WO 99/38379 - 1 - PCT/EP99/00459 Biological plant protection product with resistance-promoting action, and process for its preparation 5 The invention relates to a new and novel biological plant protection product. The plant protection product itself is based on a specific plant preparation and has not only plant-conditioning effects, but in particular a general, surprisingly highly pronounced, induction of 10 resistance in useful plants and crop plants to a wide range of pathogenic factors, in particular to attack by bacteria, fungi and even viruses. Although the product according to the invention is highly effective, it is nontoxic in all respects and thus differs from the 15 traditional plant protection products such as pesticides or fungicides, most of which are based on chemically synthesized compounds and many of which are subject to legislation in a number of countries, owing to their toxicity. 20 The invention also relates to the plant preparation on which the plant protection product is based, a process for its preparation, and an in-vitro method for the rapid identification of the bioactivity of such plant preparations or extracts. 25 The invention furthermore relates to the use of this preparation for improving polluted soils by promoting the resident microbial cultures of the soil. Finally, the invention also relates to substances found in the preparation according to the invention and which 30 the described actions can be ascribed to or which contribute to these actions. Plant diseases in crop plants or useful plants, and the control of the former, constitute an ever increasing 35 challenge. Each year, considerable yield losses are suffered all over the world owing to such diseases and to stress factors caused by the environment and/or climate such as, for example, pronounced drought periods or acid, overfertilized or otherwise polluted - 2 soils, and indeed entire ecosystems of cultured land are lost in certain circumstances. Adverse environmental and climatic factors frequently cause, and potentiate, the spread of certain diseases since 5 the plants are stressed permanently and their natural resistance is thus weakened. Plant diseases are currently predominantly controlled with conventional chemical plant protection products 10 such as, for example, pesticides or fungicides. Since such products are usually highly effective and, more over, can be produced in most cases economically and with simple technical means, so that they are of interest in particular for the third world, success is 15 initially rapid and good. However, conventional chemical plant protection products also have consider ably far-reaching disadvantages, and the general public therefore views their use with increasing skepticism, at least in the industrialized countries. For example, 20 one disadvantage of chemical plant protection is the selection of resistant strains of pathogens, and these resistant strains were and are still being generated, in the control of fungi, by introducing the systemic fungicides. Such strains can frequently only be 25 controlled with even more toxic agents. Thus, the behavior of plant fungicides is similar to antibiotics in medicine, where resistance already constitutes a very important problem. Moreover, not all plant diseases such as, for example, vascular mycoses, or 30 viruses or bacterioses, cannot be treated successfully with the agents known to date. A further disadvantage of chemical plant protection agents is their toxicity, which has already been mentioned above and which is sometimes considerable and affects not only the 35 handling characteristics of the chemical plant protec tion products, but also their effect on the environment (soils, drinking water and the like). However, they, or their metabolites, some of which are also toxic, can also be ingested by humans via the food chain and lead - 3 to physical damage or disease, some of which are irreversible. Accordingly, plant protection products need official approval in many countries and are dealt with increasingly restrictively by the authorities. 5 Biological compositions based on microorganisms, plants, plant extracts or individual isolated active compounds or active compound mixtures are considered to be a true alternative to chemical plant protection 10 products, even though they still play a minor role in practice for a variety of reasons. Examples are the treatment of cucumbers and melons against anthracnosis with extracts of Colletotrichum lagenarium (Caruso, Kuc, 1977, Phytopathology 67, 1285), which itself is 15 the causative agent of anthracnosis. In field trials, a high degree of protection against blue mold was possible in tobacco by treating the latter with spore suspensions of P. tabacina (Tuzun, Kuc, 1989, in: McKeen (Ed.), Blue mold of tobacco, 177, APS Press, St. 20 Paul Minnesota). Furthermore, it is known to use leaf extracts of Japanese knotweed (Reynoutria sachalinenis) against plant molds (Herger et al., 1988, Nachr. Blatt. Dt. Pflanzenschutzdienst 40, 56). The protective action of certain bacterial and fungal substrates on higher 25 useful plants has also been reported (Schbnbeck et al., 1980, Z. Pfl. Krankheiten, PflSchutz 87, 654). Further more, defined naturally occurring individual compounds such as salicylic acid or acetylsalicylic acid (for example Mills, Wood, 1984, Phythopathol., 111, 209) or 30 isonicotinic acid or isonicotinic acid derivatives (Kunz et al., 1988, EP 0026875) are also known and their protective action with respect to plants has been demonstrated. 