Biological product for stimulating plant growth and development, and inhibiting bacteria and fungi

Abstract

FIELD: agriculture.SUBSTANCE: complex plant protection products. The biological preparation for treating plant seeds contains 0.05–5.0 g / l of sodium lignosulfonate powder, or ammonium lignosulfonate, or potassium lignosulfonate and is an aqueous dispersion of lignosulfonate nanoparticles with a size of 20–350 nm, while the content of lignosulfonate in the original powder is at least 63% of dry matter, carbohydrates up to 12%, sodium, or ammonium, or potassium up to 17% and sulfur up to 7%.EFFECT: biopreparation has a growth-stimulating, antibacterial, and antifungal effect, plant protection from pathogenic microorganisms, increased plant resistance to environmental stress, and improved quality of agricultural plants.3 cl, 5 dwg, 4 tbl, 19 ex

Description

[0001] Technical field

[0002] The invention relates to agriculture and biotechnology, namely to complex plant protection preparations that combine the properties of biostimulating the growth and development of plants with antibacterial and antifungal activity, and can be used in plant growing for pre-sowing treatment of seeds of agricultural plants of various botanical species and use groups, improving their growth and stimulating the accumulation of photosynthetic pigments.

[0003] Technology Level

[0004] Pre-sowing seed treatment with natural-based growth regulators effectively stimulates the initial stages of plant growth and enhances seedlings' ability to withstand environmental stressors. Biopolymer-based plant growth and development stimulants, including nanostructured formulations, are environmentally safe and can be used in both open and protected fields. Agro-nanochemicals based on biopolymer nanoparticles are characterized by lower active ingredient consumption, reduced impact on biocenoses, and increased economic viability.

[0005] A polymer composition for fertilizers with fungicidal action is known (see Russian Federation Patent No. 2284705, IPC A23K 3 / 00, published on 10.10.2006), containing lignosulfonate and glycerin in an amount of 6.2-32.0% relative to the weight of lignosulfonate and an additional microelement additive (Cu or Zn) in an amount of 0.5-4.0% relative to the weight of lignosulfonate.

[0006] The disadvantages of the proposed composition are the use of highly concentrated lignosulfonate solutions (up to 30%), as well as the fact that this solution is not intended for treating seeds to stimulate plant growth.

[0007] A plant protection and / or plant fertilizer composition is known (see US application 20100068299, published 03 / 18 / 2010), comprising a soluble combination of a lignosulfonate (15-60 wt.%), a metal salt, and a phosphorus compound selected from the group consisting of phosphoric acid and its soluble salts, as well as mixtures thereof. The lignosulfonate-containing composition is recommended as a fungicide or bactericide for controlling phytopathogens, as well as a plant fertilizer. The composition can be applied to seeds, fruits, flowers or stems of plants, foliage, plant roots, or introduced into the soil or substrate.

[0008] The disadvantage of the proposed composition is its multicomponent nature and high concentration of lignosulfonate.

[0009] The use of lignosulfonates modified by nitration using concentrated nitric acid, or nitrosation using sodium nitrite and an acetic acid solution, as an organic component of a nutrient mixture for plants is known (see Russian patent 2660929, IPC C05F 11 / 00, published 11 / 10 / 2017).

[0010] The disadvantage is the need for chemical modification of lignosulfonate, which is not economically viable, since it significantly increases the cost of the finished product.

[0011] The closest to the claimed invention is an aqueous solution of lignosulfonate containing nanoparticles of 90-380 nm in size (see the article Lugovitskaya TN Sulfite lignin nanoparticles and nanovesicles as biologically active crop growth stimulants / / ACS Appl. Nano Materials. 2022. 5(6). 8048). To obtain nanoparticles, low-molecular (9.25 kDa) or high-molecular (46.30 kDa) fractions of lignosulfonates are used, isolated by preparative ultrafiltration from technical samples. Lignosulfonate is dissolved in water, after which a low-molecular electrolyte (KCl) or an organic antisolvent (ethanol / acetone) is introduced into the system. Since lignosulfonates belong to the class of polyelectrolytes, the introduction of a salt or antisolvent during the formation of nanoparticles leads to charge screening on the macroanion and, as a consequence, compression of the macromolecular coils.A solution containing nanoparticles is recommended for seed treatment and soil modification; they have a growth-promoting effect on radish (Raphanus sativus), garden cress (Lepidium sativum), garden radish (Raphanus raphanistrum subsp. sativus), and common wheat (Tríticum aestívum).

