Methods for the prevention of infections in crops and ornamental plants, particularly in viticulture, and woody plants

Description

[0001] The invention relates to a method for the prophylaxis of fungal infections, in particular oomycetes, as well as bacterial infections in crops and ornamental plants. Areas of application include vegetable, fruit, viticulture, and ornamental plant cultivation, preferably viticulture and vegetable cultivation. Significance, course and control of infections caused by oomycetes

[0002] Plant diseases cause significant annual economic losses in crops, ornamental plants, and woody plants. Fungi and oomycetes, such as Phytophthora, Pythium, and Peronospora, also play a significant role in the hydroponic cultivation of vegetables and ornamental plants in greenhouse cultures (Malathrakis & Goumas, 1999; Paulitz & Berlanger, 2001). They are of particular economic importance in vegetables (especially potatoes and tomatoes), fruit, ornamental plant, and viticulture, as well as in forestry. In 2013, potatoes were cultivated on 19.3 million hectares of agricultural land worldwide (Food and Agriculture Organization of the United Nations, Statistics Division). The most important pathogen in potato cultivation, whose importance has increased with the expansion of cultivation to warmer climates, is the oomycete Phytophthora infestans, the pathogen that causes late blight (Oerke and Steiner, 1996).Its spread can only be controlled through continuous use of fungicides (more than US$235 million annually for potato cultivation alone). The total market for fungicides alone amounts to US$5.5 billion per year (Powell & Jutsum, 1993).

[0003] In Germany, approximately 100 million euros are spent annually on plant protection in viticulture alone (Ochßner, 2009). In organic viticulture, only copper-containing pesticides are used, which, however, are environmentally hazardous and potentially toxic. For this reason, it is of great interest to establish alternative, improved pesticides that are effective against pathogens and, at the same time, environmentally friendly.

[0004] The oomycete cycle is illustrated here using the example of Plasmopara viticola, the downy mildew of grapevine. It is divided into two phases of differing epidemiological significance. In the sexual phase, the oospore is formed, which serves to overwinter the pathogen. In the asexual summer cycle, large quantities of sporangia are released. Plasmopara viticola survives the winter as oospores on the soil in the remains of heavily infected leaves. The oospores become ready to germinate during late winter and retain their viability until early summer. As soon as the soil warms and sufficient precipitation has occurred, they germinate and form primary sporangia. Oospores can germinate repeatedly until mid-June during heavy rain. Some oospores may remain dormant for over a year and not germinate until the following year.Germination and the release of zoospores from the primary sporangium usually occur when temperatures rise above 10 °C and more than 8 mm of rainfall have fallen. Under these conditions, the first leaflets of the grapevine are usually fully unfolded, allowing primary infection to occur. For primary infection by the germinated zoospores to occur, the leaves must be sufficiently moistened with water. Only during this phase can the infection be prevented or reduced if the zoospores can be damaged or inhibited.

[0005] The primary infection is the starting point of the summer cycle of Plasmopara viticola, during which the pathogen reproduces asexually with sporangia and, under favorable reproduction conditions, can cause epidemics. The primary infection is followed by the incubation period, during which the pathogen develops inside the leaf without any visible symptoms. Treatment of the infection is no longer possible at this point. The growth and development of the pathogen are highly dependent on temperature; therefore, at higher temperatures, the tissue is penetrated more quickly by the mycelium and the oil spots appear earlier than at lower temperatures. At the end of the incubation period, so-called oil spots appear as a visible sign of the fungal infection. As soon as the relative humidity rises above 95% at night and temperatures exceed 12 °C, the sporangia emerge from the stomata of the infected leaf surface.The sporangia are spread by air movement or water droplets. If they come into contact with a droplet of water on a green organ of their host plant, the zoospores hatch. Hatching of the zoospores and subsequent infection occurs within four hours under optimal conditions at 24°C. At lower or higher temperatures, the zoospores hatch more slowly and the infection process is prolonged. Plasmopara viticola can infect leaves, inflorescences including the stem framework, berries, and shoot tips if these have stomata and are moistened. Even small drops of water are sufficient for infection, but infection conditions are more favorable if the moistening with water covers a large area and lasts for a long time. Each infection is followed by an incubation period, followed by the eruption of the sporangia as soon as sufficient humidity prevails at night.Plasmopara viticola is a polycyclic pathogen and can undergo several development cycles during a growing season. If optimal conditions for the outbreak of sporangia and for infections prevail over an extended period and the incubation periods are short due to the temperature conditions, an epidemic can develop very quickly. Drought delays the spread of Plasmopara viticola and inhibits the course of epidemics. It is possible to predict phases of high infection risk locally and thus implement targeted prophylactic protective measures. Under the climatic conditions prevailing in Central Europe, infections by such pathogens can be expected every year. The extent to which these infections lead to epidemics depends greatly on the course of the annual weather and cannot be foreseen at the beginning of the growing season. Epidemics of, for example,Grape downy mildew (Plasmopara viticola) can become very severe in highly susceptible classic grape varieties within just a few days of rain. Therefore, this infection must be detected and controlled at an early stage. Subsequent control is no longer possible once the infestation has already progressed. For this reason, profitable crop production is only possible with preventative measures against such infections. A forecasting method for targeted, preventative control of Plasmopara viticola has already been developed and put into practice at the State Viticulture Institute. Numerous fungicides are currently available for conventional crop production. In viticulture alone, 29 fungicides are currently approved for use against grape downy mildew.

