Antifungal combinations

Abstract

The present invention relates to a combination of active components. Wherein the active components comprise (i) a polypeptide with cell membrane interacting properties and (ii) a polycationic polymer. The present invention further relates to mixtures and (agrochemical) compositions comprising the combination of the current invention. The invention further provides methods of application and use of the combination, mixture or (agrochemical) composition to protect or treat a plant field, plant, or part of the plant from or for an infection by a plant pathogenic fungus.

Description

[0001] Antifungal combinations

[0002] Field of the invention

[0003] The present invention relates to a combination of active components. Wherein the active components comprise (i) a polypeptide with cell membrane interacting properties and (ii) a polycationic polymer. The present invention further relates to mixtures and (agrochemical) compositions comprising the combination of the current invention. The invention further provides methods of application and use of the combination, mixture or (agrochemical) composition to protect or treat a plant field, plant, or part of the plant from or for an infection by a plant pathogenic fungus.

[0004] Therefore, the present invention is situated in the field of antifungal combinations, mixtures or compositions, preferably in the field of agrochemical combinations, mixtures or compositions. Preferably the combinations, mixture or compositions of the current invention have an antifungal activity against a plant pathogenic fungus.

[0005] Background

[0006] The current applicant has developed several proteinaceous pesticides based on heavy chain variable domain of a heavy chain antibody (VHH). For instance, VHH binding to a glucosylceramide of a fungal pest were developed showing antifungal activity (WO 2014 / 177595, WO 2014 / 191146 and WO 2016 / 071438) and more recently a VHH interaction with a lipid fraction of fungal pests have shown the ability of VHH to cause retardation of growth of a spore and even lysis of a spore of a fungal pests such as the economically important fungal plant pests Botrytis cinerea (WO 2021 / 198396). Although agrochemical compositions comprising anti-fungal agents are available that can act for instance on the cell membrane of fungal pathogens, the anti-fungal agent is not always able to (efficiently) reach its site of action due to the presence of the fungal cell wall. In addition, there remains the challenge, when employing protein based active ingredient (such as enzymes or antibodies or antibody fragments such as VHH antibodies) products in agriculture, of the relatively high cost of producing such proteins by fermentation and the high quantities that are often required to treat crops.

[0007] Antifungal properties of chitosan have been described decades ago (Carolyn and Hadwiger, 1979). Today the antifungal properties of chitosan have been widely studied and documented and are now well recognized. Ben-Shalom et al. describe treatment of grey mould caused by Botrytis cinerea in cucumber plants by means of chitosan (Ben-Shalom et al., 2003). DeGenring et al. describe the antifungal effect of chitosan against Botrytis cinerea on Petunia leaves (DeGenring et al., 2023) The specific mode of action of chitosan when applied to a fungal pest may be multifactorial. For one, due to its polycationic properties, chitosan can interfere with the plasma membrane of the cell, leading to abnormal membrane permeability (Benhamou and Lafontaine, 1992). In addition a cytological study of a Fusarium species revealed that chitosan may lead to alterations in the cell wall of the fungus through stimulating release of B-1 ,3-glucanases and chitosonases (Benhamou and Lafontaine, 1992). Chitosan may be produced by enzymatic or chemical deacetylation of chitin. One example of making chitosan from chitin derived from fungal mycelium is described in WO2023180067 Method for producing chitosan from mycelium.

[0008] The presence and persistence of pathogenic fungal infections seen in patients and animals but also in plant crops can be mainly attributed to the selective pressure of broad-spectrum antifungal active components, which are available at present. In addition, available traditional antifungal active components (e.g. synthetic organic chemicals) are increasingly under pressure by regulatory authorities and many areand will be banned from the market due to their negative impact on human and animal health and the environment.

[0009] Accordingly, there remains a need in the art to provide antifungal combinations, mixtures and compositions having a reduced negative impact on human and animal health and the environment while having improved properties, in particular with the ability to more effectively target the site of action of the anti-fungal agent, as well as reduced the dose rate required for efficient pest control, in a simple and easy to apply manner.

[0010] Summary

[0011] The present invention relates to a combination of active components. Wherein the active components comprise (i) a polypeptide with cell membrane interacting properties and (ii) a polycationic polymer. That is to say, the combination has at least two different active components: (i) a polypeptide with cell membrane interacting properties and (ii) a polycationic polymer. Preferably at least one of the active components is an antifungal active component. The inventors have surprisingly found that the combination, mixture or composition comprising said active components and according to the invention have a synergistical antifungal effect. Said surprising finding may greatly contribute to more efficient ways of treating or protecting plant fields, plants, part of plants (including crops) for or from a plant pathogenic fungal infection.

[0012] In a preferred embodiment, the polycationic polymer is chitosan, specifically a fungal chitosan as further described herein; and the polypeptide with cell membrane interacting properties is a VHH antibody with cell membrane interacting properties as further described herein. In a preferred embodiment, the combination according to the invention has an antifungal effect against a plant pathogenic fungus, more preferably the combination according to the invention has a synergistic curative antifungal effect and / or a synergistic protective antifungal effect against at least one plant pathogenic fungus.

[0013] The present invention further relates to mixtures, such as a tank mix, comprising the combination as described herein. The invention further relates to compositions comprising the combination of the current invention, such as (agrochemical) compositions comprising at least one agrochemically suitable additive.

[0014] The invention further relates to methods of applying the combination, mixture or composition according to the invention. Specifically, the invention relates to methods of applying the combination, mixture or composition according to the invention to a surface, such as the leaf of a plant or the fruit of a plant, a vegetable or a plant seed. In some embodiments, the combination, mixture or composition according to the invention are applied post-harvest. In some embodiments, the combination, mixture or composition according to the invention is applied to protect a plant field, plant, or part of the plant from an infection by a plant pathogenic fungus. In another embodiment, the combination, mixture or composition according to the invention is applied to treat a plant field, plant, or part of the plant for an infection by a plant pathogenic fungus. Therefore, the invention further relates to the use of the combination, mixture or composition according to the invention to treat and / or protect a plant field, plant, or part of the plant for or from at least one plant pathogenic fungus infection. And where said at least one plant pathogenic fungus is a plant pathogenic fungus from the genus chosen from the group comprising Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Corynespora, Diplodia, Erysiphe, Fusarium, Leptosphaeria, Gaeumannomyces, Helminthosporium, Leveillula, Macrophomina, Nectria, Oidium, Penicillium, Peronospora, Phoma, Phymatotrichum, Phytophthora, Plasmopara, Podosphaera, Puccinia, Pyrenophora, Pyricularia, Pythium, Rhizoctonia, Scerotium, Sclerotinia, Septoria, Sphaerotheca, Thielaviopsis, Uncinula,Venturia, Verticillium, Magnaporthe, Blumeria, Mycosphaerella, Ustilago, Melampsora, Phakopsora, Pseudoperonospora, Monilinia, Mucor, Rhizopus, Zymoseptoria and Aspergillus. Preferably the plant pathogenic fungus is Botrytis cinerea.

[0015] List of Figures

[0016] Figure 1 : Disease severity of Botrytis cinerea on bean leaf discs. Bars represent % area infected leaf for each treatment.

[0017] Figure 2: Visualization of bean leaf discs treated with different concentration ratios of Kitogreen and 11 A11 (checkerboard setup). Disease severity of Botrytis cinerea on bean leaf discs can be calculated from the infected leaf area for each treatment.

[0018] List of Sequences

[0019] SEQ ID NOs: 1 to 84: Polypeptides with cell membrane interacting properties as disclosed in PCT / EP2014 / 058771

[0020] SEQ ID NOs: 85 to 135: Polypeptides with cell membrane interacting properties as disclosed in PCT / EP2021 / 058548

[0021] SEQ ID NOs: 136 to 162: Polypeptides with cell membrane interacting properties as disclosed in PCT / EP2024 / 088373

[0022] Detailed description

[0023] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[0024] All documents cited in the present specification are hereby incorporated by reference in their entirety. Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0025] The present invention will be described with respect to particular embodiments but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope.

[0026] The invention relates to combinations of active components, wherein the active components comprise (i) a polypeptide with cell membrane interacting properties and (ii) a polycationic polymer. Or rather, the combination of the current invention is a combination of two or more different active components. Preferably at least one active component is an antifungal active component, more preferably both the polypeptide with cell membrane interacting properties and the polycationic polymer are antifungal active components. The current inventors have surprisingly found that a combination of a polypeptide with cell membrane interacting properties and a polycationic polymer shows an improved antifungal activity, and where said antifungal activity is improved compared to what would be expected from the mere addition of the antifungal activity of either the polypeptide with cell membrane interacting properties or the polycationic polypeptide when assessed separately or in isolation in an in vitro and / or in vivo antifungal assay. That is to say, by combining a polypeptide with cell membrane interacting properties and a polycationic polymer, a combination with improved antifungal properties is obtained. Said antifungal effect may be curative and / or preventative. Said combination may preferably be formed prior to packaging the polypeptide with cell membrane interacting properties and a polycationic polymer i.e. where the two components are present in a single container such as in the form a agrochemical composition. Alternatively, the combination of the polypeptide with cell membrane interacting properties and a polycationic polymer may be formed prior to application when two or more components are mixed such as mixed in a tank mix. Alternatively, thecombination of the polypeptide with cell membrane interacting properties and a polycationic polymer may be formed at the point of application i.e. where the two components are applied separately but in short succession so to exert the antifungal effect of both components at the same time and on the same surface, such as the surface of a leaf of a plant or the fruit of a plant, a vegetable or a plant seed. Put differently, the polypeptide with cell membrane interacting properties and a polycationic polymer may be applied separately or combined in one mixture or composition before being applied. The polypeptide with cell membrane interacting properties and a polycationic polymer may be applied simultaneously or consecutively. If the polypeptide with cell membrane interacting properties and a polycationic polymer are applied consecutively, the polycationic polymer is preferably applied prior to the application of the polypeptide with cell membrane interacting properties. Preferably, the polypeptide with cell membrane interacting properties and polycationic polymer are applied simultaneously.

[0027] Therefore, the invention further relates to mixtures comprising the combination according to the invention. The combination according to the invention may be a mixture, more specifically the combination according to the invention may be formed in a tank mix. Or rather, the combination of a polypeptide with cell membrane interacting properties and a polycationic polymer may be formed at the point where a tank mix is prepared i.e. where the two components are in for example separate containers but are combined prior to applying the components to a surface, such as the surface of a leaf of a plant or the fruit of a plant, a vegetable or a plant seed.

[0028] The invention further relates to compositions comprising the combination according to the invention. In a preferred embodiment, the combination according to the invention is comprised in an agrochemical composition, or a composition suitable for agricultural use.

[0029] In some embodiments, the application rate of the polypeptide with cell membrane interacting properties as present in the combination, mixture or composition according to the invention, is below 500g of said polypeptide with cell membrane interacting properties per hectare (ha). In specific embodiments the application rate is below 400g / ha, below 300g / ha, below 200 g / ha, below 10Og / ha, 50 g / ha, below 40 g / ha, below 35 g / ha, below 30 g / ha, below 25 g / ha, below 20 g / ha, below 15 g / ha, below 10 g / ha, below 5 g / ha, below 1 g / ha or even lower amounts of the polypeptide with cell membrane interacting properties per hectare. In a further embodiment, said upper application rate may be combined with any one of the following lower application rates to provide a suitable application rate range, namely wherein the application rate is above 0.1 g / ha, above 1 g / ha, above 2 g / ha, above 3 g / ha, above 4 g / ha, above 5 g / ha, above 6 g / ha, above 7 g / ha, above 8 g / ha, above 9 g / ha, above 10g / ha, above 15 g / ha, above 20 g / ha , above 50 g / ha, above 100 g / ha, above 150 g / ha, above 200 g / ha, above 300 g / ha, or above 400 g / ha. As is evident therefrom, in a further particular embodiment, the application rate of the polypeptide with cell membrane interacting properties is from 1 g / ha to 500 g / ha.

