METHOD AND USE OF AN ENANTIOMER OF 3,4-DIHYDROXYPHENYLALANINE (DOPA) FOR ENHANCING PLANT ATTRACTIVENESS TO BENEFICIAL INSECTS

Abstract

A method and use of an enantiomer 3,4-dihydroxyphenylalanine (DOPA) for enhancing plant attractiveness to beneficial insects by applying at least one time a composition having an effective amount of an enantiomer of DOPA selected from L-3,4-dihydroxyphenylalanine (L-DOPA) and D-3,4-dihydroxyphenylalanine (D-DOPA) or a mixture thereof to the leaves, stem and/or roots of the plant. The method provides an economical control of harmful insect pests and even allows preventive control prior to an infestation. As well as provide increases attraction of beneficial insects which in turn provide for an increased growth and propagation of the plant.

Claims

1. A method for enhancing plant attractiveness to beneficial insects, comprising applying at least one time a composition comprising an effective amount of an enantiomer of 3,4-dihydroxyphenylalanine (DOPA) selected from the group consisting of L-3,4-dihydroxyphenylalanine (L-DOPA), D-3,4-dihydroxyphenylalanine (D-DOPA) and a mixture thereof to the leaves, stem and/or roots of the plant, wherein the beneficial insects belong to the Braconidae family.

2. The method according to claim 1, wherein the beneficial insects belong to the Aphidiinae subfamily.

3. The method according to claim 2, wherein the beneficial insect is Aphidius ervi.

4. The method according to claim 1, wherein the plant is selected from the group consisting of plant species belonging to the Poaceae family, Solanaceae family, Brassicaceae family, Rutaceae family, Rosacea family, Rhamnaceae family, Apiaceae family and Leguminosae family.

5. The method according to claim 1, wherein the composition is applied to the plant before a pest infestation or after the application of a chemical agent selected from the group consisting of insecticide, fungicide, acaricide, antivirus, chemical compounds to control a pest infestation and a mixture thereof.

6. The method according to claim 1, wherein the composition is applied at least one time to the plant by spraying, spreading, fogging, dipping, drenching, dusting, injection into the plant substrate, application through the roots or adding in the irrigation water.

7. The method according to claim 1, wherein the composition is applied to plants in hydroponic or soil culture cultivation.

8. The method according to claim 1, wherein the composition comprises a further active ingredient selected from the group consisting of phytohormones, herbicides, fungicides, amino acids, humics components, organic compounds, polyphenolics, micronutrients, fertilizers, elicitors, plant growth regulators, biostimulants and combinations thereof.

9. The method according to claim 1, wherein the composition is applied to the plant at least once every three months.

10. (canceled).

11. (canceled).

12. (canceled).

13. (canceled).

Description

BRIEF DESCRIPTION OF DRAWINGS

[0053] The following figures are described below. These illustrate the exemplary embodiments and are not limiting their scope.

[0054] FIG. 1 shows the residence time (%) of Aphidius ervi females in the treatment (trt) versus control (cnt) arm of a two-way olfactometer. Treatment (trt) is a bean plant (Vicia faba) treated with L-Dopa meanwhile control (cnt) is a clean bean plant. Data were analyzed by means of a paired t-test (*: P<0.05).

[0055] FIG. 2 shows the residence time (%) of Aphidius ervi females in the treatment (trt) versus control (cnt) arm of a two-way olfactometer. Treatment (trt) is a bean plant (Vicia faba) treated with D-Dopa meanwhile control (cnt) is a clean bean plant. Data were analyzed by means of a paired t-test (*: P<0.05).

[0056] FIG. 3 shows the FID response (pA) of the volatile organic compounds (VOCs) 6-methyl-5-hepten-2one, cis-3-hexen-1-ol, cis-3-hexenyl acetate and Ocimene, produced by bean plants (Vicia faba) grown in untreated mixture of soil/vermiculite (control) and treated mixture of soil/vermiculite with L-DOPA and D-DOPA at 0.1 ppm.

