Composition containing L-carnitine and seaweed extract for alleviating abiotic stress in plants

Abstract

A method for preventing or alleviating the effects of abiotic stress in a plant involves administering to the plant, including the seed of the plant, or to the growth medium of the plant, an effective amount of L-carnitine and a seaweed extract.

Description

[Technical field]

[0001] The present disclosure relates generally to the field of methods and compositions for alleviating abiotic stress in plants. [Background technology]

[0002] Currently, various strategies are used to mitigate the effects of different forms of abiotic stress on plants. Many of these are directed at physically mitigating the effects of a particular stressor, for example by applying water or shading materials to deal with extreme temperatures or light intensities. Other approaches involve genetically engineering plants to be more resilient to stresses such as elevated salinity or extreme temperatures.

[0003] These types of measures can be labor intensive and consequently expensive, may only be useful for mitigating the effects of certain stress factors, and / or may only be applicable to certain plant species.

[0004] As a result, there remains a need for alternative methods, compounds, and compositions that are effective in mitigating the effects of abiotic stress on plants while being sustainable, cost-effective, and simple to use through current agricultural practices.

[0005] The invention disclosed herein overcomes previous deficiencies in the art by providing novel compositions and methods for the treatment and mitigation of abiotic stress in plants. Summary of the Invention

[0006] A method for preventing or alleviating the effects of abiotic stress in a plant is provided, comprising administering to the plant, to a seed of the plant, or to the growth medium of the plant an effective amount of a combination comprising L-carnitine and a seaweed extract.

[0007] Also provided is a plant biostimulant composition having L-carnitine and seaweed extract as active ingredients.

[0008] In one embodiment, the method comprises administering 50-800 g of L-carnitine per ton of seeds; and administering 50-800 g of seaweed extract per ton of seeds.

[0009] In some embodiments, the seaweed used to produce the extract is from the class Phaeophyceae, and in some embodiments, from the species Ascophyllum nodosum. The seaweed extract may be produced using alkaline hydrolysis extraction.

[0010] In some embodiments, the combination consists or consists essentially of L-carnitine and seaweed extract, and optionally, water.

[0011] In some embodiments, the plant is a cereal or legume, optionally wheat, corn, or soybean.

[0012] In some embodiments, the weight ratio of L-carnitine to seaweed extract used in the compositions or methods is between 10:1 and 1:10, optionally between 2:1 and 1:2, and in some embodiments, about 1:1.In some embodiments, the composition is an aqueous composition containing 100-250 g / L of seaweed extract and 100-250 g / L of L-carnitine.

[0013] Also provided is a seed treatment comprising or being a composition as defined herein. In one embodiment, the L-carnitine and the seaweed extract are both in particulate form.

[0014] Also provided is a kit comprising a first package containing L-carnitine and a second package containing seaweed extract, and instructions for combining L-carnitine and the seaweed extract to produce an effective amount of a plant biostimulant composition. The plant biostimulant composition is preferably a plant biostimulant composition as defined above.

[0015] During use, the method, biostimulant or seed composition may prevent or alleviate one or more of the symptoms of abiotic stress as defined herein. [Brief description of the drawings]

