Essential oil-based controlled-release nematocidal composition and uses thereof

Abstract

The present invention provides an eco-friendly, biodegradable, controlled-release pesticidal composition, comprising particular essential oils acting in synergy, adsorbed onto a silicate mineral carrier and embedded within a polysaccharide matrix, which is effective in suppressing nematode populations and enhancing plant health and growth, and methods of use.

Description

ESSENTIAL OIL-BASED CONTROLLED-RELEASE NEMATOCIDALCOMPOSITION AND USES THEREOFTECHNICAL FIELD

[0001] The present invention provides an eco-friendly, biodegradable, controlled-release pesticidal composition, comprising particular essential oils adsorbed onto a silicate mineral carrier and embedded within a polysaccharide matrix, which is effective in suppressing nematode populations and enhancing plant health and growth, and methods of use.BACKGROUND ART

[0002] Nematodes, slender worm-like organisms, are among the most varied animal phyla, with over 28,000 species cataloged, of which around 16,000 are parasitic. These pests pose a significant challenge in agriculture, as they can cause extensive damage to plant root systems, leading to reduced crop yields and substantial economic losses. Their impact is extensive, with some nematode pest families including over 30 species that affect more than 2,000 plant varieties. This broad spectrum of predation makes nematodes a major concern for global agriculture, with notable effects in regions including Israel.

[0003] Nematode damage to plants is complex, often mimicking drought conditions through symptoms like wilting and nutrient deficiencies, further diminishing productivity and plant health. The wide host range of nematodes makes them particularly harmful to essential crops such as corn, impacting both plant health and yield significantly.

[0004] The agricultural industry has traditionally relied on chemical pesticides to control nematode infestations. Although these treatments can effectively curb nematode populations and reduce damage, they come with notable disadvantages. The reliance on chemical pesticides is fraught with environmental and safety concerns, including risks to non-target species, pollution, and the development of pesticide resistance. Thus, there is an increasing demand for innovative, eco-friendly approaches to nematode management that avoid the drawbacks of conventional chemical treatments.

[0005] The nematocidal potential of essential oils is well-established in the scientific community. However, their effectiveness in the field is limited by their high volatility, which necessitates the use of carriers that can reduce evaporation and allow for controlled release, ensuring sustained pest control efficacy. Direct application in the field without protection may lead to unreliable results, as disclosed, e.g., in Oka et al. (2000), who notes the lack ofeffectiveness of limonene in controlling soil-dwelling nematodes, in contrast to the pronounced effectiveness of thyme extracts (thymol and carvacrol). This could be due to the rapid dissipation of limonene rather than an actual absence of effectiveness. Supporting this view, recent research demonstrated limonene's nematocidal efficiency in lab settings without evaporation risks (Ardakani and Hosseininejad, 2022). These findings underscore the necessity for a delivery system that can leverage the powerful nematocidal properties of essential oils like limonene and thymol, ensuring their stability and field efficacy.SUMMARY OF INVENTION

[0006] In one aspect, disclosed herein is a pesticidal composition, more specifically a nematocidal composition, comprising essential oils comprising thymol and limonene, adsorbed onto a silicate mineral carrier, wherein said mineral carrier is embedded within a polysaccharide matrix including alginate.

[0007] In certain embodiments, the nematocidal composition disclosed comprises a combination of thymol and limonene at a weight ratio of about 1: 1, adsorbed onto a silicate mineral carrier composed of kaolinite, montmorillonite and a zeolite, preferably at a weight ratio of 1: 1: 1, which is embedded within a polysaccharide matrix comprising alginate, pectin and chitosan, wherein said alginate constitutes at least 30% by weight of said polysaccharide matrix and preferably at a weight ratio of about 85: 10:5, respectively.

[0008] In another aspect, disclosed herein is a method of controlling soilborne nematodes (also referred to herein as “soil nematodes”) infection in a locus in need thereof or protecting said locus from soilborne nematodes infestation, said method comprising applying to said locus an effective amount of a composition as defined above, wherein release of said essential oils from said composition is activated primarily by, and synchronized with, soil moisture conditions including irrigation or rainfall.

[0009] In yet another aspect, disclosed herein is a method for treating a plant against soilborne nematodes infection, or protecting said plant from soilborne nematodes infestation, said method comprising applying to a locus in which said plant is cultivated an effective amount of a composition as defined above, wherein release of said essential oils from said composition is activated primarily by, and synchronized with, soil moisture conditions including irrigation or rainfall.

[0010] In still another aspect, disclosed herein is a method for promoting plant growth and resilience under biotic or abiotic stress conditions, said method comprising applying to a locusin which said plant is cultivated an effective amount of a composition as defined above, wherein release of said essential oils from said composition is activated primarily by, and synchronized with, soil moisture conditions including irrigation or rainfall.

[0011] In a further aspect, disclosed herein is use of a composition as defined above for controlling soilborne nematodes infection in a locus or protecting a locus from soilborne nematodes infestation; for treating a plant against soilborne nematodes infection or protecting a plant from soilborne nematodes infestation; or for promoting plant growth and resilience under biotic or abiotic stress conditions.BRIEF DESCRIPTION OF DRAWINGS

[0012] Fig. 1 shows nematode extraction from soil samples.

[0013] Fig. 2 shows the effect of the CRF-EO on the length of com plants. Bars labelled with different letters (a, b, c) represent statistically significant differences between treatments at p<0.05, based on two-way ANOVA. Bars sharing the same letter are not significantly different (a, b, c: denote statistically distinct concentration effects within the group treated with the thymol (Thy) + limonene (Lim) combination; a', b', c' indicate statistically distinct concentrations within the group treated with thymol alone; and a", b", c" indicate statistically distinct concentrations within the group treated with limonene alone).

[0014] Fig. 3 shows the effect of CRF-EO on plant leaf count. Bars labelled with different letters (a, b, c) represent statistically significant differences between treatments at p<0.05, based on two-way ANOVA (see Fig. 2 above).

[0015] Fig. 4 shows the effect of CRF-EO on corn fresh weight. Bars labelled with different letters (a, b, c) represent statistically significant differences between treatments at p<0.05, based on two-way ANOVA (see Fig. 2 above).

[0016] Fig. 5 shows the effect of CRF-EO on corn dry weight. Bars labelled with different letters (a, b, c) represent statistically significant differences between treatments at p<0.05, based on two-way ANOVA (see Fig. 2 above).

[0017] Fig. 6 shows the effect of CRF-EO on nematode count. Bars labelled with different letters (a, b, c, d) represent statistically significant differences between treatments at p<0.05, based on two-way ANOVA (see Fig. 2 above).

[0018] Fig. 7 shows nematode density variation under different CRF-EO treatments at varied concentrations.

[0019] Figs. 8A-8B show release rates of thymol (8A) and limonene (8B) into water from different formulations: A: Alginate-only with both active ingredients; B: Alginate -pectin with both active ingredients; C: Alginate -based formulations with doubled concentrations of either thymol or limonene.

[0020] Fig. 9 shows the number of surviving nematodes following incubation in untreated soil and soil treated with different formulations: Fl - thymol and limonene in alginate carrier; F2 - thymol and limonene in alginate -pectin carrier; F8 - thymol excess relative to limonene in alginate carrier; F9 - thymol deficiency relative to limonene in alginate carrier; F12 - limonene only in alginate carrier.DETAILED DESCRIPTION

[0021] It has now been found, in accordance with the present invention, that a biodegradable and environmentally friendly composition comprising a combination of thymol and limonene at a weight ratio of about 1:1, adsorbed onto a silicate mineral carrier consisting of kaolinite, montmorillonite and a zeolite, and embedded within a polysaccharide matrix comprising alginate, pectin and chitosan, when applied into soil rich in nematodes, releases said essential oils in a controlled-release manner through a dual mechanism of volatilization under dry conditions and dissolution under wet conditions, and consequently provides continuous nematode suppression over varying environmental conditions. As particularly found, the combination of thymol and limonene provides a synergistic nematocidal effect, which reduces nematode density and mitigates plant stress, and consequently promotes plant biomass, stem elongation, and leaf production.

