FIELD OF THE INVENTION
The present invention relates to a synergistic plant growth stimulator composition comprising phytosterols stigmasterol and campesterol, natural gibbrellins, natural cytokinins and naturally occurring auxins and a process for preparing the same.
BACKGROUND OF THE INVENTION
Phytosterols play an important role in plant growth and development, including cell division, cell elongation, embryogenesis, cellulose biosynthesis, and cell wall formation. Plant based steroids, in general, are used in very low concentration to promote cell division and cell elongation, increases tolerance under moisture and chilling stress and also potentiates the rate of photosynthesis and partitioning of assimilates in commercially important crops like fruits and vegetables, sugar crops, beverages, cereals, ornamental crops etc.
Stigmasterol is a phytosterol and considered as one of the mostly free or conjugated sterols that plays essential functions in plant growth such as structure component of lipid core of cell membrane and biogenetic precursor of numerous metabolites including steroid hormones.
Plant based steroid i.e. Stigmasterol (Chemical Name: Stigmasta-5,22-dien-3ß-ol; Mol. Formula: C29H48O; Mol. Wt.: 412.70) and Campesterol (Chemical Name: (24R)-5-Ergosten-3ß-ol; Mol. Formula: C28H48O; Mol. Wt.: 400.69) are plant-based steroids which are derived naturally. Stigmasterol and Campesterol can be described by following formula:

Stigmasterol Campesterol
Campesterol is derived from a triterpene called squaline and undergoes different reductive and oxidative pathways to form brassinolide. The active form of Brassinosteroid, Brassinolide, promotes cell elongation at nanomolar levels.
Brassinolides ((3aS,5S,6R,7aR,7bS,9aS,10R,12aS,12bS)-10-[(1S,2R,3R,4S)-2,3-dihydroxy-1,4,5-trimethylhexyl]hexadecahydro-5,6-dihydroxy-7a,9a-dimethyl-3H-benzo[c]indeno[5,4-e]oxepin-3-one) are plant based, steroids naturally derived from seeds of brassica family crops having molecular formula C28H48O6. Brassinolides can be described by following formula:

Brassinolides, in general, are used in very low concentration to promote cell division and cell elongation, increase tolerance under moisture and chilling stress and also potentiate the rate of photosynthesis and partitioning of assimilates in commercially important crops like apple, chilli, sunflower, tea, cotton, onion, tomato, okra, vegetables, grapes, mango, citrus sweet orange, flowering plants, and annual flowering plants.
Brassinolides have been shown to be involved in numerous plant processes like; promotion of cell expansion and cell elongation; works with auxin to do so, unclear role in cell division and cell wall regeneration, promotion of vascular differentiation; BR signal transduction has been studied during vascular differentiation, necessary for pollen elongation for pollen tube formation and acceleration of senescence in dying tissue cultured cells; delayed senescence in BR mutants supports that this action may be biologically relevant.
Natural gibberellins (GA4-7) are a group of plant hormones that regulate growth and influence various developmental processes, including stem elongation, germination, dormancy, flowering, sex expression, enzyme induction, and leaf and fruit senescence. Gibberellins were first recognized in 1926 by a Japanese scientist, Eiichi Kurosawa, studying bakanae, the "foolish seedling" disease in rice. It was first isolated in 1935 by Teijiro Yabuta and Sumuki, from fungal strain (Gibberella fujikuroi) provided by Kurosawa. Yabuta named the isolate as gibberellin.
Gibberellins are growth hormones that stimulate cell elongation and cause plants to grow taller. Gibberellins also have a role in other plant processes, such as stem elongation, germination, flowering, and fruit ripening. In humans, the pituitary gland secretes the human growth hormone at appropriate times; the plastids of a plant secrete gibberellin in a similar fashion. Once a plant releases gibberellins, its cells begin a process of elongation. Since, plants are composed of single cells stacked on top of one another, this elongation of thousands of individual cells results in an overall growth of the plant.
Natural gibberellins (GA4-7) are used for multiple functions such as to improve fruit setting in fruits, to elongate cluster and increase berry size in vines, breaking of dormancy in seed crops, improvement in colour in fruits and vegetables, increase in size in vegetables, to advance flowering and yield increase in strawberries etc.
Natural Cytokinins are a class of plant growth substances (phytohormones) that promote cell division, or cytokinesis, in plant roots and shoots. The examples of natural cytokinins are kinetin, zeatin, and 6-benzylaminopurine (also referred to as BAP, 6- BAP, or 6-benzyladenine).
Natural Auxins are very widely used in plant tissue culture and usually form an integral part of nutrient media. Auxins promote, mainly in combination with cytokinins, the growth of calli, cell suspensions and organs, and also regulate the direction of morphogenesis. The word auxin has a Greek origin: auxein means to enlarge or to grow. At the cellular level, auxins control basic processes such as cell division and cell elongation. Since they are capable of initiating cell division they are involved in the formation of meristems giving rise to either unorganised tissue, or defined organs. Five naturally occurring (endogenous) auxins in plants include indole-3-acetic acid, 4-chloroindole-3-acetic acid, phenylacetic acid, indole-3-butyric acid, and indole-3-propionic acid.
WO2002087329 discloses a synergistic mixture of A) abscisic; and B) at least one plant growth regulator.
CN 105052977 discloses a synergistic mixture of CPPU with plant growth promoters selected from the group consisting of gibberellic acid, thidiazuron, brassinolide and triacontanol in particular gibberellic acid and triacontanol.
CN 102613246 discloses a synergistic mixture of A) natural abscisic acid, B) gibberellic acid (GA3, or of GA4 +7), and C) an existing plant growth regulator. The existing plant growth regulator is selected from one or more than two of 6-BA (6-benzylaminopurine), thidiazuron, brassinolide, sodium nitrophenolate, potassium nitrophenolate, chlormequat chloride, naphthlcetic acid and its salt, indolebutyric acid and its salt, indoleacetic acid and its salt, ethephon, diethyl aminoethyl hexanoate, paclobutrazol, uniconazole, fulvic acid, Mepiquat chloride, nitrohumic acid, jasmonic acid and triapenthenol. However, there still exists a need for an alternative synergistic composition which facilitates plant growth.
