Patent Application
20220272990
The present invention generally relates to botanical extract for the growth and protection of plants.
There have been multiple solutions marketed to help plants grow or heal from diseases. Some of them are using chemicals wherein others are using organic components. Chemical solutions, even if they often provide the best results, may have adverse effects to the health of humans in contact with treated plants. On the other hand, organic solutions are often not as efficient as necessary. Therefore, there is a need for an organic solution that is as efficient, if not more, than most chemical solutions found on the market in order to help plants grow and heal from diseases.
Herbal extracts used in agriculture are formulated from naturally occurring plants (or other organisms) as alternatives to synthetic chemicals that could be more toxic to growers, consumers, and more harmful to the environment. Advantages such products could involve biodegradability and more eco-friendly to nature, due to no harmful residues, compared to synthetic chemical alternatives. Because of the listed advantages, agricultural industries all over the world are developing botanicals to contribute to sustainable agriculture. Testing the efficacy of these products on the initial stages of plants' growth cycle (germination and early seedling growth) is an expensive and time-consuming process. Germination rates are usually determined by petri dish assays. For early growth, another system involving hydroponics or greenhouse growth (direct germination) are usually used. A Standard Operating Procedure was developed for analysis of the impact of herbal extracts on germination and early growth (SOP). This was done in a defined system by modifying the rolled paper towel test developed by the International Seed Testing Association (ISTA, 1985). The efficacy of SOP was determined by comparing it with direct germination test. The SOP has the following advantages: (1) efficient for testing the effect of potential growth stimulants on seed germination and early growth; (2) time (2 weeks duration) and space efficient; (3) repeatable; performed under defined conditions in a growth chamber; (4) relatively simple and low-cost method that can be performed in industry by staff with minimal training and easily available materials.
The aforesaid and other objectives of the present invention are realized by generally providing a botanical composition to promote plant growth and to prevent or suppress plant diseases.
In an aspect of the present invention, a botanical composition is provided. The botanical composition comprises a granular extract of thyme leaf, an extract of Chelidonium majus roots and an extract of Chelidonium majus leaves. The composition is diluted at a concentration between 0.2% to 5% and the composition is used to promote plant growth and to prevent or supress plant diseases.
The botanical composition may comprise 0.1% to 99% of the extract of Chelidonium majus roots, 0.1% to 99% of the extract of Chelidonium majus leaves and 0.1% to 30% of the granular extract of thyme leaf.
The botanical composition may be diluted at a concentration between 0.5% and 5%. The botanical composition may have anti-bacterial and/or anti-fungal properties. The botanical composition may further comprise a tincture or seaweed. The seaweed may be Ascophyllum nodosum. The composition may comprise between 0.5 to 2 g/L of seaweed.
The composition may further comprises an additional extract of thyme leaf. The extract of thyme leaf may be a thyme leaf extract diluted in 1:2 in 50% alcohol.
The composition may further comprise thymol or an extract of yarrow leaf. The yarrow leaf extract may be diluted in 1:2 in 50% alcohol.
The botanical composition may further comprise a soil mix. The soil mix may comprise coconut husk fiber. The soil mix may further comprise peat moss and perlite or mycorrhiza.
In an other aspect of the invention, a botanical composition is provided. The botanical composition comprises an extract of yarrow leaf diluted in 1:2 in 50% alcohol the composition being used to promote plant growth and to prevent or supress plant diseases. The botanical composition may further have anti-bacterial and/or anti-fungal properties.
The extracts may be prepared using cold pressing or freeze drying. The extracts may further be prepared using a technique that involves processing using fermentation. The fermentation may be aerobic or anaerobic. The bacteria created may be lactic acid bacteria or may originate from the Bacillus species.
In yet another aspect of the invention, a method of treating plants using a botanical composition is provided. The botanical composition comprises a granular extract of thyme leaf, an extract of Chelidonium majus, concentration between 0.2% to 5%, the method further comprises soil-drenching plants using a concentration between 0.2% to 5% of the botanical composition. In such a method, the soil-drenching may be applied in a single dose or may be applied in split doses over a predetermined period of time.
In a further aspect of the invention, a method of treating plants using a botanical composition is provided where the botanical composition comprises a granular extract of thyme leaf, an extract of Chelidonium majus, concentration between 0.2% to 5%. The method comprises applying a concentration between 0.2% to 5% of the botanical composition on leaves of the plants. The application of the botanical composition may comprise dipping the leaves in the botanical composition. The application of the botanical composition may further comprise spraying the leaves with the botanical composition.
The botanical composition may be applied by spraying or dipping the leaves in a single dose or in split doses over a predetermined period of time.
The features of the present invention which are believed to be novel are set forth with particularity in the appended claims.
The above and other objects, features and advantages of the invention will become more readily apparent from the following description, reference being made to the accompanying drawings in which:
Novel methods and compositions of a botanical extract to promote and boost plant growth and prevent and suppress plant diseases will be described hereinafter. Although the invention is described in terms of specific illustrative embodiment(s), it is to be understood that the embodiment(s) described herein are by way of example only and that the scope of the invention is not intended to be limited thereby.
