CZ2021561A3 - Preparation for repelling the spruce weevil - Google Patents

Abstract

A preparation for the repellence of the spruce weevil (Ips typographus) containing a mixture of anti-attractants from the group: trans-conopthorin, 1-octen-3-ol, 3-octanol, 1-hexanol, 1,8-cineole and trans-4-thujanol, and this in an amount corresponding to the evaporation coefficient of trans-conopthorin from 0.3 to 0.6 mg/day, 1-octen-3-ol from 15 to 25 mg/day, 3-octanol from 15 to 25 mg/day, 1-hexanol from 15 to 25 mg/day, 1,8-cineole from 25 to 50 mg/day and trans-4-thujanol from 1 to 30 mg/day. In a preferred embodiment, the preparation contains trans-4-thujanol in an amount corresponding to an evaporation coefficient of 10 to 30 mg/day. The preparation does not contain verbenone and is in the form of a mixture of anti-attractants or a set of individual anti-attractants intended for joint use.

Description

Preparation for the repellence of the spruce weevil

Field of technology

The invention relates to the protection of conifers of the pine family (Pinaceae), in particular the Norway spruce (Picea abies), and specifically relates to a preparation for the repellence of the spruce weevil (Ips typographus).

Current state of the art

The spruce weevil (Ips typographus; Coleoptera) is currently the most significant economic pest of spruce stands in Central Europe. For the purposes of this application, if the text refers to "spruce gobbler" in the text, it means spruce gobbler (Ips typographus). The most endangered are above all the spruce stands formed by its preferred wood species, Norway spruce (Picea abies) grown outside the areas of natural distribution. For the purposes of this application, if "spruce" is mentioned further on in the text, it means Norway spruce (Picea abies). In most cases, the goshawk mainly attacks weakened trees, such as fells and uprooted trees, which have a lower ability to resist the attack of the goshawk. In the case of abiotic influences, such as recurring periods of drought or wind calamities, there is a significant exponential spread of this pest. This pest then also attacks healthy trees or other types of conifers. This leads not only to the death of individual trees, but also to the devastation of very large areas of spruce stands. The sap-eater is more likely to be found on mature trees, as it needs a thicker layer of sap for larval development. It attacks spruces of older age classes, i.e. 60 years and above. It passes younger growth and appears here only when there is a strong overgrowth. The gobbler communicates with members of its species by producing an aggregation pheromone. This lures both the male and the female to be able to overcome the host tree's defenses during a joint raid. The goshawk can also distinguish suitable or unsuitable habitat for reproduction by its sense of smell. Some chemical substances produced, for example, by deciduous trees, or other sources from their niche, have a repellent effect against gorse-eaters.

The prescribed way to prevent the overgrowth of the sycamore population is systematic search, marking, registration, felling of trees and timely treatment, including sanitation of infested wood. The supporting means used for the management and monitoring of the goshawk population are the concentration and extermination of the goshawk in foci of smolts and other endangered places. For this, the laying of traps is used. Lapák is a felled healthy spruce that is attractive to a goshawk attack. In addition, poisoned traps are used, where trees or logs are treated with insecticide, or pheromone traps, in which the aggregation pheromone of the goshawk is placed.

The disposal of infested wood in an attempt to prevent the next generation of sycamores from leaving the infested tree is carried out by mechanical techniques, primarily by removing such wood from the forest, by chipping it, or by removing branches, for example. In disaster situations, chemical sanitation is also applied by spraying the attacked trunks with insecticides. Due to the serious economic and ecological consequences of the overpopulation of sycamores, new methods of conifer protection are being dynamically developed.

A more ecologically acceptable alternative to the use of synthetic insecticides in the management of goshawks can be the application of so-called semiochemicals. These substances either attract goshawks, such as a pheromone, or repel them, such as specific repellents or anti-attractants naturally occurring in the ecological niche of goshawks. Forest protection methods based on a combination of both the attraction of the pheromone and the repellency of these repellent-acting anti-attractants have been developed for harmful bark beetle species on the American continent.

The purpose of anti-attractants is to repel bark beetles from healthy, uninfested trees on which these anti-attractants are applied. At least one anti-attractant is applied near the tree

- 1 CZ 2021 - 561 A3 or on it. An anti-attractant is a substance that, with its smell, repels gorse-eaters from healthy, uninfested trees. Such a solution is described, for example, in document US 6217891 Bl. The document describes a number of volatile substances with anti-attractive effects against bark beetles, especially species from the American continent Dendroctonus ponderosae, Dendroctonus rufipennis, Dendroctonus pseudotsugae, Ips pini, and Dryocoetes confusus. V documents a repellent composition containing at least one component from the group: toluene, pentanol, 2-hexanone, 3-hexanone, heptanal, benzaldehyde, 2-hydroxycyclohexanone, benzyl alcohol, (£)-ocimene, salicylaldehyde, conophthorin, guiacol, nonanal, methyl salicylate , decanal, thymol methyl ether, (£)-nerolidol or dendrolasin, at least one component of: substances from green leaves selected from: hexanal, (£)-2-hexenal, 1-hexanol, (£)-2-hexen-l-ol , (Z)-2-hexen-1-ol, and (Z)-3-hexen-1-ol, and at least one component from: verbenone and 3-methylcyclohex-2-en-1-one. Substances are used alone or in the form of a mixture. One of the components of the mentioned mixture is verbenone, which is a substance produced by trees in the event of a full occupancy of the gorse-eater or related species of bark beetles. This substance prevents other gobblers from attacking the same tree.

A study of the use of anti-attractants specifically to affect the mortality of spruce trees due to whitefly attack was described for a mixture containing substances trans-conophthorin, substances from leaves (1-hexanol) and bark of deciduous trees (l-octen-3-ol and 1-octanol) and a substance occurring when the tree is fully occupied by a bark beetle, namely verbenone (Schiebe C, et. al., Semiochemical diversity diverts bark beetle attacks from Norway spruce edges, Journal of Applied Entomology, 2011, 135: 726-737). The reduction in infestation on the plots ranged from 35 to 76% compared to the control plots, but a so-called switch effect was observed, where the application of anti-attractants, especially verbenone, can uncontrolled push the gorse-eaters onto trees outside the treated plots.

