Description:AQUEOUS NEMATICIDAL FORMULATION FOR CONTROLLING PLANT PARASITIC NEMATODES AND PREPARATION THEREOF
FIELD OF THE INVENTION
The present invention relates to the field of agricultural pest control, particularly to a nematicide for controlling plant parasitic nematodes. More particularly, the invention relates to an aqueous formulation comprising a synthetic compound, a microbial metabolite, and a bacterial culture for controlling root-knot nematode Meloidogyne incognita. The present invention further relates to a method of preparing the aqueous formulation.
BACKGROUND OF THE INVENTION
Plant-parasitic nematodes (PPNs) constitute a significant constraint to agricultural productivity, with more than 4,100 identified species responsible for extensive crop damage globally. Among these, root-knot nematodes of the genus Meloidogyne are particularly destructive, causing yield losses of up to 90% in susceptible crops. The damage arises primarily from root gall formation, disruption of normal root function, impaired uptake of water and nutrients, and increased susceptibility of host plants to secondary microbial infections. Meloidogyne incognita is a major economic threat contributing to agricultural losses amounting to billions of dollars annually worldwide
Current management practices for nematode control are dominated by several synthetic classes, including organophosphates, carbamates, halogenated fumigants, and more recently, benzamides and fluoroalkenes. Despite being long-established chemical interventions, they possess inherent technical and ecological disadvantages. Many of these synthetic agents are characterized by high environmental toxicity and a lack of non-target specificity, leading to detrimental impacts on the broader soil ecosystem and beneficial microbial communities. Furthermore, because these products generally rely on a single-compound effect often targeting only one physiological site, they are increasingly subject to reduced efficacy and the development of resistance. In many agricultural scenarios, these conventional treatments are slow in the control of nematodes, allowing the infested population to remain greater than the economic threshold level. Consequently, despite regular application, the productivity of the crops is significantly lesser than their potential due to persistent root damage and impaired nutrient uptake. Additionally, the reliance on standalone biological agents is often limited by inconsistent performance under fluctuating field temperatures and soil moisture levels, failing to provide the robust protection required in intensive cultivation.
The present invention addresses the above drawbacks of the conventional treatments by providing a nematicidal formulation comprising a synthetic compound, a microbial metabolite, and a bacterial culture having a multi-modal effect on the nematodes. It has been found that this specific combination exerts simultaneous inhibitory pressure on Adenosine Triphosphatase, Acetylcholinesterase, and Cytochrome c Oxidase 1 effectively halting the nematode's energy transport, neuromuscular signalling, and mitochondrial respiration. The synergistic interaction of the components ensures rapid suppression of Meloidogyne incognita, maintaining the infested population well below the economic damage threshold. Furthermore, by integrating biological and chemical modes of action, the formulation provides extended residual protection and minimizes the risk of resistance development. As a result, the application of this invention restores root health and crop vigour, leading to an expected higher yield for farmers and a more resilient agricultural ecosystem compared to standard synthetic applications.

OBJECT OF THE INVENTION
The principal object of the present invention is to develop a synergistic composition comprising a synthetic compound, a bioactive microbial metabolite, and a biocontrol agent for controlling plant parasitic nematodes.
Another object of the present invention is to develop a synergistic composition to target specific physiological and effector proteins to effectively inhibit egg hatching and increase juvenile mortality, thereby reducing the survival and reproductive capacity of the nematode population.
Yet another object of the present invention is to develop a synergistic nematicidal composition suitable for application to the soil or seed.
Further object of the present invention is to provide a method of preparing the nematicidal formulation.
Other objects of the inventions will be apparent from the description of the invention herein below.
SUMMARY OF THE INVENTION
In view of the foregoing, the present invention provides an aqueous nematicidal formulation comprising the synergistic blend of a synthetic compound 4-tert-butylbenzoic acid (PTBBA), a bioactive microbial metabolite 2-hydroxy-2,4,6-trimethoxychalcone, and a Bacillus subtilis culture with a suitable surfactant and stabilizer to control Meloidogyne incognita through targeted inhibition of Adenosine Triphosphatase, Acetylcholinesterase, and Cytochrome c Oxidase 1 enzymes which are essential for nematode energy metabolism and neuromuscular function.
In an aspect of the present invention, the amount of synthetic compound 4-tert-butylbenzoic acid (PTBBA) is in the range of 0.2-0.3% v/v of the total formulation.
In an aspect of the present invention, the amount of bioactive microbial metabolite 2-hydroxy-2,4,6-trimethoxychalcone is in the range of 0.2-0.3% v/v of the total formulation.
In an aspect of the present invention, the amount of biocontrol agent Bacillus subtilis culture is in the range of 10-12% v/v of the total formulation.
In an aspect of the present invention, the population of Bacillus subtilis culture ranges from of 1 x 106 -1 x 108 CFU/mL
In an aspect of the present invention, the amount of surfactant polysorbate 80 is in the range of 0.1-0.2% v/v of the total formulation.
