JP2014513919A - Proteins for biological control of phytopathogenic fungi - Google Patents

Abstract

Compositions for controlling disease symptoms caused by phytopathogenic fungi exemplified by Fusarium species, Sclerotinia species, Rhizoctonia species, Psium species and the like are provided. The composition is provided with a polypeptide molecule comprising an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. . The composition may be one of a seed treatment agent, a plant treatment agent, or a soil treatment agent. Transformed, expressing a polypeptide molecule comprising an amino acid sequence sharing at least 80% sequence identity with SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 Plant cells are provided that are resistant to infection by phytopathogenic fungi. Transformed, expressing a polypeptide molecule comprising an amino acid sequence sharing at least 80% sequence identity with SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 Providing microbial cells.

Description

The present invention relates to novel biocontrol agents, related compositions, and uses thereof that are effective for controlling fungal plant pathogens. In particular, the present invention relates to novel proteins having a mycostatic and / or mycocidal effect against phytopathogenic fungi. The present invention further relates to a biocontrol agent comprising the protein and uses, methods and compositions comprising the same.

BACKGROUND OF THE INVENTION Fusarium is a filamentous fungus that is widely distributed on plants and in soil. One Fusarium species is a phytopathogen. For example, Fusarium oxysporum causes Fusarium wilt in over 100 plant species. Fusarium oxysporum causes the disease by colonization in plant xylem that can lead to blockage and weakness. When this happens, symptoms such as leaf wilting and yellowing appear in the plant, ultimately leading to plant death. This was the main cause of the loss and disappearance of Gros Michel banana varieties from markets around the world. Recently, new strains have begun to attack the dominant Cavendish variety of plants, raising concerns that if there is no solution, this variety will disappear from the global market.

  Fusarium root rot is a major cause of seedling mortality in forest seedlings and also causes a decline in survival during the first growth period after transplantation. The disease is caused by several Fusarium species and is common in many parts of the world. Disease is a special problem in western Canada and the United States, as well as in the central and southern states. In addition, Pine pitch canker caused by the fungus Fusarium circinatum is a serious disease of pine trees and is particularly a threat to forestry in the United States and New Zealand. Radiata pine or Monterrey pine are highly susceptible to this disease, with some areas in California reaching 80% mortality in mature trees.

  Fusarium red mold disease (FHB), also known as “ear blight” or “black blight”, is a disease of wheat, barley, oats and other small grains caused by Fusarium. Corn and maize can be affected by a similar condition known as “scab”. The above conditions can reduce crop yield and grade and can contaminate cereals with mycotoxins. It is estimated that the grain industry will cost nearly $ 5 billion per year for FHB. In recent years, FHB has become a growing problem for commercially important wheat and barley crops in Western Canada. In the past, two species mainly associated with FHB have been identified as Fusarium oxysporum and Fusarium avenaceum in Canada, where yields on some affected fields have fallen by more than 30% . Harvested cereals of FHB and smut are often contaminated with mycotoxins such as deoxynivalenol (DON) trichothecenes associated with feed rejection, general digestive disorders, diarrhea, and bleeding. The emergence of the toxin-producing 3-acetyldeoxynivalenol (3-ADON) Fusarium graminearum population in North America is a fairly recent phenomenon. This is replacing the 15-ADON chemical type. Furthermore, the mycotoxin profile of F. culmorum, which is similar to F. graminearum under both laboratory and field conditions, poses an additional threat to corn, maize and wheat production worldwide. Present. Currently, there is no effective control means for FHB and related mycotoxins. In addition, there are no corn, wheat, barley, oat, or other small grain resistant varieties. Fungicides can be effective, but only temporarily control the disease.

  In addition to being a common plant pathogen, Fusarium species can opportunistically infect animals and can be agents that cause superficial and / or systemic infections in humans. Fusarium is one of the most drug-resistant fungi and makes it difficult to treat Fusarium infections. Invasive infections can be fatal.

  Sclerotinia stem rot is caused by the pathogen Sclerotinia sclerotiorum. S. sclerotiorum has a broad host and is known to infect many plant species (canola, sunflower, soybean, flax, etc.). The outbreak of disease can be particularly severe under non-rotating conditions or when the rotation includes several susceptible plant species. If the infection occurs early in the flowering period, the yield loss can reach 20%. Another widespread fungal phytopathogen is Rhizoctonia that causes “withering” or “wire-stem” symptoms in crop plants. Infection can occur at any time during the growth period, but the seedling period is most susceptible. The incidence and severity of disease during all growth periods is affected by weather, soil conditions, and inoculation levels. The cool weather favors seedling infection. Similarly, the incidence of wire stem disease is most severe in spring and autumn when the soil is wet and cold. Root rot generally occurs during periods of warm and humid weather and can affect plants at any stage of development. Seedlings affected by the withering disease cannot emerge or, if they do, decline rapidly, fall down and die. In more mature seedlings, the lower leaves turn purple and the lower part of the stem contracts near the soil surface and becomes dark brown. Other symptoms may include seed rot, corroded roots, and carcinoma on the lower petioles. Pythium ultimum is a ubiquitous soil-borne pathogen that is widely distributed throughout the world and causes plant blight and root rot. P. Ultimum has a broad host that includes many important crops and turfgrass.

  The present invention relates to novel proteins that control fungal pathogens and / or control disease symptoms caused by phytopathogenic fungi exemplified by Fusarium species, Sclerotinia species, Rhizoctonia species, Pithium species and the like. In addition, these novel proteins also have anti-mycotoxin effects on toxins produced by phytopathogenic fungi exemplified by Fusarium species, sclerotinia species, Rhizoctonia species, Psium species and the like.

  Some exemplary embodiments of the present invention provide for controlling and / or preventing spore germination of phytopathogenic fungi and / or for controlling the growth of mycelium of phytopathogenic fungi and / or phytopathogenic It relates to a novel intracellular protein produced by Sphaerodes mycoparasitica that is useful for preventing sexual fungi from infecting plant cells or for any combination thereof.

  Some exemplary embodiments of the present invention provide for controlling and / or preventing spore germination of phytopathogenic fungi and / or for controlling the growth of mycelium of phytopathogenic fungi and / or phytopathogenic It relates to a novel extracellular protein produced by S. mycoparasitica that is useful for preventing sexual fungi from infecting plant cells or for any combination thereof.

  One aspect of the invention relates to a novel extracellular protein produced by S. mycoparasitica comprising SEQ ID NO: 4 and having a molecular weight of about 79 kDa.

  One aspect of the invention relates to a novel extracellular protein produced by S. mycoparasitica comprising SEQ ID NO: 5 and having a molecular weight of about 50 kDa.

  One aspect of the invention relates to a novel extracellular protein produced by S. mycoparasitica comprising SEQ ID NO: 6 and having a molecular weight of about 36 kDa.

  One aspect of the invention relates to a novel extracellular protein produced by S. mycoparasitica comprising SEQ ID NO: 7 and having a molecular weight of about 13 kDa.

  Some exemplary embodiments of the invention relate to compositions comprising one or more novel extracellular proteins. The composition may be a seed treatment composition, a plant treatment composition, or a soil treatment composition.

  Some exemplary embodiments of the invention relate to microbial cells that have been transformed to produce one or more novel intracellular proteins and novel extracellular proteins. The transformed microbial cells can be used to control disease symptoms caused by phytopathogenic fungi exemplified by Fusarium species, Sclerotinia species, Rhizoctonia species, Psium species and the like. The transformed microbial cells can be used as seed treatment agents, and / or plant treatment agents, and / or soil treatment agents, or any combination thereof. Alternatively, transformed microbial cells can be used to produce the novel proteins. The protein can be harvested and used to prepare the composition of the present invention.

  Some exemplary embodiments of the invention relate to transformed plant cells that overexpress one or more novel proteins. Such transformed plant cells can be included in plant propagation material exemplified by seeds, somatic embryos, organogenic tissues, etc., which can be cultured to the whole plant. The entire plant, including the transformed plant cells, is resistant or at least resistant to disease symptoms caused by phytopathogenic fungi exemplified by Fusarium species, Sclerotinia species, Rhizoctonia species, Psium species, etc. is there.

  Exemplary aspects of the invention are described with reference to the following drawings.

