Note: Descriptions are shown in the official language in which they were submitted.
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
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,
bacteriocides, 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
1 S deterrent effects is known for many varieties of plants.
Essential oils (etheric 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 in oil form for thousands of years. Archaeological
studies indicate that
CA 02346763 2001-04-09
WO 00/21364 PCT/US99123399
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;
Yegen,1984; Yegen
et al. 1992; Muller-Riebau et al., 1995, 1997; Sarah and Tuns 1995a; 1995b;
Qasem and Abu-
Blan,1996; El-Gengaighi et al., 1996; Lee et al., 1997; Tuns 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 a1.1982), Mentha piperita,
Mentha citrata
and Cymbopogon pendulus (Matti et al. 1985), Achryanthus aspera (Chakravarty
and Pariya,
1977), and Peperomia pellucida (Singh et aI. 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).
-2-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
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
~0 pentachlornitrobenzol. Fungitoxic components of the extracts were
determined via thin layer
chromatography to include carvacrol and thymol.
In vitro activity of the essential oils of Satureja-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
-3-
CA 02346763 2001-04-09
WO 00/21364 PCTNS99I23399
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 al.,1986; as well as
Kivanc and Akgul ( 1990)
determined minimum inhibitory concentrations of different essential oil
extract from various
plant species against many strains ofbacteria, 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 pg/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 ~,g/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.
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
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 (Dabbah et a1.1970; Beuchat 1976; Huthanen 1980; Tharib et
a1.1983; Aktug
and Karapinar 1986; Deans and Richie 1987; Deans and Svoboda 1988, 1989). The
1 S 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 (Corner 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 throughAspergillus spp. has
advanced (Charkavarty
-S-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
and Parya, 1977; Pandey et al., 19$2; Ghosh and Nandi, 1982; Maiti et al.,
1985; Tripathi et al.,
1986; Asthana et al., 1986; Charturvedi 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.
-6-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
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 ofphytopathogens,
including ones which
are difficult to effectively control with existing pesticides, i.e.
Phytophthora sp. Essential oils
S 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 andlor 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.
CA 02346763 2001-04-09
WO 00/21364 PCTNS99/23399
Pinpinella anisum contains over 80% anethole. We have found that traps-
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
ofThymbra spicata
(50%), Satureja thymbra (30%) and Origanum spp. (20%) extracts) against
Phytophthora capsici
zoospore inoculation ( 1 x 1 OS 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 1 OS zoospores/mL).
Figure 5 shows the activity of 100-400 ppm essential oils (a mixture of
Thymbra spicata
(b0%), 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.
_g_
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
Figure 6 shows the activity of 100-400 ppm of essential oils (a mixture of
Thymbra
spicata (60%), Satureja thymbra (20%) Pinpinella anisum ( 10%) and Foeniculum
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, SO
~.1/L essential oil; 3,
-9-
CA 02346763 2001-04-09
WO 00/21364 PCTNS99/23399
40~1/L essential oil; 4, 90 ~1/L essential oil; 5, 60 pl/L essential oil; 6,
30 ~,l/L essential oil;
7, 80 ~.1/L essential oil; 8, 70 ~l/L essential oil; 9, 20 ~UL 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
(CuS04) (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 1 OS 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
withXanthomonas campestris
-10-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
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. carnpestris ( 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
1 S (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
-11-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
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. Davis,Organum saccatum
P.H. Davis,
Origanum sriacum var. bevanii (Holmes) Letswart, Origanum onites (L), and
Origanum
majorana (L). 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. Otherplant
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,~i-bisabolene,
borneol, bornyl acetate,
cadinene, camphene, camphor, O-3-carene, O-4-carene, carophyllene, carvone,
carvacrol, y-
caryophyllene, cinnamic aldehyde, citral, citronellal, cineol,1,8-cineole,p-
cymene,p-cymene-8-
ol, decanal, estragole, eugenol, eugenyl acetate, a-fenchene, fenchole,
fenchone, geranial,
geraniol, geranyl acetate, isoborneol, lavanduol, limonene, linalool, linalyl
acetate, menthol,
-12-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
menthone, menthyl acetate, cis-p-menth-2-en-1-ol, traps-p-menth-2-en-1-ol,
methoxy phenyl
acetone, methyl chavicol, methyleugenol, methylinone, 2-methylpentan-3-one,
myrcene, nerol,
nonanal, cis-(3-ocimene, traps-(3-ocimene, octanal, 3-octanol, a-pinene, (3-
pinene, a-phelladrene,
(3-phelladrene, pulegone, sabinene, cis-sabinene hydrate, traps-sabinene
hydrate, y-terminene,
terpenyl acetate, a-terpinene, y-terpinene, terpinene-4-ol, a-terpineol, ~i-
terpineol, terpinolene,
2,3,5,6-tetramethylphenol, a-thujene, thymil acetate, thymol, and tricyclene.
