Synergistic chalcone containing composition of a nematicide

Abstract

The present invention provides a synergistic composition of a nematicide comprising of a nematicidal peptide, and chalcones, wherein the nematicidal peptide is derived from CED-4 protein sequence is either, Peptide 2, or Peptide 3, or Peptide 12; and chalcones are either chalcone 17, chalcone 25, or chalcone 30. The nematicidal peptides alone show 100% nematicidal activity at a concentration of 1 mg/ml, whereas, when combined with chalcones, 100% nematicidal activity is seen at concentration as low as 0.8 mg/ml bringing a synergistic effect. Another embodiment of the invention provides a method for preparing working solutions of chalcones in aqueous solvents such as water by diluting the stock solution of chalcones, prepared in dimethyl sulfoxide, serially in ethanol and water. The final concentration of chalcone is around 10.sup.4-10.sup.5 M in the nematicidal composition. This synergistic nematicidal composition is environmentally safe and non-toxic to humans and animals.

Claims

1. A composition for controlling parasitic nematodes comprising: an effective amount of at least one peptide derived from CED-4 protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO. 2; SEQ ID NO. 3; SEQ ID NO. 4; and b) an effective amount of at least one compound of Formula 1 ##STR00038## wherein, ring A is ##STR00039## wherein, the composition has synergistic anti-nematode efficacy of 100% at concentration of the peptide about 0.8 mg/ml, and compound of Formula 1 about 10.sup.5-10.sup.6 M.

2. The composition as claimed in claim 1, wherein, the composition comprises of peptide consisting of amino acid sequence of SEQ ID NO. 2 having a length of 15 amino acids.

3. The composition as claimed in claim 1, wherein, the composition comprises of peptide consisting of amino acid sequence of SEQ ID NO. 3 having a length of 16 amino acids.

4. The composition as claimed in claim 1, wherein, the composition comprises of peptide consisting of amino acid sequence of SEQ ID NO. 4 having a length of 12 amino acids.

5. The composition as claimed in claim 1, wherein the composition further comprises at least one extender, an emulsifier and/or surfactant.

6. The composition as claimed in claim 1, wherein the composition further comprises at least one agrochemically active compound.

7. The composition as claimed in claim 6, wherein said agrochemically active compound is selected from but not limited to substances capable of treating plants, fungicides, bactericides, insecticides, acaricides, nematicides, molluscicides, safeners, plant growth regulators, plant nutrients and biological control agents.

8. The composition as claimed in claim 1, wherein, the composition comprises a peptide consisting of an amino acid sequence of SEQ ID NO. 2 and a compound of ##STR00040## wherein ring A is ##STR00041##

9. The composition as claimed in claim 1, wherein, the composition comprises a peptide consisting of an amino acid sequence of SEQ ID NO. 2 and a compound of ##STR00042## wherein ring A is ##STR00043##

10. The composition as claimed in claim 1, wherein, the composition comprises a peptide consisting of an amino acid sequence of SEQ ID NO. 2 and a compound of ##STR00044## wherein ring A is ##STR00045##

11. The composition as claimed in claim 1, wherein, the composition comprises a peptide consisting of an amino acid sequence of SEQ ID NO. 3 and a compound of ##STR00046## wherein ring A is ##STR00047##

12. The composition as claimed in claim 1, wherein, the composition comprises a peptide consisting of an amino acid sequence of SEQ ID NO. 3 and a compound of ##STR00048## wherein ring A is ##STR00049##

13. The composition as claimed in claim 1, wherein, the composition comprises a peptide consisting of an amino acid sequence of SEQ ID NO. 3 and a compound of ##STR00050## wherein ring A is ##STR00051##

14. The composition as claimed in claim 1, wherein, the composition comprises a peptide consisting of an amino acid sequence of SEQ ID NO. 4 and a compound of ##STR00052## wherein ring A is ##STR00053##

15. The composition as claimed in claim 1, wherein, the composition comprises a peptide consisting of an amino acid sequence of SEQ ID NO. 4 and a compound of ##STR00054## wherein ring A is ##STR00055##

16. The composition as claimed in claim 1, wherein, the composition comprises a peptide consisting of an amino acid sequence of SEQ ID No. 4 and a compound of ##STR00056## wherein ring A is ##STR00057##

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 depicts a graphical representation of effect of Peptide 2 in combination with chalcone 17, or chalcone 25 on nematodes in a 96-well plate experiment.

