AU2001242155A1 - Plant acaricidal compositions and methods - Google Patents

Description

TITLE OF THE INVENTION

PLANT ACARICIDAL COMPOSITIONS AND METHODS USING SAME

FIELD OF THE INVENTION

The present invention relates to acaricides. More particularly, the present invention relates to botanical acaricides. In particular, the present invention relates to compositions and methods for controlling acari with plant extracts. The invention further relates to botanical acaricides also having insecticidal activity.

BACKGROUND OF THE INVENTION

Plant feeding mites are among the most voracious phytophagous pests of crops. Synthetic insecticides or acaricides have up until now been used to control these pests; however, resistance to these products has developed with every new product put on the market. Although resistance follows a highly complex genetic and biochemical process, it can generally develop rapidly with synthetic products because their active ingredients rely on one or more molecules of the same class. The organism can therefore respond to the toxin by developing physiological, behavioral or morphological defense mechanisms to neutralize the effect of the molecule (Roush and MacKenzie, 1987).

Spider mites frequently become a problem after application of insecticides for control of insect pests because many synthetic insecticides stimulate mite reproduction. For example, mites reproduce many times faster when exposed to carbaryl, methyl parathion, or dimethoate in the laboratory than untreated populations (Flint 1990). Indeed, it is recognized that spider mites are extremely difficult to control with pesticides and that many pesticides exacerbate the pest infestation by destroying the natural predators thereof (USP 5,839,224).

Tetranychus urticae has now accumulated a considerable number of genes conferring resistance to all major classes of acaricides; it belongs to a number of especially critical cases in which nearly all of the affordable, previously effective pesticides have been depleted (Georghiou 1990).

In the recent edition of the Farm Chemical Handbook (Meister, 1999), out of a total of 2050 acaricides and insecticides listed, only 48 (2.4%) products are acaricides and 69 (3.4%) are considered both acaricides and insecticides. In addition, most available acaricides are toxic to mammals and do not meet the guidelines developed by most Integrated Pest Management programs. There are few published reports of the acaricidal properties of botanical pesticides except for experiments to assess the effect of neem extracts (Sundaram et al, 1995; Mansour et al. 1986) on the two spotted spider mite, T. urticae and on the effect of thyme oil and thymol (Mansour et al. 1986, supra) and neem extracts (El Gangaihi et al. 1996) on the spider mite predators, Phytoseiulus persimilis and Chiracanthium mildei.

There are about 270 species of Chenopodium (a plant in the Family Chenopodiaceae, Order Caryophyllales (=Centrospermae), Section Ambrina) with a wide range of genetic variability. Three important species in North America thereof are C. quinoa, C. album and C. ambrosioides. All three species are cultivated (Quarles 1992). C. quinoa has a long history of cultivation in South America and Mexico and is now being grown in England and North America as an experimental food crop (Risi and Galwey 1984).

Lambsquarters, C. album, is native to Europe and Asia, although it grows widely throughout the American continent. It contains very little essential oil and does not have the pronounced odor that C. ambrosioides and others have. C. quinoa is native to, and usually grows in the mountainous regions of South America (Quarles 1992). It is a leafy grain that is used in soups, stews, or as a porridge and has an "earthy" taste.

Though lambsquarters and quinoa contain little essential or volatile oil, other components of these plants have potential for certain pest management uses. The unsaturated fatty acids of the seed oils may act as fungicides and nematicides (Malik et al. 1985). The fiavonoids of C. album are allelopathic and possibly fungicidal, while the sesquiterpenes may be antifeedants and the steroids may affect larval growth of certain insects (Qasem and Hill, 1989; Turnock 1985). The seed saponins of quinoa may be molluscicidal or have a use in medicine (Burnouf-Radosevich 1984, 1985; Chandel and Rastogi 1980).

The species Chenopodium ambrosioides originated in Central America, and it is now distributed throughout much of the world. It has been used traditionally in Latin America as an anthelmintic (medicine for controlling internal parasites) for many years. In the early 1900s it was one of the major anthelmintics used to treat acaris and hookworms in humans, cats, dogs, horses, and pigs. Oil of Chenopodium was also called "Baltimore Oil" because of the large production facility in Baltimore that specialized in extracting the oil from the plant. Chenopodium extracts were replaced with other, more effective synthetic anthelmintics in the 1940s. In addition, the oil was used as a wood preservative for boats to protect them from colonization by barnacles.

While some anti-pest activities have been reported for Chenopodium to date, Chenopodium extracts have not been reported as having acaricidal activity. Other plant components, however, have been reported as possessing acaricidal properties.

US Patent 4,933,371 describes the use of saponins extracted from various plants (i.e. yucca, quillaja, agave, tobacco and licorice) as acaricides. Also, US Patent 4,933,371 describes the use of linalool extracted from the oil of various plants such as Ceylon's cinnamon, sassafras, orange flower, bergamot, Artemisia balchanorum, ylang ylang, rosewood and other oils extracts as acaricides. These methods however have many disadvantages: they require the extraction of the active substance from the plant which increases the cost. Furthermore, those compounds often do not meet desired levels of toxicity towards acari. Whole extracts of plants have rarely been described as having acaricidal properties. The only whole plant extract described as having acaricidal property is a neem extract described in US Patent 5,352,672, which indicates that a neem seed extract has an acaricidal effect. The neem tree only grows in tropical climates. Commercialization of neem in northern countries is therefore more difficult and costly. Furthermore, because the neem is a tree, a delay of a few years is required before the tree can be used to prepare the extract. Also, tree extracts tend to be more complex in terms of the number of different compounds that they contain. The task of determining the toxicity of extracts from tree extracts such as neem extracts may be more difficult because each compound must be tested separately.

There therefore remains a need to provide new and effective acaricidal products which overcome the problem of the acaricidal products known in the art. There also remains a need to provide an acaricidal composition which is less likely to enable a development of resistance thereto. In particular, there remains a need to provide acaricidal compositions that enable the combat of a variety of pests at different stages of plant growth. There also remains a need to provide a method to combat acari at a locus, using a composition which is not toxic to animals and especially to mammals.

The present invention seeks to meet these and other needs.

The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

An object of the present invention is therefore to provide improved acaricides that overcome the shortcomings of the prior art. More particularly, the invention relates to botanical acaricides.

In an embodiment of the present invention there is provided an acaricidal composition comprising ascaridole. In an embodiment of the present invention, there is provided a composition comprising oil extracts from Chenopodium ambrosioides for use as an acaricide. Also, there are provided formulations comprising Chenopodium ambrosioides oil extracts for use as acaricides. There is also provided a terpene-containing acaricidal composition (or extract) from C. ambrosioides.

In yet another embodiment of the present invention, there is provided a method for selecting a Chenopodium ambrosioides variety and a plant stage to produce Chenopodium crops which can generate the optimum yield of acaricidal activity of the oil, or the optimum yield of the acaricidal activity-promoting agent. In yet another embodiment of the present invention, there is provided a method for selecting a plant Chenopodium ambrosioides variety and a plant stage to produce Chenopodium plant crops generating the optimum yield of the active compound and/or compounds responsible for the acaricidal activity. In a preferred embodiment of the present invention, there is provided such a method to select a plant Chenopodium ambrosioides and a plant stage to produce Chenopodium plant crops generating the optimum yield of the active compound and/or compounds, responsible for acaricidal activity. In yet a further embodiment of the present invention, there is provided a method of identifying the active compound or compounds in the acaricidal compositions of the present invention.

