WO2026008785A1 - Agrochemical compositions - Google Patents

Abstract

The present invention relates to a composition comprising at least one antifungal compound with cell membrane interacting properties and at least one cell wall degrading enzyme, optionally wherein the at least one antifungal compound with cell membrane interacting properties is a polypeptide, optionally an antibody or fragment thereof such as a heavy chain variable domain of a heavy chain antibody (VHH) or a functional fragment thereof, which antibody or fragment thereof is capable of binding to a fungus. The invention relates to cell wall degrading enzymes, more specifically cell wall degrading enzymes according to SEQ ID NOs: 100 to 130 or mixtures thereof, and compositions or combinations thereof further comprising at least one antifungal compound with cell membrane interacting properties. The invention further relates to methods of producing, methods of using and the use of the compositions and combinations disclosed herein. The application further relates to transgenic plants, or methods for producing the same, comprising one or more of a polynucleotide according to the invention.

Description

AGROCHEMICAL COMPOSITIONS

Field of the invention

The present invention is situated in the field of antifungal compositions or combinations, preferably in the field of agrochemical compositions or combinations comprising antifungal compounds and/or cell wall degrading enzymes. More specifically, The present invention relates to a composition comprising at least one antifungal compound with cell membrane interacting properties and at least one cell wall degrading enzyme, optionally wherein the at least one antifungal compound with cell membrane interacting properties is a polypeptide, optionally an antibody or fragment thereof such as a heavy chain variable domain of a heavy chain antibody (VHH) or a functional fragment thereof, which antibody or fragment thereof is capable of binding to a fungus. The invention relates to cell wall degrading enzymes, more specifically cell wall degrading enzymes according to SEQ ID NOs: 100 to 130 or mixtures thereof, and compositions or combinations thereof further comprising at least one antifungal compound with cell membrane interacting properties. The invention further relates to methods of producing, methods of using and the use of the compositions and combinations disclosed herein. The application further relates to transgenic plants, or methods for producing the same, comprising one or more of a polynucleotide according to the invention.

Background

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 anti- fungals and the general lack of efficacy of anti-fungal agents, which are available at present.

The use of purified or transgenic plants expressing specific cell wall degrading enzymes has been suggested in the art. For instance, a bifunctional peptidoglycan/chitin hydrolase isolated from Lactobacillus was shown to prevent hyphae formation of a fungal pathogen (W02020079278), a purified chitinase from Trichoderma cell was shown to reduce disease severity caused by Botrytis cinerea on cucumber leaves (Aoki Yoshinao, Haga Sanami, Suzuki Shunji, Direct antagonistic activity of chitinase produced by Trichoderma sp. SANA20 as biological control agent for grey mould caused by Botrytis cinerea. Cogent Biology, 2020, Vol 6 (1 ), 1747903), and cell wall degrading enzymes such as glucanases, endo-chitinases and exochitinases where cloned into transgenic plant to confer them with resistance to fungal pathogens such as fusarium (W02001016353).

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 2014/191 146 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). And 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) 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.

Accordingly, there remains a need in the art to provide anti-fungal compositions 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.

Summary of the invention

The current inventors have previously found that a complex cocktail of cell wall degrading enzymes such as the commercially available cocktail Vinotaste™, derived from a fungal fermentation, can be effective at enhancing the effect of a membrane interacting substance such as a VHH antibody binding to or interacting with the cell membrane of a fungal cell, specifically a plant pathogenic fungal cell (see also co-pending, non-published international application PCT/US23/86359). Although effective, their remains a need to simplify the cocktail to a defined set of enzymes that can improve the (antifungal) effect of an antifungal compound with cell membrane interacting properties and that avoids the presence of unwanted ingredients in such fermentation extracts.

The current invention relates to compositions comprising (i) at least one antifungal compound with cell membrane interacting properties, and (ii) at least one cell wall degrading enzyme. In a particular embodiment, the current invention relates to compositions comprising (i) at least one antifungal compound with cell membrane interacting properties, and (ii) at least one recombinantly produced cell wall degrading enzyme. In a particular embodiment, the composition according to the invention comprises one or more of a glucanase and/or one or more of a chitinase. The composition according to the invention may comprise one or more of a cell wall degrading enzyme selected from the group consisting of exo-beta-1 ,3-glucanase, endo-beta-1 ,3-glucanase, exo-beta-1 ,6-glucanase, endo-beta-1 ,6-glucanase, endo-beta-1 ,3(4)-glucanase, endo-alpha-1 ,3-glucanase, exo-alpha-1 ,3- glucanase and endo-chitinase. In a preferred embodiment of the current invention the cell wall degrading enzymes are selected from the cell wall degrading enzyme comprising an amino acid sequence selected from the list consisting of SEQ ID NO: 100 to 130 or an amino acid sequence having at least about 70% sequence identity thereto. According to the current invention, when applied to a fungus or to a plant or part of a plant comprising or infected by a fungus, the cell wall degrading enzyme degrades the cell wall of said fungus, preferably said fungus is a plant pathogenic fungus. In some embodiments the antifungal compound with cell membrane interacting properties is a polypeptide, preferably an antibody, more preferably a VHH antibody or a fragment thereof. In a more preferred embodiment of the invention, the VHH antibody comprises an amino acid sequence selected from the list consisting of SEQ ID NO: 1 , 2, 6, 10 or 14 to 99 or a VHH antibody as described in US provisional applications 63/614,192 or 63/614,183 identified therein as the VHH antibody comprising SEQ ID NO: 2 (SEQ ID NO: 131 or SEQ ID NO: 132 respectively), or an amino acid sequence having at least about 80% sequence identity thereto. Therefore, in a more preferred embodiment of the invention, the VHH antibody comprises an amino acid sequence selected from the list consisting of SEQ ID NO: 1 , 2, 6, 10, 14 to 99, 131 or 132), or an amino acid sequence having at least about 80% sequence identity thereto.

The composition according to the current invention may be an agrochemical composition.

The current invention further relates to the use of the composition or agrochemical composition according the invention as an anti-fungal agent, preferably as an anti-fungal agent on plants.

The current invention further relates to a method for protecting or treating a plant or a part of said plant from an infection with a plant pathogenic fungus, at least comprising the step of applying to said plant or to a part of said plant, the composition or combination according to the invention, under conditions effective to protect or treat said plant or a part of said plant against said infection with said plant pathogenic fungus.

The current invention further relates to post-harvest treatment methods or methods for killing or inhibiting a plant pathogenic fungus on plants.

The current invention further relates to polynucleotides comprising the nucleic acid sequence capable of expressing the cell wall degrading enzymes of the current invention.

The current invention further relates to a method of producing a composition or combination according to the invention comprising the steps of: (i) cloning one or more of a polynucleotide according to the invention into a host cell, wherein the one or more polynucleotide is capable of expressing at least one cell wall degrading enzyme; (ii) recombinantly producing at least one cell wall degrading enzyme; (iii) optionally, purifying the at least one cell wall degrading enzyme optionally to a purity of 90% or higher; and (iv) combining the at least one cell wall degrading enzyme with one or more of an antifungal compound with cell membrane interacting properties and/or optionally one or more of an additive, optionally an agrochemical additive.

The current invention also relates to a combination comprising at least one antifungal compound with cell membrane interacting properties and a cell wall degrading enzyme according to the invention.

Finally, the invention also relates to transgenic plants comprising the polynucleotides according to the invention.

Description of the sequence listing

The sequence listing is depicted in Table 1 .

Description of the figures

Figure 1 : Protoplast formation by a cell wall degrading enzyme, before incubation (Figure 1 1 A), during incubation with a CWDE (Figure 1 1 B) and after purification (Figure 1 1 C). Figure 2: Antifungal leaf-disc assay showing disease repression by the VHH-88 in combination with an enzyme cocktail.

Detailed description of the invention

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.

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.

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.

Definitions

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

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.

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.

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 Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^nd ed., 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.

As used herein, the terms "polypeptide", "protein", “peptide”, and “amino acid sequence” are used interchangeably, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

As used herein, amino acid residues will be indicated either by their full name or according to the standard three-letter or one-letter amino acid code.

As used herein, the terms “nucleotide”, "nucleic acid molecule", "polynucleotide", “polynucleic acid”, “nucleic acid” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogues thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.

The current invention further discloses polynucleotides capable of expressing the amino acid sequences that define the primary structure of the polypeptides and/or cell wall degrading enzymes as disclosed herein. That is to say, the polynucleotides of the current invention comprise the coding region of the corresponding amino acid sequence of the polypeptides and/or cell wall degrading enzymes as disclosed herein. The skilled person will know how to deduce the appropriate coding region of the corresponding amino acid sequences as disclosed herein keeping into account the different codon-usage profiles each host cell may have. In addition the skilled person will know that the coding region for a polypeptide or cell wall degrading enzyme as present in the genome of species comprising said polypeptide or cell wall degrading enzyme may consist of introns and exons, that may or may not be required for successful recombinant production of the polypeptide or cell wall degrading enzyme. Such cloning strategies are common in the art and the skilled person is well equipped to, starting from the amino acid sequences as provided herein, successfully deduce the appropriate polynucleotide for expressing or recombinantly producing the amino acid sequences of the polypeptides and cell wall degrading enzymes according to the invention.

As used herein, the term “homology” denotes at least secondary structural similarity between two macromolecules, particularly between two polypeptides or polynucleotides, from same or different taxons, wherein said similarity is due to shared ancestry. Hence, the term ’’homologues” denotes so-related macromolecules having said secondary and optionally tertiary structural similarity. For comparing two or more nucleotide sequences, the '(percentage of) sequence identity' between a first nucleotide sequence and a second nucleotide sequence may be calculated using methods known by the person skilled in the art, e.g. by dividing the number of nucleotides in the first nucleotide sequence that are identical to the nucleotides at the corresponding positions in the second nucleotide sequence by the total number of nucleotides in the first nucleotide sequence and multiplying by 100% or by using a known computer algorithm for sequence alignment such as NCBI Blast. In determining the degree of sequence identity between two amino acid sequences, the skilled person may take into account so-called 'conservative' amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and which has little or essentially no influence on the function, activity or other biological properties of the polypeptide. Possible conservative amino acid substitutions will be clear to the person skilled in the art. Amino acid sequences and nucleic acid sequences are said to be “exactly the same” if they have 100% sequence identity over their entire length.

As used herein, the terms "complementarity determining region" or "CDR" within the context of antibodies refer to variable regions of either the H (heavy) or the L (light) chains (also abbreviated as VH and VL, respectively) and contain the amino acid sequences capable of specifically binding to antigenic targets. These CDR regions account for the basic specificity of the antibody for a particular antigenic determinant structure. Such regions are also referred to as "hypervariable regions." The CDRs represent non-contiguous stretches of amino acids within the variable regions but, regardless of species, the positional locations of these critical amino acid sequences within the variable heavy and light chain regions have been found to have similar locations within the amino acid sequences of the variable chains. The variable heavy and light chains of all canonical antibodies each have 3 CDR regions, each non- contiguous with the others (termed L1 , L2, L3, H1 , H2, H3) for the respective light (L) and heavy (H) chains.

The term "affinity", as used herein, refers to the degree to which a polypeptide, in particular an immunoglobulin, such as an antibody, or an immunoglobulin fragment, such as a VHH, binds to an antigen so as to shift the equilibrium of antigen and polypeptide toward the presence of a complex formed by their binding. Thus, for example, where an antigen and antibody (fragment) are combined in relatively equal concentration, an antibody (fragment) of high affinity will bind to the available antigen so as to shift the equilibrium toward high concentration of the resulting complex. The dissociation constant is commonly used to describe the affinity between the protein binding domain and the antigenic target. Typically, the dissociation constant is lower than 10⁵ M. Preferably, the dissociation constant is lower than 10^-6 M, more preferably, lower than 10^-7 M. Most preferably, the dissociation constant is lower than 10^-8 M.

The terms "specifically bind" and "specific binding", as used herein, generally refers to the ability of a polypeptide, in particular an immunoglobulin, such as an antibody, or an immunoglobulin fragment, such as a VHH, to preferentially bind to a particular antigen that is present in a homogeneous mixture of different antigens. In certain embodiments, a specific binding interaction will discriminate between desirable and undesirable antigens in a sample, in some embodiments more than about 10 to 100-fold or more (e.g., more than about 1000- or 10,000-fold).

Accordingly, an amino acid sequence as disclosed herein is said to "specifically bind to” a particular target when that amino acid sequence has affinity for, specificity for and/or is specifically directed against that target (or for at least one part or fragment thereof).

The “specificity” of an amino acid sequence as disclosed herein can be determined based on affinity and/or avidity.

An “IC50 value”, "EC50”, “IC50”, "EC50” or “half maximal inhibitory concentration” as used herein interchangeably is a measure for the potency of a polypeptide or cell wall degrading enzyme or compositions or combinations as described herein in inhibiting the growth of a fungal pathogen. In practice (also see example 1 ) the growth 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 tested polypeptide or cell wall degrading enzyme or compositions or combinations 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 the polypeptide or cell wall degrading enzyme or compositions or combinations as described herein. Said confluence 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 polypeptide or cell wall degrading enzyme or compositions 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.

The term “recombinant” or “recombinantly” or “recombinantly produced” or “recombinantly expressed” in the context of a protein refers to a protein that is produced through recombinant DNA technology or where the host cell is altered through genetic manipulation to comprise a protein that can be expressed. Thus, a recombinant polypeptide or a recombinant cell wall degrading enzyme, or a recombinantly produced polypeptide or recombinantly produced cell wall degrading enzyme may refer to a heterologous polypeptide or cell wall degrading enzyme since it may not be encoded by the wild-type genome of a host cell used to produce said polypeptide or a cell wall degrading enzyme. The polypeptide or a cell wall degrading enzyme may be a heterologous polypeptide or a cell wall degrading enzyme (i.e. the gene encoding the polypeptide or cell wall degrading enzyme is not naturally present in the genome or genetic material of the host cell of the fermentation reaction, the polypeptide or a cell wall degrading enzyme is said to be heterologous or non-native to the host cell). In a preferred embodiment, a recombinant protein is a protein produced by a host cell, wherein the protein is encoded by a recombinant gene that originates from a microorganism that is different from the host cell. In some different embodiments, the polypeptide or cell wall degrading enzyme may be a native polypeptide or a cell wall degrading enzyme but where said host cell is modified to increase the expression of a native polypeptide or cell wall degrading enzyme as compared to an unmodified host cell. In some embodiments, the polypeptide or cell wall degrading enzyme may be a native polypeptide or cell wall degrading enzyme but where the native sequence of the encoding DNA sequence or regulatory sequence such as promoter sequences has been altered to improve the expression and/or purification of the native polypeptide or cell wall degrading enzyme. For example, a promoter region may be altered or a promotor may be inserted to drive expression of the native gene or the gene expressing the native polypeptide or cell wall degrading enzyme. In another example the native polypeptide or cell wall degrading enzyme may be altered by introducing a “tag” or “peptide tag” such as a tag useful for affinity purifying the recombinant protein. Examples of such a tag may be a tag that can be used for purification or detection of the protein of interest, such as a His6, c-myc, FLAG, C-tag, 3xFLAG, His5, His10, HA, T7, strep, HSV, and/or an E-tag. In some embodiments, the at least one cell wall degrading enzyme is obtained or obtainable by recombinantly producing the cell wall degrading enzyme by fermentation of one or more host cells comprising at least one recombinant cell wall degrading enzyme. The terms “Culturing”, “cell culture”, “fermentation”, “fermenting” or “microbial fermentation” as used herein includes suspending the host cell, such as a host cell comprising one or more of a recombinant cell wall degrading enzyme, in a broth or growth medium, providing sufficient nutrients including but not limited to one or more suitable carbon source (including glucose, sucrose, fructose, lactose, avicel®, xylose, galactose, ethanol, methanol, or more complex carbon sources such as molasses or wort), nitrogen source (such as yeast extract, peptone or beef extract), trace element (such as iron, copper, magnesium, manganese or calcium), amino acid or salt (such as sodium chloride, magnesium chloride or natrium sulfate) or a suitable buffer (such as phosphate buffer, succinate buffer, HEPES buffer, MOPS buffer or Tris buffer). It can also further involve the agitation of the culture media via for example stirring of purging to allow for adequate mixing and aeration. It can further involve different operational strategies such as batch cultivation, semi-continuous cultivation or continuous cultivation and different starvation or induction regimes according to the requirements of the host cell and to allow for an efficient production of the compound of interest or a compound involved in the production of the compound of interest. Alternatively, the host cell is grown on a solid substrate in an operational strategy commonly known as solid state fermentation. In a typical fermentation reaction, a host cell is propagated in a nutrient rich broth providing the necessary nutrients, salts, minerals, oxygen etc... for the host cell to grow and multiply to reach a certain density of cells in the fermentation broth. Generally, the broth will comprise any and all nutrients required for the microbial organism to grow. The skilled person will be aware of the required components of the culture broth or fermentation broth, which may differ depending on the species of host cell being cultured. In some embodiments, the culture broth or fermentation broth may comprise a nitrogen source, such as ammonium or peptone. Suitable host cell for the recombinant production of at least one cell wall degrading enzyme and/or the polypeptides as described herein may be selected from any one specifies from Trichoderma spp., Aspergillus spp., Komagataella spp. (aka Pichia spp), Myceliophthora spp, Bacillus spp, or Escherichia spp. Non limiting examples of host cell species may be Trichoderma reesei, Aspergillus niger, Komagataella phaffii, Myceliophthora thermophilus, Bacillus subtilis, Bacillus licheniformis and Escherichia coli. In a preferred embodiment, the host cell is a microbial host cell. In other embodiments the host cell may be a mammalian host cell or insect host cell. In some embodiments the cell wall degrading enzyme and the polypeptide according to the invention are produced in the same host cell. In another embodiment the cell wall degrading enzyme and the polypeptide according to the invention are produced in different host cell. At the end of a fermentation reaction, the fermentation broths may be clarified by removing the cellular material and as such obtaining a microbial fermentation broth that is clarified. Clarification can be achieved in many ways such as commonly known filtration, centrifugation, or precipitation techniques. In some embodiments further downstream processing steps are applied to for instance further concentrate the protein content in the clarified broth by purification. Where the fermentation broth contain one or more cell wall degrading enzymes, the further downstream processing steps may be optimized to increase the concentration of said cell wall degrading enzymes. Common downstream process steps for concentrating or purifying the protein content include filtration, chromatography steps or a combination thereof. When concentrating the protein content of a clarified broth by purification, it is understood that such a purification does not necessarily need to be complete i.e. reaching for example 90% or higher purity. For instance a single filtration step may be use with a molecular weight cut-off lower than the molecular weight of the cell wall degrading enzymes to be concentrated and whereby the retentate, comprising the cell wall degrading enzymes and other larger or similar sized components, is maintained. In preferred embodiments the cell wall degrading enzyme is a recombinantly produced cell wall degrading enzyme. In a more preferred embodiment the cell wall degrading enzyme may be at least one recombinantly produced and purified cell wall degrading enzyme. In another preferred embodiment the composition or combination of the invention comprises 1 , 2, 3, 4 or 5, or more recombinantly produced and optionally purified cell wall degrading enzymes. In a preferred embodiment the composition or combination of the invention does not comprise more than 28, preferably no more than 9, preferably no more than 6, preferably no more than 3, different recombinantly produced and purified cell wall degrading enzymes and where said enzymes are selected from one or more glucanase and/or one or more chitinase. In a preferred embodiment, the recombinant cell wall degrading enzyme may be purified to a level of purity of 90% or higher. That is to say, where a cell wall degrading enzyme or a mixture thereof is produced it may be purified so that a level of purity of at least 90% is achieved before the cell wall degrading enzyme or mixture of cell wall degrading enzymes is mixed into the final composition or combination as described herein. Therefore it is understood that the total concentration of the cell wall degrading enzyme or mixture thereof may not necessarily be 90% of the total volume of the final composition or combination as described herein. In some embodiments where the cell wall degrading enzyme or mixtures have been produced in the same host cell as the antifungal compound with membrane interacting properties, all the components may jointly lead to a 90% purity when co-purified. Alternatively the cell wall degrading enzymes or mixtures may be produced in a separate fermentation reaction, purified to 90% or higher and combined with an antifungal compound with cell membrane interacting properties that may or may not have been produced through fermentation in either the same or different host cell.

Also provided herein are methods of producing a composition according to the invention and wherein the composition comprises at least one recombinantly produced cell wall degrading enzyme as described herein. The method consist of the steps of (i) cloning one or more of a polynucleotide into a host cell, wherein the one or more polynucleotide is capable of expressing at least one cell wall degrading enzyme. In some embodiments, said polynucleotide comprise the amino acid sequence selected from the group consisting of SEQ ID NO: 100 to 130. Cloning of the one or more polynucleotides may be performed using standard molecular genetic approaches well known in the art. The polynucleotides may be cloned by inserting the polynucleotide into the genome of the host cell or the polynucleotides may be presented as an episomal plasmid. Importantly the polynucleotide should be constructed according to the rules of the art such that the one or more polynucleotides are capable of expressing an amino acid sequence encoding a cell wall degrading enzyme as set out herein. In addition, the method consists of step (ii) of recombinantly producing at least one cell wall degrading enzyme in a host cell as set out above; (Hi) optionally, purifying the at least one cell wall degrading enzyme optionally to a purity of 90% or higher using the methods as set out above; and (iv) combining the at least one cell wall degrading enzyme with one or more of an antifungal compound with cell membrane interacting properties and/or optionally one or more of an additive, optionally an agrochemical additive. In some embodiments the antifungal compound with cell membrane interacting properties is a polypeptide according to the invention, such as but not limited to the polypeptides comprising an amino acid sequence according to SEQ ID NO 1 to 99, 131 or 132. In some embodiments the polypeptide is an antimicrobial peptide or AMP. In some embodiments the polypeptide is recombinantly produced such as described above in a manner similar to the recombinant production of a cell wall degrading enzyme. In some embodiments the polypeptide is recombinantly produced in a different host cell as the cell wall degrading enzyme. Therefore in some embodiments the at least one cell wall degrading enzyme and at least one polypeptide are produced in one or more separate fermentation reactions. In some embodiments the at least one cell wall degrading enzyme and at least one polypeptide are produced in the same host cell and in the same fermentation reaction.