[0012] A disadvantage is the preferential use of narrow lignosulfonate fractions and the need to use potassium chloride or ethanol with acetone to produce nanoparticles. Excessive KCl in the soil can acidify the soil colloid due to the exchangeable absorption of potassium ions, which displaces hydrogen. This can negatively affect plants, especially chlorophobic crops, and reduce the plant-available forms of nitrogen and phosphorus in the soil, thereby limiting the mineral nutrition of crops. Ethanol and acetone pollute the soil, alter its chemical composition, reduce soil fertility, and reduce crop yields. They are also toxic to soil organisms, as they can damage plant cells and disrupt metabolism. Furthermore, neutralization of the charge on the lignosulfonate macroanion by adding KCl or ethanol / acetone is accompanied by a decrease in the biological activity of the sample.

[0013] Disclosure of the essence of the invention

[0014] The technical problem of the claimed invention is the use of a biopreparation for stimulating the growth and development of plants with biologically active lignosulfonate nanoparticles of 20-350 nm in size as a biopreparation that combines the properties of biostimulating the growth and development of plants with antibacterial and antifungal activity.

[0015] The technical result is to increase the effectiveness of the biological product in relation to plants by preserving the charge on the lignosulfonate macromolecules while simplifying the method of their formation.

[0016] The technical result is achieved in that the biological product for treating plant seeds is an aqueous solution of lignosulfonate containing nanoparticles, according to the solution, the solution has a concentration of 0.05 - 5.0 g / l, the nanoparticles have a size of 20-350 nm, while the content of lignosulfonate is up to 63% of dry matter, carbohydrates up to 12%, mineral substances up to 17% and sulfur up to 7%. Sodium lignosulfonate or ammonium lignosulfonate or potassium lignosulfonate is selected as the lignosulfonate, and Na or NH4 as mineral substances + or K. The solution has a concentration of 0.05 - 5.0 g / L to stimulate plant growth and development when treating seeds. The solution has a concentration of 0.25 - 0.5 g / L to inhibit bacteria and fungi.

[0017] Brief description of drawings

[0018] The invention is explained by illustrations, where

[0019] Fig. 1 shows Table 1 - dimensional and physicochemical characteristics of an aqueous dispersion of sodium lignosulfonate nanoparticles;

[0020] Fig. 2 shows Table 2 - morphometric characteristics of seedlings of cucumber (Cucumis sativusL.) variety Nezhinsky, soft wheat (Triticum aestivumL.) variety Novoershovskaya, soybean (Glycine max(L.) Merr.) variety Natalie and lettuce (Lactuca sativaL.) variety Kucheryavets Odessky when soaking seeds in water or in a biopreparation of sodium lignosulfonate nanoparticles of different concentrations or germinating seeds in water or in a biopreparation of sodium lignosulfonate nanoparticles of different concentrations;

[0021] Fig. 3 shows Table 3 - the content of photosynthetic pigments in the leaves of cucumber (Cucumis sativusL.) of the Nezhinsky variety, soft wheat (Triticum aestivumL.) of the Novoershovskaya variety, soybeans (Glycine max(L.) Merr.) of the Natalie variety and lettuce (Lactuca sativaL.) of the Kucheryavets Odessky variety when soaking seeds in water or in a biopreparation of sodium lignosulfonate nanoparticles of different concentrations or germinating seeds in water or in a biopreparation of sodium lignosulfonate nanoparticles of different concentrations;

[0022] Fig. 4 shows Table 4 - the effect of the biopreparation of lignosulfonate nanoparticles on the in vitro growth of bacteria and fungi;

[0023] Fig. 5 shows transmission electron microscopy (TEM) photographs of sodium lignosulfonate nanoparticles;

[0024] Fig. 6 shows cucumber sprouts (Cucumis sativusL.) of the Nezhinsky variety from seeds, sprouted in water (left) or after two-hour soaking (A), and also constantly (B) in an aqueous dispersion of sodium lignosulfonate nanoparticles at a concentration of 5.0, 0.5 and 0.05 g / l (from left to right);

[0025] Fig. 7 shows sprouts of soft wheat (Triticum aestivumL.) of the Novoershovskaya variety from seeds germinated in water (left) or after two-hour soaking (A), and also constantly (B) in an aqueous dispersion of sodium lignosulfonate nanoparticles at a concentration of 5.0, 0.5 and 0.05 g / l (from left to right);

[0026] Fig. 8 shows soybean (Glycine max(L.) Merr.) variety Natalie sprouts from seeds, germinated in water (left) or after two-hour soaking (A), and also constantly (B) in an aqueous dispersion of sodium lignosulfonate nanoparticles at a concentration of 5.0, 0.5 and 0.05 g / l (from left to right);

[0027] in Fig. 9 - sprouts of lettuce (Lactuca sativa L.) of the Kucheryavets Odesskiy variety from seeds, sprouted in water (left) or after two-hour soaking (A), and also constantly (B) in an aqueous dispersion of sodium lignosulfonate nanoparticles at a concentration of 5.0, 0.5 and 0.05 g / l (from left to right).

