U.S. patent application number 14/382952 was filed with the patent office on 2015-02-05 for polyelectrolyte complexes comprising natamycine and/or phosphite for biocide enhancement.
The applicant listed for this patent is Ceradis B.V.. Invention is credited to Christiaan Gerardus Johannes Maria Jans, Wilhelmus Bernardus Albertus Hendrikus Rutten, Wilhelmus Maria van der Krieken.
Application Number | 20150038442 14/382952 |
Document ID | / |
Family ID | 47884476 |
Filed Date | 2015-02-05 |
United States Patent
Application |
20150038442 |
Kind Code |
A1 |
van der Krieken; Wilhelmus Maria ;
et al. |
February 5, 2015 |
POLYELECTROLYTE COMPLEXES COMPRISING NATAMYCINE AND/OR PHOSPHITE
FOR BIOCIDE ENHANCEMENT
Abstract
The invention relates to a composition comprising a
polyelectrolyte complex of a polyanion and a polycation, further
comprising at least one biocide comprising natamycin and/or
phosphite. A preferred composition comprises lignosulfonate and
chitosan, preferably in relative amount of from 1:2 to 60:1 (w/w).
The invention further relates to methods for generating a
composition according to the invention, and to uses of a mixture
according to the invention for protecting an agricultural plant or
plant part against a pathogen.
Inventors: |
van der Krieken; Wilhelmus
Maria; (Wageningen, NL) ; Rutten; Wilhelmus Bernardus
Albertus Hendrikus; (Wageningen, NL) ; Jans;
Christiaan Gerardus Johannes Maria; (Wageningen,
NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ceradis B.V. |
Wageningen |
|
NL |
|
|
Family ID: |
47884476 |
Appl. No.: |
14/382952 |
Filed: |
March 5, 2013 |
PCT Filed: |
March 5, 2013 |
PCT NO: |
PCT/NL2013/050144 |
371 Date: |
September 4, 2014 |
Current U.S.
Class: |
514/31 |
Current CPC
Class: |
A01N 43/90 20130101;
A01N 25/24 20130101; A01N 25/10 20130101; A01N 43/08 20130101; A01N
43/90 20130101; A01N 41/04 20130101; A01N 25/24 20130101; A01N
41/04 20130101; A01N 43/08 20130101; A01N 25/10 20130101; A01N
43/40 20130101; A01N 47/14 20130101; A01N 43/50 20130101; A01N
59/26 20130101; A01N 43/40 20130101; A01N 47/14 20130101; A01N
43/90 20130101; A01N 43/40 20130101; A01N 43/90 20130101; A01N
59/26 20130101; A01N 43/90 20130101; A01N 47/04 20130101; A01N
43/40 20130101; A01N 59/26 20130101; A01N 43/40 20130101; A01N
41/04 20130101; A01N 47/04 20130101; A01N 2300/00 20130101; A01N
59/02 20130101; A01N 47/14 20130101; A01N 59/02 20130101; A01N
43/50 20130101; A01N 47/04 20130101; A01N 43/90 20130101; A01N
43/40 20130101; A01N 47/04 20130101; A01N 47/14 20130101 |
Class at
Publication: |
514/31 |
International
Class: |
A01N 25/24 20060101
A01N025/24; A01N 43/90 20060101 A01N043/90 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 5, 2012 |
EP |
12158136.7 |
Jun 8, 2012 |
EP |
12171345.7 |
Claims
1. A composition comprising a polyelectrolyte complex of a
polyanion, preferably selected from the group consisting of a
natural polyanion such as xanthan gum, alginate, a lignin compound
such as lignosulfonate, pectin, carrageenan, humic acid, fulvic
acid, angico gum, gum Kondagogu, sodium alkyl naphtalene sulfonate,
poly-.gamma.-glutamic acid, maleic starch half-ester, carboxymethyl
cellulose, chondroitin sulphate, dextran sulphate, hyaluronic acid,
and a synthetic polyanion such as poly(acrylic acid),
polyphosphoric acid, and poly(L-lactide), and a polycation
preferably selected from the group consisting of poly-L-lysine,
epsilon-poly-L-lysine, poly-L-arginine, chitosan oligosaccharide
and chitosan, wherein said polyanion and said polycation are
present in relative amounts of from 1:2 to 60:1 (w/w), said
composition further comprising at least one biocide comprising
natamycin and/or phosphite.
2. The composition of claim 1, wherein said polyanion is selected
from the group consisting of xanthan gum, humic acid, alginate and
lignosulfonate.
3. The composition of claim 1, wherein said polycation is or
comprises chitosan.
4. The composition of claim 1, wherein said polyanion is
lignosulfonate and said polycation is or comprises chitosan.
5. The composition of claim 1, wherein said biocide is present in a
concentration of between 0.1 and 90 w/v %.
6. The composition of claim 1, wherein said polyanion and
polycation are present in relative amounts of about 5:1 (w/w).
7. The composition claim 1, wherein the biocide is a fungicide.
8. The composition of claim 1, comprising a polyelectrolyte complex
of a polyanion and a polycation and at least one biocide, wherein
the composition is a suspension concentrate.
9. The composition of claim 1, comprising a polyelectrolyte complex
of a polyanion and a polycation and at least one biocide, wherein
the composition is a water dispersible granule.
10. A method for generating a composition according to claim 1,
comprising (a) providing an aqueous solution of a polyanion
selected from the group consisting of a natural polyanion such as
xanthan gum, alginate, a lignin compound such as lignosulfonate,
pectin, carrageenan, humic acid, fulvic acid, angico gum, gum
Kondagogu, sodium alkyl naphtalene sulfonate, poly-.gamma.-glutamic
acid, maleic starch half-ester, carboxymethyl cellulose,
chondroitin sulphate, dextran sulphate, hyaluronic acid, and a
synthetic polyanion such as poly(acrylic acid), polyphosphoric
acid, and poly(L-lactide), wherein the concentration of polyanion
is 1-20% (w/v); (b) providing an acidic solution of a polycation
selected from the group consisting of poly-L-lysine,
epsilon-poly-L-lysine, poly-L-arginine, chitosan oligosaccharide
and chitosan, wherein the concentration of the polycation is 1-10%
(w/v) and the pH is below pH=5.5; (c) adding the polyanion solution
to the polycation solution, wherein said polyanion and polycation
are present in relative amounts of from 1:2 to 60:1 (w/w), whereby
a formed precipitate is crushed; and (d) adding a biocide
comprising natamycin and/or phosphite to the solution of at least
one of steps a-c.
11. The method according to claim 10, wherein said polyanion is
selected from the group consisting of xanthan gum, alginate and
lignosulfonate.
12. The method according to claim 10, wherein said polycation is or
comprises chitosan.
13. The method according to claim 10, wherein the acidic polycation
solution comprises lactate, hydrochloric acid, and/or ascorbic
acid.
14.-17. (canceled)
18. A method of protecting an agricultural plant against a
pathogen, comprising applying to said agricultural plant or to one
or more plant parts a composition according to claim 1.
19. A method of preventing, reducing and/or eliminating the
presence of a pathogen on a plant or to one or more plant parts,
comprising applying to said plant or plant part a composition
according to claim 1.
20. The method of claim 19, wherein the pathogen is Botrytis.
21. The method of claim 19, wherein the plant part comprises seed,
leaf or fruit.
22. The method of claim 1, wherein the plant part is a post-harvest
fruit.
Description
FIELD
[0001] The present invention relates to compositions comprising
natamycine and/or phosphite and a polyelectrolyte complex of a
polyanion, such as lignosulfonate, and a polycation, such as
chitosan. The invention further relates to methods for producing a
composition of the invention and to methods of preventing, reducing
and/or eliminating the presence of a pathogen on a plant or on one
or more plant parts, comprising applying a composition of the
invention to said plant or plant part.
INTRODUCTION
[0002] Plants are often threatened by various pathogenic
micro-organisms like fungi, viruses and bacteria. To overcome the
problem of infections with these micro-organisms, large quantities
of anti-microbial compounds (in particular biocides, such as
fungicides and bactericides) are applied. There is an ongoing
concern about the possible negative impact of biocides on the
environment and on human health. As a consequence, the demands with
respect to sustainability of chemical pest control are continually
increasing, as are the costs to bring new pesticides to the
market.
[0003] In principle, reduced pressure of chemical biocides on the
environment can be achieved by reduced quantities of chemicals
applied for control of pathogenic micro-organisms. It is obvious
that precise targeting and uniform distribution of the chemicals
over the intended site of application is crucial to keep the input
of chemicals to the environment as low as possible. This requires
optimal formulation and optimal application procedures. Spray drift
reduction (i.e. reduction of movement of pesticide through air to a
site other than the intended site) is a well-known example of how
on a macroscopic level substantial input reductions can be achieved
without loss of efficacy. But also on a microscopic, molecular
level targeting and distribution of the chemicals can be optimized,
allowing significant lower input of absolute quantities of
chemicals without loss of efficacy.
[0004] It is known that certain compounds of natural origin can
protect plants against pathogenic micro-organisms. These natural
biocides comprise organic substances derived from natural organisms
(e.g. plant extracts) and anorganic compounds found in the natural
environment (e.g. copperhydroxide or sulfur). The use of these
natural biocides is becoming more and more preferable since
governments aim for a reduction in the use of synthetic biocidial
compounds.
[0005] It is therefore an objective of the present invention to
provide a composition comprising plant protecting biocides that
reduces the amount of biocide that is required to protect a plant
against pathogenic micro-organisms.
SUMMARY OF THE INVENTION
[0006] The invention relates to a composition comprising a biocide
comprising natamycine and/or phosphite and a polyelectrolyte
complex of a polyanion, such as xanthan gum, alginate or a
lignin-compound, and a polycation, such as poly-L-lysine and
chitosan. Said composition is preferably used for protection of
plants or plant parts against infections with a pathogen, including
fungi. It was surprisingly found that said polyelectrolyte complex
dramatically improved the protective effect of the biocide against
a pathogen, in comparison with the same biocide without said
polyelectrolyte complex. Without being bound by theory, said
polyelectrolyte complex may provide improved, longer lasting,
adherence of the biocide to the plant or plant part and/or the
adherence to a pathogen, such as a fungus or an insect.
[0007] The polyelectrolyte complex of a polyanion and a polycation
is an irreversible insoluble complex. This complex alone does not
have biocidial efficacy. The polyelectrolyte complex has sticky
properties and contains polar parts (charges) and apolar parts. The
aromatic moieties in the complex may have affinity for the biocides
which often also contain aromatic rings. In combination with the
sticky character of the polyelectrolyte complex, the biocide will
be optimally deposited and adhered to a plant or plant part.
[0008] The invention further provides a composition comprising at
least one biocide comprising natamycine and/or phosphite and a
polyelectrolyte complex of a polyanion and a polycation. Said
polyanion is preferably selected from the group consisting of a
natural polyanion such as xanthan gum, alginate, a lignin compound
such as lignosulfonate, pectin, carrageenan, humic acid, fulvic
acid, angico gum, gum Kondagogu, sodium alkyl naphtalene sulfonate
(Morwet), poly-.gamma.-glutamic acid, maleic starch half-ester,
carboxymethyl cellulose, chondroitin sulphate, dextran sulphate,
hyaluronic acid, and a synthetic polyanion such as poly(acrylic
acid), polyphosphoric acid, and poly(L-lactide).
[0009] Said polycation is preferably selected from the group
consisting of poly-L-lysine, epsilon-poly-L-lysine,
poly-L-arginine, chitosan oligosaccharide, and chitosan, whereby
the lignin-compound and chitosan are in a relative amount of
between 1:2 and 60:1 (w/w), more preferred between 2:1 and 30:1
(w/w), most preferred about 15:1 (w/w), even more preferred about
5:1 (w/w).
[0010] Said lignin-compound preferably comprises or is
lignosulfonate, preferably a lignosulfonate salt such as, for
example, calcium lignosulfonate, sodium lignosulfonate, potassium
lignosulfonate, ammonium lignosulfonate, magnesium lignosulfonate
etc.
[0011] The biocide in a composition according to the invention is
preferably a fungicide comprising natamycine and/or phosphite.
[0012] A composition according to the invention can be a solid or,
preferably, an aqueous composition, preferably a suspension.
[0013] The invention further provides a method for producing a
composition according to the invention, comprising (a) providing an
aqueous solution of a polyanion selected from the group consisting
of a natural polyanion such as xanthan gum, alginate, a lignin
compound such as lignosulfonate, pectin, carrageenan, humic acid,
fulvic acid, angico gum, gum Kondagogu, sodium alkyl naphtalene
sulfonate, poly-.gamma.-glutamic acid, maleic starch half-ester,
carboxymethyl cellulose, chondroitin sulphate, dextran sulphate,
hyaluronic acid, and a synthetic polyanion such as poly(acrylic
acid), polyphosphoric acid, and poly(L-lactide), wherein the
concentration of said polyanion is from 0.1-60 w/v % or from 1-60
w/v %, (b) providing an aqueous acidic solution of a polycation
selected from the group consisting of poly-L-lysine,
epsilon-poly-L-lysine, poly-L-arginine, chitosan oligosaccharide,
and chitosan, wherein the concentration of said polycation is from
0.1-10 w/v % or from 1-10 w/v % and the pH is below pH=5.5, (c)
adding the solution of a polyanion to the solution of a polycation,
whereafter the formed precipitate is crushed preferably milled in
the presence of a dispersing agent and/or a wetting agent, followed
by (d) adding a biocide comprising natamycine and/or phosphite. As
an alternative, the biocide may be added to the solution of at
least one of steps a-c. If required, an acid is added to the
polycation-polyanion mixture to keep the pH of the mixture below
pH=7, preferably below pH=5.5. The relative amount of a polyanion
and a polycation in the composition is between 1:2 and 60:1 (w/w),
more preferred between 2:1 and 30:1 (w/w), most preferred about
15:1 (w/w), even more preferred about 5:1. The final pH value my be
adjusted to a pH value of between 3-12, more preferred between 5-9,
most preferred between 6-7, depending on the biocide that is used,
as is known to the skilled person.
