KR20140012681A - Proteins for biocontrol of plant pathogenic fungi - Google Patents

Abstract

A composition for controlling disease symptoms caused by phytopathogenic fungi exemplified by Fusarium species, Sclerotinia species, Lizatonia species, Pisium species and the like. The composition is provided with a polypeptide molecule comprising an amino acid sequence having at least 80% sequence homology with SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. The composition can be one of seed treatment, plant treatment or soil treatment. The transformed plant cell expresses a polypeptide molecule comprising an amino acid sequence having at least 80% sequence homology with SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 such that the plant cell is resistant to infection by plant pathogenic fungi to be. The transformed microbial cell expresses a polypeptide molecule comprising an amino acid sequence having at least 80% sequence homology with SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

Description

Protein for controlling biologically pathogenic fungi {PROTEINS FOR BIOCONTROL OF PLANT PATHOGENIC FUNGI}

The present invention relates to novel biological control agents, related compositions effective for controlling fungal plant pathogens and the use thereof. In particular, the present invention relates to novel proteins having a fungal and / or fungicidal effect on plant pathogenic fungi. The present invention also relates to biological control agents comprising proteins and uses, methods and compositions accompanying them.

Fusarium (Fusarium) is a fibrous fungi that are widely distributed in plants and soil. Certain Fusarium species are plant pathogens. For example, Fusarium oxysporum causes Fusarium wilting disease in more than 100 plants. This is caused by plant wood colonization, which can cause blockage and collapse. When this occurs, symptoms such as withering and yellowing of the leaves appear in the plant and ultimately lead to plant death. This was the main reason for the decline and disappearance of Gros Michel banana varieties from global markets. Recently, new strains have started to attack plants of the predominant Cavendish variety, raising concerns that if there is no solution, it will disappear from the world market.

Fusarium root rot is a leading cause of seedling death in forest nursery and also leads to reduced survival after outplanting during the first growing season. This disease is caused by several Fusarium species and is common in many parts of the world. The disease is a particular problem in western Canada and the United States, and in northern central and southern states. In addition, pine blight-caused by the fungus Fusarium circinatum- is a serious pine disease and is a threat to the forest industry, especially in the United States and New Zealand. Radiata or Monterey pines are very susceptible to the disease, with 80% mortality in the nature tree in some parts of California.

Fusarium Head Blight (FHB), also known as `` ear blight '' or `` scab, '' is a wheat, barley, oat and other small grain disease caused by Fusarium. to be. Corn and maize can be caused by a similar condition known as 'ear rot'. The aforementioned conditions can lower the yield and grade of the crop and potentially contaminate the grains with mycotoxins. The FHB is estimated to cost the grain industry nearly $ 5 billion annually. In recent years, FHB has become a significant problem for growing commercially important wheat and barley crops in western Canada. In the past, the Fusarium Oxysis Forum and Fusarium avenaceum have been identified as two species primarily associated with FHB in Canada, where yields in some affected areas have decreased by more than 30%. FHB and ear rot harvest grains are often contaminated with fungal toxins such as deoxinivalenol (DON) trichothecenes, which are associated with feed refusal, general digestive disorders, diarrhea and bleeding. Toxic 3-acetyldeoxynivalenol (3-ADON) Fusarium Graminearum ( Fusarium) graminearum ) military appearance is a fairly recent phenomenon. This replaces the 15-ADON chemical type. In addition, the mycotoxin profile of F. culmorum -similar to the Fusarium Graminea Room in both laboratory and field conditions-has been used in corn, maize and wheat production worldwide. Represents another threat. There is currently no effective control of FHB and related mycotoxins. Also, there are no resistant varieties of corn, wheat, barley, oats, or other small grains. Fungicides can be effective, but they only temporarily suppress the disease.

Fusarium species, as well as common plant pathogens, can infect animals opportunistically and can be a causative agent of mild and / or systemic infection in humans. Fusarium is one of the most drug resistant fungi that makes it difficult to treat a Fusarium infection. Invasive infections can be fatal.

Sclerotinia stem rot is caused by the pathogen Sclerotinia sclerotiorum . Sclerotinia sclerothiorum has a wide host range and is known to infect numerous species of plants (canola, sunflower, soybean, flax, etc.). The onset of the disease can be severe, especially under conditions that are not rotatable or when rotation involves some sensitive plant species. If the infection occurs early in the flowering period, yield reduction can reach 20%. Another widespread fungal plant pathogen is Rhizoctonia , which causes "damping off" or "wire-stem" disease symptoms in crops. Infection can occur at any time during the growing season, but seedlings are most sensitive. The incidence and severity of disease at all stages of growth is affected by weather, soil conditions and inoculum levels. Seedling infections occur well in cold weather. In addition, the incidence of wirestem disease is most severe in spring and autumn when the soil is wet and cold. Root rot generally occurs during warm, wet weather and can develop plants at any stage of development. Seedlings of the disease are not formed, and if so, they quickly shrink, fall, and die. In older tombs, the lower leaves become purple, the lower stem contracts, and dark brown near the soil surface. Other symptoms may include seed rot, root rot and canker in the lower petioles. Pythium ultimum is a widely distributed pathogen for soil transport throughout the world, which causes plant seedlings and root rot. Pisium Ultimum has a wide range of hosts, including many important agronomic crops and turfgrass.

The present invention relates to novel proteins that control fungal pathogens and / or disease symptoms caused by plant pathogenic fungi exemplified by Fusarium spp., Sclerotinia spp., Lizartonia spp., Psium spp. In addition, these novel proteins also have antifungal toxin effects on toxins produced by plant pathogenic fungi exemplified by Fusarium species, Sclerotinia species, Lizatonia species, Pisium species and the like.

Some exemplary embodiments of the invention provide germination control and / or prevention of plant pathogenic fungal spores, and / or control of proliferation of plant pathogenic fungal mycelium, and / or prevention of, or any infection thereof, of plant pathogenic fungi. A novel intracellular protein produced by Sphaerodes mycoparasitica useful for combination.

Some exemplary embodiments of the invention provide germination control and / or prevention of plant pathogenic fungal spores, and / or control of proliferation of plant pathogenic fungal mycelium, and / or prevention of, or any infection thereof, by plant pathogenic fungi. A novel extracellular protein produced by Spherodes mycoparasiticas useful in combination.

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

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

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

One aspect of the invention relates to a novel extracellular protein produced by Spherodes mycoparasitika 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 or they may be plant treatment compositions or they may be soil treatment compositions.

Some exemplary embodiments of the invention relate to microbial cells transformed to produce one or more new intracellular proteins and new extracellular proteins. Transformed microbial cells can be used to control disease symptoms caused by plant pathogenic fungi exemplified by Fusarium species, Sclerotinia species, Lizatonia species, Pisium species and the like. Transformed microbial cells can be used as seed treatment and / or plant treatment and / or soil treatment or any combination thereof. Alternatively, transformed microbial cells can be used to produce new proteins. Proteins can be harvested and used to make the compositions 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 propagules that can be cultured into intact plants, exemplified by seeds, somatic embryos, organogenic tissues, and the like. Intact plants, including transformed plant cells, are resistant or at least well resistant to disease symptoms caused by plant pathogenic fungi exemplified by Fusarium species, Sclerotinia species, Lizatonia species, Pisium species and the like.

