AU2020410966A1 - Use of limonene as an insecticide - Google Patents

Abstract

A method for curative treatment of Aleyrodidae, or white fly, on vegetables or tomatoes under cultivation, the method comprising the steps of: i. providing a diluted liquid composition, comprising limonene, in which the limonene is emulsified in an aqueous liquid; and ii. applying the diluted liquid composition as a foliar spray onto vegetables or tomatoes, the vegetables or tomatoes being infested with Aleyrodidae, or white fly, the diluted liquid composition being applied to provide a dose rate of from 1200 to 1500 grams of the limonene active ingredient per hectare of the vegetables or tomatoes. The present invention also relates to the use of limonene for the insecticidal treatment of Aleyrodidae, or white fly, without exhibiting toxicity to the whitefly predators Chrysoperla carnea and Hippodamia (= Adonia) variegata, on vegetables or tomatoes under cultivation.

Description

Use of Limonene as an Insecticide

TECHNICAL FIELD The invention pertains to the technical field of agrochemicals derived from a natural source. More in particular it pertains to the use of limonene as an insecticide and to a method of treating crops using limonene as an insecticide.

BACKGROUND

There is much interest in the replacement of synthetic insecticide-active ingredients by ingredients sourced from nature, which has created a demand for sprayable liquid formulations containing nature-derived active ingredients as active substances, and preferably the nature-derived active ingredient(s) is/are the only active ingredient(s) present in the formulation.

A candidate active ingredient for use in sprayable liquid formulations for use as an insecticide is limonene, in which the limonene is emulsified in an aqueous liquid. Limonene is obtainable from an extract of citrus fruit peel. Limonene is present in high concentrations in citrus oil, lemon oil, orange oil, lime oil, grapefruit oil and tangerine oil. In particular, orange oil has a high content of limonene, the limonene typically comprising at least 90% by weight of the orange oil.

The Applicant’s earlier WO2016062718 A1 discloses the use of limonene as an as acaricide or insecticide, and a method of treating plants and crops with a limonene comprising composition. Although WO2016062718 A1 discloses different pests and crops to be treated, nevertheless there is still a need to provide improved insect control using agrochemicals derived from a natural source, for example limonene-containing insecticide. An insecticide needs to have high efficacy against target pests, without a negative environmental impact.

Whiteflies (Hemiptera: Aleyrodidae ) are among the most important pests of tomato crops. This hemipteran insect causes severe damage directly by feeding on the leaves, which can also cause the development of honey dew. White flies are also a vector of the Geminiviridae virus (Tomato Yellow Leaf Curl Virus, TYLCV). Two species of whiteflies that affect tomato plants are, for example, Bemisia tabaci and Trialeurodes vaporariorum.

There is still a need for to provide improved treatment and protection against whiteflies (Hemiptera: Aleyrodidae) of tomato plants under cultivation using agrochemicals derived from a natural source, for example limonene-containing insecticide, which has high efficacy against whiteflies (Hemiptera: Aleyrodidae ), without a negative environmental impact.

Consequently, there is still a demand for improved insecticide formulations comprising limonene and associated applications of such insecticide formulations on particular pests and crops, in particular against whiteflies (Hemiptera: Aleyrodidae).

The present invention aims to provide a use of limonene as an insecticide which meets that demand. The present invention also aims to provide a use of limonene as an insecticide which provides improved insect control by achieving high efficacy against whiteflies (Hemiptera: Aleyrodidae ), without a negative environmental impact.

SUMMARY OF THE INVENTION

The present invention accordingly provides a method for curative treatment of Aleyrodidae, or white fly, on vegetables or tomatoes under cultivation, the method comprising the steps of: i. providing a diluted liquid composition, comprising limonene, in which the limonene is emulsified in an aqueous liquid; and ii. applying the diluted liquid composition as a foliar spray onto vegetables or tomatoes, the vegetables or tomatoes being infested with Aleyrodidae, or white fly, the diluted liquid composition being applied to provide a dose rate of from 1200 to 1500 grams of the limonene active ingredient per hectare of the vegetables or tomatoes.

The present invention also provides a use of limonene for the insecticidal treatment of Aleyrodidae, or white fly, without exhibiting toxicity to the whitefly predators Chrysoperla carnea and Hippodamia (= Adonia ) variegata, on vegetables or tomatoes under cultivation, wherein the limonene is emulsified in an aqueous liquid to form a diluted liquid composition, comprising limonene, which is applied as a foliar spray onto the vegetables or tomatoes, wherein in the diluted liquid composition the concentration of the limonene active ingredient is from 1.2 to 1.5 grams/litre.

