Antifungal polypeptide

Abstract

The present invention relates to a composition comprising at least one polypeptide, the polypeptide comprising an amino acid sequence shown in any one of SEQ ID NOs: 1 to 51 or 101 to 111, or an amino acid sequence having at least about 80% sequence identity thereto, and the polypeptide being capable of binding to a fungus. The present invention further relates to a composition comprising at least one polypeptide, the polypeptide comprising a CDR1 region having an amino acid sequence shown in any one of SEQ ID NOs: 52 to 67 or 112 to 122, a CDR2 region having an amino acid sequence shown in any one of SEQ ID NOs: 68 to 83 or 123 to 133, and a CDR3 region having an amino acid sequence shown in any one of SEQ ID NOs: 84 to 100 or 134 to 144, and the polypeptide being capable of binding to a fungus. The composition can be used as an antifungal composition.

Description

Field of the Invention

[0001] The present invention relates to a composition comprising at least one polypeptide which is capable of binding to fungi, and to said at least one polypeptide per se. The present invention also relates to the use of the composition as an antifungal agent. The present invention further relates to a method for protecting or treating a plant or a part of said plant against a fungal infection by a phytopathogenic fungus, a post-harvest treatment method for protecting or treating a harvested plant or a harvested part of said plant against a fungal infection by a phytopathogenic fungus, and a method for inhibiting the growth of or killing a phytopathogenic fungus, comprising at least the step of directly or indirectly applying the composition or the polypeptide to the plant or a part of said plant. Furthermore, the present invention relates to a method for preparing an antifungal polypeptide and a method for preparing an antifungal composition. In addition, the present invention relates to a transgenic plant, plant part, seed or plant cell. Background Art

[0002] The presence and persistence of pathogenic fungal infections in patients, animals and plant crops are mainly attributable to the selection pressure of broad-spectrum antifungal drugs and the general lack of efficacy of currently available antifungal agents.

[0003] In humans and animals, systemic fungal infections such as invasive candidiasis and invasive aspergillosis can be caused by a variety of fungal pathogens, such as the highly virulent Candida albicans, Candida tropicalis and Candida krusei, and the less virulent species Candida parapsilosis and Torulopsis glabrata (the latter sometimes referred to as Candida glabrata). Although C. albicans was once the most common fungal isolate obtained from intensive care units, subsequent studies have shown that C. tropicalis, C. glabrata, C. parapsilosis and C. krusei now account for approximately half of such isolates. The increase in non-Candida species means the emergence of Candida species resistant to conventional antifungal treatment.

[0004] Traditionally, Candida albicans, Candida tropicalis, and Candida parapsilosis have been treated with the antifungal agent amphotericin B, which is regarded as the "gold standard" for systemic antifungal therapy. Unfortunately, amphotericin B itself is highly toxic, and its use is affected by side effects, including chills, fever, myalgia, or thrombophlebitis. Other antifungal agents include oral azole drugs (miconazole, ketoconazole, itraconazole, fluconazole) and 5-fluorocytosine. However, fungal species such as Candida krusei and Torulopsis glabrata are resistant to fluconazole, and these species commonly occur in patients receiving prophylactic administration of the drug. In addition, fluconazole-resistant strains of Candida albicans have also been reported. Therefore, despite the progress made in therapeutic antifungal drugs, the need for effective drugs for treating fungal infections remains urgent.

[0005] In agriculture, crop protection relies to a large extent on the use of pesticides, which are applied to crops by spraying the pesticides onto the crops, applying them during crop watering, or incorporating them into the soil. Pesticides are generally organic chemical molecules, and their repeated application to crops poses a toxic threat to both agricultural workers during the treatment process and the environment due to spray drift, persistence in the soil, or runoff into surface water or groundwater. It would be advantageous to be able to use alternative compounds that are less toxic to humans and the environment but at the same time provide effective control of plant pests. Protein-based pesticides that are specific to a particular plant pest target may be very advantageous in this regard, as they are expected to have a short-term presence in the environment and have fewer toxic off-target effects. However, there are few known protein-based or peptide-based pesticides. Some examples are Bt toxins, lectins, defensins, fabatins, tachyplesins, bombesin, allergen proteins (see WO2010019442), pea albumin 1 subunit b (PA1b). However, these protein-based pesticides are either small, compact peptides stabilized by several disulfide bridges or larger proteins (>300 amino acids) that exist in a crystalline form (Cry toxins). It is indeed known in the agricultural field that biologics, especially proteins, are challenging structures for the development of pesticides because they generally have too low stability to maintain their pesticidal function in agrochemical formulations, especially for field applications. Summary of the Invention

[0006] The inventors have successfully developed polypeptides that have surprisingly high specificity, affinity, and potency for targets of pests (especially plant, animal, or human pathogenic pests, such as but not limited to plant, animal, or human pathogenic fungi). The polypeptides can bind to specific lipid fractions of the cell membranes of fungal spores. Merely the binding of the polypeptides is sufficient to have fungicidal activity by blocking spore growth and / or lysing and bursting the spores, thereby preventing mycelium formation. Therefore, the polypeptides can have fungicidal or fungistatic activity.

[0007] In addition, these polypeptides retain their integrity, stability and activity in the composition, and surprisingly effective pest or pathogen control can be achieved by applying the composition comprising the polypeptides disclosed in the present application to crops, animals or humans.

[0008] The efficacy and potency of the polypeptides disclosed herein indicate a lower therapeutic dose and / or the potential for more effective treatment at the same dose. This may mean a reduction in unwanted side effects and a decrease in toxicity in agrochemical and medical applications. In addition, this allows the application of lower amounts or doses of the polypeptides or compositions disclosed herein.

[0009] More specifically, the inventors have found that the molecular structure targeting pests or pathogens with the polypeptides contemplated herein allows for effective control of the pathogen.

[0010] In particular, the inventors have developed polypeptides capable of preventing, protecting, treating or curing plants, animals or humans from pathogen infection or any other biological interaction with pathogens (especially fungal pathogens). Thus, the present invention demonstrates that biomolecules (such as polypeptides or amino acid sequences) can be used to effectively protect or treat plants, animals or humans from damage caused in any way by biological interactions between plants, animals or humans and pathogens (such as through pathogen infection) or to suffer from such biological interactions.

[0011] The polypeptides and compositions of the present invention can be used as independent products, such as independent compositions, such as antifungal compositions.

[0012] The polypeptides and compositions of the present invention can be used in a prophylactic or preventive manner, i.e., the first application occurs before the appearance of the disease.

[0013] The polypeptides and compositions of the present invention can be used as contact fungicides.

[0014] Thus, according to the present invention, there is provided a composition comprising at least one polypeptide capable of binding to a fungus. The polypeptide thus causes a delay in the spore growth of the fungus and / or lysis of the spores of the fungus. That is, the binding of the polypeptide to the fungus causes a delay in the spore growth of the fungus and / or lysis of the spores of the fungus.

[0015] There is also provided a polypeptide capable of binding to a fungus. The polypeptide thus causes a delay in the spore growth of the fungus and / or lysis of the spores of the fungus. That is, the binding of the polypeptide to the fungus causes a delay in the spore growth of the fungus and / or lysis of the spores of the fungus.

[0016] The polypeptides of the present invention and those used in the present invention can (specifically) bind to the membrane of fungi or components of the fungal membrane. In some embodiments, the polypeptides of the present invention and those used in the present invention do not (specifically) bind to the cell wall of fungi or components of the cell wall. For example, in some embodiments, the polypeptides of the present invention and those used in the present invention do not (specifically) bind to glucosylceramide of fungi.

[0017] The present invention also provides a composition comprising at least one polypeptide, wherein the at least one polypeptide is capable of binding to a lipid-containing fraction of the plasma membrane of fungi (such as Botrytis cinerea or other fungi). The lipid-containing fraction can be obtained by chromatography. For example, the lipid-containing fraction can be obtained by a method comprising:

[0018] subjecting the mycelia of fungi (such as Botrytis cinerea or other fungi) to thin-layer chromatography fractionation of the total lipid extract, and selecting the fraction with a retention factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipid fraction.

[0019] The present invention also provides a polypeptide, wherein the at least one polypeptide is capable of binding to a lipid-containing fraction of the plasma membrane of fungi (such as Botrytis cinerea or other fungi). The lipid-containing fraction can be obtained by chromatography. For example, the lipid-containing fraction can be obtained by a method comprising:

[0020] subjecting the mycelia of fungi (such as Botrytis cinerea or other fungi) to thin-layer chromatography fractionation of the total lipid extract, and selecting the fraction with a retention factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipid fraction.

[0021] In addition, the present invention provides a composition comprising at least one polypeptide, the polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 51 and 101 to 111, or an amino acid sequence having at least about 80% sequence identity with any one thereof, and the polypeptide being capable of binding to fungi.

[0022] According to the present invention, there is further provided a composition comprising at least one polypeptide, the polypeptide comprising:

[0023] a CDR1 region having the amino acid sequence shown in SEQ ID NO: 52, a CDR2 region having the amino acid sequence shown in SEQ ID NO: 68, and a CDR3 region having the amino acid sequence shown in SEQ ID NO: 84, and the polypeptide being capable of binding to fungi;

[0024] A CDR1 region having the amino acid sequence shown in SEQ ID NO:53, a CDR2 region having the amino acid sequence shown in SEQ ID NO:69, and a CDR3 region having the amino acid sequence shown in SEQ ID NO:85, and the polypeptide is capable of binding to fungi;

[0025] A CDR1 region having the amino acid sequence shown in SEQ ID NO:54, a CDR2 region having the amino acid sequence shown in SEQ ID NO:70, and a CDR3 region having the amino acid sequence shown in SEQ ID NO:86, and the polypeptide is capable of binding to fungi; or

[0026] A CDR1 region having an amino acid sequence selected from SEQ ID NO:52 to 67 and 112 to 122, a CDR2 region having an amino acid sequence selected from SEQ ID NO:68 to 83 and 123 to 133, and a CDR3 region having an amino acid sequence selected from SEQ ID NO:84 to 100 and 134 to 144, and the polypeptide is capable of binding to fungi.

[0027] The present invention also provides:

[0028] A polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO:1 to 51 and 101 to 111, or an amino acid sequence having at least about 80% sequence identity with any one of them, wherein the polypeptide is capable of binding to fungi; and

[0029] A polypeptide comprising a CDRl region having the amino acid sequence shown in SEQ ID NO:52, a CDR2 region having the amino acid sequence shown in SEQ ID NO:68, and a CDR3 region having the amino acid sequence shown in SEQ ID NO:84, wherein the polypeptide is capable of binding to fungi; and

[0030] A polypeptide comprising a CDRl region having the amino acid sequence shown in SEQ ID NO:53, a CDR2 region having the amino acid sequence shown in SEQ ID NO:69, and a CDR3 region having the amino acid sequence shown in SEQ ID NO:85, and the polypeptide is capable of binding to fungi; and

[0031] A polypeptide comprising a CDRl region having the amino acid sequence shown in SEQ ID NO:54, a CDR2 region having the amino acid sequence shown in SEQ ID NO:70, and a CDR3 region having the amino acid sequence shown in SEQ ID NO:86, and the polypeptide is capable of binding to fungi; and

[0032] A polypeptide comprising a CDR1 region having an amino acid sequence selected from SEQ ID NO: 52 to 67 and 112 to 122, a CDR2 region having an amino acid sequence selected from SEQ ID NO: 68 to 83 and 123 to 133, and a CDR3 region having an amino acid sequence selected from SEQ ID NO: 84 to 100 and 134 to 144, wherein the polypeptide is capable of binding to a fungus.

[0033] The present invention also provides a polypeptide comprising, or consisting of, an amino acid sequence selected from SEQ ID NO: 1 to 51 and 101 to 111.

[0034] The present invention also provides a polypeptide comprising, or consisting of, an amino acid sequence selected from SEQ ID NO: 1 to 10, 12 to 51 and 101 to 111.

[0035] The present invention also provides a polypeptide having a CDR1 region comprising, or consisting of, a sequence selected from SEQ ID NO: 52 to 67 and 112 to 122, a CDR2 region comprising, or consisting of, a sequence selected from SEQ ID NO: 68 to 83 and 123 to 133, and a CDR3 region comprising, or consisting of, a sequence selected from SEQ ID NO: 84 to 100 and 134 to 144.

[0036] The present invention also provides a polypeptide comprising, or consisting of, the following amino acid sequence: an amino acid sequence selected from SEQ ID NO: 1 to 51, or an amino acid sequence having at most 1, at most 2, at most 3, at most 4 or at most 5 amino acid substitutions. The amino acid substitutions may increase the total charge of the polypeptide or may not change the total charge of the polypeptide.

[0037] The present invention also provides a polypeptide comprising, or consisting of, the following amino acid sequence: an amino acid sequence selected from SEQ ID NO: 1 to 10 and 12 to 51, or an amino acid sequence having at most 1, at most 2, at most 3, at most 4 or at most 5 amino acid substitutions. The amino acid substitutions may increase the total charge of the polypeptide or may not change the total charge of the polypeptide.

[0038] Any polypeptide of the present invention may be provided in a composition, such as an agrochemical composition.

[0039] The compositions disclosed herein may comprise at least one antibody or a functional fragment thereof, such as, but not limited to, a heavy chain antibody or a functional fragment thereof.

[0040] The compositions disclosed herein may comprise at least one heavy chain variable domain (V HH) or a functional fragment thereof, the heavy-chain antibody is naturally lacking a light chain, such as but not limited to the heavy-chain variable domain of a camelid heavy-chain antibody (camelid V HH ) or a functional fragment thereof.

[0041] The compositions disclosed herein may comprise at least one camelized heavy-chain variable domain (camelized V H ) or a functional fragment thereof of a conventional four-chain antibody.

[0042] The compositions disclosed herein may comprise at least one heavy-chain variable domain or a functional fragment thereof of an antibody that does not have an amino acid sequence that is identical (i.e., 100% in terms of sequence identity) to the amino acid sequence of a naturally-occurring V H domain (e.g., the amino acid sequence of a naturally-occurring V H domain from a mammal and particularly from a human).

[0043] The compositions disclosed herein may be an agrochemical composition.

[0044] The agrochemical composition disclosed herein may comprise at least one polypeptide that specifically binds to at least one plasma membrane component of a fungus.

[0045] The at least one plasma membrane component of the fungus to which the polypeptide comprised in the compositions disclosed herein binds may not be a protein.

[0046] The at least one polypeptide in the agrochemical composition disclosed herein may be present in an amount effective to protect or treat a human or an animal or a plant or any part thereof from infection by a fungal pathogen or other biological interactions with a fungal pathogen, such as but not limited to a concentration range of the polypeptide in the agrochemical composition from 0.0001 wt% to 50 wt%.

[0047] The at least one polypeptide in the agrochemical composition disclosed herein may be formulated in an aqueous solution, optionally but not limited to in combination with a suitable carrier and / or one or more suitable adjuvants (such as an agrichemically suitable carrier and / or one or more suitable adjuvants).

[0048] The agrochemical composition disclosed herein may comprise at least one polypeptide that specifically binds to a pathogenic fungus, i.e., a phytopathogenic fungus.

[0049] The agrochemical composition disclosed herein may comprise at least one polypeptide that specifically binds to a phytopathogenic fungus, such as but not limited to a phytopathogenic fungus of a genus selected from Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Diplodia, Erysiphe, Fusarium, Mycosphaerella, Gaeumannomyces, Helminthosporium, Macrophomina, Nectria, Penicillium, Peronospora, Phoma, Physoderma, Phytophthora, Plasmopara, Podosphaera, Puccinia, Pyrenophora, Pyricularia, Pythium, Rhizoctonia, Sclerotium, Sclerotinia, Septoria, Thielaviopsis, Uncinula, Venturia, Verticillium, Magnaporthe grisea, Blumeria graminis, Mycosphaerella graminicola, Ustilago, Melampsora, Puccinia graminis, Monilinia, Mucor, Rhizopus, and Aspergillus.

[0050] The agrochemical composition disclosed herein may comprise at least one polypeptide that specifically binds to a fungus that is a fungus of a plant selected from cereals, sorghum, rice, sugar beet, fodder beet, fruit, nuts, Plantaginaceae or Vitaceae, leguminous crops, oil crops, cucurbitaceous plants, fiber plants, fuel crops, vegetables, ornamental plants, shrubs, broad-leaved trees, evergreen trees, grasses, coffee, tea, tobacco, hops, pepper, rubber plants, and latex plants.

[0051] The at least one polypeptide in the agrochemical composition disclosed herein may at least comprise:

[0052] QVQLVESGGGLVQAGGSLRLSCAASRSIFSINAMDWYRQAPGKQREWVAGITRGGTTKYADSVKGRFTISRDNAKKKVYLQMNSLKPEDTAVYYCNVLRGEQPWTRDYWGQGTQVTVSS (SEQ ID NO:1);

[0053] DVQLVESGGGLVQAGGSLRLSCAASRSIFSINAMDWYRQAPGKQREWVAGITRGGTTKYADSVKGRFTISRDNAKKKVYLQMNSLKPEDTAVYYCNVLRGEQPWTRDYWGQGTQVTVSS (SEQ ID NO:2);

[0054] QVQLQESGGGLVQAGGSLRLSCAASGTIFRPTAMGWYRQAPGKERELVATITTGGSTKYADSVKGRFTISRGNAKNTVYLQMSSLKPEDTAVYYCNAQWGVRTRDYWGQGTQVTVSS (SEQ ID NO:3);

[0055] DVQLQESGGGLVQAGGSLRLSCAASGTIFRPTAMGWYRQAPGKERELVATITTGGSTKYADSVKGRFTISRGNAKNTVYLQMSSLKPEDTAVYYCNAQWGVRTRDYWGQGTQVTVSS(SEQ ID NO:4);

[0056] QVQLQESGGGLVQAGDSLRLSCAASISDRAFSRHVMGWFRQPPGKEREFVAAIGWTGRRTYYADSVKGRFTISRDNAMNTVYLQMNSLKPEDTAVYYCAASHFYSVSFEINDYDYWGQGTQVTVSS(SEQ ID NO:5); and / or

[0057] DVQLQESGGGLVQAGDSLRLSCAASISDRAFSRHVMGWFRQPPGKEREFVAAIGWTGRRTYYADSVKGRFTISRDNAMNTVYLQMNSLKPEDTAVYYCAASHFYSVSFEINDYDYWGQGTQVTVSS(SEQ ID NO:6);

[0058] or a sequence having at least about 80% sequence identity thereto, and the polypeptide is capable of binding to a fungus.

[0059] The at least one polypeptide in the agrochemical composition disclosed herein may comprise at least an amino acid sequence having a CDR1 region with the sequence RSIFSINAMD (SEQ ID NO:52), a CDR2 region with the sequence GITRGGTTK (SEQ ID NO:68), and a CDR3 region with the sequence LRGEQPWTRDY (SEQ ID NO:84), and the polypeptide is capable of binding to a fungus.

[0060] The at least one polypeptide in the agrochemical composition disclosed herein may comprise at least an amino acid sequence having a CDR1 region with the sequence GTIFRPTAMG (SEQ ID NO:53), a CDR2 region with the sequence TITTGGSTK (SEQ ID NO:69), and a CDR3 region with the sequence QWGVRTRDY (SEQ ID NO:85), and the polypeptide is capable of binding to a fungus.

[0061] The at least one polypeptide in the agrochemical composition disclosed herein can at least comprise an amino acid sequence having a CDR1 region with the sequence ISDRAFSRHV (SEQ ID NO:54), a CDR2 region with the sequence AIGWTGRRTY (SEQ ID NO:70), and a CDR3 region with the sequence SHFYSVSFEINDYD (SEQ ID NO:86), and the polypeptide is capable of binding to fungi.

[0062] The at least one polypeptide in the agrochemical composition disclosed herein can at least comprise the CDR1, CDR2, and CDR3 regions of any other polypeptide disclosed herein.

[0063] The polypeptides disclosed herein are generally capable of binding to fungi.

[0064] In a further aspect, the invention provides a composition comprising at least one polypeptide used as an antifungal agent, which specifically binds to fungi. The invention also provides a polypeptide used as an antifungal agent, which specifically binds to fungi.

[0065] Accordingly, the invention provides a composition comprising at least one polypeptide, which polypeptide comprises:

[0066] an amino acid sequence selected from SEQ ID NOs: 1 to 51 and 101 to 111, or an amino acid sequence having at least about 80% sequence identity with any one thereof, and the polypeptide is capable of binding to fungi as an antifungal agent; or

[0067] a CDR1 region comprising an amino acid sequence selected from SEQ ID NOs: 52 to 67 and 112 to 122, a CDR2 region comprising an amino acid sequence selected from SEQ ID NOs: 68 to 83 and 123 to 133, and a CDR3 region comprising an amino acid sequence selected from SEQ ID NOs: 84 to 100 and 134 to 144, and the polypeptide is capable of binding to fungi. The invention also provides the use of the composition or polypeptide disclosed herein as an antifungal agent. Such use can be as an antifungal agent on plants. Accordingly, the invention provides the use of an agrochemical composition comprising at least one polypeptide that specifically binds to fungi as an antifungal agent on plants.

[0068] In the present invention, the antifungal agent can be a fungistatic agent and / or a fungicidal agent.

[0069] The invention also provides a nucleic acid sequence encoding any of the polypeptide sequences disclosed herein.

[0070] In addition, the present invention provides methods for protecting or treating a plant or a part of a plant against a fungal plant pathogen infection, wherein the method comprises at least the step of directly or indirectly applying an agrochemical composition or a polypeptide as disclosed herein to the plant or the part of the plant. The agrochemical composition or the polypeptide may be applied under conditions effective to protect or treat the plant or the part of the plant against the fungal plant pathogen infection.

[0071] These methods may comprise directly or indirectly applying an agrochemical composition or a polypeptide as disclosed herein to a plant or a part of a plant, for example, at an application rate of more than 50 g of the agrochemical composition or the polypeptide per hectare, for example, but not limited to, an application rate of more than 75 g of the agrochemical composition or the polypeptide per hectare, for example, an application rate of more than 100 g of the agrochemical composition or the polypeptide per hectare, or in particular, an application rate of more than 200 g of the agrochemical composition or the polypeptide per hectare.

[0072] These methods may comprise directly or indirectly applying an agrochemical composition or a polypeptide as disclosed herein to a plant or a part of a plant, for example, at an application rate between 50 g and 100 g of the agrochemical composition or the polypeptide per hectare, for example, but not limited to, an application rate of 50 g to 200 g of the agrochemical composition or the polypeptide per hectare, in particular, an application rate of 75 g to 175 g of the agrochemical composition or the polypeptide per hectare, for example, an application rate of 75 g to 150 g of the agrochemical composition or the polypeptide or 75 g to 125 g per hectare.

[0073] The agrochemical composition or the polypeptide as disclosed herein may be directly or indirectly applied to a plant or a part of a plant by spraying, atomizing, foaming, fogging, hydroponic cultivation, aquaculture cultivation, coating, immersion, and / or encapsulation, optionally after harvesting.

[0074] The present invention also provides a post-harvest treatment method for protecting or treating a harvested plant or a harvested part of a plant against a fungal plant pathogen infection, which comprises at least the step of directly or indirectly applying an agrochemical composition or a polypeptide as disclosed herein to the harvested plant or the harvested part of the plant under conditions effective to protect or treat the harvested plant or the harvested part of the plant against the fungal plant pathogen infection.

[0075] The present invention also provides a method for inhibiting the growth of a fungal plant pathogen or a method for killing a fungal plant pathogen, which method comprises at least the step of directly or indirectly applying an agrochemical composition or a polypeptide as disclosed herein to a plant or a part of a plant.

[0076] In these methods, the agrochemical composition or the polypeptide as disclosed herein may be directly or indirectly applied to a plant or a part of a plant by spraying, atomizing, foaming, fogging, hydroponic cultivation, aquaculture cultivation, coating, immersion, and / or encapsulation, optionally after harvesting.

[0077] In yet another aspect, the present invention provides a method for preparing a polypeptide that specifically binds to fungi and / or has an affinity for fungi, the method comprising:

[0078] immunizing an animal with a fungal target or a suitable antigenic determinant based on or derived therefrom (such as its antigenic part, its fragment, its region, its domain, its loop or its other epitope);

[0079] obtaining a collection or sample of cells expressing a polypeptide sequence from the immunized animal;

[0080] screening the collection or sample of cells for cells expressing an amino acid sequence that binds to the fungal target and / or has an affinity for the fungal target;

[0081] (i) isolating the amino acid sequence, or (ii) isolating a nucleic acid sequence encoding the amino acid sequence from the collection or sample of cells; and

[0082] expressing the amino acid sequence,

[0083] thereby preparing a polypeptide that specifically binds to fungi and / or has an affinity for fungi.

[0084] Crude lipid extracts or total lipid extracts can be used for immunization. For example, in some embodiments, fungal hyphae and / or conidia (such as fungal hyphae and / or conidia of Fusarium oxysporum or Botrytis cinerea) can be extracted at room temperature, for example, using a chloroform:methanol ratio of 2:1 and 1:2 (v / v). The extracts thus prepared can be combined and dried to provide a crude lipid extract or TLE for immunization.

[0085] The fungal target can be a lipid-containing fraction of the plasma membrane of a fungus (such as Botrytis cinerea). The lipid-containing fraction can be obtained by chromatography. For example, the lipid-containing fraction can be obtained by a method that includes:

[0086] thin layer chromatography fractionation of the mycelium of a fungus (such as Botrytis cinerea or other fungi) with a total lipid extract, and selecting a fraction with a retention factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipid fraction.

[0087] Also provided is a method for preparing an antifungal composition, the method comprising: preparing an antifungal polypeptide according to the above method; and combining the antifungal polypeptide with one or more suitable carriers and / or one or more suitable adjuvants.

[0088] The present invention also provides a transgenic plant, plant part, seed or plant cell comprising a nucleic acid sequence encoding a polypeptide as defined herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 Lists the IncuCyte-based fungal growth monitoring in the presence of increasing doses of VHH 10G11Q.

[0090] Figure 2 . IncuCyte-based fungal growth monitoring in the presence of increasing doses of VHH 10G11Q and 41D01.

[0091] Figure 3 Lists the antifungal activity of 10G11 with single amino acid substitutions in the CDR regions of SEQ ID NOs: 17 to 50. The results are given relative to 10G11 activity.

[0092] Figure 4 Lists the antifungal activity of his-tagged variants of 10G11Q-His (SEQ ID NO: 6 and SEQ ID NOs: 17 - 50). The results are given relative to 10G11Q activity.

[0093] Figure 5 Shows the model protein structure of the 10G11 molecule with the 3 CDR regions marked. The ribbon display with selected amino acid residues is shown as a stick model.

[0094] Figure 6 Lists the antifungal activity of 10G11 charge variants. The results are given relative to 10G11 activity.

[0095] Figure 7 Shows the model protein structure of 10G11 charge variant mutant 9 (SEQ ID NO: 14) with the 3 CDR regions marked. The ribbon display with substituted amino acid residues is shown as a stick model.

[0096] Figure 8 Shows the model protein structure of 10G11 charge variant mutant 11 (SEQ ID NO: 16) with the 3 CDR regions marked. The ribbon display with substituted amino acid residues is shown as a stick model.

[0097] Figure 9 Lists the results of extended antifungal assays showing the effects of mutants 3, 9, and 11 and the 10G11 molecule.

[0098] Figure 10 Shows the thin layer chromatographic separation of 4 different fractions present in the total lipid extract.

[0099] Figure 11 Lists the results of the binding of 10G11 to liposomal vesicles with different compositions containing the fluorescent molecule DPD.

[0100] Figure 12 Lists the binding profiles of 10G11 and fraction 3 obtained by Biolayer Interferometry (BLI) compared to the reference VHH.

[0101] Figure 13 Shows the binding of 10E11Q-His (SEQ ID NO:86), 12C03Q-His (SEQ ID NO:87), and 10G11Q-His (SEQ ID NO:88) to fraction 3 obtained by ELISA.

[0102] Figure 14 Shows microscopic images of untreated and 10G11-treated Botrytis cinerea. On the right, an enlarged view of the treated Botrytis cinerea is provided.

[0103] Figure 15 Lists the evolution of % PESSEV of Asian soybean rust in the lower canopy after application of different compounds.

[0104] Figure 16 Lists the evolution of % PESSEV of Asian soybean rust in the middle canopy after application of different compounds.

[0105] Figure 17 Lists the evolution of % PESSEV of Asian soybean rust in the upper canopy after application of different compounds.

[0106] Figure 18 Lists the evolution of the severity of Colletotrichum orbiculare on pumpkins after application of different compounds.

[0107] Figure 19 Lists the evolution of the severity of Botrytis cinerea on grape bunches after application of different compounds.

[0108] Figure 20 Lists the evolution of the severity of Erysiphe necator on grape leaves after application of different compounds.

[0109] Figure 21 Lists the evolution of the severity of Erysiphe necator on grape bunches after application of different compounds.

[0110] Figure 22 Lists the evolution of the severity of Erysiphe necator on tomatoes after application of different compounds.

[0111] Figure 23 Lists the evolution of the incidence of Erysiphe necator on tomatoes after application of different compounds.

[0112] Figure 24 Lists the evolution of the severity of Erysiphe necator on strawberries after application of different compounds.

[0113] Figure 25 Lists the evolution of the incidence of powdery mildew on strawberries after application of different compounds.

[0114] Figure 26 Lists the number of strawberry fruits infected with Botrytis cinerea at harvest after application of different compounds.

[0115] Figure 27 Lists the evolution of the severity of Botrytis cinerea on strawberry fruits after harvest after application of different compounds.

[0116] Figure 28 Lists the evolution of the severity of powdery mildew on strawberries after application of different compounds.

[0117] Figure 29 Lists the ELISA absorption plots used to determine Kd.

[0118] Figure 30 Lists the sequences of some polypeptides of the present invention, indicating the SEQ ID NO of the full-length sequence and the positions of the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 regions. When the sequence in this figure is inconsistent with the sequence in the sequence listing, the sequence in the figure shall prevail.

[0119] Figure 31 Lists the antifungal activities of 10G11 with the entire CDR2 replaced by its germline sequence and 10G11 with the entire CDR3 region replaced by the CDR3 of the reference VHH. The results are given relative to the activity of 10G11.

[0120] Sequence Listing Description

[0121] The sequence listing provides at least the following sequences in Table 9:

[0122]

[0123]

[0124] Table 9: Correlation between SEQ ID NO and polypeptide and CDR sequences. Detailed Description

[0125] Any reference to prior art in this specification is not and should not be construed as an admission or any form of implication that such prior art constitutes a part of common general knowledge in any country.

[0126] All documents cited in this specification are hereby incorporated by reference in their entirety. Unless otherwise defined, all terms used to disclose the present invention, including technical and scientific terms, have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0127] The present invention will be described with respect to specific embodiments, but the invention is not limited thereto and is only limited by the claims. Any reference signs in the claims shall not be construed as limiting the scope.

[0128] Definition

[0129] When the term "comprising" is used in this specification and claims, it does not exclude other elements or steps.

[0130] In the case of using the indefinite or definite article such as "a" or "an", "the" in reference to a singular noun, this includes the plural forms of the noun, unless otherwise clearly stated.

[0131] As used herein, the term "about" when referring to a measurable value such as a parameter, quantity, duration, etc. is intended to include variations of + / −10% or less, preferably + / −5% or less, more preferably + / −1% or less and still more preferably + / −0.1% or less of the specified value, as long as such variations are suitable for carrying out the invention as disclosed. It should be understood that the value itself to which the modifier "about" refers is also specifically and preferably disclosed.

