EP2538782A2

EP2538782A2 - Plant protection agent

Info

Publication number: EP2538782A2
Application number: EP11705425A
Authority: EP
Inventors: Karl Stich; Thilo Fischer; Christian Gosch
Original assignee: Technische Universitaet Wien
Current assignee: Technische Universitaet Wien
Priority date: 2010-02-24
Filing date: 2011-02-24
Publication date: 2013-01-02
Also published as: US20120322889A1; AT509501B1; WO2011103610A3; CA2789808A1; AT509501A1; WO2011103610A2; US20140343165A1; NZ602109A

Abstract

The invention relates to the use of a compound according to formula (A) or formula (B), where R1, R4, and R5 are H, a glycoside, or an ester independently of each other, as a plant protection agent, wherein the plant protection agent comprises the compound, which is dissolved in a solvent at a concentration of 0.1 μM to 2 mM.

Description

- ι -

Pesticides

The present invention relates to a crop protection agent for controlling bacterial and / or fungal infections in plants.

In many countries, pome fruit production is threatened by bacterial and fungal infections, such as the fire blight disease caused by the bacterium Erwinia a ylovora. As a long-term perspective, the diverse breeding efforts to consider fire-blight-resistant varieties of apple and pear should be considered. In contrast, work is underway on the development of new drugs against the bacterial pathogen in a shorter perspective.

Currently, fire blight is controlled primarily by the use of the antibiotic streptomycin, which is a highly potent substance. However, resistance to the antibiotic has already been observed. In addition, ecological and toxicological considerations led to a strict regulation of the use of streptomycin against fire blight. Concerns also exist regarding the development of antibiotic-resistant human pathogenic bacteria through broad antibiotic use. Streptomycin has been used e.g. repeatedly detected in honey from application areas (and then costly destroyed).

Copper-based preparations are also active, but are increasingly restricted to prevent copper accumulation in the soil.

According to EP 2 012 589 B1, nanoscale silver can also be used to combat Erwinia amylovora, which can potentially be accumulated in the soil as an element comparable to copper.

In another approach, antagonistic bacteria are used in the flowering against fire blight, but do not achieve the efficiency of streptomycin.

Arbutin (hydroquinone glucoside) has been scientifically studied as a natural secondary metabolite of the pear (Pyrus spp.) For its potential role in the pathogen defense of the pear against Erwinia amylovora (Hildebrand and Schroth, Nature 197 (1963): 513).

For the hydrolyzed and oxidized product 1, 4-benzoquinone, which can be released from arbutin, an activity against Erwinia amylovora was found (Powell and Hildebrand, Phytopathology 60 (1970): 337-340; Jin and Sato, Phytochemistry 62 (2003): 101-107).

GB 2 159 056 discloses naphthoquinones which are suitable for use in biocides.

In EP 0 134 198 quinone derivatives are disclosed which are able to protect crops from the phytotoxic effect of herbicides.

Duroux et al. (Biochem J., 333 (1998): 275-283) deal with 1, 5-dihydroxy-4-naphthalenyl-β-D-glucopyranoside and the corresponding glucosidase.

In WO 2006/060582 compositions are disclosed which can be used to inhibit nematode damage in plants. These compositions include extracts of juglone-producing plants. The compositions described in WO 2006/060582 include juglone at a minimum concentration of 1000 ppm (1 g / L). At these concentrations, phytotoxic effects may occur in plants, especially in crops.

CN 1 781 376 relates to plant extracts which are suitable to be used as pesticides. These extracts are obtained from plants comprising juglone.

In US 2006/003894 various substances are disclosed which are suitable for controlling the growth of plant pests in the water. The compositions disclosed in this document include naphthalenedione.

In WO 2000/08495 various naphthoquinones and their derivatives are disclosed.

EP 1 051 909 relates to naphthoquinones which are suitable for combating pests.

WO 2000/56140 discloses methods and compositions for combating pests which occur in the water. These agents include, in particular, juglone and juglone analogs.

JP 4 295 402 discloses jugglucose glucosides.

Brockmann et al. (Liebigs Annalen der Chemie, 1983 (1983): 433-447) are concerned with the regioselective synthesis of various quinones.

The agents described in the prior art for controlling bacterial and fungal infections, in particular for combating Erwinia amylovora, or Erwinia infections, has a number of disadvantages. Many of the substances used can be harmful to animals and humans. On the other hand, other substances have a too low activity against fungi or bacteria, such as Erwinia, so that the economy is no longer given by an increased use of active ingredients and the environment is more heavily polluted.

It is an object of the present invention to provide means and methods by means of which Erwinia and in particular the fire blast Erwinia amylovora can be efficiently combated.

The present invention relates to the use of a compound according to the formula (A)

or the formula (B)

wherein R 1, R 4 and R 5 are independently H, a glycoside or an ester, as a pesticide, wherein the pesticide comprises the compound dissolved in a solvent at a concentration of 0.1 μM to 2 mM.

According to a preferred embodiment of the present invention, the compound is 5-hydroxy-1,4-naphthoquinone, 1,4,5-trihydroxynaphthalene and / or glycosides or esters thereof.

The present invention also relates to a crop protection agent comprising at least one compound of the general formula (I) or formula (I I):

Where R 1, R 4 and R 5 independently of one another are a glycoside radical, an added aldehyde, an added ketone, an organic acid radical, an inorganic acid radical or an aliphatic radical, and R 2, R 3, R 6, R7 and R8 are independent of each other which is a halogen or hydrogen, and addition products of S- and N-containing compounds to compounds of general formula II and polymers of both formulas and their respective derivatives in a concentration of 0.1 μΜ to 2 or 5 mM.