In an aspect of the present invention, the amount of stabilizer glycerol is in the range of 0.5 -1.5 % v/v of the total formulation.
In another aspect, the present invention provides a process for preparing the aqueous nematicidal formulation and methods for its application to control Meloidogyne incognita.
BRIEF DESCRIPTION OF THE DRAWINGS
For a better understanding of the invention, few embodiments are described below with reference to the accompanying figures, purely by way of example and non-limiting in which:
FIG. 1 illustrates the two-dimensional (2D) representation of the interaction between 4-tert-butylbenzoic acid (PTBBA) and the protein targets Acetylcholinesterase (1A) and Adenosine Triphosphatase (1B);
FIG. 2 illustrates the two-dimensional (2D) representation of the interaction between 2-hydroxy-2,4,6-trimethoxychalcone with the protein target Cytochrome c oxidase 1;
FIG. 3 illustrates the docking site of 4-tert-butylbenzoic acid (PTBBA) with the protein targets Acetylcholinesterase (3A) and Adenosine Triphosphatase (3B);
FIG. 4 illustrates the docking site of 2-hydroxy-2,4,6-trimethoxychalcone with the protein target Cytochrome c oxidase 1;
FIG.5 demonstrates the in-vitro compatibility of 4-tert-butylbenzoic acid (PTBBA) and 2-Hydroxy-2,4,6-trimethoxychalcone with Bacillus subtilis;
FIG. 6 demonstrates the effect of the aqueous formulation of the present invention on eggs of Meloidogyne incognita in which 6A shows the live nematode eggs in absolute control-T1 and 6 B shows the complete deformed eggs after treating with the present aqueous formulation-T6;
FIG. 7 demonstrates the effect of aqueous formulation of the present invention on juveniles of Meloidogyne incognita in which 7A shows the live juveniles in T1 and 7B shows the dead nematodes in T6;
FIG. 8 shows the efficacy of the aqueous formulation against Meloidogyne incognita in pot culture, where (A) shows the absolute control with severe root galling due to nematode infestation and (B) shows treatment with the aqueous formulation with significant reduction in root gall formation; and
FIG. 9 shows the field trial results demonstrating significantly reduced root galling and enhanced plant growth in treated crops compared to the untreated control.
DETAILED DESCRIPTION OF THE INVENTION
For the better understanding of the objects, technology and advantages of the present invention, the instant invention will be further explained in detail with respect to embodiments as given below. It should be understood that the specific embodiments described herein are only to be used for explaining the present invention but not used to limit the present invention.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well as the singular forms, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof.
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one having ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
In describing the invention, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefit and each can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques. Accordingly, for the sake of clarity, this description will refrain from repeating every possible combination of the individual steps in an unnecessary fashion. Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the invention and the claims.
In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It will be evident, however, to one skilled in the art that the present invention may be practiced without these specific details.
As is well known to those skilled in the art, many careful considerations and compromises typically must be made when designing for the optimal configuration of a commercial implementation of any process or formulation, and in particular, the embodiments of the present invention. A commercial implementation in accordance with the spirit and teachings of the present invention may be configured according to the needs of the particular application, whereby any aspect(s), feature(s), function(s), result(s), component(s), approach(es), or step(s) of the teachings related to any described embodiment of the present invention may be suitably omitted, included, adapted, mixed and matched, or improved and/or optimized by those skilled in the art, using their average skills and known techniques, to achieve the desired implementation that addresses the needs of the particular application
The present invention provides, in an aspect, a formulation comprising a synthetic compound 4-tert-butylbenzoic acid (PTBBA), a bioactive microbial metabolite 2-hydroxy-2,4,6-trimethoxychalcone, and a Bacillus subtilis culture, in a carrier along with a suitable stabilizer and surfactant for controlling the root-knot nematode Meloidogyne incognita.
The present invention, for the first time, discloses that the synergistic combination of 4-tert-butylbenzoic acid (PTBBA), 2-hydroxy-2,4,6-trimethoxychalcone, and Bacillus subtilis in a definite proportion is effective in inhibiting the enzymatic activities essential for nematode survival.
After large number of experimentations, the inventors of the present invention identified the synergistic blend of synthetic compound 4-tert-butylbenzoic acid (PTBBA), bioactive microbial metabolite 2-hydroxy-2,4,6-trimethoxychalcone, and Bacillus subtilis to be effective in suppressing the activities of Adenosine Triphosphatase, Acetylcholinesterase, and Cytochrome c Oxidase 1, thereby controlling the root-knot nematodes.
In an embodiment, the aqueous nematicidal formulation for controlling root-knot nematode Meloidogyne incognita comprises 4-tert-butylbenzoic acid (PTBBA), 2-hydroxy-2,4,6-trimethoxychalcone, Bacillus subtilis culture, polysorbate 80 and glycerol for controlling Root-knot nematode Meloidogyne incognita.
