WO2015058944A1 - Composition and method for plant protection - Google Patents

Abstract

13 protein mushroom extracts and 1 protein extract from mycelium with antibacterial activity against R. solanacearum have shown activity in tests. Moreover, an A. phalloides protein fraction also completely inhibited bacterial growth. The extracts and fractions not only displayed potent inhibition of bacterial multiplication but more commonly displayed bactericidal effect, rather than bacteristatic. In vivo testing of 5 selected extracts on tomato and potato plants lead to a conclusions, that C. geotropa, S. variegatus and T. saponaceum extracts lower disease occurrence and delay bacterial wilting on both tomato and potato plants. Thus, mushroom protein extracts of the present invention are an important tool to treat bacterial wilt caused by R. solanacearum. Moreover inhibition of 12 R. solanacearum strains as well as R. mannitolilytica and E. coli by mushroom protein extracts proves their broad spectrum activity, which could be beneficial in the fields of medicine, biotechnology, waste management/bioremediation and agriculture.

Description

Composition and Method for Plant Protection

DESCRIPTION Field of the invention

The present invention relates to methods for reducing, eradicating, or preventing infestation of plants and surfaces with pathogenic bacteria, to proteinaceous extracts of Basidiomycetes, protein fractions, and compositions comprising a biologically active peptide or protein. Background

Searching for new antibacterial substances is necessary since new pathogens are emerging or existing pathogens that infect humans, animals and plants are developing resistances towards currently available chemical agents. Major problems are caused by resistant bacterial strains not only in hospitals but also by resistant plant pathogenic bacteria that cause major crop losses in the fields as well as in postharvest storage. One example is the major plant pathogen, Ralstonia solanacearum (Smith, 1896), which causes bacterial wilt and devastating damage on a wide range of host plants, including important crops such as potato, tomato, banana, tobacco, ginger and egg-plants. This bacterium can also be free- living as a saprophyte in the soil or in water in the absence of host plants and thus irrigating water is a usual cause of its dissemination. The pathogen can also spread with infected tools, seeds, insects and root contacts. In spite of extensive studies on R. solanacearum and its importance in agriculture, presently there is no efficient biological or chemical agent available for its control or eradication. There are other important plant pathogenic bacteria such as Erwinia amylovora and Dickeya chrysanthemi that cause high crop losses. However, no agents are available for controlling plant diseases caused by these pathogenic bacteria.

Because of high production prices and costly registration procedures of chemically synthesized compounds, natural substances and natural products are becoming more and more interesting for commercialization. Approximately 60% of the 260 commercialized enzymes originate from fungi, although only five originate from higher fungi. One example of a mushroom protein already commercialized is a 6-phytase from Peniophora lycii, which is used as a feed additive that enhances the phosphorus and mineral uptake in monogastric animals. Hydrophobins are another important group of proteins unique to fungi that self- assemble at hydrophilic-hydrophobic interfaces in a monomolecular layer, inverting the hydrophobicity of the surface, which gives them great potential for diverse biotechnological and medical applications. The processes of bleaching, pitch control and paper coating, for which fungal enzymes (xylanases, lipases and amylases) are commercially available, could also benefit from the diversity of enzymes from higher fungi. Various hydrolases are used in organic synthesis, including nitrilases, esterases, amidases, proteases and lipases, however, none yet from higher fungi. Several mushroom-derived oxidoreductases have found application in biosynthesis, including laccases, peroxidases, and tyrosinases. Tyrosinases also have the ability to convert monophenols into diphenois, which has been used in the production of antioxidants for use as food additives or pharmaceutical drugs. Furthermore, fungal tyrosinases from aspergilli are used in the production of L-DOPA from L-tyrosine for use in treatment of early Parkinson's disease and myocardial diseases. Tyrosinases from higher fungi have been considered for enzymatic crosslinking to produce, for example, protein-polysaccharide hydrogels, which could be useful for tissue engineering, adhesives, matrices for drug delivery and skin substitutes. A summary is found in the review article "Proteins of higher fungi - from forest to application" (Erjavec et al. , 2012, Trends in Biotechnology). It was known from US 201 1/0136758 that polysaccharides extracted from specific Basidiomycetes have fungicidal and nematocidal activity. Furthermore, it was known that a Basidiomycetes extract composition can be useful for immune modulation as has been described in US 2006/0263384.

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

2) Pohleven J, Brzin J, Vrabec L, Leonardi A, Cokl A, Strukelj B, Kos J, Sabotic J (201 1 ) Basidiomycete Clitocybe nebularis is rich in lectins with insecticidal activities. Applied Microbiology and Biotechnology 91 (4):1 141 -1 148.

3) Sabotic J, Popovic 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, Wernstedt C, Antonyuk V, Klyuchivska O,

Souchelnytskyi S, Hellman U, Stoika R (2010). A new highly toxic protein isolated from the death cap Amanita phalloides is an L-amino acid oxidase. FEBS Journal 277:

1260-1269.

5) Halaouli S, Asther M, Sigoillot JC, Hamdi M, Lomascolo A (2006) Fungal tyrosinases: new prospects in molecular characteristics, bioengineering and biotechnological applications. Journal of Applied Microbiology, 100 (2): 219-232.

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 (201 1 ) Bioactive proteins from mushrooms. Biotechnology Advances, 29 (6): 667-674.

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 expressions have the following definitions: "Basidiomycetes" or "Basidiomycota" is one of two large phyla that, together with the Ascomycota, comprise the subkingdom Dikarya, often referred to as higher fungi within the kingdom Fungi.

The term "mushroom" in the present application is used for Basidiomycetes, "Mushroom material" refers to any material, fruiting body, mycelium, extract, tissue, etc. obtained from Basidiomycetes of the present invention. In the description the terms mushroom and Basidiomycetes are used interchangeably.

A "proteinaceous extract" as used in the present application refers to an extract obtained from specific species of Basidiomycetes as defined in this application, in particular extracts obtained from the fruiting bodies and/or mycelia. A proteinaceous extract of the present invention comprises proteins or protein like matter. The protein fraction shall be a part of the proteinaceous extract and specific proteins can be isolated from both the proteinaceous extract or the protein fraction. A proteinaceous extract can comprise complexes or constructs or conjugates a part of which is a protein. A method for obtaining such extracts is described below and examples are found in the experimental section. A "protein fraction" as used in the present application is a fraction obtained from the proteinaceous extract which is enriched in proteins compared to the proteinaceous extract by further purification, such as chromatography.

An "isolated protein component" refers to a protein that has been isolated from the proteinaceous extract or the protein fraction of the present invention using methods known to the skilled person, such as chromatography, or a protein that has been identified in the proteinaceous extract or the protein fraction of the present invention and has been produced by recombinant methods. An isolated protein component can be a protein or peptide having a biological activity and can also be described as biologically active protein or peptide. Examples are enzymes and lectins.

The terms "protein" and "peptide" are used interchangeably.

"Plant pathogenic bacteria" refers to bacteria that affect or damage plants, that cause wilt, and/or that are damaging to the growth or yield of plants, in particular agriculturally or horticulturally grown crops.

The term "plant" shall comprise the whole plant as well as plantal tissue or parts of a plant. Plants are in particular crop, like potato or vegetable.

"Environment" shall refer to the area surrounding a plant or object to be protected. An environment that is treated with a composition of the present invention is particular an area that is prone to infestation with pathogenic bacteria or is contaminated. Environment comprises soil around the plant as well as irrigating water.

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

The term "surface" shall comprise a hard surface as well as the surface of an object like a tool, or the surface of plant parts like roots.

"Yield" is defined as the measurable produce of economic value from a crop. This may be defined in terms of quantity and/or quality.

The term "reducing" in the context of the present invention refers to an agent or a composition that reduces and thus decreases the negative effect induced by pathogenic bacteria, e.g. by reducing the number of pathogenic bacteria infesting crop, soil, environment, surfaces etc., or by delaying progression of the disease.

The term "eradicating" in the context of the present invention means that pathogenic bacteria that have infested a plant, an environment, soil, or surfaces, are extinguished or eliminated such that multiplication is no longer possible. The term "preventing" in the context of the present invention means that by administration of the composition of the present invention no pathogenic bacteria can populate the treated area or plant. This can be achieved by the direct effect of the composition of the present invention or by strengthening the host's defence.