SUMMARY OF THE INVENTION
The present invention relates to novel plant growth stimulating compositions comprising natural phytosterols and the plant growth stimulators in synergistically effective amounts for stimulating plant growth. It further relates to the use of said compositions for stimulating plant growth, in particular enhancing crop growth with increase in yield.
Surprisingly, the inventors of the present invention have found that the plant growth regulating property of phytosterols can be significantly enhanced when combined with other plant growth promoters.
In accordance with an embodiment of the invention, there is provided a synergistic liquid fertilizer composition, comprising:
a) plant based steroids stigmasterol and campesterol;
b) natural gibberellins;
c) natural cytokinins; and
d) naturally occurring auxins
In accordance with an embodiment of the invention, the synergistic composition comprises plant-based steroids stigmasterol and campesterol (natural botanical extract) in an amount of 0.005 to 0.5%.
In accordance with an embodiment of the invention, the composition comprises natural gibberellins is preferably selected from the group consisting of GA4, GA7 and combinations thereof, present in an amount of 0.05 to 1%.
In accordance with an embodiment of the invention, the natural cytokinin is preferably selected from the group consisting of kinetin, 6-BAP, present in an amount of 0.05 to 1%.
In accordance with an embodiment of the invention, the naturally occurring auxin is preferably selected from indole-3-acetic acid, 4-chloroindole-3-acetic acid, phenylacetic acid, indole-3-butyric acid, and indole-3-propionic acid, present in an amount of 0.005 to 1%.
In accordance with an embodiment of the invention, the synergistic composition further comprises emulsifier, preservative, stabilizer, and solvent.
In accordance with an embodiment of the invention, the emulsifiers that can be used in the present invention include ionic and non-ionic emulsifying agents and are present in an amount in the range from 1 to 4%.
In accordance with an embodiment of the invention, the preservative that can be used in the present invention include 1,2-benzisothiazolin-3-one and/or 2-Methyl-2H-isothiazol-3-one or 5-chloro-2-methyl-4-isothiazolin-3-one or sodium benzoate or benzoic acid, and present in an amount in the range from 0.05 to 0.5%.
In accordance with an embodiment of the invention, the stabilizer that can be used in the present invention include carboxylic acids (citric acid, acetic acid, dodecylbenzensulfonic acid), orthophosphoric acid dodecylbenzensulfonic acid and suitable salts thereof, and present in an amount in the range from 0.1 to 5%.
In accordance with an embodiment of the invention, the solvent that can be used in the present invention include aromatic hydrocarbons, C9 aromatic solvents, isopropyl alcohol (IPA), methanol, polar (green) solvent.
In accordance with another aspect, the present disclosure provides a process for the preparation of the synergistic plant growth stimulating formulation.
In accordance with yet another aspect, the synergistic plant growth stimulating composition of the present invention is applied on orchards, crop fields, vegetables, oil- seed crops, cereal crops, flowers, other economic important crops etc.
DETAILED DECRIPTION OF THE INVENTION
The present invention relates to a synergistic liquid fertilizer composition, comprising plant-based steroids stigmasterol and campesterol (natural botanical extract), natural gibberellins, natural cytokinins and naturally occurring auxins. The composition comprises one or more plant- based steroids Stigmasterol and Campesterol (natural botanical extract), preferably present in an amount ranging from 0.005 to 0.5%. The phytosterols used for the purposes of the present invention are extracted from Soybean. Campesterol is derived from a triterpene called squaline and undergoes different reductive and oxidative pathways to form brassinolide. The active form of Brassinosteroid, Brassinolide, promotes cell elongation at nanomolar levels.
Brassinosterols promote cell division and inclination as detected in dwarf rice lamina inclination bioassay. The bending of the leaf is more prominent in young growing shoots and is a result of increased expansion of cells in the upper surface as compared to the lower surface of the petiole. Brassinosteroids (BRs) cause cell expansions at nanomolar concentrations by a slower pathway involving gene transcription. Cell expansion is caused by brassinosterol induced cell wall modifying enzymes like xyloglucan and endo-transglucosylase/hyrolases and expansis. This is followed by osmotic movement of water, microtubule organization and cell wall synthesis. Brassinosteroids causes cyclin-induced cell division in a manner similar to the cytokinins.
Brassinosteroids (BRs) are known to be required for normal root elongation. Low concentration of brassinosteroid application results in the promotion of root growth and formation of lateral roots. High concentration, of brassinosteroid applications inhibits growth of root.
Brassinosteroids (BRs) have an important role in vascular differentiation and are known to promote the formation of xylem and suppress the formation of phloem. It has been demonstrated that brassinosteroids are actively synthesized in the procambium cells which cause their differentiation into tracheary elements. A brassinosteroid dependent signaling pathway with specific receptors present in the vascular tissue has been identified in plants.
High levels of brassinosteroids are present in the pollen, where they may have some developmental and regulatory functions. Brassinosteroids are known to promote cell elongation and therefore cause the growth of pollen tube from stigma towards the embryo sac. The pollen tubes of brassinosteroid-deficient mutants of Arabidopsis cpd, do not elongate resulting in failure of seed set.
Seeds are also known to contain high levels of brassinosteroids where they promote the germination of seeds by interacting with other hormones [GA and Abscisic acid (ABA)]. Since brassinosteroids have a role in cell division and cell elongation it may facilitate seed germination and growth of the embryo.
BRs are known to increase yield in crops that are grown under stress conditions. They are also known to increase yield in agricultural crops like rice, barley and wheat. They also promote the potato tuber growth.
Natural Gibberellins can affect growth by its effect on cell growth and cell elongation. Such effects are often seen in stem growth as well as root growth. Stems and inter nodal lengths can be increased and a better more extensive root system develop. Increases in cell division can also sometimes be seen in the production of larger leaves. Natural Gibberellins leads to bigger plants with bigger shoots and leaves in many plants.
Exemplary natural gibberellins include GA4 and GA7. There are approximately 90 gibberellins, and, as used herein, all are encompassed by the general term "gibberellin", "gibberellins" or "gibberellic acid." In the formulations, either a single natural gibberellin or a combination of two or more natural gibberellins may be employed in the active medium. Natural Gibberellins are preferably present in an amount ranging from 0.05 to 1%.
