CA3060884C - Phytosanitary composition comprising essential oils that potentiate antifungal activity - Google Patents
Phytosanitary composition comprising essential oils that potentiate antifungal activity Download PDFInfo
- Publication number
- CA3060884C CA3060884C CA3060884A CA3060884A CA3060884C CA 3060884 C CA3060884 C CA 3060884C CA 3060884 A CA3060884 A CA 3060884A CA 3060884 A CA3060884 A CA 3060884A CA 3060884 C CA3060884 C CA 3060884C
- Authority
- CA
- Canada
- Prior art keywords
- inhibition
- k2co3
- concentration
- composition according
- carvacrol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The present invention relates to phytosanitary compositions with fungicidal properties that comprise one essential oil obtained from plants selected from oregano oil (Origanum vulgare) and thyme oil (Thymus vulgaris) or its active compounds carvacrol at a concentration between 0.1 and 530 ppm or thymol at a concentration between 0.31 and 530 ppm, or a combination thereof; and (2) potassium carbonate at a concentration between 3.5 and 25 mM, and an agent with known fungicidal properties for use, principally, in contact-protection against fungal infections in cultivated plants and post-harvest, and also in other antifungal applications. In said compositions, the effect of the agents that have known fungicidal properties is potentiated synergistically by the aforementioned essential oils.
Description
PHYTOSANITARY COMPOSITION COMPRISING ESSENTIAL OILS THAT
POTENTIATE ANTIFUNGAL ACTIVITY
DESCRIPTION
[0001] This invention relates to phytosanitary compositions having fungicidal properties comprising a mixture of essential oils obtained from plants, such as thyme oil (Thymus vulgalls) and oregano oil (aiganum vulgare) or mixtures thereof, and an agent having known fungicidal properties such as potassium carbonate, for use mainly in contact protection against fungicidal infections in cultivated plants and post-harvesting, and in other antifungal applications. In these compositions the effect of the agent having known fungicidal properties is synergistically potentiated by the essential oils mentioned.
POTENTIATE ANTIFUNGAL ACTIVITY
DESCRIPTION
[0001] This invention relates to phytosanitary compositions having fungicidal properties comprising a mixture of essential oils obtained from plants, such as thyme oil (Thymus vulgalls) and oregano oil (aiganum vulgare) or mixtures thereof, and an agent having known fungicidal properties such as potassium carbonate, for use mainly in contact protection against fungicidal infections in cultivated plants and post-harvesting, and in other antifungal applications. In these compositions the effect of the agent having known fungicidal properties is synergistically potentiated by the essential oils mentioned.
[0002] Essential oils are complex mixtures of natural molecules which are fundamentally obtained from plants. They are secondary metabolites which can normally be obtained by extraction with organic solvents and subsequent concentration, or by physical treatments with steam followed by separation of the water-insoluble phase. Generally they are volatile liquids soluble in organic solvents and have a density lower than that of water.
[0003] In nature they can be synthesized in different plant organs such as seeds, leaves, flowers, epidermal cells and fruits, among others, and they play an important part in protecting plants against bacterial, viral or fungal infections.
[0004] The fungicidal and bactericidal action of many plant essential oils is known, and has arrived in some case to be marketed commercially. Among these are jojoba oil (Simrnondsia californica), rosemary oil (Rosmarinus officthalis), thyme oil (T vulgaris), the clarified hydrophobic extract of neem oil (A. indica), cottonseed oil (Gossypium hirsutum) with garlic extract (Dayan et al., Bioorg.
and Med. Chem. 2009;17:4022-34).
and Med. Chem. 2009;17:4022-34).
[0005] The chemical composition of essential oils differs not only in the quantity but also in the quality and the stereochemical type of the molecules in the extracted substances. The extraction product may vary according to climate, the composition of the soil, the organ of the plant used for Page 1 of 33 extraction, and the age and stage of growth of the plant. It also depends on the extraction process used.
[0006] Furthermore, Zamani et al. showed that the potassium carbonate was effective as fungicide treatment against Penicillium digitatum in green mold on oranges (Commun.
Agric. Appl. Biol. Sci.
2007;72(4):773-7). The Patent CN107041366 discloses the use of potassium carbonate which is applied together with pesticides (including fungicides), improving the emulsification and impregnation.
Agric. Appl. Biol. Sci.
2007;72(4):773-7). The Patent CN107041366 discloses the use of potassium carbonate which is applied together with pesticides (including fungicides), improving the emulsification and impregnation.
[0007] Because of their natural origin plant essential oils are very attractive for application in agriculture in order to obtain healthy and harmless products, as this is a requirement which has been made increasingly strictly, by both consumers and regulatory authorities.
100081 There is therefore a need to find new phytosanitary compositions having antifungal properties to protect crops, including during post-harvesting, which have a minimum of secondary toxic effects and which are harmless to human beings and the environment.
[0009] The present authors have surprisingly found that some essential oils obtained from plants when mixed with other products having known antifungal properties potentiate the antifungal properties of these compounds, such as inorganic salts, for example alkali metal carbonates like potassium carbonate.
[0010] Thus one object of the present invention is to provide a phytosanitary composition having antifungal activity comprising: (1) one essential oil obtained from plants selected from oregano oil (Origanum vulgare) and thyme oil (Thymus vulgatis) or its active compounds carvacrol at a concentration between 0.1 and 530 ppm or thymol at a concentration between 0.31 and 530 ppm, or a combination thereof; and (2) potassium carbonate at a concentration between 3.5 and 25 inM.
[0011] This composition synergistically improves the antifungal properties of the agents having known antifungal activity, has a minimum of secondary toxic effects and is harmless to human beings and the environment.
Page 2 of 33 [0012] The composition according to this invention may be applied in agriculture to protect crops from the stage of germination to harvesting, and during the storage and transport of these crops, seeds, flowers or grains. Likewise, another possible application is in the elimination of fungi which attack painted surfaces and to protect carpets and fabrics in the home and in any other application against fungal attack through contact.
[0013] Among the essential oils which may be used in the phytosanitary composition according to this invention are thyme oil (Thymus vulganS) and oregano oil (Onganum vulgare) or mixtures thereof.
[0014] Without being bound to any theory in particular, it is possible that the property of the essential oils obtained from plants in potentiating antifungal activity is due to some of the compounds present in these essential oils having known activity. Thus in one embodiment of this invention the phytosanitary composition may comprise one or a mixture of active compounds isolated from the essential oils according to this invention, such as phenolic monoterpenoids such as carvacrol and thymol, and mixtures thereof, and an agent having known fungicidal properties, such as potassium carbonate. The mechanism of action of the essential oils is a multiple one due to the complex mixture of different active ingredients which they contain. However, the nature of the action of the major components in some of these oils has been described. The best described in the literature is the nature of the action of carvacrol on the growth of bacterial and yeast cells (Ultee et al., Appl. Environ.
Microbiol. 2002;68(4):1561-68). According to these studies carvacrol is capable of crossing the cell membrane when it is protonated (in acid medium) and on reaching the cytoplasm releases a proton, resulting in acidification of the cell. This manner of action does not rule out other possible modes of action such as increase in the permeability of the membrane or specific inhibiting effects on catalytic processes. Moreover, the Patent CN104642326 describes a fungicidal composition containing penflufen and carvacrol. PCT application W02014036667 discloses a continuous extraction method to produce a high content of carvacrol and thymol, which are powerful fungicides.
[0015] On the other hand, some studies showed that the essential oil of Thymus vulgths, the thyme oil, has a moderate control efficacy against Aspergillus Inger strains with its antifungal activity Page 3 of 33 resulting mainly due to killing of microorganism rather than growth inhibition. Oils on wheat seeds showed no significant phytotoxic effect in terms of seed germination or seedling growth (Kumar et al., Environ. Sci. Pollut. Res. Int. 2017;24(27):21948-59). Other important evidence is that an in vivo antifungal assay demonstrated that the maximum antifungal activity was showed by thyme oil against Pernciilium expansum and Botrytis cinerea in pear fruits (Nikkhah et al., Int.
J. Food Microbiol.
2017;18(257):285-94). Furthermore, the US09492490 Patent describes a composition for controlling a target pest comprising 0.1% to 4% isopropyl myristate, 0.1% to 15% thyme oil white, 0.1% to 2%
geraniol, and at least one additional active ingredient. The PCT W02014153042 discloses a method for treating Mycosphaerella fijiensis in crops of the Musaceae family by applying a fungicidal composition comprising garlic oil, rosemary oil, thyme oil and cinnamon oil.
[0016] Among the agents having known fungicidal properties which may be used in the composition according to the invention there are the carbonates of alkali metals, preferably of lithium, sodium or potassium. More preferably the agent having known fungicidal properties is potassium carbonate.
[0017] The concentration of thymol present in the composition according to this invention is between 0.31 and 530 ppm, preferably between 22 and 350 ppm. The concentration of carvacrol present in the composition according to this invention is between 0.1 and 530 ppm, preferably between 22 and 310 ppm. Also the concentration of the potassium carbonate having known fungicidal properties in the composition according to this invention may vary between 3.5 and 25 mM
preferably between 10 and 25 mM.
[0018] The composition according to this invention may be prepared by mixing the essential oil or oils and the agent having fungicidal properties through any method of mixing known in the art.
However, the composition may also be in solid or liquid form, such as a suspension, dispersion, emulsion, spray, microencapsulate or any other type of mixture which remains stable over time or may be incorporated in polymers, waxes or any other similar supports.
[0019] Furthermore, the phytosanitary composition according to this invention may be used as such, or may be used to formulate a phytosanitary product together with different additives used in the art Page 4 of 33 which offer different properties, such as surfactants, polymers, alkanising agents, pH-control agents, among many other additives used in the formulation of products used in the agricultural industry.
100201 The phytosanitary composition according to this invention falls within the group of contact phytosanitary agents that is the form of the protection against fungal diseases is through contact, given that the composition remains on the surface of different parts of the plant, protecting it externally against the external attack of fungi.
[0021] Being a liquid, a powder or a microencapsulate, the phytosanitary composition according to this invention can be applied by any method of application known in the art, such as spraying.
100221 The fungicidal composition according to this invention may further comprise a fertiliser, which may be selected from the group comprising compounds containing nitrogen and/or phosphorus such as urea, melamine, hexamine, dicyanodiamide, ameline, cyanuric acid, melamine nitrate, triethyl phosphite and the like or mixtures thereof.
100231 The composition according to this invention may also comprise any compound or product having chemical and/or biological activity used in agriculture, such as herbicides, insecticides, plant growth regulators and the like, or mixtures thereof.
[0024] This invention is described below in greater detail with reference to various examples.
However, these examples are not intended to restrict the technical scope of this invention.
EXAMPLES
[0025] Example 1. Inhibition of growth of the fungus Bowls cinerea by K2CO3 alone.
[0026] The fungus B. cinema was cultured in PDB (potato dextrose broth) medium with different concentrations of K2CO3. The % inhibition, representing the extent to which growth was impeded in comparison with a control which did not have the compound(s) under test, was calculated in the following way:
0 D (control) ¨ OD (x) % Inhibition = x 100 OD (control) Page 5 of 33 [0027] where "OD(control)" is the optical density of the control culture (without test compounds) and "OD(x)" is the optical density of the culture with the test substance(s). The optical density of the liquid culture was measured 24 hours after the start of culturing and the results are shown in Table I.
Table I. Inhibition of the growth of B. cinerea by K2CO3 K2CO3 concentration 0 3.5 4.25 4.5 5 (mM) Inhibition 48.2 53.3 67.2 71.0 SD (%) + 2.3 2.4 1.9 1.6 [0028] As will be seen from the table above, with a K2CO3 concentration between 3.5 and 5 mM
inhibition of the B. cinerea culture was observed.
