Tobacco alkaloid insect repellents in plants

Abstract

Use of a formulation comprising a compound of formula I for repelling or reducing infestation by hemipteran insects.

Description

[0001] The present disclosure, in its broadest aspect, relates to the use of a formulation comprising a compound of Formula I for repelling Hemipteran insects. Also provided herein is a method for repelling Hemipteran insects.

[0002] Agriculture is a key human activity, providing basic needs such as food, clothing and shelter. Agricultural productivity can be negatively affected by many factors, including insect infestations. Insect infestations interfere with the growth of crops and plants and cause damage to both cultivated and naturally growing plants. Said interference and damage can result in crop failures and failure of plants to mature. It is estimated that insect infestations undermine at least 30% to 40% of global agricultural production, posing a particular threat to the parts of the global economy that rely on agriculture.

[0003] Insects cause two main types of damage to growing crops. First, feeding insects cause direct damage to plants by eating leaves or burrowing into the stems, fruits or roots of plants. Second, indirect damage can occur when the insects themselves cause little or no harm to the plants but can transmit bacterial, viral or fungal infections to the crops.

[0004] Whiteflies are from the order Hemiptera of insects and belong to the family Aleyrodidae. They are considered a major pest of many crops because they can cause considerable damage and yield loss. They feed by sucking sap from the host plant. They are omnivorous, meaning they feed on many different plants, making them a nuisance to most crops.

[0005] The two common species of whitefly are the tobacco whitefly (Bemisia tabaci) and the greenhouse whitefly (Trialeurodes vaporariorum). Currently, the tobacco whitefly is considered to be a complex of about 40 cryptic species, distributed throughout the world, and can feed on more than 900 plant species, including a wide range of vegetables (tomato, eggplant, sweet potato, pumpkin, broccoli, cabbage, etc.), as well as cotton, tobacco and ornamental plants such as poinsettia, garden roses, lantana or lily.

[0006] The entire life cycle of whiteflies lasts 15 to 40 days, depending on the host plant and environmental conditions. For example, the number of eggs laid and the survival rate of immature stages and adults are greatly affected by temperature, with the optimal breeding temperature being about 28–30 degrees Celsius (°C) (Curnutte et al., Annals of the Entomological Society of America, Vol. 107, No. 5).

[0007] Whiteflies lay their eggs on the upper and lower sides of leaves. From the hatched eggs, the first instar nymphs (larvae) break out of their shells and move along the leaves until they find a suitable position to start feeding on sugar-rich phloem sap. The nymphs molt into second, third, and fourth instar nymphs, during which they stay in the same position and continue to feed on plants. The fourth instar is also called the pupal stage or red nymph stage, and the adults will break out of their shells from then on (Sani et al., Insects, 2020, 11, 619).

[0008] Whiteflies cause direct damage to the plant as they feed. Sucking the sap leaves discolored patches on the leaves. Additionally, as they feed on the sap, they release toxic substances into the phloem, which then diffuse throughout the plant. This causes a metabolic imbalance that leads to overall plant weakness, chlorosis, and changes in flowers and fruit.

[0009] In terms of indirect damage, the honeydew secreted by the nymphs will enable fungi such as sooty mold to form on the leaves. This mold acts as a mask and reduces the plant's photosynthetic capacity.

[0010] Finally, the most serious damage that whiteflies can cause to crops is the transmission of viruses. The tobacco whitefly complex is known to transmit more than 200 plant viruses. These viruses include TYLCV (Tomato Yellow Leaf Curl Virus), TYMV (Tomato Yellow Mosaic Virus) or the Cotton Leaf Curl Virus complex.

[0011] One of the main goals of controlling whiteflies is to prevent the crop from becoming infected with the virus. Using protective barriers such as netting and mulch are good options for preventing infestations, but they are only suitable for smaller, less mature crops.

[0012] Using control agents is the most effective way to prevent infestations throughout the life cycle of a crop.

[0013] Most insect pest control agents are synthetic pesticides. Due to the long-term use of such agents, many pests have developed resistance. Resistance is defined as a change in the sensitivity of a pest population to a pesticide, resulting in failure to properly apply the pesticide. Over the past few decades, the extensive use of pesticides to control whiteflies has led to the emergence of populations resistant to various classes of pesticides such as carbamates, organophosphates, and pyrethroids.

[0014] In addition to the difficulty of controlling these pests with current pest control agents, there is increasing evidence that some of these pest control agents are highly toxic and take many years to biodegrade. Therefore, it is desirable to develop new natural pest control agents that have a different mode of action than those known. In addition, natural pest control agents have a low impact on the environment and are generally safer and less harmful to the people who use them.

[0015] Among natural pest control agents, tobacco (Nicotiana genus spp.) plants have been used for centuries in the form of dried leaves, powders, extracts or fumigants. However, due to safety concerns about nicotine, the main alkaloid of tobacco, and the discovery of more specific and more potent synthetic pesticides, people's interest in tobacco has declined. Therefore, there are currently no commercially available tobacco derivatives or products based on nicotine.

[0016] Therefore, there is a clear need to find more plant-derived compounds that are as effective as synthetic pesticides but are more environmentally friendly, safer and have novel modes of action for inclusion in crop pest management programs. It is desirable to provide new compounds and formulations that will act in a manner other than killing insects and, in particular, that have activity as repellents.

[0017] The present disclosure relates to the use of a formulation for repelling Hemiptera insects or reducing Hemiptera insect infestation. The formulation may include a compound of formula I. The compound of formula I may be

[0018]

[0019] in

[0020] R represents hydrogen or C1–C5 alkyl, and

[0021] Represents a single bond or a double bond.

[0022] According to one aspect of the present invention, there is provided a formulation for use in repelling Hemiptera insects. According to another aspect of the present invention, there is provided a formulation for use in reducing Hemiptera insect infestation. The formulation comprises a compound of formula I. The compound of formula I is

[0023]

[0024] in

[0025] R represents hydrogen or C1–C5 alkyl, and

[0026] Represents a single bond or a double bond.

[0027] As used herein, the terms "repellent," "repellent effect," or "repellent effect" mean the effect of forcing insects away from a surface or substance and / or preventing insects from landing, walking, feeding, or laying eggs on a surface or substance.

