SI24489A - Composition and Method for Plant Protection - Google Patents

Abstract

13 protein extract from the mushroom and 1 protein extract from the mycelium showed antibacterial activity against R. solanacearum showed in various tests. In addition, the A. phalloides protein fraction also fully inhibited the growth of bacteria. The extracts and fractions showed not only a strong antibacterial activity, but also showed bactericidal effects more often than bacteristatic. In vivo testing of the five selected extracts on tomato and potato plants led to the conclusion that extracts of C. geotropa, S. variegatus and T. saponaceum inhibit the appearance of disease signs and slow down bacterial wilt both on tomato and potato plants. Mushroom extracts are such an important tool for inhibiting diseases caused by bacterium R. solanacearum. In addition, the inhibition of 12 R solanacearum strains, as well as R. mannitolilytica and E. coli, is very important in confirming a wide spectrum of action of mushroom protein extracts, which is useful in the fields of medicine, biotechnology, bioremediation / biodegradability and agriculture.

Description

PLANT COMPOSITION AND METHOD

DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION

The present invention relates to methods of reducing, eradicating or preventing infection of plants and surfaces by pathogenic bacteria, using protein extracts of higher fungi of the genus Basidiomycetes, their protein fractions and compositions containing a biologically active peptide or protein.

Background to the problem

The search for new antimicrobials is essential as new pathogens or pathogens infect humans, animals and plants, and develop resistance to the currently available chemical substances. Increasing problems are caused by resistant strains of bacteria, not only in hospitals, but also in plant-borne diseases, causing large crop losses both in the fields and in storage. One such example is the plant pathogenic bacterium Ralstonia solanacearum (Smith, 1896), which causes bacterial wilting and great economic damage to many host plants, including some of the most important crops such as potatoes, tomatoes, bananas, tobacco, ginger and eggplants. This bacterium can also be wild as a saprophyte in soil or in water, so water irrigation is usually the cause of its spread. The pathogen can also spread through infected tools, seeds, insects and across roots. Despite extensive studies on R. solanacearum and its importance in agriculture, there is currently no effective biological or chemical means to control or eradicate it. In addition to the latter, there are other important plant pathogenic bacteria, such as Ervvinia amylovora and Dickeya dadantii, which cause high crop losses. Currently, a remedy for the diseases caused by the bacteria listed above is not available.

Due to high production costs and expensive registration procedures for chemically synthesized compounds, natural substances and natural products are becoming more and more interesting for commercialization. About 60% of the 260 enzymes on the market come from fungi, though only five of them come from higher fungi. An example of a commercially available mushroom isolated protein is the enzyme 6-phytase from Peniophora lycii, which is used as a feed additive that increases the uptake of phosphorus and minerals in the gut of non-ruminants. Hydrophobins are another important group of proteins found only in the kingdom of fungi. Their characteristic is that they change the surface tension by changing the hydrophilicity or hydrophobicity of the surface, which gives them many possibilities for various biotechnological and medical applications. It could also benefit from bleaching, resin and paper production for which fungal enzymes (xylanases, lipases and amylases) are already available

a variety of enzymes from higher fungi. Various hydrolases are used in organic synthesis, including nitrilases, esterases, amidases, proteases, and lipases, but none comes from higher fungi. Many mushroom oxidoreductases play an important role in biosynthesis and related applications and include laccase, peroxidase, and tyrosinase. Tyrosinases also provide the opportunity to convert monophenols to diphenols used in the production of antioxidants as food additives or medicines. In addition, the fungal tyrosinase from aspergillus is used in the production of L-DOPA from L-tyrosine for use in the treatment of early Parkinson's disease and heart disease. Tyrosinases from higher fungi offer the possibility of producing hydrogels that could be used to produce artificial tissues, as well as in the production of adhesives, drug delivery matrices and skin substitutes. The use of mushroom protein was published in a review article by Erjavec et al. (2012).

From US patent 2011/0136758 it is known that polysaccharides derived from some bazidiomicect have fungicidal and nematicidal activity. In addition, it is known that the composition of the bazidiomycet extract may be useful for immune modulation as described in US 2006 / 0.263.384.

1) Erjavec J, Kos J, Ravnikar M, Dreo T, Sabotich J (2012) Proteins of higher fungi from forestto application. Trends in Biotechnology 30: 259-273.

2) Pohleven J, Brzin J, Sparrow L, Leonardi A, Coki A, Štrukelj B, Kos J, Sabotich J (2011) Basidiomycete Clitocybe nebularis is rich in lectins with insecticidal activities. 2011/05/11: 1141-1148. 10.1007 / s00253-011-3236-0.

3) Sabotich J, Popovich T, Puizdar V, Brzin J (2009) Macrocypins, a family of cysteine protease inhibitors from the basidiomycete Macrolepiota procera. FEBS Journal 276: 4334-4345.

4) Stasyk, T., Lutsik-Kordovsky, M., Vernstedt, C., Antonyuk, V., Klyuchivska, O., Souchelnytskyi, S., Hellman, U., and Stoika, R. (2010). A new highly toxic protein isolated from the death cap Amanita phalloides is an L-amino acid oxidase. FEBS Journal 277, 1260-9.

5) Fungal tyrosinases: new prospects in molecular characteristics, bioengineering and biotechnological applications. Journal of Applied Microbiology, 100, (2) 219232

6) Lindequist, U., Niedermeyer, T.H.J., & Julich, W.D. 2005. The Pharmacological Potential of Mushrooms. Evidence-Based Complementary and Alternative Medicine, 2, (3) 285-299

7) Xu, X., Yan, H., Chen, J., & Zhang, X. Bioactive proteins from mushrooms. Biotechnology Advances In Press, Corrected Proof. 2011.

8) MACROCYPIN P-201100304

9) MUSHROOM EXTRACTS HAVING ANTICANCER ACTIVITY US 20060057157 A1

10) L-AMINO ACID OXIDASE WO 1994025574 A1

Definitions

In the context of the present invention, the following terms have the following meanings:

Basidiomycetes or Basidiomycota is one of two large trunks that, together with the trunk of Ascomycota, form the sub-kingdom of Dikarya, often referred to as the higher fungi in the kingdom of fungi.

The term mushrooms in the present application is used instead of the term "bazidiomycetes", the material from the mushrooms refers to any material, the fruiting body, mycelium, extract, tissue, etc. obtained from the bazidiomycetta of the present invention. In the description of the invention, the terms mushrooms and basidiomycetes are used interchangeably.

The protein extract as used in the present application relates to an extract obtained from specific types of basidiomycetes as defined in this claim, from special extracts obtained from fruiting bodies and / or mycelium. The protein extract of the present invention contains proteins or protein-like substances. The protein fraction is part of a protein extract and a specific protein that can be isolated from both the protein extract and the protein fraction. A protein extract may comprise protein complexes or constructs or conjugates of which a protein is a component. The procedures for extraction are described below and examples are provided in the experiments section.

The protein fraction as used in the present application is the fraction obtained from protein extract which is richer in protein compared to protein extract due to purification processes such as e.g. chromatography.

An isolated protein component refers to a protein that has been isolated from a protein extract or protein fraction of the present invention, using methods known to one of skill in the art, such as e.g. chromatography, or a protein that has been determined in a protein extract or a protein fraction of the present invention and has been produced by the recombinant protein method. The isolated protein component can be a protein or peptide that has biological activity and can also be described as a biologically active protein or peptide. Enzymes and lectins are examples.

The terms protein and peptide are used interchangeably.

