IL281576A - Fludioxonil formulations and combinations with tea tree oil for controlling fungal plant pathogens - Google Patents

Description

38979/IL/19-ORP FLUDIOXONIL FORMULATIONS AND COMBINATIONS WITH TEA TREE OIL FOR CONTROLLING FUNGAL PLANT PATHOGENS FIELD OF THE INVENTION The present invention relates to novel fludioxonil formulations, to synergistic combinations comprising fludioxonil and tea tree oil or one or more components thereof, and to methods of using these formulations to control fungal diseases in plants.

BACKGROUND OF THE INVENTION Plants can be attacked by pathogens, such as fungi, in the field, during transport or in storage. The pathogens penetrate the plant directly through wounds caused by insects or by mechanical means. Fungal pathogens can also penetrate into its hosts through the cuticle by secreting enzymes that degrade the components of cuticle.

For example, the fungus Botrytis cinerea Pers., causes the disease "gray mold" (also commonly known as "botrytis") in flowers, fruits and stems of many fruits, vegetables and ornamental crops. This disease is very common in many export crops, and is manifested during the growing season, as well as during post-harvest storage.

Fungicides are often applied, but are usually not effective for adequate control of the disease.

Fludioxonil is a fungicide used for the treatment of crops, particularly cereals, fruits and vegetables, and ornamental plants. Fludioxonil is often used in combination with another fungicide, such as cyprodinil. Fludioxonil is used against Fusarium, Rhizoctonia, Alternaria, Penicillium and Botrytis cinerea.

Tea Tree Oil (TTO) is a natural essential oil characterized by a broad-spectrum antiseptic activity, and is known as an effective pesticide against bacteria and fungi. TTO is extracted from the foliage and terminal branches of a cultivated plant Melaleuca 138979/IL/19-ORP alternifolia, native to Australia, New Zealand and Southeast Asia. TTO contains over 100 components, mostly monoterpenes, sesquiterpenes and their alcohols.

The currently available treatments against phytopathogenic fungi do not always provide adequate disease control due to limited fungicidal activity or the emergence of tolerant isolates of the fungi.

It is therefore an object of the present invention to provide compositions and formulations of fludioxonil and tea tree oil, or one or more components of TTO, for controlling phytopathogenic fungal diseases on crops.

It is another object of the invention to provide synergistic combinations of fludioxonil and tea tree oil, or one or more components of TTO, for controlling phytopathogenic fungal diseases on plants.

It is a further object of the invention to provide methods for controlling phytopathogenic diseases on plants by applying compositions and combinations comprising fludioxonil and TTO, or one or more components of TTO.

Other objects and advantages of the invention will become apparent as the description proceeds.

SUMMARY OF THE INVENTION In one aspect, the present invention provides a method of preventing or treating a phytopathogenic fungal disease on a plant, comprising applying to the plant, or to a part thereof, a synergistically effective amount of a combination comprising an emulsifiable concentrate (EC) of fludioxonil and Tea Tree Oil (TTO) or one or more components thereof. 238979/IL/19-ORP In another aspect, the present invention provides a method of protecting a harvested plant or a part thereof against a fungal attack, comprising applying to said harvested plant or part thereof an effective amount of a combination of fludioxonil and TTO or one or more component thereof, as EC, to a fungus, a locus of a fungus, a plant or to a plant part susceptible to attack by a fungus.

According to one embodiment of the invention, the combination further comprises one or more additional active ingredient selected from a fungicide, bactericide, plant growth regulator, and plant nutrient. According to a specific embodiment, said pesticide is selected from fenhexamid, pydiflumetofen, cyprodinil, and mefenoxam.

According to another embodiment of the invention, the TTO component is selected from: α-thujene; α-pinene; sabinene; β-pinene; myrcene; phellandrene; α-terpinene; limonene; β-phellandrene; p-cymene; 1,8-cineole; γ-terpinene; terpinolene; terpinene- 4-ol; terpineol; aromadendrene; ledene (viridiflorene); δ-cadinene; globulol; viridiflorol; 1,8 cineole; α-terpinolene; and terpinen-4-ol.

In some embodiment of the invention, the plant or part thereof is treated prior or after harvest.

In yet another aspect, the present invention encompasses a fungicidal composition comprising an emulsifiable concentrate of fludioxonil and TTO or one or more components thereof.

According to one embodiment of the invention, the fungicidal composition further comprising one or more additional active ingredient selected from a fungicide, bactericide, plant growth regulator, and plant nutrient, in a synergistically effective amount. 338979/IL/19-ORP According to another embodiment of the invention, the weight ratio of fludioxonil to TTO or one or more components thereof in the fungicidal composition is between 2:1 and 1:10.

BRIEF DESCRIPTION OF THE DRAWINGS Figs. 1A-1B show the activity of various formulations of TTO + fludioxonil as emulsifiable concentrate (EC) against mycelial growth of B. cinerea.

Fig. 1A shows the mycelium diameter (MD) of B. cinerea recorded at 1, 2, and 3 days post inoculation (DPI1, DPI2, and DPI3, respectively) on potato dextrose agar supplemented with one of three EC formulations (F1, F2, and F3) of TTO + fludioxonil, wherein fludioxonil was applied at a final concentration of 1 ppm a.i., compared to untreated control (UTC).

Fig. 1B shows the mycelium diameter (MD) of B. cinerea recorded at 1, 2, and 3 days post inoculation (DPI1, DPI2, and DPI3, respectively) on potato dextrose agar supplemented with one of three EC formulations (F1, F2, and F3) of TTO + fludioxonil, wherein fludioxonil was applied at a final concentration of 0.1 ppm a.i., compared to untreated control (UTC).

Fig. 2 shows the efficacy (as percent of untreated control) of treatment with fludioxonil (SC) at 250 g/L (Fl-SC), fludioxonil (EC) at 100 g/L (Fl-EC), fludioxonil (EC) at 100 g/L and tea tree oil (TTO) at 150 g/L (Fl-EC + TTO), and fludioxonil (EC) at 100 g/L and D- Limonene (DL) at 150 g/L (Fl-EC + DL) at the indicated application rates against Botrytis cinerea (B. cinerea) strain Bc 79 inoculated on white variety of grapevine berries.

Fig. 3 shows the number of fruits infected by B. cinerea in untreated control (UTC) strawberries or fruits treated with the following mixtures (or combinations): (1) fludioxonil (175 g/ha) and cyprodinil (262 ml/ha) in SC formulation (Fl + Cypro (SC)); (2) fludioxonil (175 g/ha) and TTO (262 g/ha) in EC formulation (Fl + TTO 262 (EC)); (3) fludioxonil (175 g/ha) and TTO (340 g/ha) in EC formulation (Fl + TTO 340 (EC)); or (4) 438979/IL/19-ORP fludioxonil (175 g/ha) and TTO (340 g/ha) in ZC formulation (Fl + TTO 340 (ZC)), as determined at 7 days post the first application of treatment (7DPA1) and at 6 days post the second application of treatment (6DPA2).

Fig. 4 shows the percent of rotten oranges without sporulation (-sp) or with sporulation (+sp) of Penicillium digitatum at 33 days post treatment application with imazalil 500 ppm (Ima), pyrimethanil 500 ppm (Pyr), fludioxonil 500 ppm in SC formulation (Fl 500 (SC)), 350 ppm of a composition comprising fludioxonil (100 g/L) and TTO (150 g/L) (Fl + TTO 350 (EC)), and 500 ppm of a composition comprising fludioxonil (100 g/L) and TTO (150 g/L) (Fl + TTO 500 (EC)) or in untreated control (UTC) fruits.

Fig. 5 shows the mean incidence (%) of pomegranates with Botrytis-induced crown rot at 48 days post application of TTO (675 g/cL), fludioxonil (460 g/cL) in SC formulation (Fl (SC)), and a combination of fludioxonil (450 g/cL) and TTO (675 g/cL) in EC formulation (Fl+TTO (EC)) compared to untreated control (UTC) fruits; ap<0.005 compared to UTC or TTO alone; bp<0.005 compared to Fl (SC); the synergy factor (SF) of the combination treatment over each of the active materials administered alone was determined to be 1.71.