[0028] As used herein, the term "infestation" means a state in which abnormally high numbers of insects or other species are present on a surface or substance.

[0029] As used herein, the term "insect" means any member of the phylum Arthropoda, which is the largest class.

[0030] As used herein, "Hemiptera" means insects belonging to the taxonomic order commonly known as stink bugs, which have a common arrangement of sucking mouthparts. Thus, as used herein, the term "Hemiptera" is intended to define insects that feed on plants, using their sucking and piercing-sucking mouthparts to extract sap from plants.

[0031] The present invention is further described in conjunction with the accompanying drawings, in which:

[0032] Figure 1 The repellent effects of anatabine and DEET over time periods of 0, 1, 3 and 6 hours are shown.

[0033] Figure 2 The repellent effects of three different concentrations of anatabine over time periods of 3 and 6 hours are shown.

[0034] Figure 3 The repellent effects of anatabine, DEET and methyl benzoate over time periods of 0, 6, 24 and 48 hours are shown.

[0035] Figure 4 Shown is the mortality of whiteflies over time periods of 0, 6, 24 and 48 hours after leaves were treated with anatabine, DEET and methyl benzoate compared to whiteflies on leaves treated with a placebo solution or provided without leaves.

[0036] Figure 5 The repellent effects of anatabine and DEET over time periods of 51, 75 and 123 hours are shown.

[0037] As mentioned above, the present invention relates to the use of a formulation for repelling Hemipteran insects or reducing infestation by Hemipteran insects.

[0038] The use of formulations comprising compounds of formula I has shown an improved repellent effect against insects of the order Hemiptera, in particular whiteflies.

[0039] In particular, the use of formulations comprising anatabine has shown an improved repellent effect on Hemiptera insects, especially whiteflies.

[0040] The repellent effect of the preparation according to the present invention has been shown to be more potent and last longer than commonly used chemicals such as DEET (N, N-diethyl-m-toluamide). This has the advantage of reducing the amount and frequency of the preparation that must be applied to crops or plants. In addition, it is found that the repellent effect is more effective than methyl benzoate, which is a natural plant-derived compound known in the art that is effective against whiteflies (Mostafiz et al., PLoS ONE 13, 2018). Surprisingly, it has been found that the repellent effect of the preparation according to the present invention can be maintained at 100% over a long period of time without causing damage to crops or plants. Crops or plants are not damaged to prove that the efficacy of the preparation according to the present invention is directly attributed to the repellent of Hemiptera insects from their food source rather than destroying their food source. This repellent effect also has the additional advantage of preventing oviposition on leaves. The combined effect of preventing access to food and oviposition leads to a complete repellent effect. This complete repellent effect, which has been proven to last surprisingly long, ultimately leads to the disruption of the life cycle of whiteflies and thus reduces the whitefly population in the area where the preparation is applied.

[0041] Some tobacco plant compounds such as nicotine are known to provide a defense barrier against insect infestation. Nicotine is one of the most abundant compounds in tobacco plants, and it is reported that it is highly toxic to the vast majority of insects that feed on plants. However, whiteflies show significant tolerance to this compound and other tobacco plant compounds, so that they feed on tobacco plants, and compared with other insects, they can eat these compounds more than 40 times. Although tobacco plants are the common food source of hemiptera insects, surprisingly, natural compounds from tobacco plants, particularly anatabine, provide the repellent effect to hemiptera insects, particularly whiteflies. Advantageously, the use of anatabine and the preparation comprising anatabine provides highly effective and efficient natural repellent, while also more environmentally friendly and safer to use.

[0042] In some embodiments of the present invention, there is provided the use of a formulation according to the present invention, wherein the hemiptera insect may be a sap-feeding insect. Sap-feeding insects feed on leaves by ingesting the liquid portion. Sap-feeding insects have haustoria or sucking mouthparts that form a beak for piercing tissue and sucking from phloem tissue buried within the leaves. The nutrient-rich phloem tissue is a unique source of nutrition for sap-feeding insects, which have evolved mechanisms to obtain it. The use of a formulation according to the present invention will prevent hemiptera insects from obtaining this source of nutrition. Advantageously, this will protect the nutrient source from being destroyed by sap-feeding insects and force the sap-feeding insects to look for food elsewhere.

[0043] In yet another embodiment, there is provided a use according to the invention, wherein the Hemiptera insect may be a whitefly.

[0044] In other embodiments of the present invention, the Hemiptera insect may be selected from the family Aleyrodidae, including: European cabbage whitefly, citrus spiny whitefly, sugarcane hole whitefly, cotton whitefly, tobacco whitefly, silverleaf whitefly, citrus whitefly, bayberry whitefly, greenhouse whitefly or ash whitefly.

[0045] In other embodiments of the present invention, the Hemiptera insects can be selected from the family Aphididae, including: Aphis spp., Macrosiphum spp., Anuraphis cardui, Aulacorthum solani, Brachycaudus helichrysi, Brevicoryne brassicae, Ceratovacunalanigera, Chaetosiphon fragaefolii, Chromaphis juglandicola, Cryptomyzus ribis, Dysaphis spp., Myzus spp., Eriosoma spp., Hyalopterus arundinis, Lipaphis erysimi, Macrosiphum spp., Melanaphis spp. sacchari), Metopolophium dirhodum, Monellia costalis, Monelliopsis pecanis, Nasonovia ribisnigri, Pemphigus spp., Phloeomyzus passerinii, Phorodon humuli, Rhopalosiphum spp., Schizaphis graminum, Tinocallis caryaefoliae, Toxoptera spp., Acizzia spp., Acrida turrita, Acrogonia spp., Aeneolamia spp., Agonascena spp., Allocaridara malayensis), Amrasca spp., Aonidiella spp., Aphanostigma piri, Arboridia apicalis, Arytainilla spp., Aspididiella spp., Aspidiotus spp., Atanus spp., Balclutha spp.), Blastopsylla occidentalis, Boreoglycaspis melaleucae, Cacopsylla spp., Calligyponamarginata, Carneocephala fulgida, Cercopidae, Ceroplastes spp., Chionaspis tegalensis, Chloritaonukii, Chrysomphalus ficus, Cicadulina mbila, Coccomytilus halli, Coccus spp., Comstockaspis perniciosa, Cryptonossa, Ctenarytaina spp., Dalbulus spp.), Diaphorina citri, Diaspis spp., Drosicha spp., Dysmicoccus spp., Empoasca spp., Epiacanthus stramineus, Erythroneura spp., Eucalyptolyma spp., Euphylluras spp., Euscelis bilobatus, Ferrisia spp., Fioriniatheae, Geococcus coffeae, Glycaspis spp., Heteropsylla cubana, Heteropsylla spinulosa), Homalodiscacoagulata, Icerya spp., Idiocerus spp., Idioscopus spp., Laodelphax striatellus, Lecanium spp., Lepidosaphes spp., Macrosteles facifrons, Mahanarvaspp.), Metcalfiella spp., Nephotettix spp., Nilaparvata lugens, Oncometopia spp., Orthezia praelonga, Pachypsylla spp., Paratrioza spp., Parlatoria spp., Peregrinus maidis, Phenacoccus spp., Phylloxera spp., Pinnaspisaspidistrae, Planococcus spp., Prosopidopsylla flava, Protopulvinaria pyriformis, Pseudaonidia paeoniae, Pseudaulacaspis ssp.), Pseudococcus spp., Psyllopsis spp., Psylla spp., Pyrilla spp., Quadraspidiotus spp., Quesada gigas, Rastrococcus spp., Saissetia spp., Scaphoideus titanus, Selenaspidus articulatus, Sogata spp., Sogatella furcifera, Sogatodes spp., Stictocephala festina, Tenalaphara malayensis, Tetragonocephela spp., Tomaspis spp.), Trioza spp., Typhlocyba spp., Unaspis spp., Viteus vitifoliae or Zygina spp.