(2) FIG. 2 depicts a graphical representation of effect of Peptide 2 in combination with chalcone 30 on nematodes in a 96-well plate experiment.

(3) FIG. 3 depicts a graphical representation of effect of Peptide 3 in combination with chalcone 17, or chalcone 25 on nematodes in a 96-well plate experiment.

(4) FIG. 4 depicts a graphical representation of effect of Peptide 3 in combination with chalcone 30 on nematodes in a 96-well plate experiment.

(5) FIG. 5 depicts a graphical representation of effect of Peptide 12 in combination with chalcone 17, or chalcone 25 on nematodes in a 96-well plate experiment.

(6) FIG. 6 depicts a graphical representation of effect of Peptide 12 in combination with chalcone 30 on nematodes in a 96-well plate experiment.

(7) FIG. 7 depicts a graphical representation of Peptide 2 alone or in combination with chalcones on hatching of eggs of nematodes in a 96-well plate experiment.

(8) FIG. 8 depicts a graphical representation of Peptide 3 alone or in combination with chalcones on hatching of eggs of nematodes in a 96-well plate experiment.

(9) FIG. 9 depicts a graphical representation of Peptide 12 alone or in combination with chalcones on hatching of eggs of nematodes in a 96-well plate experiment.

DETAILED DESCRIPTION OF THE INVENTION

(10) The present invention will now be described more fully hereinafter. This invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough, and will fully convey the scope of the invention to those skilled in the art.

(11) Chalcones or chalconoids are a group of compounds that have central core comprising of an aromatic ketone and an enone. Benzylideneacetophenone is the parent member of the chalcone series. Chalcones and their derivatives are molecules with broad-spectrum of biological activities such as anti-retroviral, anti-inflammatory, anti-parasitic, anti-malarial, anti-oxidant, anti-fungal, anti-protozoal, anti-bacterial, anti-steroidal, cardioprotective, anti-tumor, anti-cancer etc. Several types of chalcones are known to exist naturally in a variety of dicotyledonous plants, and in some monocotyledonous plants, pteridophytes and gymnosperms.

(12) Chalcone, (2E)-1,3-Diphenylprop-2-en-1-one, has a chemical formula C.sub.15H.sub.12O, and other chalcones are generally derivatives of this parent compound with a structure of Formula X:

(13) ##STR00001##

(14) Chalcone structure is composed of two aromatic rings, Ring A and Ring B, that are joined by a three-carbon , -unsaturated carbonyl system. At least thirty naturally occurring chalcones are known with several types of chemical groups attached to Ring A or B.

(15) Table 1 provides the structures of thirty several types of chalcones, chalcone 1 to chalcone 30, wherein the basic structure is that provided in Formula X.

(16) TABLE-US-00002 TABLE 1 Name and structure of naturally occurring chalcones Name of chalcone and its structure (Variation in Ring B) Type 1 Name Structure of Ring B 1 2 3 4 5 6 7 8 0 9 10 11 Name of chalcone and its structure (Variation in Ring A) Type 2 Name Structure of Ring A 12 13 14 15 16 17 0 18 19 20 21 22 23 24 25 26 27 0 28 29 30

(17) Attar et. al., have shown that chalcones are known to have nematicidal activity, however, the efficacy of individual chalcones varies a lot, ranging from as low as 5% to as high as 100% nematicidal activity at different concentrations. Moreover, the effect of chalcones was tested only on the non-parasitic model nematode Caenorhabditis elegans. The activity of individual chalcones was seen at concentrations higher than 10.sup.2 M. The use of higher concentrations of compounds for minimum inhibitory effect becomes very limited due to toxicity, soil contamination, seepage to ground water, and other related problems.