In another embodiment of the present invention, there is provided a method for extracting Chenopodium ambrosioides oil containing the highest quantity of acaricidal and/or insecticidal activity. In yet another embodiment, there is provided a method to enable the production of high quantity and/or quality of oil from the genus

Chenopodium. Of course, it will be recognized by the skilled artisan to which the present invention pertains that the method of oil preparation can be adapted to other species as well as to particular needs.

The present invention further relates to a method for preventing acaricide infestation of a locus comprising an application to that locus of an effective amount of a composition of the present invention.

The present invention, in addition, relates to the use of a plant extract and/or of a composition comprising ascaridole for controlling plant-infesting acari. The method includes the step of contacting one or more part or tissue of a diseased plant or a plant susceptible to infestation by a plant acari (or pathogenic insect) within acaricidal amount of the extract and/or composition of the present invention. Non-limiting examples of tissues and parts of plants include seeds, seedlings and the plant perse.

The present invention further relates to a treatment of a locus infested with acari, comprising a contacting of the infested locus with an acaricidally effective amount of a composition or extract in accordance with the present invention. The Applicant was the first to use an extract from a plant of the Chenopodiaceae family as an acaricide. Furthermore, prior to the present invention, extracts from the Chenopodiaceae family had not been considered for use and/or validated as acaricides. In addition, the Applicant was the first to use compositions comprising the compound ascaridole as acaricides. As used herein, the term "mite" is used to refer broadly to plant acari. Similarly, the term "acari" is used to refer broadly to plant infesting acari or phytophagous acari.

A distinction is made herein between herbaceous plant extracts and woody plant extracts i.e. woody plant extracts refer to extracts taken from woody plants (trees) whereas herbaceous plant extracts are those coming from non-tree plants.

The term "ascaridole" is meant to refer herein to ascaridole and iso-ascaridole, unless otherwise provided (as when enumerating terpenes in the tables below).

The term "locus" is used herein broadly to refer to a site which is infested with an acari or which could be infested therewith.

While the locus or site which can be infested with an acari and/or insect and controlled with a composition or method of the present invention can vary, in a preferred embodiment of the present invention, such a locus is a living plant and, more particularly, a plant crop. Non-limiting examples of such plants include corn, cotton, flowers, fruits, vegetables in general, tobacco, etc.

The term "colonizing" or the like is intended to refer to an association of a pest with a material, locus or site (e.g. an organism or tissue) from which the pest derives nutrients.

The term "control" of pests (acari and/or insects) refers broadly to a significant reduction in the growth and/or activity of the pest which is targetted. The Applicant has shown a negative correlation between the quantity of the terpene ascaridole and that of alpha terpinene in plant extracts. The Applicant has also shown that plant extracts containing higher levels of α-terpinene have lower acaridal activity. The Applicant was also the first to use an herbaceous plant extract as an acaricide.

The Applicant was also the first to demonstrate that the presence of ascaridole and/or of a compound which fractionates with same in plant extracts positively correlates with the acaricidal/insecticidal activity of same. Further, upon fractionation of the C. ambrosioides extracts, it was determined that the acaricidal activity positively correlated with the presence of ascaridole and/or of a compound which fractionates with same. Thus, the present invention provides the means to identify other plants having the potential to provide acaricidal or acaricidal and insecticidal extracts, by determining the content of ascaridole in such plants and/or of a compound which can be converted into ascaridole and/or of a compound (or compounds) which fractionate with same.

In a preferred embodiment, the compositions of the present invention include at least one terpene. Non-limiting examples of terpehes that can be found in the compositions of the present invention include myrcene, alpha-terpinene, p-cymene, cineol, limonene, gamma-terpinene, t-p-mentha- 2,8-dien-1-ol, c-p-mentha-2,8-dien-1-ol, carveol, ascaridole, iso-ascaridole, carvone, t-cinnamaldehyde, pelargonic acid, thymol and carvacrol.

The mono-terpenes and the sesquiterpenes are the main components of essential oils. Monoterpenes, derived from 2 isoprene units, are 10-carbon compounds (C₁₀) and the sesquiterpenes , derived from 3 isoprene units are 15-carbon compounds (C₁₅). Both mono- and sesquiterpenes belong to the steam distillate fraction i.e. essential oils, because of their low boiling point. These compounds are known to be present in fragrant, volatile, essential oils of many plants including mints, pine, cedar, citrus, eucalyptus and spices. For example, myrcene is found in the essential oil of bay leaves and of hops. Carvone is one of the many odoriferous components of caraway seed (Carum carvi) (Brielmann 1999). The insecticidal action of monoterpenes includes neurotoxicity and insect growth regulator (IGR) activity as well as repellency and fumigation (Coats 1994).

Also, in accordance with the present invention, there is provided a composition for killing undesired phyto-acari, comprising a natural essential oil obtained from a plant, wherein the oil contributes to between about 0.125% to about 10% of the composition, and wherein the oil comprises at least one compound selected from the group consisting of ascaridole, iso-ascaridole, α-terpinene, p-cymene, limonene, thymol and carvacrol together with a suitable carrier.

In addition, in accordance with the present invention, there is provided a method of controlling plant-infesting acari, which comprises applying to a site where control is desired, a composition containing an essential oil comprising an acaricidally effective amount of a plant acaricidal composition comprising at least one compound selected from the group consisting of ascaridole, iso-ascaridole, α-terpinene, p-cymene, limonene, thymol and carvacrol together with a suitable carrier. Also, in accordance with the present invention, there is provided a method of controlling plant-infesting acari, which comprises applying to a site where control is desired, a composition according to the present invention. Further, there is provided a method of controlling acari, which comprises a contacting the acari or their habitat , with an acaricidally- effective amount of a composition of the present invention.

It has thus been found that extracts from North American plant species can be toxic to the two-spotted spider mite model system (Tetranychus urticae Koch : Tetranychidae). Several factors such as phenological age of the plant (Jackson et al. 1994), % humidity of the harvested material (Chialva et al. 1983), plant parts chosen for extraction (Jackson et al. 1994, supra; Chialva et al. 1983, supra), and the method of extraction (Perez-Souto 1992 have been identified as possible sources of variation for the chemical composition and efficacy of the extracts. Thus, extracts from a number of North American plant species are shown to exhibit significant acaricidal activity. Particularly, plant extracts from the genus Chenopodium and more particularly C. ambrosioides as well as extracts of Artemisia absinthium and Tanacetum vulgare, are shown to exhibit acaricidal activity. As exemplified herein, partially purified extracts may be employed. In addition, the extracts of the present invention are preferably mixed in with carriers or diluents (which are chosen so as to not significantly diminish the acaricidal activity of the plant extract) to produce an acaricidal composition. More preferably, the extracts are mixed in with a carrier and most preferably with an emulsifier. In a prefered embodiment, the composition is provided in a ready-to-use form. This ready-to-use composition may contain a final concentration of 0.125% to 10% by volume of oil extract, preferably between 0.25% to 2% by volume of oil extract. Of course, the skilled artisan can modify these concentrations in accordance with particular needs. For instance, a 95% concentration may be present in compositions which may be diluted before use. In another embodiment, the composition is provided as an emulsifiable concentrate comprising oil extract between 5% to 50% by volume. The practionner will adapt the concentration so that it is acaricidal yet not phytotoxic. Preferably, the extracts and compositions of the invention are acaricidal, not phytotoxic and not toxic to animals. Also preferably, the extracts and compositions further have the advantage of being insecticidal.

