Method of Producing a Biological Response

Technical Field

[1] In some embodiments, this invention relates to uses of a bioactive composition, preferably an oil extract of cannabis, which has useful bioactive properties. In some embodiments, the invention concerns a bioactive composition in the form of an oil extract of cannabis, capable of providing useful effects against biological organisms such as pesticidal effects or allelopathic effects.

Background

[2] There has been increasing global concern about the costs on human health and the environment resulting from the use of chemical pesticides, fungicides, fertilizers and other chemical products in agriculture. As a result, there is considerable interest in the development of more sustainable alternatives.

[3] One such ‘green’ alternative is the use of plant essential oils which are aromatic and volatile liquids extracted from plants. The chemicals in essential oils are called secondary metabolites, and their role in plant development appears to become more significant as research continues. Secondary metabolites have displayed a wide range of biological effects and are generally thought to be the plant’ s primary defence against a wide range of biotic factors.

[4] Secondary plant metabolites are classified according to their chemical structures into several broad classes which include phenolics, alkaloids, saponins, terpenes, lipids and carbohydrates. Of these, the terpenes appear to be the most chemically and functionally diverse. Terpenes are naturally occurring hydrocarbons produced by a wide variety of plants and are the primary constituents of essential oils. The classification of terpenes is based on five-carbon (isoprene) units as their building blocks. Different terpenes include hemiterpenes (C5), monoterpenes (CIO), sesquiterpenes (C15), diterpenes (C20), sesterterpenes (C25), triterpenes (C30), and polyterpenes (>C30).

[5] The term ‘cannabis’ refers to the plants of the species Cannabis Sativa L. Within that species are three types of cannabis plants: Cannabis Sativa, Cannabis Indica and Cannabis Ruderalis. ‘Hemp’ and cannabis are the same species of plant except that hemp produces less of the psychoactive chemical Delta-9-tetrahydrocannabinol (THC).

[6] Around 150 different terpene compounds have been identified in the cannabis plant, and each cannabis cultivar is thought to have a unique constitution and ratio of terpenes (terpene profile). Myrcene is generally the most abundant terpene in modern commercial cannabis. On average, myrcene is thought to represent over 20% of the terpene profile in most cultivars although individual cultivars do vary widely in their terpene content.

[7] Table 1 below shows six cannabis terpenes generally recognised as the most common among cultivars and various biological properties that have been associated with that terpene.

[8] Table 1

[9] Allelopathy is a process whereby an organism (usually a plant) produces a negative or positive effect on another organism (usually another plant, but sometimes an insect), by producing a chemical compound (allelochemical) which interferes with the biology of the other organism. In several studies a switch from stimulatory effects to inhibitory activity with increasing concentrations was observed (suggesting a response threshold). It is thought that the production of compounds that have an inhibitory effect on other organisms enable the producing organism to limit growth of competitors and thus facilitate access to resources.

[10] The negative or positive effects on the receiving organism appear to be due to the actions of secondary metabolites from the initiating plant. A large amount of research in allelopathy has been directed at the inhibitory effects some plant secondary metabolites can have on other plant species. For example, the effects of extracts from different parts of the initiating plant fennel (ie. vegetative, flowering, fruiting extract) have on the growth and development of lettuce seeds and seedlings (Zribi (2014)). In general, these types of studies have found (often significant) inhibitory effects on seed germination rates and seedling development. These types of studies are usually focused on developing an alternative to conventional chemical herbicides.

[11] Allelochemicals may be released from the plant through a number of mechanisms including root secretion, leaching and through volatile organic compounds (VOC’s). The VOC’s includes the terpenes and other volatile plant compounds. These compounds are given off the plant in a gaseous form (or fragrance) in normal daytime temperatures when the plant is at that stage of its life cycle. Nearby plants and insects are able to ‘sense’ and react to these VOC’s. This is known as plant volatile-mediated plant-to-plant communication. Recent research has shown how the genetic mechanisms of the receiving plant adjust as various genes are up or down regulated in response to the VOC’s (Nagashima (2018)). Examples of this include research in some vegetable species which have been found to release certain volatiles in response to attack by insect herbivores and pathogens. These induced volatiles can attract the natural enemies of insect herbivores, repel other insect herbivores and increase the resistance of tissues in both the host plant and neighbouring plants (references cited in Kang et.al 2018). Furthermore, intermittent exposure to the volatiles emitted from artificially damaged Arabidopsis has been shown to induce defensive responses in undamaged neighbouring plants (Shiojiri et al., (2012) in Kang (2018)).

[12] Hemp (cannabis) has long been observed to be effective at supressing weeds when growing in competition with other species in the field. As a result, there have been a number of studies that have investigated the potential allelopathic activity of cannabis extract. These studies were primarily focused on the potential of any inhibitory effects on other plant species and developing alternatives to chemical herbicides.

Detailed Description of the Invention

[13] The present invention, in one broad form, relates to methods of producing a biological response in a plant from the Cannabaceae family which involves administering a cannabis extract to the plant.

[14] In a first aspect of the invention, there is provided a method of producing a biological response in a plant from the Cannabaceae family, the method comprising the step of administering a bioactive oil extract to the plant to thereby produce the biological response, wherein the bioactive oil extract is a cannabis extract.

[15] The present inventors have found that by administering a cannabis derived bioactive oil extract to plants of the Cannabaceae family, it is possible to produce a range of beneficial biological responses in the subject plant, such as increased resistance to pest infestation, increased resistance to mould infection, increased resistance to bacterial, fungal or viral infection, and increased biomass production. This result was unexpected, as in the method an extract from cannabis is applied to plants of the same family. To date, there has been relatively little research directed towards the potential of positive allelopathic effects, particularly those that may be found when an extract of one plant is applied to a phylo genetically similar plant species.

[16] In some embodiments, the cannabis extract comprises, consists essentially of, or consists of the following concentrations of monoterpenes:

Terpene Profile 1 approximately 0.3-50.1 % Pinene; approximately 0.1-10.9 % Limonene; and approximately 0.2-39.0 % Ocimene.

[17] In some embodiments Terpene Profile 1 comprises, consists essentially of, or consists of the following concentrations of monoterpenes: approximately 19-31 % Pinene; approximately 4-6 % Limonene; approximately 18-21 % Ocimene.

[18] In some embodiments Terpene profile 1 comprises, consists essentially of, or consists of Pinene; Limonene; and Ocimene in a ratio of about 5:1:4.

[19] The ratio of Pinene: limonene in Terpene Profile 1 may be between about 3:1 and about 6:1.

[20] The ratio of Ocimene: limonene in Terpene Profile 1 may be between about 3:1 and about 5:1.

[21] In some embodiments Terpene Profile 1 comprises, consists essentially of, or consists of the following concentrations of monoterpenes: approximately 0.2-35.2 % a- Pinene; approximately 0.1-14.9 % P -Pinene; approximately 0.1-10.9 % D-Limonene; approximately 0.0- 1.5 % Trans-Ocimene; and approximately 0.2-37.5 % Cis-Ocimene.

[22] In some embodiments Terpene Profile 1 comprises, consists essentially of, or consists of a-Pinene; P-Pinene; D-Limonene; Trans-Ocimene; and Cis-Ocimene in a ratio of about 22:9:7:1:24. [23] In some embodiments Terpene Profile 1 comprises, consists essentially of, or consists of a-Pinene; P-Pinene; D-Limonene; Trans-Ocimene; and Cis-Ocimene in a ratio of about 19-27:9-11:6-8:1:22-29.

[24] The ratio of a-Pinene:Trans-Ocimene in Terpene Profile 1 may be between about 19:1 and about 27:1.

[25] The ratio of P-Pinene:Trans-Ocimene in Terpene Profile 1 may be between about 9:1 and about 11:1.

[26] The ratio of D-Limonene:Trans-Ocimene in Terpene Profile 1 may be between about 6:1 and about 8:1.

[27] The ratio of Cis-Ocimene:Trans-Ocimene in Terpene Profile 1 may be between about 22: 1 and about 29:1.

[28] In some embodiments, the cannabis extract comprises, consists essentially of, or consists of the following concentrations of monoterpenes:

Terpene Profile 2 approximately 4.8-45.6 % Pinene; approximately 1.0-9.0 % Limonene; approximately 3.7-35.5 % Ocimene; and approximately 1.1-9.9 % P -Myrcene.

[29] In some embodiments Terpene Profile 2 comprises, consists essentially of, or consists of the following concentrations of monoterpenes: approximately 19-31 % Pinene; approximately 4-6 % Limonene; approximately 4-6 % P-Myrcene; and approximately 18-21 % Ocimene.

[30] In some embodiments Terpene profile 2 comprises, consists essentially of, or consists of Pinene; Limonene; P-Myrcene and Ocimene in a ratio of about 5: 1:1:4.

[31] The ratio of Pinene: limonene in Terpene Profile 2 may be between about 3:1 and about 6:1.

[32] The ratio of P-Myrcene:limonene in Terpene Profile 2 may be between about 0.5:1 and about 1.5:1.

[33] The ratio of Ocimene: limonene in Terpene Profile 2 may be between about 3:1 and about 5:1.

[34] In some embodiments Terpene Profile 2 comprises, consists essentially of, or consists of the following concentrations of monoterpenes: approximately 3.4-32.0 % a- Pinene; approximately 0.1-13.6 % P -Pinene; approximately 1.1-9.9% D-Limonene; approximately 0.1- 1.4 % Trans-Ocimene; approximately 3.6-34.1 % Cis-Ocimene; and approximately 1.0-9.0 % P-Myrcene.

[35] In some embodiments Terpene Profile 2 comprises, consists essentially of, or consists of a-Pinene; P-Pinene; D-Limonene; P-Myrcene; Trans-Ocimene; and Cis-Ocimene in a ratio of 22:9:7:6: 1 :24.

[36] In some embodiments Terpene Profile 2 comprises, consists essentially of, or consists of a-Pinene; P-Pinene; D-Limonene; P-Myrcene; Trans-Ocimene; and Cis-Ocimene in a ratio of 19-27:9-11:6-8:6-7:1:22-29

[37] The ratio of a-Pinene:Trans-Ocimene in Terpene Profile 2 may be between about 19:1 and about 27:1.

[38] The ratio of P-Pinene:Trans-Ocimene in Terpene Profile 2 may be between about 9:1 and about 11:1.

[39] The ratio of D-Limonene:Trans-Ocimene in Terpene Profile 2 may be between about 6:1 and about 8:1.

[40] The ratio of P-Myrcene:Trans-Ocimene in Terpene Profile 2 may be between about 6:1 and about 7:1.

[41] The ratio of Cis-Ocimene:Trans-Ocimene in Terpene Profile 2 may be between about 22: 1 and about 29:1.

[42] In some embodiments, the cannabis extract is a liquid.

[43] In some embodiments, the cannabis extract is an essential oil extract.

[44] In some embodiments, the cannabis extract is a solid.

[45] In some embodiments, the cannabis extract is in the form of a dry powder, pellet, granules, tablets or flakes.

[46] In some embodiments, the cannabis extract is administered as part of a composition, wherein the composition comprises one or more excipients.