[0057] FIG. 4 shows the FID response (pA) of the volatile organic compounds (VOCs) 6-methyl-5-hepten-2one, cis-3-hexen-1-ol, cis-3-hexenyl acetate and Ocimene, produced by bean plants (Vicia faba) grown in untreated hydroponic medium (control) and treated hydroponic medium with L-DOPA and D-DOPA at 0.1 ppm.

[0058] FIG. 5 shows the FID response (pA) of the volatile organic compounds (VOCs) cis-3-hexenyl acetate and Ocimene, produced by bean plants (Phaseolus vulgaris) grown in untreated soil (control) and treated soil with L-DOPA and D-DOPA at 0.1 ppm.

[0059] FIG. 6 shows the FID response (pA) of the volatile organic compounds (VOCs) 6-methyl-5-hepten-2one, cis-3-hexen-1-ol, cis-3-hexenyl acetate and Ocimene produced by bean plants (Phaseolus vulgaris) grown in untreated hydroponic medium (control) and treated hydroponic medium with L-DOPA and D-DOPA at 0.1 ppm.

[0060] FIG. 7 shows the FID response (pA) of the volatile organic compound (VOC) ocimene produced by wheat plants (Triticum monococcum) grown in untreated soil (control) and treated soil with L-DOPA and D-DOPA at 0.1 ppm.

[0061] FIG. 8 shows the total phenolic content, expressed as mg gallic acid equivalent/ml, in bean plant (Vicia faba) treated with L-DOPA, D-DOPA and control (untreated).

DESCRIPTION OF EMBODIMENTS

Example 1. Behavioral Assays on Bean Plants (Vicia faba) in Soil

[0062] Broad bean seeds (Vicia faba cv Aguadulce supersimonia) were planted individually in plastic pots (669 cm) filled with Traysubstrat topsoil (Klasmann-Deilmann GmbH) or a mixture of agriperlite, vermiculite, and sand (1:1:1) and grown in an environmental chamber (242 C., 4510% RH, 14 h: 8 h L: D).

[0063] For this example, bean plants of 3-4 weeks were used. Plants were treated by soil application of the two isomers of DOPA: 3,4-dihydroxy-L-phenylalanine (Alfa Aesar, CAS Number 59-92-7) and 3,4-dihydroxy-D-phenylalanine (Sigma Aldrich, CAS Number 5796-17-8).

[0064] Untreated plants were also used as control.

[0065] The attractiveness of beneficial insects, specifically Aphidius ervi, towards a bean plants treated with the isomers of DOPA compared to clean bean plant (control) was evaluated in an olfactometer.

[0066] The behavioral response of A. ervi were evaluated over 10 min in a two-way olfactometer. Only active insects, i.e. insects that spent at least 60 s in the treatment (trt) and/or control (cnt) arm of the olfactometer were taken into account.

[0067] The results obtained in this example are expressed as a percentage of residence time in each olfactometer arm where the parasitoid remained, relative to the total time spent in the two arms. Paired t-tests were conducted using R (free software environment for statistical computing and graphics).

[0068] FIGS. 1 and 2 show the percentage of residence time of females of A. ervi attracted by bean plants treated with L-DOPA and D-DOPA, respectively, compared to clean plants. The residence time for the treated bean plants was higher (approx. +20%) than the obtained for untreated plants.

Example 2. Induction Assay on Bean Plants (Vicia faba) in Soil

[0069] Broad beans (Vicia faba cv Histal) plants were grown in a mixture of sterile soil/vermiculite (7:3) for 10 days, and treated with L-DOPA (10 g), D-DOPA (10 g) or HPLC water (control, 10 l) dissolved in 30 mL of distilled water.

[0070] After 24 h, the volatile organic compounds (VOCs) emitted from the plant were collected through a dynamic headspace.