Plant pathogenic bacteria refers to bacteria that affect or harm plants, for example, that cause wilting and / or that are detrimental to the growth or yield of plants, especially plants grown in agriculture or horticulture.

The term plant includes the whole plant, as well as plant tissues or parts of plants. The plants are mainly crops such as potatoes or vegetables.

Environment refers to the environment surrounding the plant or object we are protecting. The environment treated with the composition of the present invention is, in particular, an area that is prone to infection or contamination by pathogenic bacteria. The environment includes soil around plants as well as irrigation water.

Soil is the soil in which plants are grown and where pathogenic bacteria can live.

The term surface covers hard surfaces, as well as the surface of an object, as a tool or the surface of plant parts, such as roots.

Yield is defined as the measurable economic value of the crop. This can be defined in terms of quantity and / or quality.

The term "reducing" of the present invention refers to an agent or composition that reduces and therefore reduces the negative effect caused by pathogenic bacteria, e.g. by reducing the number of pathogenic bacteria that infect the crop, soil, environment, surfaces, etc., or slowing the progression of the disease.

The term eradication in the context of the present invention means that pathogenic bacteria that infect plants, the environment, soil or surfaces are destroyed or destroyed, for example, such that their reproduction is no longer possible.

The term "prevent" in the context of the present invention means that by adding the composition of the present invention, no pathogenic bacterium can inhabit the treated surface or plant. This can be achieved by the direct effect of the composition of the present invention or by enhancing the host defense.

The bacterial species R. solanacearum is a complex comprising four phylogotypes (I to IV) corresponding to geographical origins. Phylogotypes were determined by phylogenetic sequence analysis. Each group contains several biovars according to their biochemical properties and 5 races determined based on differences in host plants (Fegan and Prior, 2005; Budenhagen, 1962). Each filotype contains multiple sequesters, that is, groups of strains with highly conserved sequences within the genome region.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have found that the protein extract obtained from one of the specific types of mushrooms or particles or their constituents, as defined above, has antibacterial activity, especially activity against plant pathogenic bacteria. Whatever the theory, it is believed that the biologically active proteins or peptides contained in the protein extracts are responsible for the effect. Characterization of the protein components in the extract revealed that ~ 180 kDa protein complex had antibacterial activity. Further analyzes confirm at least one active ingredient, such as L - amino acid oxidase. It was already known that Amanita phalloides, a very toxic mushroom, contains L - amino acid oxidase. It has now been found that specific types of mushrooms, as defined above, comprise biologically active proteins or peptides, one of which is an amino acid oxidase that has activity against pathogenic bacteria and can therefore be used to protect plants. As can be seen from Figure 10, the L-amino acid oxidases present in the compositions of this invention have antibacterial activity; the specificity of different L-amino acid oxidases may differ.

An aspect of the present invention is a method of reducing, eliminating or preventing infection of plants or surfaces by pathogenic bacteria, using on plants or surfaces or in an environment a composition comprising a protein extract of Basidiomycet selected from Amanita phalloides, Amanita muscaria, Amanita virosa, Boletus luridiformis, Clitocybe geotropa, Gomphidius glutinosus, Tricholoma saponaceum, Hypholoma sp., Agaricus moelleri, Albatrellus ovinus, Bovista nigrescens, Suillus variegatus, Tricholoma stale or protein fractions or its constituent.

The present invention provides a composition that is effective against pathogenic bacteria and comprises an antibacterially active component. In one embodiment, the composition comprises at least a protein extract of basidiomycetes selected from Amanita phalloides, Amanita muscaria, Amanita virosa, Boletus luridiformis, Clitocybe geotropa, Gomphidius glutinosus, Tricholoma saponaceum, Hypholoma spus, Agaricusususeri, Alaricusususellus, Agaricusususellus, Su, Tricholoma rose. This extract can be obtained from mushrooms and / or mycelium. Both the cane as well as the mycelium, or both can be used to obtain the extract. The extract can be further purified or separated to obtain protein fractions or active ingredients such as enzymes. In further embodiments of the present invention, protein fractions or active components may be used in this composition of the present invention.

An extract of the present invention may be obtained by harvesting a sponge material, i.e. reeds and / or mycelium of the specific basidiomycetes described above. The extract may be obtained as is well known to the person skilled in the art, for example by using a solvent or extraction buffer or by separating the liquid from a solid material. Many of the various solvents or buffers known to the person skilled in the art can be used to prepare extracts and fractions, as long as the solvent or buffer is biocompatible and can extract protein substances. High protein extracts can be produced by growing mushrooms in vitro.

In one embodiment, the protein extract and / or part of the protein can be obtained by freezing the mushroom material at a temperature between -20 ° C and -80 ° C, thawing the frozen material and separating the liquid from solids, for example by compressing the thawed mass. In another embodiment, the mushroom material may be mechanically crushed, homogenized or biodegradable, and the resulting material may be extracted with solvent or buffer, or the material may be separated into liquid and solid fractions.

The liquid or extract obtained above is a protein extract used in the present invention containing a biologically active substance. The extract can be further purified by dialysis for example against distilled water, with low molecular weight molecules being removed. Preferably the weight limit is below 4000 Da, for example about 3000 Da. The extract can be used in liquid form or can be dried, for example by lyophilization. The extract is preferably used in the dry form, such as the lyophilized form. When used to protect plants, the dried extract can be reconstituted as is well known to one skilled in the art. An aqueous medium, preferably a buffer, especially an isotonic buffer such as Tris - HCI, can be used for preparation. The buffer preferably has a near-neutral pH, for example in the range of 6 to 8, preferably 6.8-7.6. A protein extract or reconstituted extract can be used as a concentrate and the protein concentration in the final composition can be adjusted as is well known, for example, using a buffer such as Tris - HCI buffer. The useful protein concentration is in the range of 0.1 to 10 mg / ml, preferably 0.2 to 5.0 mg / ml. Higher concentrations can also be used.

The protein extract can be further purified to give a protein-enriched fraction - the protein fraction. Protein fractions can be prepared as known to one skilled in the art. Methods for separating or isolating proteins in an extract are well known in the art, such as chromatography, such as gel chromatography or ion exchange chromatography. The protein fraction or the isolated protein component has the advantage of making the content more defined and more compatible.

As described above, it is assumed that at least part of the active substance in the protein extract or protein fraction is at least one biologically active peptide or protein such as Laminic Acid Oxidase.

From the protein fraction, the active proteins can be isolated, for example L-amino acid oxidase. The active protein has favorable properties and, in order to allow production of high quantities, it can also be obtained in purified form obtained from a sponge material or by recombinant production of a protein in E. coli or any other production organism that is well known to the person skilled in the art.

A protein extract such as the above or a protein fraction or protein component can be used as obtained. This can be sterilized if necessary to prevent contamination, for example by using sterile filtration.

Freezing is preferably used to store the protein extract, fraction or dried powder. For short storage times, temperatures in the range of -20 to -40 ° C are applicable. For long-term storage, it is better to use a storage temperature of -60 to -80 ⁰ C.

A further aspect of the present invention is a process for the production of protein extracts from mushrooms as defined above and in claim 1, by harvesting fruiting bodies and / or mycelium basidiomycet, freezing the material at a temperature between -20 ° C and - 80 ° C, thawing the material, separating them into liquid and solid particles, and returning the liquid as a protein extract.

In a further embodiment, the protein extract is the basis for isolating active proteins such as enzymes and lectins, for example L-amino acid oxidase. Isolation can be achieved by using chromatographic methods as known to one skilled in the art.

