EP1893771A1 - Method for identification of novel fungicides - Google Patents

Method for identification of novel fungicides

Info

Publication number
EP1893771A1
EP1893771A1 EP06763587A EP06763587A EP1893771A1 EP 1893771 A1 EP1893771 A1 EP 1893771A1 EP 06763587 A EP06763587 A EP 06763587A EP 06763587 A EP06763587 A EP 06763587A EP 1893771 A1 EP1893771 A1 EP 1893771A1
Authority
EP
European Patent Office
Prior art keywords
diseases
spectrin
caused
protein
proteins
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP06763587A
Other languages
German (de)
French (fr)
Inventor
Valérie TOQUIN
Jean-Luc Zundel
François BARJA
Roland Beffa
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer CropScience SA
Original Assignee
Bayer CropScience SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bayer CropScience SA filed Critical Bayer CropScience SA
Publication of EP1893771A1 publication Critical patent/EP1893771A1/en
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56961—Plant cells or fungi

Definitions

  • the invention relates to methods for the identification of fungicides and more particularly to a method for following the distribution of spectrin-like proteins.
  • the structural integrity of cells is maintained in part by the cytoskeleton, a mesh-like structure composed primarily of proteins, which lies adjacent to the inner cell surface.
  • the cytoskeleton of many cell type contains large amount of proteins such as tubulin, actin, spectrin and proteins closely related to spectrin.
  • Spectrin is the principal component of a system of structural proteins that are associated with the cytoplasmic surface of the plasma membrane in most metazoan cells (Bennet and Gilligan 1993, Ann. Rev. Cell Biol. 9: 27-66).
  • the structure and function of spectrin has been extensively investigated in mammalian erythrocytes (Marchesi and Steer 1998, Science, 159 : 203-204; Palek and Lambert 1990, Sem. Hematol. 27 : 290-332; Bennet and Gilligan 1993, Ann. Rev. Cell Biol. 9: 27-66) in which spectrin forms a meshwork responsible for maintaining the shape and mechanical property of the cell.
  • spectrin is involved in the organization of both plasma and internal membranes such as those of the Golgi apparatus, the endoplasmic reticulum and the lysosomes (Beck et al., 1994, J. Cell Biol., 127 : 707-723; 1997, J. Cell Sci., 110 : 1239-1249; Devarajan et al., 1996, J. Cell Biol. 133 : 819-830; Holleran et al., 1996, Trends Cell Biol., 8 : 26-29; Hook et al., 1997, J; Cell Biol. 136 : 1059-1070; De Matteis and Morrow, 2000, J. Cell Sci. 113 : 2331- 2343).
  • SLP Spectrin-like proteins
  • the present inventors have found that fungi presenting a redistribution of spectrin-like protein upon application of specific fungicide compounds, either inhibit their growth or die. Thus, the distribution of spectrin-like proteins is useful for identification of fungicide compounds.
  • the present inventors have found that fungi presenting a redistribution of spectrin-like proteins upon application of specific fungicide compounds, either inhibit their growth or die.
  • the redistribution of the spectrin-like proteins from the plasma membrane to the cytoplasm of the fungal cells can be observed using antibodies raised against spectrin proteins.
  • the present invention provides methods for the identification of compounds that induce a redistribution of spectrin-like proteins.
  • the methods of the invention are useful for the identification of fungicides.
  • spectrin in the present description of the invention refers to proteins characterized by the presence of spectrin repeats, actin binding domains and EF hands (calcium-binding motifs). In addition, spectrin contains specialized protein-protein interaction motifs and regions for interaction with membranes and phospholipids.
  • spectrin-like proteins in the present application refers to proteins that bind to antibodies raised against spectrin proteins.
  • the term “binds to” refers to a non-covalent or a covalent interaction, preferably non- covalent, that holds two molecules together.
  • Non-covalent interaction includes hydrogen bonding, ionic interactions among charged group, van de Waals interactions and hydrophobic interactions among non polar groups. One or more of these interactions can mediate the binding of two molecules to each other.
  • bindings to or “binding” refers more particularly to the specific interaction between antibodies raised against spectrin protein and the corresponding spectrin like proteins.
  • effective and non-phytotoxic amount and "agronomical effective” mean an amount of compound according to the invention which is sufficient to control or destroy the fungi present or liable to appear on the crops, and which does not entail any appreciable symptom of phytotoxicity for said crops. Such an amount can vary within a wide range depending on the fungus to be combated, the type of crop and the compounds used. This amount can be determined by systematic field trials, which are within the capabilities of a person skilled in the art.
  • the compounds identified by the method of treatment according to the present invention are useful to treat propagation material such as tubers or rhizomes, but also seeds, seedlings or seedling pricking out and plants or plants pricking out.
  • This method of treatment according to the invention can also be useful to treat the overground parts of the plant such as truncks, stems or stalks, buds, leaves, flowers and fruits of the concerned plant.
  • cotton Among the plants that can be protected using the compounds identified by the method according to the present invention, mention may be made of cotton; flax; vine; fruit or vegetable crops such as Rosaceae sp. (for instance pip fruit such as apples and pears, but also stone fruit such as apricots, almonds and peaches), Ribesioidae sp., Juglandaceae sp., Betulaceae sp., Anacardiaceae sp., Fagaceae sp., Moraceae sp., Oleaceae sp., Actinidaceae sp., Lauraceae sp., Musaceae sp.
  • Rosaceae sp. for instance pip fruit such as apples and pears, but also stone fruit such as apricots, almonds and peaches
  • Rosaceae sp. for instance pip fruit such as apples and pears, but also stone fruit such as apricots, almonds and pe
  • Rubiaceae sp. for instance banana trees and plantins
  • Rubiaceae sp. Theaceae sp., Sterculiceae sp., Rutaceae sp. (for instance lemons, oranges and grapefruit); Solanaceae sp. (for instance tomatoes), Liliaceae sp., Asteraceae sp. (for instance lettuces), Umbelliferae sp., Cruciferae sp., Chenopodiaceae sp., Cucurbitaceae sp., Papilionaceae sp. (for instance peas), Rosaceae sp. (for instance strawberries); major crops such as Graminae sp.
  • Asteraceae sp. for instance sunflower
  • Cruciferae sp. for instance colza
  • Fabacae sp. for instance peanuts
  • Papilionaceae sp. for instance soybean
  • Solanaceae sp. for instance potatoes
  • Chenopodiaceae sp. for instance beetroots
  • horticultural and forest crops as well as genetically modified homologues of these crops.
  • Powdery mildew diseases such as :
  • Blumeria diseases caused for example by Blumeria graminis
  • Podosphaera diseases caused for example by Podosphaera leucotricha