In an embodiment, the amount of synthetic compound 4-tert-butylbenzoic acid (PTBBA) is in the range of 0.2-0.3% v/v of the total formulation.
In an embodiment, the amount of bioactive microbial metabolite 2-hydroxy-2,4,6-trimethoxychalcone is in the range of 0.2-0.3% v/v of the total formulation.
In an embodiment, the amount of biocontrol agent Bacillus subtilis culture is in the range of 10-12% v/v of the total formulation.
In an embodiment, the Bacillus subtilis culture is used with a microbial load ranging from of 1 x 106 -1 x 108 CFU/mL
In an embodiment, the synthetic compound, microbial metabolite, and bacterial culture are solubilized with a surfactant and a stabilizer to improve the effectiveness of the formulation for use in controlling nematode infestation and root galling.
After large number of experimentations, the inventors of the present invention identified polysorbate 80 (tween-80) as a suitable surfactant and glycerol as a suitable stabilizer to ensure the homogeneity and biological viability of the formulation.
In an embodiment, the amount of polysorbate 80 is in the range of 0.1-0.2% v/v of the total formulation.
In an embodiment, the amount of glycerol is in the range of 0.5 -1.5 % v/v of the total formulation.
In one embodiment, the formulation is a liquid comprising 88 to 89% v/v distilled water based on the total volume of the formulation.
In an embodiment, the synthetic compound 4-tert-butylbenzoic acid (PTBBA), bioactive microbial metabolite 2-hydroxy-2,4,6-trimethoxychalcone, polysorbate 80 and glycerol are procured from standardized commercial vendors in Bengaluru, Karnataka. The biocontrol agent Bacillus subtilis was isolated from soil samples collected at Coimbatore, Tamil Nadu. The strain has been sequence-characterized, and the 16S rRNA gene sequence has been submitted to the NCBI GenBank database under accession number NCBI Accession No. PX599689.
The present invention, in another aspect, provides a process for preparing the aqueous nematicidal formulation comprising:
(a) preparing 4-tert-butylbenzoic acid (PTBBA) solution comprising dissolving 4-tert-butylbenzoic acid (PTBBA) in ethanol, homogenizing at 450- 500 rpm for 45-60 mins using a magnetic stirrer, wherein the amount of 4-tert-butylbenzoic acid (PTBBA) is in the range of 0.5–1.5% w/v;
(b) preparing 2-hydroxy-2,4,6-trimethoxychalcone solution comprising dissolving 2-hydroxy-2,4,6-trimethoxychalcone in ethanol homogenizing at 450- 500 rpm for 45-60 mins using a magnetic stirrer, wherein the amount of 2-hydroxy-2,4,6-trimethoxychalcone is in the range of 0.5–1.5% w/v;
(c) preparing Bacillus subtilis culture comprising inoculating Bacillus subtilis in nutrient broth, incubating and centrifuging to obtain the culture;
(d) mixing solutions obtained from steps (a) and (b) in a 1:1 volume ratio with water at 24 -26 °C, followed by addition of polysorbate 80 with continuous stirring at 600-700 rpm using a magnetic stirrer for 30-40 mins, maintaining the pH between 6.8 and 7.2;
(e) adding the culture obtained from step (c) to the mixture obtained in step (d), stirring at 140-160 rpm using a magnetic stirrer for 12-15 mins at 24 -26 °C; and
(f) adding glycerol and water at 24 -26 °C, stirring at 80-120 rpm using a magnetic stirrer and maintaining the pH between 6.8 and 7.2 for 10-12 mins to obtain the aqueous nematicidal formulation.
In an embodiment, a method for controlling the root-knot nematode Meloidogyne incognita in plants comprises applying the aqueous nematicidal formulation via soil drenching or seed treatment.
In an embodiment, the formulation is applied as a soil drench at a dosage of 2.5 to 7 mL per plant, ensuring uniform distribution of the synthetic and biological components
In an embodiment, the formulation is applied as a seed treatment, wherein seeds are coated with the synergistic formulation at a concentration of 0.5 to 2 mL per 100 g of seeds to provide early-stage protection against nematode penetration.
The above description of the invention, together with the below accompanying examples should not be construed as limiting the invention because those skilled in the art to which this invention pertains will be able to devise other forms thereof within the ambit of the appended claims.

EXAMPLES
Example 1: Selection of the compounds and study on the binding affinities and molecular docking interactions of 4-tert-butylbenzoic acid (PTBBA) and 2-Hydroxy-2,4,6-trimethoxychalcone compounds against the protein targets of Meloidogyne incognita
The synthetic compound 4-tert-butylbenzoic acid (PTBBA) and the bioactive microbial metabolite 2-Hydroxy-2,4,6-trimethoxychalcone were selected after multiple screening tests against different protein targets including Acetyl choline esterase for chemosensory roles, Adenosine Triphosphatase for energy regulation, and Cytochrome c oxidase 1 for the oxidative phosphorylation pathway.