The active medium also comprises one or more natural cytokinins, which is a class of plant growth substances (phytohormones) that promote cell division, or cytokinesis, in plant roots and shoots. The natural cytokinin can be selected from adenine-type cytokinins represented by kinetin, zeatin, and 6-benzylaminopurine (also referred to as BAP, 6- BAP, or 6-benzyladenine). In preferred embodiments the natural cytokinin is selected from the group consisting of kinetin (synthetic or derived from seaweed) or 6-BAP, preferably present in an amount ranging from 0.05 to 1%.
The active medium further comprises naturally occurring auxins. Auxins produces cell elongation. Auxin, together with cytokinins, is also involved in bud initiation and growth. Cell division seems to be regulated by the joint action of auxins and cytokinins. Auxins stimulate differentiation of vascular bundles and take part in differentiation of buds and roots. Auxins are also known for their ability to promote adventitious root formation. Examples of naturally occurring auxins used in accordance with the invention are naturally occurring endogenous auxins in plants include indole-3-acetic acid, 4-chloroindole-3-acetic acid, phenylacetic acid, indole-3-butyric acid, and indole-3-propionic acid, preferably present in an amount ranging from 0.005 to 1%.
The present invention is also related to a methods for making and using the synergistic liquid fertilizer composition.
The composition further comprises excipients like emulsifier, stabilizer, preservative and solvent in order to prepare a stable fertilizer composition.
It is desirable to have an emulsifier in the composition. One class of emulsifiers are known as "surface active agents", or surfactants. The surface-active agents can be anionic, cationic, non-ionic or polymeric in character. Typical surface active agents include salts of alkyl sulfates, such as diethanolammonium lauryl sulphate; alkylarylsulfonate salts, such as calcium dodecylbenzenesulfonate; alkylphenol-alkylene oxide addition products, such as nonylphenol-C.sub. 18 ethoxylate; alcohol-alkylene oxide addition products, such as tridecyl alcohol-C.sub. 16 ethoxylate; soaps, such as sodium stearate; alkylnaphthalenesulfonate salts, such as sodium dibutylnaphthalenesulfonate; dialkyl esters of sulfosuccinate salts, such as sodium di(2-ethylhexyl) sulfosuccinate; sorbitol esters, such as sorbitol oleate; quaternary amines, such as lauryl trimethylammonium chloride; polyethylene glycol esters of fatty acids, such as polyethylene glycol stearate; block copolymers of ethylene oxide and propylene oxide; and salts of mono and dialkyl phosphate esters, polyoxyethylene alkyl ether, present in synergistic liquid fertilizer composition in an amount in the range from 1 to 4%.
Suitable stabilizers are, for example carboxylic acids (citric acid, acetic acid), orthophosphoric acid dodecylbenzensulfonic acid and suitable salts thereof, present in synergistic liquid fertilizer composition in an amount in the range from 0.1 to 5%.
Suitable preservatives are for example 1,2-benzisothiazolin-3-one and/or 2-Methyl-2H-isothiazol-3-one or 5-chloro-2-methyl-4-isothiazolin-3-one or sodium benzoate or benzoic acid, present in synergistic liquid fertilizer composition in an amount in the range from 0.05 to 0.5%.
Solvents that can be employed include aromatic hydrocarbons, C9 aromatic hydrocarbon solvents, isopropyl alcohol (IPA), methanol, and polar (green) solvents.
The synergistic liquid fertilizer composition of present invention is storage stable and environmentally safe and also provide improved plant growth and yield. The compositions also act as plant bio-stimulant.
The embodiments of the present invention are more particularly described in the following examples that are intended as illustrations only, since numerous modifications and variations within the scope of the present invention will be apparent to those of skill in the art. Unless otherwise noted, all parts, percentages and ratios reported in the following examples are on a weight basis and all reagents used in the examples were obtained or are available from the chemical suppliers.
The invention in its broader aspects is not limited to the specific details and representative methods. The illustrative examples are described in this section in connection with the embodiments and methods provided. The invention according to its various aspects is particularly pointed out and distinctly claimed in the appended claims read in view of this specification and appropriate equivalents.
It is to be noted that, as used in the specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context
clearly dictates otherwise. Thus, for example, reference to a composition containing “a compound” includes a mixture of two or more compounds. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. The expression of various quantities in terms of “% w/w” or “%” means the percentage by weight, relative to the weight of the total solution or composition unless otherwise specified.

EXAMPLES

The unit of each value below is “% w/w” i.e. the percentage by weight, relative to the weight of the total solution or composition unless otherwise specified. The compositions illustrated in Table 1 are formulated as soluble synergistic liquid fertilizer composition (SL).
Table 1: Synergistic Liquid Fertilizer Composition
Sr. No. Ingredient Role Quantity
1 Plant based steroids Stigmasterol and Campesterol (Natural Botanical Extract) Phytosterol 0.005 to 0.5%
2 Natural Gibberellins Plant growth stimulator 0.05 to 1%
3 Natural Cytokinins Plant growth stimulator 0.05 to 1%
4 Naturally occurring auxins Plant growth stimulator 0.005 to 1%
5 Blend of ionic and Non-ionic surface active agents Emulsifier 1 to 4%
6 1,2-benzisothiazolin-3-one and/or 2-Methyl-2H-isothiazol-3-one or 5-chloro-2-methyl-4-isothiazolin-3-one or sodium benzoate or sodium benzoate or benzoic acid Preservative 0.05 to 0.5%
7 Carboxylic acids (citric acid, acetic acid, dodecylbenzensulfonic acid), orthophosphoric acid dodecylbenzensulfonic acid and suitable salts thereof Stabilizer 0.1 to 5%
8 Aromatic hydrocarbon/C9/ Isopropyl Alcohol/Methanol/Polar (green) solvent Solvent Q.S. to 100%
Q.S. = Quantity Sufficient
The aforementioned plant growth regulator compositions can be formulated as a soluble synergistic liquid fertilizer composition (SL) by the process described below.