[0006] Grape downy mildew poses a challenge for organic viticulture, as preventative treatment is essential, and currently only copper-containing products (e.g. Cuprozin) are approved. Due to the known ecotoxicological concerns regarding copper, it is urgently necessary to find alternatives to this active ingredient. However, these alternatives must be sufficiently effective even under high infestation levels. For years, trials have shown that the vast majority of products listed as plant strengtheners do not have a satisfactory effect against grape downy mildew. Some plant strengtheners are effective against grape downy mildew at low infestation levels, but in these cases, control measures would not have been necessary. At higher infestation levels, which also justify control on economic grounds, the effectiveness of the tested products was insufficient.These experiments demonstrate that biological control of grapevine downy mildew is currently not practical in organic farming. Especially in organic viticulture, with the limited ability to stop an epidemic, effective and practical concepts for biological control of epidemics are urgently needed. Significance, course and control of bacterial infections

[0007] Although the number of plant pathogenic bacteria is smaller than the number of fungal pathogens, the damage caused to crops by bacterial diseases is very high. Bacteria of the genus Xanthomonas cause diseases in all major groups of higher plants worldwide, resulting in chlorotic and necrotic lesions, wilt and rot. One example of high economic importance is black rot in cabbage varieties, which is caused by Xanthomonas campestris pv. campestris. When Xanthomonas oryzae pv. oryzae infects rice plants, it causes white leaf blotch, which is one of the most serious rice diseases and subsequently leads to major economic and social problems. Also worth mentioning are the pathovar X. axonopodis pv. citri, the causative agent of citrus canker, and X. campestris pv.Vesicatoria, the causative agent of bacterial spot disease in peppers and tomatoes, which is of economic importance especially in regions with warm and humid climates.

[0008] Another important pathogen is fire blight, caused by the notifiable pathogen Erwinia amylovora, whose host plants are Rosaceae, such as apples, pears, and quinces. E. amylovora causes the wilting of leaves and flowers of affected plants, which then turn brown or black. Another bacterial species is Pseudomonas syringae, which causes various plant diseases such as canker, wilt, and spotting in important crops such as tomatoes, peppers, and soybeans. This widespread species is of great importance in many greenhouse crops, such as tomatoes, cucumbers, and zucchini.

[0009] Most of the described bacterial plant pathogens belong to the group of Proteobacteria and are gram-negative organisms (e.g., Pseudomonas, Xanthomonas). However, there are also economically relevant gram-positive pathogens, such as Clavibacter michiganensis ssp. michiganensis, which causes bacterial tomato wilt. This quarantine pest is of great importance in warmer and drier tomato-growing regions and in greenhouses.

[0010] Plant pathogenic bacteria use various strategies to survive in the environment, for example in the soil, in plant material such as seeds, or in insects. Insects, other animals, and humans play an important role in their spread. Water, e.g. in the form of raindrops, is an important transport vector for distribution on a plant. If bacteria are transferred to a host plant, they can penetrate the plant through natural openings such as stomata or hydathodes, or through injuries. A high bacterial density, as well as external conditions such as rain, high humidity, or damaged areas on plant surfaces, facilitate the infection of a plant. Bacteria can multiply well inside the plant; they colonize the apoplast and from there damage the entire plant.They disrupt the physiology and morphology of plants and thus trigger disease symptoms such as necrotic spots, defoliation, scab formation, wilting or rot (De la Fuente and Burdman, 2011 ).

[0011] It is therefore absolutely necessary to protect crops from such bacterial infections and thereby secure their harvest. The current list of plant protection products with antibacterial effects approved in Germany includes various chemical compounds and copper compounds. Copper-containing preparations are the only agents that may be used in organic farming. Treatments with copper-containing preparations to control bacterioses are only partially effective and reach their limits once the density of the bacterial inoculum exceeds a certain threshold. Due to the known ecotoxicological effects of copper compounds and other agrochemicals, there are legitimate concerns about the use of such plant protection products. Furthermore, even in Germany, the use of plant protection products containing antibiotics such as streptomycin to control fire blight is permitted in exceptional cases.Streptomycin is a permitted treatment for bacterial infections in other countries, but at the same time, its use is highly questionable, as indiscriminate use of antibiotics can lead to undesirable environmental impacts and a loss of efficacy due to bacterial resistance. It is therefore urgently necessary to develop improved, highly effective, and economically relevant alternatives to these active substances that are more environmentally friendly and better tolerated by consumers. Current development of plant protection strategies

