Process for the prophylaxis of infections in useful plants and ornamental plants, process for the prophylaxis of infections by oomycetes, and, agent

Description

1 / 31 PROCESS FOR THE PROPHYLAXIS OF INFECTIONS IN USEFUL PLANTS AND ORNAMENTAL PLANTS, PROCESS FOR THE PROPHYLAXIS OF INFECTIONS BY OOMYCETES, AND AGENT DIVIDED FROM BR 11 2016 030114 5, deposited on 06 / 16 / 2015 DESCRIPTION

[001] The invention relates to a process for the prophylaxis of fungal infections, in particular oomycetes, as well as bacterial infections in useful and ornamental plants. The fields of application are horticulture, fruit growing, viticulture and the cultivation of ornamental plants, preferably viticulture and horticulture. Significance, Course, and Control of Oomycete Infections

[002] Plant diseases in useful and ornamental plants, as well as in woody plants, lead to large economic losses annually. Also in hydroponic cultivation of vegetables and ornamental plants under glass, fungi and oomycetes, such as Phytophthora, Phthium and Peronospora, as causes of plant diseases, play a particular role (Malathrakis & Goumas, 1999; Paulitz & Berlanger, 2001). In horticulture (in particular, potatoes and tomatoes), fruit growing, ornamental plant cultivation and viticulture, as well as in forestry, these are of particular economic importance. Potatoes were cultivated worldwide in 2013 on an area of ​​19.3 million hectares used for agriculture (Food and Agriculture Organization of the United Nations, Statistics Division).The most important pathogen in potato cultivation, whose importance has increased further due to the expansion of cultivation into warmer climates, is the oomycete Phytophthora infestans, the pathogenic agent of cabbage and tuber rot (Oerke und. Petition 870190140287, dated 12 / 27 / 2019, page 10 / 47 2 / 31 Steiner, 1996). Its spread can only be controlled by the constant use of fungicides (more than 235 million dollars annually for potato cultivation alone). The total market for fungicides alone comprises 5.5 trillion dollars per year (Powell & Jutsum, 1993).

[003] In Germany alone, approximately 100 million euros are spent annually on plant protection in viticulture (Ochener, 2009). In organic viticulture, pesticides containing exclusively copper are used, which, despite everything, are dangerous to the environment and, in certain circumstances, are poisonous. For this reason, it is of great interest to establish better alternative protection agents that are effective against pathogens and, at the same time, compatible with the environment.

[004] The oomycete life cycle is represented here by the example of Plasmopara viticola, the false downy mildew of grapevine. This branched into two sections of different epidemiological importance. In the sexual section, the oospore is formed, which serves for the hibernation of the pathogen. In the summer cycle, sporangia are released in large quantities. Plasmopara viticola survives the winter as oospores in the soil in heavily infested leaf debris. During the end of winter, the oospores are ready to germinate and reach their germination capacity by the beginning of summer. As soon as the soil warms up and sufficient rainfall occurs, they germinate and form primary sporangia. Until mid-June, with heavy rain, the oospores can germinate constantly. Some oospores may also remain dormant for a year and only germinate the following year. As a rule, germination and the release of zoospores occur from the Petition 870190140287, dated 12 / 27 / 2019, p. 11 / 47 3 / 31 primary sporangium, when temperatures rise above 10°C and more than 8 mm of precipitation has fallen. Under these conditions, the first leaves of the vine have usually developed, so primary infection can occur. For primary infection via germinated zoospores, the leaves must be sufficiently wet with water. Only at this stage, when it is possible to damage or inhibit the zoospores, can infection be prevented or reduced.

[005] Primary infection is the starting point of the summer cycle of Plasmopara viticola, in which the pathogen can multiply asexually with sporangia and cause epidemics under favorable multiplication conditions. Primary infection is followed by the incubation period, during which the pathogen develops inside the leaf without visible symptoms. Treatment of the infection at this point is no longer possible. The growth and development of the pathogen are very strongly dependent on temperature, so that at higher temperatures the tissue is penetrated more rapidly by the mycelium and oil spots appear more than at lower temperatures. At the end of the incubation period, the so-called oil spots appear as a visible sign of fungal infection.As soon as the relative humidity of the air rises above 95% during the night and temperatures above 12°C prevail, sporangia-bearing organisms emerge from the openings in the cracks of the infected leaf surface. The sporangia are spread through the movement of air or water droplets. If these reach a green organ of the host plant in a water droplet, the zoospores hatch. The hatching of the zoospores and the subsequent... Petition 870190140287, dated 12 / 27 / 2019, page 12 / 47 4 / 31 Infections occur optimally at 24°C within four hours. If lower or higher temperatures prevail, zoospores hatch later and the infection process is prolonged. Plasmopara viticola can infect leaves, inflorescences plus the peduncle structure, berries, and shoot tips when these have stomata and are moistened. Even small drops of water are sufficient for infection, but infection conditions are more favorable when water moistens over a large area for a long time. After each infection, there is another incubation period, followed by the hatching of sporangia as soon as sufficient moisture prevails overnight. Plasmopara viticola is included among polycystic pathogens and can go through a series of developmental cycles during the growing season.If optimal conditions for sporangia hatching and infection prevail for an extended period, and incubation times are short due to temperature conditions, an epidemic can develop very rapidly. Drought slows the spread of Plasmopara viticola and inhibits the course of epidemics. It is possible to predict phases with a high risk of infection and, therefore, adopt prophylactic protection measures.

