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    • 专利摘要:
    Broad-spectrum natural biocides from the endophyte fungus stemphylium solani. In the field of agriculture, organisms that are harmful to vegetation are a major problem in relation to crop productivity. The present invention relates to a fermentation product obtained from endophytic fungi of the sthemphylum solani family, which may comprise a new compound of formula (I) and a second known compound of formula (II), and which has biocidal activity simultaneously against more than one category of harmful organisms that affect plants and that are preferably selected among insect-pests, fungi and nematodes. Both the fermentation product and the compounds included in the scope of the invention are used in the preparation of a broad spectrum biocidal composition. Finally, the invention also includes the process for obtaining the fermentation product and the new compound of formula (I). (Machine-translation by Google Translate, not legally binding)
    公开号:ES2610186A2
    申请号:ES201531527
    申请日:2015-10-23
    公开日:2017-04-26
    发明作者:Azucena GONZALEZ COLOMA；M. Fe ANDRES YEVES；Carmen Elisa Diaz Hernandez；Matias REINA ARTILES；Rodney LACRET PIMIENTA；Raimundo Cabrera Perez；Cristina GIMENEZ MARIÑO；Nutan Kaushik
    申请人:ENERGY AND RESOURCES INSTITUTE (TERI)；Consejo Superior de Investigaciones Cientificas CSIC；Universidad de La Laguna；ENERGY AND RESOURCES INST (TERI)；
    IPC主号:
    专利说明:

    SECTOR OF THE INVENTION
    The invention is within the field of agriculture, and more specifically, in the field of biocides comprising substances produced or extracted from microorganisms. A fermentation product with a broad spectrum biocidal activity and a method of obtaining it from endophytic fungi of the Stemphylium solani species are proposed. The fermentation product is characterized by comprising at least one new compound of formula (1), which is also claimed, and the compound of formula (11) which gives the fermentation product its characteristic biocidal activity. Finally, the invention is also
    15 refers to the use of the fermentation product, or of the compounds included in the scope of this document, either directly or through a biocidal composition comprising them and a broad spectrum method for the control of organisms harmful to plants .
    20 STATE OF THE TECHNIQUE
    Endophytic fungi are a polyphyletic group with a high degree of diversity that
    functionally characterized by its ability to inhabit plant tissues without
    cause apparent damage. Endophytic fungal colonization may contribute to
    25 adaptation of the host plant to stress situations and in many cases the tolerance of the host plant to biotic stress has been correlated with the presence of fungal natural products (Aly et al. 2010. FungaJ Diversity 41, 1-16).
    The genus Stemphylium is composed of filamentous fungi and saprophytes of the group
    30 Hypomycetes that are widely distributed throughout the world, associated with decaying vegetation. In agriculture, Stemphylium species are responsible for diseases in many crops, to which they cause foliar damage, and are dispersed through the seeds.
    P201 531527
    From the metaballs produced by the most important phytopathogenic Stemphylium species: S. botryosum, S. herbarum, S. alfalfae, S. sarciniforme, five major compounds have been identified, stemfilin, stemphyloxin 11, stemfiperilenol, stemfol and a related compound (Barash et al. 1975. Plan! Physiol. 55, 646-651; Andersen et al. 1995. Mycol. Res. 99, 672-676; Solfrizzo et al. 1994. Nat. Toxins 2, 14-18) . On the other hand, the production of taxol by a species of Stemphylium sp. isolated as an endophyte of Taxus baccata (Mirjalili et al. 2012. FEMS Microbiol. Lett. 328, t22-9).
    More specifically, S. solani has been isolated as an endophyte in Menthapulegium (Debbab et al. 2009. J. Nat. Prod. 72, 626-31), Arabidopsis thaliana (Garcia et al. 2012. FungalDiversity.
    001: 10.1 007ls13225-012-0219-0), Vitis vinífera (González and Tello. 2011. Fungal Diversity 47: 29--42) and at the Australian endemic plant Eremophilia fongifolia (Zaferanloo et al. 2013. World J Microbiol. Biotechnol 29, 335-345). From the extracts of S. solani from M. pulegium the compounds have been identified: alterporriol G and its isomer terporriol H, altersolanol, altersolanol L, stemfipirone, 6-methoxylalaternine, macrosporine, altersolanol A, alterporriol E, alterporriol D, alterporriol A, alterporriol By altersolanol J.
    Among the aforementioned compounds, it is known that the mixture of alterporriol GYH is related to cytotoxic activity in L5178Y cells, and that the compounds 6methoxyalaternine, macrosporine, altersolanol A, as well as again the mixture of alterporriol G and H, induce a strong inhibition of protein kinase activity (Debbab et al. 2009. J. Nat. Prod. 72, 626-31).
    Agriculture constantly faces serious problems related to the incidence of pests and attacks of pathogenic organisms with the consequent reduction in productivity. Although traditionally the usual method for the control of harmful organisms that affect plants has been the application of synthetic chemicals, which in addition to their high economic price implies a serious environmental cost, the new regulatory environment, see for example both nationally As European Regulation (EC) N ° 1107/2009, has drastically limited the number of active materials and the availability of phytosanitary products for the control of diseases caused by these harmful organisms.
    Within this scenario, the search for active, alternative and effective compounds at very low doses, that have little persistence in the environment, that reduce the occurrence of cross-resistance, that do not have associated cytotoxic effects and that have their origin in for example, Endophytic fungi seems to be a good alternative. Further,
    5 it would be highly desirable for these compounds to be broad spectrum, that is, to beassets against different harmful organisms simultaneously.
    EXPLANATION OF THE INVENTION
    The present invention relates in a first aspect to a fermentation product of an endophytic fungus of the Stemphylium soJani species with broad spectrum biocidal activity, characterized in that it comprises a compound of formula (1):
    or
    "4" 2 "
    3 "R
    HO ".. ·· 6 2 5 4 3 1
    ]" 3"
    HO 2 "4" S '
    15 (1) where R is selected from H and OH,
    or an isomer, or a solvate salt thereof.
    Preferably, the fermentation product can simultaneously comprise a compound of formula (1) where Res H and another compound of formula (1) where Res OH. Even more preferably, the fermentation product also comprises another compound with broad spectrum biocidal activity of formula (11):
    OH
    4 "2 '2
    5, 2 "3" 4 "
    S "25
    (eleven )
    or an isomer, or a solvate salt thereof.
    In a second aspect, the invention relates to the new compound of formula (1)
    which is included in the fermentation product.
    In a third aspect, the invention relates to a method of obtaining the5 fermentation product comprising culturing mycelium of an endophytic fungus of theStemphylium solani species in a culture medium.
