Note: Descriptions are shown in the official language in which they were submitted.
PHYTOSANITARY COMPOSITION COMPRISING ESSENTIAL OILS THAT
POTENTIATE ANTIFUNGAL ACTIVITY
DESCRIPTION
[0001] This invention relates to phytosanitary compositions having fungicidal
properties comprising a
mixture of essential oils obtained from plants, such as thyme oil (Thymus
vulgalls) and oregano oil
(aiganum vulgare) or mixtures thereof, and an agent having known fungicidal
properties such as
potassium carbonate, for use mainly in contact protection against fungicidal
infections in cultivated
plants and post-harvesting, and in other antifungal applications. In these
compositions the effect of the
agent having known fungicidal properties is synergistically potentiated by the
essential oils mentioned.
[0002] Essential oils are complex mixtures of natural molecules which are
fundamentally obtained
from plants. They are secondary metabolites which can normally be obtained by
extraction with
organic solvents and subsequent concentration, or by physical treatments with
steam followed by
separation of the water-insoluble phase. Generally they are volatile liquids
soluble in organic solvents
and have a density lower than that of water.
[0003] In nature they can be synthesized in different plant organs such as
seeds, leaves, flowers,
epidermal cells and fruits, among others, and they play an important part in
protecting plants against
bacterial, viral or fungal infections.
[0004] The fungicidal and bactericidal action of many plant essential oils is
known, and has arrived in
some case to be marketed commercially. Among these are jojoba oil (Simrnondsia
californica),
rosemary oil (Rosmarinus officthalis), thyme oil (T vulgaris), the clarified
hydrophobic extract of
neem oil (A. indica), cottonseed oil (Gossypium hirsutum) with garlic extract
(Dayan et al., Bioorg.
and Med. Chem. 2009;17:4022-34).
[0005] The chemical composition of essential oils differs not only in the
quantity but also in the
quality and the stereochemical type of the molecules in the extracted
substances. The extraction
product may vary according to climate, the composition of the soil, the organ
of the plant used for
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extraction, and the age and stage of growth of the plant. It also depends on
the extraction process
used.
[0006] Furthermore, Zamani et al. showed that the potassium carbonate was
effective as fungicide
treatment against Penicillium digitatum in green mold on oranges (Commun.
Agric. Appl. Biol. Sci.
2007;72(4):773-7). The Patent CN107041366 discloses the use of potassium
carbonate which is
applied together with pesticides (including fungicides), improving the
emulsification and
impregnation.
[0007] Because of their natural origin plant essential oils are very
attractive for application in
agriculture in order to obtain healthy and harmless products, as this is a
requirement which has been
made increasingly strictly, by both consumers and regulatory authorities.
100081 There is therefore a need to find new phytosanitary compositions having
antifungal properties
to protect crops, including during post-harvesting, which have a minimum of
secondary toxic effects
and which are harmless to human beings and the environment.
[0009] The present authors have surprisingly found that some essential oils
obtained from plants
when mixed with other products having known antifungal properties potentiate
the antifungal
properties of these compounds, such as inorganic salts, for example alkali
metal carbonates like
potassium carbonate.
[0010] Thus one object of the present invention is to provide a phytosanitary
composition having
antifungal activity comprising: (1) one essential oil obtained from plants
selected from oregano oil
(Origanum vulgare) and thyme oil (Thymus vulgatis) or its active compounds
carvacrol at a
concentration between 0.1 and 530 ppm or thymol at a concentration between
0.31 and 530 ppm, or a
combination thereof; and (2) potassium carbonate at a concentration between
3.5 and 25 inM.
[0011] This composition synergistically improves the antifungal properties of
the agents having
known antifungal activity, has a minimum of secondary toxic effects and is
harmless to human beings
and the environment.
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[0012] The composition according to this invention may be applied in
agriculture to protect crops
from the stage of germination to harvesting, and during the storage and
transport of these crops, seeds,
flowers or grains. Likewise, another possible application is in the
elimination of fungi which attack
painted surfaces and to protect carpets and fabrics in the home and in any
other application against
fungal attack through contact.
[0013] Among the essential oils which may be used in the phytosanitary
composition according to
this invention are thyme oil (Thymus vulganS) and oregano oil (Onganum
vulgare) or mixtures
thereof.
[0014] Without being bound to any theory in particular, it is possible that
the property of the essential
oils obtained from plants in potentiating antifungal activity is due to some
of the compounds present
in these essential oils having known activity. Thus in one embodiment of this
invention the
phytosanitary composition may comprise one or a mixture of active compounds
isolated from the
essential oils according to this invention, such as phenolic monoterpenoids
such as carvacrol and
thymol, and mixtures thereof, and an agent having known fungicidal properties,
such as potassium
carbonate. The mechanism of action of the essential oils is a multiple one due
to the complex mixture
of different active ingredients which they contain. However, the nature of the
action of the major
components in some of these oils has been described. The best described in the
literature is the nature
of the action of carvacrol on the growth of bacterial and yeast cells (Ultee
et al., Appl. Environ.
Microbiol. 2002;68(4):1561-68). According to these studies carvacrol is
capable of crossing the cell
membrane when it is protonated (in acid medium) and on reaching the cytoplasm
releases a proton,
resulting in acidification of the cell. This manner of action does not rule
out other possible modes of
action such as increase in the permeability of the membrane or specific
inhibiting effects on catalytic
processes. Moreover, the Patent CN104642326 describes a fungicidal composition
containing
penflufen and carvacrol. PCT application W02014036667 discloses a continuous
extraction method to
produce a high content of carvacrol and thymol, which are powerful fungicides.
