise to chitosan solution, i nitiating the reaction. The reaction was continued for 24 hr at room temperature (20 ^"30 degree C).

T he suspension is filtered and the solid is air dried. The free flowing powder is formed by breaki ng the loose lumps mechanically and sieved using 60 mesh. The free flowing powder is then further dried under vacuum at room temperature (20 ^"30 degree C) for 24 ^" 36 hours. Solubility of the free flowing particles is checked by adding 5 gm powder slowly to 100 ml distilled water (pH 6.5) over 5 min. The mixture is stirred continuously during addition and further for 10 more min. and then the solubility is checked visibly. T he powder is freely sol ubl e i n water.

T he powder derived using corresponding control of chitosan does not dissolve completely even when the mixi ng is conti nued for 3 hrs.

E xample 9: R eaction of : pre-activated ^"chitosan with Gallic acid

Pre-activated Chitosan [60 mesh powder recovered after 3 to 5 hrs of reaction] 5 gm is taken i n a round bottom flask and 50 ml methanol (99%) is added to it. The suspension is mixed well by stirring at 100 rpm for 30 min. Acetic acid (Glacial) is added (2.5 ^" 5 ml) and the mixture is heated to 70 degree C. Gallic acid (0.1 ^" 0.5 gm) was dissolved in water to make 50 ml solution. The gallic acid solution was added to the reaction with constant stirring. The temperature of the reaction was increased to 50 degree C, and the reaction is continued with constant stirring for 1 to 5 hrs.

T he reaction of pre-activated chitosan with gall ic acid results in to a clear solution, whereas the corresponding control leads to formation of gel. The reaction mixture from pre-activated chitosan is precipitated using liquid ammonia and dried at room temperature under vacuum

EXA M PL E 10: D ET E R M INATION O F E F F E CT O F pH O N T H E SOL UTION O F C H IT OSA N DE RIVAT IV E S:

Products of Example 1, or 3, or 4, or 5, or 6, or 7, or 8, or 9 or their corresponding controls described in Example 2 and more specifically derived using chitosan of D DA value 78, and reacted for different time intervals from 1 to 24 hours (1 gm) were added to different glass beakers containing water (99 ml) and sti rred for 2-6 hrs til l a uniform clear solution is formed. All the solutions were tested for effect of pH after 24 hours of complete solubilization / final dilution. The effect of pH was determined by a drop-wise addition of solution of sodi um hydroxide (1 N) with constant sti rri ng. When a first distinct change in the solubility was noted such as turbidity, flocculation, precipitation, gel formation etc., the addition of sodium hydroxide was stopped immediately. The solution was stirred for another 30 min to confirm that the observed change in the solubility is maintained and the properties of the solution do not revert back to clear solution. At such point the pH of the solution was noted down. This pH value was considered as the pH value above which the said solution is not suitable for application. The following table (Table 1) provides pH values determined for the solutions described in this example, where change i n sol ubi I ity character was noted.

TA B L E 1 : E ffect of pH on the solubility of chitosan and its derivatives.

Not getting bound to any theory we state that the solubility of the chitosan derivatives is affected by the net charges on the molecules and surrounding solution; and hence addition of various components to the formulation also affects the solubility property. Therefore net effectiveness of such formulation(s) for promotion of plant growth, and/or building disease resistance is also regulated by the solubility of formulation in the water used by the farmer for the sai d appl i cati on.

Observation from table 1 : It is observed from the table that the derivatives of chitosan are showing better water solubility at and above pH 7. Therefore, these derivatives are preferred over the chitosan for further formulations and application in the field. EXA M PL E 11 : STA BIL IZAT IO N O F WAT E R BASE D SO L UTION OF C H IT OSA N DE RIVAT IV E USING A L C OH O L S:

T he solutions (1gnV 100 ml) of chitosan derivatives described in examples 1, or 3 to 9 were prepared individually and checked for the shelf life. The liquid product was susceptible to oxidation, and microbial degradation. T he shelf life of the solution was found to be less than 2 weeks, and discoloration of the liquid to brownish color and/or microbial growth as indicated by clear solution turning turbid, or patches of fungal growth was observed; which was an indication of spoilage of the solution. This was also associated with the decrease i n the vi scosity of the sol uti on.

T he simplest natural, organic, 100% biodegradable additive that was found to improve the shelf l ife to more than 2 years are alcohols such as methanol, ethanol, iso-propanol, butanol.

T he product of Example 1 and more specifically derived as described in Example 2 (100 ml) was added with water (80 ml) and the said alcohol - such as ethanol, methanol, i sopropanol , or butanol - ( 20 ml ) . T he sol uti on i s sti rred further for 15 mi n. T he sol uti ons remained clear when observed under stirring and / or stored without stirring for up to 2 days.