DETAILED DESCRIPTION OF THE INVENTION The disclosed invention provides emulsifiable concentrate (EC) formulations of fludioxonil and combinations thereof with Tea Tree Oil (TTO), useful in controlling various phytopathogenic fungal diseases in a wide range of agricultural crops.

In another aspect, the disclosed invention provides a method of controlling phytopathogenic fungal diseases on plants, which comprises applying to the plants, or to a part thereof, a synergistically effective amount of a combination comprising fludioxonil EC and TTO. 538979/IL/19-ORP The term "controlling" as used herein refers to all types of reducing fungal manifestation on plants, including preventing and treating phytopathogenic fungal diseases.

The inventors have found that an EC formulation comprising fludioxonil and TTO is effective in preventing and treating fungal infections during growth of crops and storage of fruits.

The inventors also show a marked synergistic action of mixtures of fludioxonil in EC formulation with TTO. Specifically, it has now been found, surprisingly, that the fungicidal mixtures of fludioxonil and TTO according to the invention not only lead to an additive effect in controlling the phytopathogen, but also provided a synergistic effect upon application. This allows, on the one hand, a reduction in the rate of application of fludioxonil, and on the other hand, a broadening of the spectrum of phytopathogens that can be controlled, including tolerant or resistant strains.

The compositions of the invention comprise the compound 4-(2,2-difluoro-1,3- benzodioxol-4-yl)-1H-pyrrole-3-carbonitrile, known as fludioxonil, having the general chemical structure of: In general, fludioxonil can be formulated in a number of formulation types, as the sole active ingredient, or in combination with one or more additional active ingredients. 638979/IL/19-ORP The present application provides a combination of fludioxonil and TTO formulated as emulsifiable concentrates (EC).

Emulsifiable concentrates (EC) of fludioxonil may be prepared by dissolving the compound in at least one organic solvent, optionally containing one or more emulsifying agents. Suitable organic solvents for use in ECs include aromatic hydrocarbons (such as alkylbenzenes or alkylnaphthalenes), ketones (such as cyclohexanone) and N- alkylpyrrolidones (such as N-methylpyrrolidone or N-octylpyrrolidone), fatty acid methyl esters (such as methyl oleate), dimethyl amides of fatty acids (such as C8-C10 fatty acid dimethylamide and N,N-dimethyloctanamide, commercially available as Rhodiasolv® ADMA 10), and lactate esters (such as n-butyl L-lactate, commercially available as PURASOLV® BL). Suitable emulsifiers for use in EC formulations of fludioxonil include but are not limited to ethoxylated castor oil (such as the commercially available Alkamus® OR/36), dodecylbenzene sulfonate (such as the commercially available Rhodacal® 60/BE) and block copolymers (such as polyalkoxylated butyl ether commercially available Ethylan® NS-500LQ).

The obtained EC product may spontaneously emulsify on addition to water, to produce an emulsion with sufficient stability to allow spray application through appropriate equipment.

Notably, fludioxonil is admixed or formulated with TTO, or one or more component thereof. The composition obtained may further be admixed with one or more additional active ingredients such as a fungicide, bactericide, plant growth regulators, and plant nutrients. The additional ingredient can be either a synthetic product or a biological product, such as a biopesticide. The particular additional active ingredient will depend upon the intended utility of the composition. 738979/IL/19-ORP The term "combination" as used herein refers to the various combinations of fludioxonil and TTO, optionally with an additional active ingredient as defined herein, for example in a single "ready-mix" form, in a combined spray mixture composed from separate formulations of the single active ingredient components, such as a "tank-mix", and in a combined use of the single active ingredients when applied in a sequential manner, i.e. one after the other with a reasonably short period, such as a few hours. The order of applying the fludioxonil, TTO, and other active ingredients is not essential for carrying out the present invention.

The weight ratio of fludioxonil to TTO, or one or more components thereof, is selected to give a synergistic activity. In general, the weight ratio of fludioxonil to TTO is between 2:1 and 1:50, specifically between 2:1 and 1:10, more specifically between 2:1 and 1:5, even more specifically between 1:1.5 and 1:2.

The fludioxonil formulations of the invention may further include one or more pesticide, as an additional active ingredient. Examples of compositions comprising fludioxonil formulations and additional pesticides include the following: Frontal (Fludioxonil and Fenhexamid); Miravis Prime (Fludioxonil and Pydiflumetofen); Switch (Fludioxonil and Cyprodinil); and Maxim XL (Fludioxonil and mefenoxam).

Examples of fungicides that are specifically effective in controlling fungal manifestation when applied in combination with fludioxonil are: TTO and TTO components, such as D- Limonene.

A composition according to the invention may include one or more additives to improve the biological performance of the composition (such as improving wetting, retention or distribution on surfaces and resistance to rain on treated surfaces). Such additives include surface active agents, spray additives based on oils, for example mineral oils or natural plant oils, and blends of these with other adjuvants which may aid or modify the 838979/IL/19-ORP action of fludioxonil. According to a specific embodiment, the composition comprises the natural essential oil Tea Tree Oil (TTO, Melaleuca alternifolia), for example, the Australian TTO, or one of more components thereof.

It should be noted that fractions of TTO, comprising components thereof and/or components in different proportions, will be effective in various degrees. Accordingly, whenever reference is made to "TTO" it should be understood to include also compositions comprising one or more TTO components as well-known in the art, and also selected from: α-thujene; α-pinene; sabinene; β-pinene; myrcene; phellandrene; α- terpinene; limonene; β-phellandrene; p-cymene; 1,8-cineole; γ-terpinene; terpinolene; terpineol; aromadendrene; ledene (viridiflorene); δ-cadinene; globulol; viridiflorol; 1,8 cineole; α-terpinolene; and terpinen-4-ol.

The contents of the various components of a standard TTO composition are detailed in ISO 4730:2017.

The synergistic activity of the combination fludioxonil and TTO is apparent from the fact that the fungicidal activity of the composition of fludioxonil + TTO is greater than the sum of the fungicidal activities of fludioxonil and TTO.

Examples of diseases and pest species which may be controlled by the compositions and formulations of the invention include: botrytis (also termed "gray mold"), caused by the necrotrophic fungus Botrytis cinerea; powdery mildew, caused by many different species of fungi in the order of Erysiphales, including Podosphaera xanthii (a.k.a.

Sphaerotheca fuliginea); white mold, caused by the fungus Sclerotinia sclerotiorum; green rot (also termed green mold), caused by the fungus Penicillium digitatum; black mold, caused by the fungus Aspergillus niger; and Fusarium wilt (a.k.a. vascular wilt), caused by the fungus Fusarium oxysporum. 938979/IL/19-ORP The compositions and combinations comprising fludioxonil according to the present invention can be applied without other adjuvants, but most often application will be of a formulation comprising one or more active ingredients with suitable carriers, diluents, and surfactants. According to the invention, a carrier is a natural or synthetic, organic or inorganic substance which is mixed or combined with fludioxonil for better applicability to plants. The carrier, which may be solid or liquid, is generally inert and is suitable for use in agriculture.

According to the invention, the composition or combination is applied to the plant at any stage of its life cycle, before or post harvest, including tissue culture, plantlet before or after hardening, vegetative growth, flowering, and fruiting.

Thus, the invention encompasses the application of a composition or combination comprising fludioxonil to any part of the plant, including the foliage, stems, branches, flowers, fruit, roots, to the seed or the meristem tissue before it is planted, or to media in which the plants are grown or are to be planted (such as soil, water or hydroponic culture systems) or to the flowers or fruits as a post-harvest treatment. The compositions and combinations of the invention may be mixed with soil, peat or other rooting media for the protection of plants against seed-borne, soil-borne or foliar fungal diseases.

The formulations and combinations according to the invention may be applied before or after infection of the plant by the phytopathogenic fungi.

