Antifungal polypeptide

Abstract

The present invention relates to 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 or 101 to 111 or an amino acid sequence having at least about 80% sequence identity to any one of them, and wherein the polypeptide is capable of binding to fungi. The present invention also relates to a composition containing at least one polypeptide, wherein the polypeptide comprises a CDR1 region having an amino acid sequence set forth in any one of SEQ ID NOS: 52 to 67 or 112 to 122, a CDR2 region having an amino acid sequence set forth in any one of SEQ ID NOS: 68 to 83 or 123 to 133, and a CDR3 region having an amino acid sequence set forth in any one of SEQ ID NOS: 84 to 100 or 134 to 144, and wherein the polypeptide is capable of binding to fungi. These compositions can be used as antifungal compositions.

Description

[Technical Field]

[0001] The present invention relates to a composition 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 composition as an antifungal agent. The present invention further relates to a method for protecting or treating a plant or a part of the plant from infection by plant pathogenic fungi, a post-harvest treatment method for protecting or treating a harvested plant or a harvested part of the plant from infection by plant pathogenic fungi, and a method for inhibiting the growth of plant pathogenic fungi or killing plant pathogenic fungi, comprising at least the step of applying the composition or the polypeptide directly or indirectly to a plant or a part of the plant. The present invention also relates to a method for producing an antifungal polypeptide and a method for producing an antifungal composition. Furthermore, 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, observed not only in patients and animals but also in cultivated plants, is likely due primarily to the selective pressure on broad-spectrum antifungal agents and the generally insufficient effectiveness 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 various fungal pathogens. Examples include the highly virulent Candida species Candida albicans, Candida tropicalis, and Candida krusei, and the less virulent species Candida parapsilosis and Torulopsis glabrata (also known as Candida glabrata). Previously, Candida albicans was the most common fungal isolate obtained from intensive care units, but subsequent reports indicate that Candida tropicalis, Candida glabrata, Candida parapsilosis, and Candida krusei now account for about half of such isolates. The rise of non-albicans Candida species signifies the emergence of Candida species resistant to conventional antifungal drugs.

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

[0005] In agriculture, crop protection heavily relies on the use of pest control agents applied to crops by spraying, irrigating, or injecting into the soil. Pest control agents are often organic chemical molecules, and repeated application to crops poses a toxic threat to both agricultural workers and the environment through drift, soil persistence, or runoff into the surface or groundwater. It would be advantageous to use alternative compounds that offer effective control of plant pests while having low toxicity to humans and the environment. Protein pest control agents with specificity to particular plant pest targets appear highly advantageous in this regard, as they are expected to have short environmental presence and low-toxicity off-target effects. However, only a small number of protein or peptide pest control agents are known. Examples include Bt toxin, lectins, defensins, favatin, tachypressin, magainin, harpin (see WO2010019442), and pea mealbumin 1 subunit b (PA1b). However, these protein pest control agents are either small molecular weight peptides with a compact structure stabilized by a few disulfide bonds, or large proteins (>300 amino acids) that exist in crystalline form (cry toxins). In fact, biological agents, especially proteins, are generally known in the agricultural field to have extremely low stability and cannot maintain their pest control function, particularly in field pesticide formulations, making their structure difficult to use in pest control agents. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] International Publication No. 2010 / 019442 [Overview of the project] [Problems that the invention aims to solve]

[0007] The inventors have succeeded in developing a polypeptide that exhibits remarkably high specificity, affinity, and efficacy against harmful organisms, particularly, but not limited to, plants, animals, or human pathogenic fungi. The polypeptide can bind to specific lipid fractions of the cell membrane of fungal spores. The binding of the polypeptide alone is sufficient to prevent mycelial formation by delaying spore growth and / or causing lysis and rupture, thereby exhibiting mycogenic activity. Therefore, the polypeptide can have either mycogenic or mycstatic activity.

[0008] Furthermore, these polypeptides can maintain their integrity, stability, and activity within the composition, and remarkably, effective control of pests or pathogens can be achieved by applying compositions containing the polypeptides disclosed herein to crops, animals, or humans.

[0009] The efficacy and efficacy of the polypeptides disclosed herein suggest that dose reduction may be possible and / or that more effective treatment may be possible at the same dose. This is expected to lead to a reduction in undesirable side effects and toxicity in both agrochemical and pharmaceutical applications. Furthermore, it may be possible to apply the polypeptides or compositions disclosed herein in smaller amounts or dosages.

[0010] More specifically, the inventors have found that by targeting and delivering the polypeptide envisioned in this application to the molecular structure of a harmful organism or pathogen, the pathogen can be efficiently controlled.

[0011] In particular, the inventors have developed polypeptides capable of preventing, protecting, treating, or treating plants, animals, or humans against infection (or outbreak) by pathogens or any other biological interaction with pathogens, especially fungal pathogens. Accordingly, 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 caused by biological interactions between plants, animals, or humans and pathogens (for example, through infection by pathogens) or against exposure to such interactions.

[0012] The polypeptides and compositions of the present invention can be used as standalone products, such as standalone compositions including antifungal compositions.

[0013] The polypeptides and compositions of the present invention can be used defensively or preventively, that is, the first application should be made before the disease is present.

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

[0015] Accordingly, the present invention provides a composition containing at least one polypeptide, wherein the polypeptide is capable of binding to a fungus. Therefore, the polypeptide causes delay in the growth of the fungal spores and / or lysis of the fungal spores. In other words, the binding of the polypeptide to the fungus results in delay in the growth of the fungal spores and / or lysis of the fungal spores.

[0016] Polypeptides are also provided, which are capable of binding to fungi. Thus, the polypeptides cause delays in the growth of the fungal spores and / or lysis of the fungal spores. In other words, the binding of the polypeptides to the fungi results in delays in the growth of the fungal spores and / or lysis of the fungal spores.

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

[0018] The present invention also provides a composition containing at least one polypeptide, the at least one polypeptide 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 obtained by chromatography. For example, the lipid-containing fraction may be obtained by a method comprising the steps of fractionating the hyphae of a fungus (e.g., Botrytis cinerea or other fungi) by total lipid extract thin-layer chromatography, and selecting a fraction having a retention factor (Rf) greater than that of the ceramide fraction and less than that of the nonpolar phospholipid fraction.

[0019] The present invention also provides polypeptides, at least one of which can bind to lipid-containing fractions of cell membranes of fungi (e.g., Botrytis cinerea or other fungi). The lipid-containing fractions may be obtained by chromatography. For example, the lipid-containing fractions may be obtained by a method comprising the steps of fractionating the hyphae 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 the ceramide fraction and less than that of the nonpolar phospholipid fraction.

[0020] Furthermore, the present invention provides a composition containing at least one polypeptide, wherein the polypeptide comprises an amino acid sequence having at least about 80% sequence identity to any one of the amino acid sequences described in SEQ ID NOs: 1-51 and 101-111, or any one thereof, and the polypeptide is capable of binding to fungi.

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

[0022] The present invention further, A polypeptide comprising an amino acid sequence described in any one of Sequence IDs 1-51 and 101-111, or an amino acid sequence having at least approximately 80% sequence identity with either of them, and capable of binding to fungi; A polypeptide comprising a CDR1 region having the amino acid sequence described in SEQ ID NO: 52, a CDR2 region having the amino acid sequence described in SEQ ID NO: 68, and a CDR3 region having the amino acid sequence described in SEQ ID NO: 84, and capable of binding to fungi; A polypeptide comprising a CDR1 region having the amino acid sequence described in SEQ ID NO: 53, a CDR2 region having the amino acid sequence described in SEQ ID NO: 69, and a CDR3 region having the amino acid sequence described in SEQ ID NO: 85, and capable of binding to fungi; A polypeptide comprising a CDR1 region having the amino acid sequence described in SEQ ID NO: 54, a CDR2 region having the amino acid sequence described in SEQ ID NO: 70, and a CDR3 region having the amino acid sequence described in SEQ ID NO: 86, and capable of binding to fungi; The present invention provides a polypeptide capable of binding to fungi, 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 having an amino acid sequence selected from the group consisting of SEQ ID NOs. 84-100 and 134-144.

[0023] The present invention further provides a polypeptide comprising or composed 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 composed 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 a polypeptide having a CDR1 region containing or composed of a sequence selected from the group consisting of SEQ ID NOs. 52-67 and 112-122, a CDR2 region containing or composed of a sequence selected from the group consisting of SEQ ID NOs. 68-83 and 123-133, and a CDR3 region containing or composed of a sequence selected from the group consisting of SEQ ID NOs. 84-100 and 134-144.

[0026] The present invention further provides 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 up to one, two, three, four, or five amino acid substitutions in the said sequence, or comprising such a sequence. The amino acid substitutions may increase the total charge of the polypeptide, or they may not change the total charge of the polypeptide.

[0027] The present invention further provides a polypeptide comprising or composed 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 one, two, three, four, or five amino acid substitutions in the said sequence. The amino acid substitutions may increase the total charge of the polypeptide, or they may not change the total charge of the polypeptide.

[0028] All polypeptides of the present invention can be provided as compositions, for example, as agricultural chemical compositions.

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

[0030] The composition disclosed herein is a heavy chain antibody that does not naturally have a light chain, and contains at least one heavy chain variable domain (V HH ) or a functional fragment thereof may, but is not limited to, a camel heavy chain antibody heavy chain variable domain (Camel V HH ) or a functional fragment thereof are examples.

[0031] The composition disclosed herein is a camelized heavy chain variable domain (camelized V) of at least one conventional quadruple-chain antibody. H ), or a functional fragment thereof may be included.

[0032] The composition disclosed herein is natural V H The amino acid sequence of the domain (for example, natural V derived from mammals, especially humans) H It may contain at least one heavy chain variable domain or functional fragment thereof of an antibody that does not have an amino acid sequence that is strictly identical (i.e., has 100% sequence identity) to the amino acid sequence of the domain.

[0033] The compositions disclosed herein may be agricultural chemical compositions.

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

[0035] The at least one fungal cell membrane component to which the polypeptide contained in the composition disclosed herein is bound may be something other than a protein.

[0036] The at least one polypeptide in the pesticide composition disclosed herein may be present in an amount effective to protect or treat a human, animal, plant, or any part thereof from infection by a fungal pathogen or other biological interaction with a fungal pathogen, and is not limited to, for example, the concentration of the polypeptide in the pesticide composition being 0.0001 to 50% by weight.

[0037] The at least one polypeptide in the pesticide composition disclosed herein can be formulated in aqueous solution with an agriculturally appropriate base and / or one or more appropriate adjuvants, but is not limited to those disclosed herein.

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

[0039] The pesticide compositions disclosed herein are not limited to those containing Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Diplodia, Erysiphe, Fusarium, Leptosphaeria, Gaeumanomyces, and Helmintspor. Helminthosporium, Macrophomina, Nectria, Penicillium, Peronospora, Phoma, Phymatotrichum, Phytophthora, Plasmopara, Podosphaera, Puccinia, Pyrenophora renophora), Pyricularia, Pythium, Rhizoctonia, Scerotium, Sclerotinia, Septoria, Thielaviopsis, Uncinula, Venturia, Verticillium, Magnaporthe, It may contain at least one polypeptide that specifically binds to plant pathogenic fungi, such as plant pathogenic fungi of genera selected from the group including Blumeria, Mycosphaerella, Ustilago, Melampsora, Phakospora, Monilinia, Mucor, Rhizopus, and Aspergillus.

[0040] The pesticide compositions disclosed herein may contain at least one polypeptide that specifically binds to fungi, which are fungi of plants selected from the group including cereals, sorghum, rice, sugar beets, fodder beets, fruit trees, nuts, Plantaginaceae or grapes, leguminous crops, oil crops, cucurbitaceae plants, fiber plants, fuel crops, vegetables, ornamental plants, shrubs, broad-leaved 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 is at least [ka] Alternatively, the polypeptide may contain a sequence having at least about 80% sequence identity to either of the above, and the polypeptide is capable of binding to fungi.

[0042] The at least one polypeptide in the pesticide composition disclosed herein may include the amino acid sequences of at least 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 fungi.

[0043] The at least one polypeptide in the pesticide composition disclosed herein may include the amino acid sequences of at least 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 fungi.

[0044] The at least one polypeptide in the pesticide composition disclosed herein may include the amino acid sequences of at least 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 fungi.

[0045] The at least one polypeptide in the pesticide composition disclosed herein may include the CDR1 region, CDR2 region, and CDR3 region of at least one 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 a composition containing at least one polypeptide that specifically binds to fungi for use as an antifungal agent. The present invention further provides polypeptides that specifically bind to fungi for use as an antifungal agent.

[0048] Therefore, the present invention provides a composition containing at least one polypeptide, wherein the polypeptide is The polypeptide comprises 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 with any of them, and the polypeptide is capable of binding to fungi for use as an antifungal agent; or, The polypeptide comprises a CDR1 region containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 52-67 and 112-122, a CDR2 region containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 68-83 and 123-133, and a CDR3 region containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 84-100 and 134-144, wherein the polypeptide is capable of binding to fungi. The present invention further provides the 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 the use of a pesticide 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 fungiostatic agent and / or a fungicide.

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

[0051] Furthermore, the present invention provides a method for protecting or treating a plant or part of a plant from infection by plant pathogenic fungi, comprising at least the step of directly or indirectly applying the pesticide composition or polypeptide disclosed herein to the plant or part of the plant. The pesticide composition or polypeptide can be applied under conditions that are effective in protecting or treating the plant or part of the plant from infection by plant pathogenic fungi.

[0052] These methods may include, but are not limited to, a step of directly or indirectly applying the pesticide composition or polypeptide disclosed herein to the plant or a portion of the plant at an application rate of, for example, more than 50 g of the pesticide composition or polypeptide per hectare, or more than 75 g of the pesticide composition or polypeptide per hectare, for example, more than 100 g of the pesticide composition or polypeptide per hectare, or in particular more than 200 g of the pesticide composition or polypeptide per hectare.

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

[0054] The pesticide compositions or polypeptides disclosed herein may be optionally applied directly or indirectly to the plants or parts thereof after harvest by spraying, atomizing, foaming, fogging, hydroculture, hydroponics, coating, immersion, and / or powder coating.

[0055] The present invention also provides a post-harvest treatment method for protecting or treating harvested plants or harvested parts of said plants from infection by plant pathogenic fungi, comprising at least the step of directly or indirectly applying the pesticide composition or polypeptide disclosed herein to the harvested plants or harvested parts of said plants under conditions effective for protecting or treating the harvested plants or harvested parts of said plants from infection by plant pathogenic fungi.

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

[0057] These methods allow for the direct or indirect application of the pesticide compositions or polypeptides disclosed herein to the plants or parts thereof, either by spraying, atomizing, foaming, fogging, hydroculture, hydroponics, coating, immersion, and / or powder coating, after selective harvesting.

[0058] In yet another embodiment, the present invention provides a method for producing polypeptides that specifically bind to and / or have affinity for fungi, the method being The process of immunizing an animal with a fungal target, or a suitable antigenic determinant based on or derived from a fungus (e.g., its antigenic portion, fragment, region, domain, loop, or other epitope); A step of obtaining a cell collection or sample expressing a polypeptide sequence from the immunized animal; A step of screening the cell collection or sample for cells that express amino acid sequences that bind to and / or have affinity for the fungal target; (i) a step of isolating the amino acid sequence, or (ii) a step of isolating a nucleic acid sequence encoding the amino acid sequence from the cell collection or sample; The process includes expressing the aforementioned amino acid sequence, This allows for the creation of polypeptides that specifically bind to and / or have affinity for fungi.

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

[0060] The fungal target can be the lipid-containing fraction of the cell membrane of a fungus (e.g., Botrytis cinerea). The lipid-containing fraction may be obtained by chromatography. For example, the lipid-containing fraction may be obtained by a method comprising the steps of fractionating the hyphae of a fungus (e.g., Botrytis cinerea or another fungus) by total lipid extract thin-layer chromatography, and selecting a fraction whose retention factor (Rf) is greater than that of the ceramide fraction and less than that of the nonpolar phospholipid fraction.

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

[0062] The present invention also provides transgenic plants, plant parts, seeds, or plant cells containing nucleic acid sequences encoding the polypeptide described in this application. [Brief explanation of the drawing]

[0063] [Figure 1] The results of monitoring fungal growth using IncuCyte in the presence of VHH10G11Q at gradually increasing doses are shown. [Figure 2] The results of monitoring fungal growth using IncuCyte in the presence of VHH10G11Q and VHH41D01 at gradually increasing doses are shown. [Figure 3] This study demonstrates the antifungal activity of 10G11 with a single amino acid substitution in the CDR region (SEQ ID NOs: 17-50). A comparison with the activity of 10G11 is also shown. [Figure 4] This paper shows the antifungal activity of his-tagged mutants of 10G11Q-His (SEQ ID NOs. 6 and 17-50) and compares it to the activity of 10G11Q. [Figure 5] The model protein structure of the 10G11 molecule is shown along with the representation of the three CDR regions. Selected amino acid residues are shown as a ribbon diagram. [Figure 6]This shows the antifungal activity of the 10G11 charged mutant. A comparison with the 10G11 activity is also shown. [Figure 7] The model protein structure of 10G11 charge mutation mutant 9 (SEQ ID NO: 14) is shown along with the indication of three CDR regions. Selected amino acid residues are shown as a ribbon diagram. [Figure 8] The model protein structure of the 10G11 charge mutation mutant 11 (SEQ ID NO: 16) is shown along with the indication of the three CDR regions. Selected amino acid residues are shown as a ribbon diagram. [Figure 9] The results of the extended antifungal assay are shown, demonstrating the effects of mutants 3, 9, and 11 and the 10G11 molecule. [Figure 10] This shows the thin-layer chromatographic separation of four different fractions present in the total lipid extract. [Figure 11] The results of binding 10G11 to liposome vesicles of various compositions containing the fluorescent molecule DPD are shown. [Figure 12] The binding profile of 10G11 to fraction 3 was determined by biolayer interferometry (BLI) and is shown in comparison to the reference VHH. [Figure 13] The results of detecting the binding of 10E11Q-His (sequence number 86), 12C03Q-His (sequence number 87), and 10G11Q-His (sequence number 88) to fraction 3 by ELISA are shown. [Figure 14] Microscopic images of Botrytis cinerea before and after treatment with 10G11 are shown. A magnified view of the treated Botrytis cinerea is shown on the far right. [Figure 15] This shows the change in %PESSEV of Asian soybean rust in the lower part of the grass canopy after application of various compounds. [Figure 16] This shows the change in %PESSEV of Asian soybean rust in the middle part of the grass canopy after application of various compounds. [Figure 17] This shows the change in %PESSEV of Asian soybean rust in the upper part of the grass canopy after application of various compounds. [Figure 18]This shows the changes in the severity of anthracnose disease in pumpkins after the application of various compounds. [Figure 19] This shows the changes in the severity of gray mold (Botrytis cinerea) on grape clusters after application of various compounds. [Figure 20] This shows the changes in the severity of powdery mildew on grape leaves after applying various compounds. [Figure 21] This shows the changes in the severity of powdery mildew on grape clusters after the application of various compounds. [Figure 22] This shows the changes in the severity of powdery mildew in tomatoes after applying various compounds. [Figure 23] This shows the change in the incidence of powdery mildew in tomatoes after applying various compounds. [Figure 24] This shows the changes in the severity of powdery mildew in strawberries after applying various compounds. [Figure 25] This shows the change in the incidence of powdery mildew in strawberries after the application of various compounds. [Figure 26] This shows the number of strawberry fruits infected with gray mold at harvest time after application of various compounds. [Figure 27] This shows the changes in the severity of gray mold disease in strawberry fruits after harvest, following the application of various compounds. [Figure 28] This shows the changes in the severity of powdery mildew in strawberries after applying various compounds. [Figure 29] The ELISA absorption plot used to determine Kd is shown. [Figure 30-1] The sequences of several examples of polypeptides of the present invention are shown, along with the sequence numbers of the full-length sequences and the positions of the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 regions. In the event of any discrepancy between the sequences in this figure and those in the sequence listing, the sequences in this figure shall prevail. [Figure 30-2] The sequences of several examples of polypeptides of the present invention are shown, along with the sequence numbers of the full-length sequences and the positions of the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 regions. In the event of any discrepancy between the sequences in this figure and those in the sequence listing, the sequences in this figure shall prevail. [Figure 30-3] The sequences of several examples of polypeptides of the present invention are shown, along with the sequence numbers of the full-length sequences and the positions of the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 regions. In the event of any discrepancy between the sequences in this figure and those in the sequence listing, the sequences in this figure shall prevail. [Figure 30-4] The sequences of several examples of polypeptides of the present invention are shown, along with the sequence numbers of the full-length sequences and the positions of the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 regions. In the event of any discrepancy between the sequences in this figure and those in the sequence listing, the sequences in this figure shall prevail. [Figure 30-5] The sequences of several examples of polypeptides of the present invention are shown, along with the sequence numbers of the full-length sequences and the positions of the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 regions. In the event of any discrepancy between the sequences in this figure and those in the sequence listing, the sequences in this figure shall prevail. [Figure 31] This paper shows the antifungal activity of 10G11 in which the entire CDR2 region is replaced with germline sequences, and 10G11 in which the entire CDR3 region is replaced with reference VHH CDR3. It also shows the results of a comparison with 10G11 activity.

[0064] Explanation of the sequence list The sequence listing shows at least the following sequences from Table 9.

[0065] [Table 1] TIFF0007844344000003.tif100166 [Modes for carrying out the invention]

[0066] Detailed description of the invention When prior art is mentioned in this specification, it should not be considered, nor should it be considered, as an indication that such prior art constitutes common knowledge in any country.

[0067] All references 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, are to be understood as they are to a person of ordinary skill in the art to which the present invention pertains.

[0068] The present invention is described below in terms of specific embodiments, but the present invention is not limited to these embodiments and is limited only to the claims. When reference numerals are included in the claims, they should not be construed as limiting the scope of the claims.

[0069] definition When the term “including” is used in this specification and in the claims, it does not exclude any other elements or processes.

[0070] When referring to a singular noun, for example, using an indefinite or definite article such as "a," "an," or "the," unless otherwise specified, it includes the plural form of that noun.

[0071] When referring to measurable values ​​such as parameters, quantities, and times, the term "approximately" as used in this application means that the variation includes ±10% or less of the specified value, preferably ±5% or less, more preferably ±1% or less, and even more preferably ±0.1% or less, provided that such variation is appropriate in the invention disclosed herein. Naturally, the numerical values ​​themselves that are "approximately" included are also disclosed as specifically preferred numerical values.

[0072] The following terms and definitions are provided solely to aid in understanding the present invention. Unless otherwise defined herein, all terms used herein have the same meaning as those used by those skilled in the art. With regard to definitions and terms in the art, the practitioner particularly refers to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 ndSee ed., Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999). The definitions used herein should not be construed as being narrower than what would be understood by a person of ordinary skill in the art.

[0073] Unless otherwise specified, all methods, processes, techniques, and operations not described in detail can and have been carried out in methods known to those skilled in the art, and are self-evident to those skilled in the art. For further information, please refer to, for example, the standard handbook, the general background art described above, and other references cited herein.

[0074] As used in this application, the terms “polypeptide,” “protein,” “peptide,” and “amino acid sequence” are used synonymously and refer to polymer-type amino acids of any length. These can include coded amino acids, non-coding amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified peptide backbone.

[0075] The amino acid residues used in this application may be represented by their full letter notation, or they may be represented according to the standard three-letter or one-letter amino acid code.

[0076] As used in this application, the terms “nucleic acid sequence,” “polynucleotide,” “polynucleic acid,” and “nucleic acid” are used synonymously and refer to nucleotides of any length in polymer form that are deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, regulatory regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecules may be linear or cyclic.

[0077] As used in this application, the term “homology” means at least secondary structural similarity between two polymers, particularly two polypeptides or polynucleotides, derived from the same or different taxones, where such similarity stems from a common ancestor. Therefore, the term “homology” means polymers of such a relationship having the aforementioned secondary structural similarity and optionally tertiary structural similarity. To compare two or more nucleotide sequences, the “percentage of sequence identity” between a first and second nucleotide sequence can be calculated using methods known to those skilled in the art, such as dividing the number of identical nucleotides in the first nucleotide sequence to the nucleotide at the corresponding position in the second nucleotide sequence by the total number of nucleotides in the first nucleotide sequence and multiplying by 100%, or 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 may consider so-called “conservative” amino acid substitutions, which can generally be described as amino acid substitutions that, when an amino acid residue is replaced with another amino acid residue of similar chemical structure, have little or no effect on the function, activity, or other biological properties of the polypeptide. Possible conserved amino acid substitutions are obvious to those skilled in the art. Amino acid sequences and nucleic acid sequences are said to be "strictly identical" if they have 100% sequence identity over their entire length.

[0078] As used herein in the context of antibodies, the term "complementary determining region" or "CDR" means the variable region of either the H (heavy) chain or the L (light) chain (abbreviated as VH and VL, respectively), and includes an amino acid sequence capable of specifically binding to an antigen target. These CDR regions determine the base specificity of the antibody for a particular antigen determinant structure. Such regions are also referred to as "hypervariable regions". CDRs correspond to discontinuous amino acid stretches within the variable region, and regardless of the molecular species, the positions of these important amino acid sequences within the heavy-chain variable region and the light-chain variable region have been found to have similar positions within the amino acid sequence of the variable region chain. The heavy-chain and light-chain variable regions of all standard antibodies each have three CDR regions (referred to as L1, L2, L3, H1, H2, H3, respectively) that are discontinuous with each other in their respective light (L) and heavy (H) chains.

[0079] As used herein, the term "affinity" means the degree to which a polypeptide, particularly an immunoglobulin such as an antibody or an immunoglobulin fragment such as VHH, binds to an antigen so as to shift the equilibrium of the antigen and the polypeptide towards the side of the existence of the complex formed by their binding. Thus, for example, when an antigen and an antibody (fragment) are combined at relatively equal concentrations, a high-affinity antibody (fragment) will bind to the available antigen so as to shift the equilibrium towards the high-concentration side of the resulting complex. The dissociation constant is widely used to represent the affinity between a protein-binding domain and an antigen target. Generally, the dissociation constant is less than 10 -5 M. The dissociation constant is preferably less than 10 -6 M, more preferably less than 10 -7 M. Most preferably, the dissociation constant is less than 10 -8 M.

