DD299150A5 - ACARICIDE AGENT AND METHOD FOR PRODUCING ITS ACTIVE SUBSTANCES - Google Patents

Abstract

The invention relates to an acaricidal agent, in particular for combating mite Varroa jacobsoni parasitizing on the honeybee (Apis mellifera). The agent contains as active ingredient the optionally liberated from the cells fermented broth or the extract of optionally liberated from the cells ferment broth or the solid (for example dried) forms of optionally liberated from the cells fermented broth of the bacillus NCAIM 001083 or the pseudomonas strain NCAIM 001086 or the mixed Micropopulation of the two strains (NCAIM National Collection of Agricultural and Industrial Microorganism, Budapest, Hungary). The agent is used for spraying the hive or frames and honeycombs, but especially, preferably mixed with sugar juice or honey, for feeding and traction of the bees {acaricide; combating; Mite; Honeybee mite; Varroa jacobsoni; fermentation broth; Fermentextrakt; Bacillus strain NCAIM 001083; Pseudomonas strain NCAIM 001086; Spray; Feed additive}

Description

The invention relates to an acaricidal agent and a process for the preparation of its active ingredients. The remedy is in particular for Combat mite Varroa jacobsoni on bees (i.e., to control varroasis). It is known that the mite Varroa jacobsoni is a parasite of the honeybee (Apis mellifera), which is native to the brood and

especially from the haemolymph of developing bees. As a result of the contamination of the hive and depending on the severity of the pollution, malformed inland appear, the people become weakened and vulnerable to

Illnesses and eventually dies. The mite Varroa jacobsoni is found in all honey-producing areas and the damage caused is very high

considerably. The infestation of a people becomes obvious at first 3-4 years, in later years 2-3 years after the infestation; the

Losses ebb off and increase again. The stick looks like it's poisoned. Various protective measures are known against varroasis:

1) The sticks are treated or fumigated with different essential oils;

2) Use of different synthetic organic agents, for example

- synergistic combination with pyrethrin piperonyl butoxide Chemical Abstracts World Index (hereinafter: WPI Acc No.) 86-212705 / 33;

Tetradifon (The Pesticide Manual, 8th Ed. The British Crop Protection Council, Registry Number (hereinafter Reg. No. 116-29-0)) and Dicofol (Reg. No. 115-32-2) as a combination; WPI Acc No. 86-037330 / 06;

Beeswax with antiparasitic agents, for example, isopropyl 4,4-dibromobenzylate, WPI Acc. 85-290439 / 47;

1-pyridylformimino-2-phenoxymethyl-2-imidazoline derivatives WPI Acc. 85-224091 / 37;

the 2-anilinomethyl-2-imidazoline derivatives WPI Acc. 85-217891 / 36

the 2-dimethylphenylimino-3-methyl-thiazoline WPI Acc. 84-284115 / 46;

- Azoxybenzene contained ^ means WPI Acc No. 82-95526E / 45

the acetone-prepared solution of aryl-N-methyl-carbamate WPI Acc. 78-82396A / 46

- Animal feed and drinking water containing synthetic pyrethroids WPI Acc. No. 87-130020 / 19th

The synthetic organic agents containing agents are introduced by sputtering or smoking in the stick (the booties). Such agents are, for example, Mitac EC (200 g / l) and Mitac WP (250-500 g / kg), which contain as active ingredient not approved in all countries Amitraze, also Ectodex EC (50g / l) (The Pesticide Manual 8th Ed. ). These preparations may only be used if the outside temperature is above 10 ^° C. If the treatment is not taken at the right time or repeated too often, it is believed that an accumulation of harmful byproducts can occur, which can be detrimental to both bees and beekeepers.

3) Lock and quenching agents

- WPI Acc No. 87-129545 / 19 and

- WPI Acc No. 84-018266 / 04;

4) other control methods, for example

- mechanical control WPI Acc No. 84-302720 / 49 and

- Sterilization of the mites by X-rays WPI Acc. 86-048672 / 08th

It has now surprisingly been found that the optionally freed from the cells fermentation broth of the strain NCAIM 001083 or strain NCAIN ¹ ') 1086 or the mixed micropopulation of the two strains or the extract

their fermentation broth, which may have been freed from the cells, or those which may have been freed from the cells

Ferment broth produced solid forms (their dried powder) against the varroasis are effective. These strains are at the National Collection of Agricultural and Industrial Microorganisms in Hungary

(National Collection of Agricultural and Industrial Microorganism, NCAIM) deposited at the Budapest University of Horticulture (Budapest, Hungary) under the numbers 001083 and 001086. Of the two strains, the Bacillus strain 001083neu, the Pseudomonas strain 001086 is known. The mixed micropopulation of the two strains is also new.

It has been found that the action against varroatosis is best when the fermentation broth or from her

obtained application forms are applied immediately after dom wintering. The toxicity for bees is about

Due to the toxicity of the commercially available amitraze, the effect is 100% administration via feed and drinking water. The latter is of particular importance because the feeding method is much easier to carry out and that

normal peoples' habits less disturbing than incense, partial disassembly of prey, etc., as shown by Example38. Also, it has never been possible to achieve a 100% effect with the very widespread Amitraze treatment. The

Importance of the novel treatment is further underlined by the fact that of the conventional means to achieve

an identical effect always higher amounts must be used, d. H. that the emergence of some resistance is observed.