[0015] On the other hand, some studies showed that the essential oil of Thymus
vulgths, the thyme
oil, has a moderate control efficacy against Aspergillus Inger strains with
its antifungal activity
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resulting mainly due to killing of microorganism rather than growth
inhibition. Oils on wheat seeds
showed no significant phytotoxic effect in terms of seed germination or
seedling growth (Kumar et al.,
Environ. Sci. Pollut. Res. Int. 2017;24(27):21948-59). Other important
evidence is that an in vivo
antifungal assay demonstrated that the maximum antifungal activity was showed
by thyme oil against
Pernciilium expansum and Botrytis cinerea in pear fruits (Nikkhah et al., Int.
J. Food Microbiol.
2017;18(257):285-94). Furthermore, the US09492490 Patent describes a
composition for controlling a
target pest comprising 0.1% to 4% isopropyl myristate, 0.1% to 15% thyme oil
white, 0.1% to 2%
geraniol, and at least one additional active ingredient. The PCT W02014153042
discloses a method
for treating Mycosphaerella fijiensis in crops of the Musaceae family by
applying a fungicidal
composition comprising garlic oil, rosemary oil, thyme oil and cinnamon oil.
[0016] Among the agents having known fungicidal properties which may be used
in the composition
according to the invention there are the carbonates of alkali metals,
preferably of lithium, sodium or
potassium. More preferably the agent having known fungicidal properties is
potassium carbonate.
[0017] The concentration of thymol present in the composition according to
this invention is between
0.31 and 530 ppm, preferably between 22 and 350 ppm. The concentration of
carvacrol present in the
composition according to this invention is between 0.1 and 530 ppm, preferably
between 22 and 310
ppm. Also the concentration of the potassium carbonate having known fungicidal
properties in the
composition according to this invention may vary between 3.5 and 25 mM
preferably between 10 and
25 mM.
[0018] The composition according to this invention may be prepared by mixing
the essential oil or
oils and the agent having fungicidal properties through any method of mixing
known in the art.
However, the composition may also be in solid or liquid form, such as a
suspension, dispersion,
emulsion, spray, microencapsulate or any other type of mixture which remains
stable over time or
may be incorporated in polymers, waxes or any other similar supports.
[0019] Furthermore, the phytosanitary composition according to this invention
may be used as such,
or may be used to formulate a phytosanitary product together with different
additives used in the art
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which offer different properties, such as surfactants, polymers, alkanising
agents, pH-control agents,
among many other additives used in the formulation of products used in the
agricultural industry.
100201 The phytosanitary composition according to this invention falls within
the group of contact
phytosanitary agents that is the form of the protection against fungal
diseases is through contact, given
that the composition remains on the surface of different parts of the plant,
protecting it externally
against the external attack of fungi.
[0021] Being a liquid, a powder or a microencapsulate, the phytosanitary
composition according to
this invention can be applied by any method of application known in the art,
such as spraying.
100221 The fungicidal composition according to this invention may further
comprise a fertiliser,
which may be selected from the group comprising compounds containing nitrogen
and/or phosphorus
such as urea, melamine, hexamine, dicyanodiamide, ameline, cyanuric acid,
melamine nitrate, triethyl
phosphite and the like or mixtures thereof.
100231 The composition according to this invention may also comprise any
compound or product
having chemical and/or biological activity used in agriculture, such as
herbicides, insecticides, plant
growth regulators and the like, or mixtures thereof.
[0024] This invention is described below in greater detail with reference to
various examples.
However, these examples are not intended to restrict the technical scope of
this invention.
EXAMPLES
[0025] Example 1. Inhibition of growth of the fungus Bowls cinerea by K2CO3
alone.
[0026] The fungus B. cinema was cultured in PDB (potato dextrose broth) medium
with different
concentrations of K2CO3. The % inhibition, representing the extent to which
growth was impeded in
comparison with a control which did not have the compound(s) under test, was
calculated in the
following way:
0 D (control) ¨ OD (x)
% Inhibition = x 100
OD (control)
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[0027] where "OD(control)" is the optical density of the control culture
(without test compounds) and
"OD(x)" is the optical density of the culture with the test substance(s). The
optical density of the
liquid culture was measured 24 hours after the start of culturing and the
results are shown in Table I.
Table I. Inhibition of the growth of B. cinerea by K2CO3
K2CO3 concentration
0 3.5 4.25 4.5 5
(mM)
Inhibition 48.2 53.3 67.2 71.0
0
SD (%) + 2.3 2.4 1.9 1.6
[0028] As will be seen from the table above, with a K2CO3 concentration
between 3.5 and 5 mM
inhibition of the B. cinerea culture was observed.
[0029] Example 2. Inhibition of growth of the fungus Botrytis cinema by
carvacrol.
[0030] The fungus B. cinema was cultured in PDB medium with different
concentrations of carvacrol.
The % inhibition was calculated as Example 1.
[0031] The optical density of the liquid culture was measured 24 hours after
the start of culturing and
the results are shown in Table II.
Table II. Inhibition of the growth of B. cinerea by carvacrol
Carvacrol concentration
0.1 0.31 1 3.1 10 31
100
(PPm)
Inhibition 10.5 13.7 22.4 21.3 51.4
74.4
0
SD (%) 7.7 4.1 3.4 5.0 5.5
1.1
[0032] Example 3. Inhibition of the fungus Botrytis cinerea by the composition
according to this
invention (K2CO3 + Carvacrol).
[0033] The fungus B. cinerea was cultured in a similar way to Example 1 with
the difference that
different concentrations of carvacrol were used in the medium and that a
constant concentration of
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K2CO3 (3.5 mM) was used throughout. The 24 hour optical density of the culture
was measured and
the results are shown in Table III.
Table III. Inhibition of B. cinerea by the composition according to this
invention (K2CO3 + Carvacrol)
K2CO3 concentration
3.5 3.5 3.5 3.5 3.5 3.5 3.5
(mM)
Carvacrol concentration
0.1 0.31 1 3.1 10 31 100
(PPm)
Inhibition 50.2 58.8 65.7 82.4 88.0 100 100
SD (%) 3.0 2.2 2.7 1.9 1.5 0 0
[0034] It will be seen how the results are improved by adding carvacrol to
K2CO3. With 10 ppm of
carvacrol (see in Table II) only 21% inhibition is achieved, and with 3.5 mM
of K2CO3 48% inhibition
is achieved. However, when the two compounds are combined inhibition of growth
of the fungus B.
cinerea is increased up to 88%.