“Plant” as used herein, means an entire plant or a part thereof, including fresh fruit, vegetables and seeds. The plant or plant part may be a live plant or part thereof. Also, the term "plant" as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest. The term "plant" also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid of interest.

The choice of suitable control plants is a routine part of an experimental setup and may include corresponding wild type plants or corresponding plants without the gene of interest. The control plant is typically of the same plant species or even of the same variety as the plant to be assessed. The control plant may also be a nullizygote of the plant to be assessed. Nullizygotes are individuals missing the transgene by segregation. A "control plant" as used herein refers not only to whole plants, but also to plant parts, including seeds and seed parts.

“Crop” as used herein means a plant species or variety that is grown to be harvested as food, livestock fodder, fuel raw material, or for any other economic purpose. As a non-limiting example, said crops can be maize, cereals, such as wheat, rye, barley and oats, sorghum, rice, sugar beet and fodder beet, fruit, such as pome fruit (e.g. apples and pears), citrus fruit (e.g. oranges, lemons, limes, grapefruit, or mandarins), stone fruit (e. g. peaches, nectarines or plums), nuts (e.g. almonds or walnuts), soft fruit (e.g. cherries, strawberries, blackberries or raspberries), the plantain family or grapevines, leguminous crops, such as beans, lentils, peas and soya, oil crops, such as sunflower, safflower, rapeseed, canola, castor or olives, cucurbits, such as cucumbers, melons or pumpkins, fibre plants, such as cotton, flax or hemp, fuel crops, such as sugarcane, miscanthus or switchgrass, vegetables, such as potatoes, tomatoes, peppers, lettuce, spinach, onions, carrots, egg-plants, asparagus or cabbage, ornamentals, such as flowers (e.g. petunias, pelargoniums, roses, tulips, lilies, or chrysanthemums), shrubs, broad-leaved trees (e.g. poplars or willows) and evergreens (e.g. conifers), grasses, such as lawn, turf or forage grass or other useful plants, such as coffee, tea, tobacco, hops, pepper, rubber or latex plants.

A “pest”, as used here, is an organism that is harmful to plants, animals, humans or human concerns, and includes, but is not limited to crop pests (as later defined), household pests, such as cockroaches, ants, etc., and disease vectors, such as malaria mosquitoes.

A “plant pest”, “plant pathogen” or “crop pest”, as used in the application interchangeably, refers to organisms that specifically cause damage to plants, plant parts or plant products, particularly plants, plant parts or plant products, used in agriculture. Note that the term “plant pest” or “crop pest” is used in the meaning that the pest targets and harms plants. Pests particularly belong to invertebrate animals (e.g. insects (including agricultural pest insects, insect pests of ornamental plants, insect pests of forests). Relevant crop pest examples include, but are not limited to, aphids, caterpillars, flies, wasps, and the like, nematodes (living freely in soil or particularly species that parasitize plant roots, such as root-knot nematode and cyst nematodes such as soybean cyst nematode and potato cyst nematode), mites (such as spider mites, thread-footed mites and gall mites) and gastropods (including slugs such as Deroceras spp., Milax spp., Tandonia sp., Limax spp., Arion spp. and Veronicella spp. and snails such as Helix spp., Cernuella spp., Theba spp., Cochlicella spp., Achatina spp., Succinea spp., Ovachlamys spp., Amphibulima spp., Zachrysia spp., Bradybaena spp., and Pomacea spp.), pathogenic fungi (including Ascomycetes (such as Fusarium spp., Thielaviopsis spp., Verticillium spp., Magnaporthe spp.), Basidiomycetes (such as Rhizoctonia spp., Phakospora spp., Puccinia spp.), and fungal-like Oomycetes (such as Pythium spp. and Phytophthora spp.), bacteria (such as Burkholderia spp. and Proteobacteria such as Xanthomonas spp. and Pseudomonas spp.), Phytoplasma, Spiroplasma, viruses (such as tobacco mosaic virus and cauliflower mosaic virus), and protozoa.

“Microbe”, as used herein, means bacterium, virus, fungus, yeast and the like and “microbial” means derived from a microbe.

“Fungus”, as used herein, means a eukaryotic organism, belonging to the group of Eumycota. The term fungus in the present invention also includes fungal-like organisms such as the Oomycota. Oomycota (or oomycetes) form a distinct phylogenetic lineage of fungus-like eukaryotic microorganisms. This group was originally classified among the fungi but modern insights support a relatively close relationship with the photosynthetic organisms such as brown algae and diatoms, within the group of heterokonts.

“Pest infection” or “pest disease” as used herein refers to any inflammatory condition, disease or disorder in a living organism, such as a plant, animal or human, which is caused by a pest.

“Fungal infection” or “fungal disease” as used herein refers to any inflammatory condition, disease or disorder in a living organism, such as a plant, animal or human, which is caused by a fungus.

“Active substance”, “active ingredient” or “active principle”, as used interchangeably herein, means any biological, biochemical or chemical element and its derivatives, fragments or compounds based thereon, including micro-organisms, having general or specific action against harmful organisms on a subject, and in particular on plants, parts of plants or on plant products, as they occur naturally or by manufacture, including any impurity inevitably resulting from the manufacturing process.

“Agrochemical”, as used herein, means suitable for use in the agrochemical industry (including agriculture, horticulture, floriculture and home and garden uses, but also products intended for non-crop related uses such as public health/pest control operator uses to control undesirable insects and rodents, household uses, such as household fungicides and insecticides and agents, for protecting plants or parts of plants, crops, bulbs, tubers, fruits (e.g. from harmful organisms, diseases or pests); for controlling, preferably promoting or increasing, the growth of plants; and/or for promoting the yield of plants, crops or the parts of plants that are harvested (e.g. its fruits, flowers, seeds etc.). Examples of such substances will be clear to the skilled person and may for example include compounds that are active as insecticides (e.g. contact insecticides or systemic insecticides, including insecticides for household use), herbicides (e.g. contact herbicides or systemic herbicides, including herbicides for household use), fungicides (e.g. contact fungicides or systemic fungicides, including fungicides for household use), nematicides (e.g. contact nematicides or systemic nematicides, including nematicides for household use) and other pesticides or biocides (for example agents for killing insects or snails); as well as fertilizers; growth regulators such as plant hormones; micro-nutrients, safeners, pheromones; repellants; insect baits; and/or active principles that are used to modulate (i.e. increase, decrease, inhibit, enhance and/or trigger) gene expression (and/or other biological or biochemical processes) in or by the targeted plant (e.g. the plant to be protected or the plant to be controlled), such as nucleic acids (e.g., single stranded or double stranded RNA, as for example used in the context of RNAi technology) and other factors, proteins, chemicals, etc. known per se for this purpose, etc. Examples of such agrochemicals will be clear to the skilled person; and for example include, without limitation: glyphosate, paraquat, metolachlor, acetochlor, mesotrione, 2, 4-D, atrazine, glufosinate, sulfosate, fenoxaprop, pendimethalin, picloram, trifluralin, bromoxynil, clodinafop, fluroxypyr, nicosulfuron, bensulfuron, imazetapyr, dicamba, imidacloprid, thiamethoxam, fipronil, chlorpyrifos, deltamethrin, lambda-cyhalotrin, endosulfan, methamidophos, carbofuran, clothianidin, cypermethrin, abamectin, diflufenican, spinosad, indoxacarb, bifenthrin, tefluthrin, azoxystrobin, thiamethoxam, tebuconazole, mancozeb, cyazofamid, fluazinam, pyraclostrobin, epoxiconazole, chlorothalonil, copper fungicides, trifloxystrobin, prothioconazole, difenoconazole, carbendazim, propiconazole, thiophanate, sulphur, boscalid and other known agrochemicals or any suitable combination(s) thereof.

An “agrochemical composition” as used herein means a composition for agrochemical use, as further defined, comprising at least one active substance, optionally with one or more additives favouring optimal dispersion, atomization, deposition, leaf wetting, distribution, retention and/or uptake of agrochemicals. It will become clear from the further description herein that an agrochemical composition as used herein includes biological control agents or biological pesticides (including but not limited to biological biocidal, biostatic, fungistatic and fungicidal agents) and these terms will be interchangeably used in the present application. Accordingly, an agrochemical composition as used herein includes compositions comprising at least one biological molecule as an active ingredient, substance or principle for controlling pests in plants or in other agro-related settings (such for example in soil). Non-limiting examples of biological molecules being used as active principles in the agrochemical compositions disclosed herein are proteins (including antibodies and fragments thereof, such as but not limited to heavy chain variable domain fragments of antibodies, including VHH’s), cell wall degrading enzymes nucleic acid sequences, (poly-) saccharides, lipids, vitamins, hormones glycolipids, sterols, and glycerolipids.

As a non-limiting example, the additives in the agrochemical compositions disclosed herein may include but are not limited to diluents, solvents, adjuvants, surfactants, wetting agents, spreading agents, oils, stickers, thickeners, penetrants, buffering agents, acidifiers, anti-settling agents, anti-freeze agents, photo-protectors, defoaming agents, biocides and/or drift control agents.

A “biostatic composition” or a “biostatic agent” as used herein means any active ingredient, substance or principle or a composition comprising any active ingredient, substance or principle for biostatic use (as further defined herein) comprising at least one active biostatic substance or ingredient, optionally combined with one or more additives favouring optimal dispersion, atomization, deposition, leaf wetting, distribution, retention and/or uptake of the active substance or ingredient. As a non-limiting examples such additives are diluents, solvents, adjuvants, (ionic) surfactants, wetting agents, spreading agents, oils, stickers, thickeners, penetrants, buffering agents, acidifiers, anti-settling agents, anti-freeze agents, photo-protectors, defoaming agents, biocides, protease inhibitors and/or drift control agents.

A “biocidal composition” or a “biocidal agent” as used herein means any active ingredient, substance or principle or a composition comprising any active ingredient, substance or principle for biocidal use (as further defined herein) comprising at least one active biocidal substance or ingredient, optionally combined with one or more additives favouring optimal dispersion, atomization, deposition, leaf wetting, distribution, retention and/or uptake of the active substance or ingredient. As a non-limiting examples such additives are diluents, solvents, adjuvants, (ionic) surfactants, wetting agents, spreading agents, oils, stickers, thickeners, penetrants, buffering agents, acidifiers, anti-settling agents, anti-freeze agents, photo-protectors, defoaming agents, biocides, protease inhibitors and/or drift control agents.

A “fungistatic composition” or a “fungistatic agent” as used herein means any active ingredient, substance or principle or a composition comprising any active ingredient, substance or principle for fungistatic use (as further defined herein) comprising at least one active fungistatic substance or ingredient, optionally combined with one or more additives favouring optimal dispersion, atomization, deposition, leaf wetting, distribution, retention and/or uptake of the active substance or ingredient. As a non-limiting examples such additives are diluents, solvents, adjuvants, (ionic) surfactants, wetting agents, spreading agents, oils, stickers, thickeners, penetrants, buffering agents, acidifiers, anti-settling agents, anti-freeze agents, photo-protectors, defoaming agents, biocides, protease inhibitors and/or drift control agents.

A “fungicidal composition” or a “fungicidal agent” as used herein means any active ingredient, substance or principle or a composition comprising any active ingredient, substance or principle for fungicidal use (as further defined herein) comprising at least one active fungicidal substance or ingredient, optionally combined with one or more additives favouring optimal dispersion, atomization, deposition, leaf wetting, distribution, retention and/or uptake of the active substance or ingredient. As a non-limiting examples such additives are diluents, solvents, adjuvants, (ionic) surfactants, wetting agents, spreading agents, oils, stickers, thickeners, penetrants, buffering agents, acidifiers, anti-settling agents, anti-freeze agents, photo-protectors, defoaming agents, biocides, protease inhibitors and/or drift control agents.

“Agrochemical use”, as used herein, not only includes the use of agrochemicals as defined above (for example, pesticides, growth regulators, nutrients/fertilizers, repellants, defoliants etc.) that are suitable and/or intended for use in field grown crops (e.g., agriculture), but also includes the use of agrochemicals as defined above (for example, pesticides, growth regulators, nutrients/fertilizers, repellants, defoliants etc.) that are meant for use in greenhouse grown crops (e.g. horticu Iture/f loriculture) or hydroponic culture systems and even the use of agrochemicals as defined above that are suitable and/or intended for 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.).

“Biostatic (effect)” or “biostatic use”, as used herein, includes any effect or use of an active substance (optionally comprised in a biostatic, biocidal, fungicidal or fungistatic composition as defined herein) for controlling, modulating or interfering with the harmful activity of a pest, such as a plant pest or a plant pathogen, including but not limited to inhibiting the growth or activity of the pest, altering the behaviour of the pest, and repelling the pest in or on plants, plant parts or in other agrorelated settings, such as for example for household uses or in soil.

“Biocidal (effect)” or “biocidal use”, as used herein, includes any effect or use of an active substance (optionally comprised in a biocidal or fungicidal composition as defined herein) for killing the pest in or on plants, plant parts or in other agro-related settings, such as for example for household uses or in soil.

“Anti-fungal” activity or effect refers to fungistatic and/or fungicidal activity or effect.

“Fungistatic (effect)” or “Fungistatic use” or “fungistatic activity”, as used herein, includes any effect or use of an active substance (optionally comprised in a fungicidal or fungistatic composition as defined herein) for controlling, modulating or interfering 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 or attracting the fungus in plants, plant parts or in other agro-related settings, such as for example for household uses or in soil.

“Fungicidal (effect)” or “Fungicidal use” or “fungicidal activity”, as used herein, includes any effect or use of an active substance (optionally comprised in a fungicidal 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.

“Pesticidal activity” or “biocidal activity”, as used interchangeably herein, means to interfere with the harmful activity of a pest, including but not limited to killing the pest.

“Biostatic activity”, as used herein, means to interfere with the harmful activity of a pest, including but not limited to inhibiting the growth or activity of the pest, altering the behaviour of the pest, or repelling the pest.

Pesticidal, biocidal, or biostatic activity of an active ingredient, substance or principle or a composition or agent comprising a pesticidal, biocidal, or biostatic active ingredient, substance or principle, can be expressed as the minimum inhibitory activity (MIC) of an agent (expressed in units of concentration such as e.g. mg/mL), without however being restricted thereto.

“Fungicidal activity”, as used herein, means to interfere with the harmful activity of a fungus, including but not limited to killing the fungus. “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.

Fungicidal or fungistatic activity of an active ingredient, substance or principle or a composition or agent comprising a pesticidal, biocidal, or biostatic active ingredient, substance or principle, can be expressed as the minimum inhibitory activity (MIC) of an agent (expressed in units of concentration such as e.g. mg/mL), without however being restricted thereto.

A “carrier”, as used herein, means any solid, semi-solid or liquid carrier in or on(to) which an active substance can be suitably incorporated, included, immobilized, adsorbed, absorbed, bound, encapsulated, embedded, attached, or comprised. Non-limiting examples of such carriers include nanocapsules, microcapsules, nanospheres, microspheres, nanoparticles, microparticles, liposomes, vesicles, beads, a gel, weak ionic resin particles, liposomes, cochleate delivery vehicles, small granules, granulates, nano-tubes, bucky-balls, water droplets that are part of an water-in-oil emulsion, oil droplets that are part of an oil-in-water emulsion, organic materials such as cork, wood or other plant-derived materials (e.g. in the form of seed shells, wood chips, pulp, spheres, beads, sheets or any other suitable form), paper or cardboard, inorganic materials such as talc, clay, microcrystalline cellulose, silica, alumina, silicates and zeolites, or even microbial cells (such as yeast cells) or suitable fractions or fragments thereof.

As used herein, the term "antibody" refers to polyclonal antibodies, monoclonal antibodies, humanized antibodies, single-chain antibodies, and fragments thereof such as Fab F(ab)2 , Fv, and other fragments that retain the antigen binding function of the parent antibody. As such, an antibody may refer to an immunoglobulin or glycoprotein, or fragment or portion thereof, or to a construct comprising an antigen-binding portion comprised within a modified immunoglobulin-like framework, or to an antigen-binding portion comprised within a construct comprising a non- immunoglobulin-like framework or scaffold.

As used herein, the term "monoclonal antibody" refers to an antibody composition having a homogeneous antibody population. The term is not limited regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is made. The term encompasses whole immunoglobulins as well as fragments such as Fab, F(ab’)2, Fv, and others that retain the antigen binding function of the antibody. Monoclonal antibodies of any mammalian species can be used in this invention. In practice, however, the antibodies will typically be of rat or murine origin because of the availability of rat or murine cell lines for use in making the required hybrid cell lines or hybridomas to produce monoclonal antibodies.

As used herein, the term "polyclonal antibody" refers to an antibody composition having a heterogeneous antibody population. Polyclonal antibodies are often derived from the pooled serum from immunized animals or from selected humans.

“Heavy chain variable domain of an antibody or a functional fragment thereof”, as used herein, means (i) the variable domain of the heavy chain of a heavy chain antibody, which is naturally devoid of light chains (also indicated hereafter as VHH), including but not limited to the variable domain of the heavy chain of heavy chain antibodies of camelids or sharks or (ii) the variable domain of the heavy chain of a conventional four-chain antibody (also indicated hereafter as VH), including but not limited to a camelized (as further defined herein) variable domain of the heavy chain of a conventional four-chain antibody (also indicated hereafter as camelized VH).

As further described hereinbelow, the amino acid sequence and structure of a heavy chain variable domain of an antibody can be considered, without however being limited thereto, to be comprised of four framework regions or “FR's”, which are referred to in the art and hereinbelow as “framework region 1 ” or “FR1 ”; as “framework region 2” or “FR2”; as “framework region 3” or “FR3”; and as “framework region 4” or “FR4”, respectively, which framework regions are interrupted by three complementary determining regions or “CDR's”, which are referred to in the art as “complementarity determining region 1 ” or “CDR1 ”; as “complementarity determining region 2” or “CDR2”; and as “complementarity determining region 3” or “CDR3”, respectively.

As also further described hereinbelow, the total number of amino acid residues in a heavy chain variable domain of an antibody (including a VHH or a VH) can be in the region of 1 10-130, is preferably 1 12-1 15, and is most preferably 1 13. It should however be noted that parts, fragments or analogs of a heavy chain variable domain of an antibody are not particularly limited as to their length and/or size, as long as such parts, fragments or analogs retain (at least part of) the functional activity, such as the pesticidal, biocidal, biostatic activity, fungicidal or fungistatic activity (as defined herein) and/or retain (at least part of) the binding specificity of the original a heavy chain variable domain of an antibody from which these parts, fragments or analogs are derived from. Parts, fragments or analogs retaining (at least part of) the functional activity, such as the pesticidal, biocidal, biostatic activity, fungicidal or fungistatic activity (as defined herein) and/or retaining (at least part of) the binding specificity of the original heavy chain variable domain of an antibody from which these parts, fragments or analogs are derived from are also further referred to herein as “functional fragments” of a heavy chain variable domain.

A method for numbering the amino acid residues of heavy chain variable domains is the method described by Chothia et al. (Nature 342, 877-883 (1989)), the so-called “AbM definition” and the so-called “contact definition”. Herein, this is the numbering system adopted.

Alternatively, the amino acid residues of a variable domain of a heavy chain variable domain of an antibody (including a VHH or a VH) may be numbered according to the general numbering for heavy chain variable domains given by Kabat et al. (“Sequence of proteins of immunological interest’, US Public Health Services, NIH Bethesda, Md., Publication No. 91 ), as applied to VHH domains from Camelids in the article of Riechmann and Muyldermans, referred to above (see for example FIG. 2 of said reference).

For a general description of heavy chain antibodies and the variable domains thereof, reference is inter alia made to the following references, which are mentioned as general background art: WO 94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel; WO 94/25591 , WO 99/37681 , WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301 , EP 1 134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of Algonomics N.V. and Ablynx NV; WO 01/90190 by the National Research Council of Canada; WO 03/025020 (=EP 1 433 793) by the Institute of Antibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551 by Ablynx; Hamers-Casterman et al., Nature 1993 Jun. 3; 363 (6428): 446-8.

Generally, it should be noted that the term “heavy chain variable domain” as used herein in its broadest sense is not limited to a specific biological source or to a specific method of preparation. For example, as will be discussed in more detail below, the heavy chain variable domains of the invention can be obtained (1 ) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by isolating the VH domain of a naturally occurring four-chain antibody (3) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (4) by expression of a nucleotide sequence encoding a naturally occurring VH domain (5) by “camelization” (as described below) of a naturally occurring Vndomain from any animal species, in particular a species of mammal, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (6) by “camelisation” of a “domain antibody” or “Dab” as described by Ward et al (supra), or by expression of a nucleic acid encoding such a camelized Vn domain (7) using synthetic or semisynthetic techniques for preparing proteins, polypeptides or other amino acid sequences; (8) by preparing a nucleic acid encoding a VHH or a VH using techniques for nucleic acid synthesis, followed by expression of the nucleic acid thus obtained; and/or (9) by any combination of the foregoing. Suitable methods and techniques for performing the foregoing will be clear to the skilled person based on the disclosure herein and for example include the methods and techniques described in more detail hereinbelow.

However, according to a specific embodiment, the heavy chain variable domains as disclosed herein do not have an amino acid sequence that is exactly the same as (i.e. as a degree of sequence identity of 100% with) the amino acid sequence of a naturally occurring Vn domain, such as the amino acid sequence of a naturally occurring Vn domain from a mammal, and in particular from a human being.

The terms "effective amount" and "effective dose", as used herein, mean the amount needed to achieve the desired result or results.

As used herein, the terms “determining”, ’’measuring”, ’’assessing”, “monitoring” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.