[0080] As used in this application, the terms "specifically bind" and "specific binding" generally refer to the ability of a polypeptide, in particular an immunoglobulin such as an antibody or an immunoglobulin fragment such as VHH, to preferentially bind to a specific antigen present in a homogeneous mixture of various antigens. In certain embodiments, the specific binding interaction will distinguish between desirable and undesirable antigens in a sample, and in certain embodiments, the distinction will be by a difference of about 10 to over 100 times or more (e.g., about 1,000 times or over 10,000 times).

[0081] Accordingly, the amino acid sequences disclosed herein are said to "specifically bind" to a target if the amino acid sequences have affinity for, specific to, and / or specific association with a particular target (or at least one part or fragment thereof).

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

[0083] The amino acid sequences disclosed herein are said to be "specific to the first target antigen compared to the second target antigen" when they bind to the first target antigen with an affinity at least five times, for example, at least ten times, for example, at least 100 times, preferably at least 1000 times, the affinity with which the amino acid sequences bind to the second target antigen. Therefore, in a given embodiment, when the amino acid sequences disclosed herein are said to be "specific to" the first target antigen compared to the second target antigen, the amino acid sequences can bind specifically to the first target antigen (as defined herein) but cannot bind to the second target antigen.

[0084] As used in this Application, the terms “inhibit,” “suppress,” and / or “interfere” mean the use of any amino acid sequence disclosed herein that specifically binds to the target antigen and inhibits, suppresses, and / or interferes with the interaction between the target antigen and its natural binding partner. The terms “inhibit,” “suppress,” and / or “interfere” may also mean the use of any amino acid sequence disclosed herein that specifically binds to the target antigen and inhibits, suppresses, and / or interferes with the biological activity of the target antigen when measured using an appropriate in vitro assay, cell assay, or in vivo assay. Therefore, “inhibit,” “suppress,” and / or “interfere” may also mean the use of any amino acid sequence disclosed herein that specifically binds to the target antigen and inhibits, suppresses, and / or interferes with one or more biological or physiological mechanisms, actions, responses, functions, pathways, or activities in which the target antigen is involved. Such antagonistic activity of the amino acid sequences disclosed herein can be measured, depending on the target antigen, using any suitable method and / or any suitable (in vitro, typically cellular, or in vivo) assay known in the art.

[0085] Therefore, more specifically, "inhibiting," "suppressing," and / or "interfering" with the amino acid sequences disclosed herein can mean inhibiting, suppressing, and / or interfering with the interaction of the target antigen and its natural binding partner, or inhibiting, suppressing, and / or interfering with the activity of the target antigen, or inhibiting, suppressing, and / or interfering with one or more biological or physiological mechanisms, actions, responses, functions, pathways, or activities involving the target antigen by, for example, at least 10%, preferably at least 20%, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% or more, compared to the activity of the target antigen measured using the same assay under the same conditions except that the amino acid sequences disclosed herein are not used. Furthermore, “inhibit,” “suppress,” and / or “interfere” may also mean inducing a decrease in the affinity, avidity, specificity, and / or selectivity of the target antigen to one or more of its natural binding partners, and / or inducing a decrease in the sensitivity of the target antigen to one or more conditions (e.g., pH, ionic strength, presence of cofactors, etc.) in the medium or environment in which the target antigen is present. In the context of the present invention, “inhibit,” “suppress,” and / or “interfere” may also mean allosteric inhibition, suppression, and / or interference of the activity of the target antigen.

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

[0087] The amino acid sequences disclosed herein are considered "essentially isolated" as used herein when they have been extracted or purified from the host cells and / or culture media from which the amino acid sequences are produced.

[0088] With respect to the amino acid sequences disclosed in this application, the terms “binding region,” “binding site,” or “interaction site” present on the amino acid sequences disclosed in this application mean a specific site, region, locus, part, or domain present on the target molecule, and such specific site, region, locus, part, or domain is involved in binding to the target molecule. Accordingly, such a binding region is essentially composed of the specific site, region, locus, part, or domain of the target molecule that is in contact with the amino acid sequence when bound to the target molecule.

[0089] As used in this application, "plant" means the whole plant or a part thereof, including fresh fruit trees, vegetables, and seeds. The plant or plant part may be a living plant or a part thereof. Furthermore, as used in this application, the term "plant" also includes the whole plant, the ancestors and descendants of the plant and plant part, and includes seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, each of which contains the relevant genes / nucleic acids. The term "plant" also includes plant cells, suspensions, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores, each of which also contains the relevant genes / nucleic acids.

[0090] The selection of an appropriate control plant is a routine part of experimental design and may include the corresponding wild-type plant or a corresponding plant that does not contain the gene in question. Generally, the control plant is the same species or variety as the plant to be evaluated. The control plant may also be a null zygote of the plant to be evaluated. A null zygote is an individual that loses the transgene upon isolation. As used in this application, "control plant" means not only the entire plant but also the plant part including the seeds and seed portion.

[0091] As used in this application, “crops” means plant species or varieties harvested for food, animal feed, fuel, or any other economic purpose. A non-limiting example of such crops includes: cereals such as maize, wheat, rye, barley, and oats, sorghum, rice, sugar beets, and fodder beets; pome fruits (e.g., apples and pears); citrus fruits (e.g., oranges, lemons, limes, grapefruit, or mandarin oranges); drupes (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 Plantaginaceae or grapes; legumes, leguminous crops such as lentils, peas, and soybeans; and oils such as sunflowers, safflower, rapeseed, canola, castor, or olives. These can include fermented crops such as cucurbitaceous plants like cucumbers, melons, or pumpkins; fiber plants such as cotton, flax, or hemp; fuel crops such as sugarcane, miscanthus, or switchgrass; vegetables such as potatoes, tomatoes, peppers, lettuce, spinach, onions, carrots, eggplants, asparagus, or cabbage; ornamental plants such as flowers (e.g., petunias, geraniums, roses, tulips, lilies, or chrysanthemums), shrubs, broad-leaved trees (e.g., poplars or willows), and evergreen trees (e.g., conifers); grasses such as lawngrass, turfgrass, or fodder grass; or other useful plants such as coffee, tea, tobacco, hops, pepper, rubber, or latex plants.

[0092] As used in this application, "harmful organisms" refers to organisms that are harmful to plants, animals, humans, or those related to humans, and is not limited to such organisms, but includes crop pests (as defined below), household pests such as cockroaches and ants, and disease-carrying organisms such as malaria mosquitoes.

[0093] In this application, the terms "plant pest," "plant pathogen," and "crop pest" are used synonymously and specifically refer to organisms that cause damage to plants, plant parts, or plant products, particularly plants, plant parts, or plant products used in agriculture. The terms "plant pest" and "crop pest" are used to mean that the aforementioned pests target and harm plants. Pests are particularly invertebrates, including, for example, insects (including agricultural pests, ornamental plant pests, and forest pests). Examples of crop pests that fall under this category include, but are not limited to, aphids, caterpillars, flies, wasps, nematodes (those that live freely in the soil, or especially cyst nematodes that parasitize plant roots, such as root-knot nematodes, soybean cyst nematodes, and potato cyst nematodes), mites (e.g., spider mites, dust mites, and gall mites), and gastropods (slugs such as species of the genera Deloceras, Milax, Tandonia, Limax, Arion, and Veronicella, as well as species of the genera Helix, Cernuella, and Theba). (including 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.)), pathogenic fungi (Ascomycetes (e.g., Fusarium spp., Thielaviopsis spp., Verticillium spp., Magnaporthe spp.)), Basidiomycetes (e.g., Rhizoctonia spp.))Examples include species of the genera Phakospora, Puccinia, and fungal-like oomycetes (e.g., Pythium and Phytophthora), bacteria (e.g., Burkholderia, Xanthomonas, and Pseudomonas), phytoplasmas, spiroplasmas, viruses (e.g., tobacco mosaic virus and cauliflower mosaic virus), and protozoa.

[0094] As used in this application, "microorganism" refers to bacteria, viruses, fungi, yeast, etc., and "microbial" means originating from microorganisms.

[0095] As used in this application, "fungus" refers to eukaryotes belonging to the phylum Eumycota. The term "fungus" in this invention also includes fungal-like organisms such as those belonging to the phylum Oomycota. The phylum Oomycota (or class Oomycetes) forms a separate phylogenetic lineage of fungal-like eukaryotic microorganisms. This group was initially classified as fungi, but recent findings have shown that it is relatively closely related to photosynthetic organisms such as brown algae and diatoms within heterokonts.

[0096] As used in this application, "harmful organism infection" or "harmful organism disease" means any inflammatory condition, disease, or disorder caused by harmful organisms in living organisms such as plants, animals, or humans.

[0097] As used in this application, "fungal infection" or "fungal disease" means any inflammatory condition, disease, or disorder caused by fungi in living organisms such as plants, animals, or humans.

[0098] In this application, "active substance," "active component," or "active main component" are used synonymously and mean any biological, biochemical, or chemical component and its derivatives, fragments, or compounds based thereon, including microorganisms, that have a general or specific effect on harmful organisms in a subject, particularly plants, plant parts, or plant products, and that may be naturally occurring or obtained by manufacture, and may inevitably contain impurities resulting from the manufacturing process.

[0099] As used in this application, "agricultural chemicals" means not only suitable for use in the agricultural chemical industry (including agriculture, horticulture, floriculture, and home and garden use), but also suitable for use in public health / pest control applications for controlling undesirable insects and rodents, household applications such as household fungicides and insecticides, and non-crop-related products such as agents for protecting plants or plant parts, crops, bulbs, tubers, and fruit trees (e.g., from pests, diseases, or insects), controlling plant growth, preferably promoting or enhancing it, and / or improving the yield of plants, crops, or plant parts (e.g., their fruits, flowers, seeds, etc.). Examples of such substances are obvious to those skilled in the art and include, for example, insecticides (e.g., contact insecticides or systemic insecticides, including household insecticides), herbicides (e.g., contact herbicides or systemic herbicides, including household herbicides), fungicides (e.g., contact fungicides or systemic fungicides, including household fungicides), nematicides (e.g., contact nematicides or systemic nematicides, including household nematicides) and other pest control agents or biocides (e.g., agents that kill insects or snails); as well as fertilizers; plant hormones and the like. Examples include growth regulators; micronutrients, phytotoxicity reducers, pheromones; repellents; insect baits; and / or active main components used to regulate (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-stranded or double-stranded RNA, as used in connection with RNAi technology) and factors, proteins, chemicals, etc., known for this purpose.Examples of such pesticides are obvious to those skilled in the art and are not limited to, but include, for example, glyphosate, paraquat, metrachlor, acetochlor, mesotrione, 2,4-D, atrazine, glufosinate, sulfocate, phenoxaprop, bendimethalin, picloram, trifluralin, bromoxynil, clodinahop, fluroxypyr, nicosulfuron, bensulfuron, imazetapyr, dicamba, imidacloprid, thiamethoxam, fipronil, chlorpyrifos, deltamethrin, lambdacihalothrin, endosulfan, and methamidophosph Examples include carbofuran, clothianidin, cypermethrin, abamectin, diflufenican, spinosad, indoxacarb, bifenthrin, tefluthrin, azoxystrobin, thiamethoxam, tebuconazole, mancozep, cyazofamide, fluazinam, pyraclostrobin, epoxyconazole, chlorothalonil, copper fungicides, trifloxystrobin, prothioconazole, difenoconazole, carbendazim, propiconazole, thiophanate, sulfur, boscalid, and other known pesticides or any suitable combination thereof.

[0100] As used in this Application, “agricultural chemical composition” means a composition for agricultural chemistry as defined in detail in this Application, which contains at least one active substance and optionally one or more additives that promote the optimal dispersion, atomization, adhesion, leaf wetting, distribution, retention and / or uptake of the pesticide. As will be apparent from the detailed description in this Application, the agricultural chemical compositions as used in this Application include biological control agents or biological pest control agents (including, but not limited to, biological biocides, biostatic agents, fungiostatic agents and fungicides), and these terms are used synonymously in this Application. Accordingly, the agricultural chemical compositions as used in this Application include compositions containing at least one biomolecule as an active ingredient, substance or main component for controlling pests in plants or other agricultural environments (e.g., in soil). Non-limiting examples of biomolecules used as active main components in the pesticide compositions disclosed herein include proteins (including, but not limited to, antibodies and their fragments, but including heavy chain variable domain fragments of antibodies such as VHH), nucleic acid sequences, (poly)saccharides, lipids, vitamins, hormonal glycolipids, sterols, and glycerolipids.

[0101] As a 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, binders, thickeners, penetrating agents, buffering agents, acidifying agents, anti-settling agents, antifreeze agents, light-protective agents, defoaming agents, biocides, and / or anti-drift agents.

[0102] As used in this application, “biostatic composition” or “biostatic agent” means any active ingredient, substance or main component for biostatic purposes (as defined in detail herein), or a composition containing any active ingredient, substance or main component, which comprises at least one biostatic active substance or component and optionally one or more additives that promote the optimal dispersion, atomization, adhesion, leaf wetting, distribution, retention and / or uptake of the said active substance or component. Non-limiting examples of such additives include diluents, solvents, adjuvants, (ionic) surfactants, wetting agents, spreading agents, oils, fixatives, thickeners, penetrating agents, buffers, acidifying agents, anti-sedimentation agents, anti-freezing agents, photoprotective agents, defoaming agents, biocides, protease inhibitors and / or anti-scatter agents.

[0103] As used in this Application, “biocidal composition” or “biocidal agent” means any active ingredient, substance or main component for biocidal purposes (as defined in detail in this Application), or a composition containing any active ingredient, substance or main component, which comprises at least one biocidal substance or component and optionally one or more additives that promote the optimal dispersion, atomization, adhesion, leaf wetting, distribution, retention and / or uptake of the active substance or component. Non-limiting examples of such additives include diluents, solvents, adjuvants, (ionic) surfactants, wetting agents, spreading agents, oils, fixatives, thickeners, penetrating agents, buffers, acidifying agents, anti-settling agents, anti-freezing agents, photoprotective agents, defoaming agents, biocidal agents, protease inhibitors and / or anti-scattering agents.

[0104] As used in this application, “fungiostatic composition” or “fungiostatic agent” means any active ingredient, substance or main component for fungi (as defined in detail herein), or a composition containing any active ingredient, substance or main component, which comprises at least one fungiostatic active substance or component and optionally one or more additives that promote the optimal dispersion, atomization, adhesion, leaf wetting, distribution, retention and / or uptake of the said active substance or component. Non-limiting examples of such additives include diluents, solvents, adjuvants, (ionic) surfactants, wetting agents, spreading agents, oils, fixatives, thickeners, penetrating agents, buffers, acidifying agents, anti-settling agents, anti-freezing agents, photoprotective agents, defoaming agents, biocides, protease inhibitors and / or anti-scattering agents.

[0105] As used in this application, “fungicidal composition” or “fungicidal agent” means any active ingredient, substance or main component for fungicidal purposes (as defined in detail in this application), or a composition containing any active ingredient, substance or main component, which comprises at least one fungicidal active substance or component and optionally one or more additives that promote the optimal dispersion, atomization, adhesion, leaf wetting, distribution, retention and / or uptake of the said active substance or component. Non-limiting examples of such additives include diluents, solvents, adjuvants, (ionic) surfactants, wetting agents, spreading agents, oils, fixatives, thickeners, penetrating agents, buffers, acidifying agents, anti-settling agents, anti-freezing agents, photoprotective agents, defoaming agents, biocides, protease inhibitors and / or anti-scattering agents.

[0106] As used in this application, "for agricultural use" includes not only the use of the above-defined pesticides (e.g., pest control agents, 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 the above-defined pesticides (e.g., pest control agents, growth regulators, nutrients / fertilizers, repellents, defoliants, etc.) for greenhouse-grown crops (e.g., horticulture / floriculture) or hydroponic systems, as well as the use of the above-defined pesticides suitable for and / or intended for non-crop uses such as for personal gardens, household use (e.g., household herbicides or insecticides), or for pest control businesses (e.g., weed control, etc.).

[0107] As used in this application, "biostatic (action)" or "for biostatic purposes" includes any action or use of an active substance (optionally included in a biostatic, biocidal, fungicidal, or fungistatic composition as defined herein) for the purpose of controlling, regulating, or interfering with the harmful activity of plant pests, plant pathogens, and other harmful organisms, for example, but not limited to, inhibiting the growth or activity of such pests in plants, plant parts, or other agricultural environments (e.g., in households or in soil), altering the behavior of such pests, and repelling or attracting such pests.

[0108] As used in this application, "biocidal (action)" or "for biocidal purposes" includes any action or use of an active substance (optionally included in a biocidal or fungicidal composition as defined in this application) for the purpose of controlling, regulating, or interfering with the harmful activity of harmful organisms such as plant pests and plant pathogens, for example, but not limited to, inhibiting the growth or activity of said pests in plants, plant parts, or other agricultural environments (e.g., in households or in soil), altering the behavior of said pests, and repelling or attracting said pests.

[0109] As used in this application, "fungiostatic (action)" or "for fungiostatic purposes" includes any action or use of an active substance (optionally included in a fungicidal or fungiostatic composition as defined herein) for the purpose of controlling, regulating or interfering with the harmful activity of fungi, for example, but not limited to, inhibiting the growth or activity of said fungi in plants, plant parts or other agricultural environments (e.g., in households or in soil), altering the behavior of said fungi, and repelling or attracting said fungi.

[0110] As used in this application, "fungicidal (action)" or "for fungicidal purposes" includes any action or use of an active substance (optionally included in a fungicidal composition as defined herein) for the purpose of controlling, regulating or interfering with the harmful activity of fungi, for example, but not limited to, killing said fungi in plants, plant parts or other agricultural environments (e.g., household or soil), inhibiting said fungi's growth or activity, altering said fungi's behavior, and repelling or attracting said fungi.

[0111] In this application, "biocidal activity" or "biocidal activity" are used synonymously and mean inhibiting the harmful activity of a pest, and include, but are not limited to, killing the pest, inhibiting the growth or activity of the pest, altering the behavior of the pest, and repelling or attracting the pest.

[0112] As used in this application, "biostatic activity" means inhibiting the harmful activity of a pest, and is not limited to, inhibiting the growth or activity of the pest, altering the behavior of the pest, or repelling or attracting the pest.

[0113] The pest-killing, bio-killing, or biostatic activity of an active ingredient, substance, or main component, or of a composition or drug containing a pest-killing, bio-killing, or biostatic active ingredient, substance, or main component, may be expressed as the minimum inhibitory activity (MIC) of the drug (expressed in units of concentration such as mg / mL), but is not limited thereto.

[0114] As used in this application, "fungicidal activity" means inhibiting the harmful activity of fungi, and is not limited to, killing fungi, inhibiting the growth or activity of fungi, altering the behavior of fungi, and repelling or attracting fungi.

[0115] As used in this application, "fungal activity" means inhibiting the harmful activity of fungi, and is not limited to, inhibiting the growth or activity of the fungi, altering the behavior of the fungi, and repelling or attracting the fungi.

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

[0117] As used in this application, “base” means any solid, semi-solid, or liquid base on which an active substance can be appropriately formulated, added, immobilized, adsorbed, absorbed, bound, encapsulated, embedded, attached to, or contained. Non-limiting examples of such bases include nanocapsules, microcapsules, nanospheres, microspheres, nanoparticles, fine particles, liposomes, vesicles, beads, gels, weak ionic resin particles, liposomes, cochleate delivery carriers, small granules, granular bodies, 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, wood or other plant-derived materials (e.g., in the form of seed shells, wood chips, pulp, spheres, beads, sheets or any other suitable form), inorganic materials such as paper or cardboard, 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 in this application, the term "antibody" means polyclonal antibodies, monoclonal antibodies, humanized antibodies, single-chain antibodies and their fragments (e.g., Fab, F(ab)2, Fv, and other fragments that maintain the antigen-binding function of the parent antibody). Therefore, an antibody may also mean an immunoglobulin or glycoprotein or a fragment or portion thereof, or a construct containing an antigen-binding portion contained within a modified immunoglobulin-like framework, or an antigen-binding portion contained within a construct containing a non-immunoglobulin-like framework or scaffold.

[0119] As used in this application, the term "monoclonal antibody" refers to an antibody composition having a homogeneous antibody population. This term is not limited in terms of antibody species or origin, nor is it limited by the method of its preparation. This term includes full-length immunoglobulins and fragments such as Fab, F(ab)2, Fv, and other fragments that maintain the antigen-binding function of the antibody. In this invention, monoclonal antibodies from any mammalian species can be used. However, in practice, since rat or mouse cell lines are readily available for producing the hybrid cell lines or hybridomas necessary to produce monoclonal antibodies, the antibodies will generally be derived from rats or mice.

[0120] As used in this application, the term "polyclonal antibody" refers to an antibody composition having a heterogeneous population of antibodies. Polyclonal antibodies are often derived from a serum pool from immunized animals or selected humans.

[0121] As used in this application, "the heavy chain variable domain of an antibody or its functional fragment" refers to (i) the heavy chain variable domain of a heavy chain antibody that does not naturally have a light chain (hereinafter, V HH (ii) means, and is not limited to, the variable domain of the heavy chain of a camel or shark heavy chain antibody, or (ii) the variable domain of the heavy chain of a conventional quadruple-chain antibody (hereinafter, V H It also means, and is not limited to, the camel-modified variable domain (hereinafter referred to as camel-modified V) of the heavy chain of conventional quadruple-chain antibodies (as defined in detail in this application). H(Also known as)

[0122] As detailed below, the amino acid sequence and structure of the antibody's heavy chain variable domain can be considered, but are not limited, to consist of 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" positioned 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) can be in the range of 110 to 130, preferably 112 to 115, and most preferably 113. Furthermore, the length and / or size of a portion, fragment, or analog of the heavy chain variable domain of an antibody is not limited, as long as such portion, fragment, or analog maintains (at least a part of) functional activity such as (as defined in this application) pesticide activity, biocidal activity, biostatic activity, fungicidal activity, or fungiostatic activity, and / or maintains (at least a part of) the binding specificity of the original heavy chain variable domain of the antibody from which these portion, fragment, or analog originates. In this application, a portion, fragment, or analog that maintains (at least a part of) functional activity such as (as defined in this application) pesticide activity, biocidal activity, biostatic activity, fungicidal activity, or fungiostatic activity, and / or maintains (at least a part of) the binding specificity of the original heavy chain variable domain of the antibody from which these portion, fragment, or analog originate is also referred to as a "functional fragment" of the heavy chain variable domain.

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

[0125] Or, (V HH or V H The amino acid residues of the variable domain of the heavy chain variable domain of the antibody (including) are derived from camel V, as described in the aforementioned paper by Riechmann and Muyldermans (see, for example, Figure 2 in the above references). HH As applied to domains, they can be numbered according to the general numbering of heavy chain variable domains described by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, Md., Publication No. 91).

[0126] For a general description of heavy chain antibodies and their variable domains, see, in particular, the following references mentioned as general background technology: WO94 / 04678, WO95 / 04079 and WO96 / 34103 under the name of Vrije Universiteit Brussel; WO94 / 25591, WO99 / 37681, WO00 / 40968, WO00 / 43507, WO00 / 65057, WO01 / 40310, WO01 / 44301, EP1134231 and WO02 / 48193 under the name of Unilever; Vlaams Instituut voor WO97 / 49805, WO01 / 21817, WO03 / 035694, WO03 / 054016 and WO03 / 055527 under the name of Biotechnologie (VIB); WO03 / 050531 under the names of Algonomics NV and Ablynx NV; WO01 / 90190 under the name of National Research Council of Canada; WO03 / 025020 (=EP1433793) under the name of Institute of Antibodies; and WO04 / 041867, WO04 / 041862, WO04 / 041865, WO04 / 041863 and WO04 / 062551 under the name of 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 in the broadest sense in this application is not limited to a specific biological origin or a specific method of production. For example, as detailed below, the heavy chain variable domain of the present invention is (1) V of a natural heavy chain antibody HH (2) Isolate the domain; (3) V of the natural quadruple-chain antibody H (3) Isolate the domain; (3) Natural V HH Express the nucleotide sequence that codes for the domain; (4) Natural V H (5) Express a nucleotide sequence that codes for the domain; (6) Natural V derived from any animal species, especially mammalian species such as humans. H To "camelize" a domain (as described below), or to "camelize" a domain in this way V HExpressing nucleic acids that encode a domain; (6) Camelizing a "domain antibody" or "Dab" as described by Ward et al. (previously cited), or such camelized V H (7) Expressing nucleic acids that encode a domain; (8) Using synthetic or semi-synthetic techniques to produce proteins, polypeptides or other amino acid sequences; (9) Using nucleic acid synthesis techniques to V HH or V H After preparing a nucleic acid encoding the above, the nucleic acid thus obtained is expressed; and / or (9) it can be obtained by any combination of the above. Suitable methods and techniques for carrying out the above methods are obvious to those skilled in the art based on the disclosure of this application, and include, for example, the methods and techniques detailed below.

[0128] On the other hand, according to one specific embodiment, the heavy chain variable domain disclosed in this application is natural V H The amino acid sequence of the domain (for example, natural V derived from mammals, especially humans) H It does not have an amino acid sequence that is exactly the same as the amino acid sequence of the domain (i.e., has 100% sequence identity).

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

[0130] The terms "measure," "actually measure," "evaluate," "monitor," and "assay" used in this application are used synonymously and include both quantitative and qualitative measurements.

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

[0132] polypeptide The polypeptides disclosed herein are generally capable of binding to fungi. Therefore, the polypeptides cause delays in the growth of the fungal spores and / or lysis of the fungal spores. That is, the binding of the polypeptides to fungi results in delays in the growth of the fungal spores and / or lysis of the fungal spores.