[0014] A composition according to the invention is preferably used
for the protection of a plant or a part of a plant against a
pathogen. Therefore, the invention also provides the use of a
composition comprising at least one biocide comprising natamycine
and/or phosphite according to the invention, whereby the
composition is sprayed over a plant or a part of a plant. Said
composition is preferably used for protection against different
forms of downy mildew, powdery mildew and Botrytis.
[0015] The invention further provides a method of protecting a
plant or a plant part against a pathogen, comprising applying to
said plant or on one or more plant parts a composition comprising
at least one biocide according to the invention. Said composition
is preferably diluted 2-1000 times, preferably about 200 times,
with water to contain between 0.001 and 1% (w/v) of the biocide.
The invention further provides a method of preventing, reducing
and/or eliminating the presence of a pathogen on a plant or on one
or more plant parts, comprising applying to said plant or plant
part a composition comprising at least one biocide according to the
invention, or a composition according to the invention that is
diluted with water to contain between 0.001 and 20%, more preferred
between 0.01 and 1% (w/v) of a biocide.
[0016] A preferred part of a plant is selected from seed, fruit and
leaf. A most preferred part of a plant comprises or is a leaf. A
further preferred part is a seed such as a vegetable seed, a bulb
or a corn, or a post-harvest fruit, such as a citrus, for example
an orange.
[0017] The invention further provides a drug delivery system
comprising a drug and a polyelectrolyte complex of polyanion
selected from the group consisting of a natural polyanion such as
xanthan gum, alginate, a lignin compound such as lignosulfonate,
pectin, carrageenan, humic acid, sodium alkyl naphtalene sulfonate,
fulvic acid, angico gum, gum Kondagogu, poly-.gamma.-glutamic acid,
maleic starch half-ester, carboxymethyl cellulose, chondroitin
sulphate, dextran sulphate, hyaluronic acid, and a synthetic
polyanion such as poly(acrylic acid), polyphosphoric acid, and
poly(L-lactide) and a polycation selected from the group consisting
of poly-L-lysine, epsilon-poly-L-lysine, poly-L-arginine, chitosan
oligosaccharide, and chitosan in a relative amount of between 1:2
and 300:1.
DETAILED DESCRIPTION
[0018] The term "'polyelectrolyte" refers to a molecule consisting
of a plurality of charged groups that are linked to a common
backbone. In the context of this application, the term "polycation"
is interchangeable with the term "positively charged
polyelectrolyte" and the term "polyanion" is interchangeable with
the term "negatively charged polyelectrolyte".
[0019] The term "polyelectrolyte complex" refers to a complex of
oppositely charged polyelectrolytes (a polyanion and a polycation)
which form strong, but reversible electrostatic links, thus
avoiding the use of covalent cross-linkers. The complex is not
soluble.
[0020] The term "lignin compound" refers to a chemical compound
that is derived from naturally occurring lignin or lignen by a
process that includes sulphonation. The resulting sulfonic acids
are strong acids and lignin compounds are therefore negatively
charged at pH values below 7.
[0021] As used herein, the term "chitosan" refers to a linear
polysaccharide composed of randomly distributed 6-(1-4)-linked
D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine
(acetylated unit). Chitosan is produced by deacetylation of chitin.
The term "chitosan" relates to chitosan, chitosan derivatives and
mixtures of chitosan and chitosan derivatives.
[0022] The term "part of a plant" indicates a part of a plant
including, but not limited to, pollen, ovule, leaf, root, flower,
fruit, stem, bulb, corn, branch and seed.
[0023] In a preferred embodiment, the invention provides a
composition comprising at least one biocide and a polyelectrolyte
complex of a lignin-compound and chitosan. Lignin-compounds such as
lignosulfonate form stable polyelectrolyte complexes with chitosan.
A composition according to the invention may comprise a mixture of
two or more lignin compounds and/or a mixture of two or more
chitosan polymers.
[0024] A preferred lignin compound is selected from Kraft lignin,
organosolv lignin and/or lignosulfonate.
[0025] A Kraft lignin is a polyphenolic product from the Kraft
pulping process for the conversion of wood into wood pulp. Included
are derivatives from Kraft lignin obtained by oxidation or other
chemical modification as is known to the skilled person.
[0026] An organosolv lignin is a polyphenolic product from
delignification processes using organic solvents. Included are
derivatives from organosolv lignin obtained by oxidation or other
chemical modification as is known to the skilled person.
[0027] Lignosulfonate (also termed lignosulphonate, lignosulfate,
lignin sulfonate, ligninsulfonate, ligninsulfonic acid,
lignosulfonic acid, lignosulfuric acid, or LST 7) is a
water-soluble anionic polymer which is, for example, formed as a
by-product in the sulphite pulping process. Lignosulfonates
generally have a wide molecular weight distribution, typically in
the range of about 500 to about 150,000. Lignosulfonates may
comprise different metal or ammonium ions as counter cations of the
sulfonate groups such as, for example, copper, zinc, calcium,
sodium, potassium, magnesium and aluminium. Suitable examples of
lignosulfonates comprise sodium lignosulfonate (e.g. sold as
BORRESPERSE NA.RTM., Borregaard LignoTech Ltd, Germany), calcium
lignosulfonate (e.g. sold as BORRESPERSE CA.RTM., Borregaard
LignoTech Ltd, Germany), ammonium lignosulfonate, potassium
lignosulfonate, modified lignosulfonate, derivatives of
lignosulfonate, or mixtures thereof. Modified lignosulfonates, and
derivatives of lignosulfonates are described in U.S. Pat. Nos.
3,639,263, 3,923,532, 4,006,779, 4,017,475, 4,019,995, 4,069,217,
4,088,640, 4,133,385, 4,181,652, 4,186,242, 4,196,777, 4,219,471,
4,236,579, 4,249,606, 4,250,088, 4,267,886, 4,269,270, 4,293,342
4,336,189, 4,344,487, 4,594,168, 4,666,522, 4,786,438, 5,032,164,
5,075,402, 5,286,412, 5,401,718, 5,446,133, 5,981,433, 6,420,602,
and 7,238,645, which are incorporated herein by reference.
[0028] A preferred lignin compound is lignosulfonate. A preferred
lignosulfonate is copper, zinc, calcium, sodium, potassium,
ammonium, magnesium and/or aluminium lignosulfonate, preferably
calcium, sodium, potassium or ammonium lignosulfonate, most
preferred calcium lignosulfonate.
[0029] The term chitosan relates to linear p-(1-4)-linked
glucosamin and N-acetylglucosamin. It may be produced from chitin
or its sodium salt (e.g. originating from shrimp) by treatment with
aqueous sodium hydroxide at elevated temperatures, or by enzymatic
treatment with, for example, a chitin deacetylase (EC 3.5.1.41).
Further sources of chitin are fungi, including Basidiomycetes,
Ascomycetes, and Phycomycetes, where it is a component of cell
walls and structural membranes of mycelia, stalks, and spores. A
most preferred chitosan is from fungi or derived from fungi.
[0030] Typically, deacetylation as determined by colloidal
titration is from 50 to 99.9%, preferably from 70 to 99.8% and most
preferably from 90 to 99.7%, as compared to chitin. Chitosan
derivatives can be prepared by reactions at the amino group (e.g.
by N-acylation, formation of N-alkylidene and N-arylidene
derivatives, N-alkylation and N-arylation) or at hydroxy groups, as
is known to the skilled person.
[0031] The polyelectrolyte complex comprises a polyanion, such as a
lignin-compound, xanthan gum and alginate, and a polycation, such
as chitosan, in a relative amount of between 1:2 and 60:1 (w/w),
more preferred between 1:1 and 50:1, more preferred between 2:1 and
30:1, such as about 10:1; about 15:1, about 20:1, about 25:1 and
about 30:1 (w/w). The relative amounts of polycation, preferably a
lignin compound, and a polyanion, preferably chitosan, in a
polyelectrolyte complex according to the invention is most
preferred about 15:1, even more preferred about 5:1 (w/w).
[0032] A preferred composition comprises a biocide comprising
natamycin and/or phosphite and a polyelectrolyte complex between a
polycation, preferably chitosan, and a polyanion, preferably
lignosulfonate. In a watery solution at a pH of about 4.5,
polycations such as chitosan polymers are positively charged and
the cationic amino groups on the glucosamine subunits can interact
electrostatically with anionic groups (usually carboxylic acid
groups) of polyanions such as lignosulfonate to form
polyelectrolyte complexes. Many different polyanions of both
natural origin, for example xanthan gum, alginate, pectin, a lignin
compound such as lignosulfonate, carrageenan, humic acid, fulvic
acid, angico gum, gum Kondagogu, sodium alkyl naphtalene sulfonate,
poly-.gamma.-glutamic acid, maleic starch half-ester, carboxymethyl
cellulose, chondroitin sulphate, dextran sulphate, and hyaluronic
acid, and synthetic origin, for example poly(acrylic acid),
polyphosphoric acid, and poly(L-lactide) can be used to form
polyelectrolyte complexes with a polycation, such as chitosan.
Preferably, said polyanion is selected from the group consisting of
xanthan gum, alginate and lignosulfonate. Most preferably, said
polyanion comprises or is lignosulfonate.
[0033] Preferably, said polycation is selected from the group
consisting of poly-L-lysine, epsilon-poly-L-lysine,
poly-L-arginine, chitosan oligosaccharide, and chitosan. Most
preferably, said polycation comprises or is chitosan. According to
the present invention, a polyelectrolyte complex that is formed
between a polyanion, preferably xanthan gum, alginate or
lignosulfonate, and a polycation, preferably chitosan, in a
relative amount of between 1:2 and 60:1 (w/w) improves the activity
of a biocide, resulting in a reduction of the amount of biocide
that is required to protect a plant or plant part against
pathogenic micro-organisms. A particularly preferred embodiment
provides a composition according to the invention comprising a
polyelectrolyte complex of lignosulfonate and chitosan, and
optionally at least one biocide.
[0034] The term "biocide" refers to a chemical substance capable of
killing living organisms. Biocides are commonly used in medicine,
agriculture, forestry, and in industry where they prevent the
fouling of, for example, water, agricultural products including
seed, and oil pipelines. A biocide can be a pesticide, including a
fungicide, herbicide, insecticide, algicide, molluscicide, miticide
and rodenticide; and/or an antimicrobial such as a germicide,
antibiotic, antibacterial, antiviral, antifungal, antiprotozoal
and/or antiparasite. A biocide of the invention comprises natamycin
(pimaricin
((1R,3S,5R,7R,8E,12R,14E,16E,18E,20E,22R,24S,25R,26S)-22-[(3-amino-3,6-di-
deoxy-D-mannopyranosyl)oxy]-1,3,26-trihydroxy-12-methyl-10-oxo-6,
11,
28-trioxatricyclo[22.3.1.05,7]octacosa-8,14,16,18,20-pentaene-25-carboxyl-
ic acid) and/or phosphite (a salt and/or ester of phosphorous acid,
and may further comprise one or more additional biocides as
indicated herein below.
[0035] A preferred additional biocide in a composition according to
the invention is a pesticidal agent including an antifungal
compound, a herbicide, an insecticide, an acaricide, and/or a
bactericide. A composition of the invention may also comprise two
or more biocides, such as two or more fungicides, two or more
herbicides, two or more insecticides, two or more acaricides, two
or more bactericides, or combinations of, for example, at least one
antifungal compound and at least one insecticide, at least one
antifungal compound and at least one herbicide, at least one
antifungal compound and at least one acaricide, at least one
antifungal compound and at least one bactericide, at least one
herbicide and at least one insecticide, at least one herbicide and
at least one acaricide, at least one herbicide and at least one
bactericide, at least one insecticide and at least one acaricide,
at least one insecticide and at least one bactericide, and at least
one acaricide and at least one bactericide. Some biocides have a
wide range of target organisms, as is known to the skilled person,
and are therefore include in more than one subgroup of biocides. A
biocide is preferably present in a concentration of between 0.1 and
90 w/v %, more preferred between 1 and 70 w/v %, more preferred
between 10 and 50 w/v %.
