91392-410896 -1- CANNABIDIVARINIC ACID AND METHODS THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Serial No.63/527,225, filed on July 17, 2023, the entire disclosures of which is incorporated herein by reference. BACKGROUND AND SUMMARY [0002] The cannabis plant contains several compounds known as cannabinoids. In particular, cannabinoids include compounds such as Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) as well as more than 100 related secondary metabolites. In addition to cannabinoids, cannabis plant species such as Cannabis sativa, Cannabis indica, and Cannabis ruderalis can possesses a range of defense mechanisms such as phenolic compounds, protease inhibitors, and terpenes. [0003] To date, regulatory agencies have provided little to no direction for use of insecticides on cannabis plants. As a result, there are a limited number of insecticide products for pest management that are permitted for use in hemp production. Currently, the few products include items such as horticultural oils, essential oils, insecticidal soaps, and a few botanically derived insecticides. Importantly, there are no currently established residue tolerances for C. sativa (in the form of hemp or marijuana) in the United States. Moreover, key pests such as cannabis aphids, hemp russet mites, and spider mites can decimate crops of cannabis plants. As a result, there exists a need for new insecticides for use in the cannabis industry. In particular, an insecticide that is identified as “organic” or as a natural biopesticide for use in the cannabis is highly desirable. [0004] Accordingly, the present disclosure provides formulations and kits comprising cannabidivarinic acid (CBDVA) that can be used for protection of plants from pests, in particular cannabis plants. Methods of applying the formulations or kits to plants are also provided. The formulations, kits, and methods of the present disclosure utilize CBDVA, which unexpectedly provides efficacy to protect plants against pests such as aphids and mites. As described herein, application of CBDVA reduced insect pest fecundity and survival in artificial feeding assays but application of CBD in artificial feeding assays increased pest insect fecundity. These results were surprising and unexpected. [0005] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the
91392-410896 -2- invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTIONS OF THE DRAWINGS [0006] The detailed description particularly refers to the accompanying figures in which: [0007] FIGURE 1 shows cannabis aphid infestation induces plant defense responses. Ten adult cannabis aphids were clip-caged on a 3-week-old cannabis single leaf. After 48 hours aphids were removed and leaves were collected and relative expression of phytohormone marker genes for salicylic acid (PR1), jasmonic acid (HEL), absicic acid (PP2C-6), and CBDA (CBDAS) were measured with RT-qPCR. Error bars represent SE of the mean of four biological replicates with two technical replicates each. Statistical analysis was performed using a t-test. Asterisks indicate significance, P < 0.05 (t-test). [0008] FIGURES 2A-2C demonstrate that cannabis aphid feeding impacts phytohormone levels. Eight week-old hemp plants were infested with 10 mixed life stages of aphids and cannabinoid levels were analyzed 20 days post-infestation. The cannabinoids include salicylic acid (SA) (FIG.2A), jasmonic acid (JA) (FIG.2B), and abscisic acid (ABA) (FIG.2C) production in infested and uninfested leaves for each cultivar. Statistical analysis was performed using a t-test. Asterisks indicate significance * P < 0.05, ** P < 0.01, *** P < 0.001. [0009] FIGURES 3A-3B show a comparison of the olivetolic and divarinic cannabinoid biosynthesis pathways. FIG.3A shows the biosynthesis pathways for cannabidiolic acid (CBDA). FIG.3B shows the biosynthesis pathways for cannabidivarinic acid (CBDVA). Both synthesis pathways begin geranyl diphosphate (GPP) and olivetolic acid (see FIG.3A) or divarinic acid (see FIG.3B) through CBGA synthase (CBGAS) activity. CBDA synthase (CBDAS) then converts CBGA (see FIG.3A) or CBGVA (see FIG.3B) to CBDA or CBDVA, respectively. [0010] FIGURES 4A-4B show cannabis aphid life performance on artificial diets. Cannabis aphids were maintained on artificial diet supplemented with either DMSO, DMSO + 1 mM CBDVA, DMSO + 0.5 mM CBDVA, DMSO + 0.1 mM CBDVA or DMSO + 1 mM CBD. A cohort of ten 8-day old adult aphids were transferred to artificial diet and allowed to feed for 4 days and the number of (FIG.4A) nymphs and (FIG.4B) surviving adults remaining each day was monitored (n = 14, n =12 for 1 mM CBD, n = 9 for 0.5 mM CBDVA and 0.1 mM CDBVA).
