COMPOSITION AND METHOD OF TREATING PLANT MATERIAL

Abstract

A biostimulant composition is provided, the composition comprising Aegle marmelos leaf particulate or an extract from Aegle marmelos leaf, the extract having been obtained with a solvent.

Claims

1. A biostimulant plant treatment composition comprising Aegle marmelos leaf particulate and/or an extract from Aegle marmelos leaf, the extract having been obtained with a solvent.

2. The composition according to claim 1 comprising at least 100 mg of the Aegle marmelos leaf particulate per liter composition.

3. (canceled)

4. (canceled)

5. The composition according to claim 1 comprising the extract from Aegle marmelos leaf extract, the Aegle marmelos extract having been obtained with an aqueous solvent, the solvent comprising at least 10 vol % water.

6. (canceled)

7. The composition according to claim 1 comprising the extract from Aegle marmelos leaf, the composition comprising from water and one or more alcohols.

8. The composition according to claim 7 comprising Aegle marmelos leaf particulate.

9. The composition according to claim 1 comprising the extract from Aegle marmelos leaf, the Aegle marmelos extract having been obtained with a deep eutectic solvent, the composition comprising a deep eutectic solvent.

10. The composition according to claim 1 comprising the extract from Aegle marmelos leaf extract, the Aegle marmelos leaf extract having been obtained with a solvent, and the composition comprising at least 0.2 wt % extract, and optionally no more than 10 wt % extract.

11. (canceled)

12. The composition according to claim 1 comprising a further biostimulant component.

13. The composition according to claim 1 comprising one or more of: orthosilicic acid; one or more weak acids; one or more amino acids; a surfactant; and a liquid carrier.

14. (canceled)

15. (canceled)

16. (canceled)

17. (canceled)

18. A method of treating an area comprising one or more plants and/or plant propagation material, the method comprising applying to said area the composition according to claim 1.

19. (canceled)

20. (canceled)

21. (canceled)

22. A pre-composition comprising Aegle marmelos leaf particulate and/or an extract from Aegle marmelos leaf, the extract having been obtained with a solvent, optionally an aqueous solvent, the pre-composition being suitable for mixing with a carrier liquid to form the composition according to claim 1.

23. A package containing a pre-composition in accordance with claim 22, the package optionally being provided with instructions for forming a biostimulant composition.

24. A package containing the composition in according to claim 1, the package optionally being provided with instructions for using the composition.

25. A substrate for supporting the growth of plants and/or plant propagation material, the substrate comprising Aegle marmelos leaf particulate and/or an extract from Aegle marmelos leaf, the extract having been obtained with a solvent, optionally an aqueous solvent.

26. A method of making the composition according to claim 1, the method comprising: producing particulate from one or more Aegle marmelos leaves, wherein the particulate comprises particles having a greatest dimension of no more than 100 nm.

27. The composition according to claim 1 comprising Aegle marmelos leaf particulate, an extract from Aegle marmelos leaf, the extract having been obtained with a solvent, and said solvent.

28. The composition according to claim 27, comprising a silicon species.

Description

DETAILED DESCRIPTION

Composition Example 1—Composition Comprising Leaf Particulate (1 g/Litre)

[0096] A mass of dry, brittle A.m. leaves were processed using a blender. A known mass of usable dry material was then added to water, and the suspension of leaf particulate in water was processed further using the blender in order to reduce the particle size further. Further water was added so that the leaf loading was 1 g of leaf per litre of water.

Composition Example 2—Composition Comprising Leaf Particulate (2 g/Litre)

[0097] Composition Example 2 was made in the same manner as Composition Example 1, but ensuring that the leaf loading was 2 g of leaf per litre of water.

Composition Example 3—Composition Comprising Leaf Particulate (4 g/l)

[0098] Composition Example 3 was made in the same manner as Composition Example 1, but ensuring that the leaf loading was 4 g of leaf per litre of water.