TABLE-US-00001 TABLE 1 Overview post-harvest fungicides Product
name Active ingredient Company Crop Penbotec pyrimethanil
PaceInternational Citrus, pome fruit Graduate fludioxonil
PaceInternational Citrus Fungaflor imazalil PaceInternational
Citrus KPhos unknown phosphite PaceInternational Citrus
Shield-Brite thiabendazole PaceInternational Citrus, pome fruit
SOPP Soap sodium ortho- PaceInternational Citrus phenylphenate
ecoFOG pyrimethanil (fogging PaceInternational Pome suitable)
ScholarTM fludioxonil PaceInternational Stone fruit, pome fruit,
kiwi and yam Philabuster 200 g/l imazalil; 200 g/l BASF Pear
pyrimethanil Pristine pyraclostrobin & boscalid BASF Grapes,
berries, stone fruit, pome fruit, tree nuts, carrots as well as
onions and other bulb vegetables Bavistine carbendazim BASF Fruit
Diabolo imazalil Certis Potato
[0036] A preferred additional antifungal compound or fungicide is a
post harvest fungicide selected from Table 1, 2-phenylphenol;
8-hydroxyquinoline sulphate; acibenzolar-5-methyl; actinovate;
aldimorph; amidoflumet; ampropylfos; ampropylfos-potassium;
andoprim; anilazine; azoxystrobin; benalaxyl; benodanil; benomyl
(methyl 1-(butylcarbamoyl)benzimidazol-2-ylcarbamate);
benthiavalicarb-isopropyl; benzamacril; benzamacril-isobutyl;
bilanafos; binapacryl; biphenyl; blasticidin-S; boscalid;
bupirimate; buthiobate; butylamine; calcium polysulphide;
capsimycin; captafol; captan
(N-(trichloromethylthio)cyclohex-4-ene-1,2-dicarboximide);
carbendazim; carboxin; carpropamid; carvone; chinomethionat;
chlobenthiazone; chlorfenazole; chloroneb; chlorothalonil;
chlozolinate;
cis-1-(4-chlorophenyl)-2-(1H-1,2,4-triazol-1-yl)-cycloheptanol;
clozylacon; a conazole fungicide such as, for example,
(RS)-1-(6-allyloxy-2,4-dichlorophenethyl)imidazole (imazalil;
Janssen Pharmaceutica NV, Belgium) and
N-propyl-N-[2-(2,4,6-trichlorophenoxyl)ethyl]imidazole-1-carboxamide
(prochloraz); cyazofamid; cyflufenamid; cymoxanil; cyprodinil;
cyprofuram; Dagger G; debacarb; dichlofluanid; dichlone;
dichlorophen; diclocymet; diclomezine; dicloran; diethofencarb;
diflumetorim; dimethirimol; dimethomorph; dimoxystrobin; dinocap;
diphenylamine; dipyrithione; ditalimfos; dithianon; dodine;
drazoxolon; edifenphos; ethaboxam; ethirimol; etridiazole;
famoxadone; fenamidone; fenapanil; fenfuram; fenhexamid; fenitrop
an; fenoxanil; fenpiclonil; fenpropidin; fenpropimorph; ferb am;
fluazinam
(3-chloro-N-(3-chloro-5-trifluoromethyl-2-pyridyl)-.alpha.,.alpha.,.alpha-
.-trifluoro-2,6-dinitro-p-toluidine); flubenzimine; fludioxonil;
flumetover; flumorph; fluoromide; fluoxastrobin; flurprimidol;
flusulfamide; flutolanil; folpet
(N-(trichloromethylthio)phthalimide); fosetyl-A1; fosetyl-sodium;
fuberidazole; furalaxyl; furametpyr; furcarbanil; furmecyclox;
guazatine; hexachlorobenzene; hymexazol; iminoctadine triacetate;
iminoctadine tris(albesilate); iodocarb; iprobenfos; iprodione;
iprovalicarb; irumamycin; isoprothiolane; isovaledione;
kasugamycin; kresoxim-methyl; mancozeb; maneb; meferimzone;
mepanipyrim; mepronil; metalaxyl; metalaxyl-M; methasulfocarb;
methfiroxam; methyl
1-(2,3-dihydro-2,2-dimethyl-1H-inden-1-yl)-1H-imidazole-5-carboxylate;
methyl
2-[[[cyclopropyl[(4-methoxyphenyl)imino]methyl]thio]-methyl]-.alph-
-a.-(methoxymethylene)benzeneacetate; methyl
2-[2-[3-(4-chlorophenyl)-1-methyl-allylideneaminooxymethyl]phenyl]-3-meth-
-oxyacrylate; metiram; metominostrobin; metrafenone; metsulfovax;
mildiomycin; monopotassium carbonate; myclozolin;
N-(3-ethyl-3,5,5-trimethylcyclohexyl)-3-formylamino-2-hydroxybenzamide;
N-(6-methoxy-3-pyridinyl)cyclopropanecarboxamide;
N-butyl-8-(1,1-dimethylethyl)-1-oxaspiro[4.5]decan-3-amine,
nitrothal-isopropyl; noviflumuron; ofurace; orysastrobin; oxadixyl;
oxolinic acid; oxycarboxin; oxyfenthiin; pencycuron; penthiopyrad;
phosdiphen; phthalide; picobenzamid; picoxystrobin; piperalin;
polyoxins; polyoxorim; procymidone; prop amocarb; prop
anosine-sodium; propineb; proquinazid; pyraclostrobin; pyrazophos;
pyrimethanil; pyroquilon; pyroxyfur; pyrrolnitrine, quinconazole;
quinoxyfen; quintozene; silthiofam; sodium tetrathiocarbonate;
spiroxamine; sulphur; tecloftalam; tecnazene; tetcyclacis; thiazole
fungicide such as, for example, 2-(thiazol-4-yl)benzimidazole
(thiabendazole; e.g. the commercial product TECTO.RTM. Flowable SC
of Syngenta, USA), thicyofen; thifluzamide; thiophanate-methyl;
thiram; tiadinil; tioxymid; tolclofos-methyl; tolylfluanid;
triazbutil; triazoxide; tricyclamide; tricyclazole; tridemorph;
trifloxystrobin; validamycin A; vinclozolin; zineb; ziram;
zoxamide;
(2S)--N-[2-[4-[[3-(4-chlorophenyl)-2-propynyl]oxy]-3-methoxyphe-
nyl]ethyl]-3-met-hyl-2-[(methylsulphonyl)amino]butanamide;
1-(1-naphthalenyl)-1H-pyrrole-2,5-dione;
2,3,5,6-tetrachloro-4-(methylsulphonyl)pyridine;
2,4-dihydro-5-methoxy-2-methyl-4-[[[[1-[3-(trifluoromethyl)phenyl]-ethyli-
-dene]amino]oxy]methyl]phenyl]-3H-1,2,3-triazol-3-one;
2-amino-4-methyl-N-phenyl-5-thiazolecarboxamide;
2-chloro-N-(2,3-dihydro-1,1,3-trimethyl-1H-inden-4-yl)-3-pyridinecarboxam-
-ide; 3,4,5-trichloro-2,6-pyridinedicarbonitrile;
3-[(3-bromo-6-fluoro-2-methyl-1H-indol-1-yl)sulphonyl]-N,N-dimethyl-1H-1,-
-2,4-triazole-1-sulphonamide; and copper salts such as Bordeaux
mixture (CuSO4.3Cu(OH)2.3CaSO4); copper hydroxide; copper
naphthenate; copper oxychloride ((CuCl2.3Cu(OH)2), tribasic copper
sulphate (CuSO4.3Cu(OH)2); cufraneb; cuprous oxide; mancopper;
oxine-copper
[0037] A most preferred fungicide is natamycin. A composition of
the invention may also comprise two or more fungicides, such as,
for example, natamycin and copper hydroxide, natamycin and a
strobilurin type of fungicides like azoxystrobin, natamycin and
phosphite, natamycin and a triazole type of fungicides like
cyproconazole, natamycin and aa succinate dehydrogenase inhibitor
type of fungicides like boscalid, natamycin and
pthalimide/pthalonitrile type of fungicides like chlorothalonil,
folpet and captan, natamycin and benzimidazole type of fungicides
like thiabendazole, natamycin and carbamate type of fungicides like
propamocarb, natamycin and carboxamide type of fungicides like
fenoxanil, natamycin and dicarboxamide type of fungicides like
iprodione, natamycin and dithiocarbamate type of fungicides like
Mancozeb, natamycin and inorganic type of fungicides like
copperhydroxide, natamycin and morpholine type of fungicides like
dimethamorph, natamycin and organophosphate type of fungicides like
fosetyl, natamycin and azole type of fungicides like
prothioconazole, natamycin and phenylamide type of fungicides like
metalaxyl, natamycin and fungicides not belonging to a specific
group of fungicides like fludioxynil.
[0038] A preferred phosphite is a phosphite salt such as
KH.sub.2PO.sub.3, K.sub.2HPO.sub.3, NaH.sub.2PO.sub.3,
Na.sub.2HPO.sub.3, (NH.sub.2).sub.2HPO.sub.3,
(NH.sub.2)H.sub.2PO.sub.3, ethyl hydrogen phosphonate, phosphorous
acid, and mixtures of these compounds. A mixture of
KH.sub.2PO.sub.3 and K.sub.2HPO.sub.3 is suitably obtained by
adding KOH or K.sub.2CO.sub.3 to a KH.sub.2PO.sub.3 composition at
a final pH of 5-9.
[0039] A further preferred additional biocide is an insecticide
and/or acaricide. Preferred insecticides include imidacloprid
(commercial product: ADMIRE.RTM., Bayer) Bacillus thuringiensis
(commercial product: TUREX.RTM., Certis USA), teflubenzuron
(commercial product: NOMOLT.RTM., BASF), pymetrozine (commercial
product: PLENUM.RTM., Syngenta) and acetamiprid (Commercial
product: GAZELLE.RTM., Certis Europe), ACTELLIC.RTM. Syngenta,
Switserland), Pyrethroids (commercial product BAYGON.RTM. (Bayer),
bifenazate (e.g. Uniroyal), dichlorvos (e.g. Amvac Chemical
Corporation), imidacloprid (e.g. Bayer), fenamiphos (e.g. Mobay
Chemical Corporation), rosemary oil, oxamyl (e.g. Dupont) and
sulfur-based insecticides. A most preferred insecticide is
pirimiphos-methyl (commercial product ACTELLIC.RTM., Syngenta,
Switserland). A composition of the invention may also comprise two
or more insecticides.
[0040] Preferred acaricides include chlofentezine (commercial
product: APOLLO.RTM., Makhteshim), acequinocyl (commercial product:
KAMEMYTE.RTM., Arysta), spirodiclofen (commercial product:
ENVIDOR.RTM., Bayer CropScience), bifenazate (commercial product:
FLORAMITE.RTM., Certis Europe) and fenbutatinoxide (commercial
product: TORQUE L.RTM., BASF). A most preferred acaricide is
spirodiclofen. A composition of the invention may also comprise two
or more acaricides.
[0041] A further preferred insecticide/acaricide is a carbamate,
for example alanycarb, aldicarb, aldoxycarb, allyxycarb, aminocarb,
bendiocarb, benfuracarb, bufencarb, butacarb, butocarboxim,
butoxycarboxim, carbaryl, carbofuran, carbosulfan, cloethocarb,
dimetilan, ethiofencarb, fenobucarb, fenothiocarb, formetanate,
furathiocarb, isoprocarb, metam-sodium, methiocarb, methomyl,
metolcarb, oxamyl, pirimicarb, promecarb, propoxur, thiodicarb,
thiofanox, trimethacarb, XMC, xylylcarb, triazamate; an
organophosphate, for example acephate, azamethiphos, azinphos
(-methyl, -ethyl), bromophos-ethyl, bromfenvinfos (-methyl),
butathiofos, cadusafos, carbophenothion, chlorethoxyfos,
chlorfenvinphos, chlormephos, chlorpyrifos (-methyl/-ethyl),
coumaphos, cyanofenphos, cyanophos, chlorfenvinphos,
demeton-5-methyl, demeton-5-methylsulphon, dialifos, diazinon,
dichlofenthion, dicrotophos, dimethoate, dimethylvinphos,
dioxabenzofos, disulfoton, EPN, ethion, ethoprophos, etrimfos,
famphur, fenamiphos, fenitrothion, fensulfothion, fenthion,
flupyrazofos, fonofos, formothion, fosmethilan, fosthiazate,
heptenophos, iodofenphos, iprobenfos, isazofos, isofenphos,
isopropyl O-salicylate, isoxathion, malathion, mecarb am,
methacrifos, methamidophos, methidathion, mevinphos, monocrotophos,
naled, omethoate, oxydemeton-methyl, parathion (-methyl/-ethyl),
phenthoate, phorate, phosalone, phosmet, phosphamidon, phosphocarb,
phoxim, pirimiphos (-methyl/-ethyl), profenofos, propaphos,
propetamphos, prothiofos, prothoate, pyraclofos, pyridaphenthion,
pyridathion, quinalphos, sebufos, sulfotep, sulprofos,
tebupirimfos, temephos, terbufos, tetrachlorvinphos, thiometon,
triazophos, triclorfon, vamidothion; a sodium channel
modulator/voltage-dependent sodium channel blocker, such as a
pyrethroid, for example acrinathrin, allethrin (d-cis-trans,
d-trans), beta-cyfluthrin, bifenthrin, bioallethrin, bioallethrin-S
cyclopentyl isomer, bioethanomethrin, biopermethrin, bioresmethrin,
chlovaporthrin, cis-cypermethrin, cis-resmethrin, cis-permethrin,
clocythrin, cycloprothrin, cyfluthrin, cyhalothrin, cypermethrin
(alpha-, beta-, theta-, zeta-), cyphenothrin, deltamethrin,
empenthrin (IR isomer), esfenvalerate, etofenprox, fenfluthrin,
fenpropathrin, fenpyrithrin, fenvalerate, flubrocythrinate,
flucythrinate, flufenprox, flumethrin, fluvalinate, flibfenprox,
gamma-cyhalothrin, imiprothrin, kadethrin, lambda-cyhalothrin,
metofluthrin, permethrin (cis-, trans-), phenothrin (IR
trans-isomer), prallethrin, profluthrin, protrifenbute,
pyresmethrin, resmethrin, RU 15525, silafluofen, tau-fluvalinate,
tefluthrin, terallethrin, tetramethrin (IR isomer), tralomethrin,
transfluthrin, ZXI 8901; an oxadiazine, for example indoxacarb; an
acetylcholine receptor agonists/antagonists; a chloronicotinyl, for
example clothianidin, dinotefuran, imidacloprid, nitenpyram,
nithiazine, thiacloprid, thiamethoxam; a nicotine such as
bensultap, cartap; an organochlorine, for example camphechlor,
chlordane, endosulfan, gamma-HCH, HCH, heptachlor, lindane,
methoxychlor, a fiproles, for example acetoprole, ethiprole,
fipronil, pyrafluprole, pyriprole, vaniliprole; a mectin, for
example avermectin, emamectin, emamectin-benzoate, ivermectin,
milbemycin; a juvenile hormone mimetics, for example diofenolan,
epofenonane, fenoxycarb, hydroprene, kinoprene, methoprene,
pyriproxifen, triprene; an ecdyson agonists/disruptors such as a
diacylhydrazines, for example chromafenozide, halofenozide,
methoxyfenozide, tebufenozide; benzoylureas, for example
bistrifluoron, chlofluazuron, diflubenzuron, fluazuron,
flucycloxuron, flufenoxuron, hexaflumuron, lufenuron, novaluron,
noviflumuron, penfluoron, teflubenzuron, triflumuron an organotin,
for example azocyclotin, cyhexatin, fenbutatin-oxide; a pyrrole,
for example chlorfenapyr; a dinitrophenol, for example binapacyrl,
dinobuton, dinocap, a tetronic acid, for example spirodiclofen,
spiromesifen; a tetramic acid, for example spirotetramat and
3-(2,5-dimethylphenyl)-8-methoxy-2-oxo-1-azaspiro[4.5]dec-3-en-4-yl
ethyl carbonate; a carboxamide, for example flonicamid; a benzoic
acid dicarboxamide, for example flubendiamide; azadirachtin, a
fumigant, for example aluminium phosphide, methyl bromide,
sulphuryl fluoride; an antifeedant, for example cryolite,
flonicamid, pymetrozine; a mite growth inhibitor, for example
clofentezine, etoxazole, hexythiazox; amidoflumet, benclothiaz,
benzoximate, bifenazate, bromopropylate, buprofezin,
quinomethionate, chlordimeform, chlorobenzilate, chloropicrin,
clothiazoben, cycloprene, cyflumetofen, dicyclanil, fenoxacrim,
fentrifanil, flubenzimine, flufenerim, flutenzin, gossyplure,
hydramethylnone, japonilure, metoxadiazone, petroleum, piperonyl
butoxide, potassium oleate, pyridalyl, sulfluramid, tetradifon,
tetrasul, triarathene, and/or verbutin.