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

[0134] The polypeptide can (specifically) bind to a lipid-containing fraction of a fungal cell membrane, such as a lipid-containing fraction of Fusarium oxysporum or another fungus. The lipid-containing fraction (of Botrytis cinerea or another fungus) may be obtained by chromatography. The chromatography can be performed on a crude lipid extract (also referred to here as a total lipid extract or TLE) obtained from fungal hyphae 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 the eluent.

[0135] For example, the lipid-containing fraction may be obtained by a method comprising the steps of fractionating the hyphae and / or conidia of a fungus (e.g., Botrytis cinerea or other fungi) by total lipid extract thin-layer chromatography, and selecting a fraction whose retention factor (Rf) is greater than that of the ceramide fraction and smaller than that of the nonpolar phospholipid fraction.

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

[0137] Alternatively, the fraction can be obtained using normal-phase flash chromatography. In such a method, the method may include the steps of fractionating the hyphae and / or conidia of a fungus (e.g., Botrytis cinerea or other fungi) by total lipid extract normal-phase flash chromatography, and selecting a fraction having a retention factor (Rf) greater than that of the ceramide fraction and less than that of the nonpolar phospholipid fraction.

[0138] In a more specific embodiment, the lipid-containing fraction may be obtained by a method comprising the steps of dissolving TLE in dichloromethane (CH2Cl2) and MeOH, fractionating the hyphae and / or conidia of a fungus (e.g., Botrytis cinerea or other fungi) by total lipid extract normal-phase flash chromatography using CH2Cl2 / MeOH (e.g., 85 / 15%, v / v) as the eluent, and then filtering the fraction through a filter.

[0139] In a more specific embodiment, the lipid-containing fraction may be obtained by a method comprising the steps of: dissolving the TLE in dichloromethane (CH2Cl2) and MeOH; loading the TLE into a normal-phase flash cartridge (e.g., a flash cartridge packed with 15 μm particles); and fractionating the hyphae and / or conidia of a fungus (e.g., Botrytis cinerea or other fungi) by total lipid extract normal-phase flash chromatography, wherein CH2Cl2 / MeOH (85 / 15%, v / v) is delivered to the column 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] The fraction from the chromatography can be processed before testing the binding of the polypeptide to the fraction or the interaction between the polypeptide and the fraction. For example, liposomes containing the fraction can be prepared. Such a method may include thin-film hydration. For example, in such a method, liposomes can be prepared using thin-film hydration with the addition of 1,6-diphenyl-1,3,5-hexatriene (DPH). The binding and / or disruption of the membrane due to polypeptide binding can be measured by the change in fluorescence before and after polypeptide binding (or by referring to an appropriate control).

[0141] Accordingly, in certain embodiments, the polypeptides of the present invention and the polypeptides used in the present invention can (specifically) bind to the lipid-containing chromatographic fraction of a fungal cell membrane, and optionally, the lipid-containing chromatographic fraction is prepared into liposomes before testing the binding of the polypeptides.

[0142] The binding of the polypeptide to the lipid-containing fraction of the fungus can be confirmed by any suitable method, such as biolayer interference. Specific interactions with the lipid-containing fraction can be tested. For example, when preparing the fraction into liposomes using a thin-film hydration method, it can be confirmed whether the polypeptide can disrupt the lipid fraction.

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

[0144] Accordingly, in certain embodiments, the polypeptide can (specifically) bind 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 fungal cell membrane can be obtained or obtained by chromatography. The chromatography can be normal-phase flash chromatography or thin-layer chromatography. The binding of the polypeptide to the lipid-containing fraction can be measured by biolayer interferometry. In certain embodiments, the chromatography step can be carried out with a crude lipid fraction obtained or obtainable by a method that includes extracting lipids from fungal hyphae and / or conidia derived from a fungal sample. The extraction step can be performed to obtain two extracts using chloroform:methanol in 2:1 and 1:2 (v / v) ratios, and these extracts can then be combined.

[0145] In a method related to thin-layer chromatography, the chromatography may include the steps of fractionating the fungal hyphae by total lipid extract thin-layer chromatography and selecting a fraction having a retention factor (Rf) greater than that of the ceramide fraction and less than that of the nonpolar phospholipid fraction.

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

[0147] In a method relating to normal-phase flash chromatography, the chromatography may include the steps of fractionating the hyphae and / or conidia of a fungus (e.g., Botrytis cinerea or other fungi) by total lipid extract normal-phase flash chromatography, and selecting a fraction having a retention factor (Rf) greater than that of the ceramide fraction and less than that of the nonpolar phospholipid fraction.

[0148] In certain methods relating to normal-phase flash chromatography, the chromatography may include the steps of: dissolving the TLE in dichloromethane (CH2Cl2) and MeOH, and fractionating the hyphae and / or conidia of a fungus (e.g., Botrytis cinerea or other fungi) by total lipid extract normal-phase flash chromatography using CH2Cl2 / MeOH (e.g., 85 / 15%, v / v) as the eluent; and then filtering the fraction.

[0149] In certain methods relating to normal-phase flash chromatography, the chromatography may include the steps of: dissolving the TLE in dichloromethane (CH2Cl2) and MeOH; loading the TLE into a normal-phase flash cartridge (e.g., a flash cartridge packed with 15 μm particles); and delivering CH2Cl2 / MeOH (85 / 15%, v / v) as the eluent to the column by total lipid extract normal-phase flash chromatography to fractionate the hyphae and / or conidia of a fungus (e.g., Botrytis cinerea or other fungi); 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 composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 51 and 101 to 111, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the said sequence.

[0151] In one embodiment, the present invention provides a polypeptide comprising or composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10, 12-51, 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 with the aforementioned sequence.

[0152] In one embodiment, the present invention provides a polypeptide comprising or composed 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 with the said sequence.

[0153] In one embodiment, the present invention provides a polypeptide comprising or composed of 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 with SEQ ID NO: 1.

[0154] In one embodiment, the present invention provides a polypeptide comprising or composed of sequence SEQ ID NO: 2, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the aforementioned sequence.

[0155] In one embodiment, the present invention provides a polypeptide comprising or composed of sequence sequence number 3, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the said sequence.

[0156] In one embodiment, the present invention provides a polypeptide comprising or composed of sequence sequence number 4, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the said sequence.

[0157] In one embodiment, the present invention provides a polypeptide comprising or composed of sequence sequence number 5, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the said sequence.

[0158] In one embodiment, the present invention provides a polypeptide comprising or composed of sequence sequence number 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 with the said sequence.

[0159] In another embodiment, the present invention is: A CDR1 region containing or composed of sequences selected from the group consisting of sequence numbers 52-67 and 112-122; A CDR2 region containing or composed of sequences selected from the group consisting of sequence numbers 68-83 and 123-133; A CDR3 region containing or composed of sequences selected from the group consisting of sequence numbers 84-100 and 134-144 Provides polypeptides containing the following:

[0160] In another embodiment, the present invention is: A CDR1 region comprising or consisting of sequences selected from the group comprising sequence numbers 52, 53, and 54; A CDR2 region containing or composed of sequences selected from the group consisting of sequence numbers 68, 69, and 70; A CDR3 region comprising or consisting of sequences selected from the group comprising sequences 84, 85, and 86 Provides polypeptides containing the following:

[0161] In another embodiment, the present invention is: A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 53, a CDR2 region containing or consisting of sequence number 69, and a CDR3 region containing or consisting of sequence number 85; A CDR1 region containing or consisting of sequence number 54, a CDR2 region containing or consisting of sequence number 70, and a CDR3 region containing or consisting of sequence number 86; A CDR1 region containing or consisting of sequence number 55, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 71, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 87; A CDR1 region containing or consisting of sequence number 55, a CDR2 region containing or consisting of sequence number 71, and a CDR3 region containing or consisting of sequence number 87; A CDR1 region containing or consisting of sequence number 56, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 88; A CDR1 region containing or consisting of sequence number 56, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 88; A CDR1 region containing or consisting of sequence number 57, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 52, and a CDR3 region containing or consisting of sequence number 89; A CDR1 region containing or consisting of sequence number 57, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 89; A CDR1 region containing or consisting of sequence number 58, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 59, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 60, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 61, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 62, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 63, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 64, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 65, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 66, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 67, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 72, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 73, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 74, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 75, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 76, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 77, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 78, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 79, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 80, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 81, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 82, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 83, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 84; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 90; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 91; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 92; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 93; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 94; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 95; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 96; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 97; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 98; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 99; A CDR1 region containing or consisting of sequence number 52, a CDR2 region containing or consisting of sequence number 68, and a CDR3 region containing or consisting of sequence number 100; A CDR1 region containing or consisting of sequence number 112, a CDR2 region containing or consisting of sequence number 123, and a CDR3 region containing or consisting of sequence number 134; A CDR1 region containing or consisting of sequence number 113, a CDR2 region containing or consisting of sequence number 124, and a CDR3 region containing or consisting of sequence number 135; A CDR1 region containing or consisting of sequence number 114, a CDR2 region containing or consisting of sequence number 125, and a CDR3 region containing or consisting of sequence number 136; A CDR1 region containing or consisting of sequence number 115, a CDR2 region containing or consisting of sequence number 126, and a CDR3 region containing or consisting of sequence number 137; A CDR1 region containing or consisting of sequence number 116, a CDR2 region containing or consisting of sequence number 127, and a CDR3 region containing or consisting of sequence number 138; A CDR1 region containing or consisting of sequence number 117, a CDR2 region containing or consisting of sequence number 128, and a CDR3 region containing or consisting of sequence number 139; A CDR1 region containing or consisting of sequence number 118, a CDR2 region containing or consisting of sequence number 129, and a CDR3 region containing or consisting of sequence number 140; A CDR1 region containing or consisting of sequence number 119, a CDR2 region containing or consisting of sequence number 130, and a CDR3 region containing or consisting of sequence number 141; A CDR1 region containing or consisting of sequence number 120, a CDR2 region containing or consisting of sequence number 131, and a CDR3 region containing or consisting of sequence number 142; A CDR1 region containing or consisting of sequence number 121, a CDR2 region containing or consisting of sequence number 132, and a CDR3 region containing or consisting of sequence number 143; or A CDR1 region containing or consisting of sequence number 122, a CDR2 region containing or consisting of sequence number 133, and a CDR3 region containing or consisting of sequence number 144. Provides polypeptides containing the following:

[0162] In certain embodiments, the CDR3 region is readily substituted without loss of activity (as demonstrated by replacing the CDR3 region with an unrelated CDR3, i.e., one that does not bind to fungi), so the polypeptide may contain only the specific CDR1 and CDR2 sequences of the polypeptide. Therefore, the CDR3 region sequence is arbitrary. Generally, the polypeptide may contain a CDR3 region (for example, to ensure structural integrity), but the sequence of the CDR3 region is not particularly important. For example, the polypeptide may contain a CDR1 region, a CDR2 region, and a CDR3 region, where the CDR1 and CDR2 regions each contain or consist of the sequences specified in this application, but the CDR3 region contains an arbitrary sequence (for example, any sequence having a length of 8 to 16 amino acids).

[0163] The polypeptide described in this application is preferably having a predetermined framework region sequence. For example, the polypeptide may include a framework region 1 (FR1) sequence that includes or is composed 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 that includes or is composed of a sequence selected from the group consisting of SEQ ID NOs: 151, 156, and 160; a framework region 3 (FR3) sequence that includes or is composed of a sequence selected from the group consisting of SEQ ID NOs: 152, 157, and 161; and a framework region 4 (FR4) sequence that includes or is composed of SEQ ID NO: 153.

[0164] In certain embodiments, for example, any polypeptide of a clone referred to in this application as 10G11 (including 10G11Q, any of mutants 1 to 11 or any of single ALA mutants 1 to 34, or any of mutants 10G11-A to 10G11K) or a polypeptide related to any polypeptide derived from the clone, and any polypeptide having any specific sequence identity or any substitution with respect to these polypeptides, may include a framework region 1 (FR1) sequence containing or composed of a sequence selected from the group consisting of SEQ ID NOs: 149 and 150, a framework region 2 (FR2) sequence containing or composed of SEQ ID NOs: 151, a framework region 3 (FR3) sequence containing or composed of SEQ ID NOs: 152, and a framework region 4 (FR4) sequence containing or composed of SEQ ID NOs: 153.

[0165] In certain embodiments, for example, any polypeptide of a clone referred to in this application as 10E11 (including 10E11Q) or a polypeptide related to any polypeptide derived from said clone, and any polypeptide having any specific sequence identity or any substitution with respect to these polypeptides, may include a framework region 1 (FR1) sequence containing or composed of sequences selected from the group consisting of SEQ ID NOs: 154 and 155, a framework region 2 (FR2) sequence containing or composed of SEQ ID NOs: 156, a framework region 3 (FR3) sequence containing or composed of SEQ ID NOs: 157, and a framework region 4 (FR4) sequence containing or composed of SEQ ID NOs: 153.

[0166] In certain embodiments, for example, any polypeptide of a clone referred to in this application as 12C03 (including 12C03Q) or a polypeptide related to any polypeptide derived from said clone, and any polypeptide having any specific sequence identity or any substitution with respect to these polypeptides, may include a framework region 1 (FR1) sequence containing or composed of sequences selected from the group consisting of SEQ ID NOs: 158 and 159, a framework region 2 (FR2) sequence containing or composed of SEQ ID NOs: 160, a framework region 3 (FR3) sequence containing or composed of SEQ ID NOs: 161, and a framework region 4 (FR4) sequence containing or composed of SEQ ID NOs: 153.

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

[0168] In certain embodiments, the polypeptide can have a length of 80 to 200 residues.

[0169] The polypeptide of the present invention can be provided as a composition (for example, a pesticide composition).

[0170] Composition containing at least one polypeptide In one embodiment, the present inventors have provided a pesticide composition comprising at least one polypeptide capable of specifically binding to a pest. Importantly, by thus interacting with the specific molecular structure of the pest, the composition disclosed herein is capable of inhibiting, interfering with, or suppressing one or more biological activities of the plant pathogen so as to inhibit, interfere with, or suppress the growth of the plant pathogen. In a given embodiment, the pesticide composition disclosed herein is capable of killing plant pests by specific interactions of at least one polypeptide contained in the composition that is capable of specifically binding to the pest.

[0171] Accordingly, the pesticide compositions disclosed herein can be used to modulate (e.g., reduce or inhibit) the biological functions of plant pests by binding to binding sites present on targets of plant pests and acting on one or more biological pathways in which the pests' natural biological activity (but not limited to, growth) and / or structural targets of the pests are involved.

[0172] Furthermore, compositions containing at least one polypeptide disclosed herein offer several additional advantages over conventional immunoglobulin and non-immunoglobulin binders known in the art. In fact, in certain embodiments, the amino acid sequences disclosed herein are isolated heavy-chain immunoglobulin variable domains, which are more potent and stable than conventional quadruple-chain antibodies, thus (1) reducing the dosage and frequency of administration, thereby reducing side effects, and (2) expanding the choice of administration routes due to improved stability. Because heavy-chain immunoglobulin variable domains are small in size, they can pass through membranes and enter physiological compartments, tissues, and organs that other larger polypeptides and proteins cannot access.

[0173] In one specific, non-limiting embodiment, the at least one polypeptide contained in the composition disclosed herein may be a polypeptide that includes an immunoglobulin fold or is capable of forming an immunoglobulin fold (by folding) under suitable conditions (e.g., physiological conditions). See, in particular, Halaby et al., J. (1999) Protein Eng. 12, 563-71. Such polypeptide sequences are preferably capable of specifically binding to a target or antigen (as defined herein) when properly folded to form an immunoglobulin fold, and having an affinity (as detailed herein) D Value (actual or apparent value), K A Value (actual or apparent value), k on Speed ​​and / or k off Speed ​​or IC 50It is more preferable that the polypeptide sequence can bind to a hazardous organism target or hazardous organism antigen (which is appropriate to measure and / or express as a value). Furthermore, it is preferable that such polypeptide sequences are selected to contain immunoglobulin folds or to form immunoglobulin folds under appropriate conditions.

[0174] In certain embodiments, the present invention provides a pesticide composition or biological pest control composition for controlling plant pests, more specifically plant fungi, wherein the composition contains at least one polypeptide or amino acid sequence of 80 to 200 amino acids as an active substance. In this application, "80 to 200 amino acids" means from 80 to 200, that is, including amounts of 80 amino acids and 200 amino acids. Therefore, "80 to 200 amino acids" can be used synonymously with "from 80 amino acids to 200 amino acids."

[0175] In another specified embodiment, the present invention provides a composition for controlling plant pests that contains at least two (different) polypeptides or at least two (different) amino acid sequences having 80 to 200 amino acids as an active substance.

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

[0177] The pesticide composition according to the present invention is a pesticide composition as defined in this application for controlling the plant pests defined above, that is, the pesticide composition, more specifically the active substance contained in the pesticide composition, can interfere with, preferably suppress or block, the harmful effects of one or more plant pests on one or more plants, preferably crops.

[0178] The polypeptides or amino acid sequences contained in the compositions disclosed herein may be natural polypeptides or amino acid sequences, may be derived from natural polypeptides, or may be entirely artificially designed or synthesized. The polypeptides or amino acid sequences may be immunoglobulin-based, or, but are not limited to, domains present in proteins such as microbial proteins, protease inhibitors, toxins, fibronectin, lipocalin, single-stranded antiparallel coiled-coil proteins, or repeat motif proteins. Non-limiting examples of such polypeptides within the amino acid length range described in this application include carbohydrate-binding domains (CBDs) (Blake et al (2006) J. Biol. Chem. 281, 29321-29329), heavy chain antibodies (hcAb), single-domain antibodies (sdAb), minibodies (Tramontano et al (1994) J. Mol. Recognition 7, 9-24), variable domains of camel heavy chain antibodies (VHH), variable domains of novel antigen receptors (VNAR), afibodies (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), and antikarin (Skerra et al. Examples include al (2008) FEBS J.275, 2677-2683), Nottin (Kolmar et al (2008) FEBS J.275, 2684-2690), and artificial CH2 domains (nanoantibodies, see Dimitrov DS (2009) mAbs 1, 26-28). In particular, the polypeptides or amino acid sequences disclosed herein consist of a single polypeptide chain and are not post-translationally modified. More specifically, the polypeptides or amino acid sequences disclosed herein are derived from the innate or adoptive immune system, preferably from proteins of the innate or adoptive immune system. Even more specifically, the polypeptides or amino acid sequences disclosed herein are derived from immunoglobulins.Most specifically, the polypeptide or amino acid sequence disclosed herein comprises four framework regions and three complementarity-determining regions, or any suitable fragment thereof (usually including at least a portion of the amino acid residues that form at least one of the complementarity-determining regions). In particular, the polypeptide or amino acid sequence disclosed herein is preferably readily produced in high yield in a microbial recombinant expression system and is convenient for subsequent isolation and / or purification. In particular, the polypeptide or amino acid sequence disclosed herein comprises DARPins, Nottin, Alpha Body and V. HH Selected from the group consisting of the following. More specifically, the polypeptide or amino acid sequence disclosed herein is an alpha body and V HH Selected from the group consisting of the following. Most specifically, the polypeptide or amino acid sequence disclosed herein is V HH That is the case.

[0179] In particular, the at least one polypeptide contained in the compositions disclosed herein may consist of a single polypeptide chain and may not be post-translationally modified. More specifically, the at least one polypeptide contained in the compositions disclosed herein may be derived from the innate immune system or the adoptive immune system, preferably from proteins of the innate or adoptive immune system. Even more specifically, the at least one polypeptide contained in the compositions disclosed herein may be derived from an immunoglobulin. Most specifically, the at least one polypeptide contained in the compositions disclosed herein may contain four framework regions and three complementarity-determining regions, or any suitable fragment thereof (which typically includes at least a portion of amino acid residues forming at least one of the complementarity-determining regions). In particular, the at least one polypeptide contained in the compositions disclosed herein is preferably readily produced in high yield in a microbial recombinant expression system and is convenient for subsequent isolation and / or purification.

[0180] According to certain embodiments, the present invention provides a number of amino acid residue stretches (i.e., low molecular weight peptides) particularly suitable for binding to harmful organism antigens or harmful organism targets (but not limited to, for example, fungal antigens or fungal targets).

[0181] These amino acid residue stretches may be present in and / or incorporated into the polypeptide, in particular, to form (part of) the antigen-binding site of the polypeptide disclosed herein. These amino acid residue stretches may be used to bind antibodies such as heavy chain antibodies produced against harmful biological targets or V H Or V HH Since these amino acid residue stretches were initially constructed as CDR sequences (or, as detailed in this application, may be based on and / or derived from such CDR sequences), these amino acid residue stretches are also generally referred to in this application 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 specific structural role or function that these amino acid residue stretches may have in the polypeptide disclosed in this application, as long as these amino acid residue stretches enable the polypeptide disclosed in this application to specifically bind to harmful organism targets such as fungal antigens or fungal targets. Therefore, the present invention, in its broadest sense, generally relates to a pesticide composition containing a polypeptide that can bind to harmful organism targets such as fungal antigens or fungal targets and includes a combination of CDR sequences as described in this application.

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

[0183] All polypeptides contained in the pesticide compositions disclosed herein, having one of these CDR sequence combinations, are selected to be able to specifically bind (as defined herein) to a pest target or pest antigen, more specifically, in particular in solution the polypeptide 10 -8 It is preferable that the compound can specifically bind to the plant pathogen target with a dissociation constant (Kd) of moles / liter or less.

[0184] The dissociation constant (Kd) can be estimated based on the ELISA results. In equilibrium analyses such as ELISA, Kd can be calculated from the equilibrium binding response. If the target antigen is coated into ELISA plate wells and the target antigen may be a lipid-containing fraction of the Botrytis cinerea membrane, such a method can further use polypeptides in a concentration range that binds to the target antigen. If ELISA generally provides quantitative adsorption measurements of each polypeptide concentration representing the binding of the polypeptide to the target antigen, the concentration range of the polypeptide can be, for example, 0.5 μM, 1 μM, 2.5 μM, 5 μM, and 10 μM. The optimal concentration range used may vary depending on the affinity of the polypeptide to the target antigen. Plotting the absorption values ​​against a logarithmic transformation of the polypeptide concentration yields a sigmoid curve from the corresponding absorption values ​​measured by ELISA. The IC50 value can be determined using this sigmoid curve. If IC50 is the polypeptide concentration and the corresponding absorption value is 50% of the saturation value estimated by the maximum value of the sigmoid function, then 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 that reaches equilibrium at 50% binding saturation. Generally, computer calculations can be used for these calculations. For example, this can be done using GraphPad. In certain embodiments, 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., a concentration (μM) that inhibits 50% of spore germination and / or mycelial growth). In certain embodiments, the polypeptide has an IC50 for inhibiting spore germination and / or mycelial growth of less than about 10 μM, for example, less than about 1 μM.

[0186] The Kd can be the Kd of binding to a lipid-containing fraction (i.e., a lipid-containing fraction obtained by chromatography, which may be derived from a fungus such as Fusarium oxysporum or Botrytis cinerea) as can be obtained as otherwise described in this application. The Kd of the polypeptide can be less than about 10 μM, for example, less than about 1 μM. The Kd can be determined by any suitable method. For example, the Kd can be determined by biolayer interference (BLI) in Octet, for example. The assay used to determine the Kd can be an ELISA assay.

[0187] The specific binding of polypeptides to harmful organism targets can be confirmed by any suitable method known in itself, such as biopanning, scatchard analysis and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assays, as well as various modifications thereof known in the art.

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

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

[0190] Also disclosed are polypeptides of 80 to 200 amino acids or subranges as disclosed above, obtained by affinity selection for specific plant pest targets, capable of inhibiting the growth and / or activity of crop pests at minimum inhibitory concentrations of approximately 0.00001 to 1 μM. In certain embodiments, the minimum inhibitory concentration is 0.0001 to 1 μM, 0.001 to 1 μM, 0.01 to 1 μM, 0.1 to 1 μM, 0.0001 to 0.1 μM, 0.001 to 0.1 μM, 0.01 to 0.1 μM, 0.00001 to 0.01 μM, 0.0001 to 0.01 μM, or 0.001 to 0.01 μM. In other specific embodiments, the minimum inhibitory concentration is approximately 0.0001 to 1 μM, approximately 0.001 to 1 μM, approximately 0.01 to 1 μM, approximately 0.1 to 1 μM, approximately 0.0001 to 0.1 μM, approximately 0.001 to 0.1 μM, approximately 0.01 to 0.1 μM, approximately 0.00001 to 0.01 μM, approximately 0.0001 to 0.01 μM, or approximately 0.001 to 0.01 μM.

[0191] The minimum inhibitory concentration (MIC) 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 to be the lowest concentration of polypeptide that inhibits the growth of fungal inoculum within 24 hours and reduces the inoculum by at least 99-90%. The MFC (minimum fungicidal concentration) can be measured on an agar plate, but it can also be measured conventionally in liquid (e.g., on a microwell plate) depending on the type of fungus and assay conditions.

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

[0193] In certain embodiments, the polypeptide in the composition disclosed herein is a heavy chain variable domain comprising, or composed of, or essentially composed of, four framework regions (FR1 to FR4, each) and three complementarity-determining regions (CDR1 to CDR3, each), or any suitable fragment of such a heavy chain variable domain (usually comprising at least a portion of amino acid residues forming at least one CDR as detailed herein). The sequences of the framework regions may be variable or specific.

[0194] The polypeptide disclosed herein may, in particular, be an antibody such as a heavy chain antibody. In other specific embodiments, the polypeptide disclosed herein may be a heavy chain variable domain sequence of an antibody derived from a conventional quadruple-chain antibody (but not limited to, for example, a V derived from a human antibody). H (Sequence) or so-called "heavy chain antibodies" (as defined in this application) derived from so-called V (as defined in this application) HH It can be an array.

[0195] In certain embodiments, the compositions disclosed herein include at least a heavy chain variable domain sequence derived from an antibody or a functional fragment thereof (but not limited to, for example, a camel heavy chain antibody or a functional fragment thereof), wherein the variable domain is, for example, the heavy chain variable domain (V) of a camel heavy chain antibody. HH ) can be done as follows.