R. solanacearum species complex is comprised of four phylotype groups (I to IV), corresponding with geographic origin. Phylotype groups are determined by phylogenetic analyses of sequence data. Each group contains several biovars according to biochemical properties and 5 races on the basis of differences in host range (Fegan and Prior, 2005; Budenhagen, 1962). Each phylotype contains several sequevars, groups of strains with highly conserved sequence within the genome area. Detailed description of the invention

The inventors found that a proteinaceous extract obtained from one of the specific mushroom species, or fractions or components thereof as defined above, has antibacterial activity, in particular activity against plant pathogenic bacteria. Without being bound by theory it is assumed that biologically active proteins or peptides that are within the proteinaceous extract are responsible for the effect. By characterization of the protein components in the extract a -180 kDa protein complex was found which was shown to have antibacterial activity. Further analyses confirmed at least one active component 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 mushroom species as defined above comprise biologically active proteins or peptides, one of which is L-amino acid oxidase, which have activity against pathogenic bacteria and, therefore, can be used for plant protection. As can be seen in Figure 10, L-amino acid oxidases present in compositions of the present invention have antibacterial activity; specificity of different L-amino acid oxidases can differ.

An aspect of the present invention is a method for reducing, eliminating, or preventing infestation of plants or surfaces with pathogenic bacteria by applying to the plant or surface or environment a composition comprising a proteinaceous extract from Basidiomycetes 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 ustale or a protein fraction or component thereof.

The present invention provides a composition that is active against pathogenic bacteria and comprises an antibacterial active component. In one embodiment the composition comprises at least a proteinaceous extract from Basidiomycetes selected from 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 ustale. This extract is obtainable from the mushrooms and/or their mycelia. Thus, the fruiting bodies as well as the mycelia or both can be used to obtain the extract. The extract can be further purified or separated to obtain a protein fraction or active components like enzymes. In further embodiments of the present invention the protein fraction or an active component thereof can be used in the composition of the present invention.

An extract of the present invention is obtainable by harvesting mushroom material, i.e. fruiting bodies and/or mycelia of the specific Basidiomycetes described above and recovering a proteinaceous extract therefrom. The extract can be recovered as is well-known to the skilled person, for example by using a solvent or buffer for extraction or by separating liquid from solid material. Many different solvents or buffers that are known to the skilled person can be used to prepare extracts and fractions, as long as the solvent or buffer is biocompatible and can take up or extract proteinaceous material. In one embodiment a proteinaceous extract and/or protein fraction can be obtained by freezing the mushroom material at a temperature between -20°C and -80°C, thawing the frozen material and separating liquid from solid material, for example by compressing the thawed mass. In another embodiment, mushroom material can be mechanically broken, homogenized or biologically digested and the material obtained can be extracted using a solvent or buffer or the material can be separated in a liquid and a solid fraction.

The liquid or extract obtained above is the proteinaceous extract used in the present invention that comprises biologically active substance. The extract can be further purified by dialysis, such as against distilled water, whereby low molecular substances are removed. Preferably the cut-off weight is below 4000 Da, such as about 3000 Da. The extract can be used as obtained in liquid form or can be dried, for example by lyophilization. For storage it is preferred to use the extract in dried form, such as lyophilized form. When used for plant protection the dried extract can be reconstituted as is well-known to the skilled person. An aqueous medium, preferably a buffer, in particular isotonic buffer, like Tris-HCI buffer can be used for reconstitution. The buffer preferably has a nearly neutral pH, for example in the range of 6 to 8, preferably 6.8 to 7.6. The proteinaceous extract or the reconstituted extract can be used as concentrate and the concentration of the proteins in the final composition can be adapted as is well-known, for example by using a buffer like Tris-HCI buffer. A 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 be used. The proteinaceous extract can be further purified to obtain a fraction that is enriched in proteins - the protein fraction. Protein fractions can be prepared as is known to the skilled person. Methods for separating or isolating proteins in an extract are well-known in the art, such as chromatography, for example size exclusion or ion exchange chromatography. A protein fraction or an isolated protein component has the advantage that the content is better defined and is better compatible.

As outlined above it is assumed that at least part of the active material in the proteinaceous extract or the protein fraction, respectively, is at least one biologically active peptide or protein, such as an L-amino acid oxidase.

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

The proteinaceous extract as above or the protein fraction or protein component can be used as obtained. It can be sterilized if this is deemed necessary to prevent contamination, for example by using sterile filtration.

For storage the protein containing extract, fraction or the dried powder preferably is frozen. For short term storage a temperature in the range of -20 to +4°C is useful. For long term storage it is preferable to use a storage temperature in the range of -60 to -80°C.

A further aspect of the present invention is a method for obtaining a proteinaceous extract from the mushrooms as defined above and in claim 1 , by harvesting fruiting bodies and/or mycelia of Basidiomycota, freezing the material at a temperature between -20°C and -80°C, thawing the material, separating liquid and solid parts, and recovering the liquid as proteinaceous extract.

In a further embodiment the proteinaceous extract obtained is used to isolate active proteins such as enzymes or lectins, for example L-amino acid oxidase. Isolation can be done using chromatographic methods as is known to the skilled person. A further aspect of the present invention is a crop protection composition comprising a proteinaceous extract, protein fraction, protein component and/or enzyme of the present invention and an agriculturally or horticulturally acceptable excipient.

The composition can comprise an agriculturally or horticulturally acceptable diluent, carrier, filler, extender or adjuvant. A composition for crop protection can in addition comprise one or more further biologically active agents like herbicides, pesticides, fungicides, plant growth agents, or fertilizers. The plant protection composition of the present invention can comprise either the proteinaceous extract as described above, or a protein fraction or isolated proteins thereof, such as enzymes or lectins.

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

It has been found that the proteinaceous extract, the protein fraction or the isolated active protein or enzyme respectively are active against plant pathogenic bacteria that are otherwise difficult to control. On the other hand, the extract does not harm the plant so that it can be used for combating bacteria.

The extract of the present invention is active against important plant pathogenic bacteria such as those of the genera Ralstonia, Erwinia, Dickeya, Pectobacterium, Xanthomonas, Agrobacterium and Escherichia. In particular, the composition of the present invention can be used to reduce, eradicate or prevent infestation with Ralstonia solanacearum, Erwinia amylovora, Dickeya chrysanthemi, Ralstonia mannitolilytica, and Escherichia coli. These bacteria infest important agricultural and horticultural plants like potato, tomato, banana, tobacco, ginger and egg-plants.

It has been found that a proteinaceous extract, protein fraction or isolated biologically active protein obtained from Agaricus moelleri, Amanita phalloides, or Tricholoma saponaceum is particularly active against Escherichia coli. Therefore, in a preferred embodiment the composition of the present invention comprising a proteinaceous extract, protein fraction or isolated biologically active protein obtained from Agaricus moelleri, Amanita phalloides, or Tricholoma saponaceum is used for reducing, eradicating or preventing infestation with E.coli.

Furthermore, it has been found that a proteinaceous extract, protein fraction or isolated biologically active protein obtained from Amanita phalloides is particularly active against R. mannitolilytica. Therefore, in a preferred embodiment the composition of the present invention comprising a proteinaceous extract, protein fraction or isolated biologically active protein obtained from Amanita phalloides is used for reducing, eradicating or preventing infestation with R. mannitolilytica. It has been found that L-amino-acid oxidase isolated from Amanita phalloides or Clitocybe geotropa is particularly active against R. solanacearum. In another preferred embodiment, thus, L-amino-acid oxidase isolated from Amanita phalloides or Clitocybe geotropa is used for reducing or eradicating R. solanacearum on plants, in the environment of plants or on surfaces or for preventing infestation with ft solanacearum.

It has been found that proteinaceous extracts obtained from Clitocybe geotropa, Suillus variegatus, and Tricholoma saponaceum are particularly active against bacterial wilt caused by R. solanacearum. Therefore, in another embodiment proteinaceous extracts obtained from Clitocybe geotropa, Suillus variegatus, and Tricholoma saponaceum are used in methods and compositions of the present invention and in particular for controlling R. solanacearum.

The composition of the present invention is also useful to treat the environment of plants, soil, water or surfaces of plants as well as hard surfaces. Thus, a method for reducing, eradicating or preventing infestation with pathogenic bacteria in the environment of plants or on surfaces is provided, by applying a composition comprising a proteinaceous extract or a protein fraction or a biologically active component as defined above to the environment of plants, the soil, the surface of plant parts or to hard surfaces. The composition of the present invention can be applied to surfaces contaminated with pathogenic bacteria to reduce or eliminate the bacteria or it can be applied to uncontaminated surfaces to protect a surface and prevent infestation with bacteria. Furthermore, the compositions of the present invention can be used for disinfection of the environment of plants, soil or surfaces.