  • Sphaerotheca diseases caused for example by Sphaerotheca fuliginea
  • Uncinula diseases caused for example by Uncinula necator
  • Rust diseases such as :
  • Gymnosporangium diseases caused for example by Gymnosporangium sabinae
  • Hemileia diseases caused for example by Hemileia vastatrix
  • Phakopsora diseases caused for example by Phakopsora pachyrhizi or Phakopsora meibomiae;
  • Puccinia diseases caused for example by Puccinia recondita
  • Uromyces diseases caused for example by Uromyces appendiculatus; Oomycete diseases such as :
  • Bremia diseases caused for example by Bremia lactucae
  • Peronospora diseases caused for example by Peronospora pisi or P. brassicae
  • Phytophthora diseases caused for example by Phytophthora infestans
  • Plasmopara diseases caused for example by Plasmopara viticola
  • Pseudoperonospora diseases caused for example by Pseudoperonospora humuli or Pseudoperonospora cubensis
  • Pythium diseases caused for example by Pythium ultimum
  • Leafspot, leaf blotch and leaf blight diseases such as :
  • Alternaria diseases caused for example by Alternaria solani;
  • Cercospora diseases caused for example by Cercospora beticola
  • Cladiosporum diseases caused for example by Cladiosporium cucumerinum
  • Cochliobolus diseases caused for example by Cochliobolus sativus
  • Colletotrichum diseases caused for example by Colletotrichum lindemuthanium;
  • Cycloconium diseases caused for example by Cycloconium oleaginum
  • Diaporthe diseases caused for example by Diaporthe citri;
  • Elsinoe diseases caused for example by Elsinoe fawcetiii; Gloeosporium diseases, caused for example by Gloeosporium laeticolor,
  • Glomerella diseases caused for example by Glomerella cingulata
  • Guignardia diseases caused for example by Guignardia bidwelli;
  • Leptosphaeria diseases caused for example by Leptosphaeria maculans; Leptosphaeria nodorum; Magnaporthe diseases, caused for example by Magnaporthe grisea;
  • Mycosphaerella diseases caused for example by Mycosphaerella graminicola; Mycosphaerella arachidicola; Mycosphaerella fijiensis; Phaeosphaeria diseases, caused for example by Phaeosphaeria nodorum;
  • Pyrenophora diseases caused for example by Pyrenophora teres
  • Ramularia diseases caused for example by Ramularia collo-cygni;
  • Rhynchosporium diseases caused for example by Rhynchosporium secalis
  • Septoria diseases caused for example by Septoria apii or Septoria lycopercisi
  • Typhula diseases caused for example by Typhula incarnata
  • Venturia diseases caused for example by Venturia inaequalis
  • Root and stem diseases such as :
  • Corticium diseases caused for example by Corticium graminearum
  • Fusarium diseases caused for example by Fusarium oxysporum
  • Gaeumannomyces diseases caused for example by Gaeumannomyces graminis;
  • Rhizoctonia diseases caused for example by Rhizoctonia solani;
  • Tapesia diseases caused for example by Tapesia acuformis
  • Thielaviopsis diseases caused for example by Thielaviopsis basicola; Ear and panicle diseases such as :
  • Altemaria diseases caused for example by Alternaria spp.;
  • Aspergillus diseases caused for example by Aspergillus flavus;
  • Cladosporium diseases caused for example by Cladosporium spp.;
  • Claviceps diseases caused for example by Claviceps purpurea
  • Fusarium diseases caused for example by Fusarium culmorum
  • Gibberella diseases caused for example by Gibberella zeae
  • Monographella diseases caused for example by Monographella nivalis; Smut and bunt diseases such as :
  • Sphacelotheca diseases caused for example by Sphacelotheca miliaria
  • Tilletia diseases caused for example by Tilletia caries
  • Urocystis diseases caused for example by Urocystis occulta
  • Ustilago diseases caused for example by Ustilago nuda; Fruit rot and mould diseases such as :
  • Aspergillus diseases caused for example by Aspergillus flavus
  • Botrytis diseases caused for example by Botrytis cinerea
  • Penicillium diseases caused for example by Penicillium expansum
  • Sclerotinia diseases caused for example by Sclerotinia sclerotiorum
  • Verticilium diseases caused for example by Verticilium alboatrum
  • Seed and soilbome decay, mould, wilt, rot and damping-off diseases Fusarium diseases, caused for example by Fusarium culmorum;
  • Phytophthora diseases caused for example by Phytophthora cactorum
  • Pythium diseases caused for example by Pythium ultimum
  • Rhizoctonia diseases caused for example by Rhizoctonia solani
  • Sclerotium diseases caused for example by Sclerotium rolfsii;
  • Microdochium diseases caused for example by Microdochium nivale; Canker, broom and dieback diseases such as : Nectria diseases, caused for example by Nectria galligena;
  • Blight diseases such as :
  • Monilinia diseases caused for example by Monilinia laxa;
  • Leaf blister or leaf curl diseases such as :
  • Taphrina diseases caused for example by Taphrina deformans; Decline diseases of wooden plants such as :
  • Esca diseases caused for example by Phaemoniella clamydospora; Diseases of flowers and Seeds such as :
  • Botrytis diseases caused for example by Botiytis cinerea
  • Diseases of tubers such as : Rhizoctonia diseases, caused for example by Rhizoctonia solani
  • fungicidal compound or “fungicide” refer to a compound that kills or inhibits the growth, viability or pathogenicity of at least one fungus, fungal cell, fungal tissue, zoospore or spore.
  • growth refers to an increases in mass, density or number of cells of said organism.
  • Some common methods for the measurement of growth include the determination of the optical density of a cell suspension, the counting of the number of cells in a fixed volume, the counting of the number of cells by measurement of cell division, the measurement of cellular mass or cellular volume, and the like.
  • fungi refers to whole fungi, fungal organs, tissues, fungal cells and the progeny thereof, it includes Ascomycetes, Basidiomycetes and Oomycetes.
  • fungal cells in the description of the present invention refer to asci, hyphae, pseudohyphae, rhizoid, sclerotia, sterigmata, spores, zoospores, sporodochia, sporangia, synnemata, conidia, ascostroma, cleistotheicia, mycelia, perithecia, basidia and the like and any fungal cells at any developmental stage.
  • the present invention provides a method for identifying fungicidal compounds comprising : a) contacting fungal cells with said compound, b) contacting said fungal cells with an antibody raised against spectrin proteins, and c) observing the distribution of spectrin-like proteins relative to a control, wherein the redistribution of the spectrin-like protein from a inner face of cell membrane to a cytosolic location identifies a candidate compound for a fungicide.
  • the method further comprises an additional step of detecting a growth inhibition, a lack of pathogenesis or the death of the fungal cell.
  • the method according to the invention can be used with compounds applied on hyphae or zoospores.
  • the compound is applied on hyphae, the growth of the hyphae is monitored by measuring the optical density at 600 nm.