[0071] The bean plants were enclosed in a glass vessel with a volume of 3.2 I. The bottom of the vessel was enclosed around the top of the pot. Air that had been purified by passage through an activated charcoal filter (BDH, 10-14 mesh) was pushed into (750 ml.Math.min.sup.1) and pulled (700 ml.Math.min.sup.1) out of the vessel. VOCs were trapped onto Porapack (Porous Polymer Adsorbent) 50/80 mesh (50 mg; Supelco, Bellefonte, USA) held in glass tubing (5 mm outer diameter) by two plugs of silanised glass wool. The Porapack tube was conditioned by washing with redistilled diethyl ether (2 ml) and heating at 130 C. for 4 h under a stream of purified nitrogen.

[0072] VOCs collected on the Porapak were eluted with 750 L of redistilled diethyl ether and the samples were stored at 20 C. until chromatographic analysis.

[0073] For the analysis, four samples (1 L) from each treatment (L-DOPA, D-DOPA and Control) were analyzed on an Agilent 7820A Gas Chromatograph (Agilent Technologies, Santa Clara, California, USA), equipped with a cool column injector, flame ionization detector (FID), and a HP-1 capillary GC column (50 m0.32 mm internal diameter0.52 m film thickness). Hydrogen was the carrier gas. The oven temperature was maintained at 30 C. for 0.1 min, then programmed to increase at 10 C..Math.min.sup.1 until 250 C., and then held for 38 min. Tentative identifications were made by Kovats' Index values and GC peak enhancement on GC columns of HP-1, using authentic samples of chemicals compounds.

[0074] The results obtained are represented in FIG. 3. A change is observed in the concentration of VOCs triggered by the action of L-DOPA and D-DOPA on the plant. However, in untreated plants (control) it is observed that the concentration is lower, and even some compounds are not emitted, as 6-methyl-5-hepten-2one.

[0075] Therefore, it is confirmed that the exogenous application of DOPA or an enantiomer thereof in plants significantly increases the emission of VOCs.

Example 3. Induction Assay on Bean Plants (Vicia faba) in Hydroponic Medium

[0076] Broad beans (Vicia faba cv Histal) were potted in sterile vermiculite and kept in a glasshouse at 23 C. After 7 days, the seedlings were gently removed from the vermiculite, the cotyledons peeled and the roots rinsed with bidistilled water, carefully brushing away all soil particles. They were then placed in glass beakers containing a hydroponic solution made with Hoagland's modified basalt salt mixture, (MP, USA), and maintained in an environmental chamber at 23 C. Each beaker, containing three seedlings, was wrapped in aluminum foil to hold the plants in position and to prevent the light from reaching the roots. Every 4 days, the hydroponic solution in each beaker was renewed. After 10 days plants were treated with L-DOPA (10 g), D-DOPA (10 g) or HPLC water (control, 10 l) dissolved in 30 mL of distilled water.

[0077] The bean plants were enclosed in a glass vessel with a volume of 3.2 I. The bottom of the vessel was enclosed around the top of the pot. Air that had been purified by passage through an activated charcoal filter (BDH, 10-14 mesh) was pushed into (750 ml.Math.min.sup.1) and pulled (700 ml.Math.min.sup.1) out of the vessel. VOCs were trapped onto Porapack (Porous Polymer Adsorbent) 50/80 mesh (50 mg; Supelco, Bellefonte, USA) held in glass tubing (5 mm outer diameter) by two plugs of silanised glass wool. The Porapack tube was conditioned by washing with redistilled diethyl ether (2 ml) and heating at 130 C. for 4 h under a stream of purified nitrogen.

[0078] VOCs collected on the Porapak were eluted with 750 L of redistilled diethyl ether and the samples were stored at 20 C. until analysis.