Composition Example 4—Composition Comprising Leaf Extract Obtained Using 80% Water, 20% Ethanol, Dilution 100:1

[0099] A known mass of dry, brittle A.m. leaf was processed using a blender. 50 g of the processed leaf was added to 320 ml of a solvent comprising a mixture of 80% water: 20% ethanol, and the solvent and leaf were agitated for about 5 hours at ambient temperature. The extract was then diluted with water, with 100 parts of water to 1 part of the extract, to provide Composition Example 4.

Composition Example 5—Composition Comprising Leaf Extract Obtained Using 20% Water, 80% Ethanol, Dilution 100:1

[0100] A known mass of dry, brittle A.m. leaf was processed using a blender. 50 g of the processed leaf was added to 320 ml of a mixture of 20% water: 80% ethanol, and the solvent and leaf were agitated for about 5 hours at ambient temperature. The extract was then diluted with water, with 100 parts of water to 1 part of the extract, to provide Composition Example 5.

Composition Example 6—Composition with Leaf Particulate, Ascorbic Acid and Orthosilicic Acid

[0101] A mass of dry, brittle A.m. leaves were processed using a blender. A known mass of usable dry material was then added to water, and the suspension of leaf particulate in water was processed further using the blender in order to reduce the particle size further. Further water was added so that the leaf loading was 33 g of leaf per litre of water. Ascorbic acid was added to a concentration of 20 g per litre of water. The solution of ascorbic acid/suspension of A.m. leaf was mixed with an equal volume of a 23 g/litre solution of liquid sodium silicate in water. The mixture was stirred and allowed to stand for at least 4 hours. A 300 micron sieve was used to filter the mixture, with material left on the sieve being discarded. The resulting liquid is stable for at least 5 days. The liquid is then diluted with water to give a leaf loading of 1 g/litre.

[0102] Without wishing to be bound by theory, it is believed that at least some of the A.m. particulate is present on a nanometer scale (approximately up to about 100 nm) and that interaction is taking place between the silicon species that are present and the nanometer scale A.m. particulate that is present. Evidence for this is that the composition is stable over a relatively long time scale, and gelling does not occur. It is thought that if no nanometer scale A.m. particulate is present, then there would be no such interaction between the silicon species and nanometer scale A.m. leaf particulate, in which case gelling of the silicon species would occur over a timescale of a few hours. Such gelling is not observed. As described below, it is further observed that Composition Example 6 demonstrates improved biostimulant activity compared to A.m. leaf particulate alone, indicating interaction between the A.m. leaf particulate and the silicon species that helps stabilise the silicon species and inhibits gelling.

Composition Example 7—Composition with Leaf Particulate and Maxstim (a Biostimulant Composition Comprising Amino Acids, Organic Acids, Trace Elements and Sugar Cane Molasses)

[0103] Two grams of A.m. leaf particulate was added to a solution comprising 2 g Maxstim biostimulant composition in approximately 250 ml of water. The resulting suspension was blended using a blender to reduce the size of the leaf particulate further. Further water was then added to make the composition up to 1 litre.

Method Example 1—CE 1 on Butterhead Lettuce (Lactuca sativa)

[0104] 6 ml of Composition Example 1 were administered on day 1 to each of five 4″ (102 mm) diameter pots filled with substrate (J Arthur Bowers compost) and provided with 5 lettuce seeds. 6 ml of Composition Example 1 were further administered on days 7, 14 and 21.

Method Example 2—CE 3 on Lettuce

[0105] 6 ml of Composition Example 3 were administered on day 1 to each of five 4″ (102 mm) diameter pots, each pot being filled with substrate and provided with five lettuce seeds. 6 ml of Composition Example 3 were further administered on days 7, 14 and 21.

Method Example 3—CE 4 on Lettuce

[0106] 6 ml of Composition Example 4 were administered on day 1 to each of five 4″ (102 mm) diameter pots, each pot being filled with substrate and provided with five lettuce seeds. 6 ml of Composition Example 4 were further administered on days 7, 14 and 21.

