WO2000021364A9 - A natural and safe alternative to fungicides, bacteriocides, nematicides and insecticides for plant protection and against household pests - Google Patents

Abstract

A composition to repel or kill insects, fungi, nematodes and bacteria, comprising, as an active ingredient, an essential oil or a component thereof, wherein said essential oil or component thereof is derived from at least one plant species in the Family Labiatae and Umbellifera is disclosed. Methods of repelling or killing insects, fungi, nematodes and bacteria using these compositions are also disclosed.

Description

A NATURAL AND SAFE ALTERNATIVE TO FUNGICIDES, BACTERIOCIDES, NEMATICIDES AND INSECTICIDES FOR PLANT PROTECTION AND AGAINST

HOUSEHOLD PESTS

This Application claims the benefit of priority to U.S. Provisional Application Serial No. 60/103,805 filed October 9, 1998.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to natural and safe compounds that are useful as insecticides, bacteπocides, fungicides and nematicides.

2. Background of the Related Art

Herbs and spices have played an important role in ancient life, as we have learned from

hieroglyphics on the walls of pyramids and the Scriptures of the Bible. Spices were ranked with

precious stones in the inventory of royal possessions and have played an important role in ancient medicine (J. S. Pruthi, 1980; Deans 1991).

While plants and plant extracts have long been used as medicaments, plants are also

known to produce compounds which have the effect of repelling or killing insects, nematodes,

bacteria and fungi that are harmful to the plants. The elaboration of natural products with

deterrent effects is known for many varieties of plants.

Essential oils (etheπc or volatile oils) are extracted from plant species by various

extraction techniques, including steam distillation including the plants belonging to Labiatae and

Umbellifera. Local populations in the Taurus Mountains in Turkey, where most of the plant

species belonging to the Labiatae Family are found growing as weeds, have traditionally used

these plants in teas or m oil form for thousands of years. Archaeological studies indicate that native plant species have been a part of common life in this area for thousands of years. The teas

are drunk from childhood to adulthood, and the people who drink them have a reputation for

vigorous health and longevity. These plants have been used to treat such ailments as stomach pains and intestinal infections. However, no experiments have been conducted to determine the

in vivo activity of these plants against plant pathogens and household pests.

The activity of essential oils has been investigated in various scientific studies. Research on the in vitro activity of essential oils against plant diseases caused by fungi, bacteria, viruses and nematodes and against insects have been initiated (Singh et al, 1983; YegenJ984; Yegen

et al. 1992; Muller-Riebau et al., 1995, 1997; Sara? and Tun<? 1995a; 1995b; Qasem and Abu-

Blan, 1996; El-Gengaighi et al, 1996; Lee et al., 1997; Tune and Sahinkaya, 1998). The works

are limited to the proof of in vitro effects and no tests to describe practical uses were conducted.

For example, in vitro inhibition of growth of different phytopathogenic fungi has been described using essential oils from Seseli indicum (Chaturvedi and Tripathi, 1989), Bifora

radians (Yegen, 1984), Hyptis suaveolens (Pandey et al. 1982), Mentha piperita, Mentha citrata

and Cymbopogon pendulus (Matti et al. 1985), Achryanthus aspera (Chakravarty and Pariya,

1977), and Peperomia pellucida (Singh et al. 1983). There are also a few reports describing in vitro anti-fungal activity of wild plant species widely present as weeds in Turkey. Akgul and

Kivanc (1989) have described in vitro activity of various Turkish spice extracts against food-

borne fungi including Aspergillus spp., Penicillium spp., Rhizopus spp. and Mucor spp.

Different plant extracts as well as their components had differential in vitro activity against

various organisms i.e. Aspergillus spp. (Moleyar and Narasimham, 1986; Asthana et al., 1986;

Farag et al, 1989b). In vitro antifungal and antibacterial activities of aqueous extracts from Thymbra spicata,

Mentha spicata, Satureja thymbra and Laurus nobilis were investigated in Petri dishes using

standard assays by Akgul and Kivanc (1988a,b). The aqueous extracts of T. spicata and S. thymbra were found to have the highest anti-bacterial activity. Our own in vitro assays have

confirmed the differential anti-fungal activity of extracts obtained from six selected plant species :

T. spicata, Satureja thymbra, L. nobilis, M. spicata, Salvia fruticosa and Inula viscosa, against

four plant-pathogenic fungi (MIC between 400-800 mg/mL medium) (Yegen et al., 1992). The

volatile phase of these extracts was also found to be active. The activity of essential oils against Phytophthora capsici was better than the activities of the fungicides carbendazim and

pentachlomitrobenzol. Fungitoxic components of the extracts were determined via thin layer chromatography to include carvacrol and thymol.

In vitro activity of the essential oils of Saturej a-types against yeast have been reported

(Conner and Beuchat, 1984a,b). While in our tests, essential oil extracts from S. thymbra and

T. spicata had the highest activity against the growth of test fungi, Akgul and Kivanc (1989) observed a negligible anti-microbial activity against food-borne fungi Aspergillus sp, . Mucor sp.,

Penicillium chrysogenum and Rhizopus sp. using the essential oil from Satureja hortensis in

comparison with oils from other spices. These studies indicate that the activity of essential oils

is affected by the species of plant. Maiti et al. (1985) have observed that the essential oils from

M. spicata and extracts of mentha-types have activity against 3 phytopathogens: Rynchosporium

oryzae, Drechsbra spp. as well as Xanthomonas campestris. The anti-microbial activity of

essential oils of other spices (i.e. sage rosemary, caraway, cumin, clove and thyme) against

bacteria and fungi has been described (Farag et al. 1989a,b; Akgul and Kivanc, 1989) indicating

broad range of activity among the spices. Also, extracts from leaves of the laurel tree were found to be active against fungi in vitro in our tests (Yegen et al. 1992) as well as others (Akgul and

Kivanc, 1989).

Singh et al. (1983), Asthana et al. (1986), Thompson and Cannon (1986), Farag et al.

{ 1989), Chaturvedi and Tripathi (1989), Tripathi et α/., 1986; as well as Kivanc and Akgul ( 1990) determined minimum inhibitory concentrations of different essential oil extract from various

plant species against many strains of bacteria, yeast and fungi being between 250 and 2000 ppm.

The minimum inhibitory concentrations of a few individual volatile chemicals that are recommended for use in the storage of wheat grains from fungal deterioration have been determined to be 100 ppm or more in vitro, and most compounds were found to be phytotoxic

at their minimum inhibitory concentrations (Ghosh and Nandi, 1989; Moleyar and Narasimham,

1986). Moreover, Moleyar and Narasimham (1987) described that the activity was higher at low

microbial concentrations in shake cultures and the activity was decreased due to rapid

detoxification of components of some essential plant oils, like menthol and citrus, by Aspergillus niger and Rhizopus stolonifer during the culturing period.

The activity of the essential oils against Phytophthora capsici was higher than both the

fungicidal compounds, carbondazim and pentachlorintrbenzal (Yegen et al. 1992). In addition

to this study, Asthana et al. (1986) found that the essential oil extracts from Ocinum adscendens, with minimum inhibitory concentrations of 300 μg/g, had greater growth retardation activity

against Aspergillus flavus when compared to a few fungicides (moderately inhibitory

concentrations were between 2,000 and 4,500 μg/g). While the activity of essential oils from the

plants belonging to Labiatae and Umbelliferae have been studied by various scientists, practical

uses in agriculture or against household pests have not yet been either studied or described. The use of pesticides as fumigants for plant protection has become a major problem in

agriculture due to soil and ground water contamination, the damaging effects of these chemicals on the ozone layer covering the Earth, and negative effects on human and animal health. Indeed,

many commonly used pesticides, i.e. the ones containing carbamizadine, have been found to be

carcinogenic and or teratogenic while others, i.e methyl bromide, have been banned from use due

to their negative effects on ozone layer. Discovering naturally derived alternatives to

petrochemically-derived pesticides has become essential to achieve sustainable crop production using current agricultural resources and production methods.

Alternative pesticides used for the preservation of food during transportation and storage

have been sought after due to the adverse effect of these chemicals on the environment and

animal health. Natural products of plants are known to be active against pathogenic organisms.

For instance, plant secondary metabolites have been shown to be active in vitro against food poisoning bacteria (Dabbahetal. 1970; Beuchat 1976; Huthanen 1980; Tharib etal. 1983; Aktug

and Karapinar 1986; Deans and Richie 1987; Deans and Svoboda 1988, 1989). The

antimicrobial activity of essential oils obtained from some of Turkey's wild growing plants, as

well as other weed species, plants or trees from around the world, are already known. Numerous

works have dealt with the usage of essential oils to kill microorganisms causing spoilage and in the conservation of food supplies (Conner and Beuchat 1984a, 1984b; Benjilal et. al. 1984; De

Boer et al. 1985; Thompson and Cannon, 1986; Moleyar and Narasimhan, 1986, 1987;

Thomson, 1986; Thompson et al., 1987; Akgul and Kivanc, 1988a, 1989a, 1989b; Farag et al.

1989a, 1989b). At the same time research regarding the use of essential oils for post-harvest prevention of the establishment of aflatoxin through Aspergillus spp. has advanced (Charkavarty and Parya, 1977; Pandey et al, 1982; Ghosh and Nandi, 1982; Maiti et al, 1985; Tripathi et al, 1986; Asthana et al, 1986; Charrurvedi and Tripathi, 1989).

While Thompson and Cannon (1986) observed inhibition of fungal growth using essential

oils fromMentha sp., Salvia sp. andLaurus sp., Thompson (1986) failed to detect activity against the germination of spores from fungi belonging to genus Aspergillus, Mucor and Rhizopus.

The partly contrary results found in the literature, concerning the antimicrobial effect of these essential oils, led us to believe that the activity of essential oils can vary drastically due to

differences not only in the plant species of origin, but between varieties and individual plants, different growing conditions, extraction procedures, as well as due to variations in strains of test

micro-organisms. Therefore, we have concentrated our efforts on a few species belonging to

Turkish natural fauna, doing extensive selection and breeding, and determining the best growing conditions and times for oil extraction experiments, which were not conducted in previous

studies. It was found that the minimum inhibitory concentration of the volatile phase of essential

oils, as well as in formulations described herein vary between about 50-200 ppm, and have the

highest biological activity of any essential oil extracts described in the literature.