[47] Preferably, the one or more excipients are selected from the group consisting of: drying agents; solvent; adhesive; aqueous or oily diluent; carrier; base; buffer; pH adjuster; bittering agent (i.e. foul-tasting agent); suspending agent; thickening agent; rheology modifier; gelling agent; viscosity increasing agent; antifreeze, emulsifier; emollient; stabilising agent; dispersing agent/dispersant; solubiliser; fragrance; preservative; surfactant; textural modifier; foaming agent; anti-foaming agent; colourant; propellant; refrigerant; and, waterproofing agent.

[48] In some embodiments, the other excipients further comprise a fertiliser.

[49] The composition may be an emulsion.

[50] In one embodiment, the emulsion is an oil-in-water emulsion. In another embodiment, the emulsion is a water-in-oil emulsion.

[51] In some embodiments, the composition is a colloid, suspension, admixture, solution or mixture.

[52] In some embodiments, the cannabis extract is an extract of cannabis flowers.

[53] In other embodiments, the cannabis extract is an extract of cannabis flowers, panicles, stem, or upper leaves, or a combination thereof.

[54] In some embodiments, the cannabis extract is an extract of a single cannabis variety. In other embodiments, the cannabis extract is an extract of more than one cannabis variety.

[55] The cannabis extract may administered during the vegetative growth stage of development of the plant.

[56] In some embodiments, the cannabis extract is administered during the seed, seedling or vegetative stages of development of the plant, or a combination thereof. In other embodiments, the cannabis extract is administered to plant cuttings, either before or after root development.

[57] In some embodiments, the biological response is increased biomass production.

[58] In some embodiments, the biological response is increased resistance to pest infestation.

[59] The pest may be selected from the group consisting of: insect and arachnid; especially from the group consisting of: mosquito, fruit fly, spider mite and aphid.

[60] In some embodiments, the pest is selected from the group consisting of: caterpillar, scale, mosquito, fly, aphid, mite, spider, cockroach, weevil and rootworm.

[61] In some embodiments, the biological response is increased resistance to bacterial, fungal or viral infection.

[62] In one embodiment, the fungal infection is a mould.

[63] In some embodiments, the biological response is stimulating the production of plant defence compounds within the plant.

[64] In some embodiments, the production of plant defence compounds provides increased resistance to pest infestation. [65] Preferably, the plant is a cannabis plant.

[66] In some embodiments, the plant is a variety of hops (Humulus lupulus) plant.

[67] In preferred embodiments, the cannabis extract is administered to the leaves of the plant.

[68] In some embodiments, the cannabis extract is administered to the roots, foliage, flower head or seed, or a combination thereof, of the plant.

[69] In some embodiments, the bioactive oil extract is a hemp extract.

[70] The method may be a prophylactic method. The method may be a preventative method.

[71] The method of producing a biological response is preferably a method of producing a beneficial biological response.

[72] In a second aspect of the invention, there is provided a method of controlling one or more properties of a plant species in the Cannabaceae family, the method comprising the step of administering to the plant a bioactive oil extract comprising the following concentrations of monoterpenes:

Terpene Profile 1 approximately 0.3-50.1 % Pinene; approximately 0.1-10.9 % Limonene; and approximately 0.2-39.0 % Ocimene, or

Terpene Profile 2 approximately 4.8-45.6 % Pinene; approximately 1.0-9.0 % Limonene; approximately 3.7-35.5 % Ocimene; and approximately 1.1-9.9 % [3-Myrcene, and wherein the one or more properties are selected from the group consisting of: stimulating the production of plant defence compounds within the plant; triggering anti-microbial defence mechanisms within the plant; activating anti-viral defence mechanisms within the plant; activating anti-bacterial defence mechanisms within the plant; eliciting plant disease resistance; enhancing plant disease resistance; promoting plant growth; increasing biomass production within the plant; increasing plant uniformity; or, use as a herbicide.

[73] The one or more properties may be stimulating the production of plant defence compounds within the plant. The production of plant defence compounds may provide increased resistance to pest infestation. [74] In a third aspect of the invention, there is provided a method of controlling one or more properties in a plant from the Cannabaceae family, the method comprising the step of administering a bioactive oil extract to the plant to thereby control the one or more properties, wherein the bioactive oil extract is a cannabis extract.

[75] In one embodiment, the cannabis extract is a fertiliser.

[76] In some embodiments, the method of producing a biological response is a method of eliciting a biological response.

[77] In some embodiments, the method of producing a biological response is method of producing an allelopathic effect.

[78] In a fourth aspect of the invention, there is provided a method of increasing biomass production in a plant from the Cannabaceae family, the method comprising the step of administering a bioactive oil extract to the plant to thereby increase biomass production, wherein the bioactive oil extract is a cannabis extract.

[79] In a fifth aspect of the invention, there is provided a method increasing resistance to pest infestation in a plant from the Cannabaceae family, the method comprising the step of administering a bioactive oil extract to the plant to thereby increase resistance to pest infestation, wherein the bioactive oil extract is a cannabis extract.

[80] In a sixth aspect of the invention, there is provided a method increasing resistance to fungal infection in a plant from the Cannabaceae family, the method comprising the step of administering a bioactive oil extract to the plant to thereby increase resistance to fungal infection, wherein the bioactive oil extract is a cannabis extract.

[81] In some embodiments, the present inventors have produced a bioactive terpene composition based on an oil extract of cannabis plants which has bioactive properties, and have further characterised a terpene profile of that extract that provides useful effects against biological organisms, such as pesticidal effects or allelopathic effects.

[82] According to a seventh aspect of the present invention, there is provided a cannabis-based bioactive terpene composition comprising the following concentrations of monoterpenes:

[83] Terpene Profile 1

[84] approximately 0.3-50.1 % Pinene;

[85] approximately 0.1-10.9 % Limonene ; and

[86] approximately 0.2-39.0 % Ocimene,

[87] or

[88] Terpene Profile 2 [89] approximately 4.8-45.6 % Pinene;

[90] approximately 1.0-9.0 % Limonene;

[91] approximately 3.7-35.5 % Ocimene; and

[92] approximately 1.1-9.9 % [3-Myrcene.

[93] According to an eighth aspect of the present invention, there is provided a bioactive terpene composition comprising a bioactive oil extract prepared from cannabis plants, wherein the composition comprises the following concentrations of monoterpenes:

[94] Terpene Profile 1

[95] approximately 0.3-50.1 % Pinene;

[96] approximately 0.1-10.9 % Limonene; and

[97] approximately 0.2-39.0 % Ocimene,

[98] or

[99] Terpene Profile 2

[100] approximately 4.8-45.6 % Pinene;

[101] approximately 1.0-9.0 % Limonene;

[102] approximately 3.7-35.5 % Ocimene; and

[103] approximately 1.1 -9.9 % P -Myrcene.

[104] Unless stated otherwise or context- specific, % means percentage volume or percentage weight. Each of those ranges includes all 0.01% incremental values between the upper and lower concentration limits.

[105] According to a ninth aspect of the present invention, there is provided a bioactive oil extract based on or prepared from cannabis plants, wherein the bioactive oil extract comprises the following concentration of monoterpenes:

[106] Terpene Profile 1

[107] approximately 0.3-50.1 % Pinene;

[108] approximately 0.1-10.9 % Limonene; and

[109] approximately 0.2-39.0 % Ocimene,

[110] or

[111] T erpene Profile 2

[112] approximately 4.8-45.6 % Pinene;

[113] approximately 1.0-9.0 % Limonene;

[114] approximately 3.7-35.5 % Ocimene; and

[115] approximately 1.1-9.9 % P-Myrcene. [116] Each of those ranges includes all 0.01% incremental values between the upper and lower concentration limits.

[117] According to a tenth aspect of the present invention, there is provided a method of preparing the bioactive terpene composition of the seventh or eighth aspect, or the bioactive oil extract of the ninth aspect, the method comprising the step of:

[118] a preparation step, comprising preparing a bioactive oil extract from cannabis plants.

[119] According to a eleventh aspect of the present invention, there is provided a cannabis-based bioactive oil extract prepared using the method according to the tenth aspect.

[120] According to a twelfth aspect of the present invention, there is provided a formulation comprising the bioactive terpene composition of the seventh or eighth aspect, or the bioactive oil extract of the ninth or tenth aspect, and, optionally, at least one additional ingredient.

[121] According to a thirteenth aspect of the present invention, there is provided use of:

[122] the bioactive terpene composition of the seventh or eighth aspect;

[123] the bioactive oil extract of the ninth or tenth aspect; or

[124] the formulation of the twelfth aspect,

[125] for controlling a biological organism.

[126] According to a fourteenth aspect of the present invention, there is provided a method of controlling a biological organism, said method comprising the step of treating the biological organism with or exposing the biological organism to:

[127] the bioactive terpene composition of the seventh or eighth aspect;

[128] the bioactive oil extract of the ninth or tenth aspect; or

[129] the formulation of the twelfth aspect,

[130] in an amount sufficient so as to control the biological organism.

[131] Plant material derived or obtained from the hemp variety can comprise flowers, panicles, stems, roots or leaves. Plant material derived or obtained from the hemp variety can comprise live plant material that can be propagated, such as cuttings or seeds.

[132] Features of the different aspects of the invention defined above are further described below. Context permitting, the following and above features apply to each and every aspect of the invention. Also, features of a product may be taken as being features of a method and vice-versa, even if not specifically stated.

[133] The bioactive terpene composition or bioactive oil extract is based on or prepared from cannabis plants of the species Cannabis Sativa L, preferably an essential oil extract. Any suitable type or types of cannabis plants can be used, including Cannabis Sativa, Cannabis Indica and/or Cannabis Ruderalis. In some embodiments, the cannabis plants can have psychoactive properties. In some embodiments, the cannabis plants can be hemp, and thus lack psychoactive properties.

[134] In some embodiments, the bioactive terpene composition or bioactive oil extract is based on or prepared from hemp, preferably an essential oil extract. In some embodiments, the bioactive terpene composition or bioactive oil extract is based on or prepared from a single cannabis plant cultivar, variety or chemovar, preferably an essential oil extract. In some embodiments, the bioactive terpene composition or bioactive oil extract is based on or prepared from more than one cannabis plant cultivar, variety or chemovar, including two, three, four or more cannabis plant cultivars, varieties or chemovars, preferably an essential oil extract.

[135] In some embodiments, the bioactive terpene composition or bioactive oil extract preferably further comprises P-Myrcene, as shown in Terpene Profile 2. The monoterpenes can each comprise one or more different isomeric forms. For example, the pinene terpene can comprise a-pinene and P-pinene. The limonene terpene can comprise D-limonene. The ocimene terpene can comprise trans-ocimene and cis-ocimene. The myrcene terpene can comprise P-myrcene.

[136] In some embodiments, the bioactive terpene composition or bioactive oil extract is further prepared by the addition of at least one oil additive, such as at least one monoterpene and/or at least one oil extract (preferably an essential oil extract), such that a terpene profile of interest is produced. In some embodiments, the oil additive is an oil extract from non-cannabis plants, preferably an essential oil extract. In some embodiments, the oil additive is an oil extract from plants in the family Cannabaceae, preferably an essential oil extract. In some embodiments, the oil additive is an oil extract from plants not in the family Cannabaceae, preferably an essential oil extract. In some embodiments, the oil additive is a naturally-derived or synthetic monoterpene. If naturally-derived, it can be produced from cannabis plants, non-cannabis plants, plants in the family Cannabaceae, or plants not in the family Cannabaceae.