91392-410896 -3- [0011] FIGURES 5A-5B show green peach aphid life performance on artificial diets. Green peach aphids were maintained on artificial diet supplemented with either DMSO, or DMSO + 0.5 mM CBDVA. A cohort of ten 8-day old adult aphids were transferred to artificial diet and allowed to feed for 3 days and the number of (FIG.5A) nymphs and (FIG.5B) surviving adults remaining each day was monitored (n = 10). [0012] FIGURES 6A-6B show western flower thrips performance of a the low- cannabinoid hemp cultivar, ‘Elite’ with CBDVA extract supplemented in the as described previously (Hayes et al.2023). The leaf petiole was submerged in a 2.5 mM CBDVA solution in a 0.2-ml Eppendorf. CBDVA was solubilized in dimethyl sulfoxide (DMSO) and an equal volume of DMSO was used as a control. Thrips were counted every other day for eight days to determine the number of eggs (FIG.6A) and larvae (FIG.6B) (n = 3 for water, n = 5 for DMSO, n = 4 for CBDVA). [0013] In an illustrative aspect, a formulation comprising cannabidivarinic acid (CBDVA) and one or more solvents is provided. In an embodiment, the solvent comprises an alcohol. In an embodiment, the alcohol comprises ethanol. In an embodiment, the alcohol comprises methanol. In an embodiment, the solvent comprises DMSO. In an embodiment, the solvent comprises water. In an embodiment, the solvent comprises acetonitrile. [0014] In an embodiment, the CBDVA is present between 0.01 mM to 10 mM. In an embodiment, the CBDVA is present between 0.01 mM to 0.1 mM. In an embodiment, the CBDVA is present between 0.1 mM to 0.5 mM. In an embodiment, the CBDVA is present between 0.5 mM to 1 mM. In an embodiment, the CBDVA is present between 1 mM to 5 mM. In an embodiment, the CBDVA is present between 5 mM to 10 mM. [0015] In an embodiment, the CBDVA is present at 0.1 mM. In an embodiment, the CBDVA is present at 0.2 mM. In an embodiment, the CBDVA is present at 0.3 mM. In an embodiment, the CBDVA is present at 0.4 mM. In an embodiment, the CBDVA is present at 0.5 mM. In an embodiment, the CBDVA is present at 0.6 mM. In an embodiment, the CBDVA is present at 0.7 mM. In an embodiment, the CBDVA is present at 0.8 mM. In an embodiment, the CBDVA is present at 0.9 mM. In an embodiment, the CBDVA is present at 1 mM. In an embodiment, the formulation further comprises a natural insecticide. [0016] In an illustrative aspect, a kit comprising cannabidivarinic acid (CBDVA) and one or more solvents is provided. In an embodiment, the solvent comprises an alcohol. In an embodiment, the alcohol comprises ethanol. In an embodiment, the alcohol comprises methanol. In an embodiment, the solvent comprises DMSO. In an embodiment, the solvent comprises water. In an embodiment, the solvent comprises acetonitrile. In an embodiment, the kit further comprises a natural insecticide.
91392-410896 -4- [0017] In an illustrative aspect, a method of protecting a plant from a pest is provided. The method comprises the step of a formulation of the present disclosure to the plant, wherein the formulation protects the plant from the pest. [0018] In an embodiment, the plant comprises one or more harvested plant parts. In an embodiment, the plant is a cannabis plant. In an embodiment, the cannabis plant is a species selected from the group consisting of Cannabis sativa, Cannabis indica, and Cannabis ruderalis. [0019] In an embodiment, the pest is an insect. In an embodiment, the insect comprises a piercing-sucking mouthpart. In an embodiment, the pest is an arthropod herbivore. [0020] In an embodiment, the pest is an aphid. In an embodiment, the aphid is a cannabis aphid. In an embodiment, the aphid is a green peach aphid. In an embodiment, the aphid is a leaf-feeding aphid. In an embodiment, the aphid is a root-feeding aphid. [0021] In an embodiment, the pest is a mite. In an embodiment, the mite is a hemp russet mite. In an embodiment, the mite is a hemp russet mite. In an embodiment, the pest is a thrips. In an embodiment, the thrips is a western flower thrips. [0022] In an embodiment, the administration is selected from the group consisting of spraying, drenching, dipping, submersing, flooding, and any combination thereof on or near the plant. In an embodiment, the method further comprises administration of sulfur to the plant. In an embodiment, the method further comprises application of a water composition to the plant. In an embodiment, the water composition comprises water and a surfactant. [0023] In an illustrative embodiment, a method of protecting a plant from a pest is provided. The method comprises the step of administering any one of the formulations described herein to the pest, wherein the formulation protects the plant from the pest. [0024] In an embodiment, the plant comprises one or more harvested plant parts. In an embodiment, the plant is a cannabis plant. In an embodiment, the cannabis plant is a species selected from the group consisting of Cannabis sativa, Cannabis indica, and Cannabis ruderalis. [0025] In an embodiment, the pest is an insect. In an embodiment, the insect comprises a piercing-sucking mouthpart. In an embodiment, the pest is an arthropod herbivore. [0026] In an embodiment, the pest is an aphid. In an embodiment, the aphid is a cannabis aphid. In an embodiment, the aphid is a green peach aphid. In an embodiment, the aphid is a leaf-feeding aphid. In an embodiment, the aphid is a root-feeding aphid. [0027] In an embodiment, the pest is a mite. In an embodiment, the mite is a hemp russet mite. In an embodiment, the mite is a hemp russet mite. In an embodiment, the pest is a thrips. In an embodiment, the thrips is a western flower thrips.
