TEPZZ_59 

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(11) EP 1 592 301 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.:

of the grant of the patent: A01N 41/04 (2006.01) A01N 59/16 (2006.01)
27.01.2016 Bulletin 2016/04 A01N 59/20 (2006.01) A01N 59/06 (2006.01)
C09K 17/02 (2006.01) A01N 43/90 (2006.01)
(21) Application number: 04705419.2 A01N 43/80 (2006.01) A01N 43/40 (2006.01)
A01N 37/10 (2006.01) A01N 37/02 (2006.01)
A01N 31/08 (2006.01) A01N 37/06 (2006.01)
(22) Date of filing: 27.01.2004

(86) International application number:

PCT/EP2004/000742

(87) International publication number:

WO 2004/067699 (12.08.2004 Gazette 2004/33)

(54) CROP PROTECTING AND IMPROVING LIGNOSULFONATE COMPOSITIONS

FELDFRÜCHTE SCHÜTZENDE UND VERBESSERNDE LIGNOSULFONAT-

ZUSAMMENSETZUNGEN
COMPOSITIONS COMPRENANT DES LIGNOSULFONATES DESTINEES A LA PROTECTION ET
A L’AMELIORATION DES CULTURES

(84) Designated Contracting States: DE-A- 3 431 565 DE-A- 4 404 860
AT BE BG CH CY CZ DE DK EE ES FI FR GB GR DE-A1- 4 404 860 DE-B- 1 199 257
HU IE IT LI LU MC NL PT RO SE SI SK TR DE-U1- 29 900 513 GB-A- 1 266 648
US-A- 2 858 250 US-A- 3 293 126
(30) Priority: 27.01.2003 PCT/EP03/00840 US-A- 3 858 354 US-B1- 6 294 571

(43) Date of publication of application: • DATABASE CA [Online] CHEMICAL ABSTRACTS

09.11.2005 Bulletin 2005/45 SERVICE, COLUMBUS, OHIO, US; XP002381805
retrieved from STN-INTERNATIONAL Database
(73) Proprietor: Ceradis B.V. accession no. 106:63015 & RO 89 310 A
6708 PW Wageningen (NL) (INTREPRINDEREA CHIMICA) 15 March 1986
(1986-03-15)
(72) Inventors: • DATABASE CA [Online] CHEMICAL ABSTRACTS
• VAN DER KRIEKEN, Wilhelmus, Maria SERVICE, COLUMBUS, OHIO, US; XP002381806
NL-6708 RE Wageningen (NL) retrieved from STN-INTERNATIONAL Database
• KOK, Cornelis, Johannes accession no. 135:241532 & RO 116 084 A
NL-6544 VT Nijmegen (NL) (INSTITUTUL DE CERCETARI) 30 October 2000
• STEVENS, Lucas, Henricus (2000-10-30)
NL-3571 WG Utrecht (NL) • DATABASE CA [Online] CHEMICAL ABSTRACTS
SERVICE, COLUMBUS, OHIO, US; XP002381807
(74) Representative: V.O. retrieved from STN-INTERNATIONAL Database
P.O. Box 87930 accession no. 133:116175 & JP 2000 204009 A
2508 DH Den Haag (NL) (HOKKO CHEMICAL INDUSTRY) 25 July 2000
(2000-07-25)
(56) References cited: • DATABASE CA [Online] CHEMICAL ABSTRACTS
EP 1 592 301 B1

EP-A- 0 480 614 WO-A-00/27220 SERVICE, COLUMBUS, OHIO, US; XP002381808

WO-A-01/35747 WO-A-01/35747 retrieved from STN-INTERNATIONAL Database
WO-A-95/22253 WO-A-95/33378 accession no. 142:88183 & IN 187 437 A
DD-A- 299 695 DD-A- 299 695 (SULPHUR MILLS) 27 April 2000 (2000-04-27)

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent
Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the
Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been
paid. (Art. 99(1) European Patent Convention).

Printed by Jouve, 75001 PARIS (FR) (Cont. next page)