Target Modeling and Validation: Protein targets for Meloidogyne incognita (Acetylcholinesterase, Adenosine Triphosphatase, and Cytochrome c oxidase 1) were retrieved from UniProt.3D homology models were constructed using MODELLER 10.4 and templates from the Protein Data Bank. Structural integrity was validated via Ramachandran Plot analysis through the Structural Analysis and Verification Server (PROCHECK), ensuring all residues were in allowed regions, while active sites were mapped using Computed Atlas of Surface Topography of proteins.
Molecular Docking: Molecular docking using the Vina Wizard (PyRx 0.8) with ligands from PubChem. Energy minimization was conducted via United Force Field (UFF). The resulting binding affinities and protein-ligand interactions were visualized in BIOVIA Discovery Studio, confirming that the present aqueous formulation provides superior synergistic inhibition on the nematode's metabolic and neuromuscular pathways.
Table 1: Binding affinities and molecular docking interactions of 4-tert-butylbenzoic acid (PTBBA) and 2-Hydroxy-2,4,6-trimethoxychalcone compounds against the protein targets of Meloidogyne incognita.
Nematicidal compound Protein targets Binding affinity (kcal/mol) Atoms involved in H bond formation
4-tert-butylbenzoic acid Acetylcholinesterase -8.3 ARG 81; GLN 82
Adenosine Triphosphatase -7.8 ALA 250; PHE 217
2-Hydroxy-2,4,6-trimethoxychalcone Cytochrome c oxidase 1 -7.2 THR 188

The two-dimensional (2D) interactions of 4-tert-butylbenzoic acid with the protein targets Acetylcholinesterase (1A) and Adenosine Triphosphatase (1B) are illustrated in Fig 1 while the two-dimensional (2D) interactions of 2-Hydroxy-2,4,6-trimethoxychalcone with the protein target Cytochrome c oxidase 1 is illustrated in Fig 2.
The specific binding sites and spatial configurations of 4-tert-butylbenzoic acid within the catalytic pockets of Acetylcholinesterase and Adenosine Triphosphatase are demonstrated in Fig (3A) and (3B). Furthermore, the structural orientation of 2-Hydroxy-2,4,6-trimethoxychalcone at the active site of the protein target Cytochrome c oxidase 1 is demonstrated in FIG. 4.
Example 2: In-vitro compatibility of the identified synthetic compound 4-tert-butylbenzoic acid (PTBBA) & the bioactive microbial metabolite 2-Hydroxy-2,4,6-trimethoxychalcone with the biocontrol agent of Bacillus subtilis.
The identified compounds (4-tert-butylbenzoic acid & 2-Hydroxy-2,4,6-trimethoxychalcone) were evaluated for their compatibility with Bacillus subtilis at different concentrations, specifically at 20, 50, 100, and 150 ppm. This study was conducted to assess the effect of the synthetic compound and microbial metabolite on the growth and viability of the bacterial culture in the formulation.
For the preparation of treatments, stock solutions of the synthetic compound 4-tert-butylbenzoic acid (PTBBA) and the bioactive microbial metabolite 2-hydroxy-2,4,6-trimethoxychalcone were prepared at a concentration of 10,000 ppm using the solvent to ensure complete solubility. To achieve standardized chemical exposure, specific volumes of these stocks were incorporated into individual Petri plates containing 20 mL of Nutrient Agar media. The Nutrient Agar media comprised Peptone (10.0 g/L), Yeast extract (3 g/L), Sodium chloride (5.0 g/L), and Agar (15.0 g/L), with the final pH adjusted to 7 at 25°C. Specifically, stock volumes of 0.04 mL, 0.1 mL, 0.2 mL, and 0.3 mL were added to the media to achieve final required test concentrations of 20 ppm, 50 ppm, 100 ppm, and 150 ppm respectively, for each compound. The Bacillus subtilis culture was streaked onto the supplemented media surface. The treatments were incubated at 26°C for 48 hours. The primary objective was to observe whether the culture remained active and continued to grow when exposed to increasing levels of 4-tert-butylbenzoic acid (PTBBA) and 2-Hydroxy-2,4,6-trimethoxychalcone.
The results (Fig 5) suggested that in all the tested concentrations (20 (A), 50 (B), 100 (C), and 150 (D) ppm), there was no inhibition in the growth of the bacteria. The culture maintained its viability and showed consistent growth throughout the observation period. This indicates the compound’s complete compatibility with the bacterial culture, confirming that 4-tert-butylbenzoic acid (PTBBA) and 2-Hydroxy-2,4,6-trimethoxychalcone can be used in combination with Bacillus subtilis without any adverse effect on its growth.

Example 3: Method of preparation of the aqueous formulation of the present invention
Step 1: Preparation of 4-tert-butylbenzoic acid (PTBBA) solution: 2 mL of 70% ethanol is pipetted into a 10 mL vial using a 100–1000 µl Accupipet. 0.02 g (1% w/v) of 4-tert-butylbenzoic acid (PTBBA) is added to achieve a 10000ppm stock. The mixture is stirred at 500 rpm for 1 hour using a magnetic stirrer until a completely homogeneous state is achieved at a controlled ambient temperature of 26°C.