[0012] 1. Chemical plant protection products are subject to ever-increasing demands regarding efficacy, selectivity, specificity, biodegradability, and effects on non-target organisms. A number of innovative plant protection products are now available that meet these requirements. The use of numerous older compounds, such as chlorinated hydrocarbons (aldrin, DDT, DDD, dieldrin, kelthane), is now prohibited. However, currently used chemical plant protection products (e.g., ortho-phenylphenol E 231 or thiabendazole E 233) are also increasingly being criticized. They exhibit numerous harmful side effects that make their use problematic.These include damage to the crop, changes in fruit flavor, toxic effects on numerous beneficial organisms, the development of resistant pest populations, incomplete degradation by microorganisms and thus excessive persistence and accumulation in the soil, as well as leaching into groundwater and accumulation in the human and animal food chain. (Source: Environmental Encyclopedia - www.umweltlexikon-online.de). 2. Biological and integrated pest management methods are becoming increasingly established, such as the use of beneficial organisms and pheromones against insects, the use of soil-borne bacterial and fungal antagonists, and the use of plant extracts. Among the most important antagonistic organism genera are Bacillus, Pseudomonas, and Streptomyces among the bacteria, and Trichoderma, Coniothyrium, and Verticillium among the fungi.Of particular importance in this context are the bacterium Bacillus subtilis, which secretes phytosanitary-effective metabolites as a "plant growth promoting rhizobacterium" (PGPR), and the fungal genus Trichoderma, whose strains are used as a "biocontrol agent" (Kücük, C. and M. Kivanc, 2002; DeMarco, JL, et al., 2003). While many animal pests can be adequately controlled by these biological methods, infections caused by oomycetes are difficult to combat. In the agricultural sector, the following plant diseases are of outstanding importance due to their infectious nature and the resulting losses (Table 1): . State of the art

[0013] It is known that glycoside-cleaving enzyme preparations of the non-starch polysaccharide hydrolase type are effective in the prophylaxis and treatment of plant-pathogenic fungi. This is based on a direct attack of the enzyme on the cell wall structures of the fungi, especially oomycetes (DE 10 2205 048 520, Biopract GmbH). However, these hydrolases can also damage the plant cell wall and are therefore only of limited use for plant protection. Dunne et al. (Microbiology (2000), 146, 2069-2078) shows that the bacterial strain Stenotrophomonas maltophilia W81 produces a serine protease that has fungicidal activity in vitro.

[0014] The use of enzymes of the non-starch polysaccharide hydrolase type for the prophylaxis and treatment of fungal phytopathogens is supported by a number of findings in other areas. For example, there is experience in the control of oomycete-based fish mycoses with complex enzyme preparations from Trichoderma spp.(WO 2004 / 002574 A1 Biopract GmbH).

[0015] United States Patent 6663860 (Tvedten, December 16, 2003) describes proteases as a pesticide against insects, bacteria, and fungi, among other things. However, their use in the prevention of fungal infections in viticulture is not envisaged.

[0016] Furthermore, various patents describe the combination of a pesticide and an enzyme or protease, although the described effect here is more likely due to the pesticide itself than to the added enzyme alone (WO 2013 / 096383 A2, CN 103461383 A, WO 1997 / 047202 A1, WO 1990 / 003732 A1). Other patents describe the combination of detergents and enzymes (US 7393528 B2), plant extracts and proteins (WO 2001 / 030161 A1), and a surfactant and an enzyme (EP 184288 A1). These publications also do not indicate that the enzyme itself is responsible for the pesticidal effect.

[0017] Finally, enzymes or enzyme combinations have been described in the past that exhibit, among other things, antifungal or antibacterial effects, such as a protease from plants (WO 1991 / 002459 A1), a protease from an earthworm (JP 2011177105A) or the culture supernatant of a Bacillus fermentation (JP 54073182 A).

[0018] None of the above-mentioned inventions describes a comparably efficient solution to the persistent problem of crop infestation by oomycetes and bacteria. The proteases described here thus represent, for the first time, a highly effective and simultaneously ecologically acceptable alternative to common pesticides. Aim of the invention

[0019] The invention aims to develop a highly effective agent for controlling infections caused by fungi, especially oomycetes, and bacterial infections in crops and ornamental plants that is safe for the plant itself and the ecosystem. The invention is based on the task of developing a method for preventing infections of crops in agriculture caused by phytopathogenic pathogens. In particular, the objective is to detect and prevent epidemics such as grapevine downy mildew in wine varieties at an early stage. The provision of suitable agents is also encompassed by the task of the invention.

[0020] This object is achieved by the measures described in the claims. The method according to the invention is characterized in that a concentrate or a ready-to-use dilution containing a protease is produced. The core of the invention is the surprising possibility of being able to provide an effective agent for controlling infections in crops and ornamental plants using proteases alone. Furthermore, the protectant can contain stabilizers, adhesion promoters, and spreading agents that improve application properties. Common rain and UV stabilizers can also be included in these mixtures. This mixture is applied to the cultivation area using conventional distribution systems at set times, which are determined based on the weather, in such a way that the entire plant is wetted. Application can take place at temperatures between 4 and 34°C and thus during the entire growing period.In greenhouse cultivation, application is largely independent of weather conditions, and temperatures range between 15°C and 25°C. This type of application ensures that the enzyme preparations are active and prevent infection of the plant by, for example, the zoospores of phytopathogenic oomycetes or by bacterial pathogens such as... Pseudomonas syringae, is prevented. The application rate per area must be determined depending on the crop. Currently, for example, in viticulture, approximately 400–800 liters of spray solution are used for one hectare. The enzyme preparations described are blended in such a way that conventional spraying techniques can continue to be used.

