Plant biostimulant formulations for promoting plant growth and managing abiotic stress

Abstract

A plant biostimulant formulation for promoting plant growth, increasing abiotic stress tolerance and increasing crop yield is described. The formulation comprises extracts of brown algae (kelp), green algae (Ulva), and wheat grass (Triticum aestivum) obtained by cold percolation process using solvents such as water-ethanol which retains polyphenols, flavonoids, tannins, antioxidants and other secondary metabolites. These bioactive compounds synergistically mitigate oxidative stress, increase membrane stability and improve root architecture, thereby increasing plant stress resistance under drought, salinity and temperature stresses. The formulation of the invention ensures higher retention of bioactive compounds and consistent efficacy. Field trials confirmed yield increase of 15-20% thereby indicating improved crop performance and climate stress resistance. The formulation is suitable for foliar spray, drenching, granular coating or seed treatment making it suitable for a variety of crops.

Description

Technical Field

[0001] This invention relates to the fields of agriculture, plant biostimulants, and crop stress management, thereby relating to overall plant health and sustainable agriculture. Specifically, this invention relates to a plant biostimulant formulation derived from a combination of seaweed and plant materials using a water-ethanol percolation process, said formulation for promoting plant growth, improving tolerance to abiotic stresses, and increasing crop yield. Background Technology

[0002] Given the significant economic, social, and environmental challenges associated with modern agriculture, including declining soil fertility, increased abiotic stress, and over-reliance on chemical fertilizers, there is an urgent need for a sustainable and effective biostimulant solution. Abiotic stresses such as drought, salinity, and temperature fluctuations can severely impact crop yield and quality; therefore, developing formulations that enhance plant stress resistance and crop performance is crucial.

[0003] Due to their benefits in promoting growth, alleviating stress, and improving soil health, seaweed-based agricultural inputs have been extensively studied. However, most commercially available formulations primarily utilize brown algae (such as *Tetranychus buergerianum*) or red algae (*Kappaella asiatica*), employing fermentation or hydrolysis-based extraction methods. Global demand for biostimulants is projected to grow exponentially, significantly pressuring the limited natural resources of key seaweeds such as *Tetranychus buergerianum* and *Kappaella asiatica*. Current estimates indicate that the biostimulant industry's seaweed demand is approximately 10 to 12 million metric tons annually, projected to grow by 30% to 40% over the next five years, driven by the increasing adoption of sustainable agricultural applications. This surge in demand could lead to raw material shortages, significantly increasing procurement costs by 20% to 25% annually, further straining the supply chain. Ensuring sustainability and exploring alternative species or synthetic solutions will be crucial to mitigating this impact.

[0004] Fermentation-based and hydrolysis-based extraction methods are the most commonly used in the biostimulant industry. These methods inherently produce inconsistent products due to the untargeted and degradative nature of the underlying processes. In fermentation, microbial enzymes break down feedstocks in an uncontrolled, pathway-dependent manner, resulting in unpredictable mixtures of metabolites that vary with microbial type, inoculum size, temperature, pH, and time. Similarly, acid or base hydrolysis leads to the indiscriminate cleavage of biomolecules (polysaccharides, proteins, lipids, phenols), resulting in over-degradation, loss of functional active ingredients, and batch-to-batch inconsistencies.

[0005] In contrast, solvent-based cold percolation is a non-destructive extraction method that preserves the natural biochemical structure. Because no enzymatic or chemical degradation occurs, the extraction selectively extracts the desired class of metabolites while maintaining their original configuration and proportions. This results in more standardized, reproducible compositions with improved retention of bioactive secondary metabolites, crucial for consistent product performance. This method ensures enhanced efficacy in stress tolerance and plant growth promotion while reducing thermal and enzymatic degradation of key metabolites.

[0006] Global agricultural practices are increasingly shifting towards sustainable and environmentally friendly alternatives to chemical fertilizers. Overuse of fertilizers leads to soil degradation, nitrogen leaching, and greenhouse gas emissions, thus necessitating solutions that can improve nutrient use efficiency (NUE) and reduce dependence on synthetic inputs.

[0007] A review of existing patents and literature reveals several biostimulant formulations utilizing seaweed extracts, each with different compositions and extraction methods. While these formulations are beneficial for plant growth and stress management, they are primarily dependent on specific seaweed species, fermentation-based extraction methods, or limited retention of bioactive compounds, creating a gap in the development of more sustainable, efficient, and broad-spectrum biostimulants.

[0008] US20150351408A1 describes a biostimulant formulation derived from the hydrolysis of alginic acid and its combination with humic / fulvic acid, focusing on delivering amino acids to alleviate plant stress and enhance nutrient absorption. However, it does not explore the role of various algae combinations or plant sources such as wheatgrass extract in improving antioxidant activity and nutrient uptake.

[0009] EP3294066A4 discloses a formulation combining seaweed juice and a hydrolysate obtained after extracting the juice from seaweed residue, intended to promote plant growth. However, this method relies on hydrolysis-based extraction and is particularly focused on the red algae Kappa algae; unlike the cold percolation water-ethanol extraction process proposed in this invention, hydrolysis-based extraction can degrade certain bioactive compounds.

[0010] US20210323885A1 discusses fertilizers in which seaweed extracts, such as those derived from one or any combination of *Kappaphycus alvarezii*, *Ascophyllum nodosum*, *Ecklonia maxima*, *Durvillea potatorum*, *Macrocystis Pyrifera*, *Sargassum spp.*, and *Laminaria digitata*, are used to increase crop yield and improve soil health. However, this document does not address direct biostimulant effects, nor does it optimize the combination of seaweed extracts with plant extracts for stress tolerance.

[0011] US10954170B2 outlines a method for producing a concentrated aqueous seaweed extract, specifically using fermentation to extract a concentrate from a Sargassum species for use as a biostimulant. While effective, this method does not employ extraction methods based on non-fermentation solvents, which ensure better retention of bioactive compounds and product consistency.

[0012] Taking all the above factors into account, it is clear that there is a need to develop a sustainable, commercially viable and effective alternative to promote plant growth, improve tolerance to abiotic stresses and reduce reliance on fertilizers, while ensuring environmental safety and agricultural sustainability.

[0013] Purpose of the invention The main objective of this invention is to provide a plant biostimulant preparation to promote plant growth, enhance tolerance to abiotic stresses, and increase crop yield.

[0014] Another object of the present invention is to provide a plant biostimulant preparation comprising a combination of brown algae extracts, such as Durvillaea potatorum extract; green algae extracts, such as Ulva lactuca extract; and plant extracts, such as Triticum aestivum extract.

[0015] Another object of the present invention is to provide a plant biostimulant formulation developed using a gentler cold percolation method with solvents such as water-ethanol, thereby ensuring the retention of key bioactive compounds for optimal plant health.

[0016] Another object of the present invention is to provide a plant biostimulant preparation that enhances the plant's resistance to stress under drought, salinity and temperature fluctuations, while also improving root development, overall plant vigor and crop productivity. Summary of the Invention

[0017] Before describing the invention, it should be understood that the invention is not limited to the specific methods and materials described, as these methods and materials can vary according to those skilled in the art. It should also be understood that the terminology used in the specification is for describing particular embodiments only and is not intended to limit the scope of the invention.

[0018] This invention relates to a plant biostimulant formulation comprising a combination of brown algae extracts, such as Durvillaea potatorum extract; green algae extracts, such as Ulva lactuca extract; and plant extracts, such as Triticum aestivum extract, for promoting plant growth, improving tolerance to abiotic stresses, and increasing crop yield.

[0019] According to one aspect of the invention, the present invention relates to a cold percolation extraction method using solvents such as water-ethanol to obtain stable and highly bioactive formulations, thereby ensuring the retention of polyphenols, flavonoids, tannins, antioxidants and other secondary metabolites that are essential for plant health.