[0035] Example 4. Inhibition of growth of the fungus Bowls cinerea by thymol
alone.
[0036] The fungus B. cinerea was cultured in a similar way to Example 1 with
different
concentrations of thymol. The 24 hour optical density of the culture was
measured and the results are
shown in Table IV.
Table IV. Inhibition of the growth of B. cinerea by Thymol
Thymol concentration
0.31 1 3.1 10 31 100
(PPm)
Inhibition 0 2.1 10.7 12.6 32.0 88.2
SD (%) 0 2.9 2.3 2.4 1.7 0.6
[0037] Example 5. Inhibition of the fungus Botrytis cinerea by the composition
according to this
invention (K2CO3 + Thymol).
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[0038] The fungus B. cinerea was cultured in a similar way to Example 1 with
the difference that
different concentrations of thymol were used in the medium and that a constant
concentration of
K2CO3 (3.5 mM) was used throughout. The 24 hour optical density of the culture
was measured and
the results are shown in Table V.
Table V. Inhibition of B. cinerea by the composition according to this
invention (K2CO3 + Thymol)
K2CO3 concentration
3.5 3.5 3.5 3.5 3.5 3.5
(mM)
Thymol concentration
0.31 1 3.1 10 31 100
(PPm)
Inhibition 49.4 62.1 73.2 81.0 93.0
100
SD (%) 2.1 2.2 1.1 1.6 1.1
[0039] It will be seen how the results are improved by adding thymol to K2CO3.
With 10 ppm of
thymol only 13% inhibition is achieved, and with 3.5 mM of K2CO3 48%
inhibition is achieved.
However, when the two compounds are combined inhibition of growth of the
fungus B. cinerea is
increased up to some 81%.
[0040] Example 6. Inhibition of growth of the fungus Alternana alternata by
K2CO3 alone.
[0041] Alternaria alternata was cultured in a similar way to Example 1 with
different concentrations
of K2CO3. The 24 hour optical density of the culture was measured and the
results are shown in Table
VI.
Table VI. Inhibition of the growth of A. alternata by K2CO3
K2CO3 concentration
0 3.5 4.25 4.5 5
(mM)
Inhibition 31.5 39.7 48.0 59.0
0
SD (%) 2.5 2.5 2.2 2.2
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[0042] As will be seen from the table above, with a K2CO3 concentration
between 3.5 and 5 mM
inhibition of the A. altemata culture was observed.
[0043] Example 7. Inhibition of growth of the fungus Alternalia alternata by
carvacrol.
[0044] The fungus A. altemata was cultured in PDB medium with different
concentrations of
carvacrol. The % inhibition was calculated as Example 1 and the results are
shown in Table VII.
Table VII. Inhibition of the growth of A. alternata by carvacrol
Carvacrol concentration
0.1 0.31 1 3.1 10 31
100
(PPm)
Inhibition 2.0 3.5 5.8 17.7 27.2
74.6
0
SD (%) 0.3 0.8 1.4 11.0 14.0
8.0
[0045] Example 8. Inhibition of the fungus Alternarin alternata by the
composition according to this
invention (K2CO3 + Carvacrol).
[0046] The fungus A. altemata was cultured in a similar way to Example 1 with
the difference that
different concentrations of carvacrol were used in the medium and that a
constant concentration of
K2CO3 (3.5 mM) was used throughout. The 24 hour optical density of the culture
was measured and
the results are shown in Table VIII.
Table VIII. Inhibition of A. alternata by the composition according to this
invention (K2CO3 +
Carvacrol)
K2CO3 concentration
3.5 3.5 3.5 3.5 3.5 3.5 3.5
(mM)
Carvacrol concentration
0.1 0.31 1 3.1 10 31 100
(PPm)
Inhibition 30.1 35.7 38.0 46.2
65.7 86.3 100
SD (%) 2.9 2.7 2.9 2.9 1.6 1.2 0
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[0047] It will be seen how the results are improved by adding carvacrol to
K2CO3. With 31 ppm of
carvacrol (see Table VII) only 27% inhibition is achieved, and with 3.5 mM of
K2CO3 32% inhibition
is achieved. However, when the two compounds are combined inhibition of growth
of the fungus A.
alternata is increased up to some 86%.
[0048] Example 9. Inhibition of growth of the fungus Alternalla alternata by
thymol alone.
[0049] The fungus A. alternata was cultured in a similar way to Example 1 with
different
concentrations of thymol. The 24 hour optical density of the culture was
measured and the
results are shown in Table IX.
Table IX. Inhibition of the growth of A. alternata by Thymol
Thymol concentration
0.31 1 3.1 10 31 100
(PPrn)
Inhibition 0 4.1 10.4 16.0 29.0 69.4
SD (%) 0 2.9 2.6 2.0 1.5
[0050] Example 10. Inhibition of the fungus Alternaria alternata by the
composition according to this
invention (K2CO3 + Thymol).
100511 The fungus A. alternata was cultured in a similar way to Example 1 with
the difference that
different concentrations of thymol were used in the medium and that a constant
concentration of
K2CO3 (3.5 mM) was used throughout. The 24 hour optical density of the culture
was measured and
the results are shown in Table X.
Table X. Inhibition of A. alternata by the composition according to this
invention (K2CO3 +
Thymol)
K2CO3 concentration (mM) 3.5 3.5 3.5 3.5 3.5 3.5
Thymol concentration
0.31 1 3.1 10 31 100
(PPm)
Inhibition 33.7 38.7 45.3 55.5 71.8
100
SD (%) 2.2 2.7 2.4 1.2 1.3
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[0052] It will be seen how the results are improved by adding thymol to K2CO3.