Method Example 4—CE 5 on Lettuce

[0107] 6 ml of Composition Example 5 were administered on day 1 to each of five 4″ (102 mm) diameter pots, each pot being filled with substrate and provided with five lettuce seeds. 6 ml of Composition Example 5 were further administered on days 7, 14 and 21.

Control for Method Examples 1-4—Water

[0108] 6 ml of water were administered on day 1 to each of five 4″ (102 mm) diameter pots, each pot being filled with substrate and provided with five lettuce seeds. 6 ml of water were further administered on days 7, 14 and 21.

Comments on Method Examples 1-4 (“AM Extracts on Lettuce”)

[0109] The growth of the lettuce plants was observed after 28 days and 40 days for Method Examples 1-4 and the associated control mentioned above. After 28 and 40 days the mass of plants generated by each of Method Examples 1-4 was noticeably greater than the mass generated by the control, as determined by eye. Furthermore, the mass of plants generated by each of Method Examples 1 and 2 was noticeably greater than the mass generated by Method Examples 3 and 4, indicating that the particulate of A.m. leaf provides superior biostimulant properties to either of the water/ethanol extracts.

Method Example 5—CE 1 on Rye Grass

[0110] 6 ml of Composition Example 1 were administered on day 1 to each of five 4″ (102 mm) diameter pots filled with substrate and which had been provided with 3 g of rye grass seeds. 6 ml of Composition Example 1 were further administered on days 7, 14 and 21.

Method Example 6—CE 2 on Rye Grass

[0111] 6 ml of Composition Example 2 were administered on day 1 to each of five 4″ (102 mm) diameter pots filled with substrate and which had been provided with 3 g rye grass seeds. 6 ml of Composition Example 2 were further administered on days 7, 14 and 21.

Method Example 7—CE 4 on Rye Grass

[0112] 6 ml of Composition Example 4 were administered on day 1 to each of five 4″ (102 mm) diameter pots filled with substrate and which had been provided with 3 g rye grass seeds. 6 ml of Composition Example 4 were further administered on days 7, 14 and 21.

Method Example 8—CE 5 on Rye Grass

[0113] 6 ml of Composition Example 5 were administered on day 1 to each of five 4″ (102 mm) diameter pots filled with substrate and which had been provided with 3 g rye grass seeds. 6 ml of Composition Example 5 were further administered on days 7, 14 and 21.

Control for Method Examples 5-8—Water

[0114] 6 ml of water were administered on day 1 to each of five 4″ (102 mm) diameter pots filled with substrate and which had been provided with 3 g rye grass seeds. 6 ml of water were further administered on days 7, 14 and 21.

Comments on Method Examples 5-8 (“AM Extracts on Rye Grass”)

[0115] The biomass was measured on day 28 for rye grass treated as described in Method Examples 5-8 and for the associated control, and the results are shown in Table 1.

TABLE-US-00001 TABLE 1 biomass generated by treatment with particulate A.m. leaf or extracts Treatment method Total biomass from the 5 pots (g) Method Example 5 69.6 Method Example 6 64.8 Method Example 7 63.3 Method Example 8 62.8 Control 46.2
The results from Table 1 show that both particulate A.m. leaf and water/ethanol extracts are effective biostimulants for rye grass. The results from Table 1 also suggest that particulate A.m. leaf is a slightly more effective stimulant that the water/ethanol extracts in relation to rye grass.

Method Example 9—CE 6 on Lettuce

[0116] 6 ml of Composition Example 6 were administered on day 1 to each of five 4″ (102 mm) diameter pots, each pot having been filled with substrate and provided with five lettuce seeds. 6 ml of Composition Example 6 were further administered on days 7, 14 and 21.

Control 1 for Method Example 9—OSA Only

[0117] 6 ml of ortho silicic acid solution (i.e. the composition of Composition Example 6 without the A.m. leaf particulate) were administered on day 1 to each of five 4″ (102 mm) diameter pots, each pot being filled with substrate and having been provided with five lettuce seeds. 6 ml of the ortho silicic acid solution were further administered on days 7, 14 and 21.