    Preferably, the endophyte fungus that is used in the process of obtaining is S. salan strain Aa22; with deposit number CECT 20941.
    In a fourth aspect, the invention relates to the use of the fermentation product, or of the compounds of formulas (1) or (11), isolated or in combination, to make a broad spectrum biocidal composition.
    In a fifth aspect, the invention relates to the broad spectrum biocidal composition comprising the fermentation product, or the compounds of formulas (1) or
    (11), isolated or in combination.
    In a sixth aspect, the invention relates to the use of the biocidal composition as
    20 broad spectrum biocidal agent for the control of at least one category of harmful organisms that affect plants, and which are selected from insect pests, fungi and nematodes, preferably all of them simultaneously.
    Detailed description of the invention
    The technical problem solved by the present invention is the search for new natural biocidal products that are simultaneously effective against several harmful organisms that affect plants, and thus represent an effective alternative to synthetic chemical biocides.
    The present invention is based on the discovery of a fermentation product of an endophytic fungus of the species Slemphylium solani, which is preferably S. salan strain Aa22; with CECT 20941 and which can simultaneously comprise at least one compound of formula (1) and a compound of formula (11), which provide it with a surprising broad-spectrum biocidal activity against phytopathogenic fungi, insect pests and the phytopathogenic nematode Me / aidagyne javanica (see Examples 4 to 6).
    The main technical advantages of the fermentation product, of its use for the control of5 harmful organisms that affect plants and their procedure, arelisted below:
    - It is broad spectrum, acting simultaneously against insects, fungi and nematodes,
    10-obtained from natural sources, by simple and economical procedures, and
    - It can be obtained on a large scale by fermentation of the fungus in bioreactors, being able to manipulate the fermentation conditions to increase the production of 15 active components.
    In a first aspect, the invention relates to a fermentation product of an endophytic fungus of the Stemphylium sa / ani species, with broad spectrum biocidal activity, hereinafter the fermentation product of the invention, comprising at least one
    20 compound of formula (1):
    or
    4 "2" 3 "R
    (one)where R is selected from H and OH,
    or an isomer, a salt solvate thereof.
    By "fermentation product" is meant a product that is the result of the action of an endophytic fungus of the Stemphylum so / ani species, which when grown on a culture medium 30 has the ability to synthesize at least one compound of formula ( one). Preferably, the endophyte fungus is S. sa / ani strain Aa22 with CECT 20941. The fermentation product
    it can be a dry extract obtained by extraction with organic solvent prior filtration, or a lyophilisate.
    In a particular embodiment, the fermentation product of the invention comprises5 simultaneously a compound of formula (1) where R is H and another compound of formula (1)where R is OH, or an isomer, or a salt or solvate thereof.
    In another more particular embodiment of the above, the fermentation product of the invention comprises another compound of formula (11):
    10 OH 4 · 2 · 2
    r
    .r 4 ··
    S ""
    (eleven)
    or an isomer, or a salt or solvate thereof.
    Although both the compounds of formula (1) and formula (11) in isolation have broad spectrum biocidal activity, their combined presence within the fermentation product of the invention provides it with a manifestly improved activity (see Examples 4 to 6).
    In general, all the compounds included in the scope of the invention can include isomers, depending on the presence of multiple bonds, including optical isomers or enantiomers, depending on the presence of chiral centers. The individual isomers, enantiomers or diastereoisomers and mixtures thereof fall within the scope of the present invention, that is, the term isomer also refers to
    25 to any mixture of isomers, such as diastereomers, racemic, etc., even to their optically active isomers or mixtures in different proportions thereof. The individual enantiomers or diastereoisomers, as well as mixtures thereof, can be separated by conventional techniques.
    Also, within the scope of this invention are the acceptable salts and solvates of all the compounds that are included in the scope of the invention or of any other compound that, when applied to an organism harmful to plants, is capable of provide (directly or indirectly) a compound as described herein.
    In another particular embodiment, the fermentation product of the invention may be incrystalline form as free compounds or as solvates. Solvation methods aregenerally known within the art.
    In a second aspect, the invention relates to a compound of formula (1):
    or
    4 "2"
    "
    3 "R
    J '4'
    2 'S'
    "
    (one )
    where R is selected from H and OH,
    15 or an isomer, a solvate salt thereof, which has broad spectrum biocidal activity, hereinafter referred to as the invention, and which is comprised in the fermentation product of the invention, either alone or in combination with at least compound of formula (11) as defined herein.
    Within the scope of the present invention, the terms "compound of formula (1) where R = H" and "Stemfolone A" are used interchangeably; similarly "compound of formula
    (1) where R = OH "and" StemfoJona B "are used interchangeably; in the same way" compound of formula (11) "and" Stemfof 'are used interchangeably.
    The isolation of the compounds that are included in the scope of the invention starting from the fermentation product of the invention is carried out by fractionation techniques.
    Fractionation techniques known to any person skilled in the art, and which allow obtaining the compounds included in the scope of the invention, are for example and without limitation, liquid vacuum chromatography (VLC), column chromatography (CC ), column chromatography (CC) using different solid phases (Silica Gel,
    Sephadex LH-20) and high performance liquid chromatography (HPLC), eluting with different polarity gradients by different combinations of organic solvents.
    5 In a third aspect, the invention relates to a method of obtaining thefermentation product of the invention, hereinafter procedure for obtaining theinvention, comprising:
    a) culturing mycelium of a fungus of the Stemphylum solani species in a culture medium 10 until a fermented product is synthesized comprising at least one compound of formula (1).
    In a particular embodiment, the process of the invention allows the fermentation product of the invention to comprise a compound of formula (1) where R is H and another compound of formula (1) where R is OH, or an isomer, or One salvate a solvate of them.
    In another more particular embodiment of the foregoing, the process of the invention achieves that additionally, the fermentation product of the invention comprises the compound
    20 of formula (11).
    Not all fungi of the Stemphylum genus have the ability to produce the fermentation product of the invention. It is fundamentally the fungus type and its interaction with the host plant that confers certain characteristics that enable the production of certain compounds. For this reason, fungal strains of the Stemphylum solan species are preferred in the present invention; that due to their interaction with the host plant they have developed the characteristics necessary to produce the compounds of the invention. Preferably it is the Aa22 strain of S. solani isolated from Artemisia absinthium leaves deposited on September 25, 2015 in the Collection
    30 Spanish of Type Crops with CECT 20941, following the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of the Patent Procedure.
    The process of the invention may include an additional stage or stage b) of drying the fermentation product of the invention through extraction with organic solvent prior filtration, or through lyophilization.