It has surprisingly been found that the compounds of the invention and the compounds having the general formulas (I) and (II) and in particular 5-hydroxy-l, 4-naphthoquinone (juglone), and 1, 4, 5-trihydroxynaphthalene

and / or derivatives or glycosides or esters thereof which are capable of releasing such are suitable, in particular, to actively inhibit growth and kill off bacterial and / or fungal plant pests such as Erwinia amylovora. Beyond the unexpectedly high and specific effect, the use of such compounds as crop protection agents was not obvious since e.g. 5-hydroxy-l, 4-naphthoquinone (juglone) is widely described in the literature as phytotoxic ("allelopathy") (Weiler, Nover 2008). However, it was surprisingly found that due to the high activity against, inter alia, Erwinia amylovora at appropriate concentrations, there is a concentration range in which the phytotoxic effect hardly or scarcely comes into play and with which, inter alia, Erwinia and in particular Erwinia amylovora are efficiently combated can. For the practical application of 5-hydroxy-l, 4-naphthoquinone (juglone) against Erwinia amylovora, the highest concentration with acceptable phytotoxicity (especially as Fruchtberostung) with single or repeated use during the flowering period is desirable.

Juglone is a substance that has been extensively studied for its phytotoxic ("allelo-pathic") effect, especially on seedlings. The native walnut tree (Juglans regia) and other Juglandaceae prevent in a natural process the emergence of competing plant growth at their location. The precursor 1, 4, 5-trihydroxynaphthalene-glucoside is formed. This is washed out of the leaves by precipitation and released via the roots and hydrolyzed in the soil by microbial glucosidases to 1,4,5-trihydroxynaphthalene. This is then oxidized to the active substance juglone (Weiler and Nover 2008). However, juglone can damage or kill even neighboring woody plants by their cumulative effect. For apple trees, this was early on as field observation. wrote (Schneiderhan 1927). According to this state of knowledge, despite the existence of publications on the general or specific antimicrobial activity of quinones (eg Didry et al., 1998 for 1, 4-naphthoquinones, Jin and Sato 2003, for benzoquinone / Erwinia amylovora), the use of juglone seems excluded from the outset. In view of the special problem of fire-firing and fruit cultivation with only very short primary infection times and thus a very limited application period for corresponding pesticides, the short-term use of substances with phytotoxic effects comes into question. This is especially true when Juglon proves to be active (more effective than other quinones, including 1, 4-naphthoquinones such as plumbagin) against Erwinia, especially Erwinia amylovora. The combination of low application concentrations with a time-limited application period represents the potential of juglone and its derivatives as plant protection products according to the invention against Erwinia amylovora.

According to the invention, the plant protection agent may also comprise interme ^¬ diary oxidation states of the compounds according to the invention, such as eg Juglon as semiquinone radical.

According to a preferred embodiment of the present invention, the glycoside residue is a glucosyl, mannosyl, galactosyl, fructosyl, ribosyl, arabinosyl, rhamnosyl or xylosyl residue.

The added aldehyde or ketone of the compound of the invention preferably contains 1 to 6 carbon atoms.

According to a further preferred embodiment of the present invention, the remainder of the organic acid is selected from the group consisting of a formyl, acetyl, propionyl, butyryl, isobutyryl, malonyl, pyruvoyl, succinyl, 2-oxo-glutaroyl , Oxaloacetic, fumaric, tartaric and citric acid groups.

The remainder of the inorganic acid is preferably a phosphoric acid or sulfuric acid radical.

The remainder of the ethers is preferably a methyl or ethyl radical.

The halogen is preferably iodine, bromine, chlorine or fluorine.

The compound of the invention may also be present as a 1,2- or 1,4-addition product of the quinone form (formula II).

The compound of the invention can also be used as a polymer present and develop a corresponding antimicrobial activity. The polymer is preferably 3,3 'bijuglon or cyclobutrolone.

The compounds according to the invention, in particular 5-hydroxy-1,4-naphthoquinone, the reduced form of which is 1,4,5-trihydroxynaphthalene and / or glycosides and esters thereof, are substances which occur naturally in nature, in particular in plants. Therefore, the plant protection agent according to the invention may comprise the compounds in synthetic form but also in the form of a plant extract. These extracts are preferably selected from the foliage or nuts of the walnut Juglans regia (or other Juglandaceae, Juglans ailanthifolia (syn. J. sieboldiana), J. cathayensis (syn. J. draconis), J. cinerea, J. hindsii, J. microcarpa (syn. J. rupestris), J. nigra, J. stenocarpa, Carya lacinosa, C. pecan (syn. C. olivaeformis), C. tomentosa; Pterocarya caucasica, P. fraxinifolia, P. hupehensis, P. rhoifolia, P. stenoptera (see Hegnauer R, Chemotaxonomy of Plants, Volume IV (1966): 281, Hegnauer R, Chemotaxonomy of Plants, Volume VIII (1989): 575), but these compounds can also be obtained from others Balsaminaceae (Hegnauer R, Chemotaxonomy of Plants, Volume VIII (1989): 101; Proteaceae: Hegnauer R, Chemotaxonomy of Plants, Volume IX (1990): 297).) To obtain the desired concentration of the compounds according to the invention in the extract, this is diluted or concentrated accordingly Of course, it is also possible to add synthetically prepared compounds of the present invention to the extract to achieve the desired concentration. Juglone or its derivatives can be determined by LC-MS (liquid chromatography-mass spectrometry) or by quinone reactions, e.g. Color reaction with leucomethylene blue (Abbul-Hajj 1978).