  • the term "compounds” is intended to mean any chemical compound or mixture of compounds, including peptides and proteins.
  • mixture of compounds refers to at least two different compounds, such as, for example, the (dia) stereoisomeres of a molecule, mixture of natural origin derived from the extraction of biological material (plants, plant tissues, bacterial cultures, yeast or fungal cultures, insects, animal tissues, etc.) or reaction mixture which are unpurified or totally or partially purified, or else mixtures of products derived from combinatorial chemistry techniques.
  • the invention provides a kit for identification of fungicidal compounds, said kit comprising a) a primary antibody raised against spectrin protein, and b) a secondary antibody conjugated to a signal-producing label, the secondary antibody being one that binds to the primary antibody.
  • the invention deals with the use of antibodies raised against spectrin proteins to identify fungicidal compounds.
  • the present invention deals also with a method of killing phytopathogenic fungi characterized in that it comprises the application on plants of an effective amount of a compound which application on fungal cells will lead to the redistribution of spectrin-like protein.
  • the invention deals with a method for preventively or curatively controlling plant disease caused by phytopathogenic fungi characterized in that it comprises the application on plants of an effective amount of a compound which application on fungi cells will lead to the redistribution of spectrin-like protein.
  • Figure 1 Localisation of spectrin-like protein(s) in hyphae of Phytophtora infestans treated with different fungicides, a) untreated control cell; b) hyphae treated for 24 hours with 10 ppm fluopicolide; c) zoxamide; d ) fenamidone; e) dimethomorph; f) metalaxyl.
  • Figure 2 kinetic of the fluopicolide effect on the distribution of the spectrin-like proteins in hyphae of Phytophthora infestans, hyphae were treated with 10 ppm fluopicolide.
  • Figure 3 Kinetic of fluopicolide effect on distribution of the spectrin-like proteins in zoospores of Phytophthora infestans, zoospores were treated with 3 ppm fluopicolide.
  • Example 1 The effect of fluopicolide (2,6-dichloro-N- ⁇ [3-chloro-5-(trifluoromethyl)-2- pyridinyl]methyl ⁇ -benzamide) on localization of spectrin-like proteins revealed a novel mode of action.
  • fluopicolide, zoxamide is used as a representative of compounds acting as inhibitors of tubulin assembly, fenamidone as an inhibitor of respiration (and more particularly of cytochrome bd), dimethomorph as acting on the cell wall synthesis and metalaxyl as an inhibitor of protein synthesis (more particularly of RNA polymerase I).
  • Phytophthora infestans strain is grown in the dark for 12 days on pea agar medium at 18°C.
  • Tissues used to study are prepared as follows: hyphae are obtained from 10-day-old V8 broth cultures inoculated with plugs of mycelium from culture plates. The differents anti-oomycetes compounds were tested using the following protocol : All fungicides tested are dissolved in dimethyl sulfoxide (DMSO) at 3 mg/ml. The fungicide solution is diluted into medium to give solution of 10 ppm and a final concentration of 1% DMSO. Control represent culture with no drug and in presence of 1% DMSO.
  • DMSO dimethyl sulfoxide
  • Mycelia from wild type strain of Phytophthora infestans treated and untreated, were fixed with 3% paraformaldehyde in 100 rtiM phosphate buffer, pH 7, for 30 min at room temperature, rinsed three times with the same buffer. Partial digestion of the cell wall was done by incubating the cells with 5 mg/ml of Novozym (Sigma) for 10 min at room temperature and stopped by rinsing the cells four times with the phosphate buffer. The cells were then permeabilized with 0.1 % Triton X-100 in the same buffer for 10 min at room temperature. Triton was removed by washing three times in phosphate buffer pH 7.
  • the fixed cells were blocked with phosphate buffer containing 3 % BSA at room temperature overnight followed by incubation with anti-chicken spectrin antibodies (Sigma) diluted to 1 :50 in 3 % BSA phosphate buffer pH 7, for 2 h at 37°C.
  • FITC Fluorescein lsothiocyanate
  • the cells were mounted in p-phenylenediamine- glycerol, with 2.5 ⁇ g/ml of 4', 6-diamidino-2-phenylindole (DAPI).
  • DAPI 6-diamidino-2-phenylindole
  • Example 2 Effect of fluopicolide on the distribution of spectrin-like protein in hyphae of P. infestans.
  • Phytophthora infestans strain is grown in the dark for 12 days on pea agar medium at 18°C.
  • Tissues used to study the effects of fluopicolide are prepared as follows: hyphae are obtained from 10-day-old V8 broth cultures inoculated with plugs of mycelium from culture plates.
  • Fluopicolide is dissolved in dimethyl sulfoxide (DMSO) at 3 mg/ml.
  • DMSO dimethyl sulfoxide
  • the fluopicolide solution is diluted into medium to give solution of 10 ppm and a final concentration of 1%.
  • Control represent culture with no drug and in presence of 1% DMSO.
  • Partial digestion of the cell wall was done by incubating the cells with 5 mg/ml of Novozym (Sigma) for 10 min at room temperature and stopped by rinsing the cells four times with the phosphate buffer. The cells were then permeabilized with 0.1 % Triton X-100 in the same buffer for 10 min at room temperature. Triton was removed by washing three times in phosphate buffer pH 7.
  • the distribution of spectrin-like proteins was examined using corresponding antibodies.
  • the fixed cells were blocked with phosphate buffer containing 3 % BSA at room temperature overnight followed by incubation with anti-chicken spectrin antibodies (Sigma) diluted to 1 :50 in 3 % BSA phosphate buffer pH 7, for 2 h at 37°C.
  • FITC Fluorescein lsothiocyanate
  • the cells were mounted in p-phenylenediamine- glycerol, with 2.5 ⁇ g/ml of 4', 6-diamidino-2-phenylindole (DAPI).
  • DAPI 6-diamidino-2-phenylindole
  • Example 3 Effect of fluopicolide on the distribution of spectrin-like protein in zoospores of P. infestans.
  • zoospores are obtained by flooding 10 day-old culture plates with 10 ml of cold water. The flooded plates were incubated for 3 h at 4 0 C to release the zoospores.
  • the fluopicolide solution is diluted into the zoospore suspension to give solution of 3 ppm and a final concentration of 1% DMSO.
  • Control represents zoospores in presence of 1% DMSO.
  • zoospores are fixed in 3,7 % formaldehyde solution after fluopicolide treatment. Centrifuged zoospores are then permeabilised and labelled with the anti-spectrin antibody as described in Examples 1 and 2.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Mycology (AREA)
  • Botany (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present invention deals with a method for identifying fungicidal compounds comprising the steps of : contacting fungal cells with said compound, contacting said fungal cells with an antibody raised against spectrin protein, and observing the distribution of spectrin-like proteins relative to a control, wherein the redistribution of the spectrin-like protein from a inner face of cell membrane to a cytosolic location identifies a fungicide candidate.