[006] Among the prevailing climatic conditions in Central Europe, infections by such pathogens must be expected every year. To what extent these infections lead to epidemics depends heavily on the course of the annual weather and is not yet predictable at the beginning of the growing season. Epidemics, for example, of grapevine downy mildew (Plasmopara viticola) can occur within a few days of very intense rainfall in classic varieties. Petition 870190140287, dated 12 / 27 / 2019, page 13 / 47 5 / 31 of grapes are highly susceptible. Therefore, this infection must be detected and controlled at an early stage. Later control is not always possible if the infestation has already progressed. For this reason, the economical cultivation of plants is only possible with preventive measures against these infections. A prognostic method, with which this preventive control can be specifically carried out, has already been developed for Plasmopara viticola at the State Institute of Viticulture (Staatlichen Weinbauinstitut) and introduced into practice.

[007] For conventional plant cultivation, numerous fungicides are currently available. In viticulture alone, 29 fungicides are currently permitted for the application of grapevine downy mildew.

[008] For organic viticulture, grapevine downy mildew represents a challenge, since it must be treated preventively, and currently only preparations containing copper (e.g., cuprozin) are authorized. Due to known ecotoxicological concerns regarding copper, it is urgently necessary to find alternatives to this active substance. However, these alternatives must also be effective at high infestation rates. For years, trials have shown that the vast majority of preparations cited as plant fortifiers do not have a satisfactory effect against grapevine downy mildew. Some plant fortifiers show an effect against grapevine downy mildew at low infestation rates, but in these cases, a control measure would not have been necessary. In the case of a higher infestation rate, which Petition 870190140287, dated 12 / 27 / 2019, page 14 / 47 6 / 31 justifies a fight also based on economic considerations, as the effect of the tested preparations was insufficient. These trials acknowledge that in organic plant cultivation today, no biological control of grapevine Peronospora can be practiced. Precisely in organic viticulture, with the limited possibility of interrupting an epidemic, effective and practically useful concepts for the biological control of epidemics are urgently needed. MEANING, COURSE AND CONTROL OF BACTERIAL INFECTIONS

[009] Although the number of phytopathogenic bacteria is smaller than the number of fungus-like pathogens, the damage to useful plants caused by bacterial diseases is very high. Bacteria of the genus Xanthomonas cause diseases worldwide in all major groups of higher plants, resulting in chlorotic and necrotic lesions, wilting, and rotting. An example of great economic importance is black rot in cabbage species, caused by Xanthomonas campestris pv. campestris. When Xanthomonas oryzae pv. oryzae infests rice plants, it leads to rice leaf pearling, which represents one of the most serious rice diseases and consequently causes major economic and social problems. Similarly, Pathovar X. axonopodis pv. citri, the pathogen of citrus canker, and X. campestris pv. can be mentioned here.Vesicatoria, the pathogen that causes bacterial spot disease in peppers and tomatoes, is of economic importance mainly in regions with warm, humid climates.

[0010] In addition, it should be mentioned here that Petition 870190140287, dated 12 / 27 / 2019, page 15 / 47 7 / 31 Bacterial fire blight, caused by the notifiable pathogen Erwinia amylovora, whose host plants are rosaceous plants such as apple, pear, and quince. E. amylovora leads to wilting of leaves and flowers of infested plants, which then turn brown or black. In addition, the bacterium Pseudomonas syringae should be mentioned, causing several plant diseases such as canker, wilting, and spots in important crops such as tomato, pepper, and soybean. This widely distributed variety is of great importance among many glass-grown crops, such as tomatoes, cucumbers, and zucchini.

[0011] Most described bacterial plant pathogens belong to the proteobacteria group and are gram-negative organisms (e.g., Pseudomonas, Xanthomonas). However, there are also economically relevant gram-positive pathogens, such as, for example, Clavibacter michiganensis ssp. michiganensis, which is the pathogen of bacterial wilt of tomato. This quarantine pest is of great importance in warmer and drier tomato-growing regions, as well as in greenhouses.

[0012] Phytopathogenic bacteria have diverse strategies for surviving 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 propagation. Water, for example, in the form of raindrops, represents an important transport vector in relation to distribution within a plant. If the bacteria are transferred to a host plant, they can penetrate it through natural openings such as stomata or hydathodes, or through lesions in the plant. Petition 870190140287, dated 12 / 27 / 2019, page 16 / 47 8 / 31 plant. A high bacterial density, as well as external conditions such as rain, high air humidity, or damaged spots on plant surfaces, facilitate plant infection. Bacteria can propagate well inside the plant, colonizing the apoplast and from there damaging the entire plant. They disrupt the physiology and morphology of the plants and, therefore, cause disease symptoms such as necrotic spots, defoliation, scar formation, wilting, or rot (De la Fuente und Burdman, 2011).