Process for preparing synergistic liquid fertilizer composition in the form of soluble synergistic liquid fertilizer composition (SL):
The process for manufacture of 100 kg batch size of composition is provided. The amounts of active ingredients presented may be greater than the values calculated taking into account the percentage purity of the active ingredients, to compensate for losses of said ingredients during the manufacturing process. It is observed that following said procedure on industrial scale the final yield of the active ingredient will be similar or same as to standardized values.
Weigh all the quality approved raw material as per the batch size. The ingredients were taken in an order to start with solvent, add emulsifier, Stigmasterol and Campesterol containing natural botanical extract, remaining adjuvants and solvent (remaining half quantity) and mix well for 2 hours and the sample is taken and analysed for quality. The quality approved finished material was filtered and the required packaging was filled in.
To determine the effectiveness of synergistic liquid fertilizer composition in the field, following experiments were conducted:-
PADDY CROP:
Bioefficacy and phytotoxicity trials of synergistic liquid fertilizer composition of present invention were conducted on paddy crop at Pundibari, Cooch Behar using Randomized Block design. The soil type in the trial site was sandy loam. The variety of paddy used in the trials was Nilanjana and trial site was irrigated.
The trials were conducted in three replicates with 8 numbers of treatments and the individual plot size was 5m * 5m with spacing of 20 * 10 cm.
The dosages of Synergistic liquid fertilizer composition used in the trials were 1.0 ml/litre of water, 1.5 ml/litre of water, 2.0 ml/litre of water and 4.0 ml/litre of water. Gibberellic Acid (GA) 0.001% L at 180 ml/ha was used as standard check and water spray was used as control.
The spray volume for Synergistic liquid fertilizer composition used was 500 l/ha and was sprayed using knapsack sprayer fitted with hollow cone nozzle after 30 days after transplanting and sprayed twice at interval of 25 days.
The trials were conducted to assess the effect of Synergistic liquid fertilizer composition on plant height, plant vigour/health and yield/hectare of paddy crop. Further, the phytotoxicity was assessed by visual observations on leaf injury, viz, epinasty, hyponasty, necrosis, vein clearing, wilting etc. and plant injury.
Treatment Details:
1. Synergistic liquid fertilizer composition @ 1.0 ml/litre of water (T1)
2. Synergistic liquid fertilizer composition @ 1.5 ml/litre of water (T2)
3. Synergistic liquid fertilizer composition @ 2.0 ml/litre of water (T3)
4. Synergistic liquid fertilizer composition @ 4.0 ml/litre of water (T4)
5. Standard Check Gibberellic Acid 0.001% L @ 180 ml/ha (T5)
6. Control (water spray) (T6)
Method of assessment for growth parameters:
• Effect on plant height: Randomly ten plants were taken from each treatment and their height was calculated.
• Effect on plant vigour/health: Effect on plant health recorded on the scale of 1-10.
• Effect on Yield/hectare: Yield/hectare was calculated and expressed as quintal/hectare (q/ha).
Method of phytotoxicity assessment:
Phytotoxicity observation on Epinasty, Hyponasty, Necrosis, Vein Clearing, wilting, etc. On 0-10 scale (as below) (X), Double (2X) and Triple (3X) doses was recorded from ten plants and then average was taken:
Score Phytotoxicity (%) Score Phytotoxicity (%)
0 No Phytotoxicity 5 41-50
1 1-10 6 51-60
2 11-20 7 61-70
3 21-30 8 71-80
4 31-40 9 and 10 Complete destruction

Table 2: Effect of Synergistic liquid fertilizer composition on Plant Height and Crop Vigour/Health in Rice crop
Sr. No. Treatments Dose Plant Height
(cm) % increase in Plant Height against control Plant vigour/Health (on 10 point scale)
1 T1 1 ml/litre of water 74.65 5.13 8.10
2 T2 1.5 ml/litre of water 74.70 5.20 8.15
3 T3 2 ml/litre of water 74.80 5.34 8.19
4 T4 4 ml/litre of water 75.30 6.04 8.30
5 T5 GA 0.001% L @ 180 ml/ha 72.35 1.89 8.00
6 T6 Water Spray 71.01 - 7.90
S Em + 0.409 - -
CD at 5% 1.013 - -
S Em + = Standard error of mean CD at 5% = Critical Difference
Table 3: Effect of Synergistic liquid fertilizer composition on yield in Rice crop
Sr. No. Treatments Dose Yield (q/ha) % Yield increase over control
1 T1 1 ml/litre of water 43.44 22.67
2 T2 1.5 ml/litre of water 44.60 25.95
3 T3 2 ml/litre of water 45.06 27.25
4 T4 4 ml/litre of water 45.10 27.36
5 T5 GA 0.001% L @ 180 ml/ha 41.77 17.96
6 T6 Water Spray 35.41
S Em + 2.4738 -
CD at 5% 7.5034 -
S Em + = Standard error of mean CD at 5% = Critical Difference
Table 4: Phytotoxicity report
Sr. No. Treatments Phytotoxicity Symptoms
Leaf tips and plant injury Wilting Vein clearing Necrosis Epinasty and hyponasty
R1 R2 R3 R1 R2 R3 R1 R2 R3 R1 R2 R3 R1 R2 R3
1 T1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2 T2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
3 T3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
4 T4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Score: 0 = No Phytotoxicity Score: 100 = 100% Phytotoxicity R = Replication
Results: The results of the field experiment conducted to assess the effect of Synergistic liquid fertilizer composition on Paddy crop is summarized below:
• Effect on Plant height
The growth attribute plant height was significantly influenced by different doses of Synergistic liquid fertilizer composition. Among the different treatments T4 (Synergistic liquid fertilizer composition @ 4.0 ml/litre of water) recorded the maximum average plant height (75.30 cm) being statistically at par with each other and also with T3 (74.80 cm), T2 (74.70 cm), T1 (74.65 cm). However, the lowest average plant height (71.01 cm) was recorded in case of T6 (Water spray) followed by T5 (GA 0.001%L @ 180 ml/ha). T6 significantly differed from all the other treatments. (Table 2).
• Effect on plant vigour/health
The results presented in Table 2 shows that the plants treated with Synergistic liquid fertilizer composition showed vigorous growth with higher height, better crop health and stand.