[0042] A further preferred additional biocide is a bactericide, for
example bronopol, dichlorophen, nitrapyrin, nickel
dimethyldithiocarbamate, kasugamycin, octhilinon, furancarboxylic
acid, oxytetracyclin, probenazole, streptomycin, tecloftalam, and
copper salts. Preferred bactericides include compounds such as
copper salts (e.g. copper hydroxide, copper oxychloride, copper
sulfate and Bordeaux mixture), sophorolipid which is a glycolipid
that is produced by yeasts such as Candida bombicola, Candida
apicola, and Wickerhamiella domercqiae and is composed of a dimeric
sugar linked with a glycosidic bond to a hydroxyl fatty acid,
streptomycin, the commercial product CITRICIDAL.RTM. (Bio/Chem
Research) and validamycin. A most preferred bactericide is copper
hydroxide. A composition of the invention may also comprise two or
more bactericides.
[0043] Some of the indicated compounds have more than one activity.
For example, copper salts (e.g. copper hydroxide) have bactericide
and fungicide activities. The activities of the individual
compounds are known to the skilled person. In addition, handbooks
and websites (e.g. www.frac.info/frac) are available to determine
the activity or activities of a compound.
[0044] A most preferred biocide in a composition according to the
invention is a fungicide, preferably natamycin and/or phosphite
and/or an insecticide, preferably rosemary oil. A further preferred
biocide is a mixture of two or more active ingredients. A preferred
mixture comprises natamycin and rosemary oil or natamycin and a
sophorolipid. Said mixture preferably comprises natamycin and
rosemary oil or a sophorolipid in ratio of between 30:1 and 1:30
(w/w), preferably in a ratio of about 1:1 (w/w).
[0045] A preferred additional biocide according to the invention is
a natural biocide. The term natural biocide comprises
micro-organisms and virusses, feromones, extracts from plants
and/or animals, and other substances such as, for example,
minerals. Preferred plant extracts are or comprise Sage extract
(=extract of Salvia officinalis), extract of Reynoutria
sachalinensis (Giant Knotweed), extract of Verticillium albo-atrum,
extract of Bacillus thuringiensis subsp. Kurstaki, extract of
Lecanicillium muscarium, laminarin, lactoperoxidase, azadirachtin,
harpin, chitosan, nisin, pythium extract (and other fungal
extracts). Preferred other substances include salicylic acid,
castor oil, carvon, cedar oil, cinnamon and cinnamon oil, citric
acid, citronella and citronella oil, cloves and clove oil, corn
gluten meal, corn oil, cottonseed oil, eugenol, garlic and garlic
oil, geraniol, geranium oil, lauryl sulphate, lemongrass oil,
linseed oil, malic acid, mint and mint oil, peppermint and
peppermint oil, 2-phenethyl propionate (2-phenylethyl propionate),
sorbate such as potassium sorbate, putrescent whole egg solids,
rosemary and rosemary oil, sesame and sesame oil, sodium chloride,
sodium lauryl sulphate, soybean oil, thyme and thyme oil, white
pepper, a sulfur-based compound, a copper-based compound,
carbamates such as sodiumdimethyldithiocarbamaat, quaternairy
ammonium compounds, benzyl-C12-16-alkyldimethyl, chlorides, sodium
dichloroisocyanurate, calcium hypochlorite, ethanol, 2-propanol,
didecyldimethylammonium chloride, ethylene oxide, sodium
dichloroisocyanurate, trichloroisocyanic acid, peracetic acid,
hydrogen peroxide, sodium-p-tolueensulfonchloramide,
glutaraldehyde, N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine,
lactic acid, sodium hypochlorite, decanoic acid, octanoic acid,
bronopol, imizalil, 1,2-benzisothiazole-3(2H)-on, dichlofluanide,
propiconazole, permethrin, boric acid, flufenoxuron,
1,3-dichloro-5,5-dimethylhydantoine,
1,3-dichloro-5-ethyl-5-methylimidazolidine,
2,2-dibroom-2-cyanacetamide, bromo
chloro-5,5-dimethylimidazolidine, difenacoum, difethialon,
bromadiolon, flocoumafen, dichloorvos, diflubenzuron, cyromazin,
bifenthrin, cyromazin, imidacloprid, deltamethrin, permethrin,
tetramethrin, d-fenothrin, cyfluthrin, piperonylbutoxide,
pyrethrine, alfa-cypermethrin, magnesiumfosfide, piperonylbutoxide,
N,N-diethyl-m-toluamide, p-menthane-3,8-diol, cybutryne, cymoxanil,
mancozeb, captan, linuron, prochloraz, glyfosate, nicosulfuron,
folpet, chloridazon, milbemectin, metamitron, metsulfuron-methyl,
gibberellic acid, azoxystrobin, spirotetramat, abamectin, indolyl
butyric acid, teflubenzuron and/or rosemary oil, and/or mixtures
thereof.
[0046] A composition according to the invention preferably further
comprises at least one additional compound selected from the group
consisting of a sticking agent, a preservative, a stabilizer, a
wax, an antioxidant, an anti-foam-forming agent, a thickening
agent, a spray oil, an UV-protectant, an anti-freezing agent, a
dispersing agent, and a flow additive.
[0047] Said sticking agent is preferably selected from latex based
products like PROLONG.RTM. (Holland Fyto B.V., The Netherlands) and
BOND.RTM. (Loveland Industries Ltd), pinolene/terpene based
products like NU-FILM.RTM. (Hygrotech Saad) and SPRAY-FAST.RTM.
(Mandops), long chain polysaccharides like gellan gum, guar gum,
succinoglycan gum (RHEOZAN.RTM.; Rhodia) and xanthan gum, or a
hydrated magnesium-aluminum silicate, for example attapulgite,
(Attagel.RTM.; BASF). Alternatively, the sticking agent may be a
polymer or co-polymer from a type of polymer such as polyacrylate
and polyethylene e.g. NEOCRYL.RTM. (DSM, The Netherlands). A
composition of the invention may also comprise two or more
different sticking agents. A sticking agent is preferably present
in an amount of between 0 to up to 20% (w/v), more preferred
between 0.1 to up to 10% (w/v), more preferred between 1 to up to
5% (w/v), more preferred about 3% (w/v).
[0048] A preservative is preferably selected from weak acid
preservatives such as sorbic acid, lactic acid, benzoic acid,
propionic acid, citric acid, acetic acid, or an alkali metal or
alkali earth metal salt thereof; inorganic acids such as
hydrochloric acid; imidazoles such as imazalil or any antifungal
compound known in the art as a preservative for food products, crop
protection or after harvest treatment of fruits, vegetables or
cereals; ethyl parabenzoate; borax; calcium bisulfite; calcium
disodium EDTA; dehydroacetic acid; isothiazoles (e.g. KATHON.RTM.
(Rohm and Haas); a quaternary ammonium salt such as, for example,
1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride (CTAC),
and antimicrobials capable of preventing bacterial growth in the
composition. A preferred preservative is a quaternary ammonium
salt, preferably CTAC stabilized with sodium bicarbonate
(Dowicil.RTM.75). A further preferred preservative is Kathon.TM.,
which is preferably present in a concentration of about 0.04
gram/liter. A composition of the invention may also comprise two or
more different biocides. A composition of the invention may also
comprise two or more different preservatives. A preservative is
preferably present in an amount between 0 to up to 20% (w/v), more
preferred between 0.01 to up to 10% (w/v), more preferred between
0.1 to up to 5% (w/v), more preferred about 0.5% (w/v).
[0049] A stabilizer, when present, is preferably selected from
xanthan gum, agar, succinoglycan gum (Rheozan), alginic acid,
alginate, a hydrated magnesium-aluminum silicate, for example
attapulgite, (Attagel.RTM.; BASF), calcium lactobionate,
carrageenan, OptiXan-D.RTM.; gellan gum, and guar gum. A
composition of the invention may also comprise two or more
different stabilizing agents. A stabilizer is preferably present in
an amount of between 0 to up to 10% (w/v), more preferred between
0.01 to up to 5% (w/v), more preferred between 0.05 to up to 0.5%
(w/v), more preferred about 0.05% (w/v).
[0050] A wax, when present, is preferably a natural or synthetic
wax selected from bee wax, carnauba wax, andelilla wax, ouricouri
wax, sugarcane wax, retamo wax, Chinese wax, jojoba oil, paraffin
wax, esparto wax, Montan wax, candelilla wax, whale spermaceti,
lanolin, and ethylene glycol diesters or triesters of long-chain
fatty acids (C18-C36). A wax is preferably present in an amount of
between 0 to up to 10% (w/v), more preferred between 0.01 to up to
5% (w/v), more preferred between 0.05 to up to 0.5% (w/v), more
preferred about 0.1% (w/v).
[0051] An antioxidant, when present, is preferably selected from
amino acids (e.g. glycine, histidine, tyrosine, tryptophan) and
their derivatives, imidazole (e.g. urocanic acid) and derivatives,
vitamin C and derivatives (such as ascorbylpalmitate and
ascorbyltetraisopalmitate, Mg-ascorbylphosphate,
Na-ascorbylphosphate, ascorbyl-acetate), tocopherol and derivates
(such as vitamin-E-acetate), mixtures of vitamin E, vitamin A and
derivatives (vitamin-A-palmitate and -acetate) as well as coniferyl
benzoate, rutinic acid and derivatives, .alpha.-glycosylrutin,
ferulic acid, furfurylideneglucitol, carnosine,
butylhydroxytoluene, butylhydroxyanisole, and
trihydroxybutyrophenone. A composition of the invention may also
comprise two or more different antioxidants. An anti-oxidant is
preferably present in an amount between 0 to of up to 20% (w/v),
more preferred between 0.1 to up to 10% (w/v), more preferred
between 1 to up to 5% (w/v), more preferred about 3% (w/v).
[0052] An anti-foam forming agent, when present, is preferably
selected from polymethylsiloxane, simethicone octanol, and silicone
oils. The composition of the invention may also comprise two or
more different anti-foam forming agents. An anti-foam agent is
preferably present in an amount of between 0 to up to 10% (w/v),
more preferred between 0.05 to up to 5% (w/v), more preferred
between 0.1 to up to 1% (w/v), more preferred about 0.05%
(w/v).
[0053] A thickening agent, when present, is preferably selected
from agar, alginic acid, alginate, carrageenan, gellan gum, xanthan
gum, succinoglycan gum, guar gum, acetylated distarch adipate,
acetylated oxidised starch, arabinogalactan, ethyl cellulose,
methyl cellulose, locust bean gum, starch sodium octenylsuccinate,
and triethyl citrate. A composition of the invention may also
comprise two or more different thickening agents. A thickening
agent is preferably present in an amount of between 0 to up to 10%
(w/v), more preferred between 0.01 to up to 5% (w/v), more
preferred between 0.02 to up to 1% (w/v), more preferred about
0.05% (w/v).
[0054] An UV-protector or UV absorbent, when present, is preferably
selected from sulphonated tannins, titaniumdioxide,
lignosulfonates, and related compounds. An UV-protector is
preferably present in amount of between 0.1 and 10% (w/v).
[0055] A composition according to the invention optionally further
comprises an additional compound selected from a spray oil, for
example, a mineral oil such as BANOLE.RTM.. In addition,
adispersing or wetting agent known to a skilled person such as, for
example, Morwet.RTM. D425, lignin sulphonate, an
alkylpolysaccharide, an styrene acrylic polymer, an acrylic
co-polymer, and ethoxylated tristyrenephenol phosphate, for example
polyethoxylated fosforic acid, and/or a wetting agent such as
di-octylsuccinate, polyoxyethylene/polypropylene and tri-stearyl
sulphonate/phosphate, is preferably present.
[0056] A further preferred composition according to the invention
comprises natamycin and a wax, a sophorolipid and a wax, or a
combination of natamycin, a sophorolipid and a wax. Said mixture
preferably comprises natamycin and a sophorolipid in ratio of
between 30:1 and 1:30 (w/w), preferably in a ratio of about 1:1
(w/w).
