Patent Application
20120129938
The present invention relates to the use of the compounds of the general formula (I) or of a salt thereof as antioomycotic, and to a method for controlling plant pathogens in which these compounds are used.
The class of the oomycetes or Peronosporomycetes (formally referred to as Oomycota or Oomycetes), which do not belong to the fungi (Fungi), comprises a varied group of saprophytic and pathogenic species. The latter include not only species which infect animals or microorganisms, but also devastating plant pathogens. Important plant pathogens are found in the genera Albugo, Bremia, Plasmopara, Peronospora and Phytophthora. These obligat-pathogenic species cause, for example, diseases such as white rust or downy mildew in a range of different plants. Within the genus Phytophthora, there are described more than 60 different species which infect predominantly dicotyledonous plants. Many of them are highly adapted to a specific host, or to a few hosts, while others are capable of colonizing many different plants.
Phytophthora infestans, the causative organism of late blight of tomato, or late blight of potato, is considered the most destructive plant pathogen worldwide. Infections are difficult to control and can lead to total yield losses since the life cycle of P. infestans only takes a few days.
Most traditional fungicides are ineffective against P. infestans and other oomycetes. The reason therefor is the lack of typical fungal target structures for the activity of many fungicides in the Peronosporomycetes.
The present invention is therefore based on the object of providing novel effective agents against plant pathogens, in particulari Peronosporomycetes.
This object is achieved by the embodiments of the present invention which are characterized in the claims.
According to the invention, there is provided in particular the use of compounds of the general formula (I) as antioomycotic, and a method of controlling plant pathogens in which these compounds are used.
Accordingly, one subject matter of the present invention relates to the use of a compound of the formula (I) or a salt thereof as antioomycotic:
in which
X is selected from among H, OR1, SR1, NR1R2+, N(OR1)(R2), N(R1)—NR1NR2 or N (R1R2R3)+A−,
Y is selected from among OR1, O−Cat+ or NR1R2,
Z is selected from among O, S, NR1, NOR1, N—CN or N—NR1R2,
R represents a substituent selected from the group consisting of (i) an unsubstituted or mono- or polysubstituted (C3-C22)-alkyl radical, (ii) an unsubstituted or mono- or polysubstituted (C3-C22)-alkenyl radical, (iii) an unsubstituted or mono- or polysubstituted (C3-C22)-alkynyl radical, (iv) an unsubstituted or mono- or polysubstituted —(CH2)m-spermine radical, (v) an unsubstituted or mono- or polysubstituted —(CH2)m-spermidine radical, (vi) an unsubstituted or mono- or polysubstituted N-methylated —(CH2)m-sperm(id)ine radical, where m is in each case an integer from 1 to 4 and where the one or the plurality of substituents in the abovementioned radicals (i) to (vi) can be selected independently of one another from among group α, consisting of a (C1-C6)-alkyl radical, a (C1-C6)-thioalkyl radical, a (C3-C7)-cycloalkyl radical which can have one or more hetero atoms such as, for example, O or S, a (C1-C6)-alkoxy radical, a hydroxyl group, a trifluoromethyl group, a triazole group, bromine, chlorine, fluorine, an unsubstituted, mono- or disubstituted phenyl, phenoxy, benzyl, benzyloxy, naphthyl or naphthoxy radical, and (vii) an ethyleneoxy group selected from among:
—CH2[OCH2CH2]n—OH,
—CH2[OCH2CH2]n—OMe,
—CH2—CH2—[OCH2CH2]n—OH
—CH2—CH2—[OCH2CH2]n—OMe
—CH2—CH2—CH2—[OCH2CH2]n—OH
—CH2—CH2—CH2—[OCH2CH2]n—OMe
—CH2—CH2—CH2—CH2—[OCH2CH2]n—OH or
—CH2—CH2—CH2—CH2—[OCH2CH2]n—OMe,
where n=1-20, preferably n=1-5,
R1, R2 and R3 independently of one another are selected from among hydrogen, a (C1-C6)-acyl radical, —CONH2, —(CO)—(CH2)0-6—COOH, a lactyl radical, a (C1-C6)-alkyl radical, a (C3-C7)-cycloalkyl radical which can have one or more hetero atoms such as, for example, O or S, an unsubstituted, mono- or disubstituted phenyl, benzyl or naphthyl radical whose substituents can be selected from among group α,
A− represents an anion selected from among halide, chlorate or carboxylate,
Cat+ represents a cation, in particular monovalent or divalent cations such as, for example, alkali metal cations (Na+, K+), alkaline-earth metal cations (Ca2+, Mg2+) or quaternary ammonium cations,
the stereoisomeric center at C5, if present, is present in the R or S form or as a racemate, and
the C2-C3 double bond is present in the E or Z form, preferably the E form (trans).