Several other scientists mentioned the need for discovery of a technique for practical

application of essential oils in agriculture (Calderone and Spivak, 1995; Mansore et. Al. 1986;

Shimoni et al, 1993). For the first time, we have demonstrated that the essential oils extracted

from the plants indicated above, when emulsified in water, are not toxic at concentrations up to

about 1000 ppm to many plant species, including rose, which is considered to be one of the more susceptible plants to toxic agents. SUMMARY OF THE INVENTION

The results of our studies clearly demonstrate tat the essential oils derived from wild-

growing Turkish plants can be used to combat a variety of phytopathogens, including ones which

are difficult to effectively control with existing pesticides, i.e. Phytophthora sp. Essential oils derived from S thymbra and T. spicata lines selected and bred for high carvacrol and thymol

content are especially effective. The essential oils extracts can be used against plant foliar

diseases caused by a broad spectrum of fungi and bacteria, as well as insects, when applied in

varying concentrations in vapor form, or sprayed as part of an aqueous emulsion in water as well as in other preparations indicated herein. The extracts can be applied to soil as a methyl bromide

replacement to kill nematodes, insects, pathogenic fungi and bacteria either alone in drip- watering systems, or together with solarization when applied in vapor form and/or by pouring

or spraying to the developing root system around the growing area of plants in formulations

indicated herein. In addition to their nematocidal, fungicidal, bactericidal and insecticidal

activity, these extracts have beneficial side effects: they can increase the concentration of

beneficial microorganisms in soils, such as fluorescent pseudomonads. The extracts also have growth promotion effects on plants such as an increased germination rate, most probably due to

an increase in photosynthesis associated with increased chlorophyll content. Treated plants are

greener and clearly dark green in color compared to light green to yellowish color in untreated

plants when the extracts are used as a foliar spray and/or in soil applications. The extracts have high thermostability, high volatile activity, a broad spectrum of activity

against fungi, bacteria, viruses, nematodes and insects can serve as an effective replacement for

the fumigant methyl bromide (which is being phased out due to its effects on stratospheric ozone,

beginning in 2000) and other, hazardous chemical pesticides in agriculture. Pinpinella anisum contains over 80% anethole. We have found that tr /w-anethole has

fumigant activity that may match or exceed methyl bromide.

It is also possible to combine the essential oils with at least one other pesticide or control

agents for other organisms as a part of an integrated pest management (IPM) strategy.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the activity of essential oil extracts from Thymbra spicata { 100-400 ppm)

against Phytophthora capsici delivered via injection with zoospores (left), or naturally infected

(no injections) from field soil (right).

Figure 2 shows the activity of 100- 1600 ppm essential oils (a mixture of ^'Thymbra spicata

(30%)), Satureja thymbra (20%>) and Origanum spp. (40%>), Pinpinella anisum (5%) and

Foeniculum vulgare (5%) extracts) against Phytophthora capsici zoospore inoculation (1 x 10⁵

zoospores/mL).

Figure 3 shows the activity of 100- 1600 ppm essential oils (a mixture oϊ Thymbra spicata

(50%), Satureja thymbra (30%>) and Origanum spp. (20%) extracts) against Phytophthora capsici zoospore inoculation (1 x 10⁵ zoospores/mL).

Figure 4 shows the activity of 100-400 ppm essential oils (a mixture of Thymbra spicata

(70%)), Satureja thymbra (20%) and Anis anisum (10%) extracts) against Phytophthora capsici

zoospore inoculation (1 x 10⁵ zoospores/mL).

Figure 5 shows the activity of 100-400 ppm essential oils (a mixture of Thymbra spicata

(60%)), Satureja thymbra (20%>) Pinpinella anisum (10%>) and Foeniculum vulgare (10%>)

extracts) or 200 ppm Dazomet in soil naturally infested with nematodes, fungi and bacteria,

including Melodoigyne spp. and Phytophthora capsici, in pots. Figure 6 shows the activity of 100-400 ppm of essential oils (a mixture of Thymbra

spicata (60%), Satureja thymbra (20%) Pinpinella anisum (10%) andFoeniculum vulgare (10%)

extracts) or 400 ppm Dazomet in soil naturally infested with nematodes, fungi and bacteria, including Melodoigyne spp. and Phytophthora capsici, in pots.

Figure 7 shows the activity of 100-400 ppm essential oils (a mixture of Thymbra spicata

(60%)), Satureja thymbra (20%) Pinpinella anisum (10%>) and Foeniculum vulgare (10%>)

extracts) or 400 ppm Dazomet in field plots naturally infested with nematodes, fungi and

bacteria, including Melodoigyne spp. and Phytophthora capsici.

Figure 8 shows the activity of a bacterium {Pseudomonas fluorescens TR97) isolated

from soils treated with essential oils, against Phytophthora capsici zoospore inoculation ( 1 x 10⁵ zoospores/mL). All seeds were treated with a mixture of chitosan (80%) and essential oil from

Thymbra spicata var. spicata (20%) and planted in sterile soil. The bacterial treatment contained

1 x 10⁶ CFU/mL P. fluorescens TR97 suspended in the chitosan/essential oil mixture.

Figure 9 shows the activity of a bacterium {Pseudomonas fluorescens TR97) isolated

from soils treated with essential oils, against Phytophthora capsici zoospore inoculation ( 1 x 10⁵

zoospores/mL). All seeds were treated with a mixture of chitosan (80%) and essential oil from

Thymbra spicata var. spicata (20%>) and planted in sterile soil. The bacterial treatment contained 1 x 10⁶ CFU/mL P. fluorescens TR97 suspended in the chitosan/essential oil mixture. After seeding, 100-400 ppm of essential oils embedded in perlite were added to the pots, which were

covered with plastic film until germination (ca. 1 week).

Figure 10 shows activity of the volatile phase of essential oils extracted from Thymbra

spicata against Erwinia amylovora on Miller- Schroth (MS) media. Droplets containing essential

oils were applied to the lids of the glass petri dishes. 1, Control; 2, 50 μl/L essential oil; 3, 40μl/L essential oil; 4, 90 μl/L essential oil; 5, 60 μl/L essential oil; 6, 30 μl/L essential oil;

7, 80 μl/L essential oil; 8, 70 μl/L essential oil; 9, 20 μl/L essential oil.

Figure 11 shows the activity of the volatile phase of essential oils extracted from

Thymbra spicata against Erwinia amylovora on Miller-Schroth (MS) media. The indicated

amount of ^'Thymbra spicata (as shown in the Figure as "thyme oil") was mixed with 0.1 mL olive oil, and droplets of the mixture were applied to the lids of the glass petri dishes.

Figure 12 shows in vivo activity of 200 ppm essential oil from Thymbra spicata or copper

sulfate (commercial preparation) against Erwinia amylovora on pear shoots. The essential oil

was applied as an aqueous emulsion to the leaves, and the shoots were inoculated with a

suspension of 1 x 10⁴ CFU/mL E. amylovora. The picture was taken seven days after inoculation. CuS indicates copper sulfate (commercial preparation).

Figure 13 shows the number of shoots exhibiting symptoms of fire blight disease in field

trials on two pear varieties (SM, Santa Maria; W, Williams), 3 and 6 weeks after an application

of essential oil (200 ppm in an aqueous emulsion) from Thymbra spicata, or copper sulfate

(CuSO₄) (commercial preparation).

Figure 14 shows the systemic activity of essential oils on Xanthomonas campestris pv.

campestris in cabbage. The soil was treated with 100-200 ppm essential oil in an aqueous

emulsion from Thymbra spicata var. spicata and plants were sprayed inoculated with X. c.

campestris (1 x 10⁵ CFU/mL). Control treatments were sprayed with 100 ppm Tween 20

(emulsifying agent) in water.

Figure 15 shows the contact activities of essential oil from Thymbra spicata emulsified

in water (100 ppm) upon foliar spray (left) or soil drench (right) applications as compared to a

pathogen control treatment (center). Plants were spray inoculated with Xanthomonas campestris

JO- pv. campestris (1 x 10⁵ CFU/mL). The control treatment was sprayed with 100 ppm Tween 20

(emulsifying agent) in water.

Figure 16 shows the contact and systemic activities of essential oil from Thymbra spicata

emulsified in water (100-250 ppm) upon foliar spray (left) or soil drench (right) applications.

Plants were spray inoculated with Xanthomonas campestris pv. campestris (1 x 10⁵ CFU/mL).

Treating soil with the emulsion resulted in better protection from X. c. campestris, indicating a systemic activity.

Figure 17 shows the effect of essential oil from Thymbra spicata emulsified in water

(100-500 ppm) on the carmine spider mite, Tetranychus cinnabarinus . Spider mites present on

nearby infested plants were allowed to naturally infest these plants. Soil was drenched with an aqueous emulsion of essential oil in concentrations indicated (control plants were treated with

water). After two weeks, the plants treated with essential oil were sprayed with a 200 ppm

aqueous emulsion of the same essential oil. After foliar treatment, no spider mites were detected

on any of the sprayed plants. The inset illustrates the difference in leaf color between the control

(leftmost) treatment and the essential oil treatments (the latter are a darker green).

Figure 18 shows the toxicity of anethole vapors to the adults of T. confusum and S.

oryzae and the larvae of E. kuehniella.

Figure 19 shows the toxicity of anethole vapors to the eggs of T. confusum and E.

kuehniella.

DETAILED DESCRIPTION OF THE INVENTION

The essentials oils covered in this application may be extracted from plant species

belonging Labiateae and Umbelliferae. Suitable plants may include specimens in the genera Thymbra, Satureja, Origanum, Corydothymus, Pinpinella and Foeniculum. Examples of plant

species in the Family Labiatae include Thymbra spicata var. spicata (L), Satureja thymbra (L),

Origanum majorana (L), Corydothymus capitatus (L.) Reichb. fil., Origanum vulgare (L) subsp.

hirtum (Link.) Let., Origanum solymicum P.H. Davis, Origanum spyleum (L), Origanum bilgeri P.H. Davis, Origanum minutiflorum O. Schwarts & P.H. Dzvis,Organum saccatum P.H. Davis,

Origanum sriacum var. bevanii (Holmes) Letswart, Origanum onites (L), and Origanum

majorana (IX). Examples of plant species in the Family Umbellifera include Pinpinella anisum

L. and Foeniculum vulgare Miller. These plant species were identified by Prof. Dr. Huseyin Sumbul, Akdeniz University according to Volumes 4 and 7 of the series of books written by L.H. Davis, entitled: Flora of Turkey and Eastern Aegean Islands. The plant species identified

herein are illustrative only and are not intended to be limiting. Other plant species containing any

one of the components of the essential oil extracts, as identified by gas chromatography

(conducted in Gottingen University, Germany, Akdeniz University, and METU, Turkey using

methods described by Shultze et al. 1986) are encompassed within the scope of the invention. The components of essential oil extracts were verified by chemical analysis (Akgul, 1986;

Arrebola et al. 1994; Capone et al. 1988; Muller-Riebau et al. 1995; Philianos et al. 1984; Ravid

and Putievski 1984, 1986; Shukla and Tripathi, 1991; Sarer et al. 1985; Schaffer et al. 1986).