[0029] Example 2. Inhibition of growth of the fungus Botrytis cinema by carvacrol.
[0030] The fungus B. cinema was cultured in PDB medium with different concentrations of carvacrol.
The % inhibition was calculated as Example 1.
[0031] The optical density of the liquid culture was measured 24 hours after the start of culturing and the results are shown in Table II.
Table II. Inhibition of the growth of B. cinerea by carvacrol Carvacrol concentration 0.1 0.31 1 3.1 10 31 (PPm) Inhibition 10.5 13.7 22.4 21.3 51.4 74.4 SD (%) 7.7 4.1 3.4 5.0 5.5 1.1 [0032] Example 3. Inhibition of the fungus Botrytis cinerea by the composition according to this invention (K2CO3 + Carvacrol).
[0033] The fungus B. cinerea was cultured in a similar way to Example 1 with the difference that different concentrations of carvacrol were used in the medium and that a constant concentration of Page 6 of 33 K2CO3 (3.5 mM) was used throughout. The 24 hour optical density of the culture was measured and the results are shown in Table III.
Table III. Inhibition of B. cinerea by the composition according to this invention (K2CO3 + Carvacrol) K2CO3 concentration 3.5 3.5 3.5 3.5 3.5 3.5 3.5 (mM) Carvacrol concentration 0.1 0.31 1 3.1 10 31 100 (PPm) Inhibition 50.2 58.8 65.7 82.4 88.0 100 100 SD (%) 3.0 2.2 2.7 1.9 1.5 0 0 [0034] It will be seen how the results are improved by adding carvacrol to K2CO3. With 10 ppm of carvacrol (see in Table II) only 21% inhibition is achieved, and with 3.5 mM
of K2CO3 48% inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus B.
cinerea is increased up to 88%.
[0035] Example 4. Inhibition of growth of the fungus Bowls cinerea by thymol alone.
[0036] The fungus B. cinerea was cultured in a similar way to Example 1 with different concentrations of thymol. The 24 hour optical density of the culture was measured and the results are shown in Table IV.
Table IV. Inhibition of the growth of B. cinerea by Thymol Thymol concentration 0.31 1 3.1 10 31 100 (PPm) Inhibition 0 2.1 10.7 12.6 32.0 88.2 SD (%) 0 2.9 2.3 2.4 1.7 0.6 [0037] Example 5. Inhibition of the fungus Botrytis cinerea by the composition according to this invention (K2CO3 + Thymol).
Page 7 of 33 [0038] The fungus B. cinerea was cultured in a similar way to Example 1 with the difference that different concentrations of thymol were used in the medium and that a constant concentration of K2CO3 (3.5 mM) was used throughout. The 24 hour optical density of the culture was measured and the results are shown in Table V.
Table V. Inhibition of B. cinerea by the composition according to this invention (K2CO3 + Thymol) K2CO3 concentration 3.5 3.5 3.5 3.5 3.5 3.5 (mM) Thymol concentration 0.31 1 3.1 10 31 100 (PPm) Inhibition 49.4 62.1 73.2 81.0 93.0 SD (%) 2.1 2.2 1.1 1.6 1.1 [0039] It will be seen how the results are improved by adding thymol to K2CO3.
With 10 ppm of thymol only 13% inhibition is achieved, and with 3.5 mM of K2CO3 48%
inhibition is achieved.
However, when the two compounds are combined inhibition of growth of the fungus B. cinerea is increased up to some 81%.
[0040] Example 6. Inhibition of growth of the fungus Alternana alternata by K2CO3 alone.
[0041] Alternaria alternata was cultured in a similar way to Example 1 with different concentrations of K2CO3. The 24 hour optical density of the culture was measured and the results are shown in Table VI.
Table VI. Inhibition of the growth of A. alternata by K2CO3 K2CO3 concentration 0 3.5 4.25 4.5 5 (mM) Inhibition 31.5 39.7 48.0 59.0 SD (%) 2.5 2.5 2.2 2.2 Pages of 33 [0042] As will be seen from the table above, with a K2CO3 concentration between 3.5 and 5 mM
inhibition of the A. altemata culture was observed.
[0043] Example 7. Inhibition of growth of the fungus Alternalia alternata by carvacrol.
[0044] The fungus A. altemata was cultured in PDB medium with different concentrations of carvacrol. The % inhibition was calculated as Example 1 and the results are shown in Table VII.
Table VII. Inhibition of the growth of A. alternata by carvacrol Carvacrol concentration 0.1 0.31 1 3.1 10 31 (PPm) Inhibition 2.0 3.5 5.8 17.7 27.2 74.6 SD (%) 0.3 0.8 1.4 11.0 14.0
100081 There is therefore a need to find new phytosanitary compositions having antifungal properties to protect crops, including during post-harvesting, which have a minimum of secondary toxic effects and which are harmless to human beings and the environment.
[0009] The present authors have surprisingly found that some essential oils obtained from plants when mixed with other products having known antifungal properties potentiate the antifungal properties of these compounds, such as inorganic salts, for example alkali metal carbonates like potassium carbonate.
[0010] Thus one object of the present invention is to provide a phytosanitary composition having antifungal activity comprising: (1) one essential oil obtained from plants selected from oregano oil (Origanum vulgare) and thyme oil (Thymus vulgatis) or its active compounds carvacrol at a concentration between 0.1 and 530 ppm or thymol at a concentration between 0.31 and 530 ppm, or a combination thereof; and (2) potassium carbonate at a concentration between 3.5 and 25 inM.
[0011] This composition synergistically improves the antifungal properties of the agents having known antifungal activity, has a minimum of secondary toxic effects and is harmless to human beings and the environment.
Page 2 of 33 [0012] The composition according to this invention may be applied in agriculture to protect crops from the stage of germination to harvesting, and during the storage and transport of these crops, seeds, flowers or grains. Likewise, another possible application is in the elimination of fungi which attack painted surfaces and to protect carpets and fabrics in the home and in any other application against fungal attack through contact.
[0013] Among the essential oils which may be used in the phytosanitary composition according to this invention are thyme oil (Thymus vulganS) and oregano oil (Onganum vulgare) or mixtures thereof.
[0014] Without being bound to any theory in particular, it is possible that the property of the essential oils obtained from plants in potentiating antifungal activity is due to some of the compounds present in these essential oils having known activity. Thus in one embodiment of this invention the phytosanitary composition may comprise one or a mixture of active compounds isolated from the essential oils according to this invention, such as phenolic monoterpenoids such as carvacrol and thymol, and mixtures thereof, and an agent having known fungicidal properties, such as potassium carbonate. The mechanism of action of the essential oils is a multiple one due to the complex mixture of different active ingredients which they contain. However, the nature of the action of the major components in some of these oils has been described. The best described in the literature is the nature of the action of carvacrol on the growth of bacterial and yeast cells (Ultee et al., Appl. Environ.
Microbiol. 2002;68(4):1561-68). According to these studies carvacrol is capable of crossing the cell membrane when it is protonated (in acid medium) and on reaching the cytoplasm releases a proton, resulting in acidification of the cell. This manner of action does not rule out other possible modes of action such as increase in the permeability of the membrane or specific inhibiting effects on catalytic processes. Moreover, the Patent CN104642326 describes a fungicidal composition containing penflufen and carvacrol. PCT application W02014036667 discloses a continuous extraction method to produce a high content of carvacrol and thymol, which are powerful fungicides.
[0015] On the other hand, some studies showed that the essential oil of Thymus vulgths, the thyme oil, has a moderate control efficacy against Aspergillus Inger strains with its antifungal activity Page 3 of 33 resulting mainly due to killing of microorganism rather than growth inhibition. Oils on wheat seeds showed no significant phytotoxic effect in terms of seed germination or seedling growth (Kumar et al., Environ. Sci. Pollut. Res. Int. 2017;24(27):21948-59). Other important evidence is that an in vivo antifungal assay demonstrated that the maximum antifungal activity was showed by thyme oil against Pernciilium expansum and Botrytis cinerea in pear fruits (Nikkhah et al., Int.
J. Food Microbiol.
2017;18(257):285-94). Furthermore, the US09492490 Patent describes a composition for controlling a target pest comprising 0.1% to 4% isopropyl myristate, 0.1% to 15% thyme oil white, 0.1% to 2%
geraniol, and at least one additional active ingredient. The PCT W02014153042 discloses a method for treating Mycosphaerella fijiensis in crops of the Musaceae family by applying a fungicidal composition comprising garlic oil, rosemary oil, thyme oil and cinnamon oil.
[0016] Among the agents having known fungicidal properties which may be used in the composition according to the invention there are the carbonates of alkali metals, preferably of lithium, sodium or potassium. More preferably the agent having known fungicidal properties is potassium carbonate.
[0017] The concentration of thymol present in the composition according to this invention is between 0.31 and 530 ppm, preferably between 22 and 350 ppm. The concentration of carvacrol present in the composition according to this invention is between 0.1 and 530 ppm, preferably between 22 and 310 ppm. Also the concentration of the potassium carbonate having known fungicidal properties in the composition according to this invention may vary between 3.5 and 25 mM
preferably between 10 and 25 mM.
[0018] The composition according to this invention may be prepared by mixing the essential oil or oils and the agent having fungicidal properties through any method of mixing known in the art.
However, the composition may also be in solid or liquid form, such as a suspension, dispersion, emulsion, spray, microencapsulate or any other type of mixture which remains stable over time or may be incorporated in polymers, waxes or any other similar supports.
[0019] Furthermore, the phytosanitary composition according to this invention may be used as such, or may be used to formulate a phytosanitary product together with different additives used in the art Page 4 of 33 which offer different properties, such as surfactants, polymers, alkanising agents, pH-control agents, among many other additives used in the formulation of products used in the agricultural industry.
100201 The phytosanitary composition according to this invention falls within the group of contact phytosanitary agents that is the form of the protection against fungal diseases is through contact, given that the composition remains on the surface of different parts of the plant, protecting it externally against the external attack of fungi.
[0021] Being a liquid, a powder or a microencapsulate, the phytosanitary composition according to this invention can be applied by any method of application known in the art, such as spraying.
100221 The fungicidal composition according to this invention may further comprise a fertiliser, which may be selected from the group comprising compounds containing nitrogen and/or phosphorus such as urea, melamine, hexamine, dicyanodiamide, ameline, cyanuric acid, melamine nitrate, triethyl phosphite and the like or mixtures thereof.
100231 The composition according to this invention may also comprise any compound or product having chemical and/or biological activity used in agriculture, such as herbicides, insecticides, plant growth regulators and the like, or mixtures thereof.
[0024] This invention is described below in greater detail with reference to various examples.
However, these examples are not intended to restrict the technical scope of this invention.
EXAMPLES
[0025] Example 1. Inhibition of growth of the fungus Bowls cinerea by K2CO3 alone.
[0026] The fungus B. cinema was cultured in PDB (potato dextrose broth) medium with different concentrations of K2CO3. The % inhibition, representing the extent to which growth was impeded in comparison with a control which did not have the compound(s) under test, was calculated in the following way:
0 D (control) ¨ OD (x) % Inhibition = x 100 OD (control) Page 5 of 33 [0027] where "OD(control)" is the optical density of the control culture (without test compounds) and "OD(x)" is the optical density of the culture with the test substance(s). The optical density of the liquid culture was measured 24 hours after the start of culturing and the results are shown in Table I.