Among the advantages of a preferred embodiment of the acaricidal compositions and of the acaricidal and insecticidal compositions of the present invention are their environmental compatibility or their environmental safety.

The compositions of the invention may be mixed with a number of known carriers and/or adjuvants. Preferably the carriers are inert. Non-limiting examples of carriers and/or adjuvants include solvents, diluents and/or surface-active agents to form emulsions, aerosol, sprays or other liquid preparations, dusts powders or solid preparations. Non-limiting examples of solvents and diluents include water, aliphatic and aromatic hydrocarbons (i.e. xylene or other petroleum fractions) and alcohols such as ethanol. Surface-active agents may be of an anionic, cationic or non-ionic type. Stabilizers, perfumes, colourings and anti-oxidants may also be included. The quantities and types of such carriers to be used in accordance with the present invention are conventionally used in pesticidal and particularly acaricidal compositions. For certain embodiments, it might be beneficial to provide time-release compositions. Such formulations could be particularly beneficial to prevent infestation of a locus or to prevent the re- infestation of same. A non-limited example of a a time-release formulation includes a composition in accordance with the present invention which has been encapsulated and or pelletized.

Emulsifiers that can be used to solubilize the extracts of the present invention in water include blends of anionic and nonionic emulsifiers. In a particular embodiment, these emulsifiers are isopropylamine salts of alkyl benzene sulfonates or the like and octylphenol ethoxylates with 9 moles of ethylene oxide or equivalent. Anionic emulsifiers are typically used in percentages of 0.125% to 5% and nonionic emulsifiers are typically used in percentages of 0.125% to 5%.

Examples of anionic emulsifiers that can be used in percentages of 0.125% to 5% in formulations of the present invention include but are not limited to the anionic emulsifiers provided in Table 1.

Table 1 : Anionic emulsifiers that can be used in accordance with the present invention

Anionic emulsifier Trade Anionic emulsifier Supplier name Chemical name

Rhodacal DS-10 Sodium dodecyl Rhone Poulenc, Cranbury benzene sulphonate NJ

Calfax DB-45 dodecyl Pilot Chem. Co. Santa Fe diphenyloxide Springs CA disulfonate

Stepanol DEA Diethanolamine Stepan Co., Northfield IL lauryl sulfate

Aerosol OT-75 Sodium dioctyl Cytec Industries Inc., sulfosuccinate Morristown NJ

Rhodacal A246L Sodium (C14-C16) Rhone Poulenc, Cranbury Olefin Sulfonate NJ

Rhodafac RE Nonoxynol-9 Rhone Poulenc, Cranbury 610 phosphate NJ

Rhodapex CO- Ammonium Rhone Poulenc, Cranbury 436 nonylphenol (4EO) NJ sulphate

Non-limiting examples of nonionic emulsifiers that can be used in formulations of the present invention (in percentages of 0.125% to 5%) are described in Table 2.

Table 2 : Nonionic emulsifiers that can be used in accordance with the present invention

Nonanionic Nonanionic emulsifier Supplier emulsifier Trade Chemical name name

Igepal CO-887 Nonylphenol Rhone Poulenc, ethoxylate (30 mol) Cranbury NJ

Macol NP-9.5 nonyl phenol (POE PPG Industries, Gurnee 9.5) ethoxylate IL

Igepal CO-430 Nonylphenol Rhone Poulenc, ethoxylate (4 mol) Cranbury NJ

Rhodasurf ON- Oleyl alcohol POE 20 Rhone Poulenc, 870 Cranbury NJ

Alkamuls EL-719 Castor oil (40mol EO) Rhone Poulenc, Cranbury NJ

Alkamuls EL-620 Castor oil (30mol EO) Rhone Poulenc, Cranbury NJ

Alkamide L9DE Lauramide DEA Rhone Poulenc, Cranbury NJ

Span 80 Sorbitan monooleate ICI Surfactants, Wilmington DE

Tween™ 80 POE 20 Sorbitan ICI Surfactants, Monooleate Wilmington DE

Alkamuls PSMO-5 POE sorbitan (5) Rhone Poulenc, monooleate Cranbury NJ

Atlas G1086 Sorbitan hexaoleate ICI Surfactants, Wilmington DE

Tween™ 20 POE 20 Sorbitan ICI Surfactants, Monolaurate Wilmington DE The emulsifiers listed above are not exhaustive; ternary mixtures of emulsifiers may also be used to produce a stable microemulsion of the plant extracts with desirable physical and chemical properties. It will become apparent to anyone skilled in the art that other stable microemulsion or macroemulsion formulation of the present invention for use as an acaricidal/insecticidal composition can be prepared. The skilled artisan, to which the present invention pertains, will be able to adapt the composition as a function of the locus which is to be treated. It should be recognized that the active agents in accordance with the present invention may be blended with known agriculturally acceptable carriers and surface-active agents commonly used for improving the dispersion of the active agents. It should be recognized that the final formulation and mode of application of the acaricidal compositions and extracts of the present invention might affect the activity thereof. Of course, the present invention provides the means to determine the acaricidal effectiveness of different types of compositions.

Spreader and sticking agents can be added to a preferred embodiment of a formulation of the present invention in percentages ranging from 0.05% to 10%. Spreader/stickers which can be used in the formulations of the present invention include but are not limited to Schercoat P110 from Scher Chemicals of Clifton NJ, Pemulen TR2 from BF Goodrich of Brecksville OH and Carboset 514H also from BF Goodrich of Brecksville OH.

In the case of a ready-to-use formulation of the present invention, water is used as a carrier to make up the volume to 100% while in an emulsifiable concentrate, carriers can be THFA, or isopropanol. Isopar is added at quantities between 50 and 90%, depending on the concentration of the extract.

Preferably, the formulation has good physical and chemical properties and is stable after storage for 6 months at RT, 37°C and 50°C.

A preferred embodiment of the ready-to-use formulation of the present invention is presented in Table 3.

Table 3: Ready - to- Use Formulation

A preferred embodiment of the emulsion formulation of the present invention is presented in Table 4.

Table 4: Emulsifiable Concentrate Formulation

It will be understood that the oils and compositions of the present invention may also contain at least one further active ingredient.

Such active ingredient may be known compounds exhibiting pesticidal activity (acaricidal or otherwise) (e.g. other terpenes). It will be understood that this at least one additional active ingredient may or may not have a synergistic effect with the active compounds of the present invention.

The oils and compositions of the present invention may be used to control pest/infestation in different types of environments, including domestic, agricultural and horticultural environments. It will be understood that depending on the environment, or locus infested with or susceptible to be infested with the acari, that the compositions can be adapted accordingly.

