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WO2015058944A1 - Composition et procédé utilisables en vue de la protection de végétaux - Google Patents

Composition et procédé utilisables en vue de la protection de végétaux Download PDF

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Publication number
WO2015058944A1
WO2015058944A1 PCT/EP2014/071216 EP2014071216W WO2015058944A1 WO 2015058944 A1 WO2015058944 A1 WO 2015058944A1 EP 2014071216 W EP2014071216 W EP 2014071216W WO 2015058944 A1 WO2015058944 A1 WO 2015058944A1
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Prior art keywords
extract
plant
plants
extracts
protein
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Inventor
Jana ERJAVEC
Tanja DREO
Jerica SABOTIC
Joze BRZIN
Janko Kos
Maja RAVNIKAR
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National Institute of Biology
Institut Jozef Stefan
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National Institute of Biology
Institut Jozef Stefan
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins

Definitions

  • 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.
  • the pathogen can also spread with infected tools, seeds, insects and root contacts.
  • R. solanacearum 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.
  • no agents are available for controlling plant diseases caused by these pathogenic bacteria.
  • 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).
  • Basidiomycetes extract composition can be useful for immune modulation as has been described in US 2006/0263384.
  • Baseidiomycetes 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.
  • Basidiomycetes 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • Basidiomycetes selected from Amanita phalloides, Amanita muscaria, Amanita virosa, Boletus luridiformis, Clitocybe geotropa, Gomphidius glutinosus, Trichol
  • the present invention provides a composition that is active against pathogenic bacteria and comprises an antibacterial active component.
  • 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.
  • Basidiomycetes selected from from Amanita phalloides, Amanita muscaria, Amanita virosa, Boletus luridiformis, Clitocybe geotropa, Gomphidius glutinosus, Tricholoma saponaceum, Hypholoma sp.,
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a protein fraction or an isolated protein component has the advantage that the content is better defined and is better compatible.
  • 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.
  • 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.
  • the protein containing extract, fraction or the dried powder preferably is frozen.
  • a temperature in the range of -20 to +4°C is useful.
  • a storage temperature in the range of -60 to -80°C is preferable to use.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Example 1 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
  • 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,
  • 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.
  • 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 ddH 2 0 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.
  • 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.
  • microwell plate assay was adopted.
  • Mixtures of 75 pl_ of YPG medium (see YPGA medium, only without added agar), 75 ⁇ 1_ of Ralstonia solanacearum suspension (10 7 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 ⁇ _ of YPG medium and 125 pl_ of 0.01 M PBS while control of extract sterility wells contained 75 ⁇ _ of YPG medium, 1 17.5 ⁇ . of 0.01 M PBS and 7.5 ⁇ _ 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 OD 595 measurements (Tecan Genios) in 24 hours. After 24 hours, 30 ⁇ _ 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
  • 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.
  • 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.
  • 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.
  • Entoloma rhodopolium no no Tapinellaceae
  • Hygrophorus eburneus no yes
  • Hydnum repandum no yes
  • Hygrophorus erubescens no no Geastrum rufescens no no
  • 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.
  • sterile needle lacogamma 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.
  • 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 nrf 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).
  • 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 ⁇ 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.
  • 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).
  • R. solanacearum and COX standard curves and NTCs (no template controls) were pipetted on each reaction plate.
  • 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.
  • 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 10 5 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.
  • variegatus and T. saponaceum were wilting, however number of completely wilted plants (grade 5) was significantly lower compared to that of positive control.
  • grade 5 number of completely wilted plants
  • 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.
  • 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.
  • AUDPC area under disease progress curve
  • 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.
  • 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.
  • Inoculation techniques may play a role when evaluating efficiency of plant protection agent.
  • bacterium and extract were mixed right before inoculation, some bacteria may have died before entering the plant.
  • 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.
  • 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.
  • 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.
  • more variation in bacterial concentration was observed in plants inoculated with R. solanacearum and mushroom extract, compared to positive control plants.
  • plants inoculated with C. geotropa, S. variegatus and T. saponaceum wilted slower and displayed milder symptoms compared to positive control plants.
  • 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.
  • Table 3 Activity of mushroom extracts after 24 hours tested against different bacteria (continued).
  • R. solanacearum strain NIB Z 30 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.
  • 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 £.
  • 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 were pooled, concentrated by ultrafiltration using molecular weight cut-off 10 kDa and dialyzed against 0.03 M BisT s, pH 6.5.
  • 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 ⁇ 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 ⁇ ! of 2M H 2 S0 4 , 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).
  • 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.
  • 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 10 5 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.
  • sterile needle laccogamma plus, 0.6mm x 25mm, Novico, Italy
  • 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.
  • 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).
  • Table 4 Comparison of relative AUDPC values 14 dpi between pathogenicity tests on tomato cv. .Moneymaker
  • 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.
  • 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 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
  • Figure 3D shows the results for a positive control.
  • Figure 4 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
  • 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.
  • 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 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.
  • LAO L-amino-acid oxidase
  • Ciitocybe geotropa lower panel
  • 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 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).