[0057] The invention further provides a method for producing a
composition according to the invention, comprising (a) providing an
aqueous solution of a polyanion selected from the group consisting
of a natural polyanion such as xanthan gum, alginate, a lignin
compound such as lignosulfonate, pectin, carrageenan, humic acid,
fulvic acid, angico gum, gum Kondagogu, poly-.gamma.-glutamic acid,
maleic starch half-ester, carboxymethyl cellulose, chondroitin
sulphate, dextran sulphate, hyaluronic acid, and a synthetic
polycation such as poly(acrylic acid), polyphosphoric acid, and
poly(L-lactide), wherein the concentration of said polyanion is
from about 0.1-70 w/v %, from about 1-70 w/v %, (b) providing an
aqueous acidic solution of a polycation selected from the group
consisting of poly-L-lysine, epsilon-poly-L-lysine, poly-L-arginine
and chitosan, wherein the concentration of said polycation is from
about 0.1-10 w/v %, from about 1-10 w/v % and the pH is below
pH=5.5, (c) adding the solution of a polycation to the solution of
a polyanion, or vice versa, whereby a precipitate is formed that is
crushed, for example by milling, preferably to an average particle
size of between 0.2 and 5 micrometer, where after (d) a biocide
comprising natamycin and/or phosphite is added. As an alternative,
the biocide is added to the solution of at least one of steps
a-c.
[0058] If required, an acid is added to the polycation-polyanion
mixture to keep the pH of the mixture below pH=7. A preferred
acidic polycation, preferably chitosan, solution comprises lactate,
phosphorous acid, hydrochloric acid, fumaric acid, and/or ascorbic
acid.
[0059] Said polyanion is preferably selected from the group
consisting of xanthan gum, alginate and a lignin compound such as
lignosulfonate. In a preferred embodiment, said polyanion comprises
or is a lignin compound, most preferably lignosulfonate. Said
polycation is preferably selected from the group consisting of
poly-L-lysine, epsilon-poly-L-lysine, poly-L-arginine and chitosan.
In a preferred embodiment, said polycation comprises or is
chitosan.
[0060] As an alternative, a method for producing a composition
according to the invention comprises (a) providing an aqueous
solution of a polyanion, preferably a lignin compound, xanthan gum
or alginate, wherein the concentration of said polyanion is from
about 0.1-70 w/v %, from about 1-70 w/v %, (b) providing a
polycation solution, preferably a chitosan solution, wherein the
concentration of polycation is from about 0.1-10 w/v %, from about
1-10 w/v %, (c) adding the polycation solution to the solution of a
polyanion or vice versa, while the pH of the mixture is kept below
pH=5.5, and (d) adding a biocide comprising natamycin and/pr
phosphite to at least one of steps a-c.
[0061] An aqueous solution of a polyanion, such as a lignin
compound, is preferably prepared by dissolving the polyanion, such
as a lignin compound, preferably lignosulfonate, in an aqueous
solution, preferably water.
[0062] A solution of a polycation, preferably chitosan, is
preferably prepared by solubilising the polycation, preferably
chitosan, in an aqueous acidic solution comprising, for example,
lactate, hydrochloric acid, phosphorous acid and/or ascorbic acid.
The amount of acid that is required to solubilise the polycation,
preferably chitosan, depends on the polycation, as is known to the
skilled person. For instance for solubilising chitosan, in general,
about 6 ml 37% hcl is required to obtain a 10 gram in 1 liter
chitosan solution in water. As an alternative, a polycation,
preferably chitosan, is dissolved in an aqueous solution,
preferably water, for example by gently shaking at 20-23.degree. C.
overnight. A salt, preferably NaCl, is preferably added at a
concentration between 1 mM and 1 M, preferably about 100 mM.
[0063] A polyanion solution, preferably a lignosulfonate, xanthan
gum, humic acid, or alginate solution, is preferably added drop
wise to the solution comprising a polycation, preferably a chitosan
compound. If required, the pH is kept below 5.5 by the addition of
an acid, preferably hydrochloric acid, lactic acid, ascorbic acid,
phosphorous acid, nonanoic acid or acetic acid. The pH is more
preferably kept below 5.0, more preferably below 4.5 during the
formation of a polyelectrolyte complex. The temperature is
preferably between 0.degree. C. and 100.degree. C., more preferred
between 10.degree. C. and 60.degree. C., more preferred ambient
temperature (15-25.degree. C.). The resulting mixture is preferably
stirred during formation of the polyelectrolyte complex and the
polyelectrolyte complex is preferably allowed to settle
overnight.
[0064] It is preferred that a polyanion, preferably a
lignosulfonate, xanthan gum, humic acid, or alginate, is added to a
solution comprising a polycation, preferably a chitosan compound.
If required, the pH is kept below 5.5 by the addition of an acid,
preferably hydrochloric acid, lactic acid, ascorbic acid,
phosphorous acid, nonanoic acid or acetic acid. The pH is more
preferably kept below 5.0, more preferably below 4.5 during the
formation of a polyelectrolyte complex. The temperature is
preferably between 0.degree. C. and 100.degree. C., more preferred
between 10.degree. C. and 60.degree. C., more preferred ambient
temperature (15-25.degree. C.). The resulting mixture is preferably
stirred during formation of the polyelectrolyte complex and the
polyelectrolyte complex is preferably allowed to settle
overnight.
[0065] Following settlement of the ply-electrolyte complex, a
dispergent and/or a wetting agent is preferably added and the
precipitate is crushed, preferably by milling for example in a bead
mill. The resultant concentrate may further comprise a sticking
agent, a preservative, a stabilizer, a wax, an antioxidant, an
anti-foam-forming agent, a thickening agent, a spray oil, an
UV-protectant, an anti-freezing agent such as, for example
monopropylene glycol, and a flow additive.
[0066] The invention further provides a use of a composition
according to the invention for the protection of a plant or a part
of a plant against a pathogen. Said composition preferably
comprises at least one biocide. Said plant preferably is a
vegetable, fruit or crop plant.
[0067] A composition according to the invention is a solid
composition, for example a granule, or a liquid, preferably
aqueous, composition, more preferably a suspension. SPrior to use,
a composition according to the invention comprising at least one
biocide comprising natamycin and/or phosphite is preferably
dissolved or dispersed in water or diluted with water to contain
between 0,001 and 1 w/v % of a biocide. If required, a sticking
agent is added to the diluted aqueous suspension. Said aqueous
composition is used, for example, to control brown rot of peaches,
powdery mildew of apples, gooseberries, hops, ornamentals, grapes,
peaches, strawberries, and sugar beets, apple scab, gall mite on
blackcurrant, peanut leafspot, mildew on roses, and mites on beans,
carrots, lucerne, melons, and tomatoes. For this, the aqueous
composition is preferably sprayed over a plant, or part thereof,
for use as a biocide, preferably as a fungicide.
[0068] A composition according to the invention comprising a
polyelectrolyte complex and at least one biocide comprising
natamycin and/or phosphite is for instance in the form of a
suspension concentrate (SC), water dispersible granules (WG), a
wettable powder (WP), a suspo emulsion (SE), an emusifiable
concentrate (EC) a dispersion concentrate (DC), a dry powder seed
treatment (DS), a water slurriable powder (WS), a flowable seed
treatment (FS) or a water dispersible granule seed treatment (WG).
Preferably, a composition of the invention is in the form of a
suspension concentrate, or in the form of water dispersible
granules. A "suspension concentrate" as used herein refers to a
suspension of solid particles in a liquid intended for dilution
with water prior to use. A "dispersion concentrate" as used herein
refers to a dispersion of solid particles in a liquid intended for
dilution with water prior to use. "Water dispersible granules" as
used herein refer to a formulation in granule form which is
dispersible in water forming a dispersion such as a suspension or
solution. A "wettable powder" as used herein refers to a powder
formulation intended to be mixed with water or another liquid prior
to use. A "water slurriable powder" as used herein refers to a
powder formulation that is made into a slurry in water prior to
use.
[0069] Alternatively, a plant of part thereof is coated with an
aqueous composition comprising at least one biocide according to
the invention by submerging the plant or part thereof in the
aqueous composition to protect the plant of part thereof against a
pathogen. A preferred part of a plant that is coated with a
composition according to the invention is a bulb, a corn, or a
seed. A further preferred part of a plant that is coated with a
composition according to the invention is a fruit such as, for
example, a citrus fruit such as orange, mandarin and lime, a pome
fruit such as apple and pear, a stone fruit such as almond,
apricot, cherry, damson, nectarine, tomato and watermelon; a
tropical fruit such as mango, lychee and tangerine. A preferred
fruit is a citrus fruit, such as orange.
[0070] A preferred part that is coated with a composition according
to the invention is a post-harvest fruit, such as a citrus fruit
such as orange, mandarin and lime, a pome fruit such as apple and
pear, a stone fruit such as almond, apricot, cherry, damson,
nectarine, tomato and watermelon; a tropical fruit such as mango,
lychee and tangerine. A preferred post-harvest fruit is a citrus
fruit, such as orange.
[0071] Said pathogen preferably is a fungus or an oomycete, esp.
Phytophthora sp. or Plasmopara vitieola. A preferred fungus is
botrytis and/or Penicillium sp.
[0072] The invention further provides a method of protecting a
plant against a pathogen, comprising applying to said plant, or on
one or more plant parts, a composition according to the invention.
Said composition is preferably dissolved or dispersed in water or
diluted with water to contain between 0.001 and 1 w/v % of a
biocide or a combination of biocides. A preferred composition for
applying to a plant or a part of a plant comprises natamycin and a
wax, a sophorolipid and a wax, or a combination of natamycin,
sophorolipid and a wax.
[0073] The invention further provides a method of preventing,
reducing and/or eliminating the presence of a pathogen on a plant
or on one or more plant parts, comprising applying to said plant or
plant part a composition according to the invention. A preferred
plant part according to the invention is selected from seed, leaf
and fruit such as, for example, a citrus fruit such as orange,
mandarin and lime, a pome fruit such as apple and pear, a stone
fruit such as almond, apricot, cherry, damson, nectarine, tomato
and watermelon; a tropical fruit such as mango, lychee and
tangerine. A preferred fruit is a citrus fruit, such as orange. A
most preferred part is a post-harvest fruit.
[0074] The invention further provides a drug delivery system
comprising a drug and a polyelectrolyte complex of a polyanion
selected from the group consisting of a natural polyanion such as a
lignin compound such as xanthan gum, alginate, lignosulfonate,
pectin, carrageenan, humic acid, fulvic acid, angico gum, gum
Kondagogu, sodium alkyl naphtalene sulfonate, poly-.gamma.-glutamic
acid, maleic starch half-ester, carboxymethyl cellulose,
chondroitin sulphate, dextran sulphate, hyaluronic acid, and a
synthetic polyanion such as poly(acrylic acid), polyphosphoric
acid, and poly(L-lactide) and a polycation selected from the group
consisting of poly-L-lysine, epsilon-poly-L-lysine,
poly-L-arginine, chitosan oligosaccharide, and chitosan, in a
relative amount of between 1:2 and 300:1. A chitosan based
polyelectrolyte complexes exhibit favourable physicochemical
properties with preservation of chitosan's biocompatible
characteristics. These complexes are therefore good candidate
excipient materials for the design of different types of dosage
forms. For example, the chitosan-based complexes can be used as
excipients in drug delivery systems such as nano- and
microparticles, beads, fibers, sponges and matrix type tablets.
Said drug delivery system can be used as a non immediate release
dosage form selected from delayed release, sustained release,
controlled release, prolonged release, and site specific
release.
[0075] Said drug delivery system is preferably prepared by a method
comprising (a) providing an polyanion or an aqueous solution of a
polyanion selected from the group consisting of a natural polyanion
such as xanthan gum, alginate, a lignin compound such as
lignosulfonate, pectin, carrageenan, humic acid, fulvic acid,
angico gum, gum Kondagogu, sodium alkyl naphtalene sulfonate,
poly-.gamma.-glutamic acid, maleic starch half-ester, carboxymethyl
cellulose, chondroitin sulphate, dextran sulphate, hyaluronic acid,
poly(acrylic acid), polyphosphoric acid, and poly(L-lactide),
wherein the concentration of said polyanion is from about 0.1-60
w/v %, from about 1-60 w/v %, (b) providing a solution of a
polycation selected from the group consisting of poly-L-lysine,
epsilon-poly-L-lysine, poly-L-arginine, chitosan oligosaccharide,
and chitosan, wherein the concentration of polycation is from about
0.1-10 w/v %, from about 1-10 w/v %, (c) adding the polyanion to
the solution of polycation, or adding the solution of the
polycation to the polyanion while the pH of the mixture is kept
below pH=5.5 by addition of an acid, whereby a precipitate is
formed that is crushed and (d) adding a drug to the solution of at
least one of steps a-c.
[0076] Nanoparticles comprising a polyelectrolyte complex according
to the invention, such as of a lignin-compound and chitosan, can be
prepared by adding an aqueous solution of calcium chloride
comprising a drug to a solution of a polyanion, preferably a lignin
compound, while stirring. A polycation, preferably chitosan,
solution is than added to the calcium polyanion pre-gel. The
resultant opalescent suspension is than equilibrated overnight to
allow nanoparticles to form with a uniform particle size.
[0077] A drug that is present in a drug delivery system according
to the invention can be any agent which is preferably not
immediately released after administration. Examples of active
agents that are preferably released at a defined time after
administration, for example in the early morning, are
anti-asthmatics (e.g. bronchodilators), anti-emetics, cardiotonics,
vasodilators, anti-vertigo and anti-meniere drugs,
anti-ulceratives, corticosteroids such as prednisone, other anti
inflammatory drugs, analgetics, anti-rheumatics, anti-arthritic
drugs; anti-angina drugs; and anti-hypertensives. In addition,
other compounds for which such formulations can be very useful to
improve patient compliance comprise sedatives such as diazepam,
antidepressants, and other CNS compounds. Other classes of agents
that are preferably formulated in drug delivery system according to
the invention are bioactive proteins, peptides, enzymes, vaccines
and oligonucleotides. These types of compounds are often not
resistant to the acidic environment of the stomach, and are
therefore preferably released in the small and/or large
intestine.