With 31 ppm of
thymol only 29% inhibition is achieved, and with 3.5 mM of K2CO3 32%
inhibition is achieved.
However, when the two compounds are combined inhibition of growth of the
fungus A. alternata is
increased up to some 72%.
[0053] Example 11. Inhibition of growth of the fungus Penicillium digitatum by
K2CO3 alone.
[0054] Penicillium digitatum was cultured in a similar way to Example 1 with
different
concentrations of K2CO3. The 24 hour optical density of the culture was
measured and the results are
shown in Table XI.
Table XI. Inhibition of the growth of P. digitatum by K2CO3
K2CO3 concentration
0 3.5 4.25 4.5 5
(mM)
Inhibition 29.1 33.0 36.9 39.5
0
SD (%) 2.1 1.7 1.6 1.8
[0055] As will be seen from the table above, with a K2CO3 concentration
between 3.5 and 5 mM
inhibition of the P. digitatum culture was observed.
100561 Example 12. Inhibition of growth of the fungus Penicillium digitatum by
carvacrol alone.
[0057] The fungus P. digitatum was cultured in 'a similar way to Example 1
with different
concentrations of carvacrol. The 24 hour optical density of the culture was
measured and the results
are shown in Table XII.
Table XII. Inhibition of the growth of P. digitatum by Carvacrol
Carvacrol
0.1 0.31 1 3.1 10 31 100
concentration (ppm)
Inhibition 32.3 34.3 37.9 52.6 57.8 86.0
100
SD (%) 2.8 2.8 3.0 2.2 2.1 1.1
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100581 Example 13. Inhibition of the fungus Penicillium digitatum by the
composition according to
this invention (K2CO3 + Carvacrol).
[0059] The fungus P. a'igitatum was cultured in a similar way to Example 1
with the difference that
different concentrations of carvacrol were used in the medium and that a
constant concentration of
K2CO3 (3.5 mM) was used throughout. The 24 hour optical density of the culture
was measured and
the results are shown in Table XIII.
Table XIII. Inhibition of P. digitatum by the composition according to this
invention (K2CO3 +
Carvacrol)
K2CO3 concentration
3.5 3.5 3.5 3.5 3.5 3.5 3.5
(mM)
Carvacrol concentration
0.1 0.31 1 3.1 10 31 100
(PPm)
Inhibition 65.9 66.2 72.4 88.6 96.5 100 100
SD (%) 2.4 2.9 2.2 1.9 1.4 0 0
[0060] It will be seen how the results are improved by adding carvacrol to
K2CO3. With 10 ppm of
carvacrol 58% inhibition is achieved, and with 3.5 mM of K2CO3 29% inhibition
is achieved.
However, when the two compounds are combined inhibition of growth of the
fungus P. digitatum is
increased up to 97%.
[0061] Example 14. Inhibition of growth of the fungus Penicillium digitatum by
thymol alone.
[0062] The fungus P. digitatum was cultured in a similar way to Example 1 with
different
concentrations of thymol. The 24 hour optical density of the culture was
measured and the results are
shown in Table XIV.
Table XIV. Inhibition of the growth of P. digitatum by Thymol
Thymol concentration
0.31 1 3.1 10 31 100 100
(PPm)
Inhibition 28.2 24.2 36.3 36.2 50.7
78.3 95.6
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SD (%) 3.9 + 6.0 2.3 2.0 2.0
2.2 0.5
[0063] Example 15. Inhibition of the fungus Penicillium digitatum by the
composition according to
this invention (K2CO3 + Thymol).
[0064] The fungus P. digitatum was cultured in a similar way to Example 1 with
the difference that
different concentrations of thymol were used in the medium and that a constant
concentration of
K2CO3 (3.5 mM) was used throughout. The 24 hour optical density of the culture
was measured and
the results are shown in Table XV.
Table XV. Inhibition of P. digitatum by the composition according to this
invention (K2CO3 +
Thymol)
K2CO3 concentration
3.5 3.5 3.5 3.5 3.5 3.5
(mM)
Thymol concentration
0.31 1 3.1 10 31 100
(PPm)
Inhibition 58.1 61.3 69.9 78.3 94.3
100
SD (%) 2.9 2.5 1.9 1.5 1.2
[0065] It will be seen how the results are improved by adding thymol to K2CO3.
With 31 ppm of
thymol (see Table XIV) only 51% inhibition is achieved, and with 3.5 mM of
K2CO3 29% inhibition is
achieved. However, when the two compounds are combined inhibition of growth of
the fungus P.
digitatum is increased up to some 94%.
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[0066] Example 16. Inhibition of growth of the fungus Cercospora beticola by
K2CO3 alone.
[0067] Cercospora beticola was cultured on PDA (potato dextrose agar) culture
medium buffered to a
pH value not exceeding 9.5. The inhibition degree, expressed as a percentage,
was determined based
on the growth relative to control that did not have the compound(s) to be
tested. The % inhibition was
calculated with the following formula:
colony diameter (control) ¨ colony diameter (x)
% Inhibition = x 100
colony diameter (control)
[0068] wherein "colony diameter (control)" is the size of the control colony
(without the compounds
to be tested) and "colony diameter (x)" is the size of the colony with the
substance(s) to be tested. A
fixed K2CO3 concentration of 7.24 mM was tested in C beticola. The results are
shown in Table XVI.
Table XVI. Inhibition of the growth of C beticola by K2CO3 alone
K2CO3 concentration
7.24
(mM)
Inhibition 25.0
SD (%) 1.2
[0069] Example 17. Inhibition of growth of the fungus Cercospora beticola by
carvacrol alone.
100701 The fungus C beticola was cultured in a similar way to Example 16 with
10 ppm of carvacrol.
The % inhibition was calculated and the results are shown in Table XVII.
