USE OF DENATONIUM BENZOATE AS A SEED TREATMENT FOR CROPS AS BIRD AND/OR INSECT REPELLENT

Abstract

The present invention relates to the protection of seed and plants from damage caused by birds and/or insects. Treatment with denatonium benzoate provides seeds which are protected against birds, especially corvids, and insects, especially wireworms.

Claims

1. A seed, treated with denatonium benzoate, wherein the denatonium benzoate is adsorbed on the seed and/or absorbed into the seed.

2. The seed according to claim 1, wherein the seed is coated with denatonium benzoate, optionally in combination with one or more further active agents and/or additives.

3. The seed according to claim 2, wherein the further active agents are selected from phytochemicals and antimicrobial agents, in particular bactericides, antiparasitics and fungicides, such as chili extract, black cumin oil, yucca extract, Mentha pulegium extract, aluminium ammonium sulfate, hops extract, fat encapsulated plant extracts, alginate encapsulated plant extracts, tefluthrin, metalaxyl, fludioxonil, prothioconazole, and combinations thereof, preferably Redigo M and/or Maxim XL, and/or wherein the additives comprise sticking agents, preferably sticking agents based on natural resins, sticking agents based on lignin sulfonate, sticking agents based on polymers and/or water-based sticking agents such as Sepiret®, Peridiam® Quality or Peridiam® Active.

4. The seed according to claim 1, wherein denatonium benzoate is present in an amount effective to provide protection against bird damage, in particular against bird from the genus Corvus (corvids) such as crows, ravens, rooks, and jackdaws, and/or protection against damage caused by insects, in particular wireworms, grain beetle or cabbage stem flea beetle.

5. The seed according to claim 1, wherein the germination capacity of the seed is comparable with untreated seed.

6. A plant or a part thereof grown from the seed according to claim 1, in particular wherein the plant or a part thereof contains denatonium benzoate, and optionally in addition one or more further active agents and/or additives selected from phytochemicals and antimicrobial agents, in particular bactericides, antiparasitics and fungicides, such as chili extract, black cumin oil, yucca extract, Mentha pulegium extract, aluminium ammonium sulfate, hops extract, fat encapsulated plant extracts, alginate encapsulated plant extracts, tefluthrin, metalaxyl, fludioxonil, prothioconazole, and combinations thereof, preferably Redigo M and/or Maxim XL, and/or wherein the additives comprise sticking agents, preferably sticking agents based on natural resins, sticking agents based on lignin sulfonate, sticking agents based on polymers and/or water-based sticking agents such as Sepiret®, Peridiam® Quality or Peridiam® Active.

7. The plant or a part thereof according to claim 6, wherein the plant is i) a seedling and contains denatonium benzoate in a mean concentration of 1 to 200 ppm, preferably 1 to 50 ppm, more preferably 5 to 30 ppm, even more preferably 10 to 22 ppm, in the entire seedling; or ii) a seedling and contains denatonium benzoate in a mean concentration of 50 to 4000 ppb, preferably 50 to 2500 ppb, more preferably 100 to 1500 ppb, even more preferably 200 to 800 ppb, in sprout tissue of the seedling; or iii) a plant in the development stage V6-V8 and contains denatonium benzoate in a mean concentration of 0.5 to 200 ppb, preferably 0.5 to 100 ppb, more preferably 1 to 50 ppb, even more preferably 5 to 20 ppb, in leaf tissue.

8. A method of using denatonium benzoate for protecting seed or plant, preferably a seedling, from damage caused by birds and/or insects, optionally in combination with one or more further active agents and/or additives, preferably as defined in claim 3.

9. A method of protecting a seed against damage caused by birds and/or insects, the method comprising contacting the seed with denatonium benzoate, optionally in combination with one or more further active agents and/or additives, preferably as defined in claim 3.

10. The method according to claim 9, wherein denatonium benzoate is used in corn in an amount of 0.1 to 70 g/50,000 seeds, 0.1 to 50 g/50,000 seeds, 0.1 to 20 g/50,000 seeds, 0.1 to 10 g/50,000 seeds, or 0.1-5 g per 50,000 seeds, in particular 0.2-2 g per 50,000 seeds or wherein denatonium benzoate is used in oilseed rape in an amount of 2 to 200 g/1.5 million oilseed rape seeds (corresponds to ˜7 kg seeds), 2 to 100 g/1.5 million oilseed rape seeds, 2 to 50 g/1.5 million oilseed rape seeds, 2 to 20 g/1.5 million oilseed rape seeds or 2 to 10 g/1.5 million oilseed rape seeds, in particular 2 to 25 g/1.5 million oilseed rape seeds; or wherein denatonium benzoate is used in rye in an amount of 10 to 1500 g/100 kg rye seeds, 10 to 1000 g/100 kg rye seeds, 10 to 750 g/100 kg rye seeds, 10 to 500 g/100 kg rye seeds or 250 g/100 kg rye seeds, in particular 10 to 75 g/100 kg rye seeds or 10-150 g/100 kg rye seeds.