T he solutions were stored further in pesticide grade H DPE bottles, closed air tight. For accelerated storage studies bottles were stored at 40°C, and for non-accelerated studies they were stored at 25 °C .

Samples were tested intermittently for the color (oxidation status), microbial growth in the solution measured as Total V iable Count (TV C), viscosity to detect degradation of the material; and its performance as indicated by the anti- microbial property. The parameters did not change significantly for the formulations in-spite of change in DDA value of chitosan or change in preservative from ethanol to methanol, to i sopropanol, or to butanol. All the four parameters described above and did not show significant deviations from those of the solution as observed when these solutions were prepared (Day zero).

Without being bound to the theory, the increase i n the shelf life may be attributed, but limited, to a hypothesis that the added alcohol fixes the movable groups attached to the chitosan by co-ordinate li nkage, which can otherwise re-adjust their position when water is used as the sole solvent. EXAMPLE 12: STABILIZATION OF WATER BASED SOLUTION OF CHITOSAN DERIVATIVE USING PRESERVATIVES:

The solution s (1gnV 100 ml) of chitosan derivatives described in example 1, or 3 to 9 showed discoloration of the liquid to brownish color and/or microbial growth as indicated by clear solution turning turbid, or patches of fungal growth was observed; which was an indication of spoilage of the solution. This was also associated with the decrease in the viscosity of the solution.

The solutions were stabilized by addition of preservatives such as sodium azide, bronopol, QUAT 188, or copper chloride in a concentration range of 0.1 % to 10% wt/ vol. to the product of Example 1 and more specifically derived as described in Example 2. Consistent improvement in the shelf life to more than 2 years was observed for concentration of the preservative exceeding 5%.

The solutions were stored further in pesticide grade HDPE bottles, closed air tight. For accelerated storage studies bottles were stored at 40°C, and for non-accelerated studies they were stored at 25 °C .

Samples were tested intermittently for the microbial growth in the solution measured as Total Viable Count (TVC), and viscosity to detect degradation of the material. The parameters did not change significantly for the formulations in-spite of change in DDA value of chitosan. However, for the concentration of preservative of up to 3% a small percentage of sampl es ( I ess than 6%) showed some mi crobi al growth.

Without being bound to the theory, the increase in the shelf life may be attributed, but limited, to a hypothesis that the added preservative gets coordinately fixed on the chitosan molecule and therefore when we use lower concentration of the preservative, pocket(s) may develop within solution where minimum inhibitory concentration is not reached. FORMULATIONS OF CHITOSAN DERIVATIVES WITH BIOMOLECULES EXAMPLES:

EXAMPLE 13: FORMULATION OF CHITOSAN DERIVATIVES WITH PROTEIN HYDROLYSATE.

In a cylindrical reaction vessel (SS 304) of capacity of 390 L and equipped with a stirrer blades and the variable stirring speed of 50 ^" 500 r.p.m., 150 L product of Example- 1, or 3, or 6, or 7, or 8 was charged. Stirring was started at about 100 revolutions per minute. Protein hydrolysate (100 L) was added slowly over 30 min. The protein hydrolysate typically contained 10 ^" 60% protein, pH of 2.2 ^"4.5, salt content less than 3% (as determined by ash), and a viscosity of about 50 -70 cps. Stirring was continued for 1 to 3 hours. After the stirring period was complete, further additions were made with constant stirring. Alcohol (30 L) was added over 30 min. Finally water (20 L) was added slowly over 30 min. In case where alcohol was not added to this formulation quantity of water was increased from 20 L to 50 L. A clear, straw yellow to brownish ^"red coloured solution of low viscosity was formed.

EXAMPLE 14: FORMULATION OF CHITOSAN DERIVATIVES WITH YEAST EXTRACT

In a cylindrical reaction vessel (SS 304) of capacity of 390 L and equipped with a stirrer blades and the variable stirring speed of 50 ^" 500 r.p. , 150 L product of Examplel, or

4, or 5, or 7, or 8 was charged. Stirring was started at about 100 revolutions per minute. Y east extract (5 L) was added slowly over 30 min. The Y east extract typically contained

30 ^" 50% TDS, pH of 3.5 ^" 4.0, salt content less than 7% (as determined by ash). Stirring was continued for 1 to 3hours. After the stirring period was complete, further additions were made with constant stirring. Alcohol (30 L) was added over 30 min. Finally water (115 L) was added slowly over 30 min. In case where alcohol was not added to this formulation quantity of water was increased from 115 L to 145 L. A clear, straw yellow to brownish ^"red coloured solution of low viscosity was formed.