 EP 1 592 301 B1

• DATABASE WPI Section Ch, Week 199442 • DATABASE CROPU [Online] XP002284153
Derwent Publications Ltd., London, GB; Class retrieved from STN-INTERNATIONAL Database
A97, AN 1994-339930 XP002381816 & SU 1 821 accession no. 2002-86537 CROPU & JP 2002
071 A1 (AS BELO GEN INORG CHEM INST) 15 029903 A (HOKKO-CHEM.IND.) 29 January 2002
June 1993 (1993-06-15) (2002-01-29)
• DATABASE WPI Section Ch, Week 198530 • DATABASE WPI Section Ch, Week 198132
Derwent Publications Ltd., London, GB; Class Derwent Publications Ltd., London, GB; Class
C03, AN 1985-179670 XP002381817 & HU 34 940 C02, AN 1981-57768D XP002284155 & JP 56
A (TOTH B) 28 May 1985 (1985-05-28) 075417 A (HOKKO CHEM IND CO LTD) 22 June
• DATABASE WPI Section Ch, Week 200053 1981 (1981-06-22) & PATENT ABSTRACTS OF
Derwent Publications Ltd., London, GB; Class JAPAN vol. 0051, no. 40 (C-070), 4 September
C01, AN 2000-567988 XP002381818 & JP 2000 1981 (1981-09-04) A
204009 A (HOKKO CHEM IND CO LTD) 25 July • DATABASE WPI Section Ch, Week 199415
2000 (2000-07-25) Derwent Publications Ltd., London, GB; Class
• DATABASE WPI Section Ch, Week 200248 A97, AN 1994-121150 XP002284156 & JP 06
Derwent Publications Ltd., London, GB; Class 065014 A (SUMITOMO FORESTRY CO LTD) 8
C03, AN 2002-447898 XP002381819 & JP 2002 March 1994 (1994-03-08)
087912 A (SUMITOMO CHEM CO LTD) 27 March • DATABASE WPI Section Ch, Week 198106
2002 (2002-03-27) Derwent Publications Ltd., London, GB; Class
• DATABASE WPI Section Ch, Week 198223 C03, AN 1981-08481D XP002284157 & JP 55
Derwent Publications Ltd., London, GB; Class 153706 A (HOKKO CHEM IND CO LTD) 29
A97, AN 1982-47074E XP002381820 & JP 57 November 1980 (1980-11-29)
070806 A (KANESHO KK) 1 May 1982 (1982-05-01) • DATABASE WPI Section Ch, Week 198936
• DATABASE WPI Section Ch, Week 199402 Derwent Publications Ltd., London, GB; Class
Derwent Publications Ltd., London, GB; Class C02, AN 1989-260878 XP002284158 & JP 01
C03, AN 1994-012153 XP002381821 & JP 05 190611 A (AJINOMOTO KK) 31 July 1989
320006 A (SDS BIOTECH CORP) 3 December 1993 (1989-07-31)
(1993-12-03) • DATABASE WPI Section Ch, Week 199129
• DATABASE CA [Online] CHEMICAL ABSTRACTS Derwent Publications Ltd., London, GB; Class
SERVICE, COLUMBUS, OHIO, US; XP002381809 C02, AN 1991-211723 XP002284159 & JP 03
retrieved from STN-INTERNATIONAL Database 133907 A (HOKKO CHEM IND CO LTD) 7 June
accession no. 134:337127 & IL 114 231 A 1991 (1991-06-07)
(JEWNIN-JOFFE INDUSTRY) 28 October 1999 • DATABASE CROPU [Online] XP002284154
(1999-10-28) retrieved from STN-INTERNATIONAL Database
• DATABASE WPI Section Ch, Week 200032 accession no. 2001-87639 CROPU & JP 2001
Derwent Publications Ltd., London, GB; Class 098295 A (SEKISU-CHEM.) 10 April 2001
A97, AN 1996-235898 XP002381856 & JP 03 (2001-04-10)
047744 B2 (MEIJI SEIKA KAISHA LTD) 5 June • S.Y.LIN ET. AL: "Ullmann’s Encyclopedia of
2000 (2000-06-05) Industrial Chemistry, Sixth Edition, 1999
• DATABASE WPI Section Ch, Week 198309 Electronic Release" 1999, WILEY-VCH ,
Derwent Publications Ltd., London, GB; Class WEINHEIM, DE , XP002284151 LIGNIN: 5.
C02, AN 1983-21027K XP002381857 & JP 58 Commercial Lignin - 9. Toxicology see 7. Uses
010504 A (HOKKO CHEM IND CO LTD) 21 January see 9. Toxicology
1983 (1983-01-21) • WEGLER R ED - WEGLER R: "Chemie der
• DATABASE CA [Online] CHEMICAL ABSTRACTS Pflanzenschutz- und
SERVICE, COLUMBUS, OHIO, US; K.KALLELA Schadlingsbekampfungsmittel, Band 6" CHEMIE
ET AL.: "The effect of grain preservatives on the DER PFLANZENSCHUTZ- UND SCHADLINGS-
growth of the fungus Fusarium graminearum and BEKAMPFUNGSMITTEL. INSEKTIZIDE.
on the quantity of zearalenone" XP002381853 BAKTERIZIDE. OOMYCETEN-FUNGIZIDE
retrieved from STN-INTERNATIONAL Database BIOCHEMISCHE UND BIOLOGISCHE
accession no. 96:179604 & ACTA VETERINARIA METHODEN. NATURSTOFFE, BERLIN,
SCANDINAVICA, vol. 22, no. 3-4, 1981, pages SPRINGER, DE, vol. BAND 6, 1981, pages
417-427, 216-218,272, XP002213754
• DATABASE CROPU [Online] XP002284152 • DATABASE WPI Section Ch, Week 198530
retrieved from STN-INTERNATIONAL Database Derwent Publications Ltd., London, GB; Class
accession no. 1999-84725 CROPU & JP 11 029418 C03, AN 1985-179670 XP002284160 & HU 34 940
A (TAKEDA) 2 February 1999 (1999-02-02) A (TOTH B) 28 May 1985 (1985-05-28)
• RÖMPP H ED - ROMPP H: "CHEMIE LEXIKON,
Band II, G-O" CHEMIE LEXIKON, STUTTGART,
FRANCKH’SCHE VERLAG, DE, vol. BAND 2,
1962, page COLUMN2950, XP002255469