[0079] For the analysis, four samples (1 L) from each treatment (L-DOPA, D-DOPA and Control) were analyzed on an Agilent 7820A Gas Chromatograph (Agilent Technologies, Santa Clara, California, USA), equipped with a cool column injector, flame ionization detector (FID), and a HP-1 capillary GC column (50 m0.32 mm internal diameter0.52 m film thickness). Hydrogen was the carrier gas. The oven temperature was maintained at 30 C. for 0.1 min, then programmed to increase at 10 C..Math.min.sup.1 until 250 C., and then held for 38 min. Tentative identifications were made by Kovats' Index values and GC peak enhancement on GC columns of HP-1, using authentic samples of chemicals compounds.

[0080] The results obtained are represented in FIG. 4. A change is observed in the concentration of 6-methyl-5-hepten-2one, cis-3-hexenyl acetate and ocimene triggered by the action of L-DOPA and D-DOPA on the plant. However, in untreated plants (control) it is observed that the concentration is lower, except for the cis-3-hexen-1-ol.

Example 4. Induction Assay on Bean Plants (Phaseolus vulgaris) in Soil

[0081] Bean (Phaseolus vulgaris cv CGB0002) plants were grown in sterile soil for 5 days, and treated with L-DOPA (10 g), D-DOPA (10 g) or HPLC water (control, 10 l) dissolved in 30 mL of distilled water.

[0082] After 24 h, the volatile organic compounds (VOCs) emitted from the plant were collected through a dynamic headspace.

[0083] The bean plants were enclosed in a glass vessel with a volume of 3.2 I. The bottom of the vessel was enclosed around the top of the pot. Air that had been purified by passage through an activated charcoal filter (BDH, 10-14 mesh) was pushed into (750 ml.Math.min.sup.1) and pulled (700 ml.Math.min.sup.1) out of the vessel. VOCs were trapped onto Porapack (Porous Polymer Adsorbent) 50/80 mesh (50 mg; Supelco, Bellefonte, USA) held in glass tubing (5 mm outer diameter) by two plugs of silanised glass wool. The Porapack tube was conditioned by washing with redistilled diethyl ether (2 ml) and heating at 130 C. for 4 h under a stream of purified nitrogen.

[0084] VOCs collected on the Porapak were eluted with 750 L of redistilled diethyl ether and the samples were stored at 20 C. until analysis.

[0085] For the analysis, four samples (1 L) from each treatment (L-DOPA, D-DOPA and Control) were analyzed on an Agilent 7820A Gas Chromatograph (Agilent Technologies, Santa Clara, California, USA), equipped with a cool column injector, flame ionization detector (FID), and a HP-1 capillary GC column (50 m0.32 mm internal diameter0.52 m film thickness). Hydrogen was the carrier gas. The oven temperature was maintained at 30 C. for 0.1 min, then programmed to increase at 10 C..Math.min.sup.1 until 250 C., and then held for 38 min. Tentative identifications were made by Kovats' Index values and GC peak enhancement on GC columns of HP-1, using authentic samples of chemicals compounds.

[0086] The results obtained are represented in FIG. 5. A change is observed in the concentration of VOCs triggered by the action of L-DOPA on the plant.

Example 5. Induction Assay on Bean Plants (Phaseolus vulgaris) in Hydroponic Medium

[0087] Beans (Phaseolus vulgaris cv CGB0002) were sown in sterile vermiculite and kept in a glasshouse at 27 C. After 17 days, the seedlings were gently removed from the vermiculite. They were then placed in glass beakers containing 150 mL of hydroponic solution made with Hoagland's modified basalt salt mixture, (MP, USA), and maintained in an environmental chamber at 27 C. Each beaker, containing one seedling, was wrapped in aluminum foil to hold the plants in position and to prevent the light from reaching the roots. After 2 days plants were treated with L-DOPA (10 g), D-DOPA (10 g) or HPLC water (control, 10 l).

[0088] After 24 h, the volatile organic compounds (VOCs) emitted from the plant were collected through a dynamic headspace.