Another article describes the testing of the effectiveness of repellent preparations for sycamores, while these repellent preparations were composed either of verbenone alone or of its mixtures with the addition of non-host alcohols, substances from leaves, or substances from the bark of deciduous trees. (Jakuš, R., et al., Overview of development of an anti-attractant based technology for spruce protection against Ips typographus: From past failures to future success, Anzeiger fur Schadlingskd., 2003, 76: 89-99). This article mainly dealt with experiments with individual substances that took place in the field with the aim of protecting trees of the pine family (Pinaceae). For the purposes of this application, if "the pine family" is mentioned further in the text, you mean the pine family (Pinaceae).

The disadvantage of the preparations described above is that they all contain verbenone, which is a substance produced when the tree is fully occupied and exhausted by the gorse. Verbenone is also formed when coniferous trees are occupied by other species of bark beetles. Therefore, if one tree is treated with this preparation, it is a signal to the sycamore that the treated tree is already occupied and exhausted and it should look for a healthy tree in the vicinity.

The task of the invention is to prepare such a preparation based on anti-attractants to protect the pine family and specifically spruces against attack by the sycamore, which would be suitable in forestry for the protection of extensive forest stands. The preparation would also be suitable for the protection of the plant walls in stands of the pine family, which are the usual first targets of the gorse-eater in the stands, and thus form foci of its spread. Furthermore, the task of the invention is to prepare a preparation against attack by the whitefly, which can be used in arboriculture or, for example, in the protection of individual spruces in public parks, or can be used to protect trees from other pine families. The preparation should have a larger radius of action in forests than the preparations used so far, i.e. it should act on trees in the wider vicinity of the application of the product, and it should be less likely to cause the emergence of new outbreaks beyond the protection radius, while its effect on repelling the gorse-eater should be maintained or increased. An increase in the radius of action would also reduce the cost of preparation and production of the preparation.

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The essence of the invention

The set task is solved with the help of a preparation for the repellency of the lycophagus, which contains at least one anti-attractant from the group: /ram-conopthorin. 1-octen-3-ol, 3-octanol, 1-hexanol and 1,8-cineole. The essence of the invention is that the preparation contains precisely all anti-attractants from the group: /ram-conopthorin. 1-octen-3-ol, 3-octanol, 1-hexanol, 1,8-cineole and that the preparation also contains 5-4-thujanol in an amount corresponding to the evaporation coefficient from 1 to 30 mg/day. The addition of just such an amount of ra«5-4-thujanol ensures an increased effect of the inhibitory effect of the preparation against the lycophagus.

The term "inhibitory effect" is defined as a percentage reduction of goshawk captures, if a defined dose of goshawk pheromone is combined with a tested mixture of anti-attractants in the pheromone trap for the same defined amount of pheromone placed in the trap separately.

In a preferred embodiment, the preparation contains /ram-conopthorin in an amount corresponding to the evaporation coefficient from 0.3 to 0.6 mg/day, it also contains an alcohol component including 1-octen-3ol, 3-octanol and 1-hexanol in an amount corresponding to the evaporation coefficient of alcohol components from 45 to 75 mg/day, it also contains 1,8-cineole in an amount corresponding to an evaporation coefficient of 25 to 50 mg/day and řraws-4-thujanol in an amount corresponding to an evaporation coefficient of 10 to 30 mg/day. The product with such an amount corresponding to the selected evaporation coefficient ensures a synergistic effect of all substances and therefore increases the protection of healthy trees of the pine family, and the radius of action of the product is further increased compared to the use of a combination of selected anti-attractants, mainly due to the addition of raws-4-thujanol and the absence of verbenone. The preparation emits a repellent signal to such an extent that the gorse does not even attack trees beyond the boundary of the protection zone.

The term "evaporation coefficient" is defined as the mass of a substance that evaporates in a day under standard conditions, i.e. an air temperature of 22 °C and a wind speed of 0.5 m/s.

The exact definition of individual anti-attractants is specified in the following tab. 1:

Tab. 1: Chemical substances used in the tree protection mixture, identified by the CAS registration number, taken from the "chemical abstract service registry number, hereinafter referred to as CAS. The table also shows the CAS enantiomers of these substances, for which the biological activity for the spruce weevil was determined.

Substance CAS of substances used for spruce protection Name of biologically active enantiomers CAS of biologically active enantiomers /ram-conopthorin 73046-13-6 (5S,7S)-[(E)-7-methyl-1,6dioxaspiro [4.5] decane] 68108-90-7 77715-03-8 1-octen-3-ol 3391-86-4 (-)-1-octen-3-ol 3684-48-7 3-octanol 589-98-0 (+)-3-octanol 22658-92-0 1-Hexanol 111-27-3 1,8-cineole (eucalyptol) 470-82-6 ørawsA-thuj anol (sabinene hydrate) 546-79-2 (+)-rra«5-4-thuj anol 7712-82-5

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In a preferred embodiment, the alcohol component of the preparation contains 1-octen-3-ol in an amount corresponding to an evaporation coefficient of 15 to 25 mg/day, 3-octanol in an amount corresponding to an evaporation coefficient of 15 to 25 mg/day, and 1-hexanol in the amount corresponding to the evaporation coefficient from 15 to 25 mg/day.

In an advantageous embodiment, the preparation is in the form of a mixture of anti-attractants intended for formulation in one container, especially for use on trees of the pine family older than 50 years. In another advantageous embodiment, the preparation is in the form of a set of individual anti-attractants intended for joint use, while the individual substances are stored in individual containers, which are subsequently placed on trees of the pine family so that containers containing each of the above are present on each selected tree of the pine family of the described substances.

In another preferred embodiment, the preparation also contains an antioxidant in an amount of from 0.3 to 0.7% by weight. The antioxidant used is 2,6-di-tert-butyl-4-methylphenol or 2-(1,1-dimethylethyl)1,4-benzenediol.

The preparation is suitable for repelling the gorse-eater from trees of the pine family. Advantageously, the preparation can be used for the repellence of the sycophant from the spruce.

The subject of the invention is also a method of application of the preparation, in which the preparation containing a mixture of anti-attractants is placed in a receptacle, or individual anti-attractants from a set of anti-attractants intended for joint use are placed in individual receptacles forming a set of receptacles. A memory stick or a set of memory sticks is then placed on the surface of individual pine tree trunks.