Accordingly, the subject of the invention is an acarricidal agent which optionally liberates as an active ingredient from cells Fermentation broth or the extract thereof optionally from the cells freed fermentation broth or solid, for example

dried forms of the optionally liberated from the cells fermentation broth of the bacillus NCAIM 001083 or the

Pseudomonas strain NCAIM 001086 or the mixed micropopulation of the two strains. The invention further relates to the Bacillus strain NCAIM 001083 and its mixtures formed with the Pseudomonas strain Micro population. The invention also relates to the agricultural use of the two strains or their mixed Micro-population, in particular for the control of mites, preferably the mite Varroa jacobsoni. In that

The control method of the invention, the honeybees or the cane or its frame with the optionally freed from the cells fermentation broth or the extract of optionally freed from the cells

Fermentation broth or the solid, for example dried, forms of the fermentation broth which may have been freed from the cells

of the bacillus strain NCAIM 001083 or the pseudomonas strain NCAIM 001086 or the mixed micropopulation of the two strains, or the bees are fed or soaked in the agent.

The invention also relates to a method for producing and maintaining the bacillus strain NCAIM 001083. For the preparation of the strain according to the invention are from the surface layer of the soil, from about 2-5cm depth Taken samples. At least one of the samples is used for a sample. The samples are sterile Removed and transported in sterile, airtight containers. The tribes are using the Isolated plate method by adding several grams of soil in approximately one hundred times the amount of physiological saline

be suspended. From the suspension with constant stirring with physiological saline a

Dilution series prepared. From the clear supernatant amounts of z. B. 1 ml pipetted into sterile Petri dishes and

filled with about ten times the amount of liquefied Nähragar the temperature of 40-45 ° C. After thorough

Stirring is allowed to solidify the Ag art. The nutrient agar may, for example, have the following composition:

a) b) meat extract 3.0 g 3.0 g peptone 10.0 g B ₁ Og agar 20.0 g 15.0 g distilled water 1000.0 ml 1000.0 ml pH 7.2 7.0

The nutrient media are sterilized at 120 ° C for 20 minutes. The Petri dishes are, preferably turned down, incubated for a few (2-6) days at 25-30X. In the course of incubation appear on the nutrient medium Kolor /; In general, the petri dishes containing about 10-20 colonies are processed further. The colonies are further isolated in a conventional manner. In this way one obtains colonies which are morphologically homogeneous or contain spontaneously formed mixed micropopulations. The spontaneously formed pure or mixed micropopulations are maintained on slant agar, which may, for example, have the following convenient composition:

meat extract 1.0g yeast extract 2.0 g peptone 5.0 g NaCl 5, ₀ agar 15.0 g distilled water 1000.0 ml pH 7.4

Sterilization at 121 ⁰ C 15 minutes. The freshly inoculated tubes are incubated at 32 ° C for 48 hours and then conveniently in the refrigerator at 4 ° C

stored.

From the surface of the slant agar stored in this way, the cells are washed off with physiological saline solution and inoculated with a nutrient solution of preferably the following composition;

peptone 7.8 g Triton 7.8 g yeast extract 7.8 g NaCl 5.6 g glucose 1.0 g distilled water 1000.0 ml pH 7.5

Sterilization at 120 ° C for 20 minutes. The cultures are 2-3 days at 30-35 ⁰ C, preferably in a shaking, or in a standing culture (sometimes under anaerobic conditions) fermentiert.Die Formentbrühe can, if desired, preferably with a membrane filter, are freed from the cells. The biological, in particular acaricidal effect of the optionally exempted from the cells Fermentbrüho is examined. If the biologically active population is a mixed population, it is added to its strains in the following manner. The population is reseeded using a nutrient medium suitable for differential diagnostic purposes. This nutrient medium may have, for example, the following composition.

a) 20.0 g peptone 5.0 g NaCl 15.0 g agar 1000.0 ml distilled water 20-5OmI defibriertesBlut

The nutrient medium is sterilized without adjustment of the pH at 120 ° C for 20 minutes, then cooled to 48 ⁰ C and verseht under sterile conditions with the defibrated blood.

b) 0.3 g meat extract 1.0g peptone 2, ög agar 1000.0 ml distilled water 10.0 m! sterile, defibrated blood

The blood is added to the liquefied, cooled to 48 ⁰ C nutrient medium. The medium is placed on a 80 ^° C. water bath for 5 minutes and then poured into plates in sterile Petri dishes.

C) 5.0 g meat extract 10.0 g peptone 5.0 g dextrose 4.0 g disodium 0.3 g EisendDsulfat 8.0 g Bismuth 0.025 g brilliant Green 20.0 g agar 1000.0 ml distilled water pH 7.7

The solution containing all components is heated with constant stirring until it foams to the neck of the flask. Then allowed to cool with constant stirring to about 50-5G ⁰ C and poured the nutrient medium into Petri dishes.

d) 10.0 g Protease p ~ oton 3.0 g yeast extract 10.0 g lactose 10.0 g sucrose 5.0 g NaCl 0.08 g phenol 0.0125 g brilliant Green 12.0 g agar 1000.0 ml distilled water pH 6.9

The nutrient medium is sterilized ⁰ C15 minutes at 12O and then poured in sterile packages.