[0132] The following terms or definitions are provided only to assist in understanding the present invention. Unless specifically defined herein, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of the present invention. For definitions and terms in the art, practitioners may be specifically 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). The definitions provided herein should not be construed as having a scope less than that understood by one of ordinary skill in the art.

[0133] Unless otherwise specified, all methods, steps, techniques and operations not specifically described in detail may be carried out and have been carried out in a manner known per se and clear to those skilled in the art. For example, again referring to standard manuals, the above general background art and further references cited therein.

[0134] 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 may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0135] As used herein, amino acid residues will be designated by their full name or according to the standard three-letter or one-letter amino acid code.

[0136] As used herein, the terms "nucleic acid molecule", "polynucleotide", "polynucleic acid", "nucleic acid" are used interchangeably and refer to a polymeric form of nucleotides of any length, which are deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of polynucleotides include genes, gene fragments, 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. Nucleic acid molecules can be linear or circular.

[0137] As used herein, the term "homology" denotes at least secondary structural similarity between two macromolecules from the same or different taxonomic units, particularly between two polypeptides or polynucleotides, where the similarity is attributable to a common ancestor. Thus, the term "homolog" denotes such related macromolecules having said secondary and optionally tertiary structural similarity. To compare two or more nucleotide sequences, the "(percent) sequence identity" between a first nucleotide sequence and a second nucleotide sequence can be calculated using methods known to those skilled in the art, such as 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 known computer algorithms for sequence alignment, such as NCBI Blast. In determining the degree of sequence identity between two amino acid sequences, those skilled in the art may consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which one amino acid residue is replaced by another amino acid residue having a similar chemical structure and having little or no effect on the function, activity, or other biological properties of the polypeptide. The possible conservative amino acid substitutions will be apparent to those skilled in the art. Multiple amino acid sequences and multiple nucleic acid sequences are said to be "identical" if they have 100% sequence identity over their entire length.

[0138] As used herein, the term "complementary determining region" or "CDR" in the context of an antibody refers to the variable region of the H (heavy) or L (light) chain (also abbreviated as VH and VL, respectively), and contains an amino acid sequence capable of specifically binding to an antigen target. These CDR regions confer the fundamental specificity of the antibody for the structure of a particular antigenic determinant. Such regions are also referred to as "hypervariable regions". CDRs represent non - contiguous stretches of amino acids within the variable region, but regardless of species, these key amino acid sequences have been found to be positioned within the variable chain amino acid sequence with similar positions within the variable heavy and light chain regions. All classical antibodies have three CDR regions in both the variable heavy and light chains, and each CDR region is discontinuous from the others (designated L1, L2, L3, H1, H2, H3) for the respective light (L) and heavy (H) chains.

[0139] As used herein, the term "affinity" refers to the degree to which a polypeptide (particularly an immunoglobulin, such as an antibody, or an immunoglobulin fragment, such as a VHH) binds to an antigen such that the equilibrium between the antigen and the polypeptide is shifted towards the formation of a complex by their binding. Thus, for example, when an antigen and an antibody (fragment) are combined at relatively equal concentrations, a high - affinity antibody (fragment) will bind to the available antigen, thereby shifting the equilibrium towards a high concentration of the resulting complex. The dissociation constant is typically used to describe the affinity between a protein - binding domain and an antigen target. Generally, the dissociation constant is below 10 -5 M. Preferably, the dissociation constant is below 10 -6 M, more preferably, below 10 -7 M. Most preferably, the dissociation constant is below 10 -8 M.

[0140] As used herein, the terms "specifically bind" and "specific binding" generally refer to the ability of a polypeptide (particularly an immunoglobulin, such as an antibody, or an immunoglobulin fragment, such as a VHH) to preferentially bind to a specific antigen present in a homogeneous mixture of different antigens. In certain embodiments, the specific binding interaction will distinguish between a desired and an undesired antigen in a sample, in some embodiments by a factor of about 10 to 100 - fold or more (e.g., by more than about 1000 or 10,000 - fold).

[0141] Thus, an amino acid sequence is said to "specifically bind" a particular target when the amino acid sequence as disclosed herein has an affinity, specificity, and / or specificity for that target (or at least a portion or fragment thereof).

[0142] The "specificity" of an amino acid sequence as disclosed herein can be determined based on affinity and / or avidity.

[0143] When the affinity of an amino acid sequence disclosed herein for binding to a first target antigen is at least 5-fold higher (such as at least 10-fold, such as at least 100-fold and preferably at least 1000-fold) than the affinity of the amino acid sequence disclosed herein for binding to a second target antigen, the amino acid sequence is said to be "specific for the first target antigen compared to the second target antigen". Thus, in certain embodiments, when an amino acid sequence as disclosed herein is said to be "specific for" a first target antigen rather than a second target antigen, it can specifically bind (as defined herein) to the first target antigen and not specifically bind to the second target antigen.

[0144] As used herein, the terms "inhibit", "reduce" and / or "prevent" can refer to an amino acid sequence as disclosed herein that specifically binds to a target antigen of interest and inhibits, reduces and / or prevents the interaction between the target antigen of interest and its natural binding partner. The terms "inhibit", "reduce" and / or "prevent" can also refer to an amino acid sequence as disclosed herein that specifically binds to a target antigen of interest and inhibits, reduces and / or prevents the biological activity of the target antigen of interest as measured using a suitable in vitro, cellular or in vivo assay. Thus, "inhibit", "reduce" and / or "prevent" can also refer to an amino acid sequence as disclosed herein that specifically binds to a target antigen of interest and inhibits, reduces and / or prevents one or more biological or physiological mechanisms, actions, reactions, functional pathways or activities involving the target antigen of interest. This action of an amino acid sequence as an antagonist as disclosed herein can be determined in any suitable manner and / or using any suitable (in vitro and typically cellular or in vivo) assay known in the art, depending on the target antigen of interest.

[0145] Thus, more specifically, "inhibiting", "reducing" and / or "preventing" using the amino acid sequences disclosed herein can mean inhibiting, reducing and / or preventing the interaction between a target antigen of interest and its natural binding partner, or inhibiting, decreasing and / or preventing the activity of the target antigen of interest, or inhibiting, reducing and / or preventing one or more biological or physiological mechanisms, actions, reactions, functions, pathways or activities involving the target antigen, e.g., inhibiting, reducing and / or preventing by at least 10%, but preferably at least 20%, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more, as measured using a suitable in vitro, cellular or in vivo assay, compared to the activity of the target antigen of interest in the same assay under the same conditions but without using the amino acid sequences disclosed herein. Additionally, "inhibiting", "reducing" and / or "preventing" can also mean inducing a decrease in the affinity, avidity, specificity and / or selectivity of the target antigen of interest for one or more of its natural binding partners, and / or inducing a decrease in the sensitivity of the target antigen of interest to one or more conditions (e.g., pH, ionic strength, presence of cofactors, etc.) in the medium or environment in which the target antigen is present, compared to the same conditions but in the absence of the amino acid sequences disclosed herein. In the context of the present invention, "inhibiting", "reducing" and / or "preventing" can also relate to allosteric inhibition, reduction and / or prevention of the activity of the target antigen of interest.

[0146] The inhibitory or antagonistic activity or enhancing or agonistic activity of the amino acid sequences disclosed herein can be reversible or irreversible, but for agrochemical, pharmaceutical and pharmacological applications, it will generally occur reversibly.

[0147] When the amino acid sequences disclosed herein have been extracted or purified from the host cell and / or culture medium in which it is produced, the amino acid sequence is considered to be in the "substantially isolated (form)" as used herein.

[0148] With respect to the amino acid sequences disclosed herein, the terms "binding region", "binding site" or "interaction site" present on the amino acid sequences disclosed herein shall have the meaning of a specific site, region, locus, portion or domain present on a target molecule that is responsible for binding to that target molecule. Such a binding region thus consists essentially of the specific site, region, locus, portion or domain of the target molecule that contacts the amino acid sequence when bound to the target molecule.

[0149] As used herein, "plant" refers to an entire plant or a part thereof, including fresh fruits, vegetables, and seeds. The plant or plant part can be a living plant or its part. Additionally, the term "plant" as used herein encompasses the entire plant, the ancestors and descendants of the plant, and plant parts, including seeds, buds, stems, leaves, roots (including tubers), flowers, and tissues and organs, each of the foregoing containing the gene / nucleic acid of interest. The term "plant" also includes plant cells, suspension cultures, callus, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores, again each of the foregoing containing the gene / nucleic acid of interest.

[0150] Selecting a suitable control plant is a routine part of experimental setup and can include the corresponding wild-type plant or the corresponding plant without the gene of interest. The control plant is typically the same plant species or even the same variety as the plant to be evaluated. The control plant can also be a nullizygote of the plant to be evaluated. A nullizygote is an individual obtained by isolating those lacking the transgene. As used herein, "control plant" refers not only to the entire plant but also to plant parts, including seeds and seed parts.

[0151] As used herein, "crop" refers to a plant species or variety grown for harvest as food, livestock feed, fuelstock, or for any other economic purpose. As non-limiting examples, the crop can be maize, cereals (such as wheat, rye, barley, and oats), sorghum, rice, sugar beet and fodder beet, fruits, such as pomaceous fruits (e.g., apples and pears), citrus fruits (e.g., oranges, lemons, limes, grapefruits, or tangerines), stone fruits (e.g., peaches, nectarines, or plums), nuts (e.g., almonds or walnuts), berries (e.g., cherries, strawberries, blackberries, or raspberries), plantains or vines, leguminous crops (such as beans, lentils, peas, and soybeans), oil crops (such as sunflowers, safflowers, rapeseed, canola, castor, or olives), cucurbitaceous plants (such as cucumbers, melons, or pumpkins), fiber plants (such as cotton, flax, or hemp), fuel crops (such as sugar cane, Miscanthus, or switchgrass), vegetables (such as potatoes, tomatoes, peppers, lettuce, spinach, onions, carrots, eggplants, asparagus, or cabbages), ornamental plants (such as flowers (e.g., petunias, geraniums, roses, tulips, lilies, or chrysanthemums)), shrubs, broad-leaved trees (e.g., poplars or willows), and evergreen trees (e.g., conifers), grasses (such as lawns, turf, or forage), or other useful plants (such as coffee, tea, tobacco, hops, pepper, rubber plants, or latex plants).

[0152] As used herein, "pest" refers to an organism that is harmful to plants, animals, humans, or human concerns, including but not limited to crop pests (as defined hereinafter), household pests (such as cockroaches, ants, etc.), and disease vectors, such as malaria mosquitoes.

[0153] "Plant pest", "plant pathogen", or "crop pest" are used interchangeably in this application and refer 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 terms "plant pest" or "crop pest" are used in the sense of pests that target plants and harm them. Pests particularly belong to invertebrates (such as insects (including agricultural pests, ornamental plant pests, forest pests)). Examples of relevant crop pests include, but are not limited to, aphids, caterpillars, flies, wasps, etc., nematodes (species that are free-living in the soil or particularly parasitic on plant roots, such as root-knot nematodes 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 reticulatum, 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., genus Zachrysia, genus Bradybaena, and Pomacea canaliculata), pathogenic fungi (including ascomycetes (such as Fusarium, Ceratocystis, Verticillium, Magnaporthe oryzae), basidiomycetes (such as Rhizoctonia, Puccinia viticola, Puccinia), and fungal-like oomycetes (such as Pythium and Phytophthora), bacteria (such as Burkholderia and proteobacteria such as Xanthomonas and Pseudomonas), phytoplasmas, spiroplasmas, viruses (such as tobacco mosaic virus and cauliflower mosaic virus), and protozoa.

[0154] As used herein, "microorganism" refers to bacteria, viruses, fungi, yeasts, etc. and "microbial" refers to being derived from a microorganism.

[0155] As used herein, "fungus" refers to eukaryotes belonging to the fungal phylum group. The term fungus in the present invention also includes fungus-like organisms, such as oomycetes. Oomycetes (or oomycota) form a unique phylogenetic lineage of fungus-like eukaryotic microorganisms. This group was initially classified as fungi, but modern insights support a relatively close relationship with photosynthetic organisms in the stramenopile group (such as brown algae and diatoms).

[0156] As used herein, "pest infection" or "pest disease" refers to any inflammatory condition, disease, or disorder in an organism (such as a plant, animal, or human) caused by a pest.

[0157] As used herein, "fungal infection" or "fungal disease" refers to any inflammatory condition, disease or disorder in a living organism (such as a plant, animal or human) caused by a fungus.

[0158] As used interchangeably herein, "active substance", "active ingredient" or "active ingredient" refers to any biological, biochemical or chemical element and its derivatives, fragments or compounds based thereon, including microorganisms, which have a general or specific effect on harmful organisms on a subject and in particular on plants, plant parts or substrate products, said harmful organisms being produced naturally or by manufacture, including any impurities inevitably produced during the manufacturing process.

[0159] As used herein, "agrochemicals" refers to products suitable for the agrochemical industry (including agriculture, horticulture, floriculture, and household and garden use) and products intended for non-crop-related uses, such as those used by public health / pest control operators to control harmful insects and rodents, household uses such as household fungicides and pesticides and reagents, for protecting plants or parts of plants, crops, bulbs, tubers, fruits (e.g., from pests, diseases or insects); for controlling, preferably promoting or increasing, the growth of plants; and / or for promoting the yield of plants, crops or harvested plant parts (e.g., their fruits, flowers, seeds, etc.). Examples of such substances will be clear to those skilled in the art and may include, for example, compounds active as insecticides (e.g., contact insecticides or systemic insecticides, including household insecticides), herbicides (e.g., contact herbicides or systemic herbicides, including household herbicides), fungicides (e.g., contact fungicides or systemic fungicides, including household fungicides), nematicides (e.g., contact nematicides or systemic nematicides, including household nematicides) and other pesticides or biocides (e.g., agents that kill insects or snails); as well as fertilizers; growth regulators such as phytohormones; micronutrients, safeners, pheromones; repellents; insect baits; and / or active ingredients for regulating (i.e., increasing, decreasing, inhibiting, enhancing and / or triggering) gene expression (and / or other characteristics or biochemical processes) in or by a target plant (e.g., a plant to be protected or a plant to be controlled), such as nucleic acids (e.g., single-stranded or double-stranded RNA, e.g., used in the context of RNAi technology) and other factors, proteins, chemicals, etc. known per se for this purpose. Examples of such agrochemicals will be clear to those skilled in the art; and include, for example, but are not limited to: glyphosate, paraquat, metolachlor, acetochlor, mesotrione, 2,4-D, atrazine, glufosinate, bialaphos, fenoxaprop-p-ethyl, pendimethalin, picloram, trifluralin, bromoxynil, clodinafop-propargyl, fluroxypyr, nicosulfuron, bensulfuron-methyl, imazethapyr, dicamba, imidacloprid, thiamethoxam, fipronil, chlorpyrifos, deltamethrin, lambda-cyhalothrin, endosulfan, methamidophos, carbofuran, clothianidin, cypermethrin, abamectin, flufenican, spinosad, indoxacarb, bifenthrin, tefluthrin, azoxystrobin, thiamethoxam, tebuconazole, mancozeb, cyazofamid, fluazinam, pyraclostrobin, epoxiconazole, chlorothalonil, copper fungicides, trifloxystrobin, prothioconazole, difenoconazole, carbendazim, propiconazole, thiophanate-methyl, sulfur, boscalid and other known agricultural chemicals or any suitable combination thereof.

[0160] As used herein, "agrochemical composition" refers to a composition for use in agrochemical applications, which, as further defined, comprises at least one active substance, optionally with one or more additives that are beneficial for the optimal dispersion, atomization, deposition, leaf wetting, distribution, retention, and / or absorption of agrochemicals. As will become clear from the further description herein, agrochemical compositions as used herein include biocontrol agents or biopesticides (including but not limited to biocides, biostatic agents, fungistatic agents, and fungicides) and these terms will be used interchangeably in this application. Thus, agrochemical compositions as used herein include compositions comprising at least one biomolecule as an active ingredient, substance, or component for controlling pests in plants or other agriculturally relevant environments (such as in the soil). Non-limiting examples of biomolecules used as active ingredients in the agrochemical compositions disclosed herein are proteins (including antibodies and their fragments, such as but not limited to heavy chain variable domain fragments of antibodies, including VHH), nucleic acid sequences, (poly)saccharides, lipids, vitamins, hormone glycolipids, sterols, and glycerides.

[0161] As non-limiting examples, additives in the agrochemical compositions disclosed herein may include but are not limited to diluents, solvents, adjuvants, surfactants, wetting agents, spreading agents, oils, adhesives, thickeners, penetrants, buffers, acidifying agents, anti-settling agents, antifreeze agents, light protectants, defoamers, biocides, and / or drift control agents.

[0162] As used herein, "biostatic composition" or "biostatic agent" refers to any active ingredient, substance, or component, or a composition comprising any active ingredient, substance, or component for use in biostatic applications (as further defined herein), which comprises at least one active biostatic substance or component, optionally in combination with one or more additives that are beneficial for the optimal dispersion, atomization, deposition, leaf wetting, distribution, retention, and / or absorption of the active substance or component. As non-limiting examples, such additives are diluents, solvents, adjuvants, (ionic) surfactants, wetting agents, spreading agents, oils, adhesives, thickeners, penetrants, buffers, acidifying agents, anti-settling agents, antifreeze agents, light protectants, defoamers, biocides, protease inhibitors, and / or drift control agents.

[0163] As used herein, "biocidal composition" or "biocide" refers to any active ingredient, substance or component, or composition comprising any active ingredient, substance or component for biocidal use (as further defined herein), which comprises at least one active biocidal substance or component, optionally in combination with one or more additives that facilitate the optimal dispersion, atomization, deposition, leaf wetting, distribution, retention and / or absorption of said active substance or component. By way of non-limiting example, such additives are diluents, solvents, adjuvants, (ionic) surfactants, wetting agents, spreading agents, oils, adhesives, thickeners, penetrants, buffers, acidifying agents, anti-settling agents, antifreeze agents, light protectants, defoamers, biocides, protease inhibitors and / or drift control agents.

[0164] As used herein, "fungistatic composition" or "fungistat" refers to any active ingredient, substance or component, or composition comprising any active ingredient, substance or component for fungistatic use (as further defined herein), which comprises at least one active fungistatic substance or component, optionally in combination with one or more additives that facilitate the optimal dispersion, atomization, deposition, leaf wetting, distribution, retention and / or absorption of said active substance or component. By way of non-limiting example, such additives are diluents, solvents, adjuvants, (ionic) surfactants, wetting agents, spreading agents, oils, adhesives, thickeners, penetrants, buffers, acidifying agents, anti-settling agents, antifreeze agents, light protectants, defoamers, biocides, protease inhibitors and / or drift control agents.

[0165] As used herein, "fungicidal composition" or "fungicide" refers to any active ingredient, substance or component, or composition comprising any active ingredient, substance or component for fungicidal use (as further defined herein), which comprises at least one active fungicidal substance or component, optionally in combination with one or more additives that facilitate the optimal dispersion, atomization, deposition, leaf wetting, distribution, retention and / or absorption of said active substance or component. By way of non-limiting example, such additives are diluents, solvents, adjuvants, (ionic) surfactants, wetting agents, spreading agents, oils, adhesives, thickeners, penetrants, buffers, acidifying agents, anti-settling agents, antifreeze agents, light protectants, defoamers, biocides, protease inhibitors and / or drift control agents.

[0166] As used herein, "agricultural and chemical use" includes not only the use of the above-mentioned agricultural chemicals (such as pesticides, growth regulators, nutrients / fertilizers, repellents, defoliants, etc.) applicable to and / or intended for field-grown crops (such as agriculture), but also the use of the above-mentioned agricultural chemicals (such as pesticides, growth regulators, nutrients / fertilizers, repellents, defoliants, etc.) intended for greenhouse-grown crops (such as horticulture / flower cultivation) or hydroponic systems, and even the use of the above-mentioned agricultural chemicals applicable to and / or intended for non-crop uses, such as for private gardens, household uses (such as herbicides or pesticides for household use) or used by pest control operators (such as weed control, etc.).

[0167] As used herein, "biostatic (effect)" or "biostatic use" includes any effect or use of an active substance (optionally contained in a biostatic, biocidal, fungicidal or antifungal composition as defined herein) for controlling, regulating or interfering with the harmful activity of pests (such as plant pests or plant pathogens), including but not limited to inhibiting the growth or activity of pests, altering the behavior of pests, and repelling or attracting pests in plants, plant parts or their agriculturally related environment, such as for household use or in the soil.

[0168] As used herein, "biocidal (effect)" or "biocidal use" includes any effect or use of an active substance (optionally contained in a biocidal or fungicidal composition as defined herein) for controlling, regulating or interfering with the harmful activity of biotic pests (such as plant pests or plant pathogens), including but not limited to killing pests, inhibiting the growth or activity of pests, altering the behavior of pests, and repelling or attracting pests in plants, plant parts or other agriculturally related environment, such as for household use or in the soil.

[0169] As used herein, "antifungal (effect)" or "antifungal use" includes any effect or use of an active substance (optionally contained in a fungicidal or antifungal composition as defined herein) for controlling, regulating or interfering with the harmful activity of fungi, including but not limited to inhibiting the growth or activity of fungi, altering the behavior of fungi, and repelling or attracting fungi in plants, plant parts or other agriculturally related environment, such as for household use or in the soil.

[0170] As used herein, "fungicidal (effect)" or "fungicidal use" includes any effect or use of an active substance (optionally contained in a fungicidal composition as defined herein) for controlling, regulating or interfering with the harmful activity of fungi, including but not limited to killing fungi, inhibiting the growth or activity of fungi, altering the behavior of fungi, and repelling or attracting fungi in plants, plant parts or other agriculturally related environment, such as for household use or in the soil.

[0171] As used interchangeably herein, "insecticidal activity" or "biocidal activity" refers to interfering with the harmful activities of pests, including but not limited to killing pests, inhibiting the growth or activity of pests, altering the behavior of pests, repelling or attracting pests.

[0172] As used herein, "biostatic activity" refers to interfering with the harmful activities of pests, including but not limited to inhibiting the growth or activity of pests, altering the behavior of pests, repelling or attracting pests.

[0173] The insecticidal, biocidal or biostatic activity of an active ingredient, substance or component, or a composition or agent containing an insecticidal, biocidal or biostatic active ingredient, substance or component, can be expressed as the minimum inhibitory concentration (MIC) of the agent (expressed in concentration units, such as mg / mL), but is not limited thereto.

[0174] As used herein, "fungicidal activity" refers to interfering with the harmful activities of fungi, including but not limited to killing fungi, inhibiting the growth or activity of fungi, altering the behavior of fungi, and repelling or attracting fungi.

[0175] As used herein, "fungistatic activity" refers to interfering with the harmful activities of fungi, including but not limited to inhibiting the growth or activity of fungi, altering the behavior of fungi, and repelling or attracting fungi.

[0176] The fungicidal or fungistatic activity of an active ingredient, substance or component, or a composition or agent containing an insecticidal, biocidal or biostatic active ingredient, substance or component, can be expressed as the minimum inhibitory concentration (MIC) of the agent (expressed in concentration units, such as mg / mL), but is not limited thereto.

[0177] As used herein, "carrier" refers to any solid, semi-solid or liquid carrier into or onto which an active substance can be suitably incorporated, included, fixed, adsorbed, absorbed, bound, encapsulated, embedded, attached or contained. Non-limiting examples of such carriers include nanocapsules, microcapsules, nanospheres, microspheres, nanoparticles, microparticles, liposomes, vesicles, beads, gels, weak ion exchange resin particles, liposomes, spiral delivery carriers, small particles, granules, nanotubes, fullerenes, water droplets as part of a water-in-oil emulsion, oil droplets as 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 husks, 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.

[0178] 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 parental antibody. Thus, an antibody can refer to an immunoglobulin or glycoprotein, or a fragment or portion thereof, or a construct that includes an antigen-binding portion within a modified immunoglobulin-like framework, or an antigen-binding portion within a construct that includes a non-immunoglobulin-like framework or scaffold.

[0179] As used herein, the term "monoclonal antibody" refers to an antibody composition having a homogeneous population of antibodies. The term is not limited by the class or source of the antibody, nor is it intended to be limited by the manner of its preparation. The term includes intact immunoglobulins as well as fragments, such as Fab, F(ab)2, Fv, and other fragments that retain the antigen-binding function of the antibody. Monoclonal antibodies of any mammalian species can be used in the present invention. However, in practice, antibodies are typically of rat or murine origin because rat or murine cell lines can be used to prepare the desired hybrid cell lines or hybridomas for the production of monoclonal antibodies.

[0180] As used herein, the term "polyclonal antibody" refers to an antibody composition having a heterogeneous population of antibodies. Polyclonal antibodies are typically derived from the pooled sera of immunized animals or selected humans.

[0181] As used herein, "the variable domain of the heavy chain of an antibody or a functional fragment thereof" means (i) the variable domain of the heavy chain of a heavy-chain antibody that is naturally devoid of a light chain (hereinafter also referred to as V HH ), including but not limited to the variable domain of the heavy chain of a heavy-chain antibody of the Camelidae family or sharks, or (ii) the variable domain of the heavy chain of a conventional four-chain antibody (hereinafter also referred to as V H ), including but not limited to the camelized (as further defined herein) variable domain of the heavy chain of a conventional four-chain antibody (hereinafter also referred to as camelized V H ).

[0182] As further described below, the amino acid sequence and structure of the variable domain of the heavy chain of an antibody can be considered, but not limited to, being composed of four framework regions or "FRs", which are respectively referred to in the art and hereinafter as "framework region 1" or "FR1"; "framework region 2" or "FR2"; "framework region 3" or "FR3"; and "framework region 4" or "FR4", and the framework regions are interrupted by three complementarity-determining regions or "CDRs", which are respectively referred to in the art as "complementarity-determining region 1" or "CDR1"; "complementarity-determining region 2" or "CDR2"; and "complementarity-determining region 3" or "CDR3".

[0183] Also as further described below, the variable domain of the heavy chain of an antibody (including V HH or VH ) The total number of amino acid residues therein can be around 110 - 130, preferably 112 - 115 and most preferably 113. However, it should be noted that the parts, fragments or analogs of the heavy chain variable domain of the antibody are not particularly limited with respect to their length and / or size, as long as these parts, fragments or analogs retain (at least in part) functional activity, such as insecticidal, biocidal, biostatic activity, fungicidal or fungistatic activity (as defined herein), and / or retain (at least in part) the binding specificity of the original heavy chain variable domain of the antibody from which these parts, fragments or analogs are derived. The parts, fragments or analogs that retain (at least in part) functional activity (such as insecticidal, biocidal, biostatic activity, fungicidal or fungistatic activity (as defined herein)) and / or retain (at least in part) the binding specificity of the original heavy chain variable domain of the antibody from which these parts, fragments or analogs are derived are also further referred to herein as "functional fragments" of the heavy chain variable domain.

[0184] The method of numbering the amino acid residues of the heavy chain variable domain is the method described by Chothia et al. (Nature 342, 877 - 883 (1989)), the so-called "AbM definition" and the so-called "contact definition". In this article, this is the numbering system adopted.

[0185] Alternatively, the amino acid residues of the heavy chain variable domain of the antibody (including the variable domain of V HH or V H ) can be numbered according to the general numbering of the heavy chain variable domain given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to the V HH domain of camelids in the article by Riechmann and Muyldermans mentioned above (see, for example, Figure 2 ) of the said reference.

[0186] For a general description of heavy chain antibodies and their variable domains, reference is made in particular to the following references, which are mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103 from 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 1134231 and WO 02 / 48193 from Unilever; WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527 from Vlaams Instituut voor Biotechnologie (VIB); WO 03 / 050531 from Algonomics N.V. and Ablynx NV; WO 01 / 90190 from National Research Council of Canada; WO 03 / 025020 (= EP 1 433 793) from Institute of Antibodies; and WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551 from Ablynx; Hamers-Casterman et al., Nature 1993 Jun. 3; 363(6428):446-8.

[0187] In general, it should be noted that the term "heavy chain variable domain" as used herein in the broadest sense is not limited to a particular biological source or a particular method of preparation. For example, as will be discussed in more detail below, the heavy chain variable domains of the present invention can be obtained by: (1) isolating the V HH domain of a naturally occurring heavy chain antibody; (2) isolating the V H domain of a naturally occurring tetrameric antibody; (3) expressing a nucleotide sequence encoding a naturally occurring V HH domain; (4) expressing a nucleotide sequence encoding a naturally occurring V H domain; (5) "camelizing" (as described below) a naturally occurring V H domain from any animal species (especially mammalian species, such as from humans), or by expressing a nucleotide sequence encoding such a camelized V HNucleic acids of the domain; (6) "camelization" of the "domain antibody" or "Dab" described by Ward et al (ibid), or by expressing nucleic acids encoding such camelized V H Nucleic acids of the domain; (7) using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences; (8) preparing nucleic acids encoding V HH or V H by using nucleic acid synthesis techniques and then expressing the nucleic acids thus obtained; and / or (9) by any combination of the foregoing. Suitable methods and techniques for carrying out the foregoing will be clear to those skilled in the art based on the disclosure herein and include, for example, the methods and techniques described in more detail below.

[0188] However, according to one specific embodiment, the heavy chain variable domain as disclosed herein does not have an amino acid sequence that is identical (i.e., has a 100% degree of sequence identity) to the amino acid sequence of a naturally occurring V H domain (e.g., the amino acid sequence of a naturally occurring V H domain from a mammal, particularly from a human).

[0189] As used herein, the terms "effective amount" and "effective dose" refer to the amount required to achieve the desired result or results.

[0190] As used herein, the terms "determine", "measure", "assess", "monitor" and "assay" are used interchangeably and include both quantitative and qualitative determinations.

[0191] All documents cited in this specification are hereby incorporated by reference in their entirety. Unless otherwise defined, all terms used to disclose the present invention, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Further guidance is provided, including term definitions, to better understand the teachings of the present invention.

[0192] Polypeptide

[0193] The polypeptides disclosed herein are generally capable of binding to fungi. The polypeptide thus causes a delay in the growth of the spores of the fungus and / or lysis of the spores of the fungus. That is, the binding of the polypeptide to the fungus causes a delay in the growth of the spores of the fungus and / or lysis of the spores of the fungus.

[0194] The polypeptides of the present invention and for use in the present invention can (specifically) bind to the membrane of the fungus or a component of the fungal membrane. In some embodiments, the polypeptides of the present invention and for use in the present invention do not (specifically) bind to the cell wall of the fungus or a component of the cell wall. For example, in some embodiments, the polypeptides of the present invention and for use in the present invention do not (specifically) bind to glucosylceramide of the fungus.

[0195] The polypeptide may be able to (specifically) bind to the lipid-containing fraction of the fungal plasma membrane, such as the lipid-containing fraction of Botrytis cinerea or other fungi. The lipid-containing fraction (of Botrytis cinerea or others) can be obtained by chromatography. Chromatography can be performed on a crude lipid extract obtained from fungal hyphae and / or conidia (also referred to herein as total lipid extract or TLE). The chromatography can be, for example, thin layer chromatography or normal phase flash chromatography. The chromatography (such as thin layer chromatography) can be performed on a substrate, such as a glass plate coated with silica gel. The chromatography can be performed using a chloroform / methanol mixture (such as 85 / 15% v / v) as the eluent.

[0196] For example, the lipid-containing fraction can be obtained by a method comprising the following:

[0197] By thin layer chromatography fractionation of the total lipid extract of the hyphae and / or conidia of a fungus (such as Botrytis cinerea or other fungi), and selecting the fraction with a retention factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipid fraction.

