AGROCHEMICAL ADJUVANTS

Abstract

A novel agrochemical formulation comprising adjuvants selected from diketopiperazines and an agrochemical active. A concentrate is also provided suitable for forming the formulation. The diketopiperazine provide adjuvancy in the concentrate and agrochemical formulations. There is also provided a method of making the formulation, and use of said diketopiperazines as adjuvants in agrochemical formulations is also provided. A method of obtaining the diketopiperazines from culturing of Metarhizium carneum, strain RKD0578 is also described. The extracted diketopiperazines may be used in a formulation for treating vegetation to control pests, or seed coating. Alternatively the strain RKD0578 itself may be used in a seed coating or in a formulation for treating vegetation to control pests.

Claims

1. An agrochemical formulation comprising: i) an adjuvant selected from a diketopiperazine according to formula (I) ##STR00007## wherein: R.sub.1 independently represents hydrogen or C.sub.1 to C.sub.4 alkyl; R.sub.2 and R.sub.3 each independently represents hydrogen, C.sub.1 to C.sub.6 alkyl, phenyl, or substituted phenyl; R.sub.4, R.sub.5, and R.sub.6 independently represents hydrogen, C.sub.1 to C.sub.4 alkyl, hydroxyl, methoxy, or ethoxy; and ii) at least one agrochemical active.

2. The formulation according to claim 1, wherein: R.sub.1 represents hydrogen, methyl, or ethyl; R.sub.2 and R.sub.3 each independently represents hydrogen, methyl, phenyl, or substituted phenyl where the substituent is methyl, ethyl, methoxy, or ethoxy; at least one of R.sub.2 and R.sub.3 represents phenyl, whilst the other represents hydrogen, methyl, or ethyl; and R.sub.4, R.sub.5, and R.sub.6 independently represents hydrogen or methyl.

3. The formulation according to claim 1, wherein: R.sub.1 represents hydrogen or methyl; R.sub.2 and R.sub.3 each independently represents hydrogen or phenyl, or substituted phenyl where the substituent is methyl, ethyl, methoxy, or ethoxy; one of R.sub.2 and R.sub.3 represents phenyl and the other represents hydrogen or methyl; and R.sub.4, R.sub.5, and R.sub.6 independently represent hydrogen.

4. The formulation according to claim 1, wherein the adjuvant is selected from diketopiperazines of formula (II): ##STR00008## wherein R.sub.1 independently represents hydrogen or C.sub.1 to C.sub.4 alkyl.

5. The formulation according to claim 1, wherein the adjuvant is selected from diketopiperazines of the following formulas: ##STR00009##

6. A concentrate formulation suitable for making an agrochemical formulation in accordance with claim 1, the concentrate comprising; i) an adjuvant selected from a diketopiperazine according to formula (I) ##STR00010## wherein: R.sub.1 independently represents hydrogen or C.sub.1 to C.sub.4 alkyl; R.sub.2 and R.sub.3 each independently represents hydrogen, C.sub.1 to C.sub.6 alkyl, phenyl, or substituted phenyl; R.sub.4, R.sub.5, and R.sub.6 independently represents hydrogen, C.sub.1 to C.sub.4 alkyl, hydroxyl, methoxy, or ethoxy; and ii) at least one agrochemical active.

7. Use of a compound selected from a diketopiperazine according to formula (I) in accordance with claim 1, ##STR00011## wherein: R.sub.1 independently represents hydrogen or C.sub.1 to C.sub.4 alkyl; R.sub.2 and R.sub.3 each independently represents hydrogen, C.sub.1 to C.sub.6 alkyl, phenyl, or substituted phenyl; R.sub.4, R.sub.5, and R.sub.6 independently represents hydrogen, C.sub.1 to C.sub.4 alkyl, hydroxyl, methoxy, or ethoxy; as an adjuvant in an agrochemical formulation comprising at least one agrochemical active.

8. A method of treating vegetation to control pests, the method comprising applying a formulation in accordance with claim 1 either to the vegetation or to the immediate environment of the vegetation.

9. A method of obtaining adjuvants in accordance with claim 1 comprising: culturing Metarhizium carneum RKDO578 in a medium under conditions which promote metabolic synthesis of an adjuvant from the Metarhizium carneum and purifying the synthesised adjuvant from the cultured medium.