The mushroom material of the present invention is particularly useful for protection of crop, including potato, tomato, egg-plant, tobacco, banana plants and ginger, preferably plants of the family Solanaceae. Widely grown plants of this family are plants of the genus Solanum, in particular tomato - S. lycopersicum, potato - S. tuberosum, and eggplant - S. melongena.

Furthermore, it has been found that the mushroom material of the present invention can also be used for treating wild hosts in particular solanaceous host plants but also wild hosts from the family Urticaceae. This is valuable, since wild host plants are a potential source of spreading the disease especially where irrigation takes place. Examples for wild hosts are Solanum dulcamara, Solanum nigrum, and Urtica dioica.

The proteinaceous extract, protein fraction and the isolated protein components of the present invention have been tested for their effect on plant growth. It was found that neither the extracts themselves nor the ingredients thereof had any negative effect on the growth of the plants to be protected. An extract was tested compared to negative and positive control plants and neither positive nor negative influence regarding the plant growth could be observed. Thus, it is evident that the compositions of the present invention are biocompatible with the plants and are not detrimental to their growth. Furthermore the antibacterial activity in vitro and in vivo has been tested. Some extracts showed less activity when applied to the plants. For other extracts the effect when applied to plants was improved. Without being bound by theory it is assumed that those extracts that have improved efficiency when applied to the plant compared to in vitro tests may also have an effect on the plant's defence system. These extracts are most preferred as by enhancing the plant defence system the plant becomes able to fight the pathogen alone and only a minimal amount of active agent is necessary.

The testing of protein mushroom extracts against major plant pathogen Ralstonia solanacearum has proven very successful. In the initial screening tests almost 18 % of extracts from different species were active in vitro. For the first time, activity against 12 R. solanacearum strains covering different phylotypes was tested. While six extracts completely inhibited R. solanacearum in vitro, extracts from Agaricus moelleri, Tricholoma u stale, Albatrellus ovinus and Clitocybe nebularis displayed more variation - from no inhibition to complete inhibition of bacterial multiplication. Since extracts were not changed during testing, it is assumed that some R. solanacearum strains are more susceptible than others, however no correlation with phylotype or biovar classification was observed. Ralstonia mannitollilytica was susceptible to the mushroom extracts of the present invention since it has very high sequence similarity to R. solanacearum. Additionally Escherichia coli has been tested, as it is often used as a production organism for recombinant proteins from fungi. T. saponaceum, A. phalloides and A. moelleri extracts completely inhibited E. coli multiplication. In general, broad spectrum activity against R. solanacearum strains is a great advantage as the active protein can be isolated and used as a plant protection agent or in any other application.

Clitocybe geotropa exhibited strong antibacterial effectivity in vitro and in vivo against different strains and in particular against 12 Ralstonia solanacearum strains. Therefore, Clitocybe geotropa extracts, protein fractions or protein components therefrom are preferred for use as plant protection agent.

The invention is further explained by way of example. The examples are not to be interpreted as restricting the invention or the 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 (NIB Z30), was mainly used in the experiments, as well as other R. solanacearum strains, Escherichia coli and Ralstonia mannitolilytica (see Table 3).

Bacteria were grown at 28 °C on yeast peptone glucose agar plates - YPGA containing 5 g/L yeast extract, 5 g/L proteose peptone, 10 g/L glucose, 12 g/L agar, with pH adjusted to 7.2-7.4. as well as on Kelman's tetrazolium medium (Kelman,

1954) to observe typical colony morphology. When grown in liquid YPGA medium, bacteria were suspended in 0.01 M phosphate buffer saline (PBS) containing 1 .071 g/L Na₂HP0₄, 0.4 g/L NaH₂P0₄ x 7H₂0, 8 g/L NaCI with pH adjusted to 7.2.

Concentration of bacteria for in vitro and in vivo tests was estimated with OD measurement at 595 nm using McFarland standard. The viable bacterial population was determined by dilution plating on YPGA medium.

Fungal culture

Live Clitocybe geotropa mycelium was isolated from fresh fruiting bodies collected in nature. Mycelium was cultured in liquid SMY medium containing 10 g sucrose, 10 g malt extract, 4 g yeast extract and 1000 mL ddH₂0 with no pH adjustment. Pieces of mycelium that was grown on solid SMY media, containing 10 g agar, were cut and transferred to 200 mL liquid SMY medium in Erlenmeyer flasks. Flasks were incubated at room temperature, in the dark and without shaking for 6 weeks. Mycelium was collected and stored at -20 °C.

Extract preparation

All mushroom material (species from the phyla Basidiomycota and Ascomycota) was collected in nature, identified and frozen at -20 °C or -80 °C. Out of 150 mushrooms, 141 were identified to the species level and 9 to the genus level. Crude mushroom extracts were prepared by homogenizing thawed fruiting bodies and centrifuging at 16000 g for 5 min to remove insoluble material. Protein extraction from the crude extract was performed using acetone precipitation by adding 4 volumes of cold (- 20°C) acetone and 40 minute-incubation on ice. Following centrifugation at 10000 g and 4°C the pellet was air-dried at 4°C and stored at -20°C. Mycelium extract was prepared by homogenization in liquid nitrogen using mortar and pestle and stored at - 20°C. Before use the protein extracts were dissolved in 0.05 M Tris-HCI buffer, containing 0.1 M NaCI, pH 7.4, centrifuged at 16000 g for 5 min to remove insoluble material and filter-sterilized through syringe driven 0.20 pm filter (Millex^®-LG) to prevent contaminations and frozen at -20 °C for short-term storage or -80 °C for long- term storage. Approximate protein content of extracts was determined using Bio-Rad Protein Assay (Bio-Rad, USA) following manufacturer's recommendations.

In vitro screening test

To observe the influence of mushroom extracts in liquid medium, microwell plate assay was adopted. Mixtures of 75 pl_ of YPG medium (see YPGA medium, only without added agar), 75 μ1_ of Ralstonia solanacearum suspension (10⁷ cells/mL), 42.5 pL of 0.01 M PBS and 7.5 pl_ of mushroom extract were prepared in a 96-well microtiter plates (U-shape wells, Golias, Labortehnika). Positive control, negative control and control of extract sterility were present on each plate. Positive control wells contained 75 pL of Ralstonia solanacearum, 75 pL of YPG medium and 50 pl_ of 0.01 M PBS. Negative control wells contained 75 μΙ_ of YPG medium and 125 pl_ of 0.01 M PBS while control of extract sterility wells contained 75 μΙ_ of YPG medium, 1 17.5 μί. of 0.01 M PBS and 7.5 μΐ_ of mushroom extract. Each sample and control was tested in at least 3 parallel wells, extract sterility was tested in 1 well. Plates were incubated in thermo shaker (PST-60HL-4, Biosan) at 28 °C and 400 rpm for 72 hours. Inhibition was monitored spectrophotometrically with several OD₅₉₅ measurements (Tecan Genios) in 24 hours. After 24 hours, 30 μΙ_ was pipetted out of each well containing mixture of R. solanacearum and extracts or only R. solanacearum (positive control) onto fresh YPG agar plates to evaluate whether effect is bactericidal (bacteria do not grow after transfer) or bacteristatic (bacteria grow after transfer). Output data was collected with software Magellan, v. 6.2. Results

In vitro screening of mushroom extracts

Mushroom fruiting bodies were collected in Slovenian forests over several seasons and identified to the species level (Table 1). Average protein content showed variability among different extracts and was 5.9 ± 2,6 mg/ml. For the initial screening of activity of mushroom extracts against Ralstonia solanacearum Z 30 (NCPPB 4156), microtiter plate method was adopted in optimized form to ensure reliable and reproducible results of target substance activity. Liquid YPG medium and incubation at 28 °C were suitable for testing against Ralstonia solanacearum, Ralstonia mannitolilytica and Escherichia coli. Absorbance was measured for 24 hours, which was determined in the preliminary experiments as the most informative time point for determination of antibacterial activity (inhibitory properties) of the extracts. Altogether 150 samples from 94 different species were tested, 148 mushrooms extracts and 2 mycelium extracts. Out of 150 extracts, 14 samples (13 mushroom extracts and 1 Clitocybe geotropa mycelium extract) inhibited R. solanacearum in vitro (Table 1). These extracts displayed different levels of inhibition against R. solanacearum Z30 compared to kinetics of positive control: complete inhibition (Amanita phalloides, Amanita muscaria, Amanita virosa, Boletus luridiformis, Clitocybe geotropa, Clitocybe geotropa mycelium extract, Gomphidius glutinosus, Tricholoma saponaceum, Hypholoma sp.), partial inhibition (Agaricus moelleri, Albatrellus ovinus, Bovista nigrescens, Suillus variegatus, Tricholoma ustale) or no inhibition (Clitocybe nebularis, Ramaria flava). Level of inhibition of representative extracts is shown in Figure 1.