The present invention also shows experiments to determine which variety, harvesting regime and distillation method could give the highest yield in oil. Experiments were also set up to compare yields of the oil of Variety 1 and Variety 2 of C. ambrosioides, and according to the stage of the plant at harvest, i.e. leaf, flower or seed stage, the humidity level of the plant material before distillation, i.e. fresh (>75%), wilted (40 to 60%) or dry

(<20%) and the extraction method used. As a further advantage of using herbaceous plant extracts instead of woody plant extracts in the present invention, the present invention includes the fact that herbaceous plants are faster and easier to grow thereby enabling the harvest of more than one crop a year. Extraction of essential oils was performed by methods that generate the volatile constituents as well as others, and the product is considered as an "extract" in the case of whole plant parts excluding the roots (Duerbeck 1993). Essential oils are generally not homogeneous. Their different constituents display variations in their chemical and physical properties. For the most part, all of these constituents are volatile in steam and relatively immiscible in water, a property which enables their separation, from a distillate mixture. There are many variations of the technique of steam distillation. Herein, a few methods are chosen for the purpose of extracting the essential oil of Chenopodium ambrosioides: Distillation in Water (DW), Direct Steam Distillation (DSD) (Duerbeck, 1993). Other extraction methods besides distillation such as solvent extraction and Microwave Assisted Process (MAP™) are described. Other known methods could be adapted by the skilled artisan. The two-spotted spider mite Tetranychus urticae is considered a model test subject in acaricide bioassays. Several articles have been written on various toxicological methods for testing the efficacy of acaricides and of their formulations and most methods used the two-spotted spider mite as a test species. A variety of methods were evaluated by Ebeling and Pence (1953), Ascher and Cwilich (1960), Dittrich (1962), Lippold (1963), Foot and Boyce (1966) and Anonymous (1968) and criteria for judging the adequacy of a method, i.e. precision, reproducibility of results and reasonable simplicity of operation were established by Busvine (1958) , all using the spider mite as a test model. Later, the spider mite was used frequently in the development of methods in detecting and measuring resistance (Walker et al. 1973; Anon. 1974; Dennehy et al. 1983; Dennehy et al. 1992).

Other objects, advantages and features of the present invention will become more apparent upon reading the following non-restrictive description of preferred embodiments which is exemplary and should not be interpreted as limiting the scope of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention therefore provides botanical acaricides which are shown to overcome a number of shortcomings associated with known acaricides. More specifically, essential oils of a number of plants are shown to display significant acaricidal properties. In addition, the compositions are shown to exhibit a significant insecticidal activity on several insect species that are serious pests of cultivated plants. The present invention provides and compares different methods of preparation of the essential oils. In one particular embodiment, the potent acaricidal activity of a botanical composition comprising C. ambrosioides extract is demonstrated and compared with known acaricidal compounds. The present invention is illustrated in further detail by the following non-limiting examples.

EXAMPLE 1 Selection of Chenopodium ambrosioides varieties

Chenopodium ambrosioides grows in temperate or subtropical areas. Aellen and Just (1943) and Voroshilov (1942) distinguish several subspecies and forms of C. ambrosioides. However, the characteristics used to distinguish the forms (i.e. stem and leaf pubescence, seed size, shape of perianth parts, inflorescence form and leafiness, shape of leaves, leaf margin characteristics) did not enable a categorization of the herein used varieties according to the classification in accordance with the prior art.

Using the Aellen and Just key for Varieties termed herein 1 and 2, the closest approximations is C. ambrosioides var. suffruticosum (Wild.) Aellen for Variety 1 and C. ambrosioides var. ambrosioides for Variety 2. In more recent taxonomic reviews, botanists do not recognize segregates from the C. ambrosioides complex. For the purposes of the present invention, regardless of whether the varieties are recognized as such, it has proven useful to select for varieties that have certain characteristics (short cycle, production of high quantity of seeds, smaller and fewer leaves, etc.) in order to maximize oil yields.

EXAMPLE 2 Crop production The plant can be successfully grown in temperate areas and a planting regime has been developed that enables the production of higher quantities of oil per kg of plant material having high percentages of active ingredients. Plants are started in the greenhouse in early March and transplanted in the field in mid-May. This regime permits the harvesting of at least two important crops of plant material for an average of 10-20 Metric Tons (MT) / ha per crop and 20 -40MT/ha per season and with a total yield production of between 20 to 100 LVha. Over the course of the present work, several varieties of Chenopodium ambrosioides were grown and these trials resulted in the production of a crop that can develop to the seed stage in 60 days to the first harvest (mid-July) and in 40 days to the second (mid to end of August) and third harvest (end of September). As mentioned above, attempts at identifying the different varieties were not conclusive. The two most promising selections were therefore identified as Variety 1 and Variety 2.

The speed in regeneration of the plant after the first and second harvests was determined through trials of differing cutting heights and it was found that a cutting height (of at least 10 cm but no more than 30 cm) permits rapid and dense plant growth producing flowers within days after harvest. This rapid regrowth is preferred in order to produce a maximum amount of plant material in the cooler temperate areas of the southwestern region of the province of Quebec, for example. Determination of oil yields using different distillation regimes and methods and determination of oil composition through chromatographic studies have resulted in the identification of a preferred variety, plant stage and plant treatment after harvest for optimum yield and quality of oil.

EXAMPLE 3

Characterization of the essential oil of Chenopodium ambrosioides

There are many compounds in the essential oil extract of a plant such as Chenopodium ambrosioides. Ascaridole and iso-ascaridole, two monoterpenes, constitute a significant proportion of the oil (between 10 - 70% and 3 - 20%, respectively). Biological activity however has been observed even when the quantities of these monoterpenes were minimal, suggesting that overall activity is due to the presence of a complex mixture of compounds. Other monoterpenes such as p-cymene, limonene, and α- terpinene are also major constituents and confer pesticidal properties to the oil. Table 5 lists the main compounds identified by gas chromatography (GC- FID) ; readings are from a DB-WAX column.

Table 5 : percentage (%) of compounds present in oil extracted from the seeds of C. abrosioides, varieties 1 & 2

The quantity of total oil extracted as well as the quantity (or %) of each compound present in the oil may vary with the variety of Chenopodium ambrosioides used, with the part of the plant harvested, age of the plant, and whether it is dried or fresh plant material, the time of the season it is picked, the climate where it is grown and the type of extraction method used.

The methods of the present invention can therefore be adapted by a skilled artisan to the different parameters which affect the yield and quality of the oil in order to achieve the type of oil which is required, and adapt the extract to particular needs.

EXAMPLE 4 Extraction method by distillation in water (DW)

The C. ambrosioides plant material was completely immersed in water which is brought to a boil by the application of heat directly to the tank containing the loosely packed plant material and water. A steam jacket or a steam coil wrapped around the tank can also be used to heat the water. The main feature in DW is that the plant material is always in contact with the boiling water and the volatile constituents of the plant are released into the water and recuperated after distillation and cooling.

EXAMPLE 5 Extraction method by direct steam distillation (DSD)

With this method, the C. ambrosioides plant material can be more densely packed in the tank as long as there is provision for the smooth passage of steam through it. The steam is generated externally with a boiler or steam generator permitting adequate control over the rate of entry of the steam. The steam, which must be partially wet, interacts with the plant material causing the essential oils within the cell membranes to diffuse out and form mixtures with the water vapor. These essential oil constituents are volatile at temperatures below 100 degrees Celsius, and hence distill with the steam over the plant material. The steam and volatiles are then condensed as they pass through a cooling unit outside the tank and the oil is then recuperated as it settles over the water collected from the condenser.

EXAMPLE 6 Organic solvent extraction Organic solvents can also be used to extract organically soluble compounds found in essential oils. Non-limiting examples of such organic solvents include methanol, ethanol, hexane, methylene chloride. These extraction methods may use extractors of different types such as the Soxlet which is a specialized glass refluxing unit in which the material is extracted over a 18 h period at a temperature of 80°C. Results from trials using methanol, ethanol, hexane and by using the Soxlet gave yields of between 0.5 and 3%. Extraction using solvents on a greater scale (i.e. for commercialization) might require an elaborate pressurized unit. Its high flammability and explosive potential could impose highly sophisticated and therefore costly safety features.