EXAMPLE 15: FORMULATION OF CHITOSAN DERIVATIVES WITH POLYSACCHARIDES.

In a cylindrical reaction vessel (SS 304) of capacity of 390 L and equipped with a stirrer blades and the variable stirring speed of 50 ^" 500 r.p.m, 150 L product of Example7, or 8, or 9 was charged. Stirring was started at about 100 revolutions per minute. Individual solution of one of the following polysaccharides (100 L) was added slowly over 60 - 90 min. The polysaccharides used were typically carboxy- methyl cellulose (1% wt/vol.)- low viscosity pH 7; Xylan and xylo-oligosaccharides (1% wt/vol.) ^" pH 8.0, Guar gum (1%) pH 5 ^" 7, cationic Guar Gum (High viscosity) pH 9.0. Stirring was continued for 1 to 3 hours. After the stirring period was complete, further additions were made with constant stirring. Alcohol (30 L) was added over 30 min. Finally water (20 L) was added slowly over 30 min. In case where alcohol was not added to this formulation quantity of water was increased from 20 L to 50 L. A clear to straw yellow coloured solutions of varying viscosity were formed.

EXA M PL E 16: FO R M U LATIO N OF C HIT OSA N DE RIVAT IV E S WIT H PLA NT EXT RACT S.

In a cylindrical reaction vessel (SS 304) of capacity of 390 L and equipped with a stirrer blades and the variable stirring speed of 50 ^" 500 r.p. , 150 L product of Examplel or 5 or 8 or 9 was charged. Stirring was started at about 100 revolutions per minute. Individual solution or combinations of one or more of the following plant extracts (together constituting 100 L) was added slowly over 60 - 90 min. The plant extracts used were typically derived by refluxi ng dry powder (5- 20 % wt/vol.) in water or 50% alcohol for 6 hours. The refluxed material was cooled, filtered, and the clear filtrate was used for formulation with chitosan derivative. The plants used for extraction were typically Aloe vera, Asparagus racemosus, and E mbelia ribes. After the extracts) was mixed with the chitosan derivative, stirring was continued for 1 to 3 hours. After the stirring period was complete, further additions were made with constant stirring. Alcohol (30 L) was added over 30 min. Finally water (20 L) was added slowly over 30 min. In case where alcohol was not added to this formulation quantity of water was increased from 20 L to 50 L. A straw yel I ow to dark brown col oured cl ear sol uti ons of I ow viscosity were formed.

EXA M PL E 17: FO R M U LAT ION O F C HIT OSA N DE RIVATIV E S WIT H L IV E M IC R O-O R GA NISM .

In a conical flask (Borosi l) of capacity of 1 L 200 ml of nutrient broth (pre-sterilized along with the flask) was inoculated with Pseudomonas aerugenosa NCIM 2200. The culture was grown on orbital shaker (100 r.p.m.) for 72 hours. The product of Example 5 1 or 3 or 4 or 5 or 7 or 8or 9 (200 ml) was added aseptically and mixed well for 10 min. Total viable count of the formulation was determined after 1,3, and 24 hours. No viable count was observed. After about 96 hours in some cases there was a precipitation of cell debris. This was removed by filtration and the clear solution was used further. EXAMPLE 18: IN-VITRO DEMONSTRATION OF ACTIVITY OF FORMULATION OF CHITOSAN DERIVATIVES AND BIOMOLECULES AGAINST BACTERIA

The Xanthomonas culture isolated from the infected Pomegranate leaves was grown overnight in Peptone (2%) yeast extract (1%) broth at 25 °C on an orbital shaker. The optical density (O.D.) of the broth was adjusted to 0.4 (about 10⁶ cells/ml density) using sterile growth medium. Ten ml of this (0.4 O.D. culture) was added with 200 ι L of 1% aqueous solution of the product derived as specified in the tableThe tubes were incubated at 25 °C without shaking for up to 7 hours. Sample (100 ι L) was drawn just after the addition of chitosan derivative, after 30 min, 120 min, 300 min, 420 min of addition of chitosan derivative and immediately spread out on nutrient agar plate (2% peptone, 1% yeast extract, 0.1% NaCI, 2% agar). The plates were incubated at 25°C for 24 hours and the colonies of Xanthomonas developed on the plates were counted. These are represented as Total Viable Count (TVC) of the bacteria after the respective treatment The results of this experiment are represented in table below (Table 2)

Table 2: Total V iable Count of Xanthomonas after various time intervals of contact with chitosan derivatives.