[137] Preferably the cannabis-based bioactive terpene composition comprises the bioactive oil extract according to the ninth aspect of the invention, but this need not be the case. The bioactive oil extract, preferably an essential oil extract, can be prepared in any suitable way. In some embodiments, the bioactive oil extract is prepared by alcohol extraction, such as ethanol extraction. In some embodiments, the bioactive oil extract is prepared by oil extraction, for example, using olive oil or coconut oil.

[138] In some embodiments, the bioactive oil extract is prepared by extraction using a hydrocarbon such as butane.

[139] In some embodiments, the bioactive oil extract is prepared by distillation. In some embodiments, the bioactive oil extract is prepared by steam distillation and/or hydrodistillation. Steam distillation and hydrodistillation procedures are well known in the art, and examples can be found in the following reference, the entire contents of which are incorporated herein by reference: Jamil et al. (2016).

[140] In some embodiments, the bioactive oil extract is prepared by using carbon dioxide (CO2) extraction technology, such as subcritical or supercritical CO2 extraction. Carbon dioxide extraction procedures are well known in the art, and examples can be found in the following reference, the entire contents of which are incorporated herein by reference: Naz et al. (2017).

[141] In some embodiments, the bioactive oil extract is prepared by distillation, preferably steam distillation, of fresh plant material or raw, frozen plant material. Any suitable part or parts of the plant can be used. Any suitable part or parts of the cannabis plant can be used, including the flowers, panicles, stem, and upper leaves of the cannabis/hemp plant. In some embodiments, the bioactive oil extract is prepared by steam distillation of cannabis flowers, preferably an essential oil extract.

[142] The bioactive terpene composition or bioactive oil extract can be in a concentrated form, undiluted/neat form or diluted form.

[143] Likewise, one or more of the monoterpenes of the bioactive terpene composition can be prepared in any suitable way. In some embodiments, the terpenes are prepared by alcohol extraction, such as ethanol extraction. In some embodiments, the terpenes are prepared by oil extraction, for example, using olive oil or coconut oil.

[144] In some embodiments, the terpenes are prepared by extraction using a hydrocarbon such as butane.

[145] In some embodiments, the terpenes are prepared by distillation. In some embodiments, the terpenes are prepared by steam distillation and/or hydrodistillation.

[146] In some embodiments, the terpenes are prepared by using carbon dioxide (CO2) extraction technology, such as subcritical or supercritical CO2 extraction. [147] In some embodiments, the terpenes are prepared by distillation, preferably steam distillation, of fresh plant material or raw, frozen plant material. Any suitable part or parts of the plant can be used. Any suitable part or parts of the cannabis plant can be used, including the flowers, panicles, stem, and upper leaves of the cannabis/hemp plant. In some embodiments, the terpenes are prepared by steam distillation of cannabis flowers, preferably an essential oil extract.

[148] In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the monoterpene concentrations of hemp variety M183, as seen in Tables 2 or 6B. In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the essential oil constituents of hemp variety M183, as seen in Tables 2 or 6B.

[149] In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the monoterpene concentrations of hemp variety M168, as seen in Tables 2 or 6D. In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the essential oil constituents of hemp variety M168, as seen in Tables 2 or 6D.

[150] In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile is produced by blending oil extracts of two or more hemp varieties together.

[151] In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the average % terpene concentration of M183 and M168 as seen in Table 2. In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the essential oil constituents of the average % terpene concentration of M183 and M168 as seen in Table 2.

[152] Table 2 (Terpene Profile 1)

[153] In some embodiments Terpene Profile 1 comprises any one or more of:

[154] approximately 0.2-35.2 % a- Pinene;

[155] approximately 0.1 - 14.9 % P -Pinene ;

[156] approximately 0.1-10.9 % D-Limonene;

[157] approximately 0.0-1.5 % Trans-Ocimene; and

[158] approximately 0.2-37.5 % Cis-Ocimene.

[159] Each of those ranges includes all 0.01% incremental values between the upper and lower concentration limits.

[160] In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the monoterpene concentrations of hemp variety Ml 83, as seen in Table 3. In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the essential oil constituents of hemp variety M183, as seen in Table 3.

[161] In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the monoterpene concentrations of hemp variety M168, as seen in Table 3. In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the monoterpene concentrations of the essential oil constituents of hemp variety M168, as seen in Table 3.

[162] In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the average % terpene concentration of M183 and M168 as seen in Table 3. In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the essential oil constituents of the average % terpene concentration of M183 and M168 as seen in Table 3.

[163] Table 3 (Terpene Profile 1)

[164] In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the monoterpene concentrations of hemp variety M183, as seen in Table 4. In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the monoterpene concentrations of the essential oil constituents of hemp variety M183, as seen in Table 4.

[165] In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the monoterpene concentrations of hemp variety M168, as seen in Table 4. In some embodiments, bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the essential oil constituents of hemp variety M168, as seen in Table 4.

[166] In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile is produced by blending the oil extracts of hemp varieties together. In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the average % terpene concentration of M183 and M168 as seen in Table 4. In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the essential oil constituents of the average % terpene concentration of M183 and M168 as seen in Table 4.

[167] Table 4 (Terpene Profile 2)

[168] In some embodiments Terpene Profile 2 comprises any one or more of:

[169] approximately 3.4-32.0 % a-Pinene;

[170] approximately 0.1 - 13.6 % P -Pinene ;

[171] approximately 1.1-9.9% D-Limonene;

[172] approximately 0.1-1.4 % Trans-Ocimene;

[173] approximately 3.6-34.1 % Cis-Ocimene; and

[174] approximately 1.0-9.0 % P-Myrcene.

[175] Each of those ranges includes all 0.01% incremental values between the upper and lower concentration limits.

[176] In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the monoterpene concentrations of hemp variety M183, as seen in Table 5. In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the essential oil constituents of hemp variety M183, as seen in Table 5.

[177] In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the monoterpene concentrations of hemp variety M168, as seen in Table 5. In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the essential oil constituents of hemp variety M168, as seen in Table 5.

[178] In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the average % terpene concentration of M183 and M168 as seen in Table 5. In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the essential oil constituents of the average % terpene concentration of M183 and M168 as seen in Table 5. [179] Table 5 (Terpene Profile 2)

[180] In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile can have monoterpene concentration ranges substantially as shown in Table 7B. Each of those ranges includes all 0.01% incremental values between the upper and lower concentration limits.

[181] In some embodiments, the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile can have terpene concentration ranges substantially as shown in Tables 4, 5 or 7A. Each of those ranges includes all 0.01% incremental values between the upper and lower concentration limits.

[182] The bioactive terpene composition or formulation can be of any suitable form. The bioactive terpene composition or formulation can be, for example, in the form of a liquid, solution, gel, creme, emulsion, paste, film, powder, foam, suspension, gas, vapour or aerosol. The bioactive oil extract can be microencapsulated using any suitable microencapsulation process.

[183] The bioactive terpene composition or formulation can comprise any suitable quantity of bioactive oil extract. In some embodiments, the ratio of bioactive oil extract to all other ingredients of the bioactive terpene composition or formulation ranges between about 1:1 to about 1:1000, and any numerical value or subrange there between. In some embodiments, the bioactive terpene composition or formulation comprises approximately 200 g/L bioactive oil extract.

[184] Depending on the form of the bioactive terpene composition or formulation, it can include one or more of the following types of ingredients: solvent; adhesive; aqueous or oily diluent; carrier; excipient; base; buffer; pH adjuster; bittering agent (i.e. foul-tasting agent); suspending agent; thickening agent; rheology modifier; gelling agent; viscosity increasing agent; antifreeze, emulsifier; emollient; stabilising agent; dispersing agent/dispersant; solubiliser; fragrance; preservative; surfactant; textural modifier; foaming agent; anti-foaming agent; colourant; propellant; refrigerant; and, waterproofing agent.

[185] In some embodiments, the bioactive terpene composition or formulation can include, for example: oleic acid or methyl ester as solvent; polymeric surfactant, oxirane, 2- methyl-, polymer with oxirane, or monobutyl ether as emulsifier; propylene glycol or propane- 1,2-diol as antifreeze; xanthum gum as rheology modifier; and, polydimethysiloxane aqueous emulsion as an anti-foaming agent.

[186] In some embodiments, the bioactive terpene composition or formulation can include, for example, alcohol or water as an excipient or carrier.

[187] Suitable oily or aqueous bases, carriers, diluents and excipients are inert and include, for example: bacteriostatic saline (saline containing benzyl alcohol), cetomacrogol, cetyl alcohol, glycerine, lanolin, petrolatum based creams, gels, hydrogels, saline, short chain alcohols and glycols (e.g. ethyl alcohol and propylene glycol), and water.

[188] Either water in oil or oil in water emulsions can be used. Examples of suitable surfactants and emulsifying agents include: vitamin E, non-ionic ethoxylated and nonethoxylated surfactants, abietic acid, almond oil PEG, beeswax, butylglucoside caprate, Cis- C36 acid glycol ester, C9-C15 alkyl phosphate, caprylic/capric triglyceride PEG-4 esters, cetomacrogol, ceteareth-7, cetereth-20, cetyl phosphate, cetyl stearyl alcohol, com oil PEG esters, DEA-cetyl phosphate, dextrin laurate, dilaureth-7 citrate, dimyristyl phosphate, glycereth-17 cocoate, glyceryl erucate, glycerol, glyceryl laurate, G.M.S. acid stable, hydrogenated castor oil PEG esters, isosteareth-11 carboxylic acid, lecithin, lysolecithin, nonoxynol-9, octyldodeceth-20, palm glyceride, PEG diisostearate, PEG stearamine, poloxamines, polyglyceryls, potassium linoleate, PPGs, raffinose myristate, sodium caproyl lactylate, sodium caprylate, sodium cocoate, sodium isostearate, sodium tocopheryl phosphate, steareths, TEA-C12-C13 pareth-3 sulfate, tri-Cn-Cis pareth-6 phosphate, and trideceths. [189] The bioactive terpene composition or formulation can comprise one or more types of preservative. A suitable preservative, for example, can be: benzalkonium chloride, benzoic acid, benzothonium chloride, benzyl alcohol, 2-bromo-2-nitropropane-l,3-diol, bronopol, butylated hydroxyanisole, butylated hydroxytoluene, butyl paraben, chlorophene, chlorphenesin, diazolidinyl urea, DMDM hydantoin, ethyl paraben, formaldehyde-releasing preservative, hydroquinone, iodopropynyl butylcarbamate, imidazolidinyl urea, methyldibromo glutaronitrile, methylhydroquinone, methylisothiazolinone, methyl paraben, nitrosamines, o-cymen-5-ol, phenoxyethanol, propyl paraben, quatemium-15, sodium benzoate, sodium dehydroacetate, sodium hydroxymethylglycinate, sodium metabisulfite, and sodium sulfite.

[190] The bioactive terpene composition or formulation can include a colourant so that its application can be verified visually. The colourant can be a pigment and/or dye.