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Abstract

13 extraits de protéines de champignon et 1 extrait de protéines de mycélium présentant une activité antibactérienne contre R. solanacearum se sont montrés actifs dans le cadre d'essais. En outre, une fraction protéique de A. phalloides a également complètement inhibé la prolifération bactérienne. Lesdits extraits et fractions n'ont pas seulement démontré une forte inhibition de la multiplication bactérienne, mais ils ont, plus généralement, montré un effet bactéricide plutôt que bactériostatique. L'essai in vivo de cinq extraits sélectionnés sur des plants de tomate et de pomme de terre nous a amenés à la conclusion que les extraits de C. geotropa, S. variegatus et T. saponaceum permettent de faire baisser les risques de survenue de la maladie et retardent le flétrissement bactérien sur les plants de tomate comme sur les plants de pomme de terre. Ainsi, les extraits de protéines de champignon de la présente invention constituent un outil important de traitement du flétrissement bactérien provoqué par R. solanacearum. En outre, l'inhibition de 12 souches deR. solanacearum, ainsi que de R. mannitolilyticaet de E. coli par des extraits de protéines de champignon démontre leur activité à large spectre, qui pourrait se révéler intéressante dans le domaine de la médecine, de la biotechnologie, de la gestion des déchets/bioréhabilitation et de l'agriculture.
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CN110563823A (zh) * 2019-10-14 2019-12-13 中华全国供销合作总社昆明食用菌研究所 一种凝集活性较高的茶褐牛肝菌凝集素的制备方法
CN113564070A (zh) * 2021-07-16 2021-10-29 江苏稼润农业开发有限公司 食用菌生防酵素发酵菌系、发酵方法及在水稻苗期抗病的应用
CN114868748A (zh) * 2020-10-29 2022-08-09 湖南艾布鲁环保科技股份有限公司 一种抑制农作物重金属污染的叶面阻控剂及其制备方法、重金属污染稻田的治理方法

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CN110563823A (zh) * 2019-10-14 2019-12-13 中华全国供销合作总社昆明食用菌研究所 一种凝集活性较高的茶褐牛肝菌凝集素的制备方法
CN114868748A (zh) * 2020-10-29 2022-08-09 湖南艾布鲁环保科技股份有限公司 一种抑制农作物重金属污染的叶面阻控剂及其制备方法、重金属污染稻田的治理方法
CN113564070A (zh) * 2021-07-16 2021-10-29 江苏稼润农业开发有限公司 食用菌生防酵素发酵菌系、发酵方法及在水稻苗期抗病的应用
CN113564070B (zh) * 2021-07-16 2023-11-28 江苏稼润农业开发有限公司 食用菌生防酵素发酵菌系、发酵方法及在水稻苗期抗病的应用

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