35 In some of the known examples of plant protection by biological means, the protection was effected, or caused, by induced resistance. The term induced resis tance of a plant or else of a microorganism is generally to be understood as meaning the phenomenon - 4 that the organism in question is enabled, by certain external treatments or substances, to prevent the action of damaging factors from taking place without altering the genetic constitution of the organism. The 5 resistance of the organism is increased generally and over a prolonged period, which leads to a reduced disease intensity and adverse effect of the remaining disease. Inter alia, induced resistance is distin guished mainly by a lacking toxicity of the resistance 10 inducing substances over the harmful substances or organisms (pathogens), and by a lacking strict, linear dose-effect relationship. Another consequence of the induced resistance in a plant can be observed at the molecular level and expresses the expression of various 15 proteins which are formed after attack by, or contact with, the pathogen or contact with the resistance inducing substance which is not pathogenic per se and which are termed PR proteins ("pathogenesis related proteins"). They include, inter alia, the PR-1 20 proteins, 0-1,3-glucanases (PR-2), chitinases (PR-3), hevein-(PR-4) and thaumatin/osmotin-like proteins (PR-5) (Schneider et al., 1996, Intern. Review of Cytology 168, 303). In many cases of induced or acquired resistance, it can be observed that the effect 25 is not localized to the site which has come into direct contact with the resistance-inducing substance or the pathogen but spreads systemically to hitherto healthy plant organs which are as yet not diseased/infested. Thus, the induction of resistance permanently activates 30 a potential of different defense mechanisms which was already latently present in the plant, and these defense mechanisms, in turn, depend on the plant species and the attacking pathogens, or the resistance inducing substance. Ultimately, this ability of the 35 plant to defend itself is somewhat comparable to the medical immunization in humans by active inoculation. Induced resistance is of considerable importance in plant protection because it widens the range of possibilities and can extend to diseases which cannot - 5 be controlled by means of traditional, abovementioned chemicals. All this is in contrast to agents which act as pure plant tonics. Plant tonics are distinguished by a 5 transient strengthening of the organism in question without having the immunizing action via acquired or induced resistance. Biological plant protection products are thus a true alternative to conventional, chemical, usually highly 10 toxic pesticides, fungicides and products with a similar action since these, as has already been mentioned, are treated increasingly restrictively with regard to the approval by the authorities and which are disliked by the ecologically minded general public. 15 However, the biological compositions known to date are frequently difficult to handle (for example when they are based on the pathogen itself, such as, for example, fungal or bacterial suspensions), can frequently not be 20 produced simply in sufficient amounts for wide use in agriculture or organic farming, are not specific enough in some cases or, very frequently, have an insufficient potency, in particular when compared with conventional plant protection products. The few biological composi 25 tions which are known are infrequently employed in particular in plants which are already diseased/ infested and damaged since they show a low activity in such cases, and if, then frequently too late. The use of such known biological plant protection products or 30 plant tonics is thus limited in practice in most cases to a prophylactic treatment only, or when the first signs of disease/infestation are visible, and, even if so, then, as a rule, only to small plant stands. 35 The aim was therefore to provide a biological plant protection product which does not have the above mentioned disadvantages of the known biological and chemical plant protection products and which, moreover, is not just a pure plant tonic. In particular, it was -6 intended to provide a product which is relatively simple to produce and which is highly effective at the lowest possible concentrations and which can be used for a broad spectrum of plants and plant diseases, even 5 in large plant stands. Such a product can be provided in accordance with the invention. It is based on a plant preparation which is simple to produce. 10 Subject matter of the invention is therefore a prepara tion based on plants or plant organs from the pink family (Caryophyllaceae) and the legume family, the percentage by weight of the powder derived from the plant family of the Caryophyllaceae amounting to over 15 80%, but less than 99%, it being possible for the preparation to be obtained by the process steps stated in the claims and specified in greater detail hereinbelow. Subject matter of the invention is also a process for 20 producing said plant- or plant-organ-based preparations which are suitable for biological plant protection, which process comprises the following steps: (a) employing plants from the pink family (Caryophyllaceae) and the legume family as starting 25 material; (b) carrying out the following steps, which must be performed separately for each plant family: (i) grinding the dried plants or plant organs of the family in question, 30 (ii) taking up the ground material in a polar organic solvent, (iii) subjecting the suspension thus obtained to extraction by stirring, (iv) removing the solvent without previously having 35 separated the solid phase from the soluble phase, drying the residue; and (c) mixing the two pulverulent products obtained as set forth in steps b (i) to b (v) in the desired ratio.