[191] The bioactive terpene composition or formulation can comprise one or more of the following adhesives, thickening agents, gelling agents, rheology-modifiers and/or viscosity increasing agents: acrylamides copolymer, agarose, amylopectin, calcium alginate, calcium carboxymethyl cellulose, carbomer, carboxymethyl chitin, castor oil derivatives, cellulose gum, cellulosic preparation, cetyl alcohol, cetostearyl alcohol, dextrin, gelatin, hydroxy cellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxpropyl starch, inert sugar, magnesium alginate, methylcellulose, microcrystalline cellulose, pectin, PEG's, polyacrylic acid, polymethacrylic acid, polyvinyl alcohol, quatemium ammonium compound of bentonite or zinc stearate, sorbitol, PPG's, sodium acrylates copolymer, sodium carrageenan, xanthum gum, and yeast beta-glucan.

[192] The bioactive terpene composition, formulation or bioactive extract can be applied or used in any suitable form. The bioactive terpene composition, formulation or bioactive extract can be applied or used in a liquid form or other free-flowing form. The bioactive terpene composition, formulation or bioactive extract can be applied or used as a spray-on liquid, spray-on gel, foam or aerosol. Alternatively, the bioactive terpene composition, formulation or bioactive extract can be applied as a gel by hand, or squeezed from a tube.

[193] A mentioned, the bioactive terpene composition, the bioactive oil extract, or the formulation aspect can be used for controlling a biological organism. Any suitable type of biological organism can be controlled and this can be achieved in any suitable way.

[194] In some embodiments “control” can mean, for example, repelling, killing or otherwise changing the normal behaviour of the biological organism. For example, the biological organism may be an insect, mite, spider or the like having a thin cuticle, or bacteria, fungus or virus.

[195] In some embodiments “control” can mean, for example, allelopathically effecting a trait of the biological organism in a positive or negative way. For example, the biological organism may be a plant.

[196] In some embodiments, the bioactive terpene composition, bioactive oil extract, or formulation is formulated for use as a pest repellant, pesticide, insect repellant, insecticide, mite repellant, miticide, arachnid repellant, acaricide, anti-fungal, fungicide, anti-bacterial, bactericide, anti-viral, viricide, and/or for plant allelopathy (positive or negative effect).

[197] In some embodiments, the bioactive terpene composition, bioactive oil extract, or formulation is capable of controlling an insect, mite, spider, or a plant. In some embodiments, the bioactive terpene composition, bioactive oil extract, or formulation is capable of controlling mosquitoes or mosquito larvae, flies or fly larvae, aphids, mites, spiders, cockroaches, weevils or rootworms.

[198] In some embodiments, the bioactive terpene composition, bioactive oil extract, or formulation is capable of controlling a fungus, bacteria or virus.

[199] In some embodiments, the bioactive terpene composition, bioactive oil extract, or formulation is capable of controlling one or more properties of a plant, either positively or negatively.

[200] In some embodiments, the bioactive terpene composition, bioactive oil extract, or formulation is capable of controlling one or more properties of plant species in the Cannabaceae family, such as cannabis, hemp or hops varieties. In some embodiments, the bioactive terpene composition, bioactive oil extract, or formulation is capable of: stimulating the production of plant defence compounds within a plant; triggering anti-microbial defence mechanisms within a plant; activating anti-viral defence mechanisms within a plant; activating anti-bacterial defence mechanisms within a plant; eliciting plant disease resistance; enhancing plant disease resistance; promoting plant growth; increasing biomass production within a plant; increasing plant uniformity; or, use as a herbicide.

[201] In some embodiments, the bioactive terpene composition, bioactive oil extract, or formulation is capable of producing an allelochemical capable of interfering with the biology of the biological organism.

[202] The step of controlling or treating the biological organism can be carried out in any suitable way. For example, the biological organism can be sprayed with the bioactive terpene composition, bioactive oil extract, or formulation, or the bioactive terpene composition, bioactive oil extract, or formulation can be applied to a surface, substrate (eg. soil) or plant surface (eg. roots, foliage, flower head, seed) on which may be found the biological organism or which may be in close proximity to the biological organism, or the bioactive terpene composition, bioactive oil extract, or formulation can be dispersed in the biological organism’s immediate vicinity or environment (eg. dispersed in the air). The plant may be sprayed with the bioactive terpene composition, bioactive oil extract, or formulation, or the bioactive terpene composition, bioactive oil extract, or formulation can be applied to a surface, substrate (eg. soil) or the bioactive terpene composition, bioactive oil extract, or formulation can be dispersed in the plant’s immediate vicinity or environment (eg. dispersed in the air).

[203] In some embodiments the bioactive terpene composition, bioactive oil extract, or formulation can be applied in an environment of the biological organism so as to elicit the desired effect. For example, the bioactive terpene composition, bioactive oil extract, or formulation can be applied to soil or a plant surface, or dispersed in the air so as to produce the desired effect on an insect, mite, spider etc.

[204] In some embodiments the bioactive terpene composition, bioactive oil extract, or formulation can be applied to a plant or dispersed in an environment of the plant, so as to invoke a defence mechanism in the plant against an insect, mite, spider, bacteria, fungus, virus etc.

[205] In some embodiments the bioactive terpene composition, bioactive oil extract, or formulation can be applied to a plant or dispersed in an environment of the plant, so as to control a property of the plant, such as eliciting plant disease resistance, enhancing plant disease resistance, promoting plant growth, increasing biomass production within the plant, or increasing plant uniformity.

[206] Any suitable quantity of bioactive terpene composition, bioactive oil extract, or formulation can be used. In some embodiments, a concentration of at least one Ippm of the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile is applied to the biological organism or surface/substrate/plant, more preferably about l-5ppm, including all 0. Ippm increments there between.

[207] The term “substantially” generally means a change of up to 10%, including all 0.01% incremental values between 0 and 10%.

[208] The term “comprise” and variants of the term such as “comprises” or

“comprising” are used herein to denote the inclusion of a stated integer or stated integers but not to exclude any other integer or any other integers, unless in the context or usage an exclusive interpretation of the term is required.

[209] The transitional phrase “consisting of’ excludes any element, step, or ingredient not specified. If in the claim, such would close the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith. When the phrase “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[210] The transitional phrase “consisting essentially of’ is used to define a composition, process or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting essentially of’ occupies a middle ground between “comprising” and “consisting of’.

[211] Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[212] Also, the indefinite articles “a” and “an” preceding an element or component of the invention are intended to be non-restrictive regarding the number of instances (i.e., occurrences) of the element or component. Therefore “a” or “an” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

[213] As used herein, with reference to numbers in a range of numerals, the terms “about”, “approximately” and “substantially” are understood to refer to the range of -10% to +10% of the referenced number, preferably -5% to +5% of the referenced number, more preferably -1 % to + 1 % of the referenced number, most preferably -0.1 % to +0.1 % of the referenced number. Moreover, with reference to numerical ranges, these terms should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, from 8 to 10, and so forth. [214] As used herein, the term “essential oil” means a concentrated liquid containing aromatic and/or volatile compounds derived from the flowers, panicles, stem, or upper leaves, or a combination thereof, of one or more plants.

[215] As used herein, the term “prophylactic” means that the method or composition is administered in order to prevent the onset of a disease or a condition such as pest infestation. As used herein, the term “prevention” or “prophylactic” does not necessarily imply that the plant will never contract a disease, disorder or condition such as a pest infestation.

“Prevention” or “prophylactic” may be considered to mean reducing the likelihood of onset of a disease, disorder or condition, or preventing or otherwise reducing the risk of developing a disease, disorder or condition.

[216] Any of the features described herein can be combined in any combination with any one or more of the other features or aspects described herein within the scope of the invention.

[217] The reference to any prior art in this specification is not, and should not be taken as an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge.

Brief Description of the Figures

[218] Figure 1. Graph showing effect of cannabis extract on plant root counts.

[219] Figure 2. Graph showing effect of cannabis extract on plant root area.

[220] Preferred features, embodiments and variations of the invention may be discerned from the following Best Modes for Carrying Out the Invention which provides sufficient information for those skilled in the art to perform the invention. The Best Modes for Carrying Out the Invention is not to be regarded as limiting the scope of the preceding Detailed Description of the Invention in any way.

[221] Best Modes for Carrying Out the Invention

[222] Example 1 - Breeding of new cannabis cultivars

[223] The inventors have been breeding new cannabis cultivars primarily with a focus on the development of phenotypes with improved pesticidal properties. The approach to this has been based on the selection of candidate plants from a broad genetic background created from crosses between several locally sourced Low THC hemp cultivars and then cultivated under commercial field conditions.

[224] Key selection criteria used was:

[225] - Insect resistance as indicated by the presence/absence of insect damage on the plant when cultivated under field conditions; [226] - The insecticidal properties of the extract when used in laboratory insect efficacy trials or applied to plants with insect infestation. The inventors have undertaken extensive testing of different extracts on a range of insect/pest species. Experimental extracts have been tested for the efficacy of their insecticidal effects on a laboratory organism (Drosophila spp.) and subsequently on several pest species (including different mite, aphids and several mosquito species (larvae stage)). In summary, all extracts tested showed efficacy as a pesticide. However, there were differences in efficacy between insect species and extract chemovars. A high resolution, rapid assay was developed with Drosophila species (melanogaster and .simulan.s) which was able to detect differences in extract efficacy between the two species, and between sexes. This assay was used as the initial screening of experimental extracts.

[227] - Presence and relative levels of key terpenes thought to be associated with insect protection.

[228] - Cuttings were taken from around 150 plants when in their vegetative state and prior planting in the field. Each plant was given a unique identification. The corresponding cuttings were also given that same identification and were maintained in a dedicated greenhouse with an extended photoperiod to ensure that cuttings remained in a vegetative state.

[229] - Plants were monitored throughout the field season and performance notes taken for each plant. In addition to the key selection traits, notes were made on general performance, extent of leaf discoloration, vigour and scent. When suitable plants were identified through their performance in the field and extract qualities, the cuttings from that plant became ‘mothers’ for that new chemovar.

[230] Example 2 - Essential oil cannabis extract preparation

[231] Essential oil extractions are generally achieved by either steam distillation or hydrodistillation. Steam distillation passes steam through a bed of plant material in a closed system. Volatile compounds are carried away in the steam and then condensed. Hydrodistillation immerses the plant material in water which is then boiled and the steam (containing the volatile compounds (terpenes)) condensed and collected.

[232] In this disclosure, bioactive essential oil extracts were prepared by steam distillation of raw, frozen material through a heated water still and two condensers.

[233] The following parameters were monitored during the extraction process:

[234] - Weights of flowers used in each distillation run

[235] - Temperature of the still (99°C) [236] - Temperature of each chiller (approx. 14°C and -10°C)

[237] - Time spent on each extraction (approx. 2.5 hours)

[238] - Final terpene yield

[239] Extraction procedure

[240] Frozen plant material was placed into the metal still. The sump in the bottom of the still was filled with water, and the lid was secured onto the still and attached to the metal condenser using a rubber ring and metal clamp. Once the lid was secured, water was poured around the lid ring to maintain a sufficient seal. The still was heated to 99°C.