[0022] In one aspect, the present invention thus provides a pesticidal composition, more specifically a nematocidal composition, comprising essential oils comprising thymol and limonene, adsorbed onto a silicate mineral carrier, wherein said mineral carrier is embedded within a polysaccharide matrix including alginate.

[0023] In certain embodiments, disclosed herein is a nematocidal composition as defined above, wherein said essential oils consist of thymol and limonene.

[0024] In certain embodiments, disclosed herein is a nematocidal composition as defined above, wherein said essential oils comprise thymol and limonene, as well as at least one essential oil having nematocidal activity such as, and without being limited to, limonin, hexanal, carvacrol, cinnamaldehyde, citral, and linalool. In certain particular such embodiments, said at least one essential oil having nematocidal activity provides synergistic nematocidal effect whencombined with thymol, and in other particular such embodiments, said at least one essential oil having nematocidal activity provides synergistic nematocidal effect when combined with limonene. Obviously, such an essential oil having nematocidal activity may provide synergistic nematocidal effect when combined with each one of thymol and limonene.

[0025] In certain embodiments, disclosed herein is a nematocidal composition according to any one of the embodiments above, wherein the ratio between said thymol and said limonene in said composition is from about 99: 1 to about 1:99, e.g., from about 80:20 to about 20:80, from about 75:25 to about 25:75, from about 70:30 to about 30:70, from about 65:35 to about 35:65, from about 60:40 to about 40:60, from about 55:45 to about 45:55, or about 1: 1, by weight, respectively. As found in accordance with the present invention, a weight ratio of approximately 1: 1 offers a favorable balance between nematocidal efficacy and formulation stability.

[0026] In certain embodiments, disclosed herein is a nematocidal composition as defined above, wherein said silicate mineral carrier is selected from a clay mineral, bentonite, a zeolite, diatomaceous earth, and a combination thereof.

[0027] The term “clay mineral” as used herein refers to a naturally occurring, fine-grained hydrous aluminum phyllosilicate, having a chemical structure built around silica (SiCU) tetrahedra and alumina (AlOe) octahedra, with water (hydroxyl groups, OH, and often molecular water, H2O) incorporated into their crystal lattice, and may further comprise significant amounts of other elements like iron, magnesium, alkali metals (sodium, potassium), and alkaline earth metals (calcium). The defining characteristic of clay minerals is their layered or sheet-like (phyllosilicate) structure. These layers are composed of one or two sheets of silica tetrahedra combined with one sheet of alumina or magnesia octahedra. More specifically, (i) 1: 1 layer clays consist of one tetrahedral sheet and one octahedral sheet (e.g., kaolinite group), and are typically held together tightly; (ii) 2: 1 layer clays consist of one octahedral sheet sandwiched between two tetrahedral sheets (e.g., smectite group like montmorillonite, illite group, vermiculite group), wherein the bonding between these T-O-T layers may vary, leading to different properties like swelling; and (iii) 2: 1: 1 layer clays include an additional hydroxide (brucite-like) sheet between the 2: 1 layers (e.g., chlorite group). Due to their unique structure and extremely small particle size (typically defined as being less than 2 pm in diameter), many clay minerals exhibit plasticity, meaning they can be molded and will retain their shape when dried. In addition, due to their large surface area (a consequence of their small particle size and layered structure), clay minerals can adsorb various ions, water molecules, and organic compounds onto their surfaces. When compacted, clays can have very low permeability to water, making them useful as liners forlandfills or ponds. Examples of clay minerals include, without limiting, kaolinite (A12Si2Os(OH)4), kaolin (China clay, a natural clay deposit predominantly composed of kaolinite), montmorillonite ((Na,Ca)o.33(Al,Mg)2(Si40io)(OH)2-nH20), hectorite (Nao.3(Mg,Li)3 Si40io(OH)2; the composition may optionally further comprise fluorine), attapulgite ((Mg,Al)2Si40io(OH)-4H20), vermiculite ((Mg,Fe2+,Fe3+)3[(Al,Si)40io](OH)2-4H20), and talc (Mg3Si40io(OH)2). The clay mineral referred to herein may also be an organically modified clay (also referred to as an organoclay or organophilic clay), i.e., a natural clay mineral such as montmorillonite, hectorite and attapulgite, that has been chemically altered (e.g., through an ion exchange process) to make its surface more compatible with organic substances, to thereby enhance the sorption of organic molecules and optimize sustained-release characteristics. Examples of organoclays include, without limiting, clays that have been modified with hexadecyltrimethylammonium (HDTMA), such as HDTMA-montmorillonite.

[0028] The term “bentonite” as used herein refers to a type of absorbent, swelling clay that is primarily composed of the mineral montmorillonite, which belongs to the smectite group of clay minerals. The most common types of bentonites are classified based on the dominant exchangeable cation present in their structure. Sodium bentonite is characterized by its high swelling capacity (when in contact with water, it can absorb many times its dry mass in water and expand up to 15-20 times its original volume, forming a viscous, gelatinous substance). Calcium bentonite has a lower swelling capacity compared to sodium bentonite; however, it possesses excellent adsorption properties, meaning it can bind to toxins, impurities, and ions on its surface (calcium bentonite can sometimes be "activated" by treating it with sodium carbonate to convert it into sodium bentonite, thereby increasing its swelling capacity). Potassium bentonite is a less common type of bentonite, where potassium is the dominant exchangeable cation.

[0029] The term “zeolite” as used herein refers to a microporous, crystalline aluminosilicate mineral, mainly consisting of silicon, aluminum and oxygen, forming a three-dimensional framework of interconnected tetrahedra (SiCE and AIO4). The most distinctive feature of zeolites is their highly porous structure. This framework contains tiny, uniform channels and cavities (or pores) of molecular dimensions, which can trap water molecules and other small molecules. The general chemical formula for zeolites may be represented as M(x / n)[(AlO2)x(SiO2)y]-zH2O, where M represents exchangeable cations such as sodium, potassium, calcium, and magnesium, 'n' is the valence of the cation, 'x' and 'y' are the number of alumina and silica tetrahedra, respectively, and 'z' is the number of water molecules in the pores. Due to their large internal surface area and the ability to selectively adsorb molecules based on size and shape, zeolites areexcellent adsorbents, which can both gases and liquids. The water molecules contained within the pores, can be removed by heating without collapsing the crystalline framework, and the dehydrated zeolite can then re-adsorb water or other small molecules.

[0030] “Diatomaceous earth”, also known as diatomite, celite or kieselguhr, is a naturally occurring, soft, siliceous sedimentary rock that can be crumbled into a fine powder, having a particle size typically ranging from 10 to 200 pm. The typical chemical composition of oven- dried diatomaceous earth is 80-90% silica, with 2-4% alumina (attributed mostly to clay minerls), and 0.5-2% iron oxide.

[0031] In particular embodiments, the silicate mineral carrier onto which the essential oils are adsorbed is a combination of at least one clay mineral and at least one zeolite, e.g., a combination of a clay mineral and a zeolite; two clay minerals and a zeolite; three clay minerals and a zeolite; a clay mineral and two zeolites; or a clay mineral and three zeolites, each at any weight ratio. In more particular such embodiments, said silicate mineral is a combination of kaolinite, montmorillonite, and a zeolite, at any weight ratio. More particular such embodiments are those wherein said silicate mineral is a combination of kaolinite, montmorillonite, and a zeolite, at a weight ratio of 1 : 1 : 1.