Step 2: Preparation of 2-Hydroxy-2,4,6-trimethoxychalcone solution: 2 mL of 70% ethanol is pipetted into a 10 mL vial using a 1000 µl Accupipet. 0.02 g (1% w/v) of 2-Hydroxy-2,4,6-trimethoxychalcone is added to achieve a 10000ppm stock. The mixture is stirred at 500 rpm for 1 hour at a controlled ambient temperature of 26°C to achieve a homogeneous dispersion.
Step 3: Preparation of Bacillus subtilis culture: A pure strain of Bacillus subtilis (NCBI Accession No. PX599689) is inoculated into 100 mL Nutrient Broth comprising Peptone (10.0 g/L), Yeast extract (3 g/L) and Sodium chloride (5.0 g/L) with the final pH adjusted to 7 in a 250 mL conical flask under aseptic conditions in a Laminar airflow chamber. The culture is incubated at 26°C with orbital shaking at 120 rpm for 48 hours. Following incubation, cells are harvested via cooling centrifugation at 4°C at 6000 rpm for 10 minutes. The supernatant is discarded, and the microbial pellet of the bacterial culture is washed twice with chilled sterile distilled water (4°C).
Step 4: Primary Dispersion: In a 1 L sterile beaker containing 500 mL of sterile distilled water maintained at 26°C, 2 mL of the 4-tert-butylbenzoic acid (PTBBA) solution and 2 mL of the 2-Hydroxy-2,4,6-trimethoxychalcone solution are slowly added using an Accupipet. Subsequently, 1 mL of polysorbate 80 is added as a surfactant to facilitate the dispersion of the insoluble compounds. The combined mixture is stirred at 600 rpm for 30 minutes using a magnetic stirrer to achieve a uniform dispersion. The pH of the dispersion should be monitored and maintained at 7 to ensure chemical stability before the addition of the biological component.
Step 5: Inoculum Incorporation: 100 mL of the standardized Bacillus subtilis culture is slowly poured down the side of the beaker into the mixture obtained in step 4 to protect the bacterial cell membranes from osmotic shock. To prevent mechanical shear stress on the bacterial cell membranes, the agitation is set to a gentle 150 rpm and stirred for 15 minutes at 26°C to ensure the microbes are evenly distributed throughout the 4-tert-butylbenzoic acid (PTBBA) and 2-Hydroxy-2,4,6-trimethoxychalcone mixture. The final cell density is adjusted to 1 x 106 CFU/mL, verified through standard plate count methods
Step 6: Final Stabilization: 10 mL of glycerol is added to the inoculated dispersion obtained in step 5 to act as a cryoprotectant and stabilizer. To ensure optimal biological activity and chemical compatibility of the active metabolites, the pH of the composition is adjusted to 7.0 using 0.1 N NaOH or 0.1 N HCl as required. The final volume is adjusted to 1 Liter by gradually adding 385 mL of sterile distilled water at 26°C. The formulation is stirred at 100 rpm for an additional 10 minutes to ensure total homogeneity. The resulting formulation is transferred into amber-colored, airtight containers and stored at 26°C for long-term stability and high-efficacy suppression of Meloidogyne incognita.
Example 4: Selection and optimization of the concentration of the synergistic formulation for control of root-knot nematode Meloidogyne incognita
To select and optimize the concentration of synergistic components of the present formulation, the following treatments were followed:
T1: Absolute control
T2: Synthetic compound 4-tert-butylbenzoic acid (PTBBA) @ 20 ppm
T3: Bioactive microbial metabolite 2-Hydroxy-2,4,6-trimethoxychalcone @ 20 ppm
T4: Bacillus subtilis @ 1×106 CFU/mL
T5: 4-tert-butylbenzoic acid (PTBBA) +2-Hydroxy-2,4,6-trimethoxychalcone @ 20 ppm each
T6: 4-tert-butylbenzoic acid (PTBBA) +2-Hydroxy-2,4,6-trimethoxychalcone @ 20 ppm each + Bacillus subtilis @ 1×106 CFU/mL
All the treatments were prepared using sterile distilled water as a carrier.
The experimental field (Capsber Global Agro Private Limited) is situated at approximately 13.29°N latitude and 77.53°E longitude, with an elevation of about 900 m above MSL. The average annual rainfall of this region is approximately 700 mm. The Meloidogyne incognita infested Tomato (Solanum lycopersicum) roots were collected and washed under a gentle stream of water. Uniformly sized egg masses were hand-picked from the galls and surface sterilized with 0.5% NaOCl for 2 minutes followed by five washings with sterile water. The egg masses were incubated at 27°C for 72 hours to allow hatching. The freshly hatched second-stage juveniles (J2) were prepared as a suspension at a concentration of 100 J2/mL counting dish under a stereomicroscope for use in further assays. Viability was verified before use and inoculations were conducted within 24 h of collection to ensure optimal activity.