2
 EP 1 592 301 B1

Description

[0001] The present invention relates to compositions for protecting crops, such as cereals like wheat, corn etc., and
flowers, fruits etc. against external threats, in particular against pathogens, such as fungi, for improving yield and/or
5 quality, to methods using these compositions and to plants or plant parts treated with the composition.
[0002] Agricultural crops are often subjected to a variety of biotic and abiotic threats, that can be induced by pathogens,
weeds, temperature, drought, light etc. These stress factors can affect the yield and/or quality of the product. In addition,
it is generally appreciated by consumers to buy produce of high quality without stress symptoms, such as leaf yellowing,
wilting or chlorosis.
10 [0003] Plants are 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 synthetic
pesticides, such as fungicides and bactericides) are applied. From an environmental and health point of view it is desirable
to reduce the amount of chemicals that are applied to the plants and the soil.
[0004] It is known that certain compounds of natural origin can protect the plant against pathogenic micro-organisms.
15 These so-called natural crop protection compounds (NCP’s) are organic substances derived from natural organisms
(e.g. pheromones, plant extracts), or anorganic compounds found in the natural environment (e.g. phosphates, sulfur).
Thus, in contrast to NCP’s, conventional pesticides are synthetic chemicals specifically designed for plant protection.
The use of these natural crop protection compounds (NCP’s) is becoming more and more preferable since governments
world-wide aim for a reduction in the use of synthetic anti-microbial compounds.
20 [0005] However, these NCP’s have limited usefulness, because they generally exhibit only modest activity. When
used at high concentrations they often have phytotoxic effects. In addition, the action of NCP’s appears to be rather
unpredictable (depending on the plant and environmental conditions). This explains why the application of NCP’s is no
general practice, despite their highly favourable environmental and toxicological properties.
[0006] It is thus a first object of the invention to improve the usefulness of NCP’s.
25 [0007] JPS5675417 discloses rice plant fungicides comprising kasugamycin and calcium lignosulfonate.
[0008] In the research that lead to the present invention it was surprisingly found that all the above-identified problems
can be solved by compositions that comprise one or more lignosulfonates and natamycin.
[0009] Lignosulfonates are a derivative of lignin and the commercially available form thereof. Lignin is a naturally
occurring component of plant cell walls (e.g. in wood), and one of nature’s most plentiful and renewable resources. The
30 lignosulfonate molecule is complex and can enter into many types of chemical reactions. This versatility allows it to be
modified into a whole family of special chemicals.
[0010] Lignosulfonate (LS) is a by-product of the paper manufacture obtained from the spent sulfite pulping liquor of
wood. It is a complex mixture of polymers with sulfonate groups attached to the molecules, and may contain a substantial
amount of reducing sugars. Particularly the sulfonate groups provide LS with cation exchange properties, e.g. for am-
35 monium and metal ions. Because lignosulfonates are a waste product of the paper industry they are generally available
and can be used in the composition at relatively low costs. In addition, lignosulfonates are biodegradable, eco-friendly
and safe for agricultural use.
[0011] According to a first aspect of the invention an antimicrobial composition is provided that comprises lignosul-
fonates (LS) and natamycin.
40 [0012] The invention thus relates to the application of natamycin in combination with one or more specific sustainable
products that provide a synergistic or additive effect, and/or protect the plant against phytotoxic activity of the NCP.
These specific sustainable products are biodegradable and eco-friendly. The sustainable product is lignosulfonate (LS)
and products derived thereof.
[0013] The composition can further comprise chemical antimicrobial compounds, in particular chemical pesticides,
45 more in particular chemical fungicides. The invention thus provides the combined application of reduced amounts of
natamycin and one or more sustainable products that provide a synergistic or additive effect, and/or prevents the plant
against phytotoxic activity of the natamycin.
Here also the sustainable product is LS.
[0014] It was thus found according to the invention that by combining the active ingredient with LS the effectiveness
50 of the active ingredient can be enhanced and/or the plant to be treated can be protected from the phytotoxicity of the
active ingredient.
[0015] It was particularly surprising to find that by using them in combination with LS some metals that are in itself
toxic to fungi and yeasts can be used in much lower amounts for achieving a comparable effect.
[0016] The metals that were found to be particularly useful in the invention are copper, zinc, aluminium, titanium, silver,
55 cobalt and manganese. The metal-LS have a particularly good activity against fungi and algae.
[0017] In a particular embodiment of the present invention either titanium (TiLS) and/or silver lignosulfonates (AgLS)
are used. The use of titanium and/or silver further enhances the anti-microbial effect of the composition. TiLS contains
TiO2. The finely distributed TiO2 catalyses in UV irradiation the formation of oxygen radicals that have biocidal activity.

