Method and plant extract composition for promoting plant growth and preventing and controlling plant diseases

Abstract

A method and a plant extract composition for promoting plant growth and preventing and controlling plant diseases are provided. [Solution] A botanical composition is applied to plants by foliar spray or drench treatment to promote plant growth and prevent or suppress plant diseases. The botanical composition contains a granular extract of thyme leaves and an extract of celandine. The composition is typically diluted to a concentration of 0.2% to 5%.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of priority to commonly assigned U.S. patent application Ser. No. 62 / 878,600, entitled "METHOD AND PLANT EXTRACT CELEXT07 COMPOSITION FOR PROMOTING PLANT GROWTH AND PREVENTING AND CONTROLING PLANT DISEASES," filed in the U.S. Patent and Trademark Office on July 25, 2019. [Technical field]

[0002] The present invention relates generally to plant extracts for plant growth and protection. [Background technology]

[0003] There are several methods on the market to help plants grow and heal from diseases. Some of them use chemicals, others use organic ingredients. Chemical solutions, even if they often provide the best results, can have adverse effects on the health of people who come into contact with the treated plants. On the other hand, organic solutions are often not as efficient as they should be. Therefore, there is a need for organic solutions that are as efficient, if not more efficient, than most chemical solutions found on the market to help plants grow and heal from diseases.

[0004] Botanical extracts used in agriculture are made from naturally occurring plants (or other organisms) as an alternative to synthetic chemicals that can be more toxic to growers, consumers, and more harmful to the environment. Advantages of such products can include being biodegradable and more eco-friendly in nature, as there are no harmful residues, compared to synthetic chemical alternatives. Due to the advantages listed above, agricultural industries around the world are developing botanical products to contribute to sustainable agriculture. Testing the effectiveness of these products for the early stages of the plant growth cycle (germination and early seedling growth) is a costly and time-consuming process. Germination rates are usually determined by Petri dish assays. For early growth, other systems are usually used, including hydroponics or greenhouse cultivation (direct germination). Standard operating procedures (SOPs) were developed for the analysis of the effect of herbal extracts on germination and early growth. This was done in a defined system by modifying the roll paper towel test developed by the International Seed Testing Association (ISTA, 1985). The effectiveness of the SOPs was determined by comparison with the direct germination test. The SOP has the following advantages: (1) it is efficient for testing the effect of potential growth stimulants on seed germination and early growth; (2) it is time (2 weeks duration) and space efficient; (3) it is repeatable; carried out under defined conditions in a growth chamber; and (4) it is a relatively simple and low-cost method that can be carried out in industry with minimally trained staff and readily available materials. Summary of the Invention [Problem to be solved by the invention]

[0005] The above and other objects of the present invention are realized by broadly providing a botanical composition for promoting plant growth and preventing or suppressing plant diseases. [Means for solving the problem]

[0006] In one aspect of the present invention, a botanical composition is provided, which comprises a granular extract of thyme leaves, an extract of Chelidonium majus roots, and an extract of Chelidonium majus leaves, the composition being diluted to a concentration of 0.2% to 5%, and the composition is used to promote plant growth and prevent or suppress plant diseases.

[0007] The plant composition may contain 0.1% to 99% of an extract of celandine root, 0.1% to 99% of an extract of celandine leaves, and 0.1% to 30% of a granular extract of thyme leaves.

[0008] The botanical composition may be diluted to a concentration of 0.5% to 5%. The botanical composition may have antibacterial and / or antifungal properties. The botanical composition may further contain a tincture or seaweed. The seaweed may be Ascophyllum nodosum. The composition may contain 0.5 to 2 g / L of seaweed.

[0009] The composition may further comprise another extract of thyme leaves, which may be an extract of thyme leaves diluted 1:2 in 50% alcohol.

[0010] The composition may further contain thymol or yarrow leaf extract, which may be diluted 1:2 in 50% alcohol.

[0011] The plant composition may further include a soil formulation. The soil formulation may include coconut husk fiber. The soil formulation may further include peat moss and perlite, or mycorrhizae.

[0012] In another aspect of the present invention, there is provided a botanical composition comprising an extract of yarrow leaves diluted 1:2 in 50% alcohol, the composition being used to promote plant growth and prevent or control plant diseases. The botanical composition may further have antibacterial and / or antifungal properties.

[0013] The extract may be prepared by cold pressing or freeze drying. The extract may also be prepared using techniques including processes using fermentation. The fermentation may be aerobic or anaerobic. The bacteria used may be lactic acid bacteria or may be from the Bacillus species.

[0014] In yet another aspect of the present invention, there is provided a method of treating a plant with a botanical composition comprising a granular extract of Thyme leaves and an extract of Chelidonium majus in a concentration of 0.2% to 5%, the method further comprising drench treating the plant with said botanical composition in a concentration of 0.2% to 5%. In such a method, the drench may be applied in a single dose or in multiple doses over a period of time.

[0015] In a further aspect of the invention, there is provided a method of treating a plant with a botanical composition comprising a granular extract of thyme leaves and an extract of Chelidonium majus in a concentration of 0.2% to 5%. The method comprises applying the botanical composition in a concentration of 0.2% to 5% to the leaves of the plant. Applying the botanical composition may comprise dipping the leaves in the botanical composition. Applying the botanical composition may further comprise spraying the leaves with the botanical composition.

[0016] The botanical composition may be applied by foliar spraying or dipping in one dose or in multiple doses over a given period of time.

[0017] The features of the present invention which are believed to be novel are set forth with particularity in the appended claims.