    5 Examples of organic solvents that are used in the scope of the invention aredichloromethane, ethyl ether and, preferably, ethyl acetate.
    In a particular embodiment, the endophyte fungus is the Aa22 strain of StemphyJum soJani isolated from plant material of Artemisia absinthium deposited in the Spanish Collection of 10 Type Crops with CECT 20941 as of September 25, 2015, grown using a YMB.
    However, strains of S. soJani can be isolated in other plants such as, for example, Mentha pulegium, Arabidopsis thaJiana, Vitis vinífera and in Eremophilia Jongifolia.
    15 Examples of culture media that allow fermentation by fungi of the Stemphylum solani species are YMB (broth yeast extract and malt extract), the media cited in the references Molitor et al. (2012. J. Nat. Prod. 75, 1265-1269) YBuckel et al. (20 13. Phytochemistry 89, 96-103); those cited in document W02002017937
    20 A1 or any other commercial culture medium for phytopathogenic fungi.
    In a fourth aspect, the invention relates to the use of the fermentation product of the invention, or of the compound of the invention, or of a compound of formula (11) either in isolation or in combination, to make a composition with biocidal activity of
    25 broad spectrum.
    "Broad spectrum biocidal activity" means the ability to simultaneously control more than one different category of organisms harmful to plants. Said control includes the prevention of the action or the direct destruction of
    30 such organisms harmful to public health and also to agriculture during production, but also extends to the storage, transport, distribution and processing of agricultural products and their derivatives.
    Examples of categories of organisms harmful to plants include, but are not limited to, pest insects, fungi or nematodes.
    Preferably, the pest insects that are included in the scope of this invention are herbivorous pest insects with different aesthetic adaptations, be they chewers or suckers (atids), and which can have a high incidence on horticultural crops causing serious economic losses, develop resistance to synthetic insecticides and
    5 present virus transmission capacity. Examples, by way of illustration and not limitation, of herbivorous pest insects with different trotic adaptations, are Spodoptera litloralis, Myzus persicae and Rhopalosiphum padi.
    The activity against these herbivorous pest insects can be determined by 10 different types of bioassays that include anti-food activity (inhibition of feeding and / or settlement in the case of aphids), repellent or toxic, among others.
    Preferably, the fungi belong to species of phytopathogenic fungi. Examples, by way of illustration and not limitation, of fungi against which the fermented product 15 of the invention is effective are Fusarium oxysporum, Fusarium moniliforme, Fusarium solani and Botrytis cinerea.
    The activity against fungi can be determined, for example, by assays of inhibition of mycelium growth in plaque.
    Preferably, the nematodes that are included in the scope of the invention are root nodule forming nematodes (Meloidogyne sp). An example of a nodule-forming nematode is the Meloidogine javanica, a polyphagous, with the ability to parasitize more than 3,000 species of crop plants, including extensive crops,
    25 horticultural and fruit trees, seriously affecting production (Agrios. 2005. Plant Pathology, Fifth edition, Elsevier / Academic, Amsterdam), and causing annual economic losses of billions of euros (Singh el al. 2013. OEPPIEPPO Bulletin 43 (2 ), 334-374).
    Activity against nematodes is related to toxicity, that is, ability to
    30 interrupt a specific phase of the nematode's life cycle preventing its development. Within the scope of the invention, nematicidal activity can be determined, for example, by determining the percentage of infective juveniles (J2) killed after 72 hours after application of the extract.
    P201 531527
    In a fifth aspect, the invention relates to a broad spectrum biocidal composition comprising the fermentation product of the invention, or at least one compound of formula (1), or to the compound of general formula (1 1) of either isolated form or in combination, hereinafter biocidal composition of the invention.
    The biocidal composition of the invention can additionally comprise various vehicles and agents that facilitate its conservation, handling and application.
    As the person skilled in the art will know, in the application of phytosanitary products, solid vehicles, liquid vehicles, gaseous vehicles, etc. are usually used, and, if necessary, surfactants and auxiliary agents for the formulation of phytosanitary compositions such as, for example, an additive for formulating forms such as concentrates emulsifies bias, wettable powders, flowable liquids, (eg, water suspension, water emulsion, etc.), powders, aerosols, ULV and the like.
    Examples of solid carriers that are included in the scope of the invention are fine powders or clay granules (eg kaolin clay, diatomaceous earth, synthetic hydrated silicon oxide, bentonite, Fubasami clay, acid clay, etc.), talc, ceramics and other inorganic minerals (eg, sericite, quartz, sulfur, active carbon, calcium carbonate, hydrated silica, etc.), commercial fertilizers (eg, ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, ammonium chloride, etc.) and Similar.
    Examples of liquid vehicles included in the scope of the invention are water, alcohols (eg, methanol, ethanol, etc.), ketones (eg, acetone, methyl ethyl ketone, etc.), aromatic hydrocarbons (eg, benzene, toluene , xylene, ethylbenzene, methylnaphthalene, etc.), aliphatic hydrocarbons (eg hexane, cyclohexane, kerosene, gas oil, etc.), esters (eg, ethyl acetate, butyl acetate, etc.), nitriles (eg, acetonitrile, isobutyronitrile , etc.), ethers (eg, diisopropyl ether, dioxane etc.), acid amides (eg, N, N-dimethylformamide, N, N-dimethylacetamide, etc.), halogenated hydrocarbons (eg, dichloroethane, trichloroethane, carbon tetrachloride, etc.), dimethyl sulfoxide, vegetable oils (eg, soybean oil, cottonseed oil, etc.) and the like.
    Examples of a gaseous vehicle that are included in the scope of the invention are spray agent, including flon gas, butane gas, LPG (liquified petroleum gas), dimethyl ether, carbon dioxide gas and the like.
    Examples of surfactant that are included in the scope of the invention are alkyl sulfates, alkyl sulfonate salts, alkyl aryl sulfonates, alkyl aryl esters, polyoxyethylene compounds thereof, polyethylene glycol esters, polyhydric alcohol esters, derivatives of sugar alcohol and the like.
    Examples of auxiliary agent for the formulation as fixing agent and dispersion agent included in the invention are casein, gelatin, polysaccharides (eg, starch powder, gum arabic, cellulose derivative, alginic acid, etc.), derivatives of lignin, bentonite, sugars, synthetic water-soluble polymers (eg, vinyl polyol,
    10 vinyl polypyrrolidone, acrylic polyacids, etc.) and the like.
    Examples of stabilizers that are included in the scope of the invention are PAP (isopropyl acid phosphate), BHT (2,6-di-tert-butyl-4-methylphenol), BHA (mixture of 2-tert-butyl-4-methoxyphenol and 3-tert-butyl-4-methoxyphenol), vegetable oils, mineral oils, agents
    15 surfactants, fatty acids or esters thereof and the like.