Table I. Inhibition of the growth of B. cinerea by K2CO3 K2CO3 concentration 0 3.5 4.25 4.5 5 (mM) Inhibition 48.2 53.3 67.2 71.0 SD (%) + 2.3 2.4 1.9 1.6 [0028] As will be seen from the table above, with a K2CO3 concentration between 3.5 and 5 mM
inhibition of the B. cinerea culture was observed.
[0029] Example 2. Inhibition of growth of the fungus Botrytis cinema by carvacrol.
[0030] The fungus B. cinema was cultured in PDB medium with different concentrations of carvacrol.
The % inhibition was calculated as Example 1.
[0031] The optical density of the liquid culture was measured 24 hours after the start of culturing and the results are shown in Table II.
Table II. Inhibition of the growth of B. cinerea by carvacrol Carvacrol concentration 0.1 0.31 1 3.1 10 31 (PPm) Inhibition 10.5 13.7 22.4 21.3 51.4 74.4 SD (%) 7.7 4.1 3.4 5.0 5.5 1.1 [0032] Example 3. Inhibition of the fungus Botrytis cinerea by the composition according to this invention (K2CO3 + Carvacrol).
[0033] The fungus B. cinerea was cultured in a similar way to Example 1 with the difference that different concentrations of carvacrol were used in the medium and that a constant concentration of Page 6 of 33 K2CO3 (3.5 mM) was used throughout. The 24 hour optical density of the culture was measured and the results are shown in Table III.
Table III. Inhibition of B. cinerea by the composition according to this invention (K2CO3 + Carvacrol) K2CO3 concentration 3.5 3.5 3.5 3.5 3.5 3.5 3.5 (mM) Carvacrol concentration 0.1 0.31 1 3.1 10 31 100 (PPm) Inhibition 50.2 58.8 65.7 82.4 88.0 100 100 SD (%) 3.0 2.2 2.7 1.9 1.5 0 0 [0034] It will be seen how the results are improved by adding carvacrol to K2CO3. With 10 ppm of carvacrol (see in Table II) only 21% inhibition is achieved, and with 3.5 mM
of K2CO3 48% inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus B.
cinerea is increased up to 88%.
[0035] Example 4. Inhibition of growth of the fungus Bowls cinerea by thymol alone.
[0036] The fungus B. cinerea was cultured in a similar way to Example 1 with different concentrations of thymol. The 24 hour optical density of the culture was measured and the results are shown in Table IV.
Table IV. Inhibition of the growth of B. cinerea by Thymol Thymol concentration 0.31 1 3.1 10 31 100 (PPm) Inhibition 0 2.1 10.7 12.6 32.0 88.2 SD (%) 0 2.9 2.3 2.4 1.7 0.6 [0037] Example 5. Inhibition of the fungus Botrytis cinerea by the composition according to this invention (K2CO3 + Thymol).
Page 7 of 33 [0038] The fungus B. cinerea was cultured in a similar way to Example 1 with the difference that different concentrations of thymol were used in the medium and that a constant concentration of K2CO3 (3.5 mM) was used throughout. The 24 hour optical density of the culture was measured and the results are shown in Table V.
Table V. Inhibition of B. cinerea by the composition according to this invention (K2CO3 + Thymol) K2CO3 concentration 3.5 3.5 3.5 3.5 3.5 3.5 (mM) Thymol concentration 0.31 1 3.1 10 31 100 (PPm) Inhibition 49.4 62.1 73.2 81.0 93.0 SD (%) 2.1 2.2 1.1 1.6 1.1 [0039] It will be seen how the results are improved by adding thymol to K2CO3.
With 10 ppm of thymol only 13% inhibition is achieved, and with 3.5 mM of K2CO3 48%
inhibition is achieved.
However, when the two compounds are combined inhibition of growth of the fungus B. cinerea is increased up to some 81%.
[0040] Example 6. Inhibition of growth of the fungus Alternana alternata by K2CO3 alone.
[0041] Alternaria alternata was cultured in a similar way to Example 1 with different concentrations of K2CO3. The 24 hour optical density of the culture was measured and the results are shown in Table VI.
Table VI. Inhibition of the growth of A. alternata by K2CO3 K2CO3 concentration 0 3.5 4.25 4.5 5 (mM) Inhibition 31.5 39.7 48.0 59.0 SD (%) 2.5 2.5 2.2 2.2 Pages of 33 [0042] As will be seen from the table above, with a K2CO3 concentration between 3.5 and 5 mM
inhibition of the A. altemata culture was observed.
[0043] Example 7. Inhibition of growth of the fungus Alternalia alternata by carvacrol.
[0044] The fungus A. altemata was cultured in PDB medium with different concentrations of carvacrol. The % inhibition was calculated as Example 1 and the results are shown in Table VII.
Table VII. Inhibition of the growth of A. alternata by carvacrol Carvacrol concentration 0.1 0.31 1 3.1 10 31 (PPm) Inhibition 2.0 3.5 5.8 17.7 27.2 74.6 SD (%) 0.3 0.8 1.4 11.0 14.0
8.0 [0045] Example 8. Inhibition of the fungus Alternarin alternata by the composition according to this invention (K2CO3 + Carvacrol).
[0046] The fungus A. altemata was cultured in a similar way to Example 1 with the difference that different concentrations of carvacrol were used in the medium and that a constant concentration of K2CO3 (3.5 mM) was used throughout. The 24 hour optical density of the culture was measured and the results are shown in Table VIII.
Table VIII. Inhibition of A. alternata by the composition according to this invention (K2CO3 +
Carvacrol) K2CO3 concentration 3.5 3.5 3.5 3.5 3.5 3.5 3.5 (mM) Carvacrol concentration 0.1 0.31 1 3.1 10 31 100 (PPm) Inhibition 30.1 35.7 38.0 46.2 65.7 86.3 100 SD (%) 2.9 2.7 2.9 2.9 1.6 1.2 0 Page 9 of 33 [0047] It will be seen how the results are improved by adding carvacrol to K2CO3. With 31 ppm of carvacrol (see Table VII) only 27% inhibition is achieved, and with 3.5 mM of K2CO3 32% inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus A.
alternata is increased up to some 86%.
[0048] Example 9. Inhibition of growth of the fungus Alternalla alternata by thymol alone.
[0049] The fungus A. alternata was cultured in a similar way to Example 1 with different concentrations of thymol. The 24 hour optical density of the culture was measured and the results are shown in Table IX.
Table IX. Inhibition of the growth of A. alternata by Thymol Thymol concentration 0.31 1 3.1 10 31 100 (PPrn) Inhibition 0 4.1 10.4 16.0 29.0 69.4 SD (%) 0 2.9 2.6 2.0 1.5 [0050] Example 10. Inhibition of the fungus Alternaria alternata by the composition according to this invention (K2CO3 + Thymol).
100511 The fungus A. alternata was cultured in a similar way to Example 1 with the difference that different concentrations of thymol were used in the medium and that a constant concentration of K2CO3 (3.5 mM) was used throughout. The 24 hour optical density of the culture was measured and the results are shown in Table X.
Table X. Inhibition of A. alternata by the composition according to this invention (K2CO3 +
Thymol) K2CO3 concentration (mM) 3.5 3.5 3.5 3.5 3.5 3.5 Thymol concentration 0.31 1 3.1 10 31 100 (PPm) Inhibition 33.7 38.7 45.3 55.5 71.8 SD (%) 2.2 2.7 2.4 1.2 1.3 Page 10 of 33 [0052] It will be seen how the results are improved by adding thymol to K2CO3.
With 31 ppm of thymol only 29% inhibition is achieved, and with 3.5 mM of K2CO3 32%
inhibition is achieved.
However, when the two compounds are combined inhibition of growth of the fungus A. alternata is increased up to some 72%.
[0053] Example 11. Inhibition of growth of the fungus Penicillium digitatum by K2CO3 alone.
[0054] Penicillium digitatum was cultured in a similar way to Example 1 with different concentrations of K2CO3. The 24 hour optical density of the culture was measured and the results are shown in Table XI.
Table XI. Inhibition of the growth of P. digitatum by K2CO3 K2CO3 concentration 0 3.5 4.25 4.5 5 (mM) Inhibition 29.1 33.0 36.9 39.5 SD (%) 2.1 1.7 1.6 1.8 [0055] As will be seen from the table above, with a K2CO3 concentration between 3.5 and 5 mM
inhibition of the P. digitatum culture was observed.
100561 Example 12. Inhibition of growth of the fungus Penicillium digitatum by carvacrol alone.
[0057] The fungus P. digitatum was cultured in 'a similar way to Example 1 with different concentrations of carvacrol. The 24 hour optical density of the culture was measured and the results are shown in Table XII.
Table XII. Inhibition of the growth of P. digitatum by Carvacrol Carvacrol 0.1 0.31 1 3.1 10 31 100 concentration (ppm) Inhibition 32.3 34.3 37.9 52.6 57.8 86.0 SD (%) 2.8 2.8 3.0 2.2 2.1 1.1 Page 11 of 33 100581 Example 13. Inhibition of the fungus Penicillium digitatum by the composition according to this invention (K2CO3 + Carvacrol).
[0059] The fungus P. a'igitatum was cultured in a similar way to Example 1 with the difference that different concentrations of carvacrol were used in the medium and that a constant concentration of K2CO3 (3.5 mM) was used throughout. The 24 hour optical density of the culture was measured and the results are shown in Table XIII.
Table XIII. Inhibition of P. digitatum by the composition according to this invention (K2CO3 +
Carvacrol) K2CO3 concentration 3.5 3.5 3.5 3.5 3.5 3.5 3.5 (mM) Carvacrol concentration 0.1 0.31 1 3.1 10 31 100 (PPm) Inhibition 65.9 66.2 72.4 88.6 96.5 100 100 SD (%) 2.4 2.9 2.2 1.9 1.4 0 0 [0060] It will be seen how the results are improved by adding carvacrol to K2CO3. With 10 ppm of carvacrol 58% inhibition is achieved, and with 3.5 mM of K2CO3 29% inhibition is achieved.
However, when the two compounds are combined inhibition of growth of the fungus P. digitatum is increased up to 97%.
[0061] Example 14. Inhibition of growth of the fungus Penicillium digitatum by thymol alone.
[0062] The fungus P. digitatum was cultured in a similar way to Example 1 with different concentrations of thymol. The 24 hour optical density of the culture was measured and the results are shown in Table XIV.
Table XIV. Inhibition of the growth of P. digitatum by Thymol Thymol concentration 0.31 1 3.1 10 31 100 100 (PPm) Inhibition 28.2 24.2 36.3 36.2 50.7 78.3 95.6 Page 12 of 33 SD (%) 3.9 + 6.0 2.3 2.0 2.0 2.2 0.5 [0063] Example 15. Inhibition of the fungus Penicillium digitatum by the composition according to this invention (K2CO3 + Thymol).
[0064] The fungus P. digitatum was cultured in a similar way to Example 1 with the difference that different concentrations of thymol were used in the medium and that a constant concentration of K2CO3 (3.5 mM) was used throughout. The 24 hour optical density of the culture was measured and the results are shown in Table XV.
Table XV. Inhibition of P. digitatum by the composition according to this invention (K2CO3 +
Thymol) K2CO3 concentration 3.5 3.5 3.5 3.5 3.5 3.5 (mM) Thymol concentration 0.31 1 3.1 10 31 100 (PPm) Inhibition 58.1 61.3 69.9 78.3 94.3 SD (%) 2.9 2.5 1.9 1.5 1.2 [0065] It will be seen how the results are improved by adding thymol to K2CO3.