To evaluate egg hatching inhibition, 2 mL of each treatment (T1–T6) was first dispensed into sterile Petri plates. Subsequently, an inoculum containing 100 freshly collected Meloidogyne incognita eggs was introduced into each and incubated at 27°C. The number of hatched juveniles was recorded at 24, 48 and 72 hours post-inoculation. Distilled water served as separate controls.
Egg hatching inhibition (%)= (Control-Treatment)/Control ×100
Treatment 24h 48h 72h
T1 87.21a 88.26a 89.76a
T2 36.64b 38.36b 38.14b
T3 33.41c 34.23c 36.83b
T4 13.35e 14.25e 14.14d
T5 18.36d 19.44d 20.63c
T6 4.26f 4.83f 5.44e
CD 0.97 0.91 1.58
SE(d) 0.44 0.41 0.72
Table 2: In vitro Evaluation of Synergistic Treatments on the Egg Hatching Inhibition of Meloidogyne incognita.
As shown in Table 2, the egg hatching inhibition assessment revealed that T2 and T3 inhibited hatching by 65% and 60% respectively, while both T4 and T5 recorded 80% inhibition. The treatment T6 which is a triple combination of synthetic compound, bioactive microbial metabolite and bacterial culture showed the highest inhibition at 95%, confirming the synergy of the blend.
To evaluate Juvenile mortality, 2 mL of each treatment was applied to sterile Petri plates containing a suspension of Meloidogyne incognita J2 suspension (100 J2/mL Plates were incubated at 27°C, and the juvenile mortality was recorded at 24, 48 and 72 hours post-inoculation. Mortality was determined based on nematode motility, with active juveniles considered alive and those that did not respond to repeated touch classified as dead. Mortality percentages were calculated using the following formula
Juvenile mortality (%)= (Number of dead juveniles)/(Total number of juveniles) ×100

Table 3: In vitro Evaluation of Synergistic Treatments on the Mortality of Meloidogyne incognita Second – stage Juveniles (J2).

Treatment 24h 48h 72h
T1 4.83e 6.13e 7.33f
T2 62.31d 63.81d 65.33e
T3 67.84c 69.77c 71.33c
T4 68.13c 68.43c 70.15d
T5 92.46b 92.77b 94.17b
T6 94.33a 95.03a 96.27a
CD 1.43 1.71 0.89
SE(d) 0.76 0.77 0.40

As shown in Table 3, the Juvenile mortality assessment revealed that T2 and T3 recorded 65% and 71% mortality respectively, while T4 and T5 recorded 70% and 94% mortality respectively. Whereas the triple combination T6 showed the highest mortality with 96% confirming the synergy of the blend. This efficacy is attributed to the synergistic action where 4-tert-butylbenzoic acid (PTBBA) disrupts egg development via Acetylcholinesterase and Adenosine Triphosphatase inhibition, while 2-Hydroxy-2,4,6-trimethoxychalcone induces mortality by targeting COX-1 proteins, further potentiated by the multifaceted biocontrol mechanisms of Bacillus subtilis.
Figures 6 and 7 demonstrate the effect of the aqueous formulation on eggs and juvenile of Meloidogyne incognita respectively in which 6A shows the live nematode eggs in absolute control-T1 6B shows the complete deformed eggs after treating with the present aqueous formulation-T6; 7A shows the live juveniles in T1 and 7B shows the dead nematodes in T6.
Example 5: In planta efficacy of aqueous formulation against Meloidogyne incognita
To evaluate the efficacy of the individual treatments which were previously assessed under in vitro bioassays against Meloidogyne incognita, pot experiments were conducted using tomato plants in a greenhouse. Fifteen-day-old tomato seedlings were transplanted into 2 kg pots containing a sterilized mixture of red soil, farmyard manure (FYM), and sand (2:1:1 ratio). Each pot (except for the healthy control) was inoculated with 2000 juveniles (J2) of Meloidogyne incognita 10 days post-planting. The greenhouse conditions were maintained at 50% shade, 26°C, with controlled irrigation.
The experiment followed a Completely Randomized Block Design (CRD) with three replications. Treatments included:
T1: Nematode inoculated (Untreated control)
T2: Uninoculated control (Healthy)
T3: 4-tert-butylbenzoic acid (PTBBA) @ 20 ppm
T4: 2-Hydroxy-2,4,6-trimethoxychalcone @ 20 ppm
T5: Bacillus subtilis @ 1×106 CFU/mL.
T6: 4-tert-butylbenzoic acid (PTBBA) +2-Hydroxy-2,4,6-trimethoxychalcone @ 20 ppm each
T7: 4-tert-butylbenzoic acid (PTBBA) +2-Hydroxy-2,4,6-trimethoxychalcone @ 20 ppm each + Bacillus subtilis @ 1×106 CFU/mL
All treatments were applied via soil drenching at a rate of 2 mL per pot. Observations on plant growth and nematode suppression were recorded 45 days after transplanting.