FIG. 1 (a) A and FIG. 1 (a) B show exemplary proteins of the invention for mycelial growth of Fusarium oxysporum (FIG. 1 (a) A) and F. graminearum (FIG. 1 (a) B) Is a micrograph of a disk diffusion assay showing the inhibitory effect of F. oxysporum (A) and F. graminearum (B) with exemplary proteins after 3 and 4 days of growth It is a figure which shows% inhibition of body growth. It is a photomicrograph of 10% non-denaturing PAGE separation into two bands of the intracellular protein produced by Spherodes mycoparasitica. 3 is a photomicrograph of 12% SDS-PAGE separation of S. mycoparasitic intracellular protein eluted from the upper and lower bands shown on the 10% non-denaturing PAGE gel shown in FIG. FIG. 4 is a photomicrograph of 10% non-denaturing PAGE separation of the extracellular protein produced by S. mycoparasitica into 4 bands. FIG. 5 is a photomicrograph of a 12% SDS-PAGE separation of S. mycoparasitic extracellular protein eluted from four bands of the 10% non-denaturing PAGE gel shown in FIG. FIG. 2 is a chromatogram showing FLPC separation of four bands of extracellular protein produced by S. mycoparasitica. FIG. 7 (a) is a photomicrograph of mycelial growth of F. oxysporum in the control treatment; FIG. 7 (b) is a photomicrograph of mycelial growth of F. oxysporum in the control treatment + buffer; 7 (c) is a photomicrograph of the inhibitory effect of a 50 kDa intracellular protein on the mycelial growth of F. oxysporum; and FIG. 7 (d) shows the 79 kDa intracellular protein on the mycelial growth of F. oxysporum. It is a microscope picture of an inhibitory effect. FIG. 8 (a) is a photomicrograph of mycelial growth of F. graminearum in the control treatment; FIG. 8 (b) is a photomicrograph of mycelium growth of F. graminealam in the control treatment + buffer; 8 (c) is a photomicrograph of the inhibitory effect of a 50 kDa intracellular protein on the mycelial growth of F. graminearum; and FIG. 8 (d) is a diagram of the 79 kDa intracellular protein on the mycelial growth of F. graminearum. It is a microscope picture of an inhibitory effect. FIG. 9 (a) is a photomicrograph showing the inhibition of mycelial growth of F. oxysporum by the 79 kDa extracellular protein (EC1) compared to the control treatment (c), and FIG. 9 (b) 2 is a photomicrograph showing the inhibition of mycelial growth of F. oxysporum by kDa extracellular protein (EC2) and 36 kDa extracellular protein (EC3). FIG. 10 (a) is a photomicrograph showing the inhibition of mycelial growth of F. graminealam by the 79 kDa extracellular protein (EC1) compared to the control treatment (c), and FIG. 10 (b) 2 is a photomicrograph showing inhibition of mycelial growth of F. graminearum by kDa extracellular protein (EC2) and 36 kDa extracellular protein (EC3). FIG. 11 (a) is a photomicrograph showing inhibition of mycelial growth of F. graminealam by 13 kDa extracellular protein (EC4) compared to control treatment (c), and FIG. 11 (b) It is a microscope picture which shows inhibition of the mycelial growth of F. oxysporum by 13 kDa extracellular protein (EC4) compared with a process (c). Figures 12 (a) and 12 (b) show the S. mycoparasitic extracellular protein fraction EC1, for germination of F. oxysporam spores (Figure 12 (a)) and F. graminearum spores (Figure 12 (b)), It is a figure which shows the effect of EC2, EC3, and EC4. FIG. 2 shows the effect of S. mycoparasitic extracellular protein fractions EC1, EC2, EC3, and EC4 on inhibition of colony growth and growth of F. oxysporum and F. graminearum. 14A (c), FIG. 14A (a), and FIG. 14A (b) show control F. oxysporum spore germination (c), inhibitory effect of 50 kDa protein on F. oxysporum spore germination (a), and 13 FIG. 14B (c), FIG. 14B (a), and FIG. 14B (b) show germination of control F. graminearum spores (c) and germination of F. graminearum spores. 2 is a photomicrograph showing the inhibitory effect (a) of a 50 kDa protein and the inhibitory effect of a 13 kDa protein. Figures 15 (a) and 15 (b) show the effect of the crystal-forming extracellular proteins EC1 and EC3 on mycelial growth of F. avenaceum (Figure 15 (b)) compared to the control (Figure 15 (a)). )). FIG. 16 (a) and FIG. 16 (c) show the colony formation and mycelium of the crystal-forming extracellular proteins EC1 and EC3 on germinating wheat seeds of F. graminearum compared to the control (FIG. 16 (a)). FIG. 16 (c) is a photomicrograph showing the effect on growth (FIG. 16 (b)), while FIG. 16 (c) shows a number of dissolved F. graminearum mycelium elements from the surface of wheat seedlings treated with the extracellular protein. It is a confocal laser scanning micrograph which shows (refer arrow). “K +” indicates that the medium contained EC1 and EC3 extracellular proteins. “K-” indicates that EC1 and EC3 extracellular proteins were not present in the medium. “K-crystals” specify the location of extracellular protein molecules. FIG. 17 (a) and FIG. 17 (b) show the effect of the crystal-forming extracellular proteins EC1 and EC3 on the mycelial proliferation of F. graminealam (FIG. 17 (b) compared to the control (FIG. 17 (a)). )). FIG. 18 (a) is a scanning electron micrograph showing the presence of protein crystals and dissolved fungal hyphae from F. graminearum treated with the crystal protein from FIG. 17 (b), FIG. 18 (b) FIG. 18 is a confocal laser scanning micrograph showing the presence of protein crystals in the mycelium of F. graminearum treated with the crystal protein from FIG. 17 (b), and FIG. 18 (c) is from FIG. 2 is a chemical force micrograph showing the presence of protein crystals and a number of broken mycelium elements and apoptotic lysed cells (arrows) from F. graminearum treated with a crystalline protein. “K crystals: locate extracellular protein molecules.” It is a figure which shows the MALDI-TOF-MS separation of the amino acid which comprises 79 kDA EC1 protein. It is a figure which shows the MALDI-TOF-MS separation of the amino acid which comprises 50 kDa EC2 protein. It is a figure which shows the MALDI-TOF-MS separation of the amino acid which comprises 36 kDa EC3 protein. It is a figure which shows the MALDI-TOF-MS separation of the amino acid which comprises 13 kDa EC3 protein. FIGS. 23 (a) -23 (f) are the following photomicrographs: (FIG. 23 (a)) Control untreated sclerotia sclerotiorum culture on PDA, (FIG. 23 (b)) S. sclerotiorum culture grown on PDA modified with extracellular protein EC1 + EC3, (FIG. 23 (c)) Control untreated Rhizoctonia solani culture, (FIG. 23 (d)) R. solani culture grown on PDA modified with extracellular protein EC1 + EC3, (FIG. 23 (e)) untreated Pisium Ultimum culture as control, and (FIG. 23 (f)) extracellular P. Ultimum culture grown on PDA modified with protein EC1 + EC3. It is a figure which shows the inhibitory effect with respect to the mycelium growth of Sclerotinia sclerotiorum, Rhizoctonia solani, and Psium ultimum of crystal formation extracellular protein EC1 and EC3.

DETAILED DESCRIPTION OF THE INVENTION Spherodes mycoparasitica (Ascomycetes Melanosporales) is isolated from isolates of Fusarium abenaceum, Fusarium graminearum, and Fusarium oxysporum that originate from wheat or asparagus fields. It is a separated fungus parasite. This species is characterized by a unique combination of ascospore size, shape (spindle and triangle), and wall decoration (reticular and smooth). In addition, conidia are produced from simple phyllides on the surface of the ascomy pericarp shell wall, on the pericytes around the hyphae, and on the irregularly branched conidia that arise from the hyphae. S. mycoparasitica has a fiaro-type anamorph and produces a simple phialide on the surface of the ascocarp shell wall or irregularly scattered on the pericycetes and on the conidia To do. S. mycoparasitica forms a hook-like structure that parasitizes the living mycelium of Fusarium.

  A sample culture of Spherodes mycoparasitica has been deposited with the International Depositary Authority of Canada Collection (1015 Arlington Street, Winnipeg, Canada, R3E 3R2) under accession number IDAC301008-01. A sample culture of Spherodes mycoparasitica that is biotrophically parasitic on Fusarium abenaceum (Teleomorph: Giberella avenacea) has been deposited with the International Depositary Authority of Canada Collection under accession number IDAC301008-02. Referred to herein as “SM-Bst”. A sample culture of Spherodes mycoparasitica that is biotrophically parasitic on Fusarium graminearum (Teleomorph: Giberella zeae), has been deposited with the International Depositary Authority of Canada Collection under accession number IDAC301008-03, In this specification, it is called “SM-Gst”.