Although all ofthese 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 %)
and F. 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
-13-
CA 02346763 2001-04-09
WO OOrll364 PGTNS99/23399
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.
-14-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
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 ca.n
be either adsorbed by
soil particles or toxic to plant roots when directly applied to soil.
Therefore, the oils have to be
-15-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
formulated in granules or powders or absorbed in a Garner, 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 O.Sg of essential oil is combined with about 10-
SOg of the carrier
substance, such as perlite, vermiculite or mixtures thereof. The applications
using the Garner
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 irngation 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
-16-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
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.
-17-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
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: S00 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
-18-
CA 02346763 2001-04-09
WO 00/21364 PCTNS99/13399
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 extracts) 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 5Ø
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
-19-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/~3399
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 S 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 1 OS 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
-20-
CA 02346763 2001-04-09
WO 00/Z1364 PCT/US99/23399
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 pg/mL essential oil. The minimum
bacterial
1 S concentration (the lowest concentration yielding fewer than 0.1 %
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 105 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 ~1, corresponding to 20 to 1280 ~g/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
-21-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
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 ofthe 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 0.25 ppm per 1000 m2. Concentrations can be increased for
application against
house pests and/or to fog greenhouses.
-22-
CA 02346763 2001-04-09
WO 00!11364 PGT/US99/23399
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
mglkg 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
-23-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
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 S-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:
-24-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
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 traps-anethole are effective, traps-anethole 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 A.
tumef'aciens than on P s. syringae and E. carotovora (Table 2, 3).
-25-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
Table 1. The activity
of the Volatile Phase
of Essential Ofls
from Thymbra spicata
against Erwinia
amylovora. A droplet
containing essential
oil was applied to
the lid of the petri
dish in the essential
oil
treatments.
Essential Oil (T. Experiment I* Experiment II
spicata) droplet Number of colonies Number of colonies
(pIIL) **
20 211.7 b 203.3 b
30 206.3 b 196.3 b
40 202.7 b 194.0 b
50 149.0 c 140.3 c
60 147.7 c 135.0 c
70 129.3 cd 126.7 cd
g0 126.0 cd 120.7 cd
90 101.3 d 93.0 d
100 0.0 a 0.0 a
200 O.Oe O.Oe
300 0.0 a 0.0 a
400 0.0 a 0.0 a
500 O.Oe O.Oe
STREPTOMYCIN 0.0 a 0.0 a
(50 ug/mL, in media)
CONTROL 256.7 a 271.0 a
lvo stgntticant 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.
-26-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99123399
Table 2.
Contact
Effect
of Essential
Oil of
Thymbra
spicata
spicata
on Plant
Pathogenic
Bacteria
TEST BACTERIA"'
(CFUImL)
Dose ~g/mLAt. C.m.m. E.c.c. E.a. P.s.s. X.a.v.
0 1.71 x 1.05 x 3.60 x 1.89 x 6.05 x 4.73 x
10a a 108 a 108 a 108 a 108 a 108 a
20 1.49 x 8.90 x 2.25 x 1.20 x 2.36 x 4.29 x
108 a 10' a 10g b l Oa 10$ b 1 O$ a
b
40 1.21x10'a7.20x10'a 1.66x108c8.33x10'c2.12x108b 2.73x108b
80 8.60 x 6.30 x 1.22 x 4.33 x 4.40 x 2.17 x
10' ab 10' ab 1 O8 10' d 10' c 10' be
ed
160 8.40 x 5.80 x 9.60 x 1.83 x 4.00 x 1.50 x
10' ab 10' ab 10' de 10' a 10' c 10 c
320 6.34x10'be1.80x10'be5.00x10'ef0.00f 3.30x10'c 7.70x102d
640 O.OOc O.OOc O.OOf O.OOf l.lOx 10'dO.OOe
1280 0.00 c 0.00 c 0.00 f 0.00 f 0.00 c 0.00 a
vanes are means of tour 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.