[0013] Therefore, it is absolutely necessary to protect useful plants against these bacterial infections and thus ensure their harvest. The current list of pesticides authorized in Germany with antibacterial effects includes several chemical compounds and copper compounds. Preparations containing copper are the only agents that can be reused in organic farming. Treatments with copper-containing preparations for the control of bacterial diseases have only a partial effect and reach their limits as soon as the density of bacterial inoculum exceeds a certain threshold. Due to the known ecotoxicological effects of copper compounds and other agrochemicals, there are legitimate concerns regarding the use of these pesticides. Furthermore, even in Germany, it is permitted, with exceptions, to use pesticides for the control of fire blight that contain antibiotics such as streptomycin.In other countries, streptomycin is an authorized agent against bacterial infections, but at the same time the use of antibiotics is highly questionable, given the inappropriate use of... Petition 870190140287, dated 12 / 27 / 2019, page 17 / 47 9 / 31 Regarding antibiotics, there are concerns about undesirable effects on the environment, as well as loss of effectiveness due to the development of bacterial resistance. Therefore, it is absolutely necessary to develop better, highly effective, and economically relevant alternatives to these active substances that are more compatible with the ecosystem and consumers. CURRENT DEVELOPMENT OF PLANT PROTECTION STRATEGIES

[0014] Increasingly stringent requirements are being placed on chemical plant protection agents with respect to efficacy, selectivity, specificity, biodegradability, and effect on non-target organisms. However, a range of modern pesticides are available that meet these requirements. The application of numerous older compounds, such as chlorinated hydrocarbons (aldrin, DDT, DDD, dieldrin, kelthan), has been banned in the meantime. Increasingly, however, currently used chemical plant protection agents (e.g., ortho-phenylphenol E 231 or thiabendazole E 233) are also the subject of criticism. These exhibit numerous harmful side effects, which make their use problematic.These include, in addition to damage to the crop plant, changes in taste in the case of fruits, toxic effects on numerous beneficial organisms, development of resistant harmful populations, incomplete decomposition by microorganisms and, consequently, very long persistence and enrichment in the soil, as well as, finally, leaching into groundwater and enrichment in the food chain of humans and animals. (source: Umweltlexikon.) Petition 870190140287, dated 12 / 27 / 2019, page 18 / 47 10 / 31 - www.umweltlexikon-online . of).

[0015] Processes of biological and integrated plant protection are increasingly being established, such as the use of beneficial organisms and pheromones against insects, the use of bacterial and fungal antagonists from the soil, as well as the use of plant extracts. The most important genera of antagonistically effective organisms include, among bacteria, Bacillus, Pseudomonas and Streptomyces, as well as among fungi, Trichoderma, Coniothyrium and Verticillium. Of particular importance in this context is obtained by the bacterium Bacillus subtilis which, as a plant growth-promoting rhizobacterium (PGPR), eliminates phytosanitarily efficient metabolic products, 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 sufficiently controlled by these biological processes, oomycete infections are only with difficulty combated through these measures. In the agricultural area, the following plant diseases are of extraordinary importance due to their risk of infection and the resulting losses (Table 1): Tax Group. Disease. Agricultural Area. Examples. Fungi. Ascomycota. True mildew. Cultivation of vegetables, cereals, fruits, wine and ornamental plants. Erysiphe necator (grapevine), Blumeria graminis (cereals). Ascomycota. Gray mold rot. Cultivation of vegetables, fruits, wine and ornamental plants. Botrytis cinerea (strawberries, grapevine and so on). Basidiomycota. Fungi of Puccinia plants. Petition 870190140287, dated 12 / 27 / 2019, page 19 / 47 11 / 31 Rusts affecting vegetables, cereals, fruits, and ornamental plants: *Graminis* (cereals), *Phakospora pachyrhizi* (soybean); Basidiomycota fire fungi affecting vegetables, cereals, fruits, and ornamental plants: *Ustilago maydis* (corn), *Ustilago hordei* (cereals); Oomycetes downy mildew affecting vegetables, fruits, vines, and ornamental plants: *Phytophthora infestans* (potato & tomato), *Plasmopara viticola* (vine). Proteobacteria bacteria causing fire spot, wilt, spots and other plant diseases in vegetables, cereals, fruits and ornamentals; Erwinia amylovorans, Pseudomonas syringae, Xanthomonas campestris; Actinomycetes causing wilt and other plant diseases in vegetables, cereals, fruits and ornamentals; Clavibacter mi chiganensis. CURRENT STATE OF THE ART

[0016] It is known that glycoside-dissociating enzyme preparations of the non-starch polysaccharide hydrolase type show effects in the prophylaxis and therapy of phytopathogenic fungi. Here, one must start from a direct attack of the enzyme on the cell wall structures of fungi, in particular, oomycetes (DE 10 2205 048 520, Biopract GmbH). But these hydrolases can also damage the plant cell wall and, therefore, are only conditionally suitable for plant protection.

[0017] The use of enzymes of the type of Petition 870190140287, dated 12 / 27 / 2019, page 20 / 47 The use of 12 / 31 non-starch polysaccharide hydrolases for the prophylaxis and therapy of fungal phytopathogens is supported by a number of results in other areas as well. For example, there are experiments in combating oomycete-based fish mycoses with complex enzymatic preparations of Trichoderma spp. (document WO 2004 / 002574 A1 Biopract GmbH).

[0018] U.S. Patent 6663860 (Tvedten, December 16, 2003) describes proteases as a pesticide against, among other things, insects, bacteria, and fungi. However, its use for the prophylaxis of fungal infestation in viticulture is not foreseen.