What is decisive for the efficacy of the plant prepara tion according to the invention is the ratio of the plant material Caryophyllaceae to Leguminosae. The Caryophyllaceae must amount to at least 80% by weight 5 based on the powder mixture, but must not exceed 99%. With the Caryophyllaceae present at between 90 and 98%, the efficacy is markedly increased the mixtures with a composition of 80 and 99%. A mixture compared with where the Caryophyllaceae amount to approximately 95% 10 and the Leguminosae to 5% is especially preferred in accordance with the invention since the efficacy as biological plant protection product with resistance inducing action shows a relatively narrow, but pronounced, maximum at this ratio. Preparations 15 composed of only one of the two plant components mentioned are, surprisingly, ineffective, as are preparations where the Caryophyllaceae amount to markedly less than 80%. The sharp rise in activity, and its surprising extent, in the preparation according to 20 the invention where the Caryophyllaceae amount to approximately 95% allows the conclusion that this is due to a synergistic action of the active compounds from the Caryophyllaceae and the Leguminosae. 25 A subject matter of the invention is thus a preferred preparation based on plants or plant organs from the family of the Caryophyllaceae and the Leguminosae, the percentage by weight of the powder derived from the plant family of the Caryophyllaceae being over 90 to 30 98%, especially preferably approximately 95% (5% Leguminosae). The Caryophyllaceae constitute a separate plant family. They are common, in particular in central Europe, and have adapted themselves, within certain limits, to a 35 wide range of soil conditions and climatic conditions. Caryophyllaceae are found on acid and limey soils, but, as a rule, they prefer poor soil. Examples of Caryo phyllaceae which are suitable for carrying out the invention are maiden pink, sweet william, Carthusians' - 8 pink, fringed pink, campion, meadow pink, German catchfly, white campion, red campion, common soapwort, rock soapwort, greater stitchwort, wood stitchwort, Moehringia trinervia. Especially preferred are the 5 meadow pink (Lychnis flos-cuculi) and German catchfly (Lychnis viscaria). It is also possible in accordance with the invention to mix various species. However, the use of only one species in each case is preferred. 10 The Leguminosae (Papilionaceae) also constitute a separate plant family; it contains a large number of subfamilies (for example Fabaceae), genera and species which are grown worldwide as useful plants or fodder plants. Their representatives are high in proteinaceous 15 matter and starch and are not particularly demanding with respect to the soil since the nitrogen-fixing bacteria, which are the root symbionts, contribute to improving the soil. Examples of suitable representatives of Leguminosae which may be mentioned 20 are many clover species, such as melilot, kidney-vetch, yellow clover, hop trefoil, bird's foot trefoil, Lotus uliginosus, Hippocrepis comosa. Also suitable are alfalfa species, milk-vetch, Tetragonolobus siliquosus, yellow vetch, common vetch. Especially suitable are the 25 alfalfa species such as, for example, sickle alfalfa or hop clover. To provide the plants for carrying out the invention, the former can also be grown systematically by sowing. 30 Preferred in accordance with the invention is the use of wild forms which have not been influenced by plant breeding methods. Subject matter of the invention is therefore a corres ponding preparation wherein the plant material employed 35 is derived from wild forms which have not been influenced by plant breeding and which preferably grew without the treatment of mineral fertilizers and/or pesticides. However, this does not mean that the plant material required can only be obtained from plants - 9 which have grown in the wild. Rather, it can also be provided by intentional sowing under the abovementioned conditions. For sowing, it is preferably recommended to choose a different site for each season; it should be 5 possible to characterize the soil of this site before hand by suitable analyses. In principle, the measures as set forth in the directive of the European Community EC 2092/91 for organic farming are sufficient to achieve satisfactory final results. The amounts of seed 10 required per hectare depend on the species to be sown and on the specific soil composition. As a rule, 20 to 30 kg of seed/hectare suffice. It is also advantageous for the efficacy of the preparation according to the invention to sow a previous crop, in particular maize 15 or wheat. In general, care should be taken that no additional stress factors such as, for example, attack by pest or contamination with pollutants are created before and during the period when the Caryophyl laceae/Leguminosae are grown. If seed is used for 20 producing the preparation according to the invention, weak or diseased seed can be eliminated by sieving, thus leading to even better results. Preparations according to the invention which have been produced from such plant material have a particularly 25 high efficacy. In some cases, a considerable plant protection effect can only be observed in starting plants treated thus. It is obvious that the original, latent defenses are already greatly reduced in those forms which are grown following the customary standard 30 methods or which have been subjected to plant breeding, so that they are no longer suitable for the purpose in accordance with the invention. In principle, all plant organs of the Caryophyllaceae 35 and Leguminosae may be used for producing the prepara tion according to the invention, for example stalks, leaves, roots, flowers or seeds. However, seeds of the species in question are preferably employed.