A botanical composition or mixture of a granular extract of thyme (Thymus vulgaris) and an extract of roots, leaves or a mixture thereof of Chelidonium majus (Chelidonium majus) to promote plant growth and prevent or suppress plant diseases is provided. The botanical composition may have different concentrations, such as but not limited to concentrations w/v 0.2%, 0.5%, 1.0%, 1.5%, 2% & 5%. The concentrations are typically prepared in distilled water and filter sterilized. In the present disclosure, unless specifically mentioned, the term thymol generally refers to thyme leaf extract that was diluted 1:2 in a 50% alcohol tincture before being added to the botanical composition or mixture. Furthermore, as in mentioned for some experiments, yarrow flower extract was diluted 1:2 in a 50% alcohol tincture before being used.
The botanical composition or mixture of a granular extract of thyme (Thymus vulgaris) and an extract of roots, leaves or a mixture thereof of Chelidonium majus (Chelidonium majus) may be used as a biostimulant, as described above. Extracts of roots, leaves or both may be in combination with a specific portion of thyme.
The relative quantity of thyme and root and leaf used in a mixture may be in the following ranges:
The extraction process of Chelidonium majus may be executed conventionally (10:1-70% ethanol) followed by spray drying, or with cold pressing or with freeze drying.
The extraction process of Chelidonium majus may further be done with various bacterias, either using aerobic or anaerobic fermentation. One such example of bacteria to use in an extraction process is the lactic acid bacteria, also referred to as LAB. Bacterias of the Bacillus species may also be used in the process.
More thyme leaves and/or yarrow leaves or any other parts of thyme or yarrow may further be added to the composition.
In a first example, roots of tomato seedlings have been treated in tissue culture with the botanical composition at different exemplary compositions (0.5%, 1% & 2%). After three weeks of treatments, tomato plants were removed from tissue culture tubes and chlorophyll content, and plant height was recorded.
The results show that after three weeks, roots of tomato seedlings treated in tissue culture with the botanical composition (0.5%, 1% & 2%) had higher chlorophyll content and the total plant height was higher compared to the control.
Referring now to
Table 1: Chlorophyll content (μg.cm-2) of control and the botanical composition treated tomato plants. Numbers in the table represent the average of two compound leaves per plant. Three separate readings per leaf was recorded.
Regarding the present exemplary experiments, lettuce seeds were sown in a soil mix, such as Agro mix® G6 (Fafard), in 200 cell trays for germination. After two weeks of growth, the lettuce plants were treated with either rhizosphere (around 1 ml/plant) water only (as control) or a concentration of 1% of the botanical extract. Each treatment had seven (7) replicates.
Referring now to
After three weeks of growth,
Referring now to Table 2, the highest chlorophyll content was found in the lettuce plants treated with 1% concentration of the botanical composition with an average of 32.2 μg.cm-2.
Referring now to Table 3, the concentration of 1.0% of the botanical extract gave the highest FW at 5.75 g. The total root length irrespective of the treatment was slightly higher in the botanical composition 1% compared to the control treatments as shown in below Table 4.
Regarding the present example, the botanical composition at different concentration rates showed a boosting impact on plants (i.e. lettuce and tobacco) and promoted their growth parameters. The botanical composition, amendment to Agro mix® G6 (Fafard), resulted in a positive boosting impact on plants and promoted their growth parameters. The components and molecules of the botanical composition may be interacting positively with the components of soil mixes, such as but not limited to the all-purpose soil mix Agro mix® G6 (Fafard). It may further be advantageous to use soil mixes comprising coconut husk fibers, fiber peat, perlite, limestone, gypsum and/or mycorrhiza to provide help with moistening, root growth and mineral retention. Soil amendment of the botanical composition with commercial soil mixes like Agro mix® may help in the production of healthier transplants as shown with lettuce and may further show similar results in other plants.
In another experiment, tobacco and lettuce seeds were sown in a first soil mix, such as the Agro mix® S4, in 200 cell trays for germination. After one (1) week of growth, the seedlings were transplanted into pots (6 inches) with second soil mix, such as commercially available Agro mix® G6 (Fafard) amended with 14-14-14 TYPE 70 nutricote NPK. The soil mix used generally comprises a high porosity that provides superior drainage and gas diffusion due to the particular peat composition of the said soil mix. The transplanted seedlings where treated with one of the following treatments: water treatment, as control or a 1% concentration of the botanical composition. Each treated pot received three single doses as soil drench (10 ml/pot) at an interval of 5 days. After 4 weeks, the plants were harvested after 4 weeks. In such an experiment, all treatments were done with 7 replicates.
Referring now to
Interestingly the chlorophyll content of the lettuce plants treated with the 1% concentration treatment was similar to the content of the lettuce plants treated with the control, as shown in below Table 6.
Based on the present experiments, the botanical composition at different concentration rates showed a boosting impact on plants (i.e. lettuce) grown in greenhouse conditions and promoted growth parameters on such plants. The botanical composition amendment to the first soil mix, such as Agro mix® G6 (Fafard), further resulted in a positive boosting impact on plants and promoted growth parameters of the same plants. Consequently, the components and the molecules of the botanical composition may positively interact with the components of the first soil mix, such as Agro mix® G6 (Fafard).