[0016] [Figure 1] An example is provided of improved vigour 21 days after sowing in winter wheat (variety Marius) seed treated with Ascophyllum nodosum extract (ANE), L-carnitine (CAR), or a combination thereof, when exposed to drought stress (50% reduced irrigation) 4 days after emergence, compared to stressed and unstressed controls according to Example 1. [Diagram 2] An example is provided of improved vigor 42 days after sowing in winter wheat (variety Marius) seed treated with ANE, CAR, or a combination thereof when exposed to drought stress (50% reduced irrigation) 4 days after emergence compared to stressed and unstressed controls according to Example 1. [Diagram 3] An example is provided in which winter wheat (variety Marius) seed-treated with ANE, CAR, or a combination thereof showed increased leaf fresh weight 50 days after sowing when exposed to drought stress (50% reduced irrigation) 4 days after emergence compared to stressed and unstressed controls according to Example 1. [Figure 4]An example is provided in which winter wheat (variety Marius) seed-treated with ANE, CAR, or a combination thereof showed increased root fresh weight 50 days after sowing when exposed to drought stress (50% reduced irrigation) 4 days after emergence compared to stressed and unstressed controls according to Example 1. [Diagram 5] An example is provided in which winter wheat (variety Marius) seed-treated with ANE, CAR, or a combination thereof showed increased plant height 22 days after sowing when exposed to salt stress (irrigation with 8 g / l NaCl) 4 days after emergence, compared to stressed and unstressed controls according to Example 2. [Figure 6] An example is provided in which winter wheat (variety Marius) seed-treated with ANE, CAR, or a combination thereof showed increased plant height 35 days after sowing when exposed to salt stress (irrigation with 8 g / l NaCl) 4 days after emergence, compared to stressed and unstressed controls according to Example 2. [Figure 7] An example is provided in which relative chlorophyll content (SPAD) was increased 29 days after sowing in winter wheat (variety Marius) seed-treated with ANE, CAR, or a combination thereof when exposed to salinity stress (irrigation with 8 g / l NaCl) 4 days after emergence, compared to stressed and unstressed controls according to Example 2. [Figure 8] An example is provided of increased plant vigor 43 days after sowing in winter wheat (variety Marius) seed-treated with ANE, CAR, or a combination thereof, when exposed to salt stress (irrigation with 8 g / l NaCl) 4 days after emergence, compared to stressed and unstressed controls according to Example 2. [Figure 9] An example is provided in which, when exposed to salt stress (irrigation with 8 g / l NaCl) 4 days after emergence, leaf biomass was increased 50 days after sowing in winter wheat (variety Marius) seed-treated with ANE, CAR, or a combination thereof, compared to stressed and unstressed controls according to Example 2. [Figure 10] An example is provided in which soybeans (variety Mungo) seed treated with ANE, CAR, or a combination thereof showed increased plant vigor 51 days after sowing when exposed to drought stress (50% reduced irrigation) 4 days after emergence compared to stressed and unstressed controls according to Example 3. [Figure 11] An example is provided in which soybean (variety Mungo) seed treated with ANE, CAR, or a combination thereof showed increased leaf fresh weight 51 days after sowing when exposed to drought stress (50% reduced irrigation) 4 days after emergence compared to stressed and unstressed controls according to Example 3. [Figure 12] An example is provided in which soybean (variety Mungo) seed-treated with ANE, CAR, or a combination thereof showed increased leaf dry weight 51 days after sowing when exposed to drought stress (50% reduced irrigation) 4 days after emergence compared to stressed and unstressed controls according to Example 3. [Figure 13] An example is provided in which, when exposed to drought stress (75% reduced irrigation) 3 days after sowing, corn (variety H140354) seed-treated with ANE, CAR, or a combination thereof showed increased root length 10 days after sowing compared to stressed and unstressed controls according to Example 4. [Figure 14] An example is provided in which when exposed to drought stress (75% reduced irrigation) 3 days after sowing, photosynthetic efficiency (Phi2) was increased 10 days after sowing in maize (variety H140354) seed-treated with ANE, CAR, or a combination thereof compared to stressed and unstressed controls according to Example 4. [Figure 15]An example is provided in which non-photochemical quenching (NPQt, an indicator of plant stress level) was increased 10 days after sowing in corn (variety H140354) seed-treated with ANE, CAR, or a combination thereof, compared to stressed and unstressed controls according to Example 4, when exposed to drought stress (75% reduced irrigation) 3 days after sowing. [Figure 16] An example is provided in which, when exposed to drought stress (75% reduced irrigation) 3 days after sowing, plant photosystem linear electron flow (LEF) was increased 10 days after sowing in maize (variety H140354) seed-treated with ANE, CAR, or a combination thereof compared to stressed and unstressed controls according to Example 4. [Figure 17] An example is provided in which photosynthetic efficiency (Phi2) was increased 10 days after sowing in maize (variety H140354) seed-treated with ANE, CAR, or a combination thereof when exposed to salinity stress (10 ml of 58 g / l NaCl) 3 days after sowing, compared to stressed and unstressed controls according to Example 5. [Figure 18] An example is provided in which the linear electron transport reaction (LEF) of the plant photosystem was increased 10 days after sowing in maize (variety H140354) seed-treated with ANE, CAR, or a combination thereof, when exposed to salt stress (10 ml of 58 g / l NaCl) 3 days after sowing, compared to stressed and unstressed controls according to Example 5. [Figure 19] An example is provided in which non-photochemical quenching (NPQt, an indicator of plant stress level) was increased 10 days after sowing in maize (variety H140354) seed-treated with ANE, CAR, or a combination thereof, compared to stressed and unstressed controls according to Example 5, when exposed to salinity stress (10 ml of 58 g / l NaCl) 3 days after sowing. [Figure 20]An example is provided in which non-photochemical quenching (NPQt, an indicator of plant stress level) was reduced 10 days after sowing in wheat (variety Skyfall, SEED-0035) seed-treated with ANE, CAR, or a combination thereof, when exposed to low temperature stress, compared to stressed and unstressed controls according to Example 6. The center line indicates the median, the box limits indicate the 25th and 75th percentiles, and the whiskers extend from the 1st / 3rd quartiles to data points less than 1.5 times the interquartile range apart. [Figure 21] 7 provides an example of reduced non-photochemical quenching (NPQt, an indicator of plant stress level) 17 days after sowing in soybean (variety Marula, SEED-003) seed-treated with ANE, CAR, or a combination thereof, when exposed to salinity stress, compared to stressed and unstressed controls according to Example 7.1. The center line indicates the median, the box limits indicate the 25th and 75th percentiles, and the whiskers extend from the 1st / 3rd quartiles to data points less than 1.5 times the interquartile range. [Figure 22] An example is provided in which, when exposed to salinity stress, soybean (variety Marula, SEED-003) seed treated with ANE, CAR, or a combination thereof exhibited increased shoot weight 17 days after sowing compared to stressed and unstressed controls according to Example 7.2. The center line indicates the median, the box limits indicate the 25th and 75th percentiles, and the whiskers extend from the 1st / 3rd quartiles to data points less than 1.5 times the interquartile range apart. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] As used herein, "abiotic stress" refers to the negative impact of non-living factors on plants. These factors can include, but are not necessarily limited to, extreme heat and cold, drought or flood water stress, high or low light intensity, elevated salinity, and soil toxicity. Thus, the results of abiotic stress are typically those that negatively affect crop yield and quality.