Table
3. Volatile
Phase
Effect
of the
Essential
Oil of
Thymbra
spicata
spicata
on Plant
Pathogenic
Bacteria
Test Bacteria*
(CFU/petri
dish)
Dose A.t. C.m.m. E.c.c. E.a. P.s.s. X.a.v.
~g/mL
air
0 133 a 195 79 a 237 a 185 a 192 a
20 71 b 76 b 61 ab 171 b 172 a 148 ab
40 26 c 40 c 57 abc 82 c 171 a 104 b
80 17c 11 d 52bc Od 169a Oc
160 Oc Od 49bc Od 137a Oc
320 Oc Od 35c Od 24b Oc
640 Oc Od Od Od 16b Oc
1280 Oc Od Od Od Ob Oc
*
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.
Clavibactermichiganensis
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.
-27-
CA 02346763 2001-04-09
WO 00/21364 PCTNS99/23399
The essential oil in the contact effect tests showed MIC ranging from 276
~,g/mL to 413
pg/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 ltg/mL to 684 pg/mL (Table 4).
Table 4. MIC of the
E.O. of T. s. spicata
to plant pathogenic
bacteria
Bacteria MIC (ltg/mL) in MIC (ug/mL air) in
contact effect tests volatile phase effect
tests
A. tumefaciens 328 9g
C. m. michiganensis 405 91
E. c. carotovora 413 569
E. amylovora 276 59
P s. syringae 344 684
X a. vesicatoria 323 41
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 Garner 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
byAscosphaera apis
-28-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
(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 ( Varrora jacobsoni) 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 ?: 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 sclerotiurrr and Phytophthora capsici) were
found to be between
300 to 800 ~,g/mL of the medium used (PDA). In addition, a droplet of
essential oils (0.1 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.
-29-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
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
1 S lesser impact on beneficial soil microflora and microfauna 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
-30-
CA 02346763 2001-04-09
WO 00/21364 PCTNS99/23399
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 SL water/mz. Soil was covered with polyethylene for a period of S 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 andXanthomonas 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 pg/mL against all bacteria tested.
MIC of the
-31-
CA 02346763 2001-04-09
WO 00121364 PCTNS99/23399
volatile phase of essential oils were ranged from 40-650 pg/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 pl/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 (Aphisgossypii) within 2-3 days
ofexposure (0.5
~1/L) under laboratory conditions. Essential oils also had very high activity
against these insects
when sprayed on to leaves as emulsion in water (I-1000 pI/L, preferably 100-
200 pl/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
-32-
CA 02346763 2001-04-09
WO 00121364 PCT/US99/Z3399
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
microfauna. 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 S' 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
p.g/mL or greater. Control suspensions reached stationary phase 24 hr after
inoculation.
The essential oil also demonstrated antibacterial activity at relatively low
concentrations;
250 p,g/mL, after 24 hrs and 500 p.g/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
-33-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
ingredients (Table 6).
Example 9: Potential Effect of the Essential Oil of Oregano in vivo
Doses of 100-1000 pg/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 pg/mL
essential oil), streptomycin sulphate {200 ~,g/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 byX.
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.
-34-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
Table 5. The Activities of Essential
Oil of Oregano on Xanthomonas
axorropodis pv. vesicatoria
in vivo
Concentration of Essential OIl Inhibition Rate (%)
(pg/mL)
0 0
1 ~ 25
250 35
45
1000 60
Streptomycin (200 pg/mL) 100
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
ug/mL
concentrations, respectively.
-3 S-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
Table
6. The
Activities
of Essential
Oil of
Oregano
on Xanthornonas
axvnopodis
pv.
vesicatoria
in vitro
ConcentratioAbsorbanceInhibitionAbsorbanceInhibitionAbsorbanceInhibition
n o f (600nm) Rate (%) (600nm) Rate (600 nm) Rate (%)
Essential (%)
Oil
(w8~ml.)
