Antifungal Polypeptides

Abstract

Compositions containing polypeptides capable of binding to fungi are provided. [Solution] A composition is provided which contains at least one polypeptide, which is capable of binding to a fungus, thereby causing retardation of spore growth and / or lysis of spores of the fungus.

Description

[Technical Field]

[0001] The present invention relates to compositions containing at least one polypeptide capable of binding to fungi, and to the at least one polypeptide itself. The present invention also relates to the use of the compositions as antifungal agents. The present invention further relates to methods for protecting or treating plants or parts of plants against infection by phytopathogenic fungi, post-harvest treatment methods for protecting or treating harvested plants or harvested parts of plants against infection by phytopathogenic fungi, and methods for inhibiting the growth of or killing phytopathogenic fungi, comprising at least the step of applying the compositions or polypeptides directly or indirectly to plants or parts of plants. The present invention also relates to methods for producing antifungal polypeptides and methods for producing antifungal compositions. The present invention also relates to transgenic plants, plant parts, seeds, or plant cells. [Background technology]

[0002] The presence and persistence of pathogenic fungal infections in cultivated plants as well as in patients and animals appears to be primarily due to the selective pressure of broad-spectrum antifungal drugs and the generally inadequate efficacy of currently available antifungal drugs.

[0003] In humans and animals, systemic fungal infections, such as invasive candidiasis and invasive aspergillosis, are thought to be caused by a variety of fungal pathogens, including the highly virulent Candida species C. albicans, C. tropicalis, and C. krusei, as well as the less virulent species C. parapsilosis and Torulopsis glabrata (also known as C. glabrata). While C. albicans was previously the most common fungal isolate obtained from intensive care units, subsequent reports suggest that C. tropicalis, C. glabrata, C. parapsilosis, and C. krusei now account for approximately half of all such isolates. The rise of non-albicans species means the emergence of Candida species that are resistant to traditional antifungal drugs.

[0004] Traditionally, Candida albicans, Candida tropicalis, and Candida parapsilosis have been treated with amphotericin B, an antifungal drug considered the "gold standard" of systemic antifungal therapy. Unfortunately, amphotericin B itself is highly toxic, and its use is limited by side effects, including chills, fever, myalgia, and thrombophlebitis. Other antifungal drugs include oral azoles (miconazole, ketoconazole, itraconazole, and fluconazole) and 5-fluorocytosine. However, fungal species such as Candida krusei and Torulopsis glabrata are resistant to fluconazole, and these species often develop in patients receiving prophylactic treatment with this drug. Furthermore, fluconazole-resistant strains of Candida albicans have also been reported. Therefore, despite advances in antifungal treatments, the need for effective treatments for fungal infections remains urgent.

[0005] In agriculture, crop protection relies heavily on the use of pesticides, which are applied to crops by spraying, irrigating, or injecting into the soil. Pesticides are often organic chemical molecules, and repeated application to crops poses a toxic threat to both agricultural workers and the environment through drift, soil residue, or runoff into surface or groundwater. It would be advantageous to have alternative compounds that provide effective control of plant pests while also offering low toxicity to humans and the environment. Protein pesticides with specificity for a given plant pest target would be highly advantageous in this regard, as they are expected to have short environmental residence times and low toxicity and off-target effects. However, only a few protein or peptide pesticides are known. Examples include Bt toxins, lectins, defensins, favatins, tachyplesins, magainins, harpins (see WO2010019442), and pea albumin 1 subunit b (PA1b). However, these protein pesticides are either small peptides with compact structures stabilized by several disulfide bonds or large proteins (>300 amino acids) that exist in crystalline form (cry toxins). In fact, biological products, especially proteins, are known in the agricultural field to be challenging structures for pesticide development because they generally have extremely low stability and cannot maintain their pesticidal function, especially in pesticide formulations for field use. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2010 / 019442 Summary of the Invention [Problem to be solved by the invention]

[0007] The present inventors have succeeded in developing polypeptides with surprisingly high specificity, affinity, and potency against targets of pests, particularly 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 in the cell membrane of fungal spores. The binding alone of the polypeptides is sufficient for fungicidal activity, preventing mycelium formation by retarding spore growth and / or lysis and rupture. Thus, the polypeptides can have fungicidal or fungistatic activity.

[0008] Furthermore, these polypeptides are able to maintain their integrity, stability and activity in compositions, and surprisingly, effective pest or pathogen control can be achieved by applying compositions containing the polypeptides disclosed herein to crops, animals or humans.

[0009] The efficacy and potency of the polypeptides disclosed herein suggest that lower dosages may be possible and / or more effective treatments may be possible at the same dose. This may lead to fewer undesirable side effects and less toxicity in both agricultural and pharmaceutical applications. Furthermore, it may also be possible to apply the polypeptides or compositions disclosed herein in lower amounts or dosages.

[0010] More specifically, the present inventors have found that targeted delivery of the polypeptides envisaged herein to the molecular structure of pests or pathogens allows for efficient control of said pathogens.

[0011] In particular, the inventors have developed polypeptides that are capable of preventing, protecting, treating or treating plants, animals or humans against (the occurrence of) infection by a pathogen or any other biological interaction with a pathogen, in particular a fungal pathogen. 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 against any damage due to biological interaction of the plant, animal or human with a pathogen (e.g. via infection by a pathogen) or against exposure to such an interaction.

[0012] The polypeptides and compositions of the present invention can be used as stand-alone products, for example stand-alone compositions such as antifungal compositions.

[0013] The polypeptides and compositions of the present invention can be used protectively or prophylactically, ie, the initial application is before disease is present.

[0014] The polypeptides and compositions of the present invention can be used as contact fungicides. [Means for solving the problem]

[0015] Thus, according to the present invention, there is provided a composition comprising at least one polypeptide, said polypeptide being capable of binding to a fungus, thereby causing a delay in the growth of and / or lysis of spores of said fungus, i.e., binding of said polypeptide to a fungus results in a delay in the growth of and / or lysis of said fungal spores.

[0016] Polypeptides are also provided, which are capable of binding to a fungus, such that the polypeptides result in the retardation of spore growth and / or lysis of the fungal spores, i.e., the binding of the polypeptides to a fungus results in the retardation of spore growth and / or lysis of the fungal spores.

[0017] The polypeptides of the present invention and used in the present invention can bind (specifically) to fungal membranes or components of fungal membranes. In certain embodiments, the polypeptides of the present invention and used in the present invention do not bind (specifically) to fungal cell walls or components of cell walls. For example, in certain embodiments, the polypeptides of the present invention and used in the present invention do not bind (specifically) to fungal glucosylceramide.

[0018] The present invention also provides compositions containing at least one polypeptide, wherein the at least one polypeptide is capable of binding to a lipid-containing fraction of the cell membrane of a fungus (e.g., Botrytis cinerea or other fungi). The lipid-containing fraction may be obtainable by chromatography. For example, the lipid-containing fraction may be obtainable by a method comprising fractionating fungal (e.g., Botrytis cinerea or other fungi) mycelia by total lipid extract thin-layer chromatography and selecting fractions having a retention factor (Rf) greater than that of a ceramide fraction and less than that of a non-polar phospholipid fraction.

[0019] The present invention also provides polypeptides, wherein the at least one polypeptide is capable of binding to a lipid-containing fraction of the cell membrane of a fungus (e.g., Botrytis cinerea or other fungi). The lipid-containing fraction can be obtainable by chromatography. For example, the lipid-containing fraction can be obtainable by a method comprising fractionating fungal (e.g., Botrytis cinerea or other fungi) mycelia by total lipid extract thin-layer chromatography and selecting fractions having a retention factor (Rf) greater than that of a ceramide fraction and less than that of a non-polar phospholipid fraction.

[0020] Furthermore, the present invention provides a composition containing at least one polypeptide, wherein the polypeptide comprises an 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 to any one of them, and the polypeptide is capable of binding to a fungus.

[0021] According to the present invention there is also provided a composition comprising at least one polypeptide, said polypeptide comprising: a CDR1 region having the amino acid sequence set forth in SEQ ID NO: 52, a CDR2 region having the amino acid sequence set forth in SEQ ID NO: 68, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 84, wherein the polypeptide is capable of binding to a fungus; a CDR1 region having the amino acid sequence set forth in SEQ ID NO: 53, a CDR2 region having the amino acid sequence set forth in SEQ ID NO: 69, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 85, wherein the polypeptide is capable of binding to a fungus; a CDR1 region having the amino acid sequence set forth in SEQ ID NO: 54, a CDR2 region having the amino acid sequence set forth in SEQ ID NO: 70, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 86, wherein the polypeptide is capable of binding to a fungus; or The polypeptide comprises a CDR1 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 52 to 67 and 112 to 122, a CDR2 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 68 to 83 and 123 to 133, and a CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 84 to 100 and 134 to 144, and the polypeptide is capable of binding to fungi.

[0022] The present invention further comprises: a polypeptide comprising an 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 to any one of them, and capable of binding to a fungus; a polypeptide comprising a CDR1 region having the amino acid sequence set forth in SEQ ID NO: 52, a CDR2 region having the amino acid sequence set forth in SEQ ID NO: 68, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 84, and capable of binding to a fungus; a polypeptide comprising a CDR1 region having the amino acid sequence set forth in SEQ ID NO: 53, a CDR2 region having the amino acid sequence set forth in SEQ ID NO: 69, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 85, and capable of binding to a fungus; a polypeptide capable of binding to a fungus, comprising a CDR1 region having the amino acid sequence set forth in SEQ ID NO: 54, a CDR2 region having the amino acid sequence set forth in SEQ ID NO: 70, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 86; Provided is a polypeptide capable of binding to fungi, which comprises a CDR1 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 52 to 67 and 112 to 122, a CDR2 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 68 to 83 and 123 to 133, and a CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 84 to 100 and 134 to 144.

[0023] The present invention further provides a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-51 and 101-111.

[0024] The present invention further provides a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10, 12-51, and 101-111.

[0025] The present invention further provides polypeptides having a CDR1 region comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 52 to 67 and 112 to 122, a CDR2 region comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 68 to 83 and 123 to 133, and a CDR3 region comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 84 to 100 and 134 to 144.

[0026] The present invention further provides polypeptides comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 51, or an amino acid sequence having up to 1, 2, 3, 4, or 5 amino acid substitutions relative to said sequence, which may increase or leave the total charge of said polypeptide unchanged.

[0027] The present invention further provides a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 10 and 12 to 51, or an amino acid sequence having up to one, two, three, four, or five amino acid substitutions relative to said sequence. The amino acid substitutions may increase the total charge of the polypeptide, or may not change the total charge of the polypeptide.

[0028] Any of the polypeptides of the present invention can be provided in a composition, for example, an agrochemical composition.

[0029] The compositions disclosed herein can contain at least one antibody or functional fragment thereof, including, but not limited to, a heavy chain antibody or functional fragment thereof.

[0030] The compositions disclosed herein comprise at least one heavy chain variable domain (V) of a heavy chain antibody that does not naturally have a light chain. HH ) or a functional fragment thereof, for example, but not limited to, the heavy chain variable domain of a camel heavy chain antibody (camel V HH ) or a functional fragment thereof.

[0031] The compositions disclosed herein comprise at least one camelized heavy chain variable domain (camelized V) of a conventional four-chain antibody. H ), or a functional fragment thereof.

[0032] The compositions disclosed herein are natural V H The amino acid sequence of the domain (e.g., a naturally occurring V domain derived from a mammal, particularly a human) H The antibody may contain at least one heavy chain variable domain or a functional fragment thereof of an antibody that does not have an amino acid sequence exactly identical (i.e., 100% sequence identity) to the amino acid sequence of the heavy chain variable domain.

[0033] The compositions disclosed herein can be pesticide compositions.

[0034] The pesticide compositions disclosed herein may contain at least a polypeptide that specifically binds to at least one cell membrane component of a fungus.

[0035] The at least one fungal cell membrane component to which the polypeptide contained in the composition disclosed herein binds may be a non-protein.

[0036] The at least one polypeptide in the pesticide composition disclosed herein can be present in an amount effective to protect or treat humans, animals, plants, or any part thereof against infection by or other biological interactions with fungal pathogens, and for example, but not limited to, the concentration of the polypeptide in the pesticide composition is 0.0001 to 50% by weight.

[0037] The at least one polypeptide in the pesticide compositions disclosed herein can optionally be formulated in an aqueous solution with a suitable base and / or one or more suitable adjuvants, such as, but not limited to, an agrochemically suitable base and / or one or more suitable adjuvants.

[0038] The pesticide compositions disclosed herein can contain at least one polypeptide that specifically binds to a pathogenic fungus, i.e., a plant pathogenic fungus.

[0039] The pesticide composition disclosed herein is effective against, but not limited to, the following species of fungi: Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Diplodia, Erysiphe, Fusarium, Leptosphaeria, Gaeumanomyces, Helminthosporum, and the like. Helminthosporium, Macrophomina, Nectria, Penicillium, Peronospora, Phoma, Phymatotrichum, Phytophthora, Plasmopara, Podosphaera, Puccinia, Pyrenophora renophora, Pyricularia, Pythium, Rhizoctonia, Scerotium, Sclerotinia, Septoria, Thielaviopsis, Uncinula, Venturia, Verticillium, Magnaporthe, The fungus may contain at least one polypeptide that specifically binds to a plant pathogenic fungus, such as a plant pathogenic fungus of a genus selected from the group including Blumeria, Mycosphaerella, Ustilago, Melampsora, Phakospora, Monilinia, Mucor, Rhizopus, and Aspergillus.

[0040] The pesticide compositions disclosed herein may comprise at least one polypeptide that specifically binds to a fungus that is a fungus of a plant selected from the group including cereals, sorghum, rice, sugar beet, fodder beet, fruit trees, nuts, plantago or grapevines, legume crops, oil crops, cucurbits, fiber plants, fuel crops, vegetables, ornamentals, shrubs, broadleaf trees, evergreen trees, grasses, coffee, tea, tobacco, hops, pepper, rubber and latex plants.

[0041] The at least one polypeptide in the pesticide composition disclosed herein comprises at least [ka] or a sequence having at least about 80% sequence identity to either thereof, wherein the polypeptide is capable of binding to a fungus.

[0042] The at least one polypeptide in the pesticide composition disclosed herein may comprise at least the amino acid sequences of a CDR1 region having the sequence RSIFSINAMD (SEQ ID NO: 52), a CDR2 region having the sequence GITRGGTTK (SEQ ID NO: 68), and a CDR3 region having the sequence LRGEQPWTRDY (SEQ ID NO: 84), and the polypeptide is capable of binding to a fungus.

[0043] The at least one polypeptide in the pesticide composition disclosed herein may comprise at least the amino acid sequences of a CDR1 region having the sequence GTIFRPTAMG (SEQ ID NO: 53), a CDR2 region having the sequence TITTGGSTK (SEQ ID NO: 69), and a CDR3 region having the sequence QWGVRTRDY (SEQ ID NO: 85), and the polypeptide is capable of binding to a fungus.

[0044] The at least one polypeptide in the pesticide composition disclosed herein may comprise at least the amino acid sequences of a CDR1 region having the sequence ISDRAFSRHV (SEQ ID NO: 54), a CDR2 region having the sequence AIGWTGRRTY (SEQ ID NO: 70), and a CDR3 region having the sequence SHFYSVSFEINDYD (SEQ ID NO: 86), and the polypeptide is capable of binding to a fungus.

[0045] The at least one polypeptide in the pesticide composition disclosed herein can comprise at least the CDR1, CDR2 and CDR3 regions of any of the other polypeptides disclosed herein.

[0046] The polypeptides disclosed herein are generally capable of binding to fungi.

[0047] In another aspect, the present invention provides compositions containing at least one polypeptide that specifically binds to fungi for use as an antifungal agent. The present invention also provides polypeptides that specifically bind to fungi for use as an antifungal agent.

[0048] Accordingly, the present invention provides a composition comprising at least one polypeptide, said polypeptide comprising: or an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-51 and 101-111, or an amino acid sequence having at least about 80% sequence identity to any of them, wherein the polypeptide is capable of binding to fungi for use as an antifungal agent; or The polypeptide comprises a CDR1 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 52-67 and 112-122, a CDR2 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 68-83 and 123-133, and a CDR3 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 84-100 and 134-144, and is capable of binding to fungi. The present invention further provides use of the composition or polypeptide disclosed herein as an antifungal agent. Such use can be as an antifungal agent for plants. Accordingly, the present invention provides use of an agrochemical composition containing at least one polypeptide that specifically binds to fungi as an antifungal agent for plants.

[0049] In the present invention, the antifungal agent may be a fungicidal and / or fungicidal agent.

[0050] The present invention further provides nucleic acid sequences encoding any of the polypeptide sequences disclosed herein.

[0051] The present invention also provides a method for protecting or treating a plant or a part of a plant against infection by a phytopathogenic fungus, comprising at least the step of directly or indirectly applying the pesticide composition or polypeptide disclosed herein to the plant or the part of the plant. The pesticide composition or polypeptide can be applied under conditions effective for protecting or treating the plant or the part of the plant against infection by the phytopathogenic fungus.

[0052] These methods can include the step of directly or indirectly applying the pesticidal composition or polypeptide disclosed herein to the plant or part of the plant, for example at a rate of more than 50 g of the pesticidal composition or polypeptide per hectare, including but not limited to, a rate of more than 75 g of the pesticidal composition or polypeptide per hectare, for example, a rate of more than 100 g of the pesticidal composition or polypeptide per hectare, or particularly a rate of more than 200 g of the pesticidal composition or polypeptide per hectare.

[0053] These methods may include a step of directly or indirectly applying the pesticide composition or polypeptide disclosed herein to the plant or a part of the plant, for example, at an application rate of 50 g to 100 g of the pesticide composition or polypeptide per hectare; examples include, but are not limited to, an application rate of 50 g to 200 g of the pesticide composition or polypeptide per hectare, particularly an application rate of 75 g to 175 g of the pesticide composition or polypeptide per hectare, for example, 75 g to 150 g of the pesticide composition or polypeptide per hectare or 75 g to 125 g per hectare.

[0054] The pesticide compositions or polypeptides disclosed herein can be applied directly or indirectly to the plants or parts of the plants, optionally after harvest, by spraying, misting, foaming, fogging, hydroponics, coating, dipping, and / or dressing.

[0055] The present invention further provides a post-harvest treatment method for protecting or treating a harvested plant or a harvested part of a plant against infection by a phytopathogenic fungus, the method comprising at least the step of applying, directly or indirectly, to the harvested plant or a harvested part of the plant an agrochemical composition or polypeptide disclosed herein under conditions effective to protect or treat the harvested plant or a harvested part of the plant against infection by the phytopathogenic fungus.

[0056] The present invention further provides a method for inhibiting the growth of or killing a plant pathogenic fungus, comprising at least the step of applying, directly or indirectly, to a plant or a part of said plant, an agrochemical composition or polypeptide disclosed herein.

[0057] These methods involve applying the pesticidal compositions or polypeptides disclosed herein directly or indirectly to the plant or plant part, optionally after harvest, by spraying, misting, foaming, fogging, hydroponics, hydroponics, coating, dipping, and / or dusting.

[0058] In yet another aspect, the present invention provides a method for producing a polypeptide that specifically binds to and / or has affinity for a fungus, said method comprising: immunizing an animal with a fungal target, or a suitable antigenic determinant based on or derived from a fungus (e.g., an antigenic portion thereof, a fragment thereof, a region thereof, a domain thereof, a loop thereof, or other epitope thereof); obtaining a cell collection or sample from said immunized animal that expresses the polypeptide sequence; screening the cell collection or sample for cells expressing an amino acid sequence that binds to and / or has affinity for the fungal target; (i) isolating said amino acid sequence, or (ii) isolating a nucleic acid sequence encoding said amino acid sequence from said cell collection or sample; and expressing the amino acid sequence, This produces a polypeptide that specifically binds to and / or has affinity for fungi.

[0059] Immunization can be performed using crude lipid extracts or total lipid extracts. For example, in certain embodiments, fungal mycelia and / or conidia (e.g., fungal mycelia and / or conidia of Fusarium oxysporum or Botrytis cinerea) can be extracted at room temperature using, for example, chloroform:methanol in a 2:1 and 1:2 (v / v) ratio. The extracts thus prepared can be combined and dried to provide crude lipid extracts or TLEs for immunization.

[0060] The fungal target can be a lipid-containing fraction of the cell membrane of a fungus (e.g., Botrytis cinerea). The lipid-containing fraction can be obtainable by chromatography. For example, the lipid-containing fraction can be obtainable by a method comprising fractionating fungal (e.g., Botrytis cinerea or other fungi) mycelia by total lipid extract thin-layer chromatography and selecting fractions having a retention factor (Rf) greater than the ceramide fraction and less than the non-polar phospholipid fraction.

[0061] A method for producing an antifungal composition is also provided, said method comprising the steps of producing an antifungal polypeptide according to the method described above and combining said antifungal polypeptide with one or more suitable bases and / or one or more suitable adjuvants.

[0062] Also provided by the present invention are transgenic plants, plant parts, seeds, or plant cells comprising a nucleic acid sequence encoding a polypeptide described herein. [Brief explanation of the drawings]

[0063] [Figure 1] Figure 1 shows fungal growth in the presence of increasing doses of VHH10G11Q monitored by IncuCyte. [Figure 2] Figure 1 shows the results of fungal growth monitored by IncuCyte in the presence of increasing doses of VHH10G11Q and VHH41D01. [Figure 3] 1 shows the antifungal activity of single amino acid substitutions (SEQ ID NOS: 17 to 50) in the CDR region of 10G11, and the results of comparison with 10G11 activity. [Figure 4] 1 shows the antifungal activity of his-tagged mutants of 10G11Q-His (SEQ ID NO: 6 and SEQ ID NOs: 17 to 50), compared with the activity of 10G11Q. [Figure 5] A model protein structure of the 10G11 molecule is shown with representations of the three CDR regions, with selected amino acid residues shown as stick models in a ribbon diagram. [Figure 6]1 shows the antifungal activity of 10G11 charge mutants, compared with the activity of 10G11. [Figure 7] The modeled protein structure of 10G11 charge mutation mutant 9 (SEQ ID NO: 14) is shown with representations of the three CDR regions, with selected amino acid residues shown as stick models in a ribbon diagram. [Figure 8] The modeled protein structure of 10G11 charge mutation mutant 11 (SEQ ID NO: 16) is shown with representations of the three CDR regions, with selected amino acid residues shown as stick models in a ribbon diagram. [Figure 9] The results of an extended antifungal assay are shown, demonstrating the effect of mutants 3, 9 and 11 and the 10G11 molecule. [Figure 10] Thin layer chromatographic separation of four different fractions present in the total lipid extract is shown. [Figure 11] 1 shows the results of binding of 10G11 to liposome vesicles of various compositions containing the fluorescent molecule DPD. [Figure 12] The binding profile of 10G11 to fraction 3 was determined by biolayer interferometry (BLI) and is shown compared to a reference VHH. [Figure 13] The results of detecting 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 by ELISA are shown. [Figure 14] Microscope images of untreated and 10G11-treated Botrytis cinerea are shown. A close-up of the treated Botrytis cinerea is shown on the far right. [Figure 15] Figure 1 shows the change in %PESSEV of Asian soybean rust in the lower part of the canopy after application of different compounds. [Figure 16] Figure 1 shows the change in %PESSEV of Asian soybean rust in the mid-canopy after application of different compounds. [Figure 17] Figure 1 shows the change in %PESSEV of Asian soybean rust in the upper canopy after application of different compounds. [Figure 18]Figure 1 shows the change in severity of anthracnose on pumpkin after application of various compounds. [Figure 19] Figure 1 shows the change in severity of Botrytis cinerea on grape bunches after application of various compounds. [Figure 20] Figure 1 shows the change in severity of powdery mildew on grape leaves after application of various compounds. [Figure 21] Figure 1 shows the change in powdery mildew severity on grape bunches after application of various compounds. [Figure 22] Figure 1 shows the change in severity of powdery mildew on tomato after application of various compounds. [Figure 23] Figure 1 shows the change in incidence of powdery mildew on tomato after application of various compounds. [Figure 24] Figure 1 shows the change in strawberry powdery mildew severity after application of various compounds. [Figure 25] Figure 1 shows the change in incidence of powdery mildew on strawberry after application of various compounds. [Figure 26] The number of strawberry fruits infected with Botrytis cinerea at harvest after application of various compounds is shown. [Figure 27] Figure 1 shows the change in severity of Botrytis cinerea on post-harvest strawberry fruit after application of various compounds. [Figure 28] Figure 1 shows the change in strawberry powdery mildew severity after application of various compounds. [Figure 29] The ELISA absorbance plot used to determine Kd is shown. [Figure 30-1] The figure shows several example sequences of the polypeptide of the present invention, including the SEQ ID NO of the full-length sequence and the locations of the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 regions. If there is a discrepancy between the sequence in this figure and the sequence in the sequence listing, the sequence in this figure takes precedence. [Figure 30-2] The figure shows several example sequences of the polypeptide of the present invention, including the SEQ ID NO of the full-length sequence and the locations of the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 regions. If there is a discrepancy between the sequence in this figure and the sequence in the sequence listing, the sequence in this figure takes precedence. [Figure 30-3] The figure shows several example sequences of the polypeptide of the present invention, including the SEQ ID NO of the full-length sequence and the locations of the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 regions. If there is a discrepancy between the sequence in this figure and the sequence in the sequence listing, the sequence in this figure takes precedence. [Figure 30-4] The figure shows several example sequences of the polypeptide of the present invention, including the SEQ ID NO of the full-length sequence and the locations of the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 regions. If there is a discrepancy between the sequence in this figure and the sequence in the sequence listing, the sequence in this figure takes precedence. [Figure 30-5] The figure shows several example sequences of the polypeptide of the present invention, including the SEQ ID NO of the full-length sequence and the locations of the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 regions. If there is a discrepancy between the sequence in this figure and the sequence in the sequence listing, the sequence in this figure takes precedence. [Figure 31] The antifungal activity of 10G11 in which the entire CDR2 was replaced with a germline sequence and 10G11 in which the entire CDR3 region was replaced with the CDR3 of a reference VHH is shown. The results are compared with the activity of 10G11.

[0064] Sequence Listing Description The sequence listing shows at least the following sequences in Table 9:

[0065] [Table 1] TIFF2026041806000003.tif100166 DETAILED DESCRIPTION OF THE INVENTION

[0066] Detailed Description of the Invention Any reference herein to prior art is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art constitutes general common general knowledge in any country.

[0067] All references cited herein are incorporated herein by reference in their entirety. Unless otherwise defined, all terms used in disclosing the present invention, including scientific and technical terms, shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0068] The present invention will be described below with respect to specific embodiments, but the present invention is not limited to these embodiments, but only by the claims. Any reference signs in the claims should not be construed as limiting the scope of the claims.

[0069] definition Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps.

[0070] Where an indefinite or definite article such as "a" or "an" or "the" is used when referring to a singular noun, this also includes a plural of that noun, unless otherwise stated.

[0071] The term "about" used herein when referring to a measurable value such as a parameter, amount, time, etc., means that the specified value can vary by ±10% or less, preferably ±5% or less, more preferably ±1% or less, and even more preferably ±0.1% or less, provided that such variations are appropriate for the invention disclosed herein. Naturally, the numerical value to which "about" is attached is also disclosed as a specifically preferred numerical value.

[0072] The following terms or definitions are intended solely to aid in the understanding of the present invention. Unless otherwise defined herein, all terms used herein have the same meaning as they would to one of ordinary skill in the art of the present invention. For definitions and terms in the art, practitioners should refer, inter alia, to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2004. nded., 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 to be narrower than would be understood by one of ordinary skill in the art.

[0073] Unless otherwise specified, all methods, steps, techniques and operations not specifically described in detail can and have been carried out in a manner known per se, as will be apparent to those skilled in the art, who again refer, for example, to standard handbooks, the general background art cited above and other references cited in this application.

[0074] As used herein, the terms "polypeptide," "protein," "peptide," and "amino acid sequence" are used interchangeably and refer to polymeric forms of amino acids of any length, and can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones.

[0075] As used herein, amino acid residues may be referred to by their full letter designation or according to the standard three-letter or one-letter amino acid code.

[0076] As used herein, the terms "nucleic acid sequence," "polynucleotide," "polynucleic acid," and "nucleic acid" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. 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, regulatory regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule can be linear or circular.

[0077] As used herein, the term "homology" refers to at least a secondary structural similarity between two polymers, particularly two polypeptides or polynucleotides, derived from the same or different taxa, where the similarity is attributable to a common ancestor. Accordingly, the term "homologous" refers to such related polymers having the secondary structural similarity and, optionally, tertiary structural similarity. To compare two or more nucleotide sequences, methods known to those skilled in the art can be used to calculate the "percentage of sequence identity" between a first nucleotide sequence and a second nucleotide sequence, such as by dividing the number of nucleotides in the first nucleotide sequence that are identical to nucleotides at 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. When determining sequence identity between two amino acid sequences, those skilled in the art can consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which the replacement of an amino acid residue with another amino acid residue of similar chemical structure has little or no effect on the function, activity, or other biological properties of the polypeptide. Possible conservative amino acid substitutions will be apparent to those skilled in the art. Amino acid and nucleic acid sequences are said to be "exactly identical" if they share 100% sequence identity over their entire length.

[0078] The term "complementarity-determining region" or "CDR" as used herein in the context of antibodies refers to the variable region of either the heavy (H) or light (L) chain (abbreviated as VH and VL, respectively), which contains amino acid sequences capable of specifically binding to antigen targets. These CDR regions determine the antibody's base specificity for a particular antigenic determinant structure. Such regions are also called "hypervariable regions." Although CDRs represent discontinuous stretches of amino acids within the variable region, the locations of these important amino acid sequences within the heavy and light chain variable regions are known to have similar positions within the amino acid sequences of the variable chains, regardless of molecular species. The heavy and light chain variable regions of all standard antibodies each contain three CDR regions (designated L1, L2, L3, H1, H2, and H3) that are discontinuous with each other in the respective light (L) and heavy (H) chains.