[0198] In a more specific embodiment, the lipid-containing fraction can be obtained by a method comprising the following:

[0199] Using a chloroform / methanol mixture (such as 85 / 15% v / v) as the eluent, fractionating the hyphae and / or conidia of a fungus (such as Botrytis cinerea or other fungi) by thin layer chromatography of the total lipid extract on a silica-coated slide, and selecting the fraction with a retention factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipid fraction.

[0200] Alternatively, the fraction can be obtained using normal phase flash chromatography. In this method, the method can comprise:

[0201] By normal phase flash chromatography fractionation of the total lipid extract of the hyphae and / or conidia of a fungus (such as Botrytis cinerea or other fungi), and selecting the fraction with a retention factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipid fraction.

[0202] In a more specific embodiment, the lipid-containing fraction can be obtained by a method comprising the following:

[0203] By normal phase flash chromatography fractionation of the hyphae and / or conidia of a fungus (such as Botrytis cinerea or other fungi) of the total lipid extract, including dissolving the TLE in dichloromethane (CH2Cl2) and MeOH and using CH2Cl2 / MeOH (such as 85 / 15%, v / v) as the eluent, and then filtering the fraction through a filter.

[0204] In more specific embodiments, the lipid-containing fraction can be obtained by a method comprising the following:

[0205] By normal-phase flash chromatography fractionation of the mycelia and / or conidia of a fungus (such as Botrytis cinerea or other fungi) from a total lipid extract (TLE), including dissolving the TLE in dichloromethane (CH2Cl2) and MeOH, loading the TLE onto a phase flash column (such as a flash column with 15 μm particles), running the column with CH2Cl2 / MeOH (85 / 15%, v / v) as the eluent, and filtering the fraction through a filter (such as a 0.45 μm syringe filter with a nylon membrane) and drying the fraction.

[0206] The fraction from chromatography can be processed before testing the binding or interaction of the polypeptide with the fraction. For example, liposomes containing the fraction can be prepared. Such a method can include using thin-film hydration. For example, in such a method, liposomes can be prepared by thin-film hydration with the addition of 1,6-diphenyl-1,3,5-hexatriene (DPH). The binding and / or disruption of the membrane by polypeptide binding can be measured by fluorescence changes before and after polypeptide binding (or by reference to a suitable control).

[0207] Thus, in some embodiments, the polypeptide of the present invention and the polypeptide for use in the present invention can (specifically) bind to the lipid-containing chromatography fraction of the fungal plasma membrane, optionally wherein the lipid-containing chromatography fraction is prepared into liposomes before testing the binding of the polypeptide thereto.

[0208] The binding of the polypeptide to the lipid-containing fraction of the fungus can be confirmed by any suitable method, such as biolayer interferometry. The specific interaction with the lipid-containing fraction can be tested. For example, when the fraction is prepared into liposomes, such as using thin-film hydration, it can be determined whether the polypeptide is able to disrupt the lipid fraction.

[0209] In a method involving chromatography, an extraction step can be carried out before the chromatography step. For example, an extraction step can be performed on the fungal mycelia and / or conidia to provide a crude lipid extract or a total lipid extract for chromatography. For example, in some embodiments, the fungal mycelia and / or conidia (such as the fungal mycelia and / or conidia of Fusarium oxysporum or Botrytis cinerea) can be extracted at room temperature, for example, using a chloroform:methanol ratio of 2:1 and 1:2 (v / v). The extracts so prepared can be combined and dried to provide a crude lipid extract or a TLE.

[0210] Thus, in some embodiments, the polypeptide may be capable of (specifically) binding to the lipid-containing fraction of the fungal plasma membrane (e.g., Fusarium oxysporum or Botrytis cinerea), wherein the lipid-containing fraction of the fungal plasma membrane is obtained or obtainable by chromatography. The chromatography can be normal-phase flash chromatography or thin-layer chromatography. The binding of the polypeptide to the lipid and the lipid-containing fraction can be determined according to biolayer interferometry. In some embodiments, the chromatography step can 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 can use a chloroform:methanol ratio of 2:1 and 1:2 (v / v) to provide two extracts, which are then combined.

[0211] In methods related to thin-layer chromatography, the chromatography can include the steps of:

[0212] Fractionating fungal hyphae by thin-layer chromatography of the total lipid extract and selecting the fraction with a retention factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipid fraction.

[0213] In some methods involving thin-layer chromatography, the chromatography can include the steps of:

[0214] On a silica-coated glass slide, using a chloroform / methanol mixture (e.g., 85 / 15% v / v) as the eluent, fractionating the hyphae and / or conidia of a fungus (e.g., Botrytis cinerea or other fungi) by thin-layer chromatography of the total lipid extract and selecting the fraction with a retention factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipid fraction.

[0215] In methods related to normal-phase flash chromatography, the chromatography can include the steps of:

[0216] Fractionating the hyphae and / or conidia of a fungus (e.g., Botrytis cinerea or other fungi) by normal-phase flash chromatography of the total lipid extract and selecting the fraction with a retention factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipid fraction.

[0217] In some methods related to normal-phase flash chromatography, the chromatography can include the steps of:

[0218] Fractionating the hyphae and / or conidia of a fungus (e.g., Botrytis cinerea or other fungi) by normal-phase flash chromatography of the total lipid extract, including dissolving the TLE in dichloromethane (CH2Cl2) and MeOH and using CH2Cl2 / MeOH (e.g., 85 / 15%, v / v) as the eluent, and then filtering the fractions through a filter.

[0219] In some methods related to normal-phase flash chromatography, the chromatography can include the steps of:

[0220] Fractionating hyphae and / or conidia of a fungus (such as Botrytis cinerea or other fungi) by normal-phase flash chromatography of total lipid extracts, including dissolving the TLE in dichloromethane (CH2Cl2) and MeOH, loading the TLE onto a phase flash column (such as a flash column with 15 μm particles), running the chromatography column with CH2Cl2 / MeOH (85 / 15%, v / v) as the eluent, and filtering the fractions through a filter (such as a 0.45 μm syringe filter with a nylon membrane) and drying the fractions.

[0221] In some aspects, the present invention provides a polypeptide comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 51 and 101 to 111, 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 to SEQ ID NOs: 1 to 51 and 101 to 111.

[0222] In one aspect, the present invention provides a polypeptide comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 10, 12 to 51 and 101 to 111, 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 to SEQ ID NOs: 1 to 10, 12 to 51 and 101 to 111.

[0223] In one aspect, the present invention provides a polypeptide comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 6, 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 to SEQ ID NOs: 1 to 6.

[0224] In one aspect, the present invention provides a polypeptide comprising or consisting of the following sequence: SEQ ID NO: 1 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 to SEQ ID NO: 1.

[0225] In one aspect, the present invention provides a polypeptide comprising or consisting of the following sequence: SEQ ID NO: 2, 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 to SEQ ID NO: 2.

[0226] In one aspect, the present invention provides a polypeptide comprising the following sequence or consisting of the following sequence: SEQ ID NO:3, 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 to SEQ ID NO:3.

[0227] In one aspect, the present invention provides a polypeptide comprising the following sequence or consisting of the following sequence: SEQ ID NO:4, 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 to SEQ ID NO:4.

[0228] In one aspect, the present invention provides a polypeptide comprising the following sequence or consisting of the following sequence: SEQ ID NO:5, 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 to SEQ ID NO:5.

[0229] In one aspect, the present invention provides a polypeptide comprising the following sequence or consisting of the following sequence: SEQ ID NO:6, 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 to SEQ ID NO:6.

[0230] In another aspect, the present invention provides a polypeptide comprising:

[0231] a CDR1 region comprising a sequence selected from SEQ ID NOs: 52 to 67 and 112 to 122 or consisting of such a sequence;

[0232] a CDR2 region comprising a sequence selected from SEQ ID NOs: 68 to 83 and 123 to 133 or consisting of such a sequence;

[0233] a CDR3 region comprising a sequence selected from SEQ ID NOs: 84 to 100 and 134 to 144 or consisting of such a sequence.

[0234] In another aspect, the present invention provides a polypeptide comprising:

[0235] a CDR1 region comprising a sequence selected from SEQ ID NOs: 52, 53 and 54 or consisting of such a sequence;

[0236] a CDR2 region comprising a sequence selected from SEQ ID NOs: 68, 69 and 70 or consisting of such a sequence; and

[0237] The CDR3 region, which comprises or consists of a sequence selected from SEQ ID NO: 84, 85 and 86.

[0238] In another aspect, the present invention provides a polypeptide comprising:

[0239] A CDR1 region comprising or consisting of SEQ ID NO: 52, a CDR2 region comprising or consisting of SEQ ID NO: 68; and a CDR3 region comprising or consisting of SEQ ID NO: 84;

[0240] A CDR1 region comprising or consisting of SEQ ID NO: 53, a CDR2 region comprising or consisting of SEQ ID NO: 69; and a CDR3 region comprising or consisting of SEQ ID NO: 85;

[0241] A CDR1 region comprising or consisting of SEQ ID NO: 54, a CDR2 region comprising or consisting of SEQ ID NO: 70, and a CDR3 region comprising or consisting of SEQ ID NO: 86;

[0242] A CDR1 region comprising or consisting of SEQ ID NO: 55, a CDR2 region comprising or consisting of SEQ ID NO: 68; and a CDR3 region comprising or consisting of SEQ ID NO: 84;

[0243] A CDR1 region comprising or consisting of SEQ ID NO: 52, a CDR2 region comprising or consisting of SEQ ID NO: 71; and a CDR3 region comprising or consisting of SEQ ID NO: 84;

[0244] A CDR1 region comprising or consisting of SEQ ID NO: 52, a CDR2 region comprising or consisting of SEQ ID NO: 68; and a CDR3 region comprising or consisting of SEQ ID NO: 87;

[0245] A CDR1 region comprising SEQ ID NO:55 or consisting of SEQ ID NO:55, a CDR2 region comprising SEQ ID NO:71 or consisting of SEQ ID NO:71; and a CDR3 region comprising SEQ ID NO:87 or consisting of SEQ ID NO:87;

[0246] A CDR1 region comprising SEQ ID NO:56 or consisting of SEQ ID NO:56, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:84 or consisting of SEQ ID NO:84;

[0247] A CDR1 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:88 or consisting of SEQ ID NO:88;

[0248] A CDR1 region comprising SEQ ID NO:56 or consisting of SEQ ID NO:56, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:88 or consisting of SEQ ID NO:88;

[0249] A CDR1 region comprising SEQ ID NO:57 or consisting of SEQ ID NO:57, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:84 or consisting of SEQ ID NO:84;

[0250] A CDR1 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52, a CDR2 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52; and a CDR3 region comprising SEQ ID NO:89 or consisting of SEQ ID NO:89;

[0251] A CDR1 region comprising SEQ ID NO:57 or consisting of SEQ ID NO:57, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:89 or consisting of SEQ ID NO:89;

[0252] A CDR1 region comprising SEQ ID NO:58 or consisting of SEQ ID NO:58, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:84 or consisting of SEQ ID NO:84;

[0253] A CDR1 region comprising SEQ ID NO:59 or consisting of SEQ ID NO:59, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:84 or consisting of SEQ ID NO:84;

[0254] A CDR1 region comprising SEQ ID NO:60 or consisting of SEQ ID NO:60, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:84 or consisting of SEQ ID NO:84;

[0255] A CDR1 region comprising SEQ ID NO:61 or consisting of SEQ ID NO:61, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:84 or consisting of SEQ ID NO:84;

[0256] A CDR1 region comprising SEQ ID NO:62 or consisting of SEQ ID NO:62, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:84 or consisting of SEQ ID NO:84;

[0257] A CDR1 region comprising SEQ ID NO:63 or consisting of SEQ ID NO:63, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:84 or consisting of SEQ ID NO:84;

[0258] A CDR1 region comprising SEQ ID NO:64 or consisting of SEQ ID NO:64, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:84 or consisting of SEQ ID NO:84;

[0259] A CDR1 region comprising or consisting of SEQ ID NO:65, a CDR2 region comprising or consisting of SEQ ID NO:68; and a CDR3 region comprising or consisting of SEQ ID NO:84;

[0260] A CDR1 region comprising or consisting of SEQ ID NO:66, a CDR2 region comprising or consisting of SEQ ID NO:68; and a CDR3 region comprising or consisting of SEQ ID NO:84;

[0261] A CDR1 region comprising or consisting of SEQ ID NO:67, a CDR2 region comprising or consisting of SEQ ID NO:68; and a CDR3 region comprising or consisting of SEQ ID NO:84;

[0262] A CDR1 region comprising or consisting of SEQ ID NO:52, a CDR2 region comprising or consisting of SEQ ID NO:72; and a CDR3 region comprising or consisting of SEQ ID NO:84;

[0263] A CDR1 region comprising or consisting of SEQ ID NO:52, a CDR2 region comprising or consisting of SEQ ID NO:73; and a CDR3 region comprising or consisting of SEQ ID NO:84;

[0264] A CDR1 region comprising or consisting of SEQ ID NO:52, a CDR2 region comprising or consisting of SEQ ID NO:74; and a CDR3 region comprising or consisting of SEQ ID NO:84;

[0265] A CDR1 region comprising or consisting of SEQ ID NO:52, a CDR2 region comprising or consisting of SEQ ID NO:75; and a CDR3 region comprising or consisting of SEQ ID NO:84;

[0266] A CDR1 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52, a CDR2 region comprising SEQ ID NO:76 or consisting of SEQ ID NO:76; and a CDR3 region comprising SEQ ID NO:84 or consisting of SEQ ID NO:84;

[0267] A CDR1 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52, a CDR2 region comprising SEQ ID NO:77 or consisting of SEQ ID NO:77; and a CDR3 region comprising SEQ ID NO:84 or consisting of SEQ ID NO:84;

[0268] A CDR1 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52, a CDR2 region comprising SEQ ID NO:78 or consisting of SEQ ID NO:78; and a CDR3 region comprising SEQ ID NO:84 or consisting of SEQ ID NO:84;

[0269] A CDR1 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52, a CDR2 region comprising SEQ ID NO:79 or consisting of SEQ ID NO:79; and a CDR3 region comprising SEQ ID NO:84 or consisting of SEQ ID NO:84;

[0270] A CDR1 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52, a CDR2 region comprising SEQ ID NO:80 or consisting of SEQ ID NO:80; and a CDR3 region comprising SEQ ID NO:84 or consisting of SEQ ID NO:84;

[0271] A CDR1 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52, a CDR2 region comprising SEQ ID NO:81 or consisting of SEQ ID NO:81; and a CDR3 region comprising SEQ ID NO:84 or consisting of SEQ ID NO:84;

[0272] A CDR1 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52, a CDR2 region comprising SEQ ID NO:82 or consisting of SEQ ID NO:82; and a CDR3 region comprising SEQ ID NO:84 or consisting of SEQ ID NO:84;

[0273] A CDR1 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52, a CDR2 region comprising SEQ ID NO:83 or consisting of SEQ ID NO:83; and a CDR3 region comprising SEQ ID NO:84 or consisting of SEQ ID NO:84;

[0274] A CDR1 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:84 or consisting of SEQ ID NO:84;

[0275] A CDR1 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:90 or consisting of SEQ ID NO:90;

[0276] A CDR1 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:91 or consisting of SEQ ID NO:91;

[0277] A CDR1 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:92 or consisting of SEQ ID NO:92;

[0278] A CDR1 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:93 or consisting of SEQ ID NO:93;

[0279] A CDR1 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:94 or consisting of SEQ ID NO:94;

[0280] A CDR1 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:95 or consisting of SEQ ID NO:95;

[0281] A CDR1 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:96 or consisting of SEQ ID NO:96;

[0282] A CDR1 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:97 or consisting of SEQ ID NO:97;

[0283] A CDR1 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:98 or consisting of SEQ ID NO:98;

[0284] A CDR1 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:99 or consisting of SEQ ID NO:99;

[0285] A CDR1 region comprising SEQ ID NO:52 or consisting of SEQ ID NO:52, a CDR2 region comprising SEQ ID NO:68 or consisting of SEQ ID NO:68; and a CDR3 region comprising SEQ ID NO:100 or consisting of SEQ ID NO:100;

[0286] A CDR1 region comprising SEQ ID NO:112 or consisting of SEQ ID NO:112, a CDR2 region comprising SEQ ID NO:123 or consisting of SEQ ID NO:123; and a CDR3 region comprising SEQ ID NO:134 or consisting of SEQ ID NO:134;

[0287] Comprising SEQ ID NO:113 and a CDR1 region consisting of SEQ ID NO:113, comprising SEQ ID NO:124 and a CDR2 region consisting of SEQ ID NO:124; and comprising SEQ ID NO:135 and a CDR3 region consisting of SEQ ID NO:135;

[0288] Comprising SEQ ID NO:114 and a CDR1 region consisting of SEQ ID NO:114, comprising SEQ ID NO:125 and a CDR2 region consisting of SEQ ID NO:125; and comprising SEQ ID NO:136 and a CDR3 region consisting of SEQ ID NO:136;

[0289] Comprising SEQ ID NO:115 and a CDR1 region consisting of SEQ ID NO:115, comprising SEQ ID NO:126 and a CDR2 region consisting of SEQ ID NO:126; and comprising SEQ ID NO:137 and a CDR3 region consisting of SEQ ID NO:137;

[0290] Comprising SEQ ID NO:116 and a CDR1 region consisting of SEQ ID NO:116, comprising SEQ ID NO:127 and a CDR2 region consisting of SEQ ID NO:127; and comprising SEQ ID NO:138 and a CDR3 region consisting of SEQ ID NO:138;

[0291] Comprising SEQ ID NO:117 and a CDR1 region consisting of SEQ ID NO:117, comprising SEQ ID NO:128 and a CDR2 region consisting of SEQ ID NO:128; and comprising SEQ ID NO:139 and a CDR3 region consisting of SEQ ID NO:139;

[0292] Comprising SEQ ID NO:118 and a CDR1 region consisting of SEQ ID NO:118, comprising SEQ ID NO:129 and a CDR2 region consisting of SEQ ID NO:129; and comprising SEQ ID NO:140 and a CDR3 region consisting of SEQ ID NO:140;

[0293] Comprising SEQ ID NO:119 and a CDR1 region consisting of SEQ ID NO:119, comprising SEQ ID NO:130 and a CDR2 region consisting of SEQ ID NO:130; and comprising SEQ ID NO:141 and a CDR3 region consisting of SEQ ID NO:141;

[0294] Comprising SEQ ID NO:120 and a CDR1 region consisting of SEQ ID NO:120, comprising SEQ ID NO:131 and a CDR2 region consisting of SEQ ID NO:131; and comprising SEQ ID NO:142 and a CDR3 region consisting of SEQ ID NO:142;

[0295] Comprising SEQ ID NO:121 and a CDR1 region consisting of SEQ ID NO:121, comprising SEQ ID NO:132 and a CDR2 region consisting of SEQ ID NO:132; and comprising SEQ ID NO:143 and a CDR3 region consisting of SEQ ID NO:143; or

[0296] Comprising SEQ ID NO:122 and a CDR1 region consisting of SEQ ID NO:122, comprising SEQ ID NO:133 and a CDR2 region consisting of SEQ ID NO:133; and comprising SEQ ID NO:144 and a CDR3 region consisting of SEQ ID NO:144.

[0297] In some embodiments, the polypeptide only comprises the designated CDR1 and CDR2 sequences of the above polypeptides because the CDR3 region may be more easily replaced without loss of activity (as demonstrated by replacing the CDR3 region with an unrelated CDR3 (i.e., a CDR3 that does not bind fungi)). Thus, the CDR3 region sequence is optional. Generally, the polypeptide may still comprise the CDR3 region (e.g., to ensure its structural integrity), but the sequence of the CDR3 region is less important. For example, the polypeptide may comprise a CDR1 region, a CDR2 region, and a CDR3 region, wherein the CDR1 and CDR2 regions each comprise the sequences designated herein or consist of the designated sequences, but the CDR3 region comprises any sequence (e.g., any sequence having a length of 8 to 16 amino acid residues).

[0298] The polypeptides defined herein may suitably have certain framework region sequences. For example, the polypeptide may comprise: a framework region 1 (FR1) sequence comprising a sequence selected from SEQ ID NO:149, 150, 154, 155, 158, and 159 or consisting of the sequence, a framework region 2 (FR2) sequence comprising a sequence selected from SEQ ID NO:151, 156, and 160 or consisting of the sequence, a framework region 3 (FR3) sequence comprising a sequence selected from SEQ ID NO:152, 157, and 161 or consisting of the sequence, and a framework region 4 (FR4) sequence comprising SEQ ID NO:153 or consisting of SEQ ID NO:153.

[0299] In some embodiments, such as those related to any polypeptide cloned as 10G11 herein or derived from such clone, including 10G11Q, any mutants 1 to 11 or any single ALA mutants 1 to 34, any mutants 10G11-A to 10G11K, and including any polypeptide having any specific sequence identity therewith or any substitution therefrom, may comprise: a framework region 1 (FR1) sequence comprising a sequence selected from SEQ ID NO:149 and 150 or consisting of such sequence, a framework region 2 (FR2) sequence comprising SEQ ID NO:151 or consisting of SEQ ID NO:151, a framework region 3 (FR3) sequence comprising SEQ ID NO:152 or consisting of SEQ ID NO:152, and a framework region 4 (FR4) sequence comprising SEQ ID NO:153 or consisting of SEQ ID NO:153.

[0300] In some embodiments, such as those related to any polypeptide cloned as 10E11 herein or derived from such clone, including 10E11Q, and including any polypeptide having any specific sequence identity therewith or any substitution therefrom, may comprise: a framework region 1 (FR1) sequence comprising a sequence selected from SEQ ID NO:154 and 155 or consisting of such sequence, a framework region 2 (FR2) sequence comprising SEQ ID NO:156 or consisting of SEQ ID NO:156, a framework region 3 (FR3) sequence comprising SEQ ID NO:157 or consisting of SEQ ID NO:157, and a sequence of a framework region 4 (FR4) sequence comprising SEQ ID NO:153 or consisting of SEQ ID NO:153.

[0301] In some embodiments, such as those related to any polypeptide cloned as 12C03 herein or derived from such clone, including 12C03Q, and including any polypeptide having any specific sequence identity therewith or any substitution therefrom, may comprise: a framework region 1 (FR1) sequence comprising a sequence selected from SEQ ID NO:158 and 159 or consisting of such sequence, a framework region 2 (FR2) sequence comprising SEQ ID NO:160 or consisting of SEQ ID NO:160, a framework region 3 (FR3) sequence comprising SEQ ID NO:161 or consisting of SEQ ID NO:161, and a framework region 4 (FR4) sequence comprising SEQ ID NO:153 or consisting of SEQ ID NO:153.

[0302] CDRs and framework regions may be defined according to the Kabat numbering system.

[0303] In some embodiments, the length of the polypeptide can be from 80 to 200 residues.

[0304] The polypeptides of the present invention can be provided in the form of a composition, such as an agrochemical composition.

[0305] Composition Comprising at Least One Polypeptide

[0306] On the one hand, the present inventors provide an agrochemical composition comprising at least one polypeptide that can specifically bind to a pest. Importantly, through this interaction with the specific molecular structure of the pest, the compositions disclosed herein are capable of inhibiting, preventing or reducing one or more biological activities of a plant pathogen, thereby inhibiting, preventing or reducing the growth of the plant pathogen. In certain embodiments, the agrochemical compositions disclosed herein are capable of killing plant pests through the specific interaction of at least one polypeptide that can specifically bind to a pest and is included in the composition.

[0307] Accordingly, the agrochemical compositions disclosed herein bind to the binding sites present on the target of the plant pest, thereby affecting 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 the pest is involved, and can thus be used to modulate (such as reduce or inhibit) the biological functions of plant pests.

[0308] In addition, compared to conventional immunoglobulins and non-immunoglobulin binding agents known in the art, compositions comprising at least one polypeptide as disclosed herein have several additional advantages. Indeed, in certain embodiments, the amino acid sequences disclosed herein are isolated heavy chain immunoglobulin variable domains, which are more effective and more stable than conventional four-chain antibodies, resulting in (1) lower formulations, lower dosing frequencies, and thus fewer side effects; and (2) increased stability, thereby providing a wider choice of administration routes. Due to their small size, heavy chain immunoglobulin variable domains have the ability to cross membranes and penetrate into physiological compartments, tissues and organs that larger polypeptides and proteins cannot enter.

[0309] In one specific but non-limiting embodiment, the at least one polypeptide comprised in the composition as disclosed herein can be a polypeptide comprising an immunoglobulin fold or capable of forming an immunoglobulin fold (i.e., by folding) under suitable conditions (such as physiological conditions). See in particular the review by Halaby et al., J. (1999) Protein Eng. 12, 563 - 71. Preferably, when properly folded to form an immunoglobulin fold, such polypeptide sequences are capable of specifically binding (as defined herein) to a target or antigen; and more preferably, capable of binding with an affinity (appropriately measured and / or expressed as a K D -value (actual or apparent), K A- value (actual or apparent), k on - rate and / or k off - rate or alternatively IC 50 value, as further described herein) binds to a pest target or pest antigen. Additionally, portions, fragments, analogs, mutants, variants, alleles, and / or derivatives of such polypeptide sequences are preferably such that they contain an immunoglobulin fold or are capable of forming an immunoglobulin fold under suitable conditions.

[0310] In a specific embodiment, the present invention provides an agrochemical composition or biopesticide composition for controlling plant pests (more particularly plant fungi), the composition comprising at least one polypeptide or amino acid sequence of 80 to 200 amino acids as an active ingredient. Note that as used herein, "80 to 200 amino acids" means from 80 to 200, i.e., including the amount of 80 amino acids and also including the amount of 200 amino acids. Thus, "80 to 200 amino acids" can be used interchangeably with "from 80 to 200 amino acids".

[0311] In certain further embodiments, the present invention provides an agrochemical composition for controlling plant pests, comprising at least two (different) polypeptides or at least two (different) amino acid sequences of 80 to 200 amino acids as active ingredients.

[0312] In a further embodiment, the present invention provides an agrochemical composition for controlling plant pests, the composition comprising at least three (different) polypeptides or at least three (different) amino acid sequences of 80 to 200 amino acids as active ingredients. Other combinations of different polypeptides are also contemplated.

[0313] The agrochemical composition according to the present invention is an agrochemical composition for controlling plant pests as defined hereinabove, meaning that the agrochemical composition, more particularly the active ingredient as defined hereinabove comprised in the agrochemical composition, is capable of interfering (preferably reducing or preventing) the harmful effects of one or more plant pests on one or more plants (preferably crops).

[0314] The polypeptides or amino acid sequences contained in the compositions disclosed herein can be naturally occurring polypeptides or amino acid sequences, which can be derived from naturally occurring polypeptides, or they can be fully designed or synthesized artificially. 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, lipocalin, single-chain anti-parallel coiled-coil proteins or repetitive motif proteins. Non-limiting examples of such polypeptides having the amino acid length ranges described herein include carbohydrate-binding domains (Blake et al (2006) J. Biol. Chem. 281, 29321-29329), heavy chain antibodies (hcAb), single-domain antibodies (sdAb), minibodies (Tramontano et al (1994) J. Mol. Recognition 7, 9-24), variable domains of camel heavy chain antibodies (VHH), variable domains of new antigen receptors (VNAR), affibodies (Nygren P.A. (2008) FEBS J. 275, 2668-2676), alphabodies (see WO2010066740), 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 (nanobodies, see Dimitrov DS (2009) mAbs 1, 26-28). In particular, the polypeptides or amino acid sequences disclosed herein consist of a single polypeptide chain and are without post-translational modifications. More particularly, the disclosed polypeptides or amino acid sequences are derived from the innate or adaptive immune system, preferably from proteins of the innate or adaptive immune system. Even more particularly, the polypeptides or amino acid sequences disclosed herein are derived from immunoglobulins. Most particularly, the polypeptides or amino acid sequences disclosed herein contain 4 framework regions and 3 complementarity-determining regions, or any suitable fragment thereof (which will typically contain at least some amino acid residues that form at least one complementarity-determining region). In particular, the polypeptides or amino acid sequences disclosed herein are easy to produce in high yields, preferably in microbial recombinant expression systems, and are amenable to subsequent isolation and / or purification. In particular, the polypeptides or amino acid sequences disclosed herein are selected from DARPins, knottins, alphabodies and V HH . More particularly, the polypeptides or amino acid sequences disclosed herein are selected from alphabodies and V HH。Most particularly, the polypeptides or amino acid sequences disclosed herein are V HH 。

[0315] Particularly, the at least one polypeptide comprised in the compositions disclosed herein may consist of a single polypeptide chain and be unmodified post-translationally. More particularly, the at least one polypeptide comprised in the compositions disclosed herein may be derived from the innate or adaptive immune system, preferably a protein from the innate or adaptive immune system. Even more particularly, the at least one polypeptide comprised in the compositions as disclosed herein may be derived from immunoglobulins. Most particularly, the at least one polypeptide comprised in the compositions disclosed herein may comprise 4 framework regions and 3 complementarity determining regions, or any suitable fragment thereof (which will generally contain at least some of the amino acid residues forming at least one complementarity determining region). Particularly, the at least one polypeptide comprised in the compositions disclosed herein is amenable to production in high yields, preferably in a microbial recombinant expression system, and is convenient for subsequent isolation and / or purification.

[0316] According to certain embodiments, the invention provides stretches of a number of amino acid residues (i.e., small peptides) that are particularly suitable for binding to a pest antigen or pest target, such as but not limited to a fungal antigen or fungal target.

[0317] These stretches of amino acid residues may be present in and / or incorporated into the polypeptides disclosed herein, particularly in such a way that they form (a part of) the antigen-binding site of the polypeptide. Since these stretches of amino acid residues are initially generated as CDR sequences of an antibody (such as a heavy chain antibody) or CDR sequences of a V H or V HH sequence generated against a pest target (or may be based on and / or derived from such CDR sequences, as further described herein), they are also generally referred to herein as "CDR sequences" (i.e., CDR1 sequence, CDR2 sequence, and CDR3 sequence, respectively). However, it should be noted that the invention in its broadest sense is not limited to the specific structural role or function that these stretches of amino acid residues may have in the polypeptides disclosed herein, as long as these stretches of amino acid residues allow the polypeptides as disclosed herein to specifically bind to a pest target (such as a fungal antigen or fungal target). Thus, generally speaking, the invention in its broadest sense relates to agrochemical compositions comprising a polypeptide capable of binding to a pest target, such as a fungal antigen or fungal target, and the polypeptide comprises a combination of CDR sequences as described herein.

[0318] Thus, in particular but non-limiting embodiments, the polypeptides disclosed herein can be polypeptides comprising at least one amino acid sequence selected from the CDR1 sequences, CDR2 sequences, and CDR3 sequences described herein. In particular, the polypeptides disclosed herein can comprise at least one antigen-binding site, wherein the antigen-binding site comprises at least one combination of the CDR1 sequences, CDR2 sequences, and CDR3 sequences described herein.

[0319] Any polypeptide comprising one of these CDR sequence combinations and included in an agrochemical composition as disclosed herein is preferably such that it can specifically bind (as defined herein) to a pest target or pest antigen, and more particularly such that it specifically binds to a target of a plant pathogen, especially where the dissociation constant (Kd) of the polypeptide in solution is 10 -8 molar / liter or lower.