A further aspect of the present invention is a crop protection composition comprising a protein extract, a protein fraction, a component and / or an enzyme of the present invention of proteins and an agricultural auxiliary.

The composition may comprise an agriculturally or horticultural acceptable solution, carrier, filler, extender or adjuvant.

The crop protection composition may additionally comprise one or more further biologically active agents such as herbicides, pesticides, fungicides, growth factors or fertilizers.

The plant protection composition of the present invention may contain either a protein extract as described above or protein fractions or isolated proteins such as enzymes and lectins.

Individual proteins can also be obtained by chromatography or electrophoresis methods, for example gel chromatography and ion exchange chromatography or native PAGE.

Protein extract, protein fraction or isolated active protein or enzyme has been found to be active against plant pathogenic bacteria that are otherwise difficult to control. On the other hand, the extract does not damage the plants so it can be used to fight bacteria.

The extract of the present invention is effective against important plant pathogenic bacteria such as those of the genera Ralstonia, Ervvinia, Dickeya, Pectobacterium, Xanthomonas, Agrobacterium and Escherichia. In particular, the composition of the present invention can be used to reduce, eliminate or prevent infection with Ralstonia solanacearum, Ervvinia amylovora, Dickeya dadantii, Ralstonia mannitolilytica and Escherichia coli. These bacteria infect important agricultural and horticultural plants such as potatoes, tomatoes, bananas, tobacco, ginger and aubergines.

Protein extract, protein fraction or isolated biologically active protein derived from Agaricus moelleri, Amanita phalloides or Tricholoma saponaceum has been found to be particularly active against Escherichia coli. Therefore, a composition of the present invention comprising a protein extract, protein fraction or isolating a biologically active protein derived from Agaricus moelleri, Amanita phalloides or Tricholoma saponaceum is used inter alia to reduce, eradicate or prevent E. coli infection.

In addition, the protein extract, protein fraction or isolated biologically active protein derived from Amanita phalloides was found to be particularly active against R. mannitolilytica. Therefore, a composition of the present invention comprising a protein extract, a protein fraction or an isolated biologically active protein derived from Amanita phalloides is used inter alia to reduce, eradicate or prevent infection with R. mannitolilytica.

L-amino acid oxidase isolated from Amanita phalloides or Clitocybe geotropa was found to be particularly active against R. solanacearum. L-amino acid oxidase isolated from Amanita phalloides or Clitocybe geotropa is thus used to reduce or eliminate R. solanacearum infection on plants, in plant environments or on surfaces, or to prevent R. solanacearum infection.

Protein extracts obtained from Clitocybe geotropa, Suillus variegatus and Tricholoma saponaceum have been found to be particularly active against bacterial wilting caused by R. solanacearum. Therefore, protein extracts obtained from Clitocybe geotropa, Suillus variegatus and Tricholoma saponaceum are used in the methods and compositions of the present invention, and in particular for the control of R. solanacearum.

The composition of the present invention is useful for treating the environment in which plants grow, soil, water or plant surfaces as well as hard surfaces. This provides a process for reducing, eradicating or preventing infection with pathogenic bacteria in plant or plant environments, by applying a composition comprising a protein extract or protein fractions or a biologically active ingredient as defined above for the plant environment, soil, plant surface or hard surfaces. The composition of the present invention can be applied to surfaces infected with pathogenic bacteria, to reduce or eliminate bacteria, or can be used to protect non-contaminated surfaces against bacterial infection. In addition, the compositions of the present invention can be used to disinfect the environment of plants, soil or surfaces.

The mushroom material of the present invention is particularly useful for protecting crops, including potatoes, tomatoes, aubergines, tobacco, bananas and ginger, preferably plants of the Solanaceae family. The more commonly grown plants of this family are plants of the genus Solanum, especially tomatoes - S. Iycopersicum, potatoes - S. tuberosum and eggplants - S. melongena.

In addition, it was found that the mushroom material of the present invention can also be used to treat wild host plants, in particular host plant plants, and wild hosts of the Urticaceae family. This is very important as wild host plants are a potential source of disease spread, especially when irrigation is taking place. Examples of wild hosts are Solanum dulcamara, Solanum nigrum, and Urtica dioica.

The protein extract, protein fractions and isolated protein components of the present invention were tested for their effect on plant growth. It was found that neither their extract or their active ingredients had any negative effect on the growth of the plants protecting them. The extracts were tested against negative and positive control plants and neither positive nor negative were observed

affects plant growth. Thus, it is apparent that the compositions of the present invention are biologically compatible with the plants and not detrimental to their growth.

Antibacterial activity was further tested in vitro and in vivo. Some extracts showed less activity when used on plants. For other extracts, the effect on plants was better than in vitro. Regardless of the theory, extracts that are more effective in vivo than in vitro are assumed to have an effect on the plant's defense system. The use of these extracts is more desirable because they strengthen the plant's natural defense system, which therefore fights the pathogenic bacterium itself and therefore requires the use of a minimal amount of active substance.

Testing the effect of protein extracts on the plant pathogen Bacterium Ralstonia solanacearum has proven to be very successful. In the initial screening tests, almost 18% of the extracts from different types of mushrooms were active in vitro. For the first time, we tested the performance of extracts against 12 strains of R. solanacearum spanning different phyllotypes. While six extracts completely inhibited the growth of R. solanacearum in vitro, more variability of inhibition was observed in extracts from Agaricus moelleri, Tricholoma ustale, Albatrellus ovinus and Clitocybe nebularis - from zero inhibition to complete inhibition of bacterial propagation. As the extracts did not change during testing, it is assumed that some strains of R. solanacearum are more sensitive than others, but no association was observed with phyllotypes or classification into biovars. Ralstonia mannitollilytica was sensitive to the mushroom extracts of the present invention because it has a very similar DNA sequence to R. solanacearum. In addition, E. coli has been tested because it is widely used as a production organism for recombinant proteins from fungi. T. saponaceum, A. phalloides and A. moelleri extracts completely inhibited E. coli in vitro. In general, a wide range of activity against R. solanacearum strains is considered to be a great advantage, since the active protein can be isolated and used as a plant protection agent or any other application.

Clitocybe geotropa was highly effective in vitro and in vivo against various strains, and in particular against 12 strains of Ralstonia solanacearum. Therefore, Clitocybe geotropic extracts, protein fractions or protein components from them are recommended for use as a plant protection product.

The invention is explained in more detail by way of examples. Cases should not be construed as limiting the invention or scope.

EXAMPLE 1

Materials and methods

Bacterial culture and inoculum preparation

Ralstonia solanacearum (Smith, 1896) Yabuuchi et al. 1996 (race 3, biovar 2), strain NCPBB 4156, was mainly used in experiments, as were other strains of R. solanacearum and Escherichia coli and Ralstonia mannitolilytica (see Table 3). The bacteria are grown at 28 ⁰ C on a solid medium containing yeast extract, peptone, glucose and agar - YPGA contains 5 g / L yeast extract, 5 g / L protease peptone, 10 g / L glucose, 12 g / L AGA, with pH adjusted to 7.2-7.4. as well as on Kelman's tetrazolium medium (Kelman, 1954) in order to observe the typical colony morphology. The bacterial growth suspension in liquid YPGA was prepared in 0.01 M phosphate buffered saline (PBS) containing 1.071 g / l Na2HPO4, 0.4 g / I NaH2PO4 χ 7H2O, 8 g / I NaCI and pH adjusted 7.2. Bacterial concentration in in vitro and in vivo assays was estimated by measuring OD at 595 nm using the McFarland standard. The concentration of live bacteria (CFU / mL) was determined by applying the dilutions to YPGA medium and counting the CFU after 48 hours.