[0018] As used herein, "plant" may refer to any organism of the plant kingdom. In preferred embodiments, the plant is a terrestrial plant. In some embodiments, a crop plant. Unless the context dictates otherwise, administering a compound or composition as described herein to a plant will be understood to include administering the compound or composition to any part of the plant and at any stage of growth, for example, to the seeds, roots, or leaves of the plant.

[0019] As used herein, "biostimulant" refers to a product composed of substances, microorganisms, and / or other materials capable of stimulating nutrient processes, independent of their nutrient content, either in the plant or in its surrounding growing environment, improving the plant's nutrient utilization efficiency, resistance to abiotic stress, and / or crop quality.

[0020] As used herein, "alleviating the effects of abiotic stress" refers to reducing, reversing, or preventing one or more symptoms of abiotic stress better than a control plant or part thereof (i.e., a plant or part thereof exposed to the same abiotic stress but not in contact with a composition of the present invention).

[0021] Methods for identifying and measuring symptoms of abiotic stress in plants are known to those of skill in the art and may include visual assessment of plant vitality, such as reduced number or size of plants or parts thereof, reduced seed germination or emergence, or reduced seedling growth rate or vigor; gravimetric assessment of biomass yield, such as fresh or dry weight of shoots or roots; optical scanner-based assessment of plant parts, such as scanning of leaves or root systems and algorithmic determination of leaf area or root length; physiological or biochemical assessments, such as cell membrane stability or relative leaf water content; and photosynthetic assessment of plant stress levels using reflectance or spectroscopy-based methods, such as determining photosynthetic efficiency, linear electron transport reactions, non-photochemical quenching, and relative chlorophyll levels.

[0022] As used herein, an "effective amount" is an amount sufficient to alleviate the effects of abiotic stress, which may be determined using one or more of the symptoms discussed above. Effective amounts vary with the type and condition of the plant, and the nature and duration of the abiotic stressor (or anticipated abiotic stressor in the case of preventative treatments).

[0023] As used herein, "growth medium" is used generically to refer to a substrate in which plants are established or grown, and may include, for example, soil or artificial media, including media suitable for hydroponic applications such as rock wool or vermiculite.

[0024] As used herein, "L-carnitine" (β-hydroxy-γ-N-trimethylaminobutyric acid) is a derivative of the amino acid lysine. It was first isolated from meat in 1905 and plays a critical role in the oxidation of long-chain fatty acids for energy production in animal tissues. Commercial uses of L-carnitine include its use as a dietary supplement and as a foaming agent in some cosmetic products.

[0025] Naturally occurring amino acids, including carnitine, phenylalanine, lysine, etc., can be obtained by extraction, fermentation, enzymatic reactions, and synthesis. Methods for the synthetic production of L-carnitine are known to those skilled in the art, and L-carnitine is commercially available from a variety of sources.

[0026] Marine algae have been used as agricultural inputs for hundreds of years. It was first used as a soil conditioner where seaweed was incorporated into the growing medium and decomposed by soil microorganisms. More recently, it has served as a raw material for the production of "seaweed extracts" used as biostimulants. Species such as Ascophyllum nodosum, Durvillaea potatorum, Ecklonia Maxima, Uiva Lactuca, and Gelidium robustum have been used in the production of plant biostimulants and, in some embodiments, may be used in the methods and compositions provided herein.

[0027] As used herein, "seaweed extract" refers to a product obtained from marine algae, preferably red, brown, or green macroalgae, through a process of extracting and concentrating biostimulatory compounds. Methods for obtaining extracts from seaweed are known to those skilled in the art, see, for example, EL Boukhari MEM, Barakate M, Bouhia Y, Lyamlouli K. Trends in Seaweed Extract Based Biostimulants: Manufacturing Process and Beneficial Effect on Soil-Plant Systems. Plants 2020, 9, 359. doi:10.3390 / plants9030359. Furthermore, in some embodiments, the seaweed extract may be prepared from a combination of seaweed species.

[0028] The processing / extraction of the seaweed can be carried out using solvents, acids, bases, enzymes, or mechanical means, eventually in any combination. Preferably, the processing / extraction is carried out by contacting the seaweed with an aqueous solution that includes an alkaline extractant.

[0029] For the purposes of the present invention, the base is preferably an inorganic base selected from NaOH, KOH, Na2CO3, K2CO3, or any combination thereof. The alkaline solvent concentration ranges from 1% to 10% w / w, more specifically, from 2% to 5% w / w.