[0046] In certain other embodiments of the present invention, the Hemiptera insects may be selected from the Heteroptera insects, such as, but not exclusively, Anasa tristis, Boisea spp., Blissus spp., Cavelerius spp., Cletus punctiger, Dasynus piperis, Diconocoris hewetti, Dysdercus spp., Eurygasters spp., Leptocorisa spp., Leptoglossus phyllopus, Macropes excavatus, Miridae, Pentastomidae, Piesma spp., Pseudacysta persea, Riptortus spp., Rhopalus spp., Scaptocoris castanea, Scotinophora spp., Stephanitis spp. or Togo hemipterus.

[0047] In some embodiments, there is provided a use according to the present invention, wherein the use may be for repelling Hemipteran insects from crops or plants or reducing infestation of Hemipteran insects on crops or plants.

[0048] In some further embodiments, there is provided a use according to the present invention, wherein the crop or plant may be susceptible to whitefly infestation, and wherein the crop or plant may be a tobacco plant, a cereal plant, a leguminous plant, an oil plant, a cucurbit plant, a fiber plant, a vegetable, a fruit or a flower.

[0049] In certain other embodiments of the present invention, the crop or plant can be selected from wheat, barley, rye, oats, corn, rice, sorghum, triticale, pome fruit, stone fruit, soft fruit, apple, grape, pear, plum, peach, apricot, pistachio, cherry, berry, strawberry, raspberry, blackberry, bell pepper, red pepper, legume, bean, lentil, pea, soybean, oilseed plant, rapeseed, mustard, sunflower, zucchini, cucumber, melon, squash, pumpkin, cotton, flowers, flax, hemp, jute, citrus, orange, mandarin, citron, citrus hybrids, chironja, tangelo, mandarin orange, grapefruit, kumquat, lemon, lime, mandarin orange, bitter orange, sweet orange, pomelo, satsuma, spinach, lettuce, asparagus, cabbage, carrot, onion, tomato, potato, red pepper, broccoli, cauliflower, sweet potato, coffee, fresh flowers, poinsettia, garden roses, lantana, lily, shrub, hardwood tree, evergreen tree, conifer or crape myrtle.

[0050] In some embodiments of the invention, a crop or plant may be present at every stage of its life cycle.

[0051] In a further embodiment, there is provided purposes according to the present invention, wherein the crops or plants can be cultivated in a greenhouse. Warm, humid conditions and abundant food in the greenhouse provide an excellent stable environment for insect development. Although Hemiptera (particularly whiteflies) thrive in temperate regions, it is known that they can easily develop in a greenhouse located in a climate zone where conditions cannot usually sustain their life cycle. Advantageously, it has been found that formulations according to the present invention can be used in a greenhouse. In fact, although the synthetic pesticides used in enclosed spaces have serious harmful consequences to staff during and after application, the use of formulations according to the present invention provides a natural alternative that is safer for the user and more sustainable for the environment.

[0052] As described above, there is provided a formulation for use in repelling Hemiptera insects or reducing Hemiptera insect infestation, wherein the formulation comprises a compound of formula I

[0053]

[0054] in

[0055] R represents hydrogen or C1–C5 alkyl, and

[0056] Represents a single bond or a double bond.

[0057] In some embodiments of the invention, in the compounds of formula I, R may represent hydrogen.

[0058] In some other embodiments of the present invention, in the compound of formula I, Can represent a double bond.

[0059] In certain embodiments of the present invention, the compound of Formula I may be anatabine.

[0060] Anatabine can exist in two enantiomeric forms: S-(-) anatabine and R-(+) anatabine. Therefore, anatabine can be present in the formulation of the present invention as any of S-(-) anatabine, R-(+) anatabine, a mixture of S-(-) anatabine and R-(+) anatabine, or a racemate of S-(-) anatabine and R-(+) anatabine. The amount of R-(+) anatabine and S-(-) anatabine can exist in any enantiomeric ratio, i.e., any enantiomeric excess (ee) of R-(+) anatabine or S-(-) anatabine.

[0061] As used herein, the term "enantiomeric ratio" of R-(+) anatabine and S-(-) anatabine is intended to refer to the weight ratio of R-(+) anatabine to S-(-) anatabine.

[0062] According to the use of the invention, the formulation may comprise an enantiomeric ratio of R-(+) anatabine to S-(-) anatabine of 10:90, 20:80, 30:70, 40:60, 60:40, 70:30, 80:20 or 90:10.

[0063] The enantiomeric ratio of R-(+) anatabine to S-(-) anatabine can be calculated using the following formula:

[0064] Ratio of R-(+) anatabine to S-(-) anatabine = weight of R-(+) anatabine / weight of S-(-) anatabine

[0065] If a solvate of R-(+) anatabine and / or S-(-) anatabine is used, the solvent may be ignored in calculating the ratio.