A further subject matter of the present invention relates to a method of controlling plant pathogens, comprising the application of an effective amount of one of the above-defined compounds or of a salt thereof to a plant, a part of the plant or the soil in which the plant grows. The method according to the invention can be used both preventatively and curatively.
In this context, the expression “plant pathogens” comprises all phytopathogenic fungi, protists, bacteria and viruses.
In a preferred embodiment of the present invention, the plant pathogens are fungi (Fungi). Especially preferred plant pathogens in this context are Colletotrichum coccodes, Colletotrichum graminicola, Septoria tritici, Fusarium graminearum, Blumeria graminis, Magnaporthe grisea, Ustilago maydis, Alternaria solani, Cladosporium fulvum, Cochliobolus heterostrophus, Pyrenophora tritici-repentis, Verticillium albo-atrum and Verticillium dahliae.
In a further preferred embodiment of the present invention, the plant pathogens are oomycetes (Peronosporomycetes, formally referred to as Oomycota or Oomycetes). Especially preferred in this context are oomycetes from among the genera Albugo, Bremia, Plasmopara, Peronospora and Phytophthora. An especially preferred plant pathogen from the genus Peronospora is Peronospora manshurica. Especially preferred plant pathogens from among the genus Phytophthora are Phytophthora sojae, Phytophthora palmivora, Phytophthora ramorum, Phytophthora cinnamomi, Phytophthora capsici and Phytophthora infestans. Very especially preferred in this context is Phytophthora infestans.
In a preferred embodiment of the present invention, the plants which are protected or treated with the method according to the invention are from the group consisting of the Fabaceae, in particular Glycine max; the Cucurbitaceae, in particular Cucurbita spp. such as Cucurbita pepo, Cucumis spp. such as Cucumis melo and Cucumis sativus, and Citrullus spp. such as Citrullus lanatus; the Brassicaceae, in particular Brassica spp. such as Brassica napus, Brassica oleracea and Brassica rapa; the Poaceae, in particular Triticum spp., Hordeum spp., Oryza sativa and Zea mays; the Solanaceae, in particular Nicotiana spp. such as Nicotiana tabacum, Capsicum spp. such as Capsicum annuum, and Solanum spp. such as Solanum tuberosum, Solanum lycopersicum and Solanum melongena; Vitis spp. such as Vitis vinifera; Beta vulgaris; and Theobroma cacao. Very especially preferred in this context is Solanum tuberosum.
Furthermore, the method according to the invention can be used for protecting or treating trees, in particular Coniferae, in particular Pinaceae, and ornamentals, in particular ornamental plants.
The concentration of the compound or of the salt thereof in the method according to the invention ranges from 1 nM to 10 nM, preferably from 10 nM to 1 mM. Especially preferred is a concentration of 100 μM.
Methods of applying, to a plant or parts of the plant, an effective amount of one of the above-defined compounds or a salt thereof are known to those skilled in the art and comprise, for example, spraying, atomizing, painting or dipping the plant.
In an especially preferred embodiment of the method according to the invention, the compounds according to the invention are present in the form of a mixture in combination with a carrier, in which mixture the active compound is present in an amount of between 0.1 and 99% by weight, preferably between 1 and 75% by weight, based on the mixture. Mixtures in combination with a carrier, for the direct use or application to the field, comprise the compounds according to the invention in an amount of between 0.0001 and 5% by weight, preferably between 0.001 and 3% by weight, based on the mixture. The method according to the invention comprises the use of formulations and compositions, which comprise mixtures of a dispersible carrier, such as a dispersible inert finely-divided solid carrier and/or a dispersible liquid carrier, such as an inert organic solvent and/or water, preferably with the inclusion of an effective amount of a surface-active carrier adjuvant and an amount of the active compounds according to the invention of between 0.0001 and 99% by weight, preferably between 0.001 and 90% by weight, preferably between 0.1 and 75% by weight. The active compounds according to the invention can be applied by customarily used methods, for example as hydraulic sprays of large amounts of liquid, sprays with low amounts of liquid, ultra-low-volume sprays, by high-pressure liquid injection, slit injection, blast-air spray, air spray or dust.
A preferred embodiment of the present invention relates to the use of a compound of the formula (I) or a salt thereof as antioomycotic, where, in formula (I),
X is selected from among H, OR1, NR1R2, N(OR1)(Ra), N(R1)—NR1R2 or N (R1r2R3)+A ,
Y is selected from among OR1 or O−Cat+,
Z is selected from O,
R represents a substituent selected from the group consisting of an unsubstituted or mono- or polysubstituted (C3-C22)-alkyl radical, preferably (C7-C12)-alkyl radical, an unsubstituted or mono- or polysubstituted (C3-C22)-alkenyl radical, —CH2[OCH2CH2]n—OH or —CH2[OCH2CH2]n—OMe, where n=1-20, preferably n=1-5,
R1, R2 and R3 independently of one another are selected from among hydrogen, a (C1-C6)-acyl radical or a (C1-C6)-alkyl radical and
the C2-C3 double bond is present in the E form.