The compounds according to the invention additionally have the advantage that they are also useful for insects, e.g. Bees, harmless, i. not eating and contact poison, are. Many crop protection agents used to date against Erwinia infections do not have this benefit and are detrimental to beneficial insects.

Preferably, in the crop protection agent according to the invention the compounds of the invention in a concentration of 0.5 μM to 4 m or 0.5 μM to 1.5 mM, preferably from 1 μM to 1 or 2 mM, more preferably from 5 μM to 0.5 or 1 mM. These amounts of active ingredient are sufficient for the desired effect when applying the plant protection product to plants, in particular to the flowers. Of course, the compounds according to the invention can also be used in concentrations of not more than 2 or 5 mM, preferably not more than 2 or 3 mM, even more preferably not more than 1 or 2 mM.

The compounds according to the invention are preferably dissolved in an aqueous and / or organic solvent in order to achieve the concentration required according to the invention in the crop protection agent. The solvents used may include, but are not limited to, water or other organic solvents such as alcohols such as ethanol. When preparing the plant protection agent according to the invention, the compounds according to the invention can be dissolved, for example, first in an alcohol such as ethanol and then diluted in water or another aqueous medium for the production of pesticides to the concentration according to the invention. In addition, in the aqueous solvent according to the invention buffer substances and the same may be present, which are usually added to crop protection agents.

The crop protection agent according to the invention can be used particularly well for controlling Erwinia amylovora in crop plants. Exemplary crops, which are frequently attacked by Erwinia amylovora, come mainly from the family Rosaceae. Particularly vulnerable is the subfamily of pome fruit (Maloideae). In the wood of pome fruits Erwinia amylovora is also able to overwinter. It is therefore particularly preferred for the plant protection agent according to the invention in rose plants, preferably in pome fruit plants, in particular in apples of the cultivars Berlepsch, Braeburn, Cox Orange, Granny Smith, Elstar, Fuji, Gala, Gloster, Gravensteiner, Idared, James Grieve, Jonagold Group, Jonathan , White Clematis, Topaz and Vista Bella, and pears of the varieties Abbate Fetel, Conference, Williams Christ, Vereinsdechants, Gellerts Butterbirne, Harrow Sweet, Clapp's Darling, Comice, Concorde, Early of Trevoux, Gute Luise, Bosc's Flaschenbirne (Kaiser Alexander) To use Pastoren pear and Passa Crassana the. In addition to cultivated plants, ornamental shrubs such as quince, medlar, vetchling, wild berry, rowanberry / rowanberry, apple berry, ornamental quince, hawthorn like hawthorn and hawthorn, loquat, dwarf medlar and stingwort may also be treated with the plant protection product according to the invention to produce Erwinia amylovora to fight on these plants.

Erwinia amylovora can be combated particularly well with the plant protection agent according to the invention. However, the plant protection product is also useful for controlling other Erwinia species which are effective as plant pathogens. These include Erwinia carotovora (causing blackleg in potato plants and wet rot in potato tubers), Erwinia billingia and Erwinia cypripedii.

The substances 5-hydroxy-l, 4-naphthoquinone (juglone) or 1, 4, 5-trihydroxynaphthalene can be obtained in various ways. On the one hand, both substances can be produced synthetically by known chemical methods, on the other hand both substances can be prepared from e.g. Leaves or nuts of Walnut Juglans regia (or other Juglandaceae) are extracted (e.g., with ethanol or an azeotropic water-ethanol mixture in a Soxhlet apparatus). The synthesis of the derivatives according to the invention can also be carried out by known processes (for example by chromate oxidation of 1,5-dihydroxynaphthalene).

The "derivatives" of 5-hydroxy-1,4-naphthoquinone and 1,4,5-trihydroxynaphthalene are "selected from the group consisting of glycosides, hemiacetals, full acetals, esters, ethers, polymers and 1,2- or 1,4 Addition products and halogen-ring-substituted derivatives ". Therefore, the term "derivative" in the context of the present invention exclusively includes substances derived from 5-hydroxy-l, 4-naphthoquinone and 1,4,5-trihydroxynaphthalene and their hydroxy or keto groups glycosidated, esterified , etherified, acetalated, polymerized, added to other molecules or their ring systems are halogenated.A particularly preferred derivative is the 1,4,5-trihydroxynaphthalene-4-glucoside incorporated in leaves and fruits of the walnut tree. 4,5-trihydroxynaphthalene-4-glucoside is enzymatically or chemically cleaved off its glucose residue and the cleavage results in the chemical compound 1,4,5- Trihydroxynaphthalene ("Hydrojuglon"). From this, after an oxidation reaction, the substance forms juglone (5-hydroxy-l, 4-naphthoquinone).

According to one embodiment of the present invention, the plant protection composition comprises 1, 4, 5-trihydroxynaphthalene or 1, ₍ 5-trihydroxynaphthalene-4-glucoside, both 1,4,5-trihydroxynaphthalene and 1,4,5-trihydroxynaphthalene-4 glucosides are converted to 5-hydroxy-1,4-naphthoquinone over time after application to the plant, this has the advantage that the plant's effect on Erwinia, for example, lasts longer, since 5-hydroxy-1, 4-naphthoquinone is released gradually.