[0032] According to the present invention, the mineral carrier onto which the essential oils are adsorbed is embedded with a polysaccharide matrix including alginate. Thus, in certain embodiments, disclosed herein is a nematocidal composition as defined above, wherein the polysaccharide matrix consists of alginate; and in other embodiments, disclosed herein is a nematocidal composition as defined above, wherein said polysaccharide matrix comprises a combination of alginate with at least one, e.g., one, two or three, additional polysaccharide, optionally wherein said alginate constitutes at least 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said polysaccharide matrix. Examples of polysaccharides that may be combined with alginate include, without being limited to, pectin, chitosan, sulfated polysaccharides such as carrageenans, e.g., kappa-carrageenan, and xanthan gum. In particular embodiments, said polysaccharide matrix comprises a combination of alginate with at least one of pectin and chitosan, i.e., a combination of alginate and pectin, a combination of alginate and chitosan, or a combination of alginate, pectin and chitosan. More particular such compositions are those wherein said alginate constitutes at least 30%, e.g., at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, by weight of said polysaccharide matrix.

[0033] In certain embodiments, disclosed herein is a nematocidal composition as defined above, wherein said polysaccharide matrix embedding said silicate mineral carrier is formulated as discrete particles. The size of such particles, in average, may be from about 0.5 mm to about 2 mm, e.g., from about 0.55 mm to about 1.9 mm, from about 0.60 mm to about 1.8 mm, from about 0.65 mm to about 1.7 mm, from about 0.70 mm to about 1.6 mm, from about 0.75 mm to about 1.5 mm, from about 0.80 mm to about 1.4 mm, from about 0.85 mm to about 1.3 mm, from about 0.90 mm to about 1.2 mm, from about 0.95 mm to about 1.1 mm, or about 1 mm.

[0034] In certain embodiments, disclosed herein is a nematocidal composition as defined above, wherein (i) said essential oils either consist of thymol and limonene, or comprise thymol, limonene and at least one additional essential oil having nematocidal activity, such as limonin, hexanal, carvacrol, cinnamaldehyde, citral, and linalool, wherein the ratio between said thymol and said limonene is from about 99: 1 to about 1:99, e.g., from about 80:20 to about 20:80, from about 75:25 to about 25:75, from about 70:30 to about 30:70, from about 65:35 to about 35:65, from about 60:40 to about 40:60, from about 55:45 to about 45:55, or about 1: 1, by weight, respectively; (ii) said silicate mineral carrier is selected from a clay mineral, bentonite, a zeolite, diatomaceous earth, and a combination thereof; and (iii) said polysaccharide matrix either consists of alginate, or comprises a combination of alginate with at least one, e.g., one, two or three, additional polysaccharide, optionally wherein said alginate constitutes at least 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said polysaccharide matrix. In particular such embodiments, (i) said essential oils consist of thymol and limonene; and / or (ii) said silicate mineral carrier is a combination of at least one clay mineral and at least one zeolite at any weight ratio; and / or (iii) said polysaccharide matrix consists of alginate; or comprises a combination of alginate with at least one of pectin and chitosan, wherein said alginate constitutes at least 30%, e.g., at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, by weight of said polysaccharide matrix. In more particular such embodiments, (i) said essential oils consist of thymol and limonene at a weight ratio of about 1: 1; and / or (ii) said silicate mineral carrier is a combination of kaolinite, montmorillonite, and a zeolite, preferably at a weight ratio of 1: 1: 1; and / or (iii) said polysaccharide matrix consists of alginate; or comprises a combination of alginate, pectin and chitosan, at a weight ratio of about 85: 10:5, respectively. In certain such nematocidal compositions, the polysaccharide matrix embedding said silicate mineral carrier is formulated as discrete particles, having an average size of, e.g., from about 0.5 mm to about 2 mm, such as from about 0.55 mm to about 1.9 mm, from about 0.60 mm to about 1.8 mm, from about 0.65 mm to about 1.7 mm, from about 0.70 mm toabout 1.6 mm, from about 0.75 mm to about 1.5 mm, from about 0.80 mm to about 1.4 mm, from about 0.85 mm to about 1.3 mm, from about 0.90 mm to about 1.2 mm, from about 0.95 mm to about 1.1 mm, or about 1 mm.

[0035] In certain embodiments, disclosed herein is a nematocidal composition according to any one of the embodiments above, wherein upon wetting, i.e., exposure to humidity, said essential oils are released from said composition in a controlled-release manner, i.e., wherein said essential oils are released or delivered gradually and predictably over a specific and extended period, and the rate and duration of said release may be regulated by each one of the specific mineral carrier and specific polysaccharide matrix used. The composition disclosed thus enables (i) gradual release of the essential oils, which are not released all at once but are made available slowly over time; (ii) predetermined release rate (the release of the essential oils is designed to occur at a specific, predictable, and often constant or near-constant rate, maintaining a steady concentration of said oils at the target site); (iii) extended release duration (due to the specific mineral carrier and polysaccharide composition used, the composition prolongs the action of the active substance, reducing the frequency of application or administration); and (iv) reproducibility (the release profile is intended to be consistent and reproducible from one application or dose to another). In particular embodiments, said controlled-release manner is achieved through a dual mechanism of volatilization under dry conditions and dissolution under wet conditions, providing continuous nematode suppression over varying environmental conditions.

[0036] As importantly found by the present inventors, under dry conditions, a low but measurable degree of volatilization of essential oils from the composition occurs. However, when stored in sealed nylon-aluminum laminated bags, volatilization is effectively minimized, preserving the composition’ s integrity and extending shelflife. Therefore, appropriate packaging mitigates unintended release during storage while still enabling functional release during application.

[0037] According to the present invention, the nematocidal composition disclosed, according to any one of the embodiments above, may be adapted for soil application, e.g., using commercial agricultural spreading, mixing, or irrigation equipment.

[0038] In another aspect, the present invention relates to a method of controlling soilborne nematodes infection in a locus in need thereof or protecting said locus from soilborne nematodes infestation (herein also referred to as “Method A”), said method comprising applying to said locus an effective amount of a nematocidal composition according to any one of theembodiments above, wherein release of said essential oils from said composition is activated primarily by, and synchronized with, soil moisture conditions including irrigation or rainfall.

[0039] In yet another aspect, the present invention relates to a method for treating a plant against soilborne nematodes infection, or protecting said plant from soilborne nematodes infestation (herein also referred to as “Method B ). said method comprising applying to a locus in which said plant is cultivated an effective amount of a nematocidal composition according to any one of the embodiments above, wherein release of said essential oils from said composition is activated primarily by, and synchronized with, soil moisture conditions including irrigation or rainfall.

[0040] The experimental section herein clearly shows the efficacy of the nematocidal composition disclosed herein in promoting plant growth and resilience under stress conditions, and specifically biotic stress conditions. Based on these data it is postulated, with a very high degree of certainty, that any biodegradable and environmentally friendly composition based on the nematocidal composition disclosed, i.e., a composition comprising one or more essential oils (such as thymol, limonene, limonin, hexanal, carvacrol, cinnamaldehyde, citral, linalool, geraniol, citronellol, p-cymene, menthol, sabinene, anethole, estragole, eugenol, and vanillin, as well as any combination thereof) adsorbed onto a silicate mineral carrier as defined herein, wherein said mineral carrier is embedded within a polysaccharide matrix including alginate as defined herein, would be effective in promoting plant growth and resilience under stress biotic or abiotic conditions.

[0041] In still another aspect, the present invention thus relates to a method for promoting plant growth and resilience under biotic or abiotic stress conditions, said method comprising applying to a locus in which said plant is cultivated an effective amount of a composition comprising one or more essential oils adsorbed onto a silicate mineral carrier, wherein said mineral carrier is embedded within a polysaccharide matrix including alginate, wherein release of said one or more essential oils from said composition is activated primarily by, and synchronized with, soil moisture conditions including irrigation or rainfall.

[0042] In a more specific such aspect, the present invention relates to a method for promoting plant growth and resilience under biotic or abiotic stress conditions (herein also referred to as “Method C”), said method comprising applying to a locus in which said plant is cultivated an effective amount of a composition according to any one of the embodiments above, wherein release of said essential oils from said composition is activated primarily by, and synchronized with, soil moisture conditions including irrigation or rainfall.