In this way, the fermentation broth or its different working-up forms (see Example 3-11), which can be used directly or in formulated form (see Examples 12-37) for combating the honeybee Apis mellifera parasitic mite Varroa jacobsoni.

According to the invention, the active ingredient (in the context of the invention, active ingredient here means the crude fermentation broth or its extract, the fermentation broth freed from the cells or its extract, which or solid, for example dried, if desired, radiation-sterilized forms of the fermentation broth) are known per se Pesticide Formulations, Wade van Valkenburg, Marcel Dekker Inc. New York 1973), for example, dusts, wettable powders, suspension concentrates, water-soluble concentrates, aerosol preparations or other, for example, suitable for feeding and watering agents.

These agents contain in addition to the active ingredient solid orflüsnige carriers, solvents, surfactants and optionally other excipients that make the drug suitable for the application (see, for example, GB1552 277). The excipients (carriers, solvents, surfactants, foam inhibitors, stabilizers, etc.) also require that they be non-toxic to the bees - or indirectly to humans.

The solid carriers and extenders may be natural or synthetic, inorganic or organic in nature. The natural solid carriers and extenders are mostly ground minerals obtained from various minerals (silica, bentonite, perlite, kaolin, dolomite, etc.). The solid carriers and diluents of synthetic origin include, for example, silicagels produced in different manners, high surface area silicas and silica gels, and the finely divided calcium carbonate produced by neutralizing lime milk, the aluminum hydrate or that produced by its heat treatment and grinding aluminum oxide. As solid organic carriers of natural origin are flour, sugar, ground material from plant waste (wood flour) in question.

Liquid carriers and solvents are wacser, organic solvents or mixtures thereof, alkananols, polyfunctional alkenols, their esters formed with different acids, for example with fatty acids, aromatic acids, hydroxy and amino acids, such as, for example, ethyl or isobutyl acetate, amyl acetate, methyl benzoate, Dioctyl phthalate, etc. Other polar organic solvents, for example, acid amides such as dimethylformamide, lactones such as gamma-butyrolactone, lactams such as N-methylpyrrolidone can be selected.

The surfactants used in the various compositions include the emulsifying, dispersing and wetting agents well known in the formulation of pesticides. The surfactants can be nonionic or ionic

The nonionic surfactants include the ether formed with alcohols or the esters of ethylene oxide formed with fatty acids, furthermore saina with Aralkylphsiiolan formed ether, its copolymers formed with propylene oxide, their

Esters and ethers, the derivatives of ethylene oxide formed with the hexitol anhydrides of fatty acids, etc. Examples are the

Nonylphonol polyglycol ether, which mentions oleic polyoxyethylene esters and the polyoxyethylene sorbitan monooleate.

The ionic surfactants may be anionic or cationic or amphoteric.

The anionic surfactants include the various organic acids, for example carboxylic acids, the sulfonic acids, the alcohol sulfates and sulfonates, the phosphate esters of the polyoxyethylene ethers and esters and their salts formed with alkali, alkaline earth or organic anions, for example soft soap, dodecylbenzenesulfonic acid -Ca, -Na, ammonium and Diisopropylammoniumsalz, Natriumdiisooctylsulfosuccinat and formed with the indicated cations salts of the polyacrylic acid.

Cationic surfactants are, for example, the halides, sulfonates of long-chain alkyiamines, stearyldimethylammonium chloride, the long-chain ethanolamines or their salts, and also the salts formed with anionic surfactants.

Amphoteric surfactants are, for example, the betaines, the lecithins, the sodium N-methylalkyltaurimide, etc.

Suitable auxiliaries are, for example, adhesion-promoting natural or synthetic polymers, for example starch, dextrin, monorod.d disaccharides, carboxymethylcellulose, hydroxyethylcellulose, polyvinylpyrrolidone, polyacrylic acid, xanthan gum, alginates. As antifoam agents, polyoxyethylene-polyoxypropylene copolymers, long-chain alkenols or special silicone oils can be used. Furthermore, attractants such as honey, mono- or disaccharides, fragrances such as isoamyl acetate can be used as adjuvants. The stabilizers, which ensure the stability of the agent, are also to be expected for the aids. Examples include benzoic acid, sodium benzoate, called the esters of p-hydroxybenzoic acid.

The active compounds can be formulated as liquid, solid or aerosol preparations.

For the preparation of liquid formulations, the active ingredient is dissolved or suspended in a suitable solvent, preferably in water, the said auxiliaries being added simultaneously or in succession.

The liquid preparations can be not only homogeneous, but also disperse. The latter are prepared by comminuting the solid active ingredient to a suitable particle size, dispersing it in a dispersion medium containing the respective surfactants, and then further milling it in, for example, a bead mill. To the resulting suspension are added, in a low-risk agitator, the antifoam and, if desired, a viscosity-enhancing agent.