[0089] The bean plants were enclosed in a glass vessel with a volume of 3.2 I. The bottom of the vessel was enclosed around the top of the pot. Air that had been purified by passage through an activated charcoal filter (BDH, 10-14 mesh) was pushed into (750 ml.Math.min.sup.1) and pulled (700 ml.Math.min.sup.1) out of the vessel. VOCs were trapped onto Porapack (Porous Polymer Adsorbent) 50/80 mesh (50 mg; Supelco, Bellefonte, USA) held in glass tubing (5 mm outer diameter) by two plugs of silanised glass wool. The Porapack tube was conditioned by washing with redistilled diethyl ether (2 ml) and heating at 130 C. for 4 h under a stream of purified nitrogen.

[0090] VOCs collected on the Porapak were eluted with 750 L of redistilled diethyl ether and the samples were stored at 20 C. until analysis.

[0091] For the analysis, four samples (1 L) from each treatment (L-DOPA, D-DOPA and Control) were analyzed on an Agilent 7820A Gas Chromatograph (Agilent Technologies, Santa Clara, California, USA), equipped with a cool column injector, flame ionization detector (FID), and a HP-1 capillary GC column (50 m0.32 mm internal diameter0.52 m film thickness). Hydrogen was the carrier gas. The oven temperature was maintained at 30 C. for 0.1 min, then programmed to increase at 10 C..Math.min.sup.1 until 250 C., and then held for 38 min. Tentative identifications were made by Kovats' Index values and GC peak enhancement on GC columns of HP-1, using authentic samples of chemicals compounds.

[0092] The results obtained are represented in FIG. 6. A change is observed in the concentration of 6-methyl-5-hepten-2one and cis-3-hexenyl acetate triggered by the action of L-DOPA and in the concentration of cis-3-hexen-1-ol and ocimene by the action of L-DOPA and D-DOPA on the plant. In untreated plants (control) it is observed that the concentration is lower, and even some compounds are not emitted, as cis-3-hexen-1ol.

Example 6. Induction Assay on Wheat Plants (Triticum monococcum) in Soil

[0093] Wheat (Triticum monococcum VAR Tocayo) plants were grown in sterile soil for 15 days (20 seeds per pot), and treated with L-DOPA (10 g), D-DOPA (10 g) or HPLC water (control, 10 l) dissolved in 30 mL of distilled water.

[0094] After 24 h, the volatile organic compounds (VOCs) emitted from the plant were collected through a dynamic headspace.

[0095] The wheat plants were enclosed in a glass vessel with a volume of 3.2 I. The bottom of the vessel was enclosed around the top of the pot. Air that had been purified by passage through an activated charcoal filter (BDH, 10-14 mesh) was pushed into (750 ml.Math.min.sup.1) and pulled (700 ml.Math.min.sup.1) out of the vessel. VOCs were trapped onto Porapack (Porous Polymer Adsorbent) 50/80 mesh (50 mg; Supelco, Bellefonte, USA) held in glass tubing (5 mm outer diameter) by two plugs of silanised glass wool. The Porapack tube was conditioned by washing with redistilled diethyl ether (2 ml) and heating at 130 C. for 4 h under a stream of purified nitrogen.

[0096] VOCs collected on the Porapak were eluted with 750 L of redistilled diethyl ether and the samples were stored at 20 C. until analysis.

[0097] For the analysis, four samples (1 L) from each treatment (L-DOPA, D-DOPA and Control) were analyzed on an Agilent 7820A Gas Chromatograph (Agilent Technologies, Santa Clara, California, USA), equipped with a cool column injector, flame ionization detector (FID), and a HP-1 capillary GC column (50 m0.32 mm internal diameter0.52 m film thickness). Hydrogen was the carrier gas. The oven temperature was maintained at 30 C. for 0.1 min, then programmed to increase at 10 C..Math.min.sup.1 until 250 C., and then held for 38 min. Tentative identifications were made by Kovats' Index values and GC peak enhancement on GC columns of HP-1, using authentic samples of chemicals compounds.