[0020] According to one aspect of the invention, the formulation utilizes the synergistic effect of extracts from marine giant kelp (Durvillaea potatorum), Ulvalactuca, and wheatgrass (Triticum aestivum), which collectively enhance root development and stress resistance, thereby increasing plant tolerance to drought, salinity, and temperature fluctuations. The formulation works by enhancing plant metabolism, root architecture, and cellular defense mechanisms, thereby improving water retention, increasing biomass accumulation, and enhancing photosynthetic efficiency. The inclusion of wheatgrass extract provides an additional source of antioxidants and tannins, which further protect the plant from oxidative stress and enhance the plant's overall physiological response to challenging environmental conditions.

[0021] According to one aspect of the invention, the main delivery methods of the biostimulant formulation include foliar spraying, root irrigation, granule coating, and seed treatment, ensuring efficient uptake and maximum efficacy across a variety of crop species. For agricultural applications, the formulation is designed for various environmental conditions, including areas experiencing water scarcity, saline-alkali stress, and temperature fluctuations. The formulation has been validated through laboratory and field studies, demonstrating yield increases of 15% to 20%, making it a viable and cost-effective alternative to traditional biostimulants. Comparative trials in different crop species, including tomatoes, rice, peppers, soybeans, and cotton, have shown significant improvements in plant vigor and total yield, further establishing the formulation's superior performance under controlled and real agricultural conditions. Attached Figure Description

[0022] The invention will be fully understood by referring to the following detailed description, which will be taken in conjunction with the accompanying drawings. The drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments of the invention and, together with the detailed description, serve to explain the principles and implementation of the invention.

[0023] Figure 1 Charts illustrating the barrel compatibility of illustrative compositions with a variety of foliar fertilizers and agrochemicals are presented. Figure 2 A chart depicting the batch consistency of the compositions produced over a three-year period is presented; Figure 3 A graph depicting high formulation stability was presented; Figures 4a to 4f The figure illustrates a schematic diagram of how the biostimulant formulation of the present invention regulates different pathways. Detailed Implementation

[0024] Before describing this invention, it should be understood that the invention is not limited to the methods described, as these methods can vary according to those skilled in the art. It should also be understood that the terminology used in this specification is for describing particular embodiments only and is not intended to limit the scope of the invention. Throughout this specification, the word “comprise” or variations such as “comprises” or “comprising” will be understood to mean including the stated elements, integers, or steps, or a set of stated elements, integers, or steps, but not excluding any other elements, integers, or steps, or any other set of elements, integers, or steps. The use of the expressions “at least” or “at least one” means the use of one or more elements, ingredients, or amounts in a manner that may be embodied in embodiments of the invention to achieve one or more of the desired purposes or results. Various embodiments of the invention are described below. However, it should be noted that the invention is not limited to these embodiments, but is intended to include obvious modifications.

[0025] This invention discloses a plant biostimulant formulation derived from extracts of brown algae (Durvillaea potatorum), green algae (Ulva lactuca), and wheatgrass (Triticum aestivum) using a cold percolation method with solvents such as water-ethanol, and a method for using this formulation to promote plant growth and yield and induce crop tolerance to abiotic stresses. Unlike traditional fermentation-based seaweed extracts, this formulation utilizes a water:ethanol solvent extraction method, ensuring the retention of key bioactive compounds such as polyphenols, flavonoids, terpenes, antioxidants, and polysaccharides. These bioactive molecules work synergistically to alleviate oxidative stress, improve membrane stability, enhance root architecture, and promote overall plant vigor. The inclusion of wheatgrass extract provides an additional antioxidant boost, further protecting plants from environmental stressors. The formulation is designed to improve stress tolerance under drought, salinity, and temperature fluctuations, while promoting plant health and yield. Comparative studies show that, compared to standard seaweed formulations, this formulation significantly improves plant water retention, antioxidant activity, and biomass accumulation. The present invention also provides a more efficient and scalable alternative to fermentation processes, ensuring consistent product quality and activity.

[0026] The raw materials used in the composition include brown algae belonging to the genus Durvillaea, which is selected from species of Durvillaea such as Durvillaea antarctica, Durvillea potatorum, Durvillaea poha, Durvillaea willana, Durvillaea fenestrate, Durvillaea amatheiae, Durvillaea incurvate, and Durvillaea chathamensis. The green algae were selected from Ulva australis, Ulva clathrata, Ulva intestinalis, Ulva linza, Ulva lactuca, Ulva compressa, Ulva fasciata, Ulvareticulata, Ulva quilonensis, Ulva gujaratensis, Ulva rigid, Ulva flexuosa, and Ulva paschima.The plant extracts are selected from common wheat (Triticum aestivum), durum wheat (Triticum durum), emmer wheat (Triticum dicoccu), round wheat (Triticum sphaerococcum), round wheat (Triticum sphaerococcum), wild emmer wheat (Triticum boeoticum), wild emmer wheat (Triticum dicoccoides), spelt wheat (riticum spelta), maca wheat (Triticum macha), Vavilov wheat (Triticum vavilovii), or other members of the Poaceae family, such as wild barley (Hordeum spontaneum), two-rowed barley (Hordeum distichum), two-rowed barley (Hordeum distichum), two-rowed barley (Hordeum distichum), wild six-rowed barley (Hordeum griocrithon), wild bottle-shaped barley (Hordeum lagunculiforme), long-bristled oats (Avena sterilis), and red oats (Avena). byzantine, wild oats (Avena fatua), white wheat (Avena sativa), millet (Panicum miliaceum), sorghum (Sorghum bicolor), and rice (Oryza sativa).

[0027] According to an embodiment of the present invention, the drying and pulverizing of seaweed and plant biomass includes the following steps. First, freshly harvested seaweed biomass is rinsed with clean water to remove surface salts, sand, and epiphytes. Freshly harvested plant biomass is first rinsed with clean water to remove sand and other impurities. The cleaned biomass is then dried under shaded conditions. The drying process is carried out in a well-ventilated shaded structure that allows sunlight and airflow to diffuse while preventing direct UV exposure, thereby ensuring the retention of bioactive compounds. The temperature is maintained below 45°C during drying and continuously monitored to avoid degradation of thermally unstable secondary metabolites. Drying continues until the moisture content of the biomass is reduced to below 10%, or until the biomass becomes brittle and crumbly to the touch. Once completely dried, the seaweed and plant biomass is ground using a hammer mill and passed through a 40-mesh sieve to obtain a fine, uniform powder suitable for solvent extraction. The resulting powder is stored at room temperature in a sealed, light-protected container until use.

[0028] According to an embodiment of the present invention, a solvent-based extraction process is performed on dried and pulverized *Ulva prostrata*, *Ulva spp.*, and wheat powder. This extraction process is designed to selectively separate secondary metabolites such as polyphenols, brown algal polyphenols, tannins, flavonoids, alkaloids, and terpenoids, while minimizing the recovery of large amounts of polysaccharides such as alginate and mannitol. First, each powder (10 kg) is pre-soaked in deionized water at 25°C at a ratio of 1:10 w / v for 24 hours for cold water pre-soaking. This hydrates the cell wall matrix and promotes the release of water-soluble impurities, thereby improving extraction efficiency. After pre-soaking, the hydrated biomass is transferred to a percolation chamber and slowly percolated using a water-alcohol solvent mixture containing 60% water and 40% ethanol (v / v) at a solvent-to-algae ratio of 10:1 (v / w). Percolation is carried out at ambient temperature under light-protected conditions to prevent oxidative degradation of photosensitive metabolites and continues for 12 to 14 days, until the percolate is visually and analytically depleted. The resulting extract was then filtered to remove particulate matter. The clarified supernatant was then stored at 25°C in the dark and then concentrated or analyzed further to ensure the retention of thermally unstable bioactive substances.