[0019] Furthermore, several patent reports describe the combination of a pesticide and an enzyme or protease, where the effect described here stems more from the pesticide than from the added enzyme itself (document WO 2013 / 096383 A2, CN 103461383 A, WO 1997 / 047202 A1, WO 1990 / 003732 A1). Other patent reports describe the combination of detergents and enzymes (document US 7393528 B2), plant extracts and proteins (document WO 2001 / 030161), as well as a surfactant and an enzyme (document EP 184288 A1). These publications also do not show that the enzyme itself is responsible for the pesticidal effect.

[0020] Finally, in the past, enzymes or combinations of enzymes have been described that, among other things, have shown antifungal or antibacterial effects, such as, for example, a plant protease (document WO 1991 / 002459 A1), a protease from an earthworm (document JP 2011177105A) or the culture supernatant of a Bacillus fermentation (document JP 54073182 A). Petition 870190140287, dated 12 / 27 / 2019, page 21 / 47 13 / 31

[0021] None of the inventions mentioned above describes a comparable efficient solution to the still persistent problem of crop plant infestation by oomycetes and bacteria. The proteases described herein therefore represent, firstly, a highly effective and at the same time ecologically justifiable alternative to common agricultural pesticides. OBJECTIVE OF THE INVENTION

[0022] The invention aims to develop a highly effective and harmless agent for the plant itself, as well as for the ecosystem, for the control of fungal infections, in particular oomycetes, and bacterial infections in useful and ornamental plants. It also aims to develop a process for the prophylaxis of infections in plants useful in agriculture through phytopathogenic pathogens. In particular, the objective should be to detect early epidemics, such as grapevine downy mildew in wine varieties, and to prevent them. The preparation of suitable media is also included within the scope of the invention.

[0023] This objective is achieved by the measures described in the claims. The process according to the invention is characterized by the fact that a concentrate or a ready-to-use dilution is prepared, containing only one protease or a combination of proteases and βglucanases and / or chitinases. The core of the invention is the surprising possibility of making available, using only proteases, an effective means for controlling infections in useful and ornamental plants.

[0024] In addition, the protection agent may Petition 870190140287, dated 12 / 27 / 2019, page 22 / 47 14 / 31 contain stabilizers, adhesion promoters, and spraying agents, which improve application properties. These mixtures may also contain rain and UV stabilizers.

[0025] This mixture is applied using standard distribution systems at specified time intervals, which are determined based on weather conditions in the growing area, in such a way that all plants are moistened. Application can be carried out at temperatures between 4 and 34°C and, in this way, throughout the growing period. In glass cultivation, application is largely independent of weather conditions and temperatures range between 15°C and 25°C. Through this type of application, it is ensured that the enzymatic preparations are active and an infection of the plants, for example, by zoospores of phytopathogenic oomycetes or by bacterial pathogens such as Pseudomonas syringae, is avoided. The amount used per area should be established according to the crop. Currently, for example, in viticulture, approximately 400 to 800 liters of spray solution are used for an area of ​​one hectare.The enzymatic preparations described are mixed in such a way that the conventional pulverization technique can be used subsequently.

[0026] The invention described represents a significant advance over the means and processes currently established.

[0027] The advantages over the current state of the art should be shown here: • Unlike copper preparations or other chemical pesticides, the use of preparations Petition 870190140287, dated 12 / 27 / 2019, page 23 / 47 15 / 31 Enzymatic substances are harmless to the ecosystem, since the active substance undergoes complete decomposition in the soil, preventing its accumulation. This avoids significant environmental pollution. • No phytotoxic reactions occur, since the proteases used according to the invention do not attack the surfaces of the plants. • Proteases and other enzymes remain effective during plant growth. They do not adhere to a point in the leaf structure, but rather break down into a liquid film on the surface. • The effectiveness of the enzymes remains for a significant period of several days despite rain and UV radiation. This stability can be optionally improved through appropriate formulations.

[0028] Proteases, also referred to as peptidases, dissociate peptide bonds in proteins and thus promote their breakdown into peptides or amino acids. Proteases are divided, based on their type of effect, into the following groups: serine proteases, EC 3.4.21.-, (S), cysteine ​​proteases (C), asparaginic acid proteases (A), metalloproteases (M), and proteases that are unknown or not classified until now (Handbook of Proteolytic Enzymes, AJ Barrett, ND Rawlings, JF Woessner (editors), Academic Press (1998)).

[0029] Proteases, which are preferably used in the sense of the descriptive report described, are primarily serine proteases. The catalytic mechanism of this class of enzymes is based on the nucleophilic hydroxyl group of the amino acid serine, which can dissociate Petition 870190140287, dated 12 / 27 / 2019, page 24 / 47 16 / 31 peptide bonds. Corresponding enzymes can be obtained from culture supernatants, for example, from microorganisms of the genera Nocardiopsis or Bacillus. The corresponding enzymes can also be produced recombinantly. Furthermore, in the case of effective proteases, these may also be mutations, variants, or fragments of the described enzymes that act in an analogous manner.

[0030] Protease activity can be determined with any detection process that uses a substrate containing the corresponding peptide bonds (e.g., casein).