(18) A group of chalcones can be collectively presented as structural Formula 1:

(19) ##STR00034##

(20) Chalcone 17 has the chemical name (2E)-1-(2,4-Dichlorophenyl)-3-phenyl-2-propen-1-one and formula as shown in Formula 2:

(21) ##STR00035##

(22) Chalcone 25 has the chemical name (2E)-1-(4-Ethoxyphenyl)-3-phenyl-2-propen-1-one and formula as shown in Formula 3:

(23) ##STR00036##

(24) Chalcone 30 has the chemical name (2E)-3-Phenyl-1-(2-thienyl)-2-propen-1-one and formula as shown in Formula 4:

(25) ##STR00037##

(26) Chalcones and chalcone-like structures exist naturally in many plants, it makes them eco-friendly; and their association with several biological activities greatly increases their potential use in agriculture. However, the major limitation of chalcones is their solubility. Chalcones are not easily soluble in aqueous solvents such as water and consequently have to be dissolved in an organic solvent such as dimethyl sulfoxide (DMSO). DMSO is considered to be toxic to humans and animals with acute oral toxicity (LD50) of 7920 mg/kg [mouse] and acute dermal toxicity (LD50) of 40000 mg/kg [rat]. Hence, chalcones dissolved in DMSO are not suitable for direct application to soils as it is hazardous to users and animals coming in touch with it. Similarly, another solvent ethanol has a acute oral toxicity (LD50) of 7060 mg/kg [rat] which is also not safe for users. Therefore, a method for using chalcones in an environmentally safe solution is required.

(27) The present invention relates to a synergistic composition which has ability to kill plant parasites, specifically, nematodes. More specifically, the present invention relates to a synergistic composition of a nematicide comprising of a nematicidal peptide, and chalcones, wherein, the peptide is derived from the CED-4 protein sequence. The combination of a nematicidal peptide and chalcones is unique, and economical compared to other existing safe methods for nematode control such as generation of transgenic lines. Though, the peptides individually show nematicidal activity, the combination with chalcones increases the efficiency and brings a synergistic effect, and also reduces the amount of peptide required for activity, thus, reducing the cost of production of the composition.

(28) In the main embodiment of the present invention, the invention provides a synergistic composition of a nematicide comprising of: 1. at least one peptide, Peptide 2, Peptide 3, or Peptide 12, derived from CED-4 protein, wherein, a) Peptide 2, a 15 amino acids long peptide comprising of the sequence DLLRPVVIAPQFSRQ (SEQ ID NO. 2), b) Peptide 3, a 19 amino acid long comprising of the sequence RQMLDRKLLLGNVPKQMTC (SEQ ID NO. 3), and c) Peptide 12, a 12 amino acids long comprising of the sequence FPKFMQLHQKFY (SEQ ID NO. 4); and 2. at least one chalcone belonging to the group of compounds, chalcones or chalconoids, wherein the chalcone is either chalcone 17 {(2E)-1-(2,4-Dichlorophenyl)-3-phenyl-2-propen-1-one}, or chalcone 25 {(2E)-1-(4-Ethoxyphenyl)-3-phenyl-2-propen-1-one.

(29) The peptides show 100% nematicidal activity at a peptide concentration of 0.8 mg/ml when combined with at least one chalcone of Formula 2-4. The chalcones also showed 100% nematicidal acidity at concentrations as low as 10.sup.4-10.sup.5 M in combination with peptides SEQ ID NO 2-4. The combination of the nematicidal peptides SEQ ID No. 2-4 with chalcones of Formula 2-4 brings a synergistic effect and the combination works at a lower concentration compared to the peptides alone.

(30) Chalcones are not easily soluble in water or other aqueous solutions which makes their usability quite limited. Dissolving chalcones in non-aqueous solutions makes them less environment-friendly. Generally, chalcones are dissolved in organic solvents such as DMSO and ethanol which are highly toxic to humans and animals.

(31) Therefore, in another embodiment of the invention, the invention provides a method for using chalcones in an environmentally safe solution with minimal DMSO and ethanol concentrations. The working solution of chalcones is made in aqueous solvent such as water in steps comprising of: 1. making a stock solution of chalcone in DMSO at the concentration of 10.sup.1 M, for e.g. by adding 13.8 mg of chalcone 17 or 12.65 mg of chalcone 25 in 1000 l of DMSO; 2. diluting the chalcone stock in 100% ethanol to prepare a chalcone solution with 10.sup.2 M concentration, for e.g. adding 100 l of chalcone stock from step 1 to 900 l of 100% ethanol; 3. diluting the chalcone solution from step 2 in 50% ethanol in water solution to get a chalcone solution with 10.sup.3 M concentration, for e.g. adding 100 l of chalcone solution from step 2 to 900 l of 50% ethanol in water solution; and 4. diluting chalcone solution from step 3 in plain distilled water to get an aqueous chalcone solution with 10.sup.4 M concentration as the working solution, for e.g adding 1 mL of chalcone solution from step 2 to 900 mL of 50% ethanol in water solution followed by further diluting the solution in distilled water for obtaining lower concentrations of chalcone solution such as 10.sup.5 M or less;
wherein, the final concentration of DMSO and ethanol in the working solution of chalcones is between 0.01-0.1% and 0.5-5%, respectively, which is significantly less than the lethal concentrations of the solvents. This method provides a dilution method for dissolving chalcones in aqueous solutions at concentrations less than 10.sup.4 M.