[0021] A first aspect of the invention relates to a method for the prophylaxis of infections in crop and ornamental plants, characterized in that the above-ground parts of the plant are sprayed with an aqueous solution of a bacterial serine protease, and characterized in that the bacterial serine protease is Bacillus sp. or Nocardiopsis sp. comes from.

[0022] In certain embodiments, the method is characterized in that the bacterial serine protease from Nocardiopsis sp. comes from.

[0023] In certain embodiments, the method is characterized in that the aqueous solution contains only the bacterial serine protease.

[0024] In certain embodiments, the method is characterized in that the aqueous solution contains a combination of bacterial serine proteases alone.

[0025] In certain embodiments, the method is characterized in that the infections are caused by oomycetes.

[0026] In certain embodiments, the method is characterized in that the oomycetes are Plasmopara viticola, Phytophthora infestans or Pseudoperonospora cubensis is.

[0027] In certain embodiments, the method is characterized in that the infections are caused by bacteria.

[0028] In certain embodiments, the method is characterized in that the bacteria are Pseudomonas syringae or Clavibacter michiganensis is.

[0029] In certain embodiments, the method is characterized in that the aqueous solution of a bacterial serine protease is applied in a dose of 0.001% - 1% when combating pathogens.

[0030] In certain embodiments, the method is characterized in that a temporal interval treatment of the plants is carried out.

[0031] In certain embodiments, the method is characterized in that the aqueous solution of a bacterial serine protease is used at pH values of 4.0 - 8.0.

[0032] In certain embodiments, the method is characterized in that the aqueous solution of a bacterial serine protease is used at temperatures of 4 to 34 °C.

[0033] In certain embodiments, the method is characterized in that the aqueous solution of a bacterial serine protease is formulated with adhesives, wetting agents and stabilizers.

[0034] The described invention represents a significant advance over currently established means and methods.

[0035] The advantages over the state of the art are presented here: In contrast to copper preparations or other chemical plant protection products, the use of enzyme preparations is harmless to the ecosystem because the active ingredient is completely degraded in the soil, rather than accumulating. This prevents significant environmental impacts. No phytotoxic reactions occur because the proteases used in the invention do not attack plant surfaces. Proteases and other enzymes remain effective throughout plant growth. They do not adhere to one spot on the leaf structure, but rather spread across the surface in a liquid film. The effectiveness of the enzymes is maintained over a relevant period of several days, despite rain and UV radiation. This stability can be improved, if necessary, through suitable formulations.

[0036] Proteases, also called peptidases, cleave peptide bonds in proteins, thus promoting their degradation into peptides or amino acids. Proteases are divided into the following groups based on their mode of action: serine proteases (EC 3.4.21.-, S), cysteine proteases (C), aspartic acid proteases (A), metalloproteases (M), and unknown or previously unclassified proteases (Handbook of Proteolysis Enzymes, AJ Barrett, ND Rawlings, JF Woessner (eds), Academic Press (1998)).

[0037] Proteases used in the context of the described invention are serine proteases. The catalytic mechanism of this enzyme class is based on the nucleophilic hydroxyl group of the amino acid serine, which can cleave peptide bonds. Corresponding enzymes are isolated from culture supernatants of microorganisms of the genera Nocardiopsis or BacillusThe corresponding enzymes can also be produced recombinantly. Furthermore, the effective proteases can also be mutations, variants, or fragments of the described enzymes that act analogously.

[0038] Protease activity can be determined using any assay that uses a substrate containing the corresponding peptide bonds (e.g. casein).

[0039] Surprisingly, it was discovered that protease preparations, used in animal feed, for example, prevent the infection of plants by phytopathogenic oomycetes and bacteria. In particular, the zoospores occurring in the oomycete cycle, which are responsible for the actual infection of the leaf tissue, are irreversibly damaged by the action of these enzymes, preventing infection of the thus protected plant. The mechanism of action against bacterial infection has not yet been elucidated. The significant effect of these enzymes was not expected to this extent, as their mechanisms of action and targets do not correspond to the described mechanism of action of β-glucanases or chitinases.

[0040] Commercially available preparations containing the described proteases are, for example, Ronozyme ®< ProAct ®< (DSM Nutritional Products AG, Application Examples 1-9: Prot III), in which a serine protease of Nocardiopsis sp. or Alcalase ® (Novozymes AG), which primarily contains a serine protease, subtilisin A, from Bacillus licheniformis. Furthermore, selected protease preparations from Lumis Enzymes (PAP 2XS), which is known to contain papain from papaya, from Dyadic (Protease Plus, Protease AP Conc), and from AB Enzymes (BlOTOUCH ROC 250LC), which is known to contain a protease from Trichoderma, demonstrated protective effects.

[0041] Following the characteristics of the specific disease, enzyme application is carried out against leaf pathogens (e.g. downy mildew or Pseudomonas syringae)by treating the above-ground parts of the plant (e.g. by spraying) with concentrations of an enzyme preparation of 0.001% - 1%.