Control 2 for Method Example 9—Water Only

[0118] 6 ml of water were administered on day 1 to each of five 4″ (102 mm) diameter pots, each pot being filled with substrate and provided with five lettuce seeds. 6 ml of water were further administered on days 7, 14 and 21

[0119] The growth of the lettuce plants was observed after 28 days and 40 days for Method Example 9, Method Example 1 and the associated two controls mentioned above. After 28 and 40 days, the mass of plants generated by each of Method Examples 1 and 9 and Control 1 for Method Example 9 was noticeably greater, when compared by eye, than the mass generated by the Control 2 for Method Example 9. Furthermore, the mass of plants generated by Method Example 9 was noticeably greater, when compared by eye, than the mass generated by Method Example 1 and Control 1 for Method Example 9, indicating that the combination of particulate A.m. leaf and ortho silicic acid provides superior biostimulant properties to particulate A.m. leaf alone.

Method Example 10—CE 6 on Rye Grass

[0120] 6 ml of Composition Example 6 were administered on day 1 to each of five 4″ (102 mm) diameter pots filled with substrate and having been provided with 3 g rye grass seeds. 6 ml of Composition Example 6 were further administered on days 7, 14 and 21.

Control 1 for Method Example 10—OSA Only

[0121] 6 ml of ortho silicic acid solution (i.e. the composition of Composition Example 6 without the A.m. leaf particulate) were administered on day 1 to each of five 4″ (102 mm) diameter pots filled with substrate and having been provided with 3 g rye grass seeds. 6 ml of the ortho silicic acid solution were further administered on days 7, 14 and 21.

Control 2 for Method Example 9—Water Only

[0122] 6 ml of water were administered on day 1 to each of five 4″ (102 mm) diameter pots filled with substrate and having been provided with 3 g rye grass seeds. 6 ml of water were further administered on days 7, 14 and 21.

[0123] The biomass was measured on day 28 for rye grass treated as described in Method Examples 10 and for the associated controls, and the results are shown in Table 2.

TABLE-US-00002 TABLE 2 biomass generated by treatment with particulate A.m. leaf and ortho silicic acid Treatment method Total biomass from the 5 pots (g) Method Example 10 68.1 Control 1 56.9 Control 2 46.2
The results from Table 2 show that particulate A.m. leaf and orthosilicic acid together provide a biostimulant effect that is superior to orthosilicic acid alone.

Method Example 11—CE 7 on Lettuce

[0124] 6 ml of Composition Example 7 were administered on day 1 to each of five 4″ (102 mm) diameter pots, each pot being filled with substrate and provided with five lettuce seeds. 6 ml of Composition Example 7 were further administered on days 7, 14 and 21.

Control 1 for Method Example 11—Maxstim Only

[0125] 6 ml of MX solution (i.e. the composition of Composition Example 7 without the A.m. leaf particulate) were administered on day 1 to each of five 4″ (102 mm) diameter pots, each pot being filled with substrate and provided with five lettuce seeds. 6 ml of the MX solution were further administered on days 7, 14 and 21.

Control 2 for Method Example 11—Water Only

[0126] 6 ml of water were administered on day 1 to each of five 4″ (102 mm) diameter pots, each pot being filled with substrate and provided with five lettuce seeds. 6 ml of water were further administered on days 7, 14 and 21

[0127] The growth of the lettuce plants was observed after 40 days for Method Example 11, Method Example 1 and the associated two controls mentioned above. After 40 days, the mass of plants generated by each of Method Examples 1 and 11 and Control 1 for Method Example 11 was noticeably greater, by eye, than the mass generated by the Control 2 for Method Example 11. Furthermore, the mass of plants generated by Method Example 11 was noticeably greater, by eye, than the mass generated by Method Example 1 and Control 1 for Method Example 11, indicating that the combination of particulate A.m. leaf and MX provides superior biostimulant properties to particulate A.m. leaf alone and to MX alone.