3
 EP 1 592 301 B1

TiO2 is not toxic for humans (it is for instance present in tooth paste). Silver lignosulfonates release Ag-ions in an aqueous
environment. Ag-ions are toxic for micro-organisms and therefore contribute to the effectivity of the composition.
[0018] Copper is known for its fungicidal activity. However, to be effective kilograms of copper compounds such as
copper sulphate or copper oxide per hectare are needed. From an environmental point of view this is far too much and
5 these copper compounds were thus banned as a crop protection agent. From the examples it follows that when combined
with lignosulfonate, the amount of copper can be reduced to tens of grams per hectare thus making copper again available
as a crop protection agent.
[0019] Natural crop protection compounds are agents that are of natural origin and protect plants or plant products
against organisms or prevent the organism’s activity; influence life processes of plants without being fertilizers; conserve
10 plant products, kill unwanted plants; or destroy plant parts or prevent or inhibit unwanted growth of plants. Lists of NCP’s
can be found on http://www.gewasbescherming.nl/index10i.html.
[0020] In the invention the one or more natural crop protection compounds is is natamycin.
[0021] Natamycin is very sensitive to light. The combination with lignosulfonates allows natamycin to be applied to
leaves, while retaining its activity.
15 [0022] Lignosulfonates not only lower the phytotoxicity of NCP’s but protect the anti-microbial compounds in the
composition against degradation thus allowing for their use in agriculture. Furthermore, lignosulfonates provide an
synergistic effect by making the plant less susceptible to the anti-microbial compounds and increasing the effect of such
compounds.
[0023] "Metal-LS" as used in this application is intended to refer to the combination of any one or more metals with
20 LS. The metal can be either complexed to LS (e.g. as counter ions) or can be used in the same composition or at the
same time as LS.
[0024] The term "acid-LS" as used in this application is intended to refer to the combination of any one or more acids
with LS. The acid can be either complexed to LS or can be used in the same composition or at the same time as LS.
[0025] "At the same time" in these definitions does not necessarily mean that the metal or acid and LS are to be
25 present during the complete same period of time but their presence in or on the plant part can also overlap only partially.
"In the same composition" does not necessarily mean that the two or more ingredients are to be present in one composition
before administration to the plant or plant part but that at some time during the treatment the two or more ingredients
are in contact. This can thus also mean that one ingredient is applied after the other.
[0026] Antimicrobial, in particular fungicidal compositions of the invention are even more effective when used in a
30 formulation that allows the active ingredient to remain in contact with the plant or plant part for a prolonged period of
time. It is for example in particular useful to administer the composition of the invention in a form that prevents the
composition from rolling of the leaves etc. to which they are administered. Compositions of the invention may thus further
comprise compounds which facilitate the spreading, the effectivity, stability, etc. of the compositions. Examples of such
compounds are detergents, buffers, chelators, spreading agents, preservatives.
35 [0027] The composition with anti-microbial function can be applied to plants as a solid, but can also be applied in
solution. The solution can be applied onto the crop plants using methods known to the person skilled in the art but is
preferably applied by spraying. Spraying allows for an even distribution of the composition.
[0028] Because of the excellent anti-microbial characteristics of the composition of the present invention, the compo-
sition can be used for protection of growing crop plants but can also be used for decontamination and subsequent
40 preservation and protection of plant parts, such as seeds and bulbs, against pathogenic micro-organisms.
[0029] The skilled person is very well capable of designing the most useful formulation for a particular application.
[0030] Lignosulfonate is in general complexed with Ca2+, but can also be complexed with other organic and inorganic
cations.
[0031] In one embodiment of the invention, the lignosulfonate component of the composition comprises at least in part
45 ammonium lignosulfonate and/or potassium lignosulfonate. These two cations are valuable nutrients for crop plants.
Because lignosulfonate is an ion exchange material it can be used to add these nutrients to the soil by exchanging the
ions with less desirable ions present in the soil or substrate. This way, nutrients can be easily added. Since ammonium
and potassium are thus slowly released from the top-layer a sustained-release formulation is provided to the growing
crop plants.
50 [0032] The metal-LS of the invention have also sustained-release properties when the metal is complexed to the LS.
It is within the general knowledge of the average skilled person to prepare complexed metal-LS.
[0033] Because the top-layer functions as a sustained-release matrix also other compounds can be added to the
formulation which enhance the growth of crop plants like trace elements like copper, molybdenum, boron, zinc, manga-
nese, cobalt; plant nutrients, such as nitrogen, potassium, magnesium; anti-microbial agents like carvacrol, azadirachtin,
55 and other NCP’s as mentioned in this application.
[0034] The invention further relates to methods for protecting plants and plant parts against pathogens wherein com-
positions of the invention are applied to soil, substrate, plant or plant part.
[0035] Lignosulfonate is typically a mixture of more or less degraded lignin residues of different sizes. This mixture