    The fermentation product of the invention, the compound of the invention, the compound of
    general formula (11) and the biocidal composition of the invention can be used
    in conjunction with at least one additional active ingredient. Ingredient examples
    Additional assets are nematicides, insecticides, acaricides, fungicides, herbicides, plant growth regulators, synergists, fertilizers, soil conditioners and animal baits.
    In a sixth aspect, the invention relates to the use as a broad biocidal agent.
    25 spectrum for the control of harmful organisms, which affect plants, hereinafter use of the invention, of the fermentation product of the invention, or of the compound of the invention, or of a compound of formula (11) either in isolation or in combination. Preferably, the use of the invention is simultaneously effective against more than one category of harmful organisms, which are selected from pest insects, fungi and
    30 minus a nematode.
    In a particular embodiment, the use of the invention as a broad-spectrum biocidal agent comprises the fermentation product of the invention and is simultaneously active against at least, insect pests, fungi and at least one nematode.
    In another particular embodiment, the use of the invention comprises at least one compound of formula (1) and is simultaneously active against pest insects, fungi and at least one nematode.
    In another particular embodiment, the use of the invention comprises the compound of formula (11)and it is active simultaneously against insect pests and fungi.
    In a final aspect, the invention relates to a broad-spectrum control method of harmful organisms affecting plants, hereinafter the control method of the invention, which comprises administering an effective dose of the biocidal composition of the invention, or of the fermentation product of the invention, or of the compound of the invention,
    or of a compound of formula (11) either in isolation or in combination. Preferably, the control method of the invention is simultaneously effective against more than one category of harmful organisms, which are selected from pest insects,
    15 fungi and at least one nematode.
    In a particular embodiment, the control method of the invention is simultaneously active against at least, insect pests, fungi and at least one nematode.
    20 The application can be directly sprayed on harmful organisms, or on the substrate in which they are found.
    The "effective dose" may optionally increase or decrease depending on whether the biocidal composition of the invention is used, or the fermentation product of the invention, or the
    The compound of the invention, or a compound of formula (11) either in isolation or in combination, or of the type of formulation, time, place and method of application, the type of harmful organism that affects the plant and the degree of damage.
    Throughout the description and the claims the word "comprises" and its variants
    30 are not intended to exclude other technical characteristics. For the person skilled in the art, other aspects, advantages and characteristics of the invention will be derived partly from the description and partly from the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.
    MODES OF REALIZATION OF THE INVENTION
    TO OBTAIN A PRODUCT OF FERMENTATION AND CHARACTERIZATION OF ITS COMPOUNDS WITH BIOCID ACTIVITY
    Example 1. Isolation and identification of the Aa22 strain of the endophyte S. solan fungus;
    The Aa22 strain of the endophytic fungus S. soJani was isolated from Artemisia absinthium leaves collected in Terceira (Azores Islands, Macaronesia region). The fresh plant material was superficially sterilized with sodium hypochlorite (65%), ethanol (75%) and sterile distilled water. Small samples of explants (leaves and stems) were incubated at 27 ° C in the dark in two culture media (PDA and YMB) in petri dishes with 50 mg / l of antibiotic to prevent contamination. The pure Aa22 strain of S. soJani was obtained by individually isolating a colony grown in YMB medium and replicated under the same conditions
    15 for maintenance.
    The morphological identification of the fungus strain was performed under a microscope according to the characteristics of the mycelium, spores and reproductive structures, staining small samples of the isolated colonies with methylene blue. Likewise, its identification was carried out at the molecular level with the amplification (PCR) and sequencing of the ribosomal ITS region of the AONr extracted from a sample of the mycelium (Arenal et al. 2000. MycoJogicaJ Research 104, 2000, 301-303 ; Giménez. 2006. Bioactive products of Canarian plants and their endophytic fungi: detection of activity and use in the control of pests and agricultural diseases. Doctoral thesis, University of La Laguna). The sequence ITS1-5.8S-ITS2 of the AONr was compared with those published in the NCBI databases (NationaJ Genter for Biotechnology Information) (GenBank accession number JF913269.1). Once the Aa22 strain of the S. solani endophyte fungus was identified, it was deposited in the Spanish Type Culture Collection (CECT) on September 25, 2015, corresponding to the CECT access number 20941, following the Budapest Treaty
    30 on the International Recognition of the Deposit of Microorganisms for the purposes of the Patent Procedure.
    Example 2. Obtaining the fermentation product of S. salan strain Aa22;
     23-1 0-2015
    From a two-week culture in YMB medium (yeast extract broth and malt extract) in petri dish of S. salani strain Aa22, six samples of fragments (2.5x2.5 cm) of fresh mycelium were taken and inoculated in twelve sterilized erlenmeyers, with 100 g of rice and 30 ml of distilled water (to prevent dehydration of rice). After three weeks of incubation in the dark at 25 ° C, the culture was extracted three times with ethyl acetate and the fermentation product obtained (hereinafter crude extract) was concentrated in vacuo (4.58 g).
    Example 3. Isolation and characterization of compounds with biocidal activity of the crude extract
    Experimental techniques
    The 1 H and 1 JC NMR spectra of the compounds identified in the different fractions of the crude extract were recorded in Bruker Advance and AMX-500 spectrometers, at 400 and 500 MHz for 1H and 100 and 125 MHz for 1JC, respectively. The products were dissolved in deuterochloroform (CDCIJ) containing tetramethylsilane (TMS) as the internal reference standard. The multiplicity of the 1JC signals were determined with broadband decoupling experiments (DEPT). The two-dimensional programs (20) used in the NMR experiments (COZY, NOESY, HSQC and HMBC) were those provided by Bruker. High and low resolution mass spectra were recorded on a Micromass AutospeC® spectrometer using the electronic impact technique (lE-MS) at 70 eV and a temperature of 220 ° C. The preparative and semi-preparative chromatographs were carried out in a flash liquid chromatography equipment (Flash Master Personal, Jones Chromatography) on silica columns (/ solute flash silica, 20 g / 70 mL, International Sorbent Technology Ud. Tucson, USA) . In liquid chromatographies under vacuum (VLC) and in column (CC) silica gel 0.025-0.04 and 0.040-0.015 mm (Macherey-Nagel GmbH & Co.KG, Düren, Germany) was used and as a support in Sephadex molecular exclusion chromatographs LH-20 (Pharmacia Fine Chemicals). The visualization of the compounds in thin layer chromatography (TLC) was performed with a solution of H, S04 (5%) and vanillin (5%) in EtOH.
    For its part, the crude extract was analyzed by LC-MS.