Referring to Table 5, the plants treated with the 1% botanical composition had higher fresh and dry weight and height compared to the plants treated with the control. In summary, there was 8% increase in dry weight, 10% increase in fresh weight, and 9.6% increase in height in the lettuce plants treated with the 1% botanical composition as compared to the lettuce plants treated with the control.
It may further be observed that there is no discernable difference between the chlorophyll content found in the plants treated with the 1.0% botanical composition and the control.
Referring now to
Referring now to Table 7, in summary, the dry weight increased by 31% increase in dry weight, the fresh weight increased by 28% and height increased 9.6% compared to the control.
Referring now to Table 8, the highest chlorophyll content was recorded with the 1.0% botanical composition treatment, a 5.42% increase as compared to control.
In conclusion, the botanical composition treatment at different concentration rates showed a boosting impact on plants, such as but not limited to tobacco plants, when grown in greenhouse conditions and promoted growth parameters of the said plants. When the botanical composition is combined to a soil mix, such as Agro mix® G6 (Fafard), the boosting impact on the plants further increased and such composition promoted the growth parameters of the said plants. The components and molecules of the botanical composition may be interacting positively with the components of the soil mix, such as Agro mix® G6 (Fafard).
In the present exemplary experiment, the SOP methodology was used to assess effect of different herbal treatment products, such as but not limited to C7, the botanical composition comprising C. majus extract and Stimulagro: A. (ST) nodosum extract. Stimulagro is a composition majorly made of algae. The herbal products were used individually and in combination, on germination and early growth of soybean and were compared to a water control treatment. The experiment further comprises measuring the physiological properties such as but not limited to growth characteristics, biomass, chlorophyll content and gas exchange parameters.
The experiments further comprise applying the SOP. The experiments comprise using the Rolled Towel Test by ISTA (1985) with some modifications. The ISTA method used dry seeds and moistened paper. The first modification involved soaking the seeds overnight (20-24 h) in double distilled water and using dry, pre-folded paper. In this way, the germination lag time was reduced, seeds stuck to the dry paper, and the paper was more readily rolled. The test was made on paper cut from a roll, such as a 60×20 cm paper cut (i.e. Classique Kraft brown, Servicorp, QC, Canada) and folded lengthwise, such as folded in 60×10 cm sections. Seeds were placed in a single row, such as 1 cm from the top and 5 cm apart. Seeds were held in position by folding the paper again lengthwise, to cover the seeds. The paper rectangle containing the seeds was rolled along the short axis and secured with glue or tape, such as scotch tape—Scotch® Magic™ Tape ¾″×1296″, MN, USA. The roll was placed vertically, with the seeds toward the top, inside a plastic container, such as a 1 L container, containing a proportion of the respective treatment solution, in this case, an exemplary 40 ml. The containers were kept in a growth chamber at predetermined conditions, such as a Conviron® environmental chamber, Winnipeg, Canada set at 16:8 h light:dark cycle with 25±2° C. temperature, 400 μM m-2 s-1 flux density of cool white and incandescent lights and 50% relative humidity. Paper towels were never allowed to dry out. Each morning, residual medium was discarded, and the same proportion (i.e. 40 ml) of fresh treatment solution was added. Germination was monitored daily. Growth (shoot length, shoot fresh and dry mass) chlorophyll content and photosynthetic rate were tested with fifteen days old seedlings.
Photosynthesis measurement: Photosynthetic rate, such as μmol m-2 s-1 CO2, was measured using a portable infra-red gas analyzer, such as IRGA-LI-6400, LI-COR Inc., Lincoln, Neb., USA. Measurement was performed on separate areas of soybean plant leaves, such as fully expanded unifoliate for seed treatments and fully expanded third trifoliate leaf for foliar applications. In such experiments, the infra-red gas analyzer (i.e. IRGA) was calibrated and zero-adjusted approx. every 30 min during the measurement period.
Chlorophyll content measurement: Leaf chlorophyll content of fully expanded leaves was estimated using a SPAD 502 meter, such as a Konica Minolta Optics, Inc., New Jersey, U.S.A., by averaging 10 readings per treatment. The results are shown in Soil Plant Analysis Development (SPAD) units.
Germination, growth characteristics and biomass measurements: Germination was monitored daily. Plant height was measured from the basal node to the shoot apex using a ruler. In an exemplary measurement process, fresh (FM) and dry (DM) shoot mass were taken on three randomly selected plants/roll (6 plants/replicate) and weighed on an analytical balance, such as Highland® balance, Adam Equipment Inc., Oxford, Conn., USA. Also, shoots were harvested just above the paper towel for FM then dried at exemplary 60° C. for 72 h and weighed for DM.