[0320] The dissociation constant (Kd) can be estimated based on the results of an ELISA. In the equilibrium analysis in an ELISA, the Kd can be calculated from the equilibrium binding reaction. In the case where the ELISA plate wells are coated with a target antigen and the target antigen can be a lipid-containing fraction of the Botrytis cinerea membrane, such a method can further use a series of concentrations of the polypeptide that binds to the target antigen. In the case where an ELISA typically provides a quantitative adsorption measurement for each polypeptide concentration representative of the binding of the polypeptide to the target antigen, the concentration range of the polypeptide can be, for example, 0.5, 1, 2.5, 5, and 10 μM. The most suitable concentration range used can vary depending on the affinity of the polypeptide for the target antigen. When the absorbance values are plotted against the logarithm of the polypeptide concentration, the corresponding absorbance values determined by ELISA can produce an S-shaped curve. This S-shaped curve can be used to define the IC 50 value. In the case where the IC 50 is the concentration of the polypeptide at which the corresponding absorbance value is 50% of the saturation value estimated by the maximum value of the S-shaped function, the Kd can be estimated by 1 / Ka, and the association constant (Ka) can be estimated as 1 / IC 50 . Thus, the Kd corresponds to the analyte concentration at which equilibrium is reached at 50% binding saturation. Conventional calculation methods can be used for these calculations. For example, this can be done using GraphPad. In some embodiments, the Kd can be determined by surface plasmon resonance (SPR).

[0321] The IC 50 can be the IC 50 that inhibits, for example, the spore germination and / or mycelial growth of Fusarium oxysporum and / or Botrytis cinerea (i.e., the concentration (μM) that inhibits 50% of the spore germination and / or mycelial growth). In some embodiments, the polypeptide has an IC 50 for inhibiting spore germination and / or mycelial growth that is less than about 10 μM (e.g., less than about 1 μM).

[0322] Kd can be the Kd that binds to the lipid-containing fraction, which can be obtained as described elsewhere herein (i.e., by chromatography and can be a lipid-containing fraction from fungi such as Fusarium oxysporum or Botrytis cinerea). The Kd of the polypeptide can be less than about 10 μM, for example less than about 1 μM. Kd can be determined according to any suitable method. For example, Kd can be determined by Biolayer Interferometry (BLI), such as on an Octet. The assay for determining Kd can be an ELISA assay.

[0323] Specific binding of the polypeptide to the pest target can be determined in any suitable manner known per se, including, for example, biopanning, Scatchard analysis, and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assays, and different variants thereof known in the art.

[0324] In a preferred embodiment, a polypeptide of 80 to 200 amino acids is obtained by affinity selection against a specific pest target molecule, and the polypeptide has a high affinity for the pest target molecule: generally, the dissociation constant of the binding between the polypeptide and its pest target molecule is less than 10 -5 M, more preferably the dissociation constant is less than 10 -6 M, even more preferably the dissociation constant is less than 10 -7 M, and most preferably the dissociation constant is less than 10 -8 M.

[0325] In a specific embodiment, the at least one polypeptide comprised in the compositions disclosed herein has a minimum inhibitory concentration (MIC) value of 1.0 μg / mL or lower for the variable domain against the phytopathogenic fungi in solution.

[0326] The present invention also discloses polypeptides having 80 to 200 amino acids or sub-ranges as previously disclosed herein, which are obtained by affinity selection for specific plant pest targets and are capable of inhibiting the growth and / or activity of crop pests at a minimum inhibitory concentration of about 0.00001 to 1 μM. In specific embodiments, the minimum inhibitory concentration is 0.0001 to 1 μM, 0.001 to 1 μM, 0.01 to 1 μM, 0.1 to 1 μM, 0.0001 to 0.1 μM, 0.001 to 0.1 μM, 0.01 to 0.1 μM, 0.00001 to 0.01 μM, 0.0001 to 0.01 μM or 0.001 to 0.01 μM. In other specific embodiments, the minimum inhibitory concentration is about 0.0001 to about 1 μM, about 0.001 to about 1 μM, about 0.01 to about 1 μM, about 0.1 to about 1 μM, about 0.0001 to about 0.1 μM, about 0.001 to about 0.1 μM, about 0.01 to about 0.1 μM, about 0.00001 to about 0.01 μM, about 0.0001 to about 0.01 μM, or about 0.001 to about 0.01 μM.

[0327] The minimum inhibitory concentration or MIC value is the lowest concentration of an agent (e.g., polypeptide) that inhibits visible growth of a crop or plant pest after incubation. For example, the minimum fungicidal concentration (MFC) is considered to be the lowest concentration of a polypeptide that prevents growth of a fungal inoculum and reduces it by at least 99.90% within 24 hours. The MFC (minimum fungal concentration) can be determined on an agar plate, but can also be conveniently assayed in liquid (e.g., in a microplate), depending on the type of fungus and the assay conditions.

[0328] In a further specific embodiment, the composition as disclosed herein comprises at least a polypeptide, which:

[0329] comprises a combination of a CDR1 region having the sequence shown in SEQ ID NO:52, a CDR2 region having the sequence shown in SEQ ID NO:68, and a CDR3 region having the sequence shown in SEQ ID NO:84 (and which is capable of binding to a fungus); or

[0330] comprises a combination of a CDR1 region having the sequence shown in SEQ ID NO:53, a CDR2 region having the sequence shown in SEQ ID NO:69, and a CDR3 region having the sequence shown in SEQ ID NO:85 (and which is capable of binding to a fungus); or

[0331] comprises a combination of a CDR1 region having the sequence shown in SEQ ID NO:54, a CDR2 region having the sequence shown in SEQ ID NO:70, and a CDR3 region having the sequence shown in SEQ ID NO:86 (and which is capable of binding to a fungus); or

[0332] a combination comprising a CDR1 region having a sequence selected from SEQ ID NOs: 52 to 67 and 112 to 122, a CDR2 region having a sequence selected from SEQ ID NOs: 68 to 83 and 123 to 133, and a CDR3 region having a sequence selected from SEQ ID NOs: 84 to 100 and 134 to 144 (and which is capable of binding to a fungus); or

[0333] an amino acid sequence selected from SEQ ID NOs: 1 to 51 or having at least about 80% sequence identity with any one thereof (and the polypeptide is capable of binding to a fungus).

[0334] In a specific embodiment, the polypeptide in the compositions disclosed herein is a heavy chain variable domain that comprises, consists of, or consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively); or any suitable fragment of such a heavy chain variable domain (which will generally contain at least some amino acid residues that form at least one CDR, as further described herein). The sequences of the framework regions can be variable or they can be specified.

[0335] The polypeptides disclosed herein can in particular be antibodies, such as heavy chain antibodies. In a further specific embodiment, the polypeptides disclosed herein can be the heavy chain variable domain sequences of antibodies derived from conventional four-chain antibodies (such as but not limited to the V H sequences) or are the so-called V HH -sequences (as defined herein) derived from so-called "heavy chain antibodies" (as defined herein).

[0336] In a specific embodiment, the compositions disclosed herein comprise at least a heavy chain variable domain sequence derived from an antibody or a functional fragment thereof, such as but not limited to a camelid heavy chain antibody or a functional fragment thereof, and the variable domain sequence can thus be, for example, the heavy chain variable domain (V HH ) of a camelid heavy chain antibody.

[0337] However, it should be noted that the present invention does not limit the source of the polypeptide (or the nucleotide sequence of the present invention for expressing it) included in the compositions disclosed herein, nor the manner in which the polypeptide or its nucleotide sequence has been (or is) produced or obtained. Thus, the polypeptides in the compositions disclosed herein can be naturally occurring polypeptides (from any suitable species) or synthetic or semi-synthetic polypeptides. In specific but non-limiting embodiments of the present 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, and immunoglobulin sequences that have been obtained by a variety of techniques such as affinity maturation (starting, for example, from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, chimerization, combining fragments from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to those skilled in the art; or any suitable combination of any of the foregoing.

[0338] The polypeptide sequences of the compositions disclosed herein can in particular be domain antibodies (or heavy chain variable domains suitable for use as domain antibodies), single domain antibodies (or heavy chain variable domains suitable for use as single domain antibodies) or "dAbs" (or heavy chain variable domains suitable for use as dAbs); other single variable domains or any suitable fragment of any of them. For a general description of (single) domain antibodies, reference is also made to the prior art cited above and EP 0 368 684. For the term "dAb", reference is made, for example, to Ward et al. (Nature 1989 Oct 12; 341(6242):544-6); Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and, for example, WO 06 / 030220, WO 06 / 003388 and other published patent applications of Domantis Ltd.

[0339] Thus, in certain embodiments, the present invention provides polypeptides having the (general) structure

[0340] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0341] wherein FR1 to FR4 refer to framework regions 1 to 4 respectively, wherein CDR1 to CDR3 refer to complementary determining regions 1 to 3 respectively, and are further as defined herein.

[0342] In particular, in some specific embodiments, the present invention provides an agrochemical composition comprising at least one polypeptide that is resistant to a pest target (such as a fungal target) and has at least 70%, at least 75%, at least 80%, preferably at least 85% (such as at least 90% or at least 95% or at least 98%) sequence identity or higher sequence identity with at least one of SEQ ID NOs: 1 to 51, as well as a nucleic acid sequence encoding such an amino acid sequence.

[0343] Some particularly preferred polypeptide sequences as disclosed herein are polypeptide sequences that can bind to and / or be resistant to pests (such as fungi) and have at least 90% (such as at least 95% or at least 97%) amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 1 to 51, wherein any changes in the sequence occur only in the CDR regions compared to the reference sequence (i.e., the designated SEQ ID NO sequence). Some particularly preferred polypeptide sequences as disclosed herein are polypeptide sequences that can bind to and / or be resistant to pests (such as fungi) and have at least 90% (such as at least 95% or at least 97%) amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 1 to 51, wherein any changes in the sequence occur only in the framework regions compared to the reference sequence (i.e., the designated SEQ ID NO sequence). In other embodiments, the changes in the sequence can occur in the CDR regions and / or framework regions compared to the reference sequence (i.e., the designated SEQ ID NO sequence). In some embodiments, the changes in the sequence can occur in the CDR3 region compared to the reference sequence (i.e., the designated SEQ ID NO sequence).

[0344] Furthermore, such polypeptides can be derived in any suitable manner and from any suitable source, and can be, for example, naturally occurring V HH sequences (i.e., from a suitable camelid species) or synthetic or semi-synthetic heavy chain variable domains, including but not limited to "camelized" immunoglobulin sequences (especially camelized heavy chain variable domain sequences), and those obtained by techniques such as affinity maturation (starting, for example, from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, chimerization, combining fragments from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques well known to those skilled in the art for engineering immunoglobulin sequences; or any suitable combination of any of the foregoing as further described herein.

[0345] It should be understood that the agrochemical or biocontrol compositions disclosed herein are stable during storage and use, which means that the integrity of the agrochemical composition is maintained under the storage and / or utilization conditions of the agrochemical composition, and such conditions may include elevated temperature, freeze-thaw cycles, changes in pH or ionic strength, UV irradiation, the presence of harmful chemicals, etc. More preferably, polypeptides having 80 to 200 amino acids and various sub-ranges as described herein remain stable in the agrochemical composition, which means that the integrity and insecticidal activity of the polypeptide are maintained under the storage and / or utilization conditions of the agrochemical composition, and such conditions may include elevated temperature, freeze-thaw cycles, changes in pH or ionic strength, UV irradiation, the presence of harmful chemicals, etc. Most preferably, the 80 to 200 amino acids and various sub-ranges of polypeptides as described herein remain stable in the agrochemical composition when the agrochemical composition is stored at ambient temperature for two years or when the agrochemical composition is stored at 54 °C for two weeks. Preferably, the agrochemical composition of the present invention retains at least about 70% of its activity, more preferably at least about 80% of its activity, and most preferably at least about 90% or higher of its activity. Optionally, the polypeptide can be incorporated in a carrier as defined to protect the polypeptide from harmful effects caused by other components in the agrochemical composition or from harmful effects during storage or application. Examples of suitable carriers include, but are not limited to, alginates, gums, starches, β-cyclodextrins, celluloses, polyureas, polyurethanes, polyesters, microbial cells, or clays.

[0346] The agrochemical composition can exist in any type of formulation, and preferred formulations are powders, wettable powders, wettable granules, water-dispersible granules, emulsions, emulsifiable concentrates, powders, suspensions, suspension concentrates, suspoemulsions (mixtures of suspensions and emulsions), capsule suspensions, aqueous dispersions, oil dispersions, aerosols, pastes, foams, slurries, or flowable concentrates.

[0347] Polypeptides of 80 to 200 amino acids and the various sub-ranges described above can be the sole active substances in the agrochemical or biocontrol compositions according to the invention; however, in addition to the polypeptide or amino acid sequence (or at least one, at least two or at least three of the disclosed polypeptide or amino acid sequences), the agrochemical compositions may also contain one or more additional agrochemicals as defined. Such additional agrochemical or biocontrol compositions may have a different effect on plant pests than the polypeptide or amino acid sequence, they may have a synergistic effect with the polypeptide or amino acid sequence, or they may even alter the activity of the polypeptide or amino acid sequence on certain plants. Suitable additional agrochemicals can be herbicides, pesticides, fungicides, nematicides, acaricides, bactericides, antivirals, plant growth regulators, safeners, etc. Such agrochemicals can be chemical substances or can be biological substances, such as microorganisms. They include but are not limited to glyphosate, paraquat, metolachlor, acetochlor, mesotrione, 2,4-D, atrazine, glufosinate, bialaphos, fenoxaprop-ethyl, pendimethalin, picloram, trifluralin, bromoxynil, clodinafop-propargyl, fluroxypyr, nicosulfuron, bensulfuron-methyl, imazethapyr, dicamba, imidacloprid, thiamethoxam, fipronil, chlorpyrifos, deltamethrin, lambda-cyhalothrin, endosulfan, methamidophos, carbofuran, clothianidin, cypermethrin, avermectin, flufenican, spinosad, indoxacarb, bifenthrin, tefluthrin, azoxystrobin, thiamethoxam, tebuconazole, mancozeb, cyazofamid, fluazinam, pyraclostrobin, epoxiconazole, chlorothalonil, copper fungicides (such as copper oxychloride, copper hydroxide), trifloxystrobin, prothioconazole, difenoconazole, carbendazim, propiconazole, thiophanate-methyl, sulfur, boscalid, tricyclazole, hexaconazole, metalaxyl, benomyl, kitazin, propineb, streptomycin sulfate and oxytetracycline and other known agrochemicals or any suitable combination thereof.

[0348] Suitable additional agrochemicals can be biological substances, such as microorganisms, such as Pseudomonas strains, Bacillus strains or Streptomyces strains.

[0349] Composition Comprising a Polypeptide Sequence Variant

[0350] In some aspects, the polypeptides included in the agrochemical compositions as disclosed herein can optionally be modified, for example, to increase the amount of positive charge (that the polypeptide has). That is, the polypeptide can be modified, typically by one or more amino acid substitutions, such that the positive charge amount of the polypeptide can be increased. Thus, an amino acid can thus be replaced with an amino acid having an increased positive charge amount (compared to the amino acid it replaces). More than one such substitution can be made, for example, two, three, four, or five such substitutions. In some embodiments, up to one, up to two, up to three, up to four, or up to five such substitutions can be made. Such substitutions can generally be made in the CDR regions, such as the CDR1 region, the CDR2 region, or the CDR3 region.

[0351] Other substitutions can also be made to the polypeptide. For example, substitutions that have no overall effect on the charge of the polypeptide can be made. Advantageously, the substitutions do not reduce the total charge of the polypeptide, as the inventors have surprisingly found that a higher positive charge may be associated with improved antifungal effects.

[0352] Other substitutions are also contemplated. For example, the polypeptide can start with a D residue or a Q residue. The inventors have surprisingly found that having a D residue at position 1 of the polypeptide (i.e., the first residue of the framework 1 region sequence) can improve the antifungal properties of the polypeptide, although Q can also be used. Thus, for any particular polypeptide sequence disclosed herein (including all peptides having any one of the sequences of SEQ ID NO: 1 to 51 or 101 to 111), the residue at position 1 can be a Q residue, or, preferably for some embodiments, a D residue. A reference polypeptide such as 10G11Q indicates that the polypeptide starts with a Q residue. A reference polypeptide such as 10G11 (without the Q suffix) indicates that the polypeptide starts with a D residue. 10G11 can alternatively be referred to herein as 10G11Q1D (an annotation indicating the substitution from Q to D at position 1).

[0353] In some embodiments, the present invention provides a polypeptide comprising or consisting of an amino acid sequence selected from: SEQ ID NO: 1 to 51, or having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% identity to any one of SEQ ID NO: 1 to 51. Any polypeptide having a specific % sequence identity to a given SEQ ID NO can have the same total charge as the reference sequence, or can have a higher positive charge. Advantageously, any polypeptide having a specified % sequence identity to a given SEQ ID NO does not have more negative charge than the reference sequence.

[0354] In some embodiments, the present invention provides a polypeptide comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 51, or an amino acid sequence having at most 1, at most 2, at most 3, at most 4, or at most 5 amino acid substitutions with respect to SEQ ID NOs: 1 to 51.

[0355] In some embodiments, the present invention provides a polypeptide comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 10 and 12 to 51, or an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% identity with any one thereof. Any polypeptide having a specified % sequence identity with a given SEQ ID NO may have the same total charge as the reference sequence, or may have a higher positive charge. Advantageously, any polypeptide having a specified % sequence identity with a given SEQ ID NO does not have more negative charge than the reference sequence.

[0356] In some embodiments, the present invention provides a polypeptide comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 10 and 12 to 51, or an amino acid sequence having at most 1, at most 2, at most 3, at most 4, or at most 5 amino acid substitutions with respect to SEQ ID NOs: 1 to 10 and 12 to 51.

[0357] In some embodiments, the present invention provides a polypeptide comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 6, or an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% identity with any one thereof. Any polypeptide having a specified % sequence identity with a given SEQ ID NO may have the same total charge as the reference sequence, or may have a higher positive charge. Advantageously, any polypeptide having a specified % sequence identity with a given SEQ ID NO does not have more negative charge than the reference sequence.

[0358] In some embodiments, the present invention provides a polypeptide comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 6, or an amino acid sequence having at most 1, at most 2, at most 3, at most 4, or at most 5 amino acid substitutions with respect thereto.

[0359] Any amino acid substitution in the polypeptide may increase the total charge of the polypeptide or may not change the total charge of the polypeptide. In some embodiments, any amino acid substitution does not decrease the total charge of the polypeptide.

[0360] Any amino acid substitution in the polypeptide can occur at any position in the polypeptide sequence. Optionally, the amino acid substitution can be limited to the CDR regions (and in such embodiments, the polypeptide retains the original framework region sequence), or can be limited to the CDR regions. In some embodiments, any substitution or variation in the sequence can occur in the CDR regions or at any residue up to 2 amino acid residues on either side of the CDR regions (as defined by the Kabat numbering system). In some embodiments, any substitution or variation in the sequence can occur in the CDR regions or at any residue up to 1 amino acid residue on either side of the CDR regions (as defined by the Kabat numbering system). In some embodiments, any substitution or variation in the sequence can occur in the CDR regions or at a residue adjacent to the N-terminus of the CDR3 region (as defined by the Kabat numbering system).

[0361] The present invention provides specific mutants, referred to herein as Mutants 1 to 5 (wherein the total charge of the polypeptide is altered to determine the functional effect on the polypeptide) and mutants referred to herein as "single ALA mutants" 1 to 34, which have been subjected to alanine scanning, although substitutions other than alanine are made at certain positions. The present invention also extends to additional mutant or variant forms of the disclosed polypeptide sequences, such as having additional substitutions or having different combinations of substitutions, or having substitutions at different positions in the polypeptide sequence. Suitably, any mutant or variant of the disclosed polypeptide sequence does not have a reduced positive charge compared to the reference sequence. In some cases, the mutant or variant of the disclosed polypeptide sequence may have an increased overall positive charge.

[0362] The total charge of the polypeptide can be calculated before and after any substitution or variation, wherein the charge of the polypeptide is calculated in an aqueous solution at the same pH before and after the substitution or variation introduced into the sequence.

[0363] The total charge of the polypeptide can be calculated according to any suitable method known to those skilled in the art. For example, the charge can be calculated according to freely available online tools, such as ExPASy–ProtParam (https: / / web.expasy.org / protparam / ).

[0364] Preferably, the charge of the polypeptide is calculated at pH 7. Generally, the polypeptide will have a total positive charge (at pH 7). The substitution or variation in the sequence preferably does not reduce the total charge (at pH 7). In some embodiments, the substitution or variation in the sequence will increase the total positive charge of the polypeptide (at pH 7).

[0365] The charge or total charge of a polypeptide will affect the isoelectric point (pI) of the polypeptide. The isoelectric point of a polypeptide is the pH at which the molecule has no net charge. Typically, polypeptides will have a pI greater than 7, indicating that they have an overall positive charge at pH 7. Substitutions or changes in the sequence are preferably not such as to lower the pI. In some embodiments, substitutions or changes in the sequence will increase the pI.

[0366] The present invention also provides polypeptides (and compositions comprising polypeptides) having certain sequences that permit mutations or substitutions at specific positions. Such mutants include those referred to herein as 10G11-A through 10G11-K.

[0367] For example, in one embodiment, the present invention provides a polypeptide comprising the following sequence:

[0368] X1VQLVESGGGLVQAGGSLRLSCAAS X2X3X4FX5INAMD WYRQAPGKQREWVA GITX6GGTTX7 YADSVKGRFTISRDNAKKKVYLQMNSLKPEDTAVYYCNV LX8GEQPX9X 10 X 11 DY WGQGTQVTVSS

[0369] wherein X1 is D or Q and each of X2 through X 11 is independently any naturally occurring amino acid (SEQ ID NO: 101, also referred to herein as 10G11-A).

[0370] In some embodiments, X1 is D or Q, and each of X2 through X 11 is independently any naturally occurring amino acid, except E or D (SEQ ID NO: 102, also referred to herein as 10G11-B).

[0371] In some embodiments, X1 is D or Q, X2, X6, X7, X8, and X 11 are each independently G, A, V, M, L, I, K, R, or H and each of X3, X4, X5, X9, and X 10 is independently any naturally occurring amino acid, except E or D (SEQ ID NO: 103, also referred to herein as 10G11-C).

[0372] In some embodiments, X1 is D or Q, X2, X6, X7, X8, and X 11 are each independently A, K, R, or H and each of X3, X4, X5, X9, and X 10 is independently any naturally occurring amino acid, except E or D (SEQ ID NO: 104, also referred to herein as 10G11-D).

[0373] In some embodiments, X1 is D or Q, and each of X2, X6, X7, X8, and X 11 is independently A, K, R, or H, each of X3, X5, and X 10 is independently a polar uncharged or positively charged amino acid (i.e., S, T, C, P, N, Q, K, R, or H), X4 is a nonpolar aliphatic or positively charged amino acid (i.e., I, G, A, V, M, L, K, R, or H), and X9 is an aromatic or positively charged amino acid (i.e., W, F, Y, K, R, or H) (SEQ ID NO: 105, also referred to herein as 10G11-E).

[0374] In some embodiments, X1 is D or Q, and each of X2, X6, X7, X8, and X 11 is independently A, K, R, or H, and each of X3 and X5 is independently S, K, R, or H, X4 is I, K, R, or H, X9 is W, K, R, or H, and X 10 is T, K, R, or H (SEQ ID NO: 106, also referred to herein as 10G11-F).

[0375] In some embodiments, X1 is D or Q, and each of X2, X6, X7, X8, and X 11 is independently, and each of X3, X4, X5, X9, and X 10 is independently (SEQ ID NO: 107, also referred to herein as 10G11-G).

[0376] In some embodiments, X1 is D or Q, and each of X2, X6, X8, and X 11 is R, and each of X3, X4, X5, X9, and X 10 is independently any naturally occurring amino acid (SEQ ID NO: 108, also referred to herein as 10G11-H).

[0377] In some embodiments, X1 is D or Q, and each of X2, X6, X7, X8, and X 11 is R, and each of X3, X4, X5, X9, and X 10 is independently any naturally occurring amino acid, except E or D (SEQ ID NO: 109, also referred to herein as 10G11-I).

[0378] In some embodiments, X1 is D or Q, and each of X2, X6, X8, and X 11 is R, and each of X3, X5, and X 10Each of them is independently a polar uncharged or positively charged amino acid (i.e., S, T, C, P, N, Q, K, R or H), X4 is a nonpolar aliphatic or positively charged amino acid (i.e., I, G, A, V, M, L, K, R or H), X7 is K, and X9 is an aromatic or positively charged amino acid (i.e., W, F, Y, K, R or H) (SEQ ID NO: 110, also referred to herein as 10G11-J).

[0379] In some embodiments, X1 is D or Q, X2, X6, X8 and X 11 are each R, X3, X5 and X 10 are each S, K, R or H, X4 is I, K, R or H, X7 is K, X9 is W, K, R or H, and X 10 is T, K, R or H (SEQ ID NO: 111, also referred to herein as 10G11-K).

[0380] In some embodiments, the present invention provides a polypeptide comprising: a CDR1 region comprising or consisting of the sequence X2X3X4FX5INAMD, a CDR2 region comprising or consisting of the sequence GITX6GGTTX7, and a CDR3 region comprising or consisting of the sequence LX8GEQPX9X 10 X 11 DY, wherein each of X2 to X 11 is independently any naturally occurring amino acid (and thus the CDR1, CDR2 and CDR3 regions have the sequences of SEQ ID NOs: 112, 123 and 134, respectively).

[0381] In some embodiments, wherein each of X2 to X 11 is independently any naturally occurring amino acid other than E or D (and thus the CDR1, CDR2 and CDR3 regions have the sequences of SEQ ID NOs: 113, 124 and 135, respectively).

[0382] In some embodiments, X2, X6, X7, X8 and X 11 are each independently G, A, V, M, L, I, K, R or H, and X3, X4, X5, X9 and X 10 are each independently any naturally occurring amino acid other than E or D (and thus the CDR1, CDR2 and CDR3 regions have the sequences of SEQ ID NOs: 114, 125 and 136, respectively).

[0383] In some embodiments, X1 is D or Q, X2, X6, X7, X8 and X 11 are each independently A, K, R or H and X3, X4, X5, X9 and X10 Each of them is independently any naturally occurring amino acid, except E or D (and thus the CDR1, CDR2, and CDR3 regions have the sequences of SEQ ID NO: 115, 126, and 137, respectively).

[0384] In some embodiments, X2, X6, X7, X8, and X 11 are each independently A, K, R, or H, X3, X5, and X 10 are each independently a polar uncharged or positively charged amino acid (i.e., S, T, C, P, N, Q, K, R, or H), X4 is a nonpolar aliphatic or positively charged amino acid (i.e., I, G, A, V, M, L, K, R, or H) and X9 is an aromatic or positively charged amino acid (i.e., W, F, Y, K, R, or H) (and thus the CDR1, CDR2, and CDR3 regions have the sequences of SEQ ID NO: 116, 127, and 138, respectively).

[0385] In some embodiments, X2, X6, X7, X8, and X 11 are each independently A, K, R, or H, and X3 and X5 are each independently S, K, R, or H, X4 is I, K, R, or H, X9 is W, K, R, or H, and X 10 is T, K, R, or H (and thus the CDR1, CDR2, and CDR3 regions have the sequences of SEQ ID NO: 117, 128, and 139, respectively). (SEQ ID NO: 106).

[0386] In some embodiments, X2, X6, X7, X8, and X 11 each independently is and X3, X4, X5, X9, and X 10 each independently is (and thus the CDR1, CDR2, and CDR3 regions have the sequences of SEQ ID NO: 118, 129, and 140, respectively). (SEQ ID NO: 107).

[0387] In some embodiments, X2, X6, X8, and X 11 each is R, and X3, X4, X5, X9, and X 10 each independently is any naturally occurring amino acid (and thus the CDR1, CDR2, and CDR3 regions have the sequences of SEQ ID NO: 119, 130, and 141, respectively). (SEQ ID NO: 108).

[0388] In some embodiments, X2, X6, X7, X8, and X 11 each is R, and X3, X4, X5, X9, and X 10Each of which is independently any naturally occurring amino acid, except E or D (and thus the CDR1, CDR2, and CDR3 regions have the sequences of SEQ ID NO: 120, 131, and 142, respectively). (SEQ ID NO: 109).

[0389] In some embodiments, each of X2, X6, X8, and X 11 is R, and each of X3, X5, and X 10 is independently a polar uncharged or positively charged amino acid (i.e., S, T, C, P, N, Q, K, R, or H), X4 is a nonpolar aliphatic or positively charged amino acid (i.e., I, G, A, V, M, L, K, R, or H), X7 is K, and X9 is an aromatic or positively charged amino acid (i.e., W, F, Y, K, R, or H) (and thus the CDR1, CDR2, and CDR3 regions have the sequences of SEQ ID NO: 121, 132, and 143, respectively). (SEQ ID NO: 110).

[0390] In some embodiments, each of X2, X6, X8, and X 11 is R, each of X3, X5, and X 10 is independently S, K, R, or H, X4 is I, K, R, or H, X7 is K, X9 is W, K, R, or H, and X 10 is T, K, R, or H (and thus the CDR1, CDR2, and CDR3 regions have the sequences of SEQ ID NO: 122, 133, and 144, respectively). (SEQ ID NO: 111).

[0391] Any polypeptide disclosed herein, including variants thereof, can be included in a composition (such as an agrochemical composition).

[0392] Generally, although the present invention extends to variants of the polypeptide, the polypeptides can retain their functional properties or can have their functional properties improved. For example, the variant may be able to (specifically) bind to a fungus. More specifically, the variant may be able to (specifically) bind to the fungal membrane or a component of the fungal membrane. In some embodiments, the variant may not bind to the fungal cell wall or a component of the cell wall. For example, in some embodiments, the variant polypeptide does not (specifically) bind to glucosylceramide of the fungus.

[0393] The variant may be able to bind to the lipid-containing fraction of the plasma membrane of a fungus (such as Botrytis cinerea or other fungi). The lipid-containing fraction can be obtained by chromatography. For example, the lipid-containing fraction can be obtained by a method that includes:

[0394] Fractionate the mycelium of a fungus (such as Botrytis cinerea or other fungi) by thin layer chromatography of the total lipid extract, and select the fraction with a retention factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipid fraction.

[0395] The variant can also cause a delay in the growth of fungal spores and / or lysis of said fungal spores. That is, the binding of the variant polypeptide to the fungus causes a delay in the growth of the fungal spores and / or lysis of said fungal spores.

[0396] In some embodiments, when measured under substantially the same conditions, the variant can cause a delay in the growth of fungal spores and / or lysis of said fungal spores, and its IC50 is equal to or less than the IC50 of the reference polypeptide. The IC50 can be the concentration (μM) that inhibits 50% of, for example, the spore germination and / or mycelial growth of Fusarium oxysporum.

[0397] In some embodiments, when measured under substantially the same conditions, the variant can (specifically) bind to a fungus (such as the membrane of the fungus or a component of the fungal membrane), and its KD is equal to or less than the KD of the reference polypeptide.

[0398] In some embodiments, when measured under substantially the same conditions, the variant can (specifically) exhibit a MIC for fungal growth that is equal to or less than the MIC of the reference polypeptide.

[0399] In certain aspects, the polypeptide contained in the agrochemical composition as disclosed herein can optionally be linked to one or more further groups, moieties or residues via one or more linkers. These one or more further groups, moieties or residues can be used to bind to other target of interest. It should be clear that such further groups, residues, moieties and / or binding sites may or may not provide further functions to the polypeptide (and / or the composition in which it is present) disclosed herein, and may or may not alter the properties of the polypeptide disclosed herein. Such groups, residues, moieties or binding units can also be, for example, chemically active groups.