- 10 Subject matter of the invention is thus a corresponding preparation wherein the plant organs used are seeds. The powder of the two abovementioned plant families which is produced in accordance with the process of the 5 invention can be stored without problems over a prolonged period and can be provided directly for use for the treatment of endangered or diseased plants, ideally in the form of aqueous suspensions. When large amounts are applied to large areas, in particular in 10 agriculture and arable farming, it is advantageous first to treat the powder with a carrier or filler. All materials which are known for such purposes are suitable, for example powdered rock, powdered magnesium silicate or powdered algae. In accordance with the 15 invention, 2 to 8 g of mixed plant powder are mixed with 98 to 92 g of the filler in question, depending on the plant species to be treated. Subject matter of the invention is thus a corresponding preparation which additionally comprises fillers or 20 carriers, the percentage by weight of the mixed powder derived from the plants preferably being 2 to 8%, based on the fillers/carriers. This ready-to-use mixture, in turn, can now be taken up in water and employed for the treatment as aqueous suspension. In accordance with the 25 invention, 50 to 400 g, preferably 100 to 200 g, of such a total mixture (plant powder plus filler/carrier) are employed per hectare. In the final analysis, however, the amount depends on various factors such as plant species, type of disease/infestation, development 30 of the disease/infestation, already existing damage, soil condition and the like. As already mentioned, the pulverulent preparation according to the invention, with or without fillers, is preferably taken up, or suspended, in water, and the 35 resulting aqueous suspension is used directly by spraying, watering, impregnating or the like. The concentration at which said mixed plant powder can, or must, be present in this aqueous solution is decisive - 11 not only for the efficacy, but also for the economy of the preparation according to the invention. Subject matter of the invention is thus a corresponding preparation which, owing to the addition of water, is 5 present as an aqueous suspension, the concentration of the plant-derived mixed powder being 0.1 mg to 2 mg/l, preferably 0.5 to 1.0 mg/l, especially preferably 0.5 to 0.6 mg/l (figures based on the plant powder only). This surprisingly low value constitutes much of the 10 inventive step of the present application. Moreover, it must be considered as surprising that higher concentra tions, for example 5 mg/l, show a lower resistance inducing action in most cases. 15 Subject matter of the invention is thus also the use of said preparation as a biological plant protection product, in particular as a product with resistance inducing action. The term "induced resistance" is defined in accordance with the invention as described 20 at the outset. The preparation according to the inven tion exhibits all the relevant features, if measured, which are of relevance for the effect of the induced resistance. The preparation at the concentrations stated is highly 25 effective in a wide range of different plants such as cereals or vegetables, but also on ornamental and woody ornamental species or silvicultural plants against a large number of pathogenic molds, including powdery mildew, and to attack by bacteria or even viruses. For 30 example, it has been demonstrated that not only is it possible to protect tobacco, after the treatment, from attack by tobacco mosaic virus (TMV), but that leaf damage is reduced greatly in tobacco plants which are already infected with TMV. 35 Subject matter of the invention is thus the use of the abovementioned preparation for achieving, and promot ing, the induced resistance in useful plants and crop plants to pathogenic and stress-related factors, in particular in the case of bacterial, fungicidal [sic] - 12 or viral infections. The terms "pathogenic and stress related factors" are also understood as meaning, in accordance with the invention, all stressors of, and damage to, the plant by environmental and soil 5 pollutants, climatic stress such as drought and cold periods, strong insolation or UV irradiation, or else unduly high and/or imbalanced fertilization. Frequently, it is precisely those factors which weaken the defenses of the plant, thus allowing attack by 10 fungi, bacteria or viruses in the first place. In addition to the action, or use, as biological, nontoxic, resistance-inducing plant protection product which is thus not subject to the official approval 15 regulations, the preparation according to the invention additionally acts as a plant tonic, and these effects can become apparent, for example, from an increased chlorophyll production, more vigorous growth, better root growth and the like. The extent of this effect, 20 however, depends greatly on the species of the plant and other external factors. It is quite possible that the effect of the induced resistance is not accompanied in some cases by a tonic effect, or by a noticeable tonic effect. To emphasize in particular the tonic 25 effect, a one-off treatment is generally insufficient, in contrast to the induction of resistance. The preparation according to the invention can be applied to, preferably sprayed onto, the growing plant, 30 inter alia. For the prophylactic treatment, the newly germinated plant, or the young plants which have just formed the first, second or third leaf are treated with the aqueous suspension. Plants which have already been attacked are treated immediately after the damage has 35 been established. As an alternative, it is also pos sible to treat the seed before sowing, either by spraying or impregnating. This method has proved to be particularly effective in preventing attack or damage.