[0079] The term "affinity" as used herein 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 in such a way as to shift the equilibrium between the antigen and the polypeptide toward the presence of a complex formed by their binding. Thus, for example, if an antigen and an antibody (fragment) are combined at relatively equal concentrations, an antibody (fragment) with high affinity will bind to the available antigen in such a way as to shift the equilibrium toward a higher concentration of the resulting complex. Dissociation constants are widely used to describe the affinity between a protein binding domain and an antigen target. Generally, dissociation constants are in the range of 10 -5 The dissociation constant is less than 10 -6 Preferably, it is less than 10 -7 More preferably, the dissociation constant is less than 10 -8 It is most preferably less than M.

[0080] 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 particular antigen present in a homogeneous mixture of different antigens. In certain embodiments, a specific binding interaction will distinguish between desired and undesired antigens in a sample, and in certain embodiments, will distinguish by about 10-fold to more than 100-fold or more (e.g., about 1000-fold or more than 10,000-fold).

[0081] Thus, an amino acid sequence disclosed herein is said to "specifically bind" to a particular target when the amino acid sequence has affinity for, specificity for, and / or specifically associates with (or at least a portion or fragment thereof).

[0082] The "specificity" of the amino acid sequences disclosed herein can be determined based on affinity and / or avidity.

[0083] An amino acid sequence disclosed herein is said to be "specific for a first target antigen of interest relative to a second target antigen of interest" if it binds to the first target antigen of interest with an affinity that is at least 5-fold, e.g., at least 10-fold, e.g., at least 100-fold, and preferably at least 1000-fold greater than the affinity with which the amino acid sequence binds to the second target antigen of interest. Thus, in certain embodiments, when an amino acid sequence disclosed herein is said to be "specific for" a first target antigen of interest relative to a second target antigen of interest, the amino acid sequence is capable of specifically binding to the first target antigen of interest (as defined herein) but not to the second target antigen of interest.

[0084] As used herein, the terms "inhibit," "suppress," and / or "interfere" refer to (the use of) an amino acid sequence disclosed herein that specifically binds to a target antigen of interest and inhibits, inhibits, and / or disrupts the interaction of the target antigen of interest with its natural binding partner. The terms "inhibit," "suppress," and / or "interfering" can also refer to (the use of) an amino acid sequence disclosed herein that specifically binds to a target antigen of interest and inhibits, inhibits, and / or disrupts a biological activity of the target antigen of interest as measured using an appropriate in vitro, cellular, or in vivo assay. Thus, "inhibit," "suppress," and / or "interfere" can also refer to (the use of) an amino acid sequence disclosed herein that specifically binds to a target antigen of interest and inhibits, inhibits, and / or disrupts one or more biological or physiological mechanisms, effects, responses, functions, pathways, or activities involving the target antigen of interest. Such antagonistic activity of the amino acid sequences disclosed herein can be measured in any suitable manner and / or using any suitable (in vitro, usually cellular or in vivo) assay known in the art, depending on the target antigen in question.

[0085] Thus, more specifically, "inhibit," "suppress," and / or "interfere," using the amino acid sequences disclosed herein, can mean inhibiting, suppressing, and / or disrupting the interaction of a target antigen of interest with its natural binding partner, or inhibiting, suppressing, and / or disrupting the activity of a target antigen of interest, or inhibiting, suppressing, and / or disrupting one or more biological or physiological mechanisms, effects, responses, functions, pathways, or activities involving the target antigen of interest, as measured using a suitable in vitro, cellular, or in vivo assay, e.g., by at least 10%, preferably at least 20%, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more, compared to the activity of the target antigen of interest measured using the same assay under the same conditions but without the amino acid sequences disclosed herein. Furthermore, "inhibit," "suppress," and / or "interfere" can also mean inducing a decrease in the affinity, avidity, specificity, and / or selectivity of a target antigen of interest for one or more of its natural binding partners, and / or inducing a decrease in the sensitivity of a target antigen of interest to one or more conditions of the medium or environment in which it is found (e.g., pH, ionic strength, presence of cofactors, etc.), compared to otherwise identical conditions without the use of the amino acid sequences disclosed herein. In the context of the present invention, "inhibit," "suppress," and / or "interfere" can also refer to allosteric inhibition, suppression, and / or interference with the activity of a target antigen of interest.

[0086] The inhibitory or antagonistic activity or the stimulatory or activating activity of the amino acid sequences disclosed herein may be reversible or irreversible, but in agrochemical, pharmaceutical and pharmacological applications, will generally be reversible.

[0087] An amino acid sequence disclosed herein is considered to be "essentially isolated" as used herein when it has been extracted or purified from the host cell and / or culture medium in which it is produced.

[0088] With respect to the amino acid sequences disclosed herein, the terms "binding region," "binding site," or "interaction site" present on the amino acid sequence disclosed herein refer to a specific site, region, locus, portion, or domain present on a target molecule, which is involved in binding to the target molecule. Thus, such a binding region essentially consists of the specific site, region, locus, portion, or domain of the target molecule that is in contact with the amino acid sequence when bound to the target molecule.

[0089] As used herein, "plant" refers to a whole plant or part thereof, including fresh fruit, vegetables, and seeds. A plant or plant part can be a living plant or part thereof. As used herein, the term "plant" also includes whole plants, ancestors and descendants of said plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, each of which contains the gene(s) / nucleic acid(s). The term "plant" also includes plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores, each of which also contains the gene(s) / nucleic acid(s).

[0090] Selection of an appropriate control plant is a routine part of the experimental setup and can include a corresponding wild-type plant or a corresponding plant that does not contain the gene of interest. The control plant is generally of the same plant species or variety as the plant being evaluated. The control plant can also be a nullzygote of the plant being evaluated. A nullzygote is an individual that lacks the transgene by segregation. As used herein, the term "control plant" refers not only to the entire plant, but also to plant parts, including seeds and seed parts.

[0091] As used herein, "crop" refers to a plant species or variety that is harvested for food, livestock feed, fuel source, or any other economic purpose. By way of non-limiting example, crops include cereals such as corn, wheat, rye, barley, and oats, sorghum, rice, sugar beet, and fodder beet; pome fruits (e.g., apples and pears); citrus fruits (e.g., oranges, lemons, limes, grapefruit, or mandarin oranges); stone fruits (e.g., peaches, nectarines, or plums); nuts (e.g., almonds and walnuts); soft fruits (e.g., cherries, strawberries, blackberries, or raspberries); fruit trees such as plantains or grapes; legumes such as beans, lentils, peas, and soybeans; oil plants such as sunflowers, safflowers, rapeseed, canola, castor, or olives. The plant may be a food crop, a cucurbit such as cucumber, melon or pumpkin, a fiber plant such as cotton, flax or hemp, a fuel crop such as sugarcane, miscanthus or switchgrass, a vegetable such as potato, tomato, pepper, lettuce, spinach, onion, carrot, eggplant, asparagus or cabbage, an ornamental plant such as a flower (e.g. petunia, geranium, rose, tulip, lily or chrysanthemum), a shrub, a broadleaf tree (e.g. poplar or willow) and an evergreen tree (e.g. conifer), a grass such as lawn grass, turf grass or forage grass, or another useful plant such as coffee, tea, tobacco, hops, pepper, rubber or latex plant.

[0092] As used herein, a "pest" is an organism that is harmful to plants, animals, humans, or humans, including, but not limited to, crop pests (defined below), household pests such as cockroaches and ants, and disease vectors such as malaria mosquitoes.

[0093] The terms "plant pest", "plant pathogen" or "crop pest" are used interchangeably herein and specifically refer to organisms that cause damage to plants, plant parts or plant products, especially plants, plant parts or plant products used in agriculture. The terms "plant pest" or "crop pest" are used in the sense that the pest targets and harms plants. Pests are particularly invertebrates, such as insects (including agricultural pests, ornamental plant pests and forest pests). Examples of relevant crop pests include, but are not limited to, aphids, caterpillars, flies, wasps, etc., nematodes (either free-living in the soil or parasitic on plant roots, particularly root-knot nematodes and cyst nematodes such as Soybean cyst nematode and Potato cyst nematode), mites (e.g. spider mites, dust mites and eriophyid mites) and gastropods (slugs such as Deroceras spp., Milax spp., Tandonia spp., Limax spp., Arion spp. and Veronicella spp., and insects such as Helix spp., Cernuella spp., Theba spp., and the like). snails such as Cochlicella spp., Achatina spp., Succinea spp., Ovachlamys spp., Amphibulima spp., Zachrysia spp., Bradybaena spp., and Pomacea spp.); pathogenic fungi (Ascomycetes (e.g., Fusarium spp., Thielaviopsis spp., Verticillium spp., Magnaporthe spp.)); spp.), Basidiomycetes (e.g., Rhizoctonia spp.), Phakospora spp., Puccinia spp.), and fungal-like Oomycetes (including, for example, Pythium spp. and Phytophthora spp.), bacteria (for example, Burkholderia spp. and Proteobacteria such as Xanthomonas spp. and Pseudomonas spp.), Phytoplasma, Spiroplasma, viruses (for example, tobacco mosaic virus and cauliflower mosaic virus), and protozoa.

[0094] As used herein, "microorganism" refers to bacteria, viruses, fungi, yeasts, etc., and "microbial" refers to originating from a microorganism.

[0095] As used herein, "fungi" refers to eukaryotic organisms belonging to the phylum Eumycota. In the present invention, the term fungi also includes fungus-like organisms such as Oomycota. Oomycota (or Oomycetes) form a distinct phylogenetic lineage of fungus-like eukaryotic microorganisms. This group was originally classified as Fungi, but recent insights support a relatively close relationship with photosynthetic organisms such as brown algae and diatoms within the heterokonts.

[0096] As used herein, "pest infection" or "pest disease" refers to any inflammatory condition, disease, or disorder caused by a pest in a living organism, such as a plant, animal, or human.

[0097] As used herein, "fungal infection" or "fungal disease" refers to any inflammatory condition, disease or disorder caused by a fungus in a living organism, such as a plant, animal or human.

[0098] The terms "active substance", "active ingredient" or "active principle" are used interchangeably herein and refer to any biological, biochemical or chemical component, derivative, fragment or compound based thereon, which has a general or specific action against a target, particularly a plant, plant part or plant product, including microorganisms, and which may be naturally occurring or obtained by manufacturing and may contain impurities inevitably resulting from the manufacturing process.

[0099] As used herein, the term "pesticidal agent" means an agent suitable for use in the agrochemical industry (including agriculture, horticulture, floriculture and home and garden applications), as well as for use by public health / pest control operators for the control of unwanted insects and rodents, for household applications such as household fungicides and insecticides, and for non-crop related products such as agents for protecting plants or plant parts, crops, bulbs, tubers, fruit trees (e.g., from pests, diseases or insects), for controlling, preferably promoting or enhancing plant growth, and / or for improving the harvest yield of plants, crops or plant parts (e.g., their fruits, flowers, seeds, etc.). Examples of such substances will be apparent to those skilled in the art and include, for example, insecticides (e.g., contact or systemic insecticides, including household insecticides), herbicides (e.g., contact or systemic herbicides, including household herbicides), fungicides (e.g., contact or systemic fungicides, including household fungicides), nematicides (e.g., contact or systemic nematicides, including household nematicides) and other pesticides or biocides (e.g., agents that kill insects or snails); as well as fertilizers; plant hormones, and the like. growth regulators; micronutrients, safeners, pheromones; repellents; insect baits; and / or active principles used to modulate (i.e. increase, decrease, inhibit, enhance and / or induce) gene expression (and / or other biological or biochemical processes) in or by target plants (e.g. plants to be protected or controlled), such as nucleic acids (e.g. single- or double-stranded RNA, as used in the context of RNAi technology) and factors, proteins, chemicals, etc. known per se for this purpose.Examples of such pesticides will be apparent to those skilled in the art and include, but are not limited to, glyphosate, paraquat, metolachlor, acetochlor, mesotrione, 2,4-D, atrazine, glufosinate, sulfosate, fenoxaprop, pendimethalin, picloram, trifluralin, bromoxynil, clodinafop, fluroxypyr, nicosulfuron, bensulfuron, imazethapyr, dicamba, imidacloprid, thiamethoxam, fipronil, chlorpyrifos, deltamethrin, lambda-cyhalothrin, endosulfan, and methamidophos. , carbofuran, clothianidin, cypermethrin, abamectin, diflufenican, spinosad, indoxacarb, bifenthrin, tefluthrin, azoxystrobin, thiamethoxam, tebuconazole, mancozep, cyazofamid, fluazinam, pyraclostrobin, epoxiconazole, chlorothalonil, copper fungicides, trifloxystrobin, prothioconazole, difenoconazole, carbendazim, propiconazole, thiophanate, sulfur, boscalid and other known pesticides or any suitable combination thereof.

[0100] As used herein, the term "pesticidal composition" refers to an agrochemical composition, as defined in detail herein, containing at least one active substance and, optionally, one or more additives that promote optimal dispersion, atomization, deposition, leaf wetting, distribution, retention, and / or uptake of the pesticide. As will be apparent from the detailed description herein, the term "pesticidal composition" as used herein includes biological control agents or biological pesticides (including, but not limited to, biological biocides, biostatic agents, fungistatic agents, and fungicides), and these terms are used interchangeably herein. Thus, the term "pesticidal composition" as used herein includes compositions containing at least one biological molecule as an active ingredient, substance, or main ingredient for controlling pests on plants or other agriculturally relevant environments (e.g., in soil). Non-limiting examples of biomolecules that may be used as active principles in the pesticide compositions disclosed herein include proteins (including antibodies and fragments thereof, including but not limited to, antibody heavy chain variable domain fragments such as VHHs), nucleic acid sequences, (poly)saccharides, lipids, vitamins, hormones, glycolipids, sterols, and glycerolipids.

[0101] By way of non-limiting example, additives in the pesticide compositions disclosed herein include, but are not limited to, diluents, solvents, adjuvants, surfactants, wetting agents, spreading agents, oils, adhesives, thickeners, penetrating agents, buffers, acidifying agents, anti-settling agents, anti-freezing agents, photoprotectants, anti-foaming agents, biocides and / or anti-scattering agents.

[0102] As used herein, the term "biostatic composition" or "biostatic agent" refers to any active ingredient, substance, or main ingredient for biostatics (as further defined herein), or a composition containing any active ingredient, substance, or main ingredient, comprising at least one biostatic active substance or component, and optionally one or more additives that promote optimal dispersion, atomization, deposition, leaf wetting, distribution, retention, and / or uptake of said active substance or component. Non-limiting examples of such additives include diluents, solvents, adjuvants, (ionic) surfactants, wetting agents, spreading agents, oils, adhesives, thickeners, penetrating agents, buffers, acidifiers, anti-settling agents, anti-freezing agents, photoprotectants, anti-foaming agents, biocides, protease inhibitors, and / or anti-scattering agents.

[0103] As used herein, the term "biocidal composition" or "biocide" refers to any active ingredient, substance, or principle for a biocide (as further defined herein), or a composition containing any active ingredient, substance, or principle, comprising at least one biocidal active substance or component, and optionally one or more additives that facilitate optimal dispersion, atomization, deposition, leaf wetting, distribution, retention, and / or uptake of said active substance or component. Non-limiting examples of such additives include diluents, solvents, adjuvants, (ionic) surfactants, wetting agents, spreading agents, oils, adhesives, thickeners, penetrating agents, buffers, acidifying agents, anti-settling agents, anti-freezing agents, photoprotectants, anti-foaming agents, biocides, protease inhibitors, and / or anti-scattering agents.

[0104] As used herein, the term "fungistatic composition" or "fungistatic agent" refers to any fungistatic active ingredient, substance, or principle (as further defined herein) or a composition containing any active ingredient, substance, or principle, comprising at least one fungistatic active substance or ingredient and, optionally, one or more additives that facilitate optimal dispersion, atomization, deposition, leaf wetting, distribution, retention, and / or uptake of said active substance or ingredient. Non-limiting examples of such additives include diluents, solvents, adjuvants, (ionic) surfactants, wetting agents, spreading agents, oils, adhesives, thickeners, penetrating agents, buffers, acidifiers, anti-settling agents, anti-freezing agents, photoprotectants, anti-foaming agents, biocides, protease inhibitors, and / or anti-scattering agents.

[0105] As used herein, the term "fungicidal composition" or "fungicide" refers to any fungicidal active ingredient, substance, or principle (as further defined herein) or a composition containing any active ingredient, substance, or principle, comprising at least one fungicidal active substance or ingredient and, optionally, one or more additives that facilitate optimal dispersion, atomization, deposition, leaf wetting, distribution, retention, and / or uptake of said active substance or ingredient. By way of non-limiting example, such additives are diluents, solvents, adjuvants, (ionic) surfactants, wetting agents, spreading agents, oils, sticking agents, thickeners, penetrating agents, buffers, acidifying agents, anti-settling agents, anti-freezing agents, photoprotectants, anti-foaming agents, biocides, protease inhibitors, and / or anti-scattering agents.

[0106] As used herein, "agricultural use" includes not only the use of agrochemicals as defined above (e.g., pesticides, growth regulators, nutrients / fertilizers, repellents, defoliants, etc.) suitable for and / or intended for use on field-grown crops (e.g., agriculture), but also the use of agrochemicals as defined above (e.g., pesticides, growth regulators, nutrients / fertilizers, repellents, defoliants, etc.) for greenhouse-grown crops (e.g., horticulture / floriculture) or hydroponic systems, as well as the use of agrochemicals as defined above suitable for and / or intended for non-crop uses, such as for personal gardens, for home use (e.g., as a household herbicide or insecticide), or for pest control professionals (e.g., for weed control, etc.).

[0107] As used herein, "biostatic" or "biostatic use" includes any action or use of an active substance (optionally contained in a biostatic, biocidal, fungicidal or fungistatic composition as defined herein) to control, regulate or interfere with the harmful activity of pests, such as plant pests or plant pathogens, for example, but not limited to, to inhibit the growth or activity of, alter the behavior of, and repel or attract such pests on plants, plant parts or other agriculturally relevant environments (e.g., in homes or soil).

[0108] As used herein, "biocidal" or "biocidal" includes any action or use of an active substance (optionally contained in a biocidal or fungicidal composition as defined herein) to control, regulate, or interfere with the harmful activity of a pest, such as a plant pest or plant pathogen, for example, but not limited to, to inhibit the growth or activity of, alter the behavior of, and repel or attract the pest on a plant, plant part, or other agriculturally relevant environment (e.g., in a home or in the soil).

[0109] As used herein, "fungistatic" or "fungistatic" includes any action or use of an active substance (optionally comprised in a fungicidal or fungistatic composition as defined herein) to control, regulate or interfere with the harmful activity of fungi, for example, but not limited to, to inhibit the growth or activity of, alter the behavior of, and repel or attract fungi on plants, plant parts or other agriculturally relevant environments (e.g., domestically or in the soil).

[0110] As used herein, "fungicide" or "fungicidal" includes any action or use of an active substance (optionally comprised in a fungicidal composition as defined herein) to control, regulate or interfere with the harmful activity of fungi, for example, but not limited to, to kill, inhibit the growth or activity of, alter the behavior of, and repel or attract fungi on plants, plant parts or other agriculturally relevant environments (e.g., domestically or in the soil).

[0111] "Pesticidal activity" or "biocidal activity" are used interchangeably herein and mean to interfere with the harmful activity of a pest, including, but not limited to, killing said pest, inhibiting the growth or activity of said pest, altering the behavior of said pest, repelling or attracting said pest.

[0112] As used herein, "biostatic activity" means interfering with the harmful activity of a pest, including, but not limited to, inhibiting the growth or activity of said pest, altering the behavior of said pest, repelling or attracting said pest.

[0113] The pesticidal, biocidal or biostatic activity of an active ingredient, substance or main ingredient, or a composition or agent containing a pesticidal, biocidal or biostatic active ingredient, substance or main ingredient, may be expressed as, but is not limited to, the minimum inhibitory activity (MIC) of the agent (expressed in concentration units such as, for example, mg / mL).

[0114] As used herein, "fungicidal activity" means interfering with the harmful activity of fungi, including, but not limited to, killing the fungi, inhibiting the growth or activity of the fungi, altering the behavior of the fungi, and repelling or attracting the fungi.

[0115] As used herein, "fungistatic activity" means interfering with the harmful activity of fungi, including, but not limited to, inhibiting the growth or activity of said fungi, altering the behavior of said fungi, and repelling or attracting said fungi.

[0116] The fungicidal or fungistatic activity of an active ingredient, substance or main ingredient, or a composition or agent containing a pesticidal, biocidal or biostatic active ingredient, substance or main ingredient, may be expressed as, but is not limited to, the minimum inhibitory activity (MIC) of the agent (expressed in concentration units such as, for example, mg / mL).

[0117] As used herein, the term "substrate" refers to any solid, semi-solid, or liquid substrate in which an active substance can be suitably formulated, loaded, immobilized, adsorbed, absorbed, bound, encapsulated, embedded, attached, or contained. Non-limiting examples of such substrates include nanocapsules, microcapsules, nanospheres, microspheres, nanoparticles, microparticles, liposomes, vesicles, beads, gels, weakly ionic resin particles, liposomes, cochleate delivery vehicles, small granules, particulates, nanotubes, buckyballs, water droplets that are part of a water-in-oil emulsion, oil droplets that are part of an oil-in-water emulsion, organic materials such as cork (e.g., in the form of seed shells, wood chips, pulp, spheres, beads, sheets, or any other suitable shape), wood, or other plant-derived materials, inorganic materials such as paper or paperboard, talc, clay, microcrystalline cellulose, silica, alumina, silicates, and zeolites, or microbial cells (e.g., yeast cells) or suitable fractions or fragments thereof.

[0118] As used herein, the term "antibody" refers to polyclonal antibodies, monoclonal antibodies, humanized antibodies, single-chain antibodies, and fragments thereof (e.g., Fab, F(ab)2, Fv, and other fragments that retain the antigen-binding function of the parent antibody). Thus, antibody can refer to an immunoglobulin or glycoprotein, or a fragment or portion thereof, or to a construct comprising an antigen-binding portion contained within a modified immunoglobulin-like framework, or to an antigen-binding portion contained within a construct comprising a non-immunoglobulin-like framework or scaffold.

[0119] As used herein, the term "monoclonal antibody" refers to an antibody composition having a homogeneous antibody population. This term is not limited with respect to the antibody species or origin, nor is it limited by its method of production. This term includes full-length immunoglobulins and 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 will generally be of rat or mouse origin due to the ready availability of rat or mouse cell lines for generating the hybrid cell lines or hybridomas necessary to produce monoclonal antibodies.

[0120] As used herein, the term "polyclonal antibody" refers to an antibody composition having a heterogeneous antibody population. Polyclonal antibodies are often derived from pooled sera from immunized animals or selected humans.

[0121] As used herein, the term "antibody heavy chain variable domain or functional fragment thereof" refers to (i) a heavy chain variable domain of a heavy chain antibody that does not naturally have a light chain (hereinafter referred to as V HH (ii) the variable domain of the heavy chain of a conventional four-chain antibody (hereinafter referred to as V H It refers to, but is not limited to, a camelized variable domain (as specifically defined herein) of the heavy chain of a conventional four-chain antibody (hereinafter referred to as camelized V H(also called) are mentioned.

[0122] As described in more detail below, the amino acid sequence and structure of an antibody heavy chain variable domain can be considered to be composed of, but not limited to, four framework regions or "FRs" referred to in the art and below as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," respectively, and three complementarity-determining regions or "CDRs" located between the framework regions and 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," respectively.

[0123] Similarly, as detailed below, (V HH or V H The total number of amino acid residues in the heavy chain variable domain of an antibody (including the heavy chain variable domain of an antibody) can range from 110 to 130, preferably 112 to 115, and most preferably 113. It should be noted that portions, fragments, and analogs of the heavy chain variable domain of an antibody are not limited in length and / or size, so long as such portions, fragments, or analogs retain (at least a portion of) a functional activity, such as pesticidal, biocidal, biostatic, fungicidal, or fungistatic activity (as defined herein), and / or retain (at least a portion of) the binding specificity of the original heavy chain variable domain of the antibody from which such portions, fragments, or analogs are derived. Portions, fragments, and analogs that retain (at least a portion of) a functional activity, such as pesticidal, biocidal, biostatic, fungicidal, or fungistatic activity (as defined herein) and / or retain (at least a portion of) the binding specificity of the original heavy chain variable domain of the antibody from which such portions, fragments, or analogs are derived are also referred to herein as "functional fragments" of the heavy chain variable domain.

[0124] The numbering system for amino acid residues in the heavy chain variable domain is the so-called "AbM definition" and so-called "contact definition" described by Chothia et al. (Nature 342, 877-883 (1989)). This numbering system is adopted in the present application.

[0125] Alternatively, (V HH or V H The amino acid residues of the variable domain of the heavy chain variable domain of the antibody (including the V HH As applied to domains, they can be numbered according to the general numbering system for heavy chain variable domains described by Kabat et al. ("Sequence of proteins of immunological interest", U.S. Public Health Services, NIH Bethesda, Md., Publication No. 91).

[0126] For a general description of heavy chain antibodies and their variable domains, the following references are mentioned in particular as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103 in the name of Vrije Universiteit Brussel; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1 134 231 and WO 02 / 48193 in the name of Unilever; WO97 / 49805, WO01 / 21817, WO03 / 035694, WO03 / 054016 and WO03 / 055527 to VIB; WO03 / 050531 to Algonomics NV and Ablynx NV; WO01 / 90190 to the National Research Council of Canada; WO03 / 025020 (=EP1433793) to the Institute of Antibodies; and WO04 / 041867, WO04 / 041862, WO04 / 041865, WO04 / 041863, WO04 / 062551 to Ablynx; Hamers-Casterman et al., Nature 1993 See Jun.3;363(6428):446-8.

[0127] In general, it should be noted that the term "heavy chain variable domain," as used herein in its broadest sense, is not limited to a particular biological origin or a particular method of production. For example, as described in more detail below, the heavy chain variable domain of the present invention may be (1) a V domain of a native heavy chain antibody; HH (2) Isolating the V domain of a natural four-chain antibody H (3) Isolate the natural V domain HH (4) expressing the nucleotide sequence encoding the natural V domain; H (5) expressing a nucleotide sequence encoding a natural V domain from any animal species, particularly a mammalian species such as a human; H "Camelizing" a domain (as described below), or creating such a camelized V H(6) "camelizing" a "domain antibody" or "Dab" as described in Ward et al., supra, or producing such a camelized V H (7) expressing nucleic acids encoding the domains; (8) using synthetic or semisynthetic techniques to produce proteins, polypeptides, or other amino acid sequences; (9) using nucleic acid synthesis techniques to produce V HH or V H and (9) producing a nucleic acid encoding the gene, and then expressing the nucleic acid thus obtained; and / or (10) any combination of the above. Suitable methods and techniques for carrying out the above methods will be apparent to those skilled in the art based on the disclosure herein, and include, for example, the methods and techniques described in detail below.

[0128] On the other hand, according to one particular embodiment, the heavy chain variable domain disclosed herein is a naturally occurring V H The amino acid sequence of the domain (e.g., a naturally occurring V domain derived from a mammal, particularly a human) H The amino acid sequence of the domain does not have an amino acid sequence that is exactly identical (i.e., 100% sequence identity) to the amino acid sequence of the domain.

[0129] As used herein, the terms "effective amount" and "effective dose" refer to an amount necessary to achieve one or more desired results.

[0130] As used herein, the terms "measuring," "measuring," "assessing," "monitoring," and "assaying" are used interchangeably and include quantitative and qualitative measurements.

[0131] All references cited herein are incorporated herein by reference in their entirety. Unless otherwise defined, all terms used to disclose the present invention, including scientific and technical terms, shall have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. For further guidance, definitions of terms are provided to better understand the teachings of the present invention.

[0132] Polypeptides The polypeptides disclosed herein are generally capable of binding to fungi, thereby causing the retardation of fungal spore growth and / or lysis of fungal spores, i.e., the binding of the polypeptides to fungi results in the retardation of fungal spore growth and / or lysis of fungal spores.

[0133] The polypeptides of the present invention and used in the present invention can bind (specifically) to fungal membranes or components of fungal membranes. In certain embodiments, the polypeptides of the present invention and used in the present invention do not bind (specifically) to fungal cell walls or components of cell walls. For example, in certain embodiments, the polypeptides of the present invention and used in the present invention do not bind (specifically) to fungal glucosylceramide.

[0134] The polypeptide can specifically bind to a lipid-containing fraction of fungal cell membranes, such as a lipid-containing fraction of Fusarium oxysporum or other fungi. The lipid-containing fraction (of Botrytis cinerea or other fungi) can be obtained by chromatography. The chromatography can be performed on a crude lipid extract (also referred to herein as a total lipid extract or TLE) obtained from fungal mycelia and / or conidia. The chromatography can be, for example, thin-layer chromatography or normal-phase flash chromatography. The chromatography (e.g., thin-layer chromatography) can be performed on a substrate, for example, on glass coated with silica gel. The chromatography can be performed using a chloroform / methanol mixture (e.g., 85 / 15% v / v) as an eluent.

[0135] For example, the lipid-containing fraction may be obtainable by a method comprising fractionating the mycelia and / or conidia of a fungus (e.g., Botrytis cinerea or other fungi) by total lipid extract thin-layer chromatography, and selecting fractions having a retention factor (Rf) greater than that of a ceramide fraction and less than that of a non-polar phospholipid fraction.

[0136] In more specific embodiments, the lipid-containing fraction may be obtainable by a method comprising fractionating fungal (e.g., Botrytis cinerea or other fungi) hyphae and / or conidia by total lipid extract thin-layer chromatography on silica-coated glass slides using a chloroform / methanol mixture (e.g., 85 / 15% v / v) as an eluent, and selecting fractions having a retention factor (Rf) greater than that of the ceramide fraction and less than that of the non-polar phospholipid fraction.