For 20 mushroom species, activity of more than one extract of the same species was analysed (Table 1). 8 Tricholoma saponaceum extracts completely inhibited R. solanacearum and 1 exhibited partial inhibition (8/1 ). Similar results were observed with Clitocybe geotropa extracts (9/3) and Amanita phalloides extracts (7/3). This is not unexpected since it has been reported that composition and content of protein and other substances may highly vary and depends on the growth substrate, weather conditions (water availability, temperature) and age of the fruiting body. Table 1 : Microtiter plate screening test: a total of 150 extracts 94 different species of higher fungi (mushrooms) representing 26 families tested in vitro.

inhibition inhibition

of of

mushroom species and family growth¹ edible mushroom species and family growth¹ edible

Basidiomycota Tricholomataceae - continued

Agaricaceae Tricholoma pardinum (2) no no

Agaricus campestris no yes Tricholomopsis rutilans (3) no yes³

Tricholoma saponaceum (8),

Agaricus tnoelleri yes no ΤΡ,,Ρ yes yes³

Agaricus silvaticus no yes Tricholoma sejunctum no yes³

Coprinus comatus (2) no yes¹ Tricholoma sp. no yes/no²

Lepiota ignivolvata no no Tricholoma terreum no yes

Leucoagaricus leucothites (2) no yes Tricholoma ustale yes no

Macrolepiota sp. no yes/no² Tricholoma vaccinum (2) no yes³

Amanitaceae Boletaceae

Amanita caesarea no yes Boletus calopus no no

Amanita lividopallescens (2) no yes³ Boletus luridiformis yes yes³

Amanita muscaria yes no Leccinum scabrum no yes

Amanita phalloides (10),TP, yes no Xerocomus badius (2) no yes³

Amanita vaginata (2) no yes³ Xerocomus chrysenteron no yes

Amanita virosa yes no Gomphidiaceae

Cortinariaceae Gomphidius glutinosus yes yes

Cortinarius caerulescens no yes³ Suillaceae

Cortinarius glaucopus no yes Suillus bovinus (6) no yes

Cortinarius sanguineus no no Suillus granulatus (3) no yes

Cortinarius sp. no yes/no Suillus lute us (3) no yes³

Hebeloma crustuliniforme no no Suillus sp. (5) no yes

Entolomataceae Suillus variegatus, TP_tiP yes yes

Entoloma rhodopolium no no Tapinellaceae

Fistulinaceae Tapinella atrotomentosa no yes³

Fistulina hepatica no yes Cantharellaceae

Hygroporaceae Cantharellus sp. no yes³

Hygrophorus chrysodon no yes Craterellus cornucopioides no yes

Hygrophorus pudorinus (2) no yes Hydnaceae

Hygrophorus eburneus no yes Hydnum repandum no yes

Hygrophorus fagi no yes Geastraceae

Hygrophorus erubescens no no Geastrum rufescens no no

Hygrocybe ovina no no Gomphaceae

Lycoperdaceae Clavariadelphus sp. no yes/no²

Bovista nigrescens, TP_t yes yes³ Ramaria botrytis no yes

Lycoperdon perlatum no yes³ Ramaria flava yes yes

Lycoperdon pyriforme no no Ramaria formosa no no

Physalacriaceae Phaliaceae

Armillaria mellea (2) no yes³ Phallus sp. no yes³

Pluteaceae Albatrellaceae

Volvariella bombycina no yes Albatrellus ovinus yes yes

Volvopluteus gloiocephallus no no Albatrellus pes-caprae no yes

Strophariaceae Russulaceae

Hypholoma fasciculare no no Lactarius blennius no no

Hypholoma sp. yes yes/no² Lactarius citriolens no no

Tricholomataceae Lactarius deliciosus no yes

Clitocybe alexandri no yes Lactarius illyricus no no

Clitocybe geotropa (12), TP _P yes yes Lactarius sp. no yes/no²

C. geotropa mycelium extract

(2) yes yes³ Russula albonigra no no

Clitocybe gibba no yes Sparassidaceae

Clitocybe nebularis yes yes Sparassis crispa no yes

Clitocybe odora no yes Thelephoraceae

Collybia confluens no yes Sarcodon imbricatus no yes³

Laccaria amethystina no yes scomycota

Lepista glaucocana (2) no yes³ Morchellaceae

Lepista inversa no yes Morchella esculenta no yes³

Lepista nuda no yes³ Tuberaceae

Mycena galericulata no no Tuber aestivum no yes

Tricholoma atrosquamosum no yes Tuber excavatum no yes

Tricholoma bufonium (3) no no Tuber fulgens no na⁴

Tricholoma imbricatum (2) no yes³ Tuber magnatum no yes

Tricholoma mutabile no na⁴

Tuber mesentericum no yes

¹ inhibition of R. solanacearum in in vitro tests

² some species in the family are edible some are not

³ edible when fruiting bodies are young and/or after cooking and/or after cutting off certain parts of the fruiting body

⁴ data not available

(No.) number of the same species tested

TP_t,p: tested in tomato and potato pathogenicity test

TP_t: tested in tomato pathogenicity test

Example 2

Materials and methods

Tomato pathogenicity test

Tomato plants (L esculentum cv. Moneymaker) were used in greenhouse experiment. Plants were potted in soil substrate in the greenhouse and kept at 21 °C in the light and in the dark with 90 pmol m^"2 s^"1 photon irradiance (L36W/77 lamp, Osram, Germany) and a 16-h photoperiod. Plants were inoculated at two to three true-leaf stage with mixed suspension of R. solanacearum and mushroom extracts. Concentration of bacteria was 10^s cfu/mL and was confirmed in preliminary experiments to be the lowest concentration which causes typical symptoms on all plants. Mushroom extract that inhibited R. solanaceraum in vitro was added to R. solanacearum suspension as 10% of the total volume of suspension. Negative control plants were inoculated with 0.01 M PBS and positive control plants were those inoculated with suspension of R. solanacearum without extract. By using sterile needle (lcogamma plus, 0.6mm x 25mm, Novico, Italy) suspension was inoculated between cotyledons, by the following procedure. Syringe was pressed until a drop of the sample appeared at the tip of the needle. Plant stem was pierced so that the needle pierced through the drop into the stem and out on the other side, where another drop was made and the needle pulled back. For each mixture of bacteria and extract 40 plants were inoculated, 42 positive control plants and 20 plants were used for negative control. Plants were observed daily for at least 14 days, at 28 °C day temperature, 20 °C night temperature, with 90 pmol nrf² s^"1 photon irradiance and a 16-h photoperiod. Severity of symptoms was evaluated following the numerical grades of Winstead and Kelman (1952): 0 (no symptoms), 1 (one leaf wilted), 2 (2-3 leaves wilted), 3 (all leaves except tip of the plant wilted), 4 (all leaves and tip of the plant wilted), 5 (plant dead).