In an advantageous embodiment of the method of application of the preparation, the memory stick or a set of memory sticks is placed on the surface of a tree of the pine family at a height of 1.5 to 5 m. need to use a ladder for an authorized person.

In another advantageous embodiment of the method of application of the preparation, the mixture of anti-attractants is placed on the surface of a tree of the pine family, which is older than 50 years and is part of an involved forest stand consisting of more than 50% trees of the pine family. The trees on which the memory sticks or a set of memory sticks are located are no more than 15 m apart.

The term "connected forest stand" is defined as a stand of trees in which their aerial parts touch each other, grow through or overlap, and the circumference of the trunk of individual trees measured at a height of 130 cm above the ground does not exceed 80 cm; if any of the trees in the stand exceeds the stated dimensions, it is always considered as an individual tree.

In another advantageous embodiment of the application of the preparation, the mixture of anti-attractants is placed on the surface of a tree of the pine family, which is older than 50 years and is part of the first or long row of the growth wall away from the clearing. The trees on the surfaces of which the traps or a set of traps are located are separated from each other by a maximum of 15 m. Such a distance provides protection for the trees of the pine family in the case of low, medium and high levels of attack of the tree stand by the sycamore in the given area.

The term "stand" is defined as an area-contiguous part of the forest, differing in age, age or spatial composition, forest category or different management. The minimum area of stands is at least 0.20 ha.

The term "growth wall" is defined as the edge of an involved forest stand, which was created as a result of the action of harmful agents, or as a result of the removal of a part of the stand, whereby the trees on this edge are exposed to changed conditions that can cause different types of stress.

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In another advantageous embodiment of the method of application of the preparation, the mixture of anti-attractants is placed in screeds or a set of screeds on the surface of individual trees of the pine family, on fragments of stands, on free-standing trees, or on displays to protect them from being attacked by a whitefly. The trees do not form a connected stand and are in any age range. Such an arrangement and distribution of the culverts is suitable in the case of prevention of free-standing trees from the pine family or vegetation fragments made up of trees of this family from being attacked by the sycamore.

The term "fragments of stands" is defined as the rest of stands that have been destroyed by harmful agents to a degree greater than 50%.

The term "exhibition" is defined as a solitary tree, intentionally left in a clearing. They are usually individuals of generative, seminal origin, extraordinary vitality, malleability and quality.

The advantages of the preparation for the repellency of the lycophagus according to the present invention consist mainly in the fact that it is based on anti-attractants, with the addition of the substance increasing the effectiveness /ram-4-thujanol. and without verbenone added so far. As a result, the preparation has a larger radius of action in forests than the preparations tested so far, i.e. it also acts on the trees in the vicinity of the application of the product, while maintaining or increasing its effect on repelling the gorse-eater. Another advantage of the mealybug repellent composition of the present invention is that the increased radius of action also reduces the cost of preparation and production of the composition. Another advantage of the product is that it is suitable for the protection of trees against attack by the sycamore in forestry, especially for the protection of involved forest stands and fresh growth walls in spruce stands, which are the usual first targets of the sycamore in the stands, and thus form foci of its spread, or for the protection of exhibitions in clearings. In addition, the product is suitable for use in arboriculture or, for example, in public parks, where it is necessary to protect individual spruces, as well as individual trees from other pine families, or fragments of stands or groups of trees of this family from being attacked by sycamores.

Clarification of drawings

Said invention will be further explained in the following drawings, where:

Fig. 1 shows a graph of averages ± standard deviations of relative captures of walleye, showing the synergistic effect of the substances of which the preparation is composed;

Fig. 2 shows a graph of the means ± standard deviations of the relative captures of walleye, showing the optimization of the appropriate dose of the addition of /ram-4-thiijanohi to the mixture of selected anti-attractants, expressed by the coefficient of evaporation;

Fig. 3 shows a graph for the number of captured individuals of the goshawk in traps containing the preparation according to the invention together with the aggregation pheromone and control traps containing the aggregation pheromone alone during the protection of forest fragments;

Fig. 4 shows a graph of the number of spruces attacked by the sycamore in 10 classes of distance from two rows of trees on fresh growth walls that were treated with the product.

Examples of implementation of the invention

General procedure for Examples 1 and 2 in which the efficacy of test substances was studied using pheromone traps

The studies were carried out at the Forest School School of the Czech University of Life Sciences in Kostelec nad Černými lesy, southeast of Prague, in the Czech Republic. The experiment took place from June 14, 2021 to

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06/28/2021, in a clearing created after the clearing of trees attacked by the lycophagus in 2020. The clearing was 300 m long and 100 m wide. The forest surrounding the clearing was mainly composed of mature spruce trees approximately 70 years old.

Ecotrap barrier pheromone traps from Fytofarm, Slovakia, were set up in a row at a distance of 15 m from each other, replicating the growth wall of a spruce forest at a distance of 30 m. The traps were mounted on wooden poles so that they were suspended at a height of 1.5 m above the ground . Goshawks were lured into the traps with a goshawk aggregation pheromone mixture consisting of Pheagr IT Extra (SciTech s.r.o., Czech Republic) and a dose of pheromone that was combined in different traps with different compositions of tested anti-attractant mixtures, which according to their effectiveness reduced the number of captured bran eaters. The tested variants of the mixtures dispensed from the dispensers, as shown in tab. 3 and tab. 6, were regularly moved from one trap to the next in order to exclude the effect of the placement of the traps on the pastures for the capture of gobblers. The rotation scheme for moving the tested substance mixtures was randomized according to the software-generated Latin Square scheme, an example is tab. 2. Designation A to G is for individual combinations of primers from which the tested mixtures were composed. Displacement of the test mixtures occurred when there were at least 15 gobblers in the traps containing the mixture of anti-attractants and pheromone. The mixture changes in the traps were the same amount as the tested mixture variants (seven for example 1 and six for example 2) and were repeated after two to five days, depending on the rate of catch of gobblers, depending on the weather and their density. Before each exchange of the mixtures in the traps, the gobies were caught in individual traps, removed and stored in bottles with ethanol. After transport to the laboratory, the number of gophers caught for individual variants of the tested mixtures was counted.

Tab. 1: Randomization of the exchange of the tested mixtures according to the scheme of the software-generated Latin square.