The 1266 bp DNA sequence from the large subunit ribosomal RNA gene (LSU) of S. mycoparasitica is shown in SEQ ID N0: 1.

The 1233 bp DNA sequence from the small subunit ribosomal RNA gene (SSU) of S. mycoparasitica is shown in SEQ ID NO: 2.

  The novel protein produced by Spherodes mycoparasitica disclosed herein is useful as an antifungal agent. These novel proteins can be used to treat, ameliorate, or otherwise control Fusarium infections. These novel proteins produced by S. mycoparasitica are used to treat, ameliorate, or otherwise control infection of Fusarium abenaceum, Fusarium graminearum, and / or Fusarium oxysporum, as well as plant It is particularly useful for improving health or growth.

  The novel S. mycoparasitica protein disclosed herein can be used as a prophylactic agent to reduce the probability that a fungal infection, particularly a fusarium infection, will occur.

  The novel S. mycoparasitic protein disclosed herein can be used to treat plants affected by Fusarium wilt or Fusarium red mold. These proteins can be used to prevent the spread of such a condition or as a preventive measure to reduce the risk of such a condition occurring.

  The novel S. mycoparasitic proteins disclosed herein can be used to treat, ameliorate, or otherwise control Fusarium infection. The protein can be applied in any suitable manner to an organism in need of treatment. For example, the protein may be used directly or as an inoculum (eg, granule composition, peat-based composition), seed application for application to a soil system or soilless growth system Inoculum (eg, powder composition, granule composition, peat-based composition, liquid composition) or inoculum for application to growing plants (eg, powder composition, liquid-based composition) ) May be further processed.

  The formulations disclosed herein include (i) one or more active ingredients, including one or more novel proteins, (ii) one or more carriers (often with a high density of activity) An inert material used to support and deliver the component to the target), and optionally (iii) one or more adjuvants (compounds that promote and maintain the function of the active component by protection from UV radiation) Compounds that ensure rain tolerance at the target, compounds that retain moisture or protect against drought, and / or Hynes and Boyetchko (2006, Research initiatives in the art and science of biopesticide formulations. Soil Biol. & Biochem. 38: 845-49), which promotes the application and dispersion of biological pesticides using standard agricultural equipment.

Internal Transcribed Spacer ribosomal DNA from S. mycoparasitica was sequenced. The 560 bp DNA sequence from the ITS region is shown in SEQ ID NO: 3.

  Whole intracellular protein extracts and whole extracellular protein extracts from Spherodes mycoparasitica demonstrate significant inhibition of germination of fungal spores of Fusarium, Sclerotinia, Rhizoctonia, and Psium species. Four novel proteins with fungicidal effects were isolated from the total protein extract, one having a molecular weight of about 79 kDa and comprising SEQ ID NO: 4, another one having a molecular weight of about 50 kDa And comprises SEQ ID NO: 5, another species has a molecular weight of about 36 kDa and comprises SEQ ID NO: 6, and the fourth species has a molecular weight of about 13 kDa and SEQ ID NO: 5. : 7 included. The present invention provides an antifungal agent comprising one or more of these proteins.

  Conventional molecular biology techniques may be used to isolate, characterize, and make each of the four novel proteins. For example, to identify the gene for this protein, S. mycoparasitica can be exposed with F. oxysporum filtrate and the upregulated mRNA can be isolated by standard Northern blot. cDNA can be prepared from mRNA by reverse transcriptase PCR (RT-PCR). The cDNA can be amplified, purified, and inserted into an appropriate vector. This vector may be inserted into a suitable host cell.

  Cloning and expression may focus on the isolation of genes encoding these novel antimicrobial proteins by designing primers for the identified proteins. The isolated gene may be cloned into a suitable expression vector (yeast or bacteria) with a suitable / efficient promoter to generate an industrial strain for large scale production of the protein. . These vectors can be purchased from Promega, Invitrogen, Clontech, or other companies. The cloned gene may be tested for its protein expression, and the expressed protein will be purified (using a His tag column or other column). The purified protein is obtained from a microtiter plate disclosed by the disk plate assay and / or Drummond et al. (2000, The development of microbiological methods for phytochemical screening. Rec. Res. Dev. Phytochem. 4: 143-152). The methods exemplified by the assay may be used to test for antifungal activity against phytopathogenic fungi. Next, rDNA techniques involving gene mutation or addition, or addition of enhancer, intron, or protein-specific promoters (GA inducible) may be used to enhance protein production.

  Sanghyun et.al. (2008, Transgenic wheat expressing a barley class II chitinase gene has enhanced resistance against Fusarium graminearum. J. Expt. Botany, 59, 2371-2378) and Tobias et. Al. (2007, Co-bombardment, integration pathogenic fungi using techniques known to those skilled in the art as exemplified by and expression of rice chitinase and thaumatin-like protein genes in barley (Hordeum vulgare cv. Conlon). Plant Cell Reports 26: 631-639). Encoding one or more or all of the 13 kDa, 36 kDa, 50 kDa, and 79 kDa proteins to create transgenic lines with antifungal activity against The selected gene may be transformed into the selected plant genome. Crop plants suitable for transformation with these novel proteins are exemplified by oil seed crops, cereal crops, legume crops, coniferous species, and deciduous species. Exemplary oilseed crops include canola, mustard, rapeseed, soybean, flax, sunflower, safflower, castor, and camelina, among others. Exemplary cereal crops include wheat, barley, oats, triticale, rye, rice, maize, sorghum, and quinoa, among others. Exemplary legume crops include, in particular, green beans, peas, chickpeas, lentils, and lupines. Exemplary conifer species include pine, spruce, fir, and hemlock, among others. Exemplary deciduous tree species include poplar, aspen, maple, oak, nutty trees, fruit trees, acacia, and eucalyptus, among others.

  The present invention relates to a 79 kDa protein (including SEQ ID NO: 4), a 50 kDa protein (including SEQ ID NO: 5), a 36 kDa protein (including SEQ ID NO: 6) produced by S. mycoparasitica And an antifungal composition comprising one or more or all of the 13 kDa proteins (including SEQ ID NO: 7). Alternatively, one or more or all of 13 kDa, 36 kDa, 50 kDa, and 79 kDa proteins are one of 13 kDa, 36 kDa, 50 kDa, and 79 kDa proteins. It may be produced by a transformed microorganism provided with one or more nucleic acid molecules encoding the species or species or all. The present invention also provides a method for controlling fungal diseases of plants and a method for detoxification of mycotoxins, hereinafter collectively referred to as “antifungal agents”, 13 kDa protein, 36 kDa protein, 50 kDa protein, and A method is provided comprising applying one or more or all of the 79 kDa proteins to a plant site.

  The composition of the present invention may comprise, for example, plant seeds and / or growth media (eg, soil or water) used to grow plants, and / or plant roots, and / or plant leaves, or You may apply to these arbitrary combinations. Exemplary crop plants that can be treated with the composition include crops such as seed crops, cereal crops, legume crops, horticultural crops, forest crops, and turfgrass.

  The present invention provides a composition comprising an antifungal agent and one or more agriculturally acceptable carriers and / or diluents that are believed to ensure the stability and / or performance of the final product. To do. Suitable carriers are in particular clay powders exemplified by kaolinite, bentonite and montmorillonite, peat granules, peat powders, carbon powders, cellulose granules and powders exemplified by sawdust, vermiculite, perlite, pumice, Illustrated by methylcellulose, talc. Suitable diluents are exemplified by water and non-viscous gels exemplified by alginate, carrageenan, agar, carboxymethylcellulose and the like. The carrier or diluent should be compatible with the active ingredient, should be agriculturally acceptable and should have excellent absorbency and appropriate volume density to allow easy particle dispersion and adhesion It is.

  The compositions herein may be applied as aqueous sprays, granules, and powders in accordance with established practices in the art. Aqueous sprays are usually prepared by mixing a wettable powder or emulsion formulation of the compound of the present invention with a relatively large amount of water to form a dispersion.

  The wettable powder may comprise a finely divided mixture of an antifungal agent, a solid carrier and a surfactant. The solid support is usually selected from attapulgite clay, kaolin clay, montmorillonite clay, diatomaceous earth, finely divided silica, purified silicate, or combinations thereof. Surfactants that may be useful herein have wetting ability, penetrating ability, and / or dispersing ability. They are typically present in an amount of about 0.5% to about 10% by weight. The surfactants herein can be selected from, for example, alkyl benzene sulfonates, alkyl sulfates, naphthalene sulfonates and condensed naphthalene sulfonates, sulfonated lignins, and nonionic surfactants.