- 13 Another method according to the invention is to water or, preferably, spray the soil surface on which the plant material to be protected is intended to grow. Thus, the germinating seed can absorb the substances of 5 the preparation according to the invention which are responsible for the effect, thus increasing the defense of the developing plant in the abovementioned sense. However, treatment of the soil with the preparation 10 according to the invention has another positive and inventive effect. This is because it was possible to demonstrate that, surprisingly, the preparation accord ing to the invention acts as a tonic on, or regener ates, the microbial cultures in the soil which are 15 located in the soil and are responsible for a healthy growth of the plants growing in it. Other use of the preparation according to the invention can thus also be extended to the remediation of polluted soils or to the improvement of depleted soils. The preparation accord 20 ing to the invention is introduced into the soil, a better distribution of the material to a certain, specific depth combined with an aeration of the soil being achieved by turning over the soil once or repeatedly. The activity of the useful soil cultures 25 can be monitored during the remediation by standard methods. Thus, especially advantageous results can be achieved by combining the treatment of the soil with treatment of plants or seeds. The surprising additional positive effect on microorganisms enables the composi 30 tion according to the invention also to be used as composting agent or composting accelerator. As has been shown, this also allows in particular harmful putrefac tive bacteria to be reduced greatly. In this context, the preparation according to the invention can also be 35 employed successfully for the prolonged keeping and preservation of, for example, cut plants, in particular cut flowers. Subject matter of the invention is thus also the use of the preparation according to the invention as a tonic for, and for the regeneration of, - 14 useful microorganisms which occur in particular in soils. The preparations according to the invention also extend to those preparations which are based on plants or 5 plant organs from the families of the Caryophyllaceae and the Leguminosae which may additionally comprise other constituents. Such constituents can be, in principle, fertilizer additives, in particular those based on organic materials, soil conditioning 10 additives, including useful microorganisms, or addi tives which are capable of promoting or regulating the action of the preparation according to the invention. Enhancing additives for the purposes of the invention 15 can be conventional plant protection products if the attack to, or the damage of, the plants in question is already severe enough for it being necessary to support the action according to the invention with such addi tives, at least temporarily, to rescue the plants in 20 the first place. In such cases, the required amount of conventional plant protection products is generally considerably less than without using the preparation according to the invention, so that the environment is markedly less polluted with such additives when the 25 preparation according to the invention is used. Regulating additives for the purposes of the invention are, preferably, plant preparations which are capable of regulating, in particular, the effects of the prepa 30 ration according to the invention as plant tonics in those cases where this is desired. Since application of the preparation according to the invention very frequently causes certain constituents to be formed in higher quantities in the treated plants, it may be 35 desired to then reduce this effect, or even to lower it to less than the normal level. Examples are the nicotine content of tobacco plants or the protein con tent of cereals for particular applications (for example malting barley). An example of such a regulat- - 15 ory additive are powders obtained in accordance with the process according to the invention from mustard plants or wild cherries. In accordance with the invention, such regulatory additives may amount to 5 between 0.5 and 10% of the total mixture. The additives are advantageously added to the plant powder according to the invention in the form of a powder and mixed with the former. In summary, the most important advantages of the 10 preparation according to the invention are those which follow: they cause induced resistance in a very wide range of treated plants. It can be observed both in healthy plants (prophylaxis) and in plants which have been 15 attacked. The efficacy in the case of the attacked plants averages between 30 and 75%, preferably between 50 and 70%, i.e. for example foliar damage after a single treatment only amounts to 25 to 70% (30 to 50%) of the original disease/infestation level. In certain 20 cases, efficacies of over 90% may also be achieved. In most cases, plant or soil material which has been treated prophylactically shows no or only a low degree of attack when the plants is [sic] exposed to the pathogen after treatment with the preparation according 25 to the invention. The preparation according to the invention allows fungal diseases such as mildew, powdery mildew, blue mold and gray mold to be controlled successfully in a large number of crop plants such as tomatoes, cucumbers, wheat and the like. 30 What must be emphasized in particular is the efficacy of the preparation against tobacco mosaic virus in tobacco plants. This is the first case where a plant based biological product is highly effective against viral attack on plants. 