According to a preferred embodiment of the present invention, the derivative of 5-hydroxy-l, 4-naphthoquinone and

1, 4, 5-trihydroxynaphthalene selected from the group

1,4,5-trihydroxynaphthalene-4-glucoside,

various 1, 4, 5-trihydroxynaphthalene-glycosides (number of radicals, 1, 4, 5-position and glycosyl radical (s) variable),

5-hydroxy-1,4-naphthoquinone-5-glucoside (but variable glycosyl),

1,4,5-trihydroxynaphthalene mono- / di- / triacetic acid ester (but

Acyl residue (s) variable),

1,4,5-trihydroxynaphthalene mono- / di- / tri-phosphoric acid ester, 1,4,5-trihydroxynaphthalene mono- / di- / tri-sulfuric acid ester, 5-hydroxy-1,4-naphthoquinone 5-acetic acid ester (but acyl residue variable),

5-hydroxy-1,4-naphthoquinone-5-phosphoric acid ester,

5-hydroxy-1,4-naphthoquinone-5-sulphate,

5-half and 5-full acetals of 5-hydroxy-l, 4-naphthoquinone, half and full acetals of 1, 4, 5-trihydroxynaphthalene (number of radicals and position (s) variable),

5-methyl and 5-ethyl ethers of 5-hydroxy-1,4-naphthoquinone

(but variable alkyl),

Methyl and ethyl ethers of 1, 4, 5-trihydroxynaphthalene (number of radicals and position (s) variable, but also variable alkyl), definable or nonspecific polymers of 5-hydroxy-l, 4-naphthoquinone or 1, 4, -trihydroxynaphthalene,

various reversible 1,2- or 1,4-addition products of 5-hydroxy-1,4-naphthoquinone with S- or N-containing compounds, on the ring system of halogenated derivatives of all the above-mentioned compounds bonds.

The following table lists the preferred substituents of the general compound of formula (I) and (II) and (A) and (B), respectively.

These active ingredients can be dissolved directly in water, or in a Pre-dissolved in organic solvent (preferably ethanol) and diluted in water before use, or, in a particularly preferred embodiment, are 1, 4, 5-trihydroxynaphthalene-4-glucoside, 1, 4, 5-trihydroxynaphthalene, 5-hydroxy-l , 4-Naphthoquinone or mixtures thereof (influenced by the nature of the isolation) from Juglandacea plant material, especially foliage of Juglans or Carya species, isolated and used as a dilute plant preparations. Another possible source of juglone is Penicillium diversum or related organisms.

The crop protection agent of the present invention may comprise surfactants which do not undergo bactericidal chemical reactions with quinones (e.g., Tween 20 (0.005-0.1% by volume), but also surfactant natural substances). The plant protection agent according to the invention particularly preferably comprises Na and K soaps, or commercial wetting agents, e.g. Agrowax 010, Break thru, Citroën, Excellent CS 7, Neo-Bet, Agronetz, Ajutol, Silwet top, Zellx CS.

A further aspect of the present invention relates to the use of 5-hydroxy-1,4-naphthoquinone, 1,4,5-trihydroxynaphthalene and / or derivatives or esters thereof selected from the group consisting of glycosides, hemiacetals, acetals, esters, ethers, Polymers, 1,2- or 1,4-addition products as well as halogen-ring-substituted derivatives as pesticides.

The plant protection agent according to the invention is particularly preferably used for combating Erwinia, preferably Erwinia amylovora.

Another aspect of the present invention relates to a method for controlling bacterial and / or fungal infections, preferably Erwinia infections, in particular Erwinia amylovora infections, in plants comprising the step of applying a plant protection agent as defined herein to flower and / or foliage the plants.

According to the invention, the plant protection agent disclosed herein can be applied to the flower and foliage of the plants to be treated by methods known in the art. Usually, the application of pesticides by spraying, spraying or dusts.

As the primary infection in pome fruit is usually due to the flowering of bees, especially when humid weather conditions conditions prevail, it is particularly preferred at this time to use the plant protection agent according to the invention. Due to the short-term application during the flowering of the flowers, cumulative phytotoxic effects on the fruit trees are avoided in this embodiment. The crop protection agent 5-hydroxy-l, 4-naphthoquinone (juglone) according to the invention has proved to be non-edible or contact-poisonous for bees in investigations carried out on behalf of the company.

It has been found according to the invention that it is advantageous to use the compounds according to the invention, in particular 5-hydroxy-1, 4-naphthoquinone, 1,4,5-trihydroxynaphthalene and / or glycosides or esters thereof, with reduced UV radiation on the plants because these compounds relatively rapidly lose antimicrobial activity, especially in the presence of UV rays. Therefore, the plant protection agent according to the invention is preferably applied to the plant in the twilight, ie reduced UV irradiation, or in the dark.

Yet another aspect of the present invention relates to a process for the preparation of a crop protection composition comprising a compound of the formula (A)

R5

or the formula

wherein R 1, R 4 and R 5 are independently H, a glycoside or an ester comprising the step of dissolving the substantially crystalline compound of the general formula (A) or (B) in a solvent in a concentration of 0.1 μM 2 mM or adjusting or diluting a plant extract comprising the compound of the general formula (A) or (B) such that the plant protection agent comprises the compound in a concentration of 0.1 μΜ to 2 mM.