[0400] In certain embodiments, these groups, moieties or residues can be linked to the polypeptide in the composition disclosed herein at the N- or C-terminus.

[0401] In certain embodiments, the polypeptide in the agrochemical composition as disclosed herein can also be chemically modified. For example, such modification can involve introducing or linking one or more functional groups, residues or moieties into or onto the heavy chain variable domain. These groups, residues or moieties can endow the polypeptide with one or more desired properties or functions. Examples of such functional groups will be clear to the person skilled in the art.

[0402] For example, introducing or attaching such functional groups to a polypeptide can result in an increase in polypeptide solubility and / or stability, a decrease in polypeptide toxicity, or the elimination or attenuation of any undesirable side effects of the polypeptide, and / or the generation of other favorable properties.

[0403] In certain embodiments, the one or more groups, residues, moieties are attached to the polypeptide via one or more suitable linkers or spacers.

[0404] In further certain embodiments, two or more of the target-specific polypeptides in the agrochemical compositions disclosed herein can be attached to each other or can be interconnected. In certain embodiments, two or more polypeptides are attached to each other via one or more suitable linkers or spacers. Suitable spacers or linkers for conjugating different heavy polypeptides as disclosed herein will be apparent to the person skilled in the art and can generally be any linker or spacer used in the art for linking peptides and / or proteins.

[0405] Some particularly suitable linkers or spacers include, for example but not limited to, polypeptide linkers such as glycine linkers, serine linkers, mixed glycine / serine linkers, glycine- and serine-rich linkers or linkers consisting of mostly polar polypeptide segments or homo- or heterobifunctional chemical crosslinking compounds such as glutaraldehyde or alternatively PEG-spaced maleimides or NHS esters.

[0406] For example, the polypeptide linker or spacer can be a suitable amino acid sequence that is from 1 to 50 amino acids in length, such as from 1 to 30 and especially from 1 to 10 amino acid residues. It should be clear that the length, degree of flexibility and / or other properties of the linker may have some effect on the properties of the polypeptide, including but not limited to the affinity, specificity or avidity for the pest target. It should be clear that when two or more linkers are used, these linkers can be the same or different. In the context and disclosure of the present invention, the person skilled in the art will be able to determine the optimal linker for conjugating the heavy chain variable domains as disclosed herein without any undue experimentation.

[0407] Composition Comprising a Fragment of a Polypeptide Sequence

[0408] The present invention also encompasses parts, fragments, analogs, mutants, variants and / or derivatives of the polypeptides contained in the compositions disclosed herein and / or polypeptides comprising one or more of such parts, fragments, analogs, mutants, variants and / or derivatives or consisting essentially of one or more of the foregoing, provided that these parts, fragments, analogs, mutants, variants and / or derivatives are suitable for the purposes contemplated herein. These parts, fragments, analogs, mutants, variants and / or derivatives according to the present invention are still capable of specifically binding to a pest target, such as a fungus, such as a phytopathogenic fungus.

[0409] Target

[0410] In certain embodiments, the polypeptides included in the compositions disclosed herein are obtained by affinity selection against a specific pest target (e.g., a fungal antigen or fungal target). Obtaining a suitable polypeptide by affinity selection against a specific pest target can be carried out, for example, by screening a group, collection or library of cells that express on their surface polypeptides that bind to the pest target molecule, which is a target of pesticides known in the art; all of these can be carried out in a manner known per se, mainly including the following non-limiting steps: a) obtaining an isolated solution or suspension of the pest target molecule, which is known to be a target of pesticides; b) biopanning phages or other cells from a polypeptide library against the target molecule; c) isolating the phages or other cells that bind to the target molecule; d) determining the nucleotide sequence encoding the polypeptide insert from a single binding phage or other cell; e) producing a quantity of the polypeptide according to the sequence using recombinant protein expression; and f) determining the affinity of the polypeptide for the pest target; and optionally g) testing the pesticidal activity of the polypeptide in a bioassay against the pest. A variety of methods can be used to determine the affinity between a polypeptide and a pest target molecule, including, for example, enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (SPR) assay, which are common practices in the art, for example, as described in Sambrook et al. (2001), Molecular Cloning, A Laboratory Manual. Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. The dissociation constant is typically used to describe the affinity between a polypeptide and its pest target molecule. Generally, the dissociation constant for the binding of a polypeptide to its pest target molecule is less than 10 -5 M, more preferably the dissociation constant is less than 10 -6 M, even more preferably the dissociation constant is less than 10 -7 M, most preferably the dissociation constant is less than 10 -8 M.

[0411] The pest target molecules disclosed herein are molecules present in or on a pest organism and which, when bound and / or inhibited, kill or prevent, inhibit or reduce the growth or pesticidal activity of said pest organism. Such suitable target molecules can be readily obtained from the existing literature or patent databases of those skilled in the art and include, but are not limited to, secreted parasitic proteins such as 16D10 (Huang et al (2006) PNAS 103:14302 - 14306) as suitable pest target molecules for root - knot nematodes, the V - ATPase proton pump (Knight AJ and Behm CA (2011) Ex. Parasitol. Sept 19) as suitable pest target molecules for Coleoptera, Hemiptera, Diptera insect species and nematodes, the transmembrane four - protein PLS1 (Gourgues et al (2002) Biochem. Biophys. Res. Commun. 297:1197) as a suitable fungal pest target molecule for Botrytis cinerea and Magnaporthe oryzae or the proton - pump ATPase as an antifungal target (Manavathu EK et al (1999) Antimicrob Agents and Chemotherapy, Dec p.2950). It should be understood that the preferred pest target molecules are accessible in the extracellular space (as opposed to intracellular pest targets).

[0412] More particularly, at least one polypeptide - binding pest target of the agrochemical compositions disclosed herein can be a plasma membrane component of the pest. As used herein, a plasma membrane component of a pest can be any component of any protein contained within or embedded in the plasma membrane phospholipid bilayer of a pest cell, or can be a part of said plasma membrane phospholipid bilayer or a part of any protein embedded therein (i.e., at least a part thereof is associated with, present in, linked to or bound to the plasma membrane phospholipid bilayer or any protein embedded therein). In a particular embodiment, the plasma membrane component of the pest can be a phospholipid, glycoprotein, carbohydrate or cholesterol.

[0413] In a particular embodiment, the plasma membrane component of the pest to which at least one polypeptide in the compositions disclosed herein specifically binds is not a protein.

[0414] Thus, in a particular embodiment, the plasma membrane component of the pest to which at least one polypeptide in the compositions disclosed herein specifically binds is a lipid, such as a phospholipid, carbohydrate or cholesterol.

[0415] In certain particular embodiments, the target to which the polypeptide in the agrochemical composition of the present invention binds is not a cell - wall component.

[0416] In certain specific embodiments, the target to which the polypeptide in the agrochemical composition of the present invention binds is not chitin.

[0417] In a preferred embodiment, the plant pests controlled by the agrochemical or biocontrol compositions disclosed herein are fungi, e.g., the phytopathogenic fungi as defined above. Fungi are highly detrimental to plants and can cause severe crop yield losses. Phytopathogenic fungi include necrotrophic and biotrophic fungi, including ascomycetes, basidiomycetes, and oomycetes.

[0418] Examples of phytopathogenic fungi are known in the art and include, but are not limited to, those selected from the following genera: Alternaria; Ascochyta; Botrytis; Cercospora; Colletotrichum; Diplodia; Erysiphe; Fusarium; Leptosphaeria; Magnaporthe; Helminthosporium; Sphaeropsis; Nectria; Oidium, Peronospora; Phakopsora; Phoma; Rhynchosporium; Phytophthora; Plasmopara; Podosphaera; Puccinia; Puthium; Pyrenophora; Pyricularia; Pythium; Rhizoctonia; Sclerotium; Sclerotinia; Septoria; Thielaviopsis; Uncinula; Venturia; and Verticillium. Specific examples of plant fungal infections that can be controlled with the agrochemical compositions of the present invention include powdery mildew and Botrytis cinerea in fruit and vegetable crops such as grapes and strawberries. Other specific examples of plant fungal infections that can be controlled with the agrochemical compositions of the present invention include: Erysiphe graminis in cereals, Erysiphe cichoracearum and Sphaerotheca fuliginea in cucurbits, Podosphaera leucotricha in apples, Podosphaera aphanis, e.g., for treating powdery mildew on, e.g., strawberries, Podosphaera xanthii, e.g., for treating powdery mildew on, e.g., cucumbers, Oidium neolycopersici, e.g., for treating powdery mildew on, e.g., tomatoes, Uncinula necator in grapevines, Puccinia graminis in cereals, Rhizoctonia solani in cotton, potatoes, rice and turf, Ustilago esculenta in cereals and sugarcane, Venturia inaequalis (scab) in apples, Helminthosporium sativum in cereals, Septoria nodorum in wheat, Septoria tritici in wheat, Rhynchosporium secalis in barley, Botrytis cinerea (gray mold) in strawberries, tomatoes and grapes, Cercospora arachidicola in peanuts, Peronospora tabacina in tobacco, or other Peronospora spp. in various crops, Pythium arrhenomanes in wheat and barley, Helminthosporium teres in barley, Magnaporthe grisea in rice, Phytophthora infestans in potatoes and tomatoes, Fusarium spp. such as Fusarium oxysporum and Verticillium spp. in various plants, Plasmopara viticola in grapes, Alternaria spp. in fruits and vegetables, Pseudoperonospora cubensis in cucumbers, Mycosphaerella fijiensis in bananas, Ascochyta ciceris in chickpeas, Leptosphaeria maculans in oilseed rape, Phakopsora spp. in various crops such as P. pachyrhizi and Colletotrichum spp., e.g., Colletotrichum orbiculare which can cause anthracnose in pumpkins. The compositions according to the invention are active against normally sensitive and resistant species and against all or some stages in the life cycle of phytopathogenic fungi.

[0419] In certain embodiments, the agrochemical compositions disclosed herein are directed against plant pathogenic fungi from genera selected from the group consisting of: Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Diplodia, Erysiphe, Fusarium, Leptosphaeria, Magnaporthe, Helminthosporium, Macrophomina, Nectria, Oidium, Penicillium, Peronospora, Phoma, Polyphagotarsonemus, Phytophthora, Plasmopara, Podosphaera, Puccinia, Pyrenophora, Pyricularia, Pythium, Rhizoctonia, Sclerotium, Sclerotinia, Septoria, Thielaviopsis, Uncinula, Venturia, Verticillium, Magnaporthe grisea, Blumeria graminis, Mycosphaerella, Ustilago, Melampsora, Phakopsora, Monilinia, Mucor, Rhizopus, and Aspergillus.

[0420] In certain specific embodiments, the compositions disclosed herein comprise at least a polypeptide that specifically binds to a fungal target from a fungal species of the genus Botrytis, Fusarium, or Penicillium, such as a plasma membrane component of the fungus.

[0421] In certain embodiments, the present invention provides an agrochemical composition comprising a polypeptide that specifically targets a structural molecular component of the plasma cell membrane of a pest.

[0422] In certain embodiments, the present invention provides an agrochemical composition comprising a polypeptide that specifically targets a structural molecular component of the plasma cell membrane of a pest, which is not a protein.

[0423] In yet another specific embodiment, the plant pest is a plant pathogenic bacterium, including but not limited to: Acidovorax avenae subsp. avenae (causing bacterial brown stripe of rice), Acidovorax avenae subsp. cattleyae (causing bacterial brown spot of Cattleya), Acidovorax konjaci Konnyaku (causing bacterial leaf blight), Agrobacterium rhizogenes (causing hairy roots of melon), Agrobacterium tumefaciens (causing crown gall), Burkholderia andropogonis (causing bacterial leaf spot of carnation), Burkholderia caryophylli (causing bacterial wilt of carnation), Burkholderia cepacia (causing bacterial brown spot of Cymbidium), Burkholderia gladioli (causing neck rot of gladiolus), Burkholderia plantarii (causing bacterial seedling blight of rice), Clavibacter michiganensis subsp. michiganensis (causing bacterial canker of tomato), Clavibacter michiganensis subsp. sepedonicus (causing potato ring rot), Clostridium spp. (causing potato soft rot), Curtobacterium flaccumfaciens (causing bacterial canker of onion), Erwinia amylovora (causing fire blight of pear), Erwinia oryzae (causing bacterial brown rot of rice), Erwinia carotovora subsp. atroseptica (causing blackleg of potato), Erwinia carotovora subsp. carotovora (causing bacterial soft rot of vegetables), Erwinia chrysanthemi (causing bacterial seedling blight of taro), Erwinia chrysanthemi pv. zeae (causing bacterial foot rot of rice), Erwinia herbicola pv. millettiae (causing bacterial gall of wisteria), Pseudomonas cichorii (causing bacterial leaf spot of chrysanthemum), Pseudomonas corrugata (causing tomato necrosis), Pseudomonas fuscovaginae (causing sheath brown rot of rice), Pseudomonas marginalis pv.marginalis) (causing soft rot of cabbage), Pseudomonas rubrisubalbicans (causing mottled stripes of sugarcane), Pseudomonas syringae pv. aptata (causing bacterial blight of sugar beet), Pseudomonas syringae pv. atropurpurea (causing ryegrass halo blight), Pseudomonas syringae pv. castaneae (causing bacterial canker of chestnut), Pseudomonas syringae pv. glycinea (causing bacterial blight of soybean), Pseudomonas syringae pv. lachrymans (causing bacterial leaf spot of cucumber), Pseudomonas syringae pv. maculicola (causing bacterial black spot of cabbage), Pseudomonas syringae pv. Mori (causing bacterial blight of mulberry), Pseudomonas syringae pv. morsprunorum (causing bacterial canker of plum), Pseudomonas syringae pv. oryzae (causing halo blight of rice), Pseudomonas syringae pv. phaseolicola (causing halo blight of bean), Pseudomonas syringae pv. pisi (causing bacterial blight of pea), Pseudomonas syringae pv. sesame (causing bacterial leaf spot of sesame), Pseudomonas syringae pv. striafaciens (causing bacterial stripe blight of oat), Pseudomonas syringae pv. syringae (causing bacterial brown spot of little red beads), Pseudomonas syringae pv. tabaci (causing tobacco wildfire), Pseudomonas syringae pv. tea (theae) (causing bacterial shoot blight of tea), Pseudomonas syringae pv. tomato (causing bacterial leaf spot of tomato), Pseudomonas viridiflava (causing bacterial brown spot of bean), Ralstonia solanacearum (causing bacterial wilt), Rathayibacter rathayi (causing bacterial head blight of orchard grass), Streptomyces scabies (causing potato scab), Streptomyces ipomoeae (causing soil rot of sweet potato), Xanthomonas albilineans (causing white stripe of sugarcane), Xanthomonas campestris pv. cerealis (causing bacterial streak of rye), Xanthomonas campestris pv. campestris (causing black rot), Xanthomonas campestris pv. citri (causing citrus canker), Xanthomonas campestris pv. cucurbitae (causing bacterial brown spot of cucumber), Xanthomonas campestris pv. glycines (causing bacterial pustule of soybean), Xanthomonas campestris pv. incanae (causing root and stem black rot), Xanthomonas campestris pv. malvacearum (causing angular leaf spot of cotton), Xanthomonas campestris pv. (causing bacterial canker of mango), Mangiferaeindicae Xanthomonas campestris pv. mellea (causing bacterial leaf spot of Wisconsin tobacco), Xanthomonas campestris pv. nigromaculans (causing bacterial leaf spot of great burdock), Xanthomonas campestris pv. phaseoli (causing bacterial pustule of bean), Xanthomonas campestris pv. pisi (causing bacterial stem rot of bean), Xanthomonas campestris pv. pruni (causing bacterial canker of peach and plum), Xanthomonas campestris pv.(Xanthomonas campestris pv. pruni) (causing bacterial leaf spot of peach), Xanthomonas campestris pv. raphani (causing bacterial leaf spot of Japanese radish), Xanthomonas campestris pv. ricini (causing bacterial leaf spot of castor oil plant), Xanthomonas campestris pv. theicola (causing tea canker), Xanthomonas campestris pv. translucens (causing bacterial blight of orchard grass), Xanthomonas campestris pv. vesicatoria (causing bacterial leaf spot of tomato), Xanthomonas oryzae pv. oryzae (causing bacterial leaf blight of rice).

[0424] In yet another embodiment, the agrochemical formulation of the present invention can also be used against plant pests encountered in agriculture, horticulture, forestry, gardens and leisure facilities, such as insects, spiders, worms, viruses, nematodes and mollusks. The compositions according to the invention are active against normally sensitive and resistant species as well as against all or certain developmental stages. These plant pests include: pests from the following phyla: Arthropoda, especially the class from Arachnida, such as Acarus spp., Aceria sheldoni, Aculops pelekassi, Aculus spp., Amblyomma spp., Tetranychus urticae, Argas spp., Boophilus spp., Brevipalpus spp., Bryobia praetiosa, Centruroides spp., Cheyletiella spp., Dermanyssus gallinae, Dermatophagoides pteronyssius, Dermatophagoides farinae, Dermacentor spp., Eotetranychus spp., Epitrimerus pyri, Eriophyes spp., Eutrombicula alfreddugesi, Hemitarsonemus spp., Hyalomma spp., Ixodes spp., Latrodectus spp., Loxosceles spp., Oligonychus spp., Nuphersa spp., Oligonychus spp., Ornithodoros spp., Phytoseiulus persimilis, Polyphagotarsonemus latus, Psoroptes spp., Sarcoptes scabiei, Scorpio maurus palmatus, Stenotarsonemus spp., Tarsonemus spp., Tetranychus spp., Vaejovis spp., Vasates lycopersici.

[0425] There are other examples from the order Phthiraptera (lice), such as Haematopinus spp., Haematopinus suis, Linognathus spp., Pediculus humanus corporis, Ptirus pubis, Trichodectes spp.

[0426] There are other examples from the Chilopoda, such as the genus Geophilus and the genus Scutigera.

[0427] There are also other examples from Coleoptera, such as the striped cucumber beetle (Acalymma vittatum), the bean weevil (Acanthoscelides obtectus), the genus Adoretus spp., the alder leaf beetle (Agelastica alni), the genus Agriotes spp., the lesser mealworm, the powder-post beetle (Amphimallon solstitialis), the furniture beetle (Anobium punctatum), the genus Anoplophora spp., the genus Anthonomus spp., the genus Anthrenus spp., the genus Apion spp., the genus Apogonia spp., the genus Atomaria spp., the genus Attagenus spp., the false bean weevil (Bruchidius obtectus), the genus Bruchus spp., the genus Cassida spp., the bean leaf beetle (Cerotoma trifurcata), the genus Ceutorrhynchus spp., the genus Chaetocnema spp., the humble asparagus weevil (Cleonus mendicus), the genus Conoderus spp., the genus Cosmopolites spp., the New Zealand grass grub (Costelytra zealandica), the genus Ctenicera spp., the genus Curculio spp., the poplar borer (Cryptorhynchus lapathi), the genus Cylindrocopturus spp., the genus Dermestes spp., the genus Diabrotica spp., the genus Dichocrocis spp., Diloboderus spp., the genus Epilachna spp., the genus Epitrix spp., the genus Faustinus spp., the spider beetle (Gibbium psylloides), the diamondback moth (Hellula undalis), the black field cockchafer (Heteronychus arator), the genus Heteronyx spp.)、Hylamorpha elegans, Hylotrupes bajulus (the old house borer), Hypera postica (the alfalfa weevil), Hypothenemus spp. (ambrosia beetles), Lachnosterna consanguinea (the sugarcane beetle), Lema spp. (the cucumber beetles), Leptinotarsa decemlineata (the Colorado potato beetle), Leucoptera spp. (the white leaf miners), Lissorhoptrus oryzophilus (the rice water weevil), Lixus spp. (the broad-nosed weevils), Luperodes spp., Lyctus spp. (powderpost beetles), Megascelis spp. (leaf beetles), Melanotus spp. (the wireworms), Meligethes aeneus (the pollen beetle), Melolontha spp. (the cockchafers), Migdolus spp., Monochamus spp. (the sawyer beetles), Naupactus xanthographus (the citrus root weevil), Niptus hololeucus (the white spider beetle), Oryctes rhinoceros (the coconut rhinoceros beetle), Oryzaephilus surinamensis (the sawtoothed grain beetle), Oryzaphagus oryzae, Otiorrhynchus spp. (the root weevils), Oxycetonia jucunda (the green flower chafer), Phaedon cochleariae (the turnip flea beetle), Phyllophaga spp. (the May / June beetles), Phyllotreta spp. (the flea beetles), Popillia japonica (the Japanese beetle), Premnotrypes spp. (the false wireworms), Prostephanus truncatus (the larger grain borer), Psylliodes spp. (the flea beetles), Ptinus spp. (the spider beetles), Rhizobius ventralis (the false ladybird), Rhizopertha dominica (the lesser grain borer), Sitophilus spp. (the weevils), Sphenophorus spp. (the billbugs), Stegobium paniceum (the drugstore beetle), Sternechus spp. (the stem borers), Symphyletes spp. (the flat-faced longhorn beetles), Tanymecus spp. (the straight-snouted weevils))), Tenebrio molitor, Tribolium spp., Trogoderma spp., Tychius spp., Xylotrechus spp., Zabrus spp.

[0428] There are also some examples from Collembola, such as Onychiurus armatus.

[0429] There are other examples from Diplopoda, such as Blaniulus guttulatus.

[0430] There are also other examples from Diptera, such as Aedes spp., Agromyza spp., Anastrepha spp., Anopheles spp., Asphondylia spp., Bactrocera spp., Bibio hortulanus, Calliphora erythrocephala, Ceratitis capitata, Chironomus spp., Chrysomyia spp., Chrysops spp., Cochliomyia spp., Contarinia spp., Cordylobia anthropophaga, Culex spp., Culicoides spp., Culiseta spp., Cuterebra spp., Dacus oleae, Dasyneura spp., Delia spp., Dermatobia hominis, Drosophila spp., Echinocnemus spp., Fannia spp., Gasterophilus spp., Glossina spp., Haematopota spp., Hydrellia spp., Hylemyia spp., Hyppobosca spp., Hypoderma spp., Liriomyza spp., Lucilia spp., Lutzomia spp., Mansonia spp., Musca spp., Nezara spp., Oestrus spp., Oscinella frit, Pegomyia spp., Phlebotomus spp., Phorbia spp., Phormia spp., Prodiplosis spp., Psila rosae, Rhagoletis spp.) Sarcophaga spp., Simulium spp., Stomoxys spp., Tabanus spp., Tannia spp., Tetanops spp., Tipula spp.

[0431] There are also other examples from the Heteroptera, such as Anasa tristis, Antestiopsis spp., Boisea spp., Blissus spp., Calocoris spp., Campylomma livida, Cavelerius spp., Cimex spp., Collaria spp., Creontiades dilutus, Dasynus piperis, Dichelops furcatus, Diconocoris hewetti, Dysdercus spp., Euschistus spp., Eurygaster spp., Heliopeltis spp., Horcias nobilellus, Leptocorisa spp., Leptoglossus phyllopus, Lygus spp., Macropes excavatus, Miridae, Monalonion atratum, Nezara spp., Oebalus spp., Pentomidae, Piesma quadrata, Piezodorus spp., Psallus spp., Pseudacysta persea, Rhodnius spp., Sahlbergella singularis, Scaptocoris castanea, Scotinophora spp., Stephanitis nashi, Tibraca spp., Triatoma spp.

[0432] There are also other examples from the Homoptera, such as Acyrthosipon spp., Acrogonia spp., Aeneolamia spp., Agonoscena spp., Aleurodes spp., Aleurolobus barodensis, Aleurothrixus spp., Amrasca spp., Anuraphis cardui, Aonidiella spp., Aphanostigma pin, Aphis spp., Arboridia apicalis, Aspidiella spp., Aspidiotus spp., Atanus spp., Aulacorthum solani, Bemisia spp., Brachycaudus helichrysii, Brachycolus spp., Brevicoryne brassicae, Calligypona marginata, Carneocephala fulgida, Ceratovacuna lanigera, Cercopidae, Ceroplastes spp., Chaetosiphon fragaefolii, Chionaspis tegalensis, Chlorita onukii, Chromaphis juglandicola, Chrysomphalus ficus, Cicadulina mbila, Coccomytilus halli, Coccus spp., Cryptomyzus ribis, Dalbulus spp., Dialeurodes spp., Diaphorina spp., Diaspis spp., Drosicha spp., Dysaphis spp., Dysmicoccus spp., Empoasca spp.)、Eriosoma spp., Erythroneura spp., Euscelis bilobatus, Ferrisia spp., Geococcus coffeae, Hieroglyphus spp., Homalodisca coagulata, Hyalopterus arundinis, Icerya spp., Idiocerus spp., Idioscopus spp., Laodelphax striatellus, Lecanium spp., Lepidosaphes spp., Lipaphis erysimi, Macrosiphum spp., Mahanarva spp., Melanaphis sacchari, Metcalfiella spp., Metopolophium dirhodum, Monellia costalis, Monelliopsis pecanis, Myzus spp., Nasonovia ribisnigri, Nephotettix spp., Nilaparvata lugens, Oncometopia spp., Orthezia praelonga, Parabemisia myricae, Paratrioza spp., Parlatoria spp., Pemphigus spp., Peregrinus maidis, Phenacoccus spp., Phloeomyzus passerinii, Phorodon humuli, Phylloxera spp., Pinnaspis aspidistrae, Planococcus spp.) Protopulvinaria pyriformis, Pseudaulacaspis pentagona, Pseudococcus spp., Psylla spp., Pteromalus spp., Pyrilla spp., Quadraspidiotus spp., Quesada gigas, Rastrococcus spp., Rhopalosiphum spp., Saissetia spp., Scaphoides titanus, Schizaphis graminum, Selenaspidus articulatus, Sogata spp., Sogatella furcifera, Sogatodes spp., Stictocephala festina, Tenalaphara malayensis, Tinocallis caryaefoliae, Tomaspis spp., Toxoptera spp., Trialeurodes spp., Trioza spp., Typhlocyba spp., Unaspis spp., Viteus vitifolii, Zygina spp..

[0433] There are also other examples from Hymenoptera, such as Acromyrmex spp., Athalia spp., Atta spp., Diprion spp., Hoplocampa spp., Lasius spp., Monomorium pharaonis, Solenopsis invicta, Tapinoma spp., Vespa spp..

[0434] There are other examples from Isopoda, such as the common pill bug (Armadillidium vulgare), the woodlouse (Oniscus asellus), and the common rough woodlouse (Porcellio scaber).

[0435] There are other examples from Isoptera, such as Coptotermes spp., Cornitermes cumulans, Cryptotermes spp., Incisitermes spp., Microtermes obesi, Odontotermes spp., and Reticulitermes spp.

[0436] There are also other examples from Lepidoptera, such as Acronicta major, Adoxophyes spp., Aedia leucomelas, Agrotis spp., Alabama spp., Amyelois transitella, Anarsia spp., Anticarsia spp., Argyroploce spp., Barathra brassicae, Borbo cinnara, Bucculatrix thurberiella, Bupalus piniarius, Busseola spp., Cacoecia spp., Caloptilia theivora, Capua reticulana, Carpocapsa pomonella, Carposina niponensis, Chematobia brumata, Chilo spp., Choristoneura spp., Clysia ambiguella, Cnaphalocerus spp., Cnephasia spp., Conopomorpha spp., Conotrachelus spp., Copitarsia spp., Cydia spp., Dalaca noctuides, Diaphania spp., Diatraea saccharalis, Earias spp., Ecdytolopha aurantium, Elasmopalpus lignosellus, Eldana saccharina, Ephestia spp., Epinotia spp., Epiphyas postvittana, Etiella spp., Eulia spp., Eupoecilia ambiguella, Euproctis spp., Euxoa spp.) Feltia spp., Galleria mellonella, Gracillaria spp., Grapholitha spp., Hedylepta spp., Helicoverpa spp., Heliothis spp., Hofmannophila pseudospretella, Homoeosoma spp., Homona spp., Hyponomeuta padella, Kakivoria flavofasciata, Laphygma spp., Laspeyresia molesta, Leucinodes orbonalis, Leucoptera spp., Lithocolletis spp., Lithophane antennata, Lobesia spp., Loxagrotis albicosta, Lymantria spp., Lyonetia spp., Malacosoma neustria, Maruca testulalis, Mamestra brassicae, Mocis spp., Mythimna separata, Nymphula spp., Oiketicus spp., Oria spp., Orthaga spp., Ostrinia spp., Oulema oryzae, Panolis flammea, Parnara spp., Pectinophora spp., Perileucoptera spp., Phthorimaea spp., Phyllocnistis citrella, Phyllonorycter spp., Pieris spp.)、Platynota stultana, Plodia interpunctella, Plusia spp., Plutella xylostella, Prays spp., Prodenia spp., Protoparce spp., Pseudaletia spp., Pseudoplusia includens, Pyrausta nubilalis, Rachiplusia nu, Schoenobius spp., Scirpophaga spp., Scotia segetum, Sesamia spp., Sparganothis spp., Spodoptera spp., Stathmopoda spp., Stomopteryx subsecivella, Synanthedon spp., Tecia solanivora, Thermesia gemmatalis, Tinea pellionella, Tineola bisselliella, Tortrix spp., Trichophaga tapetzella, Trichoplusia spp., Tuta absoluta, Virachola spp..

[0437] There are also other examples from Orthoptera, such as Acheta domesticus, Blatta orientalis, Blattella germanica, Dichroplus spp., Gryllotalpa spp., Leucophaea maderae, Locusta spp., Melanoplus spp., Periplaneta spp., Pulex irritans, Schistocerca gregaria, Supella longipalpa.

[0438] There are also other examples from the order Siphonaptera, such as Ceratophyllus spp., Ctenocephalides spp., Tunga penetrans, Xenopsylla cheopis.

[0439] There are also other examples from the class Symphyla, such as Scutigerella spp.

[0440] There are also other examples from the order Thysanoptera, such as Anaphothrips obscurus, Baliothrips biformis, Drepanothrips reuteri, Enneothrips flavens, Frankliniella spp., Heliothrips spp., Hercinothrips femoralis, Rhipiphorothrips cruentatus, Scirtothrips spp., Taeniothrips cardamoni, Thrips spp.

[0441] There are also other examples from the order Zygentoma (=Thysanura), such as Lepisma saccharina, Thermobia domestica. Such as Lepisma saccharina, Thermobia domestica.

[0442] In another embodiment, pests of the phylum Mollusca, particularly pests from the class Bivalvia, such as Dreissena, are also important plant pests.

[0443] In another embodiment, pests of the class Gastropoda are important plant pests, such as Anion spp., Biomphalaria spp., Bulinus spp., Deroceras spp., Galba spp., Lymnaea spp., Oncomelania spp., Pomacea spp., Succinea spp.