[0030] Preferably, the temperature of the extraction step is in the range of 20° C. to 100° C., and the extraction time is in the range of 30 minutes to 18 hours at a pressure in the range of 1 to 6 Bar.

[0031] If it is desired to use only the extract in the formulation, the extraction step may be followed by a further step of separating / removing the non-solubilized components. The removing / separating step is preferably carried out by decantation, filtration or centrifugation. Alternatively, a suspension containing both extracted and non-extracted components can be used.

[0032] According to a preferred embodiment, the seaweed extract used in the present invention is prepared from the brown marine algae Ascophyllum nodosum. The resulting extract may be in the form of a soluble liquid, a liquid concentrate, or a water-soluble powder.

[0033] Provided herein is a plant biostimulant composition comprising an effective amount of at least one amino acid betaine and a seaweed extract, in particular L-carnitine, and a seaweed extract, preferably from the class Phaeophyceae, more preferably from the species Ascophyllum nodosum. In some embodiments, the active ingredients of the biostimulant composition are limited to L-carnitine and a seaweed extract, preferably Ascophyllum nodosum. In some embodiments, the biostimulant composition consists or essentially consists of L-carnitine and a seaweed extract, preferably Ascophyllum nodosum, and optionally water. In this context, an "active ingredient" can be understood as an ingredient that has an observable biostimulant effect, i.e., an effect of preventing or alleviating the effects of abiotic stress, which can be identified or measured based on one or more symptoms identified herein, and which is different from an excipient that does not have such an observable effect, for example, either by its nature or as a result of minor ingredients present in the combination.

[0034] The effectiveness of the combination of ANE and L-carnitine in alleviating the effects of abiotic stress is evident from the examples.In light of the evidence of effectiveness, additional experiments were carried out to determine the synergistic effect of ANE and L-carnitine in alleviating abiotic stress.As detailed in the examples, there is evidence supporting the synergistic effect when plants are exposed to different forms of abiotic stress, including low temperature and salinity.

[0035] In one embodiment, the biostimulant composition may be in the form of a powder containing the amino acid betaine, preferably L-carnitine, and seaweed extract powder. The composition may be dispersed in a liquid medium for ease of application.

[0036] In some embodiments, the weight ratio of the combination of L-carnitine and seaweed extract used in the compositions or methods provided is between 10:1 and 1:10, and in some embodiments, between 2:1 and 1:2. In some embodiments, approximately equal amounts by weight of L-carnitine and seaweed extract are used.

[0037] In some embodiments, the biostimulant composition containing L-carnitine and Ascophyllum nodosum extract may be in the form of a liquid, which may be particularly suitable for foliar application or root irrigation.

[0038] In some embodiments, the compositions provided herein may be formulated as an aqueous concentrate containing a range of 100-250 g / l of seaweed extract, preferably Ascophyllum nodosum extract, in combination with 100-250 g / l of L-carnitine.

[0039] In some embodiments, the compositions as described herein can be used as seed treatments. Seed treatment formulations and techniques are commercially available, and methods of applying seed treatments are known to those skilled in the art and can include direct application of seed treatments to the seeds using commercially available seed treatment equipment.

[0040] Depending on the range of conditions, effective amounts typically range from 50-800 g L-carnitine per tonne of seed for seed treatments, and 50-800 g seaweed extract, preferably Ascophyllum nodosum extract, per tonne of seed.

[0041] In a preferred embodiment, the compositions described herein for use as seed treatments may be formulated as aqueous concentrates containing in the range of 100-250 g / l of Ascophyllum nodosum extract in combination with 100-250 g / l of L-carnitine.

[0042] The compositions provided herein may also contain suitable formulation excipients known to those skilled in the art and commercially available. In some embodiments, the formulation excipients are suitably selected from, but not exclusively, surfactants, wetting and dispersing agents, adhesion / binding agents, antifreeze agents, antifoam agents, preservatives, rheological aids, and, where appropriate, colorants.

[0043] The treatments described herein can also be preventative, for example, the methods and compositions provided herein can be provided as a seed treatment prior to an anticipated stressor event, such as a cold or heat wave, to mitigate the effects of the stressor.

[0044] In their broadest embodiments, the methods, compounds, and compositions described herein can be used in the cultivation of any plant, but in some embodiments, they can be used in the cultivation of commercially important plants, including, but not limited to, grains (including corn seeds, wheat, alfalfa, barley, rye, oats), vegetables (including peppers, tomatoes, lettuce, carrots, potatoes), and other important crops such as cotton, rice, soybeans, canola, rapeseed, etc.

[0045] All documents referenced herein are incorporated by reference, with the understanding that any patent, publication, or other disclosure material incorporated herein by reference is incorporated, in whole or in part, only to the extent that the incorporated material does not contradict any definitions, statements, or other disclosure material set forth in this disclosure. Thus, and to the extent necessary, the present disclosure as expressly set forth herein shall take precedence over any conflicting material incorporated herein by reference.