For the preparation of powdered agents, the active ingredient is ground in a suitable device and then homogenized with the carrier and the surfactants. It is also possible first to homogenize active ingredient, carrier and surface-active substances, then to grind and finally homogenize again. For grinding, hammer mills, blow bar mills, pin mills or air jet mills can be used. It is also possible to disperse the carrier and surfactants in the dispenser or solution containing the active ingredient and then to dry the resulting liquid in a known manner, for example by spray drying or lyophilization.

If desired, the resulting powder can be ground and homogenized.

In the preparation and use of liquid preparations, it should be remembered that the amount of liquid used can not be too large; the bees would become so wet that it would prevent their movement. Of the cell-free fermentation broth prepared according to Example 8a, preferably 5-50OmI per m ^{2 of} bee frame can be used. However, as the fermentation broth or its processing forms are not toxic to the blenny, larger or smaller doses may also be used. Frequency of treatment and size of the dose generally depend on the degree of infestation. Because of the mentioned disadvantage of the liquid preparation is generally the use of different powdered! Preparations preferred. Of these amounts are generally used, which correspond to 0.01-1 og active ingredient / m ² bee frame. A very advantageous form of application are the feeding and drinking preparations, because in their application, the prey must not be taken apart, also a uniform and lasting dosage is possible, in contrast to the spraying and dusts. In the case of feeding and drinking agents, instead of the dose, the feeding or drinking period is specified, which is required for reduction or elimination.

The invention will be further elucidated with reference to the following examples, but is not limited to these examples. The% data in the examples are% by mass.

example 1 Isolation of stems The micro-organisms were cultivated using the plate method from forest soil (Hungary, acacia groves in the county lolna, Sept,

1986). 1 g of soil sample was suspended in 99 ml of physiological saline. The suspension became a

Dilution series prepared in such a way that in each step to 3ml suspension 27ml physiological saline

were given. From each flask, 1 ml was pipetted into a sterile 4 cm diameter Petri dish and mixed with 9 ml liquefied medium at a temperature of 40-45 ⁰ C containing 0.02 mg / ml Actidion.

The culture medium was thoroughly mixed with the soil suspension by circular swirling of the Petri dish and then

stiffened. The turned down petri dishes were incubated at 28 ° C for 72 hours. For the others

Studies were carried out using Petri dishes in which 10-20 colonies had grown. These colonies were

several times separated according to methods known per se, whereby nine mutually differing, self-homogeneous

Colonies were won. In addition, a spontaneously formed mixed population had emerged. The according to the examples 3a

or 8a and b prepared fermented broths or cell-free fermented broths of the isolated pure or mixed k i i were examined for their acaricidal activity in the manner described in Examples 38-40.

Example 2 Isolation of Subcultures of Spontaneously Formed Mixed Microplatinas From the spontaneously formed mixed micropopulation were intended for differential diagnostic purposes Culture medium (chocolate agar, blood agar, brilliant green agar, bismuth sulfite agar, etc.) by re-separation and aging

two strains isolated (pure subcultures) and deposited with the NCAIM under the numbers 001083 and 001086.

One of the tribes, a Bacillus, is new; the other is known and belongs to the genus Pseudomonas. The properties of Microorganisms are the following:

a) The pseudomonas strain NCAIM 001086 forms shiny gray colonies of 0.5-1 mm in diameter on a simple nutrient agar. The growth is weak at 37 ⁰ C, better at 30 ° C.

b) The Bacillus strain NCAIM 001083 forms dull, pale gray colonies of 1-2 mm in diameter on a simple nutrient agar.

a) gram: positive b) sporulation oval c) Move positive d) Growth anaerobic positive θ) Growth on bismuth sulfite agar +/- f) on eosin methylene blue agar +/- G) in the presence of 7% NaCl positive H) Katalaso positive i) No3-NO ₂ conversion negative j) Voges-Proskauer negative k) indole negative I) Urease (Kristensen) negative m) Arginine negative n) lysine decarboxylase negative O) ornithine decarboxylase negative P) Eskulinhydrolyse positive q) starch hydrolysis negative r) Kaseinhydrolyse negative s) Lecithinhydrolyse negative t) Gelatin positive u) ammonium citrate negative ü) Acid formation on oxidation Fer- mentation medium to Hungh-Leifson oxidative v) Gas formation in peptone-water-glucose negative

Acid formation (BBS) glucose positive fructose positive lactose negative maltose negative mannitol positive ramnose negative sucrose positive xylose negative (Peptone water) arabinose negative adonitol negative ONPG positive

Example 3 Fermentation of the strain NCAIM 001083 and 001086 or their spontaneously formed mixed Mikropopulatlon

a) The cells of the mixed nutrient agar population maintained on slant nutrient agar are suspended with 5 ml of physiological saline per tube. One ml each of the suspension is added to 100 ml of nutrient medium, which is in flasks of 500 ml volume and has the following composition:

0.78% peptone

0.78% Triton

0.78% yeast extract

0.56% NaCl

0.1% glucose

(pH 7.5, sterilized at 12O ⁰ C for 20 minutes), the seeded flasks are shaken at 32 ⁰ C for 72 hours at a frequency of 300min " ¹ .

b) Working in the manner described under a) with the difference that the Pseudomonas strain NCAIM 001086 is fermented.