Table 4: Comparative efficacy of individual components and synergistic aqueous formulation on plant growth parameters and suppression of Meloidogyne incognita population under greenhouse conditions.

Treatment Shoot
length (cm) Fresh
shoot weight
(g) Root length (cm) Fresh Root weight
(g) Total nematode/100cc soil Egg mass/5g root
(g) No. of Galls/plant Root knot index
T1 36.27f 17.45e 12.33f 9.75e 142.75a 30.63a 93.71a 4.33a
T2 52.47a 27.63a 21.51e 13.23d 0.00g 0.00g 0.00g 0.00e
T3 42.67e 22.63d 28.98d 16.37c 57.43b 26.67b 36.96b 1.83b
T4 45.07d 24.43c 31.61c 16.55c 41.56c 21.71d 26.55c 1.76b
T5 46.51c 25.07bc 32.96c 17.21bc 32.55d 18.38e 24.54d 1.28c
T6 46.94c 24.97bc 35.27b 17.62ab 21.53f 25.38c 13.16e 0.71d
T7 49.81b 25.87b 37.61a 18.25a 27.28e 9.61f 4.73f 0.51d
CD (5%) 1.18 1.16 1.60 0.99 1.44 1.23 1.41 0.43
SE(d) 0.54 0.53 0.74 0.44 0.66 0.56 0.65 0.20

As shown in table 4, T7 reported an 88.9% reduction in total nematodes per 100 cc of soil, a 63% reduction in egg masses, and a 67.5% decrease in galling compared to the untreated infested control (T1). Most notably, the Root Knot Index (RKI) was reduced from a severe 4.33 in the infested control to a negligible 0.51 in the aqueous formulation treatment. These results demonstrate that the formulation effectively disrupts the nematode life cycle and root colonization, providing a comprehensive protective and curative effect on the host plant's root system.
The Nematicidal Efficacy of the Synergistic Aqueous Formulation against Meloidogyne incognita infestation in Pot Culture is shown in Fig 8.
Example 6: Field Efficacy of the aqueous formulation of the present invention against Meloidogyne incognita
Field experiments were conducted (Fig 9) to evaluate the efficacy of the formulation at varying dosage levels against Meloidogyne incognita under controlled inoculation conditions. The trials were laid out in a Randomized Block Design (RBD) consisting of four treatments. Each experimental unit was established as a 2.0 m x 2.0 m micro-plot, with a standard plant spacing of 75 x 35 cm. Fifteen-day-old tomato seedlings were transplanted into a field pre-infested with Meloidogyne incognita. To maintain experimental accuracy and mitigate border effects, all critical growth and yield parameters, including root weight and fruit yield, were recorded exclusively from the central net-plot plants.
To ensure long-term suppression of the nematode population throughout the crop cycle, a systemic protection strategy was implemented using a triple-drenching protocol. One week after transplanting, first soil drenching of the formulation was carried out using 2.5 mL, 5 mL, and 7 mL. During the crop period, the soil was then treated twice with formulation at one-month interval. At harvest, the Reproduction Factor and total fruit yield per10 plants were calculated to determine the biological and economic efficacy of the synergistic treatments.
The experiment followed a Randomized Block Design (RBD) with four treatments, each replicated five times across five distinct blocks to ensure statistical validity. The treatments included:
T1: Nematode inoculated (Control)
T2: Drenching of composition @ 2.5 mL / plant
T3: Drenching of composition @ 5 mL / plant
T4: Drenching of composition @ 7 mL / plant
Table 5: Dosage-dependent efficacy of the aqueous formulation on plant growth promotion and suppression of Meloidogyne incognita in tomato under Randomized Block Design (RBD) of field conditions.
Treatment Shoot length (cm) Root Length (cm) Fresh Root weight
(g) No. of Galls/ root No. of egg masses/ root Reproduction factor (RF) Yield
(kg/10 plants)
T1 38.03d 14.16d 16.36d 96.56a 84.64a 0.91a 4.03d
T2 51.66c 19.01c 19.91c 58.26b 50.13b 0.83b 8.34c
T3 64.76b 23.65b 25.83b 35.23c 28.55c 0.77c 14.45b
T4 75.23a 27.93a 31.37a 17.91d 12.46d 0.56d 19.45a
CD (5%) 2.53 1.46 2.64 2.15 2.05 0.09 1.17
SE(d) 1.01 0.58 1.06 0.86 0.82 0.03 0.46

As shown in Table 5, the aqueous formulation applied at 7 mL per plant (T4) reported a 90.2% reduction in the Reproduction Factor (RF) and an 81.1% reduction in root galling compared to the untreated infested control (T1). Most notably, the treatment at this concentration suppressed the formation of egg masses by 85.3%, effectively disrupting the nematode life cycle and preventing root colonization. These results demonstrate that the application at 7 mL per plant provides a comprehensive protective and curative effect, restoring the plant’s physiological health and achieving a superior yield of 19.45 kg/10 plants.