1 0.26 59.38 0.32 50 0.5 21.88
12.5 0.19 70.31 0.28 56.25 0.35 45.31
25 0.16 75 0.25 60.94 0.3 45.31
50 0.14 78.13 0.22 65.63 0.3 53.13
100 0.11 82.81 0.22 65.63 0.25 53.13
200 0.03 95.31 0.22 65.63 0.18 60.94
250 0.00 100 0.18 71.88 0.13 71.88
500 0.00 100 0.09 85.94 0.00 79.69
1000 0.00 100 0.00 100 0.00 100
2000 0.00 100 0.00 100 0.00 100
Streptomyci0.00 100 0.00 100 0.00 100
n
Control 0.64 0.00 0.64 0.00 0.64 0.00
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 l, 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
-36-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
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.
-37-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
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 SO% 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. CeII 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
-3 8-
CA 02346763 2001-04-09
WO OOI21364 PCT/US99/23399
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 ofthe 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.
Traps-anethole (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. (Tong 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
-39-
CA 02346763 2001-04-09
WO 00/21364 PCTNS99/23399
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 Iid with adhesive tape. Anethole doses
of 1.88 to 15.0 mg
diluted in 200 ~,1 acetone (corresponding to 2.9 to 23.1 mg/L air for the eggs
and adults of T.
confusum, the adults ofS. oryzae and the eggs ofE. kuehniella) and doses of
15.0 to 120.0 mg/L
air (corresponding to 23.1 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 LTSO 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 T, 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,
23.1 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 23.1 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
-40-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
(such as 89.1 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 T. confusum S, oryzae T. conjusum E. kuehniella
air) (adults) (adults) (eggs)
LTA (h) LTA LTso (h) LTA LTso (h) (eggs)
(h) LTA
(h) (h) LTso (h) LTA,
(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 ** ** ** ** ** **
T trsnmated L'l~so and L'I'B, values were too far beyond tested exposure range
to be reliable
** It was not possible to estimate LTso and LT99 values due to 100% mortality
in all exposure
periods tested.
Table 8. LTso and
LTA values for
the larvae of E.
kuehniella exposed
to various doses
of anethole
vapors at 24-96
hours
Dose (mg/L air) LTso O<) LTA (h)
23.1 323.6
46.2 22.0
92.4 4.3 89.1
184.8 ** **
*
Estimated
LTS
and
LT99
values
were
too
far
beyond
tested
exposure
range
to
be
reliable
**
It
was
not
possible
to
estimate
LTs
and
LTA
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 23.1 mg/L air was enough to achieve 100% mortality at < 24 hours in the
eggs of both species
(Fig. 19}. Based on the LTso values, the eggs ofE. kuehniella were more
sensitive than the eggs
of T. 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 LTso values, sensitivity to anethole of the species and
their different stages
-41-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
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
S toxic than its parent compound, essential oil of anise, against the species
tested. For instance,
for a 95% mortality at 24 hours, at least 108 and 135 pl 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 (Sarag and Tuna 1995) while 23.1 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 23.1 g/m' 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/m', 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
-42-
CA 02346763 2001-04-09
WO 00121364 PCT/US99/23399
the eggs, larvae, and adults of T. 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.
Table 9. An
Mixture of
Essential
Oils (80%
T. spicata,10%
P. anisum,
and 10% F.
vulgare)
are Effective
in Killing
Insects.
Insect Dose as an Results
emulsion in
water
(in PPm)
Spider Mites 100 90% killed in 1 min.
Ants 100 50% killed in 3 min.; 100% killed
in 10 min.
Aphids 200 100% killed in closed jars in 5 min.;
50% killed on plants in
10 min.
Mosquitoes 200 in emulsion100% killed when used as a spray
German 500 100% killed in 10 min.
cockroaches
Example 15: Essential Oil Compositions Repel Mosquitoes
A mixture of essential oils was prepared from in the following proportions:
40% T.
-43-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
spicata, 10% F. vulgare, 10% D. 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
rnosquitoes. 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.
-44-
CA 02346763 2001-04-09
WO 00121364 PCT/US99/23399
Table 10. Formulations for
a 1 kg skin cream containing
essential oil as an insect
repellent
Ingredient Amount in grams
Beeswax 75-100
Borax 10
Castor oil 0-20
Cocoa butter 0-20
Distilled water 313.5-443.5
Essential oil composition 6-200
Glycerin (vegetable) 15
Grapefruit seed extract** 0-10
Jojoba oil 35-50
Olive Oil 200-325
Sweet almond oil 50
Wheat germ oil 25
Shea butter 10-15
-~T ~ ms preservative may ne replaced mth a combination of 1 g/kg methyl
paraben and O.Sg/kg
propyl paraben.