EXAMPLE 7

Comparison of the effect of selected parameters on yield of oil obtained from C. ambrosioides

In order to determine which: (1) variety (Variety 1 and 2); (2) % of humidity at distillation (fresh, wilted and dry); (3) stage of plant at harvest (leaf, flower and seed); and (4) distillation method (DW and DSD) would give the highest yield in oil, a total of 30 combinations of the different variables were tested and each repeated three times for a total of 90 distillation periods. A summary of yields obtained for each variable is presented in the following table (Table 6). It is to be understood that given the different combinations used in this experiment, the averages presented here are only indications of yields that can be obtained with the different variables. When results from all 30 combinations are taken into consideration, the combinations which gave the highest yields (more than 0.5% ; Table 6) are those obtained from DW with Variety 2 at the seed stage. Fresh and wilted material gave higher yields than the dry material. Further experiments confirmed these results. DSD gave lower yield results than DW; nevertheless, DSD should be considered for a commercial extraction operation because this method is much more cost efficient and less labor intensive than DW.

Table 6: Average yield (%) in oil of the different varieties, % humidity of the distilled material, stage of plant at harvest and distillation method

Variety 2 was selected for oil production because of its highest oil yields ; This variety also has a shorter growing cycle (less than 60 days after transplanting) permitting more than one harvest per season.

It should be understood that in certain embodiments, different combinations could be more suitable to particular needs. In addition, as will be shown below, yield is not to be taken as the only important criteria. EXAMPLE 8

Determination of conditions producing oils with the highest % of acaricidal activity

Various experiments were undertaken to identify cropping and harvesting techniques and distillation methods that would optimize oil yields. The series of 30 extracts mentioned above were subjected to chromatographic analysis in order to determine the type and relative amount of each compound present in the oil.

Chromatographic separation of organic molecules was carried out. The essential oils and extracts were analyzed by capillary Gas

Chromatography (GC) equipped with a flame ionization detector (FID). GC was carried out using a Varian 6000 series Vista and peak areas were computed by a Varian DS 654 integrator. SPB-1 (30m X 0.25mm ø, 0,25μm) and Supelcowax (30m, 0.25mm 0, 0.25μm) fused silica columns were used. Compounds in the sample come off the column at different times in minutes

(Rt's or Retention Times) and these are compared to known standards and the compounds can thus be identified. When GC-FID gave ambiguous identification of certain compounds, Mass Spectrometry (MS) was used to compare the mass spectra of the compounds with a data base of known spectra.

In order to assess the contribution of ascaridole to the biological activity of the oil, the % ascaridole (readings from the Supelcowax column of the GC-FID) present in the different extracts prepared according to the above mentioned protocol (i.e. humidity levels, stages of development of the plant, variety, distillation method) were compared. Percentage obtained for ascaridole and iso-ascaridole were combined to give total ascaridole content.

Variability was considered important when the difference in amount of ascaridole present was at least greater than 10% between variables of each treatment. Therefore there was no important variability in the percentages of compounds present between oils extracted from plant material of different humidity levels (fresh, wilted, dry ; Table 7) and distillation method (DSD, DW ; Table 9). There was less ascaridole content in Variety 1 (Table 10), although the amount is still high (38.5%). The leaf stage of the plant (9.8%), however, gave much less ascaridole than the flowers (64.8%) and seeds (55.4%) (Table 8).

Table 7: Relative amount (%) of compounds present in the essential oils from plant material extracted at different levels of humidity (fresh, wilted and dry)

Table 8: Relative amount (%) of compounds present in the essential oils extracted from plant material harvested at different stages of development (leaf, flower and seed). Fresh plant material of variety 1 and extracted by DSD was used.

Table 9: Relative amount (%) of compounds present in the essential oils extracted by DSD and DW. The seed stage of fresh plant material of variety 1 was used.

Table 10: Relative amount (%) of compounds present in the essential oils extracted from plants of Variety 1 and 2. Fresh plant material at the Seed stage and extracted by DWwas used.

The thirty extracts representing the treatments and combinations mentioned above i.e. varieties, humidity levels, stage of the plant material and method of distillation were then tested for their biological activity to indicate whether the variation in ascaridole content had an effect on the degree of toxicity to the test organism. All bioassays were carried-out on the two-spotted spider mite, Tetranychus urticae (a model system for acaricide bioassays, see above).

Unfortunately, there was no significant positive correlation between the relative amount of ascaridole in an extract and its acaricidal activity. In other words, it was determined that the extracts having the highest % of ascaridole were not necessarily the most active acaricidal extracts. Thus, while ascaridole can be associated to the acaricidal activity of the oil or compositions of the present invention, other compounds appear to be necessary for same. Indeed, purified ascaridole did not exhibit the significant acaricidal activity displayed by the ascaridole-containing plant extracts of the present invention.

EXAMPLE 9 Ready-to-use (RTU) acaricidal formulations

A ready-to-use (RTU) sprayable insecticidal formulation having as the active ingredient an extract of Chenopodium was prepared. In a preferred embodiment, this formulation contains between 0.125% and 10% of Chenopodium extract, an emulsifier, a spreader and sticking agent and a carrier. Examples of nonionic and anionic emulsifiers that can be used in the formulations of the present invention have been given above.

Non-limiting examples of formulations in accordance with the present invention are given in Tables 11 and 12.

Table 11 : Example of RTU formulations without spreader/stickers

Ingredient Amount (%)

Chenopodium extract 1.00 1.00 1.00

Rhodacal lPAM 0.50 0.83 0.83

Igepal CA-630 - 0.50

Macol NP 9.5 - - 0.50

Water 98.5 97.67 97.67 Table 12 : Example of a RTU formulation with spreader/stickers

Ingredient Amount (%)

Chenopodium extract 1.00 1.00 1.00

Rhodacal IPAM 0.83 0.83 0.83

Igepal CA-630 0.50 0.50 0.50

Carboset 514H 2.00 - -

Pemulen TR2 - 0.05 -

Schercoat PHO - - 5.00

Propylene glycol - 2.00 -

Water 95.67 95.62 92.67

EXAMPLE 10 Emulsifiable concentrate (EC)

An emulsifiable concentrate formulation with an extract of Chenopodium was also prepared. The concentrate contains between 10 to 25% extract of Chenopodium, emulsifiers, a spreader/sticker and a carrier. Non-limiting examples of formulations in accordance with the present invention are given in Table 13.

EXAMPLE 11 Toxicity of a RTU formulation

To test the toxicity of a preferred formulation, thirty adult female mites were placed on their dorsum with a camel hair brush on a double-sided adhesive tape glued to a 9 cm Petri dish (after Anonymous 1968). Three dishes were prepared for each concentration of each formulations or products tested and the control, (e.g. water), for a total of 90 mites per treatment per treatment day.

One (1) ml of each preparation and of microfiltered water as control was added with a Gilson Pipetman™ P-1000 to the reservoir of the spray nozzle of a Potter Spray Tower mounted on a stand and connected to a pressure gauge set at 3 P.S.I. Petri dishes were weighed before and immediately after each application to calculate the amount of oil deposited (mg/cm²) with each sample tested.

The ready-to-use formulation (hereinafter "RTU" formulation; using Variety 2 and the DSD method) was tested according to the method mentioned above to identify the minimum concentration needed for the desired mortality (>95%) at different concentrations (0.125, 0.25, 0.5. 0.75, and 1%) in order to compare the relative efficacy of this RTU formulation and other acaricidal products (synthetic and natural) presently on the market.

The entire procedure was followed three times to give a total number of 270 mites tested with each treatment.