[0042] The effect of the enzyme preparations according to the invention, which is manifested in the prevention of the infection process, is achieved by using the proteases individually or as mixtures.

[0043] The enzymes are obtained from culture supernatants of microorganisms. The components are preferably used in an aqueous environment ranging from pH 4.5 to 8.5, preferably at pH 6.0 to 7.5, to inactivate the pathogens. They are used at a water temperature of 4°C to 34°C, preferably between 10°C and 25°C.

[0044] The invention will be further illustrated by examples below. Examples 1-7 deal with the use of proteases to protect against oomycetes, while Examples 8 and 9 describe the protective effect against bacterial infections. Ronozyme® ProAct® (DSM Nutritional Products AG) was used as protease III. Examples Protection against infection of crops by oomycetes Example 1 Infection-suppressing effect of selective foliar application with protein-splitting enzyme preparations against Plasmopara viticola on leaf discs

[0045] Leaf discs of the grapevine Vitis vinifera cv. Müller-Thurgau were treated once by spraying with different protein-cleaving enzyme preparations (protease I, II, and III) so that the underside of the leaf discs used was evenly wetted. The preparations each contained a serine protease, which was either produced by a species of the genus Nocardiopsis or BacillusThe enzyme preparations used for treatment were tested in a concentration range of 0.01% to 1% (v / v). The pH of the preparations diluted in water was between 6 and 7.5. As a control, the leaf discs were sprayed either with a copper-containing pesticide or with water. 24 hours after treatment, the leaf discs were artificially infected with Plasmopara viticola (approximately 40,000 spores per ml of water), the pathogen that causes downy mildew on grapevines. The leaf discs were then incubated on water agar plates at 22°C for six days in a plant chamber with a day-night rhythm.

[0046] The infestation severity is calculated from the ratio between the total leaf area and the infected leaf area. Image analysis software was used for the evaluation, which differentiates between the total area (number of green pixels on the leaf discs at the beginning of the experiment) and the infected area (number of white pixels at the end of the experiment). Two of the tested protease preparations (I, III) as well as the copper-containing pesticide (Cuprozin) prevented the infestation and the development of Plasmopara viticola effective (0% infestation), the third preparation, Protease II, only partially prevented the infestation (38%). Leaf discs sprayed with water, however, showed a significant infestation (see Table 2 and Figure 1 ). Table 2: Infection intensity (%) calculated from the ratio of the total area of the leaf disc to the infected area per leaf disc after treatment of leaf discs with three different protease preparations (n=36) Infestation intensity (MW) Standard deviation H2O 87% 6% Cuprozin 0% 1% Protease I 0% 1% Protease II 38% 28% Protease III 0% 0% Example 2 Infection-suppressing effect of selective foliar application with combinations of proteases, chitinases and glycoside-cleaving enzyme preparations against Plasmopara viticola on leaf discs

[0047] Leaf discs of the grapevine Vitis vinifera cv. Müller-Thurgau were treated once by spray application with an enzyme combination of protease, chitinase, and β-glucanase in a 1:1:1 ratio, ensuring that the underside of the leaf discs was evenly wetted. The concentration of the enzyme preparations used was 0.1% (v / v) in each case. The pH of the preparations diluted in water was between 6 and 7.5. As a control, the leaf discs were sprayed either with a copper-containing pesticide or with water. 24 hours after treatment, the leaf discs were artificially infected with Plasmopara viticola (approximately 40,000 spores per ml of water), the pathogen that causes downy mildew on grapevines. The leaf discs were then incubated on water agar plates at 22 °C for six days in a plant chamber with a day-night rhythm.

[0048] The infestation severity is calculated from the ratio between the total leaf area and the infected leaf area. Image analysis software was used for the evaluation, which differentiates between the total area (number of green pixels of the leaf discs at the start of the experiment) and the infected area (number of white pixels). Plasmopara viticola on the leaf discs treated with enzymes and copper-containing pesticides was effectively prevented. Example 3 Infection-suppressing effect of selective foliar application with protein-splitting enzyme preparations against Plasmopara viticola on greenhouse plants

[0049] Young vines of the variety Vitis viniferacv. Müller-Thurgau were treated once with a protein-cleaving enzyme preparation (Protease III) using a stationary application unit. The concentrations of the enzyme preparation used were 0.1, 0.2, and 0.5% (v / v). The pH values of the spray mixtures were adjusted between 6.5 and 7.5. As a control, additional potted vines were sprayed either with a copper-containing pesticide or with water. 24 hours after treatment with the protease preparation, the leaves were artificially infected with Plasmopara viticola, the pathogen that causes downy mildew on grapevines.

[0050] The plants were then incubated for one week in a greenhouse at 20°C. Infestation was determined by visually recording the percentage (%) of diseased or necrotic lesions on leaves / stems relative to the total mass of a plant per replicate (100%) and documented photographically. A rating scale with gradations of 1, 5, 10, 15, 20, 25, 30, 40, 50, 90, and 100% diseased lesions was used.