Example 7: Method of application of the aqueous nematicidal formulation of the present invention
The aqueous nematicidal formulation of the present invention is developed for versatile application to ensure the synergistic components 4-tert-butylbenzoic acid (PTBBA), 2-hydroxy-2,4,6-trimethoxychalcone, and Bacillus subtilis) effectively reach the target Meloidogyne incognita population in the rhizosphere.
For soil drenching application, the total required volume of the formulation for a given area is first diluted into a larger carrier volume of water as required to facilitate uniform field distribution. The drenching is performed using a drenching rod or a knapsack sprayer with the nozzle removed to ensure the active ingredients reach a soil depth of 10 to 15 cm. By delivering this precise concentration to the rhizosphere, the synthetic and biological components can effectively adsorb to the soil and root surfaces to significantly control Meloidogyne incognita. The formulation is applied as a soil drench at a dosage of 2.5 to 7 mL per plant.
For seed treatment application, the aqueous nematicidal formulation is applied at a concentration of 0.5 to 2 mL per 100 g of seeds. To ensure maximum adherence of the 4-tert-butylbenzoic acid (PTBBA) and microbial components, a 1% v/v solution of a sticking agent is optionally added to form a slurry. The treated seeds are then shade-dried for 2 to 4 hours to maintain the viability of the Bacillus subtilis population and the stability of the bioactive metabolites before being sown to provide early-stage protection against nematode penetration. This method ensures that the biological and synthetic agents are immediately available in the spermosphere to inhibit the nematode's enzymatic pathways as soon as the radicle emerges.
While the invention has been described in connection with an illustrative embodiment, it is not intended to limit the scope of the present disclosure to the particular form set forth, but on the contrary, it is intended to cover such alternatives, modifications, and equivalents as may be included within the scope of the present disclosure.
, Claims:We claim:
1. An aqueous nematicidal formulation comprising:
0.2-0.3% v/v 4-tert-butylbenzoic acid;
0.2-0.3% v/v 2-hydroxy-2,4,6-trimethoxychalcone;
0.1-0.2% v/v polysorbate 80;
0.5 -1.5 % v/v glycerol; and
10-12% v/v Bacillus subtilis culture with a population of 1 x 106 -1 x 108 CFU/mL
2. The formulation as claimed in claim 1, wherein the formulation is applied as a soil drench at a dosage of 2.5 to 7 mL per plant or as a seed treatment at a dosage of 0.5 to 2 mL per 100 g of seeds.
3. A process for preparing the aqueous nematicidal formulation comprising:
(a) preparing 4-tert-butylbenzoic acid solution comprising dissolving 4-tert-butylbenzoic acid in ethanol, homogenising at 450- 500 rpm for 45-60 mins using a magnetic stirrer, wherein the amount of 4-tert-butylbenzoic acid is in the range of 0.5–1.5% w/v;
(b) preparing 2-hydroxy-2,4,6-trimethoxychalcone solution comprising dissolving 2-hydroxy-2,4,6-trimethoxychalcone in ethanol homogenising at 450- 500 rpm for 45-60 mins using a magnetic stirrer, wherein the amount of 2-hydroxy-2,4,6-trimethoxychalcone is in the range of 0.5–1.5% w/v;
(c) preparing Bacillus subtilis culture comprising inoculating Bacillus subtilis in nutrient broth, incubating and centrifuging to obtain the culture;
(d) mixing solutions obtained from steps (a) and (b) in a 1:1 volume ratio with water at 24 -26°C, followed by addition of polysorbate 80 with continuous stirring at 600-700 rpm using a magnetic stirrer for 30-40 mins, maintaining the pH between 6.8 and 7.2;
(e) adding the culture obtained from step (c) to the mixture obtained in step (d), stirring at 140-160 rpm using a magnetic stirrer for 12-15 mins at 24 -26 °C; and
(f) adding glycerol and water at 24 -26 °C, stirring at 80-120 rpm using a magnetic stirrer and maintaining the pH between 6.8 and 7.2 for 10-12 mins to obtain the aqueous nematocidal formulation.
4. The process as claimed in claim 3, wherein the 4-tert-butylbenzoic acid is 0.2-0.3 % v/v of the total formulation.
5. The process as claimed in claim 3, wherein the 2-hydroxy-2,4,6-trimethoxychalcone is 0.2-0.3 % v/v of the total formulation.
6. The process as claimed in claim 3, wherein the polysorbate 80 is 0.1-0.2 % v/v and glycerol is 0.5 -1.5 % v/v of the total formulation.
7. The process as claimed in claim 3, wherein the Bacillus subtilis culture is 10-12 % v/v with a population of 1 x 106 -1 x 108 CFU/mL of the total formulation.