Fungal culture

Mycelia of the Clitocybe myotrope geotropes were isolated from fresh fruits of bodies harvested in nature. The mycelium was grown in liquid SMY medium containing 10 g of sucrose, 10 g of malt extract, 4 g of yeast extract and 1000 ml of ddH2O without pH adjustment. Mycelia pieces grown on solid SM medium (containing 10 g agar) were transferred to 200 ml of liquid SMY medium in a conical flask. They were incubated for 6 weeks at room temperature, in the dark and without shaking. The mycelium was collected and stored at −20 ° C.

Preparation of extracts

All mushroom material (species from the two thick Basidiomycota and Ascomycota) were collected in nature, labeled and frozen at -20 ° C or -80 ⁰ C. Of the 150 mushrooms, 141 were identified to species and 9 to genus. The mushroom extracts were prepared by homogenizing the thawed reeds and centrifuging at 16000 g for 5 min to remove insoluble matter. Protein extraction from the crude extract was performed by acetone precipitation by adding 4 volumes of cold (-20 ° C) acetone and incubation on ice for 40 minutes. After centrifugation at 10,000 g and 4 ⁰ C, the pellet was air dried at 4 ⁰ C and stored at -20 ° C. The mycelium extract was prepared by homogenization in liquid nitrogen using a mortar and stored at -20 ° C. Before use, protein extracts dissolved in 0.05 M Tris - HCI buffer containing 0.1 M NaCI, pH 7.4, centrifuged at 16000g for 5 min to remove insoluble matter, filtered through a sterile 0.20 μm filter (Millex ® - LG) to prevent infection and freeze at -20 ⁰ C for short-term storage or -80 ° C for long-term storage. The approximate protein content of the extracts was determined using Bio-Rad proteins (Bio-Rad, USA), as recommended by the manufacturer.

In vitro screening

To observe the effect of mushroom extracts in the liquid medium, a growth test in 96-well microtiter plates (U-shaped bottom, Golias, Labortehnika) was taken. The mixture contained: 75 pl_ of YPG medium (see YPGA medium, without agar alone), 75 pL of Ralstonia solanacearum suspension (10 ⁷ cells / ml), 42.5 pL of 0.01 M PBS and 7.5 μΙ_ of mushroom extract. Positive control, negative control, and sterility control of the extract were present on each plate. Positive controls contained 75 75 pL of Ralstonia solanacearum suspension, 75 μΙ_ YPG medium, and 50 μΐ_ 0.01 M PBS. Negative controls contain 75 μΙ_ YPG medium and 125 pL 0.01 M PBS, while extract sterility controls contain 75 pL YPG medium, 117.5 pL 0.01 M PBS and 7.5 pL mushroom extract. Each sample and control were present in two replicates and the sterility control of the extract was in one hole. The plates were incubated in a thermo shaker (PST - 60HL - 4, BIOSAN) at 28 ⁰ C and 400 rpm for 72 hours. Growth inhibition was monitored spectrophotometrically with several OD595 measurements (Tecan GENIOS) over 24 hours. After 24 hours, 30 ml were pipetted from each well onto fresh YPGA medium to evaluate whether the effect was bactericidal (bacteria do not grow after transfer) or bacteristatic (bacteria grow after transfer). Output data were collected using Magellan software, V. 6.2.

Results

In vitro screening of the antibacterial activity of olive extracts

Mushroom reeds were collected in Slovenian forests over several seasons and identified by species (Table 1). The average protein content showed variability between the different extracts and was between 5.9 ± 2.6 mg / mL. At the first examination of the performance of mushroom extracts against Ralstonia solanacearum Z 30 (NCPPB 4156), the microtiter plate screening was optimized to ensure reliable and reproducible results. Liquid PGA medium and incubation at 28 ° C are suitable for testing inhibition of R. solanacearum, R. mannitolilytica and E. coli. Absorbance was measured for 24 hours as it was determined in previous experiments as the most informative time point for determining antibacterial activity (inhibitory properties) of extracts. In total, we tested 150 samples from 94 different species, 148 mushroom extracts and 2 mycelium extracts. Of the 150 extracts, 14 samples (13 mushroom extracts and 1 mycelium C. geotropa extract) inhibited R. solanacearum growth in vitro (Table 1). These extracts showed different rates of growth inhibition of R. solanacearum Z30 compared to the growth dynamics of the positive control: complete inhibition (Amanita phalloides, Amanita muscaria, Amanita virosa, Boletus luridiformis, C. geotropa, mycelium C. geotropa extract, Gomphidius glutinosap, Trich , Hypholoma sp), partial inhibition (Agaricus moelleri, Albatrellus ovinus, Bovista nigrescens, Suillus vanegatus, Tricholoma ustale) or no inhibition (Clitocybe nebularis, Ramaria flava). The inhibition rate of representative extracts is shown in Figure 1.

The antibacterial activity of more than one extract of the same species was analyzed for 20 mushroom species (Table 1). 8 Tricholoma saponaceum extracts completely inhibited R. solanacearum and 1 showed partial inhibition (8/1). Similar results were observed for C. geotropa (9/3) and A. phalloides (7/3) extracts. This is not unexpected, as it has been reported that the composition and content of proteins and other substances can vary greatly, depending on the habitat, weather (water availability, temperature) and age of the fertile body.

Table 1: Microtiter plate screening test: 150 extracts from 94 species of fungi (mushrooms) representing 26 families were tested in vitro

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Materials and methods

Pathogenicity test on tomatoes

Tomato plants (L. esculentum cv. Moneymaker) were used in an in vivo test in a greenhouse. Prior to inoculation, plants were planted in the substrate where they grew at 21 ° C in light and in the dark with 90 pmol m ' ² with ¹ photon irradiation (L36VV / 77 light, Osram, Germany) and 16 h photoperiods. Plants were inoculated in phase two to three straight leaves with a mixture of R. solanacearum and mushroom extracts. Plants were inoculated with a bacterial concentration of 10 ⁵ cfu / ml, which in the previous experiments was confirmed as the lowest concentration that causes characteristic symptoms on all plants. Mushroom extracts that inhibited the growth of R. solanaceraum in vitro were added to the R. solanacearum suspension at a ratio of 1:10. Negative control plants were inoculated with 0.01 M PBS and positive control plants only with R. solanacearum suspension without extract. Using a sterile needle (Icogamma plus 0.6mm x 25mm, Novico, Italy), the stitches were stabbed between the germs, using the following procedure: a droplet was injected at the tip of the sterile needle and pierced the stem. When the needle pierced the stem, we made another drop of suspension on the other side and pulled the needle back. At least 40 plants, 42 positive controls and 20 negative control plants were inoculated with each bacterial and extract suspension. Plants were observed for at least 14 days at 28 ° C daytime temperature and 20 ° C nighttime temperature at 90 pmol m ' ² s' ¹ photon irradiation and 16 h photoperiods. The plants were monitored for at least 14 days, and the rate of disease signs (symptoms) was evaluated according to the numerical classes of Winstead and Kelman (1952), namely: 0 (no symptoms), 1 (one leaf uvel), 2 (2-3 leaves introduced). 3 (all leaves except the top of the plant withered), 4 (all leaves and the top withered), 5 (plant dead).