If the radical R represents a (C3-C22)-alkenyl radical, then one to three double bonds can preferably be present. The double bonds can be present in the E or in the Z form. In particular, it is possible for oligoprenyl radicals to be present, which, in turn, can optionally be substituted by one or more substituents from among group α. Examples which may be mentioned in this context are geranyl, neryl, farnesyl and geranylgeranyl.
An especially preferred embodiment of the present invention relates to the use of (E)-4-oxohexadec-2-enoic acid (formula (II)) or salts thereof, in particular alkali metal salts, as antioomycotic.
A further subject matter of the present invention relates to the use of one of the compounds according to the invention or a salt thereof as disinfectant for agricultural and/or horticultural machinery.
The figures show:
The present invention is illustrated in greater detail with reference to the following nonlimiting examples.
Materials and Methods:
General aspects. All reagents and solvents were analytical-grade or were purified with the aid of standard methods. The melting points were determined via standard methods using hotstage microscopy (Leica DM LS2) and not corrected. The reactions were monitored by means of thin-layer chromatography on silica gel 60 F254 (Merck, 0.040-0.063 mm) and detected using UV light or molybdatophosphoric acid. The solutions were concentrated under reduced pressure at 40° C. The column chromatography was performed on silica gel 60 (Merck, 0.063-0.200 mm). The 1H (300 or 400 MHz) and 13C (75.5 or 100.5 MHz) NMR spectra were recorded at room temperature (RT) using VARIAN Mercury spectrometers. For 2-dodecylfuran and (E)-4-oxohexadec-2-enal, the chemical shifts were referenced to internal TMS (δ=0 ppm, 1H) or CDCl3 (δ=77.0 ppm, 13C). Deuterated ethanol was used as the solvent for (E)-4-oxohexadec-2-enoic acid. The chemical shifts were referenced to the signals of the internal solvent methyl groups (δ=1.11 ppm, 1H, or δ=17.2 ppm, 13C). Positive and negative ESI and APCI mass spectra were obtained by an API 150Ex (Applied Biosystems) equipped with a turbo ion source. Highly-resolved positive and negative ESI mass spectra were obtained by a Bruker Apex 70e FT-ICR mass spectrometer (Bruker Daltonics) which was equipped with an Infinity™ cell, a 7.0 Tesla superconducting magnet (Bruker) an rf-only hexapole ion guide and an external electrospray ion source (Agilent). P. infestans isolate 208 m2 was grown on oat-bean medium (3.4% by weight bean flour, 1.7% by weight oat flour, 0.85% by weight sucrose, 1.5% by weight Bacto-Agar, 5 μg/ml geneticin). Measurements of GFP-emitted light were performed with the aid of a Cytofluor II plate reader (Millipore; excitation 485 nm, emission 530 nm). GFP fluorescence images were recorded using a Leica MZ FLIII fluorescence stereomicroscope (Leica Microsystems). An MRX Plate Reader 1.12 (Dynatech Laboratories) was used for measuring the P. infestans biomass. Quantitative PCR was performed as described.
Reagents and solvents. Tetrahydrofuran (THF), n-butyllithium (n-BuLi), furan, sodium hydroxide, HCl, pyridine, N-bromosuccinimide (NBS), 2-methyl-2-butene, NaClO2 and dodecyl bromide were obtained from conventional laboratory suppliers.
Synthesis of the Compounds
2-Dodecylfuran (
(E)-4-Oxohexadec-2-enal (
(E)-4-Oxohexadec-2-enoic acid (
Sodium (E)-4-oxohexadec-2-enoate (
P. infestans culture conditions. The isolate 208 m2, which harbors a GFP construct, was used for P. infestans experiments. Zoospoe solutions were prepared by growing P. infestans for 11 days on oat-bean medium at 18° C. in the dark. The mycelium was then flooded with 10 ml of deionized water, left to stand for 4 h at 4° C. to allow the release of the zoospores, and the liquid was then filtered through a layer of gauze to remove pieces of mycelium and sporangia. The solution was adjusted to 1×105 spores/ml. Sporangia solutions were prepared by flooding mycelium which had been grown for days with 10 ml of deionized water, immediate vigorous shaking to break the sporangia from the sporangiophores and adjusting the solution to 1×104 sporangia/ml.