[0031] Surprisingly, it has been found that protease preparations, which are used, for example, in animal feed, prevent plant infection by oomycetes and phytopathogenic bacteria. In particular, the zoospores that occur in the oomycete cycle, which are responsible for the infection of leaf tissue, are irreversibly damaged by the effect of these enzymes; infection of the plant protected in this way does not occur. The mechanism of action against bacterial infestation has not been clarified until now. The clear effect of these enzymes was not expected to this extent, since the mechanisms of action and points of attack of these enzymes are not in accordance with the described mechanism of action of β-glucanases or chitinases. The protective effect can be increased through combination with β-glucanases and / or chitinases.

[0032] Commercially available preparations containing the described proteases include, for example, Ronozyme®ProAct® (DSM Nutritional Petition 870190140287, dated 12 / 27 / 2019, page 25 / 47 17 / 31 Products AG, application examples 1-9: ProtIII), which contains a serine protease from Nocardiopsis sp., or Alcalase® (Novozyme AG), which contains primarily a serine protease, Subtilisin A, from Bacillus licheniformis. In addition, selected protease preparations from Lumis Enzymes (PAP 2XS), which contains, as far as is known, papain from papaya, from Dyadic (Protease Plus, Protease AP Conc), and from AB Enzymes (BIOTOUCH ROC 250LC), which, as far as is known, contains a protease from Trichoderma, have shown a protective effect.

[0033] In the case of glucanases and chitinases, these are enzymes that can hydrolyze glucans or chitin. These are classified in the EC 3.2.1 enzyme class, which contains glycosidases, therefore enzymes that dissociate O- and S-glycosidic bonds.

[0034] Depending on the specific disease characteristic, the application of the enzyme against foliar pathogens (e.g., false mildew or Pseudomonas syringae) is carried out by treating the aerial parts of the plants (e.g., by spraying) with concentrations of an enzymatic preparation of 0.001% - 1%. Preferably, proteases and glycosylases are used in different mixing ratios of the individual enzymes.

[0035] The effect according to the invention of the enzymatic preparations, which manifests itself in preventing the occurrence of infection, is obtained by the fact that the proteases are used individually or as mixtures.

[0036] The enzymes are obtained from culture supernatants of microorganisms. The components are preferably used in aqueous medium in the pH range of 4.5 Petition 870190140287, dated 12 / 27 / 2019, page 26 / 47 18 / 31 at 8.5, preferably at pH 6.0-7.5, for pathogen inactivation. These are used at a water temperature of 4°C to 34°C, preferably 10°C to 25°C.

[0037] The invention will now be explained in detail by way of examples. Examples 1 to 7 deal with the use of proteases as protection 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 cultivated plants by oomycetes. EXAMPLE 1 Suppressive efficacy against infection by spot application on leaves with enzyme preparations, with protein dissociation against Plasmopara viticola in leaf discs.

[0038] Leaf discs of the Vitis vinifera cv. Müller-Thurgau grapevine were treated once by spray application with different protein-dissociating enzyme preparations (Protease I, II, and III), so that the underside of the leaf discs used was uniformly moistened. The preparations contained a serine protease, obtained from either a type of the genera Nocardiopsis or Bacillus. The enzyme preparations used for treatment were tested in a concentration range of 0.01% to 1% (v / v). The pH value of the preparations diluted in water ranged from 6 to 7.5. For the control, the leaf discs were sprayed either with a copper-containing pesticide or with water. 24 hours after treatment, artificial infection of the discs occurred. Petition 870190140287, dated 12 / 27 / 2019, page 27 / 47 19 / 31 leaves with Plasmopara viticola (ca. 40,000 spores per ml of water), the false downy mildew pathogen in vineyards. Subsequently, the leaf discs were incubated on water agar plates at 22 °C for six days in a plant chamber with a day-night rhythm.

[0039] The intensity of infestation is calculated from the ratio between the total leaf surface area and the infected leaf surface area. For the evaluation, software was used to assess the image, comparing the total surface area (number of green dots in the image of the leaf discs at the beginning of the test) and the infected surface area (number of white dots in the image at the end of the test). Two of the protease preparations tested (I, III), as well as the copper-containing pesticide (Cuprozine), effectively prevent infestation and development of Plasmopara viticola (infestation intensity 0%), while the third preparation, Protease II, only partially prevents infestation (infestation intensity 38%). Conversely, leaf discs sprayed with water showed visible infestation (see Table 2 and Illustration 1). TABLE 2

[0040] Infestation intensity (%) calculated from the ratio of the total surface area of ​​the leaf discs to the surface area infected per leaf disc after treatment of the leaf discs with three different protease preparations (n=36) Intensity Deviation from standard infestation (MW) H2O 87% 6% Cuprozin 0% 1% Protease I 0% 1% Petition 870190140287, dated 12 / 27 / 2019, page 28 / 47 20 / 31 Protease II 38% 28% Protease III 0% 0% EXAMPLE 2 Suppressive effect of infection by spot application on leaves with combinations of proteases, chitinases and glycoside dissociating enzyme preparations against Plasmopara viticola in leaf discs.

[0041] Leaf discs of the Vitis vinifera cv. Müller-Thurgau vine were treated once by spray application with a combination of protease, chitinase, and β-glucanase enzymes in a 1:1:1 mixing ratio, so that the underside of the leaf discs used was uniformly wetted. The concentration of the enzyme preparations was 0.1% (v / v). The pH value of the preparations diluted in water ranged from 6 to 7.5. For the control, the leaf discs were either sprayed with a copper-containing pesticide or with water. 24 hours after treatment, artificial infection of the leaf discs with Plasmopara viticola (ca. 40,000 spores per ml of water), the pathogen of false downy mildew in vines, occurred. The leaf discs were then incubated on agar plates at 22 °C for six days in a day-night rhythm plant chamber.