[0028] Implementation of the invention

[0029] The biopreparation of nanostructured lignosulfonate is obtained by dissolving a sample of lignosulfonate in distilled water, followed by keeping it in a room atmosphere for 24 hours to allow associative processes to occur between the positively charged links of the lignosulfonate macrochains and counterions, accompanied by the spontaneous formation of associates in the form of nanoparticles with an increased charge density and, accordingly, increased biological activity.

[0030] For the preparation of the biopreparation, industrial samples of sodium lignosulfonate or ammonium lignosulfonate or potassium lignosulfonate of the following composition are used: lignosulfonate (LS) - at least 63% of dry matter, carbohydrates - up to 12%, mineral substances - Na / NH4 + / K up to 17%, sulfur up to 7%. Lignosulfonate samples are light yellow to brown powder. A sample of lignosulfonate is dissolved in distilled water to obtain a solution concentration of 5.0 g / L, filtered through a blue ribbon filter, and left for 24 hours until the association processes are complete and equilibrium is reached. The finished biopreparation is an aqueous dispersion of lignosulfonate nanoparticles.

[0031] To obtain working aqueous dispersions with the required concentration of lignosulfonate nanoparticles, the initial dispersion of nanoparticles is diluted with water.

[0032] The particle size (d, nm) is determined by transmission electron microscopy (TEM) on a Jeol JEM 2100 microscope at an accelerating voltage of 200 kV. The specific electrical conductivity (æ уд , S / m) of an aqueous dispersion of lignosulfonate nanoparticles is determined by measuring the resistance on a WTW inoLab Cond 7110 conductometer (WTW, Germany); the surface tension at the liquid-gas interface (σ ж-г , J / m 2 ) - stalagmometric method (glass stalagmometer, Russian Federation); hydrogen index (pH) - on a Mettler Toledo Five Easy FE20 pH meter (MTD, Singapore).

[0033] The completion of the associative processes is judged by the physicochemical parameters of the aqueous dispersion (æ уд , σ ж-г , pH), on the correspondence of the particle size to the nanometer range - according to TEM photographs.

[0034] The effectiveness of a biopreparation based on nanostructured lignosulfonate as a biostimulant on seed germination and seedling parameters of test plants is assessed. To obtain an aqueous dispersion of lignosulfonate nanoparticles for seed treatment, dilution with water is performed to obtain an aqueous dispersion of nanoparticles with a concentration of the original component of 5.0-0.05 g / L.

[0035] Experiments to assess the germination of plant seeds are carried out in accordance with GOST 12038-84 "Seeds of agricultural crops. Methods for determining germination" on filter paper. Before germination, the seeds are disinfected with a 3% hydrogen peroxide solution and washed with sterile distilled water, then soaked in a biostimulant solution or in water for 2 hours. After this, the test seeds are laid out on two or three layers of moistened paper in Petri dishes or between two layers of moistened paper, which is rolled into rolls and placed in a glass. Germination is carried out depending on the type of test seeds in the appropriate conditions: soft wheat Triticum aestivum L. variety Novoershovskaya at a temperature of 20 ° C for 7 days in the dark (in rolls); cucumber Cucumis sativus L. Nezhinsky variety at 25°C for 7 days in the dark (on paper in Petri dishes); soybean Glycine max (L.) Merr. Natalie variety at 25°C for 7 days in the dark (in rolls); lettuce (Lactuca sativa L.) of the Kucheryavets Odesskiy variety were grown at 25°C for 7 days in the dark (on paper in Petri dishes). Four replicates of 100 test seeds each were used for each experiment. Germination (laboratory) was assessed as the ratio of the number of seedlings to the total number of test seeds used for the experiment and expressed as a percentage. When counting germinated seeds, only normally germinated ones are counted. Seeds that were swollen but did not germinate normally were considered non-germinating. The seedlings were assessed for morphological characteristics: shoot length, root length, and shoot and root dry weight.