These results show that the count of actively growing bacteria of Xanthomonas sp. goes on reducing as the contact time with the said chitosan formulation is increased. It is very significant to note that this reduction in the TVC is observed in the medium that has all the required nutrients for growth. This indicates that the said formulation(s) have got capacity to kill Xanthomonas cells.

TABLE 2

Formulation TVC at different time Interval (min) of contact of Xanthomonas with chitosan derivative.

0 min 30 min 90 min 300 420

min min

Example 2 : Corresponding Δ 8000 Δ 8000 Δ 8000 About About control 1 Hr 1000 1000

Example 2 : Corresponding Δ 8000 Δ 8000 Δ 8000 About About control 7 H r 1000 1000

Further when these plates are incubated for up to 96 hours, a few colonies (less than 20) of Xanthomonas appear on the plates where zero colonies were recorded at the end of 24 hours. This indicates that the said formulation(s) have got capacity to only inhibit (and not kill) the Xanthomonas cells under certain conditions.

EXAMPLE 19: IN-VITRO DEMONSTRATION OF ACTIVITY OF FORMULATION OF CHITOSAN DERIVATIVES AND BIOMOLECULES AGAINST FUNGI.

The plant pathogenic fungal cultures obtained from the culture collections - Alternaria alternanthera MTCC 149, Cylindrocarpon MTCC 6093, C^LJJ : Dfcfii)^ a n// ¾fe □ were used for this study. Spore suspension of each of these cultures (10³ spores/ml) prepared in saline solution was used. Ten ml nutrient broth was added with 200 ι L of the spore suspension. This tube was kept as a positive control for recording the germination of spores. The product derived in Example 5, 6, 8, 9, and 10 (all derived without addition of alcohol) were added (200 ι L for Example 5 and 400 ι L for Example 6, 8, 9, and 10) i n separate tubes - each containing 10 ml of nutrient broth and 200 ι L of spore suspension. T he tubes were incubated at 25 °C with shaking for up to 7 days. The spore germination was recorded as formation of small beads of the fungal biomass. Number of beads developed, were recorded. The results of this experiment are represented in table below (Table 3). The results show that the various derivatives of chitosan inhibit the germination of spores of al I the three fungal species very effectively.

Table 3: Spore germination data for Alternaria alternanthera MT C C 149, Cylindrocarpon MT C C 6093 , F usarium oxysporum MT C C 284 i n presence of chitosan derivatives and formulations.

Formulation Germination count for fungal spores incubated with chitosan derivative.

Alternaria Cylindrocarpon

alternanthera MTCC MTCC 6093 a n/ / ¾¾

149

2 5 7 2 5 7 2 5 7

Days Days Days Days Days Days Days Days Days

No chitosan 100+ 100+ 100 100+ 100+ 100 100+ 100+ 100

+ + +

Example 2 : 50 44 100 74 100+ 100 28 100+ 100

Corresponding ; control 1 + + +

H r

Example 2 : 39 67 100 89 92 100 56 100+ 100

Corresponding ; control 7 + + +

H r

Example 2 : 47 42 100 100 100+ 100 72 100+ 100

Corresponding ; control 24 + + + +

H r

Example 1 : 1 Hr 0 0 3 0 0 0 0 0 0

Example 1 : 7 Hr 0 2 10 0 0 0 0 0 0

Example 1 : 24 Hr 0 0 7 0 0 0 0 0 0

Example 1 : 7 Hr + 0 0 0 0 0 0 0 0 0 Example 3

Example 1 : 7 Hr + 11 13 19 27 25 32 8 19 31 Example 4

Example 1 : 7 Hr+ 0 0 0 0 0 0 0 0 0 Example 5

Example 1 : 7 Hr + 4 8 7 0 0 0 0 0 0 Example 6

Example 1 : 7 Hr+ 0 0 0 0 0 0 0 0 9 Example 7

Example 1 : 7 Hr + 0 0 3 0 0 5 0 0 0 Example 8

Example 1 : 7 Hr + 0 0 0 0 0 0 0 0 4 Example 9 EXAMPLE 20: IN-VITRO DEMONSTRATION OF ACTIVITY OF FORMULATION OF CHITOSAN DERIVATIVES AND BIOMOLECULES AGAINST YEAST

The culture of yeast Candida albicans NCIM 3471 was obtained from the culture collection.