• Yield per hectare
The treatment with Synergistic liquid fertilizer composition had significantly influenced the yield per hectare. Among the different concentration of formulation used, the plots treated with T4 (Synergistic liquid fertilizer composition @ 4.0 ml/litre of water) recorded the maximum yield (45.10 q/ha) those are at par with other treatments. The control plants (T6) had registered the minimum yield 35.41 q/ha closely followed by 41.77 q/ha exhibited by T5 (GA 0.001%L @ 180 ml/ha) (Table 3).
Phytotoxicity: The result of phytotoxicity studies of Synergistic liquid fertilizer composition is presented in Table 4. There were no visual symptoms of phytotoxicity noticed in terms of leaf tips and surface injury, wilting, epinasty, necrosis and hyponasty on Paddy crop in case of all the treatments.
It is evident from the results of the field experiment on Paddy treated with Synergistic liquid fertilizer composition that all the treatments have significant effect in comparison to water spray and Gibberellic Acid (GA). However, results with different concentration on different characteristics revealed that
Significantly higher plant height was recorded with Synergistic liquid fertilizer composition treatments in comparison with control where increased height up to 6% over control was observed.
In all the treatments of Synergistic liquid fertilizer composition the results were at par with each other.
Plots treated with Synergistic liquid fertilizer composition @ 4.0 ml/litre of water recorded the maximum growth, plant health and yield per hectare.
WHEAT CROP:
Bioefficacy and phytotoxicity trials of Synergistic liquid fertilizer composition were conducted on Wheat crop at Poonthoppu Village, Tamil Nadu using Randomized Block design. The soil type in the trial site was black soil mixed with loam. The variety of wheat used in the trials was COW(w) 1 and bore well irrigation was provided.
The trials were conducted in three replicates and the individual plot size was 5m*5m with spacing of 25 cm row to row.
The dosages of Synergistic liquid fertilizer composition were 1.5 ml/litre of water, 2.0 ml/litre of water, 2.5 ml/litre of water and 3.0 ml/litre of water. WILGIB (Gibberellic Acid Technical) at 50 ppm solution was used as standard check and water spray was used as control.
The spray volume for Synergistic liquid fertilizer composition used was 500 l/ha and was sprayed using knapsack sprayer fitted with hollow cone nozzle after 35 days of sowing (DAS) and sprayed twice at interval of 15 days.
The trials were conducted to assess the effect of Synergistic liquid fertilizer composition on growth, development and yield of wheat crop by evaluating the effect on plant height and yield per hectare. Further, the phytotoxicity was assessed by visual observations on leaf injury, viz., wilting, vein clearing, necrosis, epinasty and hyponasty, and plant injury.
Method of assessment for growth parameters
The study was conducted to access the effect of Synergistic liquid fertilizer composition in comparison to the standard Gibberellic Acid Technical (WILGIB) available in the market on growth, development and yield of wheat when applied as a crop spray with three replication for following parameters:
• Effect on plant height: Randomly ten plants were taken from each treatment and their height was calculated.
• Effect on Yield/hectare: Yield per hectare was calculated and expressed as Kg/ha.
• Method of phytotoxicity assessment: Phytotoxicity for the following parameters is observed from plant after application Leaf injury*, wilting, Epinasty, Hyponasty, Necrosis, Vein Clearing and plant injury.
*Table 5: Grading for leaf injury for phytotoxicity effect on plants
Grade Score Grade Score
1 0-10 6 51-60
2 11-20 7 61-70
3 21-30 8 71-80
4 31-40 9 81-90
5 41-50 10 91-100

Treatment Details:
1. Synergistic liquid fertilizer composition @ 1.5 ml/litre of water (T7)
2. Synergistic liquid fertilizer composition @ 2.0 ml/litre of water (T8)
3. Synergistic liquid fertilizer composition @ 2.5 ml/litre of water (T9)
4. Synergistic liquid fertilizer composition @ 3.0 ml/litre of water (T10)
5. Standard Check WILGIB (Gibberellic Acid Technical) at 50 ppm solution (T11)
6. Control (water spray) (T12)
Table 6: Effect of Synergistic liquid fertilizer composition on Plant Height and Crop Vigour/Health in Wheat crop
Sr. No. Treatments Dose Plant Height
*(cm) % increase in Plant Height against control
1 T7 1.5 ml/litre of water 74.33 7.21
2 T8 2.0 ml/litre of water 74.67 7.70
3 T9 2.5 ml/litre of water 75.00 8.17
4 T10 3.0 ml/litre of water 76.00 9.62
5 T11 50 ppm solution 72.33 4.32
6 T12 Water Spray 69.33 -
S Ed 0.38 -
CD at 5% 1.82 -
*values are square root transformations
S Ed = Standard error of difference CD at 5% = Critical Difference
Table 7: Effect of Synergistic liquid fertilizer composition on yield in Wheat crop
Sr. No. Treatments Dose Yield (Kg/ha)* % increase over control
1 T7 1.5 ml/litre of water 2287 7.9
2 T8 2.0 ml/litre of water 2314 9.2
3 T9 2.5 ml/litre of water 2319 9.4
4 T10 3.0 ml/litre of water 2320 9.4
9 T11 50 ppm solution 2296 7.0
10 T12 Water Spray 2120 -
S Ed 1.99 -
CD at 5% 5.89 -
*values are square root transformations
S Ed = Standard error of difference CD at 5% = Critical Difference
Table 8: Phytotoxicity report
Sr. No. Treatments Phytotoxicity Symptoms
Leaf tips and plant injury Wilting Vein clearing Necrosis Epinasty and hyponasty
R1 R2 R3 R1 R2 R3 R1 R2 R3 R1 R2 R3 R1 R2 R3
1 T7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2 T8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
3 T9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
4 T10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Score: 0 = No Phytotoxicity Score: 100 = 100% Phytotoxicity R = Replication
Results:
The results of the field experiment conducted to assess the effect of Synergistic liquid fertilizer composition on wheat crop is summarized below:
• Effect on plant height: Among the different treatments T10 (Synergistic liquid fertilizer composition @3.0 ml/litre of water) recorded the maximum average plant height (76.00) statistically at par with majority of other treatments (Table 6). However the lowest average plant height was recorded in case of T12 (water spray) of 69.33 cm.