Exemplary embodiments of the invention are described together with reference to the following figures, wherein:
1 (a) A and 1 (a) B show the mycelial growth of the Fusarium oxysporum (1 (a) A) and Fusarium Graminearum (1 (a) B) of exemplary proteins of the invention. A micrograph of a disk diffusion assay showing the inhibitory effect, FIG. 1 (b) shows the mycelial growth of Fusarium oxysporum (A) and Fusarium gramrealum (B) with exemplary proteins three and four days after growth. A chart showing% inhibition;
FIG. 2 is a micrograph of 10% native-PAGE (native-PAGE) separated into two bands of intracellular proteins produced by Spherodes mycoparasiticas;
FIG. 3 is a micrograph of 12% SDS-PAGE isolation of Spherodes mycoparasitica intracellular protein eluted from the upper and lower bands shown in the 10% natural-PAGE gel shown in FIG. 2;
FIG. 4 is a micrograph of 10% native-PAGE separated into four bands of extracellular protein produced by Spherodes mycoparasiticas;
FIG. 5 is a micrograph of 12% SDS-PAGE isolation of Spherodes mycoparasitica extracellular protein eluted from four bands of the 10% natural-PAGE gel shown in FIG. 4;
FIG. 6 is a chromatogram showing FLPC separation of four bands of extracellular protein produced by Spherodes mycoparasiticas;
Figure 7 (a) is a micrograph of mycelial growth of the Fusarium oxysporum in the control treatment; 7 (b) is a micrograph of the mycelial growth of the Fusarium oxysporum in control treatment plus buffer; 7 (c) is a micrograph of the inhibitory effect of 50 kDa intracellular protein on mycelial growth of the Fusarium oxysporum; And 7 (d) is a micrograph of the inhibitory effect of mycelial growth of the Fusarium oxysporum of 79 kDa intracellular protein;
FIG. 8 (a) is a micrograph of the mycelial growth of Fusarium Graminearum in control treatment; 8 (b) is a micrograph of the mycelial growth of Fusarium Graminearum in control treatment plus buffer; 8 (c) is a micrograph of the inhibitory effect of 50 kDa intracellular protein on mycelial growth of Fusarium gramrearum; And 8 (d) is a micrograph of the inhibitory effect on the mycelial growth of Fusarium gramrearum of 79 kDa intracellular protein;
FIG. 9 (a) is a micrograph showing the inhibition of mycelial overgrowth of Fusarium oxysporum by 79 kDa extracellular protein (EC1) compared to control treatment (c), 9 (b) showing 50 kDa extracellular protein (EC2) and 36 kDa extracellular protein (EC3) are micrographs showing the inhibition of mycelial overgrowth of Fusarium oxysporum;
FIG. 10 (a) is a micrograph showing the inhibition of mycelial overgrowth of Fusarium Graminearum by 79 kDa extracellular protein (EC1) compared to control treatment (c), 10 (b) showing 50 kDa extracellular protein (EC2) and 36 kDa extracellular protein (EC3) are micrographs showing the inhibition of mycelial overgrowth of Fusarium Graminearum;
FIG. 11 (a) is a micrograph showing the inhibition of mycelial overgrowth of Fusarium Graminearum by 13 kDa extracellular protein (EC4) compared to control treatment (c), and 11 (b) is 13 kDa extracellular protein Micrograph showing inhibition of mycelial overgrowth of Fusarium oxis forum by (EC4) compared to control treatment (c);
12 (a) and 12 (b) show Fusarium oxysporum spores 12 (a) and Fusarium gramrearum spores 12 (b) of the spherodes mycoparasitica extracellular protein fractions EC1, EC2, EC3 and EC4. A chart showing the effect on germination;
FIG. 13 is a chart showing the effects on colony growth and developmental inhibition of Fusarium oxysForum and Fusarium gramrearum of Spherodes mycoparasitika extracellular protein fractions EC1, EC2, EC3 and EC4;
14A (c), 14A (a), 14A (b) show germination of control Fusarium oxyspores spores (c), inhibitory effect of 50 kDa protein on germination of Fusarium oxyspores spores, and It is a micrograph showing the inhibitory effect of 13 kDa protein, Figure 14B (c), 14B (a), 14B (b) is the germination of the control Fusarium Graminea spores (c), the germination of Fusarium Graminea spores Micrograph showing the inhibitory effect of 50 kDa protein on (a), and the inhibitory effect of 13 kDa protein;
15 (a) and 15 (b) are microscopes showing the effect (15 (b)) on the mycelial growth of Fusarium avenassium of crystal forming extracellular proteins EC1 and EC3 compared to control (15 (a)). Photo;
16 (a) and 16 (c) show the effect on colonization of germinated wheat seeds of the crystal forming extracellular proteins EC1 and EC3 and mycelial growth of Fusarium gramrealum compared to the control (16 (a)). (b)) is a micrograph showing 16 (c) is a confocal laser scanning micrograph showing the number of Fusarium Graminearum mycelium components (see arrow) degraded from the surface of wheat seedlings treated with extracellular proteins. "K +" indicates that the medium contained EC1 and EC3 extracellular proteins. "K-" indicates that the EC1 and EC3 extracellular proteins were not in the medium. "K-crystal" identifies the location of extracellular protein molecule (s);
17 (a) and 17 (b) are microscopes showing the effect (17 (b)) on the mycelial growth of the Fusarium Graminearum of the crystal forming extracellular proteins EC1 and EC3 compared to the control (17 (a)). Photo;
FIG. 18 (a) is a scanning electron micrograph showing the presence of degraded fungal hyphae and protein crystals from crystalline protein treated Fusarium Graminearum from FIG. 17 (b), FIG. 18 (b) shows FIG. Confocal laser scanning microscopy micrograph showing the presence of protein crystals in the mycelium of crystalline protein treated Fusarium gramrealum from b), 18 (c) is crystalline protein treated Fusarium gras from FIG. 17 (b) Chemistry microscopy micrograph showing protein crystals from Minnearum and the presence of numerous disrupted mycelial components and apoptosis lysed cells (arrows). "K determination: confirms the location of extracellular protein molecule (s)";
19 is a chart showing Malditope mass spectrometry (MALDI-TOF-MS) separation of amino acids comprising 79 kDa EC1 protein;
20 is a chart showing Malditope mass spectrometry separation of amino acids comprising 50 kDa EC2 protein;
FIG. 21 is a chart showing Malditope mass spectrometry separation of amino acids comprising 36 kDa EC3 protein; FIG.
FIG. 22 is a chart showing Malditope mass spectrometry separation of amino acids comprising 13 kDa EC3 protein; FIG.
23 (a) -23 (f) are the following micrographs: (23 (a)) control untreated culture of sclerotenia sclerotirum on PDA, (23 (b)) extracellular protein Sclerotinia sclerothiolum cultures cultured on PDA modified with EC1 plus EC3, (23 (c)) control untreated culture of Rhizoctonia solani , (23 (d)) cells Lithuanian Solani cultures cultured on PDA modified with exogenous protein EC1 plus EC3, (23 (e)) control untreated culture of Pisium Ultimum, and (23 (f)) extracellular protein EC1 plus EC3 Fish cultures cultured on modified PDA; And
FIG. 24 is a chart showing the inhibitory effect of the crystal forming extracellular proteins EC1 and EC3 on the mycelial growth of sclerotinia sclerothiorum, lizartonia solani and fishium Ultimum.

Spherodes Mycoparasitika (Saculobacterium, Melanosporales) has been isolated from isolates of Fusarium Avenacium, Fusarium Graminearum and Fusarium Oxysis Forum from wheat or asparagus fields. It is a fungus. This species is characterized by a unique combination of follicle size, shape (like fusiform and triangular) and wall pattern (reticulated and smooth). In addition, conidia are produced from simple pyaridides on the surface of the fruiting body wall of the fruiting fruit, on the fruiting body surrounding the hyphae, and on irregularly branched conidia stalks resulting from the hyphae. Spherodes mycoparasiticas have a piad asexual reproduction and produce simple piads on the surface of the fruiting fruit pericardium wall or irregularly distributed on the fruiting body surrounding the mycelia. Spherodes mycoparasitica forms a hook-like structure that parasitic to the living mycelium of Fusarium.