Preferred features of these aspects of the present invention are defined in the respective dependent claims.

The present invention is at least partly predicated on the unexpected finding by the present inventors, supported by experimental evidence as disclosed herein, that limonene can be effectively used as an insecticide on specific crops, in particular vegetables and tomatoes, and at specific dose rates of the limonene active ingredient (a.i), and optionally specific concentrations of the limonene active ingredient, for the effective treatment of Aleyrodidae (white flies) without negatively impacting on at least two specific white fly predators, Chrysoperla carnea and Hiippodamia (= Adonia) variegata.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the " foliage" of a plant is to be understood to be the shoots, stems, branches, leaves and other appendages of the stems and branches of the plant after the seed has sprouted, including the roots of the plant. It is preferable that the shoots and foliage of a plant be understood to be those parts of the plant that have grown from the seed and/or shoots of a "mother" plant.

In a first aspect, the invention provides a method for curative treatment of Aleyrodidae, or white fly, on vegetables or tomatoes under cultivation.

Typically, the white fly are at least one of the species Bemisia tabaci and Trialeurodes vaporariorum_.

The vegetables or tomatoes to be treated or protected may be in cultivation in a field, glasshouse or cultivation tunnel. In particular, the vegetables to be treated or protected may be any vegetable crop such as pepper, aubergine, courgettes, carrots and other root vegetables, cucumber and other salad vegetables, beans, peas and other legumes, etc, of any variety thereof. The tomato may be any variety of tomatoes.

The target white fly insect pest of the use and methods of present invention, i.e. insects belonging to the group of Aleyrodidae or white fly, includes the adult, nymph or larvae of any white fly species that feeds on, or damages, the shoots and foliage of the vegetable or tomato plant that is to be treated or protected by the use and method of the present invention. The foliar spray may be applied directly on the insects.

The limonene is emulsified in an aqueous liquid to form a diluted liquid composition comprising limonene.

Limonene has the chemical composition l-methyl-4-(l-methylethenyl)-cyclohexene. In a preferred embodiment, limonene is present in the composition as the enantiomeric pure form of D-limonene, CAS: 5989-27-5. In another embodiment, limonene is present in the composition as the enantiomeric pure form of L-limonene, CAS: 5989-54-8. In yet another embodiment limonene is present as a racemic mixture, also known as dipentene, CAS: 138-86-3. Most preferably, the diluted liquid composition comprises D-limonene.

Limonene can be obtained via a synthetic route, or limonene can be extracted from a natural source. As the synthetic route is too costly in view of the material available from natural sources, most commercially available limonene is from a natural source. D- Limonene can be obtained commercially from citrus fruits through two primary methods: centrifugal separation or steam distillation.

With the word “citrus fruit” is meant a fruit of the genus Citrus, preferably selected from the list of citrus, mandarin, orange, kumquat, lemon, lime, grapefruit, pomelo, citrange, and citron. In the preferred embodiment of the present invention, the limonene is comprised in orange oil.

In a preferred embodiment, limonene is obtained out of an extract of citrus fruit peels. In further embodiments said citrus fruit oil is selected from the group consisting of orange oil, lemon oil, lime oil, grapefruit oil and tangerine oil. In a preferred embodiment, said citrus fruit oil is orange oil. In a more preferred embodiment said orange oil is selected from the list of technical grade orange oil, CAS 94266-47-4; food grade orange oil, CAS 8028-48-6 or cold pressed orange oil. The person skilled in the art is familiar with orange oil and its characteristics from its listing as active substance (SANCO/12083/2013 rev 3, 2013) and the standard references ISO 3140:211 and the European Pharmacopoeia 5.0, 2005.

In a preferred embodiment, the citrus fruit oil, preferably orange oil, is directly used in the composition without the need to purify the limonene out of the oil.

Preferably, the limonene active ingredient, typically orange oil, is the only active insecticide ingredient in the diluted liquid composition.

In an alternative embodiment, other terpenes or terpenoids are present in the composition, preferably carvone, carved, limonene oxide, p-cymene, a-pinene, b- pinene, myrcene, b-ionone, linalool, geraniol or eugenol. In another alternative embodiment, oils extracted from plants, preferably mint, peppermint, spearmint, clove or rosemary are used in the composition.