Potato pathogenicity test

Tissue culture micropropagated potato plants Solanum tuberosum (cv. Desiree) were rooting in tissue culture for 4 weeks before they were planted in pots, using soil as a substrate. After 2 weeks growth in soil, under 22 °C day temperature, 20 °C night temperature with 90 μηηοΙ m^"2 s^"1 photon irradiance and a 16-h photoperiod, plants were inoculated with bacteria and extracts 1 cm above the substrate (soil) and incubated at 25 °C day and night temperature with 90 pmol m^"2 s^"1 photon irradiance and a 16-h photoperiod. For each mixture of bacteria and extract 42 plants were inoculated, 42 positive control plants and 20 plants were used for negative control. Same as in tomato, severity of symptoms was evaluated following the numerical grades of Winstead and Kelman (1952). Quantification of Ralstonia solanacearum in plants using real-time PGR To follow R. solanacearum concentration using real-time PGR method plant tissue was sampled before and after symptoms were observed. Tomato stem tissue was sampled by dissecting 5 mm long sections above inoculation point at the first and second node. Plants showing different stages of disease were sampled, with at least 3 plants sampled per symptom. If only 3 or less plants displayed certain symptom, plants were not sampled for qPCR analysis. Although plants displaying different stages of symptoms were sampled evenly between groups, plants that were not sampled (See Materials and methods) may cause less than 5 % discrepancies in per cent plants wilted, because of the lower total number of plants. Samples of plant tissue were cut in half and stored in 1.5 ml_ Eppendorf vials. Under sterile conditions 500 μΙ_ of 0.01 M PBS was added, vortexed and incubated for 10 minutes at room temperature. After incubation, 400 μΙ_ of suspension was transported and stored in sterile Eppendorf vials at -20 °C until used. Frozen tomato samples were tested for R. solanacearum using real-time (TaqMan) PGR assay. Based on preliminary experiments (data not shown) no isolation of bacterial DNA or pre-heating of samples was needed for successful amplification of target DNA. Protocol used in our experiment, including primers and probes was developed by Weller et al., 2000. 10 pL reactions were performed in 384- well reaction plates (MicroAmp, Applied Biosystems). The real-time PGR mixture for detection of both, Ralstonia solanacearum gene (16S rDNA) and cytochrome oxidase gene (COX), contained 5.0 μΐ_ of TaqMan master mix JP6251 (Applied Biosystems, 2002); 0.9 μΐ_ of 10 pmol/pL RS-I-F, RS-II-R or COX-F, COX-R; 0.2 pL of 10 pmol/pL RS-P or COX-P; 1 μΐ_ of deionized water and 2 μΙ_ of sample. Besides pathogenicity test samples, R. solanacearum and COX standard curves and NTCs (no template controls) were pipetted on each reaction plate. An ABI Prism 7900 HT Sequence Detection System was used for amplification and fluorescence measurement. All cycles began with 2 min at 50°C, continued to 10 min at 95 °C, followed by 45 two step cycles of 15 s at 95 °C and 1 min at 60 °C. Data was collected in SDS 2.2.3 software, exported and analysed with Microsoft Excel. Bacterial concentration was determined using R. solanacearum standard curve with concentrations 10° cells/mL to 10 ⁸ cells/mL

Data analysis AUDPC value (Area under Disease Progress Curve) was calculated for pathogenicity test as described by Madden et al, 2007, using R-statistical (Agricolae package). AUDPC method calculates average disease intensity between each pair of adjacent time points and therefore quantifies disease severity over time as opposed to a particular time point. Other data was analysed using either Microsoft Excel or R-statistical.

Results

Pathogenicity tests on tomato and potato plants

Ralstonia solanacearum causes bacterial wilt on many different host plants. Tomato plants are most commonly used as test plants in R. solanacearum pathogenicity tests, however potato is the primary host of R. solanacearum in the European area, therefore potato was also included to compare in vitro and in vivo effect of mushroom extract on Ralstonia solanacearum. Five extracts that were active in the early screening tests were used in pathogenicity tests on tomato and potato plants (Table 2).

The stem inoculation procedure was adopted, using low bacterial concentration of 10^s cells/mL which was determined in preliminary tests as the lowest concentration reproducibly leading to symptom development under the experimental conditions).

Amanita phalloides, Bovista nigrescens, Clitocybe geotropa, Suillus variegatus and Tricholoma saponaceum extracts that inhibited R. solanacearum in vitro were mixed with R. solanacearum suspension 10⁵ cells/mL prior to inoculation of tomato plants. Bacteria and extracts were mixed together and immediately inoculated thus lowering the effect of the extract on the starting concentration of R. solanacearum. Symptoms observed on tomato and potato plants (Lycopersion esculentum cv. Moneymaker and Solanum tuberosum cv. Desiree) inoculated with mixture of R. solanacearum and extracts were compared to those of positive control plants (plants inoculated with R. solanacearum only) and negative control plants. No symptoms were observed on negative control potato and tomato plants.

Symptoms on positive control tomato plants were observed 4 days post-inoculation, with 57 % of plants showing symptoms (Figure 4). 4 days post-inoculation 57 % and 71 % tomato plants inoculated with bacterium and A. phalloides and B. nigrescens were wilting which lead to a conclusion that A. phalloides and B. nigrescens extracts had no effect on disease progress caused by R. solanacearum on tomato plants. Tomato plants inoculated with S. variegatus and T. saponaceum started to wilt 4 days post-inoculation, but only 1 % and 27 % plants were wilting. 14 days post-inoculation 95 % and 100 % S. variegatus and T. saponaceum were wilting, however number of completely wilted plants (grade 5) was significantly lower compared to that of positive control. Similarly, only 22 % of R. solanacearum and C. geotropa infected plants had symptoms 4 days post-inoculation, compared to 57 % positive control plants. 15 days post-inoculation 98 % C. geotropa plants were wilted compared to 100% positive control plants, however severity of symptoms was much lower compared to that of positive control.

Because of the promising results on tomato plants, the experiment was repeated on potato plants. Disease progress was faster in tomato compared to potato plants. Symptoms on positive control potato plants were first observed 4 days post-inoculation, with 9 % potato plants showing symptoms (Figure 3). Symptom intensity was evaluated as 1 and/or 2 according to numerical grades of Winstead and Kelman). Symptoms on potato plants inoculated with bacterium and T. saponaceum have also appeared on the 4^th day post- inoculation, however only 3 % of plants were wilting (grade 1 ). Symptoms on potato plants inoculated with bacteria and C. geotropa and S. variegatus extracts appeared 5^th day post- inoculation with 15 % and 24 % plants showing symptoms while at that point 57 % of positive control plants were already wilting. Slower disease progression continued in potato plants inoculated with extracts compared to positive control plants, consequently 14 days post- inoculation, 92 % positive control potato plants were wilted, compared to 44 %, 67 %, and 63 % C. geotropa, S. variegatus and T. saponaceum inoculated plants, respectively.

Results of pathogenicity tests on tomato and potato plants lead to a conclusion, that C. geotropa, S. variegatus and T. saponaceum extracts lower disease occurrence and delay bacterial wilting on both tomato and potato plants, they are even more effective in potato plants.

Table 2: AUDPC (area under disease progress curve) value was calculated for tomato and potato plants infected with a mixture of R. solanacearum NIB Z30 and extracts. Symptom severity was evaluated for at least 14 days post-inoculation. AUDPC is expressed relative to positive control value (% PC).

Bovista nigrescens +^c S no 102 nt nt

Clitocybe geotropa +++ C yes 44 yes 48

Suillus variegatus ++^b S yes 75 yes 75

Trichoioma +++ C yes 77 yes 63 saponaceum

^a+++ Inhibition of bacteria - no multiplication observed (x < 15 % positive control OD₅₉₅ value) ++ Inhibition of bacteria - multiplication significantly slower compared to positive control (15% < x≤ 60 % positive control OD₅₉₅ value)

c+ Inhibition of bacteria - multiplication significantly slower compared to positive control (60% < x < 84 % positive control OD₅g₅ value)

S bacteristatic effect of the extract - bacterial growth observed when transferred onto fresh medium

C bactericidal effect - no bacterial growth observed when transferred onto fresh medium nt not tested

Number of healthy potato plants counted through the experiment correlated with AUDPC values (Table 2). Both, Amanita phalloides and Bovista nigrescens did not lower disease severity on tomato plants compared to positive control plants (AUDPC values 108 and 102 % PC), despite that they inhibited R. solanacearum in in vitro tests. Therefore they were excluded from pathogenicity test on potato plants. Trichoioma saponaceum and Suillus variegatus caused lower overall disease severity on tomato and potato plants (AUDPC values from 63 % PC to 77% PC), while Clitocybe geotropa extract significantly lowered disease severity on both, tomato and potato plants with AUDPC values 44 %PC and 48 % PC respectively. Interestingly, Suillus variegatus did not completely inhibit bacteria in vitro, but displayed similar level of inhibition in vivo than Trichoioma saponaceum which completely inhibited bacteria in vitro and had bactericidal effect. Extracts of Clitocybe geotropa and Tricholoma saponaceum fruiting bodies exhibited strong antibacterial activity in vitro and in vivo. Tricholoma saponaceum, Suillus varigatus and Clitocybe geotropa extracts delayed disease caused by R. solanacearum on tomato and potato plants, however, there were less symptomless tomato plants after 15 days post- inoculation than potato plants. Compared to positive control though, AUDPC values showed similar trends in tomato and potato pathogenicity tests except with T. saponaceum extracts, which were more effective in potato pathogenicity test. S. variegatus extract exhibited only moderate inhibitory activity in in vitro tests, while it significantly reduced symptom severity in tomato and potato plants. This confirmed previous observations that in vivo and in vitro antimicrobial activity does not always correlate. Therefore it is preferable to perform initial screening tests on plants (in vivo) rather than in vitro, not only to observe inhibitory effect but also growth-promoting effect on plants. In cases where this is not possible (due to large sample quantity, cost or test limitations), all extracts that show at least some inhibitory activity should be taken into consideration as potential plant protection agents. On the other hand, bactericidal effect in vitro does not always mean, that protein or extracts will be effective in vivo, as it has been shown for A. phalloides extract. Despite this it may still be useful for applications such as surface or water disinfection.