Sample cultures of Spherodes mycoparasitica were deposited with the Canadian International Depository (R3E 3R2 Winnipeg Arlington Street 1015, Canada) under Accession No. IDAC301008-01. Fusarium Avenacium (Full Generation: Giberella Avenasia) A sample culture of S. ferodes mycoparasitias parasiticly parasitic to avenacea )) was deposited with the Canadian International Depositary under accession number IDAC301008-02 and is referred to herein as "SM-Bst". Fusarium Graminea Room (Full Generation: Giberella A sample culture of S. ferodes mycoparasitica parasitually parasitic to zeae )) was deposited with the Canadian International Depositary under accession number IDAC301008-03 and is referred to herein as "SM-Gst".

The 1266 bp DNA sequence from the large subunit ribosomal RNA gene (LSU) of Spherodes mycoparasitika is given in SEQ ID NO: 1.

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

The novel proteins produced by the spherodes mycoparasitica disclosed herein are useful as antifungal agents. Such novel proteins can be used to treat, alleviate or otherwise control infection of Fusarium fungi. These novel proteins produced by Spherodes Mycoparasitika particularly treat, alleviate or otherwise control infections of Fusarium avenasis, Fusarium gramrealum, and / or Fusarium oxysis forums and It is useful for improving growth.

The novel Spherodes mycoparasitica protein disclosed herein can be used as a preventive agent to lower the likelihood of developing fungal infections, in particular Fusarium infections.

The novel Spherodes mycoparasitica protein disclosed herein can be used to treat plants that have developed Fusarium wilting or Fusarium upper blight. These proteins can be used as a preventive measure to prevent the spread of these conditions or otherwise reduce the risk of developing them.

The novel spherodes mycoparasitica proteins disclosed herein can be used to treat, alleviate or otherwise control infections of Fusarium fungi. The protein can be applied in any suitable way to the organism in need of treatment. For example, proteins can be used directly or alternatively they are applied to soil systems or to growth systems that do not use soil (eg, granulated compositions, peat-based compositions), to seeds (eg, powder compositions, Granule compositions, peat-based compositions, liquid compositions) or inoculum for application to growing plants (eg, powder compositions, liquid compositions).

The formulations disclosed herein comprise (i) at least one active ingredient comprising at least one novel protein, (ii) at least one carrier—an inert material used to support and deliver to the target ingredient which is often densely packed, and According to (iii) at least one adjuvant—promoting and maintaining the function of the active ingredient by ensuring protection from ultraviolet rays, ensuring targeted rain resistance, retaining moisture or protecting it from drying and / or described in Hynes and Boyetchko (2006, Research initiatives in the art and science of biopesticide formulations . Soil Biol. & Biochem. 38: 845-49) using the standard agricultural equipment such as that disclosed by the include compounds to promote the diffusion and dispersion of the organic insecticide.

Internal transcription spacer ribosomal DNA from Spherodes mycoparasitika was sequenced. The 560 bp DNA sequence from the ITS region is given in SEQ ID NO: 3.

Total intracellular protein extracts and extracellular protein extracts from Spherodes mycoparasitica demonstrate significant inhibition of fungal spore germination of Fusarium species, Sclerotinia species, Lizatonia species, and Pisium species. Having a molecular weight of about 79 kDa and comprising SEQ ID NO: 4, another having a molecular weight of about 50 kDa and comprising SEQ ID NO: 5, another having a molecular weight of about 36 kDa and comprising SEQ ID NO: 6, and the fourth is about 13 Four novel proteins with fungicidal effect, having a molecular weight of kDa and comprising SEQ ID NO: 7, were isolated from total protein extracts. The present invention provides antifungal agents comprising one or more of these proteins.

Conventional molecular biological techniques can be used to isolate, characterize and produce each of the four new proteins. For example, to identify the gene (s) for the present protein, Spherodes mycoparasiticas can be administered upregulated mRNA and Fusarium oxisforum filtrate isolated by standard Northern blots. CDNA can be produced from mRNA by reverse transcriptase PCR (RT-PCR). cDNA can be amplified, purified and inserted into a suitable vector. This vector can be inserted into a suitable host cell.

Cloning and expression can focus on the isolation of genes encoding these novel antimicrobial proteins by designing primers for the identified proteins. Isolated genes can be cloned in appropriate expression vectors (yeast or bacteria) with suitable / effective promoters to generate industrial strains for large scale production of related proteins. Such vectors can be purchased from Promega, Invitrogen, Clontech or other companies. The cloned gene can be tested for its protein expression and the expressed protein will be purified (using His-tag or other column). Purified proteins are described in Drummond et al. (2000, The development of microbiological methods for phytochemical screening . Rec. Res. Dev. Phytochem. 4: 143 - 152) using a microtiter plate assay and / or methods which are illustrated in the disc plate assay disclosed by may be tested for its antifungal activity against phytopathogenic fungi. Thereafter, rDNA techniques can be used that include the addition or deletion of gene mutations or enhancers, introns or protein specific promoters (GA inducible) to increase protein production.

Selected genes encoding one or more or all new proteins, namely 13 kDa protein, 36 kDa protein, 50 kDa protein, and 79 kDa protein, are described in 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 the method disclosed in [Tobias et. al. (2007, Co - bombardment , integration and expression of rice chitinase and thaumatin - like protein genes in barley ( Hordeum vulgare cv . Conlon ) . Plant Cell Reports 26: 631 - using known techniques to those skilled in the art, such as that illustrated by 639) for the pathogenic fungus transformed with the plant genome selected to produce a transformed cell with the antifungal activity Can be switched. Crops suitable for transformation with these novel proteins are exemplified by oilseed crops, grain crops, legume crops, coniferous species and deciduous species, among others. Exemplary oilseed crops include canola, mustard, rapeseed, soybean, flax, sunflower, safflower, castor and camellias, among others. Exemplary grain crops include wheat, barley, oats, rye wheat, rye, rice, corn, sorghum and quinoa, among others. Exemplary legume crops include legumes, peas, chickpeas, lentils, and lupins, among others. Exemplary salivary species include pine, spruce, fir, and cypress among others. Exemplary deciduous species include poplars, aspens, maples, oaks, nut producing trees, fruit trees, acacia and eucalyptus, among others.

One or more or all of the 79 kDa proteins (including SEQ ID NO: 4), 50 kDa protein (including SEQ ID NO: 5), 36 kDa protein (including SEQ ID NO: 6), and 13 kDa protein (SEQ ID NO: 6) produced by Spherodes Mycoparasitica It provides an antifungal composition comprising 7). Alternatively, one or more nucleic acids encoding one or more or all 13 kDa proteins, 36 kDa proteins, 50 kDa proteins, and 79 kDa proteins encoding one or more or all 13 kDa proteins, 36 kDa proteins, 50 kDa proteins, and 79 kDa proteins Molecules can be produced by the transformed microorganisms provided. The present invention also provides a method for controlling fungal diseases of plants, a method for detoxifying mycotoxins, which method is one or more or all 13 kDa proteins, 36 kDa proteins, 50 kDa proteins, hereafter collectively referred to herein as 'antifungal agents'. , And applying the 79 kDa protein to the site of the plant.

The compositions of the present invention may be used, for example, in seed and / or growth medium (eg, soil or water) and / or roots and / or leaves of plants, or any combination thereof, used to grow plants. Applicable Exemplary crops that can be treated with the present compositions include crops such as seed crops, grain crops, legume crops, horticultural crops, forestry crops, and turfgrass.

The present invention provides a composition comprising an antifungal agent and one or more agriculturally acceptable carriers and / or diluents which will ensure the stability and / or performance of the final product. Suitable carriers include clay dust, peat grains, peat powder, carbon powder, cellulose grains and sawdust, vermiculite, perlite, pomice, among others exemplified by kaolinite, bentonite and montmorinollite Methylcellulose, dust exemplified by talc. Suitable diluents are exemplified by water and by inviscid gels exemplified by alginate, carrageenan, agar, carboxymethylcellulose and the like. The carrier or diluent must be compatible with the active ingredient, agriculturally acceptable, have good absorption capacity and have an appropriate bulk density to facilitate particle dispersion and adhesion.