35 The preparation according to the invention is nontoxic with regard to the pathogen which triggers the plant disease, for example the bacterium, the fungus or the virus, and thus meets an important prerequisite for the presence of induced resistance. This is because it has - 16 been possible to demonstrate that the preparation according to the invention, independently of the concentration, in no way adversely affects the mycelial growth of fungi, and thus does not act as a fungicide, 5 which contrasts with the traditional plant protection products. Furthermore, it was possible to demonstrate that important PR proteins which are responsible for inducing resistance, such as, inter alia, chitinase and 10 p-1,3-glucanase, are formed in markedly greater quanti ties (increase of 30 to 50% strength) when the preparation according to the invention is applied to plants. What is also particularly remarkable is the minimal 15 effective concentration in accordance with the inven tion, which shows a relatively narrow maximum of between 0.5 mg and 1 mg/l. Plant-protecting natural extracts which act in simi larly low concentrations have not been disclosed as 20 yet. The preparation according to the invention causes the treated plant to recover or to remain healthy. At the same time, plant tonic effects can also be effective. In summary, it allows yield increases of 20 to 50% to 25 be achieved in many cases, even at field level (depend ing on the other specific conditions). These increases can even be achieved in comparison with healthy control plants (without treatment with the preparation accord ing to the invention). This means that the use of the 30 preparation according to the invention for example by means of prophylactic soil and/or seed/plant treatment results in yield increases in healthy plants with potent defenses. 35 The preparation according to the invention can be produced as follows, inclusive of obvious variants and modifications, unless otherwise stated. The treatment and culture of the plants has already been described further above.
- 17 It must be emphasized again that it is preferred to work up each representative of the two plant families according to the invention (Caryophyllaceae, Legumino sae) separately until mixing takes place if the full 5 effect according to the invention is to be achieved. If plants or plant organs are used as starting mater ials, they are first dried under mild conditions following customary methods. Temperatures of over 40*C should be avoided in this step, but also in all other 10 process steps. The dried plant material, inclusive of seeds, is ground finely in a commercially available mill. The particle size should not be less than 0.05 mm and not exceed 0.5 mm if optimal extraction effects are to be achieved. The ground material preferably has an 15 average particle size of 0.1 to 0.2 mm. The ground material is now treated with an organic polar solvent and the resulting suspension is stirred vigorously, preferably at room temperature. Temperatures of markedly less than 15*C entail a loss in action in most 20 cases and should therefore be avoided. Suitable polar organic solvents are, in particular, lower alcohols such as, for example, ethanol, n-propanol, isopropanol or n-butanol. Mixtures of various alcohols are also possible. Ethanol is especially preferred. Methanol can 25 only be employed within limits. What applies to all of the solvents used is that they should be ultrapure, that is to say should not contain, for example, any denaturants or other contaminants. The solvent may comprise 0 to 30% of water. However, a high water con 30 tent very frequently makes removal of the solvent, which is necessary at a later point in time, difficult. According to the invention, one to four parts by volume of solvent, preferably 1.5 to two parts by volume, of solvent are employed per part by volume of ground plant 35 material. This is followed by extraction by stirring over three to six, preferably four to five, days, preferably at room temperature. If appropriate, some solvent is added to the batch to compensate for evaporation effects. The - 18 solvent is subsequently removed carefully, most expediently using a rotary evaporator under slightly reduced pressure, it being recommended that tempera tures of 40"C should not be exceeded. The solvent may 5 also be left simply to evaporate at between 30 and 40*C. Thus, it is essential to the invention that substances which have dissolved during the extraction are not removed but rather be returned to the solid, "incipiently dissolved" residue. The residue thus 10 obtained, which may still be moist, is dried using a commercial dryer, it being intended to achieve a moisture content of less than 10%. This gives a product in the form of a powder which is free from residue and solvent and which can be classified as set forth in the 15 hazardous materials legislation as not hazardous to health. The resulting powders, which are obtained from plants of the Caryophyllaceae and Leguminosae, are then mixed 20 in the proportions stated above and in the claims, if appropriate with addition of the abovementioned fillers/carriers, and dissolved or suspended in water immediately prior to use, the stated concentrations being established. 