    3.A. Isolation of compounds with biocidal activity from the crude extract
    The methanol soluble fraction (4.09 g) of the crude extract (4.58 g) obtained according to Example 2, was chromatographed on silica gel (0.3 kg) by means of a liquid chromatography of vacuum (VLC) using eluent increasing mixtures in polarity of n -hexane / ethyl acetate, acetone and methanoll water giving rise to 8 fractions: H1-H2-H3-H4-H5-H6 and H8.
    The H2 fraction (n-hexanol AcüEt, 5: 1, 0.575 g) was column chromatographed (CC) with silica gel using mixtures of n-hexane ketone. Of the most polar fraction is
    10 obtained 42 mg of sternfol (Stodola et a.1973. Phytochemistry 12, 1797-1798, Marumo et al. 1985. Agríc. Biol. Chem. 49, 1521-1522). The other fractions were combined in 6 fractions (H2A-H2F). The H2E fraction (0.053 g) was again chromatographed on a silica gel column using n-hexane / dichloromethane mixtures. In the medium polarity fractions 21 mg of the stemfol were obtained.
    From the fraction H3 (n-hexane AcüEt, 4: 1, 107 mg) by Sephadex LH20 chromatography with a mixture of n-hexane / dichloromethane / methanol (2: 1: 1) the stemfol was isolated again
    (74.3 mg).
    The H6 fraction (acetone, 0.452 g) was chromatographed on a silica gel gel eluted with increasing polarity mixtures of ethyl n-hexaneacetate. In the less polar fractions obtained from this fraction 6 the stemfolone A (19 mg) was isolated and 11 mg of the stemfolone B was obtained by the most polar by Sephadex LH20 chromatography with n-hexane / dichloromethane / methanol (2: 1: 1).
    3.B. Characterization of compounds with biocidal activity
    Stemfol
    30 The stemfol was isolated from the crude extract as a white solid. The high resolution mass spectrum showed a molecular ion at 236.1772 miz (calcd. 236.1776) that was in accordance with the molecular formula C1sH24Ü 2. The absorption bands in the IR spectrum at 3300 and 1630 cm- 'indicated the presence of hydroxyl groups and double bonds, respectively. Its' H NMR spectrum showed characteristic signals of two
    35 alkyl chains (butyl and n-pentyl), the signal of a singlet that made up two protons at 86.22 (H-4, H-6) and two hydroxyl groups at 84.62 (2H, s, OH). He
    Chemical shift and multiplicity suggested the presence of a 1,3-dihydroxyphene-1-2,5-tetrasubstituted ring. This proposal was confirmed by the similarity of its spectroscopic data with those published for similar compounds (Pohanka et al. 2006. J Nat
    5 Prod. 69, 654-657).
    The 1JC NMR spectrum showed twelve signals corresponding to a methyl group, seven methylenes, a methine group and three quaternary carbons, which agreed with the correlations observed in the HSQC experiment. Signals a or 108.1, 114.1, 142.1 Y
    10 154.4 due to aromatic carbons confirmed the presence of the 1,3-dihydroxy-2,5-tetrasustiuido nyl fe group.
    The relative position of the substitutes was confirmed in the HMBC experiment, with the correlations observed between the protons at Ó 2.58 (H-1 ') and Ó 6.22 (H-4, H-6) with the 15 signals at ~ 154.4 ( C-1, C-3) and ~ 112.5 (C-2), and proton at ~ 2.44 (H-1 ··) with ~ 108.1 (C-4, C-6) and ~ 142.2 (C-5) (see Figure 1).
    These data are in agreement with the stemfol, compound previously isolated from S. majuseulum fungi (Stodola et a.1973. Phytoehemistry 12, 1797-1798) and S. botryosum
    20 (Marumo et al. 1985, Agríe, Bial. Chem. 49, 1521-1522). Its 2D NMR spectroscopic data allowed to assign all the proton and carbon signals present in the molecule (see Table 1) and identify its structure as 5-butyl-2-pentylresorcin.
    Stemlol IR (film) Vm ", 3300, 2850 1630 1588, 1430, 1270 cm · '; for data' H NMR (CDCI"
    25 500 MHz) see Table 1; for 1JC NMR data (CDCI3, 125 MHz) see Table 1; HREIMS mIz 236.1771 [Mt (calculated for C "H" O "236.1776); EIMS 70 eV miz (rel. Int.): 236 [M '] (16), 194 (16), 193 (100), 180 (45 ), 137 (7), 123 (1 1), 91 (3), 77 (4).
    Table 1. Spectroscopic data of lH NMR and 1JC NMR of the stemfol
    H / C HNMRCNMR
    one -154.4 s
    2 -112.5 s
    3 -154.4 s
    4 6.22 s
    5
    6 6.22 s
    l ' 2,581 (J = 8.0 Hz)
    2' 1.51 m
    3' 1.40 m
    4' 0.931 (J = 7.0 Hz)
    one" 2.44 1 (J = 8.0 Hz)
    2" 1.51 m
    3" 1.51 m
    4" 1.31 m
    5" 0.88 1 (J = 7.0 Hz) 108.1 d
    142.2s 108.1 d
    22.81
    31.41 22.7t 13.9q
    35.51
    30.71 31.5t
    22.51 13.9q
    Slemfo / ona A The stemfolone A was isolated from the crude extract as a colored amorphous solid
    5 brown Its molecular formula was determined as C1sH260 3 ([M] ", mIz 254.1888) (calcd. 254.1882) by high resolution mass spectrometry. The IR spectrum showed absorption bands at 3443, 1673 and 1650 cm -1 attributable to groups hydroxyls, carbonyl group and double bonds, respectively.
    10 In the 1 H-NMR spectrum, signals from two aliphatic chains were observed, comprising a total of twenty protons, a methylene group at 2.47 (1 H, dd, J = 18.0 Hz, J = 10 Hz,
    H-4ax) and O 2.61 (1 H, dd, J = 18.0Hz, J = 6.0 Hz, H-4ec), and a methine attached to an atom of oxygen at 3.98 (1 H, dd, J = 10.0 Hz, J = 6.0 Hz, H-5). Also, it was observed at low field
    the signal of an olefinic proton at o5.89 (1 H, bs, H-2), suggesting the presence of a double
    15 trisubstituted link. The NMR spectrum of lH -2D-COZY showed, as in the stemfol, the presence of aliphatic butyl and pentyl fragments that were arrested by the correlations observed in the spin systems between H-1 "-Hr 4" and H-1 ' -H3 -5 ', respectively. Another series of correlations that appear in this spectrum were in agreement with a grouping
    CH = C-CH, -CH- (O} -. Thus, e) geminal system at or 2.47 (H-4'ax) and 2.61 (H-4 ec) showed a coupling with signals at 8 3.98 (H -5) Yó 5.89 (H-2).