[0046] It will be understood that numerous modifications thereto will be apparent to those skilled in the art. Accordingly, the above description and accompanying drawings should be interpreted as illustrative of the present invention and not in a limiting sense. It will be further understood that this specification is intended to cover any variations, uses, or adaptations in accordance with the principles of the present invention, and is intended to include such departures from the present disclosure as may be applied to the essential features herein described above, as come within known or customary practice in the art to which this invention pertains, and within the scope of the appended claims as follows. EXAMPLES

[0047] Example 1: Wheat drought stress greenhouse trial. Winter wheat seeds (variety Marius) were treated with water (unstressed and stressed control treatments), or with an aqueous solution of Ascophyllum nodosum extract (ANE) at 1 ml / kg, or with an aqueous solution of L-carnitine hydrochloride (CAR) at 0.3 g / kg, or with a combination of Ascophyllum nodosum extract at 1 ml / kg with L-carnitine hydrochloride at either 0.1, 0.2, 0.3, or 0.4 g / kg, in a total applied volume of 10 ml / kg. Treated wheat seeds were sown in soil in pots with 10 biological replicates per treatment in a completely randomized block design. Four days after full emergence, all but the unstressed control treatments were drought stressed through a 50% reduced irrigation regime relative to the full irrigation received by the unstressed control.

[0048] As evident in Figure 1 and Table 1, plant vigor (rated on a scale of 0-10, with 0 being a plant with no leaves and 10 representing full vigor, which is the standard leaf vigor of a crop under normal conditions) increased by up to 17.6% in plants seed-treated with 1 ml / kg ANE and 0.4 g / kg CAR 21 days after sowing compared to stressed controls. By 42 days after sowing, this difference increased to 20.8% compared to stressed controls, as shown in Figure 2 and Table 2.

[0049] [Table 1]

[0050] [Table 2]

[0051] The improvement in plant vigor conferred by the seed treatment with the combination of ANE and CAR was translated into an increase in biomass yield assessed 50 days after sowing, with a maximum increase in leaf fresh weight of 22.1% (Figure 3, Table 3) and fresh root weight of 62% (Figure 4, Table 4) in plants seed-treated with 1 ml / kg ANE and 0.4 g / kg CAR.

[0052] [Table 3]

[0053] [Table 4]

[0054] Example 2: Wheat Salt Stress Greenhouse Trials. Winter wheat seeds (variety Marius) were treated with water (unstressed and stressed control treatments), or with an aqueous solution of Ascophyllum nodosum extract (ANE) at 1 ml / kg, or with an aqueous solution of L-carnitine hydrochloride (CAR) at 0.3 g / kg, or with a combination of Ascophyllum nodosum extract at 1 ml / kg with L-carnitine hydrochloride at either 0.1, 0.2, 0.3, or 0.4 g / kg, in a total applied volume of 10 ml / kg. Treated wheat seeds were sown in pots containing soil with 10 biological replicates per treatment in a completely randomized block design. Four days after full emergence, all treatments except the unstressed control treatments were subjected to salinity stress through irrigation with saline (8 g / l NaCl) for the remaining tests. The unstressed control treatments were irrigated with non-saline solution.

[0055] As can be seen in Figure 5 and Table 5, plants seed treated with 1 ml / kg ANE and 0.4 g / kg CAR 22 days after sowing increased plant height by up to 10.9% compared to the stressed control. By 35 days after sowing, the increase in height in this treatment group increased to 23.3% compared to the stressed control (Figure 6, Table 6).

[0056] [Table 5]

[0057] [Table 6]

[0058] At 29 days after sowing, the relative chlorophyll content (SPAD) of the plants showed a 25.5% increase in the same treatment group compared to the stressed control (Figure 7, Table 7). [Similar mitigating effects on photosynthetic rate and / or capacity as revealed by SPAD were seen in corn plants exposed to low temperature stress under protocols similar to Examples 6 and 7 below.] The increase in plant stress tolerance was similarly reflected in plant vigor assessments 43 days after sowing, with an increase of up to 16.6% over the stressed control (Figure 8, Table 8). By 50 days after sowing, an increase of up to 42.7% in fresh leaf biomass was observed in plants seed-treated with 1 ml / kg ANE and 0.3 g / kg CAR, demonstrating a strong induction of long-term salt stress tolerance from the combined treatment of ANE and CAR (Figure 9, Table 9).

[0059] [Table 7]

[0060] [Table 8]

[0061] [Table 9]

[0062] Example 3: Soybean drought stress greenhouse trial. Soybean seeds (variety Mungo) were treated with water (unstressed and stressed control treatments), or with an aqueous solution of Ascophyllum nodosum extract (ANE) at 1 ml / kg, or with an aqueous solution of L-carnitine hydrochloride (CAR) at 0.3 g / kg, or with a combination of Ascophyllum nodosum extract at 1 ml / kg with L-carnitine hydrochloride at either 0.1, 0.2, 0.3, or 0.4 g / kg, in a total applied volume of 10 ml / kg. Treated soybean seeds were sown in pots containing soil with 10 biological replicates per treatment in a completely randomized block design. Four days after full emergence, all but the unstressed control treatments were drought stressed through a 50% reduced irrigation regime relative to the full irrigation received by the unstressed control.