[0137] Alternatively, the fraction can be obtained using normal phase flash chromatography, which can include fractionating fungal (e.g., Botrytis cinerea or other fungi) mycelia and / or conidia by total lipid extract normal phase flash chromatography and selecting fractions having a retention factor (Rf) greater than the ceramide fraction and less than the non-polar phospholipid fraction.

[0138] In a more specific embodiment, the lipid-containing fraction may be obtainable by a method comprising dissolving TLE in dichloromethane (CHCl) and MeOH, fractionating the fungal (e.g., Botrytis cinerea or other fungi) mycelia and / or conidia by normal phase flash chromatography of the total lipid extract using CHCl / MeOH (e.g., 85 / 15%, v / v) as the eluent, and then filtering the fraction.

[0139] In a more specific embodiment, the lipid-containing fraction may be obtainable by a method comprising fractionating fungal (e.g., Botrytis cinerea or other fungi) mycelia and / or conidia by total lipid extract normal phase flash chromatography by dissolving the TLE in dichloromethane (CHCl) and MeOH, loading the TLE onto a normal phase flash cartridge (e.g., a flash cartridge packed with 15 μm particles), and running the column with CHCl / MeOH (85 / 15%, v / v) as the eluent; filtering the fraction through a filter (e.g., a 0.45 μm syringe filter fitted with a nylon membrane); and drying the fraction.

[0140] Before testing the binding or interaction between the polypeptide and the fraction, the fraction from the chromatography can be treated. For example, liposomes containing the fraction can be prepared. Such methods can include thin film hydration. For example, in such methods, liposomes can be prepared using thin film hydration in the presence of 1,6-diphenyl-1,3,5-hexatriene (DPH). Membrane binding and / or disruption due to polypeptide binding can be measured by changes in fluorescence before and after polypeptide binding (or by reference to an appropriate control).

[0141] Thus, in certain embodiments, the polypeptides of and used in the present invention are capable of (specifically) binding to a lipid-containing chromatographic fraction of fungal cell membranes, optionally prepared into liposomes before testing for binding of the polypeptide.

[0142] The binding of the polypeptide to the lipid-containing fraction of fungi can be confirmed by any suitable method, for example, biolayer interferometry. The specific interaction with the lipid-containing fraction can be tested. For example, it can be confirmed whether the polypeptide can disrupt the lipid fraction when the fraction is prepared into liposomes, for example, using thin film hydration.

[0143] In methods using chromatography, an extraction step can be performed before the chromatography step. For example, fungal mycelia and / or conidia can be subjected to an extraction step to obtain a crude lipid extract or a total lipid extract, and chromatography is then performed on this extract. For example, in certain embodiments, fungal mycelia and / or conidia (e.g., fungal mycelia and / or conidia of Fusarium oxysporum or Botrytis cinerea) can be extracted at room temperature using, for example, chloroform:methanol in a 2:1 and 1:2 (v / v) ratio. The extracts thus prepared can be combined and dried to form a crude lipid extract or TLE.

[0144] Thus, in certain embodiments, the polypeptide is capable of (specifically) binding to a lipid-containing fraction of the cell membrane of a fungus (e.g., Fusarium oxysporum or Botrytis cinerea), and the lipid-containing fraction of the cell membrane of the fungus is obtained or obtainable by chromatography. The chromatography can be normal-phase flash chromatography or thin-layer chromatography. Binding of the polypeptide to the lipid-containing fraction can be measured by biolayer interferometry. In certain embodiments, the chromatography step can be performed on a crude lipid fraction obtained or obtainable by a method comprising extracting lipids from fungal mycelia and / or conidia derived from a fungal sample. The extraction step can be performed using chloroform:methanol in a 2:1 and 1:2 (v / v) ratio to obtain two extracts, which can then be combined.

[0145] In methods involving thin layer chromatography, the chromatography can include fractionating the fungal mycelia by total lipid extract thin layer chromatography and selecting fractions having a retention factor (Rf) greater than a ceramide fraction and less than a non-polar phospholipid fraction.

[0146] In certain methods relating to thin layer chromatography, the chromatography can include fractionating fungal (e.g., Botrytis cinerea or other fungi) mycelia and / or conidia by total lipid extract thin layer chromatography on silica-coated glass using a chloroform / methanol mixture (e.g., 85 / 15% v / v) as an eluent, and selecting fractions having a retention factor (Rf) greater than the ceramide fraction and less than the non-polar phospholipid fraction.

[0147] In methods involving normal phase flash chromatography, the chromatography can include fractionating mycelia and / or conidia of a fungus (e.g., Botrytis cinerea or other fungi) by total lipid extract normal phase flash chromatography and selecting fractions having a retention factor (Rf) greater than the ceramide fraction and less than the non-polar phospholipid fraction.

[0148] In certain methods involving normal phase flash chromatography, the chromatography can include dissolving the TLE in dichloromethane (CHCl) and MeOH, fractionating the fungal (e.g., Botrytis cinerea or other fungi) mycelia and / or conidia by total lipid extract normal phase flash chromatography using CHCl / MeOH (e.g., 85 / 15%, v / v) as the eluent, and then filtering the fraction.

[0149] In certain methods involving normal phase flash chromatography, the chromatography can include fractionating fungal (e.g., Botrytis cinerea or other fungi) mycelia and / or conidia by total lipid extract normal phase flash chromatography by dissolving the TLE in dichloromethane (CHCl) and MeOH, loading the TLE onto a normal phase flash cartridge (e.g., a flash cartridge packed with 15 μm particles), and running the column with CHCl / MeOH (85 / 15%, v / v) as the eluent; filtering the fraction through a filter (e.g., a 0.45 μm syringe filter fitted with a nylon membrane); and drying the fraction.

[0150] In certain embodiments, the present invention provides a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-51 and 101-111, 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 thereto.

[0151] In one aspect, the present invention provides a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of 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 said sequence.

[0152] In one aspect, the present invention provides a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of 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 thereto.

[0153] In one aspect, the present invention provides a polypeptide comprising or consisting of 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 thereto.

[0154] In one aspect, the present invention provides a polypeptide comprising or consisting of 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 thereto.

[0155] In one aspect, the present invention provides a polypeptide comprising or consisting of SEQ ID NO: 3, or a sequence which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto.

[0156] In one aspect, the invention provides a polypeptide comprising or consisting of SEQ ID NO: 4, or a sequence which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto.

[0157] In one aspect, the present invention provides a polypeptide comprising or consisting of SEQ ID NO: 5, or a sequence which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto.

[0158] In one aspect, the present invention provides a polypeptide comprising or consisting of SEQ ID NO: 6, or a sequence which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity thereto.

[0159] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a CDR1 region comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 52 to 67 and 112 to 122; a CDR2 region comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 68 to 83 and 123 to 133; a CDR3 region comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 84 to 100 and 134 to 144; The present invention provides a polypeptide comprising:

[0160] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a CDR1 region comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 52, 53 and 54; a CDR2 region comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 68, 69 and 70; a CDR3 region comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 84, 85 and 86; The present invention provides a polypeptide comprising:

[0161] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: 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; 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; 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; 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; 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; 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; a CDR1 region comprising or consisting of SEQ ID NO: 55, a CDR2 region comprising or consisting of SEQ ID NO: 71, and a CDR3 region comprising or consisting of SEQ ID NO: 87; a CDR1 region comprising or consisting of SEQ ID NO: 56, a CDR2 region comprising or consisting of SEQ ID NO: 68, and a CDR3 region comprising or consisting of SEQ ID NO: 84; 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: 88; a CDR1 region comprising or consisting of SEQ ID NO: 56, a CDR2 region comprising or consisting of SEQ ID NO: 68, and a CDR3 region comprising or consisting of SEQ ID NO: 88; a CDR1 region comprising or consisting of SEQ ID NO: 57, a CDR2 region comprising or consisting of SEQ ID NO: 68, and a CDR3 region comprising or consisting of SEQ ID NO: 84; a CDR1 region comprising or consisting of SEQ ID NO: 52, a CDR2 region comprising or consisting of SEQ ID NO: 52, and a CDR3 region comprising or consisting of SEQ ID NO: 89; a CDR1 region comprising or consisting of SEQ ID NO: 57, a CDR2 region comprising or consisting of SEQ ID NO: 68, and a CDR3 region comprising or consisting of SEQ ID NO: 89; a CDR1 region comprising or consisting of SEQ ID NO: 58, a CDR2 region comprising or consisting of SEQ ID NO: 68, and a CDR3 region comprising or consisting of SEQ ID NO: 84; a CDR1 region comprising or consisting of SEQ ID NO: 59, a CDR2 region comprising or consisting of SEQ ID NO: 68, and a CDR3 region comprising or consisting of SEQ ID NO: 84; a CDR1 region comprising or consisting of SEQ ID NO: 60, a CDR2 region comprising or consisting of SEQ ID NO: 68, and a CDR3 region comprising or consisting of SEQ ID NO: 84; a CDR1 region comprising or consisting of SEQ ID NO: 61, a CDR2 region comprising or consisting of SEQ ID NO: 68, and a CDR3 region comprising or consisting of SEQ ID NO: 84; a CDR1 region comprising or consisting of SEQ ID NO: 62, a CDR2 region comprising or consisting of SEQ ID NO: 68, and a CDR3 region comprising or consisting of SEQ ID NO: 84; a CDR1 region comprising or consisting of SEQ ID NO: 63, a CDR2 region comprising or consisting of SEQ ID NO: 68, and a CDR3 region comprising or consisting of SEQ ID NO: 84; a CDR1 region comprising or consisting of SEQ ID NO: 64, a CDR2 region comprising or consisting of SEQ ID NO: 68, and a CDR3 region comprising or consisting of SEQ ID NO: 84; 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; 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; 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; 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; 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; 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; 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; a CDR1 region comprising or consisting of SEQ ID NO: 52, a CDR2 region comprising or consisting of SEQ ID NO: 76, and a CDR3 region comprising or consisting of SEQ ID NO: 84; a CDR1 region comprising or consisting of SEQ ID NO: 52, a CDR2 region comprising or consisting of SEQ ID NO: 77, and a CDR3 region comprising or consisting of SEQ ID NO: 84; a CDR1 region comprising or consisting of SEQ ID NO: 52, a CDR2 region comprising or consisting of SEQ ID NO: 78, and a CDR3 region comprising or consisting of SEQ ID NO: 84; a CDR1 region comprising or consisting of SEQ ID NO: 52, a CDR2 region comprising or consisting of SEQ ID NO: 79, and a CDR3 region comprising or consisting of SEQ ID NO: 84; a CDR1 region comprising or consisting of SEQ ID NO: 52, a CDR2 region comprising or consisting of SEQ ID NO: 80, and a CDR3 region comprising or consisting of SEQ ID NO: 84; a CDR1 region comprising or consisting of SEQ ID NO: 52, a CDR2 region comprising or consisting of SEQ ID NO: 81, and a CDR3 region comprising or consisting of SEQ ID NO: 84; a CDR1 region comprising or consisting of SEQ ID NO: 52, a CDR2 region comprising or consisting of SEQ ID NO: 82, and a CDR3 region comprising or consisting of SEQ ID NO: 84; a CDR1 region comprising or consisting of SEQ ID NO: 52, a CDR2 region comprising or consisting of SEQ ID NO: 83, and a CDR3 region comprising or consisting of SEQ ID NO: 84; 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; 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: 90; 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: 91; 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: 92; 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: 93; 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: 94; 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: 95; 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: 96; 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: 97; 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: 98; 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: 99; 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: 100; a CDR1 region comprising or consisting of SEQ ID NO: 112, a CDR2 region comprising or consisting of SEQ ID NO: 123, and a CDR3 region comprising or consisting of SEQ ID NO: 134; a CDR1 region comprising or consisting of SEQ ID NO: 113, a CDR2 region comprising or consisting of SEQ ID NO: 124, and a CDR3 region comprising or consisting of SEQ ID NO: 135; a CDR1 region comprising or consisting of SEQ ID NO: 114, a CDR2 region comprising or consisting of SEQ ID NO: 125, and a CDR3 region comprising or consisting of SEQ ID NO: 136; a CDR1 region comprising or consisting of SEQ ID NO: 115, a CDR2 region comprising or consisting of SEQ ID NO: 126, and a CDR3 region comprising or consisting of SEQ ID NO: 137; a CDR1 region comprising or consisting of SEQ ID NO: 116, a CDR2 region comprising or consisting of SEQ ID NO: 127, and a CDR3 region comprising or consisting of SEQ ID NO: 138; a CDR1 region comprising or consisting of SEQ ID NO: 117, a CDR2 region comprising or consisting of SEQ ID NO: 128, and a CDR3 region comprising or consisting of SEQ ID NO: 139; a CDR1 region comprising or consisting of SEQ ID NO: 118, a CDR2 region comprising or consisting of SEQ ID NO: 129, and a CDR3 region comprising or consisting of SEQ ID NO: 140; a CDR1 region comprising or consisting of SEQ ID NO: 119, a CDR2 region comprising or consisting of SEQ ID NO: 130, and a CDR3 region comprising or consisting of SEQ ID NO: 141; a CDR1 region comprising or consisting of SEQ ID NO: 120, a CDR2 region comprising or consisting of SEQ ID NO: 131, and a CDR3 region comprising or consisting of SEQ ID NO: 142; a CDR1 region comprising or consisting of SEQ ID NO: 121, a CDR2 region comprising or consisting of SEQ ID NO: 132, and a CDR3 region comprising or consisting of SEQ ID NO: 143; or A CDR1 region comprising or consisting of SEQ ID NO: 122, a CDR2 region comprising or consisting of SEQ ID NO: 133, and a CDR3 region comprising or consisting of SEQ ID NO: 144. The present invention provides a polypeptide comprising:

[0162] In certain embodiments, the polypeptide can comprise only the specific CDR1 and CDR2 sequences of the polypeptide, since the CDR3 region is amenable to replacement without loss of activity (as demonstrated by replacing the CDR3 region with an unrelated CDR3, i.e., one that does not bind to fungi). Thus, the sequence of the CDR3 region is arbitrary. Generally, the polypeptide can comprise a CDR3 region (e.g., to ensure structural integrity), but the sequence of the CDR3 region is not critical. For example, the polypeptide can comprise a CDR1 region, a CDR2 region, and a CDR3 region, each of which comprises or consists of a sequence as set forth herein, while the CDR3 region can comprise any sequence (e.g., any sequence having a length of 8 to 16 amino acid residues).

[0163] The polypeptides described herein suitably have predetermined framework region sequences. For example, the polypeptide may comprise a framework region 1 (FR1) sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 149, 150, 154, 155, 158 and 159, a framework region 2 (FR2) sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 151, 156 and 160, a framework region 3 (FR3) sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 152, 157 and 161, and a framework region 4 (FR4) sequence comprising or consisting of SEQ ID NO: 153.

[0164] In certain embodiments, for example, any polypeptide of or related to any polypeptide of the clone designated herein as 10G11 (including 10G11Q, any of mutants 1-11, or any of single ALA mutants 1-34, and any of mutants 10G11-A through 10G11K), or derived from said clone, and any polypeptide having any particular sequence identity to or any substitution of these polypeptides, can comprise a framework region 1 (FR1) sequence that comprises or consists of a sequence selected from the group consisting of SEQ ID NOs: 149 and 150, a framework region 2 (FR2) sequence that comprises or consists of SEQ ID NO: 151, a framework region 3 (FR3) sequence that comprises or consists of SEQ ID NO: 152, and a framework region 4 (FR4) sequence that comprises or consists of SEQ ID NO: 153.

[0165] In certain embodiments, for example, any polypeptide of the clone designated herein as 10E11 (including 10E11Q) or related to any polypeptide derived from said clone, and any polypeptide having any particular sequence identity to or any substitution of these polypeptides, can comprise a framework region 1 (FR1) sequence that comprises or consists of a sequence selected from the group consisting of SEQ ID NOs: 154 and 155, a framework region 2 (FR2) sequence that comprises or consists of SEQ ID NO: 156, a framework region 3 (FR3) sequence that comprises or consists of SEQ ID NO: 157, and a framework region 4 (FR4) sequence that comprises or consists of SEQ ID NO: 153.

[0166] In certain embodiments, for example, any polypeptide of the clone designated herein as 12C03 (including 12C03Q) or related to any polypeptide derived from said clone, and any polypeptide having any particular sequence identity to or any substitution of these polypeptides, can comprise a framework region 1 (FR1) sequence that comprises or consists of a sequence selected from the group consisting of SEQ ID NOs: 158 and 159, a framework region 2 (FR2) sequence that comprises or consists of SEQ ID NO: 160, a framework region 3 (FR3) sequence that comprises or consists of SEQ ID NO: 161, and a framework region 4 (FR4) sequence that comprises or consists of SEQ ID NO: 153.

[0167] The CDR regions and the framework regions can be defined according to the Kabat numbering system.

[0168] In certain embodiments, the polypeptide can be between 80 and 200 residues in length.

[0169] The polypeptides of the present invention can be provided as compositions (eg, pesticide compositions).

[0170] Compositions containing at least one polypeptide In one aspect, the present inventors have provided a pesticide composition comprising at least one polypeptide capable of specifically binding to a pest. Importantly, by interacting in this manner with a specific molecular structure of the pest, the composition disclosed herein can inhibit, hinder, or suppress one or more biological activities of the plant pathogen, such that the growth of the plant pathogen is inhibited, hindered, or inhibited. In certain embodiments, the pesticide composition disclosed herein can kill a plant pest due to the specific interaction of at least one polypeptide contained in the composition that is capable of specifically binding to the pest.

[0171] Thus, the pesticide compositions disclosed herein can be used to modulate (e.g., reduce or inhibit) the biological function of a plant pest by binding to a binding site present in a target of the plant pest and acting on the natural biological activity of the pest (e.g., but not limited to, growth) and / or one or more biological pathways involving a structural target of the pest.

[0172] Furthermore, compositions containing at least one polypeptide disclosed herein offer several additional advantages over conventional immunoglobulin and non-immunoglobulin binding agents known in the art. Indeed, in certain embodiments, the amino acid sequences disclosed herein are isolated heavy chain immunoglobulin variable domains, which are more potent and stable than conventional four-chain antibodies, thereby (1) allowing for lower and less frequent administration, thereby reducing side effects, and (2) improving stability, allowing for more options for administration routes. The small size of heavy chain immunoglobulin variable domains allows them to cross membranes and enter physiological compartments, tissues, and organs inaccessible to other larger polypeptides and proteins.

[0173] In one specific, non-limiting embodiment, the at least one polypeptide included in the compositions disclosed herein can be a polypeptide that comprises an immunoglobulin fold or is capable of forming (by folding) an immunoglobulin fold under appropriate conditions (e.g., physiological conditions). See, in particular, the discussion in Halaby et al., J. (1999) Protein Eng. 12, 563-71. Preferably, such a polypeptide sequence, when properly folded to form an immunoglobulin fold, is capable of specifically binding (as defined herein) to a target or antigen, and has an affinity (as defined herein) as defined herein (K as detailed herein). D value (actual or apparent), K A value (actual or apparent), k on velocity and / or k off Speed ​​or IC 50More preferably, the polypeptide sequences are capable of binding to the pest target or pest antigen at a specific binding level (suitably measured and / or expressed as a value). Portions, fragments, analogs, mutants, variants, alleles and / or derivatives of such polypeptide sequences are also preferably selected to comprise an immunoglobulin fold or to be capable of forming an immunoglobulin fold under appropriate conditions.

[0174] In a specific embodiment, the present invention provides a pesticide composition or a biological pest control composition for controlling plant pests, more particularly plant fungi, which contains at least one polypeptide or amino acid sequence of 80 to 200 amino acids as an active ingredient. As used herein, "80 to 200 amino acids" refers to 80 to 200, i.e., includes both 80 amino acids and 200 amino acids. Therefore, "80 to 200 amino acids" can be used synonymously with "80 to 200 amino acids."

[0175] In certain other embodiments, the present invention provides a pesticide composition for controlling plant pests, comprising as active substances at least two (different) polypeptides or at least two (different) amino acid sequences of 80 to 200 amino acids.

[0176] In yet another embodiment, the present invention provides an agrochemical composition for controlling plant pests, which comprises as active substances at least three (different) polypeptides or at least three (different) amino acid sequences of 80 to 200 amino acids. Other combinations of different polypeptides are also contemplated.

[0177] The pesticide composition according to the present invention is a pesticide composition as defined herein for controlling plant pests as defined above, i.e. said pesticide composition, more particularly the active substances contained in said pesticide composition, are capable of interfering with, preferably suppressing or preventing, the harmful action of one or more plant pests on one or more plants, preferably crops.

[0178] The polypeptides or amino acid sequences included in the compositions disclosed herein can be naturally occurring or derived from naturally occurring polypeptides, or can be entirely artificially designed or synthetic. The polypeptides or amino acid sequences can be based on immunoglobulins or on domains present in proteins such as, but not limited to, microbial proteins, protease inhibitors, toxins, fibronectins, lipocalins, single-stranded antiparallel coiled-coil proteins, or repeat motif proteins. Non-limiting examples of such polypeptides within the amino acid length ranges described herein include carbohydrate binding domains (CBDs) (Blake et al (2006) J. Biol. Chem. 281, 29321-29329), heavy chain antibodies (hcAbs), single domain antibodies (sdAbs), minibodies (Tramontano et al (1994) J. Mol. Recognition 7, 9-24), camelid heavy chain antibody variable domains (VHHs), variable domains of novel antigen receptors (VNARs), affibodies (Nygren PA (2008) FEBS J. 275, 2668-2676), alphabodies (see WO2010066740), artificial ankyrin repeat domains (DARPins) (Stumpp et al (2008) Drug Discovery Today 13, 695-701), anticalins (Skerra et al (2008) J. Mol. Recognition 7, 9-24), and the like. al (2008) FEBS J. 275, 2677-2683), knottins (Kolmar et al (2008) FEBS J. 275, 2684-2690) and artificial CH2 domains (nanobodies, see Dimitrov DS (2009) mAbs 1, 26-28). In particular, the polypeptides or amino acid sequences disclosed herein are composed of a single polypeptide chain and are not post-translationally modified. More particularly, the polypeptides or amino acid sequences disclosed herein 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 comprise four framework regions and three complementarity determining regions, or any suitable fragment thereof (which will usually include at least a portion of the amino acid residues forming at least one of said complementarity determining regions). In particular, the polypeptides or amino acid sequences disclosed herein are amenable to production in high yields, preferably in microbial recombinant expression systems, and conveniently subsequently isolated and / or purified. In particular, the polypeptides or amino acid sequences disclosed herein are suitable for use in DARPins, knottins, alphabodies, and V. HH More particularly, the polypeptide or amino acid sequences disclosed herein are selected from the group consisting of alphabodies and V HH Most particularly, the polypeptide or amino acid sequence disclosed herein is selected from the group consisting of V HH is.

[0179] In particular, the at least one polypeptide included in the compositions disclosed herein can consist of a single polypeptide chain and is not post-translationally modified. More particularly, the at least one polypeptide included in the compositions disclosed herein can be derived from the innate or adaptive immune system, preferably from a protein of the innate or adaptive immune system. Even more particularly, the at least one polypeptide included in the compositions disclosed herein can be derived from an immunoglobulin. Most particularly, the at least one polypeptide included in the compositions disclosed herein can comprise four framework regions and three complementarity-determining regions, or any suitable fragment thereof (usually comprising at least a portion of the amino acid residues forming at least one of the complementarity-determining regions). In particular, the at least one polypeptide included in the compositions disclosed herein is amenable to production at high yields, preferably in a microbial recombinant expression system, and subsequent isolation and / or purification.

[0180] According to certain embodiments, the present invention provides multiple stretches of amino acid residues (i.e., small peptides) that are particularly suitable for binding to pest antigens or pest targets (for example, but not limited to, fungal antigens or fungal targets).

[0181] These stretches of amino acid residues may be present and / or incorporated in the polypeptides disclosed herein in particular so as to form (part of) the antigen binding site of said polypeptides. These stretches of amino acid residues may be present in antibodies, such as heavy chain antibodies, or V-chain antibodies raised against pest targets. H or V HH Because these stretches of amino acid residues were originally created as CDR sequences of sequences (or can be based on and / or derived from such CDR sequences, as detailed herein), these stretches of amino acid residues are also generally referred to herein as "CDR sequences" (i.e., CDR1, CDR2, and CDR3 sequences, respectively). However, it should be noted that the present invention in its broadest sense is not limited to any particular structural role or function that these stretches of amino acid residues may have in the polypeptides disclosed herein, as long as they enable the polypeptides disclosed herein to specifically bind to a pest target, such as a fungal antigen or fungal target. Thus, in general, the present invention in its broadest sense relates to agrochemical compositions comprising polypeptides capable of binding to a pest target, such as a fungal antigen or fungal target, and comprising a combination of CDR sequences as described herein.

[0182] Therefore, in certain non-limiting embodiments, the polypeptides disclosed herein may comprise at least one amino acid sequence selected from the group consisting of the CDR1, CDR2, and CDR3 sequences described herein. In particular, the polypeptides disclosed herein may comprise at least one antigen-binding site, wherein the antigen-binding site comprises a combination of at least one of the CDR1, CDR2, and CDR3 sequences described herein.

[0183] Any polypeptide having one of these CDR sequence combinations and included in the pesticidal compositions disclosed herein is selected to be capable of specifically binding to a pest target or pest antigen (as defined herein), and more particularly, to the polypeptide 10 in solution. -8 Preferably, it is capable of specifically binding to a plant pathogen target with a dissociation constant (Kd) of less than or equal to moles per liter.

[0184] The dissociation constant (Kd) can be estimated based on ELISA results. In equilibrium analyses such as ELISA, Kd can be calculated from the equilibrium binding response. When ELISA plate wells are coated with a target antigen, which may be a lipid-containing fraction of Botrytis cinerea membranes, such methods can further utilize a range of polypeptide concentrations that bind to the target antigen. When ELISA provides quantitative adsorption measurements for each polypeptide concentration, which generally represent the binding of the polypeptide to the target antigen, the range of polypeptide concentrations can be, for example, 0.5 μM, 1 μM, 2.5 μM, 5 μM, and 10 μM. The optimal concentration range used can vary depending on the affinity of the polypeptide for the target antigen. When the absorbance values ​​are plotted against the logarithmic transformation of the polypeptide concentration, a sigmoidal curve is generated from the corresponding absorbance values ​​measured by ELISA. This sigmoidal curve can be used to determine the IC50 value. If this IC50 is the concentration of polypeptide and the corresponding absorption value is 50% of the saturated value estimated by the maximum of the sigmoid function, Kd can be estimated by 1 / Ka, where the association constant (Ka) can be estimated as 1 / IC50. Thus, Kd corresponds to the analyte concentration at which equilibrium is reached at 50% binding saturation. Generally, these calculations can be performed using computer calculation methods. For example, this can be performed using GraphPad. In certain embodiments, the Kd can be measured by surface plasmon resonance (SPR).

[0185] The IC50 can be, for example, the IC50 for inhibiting spore germination and / or mycelial growth of Fusarium oxysporum and / or Botrytis cinerea (i.e., the concentration (μM) that inhibits spore germination and / or mycelial growth by 50%). In certain embodiments, the polypeptide has an IC50 for inhibiting spore germination and / or mycelial growth of less than about 10 μM, e.g., less than about 1 μM.

[0186] The Kd can be the Kd for binding to a lipid-containing fraction obtainable as described elsewhere herein (i.e., obtained by chromatographic methods, which can be a lipid-containing fraction derived from a fungus such as Fusarium oxysporum or Botrytis cinerea). The Kd of the polypeptide can be less than about 10 μM, e.g., less than about 1 μM. The Kd can be determined by any suitable method. For example, the Kd can be determined by biolayer interferometry (BLI), e.g., with Octet. The assay used to determine the Kd can be an ELISA assay.

[0187] Specific binding of the polypeptide to the pest target can be confirmed by any suitable method known per se, such as biopanning, Scatchard analysis and / or competitive binding assays such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, as well as various modifications thereof known in the art.

[0188] In a preferred embodiment, a polypeptide of 80 to 200 amino acids is obtained by affinity selection for a specific pest target molecule, and the polypeptide has a high affinity for the pest target molecule, and generally, the dissociation constant of the binding between the polypeptide and the pest target molecule is 10 -5 M, and the dissociation constant is less than 10 -6 More preferably, the dissociation constant is less than 10 -7 More preferably, the dissociation constant is less than 10 -8 Less than M is most preferred.

[0189] In certain embodiments, the at least one polypeptide in the composition disclosed herein has a minimum inhibitory concentration (MIC) value against the plant pathogenic fungus of 1.0 μg / mL or less of the variable domain in solution.

[0190] Also disclosed herein are polypeptides of 80-200 amino acids or subranges as disclosed above that 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 0.00001-1 μM, 0.001-1 μM, 0.01-1 μM, 0.1-1 μM, 0.0001-0.1 μM, 0.001-0.1 μM, 0.01-0.1 μM, 0.00001-0.01 μM, 0.0001-0.01 μM, or 0.001-0.01 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.

[0191] The minimum inhibitory concentration, or MIC value, is the lowest concentration of a substance, such as a polypeptide, that inhibits the visible growth of the crop or plant pest after incubation. For example, the minimum fungicidal concentration (MFC) is considered the lowest concentration of a polypeptide that inhibits the growth of a fungal inoculum and reduces the inoculum by at least 99-90% within 24 hours. The MFC (minimum fungicidal concentration) can be measured on agar plates, but can also be measured conventionally in liquid (e.g., in microwell plates) depending on the type of fungus and the assay conditions.