Table XVII. Inhibition of the growth of C beticola by carvacrol alone
Carvacrol concentration
(PPrn)
Inhibition 17.0
SD (%) 0.8
100711 Example 18. Inhibition of growth of the fungus Cercospora beticola by
the composition
according to this invention (K2CO3 + Carvacrol).
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[0072] The fungus C beticola was cultured in a similar way to Example 16 with
a fixed concentration
of K2CO3 (7.24 mM) and carvacrol (10 ppm). The % inhibition was calculated and
the results are
shown in Table XVIII.
Table XVIII. Inhibition of the growth of C beticola by the composition
according to this
invention (K2CO3 + Carvacrol)
K2CO3 concentration
7.24
(mM)
Carvacrol concentration
(ppm)
Inhibition 65.0
SD (%) 3.2
[0073] It will be seen how the
results are improved by
adding carvacrol to K2CO3. With 10 ppm of carvacrol (see Table XVII) only 17%
inhibition is
achieved, and with 7.24 mM of K2CO3 25% inhibition is achieved. However, when
the two
compounds are combined inhibition of growth of the fungus C beticola is
increased up to some 65%.
[0074] Example 19. Inhibition of the fungus Botrytis cinerea by K2CO3 alone.
[0075] Leaves of tomato plants (var. Marmande) of 5 weeks old were treated
with different
concentrations of K2CO3, and 24 hours later, they were infected with the
fungus Botrytis cinema. Two
weeks later, fungal infection was assessed in leaves. The % inhibition,
representing the extent to
which fungal growth was impeded in comparison with a control which did not
have the compound(s)
under test, was determined in the following way:
% Infection (control) ¨ % Infection (x)
% Inhibition = ________________________________________________ x 100
Infection (control)
[0076] where "% Infection (control)" is the percentage of fungal infection of
the control leaves
(without test compounds) and "% Infection (x)" is percentage of fungal
infection of the treated leaves.
The results are shown in Table XIX.
Table XIX. Inhibition of the growth of B. cinerea by K2CO3 alone
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K2CO3 concentration
12 13 14 15 22
(mM)
Inhibition 13.4 13.5 13.8 13.8
15.3
SD (%) 2.5 2.1 2.3 3.0 2.4
[0077] Example 20. Inhibition of the fungus Botrytis cinerea by Carvacrol
alone.
[0078] Leaves of tomato plants treated with different concentrations of
carvacrol were infected with
the fungus B. cinerea in a similar way to Example 19. The % inhibition was
calculated and the results
are shown in Table XX.
Table XX. Inhibition of the growth of B. cinerea by Carvacrol alone
Carvacrol concentration
26 31 100 200 308
(PPm)
Inhibition 7.2 7.4 16.7 18.6 20.8
SD (%) 1.1 1.3 2.1 2.3 2.3
[0079] Example 21. Inhibition of growth of the fungus Bobytis cinerea by the
composition according
to this invention (K2CO3 + Carvacrol).
[0080] Leaves of tomato plants treated with different concentrations of
carvacrol and carvacrol were
infected with the fungus B. cinerea in a similar way to Example 19. The %
inhibition was calculated
and the results are shown in Table XXI.
Table XXI. Inhibition of the growth of B. cinerea by the composition according
to this
invention (K2CO3 + Carvacrol)
K2CO3 concentration (mM) 14 22 15 15 13
Carvacrol concentration
26 31 100 200 308
(PPin)
Inhibition 24.3 28.1 48.3 60.0
72.8
SD (%) 2.1 2.8 2.8 2.6 2.4
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[0081] It will be seen how the results are improved by adding carvacrol to
K2CO3. With 308 ppm
of carvacrol (see Table XX) only 21% inhibition is achieved, and with 13 mM of
K2CO3 14%
inhibition is achieved. However, when the two compounds are combined
inhibition of growth of the
fungus B. cinerea is increased up to some 73%.
[0082] Example 22. Inhibition of the fungus Botrytis cinerea by Thymol
alone.
[0083] Leaves of tomato plants treated with different concentrations of
thymol were infected
with the fungus B. cinerea in a similar way to Example 19. The % inhibition
was calculated and the
results are shown in Table XXII.
Table XXII. Inhibition of the growth of B. cinerea by Thymol alone
Thymol concentration
35 100 200 350
(PPm)
Inhibition 7.4 17.7 20.9 22.8
SD (%) 1.3 1.7 1.5 1.5
100841 Example 23. Inhibition of growth of the fungus Botlytis cinerea by the
composition according
to this invention (K2CO3 + Thymol).
[0085] Leaves of tomato plants treated with different concentrations of thymol
and K2CO3 were
infected with the fungus B. cinerea in a similar way to Example 19. The %
inhibition was calculated
and the results are shown in Table XXIII.
Table XXIII. Inhibition of the growth of B. cinerea by the composition
according to this invention
(K2CO3 + Thymol)
K2CO3 concentration
15 15 15 12
(mM)
Thymol concentration
35 100 200 350
(PPm)
Inhibition 25.6 40.8 53.5 65.0
SD (%) 2.6 2.4 2.3 2.2
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[0086] It will be seen how the results are improved by adding thymol to K2CO3.
With 350 ppm of
thymol (see Table XXII) only 23% inhibition is achieved, and with 12 mM of
K2CO3 14% inhibition
is achieved. However, when the two compounds are combined inhibition of growth
of the fungus B.
cinerea is increased up to some 65%.
100871 Example 24. Inhibition of the fungus Alternaria alternata by K2CO3
alone.
[0088] Leaves of tomato plants treated with different concentrations of K2CO3
were infected with the
fungus A. alternata in a similar way to Example 19. The % inhibition was
calculated and the results
are shown in Table XXIV.
Table XXIV. Inhibition of the growth of A. alternata by K2CO3 alone
K2CO3 concentration
12 13 14 15 22
(mM)
Inhibition 6.5 7.0 7.2 7.8 8.6
SD (%) 2.2 1.1 1.1 1.4 1.5
[0089] Example 25. Inhibition of the fungus Alternana alternata by Carvacrol
alone.