Trap position AND II III IV IN VI VII Bait exchange 1 F G C B E D AND 2 B AND F C G E D 3 D B E F AND C G 4 C D AND E B G F 5 E F G AND D B C 6 G C B D F AND E 7 AND E D G C F B

Example 1: Testing the synergistic effect of substances in a patented mixture

For the individual components of the product, experimental vaporizers with a specified nominal value were developed, and their actual evaporation coefficient was determined in the laboratory by the gravimetric method under standard conditions, i.e. temperature 22 °C, wind speed 0.5 m/s, shown in tab. 3.

In a field experiment in pheromone traps, the effectiveness of the inhibitory effect on the capture of walleyes was tested for 7 different variants of the "mixture of anti-attractants" baits, as shown in tab. 4. The bait of all AAs consisted of decoys according to tab. 3 for all selected anti-attractants and in the trap it was combined with the pheromone of the goby, consisting of the preparation Pheagr IT Extra (SciTech s.r.o., Czech Republic) and a dose of pheromone (tab. 3). Partial baits were tested in other traps, where one of the substances was always removed from this full mixture. There were four such baits, when individual substances or groups of them (-Ci, -NHV-ol, -tC, -t4ol) were taken out, and in the traps they were also combined with the goby pheromone. The composition of the individual baits tested is shown in tab. 4. The inhibitory effect of all these baits containing different combinations of anti-attractants was compared with captures on the same dose of pheromone (Pher H) placed in another trap. In the attempt

-6CZ 2021 - 561 A3 were further tested by captures in an empty trap (Blank) as a check that the gobblers did not fall into the traps randomly. According to the randomization tab. 2 according to the scheme of the Latin square, 7 relocations of the bait variants were carried out and thus 7 repetitions when the gobys were removed from the traps. The dosage of anti-attractants used was reduced to obtain more accurate data on the inhibitory effect of the mixture.

The term "all AAs" is defined as a mixture containing precisely all anti-attractants from the group: /raws-conopthorin, 3-octanol, l-octen-3-ol, 1-hexanol, 1,8-cineole and /raws-4- thujanol.

The following abbreviations are used for the individual anti-attractants in the mixture:

- /ram-conopthorin. abbreviation: tC,

- 3-octanol, abbreviation: 3ol, l-octen-3-ol, abbreviation: acetic,

- 1-hexanol, abbreviation: hex,

1,8- cineole, abbreviation: Ci,

- /raws-4-thujanol, abbreviation: t4ol.

- Non-host alcohols: NHV-ols.

Abbreviations for other lure variants:

- Pheromone of the spruce bark beetle: Pher H.

- Empty control trap: Blank.

These abbreviations will also be used in other examples.

Tab. 2: Design of precise countermeasures used in testing the synergistic effect of anti-attractants in the mixture of which the product is composed.

Semiochemicals Evaporation coefficient [mg/day The number of memory sticks used in the field trial Design of experimental memories Nominal value Laboratory means ± SD iraws-conophthorin (tC) 0.5 0.5 ±0.2 1 100 μΐ Microcaps® glass capillary sealed at one end with dental wax, filled with liquid 1 mm from the open edge, suspended horizontally NHV-ols: 1-hexanol (hex), 3-octanol (3ol), 1-octen-3-ol (oct) 3 3.5 ±1.3 1 open 730 PE bottle (9730, Kartell, Italy): 6 mm ID, 29 mm height, filled with 0.5 ml of a mixture of alcohols 1:1:1 v/v with antioxidants tert-Butylhydroquinone TBHQ 0.5% v/v, BHT - butylated hydroxytoluene 0.5% v/v 1,8-cineole (Ci) 1 0.7 ± 0.1 2 clear glass autosampler vial 2 ml screw cap with PTFE silicone septum (Agilent USA) 2 mm orifice in septum iraws-4-thujanol (t4ol) 1 1.6 ±0.3 2 cellulose wadding 2.5 x 5.5 x 0.25 cm sealed in PE film 0.1 mm, filled with 200 mg of trans-4thujanol dissolved in pentane, sealed after pentane evaporation

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2-methyl-3-buten-2ol 100 42.2 ±20 1 closed PE bottle (731, Kartell, Italy) with a 2 mm hole in the cap czs-verbenol 1.5 ±0.8 1 closed PE bottle (731, Kartell, Italy) 9 mm with a hole in the cap Pheagr IT Extra 55 ±15 1 ipsdienol ± (S)-cri-verbenol 4 ± 42 g/kg in solvent 2-methyl3-buten-2-ol/ penetration through PE membrane

Tab. 3: Composition of tested variants in baits for testing the synergistic effect of the mixture of anti-attractants from the preparation.

Semiochemicals Combination of tested anti-attractants in baits AND B C D E F G all AAs -Whose -NHV-ols -tc -t4ol PherH Blank /ram-conopthorin (tC) X X X - X - - Alcohols (NHV-ols): 1-hexanol (hex), 3-octanol (3ol), 1-octen-3-ol (oct) X X - X X - - 1,8-cineole (Ci) X - X X X - - ira«5-4-thujanol (t4ol) X X X X - - - gobbler pheromones: 2-methyl-3-buten-2-ol X X X X X X - czs-verbenol X X X X X X - PherH X X X X X X -

A total of 8,266 gobblers were captured in the entire experiment over the course of seven replicates, of which 1,102 ±1,281 (mean ± standard deviation or SD) gobblers per replicate. Relative captures were calculated as the ratio of the number of gobies caught on a specific bait variant to the sum of all gobies caught in all seven traps during one repetition/bait change. Means ± SD of relative captures are shown for comparison in Fig. 1.

Statistic methods:

Statistically, the inhibitory effects of individual bait variants containing anti-attractant to pheromone were compared. Testing of differences compared to pheromone is shown only for the full mixture or all AAs, comparison of other variants was not the aim of the experiment. The difference in mean catches for the bait variants tested was determined using their pooled standard deviations, or SD, when the uncorrected “effect size” and the “Hedges corrected effect size h” hereafter “Hedges h” (Hedges 1981; Maher et al. 2013; Nakagawa) were calculated and Cuthill 2007). The value of Hedges h, is considered strong if it is >0.8.