4
 EP 1 592 301 B1

can be fractionated and/or treated chemically. When used in this application the terms "lignosulfonate" and "lignosul-
fonates" are intended to mean both crude, untreated forms of lignosulfonate as well as more or less purified and/or
chemically modified lignosulfonate or fractions thereof.
[0036] Lignosulfonates can thus be either a mixture or isolated lignosulfonate molecules. Usually a crude mixture is
5 used, but the invention may in some applications, such as immunization, benefit from the use of pure LS. Crude mixtures
still contain 5 to 10% reducing sugars that may lead to stickiness of plant parts, such as leaves, when a solution thereof
is sprayed onto the plant or applied to the plant otherwise. Crude mixtures are however more cost-effective as they do
not require a further fractionation to remove the sugars. Lignosulfonates used according to the invention are usually Ca-
lignosulfonates or NH4-lignosulfonates.
10 [0037] As used in this application the term "active ingredient" is intended to mean any ingredient that contributes to
the function of the composition.
[0038] The invention will be further exemplified with reference to the following figures and examples. However it should
be understood that these figures and examples are not intended to limit the invention in any possible way.
[0039] The figures show:
15
Figure 1: Effect of CaLS and natamycin on development of Botrytis elliptica on lily leaf tips. At the left: control (no
treatment); in the middle: treatment with formulation; at the right: treatment with formulated natamycin. The formu-
lation contained 0.5 % (v/v) CaLS, 0.08 % EDTA and 0.007% NU-FILM-17 (a non-ionic sticker/spreader; Miller).
Figure 2: Effect of TiLS, CaLS and natamycin on development of Botrytis elliptica on lily leaf tips. Upper panel:
20 effect of TiLS and CaLS. Lower panel: effect of (combinations of) natamycin and TiLS.
Figure 3: Effect of Titanium-LS on growth of Botrytis on petri dishes with nutrient broth growing medium.
Figure 4: Effect of Silver-LS on growth of Botrytis on petri dishes with nutrient broth growing medium.
Figure 5: Petri-dishes showing the effect of different combinations of metal-LS and/or preservatives on the growh
of Botrytis cinerea.
25 Figure 5A: top row shows the effects after 3 days of water control treatment and bronopol treatments; bottom row
shows the effects after 3 days of water control treatment and sodiummethylparabenzoic acid treatments.
Figure 5B: From left to right: effects of water, 1 g/l copper-LS (Cu-LS), 1.5 g/l cobalt-LS (Co-LS) and the combination
of 1 g/l Cu-LS and 1.5 g/l Co-LS after 3 days.
Figure 5C: From left to right: effects of water, 5 g/l copper-LS (Cu-LS), 5 g/l zinc-LS (Zn-LS) and the combination
30 of 5 g/l Cu-LS and 5 g/l Zn-LS after 3 days.
Figure 5D: From left to right: effects of water, 5 g/l copper-LS (Cu-LS), 0.5 g/l formic acid-LS (Formic-LS) and the
combination of 5 g/l Cu-LS and 0.5 g/l Formic-LS after 3 days.
Figure 5E: From left to right: effects of water, 5 g/l zinc-LS (Zn-LS), 0.5 g/l formic acid-LS (Formic-LS) and the
combination of 5 g/l Cu-LS and 0.5 g/l Formic-LS after 3 days.
35 Figure 5F: Upper panel from left to right: effects of water, 5 g/l copper-LS (Cu-LS), and 5 g/l zinc-LS (Zn-LS) after
7 days; bottom row from left to right: effects of 7.5 g/l cobalt-LS (Co-LS), the combination of 5 g/l Cu-LS and 7.5 g/l
Co-LS, and the combination of 5 g/l Zn-LS and 7.5 g/l Co-LS after 7 days.
Figure 5G: Upper panel from left to right: effects of water, 5 g/l copper-LS (Cu-LS), and 5 g/l zinc-LS (Zn-LS) after
7 days; bottom row from left to right: effects of 7.5 g/l aluminium-LS (Al-LS), the combination of 5 g/l Cu-LS and 7.5
40 g/l Al-LS, and the combination of 5 g/l Zn-LS and 7.5 g/l Al-LS after 7 days.
Figure 5H: Upper panel from left to right: effects of water, and 5 g/l copper-LS (Cu-LS) after 7 days; bottom row
from left to right: effects of 0.25 g/l sodiummethylparabenzoic acid (Paraben), and the combination of 5 g/l Cu-LS
and 0.25 g/l Paraben after 7 days.
Figure 5I: Upper panel from left to right: effects of water, 5 g/l copper-LS (Cu-LS), 5 g/l zinc-LS (Zn-LS), and the
45 combination of 5 g/l Cu-LS and 5 g/l Zn-LS after 7 days; bottom row from left to right: effects of 0.5 g/l formic acid-
LS (Formic-LS), the combination of 0.5 g/l Formic-LS and.5 g/l Cu-LS, and the combination of 5 g/l Zn-LS and 0.5
g/l Formic-LS after 7 days.
Figure 6: Effects of different treatments of Phytophthora infestans leaf infections (expressed as percentage of total
leaves) in potato. Three days after spraying with the different LS-compounds the potato plants were inoculated with
50 Phytophthora. One week after inoculation the effects were monitored. The upper panel shows the effects of different
metal-LS compounds compared to the effect of a sub-optimal concentration of the synthetic fungicide Shirlan®. The
lower panel shows the effect of three concentrations of copper-LS (CuLS) in combination with three concentrations
of Shirlan®.
Figure 7: Effects of (various combinations of) formulated natamycine and the hurdle-product (see text of Example
55 11) on tulip bulbs infected with Fusarium (see Example 10 for inoculation procedure) in comparison to the effects
of control (untreated) and of treatments with 0.5 % formaldehyde.

5
 EP 1 592 301 B1

EXAMPLES

EXAMPLE 1 (comparative example, not forming part of the invention)

5 Protection of Solanum niger plants from stress induced by low dosages of herbicides

[0040] In an experiment Solanum niger plants were grown for 6 weeks after sowing. The plants were then sprayed
with a sub-optimal level of herbicide (either with 6% of the recommended dosage of Round up, containing the active
compound glyphosate, or with 10 % of the dosage recommended on the label of 2,4 D, respectively). Groups of plants
10 were sprayed with a mixture of the low dosage of the herbicide and different concentrations and types of LS (see table).
[0041] The results from table 1 show that LS induces stress tolerance to the low dosages of herbicides.