91392-410896 -5- [0028] In an embodiment, the pest is administered the formulation via food. In an embodiment, the pest is administered the via liquid. In an embodiment, the method further comprises administration of sulfur to the plant. In an embodiment, the method further comprises application of a water composition to the plant. In an embodiment, the water composition comprises water and a surfactant. [0029] The following numbered embodiments are contemplated and are non-limiting: 1. A formulation comprising cannabidivarinic acid (CBDVA) and one or more solvents. 2. The formulation of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the solvent comprises an alcohol. 3. The formulation of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the alcohol comprises ethanol. 4. The formulation of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the alcohol comprises methanol. 5. The formulation of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the solvent comprises DMSO. 6. The formulation of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the solvent comprises water. 7. The formulation of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the solvent comprises acetonitrile. 8. The formulation of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the CBDVA is present between 0.01 mM to 10 mM. 9. The formulation of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the CBDVA is present between 0.01 mM to 0.1 mM. 10. The formulation of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the CBDVA is present between 0.1 mM to 0.5 mM. 11. The formulation of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the CBDVA is present between 0.5 mM to 1 mM. 12. The formulation of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the CBDVA is present between 1 mM to 5 mM. 13. The formulation of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the CBDVA is present between 5 mM to 10 mM. 14. The formulation of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the CBDVA is present at 0.1 mM. 15. The formulation of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the CBDVA is present at 0.2 mM.
91392-410896 -6- 16. The formulation of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the CBDVA is at 0.3 mM. 17. The formulation of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the CBDVA is present at 0.4 mM. 18. The formulation of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the CBDVA is present at 0.5 mM. 19. The formulation of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the CBDVA is present at 0.6 mM. 20. The formulation of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the CBDVA is present at 0.7 mM. 21. The formulation of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the CBDVA is present at 0.8 mM. 22. The formulation of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the CBDVA is present at 0.9 mM. 23. The formulation of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the CBDVA is present at 1 mM. 24. The formulation of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the formulation further comprises a natural insecticide. 25. A kit comprising cannabidivarinic acid (CBDVA) and one or more solvents. 26. The kit of clause 25, any other suitable clause, or any combination of suitable clauses, wherein the solvent comprises an alcohol. 27. The kit of clause 26, any other suitable clause, or any combination of suitable clauses, wherein the alcohol comprises ethanol. 28. The kit of clause 26, any other suitable clause, or any combination of suitable clauses, wherein the alcohol comprises methanol. 29. The kit of clause 25, any other suitable clause, or any combination of suitable clauses, wherein the solvent comprises DMSO. 30. The kit of clause 25, any other suitable clause, or any combination of suitable clauses, wherein the solvent comprises water. 31. The kit of clause 25, any other suitable clause, or any combination of suitable clauses, wherein the solvent comprises acetonitrile. 32. The kit of clause 25, any other suitable clause, or any combination of suitable clauses, wherein the kit further comprises a natural insecticide.
91392-410896 -7- 33. A method of protecting a plant from a pest, said method comprising the step of administering the formulation of of clauses 1 to 24 to the plant, wherein the formulation protects the plant from the pest. 34. The method of clause 33, any other suitable clause, or any combination of suitable clauses, wherein the plant comprises one or more harvested plant parts. 35. The method of clause 33, any other suitable clause, or any combination of suitable clauses, wherein the plant is a cannabis plant. 36. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the cannabis plant is a species selected from the group consisting of Cannabis sativa, Cannabis indica, and Cannabis ruderalis. 37. The method of clause 33, any other suitable clause, or any combination of suitable clauses, wherein the pest is an insect. 38. The method of clause 37, any other suitable clause, or any combination of suitable clauses, wherein the insect comprises a piercing-sucking mouthpart. 39. The method of clause 33, any other suitable clause, or any combination of suitable clauses, wherein the pest is an arthropod herbivore. 40. The method of clause 33, any other suitable clause, or any combination of suitable clauses, wherein the pest is an aphid. 41. The method of clause 40, any other suitable clause, or any combination of suitable clauses, wherein the aphid is a cannabis aphid. 42. The method of clause 40, any other suitable clause, or any combination of suitable clauses, wherein the aphid is a green peach aphid. 43. The method of clause 40, any other suitable clause, or any combination of suitable clauses, wherein the aphid is a leaf-feeding aphid. 44. The method of clause 40, any other suitable clause, or any combination of suitable clauses, wherein the aphid is a root-feeding aphid. 45. The method of clause 33, any other suitable clause, or any combination of suitable clauses, wherein the pest is a mite. 46. The method of clause 45, any other suitable clause, or any combination of suitable clauses, wherein the mite is a hemp russet mite. 47. The method of clause 45, any other suitable clause, or any combination of suitable clauses, wherein the mite is a hemp russet mite. 48. The method of clause 33, any other suitable clause, or any combination of suitable clauses, wherein the pest is a thrips.