[0066] Enantiomeric ratios can be determined in a variety of ways including, but not limited to, chromatography, polarimetry of polarized light rotation, nuclear magnetic resonance spectroscopy, use of chiral compounds such as Mosher's acid derivatization of the compound followed by chromatography or nuclear magnetic resonance spectroscopy, high pressure liquid chromatography (HPLC), or direct fractional crystallization of the racemate.

[0067] According to some embodiments of the present invention, the compound of formula I can be extracted from tobacco or can be chemically synthesized. The compound of formula I can be extracted from any type of tobacco plant. As used herein, the term "extraction" or "extracted" is intended to refer to a substance obtained from a natural source or a method for obtaining a substance from a natural source. Alternatively, the compound of formula I can be chemically synthesized to make the compound identical to a naturally occurring compound.

[0068] In some embodiments, a use according to the invention is provided, wherein the formulation may comprise a concentrate of a compound of formula I.

[0069] According to the use of the present invention, the preparation may contain an amount of at least 99% by weight, at least 95% by weight, at least 90% by weight, at least 85% by weight, at least 80% by weight, at least 75% by weight, at least 70% by weight, at least 65% by weight, at least 60% by weight, at least 55% by weight, at least 50% by weight, at least 45% by weight, at least 40% by weight, at least 35% by weight, at least 30% by weight, at least 25% by weight, at least 20% by weight, at least 15% by weight, at least 10% by weight, at least 5% by weight, at least 2.5% by weight, at least 2.4% by weight, at least 2.3% by weight of the preparation. %, at least 0.9 wt %, at least 0.8 wt %, at least 0.7 wt %, at least 0.6 wt %, at least 0.5 wt %, at least 0.4 wt %, at least 0.3 wt %, at least 0.2 wt %, or at least 0.1 wt % of the compound of Formula I.

[0070] According to the use of the present invention, the preparation may contain an amount of no more than 99% by weight of the preparation, no more than 95% by weight, no more than 90% by weight, no more than 85% by weight, no more than 80% by weight, no more than 75% by weight, no more than 70% by weight, no more than 65% by weight, no more than 60% by weight, no more than 55% by weight, no more than 50% by weight, no more than 45% by weight, no more than 40% by weight, no more than 35% by weight, no more than 30% by weight, no more than 25% by weight, no more than 20% by weight, no more than 15% by weight, no more than 10% by weight, no more than 5% by weight, no more than 2.5% by weight, no more than 2.4% by weight, no more than 2.3% by weight. %, no more than 2.2 wt %, no more than 2.1 wt %, no more than 2 wt %, no more than 1.9 wt %, no more than 1.8 wt %, no more than 1.7 wt %, no more than 1.6 wt %, no more than 1.5 wt %, no more than 1.4 wt %, no more than 1.3 wt %, no more than 1.2 wt %, no more than 1.1 wt %, no more than 1 wt %, no more than 0.9 wt %, no more than 0.8 wt %, no more than 0.7 wt %, no more than 0.6 wt %, no more than 0.5 wt %, no more than 0.4 wt %, no more than 0.3 wt %, no more than 0.2 wt % or no more than 0.1 wt % of the compound of Formula I.

[0071] According to the use of the present invention, the preparation may contain about 99% by weight, about 95% by weight, about 90% by weight, about 85% by weight, about 80% by weight, about 75% by weight, about 70% by weight, about 65% by weight, about 60% by weight, about 55% by weight, about 50% by weight, about 45% by weight, about 40% by weight, about 35% by weight, about 30% by weight, about 25% by weight, about 20% by weight, about 15% by weight, about 10% by weight, about 5% by weight, about 2.5% by weight, about 2.4% by weight, about 2.3% by weight of the preparation. %, about 2.2 wt %, about 2.1 wt %, about 2 wt %, about 1.9 wt %, about 1.8 wt %, about 1.7 wt %, about 1.6 wt %, about 1.5 wt %, about 1.4 wt %, about 1.3 wt %, about 1.2 wt %, about 1.1 wt %, about 1 wt %, about 0.9 wt %, about 0.8 wt %, about 0.7 wt %, about 0.6 wt %, about 0.5 wt %, about 0.4 wt %, about 0.3 wt %, about 0.2 wt % or about 0.1 wt % of the compound of Formula I.

[0072] According to the use of the present invention, the preparation may contain an amount selected from 1% to 99% by weight, 2.5% to 95% by weight, 5% to 90% by weight, 10% to 85% by weight, 15% to 80% by weight, 20% to 75% by weight, 25% to 70% by weight, 30% to 65% by weight, 35% to 60% by weight, 40% to 55% by weight, 45% to 50% by weight of the compound of formula I. The amount of the compound of formula I may have any range from a given endpoint.

[0073] According to a further use of the present invention, the preparation may comprise an amount selected from 0.1 wt % to 5 wt %, 0.15 wt % to 4.9 wt %, 0.2 wt % to 4.8 wt %, 0.25 wt % to 4.7 wt %, 0.3 wt % to 4.6 wt %, 0.35 wt % to 4.5 wt %, 0.4 wt % to 4.4 wt %, 0.45 wt % to 4.3 wt %, 0.5 wt % to 4.2 wt %, 0.55 wt % to 4.1 wt %, 0.6 wt % to 4.0 wt %, 0.65 wt % to 3.9 wt %, 0.7 wt % to 3.8 wt %, 0.75 wt % to 3.7 wt %, 0.8 % to 2.6 wt %, 1.45 wt % to 2.5 wt %, 1.5 wt % to 2.4 wt %, 1.6 wt % to 2.3 wt %, 1.7 wt % to 2.2 wt %, 1.8 wt % to 2.1 wt % of the compound of Formula I. The amount of the compound of Formula I can have any range from the given endpoints. According to some further embodiments of the invention, the formulation may comprise a compound of formula I in an amount of 5%, 2%, 1% or 0.5% by weight of the formulation.