The compositions and combinations comprising fludioxonil according to the invention are applied as a liquid solution by foliar or soil spraying, dipping, drenching, or drip irrigating. 1038979/IL/19-ORP One method of application involves spraying a water dispersion or refined oil solution of the combination products. Compositions with spray oils, spray oil concentrations, spreader stickers, adjuvants, and other solvents, often enhance compound efficacy.

Such sprays can be applied from spray containers such as a can, a bottle or other container, either by means of a pump or by releasing it from a pressurized container, e.g., a pressurized aerosol spray can. Such spray compositions can take various forms, for example, sprays, mists, foams, fumes or fog. Such spray compositions thus can further comprise propellants, foaming agents, etc. Representative propellants include, but are not limited to, methane, ethane, propane, butane, isobutane, butene, pentane, isopentane, neopentane, pentene, hydrofluorocarbons, chlorofluorocarbons, dimethyl ether, and mixtures of the foregoing.

Typical devices for applying an effective amount of the composition or combination of the invention to the soil include a gravity flow applicator, e.g., chisel, tooth or shank type applicators; commercially available sprayers, atomizers, aerators, blowguns, low pipes; pulverizes or the like are also provided as useful applicators. Irrigating means, such as drip emitters, micro sprayers, emitter tubing, misters and the like are useful applicators.

The plants or plant parts according to the invention can be treated with a composition or combination according to the invention at any frequency, for examples one time or more, such as 2 times, 3 times, 4 times, 5 times or more. The time interval between two treatments can be chosen according to the agronomical needs. For example, the composition or combination may be applied every three days, four days, once a week, once a month, twice a month, or once a year. According to a specific embodiment, the treatment is supplemented by additional applications on various days. It should be noted that the plant may be treated with the composition or combination according to the invention for any desired period of time. In general, the number of applications and intervals can be adjusted according to a range of parameters, including the phenological 1138979/IL/19-ORP stage of the host, susceptibility of these stages, potential of infection by the pathogen, sources of inoculum, level of infection and the climatic conditions.

The amount of a composition or combination according to the invention to be applied will depend on various factors, such as the active ingredients employed; the type of fungi to be controlled; the subject of the treatment, such as, for example plants, plant parts prior or after harvest, or soil; the mode of application; or the purpose of the treatment, for example prophylactic or curative.

The present invention allows the application of the composition or combination of the invention to any crop plant, in particular monocotyledons such as cereals (wheat, millet, sorghum, rye, triticale, oats, barley, teff, spelt, buckwheat, fonio and quinoa), rice, maize (corn), and/or sugar cane; palm trees, or dicotyledon crops such as beet; fruits (for example pomegranates, grapes (grapevine berries), apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries or blackberries); leguminous plants (such as beans, soybeans, lentils, or peas); oil plants (such as rape, mustard, poppy, olives, sunflowers, coconut, castor oil plants, cocoa beans or groundnuts); cucumber plants (such as marrows, cucumbers or melons); fibre plants (such as cotton, flax, hemp or jute); citrus fruit (such as oranges, lemons, grapefruit or mandarins); vegetables (such as tomatoes, pepper, spinach, lettuce, cabbages, carrots, potatoes, or cucurbits); lauraceae (such as avocados, cinnamon or camphor); bananas; tobacco; nuts; coffee; tea; vines; hops; durian; natural rubber plants; and ornamentals (such as flowers, shrubs, broad­ leaved trees or evergreens). It should be noted that this list does not represent any limitation.

The formulations and combinations according to the invention are furthermore effective against post-harvest fungal diseases in plants, particularly against pathogens of fruits and flowers that evolve after harvest and during storage and shipping. 1238979/IL/19-ORP The invention therefore provides a method of controlling a fungus which comprises applying an effective amount of a combination of an EC formulation of fludioxonil and TTO, or one or more component thereof, to a fungus, a locus of a fungus, a plant or to a plant part susceptible to attack by a fungus.

In a further aspect, the invention provides a method of protecting a harvested plant or parts thereof against attack of fungi. The method comprises applying to said harvested plant or parts thereof an effective amount of a combination of an EC formulation of fludioxonil and TTO or one or more component thereof, to a fungus, a locus of a fungus, a plant or to a plant part susceptible to attack by a fungus.

The formulations and combinations according to the invention enable to inhibit or control a phytopathogenic fungus that occurs in plants or in parts of plants (fruit, flowers, leaves, stems, tubers, roots, seeds) in different plants, while at the same time the parts of plants which grow later are also protected from attack by the phytopathogenic fungus.

The invention will now be described with reference to specific examples and materials.

The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of specific embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

EXAMPLES Materials and methods Materials D-Limonene, a cyclic monoterpene which is a component of tea tree oil (TTO); 1338979/IL/19-ORP Fludioxonil, IUPAC Name: 4-(2,2-difluoro-1,3-benzodioxol-4-yl)-1H-pyrrole-3- carbonitrile, CAS number: 131341-86-1, having the following chemical structure in the form of a soluble concentrate or an emulsifiable concentrate: ; Imazalil (also known as Enilconazole and chloramizole), preferred IUPAC name: 1-{2- (2,4-Dichlorophenyl)-2-[(prop-2-en-1-yl)oxy]ethyl}-1H-imidazole; Pyrimethanil, IUPAC name: 4,6-Dimethyl-N-phenylpyrimidin-2-amine; Requiem® EC, contains 16.75% synthetically manufactured terpene constituents of the extract of Chenopodium ambrosioides near ambrosioides; Scholar®, contains 230 g/L fludioxonil in the form of a soluble concentrate (SC); Switch®, contains 250 g/kg fludioxonil and 375 g/kg cyprodinil in the form of water dispersible granules; Timorex Gold®, contains 222.5 g/L TTO in the form of an emulsifiable concentrate; Tea Tree Oil (oil of Melaleuca alternifolia), CAS number: 68647-73- 4.

Preparation of EC formulation of fludioxonil and TTO Formulations of fludioxonil and TTO as emulsifiable concentrate (EC) were prepared by feeding the ingredients listed in Tables 1A-1C to a container and stirring until a homogeneous solution is obtained (about half an hour at room temperature).

Table 1A. List of ingredients in a first EC formulation of fludioxonil and TTO (F1) Ingredient Function Content (g/L) Fludioxonil Tech (100%) Active ingredient 100 Tea Tree Oil (Melaleuca alternifolia) Active ingredient 150 1438979/IL/19-ORP Ingredient Function Content (g/L) Ethoxylated castor oil nonionic emulsifier Emulsifier 100 (Alkamus® OR/36) Dodecylbenzene Sulfonate (Rhodacal® 60/BE) Emulsifier 60 Methyl oleate Solvent 85 Solvent Complete to 1 L N,N-dimethyloctanamide (Rhodiasolv® ADMA 10) Table 1B. List of ingredients in a second EC formulation of fludioxonil and TTO (F2) Ingredient Function Content (g/L) Fludioxonil Tech (100%) Active ingredient 80 Tea Tree Oil (Melaleuca alternifolia) Active ingredient 150 Ethoxylated castor oil nonionic emulsifier Emulsifier 130 (Alkamus® OR/36) Polyalkoxylated butyl ether (Ethylan® NS-500LQ) Emulsifier 40 n-Butyl L-lactate (PURASOLV® BL) Solvent Complete to 1 L Table 1C. List of ingredients in a first EC formulation of fludioxonil and TTO (F3) Ingredient Function Content (g/L) Fludioxonil Tech (100%) Active ingredient 100 Tea Tree Oil (Melaleuca alternifolia) Active ingredient 200 Ethoxylated castor oil nonionic emulsifier Emulsifier 200 (Alkamus® OR/36) Polyalkoxylated butyl ether (Ethylan® NS-500LQ) Emulsifier 50 N-Methylpyrrolidone Solvent Complete to 1 L Synergy factor calculation The synergy factor (SF) between two active materials is defined as the ratio between the observed efficacy of the combined treatment and the expected efficacy of the combined treatment, such that a ratio of less than 1.0 indicates an antagonistic effect of the combined treatment, a ratio that equals 1.0 indicates an additive effect of the combined treatment and a ratio of more than 1.0 indicates a synergy between the two compound in the combination treatment. The expected efficacy (EE) of the combined treatment is determined using Colby's analysis: EE = ^ + ^ - ^^⁄100, wherein A is the efficacy of the first active material and B is the efficacy of the second active material, while the efficacy is expressed as percent of control (Colby, Weeds 15(1):20–22, 1967). 1538979/IL/19-ORP Field tests in strawberries Strawberries of Tamir variety showing preliminary symptoms of botrytis were sprayed twice at a 6 days interval with the indicated materials at a volume of 400 L/ha using Ecko Backpack sprayer with nozzle hole #2. The experiment included 4 plots for each treatment in random blocks, each plot has an area of 15 m2. The number of Botrytis- positive fruits was counted at the indicated time points and the infected fruits were removed after each count.