The compounds identified from extracts of these plant species (in alphabetical order) are: cis-

anethole, trans-anethole, anisaldehyde, anis ketone, anisole,β-bisabolene, borneol, bornyl acetate, cadinene, camphene, camphor, Δ-3-carene, Δ-4-carene, carophyllene, carvone, carvacrol, γ-

caryophyllene, cinnamic aldehyde, citral, citronellal, cineol, l,8-cineole,/)-cymene,yθ-cymene-8-

ol, decanal, estragole, eugenol, eugenyl acetate, α-fenchene, fenchole, fenchone, geranial,

geraniol, geranyl acetate, isoborneol, lavanduol, limonene, linalool, linalyl acetate, menthol, menthone, menthyl acetate, cts-p-menth-2-en-l-ol, tr< s^*-p-menth-2-en-l-ol, methoxy phenyl

acetone, methyl chavicol, methyleugenol, methylinone, 2-methylpentan-3-one, myrcene, nerol, nonanal, c/s-β-ocimene, tr< s^*-β-ocimene, octanal, 3-octanol, α-pinene, β-pinene, α-phelladrene,

β-phelladrene, pulegone, sabinene, c/s-sabinene hydrate, trαrø-sabinene hydrate, y-terminene,

terpenyl acetate, α-terpinene, γ-te inene, teφinene-4-ol, a-terpineol, β-teφineol, terpinolene,

2,3,5,6-tetramethylphenol, α-thujene, thymil acetate, thymol, and tricyclene.

Although all of these components have some biological activity, they work synergistically to provide a broad spectrum of activity. Total essential oil extracts, as extracted from plants,

have as good or often better activity than individual synthesized components, indicating that the components of essential oil extracts from these plant species act synergistically. Therefore, the

extracts can act as multisite pesticides, and their activity is relatively stable.

The major active components are: carvacrol, thymol, cymene and anethole. A suitable product for commercial use may contain various individual plant extracts or combinations of

individual plant extracts. For Example: T. spicata (10-90%>), S thymbra (10-90 %) and

Origanum spp. (5-30 %>) can be mixed to provide optimal activity against bacteria, viruses and

fungi, and T spicata (10-90 %), S thymbra (10-75), P. anisum (0.5-90 %) andF. vulgare (0.5-90

%) can be mixed to provide optimal activity against insects and nematodes. The activity of each

extract or combination of extracts against detrimental organisms varies. That is, a mixture

containing more carvacrol and thymol is more active against microbial pathogens, whereas a

mixture containing more carvacrol and anethole is more effective against insects and nematodes.

The invention also comprises chemically synthesized essential oil components which can

be used as pesticides and for pharmaceuticals. Anethole may be synthesized relatively easily, for

example, and is a effective pesticide suitably used in the invention to replace methyl bromide for fumigation, in storage applications and the like. Combinations of synthetic essential oil

components may also be produced and used within the scope of the invention.

The LD 50 values of the components of the essential oils (see Duke et al. 1992) indicate

that they are not highly toxic or teratogenic to humans and animals. Although concentrated oils are toxic to plants, and may cause a temporary painful inflammation on human skin, so far no

toxic effects to plants and/or to the environment have been recognized when the oils are used in

appropriate concentrations and in the preparations described herein.

Plants with Enhanced Essential Oil Activity

Carvacrol, thymol and anethole content was increased by up to ten-fold of the original, wild-grown plants in four selected plant species, using various selection and conventional

breeding techniques. Currently, the seedlings of obtained cuttings for Labiatae spp. or from

seeds of Umbelliformae spp. from the selected lines are growing under greenhouse conditions

for distribution to farmers, in order to prevent possible disturbances to the natural ecology of the mountains, which could be caused by the overharvesting of wild plants. None of the new

varieties of these species have been previously grown in the United States. The plant lines

selected for enriched essential oil content are Thymbra spicata var. spicata (L) Line Ant97- 364-

48, Satureja thymbra (L) Line Ant98-28-103, Pinpinella anisum (L) Line Ant98-223-137, and

Foeniculum vulgare (L) Line Ant98-89-62.

It was also determined that the highest essential oil content occurs during the flowering

period, which is late June to early July in the Mediterranean region of Turkey, for the two

perennial Labiatae species. Both Umbellifera species are annual plants and since seeds are used for extraction of essential oils, seeding period is the best time for essential oil collection. It is also possible to develop transgenic plants that have enhanced production of essential

oils or essential oil components. In particular, plants selected for enhanced production of trans-

anethole, carbacrol, and/or thymol would be expected to have more potent essential oil than

normal plants of the same species.

The essential oil extracts can be used against plant diseases caused by a broad spectrum of fungi, bacteria and nematodes as well as insects, when applied in varying concentrations mixed with other oils, in vapor form, or sprayed as part of an aqueous emulsion in water as well

as in other preparations indicated below. The extracts can be applied to soil when embedded in

perlite, in granular form, powder form, or emulsified in water and applied via drip-watering

systems. The extracts can also be used together with solarization when applied in vapor or any other form as well as by pouring or spraying aqueous emulsions around the growing area of plants in formulations indicated below. In addition to their nematicidal, fungicidal, bactericidal

and insecticidal activity, these extracts have beneficial side effects: when used as a soil and/or foliar spray and/or soil application, they can increase the concentration of beneficial

microorganisms in soils, such as fluorescent pseudomonads {Pseudomonas fluorescens), and have growth promotion effects on plants (i.e. increased germination rate, foliage production and

height). The growth promotion effects are most probably due to an increase in photosynthesis

associated with increased chlorophyll content (treated plants are greener and clearly dark green

in color compared to light green to yellowish color on untreated plants), or modifications in other

plant metabolic systems.

Essential oils as extracted are not soluble in water and are phytotoxic in undiluted oil

form, and therefore, cannot be used for direct foliar applications, and can be either adsorbed by

soil particles or toxic to plant roots when directly applied to soil. Therefore, the oils have to be formulated in granules or powders or absorbed in a carrier, such as perlite or vermiculite, for soil

applications and have to be emulsified in water for soil and foliar applications. Formulations

may be those found as commercial formulations used for other pesticides.

For soil applications, the essential oil may be mixed with a carrier substance, such as a

porous substance. Suitable porous carrier substances include perlite and vermiculite. For these

applications, at least about 0.5g of essential oil is combined with about 10-50g of the carrier substance, such as perlite, vermiculite or mixtures thereof. The applications using the carrier

substances may be applied to soil alone, or in combination with solarization. Solarization may

be conducted by covering the treated soil area with a transparent, impervious covering such as

polyethylene, and the soil kept moist for up to about 6 weeks. Plants can be transplanted into the soil immediately or several days after application of the essential oil treatment.

For application to irrigation water, essential oil concentrations may be about 10-1000 ppm. More preferably, the concentration of essential oil is about 100-1000 ppm.

For fogging applications, essential oils in emulsion form or diluted in other oils, or full

concentrate are atomized and applied over plants and/or soil in a storage area or greenhouse, for example. Typically a concentration of about 100-1000 ppm in air of the storage area is suitably

used.

The extracts obtained from the plants indicated above are also active, and being

considered non-toxic they can be used safely against, household insects, including, but not

limited to mites, spiders, dust mites, house flies, cockroaches, mosquitoes, fruit and garbage flies,

ticks and other pests, when applied in vapor or aerosol form, in dust or granular formulations,

diluted in carrier oils, extracted with solvents which dissolve essential oils or sprayed as aqueous

emulsions or in any other form onto places where insects are present, or on body parts and clothing. Essential oil extracts also can be used, when applied as a vapor or in other forms, to

control stored product insects (storage pests) to replace methyl bromide and/or other insecticides

when applied by atomizers, as vapors, i.e., from heated extracts or mixed in paint.

They can also be used as mixed with liquid paraffin as in any form indicated above for

protecting citrus and other fruits during transportation and storage, since coating orange and grapefruit fruits with paraffin containing about 0.5-90% essential oils increased storage time up

to ten fold. Although, increased concentrations of essential oils may result in an unpalatable odor, this can be eliminated by adjusting the oil content and mixture according to needs of

commercial applications. The results of current work clearly demonstrate that the essential oil of T. spicata has a

practical use as a soil fungicide and can be used as such, since good results against

Phythophthora capsici in greenhouse tests and in two field tests were achieved. In these early

tests, pure essential oil was adsorbed in perlite, which protected the essential oil from adsorption by other soil components, protected the plants against the phytotoxic effects of the undiluted oil

and ensured an equal distribution of the oil on the ground (Yegen et al, 1998). Although this

application of the essential oils proved to be effective, is was further determined that applications

of the extract in granular or dust formulations, or application as an aqueous emulsion directly to

irrigation water, in combination with covering the soil for a period of time prior to transplanting,

are more effective and practical methods. The tests where an aqueous emulsion of the essential oils poured on the soil around the plants (via irrigation or pouring by hand), or directly sprayed

on plants, provided protection against fungal, bacterial, viral pathogens, against nematodes and

insects on or around plants. We have further developed and tested practical uses against

household insects and other pests. Soil chemical fumigants and other treatments do not specifically target particular pests or pathogens. Therefore, they have negative effects on whole soil microfloral and microfaunal communities, including plant- and soil-beneficial organisms. The essential oils described in this

application, however, appear to work selectively. Detrimental organisms are targeted while

populations of soil- and plant-beneficial microflora, including fluorescent pseudomonads and

actinomycetes, are not decreased. In fact, population densities of these organisms actually increase after treatment, most likely due to the reduction in competition from phytopathogens and probable increases in available nutrients from lysed phytopathogen cells. These beneficial soil

organisms may influence plant disease resistance and growth either directly, or indirectly through

natural, microbially-mediated changes to the soil environment. A fluorescent pseudomonad

{Pseudomonas fluorescens TR97) able to degrade these essential oils has been isolated,

indicating that these oils can be metabolized. Unlike organochlorines and other recalcitrant pesticidal compounds, components of the essential oils will not accumulate in soils or in living

tissues. Some of these isolates also appear to act indirectly (as they have little direct

antimicrobial activity) to inhibit the growth of soil phytopathogens, either through the induction

of plant defense responses, or through suppression of the pathogens via competition.

The invention will now be described in further detail by reference to Examples, which

are intended to be illustrative of the invention, and not limiting. The scope of the invention is

defined in the appended claims.

EXAMPLES

1. Materials and Methods

Distillation of essential oils: 500 mL of distilled, deionized water were combined with

32 g of dried plant material or seed derived from one or several of the species indicated above and distilled with a Clevenger apparatus until 400 mL distillate was obtained. Extracted twice

with petroleum ether, the ether phase was separated and dried in a rotary evaporator at 45°C to

obtain the maximum amount of essential oils. Essential oils can easily be separated from water

using a separatory funnel. Under commercial factory conditions, about 1 kg of dried plant material or seed is generally mixed with about 10 liter of water or extracted in plant material with

vapor in commercial continuous extraction systems. It is not necessary to use petroleum ether

unless all the oil content is needed. Water and plant content may vary according to distillation technique.