[0043] The term “biotic stress conditions” as used herein refers to negative effects caused by living organisms, such as pathogens, pests, and weeds, that disrupt normal plant metabolism and growth, and may thus reduce plant vigor and productivity by causing various symptoms like leaf necrosis, chlorosis, tissue rotting, leaf spot, wilting, foliage defoliation, and root damage. In certain embodiments, the biotic stress conditions result from root-zone infestation by soilborne nematodes.

[0044] The term “abiotic stress conditions” as used herein refers to negative effects on living organisms caused by non-living environmental factors such as extreme high or low temperatures, drought or flooding, salinity, metal toxicity, and nutrient deficiencies. Abiotic stress can severely limit plant growth and productivity, impacting crop yields and food security. In certain embodiments, the abiotic stress conditions result from nutrient imbalance, reduced moisture availability, or soil compaction.

[0045] In certain embodiments, disclosed herein a method of controlling soilborne nematodes infection in a locus or protecting said locus from soilborne nematodes infestation (Method A); for treating a plant against soilborne nematodes infection or protecting said plant from soilborne nematodes infestation (Method B); or for promoting plant growth and resilience under biotic or abiotic stress conditions (Method C), each as defined above, wherein (i) said essential oils either consist of thymol and limonene, or comprise thymol, limonene and at least one additional essential oil having nematocidal activity, such as limonin, hexanal, carvacrol, cinnamaldehyde, citral, and linalool, wherein the ratio between said thymol and said limonene is from about 99: 1 to about 1:99, e.g., from about 80:20 to about 20:80, from about 75:25 to about 25:75, from about 70:30 to about 30:70, from about 65:35 to about 35:65, from about 60:40 to about 40:60, from about 55:45 to about 45:55, or about 1:1, by weight, respectively; (ii) said silicate mineral carrier is selected from a clay mineral, bentonite, a zeolite, diatomaceous earth, and a combination thereof; and (iii) said polysaccharide matrix either consists of alginate, or comprises a combination of alginate with at least one, e.g., one, two or three, additional polysaccharide, optionally wherein said alginate constitutes at least 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said polysaccharide matrix. In particular such embodiments, (i) said essential oils consist of thymol and limonene; and / or (ii) said silicate mineral carrier is a combination of at least one clay mineral and at least one zeolite at any weight ratio; and / or (iii) said polysaccharide matrix consists of alginate; or comprises a combination of alginate with at least one of pectin and chitosan, wherein said alginate constitutes at least 30%, e.g., at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least80%, by weight of said polysaccharide matrix. In more particular such embodiments, (i) said essential oils consist of thymol and limonene at a weight ratio of about 1: 1; and / or (ii) said silicate mineral carrier is a combination of kaolinite, montmorillonite, and a zeolite, preferably at a weight ratio of 1: 1: 1; and / or (iii) said polysaccharide matrix consists of alginate; or comprises a combination of alginate, pectin and chitosan, at a weight ratio of about 85: 10:5, respectively. In certain such embodiments, the polysaccharide matrix embedding said silicate mineral carrier is formulated as discrete particles, having an average size of, e.g., from about 0.5 mm to about 2 mm, such as from about 0.55 mm to about 1.9 mm, from about 0.60 mm to about 1.8 mm, from about 0.65 mm to about 1.7 mm, from about 0.70 mm to about 1.6 mm, from about 0.75 mm to about 1.5 mm, from about 0.80 mm to about 1.4 mm, from about 0.85 mm to about 1.3 mm, from about 0.90 mm to about 1.2 mm, from about 0.95 mm to about 1.1 mm, or about 1 mm.

[0046] In certain embodiments, disclosed herein a method of controlling soilborne nematodes infection in a locus or protecting said locus from soilborne nematodes infestation (Method A); for treating a plant against soilborne nematodes infection or protecting said plant from soilborne nematodes infestation (Method B); or for promoting plant growth and resilience under biotic or abiotic stress conditions (Method C), each according to any one of the embodiments above, wherein the application of said composition to said locus comprises broadcasting onto soil surface, incorporation into soil during cultivation, or mixing with irrigation water for fertigation systems.

[0047] The term “effective amount”, as used herein with respect to the nematocidal composition applied to a particular locus, refers to an amount of said composition that is sufficient, when applied once, twice or trice during an agricultural crop cycle (typically an agricultural year or season), to (a) control soilborne nematodes infection in said locus or protect said locus from soilborne nematodes infestation; (b) treat a plant cultivated in said locus against soilborne nematodes infection or protect said plant from soilborne nematodes infestation; or (c) promote plant growth and resilience under biotic or abiotic stress conditions, depending on whether the application of said composition is carried out according to Method A, Method B, or Method C. The amount must be effective to achieve the desired effect as described above, depending inter alia on the type and severity of the nematodes infection to be treated, controlled, or prevented, without causing any significant damage to the plant or locus that is treated. While referring to Method B or Method C, the amount should be specifically effective so as to improve or promote plant growth parameters including stem elongation, leaf number, plant (fresh) biomass, and / or dry weight under nematode infestation stress. The effective amount may bebased on reference standard nematicides or anthelmintics, but is preferably determined in appropriately designed trials (dose range studies), and the person versed in the art will know how to properly conduct such trials to determine said amount, i.e., the dose of said nematocidal composition that is effective. The effective amount of the composition applied according to the methods of the present invention depends on both the synergistic effect achieved when by the essential oil combination, as well as the release profile of said essential oils from said composition, which depends on the specific silicate mineral carrier composing said composition and the specific polysaccharide within which said mineral carrier is embedded. In certain embodiments, the nematocidal composition is applied to the locus treated at a rate ranging from 0.01% to 4% (by weight) of soil, corresponding to approximately 10-400 kg of composition per 106kg of soil, or 20-400 kg composition per acre depending on soil bulk density and application depth, and maintains efficacy under repeated irrigation or rainfall conditions.

[0048] in certain embodiments, disclosed herein a method of controlling soilbome nematodes infection in a locus or protecting said locus from soilborne nematodes infestation (Method A); or for treating a plant against soilborne nematodes infection or protecting said plant from soilborne nematodes infestation (Method B), each according to any one of the embodiments above, wherein said composition provides enhanced essential oil release during wet periods, ensuring targeted nematode control synchronized with nematode activity cycles.

[0049] In certain embodiments, disclosed herein a method for treating a plant against soilborne nematodes infection or protecting said plant from soilborne nematodes infestation (Method B); or for promoting plant growth and resilience under biotic or abiotic stress conditions (Method C), each according to any one of the embodiments above, wherein the release of said essential oils improves / promotes at least one plant growth parameter, such as stem elongation, leaf number, fresh weight also referred to herein as plant biomass, dry weight under nematode infestation stress, and a combination thereof.

[0050] In certain embodiments, disclosed herein a method for treating a plant against soilborne nematodes infection (Method B), wherein the release of said essential oils reduces nematode density in the soil (in the locus treated) and concurrently enhances plant biomass and elongation.

[0051] In a further aspect, the present invention relates to use of a nematocidal composition according to any one of the embodiments above for controlling soilborne nematodes infection in a locus or protecting a locus from soilborne nematodes infestation; for treating a plant against soilborne nematodes infection or protecting a plant from soilborne nematodes infestation; or for promoting plant growth and resilience under biotic or abiotic stress conditions.