Strawberries of Rocky variety (after removal of all fruits infected with B. cinerea) were sprayed twice at a 7 days interval with the indicated materials at a volume of 500 L/ha using an Ecko Backpack sprayer with nozzle hole #3. The experiment included 4 plots in for each treatment in random blocks, each plot has an area of 15 m2. The number of Botrytis-positive fruits at the northeastern part of each plot was counted at the indicated time points and the infected fruits were removed after each count.

Field tests in cucumbers Cucumber plants of Zena variety were sprayed twice at a 7 days interval with the indicated materials at a volume of 600 L/ha using an Ecko Backpack sprayer with nozzle hole #3. The experiment included 4 plots for each treatment in random blocks, each plot at an area of 16 m2. The number of powdery mildew (PM)-positive leaves was counted at the indicated time points. The percent of infected leaf area with PM (disease severity) in each of 10 leaves located at about 1.5 meters above ground was also assessed.

Cucumber plants of Boss variety (after removal of all cucumbers infected with B. cinerea) were sprayed twice at 6 days interval with the indicated materials at a volume of 500 L/ha using an Ecko Backpack sprayer with nozzle hole #3. The experiment included 4 plots for each treatment in random blocks, each plot has an area of 12 m2.

The number of Botrytis infection foci on young fruits, leaves and stems in plants located 1638979/IL/19-ORP at the center of each plot was counted at the indicated time points. The number of Sclerotinia sclerotiorum infection foci on the plants was also counted at the indicated time points. In addition, at the end of the experiment, 10 leaves of each plot located 20 cm from the plant's top were sampled and the percentage of leaf coverage with powdery mildew (diseases severity) and the number of powdery mildew infected leaves (disease incidence) were assessed.

Tests with detached grapevine berries White grapevine berries of Italia variety were surface disinfected in sodium hypochlorite and rinsed in sterile distilled water. After drying, the berries were cut lengthwise in two and the cut parts were placed in a 90 mm diameter Petri dish on sterile wet Whatman n°1 paper (5 parts per dish). The berries were wounded in their equatorial area with a sterile needle. Wounded berries were treated with sterile distilled water (untreated control, UTC), or with the indicated active agent, either alone or in mixture. Each treatment was prepared in a volume of water corresponding to 150 L/ha. Treatment was carried out with a hand sprayer at 2 bars. Three repetitions of 5 berries parts each (total 15 parts) were carried out for each treatment. After application of agents, the Petri dishes were place for 1 hour under a laminar flow hood with lid removed in order to dry the fungicides droplets. Then, the Petri dishes were incubated for 24 hours in a growth chamber executing a photoperiod of 16 hours light at 23-25 °C and a night period of 8 hours at 18 °C, with 85% relative humidity. Then, the berries were inoculated through the wound with 10 µl of a calibrated spores suspension of Botrytis cinerea of strain Bc 933, which is resistant to fludioxonil (ED50 of 0.45 a.i./ml, with Resistance Factor of 84) or 15 µl of a calibrated spores suspension of B. cinerea strain Bc 79, which is susceptible to fungicides. After inoculation, the Petri dishes were incubated again for 7 days in a growth chamber under the same conditions as described above.

At 3, 5 and 7 days post inoculation (DPI), each berry part was observed and the length of the brown lesion caused by B. cinerea was measured. The diseases intensity was 1738979/IL/19-ORP calculated in percent of the total length of the berry part analyzed. In order to determine the observed efficacy of the treatments over time, the area under the disease progress curve (AUDPC) was estimated as suggested by Shaner and Finney, Phyopathology 67:1051-1056 (1977).

Tests in post-harvested orange fruits during storage Orange fruits of Or variety were picked and placed in storage crates at 40 fruits per crate. Inoculation with Penicillium digitatum spores was carried out by wounding each fruit with a nail protruding from a cork stopper that was dipped in a spore solution (containing 104 spores) prior to the wounding. The indicated treatment compositions were prepared in a bath containing water at 56 °C. Each treatment group comprised 4 orange crates, which were sequentially immersed in the bath for 30 seconds. The bath was emptied and washed with hot water between each treatment. The number of healthy fruits, infected fruits without spores and spore-covered fruits was counted at 11, 22 and 33 days post treatment application (DPA). The infected fruits were removed from the crate after each counting.

Field tests in roses Roses of Vendela or Touch of Class varieties grown at a greenhouse (GH) in a phenological status of production affected by Botrytis were sprayed 4 times at 4-7 days interval (depending on disease potential) with the indicated materials at a volume of 720 L/ha using an electric pump with constant pressure (42 psi) with a two hollow cone­ type nozzle. The experiment included 4 plots for each treatment in completely randomized block design (CRBD), each plot has an area of 5 m2 (50 plants per plot). The pH of the sprayed solutions was confirmed to be 7.0. Phytotoxicity was evaluated one week after each application according to the European Weed Research Council (EWRC) scale presented in Table 2. Specifically, tender shoots were marked to confirm that no symptoms of phytotoxicity were observed at a later time. The severity of Botrytis was also evaluated according to the scale presented in Table 3. 1838979/IL/19-ORP Table 2. EWRC scale of phytotoxic symptoms Degree Phytotoxic symptoms % 0 Absolute absence of symptoms (healthy plants) 0 1 Very mild symptoms, some mild atrophy 0.1-3.0 2 Some mild atrophy but clearly visible symptoms 3.1-5.0 Accentuated symptoms, chlorosis, probably without 3 5.1-10.0 negative effects on the crop strong chlorosis, and/or atrophy, expected to affect the 4 10.1-50.0 crop Lethal damage to crop > 50 Table 3. Scale of Botrytis severity Degree Affected leaf area (%) 0 No sign or appreciable symptom of disease 1 Presence of small lesions on up to 5% of the leaf area 2 Presence of lesions on 6% - 10% of the leaf area 3 Presence of lesions on 11% - 25% of the leaf area 4 Presence of lesions on 26% - 50% of the leaf area Presence of lesions on > 51% of the leaf area Tests in post-harvested pomegranate fruits during storage Post-harvested pomegranate fruits of wonderful variety were punched with a toothpick and submerged in a solution of the indicated treatments for 30 seconds. Fruits were allowed to dry for 30 minutes and then packed and wrapped in export boxes (8 fruits per box), 5 boxes for treatment and were then stored at 11 °C for 48 days. The number of puncture hole areas with rot and the number of rotted crowns caused by Botrytis in each box was counted every two weeks.

In another experiment, pomegranate fruits were treated with the indicated materials (without puncture) and stored for 75 days at 11 °C. At the end of the experiment, the number of rotted crowns and rotted fruits were counted for each treatment. 1938979/IL/19-ORP Field test in grapevine berries Grapevine berries of Muscat variety were used to test the efficacy of EC formulation of fludioxonil and TTO at a concentration of 0.1%, 0.2% or 0.4%, as well as Switch® (0.1%), in the treatment of Aspergillus niger infection compared to untreated grapevines (sprayed with water). The experiment took place in a vineyard exhibiting disease symptoms. The experiment included 4 plots, each consisting of 7 vines, for each treatment in random blocks. At the beginning of the experiment, 6 healthy grape clusters per plot were labeled and 3 berries on each cluster (total of 18 berries per plot) were wounded, thereby increasing susceptibility of the berries to infection by the fungus. First application of the treatments was carried out at the same day of the wounding. 10 days post the first application (10DPA1), 3 new berries on each labeled cluster were wounded. At the same day, a second treatment was applied. The experiment ended 8 days after the second application (8DPA2). Applications of the treatments were carried out by spraying using a motorized backpack sprayer at a volume of 1000 L/ha.