Preparation of essential oils for practical use in plant protection: Essential oils from

the plants were emulsified in water. For a 1000 ppm emulsion, about 1 mL essential oil

containing extract(s) from one or several plant species was mixed with various concentrations

of Tween 20 or other commercial detergents (the optimal concentration is about 1 mL essential oil extract(s) dissolved in about 1 mL Tween 20) and added to about 1 L water. For optimal

emulsification, the water is acidic. Therefore, about 1 drop of concentrated hydrochloric acid per

L of water was used to bring down the pH of the water to approximately 5.0.

Activity of Essential Oils Against Phytophthora capsici Under Greenhouse and Field

Conditions:

(a) Soil tests: Soil samples were obtained from infected fields in Kumluca, Antalya,

Turkey, placed in the plastic pots and inoculated in the growth-chambers. Field tests were

performed at the same site where soil was obtained. 2 kg field soil was sifted through a 2 mm

sifter to a plastic container and the soil dampened with sterile distilled water to 75 % saturation.

Essential oils extracted from T. spicata and various mixtures of extracts were added in to the soil in an aqueous emulsion form by adding the emulsion to the irrigation water. Plots were covered with polyethylene plastic film to prevent evaporation of the essential oils. The activity of the

essential oils was compared to 400 mg/kg Dazomet (Basamid 980 active ingredient).

(b) Activity Against Pepper Root Rot Disease Caused by P. capsici: 500 g of sifted

soil was placed into a plastic container and dampened with distilled water to 75% to saturation.

Each container was inoculated with 2 mL zoospore suspension (1100 zoospores/mL) of P.

capsici. After 1 week 50, 100, and 200 mg essential oils of from T. spicata, or various mixtures

of essential oils as indicated above, formulated into aqueous emulsions, were mixed into the soil.

The resulting concentrations of essential oil concentrations in the soil was 100, 200, 400 and/or 1 ,600 mg/kg (See Figure Legends). The containers were covered with airtight plastic film and incubated at 25 °C for 5 days in a climate-controlled room. The film was then removed, and after

3 days of aeration, 15 pepper {Capsicum annum) seeds from a variety susceptible to P. capsici

(Demre, Vegetable seed Co. Antalya, Turkey) were seeded into each pot. The number of living

plants was determined two weeks after plantation. Field tests were conducted at a site located near Kumluca, Antalya, Turkey. Essential oil

extracts were added in water as indicated above. Parcels were covered for 5 days with plastic

film. The seeds from a susceptible pepper variety of C. annuum (Demre) were seeded in high

density equally in each plot, 8 days after the fumigation treatment. The number of diseased

plants as well as the dry weight of the plants (dried at 105 C for 24 hr), per m², was determined. The differential weight as well as the total sum of the weight of the plants were determined.

ACTIVITY OF ESSENTIAL OIL EXTRACT FROM THYMBRA SPICATA L. var.

SPICATA ON VARIOUS BACTERIA

(a) Isolation of the Essential Oil Top leaves and flowers of Thymbra spicata, Satureja thymbra, and Origanum spp. were collected from the wild at the time of flowering, while seeds were collected from Pinpinella

anisum and Foeniculum vulgare. 32 g of dried plant material or seed was steam-distilled with

500 mL of distilled water until 400 mL of condensed liquid was obtained. The separation from

the water was conducted twice by 800 mL of petroleum ether. The extract was steam-distilled

at 45°C until the petroleum ether (boiling range 60-80°C) was completely evaporated. The essential oil was stored in the dark at 4°C until further analysis.

(b) Contact and Volatile Phase Effects of the Essential Oil

Essential oil obtained from Thymbra spicata L. var. spicata was assessed for its contact

and volatile phase effects towards several economically important plant pathogens:

Agrobacterium tumefaciens, Clavibacter michiganensis subsp. michiganensis, Erwinia

amylovora, Erwinia carotovora pv. carotovora, Pseudomonas syringae pv. syringae and Xanthomonas axonopodis pv. vesicatoria. Bacteria (1 x 10⁵ CFU/mL) were incubated at 25°C

in nutrient broth (NB) containing 20-1,280 μg/mL essential oil. The minimum bacterial

concentration (the lowest concentration yielding fewer than 0 A % survivors) was determined by

plating 0.1 mL of the flask contents onto nutrient agar (NA) plates. The bacterial colonies on the NA plates were counted at 24 hr intervals for three days. Control flasks contained NB and 1 x 10⁵

CFU/mL of test bacteria.

Glass petri dishes of 100 mL capacity were used in the determination of volatile phase

effects of essential oil. The test bacteria (1 x 10³ CFU/mL) were plated onto NA, and the plates

were dried under aseptic conditions under a laminar flow hood. Different concentrations of essential oil (doses of 2-128 μl, corresponding to 20 to 1280 μg/mL air) were applied to the lids

of the petri dishes. The bottom of the petri dish was immediately placed on the lid and sealed by parafilm to prevent diffusion of essential oil from the dish. The sealed, inverted petri dishes

were incubated at 25°C for 3 days. The seals were removed after 3 days to release the volatile

essential oil. The petri dishes were incubated for an additional 3 days before determining the

minimum inhibitory concentration (MIC) of oil by counting bacterial colonies. The MSTAT

statistical program was used to compare treatments via Duncan's multiple range test (p=0.05).

The MIC was determined based on the equation of the regression analysis (Dimond et al., 1941).

Examples 1-3: Use of Essential Oils in Plant Protection:

1. Foliar Applications: (a) Spray Applications:

Essential oils extracted from plants can be emulsified in water and sprayed onto plants

at weekly intervals, or as needed, similarly to conventional pesticides. They can be used in

concentrations ranging from about 1 to about 1000 ppm according to the pest or pathogen targeted and/or to the sensitivity of the plant species (the best concentration is about 200 ppm for

most organisms and plant species). Essential oils also appear to improve plant health in the

absence of disease.

(b) Fogging with Atomizers:

Essential oils can be used in vapor form, mixed with other oils, dissolved in solvents

which dissolve essential oils, or in emulsion form to fog greenhouses or other buildings to kill

pathogens and pests using ultra low volume nozzles: about 1 -3 L per 1000 m² of greenhouse area,

down to about 0J5 ppm per 1000 m². Concentrations can be increased for application against

house pests and/or to fog greenhouses. Example 2. Soil Applications:

(a) Soil tests from the Field: Essential oil concentrations obtained from sampled field

soil and from the 2 kg field sifted field soil were between 100 to 1600 mg/kg soil in growth

chamber studies or 100-400 mg/kg soil in field tests. The application of essential oils even in

the lowest concentration (100 mg/kg) reduced the numbers of total microorganisms 7-25% even

1 day after application, compared to the controls. Populations of all three microorganisms (fungi, bacteria and actinomycetes) were significantly reduced when applied at 400 mg/kg in comparison

to the control (approximately 40%> for the fungi, 70%> for the bacteria and 40% for

actinomycetes). The populations of fungi and bacteria recovered more quickly than the

actinomycetes.

(b) Activity of Essential Oils Against Pepper Root Rot Disease: The result of growth

chamber tests were shown in the Fig. 1, 2, 3, 4, 5 and 6. The number of infected plants upon

pouring various essential oil emulsions were significantly lower compared to controls. The fresh

weight per plant and fresh weight per pot were increased compared to controls upon treatment

with essential oils (data not shown).

The total number of healthy and infected plants in the field experiments is summarized

in Fig. 7. Treatment of the soil with the aqueous solutions of essential oil extracts significantly

increased the total number of plants per m². Essential oil extracts provided better control

compared to Dazomet.

(c) Application as Imbedded in Perlite or Vermiculite:

Essential oils extracted from the plant species indicated above, alone or in combination,

can be embedded into a carrier such as perlite or vermiculite as extracted in powder or granular

formulations as formulated in commercial preparations, diluted in other oils, dissolved in solvents which dissolve essential oils or in an aqueous emulsified form. A minimum of 0.5 g of

essential oils were mixed with 10-50 g of perlite or vermiculite. The perlite or vermiculite was

then sprinkled on the soil surface or mixed to a 5-10 cm depth into soil, using a commercial fertilizer applicator, to cover an area of one (1) square meter. The surface of the soil was then

covered with a plastic sheet for at least 2 days for vaporization. Other commercially available

chemicals, such as materials known to induce systemic disease resistance in plants, including chitin and chitosan, biological control agents able to survive exposure to the oils or components

of biological control agents can be added to the same materials. Applications of perlite or vermiculite can be used alone or in combination with solarization. For solarization, the soil

surface should be covered with polyethylene and kept moist for up to six weeks. Plants can be

planted or transplanted into the soil immediately or several days after application according to

the plant species used.

(d) Application into irrigation water:

Essential oils diluted in other oils, dissolved in solvents which dissolve essential oils, or

used in an emulsified form in water as described above, can be added in a manner similar to

fertilizers to irrigation water (at about 10-1000 ppm concentrations, optimal concentrations

generally range from about 100-200 ppm), and also directly to plants in drip-water irrigated

greenhouses to reduce, minimize or completely halt the diseases caused by soil pathogenic fungi,

bacteria, or nematodes and the damage caused by insects. First irrigation can be made before

transplanting, and the soil can be covered with polyethylene to increase the vapor effect and to

allow for the integrated use of solarization. The application into irrigation water can be continued

during the growing season to increase activity.

Example 3. Storage Applications: The essential oils extracted from the plants indicated above, and the vapors of these oils,

can be used to kill storage pests and pathogens. Fogging, as indicated above, increases the

activity of the oils due to a higher distribution rate to a larger area. Essential oils may also be

used when applied as vapors, i.e., from heated extracts or mixed in paint (preferably, an oil-based paint). Heating is not required for vaporization, however, heating improves vaporization. Bombs

can be made by formulating the essential oils in preparations similar to preparations used for

methyl bromide in which the essential oil compositions are packaged in pressurized cans. Low

concentrations (about 25-1000 ppm in air volume of storage area) of vapors from essential oils can provide a good alternative to methyl bromide to kill insects or microorganisms attacking produce under storage and transportation conditions. Notably, anethole is highly effective for

this application and may be extracted from plants or is easily synthesized. We have found that

although both cis- and tra/M-anefhole are effective, trαrw-anefhole is more effective. Essential

oils can also be used as mixed with liquid paraffin as in any form indicated above for protecting citrus and other fruits during transportation and storage.

Example 4: ACTIVITY OF ESSENTIAL OIL EXTRACT FROM THYMBRA

SPICATA L. var. SPICATA ON VARIOUS BACTERIA

(a) Contact and volatile phase effect of Essential Oil

The contact and volatile phase effect of different concentrations of essential oil differed

against the various plant pathogenic bacteria tested. The number of the living bacterial cells decreased as the dose of the essential oil increased (Tables 1-3). The volatile phase of the

essential oil was more effective on E. amylovora, X. a. vesicatoria, C. m. michiganensis and

tumefaciens than on P s. syringae and E carotovora (Table 2, 3).

* No significant differences between treatments in Experiments I and II were detected using t- tests (p=0.05).

** Differences between treatments were identified using Duncan's analysis (p=0.05). Treatments followed by the same letter are not significantly different.