Results:

The full mixture of anti-attractants or "all AAs" showed the greatest inhibitory effect on goshawk captures compared to pheromone captures. Captures on “all AAs + pheromone” were 96 ±2% lower than on pheromone alone, with a high Hedges h value of -8.7.

The mixture from which ira«5-4-thujanol was removed had an inhibitory effect lower than the full mixture, where the captures for "-t4ol + pheromone" were 88 ± 4% lower than the captures for pheromone. It showed similar values

-8CZ 2021 - 561 A3 mixture from which three non-host alcohols, collectively called - NHV-ols, were extracted, while the captures for "-NHV-ols + pheromone" were lower by 84 ±8% than the captures for pheromone alone.

In the comparison of the inhibitory effect of these two least effective mixtures to all AAs, removal of - t4ol (Hedges h 2.5) and removal of - NHV-ol (Hedges h 2.4) had the greatest effect on reducing the effect. The confidence intervals of Hedges h overlapped for these mixtures, shown in tab. 5.

It was found that the mixture of anti-attractants, including the addition of irα-5-4-thujanol, has a demonstrable inhibitory effect on the captures of gophers on their aggregation pheromone. The addition of irα-5-4-thujanol significantly increases the inhibitory effect of the preparation compared to preparations that did not contain this anti-attractant.

Tab. 4: Uncorrected and corrected Hedges effect sizes for comparing the inhibitory effects of a preparation containing a mixture of anti-attractants with individual anti-attractants removed against a preparation containing a mixture of anti-attractants according to the present invention.

Tested mixtures Average relative catch (%) SD Average difference United SD Uncorrected size and effect corrected Hedges effect sizes h 95% confidence interval h p for CIs not including 0 LCI UCI all AAs 2.3 1.6 -67.6 7.1 -9.5 -8.7 12.4 -6.8 $ $ $ A mixture not containing: -Whose 4.8 1.9 2.5 1.8 1.4 1.3 0.1 2.6 * -NHV-ols 10.8 4.4 8.4 3.3 2.6 2.4 0.9 3.8 ** -tc 4.0 3.0 1.7 2.4 0.7 0.6 -0.5 1.8 NS - t4ol 8.1 2.5 5.7 2.1 2.7 2.5 1.0 4.0 ** Controlling traps PherH 70.0 10, 0 ON ON ON ON Blank 0.0 0.0 ON ON ON ON

Example 2: Optimizing the Dose of ira«5-4-thujanol

The aim was to evaluate the effect of different doses of irα-5-4-thujanol on the inhibitory effect of a mixture of anti-attractants reducing the capture of gophers on their pheromone and to determine the optimal effective dose of this substance added to the basic mixture.

The term "basic mixture" or "5AA" is defined as a mixture containing precisely all anti-attractants from the group: /ram-conopthorin. 3-octanol, 1-octen-3-ol, 1-hexanol, 1,8-cineole and does not contain /ram-4-thujanol.

For the individual components of the preparation, experimental detonators with a determined nominal value were developed, and their actual evaporation coefficient was determined in the laboratory by the gravimetric method under standard conditions, i.e. temperature 22 °C, wind speed 0.5 m/s, as shown in tab. 6.

In a field experiment in six pheromone traps, the effectiveness of six different bait variants was tested, shown in tab. 7. Four variants contained a base mix of anti-attractants "5AA": trans-conophthorin (tC), NHV-ols: 1-hexanol (hex), 3-octanol (3ol), l-octen-3-ol (oct) and 1,8-cineole (Ci), always in combination with a pheromone, composed of the preparation Pheagr IT Extra (SciTech s.r.o., Czech Republic) and a dose of pheromone (tab. 6). In one of the variants, a tank with irα-5-4-thujanol (50t4ol) was added in an amount corresponding to an evaporation coefficient of 50 mg/day, in another tank 30t4ol was added in an amount corresponding to an evaporation coefficient of 30 mg/day, and in the third 10t4ol in an amount corresponding to the coefficient evaporation 10 mg/day. In the fourth variant there was no t4ol,

-9CZ 2021 - 561 A3 contained only a basic mixture of anti-attractants in combination with a pheromone. The experiment was supplemented with a variant where the pheromone itself and an empty trap (Blank) were tested. The composition of the individual tested combinations of the mixture is shown in tab. 7. According to the randomization tab. 2 according to the scheme of the Latin square, six relocations of the bait variants were carried out, and therefore six repetitions when the gobys were removed from the traps.

Tab. 5: Accurate memories with individual anti-attractants contained in the preparation used in optimizing the dose of tra«5-4-thujanol.

Semiochemicals Evaporation coefficient [mg/day The number of memory sticks used in the field trial Design of experimental memories Nominal value Laboratory means ± SD traw-conopthorin (tC) 0.5 0.5 ±0.2 1 100 μΐ Microcaps® glass capillary sealed at one end with dental wax, filled with liquid 1 mm from the open edge, suspended horizontally NHV-ols: 1-hexanol (hex), 3-octanol (3ol), 1-octen-3-ol (oct) 3 3.5 ±1.3 1 open 730 PE bottle 9730, Kartell, Italy): 6 mm ID, 29 mm height, filled with 0.5 ml of a mixture of alcohols 1:1:1 v/v with antioxidants tertButylhydroquinone TBHQ 0.5% v/v, BHT butylated hydroxytoluene 0, 5% v/v 1,8-cineole (Ci) 1 0.7 ± 0.1 2 clear glass autosampler vial 2 ml screw cap with PTFE silicone septum (Agilent USA) 2 mm orifice in septum trans-4-thujanol 40 50 48.3 ±13.2 1 cellulose wadding 5.5 x 11 x 0.2 cm sealed in PE film 0.045 mm, filled with 800 mg transAthujanol dissolved in 12 ml pentane, sealed after evaporation of pentane trans-4-thujanol 5 30 16.1 ±5.1 2 cellulose sponge square 7.5 x 3.5 x 0.25 cm sealed in PE foil, thickness 0.1 mm, filled with 200 mg trans- of 4-thujanol dissolved in 3 ml of pentane, sealed after evaporation of the pentane

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trans-4-thujanol 2 10 9.4 ±5.5 2 cellulose sponge square 2.5x5.5x0.25 cm sealed in PE film, thickness 0.1 mm, filled with 200 mg of trans4-thujanol dissolved in 1 ml of pentane, which was evaporated before sealing gobbler pheromones: 2-methyl-3-buten-2ol 100 42.2 ±20 1 closed PE bottle (731, Kartell, Italy) with a 2 mm hole in the cap c/x-verbenol 1.5 ±0.8 1 closed PE bottle (731, Kartell, Italy) with a 9 mm hole in the cap Pheagr IT Extra 55 ±15 1 ipsdienol ± (Sj-czverbenol 4 ± 42 g/kg in solvent 2-methyl-3buten-2-ol/ penetration through PE membrane

Tab. 6: Composition of tested variants in baits for optimizing the dose of ira«5-4-thujanol in a preparation containing a mixture of anti-attractants.