11. The method according to claim 9, wherein denatonium benzoate is used as a solid powder in crystalline pure form or as a liquid composition further comprising a sticking agent.

12. The method according to claim 11, wherein the liquid composition comprises denatonium benzoate in a concentration of about 1-800 g/l, about 1-500 g/l, about 1-250 g/l, about 1-100 g/L, in particular 10-50 g/L, preferably 13-27 g/L.

13. A method of providing a plant protected against damage caused by birds and/or insects, the method comprising a) providing a seed treated with denatonium benzoate, wherein the denatonium benzoate is adsorbed on the seed and/or absorbed into the seed, or a seed obtained or obtainable according to the method of claim 9, and b) growing a plant from the seed of step a).

14. The method according to claim 13, wherein the plant in step b) is a plant or a part thereof grown from the seed, in particular wherein the plant or a part thereof contains denatonium benzoate, and optionally in addition one or more further active agents and/or additives selected from phytochemicals and antimicrobial agents, in particular bactericides, antiparasitics and fungicides, such as chili extract, black cumin oil, yucca extract, Mentha pulegium extract, aluminium ammonium sulfate, hops extract, fat encapsulated plant extracts, alginate encapsulated plant extracts, tefluthrin, metalaxyl, fludioxonil, prothioconazole, and combinations thereof, preferably Redigo M and/or Maxim XL, and/or wherein the additives comprise sticking agents, preferably sticking agents based on natural resins, sticking agents based on lignin sulfonate, sticking agents based on polymers and/or water-based sticking agents such as Sepiret®, Peridiam® Quality or Peridiam® Active.

15. A method for controlling damage caused by birds and/or insects on seeds or plants in an agricultural area, comprising the following steps: a) sowing seeds treated with denatonium benzoate, wherein the denatonium benzoate is adsorbed on the seed and/or absorbed into the seed, or seeds obtained or obtainable according to the method of claim 9 on an agricultural area, and b) growing plants from the seeds, wherein one or both steps a) and b) are performed under conditions of infestation with birds and/or insects and insect larvae; wherein the plant stand count on said agricultural area is higher than under the same conditions on a comparable agricultural area with untreated seeds or plants grown from untreated seeds, in particular wherein the plant stand count on said agricultural area is at least 5% or at least 10%, preferably at least 20% and more preferably at least 30% higher than on a comparable agricultural area with untreated seeds or plants grown from untreated seeds.

Description

FIGURES

[0067] FIG. 1A shows the plant stand counts on individual trial locations for corn seed treated with denatonium benzoate or combinations of denatonium benzoate with various common fungicides compared with corn seed treated with common bird repellents and control

[0068] FIG. 1B shows the plant stand counts summed up over all locations.

[0069] FIG. 2A shows an array of plant pots where four corn seeds have been sown per pot.

[0070] FIG. 2B shows the placing of wireworms of genus Agriotes in a central cavity of a plant pot.

[0071] FIG. 3 is a bar graph showing the plant biomass for corn plantlets that have developed from differently treated seeds (variants 1-10) in greenhouse.

[0072] 1: negative control (no seed treatment)

[0073] 2: sticker alone (control)

[0074] 3, 4: Bitrex

[0075] 5: RedigoM

[0076] 6-8: Bitrex and RedigoM

[0077] 9: RedigoM and Force 20 CS (positive control)

[0078] 10: RedigoM and Poncho (positive control)

[0079] FIG. 4: shows bar graphs of the assessment of seed damage by wireworm for corn plantlets that have developed from differently treated seeds (variants 1-10) in greenhouse (A) and climate chamber (B).

[0080] FIG. 5 shows variance analyses of plant stand count at three different location (A-C). Denatonium benzoate alone (DNB) or denatonium benzoate in combination with a solution of micronutrients (DNB+HA450) mixed with a sticker have been applied to the seeds. Three chemical references have been included in the trials: Korit, Sphere and Schwefal. Control: untreated seeds

EXAMPLES

[0081] 1. Evaluation of the Efficacy as a Bird Repellent.