Most pathogenicity tests for R. solanacearum are being done on tomato plants. Tomato plants are easy to grow and susceptible to R. solanacearum infection. When performing pathogenicity tests on tomato plants, two tomato cultivars were used. As no difference was observed between cultivars, work was continued on cv. Moneymaker, since it is routinely used in diagnostics. It was also tested, whether extracts themselves have any effect on plant growth. No positive or negative effect on plant growth was observed compared to negative and positive control plants (Figure 6).

Inoculation techniques may play a role when evaluating efficiency of plant protection agent. In the present experiments bacterium and extract were mixed right before inoculation, some bacteria may have died before entering the plant. However slightly lower concentration did not have effect on disease development otherwise it would be observed in plants inoculated with A. phalloides, which had strong bactericidal activity in vitro. A majority of bacteria thus survived and were introduced directly into plant vessels - the perfect environment for multiplication. On the contrary, as extract enters the plant, it immediately dilutes and to some extent loses the contact with bacterium. Despite this, a delay in disease progress was observed, therefore, it can be assumed that some of the extracts and their compounds enhance plant defence systems to fight the pathogen alone. Enhancing plant defences is extremely important and often more desirable compared to direct effect on the pathogen, since it induces more general resistance to several different pathogens. To confirm a pathogen-host-extract interaction, plant gene expression can be analysed using techniques like next generation sequencing (NGS). Quantification of R. solanacearum concentration in tomato tissue was determined by qPCR (Figure 1 1 ). Since the method is very sensitive, plant tissue was sampled in the early days post-inoculation in order to detect low concentration of bacteria before symptoms could be observed on the plants to see, whether slower disease progression is a consequence of lower bacteria concentration in plant tissue. This was not true in the present case, since concentration of R. solanacearum in plant tissue was very high and did not significantly vary if the plant was inoculated only with R. solanacearum or the extract was also added. However, more variation in bacterial concentration was observed in plants inoculated with R. solanacearum and mushroom extract, compared to positive control plants. Also plants inoculated with C. geotropa, S. variegatus and T. saponaceum wilted slower and displayed milder symptoms compared to positive control plants. This again suggests that instead of only directly affecting bacterial cells the extract of the present invention may also have an influence on enhancing plant defence systems and thus limit the spreading of bacteria through the plant. Concentration of R. solanacearum in tomato tissue was determined by qPCR (Figure 1 1 ). More variation in bacterial concentration was observed in plants inoculated with R. solanacearum and mushroom extract, compared to positive control plants. In all inoculated plant groups concentrations of bacteria were very high and reached over 10⁶ cells/mL even before the symptoms were observed. With symptom progression, bacterial concentration in plant did not increase significantly. Bacterial concentration was lower in node 2 compared to node 1 in all inoculated plant groups .Interestingly, no bacteria were detected in the 2^nd node of plants displaying level of symptoms 2, 3 and 5 which were inoculated with bacteria and T. saponaceum extract. Similar results were observed in A. phalloides inoculated plants, where concentration of bacteria was lower or not detected at the symptoms severity 0, 1 and 2. These results could indicate that T. saponaceum and A. phalloides extracts may restrict bacterial movement through some yet unknown mechanism of action. Table 3: Activity of mushroom extracts after 24 hours tested against different bacteria.

Table 3: Activity of mushroom extracts after 24 hours tested against different bacteria (continued).

+++ Complete inhibition of bacteria - no multiplication observed (x < 15 % positive control OD₅₉₅ value)

++ Partial inhibition of bacteria - multiplication significantly slower compared to positive control (15% < x < 60 % positive control OD_; + partial inhibition of bacteria - multiplication slower compared to positive control (60 % < < 84 % positive control OD₅₉₅ value) - No inhibition (≥ 84 % positive control OD₅₉₅ value)

S bacteristatic effect of the extract - bacterial growth observed when transferred onto fresh medium

C bactericidal effect - no bacterial growth observed when transferred onto fresh medium

nt (not tested)

na (not applicable)

/ data not available

Example 3

Results

Activity against different Ralstonia solanacearum

After a strong activity of selected mushroom extracts was observed against R. solanacearum strain NIB Z 30 in vitro and in vivo, it was tested whether the extracts show activity against other phylotypes. 12 different strains of R. solanacearum representing different phylotypes and biovars were tested against 10 mushroom extracts and 1 Amanita phalloides fraction that were active in previous tests. Moreover, Ralstonia mannitolilytica isolated from contaminated autoclave fluids was also included, since it has highest sequence similarity to R. solanacearum (Coenye et al, 2003), while Escherichia coli was chosen as an unrelated Gram negative bacterium. Moreover, R. mannitolilytica is an opportunistic human pathogen which has caused several hospital disease outbreaks in the past years.

Level of inhibition was determined after calculating percent of growth (OD₅₉₅ value) compared to positive control (% PC). Extracts that completely inhibited bacteria did not reach more than 15% PC, while extracts that did not inhibit bacteria had values in the limits of variation of positive control (at least 84 % PC). Extracts that partially inhibited Ralstonia solanacearum were distributed into 2 additional groups, those between 15 % and 60 % PC and those between 60 % and 84 % PC. 7 out of 17 samples displayed bactericidal effect, while 10 had bacteristatic effect on bacteria. Extracts of Amanita phalloides and Trichoioma saponaceum completely inhibited all R. solanacearum strains as well as R. mannitolilytica and £. coli which points to a more general mechanism of action. Besides these two, Amanita virosa, Boletus luridiformis, Clitocybe geotropa and Gomphidius glutinosus completely inhibited all Ralstonia strains, but did not inhibit E. coli. More variation of inhibition was observed with Agaricus placomyces var. terricolor, Trichoioma ustale and Albatrellus ovinus which had in all cases bacteristatic effect on bacteria. Interestingly A. moelleri did not completely inhibit most R. solanacearum strains, except Z1625, but completely inhibited £. coli. Extracts of Clitocybe nebularis and Ramaria flava were the least effective. There was no correlation between level of activity and classification into phylotypes. Moreover, out of 6 extracts that completely inhibited all Ralstonia strains, 4 of them are from edible species, of which 2 should be cooked prior consummation, which means that there was no correlation between level of inhibition and human toxins present in mushroom fruiting bodies. Example 4

Material and Methods

Isolation of the biologically active protein fraction

The biologically active protein fraction was isolated from Amanita phalloides and Clitocybe geotropa fruiting bodies and from C. geotropa cultured mycelium using size-exclusion and ion-exchange chromatographies. The extract was prepared as described in Example 1 and applied to size-exclusion chromatography using Sephacryl S-200 equilibrated in 0.02 Tris- HCI, pH 7.5 with 0.3 M NaCI. Fractions exhibiting antibacterial activity (Figure 9A) were pooled, concentrated by ultrafiltration using molecular weight cut-off 10 kDa and dialyzed against 0.03 M BisT s, pH 6.5. The sample was then applied to DEAE-Sephacel ion- exchange column equilibrated in 0.03 M BisThs, pH 6.5. Bound proteins were eluted with a gradient of 0-0.4 M NaCI in the same buffer. Inhibitory fractions were pooled and concentrated by ultrafiltration. Different affinity chromatographies may be used for further purification.