[241] An ice block was placed into the copper lined barrel for the metal condenser, and the tap feeding water into the copper piping was turned on low. The second chiller was turned on to reach - KFC, and water circulation through the condenser was initiated.

[242] After approximately 40 minutes, the still and the chillers had reached their desired temperatures. At this point the terpenes began boiling off (approximately 80-90°C). Once there was a small, steady stream pouring into the separating flask, the input power on the still was reduced.

[243] An L-shaped recovery head adapter was attached to the horizontal glass condenser to direct terpenes into a separation flask. The separation flask was then placed below with a glass funnel inside. A bucket was placed below the separation flask to recollect water that separated from the terpenes.

[244] Every half an hour, more water was added to the still, and an ice block was added to the barrel for the metal condenser. Temperatures were monitored throughout the process to maintain consistency.

[245] After approximately 2.5 hours, the extraction process was complete. The separation receiving flask was then removed and taken to a vertical separation flask. The terpenes naturally separated from the water and were poured into the vertical separation flask. This was left for a few hours to allow any water left to separate from the terpenes. Once the terpenes were completely separated from the remaining water, they were poured into an amber bottle and sealed to prevent any degradation. The distilled raw material was then taken out of the still and placed on a drying rack.

[246] Table 6

[247] Example 3 - Terpene profiles of essential oil cannabis extracts

[248] A reference sample was developed from a sample of essential oil (steam distillation) extracts of a mixed batch of the inventors’ cultivars. A gas chromatography sample was analysed by Shimadzu QP 2020 NX GCMS to identify composition and quantifying all components that were present at levels >0.1 %, based on normalised peak area of GC-FID or GC-MS response, using analytical standards or by searching against NIST El MS library database and positive matches confirmed against standard reference materials (where available).

[249] From this the terpene profiles for all experimental samples were determined by GC-FID normalised peak area and tentative component identification by retention time match against the characterised reference material.

[250] The samples were analysed on an Agilent Technologies GC 6850 gas chromatograph with a Flame Ionisation Detector. The instrument details are as follows:

Chromatographic Data System: OpenEab CDS Chemstation Edition Rev.C.0108 (210)

Column: Agilent J&W HP-5 Film Thickness: 0.25 pm

Column length: 30M Column i.d.: 0.32 mm

Carrier Gas: Nitrogen 1.2 mE/min constant flow

Oven: Initial Temp: 60 °C

Initial Time: 3.00 min

Table 6A Run Time: 60.00 min

Split/Splitless Inlet: Split Mode Split Ratio: 20:1

Temp: 220 °C Split Flow: 24.6 mL/min

Total Flow: 28.0 mL/min Saver Time: 2.00 min

Detector: Temp: 240 °C Hydrogen Flow: 30.0 mL/min

Air Flow: 300 mL/min Make Up Flow: 25.0 mL/min

Injection Volume: 1.0 pL of 1000 ppm (50 mg / 50 mL) in HPLC Grade Methanol

[251] Compositional analysis for variety 183

[252] Table 6B

[253] Determined Physicochemical properties

[254] Table 6C

[255] Compositional analysis for variety 168

[256] Table 6D

[257] Determined Physicochemical properties

[258] Table 6E

[259] Terpene profiles of developed cultivars

[260] The novel cannabis/hemp cultivars developed by the inventors were characterised by very high (in comparison to other cannabis varieties) levels of cis-ocimene. In the inventors’ varieties ocimene was the most (or second most) abundant terpene (=>20% of essential oil content).

[261] Levels of other key terpenes, being a-pinene, P-pinene and D-limonene, varied between varieties. P-myrcene was also considered a highly preferred terpene, but not necessarily a key terpene.

[262] Example 4 - Preparation of a bioactive essential oil extracts having an optimised terpene profile

[263] The inventors characterised cannabis cultivars whose active compounds included relatively high levels of the monoterpenes a-Pinene, P-Pinene, P-Myrcene, D-Limonene, Trans-Ocimene and Cis-Ocimene, and had desirable allelopathic effects.

[264] The inventors discovered that these monoterpenes were effective across a range of concentration profiles. In order to describe a useful terpene concentration range, a calculation was conducted as follows. An equal proportion (50:50) blend was created between two novel cannabis varieties (hemp varieties M168 and M183) whose extracts had been found to have beneficial allelopathic effects when applied to plants of the Cannabaceae family. Of this blend the key monoterpenes listed in the above paragraph comprised around 70% of the resulting essential oil. This left a 30% margin in which concentrations of the various monoterpenes could be increased to make 100%.

[265] This allowed the inventors to develop a range of relative active concentrations of the key monoterpenes as shown in Table 7 A below.

[266] Table 7A

[267] The above numerical values were obtained by calculating the maximum % margin increase (maximum margin increase) that kept the proportions of the terpenes the same. The upper bound was calculated by adding the maximum margin increase to the average blended result. The lower bound was calculated by subtracting the maximum margin increase from the average blended result.

[268] The inventors further developed a range of relative active concentrations of the key monoterpenes as shown in Table 7B below, but which excluded P-myrcene (which is not always a key terpene).

[269] Table 7B [270] The above numerical values were obtained by calculating the maximum % margin increase (maximum margin increase) that kept the proportions of the terpenes the same. The upper bound was calculated by adding the maximum margin increase to the average blended result. The lower bound was calculated by subtracting the maximum margin increase from the average blended result.

[271] Example 5 - Formulation containing cannabis extract

[272] This Example details the composition of an oil in water emulsion formulation containing 200 g/L of the inventors’ own hemp extracts, namely Ml 83 and Ml 68.

[273] Formulation Composition

[274] The components used in the formulation are described in Table 8 below.

[275] Table 8

[276] The extract is formulated in a batch process. The method of formulation, including the sequence of operations is described in Table 9 below.

[277] Table 9

*Note: Atlox 4916 will be semi-solid at ambient temperatures and requires melting. Heat in a suitable drum oven and maintain in a completely molten state prior to addition.

[278] Example 6 - Insecticidal properties of cannabis extract on mosquito larvae

[279] An assay was performed on 4-day old Aedes aegypti larvae. 2mL of the inventors’ own hemp extract (Mi l, M217 or M168) was mixed with 18mL 100% ethanol before the desired concentrations were produced by serial dilutions with Reverse Osmosis (RO) water. Groups of 25 larvae (4 replicates per treatment) were placed in a 250 mL glass jar, containing lOOmL of the hemp extract/water mixture. In this trial a total of five experimental extracts were tested: Ml 1 ‘indica phenotype’; Ml I D ‘indica phenotype- decarboxylated’; M21 ‘indica phenotype 2’; M168 ‘sativa phenotype - dry’, having been extracted from dry plant material; Ml 69 ‘sativa phenotype - fresh’, having been extracted from fresh plant material. [280] Table 10

[281] Observations were made at: 0 hours to make sure all larvae are alive; 24 hours; and, 48 hours.

[282] The results are shown below.

[283] Table 10A

[284] The results show high levels of larval mortality with all extracts after 48 hours. It was noted that beyond the mortality of larvae, the extract appeared to be affecting development all the way through to pupation. This means that even when an adult did emerge it was clearly weaker and not likely to be fertile.

[285] Extract Ml 68 produced the best result, but Ml 83 produced similar results to extract Ml 68 (results not shown).

[286] Example 7 - Insecticidal properties of cannabis extract on fruit flies [287] A key development in the inventors’ research was the development of a high- resolution laboratory assay capable of detecting fine scale differences between the efficacy of different extracts, and in the differential response of species to an extract. The assay used laboratory cultures of two Drosophila species (D. melanogaster and D. .simulan.s). Of each strain ten male and ten female flies were analysed per replicate at water diluted concentrations of 0 %, 20 %, 35 %, 40 %, 45 %, 50 % and 80 %. These ranges were determined in prior pilot testing. The flies were sorted by sex, anaesthetised with CO2 and placed into 60 ml glass vials at ten individuals per replicate. An agar spoon was added to each vial in order to prevent desiccation and starvation during the bioassay. A folded 70 mm Whatman® filter paper with 100 pl of each corresponding dilution was added to each vial. The vials were maintained in a 19 °C temperature chamber with a 16/8 h day/night cycle for the duration of the bioassay. Five replicates were carried out per species and sex for a total of 280 individual flies, 40 of which were negative controls.

[288] The flies tested were of uniform age. This was ensured by tipping all flies out of their breeding vials and collecting newly hatched flies after 24 hours. These flies were left to mature for three days prior to the experiment.

[289] Zero-hour baseline mortality was established immediately after commencing the assay to ensure that no flies were damaged by handling. Mortality score observations were taken at 20 h, 26 h and 50 h with 50 h being the time -point used in all downstream analysis. The flies were scored as being dead if there was no movement after agitating the vial.

[290] The results of the bioassay based on extract M183 are shown below.

[291] Table 10B

[292] The bioassay shows that the hemp extract in high concentrations is an effective insecticide for Drosophila. It also shows that the LC50 can strongly vary depending by species and even sex. Note that this is a residual assay and the relatively high concentration of extract needed to achieve LC50 is expected as the insects do not contact the extract directly. This assay has been subsequently used to test the efficacy of extracts and mixtures of extracts. [293] The research has confirmed that all the tested extracts (M183, M168 and a 50:50 blend of extracts Ml 83 and Ml 68) demonstrate insecticidal properties and that a doseresponse relationship is observed. The extracts showed differing levels of efficacy at higher concentrations in Drosphila species (although all were efficacious), and similarly at lower concentrations in mosquito larval trials.

[294] Example 8 - Miticidal properties of cannabis extract

[295] Two Spotted Spider Mites (Tetranychus urticae) are a major agricultural pest of a wide range of commercial crops including hemp and cannabis. In this study the formulated extract of Example 5 (formulation containing extract M183, as shown in Table 8) was sprayed on hemp plants with spider mite infestations. Spider mites with known genetic backgrounds were obtained from Bio21 and reared in a purpose-built rearing facility on dwarf bean plants until the numbers were sufficient for the experiment. Infested leaves of the bean plants from the pest rearing facility were removed and pinned to cannabis/hemp plants grown in a large industrial greenhouse facility and under commercial production conditions. This allowed the pests to infest the hemp plants and hence allow efficacy testing experiments.

[296] The experimental protocol consisted of four treatments (control, low concentration, medium concentration and high concentration formulated M183 extract). These concentrations were 1:5000, 1:500, 1:250. Five hemp plants were sprayed with each concentration (control was water). Prior to spraying an image was taken of leaves from each plant to estimate spider mite density. Follow up images were taken every day following spraying until Day 5 when the experiment was terminated.

[297] Results

[298] An accurate non-destructive count of spider mite population densities is notoriously difficult due to their small size and so estimates were made instead by scoring each leaf on a 1 to 10 scale of infestation. By Day 5 both the high and medium concentration treatments were showing significant population reductions in comparison to the control treatment. Eimitations with the population density estimates meant it was not possible to determine whether the low concentration treatment was significantly different from the control treatment. A formulation based on extract Ml 68 worked as well as Ml 83 (results not shown).