[0082] To evaluate the efficacy of different substances as a bird repellent, 25 locations throughout Europe have been identified with a potentially high pressure of bird damage. 17 different substances (DSM RD9103 BRP, Chili extract, Black Cumin oil, Yucca extract, Mentha pulegium extract, Aluminium-Ammonium-sulfate, Hops extract, fat encapsulated plant extracts, alginate encapsulated plant extracts, Tefluthrin, Thiram, Ziram, Methiocarb, yellow colour substance, denatonium benzoate, SatecWFO20, Redigo M, Maxim XL) have been chosen to evaluate differences on feeding behavior of crows. Three chemical references have been included in the trial: Mesurol liquid (Active substance Methiocarb), Korit (Active Substance Ziram) and TMTD (Active Substance Thiram). Mesurol liquid (Methiocarb) was the standard bird repellent in the past, with a European expiry date of the 3.sup.rd of April 2020. TMTD (Thiram) can be used until 30.sup.th of January 2020. Korit (Ziram) expires in 2021.

[0083] Denatonium benzoate has been tested in 2 different concentrations: 0.4 g/50.000 seeds in combination with standard fungicides Redigo M and Maxim XL and 0.8 g/50.000 seeds as a stand-alone. In all combinations and variants filmcoats (sticking agents) have been used to provide an even distribution of the different substances to the seed.

[0084] In one embodiment, denatonium benzoate is used as a solid powder (CAS: 3734-33-6; a.i.=3500 ppm) in crystalline pure form. In another embodiment denatonium benzoate powder is mixed with a sticker, preferably before application, and then preferably a concentration of 13.14 g per liter to 26.28 g per liter have been applied.

TABLE-US-00001 TABLE 1 Exemplary mixture of compounds in a seed treatment: fungicide Maxim XL 12.5 ml/u Metalaxyl (10 g/l) 1.05 g/ml (13.13 0.125 g a.i./u MTL FLU g/u) Fludioxonil (25 g/l) 0.313 g a.i./u fungicide RedigoM 15 ml/u Metalaxyl (20 g/l) 1.05 g/ml 16.1 g/u 0.30 g a.i./u MTL PRO Prothioconazole (100 g/l) 1.5 g a.i./u Repellent Bitrex 0.2-5 g/u Denatoniumbenzoat powder ai = 3500 ppm CAS: 3734-33-6 sticker Sepiret9290red 30 ml/u 1.20 g/ml (36 g/u)

[0085] In particular, the sticker is selected from Sepiret®, Peridiam® Quality or Peridiam® Active, especially preferred the sticker is selected from Sepiret 9290 red or Peridiam® Quality 316 https://www.seedgrowth.bayer.com/˜/media/d3ed77a4560740d8a568fa06f50129dc.ashx. Other stickers which might be used can be found on https://agriculture.basf.com/en/Crop-Protection/Seed-treatment.html.

[0086] The treating process can be based on the rotor/stator principle. The seed is accelerated by a rotating mixing cone and moves to the top of the static cylinder wall. Special bye pass blades on top of the wall guide the rising seed back down to the middle. So, a fine seed curtain is built around the chemical liquid spraying disc. The spraying disc produces a fine fog of chemical liquid which coats the inner seed curtain as well as the ascending seed at the cylinder wall.

[0087] Three different types of trial set-ups for the field trials have been placed: fully randomized microplot trials on 4 locations, non-randomized 8-row plots with three replications of the untreated control within the plots on two locations, non-randomized, 2 times replicated 1-row plots with three untreated controls on four locations and non-randomized 1-row plots with five replications of the untreated control on 16 locations.

[0088] 3 weeks after emergence, 9 locations with bird damage have been identified. Plant stand counts have been assessed and summarized. FIG. 1A shows figures on the individual trial locations, FIG. 1B shows the plant stand counts summed up over all locations.

[0089] Surprisingly, it was found that the use of denatonium benzoate as corvid repellent for corn showed an excellent effect. The efficiency was comparable or even better than the best bird repellents like Thiram, Korit and Mesurol. In addition, it has been observed that denatonium benzoate has a systemic effect on the corn plant. This results in bird protection of the seed but also the emerged young seedlings. Since the denatonium benzoate is diluted over the time of growing and ripening, the harvested crop, e.g. the cobs of corn, do not show amounts of denatonium which affect the post-harvest usage of corn, e.g. for animal feeding or silage purposes.

[0090] The bird repellent effect of denatonium benzoate has further been evaluated in additional field trials at three different location in Germany. Denatonium benzoate alone (DNB) or denatonium benzoate in combination with a solution of micronutrients (DNB+HA450) mixed with a sticker have been applied to the seeds. Denatonium benzoate has been applied in a concentration of 1 g/50.000 seeds. Three chemical references have been included in the trials: Korit (active substance: Ziram), Curb Crop Garden Powder with Sphere's formulation (active substance: aluminium ammonium sulphate) and Schwefal (active substance: organic sulfur). As negative control untreated seed has been used. All three locations showed moderate to high pressure of bird damage.