[0074] In certain embodiments according to the present invention, preparation can be water-soluble concentrate, emulsifiable concentrate, emulsion, microemulsion, oil-based suspension concentrate, flowable suspension, water-dispersible granules, water-soluble granules, wettable powder, water-soluble powder, granule, encapsulated granule, fine particle, macrogranule, aqueous suspoemulsion, microencapsulated suspension or microparticle. Advantageously, depending on the type of interaction required between preparation and crops or plants, preparation of the present invention can be provided in different forms. In fact, providing preparation of the present invention in different forms enables to select the most suitable preparation for intended use according to target crops or plants to ensure optimal biological efficiency.

[0075] According to the present invention, there is provided a use of a formulation, wherein the formulation is dilutable in water. The formulation of the present invention can be diluted in water to produce a finished product. The formulation of the present invention can be provided as a pre-diluted ready-to-use formulation or as a concentrate intended to be diluted before application. Advantageously, the pre-diluted ready-to-use formulation can be applied to plants or small crops. More advantageously, the formulation according to the present invention can be diluted just before application, thereby allowing the user to adjust the dilution ratio of the formulation to be deployed on the crops or plants according to the intended purpose of application.

[0076] According to the present invention, there is provided a use of a formulation, wherein the dilution ratio of the formulation to water can be selected from 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:600, 1:700, 1:800, 1:900 or 1:1000.

[0077] According to the present invention, there is provided the use of a diluted formulation, wherein the diluted formulation comprises a compound of formula I in an amount of at least 0.1 wt %, at least 0.2 wt %, at least 0.3 wt %, at least 0.4 wt %, at least 0.5 wt %, at least 0.6 wt %, at least 0.7 wt %, at least 0.8 wt %, at least 0.9 wt %, at least 1 wt %, at least 1.1 wt %, at least 1.2 wt %, at least 1.3 wt %, at least 1.4 wt %, at least 1.5 wt %, at least 1.6 wt %, at least 1.7 wt % or at least 1.8 wt % of the diluted formulation.

[0078] According to the present invention, there is provided the use of a diluted formulation, wherein the diluted formulation comprises a compound of formula I in an amount not exceeding 20 wt %, not exceeding 19 wt %, not exceeding 18 wt %, not exceeding 17 wt %, not exceeding 16 wt %, not exceeding 15 wt %, not exceeding 14 wt %, not exceeding 13 wt %, not exceeding 12 wt %, not exceeding 11 wt %, not exceeding 10 wt %, not exceeding 9 wt %, not exceeding 8 wt %, not exceeding 7 wt %, not exceeding 6 wt %, not exceeding 5 wt %, not exceeding 4 wt % or not exceeding 3 wt % of the diluted formulation.

[0079] According to the present invention, there is provided the use of a diluted formulation, wherein the diluted formulation comprises a compound of formula I in an amount of about 0.1% to about 20%, about 0.2% to about 19%, about 0.3% to about 18%, about 0.4% to about 17%, about 0.5% to about 16%, about 0.6% to about 15%, about 0.7% to about 14%, about 0.8% to about 13%, about 0.9% to about 12%, about 1% to about 11%, about 1.1% to about 10%, about 1.2% to about 9%, about 1.3% to about 8%, about 1.4% to about 7%, about 1.5% to about 6%, about 1.6% to about 5%, about 1.7% to about 4%, or about 1.8% to about 3% by weight of the diluted formulation.

[0080] In another embodiment of the present invention, there is provided the use of a diluted formulation, wherein the diluted formulation comprises a compound of formula I in an amount of less than about 10% by weight of the diluted formulation. In yet another embodiment of the present invention, there is provided the use of a diluted formulation, wherein the diluted formulation comprises a compound of formula I in an amount of about 5% by weight of the diluted formulation. It has been surprisingly shown that at concentrations of less than 10%, the formulation does not cause damage to crops or plants.

[0081] In a further embodiment of the present invention, a use of a formulation is provided, wherein the formulation can be applied to crops or plants by coating, spraying, soaking or irrigation. Advantageously, the selection of the application method can be carried out for different families of Hemiptera insects. For example, a method of displacing very little air by deposition closer to the leaves may be more suitable for flying insects, such as whiteflies.

[0082] In some embodiments of the present invention, a use of a formulation is provided, wherein the formulation can be applied to a crop or plant 1, 2, 3, 4, 5 or 6 times within a 24 hour period.

[0083] According to the present invention, a kind of purposes of a preparation is provided, wherein the preparation can be applied to crops or plants at a frequency of every day, every two days, every five days, every week, every two weeks (14 days) or every month. Any application frequency can be combined with the number of applications in the aforementioned 24-hour time period. According to the present invention, a kind of purposes of a preparation is provided, wherein the preparation can be applied routinely, on schedule or after evaluation after each application.

[0084] According to the present invention, there is provided a use of a formulation, wherein the formulation can be applied at the earliest detection of insects or after infestation is manifested. Alternatively, the formulation according to the present invention can be used prophylactically before the presence of insects is detected or before infestation is manifested.

[0085] According to the present invention, there is provided a use of a formulation, wherein the formulation can be applied during the cooler time period of the day, so it can be applied in the morning, evening or at night. Advantageously, it has been found that applying the formulation according to the present invention during the cooler time period of the day will reduce the chance of evaporation of the formulation, so that an effective concentration is maintained when the formulation is applied to crops or plants.

[0086] According to the present invention, there is provided a use of a formulation, wherein the formulation can be applied to crops or plants for at least 65 days, at least 60 days, at least 55 days, at least 50 days, at least 45 days, or at least 40 days, or at least 35 days, or at least 30 days, or at least 25 days or at least 20 days.

[0087] According to the present invention, there is provided a use of a formulation, wherein the formulation can be applied to crops or plants for no more than 65 days, no more than 60 days, no more than 55 days, no more than 50 days, no more than 45 days or no more than 40 days.

[0088] Advantageously, the formulation according to the invention can be applied at a determined frequency within a period of time shorter than, equal to or longer than the duration of the life cycle of insects of the order Hemiptera. More advantageously, the formulation according to the invention can be applied at a determined frequency within a period of time equal to the duration of the life cycle of whiteflies. Thus, the formulation according to the invention can be applied for a period of at least 40 days.

[0089] According to another aspect of the present invention, there is provided a method for repelling Hemiptera insects, the method comprising: preparing a concentrated formulation comprising a compound of formula I (preferably anatabine); diluting the formulation in water to a predetermined dilution ratio or concentration; applying the diluted formulation to crops or plants, preferably by spraying.