Description

Method for identification of novel fungicides
Background of the invention
The invention relates to methods for the identification of fungicides and more particularly to a method for following the distribution of spectrin-like proteins.
The structural integrity of cells is maintained in part by the cytoskeleton, a mesh-like structure composed primarily of proteins, which lies adjacent to the inner cell surface. The cytoskeleton of many cell type contains large amount of proteins such as tubulin, actin, spectrin and proteins closely related to spectrin.
Spectrin is the principal component of a system of structural proteins that are associated with the cytoplasmic surface of the plasma membrane in most metazoan cells (Bennet and Gilligan 1993, Ann. Rev. Cell Biol. 9: 27-66). The structure and function of spectrin has been extensively investigated in mammalian erythrocytes (Marchesi and Steer 1998, Science, 159 : 203-204; Palek and Lambert 1990, Sem. Hematol. 27 : 290-332; Bennet and Gilligan 1993, Ann. Rev. Cell Biol. 9: 27-66) in which spectrin forms a meshwork responsible for maintaining the shape and mechanical property of the cell. In addition to this structural role, spectrin is involved in the organization of both plasma and internal membranes such as those of the Golgi apparatus, the endoplasmic reticulum and the lysosomes (Beck et al., 1994, J. Cell Biol., 127 : 707-723; 1997, J. Cell Sci., 110 : 1239-1249; Devarajan et al., 1996, J. Cell Biol. 133 : 819-830; Holleran et al., 1996, Trends Cell Biol., 8 : 26-29; Hook et al., 1997, J; Cell Biol. 136 : 1059-1070; De Matteis and Morrow, 2000, J. Cell Sci. 113 : 2331- 2343).
Spectrin-like proteins (SLP) have been found in plants (Michaud et al., 1991, FEBS left., 294 : 77-80; De Ruijter and Emons, 1993, Cell. Biol. Int. 17 : 169-182; Faraday and Dpanswick, 1993, FEBS Lett., 318 : 313-316; Bisikirska and Sikorski, 1997, Naturforsch. C, 52 : 180- 186.), in Saccharomycθs cerevisiae (Slaninova et al., 2003, Can. J. Microbiol., 49:189-196), in the oomycete Saprolegnia ferax (Kaminskyj and Heath, 1996, Mycologia, 88 : 20-37) and the fungus Neurospora crassa (Degousee et al., 2000, Fungal Genet. Biol., 30: 33-44). In plants, spectrin-like epitopes were localized at the plasma membrane in several plant species and different cell types (Michaud et al., 1991, FEBS left., 294 : 77-80; De Ruijter and Emons, 1993, Cell. Biol. Int. 17 :169-182, Wang and Yan, 1991 , Chin. Sci. Bull, 36 : 862- 866). In yeast, spectrin-like proteins were shown to be localized in both plasma membrane and in intracellular membranes (Slaninova et al., 2003, Can. J. Microbiol., 49:189-196). Degousee et al. (2000) reported that in N. crassa, most of the SLP and actin patches were local izeded at the plasma membrane in the region of high apical expansion. In unipolar cells, labeling of spectrin-like proteins with antibodies raised against animal erythrocyte spectrin was found at the plasma membrane in the growing tips of roots hairs (Miller et al., 1997,J. Exp. Bot., 48 : 1881-1896; De Ruijter et al., 1998, Plant J., 13 : 3411- 3500), pollen tubes (Derksen et al., 1995, Acta Bot. Neerl., 44 : 93-119) and fungal hyphae (Kaminsky and Heath, 1995, J. Cell Sci., 108 : 849-856). In Ascomycetes and Oomycetes, spectrin-like proteins are believed to play a role in the maintenance of the membrane stability especially during tip extension.
The present inventors have found that fungi presenting a redistribution of spectrin-like protein upon application of specific fungicide compounds, either inhibit their growth or die. Thus, the distribution of spectrin-like proteins is useful for identification of fungicide compounds.
Summary of the invention
The present inventors have found that fungi presenting a redistribution of spectrin-like proteins upon application of specific fungicide compounds, either inhibit their growth or die. The redistribution of the spectrin-like proteins from the plasma membrane to the cytoplasm of the fungal cells can be observed using antibodies raised against spectrin proteins. Accordingly, the present invention provides methods for the identification of compounds that induce a redistribution of spectrin-like proteins. The methods of the invention are useful for the identification of fungicides.
Detailed description of the invention
Unless otherwise indicated, the following terms are intended to have the following meaning:
The term "spectrin" in the present description of the invention refers to proteins characterized by the presence of spectrin repeats, actin binding domains and EF hands (calcium-binding motifs). In addition, spectrin contains specialized protein-protein interaction motifs and regions for interaction with membranes and phospholipids.
The term "spectrin-like proteins" in the present application refers to proteins that bind to antibodies raised against spectrin proteins. The term "binds to" refers to a non-covalent or a covalent interaction, preferably non- covalent, that holds two molecules together. Non-covalent interaction includes hydrogen bonding, ionic interactions among charged group, van de Waals interactions and hydrophobic interactions among non polar groups. One or more of these interactions can mediate the binding of two molecules to each other. In the present description "binds to" or "binding" refers more particularly to the specific interaction between antibodies raised against spectrin protein and the corresponding spectrin like proteins.
The terms "effective and non-phytotoxic amount" and "agronomical effective" mean an amount of compound according to the invention which is sufficient to control or destroy the fungi present or liable to appear on the crops, and which does not entail any appreciable symptom of phytotoxicity for said crops. Such an amount can vary within a wide range depending on the fungus to be combated, the type of crop and the compounds used. This amount can be determined by systematic field trials, which are within the capabilities of a person skilled in the art.