Primary infection was assessed at 10DPA1 and 8DPA2. Secondary infection was assessed at 8DPA2. The assessment of primary infection included determining the incidence of infected berries, and a qualitative evaluation of the fungus's vitality was performed for each wounded berry according to a scale of 0-3, wherein 0 is no infection with the fungus; 1 is low infection with the fungus, characterized by low vitality (mycelia health); 2 is moderate infection with the fungus, characterized by moderate vitality; and 3 is high infection with the fungus, characterized by high vitality. The assessment of secondary infection included counting the number of infected unwounded berries on labeled clusters. In addition, the incidence of clusters with rot was also determined, as well as the incidence of the clusters with secondary infection of berries. 2038979/IL/19-ORP Statistical analysis Statistical analysis was carried out using JMP or InfoStat softwares. Tukey-Kramer post- hoc test or Newman and Keuls test were used to determine the statistical significance of differences between treatment groups, with p<0.05 considered statistically significant.

Example 1: Activity of various formulations of fludioxonil and TTO against mycelial growth of Botrytis cinerea The fungicidal activity of fludioxonil in combination with tea tree oil (TTO) was tested against B. cinerea isolate collected from infected grape vine berries. Monospore cultures of the fungus grown on potato dextrose agar (PDA) medium were used. Agar disks, 2 mm in diameter, bearing B. cinerea, were taken from the edge of a freshly growing colony and placed on PDA comprising one of three EC formulations of fludioxonil and TTO (F1, F2, and F3 as specified in Tables 1A, 1B, and 1C, respectively) in 9-cm Petri dishes, with two disks in each dish. The formulations tested were applied such that the final concentrations of fludioxonil in the PDA were 0.1, 1, 10, and 100 ppm a.i. PDA alone was used as control. Three dishes per treatment were incubated at 25°C in the dark and colony diameters were recorded at various days after inoculation.

The results obtained indicated that all three formulations at final concentrations of 10 and 100 ppm a.i of fludioxonil completely inhibited mycelial growth of B. cinerea, while growth of the fungus in untreated dishes reached 45 mm in diameter within 3 days.

Further study revealed a strong activity of all formulations at 1 ppm a.i. of fludioxonil, in which the diameter of mycelial growth in all four formulations was only 3-4 mm compared to the untreated dishes (Fig. 1A). The inhibitory effect was also observed at 0.1 ppm a.i. of fludioxonil, in which the diameter of mycelial growth reached 12-15 mm for F1 and F3 formulations, and 21 mm for F2 formulation (Figure 1B). In both concentrations, growth of the fungus in untreated dishes reached 45 mm in diameter within 3 days (Figs 1A and 1B). 2138979/IL/19-ORP The results indicate that the various formulations of fludioxonil and TTO provided a significant fungicidal activity against B. cinerea at all concentrations of fludioxonil.

Example 2: Combination of fludioxonil (SC) and TTO have a synergistic effect against Botrytis and powdery mildew Evaluation of the efficacy of fludioxonil (in the form of a soluble concentrate, SC) in combination with tea tree oil (TTO) for the treatment of Botrytis in strawberries (of Tamir variety) indicates that the combined treatment with fludioxonil (SC) and TTO has a synergistic effect. Table 4 shows that the percentage of untreated control (UTC) strawberries showing symptoms of Botrytis (namely, Botrytis-positive fruits) was higher compared to the percentage of Botrytis-positive strawberries treated with fludioxonil (SC) alone, TTO alone or the combination thereof as counted at the day of the second application of the treatments (which occurred 6 days after the first application and thus designated 6 DP1A), as well as 6 and 10 days post the second application (DP2A) of the treatments. The efficacy of each treatment at the end of the experiment presented as percent of control is also shown in Table 4. The expected efficacy of the combined fludioxonil and TTO treatment as determined by Colby's analysis is 67.6%; however, the observed efficacy of said combination as shown in Table 4 is 76.9%. The synergy factor (SF) is thus 1.1, indicating a synergistic effect of the combined treatment over the treatment with a single active material.

Table 4. Percent of Botrytis-positive strawberries and efficacy of treatments Efficacy at 10 Treatment 6 DP1A 6 DP2A 10 DP2A DP2A (%) UTC 9.0 44.7 47.7 -- Fludioxonil (SC, 253 g a.i./ha) 2.5a 22.2a 24.0a 49.7 TTO (375 g a.i. ha) 4.0a 25.7a 30.7 35.6 2238979/IL/19-ORP Efficacy at 10 Treatment 6 DP1A 6 DP2A 10 DP2A DP2A (%) Fludioxonil (SC, 253 g a.i./ha) + 11a 3.7a 10.2a 76.9 (SF=1.1) TTO (375 g a.i. ha) ap<0.05 compared to UTC of the same day.

Evaluation of the efficacy of fludioxonil (in the form of a soluble concentrate, SC) in combination with tea tree oil (TTO) for the treatment of powdery mildew (PM) in cucumber plants also indicates that combination of fludioxonil (SC) and TTO has a synergistic effect. Table 5A shows the number of untreated control (UTC) leaves of cucumber plants showing symptoms of powdery mildew (PM) compared to the number of PM-positive leaves from cucumber plants treated with fludioxonil (SC) alone, TTO alone or the combination thereof as counted 4 and 7 days post the second application (DP2A) of the treatments. The incidence efficacy of each treatment at the end of the experiment presented as percent of control is also shown in Table 5A. Table 5B shows the percent PM coverage on 10 leaves of untreated control (UTC) cucumber plants compared to the percent PM coverage of leaves from cucumber plants treated with fludioxonil (SC) alone, TTO alone or the combination thereof as counted 4 and 7 days post the second application (DP2A) of the treatments. The severity efficacy of each treatment at the end of the experiment presented as percent of control is also shown in Table 5B.

The expected incidence and severity efficacies of the combined fludioxonil and TTO treatment as determined by Colby's analysis is 31.44% and 75.6%, respectively; however, the observed efficacies of said combination as shown in Tables 5A and 5B are 45.7% and 79.7%, respectively. The synergy factor for incidence is thus 1.5, while the synergy factor for severity is 1.1. Accordingly, the synergy factors indicate a strong synergistic effect of the combined treatment over the treatment with a single active material. 2338979/IL/19-ORP Table 5A. Number of PM-positive leaves from cucumber plants and incidence efficacy of treatments Incidence efficacy at 7 Treatment 4 DP2A 7 DP2A DP2A (%) UTC 90.0 87.5 -- Fludioxonil (SC, 150 g a.i./ha) 52.5 a 70.0 20 TTO (375 g a.i. ha) 75.0 75.0 14.3 Fludioxonil (SC, 150 g a.i./ha) + TTO 22.5 a,b 47.5a 45.7 (SF=1.5) (375 g a.i. ha) ap<0.05 compared to UTC of the same day. bp<0.05 compared to fludioxonil alone of the same day.

Table 5B. Percent of PM coverage on leaves from cucumber plants and severity efficacy of treatments Severity efficacy at 7 Treatment 4 DP2A 7 DP2A DP2A (%) UTC 13.7 18.2 -- Fludioxonil (SC, 150 g a.i./ha) 4.4a 8.1a 55.5 TTO (375 g a.i. ha) 7.5a 10.0a 45.1 Fludioxonil (SC, 150 g a.i./ha) + TTO 3.7b,c 1.3c 79.7 (SF=1.1) (375 g a.i. ha) ap<0.05 compared to UTC of the same day; bp<0.05 compared to fludioxonil alone of the same day; cp<0.05 compared to TTO alone of the same day.