[0052] According to the present invention, certain nematocidal compositions for use as defined above are those wherein (i) said essential oils either consist of thymol and limonene, or comprise thymol, limonene and at least one additional essential oil having nematocidal activity, such as limonin, hexanal, carvacrol, cinnamaldehyde, citral, and linalool, wherein the ratio between said thymol and said limonene is from about 99: 1 to about 1:99, e.g., from about 80:20 to about 20:80, from about 75:25 to about 25:75, from about 70:30 to about 30:70, from about 65:35 to about 35:65, from about 60:40 to about 40:60, from about 55:45 to about 45:55, or about 1: 1, by weight, respectively; (ii) said silicate mineral carrier is selected from a clay mineral, bentonite, a zeolite, diatomaceous earth, and a combination thereof; and (iii) said polysaccharide matrix either consists of alginate, or comprises a combination of alginate with at least one, e.g., one, two or three, additional polysaccharide, optionally wherein said alginate constitutes at least 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said polysaccharide matrix. Particular such compositions are those wherein (i) said essential oils consist of thymol and limonene; and / or (ii) said silicate mineral carrier is a combination of at least one clay mineral and at least one zeolite at any weight ratio; and / or (iii) said polysaccharide matrix consists of alginate; or comprises a combination of alginate with at least one of pectin and chitosan, wherein said alginate constitutes at least 30%, e.g., at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, by weight of said polysaccharide matrix. More particular such compositions are those wherein (i) said essential oils consist of thymol and limonene at a weight ratio of about 1: 1; and / or (ii) said silicate mineral carrier is a combination of kaolinite, montmorillonite, and a zeolite, preferably at a weight ratio of 1 : 1 : 1 ; and / or (iii) said polysaccharide matrix consists of alginate; or comprises a combination of alginate, pectin and chitosan, at a weight ratio of about 85: 10:5, respectively. In certain such nematocidal compositions, the polysaccharide matrix embedding said silicate mineral carrier is formulated as discrete particles, having an average size of, e.g., from about 0.5 mm to about 2 mm, such as from about 0.55 mm to about 1.9 mm, from about 0.60 mm to about 1.8 mm, from about 0.65 mm to about 1.7 mm, from about 0.70 mm to about 1.6 mm, from about 0.75 mm to about 1.5 mm, from about 0.80 mm to about 1.4 mm, from about 0.85 mm to about 1.3 mm, from about 0.90 mm to about 1.2 mm, from about 0.95 mm to about 1.1 mm, or about 1 mm.

[0053] For purposes of clarity, and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers referring to, e.g., weight percentages or ratios, used in the present specification are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in thisdescription and claims are approximations that may vary by up to plus or minus 10% depending upon the desired properties sought to be obtained by the invention.

[0054] The invention will now be illustrated by the following non-limiting Examples.EXAMPLESStudy 1.Materials and Methods

[0055] Adsorption of essential oils onto minerals. A fraction of the essential oil, specifically thymol, limonene, or a blend of both, was dissolved in 96% technical ethanol (essential oils, solvents and chemical reagents were purchased from Sigma). This solution was then introduced to a mixture of minerals, including montmorillonite (Sigma), kaolinite (Imerys, England), and natural zeolite (Grace. Germany), in varying proportions to meet different loading capacities, frorn balanced ratios to complete omission of one mineral.

[0056] .After thoroughly mixing the slurry, residual ethanol was removed through evaporation under a nitrogen flow. The dried mixture was subsequently pulverized and uniformly blended.

[0057] Preparation of the polysaccharide solution. A polysaccharide solution was prepared using water as the solvent, incorporating three specific polysaccharides: alginate, pectin, and chitosan (all purchased from Sigma). The proportion of these polymers was tailored to achieve the target structure and porosity of the carriers, taking into account the solubility constraints of chitosan.

[0058] Preparation of formulated mixture. The process involved mixing minerals impregnated with essential oils into a polysaccharide solution. This blend was then extruded using a syringe into a container filled with a CaCfr solution. The extruded strands were chopped into smaller particles using a hand blender to reach the required size. Excess moisture was then pressed out of the particles, which were subsequently air-dried under a flow of clean, dry air.

[0059] Analysis of the final product . The concentration of essential oils within the formulation was measured using gas chromatography -mass spectrometry (GCMS). Additionally, the porous architecture of the product was examined and characterized using scanning electron microscopy (SEM).Preparation of seedlings

[0060] Corn seeds were obtained from Merhav Agro, Israel. The seed variety was PR32w86. The initial plant acquisition process (seedlings) was conducted after a germination process in the dark for 2 days on wet filter paper in a 0.5 mM CaCh solution at a temperature of 27±2°C until obtaining primary seedlings with a root length of about 1 cm and a shoot length of about 0.5 cm.

[0061] Approximately 250 primary corn plants were transferred to hydroponic growth in a controlled growth room, conditions: temperature (27±2°C), humidity (35%) and lighting (35 watt / m2), in a 0.1 Hoagland solution for an additional 3 days as previously disclosed (Asli et al., 2023). For monitoring plant growth in soil, seedlings uniform in size, number of leaves and fitness were selected.Preparation of nematodes

[0062] The nematodes used in this experiment were grown on maize plant roots within 5 kg of soil and watered for two months (60 days) before starting the experiment.Preparation of the control

[0063] The nematode reservoir was within the root system of corn plants grown in 5 kg of soil and watered for 60 days. From this soil, which is rich in nematodes, 4 pots were filled with 100 g of soil each, to which no "natural pesticide" was added and served as the control.Treatment setup

[0064] Alongside a control group, treatment groups with three distinct concentrations were established by filling four pots for each concentration with 100 grams of soil rich in nematodes. The applied concentrations of the controlled release essential oil formulation (CRF-EO) were ()% (control). 0.16%, 0.8%, and 4%. The CRF-EO comprised limonene, thymol, and their combination.

[0065] The aim was to investigate the synergistic effects of combining limonene and thymol in the CRF-EO on plant growth in nematode-infested soil. To achieve the target concentrations, 0.16. 0.8, and 4 grams of CRF-EO were mixed into individual pots containing 100 grams of the nematode-ricb soil. Each concentration level was replicated across four pots, with the experiment running from June to November 2022. The plants were watered bi-daily, with each pot receiving 50 ml of water, ensuring consistent hydration across all treatment and control groups.Plant height measurement protocol

[0066] Plant heights were recorded by measuring the length of all stems from the base just above the soil level to the top. These measurements were taken bi-weekly to track growth progression over time.Leaf counting protocol

[0067] The total number of leaves across all stems of each plant was tallied bi-weekly to monitor foliage development.Fresh weight assessment

[0068] Upon concluding the experiment, each plant was severed at the soil exit point. The fresh weight was determined after a 2-minute drying period with a paper towel to remove surface moisture.Dry weight determination

[0069] Following the fresh weight measurement, the plants subjected to various treatments were encased in labeled paper bags and oven-dried at 70°C for 48 hours. Post-drying, the plants were weighed again to obtain dry weight, offering insights into the biomass content devoid of water.Nematode extraction and counting method

[0070] The process for isolating and counting nematodes from soil involved several steps: (1) Soil sample collection: Soil samples were extracted from each treatment group and placed into individual Petri dishes; (ii) Sample preparation: The soil was then transferred onto a regular paper towel set over a mesh with 1mm diameter holes within the dish; (iii) Moisture addition: 30 ml of tap water was poured into each dish to facilitate nematode movement; (iv) Incubation period: The dishes were left undisturbed for 24 hours, allowing nematodes to migrate through the mesh with the water; (v) Nematode collection: The water that filtered through, carrying nematodes, was collected from the dishes into 30 ml test tubes; (vi) Concentration of nematodes: The test tubes were subjected to centrifugation at 5000 rpm for 5 minutes, concentrating the nematodes at the bottom; (vii) Microscopic examination: A small sample from the bottom of each test tube, where nematodes were likely to have settled, was placed on a microscope stage; and (viii) Counting nematodes: Nematodes were counted using a regular light microscope at a magnification of 0.1x4, by observing the entire area of a coverslip.ResultsEvaluating the influence of CRF-EO on plant growth

[0071] The study examining the effect of controlled release formulation of essential oils (CRF- EO) on corn plant elongation was carried out through three distinct trials: the application of limonene, thymol, and a combination of both. The experiment was designed to assess how different components and concentrations of CRF-EO treatments influenced the growth of corn plants in nematode-infested soil.

[0072] Corn plants were cultivated in pots filled with 100g of dense soil uniformly infested with nematodes. These pots were treated with diverse formulations and concentrations of "Controlled release formulation of essential oils" (CRF-EO). The growth, and specifically the stem elongation of the com plants, was observed over a 60-day period, following the methodology outlined earlier. Statistical analysis was conducted using a Two-Way ANOVA test, with different letters indicating significant differences between means at a 95% confidence level (Mean values ± Standard error, n=3).