The culture was grown overnight in Peptone (2%) yeast extract (1%) broth at 25 °C on an orbital shaker. The optical density (O.D.) of the broth was adjusted to 0.4 (about 10⁵ cells/ml density) using sterile growth medium Ten ml of this (0.4 O.D. culture) was added with 200 ι L of product derived in Example 7, The tubes were incubated at 25 °C without shaking for up to 7 hours. Sample (100 ι L) was drawn just after the addition of chitosan derivative, after 30 min, 120 min, 300 min, 420 min of addition of chitosan derivative and immediately spread out on nutrient agar plate (2% peptone, 1% yeast extract, 0.1% NaCI, 2% agar). The plates were incubated at 25°C for 24 hours and the colonies of Candida albicans NCIM 3471 developed on the plates were counted. These are represented as Total Viable Count (TVC) of the bacteria after the respective treatment The results of this experiment are represented in table below (Table 4).

Table4: Total Viable Count of Candida albicans NCIM 3471 after various time intervals of contact with chitosan derivatives.

Formulation TVC at different time Interval (min) of contact of Candida albicans

NCIM 3471 with chitosan derivative.

0 min 30 min 90 in 300 min 420 min

Example 2 Δ 8000 760 356 153 0

Example 1 + Δ 8000 205 49 0 0

Example 7

Control Δ 8000 >8000 >8000 >8000 >8000 (Chitosan with no

modification

T hese results show that the count of actively growing cells of Candida albicans NCIM 3471 sp. goes on reducing as the contact time with the said chitosan derivative formulation is increased. It is very significant to note that this reduction in the TV C is observed in the medium that has all the requi red nutrients for growth. This indicates that the said formulation(s) have got capacity to kill Candida albicans NCIM 3471 cells.

Further when these plates are incubated for up to 96 hours, a few colonies (between 10 and 70) of Candida albicans NCIM 3471 appear on the plates where zero colonies were recorded at the end of 24 hours. This indicates that the said formulation(s) have got capacity to only inhibit (and not kill) the Candida albicans NCIM 3471 cells under certain conditions.

EXA M PL E 21: IN-VIT R O DE M O NST RATION O F ACT IV ITY O F F OR M U LATION O F C HIT OSA N D E RIVATIV E S A ND BIO M O L E C U L E S AGAINST NE MAT ODE S

T he culture of a free I i vi ng nematode Panagrel I us redivivus was obtai ned from a I ocal pet shop. The culture was grown for several weeks on medium containing beer (50% vol ./vol.), and common sugar (2%) in a janvjar which was covered with a fi ne cloth so as to allow free oxygen diffusion, but inhibit insects from the entry. The culture developed bacterial growth and the nematodes started growing. When the nematode density increased rapidly, they accumulated at the edges of the jam jar near the surface. At such a stage, the I ive nematodes were careful ly ti pped off from the culture and suspended evenly in a fresh growth medium. Ten ml of this nematode suspension was added with, 400 ι L of the product (1%) derived in Example 4,6, 8,9 (all derived without addition of alcohol). T he tubes were incubated at 25 °C without shaking for 24 hours. After 24 hours all the tubes were visually inspected for the motil ity of the nematodes. The tube that did not receive any chitosan derivative showed all the nematodes moving vigorously all throughout the medium. The nematodes in the tubes containing chitosan derivatives had all collected at the bottom of the tube and the movement was either absent or very sluggish. The nematodes from the pellet at the bottom were mounted on a sl ide and observed under dissecting microscope and those showing mobility were scored. In all 200 nematodes from each tube were counted. In the tubes containing chitosan derivative as per Example no.4, 6, 8 and 9 had 31, 19, 0, 0 nematodes that were able to move, out of all the nematodes observed. This example shows that the different chitosan derivative f ormulati ons affect the nematodes negatively to different extent.

EXAMPLE 22: IN-VITRO DEMONSTRATION OF ACTIVITY OF FORMULATION OF CHITOSAN DERIVATIVES AND BIOMOLECULES IN DEGRADATION OF TOXIC PESTICIDE

Commercial pesticide formulations ^" Dimethoate 20% EC and Thiamethoxan 25% WG were procured from retail supplier of insecticide. 100 mg of each was separately suspended in 15 ml of water. After the mixture was homogeneous, the formulation of example 3(10 ml) was added. One tube was kept as control where instead of chitosan formulation water (10 ml) was added. Upon mixing of chitosan derivative formulation, we observed precipitation of the material ^" presumably complex of insecticide formulation and chitosan in 2 tubes. Dimethoate and Thiamethoxan with chitosan derivative formulation of Example 3 were i ncubated at 25 °C under 15 Watt LED light (at 8 inches^" distance from the light source) for 1 hour. After the said time period the tubes were observed for any precipitation. The suspensions were homogeneous and no precipitation was observed. The samples were spotted on silica get TLC plates and the TLC was run using acetone: cycl ox ehane (3:2) as mobile phase. The spots of pesticide were detected using iodine staining. The spots of both the pesticides were prominent near the line where they were loaded in the control samples, whereas the samples in which they were incubated with chitosan derivatives, the spots had run much further. This observation shows that the pesticide molecules treated with chitosan derivatives were reduced in their molecular weight ^" indicating the degradation of pesticides.