100901 Leaves of tomato plants treated with different concentrations of
carvacrol were infected with
the fungus A. altemata in a similar way to Example 19. The % inhibition was
calculated and the
results are shown in Table XXV.
Table XXV. Inhibition of the growth of A. alternata by Carvacrol alone
Carvacrol concentration
26 31 100 200 308
(PPm)
Inhibition 4.3 5.8 11.4 16.1 18.4
SD (%) 0.7 0.9 1.3 1.2 1.2
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[0091] Example 26. Inhibition of growth of the fungus Alternaria alternata by
the composition
according to this invention (K2CO3 + Carvacrol).
[0092] Leaves of tomato plants treated with different concentrations of
carvacrol and K2CO3 were
infected with the fungus B. cinerea in a similar way to Example 19. The %
inhibition was calculated
and the results are shown in Table XXVI.
Table XXVI. Inhibition of the growth of A. alternata by the composition
according to this
invention (K2CO3 + Carvacrol)
K2CO3 concentration
14 22 15 15 13
(mM)
Carvacrol concentration
26 31 100 200 308
(PPm)
Inhibition 18.4 17.3 43.9 60.9 70.8
SD (%) 2.1 2.4 3.2 2.8 3.5
[0093] It will be seen how the results are improved by adding carvacrol to
K2CO3. With 308 ppm of
carvacrol (see Table XXV) only 18% inhibition is achieved, and with 13 mM of
K2CO3 7% inhibition
is achieved. However, when the two compounds are combined inhibition of growth
of the fungus A.
alternata is increased up to some 71%.
[0094] Example 27. Inhibition of the fungus Altemazia alternata by Thymol
alone.
[0095] Leaves of tomato plants treated with different concentrations of thymol
were infected with the
fungus A. alternata in a similar way to Example 19. The % inhibition was
calculated and the results
are shown in Table XXVII.
Table XXVII. Inhibition of the growth of A. alternata by Thymol alone
Thymol concentration
35 100 200 350
(PPm)
Inhibition 10.0 15.2 I9.3 21.3
SD (%) 1.4 2.0 2.3 2.6
Page 19 of 33
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100961 Example 28. Inhibition of growth of the fungus Alternaria alternata by
the composition
according to this invention (K2CO3 + Thymol).
100971 Leaves of tomato plants treated with different concentrations of thymol
and K2CO3 were
infected with the fungus A. alternata in a similar way to Example 19. The %
inhibition was calculated
and the results are shown in Table XXVIII.
Table XXVIII. Inhibition of the growth of A. alternata by the composition
according to this
invention (K2CO3 + Thymol)
K2CO3 concentration
15 15 15 12
(mM)
Thymol concentration
35 100 200 350
(PPm)
Inhibition 22.4 37.3 53.7 60.8
SD (%) 2.5 2.9 2.0 2.2
100981 It will be seen how the results are improved by adding thymol to
K2CO3. With 350 ppm of
thymol (see Table XXVII) only 21% inhibition is achieved, and with 12 mM of
K2CO3 7% inhibition
is achieved. However, when the two compounds are combined inhibition of growth
of the fungus A.
alternata is increased up to some 61%.
100991 Example 29. Inhibition of the fungus Phytophthora infestans by K2CO3
alone.
1001001 Tomato plants (var. Marmande) of 5 weeks old were treated with 15 mM
of K2CO3. and 24
hours later, they were infected with the fungus Phytophthora infestans. Two
weeks later, fungal
infection was assessed in leaves. The % inhibition, representing the extent to
which growth was
impeded in comparison with a control which did not have the compounds under
test, was determined
in the following way:
% Inhibition =% Infection (control) ¨ % Infection (x)
X 100
% Infection (control)
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CA 3060884 2019-11-04
[00101] where "% Infection (control)" is the percentage of fungal infection of
the control plants
(without test compound) and "% Infection (x)" is percentage of fungal
infection of the treated plants.
The results are shown in Table XXIX.
Table XXIX. Inhibition of the growth of P. infestans by K2CO3 alone
K2CO3 concentration
(mM)
Inhibition 18.5
SD (%) 3.6
[00102] Example 30. Inhibition of the fungus Phytophthora infestans by
Carvacrol alone.
[00103] Tomato plants were treated with different concentrations of carvacrol
and subsequently
infected with the fungus P. infestans in a similar way to Example 29. The %
inhibition was calculated
and the results are shown in Table XXX.
Table XXX. Inhibition of the growth of P. infestans by Carvacrol alone
Carvacrol concentration
26 150 308
(PPm)
Inhibition 3.2 17.1 35.0
SD (%) 0.8 2.3 4.8
[00104] Example 31. Inhibition of growth of the fungus Phytophthora infestans
by the composition
according to this invention (K2CO3 + Carvacrol).
[00105] Tomato plants were treated with different concentrations of carvacrol
and a fixed
concentration of K2CO3 and subsequently infected with the fungus P. infestans
in a similar way to
Example 29. The % inhibition was calculated and the results are shown in Table
XXXI.
Table XXXI. Inhibition of the growth of P. infestans by the composition
according to this
invention (K2CO3 + Carvacrol)
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CA 3060884 2019-11-04
K2CO3 concentration
15 15 15
(mM)
Carvacrol concentration
26 150 308
(PPm)
Inhibition 25.6+ 52.2 78.3
SD (%) 3.6 4.2 4.8
[00106] It will be seen how the results are improved by adding carvacrol to
K2CO3. With 308 ppm
of carvacrol (see Table XXX) only 35% inhibition is achieved, and with 15 mM
of K2CO3 19%
inhibition is achieved. However, when the two compounds are combined
inhibition of growth of the
fungus P. infestans is increased up to some 78%.
[00107] Example 32. Inhibition of the fungus Phytophthora infestans by Thymol
alone.