Table 1
Treatment Round up 2,4-D Percentual increase in dry weight compared to
15
(glyphosate) low herbicide application
Dry weight (g) 6 weeks after Round up 2,4-D
treatment
control untreated 2.4 g 2.4 g
20
normal herbicide dosage plants died plants
died
low herbicide dosage 1.15 g 1.05 g
low herbicide dosage + 2g/l 1.9 g 1.85 g 65% 76%
25
calcium-LS
low herbicide dosage + 10g/l 1.6 1.3 39% 24%
calcium-LS
low herbicide dosage + 25g/l 2.35 1.1 104% 5%
30
calcium-LS
low herbicide dosage + 10g/l 2.05 1.25 78% 19%
iron-LS

35
EXAMPLE 2 (comparative example, not forming part of the invention)

Effect of NH4-LS and a NCP, carvacrol, on infection (lesions) of Botrytis elliptica on lily leaf tops

[0042] In all incubations the amount of NH4-LS was 5 g/l (the amount of carvacrol is given in the table).
40
[0043] In a test system, leaf tops of lily were used. For infection with pathogens, leaf tops were placed into special
square plastic trays (10 cm x 10 cm x 2 cm) which were divided in 25 small sections of 2 x 2 cm (see figure 1). At the
start of the experiments the trays were filled with water (4 ml per small section). Then the leaf tops were placed in the
trays and finally the leaf tops were sprayed with the different LS-NCP combinations. In the table the combination of LS
with carvacrol in a formulation of di-1-p-menthene and EDTA is given.
45
[0044] Twenty-four hours after treatment the leaf tops in the trays were infected with 2 ?l of Botrytis elliptica spore
suspension (approximately 500 spores/?l, see below).
[0045] Subsequently, the plastic trays were put in a transparent container with high humidity. This container was placed
in a temperature and humidity controlled greenhouse (12 hours of light, 20°C, and 400-600 ppm of CO2). Each tray
contained 15 leaf tops and all experiments were performed in triplicate.
50
[0046] For Botrytis spore production, the fungus was grown on 25 ml sterile solid medium containing liquid broth. The
spores were applied to the leaves in Gamborg B5 medium (Gamborg 3.16 g/l, Na-phosphate 10 mM pH=6.5, sucrose
10 Mm). After 3, 5 and 7 days incubation the size (mm) of the Botrytis lesions was measured.
[0047] Table 2 shows the results.
55

6
 EP 1 592 301 B1

Table 2
Treatment Lesion size (mm) after 3 Lesion size (mm) after 5 Lesion size (mm) after 7
days days days
5 Control (untreated) 6.8 8.6 14.2
Control formulation 5.3 7.5 12.5
5g/l NH4-LS/ 0.2% 0.9 1.8 2.7
carvacrol
10
5 g/l NH4-LS/ 0.8% 0.2 0.3 0.3
carvacrol
5 g/l NH4-LS/ 1.6% 0.1 0.5 1.2
carvacrol
15

EXAMPLE 3

Effect of LS and natamycin on development of Botrytis on lily leaf tips

20 [0048] The same method was used as in Example 2. The leaf tops in the left tray were treated with water, the leaf tips
in the middle were treated with the LS formulation without natamycin and the leaf tips in the incubator on the right were
treated with LS-natamycin combination.
[0049] Figure 1 demonstrates that natamycin formulated with LS protects very well against Botrytis. Natamycin-LS
also protected against other fungi. Treatment with natamycin alone did provide adequate protection but also resulted in
25 some leaf damage. The combination with LS is thus better.

EXAMPLE 4

Effect of Titanium-LS on growth of Botrytis on lily tips

30

[0050] The experiment was performed as described in Example 2 with natamycin (2 g/l) and titanium-LS (0.2 g/l and
1 g/l). The results are shown in Figure 2. The combination of natamycin and titanium-LS in an amount of 1 g/l leads to
a complete absence of lesions.

35 EXAMPLE 5 (comparative example, not forming part of the invention)

Effect of Titanium-LS on growth of Botrytis on petri dishes with nutrient broth growing medium

[0051] Botrytis spores were incubated on sterilized growing medium containing different concentrations of Ti-LS. The
40 spores were placed on the middle of the petri dish and the infection size (diameter of the Botrytis-colony) was measured
after 5 days. Figure 3 shows the results.
[0052] The outcome of the experiment was that at a concentration lower than 1.6 g/l of Ti-LS development of Botrytis
was already completely blocked.

45 EXAMPLE 6 (comparative example, not forming part of the invention)

Effect of Silver-LS on growth of Botrytis on petri dishes with nutrient broth growing medium

[0053] Botrytis spores were incubated on sterilized growing medium containing different concentrations of Ag-LS. The
50 spores were placed on the middle of the petri dish and the infection size (diameter of the Botrytis colony) was measured
after 5 days.
[0054] Figure 4 shows that at a concentration of lower than 1 g/l of Ag-LS development of Botrytis was completely
blocked.

55

7
 EP 1 592 301 B1

EXAMPLE 7 (comparative example, not forming part of the invention)

Effect of metal-lignosulfonates on the growth of Botrytis on petri dishes

5 [0055] The effect of different lignosulfonate compounds on development of Botrytis cinerea and Botrytis elliptica spores
incubated on growing medium containing malt-extract in vitro was tested. The malt extract was of the company Oxoid
B.V. (Haarlem, the Netherlands) and the method used was according to the directions of the manufacturer.
[0056] The petri dishes were incubated at 20°C for 3 days in the dark. Per petri dish 700 Botrytis cinerea and 600
Botrytis elliptica spores were incubated. The diameter of the fungus colony on the petri dish is given in table 3.
10
Table 3
Compound Diameter (mm)
B. cinerea B. elliptica
15 water (reference) 21 13
CaCl2* (g/l) (reference)
0.006 21 14
0.028 20 13
20
0.138 21 12
0.686 21 12
1.375 21 12
25
Ca-LS (g/l)
0.04 21 13
0.2 20 13