EXAMPLE 23: FIELD DEMONSTRATION OF PLANT GROWTH PROMOTION:

Out of a 3500 sq. m. plot having cauliflower plants (stage: 10 days after transplantation) 3 replicates of 8 sq. m. each were sprayed with the formulation of Example 5 at 3 ml/L concentration. The treated plants showed 30% increase in the leaf surface area and increased green colouration of leaves withi n 8 days of the spray. The yield was better on both qualitative and quantitative account. Exact data is not available.

Out of a 120000 sq. m. plot under potato sowing, 3 repl icates of 4000 sq. m. each were sprayed with the formulation of Example 6 formulated as Example 10 at 1 ml/L concentration after 30 days of sowing. The treated plants showed increase in the height and health of plants. 22% increase in the leaf surface area and increased green colouration of leaves within 10 days of the spray. The yield was better on both qualitative and quantitative account

EXA M PL E 24: FIE L D DE M O NST RATION OF PE R FO R MA NC E AGAINST T H E BACT E RIA L DISEASE :

T he product of example 3 formulated using example 14 was used at 16 farms for one complete season in pomegranate orchards where the bacterial blight disease had caused losses in the previous years. Upon use of the product, the leaves from all the fields were tested positive for induction of callose synthesis. 140 out of 162 farms had direct measurable positive impact because of the prevention of the bacterial blight. The results obtai ned for different crops are summarized in the table below.

TA B L E 5

Importance to Profit

Product farmer Implications

Fruit cracking 3 Mt extra @

prevented. Rs 25 =

Spread of 75,000. Using

Example infection product worth 3and 14 prevented. 8000.

Prevents onset

Prevents & spread of

Example 5 infection of disease in the and 10 new leaves new season.

Example 8 Gap filli ng of Rs 2000 per

and 13 is avoided. acre saved.

Application Even growth

I improves yield.

Example 6 Prevents root 30% more

and 12 fungi & growth over

nematode untreated

(affected) control.

Example 7 Plant losses at Robust growth and 11 early stage and 4- 9 days

(foliar early flowering

diseases) are (Tomato).

minimized.

EXAMPLE 25: FIELD DEMONSTRATION OF PERFORMANCE AGAINST FUNGAL DISEASE:

Out of a 400 sq. m. plot having beetle vine (stage: 5 months^" growth) ^" affected by soil borne fungal infection of phytophthora, half of the plot was treated with the formulation of Example 7. The product was diluted at 1 ml/L concentration, and this diluted solution was use to drench the soil around each of the vine. The treated plants showed signs of recovery as new spouting within 10 days.92% plants recovered completely and started producing the yield in less than 40 days. Two applications of the product were done at 10 days interval.

Out of a 1200 sq. m. plot under tomato cultivation affected by Alternaria infection, 900 sq. m. was sprayed with the formulation of Example 6 at 3 ml/L concentration. The treated plants showed greener colouration within 3 days and started growing normal within another 10 days. Two more sprays were applied at 8 days interval. The treated plants produced normal yield whereas the untreated plants died within 15 days.

EXAMPLE 26: FIELD DEMONSTRATION OF PERFROMANCE AGAINST VIRUS

Out of a 4000 sq. m. plot having capsicum (stage: 1 month s growth) ^" affected by leaf curl virus infection, 3600 sq. m of the plot was treated with the formulation of Example 7 and formulated as described in example 10. The was mixed at dilution of 2.5 ml/L of each. The product was sprayed on the affected plants. Three sprays were applied at 10 days interval. The treated plants showed signs of recovery as normal growth of curled leaves within 10 days. All plants recovered completely and started producing about 60 % more floral buds as compared with untreated plants within 25 days of initiation of the treatment EXA M PL E 27: FIE L D DE M O NST RATION OF PE R FO R MA NC E AGAINST T H E NE MAT ODE INF E CTIO N:

Pots with Gerbera plants where prevalence of soil nematodes was known were selected for the studies. T he nematode count was determi ned for the soi I samples. T he i nitial count was 22, 19, and 30 nematodes/gm of soil for the three pots. 1 ml of example 5 (formulated as described in example 14) was diluted with water to make 1 L solution. Each pot was applied with 300 ml of this dilute solution. The application was repeated every 7^th day and three such applications were made. The soil was tested again for the nematode count. The counts for the three respective pots were 4, 4, and 5 nematodes/gm of soil. The plants had also showed positive growth.