[0051] The development and spread of Plasmopara viticola on the leaves treated with Protease III and copper-containing pesticides was effectively prevented, while the leaves sprayed with water showed a high infestation ( Fig. 2 ). Example 4 Infection-suppressing effect of periodic application of protein-splitting enzyme preparations in field trials against Plasmopara viticola

[0052] Whole vines of the Vitis vinifera cv. Blauer Spätburgunder variety were repeatedly treated with a protein-splitting enzyme preparation (Prot III) at intervals of 8 to 14 days throughout the season using a tunnel sprayer, ensuring even coverage of the vine surface. The concentration of the enzyme preparation used was 0.1% (v / v). The pH values of the spray solutions were adjusted between 6.5 and 7.5. A wetting agent (TREND 90) was added to the spray solution to improve leaf wetting.

[0053] At the end of the season, the severity and frequency of downy mildew infestation on leaves and grapes was assessed. The development of Plasmopara viticola on the vines in the open field was effectively prevented. Example 5 Protective effect of protein-splitting enzyme preparations against the pathogen of late blight and brown rot (Phvtophtora infestans) on tomato plants

[0054] Red Robin tomatoes were sprayed at the 4-leaf stage with the protease preparation (Protease III, 0.1% (v / v). The pH values of the spray solutions were adjusted between 6.5 and 7.5. As additional variants, common wetting agents (T / S forte, Biomaxima, Nufilm) were added to the protease solution at a concentration of 0.02% (v / v). The commercial copper preparation Atempo and water served as internal controls. Five replicates, each with one plant, were prepared for each variant.

[0055] 24 hours after enzyme application, artificial inoculation with the pathogen was carried out Phytophthora infestanswith a sporangia concentration of 80,000 spores per ml. 6 ml of suspension was used per plant. The plants were placed in the incubator at approximately 16 °C and 100% relative humidity without lighting. After 24 hours, a lighting cycle of 16:8 hours was set, and the humidifier was turned off. Assessment took place 6 days after infection. Infection was determined by visually recording the percentage (%) of diseased or necrotic changes on leaves and stems relative to the total mass of a plant and documented photographically (Table 3). Table 3: Infestation by Phytophthora infestans and effectiveness of the protease preparation Nr. variant Infestation [%] Mean (Standard Deviation) Efficiency [%] 1 Protease III 12,00 (2,74) 87,50 2 Protease III + T / S-Forte 18,00 (2,74) 81,25 3 Protease III + BioMaxima 11,00 (5,48) 88,54 4 Protease III + Nufilm P 12,00 (2,74) 87,50 5 Atempo (copper reference agent) 1,80 (1,79) 98,13 6 Water control 96,00 (5,48) 00,0

[0056] Plants treated with Protease III showed stable protection with an efficacy of 81-87% relative to a common copper standard. Formulation excipients such as TS-Forte, BioMaxima, and NufilmP did not increase efficacy compared to variant 1. Example 6 Protective effect of protein-splitting enzyme preparations against Pseudooeronospora cubensis on cucumber plants

[0057] Cucumber plants were grown in a climate chamber. To demonstrate the protective effect of proteases, approximately 6 ml of the protease preparation (Prot III), which was available in an aqueous solution with a concentration of 0.1%, was sprayed onto the undersides of the leaves. The pH of the spray solutions was adjusted between 6.5 and 7.5. A common copper preparation (Cuprozin Progress) and water served as controls. Six replicates of each treatment, each with one plant, were prepared. One day after enzyme application, the test plants were infected with Pseudoperonospora cubensis (75,000 spores per ml). Incubation took place at room temperature in a greenhouse with a relative humidity of more than 95%. The plants were incubated in the dark for the first 48 hours, after which they were maintained in a day / night cycle of 16 / 8 hours. Assessment took place 10 days after infection. The percentage of infestation of the plants was determined.By using the preparation Prot III, the infestation was reduced to less than 4% (Table 4). Table 4: Infestation by Pseudoperonospora cubensis in % and efficiency of the protease preparation Nr. variant Infestation [%] Mean (Standard Deviation) Efficiency [%] 1 Protease III 3,6 (0,4) 94,1 5 Cuprozin Progress (copper standard) 9,00 (4,7) 85,2 6 Water 60,4 (11,9) 00,0 Example 7 Comparison of the regulatory effect of different protease preparations against Pseudoperonospora cubensis on cucumber plants