The compounds identified by the method of treatment according to the present invention are useful to treat propagation material such as tubers or rhizomes, but also seeds, seedlings or seedling pricking out and plants or plants pricking out. This method of treatment according to the invention can also be useful to treat the overground parts of the plant such as truncks, stems or stalks, buds, leaves, flowers and fruits of the concerned plant.
Among the plants that can be protected using the compounds identified by the method according to the present invention, mention may be made of cotton; flax; vine; fruit or vegetable crops such as Rosaceae sp. (for instance pip fruit such as apples and pears, but also stone fruit such as apricots, almonds and peaches), Ribesioidae sp., Juglandaceae sp., Betulaceae sp., Anacardiaceae sp., Fagaceae sp., Moraceae sp., Oleaceae sp., Actinidaceae sp., Lauraceae sp., Musaceae sp. (for instance banana trees and plantins), Rubiaceae sp., Theaceae sp., Sterculiceae sp., Rutaceae sp. (for instance lemons, oranges and grapefruit); Solanaceae sp. (for instance tomatoes), Liliaceae sp., Asteraceae sp. (for instance lettuces), Umbelliferae sp., Cruciferae sp., Chenopodiaceae sp., Cucurbitaceae sp., Papilionaceae sp. (for instance peas), Rosaceae sp. (for instance strawberries); major crops such as Graminae sp. (for instance maize, lawn or cereals such as wheat, rice, barley and triticale), Asteraceae sp. (for instance sunflower), Cruciferae sp. (for instance colza), Fabacae sp. (for instance peanuts), Papilionaceae sp. (for instance soybean), Solanaceae sp. (for instance potatoes), Chenopodiaceae sp. (for instance beetroots); horticultural and forest crops; as well as genetically modified homologues of these crops.
Among the diseases of plants or crops that can be controlled by the method according to the present invention, mention may be made of : Powdery mildew diseases such as :
Blumeria diseases, caused for example by Blumeria graminis;
Podosphaera diseases, caused for example by Podosphaera leucotricha;
Sphaerotheca diseases, caused for example by Sphaerotheca fuliginea; Uncinula diseases, caused for example by Uncinula necator,
Rust diseases such as :
Gymnosporangium diseases, caused for example by Gymnosporangium sabinae;
Hemileia diseases, caused for example by Hemileia vastatrix;
Phakopsora diseases, caused for example by Phakopsora pachyrhizi or Phakopsora meibomiae;
Puccinia diseases, caused for example by Puccinia recondita;
Uromyces diseases, caused for example by Uromyces appendiculatus; Oomycete diseases such as :
Bremia diseases, caused for example by Bremia lactucae; Peronospora diseases, caused for example by Peronospora pisi or P. brassicae;
Phytophthora diseases, caused for example by Phytophthora infestans;
Plasmopara diseases, caused for example by Plasmopara viticola;
Pseudoperonospora diseases, caused for example by Pseudoperonospora humuli or Pseudoperonospora cubensis; Pythium diseases, caused for example by Pythium ultimum;
Leafspot, leaf blotch and leaf blight diseases such as :
Alternaria diseases, caused for example by Alternaria solani;
Cercospora diseases, caused for example by Cercospora beticola;
Cladiosporum diseases, caused for example by Cladiosporium cucumerinum; Cochliobolus diseases, caused for example by Cochliobolus sativus;
Colletotrichum diseases, caused for example by Colletotrichum lindemuthanium;
Cycloconium diseases, caused for example by Cycloconium oleaginum;
Diaporthe diseases, caused for example by Diaporthe citri;
Elsinoe diseases, caused for example by Elsinoe fawcetiii; Gloeosporium diseases, caused for example by Gloeosporium laeticolor,
Glomerella diseases, caused for example by Glomerella cingulata;
Guignardia diseases, caused for example by Guignardia bidwelli;
Leptosphaeria diseases, caused for example by Leptosphaeria maculans; Leptosphaeria nodorum; Magnaporthe diseases, caused for example by Magnaporthe grisea;
Mycosphaerella diseases, caused for example by Mycosphaerella graminicola; Mycosphaerella arachidicola; Mycosphaerella fijiensis; Phaeosphaeria diseases, caused for example by Phaeosphaeria nodorum;
Pyrenophora diseases, caused for example by Pyrenophora teres;
Ramularia diseases, caused for example by Ramularia collo-cygni;
Rhynchosporium diseases, caused for example by Rhynchosporium secalis; Septoria diseases, caused for example by Septoria apii or Septoria lycopercisi;
Typhula diseases, caused for example by Typhula incarnata;
Venturia diseases, caused for example by Venturia inaequalis; Root and stem diseases such as :
Corticium diseases, caused for example by Corticium graminearum; Fusarium diseases, caused for example by Fusarium oxysporum;
Gaeumannomyces diseases, caused for example by Gaeumannomyces graminis;
Rhizoctonia diseases, caused for example by Rhizoctonia solani;
Tapesia diseases, caused for example by Tapesia acuformis;
Thielaviopsis diseases, caused for example by Thielaviopsis basicola; Ear and panicle diseases such as :
Altemaria diseases, caused for example by Alternaria spp.;
Aspergillus diseases, caused for example by Aspergillus flavus;
Cladosporium diseases, caused for example by Cladosporium spp.;
Claviceps diseases, caused for example by Claviceps purpurea; Fusarium diseases, caused for example by Fusarium culmorum;
Gibberella diseases, caused for example by Gibberella zeae;
Monographella diseases, caused for example by Monographella nivalis; Smut and bunt diseases such as :
Sphacelotheca diseases, caused for example by Sphacelotheca miliaria; Tilletia diseases, caused for example by Tilletia caries;
Urocystis diseases, caused for example by Urocystis occulta;
Ustilago diseases, caused for example by Ustilago nuda; Fruit rot and mould diseases such as :
Aspergillus diseases, caused for example by Aspergillus flavus; Botrytis diseases, caused for example by Botrytis cinerea;
Penicillium diseases, caused for example by Penicillium expansum;