[0192] In other specific embodiments, the compositions disclosed herein comprise: a polypeptide comprising a combination of a CDR1 region having the sequence set forth in SEQ ID NO: 52, a CDR2 region having the sequence set forth in SEQ ID NO: 68, and a CDR3 region having the sequence set forth in SEQ ID NO: 84 (and which is capable of binding to a fungus); or a polypeptide comprising a combination of a CDR1 region having the sequence set forth in SEQ ID NO: 53, a CDR2 region having the sequence set forth in SEQ ID NO: 69, and a CDR3 region having the sequence set forth in SEQ ID NO: 85 (and which is capable of binding to a fungus); or a polypeptide comprising a combination of a CDR1 region having the sequence set forth in SEQ ID NO: 54, a CDR2 region having the sequence set forth in SEQ ID NO: 70, and a CDR3 region having the sequence set forth in SEQ ID NO: 86 (and which is capable of binding to a fungus); or a polypeptide comprising a combination of a CDR1 region having a sequence selected from the group consisting of SEQ ID NOs: 52 to 67 and 112 to 122, a CDR2 region having a sequence selected from the group consisting of SEQ ID NOs: 68 to 83 and 123 to 133, and a CDR3 region having a sequence selected from the group consisting of SEQ ID NOs: 84 to 100 and 134 to 144 (and which is capable of binding to a fungus); or It contains at least a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 51 or an amino acid sequence having at least about 80% sequence identity to any of them (and capable of binding to fungi).

[0193] In certain embodiments, the polypeptide in the compositions disclosed herein is a heavy chain variable domain comprising, consisting of, or consisting 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 (typically comprising at least a portion of the amino acid residues forming at least one of the CDRs as detailed herein), where the sequences of the framework regions may be variable or fixed.

[0194] The polypeptides disclosed herein can be, inter alia, antibodies, such as heavy chain antibodies. In other specific embodiments, the polypeptides disclosed herein comprise an antibody heavy chain variable domain sequence derived from a traditional four-chain antibody (e.g., but not limited to, a V nucleotide sequence derived from a human antibody). H sequence) or a so-called "heavy chain antibody" (as defined herein), HH It can be an array.

[0195] In certain embodiments, the compositions disclosed herein comprise at least a heavy chain variable domain sequence derived from an antibody or functional fragment thereof (e.g., but not limited to, a camelid heavy chain antibody or functional fragment thereof), whereby the variable domain is, for example, a camelid heavy chain antibody heavy chain variable domain (V HH ) can be used.

[0196] It should be noted that the present invention is not limited with respect to the origin of the polypeptides comprised in the compositions disclosed herein (or the nucleotide sequences of the invention used to express them), nor with respect to the method by which the polypeptides or their nucleotide sequences are made or obtained (or obtained). Thus, the polypeptides in the compositions disclosed herein can be naturally occurring (from any appropriate species) or synthetic or semi-synthetic polypeptides. In certain non-limiting embodiments of the present invention, the polypeptides are naturally occurring (from any appropriate species) or synthetic or semi-synthetic immunoglobulin sequences, including, but not limited to, "camelized" immunoglobulin sequences, as well as immunoglobulin sequences obtained by techniques such as affinity maturation (e.g., starting from synthetic, random, or naturally occurring immunoglobulin sequences), CDR grafting, veneering, joining of fragments derived from various immunoglobulin sequences, PCR assembly using overlapping primers, and similar immunoglobulin sequence generation techniques well known to those skilled in the art, or any suitable combination of the above.

[0197] The polypeptide sequences of the compositions disclosed herein may 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 one of them. For a general description of (single) domain antibodies, see also the above-cited prior art and EP 0 368 684. For the term "dAb", see, for example, 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 in the name of Domantis Ltd.

[0198] Thus, in certain embodiments, the present invention provides compounds having the (generic) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 wherein FR1 to FR4 represent framework regions 1 to 4, respectively, and CDR1 to CDR3 represent complementarity determining regions 1 to 3, respectively, as defined in detail herein.

[0199] In particular, the present invention provides, in certain embodiments, pesticide compositions that are specific for a pest target, such as a fungal target, and that contain at least one polypeptide having at least 70%, at least 75%, at least 80%, preferably at least 85%, e.g., at least 90%, or at least 95%, or at least 98% or more sequence identity to at least one of the amino acid sequences of SEQ ID NOs: 1-51, and nucleic acid sequences encoding such amino acid sequences.

[0200] Certain particularly preferred polypeptide sequences disclosed herein are capable of binding to and / or specific for pests such as fungi, and have at least 90% (e.g., at least 95% or at least 97%) amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 1-51, and in which sequence variation relative to a reference sequence (i.e., the sequence of a particular SEQ ID NO) occurs only in the CDR regions. Certain particularly preferred polypeptide sequences disclosed herein are capable of binding to and / or specific for pests such as fungi, and have at least 90% (e.g., at least 95% or at least 97%) amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 1-51, and in which sequence variation relative to a reference sequence (i.e., the sequence of a particular SEQ ID NO) occurs only in the framework regions. In other embodiments, the sequence variation relative to a reference sequence (i.e., the sequence of a particular SEQ ID NO) can occur in the CDR and / or framework regions. In certain embodiments, the sequence variation relative to a reference sequence (i.e., the sequence of a particular SEQ ID NO) can occur in the CDR3 region.

[0201] Again, such polypeptides may be obtained in any suitable manner from any suitable source, for example, naturally occurring V HH The immunoglobulin sequences may be sequences (i.e. derived from an appropriate species of camel) or synthetic or semi-synthetic heavy chain variable domains, including but not limited to "camelized" immunoglobulin sequences (in particular camelized heavy chain variable domain sequences), as well as immunoglobulin sequences obtained by techniques such as affinity maturation (e.g. starting from synthetic, random or natural immunoglobulin sequences) as detailed herein, CDR grafting, veneering, joining of fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers and similar immunoglobulin sequence generation techniques well known to those skilled in the art, or any suitable combination of the above.

[0202] It should be understood that the pesticide compositions or biocontrol compositions disclosed herein are stable during both storage and use, i.e., the integrity of the pesticide composition is maintained under the storage and / or use conditions of the pesticide composition, including high temperatures, freeze-thaw cycles, changes in pH or ionic strength, UV radiation, and the presence of harmful chemicals. It is more preferred that the 80-200 amino acid and various subrange polypeptides described herein be stably maintained in the pesticide composition, i.e., the integrity and pesticidal activity of the polypeptide are maintained under the storage and / or use conditions of the pesticide composition, including high temperatures, freeze-thaw cycles, changes in pH or ionic strength, UV radiation, and the presence of harmful chemicals. It is most preferred that the 80-200 amino acid and various subrange polypeptides described herein be stably maintained in the pesticide composition when the pesticide composition is stored at ambient temperature for two years or when the pesticide composition is stored at 54°C for two weeks. Preferably, the pesticide composition of the present invention maintains at least about 70% activity, more preferably at least about 80% activity, and most preferably at least about 90% activity or more. Optionally, the polypeptide can be formulated in a carrier, as defined herein, to protect the polypeptide from adverse effects due to other components in the pesticide composition or during storage or application. Examples of suitable carriers include, but are not limited to, alginate, gum, starch, β-cyclodextrin, cellulose, polyurea, polyurethane, polyester, microbial cells, or clay.

[0203] The pesticide composition may be in any type of formulation, and preferred formulations are dust, water dispersible powder, water dispersible granule, water dispersible granule, emulsifiable concentrate, emulsifiable concentrate, dust, suspension, suspension concentrate, suspoemulsion (a mixture of suspension and emulsifiable concentrate), capsule suspension, aqueous dispersion, oil dispersion, aerosol, paste, foam, slurry or concentrated flowable formulation.

[0204] While the polypeptides of 80-200 amino acids and the various subranges described above can be the only active substance in the pesticide or biocontrol composition of the present invention, the pesticide composition can also contain one or more other pesticides in addition to the polypeptide or amino acid sequence (or at least one, at least two, or at least three polypeptides or amino acid sequences disclosed herein). Such other pesticide or biocontrol composition may have a different activity against plant pests than the polypeptide or amino acid sequence, may have a synergistic activity with the polypeptide or amino acid sequence, or may modify the activity of the polypeptide or amino acid sequence against a given plant. Suitable other pesticides can be herbicides, insecticides, fungicides, nematicides, miticides, bactericides, virucides, plant growth regulators, safeners, etc. Such pesticides can be chemicals or biological materials, such as microorganisms. Pesticides include, but are not limited to, glyphosate, paraquat, metolachlor, acetochlor, mesotrione, 2,4-D, atrazine, glufosinate, sulfosate, fenoxaprop, pendimethalin, picloram, trifluralin, bromoxynil, clodinafop, fluroxypyr, nicosulfuron, bensulfuron, imazethapyr, dicamba, imidacloprid, thiamethoxam, fipronil, chlorpyrifos, deltamethrin, lambda-cyhalothrin, endosulfan, methamidophos, carbofuran, clothianidin, cypermethrin, abamectin, diflufenican, spinosad, indoxacarb, bifen Examples of suitable fungicides include thorin, tefluthrin, azoxystrobin, thiamethoxam, tebuconazole, mancozep, cyazofamid, fluazinam, pyraclostrobin, epoxiconazole, chlorothalonil, copper fungicides (e.g., copper oxychloride, copper hydroxide), trifloxystrobin, prothioconazole, difenoconazole, carbendazim, propiconazole, thiophanate, sulfur, boscalid, tricyclazole, hexaconazole, metalaxyl, benomyl, quitazine, tebuconazole, tridemorph, propineb, streptomycin sulfate, and oxytetracycline, as well as other known pesticides or any suitable combination thereof.

[0205] Suitable other pesticides may be biological materials such as microorganisms, for example, strains of Pseudomonas, Bacillus or Streptomyces.

[0206] Compositions Comprising Polypeptide Sequence Variants In certain aspects, the polypeptides included in the pesticide compositions disclosed herein can be optionally modified, for example, to increase the amount of positive charge (carried by the polypeptide). That is, the polypeptide can be modified to increase the amount of positive charge, typically by one or more amino acid substitutions. Thus, an amino acid can be replaced with an amino acid having a greater positive charge (relative to the amino acid being replaced). Two or more such substitutions can be made, for example, two, three, four, or five such substitutions can be made. In certain embodiments, up to one, two, three, four, or five such substitutions can be made. Such substitutions can typically be made in the CDR regions, e.g., the CDR1, CDR2, or CDR3 regions.

[0207] Other substitutions may be made to the polypeptide, for example, substitutions that do not affect the overall charge of the polypeptide. Advantageously, the substitutions do not decrease the overall charge of the polypeptide, as the inventors have surprisingly found that an increase in positive charge can correlate with improved antifungal activity.

[0208] Other substitutions are also contemplated. For example, the polypeptide can start with a D or Q residue. The inventors have surprisingly discovered that, although Q can be used for residue 1 of the polypeptide (i.e., the first residue in the framework 1 region sequence), using D can improve the antifungal properties of the polypeptide. Thus, for all specific polypeptide sequences disclosed herein (including all peptides having any one of SEQ ID NOS: 1-51 or 101-111), residue 1 can be a Q residue, but in certain embodiments is preferably a D residue. A designation of a polypeptide such as 10G11Q indicates that the polypeptide starts with a Q residue. A designation of a polypeptide such as 10G11 (without the Q at the end) indicates that the polypeptide starts with a D residue. 10G11 may also be referred to herein as 10G11Q1D (a notation indicating a substitution of Q with D at position 1).

[0209] In certain embodiments, the present invention provides polypeptides comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-51, 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%, or at least about 95%, or at least about 98% identity to any of them. All polypeptides having a particular percent sequence identity to a given SEQ ID NO can have the same total charge as the reference sequence, or can have more positive charges than the reference sequence. Advantageously, all polypeptides having a particular percent sequence identity to a given SEQ ID NO will not have more negative charges than the reference sequence.

[0210] In certain embodiments, the present invention provides a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-51, or an amino acid sequence having up to 1, 2, 3, 4, or 5 amino acid substitutions relative to said sequence.

[0211] In certain embodiments, the present invention provides polypeptides comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10 and 12-51, 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%, or at least about 95%, or at least about 98% identity to any of them. All polypeptides having a particular % sequence identity to a given SEQ ID NO can have the same total charge as the reference sequence, or can have more positive charges than the reference sequence. Advantageously, all polypeptides having a particular % sequence identity to a given SEQ ID NO will not have more negative charges than the reference sequence.

[0212] In certain embodiments, the present invention provides a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10 and 12-51, or an amino acid sequence having up to 1, 2, 3, 4, or 5 amino acid substitutions relative to said sequence.

[0213] In certain embodiments, the present invention provides polypeptides comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 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%, or at least about 95%, or at least about 98% identity to any of them. All polypeptides having a particular percent sequence identity to a given SEQ ID NO can have the same total charge as the reference sequence, or can have more positive charges than the reference sequence. Advantageously, all polypeptides having a particular percent sequence identity to a given SEQ ID NO will not have more negative charges than the reference sequence.

[0214] In certain embodiments, the present invention provides a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6, or an amino acid sequence having up to 1, 2, 3, 4, or 5 amino acid substitutions relative to said sequence.

[0215] All amino acid substitutions in the polypeptide may increase the overall charge of the polypeptide or may not change the overall charge of the polypeptide, hi certain embodiments, no amino acid substitution decreases the overall charge of the polypeptide.

[0216] All amino acid substitutions in the polypeptide may occur anywhere in the polypeptide sequence. Optionally, the amino acid substitutions may be confined to the CDR regions (in such embodiments, the polypeptide maintains the original framework region sequence) or may not be confined to the CDR regions. In certain embodiments, all substitutions or mutations in the sequence may occur in any residue in the CDR regions or up to two amino acid residues on either side of a CDR region (as defined by the Kabat numbering system). In certain embodiments, all substitutions or mutations in the sequence may occur in any residue in the CDR regions or up to one amino acid residue on either side of a CDR region (as defined by the Kabat numbering system). In certain embodiments, all substitutions or mutations in the sequence may occur in the CDR regions or in residues adjacent to the N-terminus of the CDR3 region (as defined by the Kabat numbering system).

[0217] The present invention provides specific mutants, designated herein as mutants 1-5 (which alter the overall charge of the polypeptide to determine its functional impact on the polypeptide), as well as mutants designated herein as "single ALA mutants" 1-34, which have been subjected to alanine scanning, although some positions contain substitutions other than alanine. The present invention also extends to other mutants or variants of the polypeptide sequences disclosed herein, including those with other substitutions, or with various combinations of substitutions, or with substitutions at various positions within the polypeptide sequence. Suitably, any mutant or variant of the polypeptide sequences disclosed herein does not decrease the overall positive charge relative to the reference sequence. Optionally, mutants or variants of the polypeptide sequences disclosed herein may increase the overall positive charge.

[0218] The total charge of the polypeptide can be calculated before and after the substitution or mutation, and the charge of the polypeptide is calculated in aqueous solution at the same pH before and after introducing the substitution or mutation into the sequence.

[0219] 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 a free online tool such as ExPASy-ProtParam (https: / / web.expasy.org / protparam / ).

[0220] The charge of the polypeptide is preferably calculated at pH 7. Generally, the polypeptide will have a positive overall charge (at pH 7). Substitutions or mutations in the sequence preferably do not decrease the overall charge (at pH 7). In certain embodiments, substitutions or mutations in the sequence will increase the overall positive charge of the polypeptide (at pH 7).

[0221] The charge or total charge of the 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. Generally, the polypeptide is determined to have a pI greater than 7, meaning that the total charge is positive at pH 7. Preferably, the substitutions or mutations in the sequence do not decrease the pI. In certain embodiments, the substitutions or mutations in the sequence will increase the pI.

[0222] The present invention also provides polypeptides (and compositions containing the polypeptides) having predetermined sequences that allow for mutations or substitutions at specific positions. Such mutants include those referred to herein as 10G11-A through 10G11-K.

[0223] For example, in one embodiment, the present invention provides a method for producing a nucleotide sequence comprising the following sequence: [ka] In the above sequence, X1 is D or Q, and X2 to X 11 are each independently any naturally occurring amino acid (SEQ ID NO: 101, also referred to herein as 10G11-A).

[0224] In certain embodiments, X1 is D or Q, and X2 to X 11 are each independently any naturally occurring amino acid other than E or D (SEQ ID NO: 102, also referred to herein as 10G11-B).

[0225] In certain embodiments, X1 is D or Q, and X2, X6, X7, X8 and X 11 are each independently G, A, V, M, L, I, K, R, or H; X, X, X, X, and X 10 are each independently any naturally occurring amino acid other than E or D (SEQ ID NO: 103, also referred to herein as 10G11-C).

[0226] In certain embodiments, X1 is D or Q, and X2, X6, X7, X8 and X 11are each independently A, K, R, or H; X, X, X, X, and X 10 are each independently any naturally occurring amino acid other than E or D (SEQ ID NO: 104, also referred to herein as 10G11-D).

[0227] In certain embodiments, X1 is D or Q, and X2, X6, X7, X8 and X 11 are each independently A, K, R or H, and X3, X5 and X 10 are each independently an uncharged polar amino acid or a positively charged amino acid (i.e., S, T, C, P, N, Q, K, R, or H), X4 is a nonpolar aliphatic amino acid or a positively charged amino acid (i.e., I, G, A, V, M, L, K, R, or H), and X9 is an aromatic amino acid or a positively charged amino acid (i.e., W, F, Y, K, R, or H) (SEQ ID NO: 105, also referred to herein as 10G11-E).

[0228] In certain embodiments, X1 is D or Q, and X2, X6, X7, X8 and X 11 are each independently A, K, R, or H; 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 (SEQ ID NO: 106, also referred to herein as 10G11-F).

[0229] In certain embodiments, X1 is D or Q, and X2, X6, X7, X8 and X 11 are each independently X3, X4, X5, X9 and X 10 each independently (SEQ ID NO: 107, also referred to herein as 10G11-G).

[0230] In certain embodiments, X1 is D or Q, and X2, X6, X8 and X 11 are each independently R; X, X, X, X and X 10 are each independently any naturally occurring amino acid (SEQ ID NO: 108, also referred to herein as 10G11-H).

[0231] In certain embodiments, X1 is D or Q, and X2, X6, X7, X8 and X 11 are each independently R; X, X, X, X and X 10 are each independently any naturally occurring amino acid other than E or D (SEQ ID NO: 109, also referred to herein as 10G11-I).

[0232] In certain embodiments, X1 is D or Q, and X2, X6, X8 and X 11 are each independently R; X3, X5 and X 10 are each independently an uncharged polar amino acid or a positively charged amino acid (i.e., S, T, C, P, N, Q, K, R, or H), X4 is a nonpolar aliphatic amino acid or a positively charged amino acid (i.e., I, G, A, V, M, L, K, R, or H), X7 is K, and X9 is an aromatic amino acid or a positively charged amino acid (i.e., W, F, Y, K, R, or H) (SEQ ID NO: 110, also referred to herein as 10G11-J).

[0233] In certain embodiments, X1 is D or Q, and X2, X6, X8 and X 11 are R, and 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).

[0234] In certain embodiments, the invention provides 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 A polypeptide comprising a CDR3 region comprising DY or consisting of the same sequence, wherein X2 to X 11 are each independently any naturally occurring amino acid (thus the CDR1, CDR2 and CDR3 regions have the sequences of SEQ ID NOs: 112, 123 and 134, respectively).

[0235] In certain embodiments, X to X 11 are each independently any naturally occurring amino acid other than E or D (thus, the CDR1, CDR2 and CDR3 regions have the sequences of SEQ ID NOs: 113, 124 and 135, respectively).

[0236] In certain embodiments, X, X, X, X and X 11 are each independently G, A, V, M, L, I, K, R, or H; X, X, X, X, and X 10 are each independently any naturally occurring amino acid other than E or D (thus, the CDR1, CDR2 and CDR3 regions have the sequences of SEQ ID NOs: 114, 125 and 136, respectively).

[0237] In certain embodiments, X1 is D or Q, and X2, X6, X7, X8 and X 11 are each independently A, K, R, or H; X, X, X, X, and X 10 are each independently any naturally occurring amino acid other than E or D (thus, the CDR1, CDR2 and CDR3 regions have the sequences of SEQ ID NOs: 115, 126 and 137, respectively).

[0238] In certain embodiments, X, X, X, X and X 11 are each independently A, K, R or H, and X3, X5 and X 10 are each independently an uncharged polar amino acid or a positively charged amino acid (i.e., S, T, C, P, N, Q, K, R, or H), X4 is a nonpolar aliphatic amino acid or a positively charged amino acid (i.e., I, G, A, V, M, L, K, R, or H), and X9 is an aromatic amino acid or a positively charged amino acid (i.e., W, F, Y, K, R, or H) (thus, the CDR1 region, CDR2 region, and CDR3 region have the sequences of SEQ ID NOs: 116, 127, and 138, respectively).

[0239] In certain embodiments, X, X, X, X and X 11are each independently A, K, R, or H; 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 (therefore, the CDR1, CDR2 and CDR3 regions have the sequences of SEQ ID NOs: 117, 128 and 139, respectively). (SEQ ID NO: 106).

[0240] In certain embodiments, X, X, X, X and X 11 are each independently X3, X4, X5, X9 and X 10 (Accordingly, the CDR1, CDR2 and CDR3 regions have the sequences of SEQ ID NOs: 118, 129 and 140, respectively). (SEQ ID NO: 107).

[0241] In certain embodiments, X, X, X and X 11 are each independently R; X, X, X, X and X 10 are each independently any naturally occurring amino acid (thus, the CDR1, CDR2 and CDR3 regions have the sequences of SEQ ID NOs: 119, 130 and 141, respectively). (SEQ ID NO: 108).

[0242] In certain embodiments, X, X, X, X and X 11 are each independently R; X, X, X, X and X 10 are each independently any naturally occurring amino acid other than E or D (thus, the CDR1, CDR2 and CDR3 regions have the sequences of SEQ ID NOs: 120, 131 and 142, respectively). (SEQ ID NO: 109).

[0243] In certain embodiments, X, X, X and X 11 are each independently R; X3, X5 and X 10are each independently an uncharged polar amino acid or a positively charged amino acid (i.e., S, T, C, P, N, Q, K, R, or H), X4 is a nonpolar aliphatic amino acid or a positively charged amino acid (i.e., I, G, A, V, M, L, K, R, or H), X7 is K, and X9 is an aromatic amino acid or a positively charged amino acid (i.e., W, F, Y, K, R, or H) (thus, the CDR1 region, CDR2 region, and CDR3 region have the sequences of SEQ ID NOs: 121, 132, and 143, respectively). (SEQ ID NO: 110).

[0244] In certain embodiments, X, X, X and X 11 are R, and 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 (therefore, the CDR1, CDR2 and CDR3 regions have the sequences of SEQ ID NOs: 122, 133 and 144, respectively). (SEQ ID NO: 111).

[0245] All of the polypeptides disclosed herein, including variants thereof, can be incorporated into compositions (eg, pesticide compositions).

[0246] In general, the invention extends to variants of the polypeptides, which may maintain their functional properties or may have improved functional properties. For example, the variants may be capable of (specifically) binding to fungi. More specifically, the variants may be capable of (specifically) binding to fungal membranes or components of fungal membranes. In certain embodiments, the variants do not bind to fungal cell walls or cell wall components. For example, in certain embodiments, the variant polypeptides do not (specifically) bind to fungal glucosylceramide.

[0247] The mutant may be capable of binding to a lipid-containing fraction of the cell membrane of a fungus (e.g., Botrytis cinerea or other fungus). The lipid-containing fraction may be obtainable by chromatography. For example, the lipid-containing fraction may be obtainable by a method comprising fractionating fungal (e.g., Botrytis cinerea or other fungus) mycelia by total lipid extract thin-layer chromatography and selecting fractions having a retention factor (Rf) greater than that of a ceramide fraction and less than that of a non-polar phospholipid fraction.

[0248] The mutant may further cause slowing of fungal spore growth and / or lysis of the fungal spore, i.e., binding of the mutant polypeptide to a fungus results in slowing of fungal spore growth and / or lysis of the fungal spore.

[0249] In certain embodiments, the variant is capable of retarding fungal spore growth and / or lysing fungal spores with an IC50 equal to or lower than the IC50 of a reference polypeptide when measured under substantially identical conditions, where the IC50 can be, for example, the concentration (μM) that inhibits 50% of spore germination and / or mycelial growth of Fusarium oxysporum.

[0250] In certain embodiments, the variant can (specifically) bind to a fungus (e.g., a fungal membrane or a component of a fungal membrane) with a KD that is equal to or lower than the KD of the reference polypeptide when measured under substantially the same conditions.

[0251] In certain embodiments, the variant may (specifically) exhibit an MIC for fungal growth that is less than or equal to the MIC of the reference polypeptide when measured under substantially identical conditions.

[0252] In certain embodiments, the polypeptides included in the pesticide compositions disclosed herein can be operably linked to one or more other groups, moieties, or residues, optionally via one or more linkers. These one or more other groups, moieties, or residues can serve to bind to other targets of interest. Of course, such other groups, residues, moieties, and / or binding sites may or may not provide other functionality to the polypeptides disclosed herein (and / or the compositions in which they are present), and may or may not alter the properties of the polypeptides disclosed herein. Such groups, residues, moieties, or binding sites can be, for example, chemical groups that can be biologically active.

[0253] These groups, moieties, or residues may, in certain embodiments, be linked to the N-terminus or C-terminus of a polypeptide in the compositions disclosed herein.

[0254] In certain embodiments, the polypeptide in the pesticide composition disclosed herein may be chemically modified. For example, such modifications may include introducing or linking one or more functional groups, residues, or moieties to the heavy chain variable domain. These groups, residues, or moieties can confer one or more desirable properties or functions to the polypeptide. Examples of such functional groups will be apparent to those skilled in the art.

[0255] For example, introducing or linking such functional groups to a polypeptide can improve the solubility and / or stability of the polypeptide, reduce the toxicity of the polypeptide, or eliminate or reduce undesirable side effects of the polypeptide, and / or provide other advantageous properties.

[0256] In certain embodiments, the one or more groups, residues, moieties are linked to the polypeptide via one or more suitable linkers or spacers.

[0257] In certain other embodiments, two or more target-specific polypeptide molecules in the pesticide compositions disclosed herein can be linked or interconnected. In certain embodiments, the two or more polypeptide molecules are linked to each other via one or more suitable linkers or spacers. Suitable spacers or linkers for use in coupling the various heavy chain polypeptides disclosed herein will be apparent to those of skill in the art and generally can be any linker or spacer used in the art to link peptides and / or proteins.

[0258] Particularly suitable linkers or spacers include, but are not limited to, polypeptide linkers such as glycine linkers, serine linkers, mixed glycine / serine linkers, glycine-rich linkers, serine-rich linkers, or linkers composed primarily of polar polypeptide fragments or homo- or hetero-bifunctional chemical cross-linking compounds such as glutaraldehyde, maleimide or NHS esters, optionally via a PEG spacer.

[0259] For example, the polypeptide linker or spacer can be a suitable amino acid sequence having a length of 1 to 50 amino acids, such as 1 to 30, particularly 1 to 10 amino acid residues. It will be appreciated that the length, degree of flexibility, and / or other properties of the linker will have some effect on the properties of the polypeptide, including, but not limited to, affinity, specificity, or avidity for a pest target. It will be appreciated that, when two or more linkers are used, these linkers may be the same or different. In the context and disclosure of the present invention, one skilled in the art will be able to determine, without undue experimentation, the optimal linker for coupling the heavy chain variable domains disclosed herein.

[0260] Compositions containing fragments of polypeptide sequences The present invention also encompasses portions, fragments, analogs, mutants, variants, and / or derivatives of the polypeptides contained in the compositions disclosed herein, and / or polypeptides comprising or consisting essentially of one or more of such portions, fragments, analogs, mutants, variants, and / or derivatives, provided that such portions, fragments, analogs, mutants, variants, and / or derivatives are suitable for the purposes contemplated herein. Such portions, fragments, analogs, mutants, variants, and / or derivatives of the present invention are also capable of specifically binding to pest targets (e.g., fungi, such as plant pathogenic fungi).

[0261] target In certain embodiments, the polypeptides included in the compositions disclosed herein are obtained by affinity selection against a particular pest target, such as a fungal antigen or fungal target. Obtaining suitable polypeptides by affinity selection for a particular pest target can be carried out, for example, by screening a set, collection or library of cells (e.g., bacteriophage) expressing the polypeptide on their surface for binding to a pest target molecule known in the art to be a target for a pesticide, all of which can be carried out in a manner known per se and essentially comprises the following non-limiting steps: a) obtaining an isolated solution or suspension of a pest target molecule known to be a target for a pesticide; b) biopanning phage or other cells from a polypeptide library against the target molecule; c) isolating phage or other cells that bind to the target molecule; d) determining the nucleotide sequence encoding the polypeptide insert from each binding phage or other cell; e) producing a quantity of polypeptide according to this sequence using recombinant protein expression; f) measuring the affinity of the polypeptide for the pest target; and, optionally, g) testing the pesticidal activity of the polypeptide in a bioassay for the pest. Various methods can be used to measure the affinity between the polypeptide and the pest target molecule, including enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (SPR) assay, which are common methods in the art, as described in Sambrook et al. (2001), Molecular Cloning, A Laboratory Manual. Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Dissociation constants are widely used to express the affinity between a polypeptide and its pest target molecule. Generally, the dissociation constant for binding between a polypeptide and its pest target molecule is 10 -5 M, and the dissociation constant is less than 10 -6 More preferably, the dissociation constant is less than 10 -7More preferably, the dissociation constant is less than 10 -8 Less than M is most preferred.

[0262] Pest target molecules disclosed herein are molecules present in or on the surface of a pest, which when bound to and / or inhibited, kill the pest or prevent, inhibit or suppress its growth or activity. Such suitable target molecules can be easily found by a person skilled in the art from existing literature or patent databases, and examples thereof include, but are not limited to, secreted parasitic proteins such as 16D10 as a suitable pest target molecule for root-knot nematodes (Huang et al (2006) PNAS 103:14302-14306), V-ATPase proton pump as a suitable pest target molecule for insect species and nematodes of the orders Coleoptera, Hemiptera, and Diptera (Knight AJ and Behm CA (2011) Ex. Parasitol. Sept 19), and tetraspanin PLS1 as a suitable fungal pest target molecule for Botrytis cinerea and M. grisea (Gourgues et al. al (2002) Biochem. Biophys. Res. Commun. 297:1197) or antifungal targets such as proton pump ATPases (Manavathu EK et al (1999) Antimicrob Agents and Chemotherapy, December p. 2950). Naturally, preferred pest target molecules are accessible in the extracellular space (as opposed to intracellular pest targets).