[0018] The above and other objects, features and advantages of the present invention will become more readily apparent from the following description taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0019] [Figure 1A] 1A shows exemplary tomato plants, one of which is a control and the others are treated with different plant extract compositions according to the principles of the present invention, showing plant height. [Figure 1B] FIG. 1B is a diagram of the tomato plant of FIG. 1A showing chlorophyll content. [Diagram 2] FIG. 2 shows example lettuce plants grown under greenhouse conditions, where the first lettuce plant is a control and the other lettuce plants were treated with different plant extract compositions according to the principles of the present invention. [Diagram 3] FIG. 3 is a diagram showing the roots of the lettuce plant of FIG. [Figure 4A] FIG. 4A is an exemplary diagram of lettuce plants grown in individual pots, where the first lettuce plant is a control and the other lettuce plants were treated with different plant extract compositions according to the principles of the present invention. [Figure 4B] FIG. 4B shows the lettuce plant of FIG. 4A removed from the pot and without soil. [Figure 5A] FIG. 5A shows example tobacco plants grown in individual pots, with the first tobacco plant being a control and the others being treated with different plant extract compositions according to the principles of the present invention. [Figure 5B] FIG. 5B is a diagram of the tobacco plant of FIG. 4A shown removed from the pot and without soil. [Figure 6] FIG. 6 is an illustration of a standard operating procedure (SOP) for soybean germination in accordance with the principles of the present invention. [Figure 7]FIG. 7 shows exemplary cannabis buds with the same genetic characteristics, the cannabis bud on the left has been treated with a biostimulant composition of plant extract according to the principles of the present invention. [Figure 8A] FIG. 8A shows exemplary lettuce leaves infected with Botrytis plugs in a first test, where the first lettuce leaf is a control and the other lettuce leaves are treated with different plant extract compositions according to the principles of the present invention. [Figure 8B] FIG. 8B shows exemplary lettuce leaves infected with Botrytis plugs in a second trial, where the first lettuce leaf is a control and the others were treated with different plant extract compositions according to the principles of the present invention. [Figure 9A] FIG. 9A is a graph showing the Disease Index of healthy picked leaves irrigated with a water and plant extract composition according to the principles of the present invention and then infected with Botrytis cinerea, where the first leaf is a control and the other leaves are treated with different compositions including water and plant extract. [Figure 9B] FIG. 9B shows the picked leaf of FIG. 9A. [Figure 10] FIG 10A shows tomato plants drenched and treated with different plant extract compositions according to the principles of the present invention. FIG 10B shows a wet tent placed over the infected tomato plants of FIG 10A. FIG 10C is a graphical representation showing the disease index of unpicked leaves of the control plants shown in FIG 10A and tomato plants drenched and treated with different plant extract compositions. FIG 10D shows Botrytis cinerea on the leaves of the tomato plants shown in FIG 10A, where the plant on the left is a control and the plant on the right is treated with a composition comprising a 1% concentration of the plant extract. [Figure 11A] FIG. 11A shows tomato plants subjected to irrigation treatment, the first plant being a control and the others being treated with different concentrations of plant extract according to the principles of the present invention. [Figure 11B]FIG. 11B shows the disease index of the tomato plants of FIG. 11A that were subjected to irrigation treatment. [Figure 12A] FIG. 12A shows lettuce (left) and tomato (right) leaves, the top leaf being a control and the bottom leaf being soaked in a plant extract according to the principles of the present invention and treated with Botrytis cinerea. [Figure 12B] FIG. 12B shows the disease index of the lettuce and tomato leaves of FIG. 12A. [Figure 13] FIG. 13 illustrates exemplary phytotoxicity responses of plucked hop leaves treated with 1% and 2% concentrations of plant extract according to principles of the present invention, versus 1.5% bleach and water (control), showing leaves 48 hours after treatment. [Figure 14] FIG. 14 shows an example of inoculation of picked hop leaves with Botrytis cinerea, with the upper leaf treated with a 1% concentration of a plant extract according to the principles of the present invention and the lower leaf treated with water, approximately 48 hours after inoculation. [Figure 15] FIG. 15 is a diagram of an exemplary hops grown in a controlled growth chamber and treated with a botanical composition using an irrigation treatment method according to the principles of the present invention. [Figure 16A] FIG. 16A shows the percentage of necrotic lesions caused by Botrytis in hops treated by irrigation with a plant extract according to the principles of the present invention; the left portion is a software rendering of the disease severity using ImageJ software, and the right portion is the image before software rendering. [Figure 16B] FIG. 16B shows drench-treated hops treated with Botrytis cinerea using picked leaves, one leaf treated with water (control) and the others treated with different concentrations of plant extract according to the principles of the present invention. [Figure 17] FIG. 17 is a graph showing the percentage of infected hop leaf area and incidence in response to a control and different concentrations of a plant extract according to the principles of the present invention. [Figure 18] FIG. 18 shows an exemplary phytotoxicity response of cannabis plucked leaves treated with water treatment (T5) and a 1% concentration of plant extract according to the principles of the present invention, the leaves are shown 6 days after treatment. [Figure 19A] FIG. 19A illustrates a method for inoculating plucked leaves of cannabis treated with plant extract or water according to the principles of the present invention using powdery mildew (PM) infected leaves as inoculum. [Figure 19B] Figure 19B shows the leaf of Figure 19A infected with PM, 8 days after inoculation according to the principles of the present invention. Arrows indicate signs of infection. [Figure 20] FIG. 20 is a software rendering showing disease severity on cannabis leaves infected with PM and treated with a plant extract according to the principles of the present invention. [Figure 21] FIG. 21 shows a second study in which three cannabis strains were treated with a plant extract according to the principles of the invention using either a control or thymol. [Figure 22] FIG. 22 is a graph showing the percentage of infected area in cannabis plucked leaves treated with water (control) or a plant extract according to the principles of the present invention as a function of the species shown in FIG. [Diagram 23] FIG. 23 illustrates disease progression 39 DPI (days post inoculation) in different cannabis leaves treated with water (control) and 1% plant extract according to the principles of the present invention using thymol. [Figure 24] FIG. 24 is a diagram of a cannabis plant highly infected with powdery mildew. [Figure 25A] FIG. 25A is a diagram of Xanthomonas bacteria with plant extracts at concentrations of 1% to 5% according to the principles of the present invention. [Figure 25B] FIG. 25B shows Xanthomonas bacteria mixed with thyme extract (1:2 in 50% alcohol). [Figure 25C] FIG. 25C shows Xanthomonas bacteria mixed with yarrow extract (1:2 in 50% alcohol). [Figure 26A]FIG. 26A shows environmentally derived Salmonella (S22) treated with a plant extract according to the principles of the present invention together with thyme and yarrow. [Figure 26B] FIG. 26B shows environmentally derived Salmonella (S22) treated with a plant extract according to the principles of the present invention together with thyme and yarrow. [Figure 26C] FIG. 26C shows environmentally derived Salmonella (S22) treated with a plant extract according to the principles of the present invention together with thyme and yarrow. [Figure 27] FIG. 27 shows pathogenic Salmonella (S1) treated with a control and treated with thyme, yarrow extract and various concentrations of plant extracts according to the principles of the present invention. [Figure 28] FIG. 28 shows Salmonella (S31-mung bean sprout isolate) treated with thyme. [Figure 29] FIG. 29 shows Streptomyces scabies with thyme (top) and yarrow extract (bottom). [Diagram 30] FIG. 30 shows the growth of Fusarium graminearum after inoculation with different concentrations of a plant composition according to the principles of the present invention, the plate on the left is a PDA plate 24 hours after inoculation with Fusarium graminearum, and the plate on the right is a PDA plate 72 hours after inoculation with Fusarium graminearum. [Diagram 31] Figure 31 shows the growth of Fusarium graminearum in combination with thyme and yarrow extracts, the PDA plate on the left is 24 hours after inoculation with Fusarium graminearum and the PDA plate on the right is 72 hours after inoculation with Fusarium graminearum. T is thyme leaves and Y is yarrow extract, both T and Y have been developed and prepared by the applicant. T+ and Y+ are commercially available thyme and yarrow extracts, respectively. [Figure 32A]FIG. 32A shows antibacterial and MIC assays, some of which were control treated and some of which were treated with different concentrations of plant extracts according to the principles of the present invention. [Figure 32B] FIG. 32B shows antibacterial and MIC assays using mixtures of different concentrations of plant extracts and commercially available thymol according to the principles of the present invention. [Diagram 33] FIG. 33 shows an antifungal MIC assay for Aspergillus ochraceus using thymol or a mixture of a plant extract according to the principles of the present invention and commercially available thymol. [Diagram 34] FIG. 34 includes diagrams showing germination of Fusarium graminearum spores incubated for different times (24 hours to 48 hours) in different concentrations of plant extract solutions according to the principles of the present invention. [Diagram 35] FIG. 35 includes 20x magnification images showing germination of Fusarium graminearum spores incubated for different times in different concentrations of plant extract solutions according to the principles of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Novel methods and compositions of plant extracts for promoting plant growth and preventing and controlling plant diseases are described below. Although the present invention has been described with reference to certain exemplary embodiments, it should be understood that the embodiments described herein are by way of example only, and that the scope of the invention is not intended to be limited thereby.

[0021] A botanical composition or mixture of a granular extract of Thymus vulgaris and an extract of Chelidonium majus root, leaf or mixture thereof for promoting plant growth and preventing or suppressing plant diseases is provided. The botanical composition may be in different concentrations (w / v), such as, but not limited to, 0.2%, 0.5%, 1.0%, 1.5%, 2% and 5%. These concentrations are typically prepared in distilled water and filter sterilized. In this disclosure, unless otherwise stated, the term thymol generally refers to a thyme leaf extract diluted 1:2 with 50% alcohol tincture before being added to the botanical composition or mixture. Furthermore, as described for some experiments, an extract of yarrow flower was diluted 1:2 with 50% alcohol tincture before use.

[0022] As mentioned above, a botanical composition or mixture of a particulate extract of thyme (Thymus vulgaris) and an extract of Chelidonium majus root, leaf or a mixture thereof can be used as a biostimulant. The extract of the root, leaf or both can be combined with a specific amount of thyme.

[0023] The relative amounts of thyme, root and leaves used in the mixture may range from: Celandine root extract 0.1%~99%, Celandine leaf extract is 0.1%-99% and Thyme leaf granular extract 0.1%-30%

[0024] The extraction process for celandine can be carried out conventionally (10:1-70% ethanol) followed by spray drying or using cold press or freeze drying.

[0025] The extraction process of celandine may further be carried out using various bacteria, using either aerobic or anaerobic fermentation. One such example of bacteria used in the extraction process is lactic acid bacteria, also called LAB. Bacteria of the genus Bacillus may also be used in this process.

[0026] More thyme leaves and / or yarrow leaves, or any other parts of thyme or yarrow may further be added to the composition.

[0027] Example I: Growth effect of a plant composition of thyme and celandine on tomato plants grown in tissue culture:

[0028] In the first example, the roots of tomato seedlings were treated in tissue culture with plant compositions of different exemplary compositions (0.5%, 1% and 2%). Three weeks after treatment, the tomato plants were removed from the tissue culture tubes and the chlorophyll content and plant height were recorded.

[0029] The results show that after three weeks, the roots of tomato seedlings treated with the plant composition (0.5%, 1% and 2%) by tissue culture had higher chlorophyll content and were generally taller than the control.

[0030] Now, referring to FIG. 1A and FIG. 1B, exemplary tomato plants are shown, which were treated with different concentrations of the plant composition (0.0% (control), 0.5%, 1% and 2%). FIG. 1A shows the plant height, and FIG. 1B shows the chlorophyll content.

[0031] Table 1: Chlorophyll content (μg.cm-2) of control and plant composition treated tomato plants. Numbers in the table represent the average of two compound leaves per plant. Three separate measurements were recorded per leaf. [Table 1]

[0032] Example II - Effect of plant extracts on young plant growth under greenhouse conditions:

[0033] A study on plant extracts under greenhouse conditions in trays placed in a growth chamber:

[0034] For this exemplary experiment, lettuce seeds were planted and germinated in a soil mixture such as Agro mix® G6 (Fafard) using 200 cell trays. After 2 weeks of growth, the lettuce plants were either treated with watering only (as a control) in the rhizosphere (approximately 1 ml / plant) or with a 1% concentration of plant extract. Each treatment was replicated 7 times.

[0035] Referring to Figures 2 and 3, lettuce plants are shown after three weeks of growth under greenhouse conditions. From left to right, the lettuce plants are control, treated with 0.5%, 1% and 2% concentrations, respectively. Figure 2 shows the lettuce in a pot, and Figure 3 shows the lettuce plant pulled from the soil with roots emerging.