• Effect on plant vigour/health: The results presented in Table 6 shows that plants treated with Synergistic liquid fertilizer composition showed vigorous growth with greater height, better crop health and stand.
• Yield per Hectare: The treatment with Synergistic liquid fertilizer composition had significantly influenced the yield per hectare. The control plants (T12) had registered the minimum 2120 kg/ha followed by T7 (Synergistic liquid fertilizer composition @ 1.5 ml/ha). (Table 7).
• Phytotoxicity: The result of phytotoxicity studies of Synergistic liquid fertilizer composition is presented in Table 8, there were no visual symptoms of phytotoxicity noticed in terms of leaf tips and surface injury, wilting, necrosis, epinasty and hyponasty on wheat crops in all the treatment.

It is evident from the results of the field experiment on Wheat treated with Synergistic liquid fertilizer composition that all the treatments have significant effect in comparison to water spray and Gibberellic Acid (GA). However, results with different concentration on different characteristics revealed that significant higher plant height was recorded with Synergistic liquid fertilizer composition treatments in comparison with control.
Plots treated with Synergistic liquid fertilizer composition @ 3.0 ml/ha had recorded the maximum growth, plant health and yield per hectare.
CHILLI, TOMATO, BRINJAL, ONION and GRAPES CROPS:
Bioefficacy trials of Synergistic liquid fertilizer composition on management of major diseases and pests were conducted on Chilli, Tomato, Brinjal, Onion and Grapes crops at Bagalkot, Karnataka using Randomized Block design. The varieties of different crops used in the trials were Chilli Cv Sitara, Tomato Cv Ananaya, Onion Cv Bellari Red, Brinjal Cv local and Grapes Cv Thomson seedless.
The trials were conducted in four replicates with 6 numbers of treatments of Synergistic liquid fertilizer compositions.
The dosages of Synergistic liquid fertilizer composition used in the trials were 1.5 ml/litre of water, 2.0 ml/litre of water, 2.5 ml/litre of water, 3 ml/litre of water. Gibberellic Acid (GA) at 20 ppm was used as standard check and water spray was used as control.
The spray volume for Synergistic liquid fertilizer composition used was 500 l/ha and was sprayed using knapsack sprayer fitted with hollow cone nozzle as crop spray and repeated after 15 days.
Treatment Details:
1. Synergistic liquid fertilizer composition @ 1.5 ml/litre of water (T13)
2. Synergistic liquid fertilizer composition @ 2.0 ml/litre of water (T14)
3. Synergistic liquid fertilizer composition @ 2.5 ml/litre of water (T15)
4. Synergistic liquid fertilizer composition @ 3.0 ml/litre of water (T16)
5. Standard Check Gibberellic Acid at 20 ppm solution (T17)
6. Control (water spray) (T18)
In-case of Onion and Brinjal Standard check i.e. Treatment 17 (T17) used is Miraculin 2ml/l solution.
Methodology: Recommended agronomic package of practices followed. The required quantity applied as crop spray (repeated after 15 days).
Percent disease index (PDI) was calculated by using formula adopted by Horsfall and Heuberger, 1942 as follows
PDI = Sum of all disease Ratings____________________ x 100
Total no. of leaves/fruits assessed x Maximum Disease rating
Table 9. Effect of Synergistic liquid fertilizer composition on growth, pest, disease and yield of Chilli
Sr. No. Treatments Plant Height (cm) Powdery mildew (PDI) Leaf Curl index Green fruit yield (q/ha)
1 T13 133.75 39.50 11.8 89.00
2 T14 134.00 44.55 11.3 89.50
3 T15 134.68 45.00 8.8 89.25
4 T16 135.22 38.21 7.7 90.50
5 T17 128.35 42.25 8.9 87.25
6 T18 120.50 55.55 10.9 82.25
CD at 5% 0.55 3.82 1.43 2.64
CD at 5% = Critical Difference
Table 10. Effect of Synergistic liquid fertilizer composition on growth, pest, disease and yield of Tomato
Sr. No. Treatments Plant Height (cm) Early Blight (PDI) Late Blight (PDI) Sucking pest Fruit yield (q/ha)
1 T13 145.25 55.55 42.03 8.1 44.80
2 T14 145.30 55.55 38.83 9.1 45.15
3 T15 146.50 55.55 33.25 8.9 45.25
4 T16 146.25 55.55 34.26 8.8 45.59
5 T17 146.25 55.55 39.40 10.5 44.58
6 T18 140.11 60.00 53.65 9.5 33.65
CD at 5% 2.11 2.82 2.91 0.94 1.42
CD at 5% = Critical Difference
Table 11. Effect of Synergistic liquid fertilizer composition on growth, pest, disease and yield of onion
Sr. No. Treatments Plant Height (cm) Purple blotch (PDI) Thrips/leaf Yield (q/ha)
1 T13 69.24 44.44 10.52 16.10
2 T14 69.30 48.25 10.35 16.40
3 T15 69.25 46.50 8.91 16.25
4 T16 69.58 40.98 8.87 16.50
5 T17 68.88 46.25 11.11 16.25
6 T18 65.25 55.55 11.25 14.50
CD at 5% 0.52 2.87 1.14 3.27
CD at 5% = Critical Difference
Table 12. Effect of Synergistic liquid fertilizer composition on growth, pest, disease and yield of brinjal
Sr. No. Treatments Plant Height (cm) Phomopsis blight (PDI) Yield (q/ha)
1 T13 119.6 14.55 12.55
2 T14 120.5 13.58 12.60
3 T15 120.5 12.98 12.86
4 T16 120.6 13.55 12.56
5 T17 116.5 13.85 12.11
6 T18 102.8 14.11 11.40
CD at 5% 0.44 NS 0.90
CD at 5% = Critical Difference
Table 13. Effect of Synergistic liquid fertilizer composition on growth, pest, disease and yield of grapes
Sr. No. Treatments Shoot length (cm) Downy mildew (PDI) Thrips/leaf Yield (q/ha)
1 T13 88.65 46.25 5.2 16.50
2 T14 88.75 56.00 5.6 16.40
3 T15 88.98 55.55 4.9 16.25
4 T16 89.11 52.55 4.9 16.50
5 T17 86.25 55.25 5.8 16.25
6 T18 78.95 60.14 7.2 14.50
CD at 5% 2.10 3.82 1.43 3.27
CD at 5% = Critical Difference
Results: The effect of Synergistic liquid fertilizer composition on the growth, incidence of pests and diseases and yield of chilli, tomato, onion, brinjal and grape are presented in Table 9 to 13.