[0063] As can be seen in Figure 10 and Table 10, plants seed-treated with 1 ml / kg ANE and 0.2 g / kg CAR 51 days after sowing showed an increase in plant vitality of up to 25% compared to the stressed control. Correspondingly, the treatment group showed a 34.4% increase in leaf fresh weight (Figure 11, Table 11) and a 66.7% increase in leaf dry weight (Figure 12, Table 12) compared to the stressed control. This demonstrates a clear alleviation of the yield-reducing effect of induced drought stress imparted by the combined seed treatment of ANE and CAR. A similar protective effect on shoot fresh weight 17 days after sowing was observed for soybean plants germinated from seeds treated with a combination of 0.36 g ANE and 0.30 g L-carnitine exposed to drought stress under a separate protocol.

[0064] [Table 10]

[0065] [Table 11]

[0066] [Table 12]

[0067] Example 4: Corn drought stress controlled environment laboratory study. Corn seeds (variety H140354) were treated with water (unstressed and stressed control treatments), or with an aqueous solution of Ascophyllum nodosum extract (ANE) at 2 ml / kg, or with an aqueous solution of L-carnitine hydrochloride (CAR) at 0.3 g / kg, or with a combination of Ascophyllum nodosum extract at 2 ml / kg with L-carnitine hydrochloride at 0.3 g / kg, in a total applied volume of 10 ml / kg. Treated corn seeds were sown in field soil inside cell packs set up in greenhouse trays in a completely randomized block design with 16 biological replicates per treatment. Three days after sowing, all treatments except the unstressed control treatment were drought stressed through a 75% reduced irrigation regime relative to the full irrigation received by the unstressed control, and were evaluated 10 days after sowing.

[0068] As can be seen in Figure 13 and Table 13, seed treatment with 2 ml / kg ANE and 0.3 g / kg CAR resulted in a 30.6% increase in maize root length 10 days after sowing compared to the stressed control. The combined seed treatment with ANE and CAR also resulted in a 43.2% increase in the level of photosynthetic efficiency (Photosystem II efficiency, Phi2) (Figure 14, Table 14), a 40.2% reduction in non-photochemical quenching (NPQt, which is the amount of incoming light that is regulated away from the photosynthetic process to reduce damage to the plant and is an indicator of plant stress level) (Figure 15, Table 15), and a 46.2% increase in the linear electron transport reaction (LEF) of the plant photosystem (Figure 16, Table 16). In particular, linear electron transport measurements demonstrated a surprising and unexpected synergistic effect, with 2 ml / kg ANE resulting in an 18% increase in LEF and 0.3 g / kg CAR resulting in a 4.3% reduction in LEF, while the combined treatments produced a 46.2% increase. These results demonstrate the increased ability of corn plants exposed to drought stress to perform photosynthesis and maintain root growth conferred by the combined seed treatment with ANE and CAR.

[0069] [Table 13]

[0070] [Table 14]

[0071] [Table 15]

[0072] [Table 16]

[0073] Example 5: Corn Salt Stress Controlled Environment Laboratory Study. Corn seeds (variety H140354) were treated with water (unstressed and stressed control treatments) or 2 ml / kg Ascophyllum nodosum extract (ANE) in combination with either 0.2, 0.3, or 0.4 g / kg L-carnitine hydrochloride (CAR) in a total application volume of 10 ml / kg. Treated corn seeds were sown in field soil inside cell packs set up in greenhouse trays in a completely randomized block design with 16 biological replicates per treatment. Four days after sowing, all treatments except the unstressed control treatment were subjected to salinity stress through irrigation with saline (10 ml of 58 g / l NaCl). The unstressed control treatment was irrigated with non-saline solution. Evaluations were performed 11 days after sowing.

[0074] As can be seen in Figure 17 and Table 17, the combined seed treatment with 2 ml / kg ANE and 0.3 g / kg CAR resulted in an 87.8% increase in the level of photosynthetic efficiency (efficiency of photosystem II, Phi2) in salt-stressed maize plants. An increase of up to 118.5% in the linear electron transport reaction (LEF) of the plant photosystem (Figure 18, Table 18) and a reduction of up to 39.5% in the non-photochemical quenching (NPQt, an indicator of plant stress level) (Figure 19, Table 19) were further observed.

[0075] [Table 17]

[0076] [Table 18]

[0077] [Table 19]

[0078] Example 6: Exposing wheat to cold stress. A study was carried out to evaluate the effectiveness of seed treatments in mitigating the effects of a further form of abiotic stress, namely cold stress.

[0079] Seed treatment solutions were prepared according to Table 20.

[0080] [Table 20]

[0081] 0.1 mL of the treatment solution was pipetted around the rim into a 500 mL glass beaker and 10 g of wheat seeds (variety Skyfall, reference number SEED-0035) were added. The beaker was swirled by hand for 1 minute to coat the seeds, then swirled intermittently, if necessary, until dry.