Example 4 The fermentation of the spontaneously formed mixed micropopulatlon of NCAIM 001083 and 001086 and the Atomization drying of the Fermsntbruhe.

One liter of the fermentation broth obtained according to Example 3a (dry matter content 2.4%) is sputter dried with air at an inlet temperature of 12O ⁰ C and an outlet temperature of 65-75 ⁰ C. The resulting dry product has a mass of 25.5 g, its moisture content is 5.8%.

Belsplel5

Radiation sterilization of the dried fermentation product

The product obtained according to Example 4 is irradiated with gamma rays in a dose of 5 kGr. The success of radiation sterilization is controlled in a conventional manner with the plate method.

Example β

Lyophilizing the fermentation broth

100ml of the fermentation broth obtained according to Example 3a Werdan kept 24 hours at -25 ⁰ C and lyophilized in an Edwards Supermodulyo 12K type equipment within 24 hours. The dry matter of a lyophilizate is 2.3-2.5 g.

Example 7 Radiation Sterilization of the lyophilisate

The lyophilisate obtained according to Example 6 is irradiated with gamma rays in a dose of 5KGr. The success of radiation sterilization is controlled in a conventional manner with the plate method.

Example 8 Remove the cells from the ferns

a) One liter of the mixed micro-population containing fermentation broth according to Example 3a is centrifuged in a coolable centrifuge type LU 418H at a speed of 4000 min ' ¹ at 4 ° C for one hour. The clear supernatant is repeatedly filtered through membranes of pore size of 0.45, then 0.2 micron (manufacturer: Sartorius), whereby the cells are completely removed. The filtered fermentation broth has a dry matter content of 1.02%. The success of cell removal is controlled in a manner known per se with the plate method.

b) Working in the manner described in point a), but starting from the fermentation broth of the Pseudomonas strain NCAIM 001086.

Example 9 Atomization drying of cell-free fermentation broth

One liter of the Example 8 a sustaining eien cell-free fermentation broth according to (dry content matter 1.02%) is zerstäubungsgetrocknot air with an inlet temperature of 12O ⁰ C and an outlet temperature of 65-75 ⁰ C. The mass of the product obtained is 10.57 g, its moisture content 3.7%.

Example 10 Lyophiliser cell-free fermentation broth

One liter of the cell-free fermentation broth obtained according to Example 8a is kept at -25 ° C. for 48 hours and iyophilized in an Edwards Supermoduiyo 12 K apparatus within 24 hours. The mass of the lyophilisate is 10.2 g.

Example 11 Evaporation of duty free fermentation broth

One liter of the cell-free fermentation broth obtained according to Example 8a is evaporated in a film steamer at 5O ⁰ C under a pressure of 380 Hgmm to a volume of 0.33 liters.

Example 12 Lyophilized duty-free fermentation broth with added sucrose

Sucrose is added to one liter of cell-free fermentation broth obtained according to Example 8a in an amount of 1/60 of its mass. The liquid is kept for 48 hours at -25 ⁰ C and Iyophiiisiert in a device of the type Edwards Supermoduiyo 12 K within 24 hours.

Liquid preparations spray

Example 13

Active ingredient according to Example 3a or 3b 99.8%

Polyoxyethylene sorbitan monooleate 0.1%

Methylsilinol oil emulsion (10%) 0.1%

The preparation was prepared by mixing (homogenizing) the components.

Example 14 97.8% Active ingredient according to Example 8a 2.0% dextrin 0.1% polyoxyethylene sorbitan monooleate 0.1% Methylsilicone oil emulsion (10%) Preparation as in Example 13. Example 15 92.0 Vo Active ingredient according to Example 11 7.2% sucrose 0.5% polyoxyethylene sorbitan monooleate 0.3% Methylsilicone oil emulsion (10%) Preparation as in Example 13. Example 16 93.0% Active ingredient according to Example 11 6.3% sucrose Fatty alcohol polyoxyäthylenäther- 0.25% phosphatester potassium salt 0.15% polyoxyethylene sorbitan monooleate 0.25% Methylsilicone oil emulsion (10%) 0.05% isoamyl Preparation as in Example 13. Example 17 99.7% Active ingredient according to Example 3 a or b 0.1% polyoxyethylene sorbitan monooleate 0.1% Methylsilicone oil emulsion (10%) 0.1% xanthan gum Preparation as in Example 15. Example 18 30.0% Active ingredient according to Example 4 1.0% polyoxyethylene sorbitan monooleate 10.0% sucrose 0.5% Methylsilicone oil emulsion (10%) 58.0% water 0.5% isoamyl Preparation according to Example 13.

Example 19

Active ingredient according to Example 5 30.0%

Polyoxyethylene sorbitan monooleate 10%

Sucrose 10.0%

Methylsilicone oil emulsion (10%) 0.5%

Water 58.0%

Isoamyl acetate 0.5% Preparation according to Example 13.