The terms “cell wall” or “fungal cell wall”, as used herein means the outer structure of a cell such as a plant cell or fungal cell. The cell wall is present on the outside of the cell and envelopes the protoplast. The fungal cell wall is typically present as the layer outside of the cell membrane. Typically cell walls are comprised of mostly chitin, p-1 ,3-glucan and p-1 ,6-glucan polysaccharides, although many other types of polymeric (saccharide) structures, proteins or other organo-chemical molecules may be present in the (fungal) cell wall. The cell wall is a relatively rigid structure compared to the more flexible cell membrane. The cell wall protects and limits access to the cell membrane and its embedded receptors. The cell wall further provides structural integrity and rigidity to the (fungal) cell and acts as a protective barrier against environmental stresses, including osmotic pressure, mechanical damage, and immune responses. 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 compound 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.

As used herein, the phrase “degrading” or “degradation” in the context of the (fungal) cell wall relates to the activity of an agent, such as an enzyme, specifically a cell wall degrading enzyme, to alter the integrity of a cell wall. This can include, but is not limited to degradation, permeabilization, digestion, dissolving, thinning, loosening and weakening of the cell wall. The cell wall may be any cell wall, such as a bacterial cell wall, a plant cell wall or a fungal cell wall. The cell wall degrading enzyme may reduce the integrity of the cell wall. Enzymes, such as cell wall degrading enzymes, degrade the cell wall through hydrolysis of glycosidic bonds of for example chitin or glucan polymers present in the cell wall.

The term “interacting” or “interaction” or “interacts with” in the context of “cell membrane interacting properties” or “antifungal compounds with cell membrane interacting properties”, means the ability of an agent, such as an antifungal compound, to interact with or bind to the cell membrane. Alternatively, the term interacting may also refer to agents such as antifungal compounds that interact with a cellular process responsible for building or maintaining the cell membrane and whose disruption may lead to a destabilized cell membrane, for example such an antifungal compound may be an azole compound. In some aspects, and as an example where the antifungal compound may have a direct effect on the cell membrane, interacting may mean intercalating between the bilayer of the membrane. Such an agent 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 agent such as an antifungal compound may also indicate that a certain membrane protein is impeded by the antifungal compound 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 nutrient influx or hampering the generation of energy in the form of ATP in the cell, such interaction may be accomplished by binding of 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 antifungal compound with cell membrane interacting properties may be improved when the cell wall is (partially) degraded by at least one cell wall degrading enzyme, either by allowing the antifungal compound facilitated access to the cell membrane or by failing to protect the cell membrane sufficiently against antifungal compounds that may cause the cell membrane to rupture or lyse. In a preferred embodiment, the antifungal compound with cell membrane interacting properties is 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. In such a scenario, the cell wall degrading enzyme 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.

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.

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.

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.

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. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

Antifungal compounds and Polypeptides

The current invention provides compositions comprising at least one cell wall degrading enzyme and at least one antifungal compounds and wherein the antifungal compound is capable of interacting with the cell membrane, preferentially the cell membrane of a fungal cell i.e. the antifungal compound is an antifungal compound with cell membrane interacting properties. In some embodiments, the antifungal compound may disrupt the cell membrane. In some embodiments the antifungal compound with cell membrane interacting properties may lead to the rupture of the cell membrane. In some embodiments the antifungal compound with cell membrane interacting properties may lead to the rupture of the cell membrane and the subsequent spilling of cytoplasmic material into the surroundings of the cell, such as a protoplast cell, or a cell with an intact cell wall, preferably a cell with a compromised cell wall; and where said cell is a fungal cell, preferably a fungal pathogen cell, more preferably a fungal plant pathogenic cell. In another embodiment, the antifungal compound does not affect the cell membrane by may affect other cellular process through binding to a target on or in the cell membrane. By combining an antifungal compound with cell membrane interacting properties with at least one cell wall degrading enzyme the current inventors have found that the activity of the antifungal compound with cell membrane interacting properties may be improved, requiring a reduced amount, preferentially an amount that is less than the sum of the antifungal compound with cell membrane interacting properties and the at least one cell wall degrading enzyme separately (i.e. a synergistic effect is achieved), to assert an effect, such as a fungicidal effect. In a preferred embodiment, the antifungal compound with cell membrane interacting properties is selected from the polypeptides disclosed herein which are generally capable of binding to or interacting with a fungus. The polypeptides disclosed here are generally (although not exclusively) antibodies or antibody fragments such as heavy chain variable domain of an antibody or a functional fragments thereof. More specifically, a heavy chain variable domain of a heavy chain antibody (VHH) or a functional fragment thereof may be preferred. An example of an antifungal compound with cell membrane interacting properties that is not a polypeptide may be selected from the group of azole antifungal compounds that disrupts the formation of the cell membrane or nitroanilines antifungal compounds that may damage the lipid membrane, it being understood that the current invention may work with any antifungal compound that interacts with the cell membrane of a fungal species, where the fungal species is a fungal pathogen preferably a fungal plant pathogen.

In a preferred embodiment, the antifungal compound with cell membrane interacting properties is a polypeptide as described herein. The polypeptides of and used in the invention may (specifically) bind to a membrane of a fungus or a component of a membrane of a fungus. Specifically, the polypeptides may bind a component of the plasma membrane of a fungus. The polypeptides may interact with the cell membrane of a fungus. The polypeptides may specifically bind a structural molecular component of the plasma membrane of a fungus. The polypeptides used in the current invention may have, when binding to or interacting with the cell membrane, the effect of disrupting the integrity of the cell membrane, preferably the fungal cell membrane, such as by intercalating in the phospholipid bi-layer either by specifically or non-specifical interactions or binding. Preferably the polypeptides used in the invention are VHH antibodies as described herein.

The polypeptides 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.

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: 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.

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

In a more specific embodiment, the 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 normal-phase flash chromatography comprising dissolving the TLE in dichloromethane (CH2CI2) and MeOH and using CH2Cl2/MeOH (for example 85/15%, v/v) as the eluent, followed by filtration of the fractions through a filter.

In a more specific embodiment, the 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 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 CH2Cl2/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.

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). 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.

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.

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.

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.

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.

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

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

In some methods relating to normal-phase flash chromatography, the chromatography may comprise the steps of: 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 CH2Cl2/MeOH (for example 85/15%, v/v) as the eluent, followed by filtration of the fractions through a filter.

In some methods relating to normal-phase flash chromatography, the chromatography may comprise the steps of: 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 CH2Cl2/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 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, may be antibodies more specifically a VHH antibody or a fragment thereof, more specifically any one of SEQ ID NO: 1 , 2, 6, 10, 14 or 15.

Alternatively, the polypeptides may be capable of (specifically) binding to sphinglolipids 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, inositolphosphorylceramide (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.

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 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: 16 to SEQ ID NO: 99. In some aspects, the present invention provides a polypeptide comprising or consisting of the amino acid sequence set out in any one of SEQ ID NOs: 1 , 2, 6, 10, 14 to 99, 131 or 132, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto.

In some aspects, the present invention provides a polypeptide comprising or consisting of the amino acid sequence set out in any one of SEQ ID NOs: 1 , 2, 6, 10,14 to 99, 131 or 132, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto.

In some aspects, the present invention provides a polypeptide, preferably a VHH antibody, where the VHH antibody may be selected from the group consisting of SEQ ID Nos: 1 , 2, 6, 10, 14 to 99, 131 or 132.

In some embodiments, the composition disclosed herein comprises: a CDR1 comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs 3, 7 and 1 1 ; a CDR2 comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 4, 8 and 12; and a CDR3 comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 5, 9 and 13.

In some embodiments, the composition disclosed herein comprises: a CDR1 comprising or consisting of the sequence of SEQ ID NO: 3, a CDR2 comprising or consisting of the sequence of SEQ ID NO: 4 and a CDR3 comprising or consisting of the sequence of SEQ ID NO: 5; a CDR1 comprising or consisting of the sequence of SEQ ID NO: 7, a CDR2 comprising or consisting of the sequence of SEQ ID NO: 8 and a CDR3 comprising or consisting of the sequence of SEQ ID NO: 9 or a CDR1 comprising or consisting of the sequence of SEQ ID NO: 1 1 , a CDR2 comprising or consisting of the sequence of SEQ ID NO: 12 and a CDR3 comprising or consisting of the sequence of SEQ ID NO: 13.

In some embodiments, the composition comprises a VHH comprising a CDR1 comprising or consisting of the sequence of SEQ ID NO: 3, a CDR2 comprising or consisting of the sequence of SEQ ID NO: 4 and a CDR3 comprising or consisting of the sequence of SEQ ID NO: 5.

In some embodiments, the compositions disclosed herein comprises a VHH disclosed in WQ2014/177595 or WQ2014/191 146, the entire contents of which are incorporated herein by reference. More specifically the composition may comprise a VHH comprising an amino acid sequence chosen from the group consisting of SEQ ID NO's: 1 to 84 from WQ2014/177595 or WQ2014/191 146 which refer to SEQ ID Nos 16 to 99 of this application.

In some embodiments the polypeptide may be a VHH antibody as described in US provisional applications 63/614,192 or 63/614,183 identified therein as the VHH antibody comprising SEQ ID NO: 2, which refer to SEQ ID NO 131 or 132 respectively of this application, or the VHH antibody described therein as comprising CDR 1 region according to SEQ ID NO: 3, the CDR 2 region according to SEQ ID NO: 4, the CDR3 region according SEQ ID NO: 5.

The CDR and framework regions maybe defined according to the Kabat numbering system.

The polypeptides or amino acid sequences comprised in the compositions disclosed 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 or amino acid sequences as disclosed herein consist of a single polypeptide chain and are not post- translationally modified. More particularly, the polypeptides or amino acid sequences as disclosed are derived from an innate or adaptive immune system, preferably from a protein of an innate or adaptive immune system. Still more particularly, the polypeptides or amino acid sequences as disclosed herein are derived from an immunoglobulin. Most particularly, the polypeptides or amino acid sequences as disclosed herein 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, the polypeptides or amino acid sequences as disclosed herein are easy to produce at high yield, preferably in a microbial recombinant expression system, and convenient to isolate and/or purify subsequently. Particularly, the polypeptides or amino acid sequences as disclosed herein are selected from the group consisting of DARPins, knottins, alphabodies and VHH’s. More particularly, the polypeptides or amino acid sequences as disclosed herein are selected from the group consisting of alphabodies and VHH’s. Most particularly, the polypeptides or amino acid sequences as disclosed herein are VHH’s. However, it should be noted that the invention is not limited as to the origin of the polypeptides comprised in the compositions disclosed herein (or of the nucleotide sequence of the invention used to express it), nor as to the way that the polypeptides or nucleotide sequences thereof is (or has been) generated or obtained. Thus, the polypeptides in the compositions disclosed 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.

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 (1 1 ):484-490; as well as to for example WO 06/030220, WO 06/003388 and other published patent applications of Domantis Ltd.

Thus, in particular embodiments, the present invention provides compositions comprising polypeptides with the (general) structure

FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 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, and are as further defined herein.

In some aspects the polypeptide 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-helical peptides, (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 is an AMP, the AMP and the at least one cell wall degrading enzyme may act synergistically, for example to allow the IC50 to be reached at a concentration or dose lower. The combination of an AMP and a cell wall degrading enzyme may act synergistically in the same way as seen for the combination of other polypeptides and at least one cell wall degrading enzyme, for example the combination of VHHs and cell wall degrading enzymes. In this way, effective pest control can be achieved using lower doses or concentrations of each component. The polypeptides of the invention may be provided in the form of compositions, for example agrochemical compositions. The polypeptides of the invention are provided in the form of compositions further comprising at least one cell wall degrading enzyme, as described herein.

Cell Wall Degrading Enzymes

The compositions disclosed herein contain at least one cell wall degrading enzyme. The cell wall degrading enzymes disclosed herein are enzymes capable of degrading the integrity of the cell wall. More specifically, the cell wall degrading enzyme may be a fungal cell wall degrading enzyme. Therefore, the invention provides for compositions comprising at least one cell wall degrading enzyme, and methods and uses thereof. In some embodiments, the cell wall degrading enzyme is the only active ingredient in the composition.

In some embodiments, the cell wall degrading enzyme is active against a bacterial cell wall. In some embodiments, the cell wall degrading enzyme is active against a plant cell wall. In a preferred embodiment, the cell wall degrading enzyme is active against a fungal cell wall. In some embodiments, the cell wall degrading enzyme is active against a fungal cell wall but is not active against a plant cell wall.

In some embodiments, the cell wall degrading enzyme reduces the integrity of the cell wall. The reduction in cell wall integrity may allow molecules to permeate through the cell wall to the cell membrane. In some embodiments, the molecules that permeate through the cell wall are proteins. In preferred embodiments, the proteins that permeate through the cell wall comprise heavy chain variable domains of a heavy chain antibody (VHH) or a functional fragment thereof. In preferred embodiments, the molecules that permeate the cell wall are polypeptides of the composition disclosed herein. In some embodiments the cell wall degrading enzymes alone, acts as an anti-pest agent, such as for instance a biostatic agent or a pesticidal agent, including but not limited to a fungistatic or a fungicidal agent. Without wanting to be bound by theory, the reduced integrity of the cell wall may also in turn lead to the destabilization of the cell membrane. That is to say, where the cell wall has a reduced integrity the cell membrane may destabilize, disrupt and/or even rupture due to osmotic pressure on the cell membrane in the absence of the more rigid outer cell wall supporting the cell membrane. In some more preferred embodiments where the cell membrane is destabilized by for example an antifungal compound, the cell membrane will more easily destabilize, disrupt and/or rupture where the cell wall has reduced integrity. In preferred embodiments, the proteins that interact with the cell membrane comprise heavy chain variable domains of a heavy chain antibody (VHH) or a functional fragment thereof.

In some embodiments, the composition or combination disclosed herein comprises a mixture of cell wall degrading enzymes. In a preferred embodiment the cell wall degrading enzymes are active against a fungal cell wall. In some embodiments, the composition or combination comprises at least one, at least two, at least 3, at least 4 or at least 5 cell wall degrading enzymes. In some embodiments, the composition or combination comprises no more than 28 cell wall degrading enzymes, preferably no more than 9, preferably no more than 6, preferably no more than 3 cell wall degrading enzymes. In a preferred embodiment the cell wall degrading enzymes are recombinantly produced. In a more preferred embodiment the cell wall degrading enzymes are heterologous cell wall degrading enzymes. That is to say, a the cell wall degrading enzymes are produced in a host cell in which they are not native. For example, a cell wall degrading enzyme found in Trichoderma harzianum may be produced in Trichoderma reesei or Pichia pastoris where said cell wall degrading enzymes is a heterologous cell wall degrading enzyme. In an even more preferred embodiment the cell wall degrading enzymes are recombinantly produced and purified cell wall degrading enzymes. In some embodiments, the composition comprises multiple cell wall degrading enzymes or a mixture of enzymes as described below. In some embodiments, the composition or combination does not contain one or more proteases. In some embodiments, the composition or combination does not contain one or more protease inhibitors.

The cell wall degrading enzyme may be a carbohydrolase. The cell wall degrading enzyme may be a cellulase. In some embodiments the cell wall degrading enzyme is selected from the group consisting of glucanases, chitinases, mannanases, xyloglucanases, pectinases, glycosidases and xylanases. In some embodiments the cell wall degrading enzyme is selected from the group consisting of glucanases and chitinases. The invention also contemplates a composition comprising a mixture of enzymes comprising at least one enzyme selected from the group consisting of glucanases, chitinases, mannanases, xyloglucanases, pectinases, glycosidases and xylanases. In some preferred embodiments, the mixture of enzymes in the composition comprises at least one chitinase and at least one glucanase. In some embodiments, the mixture of enzymes in the composition comprises at least two chitinases and at least two glucanases. In some embodiments, the mixture of enzymes in the composition comprises at least one chitinase and at least two different glucanases, or at least two different chitinases and at least one glucanase. Mixtures of cell wall degrading enzymes are described in more detail below.

In some embodiments the cell wall degrading enzyme is a glucanase. Glucanases are also referred to as lichenases, hydrolases, glycosidases, glycosyl hydrolases, and/or laminarinases. In some embodiments the glucanase is a beta-glucanase, such as beta-1 ,3-glucanase, beta-1 ,6- glucanase, and/or alpha-1 ,3-glucanase. The glucanase may be an a glucanase selected from the group consisting of exo-beta-1 ,3-glucanase (also known as glucan 1 ,3-beta-glucosidase), endo- beta-1 ,3-glucanase (also known as glucan endo-1 ,3-beta-glucosidase), exo-beta-1 ,6-glucanase (also known as glucan 1 ,6-alpha-glucosidase), endo-beta-1 ,6-glucanase (also known as glucan endo-1 ,6-beta-glucosidase), endo-beta-1 ,3(4)-glucanase (also known as glucan endo-1 ,3(4)-beta- glucosidase), exo-beta-1 ,3(4)-glucanase (also known as glucan exo-1 ,3(4)-beta-glucosidase), endo- alpha-1 ,3-glucanase (also known as glucan endo-1 ,3-alpha-glucosidase), and exo-alpha-1 ,3- glucanase (also known as glucan exo-1 ,3-alpha-glucosidase). In some embodiments, the composition disclosed herein comprises one or more of a glucanase selected from the group consisting of exo-beta-1 ,3-glucanase, endo-beta-1 ,3-glucanase, exo-beta-1 ,6-glucanase, endo- beta-1 ,6-glucanase, endo-beta-1 ,3(4)-glucanase, exo-beta-1 ,3(4)-glucanase, endo-alpha-1 ,3- glucanase, and exo-alpha-1 ,3-glucanase and/or any combination thereof.

In some embodiments, the cell wall degrading enzyme has chitinolytic activity. The chitinase may be endo-chitinase or exo-chitinase. The exo-chitinase may target the reducing or non-reducing end of the chitin. In a preferred embodiment the chitinase is an endo-chitinase. In some embodiments, the composition disclosed herein comprises one or more of a chitinase, preferably an end-chitinase.

In some embodiments, the composition disclosed herein does not contain a cell wall degrading enzyme capable of degrading plant cell walls. In some embodiments, the composition does not contain beta-1 ,4-glucanases.

The cell wall degrading enzymes may be derived from filamentous fungi such as Trichoderma species and/or Aspergillus species. In a preferred embodiment, the cell wall degrading enzymes may be derived from Trichoderma harzianum and/or Aspergillus niger. However, the current invention is not limited to the specific sequences disclosed herein but also extends to homologs such as orthologs and paralogs thereof having the same activity as the cell wall degrading enzymes disclosed herein. That is to say, a homolog of a cell wall degrading enzyme as disclosed herein is encompassed by the invention if said homolog also comprises the same enzymatic activity as the enzyme disclosed herein. Such homologs may differ significantly at the amino acid sequence level. In some instances the difference may be a difference of around 70%, 50%, 60% or even 40% identity at the amino acid sequence level. Therefore the current invention relates to cell wall degrading enzymes that may be derived from Trichoderma harzianum and/or Aspergillus niger or cell wall degrading enzymes comprising 40% or more sequence identity thereto, provided said cell wall degrading enzymes comprising 40% or more sequence identity have the same enzymatic activity as the enzymes disclosed herein. In a more preferred embodiment, the cell wall degrading enzymes comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% to the amino acid sequences as disclosed herein, provided said cell wall degrading enzymes comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity have the same enzymatic activity as the enzymes disclosed herein.

In some embodiments, the cell wall degrading enzyme is active against a fungal cell wall. The fungal cell wall is composed of chitin (acetylglucosamine polymers), glucans, polysaccharides and mucopolysaccharides, waxes and pigments. The cell wall degrading enzyme may act against any component of the cell wall. The cell wall degrading enzyme may act against multiple components of the cell wall. Preferably, the cell wall degrading enzyme is active against chitin and/or glucans in the fungal cell wall. And where the fungus may be a pathogenic fungus, or a fungal pest. The fungus may be a plant pathogenic fungus as described herein below.

In some embodiments the cell wall degrading enzyme is a glucanase comprising an amino acid sequence selected from the list of SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto. In a more preferred embodiment the cell wall degrading enzyme is a glucanase comprising an amino acid sequence selected from the list of SEQ ID NOs: 100 to 105, 1 12 and 128 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto.