The compositions herein can be applied as aqueous sprays, granules and powder formulations according to customs in the art. Aqueous sprays are usually prepared by mixing an emulsifiable concentrate formulation or hydrating powder of a compound of the present invention with a relatively large amount of water to form a dispersion.

Hydrating powder may comprise a fine mixture of antifungal agents, solid carriers and surface active agents. The solid carrier is usually selected from attapulgite clays, kaolin clays, montmorillonite clays, diatomaceous earth, finely divided silica, purified silicates, or combinations thereof. Surfactants that may be useful herein have wetting, permeating and / or dispersing capabilities. They are typically present in amounts of about 0.5% to about 10% by weight. Surfactants herein can be selected from, for example, alkylbenzenesulfonates, alkyl sulfates, naphthalenesulfonates and condensed naphthalenesulfonates, sulfonated lignin and nonionic surfactants.

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

With respect to pre-germination protection, granule formulations or powders are sometimes more convenient than sprays when the compositions of the present invention are applied to plant debris or trash or to soil to control inoculant dispersion and contaminants.

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

The composition may comprise other active substances useful for antifungal agents. For example, the composition of the present application may be Trichoderma , sulfur, nim oil, rosemary oil, jojoba oil, Bacillus subtilis ( Bacillus) subtilis ), allylamine (eg terbinafine), antimetabolites (eg flucitocin), azoles (eg ketoconazole, itraconazole), echinocandine (eg caspofungin) And other antifungal agents, such as polyenes (eg, amphotericin B), systemic agents (eg, griseofulvin), or combinations thereof.

The compositions herein may contain about 0.1% to about 95% by weight antifungal agent and about 0.1% to about 95% by weight carrier and / or surfactant. Direct application to plant seeds prior to planting is in some instances made by mixing the seeds with the powdered solid compounds or powder formulations of the invention, which is very thin and substantially uniform, representing only 1 or 2 weight percent or less based on the weight of the seed. A coating can be obtained. However, in some instances, non-phytotoxic solvents such as methanol are conveniently used as carriers to promote uniform distribution of the compounds of the present invention on the seed surface.

The compositions herein may be useful for the treatment of fungal infections in plants. As a result, one or more or all of the disclosed herein 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 kDa protein (including SEQ ID NO: 7), and May include an acceptable carrier therefor.

The present invention is directed to an exemplary method for producing an antifungal composition comprising one or more or all of the Spherodes mycoparasitika 13 kDa protein, 36 kDa protein, 50 kDa protein and 79 kDa protein. The method includes:

(a) induction of spore formation of the spherodes mycoparasitica spores;

(b) spherodes mycoparasitica culture;

(c) protein recovery from Spherodes mycoparasitica culture;

(d) isolating and recovering one or more or all 13 kDa protein, 36 kDa protein, 50 kDa protein and 79 kDa protein from the recovered Spherodes mycoparasitica protein; And

(e) Preparation of a composition comprising at least one or all 13 kDa protein, 36 kDa protein, 50 kDa protein and 79 kDa protein recovered from Spherodes mycoparasitica 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 kDa protein (including SEQ ID NO: 7) were separated and recovered together from the recovered spherodes mycoparasitika protein. Can be. Alternatively, 13 kDa protein and / or 36 kDa protein and / or 50 kDa protein and / or 79 kDa protein or any combination thereof may be recovered from the recovered spherodes mycoparasitika protein, respectively.

The present invention includes one or more or all of the spherodes mycoparasiticas including 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 kDa protein (including SEQ ID NO: 7). It relates to another exemplary method for producing an antifungal composition. The method includes:

(f) microbial culture transformation selected with one or more genes encoding one or more or all 13 kDa protein, 36 kDa protein, 50 kDa protein, and 79 kDa protein produced by Spherodes mycoparasiticas;

(g) culture of transformed microbial culture;

(h) protein recovery from the transformed culture;

(i) isolation and recovery of 13 kDa protein and / or 36 kDa protein and / or 50 kDa protein and / or 79 kDa protein from the recovered protein;

(j) Preparation of a composition comprising at least one or all 13 kDa protein, 36 kDa protein, 50 kDa protein and 79 kDa protein.

13 kDa protein and / or 36 kDa protein and / or 50 kDa protein and / or 79 kDa protein can 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 from the recovered protein, respectively. Suitable microbial cultures capable of being transformed with one or more genes encoding one or more or all 13 kDa protein, 36 kDa protein, 50 kDa protein and 79 kDa protein produced by Spherodes Mycoparasitika are Escherichia coli ( Escherichia coli ), Pseudomonas spp.), Bacillus spp . Bacterial cultures, such as Zygosaccharomyces spp), Saccharomyces spp), Pichia species spp . ), Kluveromyces yeast cultures such as spp.), and Penicillium spp. spp.) and the like.

Example

Example  One: Spherodes From mycopara city car Intracellular  And Extracellular  Protein Production and Extraction

Spherodes mycoparasitica strains IDAC 301008-02 and / or IDAC 301008-03 have been used in all studies disclosed herein and are referred to collectively as "SM-Bst" and "SMGst", respectively. Raw cultures of SM-Bst and SM-Gst were prepared and maintained at 21 ° C. in potato dextrose broth (PDB) culture medium. About 3 ml were transferred from PDB culture to a 250-ml Erlenmeyer flask containing 50 ml PDB growth medium to prepare intracellular and extracellular proteins from each type of original culture. The flask was incubated for 7 days on a rotary shaker (150 rpm) at room temperature. The young mycelium was filtered through watmaen (Whatman) ^® No.1 filter paper (watmaen is a registered trademark of watmaen International Limited (Whatman International Ltd.) of England, Kent). In contrast with the mycelia collected on watmaen ^® No.1 filter paper for extraction of the protein in the cell, the filtered culture medium was used for the recovery of the protein outside of the cell therefrom.

Example  2: Spherodes From mycopara city car Intracellular  Protein and Extracellular  Protein Fractionation , Separation and purification

Intracellular Protein Extraction : Intracellular proteins are described in Chen et al. (2004, Heat shock proteins of thermophilic and thermotolerant fungi from Taiwan . Bot. Bull. Acad. Sin. 45: 247-257). A total of 200 mg fungal mycelium from each sample was powdered in liquid nitrogen in sterile magnetophoresis using a pestle and mixed with 1.5 ml cooled extraction buffer. Mycelium (~ 200 mg) was added to 50 mM Tris-HCl (pH 8.5); 2% sodium dodecyl sulfate (SDS); 2% β-mercaptoethanol; Grinding by sonication in a grinding tube containing 1 mL of buffer consisting of 1 mM phenylmethylsulfonyl fluoride (PMSF in 95% alcohol). The homogenate was centrifuged at 12,000 rpm at 28 ° C. for 10 minutes and the supernatant was collected. Four volumes of cold acetone (-20 ° C.) were added and samples were frozen overnight at −20 ° C. The precipitate (protein) was stored in acetone and centrifuged for 5 minutes at 12000 rpm at 28 ° C. and finally sample buffer (62.5 mM Tris-HCl (pH 6.8); 3% SDS; 10% glycerol; 5% β-mercapto Ethanol) in 50-100 μl. Protein samples were transferred to new 1.5 ml Eppendorf tubes and stored at −80 ° C. until used for further study.