Example 20

Active ingredient according to Example 11. 93.0%

Sucrose. 6.4%

Fatty alcohol polyoxyethylene ether phosphate ester 0.25%

Polyoxyethylene sorbitan monooleate 0.15%

Lecithin 0.15%

Isoamyl acetate 0.05% Preparation according to Example 13.

For feeding suitable preparations Example 21

Active ingredient according to Example 8 a 79.95%

Sucrose 20.0%

Isoamyl acetate 0.025%

Ethyl acetate 0.025%

The preparation is prepared by mixing the components.

Example 22

Active ingredient according to Example 8b 50.0%

mixed blossom honey 50.0%

Example 23

Active ingredient according to Example 11 80.0%

Sucrose 20.0%

Preparation according to Example 21.

Suitable for impregnating preparations Example 24

Active ingredient according to Example 8 a 98.0% sucrose 1.95%

Isoamyl acetate 0.025%

Ethyl acetate 0.025%

Preparation according to Example 21.

Powdered preparations Example 25

Active ingredient according to Example 4 90.0%

Silica gel with large surface area 8.0%

Sucrose 2.0%

The preparation is prepared by mixing the components and then grinding to a particle size below micron.

Example 26

Active ingredient according to Example 5 80.0%

Silica gel with large surface area 8.0%

Sucrose 2.0%

Preparation according to Example 25.

Example 27

Active ingredient according to Example 9 90.0%

Talc 9.0%

Sucrose 1.0%

Preparation according to Example 25.

Example 28

Active ingredient according to Example 6 50.0%

Silica gel with large surface area 10.0%

, Talc 35.0%

Sucrose 5.0% Preparation according to Example 25.

Example 29

Active ingredient according to Example 7 60.0%

Silica gel with large surface area 10.0%

Talc 35.0% sucrose 5.0%

Preparation according to Example 25.

Example 30

Working in the manner described in Example 29, but using the active ingredient of example wettable powder (WP preparations)

Example 31

Active ingredient according to Example 4 35.0%

Silica gel of large surface 4.0%

Sodium N-methyltaurimide 1.0%

The components are mixed together and then ground to a particle size below 40 microns.

Example 32

The procedure is as described in Example 31, but using the active ingredient according to Example

Example 33

Active ingredient according to Example 6 95.0%

Kaolin 4.0%

Sulphite lye powder 0.5%

Cg-Cg fatty alcohol sulfate Na on one

Silica gel carrier with large surface 0,5%

Preparation according to Example 31.

Example 34

The procedure is as described in Example 33, but using the active ingredient according to Example

Example 35 98.0% Active ingredient according to Example 9 0.6% sucrose 1.0% Silica gel with a large surface 0.5% polyoxyethylene sorbitan monooleate Preparation as in Example 31. Example 36 99.5% Active ingredient according to Example 12 0.5% polyoxyethylene sorbitan monooleate Preparation as in Example 31. Example 37 aerosol preparation 40.0% Active ingredient according to Example 11 3.0% sucrose 0.1% Nonylphenol polyglycol etherEO = 10 1.0% lecithin 0.1% isoamyl 0.1% butyl 55,7% propellant

The sucrose and the other additives are dissolved with stirring in the active ingredient, the solution is filled in aerosol cans. These are closed with a valve and then filled with propellant gas. Finally, the spray head is placed.

Biological effect

Example 38

Examination of the acaricidal action of the cell-free fermentation broth according to Example 8a against the honeybee (Apis mellifera) parasitic Asian bees mite Varroa jacobsoni. Three strong bee colonies in nine frames, which were moderately infected with Varroa, were treated three times, three days apart, with the cell-free fermentation broth Example 8a treated. In each case, 60 ml of broth-free fermentation broth were sprayed evenly onto the surfaces of the honeycombs and the bees there using a hand-held atomiser. Three days after the last treatment, each of the colonies was treated with an incense stick containing 30 mg of amitraz (ANTIVAR adus vet.). For comparison, three colonies were treated at the same time only with ANTIVAR incenses (30 mg Amitraze). The number of fallen mites and the dead bees in the prey were counted. In addition, the general condition, the behavior of the peoples and the queens were observed.

On the bottom plate of the prey was a made of aluminum sheet, provided with a 6mm high edge Bodoneinlage laid by a located at ground level in the wall of the prey, lockable slot could be pulled out or reinserted at any time without the prey had to be disassembled , This ground insert was used to collect the fallen mites.

The number of dead bees, maggots and pupae was determined by means of a trap. This corpse trap is a box of aluminum sheet, open at the top and at the back, with a pull-out inlay at the top, covered with 8 mm mesh at the top. This net has the task of preventing the bees from carrying their dead from the hive. Although the bees could crawl through the mesh, but not burdened with a dead bee; so the device gave the opportunity to count the dead at any time. The rear open part of the trap was placed on the exit opening of the stick.

The behavior of the bees within the hive, the "voice", the roaring of the people, the movement of the bienon on the frame, the behavior of the individual bees was observed by the removal and viewing of the frames made in each treatment.