The compounds of the invention, which may be provided in crystalline form, may be formulated inter alia by dissolving in a solvent to a crop protection agent. By using the compounds according to the invention in solid form, the plant protection agent according to the invention can be prepared in a simple manner in the required concentration range in which the weighed amount of the compounds according to the invention is mixed with an appropriate amount of solvent. If compounds according to the invention are part of a plant extract, the amount of the compounds according to the invention present in this extract is first determined in order subsequently to dilute the extract with a solvent or to add it to further compounds of the invention in solid or liquid form.

According to a particularly preferred embodiment of the present invention, the compound is 5-hydroxy-1, 4-naphthoquinone, 1, 4, 5-trihydroxynaphthalene and / or glycosides or esters thereof.

Another aspect of the present invention relates to a crop protection agent obtainable by a process of the present invention.

The present invention will be further explained with reference to the following figures and examples, but without being limited thereto.

Fig. 1A to 1D shows growth inhibition (cell density measured as optical density at 600 nm) by juglone and comparative plumbagin on suspension cultures of Erwinia amylovora: Top: the 1, 4-naphthoquinone juglone 0.01 / 0.05 / 0.1 mM, Control with equal volume of the solvent ethanol, and control with pure KB medium. Second graph: repetition with 0.01 mM juglone and lower concentrations (0.005 / 0.001 mM). Third graph: 0.1 and 1 mM plumbagin (the initial change in absorbance at 1 mM plumbagin is caused by a reaction with the medium). Fourth chart: Positive control with the conventional herbicide streptomycin (antibiotic) used against Erwinia amylovora.

Figure 2 shows the growth inhibitory effect of 0.05 and 0.1 mM juglone on Erwinia carotovora. E: Control with equal volume of the solvent ethanol, K: Control with pure KB medium.

Fig. 3 shows pear inoculation assays by preincubation with various concentrations of juglone (and streptomycin as control) and subsequent (15 min) inoculation with Erwinia amylovora. Image after 6 days incubation at 25 ° C. Culture plate as a control for the vitality of Erwinia amylovora cells used.

Fig. 4 shows the results of a study comparing the effect of representatives of three classes of quinones against Erwinia amylovora.

In the following examples, the effect of 1,4-naphthoquinones on plant pathogenic species of Erwinia was evaluated to assess their potential as crop protection agents. The 1,4-naphthoquinones juglone (5-hydroxy-1,4-naphthoquinone) and plumbagin (5-hydroxy-2-methyl-1-naphthoquinone) were tested for their activity against Erwinia amylovora and Erwinia carotovora.

Juglon Plumbagin

Example 1:

Inhibitory effect of 1, 4-naphthoquinones on Erwinia amylovora growth in in vitro suspension cultures

The 1,4-naphthoquinones juglone and plumbagin were tested for growth inhibitory effect in in vitro suspension cultures of Erwinia amylovora. Streptomycin as a conventional active against Erwinia amylovora was used as a positive control (see Figures 1A to 1D).

conclusion

From this series of experiments it can be concluded that 1, 4-naphthoquinones are active and especially active against Erwinia amylovora juglone. The latter acts like streptomycin in the sub-millimolar concentration range.

Bacteriostatic versus bactericidal effect

The growth curves of the suspension cultures in the presence of various juglone concentrations demonstrate an inhibition of the growth of Erwinia amylovora. However, these experiments give no information about the nature of the underlying effect, which could be bacteriostatic (inhibition of growth, no direct killing of cells) or bactericidal (killing of cells). However, this can be ascertained by plating out aliquots of the suspension cultures with active ingredient on active substance-free solid media. On such solid media, the drug is diluted from the small volume aliquot by diffusion below a threshold such that, if cells were only inhibited but not killed, colonies form after incubation. Such plating tests were performed with 0.1 mM juglone in the suspension culture by taking aliquots in a time series. The control was a culture containing a corresponding volume of the solvent for the Glon (ethanol) had been added. A repeat of the experiment with only 0.01 mM juglone was also performed, with serial dilutions for the respective aliquots for accurate titer determination. Only for the time points immediately after addition of juglone colony formation could be observed in both test series, after each longer incubation (after 30 min) no colony formation from surviving cells took place.

Conclusion

Juglone has a strong bactericidal effect on Erwinia amylovora.

Effect on other species of Erwinia

Several other species of Erwinia are also important phytopathogens. The bacterium Erwinia carotovora was selected as an example of another Erwinia species. The effect of 1, 4-naphthoquinone juglone on the growth of Erwinia carotovora in suspension culture was tested by the same methodology as in the case of Erwinia amylovora.

It was found that the growth of Erwinia carotovora in suspension culture is completely inhibited by 0.1 mM juglone but, unlike Erwinia amylovora, not by lower concentrations (see Figure 2).

conclusion

Juglone also has a growth-inhibiting effect on other Erwinia species in suspension culture, but at least for Erwinia carotovora this effect is weaker than for Erwinia amylovora.

In vitro infection tests with flowers of the apple (M. x thorn.)

Juglone was tested in in vitro infection tests with apple blossoms, as well as comparing the 1,4-naphthoquinone plumbagin and 1,4-benzoquinone. Streptomycin was used as a positive control.

Table 1: In vitro infection tests on apple blossoms (Malus x domestica) with juglone as well as plumbagin and 1, 4-benzoquinone. Streptomycin as a positive control.

In particular, the use of 0.01 mM juglone in the in vitro infection tests shows a high efficiency of 67%, in direct comparison to 75% for the conventional crop protection agent streptomycin. For the quinones, no formulation was yet used (mainly surfactants for wetting), which should cause an increase in the efficiency.