The experimental design was a completely randomized design (CRD). In such exemplary design, the CRD comprises 5 replicates of 20 seeds each, with 10 seeds per roll and 2 rolls per container. Data was expressed as means±standard error of the mean (SEM) and analysis of variance, such as 1-way ANOVA, was followed by post hoc Newman-Keuls Multiple Comparison Test, such as using a software GraphPad Prism version 5.01, 2007, Graf Pad Software, Inc., CA, USA. In the exemplary design, the level of significance was *P<0.05.
The treatment of soybean seeds with the 1% botanical composition alone leads to a statistically significant increase in dry weight, such as an exemplary 12.9%. Additionally, results show that the seed treatment of soybean with the combination of both the botanical composition and Stimulagro generally leads to positive enhancements in the physiological properties of the treated soybean. Amongst other treatments, the treatment with the 1% botanical composition combined to Stimulagro was observed as the most beneficial treatment. The said treatment significantly (P<0.05) increased soybean seedling (in this case 2 weeks old) shoot height (i.e. 13.2%), dry mass (i.e. 10.7%) and rate of photosynthesis (i.e. 20.3%) compared with water control (see exemplary results at Table 9).
As seen in Table 9, the results clearly show an enhancement of physiological properties, growth parameters and rate of photosynthesis of soybean when treated with the botanical composition combined with Stimulagro.
The results further underlined the discriminatory value of SOP-soybean and the potential of one herbal extract treatment in particular as a growth stimulant for soybean, as shown in
The protocol followed (SOP-Soybean) is summarized as follows:
Referring to
a) Soaking soybean seeds for a predetermined period, such as overnight (20 h), before performing the assay.
b) Folding an absorbent material in half lengthwise, such as a paper towel (i.e. 60 cm×20 cm).
c) Placing a predetermined number of pre pre-soaked seeds (i.e. 10) in single rows, such as placed 1 cm from the top, in a single row 5 cm apart, leaving 5 cm at each end.
d) Holding the seeds in place by positioning pre pre-folded paper over the seeds.
e) Folding a second time the absorbent material, such as of 1 cm, to strengthen the base of the roll.
f) Quickly rolling the dry paper containing the seeds lengthwise and securing the said paper with tape.
g) Placing vertically the rolls in a transparent container with a predetermined volume of treatment solution, such as 40 ml.
h) Maintaining the containers under defined conditions in a growth chamber, such as maintaining at 25±2° C. and 16:8 h day:night cycle with 400 μM m m-2 s-1 flux density.
i) Adding the predetermined volume of fresh treatment solution daily, such as 40 ml.
The present protocol generally aims at providing the following advantages:
The present experiment was conducted using “Candyland” hybrid cultivar. In order to identify or screen biostimulant properties and the effects on bloom and yield, a licensed patient grower having enough material to conduct the pilot trial was used.
The experiment comprises testing several concentrations, from 0.03 to 2%, of the botanical extract on Cannabis plants to evaluate the effects on bud size and yield. The same strain sharing the same genetics was used in the test. The total number of plants tested was 120. A total of 60 plants were used for the botannical compositiontreated group and 60 plants for the untreated control group. Treated plants were compared to control plants and both were grown hydroponically under conventional home greenhouse conditions. Either foliar, root drench or a combination of foliar and soil-drench were applied to the plants.
For testing the biostimulant effects, different treatments ranging from 0.03 to 2% of the botanical composition where used, such as root drench alone or root-drench combined to foliar application, with a total of 4 to 30 applications. The experimental treatments led to healthier crops and to a substantial boost and increase in the yield and size of the Cannabis flowers as shown in
According to another embodiment, the botanical composition may further be used as a biofungicide and thus help treat or stop the spread of diseases on plants.
In a first test, Botrytis cinerea plugs (5 mm) were placed on detached leaves of treated plants (single dose of the botanical composition or a combined dose of the botanical composition with yarrow (Y)) as well as control (water). Leaves were kept in trays with wet filter paper, such as Pyrex trays. The treated leaves were observed every day. The lettuce leaves infection results were recorded 72 hrs later.
Referring now
In an exemplary trial, organic tomato and lettuce seeds were germinated in a soil mix, such as Agro mix® S4 (Fafard), in predetermined number of cell trays, such as 20. After 2 weeks of growth, seedlings were transplanted into pots, such as pots having 6 inches diameter in another soil mix, such as Agromix G6 (Fafard) amended with 14-14-14 TYPE 70 nutricote NPK. Pots were placed in randomized block design (RBD) in standard greenhouse growing conditions and irrigated with an automated system. All trials were repeated twice. The appropriate data of repeated trials was pooled.
To determine whether the botanical composition can suppress disease, the botanical composition at 1% was applied using different methods: first being soil drenching and second being leaf dipping.
In a first method of treatment, 4 weeks old potted plants received, as soil drench treatment, a single dose of 1% of botanical composition, such as 10 ml or 20 ml, repeated every at a frequency, such as 24 hours for 96 hours. Control treatments instead received water. The treatments (8 in total) were applied to the soil close to the plant using different amounts as shown in Table 11. After 24 hours post-treatment of the botanical composition, detached leaves and leaves still attached to the plants were challenged with Botrytis cinerea. Furthermore, different concentrations of the botanical composition were applied as a soil drench to check for its role in disease suppression during Grey mold disease development.