[0263] More specifically, the pest target to which the at least one polypeptide of the pesticide composition disclosed herein binds can be a pest cell membrane component. The pest cell membrane component used herein can be any component contained in or part of (i.e., at least a portion of) the cell membrane phospholipid bilayer or a protein embedded in the phospholipid bilayer of the pest cell. In certain embodiments, the pest cell membrane component can be a phospholipid, a glycoprotein, a carbohydrate, or cholesterol.

[0264] In certain embodiments, the pest cell membrane component to which the at least one polypeptide in the compositions disclosed herein specifically binds is other than a protein.

[0265] Thus, in certain embodiments, the pest cell membrane component to which the at least one polypeptide in the compositions disclosed herein specifically binds is a lipid, such as, for example, a phospholipid, a carbohydrate, or cholesterol.

[0266] In certain particular embodiments, the target to which the polypeptide in the pesticide composition of the present invention binds is other than a cell wall component.

[0267] In certain specific embodiments, the target to which the polypeptide in the pesticide composition of the present invention binds is other than chitin.

[0268] In a preferred embodiment, the plant pests controlled by the pesticide or biocontrol compositions disclosed herein are fungi, such as the plant pathogenic fungi defined above. Fungi are highly harmful to plants and can result in substantial crop yield losses. Plant pathogenic fungi include necrotrophic and biotrophic fungi, including Ascomycetes, Basidiomycetes, and Oomycetes.

[0269] Examples of plant pathogenic fungi are known in the art and include, but are not limited to, the genera Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Diplodia, Erysiphe, Fusarium, Leptosphaeria, Gaeumanomyces, Helminthosporium, Macrophomina, Nectria, Oidium, Peronospora, Phakopsora, Phoma, and the like. ), Phymatotrichum, Phytophthora, Plasmopara, Podosphaera, Puccinia, Puthium, Pyrenophora, Pyricularia, Pythium, Rhizoctonia, Scerotium, Sclerotinia, Septoria, Thielaviopsis, Uncinula, Venturia, and Verticillium. Specific examples of plant fungal infections that can be controlled by the pesticide composition of the present invention include powdery mildew and gray mold (Botrytis cinerea) on fruit trees such as grapes and strawberries, and on vegetables. Other specific examples of plant fungal infections that can be controlled by the pesticide composition of the present invention include powdery mildew of cereals such as barley (Erysiphe graminis), powdery mildew of Cucurbitaceae plants (Erysiphe cichoracearum and Sphaerotheca fuliginea), and powdery mildew of apple (Podosphaeraleucotricha, for example powdery mildew of strawberry (Podosphaera aphanis), for example powdery mildew of cucumber (Podosphaera xanthii), for example powdery mildew of tomato (Oidium neolycopersici), powdery mildew of grapes (Uncinula necator), Puccinia sp. of cereals, Rhizoctonia sp. of cotton, potato, rice and lawn grass, Ustilago sp. of cereals and sugarcane, Venturia inaequalis (scab) of apple, Helminthosporium sp. of cereals, Septoria nodorum, Septoria tritici, barley scald (Rhynchosporium secalis), grey mold of strawberries, tomatoes, and grapes (Botrytis cinerea), brown spot of peanuts (Cercospora arachidicola), tobacco downy mildew (Peronospora tabacina) or downy mildew of various crops (Peronospora), eyespot of wheat and barley (Pseudocercosporella herpotrichoides), barley net blotch (Pyrenophera teres), rice blast (Pyricularia oryzae), potato and tomato late blight (Phytophthora infestans), Fusarium sp. (e.g. Fusarium wilt (Fusarium oxysporum)) and Verticillium sp. (Verticillium sp.), downy mildew of grapes (Plasmopara viticola), Alternaria sp. of fruit trees and vegetables (Alternaria sp.), downy mildew of cucumber (Pseudoperonospora cubensis), Sigatoka disease of bananas (Mycosphaerella fijiensis), Ascochyta sp. of chickpeas (Ascochyta sp.), Leptosphaeria sp. of canola and rapeseedThese include Phakopsora spp. (e.g., soybean rust (Phakopsora pachyrhizi)) and Colletotrichum spp. (e.g., Colletotrichum orbiculare, which can cause anthracnose on squash) in various crops. The compositions of the present invention are active against normally susceptible and resistant species and are active against all or some stages of the life cycle of plant pathogenic fungi.

[0270] In certain embodiments, the pesticidal compositions disclosed herein are effective against pests of the genera Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Diplodia, Erysiphe, Fusarium, Leptosphaeria, Gaeumanomyces, and the like. , Helminthosporium, Macrophomina, Nectria, Oidium, Penicillium, Peronospora, Phoma, Phymatotrichum, Phytophthora, Plasmopara, Podosphaera , Puccinia, Pyrenophora, Pyricularia, Pythium, Rhizoctonia, Scerotium, Sclerotinia, Septoria, Thielaviopsis, Uncinula, Venturia, Verticillium The fungal pathogen is specific for plant pathogenic fungi of a genera selected from the group consisting of: P. ticillium, Magnaporthe, Blumeria, Mycosphaerella, Ustilago, Melampsora, Phakopsora, Monilinia, Mucor, Rhizopus, and Aspergillus.

[0271] In certain specific embodiments, the compositions disclosed herein contain at least a polypeptide that specifically binds to a fungal target (e.g., a fungal cell membrane component) of a fungal species of the genus Botrytis, Fusarium, or Penicillium.

[0272] In certain embodiments, the present invention provides pesticide compositions containing polypeptides specific for structural molecular components of pest cell membranes.

[0273] In certain embodiments, the present invention provides pesticide compositions containing polypeptides specific for structural molecular components other than proteins of pest cell membranes.

[0274] In yet another specific embodiment, the plant pest is a plant pathogenic bacterium, including but not limited to Acidovorax avenae subsp. avenae (causing rice brown stripe), Acidovorax avenae subsp. cattleyae (causing Cattleya bacterial brown spot), Acidovorax konjaci (causing konjac leaf blight), Agrobacterium rhizogenes (causing melon hairy root disease), Agrobacterium tumefaciens (causing crown gall), Burkholderia andropogonis (causing carnation bacterial spot), and the like. andropogonis, Burkholderia caryophylli (causing carnation bacterial wilt), Burkholderia cepacia (causing Cymbidium bacterial brown spot), Burkholderia gladioli pv. gladioli (causing gladioli neck rot), Burkholderia glumae (causing rice bacterial grain rot), Burkholderia plantarii (causing rice seedling blight), and Clavibacter michiganensis subsp. michiganensis (causing tomato canker). subsp. michiganensis), Clavibacter michiganensis subsp. sepedonicus (causing potato ring rot), Clostridium spp. (causing potato slimy rot).), Curtobacterium flaccumfaciens (causing onion canker), Erwinia amylovora (causing pear fire blight), Erwinia ananas (causing rice glume browning), Erwinia carotovora subsp. atroseptica (causing potato blackleg), Erwinia carotovora subsp. carotovora (causing vegetable soft rot), Erwinia chrysanthemi (causing taro seedling bacterial blight), chrysanthemi pv. zeae (causing rice foot rot), Erwinia herbicola pv. millettiae (causing Fuji clubroot), Pseudomonas cichorii (causing chrysanthemum bacterial spot), Pseudomonas corrugata (causing tomato bacterial stem necrosis), Pseudomonas fuscovaginae (causing rice leaf sheath browning), and Pseudomonas marginalis (causing cabbage soft rot). pv. marginalis), Pseudomonas rubrisubalbicans (causing sugarcane stripe disease), Pseudomonas syringae pv. aptata (causing sugar beet bacterial spot disease), Pseudomonas syringae pv. atropurpurea (causing ryegrass halo blight disease).atropurpurea, Pseudomonas syringae pv. castaneae (causing chestnut canker), Pseudomonas syringae pv. glycinea (causing soybean bacterial leaf spot), Pseudomonas syringae pv. lachrymans (causing cucumber bacterial leaf spot), Pseudomonas syringae pv. maculicola (causing cabbage bacterial black spot), and Pseudomonas syringae pv. mori (causing mulberry bacterial leaf curl). pv.mori), Pseudomonas syringae pv.morsprunorum (causing plum canker), Pseudomonas syringae pv.oryzae (causing rice halo blight), Pseudomonas syringae pv.phaseolicola (causing bean halo blight), Pseudomonas syringae pv.pisi (causing pea bacterial spot), Pseudomonas syringae pv.sesame (causing sesame bacterial spot) pv.sesame), Pseudomonas syringae pv.striafaciens (causing oat streak blight), Pseudomonas syringae pv.syringae (causing adzuki bean brown spot), Pseudomonas syringae pv. tabaqui (causing tobacco wildfire disease).tabaci), Pseudomonas syringae pv. theae (causing tea leaf spot), Pseudomonas syringae pv. tomato (causing tomato leaf spot), Pseudomonas viridiflava (causing bean brown spot), Ralstonia solanacearum (causing bacterial wilt), Rathayibacter rathayi (causing orchardgrass yellow gum disease), and Streptomyces scabiei (causing potato scab). scabies), Streptomyces ipomoea (causing damping-off of sweet potato), Xanthomonas albilineans (causing white streak of sugarcane), Xanthomonas campestris pv. cerealis (causing bacterial rye streak), Xanthomonas campestris pv. campestris (causing black rot), Xanthomonas campestris pv. campestris (causing citrus canker), Xanthomonas campestris pv. citri (causing citrus canker) pv.citri), Xanthomonas campestris pv.cucurbitae (causing cucumber brown spot bacterial disease), Xanthomonas campestris pv.glycines (causing soybean leaf burn), Xanthomonas campestris pv.incanae (causing stock black rot).incanae), Xanthomonas campestris pv. malvacearum (causing cotton angular spot), Xanthomonas campestris pv. mangiferaeindicae (causing mango canker), Xanthomonas campestris pv. mellea (causing tobacco yellow spot), Xanthomonas campestris pv. nigromaculans (causing burdock black spot), and Xanthomonas campestris pv. phaseoli (causing bean leaf burn). pv.phaseoli), Xanthomonas campestris pv.pisi (causing kidney bean stem rot), Xanthomonas campestris pv.pruni (causing peach bore), Xanthomonas campestris pv.raphani (causing radish bacterial spot), Xanthomonas campestris pv.ricini (causing castor bean bacterial spot), and Xanthomonas campestris pv.teicola (causing tea canker). pv.theicola), Xanthomonas campestris pv.translucens (causing bacterial spot of orchardgrass), and Xanthomonas campestris pv.vesicatoria (causing bacterial spot of tomato).vesicatoria) and Xanthomonas oryzae pv. oryzae (causing rice bacterial leaf blight).

[0275] In yet another embodiment, the pesticide formulations of the present invention can also be used to control plant pests such as insects, arachnids, helminths, viruses, nematodes and mollusks encountered in agriculture, horticulture, forestry, gardens and leisure areas. The compositions according to the present invention are active against normally susceptible and resistant species and against all or some stages of development. These plant pests include those of the phylum Arthropoda, in particular arachnid pests, such as Acarus spp., Aceria sheldoni, Aculops spp., Aculus spp., Amblyomma spp., Amphitetranychus viennensis, Argas spp., Boophilus spp., Brevipalpus spp., Bryobia praetiosa, Centruroides spp., Chorioptes spp. spp.), Dermanyssus gallinae, Dermatophagoides pteronyssius, Dermatophagoides farinae, Dermacentor spp., Eotetranychus spp., Epitrimerus pyri, Eutetranychus spp., Eriophyes spp., Halotydeus destructor, Hemitarsonemus spp., Hyalomma spp., Ixodes spp., Latrodectus spp. spp.), Loxosceles spp., Metatetranychus spp.), Nuphersa spp., Oligonychus spp., Ornithodorus spp., Ornithonyssus spp., Panonychus spp., Phyllocoptruta oleivora, Polyphagotarsonemus latus, Psoroptes spp., Rhipicephalus spp., Rhizoglyphus spp., Sarcoptes spp., Israeli gold scorpion (Scorpio maurus), Stenotarsonemus spp. spp.), Tarsonemus spp., Tetranychus spp., Vaejovis spp., and Vasates lycopersici.

[0276] Further examples are the suborder Anoplura (order Phthiraptera), such as Damalinia spp., Haematopinus spp., Linognathus spp., Pediculus spp., Ptirus pubis, and Trichodectes spp.

[0277] Still other examples are the class Chilopoda, including, for example, Geophilus spp. and Scutigera spp.

[0278] Further examples are the order Coleoptera, such as Acalymma vittatum, Acanthoscelides obtectus, Adoretus spp., Agelastica alni, Agriotes spp., Alphitobius diaperinus, Amphimallon solstitialis, Anobium punctatum, Anoplophora spp., Anthonomus spp., Anthrenus spp., spp.), Apion spp., Apogonia spp., Atomaria spp., Attagenus spp., Bruchidius obtectus, Bruchus spp., Cassida spp., Cerotoma trifurcata, Ceutorrhynchus spp., Chaetocnema spp., Cleonus mendicus, Conoderus spp., Cosmopolites spp. spp.), Costelytra zealandica, Ctenicera spp., Curculio spp., Cryptorhynchus lapathi, Cylindrocopturus spp., Dermestes spp., Diabrotica spp., Dichocrocis spp., Diloboderus spp.), Epilachna spp., Epitrix spp., Faustinus spp., Gibbium psylloides, Hellula undalis, Heteronychus arator, Heteronyx spp., Hylamorpha elegans, Hylotrupes bajulus, Hypera postica, Hypothenemus spp., Lachnosterna consanguinea, Lema spp.), Colorado potato beetle (Leptinotarsa ​​decemlineata), Leucoptera spp., rice water weevil (Lissorhoptrus oryzophilus), Lixus spp., Luperodes spp., Lyctus spp., Megascelis spp., comb click beetle (Melanotus spp.), Meligethes aeneus, cockchafer beetle (Melolontha spp.), Migdolus spp., Monochamus spp., Naupactus xanthographus xanthographus, golden spider beetle (Niptus hololeucus), rhinoceros beetle (Oryctes rhinoceros), sawtoothed beetle (Oryzaephilus surinamensis), rice water weevil (Oryzaphagus oryzae), Otiorrhynchus spp., Oxycetonia jucunda, Phaedon cochleariae, Phyllophaga spp.), Phyllotreta spp., Japanese beetle (Popillia japonica), Premnotrypes spp., Prostephanus truncatus, Psylliodes spp., Ptinus spp., Rhizobius ventralis, Rhizopertha dominica, Sitophilus spp., Sphenophorus spp., Stegobium paniceum, Sternechus spp., Symphyletes spp. spp.), Tanymecus spp., Tenebrio molitor, Tribolium spp., Trogoderma spp., Tychius spp., Xylotrechus spp., and Zabrus spp. are examples of such beetles.

[0279] Yet another example is the Collembola order, such as Onychiurus armatus.

[0280] Yet another example is the class Diplopoda, which includes the snake millipede Blaniulus guttulatus.

[0281] Further examples are the order Diptera (flies), 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. 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. 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. spp.), and crane fly species (Tipula spp.).

[0282] Further examples are the suborder 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. spp.), Pentomidae, Piesma quadrata, Piezodorus spp., Psallus spp., Pseudacysta persea, Rhodnius spp., Sahlbergella singularis, Scaptocoris castanea, Scotinophora spp.), Stephanitis nashi, Tibraca spp., and Triatoma spp.

[0283] Further examples are the order Homoptera, such as Acyrthosiphon spp., Acrogonia spp., Aeneolamia spp., Agonoscena spp., Aleurodes spp., Aleurolobus barodensis, Aleurothrixus spp., Amrasca spp., Anuraphis cardui, Aonidiella spp., Aphanostigma pin, Aphis gossypii, 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.), strawberry woolly aphid (Chaetosiphon fragaefolii), Chionaspis tegalensis, Chlorita onukii, walnut aphid (Chromaphis juglandicola), Chrysomphalus ficus, corn leafhopper (Cicadulina mbila), Coccomytilus halli, Coccus spp., Cryptomyzus ribis, Dalbulus spp., Dialeurodes spp., Diaphorina spp., Diaspis spp. 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, Bayberry whitefly, Parabemisia myricae, Paratrioza spp. spp.), Parlatoria spp., Pemphigus spp., Corn planthopper (Peregrinus maidis), Cotton mealybug (Phenacoccus spp.), Poplar aphid (Phloeomyzus passerinii), Hop wart aphid (Phorodon humuli), Phylloxera spp., Pinnaspis aspidistrae, Planococcus spp., Pear scale (Protopulvinaria pyriformis), Mulberry white scale (Pseudaulacaspis pentagona), Mulberry mealybug (Pseudococcus spp.) 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, Tenalapha malayensis, Tinocallis caryaefoliae, Tomaspis spp., Toxoptera spp., Trialeurodes spp., Trioza spp. spp.), Typhlocyba spp., Unaspis spp., Viteus vitifolii, and Zygina spp.

[0284] Further examples are the Hymenoptera order, such as Acromyrmex spp., Athalia spp., Atta spp., Diprion spp., Hoplocampa spp., Lasius spp., Monomorium pharaonis, Solenopsis invicta, Tapinoma spp., and Vespa spp.

[0285] Still other examples are the order Isopoda, which includes Armadillidium vulgare, Oniscus asellus, and Porcellio scaber.

[0286] Still other examples are the order Isoptera, such as Coptotermes spp., Cornitermes cumulans, Cryptotermes spp., Incisitermes spp., Microtermes obesi, Odontotermes spp., and Reticulitermes spp.

[0287] Further examples are the order 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 pinarius, 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. spp.), Copitarsia spp., Cydia spp., Dalaca noctuides, Diaphania spp., sugarcane borer (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, Gracilaria spp. 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. spp.), Lithocolletis spp., Lithophane antennata, Lobesia spp., Loxagrotis albicosta, Lymantria spp., Lyonetia spp.), Malacosoma neustria, Bean leaf moth (Maruca testulalis), Armyworm moth (Mamestra brassicae), Mocis spp., Armyworm (Mythimna separata), Nymphula spp., Oiketicus spp., Oria spp., Orthaga spp., Ostrinia spp., Rice leaf beetle (Oulema oryzae), Pine tree moth (Panolis flammea), Skipper butterflies (Parnara spp.), Pectinophora spp., Perileucoptera spp. spp.), Phthorimaea spp., Citrus leafminer (Phyllocnistis citrella), Phyllonorycter spp., Pieris spp., Platynota stultana, Indian meal moth (Plodia interpunctella), Plusia spp., Diamondback moth (Plutella xylostella), Prays spp., Prodenia spp., Protoparce spp., Pseudaletia spp., Soybean looper (Pseudoplusia includens), European corn borer (Pyrausta nubilalis, Sunflower Looper (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, and Virachola spp.

[0288] Further examples are the Orthoptera order, such as Acheta domesticus, Blatta orientalis, Blattella germanica, Dichroplus spp., Gryllotalpa spp., Leucophaea maderae, Locusta spp., Melanoplus spp., Periplaneta spp., Pulex irritans, Schistocerca gregaria, and Supella longipalpa.

[0289] Still other examples are the order Siphonaptera, which includes, for example, Ceratophyllus spp., Ctenocephalides spp., Tunga penetrans, and Xenopsylla cheopis.

[0290] Yet another example is the order Symphyla, which includes, for example, Scutigerella spp.

[0291] Further examples are the order Thysanoptera, such as Anaphothrips obscurus, Baliothrips biformis, Drepanothris reuteri, Enneothrips flavens, Frankliniella spp., Heliothrips spp., Hercinothrips femoralis, Rhipiphorothrips cruentatus, Scirtothrips spp., Taeniothrips cardamoni, Thrips spp. spp.) are mentioned.

[0292] Still other examples are the order Zygentoma (=Thysanura), which includes, for example, the silverfish (Lepisma saccharina) and the spotted silverfish (Thermobia domestica).

[0293] In another embodiment, pests of the phylum Mollusca, particularly the class Bivalvia, such as Dreissena spp., are also important plant pests.

[0294] In another embodiment, pests from the class Gastropoda are also important plant pests, such as Anion spp., Biomphalaria spp., Bulinus spp., Deroceras spp., Galba spp., Lymnaea spp., Oncomelania spp., Pomacea spp., and Succinea spp.

[0295] In yet another embodiment, plant pests of interest are plant parasitic nematodes, which means nematodes that parasitize plants and cause damage to the plants. Plant nematodes include plant parasitic nematodes and nematodes that live in the soil. Plant-parasitic nematodes include, but are not limited to, ectoparasites such as Xiphinema spp., Longidorus spp., and Trichodorus spp.; hemiparasites such as Tylenchulus spp.; migratory endoparasites such as Pratylenchus spp., Radopholus spp., and Scutellonerna spp.; sessile parasites such as Heterodera spp., Globodera spp., and Meloidogyne spp., and Ditylenchus spp. These include stem and leaf endoparasites such as Heterodera spp., Aphelenchoides spp., and Hirshmaniella spp. Furthermore, harmful root-parasitic soil nematodes are cyst-forming nematodes of the genera 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.Further important genera that are important as plant pests include Rotylenchulus spp., Paratriclodorus spp., Pratylenchus penetrans, Radolophus simuli, Ditylenchus dispaci, Tylenchulus semipenetrans, Xiphinema spp., Bursaphelenchus spp., etc., especially Aphelenchoides spp., Bursaphelenchus spp., Ditylenchus spp., Globodera spp. spp.), Heterodera spp., Longidorus spp., Meloidogyne spp., Pratylenchus spp., Radopholus similis, Trichodorus spp., Tylenchulus semipenetrans, and Xiphinema spp.

[0296] In yet another embodiment, the plant pest is a virus and the pesticide formulation of the present invention is intended to treat viral infections or suppress viral infectivity in plants, plant viruses being selected from the group consisting of alphamoviruses, alexiviruses, alphacryptoviruses, anuraviruses, apsukaviroids, aureusviruses, avenaviruses, absinthe viroids, badnaviruses, begomoviruses, beniviruses, betacryptoviruses, betaflexiviridae, bromoviruses, bymoviruses, capilloviruses, caraviruses, carmoviruses, caulimoviruses, cavemoviruses, cheraviruses, closteroviruses, cocadoviroids, choleviroids, comoviruses, criniviruses, cucumoviruses, kurtoviruses, sitrabdoviruses, giantoviruses, enamoviruses, umbraviruses and satellite B viruses, fabaviruses, fiziviruses, flowiviruses, The virus is selected from the group consisting of rhus, hordeivirus, hostuviroid, idaeovirus, illarvirus, ipomovirus, luteovirus, macromovirus, macluravirus, marafivirus, mastrevirus, nanovirus, necrovirus, nepovirus, nucleorhabdovirus, oleavirus, ophiovirus, oryzavirus, panicovirus, pekuruvirus, petuvirus, phytoreovirus, polerovirus, pomovirus, pospiviroid, potexvirus, potyvirus, reovirus, rhabdovirus, rimovirus, sadowavirus, SbCMV-like virus, secuivirus, sobemovirus, tenuivirus, TNsatV-like satellite virus, tobamovirus, topokuvirus, tospovirus, trichovirus, tritimovirus, tungrovirus, tymovirus, umbravirus, varicosavirus, vitivirus, and waikavirus.

[0297] Form of target antigen For pesticide and biocontrol applications, it will be understood based on the present disclosure that the polypeptides of the compositions disclosed herein may be specific for or capable of specifically binding to several different forms of pest targets (e.g., fungal targets). It is also expected that the polypeptides of the compositions disclosed herein will bind to numerous natural or synthetic analogs, variants, mutants, alleles, parts, and fragments of those pest targets. More specifically, it is expected that the polypeptides of the compositions disclosed herein will bind to analogs, variants, mutants, alleles, parts, and fragments of those targets that still contain at least the binding site, part, or domain of the natural target to which they bind.

[0298] formulation The amount of polypeptide contained in the pesticide or biocontrol compositions disclosed herein can vary over a wide range, and it is generally contemplated that it will be up to the manufacturer to adjust the concentration range of a particular polypeptide depending on the particular crop pest to be controlled.

[0299] In certain embodiments, the present invention provides an agrochemical composition comprising at least one polypeptide, wherein said heavy chain variable domain is present in an amount effective to protect or treat a plant or part of said plant against infection by or other biological interaction with said plant pathogen.

[0300] In certain embodiments, the concentration of the polypeptide in the pesticide composition may be at least 0.0001% by weight.

[0301] In certain embodiments, the concentration of the polypeptide in the pesticide composition may be up to 50% by weight.

[0302] In a specific embodiment, the concentration of the polypeptide contained in the pesticide composition can be 0.0001 to 50% by weight.

[0303] In a specific embodiment, the present invention provides an agrochemical composition comprising at least one kind of polypeptide, wherein the concentration of the at least one kind of polypeptide in the agrochemical composition is 0.001 to 50% by weight.

[0304] In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition can be 0.001 to 50% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition can be 0.01 to 50% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition can be 0.1 to 50% by weight.

[0305] In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 1 to 50% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 10 to 50% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 0.0001 to 40% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 0.001 to 40% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 0.01 to 40% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 0.1 to 40% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 1 to 40% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 0.0001 to 30% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 0.001 to 30% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 0.01 to 30% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 0.1 to 30% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 1 to 30% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 0.0001 to 10% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 0.001 to 10% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 0.01 to 10% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 0.1 to 10% by weight.In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 1 to 10% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 0.0001 to 1% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 0.001 to 1% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 0.01 to 1% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 0.1 to 1% by weight.

[0306] In certain embodiments, the pesticide compositions disclosed herein contain at least one polypeptide formulated in an aqueous solution.

[0307] In other specific embodiments, the pesticide compositions disclosed herein contain at least one polypeptide and further contain an agrochemically suitable base and / or one or more suitable adjuvants.

[0308] In addition to the anti-pest polypeptide, the compositions of the present invention can contain a solid or liquid base acceptable for treating plants and / or plant parts with pests, and / or a surfactant also acceptable for treating plants and / or plant parts with pests. In particular, conventional inert bases and conventional surfactants can be used. These compositions correspond not only to compositions that can be applied immediately to the plants and / or plant parts to be treated by immersion or by using a suitable device, but also to commercially available concentrated compositions that must be diluted before application to the plants and / or plant parts.

[0309] These pesticide compositions according to the present invention may further contain any type of other ingredients, such as protective colloids, adhesives, thickeners, thixotropic agents, penetrating agents, stabilizers, sequestering agents, shape retaining agents, flavoring agents, taste enhancers, sugars, sweeteners, coloring agents, etc. More generally, any solid or liquid additive corresponding to conventional formulation techniques may be added to the active substance, i.e., the at least one heavy chain variable domain.

[0310] These pesticidal compositions according to the invention may further contain any kind of other active ingredients, such as, for example, other antibacterial or antifungal active ingredients.

[0311] The term "base" in this disclosure refers to a natural or synthetic organic or inorganic substance added to an anti-pesticide active substance to facilitate its application to a plant and / or one or more plant parts. Therefore, the base must generally be inert and agriculturally acceptable. The base may be solid (clay, natural or synthetic silicates, silica, resin, wax, solid fertilizer, etc.) or liquid (water, alcohols, especially butanol, etc.).

[0312] The surfactant can be an ionic or nonionic emulsifier, dispersant, or wetting agent, or a mixture of such surfactants. Examples include polyacrylates, lignosulfonates, phenolsulfonates, or naphthalenesulfonates, polycondensates of ethylene oxide with fatty alcohols, fatty acids, or fatty amines, substituted phenols (especially alkylphenols or arylphenols), sulfosuccinate salts, taurine derivatives (especially alkyl taurates), phosphoric acid esters of polyoxyethylenated phenols or alcohols, esters of fatty acids and polyols, and derivatives of the above compounds containing sulfuric acid, sulfonic acid, and phosphoric acid functional groups. When the inert base is water-insoluble or when the application solvent is water, the presence of at least one surfactant is generally essential.

[0313] The pesticide compositions disclosed herein may themselves be in a wide variety of solid or liquid forms.

[0314] Solid composition forms include dusts and granules (which may contain up to 100% of the active substance), in particular those obtained by extrusion, compression, impregnation of granulated carriers, or granulation using powders as starting materials (in which case the active substance content in these granules is 0.5 to 80%). Such solid compositions can optionally be used in liquid form, the viscosity of which can be increased or decreased, depending on the desired type of application, for example by dilution with water.

[0315] Liquid composition forms or forms intended to constitute a liquid composition when applied include solutions, in particular water-soluble concentrates, emulsions, concentrated suspensions, wettable powders (or sprayable powders), oils and waxes.

[0316] Concentrated suspensions that can be applied by spraying are prepared so as to obtain a stable fluid that does not form precipitates, and typically contain 10 to 75% of an active substance, 0.5 to 15% of a surfactant, 0.1 to 10% of a thixotropic agent, 0 to 10% of suitable additives (e.g., antifoaming agents, corrosion inhibitors, stabilizers, penetrating agents, and adhesives), and, as a base, water or an organic liquid in which the active substance is insoluble or sparingly soluble. Some organic solids or inorganic salts may be dissolved in the base to help prevent precipitation or to prevent water from gelling.