P. infestans bioassays and infections experiments. Spore germination experiments with P. infestans zoosporous solutions were carried out as follows. A dilution series of (E)-4-oxohexadec-2-enoic acid dissolved in 96% strength ethanol was used. The final concentrations in the spore solutions ranged from 10 nM to 100 μM and in each case 2% (v/v) 96% EtOH. Control treatments with 2% (v/v) 96% EtOH only were also carried out. After the treatment, the spores were kept at 4° C. overnight to allow germination. The percentage of germinated spores was calculated after the spores were counted on photographs taken of five nonoverlapping regions of a 10 μl drop in a hematocytometer under a light microscope. The spores were considered to be germinated when the germ tube was at least as long as the spore diameter.
The effect of (E)-4-oxohexadec-2-enoic acid on the mycelial growth of P. infestans was tested by measuring the increase in the GFP fluorescence over time. 24-well microtiter plates (Nunc A/S, Denmark) containing oat-bean medium were inoculated with 100 μl of a P. infestans sporangia solution and grown at 17° C. in the dark. After 24 h, various concentrations of (E)-4-oxohexadec-2-enoic acid were added. The final concentrations (calculated for 100 μl of sporangia solution) ranged from 10 nM to 1 mM and 1% ethanol. The growth of P. infestans was determined by measuring GFP-emitted light (excitation 485 nm, emission 530 nm).
To study the direct effect of (E)-4-oxohexadec-2-enoic acid on live P. infestans mycelium, three-week old mycelium was inoculated dropwise (10 μl) with various concentrations of (E)-4-oxohexadec-2-enoic acid. 24 h later, GFP fluorescence images of inoculated zones were recorded.
Potato plants (Solanum tuberosum L. cv. Désirée) were grown as described. Before an inoculation with a P. infestans zoospore solution, the plants were sprayed to run-off point on the abaxial leaf surface with various concentrations of sodium (E)-4-oxohexadec-2-enoate, dissolved in water. After two hours, when the sprayed leaves had dried, P. infestans was inoculated onto the abaxial leaf surface (six 10 μl drops per leaf; 1×105 spores/ml; two leaves per plant). The inoculated leaves were then covered with plastic bags to ensure 100% relative atmospheric humidity for spore germination. After three days, the inoculation sites were excised using a cork punch, and all leaf disks of a specific leaf were combined to give one sample. Determinations of the P. infestans biomass were carried out with the aid of quantitative PCR using P. infestans-specific primers.
Colletotrichum coccodes bioassay. C. coccodes (CBS369.75) was grown for five days in 50 ml of liquid soybean medium in the dark at 18° C. on a rotary shaker. In order to isolate spores, all of the culture was centrifuged for 5 min at 2100 g and 4° C. The supernatant, which contained the spores, was recentrifuged (10 min, 6500 g, 4° C.). After the supernatant had been decantered off, the pelleted spores were washed carefully in deionized water, centrifuged as above and finally the spore concentration was adjusted to 1×105 spores/ml in soybean medium. To carry out the biotest, 200 μl of this spore solution were pipetted into each well of a 96-well plate (Nunc A/S, Denmark). The plates were incubated in an incubator for 24 h at 17° C. in the dark to allow germination. Then, test concentrations of (E)-4-oxohexadec-2-enoic acid were pipetted in as described above, with final concentrations of from 0.01 μM to 100 μM and 2% (v/v) ethanol. The plates were returned into the incubator and the increase in fungal biomass was determined by daily 0D590 measurements.
A rapid and efficient three-step synthesis of highly bioactive unsaturated fatty acids was developed (
To determine the effect of (E)-4-oxohexadec-2-enoic acid (
The inhibitory effect of (E)-4-oxohexadec-2-enoic acid on the mycelial growth of a GFP-expressing P. infestans was studied in a bioassay. Various concentrations were applied to mycelium growing in multiwell plates. GFP fluorescence was recorded with a plate reader to measure the mycelial growth.
The effect of (E)-4-oxohexadec-2-enoic acid on mature mycelium was also studied. To this end, test solutions were added dropwise to three-week old mycelium growing on agar plates.
Furthermore, it was studied whether the pretreatment of potato plants with sodium (E)-4-oxohexadec-2-enoate (
To determine whether (E)-4-oxohexadec-2-enoic acid also has an inhibitory effect on the ascomycete c. coccodes, the causative organism of, inter alia, black dot disease of potato, a multiwell bioassay was set up. The growth of the fungus in liquid medium was determined on the basis of the measurement of the optical density (OD590). A medium containing soya was used for this purpose. One-day old mycelium was inoculated together with various concentrations of (E)-4-oxohexadec-2-enoic acid, and the growth of C. coccodes was determined every 24 h. As shown in
Phytophthora
Phytophtora
infestans
infestans
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2009 022 619.2 | May 2009 | DE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2010/003189 | 5/26/2010 | WO | 00 | 2/6/2012 |