With 31 ppm of thymol (see Table XIV) only 51% inhibition is achieved, and with 3.5 mM of K2CO3 29% inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus P.
digitatum is increased up to some 94%.
Page 13 of 33 [0066] Example 16. Inhibition of growth of the fungus Cercospora beticola by K2CO3 alone.
[0067] Cercospora beticola was cultured on PDA (potato dextrose agar) culture medium buffered to a pH value not exceeding 9.5. The inhibition degree, expressed as a percentage, was determined based on the growth relative to control that did not have the compound(s) to be tested. The % inhibition was calculated with the following formula:
colony diameter (control) ¨ colony diameter (x) % Inhibition = x 100 colony diameter (control) [0068] wherein "colony diameter (control)" is the size of the control colony (without the compounds to be tested) and "colony diameter (x)" is the size of the colony with the substance(s) to be tested. A
fixed K2CO3 concentration of 7.24 mM was tested in C beticola. The results are shown in Table XVI.
Table XVI. Inhibition of the growth of C beticola by K2CO3 alone K2CO3 concentration 7.24 (mM) Inhibition 25.0 SD (%) 1.2 [0069] Example 17. Inhibition of growth of the fungus Cercospora beticola by carvacrol alone.
100701 The fungus C beticola was cultured in a similar way to Example 16 with 10 ppm of carvacrol.
The % inhibition was calculated and the results are shown in Table XVII.
Table XVII. Inhibition of the growth of C beticola by carvacrol alone Carvacrol concentration (PPrn) Inhibition 17.0 SD (%) 0.8 100711 Example 18. Inhibition of growth of the fungus Cercospora beticola by the composition according to this invention (K2CO3 + Carvacrol).
Page 14 of 33 [0072] The fungus C beticola was cultured in a similar way to Example 16 with a fixed concentration of K2CO3 (7.24 mM) and carvacrol (10 ppm). The % inhibition was calculated and the results are shown in Table XVIII.
Table XVIII. Inhibition of the growth of C beticola by the composition according to this invention (K2CO3 + Carvacrol) K2CO3 concentration 7.24 (mM) Carvacrol concentration (ppm) Inhibition 65.0 SD (%) 3.2 [0073] It will be seen how the results are improved by adding carvacrol to K2CO3. With 10 ppm of carvacrol (see Table XVII) only 17%
inhibition is achieved, and with 7.24 mM of K2CO3 25% inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus C beticola is increased up to some 65%.
[0074] Example 19. Inhibition of the fungus Botrytis cinerea by K2CO3 alone.
[0075] Leaves of tomato plants (var. Marmande) of 5 weeks old were treated with different concentrations of K2CO3, and 24 hours later, they were infected with the fungus Botrytis cinema. Two weeks later, fungal infection was assessed in leaves. The % inhibition, representing the extent to which fungal growth was impeded in comparison with a control which did not have the compound(s) under test, was determined in the following way:
% Infection (control) ¨ % Infection (x) % Inhibition = ________________________________________________ x 100 Infection (control) [0076] where "% Infection (control)" is the percentage of fungal infection of the control leaves (without test compounds) and "% Infection (x)" is percentage of fungal infection of the treated leaves.
The results are shown in Table XIX.
Table XIX. Inhibition of the growth of B. cinerea by K2CO3 alone Page 15 of 33 K2CO3 concentration (mM) Inhibition 13.4 13.5 13.8 13.8 15.3 SD (%) 2.5 2.1 2.3 3.0 2.4 [0077] Example 20. Inhibition of the fungus Botrytis cinerea by Carvacrol alone.
[0078] Leaves of tomato plants treated with different concentrations of carvacrol were infected with the fungus B. cinerea in a similar way to Example 19. The % inhibition was calculated and the results are shown in Table XX.
Table XX. Inhibition of the growth of B. cinerea by Carvacrol alone Carvacrol concentration (PPm) Inhibition 7.2 7.4 16.7 18.6 20.8 SD (%) 1.1 1.3 2.1 2.3 2.3 [0079] Example 21. Inhibition of growth of the fungus Bobytis cinerea by the composition according to this invention (K2CO3 + Carvacrol).
[0080] Leaves of tomato plants treated with different concentrations of carvacrol and carvacrol were infected with the fungus B. cinerea in a similar way to Example 19. The %
inhibition was calculated and the results are shown in Table XXI.
Table XXI. Inhibition of the growth of B. cinerea by the composition according to this invention (K2CO3 + Carvacrol) K2CO3 concentration (mM) 14 22 15 15 13 Carvacrol concentration (PPin) Inhibition 24.3 28.1 48.3 60.0 72.8 SD (%) 2.1 2.8 2.8 2.6 2.4 Page 16 of 33 [0081] It will be seen how the results are improved by adding carvacrol to K2CO3. With 308 ppm of carvacrol (see Table XX) only 21% inhibition is achieved, and with 13 mM of K2CO3 14%
inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus B. cinerea is increased up to some 73%.
[0082] Example 22. Inhibition of the fungus Botrytis cinerea by Thymol alone.
[0083] Leaves of tomato plants treated with different concentrations of thymol were infected with the fungus B. cinerea in a similar way to Example 19. The % inhibition was calculated and the results are shown in Table XXII.
Table XXII. Inhibition of the growth of B. cinerea by Thymol alone Thymol concentration (PPm) Inhibition 7.4 17.7 20.9 22.8 SD (%) 1.3 1.7 1.5 1.5 100841 Example 23. Inhibition of growth of the fungus Botlytis cinerea by the composition according to this invention (K2CO3 + Thymol).
[0085] Leaves of tomato plants treated with different concentrations of thymol and K2CO3 were infected with the fungus B. cinerea in a similar way to Example 19. The %
inhibition was calculated and the results are shown in Table XXIII.
Table XXIII. Inhibition of the growth of B. cinerea by the composition according to this invention (K2CO3 + Thymol) K2CO3 concentration (mM) Thymol concentration (PPm) Inhibition 25.6 40.8 53.5 65.0 SD (%) 2.6 2.4 2.3 2.2 Page 17 of 33 [0086] It will be seen how the results are improved by adding thymol to K2CO3.
With 350 ppm of thymol (see Table XXII) only 23% inhibition is achieved, and with 12 mM of K2CO3 14% inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus B.
cinerea is increased up to some 65%.
100871 Example 24. Inhibition of the fungus Alternaria alternata by K2CO3 alone.
[0088] Leaves of tomato plants treated with different concentrations of K2CO3 were infected with the fungus A. alternata in a similar way to Example 19. The % inhibition was calculated and the results are shown in Table XXIV.
Table XXIV. Inhibition of the growth of A. alternata by K2CO3 alone K2CO3 concentration (mM) Inhibition 6.5 7.0 7.2 7.8 8.6 SD (%) 2.2 1.1 1.1 1.4 1.5 [0089] Example 25. Inhibition of the fungus Alternana alternata by Carvacrol alone.
100901 Leaves of tomato plants treated with different concentrations of carvacrol were infected with the fungus A. altemata in a similar way to Example 19. The % inhibition was calculated and the results are shown in Table XXV.
Table XXV. Inhibition of the growth of A. alternata by Carvacrol alone Carvacrol concentration (PPm) Inhibition 4.3 5.8 11.4 16.1 18.4 SD (%) 0.7 0.9 1.3 1.2 1.2 Page 18 of 33 [0091] Example 26. Inhibition of growth of the fungus Alternaria alternata by the composition according to this invention (K2CO3 + Carvacrol).
[0092] Leaves of tomato plants treated with different concentrations of carvacrol and K2CO3 were infected with the fungus B. cinerea in a similar way to Example 19. The %
inhibition was calculated and the results are shown in Table XXVI.
Table XXVI. Inhibition of the growth of A. alternata by the composition according to this invention (K2CO3 + Carvacrol) K2CO3 concentration (mM) Carvacrol concentration (PPm) Inhibition 18.4 17.3 43.9 60.9 70.8 SD (%) 2.1 2.4 3.2 2.8 3.5 [0093] It will be seen how the results are improved by adding carvacrol to K2CO3. With 308 ppm of carvacrol (see Table XXV) only 18% inhibition is achieved, and with 13 mM of K2CO3 7% inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus A.
alternata is increased up to some 71%.
[0094] Example 27. Inhibition of the fungus Altemazia alternata by Thymol alone.
[0095] Leaves of tomato plants treated with different concentrations of thymol were infected with the fungus A. alternata in a similar way to Example 19. The % inhibition was calculated and the results are shown in Table XXVII.
Table XXVII. Inhibition of the growth of A. alternata by Thymol alone Thymol concentration (PPm) Inhibition 10.0 15.2 I9.3 21.3 SD (%) 1.4 2.0 2.3 2.6 Page 19 of 33 100961 Example 28. Inhibition of growth of the fungus Alternaria alternata by the composition according to this invention (K2CO3 + Thymol).
100971 Leaves of tomato plants treated with different concentrations of thymol and K2CO3 were infected with the fungus A. alternata in a similar way to Example 19. The %
inhibition was calculated and the results are shown in Table XXVIII.
Table XXVIII. Inhibition of the growth of A. alternata by the composition according to this invention (K2CO3 + Thymol) K2CO3 concentration (mM) Thymol concentration (PPm) Inhibition 22.4 37.3 53.7 60.8 SD (%) 2.5 2.9 2.0 2.2 100981 It will be seen how the results are improved by adding thymol to K2CO3. With 350 ppm of thymol (see Table XXVII) only 21% inhibition is achieved, and with 12 mM of K2CO3 7% inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus A.
alternata is increased up to some 61%.
100991 Example 29. Inhibition of the fungus Phytophthora infestans by K2CO3 alone.
1001001 Tomato plants (var. Marmande) of 5 weeks old were treated with 15 mM
of K2CO3. and 24 hours later, they were infected with the fungus Phytophthora infestans. Two weeks later, fungal infection was assessed in leaves. The % inhibition, representing the extent to which growth was impeded in comparison with a control which did not have the compounds under test, was determined in the following way:
% Inhibition =% Infection (control) ¨ % Infection (x) % Infection (control) Page 20 of 33 [00101] where "% Infection (control)" is the percentage of fungal infection of the control plants (without test compound) and "% Infection (x)" is percentage of fungal infection of the treated plants.
The results are shown in Table XXIX.
Table XXIX. Inhibition of the growth of P. infestans by K2CO3 alone K2CO3 concentration (mM) Inhibition 18.5 SD (%) 3.6 [00102] Example 30. Inhibition of the fungus Phytophthora infestans by Carvacrol alone.
[00103] Tomato plants were treated with different concentrations of carvacrol and subsequently infected with the fungus P. infestans in a similar way to Example 29. The %
inhibition was calculated and the results are shown in Table XXX.
Table XXX. Inhibition of the growth of P. infestans by Carvacrol alone Carvacrol concentration (PPm) Inhibition 3.2 17.1 35.0 SD (%) 0.8 2.3 4.8 [00104] Example 31. Inhibition of growth of the fungus Phytophthora infestans by the composition according to this invention (K2CO3 + Carvacrol).
[00105] Tomato plants were treated with different concentrations of carvacrol and a fixed concentration of K2CO3 and subsequently infected with the fungus P. infestans in a similar way to Example 29. The % inhibition was calculated and the results are shown in Table XXXI.
Table XXXI. Inhibition of the growth of P. infestans by the composition according to this invention (K2CO3 + Carvacrol) Page 21 of 33 K2CO3 concentration (mM) Carvacrol concentration (PPm) Inhibition 25.6+ 52.2 78.3 SD (%) 3.6 4.2 4.8 [00106] It will be seen how the results are improved by adding carvacrol to K2CO3. With 308 ppm of carvacrol (see Table XXX) only 35% inhibition is achieved, and with 15 mM
of K2CO3 19%
inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus P. infestans is increased up to some 78%.