[0090] The present invention is also described by but not limited to the following examples:

[0091] Example 1: Comparative study of the repellent effects of anatabine and DEET.

[0092] The repellent effect of anatabine on adult whiteflies was evaluated in a comparative study comparing the repellent effect of anatabine with that of the standard synthetic pesticide DEET and a placebo control (in which the leaves were treated with the solvent alone).

[0093] For research purposes, tube test assays were conducted using anatabine solutions and DEET solutions prepared according to Table 1.

[0094] Table 1: Anatabine solutions and DEET solutions.

[0095]

[0096] For the purpose of the study, the leaf disk was immersed in the prepared solution for a total of 30 seconds and allowed to dry at room temperature for 10 minutes. Subsequently, the leaf disk was placed at the bottom of the glass tube and at least 20 adult whiteflies were introduced into the tube. The repellent effects of both anatabine and DEET were measured after different time points: 1 hour, 3 hours and 6 hours. The repellent effects of both anatabine and DEET are shown in Table 2 and the number of whiteflies landing on the leaves is counted by comparing with the placebo control, using the Abbott formula to calculate:

[0097] Repellent effect=1-(number of whiteflies on "placebo control" leaves / number of whiteflies on "treated" leaves)*100.

[0098] The placebo control defined the baseline, based on which the repellent effect was then calculated according to the above formula.

[0099] Table 2: Repellent effect of anatabine and DEET.

[0100]

[0101]

[0102] The comparative study found that anatabine's repellent efficacy was over 80% throughout the 6-hour evaluation period, whereas DEET's repellent efficacy dropped by nearly 40% (to around 50%) after 6 hours ( Figure 1 ).

[0103] Example 2: Study on the relationship between the repellent effect of anatabine and the concentration of anatabine.

[0104] The repellent effect of anatabine against adult whiteflies was evaluated in a study aimed at optimizing the concentration of anatabine.

[0105] For research purposes, tube test assays were performed using anatabine solutions of three different concentrations prepared according to Table 3.

[0106] The placebo control defined the baseline, based on which the repellent effect was then calculated.

[0107] Table 3: Three different concentrations of anatabine solutions.

[0108]

[0109] For the purpose of research, the leaf disk was immersed in the prepared solution for a total of 30 seconds and allowed to dry at room temperature for 10 minutes. Subsequently, the leaf disk was placed at the bottom of the glass tube, and at least 20 adult whiteflies were introduced into the tube. At different time points: the repellent effect of three different concentrations of anatabine was measured after 1 hour, 3 hours, and 6 hours. The repellent effect of different concentrations of anatabine is shown in Table 4.

[0110] Table 4: Repellent effect of three different concentrations of anatabine.

[0111]

[0112] Regardless of the concentration of anatabine, the repellent effect of anatabine remained at 100% ( Figure 2 ), however, it was also observed that anatabine at concentrations of 5% or less did not cause damage to the leaves.

[0113] Example 3: Comparative study of the repellent effects of anatabine, DEET and methyl benzoate.

[0114] The repellent effect of anatabine on adult whiteflies was evaluated in a comparative study designed to compare the repellent effects of anatabine with those of DEET and methyl benzoate, a plant-derived organic compound reported to have repellent and insecticidal properties, particularly against whiteflies.

[0115] For the purpose of the study, tube test assays were performed using anatabine solutions, DEET solutions, and methyl benzoate solutions prepared according to Table 5. A concentration of 5% was selected based on the results of Example 2. The repellent effects of anatabine, DEET, and methyl benzoate were evaluated relative to a placebo control. In the placebo control, leaves were treated with solvent only. The placebo control defined a baseline, based on which the repellent effects were then calculated.

[0116] Table 5: Anatabine solutions and DEET solutions.

[0117]

[0118] For the purpose of research, the blade disk was immersed in the prepared solution and kept for a total of 30 seconds and allowed to dry at room temperature for 10 minutes. Subsequently, the blade disk was placed at the bottom of the glass tube, and at least 20 adult whiteflies were introduced into the tube. At different time points: the repellent effect of anatabine, DEET and methyl benzoate was measured after 6 hours, 24 hours and 48 hours. The repellent effect of anatabine, DEET and methyl benzoate is shown in Table 6.

[0119] Table 6: Repellent effect of anatabine, DEET and methyl benzoate.

[0120]

[0121] Anatabine maintained 100% repellency throughout the 48-hour evaluation period. In contrast, methyl benzoate's repellency dropped by nearly 25% by the end of the 48-hour evaluation period ( Figure 3 ), the repellent effect of DEET dropped to 93%.

[0122] In addition, after a period of 48 hours, the presence and amount of eggs on the leaves were visually evaluated and the results are shown in Table 7.

[0123] Table 7: Presence of insect eggs on leaves after treatment with anatabine, DEET, methyl benzoate and placebo.

[0124]

[0125] Regardless of the repellent used, no eggs were observed on leaves treated with the repellent, but they were clearly visible on untreated leaves.

[0126] This study also evaluated whitefly mortality over the duration of the evaluation period. The results are provided in Table 8.

[0127] Table 8: Whitefly mortality study comparing leaves treated with anatabine, DEET and methyl benzoate.

[0128]

[0129] When leaves were treated with the placebo, whitefly mortality remained below 10% for the duration of the evaluation period. In contrast, when leaves were treated with the repellent to prevent whiteflies from feeding, whiteflies began to die within a day. At the end of the 48-hour evaluation period, whitefly mortality had reached about 80% for the treated leaves, as shown in Table 1. Figure 4 as shown in .

[0130] To better understand mortality, whitefly populations without access to leaves during the 48-hour evaluation period were also observed. Whiteflies without access to a food source began to die after just 24 hours, reaching 60% mortality after 48 hours.

[0131] In addition, DEET and methyl benzoate were observed to cause severe damage to the leaves during the 48-hour evaluation period. In contrast, anatabine did not cause any damage during the evaluation period, as shown in Table 9.

[0132] Table 9: Damage to leaves treated with anatabine, DEET and methyl benzoate.

[0133]

[0134]

[0135] Example 4: Comparative study of the repellent effects of anatabine and DEET.