[0030] In some embodiments, the application rate of the polycationic polymer as present in the combination, mixture or composition according to the invention, is below 1000g of said polycationic polymer per hectare. In specific embodiments the application rate is below 900g / ha, below 800g / ha, below 700 g / ha, below 600g / ha, 500 g / ha, below 400 g / ha, below 350 g / ha, below 300 g / ha, below 250 g / ha, below 200 g / ha, below 150 g / ha, below 100 g / ha, below 50 g / ha, below 10 g / ha or even lower amounts of the polycationic polymer per hectare. In a further embodiment, said upper application rate may be combined with any one of the following lower application rates to provide a suitable application rate range, namely wherein the application rate is above 0.1 g / ha, above 1 g / ha, above 2 g / ha, above 3 g / ha, above 4 g / ha, above 5 g / ha,above 6 g / ha, above 7 g / ha, above 8 g / ha, above 9 g / ha, above 10g / ha, above 15 g / ha, above 20 g / ha, above 50 g / ha, above 100 g / ha, above 150 g / ha, above 200 g / ha, above 300 g / ha, above 400 g / ha, above 500 g / ha, above 600 g / ha, above 700 g / ha, or above 800 g / ha. As is evident therefrom, in a further particular embodiment, the application rate of the polypeptide with cell membrane interacting properties is from 1 g / ha to 1000 g / ha.

[0031] It being understood that the application rate of the polypeptide with cell membrane interacting properties and / or the polycationic polymer is reduced as compared to when the combination, mixture or composition would containing either the polypeptide with cell membrane interacting properties or the polycationic polymer alone. In some embodiments, the polypeptide with cell membrane interacting properties and the polycationic polymer act synergistically to allow a reduction in one or both components in the combination, mixture or composition when applied to for example a field of plants. In some embodiments, the combined application rate of the polypeptide with cell membrane interacting properties and the polycationic polymer when applied to for example a field of plants may be lower than the equivalent combined concentrations of the polypeptide with cell membrane interacting properties and the polycationic polymer applied separately and independently, whilst achieving the same or improved antifungal effect because of the synergy seen when the two components are combined in a single application. The presence of a synergistic effect (as opposed to an additive effect), may be calculated using various mathematical solutions, usually based on assessing the EC50 value of the antifungal effect obtained by the individual active components and the EC50 value of the active components in combination. One example is the method referred to as the Wadley method (Levy et al., 1986) as is used herein in Example 3.

[0032] Therefore, the combinations, mixtures or compositions of the current invention having antifungal properties and comprising as active components a polypeptide with cell membrane interacting properties, and a polycationic polymer, will result in a decreased application rate to achieve a desired antifungal effect, or, alternatively, may result in an improved antifungal effect when the polypeptide with cell membrane interacting properties, and the polycationic polymer are applied as a combination. Where the cost of goods or cost of application is an important economic consideration, reducing the application rate required to achieve a desired antifungal effect is preferred.

[0033] In a preferred embodiment, the application rate of the polypeptide with cell membrane interacting properties, when present in a combination, mixture or composition with a polycationic polymer as described herein, may be reduced 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, or even a 50-fold reduction. In a preferred embodiment, the fold reduction of the polypeptide with cell membrane interacting properties when present in a combination, mixture or composition with a polycationic polymer as described herein, is at least 2-fold, more preferably 4-fold, even more preferably at least 10-fold reduced as compared to when the polypeptide with cell membrane interacting properties would be applied alone, i.e. in the absence of a polycationic polymer, whilst still achieving the desired antifungal effect. As a specific example for application to one hectare (ha) of crops, field of crops or field of plants, the amount of the polypeptide with cell membrane interacting properties when present in a combination, mixture or composition with a polycationic polymer as described herein, will decrease of from 500g / ha to 450g / ha, more preferably of from 500g / ha to 400g / ha, more preferably of from 500g / ha to 350g / ha, more preferably of from 500g / ha to 300g / ha, more preferably of from 500g / ha to 250g / ha, more preferably of from 500g / ha to 200g / ha, more preferably of from 150g / ha to 100g / ha, evenmore preferably of from 500g / ha to 50g / ha whilst still obtaining a desirable antifungal effect when applied to a surface, such as the surface of a leaf of a plant or the fruit of a plant, a vegetable or a plant seed, and as compared to when the polypeptide with cell membrane interacting properties would be applied alone, i.e. in the absence of a polycationic polymer.

[0034] In a preferred embodiment, the application rate of the polycationic polymer, when present in a combination, mixture or composition with a polypeptide with cell membrane interacting properties as described herein, may be reduced 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, or even a 50-fold reduction. In a preferred embodiment, the fold reduction of the polycationic polymer when present in a combination, mixture or composition with a polypeptide with cell membrane interacting properties as described herein, is at least 2-fold, more preferably 4-fold, even more preferably at least 10-fold reduced as compared to when the polycationic polymer would be applied alone, i.e. in the absence of a polypeptide with cell membrane interacting properties, whilst still achieving the desired antifungal effect. As a specific example for application to one hectare (ha) of crops, field of crops or field of plants, the amount of the polycationic polymer when present in a combination, mixture or composition with a polypeptide with cell membrane interacting properties as described herein, will decrease of from 1000g / ha to 900g / ha, more preferably of from 1000g / ha to 800g / ha, even more preferably of from 1000g / ha to 700g / ha, even more preferably of from 1000g / ha to 600g / ha, even more preferably of from 1000g / ha to 500g / ha, even more preferably of from 1000g / ha to 400g / ha, even more preferably of from 1000g / ha to 300g / ha, even more preferably of from 1000g / ha to 200g / ha, even more preferably of from 1000g / ha to 100g / ha, whilst still obtaining a desirable antifungal effect when applied to a surface, such as the surface of a leaf of a plant or the fruit of a plant, a vegetable or a plant seed, and as compared to when the polycationic polymer would be applied alone, i.e. in the absence of a polypeptide with cell membrane interacting properties.

[0035] Therefore, in an embodiment of the invention, the mixture or composition comprises the polypeptide with cell membrane interacting properties and a polycationic polymer in such amounts that when applied to a hectare of a crop, field of crops or field of plants the application rate of the polypeptide with cell membrane interacting properties is of from 50g / ha to 250g / ha and the application of the polycationic polymer is of from 150 to 600 g / ha. It being understood that an expression of application rate as an amount of active component per hectare may not be suitable for every application, for example, an expression of amount of product per leaf wall area may sometimes be more preferable, or when applying to a fruit or vegetable in a post-harvest setting, concentrations will be expressed as concentrations per liter of liquid in which the fruit or vegetable are for example submerged. Nonetheless, similar or comparable amounts or application rates will be maintained and similar gains in reduction of application amounts or rates will be observed when applying or using the combinations, mixtures or compositions according to the invention. It is understood depending on the crop and the environmental pressure of the plant pests that the farmer can vary the application rate. These application rates and variances (depending on application type, crop type, pathogen type, environment) are specified in the technical sheet delivered with the specific agrochemical composition.

[0036] When testing the active ingredients present in the combination, mixture or composition according to the current invention in in vitro or in vivo assays, such as for example the assays as described herein (Example 1 , 2 or 3), the skilled person will be able to assess the antifungal effect of the individual activecomponents as compared to the antifungal effect of the combination, mixture or composition comprising said active components according to the current invention. By assessing the EC50 value of each component and comparing said EC50 value with the EC50 value of the combination, mixture or composition, the skilled person will be able to assess the appropriate concentrations and expected effects of the combination, mixture or composition comprising a polypeptide with cell membrane interacting properties and a polycationic polymer. For instance, by using the herein described in vitro or in vivo assays, the skilled person will be able to assess the presence of a synergistic effect between a polypeptide with cell membrane interacting properties, and a polycationic polymer and further evaluate the ratio of which said components may need to be added for optimal results.

[0037] In an embodiment of the current invention, the ratio of the polypeptide with cell membrane interacting properties versus the polycationic polymer present in the combination, mixture or composition of the invention, ranges of from 100:1 to 1 :100, or from 1 :10 to 10: 1 , or from 1 :5 to 5:1 , or from 1 :2 to 2:1 , or from 0.5:1 to 2:1 . In some embodiments, the composition comprises equal amounts of the polypeptide with cell membrane interacting properties versus the polycationic polymer. Wherein the ratio of the polypeptide with cell membrane interacting properties versus the polycationic polymer is defined by the ratio of their quantities expressed in grams such as by comparing the ratio of application rates in grams per hectare or as concentrations in grams per liter.

[0038] In a preferred embodiment, the polypeptide with cell membrane interacting properties is a VHH antibody with cell membrane interacting properties, more preferably a VHH antibody capable of binding to at least one fungus as further described herein. Preferably said fungus is a plant pathogenic fungus. In a preferred embodiment, the polycationic polymer is chitosan, where said chitosan is derived from fungal, insect or crustacean chitin, more preferably from fungal chitin as further described herein. Therefore, in a preferred embodiment the current invention relates to a mixture or composition comprising a VHH antibody, as further described herein, and chitosan, as further described herein.

[0039] Definitions

[0040] Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps.

[0041] Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated.

[0042] The term "about” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier 'about' refers is itself also specifically, and preferably, disclosed.

[0043] The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrooket al., Molecular Cloning: A Laboratory Manual, 2nded., Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks, to the general background art referred to above and to the further references cited therein.

[0044] The term “cell membrane” or “fungal cell membrane” as used herein refers to the plasma membrane or cytoplasmic membrane that separates and protects the inside of the cell from the outside environment. It is largely build up from a phospholipid bilayer and is a more fluid structure compared to a more rigid cell wall that sits outside of the cell membrane. The cell membrane comprises many membrane proteins such as transmembrane proteins that can inter alia sense the environment and regulate the flux of nutrients, metabolites, catabolites and other components in and out of the cell. Additionally, the cell membrane is needed to maintaining an electrostatic potential over the membrane to allow for energy production in the cell. The cell membrane may also comprise specialized lipids such as sphingolipids for example glucosylceramide. In general the components that build up the cell membrane and can be targeted by an antifungal component such as a VHH antibody may be referred to as “plasma membrane components”. The cell membrane is generally sensitive to osmotic pressure and other physical and non-physical disturbances for which the cell wall may in some cases provide protection. When the cell membrane is ruptured, the content of the cell typically flows out into the environment a process sometimes referred to as cell lysis.

[0045] The term “VHH antibody” as defined herein refers to the variable domain of a heavy chain antibody which can be derived from an organism having as part of its immune system heavy chain antibodies (next to conventional antibodies having both heavy and light chains) such as a camelid species (referred to as a VHH or sometimes known as nanobodies) or shark species (sometimes referred to as Immunoglobulin new antigen receptors or IgNARs). Additionally the VHH antibody as defined herein can be derived from a heavy chain of a conventional antibody. The skilled person will know that when obtaining a VHH antibody from a conventional antibody certain modification may have to be made to improve solubility and / or stability of the VH chain in absence of the VL light chain.

[0046] The term “interacting” or “interaction” or “interacts with” in the context of active components such as a “polypeptide with cell membrane interacting properties” or a “polycationic polymer”, means the ability of the active component to interact with or bind to the cell membrane. Alternatively, the term interacting may also refer to active components that interact with a cellular process responsible for building or maintaining the cell membrane and whose disruption may lead to a destabilized cell membrane, such as the cell membrane of a fungal spore or fungal cell of a plant pathogenic fungus. In some aspects, and as an example where the active components may have a direct effect on the cell membrane, interacting may mean intercalating between the bilayer of the membrane. Such an active components that can intercalate in the membrane is for example an antimicrobial peptide or AMP that can disrupt the integrity of the cell membrane, which in some conditions may lead to the disruption and / or rupture or lysis of the cell, meaning that the contents of the cell are released into the environment. Interacting may also mean binding to an antigen on the surface of the membrane such as a binding to a sphingolipid for example a glucosylceramide that may influence fungal cell differentiation. Interaction with the membrane by an active component may also indicate that a certain membrane protein is impeded by the active component to perform its normal activity or function which may affect a cellular process not directly related to the cell membrane as such, for example binding of a membrane bound protein may influence cellular processes by hampering nutrientinflux or hampering the generation of energy in the form of ATP in the cell, such interaction may be accomplished by binding of for example a VHH antibody to a membrane bound protein on the surface of the cell membrane. In the context of the current invention and as explained herein, the effect or activity of the active component, specifically a polypeptide with cell membrane interacting properties, may be improved when the cell wall is (partially) degraded, disturbed or weakened, either by allowing the active component facilitated access to the cell membrane or by failing to protect the cell membrane sufficiently against active components that may cause the cell membrane to rupture or lyse. In a preferred embodiment, the antifungal component with cell membrane interacting properties is a polypeptide, more preferably a VHH antibody, in this context the term interacting means the ability of the VHH antibody to reach its target on the surface of the cell membrane. In a special case of the preferred embodiment, the VHH antibody may further lead to the destabilization of the cell membrane.