Preferably, in the diluted liquid composition the concentration of the limonene active ingredient is from 1.2 to 1.5 grams/litre.

In a preferred embodiment, the diluted liquid composition is produced from an emulsifiable concentrate composition comprising limonene, the emulsifiable concentrate having the ability to form a stable emulsion when added to an aqueous liquid such as water, so that the diluted liquid composition can be used as an insecticide. The dilution provides the desired dose rate of the limonene active ingredient. In the preferred embodiment of the present invention, the diluted liquid composition is produced by mixing a limonene-containing liquid concentrate composition comprising from 500 to 700 grams/litre of limonene active ingredient with an aqueous liquid. Typically, the limonene-containing liquid concentrate composition comprises about 600 grams/litre of limonene active ingredient.

The limonene-containing liquid concentrate composition is preferably a water- emulsifiable composition, comprising from 500 to 700 grams/litre of limonene active ingredient and an emulsifying surfactant or surfactant system, comprising one or more surfactants. The emulsifiable concentrate composition is added to an aqueous liquid such as water, whereby the limonene in combination with the surfactant(s) forms an emulsion, which can be applied to plants, crops or directly on the pest.

Preferably the concentrate composition is essentially free of solvent(s), in particular free of water and organic solvent(s), in particular aromatic solvent(s). By the term “essentially free of solvent(s)” as used in the present specification, is meant a concentrate composition with less than 10 wt% solvent based on the total weight of the concentrate composition.

Limonene, for example orange oil, is poorly soluble in water. For orange oil or limonene to be able to form a stable emulsion when added to water a surfactant is needed. The surfactant forms micelles, with a hydrophobic core and a hydrophilic outer shell. The limonene occupies the space in the hydrophobic cores of those micelles.

In the preferred embodiment of the present invention, the diluted liquid composition further comprises an ethoxylated isotridecanol nonionic surfactant. The ethoxylated isotridecanol nonionic surfactant functions as an emulsifying surfactant for the limonene in the diluted liquid composition. The weight ratio of the nonionic surfactant and the limonene active ingredient is from 0.2:1 to 0.25:1. Typically, the ethoxylated isotridecanol nonionic surfactant is isotridecanol penta-ethoxylate. Preferably, the ethoxylated isotridecanol nonionic surfactant is the only nonionic surfactant in the diluted liquid composition. In a preferred embodiment of the present invention, the ethoxylated isotridecanol nonionic surfactant has a hydrophilic-lipophilic balance (HLB) value of about 10.5. A HLB in this region is a compromise between the solubility of the surfactant in limonene and the ability to stabilize an emulsion of limonene when added to an aqueous liquid such as water, which can be considered as an oil-in-water-type system.

The ethoxylated isotridecanol nonionic surfactant is preferably present in the emulsifiable concentrate composition in an amount of 10 to 20 wt%, preferably 12- 18 wt%, based on the weight of the concentrate composition.

The ethoxylated isotridecanol nonionic surfactant is preferably present in the diluted liquid composition in an amount of 0.25 to 0.4 grams/litre, based on the weight of the diluted liquid composition.

In a preferred embodiment of the present invention, the diluted liquid composition invention comprises a wetting agent, which helps to lower the surface tension of the emulsion formed after adding the concentrate composition to an aqueous liquid such as water. This lowered surface tension helps to coat a larger surface of the leaves of the crop with the composition and this results in more insects contacting the insecticide. The need for a wetting agent is even bigger when the leaves have hydrophobic glandular hairs on their surface to repel water droplets, for example tomato leaves.

The wetting agent is commonly a water soluble anionic surfactant. The wetting agent in the diluted liquid composition is preferably an anionic surfactant with the anionic part preferably being an alkylbenzene sulfonate, more preferably a dodecylbenzene sulfonate. The cationic counter ion is preferably chosen out of the list of triethylammonium ion, triethanolammonium ion, tetrabutylammonium ion, or other tetra-alkylammonium ions, tetraphenylphosphonium ion or other tetra- alkylphosphonium ions.

In the preferred embodiment of the present invention, the diluted liquid composition therefore further comprises an anionic surfactant comprising, or consisting of, dodecylbenzene sulfonic acid or a salt thereof, wherein the weight ratio of the dodecylbenzene sulfonic acid, or salt thereof, and the limonene active ingredient is from 0.25:1 to 0.30:1. Preferably, the dodecylbenzene sulfonic acid or salt thereof is the only anionic surfactant in the diluted liquid composition.