91392-410896 -8- 49. The method of clause 48, any other suitable clause, or any combination of suitable clauses, wherein the thrips is a western 50. The method of clause 33, any other suitable clause, or any combination of suitable clauses, wherein the administration is selected from the group consisting of spraying, drenching, dipping, submersing, flooding, and any combination thereof on or near the plant. 51. The method of clause 33, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises administration of sulfur to the plant. 52. The method of clause 33, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises application of a water composition to the plant. 53. The method of clause 52, any other suitable clause, or any combination of suitable clauses, wherein the water composition comprises water and a surfactant. 54. A method of protecting a plant from a pest, said method comprising the step of administering the formulation of any one of clauses 1 to 24 to the pest, wherein the formulation protects the plant from the pest. 55. The method of clause 54, any other suitable clause, or any combination of suitable clauses, wherein the plant comprises one or more harvested plant parts. 56. The method of clause 54, any other suitable clause, or any combination of suitable clauses, wherein the plant is a cannabis plant. 57. The method of clause 56, any other suitable clause, or any combination of suitable clauses, wherein the cannabis plant is a species selected from the group consisting of Cannabis sativa, Cannabis indica, and Cannabis ruderalis. 58. The method of clause 54, any other suitable clause, or any combination of suitable clauses, wherein the pest is an insect. 59. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the insect comprises a piercing-sucking mouthpart. 60. The method of clause 54, any other suitable clause, or any combination of suitable clauses, wherein the pest is an arthropod herbivore. 61. The method of clause 54, any other suitable clause, or any combination of suitable clauses, wherein the pest is an aphid. 62. The method of clause 61, any other suitable clause, or any combination of suitable clauses, wherein the aphid is a cannabis aphid. 63. The method of clause 61, wherein the aphid is a green peach aphid. 64. The method of clause 61, wherein the aphid is a leaf-feeding aphid. 65. The method of clause 61, wherein the aphid is a root-feeding aphid.
91392-410896 -9- 66. The method of clause 54, any other suitable clause, or any combination of suitable clauses, wherein the pest is a mite. 67. The method of clause 66, wherein the mite is a hemp russet mite. 68. The method of clause 66, wherein the mite is a hemp russet mite. 69. The method of clause 54, any other suitable clause, or any combination of suitable clauses, wherein the pest is a thrips. 70. The method of clause 69, wherein the thrips is a western flower thrips. 71. The method of clause 54, any other suitable clause, or any combination of suitable clauses, wherein the pest is administered the formulation via food. 72. The method of clause 54, any other suitable clause, or any combination of suitable clauses, wherein the pest is administered the formulation via liquid. 73. The method of clause 54, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises administration of sulfur to the plant. 74. The method of clause 54, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises application of a water composition to the plant. 75. The method of clause 74, any other suitable clause, or any combination of suitable clauses, wherein the water composition comprises water and a surfactant. [0030] The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” [0031] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. EXAMPLES EXAMPLE 1 Exemplary Materials and Methods [0032] The instant example provides exemplary materials and methods utilized for the present disclosure. Insect and plant sources
91392-410896 -10- [0033] Various insect pests can be utilized for the present disclosure. For instance, cannabis aphids were collected from an facility in Loveland, CO. Insects were reared and maintained on Elite (New West Genetics, NWG Fort Collins, CO) cultivar hemp plants in 45.7 × 45.7 × 76.2 cm cage (BioQuip Products Inc., Rancho Dominguez, CA) under the greenhouse conditions with 430W HPS (High Pressure Sodium) Fixtures (P.L. Light Systems) and 400W bulbs (GE Lucalox lu400 series), photoperiod of 16:8 (L:D) hours (h) and the day: night temperature was 23:18°C. [0034] Examplary hemp cultivars were high-cannabinoid cultivar, Unicorn (Colorado Hemp Institute, CO), and low-cannabinoid cultivars, Tiborszallasi (European project Multihemp-multihemp.eu) and Elite (New West Genetics, Fort Collins, CO). Unicorn is an Association of Official Seed Certifying Agencies (AOSCA) certified high-CBD cultivar (8-9%, B. Althouse, Colorado Hemp Institute personal communication) and cloned from vegetative cuttings. Tiborszallasi is a European cultivar reported to be a low-CBD (2-3%, Glivar et al. 2020) and is grown from seed. Elite is an AOSCA certified low-CBD cultivar (2-3%, R. Flecther, New West Genetics personal communication) or grain and fiber cultivar and was grown from seed. [0035] All cultivars produce less 0.3% delta-9 THC. The high-cannabinoid cultivar was cloned from vegetative cuttings and the cuttings were rooted using liquid rooting concentrate (Dip 'n Grow). The low-cannabinoid cultivars were grown from seed. The plants were grown at Colorado State University’s Plant Growth Facilities in a greenhouse under conditions described above. All plants were fertilized with Osmocote (Scott’s Company, Marysville, OH) 15-9-12 N:P:K ratio time-released fertilizer as per label instructions and watered ad libitum. Assessing insect performance on high- and low-cannabinoid cultivars