Method Example 12—CE 7 on Rye Grass

[0128] 6 ml of Composition Example 7 were administered on day 1 to each of five 4″ (102 mm) diameter pots filled with substrate and provided with rye grass seeds. 7 ml of Composition Example 7 were further administered on days 7, 14 and 21.

Control 1 for Method Example 12—Maxstim Only

[0129] 6 ml of MX solution (i.e. the composition of Composition Example 7 without the A.m. leaf particulate) were administered on day 1 to each of five 4″ (102 mm) diameter pots filled with substrate and provided with rye grass seeds. 6 ml of the MX solution were further administered on days 7, 14 and 21.

Control 2 for Method Example 12—Water Only

[0130] 6 ml of water were administered on day 1 to each of five 4″ (102 mm) diameter pots filled with substrate and provided with rye grass seeds. 6 ml of water were further administered on days 7, 14 and 21.

[0131] The biomass was measured on day 28 for rye grass treated as described in Method Example 12 and for the associated controls, and the results are shown in Table 3.

TABLE-US-00003 TABLE 3 biomass generated by treatment with particulate A.m. leaf and MX Treatment method Total biomass from the 5 pots (g) Method Example 12 66.3 Control 1 56.3 Control 2 46.2
The results from Table 3 show that particulate A.m. leaf and MX solution together provide a biostimulant effect that is superior to MX solution alone.

[0132] Note that the methods described above illustrate the use of A.m. leaf particulate and aqueous extracts of A.m. leaf as a biostimulant.

Substrate Example 1

[0133] 5 g of dry ground A.m. leaf particulate was spread evenly over the surface of 1.5 kg (4 litres) of Westland organic compost that had been spread over a smooth surface to a depth of no more than 1 cm. The compost was then gathered up, mixed and placed into a 4 litre capacity tray.

Substrate Example 2

[0134] 10 g of dry ground A.m. leaf particulate was spread evenly over the surface of 1.5 kg (4 litres) of Westland organic compost that had been spread over a smooth surface to a depth of no more than 1 cm. The compost was then gathered up, mixed and placed into a 4 litre capacity tray.

Substrate Example 3

[0135] 20 g of dry ground A.m. leaf particulate was spread evenly over the surface of 1.5 kg (4 litres) of Westland organic compost that had been spread over a smooth surface to a depth of no more than 1 cm. The compost was then gathered up, mixed and placed into a 4 litre capacity tray.

Substrate Example 4

[0136] A master mix was made comprising 2 g per litre of ground A.m. leaf particulate per litre of 20% glycine betaine solution in water. The master mix was diluted by a factor of 40 to produce a diluted composition, and 100 ml of the diluted composition was mixed with 1.5 kg (4 litres) of Westland organic compost.

Substrate Example 5

[0137] A master mix was made comprising 2 g per litre of ground A.m. leaf particulate per litre of 20% glycine betaine solution in water. The master mix was diluted by a factor of 40 to produce a diluted composition, and 200 ml of the diluted composition was mixed with 1.5 kg (4 litres) of Westland organic compost.

Method Examples 13-17

[0138] 1.5 litres of each of Substrate Examples 1-5 were put into a 4 litre tray and 20 g of rye grass seeds was scattered over each Substrate Example. The seeds were left to germinate and were watered as required. The mass of the rye grass grown was determined after 35 days and compared to a control. The results for Method Examples 13-17 are shown below in Table 4. The control was Westland organic compost.

TABLE-US-00004 TABLE 4 effect of treated substrate on biomass of rye grass Method Example No. Substrate Example No. % difference in biomass 13 1 7 14 2 16 15 3 35 16 4 27 17 5 120

[0139] It can be seen from Method Examples 13-17 that providing a substrate with particulate A.m. leaves increases biomass.