In a preferred embodiment the wetting agent is triethanolammonium dodecylbenzene sulfonate, CAS: 27323-41-7. A combination of this anion and counter ion allows the wetting agent to be soluble in the concentrate composition without solvent other than the limonene.

The anionic surfactant wetting agent is preferably present in the emulsifiable concentrate composition in an amount of 10 to 20 wt%, preferably 15-20 wt%, based on the weight of the concentrate composition.

The anionic surfactant wetting agent is preferably present in the diluted liquid composition in an amount of 0.3 to 0.45 grams/litre, based on the weight of the diluted liquid composition.

An emulsifiable concentrate composition comprising limonene, the emulsifying surfactant and the anionic surfactant in the absence of solvent other than the limonene active ingredient was found to have good cold storage stability, at least in a temperature range between -4°C and 0 °C, and optionally at even lower temperatures, for a storage period of up to 14 days. The test was performed according to CIPAC method MT 39.1.

In a preferred embodiment, the diluted liquid composition, and thereby also the concentrate composition when used, comprises an antioxidant. Preferably the antioxidant is chosen from the list diphenylamine, ethoxyquin, BHA which is a mixture of 3-t-butyl-4-hydroxyanisole and 2-t-butyl-4-hydroxyanisole, BHT corresponding to 2,6-di-tert-butyl-p-cresol, ascorbic acid, the tocopherols and the polyphenols. The presence of an antioxidant may protect the limonene from oxidising, in particular when in concentrate form. For instance, traces of oxygen can get into the concentrate composition or the headspace of a bottle containing the composition after the bottle has been opened. The antioxidant is preferably present in an amount of less than 1 wt%, more preferably less than 0.5 wt%, most preferably less than 0.1 wt%, based on the weight of the concentrate composition.

The antioxidant is preferably present in an amount of less than 0.1 wt%, based on the weight of the diluted liquid composition.

Other conventional inactive or inert ingredients can be incorporated into the limonene compositions. Such inert ingredients include but are not limited to: conventional sticking agents, dispersing agents such as methylcellulose, polyvinyl alcohol, lecithin, polymeric dispersants such as polyvinylpyrrolidone/vinyl acetate, emulsion stabilizers, surfactants, antifreeze compounds such as urea, dyes, colorants. By including suitable additives, for example additives for improving the distribution, adhesive power and resistance to rain on treated surfaces, the limonene compositions can be better adapted for application as a foliar spray to vegetables or tomatoes.

The diluted liquid composition is applied as a foliar spray onto the vegetables or tomatoes. The diluted liquid composition is applied to provide a dose rate of from 1200 to 1500 grams of the limonene active ingredient per hectare of the vegetables or tomatoes.

Preferably, the diluted liquid composition is applied by foliar spraying at a spray rate of from 750 to 1250 litres per hectare of the vegetables or tomatoes. Typically, the spray rate is about 1000 litres per hectare of the vegetables or tomatoes.

Preferably, the foliar spray treatment is repeated within a period of from 4 to 7 days from the first foliar spray treatment.

Preferably, the foliar spray treatment has no toxicity on two whitefly predators Chrysoperla carnea and Hippodamia (= Adonid) variegata. In accordance with a second aspect, the present invention provides a use of limonene for the insecticidal treatment of Aleyrodidae, or white fly, without exhibiting toxicity to the whitefly predators Chrysoperla carnea and Hippodamia (= Adonia ) variegata, on vegetables or tomatoes under cultivation, wherein the limonene is emulsified in an aqueous liquid to form a diluted liquid composition, comprising limonene, which is applied as a foliar spray onto the vegetables or tomatoes, wherein in the diluted liquid composition the concentration of the limonene active ingredient is from 1.2 to 1.5 grams/litre. Limonene is a volatile component. Even in the form of a diluted aqueous liquid composition limonene can evaporate within 24 hours after application. This is advantageous as it leaves no residue on the treated plants or crops. This is of particular importance in biological agriculture, where use of synthetic agrochemicals is avoided. This allows the insecticide to be used until one day before harvesting the plant or crop, leaving a food safe product that can be sold or processed into a food product 24 hours after application as there is no limonene residue after this period. The pre-harvest interval is less than one day. Treating a field with insects infested crop according this method will result in a mortality of said insects higher than 80%, preferably higher than 90%. The mortality rate of the pest concerned can be measured with techniques known to the skilled person.