in whole plant assays [0036] To determine the impact of cannabinoids on insect performance (e.g., aphids), insect life histories were analyzed on high- (Unicorn) and low-cannabinoid (Tiborszallasi) cultivars. For instance, two adult aphids were placed in a 1.2 cm clip cage on the abaxial surface of the 3rd or 4th leaf, of a 5-week-old hemp plant with two clip cages were placed on each plant. Clip cages were constructed of 5-mm diameter by 15-mm long clear plastic straw sections with a removable foam top glued to a 10-cm of 20-gauge galvanized steel wire. The adults were allowed to larviposit for 24 h, the adults were removed, and remaining nymphs were left to mature. The number of nymphal instars were checked daily. Once nymphs reached 3rd instar all but one was removed. The life history of this aphid was monitored daily, and any nymphs produced were removed until the adult died. There was a total of 13 replicates for the high-cannabinoid cultivar (Unicorn) and 21 replicates for the low- cannabinoid cultivars
91392-410896 -11- (Tiborszallasi). Aphid observations were conducted in a growth chamber (Conviron, Winnipeg Canada, Model E15) where the photoperiod 10:14 (L:D) hours (h) and the day: night temperature was 22:18°C. [0037] The life history parameters analyzed are listed in Table 1 (e.g., mean fecundity, net reproductive rate (R0), mean generation time, doubling time, developmental time (days), and adult longevity (days). Assessing aphid performance with cannabinoids in artificial feeding assays [0038] To determine the role of CBD and CBDVA in aphid performance, cannabis aphids were reared on an artificial diet supplemented with CBD or CBDVA. The artificial diet was designed for green peach aphids (Myzus persicae). Ten age synchronized 1-day old adult cannabis aphids or green peach aphids were placed in an artificial feeding chamber consisting of 55 mm Petri dishes (VWR) with parafilm (Bemis) as described in Nachappa et al.2016. The artificial diet volume was 250 μL including the CBD/DMSO, CBDVA/DMSO or DMSO. Stock CBD (Cayman Chemical) solution was prepared by dissolving 10 mg of CBD with 318 μL of DMSO to make 100 mM CBD. Stock CBDVA (Cayman Chemical) was prepared by dissolving 1 mg in 30.3 μL of DMSO to make 100 mM CBDVA. Aphid populations were monitored daily for 3-4 days. Analysis of cannabinoids using Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS) [0039] For cannabinoid analysis, 14 cannabinoids were analyzed in the high- (Unicorn) and low- (Tiborszallasi) cannabinoid hemp cultivars that were either infested with cannabis aphids or left uninfested (control plants). Briefly, 8-week-old plants were infested with 10 mixed life stages of cannabis aphids by placing the aphids on the adaxial surface of nodes 9-13. The infested and uninfested plants were housed in separate cages (52 x 57 x 162 cm). Twenty days post-aphid infestation, three leaves, taken from the two uppermost fully expanded nodes, were harvested from each treatment and aphids were removed using paintbrushes. The leaves were placed in 50 mL conical tubes and stored at -20 C. There were four replicates collected for each cultivar. [0040] Samples were transported to a -80° C freezer for 2 hours immediately before lyophilization. Samples were lyophilized at 0° C for 49 h. After lyophilization, samples were stored in a -20° C freezer. Lyophilized samples were then homogenized for 5 minutes using a bead beater (Next Advance, Troy, NY, USA). After homogenization, about 40 mg tissue for each sample were weighed into 2-mL Eppendorf tubes, with 1 mL of cold 80% methanol in
91392-410896 -12- water. Samples were then vortexed vigorously for 30 min at 4°C, followed by 15-min sonication in ice bath, and another 30-min vortexing at 4°C. After mixing, samples were centrifuged at 15,000 g and 4°C for 10 min. Supernatants were recovered and diluted 10 times in cold 100% methanol. Then 50 μL of the diluted sample was mixed with 50 μL of internal standard (IS) and stored at -80°C until analysis. An aliquot (10 μL) was taken from each study sample to be pooled to generate a quality control (QC) sample. The IS used was a mixture of authentic standards of cannabinoids and labelled standard THC-d3 were purchased from Cerilliant (TX, USA). IS, 0.1 μg/mL of THC-d3 were prepared in 100% methanol. [0041] UPLC-MS/MS analysis was performed on a Waters ACQUITY UPLC coupled to a Waters Xevo TQ-S triple quadrupole mass spectrometer. Chromatographic separations were carried out on an ACQUITY UPLC HSS T3 column (1 x 100 mm, 1.8 μm, Waters, MA, USA). Mobile phases were water with 0.1% formic acid (A) and acetonitrile (B). Samples were held at 6°C in the autosampler, and the column was operated at 45°C. The capillary voltage of MS detector was set to 0.7 kV in positive mode. Inter-channel delay was set to 3 msec. Source temperature was 150°C and desolvation temperature 450°C. Desolvation gas flow was 1000 L/h, cone gas flow (nitrogen) was 150 L/h, and collision gas flow (argon) was 0.15 mL/min. Nebulizers pressure (nitrogen) was set to 7 Bar. Autodwell feature was set for the collection of 12 points-across-peak. Cone voltage and collision energy (CE) of each MRM was optimized. Several high abundance compounds (CBDVA, CBGA, CBDA) in the current sample set were analyzed using “de-optimized” cone and CE voltage. Raw data files were imported into the Skyline opensource software package (MacLean et al.2010). Each target analyte was visually inspected for retention time and peak area integration. Peak areas were extracted for target compounds detected in biological samples and normalized to the peak area of the appropriate internal standard or surrogate in each sample. Absolute quantitation (ug/g) was calculated using the linear regression equation generated for each compound from the calibration curve. [0042] Cannabinoids were analyzed using R software (version 3.6.0). Outlier tests using a Bonferroni adjustment were run for all cannabinoid levels. Once outliers were determined, instead of removing outliers by metabolite we decided to remove the entire sample from further analysis. The difference in cannabinoid levels between infested and control plants were analyzed for statistical significance using a t-test. Analysis of phytohormone- and cannabinoid-marker genes using RT-qPCR analysis [0043] Ten adult cannabis aphids were clip-caged onto the adaxial side of the most expanded leaf of 3-week-old high-cannabinoid (Unicorn) plant. The cage was positioned to allow access to both the adaxial and abaxial sides for the aphids. After 48 hours, aphids were