[0036] Figures 2 and 3 show that after 3 weeks of growth, lettuce plants treated with the plant extract were healthier and taller than the control (watered only). The combined administration of the plant extract and yarrow at a concentration of 0.5% showed healthy plant characteristics including, but not limited to, good plant height, green leaves and strong stems (data not shown). Lettuce plants treated with the plant extract at a concentration of 1% or 2% combined with thyme showed plant characteristics similar to those of the control (data not shown). [Table 2]

[0037] Referring to Table 2, the highest chlorophyll content was found in lettuce plants treated with 1% concentration of the plant composition, with an average of 32.2 μg.cm−2. [Table 3]

[0038] Referring now to Table 3, the 1.0% concentration of plant extract produced the most fresh mass (FW) at 5.75 g. As shown in Table 4 below, regardless of treatment, total root length was slightly greater in the 1% concentration of plant composition versus the control treatment. [Table 4]

[0039] In the context of this example, the plant composition at different concentrations showed a promoting effect on plants (i.e., lettuce and tobacco) and increased their growth parameters. The plant composition was an amendment to Agro mix® G6 (Fafard) and had a positive promoting effect on plants and increased their growth parameters. The components and molecules of the plant composition may interact positively with the components of soil formulations, such as, but not limited to, the universal soil formulation Agro mix® G6 (Fafard). It may be further advantageous to use soil formulations containing coconut shell fiber, fibrous peat, perlite, limestone, gypsum and / or mycorrhizae to aid in wetting, root growth and mineral retention. Amending a commercial soil formulation, such as Agro mix®, with the plant composition may help produce healthier plants, as shown in lettuce, and may also show similar results in other plants.

[0040] Experiment B on the effect of plant compositions on lettuce and tobacco grown in pots under greenhouse conditions:

[0041] In other experiments, 200 cell trays were used, and tobacco and lettuce seeds were sown and germinated in a first soil formulation (e.g., Agro mix® S4). After one week of growth, the seedlings were transplanted into pots (6 inches) containing a second soil formulation (e.g., commercial AGRO mix® G6 (Fafard) amended with 14-14-14 TYPE 70 Nutricoat NPK). The soil formulations used generally have high porosity, which provides excellent drainage and gas diffusion due to the specific peat composition of the soil formulation. The transplanted seedlings were either watered (as a control) or treated with one of the plant compositions at a concentration of 1%. Each pot received three irrigation (10 ml / pot) treatments at intervals of 5 days. After 4 weeks, the plants were harvested. In such experiments, all treatments were replicated 7 times.

[0042] Now referring to Figures 4A and 4B, exemplary lettuce plants grown in individual pots in this experiment are shown in the pot and removed from the pot with the soil washed off. The leaves of the lettuce plants treated with only the 1% concentration of the plant composition were greener, stronger, and showed no signs of nutrient deficiency compared to the control (which contained only the second soil formulation (Agro mix® G6)). The fresh and dry masses of the lettuce plants treated with the 1% concentration were substantially higher than the control treated plants, as shown in Figures 4A and 4B and Table 5 below. [Table 5]

[0043] Interestingly, the chlorophyll content of the lettuce plants treated with the 1% concentration was similar to that of the control treated lettuce plants, as shown in Table 6 below. [Table 6]

[0044] From this experiment, it can be said that the plant composition at different concentrations showed a promoting effect on plants (i.e. lettuce) grown under greenhouse conditions, increasing the growth parameters of such plants. The amendment of the first soil formulation, such as Agro mix® G6 (Fafard), with the plant composition further had a positive promoting effect on the plants, increasing the growth parameters of the plants. As a result, the components and molecules of the plant composition may positively interact with the components of the first soil formulation, such as Agro mix® G6 (Fafard).

[0045] Referring to Table 5, plants treated with the 1% botanical composition had higher fresh mass, dry mass and height compared to the control treated plants. In summary, lettuce plants treated with the 1% botanical composition had an 8% increase in dry mass, a 10% increase in fresh mass and a 9.6% increase in height compared to the control treated lettuce plants.

[0046] Furthermore, it can be observed that there is no discernible difference in chlorophyll content between plants treated with 1.0% plant composition and the control.

[0047] 5A and 5B, exemplary tobacco plants grown in individual pots are shown. Referring to FIG. 5A, tobacco plants grown in individual pots are shown, and referring to FIG. 5B, the same plants are shown removed from the pots and the soil has been washed off. The tobacco plants on the left in FIG. 5A and 5B are treated with a control (soil formulation only), and the plants on the right are treated with a 1.0% concentration of the plant composition. The tobacco plants grown in pots as controls using only soil formulation (e.g., Agro mix® G6) have normal appearance in terms of height, green color, and leaf size, but the lower leaves show signs of nutrient deficiency. The tobacco plants treated with the 1.0% concentration of the plant composition were healthier and had better growth characteristics, such as taller, greener, and much less faded leaves. The tobacco plants treated with the 1% composition had higher fresh mass, dry mass, and height than the control treated plants. [Table 7]

[0048] In summary with reference to Table 7, compared to the control, there was an increase of 31% in dry mass, 28% in fresh mass, and 9.6% in plant height. [Table 8]

[0049] Referring to Table 8, the highest chlorophyll content was recorded in the 1% botanical composition treatment, with an increase of 5.42% compared to the control.

[0050] In conclusion, when grown under greenhouse conditions, treatment with the plant composition at different concentrations showed a promoting effect on plants such as, but not limited to, tobacco, promoting the growth parameters of the plants. When the plant composition was combined with a soil formulation such as Agro mix® G6 (Fafard), the promoting effect on the plants was further increased, and such compositions promoted the growth parameters of the plants. The components and molecules of the plant composition may positively interact with the components of the soil formulation such as Agro mix® G6 (Fafard).

[0051] III- Experiments on the germination and early growth of soybeans using plant compositions and plant compositions mixed with Stimula-Gro

[0052] In this exemplary experiment, SOP methodology was used to evaluate the effect of different herbal treatment products, such as but not limited to, a botanical composition containing C7 (Celandine) extract and Stimulagro: A. (ST) nodosum extract. Stimulagro is a composition made primarily from algae. The herbal products were used alone and in combination on the germination and early growth of soybeans and compared to a control treatment of water. The experiment further included measuring physiological characteristics such as but not limited to growth characteristics, biomass, chlorophyll content, and gas exchange parameters.

[0053] The experiment further included the application of the SOP. The experiment included the use of the roll towel test according to ISTA (1985) with some modifications. In the ISTA method, dry seeds and moistened paper were used. The first modification was to soak the seeds overnight (20-24 hours) in double distilled water and to use pre-folded dry paper. This reduced the germination time lag and also allowed the seeds to adhere to the paper and make it easier to roll up the paper. The test was performed on a piece of paper (i.e. Classique Kraft brown, Servicorp, QC, Canada) cut from a roll of paper, e.g. 60 x 20 cm, folded along its length (e.g. into a 60 x 10 cm piece). Seeds were placed in a row 5 cm apart, 1 cm away from the top edge. The paper was folded again lengthwise to cover the seeds, fixing their position. The seed-containing paper rectangle is rolled along its short axis and secured with glue or tape (e.g., Scotch Magic Tape TM The seedlings were fixed with tape 3 / 4" x 1296", MN, USA). The paper rolls were placed vertically, with the seeds facing up, in plastic containers (e.g., 1 L containers) containing a portion of the respective treatment solution (e.g., 40 ml in this case). The containers were kept in a growth chamber (e.g., Conviron® Environmental Chamber, Winnipeg, Canada) set under the prescribed conditions (e.g., 25±2°C temperature, 16:8 h light:dark cycle, cool white and incandescent light with a flux density of 400 μM m-2 s-1, 50% relative humidity). The paper towels were never allowed to dry. Every morning, the remaining solution was discarded and the same proportion (i.e., 40 ml) of fresh treatment solution was added. Germination was observed daily. 15-day-old seedlings were used to measure growth (shoot length, shoot live and dry mass), chlorophyll content, and photosynthetic rate.

[0054] Photosynthesis measurements: Photosynthetic rates (e.g., μmol m-2 s-1 CO2) were measured using a handheld infrared gas analyzer (e.g., IRGA-LI-6400, LI-COR Inc., Lincoln, NB, USA). Measurements were taken on separate areas of soybean plant leaves, such as fully expanded primary leaves for seed treatments and fully expanded third true leaves for foliar sprays. In these experiments, the infrared gas analyzer (i.e., IRGA) was calibrated and zeroed approximately every 30 min during the measurement period.

[0055] Chlorophyll content measurements: Leaf chlorophyll content of fully expanded leaves was estimated by averaging 10 measurements per treatment using a SPAD 502 meter (e.g., Konica Minolta Optics, Inc., New Jersey, USA). Results are expressed in Soil Crop Analysis Development (SPAD) units.

[0056] Germination, growth characteristics and biomass measurement: Germination was observed daily. Plant height was measured from the base node to the apical bud using a ruler. In an exemplary measurement process, the fresh mass (FM) and dry mass (DM) of buds were collected from three randomly selected plants / rolls (six plants / replicate) and weighed on an analytical balance such as a Highland® balance, Adam Equipment Inc., Oxford, CT, USA. Alternatively, buds were harvested directly on a paper towel for FM, then dried for 72 hours at, for example, 60°C and weighed for DM.