REFERENCES CITED
1. Akgul, A. 1986. Studies on the essential oils from Turkish fennel seeds
(Foeniculum
vulgare M. var. Dulce). Pages 487-489. In Progress in Essential Oil Research.
Edited
by E.-J. Brunke, Walter DeGruyter & Co., New York.
2. Akgul, A. and M. Kivanc. 1988a. Inhibitory effects of six Turkish thyme-
like spices on
some common food-borne bacteria. Nahrung 32, 201-203.
3. Akgul, A. and M. Kivanc. 1988b. Inhibitory effects of selected Turkish
spices and
oregano components on some foodborne fungi. Int. J. Fd Microbiol. 6, 263-268.
4. Akgul, A. and M. Kivanc. 1989. Sensitivity of four foodborne moulds to
essential oils
from Turkish spices, herbs and citrus peel. J. Sci. Fd. Agric. 47, 129-132.
-45-
CA 02346763 2001-04-09
WO 00/21364 PCTNS99/23399
5. Anonymous. 1995. Manual of Pest Control. Ministry of Agriculture and Rural
Affairs,
Ankara (In Turkish).
6. Arrebola, M.L., M. C. Navarro, J. Jimenez and F. A. Ocana. 1994. Yield and
composition of the essential oil of Thymus serpylloides subsp. serpylloides.
Phytochemistry. 36, 67-72.
7. Asthana, A., N. N. Tropathi, S. N. Dixit: 1986. Fungitoxic and phytotoxic
studies with
essential oil of Ocimum adscendens. J. Phytopath. 117, 152-159.
8. Bazzoni, E., G. S. Passino, M. D. L. Moretti and R. Proto. 1997. Toxicity
of anethole
and its effects on egg production of Ceratitis capitata Wied. (Dip.
Tephritidae). Ann of
Appl. Biol. 131, 369-374.
9. Benjilal, B., A. T. Elakari, A. Ayadi, and M. Ihal. 1984. Method to study
antimicrobial
effects of essential oils: application to the antifungal activity of six
Moroccan essences.
J. Fd Prod. 47, 748-752.
10. Capone, W., C. Mascia, M. Melis and L. Spanneda. 1988. Determination of
terpenic
compounds in the essential oil from Satureja thymbra growing in Sardinia. J.
Chromatography 457, 427-430.
11. Chakravarty, D. K. and S. N. Pariya. 1977. Inhibition of germination of
phytopathogenic fungi in some Indian medicinal plant extracts. Z PflKranlch.
PflSchutz.
84, 221-223.
12. Chaturvedi, R. V., S. C. Tripathi: Fungitoxic, physico-chemical and
phytotoxic properties
of essential oil of Seseli indicum W. & A. J. Phytopath. 124, 316-322, 1989.
-46-
CA 02346763 2001-04-09
WO 00121364 PGT/US99/23399
13. Coats, J. R. 1994. Risks from natural versus synthetic insecticides. Ann.
Rev. Entomol.
39, 489-515.
14. Conner, D. E., L. R. Beuchat. 1984a. Sensitivity of heat-stressed yeasts
to essential oils
of plants Appl. Envir. Microbiol. 47, 229-233.
15. Conner, D. E. and L. R. Beuchat. 1984b. Effects of essential oil from
plants on growth
of food spoilage yeasts. J. Fd. Sci. 49, 429-434.
16. Deans, S.G. 1991. Evaluation of antimicrobiai activity of essential
(volatile) oils. Pages
309-320. In Essential Oils and Waxes: Modern Methods of Plant Analysis vol.
12.
Edited by H. F. Linskens and J.F. Jackson, Springer-Verlag, New York.
17. De Boer, E., W. M. Spiegelenberg, and F. W. Janssen.1985. Microbiology of
spices and
herbs. Antonie van Leeuwenhoek 51, 435-438.
18. Duke, J.A. 1992. Handbook of biologically active phytochemicals and their
activities.
CRC Press, Boca Raton, Florida USA.
19. El-Gengaihi, S.E., S.A.A. Amer and S.M. Mohamed. 1996. Biological activity
of thyme
oil against Tetranychus urticae. Koch. Anz. Schadlingskde. Pflanzenschutz,
Umweltschutz. 69, 157-159.