[0098] The results obtained are represented in FIG. 7. A change is observed in the concentration of ocimene triggered by the action of L-DOPA. It wasn't observed the emission of other VOCs (6-methyl-5-hepten-2one, cis-3-hexen-1-ol and cis-3-hexenyl acetate).

Example 7. Estimation of Total Phenolic Content in Bean Plant (Vicia faba) Leaves

[0099] The estimation of total phenolic content in bean plant leaves (Vicia faba) was based on the procedure developed by Ainsworth, E.A. et al. (2007) using Folin-Ciocalteu (F-C) reagent. F-C assay is commonly been used to assay total phenolic content. The F-C assay provides a convenient, rapid, and simple estimation of the content of total phenolics, and other oxidation substrates in plant extracts.

[0100] Broad bean (Vicia faba cv Histal) plants were grown in a mixture of sterile soil/vermiculite (7:3) for 10 days, and treated with L-DOPA (10 g), D-DOPA (10 g) or HPLC water (control, 10 l) dissolved in 30 mL of distilled water. After 24 h harvest leaves material (approximately 20 mg) in screw-capped tubes and freeze immediately in liquid nitrogen.

[0101] To homogenize the tissue, three tungsten carbide beads and 2 ml of ice-cold 95% (vol/vol) methanol were added in each sample tube and insert samples into pre-cooled Teflon adaptors. Tissue was homogenized for 5 min at 30 Hz. Then the tungsten carbide beads were removed with a magnet and the samples were incubated at room temperature for 48 h in the dark.

[0102] After that, the samples were centrifuged (13,000 g for 5 min at room temperature) and supernatant was collected in a fresh 2-ml microtube.

[0103] Subsequently, 100 l of each sample supernatant, standard or 95% (vol/vol) methanol blank were added to duplicate 2-ml microtubes.

[0104] After that, 200 l 10% (vol/vol) Folin-Ciocalteu reagent was added into each tube. All samples were thoroughly vortexed. Then, 800 l of Na.sub.2CO.sub.3 (700 mM) was added into each tube and the assay tubes were incubate at room temperature for 2 h.

[0105] Finally, a 200 ml sample, standard or blank from the assay tube were transferred to a clear 96-well micro-plate and the absorbance of each was measured at 750 nm using a spectrophotometer.

[0106] For calculate the total phenolic content, first a standard curve from the blank-corrected A.sub.750 of the gallic acid standards was calculated. Then, the regression equation between gallic acid standards and A750 was used to calculate total phenolics content. The concentration of total phenolic compounds was expressed in mg gallic acid equivalents (GAE) per ml of extract.

[0107] The results obtained are represented in FIG. 8. It is observed that the total phenolic content is higher for plants treated with L-DOPA and D-DOPA in contrast to the content obtained for untreated plants (control). Monoterpenes such as ocimene are derived biosynthetically from units of isopentenyl pyrophosphate, which is formed from an enzyme called acetyl-CoA. Phenolic compounds also are formed from that enzyme. Dopa and its enantiomers upregulated some enzymes related to monoterpene and phenol synthesis that could explain why the method of the present invention increase the phenolic content.

References

[0108] Baluka, F., Mukherjee, S., Ramakrishna, A. Neurotransmitters in Plant Signaling and Communication. [0109] Nishihara, E., Parvez, M. M., Araya, H., & Fujii, Y. (2004). Germination growth response of different plant species to the allelochemical L-3,4-dihydroxyphenylalanine (L-DOPA). Plant Growth Regulation, 42(2), 181-189. [0110] Ainsworth, E. A., & Gillespie, K. M. (2007). Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin-Ciocalteu reagent. Nature protocols, 2(4), 875-877.