  The emulsion may include the antifungal agent of the present invention in a liquid carrier that is a mixture of a water immiscible solvent and a surfactant. Solvents that may be useful herein include aromatic hydrocarbon solvents such as xylene, alkyl naphthalene, petroleum distillates, terpene solvents, ether-alcohols, organic ester solvents, or suitable combinations thereof.

  When the composition of the present invention is applied to plant debris or litter to control the dispersion of the source and inoculum, or when applied to the soil for pre-emergence protection, the granule formulation or powder is Sometimes more convenient than spray.

  In one embodiment, the antifungal agent disclosed herein is encapsulated in alginate pellets. The pellets may be prepared in any suitable manner. For example, one useful method is described in Harveson et. Al. (2002, Novel Use of a Pyrenomycetous Mycoparasite for Management of Fusarium Wilt of Watermelon. Plant Disease 86 (9): 1025-1030).

  The composition may contain other active substances that are useful in antifungal agents. For example, the compositions herein include Trichoderma, sulfur, neem oil, rosemary oil, jojoba oil, Bacillus subtilis, allylamine (eg, terbinafine), antimetabolite (eg, flucytosine), Other antifungal agents may be included such as azoles (eg ketoconazole, itraconazole), echinocandin (eg caspofungin), polyenes (eg amphotericin B), systemic agents (eg griseofulvin), or combinations thereof .

  The compositions herein may contain from about 0.1% to about 95% by weight antifungal agent, and from about 0.1% to about 95% by weight carrier and / or surfactant. Direct application to plant seeds before sowing can in some cases be very thin and provide a substantially uniform coating that accounts for only 1 or 2% by weight or less based on the weight of the seed. It may be achieved by mixing either the powdered solid compound or the powder with the seed. However, in some cases, a non-phytotoxic solvent such as methanol is conveniently used as a carrier to facilitate uniform distribution of the compounds of the invention on the surface of the seed.

  The compositions herein can be useful in the treatment of fungal infections in plants. Accordingly, the compositions herein include a 79 kDa protein (including SEQ ID NO: 4), a 50 kDa protein (including SEQ ID NO: 5), (SEQ ID NO: 6) disclosed herein. One or more or all of a 36 kDa protein (including SEQ ID NO: 7) and a 13 kDa protein (including SEQ ID NO: 7) and an acceptable carrier therefor.

The present invention relates to an exemplary method for making an antifungal composition comprising one or more or all of the 13 kDa protein, 36 kDa protein, 50 kDa protein, and 79 kDa protein of S. mycoparasitica. The method includes the following steps:
(A) inducing sporulation of S. mycoparasitica spores;
(B) culturing S. mycoparasitica;
(C) recovering the protein from the S. mycoparasitic culture,
(D) separating and recovering one or more or all of the 13 kDa protein, 36 kDa protein, 50 kDa protein, and 79 kDa protein from the recovered S. mycoparasitic protein; And (e) preparing a composition comprising one or more or all of 13 kDa, 36 kDa, 50 kDa, and 79 kDa proteins recovered from S. mycoparasitic protein .

  79 kDa protein (including SEQ ID NO: 4), 50 kDa protein (including SEQ ID NO: 5), 36 kDa protein (including SEQ ID NO: 6), and 13 (including SEQ ID NO: 7) The kDa protein may be separated and recovered together from the recovered S. mycoparasitic protein. Alternatively, the 13 kDa protein and / or the 36 kDa protein and / or the 50 kDa protein and / or the 79 kDa protein, or any combination thereof, may be individually recovered from the recovered S. mycoparasitic protein .

The invention includes a 79 kDa protein (including SEQ ID NO: 4), a 50 kDa protein (including SEQ ID NO: 5), a 36 kDa protein (including SEQ ID NO: 6), and (SEQ ID NO: 6) of S. mycoparasitica It relates to another exemplary method for making an antifungal composition comprising one or more or all of 13 kDa proteins (including ID NO: 7). The method includes the following steps:
(F) selecting a selected microbial culture from one or more of the 13 kDa, 36 kDa, 50 kDa, and 79 kDa proteins produced by S. mycoparasitica Transforming with multiple genes;
(G) culturing the transformed microbial culture;
(H) recovering the protein from the transformed culture;
(I) separating and recovering 13 kDa protein and / or 36 kDa protein and / or 50 kDa protein and / or 79 kDa protein from said recovered protein; and (j) 13 kDa protein, 36 kDa protein Preparing a composition comprising one, a plurality or all of a 50 kDa protein, and a 79 kDa protein.

  The 13 kDa protein and / or the 36 kDa protein and / or the 50 kDa protein and / or the 79 kDa protein may be separated and recovered together from the recovered protein. Alternatively, 13 kDa protein and / or 36 kDa protein and / or 50 kDa protein and / or 79 kDa protein may be recovered individually from the recovered protein. Any suitable gene that can be transformed with one or more genes encoding one or more or all of the 13 kDa, 36 kDa, 50 kDa, and 79 kDa proteins produced by S. mycoparasitica Microbial cultures include bacterial cultures such as Escherichia coli, Pseudomonas species, Bacillus species, etc., Zygosaccharomyces species, Saccharomyces species, Pichia species, and Curberomyces. Examples are yeast cultures such as (Kluveromyces) species, and Penicillium species.

Example 1: Production and extraction of intracellular and extracellular proteins from Spherodes mycoparasitica Spherodes mycoparasitica strains IDAC 301008-02 and / or IDAC 301008-03 are used in all studies disclosed herein They are called “SM-Bst” and “SMGst” respectively throughout. SM-Bst and SM-Gst stock cultures were prepared and maintained at 21 ° C. in potato dextrose broth (PDB) culture medium. To prepare intracellular and extracellular proteins from each type of stock culture, approximately 3 ml from the PDB culture was transferred to a 250-ml Erlenmeyer flask containing 50 ml PDB growth medium. The flasks were incubated for 7 days on a rotary shaker (150 rpm) at room temperature. Young mycelium was filtered through Whatman® No. 1 filter paper (Whatman is a registered trademark of Whatman International Ltd., Kent, UK). The mycelium collected on Whatman® No. 1 filter paper is used for the extraction of intracellular proteins, while the filtered culture medium is used for the recovery of extracellular proteins from the mycelium used.

Example 2: Fractionation, separation and purification of intracellular and extracellular proteins from Spherodes mycoparasitica Intracellular protein extraction:
Intracellular proteins were extracted according to the method disclosed by Chen et al. (2004, Heat shock proteins of thermophilic and thermotolerant fungi from Taiwan. Bot. Bull. Acad. Sin. 45: 247-257). A total amount of 200 mg fungal mycelium from each sample was powdered with pestle in a sterile porcelain mortar in liquid nitrogen and mixed with 1.5 ml of chilled extraction buffer. Mycelium (~ 200 mg), 50 mM Tris-HCl (pH 8.5); 2% sodium dodecyl sulfate (SDS); 2% β-mercaptoethanol; 1 mM phenylmethylsulfonyl fluoride (PMSF in 95% alcohol) Grind by sonication in a grind tube containing 1 mL buffer consisting of The homogenate was centrifuged at 12,000 rpm, 28 ° C. for 10 minutes and the supernatant was collected. Four volumes of cold acetone (−20 ° C.) was added and the sample was frozen overnight at −20 ° C. The precipitate (protein) is stored in acetone, centrifuged at 12000 rpm, 28 ° C. for 5 minutes, and finally 50-100 μl of sample buffer (62.5 mM Tris-HCl (pH 6.8); 3% SDS 10% glycerol; 5% β-mercaptoethanol). The protein sample was transferred to a new 1.5 ml Eppendorf tube and stored at -80 ° C. until used for further studies.