Mite mortality was assessed 24 and 48h after treatment. Mites that failed to respond to probing with a fine camel hair brush with movements of the legs, proboscis or abdomen were considered dead. In order to obtain LC₅₀ values (Lethal Concentration in mg/cm² is the amount of product needed to kill 50% of the test organism; therefore the lower the LC₅₀ value the more toxic the product) results of the 48h counts were subjected to Probit analysis using the POLO computer program (LeOra Software, 1987). Mortalities were entered with corresponding weighed dose (mg/cm²) to take into consideration variability in the application rate. The results obtained with these bioassays are shown in

Table 14.

Although the toxicity tests presented herein were performed with female mites, it will be clear to a person skilled in the art that those results show that the mortality that would have been observed for male mites would have been the same if not higher knowing that male mites are smaller than females.

Table 14 : Adult spider mite (Tetranychus urticae) mortality obtained with bioassays using the RTU formulation of Chenopodium ambrosioides and commercial preparations of natural and synthetic insecticides

Dose recommended on label

These tests were also performed on several insect species that are serious pests of cultivated plants. The species tested were the greenhouse whitefly, Trialeurodes vaporariorum, the Western flower thrips, Frankliniella occidentalis, the green peach aphid, Myzus persicae and the silverleaf whitefly, Bemisia argentifolii.

Results presented in Table 15 indicate that the RTU product is toxic to all organisms tested. LC₅₀ could be calculated for the greenhouse whitefly and the green peach aphid and results (LC₅₀ of 0.00131 mg/cm² and 0.0009 mg/cm² respectively) show that the product is as or more effective to these insects as to the spider mite.

Table 15. Mortality (%) following bioassays with the spider mite, using the ready to use (RTU) formulation of Chenopodium ambrosioides.

^: Insufficient data for LC₅₀ analysis EXAMPLE 12

Toxicity of the EC formulation

The Emulsifiable Concentrate (EC) formulations defined in Example 10 were tested on spider mite adults using the same methodology as with the RTU formulation. Results presented in Table 16 indicate that at a concentration of 1.0%, an EC formulation is as effective as Safer Soap and potentially more toxic because the LC₅₀ is lower for the EC product (0.009) than for Safer Soap (0.016).

Table 16. Adult spider mite mortality (%) using an Emulsifiable Concentrate formulation of Chenopodium ambrosioides and a commercial natural pesticide.

EXAMPLE 13

Effect of the acaricidal compositions of the present invention on the egg and nymphal stages of the spider mite

The RTU formulation was also tested on the egg and nymphal stages of the spider mite. Results of the test on the egg stage (Table 17) indicate that the RTU formulation has some effect on the eggs with 30% mortality using a 0.5% solution of the oil. It is expected that a higher concentration of the oil should show greater efficacy on eggs.

Table 17 : Spider mite egg (Tetranychus urticae) mortality, using the RTU formulation of Chenopodium ambrosioides oil

Similarly to the effect of the RTU formulation on the nymphal stage, even at the 0.5% concentration, the RTU gave higher results (95.8%) than the existing commercial preparations of either Avid (80.1%) or Safer (61.7%) (Table 18). Table 18 : Spider mite nymph (Tetranychus urticae) mortality, using the RTU Chenopodium extract formulation and commercial preparations of synthetic and natural products

Recommended dose

EXAMPLE 14

Residual effect of the RTU formulations of the present invention and comparison thereof with commercially available acaricidal products

The residual effect of the RTU formulation was also tested with the spider mite and compared to natural and synthetic products already on the market, (i.e. Kelthane™, Avid™, Safer's™ soap and Wilson's dormant oil). The procedure for this test involved the preparation of vials containing a nutrient solution in which individual faba bean leaves were placed. Eighteen leaves were prepared for each concentration tested and each were sprayed with the indicated concentration until run-off and allowed to dry. Ten spider mites were placed on nine of the leaves one hour after spraying and ten were placed on the other nine leaves one day following treatment. Mortality was observed 24 and 48 hr following mite introduction on the leaves. The entire procedure was repeated three times.

The results of the residual effect of the different products when the mite is introduced on the plant one hour following treatment are shown in Table 19. These results indicate that there is a residual effect of the RTU and that this effect is greater than in the Safer product. However, it is inferior to the residual effect of synthetic products such as Kelthane and Avid.

Table 19 : Residual effect of the RTU formulation and of selected synthetic and natural acaricides when the spider mite is in contact with the product 1 hour following treatment on faba bean leaves.

These results show the RTU formulation's very low persistance in the environment (about 23 mortality of spider mites when the pest is introduced on the plant one hour after treatment with the product). The RTU formulation is therefore compatible with the recommandations of an Integrated Pest Management program which supports control methods that do not harm natural enemy populations.

EXAMPLE 15

Confirmation of the acaricidal activity of the extracts on an other acari

To confirm the efficiency of the formulations of the present invention on plant infesting acari in general, certain bioassays were performed on another plant infesting mite, the European red mite,

Panonychus ulmi, a mite which shows a close taxonomical relationship with T. urticae.

The RTU formulation was thus tested on the red mite Panonychus ulmi, a pest of apple orchards, following the same protocol described for contact efficacy on adult spider mites in order to confirm its broad effect as an acaricide. The results confirm the effectiveness of Chenopodium ambrosioides extract as a contact acaricide (Table 20) which is not exclusively active on T. urticae. Table 20: red mite, Panonychus ulmi mortality, using the RTU formulation

EXAMPLE 16

Compatibility with Integrated Pest Management Programs: non-toxic effect to products developed from extracts of the present invention on mammals and on the unlikelihood of the development of resistance thereto

The compositions of the present invention are contact acaricides with low residual activity. These compositions show a low persistance in the environment (22% mortality of spider mites when the pest is introduced on the plant one hour after treatment with the product). These compositions are thus compatible with the use of biological control agents used in an Integrated Pest Management program, through "ecological selectivity" (van den Bosch and Stern, 1962), whereas selectivity can be attained if beneficial organisms survive the initial application, in tested places.

Nicotine, a botanical compound, has long been known to have little or no residual effect on beneficial forms (Ripper 1956 ; Walton and Whitehead Furthermore, the complexity of a botanical product containing more than one active ingredient such as those of the present invention also makes resistance less likely. IPM encourages the use of 'biorational pesticides' (also known as least-toxic or biopesticides). The advantages of biorational pesticides over conventional chemicals are their selectivity to a targeted pest, lower toxicity to beneficial insects and to farmer or greenhouse workers and shorter re-entry intervals of workers to the crop. Plant essential oils are a complex mixture of compounds of which many can be biologically active against insect and mite pests, the compounds acting individually or in synergy with each other, to either repel or kill the pests by contact. Because of the complexity of the mixture, it has been observed that pests do not easily develop resistance to these products as they can to synthetic pesticides. Few acaricides are available on the market and unfortunately resistance has developed to many of these. It has been indeed demonstrated that repeated treatments of pure azadirachtin (active ingredient in the extract of neem seed kernels) against the green peach aphid, led to a 9-fold resistance within 40 generations compared to an untreated group of aphids (Feng and Isman, 1995); however the repeated use of a whole neem extract gave no sign of resistance during this same period (40 generations). It follows that the compositions of the present invention offer a definite advantage to the potential long term use thereof over presently used synthetic acaricides.

Although the present invention has been described hereinabove using extracts from Chenopodium, the present invention should not be so limited. Indeed, a person skilled in the art could select other types of plants provided that they contain acaricidal activity (as exemplified hereinbelow with A. absinthium and T. vulgare).