[0196] Furthermore, the present invention is not limited with respect to the origin of the polypeptides contained in the compositions disclosed herein (or the nucleotide sequences of the present invention used to express them), nor is it limited with respect to the methods for producing or obtaining (or obtaining) such polypeptides or nucleotide sequences. Accordingly, the polypeptides in the compositions disclosed herein may be natural polypeptides (derived from any suitable species) or synthetic or semi-synthetic polypeptides. In certain non-limiting embodiments of the present invention, the polypeptides may be natural immunoglobulin sequences (derived from any suitable species) or synthetic or semi-synthetic immunoglobulin sequences, and are not limited to, but include, "camelized" immunoglobulin sequences, as well as immunoglobulin sequences obtained by techniques such as affinity maturation (starting from synthetic, random, or natural immunoglobulin sequences), CDR grafting, veneering, conjugation of fragments derived from various immunoglobulin sequences, PCR assembly using overlapping primers, and similar immunoglobulin sequence production techniques well known to those skilled in the art, or any suitable combination of the above.

[0197] The polypeptide sequences of the compositions disclosed in the present application can be, in particular, 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 prior art cited above 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, WO06 / 030220, WO06 / 003388 and other published patent applications in the name of Domantis Ltd.

[0198] Thus, in certain embodiments, the invention provides a polypeptide having the (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 wherein in the above formula, FR1 to FR4 each represent framework regions 1 to 4, and CDR1 to CDR3 each represent complementarity determining regions 1 to 3, as defined in detail in the present application.

[0199] In particular, in certain specific embodiments of the invention, the pesticidal composition contains at least one polypeptide that is specific to a pest target such as a fungal target and has at least 70%, at least 75%, at least 80%, preferably at least 85%, for example, at least 90% or at least 95% or at least 98% or more sequence identity with at least one of the amino acid sequences of SEQ ID NOs: 1 to 51, and the nucleic acid sequence encoding such an amino acid sequence is provided.

[0200] Certain particularly preferred polypeptide sequences disclosed herein are capable of binding to and / or are 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 the sequence variation compared to the reference sequence (i.e., the sequence of a specific SEQ ID NO) is present only in the CDR region. Certain particularly preferred polypeptide sequences disclosed herein are capable of binding to and / or are 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 the sequence variation compared to the reference sequence (i.e., the sequence of a specific SEQ ID NO) is present only in the framework region. In other embodiments, the sequence variation compared to the reference sequence (i.e., the sequence of a specific SEQ ID NO) can be present in the CDR region and / or the framework region. In certain embodiments, the sequence variation compared to the reference sequence (i.e., the sequence of a specific SEQ ID NO) can be present in the CDR3 region.

[0201] Again, such polypeptides can be obtained from any suitable origin by any suitable method, e.g., natural V HH sequences (i.e., those derived from a suitable species of camel), or can be synthetic or semi-synthetic heavy chain variable domains, including, but not limited to, "camelized" immunoglobulin sequences (particularly, camelized heavy chain variable domain sequences), as well as affinity maturation, CDR grafting, veneering, binding 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 immunoglobulin sequences obtained by techniques such as any suitable combination of the above, starting from synthetic, random or natural immunoglobulin sequences as detailed herein.

[0202] Naturally, the pesticide compositions or biological control compositions disclosed in this application are stable both during storage and use, that is, the integrity of the pesticide compositions is maintained under storage and / or use conditions, such as high temperatures, freeze-thaw cycles, changes in pH or ionic strength, UV irradiation, and the presence of harmful chemicals. It is more preferable that the polypeptides of 80 to 200 amino acids and the various subranges described in this application are stably maintained in the pesticide compositions, that is, the integrity of the polypeptides and their pest control activity are maintained under storage and / or use conditions, such as high temperatures, freeze-thaw cycles, changes in pH or ionic strength, UV irradiation, and the presence of harmful chemicals. It is most preferable that the polypeptides of 80 to 200 amino acids and the various subranges described in this application are stably maintained in the pesticide compositions when the pesticide compositions are stored at ambient temperature for two years or when the pesticide compositions are stored at 54°C for two weeks. The pesticide composition of the present invention preferably 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 may be incorporated into a base as defined herein to protect it from adverse effects caused by other components in the pesticide composition or adverse effects during storage or application. Suitable bases include, but are not limited to, alginates, gums, starches, β-cyclodextrins, cellulose, polyureas, polyurethanes, polyesters, microbial cells, or clay.

[0203] The aforementioned pesticide composition can be in any type of formulation, and preferred formulations include powder, wettable powder, wettable granules, water-dispersible granules, emulsion, concentrated emulsion, powder, suspension, concentrated suspension, suspend emulsion (a mixture of suspension and emulsion), capsule suspension, aqueous dispersion, oily dispersion, aerosol, paste, foam, slurry, or concentrated flowable formulation.

[0204] Polypeptides of 80 to 200 amino acids and the various subranges described above may be the sole active substance in the pesticide composition or biological control composition according to the present invention. However, the pesticide composition may 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 pesticides or biological control compositions may have a different effect on plant pests than the polypeptide or amino acid sequence, or they may have a synergistic effect with the polypeptide or amino acid sequence, or they may modify the activity of the polypeptide or amino acid sequence on a given plant. Suitable other pesticides may be herbicides, insecticides, fungicides, nematicides, acaricides, bactericides, virucidates, plant growth regulators, phytotoxicity reducers, etc. Such pesticides may be chemical substances or biomolecules, such as microorganisms. While not limited to pesticides, the following are included: glyphosate, paraquat, metrachlor, acetochlor, mesotrione, 2,4-D, atrazine, glufosinate, sulfocate, phenoxaprop, benzimethalin, picloram, trifluralin, bromoxynil, clodinahop, fluroxypyr, nicosulfuron, bensulfuron, imazetapir, dicamba, imidacloprid, thiamethoxam, fipronil, chlorpyrifos, deltamethrin, lambdasihalotrin, endosulfan, methamidophos, carbofuran, clothianidin, cypermethrin, abamectin, diflufenican, spinosad, indoxacarb, bifen Examples include trin, tefluthrin, azoxystrobin, thiamethoxam, tebuconazole, mancozep, cyazofamide, fluazinam, pyraclostrobin, epoxyconazole, chlorothalonil, copper fungicides (e.g., copper oxychloride, copper hydroxide), trifloxystrobin, prothioconazole, difenoconazole, carbendazim, propiconazole, thiophanate, sulfur, boscalid, trichalazole, hexaconazole, metalaxyl, benomyl, kitadine, tebuconazole, toridemorph, propineb, streptomycin sulfate, and oxytetracycline, as well as other known pesticides or any suitable combination thereof.

[0205] Other suitable pesticides can be biomolecules such as microorganisms, including strains of the genera Pseudomonas, Bacillus, or Streptomyces.

[0206] Composition containing polypeptide sequence variants In certain embodiments, the polypeptide contained in the pesticide composition disclosed herein can be selectively modified to increase, for example, the amount of positive charge (of the polypeptide). That is, the polypeptide can be modified by one or more amino acid substitutions to increase the amount of positive charge of the polypeptide. Accordingly, an amino acid can be substituted with an amino acid that has a larger amount of positive charge (compared to the amino acid being substituted). Two or more such substitutions can also 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 generally be made in the CDR region, for example, the CDR1 region, the CDR2 region, or the CDR3 region.

[0207] Other substitutions may be made to the polypeptide. For example, substitutions that do not affect the overall charge of the polypeptide can be made. The inventors unexpectedly found that an increase in positive charge can correlate with an improvement in antifungal activity, so it is advantageous to make substitutions that do not decrease the total charge of the polypeptide.

[0208] Other substitutions are also possible. For example, the polypeptide may begin with a D residue or a Q residue. The inventors have found that while Q can be used for the residue at position 1 of the polypeptide (i.e., the first residue of the framework 1 region sequence), using D can improve the antifungal properties of the polypeptide. Therefore, for all specific polypeptide sequences disclosed herein (including all peptides having any one of the sequences of SEQ ID NOs: 1-51 or 101-111), the residue at position 1 may be a Q residue, but in certain embodiments, it is preferable to use a D residue. The designation of a polypeptide such as 10G11Q indicates that the polypeptide begins with a Q residue. The designation of a polypeptide such as 10G11 (without Q at the end) indicates that the polypeptide begins with a D residue. In this application, 10G11 may also be referred to as 10G11Q1D (a notation indicating the substitution of Q to D at position 1).

[0209] In certain embodiments, the present invention provides polypeptides comprising or composed of amino acid sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% identity with any of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 51. All polypeptides having a specific percentage of sequence identity with a given SEQ ID NO may have the same total charge as the reference sequence, or may have more positive charge than the reference sequence. It is advantageous that all polypeptides having a specific percentage of sequence identity with a given SEQ ID NO may not have more negative charge than the reference sequence.

[0210] In certain embodiments, the present invention provides 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 up to one, two, three, four, or five amino acid substitutions in the said sequence, or comprising such a sequence.

[0211] In certain embodiments, the present invention provides polypeptides comprising or composed of amino acid sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% identity with any of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1-10 and 12-51. All polypeptides having a specific percentage of sequence identity with a given SEQ ID NO may have the same total charge as the reference sequence, or may have more positive charge than the reference sequence. It is advantageous that all polypeptides having a specific percentage of sequence identity with a given SEQ ID NO may not have more negative charge than the reference sequence.

[0212] In certain embodiments, the present invention provides a polypeptide comprising 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 in the said sequence, or comprising such a sequence.

[0213] In certain embodiments, the present invention provides polypeptides comprising or composed of amino acid sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% identity with any of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 6. All polypeptides having a specific percentage of sequence identity with a given SEQ ID NO may have the same total charge as the reference sequence, or may have more positive charge than the reference sequence. It is advantageous that all polypeptides having a specific percentage of sequence identity with a given SEQ ID NO may not have more negative charge than the reference sequence.

[0214] In certain embodiments, the present invention provides a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6, or an amino acid sequence having up to one, two, three, four, or five amino acid substitutions in the said sequence, or comprising such a sequence.

[0215] All amino acid substitutions in the polypeptide may increase the total charge of the polypeptide, or they may not change the total charge of the polypeptide. In certain embodiments, no amino acid substitutions decrease the total charge of the polypeptide.

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

[0217] The present invention provides specific mutants, referred to hereby as mutants 1 to 5, (in which the total charge of the polypeptide has been modified to investigate the functional effects on the polypeptide), and mutants, referred to hereby as "single ALA mutants" 1 to 34, which have substitutions other than alanine but were subjected to alanine scanning. The present invention also extends to other mutants or variants of the polypeptide sequence disclosed here, for example, those having other substitutions, or various combinations of substitutions, or those having substitutions at various positions in the polypeptide sequence. It is appropriate that any mutant or variant of the polypeptide sequence disclosed here does not decrease the positive charge compared to the reference sequence. In some cases, mutants or variants of the polypeptide sequence disclosed here can increase the total positive charge.

[0218] The total charge of the polypeptide can be calculated before and after substitution or mutation, and the charge of the polypeptide is calculated in an aqueous solution at the same pH before and after introducing 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 using 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 total charge (at pH 7). It is preferable that the substitution or mutation in the sequence does not decrease the total charge (at pH 7). In certain embodiments, the substitution or mutation in the sequence will increase the positive total 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 judged to have a pI greater than 7 and a positive total charge at pH 7. It is preferable that the pI does not decrease due to substitution or mutation in the sequence. In certain embodiments, the pI will increase due to substitution or mutation in the sequence.

[0222] The present invention also provides a polypeptide (and a composition containing the polypeptide) having a predetermined sequence capable of mutation or substitution at a specific position. Such mutants include those referred to as 10G11-A to 10G11-K in the present application.

[0223] For example, in one embodiment, the present invention provides the following sequence:

Chemical formula

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

[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, and X3, X4, X5, X9 and X 10 are each independently any natural amino acid other than E or D (SEQ ID NO: 103, also referred to as 10G11-C in the present application).

[0226] In certain embodiments, X1 is D or Q, and X2, X6, X7, X8 and X 11Each of these is independently A, K, R, or H, and X3, X4, X5, X9 and X 10 Each of these is an arbitrary natural amino acid other than E or D (Sequence ID 104, also referred to as 10G11-D in this application).

[0227] In certain embodiments, X1 is D or Q, and X2, X6, X7, X8 and X 11 Each of these is independently A, K, R, or H, and X3, X5 and X 10 Each of the following is an independently chargeless 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) (Sequence ID No. 105, also referred to as 10G11-E in this application).

[0228] In certain embodiments, X1 is D or Q, and X2, X6, X7, X8 and X 11 Each is independently A, K, R, or H, X3 and X5 are independently S, K, R, or H, X4 is I, K, R, or H, X9 is W, K, R, or H, X 10 is T, K, R, or H (sequence number 106, also referred to as 10G11-F in this application).

[0229] In certain embodiments, X1 is D or Q, and X2, X6, X7, X8 and X 11 Each of them is independent of X3, X4, X5, X9 and X 10 These are independent of each other (sequence number 107, also referred to as 10G11-G in this application).

[0230] In certain embodiments, X1 is D or Q, and X2, X6, X8 and X 11 Each of these is independently R, and X3, X4, X5, X9 and X 10 These are each an independent and arbitrary natural amino acid (SEQ ID NO: 108, also referred to as 10G11-H in this application).

[0231] In certain embodiments, X1 is D or Q, and X2, X6, X7, X8 and X 11 Each of these is independently R, and X3, X4, X5, X9 and X 10 Each of these is an arbitrary natural amino acid other than E or D (Sequence ID 109, also referred to as 10G11-I in this application).

[0232] In certain embodiments, X1 is D or Q, and X2, X6, X8 and X 11 Each of these is independently R, and X3, X5 and X 10 Each of the following is an independently chargeless 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) (Sequence ID 110, also referred to as 10G11-J in this application).

[0233] In certain embodiments, X1 is D or Q, and X2, X6, X8 and X 11 Each of these is R, and X3, X5, and X 10 Each of the following is S, K, R, or H, X4 is I, K, R, or H, X7 is K, X9 is W, K, R, or H, X 10 is T, K, R, or H (sequence number 111, also referred to as 10G11-K in this application).

[0234] In certain embodiments, the present invention comprises a CDR1 region containing or composed of the sequence X2X3X4FX5INAMD, a CDR2 region containing or composed of the sequence GITX6GGTTX7, and the sequence LX8GEQPX9X 10 X 11 A polypeptide is provided which contains DY or a CDR3 region composed of the same sequence, wherein in the above sequence, X2~X 11 Each of these is an independent, arbitrary natural amino acid (therefore, the CDR1, CDR2, and CDR3 regions have the sequences of SEQ ID NOs. 112, 123, and 134, respectively).

[0235] In certain embodiments, X2~X 11 Each of these is independently any natural amino acid other than E or D (therefore, the CDR1, CDR2, and CDR3 regions have the sequences of SEQ ID NOs. 113, 124, and 135, respectively).

[0236] In certain embodiments, X2, X6, X7, X8 and X 11 Each of these is independently G, A, V, M, L, I, K, R, or H, and X3, X4, X5, X9 and X 10 Each of these is independently any natural amino acid other than E or D (therefore, 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 Each of these is independently A, K, R, or H, and X3, X4, X5, X9 and X 10 Each of these is independently any natural amino acid other than E or D (therefore, the CDR1, CDR2, and CDR3 regions have the sequences of SEQ ID NOs. 115, 126, and 137, respectively).

[0238] In certain embodiments, X2, X6, X7, X8 and X 11 Each of these is independently A, K, R, or H, and X3, X5 and X 10 Each of the following is an independently chargeless 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) (therefore, the CDR1, CDR2, and CDR3 regions have the sequences of SEQ ID NOs. 116, 127, and 138, respectively).

[0239] In certain embodiments, X2, X6, X7, X8 and X 11Each is independently A, K, R, or H, X3 and X5 are independently S, K, R, or H, X4 is I, K, R, or H, X9 is W, K, R, or H, X 10 is T, K, R, or H (therefore, the CDR1, CDR2, and CDR3 regions have sequences 117, 128, and 139, respectively). (Sequence ID 106).

[0240] In certain embodiments, X2, X6, X7, X8 and X 11 Each of them is independent of X3, X4, X5, X9 and X 10 Each is independent (therefore, the CDR1 region, CDR2 region, and CDR3 region have sequences of sequence numbers 118, 129, and 140, respectively). (Sequence number 107).

[0241] In certain embodiments, X2, X6, X8 and X 11 Each of these is independently R, and X3, X4, X5, X9 and X 10 Each of these is an independent, arbitrary natural amino acid (therefore, 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, X2, X6, X7, X8 and X 11 Each of these is independently R, and X3, X4, X5, X9 and X 10 Each of these is independently any natural amino acid other than E or D (therefore, 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, X2, X6, X8 and X 11 Each of these is independently R, and X3, X5 and X 10is each independently a polar amino acid having no charge 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) (therefore, the CDR1 region, CDR2 region, and CDR3 region each have the sequences of SEQ ID NO: 121, 132, and 143, respectively). (SEQ ID NO: 110).

[0244] In certain embodiments, X2, X6, X8, and X 11 are each R, X3, X5, and X 10 are each S, K, R, or H, X4 is I, K, R, or H, X7 is K, X9 is W, K, R, or H, and X 10 is T, K, R, or H (therefore, the CDR1 region, CDR2 region, and CDR3 region each have the sequences of SEQ ID NO: 122, 133, and 144, respectively). (SEQ ID NO: 111).

[0245] All polypeptides disclosed in the present application, including their variants, can be formulated into a composition (e.g., an agrochemical composition).

[0246] Generally, the present invention also encompasses variants of the polypeptide, provided that the polypeptide can maintain its functional properties or improve its functional properties. For example, the variant may be capable of (specifically) binding to a fungus. More specifically, the variant may be capable of (specifically) binding to a fungal membrane or a component of a fungal membrane. In certain embodiments, the variant does not bind to the fungal cell wall or a component of the cell wall. For example, in certain embodiments, the variant polypeptide does not (specifically) bind to fungal glucosylceramide.

[0247] The aforementioned mutant may be able to bind to the 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 the steps of fractionating the hyphae of a fungus (e.g., Botrytis cinerea or other fungi) by total lipid extract thin-layer chromatography, and selecting a fraction having a retention factor (Rf) greater than that of the ceramide fraction and less than that of the nonpolar phospholipid fraction.

[0248] The mutant can further cause delayed growth of fungal spores and / or lysis of the fungal spores. That is, the mutant polypeptide binds to the fungus, resulting in delayed growth of the fungal spores and / or lysis of the fungal spores.

[0249] In certain embodiments, the mutant can cause delayed fungal spore growth and / or lysis of fungal spores with an IC50 less than or equal to that of a reference polypeptide when measured under substantially identical conditions. The IC50 can be, for example, a concentration (μM) that inhibits 50% of Fusarium oxysporum spore germination and / or mycelial growth.

[0250] In certain embodiments, the variant can (specifically) bind to fungi (e.g., fungal membranes or components of fungal membranes) with a KD of less than or equal to that of the reference polypeptide when measured under substantially the same conditions.

[0251] In certain embodiments, the mutant can (specifically) exhibit an MIC below that of the reference polypeptide for fungal growth when measured under substantially identical conditions.

[0252] In a predetermined embodiment, the polypeptide contained in the pesticide composition disclosed herein may optionally be functionally linked to one or more other groups, moieties, or residues via one or more linkers. These one or more other groups, moieties, or residues may play a role in binding to other applicable targets. Naturally, such other groups, residues, moieties, and / or binding sites may or may not provide other functionality to the polypeptide disclosed herein (and / or the composition in which the polypeptide is contained), nor may they alter the properties of the polypeptide disclosed herein. Such groups, residues, moieties, or binding sites may, for example, be chemical groups that may be biologically active.

[0253] These groups, parts, or residues can, in certain embodiments, be linked to the N-terminus or C-terminus of the polypeptide in the composition disclosed herein.

[0254] In certain embodiments, the polypeptide in the pesticide composition disclosed herein may be chemically modified. For example, such modification may involve introducing or linking one or more functional groups, residues, or moieties to the heavy chain variable domain. These groups, residues, or moieties can impart one or more desirable properties or functions to the polypeptide. Examples of such functional groups will be obvious 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, eliminate or mitigate undesirable side effects of the polypeptide, and / or obtain other advantageous properties.

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

[0257] In other specific embodiments, two or more target-specific polypeptide molecules in the pesticide compositions disclosed herein can be linked or bonded together. In specific embodiments, the two or more polypeptide molecules are linked together via one or more suitable linkers or spacers. Suitable spacers or linkers for use in coupling the various heavy-chain polypeptides disclosed herein are obvious to those skilled in the art and can be any linker or spacer commonly used in the art to link peptides and / or proteins.

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

[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. Naturally, the length, degree of flexibility, and / or other properties of the linker will have some influence on the properties of the polypeptide, including, but not limited to, affinity, specificity, or avidity to harmful organism targets. Naturally, if two or more linkers are used, these linkers may be the same or different. In the context and disclosure of the present invention, those skilled in the art will be able to determine the optimal linker for coupling the heavy chain variable domain disclosed herein without bearing an excessive burden of experimentation.

[0260] Composition containing a polypeptide sequence fragment The present invention also includes parts, fragments, analogs, mutants, variants, and / or derivatives of polypeptides contained in the compositions disclosed herein, and / or polypeptides containing one or more such parts, fragments, analogs, mutants, variants, and / or derivatives, provided that these parts, fragments, analogs, mutants, variants, and / or derivatives are suitable for the purposes envisioned herein. Such parts, fragments, analogs, mutants, variants, and / or derivatives according to the present invention can also specifically bind to harmful organism targets (e.g., fungi such as plant pathogenic fungi).

[0261] target In certain embodiments, polypeptides contained in the compositions disclosed herein are obtained by affinity selection for specific harmful organism targets, such as fungal antigens or fungal targets. To obtain suitable polypeptides by affinity selection for specific pest targets, this can be carried out, for example, by screening a set, collection, or library of cells (e.g., bacteriophages) expressing polypeptides on their surface for binding to pest target molecules known in the art to be targets of pest control agents. All of these steps can be carried out by methods known in themselves and essentially include the following non-limiting steps: a) obtaining an isolated solution or suspension of pest target molecules known to be targets of pest control agents; b) biopanning phages or other cells from a polypeptide library against the target molecules; c) isolating phages or other cells that bind to the target molecules; d) determining the nucleotide sequences encoding polypeptide inserts from individual binding phages or other cells; e) producing a corresponding amount of polypeptide using recombinant protein expression; f) measuring the affinity of the polypeptide to the pest target; and optionally, g) testing the pest control activity of the polypeptide in a bioassay of the pest. Various methods can be used to measure the affinity between the polypeptide and the pest target molecule. For example, enzyme immunosorbent assay (ELISA) or surface plasmon resonance (SPR) assay, which are common methods in the art, can be used, 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 the binding of a polypeptide to its pest target molecule is 10 -5 M is less than 10 -6 M is more preferably less than M, and the dissociation constant is 10 -7It is even more preferable that the dissociation constant is less than M, and the dissociation constant is 10 -8 Less than M is most preferable.

[0262] The harmful organism target molecules disclosed herein are molecules that are present inside or on the surface of harmful organisms, and when bound to and / or inhibited, cause the harmful organism to die or inhibit, suppress, or inhibit its growth or activity. Such suitable target molecules are readily searchable by those skilled in the art from existing literature or patent databases and are not limited to those listed above. Examples include secretory parasitic proteins such as 16D10 as a suitable target molecule for root-knot nematodes (Huang et al (2006) PNAS 103:14302-14306), V-ATPase proton pump as a suitable target molecule for insect species of the Coleoptera (beetles), Hemiptera (true bugs), and Diptera (flies), and nematodes (Knight AJ and Behm CA (2011) Ex. Parasitol. Sept 19), and tetraspanin PLS1 as a suitable target molecule for the fungal pests of gray mold (B. cinerea) and rice blast (M. grisea) (Gourgues et al. Examples of antifungal targets include proton pump ATPases (Manavathu EK et al (1999) Antimicrob Agents and Chemotherapy, Dec p.2950), or (2002) Biochem. Biophys. Res. Commun. 297:1197). Naturally, preferred pest target molecules are accessible in the extracellular space (in contrast to intracellular pest targets).

[0263] More specifically, the pest target to which the at least one polypeptide of the pesticide composition disclosed herein is bound may be a cell membrane component of a pest. The cell membrane component of a pest used in this application may be any component that is included in or part of either the cell membrane phospholipid bilayer or a protein embedded in the phospholipid bilayer of the pest's cell (i.e., at least a portion of which is associated with, present in, linked to, or bound to the phospholipid bilayer). In certain embodiments, the cell membrane component of a pest may be a phospholipid, glycoprotein, carbohydrate, or cholesterol.

[0264] In certain embodiments, the cell membrane component of a harmful organism to which the at least one polypeptide in the composition disclosed herein specifically binds is a non-protein.

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

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

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

[0268] In a preferred embodiment, the plant pests controlled by the pesticide composition or biological control composition disclosed herein are fungi, such as the plant pathogenic fungi defined above. Fungi are highly harmful to plants and can lead to a substantial reduction in crop yield. Examples of plant pathogenic fungi include necrotic fungi and vitrophotactic fungi, and include ascomycetes, basidiomycetes, and oomycetes.