Fungal mycelial plugs from freshly grown Botrytis fungus were placed on uniform leaves of soil drenched plants and treated with the botanical composition or treated with a water-based control. The infected leaves were covered with moist plastic bags to create a humidity tent over it, as seen in
A disease index was recorded after 72 hours of inoculation for detached leaves and for non-detached leaves. Disease index was measured as ratio of necrotic lesion area to healthy tissue area using a rendering software, such as ImageJ, following the instructions of Haliem (2012); and Steward and Macdonald (2014), and recorded as percentage. All treatments were performed with 10 replicates and the trial was repeated twice. Collected data were averaged and differences between treatments were analyzed using JMP11 (SAS-one-way ANOVA, Tukey HSD, α 0.05) and significance between treatments was indicated.
Plants receiving repeated doses of 10 ml or a single dose of 20 ml of the botanical composition 1%, see for instance T1 to T5 of below Table 11, successfully reduced lesion areas as compared to control treatment. A disease index of detached leaves of soil drenched healthy plants treated with water (control) and the botanical composition and post-infected with B. cinerea plants, from 10 ml to 40 ml, is shown in
Infection on non-detached leaves of soil-drenched plants was significantly reduced as a result of the botanical composition soil drench treatment seen in
Soil drench applications of the botanical composition at concentrations of 1% and 2% showed effective suppression of Grey mold disease on tomato during disease progression. The botanical composition at concentrations of 1% and 2% were found significantly effective in inhibiting disease on tomato leaves compared to the control plants. More so, the botanical composition at 1% was determined as being more effective than the botanical composition at 2% concentration. Referring now to
Referring now to
In another embodiment, the addition of the botanical composition to a soil mix, such as Agro mix® G6 (Fafard), enables tomato plants to become resistant to Botrytis cinerea, the causal agent of grey mold disease. The components and molecules of the botanical composition may be interacting positively with the components of the soil mix, such as Agro mix® G6 (Fafard), the resulting in inducing resistance in the plants, such as tomato plants, against disease. Thus, the botannical compositionamendment into Agro mix® G6 (Fafard) resulted in the suppression of Grey mold disease on tomato plants.
The second method of treatment comprises detaching uniform size tomato and lettuce leaves and submerging said detached leaves in 1% botanical composition for a predetermined duration, such as 30 seconds. The method further comprises placing the submerged leaves in plates, such as Pyrex plates, lined with moist filter paper. The method further comprises dipping control leaves dipped in water. The method further comprises placing B. cinerea plugs of an actively grown culture on detached leaves of control and of plants treated with the botanical composition. The trays are sealed, such as sealed using a wrapping means (i.e. Saran wrap) and incubated at room temperature, as shown in
Referring to
In yet another exemplary experiment, hops rhizomes, of the variety Willamette, were obtained. The experiment method comprises transplanting Rhizome cuttings into pots in a soil mix. As an example, the pots were 6 inches and the soil mix was Agromix G6 (Fafard). The experiment method further comprises placing in growth chambers in predetermined conditions, such as with 12/12 h of day/night, 23/21° C. day/night temperatures, 210 photons μm−2s−1, and humidity maintained at 65% throughout the entire day.
The experiment method further comprises detaching the hops leaves from two months old plants and placing the detached leaves in plates lined with wet filter papers, such as Pyrex® plates. Referring now to
Phytotoxicity occurs when a plant is exposed to an external factor that is toxic to the plant and symptoms may occur when leaf margin necrosis and browning, yellowing (chlorosis), yellow or brown or black spots (see, for instance, Kristin Getter, Michigan State University Extension, Department of Horticulture). The concentration of botanical composition was considered to be phytotoxic if treated leaves showed any of the previous symptoms when compared to the water treatment.
Still referring to
In still another experiment, two months old leaves from hops plants were detached and immersed in 3 ml of a respective treatment (various concentrations of the botannical composition) and placed Pyrex plates lined with wet filter papers for 24 hours. There were three leaves per treatment. Shown in
The aim of this experiment was to test for phytotoxic effect on potted plants, hops grown in Agromix G6 (Fafard), and leaves when the the botanical composition is soil drenched and applied as single dose or as split dose over a predetermined period of time. In this experiment, in such experiment, the period of time was three consecutive days.
Two months old hops plants, shown in
The botanical composition at a 1% concentration singularly or combined, doses, see T4 to T6 treatments in Table 12, did not cause any phytotoxicity on hops. In fact, a split dose application of the botanicals generally helped in having no harmful effects on plants.
In another experiment, the experiment comprises challenging post treatment hops plants with the fungal pathogen: B. cinerea in-vitro.
In such exemplary experiment, six uniform-sized leaves were detached from treated and control plants grown in a soil mix, such as Agromix G6 (Fafard). The experiment further comprises inoculating the detached leaves with agar plugs (i.e. 5 mm plugs) containing Botrytis cinerea being placed in the center of the detached leaves. The experiment further comprises placing the infected leaves in the trays with moist filter paper at the bottom of the tray, as seen in
The statistical analysis of the results may comprise averaging the data and analyzing the differences between treatment and control by two-way analysis of variance (ANOVA), and when necessary, by least significant differences (LSD) at P<0.05 using the SPSS statistical package v. 22.0, (IBM Corp., Armonk, N.Y., USA).