* Values are means of four replicates. Values within one column followed by different letters are significantly different at p = 0.05 (Duncan's Multiple Range Test). A.t. Agrobacterium tumefaciens; C.m.m. Clavibacter michiganensis; E.c.c. Erwinia carotovora pv. carotovora; E.a. Erwinia amylovora; P.s.s. Pseudomonas syringae pv. syringae; X.a.v. Xanthomonas axonopodis pv. vesicatoria.

* Values are means of four replicates. Values within one column followed by different letters are significantly different at p = 0.05 (Duncan's Multiple Range Test). A.t. Agrobacterium tumefaciens; C.m.m. Clavibacter michiganensis subsp. michiganensis; E.c.c. Erwinia carotovora pv. carotovora; E.a. Erwinia amylovora; P.s.s. Pseudomonas syringae pv. syringae; X.a.v. Xanthomonas axonopodis pv. vesicatoria. The essential oil in the contact effect tests showed MIC ranging from 276 μg/mL to 413 μg/mL (Table 4). In most cases, the volatile phase effect of the essential oil was more toxic to

the test bacteria than contact with the essential oil. The essential oil in the volatile phase effect tests showed an MIC ranging from 41 μg/mL to 684 μg/mL (Table 4).

Example 5. Use of essential oils against household pests:

The essential oils extracted from the plants indicated above are also active against

household pests, and could replace other pesticides or deterrents. These oils can be used as

extracted, in vapor phase, diluted in carrier oils, dissolved in solvents which dissolve essential

oils or emulsified in water to spray homes or other buildings and/or clothing to kill or deter

insects (i.e. ants, house flies, spiders, mites, fleas, mosquitoes, termites, ticks, etc.). They also

can be applied to skin in a cream or spray form to deter insects such as mosquitoes and ticks.

Example 6. Activity of essential oil extracts against honeybee parasites:

The activity of essential oils against chalkboard of honeybee caused by Ascosphaera apis (Maasen ex Claussen) Olive & Spiltoir was determined in several honeybee colonies. Aqueous

emulsions of essential oil extracts were not active against honeybees (some mortality at about

1000 ppm) although they were found to be extremely active against the parasite. Both aqueous

and volatile phase of essential oils in insecticidal preparations killed the Ascosphaera sp. under

laboratory and apiary treatments (about 100-500 ppm). In the same experiments reductions in

the natural populations of the parasitic mites {Varrorajacobsonϊ) were also observed. Essential

oils to treat honeybee parasites are used in the form of aqueous emulsions, diluted in other oils,

in the form of dust or powders, in vapor form, or any other formulation described herein.

Example 7: Activity

The minimum inhibitory concentrations (MIC) of essential oils extracted from the plants

indicated above against four fungi belonging to major plant pathogens {Fusarium moniliforme,

Rhizoctonia solani, Sclerotinia sclerotium and Phytophthora capsici) were found to be between

300 to 800 μg/mL of the medium used (PDA). In addition, a droplet of essential oils (0J mL

each) , when applied to the lid of petri dishes (10 cm diameter) completely inhibited the growth

of these fungi (indicated above), demonstrating that the active ingredient was also inhibitory in

vapour form.

The essential oils of Thymbra spicata var. spicata showed the best activity against P. capsici, the agent of pepper blight, both in greenhouse and in field studies. So far there is no

fungicide effective against P. capsici, except soil sterilization with methyl bromide, and

Phytophthora species are a major pathogen of peppers and many other crops in many areas of

the world, especially in the Mediterranean region. In greenhouse trials with naturally infested

soil, the number of healthy plants of Capsicum annuum was increased from 4 per pot to 10 per

pot after treatment with different concentrations of essential oils. The number of infected plants

was reduced, respectively, from 7 to 2 plants per pot. In field trials, the number of healthy plants

per square meter was significantly increased in treated plots. The rate of germination of pepper

seeds was 75% better than controls and 20%> better than methyl bromide (statistically significant).

The soil fumigant Dazomet (BASF, Basamid), claimed as the only broad spectrum pesticide

alternative to methyl bromide (EPA Publication on Alternatives to Methyl Bromide, U.S. EPA),

and methyl bromide were used as a positive control. Dazomet appeared to be significantly less

effective than the controls as well as treatment with the essential oil, in both greenhouse and field

trials. Investigations of the activity of the soil microflora showed that the essential oils had a

lesser impact on beneficial soil microflora and micro fauna than methyl bromide and Dazomet.

The essential oils reduced the population of beneficial soil fungi and bacteria up to 40%, while

the dehydrogenase activity was reduced only 10%. Dazomet, however, reduced the population

of beneficial soil fungi and bacteria up to 90%>, and dehydrogenase activity decreased by about 50%.

Further studies conducted under field conditions, using essential oils absorbed into

perlite, or sprayed as emulsions in water indicated that the activity of these oils is retained under

field conditions. 50 g perlite containing 10 mL of essential oil extracts was placed 5 cm deep in

the soil at equal intervals. In addition, essential oils were also applied (200-300 ppm) emulsified

in 5L water/m². Soil was covered with polyethylene for a period of 5 days for solarization and

left open for three days prior to transplanting. Presently methyl bromide is used together with

soil solarization, which takes 3-4 days or solarization is used alone, requiring for soil to be

covered with polyethylene for up to six weeks, which is too long for cut-flower production.

Aqueous emulsion application may be repeated every 15 days to increase the protection level

once the plants are established.

The antimicrobial activity of essential oil extracted from Thymbra spicata var. spicata,

and various mixtures of oils from the plant species indicated above, was also studied against

bacterial plant pathogens including: Erwinia amylovora, E. carotovora pv. carotovora,

Clavibacter michiganensis var. michiganensis, Pseudomonas syringae pv. syringae,

Agrobacterium tumefaciens and Xanthomonas axonopodis pv. vesicatoria when applied in vapor

phase or mixed in to the growing medium. Minimum inhibitory concentrations (MIC) of

essential oils in media ranged from 200-400 μg/mL against all bacteria tested. MIC of the volatile phase of essential oils were ranged from 40-650 μg/mL of air, indicating that volatile

phase was more effective to all bacteria except E. c. carotovora and P. s. syringae than its contact

effect.

We have also determined whether essential oils could replace methyl bromide for storage

applications. Essential oils from Thymbra spicata var. spicata and the other plants indicated

above, in various mixtures, were placed in a container for growing insects containing Tribolium

confusum, Stophilus oryzae and Ephestia kuehniella. Essential oils killed over 95 % of the

insects when used at a 100-200 μl/L air concentration within 1-6 days. In a similar study,

essential oils from various plant species used in vapor phase killed 99% of spider mites

{Tetranychus cinnabarinus) and cotton aphids {Aphis gossypiϊ) within 2-3 days of exposure (0.5

μl/L) under laboratory conditions. Essential oils also had very high activity against these insects

when sprayed on to leaves as emulsion in water (1-1000 μl/L, preferably 100-200 μl/L) under

greenhouse and field conditions. Essential oils also have high activity in vapor form and/or

sprayed as diluted in other oils, dissolved in solvents which dissolve essential oils, or as

emulsions in water as pesticide and/or deterrent against mosquitoes, mites, cockroaches, flies,

house flies, termites and ticks. Notably, F. vulgare and P. anisum have higher concentrations

of anethole in their essential oils than T. spicata. Therefore, essential oils derived from F.

vulgare and P. anisum are more effective, and work at lower concentrations than those derived from T. spicata. Essential oils from these plants may also be used in combination.

Extracts of plant species naturally grown in Turkey are found to be potent anti-fungal,

bactericidal, nematocidal and insecticidal agents. They also improve plant health and growth by

improving (or avoiding damage to) indigenous beneficial microflora and micro fauna. According

to EPA publications, essential oils from these species are not considered toxic to animals or to

the environment. Our discovery would replace traditionally used petrochemically derived

pesticides and fumigants, particularly methyl bromide, which will be banned from use in the early

21^st century.

Example 8: The Effect of Essential Oils from Origanum spp. Against Xanthomortus axonopodis pv. vesicatoria

Concentrations of etheric oils from Origanum spp. {in vitro and in vivo) were determined

based on spectrophotometric absorbance at 600 nm (Table 6). Streptomycin sulphate added to

a flask culture completely inhibited the growth of X. a. vesicatoria in 24 hr, and had a

bactericidal effect: living bacteria were not detected at any point up to three days following the

addition of the antibiotic. Essential oil also had a bactericidal effect at concentrations of 1,000

μg/mL or greater. Control suspensions reached stationary phase 24 hr after inoculation.

The essential oil also demonstrated antibacterial activity at relatively low concentrations;

250 μg/mL, after 24 hrs and 500 μg/mL, after 48 hrs. The decrease in the bacteriostatic activity

of the essential oil over time is probably due to the volatilization and diffusion of active volatile ingredients (Table 6).

Example 9: Potential Effect of the Essential Oil of Oregano in vivo

Doses of 100-1000 μg/mL essential oil were tested against X. axonopodis pv. vesicatoria

on the leaves of pepper plants. The plants were first kept in dew chamber in 100%> relative

humidity for 60 hrs, in order to provide water-soaked leaves on the plants. The essential oil

extracts (emulsified in water as described above, at concentrations ranging from 100- 1000 μg/mL

essential oil), streptomycin sulphate (200 μg/mL) and sterile tap water as a control were sprayed

in 5 mL water. Plants were inoculated with a bacterial suspension (100 CFU/mL) and the

percentage of water soaking was determined 7 days after inoculations. The essential oil extracts

significantly reduced the occurrence of leaf spot disease caused by X. axonopodis pv. vesicatoria

on pepper plants, compared to occurrence of the disease on the control plants and the plants

treated with streptomycin (Table 5). The inhibition rate of the disease increased as the dose of

the essential oil increased (Table 6). Streptomycin treatment completely prevented the leaf spot

diseases on leaves of pepper plants. Any level of phytotoxicity of the tested concentration of the

essential oil was not detected under the experimental conditions.

The results of this study confirmed the effectiveness of the essential oil extracts against a

bacterial pathogen of pepper. The antibacterial activity can further be increased by using

essential oil extracts in combination with low levels of various antibiotics. Essential oils from

T. spicata and various mixtures of essential oils had higher activity compared to Origanum spp.

where close to 60, 70 and 90% inhibition was detected with 250, 500 and 1000 μg/mL

concentrations, respectively.

Example 10: The Activity of Essential Oils from Thymbra spicata Against Fire Blight

Disease caused by Erwinia amylovora

Fire blight disease, caused by Erwinia amylovora, of is one of the most damaging disease of fruit trees all around the world. In vitro and in vivo activities of essential oils from Thymbra

spicata against Erwinia amylovora was determined. In order to determine in vitro activity of the

vapor phase of the essential oils, droplets containing various concentrations of essential oil were

applied to the lids of inverted petri dishes containing Miller-Schroth (MS) media, which had been

previously inoculated with E. amylovora (Table 1, Figs. 10-11). In vivo activity was determined in laboratory tests using apple tree shoots, where essential

oil extracts were sprayed in emulsions prepared as described earlier. The shoots were then sprayed with a bacterial solution containing 1 x 10⁴ CFU/mL. There was no phytotoxicity

observed with applications of up to about 1000 ppm essential oil concentration. Essential oil

extracts of Thymbra spicata (about 200 ppm) reduced disease intensity 90%> compared to controls (Fig. 12).