    The 13C NMR spectrum showed the presence of fifteen signals that were assigned to two methyl, eight methyl, two methyl and three quaternary carbons, according to an HSQC experiment. The resonances at Ó 73.9 (C-5) and (5 79.6 (C-6) indicated the existence of two two carbon atoms attached to oxygen.
    chemical shifts to 6 122.5 (C-2) and 164.2 (C-3) due to carbons of a double
    bond attached to an electron acceptor group and the signal at 201-203 (C-1) of a carbonyl group.
    10 The location of the different functional groups was established based on the correlations shown in the HMBC experiment. Thus, connectivities were observed between H-2 (15 5.89),
    H-4 (or 2.47 and 2.61 o) and H-5 (or 3.98) with C-6 (or 79.6), of Hl "(or 1.95) with Cl (or 200.1) and C6 (O 79.6), Yde Hl ' (O 2.23) with C-2 (O 122.5) YC-3 (O 164.5) (see Figure 2).
    15 The correlations of the HMBC and NMR-2D-COZY experiments of 1H suggested the presence of a 6-butyl-3-pentylcyclohexene skeleton. The stereochemistry of CS and C-6 was deduced by the NOE effects observed in the irradiation of H-4ax with H-1 "Y of H-4ec with HS. The relative position of the butyl and pentyl alkyl chains was confirmed by the effects NOE observed between protons H-2 and H-1 H-4 and H-1 '. These spectroscopic data
    t
    20 allowed to determine the structure of a compound such as 6-butyl-5,6-dihydroxy-3pentylcyclohex-2-en-1-one, not previously described in the literature and what we have named stemfolone A.
    Stemfolone A. [0] 0 +4.4 (e 0.08, CHCI,); IR (film) Vm "3443, 2957, 2930, 2861, 1673, 1650,
    25 1626, 1467, 1378, 1257, 1142, 1075 cm "; for 'H NMR (CDCI" 500 MHz) data see Table 3; for DC NMR (CDC! "125 MHz) data see Table 3; HREIMS miz 254.1888 [M] '(calculated for C" H, .O "254.1882); EIMS 70 eV miz (rel. in !.): 254 [M '] (5), 198 [MC, H,]' (35), 179 (8), 169 [M-C4H, -C, H, J '(33), 151 (21), 139 [M + H -C6H "O ~ 'fragmentation retroDiels Alder (100), 124 (22), 116 (58), 95 (20), 85 (54), 74 (55). Stemfolone B
    The stemfolone B was isolated from the crude extract as a brown amorphous solid. The molecular formula was determined as ClsH260 4 from the molecular ion amlz 270.1833 35 (calcd. 270.1831) in the high resolution mass spectrum. Spectrum absorptions
    infrared at 3418, 1673, 1651 cm "indicated the presence of hydroxyl groups, group
    carbonyl and double bonds, respectively. In its 1 H NMR spectrum, signals similar to those of stemfolone A were observed. The most significant difference between the two
    spectra was the presence of a signal at 1) 3.81 (1 H, Id, J = 12.0 Hz, J = 6.0 Hz, H-4 ') that
    5 suggested the presence of an additional hydroxyl group that could be located at C-3 or C-4. The correlation observed in the H MBC experiment between the methyl groups in 61.19 (3H, d, H-5 ') and the signal in o67.7 confirms the position of the hydroxyl group in C-4. On the other hand, the NOE effect between H-1 and H-4, as well as the coupling constants of H-3 allowed to determine the relative stereochemistry in C-2 and C-3. Based on the previous data the
    The structure of this compound was assigned as 6-butyl-5,6-dihydroxy-3- (4-hydroxypentyl) cyclohex-2-en-1-one. It is the first time that this compound is isolated as a natural product and we call it stemfolone B.
    Stemfolone B. [0] 0 +12.5 (e 0.056, CHCi,); IR (film) Vm ~ 3418, 295, 2931, 2872, 1673,
    15 1667,1651,1626,1433,1377,1260, 1138,1076 cm "; for 'H NMR (CDCi" 500 MHz) data see Table 3; for data "c NMR (CDCI" 125 MHz) see Table 3; HREIMS miz 270.1833 [Mr (calculated for C "H, .O" 270.1831); EIMS 70 eV miz (rel. In1.): 270 [M '] (7), 252 [MH, Or (14), 197 [M + HH, O-2C, H, r (35), 179 [M + H-2H, O-2C, H, r (12), 167 [MH, O-2C, H, -C, H, r (33), 155 (17), 149 (26), 137 (21), 125 (10), 116 [MC "H" O, r retro-Diels fragmentation
    20 Alder (100), 109 (28), 95 (54), 85 (85), 74 (96).
    Table 2. Spectroscopic data of 1 H NMR and 13 C NMR of stemfolone A and B.