The plants soil drenched with water only (Control Treatment) and the plants soil drenched in an application of the botanical composition at 1% were compared. The treatment included three doses over three consecutive days (T6, Day 1=20, Day 2=30, Day 3=20). The comparison resulted in a significant decrease of the percentage of disease incidence and disease severity by respectively 50% and 31%, as seen in
In another embodiment, exemplary concentrations of botanical were used on Cannabis seedlings.
In another exemplary experiment, the experiment comprises detaching Cannabis leaves from plants grown for a specific duration, such as four months. The experiment further comprises treating the detached leaves by dipping the said leaves (i.e. 6 leaves per treatment) in the following concentration of the botanical composition: T3 at 1% and T5 for water treatment, as seen in
Based on the present experiment, a single dose of the botanical composition (T3) did not cause any phytotoxic effect on Cannabis.
The trial I comprises using detached Cannabis leaves. The same leaves that were already dipped in different treatments, T3 and T5, were used (see above). Powdery mildew (PM) inoculum comprises pieces of leaves (such as 1 cm2 pieces) uniformly infected with PM conidia. Each of the leaf pieces was in the center of the botanical composition-treated (T3) or water treated (T5) leaves, as shown in
Preliminary results from trial I on Cannabis demonstrated that the botanical composition 1% alone (T3) prevented disease progression of powdery mildew (PM) and retained leaves green and healthy. Signs of infection were only observed on the water treated leaves of
This trial II was conducted with two biological replications using detached Cannabis leaves of three varieties that are known for their differential susceptibility to powdery mildew (PM). The varieties were: variety I (susceptible), variety II (susceptible) and variety III (highly susceptible). Each variety was treated with a combined dose of 1% of the botanical composition with thymol and was compared to control treatments comprising water only. The trial II comprises detaching the leaves of each of the varieties and immersing the said detached leaves in combined doses of 1% botanical composition with thymol or in doses of water (control). In the present trial II, each treatment was executed on five (5) Cannabis leaves and the experiment was repeated twice. The trial II further comprises inoculating leaves with pieces of infected leaves (such as 1 cm2). Each of the pieces comprises Powdery mildew as previously described in trial I. The trial II uses two x pieces of leaves instead of the one piece of leaves used in trial I. The trial II further comprises incubating the plates at room temperature and monitoring the disease progression over time.
The trial II comprises recording or collecting the disease severity measurements during a determined period, such as 29 days, post-infection (DPI). Disease severity was scored using a computer program, such as the software ImageJ, as shown in
The trial II is believed to successfully establish presence of the PM disease and the progression of said PM disease. The trial II further established that the presence and progression of the disease is due to the presence of two sources of inoculum and to longer incubation time, as such are believed to help in effective disease spread and successful infection, as seen in
Referring to Table 14, at 12 DPI, the percentage of disease incidence was higher in the water treated leaves, from 60 to 80%, compared to leaves treated with the combined dose of the botanical composition at 1% with thymol (20%). At 26 DPI and higher, most of the leaves, except for the variety Sachigo, showed signs of disease, incidence reached almost 100%, for both water control and the botanical treatment. The disease severity was always less in leaves treated with the botanical composition at a 1% concentration combined to thymol.
$% incidence refers to the number of infected leaves out of the total 5 that were screened.
λDisease severity decrease is calculated in relation to control.
Referring now to
Referring now to
In yet another pilot experiment comprises using “Candyland” hybrid cultivar. Referring to
The pilot experiment comprises testing several concentrations of the extract of the botanical composition, such as from 0.03 to 2%, on Cannabis plants to evaluate the preventive or curative capabilities/properties on powdery mildew. The same strain sharing the same genetics was used in the test. In such an exemplary pilot experiment for powdery mildew preventive control, the total number of plants tested was 120. The experiment comprises placing the tested plants in a room full of powdery mildew infected plants (inoculum). The exemplary experiment comprises a first group, comprising a total of 60 plants, was treated with the botanical composition and a second group, comprising 60 plants treated with the control. The experiment further comprises testing a total of 20 plants infected with powdery mildew to measure the disease elimination (curative treatment). The experiment further comprises a treatment group, the treatment group comprising 10 plants treated with the botanical composition at various concentrations, generally ranging from 0.2% to 2%. The experiment further comprises a control group. The control group comprises 10 plants treated only with water. The plants from the treatment group and the control group were always compared and plants from both groups were grown hydroponically under conventional home greenhouse conditions. The plants from the groups were treated using foliar, root drench or a combination of foliar and soil-drench.
Used in a context of disease control, the application of the botanical composition through foliar or root drench routs, or combination of both proved effective in total elimination powdery mildew from diseased plants. Concentrations ranging from 0.03 to 2% were found sufficient to eliminate the disease from treated plants. The plants that didn't receive the botanical composition showed high intensity of the disease. Also, it worth mentioning that healthy plants which received the botanical composition didn't show or develop any sign of the disease after treatment. Furthermore, two applications of the botanical composition, with a concentration ranging from 0.03 to 2%, may further be adequate to eliminate powdery mildew from the diseased plants. These results coincide with the previous tests that were performed on Cannabis and further support the biofungicidal properties of the product.