The health of the queens and their behavior were also controlled by observation. The observation extended to the behavior of the bees forming the royal court, the Queen's movement, the integrity of her wings (bites), and the manner in which suitable cells were sought for oviposition. The continuity of the brood process, from which the activity of the queen can be deduced, was controlled. The inhabited honeycomb streets were determined. As inhabited honeycomb road was the area located between two adjacent honeycomb, when the two facing honeycomb surfaces were covered to at least 70-80% with bees. By means of a manually operated air atomizer (temperature 23-24 ⁰ C) were applied to the entire surface of nine frames of the three colonies (honeycomb gas Official surface of a nation: 27216cm ²⁾ to each treatment 60ml of the undiluted, according to Example 8 freed from the cells fermentation broth sprayed whereby the bees evenly covered the honeycomb surface during the treatment. The amitraz-containing incense sticks (ANTIVAR) used in the control colonies were folded in a wedge shape, placed in the aluminum treatment dish, lighted and pushed into the casserole. The excursion opening was kept closed for one hour.

Tested parameters

a) Number of mites dropped after the treatment: determination after 24, 48 and 72 hours after treatment

b) Bee dying: Determination every three days, immediately before starting treatment

c) Behavior of peoples and queens, number of inhabited honeycomb roads: control on the occasion of the treatments, taking apart the prey.

Number of fallen mites

treatment Amitraze Time after the a total of: 2. 24 a total of: 3. 24 a total of: 4. 24 a total of: Number of fallen mites 2.VoIk 3.VoIk (30 mg) Treatment (h) 48 48 48 1.VoIk with fermentation broth 72 72 72 43 31 1. 24 74 12 10 48 5 2 0 72 1 57 41 80 52 34 • 47 1 3 6 1 1 0 54 38 53 77 47 62 9 7 12 0 1 2 86 55 76 134 94 225 4 11 24 1 0 0 139 105 2Ί9

-12- 299 150 Number of fallen mites in the reference treatment

treatment Time after the a total of: Number of fallen mites 2.VoIk 3.VoIk Treatment (h) 1.VoIk Amitraze (30 mg) 241 190 1. 24 a total of: 164 12 3 48 3 1 0 72 0 254 193 167 87 37 2. 24 a total of: 68 3 1 48 0 1 0 72 0 91 38 68 35 24 3. 24 a total of: 21 2 1 48 0 0 2 72 0 37 27 21 55 32 4. 24 57 0 4 48 4 0 1 72 1 55 37 62

Bee mortality: The number of dead bees found in the traps corresponded to the normal mortality common among peoples. A significant increase in mortality was not caused by the treatments. Behavior: In the general condition and the pitting of the peoples and queens was by the treatments no morbid To observe change. The strength of the peoples, judged by the number of inhabited lanes, deteriorated

Not.

Example 39 Toxicological studies on bees The acute Irtale toxicity was investigated: p.o. the cell-free fermentation broth produced according to Example 8a on the honeybee Apis mellifera. From the cell-free fermentation broth according to Example 8a, a dilution series was prepared with base solution (see later),

whose first member contained 1.00% fermentation broth and whose last member was 0.25% fermentation broth.

Composition of the basic solution: Sugar (granulated sugar) 20,0g

Acetone 5.0 ml

distilled water ad 100,0ml

Per 0.2 ml of solutions of dilution series the dose contained for 10 bees. Thus, 10 000 bees accounted for 500.1000

or 2000 mg.

The experimental bees were at the time of intense excursion from the brood combs of three strong bee colonies by careful Taken off and placed individually in a plastic container provided with a perforated lid. Every dose

was studied in six groups of 10 bees each. Each represented three peoples 2 groups.

The bees turned away from the brood combs and immediately transported to the laboratory were stunned with carbon dioxide, in Groups of 10 bees each and then in made of galvanized wire mesh, 10cm high cylinder of 5cm diameter

set. The wire mesh has a mesh size of 4 mm. The cylinders are closed on one side with aluminum foil, on the other with a cork covered with aluminum foil. In the middle of the cork was as a feeding place one

Well of about 1 cm in diameter, 0.4mm deep and 0.5ml in volume. About the recess were in

3-4 mm apart thin wires attached to each other.

One hour after the CO ₂ anesthesia, 0.2 ml of the broth containing the fermentation broth was added Serial dilution with injection syringe into the feeding well (6 groups per dose). The Control group received 0.2ml each of the used sugar-containing basic solution. Four hours after feeding, the Bees consumed the liquid. Then they were fed ad libitum with sugar syrup. For even distribution

The study substance was not only caused by the one-hour starvation of the bees, but also by the

Habit of the bees to feed each other in the social exchange of food. For the time of the observations, the laboratory was darkened, even at the feeding times was just just the Working necessary light intensity used. The room was not used for other purposes. The temperature was 23-24 ° C,

the relative humidity 55-70 ¹ ; '?. and this control room was checked every 3 hours.

evaluation Mortality was determined at 4.24 and 48 hours after administration of the sugar juice containing the active ingredient. There at Bees that were torn from their habitual environment, from the community of the bee colony, an increased Mortality (primarily because of the stings they offset each other) is natural Corrected doses of mortality values obtained by the mean mortality values of the control groups; in the

The following effect tables show the corrected mortality rates.