Pathogenicity test on potatoes

Micropropagated potato plants of Solanum tuberosum (cv. Desiree) were grown in tissue culture for 4 weeks before being planted in soil pots. After growing for 14 days in the soil at 22 ° C daytime temperature, 20 ° C nighttime temperature and illumination of 90 pmol m ² s' ¹ photon irradiation and 16 h photoperiods, the plants were inoculated with bacteria and extracts 1 cm above the ground (soil ) and incubated at 25 ° C day and night temperature and illumination with 90 pmol m ' ² s' ¹ photon irradiation and 16 h photoperiods. With each bacterial and extract suspension, 42 plants were inoculated, as well as 42 positive control plants (R. solanaearum only) and 20 negative control plants (0.01 M PBS). The plants were monitored for at least 14 days, and the rate of disease signs was assessed according to the numerical classes of Winstead and Kelman (1952), namely: 0 (no symptoms), 1 (one leaf uvel), 2 (2-3 leaves introduced), 3 (all leaves except the top of the plant withered), 4 (all leaves and the wilt tip), 5 (plant dead).

Quantification of R. solanacearum in plants using real-time PCR

The concentration of R. solanacearum was monitored using the real-time PCR method. Plant tissue was sampled before and after symptoms were observed. The tissue of the tomato stem was sampled by cutting 5 mm long sections above the inoculation point at the first and second nodules. We sampled plants that showed different levels of symptoms, at least 3 plants per symptom. If only 3 plants or less showed a certain degree of bz, we did not sample and include them in the qPCR analysis. Although plants displaying different levels of symptoms were sampled uniformly between groups, plants that were not sampled (see Materials and Methods) may produce less than 5% variation in the percentage of wilted plants due to the lower total number of plants.

Samples of plant tissue were cut in half and stored in 1.5 ml Eppendorf microcentrifuges. Under sterile conditions, 500 μΙ_ 0.01 M PBS was added, mixed and incubated for 10 minutes at room temperature. After incubation, 400 µL of the suspension was stored in microcentrifuges at -20 ° C until use. In these samples, the presence of R. solanacearum DNA was then determined using real-time (TaqMan) PCR. On the basis of preliminary experiments (data not shown), bacterial DNA isolation or sample warming was not required for successful amplification of the target DNA. The protocol used in our experiment, including initial oligonucleotides and probes, was developed by Weller et al., 2000. 10 ml reactions were performed in 384-well plates (MicroAmp, Applied Biosystems). Real-time PCR blend for both R. solanacearum (16S rDNA gene) and cytochrome oxidase gene (COX) contained 5.0 pl_ TaqMan master mix JP6251 (Applied Biosystems, 2002), 0.9 pL 10 pmol / pL RS- IF, RS - II - R or COX F, COX - R, 0.2 pL 10 pmol / pL L RS - P or COX - P, 1 pL of deionized water and 2 pL of sample. In addition to samples from the pathogenicity test, standard R. solanacearum and COX curves and NTC (no target control) were applied to each plate. ABI Prism Sequence Detection System 7900 HT was used to amplify and measure fluorescence. The cycles were performed according to the following protocol: 2 min at 50 ° C, 10 min at 95 ° C, followed by 45 two-step cycles of 15 seconds at 95 ⁰ C and 1 minute at 60 ⁰ C. The data were collected in SDS 2.2 software. .3, and exported and analyzed using Microsoft Excel. The bacterial concentration was determined using the R. solanacearum standard curve at concentrations of 100 cells / ml to 10 ⁸ cells / ml.

Data analysis

To evaluate disease progression on plants, we calculated the AUDPC value (Area Under Disease Progress Curve) using the R-statistical (Agricolae package; Madden et al. 2007). The procedure calculates the average disease intensity between each pair of adjacent time points and therefore the quantitative severity of the disease over time. Other data were analyzed using either Microsoft Excel or R-statistical.

Results

Pathogenicity test on tomato and potato plants

Ralstonia solanacearum causes bacterial wilting on a variety of host plants. Tomato plants most commonly used as test plants for testing the pathogenicity of R. solanacearum, but potatoes are the primary host of R. solanacearum in the European area, so we also included potato plants in our study to test the in vivo effects of mushroom extracts on R solanacearum. The five extracts active in in vitro screening were used in pathogenicity tests on tomato and potato plants (Table 2).

A. phalloides, B. nigrescens, C. geotropa, S. variegatus, and T. saponaceum extracts that inhibited R. solanacearum in vitro were mixed with R. solanacearum suspension immediately before inoculation, thereby reducing the effect of the extract on initial R concentrations. solanacearum. The rate of symptoms observed on tomato and potato plants (Lycopersion esculentum cv. Moneymaker and Solanum tuberosum cv. Desiree) inoculated with a mixture of R. solanacearum and extracts was lower compared to that of positive control plants. No symptoms of potato and tomato negative control were observed.

Symptoms were observed in positive control plants 4 days after inoculation (dpi) (57% of plants showed symptoms) (Figure 4). 4 dpi drowned 57% and 71% of the tomato plants, respectively, co-inoculated with A. phalloides and B. nigrescens extracts, which led to the conclusion that A. phalloides and B. nigrescens extracts did not inhibit diseases caused by R. solanacearum bacteria on tomato plants. Tomato plants co-inoculated with S. variegatus and T. saponaceum started to wilt 4 dpi, but only 11% and 27% of plants withered. 14 days after inoculation, the symptoms showed 95% and 100% of plants co-inoculated with S. variegatus and T. saponaceum, however, the number of fully introduced plants (class 5) was significantly lower compared to the positive control. Similarly, C. geotropa was co-inculcated with 4 dpi only 22% of the plants showing symptoms. 15 dpi, 98% of plants co-inoculated with C. geotropa showed disease signs.

Due to promising results on tomato plants, the experiment was repeated on potato plants. The disease progression in tomatoes was faster than that of potatoes. Symptoms were first observed in 4 dpi positive control plants, with 9% of potato plants showing symptoms (Figure 3). Symptom intensity was rated 1 and / or 2 by VVinstead and Kelman numerals. Likewise, 4 dpi showed symptoms in plants co-inoculated with T. saponaceum, but only 3% of plants (stage 1) wilted. Symptoms on potato plants co-inoculated with C. geotropa and S. variegatus appeared 5 dpi, at that time 15% and 24% of the plants showed symptoms, while 5 dpi evoked already 57% of the positive control plants. Slower disease progression continued in potato plants co-inoculated with extracts, with 14% dpi dropping 44%, 67% and 63% of C co-inoculated plants, respectively.

inoculated with C. geotropa, S. variegatus and T. saponaceum compared with 92% of the positive control plants.

The results of pathogenicity tests on tomatoes and potatoes lead us to conclude that extracts of C. geotropa, S. variegatus and T. saponaceum slow the progression of the disease and reduce symptom intentionality, being more effective on potato plants than on tomato plants.