[0047] In the context of the current invention, the polypeptide with cell membrane interacting properties may have an improved access to the membrane due to the activity of the polycationic polymer where the polycationic polymer may destabilise the cell wall of a plant pathogenic fungus. Additionally or alternatively, the polycationic polymer further disturbs the cell membrane through a direct interaction leading to a joint activity of the polypeptide with cell membrane interacting properties and the polycationic polymer. In such a scenario, the polycationic polymer may not only facilitate the VHH antibody to reach its target on the cell membrane more easily but may also improve the effectiveness with which the VHH antibody leads to the rupture of a fungal spore or fungal cell.

[0048] In one embodiment, the antifungal compound with cell membrane interacting properties interacts with the cell membrane by intercalating into the cell membrane. In a more preferred embodiment said antifungal compound is a polypeptide, more preferably an antimicrobial peptide or AMP.

[0049] In another preferred embodiment, the antifungal compound with cell membrane interacting properties, interacts with the cell membrane by binding to a target on the surface of the cell membrane. In a more preferred embodiment said antifungal compound is a polypeptide, more preferably an antibody, even more preferably a VHH antibody or a fragment thereof as further described herein.

[0050] In another more preferred embodiment, the antifungal compound with cell membrane interaction properties interacts with the cell membrane by binding to a target on the surface of the cell membrane and hereby causing retardation of growth of a spore or cell of a fungal pest and / or lysis of a spore or cell of a fungal pest. In a more preferred embodiment said antifungal compound is a polypeptide, more preferably an antibody, even more preferably a VHH antibody or a fragment thereof.

[0051] The term “application rate” as used herein is understood as the amount of active ingredient applied per application, an application being understood as a single event (such as a spray event) of applying the mixture or composition comprising said active ingredient on a crop, field of crops or field of plants. In a specific treatment program, multiple applications may happen over a certain time point. For instance a first application may be followed by a second application and where the time between the first application and the second application may be several days such as one week. In some embodiments up to 10 preferably up to 5 applications may be envisioned. It being understood that such a treatment plan may vary between crops and the plant pathogenic fungus targeted by the treatment.

[0052] An “IC50 value”, "EC50”, “IC50”, "EC50” or “half maximal inhibitory concentration” as used herein interchangeably is a measure for the potency of active components or combination, mixture or compositions as described herein in inhibiting the growth of a fungal pathogen. In practice (also see Example 3) thegrowth of a fungal pathogen is tracked by measuring the confluency of said fungal pathogen in for example a well of a 96-well plate or 384-well plate in the presence of a series of different concentrations of the active components or combinations thereof as described herein. The confluency data at the time-point at which the non-treated or negative control reaches for example 90% saturation is then taken for each concentration of active components or combinations as described herein. Said confluency values are then plotted against the log transformed concentration values yielding a sigmoidal curve. This sigmoid curve may serve to define an IC50 value. Where this IC50 is the concentration of the active components or combinations as described herein where the corresponding confluence value is 50% of the saturated value as estimated by the maximum of the sigmoidal function.

[0053] Polycationic polymer of the invention

[0054] The current invention relates to combinations, mixtures or compositions comprising active components, wherein the active components comprise a polypeptide with cell membrane interacting properties, and a polycationic polymer. In a preferred embodiment, said polycationic polymer is chitosan and said polypeptide with cell membrane interacting properties is a VHH antibody with cell membrane interacting properties.

[0055] Preferably the polycationic polymer is chitosan, however the current invention is not limited thereto other polycationic polymers may show antifungal properties and are expected to function well with polypeptides with membrane interacting properties, non-limiting examples of alternative polycationic polymers are polyethylenimine (branched or linear), polydiallyldimethylammonium chloride (pDADMAC), diethylaminoethyl-dextran (DEAE-dextran) or poly (amidoamine) (PANAM).

[0056] In a preferred embodiment the polycationic polymer is chitosan. Chitosan is a polycationic polymer derived from chitin. Chitin is a polymer highly abundant in the cell wall of fungi, the exoskeleton of insects, and crustaceans. The production of chitosan from chitin is well known in the art. Chitosan may be produced by deacetylation of chitin by either an enzymatical process (through the activity of deacetylase enzymes) or by alkaline conditions (such as for instance treatment by high molar concentrations of NaOH). The source of chitin may be diverse and commonly chitin is obtained (or derived or produced) from crustaceans where the waste fractions of the shellfish industry are used. Alternatively, chitosan may be obtained from the fungal cell wall. Chitosan may be obtained from the fungal cell wall through an enzymatic process. For example the process as described in EP1483299, where the fungal cell wall is first treated with a B-1 ,3-glucanase to obtain chitin and thereafter deacetylated by a chitin deacetylase. Chitosan may also be obtained from fungal mycelium through a chemical process, such as the process described in WO2023180067, where the fungal mycelium is treated with an alkaline reagent to obtain chitosan, specifically NaOH may be used. More specifically, for obtaining chitosan from Aspergillus niger, a 50% (weight / volume) of NaOH in water solution is added to the fungal mycelium at a concentration of 50% (weight / volume), liberation and deacetylation of chitin is then achieve by stirring said mixture at a preferred temperature of 105°C, preferably for 6 hours and where the pH is adjusted to pH 10. Hereafter, the mixtures is washed several times in water, to remove presence of p-glucans and other contaminants, thereafter chitosan may further be filtered whereafter the chitosan is solubilised in an aqueous solution with a pH below 6, preferably 4.5 and where the pH may be lowered by for example addition of acetic acid. Alternatively, chitosan is readily available commercially and can be purchased through suppliers such as Sigma Aldrich (Merck KGaA), Tidal Grow® AgriScience, or KitoZyme NV.In a preferred embodiment, the polycationic polymer of the mixture or composition of the current invention is chitosan and where the chitosan has an average degree of deacetylation of from 60% to 100%, such as from 65% to 100%, such as from 70% to 100%, such as from 75% to 100%, such as from 80% to 100%, such as from 85% to 100%, such as from 90% to 100% or from 95% to 100%. In the context of the present invention, a high degree of deacetylation refers to an average degree of deacetylation of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0057] In a preferred embodiment, the polycationic polymer of the combination, mixture or composition of the current invention is chitosan and where the chitosan has an average molecular weight of from 1 kDa to 2000 kDa. More preferably the chitosan has an average molecular weight of below 2000 kDa, such as below 1500kDa, below 1000 kDa, below 500 kDa, below 450 kDa, below 400 kDa, below 350 kDa below 300 kDa, below 250 kDa, below 200 kDa, below 150 kDa or even below 100 kDa. In a further embodiment, said upper limit for the average molecular weight of chitosan may be combined with any one of the following lower average molecular weight of chitosan to provide an average molecular weight range, namely wherein the average molecular weight of chitosan is above 1 kDa, above 5 kDa, above 10 kDa, above 15 kDa, above 20 kDa, above 25 kDa, above 30 kDa, above 35 kDa, above 40 kDa, above 45 kDa, above 50 kDa, above 100 kDa, above 150 kDa, , above 200 kDa, , above 250 kDa, above 300 kDa, above 350 kDa, above 400 kDa, above 450 kDa, above 500 kDa, above 600 kDa, above 700 kDa, above 800 kDa, above 900 kDa, or above 1000 kDa. As is evident therefrom, in a further particular embodiment, the average molecular weight of chitosan is from 1 kDa to 2000 kDa.

[0058] In some embodiments, low molecular weight chitosan, generally refers to chitosan with an average MW of from 1 kDA to 190 kDa. Medium molecular weight chitosan, general refers to chitosan with an average MW of from 191 kDa to 310 kDa. High molecular weight chitosan, generally refers to chitosan with an average MW of from 311 to 375 kDa or higher. In an embodiment of the invention chitosan is low molecular weight chitosan and where the chitosan is derived from fungal chitin. In another embodiment of the invention chitosan is low molecular weight chitosan and where chitosan is derived from crustacean chitin. In an embodiment of the invention chitosan is medium molecular weight chitosan and where the chitosan is derived from fungal chitin. In another embodiment of the invention chitosan is medium molecular weight chitosan and where chitosan is derived from crustacean chitin. In an embodiment of the invention chitosan is high molecular weight chitosan and where the chitosan is derived from fungal chitin. In another embodiment of the invention chitosan is high molecular weight chitosan and where chitosan is derived from crustacean chitin. Preferably a low molecular weight chitosan is used in the combinations according to the invention. For example, in a preferred embodiment, the chitosan is a low molecular weight chitosan having a high degree of deacetylation.

[0059] Where the average molecular weight of chitosan may dictate, in conjunctions with other factors such as pH, salt concentrations and / or deacetylation rate, the solubility of chitosan in an aqueous solution. Where generally lower molecular weight chitosan molecules would have an improved solubility. In a preferred embodiment the mixture or composition of the current invention comprises chitosan and where the chitosan is soluble. It being understood that solubility of chitosan should be achieved prior to application of the combination, mixture or composition according to the invention to a plant field, plant or part of a plant. That is to say, chitosan may be present in a mixture or composition according to the invention and where said mixture or composition may be in a dried, powdery or granular form and where solubilisation isachieved when for example a tank mix is prepared prior to application of the mixture or composition to a plant field, plant or part of a plant.

[0060] Solubility of chitosan may further depend on the pH of the aqueous solution. In addition, the protonation of chitosan will depend on the pH of the aqueous solution and wherein a lower pH will lead to an increased protonation of the ammonium residues and hence a positive charge of the chitosan molecules. Therefore in a preferred embodiment the mixtures and compositions according to the invention and when present in an aqueous solution, preferably have a pH of below 7, such as pH 6.5 , such as pH 6, such as pH 5.5 , such as pH 5 or even pH 4.5. In other embodiments, the pH of the mixtures and compositions according to the invention may be of from pH 7 to pH 10, such as pH 7.5, pH8, pH8.5, pH 9, pH 9.5 or pH 10.

[0061] In a preferred embodiment, the polycationic polymer of the combination, mixture or composition of the invention is chitosan. Any type of chitosan may be used in the combination, mixture or composition according to the invention as long as the chitosan is sufficiently soluble in an aqueous solution suitable for agricultural applications and shows an antifungal effect when tested in an in vitro and / or in vivo antifungal assay (such as the assays of Examples 1 , 2 or 3 as described herein). In a more preferred embodiment, the polycationic polymer of the combination, mixture or composition of the invention is chitosan, preferably a fungal chitosan, more preferably a chitosan derived from Aspergillus niger. More preferably chitosan is obtained using a chemical process as described specifically in WO2023180067, or the chitosan is the chitosan commercially available as Kitogreen as produced by Kitozyme NV, or the chitosan is the chitosan commercially available as Tidal Grow® Spectra as produced by Tidal Grow AgriScience.

[0062] Polypeptides with cell membrane interacting properties of the invention

[0063] The current invention relates to combinations, mixtures or compositions comprising active components comprising a polypeptide with cell membrane interacting properties, and a polycationic polymer. In a preferred embodiment, said polycationic polymer is chitosan and said polypeptide with cell membrane interacting properties is a VHH antibody with cell membrane interacting properties. However, as explained herein, the inventions is not limited to VHH antibodies, rather the combination as described herein may enhance the effect of any polypeptide or other type of component with cell membrane interacting properties as is explained herein.

[0064] In some embodiments, the polypeptides with cell membrane interacting properties may be capable of (specifically) binding to a lipid-containing fraction of the plasma membrane of a fungus, such as for example a lipid-containing fraction of Botrytis cinerea or other fungus. Said lipid-containing fraction (of Botrytis cinerea or otherwise) may be obtainable by chromatography. The chromatography may be performed on a crude lipid extract (also referred to herein as a total lipid extract, or TLE) obtained from fungal hyphae and / or conidia. The chromatography may be, for example, thin-layer chromatography or normal-phase flash chromatography. The chromatography (for example thin-layer chromatography) may be performed on a substrate, for example a glass plate coated with silica gel. The chromatography may be performed using a chloroform / methanol mixture (for example 85 / 15% v / v) as the eluent.

[0065] For example, said lipid-containing fraction may be obtainable by a method comprising: fractionating hyphae and / or conidia of a fungus (for example Botrytis cinerea or other fungus) by total lipid extract thin-layer chromatography and selecting the fraction with a Retention Factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipids fraction.In a more specific embodiment, the lipid-containing fraction may be obtainable by a method comprising:

[0066] fractionating hyphae and / or conidia of a fungus (for example Botrytis cinerea or other fungus) by total lipid extract thin-layer chromatography on a silica-coated glass slide using a chloroform / methanol mixture (for example 85 / 15% v / v) as the eluent and selecting the fraction with a Retention Factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipids fraction.