[0091] Plant stand counts have been assessed and summarized. FIG. 2A-C shows analysis of variance of the individual trial locations. Results of earlier field trials have been confirmed. It was found that the use of denatonium benzoate as corvid repellent for corn showed again an excellent effect. The efficiency was comparable with the best bird repellents like Korit and Sphere. In the field trails the use Schwefal on corn seeds has not shown any significant bird repellent effect.

[0092] 2. Evaluation of the Phytotoxicity

[0093] In order to evaluate the phytotoxicity of different corn seed treatments, safety tests were conducted. Germination rates of corn seeds treated with various concentrations of denatonium benzoate (Bitrex®) [in g per unit, where 1 unit is 50.000 seeds] were counted after five days at 25° C. Cold test germination rates (CT) were counted after 7 days at 10° C. and 5 days at 25° C. The treatments were compared to untreated seeds. Furthermore, the germination rates of corn seeds treated with denatonium benzoate in combination with further active agents, in particular fungicides, were examined under different environmental conditions. The results are shown in the following Table 2, with

KF=germination rates counted after 5 days at 25° C. and
CT=cold test germination rates counted after 7 days at 10° C. and 5 days at 25° C.

TABLE-US-00002 TABLE 2 Variety Chemical Additiv slurry ml/u KF ΔKF CT ΔCT Amavit no no 0 98.0% 85.5% Amavit no Bitrex 0.2 g/u 150 98.0% 0.0% 91.5% 6.0% Amavit no Bitrex 0.5 g/u 150 96.5% −1.5% 89.0% 3.5% Amavit no Bitrex 1.0 g/u 150 97.5% −0.5% 95.5% 10.0% Amavit no Bitrex 2.0 g/u 150 97.0% −1.0% 93.0% 7.5% Amavit no Bitrex 5.0 g/u 150 98.0% 0.0% 96.5% 11.0% Amavit RedigoM no 150 98.0% 97.0% Amavit RedigoM Bitrex 0.2 g/u 150 100.0% 2.0% 97.5% 0.5% Amavit RedigoM Bitrex 0.5 g/u 150 97.5% −0.5% 99.0% 2.0% Amavit RedigoM Bitrex 1.0 g/u 150 96.0% −2.0% 99.0% 2.0% Amavit RedigoM Bitrex 2.0 g/u 150 96.5% −1.5% 95.5% −1.5% Amavit RedigoM Bitrex 5.0 g/u 150 99.5% 1.5% 96.5% −0.5% Amavit MaximXL no 150 96.5% 100.0% Amavit MaximXL Bitrex 0.2 g/u 150 98.0% 1.5% 98.0% −2.0% Amavit MaximXL Bitrex 0.5 g/u 150 98.0% 1.5% 98.0% −2.0% Amavit MaximXL Bitrex 1.0 g/u 150 96.5% 0.0% 97.0% −3.0% Amavit MaximXL Bitrex 2.0 g/u 150 95.5% −1.0% 99.0% −1.0% Amavit MaximXL Bitrex 5.0 g/u 150 97.5% 1.0% 99.0% −1.0% KXB6432 no no 0 94.5% 92.5% KXB6432 no Bitrex 0.2 g/u 150 92.5% −2.0% 98.0% 5.5% KXB6432 no Bitrex 0.5 g/u 150 96.5% 2.0% 97.5% 5.0% KXB6432 no Bitrex 1.0 g/u 150 96.5% 2.0% 97.0% 4.5% KXB6432 no Bitrex 2.0 g/u 150 96.0% 1.5% 90.5% −2.0% KXB6432 no Bitrex 5.0 g/u 150 95.5% 1.0% 90.5% −2.0% KXB6432 RedigoM no 150 94.5% 97.5% KXB6432 RedigoM Bitrex 0.2 g/u 150 94.5% 0.0% 97.0% −0.5% KXB6432 RedigoM Bitrex 0.5 g/u 150 95.5% 1.0% 96.5% −1.0% KXB6432 RedigoM Bitrex 1.0 g/u 150 96.0% 1.5% 99.5% 2.0% KXB6432 RedigoM Bitrex 2.0 g/u 150 95.5% 1.0% 98.5% 1.0% KXB6432 RedigoM Bitrex 5.0 g/u 150 97.0% 2.5% 96.5% −1.0% KXB6432 MaximXL no 150 98.5% 99.0% KXB6432 MaximXL Bitrex 0.2 g/u 150 98.5% 0.0% 100.0% 1.0% KXB6432 MaximXL Bitrex 0.5 g/u 150 99.0% 0.5% 99.5% 0.5% KXB6432 MaximXL Bitrex 1.0 g/u 150 97.5% −1.0% 98.0% −1.0% KXB6432 MaximXL Bitrex 2.0 g/u 150 98.0% −0.5% 96.5% −2.5% KXB6432 MaximXL Bitrex 5.0 g/u 150 97.5% −1.0% 99.0% 0.0% ø no no 0 96.3% 89.0% ø no Bitrex 0.2 g/u 150 95.3% −1.0% 94.8% 5.8% ø no Bitrex 0.5 g/u 150 96.5% 0.3% 93.3% 4.3% ø no Bitrex 1.0 g/u 150 97.0% 0.8% 96.3% 7.2% ø no Bitrex 2.0 g/u 150 96.5% 0.3% 91.8% 2.8% ø no Bitrex 5.0 g/u 150 96.8% 0.5% 93.5% 4.5% ø RedigoM no 150 96.3% 97.3% ø RedigoM Bitrex 0.2 g/u 150 97.3% 1.0% 97.3% 0.0% ø RedigoM Bitrex 0.5 g/u 150 96.5% 0.3% 97.8% 0.5% ø RedigoM Bitrex 1.0 g/u 150 96.0% −0.2% 99.3% 2.0% ø RedigoM Bitrex 2.0 g/u 150 96.0% −0.2% 97.0% −0.2% ø RedigoM Bitrex 5.0 g/u 150 98.3% 2.0% 96.5% −0.7% ø MaximXL no 150 97.5% 99.5% ø MaximXL Bitrex 0.2 g/u 150 98.3% 0.7% 99.0% −0.5% ø MaximXL Bitrex 0.5 g/u 150 98.5% 1.0% 98.8% −0.7% ø MaximXL Bitrex 1.0 g/u 150 97.0% −0.5% 97.5% −2.0% ø MaximXL Bitrex 2.0 g/u 150 96.8% −0.7% 97.8% −1.8% ø MaximXL Bitrex 5.0 g/u 150 97.5% 0.0% 99.0% −0.5% ø ø no 0 96.7% 95.3% ø ø Bitrex 0.2 g/u 150 96.9% 0.3% 97.0% 1.8% ø ø Bitrex 0.5 g/u 150 97.2% 0.5% 96.6% 1.3% ø ø Bitrex 1.0 g/u 150 96.7% 0.0% 97.7% 2.4% ø ø Bitrex 2.0 g/u 150 96.4% −0.2% 95.5% 0.3% ø ø Bitrex 5.0 g/u 150 97.5% 0.8% 96.3% 1.1%