[0269] Examples of plant pathogenic fungi are known in the art and are not limited to, but include: Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Diplodia, Erysiphe, Fusarium, Leptosphaeria, Gaeumanomyces, Helminthosporium, Macrophomina, Nectria, Oidium, Peronospora, Phakopsora, and Phoma. Examples include those selected from the group consisting of the genera 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 with the pesticide composition of the present invention include powdery mildew and gray mold (Botrytis cinerea) of fruit trees such as grapes and strawberries, and vegetables. Other specific examples of plant fungal infections that can be controlled with the pesticide composition of the present invention include barley powdery mildew (Erysiphe graminis) of cereals, powdery mildew of cucurbitaceous plants (Erysiphe cichoracearum and Sphaerotheca fuliginea), and apple powdery mildew (Podosphaera).leucotricha), for example, powdery mildew on strawberries (Podosphaera aphanis), for example, powdery mildew on cucumbers (Podosphaera xanthii), for example, powdery mildew on tomatoes (Oidium neolycopersici), powdery mildew on grapes (Uncinula necator), Puccinia sp. on cereals, Rhizoctonia sp. on cotton, potatoes, rice and lawngrass, Ustilago sp. on cereals and sugarcane, apple scab (Venturia inaequalis) (scab disease), Helminthosporium sp. on cereals, wheat blight (Septoria nodorum), wheat leaf blight (Septoria (tritici), barley cloud spot (Rhynchosporium secalis), strawberry, tomato and grape gray mold (Botrytis cinerea), peanut brown spot (Cercospora arachidicola), tobacco downy mildew (Peronospora tabacina), or various crop downy mildews (Peronospora), wheat and barley eye spot disease (Pseudocercosporella herpotrichoides), barley net spot disease (Pyrenophera teres), rice blast (Pyricularia oryzae), potato and tomato late blight (Phytophthora infestans), various plant Fusarium sp. diseases (e.g., wilt disease (Fusarium oxysporum)) and Verticillium sp. diseases (Verticillium downy mildew of grapes (Plasmopara viticola), Alternaria sp. of fruit trees and vegetables, downy mildew of cucumbers (Pseudoperonospora cubensis), Sigatoka disease of bananas (Mycosphaerella fijiensis), Ascochyta sp. of chickpeas, Leptosphaeria sp. of canola and rapeseedExamples include Phakopsora sp., Phakopsora spp. diseases of various crops (e.g., soybean rust (Phakopsora pachyrhizi)), and Colleotrichum sp. diseases (e.g., Colleotrichum orbiculare, which can be a pathogen of pumpkin anthracnose). The compositions according to the present invention are active against susceptible and resistant species under normal conditions and are active against all or part of the life cycle of plant pathogenic fungi.

[0270] In certain embodiments, the pesticide compositions disclosed herein are for the treatment of the genera Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Diplodia, Erysiphe, Fusarium, Leptosphaeria, and Gaeumanomyces. Helminthosporium, Macrophomina, Nectria, Oidium, Penicillium, Peronospora, Phoma, Phymatotrichum, Phytophthora, Plasmopara, and Podosphaera , Puccinia, Pyrenophora, Pyricularia, Pythium, Rhizoctonia, Scerotium, Sclerotinia, Septoria, Thielaviopsis, Uncinula, Venturia, Verticillium It is specific to plant pathogenic fungi of genera selected from the group including *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 belonging to the genera Botrytis, Fusarium, or Penicillium.

[0272] In certain embodiments, the present invention provides a pesticide composition containing polypeptides specific to structural molecular components of the cell membranes of harmful organisms.

[0273] In certain embodiments, the present invention provides a pesticide composition containing polypeptides specific to structural molecular components other than proteins of the cell membranes of harmful organisms.

[0274] In yet another specific embodiment, the plant pest is a plant pathogenic bacterium, and is not limited to, *Acidovorax avenae subsp. avenae* (pathogen of rice brown stripe disease), *Acidovorax avenae subsp. cattleyae* (pathogen of cattleya bacterial brown spot disease), *Acidovorax konjaci* (pathogen of konjac leaf blight disease), *Agrobacterium rhizogenes* (pathogen of melon root blight disease), *Agrobacterium tumefaciens* (pathogen of crown gall disease), and *Burkholderia andropogonia* (pathogen of carnation bacterial spot disease). Burkholderia caryophylli (pathogen of carnation wilt), Burkholderia cepacia (pathogen of cymbidium brown spot bacterial disease), Burkholderia gladioli pv. gladioli (pathogen of gladiolus neck rot), Burkholderia glumae (pathogen of rice bacterial blight), Burkholderia plantarii (pathogen of rice seedling blight bacterial disease), Clavibacter michiganensis subspecies michiganensis (pathogen of tomato canker). Clavibacter michiganensis subsp. sepedonicus (a pathogen of potato ring rot), Clostridium spp. (a pathogen of potato slime rot)), Curtobacterium flaccumfaciens (pathogen of onion canker), Erwinia amylovora (pathogen of pear burn disease), Erwinia ananas (pathogen of rice glume browning disease), Erwinia carotovora subsp. atroseptica (pathogen of potato blackleg disease), Erwinia carotovora subsp. carotovora (pathogen of vegetable soft rot), Erwinia chrysanthemi (pathogen of taro seedling blight) Erwinia chrysanthemi pv.zeae (pathogen of rice stem rot), Erwinia herbicola pv.millettiae (pathogen of wisteria gall), Pseudomonas cichorii (pathogen of chrysanthemum bacterial spot), Pseudomonas corrugate (pathogen of tomato stem necrosis bacterial disease), Pseudomonas fuscovaginae (pathogen of rice leaf sheath browning disease), Pseudomonas marginalis pv.marginalis (pathogen of cabbage soft rot) Pseudomonas rubrisubalbicans (pathogen of sugarcane stripe disease), Pseudomonas syringae pv.aptata (pathogen of sugar beet bacterial spot), Pseudomonas syringae pv. atropurpurea (pathogen of ryegrass leaf blight)Pseudomonas syringae pv.castaneae (pathogen of chestnut canker), Pseudomonas syringae pv.glycinea (pathogen of soybean bacterial spot), Pseudomonas syringae pv.lachrymans (pathogen of cucumber bacterial spot), Pseudomonas syringae pv.maculicola (pathogen of cabbage black spot bacterial disease), Pseudomonas syringae pv.mori (pathogen of mulberry leaf curl bacterial disease) Pseudomonas syringae pv.morsprunorum (pathogen of plum canker), Pseudomonas syringae pv.oryzae (pathogen of rice leaf blight), Pseudomonas syringae pv.phaseolicola (pathogen of kidney bean leaf blight), Pseudomonas syringae pv.pisi (pathogen of pea bacterial spot), Pseudomonas syringae pv.sesame (pathogen of sesame bacterial spot) Pseudomonas syringae pv.striafaciens (pathogen of oat blight), Pseudomonas syringae pv.syringae (pathogen of adzuki bean brown spot bacterial disease), Pseudomonas syringae pv.tabaki (pathogen of tobacco wildfire)Pseudomonas syringae pv.theae (pathogen of tomato red blight), Pseudomonas syringae pv.tomato (pathogen of tomato bacterial leaf spot), Pseudomonas viridiflava (pathogen of kidney bean bacterial spot), Ralstonia solanacearum (pathogen of bacterial wilt), Rathayibacter rathayi (pathogen of orchardgrass yellow rubber disease), Streptomyces scabies (pathogen of potato scab) Streptomyces ipomoea (pathogen of sweet potato blight), Xanthomonas albilineans (pathogen of sugarcane white streak disease), Xanthomonas campestris pv. cerealis (pathogen of rye stripe bacterial disease), Xanthomonas campestris pv. campestris (pathogen of black rot), Xanthomonas campestris pv. citri (pathogen of citrus canker) Xanthomonas campestris pv.cucurbitae (pathogen of cucumber brown spot bacterial disease), Xanthomonas campestris pv.glycines (pathogen of soybean leaf blight), Xanthomonas campestris pv.incanae (pathogen of stock black rot)Xanthomonas campestris pv. malvacearum (pathogen of cottony corner blight), Xanthomonas campestris pv. mangiferaeindicae (pathogen of mango canker), Xanthomonas campestris pv. mellea (pathogen of tobacco yellow blight), Xanthomonas campestris pv. nigromaculans (pathogen of burdock black spot bacterial disease), Xanthomonas campestris pv. phaseoli (pathogen of kidney bean leaf blight) Xanthomonas campestris pv. pisi (pathogen of bean stem rot), Xanthomonas campestris pv. pruni (pathogen of peach bacterial spot), Xanthomonas campestris pv. raphani (pathogen of radish bacterial leaf spot), Xanthomonas campestris pv. ricini (pathogen of castor leaf spot), Xanthomonas campestris pv. teicola (pathogen of tea canker) Xanthomonas campestris pv.translucens (a pathogen of orchardgrass bacterial spot), Xanthomonas campestris pv. vesicatoria (a pathogen of tomato bacterial spot).Examples include *Xanthomonas oryzae pv. oryzae* (the pathogen of 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 facilities. The compositions according to the present invention are active against susceptible and resistant species under normal conditions and are active against all or some stages of development. These plant pests include those belonging to the phylum Arthropoda, particularly arachnids, such as species of the genera Acarus, Aceria sheldoni, Aculops, Aculus, Amblyomma, Amphitetranychus viennensis, Argas, Boophilus, Brevipalpus, Bryobia praetiosa, Centruroides, and Chorioptes. 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.), species of the genus Loxosceles, species of the genus Metatetranychus), Nupersa 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 Examples include species of the genera Tarsonemus, Tetranychus, Vaejovis, and Vasates lycopersici.

[0276] Further examples include the suborder Anoplura (locusts), such as species of the genera Damalinia, Haematopinus, Linognathus, Pediculus, Ptirus pubis, and Trichodectes.

[0277] Further examples include the class Chilopoda, such as species of the genera Geophilus and Scutigera.

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

[0279] Another example is the order Collembola, such as the white springtail (Onychiurus armatus).

[0280] Another example is the class Diplopoda, such as the snake millipede (Blaniulus guttulatus).

[0281] Further examples include the order Diptera (flies), such as the genera Aedes, Agromyza, Anastrepha, Anopheles, Asphondylia, Bactrocera, Bibio hortulanus, Calliphora erythrocephala, Ceratitis capitata, Chironomida, Chrysomyia, and Chrysops. spp.), Cochliomyia spp., Contarinia spp., Cordylobia anthropophaga, Culex spp., Culicoides spp., Culiceta spp., Cuterebra spp., Dacus oleae, Dasyneura spp., Delia spp., Dermatobia hominis, Drosophila spp., Echinocnemus spp., Fannia spp. spp.), botfly species (Gasterophilus spp.), tsetse species (Glossina spp.), horsefly species (Haematopota spp.), Hydrellia spp., Hylemyia spp., Hyppobosca spp., cattle fly species (Hypoderma spp.), Liriomyza spp., blowfly species (Lucilia spp.), Lutzomia spp., Mansonia spp., housefly species (Musca spp.), Nezara spp.), species of the genera Oestrus, Oscinella frit, Pegomyia, Phlebotomus, Phorbia, Phormia, Prodiplosis, Psila rosae, Rhagoletis, Sarcophaga, Simulium, Stomoxys, Tabanus, Tannia, Tetanops Examples include species of the genus Tipula (spp.), such as crane flies.

[0282] Further examples include the suborder Heteroptera, such as Anasa tristis, species of the genera Antestiopsis, Boisea, Blissus, Calocoris, Campylomma livida, Cavelerius, Cimex, Collaria, Creontiades dilutus, Dasynus piperis, Dichelops furcatus, and Diconocoris hewech. hewetti), species of the genera Dysdercus, Euschistus, Eurygaster, Heliopeltis, Horcias nobilellus, species of Leptocorisa, Leptoglossus phyllopus, species of Lygus, Macropes excavatus, Miridae, Monalonion atratum, species of Nezara, species of Oebalus (spp.), Pentomidae family, Piesma quadrata, Piezodorus spp., Psallus spp., Pseudacysta persea, Rhodnius spp., Sahlbergella singularis, Scaptocoris castanea, Scotinophora spp.Examples include the pear lace bug (Stephanitis nashi), species of the genus Tibraca, and species of the genus Triatoma.

[0283] Further examples include the suborder Homoptera, such as species of Acyrthosipon, Acrogonia, Aeneolamia, Aganoscena, Aleurodes, Aleurolobus barodensis, Aleurothrixus, Amrasca, Anuraphis cardui, Aonidiella, Aphanostigma pin, and Aphis. spp.), Arboridia apicalis, Aspidiella spp., Aspidiotus spp., Atanus spp., Aulacorthum solani, Bemisia spp., Brachycaudus helichrysii, Brachycolus spp., Brevicoryne brassicae, Calligypona marginata, Carneocephala fulgida, Ceratovacuna (lanigera), family Cercopidae, genus Ceroplastes spp.), strawberry 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), cottony cushion scales (Icerya spp.), Idiocerus spp., Idioscopus spp., small brown planthopper (Laodelphax striatellus), Lecanium spp., Lepidosaphes spp., false radish aphid (Lipaphis erysimi), Macrosiphum spp., Mahanarva spp.), Millet aphid (Melanaphis sacchari), Metcalfiella spp., Wheat aphid (Metopolophium dirhodum), Monellia costalis, Monelliopsis pecanis, Myzus spp., Lettuce aphid (Nasonovia ribisnigri), Nephotettix spp., Brown planthopper (Nilaparvata lugens), Oncometopia spp., Orthezia praelonga, Bayberry whitefly (Parabemisia myricae), Paratrioza spp. spp.), species of Parlatoria, species of Pemphigus, corn planthopper (Peregrinus maidis), species of Phenacoccus, cotton aphid (Phloeomyzus passerinii), hop aphid (Phorodon humuli), species of Phylloxera, long-legged scale insect (Pinnaspis aspidistrae), species of Planococcus, pear-shaped scale insect (Protopulvinaria pyriformis), mulberry scale insect (Pseudaulacaspis pentagona), species of Pseudococcus spp.), species of the genera Psylla, Pteromalus, Pyrilla, Quadraspidiotus, Quesada gigas, Rastrococcus, Rhopalosiphum, and Saissetia.), Scaphoides titanus, Schizaphis graminum, Selenaspidus articulatus, Sogata spp., Sogatella furcifera, Sogatodes spp., Stictocephala festina, Tenalaphala malayensis, Tinocallis caryaefoliae, Tomaspis spp., Toxoptera spp., Trialeurodes spp., Trioza Examples include species of the genera Typhlocyba, Unaspis, Viteus vitifolii, and Zygina.

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

[0285] Further examples include the order Isopoda, such as the pill bug (Armadillidium vulgare), the common woodlouse (Oniscus asellus), and the common sowbug (Porcellio scaber).

[0286] Further examples include the order Isoptera, such as species of the genera Coptotermes, Cornitermes cumulans, Cryptotermes, Incisitermes, Microtermes obesi, Odontotermes, and Reticulitermes.

[0287] Further examples include the order Lepidoptera, such as the large hawk moth (Acronicta major), species of the genus Adoxophyes, the white-backed caterpillar (Aedia leucomelas), species of the genus Agrotis, species of Alabama, the walnut moth (Amyelois transitella), species of the genus Anarsia, species of Anticarsia, species of Argyroploce, the armyworm (Barathra brassicae), the ghost skipper (Borbo cinnara), the cottonleaf perforator (Bucculatrix thurberiella), and Bupalus pinniarius. *piniarius*, *Busseola* spp., *Cacoecia* spp., *Caloptilia theivora*, *Capua reticulana*, *Carpocapsa pomonella*, *Carposina niponensis*, *Chematobia brumata*, *Chilo* spp., *Choristoneura* spp., *Clysia ambiguella*, *Cnaphalocerus* spp., *Cnephasia* spp., *Conopomorpha* spp., *Conotrachelus* spp. Copitarsia spp., Cydia spp., Dalaca noctuides, Diaphania spp., sugarcane moth (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 Lithocolletis spp., Lithophane antennata, Lobesia spp., Loxagrotis albicosta, Lymantria spp., Lyonetia spp.), Malacosoma neustria, Maruca testulalis, Mamestra brassicae, Mocis spp., Mythimna separata, Nymphula spp., Oiketicus spp., Oria spp., Orthaga spp., Ostrinia spp., Oulema oryzae, Panolis flammea, Parnara spp., Pectinophora spp., Perileucoptera spp.), Phthorimaea spp., 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), corn borer (Pyrausta) Rachiplusia nubilalis, sunflower loupe (Rachiplusia nu), Schoenobius spp., Scirpophaga spp., Scotia segetum, Sesamia spp., Sparganothis spp., Spodoptera spp., Stathmopoda spp.Examples include *Stomopteryx subsecivella*, species of *Synanthedon*, *Tecia solanivora*, *Thermesia gemmatalis*, *Tinea pellionella*, *Tineola bisselliella*, species of *Tortrix*, *Trichophaga tapetzella*, *Trichoplusia*, *Tuta absoluta*, and species of *Virachola*.

[0288] Further examples include the order Orthoptera, such as the European house cricket (Acheta domesticus), the Eastern cockroach (Blatta orientalis), the German cockroach (Blattella germanica), species of the genera Dichroplus, Grillotalpa, Leucophaea maderae, species of the genera Locusta, Melanoplus, Periplaneta, the human flea (Pulex irritans), the desert locust (Schistocerca gregaria), and the brown-banded cockroach (Supella longipalpa).

[0289] Further examples include the flea order Siphonaptera, such as species of the genera Ceratophyllus, Ctenocephalides, sand fleas (Tunga penetrans), and mouse fleas (Xenopsylla cheopis).

[0290] Another example is the order Symphyla, such as the genus Scutigerella.

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

[0292] Further examples include the silverfish order (Zygentoma (=Thysanura)), such as the European silverfish (Lepisma saccharina) and the spotted silverfish (Thermobia domestica).

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

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

[0295] In yet another embodiment, important plant pests are plant parasitic nematodes belonging to the phylum Nematoda, namely nematodes that parasitize plants and cause damage to them. Plant nematodes include plant parasitic nematodes and soil-dwelling nematodes. Plant parasitic nematodes include, but are not limited to, ectoparasites such as species of the genera Xiphinema, Longidorus, and Trichodorus; hemiparasites such as species of the genera Tylenchulus; migratory endoparasites such as species of Pratylenchus, Radopholus, and Scutellonerna; sedentary parasites such as species of Heterodera, Globodera, and Meloidogyne; and species of the genera Ditylenchus Examples of stem and leaf endoparasites include species of the genera Aphelenchoides (spp.), Hirshmaniella (spp.), and Hirshmaniella (spp.). Furthermore, harmful root-parasitic soil nematodes include cyst-forming nematodes of the genera Heterodera or Globodera, and / or root-knot nematodes of the genera Meloidogyne. Harmful species of these genera include, for example, Meloidogyne incognata, Heterodera glycines, Globodera pallida, and Globodera rostochiensis.Other important genera that are significant as plant pests include species of Rotylenculus, Paratriclodorus, Pratylenchus penetrans, Radolophus simuli, Ditylenchus dispaci, Tylenchulus semipenetrans, Xiphinema, Bursaphelenchus, and especially Aphelenchoides, Bursaphelenchus, Ditylenchus, and Globodera. Examples include species of the genera Heterodera, Longidorus, Meloidogyne, Pratylenchus, Radopholus similis, Trichodorus, Tylenchulus semipenetrans, and Xiphinema.

[0296] In yet another embodiment, the plant pest is a virus, and the pesticide formulation of the present invention aims to treat viral infections in plants or to suppress viral infectivity, and the plant viruses include alphamovirus, alexivirus, alphacryptvirus, anuravirus, apskaviroid, aureusvirus, avenavirus, absinthe viroid, badnavirus, begomovirus, benivirus, betacryptvirus, betaflexiviridae, bromovirus, baimovirus, capylovirus, karavirus, carmovirus, kaurimovirus, cabemovirus, cheravirus, clostrovirus, cocadovroid, coleviroid, comovirus, clinivirus, cucumovirus, kurutovirus, citrabudovirus, dianthovirus, enamovirus, umbravirus and type B satellite virus, fabavirus, phisivirus, floivirus Selected from Russ, Hordei virus, Phostuvirus, Idaeovirus, Iller virus, Ipomovirus, Luteovirus, Macromovirus, Macrulavirus, Malafyvirus, Mastrevirus, Nanovirus, Necrovirus, Nepovirus, Nucleorhabdovirus, Oleavirus, Ophiovirus, Oryzavirus, Panicovirus, Peculivirus, Petuvirus, Phytoreovirus, Polerovirus, Pomovirus, Pospivirus, Potexvirus, Potivirus, Reovirus, Rhabdovirus, Rimovirus, Sadwavirus, SbCMV-like virus, Sequivirus, Sobemovirus, Tenuivirus, TNsatV-like satellite virus, Tobamovirus, Topocvirus, Tospovirus, Trichovirus, Trithymovirus, Tungrovirus, Timovirus, Umbravirus, Baricosavirus, Vitivirus, or Weikavirus.

[0297] Morphology of target antigen Based on the disclosures, it will be understood that the polypeptides of the compositions disclosed herein may be specific to, or can specifically bind to, several different forms of pest targets (e.g., fungal targets) for use as pesticides and biological control agents. It will also be expected that the polypeptides of the compositions disclosed herein may bind to a number of natural or synthetic analogs, variants, mutants, alleles, parts, and fragments of those pest targets. More specifically, it will be expected that the polypeptides of the compositions disclosed herein may bind to analogs, variants, mutants, alleles, parts, and fragments of said targets that (still) contain at least the binding site, part, or domain of the natural target to which these polypeptides bind.

[0298] formulation The polypeptide content contained in the pesticide compositions or biological control compositions disclosed herein can take on a wide range, and it is generally assumed that it is left to the manufacturer to adjust the concentration range of a particular polypeptide depending on the specific crop pest to be suppressed.

[0299] In certain embodiments, the present invention provides a pesticide composition comprising at least one polypeptide, wherein the heavy-chain variable domain is present in an amount effective for protecting or treating a plant or part of the plant from infection by the plant pathogen or other biological interactions with the plant pathogen.

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

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

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

[0303] In certain embodiments, the present invention provides a pesticide composition containing at least one polypeptide, wherein the concentration of the at least one polypeptide in the pesticide composition is 0.001 to 50% by weight.

[0304] In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 0.001 to 50% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may be 0.01 to 50% by weight. In yet another specific embodiment, the concentration of the polypeptide contained in the pesticide composition may 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 0.01 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 agriculturally suitable base and / or one or more suitable adjuvants.

[0308] The compositions according to the present invention may contain, in addition to the above-mentioned pest-repellent polypeptide, a solid or liquid base acceptable for the treatment of pests on plants and / or plant parts, and / or a surfactant also acceptable for the treatment of pests on plants and / or plant parts. In particular, inert conventional bases and conventional surfactants can be used. These compositions are suitable not only for immediate application by immersion or the use of appropriate equipment to the plants and / or plant parts to be treated, but also for commercially available concentrated compositions that need to be diluted before application to plants and / or plant parts.

[0309] These pesticide compositions according to the present invention may further contain any other type of component, such as protective colloids, adhesives, thickeners, thixotropes, penetrating agents, stabilizers, metal ion chelating agents, shape-retaining agents, flavoring agents, taste enhancers, sugars, sweeteners, and colorants. 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 pesticide compositions according to the present invention may further contain any other active ingredients, such as other antibacterial or antifungal active ingredients.

[0311] In this disclosure, the term “base” means a natural or synthetic organic or inorganic substance added to an anti-pest bioactive substance to enhance its application to a plant and / or one or more plant parts. Therefore, the base must generally be inert and acceptable in the agricultural field. The base may be solid (e.g., clay, natural or synthetic silicates, silica, resins, waxes, solid fertilizers) or liquid (e.g., water, alcohols, especially butanol).

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

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

[0314] Examples of solid composition forms include powders and granules (in which the active substance content can be up to 100%), particularly those obtained by impregnation of extruded, compressed, or granulated carriers, and granulation using powder as a starting material (in these cases, the active substance content in these granules is 0.5 to 80%). Such solid compositions can optionally be used in liquid form, for example, by diluting with water to increase or decrease viscosity, depending on the desired application type.

[0315] Examples of liquid composition forms or forms intended to constitute a liquid composition when applied include solutions, particularly water-soluble concentrates, emulsions, concentrated suspensions, powdered wettable powders (or spray powders), oils, and waxes.

[0316] Concentrated suspensions that can be applied by spraying are manufactured to provide a stable fluid that does not form precipitates, and typically contain 10-75% active substance, 0.5-15% surfactant, 0.1-10% thixotrope, 0-10% suitable additives (e.g., defoamers, corrosion inhibitors, stabilizers, penetrants, and adhesives), and a base of water or an organic liquid in which the active substance is insoluble or poorly soluble. Some organic solid or inorganic salt may be dissolved in the base to facilitate the prevention of precipitation or to prevent the gelation of water.

[0317] The pesticide compositions disclosed herein can be used as is, used as formulations thereof, or used in forms manufactured from them, such as aerosol dispensers, capsule suspensions, room temperature fogging agents, high temperature fogging agents, encapsulated granules, fine granules, concentrated flowable formulations for seed treatment, immediate-use solutions, powders, concentrated emulsions, oil-in-water emulsions, water-in-oil emulsions, coarse granules, coarse granules, oil-dispersible powders, oil-miscible concentrated flowable formulations, oil-miscible liquid formulations, foaming agents, pastes, seeds coated with pesticides, concentrated suspensions (concentrated flowable formulations), suspension-emulsion-concentrator formulations, soluble concentrates, suspensions, and Soluble powders, granules, water-soluble granules or tablets, water-soluble powders for seed treatment, powdered wettable powders, natural and synthetic materials impregnated with active compounds, polymer materials for seeds and microencapsulation with jackets, microencapsulated biomaterial particles, for example, those described in WO2018 / 201160, WO2018 / 201161 and WO2019 / 060903, as well as ULV room temperature and high temperature fogging agents, (pressurized) gases, gas generators, plant rodlets, powders for dry seed treatment, solutions for seed treatment, high-concentration trace (ULV) liquids, high-concentration trace (ULV) suspensions, water-dispersible granules or tablets, and water-dispersible powders for slurry treatment.

[0318] These formulations are manufactured by known methods, which involve mixing an active compound or a combination of active compounds with conventional additives, such as conventional bulking agents, and further mixing with solvents or diluents, emulsifiers, dispersants, and / or binders or fixatives, wetting agents, water repellents, drying agents and UV stabilizers as needed, colorants, pigments, defoamers, preservatives, secondary thickeners, adhesives, gibberellin and water, and other processing aids.