[00107] Example 32. Inhibition of the fungus Phytophthora infestans by Thymol alone.
[00108] Tomato plants were treated with different concentrations of thymol and subsequently infected with the fungus P. infestans in a similar way to Example 29. The %
inhibition was calculated and the results are shown in Table XXXII.
Table XXXII. Inhibition of the growth of P. infestans by Thymol alone Thymol concentration (PPm) Inhibition 4.3 I6.2 33.6 SD (%) 0.7 3.2 5.6 [00109] Example 33. Inhibition of growth of the fungus Phytophthora infestans by the composition according to this invention (K2CO3 + Thymol).
1001101 Tomato plants were treated with different concentrations of thymol and a fixed concentration of K2CO3 and subsequently infected with the fungus P. infestans in a similar way to Example 29. The % inhibition was calculated and the results are shown in Table XXXIII.
Page 22 of 33 Table XXXIII. Inhibition of the growth of P. infestans by the composition according to this =
invention (K2CO3 + Thymol) K2CO3 concentration (mM) Thymol concentration (PPrn) Inhibition 26.3 49.3 74.2 SD (%) 2.2 3.6 5.2 [001111 It will be seen how the results are improved by adding thymol to K2CO3. With 350 ppm of thymol (see Table XXXII) only 34% inhibition is achieved, and with 15 mM of K2CO3 19% inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus P.
infestans is increased up to some 74%.
[00112] Example 34. Inhibition of the fungus Leveillula taufica by K2CO3 alone.
[00113] The fungus L. taufica was cultured in a similar way to Example 29 with different concentrations of K2CO3. The % inhibition was calculated and the results are shown in Table XXXIV.
Table XXXIV. Inhibition of the growth of L. taufica by K2CO3 alone K2CO3 concentration (mM) Inhibition 13.9 SD (%) 0.5 1001141 Example 35. Inhibition of the fungus Leveillula taufica by Carvacrol alone.
[00115] Tomato plants were treated with different concentrations of carvacrol and subsequently infected with the fungus L. taufica in a similar way to Example 29. The %
inhibition was calculated and the results are shown in Table XXXV.
Table XXXV. Inhibition of the growth of L. taufica by Carvacrol alone.
Page 23 of 33 Carvacrol concentration (PPm) Inhibition 4.0 11.2 41.5 SD (%) 1.2 1.0 1.2 [00116] Example 36. Inhibition of growth of the fungus Leveillula tautica by the composition according to this invention (K2CO3 + Carvacrol).
[00117] Tomato plants were treated with different concentrations of carvacrol and a fixed concentration of K2CO3 and subsequently infected with the fungus L. tautica in a similar way to Example 29. The % inhibition was calculated and the results are shown in Table XXX VI.
Table XXX VI. Inhibition of the growth of L. tautica by the composition according to this invention (K2CO3 + Carvacrol) K2CO3 concentration (mM) Carvacrol concentration (PPm) Inhibition 18.6 78.3 92.0 0 SD (%) 3.3 0.6 [00118] It will be seen how the results are improved by adding carvacrol to K2CO3. With 308 ppm of carvacrol (see Table XXXV) only 42% inhibition is achieved, and with 15 mM
of K2CO3 14%
inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus L. tawica is increased up to some 92%.
[00119] Example 37. Inhibition of the fungus Leveillula tautica by Thymol alone.
[00120] Tomato plants were treated with different concentrations of thymol and subsequently infected with the fungus L. taurica in a similar way to Example 29. The %
inhibition was calculated and the results are shown in Table XXX VII.
Table XXX VII. Inhibition of the growth of L. taulica by Thymol alone.
Page 24 of 33 Thymol concentration (PPm) Inhibition 10.5 38.2 51.2 SD (%) 5.4 5.5 1.0 [00121] Example 38. Inhibition of growth of the fungus Leveillula taurica by the composition according to this invention (K2CO3 + Thymol).
[00122] Tomato plants were treated with different concentrations of thymol and a fixed concentration of K2CO3 and subsequently infected with the fungus L. taurica in a similar way to Example 29. The % inhibition was calculated and the results are shown in Table XXX VIII.
Table XXX VIII. Inhibition of the growth of L. taurica by the composition according to this invention (K2CO3 + Thymol) K2CO3 concentration (mM) Thymol concentration (PPm) Inhibition 48.8 65.4 72.9 SD (%) 3.1 2.4 1.1 [00123] It will be seen how the results are improved by adding thymol to K2CO3. With 350 ppm of thymol (see Table XXXVII) only 51% inhibition is achieved, and with 15 mM of K2CO3 14%
inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus P. infestans is increased up to some 73%.
[00124] Example 39. Inhibition of the fungus Pseudoperonospora cubensis by the composition according to this invention (K2CO3 + Carvacrol).
[00125] The efficacy of the composition of the present invention (K2CO3 +
carvacrol or thymol) was tested in field assays with cucumber, tomato, lettuce or potato plants to prevent Page 25 of 33 Pseudoperonospora cubensis, Botrytis cinerea, Phytoplithora infestans or Leveillula taurica growth.
The efficacy of the fungicide was measured as follow:
% Severity (control) ¨ % Severity (x) %Efftcacy= x100 %Severity (control) 1001261 where "% Severity (control)" is the percentage of fungal severity of the control plants (without test compounds) and "% Severity (x)" is percentage of fungal severity of the treated plants.
Cucumber plants were treated with different concentrations of K2CO3 and carvacrol and subsequently infected with Pseudoperonospora cubensis. The results are shown in Table XXXIX.
Table XXXIX. Efficacy of Pseudoperonospora cubensis by the composition according to this invention (K2CO3 + Carvacrol) in cucumber K2CO3 concentration 13.5 19 6.5 13.0 22.4 (mM) Carvacrol concentration 25.5 36.5 150 300 530 (PPm) Inhibition 16.7 26.7 35.5 45.3 64.2 SD (%) 3.3 4.2 3.9 5.3 7.3 [00127] The efficacy of the inhibition of growth of the fungus P. cubensis reached 64% by combining K2CO3 and carvacrol.
[00128] Example 40. Inhibition of the fungus Pseudoperonospora cubensis by the composition according to this invention (K2CO3 + Thymol).
[00129] The efficacy of the composition of the present invention (K2CO3 +
Thymol) was tested in cucumber to prevent P. cubensis growth. The efficacy of the antifungicide was measured and the results are shown in Table XL.
Table XL. Efficacy of Pseudoperonospora cubensis by the composition according to this invention (K2CO3 + Thymol) in cucumber K2CO3 concentration 6.5 13.0 22.4 Page 26 of 33 (mM) Thymol concentration (PPm) Inhibition 31.6 41.1 53.3 SD (%) 4.6 6.1 7.0 =
[00130] The efficacy of the inhibition of growth of the fungus P. cubensis reached 53% by combining K2CO3 and thymol.
[00131] Example 41. Inhibition of the fungus Botrytis cinerea by the composition according to this invention (K2CO3 + Carvacrol).
[00132] The efficacy of the composition of the present invention (K2CO3 +
Carvacrol) was tested in tomato to prevent B. cinerea growth. The efficacy of the antifungicide was measured and the results are shown in Table XLI.
Table XLI. Efficacy of Bouytis cinerea by the composition according to this invention (K2CO3 + Carvacrol) in tomato K2CO3 concentration 13.5 19 6.5 13.0 22.4 (mM) Carvacrol concentration 25.5 36.5 150 300 530 (PM') Inhibition 21.7 27.8 38.5 69.8 78.3 SD (%) 4.4 4.1 5.6 8.3 6.5 [00133] The efficacy of the inhibition of growth of the fungus B. cinerea reached 78% by combining K2CO3 and carvacrol.
[00134] Example 42. Inhibition of the fungus BotryUs cinerea by the composition according to this invention (K2CO3 + Thymol).
Page 27 of 33 [00135] The efficacy of the composition of the present invention (K2CO3 +
Thymol) was tested in tomato to prevent B. cinerea growth. The efficacy of the antifungicide was measured and the results are shown in Table XLII.
Table XLII. Efficacy of Botrytis cinerea by the composition according to this invention (K2CO3 + Thymol) in tomato K2CO3 concentration 6.5 13.0 22.4 (mM) Thymol concentration (PPm) Inhibition 35.8 58.0 66.0 SD (%) 4.3 4.6 6.0 1001361 The efficacy of the inhibition of growth of the fungus B. cinerea reached 66% by combining K2CO3 and thymol.
[00137] Example 43. Inhibition of the fungus Phytoplithora infestans by the composition according to this invention (K2CO3+ Carvacrol).
[00138] The efficacy of the composition of the present invention (K2CO3 +
Carvacrol) was tested in lettuce to prevent P. infestans growth. The efficacy of the antifungicide was measured and the results are shown in Table XLIII.
Table XLIII. Efficacy of P. infestans by the composition according to this invention (K2CO3 +
Carvacrol) in lettuce K2CO3 concentration 5.4 12.6 21.6 (mM) Carvacrol concentration (PPm) Inhibition 58.0 72.1 80.3 SD (%) 2.4 5.2 4.3 Page 28 of 33 [00139] The efficacy of the inhibition of growth of the fungus P. infestans reached 80% by combining K2CO3 and carvacrol.
[00140] Example 44. Inhibition of the fungus Phytophthora infestans by the composition according to this invention (K2CO3+ Carvacrol).
[00141] The efficacy of the composition of the present invention (K2CO3 +
Carvacrol) was tested in potato to prevent P. infestans growth. The efficacy of the antifungicide was measured and the results are shown in Table XLIV.
Table XLIV. Efficacy of P. infestans by the composition according to this invention (K2CO3 +
Carvacrol) in potato K2CO3 concentration 5.4 12.6 21.6 (mM) Carvacrol concentration (PPm) Inhibition 68.6 71.7 86.7 SD (%) 2.9 3.4 4.1 [00142] The efficacy of the inhibition of growth of the fungus P. infestans reached 87% by combining K2CO3 and carvacrol.
[00143] Example 45. Inhibition of the fungus Leveilulla taurica by the composition according to this invention (K2CO3+ Carvacrol).
[00144] The efficacy of the composition of the present invention (K2CO3 +
Carvacrol) was tested in tomato to prevent L. taufica growth. The efficacy of the antifungicide was measured and the results are shown in Table XLV.
Table XLV. Efficacy of L. taurica by the composition according to this invention (K2CO3 +
Carvacrol) in tomato K2CO3 concentration 5.7 13.2 24.5 5.4 12.6 21.6 Page 29 of 33 CA 3060884 2019-11-04 =
(mM) Carvacrol concentration 11.0 25.6 47.5 132 309 529 (PPm) Inhibition 22.0 53.0 61.0 43.0 58.0 71.0 SD (%) 3.1 4.4 3.9 4.2 5.2 3.8 1001451 The efficacy of the inhibition of growth of the fungus L. =Ica reached 71% by combining K2CO3 and carvacrol.
Page 30 of 33
[0046] The fungus A. altemata was cultured in a similar way to Example 1 with the difference that different concentrations of carvacrol were used in the medium and that a constant concentration of K2CO3 (3.5 mM) was used throughout. The 24 hour optical density of the culture was measured and the results are shown in Table VIII.