[299] Conclusion

[300] In summary, the inventors have tested a range of its cannabis extracts for pesticidal efficacy in both laboratory and commercial production environments. These experiments confirm that cannabis extract exhibits dose/response characteristic of pesticides and appears to operate through contact toxicology rather than through residual effect. These results confirm earlier results suggesting differences between species in their susceptibility to the extract. Surprisingly, as mentioned, there appear to be differences in the susceptibility between the sexes for the two Drosophila species studied here.

[301] Example 9 - Allelopathy studies using cannabis extract

[302] The inventors have surprisingly found that when applying cannabis extracts to plants in the Cannabaceae family, the plants themselves appeared to be responding to the extract by producing unusual vegetative growth while in the flowering and fruiting phase of the life cycle. As this was likely to be some sort of allelopathic response it was decided to further investigate allelopathy in trials.

[303] To assess this small pilot studies on seed germination trials and cutting root establishment were conducted. Two preliminary seed germination trials were conducted and one cutting trial in total.

[304] A formulation containing extract Ml 83 (as shown in Table 8 of Example 5) was diluted with distilled water, 1:500 and 1:1000. Hemp seeds were soaked for six hours in the diluted formulated extract. After soaking the seeds were removed from the treatment and placed on a piece of filter paper on a plate, one each for each treatment. The seeds were kept moist with liquid from their respective treatment for a period of seven days, under lights. Photo records were taken from day 2 to assess the rate of germination. Table 11 shows results of second seed germination trial.

[305] Table 11 [306] In both seed germination trials it appeared that hemp seeds germinated faster when treated with 1:500 formulated extract/distilled water treatment. This was also apparent with the 1:1000 formulated extract/distilled water treatment. In the first trial the development of secondary roots appeared to occur faster when the 1:500 formulated extract/distilled water treatment was applied to hemp seeds. What is unclear from these trials is if this early germination and development would continue throughout the seedling development stages. The stronger treatment of 1:100 formulated extract/distilled water and 1:500 extract only treatments appeared to impede proper germination of hemp seed.

[307] Initial observations in the cutting trial indicated that the 1:500 formulated extract/distilled water treatment applied to the vegetative tops of cuttings might increase initial root development of cuttings, however, by day 13 root development under treatment appeared retarded with a possible increase in root rot detected in the treated cuttings. This suggested that any improvement in early root development from treating the cuttings with extract was not sustained as the roots developed over time.

[308] The graph of Figure 1 shows the results of root counts for both treatments on Day 13 of the experiment. The graph of Figure 2 shows the results of root area estimates for both treatments on Day 13 of the experiment.

[309] Initial observations in the cutting trial indicated that the 1:500 formulated extract/distilled water treatment applied to the vegetative tops of cuttings may increase initial root development of cuttings; however by day 13 root development under treatment was retarded with an increase in root rot detected in the treated cuttings.

[310] These initial allelopathy investigations appear to confirm the presence of allelopathic responses when the extract is applied to plants of the family Cannabaceae (such as its own species) in the seed or seedling (cutting) stage.

[311] Example 10 - Further allelopathy studies using cannabis extract

[312] Objectives

[313] It has been generally thought that allelopathic effects between species might be more pronounced the greater the phylogenetic distance between the species. That is, that the more distant species are from one another (i.e. from an evolutionary history perspective) the greater the magnitude of any inhibitory effect. Furthermore, it has been noticed that in some plant species the early developmental stages (i.e. seed - seedling) may be when that plant is most susceptible to the effects of allelochemicals (Kruse et.al 2000).

[314] Based on an identified gap in the literature and the inventors’ own research results, it was decided to undertake a full life-cycle allelopathy trial in cannabis. This was further supported by the finding that several of the inventors’ experimental cultivars were found to have very high relative levels of cis-ocimene. This terpene has been associated with triggering plant defence mechanisms. The trial examined the effects of the inventors’ cannabis extract when applied to cannabis plants across all stages of the life cycle and have a particular emphasis on assessing pest infestation.

[315] The aim of this experiment was to test an essential oil cannabis extract from a strain developed by the inventors for potential allelopathic effects when applied at different life cycle stages to cannabis plants.

[316] The allelopathic effects monitored were biomass, insect resistance and terpene profile.

[317] The developmental stages that were examined were vegetative, flowering and fruiting.

[318] This study is novel in that:

[319] - There are few studies in which the allelochemicals from a plant species are applied to plants of that species, i.e. no phylogenetic distance between initiating and receiving species.

[320] - Relatively few studies have examined the positive effects of allelopathy as these are less regularly observed.

[321] - Previous studies on allelopathy in cannabis have not used extracts developed from distillation of flowers. This study was the first to document an allelopathy study of cannabis extracts containing the highly volatile terpenes.

[322] - This study extended beyond examination of early life stages and was conducted through to plant maturity and harvesting unlike previous studies that have not extended beyond juvenile stages.

[323] - It was conducted under industrial scale commercial cultivation conditions.

[324] Materials and Methods

[325] Experiment Overview

[326] A cannabis/hemp extract full life cycle allelopathy experiment was conducted by the inventors commencing in January 2021 when cuttings were made and concluding with harvesting in June 2021. The trial consisted of foliar treatments of Ml 83 extract (formulated as per Example 5, Table 8) diluted with water at a 1:1000 ratio or water (as control) every 2-3 days on selected treatments. The final terpene profile of the bioactive essential oil extract is shown in Table 12. [327] Table 12 below presents results of extract analysis (terpene profile) of experimental treatments. Values presented are a % based on normalised peak area of GC-FID or GC-MS response.

[328] Table 12

[329] In addition to measures of biomass, pest infestation and terpene content, photographic images were taken every few days to document any visible changes in plant’s general health and/or vitality.

[330] Experimental design

[331] The main experiment considered the effect of exposure to the extract at three developmental stages: vegetative, flowering and fruiting. The experiment consisted of five different treatments each representing a different developmental stage(s), and a control treatment which was never treated with extract. Treatments and number of plants in each treatment were as in Table 13 below.

[332] Table 13

[333] Using plants that were surplus at the cuttings stage, a supplementary trial using Ml 68 formulated according to Table 8 of Example 5 was conducted to further examine the effects of treatment at the vegetation stage on final yield. The experiment consisted of two replicates of two treatments: treatment through all life cycles, and treatment in flowering and fruiting only. The difference in the treatments is then that one has been treated in the seedling and vegetation stage and the other has not. See Table 14 below.

[334] Table 14

[335] Plant establishment and maintenance

[336] Around 300 cuttings were taken from vegetating plants of various strains during the last week of January 2021. Following the inventors’ standard operating procedures (SOP) for cutting establishment, cuttings were transferred to large plastic boxes in which the cuttings are suspended with aerial parts above, and root section below in the dark and with periodical spraying with water mister. Cuttings were maintained at an 18-hour daylight diurnal cycle. [337] Cuttings were transferred into individual plastic pots with coco substrate. All plants were then transferred from the nursery to the main greenhouse where the daylight cycle was approximately 14 hours daylight/10 hours dark.

[338] Throughout the experiment, plants were maintained following normal commercial greenhouse management procedures. Plants were supplied with a varying nutrient regime in the vegetation, flowering and fruiting phases. Plants were fed according to dryness in pots and rate of nutrient uptake.

[339] At the end of the vegetative cycle, all plants were “topped” - the plant was cut at the 4 ^th to 6 ^th internode. Following this all plants were pruned (dead leaves removed).

[340] Flowering commenced with the appearance of pistils and the nutrient regime was adjusted accordingly. At the commencement of flowering plants are stripped of fan leaves and small stems growths. Flowering continued for the next six to eight weeks.

[341] Fruiting was based on 65-75% of pistils having turned brown and the calyxes becoming denser.

[342] Experimental cannabis extract treatment

[343] Treatment with the extract (to selected treatments) commenced immediately after cuttings were taken. A light foliar spray was applied every second day at a concentration of 1:1000 (extract: water). Treatments were applied using a standard pump action mist spray from approximately 30cm from the test subjects. A light but consistent coverage of extract was applied to the whole plant.

[344] Treatments not receiving extract were sprayed with approximately the same amount of water (as extract applied to test plants) at the same time as extract treatments were applied in order to provide a suitable and comparable control treatment.

[345] The main experiment used an Ml 83 extract formulated as per Table 8 of Example 5.

[346] Biomass assessment

[347] At harvesting, plants were cut at the base of the stem and each plant weighed. Flowers were then removed from stems and a total weight of flowers from each treatment was obtained. Immediately following this the flowers were bagged and frozen for twenty-four hours prior to extraction.

[348] Pest infestation assessment

[349] The pest species used in the experiment was the Black Bean Aphid (Aphis fabae), a major economic pest which has a very broad host range with over 80 host species including potato, cabbage, cauliflower, radish, celery, capsicum, eggplant, cucumber, beets, cucurbits, chilli, and grain. In addition to the direct damage that aphid feeding does to plants, aphids can transmit certain plant viruses.

[350] During the course of the experiment, Aphis fabae were only recorded in plants in the flowering and fruiting stages.

[351] Plants were evaluated for aphid infestation with a simple visual assessment. Plants were suspended upside down from a hook while the underside of the flower heads could be examined. Infestations were found in localised areas of the flowering parts of the plant, where small areas were heavily populated. When one or more densely populated sites were found on a plant it was recorded as “infested”. If a plant was found to be aphid free or with random individual aphids that do not appear to have “settled” it was recorded as “clear”.

[352] Results

[353] Biomass

[354] Gross weights

[355] Table 15 below shows the gross weight of plants in each treatment as an average weight per plant for that treatment.

[356] Table 15

[357] The results above suggest that there may be an increase in biomass in treatments that received extract treatment during the vegetative stage. While the relatively small sample size prevents this effect from reaching statistical significance and independent T-test between on the comparison between the treatment (vegetation only) and the treatment (fruiting and flowering only) approaches significance (p=0.06).

[358] In terms of the variance around biomass the result for the treatment (vegetation only) is very significant as it appears that the total variance is approximately half of the remaining treatments.

[359] Flower (yield) weights [360] Table 16 below shows flower yields (gms) average per plant, stem/stalk weight (gms) average per plant and flower ratio for main experiment.

[361] Table 16

[362] A ratio was calculated for the proportion of flower to stem (flower ratio) by dividing the average flower weight by the average stem weight for that treatment.

[363] Table 17 below shows flower yields (gms) average per plant based on a supplementary trial utilising extract Ml 68 formulated as per Table 8 of Example 5.

[364] Table 17

[365] Using the average weight per treatment a comparison of the two treatments indicate a 34% higher biomass in the treatment that received extract in the vegetative stage.

[366] Pest infestation

[367] Table 18 below reports the observed pest infestation levels in treatments of the main experiment (using Ml 83 formulated extract). Figures presented are the % of infested plants per treatment.

[368] Table 18

[369] Discussion

[370] This experiment is the first time that research has been focused on the potential of positive allelopathic effects of cannabis extract when applied to cannabis plants. Allelopathy research has largely examined the potential of negative allelopathic effects when an extract of one plant is applied to a phylogenetically distant plant species. The purpose of that research was to identify any potential for the development of “natural” herbicides. This experiment is also the first documented example of positive allelopathy in mature plants. Previous studies have not examined life stages beyond the seedling stage.