Extracellular protein extraction:
Filtered culture medium is used as a source of extracellular protein and Amicon® ultrafiltration centrifuge tube with 3000 Dalton cutoff membrane (Amicon is registered in Millipore Corp., Billerica, MA, USA) The product was concentrated by centrifugation at 4000 rpm and 4 ° C. Using a disk diffusion assay, total extracellular proteins were tested for antifungal activity against the fungal pathogens Fusarium oxysporum and Fusarium graminearum. The antifungal activity of whole extracellular proteins is described by Roberts and Selitrennikoff (1986, Isolation and partial characterization of two antifungal proteins from barley. Biochim. Biophys. Acta, 880: 161-170) with some modifications. Tested under sterile conditions by a radial disk plate diffusion assay. Tests of total extracellular protein for antifungal activity against F. oxysporum and F. graminearum were performed in petri dishes containing potato dextrose agar. Place the mycelial plug from the actively growing fungal plate in the center of the Petri dish and place a sterile filter paper disc (5 mm diameter Whatman® filter paper no. 1) from the edge of the mycelial colony on the agar surface. Placed at a distance of 0.5 cm. An aliquot (60 μL) containing 2.5 μg of extracellular protein was added to the disc (the test spot is identified as “T” in FIGS. 1 (a) A and 1 (a) B). Sterile distilled water and buffer were used as controls (control spots are identified as “C” in FIGS. 1 (a) A and 1 (a) B). The plates were then incubated for 4 days at room temperature and examined for inhibition of mycelium growth. Inhibition of fungal growth was determined by measuring the area of the mycelium colony and calculating the percent reduction in the growth area of the mycelium colony relative to the control. Total extracellular protein recovered from SMCD cultures inhibited mycelium growth in both F. oxysporum and F. graminearum (FIGS. 1 (a) A and 1 (a) B). Three days after the start of germination, about 22% inhibition in mycelial elongation of F. oxysporum and 30% inhibition in mycelial elongation of F. graminealam were observed (FIG. 1 (b)). One day later, inhibition of mycelial elongation of F. oxysporum increased to about 30%, while inhibition of mycelial elongation in F. graminearum increased to about 35% (FIG. 1b).

Non-denaturing polyacrylamide gel electrophoresis (non-denaturing PAGE):
Using the method taught by Laemmli (1970, Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227: 680-685), proteins (intracellular and extracellular) can be separated in a discontinuous buffer system. Separation was performed by 10% non-denaturing polyacrylamide gel electrophoresis (non-denaturing PAGE). Concentration gel (3.75% acrylamide / bisacrylamide 30: 0.8 v / v) has 125 mM Tris-HCL, pH 6.8 as buffer, and separation gel (7.5% acrylamide / bisacrylamide 30: 0.8 v / v) ) Had 375 mM Tris-HCl, pH 8.8 as buffer. The gel was run at 120 V overnight at 4 ° C. with Tris-glycine electrode buffer containing 25 mM Tris and 192 mM glycine, pH 8.3. After electrophoresis, the gel was stained according to the silver staining method (Bio-Rad (registered trademark) kit method).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE):
SDS-PAGE of protein eluted from non-denaturing PAGE and total protein was performed on a 12% polyacrylamide gel according to the method taught by Laemmli (1970). Proteins were analyzed by using 5% concentrating gel (pH 6.8) and 12% separating acrylamide gel (pH 8.8) in Tris-glycine buffer (pH 8.3) and appropriate markers. Prior to SDS-electrophoresis, the protein was mixed with an equal volume of sample buffer (60 mM Tris-HCl buffer, 4% SDS, pH 6.8) containing 5% β-mercaptoethanol. A mixture of standard marker proteins obtained from Bio-Rad® Laboratories Canada Ltd. (Montreal, PQ, Canada; Bio-Rad is a registered trademark of Bio-Rad Laboratories Inc., Hercules, CA, USA) used. All samples were heated at 95 ° C. for 5 minutes and cooled to room temperature. Proteins were visualized by silver staining (Bio-Rad® kit method). The molecular weight of the protein was estimated by comparison with the mobility of standard molecular weight markers.

Fast protein liquid chromatography (FPLC) of extracellular proteins:
Proteins were obtained using Superdex® 75 using the FPLC AKTA® purification system (AKTA is a registered trademark of GE Healthcare Bio-Sciences AB Ltd., Uppsala, Sweden) according to the manufacturer's instructions. Fractionation through a GL 10/30 column (Superdex is a registered trademark of GE Healthcare Bio-Sciences AB Ltd., Uppsala, Sweden). The column was pre-equilibrated with sterile water and 50 mM sodium phosphate buffer, pH 7.0 containing 0.15 M NaCl, followed by protein injection (approximately 500 μL) and protein with the same buffer at a flow rate of 1.0 ml / min. Was eluted. The protein was purified through gel filtration using 50 mM sodium phosphate buffer, pH 7.0, containing 0.15 M NaCl. Gel filtration on Superdex 75 resolved the protein into distinct peaks. 0.8 ml fractions were collected and their purity was confirmed by SDS-PAGE.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE):
SDS-PAGE (12%) of the protein recovered from the FPLC fraction was performed according to the method taught by Laemmli (1970). FPLC fractions giving all peaks were pooled and collected by precipitation in 1: 4 volume of cold acetone and stored overnight at -20 ° C. After 10 minutes centrifugation at 12,000 g, the precipitated protein (pellet) was dissolved in a minimal amount (30 μL) of assay buffer. Proteins were analyzed by SDS-PAGE with 5% concentrating gel (pH 6.8) and 12% acrylamide gel (pH 8.8) in Tris-glycine buffer (pH 8.3) and appropriate markers. Prior to SDS-electrophoresis, the protein was mixed with an equal volume of sample buffer (60 mM Tris-HCl buffer, 4% SDS, pH 6.8) containing 5% β-mercaptoethanol. A mixture of standard marker proteins (Bio-Rad® protein marker, Bio-Rad Laboratories Canada Inc.) was used. All samples were heated to 95 ° C. for 5 minutes and cooled to room temperature before loading onto the gel. Proteins were visualized by silver staining (Bio-Rad® silver staining kit, Bio-Rad Laboratories Canada Inc.). The molecular weight of the protein was estimated by comparison with the mobility of standard molecular weight markers. The molecular weight of the fractionated protein obtained from FPLC was determined by SDS-PAGE (12% and 16%). In order to separate the extracellular protein constituting the high molecular weight protein band separated by the non-denaturing PAGE process, 5% SDS-PAGE was performed.

  Non-denaturing PAGE electrophoresis of intracellular proteins extracted from SM-Bst and SMGst using a 10% non-denaturing polyacrylamide gel separated two protein bands, represented as upper and lower bands ( Figure 2). Each protein band of non-denaturing PAGE was immersed in 500-750 μl of sample buffer (62.5 mM Tris-HCl, pH 6.8; 1% PMSF, 1% EDTA) and placed at 4 ° C. overnight. The protein was recovered by centrifugation at 12,000 g for 10 minutes. For determination of the molecular weight of the separated protein eluted from non-denaturing PAGE, SDS-PAGE separation of intracellular proteins was performed using a 12% gel. Proteins eluted from the lower and upper bands of the intracellular sample indicated the presence of two separate bands of approximately 50 and 79 kDa (FIG. 3).

  Non-denaturing PAGE electrophoresis of extracellular proteins extracted from SM-Bst and SMGst using a 10% non-denaturing polyacrylamide gel separated the four protein bands designated EC1, EC2, EC3, and EC4 (Figure 4). Each protein band of non-denaturing PAGE was immersed in 500-750 μl of sample buffer (62.5 mM Tris-HCl, pH 6.8; 1% PMSF, 1% EDTA) and placed at 4 ° C. overnight. The protein was recovered by centrifugation at 12,000 g for 10 minutes. For determination of the molecular weight of the separated protein eluted from non-denaturing PAGE, SDS-PAGE separation of extracellular protein was performed using a 12% gel. Proteins eluted from the four bands have molecular weights of approximately: (i) 79 kDa (EC1), (ii) 50 kDa (CE2), (iii) 36 kDa (EC3), and 13 kDa (EC4) The presence of separated and purified protein was shown (Figure 5).

  The elution profile of four bands of extracellular protein on a Superdex 75 GL 10/30 column, determined by FPLC high-performance protein liquid chromatography using an AKTA® purification system, is shown in FIG.

Example 3: Inhibition of mycelium growth by Fusarium species by purified intracellular and purified extracellular proteins recovered from S. mycoparasitic cultures Roberts et al. (1986) with some modifications , Isolation and partial characterization of two antifungal proteins from barley. Biochim. Biophys. Acta, 880: 161-170), testing the antifungal activity of proteins under sterile conditions by a radial disc plate diffusion assay did. Isolated protein assays for antifungal activity against F. oxysporum and F. graminearum were performed in petri dishes containing potato dextrose agar. Place the mycelium plug from the actively growing fungal plate in the center of the Petri dish and place a sterile filter paper disc (Whatman® filter paper no.1 with a diameter of 5 mm) on the agar surface, at the edge of the mycelium colony. Placed at a distance of 0.5 cm. Isolated protein (60 μL) was added to the disc. Sterile distilled water and a buffer containing only antifungal protein were used as controls. The plates were then incubated at room temperature for 3-4 days and then examined for the inhibition zone around the disc, if any. In this manner, if the protein being tested is an antifungal agent, a crescent-shaped inhibition zone of fungal growth would appear around the disc. Inhibition of fungal growth was determined by measuring the area of mycelium colonies and calculating the percent reduction in area of mycelium colonies with controls (plates treated with water and buffer).