[0082] The study was set up according to the following protocol. 1. 32 8-0-6 cell packs were fitted with colored stickers to indicate five treatments, with four cell packs for the unstressed control and seven for the remaining four treatments. 2. 6 L of stockpiled field soil and 600 mL of water were mixed thoroughly in a container. 3. If the soil became too dry, an additional 300 mL of water was added, or as needed. 4. Each cell pack was filled with pre-moistened soil, tapped once on the counter to fill any gaps, filled to the brim, and leveled. The filled cell packs were placed in a 4-fold flat tray in a randomized block design. 5. Steps 3-4 were repeated in random order across treatment groups and replicates. 6. After all the cell packs were filled, a stamp was used to create holes of uniform depth in each cell pack. Three seeds per cell were planted at 1 inch and then covered with soil. 7. Each tray was watered with 750 mL by removing one cell pack and pouring water into the bottom of the tray. A 7 inch plastic dome was placed over each tray and these were grown under the following conditions: 25 / 20°C day / night, 16 / 8 hours, 70% humidity, PAR of 400 μmol m-2 s-1.

[0083] The unstressed control was watered ad libitum throughout the experiment. The unstressed control was separated from the rest of the experiment. Measurements were taken 10 days after sowing.

[0084] Plants were exposed to low temperature stress for 4 days in a growth chamber (conviron) and measurements were taken 10 days after sowing. Environmental conditions: 13 / 8°C day / night, 16 / 8 h photoperiod, 60% humidity, 600 ppm and 400 μmol m -2 s -1 PAR.

[0085] Estimates of non-photochemical quenching (NPQt), the amount of incident light that is diverted away from photosynthetic processes to reduce damage to the plant, were obtained using a MultispeQ (B-4003-AA) from PhotosynQ, following the manufacturer's instructions. A total of 12-14 plants per treatment were measured for photosynthesis measurements. Results are shown in Table 21 and Figure 20. Parametric analysis of variance (ANOVA) was performed using the aov function in the ggpubr package, and multiple comparison tests were performed with Tukey's test (alpha = 0.1) using the TukeyHSD function in the stats package in R.

[0086] [Table 21]

[0087] To supplement the evidence of observed efficacy, synergy assessment was performed. Synergy was calculated using two different methodologies, both of which are supported in the following literature: 1) Equation III from Colby, SR1967. Calculating synergistic and antagonistic responses of herbicide combinations. Weed Science 15:20-22, and 2) Equation 2 from Huang, Z., and KAFalco. 2021. Synergy assessment for plant growth by independent joint action theory. HortScience 56(5):623-626. doi:10.21273 / HORTSCI15731-21. A one-sample two-tailed t-test was used to assess the statistical significance of the difference between observed and predicted means.

[0088] Both methodologies suggested synergistic effects.

[0089] Method 1: Independent association effect (Colby 1967 Percent of control) The percent of control predicted by Colby's method for the independent associative effect of the combination of 0.36 g ANE + 0.30 g carnitine on NPQt is 68.05. The actual result was 88.83, which gives a synergy ratio of 1.31. This is greater than the predicted result, meaning that the combination performed farther from 0 than expected.

[0090] Method 2: Independent association action (Huang and Falco 2021 Plant Growth). The mean predicted value by the Huang and Falco method for the independent associative effect of the combination of 0.36 g ANE + 0.30 g carnitine on NPQt is 0.28. The observed result is 0.37, which is 30.53% greater than the predicted result.

[0091] Example 7: Exposing soybeans to salt stress. To complement the efficacy studies of Examples 2 and 5, further studies were conducted to evaluate the effectiveness of seed treatments in mitigating the effects of salt stress in soybean plants, specifically to investigate evidence of synergistic effects.

[0092] The same seed treatment solutions (Table 20) and test protocol as in Example 6 were used, but the seeds were planted at 1.5 inches. To coat the seeds, 0.3 mL of the treatment solution was pipetted around the rim into a 500 mL glass beaker and 30 g of soybean seeds were added. Marula soybean seeds from Prograin (reference number SEED-003) were used without any other treatments.

[0093] Non-photochemical quenching Estimates of NPQt were obtained using a MultispeQ™ (B-4003-AA) from PhotosynQ according to the manufacturer's instructions. A total of 12-14 plants were measured per treatment for photosynthesis measurements. Results are shown in Table 22 and Figure 21. For Example 7, parametric analysis of variance (ANOVA) was performed using the aov function in the ggpubr package, and multiple comparison tests were performed with Tukey tests (alpha = 0.1) using the TukeyHSD function in the stats package in R.

[0094] [Table 22]

[0095] Synergy was assessed using two methodologies described in Example 6. Both methodologies were suggestive of synergy.

[0096] Method 1: Independent association effect (percent of Colby 1967 control) The percent of control predicted by Colby method for the independent associative effect of the combination of 0.36 g ANE + 0.30 g carnitine on NPQt is 74.91. The actual result was 79.4, which gives a synergy ratio of 1.06, which is greater than the predicted result and means that the combination performed further from 0 than expected.