10. An organism consisting of Metarhizium carneum, strain RKDO578, Agricultural Research Service Culture Collection (NRRL) accession number NRRL-67950.

11. An extract obtained from the organism consisting of Metarhizium carneum, strain RKDO578, Agricultural Research Service Culture Collection (NRRL) accession number NRRL-67950, the extract comprising at least one of a diketopiperazine of formula (I) in accordance with claim 1.

12. A method of treating vegetation to control pests, the method comprising applying an organism in accordance with claim 10 either to the vegetation or to the immediate environment of the vegetation.

13. A seed coating composition comprising adjuvants in accordance with claim 1.

Description

EXAMPLES

[0143] In order that the present invention may be more readily understood, reference will now be made, by way of example, to the following description.

[0144] It will be understood that all tests and physical properties listed have been determined at atmospheric pressure and room temperature (i.e., 20-25° C.), unless otherwise stated herein, or unless otherwise stated in the referenced test methods and procedures.

Formation & Extraction

[0145] Bioassay-guided fractionation of the culture extracts of the fungus Metarhizium carneum RKDO578 led to the isolation of diketopiperazines. The isolate RKDO578 was cultured on YM agar (w/v: 1% malt extract, 0.2% yeast extract, 1% glucose, 2% agar) and incubated for 14 days at 22° C. Eight colony explants (approximately 3 30 mm.sup.3) were used to inoculate 15 mL of YM broth (w/v: 1% malt extract, 0.2% yeast extract, 1% glucose) in a sterile 50 mL test tube and shaken at 200 RPM, 22° C. for 5 days to create a seed inoculum. The seed culture was used to inoculate fermentation medium (w/v: 15% sucrose, 2% yeast extract, 0.05% magnesium sulphate heptahydrate and 1% L-leucine) contained in Erlenmeyer flasks.

[0146] Following 21 days at 22° C., fermentations were extracted with one volume of ethyl acetate and shaken for 60 minutes at 175 RPM. Extracts were clarified by filtering through Whatman #3 filter paper and the solvent was removed in vacuo prior to chemical purification.

[0147] RKDO578 fermentation extracts were fractionated on a Siliasep C18 flash cartridge (43 g C-18) using a gradient of 10% CH.sub.3OH:90% H.sub.2O to 100% CH.sub.3OH over 20 minutes on a Teledyne Nextgen 300.sup.+ Combiflash. Fractions were analysed on a Thermo Scientific Accela UHPLC coupled with a Thermo Exactive electrospray mass spectrometer (ESI-MS) with a SEDEX 80LT ELSD and a Thermo photodiode array (PDA) detector. Fractions containing diketopiperazines were purified using reversed-phase C-18 HPLC (Kinetex 5 μm C18 column, 10×250 mm) on a Waters HPLC system with an evaporative light scattering detector (Waters 2424) and mass spectrometer (Waters 3100). Initial purification of the diketopiperazines was carried out with an isocratic elution of 55% aqueous CH.sub.3CN with a flow rate of 3 mL/min.

[0148] The structures of the diketopiperazines were elucidated by combined mass spectrometry and NMR analysis. NMR spectra were recorded on a Bruker Avance III 400 MHz NMR spectrometer operating at 400 and 150 MHz for .sup.1H and .sup.13C, respectively. Spectra were referenced to residual undeuterated solvent peaks.

[0149] NMR analysis of the metabolites matched with literature data and confirmed the structures as diketopiperazines (Ia, Ib, Ic & Id).

ADJUVANCY EXAMPLES

[0150] The parameters ‘percent inhibition’ and ‘fold change’ will be understood to represent ad be calculated as follows;

[0151] Percent Inhibition—The percent inhibition will be understood to represent the amount the fungicide and/or adjuvant that inhibits the visible growth of the microorganism after 48 hours incubation at 22° C. relative to vehicle treated controls. This is calculated using the following formula:

[(Ø.sub.C−Ø.sub.T)/Ø.sub.C]×100%

where: [0152] Ø.sub.C=diameter of the colony grown on agar supplemented with vehicle (i.e. vehicle treated control), and [0153] Ø.sub.T=diameter of the colony grown on agar supplemented with fungicide and/or adjuvant formulated in an appropriate vehicle (i.e. solvent such as water C.sub.2H.sub.5OH, CH.sub.3OH, CH.sub.3CN, DMSO).