EXA M PL E 28: IN VIT R O ST U DIE S O N I NDUCT ION O F CA L L OSE SY NT H E SIS:

Leaves of pomegranate were sprayed with formulations developed as per Example 2, 3 and Example 7, diluted to 1 ml per L, and the solution 0.025 gm L in final concentration. Leaf samples were taken intermittently after 0 hr, 1 hr, 24 hr, 48 hr, 72 hr, 96 hr, 120 hr and 25 days. The leaves were removed from plant and incubated in a solution of DMSO: Methanol 2:1 at 60 degree C for 1 hr. These leaves lost the pigmentation and they were then washed in Phosphate buffer saline pH 9.5. T he washed leaves were stai ned by dipping in 0.005% aniline blue solution for 1 hr. The stained leaves were washed briefly in Phosphate buffer saline pH 9.5, and observed under 400 X magnification fluorescence microscope at 370 nm incident light (UV source wavelength 200 ^" 270 nnri and blue excitation filter). Callose synthesized was observed as green fluorescence (509 nm). The leaves of zero hr, did not show any synthesis of close; whereas fluorescence spots were detected from 1 hr till 21 days, which declined to negligible fluorescence on 25^th day. Callose induction was also observed in plants namely tomato, gerbera, rice, grapes after 24 hrs of spray with the modified chitosan solutions (Example 6, 8, and 9). No or very little callose synthesis was observed when chitosan solution of example 2 was used for spray. EXAMPLE 29: POT TRIAL FOR INDUCTION OF DISEASE RESISTANCE IN POMEGRANATE:

Molecules known for inducing biochemical resistance against diseases along with chemicals in vogue for management of pomegranate bacterial blight were evaluated in polyhouse against bacterial blight pathogen Xanthomonas axonopodis pv.punicae in challenge inoculation. Lowest BBD incidence was recorded in salicylic acid treatment which was at par with spray treatments with formulations containing chitosan derivative (Example 5 ,7, 8 or 9). Reduction of BBD incidence in these treatments was from 67.87- 75.97% over control having 33.50% incidence. Among other 5 molecules, significant reduction was recorded in mancozeb, carbendazim, ziram and streptocycl i ne from 45- 56%, however Fosetyl-AI did not show significant control of bacterial blight Reduction in blight severity over control was recorded from 39.82 to 54.75% with sprays of copper formulations and streptocycl ine that are at par with each other.

Claims

C LAIMS

A chitosan derivative comprising pre-activated chitosan wherein amine group is partially substituted and chemical moieties.

The derivative as claimed in claim 1, wherein said chemical moieties are selected from Formaldehyde, Succinic anhydride, Glucose, Galactose, Salicylic acid, Linoleic acid or Oleic acid and Gallic acid.

The derivative as claimed in claim 1, wherein deacetylation (DA) value of chitosan derivative is ranging from 60 to 99%.

The derivative as claimed in claim 2, wherein deacetylation (DA) value of chitosan derivative is 78%.

The derivative as claimed in claim 1, wherein pH is between 7 to 12.

The derivative as claimed in claim 1, wherein said chitosan derivatives have a molecular weight in the range of 100,000 to 300,000 Da.

A method of obtai ni ng the pre-activated chitosan comprisi ng the steps of:

i. Pre activating reaction comprising chitosan and hydrogen peroxide in a ratio of 1 :1 to 10:1 to obtain partially substituted chitosan at a temperature rangi ng from 18 to 30 degree Celsi us; ii. Adding Hydrogen peroxide and FeCI3;

iii. Adding water to uniformly mix the reagents obtained in step ii, and adding Chitosan powder (DA value 78%) to water with constant sti rri ng;

iv. Dissolving acid crystals in de-ionized water,

v. Adding the solution obtained in step iv to the chitosan with constant sti rri ng; and

vi. Termi nating the reaction of step v at time intervals ranging between 1 hour to 24 hours by precipitating the reaction mixture by adding solvents with constant stirring and obtaining pre-activated chitosan.

8. The method as claimed in claim 7, wherein deacetylation (DA) value of chitosan derivative is between 60 to 99%.

9. The method as claimed in claim 8, wherein deacetylation (DA) value of chitosan derivative is 78%.

10. The method as claimed in clai m 7, wherei n chitosan: hydrogen peroxide: FeCI3 is in the ratio 5:1 :0.001.

11. The method as claimed in claim 7, wherein acids in step iv is organic or inorganic acids.

12. T he method as cl ai med i n cl ai m 11 , wherei n organi c aci ds are sel ected from aceti c acid, citric acid, ascorbic acid and lactic acid.