[0036] Similarly obtained seedlings, but illuminated with Flora fluorescent lamps (Osram) with alternating light / dark in the 16 / 8 mode, were assessed for the content of photosynthetic pigments using the method proposed by Wellburn (Wellburn AR The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution / / J Plant Physiol. 1994. 144. P. 307-313. https: / / doi.org / 10.1016 / S0176-1617(11)81192-2). Leaves are placed in dimethyl sulfoxide at a ratio of 1 mg of leaves to 20 μl of dimethyl sulfoxide. Samples are heated in a water bath at 60°C for 30 minutes until the pigments are completely transferred into solution. The content of green photosynthetic pigments—chlorophylls a and b—and yellow pigments—carotenoids and xanthophylls—is determined by the spectral characteristics of the solution. Optical density is measured at the appropriate wavelength (665, 649, and 480 nm).When measuring the spectrum in a 1-cm cuvette, the concentration of photosynthetic pigments is calculated using the following formulas: Ca = 12.19×A. 665 - 3.45×A 649 ;Cb = 21.99×A 649 - 5.32×A 665 ;Ccar. = (1000×A 480 - 2.14×Ca - 70.16×Cb) / 220, where A 665 - optical density at 665 nm; A 649 - optical density at 649 nm; A 480 - optical density at 480 nm; Ca - chlorophyll a concentration, μg / ml; Cb - chlorophyll b concentration, μg / ml; Ccar. - carotenoid and xanthophyll concentration, μg / ml.

[0037] The results of all experiments to assess the stimulation of plant growth and development, inhibition of phytopathogens are subjected to one-way analysis of variance with determination of the reliability of differences using Fisher's F-test and calculation of the least significant difference at a significance level of 5% (p≤0.05).

[0038] Antibacterial activity is assessed by the growth of bacteria Chitinophaga polysaccharea (IBPPM 680), Azospirillum brasilense SR80 (IBPPM 24), Pseudomonas fluorescens El 2.1 (IBPPM 411), Bacillus subtilis 260 (IBPPM 406) and Acinetobacter guillouiae K2KnO2 (IBPPM 601) on LB agar medium of the following composition (g / l): peptone - 10, yeast extract - 5, NaCl - 10, agar-agar - 20. The biostimulant solution is added to final concentrations of 0.25 and 0.5 g / l. LB containing no drugs is used as a control. During the study, 5 µl of suspensions of 3-day-old bacterial cultures are plated onto Petri dishes containing LB. Cultivation is carried out in a thermostat at 28°C for 7 days, after which the diameter of the grown microbial colonies is measured.

[0039] Antifungal activity is evaluated using Lecanicillum aphanocladii IBPPM 542, Talaromycess ayulitensis IBPPM 664, and Trichodermaviride fungi on a rich basidiomycete medium of the following composition (g / L): NH4NO3, 0.724; KH2PO4, 1.0; MgSO4×7H2O, 1.0; KCl, 0.5; yeast extract, 0.5; FeSO4×7H2O, 0.01; ZnSO4×7H2O, 0.0028; CaCl2×2H2O, 0.033; glucose, 10.0; peptone, 10.0; agar-agar, 15.0; pH 6.0. The biostimulant solution is added to final concentrations of 0.25 and 0.5 g / L. The same medium, without the drug, is used as a control. The fungi are pre-cultivated on the same medium without the drug, then sterilely excised agar blocks containing mycelium, 5 mm in diameter, and transferred to experimental Petri dishes. Cultivation is carried out at 26-28°C for 7 days, after which the colony diameter is measured.

[0040] An example of obtaining an aqueous dispersion of sodium lignosulfonate or ammonium lignosulfonate or potassium lignosulfonate nanoparticles with a concentration of 5.0-0.05 g / l.

[0041] The dimensional and physicochemical characteristics of the aqueous dispersion of sodium lignosulfonate of different concentrations are given in Table 1 (Fig. 1).

[0042] Example 1. To obtain the initial aqueous dispersion of sodium lignosulfonate or ammonium lignosulfonate or potassium lignosulfonate nanoparticles with a concentration of 5.0 g / L, a sample of the LS was dissolved in distilled water with periodic stirring for 30 min, filtered through blue ribbon filter paper and left for 24 hours to allow association processes to occur and achieve equilibrium. The initial aqueous dispersion of lignosulfonate nanoparticles was diluted with distilled water to obtain a dispersion of LS nanoparticles of a lower concentration, the particle size (d), specific electrical conductivity (æ) were determined уд ), surface tension (σ ж-г ) and pH. The completion of the associative processes was assessed based on the physicochemical parameters of the aqueous dispersion of the drug, and the compliance of the drug particle size with the nanometer range was assessed using TEM photographs.

[0043] Characteristics of aqueous dispersions of sodium lignosulfonate with a concentration of 5.0-0.05 g / l: d= 20-350 nm, æ уд = 0.4-9.1⋅10- 5 cm / m, σ ж-г = 61.6-71.4, pH = 4.8-5.6. The resulting nanoparticles retain a high charge density, as evidenced by the increasing dependence of the specific electrical conductivity with decreasing concentration of sodium lignosulfonate in the dispersion. A TEM photograph of sodium lignosulfonate nanoparticles is shown in Fig. 5.

[0044] When using ammonium lignosulfonate or potassium lignosulfonate, the size and physicochemical characteristics of the aqueous dispersion of LS nanoparticles are similar.