[00108] Tomato plants were treated with different concentrations of thymol and
subsequently
infected with the fungus P. infestans in a similar way to Example 29. The %
inhibition was calculated
and the results are shown in Table XXXII.
Table XXXII. Inhibition of the growth of P. infestans by Thymol alone
Thymol concentration
35 150 350
(PPm)
Inhibition 4.3 I6.2 33.6
SD (%) 0.7 3.2 5.6
[00109] Example 33. Inhibition of growth of the fungus Phytophthora infestans
by the composition
according to this invention (K2CO3 + Thymol).
1001101 Tomato plants were treated with different concentrations of thymol and
a fixed
concentration of K2CO3 and subsequently infected with the fungus P. infestans
in a similar way to
Example 29. The % inhibition was calculated and the results are shown in Table
XXXIII.
Page 22 of 33
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Table XXXIII. Inhibition of the growth of P. infestans by the composition
according to this
=
invention (K2CO3 + Thymol)
K2CO3 concentration
15 15 15
(mM)
Thymol concentration
35 150 350
(PPrn)
Inhibition 26.3 49.3 74.2
SD (%) 2.2 3.6 5.2
[001111 It will be seen how the results are improved by adding thymol to
K2CO3. With 350 ppm of
thymol (see Table XXXII) only 34% inhibition is achieved, and with 15 mM of
K2CO3 19% inhibition
is achieved. However, when the two compounds are combined inhibition of growth
of the fungus P.
infestans is increased up to some 74%.
[00112] Example 34. Inhibition of the fungus Leveillula taufica by K2CO3
alone.
[00113] The fungus L. taufica was cultured in a similar way to Example 29 with
different
concentrations of K2CO3. The % inhibition was calculated and the results are
shown in Table XXXIV.
Table XXXIV. Inhibition of the growth of L. taufica by K2CO3 alone
K2CO3 concentration
(mM)
Inhibition 13.9
SD (%) 0.5
1001141 Example 35. Inhibition of the fungus Leveillula taufica by Carvacrol
alone.
[00115] Tomato plants were treated with different concentrations of carvacrol
and subsequently
infected with the fungus L. taufica in a similar way to Example 29. The %
inhibition was calculated
and the results are shown in Table XXXV.
Table XXXV. Inhibition of the growth of L. taufica by Carvacrol alone.
Page 23 of 33
CA 3060884 2019-11-04
Carvacrol concentration
26 150 308
(PPm)
Inhibition 4.0 11.2 41.5
SD (%) 1.2 1.0 1.2
[00116] Example 36. Inhibition of growth of the fungus Leveillula tautica by
the composition
according to this invention (K2CO3 + Carvacrol).
[00117] Tomato plants were treated with different concentrations of carvacrol
and a fixed
concentration of K2CO3 and subsequently infected with the fungus L. tautica in
a similar way to
Example 29. The % inhibition was calculated and the results are shown in Table
XXX VI.
Table XXX VI. Inhibition of the growth of L. tautica by the composition
according to this
invention (K2CO3 + Carvacrol)
K2CO3 concentration
15 15 15
(mM)
Carvacrol concentration
26 150 308
(PPm)
Inhibition 18.6 78.3
92.0 0
SD (%) 3.3 0.6
[00118] It will be seen how the results are improved by adding carvacrol to
K2CO3. With 308 ppm
of carvacrol (see Table XXXV) only 42% inhibition is achieved, and with 15 mM
of K2CO3 14%
inhibition is achieved. However, when the two compounds are combined
inhibition of growth of the
fungus L. tawica is increased up to some 92%.
[00119] Example 37. Inhibition of the fungus Leveillula tautica by Thymol
alone.
[00120] Tomato plants were treated with different concentrations of thymol and
subsequently
infected with the fungus L. taurica in a similar way to Example 29. The %
inhibition was calculated
and the results are shown in Table XXX VII.
Table XXX VII. Inhibition of the growth of L. taulica by Thymol alone.
Page 24 of 33
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Thymol concentration
35 150 350
(PPm)
Inhibition 10.5 38.2 51.2
SD (%) 5.4 5.5 1.0
[00121] Example 38. Inhibition of growth of the fungus Leveillula taurica by
the composition
according to this invention (K2CO3 + Thymol).
[00122] Tomato plants were treated with different concentrations of thymol and
a fixed
concentration of K2CO3 and subsequently infected with the fungus L. taurica in
a similar way to
Example 29. The % inhibition was calculated and the results are shown in Table
XXX VIII.
Table XXX VIII. Inhibition of the growth of L. taurica by the composition
according to this
invention (K2CO3 + Thymol)
K2CO3 concentration
15 15 15
(mM)
Thymol concentration
35 150 350
(PPm)
Inhibition 48.8 65.4 72.9
SD (%) 3.1 2.4 1.1
[00123] It will be seen how the results are improved by adding thymol to
K2CO3. With 350 ppm of
thymol (see Table XXXVII) only 51% inhibition is achieved, and with 15 mM of
K2CO3 14%
inhibition is achieved. However, when the two compounds are combined
inhibition of growth of the
fungus P. infestans is increased up to some 73%.
[00124] Example 39. Inhibition of the fungus Pseudoperonospora cubensis by the
composition
according to this invention (K2CO3 + Carvacrol).
[00125] The efficacy of the composition of the present invention (K2CO3 +
carvacrol or thymol)
was tested in field assays with cucumber, tomato, lettuce or potato plants to
prevent
Page 25 of 33
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Pseudoperonospora cubensis, Botrytis cinerea, Phytoplithora infestans or
Leveillula taurica growth.
The efficacy of the fungicide was measured as follow:
% Severity (control) ¨ % Severity (x)
%Efftcacy= x100
%Severity (control)
1001261 where "% Severity (control)" is the percentage of fungal severity of
the control plants
(without test compounds) and "% Severity (x)" is percentage of fungal severity
of the treated plants.