91392-410896 -13- removed with a fine paintbrush and leaves were collected and flash frozen in liquid nitrogen for RNA extraction. Uninfested control leaves treated in the same manner. Total RNA isolation was performed following the CTAB-C/I+RNeasy protocol described in Guerriero et al.2016. Briefly, RNA was isolated from 100 mg of tissue from three-week old hemp plants by grinding the tissue in mortar and pestle with liquid nitrogen. Next, 2.5% (% w/v) polyvinylpyrrolidone (PVP-40), 2 M NaCl, 100 mM Tris-HCl pH 8, 25 mM EDTA, 0.2% BME were added, and samples were incubated for 10 min at 60°C, vortexed, and centrifuged. The aqueous phase was extracted and precipitated with 2/3 volume of isopropanol for 1 hour at - 20°C. Samples were then loaded on RNeasy columns (Qiagen) according to the manufacturer’s instructions with the on-column DNase treatment. Elimination of genomic DNA was validated through PCR. RNA samples were quantified using a nanodrop and 2 μg of cDNA was synthesized from the RNA samples using Verso cDNA synthesis kit (Thermo Fisher) according to the manufacturer’s instructions. Genes in the CBD synthesis and phytohormone signaling pathways using primer sets were utilized. RT-qPCR was performed in a QuantStudio 3 (Thermo Fisher) with iQ SYBR Green Supermix (Bio-Rad) in a 20 ul reaction using gene specific primers with 3.5 min at 95°C, 40 cycles of 15 s at 95°C, and 60 s at 58°C. Target genes were normalized to CsClathrin and relative expression levels were calculated with 2-ΔΔCt (Livak et al.2001). Two technical replicates and four biological replicates were performed for each sample. Analysis of phytohormones using UPLC-MS/MS [0044] The phytohormone analysis was conducted as described below. Frozen samples were lyophilized and the dried samples were added with stainless steel balls and homogenized in the Bullet Blender for 2 min. The homogenate (20-30 mg) was weighed into 2-mL glass extraction vials and added with 1 mL of cold 80% methanol in water and 20 μL of internal standard mix containing 200 ng/mL of SA-D4 (Sigma-Aldrich, St. Louis, MO), 200 ng/mL of JA-D5 (Sigma-Aldrich, St. Louis, MO), 50 ng/mL of IAA-D5 (Sigma-Aldrich, St. Louis, MO), and 500 ng/mL of ABA-D6 [in 50% methanol (Santa Cruz Biotechnology, Santa Cruz, CA)]. The mixture was vigorously mixed for 0.5 h, followed by 15 min of sonication, and another 0.5 h of mixing. Then the mixture was centrifuged at 3,000 g and 4°C for 15 min. Supernatant (850 μL) was recovered. To the remaining pellets, 1 mL of acetonitrile was added and the extraction was repeated as described above. After centrifugation, supernatant (850 μL) was recovered and combined with the first aliquot of supernatant, which was then dried under nitrogen, and then resuspended in 100 μL of 50% methanol in water. An aliquot (10 uL) was taken from each
91392-410896 -14- study sample to generate a pooled quality control (QC) samples. Sample extracts and QCs were stored at -20°C until analysis. [0045] UPLC-MS/MS analysis was performed on a Waters ACQUITY Classic UPLC coupled to a Waters Xevo TQ-S triple quadrupole mass spectrometer. Chromatographic separations were carried out on a Waters UPLC T3 column (2 x 50 mm, 1.7 μM). Mobile phases were (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid. The LC gradient was as follows: time = 0 min, 1% B; time = 0.65 min, 1% B; time = 2.85 min, 99% B; time = 3.5 min, 99% B; time= 3.55 min, 1% B; time =5 min, 1% B. Flow rate was 0.5 mL/min and injection volume was 2 μL. Samples were held at 6°C in the autosampler, and the column was operated at 45°C. Mass detector was operated in ESI- mode. The capillary voltage set to 0.7 kV. Inter-channel delay was set to 3 msec. Source temperature was 150°C and desolvation gas (nitrogen) temperature 450°C. Desolvation gas flow was 1000 L/h, cone gas flow was 150 L/h, and collision gas (argon) flow was 0.15 mL/min. Nebulizer pressure (nitrogen) was set to 7 Bar. The MS acquisition functions were scheduled by retention time. Autodwell feature was set for each function and dwell time was calculated by Masslynx software (Waters) to achieve 12 points-across-peak as the minimum data points per peak. EXAMPLE 2 Cannabis aphid performance on high and low-cannabinoid cultivars in whole-plant experiments [0046] The instant example evaluated the impact of cannabinoids on cannabis aphids. Life history assays were performed on high-cannabinoid cultivar (Unicorn) and low- cannabinoid cultivar (Tiborszallasi) under greenhouse conditions. The mean fecundity and net reproductive rate (R
0) were found to be the same and they were significantly higher in low- cannabinoid cultivar, Tiborszallasi, compared to high-cannabinoid cultivar, Unicorn (Table 1). In contrast, cannabis aphid development time was shorter on high-cannabinoid cultivar compared to low-cannabinoid cultivar (Table 1). However, once the nymph reached adulthood, aphids survived longer on low-cannabinoid cultivar, compared to the high-cannabinoid cultivar (Table 1). The same trend was observed for adult longevity as well.