[0058] Cucumber plants were grown in a climate chamber. To compare the effectiveness of different protease preparations, approximately 6 ml of the respective protease preparation (Prot III - Prot IX), which was available in an aqueous solution with a concentration of 0.1%, was sprayed onto the undersides of the leaves. The pH of the spray mixtures was adjusted between 6.5 and 7.5. A common copper preparation (Cuprozin Progress) and water served as controls. Six replicates of each treatment, each with one plant, were prepared. One day after enzyme application, the test plants were infected with Pseudoperonospora cubensis. Incubation took place at room temperature in a greenhouse with a relative humidity of more than 95%. The plants were incubated in the dark for the first 48 hours, after which they were maintained on a day / night cycle of 16 / 8 hours. Assessment took place 10 days after infection.The percentage of plant infestation was determined. The effectiveness of the individual preparations is shown in Table 5. The preparation with the best effect was protease III. Proteases IV, VIII, and IX have a comparable effect. Table 5 shows the organism of origin of each protease, as far as known. Table 5: Overview of the protease preparations used in the experiment, as well as the infestation and the corresponding efficiencies. Sample ID origin product Infestation [%] Mean (Standard Deviation) Efficiency [%] Protocol III Nocaridiopsis Ronoyzme ProAct 0,4 (0,4) 98,6 Protocol IV Bacillus Alcalase 0,5 (0,4) 98,3 Protocol V Bacillus Savinase 9,8 (4,8) 65,8 Protocol VI Asperigillus Flavourzyme 35,0 (21,5) -- Protocol VIII papaya PAP 2XS 0,8 (0,3) 97,2 Protocol IX Bacillus Protease AP Conc 1,0 (0,6) 98,5 Copper fungicide - Cuprozin Progress 7,4 (6,2) 74 Protection against bacterial infection of crops Example 8 Plate test to detect the growth-inhibiting effect of protease against Clavibacter michiganensis

[0059] A culture of Clavibacter michiganensiswas grown to late log phase and diluted accordingly to OD6oonm = 1.0 in a 10mM NaCl solution. This starting culture was applied in 12 dilutions of 10 -1< to 10 -12< to nutrient agar plates containing the preparation Protease III in concentrations of 0.01-1%. Two control plates were free of Protease III and showed the maximum growth of Clavibacter michiganensis under the given conditions ( Fig. 3 , left: 10 -5< ). From a protease concentration of 0.05%, bacterial growth was significantly inhibited, as only the most concentrated dilutions grew (from 10 -1< to 10 -3< , see Fig. 3 ). The great potential of protease III as a pesticide for controlling bacterial tomato wilt ( Clavibacter michiganensis subsp. michiganensis ) becomes clear through this experiment. Example 9 Protecting tomato plants from Pseudomonas syringae infection

[0060] For this experiment, tomato plants of the "Red Robin" variety were sprayed with a 0.1% protease solution (Protease III) with and without the addition of an adhesive (NufilmP) and incubated at 22°C for 24 hours. The pH of the spray solutions was adjusted to 6.5 to 7.5. Four plants each were sprayed with tap water and a reference agent as controls. Twenty-four hours after the enzyme application, the plants were infected with Pseudomonas syringae.

[0061] The first samples of 0.7 cm 2 leaf segments were taken two hours after inoculation, with further samples taken 7, 14, and 21 days after inoculation. Four leaf segments from four individual leaves of four plants were analyzed. The number of colony-forming units (CFU) per leaf segment was 1 x 10 3 at the start of the experiment. In the control (water), the number of CFU increased within three weeks to approximately 1 x 10 6 CFU per leaf segment. Plants treated with protease III maintained the level from the start of the experiment (10 3 CFU per leaf) during the first two weeks. After three weeks, the number of CFU per leaf on the protease-treated leaves had decreased significantly to 10 CFU per leaf segment ( Fig. 4 The protease used was significantly more effective than the reference substance. Literature:

[0062] DE LA FUENTE, L. and BURDMAN, S. 2011. Pathogenic and beneficial plantassociated bacteria. In Agricultural Sciences, [Ed.Rattan Lal], in Encyclopedia of Life Support Systems (EOLSS), Developed under the Auspices of the UNESCO, Eolss Publishers, Oxford ,UK, [http: / / www.eolss.net] DE MARCO, JL; VALADARES-INGLIS, MC and CR. FELIX, 2003: Production of hydrolytic enzymes by Trichoderma isolates with antagonistic activity against Crinipellis perniciosa, the causal agent of witches' broom of cocoa. Brazilian J. Microbiol. 34, 33 - 38 KASSEMEYER H.-H. (2004) Research project for the program of the Federal Ministry of Consumer Protection, Food and Agriculture to promote research and development projects as well as technology and knowledge transfer in organic farming. "Innovations to improve the framework conditions for organic viticulture.Development of scientific approaches for the biological control of grapevine downy mildew and for strategies for its regulation in organic viticulture", project number 020E269, State Viticulture Institute Freiburg KUDO, S. and C. TESHIMA, 1991: Enzyme activities and antifungal action of fertilization envelope extract from fish eggs. The Journal of Experimental Zoology 259, 392 - 398 KUDO, S., 1992: Enzymatic basis for protection of fish embryos by the fertilization envelope. Experientia 48, 277 - 281 KUDO, S., 2000: Enzymes responsible for the bactericidal effect in extracts of vitelline and fertilization envelopes of rainbow traut eggs. Zygote 8, 257 - 265 KÜCÜK, C. and M. KIVANC 2002: Isolation of Trichoderma spp. and determination of their antifungal, biochemical and physiological features. Turk. J. Biol. 27, 247-253 MALATHRAKIS, NE, GOUMAS, DE 1999: Fungal and bacterial diseases. See Ref. 4 pp. 34-47. MÜNCH, S., NEUHAUS, JM, BOLLER, T., KEMMERLING, B. and KHKOGEL 1997: Expression of β-1,3-glucanase and chitinase in healthy, stem rust-affected and elicitor-treated near-isogenic wheat lines showing Sr5 or Sr24-specific rust resistance. Planta 201 , 235-244 OERKE, E.CH. and U. STEINER 1996: Yield losses and plant protection: The cultivation situation for the economically most important crops. Series of the German Phytomedical Society, Eugen Ulmer GmbH & Co., Stuttgart PAULITZ, TC BELANGER, RR 2001: Biological control in greenhouse systems. Annu. Rev. Phytopathol. 39, 103-133 POWELL, KA, JUTSUM, AR (1993) Technical and commercial aspects of biocontrol products. Pestic. Sei. 37, 315-321. SCALA F., SL WOO, I. GARCIA, A. ZOINA, E. FILIPPONE, J.-A. PINTOR-TORO, G. DEL SORBO, B. ALOJ and M. LORITO. 1998. Transgenic tobaeco and potato plants expressing antifungal genes from Trichoderma are resistant to several plant pathogenic fungi.7th International Congress of Plant Pathology, August 9-16, 1998, Edinburgh, Scotland, Offered Papers Abstracts - Volume 3: 5.3.10. WO 2004 / 002574 A1 Biopract GmbH, Berlin; LEIBNIZ Institute of Freshwater Ecology and Inland Fisheries in the Research Association Berlin eV Methods for the prophylaxis and therapy of mycoses in fish and invertebrates and their developmental stages. (Expired) DE 10 2205 048 520 Biopract GmbH, Berlin GmbH, Institute of Vegetable and Ornamental Plant Cultivation Großbeeren / Erfurt. Methods for the prophylaxis and therapy of mycoses in useful and ornamental plants as well as in woody plants, particularly in hydroponic systems, October 7, 2007. Key to the figures: Figure 1 : Leaf discs treated with water (A), copper-containing pesticide (B), protease I (C), protease II (D) or protease III (E). Figure 2 : Efficacy test of Prot III on Müller-Thurgau potted vines. Shown is the infestation intensity of P. viticolaIn the treated variants, Protease III levels were 0.01%-0.5% compared to the internal standards, water control, and the copper reference agent. The infestation level was effectively prevented by the use of Protease III. The increased infestation on plants treated with 0.5% Protease III (*) was caused by a spray shadow on a single leaf. The infestation level was calculated based on the percentage infestation of 6 plants with up to 6 leaves per variant. Figure 3 : Illustration of the inhibitory effect of protease III on the growth of C. michigenesis. Shown are dilution series of a bacterial culture at various concentrations of protease III. Numbers under the agar plates indicate the dilution level to which the bacteria grew. Figure 4 : Proliferation of P. syringeaBacteria on tomato leaf segments during 21 days after inoculation. Tomato plants were mock-treated (blank), sprayed with Protease III, or sprayed with Protease III in Nufilm-P. Colony-forming units (CFU) were isolated from 0.7 cm² leaf segments and counted after 48 hours of incubation.

Claims

1. Method for prophylaxis of infections in crops and ornamentals, characterized in that the above-ground parts of the plants are sprayed with an aqueous solution of a bacterial serine protease, and characterized in that the bacterial serine protease is derived from Bacillus sp. or Nocardiopsis sp.

2. Method for prophylaxis of infections in crops and ornamentals according to claim 1, characterized in that the bacterial serine protease is derived from Nocardiopsis sp.

3. Method for prophylaxis of infections in crops and ornamentals according to claim 1, characterized in that the aqueous solution contains only the bacterial serine protease.

4. Method for prophylaxis of infections in crops and ornamentals according to claim 1 or 3, characterized in that the aqueous solution contains only a combination of bacterial serine proteases.

5. Method for prophylaxis of infections in crops and ornamentals according to claims 1 to 4, characterized in that the infections are caused by oomycetes.

6. Method for prophylaxis of infections in crops and ornamentals according to claim 5, characterized in that the oomycetes are Plasmopara viticola, Phytophthora infestans, or Pseudoperonospora cubensis.

7. Method for prophylaxis of infections in crops and ornamentals according to claims 1 to 4, characterized in that the infections are caused by bacteria.

8. Method for prophylaxis of bacterial infections in crops and ornamentals according to claim 7, characterized in that the bacteria are Pseudomonas syringae or Clavibacter michiganensis.

9. Method for prophylaxis of infections in crops and ornamentals according to claims 1 to 8, characterized in that the aqueous solution of a bacterial serine protease is applied in a dose of 0.001% to 1% to combat pathogenic agents.

10. Method for prophylaxis of infections in crops and ornamentals according to claims 1 to 9, characterized in that the plants are treated at intervals.

11. Method for prophylaxis of infections in crops and ornamentals according to claims 1 to 10, characterized in that the aqueous solution of a bacterial serine protease is used at pH values ranging from 4.0 to 8.0.

12. Method for prophylaxis of infections in crops and ornamentals according to claims 1 to 11, characterized in that the aqueous solution of a bacterial serine protease is used at temperatures of 4 to 34 °C.

13. Method for prophylaxis of infections in crops and ornamentals according to claims 1 to 12, characterized in that the aqueous solution of a bacterial serine protease is formulated with adhesion and wetting agents as well as with stabilizers.