[0067] Alternatively, the fraction may be obtained using normal-phase flash chromatography. In such a method, the method may comprise:

[0068] fractionating hyphae and / or conidia of a fungus (for example Botrytis cinerea or other fungus) by total lipid extract normal-phase flash chromatography, and selecting the fraction with a Retention Factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipids fraction.

[0069] In a more specific embodiment, the lipid-containing fraction may be obtainable by a method comprising:

[0070] fractionating hyphae and / or conidia of a fungus (for example Botrytis cinerea or other fungus) by total lipid extract normal-phase flash chromatography comprising dissolving the TLE in dichloromethane (CH2CI2) and MeOH and using CH2CI2 / MeOH (for example 85 / 15%, v / v) as the eluent, followed by filtration of the fractions through a filter.

[0071] In a more specific embodiment, the lipid-containing fraction may be obtainable by a method comprising:

[0072] fractionating hyphae and / or conidia of a fungus (for example Botrytis cinerea or other fungus) by total lipid extract normal-phase flash chromatography comprising dissolving the TLE in dichloromethane (CH2CI2) and MeOH loading the TLE on to a phase flash cartridge (for example a flash cartridge with 15 pm particles), running the column with CH2CI2 / MeOH (85 / 15%, v / v) as the eluent, and filtering the fractions through a filter (for example a 0.45 pm syringe filter with a nylon membrane) and drying the fractions.

[0073] The fractions from the chromatography may be processed prior to testing of binding of the polypeptide to the fraction or of interaction with the fraction. For example, liposomes comprising the fractions may be prepared. Such a method may comprise the use of thin-film hydration. For example, in such a method, liposomes may be prepared using thin-film hydration with the addition of 1 ,6-diphenyl-1 ,3,5-hexatriene (DPH). Binding and / or disruption of the membranes by binding of the polypeptide may be measured by a change in fluorescence before and after polypeptide binding (or by reference to a suitable control).

[0074] Accordingly, in some embodiments, the polypeptides of and used in the invention may (specifically) bind to a lipid-containing chromatographic fraction of the plasma membrane of a fungus, optionally wherein the lipid-containing chromatographic fraction is prepared into liposomes prior to testing the binding of the polypeptide thereto.

[0075] Binding of the polypeptide to a lipid-containing fraction of a fungus may be confirmed by any suitable method, for example bio-layer interferometry. Specific interactions with the lipid-containing fractions may be tested. For example, it may be determined if the polypeptide is able to disrupt the lipid fraction when the fraction is prepared into liposomes, for example using thin-film hydration.

[0076] In methods involving chromatography, an extraction step may be performed prior to the step of chromatography. For example, fungal hyphae and / or conidia may be subjected to an extraction step to provide a crude lipid extract or total lipid extract on which the chromatography is performed. For example,in some embodiments, fungal hyphae and / or conidia (for example fungal hyphae and / or conidia of Fusarium oxysporum or Botrytis cinerea) may be extracted at room temperature, for example using chloroform: methanol at 2:1 and 1 :2 (v / v) ratios. Extracts so prepared may be combined and dried to provide a crude lipid extract or TLE.

[0077] Accordingly, in some embodiments, the polypeptide may be capable of (specifically) binding to a lipid-containing fraction of the plasma membrane of a fungus (such as Fusarium oxysporum or Botrytis cinerea), wherein the lipid-containing fraction of the plasma membrane of the fungus is obtained or obtainable by chromatography. The chromatography may be normal-phase flash chromatography or thin-layer chromatography. Binding of the polypeptide to the lipid to the lipid-containing fraction may be determined according to bio-layer interferometry. In some embodiments, the chromatography step may be performed on a crude lipid fraction obtained or obtainable by a method comprising extracting lipids from fungal hyphae and / or conidia from a fungal sample. The extraction step may use chloroform: methanol at 2:1 and 1 :2 (v / v) ratios to provide two extracts, and then combining the extracts.

[0078] In methods relating to thin-layer chromatography, the chromatography may comprise the steps of: fractionating hyphae of the fungus by total lipid extract thin-layer chromatography and selecting the fraction with a Retention Factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipids fraction.

[0079] In some methods relating to thin-layer chromatography, the chromatography may comprise the steps of:

[0080] fractionating hyphae and / or conidia of a fungus (for example Botrytis cinerea or other fungus) by total lipid extract thin-layer chromatography on a silica-coated glass slide using a chloroform / methanol mixture (for example 85 / 15% v / v) as the eluent and selecting the fraction with a Retention Factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipids fraction.

[0081] In methods relating to normal-phase flash chromatography, the chromatography may comprise the steps of:

[0082] fractionating hyphae and / or conidia of a fungus (for example Botrytis cinerea or other fungus) by total lipid extract normal-phase flash chromatography, and selecting the fraction with a Retention Factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipids fraction.

[0083] In some methods relating to normal-phase flash chromatography, the chromatography may comprise the steps of:

[0084] fractionating hyphae and / or conidia of a fungus (for example Botrytis cinerea or other fungus) by total lipid extract normal-phase flash chromatography comprising dissolving the TLE in dichloromethane (CH2CI2) and MeOH and using CH2CI2 / MeOH (for example 85 / 15%, v / v) as the eluent, followed by filtration of the fractions through a filter.

[0085] In some methods relating to normal-phase flash chromatography, the chromatography may comprise the steps of:

[0086] fractionating hyphae and / or conidia of a fungus (for example Botrytis cinerea or other fungus) by total lipid extract normal-phase flash chromatography comprising dissolving the TLE in dichloromethane (CH2CI2) and MeOH loading the TLE on to a phase flash cartridge (for example a flash cartridge with 15 pm particles), running the column with CH2CI2 / MeOH (85 / 15%, v / v) as the eluent, and filtering the fractions through a filter (for example a 0.45 pm syringe filter with a nylon membrane) and drying the fractions.More specifically polypeptides with cell membrane interacting properties more specifically polypeptides capable of binding to a lipid-containing fraction of the plasma membrane of a fungus, such as for example a lipid-containing fraction of Botrytis cinerea or other fungus, may be antibodies more specifically a VHH antibody or a fragment thereof, more specifically any one of SEQ ID NO: 85 to 135 corresponding to SEQ ID NO: 1 to 51 of PCT / EP2021 / 058548.

[0087] Alternatively, the polypeptides with cell membrane interacting properties may be capable of (specifically) binding to sphingolipids present in the fungal cell membrane, for instance 9-methyl 4,8-sphingadienine, glycosylceramides, glucosylceramide, monoglucosylceramides, oligoglucosylceramides, gangliosides, sulfatides, ceramides, sphingosine-1 -phosphate, ceramide-1 -phosphate, galactosylceramide, inositol-phosphorylceramide (IPC), mannosyl-inositol- phosphorylceramide (MIPC), galactosyl-inositol- phosphorylceramide, mannosyl-(inositol-phosphoryl)2-ceramide (M(IP)2C), dimannosyl-inositol- phosphorylceramide (M2IPC), galactosyl-dimannosyl-inositol- phosphorylceramide (GalM2IPC), mannosyl-di-inositol-diphosphorylceramide,di-inositol-diphosphorylceramide, trigalactosylglycosylceramide.

[0088] Non-limiting examples of sphingolipids, to which the polypeptides that may be used in the current invention may bind, are glycosylceramides, glucosylceramide, sphingomyelin, monoglycosylceramides, oligoglycosylceramides, gangliosides, sulfatides, ceramides, sphingosine-1 -phosphate and ceramide-1 -phosphate. In a preferred embodiment the polypeptide may bind to a glucosylceramide of a fungal cell, such as glucosylceramide of a Botrytis or Fusarium fungal cell. More specifically polypeptides with cell membrane interacting properties capable of binding to sphingolipids, specifically glucosylceramide may be an antibody more specifically a VHH antibody or a fragment thereof, more specifically any one of SEQ ID NO: 1 to 84 corresponding the SEQ ID NO: 1 to 84 of PCT / EP2014 / 058771 .

[0089] In some embodiments the polypeptide with cell membrane interacting properties may be a VHH antibody comprising the amino acid sequence according to SEQ ID NO: 136 to 162 corresponding to SEQ ID NOs 2, 41 to 65 and 242 of PCT / EP2024 / 088373.

[0090] The polypeptides with membrane interacting properties of the combinations, mixtures or compositions as described herein can be naturally occurring polypeptides or amino acid sequences, they can be derived from a naturally occurring polypeptide, or alternatively they can be entirely artificially designed or synthesised. The polypeptides or amino acid sequences can be immunoglobulin-based or they can be based on domains present in proteins, including but not limited to microbial proteins, protease inhibitors, toxins, fibronectin, lipocalins, single chain antiparallel coiled coil proteins or repeat motif proteins. Non-limiting examples of such polypeptides, with the herein described ranges of amino acid lengths, include carbohydrate binding domains (CBD) (Blake et al (2006) J. Biol. Chem. 281 , 29321-29329), heavy chain antibodies (hcAb), single domain antibodies (sdAb), minibodies (Tramontane et al (1994) J. Mol. Recognition 7, 9-24), the variable domain of camelid heavy chain antibodies (VHH), the variable domain of the new antigen receptors (VNAR), affibodies (Nygren P.A. (2008) FEBS J. 275, 2668-2676), alphabodies (see WQ2010066740), designed ankyrin-repeat domains (DARPins) (Stumpp et al (2008) Drug Discovery Today 13, 695-701 ), anticalins (Skerra et al (2008) FEBS J. 275, 2677-2683), knottins (Kolmar et al (2008) FEBS J. 275, 2684-2690) and engineered CH2 domains (nanoantibodies, see Dimitrov DS (2009) mAbs 1 , 26-28). In particular, the polypeptides with membrane interacting properties of the combinations, mixtures or compositions as described herein consist of a single polypeptide chain and are not post-translationally modified. More particularly, the polypeptides with membrane interacting properties of the combinations,mixtures or compositions as described herein are derived from an innate or adaptive immune system, preferably from a protein of an innate or adaptive immune system. Still more particularly, said polypeptides are derived from an immunoglobulin. Most particularly, said polypeptides comprise 4 framework regions and 3 complementary determining regions, or any suitable fragment thereof (which will then usually contain at least some of the amino acid residues that form at least one of the complementary determining regions). In particular, said polypeptides are easy to produce at high yield, preferably in a microbial recombinant expression system, and convenient to isolate and / or purify subsequently. Particularly, said polypeptides are selected from the group consisting of DARPins, knottins, alphabodies and VHH antibodies. More particularly, said polypeptides are selected from the group consisting of alphabodies and VHH antibodies. Most particularly, said polypeptides are VHH antibodies. However, it should be noted that the invention is not limited as to the origin of the polypeptides with membrane interacting properties of the combinations, mixtures or compositions as described herein, nor as to the way that said polypeptides are generated or obtained. Thus, the polypeptides with membrane interacting properties of the combinations, mixtures or compositions as described herein may be naturally occurring polypeptides (from any suitable species) or synthetic or semi-synthetic polypeptides. In a specific but non-limiting embodiment of the invention, the polypeptide is a naturally occurring immunoglobulin sequence (from any suitable species) or a synthetic or semi-synthetic immunoglobulin sequence, including but not limited to “camelized” immunoglobulin sequences, as well as immunoglobulin sequences that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing.

[0091] The polypeptide sequences of the compositions disclosed herein may in particular be a domain antibody (or an heavy chain variable domain that is suitable for use as a domain antibody), a single domain antibody (or an heavy chain variable domain that is suitable for use as a single domain antibody), or a "dAb" (or an heavy chain variable domain that is suitable for use as a dAb); other single variable domains, or any suitable fragment of any one thereof. For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684. For the term “dAb’s”, reference is for example made to Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), to Holt et al., Trends Biotechnol., 2003, 21 (11 ):484-490; as well as to for example WO 06 / 030220, WO 06 / 003388 and other published patent applications of Domantis Ltd.

[0092] Thus, in particular embodiments, the present invention provides combinations, mixtures or compositions as described herein comprising polypeptides with cell membrane interacting properties with the (general) structure

[0093] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4

[0094] in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively.