Example 2: Combination of fludioxonil (SC) and TTO has a synergistic effect in preventing Botrytis in grapevine berries Evaluation of the observed efficacy (OE) and expected efficacy (EE) of fludioxonil (in the form Scholar® which contains fludioxonil as a soluble concentrate, SC) in combination with TTO for the prevention of Botrytis in grapevine berries, as shown in Table 6, indicates that combination of fludioxonil (SC) and TTO has a synergistic effect.

Importantly, this combination exhibited a synergistic interaction at all the tested ratios 2438979/IL/19-ORP between the active agents towards B. cinerea (strain Bc 993), which is resistant to fludioxonil. However, the level of synergistic interaction between fludioxonil and TTO varies from ratio to ratio, such that the tested ratios can be classified in an ascending order of synergy as follows: 1:5 < 1:1 < 1:2 < 1:3 < 1:4.

Table 6. AUDPC and synergy factor (SF) of fludioxonil and TTO combination treatment against B. cinerea strain Bc 993 applied to white grapevine berries Treatment AUDPC OE (%) SF EE (%) UTC 398.7 - - - Fludioxonil (SC, 200 µg a.i./ml, 0.13 L/ha) 268.7a 32.6 - - 334.3a,b TTO (200 µg a.i./ml, 0.12 L/ha) 16.2 - - TTO (400 µg a.i./ml, 0.24 L/ha) 305.8a,b 23.3 - - TTO (600 µg a.i./ml, 0.36 L/ha) 303.1a,b 24.0 - - TTO (800 µg a.i./ml, 0.48 L/ha) 287.1a,b 28.0 - - TTO (1000 µg a.i./ml, 0.60 L/ha) 269.7a 32.5 - - Fludioxonil (SC, 200 µg a.i./ml, 0.13 L/ha) + 199.4a,b, c 50.0 43.4 1.15 TTO (200 µg a.i./ml, 0.12 L/ha) – ratio 1:1 Fludioxonil (SC, 200 µg a.i./ml, 0.13 L/ha) + 161.5a,b, c 59.5 48.3 1.23 TTO (400 µg a.i./ml, 0.24 L/ha) - ratio 1:2 Fludioxonil (SC, 200 µg a.i./ml, 0.13 L/ha) + 109.6a,b, c 72.5 48.8 1.49 TTO (600 µg a.i./ml, 0.36 L/ha) - ratio 1:3 Fludioxonil (SC, 200 µg a.i./ml, 0.13 L/ha) + 76.2a,b, c 80.9 51.5 1.57 TTO (800 µg a.i./ml, 0.48 L/ha) - ratio 1:4 Fludioxonil (SC, 200 µg a.i./ml, 0.13 L/ha) + 168.3a,b, c 57.8 1.06 54.5 TTO (1000 µg a.i./ml, 0.60 L/ha) - ratio 1:5 ap<0.05 compared to UTC of the same day; bp<0.05 compared to fludioxonil alone of the same day; cp<0.05 compared to TTO alone of the same day.

Example 3: Combination of fludioxonil (EC) and TTO, or fludioxonil (EC) and D-limonen, is more efficacious in preventing Botrytis in grapevine berries than fludioxonil (EC) alone The efficacy of the combination of fludioxonil in the form of EC, with TTO or D-Limonene (DL) in preventing Botrytis was evaluated at various application rates. Table 7 shows the efficacy of the indicated treatments over time by determining the AUDPC during the experiment (7 days post inoculation with B. Cinerea). Fig. 2 shows the efficacy (as 2538979/IL/19-ORP percent of control) of the different treatment rates tested. All three tested fludioxonil- based fungicide formulations exhibited a clear dose-response curve when used at 5 rates ranging from 10 µg/ml to 800 µg/ml fludioxonil (corresponding to 1.5 to 120.0 g/ha fludioxonil), towards the B. cinerea strain Bc 79, which is susceptible to fludioxonil, on grapevine berries in controlled conditions. The three fludioxonil-based fungicide formulations have the following EC50 (µg/ml fludioxonil) towards the B. cinerea strain Bc 79: fludioxonil (EC): 280 ppm, fludioxonil (EC) with TTO: 120 ppm, and fludioxonil (EC) with D-Limonen: 140 ppm. Overall, the combination of fludioxonil with TTO or D- Limonen was more efficient than the corresponding EC formulation of fludioxonil alone towards B. cinerea on grapevine berries.

Table 7. AUDPC of fludioxonil (SC or EC) alone or in combination with TTO or D- Limonene against B. cinerea strain Bc 79 applied to white grapevine berries Application rate [µg a.i./ml (g a.i./ha)] 100 Treatment 200 800 0 400 (60) (1.5) (15) (30) (120) Fludioxonil (EC), 100 413.8 151.1 504.6 322.2 270.0 235.5 g/L Fludioxonil (EC), 100 504.6 492.9 255.0 186.2a 100.4 80.0 g/L + TTO (150 g/L) Fludioxonil (EC), 100 504.6 463.3 286.1 211.0 142.5 106.3 g/L + DL (150 g/L) ap<0.05 compared to fludioxonil (EC) alone of the same dose.

Example 4: Combination of fludioxonil and TTO in EC formulation shows similar or improved efficacy in preventing diseases compared to combinations of fludioxonil and TTO in ZC formulation, or fludioxonil and cyprodinil in SC formulation A comparison of the efficacies of fludioxonil formulations as SC or EC in combination with TTO was carried out in strawberries of Rocky variety. Evaluation of the Botrytis- positive fruits was carried out at 7 days post the first application (7DPA1), and at 6 days post the second application (6DPA2). As shown in Fig. 3, the most efficacious treatment 2638979/IL/19-ORP was the combination of fludioxonil with the highest dose to TTO formulated as EC. The treatment with fludioxonil and the lower dose of TTO formulated as EC was more efficient against Botrytis than the combination of fludioxonil and TTO at a higher formulated as ZC. The commercially available combination of fludioxonil and cyprodinil formulated as WGD, which easily transforms into SC was less efficient than all the combinations of fludioxonil and TTO tested. It should be noted that the doses of active agents applied in this experiment are about 30% less than the recommended target dose for treating Botrytis. This is because the aim of the present experiment was to compare efficacies between treatment groups rather than actually treating the fruits.

Another evaluation of the efficacy of fludioxonil and TTO in EC formulation compared to other types of formulations was performed in cucumbers. Table 8A shows the number of Botrytis infection foci on young fruits, leaves and stems of the cucumber plants as counted 6 days post the first application of the treatments (6DPA1), as well as 6 and 11 days post the second application of the treatments (6DPA2 and 11DPA2, respectively).

The treatments comprising fludioxonil (Fl, 150 g/L, 1.5 L/ha) and TTO (225 or 300 g/L, 1.5 L/ha) in EC formulation were more effective in preventing Botrytis in cucumber plants than the combination of fludioxonil (Fl, 150 g/L, 1.5 L/ha) and TTO (300 g/L, 1.5 L/ha) in ZC formulation, the combination of fludioxonil (Fl, 150 g/L, 1.5 L/ha) and cyprodinil (Cypro, 225 g/L, 1.5 L/ha) in SC formulation or TTO (222.5 g/L, 1 L/ha) alone.

In addition, as shown in Table 8B, all combinations comprising fludioxonil completely abrogated Sclerotinia sclerotiorum infection compared to control treatment.

Furthermore, the combination of fludioxonil and TTO in EC formulation was effective in eliminating powdery mildew infections and in reducing the severity of the disease in a manner that is comparable to the effects of the known Switch® (a WDG/SC co­ formulation of fludioxonil and cyprodinil) (Table 8C). Application of fludioxonil and TTO in ZC formulation was less effective in reducing the frequency and severity of powdery mildew. 2738979/IL/19-ORP It should be noted that all the tested treatments mention in this example were found safe to use, and no phytotoxic effects were observed after two sequential applications.

These results indicate that the combination of fludioxonil and TTO in EC formulation has similar or improved effects in preventing and treating fungi infections in crops than other types of formulations.