Protein Characterization

Analysis of the inhibitory active protein fractions was performed by SDS-PAGE, BlueNative PAGE (Novex NativePAGE Bis-Tris Gel System (Invitrogen) and isoelectric focusing (Phast System precast pH 3-9 gradient gels (GE Healthcare) and Novex pH 3-7 IEF Gel System (Invitrogen). N-terminal sequence analysis was performed on individual bands by Edman degradation using automated amino acid sequencing in a Procise liquid pulse sequencer (Applied Biosystems) connected on-line to a model 120 A analyser after separating proteins by SDS-PAGE and electroblotting them onto a polyvinylidene difluoride membrane, and staining with Coomassie Brilliant Blue R-250. To identify individual bands proteins were analyzed by SDS-PAGE, bands excised and after in-gel trypsin digestion identified by peptide mass fingerprinting using mass spectrometry (ESI-MS/MS). Database searches were performed by Mascot in-house server using MS/MS Ion Search. Potential N-Glycosylation of the protein fraction was analysed using recombinant N-Glycosidase F (Roche) following manufacturer's recommendations.

Analysis of L -amino acid oxidase activity

L-amino acid oxidase activity was assayed as described in Kishimoto and Takahashi (2001). Briefly, the activity was assayed in microplates at 37°C and 10 μΙ of the sample was mixed with 90 pi of the substrate reaction mixture in phosphate buffer, pH 7.4 and included 5 mM L- amino acid, 2 mM o-phenylenediamine, 0.81 U/mi horseradish peroxidase. After termination of reaction by adding 50 μ! of 2M H₂S0₄, the absorbance was measured at 492 nm using 630 nm as a reference wavelength. Alternatively, absorbance was measured at 420 nm continuously in a time-course experiment. Inhibition by ascorbic acid was assayed at final concentrations ranging from 0.1 mg/ml to 5 mg/ml. pH optimum was determined by 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 antibacterial protein fraction from Clitocyhe geotropa extract was performed by size-exclusion and ion-exchange chromatographies. Strongest band observed in BlueNative PAGE showed molecular weight 180-200 kDa (Figure 8). It was excised and eluted from the gel and its antibacterial activity was confirmed in an in vitro assay. From SDS-PAGE analysis it was concluded that the protein complex consists of a major ~ 58 kDa protein band, and 5 - 6 weaker bands (Figure 8). Mass spectrometry analysis after in-gel trypsin digestion of bands excised from the SDS-PAGE identified the dihydrolypoamide dehydrogenase (ABA73359) as the most reliable hit in the -58 kDa band. This was confirmed by mass spectrometry analysis of spots excised from a 2D electrophoresis gel. N- terminal sequence was determined, however, no significant similarity with other proteins in databases was found.

Isolation of an antibacterial protein fraction from Amanita phalloides extract was performed using size-exclusion and ion-exchange chromatographies. In SDS-PAGE and isoelectric focusing analyses, a ~ 60 kDa band was observed in fractions with antibacterial activity, which showed an approximate molecular mass of 200 kDa in native PAGE and approximate pi of 6.5 (Figure 9). For the ~ 200 kDa band detected in native PAGE antibacterial activity was confirmed in vitro after elution from the excised gel band. N-terminus was determined by Edman degradation and no significant similarities with other proteins were found in available databases. The protein is N-glycosylated. After deglycosylation using recombinant N- glycosidase F the prominent band (~ 60 kDa) in SDS-PAGE was excised and after in-gel trypsin digestion mass spectrometry anaylsis was performed. The protein was identified as toxophallin (ADA58360). Toxophallin is an L-amino-acid oxidase, which was confirmed with enzymatic tests. L-amino-acid oxidase activity was also confirmed in C. geotropa fraction 18 of size exclusion chromatography (Figure 8). It was confirmed that the fractions exhibiting L- amino-acid oxidase activity also exhibited antibacterial activity in vitro. The latter can be heat- inactivated by heating at 80°C. Fractions isolated from A. phalloides and C. geotropa show L-amino-acid oxidase activities with different specificities for L-amino-acid oxidation (Figure 10). However, L-Leucine is the optimal substrate for both. Specificity pattern of the L-amino-acid oxidase activity isolated from cultured C. geotropa mycelium is the same as that isolated from the fruiting body collected in the forest. Enzyme activity was in all cases inhibited by addition of ascorbic acid. The pH optimum is at pH 5 for both C. geotropa activities and at pH 6 for the A. phalloides L- amino acid oxidase activity. Furthermore, both have a wide pH range of activity as more than 50 % enzyme activity is present in the pH range from pH 3 to pH 10.

Example 5

Materials and methods Tomato pathogenicity test

Additional pathogenicity test on tomato cv. Moneymaker plants was performed (Table 4). AUDPC was calculated for controls and plants co-inoculated with extracts. AUDPC values at 14 dpi were compared between experiments giving important information about repeatability of results, We have focused on Amanita phalloides extract and fractions due to strong LAO activity and Clitocybe geotropa extract, fraction and mycelium extract and fraction because of strong LAO activity as well as proven inhibitory properties of the extract in vivo and in vitro.

Tomato plants (L esculentum cv. Moneymaker) were used in greenhouse experiment. Plants were potted in soil substrate in the greenhouse and kept at 21 °C in the light and in the dark with 90 pmol m^"2 s^"1 photon irradiance (L36W/77 lamp, Osram, Germany) and a 16-h photoperiod. Plants were inoculated at two to three true-leaf stage with mixed suspension of R. solanacearum and mushroom extracts. Concentration of bacteria was 10⁵ cfu/mL and was confirmed in preliminary experiments to be the lowest concentration which causes typical symptoms on all plants. Mushroom extract that inhibited R. solanaceraum in vitro was added to R. solanacearum suspension as 10% of the total volume of suspension. Bacteria and extracts were mixed together and immediately inoculated into plants thus minimizing direct effect of the extracts on starting concentrations of R. solanacearum. Negative control plants were inoculated with 0.01 M PBS and positive control plants were those inoculated with suspension of R. solanacearum without extract. By using sterile needle (lcogamma plus, 0.6mm x 25mm, Novico, Italy) suspension was inoculated between cotyledons, by the following procedure. Syringe was pressed until a drop of the sample appeared at the tip of the needle. Plant stem was pierced so that the needle pierced through the drop into the stem and out on the other side, where another drop was made and the needle pulled back. For each mixture of bacteria and extract 30 to 32 plants were inoculated, 32 positive control plants and 16 plants were used for negative control. Plants were observed daily for at least 14 days, at 28 °C day temperature, 20 °C night temperature, with 90 pmol m^"2 s^"1 photon irradiance and a 16-h photoperiod. Severity of symptoms was evaluated following the numerical grades of Winstead and Kelman (1952): 0 (no symptoms), 1 (one leaf wilted), 2 (2- 3 leaves wilted), 3 (all leaves except tip of the plant wilted), 4 (all leaves and tip of the plant wilted), 5 (plant dead).

Results

Table 4: Comparison of relative AUDPC values 14 dpi between pathogenicity tests on tomato cv. .Moneymaker

tomato tomato

cv. Moneymaker 1 cv. Moneymaker 2

AUDPC value AUDPC value

name

(%PC)14 dpi (%PC)14 dpi

Amanita phalloides 98

114

extract

Amanita phalloides 93

nt

fraction

Clitocybe geotropa 75

76

extract

Clitocybe geotropa 79

nt

fraction

Clitocybe geotropa 75

nt

mycelium

Clitocybe geotropa 85

nt

mycelium fraction

Bovista nigrescens 94 nt

Suillus variegatus 68 nt

Tricholoma nt

80

saponaceum

AUDPC values of A. phalloides extract and fraction were similar to those of positive control plants (Table 4) confirming that A. phalloides does not slow or prevent disease progression on tomato plants despite its potent inhibition in vitro. AUDPC values were close to positive control values in both tomato pathogenicity tests.

AUDPC values of C. geotropa extracts were repeatable between experiments 76% and 75% PC respectively. C. geotropa mycelium extract and C. geotropa fraction has similar AUDPC values (75 % and 79% PC), while C. geotropa mycelium fraction AUDPC was 85 % PC. Lower efficacy of C. geotropa mycelium fraction is probably a consequence of a lower protein concentration present in mycelium extract and fraction.

In one of the previous experiments (Table 2) Clitocybe geotropa extract significantly lowered disease severity on tomato plants with AUDPC values 44 %PC, however concentration of R. solanacearum was lower compared to other experiments (4*10⁴ cfu/mL) and plants were already at fourth leaf stage. We believe that it is possible that lower bacterial concentration increased efficacy of the extract.