20. Farag, R. S., Z. Y. Daw, F. M. Hewedi and G. S. A. El-Baroty. 1989a.
Antimicrobial
activity of some Egyptian spice essential oils. J. Fd. Prod. 52, 665-667.
21. Farag, R. S., Z. Y. Daw and S. H. Abo-Raya. 1989b. Influence of some spice
oils on
Aspergillus parasiticus growth and production of Aflatoxin in a synthetic
medium. J. Fd.
Sci. 54, 74-76.
-47-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
22. Ghosh, J.,B. Nandi and N. Fries. 1982. Use of some volatile compounds in
the
preservation ofwheat grains from fungal deterioration in storage under Indian
conditions.
Z. PfIKrankh. PflSchutz 89, 410-418.
23. Ho, S. H., Y. Ma, and Y. Huang. 1997. Anethole, a potential insecticide
from Illicium
verum Hook F., against two stored product insects. Int. Pest Control 39, 50-
51.
24. Karapinar, M.: The effects of citrus oils and some spices on growth and
aflatoxin
production by Aspergillus parasiticus NRRL 2999. Int. J. Fd. Microbiol. 2, 239-
245,
1985.
25. Karapinar, M.: Influence of menthol and thymol on Aspergillus parasiticus
growth amid
production of aflatoxins in a synthetic medium. Mitt. Geb. Lebensmittelunters.
u. Hyg.
81, 287-295, 1990.
26. Kivanc, M.,~A. Akgiil: Mould growth on black table olives and prevention
by sorbic acid,
methyl-eugenol and spice essential oil. Nahrung 34 , 369-373, 1990
27. Klingauf, F., H. J. Bestman, O. Vostrovsky, and K. Michaelis. 1983.
Wirkung von
atherischen olen auf Schadinsekten. Mitteilungen der Deutschen Gesellschaft
fiir
Allgemeine and Angewandte Entomologie 4, 123-126.
28. Lee, S.G., LK. Soon, J.A. Young, B.K. Joon and Y.L. Boum. 1997.
Effectiveness of
carvacrol derived from Thujopsis dolabrata var. hondai sawdust against
Thecodiplosis
japonensis (Diptera: Cecidomyiidae) Pesticide Sci. 49, 119-124.
29. Maitti, D., C. R. Kole and C. Sen. 1985. Antimicrobial efficacy of some
essential oils.
- Z. PflKrankh. PflSchutz. 92, 64-68.
30. Moleyar, V., and P. Narasimham. 1986. rlntifungal activity of some
essential oil
-48-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
components. Fd. Microbiol. 3, 331-336
31. Moleyar, V., and P. Narasimham.1987. Detoxification of essential oil
components (citral
and menthol) by Aspergillus niger and Rhizopus stol onifer. J. Sci. Fd. Agric.
39, 239-
246.
32. Muller-Riebau, F. Berger, F. and O. Yegen. 1995. Chemical composition and
fungitoxic
properties to phytopathogenic fungi of essential oils of selected aromatic
plants growing
wild in Turkey. J. of Agricultural and Food Chemistry 43, 2262-2266.
33. Pandey, D. K., N.N. Tripathi, R.D. Tripathi and S.N. Dixit. 1982
Fungitoxic and
phytotoxic properties of the essential oil ofHyptis suaveolens. Z. PflKrankh.
PflSchutz.
89, 344-349.
34. Philianos, S.M., T. Andriopoulou-Athanassoula and A. Loukis.1984.
Constituents ofthe
essential oil ofSatureja thymbra L., from different regions ofGreece. Int. J.
Crude Drug
Research, 22, 145-149.
35. Pruthi, J.S. 1980. Spices and Condiments: Chemistry, Microbiology,
Technology.
Academic Press.
36. Quasem, J.R. and H.A. Abu-Blan. 1996. Fungicidal activity of some common
weed
extracts against different plant pathogenic fungi. J. Phytopath. 144, 157-161.
37. Ravid, U., and E. Putievsky,1985. Composition of essential oils of Thymbra
spicata and
Satureja thymbra. chemotypes. Planta Med. 4, 337-338.
38. Ravid, U., and E. Putievsky, 1986. Carvacrol and thymol chemotypes of East
Mediterranean wild Labiatae herbs. In Progress in Essential Oil Research.
Brunke, F.J.
(Ed.) Walter de Gruyter Berlin, 163-167.