Table 8A. Number of Botrytis infection foci on young fruits, leaves and stems of the cucumber plants at 6DPA1, 6DPA2 and 11DPA2 Fl + Fl + TTO Fl + TTO Fl + TTO Treatment UTC TTO Cypro 300 (ZC) 225 (EC) 300 (EC) (SC) 1.3a,b Young fruits 13.3 9.3 7.2 6.1a 2.9a Leaves 3.9 1.9 0.5a 0.8a 0.3a 0.7a 6DPA1 Stems 0.9 1.5 0.7 0.5 0.2 0.2 3.3a,b 2.1a,b Total 18.1 12.7 8.3a 7.5a Young fruits 17.9 10.4 7.3a 6.5a 5.0a 3.1a Leaves 8.0 4.1a 2.9 1.6a 1.0a 1.1a 6DPA2 Stems 7.6 4.5 3.1 1.8a 0.7a 1.0a .2a,b Total 33.6 19.0 13.3a 9.9a 6.6a .6a,b,c Young fruits 32.3 22.3 13.3a 12.2a 6.7a 4.0a,b 5.5a,b 1.7a,b 4.3a,b Leaves 15.4 10.7a 11DPA2 2.2a,b 2.2a,b Stems 14.9 11.2 6.7a 4.4a 22.1a,b Total 24.1a,b 24.1a,b 12.1a,b 62.6 44.2a ap<0.05 compared to UTC of the same part of plant at the same day; bp<0.05 compared to TTO alone of the part of plant at the same day; cp<0.05 compared to fludioxonil + TTO (225 g/L) of the same part of plant at the same day.

Table 8B. Number of Sclerotinia sclerotiorum infection foci in cucumber plants at 6DPA1, 6DPA2 and 11DPA2 Treatment 6DPA1 6DPA2 11DPA2 1.9 0.8 UTC 1.8 0.6 0.6 TTO (222.5 g/L), 1 L/ha 1.7 0 0.3 Fludioxonil (150 g/L) + cyprodinil (225 g/L), SC, 1.5 L/ha 0 Fludioxonil (150 g/L) + TTO (300 g/L), ZC, 1.5 L/ha 0 0 0 Fludioxonil (150 g/L) + TTO (225 g/L), EC, 1.5 L/ha 0 0 0 2838979/IL/19-ORP Treatment 6DPA1 6DPA2 11DPA2 Fludioxonil (150 g/L) + TTO (300 g/L), EC, 1.5 L/ha 0 0 0 Table 8C. Percent cucumber leaves infected with powdery mildew (frequency of disease) and percent coverage of leaves with powdery mildew (severity of diseases) Treatment Frequency Severity 82.0 5.7 UTC 75.0 4.7 TTO (222.5 g/L), 1 L/ha 0.4a,b .0a,b Fludioxonil (150 g/L) + cyprodinil (225 g/L), SC, 1.5 L/ha 1.1a,b Fludioxonil (150 g/L) + TTO (300 g/L), ZC, 1.5 L/ha 40.0a 0.3a,b 14.0a,b Fludioxonil (150 g/L) + TTO (225 g/L), EC, 1.5 L/ha 0.5a,b Fludioxonil (150 g/L) + TTO (300 g/L), EC, 1.5 L/ha 18.0a,b ap<0.05 compared to UTC; bp<0.05 compared to TTO alone.

Example 5: Combination of fludioxonil and TTO in EC formulation reduces sporulation of Penicillium digitatum during storage of orange (fruit) The effect of the combination of fludioxonil and TTO in EC formulation on sporulation of Penicillium digitatum during storage was tested in oranges. As shown in Table 9 and Fig. 4, the inoculation with Penicillium digitatum spores led to high infection rates in untreated control oranges (UTC), such that at 33 days post treatment application (DPA) about 81% of the oranges were rotten and about 50% were infected with spores. Known fungicides, i.e., imazalil (Ima, 500 ppm), pyrimethanil (Pyr, 500 ppm) and Scholar® (500 ppm) comprising fludioxonil (Fl) in SC formulation, were partially effective in reducing rotten fruits. In contrast, treatment of inoculated oranges with 350 or 500 ppm of a composition comprising fludioxonil (Fl, 100 g/L) and TTO (150 g/L) in EC formulation was most effective in maintaining the fruits asymptomatic for any rot or mold, and was specifically active against sporulation of Penicillium digitatum. 2938979/IL/19-ORP Table 9. Percent of asymptomatic fruits, rotten fruits without sporulation and rotten fruits with sporulation of Penicillium digitatum at 11, 22 and 33 DPA Fl 500 Fl + TTO Fl + TTO Treatment UTC Ima Pyr (SC) 350 (EC) 500 (EC) Asymptomatic 68.1 88.8a 96.9a 90.0a 95.6a 97.5a Rotten - no 11DPA .6 10.0 2.5a 4.4a 4.4a 2.5a sporulation Rotten - with 16.3 1.3a 0.6a 5.6a 0.0a 0.0a sporulation Asymptomatic 36.9 58.1 70.0a 72.5a 87.5a,b 87.5a,b Rotten - no 22DPA 21.9 29.4 25.0 15.6 11.3 7.5b sporulation Rotten - with 1.3a,b,c,d 1.9a,d 41.3 12.5a 7.5a 14.4a sporulation 80.0a,b,c,d 76.3a,b,c Asymptomatic 18.8 36.9 51.9a 56.3a Rotten - no 33DPA 31.9 38.8 35.6 25.6 16.3b,c 16.3b,c sporulation Rotten - with 3.8a,d 4.4a,d 49.4 18.1a 15.0a 20.6a sporulation ap<0.05 compared to UTC at the same day; bp<0.05 compared to imazalil at the same day; c p<0.05 compared to pyrimethanil at the same day; dp<0.05 compared to fludioxonil (500 ppm, SC) at the same day.

Example 6: Combination of fludioxonil and TTO in EC formulation is more efficacious in reducing the severity of Botrytis than the combination of fludioxonil and cyprodinil in SC formulation in roses Table 10A shows the mean severity of Botrytis on leaves of rose plants of Vendela variety between the different experimental plot repeats, according to the scale presented in Table 3 above. Each evaluation of severity was carried out about one week after each of the four applications of treatment (designated E1 to E4, respectively). The combination of fludioxonil and TTO in EC co-formulation at various application rates were generally more effective in reducing the severity of Botrytis in roses compared to 3038979/IL/19-ORP fludioxonil and cyprodinil SC formulation, TTO (EC) alone, or combination fludioxonil and cyprodinil (SC) with TTO (EC). After the second application of materials, the combination of fludioxonil and TTO in EC co-formulation at an application rate of 1.08 L/ha showed a statistically significant reduction in Botrytis severity compared to the untreated control (UTC) group.

Table 10A. Degree of Botrytis severity in roses of Vendela variety Treatment E1 E2 E4 E3 2.13 2.00 2.00 UTC 1.92 1.88 1.63 1.50 TTO (222.5 g/L), 1.44 L/ha 1.63 Fludioxonil (250 g/L) + cyprodinil (375 g/L), SC, 1.63 1.56 1.47 1.50 0.432 L/ha Fludioxonil (250 g/L) + cyprodinil (375 g/L), SC, 2.13 1.88 1.75 1.75 0.432 L/ha + TTO (222.5 g/L), 0.53 L/ha Fludioxonil (100 g/L) + TTO (150 g/L), EC, 0.72 1.38 1.44 1.46 1.47 L/ha Fludioxonil (100 g/L) + TTO (150 g/L), EC, 0.90 1.88 1.63 1.58 1.53 L/ha Fludioxonil (100 g/L) + TTO (150 g/L), EC, 1.08 1.25a,b 1.25 1.13a 1.13a,c L/ha ap<0.05 compared to UTC at the same evaluation time; bp<0.05 compared to fludioxonil and cyprodinil (SC) at the same evaluation time; cp<0.05 compared to fludioxonil and cyprodinil (SC) + TTO at the same evaluation time.