Table VIII. Inhibition of A. alternata by the composition according to this invention (K2CO3 +
Carvacrol) K2CO3 concentration 3.5 3.5 3.5 3.5 3.5 3.5 3.5 (mM) Carvacrol concentration 0.1 0.31 1 3.1 10 31 100 (PPm) Inhibition 30.1 35.7 38.0 46.2 65.7 86.3 100 SD (%) 2.9 2.7 2.9 2.9 1.6 1.2 0 Page 9 of 33 [0047] It will be seen how the results are improved by adding carvacrol to K2CO3. With 31 ppm of carvacrol (see Table VII) only 27% inhibition is achieved, and with 3.5 mM of K2CO3 32% inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus A.
alternata is increased up to some 86%.
[0048] Example 9. Inhibition of growth of the fungus Alternalla alternata by thymol alone.
[0049] The fungus A. alternata was cultured in a similar way to Example 1 with different concentrations of thymol. The 24 hour optical density of the culture was measured and the results are shown in Table IX.
Table IX. Inhibition of the growth of A. alternata by Thymol Thymol concentration 0.31 1 3.1 10 31 100 (PPrn) Inhibition 0 4.1 10.4 16.0 29.0 69.4 SD (%) 0 2.9 2.6 2.0 1.5 [0050] Example 10. Inhibition of the fungus Alternaria alternata by the composition according to this invention (K2CO3 + Thymol).
100511 The fungus A. alternata was cultured in a similar way to Example 1 with the difference that different concentrations of thymol were used in the medium and that a constant concentration of K2CO3 (3.5 mM) was used throughout. The 24 hour optical density of the culture was measured and the results are shown in Table X.
Table X. Inhibition of A. alternata by the composition according to this invention (K2CO3 +
Thymol) K2CO3 concentration (mM) 3.5 3.5 3.5 3.5 3.5 3.5 Thymol concentration 0.31 1 3.1 10 31 100 (PPm) Inhibition 33.7 38.7 45.3 55.5 71.8 SD (%) 2.2 2.7 2.4 1.2 1.3 Page 10 of 33 [0052] It will be seen how the results are improved by adding thymol to K2CO3.
With 31 ppm of thymol only 29% inhibition is achieved, and with 3.5 mM of K2CO3 32%
inhibition is achieved.
However, when the two compounds are combined inhibition of growth of the fungus A. alternata is increased up to some 72%.
[0053] Example 11. Inhibition of growth of the fungus Penicillium digitatum by K2CO3 alone.
[0054] Penicillium digitatum was cultured in a similar way to Example 1 with different concentrations of K2CO3. The 24 hour optical density of the culture was measured and the results are shown in Table XI.
Table XI. Inhibition of the growth of P. digitatum by K2CO3 K2CO3 concentration 0 3.5 4.25 4.5 5 (mM) Inhibition 29.1 33.0 36.9 39.5 SD (%) 2.1 1.7 1.6 1.8 [0055] As will be seen from the table above, with a K2CO3 concentration between 3.5 and 5 mM
inhibition of the P. digitatum culture was observed.
100561 Example 12. Inhibition of growth of the fungus Penicillium digitatum by carvacrol alone.
[0057] The fungus P. digitatum was cultured in 'a similar way to Example 1 with different concentrations of carvacrol. The 24 hour optical density of the culture was measured and the results are shown in Table XII.
Table XII. Inhibition of the growth of P. digitatum by Carvacrol Carvacrol 0.1 0.31 1 3.1 10 31 100 concentration (ppm) Inhibition 32.3 34.3 37.9 52.6 57.8 86.0 SD (%) 2.8 2.8 3.0 2.2 2.1 1.1 Page 11 of 33 100581 Example 13. Inhibition of the fungus Penicillium digitatum by the composition according to this invention (K2CO3 + Carvacrol).
[0059] The fungus P. a'igitatum was cultured in a similar way to Example 1 with the difference that different concentrations of carvacrol were used in the medium and that a constant concentration of K2CO3 (3.5 mM) was used throughout. The 24 hour optical density of the culture was measured and the results are shown in Table XIII.
Table XIII. Inhibition of P. digitatum by the composition according to this invention (K2CO3 +
Carvacrol) K2CO3 concentration 3.5 3.5 3.5 3.5 3.5 3.5 3.5 (mM) Carvacrol concentration 0.1 0.31 1 3.1 10 31 100 (PPm) Inhibition 65.9 66.2 72.4 88.6 96.5 100 100 SD (%) 2.4 2.9 2.2 1.9 1.4 0 0 [0060] It will be seen how the results are improved by adding carvacrol to K2CO3. With 10 ppm of carvacrol 58% inhibition is achieved, and with 3.5 mM of K2CO3 29% inhibition is achieved.
However, when the two compounds are combined inhibition of growth of the fungus P. digitatum is increased up to 97%.
[0061] Example 14. Inhibition of growth of the fungus Penicillium digitatum by thymol alone.
[0062] The fungus P. digitatum was cultured in a similar way to Example 1 with different concentrations of thymol. The 24 hour optical density of the culture was measured and the results are shown in Table XIV.
Table XIV. Inhibition of the growth of P. digitatum by Thymol Thymol concentration 0.31 1 3.1 10 31 100 100 (PPm) Inhibition 28.2 24.2 36.3 36.2 50.7 78.3 95.6 Page 12 of 33 SD (%) 3.9 + 6.0 2.3 2.0 2.0 2.2 0.5 [0063] Example 15. Inhibition of the fungus Penicillium digitatum by the composition according to this invention (K2CO3 + Thymol).
[0064] The fungus P. digitatum was cultured in a similar way to Example 1 with the difference that different concentrations of thymol were used in the medium and that a constant concentration of K2CO3 (3.5 mM) was used throughout. The 24 hour optical density of the culture was measured and the results are shown in Table XV.
Table XV. Inhibition of P. digitatum by the composition according to this invention (K2CO3 +
Thymol) K2CO3 concentration 3.5 3.5 3.5 3.5 3.5 3.5 (mM) Thymol concentration 0.31 1 3.1 10 31 100 (PPm) Inhibition 58.1 61.3 69.9 78.3 94.3 SD (%) 2.9 2.5 1.9 1.5 1.2 [0065] It will be seen how the results are improved by adding thymol to K2CO3.
With 31 ppm of thymol (see Table XIV) only 51% inhibition is achieved, and with 3.5 mM of K2CO3 29% inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus P.
digitatum is increased up to some 94%.
Page 13 of 33 [0066] Example 16. Inhibition of growth of the fungus Cercospora beticola by K2CO3 alone.
[0067] Cercospora beticola was cultured on PDA (potato dextrose agar) culture medium buffered to a pH value not exceeding 9.5. The inhibition degree, expressed as a percentage, was determined based on the growth relative to control that did not have the compound(s) to be tested. The % inhibition was calculated with the following formula:
colony diameter (control) ¨ colony diameter (x) % Inhibition = x 100 colony diameter (control) [0068] wherein "colony diameter (control)" is the size of the control colony (without the compounds to be tested) and "colony diameter (x)" is the size of the colony with the substance(s) to be tested. A
fixed K2CO3 concentration of 7.24 mM was tested in C beticola. The results are shown in Table XVI.
Table XVI. Inhibition of the growth of C beticola by K2CO3 alone K2CO3 concentration 7.24 (mM) Inhibition 25.0 SD (%) 1.2 [0069] Example 17. Inhibition of growth of the fungus Cercospora beticola by carvacrol alone.
100701 The fungus C beticola was cultured in a similar way to Example 16 with 10 ppm of carvacrol.
The % inhibition was calculated and the results are shown in Table XVII.
Table XVII. Inhibition of the growth of C beticola by carvacrol alone Carvacrol concentration (PPrn) Inhibition 17.0 SD (%) 0.8 100711 Example 18. Inhibition of growth of the fungus Cercospora beticola by the composition according to this invention (K2CO3 + Carvacrol).
Page 14 of 33 [0072] The fungus C beticola was cultured in a similar way to Example 16 with a fixed concentration of K2CO3 (7.24 mM) and carvacrol (10 ppm). The % inhibition was calculated and the results are shown in Table XVIII.
Table XVIII. Inhibition of the growth of C beticola by the composition according to this invention (K2CO3 + Carvacrol) K2CO3 concentration 7.24 (mM) Carvacrol concentration (ppm) Inhibition 65.0 SD (%) 3.2 [0073] It will be seen how the results are improved by adding carvacrol to K2CO3. With 10 ppm of carvacrol (see Table XVII) only 17%
inhibition is achieved, and with 7.24 mM of K2CO3 25% inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus C beticola is increased up to some 65%.
[0074] Example 19. Inhibition of the fungus Botrytis cinerea by K2CO3 alone.
[0075] Leaves of tomato plants (var. Marmande) of 5 weeks old were treated with different concentrations of K2CO3, and 24 hours later, they were infected with the fungus Botrytis cinema. Two weeks later, fungal infection was assessed in leaves. The % inhibition, representing the extent to which fungal growth was impeded in comparison with a control which did not have the compound(s) under test, was determined in the following way:
% Infection (control) ¨ % Infection (x) % Inhibition = ________________________________________________ x 100 Infection (control) [0076] where "% Infection (control)" is the percentage of fungal infection of the control leaves (without test compounds) and "% Infection (x)" is percentage of fungal infection of the treated leaves.
The results are shown in Table XIX.
Table XIX. Inhibition of the growth of B. cinerea by K2CO3 alone Page 15 of 33 K2CO3 concentration (mM) Inhibition 13.4 13.5 13.8 13.8 15.3 SD (%) 2.5 2.1 2.3 3.0 2.4 [0077] Example 20. Inhibition of the fungus Botrytis cinerea by Carvacrol alone.
[0078] Leaves of tomato plants treated with different concentrations of carvacrol were infected with the fungus B. cinerea in a similar way to Example 19. The % inhibition was calculated and the results are shown in Table XX.
Table XX. Inhibition of the growth of B. cinerea by Carvacrol alone Carvacrol concentration (PPm) Inhibition 7.2 7.4 16.7 18.6 20.8 SD (%) 1.1 1.3 2.1 2.3 2.3 [0079] Example 21. Inhibition of growth of the fungus Bobytis cinerea by the composition according to this invention (K2CO3 + Carvacrol).
[0080] Leaves of tomato plants treated with different concentrations of carvacrol and carvacrol were infected with the fungus B. cinerea in a similar way to Example 19. The %
inhibition was calculated and the results are shown in Table XXI.
Table XXI. Inhibition of the growth of B. cinerea by the composition according to this invention (K2CO3 + Carvacrol) K2CO3 concentration (mM) 14 22 15 15 13 Carvacrol concentration (PPin) Inhibition 24.3 28.1 48.3 60.0 72.8 SD (%) 2.1 2.8 2.8 2.6 2.4 Page 16 of 33 [0081] It will be seen how the results are improved by adding carvacrol to K2CO3. With 308 ppm of carvacrol (see Table XX) only 21% inhibition is achieved, and with 13 mM of K2CO3 14%
inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus B. cinerea is increased up to some 73%.
[0082] Example 22. Inhibition of the fungus Botrytis cinerea by Thymol alone.
[0083] Leaves of tomato plants treated with different concentrations of thymol were infected with the fungus B. cinerea in a similar way to Example 19. The % inhibition was calculated and the results are shown in Table XXII.
Table XXII. Inhibition of the growth of B. cinerea by Thymol alone Thymol concentration (PPm) Inhibition 7.4 17.7 20.9 22.8 SD (%) 1.3 1.7 1.5 1.5 100841 Example 23. Inhibition of growth of the fungus Botlytis cinerea by the composition according to this invention (K2CO3 + Thymol).