    Stemfolone A Stemfolone B
    HIC H NMRC NMRH NMRC NMR
    one -.201 .3 s-201 .3 s
    2 5.89bs122.5 d5.89 bs122.6 d
    3 __or164.2 s-163.6 s
    2.61 dd (J = 18.0, 6.0 Hz) 2.63 dd (J = 19.0, 6.0 Hz)
    4 36.1 t36.0 t
    2.47 dd (J = 18.0, 10.0 Hz) 2.47 dd (J = 19.0, 10.0Hz)
    5 3.98dd (J = 10.0, 6.0 Hz)73.9 d3.98dd (J = 10.0, 6.0 Hz)73.8 d
    6 __or79.6 s---79.6 s
    l ' 2.23dd (J = 7.0, 3.0 Hz)
    2' 1.45 m
    3' 1.30 m
    4' 1.28 m
    5' 0.881 (J = 7.0 Hz)
    one" 1.95dd (J = 13.0, 6.0 Hz)
    1.45 m
    2" 1.26 m
    3" 1.32 m
    4" 0.851 (J = 7.0 Hz) 37.7 I
    26.7 t
    31.3 I 22.4 I 13.9 q 29.3 t 24.5 I 23.1 t 13.9 q
    2.23 m 1.45 m
    1.30 m
    3.81 Id (J = 12.0, 6.0 Hz) 1.19 d (J = 6.0 Hz)
    1.95dd (J = 10.0, 6.0 Hz)
    1.45 m 1.26 m
    1.28 m
    0.85 I (J = 7.0 Hz) 37.6
    23.1
    I 38.6 I 67.7 d 23.8 q
    29.3 t
    24.5 I
    23.2 t
    13.9 q
    BI BIOCIDE ACTIVITY
    Example 4. Activity against pest insects
    The breeding and maintenance of insects was carried out in a temperature controlled chamber at 24 + 1 ° C, 60-70% relative humidity and a photoperiod of 16: 8 hours (light: dark). The larvae of S. littoralis were maintained on a semi-synthetic diet (Poitut and Bues. 1970. Ann. Zoo /. Eco /. Anim. 2, 79-91) And aphids M. persicae and Rhopalosiphum 10 padi on their host plants, Pepper -Capsicum annum L.-and barley -Hordeum vulgare L.-respectively. The trials of anti-food activity were performed with newly emerged larvae of the sixth stage of S. littoralis and aphid adult aphids. The upper surface of leaf discs (1.0 cm2) of pepper (Capsicum annum L.), were treated with 10 1-11 of a solution (10 mg / ml for extracts and 5 mg / ml for pure products). Each 15 trial consisted of 5 Petri dishes with two larvae per plate (S. littoralis) or twenty boxes (2x2 cm) with ten aphids of M. persicae or R. padi incubated in a growth chamber under the same conditions described for breeding of insects After consuming 75% of the surface of the control discs (S. littoralis) or after 24 h (M. persicae or R. padi), the consumption index (% FI) or settlement (% SI) was calculated. , respectively. % FI = [1 20 - (T I C) x 100], where T and C are the consumption of treated leaf disks and control; % of SI = [1 - (% T CI%)], where% C and% T are the percentage of aphids settled in the control and treated leaf discs (Burgueño et al. 2008. J. Chem. Eco /. 34 , 766-771). The compounds
    with an IFF IF:> 70% were tested in a dose experiment - response to calculate their relative potency (ECso, is the effective dose for a 50% reduction in feeding).
    Table 3. Insect-pest activity of the crude extract (100 iJg / cm2), of the H1-H8 fractions [100 iJg / cm2] and of the compounds with biocidal activity (50 iJg / cm2).
    Crude extract / fraction / compound S. (ittoraphisM. persicaeR. padi
    % FI % YES
    Raw Extract 97.21 ± 1.5897.23 ± 1.2740.02 ± 6.79
    H1 57.70 ± 8.8150.29 ± 10.2155.16 ± 6.27
    H2 65.22 ± 9.6492.76 ± 2.5972.57 ± 6.20
    H3 34.54 ± 11.7091 .51 ± 3.2886.57 ± 3.11
    H4 32.83 ± 16.8183.91 ± 4.0638.17 ± 7.70
    H5 54.27 ± 11.7847.54 ± 10.0668.36 ± 6.41
    H6 78.51 ± 9.5674.88 ± 6.7734.29 ± 6.90
    H7 98.22 ± 1.4493.18 ± 2.1595.46 ± 2.11
    H8 79.98 ± 7.9372.04 ± 5.6559.68 ± 6.51
    Stemfol 60.44 ± 8.785.35 ± 5.36 0_05 (0.01-0.301) '72.41 ± 5.61
    Stemfolone A 41 .8 ± 7.981.42 ± 5.61 0_15 (0.06-0_36),na
    Stemfolone B 52_9 ± 5_983.87 ± 6.89 0_02 (0.003-0.15) '45.13 ± 9.09
    'EC50 (~ g / cm)
    Example 5. Antifungal activity
    10 Phytopathogenic fungi Fusarium moniliforme (Sheldon) [CECT 2152], F. oxysporum fs. Iycopersici (Escalda) [CECT 2715] and F. solani (Mart) [CECT 2199] come from the Spanish Type Culture Collection (CET). The strain of Botrytis cinerea Pers.:Fr. (805.10) is a donation from the Department of Biochemistry of the University of La Laguna (ULL). For your
    Maintenance strains were grown in commercial solid medium PDA at 25 ° C (Fusarium) or
    room temperature (Botrytis), and subsequent conservation at -30'C in vials with glycerol at18% To determine the antifungal activity, the agar dilution method was used(Murabayashi et al. 1991. J. Pestieide Sei. 16, 419-427). Samples of dry raw extractobtained according to Example 2 were incorporated into the culture medium (5 ml) at 55 different concentrations (1,0,5,0,1,0,05 and 0,01 mg / ml). In parallel, they preparedcontrols with ethanol at a concentration of 2%. Planting of target organisms isperformed by bite (Fusarium) or with 5 mm diameter discs (B. cinerea). The coloniescultured in Petri dishes and incubated for 48 h were digitized and measuredusing the ImageJ 1.43 program. The percentage of inhibition (% 1) was calculated as:% I
    10 = (C-T / C) x 100, where C is the diameter of the control colonies and T of the colonies of the samples tested. The effective dose of growth inhibition (EC50) was determined by linear regression analysis (% inhibition of the dose log).
    Table 4. Antifungal activity of the crude extract, of the H1-H8 fractions and of the 15 compounds (using 0.5 mg / ml). 8 Tested at 0.1 mg / ml.