Conclusion

Juglone has good potential as an agent to control primary fire blight (flower infections).

Inoculation tests with pear fruits (Pyrus communis)

Inoculation of immature pear fruit with Erwinia amylovora (a test system also available after flowering) was used to test the effect of juglone on Erwinia amylovora. In contrast to the flower system, however, wounds of the plant tissue have to be worked on here. Results obtained with this system are known to be more difficult to reproduce and evaluate. The results are documented photographically in FIG. 3. Two different pear varieties were used.

The development of black necrotic lesions of the plant tissue and the formation of white bacterial mucus (exudate) were used as criteria for assessing the extent of Used infection. Necrotic lesions were observed in the experiment on both varieties (Williams Christ and Bosc's Flaschenbirne), while exudate formation was observed only in the Williams Christ variety. Although necrotic lesions were observed in both strains for the 0.01 mM low concentration of streptomycin in the positive control, these were somewhat reduced compared to inoculation with Erwinia amylovora without the antibiotic, and there was no exudate formation.

Sehlussfolgerung:

0.1 mM juglone showed approximately the same effect as 0.01 mM streptomycin in this system.

Phytotoxicity in apple and pear blossoms and for the following fruit development

The phytotoxicity of juglone and comparative plumbagin was tested on flowers of two apple varieties (Smoothy, a Golden Delicious offspring, and Idared) as well as a pear variety (Williams Christ) under growing conditions in a trellis tree planting for flowering. In each case, the concentrations were used, which had been found in the in vitro suspension cultures as fully effective (no bacterial growth). Various concentrations were tested (0.005-0.05 mM juglone and 0.1-0.5 mM plumbagin) (Table 2). Control treatments with water and with the ethanol concentrations used in the drug solutions (0.5 and 5%, to pre-dissolve the active ingredients) were performed and showed, as expected, no effects.

Table 2: Overview of phytotoxicity to flowers rsp. Fruit development of apple and pear varieties tested concentrations of juglone and plumbagin.

pear Apple

cv. Williams cv. Smoothy cv. Idared Christian

Juglone [mM] 0.005 / 0.01 0.005 / 0.01 / 0.005 / 0.01 /

0.025 / 0.05 0, 05

Plumbagin [mM] 0.1 / 0.5 0.1 / 0.5 Phytotoxicity as a browning reaction on the petals

For juglone, slightly brown spots were seen on the petals of apple blossom (Smoothy and Idared) for 0.025 mM juglone, more prominent spots for 0.05 mM. No effect could be observed on the petals of pear blossoms (Williams Christ).

In comparison, for plumbagin (0.1 and 0.5 mM), brown spots were observed on both the petals of pear (Williams Christ) and apple (Smoothy and Idared). These browning reactions were more pronounced for the higher concentration (0.5 mM) than for the lower (0.1 mM).

Phytotoxicity regarding fruit set

It could be found in the described framework of experiments no effects on the fruit set.

Phytotoxicity to fruit overgrowth

Fruit jostling could not be detected either by the juglone or the plumbagin treatments.

conclusion

It was not possible to determine any phytotoxic effects relevant to fruit production in the described framework of the experiments. The browning of short-lived petals was not accompanied by changes in fruit quantity or quality.

Discussion of the toxicity and degradability of juglone

Both 1, 4-naphthoquinones, juglone and plumbagin, are natural secondary metabolites of plants. Juglone is a well-known walnut biotactic compound (Juglans regia) (and other Juglandaceae), while plumbagin is from North American Drosera species (Drosera rotundifolia, Drosaceae), Plumbago species (Plumbaginaceae) and Diospyros species (Ebenaceae). comes. As such, both compounds are released from living (Juglans regia, Weiler Nover 2008) or, in any case, from degrading plant tissues and thereafter degraded microbially. Phytotoxic ("allelopathic") action of juglone on other plants by release of the precursor 1, 4, 5-trihydroxynaphthalene-glucoside are described for walnut (Juglans regia) (see abstract in Weiler and Nover 2008) and other Juglandaceae. With a view to use as a plant protection product according to the invention, such phytotoxic effects on the fruit trees are Molar concentrations and the extremely short period of application against primary fire blight only during the flowering period are not expected.

Both substances, juglone and plumbagin are classified as pure substances as toxic (R phrases), but juglone has a high LD50 value (rat). This is contrasted with the presence of juglone in walnut as a crop for the diet. Especially when removing the exocarps of nuts, the human is exposed to the juglone strongly over the skin (recognizable as blackening of the fingers (oxidation, polymerization)). It is also used as a component of wool dyes and for the treatment of venous diseases. Furthermore, even alcoholic beverages based on immature walnuts are traditionally and commercially produced and consumed ("nut schnapps"). Subject to a required accurate assessment as a plant protection product according to the invention, therefore, juglone is initially considered to be rather harmless.

Comparatively, it can be said about plumbagin that corresponding plant extracts have been suggested for the treatment of oral infections (Didry et al., 1998).

Example 2:

Study comparing the effect of representatives of three

Classes of quinones against Erwinia amylovora

Exemplary representatives of three classes of quinones were compared in their effect on their effects on the growth of suspension cultures of Erwinia amylovora.

Basic structures of 3 classes of quinones

1, 4-benzoquinones 1, 4-naphthoquinones anthraquinones

The focus is on the respective concentration dependence of the effect. execution

The specific members of the three classes of quinones were uniformly pre-dissolved as 1 mM solution in ethanol and then diluted to the respective final concentrations in the culture medium. Corresponding controls with equal volumes of ethanol without drug were performed (Figure 4, "KE"). In particular, a low concentration range below 100 μM was considered, in which nonspecific, weak antibacterial effects no longer apply.