[0085] Leaves of tomato plants treated with different concentrations of thymol and K2CO3 were infected with the fungus B. cinerea in a similar way to Example 19. The %
inhibition was calculated and the results are shown in Table XXIII.
Table XXIII. Inhibition of the growth of B. cinerea by the composition according to this invention (K2CO3 + Thymol) K2CO3 concentration (mM) Thymol concentration (PPm) Inhibition 25.6 40.8 53.5 65.0 SD (%) 2.6 2.4 2.3 2.2 Page 17 of 33 [0086] It will be seen how the results are improved by adding thymol to K2CO3.
With 350 ppm of thymol (see Table XXII) only 23% inhibition is achieved, and with 12 mM of K2CO3 14% inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus B.
cinerea is increased up to some 65%.
100871 Example 24. Inhibition of the fungus Alternaria alternata by K2CO3 alone.
[0088] Leaves of tomato plants treated with different concentrations of K2CO3 were infected with the fungus A. alternata in a similar way to Example 19. The % inhibition was calculated and the results are shown in Table XXIV.
Table XXIV. Inhibition of the growth of A. alternata by K2CO3 alone K2CO3 concentration (mM) Inhibition 6.5 7.0 7.2 7.8 8.6 SD (%) 2.2 1.1 1.1 1.4 1.5 [0089] Example 25. Inhibition of the fungus Alternana alternata by Carvacrol alone.
100901 Leaves of tomato plants treated with different concentrations of carvacrol were infected with the fungus A. altemata in a similar way to Example 19. The % inhibition was calculated and the results are shown in Table XXV.
Table XXV. Inhibition of the growth of A. alternata by Carvacrol alone Carvacrol concentration (PPm) Inhibition 4.3 5.8 11.4 16.1 18.4 SD (%) 0.7 0.9 1.3 1.2 1.2 Page 18 of 33 [0091] Example 26. Inhibition of growth of the fungus Alternaria alternata by the composition according to this invention (K2CO3 + Carvacrol).
[0092] Leaves of tomato plants treated with different concentrations of carvacrol and K2CO3 were infected with the fungus B. cinerea in a similar way to Example 19. The %
inhibition was calculated and the results are shown in Table XXVI.
Table XXVI. Inhibition of the growth of A. alternata by the composition according to this invention (K2CO3 + Carvacrol) K2CO3 concentration (mM) Carvacrol concentration (PPm) Inhibition 18.4 17.3 43.9 60.9 70.8 SD (%) 2.1 2.4 3.2 2.8 3.5 [0093] It will be seen how the results are improved by adding carvacrol to K2CO3. With 308 ppm of carvacrol (see Table XXV) only 18% inhibition is achieved, and with 13 mM of K2CO3 7% inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus A.
alternata is increased up to some 71%.
[0094] Example 27. Inhibition of the fungus Altemazia alternata by Thymol alone.
[0095] Leaves of tomato plants treated with different concentrations of thymol were infected with the fungus A. alternata in a similar way to Example 19. The % inhibition was calculated and the results are shown in Table XXVII.
Table XXVII. Inhibition of the growth of A. alternata by Thymol alone Thymol concentration (PPm) Inhibition 10.0 15.2 I9.3 21.3 SD (%) 1.4 2.0 2.3 2.6 Page 19 of 33 100961 Example 28. Inhibition of growth of the fungus Alternaria alternata by the composition according to this invention (K2CO3 + Thymol).
100971 Leaves of tomato plants treated with different concentrations of thymol and K2CO3 were infected with the fungus A. alternata in a similar way to Example 19. The %
inhibition was calculated and the results are shown in Table XXVIII.
Table XXVIII. Inhibition of the growth of A. alternata by the composition according to this invention (K2CO3 + Thymol) K2CO3 concentration (mM) Thymol concentration (PPm) Inhibition 22.4 37.3 53.7 60.8 SD (%) 2.5 2.9 2.0 2.2 100981 It will be seen how the results are improved by adding thymol to K2CO3. With 350 ppm of thymol (see Table XXVII) only 21% inhibition is achieved, and with 12 mM of K2CO3 7% inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus A.
alternata is increased up to some 61%.
100991 Example 29. Inhibition of the fungus Phytophthora infestans by K2CO3 alone.
1001001 Tomato plants (var. Marmande) of 5 weeks old were treated with 15 mM
of K2CO3. and 24 hours later, they were infected with the fungus Phytophthora infestans. Two weeks later, fungal infection was assessed in leaves. The % inhibition, representing the extent to which growth was impeded in comparison with a control which did not have the compounds under test, was determined in the following way:
% Inhibition =% Infection (control) ¨ % Infection (x) % Infection (control) Page 20 of 33 [00101] where "% Infection (control)" is the percentage of fungal infection of the control plants (without test compound) and "% Infection (x)" is percentage of fungal infection of the treated plants.
The results are shown in Table XXIX.
Table XXIX. Inhibition of the growth of P. infestans by K2CO3 alone K2CO3 concentration (mM) Inhibition 18.5 SD (%) 3.6 [00102] Example 30. Inhibition of the fungus Phytophthora infestans by Carvacrol alone.
[00103] Tomato plants were treated with different concentrations of carvacrol and subsequently infected with the fungus P. infestans in a similar way to Example 29. The %
inhibition was calculated and the results are shown in Table XXX.
Table XXX. Inhibition of the growth of P. infestans by Carvacrol alone Carvacrol concentration (PPm) Inhibition 3.2 17.1 35.0 SD (%) 0.8 2.3 4.8 [00104] Example 31. Inhibition of growth of the fungus Phytophthora infestans by the composition according to this invention (K2CO3 + Carvacrol).
[00105] Tomato plants were treated with different concentrations of carvacrol and a fixed concentration of K2CO3 and subsequently infected with the fungus P. infestans in a similar way to Example 29. The % inhibition was calculated and the results are shown in Table XXXI.
Table XXXI. Inhibition of the growth of P. infestans by the composition according to this invention (K2CO3 + Carvacrol) Page 21 of 33 K2CO3 concentration (mM) Carvacrol concentration (PPm) Inhibition 25.6+ 52.2 78.3 SD (%) 3.6 4.2 4.8 [00106] It will be seen how the results are improved by adding carvacrol to K2CO3. With 308 ppm of carvacrol (see Table XXX) only 35% inhibition is achieved, and with 15 mM
of K2CO3 19%
inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus P. infestans is increased up to some 78%.
[00107] Example 32. Inhibition of the fungus Phytophthora infestans by Thymol alone.
[00108] Tomato plants were treated with different concentrations of thymol and subsequently infected with the fungus P. infestans in a similar way to Example 29. The %
inhibition was calculated and the results are shown in Table XXXII.
Table XXXII. Inhibition of the growth of P. infestans by Thymol alone Thymol concentration (PPm) Inhibition 4.3 I6.2 33.6 SD (%) 0.7 3.2 5.6 [00109] Example 33. Inhibition of growth of the fungus Phytophthora infestans by the composition according to this invention (K2CO3 + Thymol).
1001101 Tomato plants were treated with different concentrations of thymol and a fixed concentration of K2CO3 and subsequently infected with the fungus P. infestans in a similar way to Example 29. The % inhibition was calculated and the results are shown in Table XXXIII.
Page 22 of 33 Table XXXIII. Inhibition of the growth of P. infestans by the composition according to this =
invention (K2CO3 + Thymol) K2CO3 concentration (mM) Thymol concentration (PPrn) Inhibition 26.3 49.3 74.2 SD (%) 2.2 3.6 5.2 [001111 It will be seen how the results are improved by adding thymol to K2CO3. With 350 ppm of thymol (see Table XXXII) only 34% inhibition is achieved, and with 15 mM of K2CO3 19% inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus P.
infestans is increased up to some 74%.
[00112] Example 34. Inhibition of the fungus Leveillula taufica by K2CO3 alone.
[00113] The fungus L. taufica was cultured in a similar way to Example 29 with different concentrations of K2CO3. The % inhibition was calculated and the results are shown in Table XXXIV.
Table XXXIV. Inhibition of the growth of L. taufica by K2CO3 alone K2CO3 concentration (mM) Inhibition 13.9 SD (%) 0.5 1001141 Example 35. Inhibition of the fungus Leveillula taufica by Carvacrol alone.
[00115] Tomato plants were treated with different concentrations of carvacrol and subsequently infected with the fungus L. taufica in a similar way to Example 29. The %
inhibition was calculated and the results are shown in Table XXXV.
Table XXXV. Inhibition of the growth of L. taufica by Carvacrol alone.
Page 23 of 33 Carvacrol concentration (PPm) Inhibition 4.0 11.2 41.5 SD (%) 1.2 1.0 1.2 [00116] Example 36. Inhibition of growth of the fungus Leveillula tautica by the composition according to this invention (K2CO3 + Carvacrol).
[00117] Tomato plants were treated with different concentrations of carvacrol and a fixed concentration of K2CO3 and subsequently infected with the fungus L. tautica in a similar way to Example 29. The % inhibition was calculated and the results are shown in Table XXX VI.
Table XXX VI. Inhibition of the growth of L. tautica by the composition according to this invention (K2CO3 + Carvacrol) K2CO3 concentration (mM) Carvacrol concentration (PPm) Inhibition 18.6 78.3 92.0 0 SD (%) 3.3 0.6 [00118] It will be seen how the results are improved by adding carvacrol to K2CO3. With 308 ppm of carvacrol (see Table XXXV) only 42% inhibition is achieved, and with 15 mM
of K2CO3 14%
inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus L. tawica is increased up to some 92%.
[00119] Example 37. Inhibition of the fungus Leveillula tautica by Thymol alone.
[00120] Tomato plants were treated with different concentrations of thymol and subsequently infected with the fungus L. taurica in a similar way to Example 29. The %
inhibition was calculated and the results are shown in Table XXX VII.
Table XXX VII. Inhibition of the growth of L. taulica by Thymol alone.
Page 24 of 33 Thymol concentration (PPm) Inhibition 10.5 38.2 51.2 SD (%) 5.4 5.5 1.0 [00121] Example 38. Inhibition of growth of the fungus Leveillula taurica by the composition according to this invention (K2CO3 + Thymol).
[00122] Tomato plants were treated with different concentrations of thymol and a fixed concentration of K2CO3 and subsequently infected with the fungus L. taurica in a similar way to Example 29. The % inhibition was calculated and the results are shown in Table XXX VIII.
Table XXX VIII. Inhibition of the growth of L. taurica by the composition according to this invention (K2CO3 + Thymol) K2CO3 concentration (mM) Thymol concentration (PPm) Inhibition 48.8 65.4 72.9 SD (%) 3.1 2.4 1.1 [00123] It will be seen how the results are improved by adding thymol to K2CO3. With 350 ppm of thymol (see Table XXXVII) only 51% inhibition is achieved, and with 15 mM of K2CO3 14%
inhibition is achieved. However, when the two compounds are combined inhibition of growth of the fungus P. infestans is increased up to some 73%.
[00124] Example 39. Inhibition of the fungus Pseudoperonospora cubensis by the composition according to this invention (K2CO3 + Carvacrol).
[00125] The efficacy of the composition of the present invention (K2CO3 +
carvacrol or thymol) was tested in field assays with cucumber, tomato, lettuce or potato plants to prevent Page 25 of 33 Pseudoperonospora cubensis, Botrytis cinerea, Phytoplithora infestans or Leveillula taurica growth.