(a) Field Tests:

Field tests were conducted at two pear orchards near Isparta, Turkey, on two different

pear varieties, disease susceptible (Santa Maria) and partially resistant (Williams). The application of essential oils from T. spicata (about 200 ppm.) was compared to the application

of copper sulfate (at commercial rates). Weekly application of essential oil extracts in an aqueous emulsion (about 200 ppm) protected both varieties significantly against fire blight disease, and

the occurrence of the disease was completely eliminated in the partial resistant variety (Fig. 13).

Example 11: Determination of Activity of Essential Oils from Thymbra spicata Against Xanthomonas campestris pv. campestris in Cabbage

Cabbage plants from a disease susceptible (Perfect Ball) variety were grown under growth

chamber conditions (22-25°C with an 18 hr photoperiod provided by sodium lamps). Seedlings

were treated with aqueous emulsions of essential oils from Thymbra spicata var. spicata at about

100, 250 and 500 ppm/plant concentrations, applied as either soil drench or foliar spray

applications (Figs 14, 15 and 16) 12 hr prior to inoculations with Xanthomonas campestris pv.

campestris (XCC), the causal agent of black rot disease of crucifers. The results indicated that

application of an emulsion of essential oils from T. spicata to the soil provided better protection

against this disease than foliar application. This may indicate that essential oils have systemic

activity in the plants against pathogenic organisms. Example 12: Activity of Essential Oils Against Carmine Spider Mite (Tetranycus cinnabarinus) in Pepper Pepper plants were treated with 100, 200 and 500 ppm concentrations of essential oil in aqueous emulsions (prepared as indicated above) from Thymbra spicata var. spicata via

initial soil drenchings, followed two weeks later by a foliar application of a 200 ppm emulsion.

The soil drenchings alone reduced infestations with mites by 60%> compared to untreated controls. Foliar application completely killed all spider mites within minutes after application

(Fig. 17), and plants remained uninfested for up to a week. The emulsions appear to work better

than any insecticide we have tested. Combinations of extracts from Thymbra spicata (60 %>),

Satureja thymbra (20 %>), Anis anisum (10%) and Foeniculum vulgare (10%>) , also killed the mites as effectively as the extract from T. spicata when used alone. The differences among the

applications may be due to differences in systemic activity.

Emulsions of essential oils have a very high contact activity, as well as a volatile phase activity, against small insects, i.e. Drosophila, spiders, mosquitoes, sugar ants and aphids.

An emulsion containing about 100-1000 ppm essential oil will kill over 50%) of the sampled insects within 0.5-3 minutes after spraying.

The antifungal and antibacterial activity of the essential oils is derived from their ability

to lyse the cell membranes of these organisms. Cell membrane lysis of zoospores and bacteria

was observed directly via optical microscopy.

Essential oils can be used against Phytophthora fragaria and nematodes infesting

strawberries and other crops dependent on methyl bromide fumigation. The fact that these oils

are derived from a natural source instead of a petrochemical one, and have no known toxicities when used in diluted form as described in this patent, would make treatment with these oils

preferable to treatment with a chemical such as Dazomet- particularly for organic growers.

Example 13. Fumigant Activity of Anethole Against Different Stages of Three

Important Stored Product Insects

We conducted a study of the fumigant activity of anethole against different stages of three

important food storage insects: eggs and adults of the confused flour beetle, Tribolium confusum,

adults of the rice weevil, Sitophilus oryzae (L.), and eggs and larvae of the Mediterranean flour

moth, Ephestia kuehniella Zeller.

Materials and methods: T. confusum were reared on a mixture of wheat flour, bran and

yeast; E. kuehniella were reared on ground wheat, and S. oryzae were reared on wheat grains.

Insect rearing and all experimental procedures were carried out at 26 ± 1°C and 65 + 5% r.h.

Tr fts-anefhole (Sigma) used in the tests was of 99% purity. Adults (< 14 days old) of T.

confusum and S. oryzae and larvae (13-16 days old) of E. kuehniella were exposed to anethole in small nylon gauze bags containing rearing food. Twenty insects were placed in each bag to

make one replicate. Three replicates for each dose and exposure time combination were taken.

Eggs (0-24 hours old) of T. confusum and E. kuehniella were exposed on cloning plates (Nunc, Denmark) modified for this purpose. (Tune et al., 1997). A set of cloning plates consisted

of a bottom plate with 60 microwells and a cover plate which had 60 holes drilled over the

microwells. A seriograph cloth was placed between two plates to avoid escape of hatched larvae

while allowing air circulation. One egg was accommodated in each microwell, for a total of 60

eggs per plate. Each plate was divided into three sections, each accommodating 20 eggs which formed one replicate. Three replicates were used for each concentration and exposure time combination. All experiments on the adults, larvae and eggs were repeated twice, thus the total

number of replicates for each dose x exposure time was six.

The test chambers were 650 mL glass jars with screw-top lids. Anethole, diluted in

acetone was applied on a blotting paper strip which measured 3 x 8 cm. The blotting paper was

attached to the lower side of the jar's lid with adhesive tape. Anethole doses of 1.88 to 15.0 mg diluted in 200 μl acetone (corresponding to 2.9 to 23 J mg/L air for the eggs and adults of T.

confusum, the adults of S. oryzae and the eggs of E. kuehniella) and doses of 15.0 to 120.0 mg/L

air (corresponding to 23J to 184.8 mg/L air for the larvae of E. kuehniella) were applied with

an automatic pipette. Only acetone was applied in control jars. Acetone was evaporated for 14 to 22 seconds before the lids were fitted to the jars. After exposure for 24, 48 or 96 hours, bags and plates containing insects were taken out of jars. Final mortality counts were taken 3 days

later for adults and larvae and 11 to 9 days later for the eggs of T. confusum and E. kuehniella,

respectively. Mortality data were corrected for natural mortality in controls and were subjected

to probit analysis to estimate LT₅₀ and LT₉₉ values (Sokal and Rohlf, 1973).

Results: Vapors of anethole were found to be toxic to all test insects: the eggs and adults

of 7, confusum, the adults of S. oryzae, and the eggs and larvae of E. kuehniella (Fig. 18).

However, the toxicity was variable among the species tested. Doses of 11.6, 23J and 184.8 mg

anethole/L air were required at varying exposure periods to achieve 100%> mortality in the adults

of S. oryzae and T. confusum, and the larvae of E. kuehniella, respectively.

The time required for 99% mortality at 23 J mg/L air was < 24 and 35.5 hours in the

adults of S oryzae and T. confusum, respectively (Table 7). A lower dose, 11.6 mg/L air would

be sufficient, however, to achieve the same mortality at a prolonged exposure time, e.g., 61.7

hours in S. oryzae. A much higher dose (such as 92.4 mg/L air) and a longer exposure period (such as 89J hours) were required for 99%> mortality in the larvae of E. kuehniella (Table 8).

Table 7. LT50 and LT99 values for T. confusum adults and eggs, S. oryzae adults and E. kuehniella eggs exposed to various doses of anethole vapors at 24-96 hours

Dose (mg/L air) T. confusum (adults) 5. oryzae (adults) T. confusum (eggs) E. kuehniella LT₅₀ (h) LT₉₉ (h) LT₅₀ (h) LT₉₉ LT₅₀ (h) LT₉₉ (eggs)

(h) (h) LT₅₀ (h) LT₉₉

(h)

2.9 988.6 229.6 208.9 40.7

5.8 134.9 29.2 29.8 2.5

11.6 10.8 875.0 13.5 61.7 2.8 218.8 1.1 117.5

23.1 3.4 35.5

* Estimated LT₅₀ and LT₉₉ values were too far beyond tested exposure range to be reliable ** It was not possible to estimate LT₅₀ and LT₉₉ values due to 100%> mortality in all exposure periods tested.

* Estimated LT₅₀ and LT₉₉ values were too far beyond tested exposure range to be reliable ** It was not possible to estimate LT₅₀ and LT₉₉ values due to 100%) mortality in all exposure periods tested.

Anethole was also toxic to the eggs of T. confusum and E. kuehniella. A concentration

of 23 J mg/L air was enough to achieve 100%) mortality at < 24 hours in the eggs of both species

(Fig. 19). Based on the LT₅₀ values, the eggs of E. kuehniella were more sensitive than the eggs of J". confusum (Table 7). These results also indicated that the eggs were more sensitive than the

other stages of the species tested (e.g. the adults in T. confusum and the larvae of E. kuehniella).

On the basis of the LT₅₀ values, sensitivity to anethole of the species and their different stages in descending order was E. kuehniella eggs, T. confusum eggs, S. oryzae adults, T. confusum

adults, and E. kuehniella larvae.

The results clearly indicate that anethole possesses a significant fumigant potential against

different stages of three important stored product insect species. Anethole apparently was more toxic than its parent compound, essential oil of anise, against the species tested. For instance,

for a 95%o mortality at 24 hours, at least 108 and 135 μl anise oil/L air (specific gravity of anise

oil was approximately 1.0) was required against the adults of S. oryzae and T. confusum, respectively (Sarac and Tune, 1995) while 23 J mg anethole/L air was sufficient to achieve the

same mortality in the adults of both species in this Example.

Ho et al. (1997) reported that anethole had fumigant activity against the adults of

Tribolium castaneum (Herbst) and Sitophilus zeamais Motschulsky. However, the way in which

the data was presented does not allow any comparison of the results of the two investigations.

Our data suggests for the first time that anethole has a fumigant activity that matches or surpasses that of methyl bromide. In experiments designed as space treatments, anethole was

capable of killing 100% of the eggs of T. confusum and E. kuehniella and the adults of S oryzae

at a dose of approximately 23 J g/m³ air and at < 24 hours exposure. The doses of methyl

bromide recommended for treatment of various commodities (e.g. for cereals, tobacco, and raisins and dried figs) in Turkey are 25, 35, and 40 g/m³, respectively, at 24 hours (Anonymous,

1995).

Certain stages of some insect species may tolerate the doses of anethole that cause 100%

mortality in other species, as exemplified by the larvae of E. kuehniella in the present Example.

It was demonstrated that this could be overcome by increasing the dose.

Apart from its fumigant activity, anethole was reported to have contact toxicity against the eggs, larvae, and adults of J! confusum and the adults of S. zeamais and exhibited a repellent effect against the adults of T. confusum (Ho et al., 1997). Anethole was also shown to be toxic

and totally inhibit the reproductive activity of a serious fruit pest, Ceratitus capitata Wied.(the

Mediterranean fruit fly) when orally administered (Bazzoni et al., 1997).