[0029] Different solvents and methods were tested for the extraction process. The extraction process was carried out continuously for 12 hours to 14 days to completely deplete the soluble metabolites in the biomass. After extraction, the solvent was removed by rotary evaporation under reduced pressure of 150 mbar and 40°C until a viscous, dark brown concentrate was obtained. The resulting extract was then standardized to a final concentration of 1,000 ppm total polyphenols (quantified as gallic acid equivalent using the Folin-Ciocalteu method) and 1,000 ppm tannins (determined using the DMBA method). The experimental results are detailed below: Water (cold soaking) 7.5 4.8 0.9 0.3 4.5 2.8 0.00 5 Low-phenol extraction, high-polysaccharide co-extraction 95% ethanol 11.2 10.6 2.3 0.8 1.2 0.6 1.20 3 It has good extraction results, high cost, and flammability issues. 80% methanol 12.0 11.4 2.6 0.9 0.9 0.5 0.90 2 It has strong extraction capabilities but also high toxicity and faces regulatory obstacles. Water-ethanol 60:40 (selected) 12.5 12.0 2.8 0.95 0.6 0.3 0.60 5 A balanced solvent system, an optimal ratio of active material to cost, and scalability. <![CDATA[Supercritical CO₂ (EtOH 5% to 10%)]]> 9.2 8.7 1.5 1.2 0.3 0.2 6 to 12 equivalent 2 High purity, expensive equipment, energy intensive Table 1 Solvent selection was guided by extraction performance and practical considerations related to safety, cost, and scalability. While safe and inexpensive, water was found ineffective for extracting most secondary metabolites, primarily producing polysaccharides. 95% ethanol offered good extraction efficiency but presented challenges due to its high cost and flammability. 80% methanol exhibited excellent solvent performance; however, its toxicity and regulatory restrictions made it unsuitable for agricultural inputs. Supercritical CO2 provided strong selectivity for certain metabolites but was ruled out due to its high operating costs and energy requirements, making it impractical for large-scale production. Ultimately, a 60:40 water-ethanol mixture was chosen as the optimal solvent system, providing high recoveries of polyphenols and brown algal polyphenols while minimizing the extraction of alginate and mannitol, offering a safe, cost-effective, and scalable solution.

[0030] According to embodiments of the present invention, the plant biostimulant formulation comprises 70% w / v brown algae (marine giant kelp) extract, 10% w / v green algae (Ulva lactuca) extract, and 20% w / v wheatgrass (common wheat) extract. The extracts are obtained using a water-ethanol solvent extraction process. The biostimulant promotes plant growth, enhances tolerance to abiotic stress, and increases crop yield. The formulation ensures the retention of key bioactive compounds, including polyphenols, flavonoids, tannins, antioxidants, and other secondary metabolites, which synergistically contribute to alleviating oxidative stress, enhancing membrane stability, improving root architecture, and increasing plant vigor.

[0031] 1. Brown algae extract Giant sea kelp Non-biological stress tolerance enhancers Improve drought resistance, salt and alkali resistance and heat resistance 70 2. Green algae extract Ulva lettuce growth stimulants Enhance plant metabolic activity and growth 10 3. Wheatgrass extract common wheat Antioxidants and metabolism boosters Enhance photosynthesis and reduce oxidative stress 20 Table 2 According to embodiments of the invention, the extract obtained using the described extraction process contains a variety of secondary metabolite classes typically ranging from 10 ppm to 50,000 ppm (w / w). Total polyphenols (in gallic acid equivalents) are recovered at high levels due to the efficiency of the 40% ethanol solvent system and are quantified using the Folin-Ciocalteu assay. Brown algal polyphenols, characteristic of brown seaweed, are similarly present in concentrations ranging from 10 ppm to 50,000 ppm and are determined using the DMBA method. Terpenoids appear in a comparable concentration range and are primarily associated with the lipophilic fraction, and are quantified by GC-MS. Flavonoids (including catechins and quercetin derivatives) are detected in concentrations ranging from 10 ppm to 20,000 ppm. In contrast, alginic acid and mannitol are present in trace to low levels (0.01% to 20,000 ppm) because their extraction is minimized by the non-acidic, low-temperature, non-fermentation solvent conditions employed, thereby reducing the presence of large amounts of polysaccharide and sugar alcohol impurities in the final extract.

[0032] Compatibility: According to embodiments of the invention, the formulation is designed for broad-spectrum compatibility with commonly used fertilizers, micronutrient blends, and crop protection products. Laboratory and field-scale evaluations have confirmed the absence of adverse interactions, ensuring consistent efficacy and performance when applied in standard agronomic programs.

[0033] The biological compositions of this invention are readily miscible in water and exhibit excellent tank compatibility with a variety of foliar fertilizers and various fertilizers (including agrochemicals). Tank compatibility was evaluated by mixing concentrated, ready-to-use dosage forms with fertilizers, micronutrients, insecticides, and pesticides. Figure 1 As illustrated, any changes in the spectral characteristics of the mixture were evaluated at 0, 2, 4, and 6 hours after mixing.

[0034] NPK, Micronutrients NPK of all levels It is even compatible with Cu, Zn, and sulfur. Incompatible with Cu and Zn Azoxystrobin Amistar Compatible Compatible Chlorpyrifos Saaf, Kavach, Bunos, Tafban, Dursban Compatible Compatible Captan Captan, Captaf, Taqat, Captol, Foltaf Compatible Compatible Manganese zinc Mancozeb, Curzate M8, Ridomil Compatible Compatible Carbendazim Carbendazim, Dhanustin, Cammando Compatible Compatible benomyl Benomyl / Benofit Compatible Compatible wettable sulfur sulfur Compatible incompatible glyphosate Round-up Compatible incompatible O,S-dimethylacetylthiophosphoramide Acephate / Asatof / Starthene Compatible incompatible Thiamethoxam Actara Compatible Compatible Imidacloprid Admire, Confidor Compatible Compatible Cypermethrin Cymbush Compatible Compatible fipronil Jump, Regent Compatible Compatible High-efficiency cyhalothrin Karate Compatible incompatible Malathion Malathion Compatible Compatible Dichlorvos Nuvan Compatible Compatible Butyl ether urea Pegasus Compatible incompatible Acetamiprid Pride / Rekord Compatible Compatible Table 3 Stability and consistency: The scale-up and manufacturing process enables the production of highly reproducible biological agents with consistent and well-defined compositions. Spectral characteristics of different batches from three different years were studied. Figure 2 The illustrated charts reveal a very high degree of adherence to spectral characteristics with minimal variation, indicating consistency across batches produced over a 3-year period. This represents a significant improvement over biostimulants based on fermented algae, which typically exhibit considerable batch-to-batch variability. Achieving consistent formulation quality is crucial, as it forms the basis for delivering reliable and reproducible field performance.

[0035] Shelf life: The formulation exhibits high formulation stability, with no significant phase separation, precipitation, or viscosity change at storage temperatures between 5°C and 45°C. Spectral characterization of individual batches was completed over four different year periods. (Example:) Figure 3 The illustrated charts reveal a high degree of adherence to spectral characteristics with minimal variation. Under recommended storage conditions, its active components retain their bioavailability for at least 4 years.

[0036] Heavy metal reduction: Compared with conventional fermentation-based algae biostimulants, the extraction method of the present invention also significantly reduces the heavy metal content in the final formulation, thereby enhancing product safety and environmental sustainability, as shown in Table 4.