[0444] In yet another embodiment, plant pests from the phylum Nematoda are important plant pests, namely plant-parasitic nematodes, which means plant-parasitic nematodes that cause damage to plants. Plant nematodes include plant-parasitic nematodes and nematodes living in the soil. Plant-parasitic nematodes include, but are not limited to, ectoparasites such as Xiphinema spp., Longidorus spp., and Trichodorus spp.; semi-parasites such as Tylenchulus spp.; migratory endoparasites such as Pratylenchus spp., Radopholus spp., and Scutellonerna spp.; sedentary parasites such as Heterodera spp., Globodera spp., and Meloidogyne spp.; and shoot and foliar endoparasites such as Ditylenchus spp., Aphelenchoides spp., and Hirshmaniella spp. In addition, harmful root-parasitic soil nematodes are cyst-forming nematodes of the genus Heterodera or Globodera, and / or root-knot nematodes of the genus Meloidogyne. Harmful species of these genera are, for example, Meloidogyne incognata, Heterodera glycines, Globodera pallida, and Globodera rostochiensis.Other important genera of plant pests include Rotylenchulus spp., Paratriclodorus spp., Pratylenchus penetrans, Radolophus simuli, Ditylenchus dispaci, Tylenchulus semipenetrans, Xiphinema spp., Bursaphelenchus spp., etc. In particular, Aphelenchoides spp., Bursaphelenchus spp., Ditylenchus spp., Globodera spp., Heterodera spp., Longidorus spp., Meloidogyne spp., Pratylenchus spp., Radopholus similis, Trichodorus spp., Tylenchulus semipenetrans, Xiphinema spp.

[0445] In yet another embodiment, the plant pest is a virus and the agrochemical formulation of the invention relates to treating or inhibiting viral infectivity in plants, said plant viruses being selected from alfamovirus, allexivirus, alphacryptovirus, anulavirus, apscaviroid, aureusvirus, avenavirus, aysunviroid, badnavirus, begomovirus, benyvirus, betacryptovirus, betaflexiviridae, bromovirus, bymovirus, capillovirus, carlavirus, carmovirus, caulimovirus, cavemovirus, cheravirus, closterovirus, cocadviroid, coleviroid, comovirus, crinivirus, cucumovirus, curtovirus, cytorhabdovirus, dianthovirus, enamovirus, umbravirus and B-type satellitevirus), Fabavirus, Fijivirus, Furovirus, Hordeivirus, Hostuviroid, Idaeovirus, Ilarvirus, Ipomovirus, Luteovirus, Machlomovirus, Macluravirus, Marafivirus, (Mastrevirus), Nanovirus, Necrovirus, Nepovirus, Nucleorhabdovirus, Oleavirus, Ophiovirus, Oryzavirus, Panicovirus, Pecluvirus, Petuvirus, Phytoreovirus, Polerovirus, Pomovirus, Pospiviroid, Potexvirus, Potyvirus, Reovirus, Rhabdovirus, Rymovirus, Sadwavirus, SbCMV-like virus, Sequivirus, Sobemovirus, Tenuivirus, TNsatV-like satellite virus, Tobamovirus, Topocuvirus, Tospovirus, Trichovirus, Tritimovirus, Tungrovirus, Tymovirus, Umbravirus, Varicosavirus, Vitivirus, or Waikavirus.

[0446] Form of the Target Antigen

[0447] It will be understood from the disclosure herein that for agrochemical and biocontrol applications, the polypeptides of the compositions disclosed herein can target or specifically bind to a variety of different forms of pest targets, such as fungal targets. It is also contemplated that the polypeptides of the compositions disclosed herein will bind to many naturally occurring or synthetic analogs, variants, mutants, alleles, parts, and fragments of their pest targets. More specifically, it is contemplated that the polypeptides of the compositions disclosed herein will bind to at least those analogs, variants, mutants, alleles, parts, and fragments of the targets that still contain the binding sites, parts, or domains of the natural targets to which those polypeptides bind.

[0448] Formulation

[0449] It is envisioned that the polypeptide content in the agrochemical or biocontrol compositions disclosed herein can vary within a wide range and is typically modified by the manufacturer according to the specific crop pests to be attenuated, with the concentration range of a specific polypeptide being adjusted accordingly.

[0450] In a particular embodiment, the present invention provides an agrochemical composition comprising at least one polypeptide, wherein the heavy chain variable domain is present in an amount effective to protect or treat a plant or a part of the plant from infection or other biological interactions with a plant pathogen.

[0451] In one specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be at least 0.0001% by weight.

[0452] In one specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be as high as 50% by weight.

[0453] In one specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 0.0001% by weight to 50% by weight.

[0454] In a particular embodiment, the present invention provides an agrochemical composition comprising at least one polypeptide, wherein the concentration range of at least one polypeptide in the agrochemical composition is from 0.001% by weight to 50% by weight.

[0455] In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 0.001% by weight to 50% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 0.01% by weight to 50% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 0.1% by weight to 50% by weight.

[0456] In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 1% to 50% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 10% to 50% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 0.0001% to 40% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 0.001% to 40% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 0.01% to 40% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 0.1% to 40% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 1% to 40% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 0.0001% to 30% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 0.001% to 30% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 0.01% to 30% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 0.1% to 30% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 1% to 30% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 0.0001% to 10% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 0.001% to 10% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 0.01% to 10% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 0.1% to 10% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 1% to 10% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 0.0001% to 1% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 0.001% to 1% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 0.01% to 1% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the agrochemical composition can be from 0.1% to 1% by weight.

[0457] In certain embodiments, the agrochemical compositions disclosed herein comprise at least one polypeptide formulated in an aqueous solution.

[0458] In further specific embodiments, the agrochemical compositions disclosed herein comprise at least one polypeptide and also comprise an agriculturally chemically suitable carrier and / or one or more suitable adjuvants.

[0459] In addition to the above-mentioned pest-resistant polypeptides, the compositions according to the invention may comprise a solid or liquid carrier acceptable in the treatment of pests of plants and / or plant parts and / or a surfactant also acceptable in the treatment of pests of plants and / or plant parts. In particular, inert and commonly used carriers and commonly used surfactants can be used. These compositions include not only compositions ready to be applied to plants and / or plant parts to be treated by dipping or using suitable devices, but also commercial concentrated compositions that must be diluted before being applied to plants and / or plant parts.

[0460] These agrochemical compositions according to the invention may also contain any other kind of ingredients, such as protective colloids, binders, thickeners, thixotropic agents, penetrants, stabilizers, chelating agents, texturing agents, flavoring agents, flavor enhancers, sugars, sweeteners, coloring agents, etc. More generally, the active substance, i.e., the at least one heavy chain variable domain, can be combined with any solid or liquid additive corresponding to usual formulation techniques.

[0461] These agrochemical compositions according to the invention may also contain any other kind of active ingredients, such as other antibacterial or antifungal active ingredients.

[0462] In the present disclosure, the term "carrier" means a natural or synthetic organic or inorganic substance with which the insecticidal active substance is combined to facilitate its application to plants and / or one or more plant parts. Thus, such a carrier is usually inert and should be acceptable in the agricultural sector. The carrier can be solid (clay, natural or synthetic silicate, silica, resin, wax, solid fertilizer, etc.) or liquid (water, alcohol, especially butanol, etc.).

[0463] The surfactant can be an ionic or non-ionic emulsifier, dispersant or wetting agent or a mixture of these surfactants. For example, mention may be made of salts of polyacrylic acid, salts of lignosulfonic acid, salts of phenol sulfonic acid or naphthalene sulfonic acid, condensates of ethylene oxide with fatty alcohols or with fatty acids or with fatty amines, substituted phenols (especially alkylphenols or arylphenols), salts of sulfosuccinates, derivatives of taurine (especially taurine alkyl esters), phosphates of polyoxyethylated phenols or alcohols, esters of fatty acids and polyols, derivatives of the above compounds containing sulfate, sulfonate and phosphate functional groups. When the inert carrier is insoluble in water and the carrier agent used for application is water, the presence of at least one surfactant is usually essential.

[0464] The agrochemical compositions disclosed herein are themselves quite diverse in solid or liquid form.

[0465] As solid composition forms, mention may be made of dustable powders (the content of active substance can be up to 100%) and granules, in particular those obtained by extrusion, by compaction, by impregnating granular carriers, by granulating using powders as starting materials (the content of active substance in these granules is 0.5% to 80% in the latter case). Such solid compositions can optionally be used in the form of a liquid, the viscosity of which more or less depends on the desired type of application, for example by dilution in water.

[0466] As liquid composition forms or forms intended to constitute a liquid composition during application, mention may be made of solutions, in particular water-soluble concentrates, emulsions, suspension concentrates, wettable powders (or spray powders), oils and waxes.

[0467] To prepare suspension concentrates that can be applied by spraying to obtain stable fluid products that do not form deposits, they generally contain 10 to 75% of active substance, 0.5 to 15% of surfactant, 0.1 to 10% of thixotropic agent, 0 to 10% of suitable additives such as defoamers, corrosion inhibitors, stabilizers, penetrants and binders, and water or an organic liquid in which the active substance is insoluble or very poorly soluble as a carrier: some organic solids or inorganic salts can be dissolved in the carrier to help prevent sedimentation or as an anti-gelling agent for water.

[0468] The agrochemical compositions disclosed herein can be used as such, in the form of their formulations, or in the form of use prepared therefrom, such as aerosol dispensers, capsule suspensions, cold fog concentrates, hot fog concentrates, encapsulated granulates, fine granulates, flowable concentrates for seed treatment, ready-to-use solutions, dusts, emulsifiable concentrates, oil-in-water emulsions, water-in-oil emulsions, macrogranules, macrogranules, oil-dispersible powders, oil-miscible flowable concentrates, oil-miscible liquid solutions, foams, pastes, seed coating pesticides, suspension concentrates (flowable concentrates), suspension-emulsion-concentrates, soluble concentrates, suspensions, soluble powders, granules, water-soluble granules or tablets, water-soluble powders for seed treatment, wettable powders, natural and synthetic materials impregnated with active compounds, microencapsulation in polymer materials and seed sheaths, microencapsulated bioparticles (such as those described in WO2018 / 201160, WO2018 / 201161 and WO2019 / 060903), and ULV - cold and hot atomization formulations, gases (pressurized), gas-generating products, plant sticks, powders for dry seed treatment, solutions for seed treatment, ultra-low volume (ULV) liquids, ultra-low volume (ULV) suspensions, water-dispersible granules or tablets, water-dispersible powders for slurry treatment.

[0469] These preparations are prepared in a known manner by mixing the active compound or combination of active compounds with conventional additives, such as conventional fillers and solvents or diluents, emulsifiers, dispersants and / or binders or fixatives, wetting agents, water repellents, (if appropriate) desiccants and UV stabilizers, colorants, pigments, defoamers, preservatives, secondary thickeners, adhesives, gibberellins and water and further processing aids.

[0470] These compositions include not only compositions ready to be applied to the plants or seeds to be treated by suitable devices (such as spraying or dusting devices), but also concentrated commercial compositions that must be diluted before application to the crop.

[0471] Method for Plant Protection or Treatment

[0472] In certain aspects, the present invention provides methods for protecting or treating plants or plant parts from infection by plant pathogens or other biological interactions, comprising at least the step of directly or indirectly applying an agrochemical composition or polypeptide as disclosed herein to the plant or plant part. The composition or polypeptide can be applied under conditions effective to protect or treat the plant or plant part from the infection or biological interaction with the plant pathogen.

[0473] In certain embodiments, these methods include directly or indirectly applying an agrochemical composition as disclosed herein to a plant or a part of the plant, for example, at an application rate of more than 50 g of the agrochemical composition per hectare, such as, but not limited to, an application rate of more than 75 g of the agrochemical composition per hectare, such as an application rate of more than 100 g of the agrochemical composition per hectare, or particularly an application rate of more than 200 g of the agrochemical composition per hectare.

[0474] In certain embodiments, these methods include directly or indirectly applying an agrochemical composition as disclosed herein to a plant or a part of the plant, for example, at an application rate of 50 g to 200 g of the agrochemical composition per hectare, such as, but not limited to, an application rate of 50 g to 200 g of the agrochemical composition per hectare, particularly an application rate of 75 g to 175 g of the agrochemical composition per hectare, such as an application rate of 75 g to 150 g of the agrochemical composition or 75 g to 125 g per hectare.

[0475] In yet another embodiment, the present invention provides a method for controlling or suppressing phytophagous pests, the method comprising applying an agrochemical or biocontrol composition according to the present invention to a plant (such as a crop) or a part of a plant or crop, for example, at an application rate of less than 50 g of polypeptide per hectare. In a specific embodiment, the application rate is less than 45 g / ha, less than 40 g / ha, less than 35 g / ha, less than 30 g / ha, less than 25 g / ha, less than 20 g / ha, less than 15 g / ha, less than 10 g / ha, less than 5 g / ha, less than 1 g / ha or even lower amounts of polypeptide / ha.

[0476] It will be understood that farmers can vary the application rate according to the environmental stress of the crop and phytophagous pests. These application rate differences are specified in the technical sheet delivered together with the specific agrochemical composition.

[0477] In yet another embodiment, the present invention provides the use of an agrochemical or biocontrol composition of the present invention in controlling or suppressing phytophagous pests.

[0478] In yet another embodiment, the present invention provides the use of a polypeptide of the present invention in controlling or suppressing phytophagous pests.

[0479] Applying an agrochemical or biocontrol composition or polypeptide according to the present invention to a crop can be accomplished using any suitable method for applying an agrochemical or biocontrol 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, immersion, smearing, atomizing, applying in the form of small droplets, mists or aerosols.

[0480] Thus, in a specific embodiment, a method for protecting or treating a plant or a plant part against infection by a phytopathogen or other biological interaction with a phytopathogen as disclosed herein comprises directly or indirectly applying the agrochemical composition to the plant or the plant part, for example, by spraying, atomizing, foaming, fogging, hydroponic culture, aquaponic culture, coating, immersion and / or encapsulation.

[0481] In certain specific embodiments, the present invention provides a method for inhibiting, preventing, reducing or controlling the growth of a phytopathogen, at least comprising the step of directly or indirectly applying an agrochemical composition as disclosed herein to a plant or a part of the plant.

[0482] In certain other embodiments, the present invention provides a method for killing a phytopathogen, at least comprising the step of directly or indirectly applying an agrochemical composition or polypeptide as disclosed herein to a plant or a part of the plant.

[0483] Alternatively, the application rate of the agrochemical composition according to the invention, i.e., the amount of the agrochemical composition applied to the crop, is such that less than 50 g, 45 g, 40 g, 35 g, 30 g, 25 g, 20 g, 15 g, 10 g, 5 g, 1 g or even less than 1 g per hectare of the polypeptide comprised in the agrochemical or biocontrol composition according to the invention is applied to the crop.

[0484] According to the method disclosed herein, the agrochemical or biocontrol composition may be applied to the crop once, or may be applied two or more times after each other at intervals between each two applications. According to the method of the invention, the agrochemical or biocontrol composition according to the invention may be applied to the crop alone or in admixture with other materials (preferably other agrochemical or biocontrol compositions); alternatively, the agrochemical or biocontrol composition according to the invention may be applied to the same crop separately at different times together with other materials (preferably other agrochemical or biocontrol compositions). According to the method of the invention, the agrochemical or biocontrol composition according to the invention may be applied prophylactically to the crop, or may be applied to the crop once a target pest has been identified on the particular crop to be treated.

[0485] The agrochemical composition as disclosed herein may be applied directly to the plant, crop or one or more parts of the plant by the methods described above, e.g., directly to the whole plant or directly to one or more parts of the plant at the pre-harvest or post-harvest stage. Pre-harvest application may have an impact on post-harvest. In some further embodiments, the agrochemical composition as disclosed herein may be applied directly to one or more parts of the plant by the methods described above, e.g., directly to the stalks, leaves, tubers, stems, buds, seeds, fruits, roots, flowers, grains, sprouts, etc.

[0486] The treatment method as disclosed herein may also be used in the field of protecting stored goods against plant pathogens. In such a treatment method, the application of the composition of the invention may be carried out before or after harvest. According to the invention, the term "stored goods" is understood to mean natural substances of plant or animal origin and their processed forms, which are taken from the natural life cycle and require long-term protection. Stored goods of plant origin, such as plants or parts thereof, e.g., stalks, leaves, tubers, seeds, fruits or grains, may be protected in the freshly harvested state or in a processed form (e.g., pre-dried, moistened, crushed, ground, pressed or roasted). Wood also falls within the definition of stored goods, whether in the form of logs, such as construction wood, power line towers and obstacles, or in the finished form, such as furniture or articles made of wood. Stored goods of animal origin are raw hides, leather, furs, hairs, etc. The combination according to the invention can prevent adverse effects, such as decay, discoloration or mildew. Preferably, "stored goods" is understood to mean natural substances of plant origin and their processed forms, more preferably fruits and their processed forms, such as pome fruits, stone fruits, soft fruits and citrus fruits and their processed forms.

[0487] The agrochemical compositions disclosed herein can also be indirectly applied to plants, crops, or one or more parts of a plant by the methods described above, for example, indirectly applied to the whole plant or indirectly applied to one or more parts of a plant at the pre-harvest or post-harvest stage. The agrochemical compositions disclosed herein can be applied close to harvest, for example, about three weeks before harvest, such as two weeks before harvest, one week before harvest, or less than one week before harvest. Pre-harvest application may have an impact on post-harvest. Thus, in certain embodiments, the agrochemical compositions disclosed herein can be indirectly applied to plants, crops, or one or more parts of a plant by the methods described above, for example, by applying the agrochemical composition to the surrounding environment or medium in which the plant or one or more parts of the plant grow or are stored, such as, but not limited to, air, soil, hydroponics, solution culture, or liquid medium, such as an aqueous liquid medium or water in which the plant or one or more parts of the plant grow or are stored.

[0488] The agrochemical compositions disclosed herein can be directly applied as part of an integrated pest management method.

[0489] Thus, in the context of the present application, it should generally be understood that treating plants and plant parts with the agrochemical compositions disclosed herein is carried out directly or by the action on their environment, habitat, or storage area by normal treatment methods, such as by watering (spraying), drip irrigation, spraying, evaporation, atomization, broadcasting, dusting, foaming, smearing, and as dusting. In addition, the composition can be applied by the ultra-low volume method, or the active compound formulation or the active compound itself can be injected into the soil.

[0490] In a particular embodiment, a method for protecting or treating a plant or a part of a plant against infection by a plant pathogen or other biological interaction with a plant pathogen, as disclosed herein, comprises directly or indirectly applying an agrochemical composition to the plant or a part of the plant at the pre-harvest or post-harvest stage.

[0491] According to a specific embodiment, the harvested product is a fruit, flower, nut, or vegetable, a fruit or vegetable with an inedible peel, preferably selected from avocado, banana, plantain, lemon, grapefruit, melon, citrus, pineapple, kiwifruit, guava, orange, mango, pumpkin, strawberry, grape, and squash, more preferably banana, citrus, lemon, and peach, especially banana. According to a further specific embodiment, the harvested product is a cut flower from an ornamental plant, preferably selected from Alstroemeria, carnation, chrysanthemum, freesia, gerbera, gladiolus, Gypsophila, sunflower, hydrangea, lily, eustoma, rose, and summer flower.

[0492] Plant species to which the agrochemical compositions disclosed herein can be applied can be, for example but not limited to, maize, soybean, alfalfa, cotton, sunflower, Brassica oilseeds such as Brassica napus (e.g., rapeseed, canola), turnip, Brassica juncea (e.g., (field) mustard) and Brassica carinata, Palmae genera (e.g., oil palm, coconut), rice, wheat, sugar beet, sugar cane, oats, rye, barley, millet and sorghum, triticale, flax, nuts, grapes and vines and various fruits and vegetables from various plant taxa, such as Rosaceae (e.g., pomaceous fruits such as apples and pears, as well as stone fruits such as apricots, cherries, almonds, plums and peaches, and berry fruits such as strawberries, raspberries, redcurrants, blackcurrants and gooseberries), Grossulariaceae (Ribesioidaesp.), Juglandaceae, Betulaceae, Anacardiaceae, Fagaceae, Moraceae, Oleaceae (e.g., olive tree), Actinidiaceae, Lauraceae (e.g., avocado, cinnamon, camphor), Musaceae (e.g., banana tree and plantation), Rubiaceae (e.g., coffee), Theaceae (e.g., tea), Sterculiaceae, Rutaceae (e.g., lemon, citrus, tangerine and grapefruit); Solanaceae (e.g., tomato, potato, pepper, chili, eggplant, tobacco), Liliaceae plants, Asteraceae (e.g., lettuce, artichoke and chicory - including chicory root, endive or common chicory), Apiaceae (e.g., carrot, parsley, celery and celeriac), Cucurbitaceae (e.g., cucumber - including gherkin, zucchini, watermelon, gourd and melon), Alliaceae (e.g., leek and onion), Brassicaceae (e.g., Chinese cabbage, red cabbage, broccoli, cauliflower, Brussels sprouts, pak choi, kohlrabi, radish, horseradish, watercress and napa cabbage), Fabaceae (e.g., peanut, pea, lentil and beans - such as common beans and broad beans), Chenopodiaceae (e.g., lamb's lettuce, fodder beet, spinach, beetroot), Linaceae (e.g., hemp), Cannabaceae (e.g., Cannabis indica), Malvaceae (such as okra, cocoa), Papaveraceae (such as poppy), Asparagaceae (such as asparagus); useful plants and ornamental plants in gardens and woods, including turf, lawn, grass and stevia; and in each case, the transgenic types of these plants.

[0493] In a preferred embodiment of the treatment method disclosed herein, the crops are selected from field crops, grass, fruits and vegetables, turf, trees and ornamental plants.

[0494] In some aspects, the present invention thus also provides a post - harvest treatment method for protecting or treating harvested plants or harvested parts of plants against infection by plant pathogens or other biological interactions with plant pathogens, comprising at least the step of directly or indirectly applying to the harvested plants or harvested parts of plants an agro - chemical composition as disclosed herein, under conditions effective to protect or treat the harvested plants or harvested parts of plants against said infection or biological interaction with plant pathogens. According to a specific embodiment, the harvested product is a fruit, flower, nut or vegetable, a fruit or vegetable with an inedible peel, preferably selected from avocado, banana, plantain, lemon, grapefruit, melon, citrus, pineapple, kiwi fruit, guava, orange, mango and squash, more preferably banana, citrus, lemon and peach, especially banana. According to a further specific embodiment, the harvested product is a cut flower from an ornamental plant, preferably selected from the genus Alstroemeria, carnation, chrysanthemum, freesia, gerbera, gladiolus, Gypsophila, sunflower, hydrangea, lily, eustoma, rose and summer flower. According to a further specific embodiment, the harvested product is mown grass or wood.

[0495] Post - harvest disorders are, for example, lenticel spot, scorch marks, senescent breakdown, bitter pit, sunburn, water core, browning, vascular breakage, CO2 injury, CO2 or O2 deficiency and softening.

[0496] Fungal diseases can be caused by, for example, the following fungi: Mycosphaerella, Mycosphaerella musae, Mycobacterium fragmentum, Mycosphaerella citri; Mucor, such as Mucor piriformis; Monilinia, such as Monilinia fructigena, Monilinia laxa; Phomopsis, Phomopsis natalensis; Colletotrichum, such as Colletotrichum musae, Colletotrichum gloeosporioides, Colletotrichum orbiculare; Verticillium, such as Verticillium theobromae; Nigrospora; Botrytis, such as Botrytis cinerea; Diplodia, such as Diplodia citri; Mycena; Alternaria, such as Alternaria citri, Alternaria alternata; Septoria, such as Septoria depressa; Venturia, such as Venturia inaequalis, Venturia nashicola; Rhizopus, such as Rhizopus stolonifer, Rhizopus oryzae; Glomerella, such as Glomerella cingulata; Sclerotinia, such as Sclerotinia fruiticola; Ceratocystis, such as Ceratocystis paradoxa; Fusarium, such as Fusarium semitectum, Fusarium moniliforme, Fusarium solani, Fusarium oxysporum; Cladosporium, such as Cladosporium fulvum, Cladosporium cladosporioides, Cladosporium cucumerinum, Cladosporium agaricinum; Penicillium, such as Penicillium funiculosum, Penicillium expansum, Penicillium digitatum, Penicillium italicum; Phytophthora, such as Phytophthora citrophthora, Phytophthora fragariae, Phytophthora cactorum, Phytophthora parasitica; Phacydiopycnis spp., such as Phacydiopycnis malirum; Gloeosporium, such as Gloeosporium album, Gloeosporium perennans, Gloeosporium fructigenum, Gloeosporium singulata; Geotrichum, such as Geotrichum candidum; Phlyctaena spp., such as Phlyctaena vagabunda; Cylindrocarpon, such as Cylindrocarpon mail; Stemphyllium spp., such as Stemphyllium vesicarium; Thielaviopsis, such as Thielaviopsis paradoxa; Aspergillus, such as Aspergillus niger, Aspergillus carbonarius; Nectria, such as Nectria galligena; Cercospora, such as Cercospora angreci, Cercospora apii, Cercospora nigrescens, Pseudocercospora musae, Cercospora zeae-maydis.

[0497] In a further aspect, the present invention provides the use of an agrochemical composition as herein disclosed as an insecticide, such as a biostatic agent or a pesticide, including but not limited to a bacteriostatic agent or a fungicide.

[0498] In a particular embodiment, the plant pests controlled by the method according to the invention are plant pathogenic fungi as defined above. As a result of applying the method according to the invention, the number of lesions, the size of the lesions and the degree of spore formation of the fungal pathogen can all be reduced.

[0499] Medical Application

[0500] In certain other embodiments, the present invention provides a method for protecting or curing a human or an animal from pests and in particular fungal infections, or a method for treating a human or an animal infected with pests and in particular fungal infections, comprising at least the step of directly or indirectly administering or dosing to the human or animal or a part of the human or animal a composition comprising at least one polypeptide of the present invention that specifically binds to the pest, such as but not limited to a fungus. The composition can be administered under conditions effective to protect or cure the human or animal from pest infestation.

[0501] Accordingly, the present invention provides a polypeptide of the present invention that specifically binds to a pest target for use in a method for preventing and / or treating in a subject at least one disease and / or disorder caused by a pest (such as a disease and / or disorder caused by a fungus). The present invention also provides a composition of the present invention for use in a method for preventing and / or treating in a subject at least one disease and / or disorder caused by a pest (such as a disease and / or disorder caused by a fungus). The present invention also provides a polypeptide of the present invention that specifically binds to a pest target for use in a method for preventing and / or treating in a subject an infection caused by a pest (such as a fungal infection). The present invention also provides a composition of the present invention that specifically binds to a pest target for use in a method for preventing and / or treating in a subject an infection caused by a pest (such as a fungal infection). In a particular embodiment, the present invention also provides a method for preventing and / or treating at least one disease and / or disorder caused by a pest, comprising administering to a subject in need a pharmaceutically active amount of one or more amino acid sequences, polypeptides and / or pharmaceutical compositions as herein disclosed. In particular, the pharmaceutically active amount can be an amount sufficient (to produce a certain level of the amino acid sequence or polypeptide in circulation) to inhibit, prevent or reduce one or more biological activities or pathways of the pest to which it binds.

[0502] Accordingly, in certain aspects, the present invention provides a composition comprising at least one polypeptide that specifically binds to a pest for use as a pest control agent in a subject (such as an animal or a human) suffering from a disease and / or disorder caused by a pest (such as a fungus).

[0503] In a specific embodiment, the pest control agent is a biological inhibitor or an insecticide. In a specific embodiment, the pest control agent is a bacteriostatic agent or a fungicide.

[0504] In addition, in certain aspects, the present invention provides a method for preventing and / or treating diseases and / or disorders caused by pests, the method comprising the steps of:

[0505] (a) providing an amino acid sequence, polypeptide or composition as disclosed herein,

[0506] (b) administering the amino acid sequence, polypeptide or pharmaceutical composition to a patient suffering from a disease and / or disorder caused by a pest.

[0507] Any suitable in vitro assay, cell-based assay, in vivo assay and / or animal model known per se or any combination thereof can be used to test the efficacy of the polypeptides and compositions containing the polypeptides disclosed herein, depending on the specific disease or disorder involved. Suitable assays and animal models, as well as those used in the following experimental sections and the prior art cited herein, will be clear to the person skilled in the art. The person skilled in the art will generally be able to select a suitable in vitro assay, cell assay or animal model to test the ability of the amino acid sequences and polypeptides disclosed herein to bind to pest targets or pest antigens or their ability to affect the activity of pest targets or pest antigens, and / or the biological mechanisms involved therewith; and their therapeutic and / or prophylactic effects on one or more diseases and disorders associated with pest antigens.

[0508] Pharmaceutical Composition

[0509] In a still further aspect, the present invention provides a pharmaceutical composition comprising one or more amino acid sequences, polypeptides and / or nucleic acid sequences as disclosed herein and optionally at least one pharmaceutically acceptable carrier (also referred to herein as the pharmaceutical composition of the present invention). According to certain specific embodiments, the pharmaceutical composition disclosed herein may further optionally comprise at least one other pharmaceutically active compound.

[0510] The pharmaceutical composition of the present invention can be used for the diagnosis, prevention and / or treatment of diseases and disorders associated with pests (such as fungi), wherein the pest target binds to the polypeptide disclosed herein.

[0511] In particular, the present invention provides a pharmaceutical composition comprising a polypeptide, which is suitable for prophylactic, therapeutic and / or diagnostic use in warm-blooded animals and especially mammals and more especially humans.

[0512] The present invention also provides pharmaceutical compositions comprising the amino acid sequences and polypeptides as disclosed herein, which can be used for veterinary purposes to prevent and / or treat or diagnose one or more diseases, disorders or conditions associated with pests (such as fungi), wherein the pest targets bind to the polypeptides disclosed herein.

[0513] Generally, for pharmaceutical use, the polypeptides disclosed herein can be formulated into pharmaceutical preparations or compositions, which comprise at least one of the polypeptides disclosed herein and at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally one or more further pharmaceutically active polypeptides and / or compounds. Such preparations can be suitable for oral, parenteral, topical or inhalational administration. Thus, the amino acid sequences or polypeptides as disclosed herein and / or the compositions comprising them can be administered, for example, orally, intraperitoneally (such as intravenously, subcutaneously, intramuscularly, transdermally, topically, by suppository, by inhalation, etc.), again, depending on the specific pharmaceutical preparation or composition to be used. The clinician will be able to select the appropriate route of administration and the appropriate pharmaceutical preparation or composition for such administration.

[0514] The pharmaceutical compositions can also comprise suitable binders, disintegrants, sweeteners or flavoring agents. Tablets, pills or capsules can be coated with, for example, gelatin, wax or sugar. In addition, the amino acid sequences and polypeptides disclosed herein can be incorporated into sustained release formulations and devices.

[0515] Pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are suitable for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions and which are optionally encapsulated in liposomes. In all cases, the final dosage form must be sterile, flowable and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or a liquid dispersion medium, comprising, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glycerides and suitable mixtures thereof. Antibacterial and antifungal agents, etc. can be optionally added.

[0516] The useful doses of the amino acid sequences and polypeptides as disclosed herein can be determined by comparing their in vitro activities and in vivo activities in animal models. Methods for extrapolating effective doses in mice and other animals to humans are known to those skilled in the art.

[0517] The amount of the amino acid sequences and polypeptides as disclosed herein required for prevention and / or treatment can vary not only with the specific amino acid sequence or polypeptide selected, but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will ultimately be determined at the discretion of the attending physician or clinician. In addition, the doses of the amino acid sequences and polypeptides as disclosed herein can vary according to the target cells, tumors, tissues, grafts or organs.

[0518] The amino acid sequences or polypeptides and / or compositions comprising them disclosed herein are administered according to a treatment regimen suitable for preventing and / or treating a disease or disorder to be predicted, diagnosed, prevented or treated. Clinicians are generally able to determine a suitable treatment regimen. Typically, the treatment regimen will involve administering one or more of the amino acid sequences or polypeptides as disclosed herein, or one or more compositions comprising them, in one or more pharmaceutically effective amounts or doses.