[0057] Experimental methods and statistical analysis: The experimental method was a completely randomized design (CRD). In such an exemplary method, the CRD was run five times with 20 seeds each, two rolls per container, and 10 seeds per roll. Data are presented as mean ± standard error of the mean (SEM) and analysis of variance (e.g., 1-way ANOVA) followed by post-hoc Newman-Keuls multiple comparison tests, for example, using software from GraphPad Prism version 5.01, 2007, Graf Pad Software, Inc., CA, USA. In this experimental method, the significance level was *P < 0.05.

[0058] Treatment of soybean seeds with 1% plant composition alone resulted in a statistically significant increase in dry mass, e.g., 12.9%. Furthermore, the experimental results showed that soybean seed treatment with both the plant composition and Stimulagro in combination generally resulted in a positive enhancement in the physiological characteristics of the treated soybeans. Among other treatment methods, treatment with 1% plant composition in combination with Stimulagro was observed as the most beneficial treatment method. This treatment method significantly (P<0.05) increased the plant height (i.e., 13.2%), dry mass (i.e., 10.7%), and photosynthetic rate (i.e., 20.3%) of soybean seedlings (in this case, 2 weeks old) compared to the water control (see exemplary results in Table 9). [Table 9]

[0059] As seen in Table 9, the experimental results clearly show that the physiological characteristics, growth parameters and photosynthetic rate of soybean are enhanced when treated with the botanical composition in combination with Stimula-Gro.

[0060] The results further highlighted the discriminatory value of SOP-Soybean and especially the potential of treatment with herbal extracts as growth stimulants for soybeans, as shown in Figure 6. SOP is used for soybean germination and growth to analyze the effect of the botanical composition and the combination of the botanical composition with Stimula-Gro on soybean germination and early growth. Also shown in Figure 6 is a modified version of the roll paper towel test developed by the International Seed Testing Association (ISTA, 1985).

[0061] The protocol followed (SOP-Soy) is summarized as follows: · SOP developed for the analysis of the effect of herbal extracts on germination and early growth of soybean (SOP-Soybean). It is a modification of the roll paper towel test developed by the International Seed Testing Association (ISTA, 1985).

[0062] Referring to FIG. 6, the protocol includes the following: a) Prior to performing the assay, soybean seeds are soaked for a predetermined period of time, for example overnight (20 hours). b) Fold an absorbent material such as a paper towel (ie, 60 cm x 20 cm) in half along its length. c) A pre-soaked number of seeds (i.e. 10) are placed in a row 5cm apart, 1cm apart from the top and leaving 5cm at each end. d) The seeds are held in place by pre-placing pre-folded paper over the seeds. e) Fold the absorbent material again, for example 1 cm, to strengthen the base of the roll. f) The dried paper containing the seeds is quickly rolled lengthwise and secured with tape. g) Place the roll of paper vertically into a transparent container containing a predetermined amount of processing solution (e.g. 40 ml). h) The container is stored in a growth chamber under the specified conditions (e.g., maintained at 25 ± 2°C, 16:8 h day / night cycle, and flow density of 400 μMm m-2 s-1). i) Provide a predetermined amount (eg 40 ml) of fresh treatment solution daily.

[0063] In general, the protocol aims to provide the following benefits: An efficient method to test the effect of potential growth stimulants on seed germination and early growth. Rapid testing (i.e., 2-week duration) and space efficient. · It is repeatable; carried out under defined growth conditions. A relatively simple and very low cost method that can be carried out in industry with minimally trained staff and materials that are readily available.

[0064] IV. Experiments to evaluate the effect of plant extracts on yield improvement in potted cannabis The experiment was conducted with a 'Candyland' hybrid cultivar, with sufficient material to conduct pilot trials by licensed and diligent growers to identify or investigate biostimulatory properties and effects on flowering and yield.

[0065] The experiment involved testing cannabis plants with plant extracts at concentrations between 0.03 and 2% to evaluate their effect on bud size and yield. The same lines with the same genetic characteristics were used in the tests. The total number of plants tested was 120. A total of 60 plants were used for treatment with the plant composition and 60 plants were used for the untreated control experimental group. The treated plants were compared to control plants, both of which were grown hydroponically under conventional home greenhouse conditions. Either foliar spray, root drench, or a combination of foliar spray and drench treatments were applied to the plants.

[0066] To test the biostimulatory effects, various treatments with the plant composition ranging from 0.03 to 2% were used (e.g., root drench alone or root drench combined with foliar sprays for a total of 4 to 30 applications). These treatments resulted in healthier crops and substantially increased the yield and size of cannabis flowers, as shown in Figure 7. Figure 7 shows, on the left, the results of buds of cannabis (Cannabis sativa) plants treated with 0.03 to 2% of the plant composition in addition to conventional fertilization and management practices. On the right side of Figure 7, the results of buds of cannabis plants that received only conventional fertilization and management practices are shown. In this experiment, the total yield was improved by 25% in plants treated with the plant extract when compared to the control treated plants. The same plant line with the same genetic characteristics was used to test the control and treatment groups.

[0067] Botanical compositions as biofungicides According to another embodiment, the botanical composition may further be used as a biofungal agent and may help combat or stop the spread of plant diseases.

[0068] I. Foliar treatment of lettuce leaves with botanical compositions alone or in combination with Yarrow to treat or suppress Botrytis cinerea

[0069] In the first test, Botrytis cinerea plugs (5 mm) were placed on plucked leaves of treated plants (one treatment with the botanical composition or one treatment with a mixture of the botanical composition and yarrow (Y) and control (water)). The leaves were held in trays (such as Pyrex trays) lined with moist filter paper. Treated leaves were observed daily. The infection results of the lettuce leaves were recorded after 72 hours.

[0070] Referring now to Figure 8 and Table 10, two test results are shown, one on the left and one on the right, for lettuce leaves infected with Botrytis plugs and treated as described above. The diameter of the lettuce leaf necrotic lesions was larger on the control treated plants and smaller on the plants treated with the botanical composition alone. [Table 10]

[0071] II - Greenhouse trials to evaluate the efficacy of plant extracts in suppressing Botrytis cinerea on tomato and lettuce

[0072] In an exemplary test, organic tomato and lettuce seeds were germinated in a soil mix (e.g., Agro mix® S4 (Fafard)) in a given number (e.g., 20) of cell trays. After two weeks of growth, the seedlings were transplanted into pots (e.g., 6-inch diameter pots) containing another soil mix (e.g., AGRO mix® G6 (Fafard) amended with 14-14-14 TYPE 70 Nutricoat NPK). The pots were placed under standard greenhouse growing conditions in a randomized block design (RBD) and irrigated with an automated system. All tests were replicated twice. Appropriate data from replicated tests was collected.

[0073] To determine whether the botanical composition could suppress the disease, 1% plant extract was applied in two different ways: drench treatment and leaf dipping.

[0074] Irrigation Treatment In the first treatment, 4-week-old potted plants were given 10 ml or 20 ml of 1% plant composition per drench treatment, which was repeated for example every 24 hours for a period of 96 hours. The control treatment was given water instead. A total of eight treatments were given in different doses in the soil near the plants, as shown in Table 11. 24 hours after the plant composition treatment, the removed leaves and the leaves still attached to the plants were challenged with Botrytis cinerea. Additionally, different concentrations of the plant composition were given by drench treatment to investigate its role in disease suppression during gray mold development.

[0075] Inoculation for diseases: Mycelium plugs from freshly grown Botrytis fungi were placed on uniform leaves of plants that had been treated with the plant composition or water by irrigation. As seen in Figure 9B and Figure 10, the infected leaves were covered with moist plastic bags and a humidity tent was placed on top. The plants were placed in separate humid growth chambers and infections were recorded after 72 hours.

[0076] Disease measurement: Disease index was recorded 72 h after inoculation for plucked and unplucked leaves. Disease index was measured as the ratio of necrotic lesion area to healthy tissue area using rendering software such as ImageJ, following the guidance of Haliem (2012), Steward and Macdonald (2014), and recorded as a percentage. All treatments were replicated 10 times, and the experiment was repeated twice. Collected data were averaged, and differences between treatments were analyzed using JMP11 (SAS-one-way ANOVA, Tukey HSD, α 0.05) to indicate significance between treatments.

[0077] Picked leaves: Plants that received multiple 10 ml applications of 1% plant composition or a single 20 ml application (see, for example, T1 to T5 in Table 11 below) successfully reduced the lesion area compared to the control treatment. Figure 9A shows the disease index of healthy, plucked leaves that were drench-treated with water (control) or 10 to 40 ml of plant composition and then infected with Botrytis cinerea. Values ​​in Figure 9A are averages for 10 leaves per treatment. Additionally, Figure 9B shows the necrotic lesions of the control and plants treated with various amounts of plant composition. As shown in Figures 9A and 9B, a single 10 ml application of 1% plant composition (see treatment T1 in Table 11) significantly (P<0.05) reduced necrotic lesions by 84%, with an average of 2% necrotic lesion area, compared to the control treatment, which had necrotic lesion area of ​​more than 15% of the leaf tissue. Additionally, multiple doses of 10 ml of the 1% botanical composition (treatments T2, T3, T4 in Table 11) were significantly effective in reducing lesion area compared to the control without being significantly different from each other.