[0095] In some aspects, the polypeptides with cell membrane interacting properties of the combinations, mixtures or compositions as described herein may be a small peptide with anti-microbial properties such as an antimicrobial peptide or AMP. AMPs usually have a length of in the range of from about 10 to about 50 amino acids. AMPs are commonly anionic or cationic (i.e. they are charged) and can be subdivided in 4 classes: (i) anionic peptides which are rich in glutamic and aspartic acids, (ii) linear cationic a-helicalpeptides, (iii) cationic peptides enriched for specific amino acid rich in proline, arginine, phenylalanine, glycine, tryptophan and (iv) anionic / cationic peptides forming disulfide bonds. More specific examples are plant derived AMPs with antimicrobial activities such as peptides composed of at least two helical domains connected by a linker / turn such as plant-derived amphipathic helix or two helices engineered into a helix-tum-helix (HTH) format in which homologous or heterogeneous helices are connected by a peptide linker. For example, as described in WO2021202476, W02020072535, W02020176224 or W02003000863. More specifically, the AMPs that may be used in the current invention may be AMPs having the ability to disrupt or destabilise the cell membrane of fungal cells. In the invention, when the polypeptide with cell membrane interacting properties is an AMP, the AMP and the polycationic polymer as described herein may act synergistically, for example to allow the antifungal effect to be reached at a concentration or dose lower than the dose that would be required when the AMP or polycationic polymer is applied alone. The combination of an AMP and a polycationic polymer may act synergistically in the same way as seen for the combination of other polypeptides and a polycationic polymer, for example the combination of VHH antibodies and polycationic polymers. In this way, effective pest control can be achieved using lower doses or concentrations of each antifungal component.

[0096] In a preferred embodiment, the combination, mixture or composition according to the invention comprises at least one VHH antibody, preferably as described above, and chitosan, preferably the chitosan as described above, and where the combination of said VHH antibody and said chitosan leads to an improved antifungal effect compared to as what would be expected based on the antifungal effect of the components individually.

[0097] Combinations

[0098] The current invention relates to combinations of active components, wherein the active components comprises a polypeptide with cell membrane interacting properties, and a polycationic polymer. In a preferred embodiment, said polycationic polymer is chitosan and said polypeptide with cell membrane interacting properties is a VHH antibody with cell membrane interacting properties. Therefore, the combinations of the current invention comprises at least two active components. The combination of the current invention has an antifungal effect when applied to a fungus, more preferably the combination of the current invention has an antifungal effect when applied to a plant pathogenic fungus. In some embodiments, the at least two active components have no or no detectable antifungal activity when applied in isolation (i.e. when present in a composition with only one active component), and where antifungal activity is observed only when at least two active components are used in combination. In some embodiments at least one active component has an antifungal activity, in some embodiments at least two active components have a detectable antifungal activity when tested in isolation. It is therefore envisioned that one active component may not be active unless a second component is added. As a specific example, a certain polypeptide with membrane interacting properties may not show activity when applied in isolation since it may not be able to reach the membrane target or (detectably) act on the membrane target unless chitosan is added. In a preferred embodiment, both the polypeptide with cell membrane interacting properties and the polycationic polymer have antifungal activity.

[0099] In an embodiment according to the invention, the active components comprise or consist of a VHH antibody with cell membrane interacting properties, and chitosan.

[0100] In an embodiment according to the invention, the active components comprise or consist of a VHH antibody comprising an amino acid sequence according to any one SEQ ID NO: 1 to 162, and chitosan.In a preferred embodiment according to the invention, the active components comprise or consist of a VHH antibody comprising an amino acid sequence according to any one SEQ ID NO: 1 to 84, and chitosan.

[0101] In a preferred embodiment according to the invention, the active components comprise or consist of a VHH antibody comprising an amino acid sequence according to any one SEQ ID NO: 85 to 135, and chitosan.

[0102] In a preferred embodiment according to the invention, the active components comprise or consist of a VHH antibody comprising an amino acid sequence according to any one SEQ ID NO: 136 to 162, and chitosan.

[0103] In a preferred embodiment according to the invention, the active components comprise or consist of a VHH antibody according to SEQ ID NO: 86, and chitosan.

[0104] In a preferred embodiment according to the invention, the active components comprise or consist of a VHH antibody according to SEQ ID NO: 136, and chitosan.

[0105] In a preferred embodiment according to the invention, the active components comprise or consist of a VHH antibody according to SEQ ID NO: 162, and chitosan.

[0106] In a preferred embodiment according to the invention, the active components comprise or consist of a VHH antibody comprising the 3 Complementary Determining Regions (CDR1 , CDR2 and CDR3) as present in any one of SEQ ID NO: 1 to 162 and as defined by the IMGT (the international ImMunoGeneTics information system) CDR definition scheme, and chitosan.

[0107] Compositions

[0108] The invention further relates to compositions comprising the combination according to the invention. In a preferred embodiment, the combination according to the invention is comprised in an agrochemical composition, or a composition suitable for agricultural used. Therefore, the composition according to the invention may comprises at least one agrochemically suitable additive and where said additive is selected from at least one of the group consisting of diluents, solvents, adjuvants, surfactants, wetting agents, spreading agents, oils, stickers, thickeners, penetrants, buffering agents, acidifiers, anti-settling agents, anti-freeze agents, photo-protectors, UV protectors, defoaming agents, biocides and drift control agents.

[0109] The compositions as disclosed herein may be in solid or liquid forms. In some embodiments, the composition may be a solid composition, more specifically the composition may be a dustable powder, granule or agglomerate, in particular those obtained by extrusion, by compacting, by impregnation of a granulated carrier, by spray-drying or by granulation through spray-drying using either a liquid or a powder as starting material such as for example described in WO2024 / 141645. Such solid compositions may be optionally used in the form of a liquid, for example by diluting or suspending in water, particularly for protecting a field of plants, plant or part of a plant from an infection by a plant pathogenic fungus or for treating a field of plants, plant or part of a plant for an infection by a plant pathogenic fungus. In some embodiments, the composition is present as a liquid composition such as water-soluble concentrates, emulsions, suspension concentrates, wettable powders (or spraying powder), oils, or self-emulsifiable concentrates such as described in WO2024 / 141638. Such liquid compositions may be optionally used in the form of a diluted liquid, for example by diluting or suspending in water, particularly for protecting a field of plants, plant or part of the plant from an infection by a plant pathogenic fungus or for treating a field of plants, plant or part of the plant for an infection by a plant pathogenic fungus. Such dilution or suspension in water may for example be done in a tank, such as a tank for spraying of a field of plants, plant or part ofthe plant, or a tank for dipping or submerging of a plant or part of the plant, here specifically a fruit of a plant, a vegetable or a plant seed. These compositions therefore include not only compositions which are ready to be applied to a field of plants, plant or part of the plant to be treated by means of a suitable device, such as a spraying, dusting, submerging device, but also concentrated commercial compositions which must be diluted before application to the crop.

[0110] Antifungal activity of the compositions

[0111] “Active components” as used herein refers to components such as agrochemicals or polypeptides, that are suitable for use in agriculture (for example, pesticides, growth regulators, nutrients / fertilizers, repellents, defoliants etc.) such as for use in a plant field, or field of crops or for use in applications on plants, part of a plant, fruits, vegetables, seeds etc, and where such active components may further be meant for use in greenhouse grown crops (e.g. horticulture / floricu Iture) or hydroponic culture systems and even the non-crop uses such as uses in private gardens, household uses (for example, herbicides or insecticides for household use), or uses by pest control operators (for example, weed control etc.). In the context of the current invention, the active components are preferably antifungal active components.

[0112] Where with “antifungal active component” is meant agrochemicals or polypeptides (such as VHH antibodies or AMP as further defined herein) or polycationic polymers (such as chitosan as further defined herein) having an antifungal effect (or activity) against a plant pathogenic fungus, where such an effect may be a fungicidal or fungistatic effect (or activity).

[0113] “Fungistatic (effect)” or “Fungistatic use” or “fungistatic activity”, as used herein, includes any effect or use of an active component (optionally comprised in a combination, mixture or composition as defined herein) for controlling, modulating or interfering with the harmful activity or infection of or by a fungus, including but not limited to inhibiting the growth or activity of the fungus, altering the behaviour of the fungus, and repelling or attracting the fungus in plants, plant parts or in other agro-related settings, such as for example for household uses or in soil.

[0114] “Fungicidal (effect)” or “Fungicidal use” or “fungicidal activity”, as used herein, includes any effect or use of an active component (optionally comprised in a combination, mixture or composition as defined herein) for killing the fungus in or on plants, plant parts or in other agro-related settings, such as for example for household uses or in soil.

[0115] “Fungistatic activity”, as used herein, means to interfere with the harmful activity of a fungus, including but not limited to inhibiting the growth or activity of the fungus, altering the behaviour of the fungus, and repelling the fungus.

[0116] In a preferred embodiment of the current invention the combination comprises at least two active components and where preferably at least one active component is an antifungal active component, more preferably at least two components are antifungal components. In a preferred embodiment, at least one active component is a polypeptide with cell membrane interacting properties as defined herein and at least one active component is a polycationic polymer as defined herein. Preferably said active components are active against a plant pathogenic fungus. The combination of active components according to the current invention leads to a synergistic effect or an effect that is greater than the effect of the components as tested in isolation (more than additive effect), said effect of the combination (or mixtures and compositions comprising said combination) as described herein may be a synergistic curative antifungal effect and / or a synergistic protective antifungal effect against a plant pathogenic fungus, preferably a synergistic protective antifungal effect against a plant pathogenic fungus. Said synergistic protective antifungal effect may be a i afungistatic of fungicidal effect, preferably the effect is a fungicidal effect. In some cases, application of the combination, mixture or composition of the invention may have both a (synergistic) curative and a (synergistic) protective effect (i.e. said combination, mixture or composition may kill the plant pathogenic fungus present in the plant field or on a plant or part of the plant and also at the same time prevent the plant pathogenic fungus of spreading to other plants or part of the plant).

[0117] In a preferred embodiment, the plant pest(s) that is / are combated by the combinations, mixtures or compositions as disclosed herein is a fungus, such as a plant pathogenic fungus, as defined below. Fungi can be highly detrimental for plants and can cause substantial harvest losses in crops. Plant pathogenic fungi include necrotrophic fungi and biotrophic fungi, and include ascomycetes, basidiomycetes and oomycetes.

[0118] Examples of plant pathogenic fungi are known in the art and include, but are not limited to, those selected from the group consisting of the Genera: Alternaria; Ascochyta; Botrytis; Cercospora; Colletotrichum; Diplodia; Erysiphe; Fusarium; Leptosphaeria; Gaeumanomyces; Helminthosporium; Macrophomina; Monilinia; Nectria; Oidium; Penicillium; Peronospora; Phakopsora; Phoma; Phymatotrichum; Phytophthora; Plasmopara; Podosphaera; Puccinia; Puthium; Pyrenophora; Pyricularia; Pythium; Rhizoctonia; Scerotium; Sclerotinia; Septoria; Thielaviopsis; Uncinula; Venturia; and Verticillium. Specific examples of plant fungi infections which may be combated by the combinations, mixtures or compositions as disclosed herein include, powdery mildew and botrytis cinerea in fruit and vegetable crops such as grapes and strawberries. Additional specific examples of plant fungi infections which may be combated by the combinations, mixtures or compositions as disclosed herein include Erysiphe graminis in cereals, Erysiphe cichoracearum and Sphaerotheca fuliginea in cucurbits, Monolinia fructicola in fruit, Penicillium expansum in apples, Podosphaera leucotricha in apples, Podosphaera aphanis, for example to treat powdery mildew, for example on strawberry, Podosphaera xanthii, for example to treat powdery mildew, for example on cucumber, Oidium neolycopersici, for example to treat powdery mildew, for example on tomatoes, Uncinula necator in vines, Puccinia sp. In cereals, Rhizoctonia sp. In cotton, potatoes, rice and lawns, Ustilago sp. In cereals and sugarcane, Venturia inaequalis (scab) in apples, Helminthosporium sp. In cereals, Septoria nodorum in wheat, Septoria tritici in wheat, Rhynchosporium secalis on barley, Botrytis cinerea (gray mold) in strawberries, tomatoes and grapes, Cercospora arachidicola in groundnuts, Peronospora tabacina in tobacco, or other Peronospora in various crops, Pseudocercosporella herpotrichoides in wheat and barley, Pyrenophera teres in barley, Pyricularia oryzae in rice, Phytophthora infestans in potatoes and tomatoes, Fusarium sp. (such as Fusarium oxysporum) and Verticillium sp. In various plants, Plasmopara viticola in grapes, Alternaria sp. In fruit and vegetables, Pseudoperonospora cubensis in cucumbers, Mycosphaerella fijiensis in banana, Ascochyta sp. In chickpeas, Leptosphaeria sp. On canola, Phakopsora spp., such as Phakopsora pachyrhizi, and Colleotrichum sp. In various crops, for example Colletotrichum orbiculare which may cause anthracnose in squash or Colletotrichum gloeosporioides causing anthracnose in peppers. The combinations, mixtures or compositions as disclosed herein are active against normally sensitive and resistant species and against all or some stages in the life cycle of the plant pathogenic fungus.