Table 2: AUDPCs were calculated for tomato and potato plants co-inoculated with mushroom extracts. AUDPC values are expressed relative to AUDPC positive controls (% PC).

in vitro pathogenicity tests tomato potato cv. Desiree

The name of the mushroom extract inhibition bactericidal / bacteristatic active (yes / no) AUDPC value (% PC) active (yes / no) AUDPC value (% PC) Amanita phalloides +++ ^a C no 114 nt nt Bovista nigrescens + ^c S no 94 nt nt Clitocybe geotropa +++ C Yes 80 Yes 48 Suillus variegatus ++ ^b S Yes 68 Yes 75 Tricholoma +++ C Yes 76 Yes 63

saponaceum ^a +++ bacterial inhibition - bacteria do not reproduce (x <15% of ₅₉₅ positive control values) ^b ++ bacterial inhibition - bacterial reproduction is much slower than in positive controls (15% <x <60% of ₅₉₅ values positive controls) ^c + bacterial inhibition - bacterial reproduction is slightly slower than positive controls (60% <x <84% OD595 positive control values)

With bacteristatic effect of extract - bacteria grow again when transferred to fresh medium

C bactericidal effect of extract- bacteria do not grow after transfer to fresh medium nt not tested

The number of healthy potato plants is consistent with the AUDPC values (Table 2). Both A. phalloides and B. nigrescens did not reduce overall disease intensity (AUDPC values 114% and 94% PC), despite inhibiting the growth of R. solanacearum in vitro. Therefore, we excluded them from the pathogenicity test on potato plants. C. geotropa, T. saponaceum and S. variegatus reduced the overall disease intensity in tomato and potato plants Interestingly, Suillus variegatus did not completely inhibit R. solanacearum in vitro, but inhibited disease progression in vivo similar to T. saponaceum which completely inhibited the bacteria in vitro and had a bactericidal effect.

C. geotropa and T. saponaceum extracts completely inhibited the bacteria in vitro and in vivo. T. saponaceum, S. vahegatus and C. geotropa extracts inhibited the disease in both tomatoes and potatoes, but there were 15 dpi fewer diseased potato plants than tomatoes. Compared to the positive control and the AUDPC values, the extracts were more effective on the potato than on the tomato. Although S. variegatus extract had only moderate inhibitory activity in vitro, it inhibited the onset and progression of disease traits in both tomato and potato plants. These results also confirmed the observations of other researchers, saying that in vivo and in vitro antimicrobial activity were not always correlated. It follows that early testing of inhibitory substances in vivo should be included, not only to observe the inhibitory effect, but also from the point of view of stimulating growth and other positive effects on plants. Likewise, any extracts showing at least partial inhibitory activity in vitro should be considered as a potential plant protection product. On the other hand, the bactericidal effect in vitro does not mean that the protein or extracts will also be effective in vivo, as has been shown in A. phalloides. Nevertheless, such extracts may still be useful for applications such as surface sterilization or water disinfection.

Most pathogenicity tests for R. solanacearum are performed on easily grown and susceptible to tomato plants. When performing pathogenicity tests on tomato plants, we used two varieties of tomatoes (Roma and Moneymaker). As no difference was observed between cultivars, work on cv continued. Moneymaker as it is routinely used in diagnostics. We have also tested whether the extracts in themselves influence the growth of the plants. Compared to negative and positive controls, no negative or positive effect on growth was observed (Figure 6).

Inoculation methods can play an important role in evaluating the effectiveness of a plant protection product. When the bacterial suspension and extract were mixed before inoculation, some bacteria could be destroyed before entering the plant. However, a slightly lower concentration would have no effect on the development of the disease, otherwise it would be observed in plants co-inoculated with A. phalloides, which had a strong antibacterial effect in vitro. Most of the bacteria are survivors and, upon entering the stem, have arrived in an ideal breeding environment. In contrast, upon entering the plant, the extract is diluted and to some extent loses contact with the bacterium. Nevertheless, we have observed that some extracts inhibit the onset and progression of the disease, leading to the conclusion that they affect the plant's defense system. In general, such an effect is very important and often more desirable than a direct effect on the bacterium, since it causes greater overall resistance to several different pathogens compared to the direct effect on the bacterium. To elucidate the mechanism of action and interactions between the plant, pathogen, and goby extract, gene expression would need to be analyzed, for example, using Next Generation Sequencing (NGS), eng. next generation sequencing).

Quantification of R. solanacearum concentration in tomato tissue was determined by qPCR (Figure 11). Because the method is very sensitive, plant tissue was sampled in the early days after infection to detect low levels of bacteria before symptoms on the plants could be observed. Thus, we would see whether the slower progression of the disease is due to a lower concentration of bacteria in the plant tissue. This is not entirely true in our case, as the concentration of R. solanacearum in plant tissue was very high and did not differ significantly if the plant was inoculated with R. solanacearum alone or an extract was also added. Nevertheless, we observed greater differences in bacterial concentrations in plants co-inoculated with R. solanacearum and mushroom extract compared with positive control plants. The concentration of R. solanacearum in the tomato tissue was determined by qPCR (Figure 11). In all inoculated groups of plants, bacterial concentrations were very high and exceeded 10 ⁶ cells / ml before symptoms were observed. As the symptoms progressed, the bacterial concentration in the plant increased significantly. Bacterial concentration was lower in the second nodium compared to the first nodium in all inoculated plant groups. Interestingly, we did not detect the bacterium in the second nodule at the rate of symptoms 2, 3, and 5 co-inoculated with T. saponaeum. Similar results were obtained for plants co-inoculated with A. phalloides, where the bacterial concentration was lower or no bacteria were detected at the symptom rate of 0, 1, and 2. These results suggest that T. saponaceum and A. phalloides extracts could limit movement of bacteria through an as yet unknown mechanism of action.

Table 3: In vitro influence of selected mushroom extracts after 24 h on R. solanacearum, R. mannitolilytica and E. coli.

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Table 3: In vitro influence of selected mushroom extracts after 24 h on R. solanacearum, R. mannitolilytica and E. coli (continued).

sijeinqdu oqAoomo ra c co ra c ra c CO., LTD ra c ra c ra c 1 + 1 1 + + 1 1 snuiAO snn9J) eqiv co co CO., LTD co co co co ra c +++ + + + +++ + + + + + + + + 1 a / eisn eLuoioqoij ± co (Oh co co co co co co +++ + + + + + + + + + + + + IJdlldOUl snoijeBv ω co co co co co co co + + + + + + + + + + + + + + snsougn / B snipiqdujOQ o o o o o o o o +++ + + + +++ + + + + + + + + + +++ + + + edo) Od6 aqAoofjio o o o υ o o o o +++ +++ + + + +++ + + + + + + +++ + + + Collection name NCPPB 4156 / CFBP 3857 NCPPB 215 / LMG 2296 (RA 1.64) NCCPB 3989 / CFBP 4613 CFBP 6783 NCCPB 4005 / CFBP 4616 CFBP 6438 CFBP 7038 NCPPB 3987 / CFBP Host plant Solanum tuberosum Lycopersicon esculentum Solanum tuberosum Heliconia caribea Zingiber (Ginger) Musa sp. cv. plantain Solanum nigrum Solanum tuberosum The name of the bacterium R. solanacearum R. solanacearum R. solanacearum \ R. solanacearum R. solanacearum R. solanacearum R. solanacearum R. solanacearum Label strain NIB Z 30 NIBZ 1625 NIB Z 1693 NIBZ 1759 NIB Z 1691 NIB Z 1779 NIB Z 1727 NIB Z 1734

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EXAMPLE 3

Results

Activity against various R. solanacearum strains

After observing the inhibitory activity of selected mushroom extracts against R. solanacearum strain NIB Z 30 in vitro and in vivo, we tested whether the extracts showed activity against other R. solanacearum phylogenies. The activity of 10 mushroom extracts was tested against 12 different strains of R. solanacearum representing different phylogotypes and biovars. In addition, we tested them against the fraction of Amanita phalloides that was active in previous tests. In addition to R. solanacearum phylogenies, we included R. mannitolilytica, which was isolated from the infected fluid after autoclaving, as it has the highest nucleotide sequence similarity to R. solanacearum (Coenye et al, 2003), whereas E. coli was selected as unrelated Gram a negative bacterium and a potential production organism for recombinant fungal proteins. In addition, R. mannitolilytica is an opportunistic human pathogenic bacterium that has caused several outbreaks of disease in hospitals in recent years.