  Both 50 kDa intracellular protein and 79 kDa intracellular protein are the hyphae of F. oxysporum (Figures 7 (a) -7 (d)) and F. graminearum (Figures 8 (a) -8 (d)). Elongation, i.e., mycelium growth, was inhibited.

  The 79 kDa protein (EC1), the 50 kDa protein (EC2), and the 36 kDa protein (EC3) are represented by F. oxysporum (Figures 9 (a) -9 (b)) and F. graminearum (Figures 10 (a)- The mycelium growth of FIG. 10 (b) was inhibited. The 13 kDa protein (EC4) inhibited the mycelial growth of F. graminearum (FIG. 11 (a)) and F. oxysporum (FIG. 11 (b)).

Example 4: Inhibition of spore germination by Fusarium species by purified extracellular protein recovered from S. mycoparasitica cultures
The inhibitory effect of four extracellular S. mycoparasitic proteins EC1, EC2, EC3, and EC4 on spore germination of Fusarium sp. 1145-1153) and Yadav et al. (2007, An antifungal protein from Escherichia coli. J. Med. Microbiol. 56: 637-644). The potential toxicity of the fractionated protein was tested in a growth inhibition assay using fungi F. oxysporum and F. graminearum. The in vitro antifungal activity of the fractionated protein was determined in 96 well microtiter plates. In microplate wells, 10 μl of potato dextrose broth (PDB; Difco Laboratories, Detroit) was mixed with 3 μl of a spore suspension of F. oxysporum and F. graminearum. 7 μL aliquots of various peaks containing protein fractions were added to the suspension in microtiter plates (12-8 wells). Water and buffer were used as negative controls. The microtiter plate was then incubated in the dark at room temperature. After 24 hours, an inverted fluorescence microscope was used to observe for inhibition of spore germination in both untreated and treated wells. % Growth inhibition was determined using the number of germinated and ungerminated spores and the percentage of the area covered by mycelium in the microscope.

  All four extracellular proteins significantly inhibited spore germination of F. oxysporum and F. graminearum (FIGS. 12 (a) and 12 (b)). In addition, all four extracellular proteins established large zones of inhibition when the organisms were separately plated on solid media for mycelial growth of F. oxysporum and F. graminearum (FIG. 13). FIG.14A (c) is a photomicrograph showing the germination of control F. oxysporam spores, FIG.14A (a) shows the inhibitory effect of the 50 kDa protein on the germination of F. oxysporam spores, and FIG. ) Shows the inhibitory effect of the 13 kDa protein on the germination of F. oxysporum spores. FIG.14B (c) is a photomicrograph showing the germination of the control F. graminearum spores, FIG.14B (a) shows the inhibitory effect of the 50 kDa protein on the germination of F. graminearum spores, and FIG. ) Shows the inhibitory effect of the 13 kDa protein on the germination of F. graminearum spores.

Example 5: Inhibitory effects of crystal-forming EC1 and EC3 proteins recovered from S. mycoparasitica culture The mycotoxin effects of crystal-forming extracellular proteins EC1 (79 KDa) and EC3 (36 KDa) are actively proliferating on PDAs Medium F. abenaceum and F. graminearum cultures were evaluated. F. Abenaceum was grown on PDA agar until the mycelium covered the surface of the plate. Half of the plate was treated with a mixture of EC1 and EC3 crystal proteins, after which the plates were incubated in the dark at 23 ° C. for 5 days. Significant mycelial damage observed on half plate treated with EC1 / EC3 protein mixture (Figure 15 (B)) compared to the untreated control side of the plate (Figure 15 (a)) It was done.

  Wheat seeds were spread throughout the surface of the PDA plate. After the seeds germinated, the plates were inoculated with F. graminearum. Half of the plate was treated with the EC1 / EC3 protein mixture, after which the plate was incubated for 5 days at 23 ° C. in the dark. On the half plate that received the EC1 / EC3 protein mixture, there was no fungal growth (Figure 16 (b)), but on the untreated side of the plate, fungal mycelium grew (Figure 16 (a) )). Examination of the surface of the agar that received the EC1 / EC3 protein mixture with a confocal laser scanning microscope showed a number of dissolved mycelium elements (see arrows in FIG. 16 (c)).

  F. Graminearum was grown on PDA agar until the mycelium covered the surface of the plate. Half of the plate was treated with a mixture of EC1 and EC3 crystal proteins, after which the plate was incubated at 21 ° C. for 5 days in the dark. Significant mycelial damage observed on half plate treated with EC1 / EC3 protein mixture (Figure 17 (B)) compared to the untreated control side of the plate (Figure 17 (a)) It was done. Examination of the surface of the agar that received the EC1 / EC3 protein mixture with a scanning electron microscope showed a number of dissolved mycelium elements (see arrows in FIG. 18 (a)). Investigation of the same surface with chemical force microscopy showed a number of broken mycelium elements and apoptotic lysed cells (see arrows in FIG. 18 (c)).

Example 6: Sequencing and putative identification of purified extracellular protein recovered from S. mycoparasitica cultures
EC1 (79 Kda), EC2 (50 Kda), EC3 (36 Kda), and EC4 (13 KDa) extracellular protein bands were excised from 5%, 12%, and 16% SDS-PAGE gels. The excised protein bands were used for sequencing and peptide characterization using a MALDI-TOF-MS instrument and for putative identification by comparing peptide sequences to UniProtKB / Swiss-Prot and NCBI databases. Sent to McGill University and Genome Quebec Innovation Center (Rm 704, 740 Dr. Penfield Avenue, Montreal, PQ, Canada).

であると決定された。
EC1の質量は、78,688 Da（79 kDa）であると決定され、その正体は、RTXタンパク質毒素および関連するCa₂₊結合タンパク質に類似していると予測された。 By MALDI-TOF-MS analysis, the amino acids constituting the EC1 protein were separated (FIG. 19). The sequence of amino acids that make up the EC1 protein is

Was determined to be.
The mass of EC1 was determined to be 78,688 Da (79 kDa) and its identity was predicted to be similar to RTX protein toxins and related Ca ₂₊ binding proteins.

EC2の質量は、49,443 Da（50 kDa）であると決定され、その正体は、β-1,4-グルカーゼタンパク質に類似していると予測された。 The MALDI-TOF-MS separation of the EC2 extracellular protein band (Figure 20) allowed the determination of the amino acid sequence that makes up the EC2 protein:

The mass of EC2 was determined to be 49,443 Da (50 kDa) and its identity was predicted to be similar to β-1,4-glucase protein.

EC3の質量は、35,578 Da（36 kDa）であると決定され、その正体は、キシラナーゼタンパク質に類似していると予測された。 By MALDI-TOF-MS separation of EC3 extracellular protein band (Fig. 21), the amino acid sequence constituting EC3 protein could be determined:

The mass of EC3 was determined to be 35,578 Da (36 kDa) and its identity was predicted to be similar to the xylanase protein.

EC4の質量は、2,563 Daであると決定され（SDS-Page分析により分子量が13 kDaであると示されているため、これは切断された断片である）、その正体は、仮想のセラトプラタニン（Cerato-platanin）毒素であると予測された。 The MALDI-TOF-MS separation of the EC4 extracellular protein band (Figure 22) allowed the determination of the amino acid sequence that makes up the EC3 protein:

The mass of EC4 was determined to be 2,563 Da (this is a cleaved fragment because the SDS-Page analysis shows that the molecular weight is 13 kDa) and its identity is hypothetical seratoplatinin (Cerato -platanin) predicted to be a toxin.