[0078] Yet another class of drugs that are preferably formulated in
drug delivery system according to the invention is probiotic
bacteria. Non-limiting examples of probiotic bacteria are Bacillus
coagulans sp., Bifidobacterium animalis, Bifidobacterium breve,
Bifidobacterium infantis, Bifidobacterium longum, Lactobacillus
acidophilus, Lactobacillus casei, Lactobacillus paracasei,
Lactobacillus fortis, Lactobacillus johnsonii, Lactobacillus
plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus,
Saccharomyces boulardii, and mixtures thereof.
[0079] Some forms of chitosan, e.g. shrimp-derived chitosan, are
generally recognized as save and have GRAS status. It is preferred
that the polyanion, such as a lignin compound, and the polycation,
such as chitosan, that are used in a drug delivery system according
to the invention are sufficiently pure and are of pharmacological
grade.
EXAMPLES
General
[0080] For all examples, the term VitoCali refers to the
polyelectrolyte complex of Ca-lignosulfonate and chitosan
Example 1
[0081] 6 young grapevines (cv Merlot) per treatment were grown
outside or under greenhouse cover (open tunnel) in pots. Plants
were treated 4 times (7 days interval) with the indicated dose rate
in a spray volume of 1000 l/ha until run-off. Four days after the
2nd fungicide application, 2 out of 6 plants were inoculated with
Plasmopara viticola. These 2 plants served as a natural inoculum
for the remaining 4 plants. Disease progression was assessed once a
week.
Results
[0082] The results of experiments with Folpet and Captan 500SC are
depicted in Tables 1 and 2.
TABLE-US-00002 TABLE 1 Effect of application of Folpet 500 SC with
and without CitoCal on of Plasmopara viticola infection on leafs of
grapevine. Presented is the % of leaf area that was infected.
Infected leaf area days after Product Dose rate infection Folpet
500 SC (L/ha) 7 d 14 d 21 d Standard Folpet 0.5 42 59 61 CitoCal*
with Folpet 0.5 30 36 39 Standard Folpet 2 29 38 46 CitoCal with
Folpet 2 5 9 14
TABLE-US-00003 TABLE 2 Effect of application of Captan 500 SC with
and without CitoCal on Plasmopara viticola infection on leafs of
grapevine. Presented is the % of leaf area infected. Infected leaf
area days after Product infection Captan 500 SC Dose rate (L/ha) 7
d 14 d 21 d Standard 0.5 39 50 66 CitoCal 0.5 21 30 35 Standard 2
35 40 46 Citocal 2 15 25 26 *CitoCal:polyelectrolyte complex of
lignosulfonate and chitosan in a 5:1 (w/w) ratio.
Example 2
Materials and methods
[0083] Tested fruit: apple from organic origin/SKAL certified. SKAL
is a semi-governmental Dutch organization that controls organic
production in the Netherlands.
[0084] Tested formulation: final concentration of 100 ppm natamycin
in different formulation backgrounds (see also example 3 and table
4).
[0085] Used pathogen: Botrytis cinerea spore-suspension containing
.about.10.sup.5 propagules (spores)/ml.
Treatments:
[0086] 1) Mock (=formulation ingredients only) 2) Lignosulfonate
(LS) and chitosan (CTS) polyelectrolyte complex (CitoCal) without
active ingredient (a.i.) 3) LS+CTS without a.i. 4) LS without a.i.
5) CTS without a.i. 6) Mock+natamycin 7) CitoCal+natamycin 8)
LS+CTS+natamycin 9) LS+natamycin 10) CTS+natamycin
[0087] Application: Fruit peel was damaged with a cork borer, depth
.about.0.5 cm into the fruit, 2 wounds per fruit. Three droplets of
a freshly prepared spore suspension of B. cinerea (estimated
10.sup.5 spores/ml) were applied by pipette into each wound.
Subsequently, the spore-suspension was allowed to air-dry for 4
hours. Treatments 1-10 were diluted 100 times in tap water and were
applied to each wound after inoculation by airbrush until run-off
(3 short bursts .about.20 cm distance of fruit surface).
[0088] All fruits were kept at room temperature (20.degree. C.).
Wounds were examined daily for 15 days or until the fruit was
completely spoiled.
[0089] Replicates: All treatments (1-10) were performed on 5
individual pieces of fruit with 2 wounds each resulting in 10
wounds per treatment.
[0090] Observation: All wounds were inspected daily for visual
symptoms of fruit rot and/or fungus growth.
[0091] Rot diameter (perpendicular) was measured at three
succeeding days. The observations were ended when all wounds of the
control formulations (1-5) showed maximal symptoms of fruit rot/and
or fungal growth.
Results
[0092] The results are depicted in Table 3.
TABLE-US-00004 TABLE 3 Apple Average diameter (cm) of infected spot
(n = 10) Product: days after infection Natamycin 2 5 6 AUDPC* Mock
1.2 4.8 6.1 14.4 Mock + CitoCal 1 4.6 5.7 13.6 Mock + LS + CTS 1.1
4.7 6 13.9 Mock + LS 1.1 4.6 5.8 13.7 Mock + CTS 1 4.5 5.8 13.3
Mock + natamycin 0.3 1.6 2.4 4.8 Mock + CitoCal + natamycin 0.3 0.4
0.7 1.6 Mock + LS + CTS + natamycin 0.3 2.4 3.6 7.1 Mock + LS +
natamycin 0.4 2.5 3.7 7.4 Mock + CTS + natamycin 0.4 3.5 4.9 9.9
*AUDPC: Area Under the Disease Progressive Curve
[0093] The sprayed mock formulation comprised 0.2 g/L
sodiumdioctylsuccinate, 0.75 g/L sophorolipid, 0.55 g/L of a 2%
xanthan and 0.01 g/L surfynol 104E
Summary & Conclusion:
Phytotoxicity
[0094] Two days after inoculation, Botrytis fruit rot was observed
on apples that were treated with a formulation that did not contain
natamycin. Apples treated with natamycin did not show any
symptoms.
[0095] Five and six days after inoculation, a clear difference was
noticeable between apples treated with and without natamycin.
Botrytis symptoms on apples not treated with natamycin was
.about.4.7 cm (5 dpi) and -5.9 cm (6 dpi). Rot on apples treated
with natamycin was clearly lower, especially the apples treated
with CitoCal and natamycin. Interestingly, apples treated with
natamycin+CaLS+CTS (non-polymer of CitoCal) was by far not as
effective as polymeric CitoCal.
Example 3
Materials and Methods
[0096] Tested crop: banana fruit (SKAL certified; SKAL is a
semi-governmental Dutch organization that controls organic
production in the Netherlands).
[0097] Application: The fruit peel was damaged with a sterile nail,
depth about 0.5 cm into the fruit, 2 wounds per fruit. Three
droplets of a freshly prepared spore suspension of Botrytis cinerea
(10.sup.5 spores/ml) was applied by pipette into each wound.
Subsequently, the applied spore-suspension was allowed to air-dry
for 4 hours. Treatments 1-10 (see table 4) were applied to each
wound after diluting 100 times in tap water by airbrush until
run-off (3 short bursts at about 20 cm distance of fruit surface).
All fruits were kept at room temperature (20.degree. C.).
[0098] Treatments: [0099] 1) Adjuvants (Control) [0100] 2)
Adjuvants plus Chitosan--Ca-Lignosulfonate polyelectrolyte complex
(CitoCaL). [0101] 3) Adjuvants plus Chitosan and Lignosulfonate (no
polyelectrolyte binding). [0102] 4) Adjuvants plus
Ca-lignosulfonate [0103] 5) Adjuvants plus chitosan [0104] 6)
Adjuvants plus natamycin [0105] 7) Adjuvants plus CitoCaLplus
Natamycin [0106] 8) Adjuvants plus Chitosan and Lignosulfonate (no
polyelectrolyte binding) plus Natamycin [0107] 9) Adjuvants plus
Ca-lignosulfonate plus natamycin [0108] 10) Adjuvants plus chitosan
plus natamycin
[0109] All treatment compositions are presented in table 4. All
numbers express g/L
TABLE-US-00005 TABLE 4 Treatment: Formulation (g/L): #1 #2 #3 #4 #5
#6 #7 #8 #9 #10 Water 664 664 664 664 664 664 664 664 664 664 Ca-LS
xx 250 250 250 xx xx 250 250 250 xx Chitosan xx 10 10 xx 10 xx 10
10 xx 10 H3PO3 xx 5 xx 5 5 xx 5 xx 5 5 Sodium dioctylsuccinate 40
40 40 40 40 40 40 40 40 40 (50%) Sophorolipid 75 75 75 75 75 75 75
75 75 75 Xanthan (2% in H2O) 55 55 55 55 55 55 55 55 55 55 Surfynol
104E 1 1 1 1 1 1 1 1 1 1 Natamycin (technical 95%) xx xx xx xx xx
10 10 10 10 10
[0110] Replicates: All treatments (1-10) were performed on 5
individual pieces of fruit with 2 wounds each resulting in 10
wounds per treatment.
[0111] Observation: All wounds were inspected daily for visual
symptoms of fruit rot and/or fungus growth.
[0112] Rot diameter (perpendicular) was measured at three
succeeding days. The observations were ended when all wounds of the
control formulations (1-5) showed maximal symptoms of fruit rot/and
or fungal growth.
TABLE-US-00006 Crop: Banana Average diameter (cm) of infected spot
(n = 10) days after Active ingredient: infection Natamycin 5 6 7
Treatment 1 2.6 3.5 4.6 Treatment 2 2.9 4.0 5.3 Treatment 3 2.7 3.1
4.8 Treatment 4 2.9 3.5 4.6 Treatment 5 2.8 3.9 5.0 Treatment 6 1.4
2.1 2.8 Treatment 7 1.3 1.8 2.5 Treatment 8 1.7 2.3 3.2 Treatment 9
1.2 1.9 2.7 Treatment 10 2.0 3.4 4.2
Conclusion Example 3
[0113] Example 3 demonstrates that neither the Adjuvants plus
Ca-lignosulfonate (treatment 4), nor the Adjuvants plus chitosan
(treatment 5), nor adjuvants plus Chitosan and Lignosulfonate (no
polyelectrolyte binding--treatment 3), nor the adjuvants plus
Chitosan Ca-Lignosulfonate complex (CitoCal--treatment 2) have
antifungal properties (compare treatment 1 with treatment 2 to 5).
Example 3 also demonstrates that CitoCal provides a synergistic
effect to the used active ingredient (i.e. natamycin) for the
prevention of Botrytis cinerea fruit rot on banana when applied
post-harvest, whereas, incubations without CitoCal do not (compare
treatment 6 with 8 to 10).
[0114] Treatment 7, in which the polyelectrolytes Chitosan and
Ca-lignosulfonate are bound, results in in a decrease of the
Botrytis infection compared to incubation 8 in which the chitosan
and ca-lignosulfonate are not bound. This shows the unique effect
of the polyelectrolyte complex on disease development.
Example 4
Materials and Methods
[0115] As example 3 (treatment numbers are explained in example 3),
but in example 4 only treatment 1, 2, 6 and 7 were performed. Apple
fruit was used instead of banana fruit.
Results Example 4
TABLE-US-00007 [0116] Crop: Apple Average diameter (cm) of infected
spot (n = 10) days after Active ingredient: infection Natamycine 3
4 5 Treatment 1 3.9 10 22 Treatment 2 3.2 7.7 13 Treatment 6 0.8
1.6 4.0 Treatment 7 0.3 0.9 2.9
Conclusion:
[0117] Example 4 demonstrates that the polyelectrolyte bound
Chitosan and Ca-lignosulfonate (treatment 7) provides a synergistic
effect to the used active ingredient (i.e. natamycin) for the
prevention of Botrytis cinerea fruit rot on apple when applied
post-harvest. This shows the unique effect of the polyelectrolyte
complex on disease development.
Example 5
Materials and Methods
[0118] As example 3 (treatment numbers are explained in example 3),
but in example 5 only treatment 1, 2, 6 and 7 were performed.
Results Example 5
TABLE-US-00008 [0119] Crop: Banana Average diameter (cm) of
infected spot (n = 10) days after Active ingredient: infection
Natamycine (100 ppm) 4 5 6 Treatment 1 1.9 2.8 4.0 Treatment 2 1.7
2.7 3.8 Treatment 6 1.6 2.5 3.9 Treatment 7 1.4 2.1 2.9
Conclusion Example 5
[0120] Example 5 demonstrates that the polyelectrolyte bound
Chitosan and Ca-lignosulfonate provides a synergistic effect to the
used active ingredient (i.e. natamycin) for the prevention of
Botrytis cinerea fruit rot on banana when applied post-harvest.
This shows the unique effect of the polyelectrolyte complex on
disease development.
Example 6
Materials and Methods
[0121] As example 3 (treatment numbers are explained in example 3),
except that the tested crop is tomato fruit of organic origin and
the tested formulations were diluted 200 times instead of 100
times
Results Example 6
TABLE-US-00009 [0122] Tomato Average diameter (cm) of infected spot
(n = 10) days after Product: infection Natamycin 3 4 Treatment 1
2.7 4.3 Treatment 2 2.8 4.3 Treatment 6 2.4 3.9 Treatment 7 1.5
3.4
Conclusion Example 6
[0123] Example 6 demonstrates that the polyelectrolyte bound
Chitosan and Ca-lignosulfonate provides a synergistic effect to the
used active ingredient (i.e. natamycin) for the prevention of
Botrytis cinerea fruit rot on tomato when applied post-harvest.
This shows the unique effect of the polyelectrolyte complex on
disease development.
Example 7
Materials and Methods
[0124] Method as in Example 3, except that the active ingredient is
imazalil instead of natamycin and the polyelectrolyte of
Ca-lignosulfonate and chitosan was added separately (as a `tank
mix`) before spraying.