91392-410896 -15- Table 1. Cannabis aphid reproduction and life table parameters on high and low-cannabinoid hemp cultivars. Life history parameters Cultivars High-cannabinoid (Unicorn) Low-cannabinoid (Tiborszallasi)
Cannabis aphid impact on cannabinoid production [0047] The instant example evaluated the impact of cannabis aphid feeding on cannabinoid production. Cannabinoid production in leaf tissues in aphid-infested and control (uninfested) or healthy plants was analyzed. A total of 14 cannabinoids were analyzed out of which nine were detected and quantified as described in Table 2. Both CBD and THC had slight but not significant increases in concentrations in the high-cannabinoid cultivar (Unicorn) in response to cannabis aphid infestation. There was no impact of aphid infestation on cannabinoid levels in the low-cannabinoid cultivar (Tiborszallasi) (Table 2). Table 2. Cannabis aphid impacts on cannabinoid levels in high-cannabinoid cultivar (Unicorn) and low-cannabinoid cultivar (Tiborszallasi).
91392-410896 -16- Cultivar Treatment Mean ± SE t- P- (ug/g) test
(df) value
candidate cannabinoids that potentially affected aphid performance on whole plants were identified by comparing the cannabinoid profiles in the leaf tissues of healthy or uninfested high- and low-cannabinoid cultivars. Of the nine cannabinoids detected, five were statistically different between the uninfested high- and low-cannabinoid cultivars (Table 3). The three cannabinoids that were significantly higher in the high-cannabinoid cultivar were
91392-410896 -17- cannabidivarinic acid (CBDVA), tetrahydrocannabivarinic acid (THCVA) and cannabigerol (CBG) (Table 3). Surprisingly, both CBD and THC levels were significantly higher in the low- cannabinoid cultivar (Table 3). This is in contrast to reported CBD levels in inflorescence of Unicorn (8-9%) and in Tiborszallasi. Table 3. Difference in cannabinoid levels between uninfested controls (healthy) high- cannabinoid cultivar (Unicorn) and low-cannabinoid cultivar (Tiborszallasi). Cannabinoid Treatment Mean ± SE (ug/g) t-test
(df) P-value Unicorn 8644 ± 505.57 1
1
EXAMPLE 4 Cannabis aphid performance in artificial feeding assays supplemented with cannabinoids [0049] The example conducted analyses to determine the effect of CBD and CBVA, the two major cannabinoids that were found to be different between the healthy controls of the high- (Unicorn) and low- (Tiborszallasi) cannabinoid cultivars via artificial feeding assays. Ten age-synchronized adult aphids were placed on each diet and their fecundity and survival were monitored for 4 days. [0050] In contrast to CBD supplementation, the addition of CBDVA in the artificial diet had a negative impact on cannabis aphid performance (FIGS.4A-4B). Cannabis aphids feeding on all three concentrations of CBDVA screened (0.1 mM, 0.5 mM, and 1 mM) all had negative impacts on aphid performance compared to diet and DMSO alone. The addition of 1 mM
91392-410896 -18- CBDVA to artificial diets significantly reduced fecundity compared to diet at all timepoints (Day 1: F = 45.64, df = 5, P < 0.0001, Day 4: F = 22.47, df=5, P<0.0001) and DMSO alone (Day 1: F = 45.64, df = 5, P < 0.0001, Day 4: F = 22.47, df = 5, P = 0.007) (FIG.4B). [0051] The addition of 0.5 mM CBDVA also had a significant negative impact on adult survival compared to diet after two days (Day 3: F = 10.47, df = 5, P < 0.0001) and DMSO alone (Day 3: F = 10.47, df = 5, P = 0.0001) FIG.4A). The addition of 0.1 mM CBDVA to the artificial diet led to significant reduced fecundity compared to diet from days 1 (Day 1: F = 45.64, df = 5, P < 0.0001) to day 3 (Day 3: F = 28.55, df = 5, P = 0.001) and compared to DMSO alone on day 1 (Day 1: F = 45.65, df = 5, P = 0.0051) (FIG.4B). [0052] CBDVA supplementation in the artificial diet for green peach aphids also had a negative impact on aphid performance (FIGS.5A-5B). Green peach aphid survival was also significantly reduced in the for aphids feeding on the 0.5 mM CBDVA treatment after two days compared to diet (Day 2: F = 8.44, df = 2, P = 0.0026, Day 3: F = 17.23, df = 2, P < 0.0001) and DMSO alone (Day 2: F = 8.44, df = 2, P = 0.0054, Day 3: F= 17.23, df = 2, P < 0.0001) (FIG.5A). Green peach aphid adult fecundity was significantly reduced compared to diet (Day 1: F = 10.47, df = 2, P = 0.0004, Day 3: F = 34.65, df = 2, P < 0.0001) and DMSO alone (Day 1: F = 10.47, df = 2, P = 0.0079, Day 3: F = 34.65, df = 2, P < 0.0001) for aphids feeding on 0.5 mM CBDVA treatment for all timepoints (FIG.5B). EXAMPLE 5 Cannabis aphid feeding impacts phytohormone signaling [0053] To determine aphid-induced changes in phytohormone and cannabinoid levels, RT-qPCR analysis was performed to determine expression