Sclerotinia diseases, caused for example by Sclerotinia sclerotiorum;
Verticilium diseases, caused for example by Verticilium alboatrum; Seed and soilbome decay, mould, wilt, rot and damping-off diseases : Fusarium diseases, caused for example by Fusarium culmorum;
Phytophthora diseases, caused for example by Phytophthora cactorum;
Pythium diseases, caused for example by Pythium ultimum; Rhizoctonia diseases, caused for example by Rhizoctonia solani;
Sclerotium diseases, caused for example by Sclerotium rolfsii;
Microdochium diseases, caused for example by Microdochium nivale; Canker, broom and dieback diseases such as : Nectria diseases, caused for example by Nectria galligena;
Blight diseases such as :
Monilinia diseases, caused for example by Monilinia laxa; Leaf blister or leaf curl diseases such as :
Taphrina diseases, caused for example by Taphrina deformans; Decline diseases of wooden plants such as :
Esca diseases, caused for example by Phaemoniella clamydospora; Diseases of flowers and Seeds such as :
Botrytis diseases, caused for example by Botiytis cinerea; Diseases of tubers such as : Rhizoctonia diseases, caused for example by Rhizoctonia solani
The terms "fungicidal compound" or "fungicide" refer to a compound that kills or inhibits the growth, viability or pathogenicity of at least one fungus, fungal cell, fungal tissue, zoospore or spore.
As used in this disclosure, the terms "growth", "cell growth" of an organism refers to an increases in mass, density or number of cells of said organism. Some common methods for the measurement of growth include the determination of the optical density of a cell suspension, the counting of the number of cells in a fixed volume, the counting of the number of cells by measurement of cell division, the measurement of cellular mass or cellular volume, and the like.
The term "fungi" refers to whole fungi, fungal organs, tissues, fungal cells and the progeny thereof, it includes Ascomycetes, Basidiomycetes and Oomycetes. The terms "fungal cells" in the description of the present invention refer to asci, hyphae, pseudohyphae, rhizoid, sclerotia, sterigmata, spores, zoospores, sporodochia, sporangia, synnemata, conidia, ascostroma, cleistotheicia, mycelia, perithecia, basidia and the like and any fungal cells at any developmental stage.
The present invention provides a method for identifying fungicidal compounds comprising : a) contacting fungal cells with said compound, b) contacting said fungal cells with an antibody raised against spectrin proteins, and c) observing the distribution of spectrin-like proteins relative to a control, wherein the redistribution of the spectrin-like protein from a inner face of cell membrane to a cytosolic location identifies a candidate compound for a fungicide.
In another aspect of the invention the method further comprises an additional step of detecting a growth inhibition, a lack of pathogenesis or the death of the fungal cell.
The method according to the invention can be used with compounds applied on hyphae or zoospores.
In the embodiment of the invention, the compound is applied on hyphae, the growth of the hyphae is monitored by measuring the optical density at 600 nm.
According to the invention, the term "compounds" is intended to mean any chemical compound or mixture of compounds, including peptides and proteins.
The terms "mixture of compounds" refers to at least two different compounds, such as, for example, the (dia) stereoisomeres of a molecule, mixture of natural origin derived from the extraction of biological material (plants, plant tissues, bacterial cultures, yeast or fungal cultures, insects, animal tissues, etc.) or reaction mixture which are unpurified or totally or partially purified, or else mixtures of products derived from combinatorial chemistry techniques.
In another aspect the invention provides a kit for identification of fungicidal compounds, said kit comprising a) a primary antibody raised against spectrin protein, and b) a secondary antibody conjugated to a signal-producing label, the secondary antibody being one that binds to the primary antibody.
In a still further aspect the invention deals with the use of antibodies raised against spectrin proteins to identify fungicidal compounds.
The present invention deals also with a method of killing phytopathogenic fungi characterized in that it comprises the application on plants of an effective amount of a compound which application on fungal cells will lead to the redistribution of spectrin-like protein. In a still further embodiment, the invention deals with a method for preventively or curatively controlling plant disease caused by phytopathogenic fungi characterized in that it comprises the application on plants of an effective amount of a compound which application on fungi cells will lead to the redistribution of spectrin-like protein.
Brief description of the figures :
Figure 1 : Localisation of spectrin-like protein(s) in hyphae of Phytophtora infestans treated with different fungicides, a) untreated control cell; b) hyphae treated for 24 hours with 10 ppm fluopicolide; c) zoxamide; d ) fenamidone; e) dimethomorph; f) metalaxyl.
Figure 2 : kinetic of the fluopicolide effect on the distribution of the spectrin-like proteins in hyphae of Phytophthora infestans, hyphae were treated with 10 ppm fluopicolide. a) untreated control cell; b) hyphae treated for 3 min; c) 10 min; d) 2 hours; e) 24 hours. Figure 3 : Kinetic of fluopicolide effect on distribution of the spectrin-like proteins in zoospores of Phytophthora infestans, zoospores were treated with 3 ppm fluopicolide. a) untreated control cell; b) hyphae treated for 1 min; c) 5 min; d) 10 min; e) 20 min.
The following examples are meant to be illustrative of the present invention; however, the practice of the invention is not limited or restricted in any way by them.