[0042] The intensity of infestation is calculated from the ratio between the total leaf surface area and the surface area of ​​the infected leaf. For the evaluation, image evaluation software was used, which differentiates between the total surface area (number of green pixels on the leaf discs at the beginning of the test) and the infected surface area (number of white dots in the image). The development of Plasmopara viticola on leaf discs treated with enzymes and Petition 870190140287, dated 12 / 27 / 2019, page 29 / 47 21 / 31 The agricultural pesticide containing copper was effectively prevented. EXAMPLE 3 Suppressive effect of infection by spot application on leaves with protein-dissociating enzyme preparations against Plasmopara viticola in greenhouse plants.

[0043] Young vines of the Vitis vinifera cv. Müller-Thurgau variety were treated once, completely, using a stationary application unit, with a protein-dissociating enzyme preparation (protease III). The concentration of the enzyme preparation used was 0.1, 0.2, and 0.5% (v / v). The pH values ​​of the spray solutions were adjusted between 6.5 and 7.5. For control, other vines in pots were sprayed either with a copper-containing pesticide or with water. 24 hours after treatment with the protease preparation, artificial infection of the leaves with Plasmopara viticola, the pathogen of false downy mildew in the vineyard, occurred.

[0044] The plants were then incubated at 20°C for one week in an incubator. Infestation was established by visual assessment of the proportion (%) of infestation changes or necrotic changes in leaves / stems in the total mass of one plant per replicate (100%) and photographically documented. An evaluation scale was used with the gradations 1, 5, 10, 15, 20, 25, 30, 40, 50, 90, 100% of infestation changes.

[0045] The development and spread of Plasmopara viticola on leaves treated with protease III and copper-containing pesticides was effectively prevented, while leaves sprayed with water showed a high infestation (Fig. 2). Petition 870190140287, dated 12 / 27 / 2019, pp. 30 / 47 22 / 31 EXAMPLE 4 Suppressive effect of infection by periodic application of protein-dissociating enzyme preparations in field trials against Plasmopara viticola.

[0046] Whole vineyards of the Vitis vinifera cv. Pinot Noir variety were repeatedly treated with the aid of a tunnel sprayer device with a protein-dissociating enzyme preparation (Prot III) throughout the season in periods of 8 to 14 days, so that the surface of the vine was uniformly wetted. The concentration of the enzyme preparation used was 0.1% (v / v). The pH value of the spray solution was adjusted between 6.5 and 7.5. For better wetting of the leaves, a wetting agent (TREND 90) was also added to the spray solution.

[0047] At the end of the season, the intensity and frequency of infestation by false downy mildew on leaves and grapes were assessed. The development of Plasmopara viticola in the vineyards in the field was effectively prevented. EXAMPLE 5 Protective efficacy of protein dissociating enzyme preparations against the late blight (brown rot) pathogen (Phytophthora infestans) in tomato plants.

[0048] Red Robin variety tomatoes were sprayed at leaf stage 4 with the protease preparation (Protease III, 0.1% (v / v)). The pH values ​​of the spray broths were adjusted between 6.5 and 7.5. As additional variants to the protease solution, usual humectants (T / S forte, Biomaxima, Nufilm) were added at a concentration of Petition 870190140287, dated 12 / 27 / 2019, pp. 31 / 47 23 / 31 0.02% (v / v). The commercial copper preparation Atempo and water were used as an internal control. Five replicates were performed for each plant variant.

[0049] 24 hours after enzyme application, artificial inoculation with the pathogen Phytophthora infestans was performed with a sporangium concentration of 80,000 spores per ml. 6 ml of suspension was used for each plant. The plants were placed in an incubator at approximately 16 °C and 100% relative humidity without lighting. After 24 hours, a lighting schedule of 16:8 hours was adjusted and the humidifier was turned off.

[0050] The assessment took place 6 days after infection. Infestation was indicated by visual detection of the proportion (%) of diseased or necrotic leaves and stems in the total mass of a plant, and was photographically documented (Table 3). TABLE 3

[0051] Phytophthora infestans infestation and degree of effectiveness of the protease preparation Variant No. Infestation [%] Average Value (standard deviation) Degree of effectiveness [%] 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 medium) 1.80 (1.79) 98.13 6 Water - Control 96.00 (5.48) 0.00

[0052] Plants that have been treated with Protease Petition 870190140287, dated 12 / 27 / 2019, pp. 32 / 47 24 / 31 III, showed stable protection with an efficacy rate of 81 to 87% based on the efficacy rate of a conventional copper standard. Formulation adjuvants, such as TS-Forte, BioMaxima, and NufilmP, did not lead to an increase in efficacy compared to Variant 1. EXAMPLE 6 PROTECTIVE EFFICACY OF PROTEIN DISSOCIATING ENZYME PREPARATION AGAINST PSEUDOPERONOSPORA CUBENSIS IN CUCUMBER PLANTS