The present invention will be now described in more details, referring to examples.

Example 1: Concentrated orange oil composition

A concentrated orange oil composition having the composition as shown in Table 1 was prepared.

Table 1

The concentrated orange oil composition of Table 1 was diluted in an aqueous liquid, in particular water, to form form a diluted liquid composition, comprising limonene, which is to be applied as a foliar spray onto vegetables or tomatoes at a dose rate of from 1200 to 1500 grams of the limonene active ingredient per hectare of the vegetables or tomatoes, the limonene being emulsified in the aqueous liquid. In order to provide a dose rate of from 1200 to 1500 grams of the limonene active ingredient per hectare of the vegetables or tomatoes, when the spray volume is set at 1000 litres/hectare the diluted liquid composition to be sprayed comprises from 2 to 2.5 litres of the concentrated orange oil composition per hectare of crop to be sprayed.

Example 2: Use of the orange oil composition according to Example 1 against whiteflies on tomatoes

In this Example, the concentrate composition of Example 1 was tested to evaluate the efficacy of a limonene-containing diluted aqueous liquid composition as an insecticide against two species of adults of whiteflies, namely Bemisia tabaci and Trialeurodes vaporariorum.

The crop was a tomato cultivar, Calvi, cultivated in a greenhouse, of the “Canary-type”, in a farm in the locality of Agadir, Morocco. The total area of the greenhouse was about 5 Ha.

At the start of the test the tomato plants had a high level of infestation by adults of whiteflies of two species of whiteflies, namely Bemisia tabaci and Trialeurodes vaporariorum

The concentrate composition of Example 1 was diluted in water to provide three diluted aqueous treatment compositions having different concentrations of limonene. The treatment compositions were applied to the tomato plants as a foliar spray, using a backpack sprayer, at a spray volume of 1000 litres/hectare (1/ha).

The treatment compositions were applied as shown in Table 2. Table 2

In addition to the three Treatment Compositions 1-3 comprising limonene, a positive control was tested, called Control Composition 1. The positive control comprised the commercial insecticide known as DECIS EXPERT (trade mark), which has the active insecticide ingredient Deltamethrine. The positive control used was sprayed as a foliar spray at a concentration of 10 cc/hl using the backpack sprayer at a spray volume of 1000 litres/hectare (1/ha).

The experimental design was as follows. The three Treatment Compositions 1-3 and the Control Composition 1 were applied by dividing the treatment area into a series of 4 blocks and within each block a random arrangement of a series of 4 zones was provided, each zone corresponding to a respective one of the 4 compositions consisting of the three Treatment Compositions 1-3 and the Control Composition 1.

The infested leaves was sampled before treatment (Day 0), and on Days 1, 3 and 5 after treatment. For each sample 4 infested leaves were checked.

The mortality rate of adults of whiteflies (moribund and dead insects) was determined for each sample. The mortality data was subjected to ANOVA at P < 0.01 with interactions tested between the various factors (time after treatment and insecticides) for adult whiteflies mortality rates and resulting means were compared using the Newman- Keuls test. The results are summarised in Table 3.

Table 3

It may be seen from Table 3 that the mortality rates observed at 24h, 3 days and 5 days after treatment were consistently higher for Treatment Compositions 2 and 3 as compared to either Treatment Composition 1 or Control Composition 1.

At 24 hours after treatment, there was statistically (P<0,001) no difference of mortality rates caused by the medium and higher doses (2 and 2.5 L/ha of the concentrate of Example 1) of Treatment Compositions 2 and 3. These rates were statistically different and higher than mortality observed by lower dose of Treatment Composition 1 (1.5 L/ha).

At 3 days after treatment, the mortality rates caused by the medium and higher doses (2 and 2.5 L/ha of the concentrate of Example 1) of Treatment Compositions 2 and 3 were relatively higher than those observed 24h after treatment, and were also statistically different and higher than mortality observed by lower dose of Treatment Composition 1 (1.5 L/ha). At 5 days after treatment, the toxicity of the limonene-containing composition was relatively lower than the toxicity observed 3 days after treatment, but nevertheless more than 85% for the medium and higher doses (2 and 2.5 L/ha of the concentrate of Example 1) of Treatment Compositions 2 and 3. Regarding the lower dose (1.5 L/ha of the concentrate of Example 1) of Treatment Composition 1, the mortality rate was moderate and caused 68% of mortality 5 days after treatment. No phytotoxicity i.e. toxic effect on the tomato plants, was observed at the higher dose on tomato plants during this trial.