Example 14: Mixtures of Essential Oils from T. spicata, P. anisum and F. vulgare Are

Effective in Killing Insects

Mixtures of essential oils were applied in various concentrations in different formulations to test efficacy in killing insects. Efficacy can be found in the range of about 50 to 1000 ppm. When essential oil is derived from Umbelliferae alone (such as from P. anisum) as little as about

1 ppm is effective in killing a variety of insects.

Tests were conducted in jars, on plants and by spraying in air. The results are summarized in Table 9.

Example 15: Essential Oil Compositions Repel Mosquitoes

A mixture of essential oils was prepared from in the following proportions: 40%> T. spicata, 10% F. vulgare, 10%> O. ssp., 30%) S. thymbra. The mixture was formulated in

compositions comprising 10-50% essential oils in olive oil. Application of the essential oil composition was effective in repelling mosquitoes for 3-4 hours. No bites were received in the

time period tested. Olive oil controls were not effective in repelling mosquitoes. The 50%> composition was the most effective, but slight burning sensation was reported. A 30%>

composition formulated in a cream was effective for repelling mosquitoes for 1.5 to 2 hours. An

example of a cream formula composition containing the essential oils described above was formulated as shown in Table 10.

** This preservative may be replaced with a combination of 1 g/kg methyl paraben and 0.5g/kg

propyl paraben.

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Claims

1. A composition to repel or kill insects, fungi, nematodes and bacteria, comprising, as an

active ingredient, an essential oil or a component thereof, wherein said essential oil or

component thereof is derived from at least one plant species in the Family Labiatae and Umbellifera.

2. The composition of Claim 1 wherein said plant species is at least one species of a genera

selected from the group consisting of Thymbra, Satureja, Origanum, Corydothymus,

Pinpinella and Foeniculum.

3. The composition of Claim 1 wherein said plant species is selected from the group consisting of Thymbra spicata var. spicata, Satureja thymbra, Origanum majorana,

Corydothymus capitatus, Origanum vulgare, Origanum solymicum, Origanum spyleum,

Origanum bilgeri, Origanum minutiflorum, Organum saccatum, Origanum sriacum, Origanum onites, Origanum majorana, Pinpinella anisum, and Foeniculum vulgare.

4. The composition of Claim 3 wherein said plant species is selected from the group

consisting of ^'Thymbra spicata var. spicata (L) Line Ant97- 364-48, Satureja thymbra (L)

Line Ant98-28- 103 , Pinpinella anisum (L) Line Ant98-223- 137, and Foeniculum vulgare (L) Line Ant98-89-62.

5. The composition of Claim 1 wherein said essential oil or component thereof comprises

at least one compound selected from the group consisting of czs-anefhole, tra«,s^*-anefhole, anisaldehyde, anis ketone, anisole, β-bisabolene, borneol, bornyl acetate, cadinene,

camphene, camphor, Δ-3-carene, Δ-4-carene, carophyllene, carvone, carvacrol, γ-

caryophyllene, cinnamic aldehyde, citral, citronellal, cineol, 1,8-cineole, -cymene, p- cymene-8-ol, decanal, estragole, eugenol, eugenyl acetate, α-fenchene, fenchole,

fenchone, geranial, geraniol, geranyl acetate, isoborneol, lavanduol, limonene, linalool,

linalyl acetate, menthol, menthone, menthyl acetate, c/s-p-menth-2-en-l-ol, trans-n-

menth-2-en-l-ol, methoxy phenyl acetone, methyl chavicol, methyl eugenol, methylinone, 2-methylpentan-3-one, myrcene, nerol, nonanal, ^'s-β-ocimene, trαfts-β-ocimene, octanal, 3-octanol, α-pinene, β-pinene, α-phelladrene, β-phelladrene, pulegone, sabinene, cis-

sabinene hydrate, trαrcs-sabinene hydrate, y-terminene, te enyl acetate, α-terpinene, γ-

teφinene, teφinene-4-ol, a-teφineol, β-teφineol, teφinolene, 2,3,5,6-tetramethylphenol, α-thujene, thymil acetate, thymol, and tricyclene.

6. The composition of Claim 4, wherein said essential oil or component thereof is at least

one compound selected from the group consisting of carvacrol, thymol, cymene and

anethole.

7. The composition of Claim 6 wherein said component is anethole.

8. The composition of Claim 7 wherein said component is trαws-anefhole.

9. The composition of Claim 1 wherein said essential oil or component thereof is present

in an amount of at least about 1 ppm.

10. The composition of Claim 1 further comprising a carrier component for soil application.

11. The composition of Claim 8 wherein said carrier component is vermiculite or perlite.

12. The composition of Claim 1 in a liquid form.

13. The composition of Claim 12 wherein said active ingredient is emulsified in water.

14. The composition of Claim 1 wherein said active ingredient is present in a paint.

15. The composition of Claim 1 formulated for vaporization.

16. The composition of Claim 1 formulated as an aerosol.

17. The composition of Claim 1 formulated as a cream.

18. The composition of Claim 1 formulated as a powder.

19. The composition of Claim 1 formulated as a dilution in a carrier oil.

20. The composition of Claim 1 formulated with paraffin.

21. The composition of Claim 1 wherein said essential oil is present in an amount of about 0.5 to 90%.

22. The composition of Claim 1 further comprising a detergent for emulsification.

23. The composition of Claim 22 wherein said detergent is Tween 20.

24. A method of protecting a plant against pathogenic or parasitic organisms comprising

applying to said plant a composition comprising, as an active ingredient, an essential oil

or at least one component thereof in an amount sufficient to prevent adverse effects to the

plant caused by said pathogenic organisms.

25. The method of Claim 24 wherein said pathogenic or parasitic organisms are at least one

organism selected from the group consisting of nematodes, bacteria, fungi and insects.

26. The method of Claim 25 wherein said insects are selected from the group consisting of

mites, ants, aphids, and termites.

27. The method of Claim 26 wherein said insects are at least one species selected from the

group consisting of Tetranychus.

28. The method of Claim 27 wherein said insects are Tetranychus cinnabarinus.

29. The method of Claim 24 wherein said bacteria are at least one species selected from the

group consisting of Erwinia, Xanthomonas, Pseudomonas, Clavibacter, and

Agrobacterium.

30. The method of Claim 29 wherein said bacteria are at least one species selected from the

group consisting of Agrobacterium tumifaciens, Clavibacter michiganensis, Erwinia

amylovora, Erwinia carotovora, Pseudomonas syringae, and Xanthomonas axonopodis.

31. The method of Claim 30 wherein said fungi are at least one species selected from the group consisting of Fusarium, Rhizoctonia, Sclerotinia, Ascophaera, and Phytophthora.

32. The method of Claim 31 wherein said fungi are at least one species selected from the

group consisting of Fusarium monoliforme, Rhizoctonia solani, Sclerotinia sclerotium, Ascosphaera apis, Phytophthora capsici and Phytophthora fragaria.

33. The method of Claim 24 wherein said essential oil or component thereof is derived from

at least one essential oil producing plant species of a genera selected from the group

consisting of ^'Thymbra, Satureja, Origanum, Corydothymus, Pinpinella andFoeniculum.

34. The method of Claim 24 wherein said essential oil producing plant species is selected

from the group consisting of Thymbra spicata var. spicata, Satureja thymbra, Origanum

majorana, Corydothymus capitatus, Origanum vulgare. Origanum solymicum, Origanum

spyleum, Origanum bilgeri, Origanum minutiflorum, Organum saccatum, Origanum sriacum, Origanum onites, Origanum majorana, Pinpinella anisum, and Foeniculum

vulgare.

35. The method of Claim 24 wherein said essential oil producing plant species is selected from the group consisting of Thymbra spicata var. spicata (L) Line Ant97- 364-48,

Satureja thymbra (L) Line Ant98-28-103, Pinpinella anisum (L) Line Ant98-223-137, and Foeniculum vulgare (IX) Line Ant98-89-62.

36. The method of Claim 24 wherein said essential oil or component thereof comprises at

least one compound selected from the group consisting of cis-anethole, trans-anethole, anisaldehyde, anis ketone, anisole, β-bisabolene, boraeol, bornyl acetate, cadinene, camphene, camphor, Δ-3-carene, Δ-4-carene, carophyllene, carvone, carvacrol, γ-

caryophyllene, cinnamic aldehyde, citral, citronellal, cineol, 1,8-cineole, -cymene, -

cymene-8-ol, decanal, estragole, eugenol, eugenyl acetate, α-fenchene, fenchole, fenchone, geranial, geraniol, geranyl acetate, isoborneol, lavanduol, limonene, linalool,

linalyl acetate, menthol, menthone, menthyl acetate, c/s-p-menth-2-en-l-ol, trans-p-

menth-2-en-l-ol, methoxy phenyl acetone, methyl chavicol, methyleugenol, methylinone,

2-methylpentan-3-one, myrcene, nerol, nonanal, cw-β-ocimene, tra«5^,-β-ocimene, octanal,

3-octanol, α-pinene, β-pinene, α-phelladrene, β-phelladrene, pulegone, sabinene, cis- sabinene hydrate, trøns-sabinene hydrate, y-terminene, teφenyl acetate, α-teφinene, γ-

teφinene, teφinene-4-ol, a-teφineol, β-teφineol, teφinolene, 2,3,5,6-tetramethylphenol,

α-thujene, thymil acetate, thymol, and tricyclene.

37. The method of Claim 24 wherein said essential oil or component thereof is at least one

compound selected from the group consisting of carvacrol, thymol, cymene and anethole.

38. The method of Claim 24 wherein said component is anethole.

39. The method of Claim 24 wherein said component is trans-anet ole.

40. The method of Claim 24 wherein said essential oil or component thereof is present in an

amount of at least about 1 ppm.

41. A method of protecting plants from pathogens comprising inoculating the soil

surrounding said plants with a Pseudomonas fluorescens TR97.

42. The method of claim 24 wherein said composition is applied by spraying.

43. The method of Claim 42 further comprising solarization.

44. The method of Claim 24 wherein said composition is applied by fogging.

45. The method of Claim 24 wherein said composition is applied in irrigation water.

46. The method of Claim 24 wherein said composition further comprises a carrier, and said

composition is applied in the soil around the plant.

47. The method of Claim 46 wherein said carrier is selected from the group consisting of

perlite, commercially available dust preparations, commercially available granule

preparations and vermiculite.

48. The method of Claim 24 wherein said composition further comprises paint.

49. A fungicide composition comprising an essential oil, or at least one component thereof

from Laurus nobilis.

50. A method of inhibiting fungal infections of plants comprising applying a composition

comprising an essential oil or at least one active component thereof to the plant, wherein

said essential oil or active component thereof is from Laurus nobilis.