[0078] Non-picked leaves: Infection of intact leaves of drench-treated plants was significantly reduced as a result of drench treatment with the botanical composition shown in FIG. 10. It was also more effective as compared to in vitro plucked leaves as shown in FIG. 9A and FIG. 9B. Referring to FIG. 10, the drench treatment method and the results of the method are shown. In step A, the drench treatment is applied to tomato plants in pots. In step B, a wet tent is placed on the infected tomato leaves. In step C, a disease index is generated showing the number of infected and intact leaves of the control and tomato plants treated with botanical compositions ranging from 10 ml to 40 ml. In step D, Botrytis cinerea on the leaves of the control and tomato plants treated with 1% botanical composition is shown. The disease index was reduced by almost 90% as compared to the control treatment. The disease index was significantly lower for all four treatments, i.e., T1 to T4, shown in Table 11. [Table 11]

[0079] Drench treatment of the plant composition at 1% and 2% concentrations showed effective suppression of gray mold on tomatoes during disease progression. The plant composition at 1% and 2% concentrations was found to be significantly more effective in suppressing disease on tomato leaves compared to the control plants. Furthermore, the plant composition at 1% was determined to be more effective than the plant composition at 2% concentration. Now referring to FIG. 11A, tomato plants are treated with the plant composition by drench treatment. From left to right, the plant composition is treated with the control, the plant composition at 1.0% concentration, and the plant composition at 2.0% concentration. In FIG. 11B, the disease index of the tomato plants treated with the plant composition by drench treatment is shown.

[0080] In another embodiment, the addition of the botanical composition to a soil formulation such as Agro mix® G6 (Fafard) allows tomato plants to acquire resistance against Botrytis cinerea, the causative agent of gray mold disease. The components and molecules of the botanical composition may interact positively with the components of a soil formulation such as Agro mix® G6 (Fafard). As a result, resistance to the disease can be induced in plants such as tomato plants. Thus, the amendment of Agro mix® G6 (Fafard) with the botanical composition suppressed gray mold disease in tomato plants.

[0081] Leaf soaking method: The second treatment method involves picking uniformly sized tomato and lettuce leaves and immersing the picked leaves in 1% botanical composition for a predetermined time (e.g., 30 seconds). The method further involves placing the immersed leaves on a plate (e.g., a Pyrex plate) lined with moist filter paper. The method further involves immersing control leaves in water. The method further involves placing Botrytis cinerea plugs of actively growing cultures on the plucked leaves from the control and botanical composition treated plants. The plates are sealed using wrapping means (i.e., Saran Wrap) as shown in FIG. 12A and incubated at room temperature. Disease index was recorded after 72 hours of incubation.

[0082] Referring to FIG. 12A, a lettuce leaf is shown on the left and a tomato leaf is shown on the right. In this exemplary experiment, the bottom row was either briefly dipped or submerged in 1% botanical composition and dosed with Botrytis cinerea plugs, while the top row was dipped in a water-based control. Referring now to FIG. 12B, the disease index is shown graphically. In these examples, the disease index was calculated based on 10 biological replicates. Significant disease reduction was observed with the botanical composition treatments, with a 33% reduction in lettuce and up to 45% reduction in tomato, compared to the control treatment.

[0083] III. Efficacy of botanical compositions of plant extracts in suppressing fungal diseases on cannabis and hops grown under greenhouse production systems

[0084] 1. Biological activity of plant extract compositions in hops (Humulus lupulus): Phytotoxicity tests on plucked hop leaves after foliar application: In yet another exemplary experiment, Willamette hop rootstocks were obtained. The experimental method included transplanting rootstock segments into pots containing a soil mix. In one example, the pots were 6 inches and the soil mix was Agro mix® G6 (Fafard). The experimental method included transplanting the rootstock segments under predetermined conditions (e.g., 12 / 12 hour day / night, 23 / 21°C day / night temperature, 210 photon μm- 2 s- 1 This involves placing the plants in a growth chamber under constant temperature (at 65° C. and humidity level maintained at 65% throughout the day).

[0085] The experimental method further includes plucking hop leaves from two-month-old plants and placing the plucking leaves on a plate (e.g., a Pyrex plate) lined with moist filter paper. Referring now to FIG. 13, the method further includes immersing the leaves in different concentrations of the botanical composition (e.g., 3 ml of the botanical composition). In such experiments, three leaves from three separate plants were used for each treatment and incubated at room temperature. Control treatments included 1.5% bleach as a positive control and water as a negative control.

[0086] Phytotoxicity occurs when plants are exposed to a toxic external agent and may manifest as necrosis, browning, yellowing (chlorosis) of the leaf margins, or yellow, brown, or black spots (see, for example, Kristin Getter, Michigan State University Extension, Department of Horticulture). If the treated leaves showed any of the above symptoms compared to the water treatment, the plant composition at that concentration was considered to be phytotoxic.

[0087] Referring again to Figure 13, the phytotoxicity response of picked hop leaves treated with the plant extracts is shown two days after treatment. The leaves were immersed in either a given volume (i.e., 3 ml) of two different concentrations of the plant composition or the same given volume (i.e., 3 ml) of bleach or water (control). The experiment further included coating the picked leaves with 1% and 2% treatments of the plant composition. The coating did not produce phytotoxicity on the hops. Thus, foliar applications of the plant composition from 0.2% to 2% are considered safe when used commercially under greenhouse growing conditions.

[0088] Fungicidal activity of botanical compositions against gray mold after foliar application: In yet another experiment, two-month-old leaves were picked from hop plants, immersed in 3 ml of treatment solution (various concentrations of plant composition) each, and placed in Pyrex plates lined with moist filter paper for 24 hours. Three leaves per treatment. As shown in Figure 14, agar plugs containing colonies of Botrytis cinerea that had been actively growing for one week were inoculated into the center of the leaves, and the plates were incubated at room temperature for 48 hours. In the control treatment, leaves were treated with water only. Also in Figure 14, the upper leaves were treated with 1% concentration of plant composition, and the lower leaves were treated with water, along with Botrytis cinerea. It can be observed that a single foliar application of 1% of the plant composition resulted in disease suppression of gray mold on hops.

[0089] Phytotoxicity testing of hop leaf samples following drench application: The purpose of this experiment was to test the phytotoxic effect of the botanical composition on potted plants, i.e. hops grown in Agromix G6 (Fafard), and its leaves, when irrigated once or divided into several times over a given period of time, in this experiment the duration of such experiment was 3 consecutive days.

[0090] Two-month-old hop plants were treated with either water alone or a 1% concentration botanical composition, as shown in Figure 15. The required amount of botanical material was applied by pipetting 3 cm deep around the crown region of each plant, as shown in Table 12. Two potted plants were treated for each treatment. A total of six leaves per treatment were observed for phytotoxic symptoms 24 hours after treatment. [Table 12]

[0091] The botanical composition at a concentration of 1% did not cause any phytotoxicity in hops when applied in single or multiple applications (see treatments T4-T6 in Table 12). In fact, split applications of the botanical material generally contributed to no detrimental effects on the plants.

[0092] Fungicidal activity against gray mold in plant compositions after drench treatment: In other experiments, the experiment involves administering the fungal pathogen Botrytis cinerea to treated hop plants in vitro.

[0093] In such an exemplary experiment, six uniformly sized leaves were picked from treated and control plants grown in a soil formulation such as Agromix G6 (Fafard). The experiment further included inoculating the center of the picked leaf with an agar plug (i.e., a 5 mm plug) containing Botrytis cinerea. The experiment further included placing the infected leaves in a tray with a moist filter paper on the bottom as shown in FIG. 16A. The placed infected leaves are then incubated at room temperature for a specific period of time (e.g., 5 days). The experiment further included establishing a control leaf. The control treatment included treating the leaves with water only. The experiment may further include assessing disease severity using a computer program. In such experiments, the software ImageJ was used to score or map the severity as shown in FIG. 16A (Haliem, 2012; Steward and Macdonald, 2014). In these experiments, disease severity was defined as the percentage of infected leaf area to total leaf area.

[0094] statistical analysis Statistical analysis of the results may include averaging the data and analyzing differences between treatments and controls by two-way analysis of variance (ANOVA) and, where appropriate, least significant difference (LSD) at P<0.05 using the SPSS statistical package v.22.0 (IBM Corp., Armonk, NY, USA).

[0095] Plants irrigated with water only (control treatment) were compared to plants irrigated with 1% plant composition. Treatment included three doses on three consecutive days (T6, day 1=20, day 2=30, day 3=20). The comparison showed a significant reduction in disease incidence and disease severity of 50% and 31%, respectively, as seen in Figure 16B, Figure 17, and Table 13 below. Referring now to Figure 17, the percentage of infected area on plucked hop leaves for control and treated plants is shown. Disease severity was calculated for infected leaves only (infected leaf area / total leaf area). Disease incidence was calculated for six leaves (number of infected leaves / total number of leaves). "*" indicates significant difference between treatments and water control by least significant difference (LSD) (P<0.05) test. [Table 13]

[0096] 1. Biological activity of plant composition extracts from Cannabis:

[0097] In other embodiments, different exemplary concentrations of botanical matter were used on Cannabis seedlings.