[0045] A group of examples of evaluating the germination of test seeds and morphometric parameters of seedlings.

[0046] A comparative assessment of the effect of a biostimulant compared to water on the germination of test seeds and the morphometric parameters of plant crop seedlings is given in Table 2 (Fig. 2).

[0047] Example 2. The original and water-diluted dispersion of sodium lignosulfonate nanoparticles obtained in Example 1 were used to soak test seeds of cucumber (Cucumis sativusL.) variety Nezhinsky, followed by their germination on wet filter paper in Petri dishes under controlled conditions.

[0048] Test seeds of cucumber (Cucumis sativus L.) variety Nezhinsky, soaked for 2 hours in water (1) or in the biopreparation LS at a concentration of 5.0 g / l (2), 0.5 g / l (3) and 0.05 g / l (4), were germinated in Petri dishes on filter paper moistened with distilled water at a temperature of 25 ° C for 7 days in the dark. The shoot length in the control sample (1) was 4.8 cm, in the experimental samples (2) - 7.0 cm, (3) - 6.8 cm, (4) - 7.9 cm, which significantly exceeds the control. Dry root weight in sample (1) was 7.6 mg, in sample (3) - 17.5 mg, which significantly exceeded the control. Seedlings do not differ in other characteristics.

[0049] Cucumber (Cucumis sativusL.) sprouts of the Nezhinsky variety from test seeds germinated in water (left), soaked in an aqueous dispersion of sodium lignosulfonate nanoparticles at a concentration of 5.0, 0.5 and 0.05 g / l (from left to right) are shown in Fig. 6(A).

[0050] Example 3. Conducted similarly to Example 2. The difference is that the cucumber test seeds were germinated in water (1) or biopreparation solutions (2-4) for the entire 7 days. The root length in sample (1) is 7.3 cm, while in sample (3) it is 9.6 cm, significantly exceeding the control. The seedlings were indistinguishable in other respects.

[0051] Cucumber (Cucumis sativusL.) sprouts of the Nezhinsky variety from test seeds germinated in water (left) and in an aqueous dispersion of sodium lignosulfonate nanoparticles at a concentration of 5.0, 0.5 and 0.05 g / l (from left to right) are shown in Fig. 6(B).

[0052] Example 4. Performed similarly to Example 2. The difference is that the test seeds of common wheat (Triticum aestivum L.) of the Novoershovskaya variety were germinated. The shoot dry weight in sample (1) is 8.9 mg, in sample (2) - 16.8 mg, in sample (3) - 21.0 mg, and in sample (4) - 17.7 mg, which significantly exceeds the control. The seedlings do not differ in other characteristics.

[0053] Sprouts of soft wheat (Triticum aestivumL.) variety Novoershovskaya from test seeds germinated in water (left), soaked in an aqueous dispersion of sodium lignosulfonate nanoparticles at a concentration of 5.0, 0.5 and 0.05 g / l (from left to right) are shown in Fig. 7(A).

[0054] Example 5. Performed similarly to example 3. The difference is that the test seeds of soft wheat (Triticum aestivum L.) of the Novoershovskaya variety were germinated. The shoot length in sample (1) is 8.0 cm, in sample (3) - 11.6 cm, which significantly exceeds the control. The root length in sample (1) is 11.0 cm, in sample (3) - 12.5 cm, which significantly exceeds the control. The dry root weight in sample (1) is 14.5 mg, in sample (3) - 21.3 mg, which significantly exceeds the control, in sample (4) - 22.7 mg, which also significantly exceeds the control. The seedlings do not differ in other characteristics.

[0055] Sprouts of soft wheat (Triticum aestivumL.) of the Novoershovskaya variety from test seeds germinated in water (left) and in an aqueous dispersion of sodium lignosulfonate nanoparticles at a concentration of 5.0, 0.5 and 0.05 g / l (from left to right) are shown in Fig. 7(B).

[0056] Example 6. Performed similarly to Example 2. The difference is that test seeds of soybean (Glycine max (L.) Merr.) variety Natalie were used, and germination was carried out in filter paper rolls. The root length in sample (1) is 10.5 cm, in experimental samples (2) - 15.1 cm, (3) - 14.8 cm, (4) - 14.5 cm, which significantly exceeds the control. The dry root weight in sample (1) is 11.2 mg, in experimental samples (2) - 19.3 mg, (3) - 17.8 mg, (4) - 16.7 mg, which significantly exceeds the control. The seedlings do not differ in other characteristics.

[0057] Soybean (Glycine max(L.) Merr.) sprouts of the Natalie variety from test seeds germinated in water (left), soaked in an aqueous dispersion of sodium lignosulfonate nanoparticles at a concentration of 5.0, 0.5 and 0.05 g / l (from left to right) are shown in Fig. 8(A).