In a more specific embodiment, the cell wall degrading enzyme comprises a glucanase comprising an amino acid sequence according to SEQ ID NO: 100, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises glucanase activity. In a more specific embodiment the glucanase is the endo-beta-1 ,3(4)-glucanase according to UniProt ID: A0A2T4A0S6. In a more specific embodiment, the cell wall degrading enzyme comprises a glucanase comprising an amino acid sequence according to SEQ ID NO: 101 , or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises glucanase activity. In a more specific embodiment the glucanase is endo-beta-1 ,3-glucanase according to UniProt ID: A0A2T4A5D1 . In a more specific embodiment, the cell wall degrading enzyme comprises a glucanase comprising an amino acid sequence according to SEQ ID NO: 102, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises glucanase activity. In a more specific embodiment the glucanase is a exo-beta-1 ,3-glucanase according to UniProt ID: A0A2T4ASK4. In a more specific embodiment, the cell wall degrading enzyme comprises a glucanase comprising an amino acid sequence according to SEQ ID NO: 103, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises glucanase activity. In a more specific embodiment the glucanase is the endo-beta-1 ,6-glucanase according to UniProt ID: A0A2T4A8W0. In a more specific embodiment, the cell wall degrading enzyme comprises a glucanase comprising an amino acid sequence according to SEQ ID NO: 104, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises glucanase activity. In a more specific embodiment the glucanase is the endo-beta-1 ,6-glucanase according to UniProt ID: A0A1 17DVW2. In a more specific embodiment, the cell wall degrading enzyme comprises a glucanase comprising an amino acid sequence according to SEQ ID NO: 105, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises glucanase activity. In a more specific embodiment the glucanase is the exo-beta-1 ,3-glucanase according to UniProt ID: A0A1 17DW84. In a more specific embodiment, the cell wall degrading enzyme comprises a glucanase comprising an amino acid sequence according to SEQ ID NO: 106, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises glucanase activity. In a more specific embodiment the glucanase is the endo-beta-1 ,3-glucanase according to UniProt ID: A0A2T4AM74. In a more specific embodiment, the cell wall degrading enzyme comprises a glucanase comprising an amino acid sequence according to SEQ ID NO: 107, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises glucanase activity. In a more specific embodiment the glucanase is the beta-1 ,3-glucanase according to UniProt ID: A0A2T3ZSZ2. In a more specific embodiment, the cell wall degrading enzyme comprises a glucanase comprising an amino acid sequence according to SEQ ID NO: 108, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises glucanase activity. In a more specific embodiment the glucanase is the endo-beta-1 ,3-glucanase according to UniProt ID: A0A2T3ZV04. In a more specific embodiment, the cell wall degrading enzyme comprises a glucanase comprising an amino acid sequence according to SEQ ID NO: 109, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises glucanase activity. In a more specific embodiment the glucanase is the endo-beta-1 ,3-glucanase according to UniProt ID: A0A2T4AKQ4. In a more specific embodiment, the cell wall degrading enzyme comprises a glucanase comprising an amino acid sequence according to SEQ ID NO: 110, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises glucanase activity. In a more specific embodiment the glucanase is the endo-beta-1 ,6-glucanase according to UniProt ID: A0A2T4A2Q3. In a more specific embodiment, the cell wall degrading enzyme comprises a glucanase comprising an amino acid sequence according to SEQ ID NO: 111 , or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises glucanase activity. In a more specific embodiment the glucanase is the endo-beta-1 ,3-glucanase according to UniProt ID: A0A2T4AUZ7. In a more specific embodiment, the cell wall degrading enzyme comprises a glucanase comprising an amino acid sequence according to SEQ ID NO: 112, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises glucanase activity. In a more specific embodiment the glucanase is the endo-alpha-1 ,3-glucanase according to UniProt ID: A0A2T3ZUS3. In a more specific embodiment, the cell wall degrading enzyme comprises a glucanase comprising an amino acid sequence according to SEQ ID NO: 113, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises glucanase activity. In a more specific embodiment the glucanase is the endo-alpha-1 ,3-glucanase according to UniProt ID: A0A2T4ATW4. In a more specific embodiment, the cell wall degrading enzyme comprises a glucanase comprising an amino acid sequence according to SEQ ID NO: 114, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises glucanase activity. In a more specific embodiment the glucanase is the endo-alpha-1 ,3-glucanase according to UniProt ID: A0A2T4A4Y9. In a more specific embodiment, the cell wall degrading enzyme comprises a glucanase comprising an amino acid sequence according to SEQ ID NO: 115, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises glucanase activity. In a more specific embodiment the glucanase is the endo-alpha-1 ,3-glucanase according to UniProt ID: A0A2T4A5I0. In a more specific embodiment, the cell wall degrading enzyme comprises a glucanase comprising an amino acid sequence according to SEQ ID NO: 128, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises glucanase activity. In a more specific embodiment the glucanase is the exo-beta-1 ,3-glucanase according to UniProt ID: A0A2T4APU2. In a more specific embodiment, the cell wall degrading enzyme comprises a glucanase comprising an amino acid sequence according to SEQ ID NO: 129, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises glucanase activity. In a more specific embodiment the glucanase is the exo-beta-1 ,3-glucanase according to UniProt ID: A0A2T4ADC1 . In a more specific embodiment, the cell wall degrading enzyme comprises a glucanase comprising an amino acid sequence according to SEQ ID NO: 130, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises glucanase activity. In a more specific embodiment the glucanase is the exo-beta-1 ,3-glucanase according to UniProt ID: A0A2T4A2N7.

In a preferred embodiment, the composition according to the invention comprises a single cell wall degrading enzyme. In a preferred embodiment, the composition according to the invention comprises a single cell wall degrading enzyme and that cell wall degrading enzyme is selected from the group of glucanase comprising an amino acid sequence selected from the group of SEQ ID NO: 100 to 1 15 and 128 to 130, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto. In a more preferred embodiment, the composition according to the invention comprises a single cell wall degrading enzyme and that cell wall degrading enzyme is selected from the group of glucanase comprising an amino acid sequence selected from the group of SEQ ID NOs: 100 to 1 15 and 128 to 130. In a more preferred embodiment, the composition according to the invention comprises a single cell wall degrading enzyme and that cell wall degrading enzyme is selected from the group of glucanase comprising an amino acid sequence selected from the group of SEQ ID NO: 100 to 105, 1 12 and 128. In a more preferred embodiment, the composition according to the invention comprises a single cell wall degrading enzyme and that cell wall degrading enzyme is a exo-beta-1 ,3-glucanase. In a more preferred embodiment, the composition according to the invention comprises a single cell wall degrading enzyme and that cell wall degrading enzyme is selected from the group of glucanase comprising an amino acid sequence selected from the group of SEQ ID NO: 102, 105, and 128. In a more preferred embodiment, the composition according to the invention comprises a single cell wall degrading enzyme and that cell wall degrading enzyme is SEQ ID NO: 128.

In some embodiments the cell wall degrading enzyme is a chitinase, preferably an endochitinase comprising an amino acid sequence selected from the list of SEQ ID NOs: 1 16 to 124 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto. In a more preferred embodiment the cell wall degrading enzyme is a chitinase comprising an amino acid sequence selected from the list of SEQ ID NOs: 1 16 to 1 19 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto. In some embodiments the cell wall degrading enzyme is a chitinase of the chitinase-like protein from the glycoside hydrolase family 18 comprising an amino acid sequence selected from the list of SEQ ID NOs: 125 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto. In a more preferred embodiment the cell wall degrading enzyme is a chitinaselike protein from the glycoside hydrolase family 18 comprising an amino acid sequence according to SEQ ID NO: 125 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto.

In a more specific embodiment, the cell wall degrading enzyme comprises a chitinase comprising an amino acid sequence according to SEQ ID NO: 1 16, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises chitinolytic activity. In a more specific embodiment the chitinase is the endo-chitinase according to UniProt ID: A0A2T3ZT47. In a more specific embodiment, the cell wall degrading enzyme comprises a chitinase comprising an amino acid sequence according to SEQ ID NO: 1 17, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises chitinolytic activity. In a more specific embodiment the chitinase is the endo-chitinase according to UniProt ID: A0A2T4AMS9. In a more specific embodiment, the cell wall degrading enzyme comprises a chitinase comprising an amino acid sequence according to SEQ ID NO: 1 18, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises chitinolytic activity. In a more specific embodiment the chitinase is the endo-chitinase according to UniProt ID: A0A2T4AIG1 . In a more specific embodiment, the cell wall degrading enzyme comprises a chitinase comprising an amino acid sequence according to SEQ ID NO: 1 19, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises chitinolytic activity. In a more specific embodiment the chitinase is the endo-chitinase according to UniProt ID: A0A2T3ZS1 1 . In a more specific embodiment, the cell wall degrading enzyme comprises a chitinase comprising an amino acid sequence according to SEQ ID NO: 120, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises chitinolytic activity. In a more specific embodiment the chitinase is the endo-chitinase according to UniProt ID: A0A2T4AFS6. In a more specific embodiment, the cell wall degrading enzyme comprises a chitinase comprising an amino acid sequence according to SEQ ID NO: 121 , or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises chitinolytic activity. In a more specific embodiment the chitinase is the endo-chitinase according to UniProt ID: A0A2T3ZRX9. In a more specific embodiment, the cell wall degrading enzyme comprises a chitinase comprising an amino acid sequence according to SEQ ID NO: 122, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises chitinolytic activity. In a more specific embodiment the chitinase is the endo-chitinase according to UniProt ID: A0A2T3ZVR8. In a more specific embodiment, the cell wall degrading enzyme comprises a chitinase comprising an amino acid sequence according to SEQ ID NO: 123, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises chitinolytic activity. In a more specific embodiment the chitinase is the endo-chitinase according to UniProt ID: A0A1 17DXS9. In a more specific embodiment, the cell wall degrading enzyme comprises a chitinase comprising an amino acid sequence according to SEQ ID NO: 124, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises chitinolytic activity. In a more specific embodiment the chitinase is the endo-chitinase according to UniProt ID: A0A2T3ZTE3. In a more specific embodiment, the cell wall degrading enzyme comprises a chitinase comprising an amino acid sequence according to SEQ ID NO: 125, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises chitinolytic activity. In a more specific embodiment the chitinase is the chitinase-like protein according to UniProt ID: A0A2T4A8C1 . In a more specific embodiment, the cell wall degrading enzyme comprises a chitinase comprising an amino acid sequence according to SEQ ID NO: 126, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises chitinolytic activity. In a more specific embodiment the chitinase is the chitinase-like protein according to UniProt ID: A0A2T4ASQ3. In a more specific embodiment, the cell wall degrading enzyme comprises a chitinase comprising an amino acid sequence according to SEQ ID NO: 127, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and where said amino acid sequence comprises chitinolytic activity. In a more specific embodiment the chitinase is the chitinase-like protein according to UniProt ID: A0A2T3ZUN3.

In a preferred embodiment, the composition according to the invention comprises a single cell wall degrading enzyme. In a preferred embodiment the composition according to the invention comprises a single cell wall degrading enzyme and that cell wall degrading enzyme is selected from the group of chitinase comprising an amino acid sequence selected from the group of SEQ ID NO: 1 16 to 127, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto. In a more preferred embodiment the composition according to the invention comprises a single cell wall degrading enzyme and that cell wall degrading enzyme is selected from the group of chitinase comprising an amino acid sequence selected from the group of SEQ ID NOs: 1 16 to 126. In a more preferred embodiment the composition according to the invention comprises a single cell wall degrading enzyme and that cell wall degrading enzyme is selected from the group of chitinase comprising an amino acid sequence selected from the group of SEQ ID NO: 1 16 to 1 19 and 125. In a more preferred embodiment the composition according to the invention comprises a single cell wall degrading enzyme and that cell wall degrading enzyme is a endo-chitinase. In a more preferred embodiment the composition according to the invention comprises a single cell wall degrading enzyme and that cell wall degrading enzyme is selected from the group of chitinase comprising an amino acid sequence selected from the group of SEQ ID NO: 1 16, 1 19 and 125. In a more preferred embodiment the composition according to the invention comprises a single cell wall degrading enzyme and that cell wall degrading enzyme is SEQ ID NO: 1 19.

A mixture of cell wall degrading enzymes

In a preferred embodiment of the invention the cell wall degrading enzyme is a mixture of two or more cell wall degrading enzymes. Therefore when referring herein to a cell wall degrading enzyme, or at least one cell wall degrading enzyme, in a preferred embodiment a mixture of two or more cell wall degrading enzymes is used. The mixture may contain at least two cell wall degrading enzyme selected from a glucanase and/or a chitinase and where the glucanase and chitinase are as described above. The mixture may comprise at least 2, at least 3, at least 4 or at least 5 glucanase and/or chitinases. Preferably the mixture does not comprise more than 28, preferably no more than 9, preferably no more than 6, preferably no more than 3 glucanases and/or chitinases. The mixture may comprise at least one glucanase, such as a glucanase selected from the group consisting of exo-beta-1 ,3-glucanase, endo-beta-1 ,3-glucanase, exo-beta-1 ,6-glucanase, endo-beta-1 ,6- glucanase, endo-beta-1 ,3(4)-glucanase, endo-alpha-1 ,3-glucanase, and exo-alpha-1 ,3-glucanase or any combination thereof; and/or at least one chitinase. In a more preferred embodiment, the mixture may comprise at least one of a cell wall degrading enzyme selected from the list consisting of an endo-1 ,3(4)-beta-glucanase, endo-beta-1 ,3-glucanase, exo-beta-1 ,3-glucanase, endo-beta- 1 ,6-glucanase, and endo-alpha-1 ,3-glucanase; and/or at least one chitinase. In some embodiments the mixture does not comprise a chitinase but comprises 2 or more of a glucanase as described herein. In some embodiments the mixture may comprise 2 or more of a different enzyme comprising the same enzymatic activity. That is to say, two different enzymes may have the same enzymatic activity but may differ in for example their amino acid sequence, substrate specificity, optimal temperature range, or optimal pH range for activity.

In a preferred embodiment the cell wall degrading enzyme is a mixture of two or more cell wall degrading enzymes. In one such an embodiment the mixture comprises two glucanases and where the two glucanases are selected from the list of endo-beta-1 ,3(4)-glucanase, endo-beta-1 ,3- glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6-glucanase and endo-alpha-1 ,3-glucanase. In another such an embodiment the mixture comprises three glucanases and where the three glucanases are selected from the list of endo-beta-1 ,3(4)-glucanase, endo-beta-1 ,3-glucanase, exo- beta-1 ,3-glucanase, endo-beta-1 ,6-glucanase and endo-alpha-1 ,3-glucanase. In another such an embodiment the mixture comprises four glucanases and where the four glucanases are selected from the list of endo-beta-1 ,3(4)-glucanase, endo-beta-1 ,3-glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6-glucanase and endo-alpha-1 ,3-glucanase. In another such an embodiment the mixture comprises five glucanases and where the five glucanases are selected from the list of endo- beta-1 ,3(4)-glucanase, endo-beta-1 ,3-glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6- glucanase and endo-alpha-1 ,3-glucanase. In another such an embodiment the mixture comprises six glucanases and where the six glucanases are selected from the list of endo-beta-1 ,3(4)- glucanase, endo-beta-1 ,3-glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6-glucanase and endo- alpha-1 ,3-glucanase. In another such an embodiment the mixture comprises seven glucanases and where the seven glucanases are selected from the list of endo-beta-1 ,3(4)-glucanase, endo-beta- 1 ,3-glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6-glucanase and endo-alpha-1 ,3-glucanase.

In a more preferred embodiment, the mixture comprises at least one glucanase and at least one chitinase. In one such embodiment the mixture comprises one glucanase selected from the list of endo-beta-1 ,3(4)-glucanase, endo-beta-1 ,3-glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6- glucanase and endo-alpha-1 ,3-glucanase; and a chitinase preferably an endo-chitinase. In a more preferred embodiment, the mixture comprises at least two glucanase and at least one chitinase. In one such embodiment the mixture comprises two glucanases selected from the list of endo-beta- 1 ,3(4)-glucanase, endo-beta-1 ,3-glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6-glucanase and endo-alpha-1 ,3-glucanase; and a chitinase preferably an endo-chitinase. In a more preferred embodiment, the mixture comprises at least three glucanase and at least one chitinase. In one such embodiment the mixture comprises three glucanases selected from the list of endo-beta-1 ,3(4)- glucanase, endo-beta-1 ,3-glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6-glucanase and endo- alpha-1 ,3-glucanase; and a chitinase preferably an endo-chitinase. In a more preferred embodiment, the mixture comprises at least four glucanase and at least one chitinase. In one such embodiment the mixture comprises four glucanases selected from the list of endo-beta-1 ,3(4)-glucanase, endo- beta-1 ,3-glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6-glucanase and endo-alpha-1 ,3- glucanase; and a chitinase preferably an endo-chitinase. In a more preferred embodiment, the mixture comprises at least five glucanase and at least one chitinase. In one such embodiment the mixture comprises five glucanases selected from the list of endo-beta-1 ,3(4)-glucanase, endo-beta- 1 ,3-glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6-glucanase and endo-alpha-1 ,3-glucanase; and a chitinase preferably an endo-chitinase. In a more preferred embodiment, the mixture comprises at least six glucanase and at least one chitinase. In one such embodiment the mixture comprises six glucanases selected from the list of endo-beta-1 ,3(4)-glucanase, endo-beta-1 ,3- glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6-glucanase and endo-alpha-1 ,3-glucanase; and a chitinase preferably an endo-chitinase. In a more preferred embodiment, the mixture comprises at least seven glucanase and at least one chitinase. In one such embodiment the mixture comprises a seven glucanases selected from the list of endo-beta-1 ,3(4)-glucanase, endo-beta-1 ,3-glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6-glucanase and endo-alpha-1 ,3-glucanase; and a chitinase preferably an endo-chitinase.

In a more preferred embodiment, the mixture comprises at least one glucanase comprising an amino acid sequence selected from the list of SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto and/or at least one chitinase comprising an amino acid sequence selected from the list of SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto. In an even more preferred embodiment, the mixture comprises at least one glucanase comprising an amino acid sequence selected from the list of SEQ ID NOs: 100 to 1 15 and 128 to 130 and/or at least one chitinase comprising an amino acid sequence selected from the list of SEQ ID NOs: 1 16 to 127. In an even more preferred embodiment, the mixture comprises at least one glucanase comprising an amino acid sequence selected from the list of SEQ ID NOs: 100 to 105, 1 12 and 128 and/or at least one chitinase comprising an amino acid sequence selected from the list of SEQ ID NOs: 1 16 to 1 19 and 125.

In some embodiments, the mixture does not comprise a pectinase and/or a arabinase and/or a protease and/or a protease inhibitor.

In a more preferred embodiment the cell wall degrading enzyme is a mixture of cell wall degrading enzymes and where the cell wall degrading enzymes are chosen from at least one glucanase and at least one chitinase as described herein above. More specifically the mixture comprises at least one glucanase comprising an amino acid sequence selected from the list of SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and at least one chitinase comprising an amino acid sequence selected from the list of SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto. In an even more preferred embodiment the mixture comprises at least one glucanase comprising an amino acid sequence selected from the list of SEQ ID NOs: 100 to 105, 1 12 and 128 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and at least one chitinase comprising an amino acid sequence selected from the list of SEQ ID NOs: 1 16 to 1 19 and 125 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto. In an even more preferred embodiment the mixture comprises at least one glucanase comprising an amino acid sequence selected from the list of SEQ ID NOs: 100 to 105, 1 12 and 128; and at least one chitinase comprising an amino acid sequence selected from the list of SEQ ID NOs: 1 16 to 1 19 and 125.

In a preferred embodiment the cell wall degrading enzyme is a mixture of two or more cell wall degrading enzymes. In one embodiment the mixture comprises two glucanases and where the two glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the two glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the two glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128. In another embodiment the mixture comprises three glucanases and where the three glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the three glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the three glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128. In another embodiment the mixture comprises four glucanases and where the four glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the four glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15; even more preferably the four glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128. In another embodiment the mixture comprises five glucanases and where the five glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the five glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the five glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128. In another embodiment the mixture comprises six glucanases and where the six glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the six glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the six glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105,1 12 and 128. In another embodiment the mixture comprises seven glucanases and where the seven glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the seven glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the seven glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128.

In a more preferred embodiment, the mixture comprises at least one glucanase and at least one chitinase. In one such embodiment the mixture comprises one glucanase selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the one glucanase is selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the one glucanase is selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and one chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125. In another such embodiment the mixture comprises two glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the two glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the two glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and one chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125. In another such embodiment the mixture comprises three glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the three glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the three glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and one chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125. In another such embodiment the mixture comprises four glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the four glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the four glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and one chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125. In another such embodiment the mixture comprises five glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the five glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the five glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and one chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125. In another such embodiment the mixture comprises six glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the six glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the six glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and one chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125. In another such embodiment the mixture comprises seven glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the seven glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the seven glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and one chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125.

In another such embodiment the mixture comprises one glucanase selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the one glucanase is selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the one glucanase is selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and two chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125. In another such embodiment the mixture comprises two glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the two glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the two glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and two chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125. In another such embodiment the mixture comprises three glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the three glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the three glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and two chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 116 to 119 and 125. In another such embodiment the mixture comprises four glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 115 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the four glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 115 and 128 to 130; even more preferably the four glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 112 and 128; and two chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 116 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 116 to 127; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 116 to 119 and 125. In another such embodiment the mixture comprises five glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 115 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the five glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 115 and 128 to 130; even more preferably the five glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 112 and 128; and two chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 116 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 116 to 127; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 116 to 119 and 125. In another such embodiment the mixture comprises six glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 115 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the six glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 115 and 128 to 130; even more preferably the six glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 112 and 128; and two chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 116 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 116 to 127; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125. In another such embodiment the mixture comprises seven glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the seven glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the seven glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and two chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125.