Chilli: Plant height was significantly highest (135.22 cm) in Synergistic liquid fertilizer composition @3.0 ml/litre (T16) of water than all the treatments. This treatment also recorded least PDI (38.21) for powdery mildew, least leaf curl index for sucking pest (7.7) and highest green fruit yield (90.50 q/ha) and also found at par with its lower doses (Table 9).
Tomato: Highest plant height (146.25 cm) and yield of tomato (45.59 q/ha) was recorded in Synergistic liquid fertilizer composition @ 3.0 ml/litre of water (T16) which was significantly higher than control and at par with its lower doses. This treatment also recorded significantly least PDI for early blight, late blight and sucking pest population. (Table 10).
Onion: Treatments differed non-significantly with respect to plant height, purple blotch, thrips count and yield. Plant height was significantly highest in Synergistic liquid fertilizer composition @ 3.0 ml/litre of water (T16) (69.58), than all the treatments. Synergistic liquid fertilizer composition @ 3.0 ml/litre of water (T16) recorded least incidence with respect to the purple blotch (40.98) and thrips incidence (8.87/leaf) than all other treatments. This treatment also recorded highest yield of 16.50 q/ha (Table 11).
Brinjal: Plant height was significantly highest (120.6 cm) in Synergistic liquid fertilizer composition @ 3.0 ml/litre of water (T16) than all the treatments. However there was no significant difference with respect phomopsis blight. Highest fruit yield was recorded in Synergistic liquid fertilizer composition @ 2.5 ml/litre of water (T15) (12.86 q/ha) than all the treatments, but on par with Synergistic liquid fertilizer composition @ 3.0 ml/litre of water (T16) (12.56 q/ha) and its lower doses (Table 12).
Grapes: There was increase in shoot length, decrease in incidence of powdery mildew and increase in yield in treatment with Synergistic liquid fertilizer composition @ 3.0 ml/litre of water (T16) with significant increase in shoot length (89.11) and yield (16.50) in comparison to control (Table 13).
Conclusion: Application of Synergistic liquid fertilizer composition @ 1.5 ml to 3.0 ml/litre of water has significantly improved the growth attributes in crops viz. chilli, tomato, brinjal, onion and grapes. Not only that, but the treatments also improved the crop health which was clearly evident from fewer incidences of pests and diseases in the respective crop treated with Synergistic liquid fertilizer composition. Significant increase in yield was also recorded due to the application of Synergistic liquid fertilizer compositions.

COTTON CROP:
Bioefficacy and phytotoxicity trials of Synergistic liquid fertilizer composition were conducted on cotton crop at Poonthoppu Village, Tamil Nadu using Randomized Block Design. The soil type in the trial site was black to mixed loam. The variety of cotton used in the trials was MCU 5 and bore well irrigation was provided.
The trials were conducted in three replicates and the individual plot size was 5m * 5m with spacing of 75 cm between ridges and 30 cm between plants.
The dosages of Synergistic liquid fertilizer composition used in the trial were 1.5 ml/litre of water, 2.0 ml/litre of water, 2.5 ml/litre of water and 3.0 ml/litre of water. WILGIB (Gibberellic Acid Technical) at 50 ppm solution was used as standard check and water spray was used as control.
The spray volume for Synergistic liquid fertilizer composition used was 500 l/ha and was sprayed using knapsack sprayer fitted with hollow cone nozzle after 35 days of sowing (DAS) and sprayed twice at interval of 15 days.
The trials were conducted to assess the effect of Synergistic liquid fertilizer composition on growth, development and yield of cotton crop by evaluating the effect on plant height, health based on leaf colour and yield per hectare. Further, the phytotoxicity was assessed by visual observations on leaf injury, viz., wilting, vein clearing, necrosis, epinasty and hyponasty and plant injury.
Method of assessment for growth parameters
The study was conducted to access the effect of Synergistic liquid fertilizer composition in comparison to the standard Gibberellic Acid Technical (WILGIB) available in the market on Growth, Development and Yield of cotton when applied as a crop spray with three replication for following parameters:
• Effect on Plant height: Randomly ten plants were taken from each treatment and their height was calculated.
• Effect on health: Effect of on plant health based on leaf colour (visual).
• Effect on Yield/hectare: Yield per hectare was calculated and expressed as q/ha.
Method of phytotoxicity Assessment
Phytotoxicity assessment for the following parameters was observed from the plant after application Leaf injury*; Wilting; Vein clearing; Necrosis; Epinasty; Hyponasty; and plant injury.
* Grading for leaf injury for phytotoxicity effect on plants as enumerated in Table 5.
Treatment Details:
1. Synergistic liquid fertilizer composition @ 1.5 ml/litre of water (T19)
2. Synergistic liquid fertilizer composition @ 2.0 ml/litre of water (T20)
3. Synergistic liquid fertilizer composition @ 2.5 ml/litre of water (T21)
4. Synergistic liquid fertilizer composition @ 3.0 ml/litre of water (T22)
5. Standard Check WILGIB (Gibberellic Acid Technical) at 50 ppm solution (T23)
6. Control (water spray) (T24)
Table 14: Effect of Synergistic liquid fertilizer composition on Plant height and leaf colour in cotton crop.
Sr. No. Treatments No. Dose Plant height* (cm) Leaf colour
1 T19 1.5 ml/litre of water 123.83 Dark Green
2 T20 2.0 ml/litre of water 124.33 Dark Green
3 T21 2.5 ml/litre of water 124.67 Dark Green
4 T22 3.0 ml/litre of water 125.00 Dark Green
5 T23 50 ppm solution 123.67 Dark Green
6 T24 Water Spray 115.00 Green
S Ed 0.47
CD at 5% 1.37
*values are square root transformations.