The mortality rate caused by the DECIS insecticide Control Composition 1 after treatment was only moderate (44, 52 or 55%) after each treatment period, and consistently worse that using the limonene-containing compositions of Treatment Compositions 1, 2 and 3.

It can be concluded from this data that the efficacy of the limonene-containing compositions of Treatment Compositions 2 and 3, having limonene dose rates of 1200 grams/hectare and 1500 grams/hectare respectively, to provide a highly toxic effect on the adults of whiteflies on tomato crops was confirmed, consistently exhibiting a mortality rate of more than 85%.

In contrast, the lower limonene dose rate of 900 grams/hectare of Treatment Composition 1, and the Control Composition 1 comprising DECIS, had only a moderate toxic effect on the adults of whiteflies.

Since the mortality rate started to decrease 5 days after treatment on the tomato crops by Treatment Compositions 2 and 3, preferably for continued treatment against whiteflies the foliar spray treatment should be repeated within a period of from 4 to 7 days from the first foliar spray treatment.

Example 3: Use of the orange oil composition according to Example 1 against whiteflies on peppers

In this Example, the concentrate composition of Example 1 was tested to evaluate the efficacy of a limonene-containing diluted aqueous liquid composition as an insecticide against a species of adults of whiteflies, namely Bemisia tabaci.

The crop was a pepper cultivar, Classico, cultivated in a cultivation tunnel, in a farm in Southern Italy. The crop height was 80-90 cm and 100-150 cm. At the start of the test, the pepper plants had an infestation by adults of whiteflies of the species Bemisia tabaci.

The concentrate composition of Example 1 was diluted in water to provide four diluted aqueous treatment compositions having different concentrations of limonene. The treatment compositions were applied to the pepper plants as a foliar spray, using a backpack sprayer, at a spray volume of 1000 litres/hectare (1/ha).

The treatment compositions were applied as shown in Table 4.

Table 4 It may be seen that the three Treatment Compositions 1-3 are the same as used in Example 1, but that the Treatment Composition 4 has a lower limonene-concentration than Treatment Compositions 1-3.

In addition to the four Treatment Compositions 1-4 comprising limonene, two positive controls were tested, called Control Compositions 2 and 3.

The positive control of Control Composition 2 comprised the commercial insecticide known as Epik SL (trade mark), which has the active insecticide ingredient Acetamiprid, and was sprayed as a foliar spray at a dose rate of 200ml/100L of spray volume (lOg a.i./lOOL) using the backpack sprayer at a spray volume of 1000 litres/hectare (1/ha). The positive control of Control Composition 3 comprised the commercial insecticide known as Prev-Am Plus (registered trade mark) available in commerce from Ori Agro International Ltd.. Prev-Am Plus is an orange oil (i.e. limonene) containing insecticide composition and comprises the active insecticide ingredient orange oil at a concenration of 60 g/litre. The data sheet from Ori Agro International Ltd. on the Prev-Am Plus product states a dose rate of 2 litres/hectare, i.e. 120 grams/hectare of orange oil active ingredient for the treatment of tomatoes and peppers. The Prev-Am Plus product, after dilution with water, was sprayed as a foliar spray at a dose rate of 400ml/100L of spray volume (24g a.i./lOOL) using the backpack sprayer at a spray volume of 1000 litres/hectare (1/ha). All products tested in the trial were sprayed twice with a 7 day interval between the two spray applications.

The experimental design individually treated the pepper crops with the respective four Treatment Compositions 1-4 and two Control Compositions 2-3. There were two successive treatment days, at day 0 and day 7.

The infested leaves was sampled to count the number of adult whiteflies on the top 20 pepper leaves before treatment (Day 0), and on Days ¼, 1 , 3 and 7 after the first treatment and on Days 1, 3 and 7 after the second treatment. The test therefore took two weeks in total. After the second treatment, on Days 3 and 7 after the second treatment the number of whitefly nymphs on the top 20 pepper leaves were also counted.

The results are summarised in Tables 5a and 5b.