The current invention relates to compositions comprising (i) at least one antifungal compound with cell membrane interacting properties, and (ii) at least one cell wall degrading enzyme. In a particular embodiment, the current invention relates to compositions comprising (i) at least one antifungal compound with cell membrane interacting properties, and (ii) at least one recombinantly produced cell wall degrading enzyme. In a preferred embodiment, the composition according to the invention comprises a mixture of cell wall degrading enzyme and said mixture consist of two cell wall degrading enzymes, preferably recombinantly produced cell wall degrading enzymes, and wherein the first cell wall degrading enzyme is the exo-beta-1 ,3-glucanase comprising an amino acid sequence according to SEQ ID NO: 105, and the second cell wall degrading enzyme is the endochitinase comprising an amino acid according to SEQ ID NO: 1 16. In another preferred embodiment, the composition according to the invention comprises a mixture of cell wall degrading enzyme and said mixture consist of two cell wall degrading enzymes, and wherein the first cell wall degrading enzyme is the exo-beta-1 ,3-glucanase comprising an amino acid sequence according to SEQ ID NO: 105, and the second cell wall degrading enzyme is the endo-chitinase comprising an amino acid according to SEQ ID NO: 1 19. In another preferred embodiment, the composition according to the invention comprises a mixture of cell wall degrading enzyme and said mixture consist of two cell wall degrading enzymes, and wherein the first cell wall degrading enzyme is the exo-beta-1 ,3-glucanase comprising an amino acid sequence according to SEQ ID NO: 128, and the second cell wall degrading enzyme is the endo-chitinase comprising an amino acid according to SEQ ID NO: 1 16. In another preferred embodiment, the composition according to the invention comprises a mixture of cell wall degrading enzyme and said mixture consist of two cell wall degrading enzymes, and wherein the first cell wall degrading enzyme is the exo-beta-1 ,3-glucanase comprising an amino acid sequence according to SEQ ID NO: 128, and the second cell wall degrading enzyme is the endo- chitinase comprising an amino acid according to SEQ ID NO: 1 19. In another preferred embodiment, the composition according to the invention comprises a mixture of cell wall degrading enzyme and said mixture consist of two cell wall degrading enzymes, and wherein the first cell wall degrading enzyme is the endo-chitinase comprising an amino acid sequence according to SEQ ID NO: 1 16, and the second cell wall degrading enzyme is the endo-beta-1 ,3(4)-glucanase comprising an amino acid according to SEQ ID NO: 100. In another preferred embodiment, the composition according to the invention comprises a mixture of cell wall degrading enzyme and said mixture consist of three cell wall degrading enzymes, and wherein the first cell wall degrading enzyme is the exo-beta-1 ,3- glucanase comprising an amino acid sequence according to SEQ ID NO: 105, the second cell wall degrading enzyme is the endo-chitinase comprising an amino acid according to SEQ ID NO: 1 16, and the third cell wall degrading enzyme is the endo-beta-1 ,3-glucanase comprising an amino acid sequence according to SEQ ID NO: 101 . In another preferred embodiment, the composition according to the invention comprises a mixture of cell wall degrading enzyme and said mixture consist of three cell wall degrading enzymes, and wherein the first cell wall degrading enzyme is the exo-beta-1 ,3-glucanase comprising an amino acid sequence according to SEQ ID NO: 128, the second cell wall degrading enzyme is the endo-chitinase comprising an amino acid according to SEQ ID NO: 1 16, and the third cell wall degrading enzyme is the endo-beta-1 ,3-glucanase comprising an amino acid sequence according to SEQ ID NO: 101 . In another preferred embodiment, the composition according to the invention comprises a mixture of cell wall degrading enzyme and said mixture consist of three cell wall degrading enzymes, and wherein the first cell wall degrading enzyme is the exo-beta-1 ,3-glucanase comprising an amino acid sequence according to SEQ ID NO: 105, the second cell wall degrading enzyme is the endo-chitinase comprising an amino acid according to SEQ ID NO: 1 16, and the third cell wall degrading enzyme is the endo-beta-1 ,3(4)-glucanase comprising an amino acid sequence according to SEQ ID NO: 100. In another preferred embodiment, the composition according to the invention comprises a mixture of cell wall degrading enzyme and said mixture consist of three cell wall degrading enzymes, and wherein the first cell wall degrading enzyme is the exo-beta-1 ,3-glucanase comprising an amino acid sequence according to SEQ ID NO: 128, the second cell wall degrading enzyme is the endo-chitinase comprising an amino acid according to SEQ ID NO: 1 16, and the third cell wall degrading enzyme is the endo-beta-1 ,3(4)- glucanase comprising an amino acid sequence according to SEQ ID NO: 100.

When one, two, three or more cell wall degrading enzymes are selected from a list or group of cell wall degrading enzymes or cell wall degrading enzymes comprising an amino acid sequence according to the amino acid sequence as disclosed herein, it is said that a first, second, third or higher number of cell wall degrading enzymes are selected from a group or list. Such a selection may occur without or with repetition, that is to say, where a selection is made from a list of enzymes with similar enzymatic functions, an enzymatic function may be chosen more than once and is thus chosen with repetition. For instance, a mixture according to the invention may comprise 2 or more different cell wall degrading enzymes having endo-beta-1 ,3-glucanase activity. When a selection occurs from a list of specific amino acid sequences, such a selection may be without repetition.

In a preferred embodiment, the mixture of enzymes disclosed herein comprise recombinantly produced and purified cell wall degrading enzymes. In an even more preferred embodiment the mixture comprises 1 , 2, 3, 4 or 5 or more recombinantly produced and purified cell wall degrading enzymes. Most preferably, the mixture described herein does not comprise more than 28, preferably no more than 9, preferably no more than 6, preferably no more than 3 recombinantly produced and optionally purified cell wall degrading enzymes and where said cell wall degrading enzymes are selected from one or more of a glucanase and/or one or more of a chitinase as described herein above. In a particular embodiment, the compositions, combinations and mixtures of the invention comprise from 1 up to 9, such as from 1 up to 5, cell wall degrading enzymes. In a further embodiment, the compositions, combinations and mixtures of the invention comprise from 1 up to 3, such as 1 or 2, cell wall degrading enzymes.

Compositions, agrochemical compositions and combinations

In a preferred embodiment of the current invention, the above described cell wall degrading enzymes or mixtures thereof are further combined with at least one antifungal compound, preferably at least one antifungal compound with cell membrane interacting properties as defined herein, more preferably said antifungal compound with cell membrane interacting properties is a polypeptide, most preferably the antifungal compound with cell membrane interacting properties is an antibody or VHH antibody as described herein above. In a preferred embodiment, the at least one cell wall degrading enzyme and at least one VHH antibody are present in a composition, optionally an agrochemical composition. In another preferred embodiment, the at least one cell wall degrading enzyme and at least one VHH antibody are present in a combination. In a more preferred embodiment the composition or combination comprises a mixture of cell wall degrading enzymes as described herein above; and at least one antifungal compound with membrane interacting properties. The current invention also relates to combinations of cell wall degrading enzymes and antifungal compounds with cell membrane interacting properties as described herein.

Therefore, in a preferred embodiment, the composition or agrochemical compositions according to the invention comprise a cell wall degrading enzyme as described herein, preferably the cell wall degrading enzyme is a mixture of enzymes as described herein, and the at least one antifungal compound with cell membrane interacting properties is a polypeptide as described herein, where the polypeptide preferably is a VHH antibody, even more preferably a VHH antibody comprising or consisting of the amino acid sequence set out in any one of SEQ ID NOs: 1 , 2, 6, 10 ,14 to 99, 131 or 132, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto. In another embodiment, the antifungal compound with cell membrane interacting properties is an azole antifungal or a nitroaniline antifungal. In yet another embodiment, the antifungal compound with cell membrane interacting properties is an anti-microbial peptide or AMP.

In some embodiments, the antifungal compound with cell membrane interacting properties and cell wall degrading enzyme disclosed herein targets the same fungal spore. In some embodiments, the antifungal compound with cell membrane interacting properties and cell wall degrading enzyme disclosed herein targets the same fungal cell. In some embodiments, the antifungal compound with cell membrane interacting properties and cell wall degrading enzyme disclosed herein targets the same fungal mycelium. In a preferred embodiment, the antifungal compound with cell membrane interacting properties and the cell wall degrading enzyme target the same fungal species. Therefore, in a particular embodiment, the antifungal compound with cell membrane interacting properties is able to interact with a plant pathogenic fungus as described herein and the cell wall degrading enzyme is able to degrade the cell wall of the same plant pathogenic fungus. In a further embodiment, the antifungal compound with cell membrane interacting properties is a polypeptide, preferably a VHH, that is able to bind to a cell membrane of a plant pathogenic fungus as described herein; and the cell wall degrading enzyme is able to degrade the cell wall of the same plant pathogenic fungus. In an even further embodiment, the antifungal compound with cell membrane interacting properties is a polypeptide, preferably a VHH, that is able to bind to a cell membrane of Botrytis cinerea, and the cell wall degrading enzyme is able to degrade the cell wall of Botrytis cinerea.

The invention also provides agrochemical compositions comprising at least one antifungal compound with cell membrane interacting properties as described herein and at least one cell wall degrading enzyme as described herein.

The invention also provides combinations comprising at least one antifungal compound with cell membrane interacting properties as described herein and at least one cell wall degrading enzyme or a mixture of cell wall degrading enzymes as described herein.

Accordingly, the composition or combination as disclosed herein can be used to modulate, such as to decrease or inhibit, the biological function of a plant pest. The modulation of plant pest function may be enhanced when the composition or combination is used compared to individual constituents of the composition. The modulation may affect the natural biological activities (such as, but not limited to, growth) of the pest and/or one or more biological pathways in which the structural target of that pest is involved.

Furthermore, the composition or combination comprising at least one antifungal compound with cell membrane interacting properties and at least one cell wall degrading enzyme as disclosed herein may have several additional advantages over the traditional immunoglobulin and nonimmunoglobulin binding agents known in the art. Indeed, in certain embodiments, the antifungal compound with cell membrane interacting properties as disclosed herein have an improved activity when combined with at least one cell wall degrading enzyme. Without being bound by theory, it is envisioned that the cell wall degrading enzyme provide an even more facilitated access of the antifungal compound with cell membrane interacting properties to targets at the cell membrane of the pest or targets inside the pest, where the pest may be a fungal pest, such as a plant fungal pathogen as described herein. Additionally or alternatively, the cell membrane in the absence of a fully intact cell wall may be destabilized and more prone to rupture or destabilization by the effect of an antifungal compound with cell membrane interacting properties. In a preferred embodiment, the antifungal compound with cell membrane interacting properties is a polypeptide, more preferably an antibody or VHH antibody or a fragment thereof as described herein.

The antifungal compound with cell membrane interacting properties disclosed herein generally binds or interacts to a plasma membrane components of a target or a cellular process involved in building or maintaining the integrity of the plasma membrane. Preferably, the target is a fungus, fungal pest, pathogen fungus and/or plant pathogenic fungus as described herein. The compositions disclosed herein comprise an antifungal compound with cell membrane interacting properties as disclosed herein and a cell wall degrading enzyme (as disclosed herein).

The cell wall degrading enzyme may disrupt the cell wall of the target. More specifically, the cell wall degrading enzyme may disrupt the cell wall of a fungal target. Disrupting the cell wall may increase permeability of substances or compositions through the cell wall. In preferred embodiments, the cell wall degrading enzyme disrupts the cell wall to allow the antifungal compound with cell membrane interacting properties to permeate through the cell wall. In some embodiments, the cell wall degrading enzyme may allow increased quantities of antifungal compound with cell membrane interacting properties to permeate through the cell wall. In some embodiments, the cell wall degrading enzyme may allow the antifungal compound with cell membrane interacting properties to permeate the cell wall faster. In some embodiments, the cell wall degrading enzyme may allow the antifungal compound with cell membrane interacting properties to have an increased antifungal effect due to the destabilisation of the cell membrane in the absence of a (fully) intact cell wall. In some embodiments, the composition as disclosed herein has greater efficacy as a plant pest agent than either the cell wall degrading enzyme or antifungal compound with cell membrane interacting properties alone. In some embodiments, the effect of the composition is synergistic compared to the cell wall degrading enzyme and antifungal compound with cell membrane interacting properties alone at comparable concentrations. In some embodiments the effect of the composition is additive compared to the cell wall degrading enzyme and antifungal compound with cell membrane interacting properties alone at comparable concentrations. In some embodiments the IC50 of the antifungal compound with cell membrane interacting properties in the composition as disclosed herein is reduced compared to the antifungal compound with cell membrane interacting properties alone, or compared to when the polypeptide is in a composition that does not contain a cell wall degrading enzyme. The reduction in the IC50 may be at least 2-fold, at least 5-fold, at least 10-fold, at least 100-fold, or greater, compared to the antifungal compound with cell membrane interacting properties used alone, or compared to a composition comprising the antifungal compound with cell membrane interacting properties that does not contain a cell wall degrading enzyme.

In some embodiments the IC50 of the cell wall degrading enzyme in the composition as disclosed herein is reduced compared to the cell wall degrading enzyme alone, or compared to when the cell wall degrading enzyme is in a composition that does not contain an antifungal compound with cell membrane interacting properties. The reduction in the IC50 may be at least 2-fold, at least 5-fold, at least 10-fold, at least 100-fold, or greater, compared to the cell wall degrading enzyme used alone, or compared to a composition comprising the cell wall degrading enzyme that does not contain an antifungal compound with cell membrane interacting properties.

In some embodiments the IC50 of both the cell wall degrading enzyme and the antifungal compound with cell membrane interacting properties in the composition as disclosed herein is reduced compared to the cell wall degrading enzyme or antifungal compound with cell membrane interacting properties alone, or compared to when the cell wall degrading enzyme or antifungal compound with cell membrane interacting properties is in a composition that does not contain an antifungal compound with cell membrane interacting properties or cell wall degrading enzyme respectively. In other words, the IC50 of the composition may be reduced compared to the IC50 of a composition comprising only one of the cell wall degrading enzyme and the antifungal compound with cell membrane interacting properties. The reduction in the IC50 may be at least 2-fold, at least 5-fold, at least 10-fold, at least 100-fold, or greater, compared to the cell wall degrading enzyme or antifungal compound with cell membrane interacting properties used alone, or compared to a composition comprising the antifungal compound with cell membrane interacting properties or the cell wall degrading enzyme that does not respectively contain a cell wall degrading enzyme or antifungal compound with cell membrane interacting properties.

In a preferred embodiment of the invention the composition comprises a cell wall degrading enzyme and at least one antifungal compound with cell membrane interacting properties, preferably the cell wall degrading enzyme is a mixture of two or more cell wall degrading enzymes as described herein. Therefore, in a preferred embodiment, the composition may comprise at least two cell wall degrading enzyme selected from a glucanase and/or a chitinase and where the glucanase and chitinase are as described above, and at least one antifungal compound with cell membrane interacting properties as described above. The composition may comprise at least 2, at least 3, at least 4 or at least 5 glucanase and/or chitinases; and at least one antifungal compound with cell membrane interacting properties as described above. Preferably the composition does not comprise more than 28, preferably no more than 9, preferably no more than 6, preferably no more than 3 glucanase and/or chitinases. The composition may comprise at least one glucanase, such as a glucanase selected from the group consisting of exo-beta-1 ,3-glucanase, endo-beta-1 ,3-glucanase, exo-beta-1 ,6-glucanase, endo-beta-1 ,6-glucanase, endo-beta-1 ,3(4)-glucanase, endo-alpha-1 ,3- glucanase, and exo-alpha-1 ,3-glucanase or any combination thereof; and/or at least one chitinase; and at least one antifungal compound with cell membrane interacting properties as described above. In a more preferred embodiment, the composition may comprise at least one of a cell wall degrading enzyme selected from the list consisting of an endo-1 ,3(4)-beta-glucanase, endo-beta-1 ,3- glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6-glucanase, and endo-alpha-1 ,3-glucanase; and/or at least one chitinase; and at least one antifungal compound with cell membrane interacting properties as described above. In some embodiments the composition does not comprise a chitinase but comprises 2 or more of a glucanase as described herein; and at least one antifungal compound with cell membrane interacting properties as described above. In some embodiments the composition may comprise 2 or more of a different enzyme comprising the same enzymatic activity; and at least one antifungal compound with cell membrane interacting properties as described above. That is to say, two different enzymes may have the same enzymatic activity but may differ in for example their amino acid sequence, substrate specificity, optimal temperature range, or optimal pH range for activity. In a preferred embodiment, the at least one antifungal compound with cell membrane interacting properties as present in the composition of the invention is a polypeptide, more preferably a VHH antibody or a fragment thereof. In a more preferred embodiment the at least one antifungal compound with cell membrane interacting properties as present in the composition of the invention is a VHH antibody comprising or consisting of the amino acid sequence set out in any one of SEQ ID NOs: 1 , 2, 6, 10, 14 to 99, 131 or 132, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto. In a more preferred embodiment the composition as described herein preferably comprises a VHH antibody comprising or consisting of the amino acid sequence according to SEQ ID NO: 131 . In an even more preferred embodiment the composition as described herein preferably comprises a VHH antibody comprising or consisting of the amino acid sequence according to SEQ ID NO: 132. In a more preferred embodiment the composition as described herein most preferably comprises a VHH antibody comprising or consisting of the amino acid sequence according to SEQ ID NO: 2.

In a most preferred embodiment, the composition according to the invention is an agrochemical composition. In some preferred embodiments, the composition or agrochemical composition does not comprise a pectinase and/or a arabinase and/or a protease and/or a protease inhibitor.

In a more preferred embodiment the composition comprises a cell wall degrading enzyme, preferably a mixture of cell wall degrading enzymes and where the cell wall degrading enzymes are chosen from at least one glucanase and at least one chitinase as described herein above; and at least one antifungal compound with cell membrane interacting properties. Therefore the composition according to the invention comprises at least one glucanase comprising an amino acid sequence selected from the list of SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and at least one chitinase comprising an amino acid sequence selected from the list of SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and at least one antifungal compound with cell membrane interacting properties. In an even more preferred embodiment the composition comprises at least one glucanase comprising an amino acid sequence selected from the list of SEQ ID NOs: 100 to 105, 1 12 and 128 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and at least one chitinase comprising an amino acid sequence selected from the list of SEQ ID NOs: 1 16 to 1 19 and 125 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; and at least one antifungal compound with cell membrane interacting properties. In an even more preferred embodiment the composition comprises at least one glucanase comprising an amino acid sequence selected from the list of SEQ ID NOs: 100 to 105, 1 12 and 128; and at least one chitinase comprising an amino acid sequence selected from the list of SEQ ID NOs: 1 16 to 1 19 and 125; and at least one antifungal compound with cell membrane interacting properties.

In one such an embodiment the composition comprises two glucanases and where the two glucanases are selected from the list of endo-beta-1 ,3(4)-glucanase, endo-beta-1 ,3-glucanase, exo- beta-1 ,3-glucanase, endo-beta-1 ,6-glucanase and endo-alpha-1 ,3-glucanase; and at least one antifungal compound with cell membrane interacting properties. In another such an embodiment the composition comprises three glucanases and where the three glucanases are selected from the list of endo-beta-1 ,3(4)-glucanase, endo-beta-1 ,3-glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6- glucanase and endo-alpha-1 ,3-glucanase; and at least one antifungal compound with cell membrane interacting properties. In another such an embodiment the composition comprises four glucanases and where the four glucanases are selected from the list of endo-beta-1 ,3(4)-glucanase, endo-beta- 1 ,3-glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6-glucanase and endo-alpha-1 ,3-glucanase; and at least one antifungal compound with cell membrane interacting properties. In another such an embodiment the composition comprises five glucanases and where the five glucanases are selected from the list of endo-beta-1 ,3(4)-glucanase, endo-beta-1 ,3-glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6-glucanase and endo-alpha-1 ,3-glucanase; and at least one antifungal compound with cell membrane interacting properties. In another such an embodiment the composition comprises six glucanases and where the six glucanases are selected from the list of endo-beta-1 ,3(4)- glucanase, endo-beta-1 ,3-glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6-glucanase and endo- alpha-1 ,3-glucanase; and at least one antifungal compound with cell membrane interacting properties. In another such an embodiment the composition comprises seven glucanases and where the seven glucanases are selected from the list of endo-beta-1 ,3(4)-glucanase, endo-beta-1 ,3- glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6-glucanase and endo-alpha-1 ,3-glucanase; and at least one antifungal compound with cell membrane interacting properties.

In a more preferred embodiment, the composition comprises at least one glucanase and at least one chitinase. In one such embodiment the composition comprises one glucanase selected from the list of endo-beta-1 ,3(4)-glucanase, endo-beta-1 ,3-glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6-glucanase and endo-alpha-1 ,3-glucanase; and a chitinase preferably an endochitinase; and at least one antifungal compound with cell membrane interacting properties. In a more preferred embodiment, the composition comprises at least two glucanase and at least one chitinase. In one such embodiment the composition comprises two glucanases selected from the list of endo- beta-1 ,3(4)-glucanase, endo-beta-1 ,3-glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6- glucanase and endo-alpha-1 ,3-glucanase; and a chitinase preferably an endo-chitinase; and at least one antifungal compound with cell membrane interacting properties. In a more preferred embodiment, the composition comprises at least three glucanase and at least one chitinase. In one such embodiment the composition comprises three glucanases selected from the list of endo-beta- 1 ,3(4)-glucanase, endo-beta-1 ,3-glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6-glucanase and endo-alpha-1 ,3-glucanase; and a chitinase preferably an endo-chitinase; and at least one antifungal compound with cell membrane interacting properties. In a more preferred embodiment, the composition comprises at least four glucanase and at least one chitinase. In one such embodiment the composition comprises four glucanases selected from the list of endo-beta-1 ,3(4)-glucanase, endo-beta-1 ,3-glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6-glucanase and endo-alpha-1 ,3- glucanase; and a chitinase preferably an endo-chitinase; and at least one antifungal compound with cell membrane interacting properties. In a more preferred embodiment, the composition comprises at least five glucanase and at least one chitinase. In one such embodiment the composition comprises five glucanases selected from the list of endo-beta-1 ,3(4)-glucanase, endo-beta-1 ,3- glucanase, exo-beta-1 ,3-glucanase, endo-beta-1 ,6-glucanase and endo-alpha-1 ,3-glucanase; and a chitinase preferably an endo-chitinase; and at least one antifungal compound with cell membrane interacting properties. In a more preferred embodiment, the composition comprises at least six glucanase and at least one chitinase. In one such embodiment the composition comprises six glucanases selected from the list of endo-beta-1 ,3(4)-glucanase, endo-beta-1 ,3-glucanase, exo- beta-1 ,3-glucanase, endo-beta-1 ,6-glucanase and endo-alpha-1 ,3-glucanase; and a chitinase preferably an endo-chitinase; and at least one antifungal compound with cell membrane interacting properties. In a more preferred embodiment, the composition comprises at least seven glucanase and at least one chitinase. In one such embodiment the composition comprises a seven glucanases selected from the list of endo-beta-1 ,3(4)-glucanase, endo-beta-1 ,3-glucanase, exo-beta-1 ,3- glucanase, endo-beta-1 ,6-glucanase and endo-alpha-1 ,3-glucanase; and a chitinase preferably an endo-chitinase; and at least one antifungal compound with cell membrane interacting properties.