Example 3: Determination of Systemic Activity of Denatonium Benzoate

[0094] To determine, if denatonium benzoate could be systemic (taken up by the plant), a Liquid Chromatography with tandem mass spectrometry (LC-MS-MS) was performed, which is a powerful analytical technique that combines the separating power of liquid chromatography with the highly sensitive and selective mass analysis capability of triple quadrupole mass spectrometry. Therefore, we used 1) seedlings just after field emergence to determine the content of denatonium benzoate in the complete plant and in the sprout separately (Table 3), as well as 2) middle-aged plants (V6-V8), where the leaves have been investigated (Table 4).

TABLE-US-00003 TABLE 3 Results of denatonium benzoate determination in seedlings just after field emergence Weight Final Conc. μg DNB/plant or Sample Description mg mg/g ppm/μg/g ppb/ng/g sprout 1 5 complete 3245.54 0.009 9.047 9047.3 5.87 2 4 complete 2726.9 0.021 20.920 20920.4 14.26 3 4 sprout 813.36 0.001 0.782 782.1 0.16 4 4 sprout 1008.56 0.000 0.231 231.5 0.06

TABLE-US-00004 TABLE 4 Results of denatonium benzoate determination in middle-aged plants Weight Final Conc. Sample Description mg ppb/ng/g 14 DNB 0.8g_P1 leaves 3039.6 5.49 14 DNB 0.8g_P2 3098.6 5.34 14 DNB 0.8g_P3 3053.9 5.46 20 DNB 0.4g_P1 leaves 3027.6 6.61 20 DNB 0.4g_P2 3022.0 20.03 20 DNB 0.4g_P3 3021.5 12.04 22 DNB 0.4g_P1 leaves 3087.0 4.74 22 DNB 0.4g_P2 3072.1 17.26 22 DNB 0.4g_P3 3044.4 13.72

[0095] Denatonium benzoate was taken up by the plant and shows concentration differences between different plant sections/organs and development stadiums:

[0096] In early stage denatonium benzoate is selective detectable. The concentration in the complete plant is in the lower ppm range (˜10-20 ppm) and in sprouts is in the upper ppb range (˜200-800 ppb). The variation in concentration is high (Table 3). In middle-aged corn plants the concentration in leaves is in the lower ppb range (˜5-20 ppb). Variation in concentration differs between type (Inter) and plant (Intra) and concentration of DNB decrease from early stage (200-800 ppb in sprout) to mid stage (5-20 ppb in leaves) significantly.