[0037] Chromium (IV), calculated as Cr <0.1 mg / L Below the detection limit Below the detection limit lead <0.1 mg / L Below the detection limit Above the threshold Arsenic, calculated as (As2O3) <0.1 mg / L Below the detection limit Above the threshold cadmium <0.1 mg / L Below the detection limit Above the threshold copper <0.1 mg / L Below the detection limit Below the detection limit Zinc <0.1 mg / L Below the detection limit Below the detection limit mercury <0.1 mg / L Quarterly testing limit Above the threshold Table 4 toxicity: Toxicological evaluations have confirmed that the biostimulant of this invention is non-toxic to humans, animals, and beneficial non-target organisms when used according to label instructions. As illustrated in Table 5, the formulation is classified as low-risk with no reported phytotoxicity in the test crops. It complies with the safety standards for the use of biostimulants under current agricultural regulations.

[0038] Oral LD50 (rat) Non-toxic (maximum test dose: 5,000 mg / kg) Non-toxic (maximum test dose: 5,000 mg / kg) Transdermal LD50 (rat) Non-toxic (maximum test dose: 2,000 mg / kg) Non-toxic (maximum test dose: 2,000 mg / kg) Inhaled LC50 (rat) Non-toxic (maximum test dose: 10.04 ml / L) Non-toxic (maximum test dose: 10.04 ml / L) Eye irritation (rabbit) Minimal irritation (rating index: 2) Irritation, with mild conjunctival congestion reported (score index: 4). Skin irritation (rabbit) Non-irritating mild irritation Fish toxicity (96h LC50, tilapia) Non-toxic (maximum test dose: 100 mg / L) Due to its high pH, ​​it is less toxic. Poultry toxicity (oral LC50, domestic chickens) Non-toxic (maximum test dose: 5 ml / kg) Non-toxic (maximum test dose: 5 ml / kg) Bee toxicity (contact LD50, Western honeybee) Non-toxic (maximum test dose: 100 µl / bee) Non-toxic (maximum test dose: 100 µl / bee) Earthworm toxicity (contact LC50, Eisenia fetida) Non-toxic (maximum test dose: 1,000 ml / kg soil) Non-toxic (maximum test dose: 1,000 ml / kg soil) Table 5 Applications: Materials and Methods: According to embodiments of the present invention, the main delivery methods of the biostimulant formulation include foliar spraying, root irrigation, granule coating, and seed treatment, ensuring efficient uptake and maximum efficacy across a variety of crop types. The formulation can also be mixed with other agrochemicals (such as fertilizers, micronutrients, hormones, amino acids, protein hydrolysates, humic or fulvic acid, vitamins, insecticides, pesticides, etc.) for comprehensive application. For agricultural applications, the formulation is designed for various environmental conditions, including areas experiencing water scarcity, salinity stress, and temperature fluctuations. The formulation has been validated through laboratory and field studies, demonstrating its stress tolerance, growth, and yield enhancement.

[0039] According to embodiments of the present invention, comparative trials conducted in different crop species (including tomatoes, rice, peppers, soybeans, and cotton) for field application showed significant improvements in plant vigor and total yield, further establishing the superior performance of the formulation under controlled and real agricultural conditions. The biostimulant formulation works by enhancing plant metabolism, root architecture, and cellular defense mechanisms, thereby improving water retention, increasing biomass accumulation, and enhancing photosynthetic efficiency. The inclusion of wheatgrass extract provides an additional source of antioxidants and tannins, which further protect plants from oxidative stress and enhance the overall physiological response of plants under challenging environmental conditions.

[0040] Plant material and growing conditions: Rice (Oryza sativa L., variety: DRR Dhan 65) and tomato (Solanum lycopersicum) seedlings were planted in 12-inch pots containing a sterilized soil-sand-compost mixture in a 2:1:1 ratio. The plants were maintained in a controlled greenhouse environment with a temperature of 28°C, relative humidity of 60% to 70%, and a photoperiod of 14 hours light / 10 hours dark. After 21 days of growth under these conditions, stress treatment was initiated.

[0041] Biostimulant Preparation and Application: This study evaluated two biostimulant formulations. The first was a seaweed-plant combination consisting of extracts from brown and green algae, along with plant components; this formulation was applied as a foliar spray at a concentration of 4 mL / L, equivalent to 800 mL per acre. The second treatment consisted of a commercially available fermented *Lycopodium clavatum* extract, characterized by its high polysaccharide content, and was applied at the manufacturer's recommended dose of 2.5 mL / L. Both biostimulants were applied twice, once 15 days after sowing (DAS) and again at 21 DAS (i.e., three days before the application of the stress condition).

[0042] Stress treatment: After acclimatization, plants were placed under abiotic stress conditions for seven days. Drought stress was induced by stopping irrigation and maintaining soil moisture below 15% (v / v). Salt-alkali stress was applied by irrigating plants daily with a 150 mM NaCl solution. Heat stress was applied by transferring plants to a controlled environment chamber maintained at 45℃ ± 1℃ during the light period for six hours daily. Control plants were maintained under normal irrigation conditions without any stress.

[0043] Morphological and physiological measurements a. Root architecture: Plants were harvested seven days after the onset of stress, and the root system was analyzed using the WinRHIZO root scanning system or by manual imaging. Quantitative parameters recorded included total root length, root surface area, root volume, and total number of root tips.

[0044] b. Growth Indicators: Branch length, fresh weight, and dry weight of all experimental plants were measured. Relative water content (RWC) was calculated using the following formula: RWC = [(FW – DW) / (TW – DW)] × 100, where FW represents fresh weight, TW represents saturated fresh weight obtained after hydration for four hours, and DW represents dry weight measured after drying the sample at 70°C for 48 hours.

[0045] c. Membrane Stability Index (MSI): Leaf discs with a diameter of 0.5 cm were cut from treated and control plants and incubated in 10 mL of distilled water at 25°C. The conductivity of the solution was measured after four hours to obtain the initial conductivity value (C1). The samples were then boiled at 100°C for ten minutes, cooled, and measured again to obtain the total conductivity (C2). The membrane stability index was calculated as follows: MSI(%) = [1 – (C1 / C2)] × 100.

[0046] Detection of reactive oxygen species (ROS) in leaves a. DAB staining with hydrogen peroxide (H2O2): Leaves were immersed in a 1 mg / mL solution of 3,3′-diaminobenzidine (DAB) (pH adjusted to 3.8) and incubated under light for six hours. After staining, chlorophyll was removed by bleaching with ethanol, and the leaves were imaged under bright field conditions. The formation of a brown precipitate served as an indicator of H2O2 accumulation.

[0047] b. NBT staining for superoxide radicals: To assess superoxide levels, leaves were incubated in the dark for four hours in a 0.1% nitroblue tetrazolium (NBT) solution (prepared with 10 mM potassium phosphate buffer at pH 7.8). After staining, the leaves were destained in ethanol, and the formazan blue deposition was imaged. The staining intensity was digitally quantified using ImageJ software.

[0048] Statistical analysis: All experimental treatments were performed in at least five biological replicates. Analysis of variance (ANOVA) was performed on the data, and Tukey's HSD post-hoc test was used to determine significant differences between treatments at a significance threshold of p < 0.05. Where applicable, root structure characteristics and ROS staining intensity were quantified using digital image analysis tools.