30
1 20 11
5 15 9
10 15 8
Zn-LS (g/l)
35
0.04 20 14
0.2 20 13
1 17 12

40 5 8 5
10 0 1
Cu-LS (g/l)
0.04 22 14
45
0.2 20 15
1 18 12
5 13 7

50 10 4 0
Co-LS (g/l)
0.04 20 13
0.2 20 13
55
1 13 10
5 0 2

8
 EP 1 592 301 B1

(continued)

Co-LS (g/l)
10 0 1
5
Al-LS (g/l)
0.04 22 13
0.2 20 13
10 1 19 12
5 16 10
10 0 1
formic acid/propionic acid-LS (g/l)
15
0.04 20 13
0.2 17 12
1 0 0
20 5 0 0
10 0 0
Na-methyl-paraben (g/l) (reference)
1 0 0
25
5 0 0
25 0 0
Bronopol (mg/l) (reference)
30
2 20 12
10 20 12
50 14 10

35
*The Ca concentration in CaCl2 is equal to the Ca concentration in Ca-LS: the proportion of calcium in Ca-LS was
approximately 5%.
Ca-LS = calcium lignosulfonate; CaCl2 = calcium chloride; Zn-LS = zinc lignosulfonate; Cu-LS = copper lignosulfonate;
Co-LS = cobalt lignosulfonate; Al-LS = aluminum lignosulfonate; formic acid/propionic acid LS = 34% formic acid +
7% propionic acid + 30% lignosulfonate + 29% water; Na-methylparaben = sodium methyl para benzoic acid; Bronopol
40
= 2-bromo-2-nitro-1,3-propane diol.

[0057] The results show that the metal-LS compounds inhibit growth of both Botrytis cinerea and Botrytis elliptica.

EXAMPLE 8 (comparative example, not forming part of the invention)

45
Effect of combinations of metal-lianosulfonates on the growth of Botrytis on petri dishes

[0058] The same experiment as described in Example 7 was performed with combinations of metal- or acid-lignosul-
fonates with each other or with known fungicides. Figure 5 shows the results after 3 (Figures 5A-E) and 7 days (Figures
50 5F-I).
[0059] It follows from this figure that combinations of two metal-LS or a metal-LS with a known fungicide or with an
acid-LS can completely abolish growth of Botrytis cinerea in vitro.

EXAMPLE 9 (comparative example, not forming part of the invention)

55
Metal lignosulfonate for controlling Phytophthora infestans in potato

[0060] Five potato plants were treated with the following lignosulfonate solutions in water:

9
 EP 1 592 301 B1

1) 1 g/l calcium lignosulfonate (CaLS) (reference);

2) 1 g/l aluminium lignosulfonate (AlLS) ;
3) 1 g/l copper lignosulfonate (CuLS);
3a) 2.5 g/l copper lignosulfonate (CuLS);
5 4) 1 g/l titanium lignosulfonate (TiLS);

Three days after treatment the 5-10 leaves of each plant were inoculated at five locations with Phytophthora infestans.
One week later the plants were evaluated. The results are found in table 4.

10 Table 4
No. metal-LS (g/l) % infection
control - 98.8
1 (reference) CaLS (1) 98.8
15
2 AlLS (1) 81.3
3 CuLS (1) 12.5
3a CuLS (2.5) 5.6
20 4 TiLS (1) 100

It follows that CuLS alone significantly lowers the infection and is thus active on its own as a fungicide.
[0061] The effect of these lignosulfonates on the activity of the commercial fungicide Shirlan® (also known as fluazinam
25
or 2,6-dinitroaniline, or 3-chloro-N-[3-chloro-2,6-dinitro-4-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2-pyridinamine; ob-
tainable from Syngenta) was also tested. The experiment was performed as described above with the same metal
lignosulfonates in the same concentration and in addition 0.0032 l/ha or 0.016 l/ha Shirlan®. The results are shown in
table 5 and Figure 6.

Table 5
30
No. metal-LS (g/l) % infection
0 l/ha Shirlan® 0.0032 1/ha Shirlan® 0.016 l/ha Shirlan®
control - 98.1 93.1 64.5
35 1 (reference) CaLS (1) 98.8 98.8 62.9
2 AlLS (1) 81.3 66.3 32.5
3 CuLS (1) 12.5 13.8 3.8
3a CuLS (2.5) 5.6 6.3 2.5
40
4 TiLS 100.0 85.0 40.0

It follows from table 5 and Figure 6 that the metal-LS significantly enhances the fungicidal activity of Shirlan®, which is
45
a clear synergistic effect.

EXAMPLE 10 (comparative example, not forming part of the invention)

Use of acid-LS in protection against Fusarium in tulip bulbs

50
[0062] A composition containing 30% formic acid, 6% propionic acid, 20% LS and 44% water was used to treat tulip
bulbs infected with Fusarium.
[0063] Five pots were filled with potting soil and 10 bulbs of the tulip cultivar Prominence, size 12/13. The bulbs were
inoculated with Fusarium by a 15 minute dip in a solution heavily infected with Fusarium.
55
[0064] The results of the test are summarized in table 6.