Example 6: Effect of purified extracellular protein recovered from S. mycoparasitica cultures on mycelial growth and growth of Sclerotinia sclerotiorum, Rhizoctonia solani, and Psium Ultimum
S. Mycoparasitica recovered three common and widely used fungal and mycotoxin effects of purified extracellular proteins EC1 (79 KDa) and EC3 (36 KDa) that cause significant damage to crops Of additional plant pathogens. Ghosh (2006) and Yadav et al. (2007) disclose the inhibitory effect of a mixture of EC1 + EC3 extracellular S. mycoparasitic protein (1: 1) on S. sclerotiolum, R. solani, and P. Ultimum Was evaluated using a microtiter plate assay. According to the method taught by Strom et al. (2005, Co-cultivation of antifungal Lactobacillus plantarum MiLAB 393 and Aspergillus nidulans, evaluation of effects on fungal growth and protein expression. FEMS Microb Let, 246: 119-124) After growth, the mycelium was collected and the biomass was weighed. FIG. 23 (a), FIG. 23 (c), and FIG. 23 (e) are S. sclerotiolum, R. solani, and P. Ultima grown on PDA for 4 days in the dark at 26 ° C., respectively. 2 is a photomicrograph of a control culture. Figure 23 (b), Figure 23 (d), and Figure 23 (f) corrected with a mixture of EC1 (79 kDa) and EC3 (36 kDa) extracellular proteins produced by S. mycoparasitica (1: 1) 2 are photomicrographs of S. sclerotiorum, R. solani, and P. Ultimum grown on cultured PDAs, respectively. The extracellular protein mixture caused significant mycelial damage to all three plant pathogen fungal cultures (FIGS. 23 (b), 23 (d), and 23 (f)). The data in FIG. 24 shows that the extracellular protein mixture caused (i) about 80% inhibition in growth of S. sclerotiorum compared to untreated controls, and (ii) in growth of R. solani. Demonstrated that it caused about 85% inhibition compared to untreated controls, and (iii) caused about 95% inhibition in P. Ultimum growth compared to untreated controls. doing. These data demonstrate that the extracellular protein of S. mycoparasitica disclosed herein has a bacteriostatic and mycotoxin effect against a wide range of soil and phytopathogenic fungi.

  Studies disclosed herein have revealed that a 79 kDa protein comprising SEQ ID NO: 4, a 50 kDa protein comprising SEQ ID NO: 5, a 36 kDa protein comprising SEQ ID NO: 6, and SEQ ID NO: 7 An extracellular protein derived from S. mycoparasitica, identified as a 13 kDa protein containing, is useful for inhibiting spore germination of various phytopathogenic fungi such as Fusarium, Sclerotinia, Rhizoctonia, and Psium That is clearly demonstrated. According to the studies disclosed herein, contacting the mycelium of a phytopathogenic fungus with one or more of these extracellular proteins causes cessation of mycelium growth and leads to lysis, Further demonstrated. Thus, one or more polypeptides comprising an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 Amino acids disclosed in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 by transforming plant cells with one or more nucleic acid molecules encoding It is believed that one or more polypeptide molecules comprising an amino acid sequence having at least 80% sequence identity with the sequence can be produced in plant cells. Thus, when such transformed cells are cultured in plants, the plant containing the cells is thought to express a polypeptide that results in the formation of extracellular proteins within the cells. Are phytopathogenic fungi that are in close proximity to and / or in contact with extracellular proteins inhibited during the process of colonizing and / or infecting the surface of such plants? Or it is considered dissolved.

Some exemplary embodiments of the invention relate to transformed plant cells that overexpress one or more novel proteins. Such transformed plant cells can be included in plant propagation material exemplified by seeds, somatic embryos, organogenic tissues, etc., which can be cultured to the whole plant. The entire plant, including the transformed plant cells, is resistant or at least resistant to disease symptoms caused by phytopathogenic fungi exemplified by Fusarium species, Sclerotinia species, Rhizoctonia species, Psium species, etc. is there.
[Invention 1001]
A composition for controlling disease symptoms caused by one or more phytopathogenic fungi comprising SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 A composition comprising a polypeptide molecule comprising an amino acid sequence sharing at least 80% sequence identity with an amino acid sequence selected from and a carrier.
[Invention 1002]
The composition of the present invention 1001, wherein the polypeptide molecule comprises an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 4.
[Invention 1003]
The composition of the present invention 1001 wherein the polypeptide molecule comprises SEQ ID NO: 4.
[Invention 1004]
The composition of the invention 1001 wherein the polypeptide molecule comprises an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 5.
[Invention 1005]
The composition of the present invention 1001 wherein the polypeptide molecule comprises SEQ ID N0: 5.
[Invention 1006]
The composition of the invention 1001 wherein the polypeptide molecule comprises an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 6.
[Invention 1007]
The composition of the invention 1001 wherein the polypeptide molecule comprises SEQ ID N0: 6.
[Invention 1008]
The composition of the invention 1001 wherein the polypeptide molecule comprises an amino acid sequence sharing at least 80% sequence identity with SEQ ID NO: 7.
[Invention 1009]
The composition of the invention 1001 wherein the polypeptide molecule comprises SEQ ID N0: 7.
[Invention 1010]
The composition according to any one of the present inventions 1001 to 1009, which is a seed treatment composition.
[Invention 1011]
The composition according to any one of the present inventions 1001 to 1009, which is a plant treatment composition.
[Invention 1012]
The composition according to any one of the present inventions 1001 to 1009, which is a soil treatment composition.
[Invention 1013]
The composition of any of the inventions 1001-1012, wherein the phytopathogenic fungus is one of a Fusarium species, a Sclerotinia species, a Rhizoctonia species, or a Pithium species.
[Invention 1014]
Use of a composition according to any of the invention 1001 to 1013 for controlling disease symptoms in a plant caused by one of Fusarium species, Sclerotinia species, Rhizoctonia species, or Psium species.
[Invention 1015]
Use of the present invention 1014 comprising one or more of treating seeds, treating plants, and treating plant growth substrates.
[Invention 1016]
Amino acids selected from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 for controlling disease symptoms caused by one or more phytopathogenic fungi Use of a microbial cell that expresses a polypeptide molecule comprising an amino acid sequence that shares at least 80% sequence identity with the sequence.
[Invention 1017]
The use of the present invention 1016 wherein the polypeptide molecule comprises an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 4.
[Invention 1018]
The use of the present invention 1016 wherein the polypeptide molecule comprises SEQ ID N0: 4.
[Invention 1019]
The use of the invention 1016 wherein the polypeptide molecule comprises an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 5.
[Invention 1020]
The use of the present invention 1016 wherein the polypeptide molecule comprises SEQ ID N0: 5.
[Invention 1021]
The use of the invention 1016 wherein the polypeptide molecule comprises an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 6.
[Invention 1022]
The use of the present invention 1016 wherein the polypeptide molecule comprises SEQ ID N0: 6.
[Invention 1023]
The use of the present invention 1016 wherein the polypeptide molecule comprises an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 7.
[Invention 1024]
The use of the present invention 1016 wherein the polypeptide molecule comprises SEQ ID N0: 7.
[Invention 1025]
The use of any of the present invention 1016-1024, wherein the microbial cells are fungal cells, yeast cells or bacterial cells.
[Invention 1026]
Use of the invention 1025, wherein the fungal cell is a cell of Sphaerodes mycoparasitica or a cell of the Penicillium species.
[Invention 1027]
Use of Invention 1025, wherein the yeast cell is a Zygosaccharomyces species cell, a Saccharomyces species cell, a Pichia species cell, or a Kluveromyces species cell.
[Invention 1028]
Use of the invention 1025 wherein the bacterial cell is an Escherichia coli cell, a Pseudomonas species cell, or a Bacillus species cell.
[Invention 1029]
Express a polypeptide molecule comprising an amino acid sequence that shares at least 80% sequence identity with an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. A transformed plant cell that is resistant to infection by Fusarium species.
[Invention 1030]
The transformed plant cell of the present invention 1029, wherein the polypeptide molecule comprises an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 4.
[Invention 1031]
The transformed plant cell of the present invention 1029, wherein the polypeptide molecule comprises SEQ ID NO: 4.
[Invention 1032]
The transformed plant cell of the present invention 1029, wherein the polypeptide molecule comprises an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 5.
[Invention 1033]
The transformed plant cell of the present invention 1029, wherein the polypeptide molecule comprises SEQ ID NO: 5.
[Invention 1034]
The transformed plant cell of the present invention 1029, wherein the polypeptide molecule comprises an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 6.
[Invention 1035]
The transformed plant cell of the present invention 1029, wherein the polypeptide molecule comprises SEQ ID NO: 6.
[Invention 1036]
The transformed plant cell of the present invention 1029, wherein the polypeptide molecule comprises an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 7.
[Invention 1037]
The transformed plant cell of the present invention 1029, wherein the polypeptide molecule comprises SEQ ID NO: 7.
[Invention 1038]
The transformed plant cell of any of the present invention 1029-1037, which is an oilseed plant cell, or a cereal plant cell, or a legume plant cell.
[Invention 1039]
An isolated protein for controlling disease symptoms caused by one or more phytopathogenic fungi, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID An isolated protein comprising a polypeptide molecule comprising an amino acid sequence sharing at least 80% sequence identity with an amino acid sequence selected from NO: 7.
[Invention 1040]
The isolated protein of the invention 1039, wherein the polypeptide sequence comprises an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 4.
[Invention 1041]
An isolated protein of the present invention 1039 comprising SEQ ID NO: 4.
[Invention 1042]
An isolated protein of the invention 1039 comprising an amino acid sequence sharing at least 80% sequence identity with SEQ ID NO: 5.
[Invention 1043]
An isolated protein of the present invention 1039 comprising SEQ ID NO: 5.
[Invention 1044]
An isolated protein of the invention 1039 comprising an amino acid sequence sharing at least 80% sequence identity with SEQ ID NO: 6.
[Invention 1045]
An isolated protein of the present invention 1039 comprising SEQ ID NO: 6.
[Invention 1046]
An isolated protein of the invention 1039 comprising an amino acid sequence sharing at least 80% sequence identity with SEQ ID NO: 7.
[Invention 1047]
An isolated protein of the present invention 1039 comprising SEQ ID NO: 7.
[Invention 1048]
An isolated nucleic acid molecule encoding the protein of any of the present invention 1039-1047.
[Invention 1049]
A microbial cell transformed with the nucleic acid molecule of the present invention 1048.
[Invention 1050]
The microbial cell of the present invention 1049, wherein the nucleic acid molecule is operably linked to a promoter.
[Invention 1051]
The microbial cell of the invention 1049 or 1050 which is a fungal cell, a yeast cell or a bacterial cell.
[Invention 1052]
The microbial cell of the present invention 1051, wherein the fungal cell is a Sphaerodes mycoparasitic cell or a Penicillium species cell.
[Invention 1053]
1051. The microbial cell of the invention 1051 wherein the yeast cell is a cell of Digosaccharomyces spp., A cell of Saccharomyces spp., A cell of Pichia spp.
[Invention 1054]
1051. The microbial cell of the invention 1051 wherein the bacterial cell is an E. coli cell, Pseudomonas species cell, or Bacillus species cell.
[Invention 1055]
A plant cell transformed with the nucleic acid molecule of the present invention 1048.
[Invention 1056]
The plant cell of the present invention 1055, wherein the nucleic acid molecule is operably linked to a promoter.
[Invention 1057]
The plant cell of the present invention 1055 or 1056, which is an oilseed plant cell, a cereal plant cell, or a legume plant cell.