GCMS Analysis of Particulate A.m. Leaves, Water/Alcohol Extract from A.m. Leaves and Water Extract from A.m. Leaves

[0140] GCMS analysis of particulate A.m. leaves, water/alcohol extract from A.m. leaves and water extract from A.m. leaves was undertaken and compared to GCMS results from A.m. essential oils.

Sample Preparation

[0141] The water and alcohol/water extraction samples were centrifuged at 4500 rpm for 10 min at 4° C., before filtering through a 0.45 μm syringe filter (Whatman).

[0142] The GCMS samples from particulate leaf were prepared as follows. 1 g of particulate leaves were extracted as received by macerating with 80% methanol, 1% acetic acid followed by centrifuging at 4500 rpm for 10 min at 4° C. The sample was then reextracted and the extracts combined and made up to a known volume, before being passed through a 1 μm syringe filter. A dry matter analysis was also carried out on this sample.

[0143] Total polyphenol content was determined as follows. Samples were diluted to an appropriate concentration in methanol and analysed in triplicate by adding in 20 μl aliquots to individual wells in a 96 well plate. 100 μl of 2M Folin & Ciocalteu's reagent pre diluted 1:4 was then added to all wells and the plate shaken using a plate shaker for 4 min. 75 μl of 100 g/l Sodium Carbonate solution was then added to all wells and the plate shaken for a further 1 min using a plate shaker before an adhesive lid was applied.

[0144] The microplate was then incubated in the dark at room temperature for 2 hours before reading at 750 nm using a spectrophotometer. Results are expressed against a Gallic acid standard as mg GAE/ml in the case of the extracts and as mg GAE/g in the case of the particulate leaves.

[0145] DPPH TROLOX equivalent free radical scavenging capacity was measured as follows. Samples were diluted to an appropriate concentration in 80% methanol and analysed in triplicate by adding in 20 μl aliquots to individual wells in a 96 well plate. 2800 of 150 μmol/l DPPH radical working solution was then added to all wells and an adhesive lid applied before the plate was shaken using a plate shaker set at 350±50 rpm for 45 min.

[0146] The microplate was then read at 515 nm using a spectrophotometer. Antioxidant capacity was calculated as percentage of DPPH quenched relative to the reactivity of TROLOX as a standard under the same conditions. Results are expressed as μmol TROLOX eq/ml in the case of the extracts and as μmol TROLOX eq/g in the case of the particulate leaves.

[0147] ABTS TROLOX equivalent free radical scavenging capacity was determined as follows. Samples were diluted to an appropriate concentration in methanol and analysed in triplicate by adding in 20 μl aliquots to individual wells in a 96 well plate. 280 μl of ABTS radical cation working solution was then added to all wells and the plate shaken using an instrumental shaking method for 4 min. An adhesive lid was applied and the microplate incubated in the instrument at 28° C. for 30 min before reading at 734 nm. Antioxidant capacity was then calculated as percentage inhibition of ABTS relative to the reactivity of TROLOX as a standard under the same conditions. Results are expressed as μmol TROLOX eq/ml in the case of the extracts and as μmol TROLOX eq/g in the case of the particulate leaves.

[0148] LC-DAD-QTOF polyphenolic analysis was performed as follows. Polyphenolic analysis were performed on an Agilent 6510 QTOF mass spectrometer/Agilent 1200 HPLC system equipped with a diode array detector (DAD). Separation was achieved using a Phenomenex Luna 5μ C18(2) 100 Å LC (150 mm×2.0 mm) column operated at 30° C. The mobile phase consisted of 100% deionised water containing 1% formic acid (mobile phase A) and 100% Acetonitrile containing 1% formic acid (mobile phase B). A gradient program was employed where % B ranged from 1% to 100% over a runtime of 81 min. The flow rate was held at 0.2 ml/min and 50 of each sample was injected. Simultaneous monitoring of UV signals at 280, 320, 360 and 530 nm was carried out. Accurate mass data was also collected in negative ESI mode over a mass range of 100-1000 Da at a rate of 1.5 spectra/s.