[0319] These compositions include not only compositions that can be immediately applied to the plants or seeds to be treated using appropriate equipment such as sprayers or powderers, but also commercially available concentrated compositions that need to be diluted before application to crops.

[0320] Plant protection or treatment methods In a particular embodiment, the present invention provides a method for protecting or treating a plant or part of a plant from infection by a plant pathogen or other biological interactions with a plant pathogen, comprising the step of directly or indirectly applying at least one of the pesticide compositions or polypeptides disclosed herein. The composition or polypeptide can be applied under conditions effective for protecting or treating the plant or part of the plant from infection by a plant pathogen or other biological interactions with a plant pathogen.

[0321] In certain embodiments, these methods include applying the pesticide composition disclosed herein to the plant or a portion of the plant directly or indirectly at an application rate of, for example, more than 50 g of the pesticide composition per hectare, but not limited to, for example more than 75 g of the pesticide composition per hectare, for example more than 100 g of the pesticide composition per hectare, or in particular more than 200 g of the pesticide composition per hectare.

[0322] In certain embodiments, these methods include applying the pesticide composition disclosed herein to the plant or a portion of the plant directly or indirectly at an application rate of, for example, 50 g to 200 g of the pesticide composition per hectare, but not limited to, for example, 50 g to 200 g of the pesticide composition per hectare, particularly 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 of the pesticide composition per hectare.

[0323] In yet another embodiment, the present invention provides a method for controlling or suppressing plant pests, comprising the step of applying the pesticide composition or biological control composition according to the present invention to a plant such as a crop or a part of a plant or crop at an application rate of less than 50 g of polypeptide per hectare. In a particular embodiment, the application rate is less than 45 g / ha, less than 40 g / ha, less than 35 g / ha, less than 30 g / ha, less than 25 g / ha, less than 20 g / ha, less than 15 g / ha, less than 10 g / ha, less than 5 g / ha, less than 1 g / ha, or less than or equal to the polypeptide.

[0324] Naturally, farmers can adjust application rates in response to environmental pressures on crops and plant pests. These rate variations are specified in the technical sheet accompanying the specific pesticide composition.

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

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

[0327] The pesticide composition or biological control composition or polypeptide according to the present invention can be applied to crops using any method suitable for applying the pesticide composition or biological control composition to crops, but is not limited to, spraying (low-concentration high-volume (HV), high-concentration low-volume (LV), and high-concentration low-volume (ULV) spraying), brush application, bandaging, dripping, coating, dipping, immersion, spraying, fogging, application as droplets, mist, or aerosol.

[0328] Accordingly, in certain embodiments, methods for protecting or treating a plant or part of a plant disclosed herein from infection by a plant pathogen or other biological interaction with a plant pathogen include, for example, the step of applying the pesticide composition directly or indirectly to the plant or part of the plant by spraying, atomizing, foaming, fogging, hydroculture, hydroponics, coating, immersion, and / or powder coating.

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

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

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

[0332] According to the method disclosed herein, the pesticide composition or biological control composition can be applied to a crop once, or it can be applied two or more times with intervals between each application. According to the method of the present invention, the pesticide composition or biological control composition according to the present invention can be applied to the crop alone, or it can be applied as a mixture with other materials, preferably other pesticide compositions or biological control compositions, or the pesticide composition or biological control composition according to the present invention can be applied separately to the same crop at different times with other materials, preferably other pesticide compositions or biological control compositions. According to the method of the present invention, the pesticide composition or biological control composition according to the present invention can be applied to the crop preventively, or it can be applied after the target pest has been identified in the specific crop to be treated.

[0333] The pesticide compositions disclosed herein can be applied directly to plants, crops, or one or more parts of said plants by the method described above, for example, to the entire plant or to one or more parts of said plant before or after harvest. Application before harvest may produce an effect after harvest. In other specified embodiments, the pesticide compositions disclosed herein can be applied directly to one or more parts of said plants by the method described above, for example, to petioles, leaves, tubers, stems, shoots, seeds, fruits, roots, flowers, grains, buds, etc.

[0334] The treatment methods disclosed herein can also be used in the field of protecting stored materials from attacks by plant pathogens. In these treatment methods, the composition of the present invention may be applied before or after harvest. According to the present invention, the term “stored materials” is considered to mean natural substances of plant or animal origin, and processed products thereof that have been taken out of their natural life cycle and for which long-term protection is desired. Plant-derived stored materials such as plants or parts thereof (e.g., petioles, leaves, tubers, seeds, fruits, or grains) can be protected in their freshly harvested state, or they can be protected in processed forms such as pre-drying, wetting, crushing, grinding, pressing, or roasting. Wood is also included in the definition of stored materials and may be unprocessed wood such as building timber, power transmission towers, and fences, or finished products such as furniture or wood products. Animal-derived stored materials include animal hides, leather, fur, and hair. The composition of the present invention can prevent adverse effects such as corrosion, discoloration, or mold. It is preferable to consider "stored goods" to mean natural substances of plant origin and their processed products, and more preferably to mean fruit trees such as pome fruits, drupes, soft fruits, and citrus fruits and their processed products.

[0335] The pesticide compositions disclosed herein can also be applied indirectly to a plant, crop, or one or more parts of the plant by the method described above, either before or after harvest, for example, indirectly to the entire plant or to one or more parts of the plant. The pesticide compositions disclosed herein can be applied just before harvest, for example, about three weeks before harvest, for example, two weeks before harvest, one week before harvest, or within one week before harvest. Pre-harvest application can produce effects after harvest. Accordingly, in a given embodiment, the pesticide compositions disclosed herein can be applied indirectly to a plant, crop, or one or more parts of the plant by the method described above, for example, by applying the pesticide composition to the environment or medium (but not limited to, air, soil, hydroponics, hydroculture) in which the plant or the one or more parts of the plant are growing or stored, or by applying it to the liquid medium (e.g., aqueous liquid medium or water) in which the plant or the one or more parts of the plant are growing or stored.

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

[0337] Therefore, naturally in the context of this application, treatment of plants or plant parts with the pesticide compositions disclosed herein may be carried out directly, or it may be carried out by treating the environment, habitat or storage area by conventional treatment methods, such as irrigation (drenching), drip irrigation, spraying, vaporization, atomization, scattering, powdering, foaming, and as a powder. Furthermore, it is also possible to apply the compositions by high-concentration micro-methods, and it is also possible to inject active compound formulations or the active compounds themselves into the soil.

[0338] In certain embodiments, a method for protecting or treating a plant or part of a plant disclosed herein from infection by a plant pathogen or other biological interactions with a plant pathogen includes the step of applying the pesticide composition directly or indirectly to the plant or part of the plant at a pre-harvest or post-harvest stage.

[0339] According to a particular embodiment, the harvested product is a fruit tree, flower, nut, or vegetable, preferably a fruit tree or vegetable with an inedible peel, preferably selected from avocado, banana, plantain, lemon, grapefruit, melon, orange, pineapple, kiwifruit, guava, mandarin orange, mango, pumpkin, strawberry, grape, and rib, more preferably banana, orange, lemon, and peach, and especially banana. According to other specific embodiments, the harvested product is preferably cut flowers of ornamental plants, selected from the genera Alstroemeria, carnation, chrysanthemum, freesia, gerbera, gladiolus, gypsophila, sunflower, hydrangea, lily, lisianthus, rose, and summer flowers.

[0340] The plant species to which the pesticide compositions disclosed herein can be applied include, but are not limited to, corn, soybeans, alfalfa, cotton, sunflowers, rapeseed oil seeds such as corn, soybeans, alfalfa, canola, sunflowers, turnips (Brassica napus) (e.g., canola), turnips (Brassica rapa), mustard (B. juncea) (e.g., (field) mustard), and Abyssinian mustard (Brassica carinata), palm species (Arecaceae sp.) (e.g., oil palm, coconut), rice, wheat, sugar beets, sugarcane, oats, rye, barley, millet and sorghum, rye, flax, nuts, grapes and climbing plants, as well as various fruit trees and vegetables belonging to various botanical classifications, for example, Rosaceae species. sp.) (For example, in addition to pome fruits such as apples and pears, drupes such as apricots, cherries, almonds, plums and peaches, and berries such as strawberries, raspberries, red currants, black currants and European currants), Ribesioidae sp., Juglandaceae sp., Betulaceae sp., Anacardiaceae sp., Fagaceae sp., Moraceae sp., Oleaceae sp. (for example, olive trees), Actinidaceae sp., Lauraceae sp. (for example, avocados, cinnamon, camphor), Musaceae sp. (for example, banana trees and plantains), Rubiaceae sp. 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, chili pepper, paprika, eggplant, tobacco), Liliaceae sp., Asteraceae sp.)(e.g., lettuce, artichoke and root chicory, endive or chicory including chicory), Apiaceae sp. (e.g., carrots, parsley, celery and celeriac), Cucurbitaceae sp. (e.g., cucumbers including gherkins, pumpkins, watermelons, calabash and melons), Alliaceae sp. (e.g., leeks and onions), Cruciferae sp. (e.g., white cabbage, red cabbage, broccoli, cauliflower, Brussels sprouts, bok choy, kohlrabi, radishes, horseradish, cress and Chinese cabbage), Leguminosae sp. (e.g., peas, peas, lentils and legumes, e.g., green beans and broad beans), Chenopodiaceae This includes useful and ornamental plants in gardens and forests, including (e.g., Swiss chard, fodder beet, spinach, beets), Linaceae sp. (e.g., hemp), Cannabeaceae sp. (e.g., cannabis), Malvaceae sp. (e.g., okra, cacao), Papaveraceae (e.g., poppy), Asparagaceae (e.g., asparagus), turfgrass, lawngrass, pasture grass, and stevia (Stevia rebaudiana), as well as genetically modified varieties of these plants in each case.

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

[0342] Accordingly, in a particular embodiment, the present invention also provides a post-harvest treatment method for protecting or treating harvested plants or harvested parts of said plants from infection by plant pathogens or other biological interactions with plant pathogens, comprising the step of applying the pesticide composition disclosed herein directly or indirectly to said harvested plants or harvested parts of said plants under conditions effective for protecting or treating said harvested plants or harvested parts of said plants from infection by plant pathogens or other biological interactions with plant pathogens. According to a particular embodiment, said harvested products are fruit trees, flowers, nuts or vegetables, and fruit trees or vegetables with inedible peels are preferred, selected from avocado, banana, plantain, lemon, grapefruit, melon, orange, pineapple, kiwifruit, guava, mandarin orange, mango and pumpkin, more preferably banana, orange, lemon and peach, and especially banana. According to other specific embodiments, the harvested product is cut flowers of ornamental plants, preferably selected from the genera Alstroemeria, carnation, Chrysanthemum, Freesia, Gerbera, Gladiolus, Gypsophila, Helianthus, Hydrangea, Lilium, Lisianthus, rose, and summer flowers. According to other specific embodiments, the harvested product is mowed pasture or trees.

[0343] Post-harvest problems include, for example, lenticels, leaf burn, senescence damage, bitter pit, scorching, honeycombing, browning, vascular damage, CO2 damage, CO2 or O2 deficiency, and softening.

[0344] Fungal diseases are caused by the following fungi, for example: Mycosphaerella species (e.g., Mycosphaerella musae, Mycosphaerella fraga ae, Mycosphaerella citri); Mucor species (e.g., Mucor piriformis); Monilinia species (e.g., Monilinia fructigena, Monilinia laxa); Phomopsis species (e.g., Phomopsis natalensis); and Colletotrichum species. 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 *Citria*, *Alternaria alternata*; *Septoria* spp., for example, *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.), 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* species (spp.), for example, Phytophthora citrophthora, Phytophthora fragariae, Phytophthora cactorum, Phytophthora parasitica; Phacidiopycnis spp., for example, Phacidiopycnis malirum; Gloeosporium spp., for example, Gloeosporium album, Gloeosporium perennans, Gloeosporium fructigenum, Gloeosporium singulata singulata; Geotrichum spp., e.g., Geotrichum candidum; Phlyctaena spp., e.g., Phlyctaena vagabunda; Cylindrocarpon spp., e.g., Cylindrocarpon mail; Stemphyllium spp., e.g., Stemphyllium vesica um; Thielaviopsis spp., e.g., Thielaviopsis paradoxy; Aspergillus spp., e.g., Aspergillus niger niger), Aspergillus carbonarius; Nectria spp. species, for example, Nectria galligena; Cercospora spp. species.For example, Cercospora angreci, Cercospora apii, Cercospora atrofiliformis, Cercospora musae, and Cercospora zeaemaydis can be pathogenic.

[0345] In another embodiment, the present invention provides the use of the pesticide compositions disclosed herein as pest control agents, such as biostatic agents or pest control agents (including, but not limited to, fungiostatic agents or fungicidal agents).

[0346] In certain embodiments, the plant pests controlled by the method according to the present invention are the plant pathogenic fungi defined above. As a result of applying the method according to the present invention, the number of lesions, lesion size, and the degree of spore formation of the fungal pathogen can all be reduced.

[0347] Medical use In other specified embodiments, the present invention provides a method for protecting or treating a human or animal from infection by a harmful organism, particularly fungi, or a method for treating an infection of a human or animal caused by a harmful organism, particularly fungi, comprising the step of directly or indirectly applying or administering a composition containing at least one polypeptide of the present invention that specifically binds to at least a harmful organism (but not limited to fungi) to the human or animal or a part of the human or animal. The composition can be applied under conditions that are effective in protecting or treating the human or animal from the harmful organism.

[0348] Accordingly, the present invention provides polypeptides of the present invention that specifically bind to pest targets for use in methods for preventing and / or treating at least one disease and / or disorder caused by pests in a subject (e.g., a disease and / or disorder caused by fungi). The present invention further provides compositions of the present invention for use in methods for preventing and / or treating at least one disease and / or disorder caused by pests in a subject (e.g., a disease and / or disorder caused by fungi). The present invention further provides polypeptides of the present invention that specifically bind to pest targets for use in methods for preventing and / or treating infectious diseases caused by pests in a subject (e.g., fungal infections). The present invention further provides compositions of the present invention that specifically bind to pest targets for use in methods for preventing and / or treating infectious diseases caused by pests in a subject (e.g., fungal infections). In certain embodiments, the present invention further provides a method for preventing and / or treating at least one disease and / or disorder caused by pests, comprising the step of administering a pharmaceutically active amount of one or more amino acid sequences, polypeptides and / or pharmaceutical compositions disclosed herein to a subject in need of treatment, etc. In particular, the pharmaceutically active amount can be sufficient to inhibit, interfere with, or suppress the biological activity or pathway of one or more of the conjugated harmful organisms (to produce a circulating concentration of the amino acid sequence or polypeptide).

[0349] Accordingly, in a given embodiment, the present invention provides a composition containing at least one polypeptide that specifically binds to harmful organisms, for use as a pest control agent in animals or humans suffering from diseases and / or disorders caused by harmful organisms (e.g., fungi).

[0350] In certain embodiments, the pest control agent is a biostatic agent or a pest control agent. In certain embodiments, the pest control agent is a fungiostatic agent or a fungicide.

[0351] Furthermore, in a predetermined embodiment, the present invention provides a method for preventing and / or treating diseases and / or disorders caused by harmful organisms, (a) A step of preparing an amino acid sequence, polypeptide, or composition disclosed herein, (b) A method comprising the step of administering the amino acid sequence, polypeptide, or pharmaceutical composition to a patient suffering from the disease and / or disorder caused by a pest.

[0352] The efficacy of the polypeptides disclosed herein and compositions containing such polypeptides can be tested using any suitable in vitro assay, cell assay, in vivo assay and / or animal model known in itself, or any combination thereof, depending on the specific disease or disorder being addressed. Suitable assays and animal models are obvious to those skilled in the art, and assays and animal models used in the experimental section below and in the prior art referenced herein are available. Those skilled in the art can generally select a suitable in vitro assay, cell assay or animal model to test the amino acid sequences and polypeptides disclosed herein for their binding to or ability to affect the activity of harmful organism targets or antigens, and / or the biological mechanisms in which they are involved, as well as their therapeutic and / or prophylactic effects on one or more diseases and disorders associated with harmful organism antigens.

[0353] Pharmaceutical composition In yet another embodiment, the present invention provides a pharmaceutical composition (hereinafter also referred to as the pharmaceutical composition of the present invention) comprising one or more amino acid sequences, polypeptides and / or nucleic acid sequences disclosed herein and at least one pharmaceutically acceptable base material optionally. According to a certain particular embodiment, the pharmaceutical composition disclosed herein may further optionally contain at least one other pharmaceutically active compound.

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

[0355] In particular, the present invention provides a pharmaceutical composition containing a polypeptide suitable for prevention, treatment and / or diagnosis in warm-blooded animals, especially mammals, and more specifically, humans.

[0356] The present invention further provides a pharmaceutical composition containing the amino acid sequence and polypeptide disclosed herein, which can be used for veterinary purposes in preventing and / or treating or diagnosing one or more diseases, disorders, or conditions related to harmful organisms, such as fungi, by conjugating the polypeptide disclosed herein with a pest target.

[0357] In general, for pharmaceutical use, the polypeptides disclosed herein can be formulated as pharmaceutical preparations or compositions comprising at least one polypeptide disclosed herein, at least one pharmaceutically acceptable base, diluent, or excipient and / or adjuvant, and optionally one or more other pharmaceutically active polypeptides and / or compounds. Such preparations are likely to be suitable for oral, parenteral, topical, or inhalation administration. Accordingly, the amino acid sequences or polypeptides disclosed herein and / or compositions containing them can also be administered, for example, orally, intraperitoneally, intravenously, subcutaneously, intramuscularly, transdermally, topically, suppositories, or by inhalation, depending on the specific pharmaceutical preparation or composition used. Clinicians may select an appropriate route of administration and a pharmaceutical preparation or composition suitable for use in such administration.

[0358] The pharmaceutical composition may further contain a suitable binder, disintegrant, sweetener, or flavoring agent. Tablets, pills, or capsules may be coated with, for example, gelatin, wax, or sugar. Furthermore, the amino acid sequences and polypeptides disclosed herein may be incorporated into sustained-release formulations and devices.

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

[0360] The useful doses of the amino acid sequences and polypeptides disclosed herein can be determined by comparing their in vitro and in vivo activities in animal models. Methods for extrapolating effective doses in mice and other animals to humans are known to those skilled in the art.

[0361] The amount of amino acid sequences and polypeptides disclosed herein required for prevention and / or treatment will likely vary depending not only on the specific amino acid sequence or polypeptide selected, but also on the route of administration, the type of disease being treated, and the patient's age and health condition, and will ultimately be left to the discretion of the attending physician or clinician. The dosage of amino acid sequences and polypeptides disclosed herein may also vary depending on the target cells, tumors, tissues, transplanted tissues, or organs.

[0362] The amino acid sequences or polypeptides and / or compositions containing them disclosed herein are administered in accordance with a therapeutic regimen suitable for the prevention and / or treatment of a disease or disorder that is subject to prognosis, diagnosis, prevention, or treatment. A clinician can generally determine an appropriate therapeutic regimen. Generally, a therapeutic regimen would involve administering one or more amino acid sequences or polypeptides or one or more compositions containing them disclosed herein in one or more pharmaceutically effective amounts or doses.

[0363] The desired dose can be appropriately provided as a single dose or in multiple doses at appropriate intervals (each dose may be further divided into multiple doses). The administration regimen may include long-term administration (i.e., at least two weeks, e.g., several months or several years) or daily administration.

[0364] The amino acid sequences or polypeptides disclosed herein will be administered in amounts determined by healthcare professionals, particularly based on the severity of the condition and the patient being treated. Generally, for each indication, an optimal dose will be determined, specifying the amount to be administered per kg of body weight per day, either continuously (e.g., by intravenous fluid) or once daily or in multiple doses throughout the day. Clinicians can generally determine an appropriate daily dose depending on the factors referred to herein. It is also obvious that in specific cases, clinicians may choose to deviate from these amounts, for example, based on the above factors and their own professional judgment.

[0365] In particular, the amino acid sequences or polypeptides disclosed herein may be used or may be used in combination with other pharmaceutically active compounds or active ingredients used for the prevention and / or treatment of the diseases and disorders referred to herein, whether or not a synergistic effect is resulting. Examples of such compounds and active ingredients, as well as their routes of administration and pharmaceutical formulations or compositions, are obvious to clinicians.

[0366] The compositions of the present invention can be used in combination with known antifungal agents. Suitable antifungal agents include, but are not limited to, azoles (e.g., fluconazole, itraconazole), polyenes (e.g., amphotericin B), flucytosine, and squalene epoxidase inhibitors (e.g., terbinafine) [see also ref57]. The composition may be used in combination with known antiviral agents, for example, 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, and 1,3-dioxolane derivatives (e.g., 2',3'-dideoxyl-3'-thiacytidine). Examples include oxetanosine analogs and their carbocyclic derivatives (e.g., cyclobuto-G), as well as derivatives of 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and 9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA), tetrahydroimidazo[4,5,1jk][1,4]-benzodiazepine-2(1H)one (TIBO), 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT), dipyrido[3,2-b:2',3'-e]-[1,4]diazepine-6-one (nevirapine) and pyridine-2(1H)one derivatives, 3TC, etc.

[0367] The aforementioned amino acid sequence, polypeptide, and pharmaceutical composition are used to control Candida species such as Candida albicans; Cryptococcus species such as Cryptococcus neoformans; Enterococcus species such as Enterococcus faecalis; Streptococcus pneumoniae, Streptococcus mutans, and Streptococcus agara in animals or humans. Streptococcus species such as S. agalactiae and S. pyogenes; Leishmania species such as Leishmania major and Leishmania infantum; Acanthamoeba species such as Acanthamoeba castellani; Aspergillus fumigatus and Aspergillus flavus (A. Aspergillus species such as Aspergillus flavus; Pneumocystis species such as Pneumocystis carinii; Mycobacterium species such as Mycobacterium tuberculosis; Pseudomonas species such as Pseudomonas aeruginosa; Staphylococcus species such as Staphylococcus aureus * 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 in treating infections caused by Histoplasma species (such as capsulatum), Paracoccidioides species (such as P. brasiliensis), Pythium species (such as P. insidiosum), and Escherichia species (such as E. coli). The aforementioned amino acid sequences, polypeptides, and pharmaceutical compositions are, but are not limited, particularly useful in the treatment of diseases such as candidiasis, aspergillosis, cryptococcosis, cutaneous mycoses, sporotrichosis and other deep-seated mycoses, blastomycosis, histoplasmosis, coccidioidomycosis, paracoccidioidomycosis, Pneumocystis pneumonia, thrush, tuberculosis, mycobacterial infections, respiratory infections, scarlet fever, pneumonia, impetigo, rheumatic fever, sepsis, sepsis with bacteremia, cutaneous and visceral leishmaniasis, Acanthamoeba keratitis, keratitis, cystic fibrosis, typhoid fever, gastroenteritis, and hemolytic uremic syndrome. Anti-Candida albicans (C. albicans) activity is particularly useful in the treatment of infections in AIDS patients.

[0368] Production and manufacturing method of the polypeptide The present invention further provides methods for preparing or generating the polypeptide sequences, nucleic acids encoding these polypeptide sequences, and methods for producing host cells, products, and compositions containing these polypeptide sequences. Preferred non-limiting examples of such methods will be apparent from the detailed description of this application.

[0369] As will be obvious to those skilled in the art, one example of a particularly useful method for producing the polypeptide sequences disclosed herein is generally, (a) A step of expressing a nucleotide sequence encoding a polypeptide sequence disclosed herein or a vector or gene construct comprising a nucleotide sequence encoding the polypeptide, (b) optionally comprising the step of isolating and / or purifying the polypeptide sequence.

[0370] In a particular embodiment envisioned in this application, the pest-specific polypeptide sequence can be obtained by a method comprising the steps of preparing a random library of amino acid sequences and screening this library for amino acid sequences that can specifically bind to a pest target.

[0371] Therefore, in a particular embodiment, the method for producing the polypeptide sequence disclosed herein is a) A step of preparing a set, collection, or library of amino acid sequences; b) A step of screening the set, collection or library for amino acid sequences that bind to and / or have affinity for the pest target; c) The process includes the step of isolating an amino acid sequence that binds to and / or has affinity for the pest target.

[0372] In such a method, the set, collection, or library of polypeptide sequences may be any suitable set, collection, or library of amino acid sequences. For example, the set, collection, or library of amino acid sequences may be a set, collection, or library of immunoglobulin fragment sequences (as described in this application), such as a naive set, collection, or library of immunoglobulin fragment sequences; a synthesized or semi-synthetic set, collection, or library of immunoglobulin fragment sequences; and / or a set, collection, or library of immunoglobulin fragment sequences subjected to affinity maturation.

[0373] In certain embodiments of this method, the set, collection, or library of amino acid sequences may be, for example, an immunization set, collection, or library of immunoglobulin fragment sequences derived from mammals appropriately immunized with a hazardous organism target or a suitable antigenic determinant (e.g., its antigenic portion, fragment, region, domain, loop, or other epitope) based on or derived from a hazardous organism target. In one particular embodiment, the antigenic determinant may be an extracellular portion, region, domain, loop, or other extracellular epitope.

[0374] In the above method, the set, collection, or library of polypeptide sequences can be presented on the surface of a phage, phagemid, ribosome, or suitable microorganism (e.g., yeast) for purposes such as facilitating screening. Suitable methods, techniques, and host organisms for presenting and screening amino acid sequences (sets, collections, or libraries) are obvious to those skilled in the art, for example, based on the detailed disclosure of this application. See also the article by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

[0375] In other embodiments, the method for producing the polypeptide sequence disclosed herein is: a) A step of preparing a cell collection or sample expressing a polypeptide sequence; b) A step of screening the cell collection or sample for cells that express amino acid sequences capable of binding to and / or having affinity for harmful organism targets; c) The method includes at least the steps of (i) isolating the amino acid sequence, or (ii) expressing the amino acid sequence after isolating the nucleic acid sequence encoding the amino acid sequence from the cell.