Claims (57)

  A composition for controlling disease symptoms caused by one or more phytopathogenic fungi comprising SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 A composition comprising a polypeptide molecule comprising an amino acid sequence sharing at least 80% sequence identity with an amino acid sequence selected from and a carrier.   2. The composition of claim 1, wherein the polypeptide molecule comprises an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 4.   2. The composition of claim 1, wherein the polypeptide molecule comprises SEQ ID NO: 4.   2. The composition of claim 1, wherein the polypeptide molecule comprises an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 5.   2. The composition of claim 1, wherein the polypeptide molecule comprises SEQ ID N0: 5.   2. The composition of claim 1, wherein the polypeptide molecule comprises an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 6.   2. The composition of claim 1, wherein the polypeptide molecule comprises SEQ ID N0: 6.   2. The composition of claim 1, wherein the polypeptide molecule comprises an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 7.   2. The composition of claim 1, wherein the polypeptide molecule comprises SEQ ID N0: 7.   The composition according to any one of claims 1 to 9, which is a seed treatment composition.   The composition according to any one of claims 1 to 9, which is a plant treatment composition.   The composition according to any one of claims 1 to 9, which is a soil treatment composition.   13. The phytopathogenic fungus according to any one of claims 1 to 12, wherein the phytopathogenic fungus is one of a Fusarium species, a Sclerotinia species, a Rhizoctonia species, or a Pithium species. Composition.   14. Use of a composition according to any one of claims 1 to 13 for controlling disease symptoms in a plant caused by one of Fusarium species, Screrotinia species, Rhizoctonia species, or Psium species.   15. Use according to claim 14, comprising one or more of treating seed, treating a plant, and treating a plant growth substrate.   Amino acids selected from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 for controlling disease symptoms caused by one or more phytopathogenic fungi Use of a microbial cell that expresses a polypeptide molecule comprising an amino acid sequence that shares at least 80% sequence identity with the sequence.   17. Use according to claim 16, wherein the polypeptide molecule comprises an amino acid sequence sharing at least 80% sequence identity with SEQ ID NO: 4.   17. Use according to claim 16, wherein the polypeptide molecule comprises SEQ ID N0: 4.   17. Use according to claim 16, wherein the polypeptide molecule comprises an amino acid sequence sharing at least 80% sequence identity with SEQ ID NO: 5.   17. Use according to claim 16, wherein the polypeptide molecule comprises SEQ ID N0: 5.   17. Use according to claim 16, wherein the polypeptide molecule comprises an amino acid sequence sharing at least 80% sequence identity with SEQ ID NO: 6.   17. Use according to claim 16, wherein the polypeptide molecule comprises SEQ ID N0: 6.   17. Use according to claim 16, wherein the polypeptide molecule comprises an amino acid sequence sharing at least 80% sequence identity with SEQ ID NO: 7.   17. Use according to claim 16, wherein the polypeptide molecule comprises SEQ ID N0: 7.   25. Use according to any one of claims 16 to 24, wherein the microbial cell is a fungal cell, a yeast cell or a bacterial cell.   26. Use according to claim 25, wherein the fungal cell is a cell of Sphaerodes mycoparasitica or a cell of the Penicillium species.   26. The use of claim 25, wherein the yeast cell is a Zygosaccharomyces species cell, a Saccharomyces species cell, a Pichia species cell, or a Kluveromyces species cell.   26. The use according to claim 25, wherein the bacterial cell is an Escherichia coli cell, a Pseudomonas species cell, or a Bacillus species cell.   Express a polypeptide molecule comprising an amino acid sequence that shares at least 80% sequence identity with an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. A transformed plant cell that is resistant to infection by Fusarium species.   30. The transformed plant cell of claim 29, wherein the polypeptide molecule comprises an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 4.   30. The transformed plant cell of claim 29, wherein the polypeptide molecule comprises SEQ ID NO: 4.   30. The transformed plant cell of claim 29, wherein the polypeptide molecule comprises an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 5.   30. The transformed plant cell of claim 29, wherein the polypeptide molecule comprises SEQ ID NO: 5.   30. The transformed plant cell of claim 29, wherein the polypeptide molecule comprises an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 6.   30. The transformed plant cell of claim 29, wherein the polypeptide molecule comprises SEQ ID NO: 6.   30. The transformed plant cell of claim 29, wherein the polypeptide molecule comprises an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 7.   30. The transformed plant cell of claim 29, wherein the polypeptide molecule comprises SEQ ID NO: 7.   38. A transformed plant cell according to any one of claims 29 to 37, which is an oilseed plant cell, or a cereal plant cell, or a legume plant cell.   An isolated protein for controlling disease symptoms caused by one or more phytopathogenic fungi, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID An isolated protein comprising a polypeptide molecule comprising an amino acid sequence sharing at least 80% sequence identity with an amino acid sequence selected from NO: 7.   40. The isolated protein of claim 39, wherein the polypeptide sequence comprises an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 4.   40. The isolated protein of claim 39, comprising SEQ ID NO: 4.   40. The isolated protein of claim 39, comprising an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 5.   40. The isolated protein of claim 39, comprising SEQ ID NO: 5.   40. The isolated protein of claim 39, comprising an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 6.   40. The isolated protein of claim 39, comprising SEQ ID NO: 6.   40. The isolated protein of claim 39, comprising an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 7.   40. The isolated protein of claim 39, comprising SEQ ID NO: 7.   48. An isolated nucleic acid molecule encoding the protein of any one of claims 39-47.   49. A microbial cell transformed with the nucleic acid molecule of claim 48.   50. The microbial cell of claim 49, wherein the nucleic acid molecule is operably linked to a promoter.   51. The microbial cell according to claim 49 or 50, which is a fungal cell, a yeast cell, or a bacterial cell.   52. The microbial cell according to claim 51, wherein the fungal cell is a Sphaerodes mycoparasitic cell or a Penicillium species cell.   52. The microbial cell of claim 51, wherein the yeast cell is a cell of Digosaccharomyces spp., A cell of Saccharomyces spp., A cell of Pichia spp.   52. The microbial cell according to claim 51, wherein the bacterial cell is an E. coli cell, a Pseudomonas species cell, or a Bacillus species cell.   49. A plant cell transformed with the nucleic acid molecule of claim 48.   56. The plant cell of claim 55, wherein the nucleic acid molecule is operably linked to a promoter.   57. The plant cell of claim 55 or 56, which is an oilseed plant cell, a cereal plant cell, or a legume plant cell.

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