[0049] The comparison of the effects of biostimulants under drought conditions (combined biostimulants and single-source biostimulants) is illustrated in Table 6: Branch length (cm) 20 ± 0.6 10.4 ± 0.7 11.7 ± 0.4 15.9 ± 0.7 14.6 ±0.4 12.7 ±0.2 17.7 ± 0.6 Branch dry weight (g) 1.1 ±0.1 0.6 ± 0.2 0.63 ± 0.3 0.88 ± 0.4 0.75 ±0.6 0.8 ±0.5 0.97 ± 0.7 Crown area (cm2) 55.6 ±0.3 15.73 ± 0.9 34.13 ± 1.1 40.59 ±1.3 39.1±0.8 35.03 ±1.5 48.59 ± 1.3 Relative moisture content (%) 85.7 ±1.1 33.5 ± 2.1 41.3 ± 1.7 63.4 ± 1.9 58.73 ±2.2 55.9 ±1.8 75.3 ± 1.5 Membrane stability index (MSI%) 87.7 ±4.2 42.5 ± 2.3 51.6 ± 2.7 64.1±3.1 68.29 ±2.7 62.3 ±2.1 77.1±1.9 Leaf curl index (%) 0 25.24 ± 1.1 41.47±1.3 15.46 ±1.4 13.45 ±1 12.24 ±1.3 11.06 ± 0.9 Chlorophyll content 42.62 ±1.4 49.41 ± 1.1 43.82 ± 1.6 52.52±1.9 56 ± 2.0 58.70 ±1.8 63.63 ± 2.2 DAB staining intensity (AU) - 210 ± 9 175 ± 11 121 ± 14 115 ± 10 95 ± 9 85 ± 5.4 NBT staining intensity (AU) - 198 ± 9 152 ± 12 108 ± 7 99 ± 2 90 ± 6 88 ± 3 Seedling survival rate (%) 95 ± 2 21 ± 5 33 ± 5 75 ± 4 80 ± 6 86 ± 3 91 ± 1 Table 6 The comparison of the effects of biostimulants (combined biostimulants and single-source biostimulants) under saline-alkali conditions is illustrated in Table 7: Branch length (cm) 21 ±0.7 10.8 ± 0.5 12.7 ± 0.4 15.9 ± 0.9 16.6 ±0.4 15.7 ±0.2 18.5 ± 0.3 Branch dry weight (g) 1.1 ±0.2 0.6 ± 0.4 0.82 ± 0.3 0.98 ± 0.6 1.03 ±0.5 0.98 ±0.4 1.05 ± 0.9 Crown area (cm2) 62.8 ±0.2 39.73 ± 1.3 41.13 ± 0.8 48.59 ±1.1 49.1±0.6 44.03 ±1.2 59.4 ± 1.1 Relative moisture content (%) 90.7 ±2.2 73.5 ± 2.4 76.3 ± 1.7 83.4 ± 2.1 85.63 ±1.6 85.9 ±1.7 89.3 ± 2.1 Membrane stability index (MSI%) 80.7 ±3.1 41.9 ± 2.8 48.2 ± 2.1 61.4±2.4 58.29 ±1.9 65.3 ±1.5 71.1±1.2 Chlorophyll content 40.6 ±1.9 25.1 ± 1.6 27.82 ± 1.3 33.5 ± 1.5 36 ± 1.9 35 ± 2.1 39 ± 2.3 DAB staining intensity (AU) - 201 ± 11 155 ± 8 131 ± 9 115 ± 12 101 ± 10 89 ± 6 NBT staining intensity (AU) - 189±9 108 ± 8 87 ± 7 90 ± 5 80 ± 6 78 ± 4 Seedling survival rate (%) 96 ± 1 15 ± 3 43 ± 6 81 ± 7 84 ± 5 87 ± 2 92 ± 3 Table 7 The comparison of the effects of biostimulants (combined biostimulants and single-source biostimulants) under heating conditions is illustrated in Table 8: Branch length (cm) 26.9 ±0.6 17.4 ± 0.5 19.7 ± 0.4 22.1 ± 0.6 22.6 ±0.8 21.7 ±0.3 24.7 ± 0.2 Branch dry weight (g) 1.1 ±0.2 0.56 ± 0.1 0.61 ± 0.2 0.77 ± 0.3 0.85 ±0.4 0.83 ±0.6 0.91 ± 0.3 Crown area (cm2) 52.7 ±0.5 35.3 ± 1.1 38.2 ± 1.2 45.9 ± 0.9 48.1 ±0.3 40.3 ±1.2 50.9 ± 1.1 Relative moisture content (%) 96.7 ±1.6 69.5 ± 2.3 76.3 ± 1.5 85.4 ± 2.1 92.63 ±1.7 90.9 ±1.8 95.3 ± 1.5 Membrane stability index (MSI%) 82.4 ±3.3 55.9 ± 3.0 59.2 ± 2.4 71.4±2.1 67.29 ±2.3 72.3 ±2.8 77.1±2.2 Chlorophyll content 41.32 24.31 ± 2.3 23.82 ± 1.6 30.52 ±2.1 31.5 ±3.9 32 ± 5.2 35 ± 3.5 DAB staining intensity (AU) - 168 ± 9 145 ± 7 109 ± 11 111 ± 5 95 ± 3 85 ± 2 NBT staining intensity (AU) - 149 ± 6 116 ± 9 101 ± 11 98 ± 7 86 ± 3 75 ± 2 Seedling survival rate (%) 95 ± 3 19 ± 2 68 ± 4 79 ± 2 82 ± 3 84 ± 2 91 ± 1 Table 8 Field studies: Comparative studies of combined biostimulants versus single-source biostimulants in tomatoes, cotton, soybeans, and peppers. Objective: This study aimed to evaluate and compare the agronomic performance, physiological response, and yield and quality attributes of various crop species (including tomato, cotton, soybean, and pepper) treated with two different classes of biostimulants. The first treatment consisted of a polyphenol-rich solvent extract of *Clerodendrum thomsoniae*, while the second treatment consisted of a traditionally fermented extract of *Clerodendrum thomsoniae* rich in polysaccharides. An untreated control group was included to establish a baseline for physiological and yield performance. The primary objective of this study was to determine how well the combination of biostimulants promoted plant vigor, stress tolerance, and crop productivity compared to traditional single-source biostimulants.

[0050] Experimental Design: A randomized complete block design was used in the field trial with cucumber as the model crop, with five replicates for each treatment. Each plot was measured to be 3 m × 2 m and contained ten evenly distributed plants. Three treatments were administered: a control group that did not receive biostimulants; a foliar spray of fermented *Lycopodium clavatum* extract at 5 mL / L; and a combination of biostimulants applied at the same concentration. Applications were made via foliar spray and root irrigation at 15, 30, 45, and 60 days post-transplanting. The trial was conducted in three agroclimatic zones: Dapoli, Dharwad, and Rahuri in India, representing diverse environmental conditions to ensure robustness of the surface results. All plots received uniform irrigation and fertilization (including standardized NPK input) and were maintained according to recommended cultivation methods suitable for each location.

[0051] Measurement parameters: Morphological, physiological, and yield-related parameters were recorded at multiple growth stages to quantify treatment effects. Plant height was measured at 30, 45, and 60 days post-transplantation (DAT) by recording the distance from the soil surface to the apical meristem. Crown spread was assessed at 45 and 60 DAT by measuring the maximum horizontal diameter of the plant. Chlorophyll concentration was measured at the same time points using a SPAD-502 chlorophyll meter. Reproductive development was monitored by recording 50% flowering days daily and manually calculating the number of flowers produced per plant between 45 and 60 DAT. Yield was assessed at harvest by determining the total fruit weight per plant and calculating the corresponding value in tons per hectare. Post-harvest fruit quality parameters were assessed by: total soluble solids (TSS) measured using a digital refractometer; hardness measured using a needle hardness tester; color measured using a visual rating or colorimeter; and titratable acidity determined by standard acid-base titration. These comprehensive assessments enable a full comparison of the performance of combined biostimulants with single-source biostimulants and untreated control groups.

[0052] The results of the field studies are presented in Table 9 below as the average of all studies. The seaweed-plant combination was statistically significantly superior to application of seaweed or plant extracts alone, with improvements observed in vegetative growth, early maturity, yield, and fruit quality. On average, the combination achieved a 13.6% improvement compared to the best-performing single component, indicating a synergistic rather than additive effect.