[0376] The cell collection or sample may be, for example, a collection or sample of B cells. Furthermore, in this method, the cell sample may be derived from mammals appropriately immunized with a fungal target or a suitable antigenic determinant (e.g., its antigenic portion, fragment, region, domain, loop, or other epitope) based on or derived from a fungal target. In one particular embodiment, the antigenic determinant may be an extracellular portion, region, domain, loop, or other extracellular epitope.

[0377] In another embodiment, a method for producing polypeptide sequences specific to harmful organism targets is: a) the step of preparing a set, collection, or library of nucleic acid sequences encoding polypeptides or amino acid sequences; b) A step of screening the set, collection, or library of nucleic acid sequences for nucleic acid sequences that can bind to and / or have affinity for the pest target; c) The process may include at least the step of expressing the amino acid sequence after isolating the nucleic acid sequence.

[0378] In the above method, the harmful organism target can be the lipid-containing fraction of the cell membrane of a fungus (e.g., Botrytis cinerea or other fungi). The lipid-containing fraction may be obtained by chromatography. For example, the lipid-containing fraction may be obtained by a method comprising the steps of fractionating the hyphae of a fungus (e.g., Botrytis cinerea or other fungi) by total lipid extract thin-layer chromatography, and selecting a fraction whose retention factor (Rf) is greater than that of the ceramide fraction and less than that of the nonpolar phospholipid fraction.

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

[0380] In particular, in such a method, the set, collection or library of nucleic acid sequences encodes a set, collection or library of polypeptides (such as V H domains or V HH domains). 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 sequences.

[0381] In the above method, for the purpose of facilitating screening, etc., the set, collection or library of nucleotide sequences can be displayed on the surface of phage, phagemid, ribosome or a suitable microorganism (such as yeast). Methods, techniques and host organisms suitable for presenting and screening nucleotide sequences (sets, collections or libraries) encoding amino acid sequences will be apparent to those skilled in the art based on, for example, the detailed disclosure of the present application. See also the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

[0382] The present invention further relates to polypeptide sequences that can be obtained by the above method, or comprising one of the above methods, and further comprising the steps of: determining at least the nucleotide sequence or amino acid sequence of the immunoglobulin sequence; and expressing or synthesizing the amino acid sequence by a method known in itself, such as expression in a host cell or host organism or by a chemical synthesis method.

[0383] Isolation of polypeptide sequences Depending on the circumstances, the method for producing amino acid sequences that specifically bind to fungal targets as envisioned in this application may further include the step of isolating from the amino acid sequence library at least one polypeptide having detectable binding affinity or detectable in vitro activity to a harmful organism target.

[0384] These methods may further include a step of amplifying a sequence encoding at least one polypeptide that has a detectable binding affinity to a toxic organism target or a detectable in vitro activity against that target. For example, a phage clone presenting a specific amino acid sequence obtained from the selection step of the methods described herein can be amplified by reinfection with a host bacterium and incubation in a growth medium.

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

[0386] When polypeptide sequences contained in a set, collection, or library of amino acid sequences are presented on the surface of a suitable cell, phage, or particle, it is possible to isolate the nucleotide sequences encoding the amino acid sequences from the cell, phage, or particle. Thus, the nucleotide sequences of selected amino acid sequence library members can be determined by routine sequencing methods.

[0387] In other specific embodiments, the polypeptide production method envisioned in this application includes the step of expressing the nucleotide sequence in a host organism under appropriate conditions to obtain the actual desired amino acid sequence. This step can be carried out by methods known to those skilled in the art.

[0388] Furthermore, polypeptide sequences thus obtained that have detectable binding affinity to harmful organism targets or detectable in vitro activity against them can be optionally synthesized as soluble protein constructs after their sequences have been identified.

[0389] For example, polypeptide sequences obtained, obtainable, or selected by the above method can be synthesized using recombinant or chemical synthesis methods known in the art. Furthermore, amino acid sequences obtained, obtainable, or selected by the above method can also be produced by genetic recombination technology. Therefore, a method for synthesizing polypeptide sequences obtained, obtainable, or selected by the above method may include a step of transforming or infecting host cells with nucleic acids or vectors encoding amino acid sequences that have a detectable binding affinity to a pest target or a detectable in vitro effect on its activity. Thus, amino acid sequences that have a detectable binding affinity to a pest target or a detectable in vitro effect on its activity can be produced by recombinant DNA methods. DNA encoding amino acid sequences can be easily synthesized using conventional procedures. Once the DNA is produced, it can be introduced into an expression vector, and then the vector can be transformed or transfected into host cells such as E. coli or any suitable expression system to obtain expression of the amino acid sequence in recombinant host cells and / or in a culture medium containing these recombinant host cells.

[0390] Naturally, as is well known to those skilled in the field of protein expression and purification, polypeptides produced from expression vectors using a suitable expression system may be tagged with, for example, a His tag or other sequence tags to facilitate purification (generally at the N-terminus or C-terminus of the amino acid sequence).

[0391] Transformation or transfection of host cells with nucleic acids or vectors can be carried out by various means known to those skilled in the art, including calcium phosphate DNA coprecipitation, DEAE-dextran transfection, polyblen transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and particle gun methods.

[0392] Suitable host cells for expressing 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), regardless of whether they are located in vitro or in vivo. For example, the host cell can be located in a transgenic plant or animal.

[0393] Accordingly, the present invention further provides a method for producing a polypeptide sequence having a detectable binding affinity to a harmful organism target or a detectable in vitro activity against its activity, comprising the steps of 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 invention further provides a method for producing a polypeptide sequence having a detectable binding affinity to a harmful organism target or a detectable in vitro activity against its activity, comprising the steps of preparing a host cell into which the nucleic acid sequence or vector encoding the polypeptide has been introduced, and expressing the polypeptide under appropriate conditions. The method of the present invention may further include the step of isolating the polypeptide, for example, isolating the polypeptide from a cell culture medium or fermentation broth or from within the host cell (for example, after the step of lysing the host cell).

[0394] In yet another embodiment, the present invention further provides a method for producing (synonymously, "or manufacturing") the pesticide composition or biological control composition disclosed herein.

[0395] In certain embodiments, the present invention provides a method for producing the pesticide composition disclosed herein, - A step of obtaining at least one polypeptide that specifically binds to harmful organisms, The present invention provides a method comprising at least the step of formulating the polypeptide or a functional fragment thereof into a pesticide composition.

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

[0397] In other specific embodiments of these methods, the step of obtaining at least one polypeptide that specifically binds to a pest is: a) the process of preparing a set, collection, or library of polypeptide sequences; b) A step of screening the set, collection or library of polypeptide sequences for sequences that specifically bind to and / or have affinity for harmful organisms, and optionally, c) The process includes isolating the polypeptide sequence which specifically binds to and / or has affinity for harmful organisms.

[0398] The present application further provides a method for producing (or manufacturing) the pesticide composition or biological control composition disclosed herein, comprising the step of formulating an amino acid sequence or polypeptide of 80 to 200 amino acids or other suitable subranges defined above, which has pest control activity, together with at least one conventional pesticide adjuvant.

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

[0400] Specifically, the 80-200 amino acid sequences or polypeptides disclosed herein, or other suitable subranges defined above, can be produced by chemical synthesis.

[0401] Furthermore, it is disclosed that amino acid sequences or polypeptides of 80 to 200 amino acids or other suitable subranges defined above can be prepared in vitro using a recombinant microbial expression system 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, decontaminated, buffered and / or further processed before formulation with conventional pesticide adjuvants.

[0402] Specifically, recombination generally involves inserting a DNA molecule expressing a target amino acid sequence, protein, or polypeptide into a heterologous expression system (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. DNA transcription depends on the presence of a promoter. Similarly, mRNA translation in prokaryotic cells depends on the presence of appropriate prokaryotic signals that differ from eukaryotic signals. For a discussion on maximizing gene expression, see Roberts and Lauer, Methods in Enzymology 68:473 (1979). Regardless of the specific regulatory sequence used, the DNA molecule is cloned into the vector 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 is cloned into an expression system, it is ready for uptake by a host cell. Such uptake can be carried out 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, and plants. Optionally, the recombinant host cell may be a host cell expressing a native or recombinant functional type III secretory system. This is described in detail in US6,596,509. As a result of expressing a functional type III secretory system, the cell will express the polypeptide and subsequently secrete the protein into the culture medium. This simplifies the isolation and purification of the polypeptide. The recombinant host cell can be grown in a suitable fermentation chamber, preferably under temperature and nutritional 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 times its volume of buffer 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 include, for example, potassium phosphate buffer and Tris-EDTA buffer. The concentration of the buffer 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 to 30 minutes. The heated suspension is then cooled. A suitable cooling temperature is not limited, but is about 35 to 55°C, preferably about 45°C. After cooling, the bacterial cells in the bacterial suspension are lysed as needed to release polypeptides. Cell lysis can be carried out, for example, by contacting the bacterial suspension with lysozyme. The concentration of lysozyme can be about 2 ppm to 100 ppm. Alternatively, cell lysis may be performed by non-chemical methods such as high pressure or sonication, both of which are well known to those with ordinary skill in the art. It is desirable to incubate the bacterial suspension after cell lysis. The appropriate incubation time can vary. For example, it is 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 cell debris and denatured proteins resulting from the heat treatment in the preceding step. In one embodiment, the extract is centrifuged for about 10-20 minutes to remove some of the cell debris. The appropriate centrifugal speed can be about 4,000-20,000 rpm, and the spin-down time can be about 10-20 minutes. Subsequently, further cell debris is removed by heat treatment and centrifugation of the supernatant, and a liquid extract substantially free of cell debris is obtained by removing more than 60%, 70%, 80%, 90%, or 95% of the total solids. The subsequent heat treatment can be carried out at a temperature of approximately 60°C for up to approximately 2 hours, at approximately 100°C for approximately 10 minutes, or at approximately 121°C for approximately 5 minutes under a pressure of 15 psi. These temperatures and times may vary depending on other conditions.A method for producing a stable liquid composition containing the amino acid sequence or polypeptide disclosed herein further comprises the steps of introducing a biocide and, optionally, one or both of a protease inhibitor and a nonionic surfactant into the liquid extract to obtain a liquid composition containing the polypeptide. In one embodiment, the protease inhibitor is introduced into the liquid extract without the addition of a nonionic surfactant. In another embodiment, the nonionic surfactant is introduced into the liquid extract without the addition of a protease inhibitor. In yet another embodiment, both the protease inhibitor and the nonionic surfactant are introduced into the liquid extract. In yet another embodiment, neither the protease inhibitor nor the nonionic surfactant is introduced into the liquid extract. Alternatively, the stability of the liquid composition disclosed herein can be evaluated 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 composition disclosed herein can be measured by comparing the amount of protein in the matured liquid composition with the amount in the freshly prepared liquid composition or a previous quantitative determination performed with the same composition. Measuring protein stability is strongly correlated with maintaining its activity.

[0403] Conventional pesticide additives are well known in the art and include, but are not limited to, aqueous or organic solvents, buffers, acidifiers, surfactants, wetting agents, spreading agents, tackifiers, fixatives, bases, fillers, thickeners, emulsifiers, dispersants, metal ion chelating agents, anti-settling agents, fusion aids, rheological modifiers, defoamers, photoprotective agents, 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 the subranges disclosed above, which are obtained by affinity selection for a predetermined plant pest target and are capable of inhibiting the growth and / or activity of plant pests at a minimum inhibitory concentration of about 0.00001 to 1 μM.

[0405] In certain embodiments of methods for protecting, preventing, treating or treating plants disclosed herein against fungal infections, the polypeptides or compositions disclosed herein are applied directly or indirectly to the plants by spraying, atomizing, foaming, fogging, hydroponics, coating, immersion, and / or powdering.

[0406] nucleic acid sequence In another embodiment, the present invention provides nucleic acid sequences encoding polypeptide sequences (or suitable fragments thereof) disclosed herein. These nucleic acid sequences may also be in the form of vectors, gene constructs, or polynucleotides. The nucleic acid sequences disclosed herein may be synthetic or semi-synthetic sequences, nucleotide sequences isolated from libraries (particularly expression libraries), nucleotide sequences prepared by PCR using overlapping primers, or nucleotide sequences prepared using DNA synthesis techniques known in themselves.

[0407] The present invention includes nucleic acid sequences encoding any polypeptide 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 (for example, those having amino acid substitutions or a predetermined percentage of identity with respect to the sequence).

[0408] Construct, vector, host cell The gene construct disclosed herein may be DNA or RNA, but double-stranded DNA is preferred. The gene construct of the present invention may further be in a form suitable for transformation of the target host cell or host organism, in a form suitable for integration into the genomic DNA of the target host cell, or in a form suitable for independent replication, maintenance and / or inheritance in the target host organism. For example, the gene construct 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 that enables in vitro and / or in vivo expression (for example, in a suitable host cell, host organism and / or expression system).

[0409] Accordingly, in yet another embodiment, the present invention also provides a vector comprising one or more nucleic acid sequences of the present invention.

[0410] In yet another embodiment, the present invention provides a host or host cell that expresses or is capable of expressing one or more amino acid sequences disclosed herein. Examples of hosts or host cells suitable for expressing the amino acid sequences and polypeptides of the present invention will be obvious to those skilled in the art.

[0411] The present application further discloses that polypeptides of 80 to 200 amino acids or the subranges described above are stably maintained in the pesticide composition or biological control composition as defined in the present application, that is, the integrity of the polypeptides and the pest control activity as defined in the present application are maintained under the storage and / or use conditions of the pesticide composition, such conditions include high temperature, freeze-thaw cycles, changes in pH or ionic strength, UV irradiation, and the presence of harmful chemicals. It is most preferable that these polypeptides of 80 to 200 amino acids 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 polypeptides of 80 to 200 amino acids contained in the pesticide composition maintain at least about 70% of their activity after storage at ambient temperature in the pesticide composition for two years, or when the pesticide composition containing the polypeptides is stored at 54°C for two weeks, and more specifically, maintain at least about 70% to 80% of their activity, and most specifically, maintain about 80% to 90% of their activity.

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

[0413] We also disclose a chimeric gene comprising the following functionally linked DNA components, namely a) a plant expression promoter, b) a DNA region that, when transcribed, becomes an mRNA molecule capable of being translated into a polypeptide, and c) a 3' terminal region containing a transcription termination and polyadenylation signal that functions within the plant cell.

[0414] A "chimeric gene" or "chimeric construct" is a recombinant nucleic acid sequence in which a promoter (e.g., a plant expression promoter) or regulatory nucleic acid sequence is functionally linked or bound to a nucleic acid sequence encoding mRNA, and when the regulatory nucleic acid sequence is introduced into a cell such as a plant cell, it is configured to regulate the transcription or expression of the bound nucleic acid coding sequence. In nature, the regulatory nucleic acid sequence of a chimeric gene and the nucleic acid sequence to which it is bound in this way are not usually functionally linked.

[0415] In the present invention, the "plant promoter" includes regulatory components that mediate the expression of coding sequence segments in plant cells. To be expressed in plants, nucleic acid molecules must be functionally linked to or contain a suitable promoter that expresses the gene in the required spatial expression pattern at the correct time.

[0416] As used in this application, the term "functionally linked" means that the promoter sequence and the target gene are functionally linked so that the promoter sequence can initiate transcription of the target gene.

[0417] Plant expression promoters contain nucleic acid sequences capable of inducing transgene expression in plants. Examples of plant expression promoters include constitutive promoters that are transcriptionally active under most environmental conditions for most, if not all, of the growth and developmental stages in at least one cell, tissue, or organ; inductive promoters; tissue-specific promoters; and abiotic stress-inducible promoters.

[0418] When a chimeric gene (or expression cassette) is transformed into a plant, it expresses nucleic acids, which in turn express proteins.

[0419] Recombinant vectors containing the expression cassette (or chimeric gene) described above are also disclosed.

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

[0421] A “selection marker,” “selection marker gene,” or “reporter gene” includes any gene that confers a phenotype to cells expressing them, in order to facilitate the identification and / or selection of cells to be transfected or transformed with the nucleic acid construct of the present invention. These marker genes enable confirmation of the successful introduction of nucleic acid molecules by a series of different principles. Appropriate markers may be selected from markers that confer antibiotic or herbicide resistance, markers that introduce novel metabolic traits, or markers that enable visual selection. Examples of selection 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 bleomycin, streptomycin, tetracycline, chloramphenicol, ampicillin, gentamicin, genethicin (G418), spectinomycin, or blasticidine), genes that confer herbicide resistance (e.g., bar, which provides resistance to Basta(R), aroA or gox, which provides resistance to glyphosate, or genes that confer resistance to imidazolinone, phosphinothricin, or sulfonylurea), or genes that provide metabolic traits (e.g., manA, which enables plants to use mannose as a sole carbon source, or xylose isomerase, which enables the utilization of xylose, or non-nutritional markers such as resistance to 2-deoxyglucose). When a visual marker gene is expressed, it can produce coloration (e.g., β-glucuronidase GUS or β-galactosidase and its coloring agent, e.g., X-Gal), luminescence (e.g., luciferin / luciferase system), or fluorescence (green fluorescent protein GFP and its derivatives). The above examples represent only a small fraction of possible markers. Experienced operators are familiar with such markers. Different markers are preferred depending on the organism and the selection method.

[0422] When nucleic acids are stably or transiently incorporated into plant cells, a small number of these cells are known to take up this foreign DNA and, if necessary, incorporate it into their genome depending on the expression vector and transfection technique used. To identify and select these incorporated cells, genes encoding selection markers (such as those described above) are typically introduced into host cells along with the target gene. These markers can be used, for example, in mutants in which these genes are non-functional due to deletions, for example, by conventional methods. Furthermore, nucleic acid molecules encoding selection markers can be introduced into host cells using the same vector containing the polypeptide of the present invention or the polypeptide sequence used in the method of the present invention, or they can be introduced using a different vector. Cells that have been stably transfected with the introduced nucleic acid can be identified, for example, by selection (e.g., cells that have incorporated the selection marker survive, while cells that have not incorporated it die).

[0423] If nucleic acid introduction is successful, marker genes, particularly those for antibiotic and herbicide resistance, become unnecessary or undesirable in transgenic host cells. Therefore, the nucleic acid introduction method according to the present invention is advantageous when it utilizes a technique that allows for the removal or excision of these marker genes. One example of such a method is called co-transformation. The co-transformation method uses two vectors simultaneously for transformation: a vector carrying the nucleic acid according to the present invention and a second vector carrying the marker gene. A high percentage of transformants (up to 40% or more of transformants) receive both vectors, or in the case of plants, they contain both vectors. In the case of transformation by Agrobacterium, the transformant usually receives only a portion of the vector, i.e., the sequence flanked by T-DNA, which usually corresponds to the expression cassette. Subsequently, the marker gene can be removed from the transformed plant by crossbreeding. In another method, a marker gene incorporated into a transposon is used for transformation along with the desired nucleic acid (called the Ac / Ds technique). Transformants can be crossed with a transposase source, or they can be transformed with a nucleic acid construct that transiently or stably confers transposase expression. In some cases (about 10%), after successful transformation, the transposon is lost, having jumped out of the host cell's genome. More often, the transposon jumps to another location. In these cases, it is necessary to eliminate the marker gene by crossing. Microbiology has developed techniques that enable or facilitate the detection of such events. A more advantageous method relies on a method called a recombinant system, whose advantage is that it eliminates the need for elimination by crossing. The best-known system of this type is called the Cre / lox system. Cre1 is a recombinase that removes sequences located between loxP sequences. If the marker gene is incorporated between loxP sequences, it is removed by the expression of the recombinase after successful transformation.Other recombination systems include the HIN / HIX, FLP / FRT, and REP / STB systems (Tribble et al., J. Biol. Chem., 275, 2000: 22255-22267; Velmurugan et al., J. Cell Biol., 149, 2000: 553-566). The nucleic acid sequence according to the present invention can be incorporated into the plant genome in a site-specific manner.

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

[0425] Therefore, for the purposes of the present invention, transgenic plants are considered to mean 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 is not present in its natural locus, and the nucleic acid can be expressed in the same species or in different species. However, as stated above, transgenic also means that the nucleic acid according to the present invention or the nucleic acid used in the method of the present invention is in its natural position in the genome of the plant, but its sequence is modified from the natural sequence, and / or the regulatory sequence of the natural sequence is modified. It is preferable to consider transgenic as meaning that the expression of the nucleic acid according to the present invention occurs at a non-natural locus in the genome, that is, that the nucleic acid is expressed in the same species or in different species. Preferred transgenic plants are referred to in this application.

[0426] The terms "expression" or "gene expression" mean the transcription of one or more specific genes or gene constructs. Specifically, "expression" or "gene expression" means that one or more genes or gene constructs are transcribed into structural RNA (rRNA, tRNA) or mRNA, which may or may not be translated into protein. This process includes the transcription of DNA and the processing of the resulting mRNA product.

[0427] As used in this application, the terms “increased expression” or “overexpression” refer to any form of expression added to the original wild-type expression level. For the purposes of this invention, the original wild-type expression level may be zero, i.e., expression may be absent, or expression may be unmeasurable.

[0428] Methods for increasing the expression of a gene or gene product are clearly described in the literature in the art, and include, for example, overexpression driven by a suitable promoter (as described above), the use of a transcriptional enhancer, or a translational enhancer. Isolated nucleic acids that function as promoter or enhancer elements can be introduced at a suitable position (generally upstream) of a non-heterogeneous polynucleotide to upregulate the expression of the nucleic acid encoding the polypeptide of interest. When polypeptide expression is desired, it is generally desirable to add a polyadenylated region to the 3' end of the polynucleotide coding region. The polyadenylated region can be derived from a native gene, various other plant genes, or T-DNA. The 3' end sequence to be added can be derived, for example, from a nopalin synthase or octopine synthase gene, or another plant gene, or, less preferably, any other eukaryotic gene.

[0429] To increase the amount of maturation messages accumulated in the cytosol, intron sequences may be added to the 5' untranslated region (UTR) of a partial coding sequence or to the coding sequence itself. Adding 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 maximized when the introns are located 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 is 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 “introduction” or “transformation” refer to the introduction of an exogenous polynucleotide or chimeric gene (or expression cassette) into host cells, regardless of the method used for introduction. Plant tissues capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, can be transformed with the gene constructs of the present invention, resulting in the regeneration of a complete plant. The specific tissues selected depend on the available and optimal clonal propagation system for the specific species to be transformed. Typical tissue targets include leaf discs, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristems (e.g., apical meristem, axillary buds, and root meristems), and induced meristems (e.g., cotyledonous meristems and hypocotyl meristems). Polynucleotides can be introduced into host cells transiently or stably, and may remain unintegrated, for example, as a plasmid. Alternatively, they may be integrated into the host genome. The resulting transformed plant cells can then be used to regenerate transformed plants in methods known to those skilled in the art.

[0431] The process of introducing foreign genes into the genome of a plant is called transformation. Plant transformation is now a fairly common technique. It has the advantage that any of several transformation methods can be used to introduce the target gene into the appropriate ancestral cells. Methods described for the transformation and regeneration of plants from plant tissue 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 gun methods, transformation methods using viruses or pollen, and microinjection methods. Methods such as 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 DNA or RNA-coated particles (Klein TM et al., (1987) Nature 327:70); and infection with (non-integrated) viruses can be selected. Transgenic plants, including transgenic cultivated plants, are preferably produced by transformation using Agrobacterium. The advantageous transformation method is transformation in the plant body. For this purpose, for example, Agrobacterium can be applied to plant seeds or inoculated into plant meristems. 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 to the primordia. The plants are then grown until seeds of the treated plants are obtained (Clough and Bent, Plant J. (1998) 16, 735-743).Well-known rice transformation methods using Agrobacterium include those described in the following documents, namely, European Patent Application EP1198985, Aldemita and Hodges (Planta 199:612-617, 1996), Chan et al. (Plant Mol Biol 22(3):491-506, 1993), and Hiei et al. (Plant J 6(2):271-282, 1994), and the entire disclosure thereof is incorporated into this application. In the case of maize transformation, preferred methods are those described in either Ishida et al. (Nat. Biotechnol 14(6):745-50, 1996) or Frame et al. (Plant Physiol 129(1):13-22, 2002), and the entire disclosure thereof is incorporated into this application. The aforementioned method is also described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. SDKung and R. Wu, Academic Press (1993) 128-143, and Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). It is preferable to clone the nucleic acid or construct to be expressed into a vector suitable for transforming Agrobacterium tumefaciens (e.g., pBin19) (Bevan et al (1984) Nucl. Acids Res. 12-8711).Subsequently, Agrobacterium transformed with such vectors can be used for plant transformation in known methods. For example, by immersing crushed or shredded leaves in an Agrobacterium solution and then culturing them in a suitable medium, it can be used in plants used as models, such as Arabidopsis (Arabidopsis thaliana is not considered a cultivated plant within the scope of this invention), or in cultivated plants such as tobacco. The method of plant transformation using Agrobacterium tumefaciens is known, for example, from Hofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877, or in particular from FF White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. SDKung and R. Wu, Academic Press, 1993, pp. 15-38.

[0432] In addition to the transformation of somatic cells that need to be regenerated into intact plants later, it is also possible to transform cells of plant meristems, particularly cells that will grow into gametes. In this case, the transformed gametes will undergo natural plant development, producing transgenic plants. For example, Arabidopsis seeds can be treated with Agrobacterium, and seeds can be obtained from developing plants in which a predetermined proportion have been transformed into transgenic plants [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]. Another method involves repeatedly removing the inflorescence and incubating the excised central rosette with transformed Agrobacterium to obtain similarly transformed seeds at a later stage (Chang (1994). Plant J. 5:551-558; Katavic (1994). Mol Gen Genet, 245:363-370). However, particularly effective methods are the vacuum immersion method and its variations, such as the "floral dip" method. In the vacuum immersion method for Arabidopsis, intact plants are treated with an Agrobacterium suspension under reduced pressure [Bechthold, N (1993). CR Acad Sci Paris Life Sci, 316: 1 194-1 199], while in the "floral dip" method, developing floral tissue is briefly incubated with an Agrobacterium suspension treated with a surfactant [Clough, SJ and Bent AF (1998) The Plant J. 16, 735-743]. In both cases, a predetermined proportion of transgenic seeds are harvested, and these seeds can be distinguished 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, and the risk of transgene leakage via pollen is reduced or eliminated.Chloroplast genome transformation is generally performed by the method schematically shown in Klaus et al., 2004 [Nature Biotechnology 22(2), 225-229]. In summary, the sequence to be transformed is cloned along with a selection marker gene between homologous flanking sequences of the chloroplast genome. These homologous flanking sequences induce site-specific integration into the chloroplast genome (plastome). Plastiid transformation methods have been described for a wide variety of plant species, and reviews are available 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, recent biotechnological advances have been reported in the production of marker-free plastid transformants that can be produced by transiently and simultaneously incorporated marker genes (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229).