Treatments:
[0125] Treatment 1: 4 g/L Adjuvants (control); see table 5.
Treatment 2: 4 g/L Adjuvants plus 8 g/L Chitosan Ca-Lignosulfonate
complex (CitoCaL); see table 5 and 6. Treatment 3: 4 g/L Imazalil;
see table 7. Treatment 4: 4 g/L Imazalil plus 8 g/L Chitosan
Ca-Lignosulfonate complex (CitoCaL); see table 6 and 7.
Formulations:
TABLE-US-00010 [0126] TABLE 5 Adjuvants formulation: Methyloleaat
7.53 g Ethanolamine 2.5 g Zephrym 3300B 15.12 g Sophorolipide 6.00
g polyoxyethylene sorbitol 9.02 g oleate Water 62.38 g
TABLE-US-00011 TABLE 6 Chitosan Ca-Lignosulfonate complex (CitoCaL)
formulation: Calciumligninsulfonaat 250.00 Chitosan 10.00 H3PO3
5.00 NaOH 35% 13.30 Sophorolipide 75.00 Surfynol 104E 1.00 Xanthan
1.10 sodiumdioctylsulfosuccinate 40.00 Water 704.60
TABLE-US-00012 TABLE 7 Imazalil formulation: Imazalil 97% 10.3 g
Methyloleaat 7.53 g Ethanolamine 2.5 g Zephrym 3300B 15.12 g
Sophorolipide 6.00 g polyoxyethylene sorbitol 9.02 g oleate Water
51.98 g
TABLE-US-00013 Crop: Apple Average diameter (cm) of infected spot
(n = 10) days after Active ingredient: infection Imazalil 4 5 6
Treatment 1 2.8 3.4 5.3 Treatment 2 2.9 4.2 5.1 Treatment 3 2.4 3.6
4.6 Treatment 4 1.0 2.0 3.1
[0127] Example 7 demonstrates that the polyelectrolyte bound
Chitosan and Ca-lignosulfonate provides a synergistic effect to the
used active ingredient (i.e. imazalil) for the prevention of
Botrytis cinerea fruit rot on apple when applied post-harvest. This
shows the unique effect of the polyelectrolyte complex on disease
development.
Example 8
Materials and Methods
Application:
[0128] Cultivated potato plants (cv. Bintje) were grown in pots.
The pots with a content of 5 litre were filled with soil and the
potato tubers were placed at a depth of 10 cm. From emergence until
inoculation the plants were placed outside to create short and
strong stems. In the greenhouse with high temperatures the stems
would be long and weak. After spraying the plants were placed in
the greenhouse. Spraying was carried out when the potato plants
reached a height of .about.10 cm. Potato plants were sprayed in a
spraying cabin developed by Applied Plant Research (PPO)--The
Netherlands. The fungicides were sprayed using a spray boom with
three spray nozzles, placed 50 cm apart, which was moving
approximately 40 cm over the top of the potato plants. Spray volume
was 250 l/ha. Potato plants were sprayed four times; on 15 May, 22
May, 31 May and 7 Jun. 2012. A P. infestans strain was cultivated
on agar plates and potato slices. An inoculum suspension was made
by rinsing a one week old culture of P. infestans with tap water.
The inoculum density was set at approximately 10,000 sporangia per
ml. Inoculation was carried out by spraying potato plants over head
with approximately 8-10 ml of inoculum. Inoculation was carried out
on air dry plants on 30 May 2012. After inoculation plants were
incubated at a relative humidity of 100% for one night during
approximately 16 hours. Disease observations were carried out 6, 9
and 12 days after inoculation. Percentage necrotic foliage per
plant was estimated. Replicates: 4 replicates of 3 plants per
replicate
Treatments:
[0129] 1) Untreated control 2) CitoCal (equivalent to 1.5 l/ha); 3)
Dithane (equivalent to 2 kg/ha) 4) Dithane+CitoCal (equivalent to 2
kg/ha and 1.5 l/ha, respectively)
Formulations:
[0130] CitoCal: see example 7, table 6.
[0131] Dithane: Commercial available fungicide by Dow AgroSciences
containing 75% mancozeb
Results Example 8
TABLE-US-00014 [0132] Crop: Potato Average percentage disease
severity Active ingredient: days after infection Mancozeb 6 9 12
UTC 37.5 80.3 87.4 Citocal 39.4 78.8 86.8 Dithane 5.0 9.4 14.9
Dithane + Citocal 2.6 5.1 11.0
Conclusion Example 8
[0133] Example 8 demonstrates that the polyelectrolyte bound
Chitosan and Ca-lignosulfonate provides a synergistic effect to the
used active ingredient (i.e. mancozeb) for the prevention of
Phytophthora infestans late blight disease on potato when applied
to whole plants. This shows the unique effect of the
polyelectrolyte complex on disease development.
Example 9
Materials and Methods
[0134] As example 8, except Shirlan was used instead of Dithane
Treatments:
[0135] 1) Untreated control 2) Citocal (equivalent to 1.5 l/ha) 3)
Shirlan (equivalent to 0.4 l/ha) 4) Shirlan+CitoCal (equivalent to
0.4 l/ha and 1.5 l/ha, respectively)
Formulations:
[0136] CitoCal: see example 7, table 6.
[0137] Shirlan: Commercial available fungicide by Syngenta
containing 39% fluazinam
Results Example 9
TABLE-US-00015 [0138] Crop: Potato Average percentage disease
severity Active ingredient: days after infection Fluazinam 6 9 12
UTC 37.5 80.3 87.4 Citocal 39.4 78.8 86.8 Shirlan 5.6 14.1 16.5
Shirlan + Citocal 3.4 7.5 12.3
Conclusion Example 9
[0139] Example 9 demonstrates that the polyelectrolyte bound
Chitosan and Ca-lignosulfonate provides a synergistic effect to the
used active ingredient (i.e. fluazinam) for the prevention of
Phytophthora infestans late blight disease on potato when applied
to whole plants. This shows the unique effect of the
polyelectrolyte complex on disease development.
Example 10
Materials and Methods
[0140] The cultivated grapevine plants (cv. merlot) were grown in
pots. The pots were filled with potting soil and grapevine stems
were planted. All stems contained 2 buds. Plants were grown outside
in South West France.
[0141] Spraying was carried out when the plants had developed 4
leaves with a lance under gas pressure with an interval of 8+/-2
days until run off (equivalent of 1000 l/ha). In total, 6 plants
per replicate were used, each treatment contained 3 replicates. 2
plants were artificially inoculated with 40.000 spores per ml of
Plasmopara viticola and kept at 100% RH for 12 hours. These 2
plants served as a natural inoculum for the other 4 plants.
Assessment of percentage infected leaves was done at 6, 9, 15 and
22 days after inoculation.
Treatments:
[0142] 1. Commercial captan: a fungicide containing 500 g/L Captan
[0143] 2. Captan formulation (containing the polyelectrolyte bound
Chitosan and Ca-lignosulfonate (CitoCal)); see table 9.
TABLE-US-00016 [0143] TABLE 9 Captan formulation (g/L) Captan
techn. 510 Water (tap) 541.3 polyethoxylated fosforic acid 40
Calciumligninsulfonate 100 polydimethylsiloxane 3 Chitosan 5 Citric
acid 8 NaOH techn. 2 Xanthan gum 0.7 Totals 1210
Results Example 10
TABLE-US-00017 [0144] Crop: Grapevine % Infected leaf area Active
ingredient: Dose rate days after infection Captan (L/ha) 6 9 15 22
UTC -- 34 54 72 76 Commercial captan 0.5 15 21 37 47 Captan
formulation 0.5 8 12 22 22 Commercial captan 2 16 29 29 35 Captan
formulation 2 10 15 20 18
Summary and Conclusion:
[0145] Example 10 clearly demonstrates that the polyelectrolyte
bound chitosan and Ca-lignosulfonate provides a synergistic effect
to the used active ingredient (i.e. captan) for the prevention of
Plasmopara viticola, the causal agent of downy mildew on grapevine.
This shows the unique effect of the polyelectrolyte complex on
disease development.
Example 11
Materials and Methods
[0146] As example 10, however, in this experiment folpet was used
instead of captan.
Treatments:
[0147] 1. Commercial folpet: a fungicide containing 500 g/L folpet
[0148] 2. Folpet formulation (containing polyelectrolyte bound
Chitosan and Ca-lignosulfonate (CitoCal)); see table 10.
TABLE-US-00018 [0148] TABLE 10 Folpet formulation (g/L): Folpet
techn. 510 Water (tap) 536.3 polyethoxylated fosforic acid 40
Calciumligninsulfonate 100 polydimethylsiloxane 3 Chitosan 5
Fumaarzuur 3 NaOH techn. 2 Xanthan gum 0.7 Total 1200
Results Example 11
TABLE-US-00019 [0149] Crop: Grapevine % Infected leaf area days
after Active ingredient: Dose rate infection Folpet (L/ha) 6 9 15
22 UTC -- 34 54 72 76 Commercial captan 0.5 17 39 45 44 Folpet +
CitoCal 0.5 12 27 28 27 Commercial captan 2 10 36 27 35 Folpet +
CitoCal 2 3 17 13 15
Summary and Conclusion:
[0150] Example 11 clearly demonstrates that the polyelectrolyte
bound Chitosan and Ca-lignosulfonate provides a synergistic effect
to the used active ingredient (i.e. folpet) for the prevention of
Plasmopara viticola, the causal agent of downy mildew on grapevine.
This shows the unique effect of the polyelectrolyte complex on
disease development.
Example 12
Materials and Methods
[0151] An open-field experiment was carried out according to the
harmonised protocol according to the "European network on Potato
Late Blight` in Talinn (2005), Bologna (2007), Hamar (2008) and
Arras (2010). The protocol can be found on the Euroblight website
(http://www.euroblight.net/Euroblight.asp). The trial conformed to
local good agriculutural practice, trials were carried out in four
replicates and sprays against P. infestans were carried out in a
more or less weekly schedule.
[0152] The fungicide applications were carried out using a AZO
field sprayer with 6 XR TeeJet 80015Vs nozzles approximately 50 cm
above the foliage. Spraying were carried out with 300 l/ha. Potato
plants were sprayed for the first time at 100% emergence.
Fungicides were sprayed in a weekly scheme, according to the agreed
protocol. Dose was 2.0 kg/ha Dithane DG (Dow AgroSciences) for the
first application and 2.25 kg/ha thereafter. CitoCal (for
formulation, see example 7, table 6) was added as a tankmix at an
dose equivalent of 1.5 l/ha
[0153] Tested crop: Potato cv. Bintje (field grown).
[0154] Pathogen: Natural occurring Phytophthora infestans
Treatments:
[0155] 1) Dithane (equivalent to 2.0-2.25 kg/ha) 2) Dithane+CitoCal
(equivalent to 2.0-2.25 kg/ha and 1.5 l/ha, respectively)
[0156] Observation: Percentage of disease severity was assessed
throughout the trial. After the trial, stAUDPC48 was calculated
based on late blight observations from Jul. 18 2012 until 28 Aug.
2012.
Results Example 12
TABLE-US-00020 [0157] Crop: Potato stAUDPC48 Dithane 0.84 Dithane +
Citocal 0.64
Summary and Conclusion:
[0158] Example 12 clearly demonstrates that CitoCal provides a
synergistic effect to the used active ingredient (i.e. mancozeb)
for the prevention of Phytophthora infestans, the causal agent of
late blight on field-grown potato.
Example 13
Materials and Methods
[0159] The experiment was performed with a similar setup as in
example 10. Unicula necator, the causal agent of powdery mildew on
grapevine, was used instead of Plasmopara viticola.
[0160] Observation: Assessment of percentage infected leaves at 6,
11, 19, 28 and 42 days after inoculation
Formulation:
TABLE-US-00021 [0161] TABLE 11 Sulfur with and without
polyelectrolyte of Ca-lignosulfonate and chitosan (CitoCal) (in
g/L) Sulfur + CitoCal Sulfur - CitoCal 80% sulfur 800.00 800.00
Water 627.50 666.50 Chitosan 5.00 -- HCL 37% 2.50 -- Na-LS 50%
70.00 70.00 polydimethylsiloxane 5.00 5.00
Results Example 13
TABLE-US-00022 [0162] Crop: Grapevine Dose rate (kg/ha % Infected
leaf area elemental days after infection Active ingredient: S) 6 11
19 28 42 UTC -- 40 86.3 100 100 100 Sulfur - CitoCal 1.5 7.5 6.3
2.5 18.8 16.3 Sulfur + CitoCal 1.5 2.5 6.3 1.3 7.5 11.3
Summary and Conclusion:
[0163] Example 13 clearly demonstrates that CitoCal provides a
synergistic effect to the used active ingredient (i.e. sulfur) for
the prevention of Uncinula necator, the causal agent of powdery
mildew on grapevine when applied to whole plants.
Examples 14, 15, 16 And 17
[0164] General. In this experiment we show that not only
polyelectrolyte combinations of chitosan and lignosulfonate are
effective in enhancement of fungicidal efficacy but also
polyelectrolytre combinations of other polyanion molecules (humic
acid and the synthetic compound Morwet) in combination with
chitosan. In example 14 the polyelectrolyte combination is
lignosulfonate--chitosan; in example 15 humic acid--chitosan and in
example 16 Morwet--chitosan. In example 17, a polyelectrolyte
combination of lignosulfonate and oligo-chitosan was tested on
tomato fruit.
Materials and Methods:
[0165] The used protocol was based on: Dik et al., 1999. Europ J
Plant Path 105: p115-122. Briefly, main stems of 9 individual about
50 cm tall tomato plants were cut in to 3-4 cm pieces, and
randomized. Stem pieces were placed in glass reaction-tube (in a
tube rack), partially filled with glass beads so 1-2 cm stem would
raise above the top. All treatments were performed on 8 individual
stem pieces.