-4.9-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/23399
39. Sara, A. and I. Tunr. 1995a. Toxicity of essential oil vapours to stored-
product insects.
Z. PflKrankh. PflSchutz 102, 69-74.
40. Sara, A. and I. Tuns. 1995b. Residual toxicity and repellency of essential
oils to stored-
product insects. Z. PflKrankh. PflSchutz 102, 429-434.
41. Sarer, E., J.J.C. Scheffer, A.M. Janssen, A. Baerheim-Svendsen. 1985.
Composition of
the essential oil of Origanum majorana grown in different localities in
Turkey. Pages
209-212. In Essential Oils and Aromatic Plants, edited by A. Baerheim-Svendsen
and
J.J.C. Scheffer, Nijhoff/Junk, Dordrecht, the Netherlands.
42. Scheffer, J.J.C., A. Looman, A. Baerheim-Svendsen and E. Sarer. 1986. The
essential
oils of three Origanum species grown in Turkey. Pages 151-156 in Progress in
Essential
Oil Research. Brunke, F.J. (Ed.) Walter de Gruyter, Berlin.
43. Shaaya, E.r U. Ravid, N. Paster, B. Juven, U. Zisman, and V. Pissarev.
1991. Fumigant
toxicity of essential oils against four major stored-product insects. J. Chem.
Ecol. 17,
499-504.
44. Shukla, H.S. and S.C. Tripathi. 1987. Antifungal substance in the
essential oil of anise
(Pinpinella anisum L.) Env. Biol. Chem. 51, 1991-1993.
45. Singh, D. K., R. Dikshit, S. N. Dixit: Antifungal studies ofPeperonin
pellucida. Beitr.
Biol. Pfl. 58, 357-368, 1983.
46. Sokal, R. R. and F. J. Rohlf. 1973. Introduction to Biostatistics. Freeman
and Company,
San Fransisco, California.
47. Thompson, D. P: 1986. Effect of essential oils on spore germination
ofRhizopus, Mucor
-50-
CA 02346763 2001-04-09
WO 00/21364 PCT/US99/Z3399
and Aspergillus species. Mycologia 78, 482-485.
48. Thompson, D. P. and C. Cannon. 1986. Toxicity of essential oils on
toxigenic and
nontoxigenic fungi. Bull. Envir. Contain. Toxicol. 36, 527-532.
49. Thompson, D. P., C. Cannon, G. Parter and T. Tsefamichael. 1987. Mycoassay
of
fluorescent fractions from seven essential oils. Bull. Environ. Contain.
Toxicol. 39, 688-
695.
50. Tripathi, S. C., S. P. Singh and S. Dube. 1986. Studies on antifungal
properties of
essential oil of Trachyspermum ammi (L.) Sprague. J. Phytopath. 116, 113-120.
51. Tuna L, S. Sahinkaya. 1998. Sensitivity of two greenhouse pests to vapor
of essential
oils. Entomologia Experimentalis et Applicata 86, 183-187.
52. Tuns, L, F. Erler, F. Dagli and O ~ali~. 1997. Insecticidal activity of
acetone vapours.
J. Stored Prod. Res. 33, 181-185.
53. S. Sahinkaya. 1998. Sensitivity of two greenhouse pests to vapor of
essential oils.
Entomologia Experimentalis et Applicata 86, 183-187.
54. Yegen, O.: Kokarot (Bifora radians) Bunyesindeki Ucucu Yagin Fungitoksik
Potansiyeli.
Ankara Universitesi Ziraat Fakultesi Yayinlar 909, Bil. Ar. ve Incel. 53I, 24,
1984.
55. Yegen, O., B. Berger, R. Heitefuss, 1992. Investigations on the
fungitoxicity of extracts
of six selected plants from Turkey against Phytopathogenic fungi. Zeitschrift
fur
Pflanzenkrankheiten and Pflanzenschutz. Journal of Plant Diseases and
Protection 99,
349-359.
56. Yegen, O., A. Unlu and B.M. Berger. 1998. Einsatz and nebenwirkungen auf
-51-
CA 02346763 2001-04-09
WO 00/Z1364 ' PCT/US99/23399
bodenmikrobielle aktivitaten des etherischen ols aus Thymbra spicata bei der
bekamfung
der wurzelhalskrankheit an paprika Phythophthora capsici. Z. PflKrankh.
PflSchutz. l O5,
602-610.
-52-