[371] In summary the results of the present experiment suggest that treatment with high ocimene cannabis extract in the vegetative stage appears to provide the plant with lifetime insect defence, increase yield at harvest by somewhere between 10 and 35%, with a higher flower ratio and reduced variance. Additionally, observations of the experiment note that although not quantified there were very low levels of fungal infections in treated plants.

[372] The differences in biomass are approaching statistical significance despite the relatively small sample sizes suggesting a strong effect. This is the first experiment that the inventors are aware of that demonstrates a potential increase in biomass (yield) in mature plants through allelopathic effects. Additionally, the large reduction in variance around the average yield in the vegetation only treatment requires further investigation. If the extract was acting to reduce or narrow the range of environmental effects in the plant’ s phenotypic development, that would be of significant interest to the broader agricultural and scientific community. The same also applies to the apparent increase in the flower ratio.

[373] The results suggest that there may be a “threshold effect” as treatment with the extract in the late flowering and fruiting stages appearing detrimental. It is known that cannabis plants are susceptible to damage in the fruiting stage and some cultivators avoid treating plants as the trichomes are fragile and easily damaged.

[374] Example 11 - Randomised trial using cannabis extract

[375] Experimental Objectives [376] This experiment describes a cannabis extract trial conducted over the course of 13.5 weeks (24 January 2022 - 10 June 2022) to observe effects on population densities of aphids, spider mites, caterpillars, scale, mould and other fungal infections as a result of treatment with the cannabis extract during the cutting and vegetation stages of plant development in cannabis plants.

[377] Description of treatments and combinations

[378] The trial consisted of two extracts (from varieties M168 and M183) and a blend of the two (i.e. 50:50 M168:M183) - a total of three cannabis extracts. These were applied to the leaves through foliar application to cuttings and in the vegetative stages of cannabis plants of the Ml 68 and Ml 83 variety. A control treatment for each variety was included by treating plants with a foliar spray of water only. This resulted in a total of eight treatments (see table 19 below). Each treatment had 30 plants giving a total of 240 plants in the experiment in total.

[379] Table 19

[380] Extracts and Application

[381] The two cannabis extracts used in the experiment were an extract from the flowers of varieties M168 and M183. The methods of extraction and subsequent formulation of the cannabis extracts are as previously described. A third extract was made from an equal parts blend of the M168 and M183 extracts. [382] Foliar treatments were applied using a standard pump action mist spray - administered approximately 30cm from the test subjects in each bay twice a week during the cutting and vegetative stages.

[383] Trial preparation and test subject maintenance during trial

[384] Cuttings of each variety were taken off mother plants and placed into a propagation tray to root for two weeks on a 18/6 light cycle. Plants were sprayed with assigned foliar treatments while in the propagating stage. Once rooted, clones were repotted into 5L pots (75% coconut coir/25% perlite) with Great White Premium Mycorrhizae added to the medium.

[385] Bays were individually cleaned by removing residual insects and other matter from the flood tray.

• Bays were cleaned using brush and pan to remove excess plant matter to prevent blocking irrigation pipe.

• Flood trays were sprayed with vinegar and power washed thoroughly, metal frames were wiped down to remove all mould and unwanted dust/dirt.

• The nutrient reservoir was cleaned and sanitized using hydrogen peroxide, then rinsed to remove remaining chemicals.

[386] The experimental plants were maintained with the nutrients Byron Bay Gold Grow A&B, Bloom A&B and Fruit A&B (obtained from www.byronbaygold.com). Plants were fed according to dryness in pots and rate of nutrient uptake.

[387] The above-mentioned nutrient solutions contain the following nutrient profiles.

[388] Table 20

389] 30 plants were placed in each tray according to variety and foliar treatments continued for approximately 5 weeks after cutting from mother plants. Plants began to flower approximately one week later. Flowering continued for another 8-10 weeks at which stage the experiment was terminated.

[390] Labelling and Plant Randomization Process

[391] The pot of each experimental plant was labelled with a random number from 1 to 240 while the plants were in their respective bays during the foliar treatment phase. A master list was made which recorded what treatment each pot had received. Following the commencement of flowering and the cessation of foliar treatment, all pots were transferred to the greenhouse floor and randomly mixed before being returned to the tables. This meant that untreated plants were now mingled with treated plants and that experimental scoring for fungal and insect infestation levels would be conducted blind i.e. with no knowledge of the treatment history of the plant under consideration.

[392] Insect Infestation and Mould Estimation

[393] The experiment was terminated on the 10th of June 2022 at which time all plants were processed for insect infestation levels.

[394] Insect infestation levels were recorded for aphids, spider mites, caterpillars and scale. However, caterpillar and scale numbers were so low that these results have not been included. Aphid and spider mite populations were scored for each plant against the criteria in the table below and a score from 0 to 5 was given.

[395] Table 21 [396] Mould and/or fungal infection was observed in low numbers across the experimental subjects and was recorded as a simple presence/absence.

[397] Results

[398] Spider Mites

[399] The table below presents the combined plant scores for spider mites for each experimental treatment.

[400] Table 22

[401] Aphids

[402] The table below presents the combined plant scores for aphids for each experimental treatment.

[403] Table 23

[404] Mould (fungal infections)

[405] The table below presents the combined plant scores for mould/fungal infections for each experimental treatment.

[406] Table 24

[407] Conclusion/Discussion

[408] Experimental conditions and limitations

[409] A key challenge in experiments that attempt to investigate the effects of volatile organic compounds such as terpenes is to ensure that treatments are kept separate (so that there is sufficient delineation between treated and untreated plants) but at the same time to ensure that experimental treatments are receiving the same base conditions. For this reason, the plants were kept separate during the experimental phases in which they were receiving treatment and on the completion of this stage the plants were mixed to ensure that all were grown under the same micro conditions.

[410] Spider Mite Results

[411] In general, there were low levels of pest infestation across all treatments, considering that no pest insecticides were used and in comparison to infestation levels observed in previous years. Spider mite infestations were only occasional and were generally on the lower levels of infestation when recorded.

[412] Despite this there is a clear trend supporting the previous findings that a blend of cannabis extracts (in the 50:50 M168, M183 mixture) provides superior protection against spider mites with the blended extract showing the lowest level of spider mite infestation for both extracts and both plant varieties.

[413] Aphid scores

[414] The aphid species studied was the Black Bean Aphid, a major pest of bean crops but with a wide host plant range with over 80 species known to be affected. It appears that the major impact of this species is its ability to transmit plant viruses in addition to damage from feeding. It is thought that not all life stages of this species actively feed off the plant itself.

[415] The results were broadly in line with the spider mite trial, with the lowest levels of infestation observed on the plants receiving the blended extract.

[416] Mould

[417] Very low levels of mould/fungal infection were observed across all experimental treatments and the highest levels were seen in untreated plants.

[418] This was very surprising given the amount of mould and fungal growth present in the broader environment at the time and to levels observed in previous seasons. [419] General Observations

[420] In general, the results support a finding that treatment during the cutting and vegetation stages with a cannabis extract provides the plant with an increased resistance to pest infestation, and mould/fungal infections. Additionally, the data show that a blend of cannabis varieties in the extract results in superior protection compared to single variety extracts.

[421] Potential applications of the bioactive essential oil extract

[422] Concerns about potential impact on human health arising from the use of chemical pesticides etc. are elevated for products in which extractions are made for food or medicine purposes as any chemical residues left on the plants will be retained and concentrated in the extraction.

[423] The bioactive essential oil extract of the present invention may be applied to and should be effective on all plants within the Canabaceae family. Primarily, this applies to cannabis, hemp and hops varieties.

[424] Cannabis and hemp

[425] Pesticide residue has become a major issue for the hemp and medicinal cannabis industries. There are a number of reasons for this. Firstly, the concentration of plant chemicals through the extraction process means that any pesticide residue on the raw material is concentrated in the extract. As these extracts are used in food and medicines there has been growing concern that they may have serious impacts on human health. This has occurred in the context of a broader community concern about the longer - term impacts of chemical pesticides on bees and the wider environment.

[426] With more states in the US and Canada enacting medicinal and adult recreational cannabis laws each year, health officials have increasingly warned about the potential hazards of products made from crops treated with certain chemicals. In particular, chemical pesticides have been identified as a threat to cannabis consumers' health, with potential risks that can vary depending on whether products are eaten, smoked, vaped, or topically applied. Another reason for the lack of approved pesticides (at least for the US market) is that pesticides are regulated at the federal level, however, cannabis has not been deregulated at a federal level in the US and the industry is existing largely under State laws.

[427] See for example:

[428] https://www.cannabisbusinesstimes.com/article/purdue-researc hers-cannabis- pesticide-problem/ [429] https://www.purdue.edu/newsroom/releases/2018/O4/legal-hemp- raises- questions-about-pesticides.html

[430] https://cen.acs.org/business/agriculture/Nurturing-cannabis/ 96/i21

[431] Hops

[432] Hops are closely related to cannabis and are in the same family Canabaceae. As with cannabis, hops products are made using various forms of extraction and are hence susceptible to residue from chemical insecticides, fungicides etc.

[433] The female flowering hop plant (Humulus lupulus) produces cones that are also called seed cones or strobiles. Non-fertilized cones are used as a bittering and flavouring agent in beer. Global demand for beers with high hop content continues to grow and craft brewers are proving to be innovative in the ways they are introducing high volumes of hops into their brews. Hops are increasingly being added later in the brewing process and many craft breweries are using the addition of green, undried, high-moisture hops to the brewing process. As with other crops, hop cultivation encounter a range of challenges including bacterial disease, fungus and mildew, viral disease, pests and parasitic invasion. The use of chemical agricultural products to counter these challenges can result in the accumulation of chemical residue and hence the concerns over human and environmental health.

[434] As a result, there are many restrictions and few approved products for the control of insects and fungal issues in hops. For example, there are currently no antifungals registered or permitted for use on hops anywhere in Australia.

[435] Advantages of the present invention as exemplified

[436] The inventors’ have developed a bioactive cannabis/hemp (essential) oil extract that addresses a significant need in the hemp and medicinal cannabis industry, which is for a safe, residue free pest control product. No pesticide product labels currently list cannabis crops (nor are any approved by regulators) as an allowable application, and only a few are currently approved for hemp fiber (many of which are neem oil/azadarichtin formulations).

[437] Clearly, a pesticide made from cannabis will not have any residues (i.e. residue will be indistinguishable from existing plant compounds) on cannabis plants, particularly since preferred embodiments of the invention, increase pest resistance by producing a biological response in the subject plant. Accordingly, pest resistance is preferably achieved by way of increasing production of plant defence compounds, which are native to the subject plant and therefore do not leave any residue. The inventors believe that their extract will service a major gap in the market. [438] The inventors believe that their extract can be used for a wide range of pests on cannabis and other plants in the Cannabaceae family.

[439] Some embodiments of the invention are described in the paragraphs below.

[440] 1. A cannabis-based bioactive terpene composition comprising the following concentrations of monoterpenes:

[441] Terpene Profile 1

[442] approximately 0.3-50.1 % Pinene;

[443] approximately 0.1-10.9 % Limonene; and

[444] approximately 0.2-39.0 % Ocimene,

[445] or

[446] Terpene Profile 2

[447] approximately 4.8-45.6 % Pinene;

[448] approximately 1.0-9.0 % Limonene;

[449] approximately 3.7-35.5 % Ocimene; and

[450] approximately 1.1-9.9 % [3-Myrcene.