A similar experiment was carried out on roses of Touch of Class variery. As shown in Table 10B, the EC co-formulation of fludioxonil and TTO applied at a rate of 0.90 L/ha was most effective in treating Botrytis.

Table 10B. Degree of Botrytis severity in roses of Touch of Class variety Treatment E1 E2 E4 E3 3.13 2.63 2.53 UTC 2.58 1.88a 1.75a 2.03 TTO (222.5 g/L), 1.44 L/ha 2.21 Fludioxonil (250 g/L) + cyprodinil (375 g/L), 1.75a 1.69a 2.03 2.13 SC, 0.432 L/ha Fludioxonil (250 g/L) + cyprodinil (375 g/L), 2.63 2.13 2.25 2.16 SC, 0.432 L/ha + TTO (222.5 g/L), 0.53 L/ha 3138979/IL/19-ORP Treatment E1 E2 E4 E3 Fludioxonil (100 g/L) + TTO (150 g/L), EC, 1.63a 1.63a 1.63a 1.53a 0.72 L/ha Fludioxonil (100 g/L) + TTO (150 g/L), EC, 1.38a,b,c 1.34a,b,c,d 1.50a 1.38a 0.90 L/ha Fludioxonil (100 g/L) + TTO (150 g/L), EC, 1.88a 1.56a 1.67a 1.66a 1.08 L/ha ap<0.05 compared to UTC at the same evaluation time; bp<0.05 compared to TTO at the same evaluation time; cp<0.05 compared to fludioxonil and cyprodinil (SC) at the same evaluation time; dp<0.05 compared to fludioxonil and cyprodinil (SC) + TTO at the same evaluation time.

It should be noted that no phytotoxic effects were observed during the experiments described above.

Example 7: Combination of fludioxonil and TTO in EC formulation reduces Botrytis-induced rot during storage of pomegranates The effect of the combination of fludioxonil and TTO in EC formulation on crown rot caused by Botrytis, as well as rot in the area of a punched hole, was tested in pomegranates during storage. As shown in Table 11A, after 48 days of storage, TTO alone partially prevented rot in the area of the punched hole compared to untreated control (UTC) fruits. By contrast, the present of fludioxonil in the solution, either in SC or EC formulation completely prevented the formation of rot in the hole area.

Furthermore, while TTO alone had no effect on Botrytis-induced crown rot, fludioxonil in SC formulation reduced Botrytis-mediated rot to about 40% of the fruits in storage, and the combination of fludioxonil and TTO in EC co-formulation reduced Botrytis- mediated rot to only about 10% of the fruits in storage (Fig. 5). The synergy factor calculated for the efficacy of the fludioxonil (EC) and TTO combination in reducing crown rot incidence compared to the efficacies of each of the active ingredients alone is 1.71. 3238979/IL/19-ORP Table 11A. Mean number of puncture hole areas with rot # of puncture hole areas with rot Treatment (mean of 5 repeats) 2.2 UTC 1.2 TTO (675 g/cL) 0 Fludioxonil (460 g/cL), SC Fludioxonil (450 g/cL) + TTO (675 g/cL), EC 0 In a similar experiment, pomegranate fruits (unwounded) were stored for 75 days and the rot incidence was qualitatively assessed (rotted or not rotted fruits). As shown in Table 11B, fludioxonil alone in SC formulation had no effect on rot incidence. In addition, TTO alone has only a partial effect on rot incidence. Surprisingly, almost all the fruits treated with the combination of fludioxonil in EC formulation with TTO remained healthy after 75 days. The synergy factor calculated for the efficacy of the fludioxonil (EC) and TTO combination in reducing rot incidence compared to the efficacies of each of the active ingredients alone is 6.32.

Table 11B. Mean number of puncture hole areas with rot Treatment Efficacy (%) Incidence (%) UTC 97.5 0 Fludioxonil (450 ppm), SC 97.5 .4 TTO (675 ppm) 82.5 Fludioxonil (450 ppm) + TTO (675 ppm), EC 2.5 97.4 Example 8: Combination of fludioxonil and TTO in EC formulation reduces infection by Aspergillus niger in grapevine berries The effect of the combination of fludioxonil and TTO in EC formulation on infection by Aspergillus niger was tested in grapevine berries that were wounded in order to increase their susceptibility to infection by the fungus. As shown in Table 12, the EC formulation of fludioxonil and TTO significantly reduced the incidence as well as the severity of the infection in the wounded grapevine berries compared to untreated fruit 3338979/IL/19-ORP after both the first and second applications of the treatments. The EC formulation of fludioxonil and TTO showed comparable results to those obtained by treatment with Switch (Fludioxonil and Cyprodinil in SC formulation).

Furthermore, 8 days after the second application of the treatments shown in Table 12, rot in unwounded berries was observed in 75% of the untreated labeled grape clusters that contained wounded grapes, with an average of 2.5 rotten berries per labeled cluster. The EC formulation of fludioxonil and TTO (at a concentration of 0.2% or more) resulted in decreased secondary infection, namely, decreased number of rotten unwounded berries, with an average number of about one rotten berry per labeled cluster. Furthermore, about 16% to 35% of the labeled treated clusters were actually infected by Aspergillus niger, while in the untreated group about twice as much labeled clusters were infected by the fungus.

The effects of the EC formulation of fludioxonil and TTO on primary and secondary infection by Aspergillus niger in grapevines berries were comparable to those obtained by treatment with Switch (Fludioxonil and Cyprodinil in SC formulation).

Table 12. Incidence and severity of Aspergillus niger infection in grapevine berries 10DPA1 8DPA2 Treatment Incidence Incidence Severity Severity (%) (%) .6 0.5 0.9 UTC 58.6 Fludioxonil (250 g/L) + cyprodinil 2.9a 0.0a 0.2a 19.4a (375 g/L), SC (Switch®, 0.1%) Fludioxonil + TTO, EC (0.1%) 10.0a 0.1 24.4a 0.4a Fludioxonil + TTO, EC (0.2%) 9.2a 0.2 30.1a 0.5a Fludioxonil + TTO, EC (0.4%) 2.8a 0.0a 19.4a 0.2a ap<0.05 compared to UTC at the same evaluation time. 34

Claims (10)

38979/IL/19-ORP CLAIMS

1. A method of preventing or treating a phytopathogenic fungal disease on a plant, comprising applying to the plant, or to a part thereof, a synergistically effective amount of a combination comprising an emulsifiable concentrate (EC) of fludioxonil and Tea Tree Oil (TTO) or one or more components thereof.

2. A method of protecting a harvested plant or a part thereof against a fungal attack, comprising applying to said harvested plant or part thereof an effective amount of a combination of fludioxonil and TTO or one or more component thereof, as EC, to a fungus, a locus of a fungus, a plant or to a plant part susceptible to attack by a fungus.

3. The method according to claim 1 or 2, wherein the combination further comprises one or more additional active ingredient selected from a fungicide, bactericide, plant growth regulator, and plant nutrient.

4. The method according to claim 3, wherein said pesticide is selected from fenhexamid, pydiflumetofen, cyprodinil, and mefenoxam.

5. The method according to claim 1 or 2, wherein said component is selected from: α- thujene; α-pinene; sabinene; β-pinene; myrcene; phellandrene; α-terpinene; limonene; β-phellandrene; p-cymene; 1,8-cineole; γ-terpinene; terpinolene; terpinene-4-ol; terpineol; aromadendrene; ledene (viridiflorene); δ-cadinene; globulol; viridiflorol; 1,8 cineole; α-terpinolene; and terpinen-4-ol.

6. The method according to claim 1, wherein the plant or part thereof is treated prior or after harvest. 3538979/IL/19-ORP

7. A fungicidal composition comprising an emulsifiable concentrate of fludioxonil and TTO or one or more components thereof.

8. The fungicidal composition according to claim 7, further comprising one or more additional active ingredient selected from a fungicide, bactericide, plant growth regulator, and plant nutrient, in a synergistically effective amount.

9. The fungicidal composition according to claim 7, wherein the weight ratio of fludioxonil to TTO or one or more components thereof is between 2:1 and 1:

10. 36