Extracellular Protein extraction : The filtered culture medium was used as a source of extracellular protein and Amicon as a 3000-Dalton cutoff membrane.^® Concentrated by centrifugation at 4000 rpm at 4 ° C. in an ultrafiltration centrifuge tube (Amicon is a registered trademark of Billyrica Millipore Corp., Mass., USA). Total extracellular proteins were tested for antifungal activity against fungal pathogens, Fusarium oxysporum and Fusarium gramrearum using a disk diffusion assay. Antifungal activity of total 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) were modified under sterile conditions by a radial disc plate diffusion assay with some modifications. Total extracellular protein tests for antifungal activity against the Fusarium oxime forum and the Fusarium gramrearum were performed in a Petri dish containing potato dextrose agar. Mycelial plugs from actively growing fungal plates were placed in the center of the Petri dish and sterile filter paper discs (5 mm diameter Whatman)^® Filter paper no. 1) was placed on the agar surface at a distance of 0.5 cm from the edge of the mycelia colonies. Aliquots (60 μl) containing 2.5 μg extracellular protein were added to the discs (test spots are indicated as “T” in FIGS. 1 (a) A and 1 (a) B). Sterile distilled water and buffer were used as controls (control spots are indicated as "C" in Figures 1 (a) A and 1 (a) B). Plates were then incubated for 4 days at room temperature and examined for mycelial overgrowth inhibition. The area of mycelial colonies was measured and fungal growth inhibition was determined by calculating the percentage of reduction of the mycelial colony overgrowth area compared to the control. Total extracellular protein recovered from the SMCD culture inhibited mycelial overgrowth in both Fusarium oxysporum and Fusarium gramrearum (FIGS. 1 (a) A and 1 (a) B). About 22% inhibition in mycelial kidney of Fusarium oxime forum and 30% inhibition in mycelial kidney of Fusarium gramrealum was observed 3 days after germination (FIG. 1 (b)). After 1 day, the mycelial suppression of Fusarium oxime forum increased by about 30%, while the mycelial suppression of Fusarium Graminearum increased by about 35% (FIG. 1B).

Natural polyacrylamide gel electrophoresis (natural- PAGE ): proteins (intracellular and extracellular) were described in Laemmli (1970, Cleavage). of structural protein during the assembly of the head of bacteriophage T4 . Nature 227 : 680-685) was isolated by 10% natural polyacrylamide gel electrophoresis (natural-PAGE) in a discrete buffer system. Accumulation gel (3.75% acrylamide / bis-acrylamide 30: 0.8 v / v) contained 125 mM Tris-HCl, pH 6.8 as buffer and separation gel (7.5% acrylamide / bis-acrylamide 30: 0.8 v / v). v) included 375 mM Tris-HCl, pH 8.8 as buffer. The gel was operated with Tris-glycine electrode buffer containing 25 mM Tris and 192 mM Glycine, pH 8.3 at 120 V overnight at 4 ° C. After electrophoresis, the gels were stained according to the silver staining method (Bio-Rad ^® kit method).

Sodium dodecyl Sulfate - Polyacrylamide Gel Electrophoresis ( SDS - PAGE ): SDS-PAGE of protein eluted from natural-PAGE and total protein was performed on a 12% polyacrylamide gel according to the method given in Laemmli (1970) It was. Proteins were analyzed using 5% accumulation gel (pH 6.8) and 12% isolated acrylamide gel (pH 8.8) and appropriate markers in Tris-glycine buffer (pH 8.3). Prior to SDS-electrophoresis, the proteins were mixed with an equal volume of sample buffer containing 60% β-mercaptoethanol (60 mM Tris-HCl buffer, 4% SDS, pH 6.8). Bio-Rad Laboratories ^® Tori Canada Limited ^{(Bio-Rad ® Laboratories Canada Ltd.} ) ( Montreal, PQ, Canada; Bio-Rad US heokyu CA Leasing Bio-Rad Laboratories is a registered trademark of Torrey Inc.) standard protein markers obtained from A mixture of was 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). Protein molecular mass was estimated by comparing the mobility of standard molecular mass markers.

Fast Protein Liquid Chromatography of Extracellular Proteins ( FPLC ): FPLC AKTA ^® Purifier System (AKTA is a registered trademark of GE Healthcare Bio-Sciences AB Ltd., Uppsala, Sweden, as directed by the manufacturer. ) were super dex (Superdex) ^® 75 GL 10/30 column (GE Healthcare, Uppsala, Sweden Super Dex using bio-protein fractions were angry over a registered trademark of AB Science Ltd.). The column was previously equilibrated with 50 mM sodium phosphate buffer, pH 7.0 containing sterile water and 0.15 M NaCl, and then the protein was injected (about 500 μl) and the protein was eluted with the same buffer at a flow rate of 1.0 ml / min. Protein was purified via gel filtration using 50 mM sodium phosphate buffer, pH 7.0 containing 0.15 M NaCl. Proteins were analyzed as separate peaks on gel filtration on Superdex 75 phase. 0.8-ml fractions were collected and their purity was checked 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 given in Laemmli (1970). All peaks representing the FPLC fractions were collected and recovered by precipitation in cold acetone 1: 4 volume and kept at -20 ° C overnight. After centrifugation at 12,000 g for 10 minutes, the precipitated protein (pellets) was dissolved in a minimal amount (30 μl) of assay buffer. Proteins were analyzed by SDS-PAGE with 5% accumulation gel (pH 6.8) and 12% isolated acrylamide gel (pH 8.8) and appropriate markers in Tris-glycine buffer (pH 8.3). 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 at 95 ° C. for 5 minutes and cooled to room temperature before loading into the gel. Proteins were visualized by silver staining (Bio-Rad ^® Silver Staining Kit, Bio-Rad Laboratories Canada Inc.). Protein molecular mass was estimated by comparing the mobility of standard molecular mass markers. The molecular mass of the fractionated protein obtained from FPLC was determined by SDS-PAGE (12% and 16%). 5% SDS-PAGE was performed to separate extracellular proteins comprising large weight protein bands separated by the natural-PAGE method.

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

Natural-PAGE electrophoresis using 10% natural polyacrylamide gels of extracellular proteins extracted from SM-Bst and SMGst separated four bands of proteins designated EC1, EC2, EC3, and EC4 (FIG. 4). Each protein band of native-PAGE was immersed in 500-750 μl of sample buffer (62.5 mM Tris-HCl, pH 6.8; 1% PMSF, 1% EDTA) and left overnight at 4 ° C. The protein was recovered by centrifugation at 12,000 g for 10 minutes. SDS-PAGE separation of extracellular protein was performed using a 12% gel to determine the molecular weight of the isolated protein eluted from native-PAGE. Eluted proteins from the four bands showed the presence of isolated and purified proteins having the following molecular weights: (i) 79 kDa (EC1), (ii) 50 kDa (EC2), (iii) 36 kDa (EC3) , And 13 kDa (EC4) (FIG. 5).

^® AKTA purifier FPLC Fast protein shows the elution profile of the four bands of the super dex 75 GL 10/30 column a extracellular protein by determining by means of liquid chromatography using the system in FIG.

Example  3: In the Fusarium  by Spherodes My Co-Para City Car  Purified recovered from culture Intracellular  Protein and Purification Extracellular  Inhibition of mycelial overgrowth by protein

Some modifications of the antifungal activity of proteins are described in Roberts et al. (1986, Isolation and partial characterization of two antifungal proteins from barley . Biochim. Biophys. Acta, 880: 161-170) was tested under sterile conditions by radial disc plate diffusion assay. Analysis of isolated proteins was performed for antifungal activity against Fusarium oxysporum and Fusarium graminearum in Petri dishes containing potato dextrose agar. Mycelial plugs from actively growing fungal plates were placed in the center of the Petri dish and a sterile filter paper disc (Watman ^® filter paper no. 1 of 5 mm diameter) was placed on the agar surface 0.5 cm from the edge of the mycelia colonies. Isolated protein (60 μl) was added to the disc. Sterile distilled water and buffer without only antifungal protein were used as controls. Plates were then incubated for 3-4 days at room temperature and then examined for inhibition zones, if any, around the disc. In this way, if the protein being tested was an antifungal agent, an inhibitory zone of crescent form fungal growth may have occurred around the disc. The area of mycelial colonies was measured and fungal growth inhibition was determined by calculating the% reduction of the mycelial colony area with a control (water and buffer treated plates).

Both the 50 kDa intracellular protein and the 79 kDa intracellular protein both mycelial elongation, ie, Fusarium oxysporum (Figs. 7 (a) -7 (d)) and Fusarium Graminearum (Fig. 8 (a) -8 (d) Mycelial growth of)) was inhibited.