51. A method of preserving food for storage by repelling or killing insects comprising

applying a composition comprising, as an active ingredient, an essential oil or a

component thereof, wherein said essential oil or component thereof is derived from at

least one plant species in the Family Labiatae and Umbellifera.

52. The method of Claim 51 wherein said plant species is at least one species of a genera

selected from the group consisting of Thymbra, Satureja, Origanum, Corydothymus,

Pinpinella and Foeniculum.

53. The method of Claim 51 wherein said plant species is selected from the group consisting

of Thymbra spicata var. spicata, Satureja thymbra, Origanum majorana, Corydothymus capitatus, Origanum vulgare, Origanum solymicum, Origanum spyleum, Origanum

bilgeri, Origanum minutiflorum, Organum saccatum, Origanum sriacum, Origanum

onites, Origanum majorana, Pinpinella anisum, and Foeniculum vulgare.

54. The method of Claim 51 wherein said plant species is selected from the group consisting of Thymbra spicata var. spicata (L) Line Ant97- 364-48, Satureja thymbra (L) Line Ant9S-2S-l03, Pinpinella anisum (L) Line Ant98-223-137, andFoeniculum vulgare (L)

Line Ant98-89-62.

55. The method of Claim 51 wherein said essential oil or component thereof comprises at least one compound selected from the group consisting of c/s-anefhole, trαws-anethole,

anisaldehyde, anis ketone, anisole, β-bisabolene, borneol, bornyl acetate, cadinene,

camphene, camphor, Δ-3-carene, Δ-4-carene, carophyllene, carvone, carvacrol, γ-

caryophyllene, cinnamic aldehyde, citral, citronellal, cineol, 1,8-cineole, -cymene, p- cymene-8-ol, decanal, estragole, eugenol, eugenyl acetate, α-fenchene, fenchole,

fenchone, geranial, geraniol, geranyl acetate, isoborneol, lavanduol, limonene, linalool,

linalyl acetate, menthol, menthone, menthyl acetate, cw-p-menth-2-en-l-ol, trans-n-

menth-2-en- 1 -ol, methoxy phenyl acetone, methyl chavicol, methyleugenol, methylinone,

2-methylpentan-3-one, myrcene, nerol, nonanal, cts-β-ocimene, trαrø-β-ocimene, octanal,

3-octanol, α-pinene, β-pinene, α-phelladrene, β-phelladrene, pulegone, sabinene, cis- sabinene hydrate, trans-sabinene hydrate, y-terminene, teφenyl acetate, α-teφinene, γ- teφinene, teφinene-4-ol, a-teφineol, β-teφineol, teφinolene, 2,3,5,6-tetramethylphenol,

α-thujene, thymil acetate, thymol, and tricyclene.

56. The method of Claim 51 , wherein said essential oil or component thereof is at least one

compound selected from the group consisting of carvacrol, thymol, cymene and anethole.

57. The method of Claim 51 wherein said component is anethole.

58. The method of Claim 51 wherein said component is tn s-anethole.

59. The method of Claim 51 wherein said composition further comprises paraffin.

60. The method of Claim 51 wherein said insects are at least one species of the genera

selected from the group consisting of Tribolium, Sitophilus, Ephestia and Ceratitus.

61. The method of Claim 51 wherein said insects are selected from the group consisting of

Tribolium confusum, Sitophilus zeamais, Sitophilus oryzae, Ephestia kuehniella and

Ceratitus capita.

62. A method of treating pepper root rot disease caused by Phytophthora capsici in an

affected plant comprising administering an aqueous emulsion comprising, as an active

ingredient, an essential oil or at least one component thereof, to the soil about said

affected plant.

63. The method of Claim 62 wherein said essential oil is derived from a plant of the Family

selected from the group consisting of Labiatae and Umbelliferae.

64. The method of Claim 62 wherein said plant species is selected from the group consisting

of Thymbra spicata var. spicata, Satureja thymbra, Origanum majorana, Corydothymus

capitatus, Origanum vulgare, Origanum solymicum, Origanum spyleum, Origanum bilgeri, Origanum minutiflorum, Organum saccatum, Origanum sriacum, Origanum

onites, Origanum majorana, Pinpinella anisum, and Foeniculum vulgare.

65. The method of Claim 62 wherein said plant species is selected from the group consisting of Thymbra spicata var. spicata (L) Line Ant97- 364-48, Satureja thymbra (L) Line

Ant98-28-103, Pinpinella anisum (L) Line Ant98-223-137, andFoeniculum vulgare (L)

Line Ant98-89-62.

66. The method of Claim 62 wherein said component of said essential oil is at least one

compound selected from the group consisting of cis-anethole, trans-anethole,

anisaldehyde, anis ketone, anisole, β-bisabolene, borneol, bornyl acetate, cadinene, camphene, camphor, Δ-3-carene, Δ-4-carene, carophyllene, carvone, carvacrol, γ-

caryophyllene, cinnamic aldehyde, citral, citronellal, cineol, 1,8-cineole, /j-cymene, p- cymene-8-ol, decanal, estragole, eugenol, eugenyl acetate, α-fenchene, fenchole,

fenchone, geranial, geraniol, geranyl acetate, isoborneol, lavanduol, limonene, linalool,

linalyl acetate, menthol, menthone, menthyl acetate, cw-p-menth-2-en-l-ol, trans-p- menth-2-en-l-ol, methoxy phenyl acetone, methyl chavicol, methyleugenol, methylinone,

2-methylpentan-3-one, myrcene, nerol, nonanal, c/s-β-ocimene, trαrø-β-ocimene, octanal,

3-octanol, α-pinene, β-pinene, α-phelladrene, β-phelladrene, pulegone, sabinene, cis- sabinene hydrate, trαns-sabinene hydrate, y-terminene, teφenyl acetate, α-teφinene, γ-

teφinene, teφinene-4-ol, a-teφineol, β-teφineol, teφinolene, 2,3,5,6-tetramethylphenol, -thujene, thymil acetate, thymol, and tricyclene.

67. The method of Claim 62 wherein said active ingredient is at least one compound selected from the group consisting of carvacrol, thymol, cymene and anethole.

68. The method of Claim 62 wherein said active ingredient is anethole.

69. The method of Claim 62 wherein said active ingredient is trα«5-anethole.

70. A method of repelling or killing insects comprising applying a composition to an area,

wherein said composition comprises, as an active ingredient, an essential oil from at least

one plant selected from the genera Labiate and Umbellifera.

71. The method of Claim 70 wherein said plant species is selected from the group consisting

of Thymbra spicata var. spicata, Satureja thymbra, Origanum majorana, Corydothymus

capitatus, Origanum vulgare, Origanum solymicum, Origanum spyleum, Origanum bilgeri, Origanum minutiflorum, Organum saccatum, Origanum sriacum, Origanum

onites, Origanum majorana, Pinpinella anisum, and Foeniculum vulgare.

72. The method of Claim 70 wherein said plant species is selected from the group consisting

of Thymbra spicata var. spicata (L) Line Ant97- 364-48, Satureja thymbra (L) Line

Ant98-28-103,Pt«p «β//α anisum (L) Line Ant98-223-137, andFoeniculum vulgare (L) Line Ant98-89-62.

73. The method of Claim 70 wherein said component of said essential oil is at least one

compound selected from the group consisting of cis-anethole, trans-anethole,

anisaldehyde, anis ketone, anisole, β-bisabolene, borneol, bornyl acetate, cadinene, camphene, camphor, Δ-3-carene, Δ-4-carene, carophyllene, carvone, carvacrol, γ-

caryophyllene, cinnamic aldehyde, citral, citronellal, cineol, l,8-cineole, -cymene, p- cymene-8-ol, decanal, estragole, eugenol, eugenyl acetate, α-fenchene, fenchole,

fenchone, geranial, geraniol, geranyl acetate, isoborneol, lavanduol, limonene, linalool,

linalyl acetate, menthol, menthone, menthyl acetate, cw-p-menth-2-en-l-ol, trans-p-

menth-2-en- 1 -ol, methoxy phenyl acetone, methyl chavicol, methyleugenol, methylinone,

2-methylpentan-3-one, myrcene, nerol, nonanal, cw-β-ocimene, trα«s-β-ocimene, octanal,

3-octanol, α-pinene, β-pinene, α-phelladrene, β-phelladrene, pulegone, sabinene, cis-

sabinene hydrate, trαrø-sabinene hydrate, y-terminene, teφenyl acetate, α-teφinene, γ- teφinene, teφinene-4-ol, a-teφineol, β-teφineol, teφinolene, 2,3,5,6-tetramethylphenol,

α-thujene, thymil acetate, thymol, and tricyclene.

74. The method of Claim 70 wherein said active ingredient is at least one compound selected

from the group consisting of carvacrol, thymol, cymene and anethole.

75. The method of Claim 70 wherein said active ingredient is anethole.

76. The method of Claim 70 wherein said active ingredient is trcms-anefhole.

77. The method of Claim 70 wherein said active ingredient is emulsified in water, wherein said composition is formulated as a spray, and wherein said active ingredient is present in a concentration of at least about 1 ppm.

78. The method of Claim 70 wherein said active ingredient is combined with at least one inactive oil, wherein said composition is formulated as a fogging vapor, and wherein said

essential oil is atomized to a concentration of about 0J5 to 1000 ppm/m² of area.

79. The method of Claim 70 wherein said composition further comprises a carrier.

80. The method of Claim 79 wherein said carrier is selected from the group consisting of perlite, commercially available dust preparations, commercially available granule

preparations, and vermiculite.

81. The method of Claim 70 wherein said composition is formulated as a cream.

82. The method of Claim 70 wherein said composition is formulated as a powder.

83. The method of Claim 70 wherein said composition is formulated in paraffin.

84. The method of Claim 70 wherein said composition is formulated in paint.

85. The method of Claim 84 wherein said paint is an oil-based paint.

86. The method of Claim 70 wherein said insects are at least one selected from the group consisting of flies, mosquitoes, aphids, fleas, ticks, spiders, cockroaches, ants, termites,

and mites.

87. A method of protecting plants from pathogenic or parasitic organisms comprising treating

seeds of said plants with a composition comprising at least one essential oil, at least one

material that induces systemic disease resistance in plants and Psuedomonas fluorescens, and thereafter cultivating said seeds.

88. The method of Claim 87 wherein said material that induces systemic disease resistance in plants is selected from the group consisting of chitin and chitosan.

89. The composition of Claim 1 further comprising at least one other pesticide.

90. The composition of Claim 49 wherein said composition further comprises at least one

other fungicide.

91. The method of Claim 24 wherein said composition further comprises at least one other

pesticide.

92. The method of Claim 41 further comprising the application of at least one other pesticide.

93. The method of Claim 50 wherein said composition further comprises at least one other

fungicide.

94. The method of Claim 51 wherein said composition further comprises at least one other

pesticide.

95. The method of Claim 62 wherein said aqueous emulsion further comprises at least one

other pesticide.

96. The method of Claim 70 wherein said composition further comprises at least one other

pesticide.