In one embodiment the composition comprises two glucanases and at least one antifungal compound with cell membrane interacting properties; and where the two glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the two glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the two glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128. In another embodiment the composition comprises three glucanases and at least one antifungal compound with cell membrane interacting properties; and where the three glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the three glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the three glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105,1 12 and 128. In another embodiment the composition comprises four glucanases and at least one antifungal compound with cell membrane interacting properties; and where the four glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the four glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the four glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128. In another embodiment the composition comprises five glucanases and at least one antifungal compound with cell membrane interacting properties; and where the five glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the five glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the five glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128. In another embodiment the composition comprises six glucanases and at least one antifungal compound with cell membrane interacting properties; and where the six glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the six glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the six glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128. In another embodiment the composition comprises seven glucanases and at least one antifungal compound with cell membrane interacting properties; and where the seven glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the seven glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the seven glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128.

In a more preferred embodiment, the composition comprises at least one glucanase, at least one chitinase and at least one antifungal compound with membrane interacting properties. In one such embodiment the composition comprises one glucanase selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the one glucanase is selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the one glucanase is selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and one chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125; and at least one antifungal compound with membrane interacting properties. In another such embodiment the composition comprises two glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the two glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the two glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and one chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125; and at least one antifungal compound with membrane interacting properties. In another such embodiment the composition comprises three glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the three glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the three glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and one chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125; and at least one antifungal compound with membrane interacting properties. In another such embodiment the composition comprises four glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the four glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the four glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and one chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125; and at least one antifungal compound with membrane interacting properties. In another such embodiment the composition comprises five glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the five glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the five glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and one chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125; and at least one antifungal compound with membrane interacting properties. In another such embodiment the composition comprises six glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the six glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the six glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and one chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125; and at least one antifungal compound with membrane interacting properties. In another such embodiment the composition comprises seven glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the seven glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the seven glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and one chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the one chitinase is selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125; and at least one antifungal compound with membrane interacting properties.

In another such embodiment the composition comprises one glucanase selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the one glucanase is selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the one glucanase is selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and two chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125; and at least one antifungal compound with membrane interacting properties. In another such embodiment the composition comprises two glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the two glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the two glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and two chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125; and at least one antifungal compound with membrane interacting properties. In another such embodiment the composition comprises three glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the three glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the three glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and two chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125; and at least one antifungal compound with membrane interacting properties. In another such embodiment the composition comprises four glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the four glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the four glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and two chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125; and at least one antifungal compound with membrane interacting properties. In another such embodiment the composition comprises five glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the five glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the five glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and two chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125; and at least one antifungal compound with membrane interacting properties. In another such embodiment the composition comprises six glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the six glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the six glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105, 1 12 and 128; and two chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125; and at least one antifungal compound with membrane interacting properties. In another such embodiment the mixture comprises seven glucanases selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the seven glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 1 15 and 128 to 130; even more preferably the seven glucanases are selected from the list of glucanases comprising an amino acid sequence according to SEQ ID NOs: 100 to 105,1 12 and 128; and two chitinase selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 127; more preferably the two chitinases are selected from the list of chitinases comprising an amino acid sequence according to SEQ ID NOs: 1 16 to 1 19 and 125; and at least one antifungal compound with membrane interacting properties.

In a preferred embodiment, the cell wall degrading enzymes comprised in the compositions described above are recombinantly produced cell wall degrading enzymes. In a more preferred embodiment, the cell wall degrading enzymes comprised in the compositions described above are recombinantly produced and purified cell wall degrading enzymes. In an even more preferred embodiment the mixture comprises 1 , 2, 3, 4 or 5 or more recombinantly produced and optionally purified cell wall degrading enzymes. Most preferably, the mixture described herein does not comprise more than 28, preferably no more than 9, preferably no more than 6, preferably no more than 3 recombinantly produced and optionally purified cell wall degrading enzymes and where said cell wall degrading enzymes are selected from one or more of a glucanase and/or one or more of a chitinase as described herein above.

In a more preferred embodiment, the antifungal compound with cell membrane interacting properties is a VHH antibody as disclosed in US provisional applications 63/614,192. Therefore, in a more preferred embodiment the composition as described herein preferably comprises a VHH antibody comprising or consisting of the amino acid sequences as described in US provisional applications 63/614,192; particularly a VHH antibody according to the claims of US provisional applications 63/614,192; more particularly a VHH antibody comprising SEQ ID NO: 2 of US provisional applications 63/614,192, herein referred to as SEQ ID NO: 131 .

In a more preferred embodiment, the antifungal compound with cell membrane interacting properties is a VHH antibody as disclosed in US provisional applications 63/614,183. Therefore, in an even more preferred embodiment the composition as described herein preferably comprises a VHH antibody comprising or consisting of the amino acid sequences as described in US provisional applications 63/614,183; particularly a VHH antibody according to the claims of US provisional applications 63/614,183; more particularly a VHH antibody comprising SEQ ID NO: 2 of US provisional applications 63/614,183, herein referred to as SEQ ID NO: 132. In a most preferred embodiment, the antifungal compound with cell membrane interacting properties is a VHH antibody as disclosed in the current application. Therefore, in a most preferred embodiment the composition as described herein most preferably comprises a VHH antibody comprising or consisting of the amino acid sequence according to SEQ ID NO:2.

In a most preferred embodiment, the composition according to the invention is an agrochemical composition.

In some preferred embodiments, the composition or agrochemical composition does not comprise a pectinase and/or a arabinase and/or a protease and/or a protease inhibitor. The disclosure also covers a composition comprising at least one cell wall degrading enzyme or mixtures that do not contain an antifungal compound with membrane interacting properties. In some embodiments, the composition comprises only a cell wall degrading enzyme or a mixture thereof as the active ingredient.

Testing cell wall degrading enzymes

A cell wall degrading enzyme, either an individual cell wall degradubg enzyme or a mixture of two or more cell wall degrading enzymes, may be identified by subjecting a fungal species, such as a plant pathogenic species, to increasing amounts of the cell wall degrading enzyme and observing said fungal species using standard brightfield or phase contrast microscopy with a magnification factor of 200x or more. A cell wall degrading enzyme may lead at a sufficiently high concentration to the formation of protoplasts, indicating that the cell wall has been removed, protoplasts may be maintained in a correct buffer solution with a isotonic osmotic pressure (as opposed to a hypotonic buffer solution leading to collapse of the protoplasts or a hypertonic buffer solution leading to the rupture of protoplasts). A specific example of a buffer solution that may be used is the FF1 buffer consisting of 29.58 g MgSO4.7H2O, 1 .37 g NaH2PO4.2H2O, 100 ml MQ water (pH 5.8). This buffer may for example be used to assess protoplasts formation of Botrytis cinerea, but the skilled person will know that many other buffers may be used as long as they are isotonic relative to the interior of the protoplasts and allow for the cell wall degrading enzyme to be active. This assay is exemplified in Example 3 for Botrytis cinerea. The concentration required to obtain protoplasts may not be the concentration that is required in the compositions of the current invention. That is to say, for assessing the ability of a cell wall degrading enzyme to disrupt the fungal cell wall, a high concentration may be required to observe protoplast formation, whereas in practice the concentration to observe an effect such as an anti-fungal effect may be lower. Without wanting to be bound by theory, it may be proposed that at lower concentrations of the cell wall degrading enzyme, smaller disruptions of the cell wall may already be sufficient to allow the polypeptide to have an improved chance of acting on the cell membrane (for example a VHH binding to a target on the cell membrane) to show an improved efficacy.

In some embodiments, the cell wall degrading enzyme is active across temperatures of from about 0 to about 60^eC. In some embodiments, the cell wall degrading enzyme is active at temperatures above 0^eC. In some embodiments, the cell wall degrading enzyme is active at temperatures from about 0 to about 10 ^eC. In some embodiments, the cell wall degrading enzyme is active at temperatures from about 0 to about 20^eC. In some embodiments, the cell wall degrading enzyme is active at temperatures from about 0 to about 30^eC. In some embodiments, the cell wall degrading enzyme is active at temperatures from about 0 to about 40 ^eC. In some embodiments, the cell wall degrading enzyme is active at temperatures from about 0 to about 50 ^eC. In some embodiments, the cell wall degrading enzyme is active during cold storage (from about 0 to about 10 ^eC) and at warmer temperatures up to 50 ^eC. In some embodiments the cell wall degrading enzyme is resistant to denaturation. In some embodiments, the cell wall degrading enzyme is not temperature sensitive. In some embodiments the activity of the cell wall degrading enzyme is not affected by temperature. In some embodiments the activity of the cell wall degrading enzyme peaks at around 10^eC, around 20^eC, around 30^eC, around 40^eC, or around 50^eC.

In some embodiments, the cell wall degrading enzyme is active during cold storage (below about 10 ^eC), at room temperature (from about 10 to about 25 ^eC), and under sunny growing conditions, such as about 25 to about 35 ^eC.

In some embodiments, the cell wall degrading enzyme is an enzyme. In some embodiments, the cell wall degrading enzyme is a cell wall degrading enzyme. In some embodiments the cell wall degrading enzyme is active across temperatures of from about 0 to about 60^eC. In some embodiments the cell wall degrading enzyme is active across temperatures of from about 0 to about 50^eC. In some embodiments, the cell wall degrading enzyme is active at temperatures above 0^eC. In some embodiments, the cell wall degrading enzyme is active at temperatures from about 0 to about 10 ^eC. In some embodiments, the cell wall degrading enzyme is active at temperatures from about 0 to about 20^eC. In some embodiments, the cell wall degrading enzyme is active at temperatures from about 0 to about 30^eC. In some embodiments, the cell wall degrading enzyme is active at temperatures from about 0 to about 40 ^eC. In some embodiments, the cell wall degrading enzyme is active at temperatures from about 0 to about 50 ^eC. In some embodiments, the cell wall degrading enzyme is active at temperatures above about 5^eC. In some embodiments, the cell wall degrading enzyme is active at temperatures from about 5 to about 10 ^eC. In some embodiments, the cell wall degrading enzyme is active at temperatures from about 5 to about 20^eC. In some embodiments, the cell wall degrading enzyme is active at temperatures from about 5 to about 30^eC. In some embodiments, the cell wall degrading enzyme is active at temperatures from about 5 to about 40 ^eC. In some embodiments, the cell wall degrading enzyme is active at temperatures from about 5 to about 50 ^eC.

In some embodiments the cell wall degrading enzyme is resistant to denaturation at temperatures of from about 0 to about 60^eC. In some embodiments the cell wall degrading enzyme is resistant to denaturation at temperatures of from about 0 to about 50^eC. In some embodiments, the cell wall degrading enzyme is resistant to denaturation at temperatures above 0^eC. In some embodiments, the cell wall degrading enzyme is resistant to denaturation at temperatures from about 0 to about 10 ^eC. In some embodiments, the cell wall degrading enzyme is resistant to denaturation at temperatures from about 0 to about 20^eC. In some embodiments, the cell wall degrading enzyme is resistant to denaturation at temperatures from about 0 to about 30^eC. In some embodiments, the cell wall degrading enzyme is resistant to denaturation at temperatures from about 0 to about 40 ^eC. In some embodiments, the cell wall degrading enzyme is resistant to denaturation at temperatures from about 0 to about 50 ^eC. In some embodiments, the cell wall degrading enzyme is resistant to denaturation at temperatures above about 5^eC. In some embodiments, the cell wall degrading enzyme is resistant to denaturation at temperatures from about 5 to about 10 ^eC. In some embodiments, the cell wall degrading enzyme is resistant to denaturation at temperatures from about 5 to about 20^eC. In some embodiments, the cell wall degrading enzyme is resistant to denaturation at temperatures from about 5 to about 30^eC. In some embodiments, the cell wall degrading enzyme is resistant to denaturation at temperatures from about 5 to about 40 ^eC. In some embodiments, the cell wall degrading enzyme is resistant to denaturation at temperatures from about 5 to about 50 ^eC.

In some embodiments, the cell wall degrading enzyme is active during cold storage (from about 0 to about 10 ^eC) and at warmer temperatures up to 50 ^eC. In some embodiments the cell wall degrading enzyme is resistant to denaturation. In some embodiments, the cell wall degrading enzyme is not temperature sensitive. In some embodiments the activity of the cell wall degrading enzyme is not affected by temperature. In some embodiments the activity of the cell wall degrading enzyme peaks at around 10^eC, around 20^eC, around 30^eC, around 40^eC, or around 50^eC.

In some embodiments, the cell wall degrading enzyme is active during cold storage (below about 10 ^eC), at room temperature (from about 10 to about 25 ^eC), and under sunny growing conditions, such as about 25 to about 35 ^eC.

In some embodiments, the cell wall degrading enzyme is active at a pH of from 2 to about 12. In some embodiments, the cell wall degrading enzyme is active at a pH of from 3 to about 1 1 . In some embodiments, the cell wall degrading enzyme is active at a pH of from 4 to about 10. In some embodiments, the cell wall degrading enzyme is active at a pH of from 5 to about 9. In some embodiments, the cell wall degrading enzyme is active at a pH of from 5 to 7. In some embodiments, the cell wall degrading enzyme is active at a pH of from 7 to 9. In a preferred embodiment the cell wall degrading enzyme is active at a pH of from 4.5 to 9.5. In a more preferred embodiment the cell wall degrading enzyme is active at a pH of from 5.5 to 8.5. In an even more preferred embodiment the cell wall degrading enzyme is active at a pH of from 6.5 to 7.5. The cell wall degrading enzyme as described herein may be provided in the form of compositions, for example agrochemical compositions. The cell wall degrading enzyme may be provided in the form of a combination. The cell wall degrading enzyme of the invention are provided in the form of compositions or combinations further comprising antifungal compound with cell membrane interacting properties, as described herein. The cell wall degrading enzyme may be co-administered with the antifungal compound with cell membrane interacting properties as described herein. Co-administration occurs by administering a composition comprising both an antifungal compound with cell membrane interacting properties and a cell wall degrading enzyme.

Antifungal activity of the compositions

In a preferred embodiment, the plant pest(s) that is/are combated by the cell wall degrading enzymes, mixtures, compositions, agrochemical compositions or combinations 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. 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 cell wall degrading enzymes, mixtures, compositions, agrochemical compositions or combinations 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 cell wall degrading enzymes, mixtures, compositions, agrochemical compositions or combinations 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 compositions or combinations according to the invention are active against normally sensitive and resistant species and against all or some stages in the life cycle of the plant pathogenic fungus.

In particular embodiments, the cell wall degrading enzymes, mixtures, compositions, agrochemical compositions or combinations 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. In a more preferred embodiment, the cell wall degrading enzymes, mixtures, compositions, agrochemical compositions or combinations 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.

Forms of Target Antigen of the Polypeptides

It will be appreciated based on the disclosure herein that for agrochemical and biological control applications, the polypeptides of the compositions as disclosed herein may be directed against or specifically bind to several different forms of the pest target, for example a fungal target.

It is also expected that the polypeptides of the compositions as disclosed herein will bind to a number of naturally occurring or synthetic analogues, variants, mutants, alleles, parts and fragments of their pest target. More particularly, it is expected that the polypeptides of the compositions as disclosed herein will bind to at least to those analogues, variants, mutants, alleles, parts and fragments of the target that (still) contain the binding site, part or domain of the natural target to which those polypeptides bind. In preferred embodiments the polypeptides of the invention bind a fungal plasma membrane component.

Formulations

It is understood that the compositions or agrochemical compositions as disclosed herein are stable, both during storage and during utilization, meaning that the integrity of the composition or agrochemical composition is maintained under storage and/or utilization conditions of the composition or agrochemical composition, which may include elevated temperatures, freeze-thaw cycles, changes in pH or in ionic strength, UV-irradiation, presence of harmful chemicals and the like. More preferably, the antifungal compound with cell membrane interacting properties, and the cell wall degrading enzyme remain stable in the agrochemical composition, meaning that the integrity and the pesticidal activity of the substances is maintained under storage and/or utilization conditions of the agrochemical composition, which may include elevated temperatures, freeze-thaw cycles, changes in pH or in ionic strength, UV-irradiation, presence of harmful chemicals and the like. Most preferably, said antifungal compound with cell membrane interacting properties and the cell wall degrading enzyme, remain stable in the composition or agrochemical composition when the composition or agrochemical composition is stored at ambient temperature for a period of two years. Preferably, the composition or agrochemical composition of the present invention retains at least about 70% activity, more preferably at least about 80% activity, most preferably at least about 90% activity or more. Optionally, the antifungal compound with cell membrane interacting properties and/or the cell wall degrading enzyme may be comprised in a carrier, as defined, to protect the substances from harmful effects caused by other components in the agrochemical composition or from harmful effects during storage or during application. Examples of suitable carriers include, but are not limited to alginates, gums, starch, p-cyclodextrins, celluloses, polyurea, polyurethane, polyester, microbial cells or clay.

The composition or agrochemical composition may occur in any type of formulation, preferred formulations are powders, wettable powders, wettable granules, water dispersible granules, emulsions, emulsifiable concentrates, dusts, suspensions, suspension concentrates, suspoemulsions (mixtures of suspensions and emulsions), capsule suspensions, aqueous dispersions, oil dispersions, aerosols, pastes, foams, slurries or flowable concentrates.

The antifungal compound with cell membrane interacting properties and/or cell wall degrading enzyme, may be the only active substances in the composition or agrochemical composition according to the invention; however, it is also possible that the composition comprises one or more additional agrochemicals, as defined, in addition to the polypeptide and cell wall degrading enzyme. Such additional agrochemicals or biological control compositions may have a different effect on plant pests as the antifungal compound with cell membrane interacting properties or cell wall degrading enzyme, they may have a synergistic effect with the antifungal compound with cell membrane interacting properties or and cell wall degrading enzyme, or they may even modify the activity of the antifungal compound with cell membrane interacting properties or cell wall degrading enzyme on certain plants. Suitable additional agrochemicals can be herbicides, insecticides, fungicides, nematicides, acaricides, bactericides, viricides, plant growth regulators, safeners and the like. Such agrochemicals may be chemicals or may be biological substances, for example a microbial. They include, but are not limited to glyphosate, paraquat, metolachlor, acetochlor, mesotrione, 2, 4-D, atrazine, glufosinate, sulfosate, fenoxaprop, pendimethalin, picloram, trifluralin, bromoxynil, clodinafop, fluroxypyr, nicosulfuron, bensulfuron, imazetapyr, dicamba, imidacloprid, thiamethoxam, fipronil, chlorpyrifos, deltamethrin, lambda-cyhalotrin, endosulfan, methamidophos, carbofuran, clothianidin, cypermethrin, abamectin, diflufenican, spinosad, indoxacarb, bifenthrin, tefluthrin, azoxystrobin, thiamethoxam, tebuconazole, mancozeb, cyazofamid, fluazinam, pyraclostrobin, epoxiconazole, chlorothalonil, copper fungicides (for example copper oxychloride, copper hydroxide), trifloxystrobin, prothioconazole, difenoconazole, carbendazim, propiconazole, thiophanate, sulphur, boscalid, tricyclazole, hexaconazole, metalaxyl, benomyl, kitazin, tebuconazole, tridemorph, propineb, streptomycin sulfate and oxytetracycline and other known agrochemicals or any suitable combination(s) thereof.

Suitable additional agrochemicals may be a biological substance, such as a microbial, for example a Pseudomonas strain, a Bacillus strain or a Streptomyces strain.

In some embodiments, the composition or agrochemical composition disclosed herein, when applied to a fungus or to a plant or part of a plant comprising or infected by a fungus, the cell wall degrading enzyme disrupts the cell wall of said fungus and antifungal compound with cell membrane interacting properties disrupts the cell membrane of said fungus.

It is envisaged that the antifungal compound with cell membrane interacting properties and/or cell wall degrading enzyme content contained in the composition as disclosed herein may vary within a wide range and it is generally up to the manufacturer to modify the concentration range of particular active substances according to specific crop pest which is to be attenuated.

The composition of the invention may be formulated into any type of formulation, preferred formulations are powders, wettable powders, wettable granules, water dispersible granules, emulsions, emulsifiable concentrates, dusts, suspensions, suspension concentrates, suspoemulsions (mixtures of suspensions and emulsions), capsule suspensions, aqueous dispersions, oil dispersions, aerosols, pastes, foams, slurries or flowable concentrates.

In particular embodiments, the present invention provides agrochemical compositions comprising at least one antifungal compound with cell membrane interacting properties and at least one cell wall degrading enzyme. The antifungal compound with cell membrane interacting properties and/or cell wall degrading enzyme are present in an amount effective to protect or treat a plant or a part of said plant from an infection or other biological interaction with said plant pathogen.

In a specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be at least 0.0001 % by weight.

In a specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be up to 50% by weight.

In a specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 0.0001 % to 50% by weight.

In particular embodiments, the present invention provides agrochemical compositions comprising at least one antifungal compound with cell membrane interacting properties, wherein the concentration of the at least one polypeptide in the agrochemical composition ranges from 0.001 % to 50% by weight.

In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 0.001 % to 50% by weight. In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 0.01 % to 50% by weight. In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 0.1 % to 50% by weight.

In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 1 % to 50% by weight. In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 10% to 50% by weight. In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 0.0001 % to 40% by weight. In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 0.001 % to 40% by weight. In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 0.01 % to 40% by weight. In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 0.1 % to 40% by weight. In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 1 % to 40% by weight. In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 0.0001 % to 30% by weight. In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 0.001 % to 30% by weight. In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 0.01 % to 30% by weight. In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 0.1 % to 30% by weight. In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 1 % to 30% by weight. In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 0.0001 % to 10% by weight. In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 0.001 % to 10% by weight. In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 0.01 % to 10% by weight. In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 0.1 % to 10% by weight. In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 1 % to 10% by weight. In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 0.0001 % to 1 % by weight. In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 0.001 % to 1 % by weight. In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 0.01 % to 1 % by weight. In yet another specific embodiment the concentration of the antifungal compound with cell membrane interacting properties contained in the agrochemical composition may be from 0.1 % to 1 % by weight.