EXAMPLE 1

A) M. incognita Propagation

(32) Tomato plants were inoculated with M. incognita juveniles and maintained in a growth chamber. After at least two months, the M. incognita eggs were extracted from the roots for experiments. The procedure followed for extracting M. incognita eggs is explained below.

(33) The root tissues were either chopped by hand using a surgical blade and a watch glass, or it was chopped up using a food processor. The chopped tissue was then placed in a bottle and washed with a 10% dilution of bleach. Under sterile conditions, the root solution was then poured through sieves (60 count sieve on top, 500 count sieve on the bottom). The crude egg collection was collected from the bottom of the 500 count sieve into 5 mL each of bleach and egg mixture in 15 mL Falcon tubes. 5 mL of 70% sucrose solution was then placed in each Falcon tube. A 1 mL layer of double distilled sterile water was then gently placed on top of the sucrose mixtures in each Falcon tube. The samples were then centrifuged for 5 minutes at 1200 rpm. The embryos that were suspended between the sucrose solution and the 1 mL water layer were collected in a total of 3 mL (top layer of 3 mL of the solution) from each Falcon tube into fresh 15 mL Falcon tubes. 10 mL of a 5% bleach solution was added and the eggs were vortexed for 10 minutes. The Falcon tubes were then centrifuged for 5 minutes at 2000 rpm. The supernatant was then removed, and the eggs were rinsed in 10 mL of sterile double distilled water and re-centrifuged for 5 minutes at 2000 rpm. This process was repeated two more times. After the last wash, 5 mL of supernatant was removed, while the remaining 5 mL of water was mixed with the eggs and placed into a 5 mL Petri dish. The eggs were then placed in an incubator at 25-27 C., and juvenile worms (J2 stage) hatched after about 10 days. The worms were kept in a 25-27 C. incubator for storage.

B) Preparation of Testing Solutions and Placement of Nematodes

(34) The chalcone to be tested was placed in a 1.5 mL eppendorf tube, and 1 mL of dimethyl sulfoxide (DMSO) was added to make a 10.sup.1 M solution. This stock solution was used for further dilutions in sterile double distilled water (10.sup.4 M to 10.sup.5 M). For instance, 10 L of 10-.sup.1 M chalcone in DMSO solution was added to 9990 L of sterile double distilled water to make a 10.sup.4 M solution for assays with the nematodes. 100 L of the dilutions in water were pipetted into 30 wells of a 96 well plate and one worm was transferred into each well from a stock of extracted J2 M. incognita. For a negative control, to confirm that the concentration of DMSO was not killing the nematodes, 30 worms were placed in 100 L of 1% DMSO in sterile double distilled water for each experiment (past work has demonstrated that C. elegans worms can survive at this concentration of DMSO (Attar et al., 2011).

C) Monitoring Nematodes

(35) The bioassay is designed to test the ability of the peptides, and chalcones, to kill the worms (% mortality). Each test was performed in a 96-well plate with one nematode in each well (30 wells total). The nematodes were incubated in the treatment solutions for 5 days. Viability of the nematodes was tested under a dissecting microscope by examining each for movement after disturbance with a probe.

D) Egg Hatching Experiments

(36) The bioassay is designed to test the ability of the mixture of peptides and chalcone to prevent egg hatching (% unhatched eggs). Each test was performed in a 96-well plate with 10 eggs per well. The eggs were incubated in each treatment for 16 days. Hatched vs unhatched eggs were determined by microscopic images.

EXAMPLE 2

Efficacy of Nematicidal Composition Comprising of Peptides and Chalcones

(37) As described earlier, equal number nematodes were incubated in various treatment solutions in a 96-well plate for 5 days and the potency of different combination of nematicides was tested by counting the number of nematodes dead or surviving on Day 5. Similarly, the effect of different combination of nematicide composition on hatching of nematode eggs was also counted.