[0053] Plant height 12.97 14.2 15.5 15.5 15.1 16.42 6.34 -2.58 -2.58 Leaves / Plants 4.7 5 5.1 5.1 5.3 12.77 6.00 3.92 3.92 Male flowers / plants 8.55 6.8 7.1 6.5 10.8 26.32 58.82 52.11 66.15 female flowers / plants 4.4 4.65 4.7 5.2 5.85 32.95 25.81 24.47 12.50 50% of the flowering period days 38.25 37.5 38.5 39 35.5 -7.19 -5.33 -7.79 -8.97 Fruits / plants 7.15 7.3 7 7.32 8.9 24.48 21.92 27.14 21.58 Fruit length 14.35 14.77 14.9 15.16 16.81 17.14 13.81 12.82 10.88 Fruit circumference 30.18 30.58 30.1 31.23 32.72 8.42 7.00 8.70 4.77 Single fruit weight 72 81.5 73.5 102.5 115.5 60.42 41.72 57.14 12.68 Yield / acre 33.32 37.31 36.1 36.91 41.79 25.42 12.01 15.76 13.22 Table 9 Transcriptomics Research - Mode of Action Methods for sample collection and transcriptomic studies (RNA-Seq) of tomato plants treated with biostimulants: To compare transcriptomic changes in tomato plants treated with solvent-extracted formulations and fermented *Alternaria solani* biostimulants under field conditions, and to identify differentially expressed genes (DEGs) associated with growth, stress response, hormone signaling, and secondary metabolism. Same treatments as outlined in the field design: • T1—Untreated control group • T2 – Laminaria japonica extract – Traditional formulation • T3 - Combination Extract - Formulation of the Invention Sample collection for transcriptomics: Organization type The fifth leaf that has fully unfolded from the apex (vegetative growth stage) and the young flower bud (reproductive growth stage). Sampling time Ideally, this should occur 48 hours after the last application of the biostimulant (peak transcriptomic response), between 10:00 and 11:00 AM. Biological Repetition Three plants were processed each time (n=3), and samples were taken independently. Disposal Immediately cut leaf tissue (approximately 100 mg) and flash-freeze it in liquid nitrogen in the field. store Transported in dry ice and stored at -80°C until RNA extraction. RNA extraction: Total RNA was extracted using the Qiagen RNeasy Plant Mini Kit or an equivalent kit. This included on-column DNase treatment to remove genomic DNA. RNA quality was assessed using Nanodrop (260 / 280 and 260 / 230 ratios), Agilent Bioanalyzer, or TapeStation (RIN > 7.5), and quantified using the QubitRNA HS assay.

[0054] Library construction and sequencing: mRNA enrichment was performed using poly(A) selection; library construction: the RNA LibraryPrep Kit or an equivalent kit was used. All samples and pools were indexed. Paired-end sequencing (150 bp) was performed on an Illumina NovaSeq or HiSeq platform. The goal was approximately 30 million reads per sample.

[0055] Bioinformatics Analysis Quality inspection FastQC, Trimmomatic Read length comparison Use HISAT2 or STAR to compare to the tomato (Tomato) reference genome. Transcript assembly and quantification StringTie or featureCounts Differential expression analysis DESeq2 or edgeR Functional annotations Blast2GO, KEGG Mapper Pathway enrichment GO, MapMan, PlantReactome analyze: Transcriptomic analysis of the treated plants revealed different pathways of regulation of the formulations of the present invention, as illustrated in Table 10.

[0056] Photosynthesis (PSII / PSI) Increase PSII (10×) to maintain light collection (LHCB1↑), decrease PSI to limit ROS. Upregulation of PSII (3.3x) and PSI (3.3x) increases energy production through upregulation of 3X ATP synthase. Increase ATP synthase (7×), complex I / V (6.7×), and PSII (3×), and decrease PET5 to control ROS. The combined effect enhances light harvesting and optimizes photophosphorylation while maintaining ROS balance. Chlorophyll / Proheme Synthesis – – Heme ↑20× Maximize chlorophyll production and energy transfer Ribulose-1,6-bisphosphate carboxylase / carbon fixation – ↑2.5x ↑3x <![CDATA[Stabilizing CO2 Assimilation Across Growth Stages]]> Sugar transport Increased sucrose transport (10x) shifts carbon from lipids to polyphenols; limits saccharification. – Malate and glycerides increased by 10x, enhancing the glyoxylate cycle, carbon fixation (7.4×), and photorespiration-related recovery. Sugar transport for dual control of carbon for active growth: Fortisia preservation, 7525 recycling cycles – improving the carbon economy and replenishment recovery. Oxidative phosphorylation Complex I / V ↑ 10x, ↓ ROS complex ETC↑ (cytochrome bc1 / c oxidase) ↑Cross-key ETC enzymes ATP is continuously produced during growth and reproduction. GABA shunting / redox regulation GABA↑20x, putrescine↑9x – GABA↑3x, Dioxide reduction and pH buffering under stress Amino acid reprogramming (BCAA, Thr) – BCAA↑、Thr↑3x BCAA↑3x、Thr↑3x Energy supply and reproductive tissue support enhance osmotic protection, detoxification, and cellular integrity under stress. Arginine / Polyamine Pathway Putrescine ↑9x (via SAM) Arginine, ornithine, citrulline ↑ active Fertility support + Osmosis protection nitrogen flow Methionine cycle / SAM Homocysteine ​​↑2x, SAM ↑>20x – active Epigenetic control + cell stability EF Tu (protein synthesis) – ↑ ↑ Support the high protein requirements during fruit setting. Hormone regulation (GA / IAA / Zaatin) GA↑10x, Support 1 / GH3↑11x GA↑20x, IAA↑3.5x, Zeatin↑6.6x GA↑10x, Zeatin↑7x, IAA transport↑20x, Brassinosteroids↑2X, ABA-regulated stress↑10x Hormonal Rebalancing: Fortisia Supports Growth; 7525 Enhances Rooting and Stress Initiation—Together Providing Adaptive Plasticity MAPK – – MAPK18 Upregulation of mitigation pathways for drought stress, salinity stress, and heat stress Ethylene regulation – Inhibition ↑2.5x active Delay aging and extend shelf life Volatile organic compounds (VOCs) – 3,6-Nonadien-1-ol↑2.5x Uncertain, but exhibits BCAA activity. The aroma enhancement comes from Chiron Fruit softening / ripening enzymes – PG↑7x, Pectin↑3x – Chiron input improves texture and ripeness. Lipid signaling (PG / PA) – PG & PA↑ PG & PA↑ Fragrance transport, gamete formation, embryo development Phenylacetane (PAL active) <![CDATA[PAL↑10¹ 5 x→Shift towards soluble phenols]]> Moderate (via PR1) Chlorone synthase ↑7x Fortisea's ROS protection + Chiron's flavonoid signaling → dual function of antioxidation and regulation Lignin biosynthesis ↓ (Bypass to soluble pool) ↑7.2x balance Maintain fruit firmness without sacrificing elasticity Flavonoids (quercetin, kaempferol) Downward adjustment (↓9x) ↑ (Multiple flavonoids) ↑↑ (Multiple active categories) UV protection, auxin regulation, and flower induction support Anthocyanins / carotenoids Alphazoline and geraniol increased; astaxanthin increased by 4x Indirect ripening Activated through inheritance Synergistic effect of pigmentation and ROS defense Plant antitoxins / PR proteins – PR1↑5.1x, phytoalexin↑ PR1+ Chalcone-derived phytoalexin ↑ Systemic immunity + local defense + antioxidant activation PAMP-triggered immunity (PTI) – – flagellin and fungal PAMP↑20x Pathogen defense combines passive (chemical) and active (immune) defenses—from early identification to late-stage enhancement—to provide broad-spectrum protection. Epidermal wax cell wall – – Phospholipids, MECR↑2x Thicker stratum corneum, reinforced cell walls, and wax coating Table 10 like Figures 4a to 4f As illustrated, the biostimulant formulations of the present invention regulate different pathways. Figure 4a —Biostimulants significantly enhance photosynthetic performance by increasing chlorophyll synthesis sevenfold and photosynthetic system I and II activities threefold and sevenfold, respectively, resulting in greater light capture and stronger electron transport. This leads to increased ATP and NADPH production, while ribulose-1,2-bisphosphate carboxylase levels double and proheme synthesis increases twentyfold, thereby collectively strengthening carbon fixation and chlorophyll biosynthesis.