Example 1 : The effect of fluopicolide (2,6-dichloro-N-{[3-chloro-5-(trifluoromethyl)-2- pyridinyl]methyl}-benzamide) on localization of spectrin-like proteins revealed a novel mode of action. Different anti-oomycetes compounds were tested : fluopicolide, zoxamide is used as a representative of compounds acting as inhibitors of tubulin assembly, fenamidone as an inhibitor of respiration (and more particularly of cytochrome bd), dimethomorph as acting on the cell wall synthesis and metalaxyl as an inhibitor of protein synthesis (more particularly of RNA polymerase I). Phytophthora infestans strain is grown in the dark for 12 days on pea agar medium at 18°C. Tissues used to study are prepared as follows: hyphae are obtained from 10-day-old V8 broth cultures inoculated with plugs of mycelium from culture plates. The differents anti-oomycetes compounds were tested using the following protocol : All fungicides tested are dissolved in dimethyl sulfoxide (DMSO) at 3 mg/ml. The fungicide solution is diluted into medium to give solution of 10 ppm and a final concentration of 1% DMSO. Control represent culture with no drug and in presence of 1% DMSO. Mycelia, from wild type strain of Phytophthora infestans treated and untreated, were fixed with 3% paraformaldehyde in 100 rtiM phosphate buffer, pH 7, for 30 min at room temperature, rinsed three times with the same buffer. Partial digestion of the cell wall was done by incubating the cells with 5 mg/ml of Novozym (Sigma) for 10 min at room temperature and stopped by rinsing the cells four times with the phosphate buffer. The cells were then permeabilized with 0.1 % Triton X-100 in the same buffer for 10 min at room temperature. Triton was removed by washing three times in phosphate buffer pH 7.
The fixed cells were blocked with phosphate buffer containing 3 % BSA at room temperature overnight followed by incubation with anti-chicken spectrin antibodies (Sigma) diluted to 1 :50 in 3 % BSA phosphate buffer pH 7, for 2 h at 37°C.
Following a rinse in phosphate buffer, the samples were incubated for 1 h at 37°C with Fluorescein lsothiocyanate (FITC) conjugated with corresponding immunoglobulin, diluted to 1 :50 (Sigma).
After a final rinse in phosphate buffer, the cells were mounted in p-phenylenediamine- glycerol, with 2.5 μg/ml of 4', 6-diamidino-2-phenylindole (DAPI).
Confocal immunofluorescence microscopy of the untreated mycelium reveals that spectrin- like proteins are stained in the peripheral regions of the hyphae. After fluopicolide treatment, only spherical cytoplasmic dots are observed showing that fluopicolide induces a change of spectrin-like proteins distribution from the membrane to the cytoplasm. This delocalisation does not exist when hyphae are treated with zoxamide, fenamidone, dimethomorph or metalaxyl (Figure 1).
Example 2 : Effect of fluopicolide on the distribution of spectrin-like protein in hyphae of P. infestans.
Phytophthora infestans strain is grown in the dark for 12 days on pea agar medium at 18°C. Tissues used to study the effects of fluopicolide are prepared as follows: hyphae are obtained from 10-day-old V8 broth cultures inoculated with plugs of mycelium from culture plates.
Fluopicolide is dissolved in dimethyl sulfoxide (DMSO) at 3 mg/ml. The fluopicolide solution is diluted into medium to give solution of 10 ppm and a final concentration of 1%. Control represent culture with no drug and in presence of 1% DMSO. Mycelia, from wild type strain of Phytophthora infestans treated with fluopicolide and untreated, were fixed with 3% paraformaldehyde in 100 mM phosphate buffer, pH 7, for 30 min at room temperature, rinsed three times with the same buffer. Partial digestion of the cell wall was done by incubating the cells with 5 mg/ml of Novozym (Sigma) for 10 min at room temperature and stopped by rinsing the cells four times with the phosphate buffer. The cells were then permeabilized with 0.1 % Triton X-100 in the same buffer for 10 min at room temperature. Triton was removed by washing three times in phosphate buffer pH 7.
The distribution of spectrin-like proteins was examined using corresponding antibodies. The fixed cells were blocked with phosphate buffer containing 3 % BSA at room temperature overnight followed by incubation with anti-chicken spectrin antibodies (Sigma) diluted to 1 :50 in 3 % BSA phosphate buffer pH 7, for 2 h at 37°C.
Following a rinse in phosphate buffer, the samples were incubated for 1 h at 37°C with Fluorescein lsothiocyanate (FITC) conjugated with corresponding immunoglobulin, diluted to 1 :5 (Sigma).
After a final rinse in phosphate buffer, the cells were mounted in p-phenylenediamine- glycerol, with 2.5 μg/ml of 4', 6-diamidino-2-phenylindole (DAPI).
Confocal immunofluorescence microscopy of the untreated mycelium reveals that spectrin- like proteins are stained in the peripheral regions of the hyphae. After fluopicolide treatment, only spherical cytoplasmic dots are observed showing that fluopicolide induces a change of spectrin-like proteins distribution from the membrane to the cytoplasm. This delocalisation is observed within 3 minutes and is maintained after 24 hours of treatment (Figure 2)
Example 3 : Effect of fluopicolide on the distribution of spectrin-like protein in zoospores of P. infestans.
For the assay, zoospores are obtained by flooding 10 day-old culture plates with 10 ml of cold water. The flooded plates were incubated for 3 h at 4 0C to release the zoospores. The fluopicolide solution is diluted into the zoospore suspension to give solution of 3 ppm and a final concentration of 1% DMSO. Control represents zoospores in presence of 1% DMSO. For immunofluorescence experiments zoospores are fixed in 3,7 % formaldehyde solution after fluopicolide treatment. Centrifuged zoospores are then permeabilised and labelled with the anti-spectrin antibody as described in Examples 1 and 2.
In untreated zoospores spectrin-like protein are localized in the cell periphery. Upon addition of fluopicolide, a significant dispersion of spectrin-like proteins throughout the cytoplasm is observed (Figure 3). This effect is extremely fast since it is observed within 1 minute after treatment.