(38) As depicted in FIG. 1-6, incubation of J2 stage juvenile nematodes in water, or 1% DMSO caused death of 10-15% nematodes on Day 5, and these treatments served as negative controls. Whereas, incubation of juvenile nematodes in a chemical mixture comprising of chlorpyriphos and imidacloprid (1:1 ratio) resulted in around 50% death on Day 5 which served as a positive control. Treatment of nematodes with chalcone 17, chalcone 25, or chalcone 30 alone in concentration between 10.sup.4-10.sup.5 M caused between 70-85% death of juvenile nematodes.

(39) Similarly, as depicted in FIG. 7, FIG. 8 and FIG. 9, incubation of eggs in water resulted in hatching of 70% of the eggs which was used as a negative control. Treatment of nematode eggs with either chalcone 17 or chalcone 25 at a concentration of 10.sup.4-10.sup.5 M caused around only 50% of hatched eggs by Day 16.

(40) As depicted in FIGS. 1 and 2, incubation of juvenile nematodes with Peptide 2 alone at a concentration of 0.8 mg/ml resulted in around 60-80% death on Day 5. However, the combination of Peptide 2 with either chalcone 17, chalcone 25, or chalcone 30 resulted in nearly 100% death of juvenile nematodes on Day 5, wherein the concentration of the peptide was 0.8 mg/ml and the concentration of the chalcones was as low as 10.sup.4-10.sup.5 M. Moreover, as depicted in FIG. 2, the combination of Peptide 2 (0.8 mg/ml), and chalcone 17 and chalcone 30 at a ratio of 1:1 with a concentration of 10.sup.4-10.sup.5 M was highly effective.

(41) Similarly, FIGS. 3 and 4 depict that Peptide 3 alone is less effective in killing juvenile nematodes as compared to its combination with chalcones. In case of Peptide 3, the combination with chalcone 25 or chalcone 30 is more effective than combination with chalcone 17. Moreover, as depicted in FIG. 4, the combination of Peptide 3 (0.8 mg/ml), and chalcone 17 and chalcone 30 at a ratio of 1:1 with a concentration of 10.sup.4-10.sup.5 M was immensely effective.

(42) FIGS. 5 and 6 depict that Peptide 12 alone at a concentration of 0.8 mg/ml is highly effective in killing juvenile nematodes, nevertheless, the combination of the peptide with at least one chalcone significantly enhances the potency of the nematicide composition.

(43) As depicted in FIG. 7, the treatment of nematode eggs with Peptide 2 at 0.8 mg/ml concentration caused around 30% of hatched eggs by Day 16, whereas, the combination of Peptide 2 (0.8 mg/ml) and chalcone (10.sup.4-10.sup.5 M) resulted in 10-25% of hatched eggs by Day 16.

(44) As depicted in FIG. 8, the treatment of nematode eggs with Peptide 3 at 0.8 mg/ml concentration caused around 10% of hatched eggs by Day 16, whereas, the combination of Peptide 3 (0.8 mg/ml) and chalcone (10.sup.4-10.sup.5 M) resulted in 10-20% of hatched eggs by Day 16.

(45) As depicted in FIG. 9, the treatment of nematode eggs with Peptide 12 at 0.8 mg/ml concentration caused around 20% of hatched eggs by Day 16, whereas, the combination of Peptide 12 (0.8 mg/ml) and chalcone (10.sup.4-10.sup.5 M) resulted in 10-15% of hatched eggs by Day 16.

(46) These results further suggest that the peptides, Peptide 2 or Peptide 3, or Peptide 12, derived from CED-4 protein, and chalcones have efficient nematicidal activities and have the ability of killing the nematodes and also destroying their eggs.

(47) However, the combination of these peptides with chalcones, specifically, with chalcone 30 showed severe synergistic effect on killing nematodes.

(48) These results suggest that the combination of nematicidal peptides, Peptide 2 or Peptide 3, or Peptide 12 at a concentration of 0.8 mg/ml, with chalcones, either chalcone 17, chalcone 25, or chalcone 30 at a concentration as low as 10.sup.4-10.sup.5 M has a synergistic effect on killing nematodes and are highly potent as nematicidal compositions.