EXAMPLE 17 Other plant extracts having acaricidal activity

Whole plants of A. absinthium and of T. vulgare were harvested in full bloom in the fall of 1993 from a cultivated plot at the Agriculture and Agri-Food Canada experimental farm at L' Acadie (45° 18 ' N, 73° 20 'W), Quebec, Canada. A Microwave Assisted Process (MAP™) and two variants of steam distillation i.e. Distillation in Water (DW) and Direct Steam Distillation (DSD) (Duerbeck, K., 1993), were used to extract the fresh plant material.

The MAP process uses microwaves to excite water molecules in the plant tissue which causes plant cells to rupture and release the essential oils trapped in the extracellular tissues of the plant (Belanger et al., 1991) For this method, whole plant parts were shredded and 20 g were immersed in 100ml of hexane and irradiated at 2450 Mhz for 90 seconds at an intensity of 675 W.

Distillation in water (DW) and DSD were carried out as previously described. Briefly, a 380 L distillator with a capacity for processing ca. 20 kg of plant material was used. During the process of DW, plant material was completely immersed in an appropriate volume of water which was then brought to a boil by the application of heat with a steam coil located at the base of the still body. In DSD, the plant material was supported within the still body and packed uniformly and loosely to provide for the smooth passage of steam through it. Steam was produced by an external generator and allowed to diffuse through the plant material from the bottom of the tank. The rate of entry of the steam was set at (300 ml/min). With both methods, the oil constituents are released from the plant material and with the water vapor are allowed to cool in a condenser to separate into two components, oil and water.

The essential oils and extracts were analyzed by capillary gas chromatography (GC). GC was carried out using a Varian 6000 series Vista equipped with two flame ionization detectors. Peak areas were computed by a Varian DS 654 integrator. SPB-1 (30m X 0.25mm ø, 0,25μm) and Supelcowax (30m, 0.25mm ø, 0.25μm) fused silica columns were used with helium as a carrier gas at a velocity of 30 cm/sec (1.5 ml/min). The oven temperature was programmed from 40°C to 240°C at 2°C/min and the injector and detector temperatures were set at 230°C and 250°C respectively.

The procedure described below was followed separately for each plant species. Three concentrations of each extract, i.e. from MAP, DW and DSD, were tested on the spider mite. Emulsions were made by first preparing a 300 ml stock solution of emulsifier (0.32% of Alkamul EL-620), denatured ethanol (9%) and microfiltered water. Microfiltered water was used for the control. Forty, 80, 160 and 320μl of oil were completed with the stock solution to 4 ml to give the 1 , 2, 4 and 8% solutions respectively. The 8% concentrations of the MAP extracts for both A. absinthium and T. vulgare were not prepared because of insufficient quantities of these oils.

Thirty adult female mites were placed on their dorsum with a camel hair brush on a double-sided adhesive tape glued to a 9 cm Petri dish (Anonymous, 1968, supra). Three dishes were prepared for each concentration of the oil extracted by the three methods and the control, i.e. water, for a total of 90 mites per extraction method per treatment day.

For each application (one per Petri dish), 1ml of each preparation and of microfiltered water for the control was added with a Gilson Pipetman^® P-1000 to the reservoir of the spray nozzle of a Potter Spray Tower mounted on a stand and connected to a pressure gauge set at 3 PSI. Petri dishes were weighed before and immediately after each application and, on average, 205mg (± 42; n=50) of solution was deposited on each dish, representing 2.1 (1%), 4.1 (2%), 8.2 (4%) and 16.4 mg/cm² (8%) of oil deposited with each concentration.

The entire procedure was followed twice (1 and 2% of A. absinthium MAP and 4% of T. vulgare MAP solutions) and three times (the remaining MAP and all DW and DSD solutions of both plant species). The third tests using MAP extracts were not done because of insufficient quantities of the oil.

Mite mortality was assessed 24 and 48h after treatment. As previously, mites that failed to respond to probing with a fine camel hair brush with movements of the legs, proboscis or abdomen were considered dead. Results of the 48h counts were subjected to Probit analysis using the POLO computer program (LeOra Software, 1987). Mortalities were entered with corresponding weighed dose (mg/cm²) to take into consideration variability in application rate. The significance of differences in LC₅₀ values was determined by comparing the 95% confidence intervals computed by POLO (LeOra Software, 1987, supra). Analysis of the oils

Chromatographic analysis of the oils extracted from A. absinthium indicated differences in chemical composition between extraction methods (Table19). Both sabinene and α-thujone were absent in the DSD oil and present in the MAP and DW oils and a compound identified as a C₁₅H₂₄ was present in DSD but absent in MAP and DW.

Table 21: Major compounds present in Artemisia absinthium oil extracted by MAP, DW and DSD

Relative amount of compounds (%)

Compounds with each extraction method

present K.l.a¹ K.l.p² MAP DW DSD

Sabinene 3 1101 2.4 4.5 0 α-thujone 1086 1380 1.4 2.9 0 β-thujone 1091 1399 11.5 32.1 12.3

C₁₀H₁₆O 1113 1439 54.1 36.1 49.1

Unknown #1 1465 1832 2.1 2.1 3.4

¹Kovat's Indice on apolar DB-1 column ²Kovat's Indice on polar Supelcowax column ³unknown

In T. vulgare extracts, β-thujone was the major component of all three extraction techniques (MAP : 92.2% ; DW : 87.6% ; DSD :91.9%) (Table 22). Terpin-4-ol and α-cubebene were present in the DW extract and absent in MAP and DSD. Table 22: Major compounds present in Tanacetum vulgare oil extracted by MAP, DW and DSD

Relative amount of compounds (%)

Compounds with each extraction method

present K.l.a¹ K.I.p² MAP DW DSD

α-thujone 1083.4 1378 0.357 0.935 1.058 β-thujone 1094 1400 92.203 87.635 91.875

Camphor 1118 1622 1.064 0.963 0

Terpin-4-ol 1158.3 1559 0 0.552 0 α-cubebene 1460 1663 0 5.063 0

¹Kovat's Indice on apolar DB-1 column ²Kovat's Indice on polar Supelcowax column ³On apolar DB-1 column

Bioassay results

After 48h, all three extracts (MAP, DW and DSD) of A. absinthium were lethal to T. urticae (Table 23). However, there was variability in the degree of toxicity of the extracts to the two-spotted spider mite. Thus, at 4% concentration, oil extracted by the MAP and the DW methods caused 52.7 and 51.1% mortality respectively, whereas oil extracted by DSD resulted in 83.2% mortality. LC₅₀ values obtained for oil extracted by MAP (0.134 mg/cm²) and with the DW (0.130 mg/cm²) whereas the LC₅₀ of the oil extracted by DSD was significantly lower (0.043 mg/cm²) (Table 24).