Claims

Claims
1. A method for identifying fungicidal compounds comprising : a) contacting fungal cells with said compound, b) contacting said fungal cells with an antibody raised against spectrin protein, and c) observing the distribution of spectrin-like proteins relative to a control, wherein the redistribution of the spectrin-like protein from a inner face of cell membrane to a cytosolic location identifies a fungicide candidate.
2. The method of claim 1 further comprising : contacting fungal cells with said compound and detecting a growth inhibition, a lack of pathogenesis or death.
3. The method according to claim 1 or 2 in which the compound is applied on hyphae.
4. The method according to claim 1 or 2 in which the compound is applied on zoospores.
5. The method according to claim 3 in which the growth inhibition is monitored by measuring the optical density at 600 nm.
6. Kit for identification of fungicidal compounds said kit comprising d) a primary antibody raised against spectrin protein, and e) a secondary antibody conjugated to a signal-producing label, the secondary antibody being one that binds to the primary antibody.
7. Use of antibodies raised against spectrin protein to identify fungicidal compounds.
EP06763587A 2005-06-09 2006-06-08 Method for identification of novel fungicides Ceased EP1893771A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US68913205P 2005-06-09 2005-06-09
PCT/EP2006/063016 WO2006131546A1 (en) 2005-06-09 2006-06-08 Method for identification of novel fungicides

Publications (1)

Publication Number Publication Date
EP1893771A1 true EP1893771A1 (en) 2008-03-05

Family

ID=36940632

Family Applications (1)

Application Number Title Priority Date Filing Date
EP06763587A Ceased EP1893771A1 (en) 2005-06-09 2006-06-08 Method for identification of novel fungicides

Country Status (3)

Country Link
EP (1) EP1893771A1 (en)
JP (1) JP2008545982A (en)
WO (1) WO2006131546A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117368146B (en) * 2023-12-08 2024-03-12 苏陀科技(北京)有限公司 Rapid detection method for mycelium protein content

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995028159A1 (en) * 1994-04-18 1995-10-26 Faezeh Sarfarazi Compositions useful for glaucoma treatment
JP2004248596A (en) * 2003-02-20 2004-09-09 Osaka Industrial Promotion Organization Screening method of antibacterial agent and microorganism used for it

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2006131546A1 *

Also Published As

Publication number Publication date
WO2006131546A1 (en) 2006-12-14
JP2008545982A (en) 2008-12-18

Similar Documents

Publication Publication Date Title
Geddes et al. Differential expression of proteins in response to the interaction between the pathogen Fusarium graminearum and its host, Hordeum vulgare
RU2762235C2 (en) Application of a cellular extract of one or more microscopic algae of the amphidinium genus for fungicidal and/or bactericidal activity thereof against fungi, oomycetes and/or pathogenic bacteria of plants and crop seeds
Schibler et al. The colR4 and colR15 beta-tubulin mutations in Chlamydomonas reinhardtii confer altered sensitivities to microtubule inhibitors and herbicides by enhancing microtubule stability.
JP5356247B2 (en) Pesticide active compositions containing synthetic and fungicidal compounds useful as nodulation factors for legumes
Zottich et al. Purification, biochemical characterization and antifungal activity of a new lipid transfer protein (LTP) from Coffea canephora seeds with α-amylase inhibitor properties
Ribeiro et al. Isolation and characterization of novel peptides from chilli pepper seeds: antimicrobial activities against pathogenic yeasts
Tóth et al. The potential use of the Penicillium chrysogenum antifungal protein PAF, the designed variant PAFopt and its γ‐core peptide Pγopt in plant protection
JP7248663B2 (en) Use of amphidinol for its fungicidal and/or bactericidal activity against fungi, oomycetes and/or pathogenic bacteria in crop plants and seeds
KR20170129699A (en) Fungicide enhancers effective in treating plants infected with fungal pathogens
Hatamoto et al. Aminopyrifen, a novel 2-aminonicotinate fungicide with a unique effect and broad-spectrum activity against plant pathogenic fungi
Ribeiro et al. Capsicum annuum L. trypsin inhibitor as a template scaffold for new drug development against pathogenic yeast
Yemets et al. Cadmium, nickel, copper, and zinc influence on microfilament organization in Arabidopsis root cells
Sambasivam et al. Evidence of recent increased pathogenicity within the Australian Ascochyta rabiei population
Bertoldi Human peroxiredoxins 1 and 2 and their interacting protein partners; through structure toward functions of biological complexes
WO2006131546A1 (en) Method for identification of novel fungicides
Fischer et al. The direct toxicity of BABA against grapevine ecosystem organisms
JP4434643B2 (en) Methods for identifying inhibitors of 20S and 26S proteasomes
Noll et al. Allicin From Garlic Disrupts the Cytoskeleton in Plants
US11589588B2 (en) Methods of identification and use of nematicide compounds
Fourie Metalaxyl sensitivity status of downy mildew populations in Western Cape vineyards
Kononova et al. Suppression of development of vancomycin-resistant Staphylococcus epidermidis by low-molecular-weight cationic peptides of the lantibiotic family
Nanni et al. Dimethomorph activity and its effect on morphology in different oomycete species of economic and veterinary interest
Masler Effects of catechin polyphenols and preparations from the plant–parasitic nematode Heterodera glycines on protease activity and behaviour in three nematode species
三谷滋 et al. Biological Properties of Fungitoxic Propargyl N-(6-ethyl-5-iodo-2-pyridyl) carbamate
Deepak et al. Developmental stage response of pearl millet downy mildew (Sclerospora graminicola) to fungicides

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20080109

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI SK TR

DAX Request for extension of the european patent (deleted)
17Q First examination report despatched

Effective date: 20080530

DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN REFUSED

18R Application refused

Effective date: 20090925