[0097] Beside above experiments the concentration of Bitrex in the first leaf of the seedling have been measured wherein different dosages of Bitrex have been applied to the seed. The higher the application rate, the higher was the Bitrex concentration in the tested leaf tissue.

TABLE-US-00005 TABLE 4a Results of denatonium benzoate determination in leaf of seedling 0.5 g/unit 0.8 g/unit 1 g/unit 2 g/unit Bitrex 22.8 36.8 88.7 178.2 concentration 10.6 75.2 155.6 in ng/g 55.0 70.4 97.1 96.2 88.7

[0098] Further experiments on system uptake of Bitrex have been conducted also on oilseed rape (Brassica napus). Seeds of Brassica napus have been treated with concentration of Bitrex from 0.15 g/unit to 75 g/unit. Seeds have been sown and leaf samples have been taken 6 days after emergence and 22 days after emergence. By means of Liquid Chromatography with tandem mass spectrometry (LC-MS-MS) as described above Bitrex amount in leaf tissue has been determined with 4 repetition per Bitrex concentration. Leave samples have been prepared as follows: Leaf material has been harvested, cut in small pieces and put in 3-5 ml ethanol. After 45 min vortexing, centrifuging and taking aliquot of 1 ml for further analysis.

TABLE-US-00006 TABLE 4b Results of denatonium benzoate determination in seedlings. One unit of oilseed rape is 1.5 million viable seeds, typically this is ~7 kg seeds per unit. Bitrex concentation Bitrex concentation 6 days after 22 days after Application rate Repetition emergence [ng/g] emergence [ng/g] Bitrex 0.15 g/unit 1 444.74 5.58 2 633.25 7.75 3 247.30 11.03 4 215.97 10.65 Bitrex 0.75 g/unit 1 193.92 0.55 2 456.84 1.45 3 233.53 31.4 4 212.19 8.31 Bitrex 1.5 g/unit 1 487.17 4.70 2 268.41 6.71 3 359.84 13.22 4 232.40 4.93 Bitrex 7.5 g/unit 1 354.65 6.20 2 194.60 6.75 3 266.85 6.43 4 251.41 2.77 Bitrex 15 g/unit 1 484.53 1.32 2 621.16 2.74 3 564.00 1.26 4 1200.81 6.12 Bitrex 75 g/unit 1 2791.25 2.78 2 4414.30 12.52 3 2193.04 12.41 4 2358.35 6.18

[0099] The results (Table 4b) show that Bitrex has been taken up into the tissues of the seedling. The amount of Bitrex in the leaf tissue was dependent on the amount of Bitrix applied to the seeds. Interestingly after 6 days at concentrations from 0.15-7.5 g/unit the uptake of Bitrex is quite comparable. At concentrations of 15 g/unit and 75 g/unit a significant higher amount of Bitrex have been observed. After 22 days for all applications Bitrex was still detectable but at a low amount. That indicates that Bitrex is further distributed in the plantlets and also degraded. However, in particular in the early seedling phase the amount of Bitrex in the plants is quite high and provides the desired repellent effect against birds and insects and reduces respective damages. Simultaneously, due to further distribution and degradation the adult plant is almost free of Bitrex.

[0100] Additional experiments in hybrid rye show also excellent systemic uptake and distribution of Bitrex. Seeds of a hybrid rye variety have been treated in application A with KintoPlus 150 ml/100 kg seeds and Bitrex 75 g/100 kg seeds and in application B with KintoPlus 150 ml/100 kg seeds and Bitrex 150 g/100 kg seeds. Seeds have been sown in longitudinal plots of 15 qm in four repetitions. 3 weeks after sowing seedlings have been harvested (40 plants per plot) and leaf material and root tissue have been analyzed using Liquid Chromatography with tandem mass spectrometry (LC-MS-MS) as described above.