[0057] Figure 4b Significant increases in oxidative phosphorylation (6-fold), malate (6-fold to 8-fold), and D-glyceroate (7-fold to 8-fold) were observed, thereby improving ATP synthesis and reducing metabolic waste. Malate, as a redox carrier and carbon carrier, plays a crucial role in bridging organelles and supporting C4 / CAM carbon concentration strategies. This metabolic rebalancing replenishes TCA intermediates and enhances stress tolerance by balancing carbon and nitrogen flows.

[0058] Figure 4c—This illustrates how amino acid metabolism can be reprogrammed to enhance stress resistance and reproductive success. Key amino acids—serine, glycine, cysteine; valine, leucine, isoleucine; and glutamate—all showed a threefold increase, supporting energy production, protein synthesis, and flowering. Threonine conversion promoted the synthesis of branched-chain amino acids (BCAAs), while the phosphoserine pathway enhanced glutathione synthesis and redox balance, both crucial for flower development and stress tolerance.

[0059] Figure 4d —This demonstrates how hormone reprogramming enables plants to grow under stress conditions such as drought and salinity. Key changes include a 7-fold increase in zeatin content, which is used for cell division and root development, and a 20-fold increase in the content of auxin transporter ABCB1, which enhances growth directionality and vascular flow. The stress-responsive MAPK pathway is also activated, while ABA signaling increases 10- to 12-fold to enhance tolerance. These shifts collectively contribute to resistance and sustained growth under adverse conditions.

[0060] Figure 4e The study highlights how phenylpropane and flavonoid pathways are reprogrammed to enhance plant defense and stress adaptation. Key changes include a twofold increase in chalcon synthase activity and mobile antioxidants, as well as increased production of pigments such as flavonoids. Metabolites such as luteolin, quercetin, and naringenin enhance UV protection and antioxidant capacity, while changes in alcohol derivatives favor mobile defense rather than structural enhancement. These biochemical modifications support growth regulation and resistance.

[0061] Figure 4f This study demonstrates how upregulation of the MECR enhances plant lipid metabolism, thereby improving stress resistance. MECR activation (2.9-fold) stimulates mitochondrial fatty acid synthesis, leading to increased palmitic acid and phospholipids. This results in improved membrane fluidity, epidermal wax biosynthesis, and structural lipid production, thereby enhancing drought tolerance, pathogen defense, and post-stress recovery. Lipid signaling and membrane stability are key outcomes of this metabolic reprogramming.

[0062] The biostimulant formulations of this invention exhibit a range of functional properties that exceed those of traditional brown algae extracts, green algae extracts, or single-source plant preparations. These enhanced properties stem from the synergistic effects between the constituent classes of their metabolites, resulting in coordinated physiological effects that no single-source extract could produce. One of the defining characteristics of the composition is its ability to support the continuous transition between the vegetative and reproductive stages of plant development. This is achieved through the integrated delivery of metabolic energy substrates, biosynthetic precursors, and developmental signaling molecules, which work together to maintain uninterrupted physiological activity as the plant transitions from biomass accumulation to flowering and fruit germination.

[0063] Another notable characteristic of biostimulants is their ability to combine reactive oxidative (ROS) defense mechanisms with immune-initiating activity. The synergistic presence of phenolic antioxidants, pathogenesis-related (PR) proteins, and pattern-associated molecular pattern (PAMP) cues enhances basal immunity while modulating oxidative stress. This dual effect enhances stress tolerance without triggering a harmful hypersensitive response, thus contributing to sustained growth under both abiotic and biotic stress conditions.

[0064] The composition further supports organogenesis through finely regulated flavonoid-auxin interactions mediated by compounds such as luteolin, vitexin, and quercetin. These bioactive substances regulate auxin transport and sensitivity, thereby promoting precise angiogenesis and flower tissue development. Therefore, the formulation enables more uniform reproductive initiation and improves the structural integrity of developing organs. Importantly, the formulation avoids the hormonal overstimulation often encountered with single-source GA-based or auxin-based products, instead producing a controlled expansion of endogenous hormonal pathways. This mild hormonal response prevents abnormal growth while maintaining optimal metabolic activation.

[0065] Furthermore, the formulation enhances sensory and post-harvest quality characteristics through unique interactions of polysaccharide-regulated enzymes, lignin precursors, volatile organic compounds (VOCs), and antioxidant pigments. This synergistic effect contributes to improved fruit firmness, aroma characteristics, and shelf stability—attributes typically not observed when using single-source extracts lacking multi-pathway activity.

[0066] In summary, these features indicate that the biostimulant formulation of the present invention represents a novel, non-obvious, and synergistic biostimulant composition that integrates an alginate-derived formulation that confers vegetative growth vigor with a plant component that promotes reproductive development. The formulation uniquely combines redox stability with hormone homeostasis, photosynthetic efficiency with developmental signaling, and immune initiation with improved fruit quality. These multilayered effects are associated with different ploidy kinetics in primary and secondary metabolic pathways, responses that cannot be reproduced by either the alginate or plant component when applied independently. Therefore, the formulation is a first-class broad-spectrum biostimulant with significant functional differentiation and strong commercial potential.

[0067] In summary, the biostimulant formulations disclosed in this invention provide a scientifically validated, scalable, and sustainable solution for modern agriculture. By combining the unique bioactivities of extracts from marine giant kelp, Ulva lactuca, and wheatgrass, and utilizing a non-fermentation, cold percolation process, this invention sets a new benchmark for plant biostimulant efficacy, providing enhanced stress resistance, improved climate change resilience, higher yields, and long-term agricultural sustainability.

[0068] Novelty and difference of the present invention: • Seaweed Combination: Most commercially available seaweed-based formulations use fermented *Cladosporium* (brown algae) or red algae. However, this invention uses *Klephalopoda* (a less commercially available brown algae) and *Ulva* (green algae), creating a unique seaweed combination. A synergistic effect was observed between *Cladosporium*'s tolerance to abiotic stress and *Ulva*'s growth-promoting effect.

[0069] • Addition of Plant (Wheatgrass) Extract: The inclusion of wheatgrass extract is novel. Wheatgrass is known for its high content of antioxidants (vitamins, minerals, amino acids, and chlorophyll), which may enhance stress relief in plants through synergistic effects. Few (if any) commercially available agricultural formulations use wheatgrass extract. The combination of seaweed and plant extracts is very novel, resulting in a more synergistic formulation that combines the bioactive substances of seaweed and terrestrial plants. The addition of wheatgrass significantly improves antioxidant effects, reduces oxidative damage, and enhances photosynthetic efficiency under stress conditions.

[0070] • Extraction Methods (Water:Ethanol-Based Extraction): Popular extraction processes rely on acid / alkali treatment of biomass, followed by fermentation-based processes for *Laminaria japonica*, resulting in formulations with high pH values ​​of 8 to 9. In contrast, cold percolation extraction of marine giant kelp, *Ulva prostrata*, and wheatgrass using water:ethanol-based solvents stands out for its clear, free-flowing nature and low pH of 3 to 4. This method offers advantages such as higher recovery rates of bioactive compounds, faster processing, and consistency of extract composition. Solvent-based extraction methods retain specific bioactive compounds, particularly secondary metabolites that are degraded or modified during the fermentation process.