10
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Table 6
composition infection plant status after 1 % bloom after 6 % Fusarium on bulb
month* weeks
5 heavy light total
infection infection
not treated not infected 7.2 78 30 54 84**
not treated infected 3.4 0 100 0 100
10 0.5% formaldehyde infected 10 100 0 22 22
(reference)
1% composition of the infected 9.4 96 8 42 50
invention
15 * plant status varies from 0 (bad) to 10 (good)
** Fusarium infection could be naturally occurring or induced by dipping the bulb in a Fusarium solution

[0065] No phytotoxic symptoms were found during culture. The composition of the invention is very effective in treating
20
Fusarium.

EXAMPLE 11

Use of Cu-LS in combination with natamycin in protection against Fusarium on tulip bulbs
25
[0066] The experiment was performed as described in Example 10 but with different treatments as summarized in
table 7.

Table 7
30 Number Treatment
1 (control) water
2 (reference) 55 ppm natamycin in formulation
3 (reference) 110 ppm natamycin in formulation
35
4 (reference) 220 ppm natamycin in formulation
6 55 ppm natamycin in formulation + 50% hurdle*
7 110 ppm natamycin in formulation + 50% hurdle*
40 8 220 ppm natamycin in formulation + 50% hurdle*
11 110 ppm natamycin in formulation + 100% hurdle*
15 formaldehyde
*100% Hurdle = Cu-LS (5g LS + 0.25g Cu per liter) + 15 ml/l 5-chloro-2-methyl-4-isothiazolin-3-one/2-methyl-4-
45
isothiazolin-3-one (CIT/MIT in a 3:1 ratio) + 50 mg/l bronopol.

[0067] The results are shown in Figure 7. Tulips that were treated with natamycin alone show some yellow spots on
the leaves and along the leaf edges indicating that the bulbs are infected. Leaves of tulips treated with natamycin and
50 Cu-LS did not show such spots.

Claims

55 1. Composition for protecting an agricultural crop against biotic stress caused by microorganisms, in particular fungi,
which composition comprises one or more lignosulfonates and natamycin.

2. Composition according to claim 1, wherein the one or more lignosulfonates are metal-lignosulfonates.

11
 EP 1 592 301 B1

3. Composition according to claim 2, wherein the metal-lignosulfonates are selected from the group consisting of
titanium lignosulfonates, copper lignosulfonates, cobalt lignosulfonates, zinc lignosulfonates, aluminium lignosul-
fonates, manganese lignosulfonates and silver lignosulfonates.

5 4. Composition according to any of the claims 2 to 3, wherein the metallignosulfonates are copper-lignosulfonate.

5. Composition as claimed in any one of the claims 1-4, wherein the composition is in a solid form, in particular a
powder, flakes, granules, pellets, wettable powder.

10 6. Method for protecting plants or plant parts against biotic stress which method comprises applying a composition
according to claims 1-5 to plants or plant parts.

Patentansprüche
15
1. Zusammensetzung zum Schutz einer landwirtschaftlichen Kulturpflanze gegen von Mikroorganismen, insbesondere
von Pilzen, verursachten biotischen Stress, wobei die Zusammensetzung ein oder mehrere Ligninsulfonate und
Natamycin umfasst.

20 2. Zusammensetzung nach Anspruch 1, wobei einoder mehrere Ligninsulfonate Metall-Ligninsulfonate sind.

3. Zusammensetzung nach Anspruch 2, wobei die Metall-Ligninsulfonate ausgewählt sind aus der Gruppe bestehend
aus Titan-Ligninsulfonaten, Kupfer-Ligninsulfonaten, Kobalt-Ligninsulfonaten, Zink-Ligninsulfonaten, Aluminium-
Ligninsulfonaten, Mangan-Ligninsulfonaten und Silber-Ligninsulfonaten.
25
4. Zuasmmensetzung nach einem der Ansprüche 2 bis 3, wobei die Metall-Ligninsulfonate Kupfer-Ligninsulfonate sind.

5. Zusammensetzung nach einem der Ansprüche 1-4, wobei die Zusammensetzung in fester Form, insbesondere als
Pulver, Flocken, Granulat, Pellets und benetzbares Pulver vorliegt.
30
6. Verfahren zum Schutz von Pflanzen oder Pflanzenteilen gegen biotischen Stress, wobei das Verfahren die Anwen-
dung der Zusammensetzung nach den Ansprüchen 1 - 5 auf Pflanzen oder Pflanzenteile umfasst.

35 Revendications

1. Composition pour protéger une récolte agricole contre le stress biotique provoqué par des micro-organismes, en
particulier les champignons, laquelle composition comprend un ou plusieurs lignosulfonates et de la natamycine.

40 2. Composition selon la revendication 1, dans laquelle les un ou plusieurs lignosulfonates consistent en lignosulfonates
de métaux.

3. Composition selon la revendication 2, dans laquelle les lignosulfonates de métal comprennent les lignosulfonates
de titane, les lignosulfonates de cuivre, les lignosulfonates de cobalt, les lignosulfonates de zinc, les lignosulfonates
45 d’aluminium, les lignosulfonates de manganèse et les lignosulfonates d’argent.

4. Composition selon l’une quelconque des revendications 2 ou 3, dans laquelle les lignosulfonates de métal consistent
en lignosulfonates de cuivre.

50 5. Composition telle que revendiquée dans l’une quelconque des revendications 1-4, dans laquelle la composition est
sous une forme solide, en particulier de poudre, de flocons, de granulés, de pellets, de poudre mouillable.

6. Procédé de protection des plantes ou de parties de plantes contre le stress biotique, laquelle méthode comprend
l’application aux plantes ou parties de plantes d’une composition selon les revendications 1 à 5.
55

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