    Crude extract / compound fraction Antifungal activity (0.5 mg / ml) EC5Q mg / ml (95% e L)
    Fusarium oxysporum F. moniliformF. salaniBatrytis cinerea
    Raw extract 85.5tO.89 0.144 (0.113-0.183)80,921 ± 1,67477.78t1.09 0.08 (0.07-0.08)61 .176t3.793
    0.196 (0.195-0.196) 0.30 (0.310.30)
    H1 10.90t2.593.52tO.781,126t1,6259.61 ± 3.66
    H2 50.44 ± 9.40 0.464 (0.069-0.952)32.79t1.7246.79t6.899.44t3.77a
    H3 69.38 ± 1 .17 0.179 (0.147-0.218)56.26 ± 1 .69a82.55 ± 2.9a54.82 ± 1 .97a
    H4 3.61 ± 1 .18OR15,262 ± 2,7821.03 ± 1.13
    H5 12.79t1.8412.87t2.8615,328t3,00616.03t2.30
    H6 22.68 ± 2.6222.93 ± 3.4836,459 ± 3,54522.83 ± 2.72
    H7 OROR4 .629t1.937O.24t2.67
    H8 O.23t1 .65OR4,585t2,4323.87t2.B1
    Sternfol 70.22 ± 2.04 0.01 (0.005-0.024)67.35 ± 2.0547,093 ± 3,57844.74 ± 1.35
    0.02 (0.020-0.021) 0.53 (0.530.54)
    Stemfolone nanana56.21 ± 1.50
    A2 0.43 (0.42 · 0.43)
    Sternfolone B ---4.20 ± 1 .14a49.94 ± 4.40 0.21 (0.21 · 0.22)11.68 ± 3.67
    Example 6. Nematicidal activity
    The nematode population (M. javanica) remained in growth chambers on
    5 tomato plants - Lycopersicon esculentum (var. Marmande) - at 25 oC and a relative humidity of 70%. The tests were performed according to the methodology described for M. javanica (Andres et al. 20 12. Phytochem. Rev. 11, 371-390) using the biological phase of infective juveniles (J2). The activity of the dry crude extract obtained according to Example 2, of its fractions and of the compounds with biocidal activity was quantified at a final concentration
    10 per well of 1.0 / 0.5 and 0.25 mg / ml, respectively. Each treatment was repeated four times and the nematicidal activity was determined from the percentage of infectious juveniles killed after 72h. In cases where a mortality rate> 99% was determined, dose-response experiments were performed to determine LCso and LC9Q
    15 Table 5. Nematicidal activity of the crude extract, H1-H8 fractions and compounds
    Crude extract / fraction / compound mg / mlM. javanica% paralyzed
    Raw extract one94.82 ± 0.75
    H1 one0.41 ± 1.60
    H2 one7.46 ± 1.79
    H3 one0.00 ± 0.00
    H4 one0.00 ± 0.00
    H5 one10.39 ± 1.87
    H6 one11.14 ± 3.23
    H7 one0.98 ± 1.42
    He has one1.80 ± 0.65
    SternfoJ 0.51.24 ± 0.68
    Stemfolone A 0.583.05 ± 3. 15
    0.25 2.8 ± 0.5
    Stemfolone B 0.51.24 ± 0.9
    权利要求:
    Claims (12)
    [1]
    1.-Fermentation product of an endophytic fungus of the Stemphylium soJani species with broad spectrum biocidal activity, characterized in that it comprises a compound of 5 formula (1):
    OR
    R
    l · 3 ·
    2 · 4 · S ·
    (one)
    where R is selected from H and OH, 10
    or an isomer, or a solvate salt thereof.
    [2]
    2. Fermentation product according to claim 1, characterized in that it simultaneously comprises a compound of formula (1) wherein R is H, and another compound of formula (1) where R is OH, or an isomer, or a salt a solvate of them.
    [3]
    3. Fermentation product according to any of claims 1 and 2, characterized in that it additionally comprises another compound with a broad spectrum biocidal activity of formula (11):
    OH 4 · 2 '2
    r
    2 ·· 4 "
    (eleven )
    25 or an isomer, or a solvate thereof.
    [4]
    4.-Compound of formula (1):
    or
    4 "2"
    "
    3 "R
    3. 4'
    2 'S'
    "
    (one)where R is selected from H and OH,
    5 or an isomer, or a solvate thereof.
    [5]
    5. Process for obtaining the fermentation product defined according to any of claims 1 to 3, characterized in that it comprises culturing mycelium of an endophytic fungus of the Stemphylium solan species; In a culture medium.
    [6]
    6. Method of obtaining according to claim 5, characterized in that it additionally comprises a drying step using a technique that is selected from extraction with organic solvent prior filtration or lyophilization.
    7. Method according to claim 6, characterized in that the endophytic fungus is Stemphylium salan strain Aa22; with deposit number CECT 20941.
    [8]
    8. Use of a fermentation product as defined according to claims 1 to 3, of a compound of general formula (1) as defined in claim 4, or of a compound of formula (11) as and as defined in claim 3 for making a broad spectrum biocidal composition.
    [9]
    9. A broad spectrum biocidal composition comprising a fermentation product as defined according to claims 1 to 3, or a compound of general formula (1) as defined in claim 4, or a compound of formula ( 11) as defined in claim 3.
    [10]
    10. Use of the biocidal composition according to claim 9 as a broad spectrum biocidal agent for the simultaneous control of more than one category of harmful organisms that affect plants.
    [11]
    11. Use according to claim 10, characterized in that the categories of harmful organisms that affect plants are selected from insect pests, fungi and nematodes.
    12. 12. Use according to any of claims 10 and 11, characterized in that thecategories of harmful organisms are simultaneously pest insects, fungi andnematodes
    [13]
    13.-Method for the simultaneous control of more than one category of harmful organisms
    10 which affect plants, which comprises administering an effective dose of a fermentation product as defined according to claims 1 to 3, or of a compound of general formula (1) as defined in claim 4, or of a compound of formula (11) as defined in claim 3.
    14. Control method according to claim 13, characterized in that the categories of harmful organisms that affect plants are selected from insect-pests, fungi and nematodes.
    [15]
    15. Control method according to any of claims 13 and 14, characterized by
    20 that the categories of harmful organisms are simultaneously pest insects, fungi and nematodes.
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    同族专利:
    公开号 | 公开日
    CA3002123A1|2017-04-27|
    WO2017068223A8|2018-05-11|
    AR106443A1|2018-01-17|
    EP3366136A1|2018-08-29|
    MA44477A|2021-04-07|
    US20180312880A1|2018-11-01|
    WO2017068223A1|2017-04-27|
    UY36960A|2017-05-31|
    ES2610186B1|2018-09-06|
    ES2610186R1|2017-09-15|
    BR112018007820A2|2018-10-30|
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    US4036629A|1971-06-14|1977-07-19|Mobil Oil Corporation|3-butyl esters|
    US4036639A|1973-09-10|1977-07-19|Sherritt Gordon Mines Limited|Production of copper|
    US6544524B2|2000-09-01|2003-04-08|Fundacao Oswaldo Cruz - Fiocruz|Ethyl acetate extract from guignardia used to treat an individual infected by fungi or bacteria|
    DE102009022618A1|2009-05-26|2010-12-02|Leibniz-Institut Für Pflanzenbiochemie|New hydropyrone derivatives useful as biocide, antibiotics, fungicide, antioomycetes, inhibitor for enzymes, disinfectant, textile modifier and wood preservative|CN107915616A|2017-10-18|2018-04-17|浙江大学|A kind of compound and its application|
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    US15/769,674| US20180312880A1|2015-10-23|2016-10-21|Natural broad-spectrum biocides|
    ARP160103222A| AR106443A1|2015-10-23|2016-10-21|WIDE SPECTRO NATURAL BIOCIDES|
    EP16805478.1A| EP3366136A1|2015-10-23|2016-10-21|Natural broad-spectrum biocides|
    BR112018007820-4A| BR112018007820A2|2015-10-23|2016-10-21|broad-spectrum natural biocides|
    MA044477A| MA44477A|2015-10-23|2016-10-21|WIDE SPECTRUM NATURAL BIOCIDES|
    CA3002123A| CA3002123A1|2015-10-23|2016-10-21|Natural broad-spectrum biocides|
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