The inhibition rate was determined after this calculation of the percentage of growth (A595) compared to the positive control (% PC). Extracts that completely inhibited bacterial growth did not reach more than 15% PC growth, whereas extracts that did not inhibit bacterial growth had values within the limits of positive control variation (at least 84% PC). The extracts that partially inhibited the growth of R. solanacearum were divided into 2 additional groups, those between 15% and 60% PC and those between 60% and 84% PC. Amanita phalloides and Tricholoma saponaceum extracts completely inhibited the growth of all R. solanacearum strains as well as R. mannitolilytica and E. coli, indicating a more general mechanism of action. In addition to these two, Amanita virosa, Boletus luridiformis, Clitocybe geotropa and Gomphidius glutinosus completely inhibited all tested Ralstonia species but not E. coli. Several variations of inhibition were observed with Agaricus placomyces var. terricolor, Tricholoma ustale and Albatrellus ovinus, which in all cases had a bacteristatic effect on the bacteria. Interestingly, A. moelleri did not completely inhibit most R. solanacearum strains except Z1625 and Z1754, but did inhibit E. coli. Clitocybe nebularis and Ramaria flava extracts were least effective. We found that in our case there was no correlation between activity level and classification in the phylogotype In addition, of the 6 extracts that completely inhibited all Ralstonia strains, 4 of them are from edible species, 2 of which must be pre-ingested cook.

EXAMPLE 4

Materials and methods

Isolation of biologically active proteins

The biologically active protein fraction was isolated from Amanita phalloides and Clitocybe geotropes using gel and ion exchange chromatography. The extract was prepared as described in Example 1 and used for gel chromatography using Sephacryl S-200, balanced in 0.02 M Tris-HCl, pH 7.5 with 0.3 M NaCl. The fractions showing antibacterial activity (Figure 9a) were collected, concentrated by ultrafiltration, with a molecular weight cut-off of 10 kDa and dialyzed against 0.03 M BisTris, pH 6.5. The sample was then applied to a DEAE-Sephacel ion exchange column equilibrated in 0.03 M BisTris, pH 6.5. The bound proteins were eluted with a 0-0.4 M NaCI gradient in the same buffer. The inhibitory fractions were collected and concentrated by ultrafiltration.

Protein characterization

Analysis of bacterial inhibitory protein fractions was performed with SDS-PAGE, BlueNative PAGE (Novex NativePAGE), Bis-Tris Gel System (Invitrogen), and Iselectric Focusing (Phast System, Prepared pH 3-9 Gradient Gels - GE Healthcare and Novex pH 3-7 IEF Gel System (Invitrogen) .N-terminal sequence analysis was performed on individual spots by Edman decomposition and use of an automated amino acid sequence sequencer with Procise Liquid Pulse (Applied Biosystems). separation of proteins by SDS-PAGE and electroblotting on a polyvinylidene difluoride membrane and staining with Coomassie Brilliant Blue 250. To identify individual spots, proteins were analyzed by SDS-PAGE, excised, digested with trypsin, and analyzed by peptide mass by mass spectrometry (ESI- MS / MS) Database searches were performed using Mascot internally using the MS / MS Ion Search server Potentially N-glycosyl Protein fractions were analyzed using recombinant N - Glycolidase F (Roche), as recommended by the manufacturer.

Analysis of L-aminooxidase activity

L-amino acid oxidase activity was analyzed as described in Kishimoto and Takahashi (2001). The activity was tested in microtiter plates at 37 ⁰ C. 10 μl of the sample was mixed with 90 μl of the substrate of the reaction mixture in phosphate buffer, pH 7.4, and 5 mM L-amino acid, 2 mM O-phenylenediamine, 0.81 U / was added. ml of horseradish peroxidase. The reaction was stopped by the addition of 50 μl of 2M H2SO4 and the absorbance at 492 nm was measured using 630 nm as the reference wavelength. Alternatively, the absorbance at 420 nm can be measured during the experiment. Ascorbic acid inhibition was tested at final concentrations in the range of 0.1 mg / ml to 5 mg / ml. The pH optimum was determined using citrate phosphate buffer (pH 2.6 - pH 7.6), phosphate buffer (pH 6 - pH 9) and (bi) carbonate buffer (pH 9 - pH 11).

Results

Isolation of the protein fraction with antibacterial activity from Clitocybe geotrop extract was performed by gel and ion exchange chromatography. The strongest lysis was obtained using BlueNative PAGE, with a molecular weight of 180–200 kDa (Fig. 8). The fox was excised and eluted from the gel and its antibacterial activity confirmed in vitro. SDS-PAGE analysis revealed that the protein complex is composed of a protein spot of ~ 58 kDa large and 5-6 weaker spots (Figure 8). Analysis of ~ 58kDa stain mass spectrometry identified the stain as dihydrolipoamide dehydrogenase (ABA73359) as the most reliable hit. This was also confirmed by the mass spectrometry analysis of pixels excised from 2D gel electrophoresis. Nterminal sequencing was determined, but we found no similarity with other proteins in the databases.

Isolation of the protein fraction with antibacterial activity from Amanita phalloides extract was performed by gel and ion exchange chromatography. After analysis by SDS-PAGE and isoelectric focusing, ~ 60 kDa lysis was observed in the fractions with antibacterial activity, showing an approximate molecular weight of 200 kDa and pl 6.5 at native PAGE (Figure 9). Antibacterial activity and vit were confirmed for ~ 200 kDa spots after rinsing the excised gel stain. The N -terminal sequence was determined by Edman degradation, but we found no similarity with other proteins in the databases. The protein is N-glycosylated. After deglycosylation using recombinant N glycosidase F, the - 60 kDa lysis was excised from SDSPAGE, cut with trypsin, and the sample prepared for analysis by mass spectrometry. The protein has been identified as toxophallin (ADA58360), which is an oxidase of L-amino acids and has been confirmed by enzymatic assays. L-amino acid oxidase activity was also confirmed in C. geotropa (fractions 18) obtained by gel chromatography (Figure 8).

The fractions isolated from A. phalloides and C. geotropa show L-amino acid oxidase activity with different specificities for L-amino acids (Figure 10). The amino acid L-leucine is the optimal substrate for both. Enzymatic activity was prevented in both cases by the addition of ascorbic acid. The pH optimum for enzymatic activity is at pH 5 for C. geotropa and at pH 6 for A. phalloides. In addition, both have a wide range of pH activity, since they both have more than 50% enzymatic activity in the range of pH 3 to pH 10.

DESCRIPTION OF THE DRAWINGS OF THE INVENTION

Figure 1 shows the effects of representative protein extracts of mushrooms with antibacterial activity against R. solanacearum Z30 in vitro. Three levels of inhibition were determined: complete inhibition of bacterial growth (value within the limits of negative control variation), partial inhibition of bacterial growth (bacteria grow slower than positive control), and no inhibition of bacterial growth (value within the limits of positive control variation).

Figure 2 (AD) shows the progression of disease signs on potato cv. Desirees inoculated with R. solanacearum and various mushroom extracts from day 1 to day 14 after inoculation. Symptoms were evaluated by numerical ratings according to Winstead and Kelman (1952): 0 (no symptoms), 1 (one leaf of oysters), 2 (2-3 leaves of oysters), 3 (all leaves except the top of the plant oysters) 4 (all leaves and the top of the ram plant), 5 (death of the plant). Figure 2A shows the results obtained from extracts from Suillus variegatus, Figure 2B shows the results obtained from extracts from Tricholoma saponaceum, Figure 2C shows the results obtained with extract from Clitocybe geotrop and Figure 2D shows the results for positive control.