79 kDa protein (EC1), 50 kDa protein (EC2), and 36 kDa protein (EC3) were found in the Fusarium oxisforum (Figs. 9 (a) -9 (b)) and Fusarium Graminearum (Fig. 10 (a) Mycelial overgrowth of) -10 (b)) was inhibited. 13 kDa protein (EC4) inhibited mycelial overgrowth of Fusarium Graminearum (FIG. 11 (a)) and Fusarium Oxy Forum (FIG. 11 (b)).

Example  4: In the Fusarium  Caused by spore germination Spherodes My Co-Para City Car  Purified recovered from culture Extracellular  Inhibition by Protein

Inhibitory effect of four extracellular spherodes mycoparasitika proteins EC1, EC2, EC3 and EC4 on Fusarium spore germination [Ghosh (2006, Antifungal) Properties of Haem Peroxidase from Acorus calamus . Ann. Bot. 98: 1145-1153) and Yadav et al . (2007, An antifungal protein from Escherichia coli . J. Med. Microbiol. 56: 637-644). Possible toxicity of the fractionated proteins was tested in percent growth inhibition assays using the fungal Fusarium oxysForum and Fusarium gramrearum. In vitro antifungal activity of fractionated proteins was determined in 96-well microtiter plates. 10 μl of potato dextrose broth (PDB; Difco Laboratories of Detroit) was mixed with 3 μl of spore suspension of Fusarium oxysporum and Fusarium gramrearum in microplate wells. 7 μl aliquots of different peaks containing were added to the suspension in the microtiter plate (12-8 wells) Water and buffer were used as negative controls The microtiter plates were then incubated at room temperature in the dark. Inverted and fluorescence microscopy were used to observe spore germination inhibition in both untreated and treated wells after 24 h.The number of germinated and ungerminated spores in the microscope and the percentage of areas covered by mycelium inhibited growth. It was used to determine the percentage of.

All four extracellular proteins significantly inhibited the spore germination of the Fusarium oxysporum and the Fusarium Graminearum (FIGS. 12 (a) and 12 (b)). In addition, all four extracellular proteins formed large zones (when these organisms were plated separately in solid medium) in the large zone of mycelial growth inhibition of the Fusarium oxysporum and the Fusarium Graminearum (FIG. 13). 14A (c) is a micrograph showing the germination of control Fusarium oxysporum spores and FIG. 14A (a) shows the inhibitory effect on the germination of Fusarium oxysporum spores of 50 kDa protein and FIG. 14A (b) ) Shows the inhibitory effect of the 13 kDa protein on the germination of the Fusarium oxysporum spores. FIG. 14B (c) is a micrograph showing the germination of control Fusarium Graminear spores and FIG. 14B (a) shows the inhibitory effect on the germination of Fusarium Graminerum spores of 50 kDa protein, FIG. 14 (b) ) Shows the inhibitory effect of the 13 kDa protein on the germination of Fusarium Graminea spores.

Example  5: Spherodes My Co-Para City Car  Crystal Formation Recovered from Culture EC1  And EC3  Inhibitory Effects of Proteins

The fungal toxic effects of the crystal-forming extracellular proteins EC1 (79 kDa) and EC3 (36 kDa) were assessed with the Fusarium avenacium and Fusarium Graminearum cultures actively growing on PDA. Fusarium avenasium 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 crystalline proteins and the plates were then incubated at 23 ° C. for 5 days in the dark. Significant mycelial damage was observed in half of the plates treated with EC1 / EC3 protein mixture (FIG. 15 (B)) compared to the untreated control side of the plate (FIG. 15 (A)).

Sprinkle wheat seeds across the PDA plate surface. After seed germination, Fusarium Graminearum was inoculated on the plate. Half of the plate was treated with the EC1 / EC3 protein mixture and then the plate was incubated at 23 ° C. for 5 days in the dark. The EC1 / EC3 protein mixture had no fungal growth on half of the given plate (FIG. 16 (b)), while fungal mycelia grew on the untreated side of the plate (FIG. 16 (a)). Confocal laser scanning microscopy of the agar surface given the EC1 / EC3 protein mixture showed a number of degraded mycelial components (see arrow in FIG. 16 (c)).

Fusarium 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 crystalline proteins and the plates were then incubated at 21 ° C. for 5 days in the dark. Significant mycelial damage was observed in half of the plates treated with EC1 / EC3 protein mixture (FIG. 17 (B)) compared to the untreated control side of the plate (FIG. 17 (A)). Scanning electron microscopy of the agar surface given the EC1 / EC3 protein mixture showed a number of degraded mycelial components (see arrow in FIG. 18 (a)). Same surface examination with chemical force microscopy showed destroyed mycelial components and apoptosis lysed cells (see arrow in FIG. 18 (c)).

Example  6: Spherodes My Co-Para City Car  Purified recovered from culture Extracellular  Sequence Analysis and Presumptive Identification of Proteins

Extracellular protein bands EC1 (79 kDa), EC2 (50 kDa), EC3 (36 kDa), and EC4 (13 kDa) were cut from 5%, 12% and 16% SDS-PAGE gels. The cleaved protein bands were subjected to McGill's for sequencing using the Malditops mass spectrometry and for speculative characterization by peptide characterization and comparison of the peptide sequences with UniProtKB / Swiss-Prot and the NCBI database. McGill University and Genome Quebec Innovation Center (Canada PQ Montreal Drive Penfield Avenue 740 Rm 704).

Malditops mass spectrometry allowed the separation of amino acids including EC1 protein (FIG. 19). The sequence of amino acids comprising the EC1 protein was found to be:

YLPGGGGGRDEPPPR (SEQ ID NO: 4)

The mass of EC1 have been found to 78,688 Da (79 kDa) that identity RTX toxin protein and associated Ca _{2 +} - was expected to be similar to the binding protein.

Malditope mass spectrometry separation of the EC2 extracellular protein band (FIG. 20) made it possible to determine the amino acid sequence comprising the EC2 protein:

LHVQFMSSK (SEQ ID NO: 5)

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

Malditope mass spectrometry separation of the EC3 extracellular protein band (FIG. 21) made it possible to determine the amino acid sequence comprising the EC3 protein:

EIAVTELDIAGASSTDYVEVVEACLNQPK (SEQ ID NO: 6)

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

Malditope mass spectrometry separation of the EC4 extracellular protein band (FIG. 22) made it possible to determine the amino acid sequence comprising the EC3 protein:

TTVSYDTGYDDK (SEQ ID NO: 7)

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

Example  6: Spherodes My Co-Para City Car  Purified from Culture Extracellular  Protein Sclerotinia Sclero Room , Liqtonia Solani , And Pisium Ultimum  Influence on Mycelial Growth and Development

The fungal and fungal toxic effects of the purified extracellular proteins EC1 (79 kDa) and EC3 (36 kDa) recovered from Spherodes mycoparasitika cultures were evaluated with three additional widespread plant pathogens causing significant damage to crops. . The inhibitory effect of a mixture of EC1 + EC3 extracellular Spherodes mycoparasitica protein (1: 1) on sclerotenia sclerotirum, lizartonia sorani, and fishium Ultimum is described by Ghosh (2006). ) And Yadav et. al . (2007) using the microtiter plate analysis method disclosed. Mycelia were collected and 36 hours after growth in 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). Figures 23 (a), 23 (c), and 23 (e) show sclerotnia sclerothiorum, lisartonia solani, and pisium ultimum grown on PDA at 26 ° C. for 4 days in the dark, respectively. Micrographs of control cultures. Figures 23 (b), 23 (d), and 23 (f) are (1: 1) mixtures of EC1 (79 kDa) and EC3 (36 kDa) extracellular proteins produced by Spherodes mycoparasiticas, respectively. Micrographs of sclerotinia sclerotiorum, lizartonia solani, and fishium Ultimum grown on fertilized PDA. The extracellular protein mixture caused significant mycelial damage to all three plant pathogen fungal cultures (Figures 23 (b), 23 (d), 23 (f)). The data in FIG. 24 demonstrate that the extracellular protein mixture resulted in: (i) about 80% inhibition of sclerotnia sclerothiorum growth compared to the untreated control, (ii) untreated control Inhibition of about 85% of liqtonia solanie growth in comparison with, and (iii) Inhibition of about 95% of pisium Ultimum growth in comparison to the untreated control. These data demonstrate that the spherodes mycoparasitica extracellular protein disclosed herein has a fungal and fungal toxic effect on a wide range of soil and plant pathogenic fungi.