[0053] Cucumber plants were grown in a climate-controlled environment. To verify the protective efficacy of proteases, approximately 6 ml of the protease preparation (ProtIII), which was present in an aqueous solution at a concentration of 0.1%, was sprayed onto the undersides of the leaves. The pH values ​​of the spray solution were adjusted between 6.5 and 7.5. A conventional copper preparation (Cuprozin Progress) and water served as a control. Six replicates were performed for each plant variant. One day after enzyme application, the plants in the assay were infected with Pseudoperonospora cubensis (75,000 spores per ml). Incubation took place at room temperature in an incubator with a relative humidity above 95%. For the first 48 hours, the plants were incubated in the dark, then they were kept on a 16 / 8 hour day / night cycle. The evaluation occurred 10 days after infection. Here, the percentage infestation of the plants was determined.By using the Prot III preparation, the infestation can be reduced to less than 4% (Table 4). TABLE 4. Pseudoperonospora cubensis infestation in % and Petition 870190140287, dated 12 / 27 / 2019, pp. 33 / 47 25 / 31 DEGREE OF EFFECTIVENESS OF THE PROTEASE PREPARATION Variant No. Infestation [%] Average Value (standard deviation) Degree of effectiveness [%] 1 Protease III 3.6 (0.4) 94.1 2 Cuprosin Progress (standard copper) 9.00 (4.7) 85.2 3 Water 60.4 (11.9) 0.0 EXAMPLE 7 Comparison of the regulatory efficacy of various protease preparations against Pseudoperonospora cubensis in cucumber plants.

[0054] Cucumber plants were grown in a climate-controlled environment. To compare the effectiveness of various protease preparations, approximately 6 ml of the respective protease preparation (Prot III to Prot IX), present in an aqueous solution with a concentration of 0.1%, were sprayed onto the underside of the leaves. The pH of the spray solutions was adjusted between 6.5 and 7.5. A conventional copper preparation (Cuprozin Progress) and water were used as controls. Six replicates were performed for each plant variant. One day after enzyme application, the plants in the assay were infected with Pseudoperonospora cubensis. Incubation took place at room temperature in an incubator with a relative humidity above 95%. For the first 48 hours, the plants were incubated in the dark; after that, the plants were kept on a 16 / 8 hour day / night cycle. The evaluation occurred 10 days after infection.Thus, the percentage infestation of the plants was determined. The degree of effectiveness of the individual preparation is... Petition 870190140287, dated 12 / 27 / 2019, pp. 34 / 47 26 / 31 presented in Table 5. The preparation with the best efficacy was Protease III. Proteases IV, VIII, and IX have comparable efficacy. Table 5 illustrates the organism of origin of the respective Protease known so far. TABLE 5:

[0055] Overview of the protease preparations used in the experiment, as well as the degree of infestation and the corresponding degree of effectiveness. Sample ID Origin Product Infestation [%] Average Value (Standard Deviation) Degree of Efficacy [%] Protein III Nocardiopsis Ronozyme ProAct 0.4 (0.4) 98.6 Protein IV Bacillus Alcalase 0.5 (0.4) 98.3 Protein V Bacillus Savinase 9.8 (4.8) 65.8 Protein VI Aspergillus Flavourzyme 35.0 (21.5) — Protein VIII Papaya PAP 2XS 0.8 (0.3) 97.2 Protein IX Bacillus Protease AP Conc 1.0 (0.6) 98.5 Copper Fungicide — Cuprozin Progress 7.4 (6.2) 74 Protection against bacterial infection in cultivated plants. EXAMPLE 8 PLATE TEST TO VERIFY THE EFFECTIVENESS OF Inhibition of protease growth compared to Clavibacter michiganensis.

[0056] A culture of Clavibacter michiganensis was grown to the late logarithmic phase and then diluted to OD600nm = 1.0 in a 10mM NaCl solution. This starting culture was applied to Petition 870190140287, dated 12 / 27 / 2019, pages 35 / 47 27 / 31 respectively 12 dilutions from 10⁻¹ to 10⁻¹² on nutrient agar plates, which contained the Protease III preparation at concentrations of 0.01 to 1%. Two control plates were free of Protease III and showed maximum growth of Clavibacter michiganensis under the indicated conditions (illustration 3, left: 10⁻⁵). From a protease concentration of 0.05%, bacterial growth was visibly inhibited, as only the most highly concentrated dilutions grew (from 10⁻¹ to 10⁻³, see illustration 3). The greater potential of Protease III as an agricultural pesticide for the control of bacterial wilt of tomato (Clavibacter michiganensis subsp. michiganensis) is elucidated through this assay. EXAMPLE 9 Protection of tomato plants against infection by Pseudomonas syringae

[0057] For this trial, “Red Robin” tomato plants 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 broth was adjusted from 6.5 to 7.5. Four plants were used as a control, each sprayed with tap water or a comparative medium. 24 hours after enzyme application, the plants were infected with Pseudomonas syringae using a syringe.