[0073] Fig. 2 illustrates the impact of varying concentrations of the CRF-EO on the elongation of corn plants. The data indicates that the combined application of limonene and thymol at diverse concentrations significantly enhances plant growth compared to the control group. Similarly, limonene as a standalone treatment also promotes plant elongation, but notably at higher concentrations. The findings suggest that the synergistic effect of limonene and thymol together facilitates plant growth recovery even at lower doses, whereas limonene requires higher levels to be effective. Thymol alone, however, does not show a notable improvement in plant growth when compared to the control. Significant statistical differences were noted in treatments with observable growth enhancement, as confirmed by a Two-Way ANOVA test, detailed in Table 1, with a 95% confidence interval.Impact of CRF-EO on leaf count

[0074] Corn plants were cultivated in 100g pots of dense soil uniformly infused with nematodes, treated with varied concentrations of CRF-EO. The branching patterns of the corn plants were observed over a 60-day period, as outlined in the methodology section. Statistical analyses, specifically Two-Way ANOVA tests, indicated significant differences between treatments, denoted by distinct letters, at a 95% confidence level (Mean values ± Standard error, n=3).Table 1: Statistical analysis of CRF-EO's impact on corn plant length

[0075] Fig. 3 illustrates the variation in leaf count across plants treated with different concentrations of the CRF-EO. The data highlights a notable increase in leaf production for plants treated with a combination of thymol and limonene, even at lower concentrations, in comparison to the control group. Limonene, when used independently, also enhanced leaf count but required higher concentrations to achieve a similar effect. The most significant increase in foliage was observed in plants treated with the synergistic blend of thymol and limonene. Conversely, thymol alone showed a minimal impact on leaf production, and low concentrations of limonene resulted in a reduced number of leaves. However, increasing the concentration of limonene alone led to a marked improvement in leaf count. This suggests that while the combination of thymol and limonene effectively boosts leaf growth at lower doses, limonene's efficacy as a standalone treatment emerges at higher concentrations. Thymol, on its own, does not significantly affect leaf count compared to the control.Impact of CRF-EO on Corn Fresh Weight

[0076] Corn plants were cultivated in 100g pots of dense, nematode-infested soil, treated with various concentrations of CRF-EO. Over a 60-day observation period, the fresh weight of the corn plants was measured, adhering to the outlined methodology. Statistical significance among different treatments was determined using a Two-Way ANOVA test, with distinct letters indicating significant differences at a 95% confidence level (Mean values ± SE, n=3).

[0077] Fig. 4, illustrating the fresh weight of corn plants under different CRF-EO treatments, reveals that the combined application of thymol and limonene within the CRF-EO notably enhanced the plants' fresh weight. This suggests that the synergistic effect of these two essentialoils, when used together in the CRF-EO formulation, positively contributes to the biomass accumulation in corn plants grown in nematode -rich soil.Table 2: The effect of natural pesticide on the number of leaves in the plant - statistical analysisImpact of CRF-EO on corn dry weight

[0078] Corn plants were cultivated in 100g pots filled with dense, nematode-infested soil and treated with different concentrations of CRF-EO. The dry weight of the corn was measured after 60 days, following the methodological guidelines previously described. Statistical analysis, conducted using a Two-Way ANOVA test, identified significant differences in dry weight across treatments, denoted by different letters at a 95% confidence level (Mean values ± SE, n=3).Table 3: The effect of the natural pesticide on the fresh weight of the corn - statistical analysis

[0079] The graphical representation in Fig. 5 demonstrates the dry weight outcomes for corn plants under various CRF-EO treatments. The data clearly shows that the incorporation of a CRF- EO mixture containing both Thymol and Limonene significantly enhanced the plants' dry weight. High concentrations of limonene alone also yielded a similar increase in dry weight. The most pronounced improvement in dry biomass was observed with the combined treatment of thymol and limonene, suggesting a synergistic effect that maximizes growth. In contrast, thymol applied independently resulted in a lesser increase in dry weight compared to the control group. The observed augmentation in dry weight with the combined essential oils treatment, even at lower concentrations, suggests a potential reduction in nematode impact, highlighting the efficacy of the CRF-EO in promoting plant growth and resilience against nematode infestation.Impact of CRF-EO on nematode population

[0080] Corn plants were grown in 100g pots of dense soil uniformly infested with nematodes and treated with varying concentrations of CRF-EO. Nematode counts were conducted after 60 days following the outlined methods. Statistical differences among treatments were determined using a Two-Way ANOVA test, with distinct letters indicating significant variances at a 95% confidence level (Mean values ± SE, n=3).

[0081] The data presented in Fig. 6 illustrates the nematode populations under different CRF- EO treatments. Applying a combination of thymol and limonene significantly reduced nematode numbers compared to the control, contributing to decreased plant damage and enhanced growth. High concentrations of limonene alone also effectively lowered nematode densities. A trend was observed across all treatments where increasing pesticide concentrations corresponded to reduced nematode populations, as further detailed in Fig. 7. The most substantial decline in nematode numbers was noted with the combined use of thymol and limonene, whereas thymol alone was less effective, with nematode counts remaining high. Low concentrations of limonene were initially ineffective in reducing nematode numbers, but as concentrations increased, a significant decrease in nematode density was observed, mirroring the efficacy of the combined essential oil treatment. The dual essential oil treatment proved beneficial at lower concentrations by significantly reducing soil nematode levels, thereby supporting plant growth. Conversely, limonene's effectiveness as a standalone treatment emerged at higher concentrations, while Thymol required very high concentrations to match the control's efficacy in reducing nematode counts.Table 4: The effect of the CRF-EO on the dry weight of the corn - statistical analysisTable 5: Statistical analysis of CRF-EO impact on nematode populationRelease mechanism in soil

[0082] The release of essential oils from a polymer-mineral carrier (CRF-EO) to combat nematodes, can occur through two possible mechanisms:1. Volatilization: This mechanism is predominant under dry conditions and is primarily influenced by the vapor pressure of the essential oil. It becomes active when there is insufficient moisture in the environment to trigger the dissolution mechanism. In this state, the essential oil molecules transition from the solid or liquid phase to the gas phase, diffusing into the surrounding environment where they can exert their nematocidal effects.2. Dissolution: This mechanism operates under moist conditions and is chiefly affected by the solubility of the essential oil. When the formulation is moistened, water penetrates the carrier, initiating the dissolution of the essential oils. This process facilitates the diffusion of the essential oil out of the carrier, promoting an effective nematocide action against the nematodes.

[0083] The release mechanisms of essential oils from the polymer-mineral carrier for nematode control in our invention are significantly influenced by the essential oil's binding strength to the mineral component and the porosity of the carrier, including pore size and shape. To guarantee the efficient dispersal of essential oils for nematode eradication, it is critical to irrigate the soil following the deployment of the formulation, or alternatively, apply it during the rainy season. This watering facilitates the dissolution process, ensuring the active compounds are effectively liberated. In contrast, the volatilization mechanism is deemed ineffective for this purpose, as it fails to achieve the necessary concentration levels of active components, such as limonene and thymol, required for the successful elimination of nematodes.

[0084] Our findings presented here suggest that under normal conditions, plants naturally progress through growth stages, including elongation, leaf production, and biomass accumulation. However, the presence of nematodes in the root zone can significantly impede these growth processes. An effective pesticide is characterized by its ability to mitigate nematode populations, thereby facilitating improved plant growth.

[0085] The application of Controlled Release Formulation (CRF) containing a combination of limonene and thymol to the root environment markedly enhanced plant elongation and leaf count compared to control plants, demonstrating a potent synergistic effect between these two essential oils. This combination proved beneficial for plant biomass at relatively low concentrations, whereas CRF formulations containing only limonene required higher concentrations to exhibit similar growth-promoting effects. Conversely, CRF with thymol alone did not significantly impact plant biomass.