Table 23: Percent adult Tetranychus urticae mortality 48h following treatments with Artemisia absinthium oil extracted by MAP, DW and DSD

Concentration of oil (% 0/„)\1

Extraction 0 1 2 4 8 method

MAP 5.3 15.7² 19.5² 52.7 — ³

DW 2.8 20.5 28.2 51.1 65.6

DSD 2.6 42.1 71.3 83.2 92.8

¹ n = 270 mites for each concentration except where specified

² n = 180 mites

³ Quantity of MAP oil was insufficient for bioassays at this concentration Table 24: Probit analysis of adult Tetranychus urticae mortalities 48 h following treatments with Artemisia absinthium oil extracted by MAP, DW and DSD

Extraction n Intercept Slope t ratio* LC₅₀ 99% confidence method ± SEM ± SEM (mg/c²) limits of LC^,

MAP 900 2.12+0.31 2.44+0.30 8.06 0.134 0.096-0.280

DW 1260 1.72+0.15 1.94+0.16 11.78 0.130 0.081-0.205

DSD 1260 2.93+0.18 2.15+0.16 13.65 0.043 0.028-0.057

* (t values > 1.96 are significant at P=0.01)

The T. vulgare extracts were also lethal to the two-spotted spider mite (Table 25), though extracts obtained by DW and DSD had greater acaricidal effect than the extract obtained by the MAP process. At 4% concentration, the oil extracted by the DW and DSD methods caused 60.4 and 75.6% mortality respectively, while oil extracted by MAP gave 16.7% mortality. Table 25: Percent adult Tetranychus urticae mortality 48h following treatments with Tanacetum vulgare oil extracted by MAP, DW and DSD

Concentration of oil (% 0/Λ)1

Extraction 0 1 2 4 8 method

MAP 6.3 17.8 11.1 16.7² — ³

DW 5.6 48.1 64.9 60.4 89.3

DSD 5.0 52.9 64.1 75.6 95.6

¹ n = 270 mites for each concentration except where specified

² n = 180 mites

³ Quantity of MAP™ oil was insufficient for assays at this concentration

Probit analysis of mortality data obtained from bioassays with the DW and DSD methods could be compared; however analysis of the MAP mortality data gave unreliable results because of the high variation in % mortality values between replicates treated at the same concentration (Table 26). It is likely that this variation is due to the physical properties of the MAP extract. During this process, organic compounds such as waxes and resins were released from plant cells along with the essential oils. These products may not have been adequately mixed by the Alkamuls-EL620 emulsifier resulting in a heterogenous solution.

Table 26: Probit analysis of adult Tetranychus urticae mortalities 48 h following treatments with Tanacetum vulgare oil extracted by DW and DSD

Extraction n Intercept Slope t ratio* LC₅₀ 99% confidence method ± SEM ± SEM (mg/c²) limits of LG;₀

DW 1350 1.81±0.15 1.42+0.14 10.08 0.054 0.013-0.088

DSD 1350 2.50±0.17 1.86+0.15 12.43 0.046 0.022-0.066

* (t values > 1.96 are significant at P=0.01)

DISCUSSION

While some variation has been observed in the bioassays with A. absinthium and T. vulgare extracts, the present invention has nevertheless shown that A. absinthium oil extracted by DSD is more effective at controlling the spider mite than the A. absinthium oils extracted by the other methods. The sesquiterpene C₁₅H₂₄ compound, present at 4.2% in DSD and absent in the other two extracts (Table 21), may be responsible for the higher degree in biological activity. However, identification of the unknown C₁₅H₂₄ compound in A. absinthium, and bioassays with individual compounds using the same three extraction methods, will be necessary for the determination of the active ingredients found in A. absinthium oil.

The similarity in biological response between the oil of tansy extracted by DW and DSD, implies that terpin-4-ol and α-cubebene (present in DW and not in DSD) contribute very little to the acaricidal activity of the oil extracted by DW. Because of the considerably high % of β-thujone in all three extracts, this component is likely to be the main active ingredient (a.i.) with negligeable activity attributable to the other chemical constituents. This would explain the similar results obtained from DW extracts at 4% concentration (60.4% mortality and 87.6% β-thujone) and DSD extracts (75.5% mortality and 91.88% β-thujone) but does not account for the low mortality with the MAP extract (16.7 % mortality and 92.2 β-thujone). The MAP extract may not have been adequately emulsified in the solution due to the presence of waxes and resins.

Identification of the active ingredient(s) in an extract is essential for registration when developing a botanical pesticide. Variability in response from a series of essential oil extracts must be minimized in order to obtain consistency in toxicity of a product. In addition, other variables such as phenological age of the plant, % humidity of the harvested material and plant parts selected for the extraction must be considered for the extraction of oils with the highest biological activity (as seen above). DSD is the most widely accepted method for the production of essential oils on a commercial scale and should be considered for large-scale production of a biologically active oil because, besides producing oil of greater toxicity in the case of A. absinthium, it is less expensive and yields are comparable to that of the other extraction methods (Chiasson and Belanger, unpublished results). The amount of energy required to generate steam in DSD is considerably lower than that required to boil water for the DW process. MAP is still experimental, and cannot yet be considered for large scale production.

CONCLUSION

Thus, the present invention describes botanical acaricides, oil extracts and acaricidal compositions that display a potent activity on two species of plant infesting mites. These compositions are also shown to display acaricidal activity at different stages on the life cycle of the mites.

Furthermore, these compositions are shown to have a moderate residual effect on the spider mite one hour after introduction of treated leaves, such that they are compatible with an Integrated Pest Managament Program. Taken together, the acaricidal compositions of the present invention show a number of advantages over the known acaricidal products.

As shown in a preferred embodiment of the present invention, the botanical extracts of the present invention having an acaricidal activity on plant acari, comprise terpenes like p-cymene, α-terpinene, limonene, thymol, carvacrol and ascaridole. Furthermore, extracts of the present invention are shown to contain a dual acaricidal and insecticidal activity.

Although the present invention has been described hereinabove by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

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Claims (20)

WHAT IS CLAIMED IS:

1. A herbaceous plant extract having an acaricidal activity against plant acari.

2. The plant extract according to claim 1 , wherein said extract is from Chenopodium ambrosioides.

3. The plant extract according to claim 2, wherein said acari is from the Tetranychus genera.

4. The extract according to claim 3, wherein the acari is the two-spotted spider mite.

5. The extract according to claim 1, further having an insecticidal activity on plant-infesting insects.

6. A composition comprising the plant extract according to claim 1 wherein said plant extract contributes to between about 0.125% to about 10% of said composition together with a suitable carrier.

7. The composition of claim 6, wherein said carrier is a suitable emulsifier.

8. The composition of claim 7, wherein the emulsifier is a blend of at least one non-anionic emulsifier and at least one anionic emulsifier.

9. The composition of claim 7, wherein the emulsifier is a non-anionic emulsifier.

10. The composition of claim 7, wherein the emulsifier is an anionic emulsifier.

11. The composition according to claim 7, comprising about 0.125 to 5% of emulsifier.

12. The composition according to claim 6, further comprising a spreader/sticking agent.

13. An acaricidal composition against plant-infesting acari comprising between about 5 and 60% of α-terpinene, between about 5 and 30% of p-cymene, between about 1 and 20% of limonene, between about 10 and 70% of ascaridole, between about 3 and 20% of iso-ascaridole, between about 0.25 to 10% of thymol and between about 0.25 and10% of carvacrol.

14. The composition according to claim 13, wherein said acari is from the Tetranychus genera.

15. The composition according to claim 13, further comprising a suitable emulsifier.

16. A method of controlling plant-infesting acari, which comprises applying to a site where control is desired, a composition containing an acaricidally effective amount of a Chenopodium plant extract, together with a suitable carrier.

17. The method of claim 16, wherein said Chenopodium is of the ambrosioides species.

18. The method of claim 17, wherein said carrier is a suitable emulsifier.

19. The method of claim 17, wherein said acari is from the Tetranychus species.

20. A method of controlling plant-infesting acari, which comprises applying to a site where control is desired, the composition according to claim 6 containing an acaricidally effective amount of a Chenopodium plant extract, together with a suitable carrier.