[0071] • Synergistic Phase Bridging: Synthesis supports the continuity between the vegetative and reproductive growth stages by integrating energy metabolism, biosynthesis, and signal transduction pathways. Neither algae nor plants can achieve this seamless transition alone, thus helping to reduce flower drop and improve fruit set.

[0072] • Integrated stress on the immune system: The formulation triggers a dual defense mechanism: scavenging phenolic ROS and immune activation via PR protein + PAMP-like signaling. Quantifiable results include reduced biomarkers of oxidative stress and enhanced disease tolerance.

[0073] • Cross-talk between flavonoids and auxin on organogenesis: Specific flavonoids (luteolin, vitexin, quercetin) regulate auxin transport and signal transduction, thereby promoting vascular tissue integrity, flower differentiation, and fruit germination. Precise flavonoids derived from plant components and seaweed matrix are novel.

[0074] • Controlled Hormone Surge: The combination avoids the harmful hormone surges (GA, auxin) commonly found in single-source products. Therefore, this combination is a balanced hormone regulation system, ensuring stable yields and reducing physiological imbalances.

[0075] Post-harvest quality enhancement: It offers unique synergistic effects in extending shelf life and sensory characteristics, such as pectin softening via PG enzymes, structural compactness achieved through lignin deposition, volatile organic compounds (VOCs) that contribute to aroma, and pigments and antioxidants that contribute to color stability and nutritional quality.

[0076] • Superiority over single inputs: When used alone, the combination consistently delivers greater than 13% to 15% better performance in terms of growth, yield, or quality compared to using seaweed or plant extracts alone.

[0077] advantage: The advantages of this invention are as follows: • The synergistic combination of extracts from brown algae (marine giant kelp), green algae (ulnatum lettuce), and wheatgrass (common wheat) enhanced plant stress tolerance and improved growth efficiency.

[0078] • Retaining key bioactive compounds (including polyphenols, flavonoids, tannins, antioxidants, and other secondary metabolites) ensures that plants receive optimal support for mitigating oxidative stress, improving membrane stability, and enhancing root development.

[0079] • Increased biomass accumulation, better water retention, and higher photosynthetic efficiency (specifically under drought, salinity, and temperature stress conditions).

[0080] • The use of cold percolation processes with solvents such as water-ethanol provides a highly stable and effective alternative for fermentation-based seaweed extracts, ensuring consistent retention of bioactive compounds and greater efficacy in promoting plant growth.

[0081] • Unlike traditional fermentation methods that may degrade essential bioactive substances, this process preserves and maximizes the potency of secondary metabolites that are crucial for plant resistance.

[0082] • The inclusion of wheatgrass extract further enhances the antioxidant activity of the formulation, providing better stress protection and thus improving the overall performance of the crop.

[0083] In addition, the formulation can be applied as a foliar spray, root drenching, granule coating, or seed treatment, making it a versatile and flexible solution suitable for a variety of agricultural applications.

[0084] Although the components and elements of the preferred embodiments have been emphasized to a considerable extent herein, it should be understood that various embodiments and modifications may be made to the preferred embodiments without departing from the principles of this disclosure. These and other changes to the preferred embodiments of this disclosure will be apparent to those skilled in the art from the disclosure herein, and it should be clearly understood that the foregoing descriptions should be interpreted as illustrative only and not limiting.

Claims

1. A plant biostimulant preparation for promoting plant growth and managing abiotic stress. Its features are, The biological composition comprises 70% w / v of *Blastophyta spp.* extract, 10% w / v of *Ulva prostrata* extract, and 20% w / v of wheatgrass extract; and Extraction is performed using a cold percolation method with a water-ethanol solvent at a ratio of 60:40 v / v to maximize the recovery of active metabolites; and the preparation method of the formulation includes the following steps: Brown and green algae biomass is cleaned by rinsing freshly harvested seaweed with clean water to remove surface salt, sand, and epiphytes. Cleaning plant biomass involves rinsing freshly harvested plant material with clean water to remove sand and other impurities. In a well-ventilated shaded structure, the washed biomass is dried in a cool place, the well-ventilated shaded structure preventing direct ultraviolet radiation while allowing diffused sunlight to pass through, maintaining the temperature below 45°C, until the moisture content drops below 10% or the biomass becomes brittle and fragile. The dried biomass was pulverized using a hammer mill, and the resulting material was passed through a 40-mesh sieve to obtain a fine, uniform powder. The powdered biomass was stored in a sealed, light-proof container at room temperature. Using a water-ethanol solvent system, at a volume ratio of approximately 60:40 (v / v) and a solvent-to-biomass ratio of approximately 10:1, bioactive compounds were extracted from the powdered biomass by cold percolation at ambient temperature over a period of 12 hours to 14 days. The solvent was removed from the extract by rotary evaporation under reduced pressure at a temperature between 40°C and 45°C. The extract was standardized to contain not less than 1,000 ppm of polyphenols and not less than 1,000 ppm of tannins.

2. The biological composition according to claim 1, wherein the brown algae extract improves drought resistance, salt and alkali resistance and heat resistance, the green algae extract enhances plant metabolic activity and growth, and the wheatgrass extract enhances photosynthesis and reduces oxidative stress.

3. The biological composition according to claim 1, wherein the use of a water-ethanol solvent for cold percolation extraction of *Ulva prostrata*, *Ulva spp.*, and wheatgrass achieves higher recovery rates of bioactive compounds, faster processing, and consistency of extract composition, and the cold percolation extraction process retains specific bioactive compounds, particularly secondary metabolites, that are degraded or modified during fermentation.

4. The biological composition according to claim 1, wherein the formulation has a low pH of 3 to 4 and is readily soluble and miscible in water, and has good tank compatibility with a variety of foliar fertilizers and fertilizers including agrochemicals.

5. The biological composition of claim 1, wherein the composition has a shelf life of at least four years when maintained in a temperature range of about 5°C to 45°C.

6. The biological composition of claim 1, wherein the formulation supports continuity between the vegetative growth phase and the reproductive growth phase by integrating energy metabolism, biosynthesis and signal transduction pathways.

7. The biological composition of claim 1, wherein the formulation induces a dual defense mechanism, including ROS scavenging mediated by phenols and immune activation mediated by PR proteins and PAMP-like signaling, thereby exhibiting reduced oxidative stress markers and enhanced disease tolerance.

8. The biological composition according to claim 1, wherein the formulation contains a specific spectrum of flavonoid compounds, including luteolin, vitexin, and quercetin, and regulates auxin transport and signal transduction, thereby promoting vascular tissue integrity, flower differentiation, and fruit germination.

9. The biological composition of claim 1, wherein the formulation avoids a surge in harmful hormones such as GA and auxin, and the formulation is a balanced hormone regulation system that ensures stable yield and reduces physiological imbalances.

10. The biological composition of claim 1, wherein the formulation provides unique synergistic effects in terms of extended shelf life and sensory properties, such as pectin softening via PG enzymes, structural compactness achieved through lignin deposition, volatile organic compounds (VOCs) that contribute to aroma, and pigments and antioxidants that contribute to color stability and nutritional quality.

11. The biological composition according to claim 1, wherein the main delivery methods of the formulation include foliar spraying, root irrigation, granule coating and seed treatment, ensuring efficient uptake and maximum efficacy in a variety of crop species.

12. The biological composition of claim 1, wherein the formulation consistently exhibits a performance improvement of more than 13% to 15% in terms of growth, yield, or quality compared to the use of seaweed or plant extracts alone.

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