In a specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be at least 0.0001 % by weight. In a specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be up to 50% by weight.

In a specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 0.0001 % to 50% by weight.

In particular embodiments, the present invention provides agrochemical compositions comprising at least one cell wall degrading enzyme, wherein the concentration of the at least one cell wall degrading enzyme in the agrochemical composition ranges from 0.001 % to 50% by weight.

In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 0.001 % to 50% by weight. In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 0.01 % to 50% by weight. In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 0.1 % to 50% by weight.

In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 1 % to 50% by weight. In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 10% to 50% by weight. In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 0.0001 % to 40% by weight. In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 0.001 % to 40% by weight. In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 0.01 % to 40% by weight. In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 0.1 % to 40% by weight. In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 1 % to 40% by weight. In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 0.0001 % to 30% by weight. In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 0.001 % to 30% by weight. In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 0.01 % to 30% by weight. In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 0.1 % to 30% by weight. In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 1 % to 30% by weight. In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 0.0001 % to 10% by weight. In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 0.001 % to 10% by weight. In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 0.01 % to 10% by weight. In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 0.1 % to 10% by weight. In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 1 % to 10% by weight. In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 0.0001 % to 1 % by weight. In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 0.001 % to 1 % by weight. In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 0.01 % to 1 % by weight. In yet another specific embodiment the concentration of the cell wall degrading enzyme contained in the agrochemical composition may be from 0.1 % to 1 % by weight.

In a preferred embodiment the total concentration of both the antifungal compound with cell membrane interacting properties (such as the VHH antibody) and cell wall degrading enzyme contained in the agrochemical composition is between 5% and 50% by weight. In a more preferred embodiment the total concentration of both the antifungal compound with cell membrane interacting properties (such as the VHH antibody) and cell wall degrading enzyme contained in the agrochemical composition is between 5% and 20% by weight. Compositions that comprise a combination of an antifungal compound with cell membrane interacting properties and a cell wall degrading enzyme may allow for a reduction in the amount of one or both of the components since they may work synergistically, for example to achieve an IC50 at lower doses. In some cases the combination of the antifungal compound with cell membrane interacting properties and the cell wall degrading enzyme allows the concentration of one or both of the components to be reduced by up to 50% (or more) whilst achieving the same or even a higher level of pest control.

In particular embodiments, the agrochemical compositions disclosed herein comprise at least one antifungal compound with cell membrane interacting properties, which is formulated in an aqueous solution. In particular embodiments, the agrochemical compositions disclosed herein comprise at least one cell wall degrading enzyme, which is formulated in an aqueous solution.

In embodiments in which the composition or combination comprises an antifungal compound with cell membrane interacting properties and a cell wall degrading enzyme the composition or combination may comprise the two components in a variety of different ratios. In some embodiments, the compositions comprise a cell wall degrading enzyme and an antifungal compound with cell membrane interacting properties in a range of froml :100 to 100:1 , 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 cell wall degrading enzyme and antifungal compound with cell membrane interacting properties. Wherein the ratio of the cell wall degrading enzyme over the antifungal compound with cell membrane interacting properties is defined by the ratio of their quantities expressed in moles or as molar concentrations.

In further particular embodiments, the agrochemical compositions disclosed herein comprise at least one antifungal compound with cell membrane interacting properties and at least one cell wall degrading enzyme and further comprises an agrochemically suitable carrier and/or one or more suitable adjuvants. The compositions according to the invention may comprise, in addition to the antifungal compound with cell membrane interacting properties described above and the cell wall degrading enzyme, solid or liquid carriers which are acceptable in the pest treatment of plants and/or parts of plants and/or surfactants which are also acceptable in the pest treatment of plants and/or parts of plants. In particular, there may be used inert and customary carriers and customary surfactants. These compositions cover not only compositions ready to be applied to the plants and/or parts of plants to be treated by immersion or using a suitable device, but also the commercial concentrated compositions which have to be diluted before application to the plants and/or parts of plants.

These agrochemical compositions according to the invention may also contain any sort of other ingredients such as, for example, protective colloids, adhesives, thickeners, thixotropic agents, penetrating agents, stabilizers, sequestrants, texturing agents, flavoring agents, taste enhancers, sugars, sweeteners, colorants and the like. More generally, the active substances, i.e. the at least one antifungal compound with cell membrane interacting properties and cell wall degrading enzyme, may be combined with any solid or liquid additives corresponding to the usual formulation techniques.

These agrochemical compositions according to the invention may also contain any sort of other active ingredient such as, for example, other anti-bacterial or anti-fungal active ingredients.

The term “carrier”, in the present disclosure, denotes a natural or synthetic organic or inorganic substance with which the anti-pest active substance is combined to facilitate its application to plants and/or one or more plant parts. This carrier is therefore generally inert and should be acceptable in the agri-sector. The carrier may be solid (clays, natural or synthetic silicates, silica, resins, waxes, solid fertilizers, and the like) or liquid (water, alcohols, in particular butanol, and the like).

The surfactant may be an emulsifying agent, a dispersing agent or a wetting agent of the ionic or nonionic type or a mixture of such surfactants. There may be mentioned, for example, salts of polyacrylic acids, salts of lignosulphonic acids, salts of phenolsulphonic or naphthalenesulphonic acids, polycondensates of ethylene oxide with fatty alcohols or with fatty acids or with fatty amines, substituted phenols (in particular alkylphenols or arylphenols), salts of esters of sulphosuccinic acids, derivatives of taurine (in particular alkyl taurates), phosphoric esters of polyoxyethylated phenols or alcohols, esters of fatty acids and polyols, sulphate, sulphonate and phosphate functional group- containing derivatives of the above compounds. The presence of at least one surfactant is generally essential when the inert carrier is not soluble in water and when the vector agent for application is water.

The agrochemical compositions as disclosed herein are themselves in fairly diverse, solid or liquid, forms.

As solid composition forms, there may be mentioned dustable powders (content of active substance which may be up to 100%) and granules, in particular those obtained by extrusion, by compacting, by impregnation of a granulated carrier, by granulation using a powder as starting material (the content of active substance in these granules being between 0.5 and 80% for these latter cases). Such solid compositions may be optionally used in the form of a liquid which is viscous to a greater or lesser degree, depending on the type of application desired, for example by diluting in water.

As liquid composition forms or forms intended to constitute liquid compositions during application, there may be mentioned solutions, in particular water-soluble concentrates, emulsions, suspension concentrates, wettable powders (or spraying powder), oils and waxes.

The suspension concentrates, which can be applied by spraying, are prepared so as to obtain a stable fluid product which does not form a deposit and they usually contain from 10 to 75% of active substance, from 0.5 to 15% of surfactants, from 0.1 to 10% of thixotropic agents, from 0 to 10% of appropriate additives, such as antifoams, corrosion inhibitors, stabilizers, penetrating agents and adhesives and, as carrier, water or an organic liquid in which the active substance is not or not very soluble: some organic solids or inorganic salts may be dissolved in the carrier to help prevent sedimentation or as antigels for water.

The agrochemical compositions as disclosed herein can be used as such, in form of their formulations or as the use forms prepared therefrom, such as aerosol dispenser, capsule suspension, cold fogging concentrate, hot fogging concentrate, encapsulated granule, fine granule, flowable concentrate for seed treatment, ready-to-use solutions, dustable powder, emulsifiable concentrate, emulsion oil in water, emulsion water in oil, macrogranule, macrogranule, oil dispersible powder, oil miscible flowable concentrate, oil miscible liquid, froths, paste, seed coated with a pesticide, suspension concentrate (flowable concentrate), suspensions-emulsions-concentrates, soluble concentrate, suspensions, soluble powder, granule, water soluble granules or tablets, water soluble powder for seed treatment, wettable powder, natural and synthetic materials impregnated with active compound, micro-encapsulation in polymeric materials and in jackets for seed, microencapsulation biological particles, for example those described in WO2018/201 160, WO2018/201 161 and WO2019/060903, as well as ULV-cold and hot fogging formulations, gas (under pressure), gas generating product, plant rodlet, powder for dry seed treatment, solution for seed treatment, ultra-low volume (ULV) liquid, ultra-low volume (ULV) suspension, water dispersible granules or tablets, water dispersible powder for slurry treatment.

These formulations are prepared in a known manner by mixing the active compounds or active compound combinations with customary additives, such as, for example, customary extenders and also solvents or diluents, emulsifiers, dispersants, and/or bonding or fixing agent, wetting agents, water repellents, if appropriate siccatives and UV stabilisers, colorants, pigments, defoamers, preservatives, secondary thickeners, adhesives, gibberellins and water as well further processing auxiliaries.

These compositions include not only compositions which are ready to be applied to the plant or seed to be treated by means of a suitable device, such as a spraying or dusting device, but also concentrated commercial compositions which must be diluted before application to the crop.

Methods Of Plant Protection Or Treatment

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 to the plant or to a part of the plant, an agrochemical composition as disclosed herein. The composition may be applied under conditions effective to protect or treat the plant or a part of the plant against that infection or biological interaction with the plant pathogen.

In particular embodiments, these methods comprise applying to the plant or to a part of the plant an agrochemical composition as disclosed herein for example at an application rate higher than 50g of the agrochemical composition per hectare, such as but not limited to an application rate higher than 75g of the agrochemical composition per hectare, such as an application rate higher than 100g of the agrochemical composition per hectare, or in particular an application rate higher than 200g of the agrochemical composition per hectare.

In particular embodiments, these methods comprise applying to the plant or to a part of the plant an agrochemical composition as disclosed herein for example at an application rate between 50g and 1000g of the agrochemical composition per hectare, such as but not limited to an application rate of between 50g and 800g of the agrochemical composition per hectare, in particular an application rate of between 75g and 500g of the agrochemical composition per hectare, such as between 75g and 200g of the agrochemical composition per hectare or between 75g and 150g per hectare.

In yet another embodiment, the invention provides methods for combating or inhibiting plant pests, which methods comprise applying an agrochemical or biological control composition according to the invention to a plant, such as a crop, or a part of a plant or a crop, for example at an application rate below 100g of said polypeptide per hectare. In specific embodiments the application rate is below 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 antifungal compound with cell membrane interacting properties /ha and where said antifungal compound with cell membrane interacting properties is present in the composition together with at least one cell wall degrading enzyme. In a more preferred embodiment, the application rate of the antifungal compound with cell membrane interacting properties when present in the composition according to the invention is between 5 g/ha and 50 g/ha and where said antifungal compound with cell membrane interacting properties is present in the composition together with at least one cell wall degrading enzyme.

Similarly, the invention provides methods for combating or inhibiting plant pests, which methods comprise applying an agrochemical or biological control composition according to the invention to a plant, such as a crop, or a part of a plant or a crop, for example at an application rate below 100g of said cell wall degrading enzyme per hectare. In specific embodiments the application rate is below 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 cell wall degrading enzyme/ha and where said cell wall degrading enzyme is present in the composition together with at least one antifungal compound with cell membrane interacting properties. In a more preferred embodiment, the application rate of the cell wall degrading enzyme when present in the composition according to the invention is between 5 g/ha and 50 g/ha and where said cell wall degrading enzyme is present in the composition together with at least one antifungal compound with cell membrane interacting properties. Therefore, the invention provides methods for combating or inhibiting plant pests, which methods comprise applying an agrochemical or biological control composition according to the invention to a plant, such as a crop, or a part of a plant or a crop, for example at an application rate of below 200g of total weight of said antifungal compound with cell membrane interacting properties and cell wall degrading enzyme per hectare. In specific embodiments the application rate is below 100 g/ha, below 80 g/ha, below 70 g/ha, below 60 g/ha, below 50 g/ha, below 40 g/ha, below 30 g/ha, below 20 g/ha, below 10 g/ha, below 2 g/ha or even lower amounts of total weight of the polypeptide and cell wall degrading enzyme/ha. In a more preferred embodiment, the application rate of the antifungal compound with cell membrane interacting properties and cell wall degrading enzyme when present in the composition according to the invention is between 10 g/ha and 100 g/ha.

In some embodiments the cell wall degrading enzyme is applied to a crop, or a part of a plant or a crop without the polypeptide being present in the agrochemical composition. It is understood that more than one cell mebrane interacting substance may be present in the agrochemical composition 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 variances are specified in the technical sheet delivered with the specific agrochemical composition.

In yet another embodiment, the invention provides the use of the agrochemical or biological control compositions of the invention in combating or inhibiting plant pests. In yet another embodiment, the invention provides the use of the agrochemical compositions of the invention in a method of combating or inhibiting plant pests.

In yet another embodiment, the invention provides the use of the antifungal compound with cell membrane interacting properties in combination with at least one cell wall degrading enzyme of the invention in combating or inhibiting plant pests. Thus, the present invention also provides a combination comprising at least one antifungal compound with cell membrane interacting properties as described herein and at least one cell wall degrading enzyme as described herein. As is clear from the disclosures herein, the present invention thus also provides, for example, methods comprising applying at least one antifungal compound with cell membrane interacting properties as described herein and a cell wall degrading enzyme as described herein. The at least one antifungal compound with cell membrane interacting properties and at least one cell wall degrading enzyme may be applied separately or combined in one composition. The at least one antifungal compound with cell membrane interacting properties and at least one cell wall degrading enzyme may be applied simultaneously or consecutively. If the at least one antifungal compound with cell membrane interacting properties and at least one cell wall degrading enzyme are applied consecutively, the at least one cell wall degrading enzyme is preferably applied prior to the application of the VHH antibody. Preferably, the VHH antibody and at least one cell wall degrading enzyme are applied simultaneously.

Applying an agrochemical composition or polypeptide according to the invention to a crop may be done using any suitable method for applying an agrochemical composition to a crop, 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.

Thus, in particular embodiments, the methods for protecting or treating a plant or a part of a plant from an infection or other biological interaction with a plant pathogen as disclosed herein, comprise applying the agrochemical composition 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.

In certain particular embodiments, the present invention provides methods of inhibiting, preventing, reducing or controlling the growth of a plant pathogen, comprising at least the step of applying to a plant or to a part of said plant, an agrochemical composition as disclosed herein.

In certain other embodiments, the present invention provides methods for of killing a plant pathogen, comprising at least the step of applying to a plant or to a part of said plant, an agrochemical composition as disclosed herein.

Alternatively, the application rate of the agrochemical composition according to the invention, meaning the amount of the agrochemical composition that is applied to the crop, is such that less than 100g, 50 g, 40 g, 35 g, 30 g, 25 g, 20 g, 20 g, 15 g, 10 g, 5 g, 1 g or even lower than 1 g but preferably between 5 g and 50 g of the antifungal compound with cell membrane interacting properties and 100g, 50 g, 40 g, 35 g, 30 g, 25 g, 20 g, 20 g, 15 g, 10 g, 5 g, 1 g or even lower than 1 g but preferably between 5 g and 50 g of the cell wall degrading enzyme, comprised in the agrochemical composition according to the invention, is applied to the crop per hectare. It being understood that the amount of the at least one antifungal compound with cell membrane interacting properties and/or the at least one cell wall degrading enzyme is reduced as compared to when the agrochemical composition would containing either the at least one antifungal compound with cell membrane interacting properties or the at least one cell wall degrading enzyme alone. In some embodiments, the antifungal compound with cell membrane interacting properties and the cell wall degrading enzyme act synergistically to allow a reduction in one or both components in the composition. In some embodiments, the combined concentration of the antifungal compound with cell membrane interacting properties and the cell wall degrading enzyme when present in a single composition may be lower than the equivalent combined concentrations of the antifungal compound with cell membrane interacting properties and the cell wall degrading enzyme when present in separate compositions, because of the synergy seen when the two components are combined in a single composition.

According to the methods as disclosed herein, the agrochemical composition 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 agrochemical composition according to the invention can be applied alone or in mixture with other materials, preferably other agrochemical compositions, to the crop; alternatively, the agrochemical composition according to the invention can be applied separately to the crop with other materials, preferably other agrochemical compositions, applied at different times to the same crop. According to the method of the present invention, the agrochemical composition according to the invention may be applied to the crop prophylactically, or alternatively, may be applied once target pests have been identified on the particular crop to be treated.

The agrochemical compositions 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 agrochemical compositions 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. .

The method of treatment 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 agrochemical 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.

The agrochemical compositions as disclosed herein 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 postharvest stage. The agrochemical compositions as disclosed 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 agrochemical compositions as disclosed herein 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 agrochemical composition 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.

The agrochemical compositions as disclosed herein can be applied directly as a component of an integrated pest management approach. It thus should be generally understood in the context of this application that the treatment of plants and plant parts with the agrochemical compositions as disclosed herein 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 compositions by the ultra-low volume method, or to inject the active compound preparation or the active compound itself into the soil.

In particular embodiments, the methods for protecting or treating a plant or a part of a plant from an infection or other biological interaction with a plant pathogen as disclosed herein, comprise applying the agrochemical composition to the plant or to a part of the plant either in a pre-harvest or in a post-harvest stage.

According to specific embodiments, the harvested produce is a fruit, flower, nut or vegetable, a fruit or vegetable with inedible peel, preferably selected from avocados, bananas, plantains, lemons, grapefruits, melons, oranges, pineapples, kiwi fruits, guavas, mandarins, mangoes, squash, strawberries, grapes, pumpkin and peaches. According to further specific embodiments, the harvested produce is a cut flower from ornamental plants, preferably selected from Alstroemeria, Carnation, Chrysanthemum, Freesia, Gerbera, Gladiolus, baby's breath (Gypsophila spec), Helianthus, Hydrangea, Lilium, Lisianthus, roses and summer flowers.

The plant species to which the agrochemical compositions as disclosed herein can be applied can for example be but are not limited to maize, soya bean, alfalfa, cotton, sunflower, Brassica oil seeds such as Brassica napus (e.g. canola, rape- seed), Brassica rapa, B. juncea (e.g. (field) mustard) and Brassica carinata, Arecaceae sp. (e.g. oilpalm, coconut), rice, wheat, sugar beet, sugar cane, oats, rye, barley, millet and sorghum, triticale, flax, nuts, grapes and vine and various fruit and vegetables from various botanic taxa, e.g. Rosaceae sp. (e.g. pome fruits such as apples and pears, but also stone fruits such as apricots, cherries, almonds, plums and peaches, and berry fruits such as strawberries, raspberries, red and black currant and gooseberry), Ribesioidae sp., Juglandaceae sp., Betulaceae sp., Anacardiaceae sp., Fagaceae sp., Moraceae sp., Oleaceae sp. (e.g. olive tree), Actinidaceae sp., Lauraceae sp. (e.g. avocado, cinnamon, camphor), Musaceae sp. (e.g. banana trees and plantations), Rubiaceae sp. (e.g. coffee), Theaceae sp. (e.g. tea), Sterculiceae sp., Rutaceae sp. (e.g. lemons, oranges, mandarins and grapefruit); Solanaceae sp. (e.g. tomatoes, potatoes, peppers, capsicum, aubergines, tobacco), Liliaceae sp., Compositae sp. (e.g. lettuce, artichokes and chicory - including root chicory, endive or common chicory), Umbelliferae sp. (e.g. carrots, parsley, celery and celeriac), Cu- curbitaceae sp. (e.g. cucumbers - including gherkins, pumpkins, watermelons, calabashes and melons), Alliaceae sp. (e.g. leeks and onions), Cruciferae sp. (e.g. white cabbage, red cabbage, broccoli, cauliflow- er, Brussels sprouts, pak choi, kohlrabi, radishes, horseradish, cress and Chinese cabbage), Leguminosae sp. (e.g. peanuts, peas, lentils and beans - e.g. common beans and broad beans), Chenopodiaceae sp. (e.g. Swiss chard, fodder beet, spinach, beetroot), Linaceae sp. (e.g. hemp), Cannabeacea sp. (e.g. cannabis), Malvaceae sp. (e.g. okra, cocoa), Papaveraceae (e.g. poppy), Asparagaceae (e.g. asparagus); useful plants and ornamental plants in the garden and woods including turf, lawn, grass and Stevia rebaudiana; and in each case genetically modified types of these plants.

In a preferred embodiment of the treatment methods disclosed herein, the crop is selected from the group consisting of field crops, grasses, fruits and vegetables, lawns, trees and ornamental plants.

In certain aspects, the present invention thus also provides post-harvest treatment methods for protecting or treating a harvested plant or a harvested part of the plant from an infection or other biological interaction with a plant pathogen, at least comprising the step of applying to the harvested plant or to a harvested part of the plant, an agrochemical composition as disclosed herein, under conditions effective to protect or treat the harvested plant or a harvested part of the plant against the infection or biological interaction with the plant pathogen. According to specific embodiments, the harvested produce is a fruit, flower, nut or vegetable, a fruit or vegetable with inedible peel, preferably selected from avocados, bananas, plantains, lemons, grapefruits, melons, oranges, pineapples, kiwi fruits, guavas, mandarins, mangoes, pumpkin and peaches. According to further specific embodiments, the harvested produce is a cut flower from ornamental plants, preferably selected from Alstroemeria, Carnation, Chrysanthemum, Freesia, Gerbera, Gladiolus, baby's breath (Gypsophila spec), Helianthus, Hydrangea, Lilium, Lisianthus, roses and summer flowers. According to further specific embodiments, the harvested produce is cut grass or wood.

Post-harvest disorders are e.g. lenticel spots, scorch, senescent breakdown, bitter pit, scald, water core, browning, vascular breakdown, CO2 injury, CO2 or O2 deficiency, and softening.

In further aspects, the present invention provides uses of the agrochemical compositions as disclosed herein as an anti-pest agent, such as for instance a biostatic agent or a pesticidal agent, including but not limited to a fungistatic or a fungicidal agent.

In a particular embodiment, the plant pests combated by the method according to the present invention are plant pathogenic fungi, as defined before. Lesion number, lesion size, and extent of sporulation of fungal pathogens may all be decreased as a result of the application of the method according to the present invention.