[0317] The pesticide composition disclosed in the present application can be used as it is, or can be used in the form of a formulation thereof, or can be used in a use form prepared therefrom, and examples thereof include aerosol dispensers, capsule suspensions, room temperature aerosols, hot aerosols, encapsulated granules, fine granules, concentrated flowables for seed treatment, ready-to-use solutions, dusts, emulsifiable concentrates, oil-in-water emulsions, water-in-oil emulsions, coarse granules, coarse granules, oil-dispersible powders, oil-miscible concentrated flowables, oil-miscible liquids, foams, pastes, pesticide-coated seeds, concentrated suspensions (concentrated flowables), suspension-emulsion-concentrates, soluble concentrates, suspensions, soluble concentrates, and the like. Water-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 polymeric materials and jackets for seeds, microencapsulated biomaterial particles, such as those described in WO2018 / 201160, WO2018 / 201161 and WO2019 / 060903, as well as ULV room temperature and high temperature aerosols, (pressurized) gases, gas generants, plant rodlets, dry seed treatment powders, seed treatment solutions, ultra-low volume (ULV) liquids, ultra-low volume (ULV) suspensions, water-dispersible granules or tablets, water-dispersible powders for slurry treatment.

[0318] These formulations are prepared in known manner by mixing the active compound or combinations of active compounds with customary additives, such as, for example, customary extenders, and further with solvents or diluents, emulsifiers, dispersants, and / or binders or fixatives, wetting agents, water repellents, if necessary drying agents and UV stabilizers, colorants, pigments, antifoaming agents, preservatives, secondary thickeners, adhesives, gibberellins and water, and other processing aids.

[0319] These compositions include compositions which can be applied immediately to the plants or seeds to be treated by suitable equipment, such as spraying or dusting equipment, as well as commercially available concentrated compositions which must be diluted before application to the crop.

[0320] Plant protection or treatment methods In certain embodiments, the present invention provides a method for protecting or treating a plant or a part of a plant against infection by or other biological interaction with a plant pathogen, the method comprising the step of directly or indirectly applying to the plant or part of the plant at least one of an agrochemical composition or polypeptide disclosed herein, wherein the composition or polypeptide can be applied under conditions effective to protect or treat the plant or part of the plant against infection by or other biological interaction with the plant pathogen.

[0321] In certain embodiments, these methods comprise the step of applying, directly or indirectly, an agrochemical composition disclosed herein to the plant or part of the plant, for example, at a rate of more than 50 g of the agrochemical composition per hectare, for example, but not limited to, at a rate of more than 75 g of the agrochemical composition per hectare, for example, at a rate of more than 100 g of the agrochemical composition per hectare, or particularly at a rate of more than 200 g of the agrochemical composition per hectare.

[0322] In certain embodiments, these methods comprise the step of applying, directly or indirectly, the pesticide composition disclosed herein to the plant or part of the plant, for example, but not limited to, at an application rate of 50 g to 200 g of the pesticide composition per hectare, for example, but not limited to, at an application rate of 50 g to 200 g of the pesticide composition per hectare, particularly at an application rate of 75 g to 175 g of the pesticide composition per hectare, for example, 75 g to 150 g of the pesticide composition per hectare or 75 g to 125 g per hectare.

[0323] In yet another embodiment, the invention provides a method for controlling or suppressing plant pests, comprising applying the pesticide or biocontrol composition of the invention to a plant, such as a crop, or part of a plant or crop, e.g., at a rate of less than 50 g of the polypeptide per hectare. In certain embodiments, the rate is less than 45 g, 40 g, 35 g, 30 g, 25 g, 20 g, 15 g, 10 g, 5 g, 1 g, or less than 1 g of polypeptide per hectare.

[0324] Naturally, farmers can vary application rates depending on the crop and environmental pressures on plant pests. These rate variations are specified in the technical sheets that accompany particular pesticide compositions.

[0325] In yet another embodiment, the present invention provides the use of the pesticide or biocontrol composition of the present invention in controlling or suppressing plant pests.

[0326] In yet another embodiment, the present invention provides the use of a polypeptide of the present invention in the control or suppression of plant pests.

[0327] Application of the pesticide or biocontrol composition or polypeptide of the present invention to crops can be carried out using any method suitable for applying pesticide or biocontrol compositions to crops, including, but not limited to, spraying (high volume (HV), low volume (LV) and ultra-low volume (ULV) sprays), brushing, bandaging, dripping, coating, dipping, soaking, dusting, fogging, applying as droplets, misting or aerosols.

[0328] Thus, in certain embodiments, the methods disclosed herein for protecting or treating a plant or plant part against infection by or other biological interactions with a plant pathogen comprise applying the pesticide composition directly or indirectly to the plant or plant part, for example, by spraying, misting, foaming, fogging, hydroponics, coating, dipping, and / or dressing.

[0329] In certain specific embodiments, the present invention provides a method for inhibiting, hindering, suppressing or controlling the growth of a plant pathogen, the method comprising the step of applying, directly or indirectly, to a plant or part of said plant at least one of the pesticide compositions disclosed herein.

[0330] In certain other embodiments, the present invention provides a method for killing plant pathogens, comprising the step of applying, directly or indirectly, to a plant or part of said plant, at least one of the pesticide compositions or polypeptides disclosed herein.

[0331] Alternatively, the application rate of the pesticide composition of the present invention, i.e., the amount of the pesticide composition applied to the crop, is such that less than 50 g, less than 45 g, less than 40 g, less than 35 g, less than 30 g, less than 25 g, less than 20 g, less than 20 g, less than 15 g, less than 10 g, less than 5 g, less than 1 g, or less than 1 g of polypeptide contained in the pesticide composition or biological control composition of the present invention is applied to the crop per hectare.

[0332] According to the methods disclosed herein, the pesticide or biocontrol composition can be applied to a crop once, or two or more times at intervals. According to the methods of the present invention, the pesticide or biocontrol composition of the present invention can be applied to the crop alone, or as a mixture with other materials, preferably other pesticide or biocontrol compositions, or the pesticide or biocontrol composition of the present invention can be applied separately from other materials, preferably other pesticide or biocontrol compositions, at different times to the same crop. According to the methods of the present invention, the pesticide or biocontrol composition of the present invention can be applied to the crop preventively, or can be applied after the target pest has been identified in the particular crop to be treated.

[0333] The pesticide compositions disclosed herein can be applied directly to a plant, crop, or one or more parts of the plant by the methods described above, for example, directly to the entire plant or to one or more parts of the plant at a pre- or post-harvest stage. Pre-harvest application can be effective after harvest. In certain other embodiments, the pesticide compositions disclosed herein can be applied directly to one or more parts of the plant by the methods described above, for example, directly to petioles, leaves, tubers, stems, shoots, seeds, fruits, roots, flowers, grains, sprouts, etc.

[0334] The treatment method disclosed herein can also be used in the field of protecting stored materials against attack by plant pathogens. In this treatment method, the application of the composition of the present invention can be carried out either before or after harvest. According to the present invention, the term "stored materials" is considered to mean natural materials of plant or animal origin, as well as processed materials thereof that have been removed from their natural life cycle and for which long-term protection is desired. Stored materials of plant origin, such as plants or their parts (e.g., petioles, leaves, tubers, seeds, fruits, or grains), can be protected in their freshly harvested state or in processed forms such as pre-drying, wetting, crushing, grinding, pressing, or roasting. Wood is also included in the definition of stored materials, and can be raw wood, such as building timber, electricity transmission towers, and fences, or finished products, such as furniture or wooden products. Stored materials of animal origin include hides, leather, fur, fur, etc. The compositions of the present invention can prevent adverse effects such as decay, discoloration, or mold. "Storage products" is preferably understood to mean natural substances of plant origin and processed products thereof, more preferably fruit trees such as pome fruits, stone fruits, soft fruits and citrus fruits and processed products thereof.

[0335] The pesticide compositions disclosed herein can be indirectly applied to a plant, crop, or one or more parts of the plant by the above-described methods at a pre- or post-harvest stage, for example, indirectly applied to the entire plant or to one or more parts of the plant. The pesticide compositions disclosed herein can be applied shortly before harvest, for example, about three weeks before harvest, for example, two weeks before harvest, one week before harvest, or up to one week before harvest. Pre-harvest applications can be effective after harvest. Thus, in certain embodiments, the pesticide compositions disclosed herein can be indirectly applied to a plant, crop, or one or more parts of the plant by the above-described methods, for example, by applying the pesticide composition to the environment or medium (e.g., air, soil, hydroponics, hydroculture) in which the plant or one or more parts of the plant are growing or stored, or by applying the pesticide composition to a liquid medium (e.g., an aqueous liquid medium or water) in which the plant or one or more parts of the plant are growing or stored.

[0336] The pesticidal compositions disclosed herein can be applied directly as part of an integrated pest management approach.

[0337] It is therefore understood in the context of the present application that the treatment of plants or plant parts with the agrochemical compositions disclosed herein can be carried out directly or by treating their environment, habitat or storage area by conventional application methods, for example by watering (drenching), drip irrigation, spraying, vaporizing, atomizing, scattering, dusting, foaming, spreading and as a powder. Furthermore, the compositions can also be applied by the high-concentration method, or active compound formulations or the active compounds themselves can be injected into the soil.

[0338] In certain embodiments, the methods disclosed herein for protecting or treating a plant or plant part against infection by or other biological interaction with a plant pathogen comprise applying, directly or indirectly, the pesticide composition to the plant or plant part at a pre- or post-harvest stage.

[0339] According to a particular embodiment, the harvested produce is a fruit tree, flower, nut or vegetable, preferably a fruit tree or vegetable with an inedible skin, preferably selected from avocado, banana, plantain, lemon, grapefruit, melon, orange, pineapple, kiwifruit, guava, mandarin orange, mango, pumpkin, strawberry, grape and pumpkin, more preferably banana, orange, lemon and peach, especially banana. According to another particular embodiment, the harvested product is cut flowers of ornamental plants, preferably selected from the group consisting of Alstroemeria, carnation, Chrysanthemum, Freesia, Gerbera, Gladiolus, Gypsophila species, Helianthus, Hydrangea, Lilium, Lisianthus, roses and summer flowers.

[0340] Plant species to which the pesticide compositions disclosed herein can be applied include, but are not limited to, corn, soybean, alfalfa, cotton, sunflower, rapeseed oilseeds such as Brassica napus (e.g., canola), Brassica rapa, B. juncea (e.g., (field) mustard), and Brassica carinata, Arecaceae species (e.g., oil palm, coconut), rice, wheat, sugar beet, sugarcane, oats, rye, barley, millet, and sorghum, triticale, flax, nuts, grapes, and vines, as well as various fruit trees and vegetables belonging to various botanical classes, such as Rosaceae species. species) (e.g., pome fruits such as apples and pears, as well as stone fruits such as apricots, cherries, almonds, plums and peaches, and berries such as strawberries, raspberries, red currants, black currants and gooseberries), Ribesioidae species, Juglandaceae species, Birch species (Betulaceae species), Anacardiaceae species, Fagaceae species, Moraceae species, Oleaceae species (e.g., olive trees), Actinidaceae species, Lauraceae species (e.g., avocados, cinnamon, camphor), Musaceae species (e.g., banana trees and plantains), Rubiaceae species sp.) (e.g. coffee), Theaceae sp. (e.g. tea), Sterculiceae sp., Rutaceae sp. (e.g. lemon, orange, mandarin orange and grapefruit), Solanaceae sp. (e.g. tomato, potato, pepper, paprika, eggplant, tobacco), Liliaceae sp., Compositae sp.) (e.g., lettuce, artichoke, and chicory root, including endive or chicory), Umbelliferae species (Umbelliferae species) (e.g., carrot, parsley, celery, and celeriac), Cucurbitaceae species (Cucurbitaceae species) (e.g., cucumber, including gherkin, pumpkin, watermelon, calabash, and melon), Alliaceae species (e.g., leek and onion), Cruciferae species (Cruciferae species) (e.g., white cabbage, red cabbage, broccoli, cauliflower, Brussels sprouts, bok choy, kohlrabi, radish, horseradish, watercress, and Chinese cabbage), Leguminosae species (Leguminosae species) (e.g., peanuts, peas, lentils, and legumes, such as kidney beans and fava beans), Chenopodiaceae species Useful and ornamental plants in gardens and forests, including Linaceae species (e.g., hemp), Cannabeaceae species (e.g., cannabis), Malvaceae species (e.g., okra, cocoa), Papaveraceae (e.g., poppy), Asparagaceae (e.g., asparagus), turfgrass, lawn grass, pasture grass and Stevia rebaudiana, and in each case genetically modified varieties of these plants, are also included.

[0341] In a preferred embodiment of the treatment method disclosed herein, the crop is selected from the group consisting of field crops, herbs, fruit trees and vegetables, lawn grasses, trees and ornamentals.

[0342] Thus, in certain aspects, the present invention also provides a post-harvest treatment method for protecting or treating a harvested plant or a harvested portion of the plant against infection by or other biological interactions with a plant pathogen, the method comprising applying, directly or indirectly, to the harvested plant or a harvested portion of the plant, an agrochemical composition as disclosed herein under conditions effective to protect or treat at least the harvested plant or the harvested portion of the plant against infection by or other biological interactions with the plant pathogen. According to certain embodiments, the harvested product is a fruit tree, flower, nut, or vegetable, preferably a fruit tree or vegetable with an inedible skin, selected from avocado, banana, plantain, lemon, grapefruit, melon, orange, pineapple, kiwifruit, guava, mandarin orange, mango, and pumpkin, more preferably banana, orange, lemon, and peach, especially banana. According to another particular embodiment, the harvested product is cut flowers of ornamental plants, preferably selected from Alstroemeria, carnation, Chrysanthemum, Freesia, Gerbera, Gladiolus, Gypsophila species, Helianthus, Hydrangea, Lilium, Lisianthus, roses and summer flowers. According to another particular embodiment, the harvested product is grass cuttings or tree cuttings.

[0343] Post-harvest disorders include, for example, lenticel spot, leaf scorch, senescence damage, bitter pit, scald, honeydew, browning, vascular damage, CO2 damage, CO2 or O2 deficiency, and softening.

[0344] Fungal diseases may be caused, for example, by the following fungi: Mycosphaerella spp., such as Mycosphaerella musae, Mycosphaerella fraga ae, Mycosphaerella citri; Mucor spp., such as Mucor piriformis; Monilinia spp., such as Monilinia fructigena, Monilinia laxa; Phomopsis spp., such as Phomopsis natalensis; Colletotrichum spp. spp., for example, Colletotrichum musae, Colletotrichum gloeosporioides, Colletotrichum coccodes; Verticillium spp., for example, Verticillium theobromae; Nigrospora spp.; Botrytis spp., for example, Botrytis cinerea; Diplodia spp., for example, Diplodia citri; Pezicula spp.; Alternaria spp., for example, Alternaria citri citri, Alternaria alternata; Septoria spp., e.g., Septoria depressa; Venturia spp.), for example, Venturia inaequalis, Venturia pyrina; Rhizopus spp., for example, Rhizopus stolonifer, Rhizopus oryzae; Glomerella spp., for example, Glomerella cingulata; Sclerotinia spp., for example, Sclerotinia fruiticola; Ceratocystis spp., for example, Ceratocystis paradoxa; Fusarium spp. spp., for example, Fusarium semitectum, Fusarium moniliforme, Fusarium solani, Fusarium oxysporum; Cladosporium spp., for example, Cladosporium fulvum, Cladosporium cladosporioides, Cladosporium cucumerinum, Cladosporium musae; Penicillium spp., for example, Penicillium funiculosum funiculosum, Penicillium expansum, Penicillium digitatum, Penicillium italicum; Phytophthora spp., for example, Phytophthora citrophthora, Phytophthora fragariae, Phytophthora cactorum, Phytophthora parasitica; Phacydiopycnis spp., for example, Phacydiopycnis malirum; Gloeosporium spp., for example, Gloeosporium album, Gloeosporium perennans, Gloeosporium fructigenum, Gloeosporium cingulata singulata; Geotrichum spp., for example, Geotrichum candidum; Phlyctaena spp., for example, Phlyctaena vagabunda; Cylindrocarpon spp., for example, Cylindrocarpon mail; Stemphyllium spp., for example, Stemphyllium vesica um; Thielaviopsis spp., for example, Thielaviopsis paradoxy; Aspergillus spp., for example, Aspergillus niger niger, Aspergillus carbonarius; Nectria spp., e.g., Nectria galligena; Cercospora spp.The pathogens include, for example, Cercospora angreci, Cercospora apii, Cercospora atrofiliformis, Cercospora musae, and Cercospora zeaemaydis.

[0345] In another aspect, the present invention provides the use of the pesticide compositions disclosed herein as a pest control agent, such as a biostatic agent or pesticide (including but not limited to a fungistatic agent or fungicide).

[0346] In a particular embodiment, the plant pest controlled by the method of the present invention is a plant pathogenic fungus as defined above. As a result of applying the method of the present invention, the number of lesions, the size of the lesions, and the degree of sporulation of the fungal pathogen can all be reduced.

[0347] Medical Use In certain other embodiments, the present invention provides a method for protecting or treating a human or animal against infection by a pest, particularly a fungus, or for treating an infection of a human or animal by a pest, particularly a fungus, comprising the step of applying or administering, directly or indirectly, to said human or animal or to a part of said human or animal, a composition comprising at least one polypeptide of the present invention that specifically binds to at least a pest (such as, but not limited to, a fungus), said composition being applicable under conditions effective to protect or treat said human or animal against said pest.

[0348] Accordingly, the present invention provides polypeptides of the present invention that specifically bind to a pest target for use in a method for preventing and / or treating at least one disease and / or disorder caused by a pest (e.g., a disease and / or disorder caused by a fungus) in a subject. The present invention further provides compositions of the present invention for use in a method for preventing and / or treating at least one disease and / or disorder caused by a pest (e.g., a disease and / or disorder caused by a fungus) in a subject. The present invention further provides polypeptides of the present invention that specifically bind to a pest target for use in a method for preventing and / or treating an infection caused by a pest (e.g., a fungal infection) in a subject. The present invention further provides compositions of the present invention that specifically bind to a pest target for use in a method for preventing and / or treating an infection caused by a pest (e.g., a fungal infection) in a subject. In certain embodiments, the present invention further 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 thereof a pharmaceutically active amount of one or more amino acid sequences, polypeptides and / or pharmaceutical compositions disclosed herein. In particular, the pharmaceutically active amount can be an amount sufficient (to produce circulating concentrations of the amino acid sequence or polypeptide) to inhibit, interfere with or suppress one or more biological activities or pathways of the bound pest.

[0349] Thus, in certain embodiments, the present invention provides compositions comprising at least one polypeptide that specifically binds to a pest (e.g., a fungus) for use as a pest control agent in a subject, such as an animal or human, suffering from a disease and / or disorder caused by the pest.

[0350] In certain embodiments, the pesticide is a biostatic agent or a pesticide. In certain embodiments, the pesticide is a fungicide or a fungicide.

[0351] In addition, in certain embodiments, the present invention provides a method for preventing and / or treating diseases and / or disorders caused by pests, comprising: (a) providing an amino acid sequence, polypeptide, or composition disclosed herein; (b) administering said amino acid sequence, polypeptide or pharmaceutical composition to a patient suffering from said disease and / or disorder caused by a pest.

[0352] The efficacy of the polypeptides and compositions containing said polypeptides disclosed herein can be tested using any suitable in vitro assay, cellular assay, in vivo assay, and / or animal model, or any combination thereof, known per se, depending on the particular disease or disorder of interest. Suitable assays and animal models will be apparent to those skilled in the art, and include those used in the experimental section below and the prior art cited herein. Those skilled in the art will generally be able to select an appropriate in vitro assay, cellular assay, or animal model to test the amino acid sequences and polypeptides disclosed herein for their ability to bind to or affect the activity of pest targets or pest antigens, and / or the biological mechanisms involved, as well as their therapeutic and / or preventive effects against one or more diseases and disorders associated with pest antigens.

[0353] Pharmaceutical Composition In yet another aspect, the present invention provides pharmaceutical compositions (also referred to herein as pharmaceutical compositions of the present invention) containing one or more amino acid sequences, polypeptides, and / or nucleic acid sequences disclosed herein, and optionally at least one pharmaceutically acceptable carrier. According to certain specific embodiments, the pharmaceutical compositions disclosed herein can further optionally contain at least one other pharmaceutically active compound.

[0354] The pharmaceutical compositions of the present invention bind a pest target to a polypeptide disclosed herein and can be used to diagnose, prevent and / or treat diseases and disorders associated with pests such as fungi.

[0355] In particular, the present invention provides pharmaceutical compositions containing the polypeptides which are suitable for prophylactic, therapeutic and / or diagnostic use in warm-blooded animals, particularly mammals, and more particularly humans.

[0356] The present invention further provides pharmaceutical compositions containing the amino acid sequences and polypeptides disclosed herein that bind the polypeptides and pest targets and can be used for veterinary purposes, for example in the prevention and / or treatment or diagnosis of one or more diseases, disorders or conditions associated with pests, such as fungi.

[0357] Generally, for pharmaceutical use, the polypeptides disclosed herein can be formulated as pharmaceutical formulations or compositions containing at least one polypeptide disclosed herein, at least one pharmaceutically acceptable carrier, diluent, or excipient and / or adjuvant, and optionally one or more other pharmaceutically active polypeptides and / or compounds. Such formulations may be suitable for oral, parenteral, topical, or inhalation administration. Thus, the amino acid sequences or polypeptides disclosed herein and / or compositions containing them can be administered, for example, orally, intraperitoneally, intravenously, e.g., subcutaneously, intramuscularly, transdermally, topically, by suppository, or by inhalation, again depending on the particular pharmaceutical formulation or composition used. The clinician will be able to select an appropriate route of administration and a suitable pharmaceutical formulation or composition for use in such administration.

[0358] The pharmaceutical compositions may further comprise suitable binders, disintegrating agents, sweetening agents, or flavoring agents. Tablets, pills, or capsules may be coated, for example, with gelatin, wax, or sugar. Furthermore, the amino acid sequences and polypeptides disclosed herein may be incorporated into sustained-release preparations and devices.

[0359] Pharmaceutical dosage forms suitable for injection or infusion include sterile aqueous solutions or dispersions, or sterile powders containing the active ingredient, optionally encapsulated in liposomes, suitable for the extemporaneous preparation of sterile injectable or infusion solutions or dispersions. In all cases, the final dosage form must be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid base or carrier can be, for example, a solvent or liquid dispersion medium containing water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. Optionally, antibacterial and antifungal agents can be added.

[0360] Useful dosages of the amino acid sequences and polypeptides disclosed herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the skilled artisan.

[0361] The amount of the amino acid sequences and polypeptides disclosed herein required for prophylactic and / or therapeutic use will vary depending not only on the particular amino acid sequence or polypeptide selected, but also on the route of administration, the type of condition being treated, and the age and health of the patient, and will ultimately be at the discretion of the attending physician or clinician. The dosage of the amino acid sequences and polypeptides disclosed herein may also vary depending on the target cell, tumor, tissue, transplant tissue, or organ.

[0362] The amino acid sequences or polypeptides disclosed herein and / or compositions containing them are administered according to a therapeutic regimen appropriate for the prevention and / or treatment of the disease or disorder being prognosed, diagnosed, prevented, or treated. A clinician will generally be able to determine an appropriate therapeutic regimen. In general, a therapeutic regimen will involve the administration of one or more pharmaceutically effective amounts or doses of one or more amino acid sequences or polypeptides disclosed herein or one or more compositions containing them.

[0363] The desired dose may be suitably provided as a single dose or as multiple doses administered at appropriate intervals (each of which may be further divided into multiple doses). Dosage regimens may include long-term (i.e., for at least two weeks, e.g., for several months or years) or daily administration.

[0364] The amino acid sequences or polypeptides disclosed herein will be administered in amounts determined by a medical professional based, inter alia, on the severity of the condition and the patient being treated. Generally, an optimal dosage will be determined for each indication, specifying the amount per kilogram of body weight per day to be administered continuously (e.g., by infusion) or once daily or in multiple doses throughout the day. Clinicians will generally be able to determine an appropriate daily dose depending on the factors mentioned herein. It is understood that in certain cases, clinicians may choose to deviate from these amounts, for example, based on the factors mentioned above and their own professional judgment.

[0365] In particular, the amino acid sequences or polypeptides disclosed herein can be used in combination with other pharmaceutically active compounds or principles that are or can be used in the prevention and / or treatment of the diseases and disorders mentioned herein, which may or may not result in a synergistic effect. Examples of such compounds and principles, as well as their routes of administration and pharmaceutical formulations or compositions, will be readily apparent to the clinician.

[0366] The compositions of the present invention can be used in combination with known antifungal agents, including, but not limited to, azoles (e.g., fluconazole, itraconazole), polyenes (e.g., amphotericin B), flucytosine, and squalene epoxidase inhibitors (e.g., terbinafine) [see also ref. 57]. The compositions may be used in combination with known antiviral agents, such as HIV protease inhibitors, 2',3'-dideoxynucleosides (e.g., DDC, DDI), 3'-azido-2',3'-dideoxynucleosides (AZT), 3'-fluoro-2',3'-dideoxynucleosides (FLT), 2',3'-didehydro-2',3'-dideoxynucleosides (e.g., D4C, D4T) and their carbocyclic derivatives (e.g., carbovir), 2'-fluoro-ara-2',3'-dideoxynucleosides, 1,3-dioxolane derivatives (e.g., 2',3'-dideoxyl-3'-thiacytidine), ), oxetanocin analogs and carbocyclic derivatives thereof (e.g., cyclobut-G) and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and 9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) derivatives, tetrahydroimidazo[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, and the like.

[0367] The amino acid sequences, polypeptides and pharmaceutical compositions can be used to treat Candida species such as Candida albicans (C. albicans); Cryptococcus species such as C. neoformans (C. neoformans); Enterococcus species such as E. faecalis (E. faecalis); Streptococcus pneumoniae (S. pneumoniae), Streptococcus mutans (S. mutans), Streptococcus agarose (S. agarose), and the like in animals or humans. Streptococcus species such as S. agalactiae and S. pyogenes; Leishmania species such as L. major and L. infantum; Acanthamoeba species such as A. castellani; Aspergillus fumigatus and Aspergillus flavus; Aspergillus species such as Pneumocystis carinii (P. carinii); Mycobacterium species such as Mycobacterium tuberculosis (M. tuberculosis); Pseudomonas species such as Pseudomonas aeruginosa (P. aeruginosa); Staphylococcus species such as Staphylococcus aureus (S. aureus); taphylococcus species; Salmonella species such as Salmonella typhimurium; Coccidioides species such as Coccidioides iminitis; Trichophyton species such as Trichophyton verrucosum; Blastomyces species such as Blastomyces dermatidis; Histoplasma capsulatum (H.It is particularly useful for treating infections caused by Histoplasma species such as P. capsulatum; Paracoccidioides species such as P. brasiliensis; Pythium species such as P. insidiosum; and Escherichia species such as E. coli. The amino acid sequences, polypeptides, and pharmaceutical compositions are particularly useful in treating diseases such as, but not limited to, candidiasis, aspergillosis, cryptococcosis, dermatomycosis, sporotrichosis and other deep mycoses, blastomycosis, histoplasmosis, coccidioidomycosis, paracoccidioidomycosis, Pneumocystis pneumonia, thrush, tuberculosis, mycobacteriosis, respiratory infections, scarlet fever, pneumonia, impetigo, rheumatic fever, sepsis, sepsis with bacteremia, cutaneous and visceral leishmaniasis, Acanthamoeba corneal infection, keratitis, cystic fibrosis, typhoid fever, gastroenteritis, and hemolytic uremic syndrome. The anti-Candida albicans (C. albicans) activity is particularly useful in treating infections in AIDS patients.

[0368] Method for producing the polypeptide The present invention further provides methods for making or generating said polypeptide sequences, and methods for producing nucleic acids encoding these polypeptide sequences, as well as host cells, products and compositions comprising these polypeptide sequences, preferred, non-limiting examples of which will be apparent from the detailed description herein.

[0369] As will be apparent to those skilled in the art, one particularly useful method for producing the polypeptide sequences disclosed herein generally comprises: (a) expressing a nucleotide sequence encoding a polypeptide sequence disclosed herein or a vector or genetic construct comprising a nucleotide sequence encoding said polypeptide; (b) optionally isolating and / or purifying said polypeptide sequence.

[0370] In certain embodiments contemplated herein, the pest-specific polypeptide sequence can be obtained by a method comprising the steps of generating a random library of amino acid sequences and screening this library for amino acid sequences capable of specifically binding to a pest target.

[0371] Thus, in certain embodiments, the methods for producing the polypeptide sequences disclosed herein comprise: a) providing a set, collection or library of amino acid sequences; b) screening said set, collection or library for amino acid sequences that bind to and / or have affinity for said pest target; c) isolating amino acid sequences that bind to and / or have affinity for said pest target.

[0372] In such methods, the set, collection, or library of polypeptide sequences can be any suitable set, collection, or library of amino acid sequences. For example, the set, collection, or library of amino acid sequences can be a set, collection, or library of immunoglobulin fragment sequences (as described herein), including a naive set, collection, or library of immunoglobulin fragment sequences; a synthetic or semi-synthetic set, collection, or library of immunoglobulin fragment sequences; and / or a set, collection, or library of immunoglobulin fragment sequences that have been subjected to affinity maturation.

[0373] In certain embodiments of this method, the set, collection, or library of amino acid sequences can be, for example, an immunized set, collection, or library of immunoglobulin fragment sequences from a mammal suitably immunized with a pest target or a suitable antigenic determinant based on or derived from a pest target (e.g., an antigenic portion, fragment, region, domain, loop, or other epitope thereof). In a particular aspect, the antigenic determinant can be an extracellular portion, region, domain, loop, or other extracellular epitope.

[0374] In the above methods, the set, collection, or library of polypeptide sequences can be displayed on the surface of a phage, phagemid, ribosome, or suitable microorganism (e.g., yeast), for example, to facilitate screening. Suitable methods, techniques, and host organisms for displaying and screening (sets, collections, or libraries) of amino acid sequences will be apparent to those skilled in the art, for example, based on the detailed disclosure herein. See also the discussion by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

[0375] In another embodiment, the method for producing the polypeptide sequences disclosed herein comprises: a) providing a cell collection or sample expressing a polypeptide sequence; b) screening the cell collection or sample for cells expressing amino acid sequences capable of binding to and / or having affinity for a pest target; c) (i) isolating the amino acid sequence, or (ii) isolating a nucleic acid sequence encoding the amino acid sequence from the cell, followed by expressing the amino acid sequence.