[0149] The GCMS data showed that the A.m. essential oils had a very different chemical composition to the particulate A.m. leaves, water/alcohol extract from A.m. leaves and water extract from A.m. leaves. In this connection, the GCMS data obtained from the A.m. essential oils indicated the presence of significant amounts of terpenes (such as beta-carophylene) and flavonoid aglycones. The GCMS data obtained from particulate A.m. leaves and water/alcohol extract from A.m. leaves indicated the presence of a significant amount of polyphenols. In this connection, the water/alcohol extract yielded a total phenolic content as determined in gallic acid equivalents of 3.50 mg/ml, a DPPH TROLOX equivalent free radical scavenging capacity of 18.39 micromol/ml and an ABTS TROLOX equivalent anti-oxidant capacity of 20.74 micromol/ml. The particulate leaf (12.3% dry matter sample) yielded a total phenolic content as determined in gallic acid equivalents of 4.51 mg/ml, a DPPH TROLOX equivalent free radical scavenging capacity of 19.10 micromol/ml and an ABTS TROLOX equivalent anti-oxidant capacity of 38.43 micromol/ml. A water extract yielded a total phenolic content as determined in gallic acid equivalents of 0.35 mg/ml, a DPPH TROLOX equivalent free radical scavenging capacity of 1.65 micromol/ml and an ABTS TROLOX equivalent anti-oxidant capacity of 3.32 micromol/ml. The results obtained from the water/alcohol extract and the leaf particulate are particularly impressive, not least when compared to results generated from Bramley apple, a recognised source high in polyphenols (a 13.3% dry matter sample yielding a total phenolic content as determined in gallic acid equivalents of 1.50 mg/ml, a DPPH TROLOX equivalent free radical scavenging capacity of 9.21 micromol/ml and an ABTS TROLOX equivalent anti-oxidant capacity of 12.57 micromol/ml).

Composition Example 8

[0150] A mass of dry, brittle A.m. leaves were processed using a blender. A known mass of usable dry material was then added to a volume of solvent comprising 20% ethanol:80% water mixture (vol:vol). The suspension of leaf particulate in solvent was processed further using the blender in order to reduce the particle size further. Further solvent was added to a dispersion concentration of 2 g of A.m. leaves per 100 ml solvent. The dispersion was then diluted by water 1000 parts water:1 part dispersion (vol:vol.), ready for use.

Composition Example 9

[0151] Composition Example 9 was made as per Composition Example 8, but with 4 g of A.m. leaves per 100 ml solvent.

Composition Example 10

[0152] Composition Example 10 was made as per Composition Example 8, but with 10 g of A.m. leaves per 100 ml solvent.

Composition Example 11

[0153] A mass of dry, brittle A.m. leaves were processed using a blender. A known mass of usable dry material was then added to a volume of solvent comprising an emulsion of orange terpenes and water (2:1 vol.:vol.). The suspension of leaf particulate in solvent was processed further using the blender in order to reduce the particle size further. The sample was subject to 10 mins in an ultrasound bath to improve extraction. Further solvent was added to a dispersion concentration of 2 g of A.m. leaves per 100 ml solvent. The dispersion was then diluted by water 1000 parts water:1 part dispersion (vol:vol.), ready for use.

Composition Example 12

[0154] A mass of dry, brittle A.m. leaves were processed using a blender. A known mass of usable dry material was then added to a volume of deep eutectic solvent. The deep eutectic solvent was made from choline bitartrate, glycerol and water mixed at the ratio 42:45:13 wt:wt. This was heated at 40° C. for 30 minutes to form a clear liquid. The suspension of leaf particulate in solvent was processed further using the blender in order to reduce the particle size further. The sample was subject to 10 mins in an ultrasound bath to improve extraction. Further solvent was added to a dispersion concentration of 2 g of A.m. leaves per 100 ml solvent. 2 ml of yucca extract was also added per 100 ml of solvent as a surfactant. The dispersion was then diluted by water 1000 parts water:1 part dispersion (vol:vol.), ready for use.