[0058] The first samples of leaf segments measuring 0.7 cm² were taken every two hours after inoculation; other samples were taken 7, 14, and 21 days after inoculation. The analysis included, respectively, 4 individual leaf segments of 4 Petition 870190140287, dated 12 / 27 / 2019, pp. 36 / 47 28 / 31 plants. The number of colony-forming units (KBE) per leaf segment at the initial baseline was 1 x 10³. In the control (water), the number of KBE increased over three weeks to approximately 1 x 10⁶ KBE per leaf segment. With Protease III, the treated plants maintained the initial test level (10³ KBE per leaf) during the first two weeks. After three weeks, the number of KBE per leaf in the Protease-treated leaves decreased significantly to 10 KBE per leaf segment (Illustration 4). The protease used was visibly more effective than the comparator substance. LITERATURE: • DE LA FUENTE, L. and BURDMAN, 2011. “Pathogenic and beneficial plant-associated bacteria” In Agricultural Sciences, [Ed. Rattan Lal], in the Encyclopedia of Life Support Systems (EOLSS), developed under the auspices of 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 to 38 • KASSEMEYER H.-H. (2004) Research plan for the Federal Ministry's program for Consumer Protection, Nutrition and Agriculture to promote research and development plans, as well as technology and science transfer in ecological agriculture “Innovations to improve boundary conditions for ecological viticulture. Development of scientific approaches for the Petition 870190140287, dated 12 / 27 / 2019, pages 37 / 47 29 / 31 Biological control of Rebenperonospora and strategies for its regulation in organic viticulture”, Project number 020E269, State Institute for Viticulture, Freiburg. • KUDO, S. and C. TESHIMA, 1991: Enzymatic activities and antifungal action of the extract of the fertilization envelope of fish eggs. The Journal of Experimental Zoology 259, 392-398 • KUDO, S., 1992: Enzymatic basis for the 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. Türk. J. Biol. 27, 247 to 253 • MALATHRAKIS, NE, GOUMAS, DE 1999: Fungal and bacterial diseases. See ref. 4, pp. 34 to 47. • MÜNCH, S., NEUHAUS, JM, BOLLER, T., KEMMERLING, B. and KH KOGEL 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 to 244. • OERKE, E.CH. and U. STEINER 1996: “Productivity losses and plant protection: The situation of cultivation for the most economically important crops”. Publication series of the German Society for Plant Medicine, Eugen Ulmer GmbH & Co., Stuttgart Petition 870190140287, dated 12 / 27 / 2019, pp. 38 / 47 30 / 31 • PAULITZ, TC BELANGER, RR 2001: Biological control in greenhouse systems. Annu. Rev. Phytopathol. 39, 103 to 133 • POWELL, KA, JUTSUM, AR (1993) Technical and commercial aspects of biocontrol products. Pestic. pages. 37, 315 to 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 tobacco 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 for Aquatic Technology and Fishing Activities at the Berlin eV Research Cooperative “Process for the prophylaxis and therapy of mycoses in fish and invertebrates and their developmental stages”. (out of print) • DE 10 2205 048 520 Biopract GmbH, Berlin GmbH, Institute for Planting Vegetables and Ornamental Plants GroLbeeren / Erfurt. “Process for the prophylaxis and therapy of mycoses in useful and ornamental plants, as well as in trees, particularly in hydroponic systems”, 07.10.2007 CAPTIONS FOR THE ILLUSTRATIONS

[0059] Illustration 1: Discs of leaves treated with water (A), pesticide containing copper (B), discs of leaves treated with Protease I (C), Protease II (D) or Protease III (E).

[0060] Illustration 2: Prot efficacy test Petition 870190140287, dated 12 / 27 / 2019, pages 39 / 47 31 / 31 III in Müller-Thurgau potted vines. The intensity of P. viticola infestation in variants treated with Protease III 0.01% to 0.5% is illustrated in comparison with internal standards of water control and copper reference agent. The intensity of infestation was effectively avoided by the use of Prot III. High infestation of plants treated with 0.5% Prot III (*) was caused by a spray shadow on a single leaf. The intensity of infestation was calculated based on the percentage infestation of 6 plants with up to 6 leaves per variant.

[0061] Illustration 3: Illustration of the inhibitory efficacy of Protease III on the growth of C. michigenesis. The dilution series of a bacterial culture at various concentrations of Protease III are illustrated. Numbers between the Agar plates clarify the dilution step to which the bacteria grew.

[0062] Illustration 4: Proliferation of P. syringea bacteria in tomato leaf segments during 21 days after inoculation. Tomato plants were sham-treated (Blank), either with Protease III or sprayed with Protease III on Nufilm-P. Colony-forming units (KBE) were isolated from leaf segments in 0.7 cm2 and counted after a 48-hour incubation. Petition 870190140287, dated 12 / 27 / 2019, pages 40 / 47

Claims

1 / 1 CLAIMS 1. A method for the prophylaxis of Phytophthora infestans infection in crops and ornamental plants comprising spraying the plants with an aqueous solution of a bacterial serine protease, characterized in that said bacterial serine protease is derived from Nocardiopsis sp., that said aqueous solution of a bacterial serine protease for pathogen control is applied at a dosage from 0.001% to 1%, and that a time-interval treatment of the plants is carried out by spraying the aerial parts of the plants.

2. Method according to claim 1, characterized in that said aqueous solution of a bacterial serine protease is applied at a pH of 4.0 to 8.

0.

3. A method according to any one of claims 1 to 2, characterized in that said aqueous solution of a bacterial serine protease is applied at temperatures of 4 °C to 34 °C.

4. A method according to any one of claims 1 to 3, characterized in that said aqueous solution of a bacterial serine protease is formulated with adhesive and wetting agents, as well as stabilizers.

5. Method, according to any one of claims 1 to 4, characterized in that said aqueous solution consists of said bacterial serine protease. Petition 870220051252, dated 10 / 06 / 2022, p. 96 / 96

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