DESCRIPTION OF DRAWINGS

Figure 1 shows the effects of representative protein mushroom extracts on Ralstonia solanacearum Z30 observed in in vitro testing. Three levels of inhibition were determined: complete inhibition of multiplication of bacteria (values within the variation of negative control), delay of multiplication of bacteria (bacteria multiply slower compared to positive control), no inhibition of multiplication of bacteria (values within the variation of positive control).

Figure 2 shows Clitocybe geotropa mycelium cultivation on solid (A) and in liquid (B) medium.

Figure 3 (A-D) shows progression of disease symptoms on potato cv. Desiree plants inoculated with Ralstonia solanacearum and different mushroom extracts 3 to 15 day post-inoculation. Symptoms were evaluated according to the numerical grades of Winstead and Kelman (1952): 0 (no symptoms), 1 (one leaf wilted), 2 (2-3 leaves wilted), 3 (all leaves except tip of the plant wilted), 4 (all leaves and tip of the plant wilted), 5 (plant death). Figure 3A) shows the results obtained with extract from Suillus variegatus, Figure 3B) shows the results obtained with extract from Tricholoma saponaceum, Figure 3C) shows the results obtained with extract from Clitocybe geotropa, and Figure 3D) shows the results for a positive control.

Figure 4 (A-F) shows progression of disease symptoms on tomato cv. Moneymaker plants inoculated with Ralstonia solanacearum and different mushroom extracts 3 to 14 days post-inoculation. Symptoms were evaluated according to numerical grades of Winstead and Kelman (1952): 0 (no symptoms), 1 (one leaf wilted), 2 (2-3 leaves wilted), 3 (all leaves except tip of the plant wilted), 4 (all leaves and tip of the plant wilted), 5 (plant death). Figure 4A) shows the results obtained with extract from Amanita phalloides; Figure 4B) shows the results obtained with extract from Bovista nigrescens; Figure 4C) shows the results obtained with extract from Suillus variegatus; Figure 4D) shows the results obtained with extract from Tricholoma saponaceum; Figure 4E) shows the results obtained with extract from Clitocybe geotropa and Figure 4F) shows the results for a positive control.

Figure 5 shows tomato plants inoculated with mushroom extracts and R. solanacearum. Symptoms were evaluated 10 days post-inoculation.

Figure 6 shows tomato plants inoculated with Clitocybe geotropa extract and R.solanacearum. Symptoms were evaluated 1 1 days post-inoculation.

Figure 7 shows potato plants in different stages of disease progress, evaluated according to Winstead and Kelman numerical grades.

Figure 8 shows the results of SDS-PAGE and Blue-native PAGE for a Clitocybe geotropa extract and some fractions thereof. Isolation of antibacterial protein from C. geotropa extract was performed by size-exclusion and ion-exchange chromatographies and fractions analysed by SDS-PAGE (left) and blue native PAGE (right): lane 1 , molecular mass standard; lane 2, C. geotropa extract; lane 3, fractions 71-85 of ion-exchange on DEAE-Sephacel™ performed at pH 6.5: lane 4, fraction 18 of the size-exclusion chromatography on Sephacryl S-200; lane 5, fractions 86-105 of ion-exchange on DEAE-Sephacel™ performed at pH 6.5.

Figure 9 (A-C) shows the results of SDS-PAGE and Blue-native PAGE for Amanita phalloides. Antibacterial protein was isolated from Amanita phalloides extract using size-exclusion chromatography of A. phalloides extract. Figure 9A): Size-exclusion chromatography and analysis of antibacterial activity in fractions. Figure 9B): SDS- PAGE and Figure 9C): isoelectric focusing analysis of fractions from size-exclusion chromatography in panel A. Numbers above lanes in panels B and C correspond to fractions in panel A; lane M denotes the molecular mass marker in panel B and pi marker in panel C. Figure 10 (A-B) shows the results of an analysis of specificity of L-amino-acid oxidase (LAO) activity for Amanita phalloides (10A) and Ciitocybe geotropa (10B) protein fractions. The specificity of L-amino-acid oxidase (LAO) activity for Amanita phalloides (upper panel) and Ciitocybe geotropa (lower panel) protein fractions was analysed. Fractions were diluted 5-times which was determined as an appropriate concentration in previous experiments (not shown). All L-amino-acids were included in the test and urea for negative control. Buffer pH was set to 7.5 and tests were performed at 37°C. Results for A. phalloides are shown at 30 minutes of incubation and for C. geotropa at 60 min of incubation. Values were normalized to Leu, which is the optimal substrate for both oxidases.

Figure 1 1 (A-B) shows concentration of R. solanacearum in tomato plant tissue (logarithmic scale) at different stages of symptoms (0-5). Bacterial concentration was determined in the first node (A) and in the second node (B).

Claims

1. A method for reducing, eliminating, or preventing infestation of plants or surfaces with pathogenic bacteria and for reducing or preventing bacterial wilt by applying to a plant, an environment of a plant, water, or soil, or to a surface, a composition comprising a proteinaceous extract from Basidiomycetes 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 ustale or a protein fraction or a biologically active protein thereof.

2. The method of claim 1 wherein the pathogenic bacterium is selected from one of the genera Ralstonia, Erwinia, Dickeya, Pectobacterium, Xanthomonas, Agrobacterium, Enterobacter and Escherichia.

3. The method of claim 2, wherein the pathogenic bacterium is selected from Ralstonia solanacearum, Ralstonia mannitolilytica, Agrobacterium tumefaciens, Escherichia coli, Dickeya chrysanthemi, Pectobacterium subsp. atrospecticum, Erwinia amylovora, Pectobacterium carotovorum subsp. carotovorum, Xanthomonas arboricola pv. pruni, Enterobacter sp. .

4. The method of one of the preceding claims, wherein the pathogenic bacterium is Ralstonia solanacearum, Ralstonia mannitolilytica, or Escherichia coli.

5. The method of one of the preceding claims, wherein the plant is crop, in particular a plant of family Solanaceae or Urticaceae.

6. 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. lycopersicum, S. tuberosum, S. melongena,

8. The method of claim 5, wherein the plant is Solanum dulcamara, Solanum nigrum, or Urtica dioica.

9. The method of one of the preceding claims, wherein the extract has been obtained from the fruiting bodies of Basidiomycetes and/or from Basidiomycetes mycelium.

10. The method of one of the preceding claims, wherein a protein fraction of the proteinaceous extract is used.

11. The method of one of the preceding claims, wherein the protein fraction comprises at least L-amino acid oxidase.

12. The method of one of the preceding claims, wherein the composition is applied to a hard surface contaminated with pathogenic bacteria for reducing or eliminating the bacteria or to an uncontaminated hard surface to prevent infestation.

13. The method of one of the preceding claims, wherein the composition is applied to soil and/or to irrigating water.

14. A method for obtaining a proteinaceous extract by harvesting fruiting bodies and/or mycelia of Basidiomycota, freezing the material at a temperature between -20°C and - 80°C, thawing the material, separating liquid and solid parts, and recovering the liquid as proteinaceous extract.

15. The method of claim 14, wherein the proteinaceous extract or the protein fraction is subjected to dialysis and/or a drying step.

16. The method of one of claims 14 or 15, wherein in a further step the proteinaceous extract is further purified to obtain a protein fraction.

17. Plant protection composition comprising a proteinaceous extract from fruiting bodies and/or mycelia of Basidiomycetes 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, and Tricholoma ustale or a protein fraction therefrom or a biologically active protein such as an L-amino acid oxidase therefrom, and optionally at least one agriculturally or horticulturally acceptable excipient, diluent, carrier, filler, extender and/or adjuvant.

18. The composition according to claim 17, wherein the extract or fraction comprises L- amino acid oxidase.

19. The composition according to claim 17 comprising L-amino acid oxidase isolated from Clitocybe geotropa or A. phalloides .

20. The composition according to claim 17 or 18, comprising one or more further biologically active agents such as a herbicide, pesticide, fungicide, plant growth agent, or fertilizer.

21. Proteinaceous Basidiomycetes extract obtainable by the method of one of claims 14 to 16.

22. The method of one of claims 1 to 1 1 wherein the composition to be applied is a composition according to one of claims 17 to 20.

23. Use of a composition of one of claims 17 to 21 for eliminating, reducing or preventing infestation with Ralstonia solanacearum.