[0119] In particular embodiments, the combinations, mixtures or compositions as disclosed herein may be effective or active against plant pathogenic fungus from the genus chosen from the group comprising Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Corynespora, Diplodia, Erysiphe, Fusarium, Leptosphaeria, Gaeumannomyces, Helminthosporium, Leveillula, Macrophomina, Nectria, Oidium,Penicillium, Peronospora, Phoma, Phymatotrichum, Phytophthora, Plasmopara, Podosphaera, Puccinia, Pyrenophora, Pyricularia, Pythium, Rhizoctonia, Scerotium, Sclerotinia, Septoria, Sphaerotheca, Thielaviopsis, Uncinula, Venturia, Verticillium, Magnaporthe, Blumeria, Mycosphaerella, Ustilago, Melampsora, Phakopsora, Pseudoperonospora, Monilinia, Mucor, Rhizopus, Zymoseptoria and Aspergillus.

[0120] In a more preferred embodiment, the combinations, mixtures or compositions as disclosed herein may be effective or active against a plant pathogenic fungus according to the species chosen from the group comprising Alternaria alternata, Alternaria aroborescens, Alternaria solani, Alternaria brassicicola, Alternaria brassicae, Blumeria graminis, Botrytis cinerea, Cercospora beticola, Cercospora canescens, Cercospora soijina, Cercospora zeae-maydis, Cercospora kikuchii, Cercospora nicotianae, Cercospora cf. flagellaris, Cercospora cf. sigesbeckiae, Colletotrichum orbiculare, Colletotrichum gloeosporioides, Colletotrichum lindemuthianum, Colletotrichum coccodes, Colletotrichum Musea, Colletotrichum Fruticola, Corynespora cassiicola, Erysiphe cichoracearum, Erysiphe necator, Fusarium graminearum, Fusarium culmorum, Fusarium oxysporum, Gaeumannomyces tritici, Leveillula Taurica, Magnaporthe grisea, Monilinia fructicola, Penicillium expansum, Penicillium digitatum, Penicillium Italicum, Peronospora destructor, Phakopsora pachvrhizi, Phytophthora infestans, Phytophthora parasitica, Phytophthora capsica, Plasmopara viticola, Pseudoperonospora cubensis, Uncinula necator, Venturia inaequalis, Venturia pirina, Oidium Neolycopersici, Podosphaera aphanis, Podosphaera xanthii, Sphaerotheca fuliginea, Sclerotinia sclerotiorum, Zymoseptoria tritici. Preferably, the plant pathogenic fungus is Botrytis cinerea.

[0121] Methods of application

[0122] The combinations, mixtures and compositions of the invention beneficially provide antifungal activity when applied to a fungus, such as a plant pathogenic fungus. Therefore, in a particular embodiment, the present invention provides the use of the combinations, mixtures and compositions as described herein for use as an antifungal agent. Methods and uses of the invention may comprise applying the combinations, mixtures and compositions as described herein to a surface. “Surface” in the context of application of the invention refers to any external or internal substrate or area where the combinations, mixtures and compositions as described herein can be applied to. The term encompasses, but is not limited to, human and animal surfaces, such as the skin, mouth, mucous membranes, or respiratory organs (lungs); plant surfaces, such as seeds, shoots, stems, leaves, roots (including tubers), flowers, fruits, vegetables, and tissues and organs; inanimate surfaces, such as the surfaces of objects or equipment, such as agricultural equipment, storage facilities and household surfaces (including countertops, floors, walls, and other areas that may require decontamination from fungal contaminants). In a preferred embodiment, said surface is a plant surface, such as the surface of leaves, fruits, vegetables or seeds.

[0123] In certain aspects, the present invention provides methods comprising applying directly or indirectly the combinations, mixtures or compositions as described herein to a plant or a part of the plant, wherein “part of the plant” may refer to, but is not limited thereto, leaves, stems, roots, fruits, vegetables or seeds. The methods may be performed to a plant field (or field of crops; and where said plants or crops may be either in open air or in greenhouse environments). Thus, the present invention also provides methods comprising applying directly or indirectly the combinations, mixtures or compositions as described herein to a plant field. In a particular further embodiment, the combinations, mixtures or compositions as describedherein is applied to the plant, part of the plant or plant field at an application rate or concentration as detailed herein.

[0124] In certain aspects, the present invention provides methods for protecting or treating a plant or a part of a plant from an infection or other biological interaction with a plant pathogen, at least comprising the step of applying directly or indirectly to the plant or to a part of the plant, the combinations, mixtures and compositions as described herein. The combinations, mixtures and compositions as described herein may be applied under conditions effective to protect, treat or cure the plant or a part of the plant from or for an infection or biological interaction with a plant pathogen, preferably a plant pathogenic fungus as described herein.

[0125] The methods and uses of the combinations, mixtures and compositions as described herein, will include applying the active components as described herein with certain beneficial and improved application rates, said application rates are described herein above.

[0126] In yet another embodiment, the invention provides the use of the the combinations, mixtures and compositions as described herein in combating or inhibiting plant pests.

[0127] Applying a combination, mixture or composition according to the invention to a crop or field of plants may be done using any suitable method for applying a combination, mixture or composition to a plant field, plant part of the plant or surface, including, but not limited to spraying (including high volume (HV), low volume (LV) and ultra-low volume (ULV) spraying), brushing, dressing, dripping, coating, dipping, immersing, spreading, fogging, applying as small droplets, a mist or an aerosol.

[0128] Thus, in particular embodiments, the methods for protecting or treating a plant field, plant or a part of a plant from an infection or other biological interaction with a plant pathogen as disclosed herein, comprise applying the combination, mixture or composition as described herein directly or indirectly to the plant or to a part of the plant for example by spraying, atomizing, foaming, fogging, culturing in hydroculture, culturing in hydroponics, coating, submerging, and / or encrusting.

[0129] In certain particular embodiments, the present invention provides methods of inhibiting, preventing, reducing or controlling the growth of a plant pathogen (such as a plant pathogenic fungus), comprising at least the step of applying directly or indirectly to a plant or to a part of said plant, a combination, mixture or composition as disclosed herein.

[0130] In certain other embodiments, the present invention provides methods for of killing a plant pathogen, comprising at least the step of applying directly or indirectly to a plant field, a plant or to a part of said plant, a combination, mixture or composition as disclosed herein.

[0131] According to the methods as disclosed herein, the combination, mixture or composition of the invention can be applied once to a crop, or it can be applied two or more times after each other with an interval between every two applications. According to the method of the present invention, the combination, mixture or composition of the invention can be applied alone or in mixture with other materials, to the plant field, plant or part of the plant; alternatively, the combination, mixture or composition of the invention can be applied separately to the crop with other materials, applied at different times to the same crop. According to the method of the present invention, the combination, mixture or composition of the invention may be applied to the crop prophylactically (where it is said that the effect may be a protective effect), or alternatively, may be applied once target pests have been identified on the particular crop to be treated (where it is said that the effect may be a curative effect).The combination, mixture or composition of the invention as disclosed herein can be applied directly to a plant, a crop or to one or more parts of the plant by the above mentioned methods, such as directly to the entire plant or directly to one or more parts of the plant, either in a pre-harvest or in a post-harvest stage. Pre-harvest application may have an effect post-harvest. In certain further embodiments, the combination, mixture or composition as disclosed herein can be applied directly to one or more parts of the plant by the above mentioned methods, such as directly to the stalks, leaves, tubers, stems, shoots, the seeds, the fruits, the roots, the flowers, grains, the buds etc.

[0132] The method of application as disclosed herein can also be used in the field of protecting storage goods against attack of plant pathogens. In this method of treatment, application of a combination, mixture or composition of the invention may be pre-harvest or post-harvest. According to the present invention, the term "storage goods" is understood to denote natural substances of vegetable or animal origin and their processed forms, which have been taken from the natural life cycle and for which long-term protection is desired. Storage goods of vegetable origin, such as plants or parts thereof, for example stalks, leaves, tubers, seeds, fruits or grains, can be protected in the freshly harvested state or in processed form, such as pre-dried, moistened, comminuted, ground, pressed or roasted. Also falling under the definition of storage goods is timber, whether in the form of crude timber, such as construction timber, electricity pylons and barriers, or in the form of finished articles, such as furniture or objects made from wood. Storage goods of animal origin are hides, leather, furs, hairs and the like. The combinations according the present invention can prevent disadvantageous effects such as decay, discoloration or mold. Preferably "storage goods" is understood to denote natural substances of vegetable origin and their processed forms, more preferably fruits and their processed forms, such as pomes, stone fruits, soft fruits and citrus fruits and their processed forms.

[0133] The combination, mixture or composition of the invention can also be applied indirectly to a plant, a crop or to one or more parts of the plant by the above mentioned methods, such as indirectly to the entire plant or indirectly to one or more parts of the plant, either in a pre-harvest or in a post-harvest stage. The combination, mixture or composition of the invention herein can be applied close to harvest, such as about three weeks pre-harvest, for example two weeks pre-harvest or one week prior to harvest or less than one week pre-harvest. Pre-harvest application may have an effect post-harvest. Thus, in certain embodiments, the combination, mixture or composition of the invention can be applied indirectly to a plant, a crop or to one or more parts of the plant by the above mentioned methods, such as by applying the combination, mixture or composition of the invention to the surroundings or to the medium in which the plant or the one or more parts of the plant are growing or are stored, such as for instance but not limited to the air, the soil, the hydroponic culture, the hydroculture, or the liquid medium, such as for instance the aqueous liquid medium or water, in which the plant or the one or more parts of the plant are growing or are stored.

[0134] The combination, mixture or composition of the invention can be applied directly as a component of an integrated pest management approach.

[0135] It thus should be generally understood in the context of this application that the treatment of plant fields, plants and plant parts with the combination, mixture or composition of the invention is carried out directly or by action on their environment, habitat or storage area by means of the normal treatment methods, for example by watering (drenching), drip irrigation, spraying, vaporizing, atomizing, broadcasting, dusting, foaming, spreading-on, and as a powder. It is furthermore possible to apply the combination,mixture or composition of the invention by the ultra-low volume method, or to inject the active compound preparation or the active compound itself into the soil or into the plants.

[0136] In particular embodiments, the combination, mixture or composition of the invention is applied to protect a plant field, plant, or part of the plant from an infection by a plant pathogenic fungus. In another embodiment, the combination, mixture or composition of the invention is applied to treat a plant field, plant, or part of the plant from an infection by a plant pathogenic fungus. And wherein said applications may be applications at the pre or post-harvest stage. Therefore, the current invention envisions the use of the combination, mixture or composition according to the invention for treating or protecting a plant field, plant, or part of the plant for or from a plant pathogenic fungus infection.

[0137] The present invention will now be illustrated by way of the following non-limiting Examples.

[0138] Examples

[0139] Example 1: PDA plate assay

[0140] The antifungal activity of a VHH antibody (04D12 and 11A11) and chitosan (here a fungal derived chitosan was used, commercially available as Kitogreen produced by Kitozyme NV, Belgium), either alone or in combination, was assessed in a PDA plate assay against the plant pathogenic fungus Botrytis cinerea. In a PDA plate assay, one or more antifungal compounds (VHH antibody, chitosan or a combination of VHH antibody and chitosan) were co-incubated with 1 E+05 Botrytis cinerea spores per ml in liquid PDB (Potato Dextrose Broth; autoclaved) medium (1 / 2x PDB final), preferably in a 96-well format. After a 72h incubation period at 21 °C, 10pl of this incubated spore solution was spotted in the middle of a PDA plate (Potato Dextrose Agar; autoclaved) (1 / 4x PDA final). Each combination was run in duplicate on the PDA plates. After 3 days of incubation at 21 °C, the mycelial diameter on each plate was measured. The mycelial diameter reflects degree of spore germination and spore viability, which itself is affected by the antifungal activity of the added compound (or combinations thereof). As a positive control, Teldor® (Bayer; 50% fenhexamid) was included.