[0058] Example 7. The same procedure as Example 3. The difference is that test seeds of soybean (Glycine max(L.) Merr.) of the Natalie variety were germinated. The seedlings from the test seeds did not differ significantly in any way.

[0059] Soybean (Glycine max(L.) Merr.) variety Natalie sprouts from test seeds germinated in water (left) or in an aqueous dispersion of sodium lignosulfonate nanoparticles at concentrations of 5.0, 0.5, and 0.05 g / l (left to right) are shown in Fig. 8(B).

[0060] Example 8. Performed similarly to Example 2. The difference is that the test seeds of lettuce (Lactuca sativa L.) of the Kucheryavets Odessky variety were germinated. The sprout length in sample (1) is 5.7 cm, in experimental samples (2) - 7.1 cm, (3) - 7.0 cm, (4) - 7.1 cm, which significantly exceeds the control. The dry weight of the sprout in sample (1) is 0.8 mg, in experimental samples (2) - 1.4 mg, (3) - 3.0 mg, which significantly exceeds the control. The sprouts do not differ in other characteristics.

[0061] Lettuce sprouts (Lactuca sativa L.) of the Kucheryavets Odesskiy variety from test seeds germinated in water (left), soaked in an aqueous dispersion of sodium lignosulfonate nanoparticles at a concentration of 5.0, 0.5 and 0.05 g / l (from left to right) are shown in Fig. 9(A).

[0062] Example 9. Performed similarly to Example 3. The difference is that the test seeds of lettuce (Lactuca sativa L.) of the Kucheryavets Odessky variety were germinated. The seed germination rate in sample (1) is 85.3%, in sample (4) - 100.0%. The seedling length in sample (1) is 5.8 cm, in the experimental ones (3) - 7.4 cm, (4) - 7.6 cm, which significantly exceeds the control. The dry weight of the seedling in sample (1) is 0.8 mg, in the experimental sample (3) - 2.6 mg, which significantly exceeds the control. The seedlings do not differ in other respects.

[0063] Sprouts of lettuce (Lactuca sativa L.) of the Kucheryavets Odesskiy variety from test seeds germinated in water (left) and in an aqueous dispersion of sodium lignosulfonate nanoparticles at a concentration of 5.0, 0.5 and 0.05 g / l (from left to right) are shown in Fig. 9(B).

[0064] When using ammonium lignosulfonate or potassium lignosulfonate, the effect of the biostimulant on the germination of test seeds and the morphometric parameters of plant crop seedlings is similar compared to water.

[0065] Group of examples of assessing the content of photosynthetic pigments in the leaves of seedlings of test seeds of plant crops.

[0066] A comparative assessment of the effect of a biostimulant compared to water on the content of photosynthetic pigments in the leaves of seedlings of test seeds of plant crops is given in Table 3 (Fig. 3).

[0067] Example 10. Performed similarly to Example 2. The difference is that the test cucumber seeds were germinated in the light. The chlorophyll a content in the leaves of the control sample (1) is 730.6 μg / g, in the test samples (2) - 489.6 μg / g, (3) - 560.3 μg / g, which is significantly lower than the control. The chlorophyll b content in the leaves of sample (1) is 277.4 μg / g, in sample (4) - 330.0 μg / g, which is significantly higher than the control. The carotenoid content in the leaves of the control sample (1) is 84.46 μg / g, in the test samples (2) - 54.1 μg / g, (3) - 57.4 μg / g, which is significantly lower than the control. In the remaining samples, the seedlings did not differ in terms of the content of photosynthetic pigments.

[0068] Example 11. Performed similarly to Example 3. The difference is that the test cucumber seeds were germinated in the light. The chlorophyll-a content in the leaves of sample (1) is 730.6 μg / g, in sample (2) - 1019.8 μg / g, which is significantly higher than the control. The chlorophyll-b content in the leaves of the control sample (1) is 277.4 μg / g, in the test samples (2) - 420.4 μg / g, in sample (3) - 336.5 μg / g, which is significantly higher than the control. The carotenoid content in the leaves of the control sample (1) was 84.46 μg / g, in the experimental samples (2) - 322.9 μg / g, (3) - 105.3 μg / g, which is significantly higher than the control. In the remaining samples, the seedlings did not differ in terms of photosynthetic pigment content.

[0069] Example 12. Conducted similarly to Example 4. The difference is that the test wheat seeds were germinated in the light. The seedlings in all samples showed no differences in photosynthetic pigment content.

[0070] Example 13. Conducted similarly to Example 5. The difference is that the test wheat seeds were germinated in the light. The seedlings in all samples showed no differences in photosynthetic pigment content.