13. The method as claimed in claim 11, wherein inorganic acids is hydrochloric acid or phosphorus acid.

14. The method as claimed i n claim 7, wherein said solvents in step vi are selected from methanol, butanol, isopropanol and Sodium hydroxide.

15. The method as claimed in claim 7, wherein said chitosan: acid is present in a ratio ranging from 8:1 to 1 :1.

16. The method as claimed in claim 15, wherein said chitosan: acid is present in a ratio 2:1.

17. A method for preparing the chitosan derivative of claim 1 comprising steps of

Step I-obtai ni ng the pre-activated chitosan

i. Pre activating reaction comprising chitosan and hydrogen peroxide in a ratio of 1 :1 to 10:1 to obtain partially substituted chitosan at a temperature ranging from 18 to 30 degree Celsius;

ii. Adding Hydrogen peroxide and FeCI3;

iii. Adding water to uniformly mix the reagents obtained in step ii, and adding Chitosan powder (DA value 78%) to water with constant sti rri ng; iv. Dissolving acid crystals in de- ionized water,

v. Adding the solution obtained in step iv to the chitosan with constant stirring; and

vi . T ermi nati ng the reacti on of step v at ti me i ntervals rangi ng between 1 hour to 24 hours by precipitating the reaction mixture by adding solvents with constant stirring and obtaining pre-activated chitosan.

Step II- i. Reacting the pre-activated chitosan obtained in step I with chemical moieties to form a solution at a temperature ranging from 20 to 95, degree C, pH between 2-5.5 and Time between 3^" 10 hrs. and

ii. Obtaining chitosan derivatives.

18. The method as claimed in claim 17, wherein said chemical moieties are selected from Formaldehyde, Succinic anhydride, Glucose, Galactose, Salicylic acid, Linoleic acid or Oleic acid and Gallic acid.

19. The method as claimed in claim 17, further comprising the steps of stabilizing the chitosan derivative by dissolving chitosan derivatives in water and adding water miscible organic solvents or preservatives.

20. The method as claimed in claim 19, wherein said water misci ble organic solvents are selected from methanol, ethanol, isopropanol, and butanol.

21. The method as claimed in claim 19, wherein said water misci ble organic solvents is present i n a range from 1 - 30%.

22. The method as claimed in claim 19, wherein said composition is stable at from pH 7 to pH 12.

23. The method as claimed in claim 19, wherein said preservatives are selected from sodium azide, bronopol, QUAT 188, and copper chloride.

24. The method as claimed in claim 19, wherein said preservatives is present in a range from 0.1 % to 10% wt/vol.

25. The method as claimed in claim 19, wherein said preservatives is present in an amount of 5% wt/vol.

26. A formulation for disease resistance and growth of plants comprising chitosan derivatives of claim 1 and one or more biologically active substances.

27. The formulation as claimed in claim 26, wherein said one or more biologically active substances are selected from group consisting of oligosaccharides, protein hydrolyzate, carboxylic acids, inorganic salts, chemically complex plant and microbial extracts, commercial yeast extract herbal extracts, protein hydrolysate, and extracts of micro-organisms.

28. The formulation as claimed in claim 26, wherein said one or more biologically active substances are present i n an amount range 1 -10% Wt/vol.

29. The formulation as claimed in clai m 26, wherein said chitosan derivatives is present i n an amount range 1 -10% Wt/wt.

30. Use of the chitosan derivatives as defined in claim 1 for an improved rate of germination.

31. Use of chitosan derivatives as defined in claim 1 for an improved growth of leaf surface area.

32. Use of chitosan derivatives as defined in claim 1 for an increase i n the chlorophyll content.

33. Use of chitosan derivatives as defined in claim 1 for callose bio- synthesis.

34. Use of chitosan derivatives as defined in claim 1 for inhibition of microbial growth.

35. Use of chitosan derivatives as defi ned in claim 1 for inhibition of microbicide activity.

36. An anti-anti-microbial formulation comprising chitosan derivative and one or more biomolecules.

37. An anti-fungal formulation comprising chitosan derivative and one or more biomolecules.

38. An anti-viral formulation comprising chitosan derivative and one or more biomolecules.

39. An improved composition comprising chitosan derivative and one or more biomolecules for degradation of toxic pesticide.

40. An improved composition comprising chitosan derivative and one or more biomolecules having shelf life more than 2 years.

41. An in-vitro method for callose induction in plants comprising the steps of treating the plant tissues with formulation comprising chitosan derivative and one or more biomolecules.

42. A method of disease resistance and growth of plants comprising contacting plants with formulation comprising chitosan derivative and one or more biomolecules.