[0097] Method 2: Independent association action (Huang and Falco 2021 Plant Growth). The mean predicted value by the Huang and Falco method for the independent associative effect of the combination of 0.36 g ANE + 0.30 g carnitine on NPQt is 0.65. The observed value is 0.69, which is 6% greater than the predicted value.

[0098] Shoot Weight Shoot weight was determined to assess whether the positive effects observed in NPQt were reflected in more robust plant growth. Seven cell packs per treatment were measured for shoot weight. Although sample size was limited, the combination showed a positive effect on growth consistent with the positive effects observed in NPQt.

[0099] The results are shown in Table 23 and Figure 22.

[0100] [Table 23]

[0101] Synergy was assessed using two methodologies described in Example 6. Both methodologies were suggestive of synergy.

[0102] Method 1: Independent association effect (percent of Colby 1967 control) The percent of control predicted by Colby method for the independent associative effect of the combination of 0.36g ANE + 0.30g carnitine on shoot fresh weight is 90.93. The actual result was 102.54, which gives a synergy ratio of 1.13, which is greater than the predicted result and means that the combination performed farther from 0 than expected.

[0103] Method 2: Independent association action (Huang and Falco 2021 Plant Growth). The mean predicted value by the Huang and Falco method for the independent associative effect of the combination of 0.36 g ANE + 0.30 g carnitine on shoot fresh weight was 1.5. The actual value was 1.69, which was 12.78% greater than the predicted value (P = 0.14).

[0104] It is to be understood, therefore, that the above described and illustrated embodiments are intended to be exemplary only, the scope being indicated by the appended claims.

[0105] Many additional features and combinations of improvements of the invention will become apparent to those of skill in the art upon reading this disclosure.

Claims

1. A method for preventing or mitigating the effects of abiotic stress in a plant, comprising administering an effective amount of a combination comprising L-carnitine and seaweed extract to the plant, the seeds of the plant, or the growing medium of the plant.

2. The method according to claim 1, wherein the method is a seed treatment agent, and the method comprises administering 50 to 800 g of L-carnitine per ton of seeds and administering 50 to 800 g of seaweed extract per ton of seeds.

3. The method according to claim 1, wherein the seaweed used to produce the extract is from the class Phaeophyceae.

4. The method according to claim 1, wherein the seaweed extract is produced from the species Ascophyllum nodosum.

5. The seaweed extract is produced using alkaline hydrolysis extraction, as described in claim 4. Method of loading.

6. The method according to claim 1, wherein the combination comprises L-carnitine and seaweed extract, and optionally water, or is essentially composed of them.

7. Mitigating abiotic stress is effective compared to control plants exposed to abiotic stress but not administered the combination, or crops grown under similar environmental conditions that encountered abiotic stress but were not administered the combination. a) Increase in plant vitality, b) Increased root growth and development, c) Increased growth and development of shoots, d) Increase in plant growth rate, e) Increase in the rate and capacity of photosynthesis, f) Improvement of yield, The method according to claim 1, comprising one or more of the following.

8. The method according to claim 1, wherein the plant is a cereal or a legume, optionally wheat, corn, or soybean.

9. A plant biostimulant composition comprising L-carnitine and seaweed extract as active ingredients, and optionally water.

10. The composition according to claim 9, wherein the seaweed extract is from the class Phaeophyceae.

11. The composition according to claim 10, wherein the seaweed extract is from the species Ascophyllum nodosum.

12. The composition according to claim 9, wherein the weight ratio of L-carnitine to the seaweed extract in the composition is 10:1 to 1:

10.

13. The composition according to claim 12, wherein the weight ratio of L-carnitine to the seaweed extract in the composition is 2:1 to 1:

2.

14. The composition according to claim 9, comprising approximately equal weights of L-carnitine and the seaweed extract.

15. The composition according to claim 9, wherein the composition is an aqueous composition containing 100 to 250 g / L of seaweed extract and 100 to 250 g / L of the L-carnitine. thing.

16. When applied in an effective amount to a plant, the seeds of the plant, or the growing medium of the plant, the composition alleviates abiotic stress in the plant, compared to a control plant that was not administered the composition and was exposed to the same or equivalent abiotic stress as the plant. a) Increase in plant vitality, b) Increased root growth and development, c) Increased growth and development of shoots, d) Increase in plant growth rate, e) Increase in the rate and capacity of photosynthesis, f) Improvement of yield, The composition according to claim 9, comprising one or more of the following.

17. A seed treatment agent comprising the composition described in claim 9.

18. The composition according to claim 9 or the seed treatment agent according to claim 17, wherein both L-carnitine and the seaweed extract are in the form of fine particles.

19. A kit comprising a first package containing L-carnitine, a second package containing a seaweed extract, and instructions for combining the L-carnitine and the seaweed extract to produce an effective amount of a plant biostimulant composition.

20. The kit according to claim 19, wherein the plant biostimulant composition is the plant biostimulant composition according to claim 9.

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