Table 5a

Before

0.25 days 1 day after 3 days 7 days 1 day after 3 days 7 days first

Treatment Overall - after 1st 1st after 1st after 1st 2nd after 2nd after 2nd treatment number/category treatment treatment treatment treatment treatment treatment treatment - day 0

5

10

15 Table 5b

Before

0.25 days 1 day after 3 days 7 days 1 day after 3 days 7 days first Overall -

Treatment after 1st 1st after 1st after 1st 2nd after 2nd after 2nd treatment number/category treatment treatment treatment treatment treatment treatment treatment - day 0

It may be seen from Table 5a that Treatment Compositions 1-4 were effective against adult whiteflies of the species Bemisia tabaci on a vegetable such as pepper. There is a 5 reduction in the adult population within 24 hours of the initial treatment. The treatment efficacy was highest for Treatment Composition 3 as compared to Treatment Compositions 1, 2 and 4. The efficacy of Treatment Composition 3 was statistically better than Control Composition 3, which also comprised orange oil in the composition Prev-Am Plus, and numerically better than Control Composition 2, which comprised 10 Epik SL. The adult population tended to increase again 7 days after the initial treatment, which showed that to maintain efficacy persistence the treatment should be repeated within 7 days or fewer.

It may be seen from Table 5b that Treatment Compositions 2 and 3 were most effective 15 against whitefly nymphs of the species Bemisia tabaci on a vegetable such as pepper, as compared to Treatment Compositions 1 and 4. It was also found that the foliar spray treatment had no toxicity on two whitefly predators Chrysoperla carnea and Hippodamia (= Adonia ) variegata during the two-week trial. This is an important finding because this data shows that the compositions used in accordance with the present invention can use limonene as an insecticide to provide improved insect control by achieving high efficacy against whiteflies (Hemiptera: Aleyrodidae ), without a negative environmental impact, in particular without a negative environmental impact on the two whitefly predators Chrysoperla carnea and Hippodamia (= Adonia ) variegata. It can be concluded from this data that the efficacy of the limonene-containing compositions of Treatment Compositions 2 and 3, having limonene dose rates of 1200 grams/hectare and 1500 grams/hectare respectively, to provide a highly toxic effect on the adults and nymphs of whiteflies on pepper crops was confirmed. In contrast, the lower limonene dose rates of 600 and 900 grams/hectare of Treatment Compositions 4 and 1 respectively, had lower efficacy. The limonene-containing compositions of Treatment Compositions 2 and 3 had similar efficacy as compared to the Control Composition 2 comprising Epik SL, and improved efficacy as compared to the Control Composition 3comprising a commercial orange oil composition Prev-Am Plus. Preferably for continued treatment against whiteflies the foliar spray treatment should be repeated within a period of from 4 to 7 days from the first foliar spray treatment.

Claims (27)

1. A method for curative treatment of Aleyrodidae, or white fly, on vegetables or tomatoes under cultivation, the method comprising the steps of: i. providing a diluted liquid composition, comprising limonene, in which the limonene is emulsified in an aqueous liquid; and ii. applying the diluted liquid composition as a foliar spray onto vegetables or tomatoes, the vegetables or tomatoes being infested with Aleyrodidae, or white fly, the diluted liquid composition being applied to provide a dose rate of from 1200 to 1500 grams of the limonene active ingredient per hectare of the vegetables or tomatoes.

2. The method according to claim 1, wherein in the diluted liquid composition the concentration of the limonene active ingredient is from 1.2 to 1.5 grams/litre.

3. The method according to claim 1 or claim 2 wherein the diluted liquid composition is applied by foliar spraying at a spray rate of from 750 to 1250 litres per hectare of the vegetables or tomatoes.

4. The method according to claim 3 wherein the spray rate is about 1000 litres per hectare of the vegetables or tomatoes.

5. The method according to any one of claims 1 to 4 wherein the diluted liquid composition is produced by mixing a limonene-containing liquid concentrate composition comprising from 500 to 700 grams/litre of limonene active ingredient with an aqueous liquid.

6. The method according to claim 5 wherein the limonene-containing liquid concentrate composition comprises about 600 grams/litre of limonene active ingredient.

7. The method according to any one of claims 1 to 6 wherein the diluted liquid composition further comprises an ethoxylated isotridecanol nonionic surfactant, wherein the weight ratio of the nonionic surfactant and the limonene active ingredient is from 0.2:1 to 0.25:1.