[0136] The repellent effect of anatabine on adult whiteflies was evaluated in a further comparative study, in which additional whiteflies and freshly treated leaves were introduced at set evaluation time points. For the purpose of the study, tube test assays were performed using anatabine solutions at a concentration of 5% and DEET solutions as well as placebo solutions, as shown in Table 10. The placebo control defined the baseline, based on which the repellent effect was then calculated.

[0137] Table 10: Anatabine solutions and DEET solutions.

[0138]

[0139] Whiteflies were introduced into the tubes at infestation time points: 48, 72 and 120 hours, and the repellent effect was measured 3 hours later at evaluation time points: 51, 75 and 123 hours. The results are shown in Table 11.

[0140] Table 11: Repellent effect of anatabine and DEET

[0141]

[0142] Anatabine was shown to have 100% efficacy 48 hours after treatment, followed by over 80% repellency for the remaining 5 days of the evaluation period (up to 123 hours). In contrast, DEET's efficacy dropped by nearly 50% after the first 48 hours and remained below 50% for the remaining 5 days of the evaluation period (up to 123 hours). Figure 5 ).

[0143] The presence of eggs was visually monitored 24 hours after each infestation time point. The results are provided in Table 12.

[0144] Table 12: Presence of insect eggs on leaves after treatment with anatabine, DEET and placebo.

[0145]

[0146] The number of eggs observed on leaves treated with anatabine remained low up to the 6-day (144-hour) evaluation period. In contrast, eggs were observed on leaves treated with DEET after only 3 days (72 hours), and the number of eggs was close to that on leaves treated with placebo at the end of the 6-day (144-hour) evaluation period.

[0147] The invention is defined in the claims, however, a non-exhaustive list of non-limiting aspects is provided below.Any one or more features of these aspects may be combined with any one or more features of another aspect described herein.

[0148] Aspect 1: Use of a formulation for repelling Hemiptera insects or reducing the infestation of Hemiptera insects, the formulation comprising a compound of formula I,

[0149]

[0150] in

[0151] R represents hydrogen or C1–C5 alkyl, and

[0152] Represents a single bond or a double bond.

[0153] Aspect 2: The use according to aspect 1, wherein the Hemiptera insect is a sap-sucking insect.

[0154] Aspect 3: The use according to aspect 1 or aspect 2, wherein the Hemiptera insect is a sap-sucking insect that feeds on plants.

[0155] Aspect 4: The use according to aspect 1 to aspect 3, wherein the Hemiptera insect is a sap-sucking insect that sucks sap from a plant.

[0156] Aspect 5: The use according to aspect 1 to aspect 4, wherein the Hemiptera insect is whitefly.

[0157] Aspect 6: The use according to aspects 1 to 5, for repelling Hemiptera insects from crops or plants or reducing the infestation of Hemiptera insects on crops or plants.

[0158] Aspect 7: The use according to aspects 1 to 6, for repelling whiteflies from crops or plants or reducing the infestation of whiteflies on crops or plants.

[0159] Aspect 8: The use according to aspect 6 or aspect 7, wherein the crop or plant is susceptible to whitefly infestation.

[0160] Aspect 9: The use according to aspect 6 to aspect 8, wherein the crop or plant is selected from tobacco plants, cereal plants, leguminous plants, oil plants, cucurbitaceous plants, fiber plants, vegetables, fruits, flowers and ornamental plants.

[0161] Aspect 10: The use according to aspect 6 to aspect 9, wherein the crop or plant is a tobacco plant.

[0162] Aspect 11: The use according to aspect 6 to aspect 10, wherein the crop or plant is cultivated in a greenhouse.

[0163] Aspect 12: The use according to aspects 1 to 11, wherein R in the compound of formula I represents hydrogen.

[0164] Aspect 13: The use according to aspects 1 to 12, wherein the compound of formula I Represents a double bond.

[0165] Aspect 14: The use according to Aspect 1 to Aspect 13, wherein the compound of Formula I is selected from anatabine, S-(-) anatabine, R-(+) anatabine, a mixture of S-(-) anatabine and R-(+) anatabine, or a racemate of S-(-) anatabine and R-(+) anatabine.

[0166] Aspect 15: The use according to aspects 1 to 14, wherein the Hemiptera insect is a sap-sucking insect that feeds on tobacco plants, and wherein the compound of formula I is anatabine.

[0167] Aspect 16: The use according to aspects 1 to 15, wherein the Hemiptera insect is whitefly, and wherein the compound of formula I is anatabine.

[0168] Aspect 17: The use according to Aspect 1 to Aspect 16, wherein the compound of Formula I is extracted from tobacco or chemically synthesized.

[0169] Aspect 18: The use according to Aspect 1 to Aspect 17, wherein the preparation comprises a concentrate of the compound of Formula I.

[0170] Aspect 19: The use according to Aspect 1 to Aspect 18, wherein the preparation contains the compound of Formula I in an amount of at least 99% by weight, at least 95% by weight, at least 90% by weight, at least 85% by weight, at least 80% by weight, at least 75% by weight, at least 70% by weight, at least 65% by weight, at least 60% by weight, at least 55% by weight, at least 50% by weight, at least 45% by weight, at least 40% by weight, at least 35% by weight, at least 30% by weight, at least 25% by weight, at least 20% by weight, at least 15% by weight, at least 10% by weight, at least 5% by weight, at least 2.5% by weight or at least 1% by weight of the preparation.

[0171] Aspect 20: The use according to Aspect 1 to Aspect 19, wherein the formulation is a water-soluble concentrate, an emulsifiable concentrate, a microemulsion, an oil-based suspension, a flowable suspension, water-dispersible granules, water-soluble granules, a wettable powder, a water-soluble powder, encapsulated granules, large granules, an aqueous suspoemulsion, a microencapsulated suspension or microparticles.

[0172] Aspect 21: The use according to aspect 1 to aspect 20, wherein the preparation is diluted in water.

[0173] Aspect 22: The use according to Aspect 21, wherein the dilution ratio of the formulation to water is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:600, 1:700, 1:800, 1:900 or 1:1000.