[451] 2. A bioactive terpene composition comprising a bioactive oil extract prepared from cannabis plants, wherein the composition comprises the following concentrations of monoterpenes:

[452] Terpene Profile 1

[453] approximately 0.3-50.1 % Pinene;

[454] approximately 0.1-10.9 % Limonene; and

[455] approximately 0.2-39.0 % Ocimene,

[456] or

[457] Terpene Profile 2

[458] approximately 4.8-45.6 % Pinene;

[459] approximately 1.0-9.0 % Limonene;

[460] approximately 3.7-35.5 % Ocimene; and

[461] approximately 1.1-9.9 % [3-Myrcene.

[462] 3. A bioactive oil extract based on or prepared from cannabis plants, wherein the bioactive oil extract comprises the following concentration of monoterpenes:

[463] Terpene Profile 1

[464] approximately 0.3-50.1 % Pinene;

[465] approximately 0.1-10.9 % Limonene; and

[466] approximately 0.2-39.0 % Ocimene, [467] or

[468] Terpene Profile 2

[469] approximately 4.8-45.6 % Pinene;

[470] approximately 1.0-9.0 % Limonene;

[471] approximately 3.7-35.5 % Ocimene; and

[472] approximately 1.1-9.9 % [3-Myrcene.

[473] 4. A method of preparing the bioactive terpene composition of the first or second paragraph, or the bioactive oil extract of the third paragraph, the method comprising the step of:

[474] a preparation step, comprising preparing a bioactive oil extract from cannabis plants.

[475] 5. A cannabis-based bioactive oil extract prepared using the method according to the paragraph 4.

[476] 6. A formulation comprising the bioactive terpene composition of the first or second paragraph, or the bioactive oil extract of the third or fifth paragraph, and, optionally, at least one additional ingredient.

[477] 7. Use of:

[478] the bioactive terpene composition of the first or second paragraph;

[479] the bioactive oil extract of the third or fifth paragraph; or

[480] the formulation of the sixth paragraph,

[481] for controlling a biological organism.

[482] 8. A method of controlling a biological organism, said method comprising the step of treating the biological organism with or exposing the biological organism to:

[483] the bioactive terpene composition of the first or second paragraph;

[484] the bioactive oil extract of the third or fifth paragraph; or

[485] the formulation of the sixth paragraph,

[486] in an amount sufficient so as to control the biological organism.

[487] 9. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition or bioactive oil extract is based on or prepared from cannabis plants of the species Cannabis Sativa L.

[488] 10. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the oil extract is an essential oil extract.

[489] 11. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the cannabis plants are hemp. [490] 12. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition or bioactive oil extract is prepared from a single cannabis plant cultivar, variety or chemovar.

[491] 13. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition or bioactive oil extract is prepared from more than one cannabis plant cultivar, variety or chemovar, preferably two.

[492] 14. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the pinene terpene comprises a-pinene and P-pinene.

[493] 15. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the limonene terpene comprises D-limonene.

[494] 16. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the ocimene terpene comprises trans-ocimene and cis-ocimene.

[495] 17. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the myrcene terpene comprises P-myrcene.

[496] 18. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein at least one of the monoterpenes of the bioactive terpene composition or the bioactive oil extract is prepared by distillation.

[497] 19. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein at least one of the monoterpenes of the bioactive terpene composition or the bioactive oil extract is prepared by steam distillation and/or hydrodistillation.

[498] 20. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein at least one of the monoterpenes of the bioactive terpene composition or the bioactive oil extract is prepared by using carbon dioxide (CO2) extraction technology, such as subcritical or supercritical CO2 extraction.

[499] 21. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein at least one of the monoterpenes of the bioactive terpene composition or the bioactive oil extract is prepared by distillation, preferably steam distillation, of fresh plant material or raw, frozen plant material.

[500] 22. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein at least one of the monoterpenes of the bioactive terpene composition or the bioactive oil extract is prepared by steam distillation of cannabis flowers.

[501] 23. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the monoterpene concentrations of hemp variety M183, as seen in Tables 2 or 6B.

[502] 24. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the essential oil constituents of hemp variety M183, as seen in Tables 2 or 6B.

[503] 25. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the monoterpene concentrations of hemp variety M168, as seen in Tables 2 or 6D.

[504] 26. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the essential oil constituents of hemp variety M168, as seen in Tables 2 or 6D.

[505] 27. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile is produced by blending the oil extracts of hemp varieties together.

[506] 28. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the average % terpene concentrations of M183 and M168 as seen in Table 2.

[507] 29. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the essential oil constituents of the average % terpene concentration of M183 and M168 as seen in Table 2.

[508] 30. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein Terpene Profile 1 comprises any one or more of:

[509] approximately 0.2-35.2 % a- Pinene;

[510] approximately 0.1-14.9 % P-Pinene;

[511] approximately 0.1- 10.9 % D-Limonene;

[512] approximately 0.0- 1.5 % Trans-Ocimene; and

[513] approximately 0.2-37.5 % Cis-Ocimene.

[514] 31. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the monoterpene concentrations of hemp variety Ml 83, as seen in Table 3.

[515] 32. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the essential oil constituents of hemp variety M183, as seen in Table 3.

[516] 33. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the monoterpene concentrations of hemp variety M168, as seen in Table 3.

[517] 34. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the essential oil constituents of hemp variety M168, as seen in Table 3.

[518] 35. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the average % terpene concentration of M183 and M168 as seen in Table 3.

[519] 36. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the essential oil constituents of the average % terpene concentration of M183 and M168 as seen in Table 3.

[520] 37. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the monoterpene concentrations of hemp variety M183, as seen in Table 4.

[521] 38. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the essential oil constituents of hemp variety M183, as seen in Table 4.

[522] 39. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the monoterpene concentrations of hemp variety M168, as seen in Table 4. [523] 40. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the essential oil constituents of hemp variety M168, as seen in Table 4.

[524] 41. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the average % terpene concentration of M183 and M168 as seen in Table 4.

[525] 42. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the essential oil constituents of the average % terpene concentration of M183 and M168 as seen in Table 4.

[526] 43. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein Terpene Profile 2 comprises any one or more of:

[527] approximately 3.4-32.0 % a- Pinene;

[528] approximately 0.1-13.6 % P-Pinene;

[529] approximately 1.1-9.9% D-Limonene;

[530] approximately 0.1- 1.4 % Trans-Ocimene;

[531] approximately 3.6-34.1 % Cis-Ocimene; and

[532] approximately 1.0-9.0 % P-Myrcene.

[533] 44. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the monoterpene concentrations of hemp variety M183, as seen in Table 5.

[534] 45. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the essential oil constituents of hemp variety M183, as seen in Table 5.

[535] 46. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the monoterpene concentrations of hemp variety M168, as seen in Table 5.

[536] 47. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the essential oil constituents of hemp variety M168, as seen in Table 5.

[537] 48. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the average % terpene concentration of M183 and M168 as seen in Table 5.

[538] 49. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the essential oil constituents of the average % terpene concentration of M183 and M168 as seen in Table 5.

[539] 50. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the monoterpene concentration ranges substantially as shown in Table 7B.

[540] 51. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, bioactive oil extract, formulation, or terpene profile comprises or substantially comprises the terpene concentration ranges substantially as shown in Tables 4, 5 or 7A.

[541] 52. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition or formulation is in the form of a liquid, solution, gel, creme, emulsion, paste, film, powder, foam, suspension, gas, vapour or aerosol.

[542] 53. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, formulation or bioactive oil extract is formulated for use as a pest repellant, pesticide, insect repellant, insecticide, mite repellant, miticide, arachnid repellant, acaricide, anti-fungal, fungicide, anti-bacterial, bactericide, antiviral, viricide, and/or for plant allelopathy (positive or negative effect).

[543] 54. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, formulation or bioactive oil extract is capable of controlling an insect, mite, spider, or a plant.

[544] 55. The preferred embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, formulation or bioactive oil extract is capable of controlling mosquitoes or mosquito larvae, flies or fly larvae, aphids, mites, spiders, cockroaches, weevils or rootworms. [545] 56. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, formulation or bioactive oil extract is capable of controlling a fungus, bacteria or virus.

[546] 57. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, formulation or bioactive oil extract is capable of controlling one or more properties of a plant, either positively or negatively.

[547] 58. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, formulation or bioactive oil extract is capable of controlling one or more properties of plant species in the Cannabaceae family, such as cannabis, hemp or hops varieties.

[548] 59. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, formulation or bioactive oil extract is capable of: stimulating the production of plant defence compounds within a plant; triggering anti-microbial defence mechanisms within a plant; activating anti-viral defence mechanisms within a plant; activating anti-bacterial defence mechanisms within a plant; eliciting plant disease resistance; enhancing plant disease resistance; promoting plant growth; increasing biomass production within a plant; increasing plant uniformity; or, use as a herbicide.

[549] 60. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, formulation or bioactive oil extract is capable of producing an allelochemical capable of interfering with the biology of the biological organism.

[550] 61. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the biological organism is sprayed with the bioactive terpene composition, formulation or bioactive oil extract.

[551] 62. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, formulation or bioactive oil extract is applied to a surface, substrate (eg. soil) or plant surface (eg. roots, foliage, flower head, seed) on which may be found the biological organism or which may be in close proximity to the biological organism, or the bioactive terpene composition, formulation or bioactive oil extract is dispersed in the biological organism’s immediate vicinity or environment (eg. dispersed in the air).

[552] 63. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein if the biological organism needing controlling is a plant, then the plant is sprayed with the bioactive terpene composition, formulation or bioactive oil extract, or the bioactive terpene composition, formulation or bioactive oil extract is applied to a surface, substrate (eg. soil) or the bioactive terpene composition, formulation or bioactive oil extract is dispersed in the plant’s immediate vicinity or environment (eg. dispersed in the air).

[553] 64. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, formulation or bioactive oil extract is directly applied to the biological organism so as to elicit the desired effect.

[554] 65. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, formulation or bioactive oil extract is applied in an environment of the biological organism so as to elicit the desired effect.

[555] 66. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, formulation or bioactive oil extract is applied to a plant or dispersed in an environment of the plant, so as to invoke a defence mechanism or allelogenic chemical in the plant against an insect, mite, spider, bacteria, fungus or virus.

[556] 67. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein the bioactive terpene composition, formulation or bioactive oil extract is applied to a plant or dispersed in an environment of the plant, so as to control a property of the plant, such as eliciting plant disease resistance, enhancing plant disease resistance, promoting plant growth, increasing biomass production within the plant, or increasing plant uniformity.

[557] 68. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein plant material derived or obtained from the hemp variety comprises flowers, panicles, stems, roots or leaves.

[558] 69. The embodiment as described in any one of the preceding paragraphs (context permitting), wherein plant material derived or obtained from the hemp variety comprises live plant material that can be propagated, such as cuttings or seeds. List of References

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Published on December 28, 2018 as Manuscript RAI 18.005843. The latest version is at http://www.ibc.org/cgi/doi/10.1074/ibc.RA118.005843.

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