[0141] Table 1 sets out the results of a PDA plate assay using different combinations of Kitogreen and the VHH antibody 11 A11 (SEQ ID NO: 136). The assays illustrate that Kitogreen or 11 A11 in isolation do not reach the level of antifungal activity as may be reached when combining both active ingredients, where a complete inhibition of fungal outgrowth was observed. See for example object 5 of Table 1 showing full inhibition, compared to objects 9 and 10 of Table 1 showing only a slight inhibition of fungal outgrowth by respectively 11 A11 and Kitogreen in isolation and at the same concentrations.

[0142] Table 1 : Average mycelial diameter on 1 / 4x PDA (in mm) for Kitogreen x 11A11 combinations, Kitogreen alone and 11 A11 alone.

[0143]

[0144]

[0145]

[0146] Table 2 sets out the results of a PDA plate assay using different combinations of Kitogreen and the VHH antibody 04D12 (SEQ ID NO: 162). The assays illustrate that Kitogreen or 04D12 in isolation do not reach the level of antifungal activity as may be reached when combining both active ingredients, where a complete inhibition of fungal outgrowth was observed. See for example object 3 of Table 2 showing full inhibition, compared to objects 8 and 10 of Table 2 showing only a slight inhibition of fungal outgrowth by respectively 04D12 and Kitogreen in isolation and at the same concentrations.

[0147] Table 2: Average mycelial diameter on 1 / 4x PDA (in mm) for Kitogreen x 04D12 combinations, Kitogreen alone and 04D12 alone.

[0148]

[0149] Example 2: Leaf disc assays

[0150] To evaluate the efficacy of a mixtures of VHH antibody 11 A11 (SEQ ID NO: 136) and chitosan against Botrytis cinerea on bean leaf discs the following protocol was used: Leaf discs (2 cm diameter) were prepared from 2-weeks old bean plants (Variety: Saxa) and were kept moist on filter paper in square petri dishes (240 mm x 240 mm) containing 3 filter papers with 80mL of water. In total, 64 leaf discs were placedper petri dish / repetition and 3 replicate petri dishes were prepared per treatment. Botrytis cinerea spores were harvested from 2 week old PDA plates and spore suspension was stored at 4°C before usage. A final spore concentration of 2E+5 spores / mL was prepared in 1xPDB. The test product (10 mL) was prepared at a concentration 2x the test concentration in the leaf disc assay in a solution containing 10 mM HEPES pH7. In total, 150 pL of the product solution (2x) was added in 96-well plate and mixed with 150 pL of spore suspension (2E+5 spores / mL) to create the final spore-product solution. Teldor (50% Fenhexamid) was used as chemical reference and positive control. The different spore-product solutions were then coincubated at 21 °C for 24h in the dark. After incubation, leaf discs were inoculated with 10 pL spore-product solution. Inoculated leaf discs were kept in a growth chamber and grown under a light regime of 16h day / 8h night at a temperature of 21 °C. Three days after inoculation disease symptoms were scored based on the diseased leaf area by processing images taken from the leaf disc by the software Imaged. This software measures the leaf area and the lesion area and computes the diseased leaf area (DLA) using following formula: DLA = (lesion area / leaf area)*100.

[0151] Figure 1 shows disease severity of Botrytis cinerea on bean leaf discs after 3 days of incubation at 21 °C with 16h I ig ht / 8h dark regime, a sample of the corresponding infected leaf discs is provided in Figure 2. The results show the antifungal effect of Chitosan (here a fungal derived chitosan was used, commercially available as Kitogreen produced by Kitozyme NV, Belgium) in a dose response curve from 40 mg / l to 10 mg / ml. The tested VHH antibody (11 A11 ) was tested in a concentration range from 10Omg / l to 25mg / l (7pM to 1 ,75 pM). The ratios of chitosan over VHH used were ranging from 0,8 to 0,1. Surprisingly, when combining both chitosan and a VHH antibody in a single application with a concentration for chitosan of 40mg / L and for the VHH antibody 25mg / l to 50mg / l, an effect that was greater than the mere sum of the individual applications was observed.

[0152] Example 3: in vitro assays AFA

[0153] The antifungal activity of a VHH antibody 11 A11 (SEQ ID NO: 136) or 10G11 (SEQ ID NO: 86), and chitosan (here both a fungal derived chitosan, commercially available as Kitogreen® produced by Kitozyme NV, Belgium and a low MW chitosan derived from shrimp shells (Sigma-Aldrich; Prod nr. 448869)), either alone or in combination were assessed in an in vitro assay against the plant pathogenic fungus Botrytis cinerea. The in vitro assay is a standard microtiter plate-based antifungal assay against Botrytis cinerea based on confluence analysis using an IncuCyte S3 System. The media used is 1 / 2x PDB (Potato Dextrose Broth; autoclaved, no pH adjustment). The VHHs were added in a fixed 2-fold dilution series between 400mg / l and 6mg / l, with 20mM HEPES buffer pH 7. Fungal chitosan (formulated as Kitogreen®) was added in a fixed 2-fold dilution series between 200 mg / l and 3mg / l in 20mM HEPES with final pH of 6. Low MW chitosan derived from shrimp shells was added in a fixed 2-fold dilution series between 80 mg / l and 1 mg / l in 10mM acetate buffer + 50mM HEPES with final pH of 5. As a buffer control 50pl of 20mM HEPES buffer pH 7 was added to the control wells. As a positive control (chemical control) 50 pl 80 pM FCCP was added to the control wells. To all wells, 50pl of a Botrytis cinerea cell suspension in 1 / 2x PDB medium at 7,5E+3 spores / 50pl was added. For this Botrytis cinerea spores were harvested from 2-week-old PDA (Potato Dextrose Agar) plates and spore suspension was stored at 4°C in UP water before usage. Spore suspensions were diluted to 1 ,5E+5 spores / ml in 1 / 2x PDB before use.

[0154] Tables 3 to 6 show the observed or expected IC50 values of each compound or combinations thereof after 48h of incubation with Botrytis cinerea at 21 °C. Based on these IC50 values, synergism ratio (R) was calculated using Wadley's method for binary mixes (Levy et al., 1986). The results show for all testedcombinations (10G11 x Kitogreen; 10G11 x low MW chitosan; 11A11 x Kitogreen and 11A11 x low MW chitosan) an R-value higher than 1 , indicating that the experimentally observed IC50 of both compounds is lower than the expected IC50. This points at a synergetic effect upon combination of both compounds. In conclusion these result show the surprising effect of combining both a VHH antibody with cell membrane interacting properties and the polycationic polymer chitosan. Moreover these results further illustrate that the effect is not limited to chitosan derived from fungal or Aspergillus niger chitosan.

[0155] Table 3: Observed (o) and expected (e) IC50 values of Kitogreen and 10G11, either alone or in combination. The resulting synergism index R is calculated by dividing the expected IC50 value of both compounds combined by the observed IC50 value of both compounds combined. If R > 1 : synergy; if R = 1 : additivity; if R < 1 : antagonism

[0156] A(o) observed IC50 compound A alone

[0157] B(o) observed IC50 compound B alone

[0158] A(B;o) observed IC50 compound Ain combination with compound B B(A;o) observed IC50 compound B in combination with compound A A+B(o) = A(B;o) + B(A;o)

[0159] A+B(e) = A(B;o) + B(A;o) / (A(B;o) / A(o) + B(A;o) / B(o))

[0160] R = A+B(e) / A+B(o)

[0161]

[0162] Table 4: Observed (o) and expected (e) IC50 values of Kitogreen and 11A11, either alone or in combination. The resulting synergism index R is calculated by dividing the expected IC50 value of both compounds combined by the observed IC50 value of both compounds combined. If R > 1 : synergy; if R = 1 : additivity; if R < 1 : antagonism

[0163] If R > 1 : synergy; if R = 1 : additivity; if R < 1 : antagonism

[0164] A(o) observed IC50 compound A alone

[0165] B(o) observed IC50 compound B alone

[0166] A(B;o) observed IC50 compound Ain combination with compound B

[0167] B(A;o) observed IC50 compound B in combination with compound A

[0168] A+B(o) = A(B;o) + B(A;o)

[0169] A+B(e) = A(B;o) + B(A;o)Z (A(B;o) / A(o) + B(A;o) / B(o))

[0170] R = A+B(e) / A+B(o)

[0171]

[0172] Table 5: Observed (o) and expected (e) IC50 values of Chitosan low MW and 10G11, either alone or in combination. The resulting synergism index R is calculated by dividing the expected IC50 value of both compounds combined by the observed IC50 value of both compounds combined. If R > 1 : synergy; if R = 1 : additivity; if R < 1 : antagonism

[0173]

[0174] A(o) observed IC50 compoundAalone observed IC50 compound B alone

[0175] observed IC50 compound Ain combination with compound B observed IC50 compound B in combination with compound A

[0176]

[0177] A+B(e) / A+B(o)

[0178]

[0179] Table 6: Observed (o) and expected (e) IC50 values of Chitosan low MW and 11 A11 , either alone or in combination. The resulting synergism index R is calculated by dividing the expected IC50 value of both compounds combined by the observed IC50 value of both compounds combined. If R > 1 : synergy; if R = 1 : additivity; if R < 1 : antagonism.

[0180]

[0181] Example 4: Further in vitro assays AFA

[0182] The antifungal activity of a VHH antibody 11 A11 (SEQ ID NO: 136) or 10G11 (SEQ ID NO: 86), and a chitosan different from Example 3 (here a crustacean derived chitosan, commercially available as Tidal Grow Spectra® produced by Tidal Grow AgriScience) either alone or in combination were assessed in an in vitro assay against the plant pathogenic fungus Botrytis cinerea. The in vitro assay is a standard microtiter plate-based antifungal assay against Botrytis cinerea based on confluence analysis using an IncuCyte S3 System. The media used is 1 / 2x PDB (Potato Dextrose Broth; autoclaved, no pH adjustment). The VHHs were added in a fixed 2-fold dilution series between 400mg / l and 6.25mg / l, with 20mM HEPES buffer pH 7 as the dilution buffer. Crustacean chitosan (formulated as Tidal Grow Spectra®) was added in a fixed 2-fold dilution series between 80 mg / l and 1 .25mg / l with 20mM HEPES pH7 as the dilution buffer. As a buffer control 50pl of 20mM HEPES buffer pH 7 was added to the control wells. As a positive control (chemical control) 50 pl 40 pM FCCP was added to the control wells. To all wells, 50pl of a Botrytis cinerea cell suspension in 1 / 2x PDB medium at 7,5E+3 spores / 50pl was added. For this Botrytis cinerea spores were harvested from 2-week-old PDA (Potato Dextrose Agar) plates and spore suspension was stored at 4°C in UP water before usage. Spore suspensions were diluted to 1 ,5E+5 spores / ml in 1 / 2x PDB before use.

[0183] Tables 7 and 8 show the observed or expected IC50 values of each compound or combinations thereof after 48h of incubation with Botrytis cinerea at 21 °C. Based on these IC50 values, synergism ratio (R) was calculated using Wadley's method for binary mixes (Levy et al., 1986). The results show for all tested combinations (10G11 x Tidal Grow Spectra; and 11A11 x Tidal Grow Spectra) had R-value higher than 1 , indicating that the experimentally observed IC50 of both compounds is lower than the expected IC50. This points at a synergetic effect upon combination of both compounds. In conclusion these resultshow the surprising effect of combining both a VHH antibody with cell membrane interacting properties and the polycationic polymer chitosan.