Semiochemicals Combination of tested anti-attractants in baits AND B C D E F 50t4ol±5A A±Phe 30t4ol±5A A±Phe 10t4ol±5A A±Phe 5AA±Phe Phew Blank /ram-conopthorin (tC) X X X X - - Alcohols (NHV-ols): 1-hexanol (hex), 3-octanol (3ol), 1-octen-3-ol (oct) X X X X - - 1,8-cineole (Ci) X X X X - - ira«5-4-thujanol 50 X - - - - - ira«5-4-thujanol 30 - X - - - - ira«5-4-thujanol 10 - - X - - - gobbler pheromones: 2-methyl-3-buten-2-ol X X X X X - czs-verbenol X X X X X - PherH X X X X X -

The /ram-4-thujanohi dosage optimizations are shown in Fig. 2, where the relative catches ± SD of gobblers on individual bait variants are shown. 5AA: a combination of five memory recallers with anti-attractants /ram-conophthorin (tC), NHV-ols: 1-hexanol (hex), 3-octanol (3ol), l-octen-3-ol (oct) and 1,8-cineole ( Ci) in combination with the gobbler pheromone; 10t4ol: addition of repellents with trans-410 thujanol lOmg/day to the 5AA mixture in combination with the goby pheromone; 30t4ol: addition of memory boosters with íraws-4-thujanol 30 mg/day to the 5AA mixture in combination with gobbler pheromone; 50t4ol: the addition of memory boosters with /ram-4-thujanolcm 50 mg/day in combination with the pheromone of the gobbler; Pher H: gobbler pheromone, Blank: empty trap.

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Tab. 7: Uncorrected values of the effect of the addition of tra«5-4-thujanol to optimize the amount of added dose of /ram-4-thujanol to the mixture of five other anti-attractants from the preparation.

Variant vs. Pher H Variant with t4ol vs 5AA Bait variant Average relative catch (%) SD Average difference United SD Effect size Average difference United SD Effect size 50 t4ol 1.60 1.00 -77.90 9.46 -8.24 -11.00 6.30 -1.80 30 t4ol 1.60 1.20 -78.00 9.55 -8.17 -11.10 6.40 -1.70 10t4ol 4.60 5.70 -75.00 11.80 -6.40 -8,10 8.60 -0.90 0t4ol (AA=5) 12.60 11.50 -66.90 14.70 -4.60 0.00 5.70 0.00 PherH 79.50 17.90 ON ON Blank 0.00 0.10 ON ON

After comparing the captures of gobblers on the individual tested bait variants, a significantly stronger inhibitory effect against the pheromone was shown for preparations containing a mixture of anti-attractants with the addition of a certain amount of /ram-4-thujanol than anti-attractants without it. Captures of anti-attractants with the addition of /ram-4-thujanol with an evaporation coefficient of 50 mg/day 50t4ol decreased by 98 ±1%, in the variant with the addition of 30 mg/day 30 t4ol by 97 ±3% and in the variant with the addition of 10 mg/ day 10 t4ol by 92 ±12% of captured gobblers compared to gobblers caught on pheromone. On the anti-attractants alone without the addition of /ram-4-thujanol 5AA, captures were only 80 ±29% lower than on the pheromone.

The results were statistically evaluated after comparing the calculated uncorrected "effect sizes", i.e. both from the cumulative standard deviations of captures on mixtures with anti-attractants compared to captures on pheromone, as well as on the comparison of only mixtures of anti-attractants with the addition of trans-4thujanol against the mixture of anti-attractants alone, shown in tab. 8.

All variants containing anti-attractants had high uncorrected "effect sizes" compared to the pheromone and thus have a demonstrable inhibitory effect. When comparing the effectiveness of the variants with the addition of /ram-4-thujanol to the mixture of anti-attractants alone, the highest uncorrected "effect size" value of -1.8 and -1.7 was calculated for the variants with the addition of trans-4-thujanol 50 and 30 mg/day. Catches for these two variants did not differ significantly from each other. Lower "effect sizes" of -0.9 were shown by the variant with the addition of 10 mg/day /ram-4-thujanol.

After evaluating these findings, it was summarized that adding an amount of /ram-4-thujanol with an evaporation coefficient of 10 mg/day to a mixture of five other anti-attractants significantly improved the inhibitory effect, adding 3 and 5 times higher doses of /ram-4-thujanol continued increased the inhibitory effect, whereas the inhibitory effect of the two higher doses was not statistically different, so the addition of more than 30 mg/day /ram-4-thujanol did not increase the inhibitory effect of the resulting mixture contained in the preparation.

Based on these findings, the optimal dose of /ram-4-thujanol added to the preparation was determined, i.e. in an amount corresponding to the evaporation coefficient in the range of 10 mg/day to 30 mg/day.

Example 3: Testing the protection of fragments of young pine trees

Twelve fragments of forest stands with 251th spruce were selected, which were surrounded by sufficient space for the flight of the goshawk. At each location, two Theysohn barrier pheromone traps were placed against the vegetation wall of each fragment at a distance of less than 1 m from the line of exposed trees, and at a distance of 15 to 20 m from each other. In one pheromone trap, a bait with only the pheromone Pheroprax A® was placed. BASF AG (GE) in the second trap then a combination of the same dose of pheromone in combination with a selected mixture of anti-attractants

-12 CZ 2021 - 561 A3 contained in the preparation, namely: /ram-conopthorin. 1-octen-3-ol, 3-octanol, 1-hexanol, 1,8-cineole and irα-5-4-thujanol, which were stored in individual containers, according to tab. 9, to evaluate their protective effect.