The studies disclosed herein were made from spherodes mycoparasitika identified as 79 kDa protein comprising SEQ ID NO: 4, 50 kDa protein comprising SEQ ID NO: 5, 36 kDa protein comprising SEQ ID NO: 6 and 13 kDa protein comprising SEQ ID NO: 7. The extracellular proteins derived clearly demonstrate that they are useful for inhibiting spore germination of various plant pathogenic fungi such as Fusarium species, Sclerotinia species, Lizatonia species, and Pisium species. The studies disclosed herein also demonstrate that contacting the mycelium of plant pathogenic fungi with one or more such extracellular proteins causes disruption of mycelial growth and leads to degradation. Thus, transforming a plant cell with at least one nucleic acid molecule encoding at least one polypeptide comprising an amino acid sequence having at least 80% sequence homology with SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 indicates that the plant cell is SEQ ID NO: 4. It will be possible to produce one or more polypeptide molecules comprising an amino acid sequence having at least 80% sequence homology with the amino acid sequence set forth in SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. Thus, when such transformed cells are cultured into plants, the plant comprising the cells will express a polypeptide that results in the formation of extracellular proteins in the cells. During the course of surface colonization of these plants and / or plant infections, plant pathogenic fungi which come into contact with and / or come into contact with extracellular proteins will be inhibited and / or degraded.

IDAC IDAC301008-01 20081030 IDAC IDAC301008-02 20081030 IDAC IDAC301008-03 20081030

                         SEQUENCE LISTING <110> University of Saskatchewan        Vujanovic, Vladimir   <120> PROTEINS FOR BIOCONTROL OF PLANT PATHOGENIC FUNGI <130> 13764-P39401PC01 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 1266 <212> DNA <213> Sphaerodes mycoparasitica <400> 1 atagggagaa gaagcactgc gattgcccta gtaacggcga gtgaagcggc agcagcccag 60 atttggaatc tggtcctttt ggggcccgag ttgtaatctg cagaggaagc gtctggtgcg 120 gtgccggcct agttccctgg aacgggacgc cgtagagggt gacagccccg tacggtcggc 180 caccaaacct gtgtgtcgct ccttcgaaga gtcgcgtagt ttgggaatgc tgcgtaaagt 240 gggaggtatg ctcctcctaa ggctaaatac cggccagaga ccgatagcgc acaagtagag 300 tgatcgaaag atgaaaagca ccttgaaaat ggggttaaaa agtacgtgaa attgccaaag 360 gggaagcgct cgtggccaga ctcgtgcctt atggatcatc cggctatttc gccggtgcac 420 tccattaggc tcgggccagc gtcggtcggc gccggtacta aaagacagcg cgaacgtggc 480 tctcttcggg gagtgttata gcgcgctgtg taatgtgctg gcgccgtccg aggaccgcgc 540 atttatgcaa ggacgctggc gtaatggcca ctagcgaccc gtcttgaaac acggaccaag 600 gagtcgccca gagacgcgag tgtgcgggtg acaaacccct gcgcgaagtg aaagcgaacg 660 ctggtgggaa ccctcacggg tgcaccaccg accgatcctg atgtcttcgg atggatttga 720 gtatgagcgt ttctggtcgg acccgaaaga gggtgaacta tgcttgggta gggtgaagcc 780 agaggaaact ctggtggagg ccccgtttgg gttctgacgt gcaaatcgat ccataaacct 840 gggcatagcg gcgaaagact aatcgaacct tctagtagct ggttcgcatt ctctctctcg 900 cacgagagag agaaaacctc tgtgatatca cgattatcag tgaaaaccac accgagaccc 960 aacggagttc ttctggattt cctcatgctt caattaccac gcctagtgga cctacctgga 1020 gcgctacaat aaagtcatac gaaaatctcg aagatcgggg tgacggtgag ggatcctaag 1080 gttctctcgt tgagtgcgtt ggacgggcat ggccgtcagc gatctggggc gaccgttgcc 1140 ggatcataag ggctttagtg cttaggctat tggtattgag gggtctgaag acggtaatct 1200 gaaaccaaag gctttattct aaacccgcgc agcatgggcg tagtaggaag agacagcgaa 1260 gtctag 1266 <210> 2 <211> 1233 <212> DNA <213> Sphaerodes mycoparasitica <400> 2 agtgcggcat gttgtagcct aagcaattat acagcgaaac tgcgaatggc tcattatata 60 agttatcgtt tatttgatag tgccttacta cttggataac cgtggtaatt ctagagctaa 120 tacatgctga aagccccgac ttacggaggg gcgtatttat tagattaaaa accaatgccc 180 ttcggggctc tttggtgatt catgataact tctcgaatcg cacggccttg cgccggcgat 240 ggttcattca aatttcttcc ctatcaactt tcgatgtttg ggtagtggcc aaacatggtg 300 gcaacgggta acggagggtt agggctcgac cccggagaag gagcctgaga aacggctcct 360 acatccaagg aaggcagcag gcgcgcaaat tacccaatct caactcgagg aggtagtgac 420 aataaatacc gatgcagggc tctttagggt cttgcaattg gaatgagtac aatttaaatc 480 ccttaacgag gaacaattgg agggcaagtc tggtgccagc agccgcggta actccagctc 540 caatagcgta tattaaagtt gttgtggtta aaaagctcgt agttgaacct tgggcctggc 600 cggctggtcc gcctaacagc gtgcactggt gcggccgggt cttcccaccg cggagccgca 660 tgtccttcac tgggcgtgtc ggggaagcgg tacttttact gtgaaaaaat tagagtgctc 720 taagcaggcc tatgctcgaa tacattagca tggaataata gaataggaca gtcgttctat 780 tttgttggtt tctaggacgt ctgtaatgat taacagaaac aatcgggggc gtcagtattg 840 catcgtcaga ggtgaaattc ttagatcgat gcaagactaa ctactgcgaa agcattcgcc 900 aagggtgttt tcattaatca ggaacgaaag ttaggggatc gaagacgatc agataccgtc 960 gtagtcttaa ccataaacta tgccgactag ggatcgggcg gtgtaatttt gacccgctcg 1020 gcacttacga gaaatcttaa gtgcttgggc tccaggggag tatggtcgca aggctgaaac 1080 ttaaagaaat gacgaagggc accaccaagg gtgaacctgc ggcctagttg actcacacgg 1140 gaaactcacg aggtaggaat atgtagatga cggatggagc cttcagaata catatgggca 1200 tgcgctctta ataccggtat tgaaaatggg cag 1233 <210> 3 <211> 560 <212> DNA <213> Sphaerodes mycoparasitica <400> 3 ttcgtttctt atcgcattgg tgaccagcgg agggtcatta cgaatcggac catttatgtc 60 atggctctgc caaccctgtg aactttatac ttgtacgttg cctcggcgga acctgccttt 120 tcggcaggcc gccggccggc atatacgcaa acgctctgaa aaagctccgc gctctatctg 180 aataataaaa ctttaacgag taaaaacttt tggcaacgga tctcttggct ctggcatcga 240 tgaaaaacgc agcgaaatgc gatacgtaat gtgaattgca gaattcagtg aaccatcgaa 300 tctttgaacg caccttgcgg ccgccggtaa tccggcggcc atgcccgtcc gagcgtcgtt 360 tccaccctcg ggagttctcc tcctaagaaa atttctcccg gccttgggcc agcgcgttgc 420 gcggctgccc gaccaacggc ggcaggaccg gcgatgtcct ctgtgccctg catttatata 480 aaactcgcat tggtccccgg taaggcttgc cttgcaacca acttctttag gtcgacctca 540 gatcggatag ggatacccgc 560 <210> 4 <211> 15 <212> PRT <213> Sphaerodes mycoparasitica <400> 4 Tyr Leu Pro Gly Gly Gly Gly Gly Arg Asp Glu Pro Pro Pro Arg 1 5 10 15 <210> 5 <211> 9 <212> PRT <213> Sphaerodes mycoparasitica <400> 5 Leu His Val Gln Phe Met Ser Ser Lys 1 5 <210> 6 <211> 29 <212> PRT <213> Sphaerodes mycoparasitica <400> 6 Glu Ile Ala Val Thr Glu Leu Asp Ile Ala Gly Ala Ser Ser Thr Asp 1 5 10 15 Tyr Val Glu Val Val Glu Ala Cys Leu Asn Gln Pro Lys             20 25 <210> 7 <211> 12 <212> PRT <213> Sphaerodes mycoparasitica <400> 7 Thr Thr Val Ser Tyr Asp Thr Gly Tyr Asp Asp Lys 1 5 10