The invention further relates to transgenic plants or methods of producing transgenic plants. More specifically here disclosed are transgenic plants, plant parts, seeds or plants cell comprising one or more of a polynucleotide comprising at least one nucleic acid sequence encoding a cell wall degrading enzyme according to the invention and at least one nucleic acid sequence encoding a polypeptide according to the invention. The invention further relates to methods for generating a transgenic plant comprising a step of introducing one or more of a polynucleotide comprising at least one nucleic acid sequence encoding a cell wall degrading enzymes according to the invention and one or more of a polynucleotide comprising at least one nucleic acid sequence encoding a polypeptide according to the invention, into a plant cell.

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

Examples

Example 1 : In vitro antifungal assay The antifungal activity of a composition comprising at least one polypeptide and at least one cell wall degrading enzyme, wherein the at least one polypeptide is a heavy chain variable domain of a heavy chain antibody (VHH) or a functional fragment thereof is assessed in vitro against a plant pathogenic fungus such as Botrytis cinerea R16.

Two-fold dilutions of said composition and two-fold dilutions of the individual VHH and cell wall degrading enzymes are prepared separately in 96-well microtiter plates. Either 20 pl of these dilutions or 20 pl of water as a control, are added to 80 pl of fungal spore suspension (1 E+05 spores/ml in half strength potato dextrose broth (PDB)), starting with a concentration of 10 pM. The test plates are incubated for 36h at 25°C using the IncuCyte Zoom live cell imaging system. All tests are performed in at least 2 replicates.

The results of the antifungal activity assay indicate a clear dose-dependent growth inhibition pattern. Furthermore the combination of a VHH and a cell wall degrading enzyme show an increased effect as opposed to a VHH or cell wall degrading enzyme alone. Activities are expressed as the % fungal growth as a function of the concentration of the composition (pM).

Example 2: CWDEs digest fungal cell walls and cause the formation of protoplasts.

In order to test whether cell wall degrading enzymes or mixtures of cell wall degrading enzymes comprise CWDEs, a test is set-up. Therefore Botrytis cinerea mycelium is cultured overnight in YPD broth (10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose), filtered, and washed with FF1 buffer (1 .2 M MgSOp/HzO, 7mM NaHzPO^HzO, pH 5.8). Washed mycelium is added to the CWDE solution (1 .5 g in 15 mL FF1 buffer, pH 7.0) and incubated 2 to 4 hours at 28°C, 70 rpm. Protoplasts are separated from mycelium by filtration. Protoplasts are then centrifuged 15 min at 4000 rpm at 4°C with FF2 buffer (0.1 M Tris-HCI, 0.6M D-sorbitol). The interphase is collected and transferred to a new tube. FF3 buffer (10 mM Tris-HCI, 10mM CaCIz, 1 .2M D-sorbitol, pH 7.5) is added to the sample and centrifuged 5 min at 2500 rpm, 4°C. Supernatant is discarded and the pellet is resuspended in 1 mL of FF3 buffer. Pictures are captured in bright field after the incubation in CWDE solution or in FF2 buffer for negative controls. Purified protoplast pictures are captured after the last purification step. An example of the effect of a cell wall degrading enzyme on fungal mycelium is illustrated in Figure 1 .

Example 2: leaf disk assays

In order to show that an enzyme cocktail having multiple cell wall degrading enzymes (including enzymes with glucanase and chitinase activity) can act with a synergistically antifungal effect with an antifungal compound with cell membrane interacting properties (in this example VHH- 88), a leaf disc assay was set up.

Leaf discs (1 cm diameter) were prepared from 2-weeks old bean plants (Variety: Jupiter) and were kept moist on filter paper in square petri dishes (= 3 filter papers with 25mL of water). In total, 16 leaf discs were placed per petri dish. The experiment was prepared twice and data were aggregated across experiments.

Botrytis cinerea spores were harvest from 2 week old PDA plates and spore solution was stored at 4°C before usage. Spore solutions were diluted to 1 x10^A6 cfu/ml in 1 x PDB. Subsequently, 1 mL of spore solution was added to 1 mL of VHH and/or enzyme cocktail diluted in 10mM HEPES to have a final concentration of 150 pM VHH and/or of 50mg ai/L of enzyme cocktail (where ai/L of enzyme cocktail refers to the amount of enzyme active against the fungal cell wall contained therein).

Leaf discs were treated with VHH-88 (SEQ ID NO: 131 ) at 200pM and an enzyme cocktail at 50mg ai/L either separately or in combination. A buffer control without VHH nor enzyme cocktail was included. These samples were incubated in the dark for 21 h at 21 °C on a rotor (20 rpm). After incubation, leaf discs were inoculated. For that, each punch was inoculated with a 10pL droplet. 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 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.

Figure 2 shows disease severity of Botrytis cinerea on bean leaf discs after 3 days of incubation at 21 °C with 16h light/8h dark regime. Boxplots show Tukey-style data distributions for each treatment. Boxes show the inner quartile range (IQR), with the median as the middle line; hinges extend up to 1 .5*IQR past the boxplot, or the edge of the data, whichever is closer. Overlaid points are means of the data distributions. The individual components VHH and enzyme cocktail show no effect at the tested concentrations whereas the combination of the VHH and the enzyme cocktail clearly show a decreased in disease severity (75% suppression of disease p-value: 1 .5E- 1 1 ). These results show a clear synergistic antifungal effect of the combination of cell wall degrading enzymes with the VHH antibodies as disclosed herein.

The current invention provides a specific set of one or more cell wall degrading enzymes that may act synergistically with an antifungal compound with cell membrane interacting properties such as the VHH antibodies as described herein.

Claims

Claims

1 . A composition comprising

(i) at least one antifungal compound with cell membrane interacting properties, and

(ii) at least one recombinantly produced cell wall degrading enzyme.

2. The composition according to claim 1 , wherein the composition comprises one or more of a glucanase and/or one or more of a chitinase.

3. The composition according to any one of the claims above, wherein the at least one recombinantly produced cell wall degrading enzyme is selected from the group consisting of exo-beta-1 ,3-glucanase, endo-beta-1 ,3-glucanase, exo-beta-1 ,6-glucanase, endo-beta-1 ,6- glucanase, endo-beta-1 ,3(4)-glucanase, endo-alpha-1 ,3-glucanase, exo-alpha-1 ,3-glucanase and endo-chitinase.

4. The composition according to any one of the claims above, wherein the at least one recombinantly produced cell wall degrading enzyme is selected from the group consisting of endo-1 ,3(4)-beta-glucanase, endo-beta-1 ,3-glucanase, exo-beta-1 ,3-glucanase, endo-beta- 1 ,6-glucanase, endo-alpha-1 ,3-glucanase and endo-chitinase.

5. The composition according to any one of the claims above, wherein the at least one recombinantly produced cell wall degrading enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 100 to 130 or an amino acid sequence having at least about 70% sequence identity thereto.

6. The composition according to any one of the claims above, wherein the at least one recombinantly produced cell wall degrading enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 100 to 105, 1 12, 1 16 to 1 19,125 and 128 or an amino acid sequence having at least about 70% sequence identity thereto.

7. The composition according to any one of the claims above, wherein the composition comprises at least two recombinantly produced cell wall degrading enzymes, and wherein a first recombinantly produced cell wall degrading enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least about 70% sequence identity thereto, and a second recombinantly produced cell wall degrading enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 16 to 127 or an amino acid sequence having at least about 70% sequence identity thereto.

8. The composition according to any one of the claims above, wherein the composition comprises at least two recombinantly produced cell wall degrading enzymes, and wherein a first recombinantly produced cell wall degrading enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 100 to 105, 1 12 and 128 or an amino acid sequence having at least about 70% sequence identity thereto, and a second recombinantly produced cell wall degrading enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 16 to 1 19 and 125 or an amino acid sequence having at least about 70% sequence identity thereto.

9. The composition according to any one of the claims above, wherein the composition comprises at least three recombinantly produced cell wall degrading enzymes, and wherein a first and a second recombinantly produced cell wall degrading enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 100 to 1 15 and 128 to 130 or an amino acid sequence having at least about 70% sequence identity thereto, and a third recombinantly produced cell wall degrading enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 16 to 127 or an amino acid sequence having at least about 70% sequence identity thereto.

10. The composition according to any one of the claims above, wherein the composition comprises at least three recombinantly produced cell wall degrading enzymes, and wherein a first and a second recombinantly produced cell wall degrading enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 100 to 105, 1 12 and 128, or an amino acid sequence having at least about 70% sequence identity thereto, and a third recombinantly produced cell wall degrading enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 16 to 1 19 and 125 or an amino acid sequence having at least about 70% sequence identity thereto.

1 1 . The composition according to any one of the claims above, wherein the composition comprises no more than 28, preferably no more than 9, preferably no more than 6, preferably no more than 3 recombinantly produced cell wall degrading enzymes.

12. The composition according to any one of claims 1 to 1 1 , wherein the cell wall degrading enzyme is a mixture consisting of two recombinantly produced cell wall degrading enzymes, and wherein the first cell wall degrading enzyme is the exo-beta-1 ,3-glucanase comprising an amino acid sequence according to SEQ ID NO: 105, and the second cell wall degrading enzyme is the endo-chitinase comprising an amino acid according to SEQ ID NO: 1 16.

13. The composition according to any one of claims 1 to 1 1 , wherein the cell wall degrading enzyme is a mixture consisting of two recombinantly produced cell wall degrading enzymes, and wherein the first cell wall degrading enzyme is the exo-beta-1 ,3-glucanase comprising an amino acid sequence according to SEQ ID NO: 105, and the second cell wall degrading enzyme is the endo-chitinase comprising an amino acid according to SEQ ID NO: 1 19.

14. The composition according to any one of claims 1 to 1 1 , wherein the cell wall degrading enzyme is a mixture consisting of two recombinantly produced cell wall degrading enzymes, and wherein the first cell wall degrading enzyme is the exo-beta-1 ,3-glucanase comprising an amino acid sequence according to SEQ ID NO: 128, and the second cell wall degrading enzyme is the endo-chitinase comprising an amino acid according to SEQ ID NO: 1 16.

15. The composition according to any one of claims 1 to 1 1 , wherein the cell wall degrading enzyme is a mixture consisting of two recombinantly produced cell wall degrading enzymes, and wherein the first cell wall degrading enzyme is the exo-beta-1 ,3-glucanase comprising an amino acid sequence according to SEQ ID NO: 128, and the second cell wall degrading enzyme is the endo-chitinase comprising an amino acid according to SEQ ID NO: 1 19.

16. The composition according to any one of claims 1 to 1 1 , wherein the cell wall degrading enzyme is a mixture consisting of two recombinantly produced cell wall degrading enzymes, and wherein the first cell wall degrading enzyme is the endo-chitinase comprising an amino acid sequence according to SEQ ID NO: 1 16, and the second cell wall degrading enzyme is the endo-beta- 1 ,3(4)-glucanase comprising an amino acid according to SEQ ID NO: 100.

17. The composition according to any one of claims 1 to 1 1 , wherein the cell wall degrading enzyme is a mixture consisting of three recombinantly produced cell wall degrading enzymes, and wherein the first cell wall degrading enzyme is the exo-beta-1 ,3-glucanase comprising an amino acid sequence according to SEQ ID NO: 105, the second cell wall degrading enzyme is the endo-chitinase comprising an amino acid according to SEQ ID NO: 1 16, and the third cell wall degrading enzyme is the endo-beta-1 ,3-glucanase comprising an amino acid sequence according to SEQ ID NO: 101 .

18. The composition according to any one of claims 1 to 1 1 , wherein the cell wall degrading enzyme is a mixture consisting of three recombinantly produced cell wall degrading enzymes, and wherein the first cell wall degrading enzyme is the exo-beta-1 ,3-glucanase comprising an amino acid sequence according to SEQ ID NO: 128, the second cell wall degrading enzyme is the endo-chitinase comprising an amino acid according to SEQ ID NO: 1 16, and the third cell wall degrading enzyme is the endo-beta-1 ,3-glucanase comprising an amino acid sequence according to SEQ ID NO: 101 .

19. The composition according to any one of claims 1 to 1 1 , wherein the cell wall degrading enzyme is a mixture consisting of three recombinantly produced cell wall degrading enzymes, and wherein the first cell wall degrading enzyme is the exo-beta-1 ,3-glucanase comprising an amino acid sequence according to SEQ ID NO: 105, the second cell wall degrading enzyme is the endo-chitinase comprising an amino acid according to SEQ ID NO: 1 16, and the third cell wall degrading enzyme is the endo-beta-1 ,3(4)-glucanase comprising an amino acid sequence according to SEQ ID NO: 100.

20. The composition according to any one of claims 1 to 1 1 , wherein the cell wall degrading enzyme is a mixture consisting of three recombinantly produced cell wall degrading enzymes, and wherein the first cell wall degrading enzyme is the exo-beta-1 ,3-glucanase comprising an amino acid sequence according to SEQ ID NO: 128, the second cell wall degrading enzyme is the endo-chitinase comprising an amino acid according to SEQ ID NO: 1 16, and the third cell wall degrading enzyme is the endo-beta-1 ,3(4)-glucanase comprising an amino acid sequence according to SEQ ID NO: 100.

21 . The composition according any one of the claims above, wherein the recombinantly produced cell wall degrading enzyme is purified to a purity of 90% or higher.

22. The composition according to any one of the claims above, wherein the recombinantly produced cell wall degrading enzyme degrades a fungal cell wall, preferably a plant pathogenic fungal cell wall.

23. The composition according to any preceding statement, wherein, when applied to a fungus or to a plant or part of a plant comprising or infected by a fungus, the recombinantly produced cell wall degrading enzyme degrades the cell wall of said fungus, preferably said fungus is a plant pathogenic fungus.

24. The composition according to any one of the claims above, wherein the recombinantly produced cell wall degrading enzyme does not degrade a plant cell wall.

25. The composition according to any one of the claims above, wherein the composition does not comprise one or more of a protease, one or more of a pectinase, one or more of an arabinase and/or one or more of a protease inhibitor.

26. The composition according to any one of the claims above, wherein the composition does not comprise a beta-1 ,4-glucanase.

27. The composition according to any one of the claim above, wherein the recombinantly produced cell wall degrading enzyme is active at a pH of from 4.5 to 9.5.

28. The composition according to any one of the claims above, wherein the antifungal compound with cell membrane interacting properties, when applied to a fungus or to a plant or part of a plant comprising or infected by a fungus, interacts with a cell membrane, preferably a plant pathogenic fungal cell membrane.

29. The composition according to any one of the claims above, wherein the at least one antifungal compound with cell membrane interacting properties is an azole or nitroaniline antifungal compound.

30. The composition according to any one of the claims above, wherein the at least one antifungal compound with cell membrane interacting properties is a polypeptide.

31 . The composition according to claim 30, wherein the polypeptide is an antibody.

32. The composition according to claim 31 , wherein the antibody is a VHH antibody or a fragment thereof.

33. The composition according to any one of claims 30 to 32, wherein the polypeptide specifically binds to a fungus, preferably a plant pathogenic fungus.

34. The composition according to any one of claims 30 to 33, wherein the polypeptide specifically binds to at least one plasma membrane component of the fungus.

35. The composition according to any one of claims 30 to 34, wherein the polypeptide 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

36. The composition according to claim 34, wherein the plasma membrane component is a sphingolipid.

37. The composition according to claim 36, wherein the sphingolipid is a glucosylceramide.

38. The composition according to any one of claims 30 to 35, wherein the polypeptide is a VHH antibody and wherein the VHH antibody comprises an amino acid sequence selected from the list consisting of SEQ ID Nos 1 , 2, 6, 10, 14 or 15 or an amino acid sequence having at least about 80% sequence identity thereto.

39. The composition according to claims 30 to 34, 36 or 37, wherein the polypeptide is a VHH antibody and wherein the VHH antibody comprises an amino acid sequence selected from the list consisting of SEQ ID Nos 16 to 99 or an amino acid sequence having at least about 80% sequence identity thereto.

40. The composition according to any one of claims 30 to 34, wherein the polypeptide is a VHH antibody and wherein the VHH antibody comprises an amino acid sequence selected from the list consisting of SEQ ID Nos 1 , 2, 6, 10, 14 to 99, 131 or 132 or an amino acid sequence having at least about 80% sequence identity thereto.

41 . The composition of any one of claims 30 to 35, wherein the polypeptide is a VHH antibody and wherein the VHH antibody comprises an amino acid sequence according to SEQ ID NO: 2 or an amino acid sequence having at least about 80% sequence identity thereto.

42. The composition of any one of claims 30 to 34, wherein the polypeptide is a VHH antibody and wherein the VHH antibody comprises an amino acid sequence according to SEQ ID NO: 132 or an amino acid sequence having at least about 80% sequence identity thereto.

43. The composition of any one of claims 30 to 34, wherein the polypeptide is a VHH antibody and wherein the VHH antibody comprises an amino acid sequence according to SEQ ID NO: 131 or an amino acid sequence having at least about 80% sequence identity thereto.

44. The composition according to claim 30, where the polypeptide is an antimicrobial peptide or AMP.

45. The composition according to any one of the claims above, wherein the composition is an agrochemical composition, wherein the agrochemical composition optionally further comprises one or more of an agrochemically suitable carrier and/or one or more suitable adjuvants.

46. Use of a composition or agrochemical composition according to any of one of the preceding claims as an anti-fungal agent.

47. The use according to claim 46 as an anti-fungal agent on plants.

48. Use of a composition according to any one of claims 1 to 45 in a method of treating or preventing a plant or a part of said plant from an infection with a plant pathogenic fungus.

49. A method for protecting or treating a plant or a part of said plant from an infection with a plant pathogenic fungus, at least comprising the step of applying to said plant or to a part of said plant, a composition according to any one of claims 1 to 45, under conditions effective to protect or treat said plant or a part of said plant against said infection with said plant pathogenic fungus.

50. A post-harvest treatment method for protecting or treating a harvested plant or a harvested part of said plant from an infection with a plant pathogenic fungus, at least comprising the step of applying to said harvested plant or to a harvested part of said plant, a composition according to any one of claims 1 to 45, under conditions effective to protect or treat said harvested plant or a harvested part of said plant against said infection with said plant pathogenic fungus.

51 . A method of inhibiting or killing the growth of a plant pathogenic fungus, comprising at least the step of applying to a plant or to a part of said plant, a composition according to any of one of claims 1 to 45.

52. The method or use according to any one of claims 46 to 51 , wherein the composition is applied at an application rate of below 200g of total weight of said at least one antifungal compound with cell membrane interacting properties and at least one recombinantly produced cell wall degrading enzyme per hectare.

53. The method or use of claim 52, wherein the application rate is between 10 g/ha and 100 g/ha.

54. A polynucleotide comprising a nucleic acid sequence capable of expressing an amino acid sequence selected from the list consisting of SEQ ID NO: 100 to 130.

55. A method of producing a composition according to any one of claims 1 to 45 or a combination according to claim 59 comprising the steps of:

(i) cloning one or more of a polynucleotide into a host cell, wherein the one or more polynucleotide is capable of expressing at least one cell wall degrading enzyme;

(ii) recombinantly producing at least one cell wall degrading enzyme in a host cell;

(Hi) optionally, purifying the at least one cell wall degrading enzyme, optionally to a purity of 90% or higher;

(iv) combining the at least one cell wall degrading enzyme with one or more of an antifungal compound with cell membrane interacting properties and/or optionally one or more of an additive, optionally an agrochemical additive.

56. The method according to claim 55, wherein the polynucleotide is the polynucleotide according to claim 44.

57. The method according to claim 55 or 56, wherein the antifungal compound with cell membrane interacting properties is a polypeptide according to any one of claims 30 to 44.

58. The method according to any one of claims 55 to 57, wherein the antifungal compound with cell membrane interacting properties is recombinantly produced in the same or different host cell.

59. A combination comprising at least one antifungal compound with cell membrane interacting properties according to any one of statements 28 to 44 and a cell wall degrading enzyme according to any one of statements 2 to 27.

60. The method or use claim according to any one of claims 46 to 53, wherein the method comprises applying the combination according to claim 59.

61 . The composition according to claims 22, 23, 28, 33 and 48 to 51 , wherein the genus of said plant pathogenic fungus is 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.

62. The composition according to any one of claims 1 to 45 or the combination according to claim 59, wherein the antifungal compound with cell membrane interacting properties is able to interact with a plant pathogenic fungus chosen from the group defined in claim 61 and the cell wall degrading enzyme is able to degrade the cell wall of the same plant pathogenic fungus.

63. The composition or combination according to claim 62, wherein the antifungal compound with cell membrane interacting properties is a polypeptide, preferably a VHH antibody, that is able to bind to a cell membrane of a plant pathogenic fungus chosen from the group defined in claim 61 ; and wherein the cell wall degrading enzyme is able to degrade the cell wall of the same plant pathogenic fungus.

64. The composition or combination according to claims 62 or 63, wherein the plant pathogenic fungus is Botrytis cinerea.

65. The composition according to any one of claims 1 to 45 or the combination according to claim 59, wherein the antifungal compound with cell membrane interacting properties and the cell wall degrading enzyme have been produced in the same host cell.

66. A transgenic plant, plant part, seed, or plant cell comprising one or more of a polynucleotide comprising at least one nucleic acid sequence encoding a cell wall degrading enzyme as defined in any one of claims 2 to 27 and one or more of a polynucleotide comprising at least one nucleic acid sequence encoding a polypeptide according to any one of claims 30 to 44.

67. A method for generating a transgenic plant comprising a step of introducing one or more of a polynucleotide comprising at least one nucleic acid sequence encoding a cell wall degrading enzymes as defined in any one of claims 2 to 27 and one or more of a polynucleotide comprising at least one nucleic acid sequence encoding a polypeptide according to any one of claims 30 to 44, into a plant cell.

68. The transgenic plant, plant part, seed, plant cell or method of claim 66 or 67, where the polynucleotide comprising at least one nucleic acid sequence encoding a cell wall degrading enzyme is the polynucleotide according to claim 54.