[0071] Example 14. Performed similarly to Example 6. The difference is that the test soybean seeds were germinated in the light. The chlorophyll-a content in the leaves of sample (1) was 1708.9 μg / g, while in sample (4) it was 2212.3 μg / g, which is significantly higher than the control. The seedlings in the other samples did not differ in terms of photosynthetic pigment content.

[0072] Example 15. Performed similarly to Example 7. The difference is that the test soybean seeds were germinated in the light. The chlorophyll a content in the leaves of the control sample (1) is 1708.9 μg / g, in the test samples (3) - 3132.5 μg / g, (4) - 2258.7 μg / g, which is significantly higher than the control. The chlorophyll b content in the leaves of the control sample (1) is 615.7 μg / g, in the test samples (3) - 1059.5 μg / g, (4) - 822.8 μg / g, which is significantly higher than the control. The carotenoid content in the leaves of the control sample (1) was 343.5 μg / g, in the experimental samples (3) - 597.6 μg / g, (4) - 467.8 μg / g, which is significantly higher than the control. In the remaining samples, the seedlings did not differ in terms of photosynthetic pigment content.

[0073] Example 16. Performed similarly to Example 8. The difference is that the test lettuce seeds were germinated in the light. The chlorophyll a content in the leaves of sample (1) is 338.8 μg / g, while in sample (3) it is 400.9 μg / g, which is significantly higher than the control. The chlorophyll b content in the leaves of sample (1) is 90.3 μg / g, while in sample (3) it is 139.6 μg / g, which is significantly higher than the control. The carotenoid content in the leaves of sample (1) is 56.6 μg / g, while in sample (3) it is 80.6 μg / g, which is significantly higher than the control. In the remaining samples, the seedlings did not differ in the content of photosynthetic pigments.

[0074] Example 17. Performed similarly to example 9. The difference is that the test lettuce seeds were germinated in the light. The chlorophyll a content in the leaves of sample (1) is 167.6 μg / g, while in sample (3) it is 274.9 μg / g, which is significantly higher than the control. The chlorophyll b content in the leaves of sample (1) is 90.3 μg / g, while in sample (2) it is 65.1 μg / g, which is significantly lower than the control. The carotenoid content in the leaves of sample (1) is 31.4 μg / g, while in sample (3) it is 54.4 μg / g, which is significantly higher than the control. In the remaining samples, the seedlings did not differ in the content of photosynthetic pigments.

[0075] Group of examples of evaluation of antibacterial and antifungal activity of a biological product.

[0076] The effect of the biopreparation of lignosulfonate nanoparticles on the in vitro growth of bacteria and phytopathogenic fungi is shown in Table 4 (Fig. 4).

[0077] Example 18. A dispersion of sodium lignosulfonate nanoparticles diluted with water, obtained according to Example 1, was taken to evaluate the antibacterial activity of the biopreparation.

[0078] A dispersion of lignosulfonate nanoparticles at final concentrations of 0.25 and 0.5 g / L was added to LB agar medium autoclaved at 120°C for 30 minutes and poured into Petri dishes. LB medium without the addition of lignosulfonate nanoparticles was used as a control. Suspensions of 3-day-old agar bacterial cultures of Acinetobacter guillouiae (1), Azospirillum brasilense (2), Bacillus subtilis (3), Chitinophaga polysaccharea (4), and Pseudomonas fluorescens (5) were plated at 5 μL per Petri dish with LB with or without the addition of LS. Cultivation was carried out in a thermostat at 28°C for 7 days, after which the diameter of the grown colonies of microorganisms was measured. Inhibition of bacterial growth was calculated in comparison with the control variant (without drugs) and was expressed as a % relative to the control.

[0079] The inhibition of bacterial growth in sample (1) was 43.3%, in sample (2) - 21.7-26.1%, in sample (3) - 50.0-54.5%, in sample (4) - 41.7-45.8%, in sample (5) - 24.2-33.3%, which is significantly lower than the control.

[0080] Example 19. A dispersion of sodium lignosulfonate nanoparticles diluted with water, obtained according to Example 1, was taken to evaluate the antifungal activity of the biopreparation.

[0081] A dispersion of lignosulfonate nanoparticles at final concentrations of 0.25 and 0.5 g / L was added to a rich basidiomycete medium autoclaved at 120°C for 20 minutes and poured into Petri dishes. A rich basidiomycete medium without the addition of lignosulfonate nanoparticles was used as a control. The fungi Lecanicillum aphanoclodii (1), Talaromycess ayulitensis (2), and Trichodermaviride (3) were pre-cultivated on the same medium without the addition of LS. Agar blocks containing 5 mm diameter mycelium were then sterilely excised and transferred to experimental Petri dishes. Cultivation was carried out at 26-28°C for 7 days, after which the colony diameter was measured. Inhibition of fungal growth was calculated in comparison with the control variant (without drugs) and was expressed as a % relative to the control.