In still another experiment method, various concentrations of the botanical composition, thyme leaf extract, yarrow extract, thymol and combination of the botanical composition and the previous were used to screen for their antimicrobial properties.
A method for preparing a single dose of a botanical extract of the botanical composition is provided. The method for preparing a single dose comprises dissolving in water the botanical composition and serially diluting to achieve solutions of 0.5%, 1.0%, and 2.0% (w/v). The method may further comprise serially diluting up to 5.% (w/v).
The method further comprises preparing thymol at concentration of 1:256, 1:512, and 1:768.
The method also comprises preparing thyme leaf and yarrow extracts in the following proportion: 1:2 in 50% Alcohol.
A method for preparing a combined dose of a botanical extract of the botanical composition is also provided.
The method comprises diluting each of the botanical composition and mixing the diluted botanical composition to a specific concentration of thymol.
The method further comprises using water and PDB as negative controls and using Ethanol as a positive control.
MIC Determinations: The minimum inhibitory concentrations (MIC) of the botanical products was established for cultured pathogens.
Burkholder's Assay: Referring to
The preparation of spores or conidia comprises growing B. cinerea and Fusarium equiseti on PDA for 3 to 4 weeks. The preparation further comprises flooding the surface of the culture plate with a volume of PDB, such as 5 ml of ¼ strength PDB passed through sterile gauze, to collect spores or conidia. In an exemplary preparation, the spore concentration/mL was adjusted to 106/ml.
The determination comprises placing in duplicate a determined volume (i.e. 5 μl) of each treatment or control onto the surface of PDA plates and incubating the placed duplicates for 48 hours to measure hyphal growth.
Spore collection: Target pathogens: B. cinerea and F. graminearum.
Preparation of serial dilution of botanicals mixed with spores: the powdered botanical composition was measured and diluted into solutions of 0.5%, 1.0%, and 2.0% concentration. The botanical composition may have further been diluted into solutions of up to 5.0% in other embodiments. The thyme and yarrow extract were not diluted but were filtered.
96-wells incubation: All the solutions were placed in a 96-microtiter plate and spores of F. graminearum and B. cinerea were added to the solution. Then the plate was incubated for 24 h and 48 hours. It is from that same plate that the samples for the haemocytometer and the PDA plates were taken.
Inoculation of PDA with botanical extract mixed with fungal spores: PDA plates are inoculated at 4 or 5 specific points. Each point represents a different solution of extract and fungal spores or a different concentration of the botanical composition solution mixed with fungal spores.
To test antifungal or antimicrobial efficacy of three concentrations of extracts test were done on the following organisms:
Referring now to Table 15 and
Referring now to
Referring now to
Referring now to
Referring now to
Xanthomonas
compestris
Salmonella
Salmonella
Salmonella
Streptomyces
scabies
Fusarium graminearum inoculated with 1.0% and 2.0% the botanical composition (and somewhat 0.5%) seem to have reduced growth compared to the control. No growth of fungi is observed on fungi treated with the thyme mix solutions. Both the botanical composition and the tincture inhibit the growth of hyphae.
Referring to
Fusarium graminearum
Referring to Table 16, the botanical composition is believed to allows thymol to be effective as an antimicrobial or antifungal agent in concentrations that are not effective when thymol is used alone on the following bacteria/fungi:
A synergistic effect is also contemplated with other commercial botanicals, such as thymol. Referring to
Referring now to
Referring now to
Referring now to
Referring now to
Botrytis cinerea
Fusarium graminearum
In summary and referring to Table 17 and to
At concentration rates below 2% of the botanical composition, the herbal extract does not cause any phytotoxic response on leaves of lettuce, tomato, tobacco, hops and Cannabis when applied as foliar or soil drench treatments. The botanical composition is believed to be a safe option for the application in agricultural practices. The application of the botanical composition induced a significant increase in chlorophyll content (tomato and lettuce seedlings and tobacco mature plants), height (tomato seedlings, lettuce and tobacco) and fresh & dry weight (lettuce and tobacco) compared to controls.
The pilot experiment on Cannabis plants showed that soil-drench, foliar, or a combination (soil-drench and foliar) of concentrations ranging from 0.03 to 2% of the botanical composition lead to enhanced bud size and yield. Furthermore, the treatment of soybean seeds with a concentration of 1% of the botanical composition and/or with a combination of the botanical composition and the seaweed algae Stimulagro lead to significant enhancement on soybean growth and early growth as compared to water control. Although Stimulagro was used in these tests, other types of seaweeds or seaweed compositions may be used in combination with the botanical composition. One such example of seaweed to be used may be Ascophyllum nodosum. The botanical composition was found to have synergy with the sea algae or seaweed, irrespetive of the extraction or processing method of such sea algae or seaweed. The results mean that the botanical composition may be used as a biostimulant agent to promote the growth of plants. More so, the amendment of the botanical composition with a soil mix, such as the commercially available Agro mix® G6 (Fafard), resulted in a positive boosting impact on plants, such as but not limited to lettuce and tobacco, and promoted the growth parameters, such as but not limited to chlorophyll content, height, fresh and dry weight of such plants. The amended botanical composition further suppresses grey mold disease. Hence, soil amendment of the botanical composition with soil mixes, such as but not limited to Agro mix® (lettuce and tobacco) may help in the production of healthier plants as shown with lettuce and tobacco and is speculated to show similar results on other plants. The botanical composition components and molecules may be interacting positively with the components of a soil mix, such as Agro mix® G6 (Fafard).