in marker genes. After a 48-hour infestation period of 3-week-old high-cannabinoid cultivar (Unicorn), the expression of the SA marker gene, PR1 was significantly increased in the aphid infested leaf tissues compared to the uninfested controls (FIG.1; P = 0.02). While not statistically significant, the JA marker gene HEL was also expressed higher at 48 hours (FIG.1, P = 0.06) in the aphid infested samples compared to the uninfested controls. There was no effect of aphid infestation on another stress- related phytohormone, abscisic acid (ABA) marker gene PP2C-6 (FIG.1, P = 0.21) and Cannabidiolic acid synthase (CBDAS) expression (FIG.1, P = 0.20) at 48-hour post-infestation. [0054] To assess the impact of long-term aphid feeding (20 days) on phytohormone production, the levels of two classical defense phytohormones, SA and JA, as well as ABA, were monitored on 8-week-old plants (FIGS.2A-2C). In the high-cannabinoid cultivar (Unicorn), the aphid infested plants had significantly higher levels of SA compared to the
91392-410896 -19- uninfested controls (P = 0.0048, FIG.2A). This result is consistent with the impact of early aphid infestation as determined by RT- The high-cannabinoid cultivar also had higher JA (P = 0.03) and ABA (P = 0.008) levels relative to the uninfested controls (FIGS.2B, 2C). In the low-cannabinoid cultivar (Tiborszallasi), aphid infestation only SA levels were significantly impacted by aphid infestation relative to uninfested controls (FIG.2A, P = 0.007). EXAMPLE 6 Hemp russet mite performance on high and low-cannabinoid cultivars in detached leaf experiments [0055] The instant example evaluated the impact of CBDVA on hemp russet mites, in particular hemp russet mite life performance on high-CBDVA cultivar (Unicorn) and low- CBDVA cultivar (Elite) detached leaves under lab conditions. As shown in Table 4, hemp russet mites reared on the low-CBDVA leaves had a significantly faster development time (t = 6.75, df = 36, P < 0.0001) compared to mites reared on the high-CBDVA leaves. The fecundity of the mites reared on the low-CBDVA leaves was also significantly higher (t = 4.29, df = 37, P = 0.0001) than the mites reared on the high-CBDVA leaves. Table 4. Hemp russet mite lifer performance on high and low-CBDVA hemp leaves. Mean ± SE offspring Mean ± SE developmental Mean ± SE adult f 7 i l i
EXAMPLE 7 Thrips performance on performance in detached leaf assay supplemented with cannabinoids [0056] To determine the effect of CBDVA on western flower thrips performance, five adult thrips were monitored in a detached leaf assay of a low-cannabinoid hemp cultivar, ‘Elite’ with CBDVA extract supplemented. The leaf petiole was submerged in a 2.5 mM CBDVA solution in a 0.2-ml Eppendorf tube. [0057] CBDVA (Cayman Chemical) was solubilized in dimethyl sulfoxide (DMSO) and an equal volume of DMSO (Supelco) was used as a control. Thrips were counted every other day for eight days to determine the survival of the adults and number of offspring. There were 3-5 replicates per treatment (n = 3 for water, n = 5 for DMSO, n = 4 for CBDVA).
91392-410896 -20- [0058] The addition of supplemental CBDVA had a significant impact on thrips egg production after 4 days compared to water (F = 6.34, df = 9, P = 0.03, FIGS 6A-6B). The addition of CBDVA did not have a difference at any of these timepoints with the DMSO control (Day 4 F = 6.34, df = 9, P = 0.99; Day 6: F = 13.19, df = 9, P = 0.81; Day 8: F = 17.18, df = 9, P = 0.80, FIG.6A). While not significant, more thrips larvae appeared on day 4 (F = 2.36, df = 9, P = 0.15, FIG.6B) and day 6 (F = 0.83, df = 9, P = 0.47, FIG.6B) on the DMSO control than either the water control or CBDVA treatment.
candidate cannabinoids that potentially affected aphid performance on whole plants were identified by comparing the cannabinoid profiles in the leaf tissues of healthy or uninfested high- and low-cannabinoid cultivars. Of the nine cannabinoids detected, five were statistically different between the uninfested high- and low-cannabinoid cultivars (Table 3). The three cannabinoids that were significantly higher in the high-cannabinoid cultivar were
91392-410896 -17- cannabidivarinic acid (CBDVA), tetrahydrocannabivarinic acid (THCVA) and cannabigerol (CBG) (Table 3). Surprisingly, both CBD and THC levels were significantly higher in the low- cannabinoid cultivar (Table 3). This is in contrast to reported CBD levels in inflorescence of Unicorn (8-9%) and in Tiborszallasi. Table 3. Difference in cannabinoid levels between uninfested controls (healthy) high- cannabinoid cultivar (Unicorn) and low-cannabinoid cultivar (Tiborszallasi). Cannabinoid Treatment Mean ± SE (ug/g) t-test(df) P-value Unicorn 8644 ± 505.57 1 1