It can be seen from the table that the po LD _M value is more than 200.0 micrograms / bee (this corresponds to a value of

more than 2.0 g / kg bee weight). The LD _W value of the active substance Amitraze is 12 micrograms / bee.

Acute oral toxicity of the cell-free fermentation broth according to Example 8a for Apis mellifera

dose Konz.der group dead bees in the Dead per 10 animals corrected Mg / 10 fermentation broth No. individual groups in 48 h mortality bees in the sugar juice 4 24 48 (Average) w / v% Hours after feeding %

0.25

0.67

0.00

0.50

0 0 0 0 0 0

0 0 0 0 0 0

0.83

1.7! "

1.00

1 0 0 0 1 1

1.00

3.57

control

0.00

1 0 0 0 0 0

1 0 0 0 1 0

0.67

Example 40

The effect of the fermentation broth against the mite Varroa jacobsonl was studied on mites infested with mites (Apis mellifera). For this purpose, a solution was prepared from the fermentation broth according to Example 3 b with 50% sugar solution containing 2.5% fermentation broth. The bees for the experiment were collected at the time of intense excursion from the brood combs of strong bee colonies by gentle sweeping in plastic containers provided with a perforated lid. The bees were immediately taken to the laboratory and anesthetized with carbon dioxide. The bees were examined with the magnifying glass and the bees attacked by the Milb9 were placed in a 10 cm high, 8 cm diameter, cylinder made of galvanized wire mesh of 4 mm mesh closed at one end with aluminum foil. At the bottom of the zvlindor was a 3-5cm long honeycomb strip. On this, the above-mentioned reading was applied with an injection syringe in an amount of 5-1OmI. The bees could eat it ad libitum. For the duration of the experiment, the laboratory was darkened and not used for other purposes. The temperature was 23-24 ⁰ C, the relative humidity 55-70%.

evaluation

12 hours after administration of the active substance-containing sugar solution, the fallen mites were counted and the bees after stunning with CO ₂ for still adhering mites examined. It was found that all mites had been destroyed and dropped to the bottom of the cage. The sugar solution containing the fermentation broth according to Example 3 b, used for feeding, had killed the mites 100% within 12 hours.

Claims (9)

1. Acaricidal agent, characterized in that it contains as active ingredient optionally liberated from the cell fermentation broth or the extract of the optionally liberated from the cell fermentation broth or the solid (for example dried) forms of optionally liberated from the cells fermentation broth of the bacillus NCAIM 001083 or of the pseudomonas strain NCAIM 001086 or the mixed micropopulation of the two strains (NCAIM - National Collection of Agricultural and Industrial Microorganism, Budapest, Hungary), if desired in sterilized form, optionally together with solid and / or liquid diluents and surfactants. Agent according to claim 1, characterized in that it contains as active ingredient the Bacillus strain NCAIM001083. Agent according to claim 1, characterized in that it contains as active ingredient the pseudomonas strain NCAIM 001086. 4. Bacillus strain, deposited under the number 001083 at the National Collection of Agricultural and Industrial Microorganism, Budapest, Hungary, characterized in that it forms dull light gray colonies of 1-2 mm on simple nutrient agar and has the following properties: z) ONPG positive. 5. Mixed micropopulation, characterized in that it contains the Bacillus strain NCAIM 001083 according to claim 4 and the pseudomonas strain NCAIM 001086. 6. A process for the production of an acaricidal active ingredient by aerobic or partially anaerobic fermentation carried out in the presence of carbohydrates, inorganic nitrogen and other additives known per se in a liquid nutrient medium characterized in that the mixed micropopulation of the strains NCAIM 001083 and 001086 is fermented. 7. A process for the preparation of an acaricidal active ingredient by in the presence of carbohydrates, inorganic nitrogen and other additives known per se in a liquid nutrient medium made aerobic or partially anaerobic fermentation, characterized in that fermenting the Bacillus strain NCAIM 001083. 8. A process for the preparation and maintenance of the Bacillus strain NCAIM 001083 or the mixed micropopulation of this strain formed with the Pseudomonas strains, characterized in that - extracts soil samples with physiological saline, - transfers the extract to a source of carbon and nitrogen, mineral salts and, where appropriate, additives containing nutrient agar (nutrient agar), - incubated at 25-30 ⁰ C 2-6 days - the resulting colonies are isolated, if desired, the resulting mixed micropopulation is separated or - isolated the individual strains (subcultures) from the mixed micropopulation on differential diagnostic media by re-separation and maintained in a conventional manner. 9. A method for the destruction of mites, characterized in that the soil or , livestock or other animals with one of the means according to claims 1-3 or treated with their diluted forms of application. 10. The method of claim 9 for combating mites, in particular for combating Varroa jacobsoni, characterized in that treating the honeybee or its stick or the frame of the stick with one of the means according to claims 1-3 or their diluted forms of application. 11. The method according to claim 10, characterized in that one feeds or soaks the honeybees with an agent according to any one of claims 1-3 containing impregnation or feeding means, preferably evenly and over a long time.