Used representatives of the 3 classes of quinones

1, 4-benzoquinone 1, 4-naphthoquinones: anthraquinones:

Juglon anthraquinone

alizarin

Fig. 4 shows the progressions of the growth curves in the presence of the various members of the classes as a function of the concentration (50/25/125/10/5/2.5 μM). It is clear (it should be noted the courses for 10 μM to 25 μM) that in this concentration range no representative of other quinone classes has an effect that is approximately comparable to the juglone. Only at the low concentration of 5 μM juglone does growth reappear after a long incubation period. 10 μM of juglone, corresponding to 1.74 mg / l, was still completely bactericidal.

(The interpretation of the growth patterns for 50 μΜ is hampered by two overlapping effects: on the one hand, the self-absorption of the colored active substances alizarine and anthraquinone comes into play at this higher concentration (see time 0, curves postponed), and 5% shows ethanol in the Medium this one inhibitory effect on the bacteria, but which (probably after degradation of the ethanol) can be overcome by the culture). Taking these effects into account, 50 μg of juglone as the only test substance shows a clear effect.)

conclusion

Juglon has against Erwinia amylovora against other representatives tern of quinones, in each case described effect against various microorganisms, a beyond, special (bactericidal) effect.

material and methods

Erwinia amylovora strain

For the experiments, the Austrian standard strain Erwina amylovora strain 295/93 (AGES) was used.

In vitro suspension cultures and plating medium

Erwinia amylovora was inoculated in 5 ml of KB medium and cultured overnight at 28 ° C and 200 rpm. This culture was diluted to ODgoo ⁼ 0.3 with KB medium and 2 ml thereof were further cultured directly in sealed cuvettes in the presence of various drug concentrations. Juglone and plumbagin were pre-dissolved in ethanol and then added according to the final concentration to be achieved in the medium. Two controls were performed: unchanged medium, and medium each with the same ethanol concentration as in the test batches. The optical densities of the cultures were measured over time at fixed intervals.

KB Medium:

20 g peptone

1.5 g K ₂ HP0 ₄

1.5 g MgS0 ₄ x 7 H ₂ 0

10 ml of glycerol

pH: 7.2

to 1 1 with water

for the solid medium with an additional 14 g agar / 1 added

Bacteriostatic versus bactericidal effect

The experiment was performed for growth inhibition curves with 0.1 or 0.01 mM juglone (20 μΐ 10 mM and 1 mM juglone in ethanol to 2 ml of suspension cultures). 10 μΐ aliquots of the cultures were taken immediately after addition of the juglone solution and mixing, as well as at regular time intervals and plated on juglone-free solid medium KB. As a control aliquots were taken from the suspension culture with an equivalent concentration of ethanol and also plated out. After two days incubation of the plates at 25 ° C, the respective colony formation was documented. Phytotoxizi did in flowers and for the subsequent fruit development

For the applications, juglone and plumbagin were pre-dissolved in ethanol (65 ° C) and then diluted to the respective test concentrations in water. Maximum final concentration for the solvent ethanol in the treatment solutions was 5%, corresponding control treatments with 0.5 or 5% ethanol in water were performed. For each variant (active ingredient, concentration, variety) two larger branches were treated. The tests took place in the experimental fruit plant of the University of Natural Resources and Life Sciences in Jedlersdorf). All trees in a series were of the same age, developmental and observable physiological state. The treatments were in the morning from 8:00 to 9:30 at a rel.

Humidity of 68-75% and temperatures of 12-13 ° C performed. On all treatment days, the sprayed flowers were then exposed to direct sunlight. On the days following the treatment, phytotoxic effects were assessed. In the following months, fruit set and development were observed and compared with those of the control plants. Special attention was paid to visible changes in the fruit pidermis.

In vitro infection tests with flowers of the apple (M. x thorn.)

The in vitro infection tests with apple blossoms reflecting the natural route of infection were carried out as follows: Separated blooming apple blossoms were further cultured in sterile sugar solution standing in transparent plastic boxes. The stigmas of the flowers were inoculated with 10 ⁴ cfu Erwinia amylovora cells and incubated for 2 hours at room temperature. Thereafter, the drug solutions to be tested were sprayed on. After a further 35 hours of incubation, it was moistened by spraying water. The infection rate was determined after 8-10 days of incubation at 22 ° C and 70% relative humidity.

Inoculation tests with pear fruits (Pyrus communis)

Erwinia amylovora was inoculated in 5 ml of KB medium and cultured overnight at 28 ° C and 200 rpm. After reaching a cell density corresponding to OD6oo (diluted ^1: 1) = 0.17-0.20, 1 ml of the undiluted culture was centrifuged and the bacterial pellet resuspended in PBS (phosphate buffered saline). Unripe pear fruits (July) from experimental cultivation (Jedlersdorf, BOKU) were harvested, washed with water, dried and used for inoculation (EPPO protocol 2004). For inoculation were Drill two holes each about 10 μΐ volume into each fruit with the tip of a pipette, pipette 10 μΐ of the test solution into the cavities and then incubate the fruits for 15 minutes. After absorption of the test solutions into the tissue, each wound was inoculated with 10 μΐ Erwinia amylovora suspension in PBS. As a positive control, fruits were directly inoculated without prior active ingredient additions. For negative control, sterile water was used instead of drug solution. As a positive control, a 0.01 mM streptomycin solution was used. The inoculated fruits were incubated at 25 ° C and 100% humidity for 3-7 days.