25 Subject matter of the invention is furthermore an in vitro rapid-test method for determining the bioactivity of the preparation specified above and in the claims as biological plant protection product with a resistance 30 inducing action, which comprises treating a sample of said preparation with a microorganism culture capable of fermentation and measuring under the customary conditions the evolution of C02 as a measure for the cell division rate induced by the active substances in 35 comparison with the control. This is because it has emerged, surprisingly, that the resistance-inducing action of the preparation according to the invention, which can only be detected in vivo in higher plants at a later point in time by complicated experiments, can - 19 be made identifiable in an early stage by means of a microorganism which is easy to handle, for example brewer's yeast. Thus, at least with respect to the preparation according to the invention, a direct 5 relationship was established between the resistance induced in higher plants and an increased cell division rate in microorganisms, it being possible for the increased cell division rate to be measured easily by the greater evolution of CO 2 . However, the greater 10 evolution of C02 is relatively not very pronounced in many cases, so that conventional fermentation saccharometers only allow inaccurate findings. If the measurement via the simple, known fermentation tubes, which are easy to handle, is still to be carried out, 15 these fermentation tubes must have larger volumes and a corresponding detailed and long graduation. In accordance with the invention, suitable fermentation tubes have volumes of at least 50 ml, preferably 60 to 80 ml. The graduated measurement tube as component of 20 the fermentation saccharometer should have a diameter of between 0.5 and 1.5 cm, preferably 0.8 to 1.2 cm, over a length of 10 to 30 cm. When the preparations according to the invention were 25 examined for active-compound-containing substances, it was found that, in addition to a series of phyto sterols, at least three brassinosteroid compounds are also present, of which one compound has not been iden tified as yet, one is 24-epi-castasterone 30 ((22R,23R,24R)-2,3,22,23-tetrahydroxy-24-methyl- 5 a cholestan-6-one) and the other is 24-epi-secasterone ((22R,23R,24R) -22,23-dihydroxy-2 3 ,3/-epoxy-24-methyl 5a-cholestan-6-one). The latter has not been known as yet as a natural substance. The invention thus also 35 relates to the use of 24-epi-secasterone and 24-epi castasterone as resistance-promoting agents for plants. Whether it is these compounds alone which are respon sible for the resistance-promoting action or whether other substances also play an additional role or - 21 If appropriate, the residue is dried further until a fine powder is present. Example 2: 5 Sickle alfalfa seeds (1.2 g) are ground and extracted by stirring as described in Example 1. Again, this gives a dried powder. Example 3: 10 950 mg of the powder produced as described in Example 1 and 50 mg of the powder produced as described in Example 2 are mixed intimately. 50 mg of this mixture are placed in 200 1 of water and the mixture is mixed thoroughly. The solution can be employed directly for 15 spraying a field of approx. 100 m 2 Example 4: 6 g of a mixture obtained from meadow-pink leaves and alfalfa leaves are produced as described (5.7 g of 20 powdered pinks, 0.3 g of powdered alfalfa). This quantity is added to, or mixed intimately with, 96 g of powdered rock. In a stirred tank with a volume of approx. 15,000 1, 10,000 1 of water are added to this mixture. This quantity is sufficient for treating 1 25 hectare of arable land. Example 5: In a greenhouse, tobacco plants of the variety "Xanthi NN", which shows a hypersensitive reaction, are grown. 30 Some of the plants are sprayed with water to which 0.125% of Tween 20 has been added as wetter. These plants constitute the control. The other plants are sprayed with a solution which comprises 1 mg/l powdered plant (95/5 mixture of pinks/alfafa) and also Tween 20 35 as wetter (0.125%). After 5 days of incubation, the plants are inoculated with tobacco mosaic virus. To this end, plant material which was already infected with TMV was ground with a pestle and mortar in 0.1 M sodium phosphate buffer (pH = 6.8) and a small amount - 22 of carborundum was added to act as abrasive. This suspension is subsequently rubbed onto the second and third true leaves (starting at the bottom) of the treated and the control plants, using a brush. After 6 5 weeks, the symptoms, or the disease level, were assessed (% of the leaf area, diameter of the lesions in mm). Table 1 10 Effect of different concentrations of the preparations according to the invention on TMV-infected leaves of tobacco plants. The data shown are the leaf damage in based on the control (untreated plants = 100%). Concentration of the 0 mg / 1 0.5 mg / 1 1.0 mg / 1 2.0 mg / 1 preparation / tobacco (control) leaves leaf 2 100% 80% 50-60% 63-80% leaf 3 100% n.d. 35-62% 65-75% 15 Example 6: As described in Example 5, wheat plants are treated with the preparation according to the invention (control plants: only water) and then infected with 20 powdery mildew and the leaf damage is assessed. The results can be seen from Table 2. Table 2: Effect of different concentrations of the preparations 25 according to the invention on wheat plants which are infected with powdery mildew. The data shown are the leaf damage in % based on the control (untreated plants = 100%) . concentration of the 0 mg /i 0.25 0.5 1.0 2.0 preparation/ wheat (control) mg / 1 mg / 1 Mg / l mg / l leaf 2 100% 70-90% 65-75% 85-95% 74-87% leaf 3 100% 78-85% 70-80% 80-90% 75-85% 30 - 24 six weeks, various parameters such as fresh weight, leaf area, shoot length and chlorophyll content are determined by customary standard methods and compared with those of the control plant [sic]. 