[0154] Fold Change—The fold change is a measure of the adjuvant/fungicide combination in inhibiting a microorganism compared to the fungicide alone. This indicates how the adjuvant performs relative to the fungicide alone. This is calculated using the following formula:

INH.sub.AF/INH.sub.F

where: [0155] INH.sub.AF=percent inhibition of fungal growth when treated with fungicide and adjuvant, and [0156] INH.sub.F=percent inhibition of fungal growth when treated with fungicide alone.

Examples—Adjuvancy

[0157] Botrytis cinerea (ATCC 90479) was cultured on Difco Potato Dextrose Agar (PDA) for 7 days with diurnal UV cycles (12 h UV light and 12 h dark). Spores were harvested in a buffered, sterile saline solution (w/v: 0.9% NaCl with 1% Tween 80) and counted using a haemocytometer. The spore suspension was adjusted to a final concentration of 8.5×10.sup.6 spores/mL to create a standardised inoculum.

[0158] To prepare hyphal fragments for adjuvant testing, 8.5×10.sup.4 spores were used to inoculate 10 mL of Difco Potato Dextrose Broth in a 150×25 mm tube. The tube was incubated at 220 RPM, 22° C. for 48 hours. To create hyphal fragments from the culture, the culture was transferred to a 50 mL plastic conical tube containing approximately 20 sterile 5 mm diameter glass beads and vortexed for 5 min. After vortexing the tube was allowed to stand for 5 min to allow large mycelial clumps to settle and then the top layer containing hyphal fragments was removed and used as inocula for growth inhibition assays.

[0159] Fungicides and adjuvants were dissolved in methanol and added to molten PDA (˜50° C.) and then the agar was distributed in the wells of 12 well multiwell plates (1 mL/well). The plates were cooled to room temperature and 10 μL of hyphal inoculum was added to the centre of each well. The plates were incubated at 22° C. for 48 hours and then the colony diameter was measured using a digital caliper. The biological growth control consisted of hyphae and vehicle (0.07% methanol), the negative control was media and vehicle (0.07% methanol).

[0160] The results of the adjuvant actives of the invention are shown in Tables 1 and 2. Adjuvant activity with pyraclostrobin against B. cinerea was observed for each of the compounds tested, most notably diketopiperazine Ia and diketopiperazine Ic which showed a 3.31 and 3.14 fold increase in fungicidal activity compared to pyraclostrobin alone at 0.03 μg/mL. Diketopiperazine Ia showed a 4.39 fold increase in fungicidal activity compared to tebuconazole alone at 0.19 μg/mL. Diketopiperazine (Ia, Ib, Ic & Id), did not demonstrate any inherent fungicidal activity on their own at any of the concentrations tested.

TABLE-US-00002 TABLE 1 Effect of adjuvants on fungicidal activity of pyraclostrobin (Pyra). Fold Change in Growth Inhibition Adjuvant Pyra Pyra Pyra Concentration 0.0625 0.0313 0.0156 Adjuvant (μg/mL) μg/mL μg/mL μg/mL None 0 1.00 1.00 1.00 DKP (Ia) 128 1.87 3.31 2.18 DKP (Ia 128 1.69 2.88 2.79 and Ib) DKP (Ic) 128 1.48 3.14 1.52 DKP (Ic 128 1.69 3.01 1.86 and Id)

TABLE-US-00003 TABLE 2 Effect of adjuvants on fungicidal activity of tebuconazole (Teb). Adjuvant Fold Change in Growth Inhibition Concentration Teb Teb Adjuvant (μg/mL) 0.19 μg/mL 0.095 μg/mL None 0 1.00 1.00 DKP (Ia) 100 2.76 2.00 50 1.78 1.78 25 4.39 1.97

[0161] The effect of the adjuvants on fungicidal activity is represented as fold increase in growth inhibition. A value of 1 indicates no increase in fungicidal activity. A value less than 1 indicates reduced fungicidal activity and a value greater than 1 indicates increased fungicidal activity.