TABLE-US-00007 TABLE 4c Results of denatonium benzoate determination in seedlings. One unit of oilseed rape is 1.5 million viable seeds, typically this is ~7 kg seeds per unit. application repetition tissue Mean Bitrex concentration (ng/g) A 1 leaf 6099.5 root 10362.9 2 leaf 3941.9 root 4508.1 3 leaf 2932.7 root 5310.3 4 leaf 3041.6 root 8439.4 kernel 460473.8 B 1 leaf 12287.9 root 21169.7 2 leaf 17191.9 root 25378.0 3 leaf 29225.6 root 18336.0 4 leaf 13195.0 root 15826.3

[0101] Results in Table 4c shows that rye plant take up Bitrex in tissues like root and leaf. The application dosage of Bitrex on the seeds determines the subsequent concentration in the tissues. This indicates clearly that also in rye Bitrex is systemic distributed. In this experiment concentration of Bitrex between 3,000 ng/g to 30,000 ng/g in leaves and between 4,500 ng/g and 25,000 ng/g in roots have been measured. As expected in kernels the concentration was even higher with up to 460,000 ng/g.

Example 4: Evaluation of the Efficacy of Denatonium Benzoate as a Repellent for Wireworms in Greenhouse and Climate Chamber

[0102] The following set up of experimental approaches as shown in Table 5 was applied. The denatonium benzoate (Bitrex) was tested in concentrations of 0.5 g/unit (wherein 1 unit=50.000 seeds), 1 g/unit and 5 g/unit, either without any other plant protection product (variants 3 and 4) or with RedigoM (active ingredients 100 g/l Prothioconazole (PRO) and 20 g/l Metalaxyl (MTL); (variants 6-8). Sticker was always Sepiret 9290 red, whereby 30 ml/unit has been applied. Variant 1 is the negative control wherein no treatment of the seeds took place, variant 2 is another control only treated with the sticker. Variant 5 is RedigoM and sticker without Bitrex. Variants 9 and 10 serve as positive control. Beside RedigoM and the sticker, the seeds have been treated with either with Force 20 CS (active ingredient: tefluthrin (TEF); or with Poncho® 600 (active ingredient: clothianidin (CLO). Both Force 20 CS and Poncho® 600 are standard treatments against wireworm infestation. All testing were conducted on corn variety Amavit of KWS SAAT SE & Co. KGaA.

TABLE-US-00008 TABLE 5 Set for wireworm repellent testing. Variants 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 has been tested in greenhouse and climate chamber, variant 4b only in climate chamber. Thousand kernel Plant mass protection active Variant (g/1tk) product compound additive sticker 1 293 none none none none 2 293 none none none Seperit 9290 red 30 ml 3 293 none none Bitrex Seperit 9290 0.5 g/u red 30 ml 4 293 none none Bitrex Seperit 9290 1 g/u red 30 ml 4b 293 none none Bitrex Seperit 9290 5 g/u red 30 ml 5 293 RedigoM MTL; none Seperit 9290 PRO red 30 ml 6 293 RedigoM MTL; Bitrex Seperit 9290 PRO 0.5 g/u red 30 ml 7 293 RedigoM MTL; Bitrex Seperit 9290 PRO 1 g/u red 30 ml 8 293 RedigoM MTL; Bitrex Seperit 9290 PRO 5 g/u red 30 ml 9 293 RedigoM MTL; none Seperit 9290 Force PRO red 30 ml 20 CS TEF 10 293 RedigoM MTL; none Seperit 9290 Poncho PRO red 30 ml CLO

[0103] Testing took place in pot assays. Per pot four corn seeds were sown (see FIG. 2A). In each pot four wireworm of genus Agriotes were placed in a central cavity of 5 cm depth (see FIG. 2B). Each treatment variant 1-10 was replicated seven times. The seeds in the pots were germinated and seedlings were grown under controlled conditions in greenhouse.

[0104] Eight days after sowing the plant biomass (FIG. 3; Table 6) as well as seed damage (FIG. 4A; Table 6) were assessed. Treatment with Bitrex had a positive effect on the development of biomass. Plantlets without treatment of plant protection product(s) (variants 1 and 2) suffered infestation and showed slower early plant development. The treatment only with the sticker Seperit 9290 red (variant 2) has no effect on plant development. Plantlets treated with Bitrex showed comparable or even more biomass than control plants without wireworm infestations (′control (no WW)′) and comparable with variants 9 and 10 treated with the standard plant protection products Force 20 CS and Poncho.

[0105] The seed damages and seedling damages, preferably on the roots, that were evaluated are significantly reduced if Bitrex has been applied. Non-treated seed (variant 1) showed damages on average on 3 seeds out of the four sown seeds per pot (mean damage: 75%). As positive control Force 20 CS and Poncho showed no seed damage at all (mean damage: 0%). Application of Bitrex, in particular in concentrations higher than 1 g/unit, significantly increased the tolerance towards wireworms (see variant 4, 7 and 8). The seed damage could be reduced to a mean of 21.5% in variant 8 (corresponding to on average 0.86 damaged seeds out of four seeds per pot).