Cucumber plants were treated with different concentrations of K2CO3 and
carvacrol and subsequently
infected with Pseudoperonospora cubensis. The results are shown in Table
XXXIX.
Table XXXIX. Efficacy of Pseudoperonospora cubensis by the composition
according to this
invention (K2CO3 + Carvacrol) in cucumber
K2CO3 concentration
13.5 19 6.5 13.0 22.4
(mM)
Carvacrol concentration
25.5 36.5 150 300 530
(PPm)
Inhibition 16.7 26.7 35.5 45.3
64.2
SD (%) 3.3 4.2 3.9 5.3 7.3
[00127] The efficacy of the inhibition of growth of the fungus P. cubensis
reached 64% by
combining K2CO3 and carvacrol.
[00128] Example 40. Inhibition of the fungus Pseudoperonospora cubensis by the
composition
according to this invention (K2CO3 + Thymol).
[00129] The efficacy of the composition of the present invention (K2CO3 +
Thymol) was tested in
cucumber to prevent P. cubensis growth. The efficacy of the antifungicide was
measured and the
results are shown in Table XL.
Table XL. Efficacy of Pseudoperonospora cubensis by the composition according
to this invention
(K2CO3 + Thymol) in cucumber
K2CO3 concentration 6.5 13.0 22.4
Page 26 of 33
CA 3060884 2019-11-04
(mM)
Thymol concentration
150 300 530
(PPm)
Inhibition 31.6 41.1 53.3
SD (%) 4.6 6.1 7.0
=
[00130] The efficacy of the inhibition of growth of the fungus P. cubensis
reached 53% by
combining K2CO3 and thymol.
[00131] Example 41. Inhibition of the fungus Botrytis cinerea by the
composition according to this
invention (K2CO3 + Carvacrol).
[00132] The efficacy of the composition of the present invention (K2CO3 +
Carvacrol) was tested in
tomato to prevent B. cinerea growth. The efficacy of the antifungicide was
measured and the results
are shown in Table XLI.
Table XLI. Efficacy of Bouytis cinerea by the composition according to this
invention (K2CO3
+ Carvacrol) in tomato
K2CO3 concentration
13.5 19 6.5 13.0 22.4
(mM)
Carvacrol concentration
25.5 36.5 150 300 530
(PM')
Inhibition 21.7 27.8 38.5 69.8 78.3
SD (%) 4.4 4.1 5.6 8.3 6.5
[00133] The efficacy of the inhibition of growth of the fungus B. cinerea
reached 78% by
combining K2CO3 and carvacrol.
[00134] Example 42. Inhibition of the fungus BotryUs cinerea by the
composition according to this
invention (K2CO3 + Thymol).
Page 27 of 33
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[00135] The efficacy of the composition of the present invention (K2CO3 +
Thymol) was tested in
tomato to prevent B. cinerea growth. The efficacy of the antifungicide was
measured and the results
are shown in Table XLII.
Table XLII. Efficacy of Botrytis cinerea by the composition according to this
invention
(K2CO3 + Thymol) in tomato
K2CO3 concentration
6.5 13.0 22.4
(mM)
Thymol concentration
150 300 530
(PPm)
Inhibition 35.8 58.0 66.0
SD (%) 4.3 4.6 6.0
1001361 The efficacy of the inhibition of growth of the fungus B. cinerea
reached 66% by
combining K2CO3 and thymol.
[00137] Example 43. Inhibition of the fungus Phytoplithora infestans by the
composition according
to this invention (K2CO3+ Carvacrol).
[00138] The efficacy of the composition of the present invention (K2CO3 +
Carvacrol) was tested in
lettuce to prevent P. infestans growth. The efficacy of the antifungicide was
measured and the results
are shown in Table XLIII.
Table XLIII. Efficacy of P. infestans by the composition according to this
invention (K2CO3 +
Carvacrol) in lettuce
K2CO3 concentration
5.4 12.6 21.6
(mM)
Carvacrol concentration
132 309 529
(PPm)
Inhibition 58.0 72.1 80.3
SD (%) 2.4 5.2 4.3
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[00139] The efficacy of the inhibition of growth of the fungus P. infestans
reached 80% by
combining K2CO3 and carvacrol.
[00140] Example 44. Inhibition of the fungus Phytophthora infestans by the
composition according
to this invention (K2CO3+ Carvacrol).
[00141] The efficacy of the composition of the present invention (K2CO3 +
Carvacrol) was tested in
potato to prevent P. infestans growth. The efficacy of the antifungicide was
measured and the results
are shown in Table XLIV.
Table XLIV. Efficacy of P. infestans by the composition according to this
invention (K2CO3 +
Carvacrol) in potato
K2CO3 concentration
5.4 12.6 21.6
(mM)
Carvacrol concentration
132 309 529
(PPm)
Inhibition 68.6 71.7 86.7
SD (%) 2.9 3.4 4.1
[00142] The efficacy of the inhibition of growth of the fungus P. infestans
reached 87% by
combining K2CO3 and carvacrol.
[00143] Example 45. Inhibition of the fungus Leveilulla taurica by the
composition according to
this invention (K2CO3+ Carvacrol).
[00144] The efficacy of the composition of the present invention (K2CO3 +
Carvacrol) was tested in
tomato to prevent L. taufica growth. The efficacy of the antifungicide was
measured and the results
are shown in Table XLV.
Table XLV. Efficacy of L. taurica by the composition according to this
invention (K2CO3 +
Carvacrol) in tomato
K2CO3 concentration
5.7 13.2 24.5 5.4 12.6 21.6
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CA 3060884 2019-11-04 =
(mM)
Carvacrol concentration
11.0 25.6 47.5 132 309 529
(PPm)
Inhibition 22.0 53.0 61.0 43.0 58.0
71.0
SD (%) 3.1 4.4 3.9 4.2 5.2 3.8
1001451 The efficacy of the inhibition of growth of the fungus L. =Ica reached
71% by
combining K2CO3 and carvacrol.
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