[0098] Phytotoxicity testing of hop leaves following foliar sprays: In another exemplary experiment, the experiment includes picking cannabis leaves from plants that have been grown for a certain period of time (e.g., 4 months). The experiment further includes treating the picked leaves by immersing the leaves (i.e., 6 leaves per treatment) in the plant composition at the following concentrations (T3: 1%, T5: water), as shown in Figure 18. The experiment further includes placing the leaves on a plate (e.g., a Pyrex plate) lined with moist filter paper and incubating at room temperature. Referring to Figure 16, the leaves are shown 6 days after the above-mentioned treatment.

[0099] Based on this experiment, a single application of the botanical composition (T3) did not produce any phytotoxic effects on cannabis.

[0100] Study I: Efficacy of plant extracts in preventing powdery mildew in cannabis (foliar application) Test I involves the use of harvested cannabis leaves. Leaves previously soaked in the different treatments (T3 and T5) were used (see above). Powdery mildew (PM) inoculum was prepared by inoculating leaf pieces (e.g. 1 cm2) uniformly infected with PM conidia. 2 The test included plates of 100 x 100 mm (leaf pieces of 100 x 100 mm) of 100 x 100 mm plant material (T3) or 100 x 100 mm plant material (T5)). Each leaf piece was placed in the center of a leaf that had been treated with either the botanical composition (T3) or water (T5) as shown in Figure 19A. The test further included incubating the plates at room temperature and observing disease progression over time (e.g., 8 days after inoculation). The test further included evaluating disease severity and treatment efficacy by comparing with the results of the control treatment T5, which was water only.

[0101] Preliminary results of Trial I on cannabis demonstrated that 1% plant composition alone (T3) inhibited the disease progression of powdery mildew (PM) and kept the leaves green and healthy. Signs of infection were only observed in the water-treated leaves in Figures 19A and 19B. Figure 19B shows the leaves after inoculation (8 days) with the arrows indicating the signs or symptoms of infection. As Trial I was a preliminary screening experiment, another trial, Trial II, was conducted to confirm the results and to extend the experiment to include different cannabis varieties that exhibit different levels of susceptibility to powdery mildew.

[0102] Study II: Efficacy of plant extracts (foliar application) in preventing powdery mildew in three cannabis varieties This test II was carried out using two biological replicates with plucked leaves of three cannabis cultivars known to exhibit different susceptibility to powdery mildew (PM). The cultivars were cultivar I (susceptible), cultivar II (susceptible) and cultivar III (highly susceptible). Each cultivar was treated with an application of 1% of the plant composition in combination with thymol and compared to a control treatment containing water only. Test II involved plucking leaves of each cultivar and immersing the plucked leaves in either 1% of the plant composition in combination with thymol or in water (control). In this test II, each treatment was carried out on five cannabis leaves and the experiment was repeated twice. Test II was further carried out by cutting infected leaves (e.g. 1 cm 2 ) onto the leaves. Each section contains powdery mildew as previously described in Test I. Test II uses twice as many leaf sections instead of the single leaf section used in Test I. Test II further involves incubating the plates at room temperature and observing disease progression over time.

[0103] Test II involves recording or collecting disease severity measurements during a given period (e.g., 29 days post infection (DPI)). Disease severity was scored using a computer program (e.g., software ImageJ) as shown in FIG. 20. Disease severity is defined as the percentage of infected leaf area relative to total leaf area. In this test II, disease incidence was recorded or collected at 12, 18, 26, and 29 DPI. Analysis further involves averaging the collected data and analyzing the differences between treatments and controls. An exemplary analysis uses a two-way analysis of variance (ANOVA) and, where necessary, least significant difference (LSD) at P<0.05 using the SPSS statistical package v.22.0 (IBM Corp., Armonk, NY, USA).

[0104] Test II is believed to have established the presence and progression of PM disease. Test II further established that the presence and progression of this disease was due to the presence of the two inocula and the longer incubation time, as they likely aided in effective disease spread and successful infection, as shown in Figure 21.

[0105] Referring to Table 14, the percentage of disease incidence at 12 DPI was higher in leaves treated with water (60-80%) compared to leaves treated with a combination application of 1% botanical composition and thymol (20%). At 26 DPI and above, most leaves showed disease symptoms, except for cultivar Sachigo, where the incidence reached nearly 100% in both the water control and botanical material treatments. Disease severity was always lower in leaves treated with 1% concentration of botanical composition in combination with thymol. [Table 14]

[0106] Referring now to Figure 22, the percentage area of ​​infected harvested cannabis leaves in water-treated control (cont) and treated plants with 1% plant composition in combination with thymol (Trt) is shown. Disease severity was calculated by dividing infected leaf area by total leaf area. The term "*" indicates a significant difference between treatment and water control using the least significant difference (LSD) test (P<0.05).

[0107] Now referring to Figure 23, PM infection at 39 DPI appears to be limited to limited necrotic areas in leaves treated with a combined application of 1% botanical composition and thymol, compared to symptoms that cover most of the leaves in the water control. Still referring to Figure 23, the circle shown indicates fungal containment in leaves treated with a 1% concentration of botanical composition in combination with thymol, while the black arrow indicates yellowing in water-treated leaves, meaning that the fungus is still viable. As shown, the grey arrow indicates PM symptoms.

[0108] IV. Pilot Study to Evaluate the Efficacy of Plant Extracts in Suppressing Powdery Mildew in Potted Cannabis Yet another pilot study involved using the "Candyland" hybrid cultivar. Referring to Figure 24, powdery mildew was present at high intensity on most plants.

[0109] The pilot experiment involves testing several concentrations of the extract of the botanical composition (e.g., 0.03-2%) on cannabis plants to evaluate their powdery mildew preventive or curative abilities / properties. The same lines with the same genetic characteristics were used for the tests. In such an exemplary pilot experiment for powdery mildew preventive management, the total number of plants tested was 120. The experiment involves placing the plants to be tested in a room filled with powdery mildew infected plants (inoculum). The exemplary test involves a first group including a total of 60 plants treated with the botanical composition and a second group including 60 plants treated with a control. The experiment further involves testing a total of 20 plants infected with powdery mildew to measure disease elimination (curative treatment). The experiment further involves a treatment group, which includes 10 plants treated with various concentrations of the botanical composition (generally ranging from 0.2% to 2%). The experiment further involves a control group. A control group included 10 plants that were treated with water only. Treatment and control plants were always compared, and both groups of plants were grown hydroponically under conventional home greenhouse conditions. Both groups of plants were treated with either foliar spray, root irrigation, or a combination of foliar spray and irrigation treatments.

[0110] When used in disease control situations, application of the botanical composition via foliar, root drench, or a combination of both proved to be effective in completely eliminating powdery mildew from diseased plants. Concentrations ranging from 0.03-2% were found to be sufficient to eliminate disease from treated plants. Plants that were not given the botanical composition showed a high degree of disease. It is also worth mentioning that healthy plants administered the botanical ingredient did not show or develop any disease symptoms after treatment. Furthermore, two applications of the botanical composition in the range of concentrations 0.03-2% may be even more suitable to eliminate powdery mildew from diseased plants. These results are consistent with previous tests conducted on cannabis and further support the fungicidal properties of the product.

[0111] V. Evaluation of the antibacterial properties of combinations of botanical compositions with thymol for inhibiting the growth of various plant and foodborne pathogens In yet another experimental method, various concentrations of the botanical composition, thyme leaf extract, yarrow extract, thymol, and combinations of the botanical composition with the above were used and tested for their antibacterial properties.

[0112] Preparation of plant extracts:

[0113] Single-dose preparation: A method for preparing a single application of a plant extract of a botanical composition is provided. The method for preparing the single application includes dissolving the botanical composition in water and serially diluting to obtain 0.5%, 1.0%, and 2.0% (w / v) solutions. The method may further include serially diluting to 5% (w / v).

[0114] The method further includes preparing thymol at concentrations of 1:256, 1:512, and 1:768.

[0115] The method also includes preparing a thyme leaf extract and a yarrow extract in the following ratio: 1:2 in 50% alcohol.

[0116] Preparation of the combined dose: Methods for preparing combined applications of plant extracts of botanical compositions are also provided.

[0117] The method involves diluting each of the botanical compositions and mixing the diluted botanical compositions with a particular concentration of thymol.

[0118] The method further includes using water and PDB as negative controls, and ethanol as a positive control.

[0119] MIC determination: The minimum inhibitory concentration (MIC) of the plant products was determined for cultured pathogens.

[0120] Burkholder's Assay: Referring to Figures 1A-5B, to perform bioassays with plant extracts of botanical compositions, thymol, thyme leaf extract and yarrow leaf extract, a given amount (e.g. 10 μl) of each concentration of dissolved extract was dropped onto the overlay of bacteria / fungi. As a negative control, a drop of water of the same given amount was used. Growth inhibition in the form of a clear zone was observed after a given time (e.g. 48 hours) as shown in Figures 1A-5B.