8. The method according to claim 7 wherein the ethoxylated isotridecanol nonionic surfactant is isotridecanol penta-ethoxylate.

9. The method according to claim 7 or claim 8, wherein in the diluted liquid composition the concentration of the ethoxylated isotridecanol nonionic surfactant is from 0.25 to 0.4 grams/litre, based on the weight of the diluted liquid composition.

10. The method according to any one of claims 1 to 9 wherein the diluted liquid composition further comprises an anionic surfactant comprising, or consisting of, dodecylbenzene sulfonic acid or a salt thereof, wherein the weight ratio of the dodecylbenzene sulfonic acid, or salt thereof, and the limonene active ingredient is from 0.25:1 to 0.30:1.

11. The method according to claim 10, wherein in the diluted liquid composition the concentration of the anionic surfactant is from 0.3 to 0.45 grams/litre, based on the weight of the diluted liquid composition.

12. The method according to claim 10 or claim 11 when dependent on any one of claims 7 to 9 wherein the ethoxylated isotridecanol nonionic surfactant is the only nonionic surfactant in the diluted liquid composition and the dodecylbenzene sulfonic acid or salt thereof is the only anionic surfactant in the diluted liquid composition.

13. The method according to any one of claims 1 to 12 wherein the limonene active ingredient is the only active insecticide ingredient in the diluted liquid composition.

14. The method according to any one of claims 1 to 13 wherein the limonene is comprised in orange oil.

15. The method according to any one of claims 1 to 14 wherein the foliar spray treatment is repeated within a period of from 4 to 7 days from the first foliar spray treatment

16. The method according to any one of claims 1 to 15 wherein the white fly are at least one of the species Bemisia tabaci and Trialeurodes vaporariorum_.

17. The method according to any one of claims 1 to 16 wherein the foliar spray treatment is also applied to, but has no toxicity on, two whitefly predators Chrysoperla carnea and Hippodamia (= Adonid) variegata.

18. Use of limonene for the insecticidal treatment of Aleyrodidae, or white fly, without exhibiting toxicity to the whitefly predators Chrysoperla carnea and Hippodamia (= Adonid) variegata, on vegetables or tomatoes under cultivation, wherein the limonene is emulsified in an aqueous liquid to form a diluted liquid composition, comprising limonene, which is applied as a foliar spray onto the vegetables or tomatoes, wherein in the diluted liquid composition the concentration of the limonene active ingredient is from 1.2 to 1.5 grams/litre.

19. Use according to claim 18 wherein the diluted liquid composition further comprises an ethoxylated isotridecanol nonionic surfactant, wherein the weight ratio of the nonionic surfactant and the limonene active ingredient is from 0.2: 1 to 0.25:1.

20. Use according to claim 19 wherein the ethoxylated isotridecanol nonionic surfactant is isotridecanol penta-ethoxylate.

21. Use according to claim 19 or claim 20, wherein in the diluted liquid composition the concentration of the ethoxylated isotridecanol nonionic surfactant is from 0.25 to 0.4 grams/litre, based on the weight of the diluted liquid composition.

22. Use according to any one of claims 18 to 21 wherein the diluted liquid composition further comprises an anionic surfactant comprising, or consisting of, dodecylbenzene sulfonic acid or a salt thereof, wherein the weight ratio of the dodecylbenzene sulfonic acid, or salt thereof, and the limonene active ingredient is from 0.25:1 to 0.30:1.

23. Use according to claim 22 wherein in the diluted liquid composition the concentration of the anionic surfactant is from 0.3 to 0.45 grams/litre, based on the weight of the diluted liquid composition.

24. Use according to claim 22 or claim 23 when dependent on claim 21 or claim 22 wherein the ethoxylated isotridecanol nonionic surfactant is the only nonionic surfactant in the diluted liquid composition and the dodecylbenzene sulfonic acid or salt thereof is the only anionic surfactant in the diluted liquid composition.

25. Use according to claim any one of claims 18 to 24 wherein the limonene active ingredient is the only active insecticide ingredient in the diluted liquid composition.

26. Use according to any one of claims 18 to 25 wherein the foliar spray treatment is repeated within a period of from 4 to 7 days from the first foliar spray treatment

27. Use according to any one of claims 18 to 26 wherein the white fly are at least one of the species Bemisia tabaci and Trialeurodes vaporariorum_.