[0519] The required dose can conveniently be administered as a single dose or in divided doses (which may be further divided) at appropriate time intervals. The dosing regimen can include long-term (i.e., at least two weeks, such as several months or years) or daily treatment.

[0520] The dosage of the amino acid sequences or polypeptides disclosed herein will be determined by a medical practitioner, in particular, based on the severity of the condition and the patient to be treated. Typically, for each disease indication, an optimal dose will be determined, specifying the amount to be administered per kg body weight per day, either continuously (e.g., by infusion) as a single daily dose or as multiple divided doses during the day. Depending on the factors mentioned herein, clinicians are generally able to determine a suitable daily dose. It is also clear that in certain circumstances, the clinician may choose to deviate from these amounts, for example, based on the above factors and his expert judgment.

[0521] In particular, the amino acid sequences or polypeptides disclosed herein can be used in combination with other pharmaceutically active compounds or ingredients that are used or can be used for preventing and / or treating the diseases and disorders cited herein, and as a result, a synergistic effect may or may not be obtained. Examples of such compounds and ingredients, as well as the routes, methods and pharmaceutical formulations or compositions for administering them, are clear to clinicians.

[0522] The compositions of the present invention can be used in combination with known antifungal agents. Suitable antifungal agents include, but are not limited to, azoles (e.g., fluconazole, itraconazole), polyenes (e.g., amphotericin B), flucytosine, and squalene epoxidase inhibitors (e.g., terbinafine) [see also reference 57]. The compositions can also be used in combination with known antiviral drugs, such as HIV protease inhibitors, 2',3'-dideoxynucleosides (e.g., DDC, DDI), 3'-azido-2',3'-dideoxyribonucleoside (AZT), 3'-fluoro-2',3'-dideoxyribonucleoside (FLT), 2',3'-didehydro-2',3'-dideoxyribonucleoside (e.g., D4C, D4T) and their carbocyclic derivatives (e.g., carbovir), 2'-fluoro-ara-2',3'-dideoxyribonucleoside, 1,3-dioxolane derivatives (e.g., 2',3'-dideoxy-3'-thiacytidine), oxetanocin analogs and their carbocyclic derivatives (e.g., cyclobut-G), and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and 9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) derivatives, tetrahydro-imidazo[4,5,1jk][1,4]-benzodiazepin-2(1H)-one (TIBO), 1-[(2-hydroxyethoxy)-methyl]-6-(phenylthio)thymine (HEPT), dipyrido[3,2-b:2',3'-e]-[1,4]diazepin-6-one (nevirapine) and pyridin-2(1H)-one derivatives, 3TC, etc.

[0523] The amino acid sequences, polypeptides, and pharmaceutical compositions are particularly useful for treating infections in animals and humans caused by the following species: Candida species, such as Candida albicans; Cryptococcus species, such as Cryptococcus neoformans; Enterococcus species, such as Enterococcus faecalis; Streptococcus species, such as Streptococcus pneumoniae, Streptococcus mutans, Streptococcus agalactiae, and Streptococcus pyogenes; Leishmania species, such as Leishmania major and Leishmania infantum; Acanthamoeba species, such as Acanthamoeba castellanii; Aspergillus species, such as Aspergillus fumigatus and Aspergillus flavus; Pneumocystis species, such as Pneumocystis carinii; Mycobacterium species, such as Mycobacterium tuberculosis; Pseudomonas species, such as Pseudomonas aeruginosa; Staphylococcus species, such as Staphylococcus aureus; Salmonella species, such as Salmonella typhimurium; Coccidioides species, such as Coccidioides immitis; Trichophyton species, such as Trichophyton verrucosum; Blastomyces species, such as Blastomyces dermatitidis; Histoplasma species, such as Histoplasma capsulatum; Paracoccidioides species, such as Paracoccidioides brasiliensis; Pythium species, such as Pythium insidiosum; and Escherichia coli species, such as Escherichia coli. The amino acid sequences, polypeptides, and pharmaceutical compositions are particularly useful for treating diseases including, but not limited to: candidiasis, aspergillosis, cryptococcosis, dermatomycosis, sporotrichosis and other subcutaneous mycoses, blastomycosis, histoplasmosis, coccidioidomycosis, paracoccidioidomycosis, pneumocystosis, thrush, tuberculosis, mycobacteriosis, respiratory infections, scarlet fever, pneumonia, impetigo, rheumatic fever, septicemia, sepsis, cutaneous and visceral leishmaniasis, Acanthamoeba keratitis, keratitis, cystic fibrosis, typhoid, gastroenteritis, and hemolytic uremic syndrome. Anti-Candida albicans activity is particularly useful for treating infections in AIDS patients.

[0524] Method for Production and Manufacture of Polypeptides

[0525] The present invention further provides methods for preparing or generating polypeptide sequences, as well as methods for generating nucleic acids encoding these polypeptides and host cells, products, and compositions containing these polypeptide sequences. Some preferred but non-limiting examples of such methods will become clear from the further description herein.

[0526] As will be clear to those skilled in the art, a particularly useful method for preparing the polypeptide sequences disclosed herein generally includes the steps of:

[0527] (a) expressing a nucleotide sequence encoding the polypeptide sequence disclosed herein or a vector or genetic construct containing the nucleotide sequence encoding the polypeptide sequence, and

[0528] (b) optionally isolating and / or purifying the polypeptide sequence.

[0529] In the specific embodiments contemplated herein, the pest-specific polypeptide sequences can be obtained by a method that includes generating a random library of amino acid sequences and screening the library for amino acid sequences that specifically bind to a pest target.

[0530] Thus, in certain embodiments, a method of preparing a polypeptide sequence as disclosed herein includes the steps of:

[0531] a) providing a group, collection, or library of amino acid sequences; and

[0532] b) screening the group, collection, or library for amino acid sequences that bind to and / or have affinity for a pest target, and

[0533] c) isolating the amino acid sequences that bind to and / or have affinity for a pest target.

[0534] In such a method, the group, collection, or library of polypeptide sequences can be any suitable group, collection, or library of amino acid sequences. For example, the group, collection, or library of amino acid sequences can be a group, collection, or library of immunoglobulin fragment sequences (as described herein), such as a naive group, collection, or library of immunoglobulin fragment sequences; a synthetic or semi-synthetic group, collection, or library of immunoglobulin fragment sequences; and / or a group, collection, or library of immunoglobulin fragment sequences that have undergone affinity maturation.

[0535] In a particular embodiment of the method, the group, collection, or library of amino acid sequences can be an immune group, collection, or library of immunoglobulin fragment sequences, such as derived from a mammal that has been suitably immunized with a pest target or with a suitable antigenic determinant based on or derived from it (e.g., its antigenic portion, fragment, region, domain, loop, or other epitope). In a particular aspect, the antigenic determinant can be an extracellular portion, region, domain, loop, or other extracellular epitope.

[0536] In the methods described above, the group, collection, or library of polypeptide sequences can be displayed on a phage, phagemid, ribosome, or suitable microorganism (e.g., yeast) to facilitate screening. Suitable methods, techniques, and host organisms for displaying and screening amino acid sequences (groups, collections, or libraries) will be apparent to those skilled in the art, e.g., based on further disclosure herein. Also refer to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

[0537] In other embodiments, a method for generating a polypeptide sequence as disclosed herein includes at least the steps of:

[0538] a) providing a collection or sample of cells that express the polypeptide sequence;

[0539] b) Screen the cells in the cell population or cell sample that express an amino acid sequence that can bind to and / or has an affinity for a pest target; and

[0540] c) (i) Isolate the amino acid sequence; or (ii) Isolate the nucleic acid sequence encoding the amino acid sequence from the cells and then express the amino acid sequence.

[0541] The cell population or sample can be, for example, a population or sample of B cells. Additionally, in this method, the cell sample can be derived from a mammal that has been appropriately immunized with a fungal target or a suitable antigenic determinant based on or derived from it (such as its antigenic part, fragment, region, domain, loop, or other epitope). In a specific embodiment, the antigenic determinant can be an extracellular part, region, domain, loop, or other extracellular epitope.

[0542] In other embodiments, a method for generating a polypeptide sequence against a pest target can at least include the steps of:

[0543] a) Provide a group, collection, or library of nucleic acid sequences encoding polypeptide amino acid sequences;

[0544] b) Screen the nucleic acid sequences in the group, collection, or library of nucleic acid sequences that encode an amino acid sequence that can bind to and / or has an affinity for a pest target; and

[0545] c) Isolate the nucleic acid sequence and then express the amino acid sequence.

[0546] In the above method, the pest target can be a lipid-containing fraction of the plasma membrane of a fungus (such as Botrytis cinerea or other fungi). The lipid-containing fraction can be obtained by chromatography. For example, the lipid-containing fraction can be obtained by a method including:

[0547] Thin layer chromatography fractionation of the mycelium of a fungus (such as Botrytis cinerea or other fungi) from a total lipid extract and selecting the fraction with a retention factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipid fraction.

[0548] In the above method, the group, collection, or library of nucleic acid sequences encoding an amino acid sequence can be, for example, a group, collection, or library of nucleic acid sequences of a naive group, collection, or library encoding immunoglobulin fragment sequences; a group, collection, or library of nucleic acid sequences of a synthetic or semi-synthetic group, collection, or library encoding immunoglobulin fragment sequences; and / or a group, collection, or library of nucleic acid sequences of a group, collection, or library encoding immunoglobulin fragment sequences that have undergone affinity maturation.

[0549] In particular, in such a method, the group, collection or library of nucleic acid sequences encodes a group, collection or library of polypeptides (such as V H domain or V HH domain). For example, the group, collection or library of nucleic acid sequences may encode a group, collection or library of domain antibodies or single domain antibodies, or a group, collection or library of amino acid sequences capable of acting as domain antibodies or single domain antibodies. In a specific embodiment, the group, collection or library of nucleotide sequences encodes a group, collection or library of V HH sequences.

[0550] In the above method, the group, collection or library of nucleotide sequences can be displayed on phage, phagemid, ribosome or a suitable microorganism (such as yeast) for easy screening. Suitable methods, techniques and host organisms for displaying and screening nucleotide sequences (groups, collections or libraries) encoding amino acid sequences will be clear to those skilled in the art, for example based on further disclosure herein. Also refer to the review by Floogenboom in Nature Biotechnology, 23, 9, 1105 - 1116 (2005).

[0551] The present invention also relates to polypeptide sequences which can be obtained or have been obtained by the above method, or which can be obtained or have been obtained by a method comprising one of the above methods and additionally at least the following steps: determining the nucleotide sequence or amino acid sequence of the immunoglobulin sequence; and expressing or synthesizing the amino acid sequence in a manner known per se, for example by expression in a suitable host cell or host organism or by chemical synthesis.

[0552] Isolation of Polypeptide Sequences

[0553] In some cases, a method for generating an amino acid sequence that specifically binds to a fungal target as contemplated herein may further comprise the step of isolating at least one polypeptide from an amino acid sequence library, the at least one polypeptide having a detectable binding affinity for a pest target or a detectable in vitro effect on a pest target.

[0554] These methods may further comprise the step of amplifying the sequence encoding at least one polypeptide having a detectable binding affinity or a detectable in vitro effect on the activity of a pest target. For example, a phage clone displaying a specific amino acid sequence obtained from the selection step of the methods described herein can be amplified by reinfection of the host bacterium and incubation in a growth medium.

[0555] In a specific embodiment, these methods may include determining the sequences of one or more amino acid sequences capable of binding to a pest target.

[0556] In the case where a polypeptide sequence contained in a group, collection or library of amino acid sequences is displayed on a suitable cell or phage or particle, the nucleotide sequence encoding that amino acid sequence can be isolated from the cell or phage or particle. In this way, the nucleotide sequence of a selected member of the amino acid sequence library can be determined by conventional sequencing methods.

[0557] In a further specific embodiment, a method for producing a polypeptide as contemplated herein includes the step of expressing the nucleotide sequence in a host organism under suitable conditions to obtain the actually desired amino acid sequence. This step can be carried out by methods known to those skilled in the art.

[0558] Furthermore, polypeptide sequences that have a detectable binding affinity or a detectable in vitro effect on the activity against a pest target, optionally after their sequences have been identified, can be synthesized into soluble protein constructs.

[0559] For example, polypeptide sequences obtained, obtainable or selected by the above methods can be synthesized using recombinant methods or chemical synthesis methods known in the art. In addition, amino acid sequences obtained, obtainable or selected by the above methods can be produced by genetic engineering techniques. Thus, a method for synthesizing a polypeptide sequence obtained, obtainable or selected by the above methods can include: transforming or infecting a host cell with a nucleic acid or vector encoding an amino acid sequence that has a detectable binding affinity or a detectable in vitro effect on the activity against a pest target. Thus, the amino acid sequence that has a detectable binding affinity or a detectable in vitro effect on the activity against a pest target can be prepared by recombinant DNA methods. DNA encoding an amino acid sequence can be easily synthesized using conventional methods. Once prepared, the DNA can be introduced into an expression vector and then can be transformed or transfected into a host cell (such as Escherichia coli) or any suitable expression system to obtain expression of the amino acid sequence in the recombinant host cell and / or in the medium in which these recombinant host cells are located.

[0560] It should be understood that, as known to those skilled in the art of protein expression and purification, polypeptides produced from an expression vector using a suitable expression system can be labeled (usually at the N-terminus or C-terminus of the amino acid sequence) with, for example, a His-tag or other sequence tags that facilitate purification.

[0561] Transformation or transfection of a nucleic acid or vector into a host cell can be accomplished by a variety of methods known to those skilled in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, liposome transfection, protoplast fusion, retroviral infection and gene gun.

[0562] Suitable host cells for expressing the desired polypeptide sequence can be any eukaryotic or prokaryotic cell (e.g., bacterial cells such as Escherichia coli, yeast cells, mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo. For example, the host cells can be located in transgenic plants or animals.

[0563] Accordingly, the present application also provides methods for producing polypeptide sequences having a detectable binding affinity for a pest target or a detectable in vitro effect on the activity of a pest target, comprising transforming, transfecting, or infecting a host cell with a nucleic acid sequence or vector encoding such an amino acid sequence and expressing the amino acid sequence under suitable conditions. The present application also provides methods for producing polypeptide sequences having a detectable binding affinity for a pest target or a detectable in vitro effect on the activity of a pest target, comprising providing a host cell comprising a nucleic acid sequence or vector encoding the polypeptide and expressing the polypeptide under suitable conditions. The methods of the present invention may further comprise isolating the polypeptide, e.g., from the cell culture medium or fermentation broth or from inside the host cell (e.g., after the step of lysing the host cell).

[0564] In yet another embodiment, the present invention further provides a method for manufacturing (or the equivalent phrase 'producing') an agrochemical or biocontrol composition as disclosed herein.

[0565] In a particular embodiment, the present invention provides a method for producing an agrochemical composition as disclosed herein, comprising at least the steps of:

[0566] - obtaining at least one polypeptide that specifically binds to a pest, and

[0567] - formulating the polypeptide or a functional fragment thereof into an agrochemical composition.

[0568] In a particular embodiment of these methods, the step of obtaining at least one polypeptide that specifically binds to a pest comprises:

[0569] (a) expressing a nucleotide sequence encoding a polypeptide that specifically binds to a pest, and optionally

[0570] (b) isolating and / or purifying the polypeptide.

[0571] In other particular embodiments of these methods, the step of obtaining at least one polypeptide that specifically binds to a pest comprises:

[0572] a) providing a group, collection, or library of polypeptide sequences;

[0573] b) screening the group, collection, or library of polypeptide sequences for sequences that specifically bind to a pest and / or have an affinity for a pest, and optionally

[0574] c) Isolate the polypeptide sequence that specifically binds to pests and / or has an affinity for pests.

[0575] The present application further discloses a method for manufacturing (or the equivalent phrase 'producing') an agrochemical or biocontrol composition as disclosed herein, comprising formulating an amino acid sequence or polypeptide having pesticidal activity of 80 to 200 amino acids or other suitable sub-ranges as defined above using at least one conventional agrochemical adjuvant.

[0576] Suitable manufacturing methods are known in the art and include, but are not limited to, high or low shear mixing, wet or dry grinding, drop casting, encapsulation, emulsification, coating, encapsulation, pill making, extrusion granulation, fluidized bed granulation, co-extrusion, spray drying, spray cooling, atomization, addition or condensation polymerization, interfacial polymerization, in-situ polymerization, coacervation, spray encapsulation, cooling molten dispersion, solvent evaporation, phase separation, solvent extraction, sol-gel polymerization, fluidized bed coating, pan coating, melting, passive or active absorption or adsorption.

[0577] Specifically, an amino acid sequence or polypeptide of 80 to 200 amino acids or other suitable sub-ranges as disclosed herein can be prepared by chemical synthesis.

[0578] Further disclosed are amino acid sequences or polypeptides of 80 to 200 amino acids or other suitable sub-ranges as defined above, which can be prepared and isolated by an in vitro recombinant microbial expression system for further use. Such amino acid sequences or polypeptides can be in crude cell lysates, suspensions, colloids, etc., or can be purified, refined, buffered, and / or further processed before formulating with conventional agrochemical adjuvants.

[0579] Specifically, the recombinant method generally involves inserting a DNA molecule expressing a desired amino acid sequence, protein or polypeptide into an expression system that is heterologous to the DNA molecule (i.e., not normally present in the host). The heterologous DNA molecule is inserted into the expression system or vector in the appropriate sense orientation and correct reading frame. The vector contains the elements necessary for transcription and translation of the inserted protein-coding sequence. Transcription of DNA depends on the presence of a promoter. Similarly, translation of mRNA in prokaryotes depends on the presence of appropriate prokaryotic signals that are different from those in eukaryotes. For a review on maximizing gene expression, see Roberts and Lauer, Methods in Enzymology 68:473 (1979). Regardless of the specific regulatory sequences employed, the DNA molecule is cloned into the vector using standard cloning procedures in the art, as described in Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989). Once the isolated DNA molecule encoding the protein is cloned into the expression system, it can be integrated into the host cell. This incorporation can be carried out by various forms of transformation, depending on the vector / host cell system. Suitable host cells include, but are not limited to, bacteria, viruses, yeast, mammalian cells, insects, plants, etc. Optionally, the recombinant host cell can be a host cell that expresses a native or recombinant functional type III secretion system. This is described in detail in US 6,596,509. As a result of expressing the functional type III secretion system, the cell will express the polypeptide and then secrete the protein into the culture medium. This can simplify the isolation and purification of the polypeptide. The recombinant host cells can be grown in a suitable fermentation chamber, preferably under temperature and nutrient conditions that optimize host cell growth and polypeptide expression. Those skilled in the art are able to determine the optimal conditions for a particular host cell. After fermentation, for example, the bacterial suspension can be diluted in, for example, about 2 to 5 volumes of buffer to adjust the pH to about 5.5 to 10, more preferably to about 7 to 9, and even more preferably to a pH of about 8.0. Suitable buffers are well known in the art and can include, for example, potassium phosphate buffer or Tris-EDTA buffer. The concentration of the buffer can be from about 0.001 mM to about 0.5 M. After pH adjustment, the (bacterial) suspension solution is heat-treated to a temperature of about 60 - 130°C, preferably about 95 - 125°C. The heat treatment can be carried out for any suitable time. In one embodiment, the heat treatment is carried out for about 5 minutes to about 30 minutes. Then the heated suspension is cooled. Suitable cooling temperatures are, but are not limited to, about 35 - 55°C, preferably about 45°C. After cooling, if necessary, the bacterial cells in the bacterial suspension are lysed to release the polypeptide.Cell lysis can be carried out, for example, by contacting a bacterial suspension with lysozyme. The concentration of lysozyme can be from about 2 ppm to 100 ppm. Alternatively, cell lysis can involve non-chemical methods such as high pressure or sonication, both of which are well known to those of ordinary skill in the art. It may be necessary to incubate the bacterial suspension after cell lysis. The appropriate incubation time may vary. For example, it may be necessary to incubate the bacterial suspension at a temperature of about 40 - 42 °C for about 30 - 45 minutes. After lysis, the desired polypeptide can be further extracted by removing cell debris and denatured proteins produced by the previous heat treatment step. In one embodiment, the extract is centrifuged for about 10 - 20 minutes to remove some cell debris. Suitable centrifugation speeds can be from about 4,000 to 20,000 rpm and the spin-down time can be from about 10 minutes to 20 minutes. Then more cell debris can be removed by heat treatment and centrifuging the supernatant to obtain a liquid extract substantially free of cell debris by removing more than about 60%, 70%, 80%, 90% or 95% of the total solids. This subsequent heat treatment can be carried out at a temperature of about 60 °C for up to about 2 hours, at about 100 °C for about 10 minutes, or at about 121 °C and 15 psi pressure for about 5 minutes. These temperatures and times may vary depending on other conditions. The method of preparing a stable liquid composition containing an amino acid sequence or polypeptide as disclosed herein further involves introducing a biocide and optionally one or both of a protease inhibitor and a non-ionic surfactant into the liquid extract, thereby obtaining a liquid composition containing the polypeptide. In one embodiment, the protease inhibitor is introduced into the liquid extract in the absence of a non-ionic surfactant. In another embodiment, the non-ionic surfactant is introduced into the liquid extract in the absence of a protease inhibitor. In a further embodiment, both the protease inhibitor and the non-ionic surfactant are introduced into the liquid extract. In yet another embodiment, neither the protease inhibitor nor the non-ionic surfactant is introduced into the liquid extract. Alternatively, the stability of the liquid composition as disclosed herein can be evaluated using, for example, HPLC analysis or other suitable procedures that can identify the amount of a specific protein or polypeptide. The stability of the amino acid sequence or polypeptide in the composition as disclosed herein can be determined by comparing the amount of protein in an aged liquid composition with that in a recently prepared liquid composition, or with a previous quantification of the same composition. The measurement of protein stability is closely related to the retention of its activity.

[0580] Conventional agrochemical adjuvants are well known in the art and include, but are not limited to, aqueous or organic solvents, buffers, acidifying agents, surfactants, wetting agents, spreading agents, tackifiers, adhesives, carriers, fillers, thickeners, emulsifiers, dispersants, chelating agents, anti-settling agents, coalescing agents, rheology modifiers, defoaming agents, light protectants, antifreeze agents, biocides, penetrants, mineral or vegetable oils, pigments, and drift control agents or any suitable combination thereof.

[0581] In yet another embodiment, the present invention provides a polypeptide having 80 to 200 amino acids or a sub-range disclosed hereinbefore, which is obtained by affinity selection against a plant pest target and is capable of inhibiting the growth and / or activity of plant pests at a minimum inhibitory concentration of about 0.00001 to 1 μM.

[0582] In a specific embodiment of the method for protecting, preventing, curing, or treating plants from fungal infections disclosed herein, the polypeptides or compositions disclosed herein are directly or indirectly applied to the plants by spraying, atomizing, foaming, fogging, solution culture / hydroponics, coating, immersion, and / or encapsulation.

[0583] Nucleic Acid Sequence

[0584] In a further aspect, the present invention provides nucleic acid sequences encoding the polypeptide sequences (or suitable fragments thereof) disclosed herein. These nucleic acid sequences can also be in the form of vectors, gene constructs, or polynucleotides. The nucleic acid sequences disclosed herein can be synthetic or semi-synthetic sequences, nucleotide sequences isolated from libraries (especially expression libraries), nucleotide sequences prepared by PCR using overlapping primers, or nucleotide sequences prepared using DNA synthesis techniques known per se.

[0585] The present invention includes nucleic acid sequences encoding any of the polypeptides disclosed herein. For example, the present invention includes nucleic acid sequences encoding polypeptides comprising amino acid sequences selected from SEQ ID NOs: 1 to 161 and variants thereof (e.g., those sequences having amino acid substitutions or a specific percentage identity thereto).

[0586] Construct, Vector, Host Cell

[0587] The genetic constructs disclosed herein can be DNA or RNA, and are preferably double-stranded DNA. The genetic constructs of the present invention can also be in a form suitable for transforming a target host cell or host organism, in a form suitable for integration into the genomic DNA of the target host cell, or in a form suitable for independent replication, maintenance, and / or inheritance in the intended host organism. For example, the genetic constructs of the present invention can be in the form of a vector, such as a plasmid, cosmid, YAC, viral vector, or transposon. In particular, the vector can be an expression vector, i.e., a vector that can provide expression in vitro and / or in vivo (e.g., in a suitable host cell, host organism, and / or expression system).

[0588] Accordingly, in a further aspect, the present invention also provides a vector comprising one or more nucleic acid sequences of the present invention.

[0589] In a still further aspect, the present invention provides a host or host cell that expresses or is capable of expressing one or more amino acid sequences as disclosed herein. Suitable examples of hosts or host cells for expressing the amino acid sequences and polypeptides of the present invention will be apparent to those skilled in the art.

[0590] The present application also discloses that polypeptides having 80 to 200 amino acids or the sub-ranges discussed previously herein remain stable in agrochemical or biocontrol compositions as defined, which means that the integrity of the polypeptide and the pesticidal activity as defined are maintained under the storage and / or use conditions of the agrochemical composition, which may include conditions such as elevated temperature, freeze-thaw cycles, changes in pH or ionic strength, UV irradiation, the presence of harmful chemicals, etc. Most preferably, these polypeptides of 80 to 200 amino acids remain stable in the agrochemical composition when the agrochemical composition is stored at ambient temperature for two years or when the agrochemical composition is stored at 54 °C for two weeks. In particular, when stored in the agrochemical composition at ambient temperature for two years or when the agrochemical composition containing the polypeptide is stored at 54 °C for two weeks, the polypeptide having 80 to 200 amino acids contained in the agrochemical composition retains at least about 70% of its activity after storage, more particularly at least about 70% to 80% of its activity, and most particularly about 80% to 90% of its activity.

[0591] In yet another embodiment, for use in the methods disclosed herein, the present application discloses a nucleic acid sequence encoding a polypeptide having 80 to 200 amino acids, wherein the polypeptide is obtained by affinity selection against a specific plant pathogen target and is capable of inhibiting the growth and / or activity of a crop pest at a minimum inhibitory concentration of about 0.00001 to 1 μM.

[0592] Also disclosed is a chimeric gene comprising the following operably linked DNA elements: a) a plant-expressible promoter, b) a DNA region which, when transcribed, gives rise to an mRNA molecule capable of being translated into a polypeptide, and c) a 3'-terminal region comprising a transcription termination and polyadenylation signal functional in the cells of said plant.

[0593] A "chimeric gene" or "chimeric construct" is a recombinant nucleic acid sequence in which a promoter (e.g., a plant-expressible promoter) or regulatory nucleic acid sequence is operably linked or associated with a nucleic acid sequence encoding an mRNA such that, when introduced into a cell (such as a plant cell), the regulatory nucleic acid sequence is capable of regulating the transcription or expression of the associated nucleic acid coding sequence. The regulatory nucleic acid sequence of a chimeric gene is generally not operably linked to the associated nucleic acid sequence found in nature.

[0594] In the present invention, a "plant promoter" comprises regulatory elements which mediate the expression of a coding sequence segment in a plant cell. For expression in a plant, a nucleic acid molecule must be operably linked to or comprise a suitable promoter which expresses the gene at the correct time point and in the desired spatial expression pattern.

[0595] As used herein, the term "operably linked" refers to a functional linkage between a promoter sequence and a gene of interest such that the promoter sequence is capable of initiating transcription of the gene of interest.

[0596] A plant-expressible promoter comprises a nucleic acid sequence capable of directing the expression of a transgene in a plant. Examples of plant-expressible promoters are constitutive promoters which are transcriptionally active in at least one cell, tissue or organ during most but not necessarily all stages of growth and development and under most environmental conditions, other promoters are inducible promoters, other examples are tissue-specific promoters, and still other examples are abiotic stress-inducible promoters.

[0597] When transformed in a plant, a chimeric gene (or expression cassette) expresses a nucleic acid which results in the expression of a protein.

[0598] Also disclosed is a recombinant vector comprising an expression cassette (or chimeric gene) as described hereinabove.

[0599] The term "terminator" includes a control sequence which is a DNA sequence at the end of a transcription unit which signals the 3'-processing and polyadenylation of the primary transcript and the termination of transcription. Terminators can be derived from natural genes, from a variety of other plant genes or from T-DNA. The terminator to be added can be derived, for example, from the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.

[0600] "Selectable marker", "selectable marker gene" or "reporter gene" includes any gene that confers a phenotype on the cells expressing it to facilitate the identification and / or selection of cells transfected or transformed with a nucleic acid construct of the present invention. These marker genes are capable of identifying the successful transfer of nucleic acid molecules by a variety of different principles. Suitable markers can be selected from those that confer antibiotic or herbicide resistance, introduce new metabolic traits or allow visual selection. Examples of selectable marker genes include genes that confer resistance to antibiotics (e.g., nptII that phosphorylates neomycin and kanamycin, or hpt that phosphorylates hygromycin, or genes that confer resistance to, for example, bleomycin, streptomycin, tetracycline, chloramphenicol, ampicillin, gentamicin, geneticin (G418), spectinomycin or blasticidin), genes for herbicide resistance (e.g., bar that provides resistance to ; aroA or gox that provides resistance to glyphosate, or genes that confer resistance to, for example, imidazolinone, phosphinothricin or sulfonylurea) or genes that provide metabolic traits (e.g., manA for xylose isomerase that allows plants to use mannose as the sole carbon source or for utilization of xylose, or antinutritional markers, such as resistance to 2-deoxyglucose). Expression of visual marker genes results in the formation of color (e.g., β-glucuronidase, GUS or β-galactosidase and their colored substrates, such as X-Gal), luminescence (e.g., luciferin / luciferase system) or fluorescence (green fluorescent protein, GFP and its derivatives). This list represents only a few of the possible markers. Those skilled in the art are familiar with these markers. Depending on the organism and the selection method, different markers are preferred.

[0601] It is known that after the stable or transient integration of nucleic acids into plant cells, only a small number of cells take up the foreign DNA and, if desired, integrate it into their genome, depending on the expression vector used and the transfection technique employed. To identify and select these integrants, a gene encoding a selectable marker (such as those described above) is usually introduced into the host cell together with the gene of interest. For example, these markers can be used in mutants in which these genes are non-functional due to deletion by, for example, conventional methods. In addition, the nucleic acid molecule encoding the selectable marker can be introduced into the host cell on the same vector containing the sequence encoding the polypeptide of the present invention or on the same vector used in the method of the present invention or in a separate vector. Cells that have been stably transfected with the introduced nucleic acid can be identified, for example, by selection (e.g., survival of cells that have integrated the selectable marker while other cells die).

[0602] Since once a nucleic acid has been successfully introduced, marker genes are no longer required or are dispensable in the transgenic host cell, in particular genes that confer resistance to antibiotics and herbicides, the method for introducing a nucleic acid according to the invention advantageously employs techniques that enable the removal or excision of these marker genes. One such method is so-called co-transformation. The co-transformation method uses two vectors for transformation simultaneously, one vector carrying the nucleic acid according to the invention and the second vector carrying the marker gene. Most transformants receive or, in the case of plants, contain (up to 40% or more of the transformants) both vectors. In the case of transformation with Agrobacterium, the transformants usually only receive a part of the vector, namely the sequence flanked by the T-DNA, which usually represents the expression cassette. Subsequently, the marker gene can be removed from the transformed plants by performing a cross. In another method, the marker gene integrated into a transposon is used for transformation together with the desired nucleic acid (so-called Ac / Ds technology). The transformants can be crossed with a source of transposase, or the transformants are transformed with a nucleic acid construct that confers transient or stable expression of the transposase. In some cases (about 10%), once the transformation is successful, the transposon jumps out of the genome of the host cell and is lost. In more cases, the transposon jumps to a different location. In these cases, the marker gene must be eliminated by performing a cross. In microbiology, techniques have been developed that make it possible or facilitate the detection of such events. Another advantageous method relies on so-called recombination systems; the advantage is that the cross-out can be dispensed with. The most well-known system of this type is the so-called Cre / lox system. Cre1 is a recombinase that removes the sequence located between the loxP sequences. If the marker gene is integrated between the loxP sequences, it is removed once the transformation is successful, i.e., by the expression of the recombinase. Further recombination systems are the HIN / HIX, FLP / FRT, and REP / STB systems (Tribble et al., J. Biol. Chem., 275, 2000:22255-22267; Velmurugan et al., J. Cell Biol., 149, 2000:553-566). The nucleic acid sequence according to the invention can be site-specifically integrated into the plant genome.