5 With one exception, an increase in the abovementioned parameters was observed in all cases: Fresh weight: increase by 21 (wheat) to 57% (tomatoes); Shoot length: increase by 15 (beans) to 34% (tomatoes); Chlorophyll content: increase by 8 (wheat) and 76% 10 (cucumbers) or 90% (beans); only in tomatoes the chlorophyll content was not found to be significantly increased. Example 10: 15 10 liters of a solution of the preparation according to the invention are sprayed onto the surface of a culti vated soil (area: approx. 10 M 2 ) in the greenhouse at a concentration of 0.6 mg/l. Wheat, tomatoes and bean plants are grown in this soil. Some of the plants are 20 grown on untreated soil, but under otherwise identical conditions, to act as controls. After six weeks, various parameters such as fresh weight, leaf area, shoot length and chlorophyll content are determined and compared with those of the control plant [sic]. 25 While no significant increases in the abovementioned parameters were observed in wheat and tomatoes under these conditions, the fresh weight of bean plants was increased by an average of 21%, the leaf area by 18%, the shoot length by 25% and the chlorophyll content by 30 5% in comparison with that of the respective control plants. Example 11: The mycelial growth of the potato blight pathogen 35 Phytophthora infestans was measured on V8 agar in the presence of various concentrations of the preparation according to the invention. It was demonstrated that the mycelial growth is not affected significantly over the entire concentration range measured between 0.5 and - 25 1000 mg/i of the preparation according to the inven tion. This means that the preparation according to the invention has no toxic effect on the fungus. The action according to the invention can therefore not be 5 explained by a fungicidal effect, as is the case in conventional plant protection products. Example 12: Isolation and identification of brassinosteroids: 10 Powder (25 g) which has been produced as described in Example 1 was extracted three times with 200 ml of methanol over the course of one week, and then three times with 200 ml of a methanol/ethyl acetate mixture (1:1 v/v), also for one week. The combined extracts 15 were concentrated in vacuo. The residue was extracted by shaking with water / ethyl acetate (1:1 v/v) . The combined ethyl acetate phases were concentrated in vacuo and the residue was extracted by shaking three times with a methanol (90%)/ n-hexane mixture. The 20 combined aqueous methanol phases were concentrated and extracted by shaking with a mixture of ethyl acetate / saturated sodium hydrogencarbonate solution (1:1 v/v). The residue of the ethyl acetate phases was chromatographed on a silica gel column (20 cm x 1 cm, 25 35-70 mesh Kieselgel, Merck, Germany). The column was eluted successively with chloroform (50 ml) and then with methanol in chloroform (2, 5, 10, 15, 20, 100 v/v %, in each case 50 ml). The eluate was collected in 25-ml fractions. The biological activity 30 was measured with the rice laminar inclination test. This test was developed specifically for the phytohormone group of the brassinosteroids (Arima et al., 1984, Phytochemistry 23, 1587). The fraction which showed biological activity (fraction 5) was purified 35 further by means of preparative HPLC chromatography (4.6 x 250 mm Ultrasphere ODS RP-18 column). The mobile phase was composed of A (water, 0.1% TFA v/v) and B (methanol) at a flow rate of 1 ml/min. The gradient profile was: 0-1 min: 100% A, 1-10 min: 0-40% B - 26 (linear), 10-15 min: 40 B, 15-27 min: 40-100% B (linear), 27-35 min: 100% B, 35-37 min: 0-100% A (linear). The active fractions had a retention time of 25.5 - 29.5 minutes and were concentrated and deriva 5 tized for the purposes of GC-MS analysis. The following conditions were used: EI (70 eV), 200 0 C source temperature, column: DB-5MS (J&W, 15 m x 0.32 mm, 0.25 prm film thickness), injection temperature: 260*C, interface temperature: 3000C, carrier gas: helium, flow 10 rate: 1 ml/min; column temperature program: 170 0 C for 1 minute, heating up to 290*C at 30 0 C/min, 20 min at 290*C. Derivatization was carried out by standard methods. To identify 24-epi-castasterone and 24-epi secasterone, authentic samples were also analyzed on 15 the GC-MS system. Purification and identification of phytosterols: The n-hexane fraction obtained above was concentrated, divided into two portions, and chromatographed on two identical silica gel columns (20 cm x 1 cm, 35-70 mesh, 20 Merck). The columns were eluted stepwise with n-hexane (50 ml) and then with ethyl acetate in n-hexane (10, 30, 50, 70, 90 v/v %, in each case 50 ml). The eluate was collected in 6-ml fractions and separated by means of DC (mobile phase: chloroform/methanol, 95:5 v/v). 25 The phytosterols were visualized by spraying with 5% sulfuric acid in acetic anhydride and heating the DC plate to 1500C. The fractions containing phytosterols were purified further by means of silica gel columns, dichloromethane/heptane (1:1 v/v) being employed as 30 eluent. The sterol-containing fraction was acetylated and analyzed by GC-MS following standard methods.