[0184] Table 7: Observed (o) and expected (e) IC50 values of Tidal Grow Spectra and 10G11 , either alone or in combination. The resulting synergism index R is calculated by dividing the expected IC50 value of both compounds combined by the observed IC50 value of both compounds combined. If R > 1 : synergy; if R = 1 : additivity; if R < 1 : antagonism

[0185] A(o) observed IC50 compound A alone

[0186] B(o) observed IC50 compound B alone

[0187] A(B;o) observed IC50 compound Ain combination with compound B B(A;o) observed IC50 compound B in combination with compound A

[0188]

[0189] R = A+B(e) / A+B(o)

[0190]

[0191] Table 8: Observed (0) and expected (e) IC50 values of Tidal Grow Spectra and 11 A11 , either alone or in combination. The resulting synergism index R is calculated by dividing the expected IC50 value of both compounds combined by the observed IC50 value of both compounds combined. If R > 1 : synergy; if R = 1 : additivity; if R < 1 : antagonism

[0192] A(o) observed IC50 compound A alone

[0193] B(o) observed IC50 compound B alone

[0194] A(B;o) observed IC50 compound Ain combination with compound B B(A;o) observed IC50 compound B in combination with compound A A+B(o) = A(B;o) + B(A;o)

[0195] A+B(e) = A(B;o) + B(A;o)Z (A(B;o) / A(o) + B(A;o) / B(o))

[0196] R = A+B(e) / A+B(o)

[0197]

[0198] Bibliography

[0199] Benhamou, N. 1992. Ultrastructural and cytochemical characterization of elicitor-induced structural responses in tomato root tissues infected by Fusarium oxysporum f.sp. radicis-lycopersici. Cytology and Histology 82, 1185-1193. https: / / d0i.0rg / l 0.1007 / bf00239944

[0200] Ben-Shalom, N., .Ardi, R., .Pinto, R., .Aki, C., . Fallik, E., 2003. Controlling gray mould caused by Botrytis cinerea in cucumber plants by means of chitosan. Crop Prot 22, 285-290. https: / / doi.Org / 10.1016 / S0261 -2194(02)00149-7

[0201] Carolyn, A.R., Hadwiger, L.A., 1979. The fungicidal effect of chitosan on fungi of varying cell wall composition. Fungal Genet Biol 3, 285-287. https: / / doi.org / 10.1016 / S0147-5975(79)80054-7DeGenring, L., Dickson, R., Poleatewich, A., 2023. Inhibition of Botrytis cinerea Growth and Suppression of Gray Mold on Petunia Leaves Using Chitosan. Plant Dis 107, 840-848. https: / / doi.Org / 10.1094 / PDIS-07-22-1628-RE

[0202] Levy, Y., Benderly, M., Cohen, Y., Gisi, U., Bassand, D., 1986. The joint action of fungicides in mixtures: comparison of two methods for synergy calculation. Bull OEPP 16, 651-657. https: / / doi.Org / 10.1111 / j.1365-2338.1986.tb00338.x

[0203] Statements (features) and embodiments of the combinations, mixtures, compositions, methods and uses as disclosed herein are set herebelow. Each of the statements and embodiments as disclosed by the invention so defined may be combined with any other statement and / or embodiment unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0204] Embodiments

[0205] 1 . A combination of active components, wherein the active components comprise (i) a polypeptide with cell membrane interacting properties and (ii) a polycationic polymer.

[0206] 2. The combination according to embodiment 1 , wherein the polycationic polymer is chitosan.

[0207] 3. The combination according to embodiment 2, wherein chitosan is derived from fungal, insect or crustacean chitin, preferably fungal chitin.

[0208] 4. The combination according to embodiment 3, wherein the fungal chitosan is chitosan derived from Aspergillus niger.

[0209] 5. The combination according to any one of embodiments 2 to 4, wherein the chitosan is soluble at a pH of from pH 5 to pH 7.

[0210] 6. The combination according to any one of embodiments 2 to 5, wherein the chitosan has a molecular weight of 2000 kDa or lower.

[0211] 7. The combination according to any one of embodiment 2 to 6, wherein the chitosan has a degree of deacetylation of from 60% to 100%.

[0212] 8. The combination according to any one of the embodiments above, wherein the polypeptide is a VHH antibody.

[0213] 9. The combination according to embodiment 8, wherein the VHH antibody is capable of binding to at least one fungus, preferably at least one plant pathogenic fungus.

[0214] 10. The combination according to any one of the embodiments above, wherein the polypeptide specifically binds to at least one plasma membrane component of the fungus.

[0215] 11 . The combination according to embodiment 10, wherein the VHH antibody is capable of binding to a lipid-containing fraction of the plasma membrane of Botrytis cinerea, said lipid-containing fraction being obtainable by a method comprising: fractionating hyphae of Botrytis cinerea by total lipid extract thin-layer chromatography and selecting the fraction with a Retention Factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipids fraction.

[0216] 12. The combination according to embodiment 10, wherein the plasma membrane component is a sphingolipid.

[0217] 13. The combination according to embodiment 12, wherein the sphingolipid is a glucosylceramide.14. The combination according to any one of embodiments 8 to 11 , wherein the VHH antibody is a VHH antibody comprising the amino acid sequence according to SEQ ID NO: 85 to 135.

[0218] 15. The combination according to any one of embodiments 8 to 10, wherein the VHH antibody is a VHH antibody comprising the amino acid sequence according to SEQ ID NO: 136 to 162

[0219] 16. The combination according to any one of embodiments 8 to 10, 12 or 13, wherein the VHH antibody is a VHH antibody comprising the amino acid sequence according to SEQ ID NO: 1 to 84.

[0220] 17. The combination according to any one of embodiments 1 to 7, wherein the polypeptide is an antimicrobial peptide or AMP, wherein the antimicrobial peptide or AMP is capable of interacting with the cell membrane of at least one plant pathogenic fungus

[0221] 18. The combination according to any one of the embodiments above, wherein the ratio of the polypeptide over the polycationic polymer is in a range of from 1 :100 to 100:1 .

[0222] 19. The combination according to any one of the embodiments above, wherein at least one of the active components is an antifungal active component.

[0223] 20. The combination according to any one of the embodiments above, wherein the combination has an antifungal effect against at least one plant pathogenic fungus.

[0224] 21 . The combination according to any one of the embodiments above, wherein the combination has a synergistic curative antifungal effect and / or a synergistic protective antifungal effect against at least one plant pathogenic fungus.

[0225] 22. A mixture comprising the combination of any one of embodiments 1 -21 .

[0226] 23. The mixture of embodiment 22, wherein the mixture is a tank mix.

[0227] 24. A composition comprising the combination of any one of embodiments 1 -21 .

[0228] 25. The composition according to embodiment 24, further comprising an agrochemically suitable additive.

[0229] 26. The composition according to embodiment 25, wherein the agrochemically suitable additive is selected from at least one of the group consisting of diluents, solvents, adjuvants, surfactants, wetting agents, spreading agents, oils, stickers, thickeners, penetrants, buffering agents, acidifiers, antisettling agents, anti-freeze agents, photo-protectors, UV protectors, defoaming agents, biocides and drift control agents.

[0230] 27. A method comprising applying the combination, mixture or composition according to any one of the embodiments above, to a surface.

[0231] 28. The method according to embodiment 27, wherein said surface is the leaf of a plant or the fruit of a plant, a vegetable or a plant seed.

[0232] 29. The method according to embodiments 27 or 28, wherein the polypeptide is applied with a rate of from 1 g / ha to 500 g / ha and the polycationic compound is applied at a rate of from 1 g / ha to 1000 g / ha.

[0233] 30. The method according to any one of embodiments 27 to 29, wherein the application rate of the active components is so that the ratio of the polypeptide over the polycationic polymer is in a range of from 1 :100 to 100:1.

[0234] 31. The method according to any one of embodiments 27 to 30, wherein the combination, mixture or composition is applied post-harvest.The method according to any one of embodiments 27 to 31 , wherein the combination, mixture or composition is applied to protect a plant field, plant, or part of the plant from an infection by at least one plant pathogenic fungus.

[0235] The method according to any one of embodiments 27 to 31 , wherein the combination, mixture or composition is applied to treat a plant field, plant, or part of the plant for an infection by at least one plant pathogenic fungus.

[0236] Use of the combination, mixture or composition according to any one of embodiments 1 to 26 for treating a plant field, plant, or part of the plant for at least one plant pathogenic fungus infection. Use of the combination, mixture or composition according to any one of embodiments 1 to 26 for protecting a plant field, plant, or part of the plant from at least one plant pathogenic fungus infection. The combination according to embodiments 9 to 17, 20 or 21 , the mixture according to embodiment 22 or 23, the composition according to embodiments 24 to 26, the method according to embodiment 32 or 33, or the use according to embodiment 34 or 35, wherein the plant pathogenic fungus is a plant pathogenic fungus from the genus chosen from the group comprising Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Corynespora, Diplodia, Erysiphe, Fusarium, Leptosphaeria, Gaeumannomyces, Helminthosporium, Leveillula, Macrophomina, Nectria, Oidium, Penicillium, Peronospora, Phoma, Phymatotrichum, Phytophthora, Plasmopara, Podosphaera, Puccinia, Pyrenophora, Pyricularia, Pythium, Rhizoctonia, Scerotium, Sclerotinia, Septoria, Sphaerotheca, Thielaviopsis, Uncinula, Venturia, Verticillium, Magnaporthe, Blumeria, Mycosphaerella, Ustilago, Melampsora, Phakopsora, Pseudoperonospora, Monilinia, Mucor, Rhizopus, Zymoseptoria and Aspergillus.

[0237] The combination, mixture, composition, method or use according to embodiment 36, wherein the plant pathogenic fungus is Botrytis cinerea.

Claims

1. Claims1. A combination of active components, wherein the active components comprise (i) a polypeptide with cell membrane interacting properties and (ii) chitosan.

2. The combination according to claim 1 , wherein chitosan is derived from fungal, insect or crustacean chitin, preferably fungal chitin, preferably fungal chitin from Aspergillus niger.

3. The combination according to any one of the claims above, wherein the polypeptide is a VHH antibody.

4. The combination according to claim 3, wherein the VHH antibody is capable of binding to at least one fungus, preferably at least one plant pathogenic fungus, and wherein the VHH antibody specifically binds to at least one plasma membrane component of the fungus.

5. The combination according to claim 1 or 2, wherein the polypeptide is an antimicrobial peptide or AMP, wherein the antimicrobial peptide or AMP is capable of interacting with the cell membrane of at least one plant pathogenic fungus.

6. The combination according to any one of the claims above, wherein at least one of the active components is an antifungal active component.

7. The combination according to any one of the claims above, wherein the combination has a synergistic curative antifungal effect and / or a synergistic protective antifungal effect against at least one plant pathogenic fungus.

8. A mixture comprising the combination of any one of claims 1 -7.

9. The mixture of claim 8, wherein the mixture is a tank mix.

10. A composition comprising the combination of any one of claims 1 -7.

11. The composition according to claim 10, further comprising an agrochemically suitable additive, optionally wherein the agrochemically suitable additive is selected from at least one of the group consisting of diluents, solvents, adjuvants, surfactants, wetting agents, spreading agents, oils, stickers, thickeners, penetrants, buffering agents, acidifiers, anti-settling agents, anti-freeze agents, photo-protectors, UV protectors, defoaming agents, biocides and drift control agents.

12. A method comprising applying the combination, mixture or composition according to any one of the claims above, to a surface, such as the surface of a leaf of a plant or the fruit of a plant, a vegetable or a plant seed.

13. The method according to claim 12, wherein the combination, mixture or composition is applied to treat or protect a plant field, plant, or part of the plant for or from an infection by at least one plant pathogenic fungus.

14. Use of the combination, mixture or composition according to any one of claims 1 to 11 for treating or protecting a plant field, plant, or part of the plant for or from at least one plant pathogenic fungus infection.

15. The combination according to claims 4, 5 or 7, the mixture according to claim 8 or 9, the composition according to claim 10 or 11 , the method according to claim 12 or 13, or the use according to claim 14, wherein the plant pathogenic fungus is a plant pathogenic fungus from the genus chosen from the group comprising Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Corynespora, Diplodia, Erysiphe, Fusarium, Leptosphaeria, Gaeumannomyces, Helminthosporium, Leveillula, Macrophomina, Nectria, Oidium, Penicillium, Peronospora, Phoma, Phymatotrichum, Phytophthora, Plasmopara, Podosphaera, Puccinia, Pyrenophora, Pyricularia, Pythium, Rhizoctonia, Scerotium,33Sclerotinia, Septoria, Sphaerotheca, Thielaviopsis, Uncinula, Venturia, Verticillium, Magnaporthe, Blumeria, Mycosphaerella, Ustilago, Melampsora, Phakopsora, Pseudoperonospora, Monilinia, Mucor, Rhizopus, Zymoseptoria and Aspergillus, preferably the plant pathogenic fungus is Botrytis cinerea.

Images