[0603] For the purposes of the present invention, "transgenic", "transgene" or "recombinant" means, for example, a nucleic acid sequence, an expression cassette comprising said nucleic acid sequence, a gene construct or a vector, or an organism transformed with a nucleic acid sequence, an expression cassette or a vector according to the invention.

[0604] Thus, transgenic plants for the purposes of the present invention are understood as described above, meaning that the nucleic acid used in the method of the present invention is not present in or does not originate from the genome of said plant, or is present in the genome of said plant but not at its natural locus in the genome of said plant, and the nucleic acid can be expressed homologously or heterologously. However, as described above, transgenic also means that although the nucleic acid according to the present invention or used in the method of the present invention is in its natural position in the plant genome, the sequence has been modified relative to the natural sequence, and / or the regulatory sequences of the natural sequence have been modified. Transgenic is preferably understood to mean that the nucleic acid according to the present invention is expressed at a non-natural locus in the genome, i.e., homologous or heterologous expression of the nucleic acid occurs. Preferred transgenic plants are mentioned herein.

[0605] The terms "expression" or "gene expression" refer to the transcription of a particular gene or a plurality of particular genes or a particular gene construct. The terms "expression" or "gene expression" particularly refer to the transcription of one or more genes or genetic constructs into structural RNAs (rRNA, tRNA) or mRNA, followed by the translation of the latter into a protein or not into a protein. The process includes the transcription of DNA and the processing of the resulting mRNA product.

[0606] As used herein, the terms "increased expression" or "overexpression" refer to any form of expression in addition to the original wild-type expression level. For the purposes of the present invention, the original wild-type expression level may also be zero, i.e., there is no expression or measurable expression.

[0607] Methods for increasing the expression of a gene or gene product are well documented in the art and include, for example, overexpression driven by a suitable promoter (as described hereinabove), the use of transcriptional enhancers or translational enhancers. The isolated nucleic acid used as a promoter or enhancer element can be introduced into the appropriate position (usually upstream) of the polynucleotide in a non-heterologous form to upregulate the expression of the nucleic acid encoding the polypeptide of interest. If polypeptide expression is desired, a polyadenylation region is usually required at the 3'-end of the polynucleotide coding region. The polyadenylation region can be derived from a natural gene, from a variety of other plant genes, or from T-DNA. The 3'-end sequence to be added can be derived, for example, from the nopaline synthase or octopine synthase gene, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.

[0608] Intron sequences can also be added to the 5' untranslated region (UTR) or to the coding sequence of a partial coding sequence to increase the amount of mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit of plant and animal expression constructs has been shown to increase gene expression up to 1000-fold at the mRNA and protein levels (Buchman and Berg (1988) Mol. Cell biol. 8:4395-4405; Callis et al. (1987) Genes Dev 1:1183-1200). This intron enhancement of gene expression is generally greatest when near the 5' end of the transcription unit. The use of maize introns Adh1-S introns 1, 2, and 6, Bronze-1 intron is known in the art. For general information, see: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).

[0609] As used herein, the terms "introducing" or "transforming" include the transfer of an exogenous polynucleotide or chimeric gene (or expression cassette) into a host cell, regardless of the method used for transfer. Plant tissues capable of subsequent clonal propagation by organogenesis or embryogenesis can be transformed with the gene constructs of the present invention and whole plants regenerated therefrom. The particular tissue chosen will vary depending on the clonal propagation system available for and most suitable to the particular species to be transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, female gametophytes, callus, existing meristems (e.g., apical meristems, axillary buds, and root meristems), and induced meristems (e.g., cotyledonary meristems and hypocotyl meristems). The polynucleotide can be introduced into the host cell transiently or stably and can remain non-integrated, such as as a plasmid. Alternatively, it can integrate into the host genome. The resulting transformed plant cells can then be used to regenerate transformed plants in a manner known to those skilled in the art.

[0610] The transfer of foreign genes into the plant genome is called transformation. Transformation of plant species is now a rather routine technique. Advantageously, any of a variety of transformation methods can be used to introduce a gene of interest into a suitable progenitor cell. The methods described for transforming and regenerating plants from plant tissues or plant cells can be used for transient or stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase the uptake of free DNA, direct injection of DNA into plants, particle gun bombardment, transformation using viruses or pollen, and microprojection. The methods can be selected from the calcium / polyethylene glycol method for protoplasts (Krens, F.A. et al., (1982) Nature 296, 72 - 74; Negrutiu I et al. (1987) Plant Mol Biol 8:363 - 373); electroporation of protoplasts (Shillito R.D. et al. (1985) Bio / Technol 3, 1099 - 1102); microinjection into plant material (Crossway A et al., (1986) Mol. Gen Genet 202:179 - 185); particle bombardment with DNA or RNA-coated particles (Klein T.M. et al., (1987) Nature 327:70), infection with (non-integrative) viruses, etc. Transgenic plants, including transgenic crop plants, are preferably produced by Agrobacterium-mediated transformation. One advantageous transformation method is transformation in planta. For this purpose, for example, Agrobacterium can be allowed to act on plant seeds or the plant meristem can be inoculated with Agrobacterium. According to the present invention, it has been shown to be particularly advantageous to allow a suspension of transformed Agrobacterium to act on a whole plant or at least on floral primordia. The plant is then allowed to grow until seeds of the treated plant are obtained (Clough and Bent, Plant J. (1998) 16, 735 - 743). Methods for Agrobacterium-mediated transformation of rice include well-known rice transformation methods, such as those described in any of the following: European Patent Application EP1198985, Aldemita and Hodges (Planta 199:612 - 617, 1996); Chan et al. (Plant Mol Biol 22(3):491 - 506, 1993), Hiei et al. (Plant J 6(2):271 - 282, 1994), the disclosures of which are incorporated herein by reference as if fully set forth.In the case of maize transformation, preferred methods are as described in Ishida et al. (Nat. Biotechnol 14(6):745-50, 1996) or Frame et al. (Plant Physiol 129(1):13-22, 2002), the disclosures of which are incorporated herein by reference as if fully set forth. For example, the methods are further described in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press (1993) 128-143 and Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The nucleic acid or construct to be expressed is preferably cloned into a vector suitable for transformation of Agrobacterium tumefaciens, such as pBin19 (Bevan et al (1984) Nucl. Acids Res. 12-8711). Agrobacterium transformed by such a vector can then be used in a known manner to transform plants, such as plants used as models, such as Arabidopsis (Arabidopsis thaliana is not considered a crop plant within the scope of the present invention) or crop plants, such as tobacco plants, for example by dipping wounded or shredded leaves into an Agrobacterium solution and then culturing them in a suitable medium. For example, Hofgen and Willmitzer describe the transformation of plants by Agrobacterium tumefaciens in Nucl. Acid Res. (1988) 16, 9877, or are especially known from F.F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.

[0611] In addition to transformed somatic cells (which then have to be regenerated into whole plants), it is also possible to transform cells of the plant meristem, especially those that develop into gametes. In this case, the transformed gametes follow the natural plant development, giving rise to transgenic plants. Thus, for example, seeds of Arabidopsis thaliana are treated with Agrobacterium and seeds are obtained from the developing plants, a certain proportion of which are transformed and thus transgenic [Feldman, K A and Marks M D (1987). Mol Gen Genet 208:1-9; Feldmann K (1992). In: C Koncz, N-H Chua and J Shell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore, pp. 274-289]. An alternative method is based on repeatedly removing the inflorescence and culturing the excised site at the center of the rosette leaves with transformed Agrobacterium, whereby transformed seeds can likewise be obtained at a later time point (Chang (1994). Plant J. 5:551 -558; Katavic (1994). Mol Gen Genet, 245:363-370). However, a particularly efficient method is the vacuum infiltration method and its improvements, such as the "floral dip method". In the case of vacuum infiltration of Arabidopsis thaliana, intact plants are treated with an Agrobacterium suspension under reduced pressure [Bechthold, N (1993). CR Acad Sci Paris Life Sci, 316:1 194-1 199], while in the case of the "floral dip method", the developing floral tissue is briefly incubated with a surfactant-treated Agrobacterium suspension [Clough, S J and Bent A F (1998) The Plant J. 16, 735-743]. A certain proportion of transgenic seeds are harvested in both cases, and these seeds can be distinguished from non-transgenic seeds by growing them under the above selective conditions. In addition, stable transformation of plastids is advantageous because plastids are maternally inherited, reducing or eliminating the risk of transgene flow through pollen in most crops. Transformation of the chloroplast genome is generally achieved by the process schematically shown in Klaus et al., 2004 [Nature Biotechnology 22(2), 225-229]. Briefly, the sequence to be transformed is cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site-specific integration into the plastid genome.Plastid transformation has been described for many different plant species and has been reviewed by Bock (2001) Transgenic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep 21; 312(3):425 - 38 or Maliga, P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21, 20 - 28. Further biotechnological advances have recently been reported in the form of marker - free plastid transformants, which can be generated from transiently co - integrated marker genes (Klaus et al., 2004, Nature Biotechnology 22(2), 225 - 229).

[0612] Genetically modified plant cells can be regenerated by all methods familiar to the person skilled in the art. Suitable methods can be found in the aforementioned publications by S.D. Kung and R.Wu, Potrykus or Hofgen and Willmitzer.

[0613] Typically, after transformation, plant cells or cell populations are selected for the presence of one or more markers encoded by plant - expressible genes co - transferred with the gene of interest, and the transformed material is then regenerated into whole plants. To select transformed plants, the plant material obtained in the transformation is usually subjected to selective conditions so that transformed plants can be distinguished from non - transformed plants. For example, seeds obtained in the above - mentioned manner can be planted and, after the initial growth period, appropriate selection can be carried out by spraying. Another possibility is, if appropriate, after sterilization, to culture the seeds on agar plates with a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, transformed plants can be screened for the presence of a selectable marker, such as the markers mentioned above.

[0614] After DNA transfer and regeneration, putative transformed plants can also be evaluated, for example using Southern analysis, to assess the presence of the gene of interest, copy number and / or genomic organization. Alternatively or additionally, Northern and / or Western analysis can be used to monitor the expression level of the newly introduced DNA, both of which techniques are well - known to the person skilled in the art.

[0615] The resulting transformed plants can be propagated in a variety of ways, such as by clonal propagation or classical breeding techniques. For example, the first generation (or T1) transformed plants can be self-pollinated and homozygous second generation (or T2) transformants selected, and then the T2 plants can be further propagated by classical breeding techniques. The resulting transformed organisms can take a variety of forms. For example, they can be chimeras of transformed and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and non-transformed tissue (e.g., in plants, a transformed rootstock is grafted onto an untransformed scion).

[0616] The invention will now be illustrated by the following non-limiting examples.

[0617] Example

[0618] Example 1: Preparation of the Folch Lower Phase Antigen of Fusarium oxysporum for Use as an Immunogen, Phage Display, and Screening Assays

[0619] Intact mycelia and conidia of Fusarium oxysporum were successively extracted at room temperature with chloroform:methanol in 2:1 and 1:2 (v / v) ratios. The extracts were combined and dried, and the crude lipid extract was partitioned as described by Folch et al (1957(1957).Asimple method forthe isolation and purification of total lipids from animaltissues.J.Biol.Chem.226,497–509.). Lipids recovered from the Folch lower phase were used for immunization.

[0620] Example 2: Preparation of the Ceramide Monohexoside Fraction of Fusarium oxysporum for Phage Display and Screening Assays

[0621] A fraction of monoglucosylceramides from Fusarium oxysporum was prepared as described in the method from Eliana Barreto-Bergter in Barreto-Bergter et al. (2011Barreto-Bergter E,Sassaki G and,de Souza LM.(2011).Structural analysis of fungalcerebrosides.Front Microbiol.2:239.).

[0622] Example 3: Identification of the Folch Lower Phase Antigen of Fusarium oxysporum Binding to VHH

[0623] 3.1 Immunization

[0624] VHHs were generated from llamas immunized with Folch lower phase extracts from Fusarium oxysporum. According to standard protocols, Folch lower phase extracts were spotted on thin layer chromatography (TLC) plates from Fusarium oxysporum and the llamas were boost immunized six times. Silica gel that had adsorbed the Folch lower phase extracts was scraped from the plates and suspended in phosphate buffer. The suspension was sonicated, mixed with Freund's incomplete adjuvant, and used for subcutaneous injection. All llamas remained healthy throughout the immunization process and blood samples were taken before and after immunization.

[0625] 3.2 Library construction

[0626] For library construction, peripheral blood mononuclear cells were prepared from blood samples of immunized llamas using Ficoll-Hypaque according to the manufacturer's instructions. Total RNA was extracted from these cells and used as starting material for RT-PCR to amplify VHH-encoding gene fragments. These fragments were cloned into the phagemid vector pASF20. pASF20 is an expression vector derived from pUC119 that contains a lacZ promoter, a synthetic leader sequence, a multiple cloning site, the coding sequence for the Escherichia coli bacteriophage pIII protein, an ampicillin resistance gene, and an M13 bacteriophage origin for single-strand production. Within the framework of the VHH-encoding sequence, this vector encodes a C-terminal (His)6 peptide tag and a c-myc peptide tag. Phage were prepared according to standard methods (Phage Display of Peptides and Proteins: A Laboratory Manual; Brian K. Kay, Jill Winter, Dr. John McCafferty). A library with a clone diversity equal to or greater than 1E+08 was obtained and phage were generated to ensure that antibody diversity was presented.

[0627] 3.3 Selection

[0628] Two rounds of panning were performed as follows: The fungal lipid fraction was always coated on polystyrene Maxisorp microtiter plates in 5% chloroform / methanol. In the first round of selection, 25 μl of phage (1.00E+11 phage / selection condition) were selected from the library using four different conditions: two different lipid fraction (fungal; Fusarium oxysporum) concentrations (50 μg / ml and 5 μg / ml) and two different blank conditions (5% CHCl3 / MeOH and PBS) for background control. For the second round of selection, in addition to the conditions of the first round of selection, a lower antigen concentration (0.5 μg / ml) was included. For the second round of selection, the phage input was reduced 10-fold compared to the input of the first round of selection, which reduced the selection of non-specific phage binding.

[0629] Good enrichment was observed in the first round of selection, especially for the output from the 50 μg / ml selection condition. The phage output from this selection was rescued, precipitated, and used as input for the second round of selection. In this round of selection, significantly higher enrichment was observed for the antigen-coated conditions, except for the 0.5 μg / ml selection condition.

[0630] Individual colonies of E. coli TG1 cells infected with the selected eluted phage pool obtained after the second round of panning were picked into 96-well plates, with the master plate (MP) containing 100 μl of 2xTY medium with 2% glucose and 100 μg / ml carbenicillin per well and incubated overnight at 37 °C. The master plate was stored at -80 °C in 20% glycerol and used for periplasmic extract production, screening, and sequencing.

[0631] Example 4: Screening of VHH Binding to the Lipid Fraction of Fusarium oxysporum

[0632] To verify whether the VHH binds to the Fusarium oxysporum lipid fraction, binding was evaluated by ELISA. Each individual clone was screened for binding to wells coated with 10 μg / ml fungal lipid fraction in 5% CHCl3 / MeOH and wells coated with 5% CHCl3 / MeOH only. A clone was considered positive when the ratio between the OD450nm binding signal of the antigen-coated well and the corresponding blank well was higher than 2-fold. Based on the defined cut-off criteria applied to the 360 individual clones tested, a total hit rate of 14.2% was obtained.

[0633] Example 5: VHH 10G11 Inhibits the Growth of Botrytis cinerea in a Dose-Dependent Manner in an In Vitro Antifungal Assay

[0634] The antifungal activity of VHH 10G11Q (SEQ ID NO:1) against the phytopathogenic fungus Botrytis cinerea R16 was evaluated in vitro.

[0635] Two-fold dilutions of purified VHH 10G11Q were prepared in 96-well microtiter plates. To 20 μl of these dilutions and 20 μl of water as a control, 80 μl of a fungal spore suspension (1E+05 spores / ml, in half-strength potato dextrose broth (PDB)) was added, starting from a final VHH 10G11Q concentration of 10 μM. The test plates were incubated at 25 °C for 36 h using an IncuCyte Zoom live cell imaging system. All tests were performed with at least 2 replicates.

[0636] Figure 1 The results of the antifungal activity assay shown indicated a distinct dose-dependent growth inhibition pattern, expressed as % fungal growth (total green object area) as a function of VHH 10G11Q concentration (μM).

[0637] Example 6: VHH 10G11 Is More Effective in Inhibiting the Growth of Botrytis cinerea in an In Vitro Antifungal Assay Compared to VHH 41D01 Binding to Glycosylceramide in Inhibiting the Growth of Botrytis cinerea

[0638] WO2014 / 177595A1 and WO2014 / 191146A1 describe anti-glucosylceramide-binding VHHs, where VHH41D01 shows the most significant antifungal activity against a variety of test strains, including Botrytis cinerea R16.

[0639] The growth inhibitory properties of VHH 10G11Q (SEQ ID NO:1) were compared with those of VHH 41D01 against the phytopathogenic fungus Botrytis cinerea R16 in vitro.

[0640] Two-fold dilutions of purified VHH 10G11Q and 41D01 were prepared in 96-well microtiter plates. To 20 μl of these dilutions and 20 μl of water as a control, 80 μl of a fungal spore suspension (1E+05 spores / ml, in half-strength potato dextrose broth (PDB)) was added, starting from a final VHH 10G11Q concentration of 40 μM. Using an IncuCyte Zoom live cell imaging system, the test plates were incubated at 25 °C for 48 hours. All tests were performed with at least 2 replicates.

[0641] Figure 2 The results of the antifungal activity assay shown surprisingly indicate that the growth inhibitory pattern of VHH10G11Q is more significant compared to VHH bioactivity 41D01.

[0642] Example 7: Site-Directed Mutagenesis

[0643] Site-directed mutagenesis of VHH was performed as follows. The nucleotide sequence encoding the VHH sequence variant was synthesized and constructed as a gene fragment, and further cloned into the pPpT4GAPαS plasmid suitable for transformation into Pichia pastoris ( et al(2012),Plos One,7(6):e39720). This plasmid contains the P AOX promoter to drive the expression of the cloned gene fragment. Standard cloning techniques were used during the construction of the plasmid containing the desired VHH sequence variant. Successful clones were sequenced to confirm the presence and correct cloning of the desired VHH sequence variant. Thereafter, the linearized plasmid was transformed into competent Pichia pastoris ATCC 76273 TM cells using standard electroporation protocols. Subsequently, the successful transformants were used to produce the VHH sequence variant. First, the transformants were cultured in BMGY (buffered glycerol-complex medium), and then transferred to BMMY (buffered methanol-complex medium) medium to initiate induction (Weidner et al(2020),J Vis Exp,36:1862). Subsequently, the VHH was purified using filtration and / or chromatography techniques well known in the art.

[0644] Example 8: Ala Scanning of VHH 10G11

[0645] All three CDR regions of VHH 10G11 were evaluated by Ala scanning to determine the effect of single amino acid substitutions on the antifungal effect of VHH 10G11 (SEQ ID NOs: 18 to 51). Variants of 10G11 were constructed and produced as described in Example 7, and subsequent antifungal assays were performed as described in Example 5. Surprisingly, in most cases where a single amino acid in the CDR region was replaced with alanine, 10G11 showed retained performance, as clearly demonstrated by the retained antifungal effect of the mutants ( Figure 3 ). Additional single amino acid substitutions were constructed using glycine (R26G; SEQ ID NO: 18 and D35G; SEQ ID NO: 27), serine (R53S; SEQ ID NO: 32 and T56S; SEQ ID NO: 36), or asparagine (K58N; SEQ ID NO: 39) in place of alanine, with similar results ( Figure 3 ).

[0646] Example 9: The His-Tagged Version of VHH 10G11 Has Enhanced Antifungal Activity

[0647] A His-tag is generally added to a polypeptide for purification using affinity chromatography. During routine testing using the antifungal assay described in Example 5, it was surprisingly found that the 10G11Q polypeptide with a 6x His-tag (SEQ ID NO: 148) added to the C-terminus had significantly enhanced antifungal activity ( Figure 4 ). Since the His-tag consists of six positively charged histidine amino acids, this suggests that positive charge may facilitate the antifungal interaction of 10G11.

[0648] Example 10: Computer Modeling of VHH 10G11Q

[0649] To better understand the 10G11 molecule, its 3-D structure was modeled computationally. A BLAST search of the Protein Data Bank (PDB) was performed to obtain the structural coordinates of antibodies with the same CDR length as the 10G11 molecule to be modeled. The Blosum 62 matrix was used with a gap cost of 13. Among the top 250 hits, each structure with the same CDR length as the 10G11 molecule under study was used for homology modeling attempts. First, the residues that differed between the obtained PDB structures and the 10G11 molecule were mutated. Then, as found in the Dunbrack 2010 rotamer library (Shapovalov and Dunbrack, (2011), Structure, 19(6):844-58), the side chains of these residues were modeled according to the observed preferences. If appropriate, the side chains in the immediate vicinity were also modeled to accommodate the modeled mutations. The model was used as a guide to identify the important amino acid residues of the molecule.

[0650] Figure 5 The resulting model with the indicated 3 CDR regions is shown. A selection of exposed side chains is shown on the ribbon diagram of 10G11, and the amino acids are indicated according to Kabat numbering ( Figure 5 The germline sequence is also shown and the substitutions leading to the 10G11 sequence are indicated. It also provides the corresponding Kabat number). These amino acid residues were selected as the best candidates for directed amino acid substitutions because the side chains are exposed and there is suspected to be minimal structural interference.

[0651] Example 11: Antifungal Activity of 10G11 Charge Variants

[0652] From Figure 3 the assay results shown, four-fifths of the mutants with reduced antifungal activity were characterized by the replacement of positively charged amino acids with uncharged variants. Along with the observation that the positively charged His-tagged variant had increased antifungal activity ( Figure 4 ), the effect of charge was further investigated. For this purpose, different 10G11 charge variants were generated as described in Example 7 and their antifungal activities were tested as described in Example 5. The following substitutions were designed to reduce the total charge of the 10G11 molecule at the antigen-binding interface of the molecule:

[0653] · Mutant 1: R26A substitution in CDR1 (SEQ ID NO:7)

[0654] · Mutant 2: R53A and K58A substitutions in CDR2 (SEQ ID NO:8)

[0655] · Mutant 3: R96A and R100cA substitutions in CDR3 (SEQ ID NO:9)

[0656] · Mutant 4: R26A substitution in CDR1, R53A and K58A substitutions in CDR2, and R96A and R100cA substitutions in CDR3 (SEQ ID NO:10)

[0657] · Mutant 5: R26A substitution in CDR1, R53A and K58A substitutions in CDR2, R96A and R100cA substitutions in CDR3, and K76N and K77T substitutions in the constant region (SEQ ID NO:11).

[0658] These mutants with reduced total charge showed reduced or absent antifungal activity ( Figure 6 ). This further emphasizes the speculation that positive charge is important for the activity of 10G11. Therefore, the following positively charged variants were designed based on the structural analysis described in Example 10:

[0659] · Mutant 6: S27H, I28H, and S30H substitutions in CDR1 (SEQ ID NO:12)

[0660] · Mutant 7: W100aH and T100bH substitutions in CDR3 (SEQ ID NO:13)

[0661] · Mutant 8: S27H, I28H, and S30H substitutions in CDR1 and W100aH and T100bH substitutions in CDR3 (SEQ ID NO:14)

[0662] · Mutant 9: S27K, I28K, and S30K substitutions in CDR1 (see Figure 7 )(SEQ ID NO:15)

[0663] · Mutant 10: W100aK and T100bK substitutions in CDR3 (SEQ ID NO:16)

[0664] · Mutant 11: S27K, I28K, and S30K substitutions in CDR1 and W100aK and T100bK substitutions in CDR3 (see Figure 8 )(SEQ ID NO:17)

[0665] Figure 6 It was shown that mutants 6 and 7 had antifungal activity comparable to that of 10G11. Mutants 8, 9, 10, and 11 showed significantly improved antifungal activity, with the antifungal activity of mutant 11 being 5-fold higher than that of 10G11.

[0666] Finally, an extended incubation antifungal assay was performed, in which the experimental setup as described in Example 5 was carried out for a period of 14 days, showing the sustained effect of mutants 9 and 11 in preventing spore growth over the evaluated time, in contrast to 10G11, which, although highly effective, allowed spore growth to partially resume after 3 days( Figure 9 ).

[0667] Example 12: 10G11 Binds to Fraction 3 of the Folch Lower Phase Extract

[0668] To investigate the putative interaction partners of 10G11 and its variants, lipids were extracted from Botrytis cinerea mycelia as described in Example 1. For further fractionation of the total lipid extract, thin layer chromatography (TLC; Skipski et al (1965), Biochimica et Biophysica Acta, 106(2):386-396) was used. The extract was spotted on a silica gel glass plate and run in a chromatography tank containing 100 mL of a chloroform / methanol mixture (85 / 15, v / v). Lipid bands separated by TLC were stained with α-naphthol, enabling visual detection of the lipids. Four fractions were identified( Figure 10 ): fraction 1, a fraction of polar lipids; fraction 2, with a retention factor (Rf) similar to that of a glucosylceramide reference standard (GlcCer from Tamogitake), likely a ceramide; fraction 3, with an Rf slightly higher than the reference standard; and fraction 4, non-polar phospholipids (PL). To obtain sufficient material from the individual fractions, normal-phase flash chromatography (Gorden, M.H. (2015). Encyclopedia of Analytical Science (Second edition), Elsevier) was used. Thus, the TLE was dissolved in CH2Cl2 by adding a few drops of MeOH and loaded onto a 4 g normal-phase flash column with 15 μm particles. The column was run with CH2Cl2 / MeOH (85 / 15, v / v) as the eluent. Finally, the fractions were filtered through a 0.45 μm syringe filter (nylon membrane) and dried. Thereafter, liposomes were prepared from the true lipid extract and its individual fractions by the thin film hydration method with 1,6-diphenyl-1,3,5-hexatriene (DPFI) (Trucillo et al, 2020. Processes 8(9):1022; Zhang, 2017. Liposomes 1522:1 7-22). DPH is almost non-fluorescent in aqueous media and exhibits strong fluorescence when it is incorporated into the lipid membrane. Any disruption of the lipid membrane results in a decrease in the fluorescence signal. Thereafter, the interaction of 10G11 with these liposomes was tested. Figure 11The results in indicate that 10G11 interacts with the lipids present in the total lipid extract (TLE), fraction 3, and fraction 4. Additionally, this suggests that 10G11 causes vesicle rupture, which points to an interesting effect of 10G11 on membrane disruption.

[0669] The binding of 10G...

Claims

1. A composition comprising at least one polypeptide, wherein, The polypeptide comprises the amino acid sequence listed in SEQ ID NO: 1 or SEQ ID NO: 2, and wherein the polypeptide comprises a CDR1 region having the amino acid sequence according to SEQ ID NO: 52, a CDR2 region having the amino acid sequence according to SEQ ID NO: 68, and a CDR3 region, optionally wherein the CDR3 region has the sequence according to SEQ ID NO: 84, and the polypeptide is capable of binding to a fungus, wherein the at least one polypeptide is an antibody or a functional fragment thereof.

2. The composition according to claim 1, wherein, The polypeptide is capable of binding to a fungus, thereby causing a delay in the growth of the spores of the fungus and / or lysis of the spores of the fungus.

3. The composition according to claim 1, wherein The at least one polypeptide is capable of binding to a lipid-containing fraction of the plasma membrane of Botrytis cinerea, and the lipid-containing fraction can be obtained by a method comprising: fractionating the mycelium of Botrytis cinerea by thin layer chromatography of the total lipid extract, and selecting the fraction having a retention factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipid fraction.

4. The composition according to claim 1, wherein The at least one polypeptide is a heavy chain variable domain of an antibody or a functional fragment thereof.

5. The composition according to claim 1, wherein, The at least one polypeptide specifically binds to at least one plasma membrane component of the fungus.

6. The composition according to claim 5, wherein, The at least one polypeptide is capable of binding to a lipid-containing fraction of the plasma membrane of Botrytis cinerea, and the lipid-containing fraction can be obtained by a method comprising: fractionating the mycelium of Botrytis cinerea by thin layer chromatography of the total lipid extract, and selecting the fraction having a retention factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipid fraction.

7. The composition according to claim 1, wherein The concentration range of the at least one polypeptide in the composition is from 0.0001% by weight to 50% by weight.

8. The composition according to claim 1, wherein The fungus is a phytopathogenic fungus.

9. The composition according to claim 8, wherein The genus of the phytopathogenic fungus is selected from Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Diplodia, Erysiphe, Fusarium, Mycosphaerella, Gaeumannomyces, Helminthosporium, Macrophomina, Nectria, Penicillium, Peronospora, Phoma, Physalospora, Phytophthora, Plasmopara, Podosphaera, Puccinia, Pyrenophora, Pyricularia, Pythium, Rhizoctonia, Sclerotium, Sclerotinia, Septoria, Thielaviopsis, Uncinula, Venturia, Verticillium, Magnaporthe grisea, Blumeria graminis, Mycosphaerella graminicola, Ustilago, Puccinia graminis, Phakopsora pachyrhizi, Monilinia, Mucor, Rhizopus, and Aspergillus.

10. The composition according to any one of the preceding claims, which is an agrochemical composition.

11. The composition according to claim 10, which further comprises an agriculturally chemically suitable carrier and / or one or more suitable adjuvants.

12. Use of the composition according to claim 11 as an antifungal agent.

13. Use according to claim 12, as an antifungal agent on plants.

14. A method for protecting or treating a plant or a part of the plant against infection by a phytopathogenic fungus, comprising at least the step of directly or indirectly applying the composition according to any one of claims 1 to 11 to the plant or the part of the plant under conditions effective to protect or treat the plant or the part of the plant against the infection by the phytopathogenic fungus.

15. A post-harvest treatment method for protecting or treating a harvested plant or a harvested part of said plant against fungal infections caused by phytopathogenic fungi, wherein, Comprising at least the step of directly or indirectly applying the composition according to any one of claims 1 to 11 to the harvested plant or the harvested part of the plant under conditions effective to protect or treat the harvested plant or the harvested part of the plant against the infection by the phytopathogenic fungus.

16. A method for inhibiting or killing the growth of phytopathogenic fungi, wherein, Comprising at least the step of directly or indirectly applying the composition according to any one of claims 1 to 11 to the plant or the part of the plant.

17. A polypeptide as defined in any one of claims 1 to 9.

18. A polynucleotide comprising a nucleic acid encoding the polypeptide according to claim 17.

19. A method for producing a transgenic plant, comprising introducing the polynucleotide according to claim 18 into a plant cell.

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