[0121] Preparation of spores or conidia: The preparation of spores or conidia includes growing B. cinerea and Fusarium equiseti on PDA for 3-4 weeks. The preparation further includes pouring a volume of PDB (e.g., 5 ml of 1 / 4 PDB passed through sterile gauze) onto the surface of a culture plate and harvesting the spores or conidia. In an exemplary preparation, the spore concentration / mL was adjusted to 106 / mL.

[0122] Spore inhibition rate and MIC determination: This involves depositing a predetermined amount (ie 5 μl) of each treatment or control onto the surface of a PDA plate in duplicate, incubating the bilayers for 48 hours and measuring mycelial growth.

[0123] Possible inhibition of fungal spore germination using 96-well plates and on growth media.

[0124] Spore collection Target pathogens: Botrytis cinerea (B. cinerea) and Fusarium graminearum (F. graminearum)

[0125] Preparation of serial dilutions of plant material mixed with spores : The powdered botanical compositions were measured and diluted to solutions of 0.5%, 1.0%, and 2.0% concentration. In other embodiments, the botanical compositions may be further diluted to solutions of up to 5.0%. Thyme and yarrow extracts were undiluted and filtered.

[0126] 96-well culture All the solutions were placed in a 96 well microtiter plate and spores of F. graminearum and B. cinerea were added to the solutions. The plates were then incubated for 24 and 48 hours. Samples for hemocytometer and PDA plates were taken from the same plate.

[0127] Inoculation of PDA with plant extracts mixed with fungal spores : Inoculate 4 or 5 specific spots on the PDA plate, each spot representing a different extract solution and fungal spore mixture, or a different concentration of plant composition solution and fungal spore mixture.

[0128] To test the antifungal or antibacterial effect of the extract at three concentrations, the following organisms were tested:

[0129] Bacteria: 1-Xanthomonas campestris Bacterial spot disease

[0130] 2-Salmonella spp. (S531) Food isolates (weak)

[0131] 3-Salmonella spp. (S22) Environmental origin (medium)

[0132] 4-Salmonella spp. (SL1) Human pathogens (strong)

[0133] fungi: 1- Botrytis cinerea (B. cinerea) Gray mold

[0134] 2- Fusarium graminearum - Red mold disease of wheat and barley

[0135] 3- Fusarium equiseti Damping off and root rot

[0136] 4- Fusarium graminearum (Fusarium graminearum) Red mold disease of wheat and barley

[0137] 5-Aspergillus ochraceus Ochratoxin A

[0138] MIC Assays to Determine the Antibacterial Potential of Botanical Compositions and Other Extracts : Now referring to Table 15 and Figures 25A-23, C. majus is known to have direct antibacterial and antifungal properties (Moricz et al., 2015; Parvu et al., 2008;), but the botanical composition diluted in water to 0.2-5% only showed direct efficacy against Fusarium graminearum. Now referring to Table 15 and Figures 25A-25C, thyme leaf extract, yarrow leaf extract and thymol were highly effective against the screened bacteria and fungi, as seen in Table 15 and Figures 25A-25C. Now referring to Figure 25A, Xanthomonas combined with botanical composition extracts having concentrations ranging from 1-5% are shown. Now referring to Figure 25B, Xanthomonas combined with thyme extract 1:2 in 50% alcohol are shown. Referring to Figure 25C, Xanthomonas combined with 1:2 yarrow extract in 50% alcohol is shown. Bioassays were performed using plant extracts (plant composition, thymol, thyme leaf and yarrow leaf extracts). A fixed amount (e.g. 10 μl) of each concentration of dissolved extract was dropped onto the bacterial overlay.

[0139] Referring now to Figures 26A-26C, environmentally derived Salmonella (S22) treated with thyme, yarrow, botanical composition and a control (C) are shown. Bioassays were performed using botanical extracts (botanical composition, thymol, thyme leaf and yarrow leaf extracts). A fixed amount (i.e. 10 μl) of each concentration of dissolved extract was dropped onto the bacterial coverage.

[0140] Referring now to Figure 27, the diagram on the left shows virulent Salmonella treated with a control (S1) and the diagram on the right shows Salmonella treated with thyme extract, yarrow extract and botanical composition (S1). Bioassays were performed with the botanical extracts (botanical composition, thymol, thyme leaf and yarrow leaf extracts) by dropping a fixed amount (i.e. 10 μl) of each concentration of dissolved extract onto the bacterial coverage.

[0141] Referring to Figure 28, Salmonella (S31-mung bean sprout isolate) treated with thyme is shown. Such treatment appears to be the most effective. Bioassays were performed with plant extracts (plant composition, thymol, thyme leaf and yarrow leaf extracts) and a given amount (i.e. 10 μl) of each concentration of dissolved extract was dropped onto the bacterial coverage.

[0142] Referring to Figure 29, Streptomyces scabies treated with thyme extract (upper image) and yarrow extract (lower image) are shown. Bioassays were performed with plant extracts (plant composition, thymol, thyme leaf and yarrow leaf extracts) and a given amount (i.e. 10 μl) of each concentration of dissolved extract was dropped onto the bacterial coverage. [Table 15]

[0143] Fusarium graminearum inoculated with 1.0% and 2.0% of the plant composition (some at 0.5%) appeared to have reduced growth compared to the control. No fungal growth was observed in fungi treated with the thyme mixture solution. Both the plant composition and the tincture inhibited mycelial growth.

[0144] With reference to Figures 30, 31 and Table 16, yarrow extract appears to inhibit the growth of Fusarium pathogens more efficiently than the commercially available product control. With reference now to Figure 30, the growth of Fusarium graminearum is shown after inoculation with exemplary concentrations of the plant composition of 0.5%, 1.0% and 2.0%. In Figure 30, the PDA on the left plate shows 24 hours after inoculation with Fusarium graminearum, and the PDA on the right plate shows 72 hours after inoculation with Fusarium graminearum. With reference to Figure 31, the growth of Fusarium graminearum is shown after inoculation with thyme and yarrow extract. In Figure 31, the PDA plate on the left shows 24 hours after inoculation with Fusarium graminearum, and the PDA plate on the right shows 48 hours after inoculation with Fusarium graminearum. The term "T" refers to thyme leaf extract and the term "Y" refers to yarrow extract, such terms "T" and "Y" both refer to thyme leaf and yarrow extracts developed and prepared by the applicant. The term "T+" refers to thyme extract and the term "Y+" refers to yarrow extract, both terms refer to thyme and yarrow extracts available on the market. [Table 16]

[0145] With reference to Table 16, it is believed that the botanical composition allows thymol to be effective as an antibacterial or antifungal agent at concentrations in which the thiol is not effective when used alone against the following bacteria / fungi:

[0146] 1-Xanthomonas campestris Bacterial spot disease

[0147] 2-Salmonella spp. (S531) Food isolates (weak)

[0148] 3-Salmonella spp. (S22) Environmental origin (medium)

[0149] 4-Salmonella spp. (SL1) Human pathogens (strong)

[0150] 5- Aspergillus ochraceus

[0151] Synergistic effects are also possible with other commercially available botanical substances such as thymol. With reference to Figures 32A-33, such combinations resulted in improved antimicrobial properties. The botanical composition at a concentration of 2% was highly effective against the above bacteria (1-4) in combination with thymol 1:512, whereas thymol 1:512 applied alone was ineffective. A similar pattern was observed for pathogenic strains of E. coli.

[0152] Now referring to FIG. 32, it was observed that for Aspergillus ochraceus, thymol 1:768 alone was not effective, whereas 1% concentration of the botanical composition in combination with thymol 1:768 was sufficient to effectively stop the growth of Aspergillus ochraceus bacteria. Now referring to FIG. 32A, antibacterial and MIC assays using the botanical composition are shown. Referring to FIG. 32B, the combination of the botanical composition with commercial thymol is shown. Xanthomonas campestris (XP), Salmonella spp. (S531), Salmonella spp. (S22) and Salmonella spp. (SL1) were treated with either the botanical composition alone or the combination of the botanical composition and commercial thymol. Bioassays were performed using the above plant extracts. The bioassays involved dropping a given amount (i.e., 10 μl) of each concentration of dissolved extract onto a surface of bacteria.

[0153] Referring now to Figure 33, antifungal and MIC assays against Aspergillus ochraceus using the botanical composition or a combination of the botanical composition and commercial thymol are shown. Aspergillus ochraceus were treated with the botanical composition alone or a combination of the botanical composition and commercial thymol. Bioassays were performed by dropping a given amount (i.e., 10 μl) of each concentration of dissolved extract onto the fungus covering.

[0154] Referring now to Figures 34, 35 and Table 17, in vitro studies were conducted to evaluate the germination rate of Botrytis cinerea and Fusarium graminearum. The in vitro studies showed that the botanical composition had a promoting effect on spore germination. Regardless of the target pathogen, the botanical composition appears to have a promoting effect on spore germination as the concentration increases after 24 hours of exposure. Interestingly, the germination rate (%) after 48 hours of exposure was similar in all treatments, including the control. After 48 hours, Fusarium graminearum grown in PDB had 53% spores that germinated when treated with yarrow and thyme.