The efficacy of the fungicide was measured as follow:
% Severity (control) ¨ % Severity (x) %Efftcacy= x100 %Severity (control) 1001261 where "% Severity (control)" is the percentage of fungal severity of the control plants (without test compounds) and "% Severity (x)" is percentage of fungal severity of the treated plants.
Cucumber plants were treated with different concentrations of K2CO3 and carvacrol and subsequently infected with Pseudoperonospora cubensis. The results are shown in Table XXXIX.
Table XXXIX. Efficacy of Pseudoperonospora cubensis by the composition according to this invention (K2CO3 + Carvacrol) in cucumber K2CO3 concentration 13.5 19 6.5 13.0 22.4 (mM) Carvacrol concentration 25.5 36.5 150 300 530 (PPm) Inhibition 16.7 26.7 35.5 45.3 64.2 SD (%) 3.3 4.2 3.9 5.3 7.3 [00127] The efficacy of the inhibition of growth of the fungus P. cubensis reached 64% by combining K2CO3 and carvacrol.
[00128] Example 40. Inhibition of the fungus Pseudoperonospora cubensis by the composition according to this invention (K2CO3 + Thymol).
[00129] The efficacy of the composition of the present invention (K2CO3 +
Thymol) was tested in cucumber to prevent P. cubensis growth. The efficacy of the antifungicide was measured and the results are shown in Table XL.
Table XL. Efficacy of Pseudoperonospora cubensis by the composition according to this invention (K2CO3 + Thymol) in cucumber K2CO3 concentration 6.5 13.0 22.4 Page 26 of 33 (mM) Thymol concentration (PPm) Inhibition 31.6 41.1 53.3 SD (%) 4.6 6.1 7.0 =
[00130] The efficacy of the inhibition of growth of the fungus P. cubensis reached 53% by combining K2CO3 and thymol.
[00131] Example 41. Inhibition of the fungus Botrytis cinerea by the composition according to this invention (K2CO3 + Carvacrol).
[00132] The efficacy of the composition of the present invention (K2CO3 +
Carvacrol) was tested in tomato to prevent B. cinerea growth. The efficacy of the antifungicide was measured and the results are shown in Table XLI.
Table XLI. Efficacy of Bouytis cinerea by the composition according to this invention (K2CO3 + Carvacrol) in tomato K2CO3 concentration 13.5 19 6.5 13.0 22.4 (mM) Carvacrol concentration 25.5 36.5 150 300 530 (PM') Inhibition 21.7 27.8 38.5 69.8 78.3 SD (%) 4.4 4.1 5.6 8.3 6.5 [00133] The efficacy of the inhibition of growth of the fungus B. cinerea reached 78% by combining K2CO3 and carvacrol.
[00134] Example 42. Inhibition of the fungus BotryUs cinerea by the composition according to this invention (K2CO3 + Thymol).
Page 27 of 33 [00135] The efficacy of the composition of the present invention (K2CO3 +
Thymol) was tested in tomato to prevent B. cinerea growth. The efficacy of the antifungicide was measured and the results are shown in Table XLII.
Table XLII. Efficacy of Botrytis cinerea by the composition according to this invention (K2CO3 + Thymol) in tomato K2CO3 concentration 6.5 13.0 22.4 (mM) Thymol concentration (PPm) Inhibition 35.8 58.0 66.0 SD (%) 4.3 4.6 6.0 1001361 The efficacy of the inhibition of growth of the fungus B. cinerea reached 66% by combining K2CO3 and thymol.
[00137] Example 43. Inhibition of the fungus Phytoplithora infestans by the composition according to this invention (K2CO3+ Carvacrol).
[00138] The efficacy of the composition of the present invention (K2CO3 +
Carvacrol) was tested in lettuce to prevent P. infestans growth. The efficacy of the antifungicide was measured and the results are shown in Table XLIII.
Table XLIII. Efficacy of P. infestans by the composition according to this invention (K2CO3 +
Carvacrol) in lettuce K2CO3 concentration 5.4 12.6 21.6 (mM) Carvacrol concentration (PPm) Inhibition 58.0 72.1 80.3 SD (%) 2.4 5.2 4.3 Page 28 of 33 [00139] The efficacy of the inhibition of growth of the fungus P. infestans reached 80% by combining K2CO3 and carvacrol.
[00140] Example 44. Inhibition of the fungus Phytophthora infestans by the composition according to this invention (K2CO3+ Carvacrol).
[00141] The efficacy of the composition of the present invention (K2CO3 +
Carvacrol) was tested in potato to prevent P. infestans growth. The efficacy of the antifungicide was measured and the results are shown in Table XLIV.
Table XLIV. Efficacy of P. infestans by the composition according to this invention (K2CO3 +
Carvacrol) in potato K2CO3 concentration 5.4 12.6 21.6 (mM) Carvacrol concentration (PPm) Inhibition 68.6 71.7 86.7 SD (%) 2.9 3.4 4.1 [00142] The efficacy of the inhibition of growth of the fungus P. infestans reached 87% by combining K2CO3 and carvacrol.
[00143] Example 45. Inhibition of the fungus Leveilulla taurica by the composition according to this invention (K2CO3+ Carvacrol).
[00144] The efficacy of the composition of the present invention (K2CO3 +
Carvacrol) was tested in tomato to prevent L. taufica growth. The efficacy of the antifungicide was measured and the results are shown in Table XLV.
Table XLV. Efficacy of L. taurica by the composition according to this invention (K2CO3 +
Carvacrol) in tomato K2CO3 concentration 5.7 13.2 24.5 5.4 12.6 21.6 Page 29 of 33 CA 3060884 2019-11-04 =
(mM) Carvacrol concentration 11.0 25.6 47.5 132 309 529 (PPm) Inhibition 22.0 53.0 61.0 43.0 58.0 71.0 SD (%) 3.1 4.4 3.9 4.2 5.2 3.8 1001451 The efficacy of the inhibition of growth of the fungus L. =Ica reached 71% by combining K2CO3 and carvacrol.
Page 30 of 33
Claims (12)
1. A phytosanitary composition having antifungal activity comprising: (1) one essential oil obtained from plants selected from oregano oil (Origanum vulgare) and thyme oil (Thymus vulgaris) comprising its active compounds carvacrol, which is present at a concentration between 0.1 and 308 ppm, and thymol, which is present at a concentration between 0.31 and 350 ppm, respectively, or a combination thereof; and (2) potassium carbonate at a concentration between 3.5 and 25 mM.
2. A phytosanitary composition according to claim 1, characterised in that the carvacrol is at a concentration between 22 and 308 ppm.
3. A phytosanitary composition according to claim 1, characterised in that the thymol is at a concentration between 22 and 350 ppm.
4. A phytosanitary composition according to claim 1, characterised in that the potassium carbonate is at a concentration between 10 and 25 mM.
5. A phytosanitary composition according to any one of claims 1 to 4, characterised in that the composition is in liquid or solid fonnwhich remains stable over time or is incorporated in polymers, waxes or a support.
6. A phytosanitary composition according to claim 5, characterised in that the liquid or solid form is selected from the group consisting of suspension, dispersion, emulsion, spray, and microencapsulate.
7. A phytosanitary composition according to any one of claims 1 to 6, characterised in that the phytosanitary composition is formulated together with additives.
Date Recue/Date Received 2021-09-02
Date Recue/Date Received 2021-09-02
8. A phytosanitary composition according to claim 7, characterised in that the additives is selected from the group consisting of surfactants, polymers, alkalinising agents, and pH-controlling agents.
9. A phytosanitary composition according to any one of claims 1 to 8, which further comprises a fertiliser selected from the group consisting of compounds containing nitrogen, phosphorus, and mixtures thereof.
10. A phytosanitary composition according to claim 9, characterised in that the fertiliser is selected from the group consisting of urea, melamine, hexamine, dicyanodiamide, ameline, cyanuric acid, melamine nitrate, and triethyl phosphate.
11. A phytosanitary composition according to any one of claims 1 to 10, which further comprises a compound or product having chemical and/or biological activity used in agriculture.
12. A phytosanitary composition according to claim 11, characterised in that the compound or product is selected from the group consisting of herbicides, insecticides, plant growth regulators and mixtures thereof.
Date Recue/Date Received 2021-09-02
Date Recue/Date Received 2021-09-02
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US16231731 | 2018-12-24 | ||
| US16/231,731 US10820597B2 (en) | 2011-03-18 | 2018-12-24 | Phytosanitary composition comprising essential oils that potentiate antifungal activity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA3060884A1 CA3060884A1 (en) | 2020-06-24 |
| CA3060884C true CA3060884C (en) | 2022-11-15 |
Family
ID=71141390
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA3060884A Active CA3060884C (en) | 2018-12-24 | 2019-11-04 | Phytosanitary composition comprising essential oils that potentiate antifungal activity |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA3060884C (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117882717A (en) * | 2022-10-25 | 2024-04-16 | 上海市农业科学院 | Rhizoctonia solani growth inhibitor |
-
2019
- 2019-11-04 CA CA3060884A patent/CA3060884C/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CA3060884A1 (en) | 2020-06-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10863749B2 (en) | Phytosanitary composition comprising essential oils that potentiate antifungal activity | |
| KR20210145122A (en) | Antibacterial Nano Emulsion | |
| CN104604938A (en) | Fungicidal composition and the use thereof | |
| AU2016282319B2 (en) | Antimicrobial compositions comprising food approved antimicrobials and zinc pyrithione | |
| RU2027367C1 (en) | Fungicidal composition for plants | |
| CN104430357A (en) | Bactericide composition containing coumoxystrobin with synergistic effect | |
| UA72041C2 (en) | FUNGICIDAL COMPOSITION CONTAINING N-(a-CYANO-2-THENYL)-4-ETHYL-2-(ETHYLAMINO)-5-THIAZOLECARBOXAMIDE, A METHOD OF ENHANCING THE FUNGICIDAL ACTIVITY AND A METHOD OF CONTROLLING HARMFUL PHYTOPATHOGENS | |
| KR100334348B1 (en) | Fungicide Composition | |
| CA3060884C (en) | Phytosanitary composition comprising essential oils that potentiate antifungal activity | |
| CN109122677A (en) | A kind of agricultural spray adjuvants | |
| US10820597B2 (en) | Phytosanitary composition comprising essential oils that potentiate antifungal activity | |
| WO2015135422A1 (en) | Method for protecting plant against plant disease | |
| KR101232936B1 (en) | Simultaneous Controlling Agent for Plant Diseases Comprising Strawberry Pests Comprising Botanical Extract and Controlling Method Using The Same | |
| AU2011210381A1 (en) | A botanical pesticide for agriculture/horticulture crops | |
| CN1311746C (en) | Bactericidal compositions of flumorph and enostroburin and synergist | |
| CN102715182A (en) | Sterilization composition containing active components Bellkute and imazalil | |
| Rismayani et al. | Bioassay of botanical insecticide formulas against Nilaparvata lugens Stahl (Hemiptera: Delphacidae) | |
| JP2009019011A (en) | Powdery mildew controller and control method | |
| CZ304990B6 (en) | Plant protection formulation | |
| Nurmansyah et al. | Antifungal effectiveness of some essential oils and their mixtures against Fusarium oxysporum f. sp cubense that causes fusarium wilt disease of banana plants | |
| CN116349699A (en) | Stable pesticide synergist and preparation method thereof | |
| CN103875689B (en) | A kind of bactericidal composition and preparation thereof | |
| KR20140045251A (en) | Bacterial wilt preventing composition and the preparation method thereof | |
| KR20090059729A (en) | Composition of essential oils for controlling weeds in crops and method of controlling weeds using the same | |
| JP2002154909A (en) | Disease and insect damage exterminating agent |