[0174] Aspect 23: The use according to Aspect 21 or 22, wherein the amount of the compound of Formula I is 0.1% to 20% by weight, 0.2% to 19% by weight, 0.3% to 18% by weight, 0.4% to 17% by weight, 0.5% to 16% by weight, 0.6% to 15% by weight, 0.7% to 14% by weight, 0.8% to 13% by weight, 0.9% to 12% by weight, 1% to 11% by weight, 1.1% to 10% by weight, 1.2% to 9% by weight, 1.3% to 8% by weight, 1.4% to 7% by weight, 1.5% to 6% by weight, 1.6% to 5% by weight, 1.7% to 4% by weight, or 1.8% to 3% by weight of the diluted formulation.

[0175] Aspect 24: The use according to aspect 1 to aspect 23, wherein the preparation is applied to crops or plants by coating, spraying, soaking or irrigation.

[0176] Aspect 25: The use according to Aspect 24, wherein the formulation is applied to the crop or plant by spraying.

[0177] Aspect 26: The use according to aspects 1 to 25, wherein the formulation is applied to the crop or plant 1, 2, 3, 4, 5 or 6 times within a 24-hour period.

[0178] Aspect 27: The use according to aspect 1 to aspect 26, wherein the formulation is applied to crops or plants at a frequency of daily, every two days, every five days, weekly, every two weeks or monthly.

[0179] Aspect 28: The use according to aspects 26 and 27, wherein the formulation is applied to the crop or plant once every five days.

[0180] Aspect 29: The use according to Aspect 1 to Aspect 28, wherein the formulation is applied to the crop or plant for at least 65 days, at least 60 days, at least 55 days, at least 50 days, at least 45 days, or at least 40 days, or at least 35 days, or at least 30 days, or at least 25 days or at least 20 days.

[0181] Aspect 30: The use according to aspect 29, wherein the formulation is applied to the crop or plant for at least 40 days.

[0182] Aspect 31: Use of a compound of formula I for repelling Hemiptera insects or reducing infestation by Hemiptera insects,

[0183]

[0184] in

[0185] R represents hydrogen or C1–C5 alkyl, and

[0186] Represents a single bond or a double bond.

[0187] Aspect 32: The use according to Aspect 31, wherein the compound of Formula I is anatabine, S-(-) anatabine, R-(+) anatabine, a mixture of S-(-) anatabine and R-(+) anatabine, or a racemate of S-(-) anatabine and R-(+) anatabine.

[0188] Aspect 33: The use according to Aspect 31 or 32, wherein the Hemiptera insect is whitefly.

[0189] Aspect 34: A method for repelling Hemiptera insects, the method comprising:

[0190] a. preparing a concentrated formulation comprising a compound of formula I, preferably anatabine;

[0191]

[0192] in

[0193] R represents hydrogen or C1–C5 alkyl, and

[0194] Represents a single bond or a double bond

[0195] b. diluting the formulation in water to a predetermined dilution ratio or concentration;

[0196] c. Applying the diluted formulation to crops or plants, preferably by spraying.

[0197] Aspect 35: The method according to aspect 34, wherein the Hemiptera insect is whitefly.

[0198] For the purpose of this specification and the appended claims, unless otherwise stated, all numbers representing amounts, quantities, percentages, ratios, etc. should be understood to be modified by the term "about" in all cases. In addition, all ranges include the maximum and minimum points disclosed, and include any intermediate ranges therein that may or may not be specifically listed herein. Therefore, in this context, the number A is understood to be A±5%A. In this context, the number A may be regarded as including a numerical value within the general standard error for the measurement of the attribute modified by the number A. In certain cases used in the appended claims, the number A may deviate from the percentages listed above, provided that the amount of A deviation does not substantially affect the basic and novel features of the invention claimed for protection. In addition, all ranges include the maximum and minimum points disclosed, and include any intermediate ranges therein that may or may not be specifically listed herein.

Claims

1. A preparation for repelling Hemiptera insects or reducing the infestation of Hemiptera insects, the preparation comprising a compound of formula I, in R represents hydrogen or C1–C5 alkyl, and Represents a single bond or a double bond.

2. The use according to claim 1, wherein the Hemiptera insect is a sap-sucking insect.

3. The use according to claim 1 or 2, wherein the Hemiptera insect is whitefly.

4. Use according to any preceding claim for repelling hemipteran insects from crops or plants or reducing infestation of crops or plants by hemipteran insects.

5. The use according to claim 4, wherein the crop or plant is susceptible to whitefly infestation.

6. The use according to claim 4 or 5, wherein the crop or plant is selected from tobacco plants, cereals, leguminous plants, oil plants, cucurbitaceous plants, fiber plants, vegetables, fruits, flowers and ornamental plants.

7. Use according to claims 4 to 6, wherein the crop or plant is a tobacco plant.

8. The use according to claims 4 to 7, wherein the crops or plants are cultivated in a greenhouse.

9. The use according to any preceding claim, wherein the compound of formula I is selected from anatabine, S-(-) anatabine, R-(+) anatabine, a mixture of S-(-) anatabine and R-(+) anatabine, or a racemate of S-(-) anatabine and R-(+) anatabine.

10. Use according to any preceding claim, wherein the formulation comprises a concentrate of the compound of formula I.

11. The use according to any preceding claim, wherein the formulation comprises the compound of formula I in an amount of at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, at least 5%, at least 2.5% or at least 1% by weight of the formulation.

12. Use according to any preceding claim, wherein the formulation is diluted in water.

13. The use according to claim 12, wherein the amount of the compound of formula I is 0.1% to 20%, 0.2% to 19%, 0.3% to 18%, 0.4% to 17%, 0.5% to 16%, 0.6% to 15%, 0.7% to 14%, 0.8% to 13%, 0.9% to 12%, 1% to 11%, 1.1% to 10%, 1.2% to 9%, 1.3% to 8%, 1.4% to 7%, 1.5% to 6%, 1.6% to 5%, 1.7% to 4%, or 1.8% to 3% of the diluted formulation.

14. Use according to any preceding claim, wherein the formulation is applied to crops or plants by coating, spraying, soaking or irrigation.

15. A method for repelling Hemiptera insects, the method comprising: a. preparing a concentrated formulation comprising a compound of formula I, preferably anatabine; in R represents hydrogen or C1–C5 alkyl, and Represents a single bond or a double bond b. diluting the formulation in water to a predetermined dilution ratio or concentration; c. Applying the diluted formulation to crops or plants, preferably by spraying.

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