For the individual components of the preparation, experimental vaporizers with a determined nominal value were developed, and their actual evaporation coefficient was determined in the laboratory by the gravimetric method under standard conditions, i.e. temperature 22 °C, wind speed 0.5 m/s, shown in tab. 9.

Both baits, the aggregation pheromone and the product, were placed in the traps on 07/06/2021, the baits of the product containing anti-attractants were replaced on 26/07/2021. shorter, in the wet season relatively longer Fig. 3, but always after a maximum of 1 week.

The experiment took place at the School Forestry of the Czech University of Life Sciences in Kostelec nad Černými lesy in the southeast of Prague, in the Czech Republic, part of Bohumile, forest stand 442G2, in the period from 06/07 to 07/20/2021. It involved an extensive spruce monoculture with an area of 8 ha, which was created after afforestation of the wind-damaged area. The age of the tree in the stand was 25 years, the average height was 15 m, the diameter at breast height was approximately 15 cm, while in the selected section there are also several sections with younger trees. The 442G2 stand area was not entirely forested, but was divided by a mosaic of forest meadows and logging roads.

Two criteria were evaluated:

1. Number of infested trees located near pheromone traps versus near pheromone traps in combination with the product.

2. Number of gobblers caught in traps with only pheromone versus traps with pheromone-inhibited preparation.

Tab. 8: Precise decoys with individual anti-attractants contained in the preparation used to protect fragments of young growth of the pine family.

Semiochemicals Evaporation coefficient [mg/day Memory foam design Nominal Laboratory means ± SD trans-conophthorin (tC) 0.5 0.5 ±0.2 100 μΐ Microcaps® glass capillary sealed at one end with dental wax, filled with liquid 1 mm from the open edge, suspended horizontally NHV-ols: 1-hexanol (hex), 3-octanol (3ol), 1-octen-3-ol (oct) 70 66.1 ±1.5 cellulose wadding 10 x 10 x 0.25 cm sealed in PE film 0.1 mm, filled with 12 ml of a mixture of alcohols 1:1:1 v/v with antioxidants tertButylhydroquinone TBHQ 0.5% v/v, BHT butylated hydroxytoluene 0.5% v/v 1,8-cineole (Ci) 30 45.9 ±4.7 cellulose wadding 5 x 2.5 x 0.25 cm sealed in a 0.045 mm microtene film, filled with 2 ml of 1,8-cineole with antioxidants tert- Butylhydroquinone TBHQ 0.5% v/v, BHT Butylated hydroxytoluene 0.5% v/v

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iraws-4-thujanol (t4ol) 10 2x5.1 ±1.6 cellulose wadding 7.5 x 3.5 x 0.25 cm sealed in PE film 0.1 mm, filled with 200 mg transAthujanol dissolved in 3 ml pentane, sealed after pentane evaporation gobbler pheromones: Pheroprax A®, BASF AG (GE) 50 55 ±1 ipsdienol (3.56 g/kg) + (S)-cA-verbenol (35.59 g/kg) in solvent 2-methyl-3buten-2-ol in ampoules, penetrates the plastic wall of the ampoule

A total of nine inspections were carried out: 11.6.; 17.6.; 21.6.; 24.6.; 28.6.; 1.7.; 8.7.; 15.7. and 20.7. Wrens caught in individual traps in these data were selected from the traps and stored in 95% ethanol for later counting in the laboratory.

In the same data, the number of trees attacked by the sycamore in a 15 m radius around both traps was also evaluated. Infestation was checked by looking for sinkholes of gorse eaters on the trunk and checking for food in the relevant part of the goblet.

A generalized linear mixed model, or GLMM, was used for statistical evaluation, and a random regression line was created for each site. The comparison of the influence of the bait on the number of captured goby was carried out with regard to the specificity of each location. The negative binomial distribution, or NB, was used for the dependent variable, which was taking on a frequency character, due to the considerable heterogeneity of the variance, which is not allowed by the use of the Poisson distribution (Zuur & léno 2015). The analysis was performed in the R software environment (R Core Team 2020). The glmmTMB package (Brooks et al. 2017) was used to build the regression model. The procedure of Nakagawa & Schielzeth (2013) was used to calculate the conditional and group coefficient of determination for the generalized linear mixed model.

During the entire experiment, 74,422 goshawk adults were captured in Theysohn traps, of which 74,403 individuals were caught in traps containing aggregation pheromone alone, and only 19 goshawks were caught in traps containing anti-attractant, which is less than 1%. The highest capture in pheromone traps (32,729 individuals) was detected in the period between 18.6. and 21.6., when the second phase of the spring swarming of the goshawk took place. An overview of the captures according to the individual control dates is shown in Fig. 3. The number of gophers captured in traps containing a mixture of anti-attractants was significantly lower than in traps containing aggregation pheromone alone (GLMM NB: N = 216, df = 4, p < 0.001 , R ² = 0.988).

A total of 18 trees infested with lycophagus were found in a 5 m radius around the traps baited with the aggregation pheromone alone, and no infested trees were found near the traps with a combination of pheromone and preparation. Infested trees were found during an inspection on 17.6., when 11 trees were involved, during an inspection on 21.6. then another 7 trees were found.

Less than 1% of the total number of caught goby was caught in traps containing the preparation according to the invention. The difference in the number of captured individuals between traps with the preparation according to the invention and traps containing only aggregation pheromone was statistically significant. Likewise, the number of infested trees in the vicinity. The preparation according to the invention was able to reliably protect standing young spruces in a fragmented stand from attack by the whitefly.

Example 4: Testing the protection of pine trees by placing a mixed repellent with anti-attractants containing a solution of all active substances on tree trunks on the growth wall

The testing took place in a pre-selected area in forest stands with a predominance of spruce as described in the introduction. For the experiment, south-facing forest walls were selected, which remained after the logging carried out in the previous year in 2020 in mature forest spruce stands (older than 80 years).

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The vegetation walls were located in an area with a high probability of being attacked by a lycophagus. Nine experimental blocks were selected, spaced from several hundred meters to several kilometers apart, with a total of 190 trees treated with anti-attractant decoys. The coordinates of individual locations are shown in tab. 10. In one experimental block, there were ten trees in the first row and ten trees in the second row on fresh growth walls at the edge of the forest. For each tree selected for treatment, one trap with the anti-attractant mixture was installed on the north side of the trunk at a height of 2 m from the ground.