[0166] Stem pieces were sprayed with 10.sup.5 spores/ml suspension
of a freshly grown Botrytis cinereaculture. Subsequently, stem
pieces were allowed to dry for 30 minutes. Different formulations
were prepared by adding 0.75 gram of formulation (see table 12) to
100 ml water, resulting in approx. 75 ppm natamycin (equivalent)
per formulation. Stem parts were thoroughly sprayed from all
directions, and placed in a carton box containing a wet towel. For
high humidity, a semi-transparent bag was placed over the carton
box. Boxes with stem pieces were kept at room temperature
(.about.19.degree. C.) and checked frequently for Botrytis growth .
. . .
Formulations:
TABLE-US-00023 [0167] TABLE 12 formulations (g/L) Treatment: #10 #5
#7 #8 #9 Adju- #2 #3 #4 Adju- #6 Adju- Adju- Adju- vants + #1 Adju-
Adju- Adju- vants + Adju- vants + vants + vants + oligo- Adju-
vants + vants + vants + oligo- vants + citocal + CitocalHumic +
CitocalMorwet + Citocal + vants citocal CitocalHumic CitocalMorwet
Citocal ai ai ai ai ai Water 664 664 664 664 664 664 664 664 664
664 Ca- xx 250 250 xx 250 250 Lignosulfonate Potassium humate 100
100 Morwet (Akzo) 83.3 83.3 Chitosan xx 10 10 10 xx 10 10 10
Oligomers of 10 10 Chitosan H3PO3 xx 5 5 5 5 xx 5 5 5 5 Sodium- 40
40 40 40 40 40 40 40 40 40 dioctylsuccinate (50%) Sophorolipid 75
75 75 75 75 75 75 75 75 75 Xanthan gum 55 55 55 55 55 55 55 55 55
55 (2% in H2O) poly- 1 1 1 1 1 1 1 1 1 1 dimethylsilox- ane
Natamycin xx xx xx xx xx 10 10 10 10 10 Active ingredient (A.I.) =
natamycin
Assessment:
[0168] Stem pieces were scored for disease incidence:
1=less than 1/2 of the stem piece infected 2=1/2-3/4 of the stem
piece infected 3=3/4-full infection 4=full infection
[0169] Disease incidence was calculated with the formula as
described in Dik et. al. 1999.
[0170] A higher number relates to a higher infection rate.
Results Example 14
[0171] See table 12 for formulation composition
TABLE-US-00024 Days post infection Tomato plant (stems) 7 8 9
Treatment 1 (Adjuvants) 2.500 3.500 3.750 Treatment 2 (Adjuvants +
CitoCal) 2.875 3.125 3.375 Treatment 6 (Adjuvants + ai) 2.300 2.625
2.875 Treatment 7 (Adjuvants + CitoCal + ai) 2.286 2.571 2.429
Summary and Conclusion:
[0172] Example 14 clearly demonstrates that CitoCal provides a
synergistic effect to the used active ingredient (i.e. natamycin)
for the prevention of Botrytis cinerea stem rot on tomato plants
(stem parts).
Results Example 15
[0173] See table 12 for formulation composition
TABLE-US-00025 Days post infection Tomato plant (stems) 7 8 9
Treatment 1 (Adjuvants) 2.500 3.500 3.750 Treatment 3 (Adjuvants +
humic acid-cal) 2.500 2.750 3.125 Treatment 6 (Adjuvants + ai)
2.300 2.625 2.875 Treatment 8 (Adjuvants + Humic-acid-cal + ai)
2.167 2.286 2.125
Summary and Conclusion:
[0174] Example 15 clearly demonstrates that that polyelectrolyte
complex of humic acid and chitosan (Humic acid-cal) provides a
synergistic effect to the used active ingredient (i.e. natamycin)
for the prevention of Botrytis cinerea stem rot on tomato.
Results Example 16
[0175] See table 12 for formulation composition.
TABLE-US-00026 TABLE 12 Days post infection Tomato plant (stems) 7
8 9 Treatment 1 (Adjuvants) 2.500 3.500 3.750 Treatment 4
(Adjuvants + morwett-cal) 2.750 3.375 3.375 Treatment 6 (Adjuvants
+ ai) 2.300 2.625 2.875 Treatment 9 (Adjuvants + morwett-cal + ai)
2.286 2.429 2.625
Summary and Conclusion:
[0176] Example 16 clearly demonstrates that that polyelectrolyte
complex of morwet and chitosan (morwet-cal) provides a synergistic
effect to the used active ingredient (i.e. natamycin) for the
prevention of Botrytis cinerea stem rot on tomato.
Example 17
Materials and Methods
[0177] As described in example 3, except tomato fruits were used
instead of banana fruit.
Formulations:
[0178] As in example 14 (see table 12)
Results Example 17
TABLE-US-00027 [0179] Crop: tomato fruit Average diameter (cm) of
infected spot (n = 10) days after Active ingredient: infection
natamycin 3 4 5 Adjuvants 1 2.3 3.2 Adjuvants + oligo-Citocal 0.8
1.9 2.8 Adjuvants + ai 1 1.9 2.9 Adjuvants + oligo-Citocal + ai 0.7
1.6 2.5
Summary and Conclusion:
[0180] Example 17 clearly demonstrates that the combination of
lignosulfonate and oligo-chitosan provides a synergistic effect to
the used active ingredient (i.e. natamycin) for the prevention of
Botrytis cinerea fruit rot on tomato fruit.
Example 18
Method
[0181] As in example 3, except apple fruit was used instead of
banana fruit. The used formulations are:
Formulations:
TABLE-US-00028 [0182] TABLE 13 Formulations (g/L) Adjuvants
Adjuvants + Adjuvants + Citocal + + Adjuvants Citocal natamycin
natamycin g/L g/L g/L g/L water 882.6 830.0 789.4 836.8 chitosan
10.1 9.6 HCl (37%) 5.0 4.8 Calciumligninsulfonate 50.4 48.0
NaOH(aq) monopropyleenglycol 71.5 67.2 64.0 67.8
polydimethylsiloxane 7.2 6.7 6.4 6.8 Atlox 4894 35.8 33.6 32.0 33.9
Atlox 4913 18.6 17.5 16.6 17.6 polyvinylpyrrolidone 5.7 5.4 5.1 5.4
(30%) Natamycin (95%) 53.8 57.1 Xanthan gum (2%) 78.7 74.0 70.3
74.6
Results Example 18
TABLE-US-00029 [0183] Crop: Apple fruits Average diameter (cm) of
infected spot (n = 10) days after Active ingredient: infection
Natamycin (100 ppm) 3 4 5 Adjuvants 2.9 3.6 4.8 Adjuvants + Citocal
2.8 3.8 4.7 Adjuvants + natamycin 2.4 3.6 4.6 Adjuvants + Citocal +
natamycin 1.7 2.6 3.6
Conclusion Example 18
[0184] Example 18 clearly demonstrates that CitoCal has no
antifungal properties. Example 17 also clearly demonstrates that
CitoCal provides a synergistic effect to the used active ingredient
(i.e. natamycin) for the prevention of Botrytis cinerea fruit rot
on apple when applied post-harvest.
Example 19
[0185] We show in this experiment the efficacy of the different
polyelectrolytes on (apple) fruits.
Method:
[0186] Material and methods are as in example 3, except apple fruit
was used instead of banana fruit.
Treatments:
1=Adjuvants
[0187] 2=Adjuvants+citocal
3=Adjuvants+CitocalHumicAcid
4=Adjuvants+CitocalMorwet
[0188] 5=Adjuvants+oligo-Citocal 6=Adjuvants+folpet
7=Adjuvants+citocal+folpet 8=Adjuvants+CitocalHumic+folpet
9=Adjuvants+CitocalMorwet+folpet
10=Adjuvants+oligo-Citocal+folpet
Formulations:
TABLE-US-00030 [0189] TABLE 14 Formulations (g/L) Treatments #10 #5
#7 #8 #9 Adju- #2 #3 #4 Adju- #6 Adju- Adju- Adju- vants + #1 Adju-
Adju- Adju- vants + Adju- vants + vants + vants + oligo- Adju-
vants + vants + vants + oligo- vants + citocal + CitocalHumic +
CitocalMorwet + Citocal + vants citocal CitocalHumic CitocalMorwet
Citocal ai ai ai ai ai Water 528.5 528.5 528.5 528.5 528.5 528.5
528.5 528.5 528.5 528.5 Ca-LS 35.0 35.0 35.0 35.0 KHumaat 35.0 35.0
Morwet D425 35.0 35.0 Chitosan 5.0 5.0 5.0 5.0 5.0 5.0 Chs oligo
5.0 5.0 Fumaric acid 29.0 29.0 29.0 29.0 29.0 29.0 29.0 29.0 29.0
29.0 NaOH 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0
Sophrophor FL 40.0 40.0 40.0 40.0 40.0 40.0 40.0 40.0 40.0 40.0
ATLOX 4913 40.0 40.0 40.0 40.0 40.0 40.0 40.0 40.0 40.0 40.0
Xanthan opl 37.5 37.5 37.5 37.5 37.5 37.5 37.5 37.5 37.5 37.5 poly-
5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 dimethylsilox- ane Folpet
techn. 510.0 510.0 510.0 510.0 510.0 Totals 700.0 740.0 740.0 740.0
740.0 1210.0 1250.0 1250.0 1250.0 1250.0 Active ingredient (A.I.) =
folpet
Results Example 19
TABLE-US-00031 [0190] Crop: Apple fruit Average diameter (cm) of
infected spot (n = 10) days after Active ingredient: infection
Folpet (1 g/L) 4 Treatment 1 (Adjuvants) 2.2 Treatment 2 (Adjuvants
+ Citocal) 2.4 Treatment 3 (Adjuvants + CitocalHumicAcid) 2.5
Treatment 4 (Adjuvants + CitocalMorwet) 2.6 Treatment 5 (Adjuvants
+ oligo-Citocal) 2.3 Treatment 6 (Adjuvants + folpet) 1.5 Treatment
7 (Adjuvants + Citocal + folpet) 1.2 Treatment 8 (Adjuvants +
CitocalHumic + 1.2 folpet) Treatment 9 (Adjuvants + CitocalMorwet +
1.3 folpet) Treatment 10 (Adjuvants + oligo-Citocal + 1.1
folpet)
Conclusions Example 18
[0191] Example 19 clearly demonstrates that none of the
polyelectrolytes have antifungal properties. Example 19 also
demonstrates that different polyelectrolyte complexes provide a
synergistic effect to the used active ingredient (i.e. folpet) for
the prevention of Botrytis cinerea on apple when applied
post-harvest.
Example 20
Method
[0192] As example 14, except formulations as in example 18; see
table 13.
Results:
TABLE-US-00032 [0193] Crop: Tomato plant (stems) dpi 7 8 9
Adjuvants 3.2 3.6 3.9 Adjuvants + CitoCal 3.3 3.5 3.9 Adjuvants +
natamycin 3.1 3.6 3.9 Adjuvants + CitoCal + 2.6 2.8 3 natamycin
Conclusion Example 20
[0194] Example 20 clearly demonstrates that CitoCal provides a
synergistic effect to the used active ingredient (i.e. natamycin)
for the prevention of Botrytis cinerea stem rot on tomato. Example
20 also clearly demonstrates that CitoCal has no antifungal
properties by itself.
Example 21
Formation of Chitosan-Lignosulfonate Polyelectrolyte Complex
[0195] The following experiments is a repeat from the experiments
described in the Pakistan Journal of Biological Sciences 11 (19);
2291-2299, 2008.
[0196] Chitosan (3 gr) was added to 100 mL of water containing 1%
acetic acid. The semi-clear solution was stirred. Than 1% solution
of Calcium-lignosulfonate in 100 mL water was added to the chitosan
solution. This resulted in a milky solution. However, no
precipitation of solids was observed. Furthermore, if this solution
was diluted with water no precipitation was observed.
Example 22
[0197] 10 gram of Chitosan was suspended in 885 mL water, 5 gram
37% HCl was added to completely dissolve the chitosan. Then 100
gram Calcium-Lignosulfonate was added portion wise to the solution.
A milky solution appeared immediately and solids precipitated from
the solution. When a further 40-50 grams of the Ca-LS was added a
rise in viscosity was observed and the aqueous solution thickened
and the polyelectrolyte clearly separated as a solid from the
aqueous phase. By addition of the remaining 50 gr Ca-LS the aqueous
phase became less viscous. The solid was settled overnight and
filtered over a Buchner funnel. The filter was washed several times
to remove the excess water soluble Ca-LS. The solid was dried in
order to measure the weight of the reaction product. The weight of
the solid after drying was 62.4 gram. 10 grams of this complex is
chitosan, this means that slightly more than 50 grams is
Lignosulfonate. The ratio Chitosan:Ca-LS is therefore about 1:5
(w/w).
Example 23
[0198] When the same experiment was performed with 20 gram chitosan
in 500 mL water (more concentrated), the viscosity rised
dramatically and the jelly solution was hardly stirrable after
addition of 35 gram Ca-LS. However, after an additional 125 grams
of Ca-LS the aqueous phase became less viscous. The solid was
settled overnight and filtered over a Buchner funnel. The filter
was washed several times to remove the excess water soluble Ca-LS.
The solid was dried in order to measure the weight of the reaction
product. The ratio was determined as described above and appeared
to be Chitosan:-Ca-LS 1:3.
[0199] Conclusion: The complex formed in experiment 20 mainly
contained protonated chitosan and therefore remained in solution.
The complex formed in experiment 23 contained less Lignosulfonate
because the partly formed complex already precipitated from the
aqueous phase before the whole neutral complex was formed. Clearly
not enough water was available to keep the protonated chitosan
amine groups in solution and therefore prevented the reaction with
the excess Ca-Lignosulfonate to completion. In experiment 22 enough
water was available to completely form the whole neutral
Chitosan-Lignosulfonate polyelectrolyte complex.
* * * * *
References