[0376] The cell collection or sample can be, for example, a collection or sample of B cells. Also, 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 a fungal target (e.g., an antigenic portion, fragment, region, domain, loop, or other epitope thereof). In a particular embodiment, the antigenic determinant can be an extracellular portion, region, domain, loop, or other extracellular epitope.

[0377] In another embodiment, the method for generating a polypeptide sequence specific for a pest target comprises: a) providing a set, collection or library of nucleic acid sequences encoding polypeptide or amino acid sequences; b) screening said set, collection or library of nucleic acid sequences for nucleic acid sequences encoding amino acid sequences capable of binding to and / or having affinity for said pest target; c) isolating the nucleic acid sequence and then expressing the amino acid sequence.

[0378] In the above method, the pest target can be a lipid-containing fraction of the cell membrane of a fungus (e.g., Botrytis cinerea or other fungus). The lipid-containing fraction can be obtainable by chromatography. For example, the lipid-containing fraction can be obtainable by a method comprising fractionating fungal (e.g., Botrytis cinerea or other fungus) mycelia by total lipid extract thin-layer chromatography and selecting fractions having a retention factor (Rf) greater than that of a ceramide fraction and less than that of a non-polar phospholipid fraction.

[0379] In the above methods, the set, collection or library of nucleic acid sequences encoding amino acid sequences can be, for example, a set, collection or library of nucleic acid sequences encoding a naive set, collection or library of immunoglobulin fragment sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of immunoglobulin fragment sequences; and / or a set, collection or library of nucleic acid sequences encoding a set, collection or library of immunoglobulin fragment sequences that have been subjected to affinity maturation.

[0380] In particular, in such methods, the set, collection or library of nucleic acid sequences comprises: H Domain or V HH For example, the set, collection, or library of nucleic acid sequences can encode a set, collection, or library of domain antibodies or single domain antibodies, or a set, collection, or library of amino acid sequences capable of functioning as domain antibodies or single domain antibodies. In certain embodiments, the set, collection, or library of nucleotide sequences encodes a set, collection, or library of V HH It encodes a set, collection or library of sequences.

[0381] In the above-mentioned methods, the set, collection, or library of nucleotide sequences can be displayed on the surface of a phage, phagemid, ribosome, or suitable microorganism (e.g., yeast), for example, to facilitate screening. Suitable methods, techniques, and host organisms for displaying and screening (sets, collections, or libraries) of nucleotide sequences encoding amino acid sequences will be apparent to those skilled in the art, for example, based on the detailed disclosure herein. See also the discussion by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

[0382] The present invention further relates to a polypeptide sequence obtainable or obtained by the above-described method or by a method comprising one of the above-described methods, further comprising the steps of determining the nucleotide or amino acid sequence of at least said immunoglobulin sequence; and expressing or synthesizing said amino acid sequence in a manner known per se, such as, for example, by expression in a host cell or host organism or by chemical synthesis methods.

[0383] Isolation of Polypeptide Sequences Optionally, the method for producing an amino acid sequence that specifically binds to a fungal target as envisaged herein may further comprise the step of isolating from the library of amino acid sequences at least one polypeptide that has a detectable binding affinity or a detectable in vitro effect on the pest target.

[0384] These methods can further include amplifying the sequence encoding at least one polypeptide that has a detectable binding affinity for the pest target or a detectable in vitro effect on its activity. For example, phage clones displaying specific amino acid sequences obtained from the selection step of the methods described herein can be amplified by re-infection of host bacteria and incubation in growth medium.

[0385] In certain embodiments, these methods can include determining the sequence of one or more amino acid sequences capable of binding to a pest target.

[0386] When the polypeptide sequences contained in a set, collection or library of amino acid sequences are displayed on the surface of a suitable cell or phage or particle, the nucleotide sequences encoding said amino acid sequences can be isolated from said cell or phage or particle, and the nucleotide sequences of selected amino acid sequence library members can then be determined by routine sequencing methods.

[0387] In another particular embodiment, the method for producing the polypeptides contemplated herein comprises the step of expressing said nucleotide sequence in a host organism under appropriate conditions to obtain the actual desired amino acid sequence, which can be carried out by methods known to those skilled in the art.

[0388] Furthermore, the resulting polypeptide sequences that have a detectable binding affinity for the pest target or a detectable in vitro effect on its activity can optionally be synthesized as soluble protein constructs after the sequences have been identified.

[0389] For example, the polypeptide sequence obtained, obtainable, or selected by the above method can be synthesized using recombinant or chemical synthesis methods known in the art. The amino acid sequence obtained, obtainable, or selected by the above method can also be produced by recombinant genetic engineering. Thus, a method for synthesizing the polypeptide sequence obtained, obtainable, or selected by the above method 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 for a pest target or a detectable in vitro effect on its activity. Thus, an amino acid sequence that has a detectable binding affinity for a pest target or a detectable in vitro effect on its activity can be produced by recombinant DNA techniques. DNA encoding the amino acid sequence can be readily synthesized using conventional procedures. Once the DNA is produced, it can be introduced into an expression vector, which can then be transformed or transfected into a host cell, such as E. 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 reside.

[0390] Of course, as known to those skilled in the art of protein expression and purification, polypeptides produced from expression vectors using appropriate expression systems may be tagged (generally at the N- or C-terminus of the amino acid sequence) with, for example, a His tag or other sequence tag to facilitate purification.

[0391] Transformation or transfection of a host cell with a nucleic acid or vector can be carried out by various means known to those skilled in the art, including calcium phosphate DNA co-precipitation, DEAE-dextran transfection, polybrene transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and particle gun technology.

[0392] Suitable host cells for expression of the desired polypeptide sequence can be any eukaryotic or prokaryotic cell (e.g., bacterial cells such as E. 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 cell can be located in a transgenic plant or animal.

[0393] Thus, the present application further provides a method for producing a polypeptide sequence that has a detectable binding affinity for a pest target or that has a detectable in vitro effect on its activity, 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 appropriate conditions. The present application further provides a method for producing a polypeptide sequence that has a detectable binding affinity for a pest target or that has a detectable in vitro effect on its activity, comprising providing a host cell into which a nucleic acid sequence or vector encoding the polypeptide has been introduced, and expressing the polypeptide under appropriate conditions. The methods of the present invention may further comprise the step of isolating the polypeptide, for example, from the cell culture medium or fermentation broth or from within the host cell (e.g., after lysing the host cell).

[0394] In yet another embodiment, the present invention further provides methods for making (synonymously "producing") the pesticide or biocontrol compositions disclosed herein.

[0395] In certain embodiments, the present invention provides a method for producing the pesticide composition disclosed herein, comprising: - obtaining at least one polypeptide that specifically binds to a pest; - formulating said polypeptide or a functional fragment thereof into an agrochemical composition.

[0396] In certain embodiments of these methods, obtaining at least one polypeptide that specifically binds to a pest comprises: (a) expressing a nucleotide sequence encoding a polypeptide that specifically binds to a pest; and optionally (b) isolating and / or purifying the polypeptide.

[0397] In other particular embodiments of these methods, the step of obtaining at least one polypeptide that specifically binds to a pest comprises: a) providing a set, collection or library of polypeptide sequences; b) screening said set, collection or library of polypeptide sequences for sequences that specifically bind to and / or have affinity for pests; and optionally c) isolating said polypeptide sequences that specifically bind to and / or have affinity for pests.

[0398] The present application further provides a method for producing (synonymously, "producing") the pesticidal or biocontrol compositions disclosed herein, comprising formulating an amino acid sequence or polypeptide of 80 to 200 amino acids, or any other suitable subrange as defined above, having pesticidal activity, with at least one conventional pesticide adjuvant.

[0399] Suitable manufacturing methods are known in the art and include, but are not limited to, high or low shear mixing, wet or dry milling, drip casting, encapsulation, emulsification, coating, powder coating, pilling, extrusion granulation, fluidized bed granulation, coextrusion, spray drying, spray cooling, atomization, addition polymerization or polycondensation processes, interfacial polymerization, in situ polymerization, coacervation, spray encapsulation, melt dispersion cooling, solvent evaporation, phase separation, solvent extraction, sol-gel polymerization, fluidized bed coating, pan coating, melting, passive or active absorption or adsorption.

[0400] Specifically, the amino acid sequences or polypeptides of 80 to 200 amino acids or any other suitable subrange as defined above disclosed herein can be produced by chemical synthesis methods.

[0401] It is further disclosed that amino acid sequences or polypeptides of 80-200 amino acids, or any other suitable subrange as defined above, can be produced in vitro in recombinant microbial expression systems and isolated for subsequent use. Such amino acid sequences or polypeptides may be in the form of crude cell lysates, suspensions, colloids, etc., or may be purified, purified, buffered, and / or further processed prior to formulation with conventional agricultural adjuvants.

[0402] Specifically, recombinant methods generally involve inserting a DNA molecule expressing a desired amino acid sequence, protein, or polypeptide into an expression system heterologous to the DNA molecule (i.e., such a DNA molecule is not normally present in the host). The heterologous DNA molecule is inserted into the expression system or vector in the correct orientation and reading frame. The vector contains the components necessary for the transcription and translation of the inserted protein-coding sequence. Transcription of DNA is dependent on the presence of a promoter. Similarly, translation of mRNA in prokaryotic cells is dependent on the presence of proper prokaryotic signals, which differ from eukaryotic signals. For a discussion of maximizing gene expression, see Roberts and Lauer, Methods in Enzymology 68:473 (1979). Regardless of the particular regulatory sequences utilized, DNA molecules are cloned into vectors using standard cloning procedures in the art, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, NY (1989). Once the isolated DNA molecule encoding the protein has been cloned into an expression system, it is ready to be incorporated into a host cell. This can be accomplished by various transformation methods, 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 expressing a functional native or recombinant type III secretion system, as described in detail in U.S. Pat. No. 6,596,509. As a result of expressing a functional type III secretion system, the cell will express the polypeptide and subsequently secrete the protein into the culture medium. This can simplify polypeptide isolation and purification. The recombinant host cell 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 can determine the optimal conditions for a particular host cell.After fermentation, the bacterial suspension can be diluted with, for example, about 2 to 5 volumes of a buffer solution to adjust the pH to about 5.5 to 10, more preferably about 7 to 9, and even more preferably about 8.0. Suitable buffers are well known in the art, and examples include potassium phosphate buffer and Tris-EDTA buffer. The buffer concentration can be about 0.001 mM to about 0.5 M. After pH adjustment, the (bacterial) suspension is heat-treated to a temperature of about 60 to 130°C, preferably about 95 to 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. The heated suspension is then cooled. Suitable cooling temperatures include, but are not limited to, about 35 to 55°C, preferably about 45°C. After cooling, bacterial cells in the bacterial suspension can be lysed as needed to release polypeptides. Cell lysis can be carried out, for example, by contacting the bacterial suspension with lysozyme. The lysozyme concentration can be about 2 ppm to 100 ppm. Alternatively, cell lysis may be achieved by non-chemical methods, such as high pressure or sonication, both of which are well known to those skilled in the art. It may be desirable to incubate the bacterial suspension after cell lysis. Suitable incubation times may vary. For example, it may be desirable 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 cellular debris and denatured proteins resulting from the previous heat treatment. In one embodiment, the extract is centrifuged for about 10-20 minutes to remove some of the cellular debris. A suitable centrifugation speed may be about 4,000-20,000 rpm, and the spin-down time may be about 10-20 minutes. Further removal of cellular debris is then achieved by heat treatment and centrifugation of the supernatant, resulting in removal of more than about 60%, more than 70%, more than 80%, more than 90%, or more than 95% of the total solids, yielding a liquid extract substantially free of cellular debris. This post-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. under a pressure of 15 psi for about 5 minutes. These temperatures and times may vary depending on other conditions.The method for producing a stable liquid composition containing the amino acid sequence or polypeptide disclosed herein further comprises the step of introducing a biocide and, optionally, one or both of a protease inhibitor and a non-ionic surfactant into the liquid extract to obtain a liquid composition containing the polypeptide. In one embodiment, a protease inhibitor is introduced into the liquid extract without the addition of a non-ionic surfactant. In another embodiment, a non-ionic surfactant is introduced into the liquid extract without the addition of a protease inhibitor. In another embodiment, both a protease inhibitor and a non-ionic surfactant are introduced into the liquid extract. In yet another embodiment, neither a protease inhibitor nor a non-ionic surfactant is introduced into the liquid extract. Alternatively, the stability of the liquid compositions disclosed herein can be assessed using, for example, HPLC analysis or other suitable methods capable of quantifying the amount of a particular protein or polypeptide. The stability of the amino acid sequence or polypeptide in the compositions disclosed herein can be determined by comparing the amount of protein in the liquid composition after aging with the amount in a freshly prepared liquid composition or with a previous quantification performed on the same composition. Measurement of protein stability correlates strongly with the maintenance of its activity.

[0403] Conventional agricultural chemical 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, bases, fillers, thickeners, emulsifiers, dispersing agents, sequestrants, anti-settling agents, coalescing agents, rheology modifiers, antifoaming agents, photoprotectants, antifreeze agents, biocides, penetrating agents, mineral or vegetable oils, pigments and anti-scattering agents or any suitable combination thereof.

[0404] In yet another embodiment, the present invention provides polypeptides of 80 to 200 amino acids or subranges disclosed above, obtained by affinity selection against a predetermined plant pest target, capable of inhibiting the growth and / or activity of a plant pest at a minimum inhibitory concentration of about 0.00001 to 1 μM.

[0405] In certain embodiments of the methods disclosed herein for protecting against, preventing, treating, or treating a plant against fungal infection, a polypeptide or composition disclosed herein is applied directly or indirectly to the plant by spraying, atomizing, foaming, fogging, hydroponics, coating, dipping, and / or dressing.

[0406] Nucleic acid sequence In another aspect, the present invention provides nucleic acid sequences encoding the polypeptide sequences disclosed herein (or suitable fragments thereof). These nucleic acid sequences may be in the form of a vector or genetic construct or polynucleotide. The nucleic acid sequences disclosed herein may be synthetic or semi-synthetic sequences, nucleotide sequences isolated from a library (particularly an expression library), nucleotide sequences generated by PCR using overlapping primers, or nucleotide sequences generated using DNA synthesis techniques known per se.

[0407] 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 161, and variants thereof (e.g., those having amino acid substitutions or a specified percentage of identity to the aforementioned sequences).

[0408] Constructs, vectors, and host cells The genetic constructs disclosed herein may be DNA or RNA, with double-stranded DNA being preferred. The genetic constructs of the present invention may also be in a form suitable for transformation of a desired host cell or host organism, for integration into the genomic DNA of a desired host cell, or for independent replication, maintenance, and / or inheritance in a desired host organism. For example, the genetic constructs of the present invention may be in the form of a vector, such as a plasmid, cosmid, YAC, viral vector, or transposon. In particular, the vector may be an expression vector, i.e., a vector capable of in vitro and / or in vivo expression (e.g., in a suitable host cell, host organism, and / or expression system).

[0409] Thus, in a further aspect, the present invention also provides a vector comprising one or more nucleic acid sequences of the present invention.

[0410] In yet another aspect, the present invention provides hosts or host cells that express or are capable of expressing one or more of the amino acid sequences disclosed herein. Examples of hosts or host cells suitable for expressing the amino acid sequences, polypeptides of the present invention will be apparent to those skilled in the art.

[0411] The present application further discloses that polypeptides of 80 to 200 amino acids or subranges thereof described above are stably maintained in the pesticide or biocontrol composition defined herein, i.e., the integrity of the polypeptide and the pesticidal activity defined herein are maintained under the storage and / or use conditions of the pesticide composition, including high temperatures, freeze-thaw cycles, changes in pH or ionic strength, UV radiation, and the presence of harmful chemicals. Most preferably, these 80 to 200 amino acid polypeptides are stably maintained in the pesticide composition when the pesticide composition is stored at ambient temperature for two years or when the pesticide composition is stored at 54°C for two weeks. In particular, the 80 to 200 amino acid polypeptide contained in the pesticide composition maintains at least about 70% of its activity, more particularly at least about 70% to 80% of its activity, and most particularly about 80% to 90% of its activity, after two years of storage in the pesticide composition at ambient temperature or after two weeks of storage of a pesticide composition containing the polypeptide at 54°C.

[0412] In yet another embodiment for use in the methods disclosed herein, the present application discloses a nucleic acid sequence encoding a polypeptide of 80-200 amino acids, wherein the polypeptide is obtained by affinity selection for a specific plant pathogenicity target, and wherein the polypeptide is capable of inhibiting the growth and / or activity of a crop pest at a minimum inhibitory concentration of about 0.00001-1 μM.

[0413] Also disclosed is a chimeric gene comprising the following operably linked DNA components: a) a plant-expressible promoter; b) a DNA region that, when transcribed, results in an mRNA molecule that can be translated into a polypeptide; and c) a 3' terminal region that includes transcription termination and polyadenylation signals that function in cells of the plant.

[0414] 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 the regulatory nucleic acid sequence, when introduced into a cell, such as a plant cell, is capable of regulating the transcription or expression of the associated nucleic acid coding sequence. The regulatory nucleic acid sequence of a chimeric gene and the nucleic acid sequence to which it is so linked are not normally operably linked in nature.

[0415] In the present invention, a "plant promoter" comprises a regulatory component that mediates the expression of a coding sequence segment in plant cells. To be expressed in plants, a nucleic acid molecule must be operably linked to or contain an appropriate promoter that expresses the gene at the correct time and in the required spatial expression pattern.

[0416] As used herein, the term "operably linked" means that a promoter sequence and a gene of interest are operably linked such that the promoter sequence can initiate transcription of the gene of interest.

[0417] Plant-expressible promoters include nucleic acid sequences capable of directing expression of a transgene in a plant. Examples of plant-expressible promoters are constitutive promoters that are transcriptionally active in at least one cell, tissue, or organ under most environmental conditions during most, if not all, stages of growth and development; other promoters are inducible promoters; other examples are tissue-specific promoters; and still other examples are abiotic stress-inducible promoters.

[0418] The chimeric gene (or expression cassette), when transformed into a plant, expresses the nucleic acid, which results in expression of the protein.

[0419] Also disclosed are recombinant vectors containing the above-described expression cassettes (or chimeric genes).

[0420] The term "terminator" refers to a regulatory sequence, a DNA sequence at the end of a transcription unit that directs 3' processing and polyadenylation of the primary transcript and transcription termination. Terminators can be derived from the native gene, from a variety of other plant genes, or from T-DNA. Additional terminators can be derived, for example, from the nopaline synthase or octopine synthase gene, or from another plant gene, or, less preferably, from any other eukaryotic gene.

[0421] "Selection marker," "selection marker gene," or "reporter gene" includes any gene that confers a phenotype on cells in which it is expressed, so as to facilitate the identification and / or selection of cells transfected or transformed with a nucleic acid construct of the invention. These marker genes allow for confirmation of successful introduction of a nucleic acid molecule by a range of different principles. Suitable markers can be selected from markers that confer antibiotic or herbicide resistance, markers that introduce novel metabolic traits, or markers that allow visual selection. Examples of selectable marker genes include genes that confer antibiotic resistance (e.g., nptll, which phosphorylates neomycin and kanamycin, or hpt, which phosphorylates hygromycin, or genes that confer resistance to, for example, bleomycin, streptomycin, tetracycline, chloramphenicol, ampicillin, gentamicin, geneticin (G418), spectinomycin, or blasticidin), genes that confer herbicide resistance (e.g., bar, which provides resistance to Basta®, aroA or gox, which provide resistance to glyphosate, or genes that confer resistance to, for example, imidazolinones, phosphinothricin, or sulfonylureas), or genes that provide metabolic traits (e.g., manA, which enables plants to use mannose as a sole carbon source, or xylose isomerase for xylose utilization, or non-nutritional markers such as resistance to 2-deoxyglucose). The expression of visual marker genes can result in color (e.g., β-glucuronidase GUS or β-galactosidase and its coloring substances, e.g., X-Gal), luminescence (e.g., the luciferin / luciferase system), or fluorescence (green fluorescent protein GFP and its derivatives). These examples are only a small selection of possible markers. Skilled workers are familiar with such markers. Different markers are preferred depending on the organism and the selection method.

[0422] It is known that when a nucleic acid is stably or transiently integrated into a plant cell, a small percentage of the cells will take up the foreign DNA and, if necessary, integrate it into their genome, depending on the expression vector and transfection technique used. To identify and select these integrated cells, a gene encoding a selectable marker (such as those described above) is typically introduced into the host cells along with the gene of interest. These markers can be used, for example, in mutants in which the gene has been rendered nonfunctional, e.g., by deletion using conventional methods. Furthermore, a nucleic acid molecule encoding a selectable marker can be introduced into the host cell using the same vector containing a sequence encoding a polypeptide of the invention or a polypeptide used in a method of the invention, or it can be introduced using a separate vector. Cells stably transfected with the introduced nucleic acid can be identified, for example, by selection (e.g., cells that have integrated the selectable marker will survive, while cells that have not will die).

[0423] Because successful nucleic acid introduction renders marker genes, particularly genes for antibiotic and herbicide resistance, unnecessary or undesirable in transgenic host cells, the nucleic acid introduction method of the present invention advantageously utilizes techniques that allow for the removal or excision of these marker genes. One example of such a method is called co-transformation. Co-transformation involves the simultaneous use of two vectors: one vector carrying the nucleic acid of the present invention and a second vector carrying a marker gene. A high percentage of transformants (up to 40% or more of the transformants) receive both vectors, or, in the case of plants, contain both vectors. In the case of Agrobacterium-mediated transformation, transformants typically receive only a portion of the vector, i.e., sequences flanked by T-DNA, typically corresponding to the expression cassette. The marker gene can then be removed from the transformed plant by crossing. Alternatively, a marker gene incorporated into a transposon is used for transformation along with the desired nucleic acid (known as the Ac / Ds technique). Transformants can be mated with a transposase source or transformed with a nucleic acid construct that confers transient or stable expression of the transposase. In some cases (approximately 10%), successful transformation results in the transposon being expelled from the host cell genome and lost. In many more cases, the transposon jumps to another location. In these cases, it is necessary to eliminate the marker gene by mating. In microbiology, techniques have been developed that allow or facilitate the detection of such events. More advantageous methods rely on a method called a recombination system, which has the advantage of eliminating the mating step. The most well-known system of this type is the Cre / lox system. Cre1 is a recombinase that removes sequences located between loxP sequences. If the marker gene is integrated between the loxP sequences, it is removed by expression of the recombinase after successful transformation.Other 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 of the present invention can be integrated site-specifically into the plant genome.

[0424] For the purposes of the present invention, "transgenic", "transgene" or "recombinant" means, for example, in the case of a nucleic acid sequence, an expression cassette, genetic construct or vector comprising said nucleic acid sequence, or an organism transformed with a nucleic acid sequence, expression cassette or vector according to the invention.

[0425] Thus, for the purposes of the present invention, a transgenic plant is considered to mean, as described above, that the nucleic acid used in the method of the present invention is not present in or derived from the genome of the plant, or is present in the genome of the plant but not at its natural locus in the genome of the plant, and the nucleic acid can be expressed homologously or heterologously. However, as described above, transgenic also means that the nucleic acid of the present invention or the nucleic acid used in the method of the present invention is in its natural location in the genome of the plant, but the sequence has been modified relative to the natural sequence and / or the regulatory sequences of the natural sequence have been modified. Preferably, transgenic is considered to mean that the expression of the nucleic acid of the present invention is at a non-native locus in the genome, i.e., that the nucleic acid is expressed homologously or heterologously. Preferred transgenic plants are referred to in the present application.

[0426] The term "expression" or "gene expression" refers to the transcription of a specific gene or genes or a specific gene construct. The term "expression" or "gene expression" specifically refers to the transcription of a gene or genes or gene constructs into structural RNA (rRNA, tRNA) or mRNA, which may or may not then be translated into a protein. This process includes transcription of DNA and processing of the resulting mRNA product.

[0427] As used herein, the term "increased expression" or "overexpression" refers to any form of expression that is in addition to the original wild-type expression level. For purposes of the present invention, the original wild-type expression level may be zero, i.e., no expression or no measurable expression.

[0428] Methods for increasing gene or gene product expression are well documented in the art, including, for example, overexpression driven by an appropriate promoter (as described above) or the use of transcriptional or translational enhancers. An isolated nucleic acid functioning as a promoter or enhancer element can be introduced into a suitable position (typically upstream) of a non-heterologous polynucleotide to upregulate expression of a nucleic acid encoding a polypeptide of interest. When polypeptide expression is desired, it is generally desirable to add a polyadenylation region to the 3' end of the polynucleotide coding region. The polyadenylation region can be derived from the native gene, various other plant genes, or T-DNA. The added 3' terminal sequence can be derived, for example, from the nopaline synthase or octopine synthase gene, or another plant gene, or, less preferably, from any other eukaryotic gene.

[0429] To increase the amount of mature message that accumulates in the cytosol, intron sequences may be added to the 5' untranslated region (UTR) of a partial coding sequence or to the coding sequence. Addition of splicable introns to transcription units in both plant and animal expression constructs has been shown to increase gene expression by up to 1000-fold at both the mRNA and protein levels (Buchman and Berg (1988) Mol. Cell Biol. 8:4395-4405; Callis et al. (1987) Genes Dev 1:1 183-1200). Such intron enhancement of gene expression is generally greatest when placed near the 5' end of the transcription unit. The use of maize introns Adh1-S introns 1, 2, and 6, and the Bronze-1 intron, are known in the art. For general information, see The Maize Handbook, Chapter 1 16, Freeling and Walbot, Eds., Springer, NY (1994).

[0430] As used herein, the terms "transduction" and "transformation" refer to the introduction of an exogenous polynucleotide or chimeric gene (or expression cassette) into a host cell, regardless of the method used for the introduction. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, can be transformed with the genetic constructs of the present invention and subsequently regenerated into whole plants. The particular tissue selected will depend on the clonal propagation system available and optimal for the particular species being transformed. Typical tissue targets include leaf discs, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, pre-existing meristems (e.g., apical meristem, axillary bud, and root meristem), and derived meristems (e.g., cotyledonary meristem and hypocotyl meristem). Polynucleotides can be transiently or stably introduced into host cells and can remain unintegrated, for example, as a plasmid. Alternatively, they can be integrated into the host genome. The resulting transformed plant cells can then be used to regenerate transformed plants using methods known to those skilled in the art.

[0431] Transformation refers to the introduction of a foreign gene into the genome of a plant. Transformation of plant species is now a fairly routine technique. Advantageously, any of several transformation methods can be used to introduce the gene of interest into suitable ancestor cells. Methods described for the transformation and regeneration of 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 enhance free DNA uptake, direct injection of DNA into plants, particle bombardment, viral or pollen-mediated transformation, and microinjection. The method can be selected from the calcium / polyethylene glycol method for protoplasts (Krens, FA et al., (1982) Nature 296, 72-74; Negrutiu I et al., (1987) Plant Mol Biol 8:363-373); electroporation of protoplasts (Shillito RD et al., (1985) Bio / Technol 3, 1099-1 102); microinjection into plant material (Crossway A et al., (1986) Mol. Gen Genet 202:179-185); particle gun method using particles coated with DNA or RNA (Klein TM et al., (1987) Nature 327:70); infection with (non-integrating) viruses, etc. Preferably, transgenic plants, including transgenic cultivated plants, are produced by Agrobacterium-mediated transformation. An advantageous transformation method is in planta. For this purpose, for example, Agrobacterium can be applied to plant seeds or plant meristems can be inoculated with Agrobacterium. According to the present invention, it has been found to be particularly advantageous to apply a suspension of transformed Agrobacterium to intact plants or at least flower primordia. The plants are then grown until treated plant seeds are obtained (Clough and Bent, Plant J. (1998) 16, 735-743).Agrobacterium-mediated transformation of rice includes well-known rice transformation methods such as those described in European Patent Application EP 1198985, Aldemita and Hodges (Planta 199:612-617, 1996), Chan et al. (Plant Mol Biol 22(3):491-506, 1993), or Hiei et al. (Plant J 6(2):271-282, 1994), the disclosures of which are incorporated herein by reference in their entireties. For maize transformation, a preferred method is that described in either 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 in their entireties. The method is also described, for example, in B. Jenez 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. Preferably, the nucleic acid or construct to be expressed is cloned into a vector (e.g., pBin19) suitable for transforming Agrobacterium tumefaciens (Bevan et al. (1984) Nucl. Acids Res. 12-8711).Agrobacterium transformed with such vectors can then be used to transform plants in a known manner, for example, by immersing crushed or shredded leaves in an Agrobacterium solution followed by culturing in a suitable medium, for example, in plants used as models such as Arabidopsis (Arabidopsis thaliana is not considered a cultivated plant within the scope of the present invention), or in cultivated plants such as tobacco. Methods for transforming plants with Agrobacterium tumefaciens are described, for example, by Hofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877, or are known in particular 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.