The botanical extract of the botanical composition is free of direct inhibitory effect on bacteria and fungi. Only antifungal properties were observed on F. graminarium. In-vitro trails showed a potential synergistic effect with other botanicals, such as but not limited to thyme leaf and yarrow extracts and thymol, lead to improved antimicrobial properties against important bacterial or fungal Phytopathogens. The in-vitro study assessing the germination rate of Botrytis cinerea and Fusarium graminearum showed that the botanical composition had a stimulatory effect on spore germination.
Based on the repeated demonstration of a preventive and treatment antifungal effect, the botanical composition generally induces a plant-based response against the infections including potentially a type of resistance. Also, the capacity of the botanical composition to cure (e.g. powdery mildew on Cannabis) and prevent fungal diseases e.g. powdery mildew on Cannabis and grey mold on tomatoes, lettuce, and hops through foliar application or soil drench and powdery mildew on Cannabis through foliar application are generally improved over control. The botanical composition may be used as a biopesticide agent to promote the resistance of plants to fight diseases.
In yet another experiment, a composition comprising botanical plant extracts in accordance with the embodiments presented above has been analysed in order to determine the metabolites present components.
In such an experiment, two series of tests were conducted, a first on leaf extracts and a second on root extracts. In such an exemplary experiment, the analysis methods comprised QE Orbitrap MS (LC/MS/MS) and GC/EI/MS metabolite profiling.
In the tests involving leaf extracts, the identified bioactive metabolites were found to be selected amongst the followings: apigenin, genistein, genipin, p-coumaroyltyramine 18-hydroxyoleate, 4-coumaroylquinate, chlorogenic acid, genistin, caffeoylshikimate, 15-HETE, p-Hydroxybenzoic acid, Succinic acid, Tyrosol, γ-Hydroxybutyric acid, Vanillic acid, 3-Hydroxybenzoic acid, Acetic acid, Caffeic acid, Phenylacetic acid, Phosphoric acid.
Still referring to the leaf extracts tests, the following metabolites were found to have different roles in the physiology of the plants: traumatic acid, Abscisic acid, Epijasmonic acid, jasmonate, salicyl-HCH, traumatin, gibberellin, 7-isomethyl-jasmonate, a-linolenate, indole-3-acetate, a,a-Trehalose, a-Linolenic acid, D-Fructose.
In the tests involving leaf extracts, the identified bioactive metabolites were found to be selected amongst the followings: 13-epoxyoctadeca-9; 11-dienoate, ferulate, 9(S); 12(S); 13(S)-trihydroxy-10(E)-octadecenoic acid, apigenin, genistein, genipin, p-coumaroyltyramine 18-hydroxyoleate, 4-coumaroylquinate, chlorogenic acid, genistin, caffeoylshikimate, vanillin, Succinic acid, Tyrosol, γ-Hydroxybutyric acid, Vanillic Acid, 4-Coumaric acid, Acetic acid, Caffeic acid, Phenylacetic acid, Phosphoric acid, Pantothenic acid.
Still referring to the leaf extracts tests, the following metabolites were found to have different roles in the physiology of the plants: traumatic acid, Abscisic acid, Epijasmonic acid, 7-iso-jasmonate, jasmonate, salicyl-HCH, traumatin, gibberellins, 7-isomethyl-jasmonate, a-linolenate, indole-3-acetate, a,a-Trehalose, glutathione, salicylate, a-tocopherol, a-Linolenic acid, D-Fructose, GABA.
A metabolomics analysis was further conducted. The results are presented in the Table 18, showing the relative exemplary concentration of alkaloids in the compound. Alkaloids may have numerous advantages in the protection and growth of plants.
While illustrative and presently preferred embodiment(s) of the invention have been described in detail hereinabove, it is to be understood that the inventive concepts may be otherwise variously embodied and employed and that the appended claims are intended to be construed to include such variations except insofar as limited by the prior art.
The present patent application claims the benefits of priority of commonly assigned U.S. Patent Application No. 62/878,600 entitled “METHODS AND COMPOSITIONS OF THE BOTANICAL EXTRACT CELEXT07 TO PROMOTE AND BOOST PLANT GROWTH AND PREVENT AND SUPRESS PLANT DISEASES” and filed at the United States Patent and Trademark Office on Jul. 25, 2019.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2020/051030 | 7/27/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62878600 | Jul 2019 | US |