[0086] Furthermore, the CRF containing both essential oils effectively reduced nematode density in the soil at low concentrations, which is conducive to plant elongation. In contrast, CRF with limonene alone was only effective at higher concentrations. CRF with thymol alone, despite its apparent ability to reduce nematode numbers, did not positively affect plant growth. This discrepancy raises questions about thymol's potential toxicity at certain concentrations, which may harm nematodes without fostering plant recovery.

[0087] Given these observations, the combination of limonene and thymol is recommended for use, as it likely mitigates any toxic effects that might be present when these components are used individually at high doses. This synergistic blend offers a balanced approach to controlling nematode populations while supporting plant health and development.Study 2.

[0088] The present study evaluated controlled-release formulations for soil nematode control, focusing on optimizing the combination of thymol and limonene essential oils within polysaccharide-mineral carrier systems. Mechanical, chemical, and biological tests demonstrated that a balanced thymol-to-limonene ratio, supported by an alginate -based carrier supplemented with complementary minerals, is critical for achieving optimal stability, controlled release, and nematocidal efficacy. Among the tested formulations, those based solely on alginate exhibited the most favorable release profiles and sustained active ingredient concentrations under both aqueous and soil field-simulated conditions. Unlike conventional formulations, the current system achieves prolonged active retention under dynamic soil conditions through mineral- stabilized alginate matrices. Deviations from the optimal thymol-limonene ratio, or the use of less effective carrier matrices such as alginate -pectin, resulted in significantly reduced nematocidal performance. Overall, the results highlight the crucial role of formulation design in regulating active ingredient release, enhancing biological activity, and improving resilience under dynamic environmental conditions. This delivery system offers a promising, sustainable platform for the targeted control of soilborne nematodes in agricultural applications.Polysaccharide ratios

[0089] The three polysaccharides, alginate, pectin and chitosan, were evaluated in various compositions, with alginate constituting at least 30% of the total polysaccharide content in all formulations.

[0090] The initial evaluation of the formulations was carried out through yield and efficiency assessments, designed to determine the capacity to produce a stable system amenable to molding during preparation and subsequent modification during final shaping. These assessments served as critical indicators of gelation performance and the extent of polysaccharide interactions within the formulation.

[0091] Findings. Incorporating approximately 30% pectin into the alginate matrix enhanced the stability of the formulation and increased the production yield by approximately 20%.Conversely, the addition of chitosan to the mixture resulted in a substantial reduction in yield, approximately 70% in alginate -based formulations and about 50% in alginate -pectin formulations. This decline is attributed to insufficient gelation, leading to polysaccharide loss alongside the gelling salt solution. The moisture content was consistent across all formulations, averaging around 50%, with differences in yield primarily attributed to losses in solid material.Mineral ratios

[0092] Three minerals, montmorillonite, kaolinite and zeolite, were evaluated in combination with polysaccharides, using product yield and formulation stability as the primary assessment criteria.

[0093] Findings. Montmorillonite exhibited a clear advantage, resulting in approximately 10% higher yield compared to zeolite and around 20% higher yield compared to kaolinite. A combination of all three minerals in equal proportions further improved yield by an additional 10% relative to montmorillonite alone.Essential oil ratios

[0094] The amount and proportion of essential oils did not significantly affect the carrier’s efficiency or stability, as the quantities used remained below the adsorption capacity of the mineral-polysaccharide matrix. It is noteworthy that the concentration of active ingredients in the final product was approximately 5% by weight.Field stability testing of the formulations

[0095] To evaluate the long-term stability of the formulations under field-like conditions, representative products that passed the initial yield tests were incubated in exposed loess soil under conditions of increased airflow and repeated wetting and drying cycles for two weeks.

[0096] Following the incubation period, both the physical integrity of the formulations and the retention of essential oils within the carriers were assessed. The concentrations of essential oils (thymol and limonene) were quantified in both the residual formulation and the surrounding soil. This was accomplished by extracting soil samples with a water-ethyl acetate mixture, isolating the organic phase, and analyzing the oil content using GC-MS, relative to the initial concentrations.

[0097] Findings. The primary formulation, composed of alginate combined with a blend of three minerals, exhibited high stability under the simulated field conditions, maintaining a substantial portion of the active ingredient content. After two weeks, approximately 50% of theactive compounds remained within the formulation and the surrounding soil, indicating a controlled release profile and resilience to environmental fluctuations. In contrast, other formulations, particularly those containing only limonene, demonstrated markedly reduced stability, with minimal to negligible retention of active materials after two weeks.Chemical release tests in water

[0098] Based on mechanical stability and production yield results, four representative formulations were selected for chemical release evaluation in an aqueous environment. These tests provided a rapid and efficient means for comparing the release profiles of different formulations. The testing protocol involved measuring the release rates of essential oils from the controlled-release carriers into water. Oil concentrations were determined using GC-MS analysis with headspace injection, allowing the volatile components to be analyzed directly without additional solvent extraction.

[0099] Findings. Figs. 8A-8B present the release profiles of thymol and limonene from the various systems into water. Formulation A (alginate only) exhibited a controlled and consistent release of both active compounds, maintaining relative stability over time. Formulation B (alginate-pectin) demonstrated an initially slower release rate, followed by a sharp increase after 24 hours, likely due to water infiltration into the porous matrix, which accelerated diffusion pathways and enhanced the release of the active ingredients. This behavior highlights the role of pectin in promoting late-stage release acceleration. Formulation C, which was tested in two versions (one with doubled thymol and one with doubled limonene), showed pronounced mutual interactions between the components. The presence of one compound significantly influenced the stabilization and release dynamics of the other, underscoring the critical importance of balancing active ingredient composition to achieve a consistent and controlled release profile.

[0100] Additional observations. The concentrations of limonene released were consistently and significantly lower than those of thymol across all formulations, with limonene levels remaining below one-tenth of the corresponding thymol concentrations throughout the release period. The alginate-only formulation containing a balanced ratio of thymol and limonene demonstrated the most favourable performance, achieving both a delayed release profile and sustained concentration stability over time.Release rates in soil

[0101] Evaluation of controlled-release formulations for nematode control in soil. The objective of the experiment was to evaluate the effectiveness of various controlled-releaseformulations for nematode control in soil. The study was conducted in the laboratory of Dr. Sigal Brown at the Volcani Institute (Israel), a specialized research facility dedicated to the evaluation of nematocidal activity at both experimental and pre-commercial stages.

[0102] Experiment description. The experiment was conducted using 60 mL plastic cups filled with loess soil, with each cup inoculated with 500 second-stage juveniles (J2) of Meloidogyne javanica (root-knot nematodes). A fixed amount of the tested formulation was added to each cup, with six replicates prepared for each treatment group. Following a 48-hour incubation period, nematodes were extracted from the soil using the Baermann funnel method, which allowed live nematodes to migrate through a mesh filter. The surviving nematodes were then counted using an optical microscope.

[0103] Findings. Fig. 9 illustrates a clear "golden ratio", a thymol-to-limonene ratio of 1: 1, associated with optimal nematocidal activity (Fl). Deviations from this ratio (F8, F9) resulted in a marked decrease in nematode-control efficiency. A formulation containing limonene alone without thymol (F12) showed no activity. Furthermore, the carrier composition significantly influenced the release rate and stability of the oils in the soil. For instance, formulation F2 (alginate-pectin carrier) showed a marked decrease in efficacy.Assessment of different essential oil blends for soil nematode control

[0104] The effectiveness of various essential oil mixtures for controlling soil nematodes was evaluated using the same biological system described above. The essential oil blends were adsorbed onto a mineral matrix (without alginate) to facilitate homogeneous mixing into the soil and to enable accurate measurement of nematocidal efficacy.

[0105] The following essential oil combinations were tested: thymol, limonene, thymollimonene, thymol-linalool, thymol-cinnamaldehyde.Summary and conclusions

[0106] The biological test results were fully consistent with the chemical and physical evaluations. Collectively, the data consistently emphasize the critical importance of maintaining a balanced ratio of thymol and limonene within a controlled-release system; as well as the distinct advantage of adsorbing the active compounds onto an alginate -based carrier supplemented with a balanced mixture of minerals.