To protect the vegetation walls, 7.5 x 15 cm hoppers with a plastic carrier for soaking compounds were used, while the permeable membrane was a polyethylene film. The traps were filled with a liquid mixture of anti-attractants with a total evaporation coefficient of all substances of 90 mg/day. The evaporation coefficient was determined gravimetrically under the experimental conditions. The memory filling mixture consisted of 1,8-cineole (Ci) in the amount of 1516 mg, racemic /ram-conophthorin (tC) in the amount of 15.37 mg, NHV-ols (1-hexanol (hex) in the amount of 1012 mg, 1-octen-3-ol (oct) in the amount of 1012 mg and 3-octanol (3ol) in the amount of 1516 mg) and ira«5-4-thujanol (t4ol) in the amount of 51 mg, all in the ratio hex:oct:3ol: C1:tC:t4ol 0.2:0.2:0.3:0.003.

From this ratio, the estimated evaporation coefficient of the individual components of the mixture is 63 mg/day for NHV-ols (1-hexanol 18 mg/day, l-octen-3-ol 18 mg/day; 3-octanol 27 mg/day), 1, 8-cineole 27 mg/day, /ram-conophthorin 0.3 mg/day with ira«5-4-thujanol 1 mg/day.

Tab. 9: GPS coordinates of experimental locations.

Latitude Longitude 1. 49°58'10.44N 14°49'42.95 E 2. 49°58'8.17N 14°51T8.46E 3. 49°57'34.62 N 14°49'37.90 E 4. 49°55'37.17 N 14°50'59.90 E 5. 49°54'56.07 N 14°52'38.25 E 6. 49°56'4.48N 14°52'36.26 E 7. 49°56'14.61N 14°52'10.08 E 8. 49°56'18.75N 14°54'48.33 E 9. 49°55'28.35 N 14°53'4.15E

The experiment was started before the swarming of the goshawk on 07/05/2021 and ended on 30/07/2021. Goshawk raids were monitored at several weekly intervals in the vicinity at a distance of 100 m from the treated trees.

The results in Fig. 4 show that the treatment with the preparation was effective in protecting against the attack of lycophagus up to a distance of 40 m from the installed dispensers containing the preparation composed of anti-attractants according to the invention. Unlike the previous experiments, there was no significant "switch" effect. Treated trees and trees within 40 m of treated trees were not attacked. The most attacked trees were monitored at a distance of 60 to 70 m from the trees treated with the preparation according to the invention.

Industrial applicability

The preparation for repellent of the sycamore according to the present invention can be used in forest stands and isolated trees or fragments of stands containing trees of the pine family, as a protection of healthy trees, especially spruce, against attack by the sycamore.

Claims (13)

PATENT CLAIMS 1. Preparation for the repellence of the spruce weevil Ips typographus containing at least one anti-attractant from the group: /ram-conopthorin. 1-octen-3-ol, 3-octanol, 1-hexanol and 1,8-cineole, characterized by the fact that it contains precisely all anti-attractants from the group: /ram-conopthorin. 3-octanol, 1-octen-3-ol, 1-hexanol, 1,8-cineole and also contains trans·-4-thujanol in an amount corresponding to the evaporation coefficient from 1 to 30 mg/day. 2. The preparation according to claim 1, characterized in that it contains: /rans-conopthorin in an amount corresponding to the evaporation coefficient from 0.3 to 0.6 mg/day, an alcohol component including l-octen-3-ol, 3-octanol and 1-hexanol in the amount corresponding to the evaporation coefficient of the alcohol component from 45 to 75 mg/day, 1,8-cineole in the amount corresponding to the evaporation coefficient from 25 to 50 mg/day, and ira«5-4-thujanol in the amount corresponding to the evaporation coefficient from 10 to 30 mg/day. 3. The preparation according to claim 1 to 2, characterized in that the alcohol component includes l-octen-3-ol in an amount corresponding to an evaporation coefficient of 15 to 25 mg/day, 3-octanol in an amount corresponding to an evaporation coefficient of 15 to 25 mg /day and 1-hexanol in the amount corresponding to the evaporation coefficient from 15 to 25 mg/day. 4. The preparation according to claims 1 to 3, characterized in that it has the form of a mixture of anti-attractants or a set of individual anti-attractants intended for joint use. 5. The preparation according to one of claims 1 to 4, characterized in that it also contains the antioxidant 2,6-di-tert-butyl-4-methylphenol or 2-(1,1-dimethylethyl)-1,4-benzenediol. 6. The preparation according to claim 5, characterized in that the antioxidant is contained in an amount of from 0.3 to 0.7% by weight. 7. Use of the preparation according to one of claims 1 to 6 to repel the spruce weevil Ips typographus in trees of the pine family Pineacea. 8. Use according to claim 7, for the repellence of the spruce borer Ips typographus in Picea ahies spruce trees. 9. Method of application of the preparation according to one of claims 1 to 6, characterized in that the mixture of anti-attractants is placed in a receptacle, or individual anti-attractants from a set of anti-attractants intended for joint use are placed in individual receptacles forming a set of receptacles, and subsequently the memory stick or a set of memory sticks is placed on the surface of a tree of the pine family Pinaceae. 10. The method of application of the preparation according to claim 9, characterized in that the reminder or a set of reminders is placed on the surface of a tree of the pine family Pinaceae at a height of 1.5 m to 5 m from the ground. 11. The method of application of the preparation according to claims 9 to 10, characterized in that the reminder or a set of reminders is placed on the surface of a tree older than 50 years of the pine family Pinaceae, which is part of an involved forest stand containing more than 50% of trees of the pine family Pinaceae, while the trees , on which the memory devices are located, are no more than 15 m apart. 12. The method of application of the preparation according to claims 9 to 10, characterized in that the scavenger or set of scavengers is placed on the surface of a tree older than 50 years of the pine family Pinaceae, which is part of the first or long row of the vegetation wall away from the clearing, while the trees on which memory sticks are located, they are separated by a maximum of 15 m. - 16CZ 2021 - 561 A3 13. Method of application of the preparation according to claims 9 to 10, characterized in that the repellent or a set of repellents is placed on the surface of selected isolated trees, or fragments of stands or on a group of stand-alone trees, while the trees on which the repellents are placed do not form a connected stand .