[0432] In addition to transforming somatic cells, which subsequently must be regenerated into intact plants, it is also possible to transform cells of plant meristems, particularly those cells that will develop into gametes. In this case, the transformed gametes will follow natural plant development and give rise to transgenic plants. Thus, for example, Arabidopsis seeds can be treated with Agrobacterium and seeds obtained from developing plants of which a predetermined percentage have been transformed and become transgenic [Feldman, KA and Marks MD (1987). Mol Gen Genet 208:1-9; Feldmann K (1992). In: C Koncz, NH Chua and J Shell, eds., Methods in Arabidopsis Research. Word Scientific, Singapore, pp. 274-289]. Alternatively, transformed seeds can be obtained at later time points by repeatedly removing the inflorescence and incubating the excised portion at the center of the rosette with transformed Agrobacterium (Chang (1994). Plant J. 5:551-558; Katavic (1994). Mol Gen Genet, 245:363-370). However, particularly effective methods are vacuum infiltration and its variants, such as the "floral dip" method. In the case of vacuum infiltration of Arabidopsis, intact plants are treated with an Agrobacterium suspension under reduced pressure (Bechthold, N (1993). CR Acad Sci Paris Life Sci, 316:1194-1199). In the case of "floral dip," developing floral tissue is briefly incubated with a detergent-treated Agrobacterium suspension (Clough, SJ and Bent AF (1998) The Plant J. 16:735-743). In either case, a proportion of transgenic seeds are harvested and can be differentiated from non-transgenic seeds by growing them under the selection conditions described above. Furthermore, stable transformation of plastids is advantageous because plastids are maternally inherited in most crops, reducing or eliminating the risk of transgene shedding through pollen.Transformation of the chloroplast genome is generally carried out according to the method diagrammed in Klaus et al., 2004 [Nature Biotechnology 22(2), 225-229]. Briefly, the sequence to be transformed is cloned with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site-specific integration into the chloroplast genome (plastome). Plastid transformation methods have been described for a wide variety of plant species and are reviewed in 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. Furthermore, biotechnological advances have recently been reported in the form of marker-free plastid transformants that can be produced by transiently co-integrating marker genes (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229).

[0433] Genetically modified plant cells can be regenerated by any method familiar to the skilled worker: for suitable methods see the publications by S.D. Kung and R. Wu, Potrykus or Hofgen and Willmitzer, supra.

[0434] Generally, after transformation, plant cells or cells are selected for the presence of one or more markers encoded by the plant-expressible gene transfected with the gene of interest, and the transformed material is then regenerated into whole plants. To select transformed plants, the plant material obtained by transformation is generally subjected to selection conditions so that transformed plants can be distinguished from non-transformed plants. For example, seeds obtained as described above can be sown and, after an initial growth period, subjected to appropriate selection by spraying. Alternatively, seeds can be grown on agar plates, optionally after sterilization, using an appropriate selection agent so that only transformed seeds can develop into plants. Alternatively, transformed plants can be screened for the presence of selection markers such as those described above.

[0435] After DNA introduction and regeneration, putatively transformed plants may be evaluated for the presence, copy number, and / or genomic organization of the gene of interest using, for example, Southern analysis. Alternatively, or additionally, expression levels of the newly introduced DNA may be monitored by Northern and / or Western analysis, both techniques well known to those of ordinary skill in the art.

[0436] The resulting transformed plants can be propagated by various means, including clonal propagation and classical breeding techniques. For example, first-generation (or T1) transformed plants can be self-fertilized, homozygous second-generation (or T2) transformants selected, and the T2 plants can then be further propagated by classical breeding techniques. The resulting transformed organisms can take various forms, including chimeras of transformed and untransformed cells, clonal transformants (e.g., whole cells transformed to contain the expression cassette), and grafts of transformed and untransformed tissue (e.g., in plants, transformed rootstock grafted onto untransformed scions).

[0437] The present invention will now be described in detail with reference to the following non-limiting examples. [Example]

[0438] Example 1: Preparation of Folch-phase antigens of Fusarium oxysporum for immunization, phage display, and screening assays Intact hyphae and conidia of Fusarium oxysporum were extracted sequentially at room temperature using chloroform:methanol in a 2:1 and 1:2 (v / v) ratio. The extracts were combined, dried, and the crude lipid extract was partitioned as described by Folch et al. (1957). A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497-509.) The lipids from the lower layer of the Folch procedure were collected and used for immunization.

[0439] Example 2: Preparation of a ceramide monohexoside fraction from Fusarium oxysporum for phage display and screening assays A ceramide monohexoside fraction from Fusarium oxysporum, prepared as described by Barreto-Bergter et al. (2011 Barreto-Bergter E, Sassaki G and de Souza LM. (2011) Structural analysis of fungal cerebrosides. Front Microbiol. 2:239.), was obtained from Eliana Barreto-Bergter.

[0440] [Example 3] Identification of VHHs that bind to the Folch-type lower phase antigen of Fusarium oxysporum 3.1 Immunology VHHs were generated from llamas immunized with a Folch lower-phase extract derived from Fusarium oxysporum. Following standard protocols, llamas were boosted six times with thin-layer chromatography (TLC) spots of the Folch lower-phase extract derived from Fusarium oxysporum. The silica particles adsorbed with the Folch lower-phase extract were scraped from the plate and suspended in phosphate buffer. The suspension was sonicated, mixed with incomplete Freund's adjuvant, and used for subcutaneous injection. All llamas remained healthy throughout the immunization process, and blood samples were collected before and after immunization.

[0441] 3.2 Library construction 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 and contains a lacZ promoter, a synthetic leader sequence, a multiple cloning site, a coliphage pIII protein coding sequence, an ampicillin resistance gene, and an M13 phage origin for single-strand production. This vector encodes a C-terminal (His)6 peptide tag and a c-myc peptide tag in frame with the VHH-coding sequence. Phages were generated 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 of more than 1E+08 was obtained, confirming that phages were produced and exhibited antibody diversity.

[0442] 3.3 Selection Two rounds of panning selection were performed as follows. Fungal lipid fractions were coated onto polystyrene Maxisorp multiwell plates, always in 5% chloroform / methanol. In the first round of selection, 25 μl of phages from the library (1,00E+11 phages / selection condition) were selected using four different conditions: two different lipid fraction (fungal; Fusarium oxysporum) concentrations (50 μg / ml and 5 μg / ml) and two different blank conditions for background control (5% CHCl3 / MeOH and PBS). In the second round of selection, in addition to the conditions used in the first round of selection, an even lower antigen concentration (0.5 μg / ml) was used. In the second round of selection, the phage input was diluted 10-fold compared to the input from the first round of selection to reduce the selection of nonspecific phage binding.

[0443] The first round of selection showed good enrichment, 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 selection round, significantly higher enrichment was observed in the antigen-coated conditions, except for the 0.5 μg / ml selection condition.

[0444] The selected eluted phage pool obtained after the second round of panning selection was infected into E. coli TG1 cells, and individual colonies were picked and transferred to a 96-well plate containing 100 μl of 2xTY medium supplemented with 2% glucose and 100 μg / ml carbenicillin per well as a master plate (MP), where they were grown overnight at 37°C. The master plate was stored in 20% glycerol at -80°C and used for periplasmic extract production, screening, and sequencing.

[0445] [Example 4] Screening of VHHs that bind to lipid fractions of Fusarium oxysporum To verify whether VHHs bind to the lipid fraction of Fusarium oxysporum, binding was assessed by ELISA. Individual clones were screened for binding to wells coated with 10 μg / ml of the fungal lipid fraction in 5% CHCl3 / MeOH and wells coated with 5% CHCl3 / MeOH alone. Clones were considered positive if the OD450nm binding signal in the antigen-coated wells was greater than twofold that of the corresponding blank wells. According to the defined cutoff criteria applied to the 360 ​​individual clones tested, an overall hit rate of 14.2% was obtained.

[0446] [Example 5] VHH10G11 dose-dependently inhibits the growth of Botrytis cinerea in an in vitro antifungal assay.

[0447] The antifungal activity of VHH10G11Q (SEQ ID NO: 1) against the plant pathogenic fungus Botrytis cinerea R16 was evaluated in vitro.

[0448] Two-fold dilutions of purified VHH10G11Q were prepared in 96-well microtiter plates. Starting with a final VHH10G11Q concentration of 10 μM, 20 μl of these dilutions, plus 20 μl of water as a control, were supplemented with 80 μl of fungal spore suspension (1E+05 spores per ml of half-strength potato dextrose broth (PDB)). Test plates were incubated at 25°C for 36 hours using an IncuCyte Zoom live-cell imaging system. All tests were performed at least in duplicate.

[0449] The results of the antifungal activity assay, shown in Figure 1, revealed a clear dose-dependent growth inhibition pattern when expressed as % fungal growth (total area of ​​green objects) versus VHH10G11Q concentration (µM).

[0450] [Example 6] VHH10G11 more potently inhibits the growth of Botrytis cinerea compared to glycosylceramide-binding VHH41D01 in an in vitro antifungal assay.

[0451] WO2014 / 177595A1 and WO2014 / 191146A1 describe several anti-glucosylceramide-binding VHHs, among which VHH41D01 shows the most significant antifungal activity against several test strains, including Botrytis cinerea R16.

[0452] The growth inhibitory properties of VHH10G11Q (SEQ ID NO: 1) against the plant pathogenic fungus Botrytis cinerea R16 were compared in vitro with those of VHH41D01.

[0453] Two-fold dilutions of purified VHH10G11Q and 41D01 were prepared in 96-well microtiter plates. Starting with a final VHH10G11Q concentration of 40 μM, 20 μl of these dilutions, plus 20 μl of water as a control, were supplemented with 80 μl of fungal spore suspension (1E+05 spores per ml of half-strength potato dextrose broth (PDB)). Test plates were incubated at 25°C for 48 hours using an IncuCyte Zoom live-cell imaging system. All tests were performed at least in duplicate.

[0454] The results of the antifungal activity assay shown in Figure 2 surprisingly revealed a more pronounced growth inhibition pattern for VHH10G11Q compared to the biologically active VHH41D01.

[0455] [Example 7] Site-directed mutagenesis Site-directed mutagenesis of VHHs was performed as follows. Nucleotide sequences encoding VHH sequence variants were synthetically constructed as gene fragments and cloned into the plasmid pPpT4GAPαS (Naatsaari et al. (2012), Plos One, 7(6):e39720), suitable for transformation of Pichia pastoris. This plasmid contains P to induce expression from the cloned gene fragments. AOXPlasmids containing the desired VHH sequence variants were constructed using standard cloning techniques. Successful clones were sequenced to confirm the presence and correct cloning of the desired VHH sequence variants. The linearized plasmids were then transformed into competent Pichia pastoris (Pichia pastoris) ATCC76273™ cells using a standard electroporation protocol. Successful transformants were then used to generate VHH sequence variants. Transformants were first cultured in BMGY (buffered glycerol complex medium) and then transferred to BMMY (buffered methanol complex medium) to initiate induction (Weidner et al. (2020), J Vis Exp, 36:1862). The VHHs were then purified using filtration and / or chromatography techniques commonly known in the art.

[0456] [Example 8] Ala scanning of VHH10G11 All three CDR regions of VHH10G11 were evaluated by Ala scanning to examine the effect of single amino acid substitutions on the antifungal activity of VHH10G11 (SEQ ID NOS: 18-51). 10G11 mutants were constructed and generated as described in Example 7, followed by antifungal assays as described in Example 5. Surprisingly, 10G11 maintained its performance in most mutants where individual amino acids in the CDR regions were substituted with alanine, as evidenced by the maintenance of antifungal activity of the mutants (Figure 3). Similar results were obtained when other single amino acid substitution mutants were constructed using glycine (R26G; SEQ ID NOS: 18 and D35G; SEQ ID NOS: 27), serine (R53S; SEQ ID NOS: 32 and T56S; SEQ ID NOS: 36), or asparagine (K58N; SEQ ID NOS: 39) instead of alanine (Figure 3).

[0457] [Example 9] Improvement of antifungal activity of His-tagged VHH10G11 His tags are commonly added to polypeptides for purification using affinity chromatography. During routine testing using an antifungal assay such as that described in Example 5, it was surprisingly found that the 10G11Q polypeptide (SEQ ID NO: 148) with a 6xHis tag added to its C-terminus exhibited significantly improved antifungal activity (Figure 4). Because the His tag is composed of six positively charged histidine amino acids, the positive charge appeared to be beneficial for the antifungal interaction of 10G11.

[0458] [Example 10] In silico modeling of VHH10G11Q To better understand the 10G11 molecule, we modeled its three-dimensional structure using an in silico approach. A BLAST search was performed on the Protein Data Bank (PDB) to obtain the structural coordinates of an antibody with the same CDR length as the 10G11 molecule to be modeled. A Blosum 62 matrix was used with a Gap cost of 13. Of the top 250 hits, each structure with the same CDR length as the 10G11 molecule under investigation was used for homology modeling trials. First, residues that differed between the resulting PDB structure and the 10G11 molecule were mutated. Next, the side chains of these residues were modeled according to their observed preferences as detected by the Dunbrack 2010 rotamer library (Shapovalov and Dunbrack, (2011), Structure, 19(6):844-58). Where necessary, nearby side chains were also modeled to accommodate modeling mutations. Using this model as a guide, we determined the critical amino acid residues of the molecule.

[0459] The resulting model, with a representation of the three CDR regions, is shown in Figure 5. A selection of exposed side chains is visualized on a ribbon diagram of 10G11 and displayed according to Kabat numbering (Figure 5 also shows the germline sequence and displays the substitutions that resulted in the 10G11 sequence. Additionally, the corresponding Kabat numbering is shown). These amino acid residues were selected as the best candidates for specific amino acid substitutions because their side chains were exposed and appeared to present minimal structural disturbance.

[0460] [Example 11] Antifungal activity of 10G11 charge mutants As confirmed by the assay results shown in Figure 3, four of the five mutants that showed reduced antifungal activity were characterized by the substitution of positively charged amino acids with uncharged variants. Given the results showing that the positively charged His-tagged variants had increased antifungal activity (Figure 4), the effect of charge was further investigated. To this end, various 10G11 charge variants were generated as described in Example 7, and their antifungal activity was tested as described in Example 5. The following substitutions were designed to reduce the overall charge of the 10G11 molecule at the antigen-binding interface of the molecule. Mutant 1: R26A substitution in CDR1 (SEQ ID NO: 7) Mutant 2: R53A and K58A substitutions in CDR2 (SEQ ID NO: 8) Mutant 3: R96A and R100cA substitutions in CDR3 (SEQ ID NO: 9) Mutant 4: R26A substitution in CDR1, R53A and K58A substitutions in CDR2, and R96A and R100cA substitutions in CDR3 (SEQ ID NO: 10) 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).

[0461] These mutants with reduced net charge exhibit reduced or absent antifungal activity (Figure 6), further supporting the hypothesis that a positive charge is important for the activity of 10G11. Therefore, we designed the following positively charged mutants based on the structural analysis described in Example 10. Mutant 6: S27H, I28H and S30H substitutions in CDR1 (SEQ ID NO: 12) Mutant 7: W100aH and T100bH substitutions in CDR3 (SEQ ID NO: 13) Mutant 8: S27H, I28H and S30H substitutions in CDR1 and W100aH and T100bH substitutions in CDR3 (SEQ ID NO: 14) Mutant 9: S27K, I28K and S30K substitutions in CDR1 (see Figure 7) (SEQ ID NO: 15) Mutant 10: W100aK and T100bK substitutions in CDR3 (SEQ ID NO: 16) Mutant 11: S27K, I28K and S30K substitutions in CDR1 and W100aK and T100bK substitutions in CDR3 (see Figure 8) (SEQ ID NO: 17).

[0462] Figure 6 shows that mutants 6 and 7 have antifungal activity comparable to that of 10G11. Mutants 8, 9, 10 and 11 show significant increases in antifungal activity, with mutant 11 reaching five times the antifungal activity of 10G11.

[0463] Finally, an extended incubation antifungal assay was performed, extending the experimental setup as described in Example 5 to 14 days. 10G11, although highly potent, partially resumed spore growth after 3 days, whereas mutants 9 and 11 were found to have a sustained effect in preventing spore growth throughout the evaluation period (Figure 9).

[0464] [Example 12] 10G11 binds to fraction 3 of the Folch lower phase extract. To investigate the putative interacting partners of 10G11 and its mutants, lipids were extracted from Botrytis cinerea mycelia as described in Example 1. Thin-layer chromatography (TLC; Skipski et al. (1965), Biochimica et Biophysica Acta, 106(2):386-396) was used to further fractionate the total lipid extract. The extract was spotted onto a silica gel glass plate and developed in a chromatography tank filled with 100 mL of a chloroform / methanol mixture (85 / 15, v / v). Lipid bands separated by TLC were stained with α-naphthol to allow visual detection of lipids. Four fractions were identified: fraction 1, which was a polar lipid fraction; fraction 2, which had a retention factor (Rf) similar to that of a glucosylceramide reference standard (GlcCer from Pleurotus cornucopiae) and was likely ceramide; fraction 3, which had an Rf slightly higher than that of the reference standard; and fraction 4, which was a nonpolar phospholipid (PL) (Figure 10). To obtain sufficient material from each fraction, normal-phase flash chromatography can be used (Gorden, M.H. (2015). Encyclopedia of Analytical Science (Second edition), Elsevier). Therefore, TLE was dissolved in CHCl by adding a few drops of MeOH and loaded onto a 4 g normal-phase flash cartridge packed with 15 μm particles. CHCl / MeOH (85 / 15, v / v) was used as the eluent. Finally, the fractions were filtered through a 0.45 μm syringe filter (nylon membrane) and dried. Liposomes were then prepared from the true lipid extract and its individual fractions by thin-film hydration in the presence of 1,6-diphenyl-1,3,5-hexatriene (DPH) (Trucillo et al., 2020. Processes 8(9):1022; Zhang, 2017. Liposomes 1522:1 7-22). DPH is nearly non-fluorescent in aqueous media, but exhibits strong fluorescence when inserted into lipid membranes. Disruption of the lipid membrane would lead to a decrease in the fluorescent signal. We then examined the interaction of 10G11 with these liposomes.The results in Figure 11 show that 10G11 interacts with lipids present in the total lipid extract (TLE), fraction 3, and fraction 4. Furthermore, 10G11 causes vesicle disruption, leading to the interesting effect of membrane disruption by 10G11.

[0465] The binding of 10G11 to Fraction 3 was further confirmed by biolayer interferometry (BLI), a label-free optical analysis technique for measuring biomolecular interactions (Kamat et al., 2017. Analytical biochemistry 536:16-31; Wallner et al., 2013. Journal of Pharmaceutical and Biomedical Analysis 72:150-154). This analysis confirmed the binding of 10G11 to Fraction 3 (Figure 12).

[0466] [Example 13] Other molecules that bind to fraction 3 The liposome formation method described above (Example 12) was modified by the addition of a biotinylated lipid (12:0 biotinyl MG (1-(12-N-biotin) aminododecanoyl-rac-glycerol) (Sigma-Aldrich) to enable the use of these liposomes in an ELISA using streptavidin-coated plates (Pierce™, a Thermo Fisher Scientific product, high-binding capacity clear 96-well plates coated with streptavidin and blocked with SuperBlock™ buffer). This method was used to screen for other molecules that bind to Fraction 3, and two additional molecules that bind to Fraction 3 were identified: SEQ ID NO:3 (10E11Q) and SEQ ID NO:5 (12C03Q), as shown in Figure 13.

[0467] [Example 14] Mode of action The effect of 10G11 on Botrytis cinerea spores was observed to the extent that the spores were inhibited from initiating growth after spore germination, and surprisingly, a significant proportion of the spores were also lysed by 10G11 activity, as confirmed by a rapid decline in structural integrity during microscopic time-lapse imaging with an IncuCyte Zoom live-cell imaging system (Figure 14). The ability of 10G11 to disrupt liposomes (Example 12) further supports the role of 10G11 in spore lysis.

[0468] Example 15: Control of soybean rust caused by Phakopsora pachyrhizi in greenhouses Four pots of potted soybean seedlings (four plants per pot) were sprayed with 125 ml of an aqueous suspension containing the active ingredient at the concentrations listed in Table 1. Two applications were performed, 14 days apart. Two days after the first application of active ingredient (Application A), the seedlings were inoculated once with Phakopsora pachyrhizi. The growth inhibition properties of 10G11Q were compared with those of a non-antifungal reference VHH molecule, an untreated control, and the systemic chemical pesticide Quadris Top SBX (azoxystrobin, difenoconazole).

[0469] [Table 2]

[0470] Disease severity is recorded as a percentage of severity on a scale of 0 to 100%. Recording begins when the first symptoms appear, and is then assessed weekly until at least three weeks after the second application of active ingredient (Application B). Percentage severity is assessed based on seedling location as percentage lower canopy severity, percentage mid-canopy severity, and percentage upper canopy severity, to adequately track disease progression after natural occurrence of the disease in the field.

[0471] The results, shown in Figure 15, show a significant dose-related pattern of growth inhibition in the lower canopy for 10G11 compared to the reference VHH and the untreated inoculated control up to 3 weeks after the second application.

[0472] The time indication on the x-axis of the graph represents the time point at which disease severity or incidence was recorded. These are relative values ​​and are expressed as the number of days after application at the time the recording was made. For example, a recording made 13 days after the second application (Application B) would be represented as 13DAB. This code applies to Figures 15-28.

[0473] The results, shown in Figure 16, show a significant dose-related pattern of growth inhibition in the mid-canopy for 10G11 compared to the reference VHH and the untreated inoculated control up to 3 weeks after the second application.

[0474] The results, shown in Figure 17, show a significant dose-related pattern of growth inhibition in the upper canopy for 10G11 compared to the reference VHH and the untreated inoculated control up to 3 weeks after the second application.

[0475] In general, 10G11 at 750 gai / ha often exhibits "equivalent" activity to the reference chemical pesticide Quadris Top SBX.

[0476] [Example 16] Control of pumpkin anthracnose caused by Colletotrichum orbiculare in a greenhouse Four pots of potted squash seedlings (two plants per pot) were sprayed with 125 ml of an aqueous suspension containing the active ingredient at the concentration listed in Table 2. Four applica...

Claims

1. A composition comprising at least one polypeptide, which is capable of binding to a fungus, thereby causing retardation of spore growth and / or lysis of spores of the fungus.

2. The composition of claim 1 , wherein the at least one polypeptide specifically binds to at least one cell membrane component of the fungus.

3. 1. A composition comprising at least one polypeptide capable of binding to a lipid-containing fraction of the cell membrane of Botrytis cinerea, wherein the lipid-containing fraction is obtainable by a method comprising the steps of fractionating mycelia of Botrytis cinerea by total lipid extract thin-layer chromatography and selecting a fraction having a retention factor (Rf) greater than that of a ceramide fraction and less than that of a non-polar phospholipid fraction.

4. 1. A composition comprising at least one polypeptide, wherein the polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-51 or 101-111, or an amino acid sequence having at least about 80% sequence identity to any of them, and wherein the polypeptide is capable of binding to a fungus.

5. 1. A composition comprising at least one polypeptide, said polypeptide comprising: a CDR1 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 52-67 and 112-122; a CDR2 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 68-83 and 123-133; and a CDR3 region, optionally wherein said CDR3 region has a sequence selected from the group consisting of SEQ ID NOs: 84-100 and 134-144; and wherein said polypeptide is capable of binding to a fungus.

6. A composition comprising at least one polypeptide, said polypeptide comprising a CDR1 region having the amino acid sequence set forth in SEQ ID NO:52, a CDR2 region having the amino acid sequence set forth in SEQ ID NO:68, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO:84, said polypeptide being capable of binding to a fungus.

7. A composition comprising at least one polypeptide, said polypeptide comprising a CDR1 region having the amino acid sequence set forth in SEQ ID NO:53, a CDR2 region having the amino acid sequence set forth in SEQ ID NO:69, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO:85, said polypeptide being capable of binding to a fungus.

8. A composition comprising at least one polypeptide, said polypeptide comprising a CDR1 region having the amino acid sequence set forth in SEQ ID NO:54, a CDR2 region having the amino acid sequence set forth in SEQ ID NO:70, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO:86, said polypeptide being capable of binding to a fungus.

9. 1. A composition comprising at least one polypeptide, wherein the polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-51 or 101-111, or an amino acid sequence having at least about 80% sequence identity thereto, wherein the polypeptide is capable of binding to a fungus, and wherein the polypeptide comprises one or more substitutions that result in an increased positive charge compared to any one of SEQ ID NOs: 1-51 or 101-111.

10. The composition according to any one of claims 1 to 9, wherein the at least one polypeptide is an antibody or a functional fragment thereof.

11. The at least one polypeptide comprises a heavy chain variable domain (V Ｈ or V ＨＨ 11. The composition according to any one of claims 1 to 10, which is a functional fragment thereof.

12. The composition of any one of claims 4 to 11, wherein the at least one polypeptide specifically binds to at least one cell membrane component of the fungus.

13. 13. The composition of claim 12, wherein the at least one polypeptide is capable of binding to a lipid-containing fraction of the cell membrane of Botrytis cinerea, and the lipid-containing fraction is obtainable by a method comprising fractionating the mycelia of Botrytis cinerea by total lipid extract thin-layer chromatography and selecting a fraction having a retention factor (Rf) greater than that of a ceramide fraction and less than that of a non-polar phospholipid fraction.

14. The composition according to any one of claims 1 to 13, wherein the concentration of said at least one polypeptide in said composition is from 0.0001 to 50% by weight.

15. The composition according to any one of claims 1 to 14, which is an agrochemical composition.

16. 16. A composition according to claim 15, further comprising an agrochemically suitable base and / or one or more suitable adjuvants.

17. The composition according to any one of claims 1 to 16, wherein the fungus is a plant pathogenic fungus.

18. The genus of the plant pathogenic fungus is selected from the group consisting of Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Diplodia, Erysiphe, Fusarium, Leptosphaeria, Gaeumannomyces, Helminthium, and the like. The genera Helminthosporium, Macrophomina, Nectria, Penicillium, Peronospora, Phoma, Phymatotrichum, Phytophthora, Plasmopara, Podosphaera, Puccinia, nia), Pyrenophora, Pyricularia, Pythium, Rhizoctonia, Sclerotium, Sclerotinia, Septoria, Thielaviopsis, Uncinula, Venturia, Verticillium 18. The composition of claim 17, wherein the fungus is selected from the group comprising the genera Mycosphaerella, Magnaporthe, Blumeria, Mycosphaerella, Ustilago, Melampsora, Phakospora, Monilinia, Mucor, Rhizopus, and Aspergillus.

19. 1. A composition comprising at least one polypeptide, wherein the polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-51 or 101-111, or an amino acid sequence having at least about 80% sequence identity to any of them, and wherein the polypeptide is capable of binding to fungi for use as an antifungal agent.

20. 1. A composition comprising at least one polypeptide, said polypeptide comprising: a CDR1 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 52-67 and 112-122; a CDR2 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 68-83 and 123-133; and a CDR3 region, optionally wherein said CDR3 region has a sequence selected from the group consisting of SEQ ID NOs: 84-100 and 134-144, wherein said polypeptide is capable of binding to fungi for use as an antifungal agent.

21. A composition comprising at least one polypeptide, said polypeptide comprising a CDR1 region having the amino acid sequence set forth in SEQ ID NO:52, a CDR2 region having the amino acid sequence set forth in SEQ ID NO:68, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO:84, said polypeptide being capable of binding to fungi for use as an antifungal agent.

22. A composition comprising at least one polypeptide, said polypeptide comprising a CDR1 region having the amino acid sequence set forth in SEQ ID NO:53, a CDR2 region having the amino acid sequence set forth in SEQ ID NO:69, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO:85, said polypeptide being capable of binding to fungi for use as an antifungal agent.

23. A composition comprising at least one polypeptide, said polypeptide comprising a CDR1 region having the amino acid sequence set forth in SEQ ID NO:54, a CDR2 region having the amino acid sequence set forth in SEQ ID NO:70, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO:86, said polypeptide being capable of binding to fungi for use as an antifungal agent.

24. Use of a composition according to any one of claims 1 to 23 as an antifungal agent.

25. 25. Use according to claim 24 as an antifungal agent in plants.

26. 24. A method for protecting or treating a plant or a part of a plant against infection by a phytopathogenic fungus, said method comprising at least the step of applying directly or indirectly to said plant or said part of a plant a composition according to any one of claims 1 to 23 under conditions effective to protect or treat said plant or said part of a plant against said infection by said phytopathogenic fungus.

27. 24. A post-harvest treatment method for protecting or treating a harvested plant or a harvested part of said plant against infection by a phytopathogenic fungus, said method comprising at least the step of applying, directly or indirectly, to said harvested plant or a harvested part of said plant a composition according to any one of claims 1 to 23 under conditions effective to protect or treat said harvested plant or a harvested part of said plant against said infection by said phytopathogenic fungus.

28. 24. A method for inhibiting the growth of or killing plant pathogenic fungi, said method comprising at least the step of applying, directly or indirectly, to a plant or part of said plant a composition according to any one of claims 1 to 23.

29. A polypeptide according to any one of claims 1 to 23.

30. 1. A method for producing a polypeptide that specifically binds to and / or has affinity for a fungus, comprising: immunizing an animal with a fungal target or a suitable antigenic determinant based on or derived from a fungal target (e.g., an antigenic portion thereof, a fragment thereof, a region thereof, a domain thereof, a loop thereof, or other epitope thereof); obtaining a cell collection or sample from the immunized animal that expresses the polypeptide sequence; screening the cell collection or sample for cells expressing an amino acid sequence that binds to and / or has affinity for the fungal target; (i) isolating said amino acid sequence, or (ii) isolating a nucleic acid sequence encoding said amino acid sequence from said cell collection or sample; expressing the amino acid sequence; thereby producing a polypeptide that specifically binds to and / or has affinity for a fungus.

31. 31. The method of claim 30, wherein the fungal target is a lipid-containing fraction of the cell membrane of Botrytis cinerea, and the lipid-containing fraction is obtainable by a method comprising fractionating the mycelia of Botrytis cinerea by total lipid extract thin layer chromatography and selecting a fraction having a retention factor (Rf) greater than that of a ceramide fraction and less than that of a non-polar phospholipid fraction.

32. 1. A method for producing an antifungal composition, comprising: Producing an antifungal polypeptide according to the method of claim 30 or 31; combining said antifungal polypeptide with one or more suitable bases and / or one or more suitable adjuvants; The method comprising:

33. A transgenic plant, plant part, seed or plant cell comprising a nucleic acid sequence encoding a polypeptide according to any one of claims 1 to 23.

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