Claims (57)

Disease symptoms caused by one or more plant pathogenic fungi comprising a polypeptide molecule and a carrier comprising an amino acid sequence having at least 80% sequence homology 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 composition for controlling it. The composition of claim 1, wherein the polypeptide molecule comprises an amino acid sequence having at least 80% sequence homology with SEQ ID NO: 4. The composition of claim 1, wherein the polypeptide molecule comprises SEQ ID NO: 4. The composition of claim 1, wherein the polypeptide molecule comprises an amino acid sequence having at least 80% sequence homology with SEQ ID NO: 5. The composition of claim 1, wherein the polypeptide molecule comprises SEQ ID NO: 5. The composition of claim 1, wherein the polypeptide molecule comprises an amino acid sequence having at least 80% sequence homology with SEQ ID NO: 6. The composition of claim 1, wherein the polypeptide molecule comprises SEQ ID NO: 6. The composition of claim 1, wherein the polypeptide molecule comprises an amino acid sequence having at least 80% sequence homology with SEQ ID NO: 7. The composition of claim 1, wherein the polypeptide molecule comprises SEQ ID NO: 7. The composition according to any one of claims 1 to 9, which is a seed treatment composition. The composition of claim 1, which is a plant treatment composition. The composition of claim 1, which is a soil treatment composition. 13. The composition of any one of the preceding claims, wherein the plant pathogenic fungus is any of Fusarium species, Sclerotinia species, Lizatonia species, or Pisium species. Use of the composition of any one of claims 1 to 13 for controlling a disease symptom in a plant caused by any of Fusarium species, Sclerotinia species, Lizartonia species, or Pisium species. Use according to claim 14, which comprises at least one of seed treatment, plant treatment, and plant growth substrate treatment. A polypeptide molecule comprising an amino acid sequence having at least 80% sequence homology with an amino acid sequence 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 plant pathogenic fungi. Use of microbial cells to express. The use of claim 16, wherein the polypeptide molecule comprises an amino acid sequence having at least 80% sequence homology with SEQ ID NO: 4. The use of claim 16, wherein the polypeptide molecule comprises SEQ ID NO: 4. The use of claim 16, wherein the polypeptide molecule comprises an amino acid sequence having at least 80% sequence homology with SEQ ID NO: 5. The use of claim 16, wherein the polypeptide molecule comprises SEQ ID NO: 5. The use of claim 16, wherein the polypeptide molecule comprises an amino acid sequence having at least 80% sequence homology with SEQ ID NO: 6. The use of claim 16, wherein the polypeptide molecule comprises SEQ ID NO: 6. The use of claim 16, wherein the polypeptide molecule comprises an amino acid sequence having at least 80% sequence homology with SEQ ID NO: 7. The use of claim 16, wherein the polypeptide molecule comprises SEQ ID NO: 7. The use according to any one of claims 16 to 24, wherein the microbial cells are fungal cells, yeast cells, or bacterial cells. The use according to claim 25, wherein the fungal cell is a Spherodes mycoparasitica cell or a penicillium species cell. The use according to claim 25, wherein the yeast cells are Zaigo Saccharomyces spp. Cells, Saccharomyces spp. Cells, Pichia spp. Cells or Cluberomyces spp. Cells. The use according to claim 25, wherein the bacterial cell is Escherichia coli cell, Pseudomonas cell or Bacillus cell. Transformed plant cells resistant to infection by Fusarium species expressing a polypeptide molecule comprising an amino acid sequence having at least 80% sequence homology with an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 . The transformed plant cell of claim 29, wherein the polypeptide molecule comprises an amino acid sequence having at least 80% sequence homology with SEQ ID NO: 4. The transformed plant cell of claim 29, wherein the polypeptide molecule comprises SEQ ID NO: 4. The transformed plant cell of claim 29, wherein the polypeptide molecule comprises an amino acid sequence having at least 80% sequence homology with SEQ ID NO: 5. The transformed plant cell of claim 29, wherein the polypeptide molecule comprises SEQ ID NO: 5. The transformed plant cell of claim 29, wherein the polypeptide molecule comprises an amino acid sequence having at least 80% sequence homology with SEQ ID NO: 6. The transformed plant cell of claim 29, wherein the polypeptide molecule comprises SEQ ID NO: 6. The transformed plant cell of claim 29, wherein the polypeptide molecule comprises an amino acid sequence having at least 80% sequence homology with SEQ ID NO: 7. The transformed plant cell of claim 29, wherein the polypeptide molecule comprises SEQ ID NO: 7. 38. The transformed plant cell of any one of claims 29-37, wherein the plant cell is an oil seed plant cell or grain plant cell or a legume plant cell. Controlling a disease condition caused by one or more plant pathogenic fungi comprising a polypeptide molecule comprising an amino acid sequence having at least 80% sequence homology with an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 Isolated protein for. The isolated protein of claim 39, wherein the polypeptide sequence comprises an amino acid sequence having at least 80% sequence homology with SEQ ID NO: 4. The isolated protein of claim 39 comprising SEQ ID NO: 4. The isolated protein of claim 39 comprising an amino acid sequence having at least 80% sequence homology with SEQ ID NO: 5. The isolated protein of claim 39, comprising SEQ ID NO: 5. The isolated protein of claim 39 comprising an amino acid sequence having at least 80% sequence homology with SEQ ID NO: 6. The isolated protein of claim 39 comprising SEQ ID NO: 6. The isolated protein of claim 39 comprising an amino acid sequence having at least 80% sequence homology with SEQ ID NO: 7. The isolated protein of claim 39 comprising SEQ ID NO: 7. An isolated nucleic acid molecule encoding a protein of any one of claims 39-47. A microbial cell transformed with the nucleic acid molecule of claim 48. The microbial cell of claim 49, wherein the nucleic acid molecule is operably linked to a promoter. 51. The microbial cell of claim 49 or 50, which is a fungal cell, yeast cell or bacterial cell. 52. The microbial cell of claim 51 wherein the fungal cell is a Spherodes mycoparasitica cell or a penicillium species cell. 52. The microbial cell according to claim 51, wherein the yeast cell is a Zagoscharomyces spp. Cell, Saccharomyces spp. Cell, Pichia spp. Cell or Cluberomyces spp. 52. The microbial cell of claim 51 wherein the bacterial cell is Escherichia coli cell, Pseudomonas cell or Bacillus cell. A plant cell transformed with the nucleic acid molecule of claim 48. The plant cell of claim 55, wherein the nucleic acid molecule is operably linked to a promoter. 59. The plant cell of claim 55 or 56, which is an oil seed plant cell, grain plant cell or a legume plant cell.

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