[0155] Referring now to FIG. 35 and Table 17, the thyme and yarrow treatments, as well as the 35% ethanol treatment, showed complete growth inhibition. Such results indicate that 35% ethanol is toxic. Referring now to FIG. 34, a diagram (20x magnification) of Fusarium graminearum spores germinated after incubation in a plant composition solution is shown. FIG. 35 shows, clockwise from the top left, a 2% plant composition solution at 24 hours, a 1% concentration plant composition solution at 24 hours, a plant composition control solution at 24 hours, and a 1% plant composition solution at 48 hours. It can be observed that the Fusarium graminearum spores germinated regardless of the concentration of the plant composition. Referring further to FIG. 35, a diagram (20x magnification) of Fusarium graminearum spores germinated after incubation in a yarrow extract solution is shown. FIG. 35 shows, clockwise from the top left, a positive control at 24 hours, a yarrow product according to the principles of the present invention at 48 hours, a commercial yarrow product at 48 hours, and a negative control at 48 hours. [Table 17]

[0156] In summary, with reference to Table 17 and Figures 32A-35, in vitro testing showed that the botanical composition diluted with water alone had no direct antifungal or antibacterial activity. Antifungal properties were observed only for Fusarium graminearum. Furthermore, in vitro testing showed that thyme and yarrow extracts were the only plant extracts with antifungal and antibacterial inhibitory properties. Meanwhile, the botanical composition was shown to improve the antifungal and antibacterial properties of thyme leaf extract or thymol when used in combination. It is believed that the botanical composition allows thymol to be effective as an antibacterial or antifungal agent at concentrations where thymol is not effective when used alone. Finally, in vitro testing evaluating the germination rates of Botrytis cinerea and Fusarium graminearum showed that the botanical composition had a promoting effect on spore germination.

[0157] When applied as foliar spray or drench treatment, the botanical composition at a concentration of less than 2% does not cause phytotoxic responses in the leaves of lettuce, tomato, tobacco, hops and cannabis. The botanical composition is considered to be a safe option for application in agricultural practices. Application of the botanical composition resulted in a significant increase in chlorophyll content (tomato and lettuce seedlings, and tobacco mature plants), plant height (tomato seedlings, lettuce and tobacco), and fresh and dry mass (lettuce and tobacco) compared to the control.

[0158] Pilot experiments on cannabis plants showed that irrigation, foliar spray, or a combination (irrigation and foliar spray) of the botanical composition at concentrations ranging from 0.03 to 2% resulted in increased bud size and yield. Furthermore, treatment of soybean seeds with a 1% concentration of the botanical composition and / or a combination of the botanical composition and the seaweed Stimulagro results in a significant enhancement in soybean growth and early growth compared to a water control. In these tests, Stimulagro was used, but other types of seaweed or seaweed compositions can be used in combination with the botanical composition. One example of a seaweed used can be Ascophyllum nodosum. The botanical composition was found to have a synergistic effect with the seaweed, regardless of the extraction or processing method of the seaweed. The results imply that the botanical composition can be used as a biostimulant to promote plant growth. Furthermore, amendment of soil formulations such as the commercially available Agro mix® G6 (Fafard) with the botanical composition has a positive promoting effect on plants such as, but not limited to, lettuce and tobacco, promoting growth parameters such as, but not limited to, chlorophyll content, plant height, fresh mass and dry mass in such plants. The improved botanical composition also inhibits gray mold disease. Thus, amendment of soil formulations such as Agro mix® (lettuce and tobacco) with the botanical composition can help produce healthier plants, as shown with lettuce and tobacco, and is suspected to show similar results for other plants. The components and molecules of the botanical composition may positively interact with the components of soil formulations such as Agro mix® G6 (Fafard).

[0159] The plant extracts of the botanical composition have no direct inhibitory effect on bacteria and fungi. Antifungal properties were observed only for Fusarium graminearum. In vitro studies showed that potential synergistic effects with other botanical substances, such as, but not limited to, extracts of thyme leaves and yarrow, as well as thymol, could result in improved antibacterial properties against important bacterial or fungal plant pathogens. In vitro studies evaluating the germination rate of Botrytis cinerea and Fusarium graminearum showed that the botanical composition has a promoting effect on spore germination.

[0160] Based on repeated demonstrations of preventive and therapeutic antifungal effects, the botanical compositions generally induce a plant-derived response to infection, potentially including a form of resistance. Also, the ability of the botanical compositions to cure (e.g., cannabis powdery mildew) or prevent fungal diseases (e.g., cannabis powdery mildew and gray mold of tomato, lettuce and hops) by foliar spray or drench treatment, or cannabis powdery mildew by foliar spray, is generally improved over control. The botanical compositions can be used as biopesticides to promote plant resistance to combat disease.

[0161] VI. Metabolite composition and bioactivity analysis of plant extracts

[0162] In yet other experiments, compositions containing plant extracts according to the embodiments set forth above were analyzed to determine the metabolites present in the components.

[0163] In such experiments, two series of studies were performed, the first on the leaf extract and the second on the root extract. In such exemplary experiments, analytical methods included QE Orbitrap MS (LC / MS / MS) and GC / EI / MS metabolite profiling.

[0164] leaf extract In studies on leaf extracts, the identified bioactive metabolites were found to be selected from among the following: apigenin, genistein, genipin, p-coumaroyltyramine 18-hydroxyoleate, 4-coumaroylquinate, chlorogenic acid, genistin, caffeoylshikimate, 15-HETE, p-hydroxybenzoic acid, succinic acid, tyrosol, gamma-hydroxybutyric acid, vanillic acid, 3-hydroxybenzoic acid, acetic acid, caffeic acid, phenylacetic acid, phosphoric acid.

[0165] Further referring to the leaf extract studies, traumatic acid, abscisic acid, epijasmonic acid, jasmonic acid, salicyl-HCH, traumatin, gibberellins, 7-isomethyl-jasmonic acid, a-linolenate, indole-3-acetic acid, a,a-trehalose, a-linolenic acid, and D-fructose were found to have different roles in plant physiology.

[0166] root extract In studies on leaf extracts, the identified bioactive metabolites were found to be selected from among 13-epoxyoctadeca-9;11-dienoic acid, ferulic acid, 9(S);12(S);13(S)-trihydroxy-10(E)-octadecenoic acid, apigenin, genistein, p-coumaroyltyramine 18-hydroxyoleic acid, 4-coumaroylquinate, chlorogenic acid, genistin, caffeoylshikimate, vanillin, succinic acid, tyrosol, gamma-hydroxybutyric acid, vanillic acid, 4-coumaric acid, acetic acid, caffeic acid, phosphoric acid and pantothenic acid.

[0167] With further reference to the leaf extract studies, the following metabolites were found to have different roles in plant physiology: traumatic acid, abscisic acid, epijasmonic acid, 7-iso-jasmonic acid, jasmonic acid, salicyl-HCH, traumatin, gibberellin, 7-isomethyl-jasmonic acid, a-linolenic acid, indole-3-acetic acid, a,a-trehalose, glutathione, salicylic acid, a-tocopherol, a-linolenic acid, D-fructose, and GABA.

[0168] Metabolomic analysis Further metabolomics analysis was performed. The results are shown in Table 18. Table 18 shows the relative and exemplary concentrations of alkaloids in the compounds. Alkaloids may have many benefits in plant protection and growth. [Table 18]

[0169] While illustrative and presently preferred embodiments of the invention have been described in detail above, it is to be understood that the inventive concepts may be embodied and used in various other ways, and the appended claims are intended to be construed to include such variations except insofar as limited by the prior art.

Claims

1. A granular extract of thyme leaves, Extract of the roots of Chelidonium majus, Celandine leaf extract, A botanical composition comprising: The composition is diluted to a concentration of 0.2% to 5%, and is used to promote plant growth and prevent or suppress plant diseases.

2. The plant composition described in claim 1, wherein the plant composition before dilution contains 0.1% to 99% extract of celandine root, 0.1% to 99% extract of celandine leaf, and 0.1% to 30% granular extract of thyme leaves.

3. 10. The botanical composition of claim 1, which is diluted to a concentration of 0.5% to 5%.

4. 2. The plant composition of claim 1, having antibacterial and / or antifungal properties.

5. The botanical composition of claim 1 further comprising a tincture.

6. The botanical composition of claim 1 further comprising seaweed.

7. 7. The botanical composition of claim 6, wherein the seaweed is Ascophyllum nodosum.

8. The plant composition described in claim 6, wherein the plant composition after dilution contains 0.5 to 2 g / L of the seaweed.

9. 10. The botanical composition of claim 1 further comprising thymol.

10. 10. The botanical composition of claim 1 further comprising an extract of yarrow leaves.

11. 11. The botanical composition of claim 10, wherein the yarrow leaf extract is a yarrow leaf extract diluted 1:2 in 50% alcohol.

12. 10. The botanical composition of claim 1 further comprising a soil formulation.

13. 13. The botanical composition of claim 12, wherein the soil formulation comprises coconut husk fiber.

14. 13. The botanical composition of claim 12, wherein the soil mix comprises peat moss and perlite.

15. The plant composition of claim 12, wherein the soil formulation comprises mycorrhizae.

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