Literature:

Abbul-Hajj JBC 253, (7) (1978): 2356-60

Didry, N, et al. , Journal of Ethnopharmacology 60 (1998): 91-96

EPPO Standard Protocols: Diagnostic Protocols for regulated pests PM 7/20. OEPP / EPPO Bulletin 34, (2004): 155-157

Hegnauer R, Chemotaxonomy of Plants, Volume IV (1966) Birkhäuser, Basel

Hegnauer R, Chemotaxonomy of Plants, Volume VIII (1989) Birkhäuser, Basel

Hildebrand DC, Schroth MN, Nature 197, (1963): 513

Jin S, Sato N, Phytochemistry 62 (2003): 101-107

Powell CC, Hildebrand DC, Phytopathology 60 (1970): 337-340

Schneiderhan FJ, hytopathology 17 (1927): 529

Weiler E, Nover L, General and Molecular Botany

(2008): 900 p. , Georg Thieme, Stuttgart, New York

Claims

Claims:

1. Use of a compound according to the formula (A)

or the formula (B)

wherein R 1, R 4 and R 5 are independently H, a glycoside or an ester, as crop protection agents, wherein the crop protection agent comprises the compound dissolved in a solvent in a concentration of 0.1 μM to 2 mM.

2. Use according to claim 1, characterized in that the compound is 5-hydroxy-l, 4-naphthoquinone, 1,4,5-trihydroxynaphthalene and / or glycosides or esters thereof.

3. Use according to claim 1 or 2, characterized in that the compound 5 in a concentration of 0.5 uM to 1.5 mM, preferably from 1 uM to 1 or 2 mM, even more preferred from 5 μΜ to 0.5 mM, is present in the plant protection product.

4. Use according to any one of claims 1 to 3, characterized in that the glycoside residue of the glycoside is a glucosyl, mannosyl, galactosyl, fructosyl, ribosyl, arabinosyl, rhamnisoyl or xylosyl residue.

5. Use according to any one of claims 1 to 4, characterized in that the plant protection product comprises a plant extract comprising a compound as defined in any one of claims 1 to 4.

6. Use according to claim 5, characterized in that the plant extract is obtained from Juglandaceae, preferably Julian's ailanthifolia (syn. Sieboldiana), Juglans cathayensis (Syn. Draconis), Juglans cinerea, Juglans hindsii, Juglans microrocarpa (syn. rupestris), Juglans nigra, Juglans regia and / or Juglans stenocarpa, Carya lacinosa, Carya pecan (Synonym oliva Foremis), Caryta tomentosa, Pterocarya caucasica, Pterocarya fraxinifolia, Pterocarya hupehensis, Pterocarya rhoifolia and Pterocarya stenoptera.

7. Use according to one of claims 1 to 6, characterized in that the plant protection agent is used for controlling bacterial and / or fungal infections in plants.

8. Use according to claim 7, characterized in that the bacterial infection is a Erwinia infection, preferably an Erwinia amylovora infection.

9. A method for controlling bacterial and / or fungal infections, preferably Erwinia infections, in particular Erwinia amylovora infections, in plants comprising the step of applying a plant protection product as defined in any one of claims 1 to 6 to flowering and / or foliage the plants.

10. The method according to claim 9, characterized in that the pesticide in the dusk or in the dark the plant is applied.

11. A process for the preparation of a plant protection product comprising a compound according to the formula (A)

or the formula (B)

wherein R 1, R 4 and R 5 are independently H, a glycoside or an ester, comprising the step of dissolving the substantially crystalline compound of the general formula (A) or (B) in a solvent in a concentration of 0.1 μM 2 mM or adjusting or diluting a plant extract comprising the compound of general formula (A) or (B) such that the plant protection agent comprises the compound in a concentration of 0.1 μM to 2 mM.

12. The method according to claim 11, characterized in that the Compound 5-hydroxy-1, 4-naphthoquinone, 1, 4, 5-trihydroxynaphthalene and / or glycosides or esters thereof.

13. A method according to claim 11 or 12, characterized in that the compound is dissolved in a concentration of from 0.5 μM to 1.5 mM, preferably from 1 μM to 1 or 2 mM, even more preferably from 5 μM to 0.5 mM ,

14. Method according to one of claims 11 to 13, characterized in that the glycoside residue of the glycoside a

Glucosyl, mannosyl, galactosyl, fructosyl, ribosyl,

Arabinosyl, rhamnosyl or xylosyl residue.

15. Method according to one of claims 11 to 14, characterized in that the plant protection agent comprises a plant extract comprising a compound as defined in any one of claims 11 to 14.

16. A method according to claim 15, characterized in that the plant extract is obtained from Juglandaceae, preferably Juglans ailanthifolia (syn. Sieboldiana), Juglans cathayensis (Syn. Draconis), Juglans cinerea, Juglans hindsii, Juglans microrocarpa (syn. Rupestris). , Juglans nigra, Juglans regia and / or Juglans stenocarpa, Carya lacinosa, Carya pecan (Synonym oliva foremis), Caryta tomentosa, Pterocarya caucasica, Pterocarya fraxinifolia, Pterocarya hupehensis, Pterocarya rhoifolia and Pterocarya stenoptera.

17. A crop protection agent obtainable by a process according to any one of claims 11 to 16.