[0082] The inhibition of fungal growth in sample (1) was 9.3-11.1%, in sample (2) - 40.8-42.9%, in sample (3) - 22.2-24.4%, which is significantly lower than the control.

[0083] It has been experimentally established that the technical result of the invention is achieved when using a biological product in the form of an aqueous dispersion of lignosulfonate nanoparticles, according to the claimed examples 1-17. Example 1 establishes the formation of aqueous dispersions of lignosulfonate nanoparticles with sizes of 20-350 nm. Examples 2-9 demonstrate stimulation of the growth parameters of seedlings of cereals, legumes, greens, and vegetables. Examples 10-17 demonstrate the activity of the biological product in relation to the content of photosynthetic pigments in leaves, which positively influences the accumulation of plastic substances, as well as provitamin A (β-carotene). Examples 18-19 demonstrate the antibacterial and antifungal activities of the biological product.

[0084] Thus, the invention demonstrates high growth-promoting activity of the lignosulfonate nanoparticle biopreparation when evaluating the germination of test seeds, growth, and vegetation of test plants. This activity is reflected in increased seedling growth rates, weight, and accumulation of plastic substances, including provitamin A, compared to the control. This growth-promoting activity is combined with antibacterial and antifungal properties, resulting in suppression of the growth of test bacteria and fungi.

[0085] This biopreparation, an aqueous dispersion of lignosulfonate nanoparticles, is easy to produce due to the good solubility of lignosulfonate in water and the relative simplicity of the self-association processes of charged macrotangles into nanoparticles with a high charge density. The biopreparation is also easy to use. The lignosulfonate used to produce the biopreparation belongs to hazard class 4 (low-hazard substances that do not harm the environment, animals, or humans). In low concentrations, it exhibits a noticeable biostimulating effect. It is produced from inexpensive and annually renewable natural raw materials.

[0086] The combination of properties of biologically active lignosulfonate nanoparticles included in the biopreparation provides not only high growth-stimulating activity and activity in relation to the content of photosynthetic pigments in leaves, but also protection against a number of soil bacteria and fungi, which positively affects the adaptive responses of plants to various environmental factors, the accumulation of plastic substances, as well as provitamin A (β-carotene).

[0087] Another advantage is the stimulation of growth parameters for seedlings of cereals, legumes, greens, and vegetables, as well as the biopreparation's activity in relation to the content of photosynthetic pigments in leaves. Another advantage is the suppression of bacterial and fungal growth.

[0088] Thus, the authors obtained a nanostructured natural polyelectrolyte - anionic lignosulfonate, which spontaneously forms nanoassociates in the form of nanoparticles with an increased charge density and, accordingly, increased biological activity in an aqueous medium.

[0089] Additional Literature

[0090] 1. Lugovitskaya TN, Kolmachikhina EB Associative behavior of lignosulphonates in moderately concentrated water, water-salt, and water-alcoholic media / / Biomacromolecules. 2021. 22(8). 3323.

[0091] 2. Lugovitskaya TN, Ulitko MV, Kozlova NS, Rogozhnikov DA, Mamyachenkov SV Self-assembly polymersomes based on sulfite lignins with biological activity. / / Rus. J. Phys. Chem. A. 2023. 97(3). 534.

[0092] 3. Lugovitskaya TN, Rogozhnikov DA Construction of lignosulphonate-containing polymersomes and prospects for their use for elemental sulfur encapsulation / / J. Mol. Liq. 2024. 400. 124612.

[0093] 4. Lugovitskaya T.N., Rogozhnikov D.A., Mamyachenkov S.V. Preparation of lignosulphonate nanoparticles and their applications in dye removal and as plant growth stimulators / / J. Mol. Liq. 2025. 417. 126693.

Claims

1. A biological preparation for treating plant seeds, characterized by the fact that it contains 0.05-5.0 g / l of sodium lignosulfonate powder, or ammonium lignosulfonate, or potassium lignosulfonate and is an aqueous dispersion of lignosulfonate nanoparticles with a size of 20-350 nm, while the content of lignosulfonate in the original powder is at least 63% of dry matter, carbohydrates up to 12%, sodium, or ammonium, or potassium up to 17% and sulfur up to 7%.

2. A biological product according to paragraph 1, characterized in that it is used in a concentration of 0.05-5.0 g / l to stimulate the growth and development of plants when treating seeds.

3. A biological product according to paragraph 1, characterized in that it is used in a concentration of 0.25-0.5 g / l to inhibit bacteria and fungi when treating seeds.

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