[0106] Testing has been repeated in climate chambers in order to ensure high humidity and moderate temperatures which results in optimal environmental condition for the wireworm. Thereby, a high level of insect pressure on the seeded corn should be simulated. In each pot four wireworm of genus Agriotes were placed in a central cavity of 5 cm depth (see FIG. 2B). Each treatment variant 1-10 was replicated eight times (8 pots each with 4 seeds). The seeds in the pots were germinated and seedlings were grown under climate chamber conditions: 18° C. day/12° C. night.

[0107] Eighteen days after sowing the plant biomass (Table 6) as well as seed damage (FIG. 4B; Table 6) were assessed. The positive effect of Bitrex on the development of biomass as well as on seed damages and seedling damages has been confirmed even under such enhanced biotic stress conditions.

TABLE-US-00009 TABLE 6 Results of testing in greenhouse (GH) and climate chamber (CC) Number of No. plants feeding with feeding marks Number of Emerged damage on on roots/ wireworms plants Biomass (g) seed plant Variant GH* CC* GH CC GH CC GH CC GH CC 1 4.00 4.00 3.86 4.00 11.05 6.81 3.00 2.63 5.57 5.38 2 4.00 3.88 3.57 3.38 11.19 7.02 2.71 2.13 2.86 5.63 3 3.43 3.88 4.00 3.88 13.27 8.13 2.57 1.88 4.14 7.50 4 4.00 3.63 3.86 3.88 15.16 7.12 1.71 1.25 4.86 4.50  4b — 3.75 — 3.88 — 7.34 — 0.88 — 3.25 5 4.00 3.75 4.00 3.50 15.15 6.92 2.71 1.50 4.29 3.00 6 3.71 3.50 3.71 3.88 15.85 7.05 2.14 1.88 5.00 6.38 7 3.57 4.13 4.00 3.38 16.51 6.03 1.57 1.13 2.57 4.63 8 3.86 3.88 3.71 3.75 15.74 7.41 0.86 0.88 7.71 3.75 9 3.57 3.38 4.00 4.00 15.99 9.42 0.00 0.00 3.57 2.00 10  2.14 3.13 4.00 4.00 14.85 9.40 0.00 0.00 1.29 1.13

Example 5: Evaluation of the Efficacy of Denatonium Benzoate as a Repellent for Wireworms in Field Trials

[0108] To determine, whether the positive effect of Bitrex on the development of biomass as well as on seed damages and seedling damages can be also expected in the field, a comparable experimental set-up as described for greenhouse and climate chamber above has been used except the amount of applied Bitrix was different. Seeds have been treated with 5 g Bitrex per 50,000 seeds, 10 g Bitrex per 50,000 seeds and 20 g Bitrex per 50,000 seeds. Each variant has been sown in plots of 4 rows (21 m.sup.2) at a location in Italy where the soil shows high exposure of wireworm of genus Agriotes. Evaluation took place at emergence (E), at V3 stage (=3 leaves unfolded) and at V5 stage (=5 leaves unfolded). The following characteristics have been phenotyped: phytotoxicity (PHYGEN) at stage E, V3 and V5 in % per plot, vigor (VIGOR) at stage E, V3 and V5 in ratings from 1-10 (10 is best), insect damage (DAMINS) at stage V5 in plants per row (left DAMINS column in Table 7) and in % per row (right DAMINS column in Table 7). As positive control again Force 20 CS (60 ml/50,000 seeds) and as negative control untreated seeds (control) has been used.

TABLE-US-00010 TABLE 7 Results of testing of field trials. Characteristic PHYGEN VIGOR DAMINS Stage E V3 V5 E V3 V5 V5 V5 Control 0 0 0 10 10 10 10.5 8.60 5 g Bitrex 0 0 0 10 10 10 6.8 5.62 10 g Bitrex 0 0 0 10 10 10 7.3 6.07 20 g Bitrex 0 0 0 10 10 10 5.8 4.79 Force 20 CS 0 0 0 10 10 10 0.5 0.42

[0109] Field trial showed that the treatment of seeds with Bitrex provides a repellent effect towards wireworms also under natural conditions. In this experiment the application of 5-20 g Bitrex reduced the number of maize plants with wireworm damages by approximately 35%-45%. A negative effect on plant vitality with respect to phytotoxicity and plant vigor has not been observed even though Bitrex has been applied at high concentration. Thereby, the trials showed that Bitrex is usable as insect repellent also under field conditions what opens up a new ecologically safe alternative to other chemical substances which are potentially environmentally hazardous.