S Ed = Standard error of difference CD at 5% = Critical Difference
Table 15: Effect of Synergistic liquid fertilizer composition on yield in cotton crop
Sr. No. Treatments No. Dose Yield (q/ha)* % increase
1. T19 1.5 ml/Litre of water 18.0 7.1
2. T20 2.0 ml/Litre of water 18.2 8.3
3. T21 2.5 ml/Litre of water 18.4 9.5
4. T22 3.0 ml/Litre of water 18.6 10.7
9. T23 50 ppm solution 18.3 8.9
10. T24 Water Spray 16.8
S Ed 0.10
CD at 5% 0.33
*values are square root transformations.
S Ed = Standard error of difference CD at 5% = Critical Difference
Table 16: Phytotoxicity report
Sr. No. Treatments Phytotoxicity Symptoms
Leaf tips and plant injury Wilting Vein clearing Necrosis Epinasty and hyponasty
R1 R2 R3 R1 R2 R3 R1 R2 R3 R1 R2 R3 R1 R2 R3
1 T19 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2 T20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
3 T21 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
4 T22 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Score: 0 = No Phytotoxicity 100 = 100% Phytotoxicity R = Replication
Results: The results of the field experiment conducted to assess the effect of Synergistic liquid fertilizer composition on cotton crop is summarized below:
• Effect on plant height: Among the different treatments T22 (Synergistic liquid fertilizer composition @ 3.0 ml/litre of water) recorded the maximum average plant height (125 cm) statistically at par with T21, T20, T19 and T23 (Table 14). However the lowest average plant height was recorded in case of T24 (Water spray) of 115 cm.
• Effect on plant health: The results presented in Table 14 shows that plants treated with Synergistic liquid fertilizer composition showed dark green colour with better crop growth. Results also showed that the plots treated with GA @ 50 ppm (T23) showed better leaf colour and crop growth in comparison to control.
• Yield per Hectare: The Synergistic liquid fertilizer composition treatment had significantly influenced the yield per hectare. Among the different concentration of Synergistic liquid fertilizer composition used, the plots treated with Synergistic liquid fertilizer composition @ 3.0 ml/litre of water (T22) showed significantly the maximum of 18.6 quintal of yields per hectare which is at par with other treatments. The control plants (T24) had registered the minimum 16.8 quintal per hectare (Table 15).
Phytotoxicity: The result of phytotoxicity studies of Synergistic liquid fertilizer composition is presented in Table 16, there were no visual symptoms of phytotoxicity noticed in terms of leaf tips and surface injury, wilting, necrosis, epinasty and hyponasty on cotton crops in all the treatments.
It is evident from the results of the field experiment on cotton treated with Synergistic liquid fertilizer composition that all the treatments have significant effect in comparison to water spray and Gibberellic Acid (GA). However, results with different concentration on different characteristics revealed that Synergistic liquid fertilizer composition has significant effect on the plant height and growth in cotton. In all the treatments of Synergistic liquid fertilizer composition the results were at par with each other. Plots treated with Synergistic liquid fertilizer composition @ 3.0 ml/ha had recorded the maximum growth, plant health and yield per hectare which is at par with other doses.
From the foregoing it will be observed that numerous modifications and variations can be effectuated without departing from the true spirit and scope of the novel concepts of the present invention. It is to be understood that no limitations with respect to the specific embodiments illustrated is intended or should be inferred. It should be understood that all such modifications and improvements have been deleted herein for the sake of conciseness and readability but are properly within the scope of the following claims.

We claim:
1. A synergistic liquid fertilizer composition, comprising:
extracts of stigmasterol and campesterol;
natural gibberellins;
natural cytokinins;
naturally occurring auxins; and
inert ingredients.

2. The synergistic liquid fertilizer composition as claimed in claim 1, wherein the extract of stigmasterol and campesterol are present in an amount ranging from 0.005 to 0.5%.
3. The synergistic liquid fertilizer composition as claimed in claim 1, wherein the natural gibberellin is selected from the group consisting of GA4, GA7 and combinations thereof, preferably present in an amount ranging from 0.05 to 1%.
4. The synergistic liquid fertilizer composition as claimed in claim 1, wherein the natural cytokinin is selected from the group consisting of kinetin or 6-BAP, and present in an amount ranging from 0.05 to 1%.
5. The synergistic liquid fertilizer composition as claimed in claim 1, wherein the naturally occurring auxin is selected from indole-3-acetic acid, 4-chloroindole-3-acetic acid, phenylacetic acid, indole-3-butyric acid or indole-3-propionic acid, and present in an amount ranging from 0.005 to 1%.
6. The synergistic liquid fertilizer composition as claimed in claims 1 to 5, wherein the composition comprises inert ingredients selected from the group consisting of emulsifier, preservative, stabilizer and solvent.
7. The synergistic liquid fertilizer composition as claimed in claim 6, wherein the emulsifier comprises a blend of ionic and non-ionic surfactants, and present in an amount ranging from 1 to 4%.
8. The synergistic liquid fertilizer composition as claimed in claim 6, wherein the preservative is selected from the group consisting of 1,2-benzisothiazolin-3-one and/or 2-Methyl-2H-isothiazol-3-one or 5-chloro-2-methyl-4-isothiazolin-3-one or sodium benzoate or benzoic acid, and present in an amount ranging from 0.05 to 0.5%.
9. The synergistic liquid fertilizer composition as claimed in claim 6, wherein the stabilizer is selected from the group consisting of carboxylic acids such as citric acid, acetic acid, dodecylbenzensulfonic acid, orthophosphoric acid dodecylbenzensulfonic acid and suitable salts thereof, and present in an amount ranging from 0.1 to 5%.
10. The synergistic liquid fertilizer composition as claimed in claim 6, wherein the solvent is selected from C9 aromatic solvents, isopropyl alcohol, methanol, and polar green solvent.
11. The synergistic liquid fertilizer composition as claimed in claims 1 to 10, wherein the composition is storage stable and environmentally safe and also provides improved plant growth and yield.
12. The synergistic liquid fertilizer composition as claimed in claims 1 to 11, wherein the composition also serves as an efficient plant bio-stimulant.