Figure 3 (AF) shows the progression of symptoms on tomato cv. Moneymaker inoculated with R. solanacearum and various mushroom extracts 3-14 days after inoculation. Symptoms were evaluated by numerical ratings according to Winstead and Kelman (1952): 0 (no symptoms), 1 (one leaf of oysters), 2 (2-3 leaves of oysters), 3 (all leaves except the top of the plant oysters) 4 (all leaves and the top of the ram plant), 5 (death of the plant). Figure 3A shows the results obtained with an extract from Amanita phalloides ·, Figure 3B shows the results obtained with an extract from Bovista nigrescens ·, Figure 3C shows the results obtained with an extract from Suillus variegatus ·, Figure 3D shows the results obtained with an extract from Tricholoma saponaceum ·, Figure 3E shows the results obtained from an extract from the Clitocybe geotrop and Figure 3F shows the results for the positive control.

Figure 4 shows the results of SDS-PAGE and Blue-Native PAGE for the extract of C. geotrop and some of its fractions. Isolation of the antibacterial protein from C. geotropa extract was performed by gel and ion exchange chromatography. Fractions analyzed by SDS-PAGE (left) and Blue-Native PAGE (right): column 1, standard, column 2, C. geotropa extract ·, column 3, fractions 71-85 ion exchange chromatography on DEAE-Sephacel ™ at pH 6 , 5: column 4, fraction 18 by gel chromatography on Sephacryl S-200; column 5, fractions 86-105 ion exchange chromatography on DEAE-Sephacel ™ at pH 6.5.

Figure 5 (AC) shows the results of SDS-PAGE and Blue-Native PAGE for Amanita phalloides. The antibacterial protein was isolated from A. phalloides extract using gel chromatography. Figure 5Α): gel chromatography and analysis of antibacterial activity in fractions. Figure 5Β): SDS-PAGE and 5C); isoelectric focusing and analysis of fractions from gel chromatography (A). The numbers above the columns above (B) and (C) correspond to the fractions in (A); Column M is indicated by the molecular weight of the marker in (B) and the pl marker in (C).

Μ • · · ·

Figure 6 (ΑΒ) shows the results of the analysis of the specificity of the L-amino oxidase activity (LAO) of the protein fractions of Amanita phalloides (6A) and Clitocybe geotropa (6B). Specificity of LAO activity of protein fractions of A. phalloides (upper image) and C. geotropa (lower image). We diluted the fractions 5 times. Dilution was found to be most appropriate in previous experiments (not shown). All L-amino acids and urea for negative control were included in the test. The pH of the buffer was 7.5 and the tests were performed at 37 ° C. Results for A. phalloides are shown after 30 minutes of incubation and C. geotropa after 60 minutes of incubation. The values were normalized to Leo, which is the optimal substrate for both oxidases.

Figure 7 (AB) shows the concentration of R. solanacearum in tomato tissue at different levels of symptoms (0-5). The bacterial concentration was determined in the first (A) and in the second nodium (B).

Claims (23)

PATENT APPLICATIONS 1. A method for reducing, eliminating or preventing infection of plants or surfaces by pathogenic bacteria, or for reducing or preventing bacterial wilting by application to a plant, to a plant, water or soil, or to a surface, containing compositions of Basidiomycet protein extracts selected from Amanita phalloides, Amanita muscaria, Amanita virosa, Boletus luridiformis, Clitocybe geotropa, Gomphidius glutinosus, Tricholoma saponaceum, Hypholoma sp., Agaricus moelleri, Albatrellus ovinus, Bovista nigrescens, Su Bovista nigrescens, Suv. The method of claim 1 wherein the pathogenic bacterium is selected from one of the following genera: Ralstonia, Ervvinia, Dickeya, Pectobacterium, Xanthomonas, Agrobacterium, Enterobacter and Escherichia. The method of claim 2, wherein the pathogenic bacterium is selected from Ralstonia solanacearum, Ralstonia mannitolilytica, Agrobacterium tumefaciens, Escherichia coli, Dickeya dadantii, Pectobacterium subsp. atrospecticum, Ervvinia amylovora, Pectobacterium carotovorum subsp. carotovorum, Xanthomonas arboricola pv. pruni, Enterobacter sp. The method of one of the preceding claims, wherein the pathogenic bacterium is Ralstonia solanacearum, Ralstonia mannitolilytica or Escherichia coli. The method of one of the preceding claims, wherein the plant is a crop, in particular a plant of the family Solanaceae or Urticaceae. The method of one of the preceding claims, wherein the plant is a plant of the genus Solanum. 7. The method of claim 5, wherein the plant is selected from S. Iycopersicum, S. tuberosum, S. melongena, The method of claim 5, wherein the plant is Solanum dulcamara, Solanum nigrum, or Urtica dioica. The method of one of the preceding claims, wherein the extract is obtained from the fruiting bodies of Basidiomycetes and / or from the mycelium of Basidiomycetes. The method of one of the preceding claims, using a protein fraction or a protein extract. The method of one of the preceding claims, wherein the protein fraction contains at least L-amino acid oxidase (LAO). The method of one of the preceding claims, wherein the composition is applied to a solid surface infected with pathogenic bacteria for the purpose of reducing or eliminating the bacteria or to an uninfected solid surface to prevent infection. The method of one of the preceding claims, wherein the composition is applied to soil and / or irrigation water. 14. A method for obtaining protein extracts by harvesting fruiting bodies and or mycelium of Basidiomycota trunk fungi, freezing the material at temperatures between -20 ° C and -80 ° C, grinding the material, separating the liquid and solid particles, obtaining the liquid as a protein extract. 15. The method of claim 14, wherein the protein extract or protein fraction is treated by dialysis and / or drying step. 16. The method of claims 14 or 15, the next step being to further purify the protein extract in order to obtain the protein fraction. 17. A plant protection composition comprising a protein extract from plonose bodies and / or a mycelium of Basidiomicet selected from Amanita phalloides, Amanita muscaria, Amanita virosa, Boletus luridiformis, Clitocybe geotropa, Gomphidius glutinosus, Tricholoma saponaceus, Hypholomausellell, Hypholomausellus, Hypholomausellus, Hypholomausellus, Hypholomausellus, Hypholomausellellus, Hypholomausellus, Hypholomausellellus, Hypholomausellellus, Hypholomausellellus, Hypholomausellellus, Hypholomausellellus, Hypholomauselleri ovinus, Bovista nigrescens, Suillus variegatus, and Tricholoma stagnants or their protein fractions or biologically active protein, such as L-amino acid oxidase or, optionally, at least one agricultural or horticultural adjuvant, extender, solvent, carrier, filler, and / or adjuvant. The composition according to claim 17, wherein the extract contains L-amino acid oxidase. 19. The composition according to claim 17, wherein the extract contains L contains Laminic acid oxidase isolated from Clitocybe geotropa or A. phalloides. 20. A composition according to claim 17 or 18, containing one or more biologically active agents, for example a herbicide, pesticide, fungicide, plant growth promoter or fertilizer. 21. Basidiomycetes protein extract obtained by the method of one of claims 14 to 16. The method of one of claims 1 to 11 wherein the application composition is a composition according to one of claims 17 to 20. Use of the composition of one of claims 17 to 21 for the elimination, reduction or prevention of infection with Ralstonia solanacearum. co