[0107] This delivery system enables: (i) controlled and uniform release of active substances over time, while preserving their stability within the soil environment; (ii) regulation of diffusionrates through the carrier matrix in response to environmental conditions, thereby minimizing rapid and uncontrolled release; and (iii) enhanced biological efficacy in nematode control by achieving an optimal balance between immediate availability and sustained activity.

[0108] Conclusions. The full realization of the chemical and biological potential of the formulation in complex soil systems can only be achieved through the correct combination of thymol and limonene in a balanced ratio, supported by a high-quality alginate-based carrier integrated with complementary minerals.REFERENCESArdakani, A.S.; Hosseininejad, S.A., Identification of chemical components from essential oils and aqueous extracts of some medicinal plants and their nematicidal effects on Meloidogyne incognita. Journal of Basic and Applied Zoology, 2022, 83, 14Asli, S.; Diab, M.; Hugerat, M.; Haj-Zaroubi, M., Hydroxy tyro sol increases salt tolerance of maize and wheat by expanding the pore diameter on root cell wall. Theor. Exp. Plant Physiol., 2023, 35, 287-298Oka, Y.; Nacar, S.; Putievsky, E.; Ravid, U.; Yaniv, Z.; Spiegel, Y., Nematicidal activity of essential oils and their components against the root-knot nematode. Phytopathology, 2000, 90(7), 710-715

Claims

CLAIMS1. A nematocidal composition comprising essential oils comprising thymol and limonene, adsorbed onto a silicate mineral carrier, wherein said mineral carrier is embedded within a polysaccharide matrix including alginate.

2. The composition of claim 1, wherein said essential oils consist of thymol and limonene.

3. The composition of claim 1, wherein said essential oils further comprise at least one essential oil having nematocidal activity, such as limonin, hexanal, carvacrol, cinnamaldehyde, citral, and linalool.

4. The composition of claim 3, wherein said at least one essential oil having nematocidal activity provides synergistic nematocidal effect when combined with either thymol or limonene.

5. The composition of any one of claims 1-4, wherein the ratio between said thymol and said limonene in said composition is from about 99: 1 to about 1:99, e.g., from about 80:20 to about 20:80, from about 75:25 to about 25:75, from about 70:30 to about 30:70, from about 65:35 to about 35:65, from about 60:40 to about 40:60, from about 55:45 to about 45:55, or about 1: 1, by weight, respectively.

6. The composition of claim 1, wherein said silicate mineral carrier is selected from a clay mineral such as kaolin (kaolinite), montmorillonite, attapulgite, vermiculite, and talc, bentonite, a zeolite, diatomaceous earth, and a combination thereof.

7. The composition of claim 6, wherein said silicate mineral carrier is a combination of kaolinite, montmorillonite, and a zeolite, at any weight ratio.

8. The composition of claim 7, wherein said mineral carrier is a combination of kaolinite, montmorillonite, and a zeolite, at a weight ratio of 1: 1: 1.

9. The composition of claim 1, wherein said polysaccharide matrix consists of alginate.

10. The composition of claim 1, wherein said polysaccharide matrix comprises a combination of alginate with at least one of pectin and chitosan.

11. The composition of claim 10, wherein said alginate constitutes at least 30% by weight of said polysaccharide matrix.

12. The composition of claim 1, wherein said polysaccharide matrix embedding said silicate mineral carrier is formulated as discrete particles.

13. The composition of claim 1, wherein said essential oils either consist of thymol and limonene, or comprise thymol, limonene and at least one additional essential oil having nematocidal activity, such as limonin, hexanal, carvacrol, cinnamaldehyde, citral, and linalool, wherein the ratio between said thymol and said limonene is from about 99: 1 to about 1:99, e.g., from about 80:20 to about 20:80, from about 75:25 to about 25:75, from about 70:30 to about 30:70, from about 65:35 to about 35:65, from about 60:40 to about 40:60, from about 55:45 to about 45:55, or about 1: 1, by weight, respectively; said silicate mineral carrier is selected from a clay mineral, bentonite, a zeolite, diatomaceous earth, and a combination thereof; and said polysaccharide matrix either consists of alginate, or comprises a combination of alginate with at least one additional polysaccharide, optionally wherein said alginate constitutes at least 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said polysaccharide matrix.

14. The composition of claim 13, wherein(i) said essential oils consist of thymol and limonene;(ii) said silicate mineral carrier is a combination of at least one clay mineral and at least one zeolite at any weight ratio; and / or(iii) said polysaccharide matrix consists of alginate; or comprises a combination of alginate with at least one of pectin and chitosan, wherein said alginate constitutes at least 30% by weight of said polysaccharide matrix.

15. The composition of claim 14, wherein:(i) said essential oils consists of thymol and limonene at a weight ratio of about 1: 1;(ii) said silicate mineral carrier is a combination of kaolinite, montmorillonite and a zeolite, preferably at a weight ratio of 1 : 1 : 1 ; and / or(iii)said polysaccharide matrix consists of alginate; or comprises a combination of alginate, pectin and chitosan at a ratio of about 85:10:5, by weight, respectively.

16. The composition of any one of claims 13-15, wherein said polysaccharide matrix embedding said silicate mineral carrier is formulated as discrete particles.

17. The composition of any one of claims 1-16, wherein upon wetting, said essential oils are released from said composition in a controlled-release manner.

18. The composition of any one of claims 1-16, wherein said essential oils are released from said composition in a controlled-release manner through a dual mechanism of volatilization under dry conditions and dissolution under wet conditions.

19. The composition of any one of claims 1-16, wherein said composition is adapted for soil application, e.g., using commercial agricultural spreading, mixing, or irrigation equipment.

20. A method of controlling soilborne nematodes infection in a locus in need thereof or protecting said locus from soilborne nematodes infestation, said method comprising applying to said locus an effective amount of a composition according to any one of claims 1-19, wherein release of said essential oils from said composition is activated primarily by, and synchronized with, soil moisture conditions including irrigation or rainfall.

21. A method for treating a plant against soilborne nematodes infection, or protecting said plant from soilborne nematodes infestation, said method comprising applying to a locus in which said plant is cultivated an effective amount of a composition according to any one of claims 1- 19, wherein release of said essential oils from said composition is activated primarily by, and synchronized with, soil moisture conditions including irrigation or rainfall.

22. A method for promoting plant growth and resilience under biotic or abiotic stress conditions, said method comprising applying to a locus in which said plant is cultivated an effective amount of a composition according to any one of claims 1-19, wherein release of said essential oils from said composition is activated primarily by, and synchronized with, soil moisture conditions including irrigation or rainfall.

23. The method of claim 22, wherein said biotic stress conditions result from root-zone infestation by soilborne nematodes.

24. The method of claim 22, wherein said abiotic stress conditions result from nutrient imbalance, reduced moisture availability, or soil compaction.

25. The method of any one of claims 20-24, wherein the application of said composition to said locus comprises broadcasting onto soil surface, incorporation into soil during cultivation, or mixing with irrigation water for fertigation systems.

26. The method of claim 20 or 21, wherein said composition provides enhanced essential oil release during wet periods, ensuring targeted nematode control synchronized with nematode activity cycles.

27. The method of any one of claims 21-24, wherein the release of said essential oils improves / promotes at least one plant growth parameter.

28. The method of claim 27, wherein said plant growth parameter is selected from stem elongation, leaf number, fresh weight, dry weight under nematode infestation stress, and a combination thereof.

29. The method of claim 21, for treating a plant against soilborne nematodes infection, wherein the release of said essential oils reduces nematode density and concurrently enhances plant biomass and elongation.

30. Use of a composition according to any one of claims 1-19 for controlling soilborne nematodes infection in a locus or protecting a locus from soilborne nematodes infestation, for treating a plant against soilborne nematodes infection or protecting a plant from soilborne nematodes infestation, or for promoting plant growth and resilience under biotic or abiotic stress conditions.

Images