Composition Example 13

[0155] Composition Example 13 was made as per Composition Example 12, but with 4 g of A.m. leaves per 100 ml solvent.

Method Examples 18 to 23

[0156] 6 ml of Composition Examples 8 to 13 were sprayed onto a pot comprising grass seeds in a growth substrate, once immediately after sowing, then 1 week post-sowing and 2 weeks post-sowing. Five pots were used per Composition Example. After several weeks, the leaf biomass of the five pots was measured by removing and weighing the leaves from the five pots. Root biomass was estimated visually for each pot on a scale of 1-5, and the scale estimations for the five pots were aggregated to give an overall score from 5 to 25. The results are shown below in Table 5.

TABLE-US-00005 TABLE 5 effect of treatment on leaf and root biomass Composition Method Example Leaf biomass of five Root biomass Example No. No. pots (g) (5-25) 8 18 23.3 21 9 19 28.2 18 10 20 68.7 22 11 21 56.0 13 12 22 46.5 9 13 23 46.2 8 Control (water only) N/A 38.7 9

[0157] Table 5 shows that the use of higher amounts of A.m. leaf (Composition Example 10), the use of A.m. leaf with orange terpenes and the use of a deep eutectic solvent are effective. Furthermore, Table 5 also shows that treatment with smaller amounts of A.m. leaf may produce high root biomass (Composition Examples 8 and 9).

[0158] The total polyphenol content, DPPH TROLOX equivalent free radical scavenging capacity and ABTS TROLOX equivalent free radical scavenging capacity of the pre-compositions that were diluted to form Composition Examples 8-13 were measured and the results are shown below in Table 6. The pre-compositions were 1000 times more concentrated than the Composition Examples.

TABLE-US-00006 TABLE 6 measurement of some active components of pre-compositions that were diluted to form some of Composition Examples 8-13 DPPH ABTS Total polyphenols μmol TROLOXeq μmol TROLOXeq Composition mg GAE/ml in pre- /ml in pre- /ml in pre- Example No. composition composition composition 8 0.76 3.86 7.18 9 1.15 6.19 11.30 10 2.63 13.55 24.31 12 n/a 7.48 17.52 13 n/a 13.03 26.90

[0159] Whilst the present invention has been described and illustrated with reference to particular embodiments, it will be appreciated by those of ordinary skill in the art that the invention lends itself to many different variations not specifically illustrated herein. By way of example only, certain possible variations will now be described.

[0160] In the examples above, Indian A.m. leaves were used. Those skilled in the art will realise that A.m. leaves from other countries may be used.

[0161] In the examples above the leaf particulate is formed using a blender. Those skilled in the art will realise that other techniques may be used to form the particulate. For example, the leaf particulate may be formed by grinding.

[0162] In the examples above, the aqueous extract is formed using a solvent comprising water and ethanol. Those skilled in the art will realise that other solvents may be used. The solvent may typically comprise one or more alcohols.

[0163] In the examples above, lettuce and rye grass were treated. Those skilled in the art will realise that other plants may be treated.

[0164] The treatment methods described above used a regime of treatment at days 1, 7, 14 and 21 days (i.e. weekly). Those skilled in the art will realise that other treatment regimes are possible.

[0165] The examples above describe biostimulant compositions optionally comprising orthosilicic acid. Those skilled in the art will realise that other components may be added to the composition.

[0166] Where in the foregoing description, integers or elements are mentioned which have known, obvious or foreseeable equivalents, then such equivalents are herein incorporated as if individually set forth. Reference should be made to the claims for determining the true scope of the present invention, which should be construed so as to encompass any such equivalents. It will also be appreciated by the reader that integers or features of the invention that are described as preferable, advantageous, convenient or the like are optional and do not limit the scope of the independent claims. Moreover, it is to be understood that such optional integers or features, whilst of possible benefit in some embodiments of the invention, may not be desirable, and may therefore be absent, in other embodiments.