[0433] Genetically modified plant cells can be regenerated using all methods familiar to skilled operators. For appropriate methods, please refer to the publications by SDKung and R. Wu, Potrykus or Hofgen and Willmitzer mentioned above.

[0434] Generally, after transformation, plant cells or cell groups are selected for the presence of one or more markers encoded by plant-expression genes transfected with the target gene, and then the transformed material is regenerated into a complete plant. 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, the seeds obtained as described above can be sown and subjected to appropriate selection by spraying after the initial growth period. Alternatively, the seeds may be grown on agar plates using an appropriate selective agent after sterilization as needed, so that only transformed seeds can grow into plants. Or, transformed plants can be screened for the presence of selection markers such as those described above.

[0435] After DNA introduction and regeneration, the plants presumed to be transformed may be evaluated for the presence, copy number, and / or genome composition of the target gene, for example, using Southern blot analysis. Alternatively, or in addition to this, the expression level of the newly introduced DNA may be monitored by Northern blot analysis and / or Western blot analysis, both of which are well known to those with ordinary skill in the art.

[0436] The transformed plants can be propagated by various means, such as clonal propagation and classical breeding techniques. For example, first-generation (or T1) transformed plants can be self-fertilized, homozygous second-generation (or T2) transformed plants can be selected, and then the T2 plants can be further propagated using classical breeding techniques. The transformed organisms can take various forms. For example, they can be chimeras of transformed and untransformed cells, clonal transformeds (e.g., whole cells transformed to include an expression cassette), or grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock is grafted onto an untransformed scion).

[0437] The present invention will be specifically described below with reference to non-limiting embodiments. [Examples]

[0438] [Example 1] Preparation of Fusarium oxysporum Folch-phase antigen for immunotherapy, phage display, and screening assays Intact hyphae and conidia of Fusarium oxysporum were sequentially extracted at room temperature using chloroform:methanol in 2:1 and 1:2 (v / v) ratios. 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.). Lipids derived from the lower layer of the Folch method were recovered and used for immunization.

[0439] [Example 2] Preparation of ceramide monohexoside fraction of Fusarium oxysporum for phage display and screening assays Ceramide monohexoside fractions derived from Fusarium oxysporum, prepared according to the method 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.), were obtained from Eliana Barreto-Bergter.

[0440] [Example 3] Identification of VHH binding to the lower-phase antigen of Fusarium oxysporum using the Forti method. 3.1 Immunology VHH was prepared from llamas immunized with a Fusarium oxysporum-derived Folzhi subphase extract. Following a standard protocol, llamas were boost-immunized six times with a Folzhi subphase extract spotted on thin-layer chromatography (TLC). Silica adsorbed with the Folzhi subphase extract was 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 immunized llama blood samples using Ficoll-Hypaque according to the manufacturer's instructions. Total RNA was extracted from these cells and used as a starting material for RT-PCR to amplify VHH encoding gene fragments. These fragments were cloned into the phagemide vector pASF20. pASF20 is an expression vector derived from pUC119 and contains the lacZ promoter, synthetic leader sequence, multicloning site, colyphage pIII protein coding sequence, ampicillin resistance gene, and M13 phage origin for single-strand production. This vector encodes the C-terminal (His)6 peptide tag and c-myc peptide tag in frame with the VHH coding sequence. Phages were constructed according to standard methods (Phage Display of Peptides and Proteins: A Laboratory Manual; Brian K. Kay, Jill Winter, Dr. John McCafferty). Libraries with clonal diversity of 1E+08 or higher were obtained, confirming phage production and antibody diversity.

[0442] 3.3 Selection Two rounds of panning selection were performed as follows. The fungal lipid fraction was coated onto polystyrene Maxisorp multiwell plates using 5% chloroform / methanol as the solvent in both rounds. In the first round of selection, 25 μl of phages (1,000E+11 phages / selection condition) were selected from the library using four different conditions: two different lipid fraction (fungal; Fusarium oxysporum) concentrations (50 μg / ml and 5 μg / ml) and two different blank conditions for background control (5% CHCl3 / MeOH and PBS). In the second round of selection, in addition to the conditions of the first round, 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 non-specific phage binding.

[0443] In the first round of selection, good enrichment was observed, particularly from the 50 μg / ml selection condition. The phage output from this selection was rescued, precipitated, and used as input in the second round of selection. In this selection round, significantly higher enrichment was observed under the antigen-coated conditions, with the exception of the 0.5 μg / ml selection condition.

[0444] After the second round of panning selection, the selected eluted phage pool was used to infect E. coli TG1 cells. Individual colonies were picked and transferred to a 96-well plate containing 100 μl each of 2×TY medium supplemented with 2% glucose and 100 μg / ml carbenicillin to create a master plate (MP). The cells were grown overnight at 37°C. The master plate was stored in 20% glycerol at -80°C and used for periplasm extract production, screening, and sequencing.

[0445] [Example 4] Screening of VHHs that bind to the lipid fraction of Fusarium oxysporum To verify whether VHH binds to the lipid fraction of Fusarium oxysporum, binding was evaluated by ELISA. Each clone was screened for binding to wells coated with 10 μg / ml of fungal lipid fraction dissolved in 5% CHCl3 / MeOH, and to wells coated with 5% CHCl3 / MeOH alone. A clone was considered positive if the ratio of the OD450nm binding signal in the antigen-coated well to the corresponding blank well was greater than 2. An overall hit rate of 14.2% was obtained using the standard cutoff criteria applied to the 360 ​​individual clones tested.

[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] A 2-fold dilution of purified VHH10G11Q was prepared in a 96-well microtiter plate. Starting with a final VHH10G11Q concentration of 10 μM, 20 μl of these dilutions and 20 μl of water as a control were mixed with 80 μl of fungal spore suspension (1E+05 spores per 1 ml of 1 / 2 concentration potato modextrose broth (PDB)). The test plates were incubated at 25°C for 36 hours using the IncuCyte Zoom live cell imaging system. At least two plates were used for each test.

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

[0450] [Example 6] VHH10G11 more strongly 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, of which VHH41D01 shows the most significant antifungal activity among 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] Purified VHH10G11Q and 41D01 were prepared in 2-fold dilutions in 96-well microtiter plates. Starting with a final VHH10G11Q concentration of 40 μM, 20 μl of these dilutions and 20 μl of water as a control were mixed with 80 μl of fungal spore suspension (1E+05 spores per 1 ml of 1 / 2 concentration potato modextrose broth (PDB)). The test plates were incubated at 25°C for 48 hours using the IncuCyte Zoom live cell imaging system. At least two plates were used for each test.

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

[0455] [Example 7] Site-directed mutagenesis Site-directed mutagenesis of VHH was performed as follows. The nucleotide sequence encoding the VHH sequence mutant was synthesized as a gene fragment and then cloned into the plasmid pPpT4GAPαS (Naatsaari et al (2012), Plos One, 7(6):e39720), which is suitable for transformation of Pichia pastoris (Pichia yeast). This plasmid was then used to induce expression from the cloned gene fragment. AOXThe promoter was included. Standard cloning techniques were used to construct the plasmid containing the target VHH sequence variant. The successfully generated clones were sequenced to confirm the presence of the target VHH sequence variant and that they were correctly cloned. Subsequently, the linearized plasmid was transformed into competent Pichia pastoris (Pichia yeast) ATCC76273(TM) cells using a standard electroporation protocol. Then, the successfully generated transformants were used to produce the VHH sequence variant. First, the transformants were cultured in BMGY (buffered glycerol complex medium), then transferred to BMMY (buffered methanol complex medium) to initiate induction (Weidner et al (2020), J Vis Exp, 36:1862). Subsequently, VHH was purified using filtration and / or chromatography techniques widely known in the art.

[0456] [Example 8] Ala scanning of VHH10G11 All three CDR regions of VHH10G11 were evaluated by Ala scanning to investigate the effect of single-amino acid substitutions on the antifungal activity of VHH10G11 (SEQ ID NOs: 18-51). After constructing and preparing mutants of 10G11 as described in Example 7, antifungal assays were performed as described in Example 5. Surprisingly, as clearly demonstrated by the maintenance of antifungal activity in the mutants, 10G11 maintained its performance in most mutants in which individual amino acids in the CDR region were substituted with alanine (Figure 3). Similar results were obtained when other single-amino acid substitution mutants were constructed using glycine (R26G; SEQ ID NO: 18 and D35G; SEQ ID NO: 27), serine (R53S; SEQ ID NO: 32 and T56S; SEQ ID NO: 36), or asparagine (K58N; SEQ ID NO: 39) instead of alanine (Figure 3).

[0457] [Example 9] Improvement of antifungal activity of VHH10G11 with His tag attached The His tag is commonly added to polypeptides for purification using affinity chromatography. Surprisingly, during routine testing using an antifungal assay as described in Example 5, the 10G11Q polypeptide (SEQ ID NO: 148) with a 6×His tag added to its C-terminus showed significantly improved antifungal activity (Figure 4). Since the His tag consists of six positively charged histidine amino acids, it was thought that the positive charge was beneficial for the antifungal interaction of 10G11.

[0458] [Example 10] In silico modeling of VHH10G11Q To better understand the 10G11 molecule, its three-dimensional structure was modeled using an in silico approach. A BLAST search was performed in the Protein Data Bank (PDB) to obtain the structural coordinates of antibodies with the same CDR length as the 10G11 molecule to be modeled. A Blosum 62 matrix was used with a gab cost of 13. Of the top 250 hits, each structure with the same CDR length as the target 10G11 molecule was used for homology modeling trials. First, residues that differed between the obtained PDB structure and the 10G11 molecule were mutated. Next, the side chains of these residues were modeled according to their experimental priority, as detected in the Dunbrack 2010 rotational isomer library (Shapovalov and Dunbrack, (2011), Structure, 19(6):844-58). Where necessary, the immediate side chain was also modeled to adopt the modeling mutation. Using this model as a guide, the important amino acid residues of the molecule were determined.

[0459] The resulting model is shown in Figure 5, along with the display of three CDR regions. The selection of exposed side chains is visualized on the 10G11 ribbon diagram and displayed according to Kabat numbering (Figure 5 also shows the germline sequence and the substitutions that result in the 10G11 sequence. Furthermore, the corresponding Kabat numbering is also shown). Since the side chains were exposed and structural damage appeared to be minimal, these amino acid residues were selected as the best candidates for specific amino acid substitutions.

[0460] [Example 11] Antifungal activity of the 10G11 charge mutant As confirmed by the assay results shown in Figure 3, four of the five mutants that showed decreased antifungal activity were characterized by the substitution of a positively charged amino acid with an uncharged mutant. Considering the result that the positively charged His-tagged mutant showed increased antifungal activity (Figure 4), the effect of charge was further investigated. For this purpose, various 10G11 charge mutants were prepared as described in Example 7, and their antifungal activity was tested as described in Example 5. The following substitutions were designed to reduce the total charge of the 10G11 molecule at the antigen-binding interface. • 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 R10...

Claims

1. CDR1 region containing the amino acid sequence of SEQ ID NO: 52, CDR2 region containing the amino acid sequence of SEQ ID NO: 68, and CDR3 region containing the amino acid sequence of SEQ ID NO:

84. The CDR1 region contains the amino acid sequence of SEQ ID NO: 53, the CDR2 region contains the amino acid sequence of SEQ ID NO: 69, and the CDR3 region contains the amino acid sequence of SEQ ID NO:

85. CDR1 region containing the amino acid sequence of SEQ ID NO: 54, CDR2 region containing the amino acid sequence of SEQ ID NO: 70, and CDR3 region containing the amino acid sequence of SEQ ID NO:

86. The CDR1 region contains the amino acid sequence of SEQ ID NO: 55, the CDR2 region contains the amino acid sequence of SEQ ID NO: 68, and the CDR3 region contains the amino acid sequence of SEQ ID NO:

84. CDR1 region containing the amino acid sequence of SEQ ID NO: 52, CDR2 region containing the amino acid sequence of SEQ ID NO: 71, and CDR3 region containing the amino acid sequence of SEQ ID NO: 84, CDR1 region containing the amino acid sequence of SEQ ID NO: 52, CDR2 region containing the amino acid sequence of SEQ ID NO: 68, and CDR3 region containing the amino acid sequence of SEQ ID NO: 87, CDR1 region containing the amino acid sequence of SEQ ID NO: 55, CDR2 region containing the amino acid sequence of SEQ ID NO: 71, and CDR3 region containing the amino acid sequence of SEQ ID NO:

87. CDR1 region containing the amino acid sequence of SEQ ID NO: 56, CDR2 region containing the amino acid sequence of SEQ ID NO: 68, and CDR3 region containing the amino acid sequence of SEQ ID NO:

84. CDR1 region containing the amino acid sequence of SEQ ID NO: 52, CDR2 region containing the amino acid sequence of SEQ ID NO: 68, and CDR3 region containing the amino acid sequence of SEQ ID NO: 88, CDR1 region containing the amino acid sequence of SEQ ID NO: 56, CDR2 region containing the amino acid sequence of SEQ ID NO: 68, and CDR3 region containing the amino acid sequence of SEQ ID NO: 88, CDR1 region containing the amino acid sequence of SEQ ID NO: 57, CDR2 region containing the amino acid sequence of SEQ ID NO: 68, and CDR3 region containing the amino acid sequence of SEQ ID NO:

84. CDR1 region containing the amino acid sequence of SEQ ID NO: 52, CDR2 region containing the amino acid sequence of SEQ ID NO: 68, and CDR3 region containing the amino acid sequence of SEQ ID NO: 89, The CDR1 region contains the amino acid sequence of SEQ ID NO: 57, the CDR2 region contains the amino acid sequence of SEQ ID NO: 68, and the CDR3 region contains the amino acid sequence of SEQ ID NO:

89. CDR1 region containing the amino acid sequence of SEQ ID NO: 58, CDR2 region containing the amino acid sequence of SEQ ID NO: 68, and CDR3 region containing the amino acid sequence of SEQ ID NO:

84. The CDR1 region contains the amino acid sequence of SEQ ID NO: 59, the CDR2 region contains the amino acid sequence of SEQ ID NO: 68, and the CDR3 region contains the amino acid sequence of SEQ ID NO:

84. CDR1 region containing the amino acid sequence of SEQ ID NO: 60, CDR2 region containing the amino acid sequence of SEQ ID NO: 68, and CDR3 region containing the amino acid sequence of SEQ ID NO: 84, The CDR1 region contains the amino acid sequence of SEQ ID NO: 61, the CDR2 region contains the amino acid sequence of SEQ ID NO: 68, and the CDR3 region contains the amino acid sequence of SEQ ID NO:

84. CDR1 region containing the amino acid sequence of SEQ ID NO: 62, CDR2 region containing the amino acid sequence of SEQ ID NO: 68, and CDR3 region containing the amino acid sequence of SEQ ID NO: 84, CDR1 region containing the amino acid sequence of SEQ ID NO: 63, CDR2 region containing the amino acid sequence of SEQ ID NO: 68, and CDR3 region containing the amino acid sequence of SEQ ID NO: 84, CDR1 region containing the amino acid sequence of SEQ ID NO: 64, CDR2 region containing the amino acid sequence of SEQ ID NO: 68, and CDR3 region containing the amino acid sequence of SEQ ID NO: 84, CDR1 region containing the amino acid sequence of SEQ ID NO: 65, CDR2 region containing the amino acid sequence of SEQ ID NO: 68, and CDR3 region containing the amino acid sequence of SEQ ID NO: 84, CDR1 region containing the amino acid sequence of SEQ ID NO: 66, CDR2 region containing the amino acid sequence of SEQ ID NO: 68, and CDR3 region containing the amino acid sequence of SEQ ID NO:

84. The CDR1 region contains the amino acid sequence of SEQ ID NO: 67, the CDR2 region contains the amino acid sequence of SEQ ID NO: 68, and the CDR3 region contains the amino acid sequence of SEQ ID NO:

84. CDR1 region containing the amino acid sequence of SEQ ID NO: 52, CDR2 region containing the amino acid sequence of SEQ ID NO: 72, and CDR3 region containing the amino acid sequence of SEQ ID NO: 84, The CDR1 region contains the amino acid sequence of SEQ ID NO: 52, the CDR2 region contains the amino acid sequence of SEQ ID NO: 73, and the CDR3 region contains the amino acid sequence of SEQ ID NO:

84. The CDR1 region contains the amino acid sequence of SEQ ID NO: 52, the CDR2 region contains the amino acid sequence of SEQ ID NO: 74, and the CDR3 region contains the amino acid sequence of SEQ ID NO:

84. CDR1 region containing the amino acid sequence of SEQ ID NO: 52, CDR2 region containing the amino acid sequence of SEQ ID NO: 75, and CDR3 region containing the amino acid sequence of SEQ ID NO:

84. CDR1 region containing the amino acid sequence of SEQ ID NO: 52, CDR2 region containing the amino acid sequence of SEQ ID NO: 76, and CDR3 region containing the amino acid sequence of SEQ ID NO: 84, The CDR1 region contains the amino acid sequence of SEQ ID NO: 52, the CDR2 region contains the amino acid sequence of SEQ ID NO: 77, and the CDR3 region contains the amino acid sequence of SEQ ID NO:

84. CDR1 region containing the amino acid sequence of SEQ ID NO: 52, CDR2 region containing the amino acid sequence of SEQ ID NO: 78, and CDR3 region containing the amino acid sequence of SEQ ID NO: 84, The CDR1 region contains the amino acid sequence of SEQ ID NO: 52, the CDR2 region contains the amino acid sequence of SEQ ID NO: 79, and the CDR3 region contains the amino acid sequence of SEQ ID NO:

84. CDR1 region containing the amino acid sequence of SEQ ID NO: 52, CDR2 region containing the amino acid sequence of SEQ ID NO: 80, and CDR3 region containing the amino acid sequence of SEQ ID NO: 84, CDR1 region containing the amino acid sequence of SEQ ID NO: 52, CDR2 region containing the amino acid sequence of SEQ ID NO: 81, and CDR3 region containing the amino acid sequence of SEQ ID NO: 84, CDR1 region containing the amino acid sequence of SEQ ID NO: 52, CDR2 region containing the amino acid sequence of SEQ ID NO: 82, and CDR3 region containing the amino acid sequence of SEQ ID NO: 84, The CDR1 region contains the amino acid sequence of SEQ ID NO: 52, the CDR2 region contains the amino acid sequence of SEQ ID NO: 83, and the CDR3 region contains the amino acid sequence of SEQ ID NO:

84. CDR1 region containing the amino acid sequence of SEQ ID NO: 52, CDR2 region containing the amino acid sequence of SEQ ID NO: 68, and CDR3 region containing the amino acid sequence of SEQ ID NO:

84. CDR1 region containing the amino acid sequence of SEQ ID NO: 52, CDR2 region containing the amino acid sequence of SEQ ID NO: 68, and CDR3 region containing the amino acid sequence of SEQ ID NO: 90, CDR1 region containing the amino acid sequence of SEQ ID NO: 52, CDR2 region containing the amino acid sequence of SEQ ID NO: 68, and CDR3 region containing the amino acid sequence of SEQ ID NO: 91, The CDR1 region contains the amino acid sequence of SEQ ID NO: 52, the CDR2 region contains the amino acid sequence of SEQ ID NO: 68, and the CDR3 region contains the amino acid sequence of SEQ ID NO:

92. The CDR1 region contains the amino acid sequence of SEQ ID NO: 52, the CDR2 region contains the amino acid sequence of SEQ ID NO: 68, and the CDR3 region contains the amino acid sequence of SEQ ID NO:

93. CDR1 region containing the amino acid sequence of SEQ ID NO: 52, CDR2 region containing the amino acid sequence of SEQ ID NO: 68, and CDR3 region containing the amino acid sequence of SEQ ID NO: 94, CDR1 region containing the amino acid sequence of SEQ ID NO: 52, CDR2 region containing the amino acid sequence of SEQ ID NO: 68, and CDR3 region containing the amino acid sequence of SEQ ID NO:

95. The CDR1 region contains the amino acid sequence of SEQ ID NO: 52, the CDR2 region contains the amino acid sequence of SEQ ID NO: 68, and the CDR3 region contains the amino acid sequence of SEQ ID NO:

96. CDR1 region containing the amino acid sequence of SEQ ID NO: 52, CDR2 region containing the amino acid sequence of SEQ ID NO: 68, and CDR3 region containing the amino acid sequence of SEQ ID NO: 97, The CDR1 region contains the amino acid sequence of SEQ ID NO: 52, the CDR2 region contains the amino acid sequence of SEQ ID NO: 68, and the CDR3 region contains the amino acid sequence of SEQ ID NO:

98. The CDR1 region containing the amino acid sequence of SEQ ID NO: 52, the CDR2 region containing the amino acid sequence of SEQ ID NO: 68, and the CDR3 region containing the amino acid sequence of SEQ ID NO: 99, or CDR1 region containing the amino acid sequence of SEQ ID NO: 52, CDR2 region containing the amino acid sequence of SEQ ID NO: 68, and CDR3 region containing the amino acid sequence of SEQ ID NO: 100 A heavy chain variable domain (VHH) of a heavy chain antibody, The aforementioned VHH can bind to the lipid-containing fraction of the cell membrane of Botrytis cinerea, and the aforementioned lipid-containing fraction is A process of fractionating the mycelium of Botrytis cinerea by total lipid extract thin-layer chromatography, A step of selecting a fraction in which the retention coefficient (Rf) is greater than that of the ceramide fraction and less than that of the nonpolar phospholipid fraction. VHH obtained by a method comprising the following, wherein the VHH delays the growth of fungal spores and / or causes lysis of the fungal spores.

2. The VHH according to claim 1, comprising the amino acid sequence described in any one of Sequence IDs 1 to 51.

3. A composition comprising at least one VHH as described in claim 1 or 2.

4. The composition according to claim 3, wherein the concentration of at least one VHH in the composition is 0.0001 to 50% by weight.

5. The composition according to claim 3 or 4, which is a pesticide composition.

6. The composition according to claim 5, further comprising an agriculturally chemistry-appropriate base and / or one or more appropriate adjuvants.

7. The aforementioned fungi are plant pathogenic fungi, and the genera of the plant pathogenic fungi are Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Diplodia, Erysiphe, Fusarium, Leptospheria, and Gaeumanomyces. ), Helminthosporium, Macrophomina, Nectria, Penicillium, Peronospora, Phoma, Phymatotricum, Phytophthora, Plasmopara, Podosphera, Puccinia , Pyrenophora, Pyricularia, Pythium, Rhizoctonia, Sclerotium, Sclerotinia, Septoria, Thielaviopsis, Uncinula, Venturia, Verticillium, Magnaporte (M A VHH according to claim 1 or 2, or a composition according to any one of claims 3 to 6, selected from the group consisting of agnaporthe, Blumeria, Mycosphaerella, Ustilago, Melampsora, Phakospora, Monilinia, Mucor, Rhizopus, and Aspergillus.

8. A composition according to any one of claims 3 to 7, or a VHH according to any one of claims 1, 2, or 7, for use as an antifungal agent.

9. The composition or VHH according to claim 8 for use as an antifungal agent in plants.

10. A method for protecting or treating a plant or a part of the plant from infection by a plant pathogenic fungus, comprising at least the step of directly or indirectly applying the VHH according to any one of claims 1, 2, or 7 or the composition according to any one of claims 3 to 7 to the plant or a part of the plant under conditions effective for protecting or treating the plant or a part of the plant from the infection by the plant pathogenic fungus.

11. A post-harvest treatment method for protecting or treating a harvested plant or a harvested part of the plant from infection by a plant pathogenic fungus, comprising at least the step of directly or indirectly applying a VHH according to any one of claims 1, 2, or 7 or a composition according to any one of claims 3 to 7 to the harvested plant or a harvested part of the plant under conditions effective for protecting or treating the plant pathogenic fungus from the infection.

12. A method for inhibiting the growth of plant pathogenic fungi or killing plant pathogenic fungi, comprising at least the step of directly or indirectly applying a VHH according to any one of claims 1, 2, or 7 or a composition according to any one of claims 3 to 7 to a plant or a part of the plant.

13. A method for producing a VHH that specifically binds to and / or has affinity for fungi, The process of immunizing camelid animals with fungal targets; A step of obtaining a cell collection or sample expressing VHH from the immunized camelid; A step of screening the cell collection or sample for cells that express VHH that bind to and / or have affinity for the fungal target; (i) a step of isolating the amino acid sequence of the VHH, or (ii) a step of isolating a nucleic acid sequence encoding the amino acid sequence of the VHH from the cell collection or sample; The process of expressing the amino acid sequence of VHH Includes, This allows for the production of VHH that specifically binds to and / or has affinity for fungi. The method is characterized in that the fungal target is a lipid-containing fraction of the cell membrane of Botrytis cinerea, and the lipid-containing fraction is obtained by a method comprising the steps of fractionating the hyphae of Botrytis cinerea by total lipid extract thin-layer chromatography and selecting a fraction having a retention factor (Rf) greater than that of the ceramide fraction and less than that of the nonpolar phospholipid fraction.

14. A method for producing an antifungal composition, A step of producing an antifungal VHH according to the method of claim 13; The process of combining the aforementioned antifungal VHH with one or more suitable bases and / or one or more suitable adjuvants. Methods that include...

15. A nucleotide encoding VHH according to any one of claims 1, 2, or 7, which is contained in a transgenic plant, a part of a plant, a species, a plant cell, a vector, a plasmid, or a host cell containing the vector or plasmid.

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