Examples—Cytotoxicity

[0162] The cytotoxicity of the diketopiperazines was assessed in vitro against African green monkey Vero kidney cells (ATCC CCL-81). Cells were cultured in 15 mL of Eagle's minimal essential medium (Sigma) supplemented with 10% (v/v) fetal bovine serum (VWR), 100 μU penicillin and 0.1 mg/mL streptomycin in T75 cm.sup.2 cell culture flasks. Cells were incubated for 24 hours at 37° C. in a humidified atmosphere of 5% CO.sub.2. Culture medium was refreshed every 2-3 days and cells were not allowed to exceed 80% confluency.

[0163] At 80% confluency the cells were counted, diluted and plated into 96 well flat bottom cell culture plates (Corning) at a cell density of 10,000 cells per well in 90 μL of growth medium. The plates were incubated at 37° C. in a humidified atmosphere of 5% CO.sub.2 for 24 hours to allow cells to adhere to the plate before treatment. After 24 hours, adjuvants were solubilised in DMSO, serially diluted and added to the wells at final concentrations ranging from 1 μg/mL to 128 μg/mL. DMSO was used as the vehicle at a final concentration of 1% in the wells.

[0164] The plates were incubated at 37° C. in a humidified atmosphere of 5% CO.sub.2 for 24 hours after which alamarBlue (Invitrogen) was added to each well at 10% of the culture volume. Fluorescence was monitored using a Thermo Scientific Varioskan Flash plate reader at 560/12 excitation, 590 nm emission both at time zero and 4 hrs after alamarBlue addition. After subtracting the time zero emission 590 nm measurement from the final reading, the inferred percentage of cell viability relative to the vehicle control wells was calculated.

[0165] Diketopiperazines (Ia, Ib, Ic & Id) did not exhibit any cytotoxicity against Vero cells at the highest concentration tested (128 μg/mL) indicating that the adjuvants are not toxic to mammalian cells at the concentration tested.

Examples—Phytotoxicity

[0166] Phytotoxicity of the diketopiperazines was assessed on soybean and butterhead lettuce leaves. Soybean seeds were inoculated with Bradyrhizobium japnicum and sown in Levington's M3 compost in 9 cm.sup.2 plastic pots. Soybean plants were given supplementary lighting with SON-T bulbs. Biological control was used to prevent thrip damage (Biolone—Amblyseius cucumeris). The glasshouse temperatures were set to maintain 22° C.±2 during the day and 19° C.±2 at night. Lettuce plants were grown in the same manner without rhizobacteria inoculation. Supplementary lighting was provided by LED bulbs. All plants were maintained as well watered and grown for fourteen days.

[0167] Test compounds/extracts were resuspended in 2% DMSO (0.10%, 1.0 g/L). Synperonic Al1 LQ was used a positive control and UEP-100 as a negative control. Treatments were applied as six 10 μL droplets on the leaf's surface. Phytotoxic tissue damage was assessed 1- and 7-days post-treatment and on a scale of 0 to 3.

[0168] Diketopiperazine Ia did not exhibit any phytotoxicity against soybean or butterhead lettuce leaves after 7 days. RKDO578 crude extract exhibited very minor phytotoxicity after 7 days against soybean and butterhead lettuces (scores 0.4 and 0.2, respectively).

TABLE-US-00004 TABLE 3 Phytotoxic effect of adjuvants on soybean and butterhead lettuce. Mean Score Day 1 Day 7 Treatment Soy Lettuce Soy Lettuce Synperonic A11 (positive 2.3 0.73 2.4 2 control) UEP (negative control) 0 0 0.5 0 DKP (1a) 0 0 0 0 RKDO578 Crude Extract 0 0 0.4 0.2

[0169] The phytotoxic effect of the compounds/extract at 1 g/L is represented as a mean phytotoxic necrosis score, where 0=no necrosis; 1=slight spot-like necrosis of the inoculation site; 2=ring-shaped necrosis; 3=extended necrosis.

[0170] It is to be understood that the invention is not to be limited to the details of the above embodiments, which are described by way of example only. Many variations are possible.